3-Aminoazetidin-2-one derivatives as N-acylethanolamine acid amidase (NAAA) inhibitors suitable for systemic administration

Article abstract of DOI:10.1002/cmdc.201300546

N-Acylethanolamine acid amidase (NAAA) is a cysteine hydrolase that catalyzes the hydrolysis of endogenous lipid mediators such as palmitoylethanolamide (PEA). PEA has been shown to exert anti-inflammatory and antinociceptive effects in animals by engaging peroxisome proliferator-activated receptor α (PPAR-α). Thus, preventing PEA degradation by inhibiting NAAA may provide a novel approach for the treatment of pain and inflammatory states. Recently, 3-aminooxetan-2-one compounds were identified as a class of highly potent NAAA inhibitors. The utility of these compounds is limited, however, by their low chemical and plasma stabilities. In the present study, we synthesized and tested a series of N-(2-oxoazetidin-3-yl)amides as a novel class of NAAA inhibitors with good potency and improved physicochemical properties, suitable for systemic administration. Moreover, we elucidated the main structural features of 3-aminoazetidin-2-one derivatives that are critical for NAAA inhibition. Stability is the key: α-Amino-β-lactams were synthesized as amide derivatives, and the effect of the azetidin-2-one ring, the stereochemistry at the α-position, and the functionalization of the α-amino group were studied with regard to N-acylethanolamine acid amidase inhibitory potency and hydrolytic and plasma stability.

Full text of DOI:10.1002/cmdc.201300546

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DOI: 10.1002/cmdc.201300546
3-Aminoazetidin-2-one Derivatives as N-Acylethanolamine
Acid Amidase (NAAA) Inhibitors Suitable for Systemic
Administration
Annalisa Fiasella,[a] Andrea Nuzzi,[a] Maria Summa,[a] Andrea Armirotti,[a] Glauco Tarozzo,[a]
Giorgio Tarzia,[b] Marco Mor,[c] Fabio Bertozzi,[a] Tiziano Bandiera,*[a] and Daniele Piomelli*[a, d]
N-Acylethanolamine acid amidase (NAAA) is a cysteine hydro-
lase that catalyzes the hydrolysis of endogenous lipid media-
tors such as palmitoylethanolamide (PEA). PEA has been
shown to exert anti-inflammatory and antinociceptive effects
in animals by engaging peroxisome proliferator-activated re-
ceptor a (PPAR-a). Thus, preventing PEA degradation by inhib-
iting NAAA may provide a novel approach for the treatment of
pain and inflammatory states. Recently, 3-aminooxetan-2-one
compounds were identified as a class of highly potent NAAA
inhibitors. The utility of these compounds is limited, however,
by their low chemical and plasma stabilities. In the present
study, we synthesized and tested a series of N-(2-oxoazetidin-
3-yl)amides as a novel class of NAAA inhibitors with good po-
tency and improved physicochemical properties, suitable for
systemic administration. Moreover, we elucidated the main
structural features of 3-aminoazetidin-2-one derivatives that
are critical for NAAA inhibition.
Introduction
Fatty acid ethanolamides (FAEs) are a family of lipid-derived
messengers that play important roles in the control of pain, in-
flammation, and energy balance.[1] Two structurally and func-
tionally distinct classes of FAEs have been described: polyunsa-
turated FAEs, such as arachidonoylethanolamide (anandamide),
which are endogenous ligands for cannabinoid receptors and,
among other functions, participate in the control of stress
coping responses and pain initiation;[2] and monounsaturated
and saturated FAEs, such as oleoylethanolamide (OEA) and pal-
mitoylethanolamide (PEA), which are endogenous ligands for
peroxisome proliferator-activated receptor a (PPAR-a) and are
involved in energy balance, pain, and inflammation.[1a]
Within the latter class, PEA, the endogenous amide of pal-
mitic acid and ethanolamine, has been shown to inhibit pe-
ripheral inflammation and mast cell degranulation[3] and to ex-
hibit antinociceptive properties in rat and mouse models of
acute and chronic pain.[3b,4] Those properties depend on PPAR-
a activation because they are absent in PPAR-a-null mice, are
mimicked by synthetic PPAR-a agonists, and are blocked by
PPAR-a antagonists.[5] Furthermore, PEA has been reported to
suppress pain behaviors induced by tissue injury, nerve
damage, or inflammation in mice[5c] and to attenuate skin in-
flammation and neuropathic pain in humans.[6]
Tissue FAE levels are regulated at the level of biosynthesis
and degradation.[7] A molecularly unique phospholipase d re-
leases FAEs from precursor N-acylphosphatidylethanolamines
(NAPEs) present in cell membranes.[8] Degradation of FAEs to
the corresponding free fatty acids and ethanolamine is carried
out by either fatty acid amide hydrolase (FAAH) or N-acyletha-
nolamine acid amidase (NAAA).[9] These two intracellular lipid
amidases show different cellular localization and substrate se-
lectivity, with NAAA preferentially catalyzing the hydrolysis of
PEA and OEA in innate immune cells.[4b]
NAAA[10] is a cysteine amidase that belongs to the N-terminal
nucleophile (Ntn) family of enzymes,[11] and is characterized by
a conserved amino acid catalytic triad Cys, Arg, and Asp. The
primary structure of NAAA has no homology with that of
FAAH,[11] while it shares 33–34% identity and 70% similarity
with acid ceramidase (AC), a cysteine amidase involved in the
hydrolysis of the sphingolipid messenger ceramide. NAAA is
thought to be localized to lysosomes[12] and shares common
features with other lysosomal hydrolases, that is, N-glycosyla-
tion, autoproteolytic cleavage,[13] and activation at acidic pH
(around 4.5–5.0).[9a] After its maturation, the N-terminal cys-
[a] Dr. A. Fiasella,+ Dr. A. Nuzzi,+ Dr. M. Summa, Dr. A. Armirotti, Dr. G. Tarozzo,
Dr. F. Bertozzi, Dr. T. Bandiera, Prof. D. Piomelli
Drug Discovery and Development, Istituto Italiano di Tecnologia
Via Morego 30, 16163 Genova (Italy)
Fax: (+39)010-71781228
E-mail: tiziano.bandiera@iit.it
[b] Prof. G. Tarzia
Dipartimento di Scienze Biomolecolari
Universitꢀ degli Studi di Urbino “Carlo Bo”
Piazza del Rinascimento 6, 61029 Urbino (Italy)
[c] Prof. M. Mor
Dipartimento di Farmacia, Universitꢀ degli Studi di Parma
Viale della Scienze 27A, 43124 Parma (Italy)
[d] Prof. D. Piomelli
Departments of Anatomy & Neurobiology, Pharmacology, and Biological
Chemistry, University of California, Irvine
3216 Gillespie Neuroscience Facility, Irvine, CA 92697-4621 (USA)
E-mail: daniele.piomelli@iit.it
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201300546.
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teine residue (Cys131 in rodents; Cys126 in humans) is respon-
sible for the catalytic cleavage of nonpeptide CꢀN bonds in
linear amides;[14] for this reason, NAAA is also linked to the
choloylglycine hydrolase superfamily.[11] Site-directed mutagen-
esis[15] and mass spectrometry studies[16] have unambiguously
identified human Cys126 as the nucleophile responsible for
both autoproteolysis and activity.
to 3-aminooxetan-2-one, to test whether replacement of the b-
lactone core with a b-lactam retained inhibitory activity toward
NAAA, while increasing chemical and plasma stabilities.
In the present work, we report the discovery of 3-aminoaze-
tidin-2-ones as a new class of NAAA inhibitors endowed with
good potency and promising chemical and plasma stabilities,
which may make these derivatives suitable for systemic admin-
istration. In particular, we prepared a small series of b-lactam
analogues and evaluated their inhibitory activities toward
human NAAA (h-NAAA). This study allowed us to highlight key
structural features for NAAA inhibition and derive an initial
structure–activity relationship (SAR).
Recently, inhibition of NAAA activity has attracted increasing
interest as a strategy to sustain endogenous PEA and OEA
levels, and this enzyme has been envisaged as a new potential
therapeutic target for inflammation.[5a] So far, only a limited
number of natural[17] or synthetic[18] NAAA inhibitors have been
reported in the literature. Among these, a-amino-b-lactone (3-
aminooxetan-2-one) derivatives have shown considerable
promise because of their high inhibitory potency and target
selectivity.[19] In particular, in vitro and in vivo studies have
demonstrated that (S)-N-(2-oxo-3-oxetanyl)-3-phenylpropiona-
mide ((S)-OOPP, 1), which inhibits rat-NAAA (r-NAAA) with
Results and Discussion
Chemistry
Differently substituted 3-aminoazetidin-2-one derivatives were
efficiently accessed in an enantioselective fashion, starting
from the corresponding d- or l-N-protected-serine derivatives.
As reported in Scheme 1, the designed synthetic route envis-
aged both the (S)- and (R)-2-oxoazetidin-3-ylammonium ace-
tate (9 and 10)[21] as key intermediates for the synthesis of 3-
aminoazetidin-2-one amide derivatives bearing different side
chains. Notably, by modifications and optimization of pub-
lished procedures,[22] compounds 9 and 10 were prepared on
a multigram scale, with 40–44% overall yield over four steps.[23]
First, benzyloxycarbonyl (Cbz)-protected azetidinones 7 and
8 were prepared following a three-step sequence.[22a] Amide
coupling between p-anisidine and N-[(benzyloxycarbonyl)]-l-
and d-serine afforded intermediates 3 and 4, which were con-
a median inhibitory concentration (IC50) of 0.42 mm,[19a,f] blocks
FAE hydrolysis in activated inflammatory cells and dampens
tissue reactions to various pro-inflammatory stimuli.[19b] More
recently, 5-phenylpentyl N-[(2S,3R)-2-methyl-4-oxo-oxetan-3-yl]-
carbamate (ARN077, 2; r-NAAA IC50 =0.050 mm),[19c,d,f] a new
potent and selective b-lactone NAAA inhibitor, was shown to
elevate PEA and OEA levels in
mouse skin and sciatic nerve tis-
sues and to attenuate nocicep-
tion in mice and rats following
topical administration.[20]
Despite their high potency
and efficacy, b-lactone-based
NAAA inhibitors suffer from lim-
ited chemical and plasma stabili-
ties,[19c,e] due to facile ring-open-
ing to the corresponding b-hy-
droxy acid. Their low chemical
stability makes these derivatives
suitable for topical applica-
tions[19c,e] but prevents their use
as systemic agents.
To further explore the thera-
peutic utility of NAAA inhibitors,
we started a program aimed at
identifying new chemical series
that might be suitable for sys-
temic administration. Within this
Scheme 1. Synthetic pathway for the preparation of amide derivatives 11 a–q and 12h. Reagents and conditions:
a) p-anisidine, EDC, THF/CH2Cl2 (3:1), RT, 16 h, 85–92%; b) ImSO2Im, 08C, 1 h, then NaH, DMF, ꢀ208C, 1 h, 75%;
c) CAN, MeCN/H2O (1:1), 08C, 1 h, 80–85%; d) 1,4-cyclohexadiene, 10% Pd on charcoal, EtOH, RT, 12 h, then AcOH,
EtOAc, 79%; Method A [for 11 a, 11 f–h, 11 l, 11 o, and 12h]: RCOCl, Et3N, CH2Cl2 or CH2Cl2/DMF (3:1), RT, 16 h,15–
program, we investigated func-
tionalized a-amino-b-lactam (3-
amidoazetidin-2-one) derivatives,
a chemotype structurally similar 65%; Method B [for 11 b–e, 11 i–k, 11 m–n, and 11 p–q]: RCOOH, TBTU, Et3N, CH2Cl2/DMF (3:1), RT, 16 h, 30–62%.
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verted into the corresponding N-protected a-amino-
b-lactams 5 and 6 by a one-pot cyclization reaction,
which represents the key step in this synthesis. Sub-
sequent p-methoxyphenyl deprotection with ceric
ammonium nitrate in an acetonitrile/water (1:1) mix-
ture led to b-lactams 7 and 8. Cbz deprotection[22b]
was then carried out by catalytic transfer hydrogena-
tion, using 1,4-cyclohexadiene as a hydrogen source
in the presence of 10% palladium on charcoal.[24] The
resulting unprotected 3-aminoazetidin-2-ones were
immediately trapped as acetate salts 9 and 10, which
could be easily stored for several months.
Coupling reactions of salt 9 with the suitable acyl
chloride in the presence of triethylamine afforded
amides 11 a, 11 f–h, 11 l, and 11 o, while reaction of
enantiomer 10 with nonanoyl chloride led to com-
pound 12h (Method A, Scheme 1). Alternatively, the
reaction of compound 9 with the appropriate carbox-
ylic acid, activated with TBTU and triethylamine, gave
amides 11 b–e, 11 i–k, 11 m–n, and 11 p–q (Method B,
Scheme 1). In all coupling reactions, no racemization
was detected at the stereogenic a-center.
Scheme 2. Syntheses of carboxylic acids 13, 14, and 15. Reagents and conditions: a) LiOH,
THF/MeOH/H2O (1:1:1), RT, 1 h, quant.; b) Dess–Martin periodinane, CH2Cl2, 08C, 1 h, then
RT, 1 h, 94%; c) NaClO2, NaH2PO4, 2-methyl-2-butene, tBuOH/H2O 4:1, RT, 1.5 h, quant.;
d) DMSO, oxalyl chloride, CH2Cl2, ꢀ788C, 15 min then 17, ꢀ788C, 1 h, then Et3N, RT,
quant.; e) (EtO)3POCH2CO2Et, NaH (95%), THF, 08C!RT, 16 h, 77%; f) H2, 10% Pd/C car-
tridge (H-Cube, EtOH, 458C, 20 bar, flow: 1.0 mLminꢀ1), 92%; g) LiOH, THF/MeOH/H2O
(1:1:1), RT, 1 h, quant.
All of the employed acyl chlorides and most of the
carboxylic acids were commercially available; carbox-
ylic acids 13, 14, and 15 were prepared as reported
in Scheme 2. Compound 13 was synthesized by
saponification of commercially available methyl (E)-3-
nonenoate (Scheme 2). The preparation of (Z)-non-3-
enoic acid (14) was accomplished in two steps start-
ing from commercially available (Z)-non-3-en-1-ol. Ox-
idation with Dess–Martin periodinane[25] led to the
exclusive formation of (Z)-non-3-enal (16) (Scheme 2).
Further oxidation under Pinnick reaction conditions,
in the presence of 2-methyl-2-butene as a scavenger,
selectively yielded acid 14 as a single diastereomer.
Carboxylic acid 15 was prepared starting from 5-
cyclohexylpentan-1-ol (17),[26] which was first oxidized
to aldehyde 18. Subsequent Horner–Emmons olefina-
tion afforded the a,b-unsaturated ester 19, which
was subjected to hydrogenation, followed by hydro-
lysis, to furnish compound 15 (Scheme 2).
Scheme 3. Syntheses of compounds 21h–24h. Reagents and conditions: a) 1n NaOH,
35%, H2O2, 60% tBuOH·H2O, RT, 16 h, 82%; b) 2n HCl·Et2O, 1,2-bis(trimethylsiloxy)cyclo-
butene, THF, reflux, 3 h, 37%; c) nonanoyl chloride, Et3N, CH2Cl2, RT, 2 h, 94%; d) TFA/
CH2Cl2 (1:3), 08C, 30 min, then RT, 45 min, quant.; e) 2n HCl, RT, 30 min, then THF, RT,
16 h, 79%; Method A: nonanoyl chloride, Et3N, CH2Cl2, RT, 16 h, 83%.
Compounds 21h–24h were synthesized as report-
ed in Scheme 3. Cyclobutanone derivative 21h was
prepared by the acid-catalyzed reaction of 1,2-bis(tri-
methylsilanoxy)cyclobutene with amide 25, obtained
by alkaline hydrolysis of nonanenitrile (Scheme 3).[19c]
The synthesis of compound 22h was accomplished starting
from commercially available N-Boc-3-amino-azetidine by stan-
dard amide coupling with nonanoyl chloride, followed by Boc
deprotection with TFA/CH2Cl2 (Scheme 3). Acid-mediated ring-
opening of 11 h smoothly delivered compound 23h as a hydro-
chloride salt in good yield (Scheme 3). Finally, standard amide
coupling of nonanoyl chloride with commercially available
(3S)-3-aminopyrrolidin-2-one led to derivative 24h (Scheme 3).
N-methylated amides 27h and 28h were prepared as re-
ported in Scheme 4. Alkylation of azetidinone 11 h with methyl
iodide in the presence of sodium hydride in DMF selectively
occurred on the N1 endocyclic nitrogen, providing compound
27h (Scheme 4). Alkylation of protected b-lactam 5 with
methyl iodide under standard conditions afforded compound
29 in good yield. Cleavage of the p-anisyl group, followed by
catalytic hydrogenation, led to N-methyl acetate salt 31, which
gave N-methyl derivative 28h after amide coupling with nona-
noyl chloride using Method A (Scheme 4).
Acetate salt 9 was used to access amine, urea, and carba-
mate derivatives 32, 33, and 34, respectively, as reported in
Scheme 5. Amine derivative 32 was synthesized by reductive
amination of aldehyde 35, obtained by oxidation of n-nonanol
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hydrolysis of N-(4-methyl-2-oxo-chromen-7-yl)hexadecanamide
(PAMCA) by recombinant h-NAAA heterologously expressed in
HEK293 human embryonic kidney cells (see Experimental Sec-
tion).[16] Median inhibitory concentration (IC50) values are re-
ported in Tables 1, 3, and 4. A few representative compounds
were also evaluated for their chemical stability in buffer at
pH 7.4 and 5.0[28] and for their stability in mouse plasma
(Table 2).
We used b-lactam derivative 11 a (Table 1), an analogue of
the known b-lactone NAAA inhibitor (S)-OOPP (1, h-NAAA IC50:
1.29 mm), as the starting point for our initial SAR work. Disap-
pointingly, 11 a turned out to be inactive. However, the com-
pound showed substantially higher hydrolytic stability (t1/2
>
1440 min at both pH 7.4 and pH 5.0) and promising mouse
Scheme 4. Syntheses of N-methylated compounds 27h–28h. Reagents and
conditions: a) NaH (60% in mineral oil), MeI, THF, 08C, 1 h then RT, 4 h, 25%;
b) NaH (95%), MeI, THF, 08C, 1 h then RT, 1 h, 90%; c) CAN, MeCN/H2O (1:1),
08C, 1 h, 92%; d) H2, 10% Pd/C cartridge (H-Cube, EtOAc, 308C, 1.0 bar, flow:
1.0 mLminꢀ1), then AcOH, EtOAc, 78%; Method A: nonanoyl chloride, Et3N,
CH2Cl2, RT, 16 h, 33%.
plasma stability (t1/2 >120 min) and rat plasma stability (t1/2
120 min) relative to (S)-OOPP (t1/2 <1 min) (Table 2).
>
Based on previous SAR results for the b-lactone class,[19d] we
hypothesized that activity might be recovered by increasing
the length of the amide side chain. Therefore, a small set of
analogues was prepared in which the length of the phenylalkyl
chain of 11 a was progressively increased from two to six
methylene units (11 b–e, Table 1). Confirming our hypothesis,
NAAA inhibitory activity, albeit in the high micromolar range,
Table 1. Inhibitory potencies of compounds 11 a–h against human N-acy-
lethanolamine acid amidase (NAAA).
Compd
Structure
IC50 [mm][a]
11 a
n.a.[b]
11 b
11 c
11 d
11 e
73.36ꢁ10.68
27.16ꢁ1.89
12.06ꢁ2.18
0.60ꢁ0.17
Scheme 5. Syntheses of amine, urea, and carbamate derivatives 32–34. Re-
agents and conditions: a) DMSO, oxalyl chloride, CH2Cl2, ꢀ788C, 15 min, then
n-nonanol ꢀ788C, 1 h, then Et3N, RT, quant.; b) 9, Et3N, dichloroethane,
10 min, then Na(OAc)3BH, RT, 1.5 h, 21%; c) nonyl isocyanate, DMAP, pyri-
dine, RT, 16 h, 59%; d) DMAP, 2-DPC, CH2Cl2, RT, 16 h, quant.; e) 9, DIPEA,
CH2Cl2, RT, 16 h, 36%.
with salt 9, while urea 33 was obtained by treating 9 with
nonyl isocyanate in the presence of catalytic DMAP
(Scheme 5). Carbamate 34 was synthesized in a two-step se-
quence. n-Heptanol was reacted with 2-pyridyl carbonate (2-
DPC)[19d,27] and catalytic DMAP to give an isomeric mixture of
2-pyridyl carbonate 36 and 2-oxopyridine-1-carboxylate 37
(65:35 ratio), which was then coupled with 9 to afford desired
compound 34 in good yield (Scheme 5).
11 f
11 g
11 h
27.02ꢁ6.15
1.91ꢁ0.28
0.34ꢁ0.03
Structure–activity relationship (SAR) and stability studies
The aim of the present study was to explore substituted 3-ami-
noazetidin-2-one derivatives as a novel class of NAAA in-
hibitors potentially suitable for systemic administration. The
new compounds were tested for their ability to inhibit the
[a] Values are the meanꢁSEM of three or more determinations. [b] Not
active (n.a.): <10% inhibition at 50 mm.
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Table 2. Stability data of (S)-OOPP (1) and compounds 11 a and 11 h at
various pH values and in mouse (m) and rat (r) plasma.[a]
Table 3. Inhibitory potencies of analogues of compound 11 h against
human N-acylethanolamine acid amidase (NAAA).
Compd
t1/2 [min][b]
t1/2 [min]
Compd
Structure
IC50 [mm][a]
pH 7.4
30
pH 5.0
m plasma[c]
r plasma[d]
1
11 a
11 h
45
<1
>120
103
n.t.[e]
>120
>120
12h
74.52ꢁ9.08
>1440 (92)
>1440 (97)
>1440 (98)
>1440 (100)
[a] All stability values are the meanꢁSEM of three or more determina-
tions. [b] Chemical stability measured after 24 h incubation at 378C in
PBS+10% MeCN, at pH 7.4 and 5.0; values in parentheses represent the
compound remaining (%) after 24 h (1440 min). [c] Measured in 100%
mouse plasma. [d] Compounds tested at 2.5 mm in rat plasma with 2.5%
DMSO. [e] Not tested (n.t.); compound 1 is reported to have t1/2 <10 s in
80% rat plasma.[19c]
21h
22h
n.a.[b]
n.a.[b]
23h
24h
27h
n.a.[b]
n.a.[b]
n.a.[b]
was observed for compounds 11 b (IC50 =73.36 mm) and 11 c
(IC50 =27.16 mm), while 6-phenylhexyl analogue 11 e showed
sub-micromolar potency (IC50 =0.60 mm). Removal of the
phenyl group from 11 e led to compound 11 f (IC50 =
27.02 mm), which showed a greater than 40-fold drop in poten-
cy relative to 11 e. Good inhibitory activity against NAAA was
again recovered by increasing the length of the alkyl chain
from six to eight carbon atoms, as in analogue 11 h (IC50 =
0.34 mm), which turned out to be the most potent derivative
within this small series of compounds. Notably, b-lactam 11 h
retained a favorable chemical and plasma stability profile
28h
32
n.a.[b]
n.a.[b]
(Table 2), proving stable to chemical hydrolysis over 24 h (t1/2
>
1440 min) and showing an acceptable half-life in mouse and
rat plasma (t1/2 =103 min and> 120 min, respectively). These
initial results suggested that N-(2-oxoazetidin-3-yl)amide deriv-
atives may have potential as a new potent and stable class of
NAAA inhibitors.
[a] Values are the meanꢁSEM of three or more determinations. [b] Not
active (n.a.): <10% inhibition at 50 mm.
Encouraged by these results, we considered compound 11 h
as the prototype for this novel class of inhibitors and synthe-
sized an initial set of analogues to identify the key structural
features for NAAA inhibition. We first modified the 3-aminoaze-
tidin-2-one scaffold while maintaining fixed the amide chain;
the stereochemistry at the C3 position was inverted, the nitro-
gen and the carbonyl group of the azetidinone ring were re-
placed by a methylene, and the b-lactam ring was hydrolyzed
and expanded to the five-atom g-lactam homologue. Finally,
the endocyclic nitrogen was methylated to assess the stereo-
electronic requirements at this position.
We then focused our attention on the amide functionality at
the C3 position of the azetidinone ring. The exocyclic nitrogen
was methylated, leading to compound 28h, and the amide
was replaced by an n-nonylamino group, as in compound 32.
Both derivatives turned out to be inactive. A secondary amide
at the C3 position of the azetidinone ring appears, therefore,
to be required for NAAA inhibition.
As the next step in our SAR study, we directed our attention
to the amide side chain and explored the importance of the
conformational flexibility, substitution, and length of the alkyl
chain. First, we introduced a steric constraint in compounds
11 i–j (Table 4), two analogues of 11 h that contain a double
bond in the (E) and (Z) configuration, respectively. Both
changes resulted in a 10-fold drop in potency, with no prefer-
ence for the alkene configuration (11 i, IC50 =3.09 mm; 11 j,
IC50 =3.90 mm). Further reduction of the side chain flexibility by
introduction of a para-substituted phenyl ring, as in com-
pounds 11 k–l, led to a decrease (11 k, IC50 =13.85 mm) or total
loss (11 l) of inhibitory activity. These findings indicated that
the insertion of sterically constrained amide chains is detrimen-
tal for activity, contrary to what was observed for b-lactone
amides.[19c]
The importance of the S configuration for the substituent at
the C3 position was clearly demonstrated by the greater than
200-fold loss in potency observed for compound 12h (IC50 =
74.52 mm, Table 3), the R enantiomer of 11 h. This stereochemi-
cal preference is in agreement with previous observations of
serine-derived b-lactone inhibitors.[19a,b] Replacement of the en-
docyclic nitrogen with a methylene (21h), reduction of the
azetidinone to an azetidine ring (22h), ring-opening of the
azetidinone (23h), and ring-expansion to a g-lactam (24h) all
led to loss of activity, as previously observed in the b-lactone
series.[19a,b] Methylation of the N1 endocyclic nitrogen (27h)
abolished NAAA inhibitory activity. These findings unambigu-
ously indicated that an intact b-lactam moiety was mandatory
for NAAA inhibition.
We also synthesized compounds bearing a branched aliphat-
ic side chain (11 m and 11 n). A single methyl group close to
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The insertion of a terminal cyclohexyl moiety, as in amide
11 q (IC50 =0.28 mm), resulted in a slight decrease in potency
compared with 11 p (Table 4) but a 2-fold potency increase
with respect to phenyl analogue 11 e (Table 1). Finally, we in-
vestigated the impact of the functionality at the N3 exocyclic
amino group of the b-lactam on NAAA inhibition by synthetiz-
ing the corresponding urea (33) and carbamate (34) analogues
of amide 11 h (Table 4). Replacement of the amide moiety with
a urea, as in derivative 33 (IC50 =5.76 mm), resulted in a marked
decrease in potency. By contrast, carbamate 34 (IC50 =0.24 mm)
was notably more potent than amide 11 h.
Table 4. Inhibitory potencies of compounds 11 i–q, 33, and 34 against
human N-acylethanolamine acid amidase (NAAA).
Compd
Structure
IC50 [mm][a]
11 i
3.09ꢁ0.50
11 j
3.90ꢁ0.86
NAAA shows 33–34% identity and 70% similarity to AC,
a cysteine amidase that catalyzes the deactivating hydrolysis of
the pro-inflammatory lipid messenger, ceramide. We therefore
tested the most representative compounds identified in our
study (11 h, its enantiomer 12h, and 11 p) for their selectivity
against human AC and FAAH. The selectivity of compounds
11 h, 12h, and 11 p versus AC was evaluated using a UPLC–
MS-based assay[19a,29] to measure IC50 values under similar ex-
perimental conditions. The results are reported in Table 5.
11 k
13.85ꢁ3.41
11 l
n.a.[b]
11 m
11 n
11 o
11 p
0.22ꢁ0.03
0.76ꢁ0.26
0.24ꢁ0.03
0.10ꢁ0.02
Table 5. Inhibition of human N-acylethanolamine acid amidase (NAAA),
human acid ceramidase (AC), and human fatty acid amide hydrolase
(FAAH) by compounds 11 h, 11 p, and 12h.
Compd
IC50 [mm][a]
Inhib. [%][b]
h-NAAA
h-AC
h-FAAH
11 h
12h
11 p
0.13ꢁ0.03
2.53ꢁ0.06
27.81ꢁ5.38
0.33ꢁ0.10
n.a.
n.a.
n.a.
45.54ꢁ19.94
0.056ꢁ0.010
[a] Values were determined by LC–MS; data are the meanꢁSD of three
or more determinations. [b] Percent inhibitory activity; not active (n.a.):
<10% inhibition at 100 mm.
11 q
0.28ꢁ0.07
33
34
5.76ꢁ0.90
0.24ꢁ0.02
Compound 11 h showed ~19-fold selectivity for NAAA vs.
AC (h-NAAA IC50 =0.13 mm; h-AC IC50 =2.53 mm) while its enan-
tiomer, 12h, displayed low potency toward the two enzymes
(h-NAAA IC50 =45.5 mm; h-AC IC50 =27.8 mm). Interestingly,
lengthening the amide alkyl chain, as in compound 11 p, led to
an approximate 7-fold increase in potency toward h-AC (IC50 =
0.33 mm) and an approximate 2-fold increase in potency
toward h-NAAA (IC50 =0.056 mm). Lengthening of the alkyl
chain appeared, therefore, to have a more pronounced effect
on AC than on NAAA, as indicated by the drop in selectivity
toward AC for analogue 11 p versus 11 h. None of the three se-
lected compounds showed inhibitory activity toward h-FAAH.
The higher selectivity toward AC of 11 h with respect to
11 p, coupled with its greater chemical and plasma stability,
make this compound a potential probe for further exploration
of the functional role of NAAA. To test whether 11 h could be
administered systemically, we dosed the compound intrave-
nously (3.0 mgkgꢀ1) and orally (10 mgkgꢀ1) in rats and deter-
mined its pharmacokinetic (PK) profile. Relevant PK parameters
are reported in Table 6. Although 11 h showed a relatively high
clearance (Cl=702 mLminꢀ1 kgꢀ1) by the intravenous route,
the oral PK profile was characterized by a maximal plasma con-
[a] Values are the meanꢁSEM of three or more determinations. [b] Not
active (n.a.): <10% inhibition at 50 mm.
the amide functionality appeared to be well accommodated as
compound 11 m (IC50 =0.22 mm), even as a mixture of diaste-
reomers, showed a slight increase in potency relative to com-
pound 11 h. However, the introduction of a gem-dimethyl
group in the same position, as in derivative 11 n (IC50 =
0.76 mm), was detrimental for potency.
We then turned our attention to analogues with longer alkyl
chain and prepared compounds containing a decanamide
(11 o) and an undecanamide residue (11 p). These compounds
showed improved NAAA inhibitory activity (11 o, IC50 =
0.24 mm; 11 p, IC50 =0.10 mm). This result supports the idea that
linear lipophilic moieties, which resemble the natural enzyme
substrate, are well accommodated in the active site.
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Experimental Section
Table 6. Plasma pharmacokinetic parameters of compound 11 h after
single intravenous (i.v.) or oral (p.o.) administration in rats.[a]
Chemistry
Parameter
3.0 mgkgꢀ1 (i.v.)
10mgkgꢀ1 (p.o.)[a]
Chemicals, materials and methods: All commercially available re-
agents and solvents were used as purchased from vendors without
further purification. Dry solvents (CH2Cl2, THF, DMF, pyridine, di-
chloroethane) were purchased from Sigma–Aldrich. Optical rota-
tions were measured on a Rudolf Research Analytical Autopol II au-
tomatic polarimeter using a sodium lamp (589 nm) as the light
source; concentrations are expressed in g/100 mL using MeOH as
solvent and a 1 dm cell. Automated column chromatography puri-
fications were done using a Teledyne ISCO apparatus (CombiFlash
Rf) with pre-packed silica gel columns (4 g). Mixtures of increasing
polarity of cyclohexane (Cy) and ethyl acetate (EtOAc) were used
as eluents. Column chromatography was performed manually on
pre-packed silica cartridges (2 g or 5 g) from Biotage or on glass
columns using Merck silica gel 60 (230–400 mesh) as the stationary
phase. Purifications by preparative HPLC–MS were run on a Waters
Autopurification system consisting of a 3100 single quadrupole
mass spectrometer equipped with an electrospray ionization inter-
face and a 2998 photodiode array detector. The HPLC system in-
cluded a 2747 sample manager, a 2545 binary gradient module,
a system fluidic organizer, and a 515 HPLC pump. The PDA range
was 210–400 nm. Purifications were performed on an XBridgeTM
Prep C18 OBD (100ꢁ19 mm i.d., particle size 5 mm) with a XBridgeTM
Prep C18 (10ꢁ19 mm i.d., particle size 5 mm) Guard Cartridge. The
mobile phase was 10 mm NH4OAc in MeCN/H2O (95:5) at pH 5.
Electrospray ionization (ESI) was used in positive and negative
mode. Hydrogenation reactions were performed using H-Cube
continuous hydrogenation equipment (SS-reaction line version),
employing disposable catalyst cartridges (CatCart) preloaded with
the required heterogeneous catalyst. NMR experiments were run
on a Bruker Avance III 400 system (400.13 MHz for 1H, and
100.62 MHz for 13C) equipped with a BBI probe and Z-gradients.
Spectra were acquired at 300 K using deuterated dimethyl sulfox-
ide ([D6]DMSO) or deuterated chloroform (CDCl3) as solvents.
UPLC–MS analyses were run on a Waters Acquity UPLC–MS system
consisting of a SQD (single quadrupole detector) mass spectrome-
ter equipped with an electrospray ionization interface and a photo-
diode array detector. The PDA range was 210–400 nm. Analyses
were performed on an Acquity UPLC HSS T3 C18 column (50ꢁ
2.1 mm i.d., particle size 1.8 mm) with a VanGuard HSS T3 C18 pre-
column (5ꢁ2.1 mm i.d., particle size 1.8 mm). The mobile phase
was either 10 mm NH4OAc in H2O at pH 5, adjusted with AcOH (A)
and 10 mm NH4OAc in MeCN/H2O (95:5) at pH 5 (B). ESI was ap-
plied in positive and negative mode. Accurate mass measurement
(HMRS) was performed on a Synapt G2 quadrupole-Tof instrument
(Waters, USA), equipped with an ESI ion source.
Cmax [ngmLꢀ1
]
397
0.08
111
702
–
570
0.25
247
–
T
max [h]
AUC [nghmLꢀ1
]
Cl [mLminꢀ1 kgꢀ1
]
F [%]
67
[a] Maximum observed concentration (Cmax); cumulative area under curve
(AUC) for experimental time points (0–24 h); systemic clearance (Cl)
based on observed data points (0–24 h); bioavailability (F). Compound
was dosed in 10% PEG400/10% Tween 80/80% saline solution; three ani-
mals per dosage were treated.
centration (Cmax) of 570 ngmLꢀ1 at 15 min post-dosing and
a bioavailability of 67% (Table 6). These results support the use
of 11 h as an NAAA inhibitor for systemic administration.
Conclusions
In the present work, we report the discovery of 3-aminoazeti-
din-2-one derivatives as a novel class of NAAA inhibitors. A
series of N-(2-oxoazetidin-3-yl)amides were synthesized and
tested to identify key structural features for NAAA inhibition.
Our results showed that the b-lactam moiety is mandatory for
activity, and that alkylation of the endocyclic nitrogen is not
tolerated. The S configuration of the acylamino substituent at
C3 position is strongly preferred over the R configuration. The
potency of N-(2-oxoazetidin-3-yl)amides as NAAA inhibitors is
modulated by the amide alkyl chain, with long and flexible
alkyl residues being preferred over sterically constrained
chains. Interestingly, we found out that the amino group on
the 3-aminoazetidin-2-one scaffold could be further derivatized
as a carbamate (34) with retention of NAAA inhibitory activity
(Figure 1). Further studies on this new series of derivatives are
Figure 1. Summary of key structural features for b-lactam-based NAAA
All final compounds (11 a–q, 12h, 21h–24h, 27h, 28h, and 32–
34) showed ꢂ95% purity by NMR and UPLC–MS analysis. The syn-
theses of reaction intermediates 9, 10, 13–15, 25, 26h, 31, and
35–37 are described in the Supporting Information.
inhibitors.
currently ongoing. Among the synthesized compounds, N-[(S)-
2-oxoazetidin-3-yl]nonanamide (11 h) showed good inhibitory
potency (IC50 =0.34 mm) against NAAA, ~19-fold selectivity
versus AC (a closely related cysteine amidase), excellent chemi-
cal stability in buffer, and acceptable plasma stability. Impor-
tantly, this compound showed good oral bioavailability in rats.
Compound 11 h and other potent N-(2-oxoazetidin-3-yl)-
amide analogues represent promising probes that may help
further characterize the functional roles of NAAA and assess
the therapeutic potential of systemically available NAAA inhibi-
tors.
General procedures for the synthesis of amide derivatives 11a,
11 f–h, 11 l, 11o and 12h via Method A (Scheme 1): Under nitro-
gen atmosphere, dry Et3N (2.1 equiv) and the suitable acid chloride
(1.1 equiv) were added to a cooled (08C), stirred suspension of (2-
oxoazetidin-3-yl)ammonium acetate (9 or 10, 1.0 equiv) in dry
CH2Cl2 (0.07 m solution) or to a mixture of dry CH2Cl2/DMF (3:1;
0.07 m solution). The resulting mixture was stirred at room temper-
ature for 16 h, then diluted with CH2Cl2 and washed with saturated
NH4Cl solution, saturated NaHCO3 solution, and brine. The organic
phase was dried over Na2SO4, filtered, concentrated to dryness,
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and purified according to the specific conditions described in each
example.
[MꢀH]ꢀ; HRMS-ESI: m/z [M+H]+ calcd for C12H23N2O2: 227.1760,
found: 227.1771.
3-Phenyl-N-[(S)-2-oxoazetidin-3-yl]propanamide (11a): The reac-
tion was carried out following Method A, using salt 9 (0.050 g,
0.34 mmol), commercially available hydrocinnamoyl chloride
(0.056 mL, 0.38 mmol), and dry Et3N (0.10 mL, 0.71 mmol) in dry
N-[(S)-2-Oxoazetidin-3-yl]-4-phenylbenzamide (11l): The reaction
was carried out following Method A, using salt
9 (0.13 g,
0.89 mmol), commercially available 4-phenylbenzoyl chloride
(0.21 g, 0.98 mmol), and dry Et3N (0.26 mL, 1.87 mmol) in dry
CH2Cl2/DMF (4.0 mL). After workup, trituration with EtOAc afforded
compound 11 l (0.10 g, 42%) as a white solid: Rt =1.89 min;
1H NMR (400 MHz, [D6]DMSO): d=9.14 (d, 1H, J=8.5 Hz), 8.05 (bs,
1H), 7.97 (d, 2H, J=8.4 Hz), 7.79 (d, 2H, J=8.4 Hz), 7.74 (d, 2H, J=
7.4 Hz), 7.50 (t, 2H, J=7.6 Hz), 7.45–7.38 (m, 1H), 5.09 (ddd, 1H,
J=8.5, 5.2, 2.5 Hz), 3.49 (t, 1H, J=5.2 Hz), 3.27 ppm (dd, 1H, J=
5.2, 2.5 Hz); 13C NMR (100 MHz, [D6]DMSO): d=168.6, 166.1, 143.5,
139.5, 132.8, 129.4, 128.5, 127.3, 126.9, 58.5, 43.3; MS (ESI, +) m/z:
267 [M+H]+, 289 [M+Na]+; MS (ESI,ꢀ) m/z: 265 [MꢀH]ꢀ; HRMS-
ESI: m/z [M+H]+ calcd for C16H15N2O2: 267.1134, found: 267.1133.
CH2Cl2 (5.0 mL). After workup, trituration with EtOAc afforded com-
25
pound 11 a (0.080 g, 15%) as a white solid: Rt =1.40 min; ½aꢃD
=
ꢀ10.8 cm3 gꢀ1 dmꢀ1 (c=0.09 in MeOH); 1H NMR (400 MHz,
[D6]DMSO): d=8.51 (d, 1H, J=8.2 Hz), 7.96 (bs, 1H), 7.29–7.24 (m,
2H), 7.22–7.14 (m, 3H), 4.87–4.80 (m, 1H), 3.38 (t, 1H, J=5.4 Hz),
2.99 (dd, 1H, J=5.4, 2.6 Hz), 2.81 (t, 2H, J=7.9 Hz), 2.41 ppm (t,
2H, J=7.9 Hz); 13C NMR (100 MHz, [D6]DMSO): d=171.4, 168.0,
141.1, 128.3, 128.2, 125.4, 56.9, 42.9, 36.8, 30.9 ppm; MS (ESI, +) m/
z: 219 [M+H]+, 241 [M+Na]+, 257 [M+K]+; MS (ESI,ꢀ) m/z: 217
[MꢀH]ꢀ; HRMS-ESI: m/z [M+H]+ calcd for C12H15N2O2: 219.1134,
found: 219.1136.
N-[(S)-2-Oxoazetidin-3-yl]decanamide (11o): The reaction was car-
ried out following Method A, using salt 9 (0.050 g, 0.34 mmol),
commercially available decanoyl chloride (0.077 mL, 0.38 mmol),
and dry Et3N (0.11 mL, 0.71 mmol) in dry CH2Cl2 (6.0 mL). After
workup, purification by typical silica gel flash chromatography
N-[(S)-2-Oxoazetidin-3-yl]heptanamide (11 f): The reaction was
carried out following Method A, using salt 9 (0.050 g, 0.34 mmol),
commercially available heptanoyl chloride (0.058 mL, 0.38 mmol),
and dry Et3N (0.1 mL, 0.71 mmol) in dry CH2Cl2 (5.0 mL). After
workup, purification by typical silica gel flash chromatography
(CH2Cl2/MeOH, from 100:0 to 96:4) afforded compound 11 o
25
(CH2Cl2/MeOH, from 100:0 to 96:4) afforded compound 11 f
(0.051 g, 63%) as
a
white solid: Rt =2.31 min; ½aꢃD
=
25
(0.023 g, 34%) as
a
white solid: Rt =1.63 min; ½aꢃD
=
ꢀ16.2 cm3 gꢀ1 dmꢀ1 (c=0.07 in MeOH); 1H NMR (400 MHz,
[D6]DMSO): d=8.43 (d, 1H, J=8.4 Hz), 7.94 (s, 1H), 4.82 (ddd, 1H,
J=8.4, 5.4, 2.7 Hz), 3.38 (t, 1H, J=5.4 Hz), 3.02 (dd, 1H, J=5.4,
2.7 Hz), 2.08 (t, 2H, J=7.5 Hz), 1.53–1.42 (m, 2H), 1.33–1.16 (m,
12H), 0.86 ppm (t, 3H, J=7.1 Hz); 13C NMR (100 MHz, [D6]DMSO):
d=172.7, 168.7, 57.3, 43.3, 35.6, 31.7, 29.3, 29.2, 29.1, 29.0, 25.5,
22.6, 14.4 ppm; MS (ESI, +) m/z: 241 [M+H]+, 263 [M+Na]+, 279
[M+K]+; MS (ESI,ꢀ) m/z: 239 [MꢀH]ꢀ; HRMS-ESI: m/z [M+H]+
calcd for C13H25N2O2: 241.1916, found: 241.1920.
ꢀ20.3 cm3 gꢀ1 dmꢀ1 (c=0.07 in MeOH); 1H NMR (400 MHz,
[D6]DMSO): d=8.43 (d, 1H, J=8.3 Hz), 7.94 (bs, 1H), 4.82 (ddd, 1H,
J=8.3, 5.4, 2.7 Hz), 3.38 (t, 1H, J=5.4 Hz), 3.02 (dd, 1H, J=5.4,
2.7 Hz), 2.08 (t, 2H, J=7.4 Hz), 1.53–1.42 (m, 2H), 1.32–1.17 (m,
6H), 0.85 ppm (t, 3H, J=7.0 Hz); 13C NMR (100 MHz, [D6]DMSO):
d=172.7, 168.7, 57.3, 43.3, 35.6, 31.5, 28.7, 25.5, 22.4, 14.4 ppm;
MS (ESI, +) m/z: 199 [M+H]+, 221 [M+Na]+, 237 [M+K]+; MS
(ESI,ꢀ) m/z: 197 [MꢀH]ꢀ; HRMS-ESI: m/z [M+H]+ calcd for
C10H19N2O2: 199.1447, found: 199.1449.
N-[(R)-2-Oxoazetidin-3-yl]nonanamide (12h): The reaction was
carried out following Method A, using salt 10 (0.090 g, 0.62 mmol),
commercially available nonanoyl chloride (0.13 mL, 0.68 mmol),
and dry Et3N (0.18 mL, 1.03 mmol) in dry CH2Cl2 (9.0 mL). After
workup, trituration with EtOAc afforded compound 12h (0.074 g,
53%) as a white solid: Rt =2.13 min; ½aꢃ2D5 = +14.3 cm3 gꢀ1 dmꢀ1
N-[(S)-2-Oxoazetidin-3-yl]octanamide (11g): The reaction was car-
ried out following Method A, using salt 9 (0.060 g, 0.41 mmol),
commercially available octanoyl chloride (0.076 mL, 0.45 mmol),
and dry Et3N (0.11 mL, 0.86 mmol) in dry CH2Cl2 (6.0 mL). After
workup, trituration with EtOAc afforded compound 11 g (0.019 g,
22%) as a white solid: Rt =1.88 min; ½aꢃ2D5 =ꢀ10.5 cm3 gꢀ1 dmꢀ1 (c=
1
(c=0.07 in MeOH); H NMR (400 MHz, [D6]DMSO): d=8.42 (d, 1H,
1
0.07 in MeOH); H NMR (400 MHz, [D6]DMSO): d=8.43 (d, 1H, J=
J=8.3 Hz), 7.94 (bs, 1H), 4.83 (ddd, 1H, J=8.3, 5.3, 2.7 Hz), 3.38 (t,
1H, J=5.3 Hz), 3.02 (dd, 1H, J=5.3, 2.7 Hz), 2.08 (t, 2H, J=7.3 Hz),
1.53–1.42 (m, 2H), 1.31–1.18 (m, 10H), 0.86 ppm (t, 3H, J=6.8 Hz);
13C NMR (100 MHz, [D6]DMSO): d=172.2, 168.2, 56.8, 42.8, 35.1,
31.2, 28.7, 28.6, 28.5, 25.1, 22.1, 13.9 ppm; MS (ESI, +) m/z: 227
[M+H]+, 249 [M+Na]+, 265 [M+K]+; MS (ESI,ꢀ) m/z: 225
[MꢀH]ꢀ; HRMS-ESI: m/z [M+H]+ calcd for C12H23N2O2: 227.1760,
found: 227.1766.
8.2 Hz), 7.94 (bs, 1H), 4.82 (ddd, 1H, J=8.2, 5.4, 2.4 Hz), 3.38 (t, 1H,
J=5.4 Hz), 3.02 (dd, 1H, J=5.4, 2.4 Hz), 2.08 (t, 2H, J=7.4 Hz),
1.53–1.42 (m, 2H), 1.32–1.17 (m, 8H), 0.85 ppm (t, 3H, J=7.0 Hz);
13C NMR (100 MHz, [D6]DMSO): d=172.2, 168.2, 56.8, 42.8, 35.1,
31.1, 28.5, 28.4, 25.1, 22.0, 13.9 ppm; MS (ESI, +) m/z: 213 [M+H]+,
251 [M+K]+; HRMS-ESI: m/z [M+H]+ calcd for C11H21N2O2:
213.1603, found: 213.1611.
N-[(S)-2-Oxoazetidin-3-yl]nonanamide (11h): The reaction was
carried out following Method A, using salt 9 (0.60 g, 4.1 mmol),
commercially available nonanoyl chloride (0.85 mL, 4.51 mmol),
and dry Et3N (1.2 mL, 8.6 mmol) in dry CH2Cl2 (60 mL). After
workup, trituration with EtOAc afforded compound 11 h (0.60 g,
65%) as a white solid: Rt =2.13 min; ½aꢃ2D5 =ꢀ18.6 cm3 gꢀ1 dmꢀ1 (c=
General procedures for the synthesis of amide derivatives 11b–
e, 11 i–k, 11 m–n, and 11p–q via Method B (Scheme 1): Under ni-
trogen atmosphere, dry Et3N (2.2 equiv) was added to a cooled
(08C) solution of the suitable carboxylic acid (1.1 equiv) in dry
CH2Cl2 (0.07 m solution) or in a 3:1 mixture of dry CH2Cl2/DMF
(0.07 m solution), followed by addition of N,N,N’,N’-tetramethyl-O-
(benzotriazol-1-yl)uronium tetrafluoroborate (TBTU, 1.1 equiv). The
resulting reaction mixture was stirred for 10 min, then (S)-2-oxoaze-
tidin-3-yl)ammonium acetate (9, 1.0 equiv) was added. The reaction
mixture was stirred at room temperature for 16 h, then diluted
with CH2Cl2 and washed with saturated NH4Cl solution, saturated
NaHCO3 solution, and brine. The organic phase was dried over
Na2SO4, filtered, concentrated to dryness, and purified according to
the specific conditions described in each example.
1
0.07 in MeOH); H NMR (400 MHz, [D6]DMSO): d=8.42 (d, 1H, J=
8.3 Hz), 7.94 (bs, 1H), 4.83 (ddd, 1H, J=8.3, 5.3, 2.7 Hz), 3.38 (t, 1H,
J=5.3 Hz), 3.02 (dd, 1H, J=5.3, 2.7 Hz), 2.08 (t, 2H, J=7.3 Hz),
1.53–1.42 (m, 2H), 1.31–1.18 (m, 10H), 0.86 ppm (t, 3H, J=6.8 Hz);
13C NMR (100 MHz, [D6]DMSO): d=172.2, 168.2, 56.8, 42.8, 35.1,
31.2, 28.7, 28.6, 28.5, 25.1, 22.1, 13.9 ppm; MS (ESI, +) m/z: 227
[M+H]+, 249 [M+Na]+, 265 [M+K]+; MS (ESI,ꢀ) m/z: 225
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N-[(S)-2-Oxoazetidin-3-yl]-4-phenylbutanamide (11b): The reac-
tion was carried out following Method B, using salt 9 (0.060 g,
0.41 mmol), commercially available 4-phenylbutanoic acid (0.074 g,
0.45 mmol), TBTU (0.144 g, 0.45 mmol), and dry Et3N (0.12 mL,
0.90 mmol) in dry CH2Cl2/DMF (6.0 mL). After workup, purification
by silica gel column chromatography (Cy/EtOAc, from 100:0 to
10:90) afforded compound 11 b (0.032 g, 34%) as a white solid:
Rt =1.62 min; ½aꢃD25 =ꢀ10.8 cm3 gꢀ1 dmꢀ1 (c=0.09 in MeOH);
1H NMR (400 MHz, [D6]DMSO): d=8.46 (d, 1H, J=8.4 Hz), 7.94 (bs,
1H), 7.33–7.24 (m, 2H), 7.20–7.16 (m, 3H), 4.82 (ddd, 1H, J=8.4,
5.4, 2.5 Hz), 3.39 (t, 1H, J=5.4 Hz), 3.03 (dd, 1H, J=5.4, 2.5 Hz),
2.55 (t, 2H, J=7.5 Hz), 2.12 (t, 2H, J=7.5 Hz), 1.79 ppm (p, 2H, J=
7.5 Hz); 13C NMR (100 MHz, [D6]DMSO): d=171.9, 168.4, 141.7,
128.9, 126.0, 125.7, 56.9, 43.1, 34.5, 34.6, 26.9 ppm; MS (ESI, +) m/z:
233 [M+H]+, 250 [M+Na]+, 271 [M+K]+; HRMS-ESI: m/z [M+H]+
calcd for C13H17N2O2: 233.129, found: 233.1299.
K]+; HRMS-ESI: m/z [M+H]+ calcd for C16H23N2O2: 275.1760, found:
275.1766.
(E)-N-[(S)-2-Oxoazetidin-3-yl]non-3-enamide (11i): The reaction
was carried out following Method B, using salt
9 (0.050 g,
0.34 mmol), (E)-3-nonenoic acid (13) (0.058 g, 0.37 mmol), TBTU
(0.12 g, 0.37 mmol), and dry Et3N (0.10 mL, 0.75 mmol) in dry
CH2Cl2 (5.0 mL). After workup, trituration with EtOAc afforded com-
25
pound 11 i (0.035 g, 46%) as a white solid: Rt =2.04 min; ½aꢃD
=
ꢀ16.5 cm3 gꢀ1 dmꢀ1 (c=0.07 in MeOH); 1H NMR (400 MHz,
[D6]DMSO): d=8.45 (d, 1H, J=8.4 Hz), 7.96 (bs, 1H), 5.54–5.41 (m,
2H), 4.81 (ddd, 1H, J=8.4, 5.5, 2.6 Hz), 3.38 (t, 1H, J=5.5 Hz), 3.03
(dd, 1H, J=5.5, 2.6 Hz), 2.83 (d, 2H, J=5.6 Hz), 2.01–1.93 (m, 2H),
1.37–1.19 (m, 6H), 0.86 ppm (t, 3H, J=7.1 Hz); 13C NMR (100 MHz,
[D6]DMSO): d=171.0, 168.5, 133.5, 124.0, 57.3, 43.2, 39.6, 32.3, 31.3,
28.9, 22.4, 14.4 ppm; MS (ESI, +) m/z: 225 [M+H]+, 247 [M+Na]+,
263 [M+K]+; HRMS-ESI: m/z [M+H]+ calcd for C12H21N2O2:
225.1603, found: 225.1614.
5-Phenyl-N-[(S)-2-oxoazetidin-3-yl]pentanamide (11c): The reac-
tion was carried out following Method B, using salt 9 (0.050 g,
0.34 mmol), commercially available 5-phenylpentanoic acid
(0.067 g, 0.38 mmol), TBTU (0.12 g, 0.38 mmol), and dry Et3N
(0.10 mL, 0.71 mmol) in dry CH2Cl2 (6.0 mL). After workup, tritura-
tion with Et2O afforded compound 11 c (0.028 g, 33%) as a white
solid: Rt =1.79 min; ½aꢃ2D5 =ꢀ40.2 cm3 gꢀ1 dmꢀ1 (c=0.07 in MeOH);
1H NMR (400 MHz, [D6]DMSO): d=8.46 (d, 1H, J=8.3 Hz), 7.94 (bs,
1H), 7.30–7.23 (m, 2H), 7.21–7.13 (m, 3H), 4.82 (ddd, 1H, J=8.3,
5.4, 2.6 Hz), 3.38 (t, 1H, J=5.4 Hz), 3.02 (dd, 1H, J=5.4, 2.6 Hz),
2.56 (t, 2H, J=7.2 Hz), 2.12 (t, 2H, J=6.8 Hz), 1.60–1.45 ppm (m,
4H); 13C NMR (100 MHz, [D6]DMSO): d=172.6, 168.6, 142.5, 128.7,
128.6, 126.1, 57.3, 43.31, 35.4, 35.3, 30.9, 25.2 ppm; MS (ESI, +) m/z:
247 [M+H]+, 269 [M+Na]+, 285 [M+K]+; MS (ESI,ꢀ) m/z: 245
[MꢀH]ꢀ; HRMS-ESI: m/z [M+H]+ calcd for C14H19N2O2: 247.1447,
found: 247.1458.
(Z)-N-[(S)-2-Oxoazetidin-3-yl]non-3-enamide (11j): The reaction
was carried out following Method B, using salt
9 (0.050 g,
0.34 mmol), (Z)-non-3-enoic acid (14; 0.059 g, 0.38 mmol), TBTU
(0.12 g, 0.38 mmol), and dry Et3N (0.10 mL, 0.71 mmol) in dry
CH2Cl2 (3.0 mL). After workup, trituration with EtOAc afforded com-
25
pound 11 j (0.034 g, 45%) as a white solid: Rt =1.98 min; ½aꢃD
=
ꢀ7.6 cm3 gꢀ1 dmꢀ1 (c=0.07 in MeOH); 1H NMR (400 MHz,
[D6]DMSO): d=8.50 (d, 1H, J=8.3 Hz), 7.97 (bs, 1H), 5.52–5.42 (m,
2H), 4.82 (ddd, 1H, J=8.3, 5.4, 2.5 Hz), 3.39 (t, 1H, J=5.4 Hz), 3.03
(dd, 1H, J=5.4, 2.5 Hz), 2.94–2.86 (m, 2H), 2.04–1.96 (m, 2H), 1.37–
1.18 (m, 6H), 0.86 ppm (t, 3H, J=7.1 Hz); 13C NMR (100 MHz,
[D6]DMSO): d=170.4, 168.0, 131.8, 122.9, 56.9, 42.7, 34.0, 30.8, 28.5,
26.8, 21.9, 13.9 ppm; MS (ESI, +) m/z: 225 [M+H]+, 247 [M+Na]+,
263 [M+K]+; MS (ESI,ꢀ) m/z: 223 [MꢀH]ꢀ; HRMS-ESI: m/z [M+H]+
calcd for C12H21N2O2: 225.1603, found: 225.1612.
N-[(S)-2-Oxoazetidin-3-yl]-6-phenylhexanamide (11d): The reac-
tion was carried out following Method B, using salt 9 (0.060 g,
0.41 mmol), commercially available 6-phenylhexanoic acid
(0.084 mL, 0.45 mmol), TBTU (0.144 g, 0.45 mmol), and dry Et3N
(0.12 mL, 0.90 mmol) in dry CH2Cl2/DMF (6.0 mL). After workup, trit-
uration with Et2O afforded compound 11 d (0.032 g, 30%) as
a white solid: Rt =1.98 min; ½aꢃ2D5 =ꢀ12.9 cm3 gꢀ1 dmꢀ1 (c=0.07 in
4-Butyl-N-[(S)-2-oxoazetidin-3-yl]benzamide (11k): The reaction
was carried out following Method B, using salt
9 (0.060 g,
0.41 mmol), commercially available 4-butylbenzoic acid (0.080 g,
0.45 mmol), TBTU (0.144 g, 0.45 mmol), and dry Et3N (0.12 mL,
0.90 mmol) in dry CH2Cl2 (6.0 mL). After workup, trituration with
Et2O afforded compound 11 k (0.029 g, 29%) as a white solid: Rt =
2.07 min; ½aꢃD25 =ꢀ7.5 cm3 gꢀ1 dmꢀ1 (c=0.07 in MeOH); 1H NMR
(400 MHz, [D6]DMSO): d=8.99 (d, 1H, J=8.4 Hz), 8.02 (bs, 1H), 7.78
(d, 2H, J=8.2 Hz), 7.29 (d, 2H, J=8.2 Hz), 5.05 (ddd, 1H, J=8.4,
5.4, 2.6 Hz), 3.46 (t, 1H, J=5.4 Hz), 3.24 (dd, 1H, J=5.4, 2.6 Hz),
2.63 (t, 2H, J=7.5 Hz), 1.63–1.50 (m, 2H), 1.37–1.20 (m, 2H),
0.89 ppm (t, 3H, J=7.4 Hz); 13C NMR (100 MHz, [D6]DMSO): d=
168.3, 166.0, 146.3, 131.1, 128.3, 127.4, 57.3, 42.5, 34.6, 32.8, 217,
13.7 ppm; MS (ESI, +) m/z: 247 [M+H]+, 269 [M+Na]+, 285 [M+
K]+; HRMS-ESI: m/z [M+H]+ calcd for C14H19N2O2: 247.1447, found:
247.1457.
1
MeOH); H NMR (400 MHz, [D6]DMSO): d=8.44 (d, 1H, J=8.2 Hz),
7.94 (bs, 1H), 7.29–7.23 (m, 2H), 7.20–7.13 (m, 3H), 4.82 (ddd, 1H,
J=8.2, 5.4, 2.5 Hz), 3.38 (t, 1H, J=5.4 Hz), 3.01 (dd, 1H, J=5.4,
2.5 Hz), 2.58–2.52 (m, 2H), 2.08 (t, 2H, J=7.4 Hz), 1.60–1.42 (m,
4H), 1.32–1.20 ppm (m, 2H); 13C NMR (100 MHz, [D6]DMSO): d=
172.5, 168.5, 142.2, 128.3, 128.2, 125.6, 56.8, 42.8, 35.5, 35.4, 30.7,
28.2, 24.9 ppm; MS (ESI, +) m/z: 261 [M+H]+, 283 [M+Na]+, 299
[M+K]+; HRMS-ESI: m/z [M+H]+ calcd for C15H21N2O2: 261.1603,
found: 261.1603.
7-Phenyl-N-[(S)-2-oxoazetidin-3-yl]heptanamide (11e): The reac-
tion was carried out following Method B, using salt 9 (0.030 g,
0.21 mmol), commercially available 7-phenylheptanoic acid
(0.065 mL, 0.23 mmol), TBTU (0.073 g, 0.23 mmol), and dry Et3N
(0.06 mL, 0.46 mmol) in dry CH2Cl2 (3.0 mL). After workup, tritura-
tion with EtOAc afforded compound 11 e (0.022 g, 38%) as a white
solid: Rt =2.19 min; ½aꢃ2D5 =ꢀ17.6 cm3 gꢀ1 dmꢀ1 (c=0.07 in MeOH);
1H NMR (400 MHz, [D6]DMSO): d=8.43 (d, 1H, J=8.4 Hz), 7.94 (bs,
1H), 7.29–7.23 (m, 2H), 7.20–7.13 (m, 3H), 4.82 (ddd, 1H, J=8.4,
5.3, 2.6 Hz), 3.38 (t, 1H, J=5.3 Hz), 3.02 (dd, 1H, J=5.3, 2.6 Hz),
2.59–2.53 (m, 2H), 2.08 (t, 2H, J=7.4 Hz), 1.60–1.42 (m, 4H), 1.32–
1.21 ppm (m, 4H); 13C NMR (100 MHz, [D6]DMSO): d=172.7, 168.6,
142.8, 128.7, 128.6, 126.0, 57.4, 43.3, 35.6, 35.5, 31.4, 28.9, 28.8,
25.5 ppm; MS (ESI, +) m/z: 275 [M+H]+, 297 [M+Na]+, 313 [M+
(2R)- and (2S)-2-Methyl-N-[(3S)-2-oxoazetidin-3-yl]nonanamide
(11m): The reaction was carried out following Method B, using salt
9
(0.045 mg, 0.31 mmol), 2-methylnonanoic acid (0.059 mg,
0.34 mmol), TBTU (0.109 mg, 0.34 mmol), and dry Et3N (0.090 mL,
0.68 mmol) in dry CH2Cl2 (4.5 mL). After workup, purification by
silica gel flash chromatography using a Teledyne ISCO apparatus
(Cy/EtOAc from 90:10 to 0:100) afforded compound 11 m (0.024 g,
32%) as a clear liquid 1:1 mixture of isomers: Rt =2.28 min;
1H NMR (400 MHz, [D6]DMSO): d=8.41 (t, 2H, J=8.0 Hz), 7.94 (bs,
2H), 4.85–4.78 (m, 2H), 3.44–3.36 (m, 2H), 3.01 (ddd, 2H, J=8.0,
5.2, 2.7 Hz), 2.30–2.15 (m, 2H), 1.50–1.44 (m, 2H), 1.23 (s, 22H),
0.99–0.97 (m, 6H), 0.85 ppm (t, 6H, J=6.8 Hz); 13C NMR (100 MHz,
[D6]DMSO): d=175.8, 168.2, 56.9, 43.0, 42.9, 33.8, 31.2, 29.0, 28.9,
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28.7, 26.9, 26.8, 22.1, 17.9, 17.8, 14.0 ppm; MS (ESI, +) m/z: 241
[M+H]+, 258 [M+NH4]+, 279 [M+K]+; HRMS-ESI: m/z [M+H]+
calcd for C13H25N2O2: 241.1916, found: 241.1923.
2H), 1.32–1.17 (m, 10H), 0.86 ppm (t, 3H, J=6.8 Hz); 13C NMR
(100 MHz, [D6]DMSO): d=207.5, 172.3, 64.0, 41.6, 35.2, 31.7, 29.2,
29.1, 29.0, 25.5, 22.5, 18.8, 14.4 ppm. MS (ESI, +) m/z: 226 [M+H]+,
248 [M+Na]+, 264 [M+K]+; HRMS-ESI: m/z [M+H]+ calcd for
C13H24NO2: 226.1807, found: 226.1814.
2,2-Dimethyl-N-[(S)-2-oxoazetidin-3-yl]nonanamide (11n): The re-
action was carried out following Method B, using salt 9 (0.045 mg,
0.31 mmol), commercially available 2,2-dimethylnonanoic acid
(0.064 mg, 0.34 mmol), TBTU (0.144 g, 0.45 mmol), and dry Et3N
(0.090 mL, 0.68 mmol) in dry CH2Cl2/DMF (3:1, 4.4 mL). After
N-(Azetidin-3-yl)nonanamide (22h): A mixture of TFA/CH2Cl2 (1:3;
4.0 mL) was added dropwise to a cooled (08C), stirred solution of
Boc-derivative 26 (0.060 g, 0.19 mmol) in CH2Cl2 (3.0 mL). The reac-
tion mixture was stirred at 08C for 30 min, then at room tempera-
ture for an additional 30 min. The solution was diluted with sat.
Na2CO3 solution until neutralization. The organic phase was sepa-
rated, and the aqueous phase was extracted from CH2Cl2 (15.0 mL)
and EtOAc (15.0 mL). The combined organic phases were dried
over Na2SO4, filtered, and concentrated to dryness, affording com-
pound 22h (0.095 g, quant.), as a colorless oil: Rt =1.92 min;
1H NMR (400 MHz, [D6]DMSO): d=8.62 (bs, 1H), 8.50 (d, 1H, J=
6.5 Hz), 4.55 (sex, 1H, J=15.1, 7.6 Hz), 4.11–4.04 (m, 1H), 3.92–3.85
(m, 1H), 2.08 (t, 2H, J=7.4 Hz), 1.55–1.40 (m, 2H), 1.32–1.17 (m,
10H), 0.86 ppm (t, 3H, J=6.8 Hz); 13C NMR (100 MHz, [D6]DMSO):
d=172.9, 52.6, 41.4, 35.6, 31.7, 29.2, 29.1, 29.0, 25.4, 22.5,
14.4 ppm; MS (ESI, +) m/z: 213 [M+H]+, 235 [M+Na]+; HRMS-ESI:
m/z [M+H]+ calcd for C12H25N2O: 213.1967, found: 213.1977.
workup, purification by preparative HPLC afforded compound 11 n
25
(0.026 g, 33%) as
a
clear liquid: Rt =2.47 min; ½aꢃD
=
ꢀ3.50 cm3 gꢀ1 dmꢀ1 (c=0.10 in MeOH); 1H NMR (400 MHz,
[D6]DMSO) d=8.01 (d, 1H, J=8.4 Hz), 7.88 (bs, 1H), 4.79 (ddd, 1H,
J=8.4, 5.4, 2.8 Hz), 3.34 (t, 1H, J=5.4 Hz), 3.09 (dd, 1H, J=5.4,
2.8 Hz), 1.45–1.36 (m, 2H), 1.29–1.07 (m, 10H), 1.04 (s, 6H), 0.84 (t,
3H, J=6.9 Hz); 13C NMR (100 MHz, [D6]DMSO): d=177.0, 168.6,
57.1, 42.4, 41.5, 40.6, 31.3, 29.6, 28.7, 25.3, 25.2, 24.2, 22.1,
14.0 ppm; MS (ESI, +) m/z: 255 [M+H]+, 272 [M+NH4]+, 277 [M+
Na]+; HRMS-ESI: m/z [M+H]+ calcd for C14H27N2O2: 255.2073,
found: 255.2084.
N-[(S)-2-Oxoazetidin-3-yl]undecanamide (11p): The reaction was
carried out following Method B, using salt 9 (0.060 g, 0.41 mmol),
commercially available undecanoic acid (0.084 g, 0.45 mmol), TBTU
(0.144 g, 0.45 mmol), and dry Et3N (0.12 mL, 0.90 mmol) in dry
[(S)-3-Hydroxy-2-(nonanoylamino)-3-oxo-propyl]ammonium
chloride (23h): b-lactam amide 11 h (0.040 g, 0.18 mmol) was sus-
pended in 2n HCl (3.0 mL), the mixture was vigorously stirred at
room temperature for 30 min. THF (2.0 mL) was then added to the
suspension to dissolve any insoluble residue. The resulting solution
was stirred at room temperature for 16 h, the solvents were evapo-
rated, and trituration with EtOAc afforded compound 23h (0.040 g,
79%) as a white solid: Rt =1.68 min; ½aꢃ2D5 =ꢀ10.0 cm3 gꢀ1 dmꢀ1 (c=
0.11 in MeOH); 1H NMR (400 MHz, [D6]DMSO): d=13.00 (bs, 1H),
8.31 (d, 1H, J=8.0 Hz), 8.17 (bs, 3H), 4.47 (dt, 1H, J=8.0, 5.2 Hz),
3.19 (dd, 1H, J=13.0, 5.2 Hz), 3.00 (dd, 1H, J=13.0, 8.9 Hz), 2.15 (t,
2H, J=7.6 Hz), 1.56–1.46 (m, 2H), 1.33–1.19 (m, 10H), 0.90–
0.82 ppm (m, 3H); 13C NMR (100 MHz, [D6]DMSO): d=173.4, 171.3,
50.4, 35.7, 31.7, 29.3, 29.1, 25.4, 22.6, 14.4 ppm; MS (ESI, +) m/z:
245 [M+H]+, 267 [M+Na]+, 283 [M+K]+; HRMS-ESI: m/z [M+H]+
calcd for C12H25N2O3: 245.1865, found: 245.1873.
CH2Cl2 (6.0 mL). After workup, trituration with EtOAc afforded com-
25
pound 11 p (0.065 g, 62%) as a white solid: Rt =2.54 min; ½aꢃD
=
ꢀ15.6 cm3 gꢀ1 dmꢀ1 (c=0.08 in MeOH); 1H NMR (400 MHz,
[D6]DMSO): d=8.43 (d, 1H, J=8.3 Hz), 7.94 (bs, 1H), 4.82 (ddd, 1H,
J=8.3, 5.4, 2.5 Hz), 3.38 (t, 1H, J=5.4 Hz), 3.02 (dd, 1H, J=5.4,
2.5 Hz), 2.08 (t, 2H, J=7.4 Hz), 1.54–1.42 (m, 2H), 1.33–1.18 (m,
14H), 0.84 ppm (t, 3H, J=6.4 Hz); 13C NMR (100 MHz, [D6]DMSO):
d=172.3, 168.2, 55.9, 42.9, 35.2, 31.3, 29.0, 28.9, 28.8, 28.7, 28.6,
25.1, 22.1, 14.0 ppm; MS (ESI, +) m/z: 255 [M+H]+, 293 [M+K]+;
HRMS-ESI: m/z [M+H]+ calcd for C14H27N2O2: 255.2073, found:
255.2083.
6-Cyclohexyl-N-[(S)-2-oxoazetidin-3-yl]hexanamide (11q): The re-
action was carried out following Method B, using salt 9 (0.050 g,
0.34 mmol), 7-cyclohexylheptanoic acid (15) (0.08 g, 0.38 mmol),
TBTU (0.12 g, 0.38 mmol), and dry Et3N (0.100 mL, 0.75 mmol) in
dry CH2Cl2 (6.0 mL). After workup, trituration with Et2O afforded
compound 11 q (0.033 g, 35%) as a white solid: Rt =2.73 min;
½aꢃD25 =ꢀ14.5 cm3 gꢀ1 dmꢀ1 (c=0.07 in MeOH); 1H NMR (400 MHz,
[D6]DMSO): d=8.42 (d, 1H, J=8.3 Hz), 7.94 (bs, 1H), 4.85–4.79 (m,
1H), 3.38 (t, 1H, J=5.3 Hz), 3.02 (dd, 1H, J=5.3, 2.7 Hz), 2.08 (t,
2H, J=7.4 Hz), 1.70–1.55 (m, 5H), 1.53–1.42 (m, 2H), 1.28–1.07 (m,
12H), 0.89–0.77 ppm (m, 2H); 13C NMR (400 MHz, [D6]DMSO) d=
172.7, 168.6, 57.3, 43.3, 37.5, 37.4, 35.6, 33.4, 29.6, 29.1, 26.7, 26.6,
26.3, 25.5 ppm; MS (ESI, +) m/z: 281 [M+H]+, 303 [M+Na]+, 319
[M+K]+; MS (ESI,ꢀ) m/z: 279 [MꢀH]ꢀ; HRMS-ESI: m/z [M+H]+
calcd for C16H29N2O2: 281.2229, found: 281.2237.
N-[(3S)-2-Oxopyrrolidin-3-yl]nonanamide (24h): The reaction was
carried out following Method A, using commercially available (3S)-
3-aminopyrrolidin-2-one (0.100 g, 1.0 mmol), commercially avail-
able nonanoyl chloride (0.24 mL, 1.10 mmol), and dry Et3N
(0.15 mL, 1.10 mmol) in dry CH2Cl2 (8 mL). After workup, trituration
with Et2O afforded compound 24h (0.20 g, 83%) as a white solid:
Rt =2.09 min; 1H NMR (400 MHz, [D6]DMSO): d=7.99 (d, 1H, J=
8.3 Hz), 7.76 (bs, 1H), 4.27 (dt, 1H, J=10.3, 8.3 Hz), 3.20–3.11 (m,
2H), 2.32–2.23 (m, 1H), 2.07 (t, 2H, J=7.4 Hz), 1.81–1.69 (m, 1H),
1.53–1.43 (m, 2H), 1.31–1.20 (m, 10H), 0.85 ppm (t, 3H, J=6.6 Hz);
13C NMR (100 MHz, [D6]DMSO): d=174.5, 172.2, 49.3, 38.0, 35.2,
31.2, 28.7, 28.6, 28.5, 25.2, 22.1, 13.9 ppm; MS (ESI, +) m/z: 241
[M+H]+; MS (ESI,ꢀ) m/z: 239 [MꢀH]ꢀ; HRMS-ESI: m/z [M+H]+
calcd for C13H25N2O2: 241.1916, found: 241.192.
N-(2-Oxocyclobutyl)nonanamide (21h): Under nitrogen atmos-
phere, amide 25 (0.210 g, 1.34 mmol) was dissolved in dry THF
(15.0 mL), and commercially available 2n HCl·Et2O solution
(15.0 mL) was added at 08C. Commercially available bis(trimethylsi-
lyloxy)cyclobutene (0.33 mL, 1.27 mmol) was then added in one
portion. The solution was refluxed for 3 h and then concentrated
to dryness. Purification by typical silica flash chromatography (Cy/
EtOAc, 60:40), and preparative HPLC afforded compound 21h
(0.110 g, 37%) as a white solid: Rt =2.48 min; 1H NMR (400 MHz,
[D6]DMSO): d=8.27 (d, 1H, J=7.9 Hz), 4.77 (dt, 1H, J=10.3,
7.9 Hz), 2.93–2.80 (m, 1H), 2.79–2.68 (m, 1H), 2.19 (qd, 1H, J=10.3,
4.5 Hz), 2.06 (t, 2H, J=7.3 Hz), 2.02–1.91 (m, 1H), 1.52–1.40 (m,
N-[(S)-1-methyl-2-oxo-azetidin-3-yl]nonanamide (27h): Under ni-
trogen atmosphere, a suspension of NaH (60% mineral oil, 0.015 g,
0.362 mmol) in dry THF (4.0 mL) was added dropwise to a cooled
(08C) solution of amide 11 h in dry THF (2.5 mL). The mixture was
warmed to room temperature, stirred for an additional 20 min, and
cooled again to 08C. MeI (0.022 mL, 0.362 mmol) was added drop-
wise, and the resulting reaction mixture was maintained at 08C for
3 h, warmed to room temperature, and stirred for additional 2 h
and 30 min. The reaction mixture was diluted with CH2Cl2 (25 mL)
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and water (2.0 mL). The aqueous layer was extracted with CH2Cl2 (3
x10 mL), and the combined organic layers were dried over Na2SO4,
filtered, and concentrated to dryness. Purification by preparative
HPLC afforded pure compound 27h (0.020 g, 25% yield) as
a white solid: Rt =2.23 min; ½aꢃ2D5 =ꢀ6.7 cm3 gꢀ1 dmꢀ1 (c=0.07 in
Heptyl N-[(S)-2-oxoazetidin-3-yl]carbamate (34): Under nitrogen
atmosphere, DIPEA (0.053 mL, 0.32 mmol) was added dropwise to
a suspension of salt 9 (0.040 g, 0.27 mmol) in dry CH2Cl2 (4.0 mL).
Subsequently, the crude mixture (0.179 g) containing 37 (0.064 g,
0.27 mmol) in dry CH2Cl2 (2.0 mL) was added. The reaction mixture
was stirred at room temperature for 15 h, then concentrated to
dryness. Purification by typical silica gel flash chromatography (Cy/
EtOAc, from 100:0 to 40:60) afforded compound 34 (0.022 g, 36%)
as a white solid: Rt =2.18 min; ½aꢃ2D5 =ꢀ15.1 cm3 gꢀ1 dmꢀ1 (c=0.05
1
MeOH); H NMR (400 MHz, [D6]DMSO): d=8.42 (d, 1H, J=8.1 Hz),
4.81 (ddd, 1H, J=8.1, 5.2, 2.4 Hz), 3.46 (t, 1H, J=5.2 Hz), 3.08 (dd,
1H, J=5.2, 2.4 Hz), 2.73 (s, 3H), 2.07 (t, 2H, J=7.4 Hz), 1.55–1.42
(m, 2H), 1.33–1.17 (m, 10H), 0.86 ppm (t, 3H, J=6.8 Hz); 13C NMR
(100 MHz, [D6]DMSO: d=172.2, 167.1, 56.0, 49.0, 35.1, 31.2, 28.7,
28.6, 28.5, 28.1, 25.1, 22.1, 13.9 ppm; MS (ESI, +) m/z: 241 [M+H]+,
263 [M+Na]+, 279 [M+K]+; HRMS-ESI: m/z [M+H]+ calcd for
C13H25N2O2: 241.1916, found: 241.1918.
1
in MeOH); H NMR (400 MHz, [D6]DMSO): d=7.90 (bs, 1H), 7.78 (d,
1H, J=8.6 Hz), 4.58–4.62 (m, 1H), 3.95 (t, 2H, J=6.7 Hz), 3.37 (t,
1H, J=5.4 Hz), 3.07 (dd, 1H, J=5.4, 2.7 Hz), 1.59–1.48 (m, 2H),
1.35–1.21 (m, 8H), 0.86 ppm (t, 3H, J=6.9 Hz); 13C NMR (100 MHz,
[D6]DMSO): d=168.2, 155.6, 64.1, 58.3, 42.6, 31.2, 28.6, 28.3, 25.3,
22.0, 13.9 ppm; MS (ESI, +) m/z: 229 [M+H]+, 251 [M+Na]+, 267
[M+K]+; HRMS-ESI: m/z [M+Na]+ calcd for C11H20N2O3Na:
251.1372, found: 251.1374.
N-Methyl-N-[(S)-2-oxoazetidin-3-yl]nonanamide (28h): The reac-
tion was carried out following Method A, using salt 31 (0.050 g,
0.31 mmol), commercially available nonanoyl chloride (0.076 mL,
0.34 mmol), and dry Et3N (0.091 mL, 0.66 mmol) in dry CH2Cl2
(5.0 mL). After workup, purification by preparative HPLC afforded
compound 28h (0.027 g, 33%) as an oil in a 1:1 mixture of two ro-
tamers: Rt =2.31 min; ½aꢃ2D5 =ꢀ16.3 cm3 gꢀ1 dmꢀ1 (c=0.12 in
MeOH); 1H NMR (400 MHz, [D6]DMSO): d=8.14 (bs, 1H), 8.07 (bs,
1H), 5.50–5.45 (m, 1H), 5.33–5.27 (m, 1H), 3.43 (t, 1H, J=5.8 Hz),
3.35 (t, 1H, J=5.8 Hz), 3.22 (dd, 1H, J=5.8, 2.5 Hz), 3.17 (dd, 1H,
J=5.8, 2.5 Hz), 2.90 (s, 3H), 2.74 (s, 3H), 2.42–2.23 (m, 4H), 1.53–
1.40 (m, 4H), 1.33–1.16 (m, 20H), 0.86 ppm (t, 6H, J=7.0 Hz);
13C NMR (100 MHz, [D6]DMSO): d=172.6, 172.2, 167.4, 166.8, 64.6,
62.0, 32.6, 32.4, 31.4, 31.2, 28.8, 28.7, 28.6, 28.0, 24.9, 24.4, 22.1,
14.0 ppm; MS (ESI, +) m/z: 241 [M+H]+, 263 [M+Na]+, 279 [M+
K]+; HRMS-ESI: m/z [M+H]+ calcd for C13H25N2O2: 241.1916, found:
241.1918.
Pharmacology
Fluorogenic h-NAAA assay: HEK293 human embryonic kidney cells
stably transfected with the human NAAA coding sequence cloned
from a human spleen cDNA library were used as the enzyme
source. Recombinant HEK-h-NAAA pellets were resuspended in ho-
mogenizing buffer and sonicated. Samples were centrifuged at
800 g for 15 min at 48C, and the resulting supernatants were then
ultracentrifuged at 12000 g for 30 min at 48C. The pellets were re-
suspended in PBS at pH 7.4 on ice and subjected to two freeze/
thaw cycles at ꢀ808C. The suspension was finally centrifuged at
105000 g for 1 h at 48C. Protein concentration was measured, and
samples were aliquoted and stored at ꢀ808C until use. The assay
was run in Optiplate 96-well black plates (PerkinElmer Inc., Boston,
MA, USA) in a total reaction volume of 200 mL. NAAA protein prep-
aration (20 mg) was pre-incubated for 10 min with various concen-
trations of test compounds or vehicle control (5% DMSO) in
100 mm citrate/phosphate buffer (pH 4.5) containing 3 mm DTT,
0,1% Triton X-100, 0.05% BSA, and 150 mm NaCl. N-(4-methyl-2-
oxo-chromen-7-yl)hexadecanamide (PAMCA)[16] was used as a sub-
strate (5 mm), and the reaction was carried out over 30 min at
378C. The samples were then read in a PerkinElmer Envision plate
reader (PerkinElmer Inc., Boston, MA, USA) using an excitation
wavelength of 360 nm and emission of 460 nm. IC50 values were
calculated by non-linear regression analysis of log[concentration]/
inhibition curves using GraphPad Prism 5 (GraphPad Software Inc.,
CA, USA) applying a standard slope curve fitting.
(S)-3-(Nonylamino)azetidin-2-one (32): Under nitrogen atmos-
phere, dry Et3N (0.050 mL, 0.38 mmol) was added to a stirred sus-
pension of salt 9 (0.050 g, 0.34 mmol) in dry dichloroethane
(3.5 mL). The resulting suspension was stirred for 10 min, then a so-
lution of aldehyde 35 (0.049 g, 0.34 mmol) in dry dichloroethane
(0.5 mL) and Na(OAc)3BH (0.100 g, 0.48 mmol) were added to the
reaction mixture. The solution was stirred for 90 min, then diluted
with EtOAc (20.0 mL) and washed with saturated NaHCO3 solution
(10.0 mL). The organic phase was dried over Na2SO4, filtered, and
concentrated to dryness. Purification by typical silica flash chroma-
tography (CH2Cl2/MeOH, from 97:3 to 95:5) afforded compound 32
(0.015 g, 21%) as a white solid: Rt =2.37 min; 1H NMR (400 MHz,
[D6]DMSO): d=7.72 (bs, 1H), 4.00 (bs, 1H), 3.24 (t, 1H, J=5.2 Hz),
2.92 (dd, 1H, J=5.6, 2.4 Hz), 2.63–2.52 (m, 2H), 1.42–1.16 (s, 14H),
0.86 ppm (d, 3H, J=7.0 Hz); 13C NMR (100 MHz, [D6]DMSO): d=
168.5, 67.5, 46.5, 43.0, 31.8, 30.3, 29.5, 29.4, 29.1, 27.2, 22.6,
14.4 ppm; MS (ESI, +) m/z: 213 [M+H]+, 235 [M+Na]+; HRMS-ESI:
m/z [M+H]+ calcd for C12H25N2O: 213.1967, found: 213.1977.
In vitro chemical stability: Chemical stability of selected compounds
was evaluated under physiological pH conditions (0.01 m phos-
phate-buffered saline, pH 7.4) and acidic pH conditions (0.01 m
phosphate buffer, pH 5.0) for up to 24 h. Both buffers were added
with 10% of CH3CN. Stock solutions of each compound (10 mm)
were prepared freshly in DMSO. Each compound was incubated at
a final concentration of 1 mm (1% DMSO) in both preheated buf-
fers (378C). The sample solutions were divided into aliquots in
glass vials (preheated at 378C) for each time point. The samples
were maintained at 378C in the UPLC–MS autosampler during the
study (without shaking). A reference solution of each compound
(final concentration: 1 mm at 1% DMSO) in preheated CH3CN
(378C) was prepared from the stock solutions (10 mm in DMSO)
and maintained at 378C in the UPLC–MS autosampler during the
study (without shaking). The analyses were performed on a Waters
Acquity UPLC–MS triple quadrupole detector (TQD) system consist-
ing of a TQD mass spectrometer equipped with an ESI interface
and a photodiode array detector. The 24 h time course analyses for
1-Heptyl-3-[(S)-2-oxoazetidin-3-yl]urea (33): DMAP (0.048 g,
0.39 mmol) was added to a solution of salt 9 (0.047 g, 0.32 mmol)
in dry pyridine (4.0 mL), followed by the addition of heptyl isocya-
nate (0.057 mL, 0.36 mmol). The reaction mixture was stirred at
room temperature for 16 h and concentrated to dryness. Tritura-
tion with CH2Cl2 afforded compound 33 (0.040 g, 59%) as a white
solid: Rt =1.90 min; ½aꢃ2D5 = + 2.07 cm3 gꢀ1 dmꢀ1 (c=0.08 in MeOH);
1H NMR (400 MHz, [D6]DMSO): d=7.83 (bs, 1H), 6.50 (d, 1H, J=
8.4 Hz), 5.94 (t, 1H, J=5.4 Hz), 4.80–4.63 (m, 1H), 3.34 (t, 1H, J=
5.4 Hz), 3.03–2.99 (m, 1H), 2.99–2.92 (m, 2H), 1.31–1.14 (m, 10H),
0.94–0.81 ppm (m, 3H); 13C NMR (100 MHz, [D6]DMSO): d=169.4,
157.0, 57.9, 43.8, 31.3, 29.9, 28.4, 26.3, 22.0, 13.9 ppm; MS (ESI, +)
m/z: 228 [M+H]+, 250 [M+Na]+; HRMS-ESI: m/z [M+H]+ calcd
for C11H22N3O2: 228.1712, found: 228.1718.
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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1, 11 a, and 11 h were carried out by UPLC-UV on the same instru-
ment described above. A calibration curve in the 0.2–50 mm con-
centration range was prepared for each parent compound by
serial dilution in CH3CN (R2 values were >0.999 for all compounds).
The concentrations of the rearranged and hydrolyzed products of
each parent were then calculated on the corresponding calibration
curve, assuming no changes in the molar absorptivity values (e). To
test this assumption, each parent was fully hydrolyzed with 1 m
NaOH solution, and the concentration of the corresponding prod-
uct was calculated on the parent compound calibration curve. The
measured concentration matched the expected 30 mm value. The
analyses were run on an ACQUITY UPLC BEH C18 1.7 mm 2.1ꢁ
50 mm column with a VanGuard BEH C18 1.7 mm pre-column at
408C. The mobile phase was 0.1% HCOOH in H2O (A) and 0.1%
HCOOH in CH3CN (B) using the following gradient: 0–0.5 min:
5% B, 0.5–2.5 min: 5–100% B, 2.5–2.7 min: 100% B, 2.7–2.8 min:
pounds disclosed in this paper was filed by the following authors:
Daniele Piomelli, Marco Mor, Giorgio Tarzia, Fabio Bertozzi,
Andrea Nuzzi, Annalisa Fiasella, Tiziano Bandiera.
Keywords: N-acylethanolamine acid amidase · b-lactams ·
cysteine hydrolase · inhibitors · structure–activity relationships
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In vitro mouse plasma stability: Compounds were diluted in mouse
plasma with 5% DMSO added to help solubilization. Plasma was
pre-heated at 378C (10 min). The final compound concentration
was 2 mm. At selected time points (immediately after dilution, 5,
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In vitro rat plasma stability: Compounds were added to rat plasma
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points (0, 5, 15, 30, 60, and 120 min), and 150 mL of CH3CN spiked
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Acknowledgements
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The authors thank Dr. Giuliana Ottonello and Sine Mandrup Ber-
tozzi of the Italian Institute of Technology (IIT) for determination
of compound stability in buffer and in mouse and rat plasma,
Dr. Federica Vacondio (University of Parma) for providing the ex-
perimental protocol for chemical stability studies, Luca Goldoni
(IIT) for NMR technical support, Silvia Venzano (IIT) for compound
handling, Luisa Mengatto (IIT) for compound testing on h-NAAA,
and Dr. Natalia Realini and Clara Albani (IIT) for compound test-
ing on h-AC and h-FAAH, respectively. This study was partially
supported by a grant from the National Institute on Drug Abuse
(RO1-DA-012413 to D.P.). The authors declare the following com-
peting financial interest: A patent protecting the class of com-
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Received: December 20, 2013
Revised: April 3, 2014
Published online on && &&, 0000
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A. Fiasella, A. Nuzzi, M. Summa,
Stability is the key: a-Amino-b-lactams
were synthesized as amide derivatives,
and the effect of the azetidin-2-one
ring, the stereochemistry at the a-posi-
tion, and the functionalization of the a-
amino group were studied with regard
to N-acylethanolamine acid amidase in-
hibitory potency and hydrolytic and
plasma stability.
A. Armirotti, G. Tarozzo, G. Tarzia, M. Mor,
F. Bertozzi, T. Bandiera,* D. Piomelli*
&& – &&
3-Aminoazetidin-2-one Derivatives as
N-Acylethanolamine Acid Amidase
(NAAA) Inhibitors Suitable for
Systemic Administration
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