Synthesis, structure-activity, and structure-stability relationships of 2-substituted-n-(4-oxo-3-oxetanyl) n-acylethanolamine acid amidase (NAAA) inhibitors

Article abstract of DOI:10.1002/cmdc.201300416

N-Acylethanolamine acid amidase (NAAA) is a cysteine amidase that preferentially hydrolyzes saturated or monounsaturated fatty acid ethanolamides (FAEs), such as palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), which are endogenous agonists of nu

Full text of DOI:10.1002/cmdc.201300416

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DOI: 10.1002/cmdc.201300416
Synthesis, Structure–Activity, and Structure–Stability
Relationships of 2-Substituted-N-(4-oxo-3-oxetanyl)
N-Acylethanolamine Acid Amidase (NAAA) Inhibitors
Romina Vitale,[a] Giuliana Ottonello,[a] Rita Petracca,[a] Sine Mandrup Bertozzi,[a]
Stefano Ponzano,[a] Andrea Armirotti,[a] Anna Berteotti,[a] Mauro Dionisi,[a] Andrea Cavalli,[a, b]
Daniele Piomelli,[a, c] Tiziano Bandiera,*[a] and Fabio Bertozzi*[a]
N-Acylethanolamine acid amidase (NAAA) is a cysteine amidase
that preferentially hydrolyzes saturated or monounsaturated
fatty acid ethanolamides (FAEs), such as palmitoylethanolamide
(PEA) and oleoylethanolamide (OEA), which are endogenous
agonists of nuclear peroxisome proliferator-activated receptor-
a (PPAR-a). Compounds that feature an a-amino-b-lactone
ring have been identified as potent and selective NAAA inhibi-
tors and have been shown to exert marked anti-inflammatory
effects that are mediated through FAE-dependent activation of
PPAR-a. We synthesized and tested a series of racemic, diaste-
reomerically pure b-substituted a-amino-b-lactones, as either
carbamate or amide derivatives, investigating the structure–ac-
tivity and structure–stability relationships (SAR and SSR) follow-
ing changes in b-substituent size, relative stereochemistry at
the a- and b-positions, and a-amino functionality. Substituted
carbamate derivatives emerged as more active and stable than
amide analogues, with the cis configuration being generally
preferred for stability. Increased steric bulk at the b-position
negatively affected NAAA inhibitory potency, while improving
both chemical and plasma stability.
Introduction
Palmitoylethanolamide (PEA), the endogenous amide of pal-
mitic acid and ethanolamine, belongs to the family of fatty
acid ethanolamides (FAEs), a class of lipid-derived messengers
that participate in the control of multiple physiological func-
tions, including pain and inflammation.[1] PEA is produced by
most mammalian cells,[2] and 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.[4] It has also been suggested that PEA
might attenuate skin inflammation and neuropathic pain in
humans.[5] These effects are mainly attributed to the ability of
PEA to activate peroxisome proliferator-activated receptor-
a (PPAR-a), a member of the steroid/nuclear receptor super-
family.[6]
FAEs such as PEA and its monounsaturated analogue oleoy-
lethanolamide (OEA) are produced on demand and their en-
dogenous levels are regulated by enzymes responsible for
their formation and degradation.[7] They are enzymatically bio-
synthesized from membrane glycerophospholipids via their
corresponding N-acylphosphatidylethanolamines (NAPEs).[8]
The major pathway for degradation of FAEs is the hydrolysis to
free fatty acids and ethanolamine, which is carried out by two
intracellular lipid amidases: fatty acid amide hydrolase (FAAH)
and N-acylethanolamine acid amidase (NAAA).[9] Although
NAAA and FAAH share the ability to cleave lipid amide bonds,
they show major differences in primary structure, substrate se-
lectivity, and cellular localization. NAAA is a cysteine amidase
that belongs to the N-terminal nucleophile (Ntn) family of en-
zymes,[9c,10] preferentially hydrolyzing saturated or monounsa-
turated FAEs that activate PPAR-a, such as PEA and OEA, over
the polyunsaturated endocannabinoid anandamide.[9c,11] NAAA
has no sequence homology to FAAH,[9c] but is linked to the
choloylglycine hydrolase family of enzymes, members of which
are characterized by the ability to cleave carbon–nitrogen
bonds in linear amides, with the exception of peptide
bonds.[12] Like other Ntn enzymes, NAAA is produced as an in-
active pro-enzyme and is activated at acidic pH by autocatalyt-
ic cleavage at a specific site of the peptide chain.[13] Site-direct-
ed mutagenesis experiments have univocally identified Cys131
(in mice) and Cys126 (in humans) as the catalytic residue re-
sponsible for both auto-proteolysis and FAE hydrolysis,[12,14]
[a] R. Vitale,+ Dr. G. Ottonello,+ R. Petracca, S. M. Bertozzi, Dr. S. Ponzano,
Dr. A. Armirotti, Dr. A. Berteotti, Dr. M. Dionisi, Prof. A. Cavalli,
Prof. D. Piomelli, Dr. T. Bandiera, Dr. F. Bertozzi
Drug Discovery & Development
Italian Institute of Technology (IIT), Via Morego 30, 16163 Genova (Italy)
E-mail: tiziano.bandiera@iit.it
fabio.bertozzi@iit.it
[b] Prof. A. Cavalli
Department of Pharmacy & Biotechnology
University of Bologna, Via Belmeloro 6, 40126 Bologna (Italy)
[c] Prof. D. Piomelli
Departments of Anatomy & Neurobiology,
Pharmacology & Biological Chemistry
University of California, Irvine, Neuroscience Research Facility
3216 Gillespie Building, Irvine, CA 92697-4621 (USA)
[+] 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.201300416.
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and have indicated Cys126, Arg142, and Asp154 as the resi-
dues that constitute the catalytic triad of human NAAA.[14a]
Because NAAA has been shown to primarily deactivate PEA
and OEA,[9c,11] selective NAAA inhibitors could be envisaged to
increase local levels of these FAEs, which may lead to anti-in-
flammatory and analgesic effects via PPAR-a signaling. This hy-
pothesis was confirmed experimentally.[15] As the discovery and
initial characterization of NAAA are quite recent, only a limited
number of NAAA inhibitors have been reported. Among them,
palmitic acid derivatives,[16] lipophilic amides[17] and amines[18]
were reported to inhibit NAAA activity with medium-to-low
micromolar potencies. Recently, compounds featuring an a-
amino-b-lactone ring as a reactive electrophilic warhead were
identified as the first class of potent and selective NAAA inhibi-
tors.[19]
tions aiming at a structure–activity relationship (SAR) expan-
sion of a-amino-b-lactone derivatives examined the effects of
side chain modifications on NAAA inhibition and the stereo-
chemical requirements for introduction of a methyl group in
the b-position. These studies led to the identification of com-
pounds that are highly potent at inhibiting both rNAAA and
human NAAA (hNAAA), such as b-lactones 6 (rNAAA: IC50 =
0.050 mm, hNAAA: IC50 =0.007 mm)[19c,28] and 7[28b] (r and
hNAAA: IC50 =0.007 mm; Figure 1). The in vitro characterization
of 6 proved that this compound inhibits rNAAA through
a mechanism that is rapid, noncompetitive, and fully reversible
after overnight dialysis.[29] Furthermore, topical administration
of 6 was shown to elevate FAE levels in mouse skin and sciatic
nerve tissues, and to attenuate nociception in mice and rats
through a mechanism engaging PPAR-a activation.[29]
The b-lactone ring is present in many biologically active nat-
ural products such as lipstatin 1,[20] the clasto-lactacystin b-lac-
tone (omuralide) 2,[21] salinosporamide A 3,[22] and obafluorin
4[23] (Figure 1) and represents a promising privileged structure
The b-lactone moiety, which is crucial for NAAA inhibitory
activity, displays low chemical and plasma stability, thus limit-
ing the therapeutic applications of these compounds.[19c,30]
However, threonine-derived b-lactones, which bear a b-methyl
substituent, are known to have enhanced stability in aqueous
media relative to their serine-derived b-lactone counterparts,
which are readily hydrolyzed to the corresponding b-hydroxy
acids.[19c,24b,30]
In the present work, we focused our attention on b-substi-
tuted a-amino-b-lactones, envisioning that the presence of
a bulkier substituent at the b-position of the oxetan-2-one
scaffold could improve the hydrolytic stability in aqueous
media and provide new insight into substitution patterns that
may lead to improved potency. To address the SARs as well as
the structure–stability relationships (SSRs), we synthesized and
tested a series of racemic, diastereomerically pure b-substitut-
ed a-amino-b-lactones as cis- and trans-carbamic acid esters
and amide derivatives and examined both their potency and
stability. In particular, we investigated the effect of the b-sub-
stituent size (Me, Et, iPr, tBu) and relative stereochemistry at
positions 2 and 3 on NAAA inhibitory activity and chemical
and plasma stability. Finally, quantum chemical calculations at
the density functional theory (DFT) level were carried out to
help explain the results of our experimental SSR studies.
Figure 1. Naturally occurring b-lactones 1–4 and a-amino-b-lactone NAAA
inhibitors 5–7.
Results and Discussion
that can react covalently with the active sites of certain en-
zymes, such as lipases,[20b] cysteine[24] and serine[25] proteases,
and the proteasome.[22b,23a,26] Over the last 20 years naturally
occurring b-lactone (oxetan-2-one) derivatives have been ex-
tensively studied as potential drug candidates for several
human disease conditions, including hypercholesterolemia and
cancer.[27] Among them, a-amino-b-lactone derivatives have
been studied as proteasome inhibitors and have progressed to
clinical studies as novel anticancer agents.[22a,23a,27d]
Chemistry
The synthesis of b-substituted a-amino-b-lactones as carba-
mate 23a–c–26a–c and amide 27a–c–30a–c derivatives was
undertaken following a racemic, diastereoselective synthetic
pathway, which allowed the independent generation of cis-
and trans-stereoisomers (Scheme 1).
Racemic cis-b-substituted a-amino-b-lactones 23a–c and
25a–c were obtained by [benzotriazol-1-yloxy(dimethylamino)-
methylidene]dimethylazanium tetrafluoroborate (TBTU)-medi-
ated cyclization from the corresponding erythro-a-substituted
b-hydroxy acids 15a–c–16a–c, prepared following a previously
reported protocol by Guanti et al. (Scheme 1).[31] Microwave ir-
radiation of a mixture of dibenzylamine and ethyl chloroace-
tate gave dibenzylamino ethyl acetate (8),[32] which was acylat-
Within this class of compounds, (S)-N-(2-oxo-3-oxetanyl)-3-
phenylpropionamide [(S)-OOPP, 5, Figure 1] was reported to be
a relatively potent NAAA inhibitor [rat NAAA (rNAAA): IC50 =
0.42 mm], and was shown to increase PEA and OEA levels in ac-
tivated leukocytes and decrease responses induced by inflam-
matory stimuli both in vitro and in vivo.[19a,b] Further investiga-
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Scheme 1. General strategy for the synthesis of cis- and trans-b-substituted a-amino-b-lactones, as carbamates (23–26) and amides (27–30). Reagents and
conditions: a) LDA, THF, ꢀ788C, 30 min; b) R1COCl, ꢀ788C, 10 min, 70–80%; c) NaBH4, NH4Cl, EtOH/H2O 4:1, RT, 3 h, 55–86%; d) H2, 10% Pd(OH)2/C, Boc2O (H-
Cube, EtOH, 708C, 1.0 bar), 55%–quant.; e) NaBH4, THF/EtOH 1:1, RT, 1 h, 48–83%; f) H2, 10% Pd(OH)2/C (H-Cube, EtOH, 708C, 1.0 bar), 70%–quant.; g) 6.0m
HCl, microwave, 1308C, 30 min, 81–93%; h) mixture of 41–42 [R2 =(CH2)5Ph] or 43–44 [R2 =CH2Ar], NaHCO3, H2O/THF 1:1, RT, 16 h, 32–93%; i) 1.0m NaOH,
acetone/DMF 6:1, RT, 1 h; j) TBTU, Et3N, CH2Cl2, RT, 16 h, 22–92%; l) CF3COOH, p-TsOH, 08C, 30 min, 83%–quant.; m) TBTU, Et3N, CH2Cl2, RT, 16 h, 10–65%;
n) [R3 =(CH2)6Ph (27a–c, 28a–c)] R3COOH, TBTU, Et3N, CH2Cl2, RT, 16 h, 30–46%; [R3 =PhOBn (29a–c, 30a–c)] R3COCl, Et3N, CH2Cl2, 08C, 2 h, 8.5–56% [Ar=4-
phenylphenyl].
ed with the desired acyl chloride (R1 =Et, iPr, tBu) to give the
corresponding b-keto esters 9a–c. Acidic chemoselective re-
duction of the keto functionality with sodium borohydride in
the presence of ammonium chloride[31] allowed complete ste-
reoselective synthesis of racemic erythro-b-hydroxyamino
esters 10a–c. After deprotection of the amino functionality by
hydrogenation, hydrolysis of the corresponding ester moiety
of 14a–c followed by carbamoylation of the free amino group
with activated alcohols 41–44[28b](see Supporting Information)
led to the desired carbamic acid ester intermediates 15a–c–
16a–c.
Diastereomeric threo-b-substituted a-amino-b-hydroxy acids
17a–c–18a–c were obtained by a modification of the proce-
dure described above. One-pot deprotection of dibenzylamino
derivatives 9a–c, followed by in situ Boc protection of the free
amino group in the H-Cube, furnished b-keto esters 11 a–c in
good yields (55–75%). As previously reported,[33] sodium boro-
hydride reduction of compounds 11 a–c led to threo/erythro
mixtures of the corresponding b-hydroxy esters 13a–c in ratios
of 8:2 (R1 =Et and iPr) to 9:1 (R1 =tBu). After one-pot acidic N-
Boc deprotection and hydrolysis of esters 13a–c,[34] isomeric
mixtures of compounds 17a–c–18a–c were obtained by N-car-
bamoylation with activated alcohols 41–44. Cyclization per-
formed in the presence of TBTU led to the final b-substituted
a-amino-b-lactones as a 8:2–9:1 mixtures of trans/cis isomers,
which after chromatographic purification afforded the diaste-
reomerically pure trans-b-lactone carbamates 24a–c and 26a–
c. Because the cyclization of N-acylated-b-hydroxyamino acids
to b-lactones has been reported to mainly produce the corre-
sponding five-membered ring azalactones,[35] an alternative
route to racemic cis- and trans-b-substituted a-amino-b-lactone
amide derivatives 27a–c–30a–c was envisaged, starting from
intermediates 10a–c and 13a–c, respectively. Concerning cis-
amide derivatives, in situ one-pot hydrogenation/N-Boc protec-
tion of intermediates 10a–c provided diastereochemically pure
erythro-b-hydroxy esters 12a–c, which, after basic hydrolysis
and TBTU cyclization, gave N-Boc-a-amino-b-lactones 19a–
c (Scheme 1). As already reported for similarly protected a-
amino-b-lactones,[19b,24b] reaction of 19a–c with p-toluenesul-
fonic acid in TFA gave the corresponding toluenesulfonate
salts 21a–c, which could be functionalized by coupling reac-
tion with the desired carboxylic acid or acyl chloride in the
presence of TBTU to give the final amides 27a–c and 29a–c.
trans-Amide analogues 28a–c and 30a–c were prepared by
following a similar procedure as for cis derivatives, starting
from intermediates 13a–c. Diastereomerically pure trans-b-lac-
tone amides were isolated after chromatographic purification
of the corresponding mixtures of trans/cis isomers.
The preparation of (2S,3R)-2-methyl-4-oxo-oxetan-3-yl-carba-
mates 6,[19c,28b] 7[28b] and amides 35g,[28a] h, and (2S,3S)-2-
methyl-4-oxo-oxetan-3-yl-carbamates 38d,[28b] e was accom-
plished in a similar manner as the above-described b-substitut-
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Scheme 2. Preparation of (2S,3R)-2-methyl-4-oxo-oxetan-3-yl-carbamates 6, 7 and amides 35g,h, and (2S,3S)-2-methyl-4-oxo-oxetan-3-yl-carbamates 38d,e.
Reagents and conditions: a) mixture of 41–42 [R2 =(CH2)5Ph] or 43–44 [R2 =CH2Ar] or Boc2O [R2 =tBu], NaHCO3, H2O/THF 1:1, RT, 16 h, 88–92%; b) TBTU, Et3N,
CH2Cl2, RT, 16 h, 17–30%; c) [R2 =tBu (33 f)] CF3COOH, p-TsOH, 08C, 30 min, 95%; d) [R3 =(CH2)6Ph (35g)] R3COOH, TBTU, Et3N, CH2Cl2, RT, 16 h, 40%;
[R3 =PhOBn (35h)] R3COCl, Et3N, CH2Cl2, 08C, 2 h, 36% [Ar=4-phenylphenyl].
ed analogues, starting from d- (31) and l-allo-threonine (36),
respectively (Scheme 2). Functionalization of the amino group
as for carbamates 15a–c–18a–c was performed according to
a previously reported protocol[28b,36] involving the activation of
the desired alcohols 39 and 40 with di-2-pyridyl carbonate
(DPC) followed by coupling with the b-hydroxy-a-amino acid
(see Supporting Information).
the hydrolysis of N-(4-methyl-2-oxo-chromen-7-yl)hexadecana-
mide (PAMCA) by recombinant hNAAA heterologously ex-
pressed in HEK293 cells (see Experimental Section below).[14b,c]
IC50 values are listed in Tables 1 and 2.
We first focused on the carbamate series, investigating the
role of the alkyl groups—Et, iPr, and tBu—at position 2 of the
b-lactone ring. Compound 23a, the racemic cis-2-ethyl stereo-
isomer carrying a 5-phenylpentyl side chain, turned out to be
a nanomolar inhibitor of hNAAA (IC50 =0.009 mm), showing
similar potency to 6 (IC50 =0.014 mm), the (2S,3R)-2-methyl ana-
logue (Table 1). Increasing the steric bulk at the b-position
while maintaining a cis configuration led to compounds 23b
(R1 =iPr, IC50 =0.063 mm) and 23c (R1 =tBu, IC50 =0.302 mm),
which respectively showed 6- and 30-fold lower potency than
23a. We then investigated the effect of stereochemistry on
Structure–activity relationship (SAR)
In our previous studies on serine- and threonine-derived a-
amino-b-lactones, we found that the introduction of a methyl
group at the b-position of the oxetan-2-one moiety proved to
be beneficial for potency on both rat and human NAAA.[19c,28b]
Furthermore, the type of functionalization of the amino group
of the b-lactone, that is, amide
versus carbamate, proved to be
important in terms of activity
and stability.[19b,c,28b]
Table 1. Inhibitory potency toward hNAAA activity, and in vitro chemical and rat plasma stabilities of carba-
mates 6,[a] 7,[a] 23a–c–26a–c[b] and 38d,e.[a]
Here, to investigate the possi-
ble influence of various alkyl
groups at the b-position on po-
tency, a series of b-substituted
a-amino-b-lactone
derivatives
Compd
Config
R1
R2
IC50 [mm][c]
t1/2 [min][d]
pH 7.4[e]
pH 5.0[f]
RP[g]
<5
<5
<5
<5
<5
<5
<5
were designed and synthesized
as carbamic acid esters (here-
after referred to as the “carba-
mate series”) and amides (here-
after referred to as the “amide
series”). The amino group was
functionalized with different
chains, selected among those
previously disclosed in potent b-
lactone NAAA inhibitors. In par-
ticular, we selected the 5-phenyl-
pentyl[19c,28b] and the 4-phenyl-
phenylmethyl[28b] chains for the
carbamate series (Table 1), and
the 4-benzyloxyphenyl[19b,c] and
6-phenylhexyl[28a] chain for the
amide analogues (Table 2).
6
cis-(2S,3R)
cis
Me
Et
iPr
tBu
Me
Et
iPr
tBu
Me
Et
iPr
tBu
Me
Et
(CH2)5Ph
(CH2)5Ph
(CH2)5Ph
(CH2)5Ph
(CH2)5Ph
(CH2)5Ph
(CH2)5Ph
(CH2)5Ph
CH2Ar[i]
0.014ꢁ0.006
0.009ꢁ0.003
0.063ꢁ0.015
0.302ꢁ0.064
0.008ꢁ0.001
0.004ꢁ0.001
0.019ꢁ0.004
0.234ꢁ0.054
0.015ꢁ0.004
0.015ꢁ0.003
0.039ꢁ0.004
0.576ꢁ0.128
0.008ꢁ0.002
0.006ꢁ0.001
0.023ꢁ0.007
0.507ꢁ0.196
215ꢁ26
223ꢁ9
522ꢁ44
550ꢁ64
23a
23b
23c
38d
24a
24b
24c
7
25a
25b
25c
38e
26a
26b
26c
cis
cis
354ꢁ19
611ꢁ42
184ꢁ15
145ꢁ4
>1440 (76)[h]
>1440 (73)[h]
340ꢁ28
trans-(2S,3S)
trans
trans
trans
cis-(2S,3R)
cis
339ꢁ46
259ꢁ13
235ꢁ40
201ꢁ41
183ꢁ28
305ꢁ36
605ꢁ53
159ꢁ20
151ꢁ22
191ꢁ35
181ꢁ12
1258ꢁ83
>1440 (68)[h]
491ꢁ65
5.3ꢁ1.0
<5
<5
<5
<5
<5
<5
<5
CH2Ar[i]
415ꢁ91
cis
cis
CH2Ar[i]
>1440 (65)[h]
>1440 (65)[h]
395ꢁ45
CH2Ar[i]
trans-(2S,3S)
trans
trans
CH2Ar[i]
CH2Ar[i]
457ꢁ37
iPr
tBu
CH2Ar[i]
1121ꢁ201
>1440 (56)[h]
trans
CH2Ar[i]
6.5ꢁ1.8
[a] Compounds tested as pure enantiomers. [b] Compounds tested as racemic mixtures. [c] Values are the
meanꢁSD of three or more determinations. [d] Stability values are the meanꢁSD of three or more determina-
tions. [e] PBS (0.01m) + 10% CH3CN, pH 7.4, 378C. [f] Phosphate buffer (0.01m) + 10% CH3CN, pH 5.0, 378C.
[g] Rat plasma (80% v/v) + 20% PBS, 5% DMSO, pH 7.4. [h] Compound remaining (%) after 24 h (1440 min).
[i] Ar=4-phenylphenyl.
All the compounds were
tested for their ability to inhibit
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NAAA inhibition, while keeping the same substituents on the
a-amino group. Along with a generally slight increase in inhibi-
tory activity, compounds with a trans configuration displayed
an analogous pattern to that of the corresponding cis stereo-
isomers, showing a decrease in potency for more sterically hin-
dered b-substituted compounds (IC50 values from 0.004 mm for
24a to 0.234 mm for 24c).
Moving to compounds 25a–c and 26a–c, which carry a 4-
phenylphenylmethyl chain, we similarly observed a positive in-
fluence on NAAA inhibitory potency of a trans relationship be-
tween the substituents at positions a and b, with compounds
26a–c (IC50 =0.006–0.507 mm) slightly more active than stereo-
isomers 25a–c (IC50 =0.015–0.576 mm). The detrimental effect
of bulky substituents at the b-position was also evident in this
subset of compounds. Notably, increasing steric hindrance at
the b-position (from methyl to tert-butyl) led to a progressive
decrease in NAAA inhibition of this series of compounds, inde-
pendently from the carbamate functionalization (5-phenylpen-
tyl or 4-phenylphenylmethyl).
0.379 mm, respectively), a significant drop in potency was ob-
served in the case of the tert-butyl group in b-position (27c,
IC50 =2.87 mm), confirming the trend observed in the carba-
mate series. Although the previously described l-serine-derived
amide carrying a 4-benzyloxyphenyl chain had shown nano-
molar potency against NAAA,[19b] all substituted cis-amides
29a–c and 35h disappointingly turned out to be poorly active
(IC50 >100 mm). The data obtained for d-threonine analogue
35h suggests that the low activity observed for this set of
compounds relative to the l-serine-derived amide, could be as-
cribed to the opposite configuration at a-position (R vs. S con-
figuration) and/or the substitution at the b-position (Me vs. H).
Moving to amides with the trans configuration, whereas ana-
logues 28a,b and 30a could not be tested due to their low in-
trinsic chemical stability (see the following section), com-
pounds 28c and 30b,c showed moderate NAAA inhibition
(IC50 values from 0.277 mm for 28c to 1.98 mm for 30c).
Chemical stability
Replacement of the carbamate function with an amide, as in
derivatives 27a–c–30a–c and 35g,h, resulted in an evident
decrease in potency (Table 2). A >40-fold drop in NAAA inhibi-
tory activity was observed for the b-lactone amide 27a (IC50 =
0.408 mm) relative to the corresponding carbamate analogue
23a (IC50 =0.009 mm). A more pronounced effect was observed
for the enantiomerically pure threonine-derived compounds 6
(IC50 =0.014 mm) and 35g (IC50 =1.56 mm), which showed
a ~100-fold difference in potency.
b-Lactones are known to exhibit limited stability in water due
to ring-opening to the corresponding b-hydroxy acids, which
relieves the strain of the four-membered ring. In a-amino-b-
lactone derivatives, it has been reported that a methyl group
at the b-position of the oxetanone ring, as in threonine-de-
rived b-lactones, improves the hydrolytic stability of such com-
pounds with respect to the corresponding unsubstituted
serine-derived analogues.[19c,24b]
While the activity of b-ethyl (27a) and b-isopropyl (27b) an-
alogues turned out to be quite similar (IC50 values of 0.408 and
On the other hand, replacement of the substituted benzyl
group in the naturally occurring obafluorin (4, Figure 1) with
a less bulky methyl led to a de-
crease in the hydrolytic stability
of the b-lactone ring.[30] These
Table 2. Inhibitory potency toward hNAAA activity, and in vitro chemical and rat plasma stabilities of amides
27a–c–30a–c[a] and 35g,h.[b]
findings prompted us to expand
previous studies on the role of
the b-substituent on the hydro-
lytic stability of a-amino-b-lacto-
nes.[19c] We focused our attention
on the effect of the size of the
b-substituent and on the role of
the relative stereochemistry at
positions 2 and 3. The role of
the amino group functionaliza-
tion, as amides and carbamic
acid esters, was also investigated
with the aim to understand the
contribution of this modification
to the susceptibility of disubsti-
tuted a-amino-b-lactones to
chemical hydrolysis.
Compd
Config
R1
R3
IC50 [mm][c]
t1/2 [min][d]
pH 7.4[e]
pH 5.0[f]
RP[g]
<5
<5
<5
35g
27a
27b
27c
28a
28b
28c
35h
29a
29b
29c
30a
30b
30c
cis-(2S,3R)
cis
cis
Me
Et
iPr
tBu
Et
iPr
tBu
Me
Et
iPr
tBu
Et
(CH2)6Ph
(CH2)6Ph
(CH2)6Ph
(CH2)6Ph
(CH2)6Ph
(CH2)6Ph
(CH2)6Ph
PhOBn
PhOBn
PhOBn
PhOBn
PhOBn
1.56ꢁ0.208
0.408ꢁ0.122
0.379ꢁ0.124
2.874ꢁ0.176
Unst.[i]
109ꢁ9
127ꢁ10
136ꢁ21
356ꢁ47
n.a.[j]
229ꢁ20
387ꢁ34
>1440 (54)[h]
>1440 (73)[h]
n.a.[j]
cis
8.3ꢁ2.3
trans
trans
trans
cis-(2S,3R)
cis
cis
cis
trans
trans
trans
Unst.[i]
n.a.[j]
n.a.[j]
0.277ꢁ0.022
152ꢁ15
50ꢁ4
573ꢁ51
175ꢁ5
<5
10.3ꢁ1.2
6.3ꢁ1.8
<5
10.2ꢁ1.0
>100[k]
>100[k]
>100[k]
>100[k]
54ꢁ8
298ꢁ7
62ꢁ3
1345ꢁ85
>1440 (69)[h]
n.a.[j]
98ꢁ8
Unst.[i]
0.581ꢁ0.027
1.98ꢁ0.259
n.a.[j]
iPr
tBu
PhOBn
PhOBn
20ꢁ2
24ꢁ2
<5
<5
Chemical stability data for 2-
substituted N-(4-oxo-3-oxetanyl)-
carbamic acid esters 6, 7, 23a–
c–26a–c, 38d,e and amides
27a–c–30a–c, 35g,h are sum-
marized in Tables 1 and 2, re-
spectively. Compound stability
156ꢁ17
494ꢁ53
[a] Compounds tested as pure enantiomers. [b] Compounds tested as racemic mixtures. [c] Values are the
meanꢁSD of three or more determinations. [d] Stability values are the meanꢁSD of three or more determina-
tions. [e] PBS (0.01m) + 10% CH3CN, pH 7.4, 378C. [f] Phosphate buffer (0.01m) + 10% CH3CN, pH 5.0, 378C.
[g] Rat plasma (80% v/v) + 20% PBS, 5% DMSO, pH 7.4. [h] Compound remaining (%) after 24 h (1440 min).
[i] Unstable. [j] Not available (t1/2 <10 min). [k] IC50 >100 mm refers to compounds showing <30% inhibition at
100 mm.
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was evaluated as the half-life (t1/2), in minutes, in buffered solu-
tions at pH 7.4 (physiological pH) and 5.0 (pH of the hNAAA
assay), at 378C, by measuring the disappearance of each
tested compound at various time points.
rearrangement followed by acid-mediated hydrolysis of the ox-
azoline-carboxylic acid intermediate (C) (Scheme 3).[19c,30,37]
To determine whether a similar degradation mechanism also
occurred to b-substituted higher homologues, we selected the
two diastereomeric amides 29b and 30b, and we carried out
a time course UPLC–UV analysis, at pH 7.4 and 5.0, of their
chemical degradation. Similar to what we observed with
serine- and threonine-derived b-lactones, both b-isopropyla-
mide stereoisomers 29b and 30b showed the formation of
a consistent amount of degradation products deriving from an
intramolecular nucleophilic attack of the amide carbonyl group
to the b-position of the lactone ring (see Supporting Informa-
tion, figures S1 and S2).[19c,24b,30] Whereas at pH 7.4 trans-isomer
30b (t1/2 =20 min; Table 2) converted in a time dependent
manner over 24 h, mainly (~70% mol) into the corresponding
4,5-dihydro-2-(4-benzyloxyphenyl)-5-isopropyl-4-oxazolecarbox-
ylic acid (C, R1 =iPr; Scheme 3), the O-4-benzyloxybenzoyl-b-
hydroxyamino acid D (R1 =iPr, Scheme 3) was the only species
detected at pH 5.0 (Supporting Information, figure S1).[19c]
Interestingly, unlike the trans stereoisomer, cis-b-lactone
amide 29b (t1/2 =62 min; Table 2) produced, both at pH 7.4
and 5.0, a consistent amount of diastereomeric N-acyl-b-hy-
droxy acid B (R1 =iPr, Scheme 3; 40 and 25% mol at pH 7.4
and 5.0, respectively), along with the products deriving from
intramolecular rearrangement (Supporting Information, fig-
ure S2). These results clearly indicated for cis-amide 29b the
presence of two competitive degradation pathways relative to
trans analogue 30b.
Previous stability studies had shown (2S,3R)-2-methyl-4-oxo-
3-oxetanylcarbamic acid ester derivative 6 to have an im-
proved chemical stability profile relative to serine-derived b-
lactone analogues.[19c] These results led us to consider 6 as the
starting point for our SSR investigations.
Replacement of the 5-phenylpentyl side chain with a 4-phe-
nyphenylmethyl residue, as in compound 7, or inversion of the
stereochemistry at the a-position on both 6 and 7, leading to
compounds 38d and 38e, did not have major impacts on hy-
drolytic stability (Table 1). Conversely, amides 35g,h turned
out to be considerably less stable (pH 7.4: t1/2 =50–109 min,
pH 5.0: t1/2 =175–229 min) than carbamate 6 (pH 7.4: t1/2
215 min, pH 5.0: t1/2 =522 min) (Table 2).
=
The effect of the relative stereochemistry at positions 2 and
3 of the b-lactone ring, carrying respectively the alkyl substitu-
ent and the functionalized amino group, was initially investi-
gated. Whereas for the carbamate series, derivatives with a cis
configuration (23a–c: t1/2 =223–611 min, 25a–c: t1/2 =183–
605 min) showed slightly higher hydrolytic stability at pH 7.4
than the corresponding trans counterparts (24a–c: t1/2 =145–
235 min, 26a–c: t1/2 =151–181 min; Table 1), for the amide an-
alogues the relative stereochemistry had a more pronounced
impact on the stability depending on the b-substitution. In
fact, although cis-amides (27a–c: t1/2 =127–356 min, 29a–c:
t
1/2 =54–98 min; Table 2) displayed an overall higher tendency
Considering that the functionalization of the amino group of
a-amino-b-lactones as carbamate has been reported to posi-
tively affect the chemical stability of such derivatives,[19c] a simi-
lar study was also conducted on cis- and trans-carbamate ste-
reoisomers 25b and 26b. At pH 7.4, both b-lactones (25b:
toward hydrolysis than the carbamate analogues, a significant
effect of the size of the b-substituent was observed with trans-
substituted compounds. Replacement of the less sterically hin-
dered ethyl group with the bulkier tert-butyl moiety resulted in
a considerable increase in chemical stability (28a, 30a: t1/2
<
t1/2 =305 min, 26b: t1/2 =191 min; Table 1) afforded exclusively
10 min, 28c, 30c: t1/2 =152–156 min; Table 2). A rationalization
of the lower stability of a-amino-b-lactone amides with respect
to the corresponding carbamic acid ester derivatives has been
previously provided for serine- and threonine-derived b-lac-
tone amides (A, R1 =H, Me; Scheme 3). This type of compound
has been described to undergo two possible degradation
pathways, leading to either N-acylhydroxy acids (B), resulting
from the hydrolytic opening of the b-lactone ring, or the corre-
sponding O-acyl isomers (D), as a product of an intramolecular
the corresponding diastereomeric N-(4-phenylphenyl)methoxy-
carbonyl-b-hydroxyamino acids 16b and 18b. This indicates
the hydrolytic opening of the b-lactone ring, leading to adduct
B (Scheme 3), as the main degradation pathway for this kind of
compound (Supporting Information, figures S3 and S4).
The peculiar behavior observed with a-amino-b-lactone
amide derivatives could be reasonably explained by two possi-
ble competitive nucleophilic attacks occurring at different reac-
tion rates, that is, intramolecular attack of the amide carbonyl
moiety to the b-position of the b-lactone, versus attack of an
external nucleophile (water) to the electrophilic endocyclic car-
bonyl moiety. The intramolecular pathway is expected to pro-
ceed more rapidly in trans isomers as a result of an antiperipla-
nar attack of the amide carbonyl group at the b-position rela-
tive to cis derivatives.[30,38] This degradation pathway, although
possible also for the carbamate analogues, is expected to be
more favored in amides due to the higher nucleophilicity of
their carbonyl oxygen atom, as previously described.[19c] There-
fore, the overall higher stability of b-lactone carbamate deriva-
tives than amide analogues might be explained by the weak
nucleophilic character of the carbamate carbonyl moiety, re-
sulting in only one degradation pathway, that is, water attack
with formation of the b-hydroxy acid.[39]
Scheme 3. Substituted b-lactone amides (A) degradation products: N-acyl-b-
hydroxy acids (B), and O-acyl-a-amino acids (D).
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The influence of the various b-alkyl substitutions on stability
was examined on both the carbamate and amide series of a-
amino-b-lactones. A consistent trend was observed in the sta-
bility at pH 7.4 and 5.0 for carbamate and amide derivatives
with the cis configuration. Increasing the steric demand of the
alkyl group at the b-position led to an increase in the stability
of such compounds. In fact, cis-amides 35g,h, 27a–c and
29a–c bearing simple methyl to larger tert-butyl groups, dis-
played half-life (t1/2) values ranging from 50 to 356 min at
pH 7.4 and from 229 to more than 1440 min at pH 5.0
(Table 2). Interestingly, compounds 29a–c and 35h, featuring
a benzamide side chain (R2 =4-benzyloxyphenyl) generally ex-
hibited a lower half-life than the other substituted amides
27a–c and 35g having an aliphatic side chain (R2 =6-phenyl-
hexyl).
referred to as “syn” and “anti” attack with respect to the carba-
mate moiety; see Supporting Information). The reaction mech-
anism turned out to be quite similar for both the syn and anti
attacks.
Starting from the noncovalent complex, the reactants (R),
the oxygen atom of one of the two water molecules (O3, ac-
cording to the numbering reported in Scheme 4) approached
the carbonyl carbon of the lactone ring (C1). The C1ꢀO2 bond
length increased, while the distance between O2 and H4 (i.e.,
a hydrogen atom of the second water molecule) decreased. O4
was thus able to accept a proton (H2) from the water carrying
the nucleophilic attack. The main geometrical features charac-
terizing the reaction mechanism are reported in the Support-
ing Information (table S2). All eight reactions occurred through
a concerted mechanism, that is, at the transition state (TS) the
nucleophilic attack and the proton transfers occurred simulta-
neously, leading to hydrolysis of the lactone ring (Scheme 4).
The b-lactone carbamates with cis configuration—6, 7, 23a–
c and 25a–c—showed a similar trend concerning the effect of
b-substitution (Table 1). Whereas methyl and ethyl substitution
resulted in similar chemical stabilities (6, 23a: t1/2: ~220 min),
increasing the size of the alkyl group at the b-position led to
Table 3. In vitro chemical stabilities, representation of the truncated
forms of compounds listed as determined by computational studies, and
activation energy barriers for both the syn and anti water attacks.
a significant improvement in hydrolytic stability (23b: t1/2
=
354 min and 23c: t1/2 =611 min), particularly at pH 7.4. Unlike
amide derivatives, for cis-carbamates no relevant differences in
hydrolytic stability were observed among compounds with dif-
ferent chains linked to the carbamate function.
Compd
t1/2 [min][a]
Forms
Ea [kcalmolꢀ1
]
syn
anti
Interestingly, the effect of the alkyl substituent at the b-posi-
tion was much less evident for the series of trans-carbamates.
In fact, contrary to what previously observed for cis-analogues,
in compounds 24a–c, 26a–c, and 38d,e the evident impact of
steric hindrance at the b-position on degradation was some-
how lost at pH 7.4. All trans-stereoisomers, regardless of the
alkyl group at the b-position, showed a similar t1/2 (38d, 24a–
c: t1/2 =184–235 min), with this result being more evident for
the 4-phenylphenylmethyl side chain (38e, 26a–c: t1/2 =159–
181 min; Table 1).[40]
6
215
37.5
33.5
23c
38d
24c
611
184
235
42.2
36.9
37.0
33.9
36.0
37.7
To provide a rational explanation of the variation in stability
of the carbamate series at pH 7.4, we carried out DFT-based
calculations simulating lactone hydrolysis in water of the trun-
cated form of compounds 6, 23c, 24c and 38d, where the
side chain of the carbamate function was replaced by a methyl
group. In detail, we studied the nucleophilic attack of a water
molecule at the carbonyl carbon of the b-lactone ring
(Scheme 4). The hydrolysis was studied for both cis (6 and
23c) and trans (24c and 38d) configurations, simulating the
water nucleophilic attack from both sides of the ring (hereafter
[a] Determined at pH 7.4.
In Table 3, we report the in vitro chemical stability, the 2D
representation of the truncated forms of compounds of
column 1 studied by means of computational studies, and the
activation energy (Ea) barriers for both the syn and anti attacks.
For compounds bearing a cis configuration (6 and 23c), the
difference between syn and anti attacks was higher than the
same difference for compounds with a trans configuration
(24c and 38d). This outcome could be explained by consider-
ing that for 6 and 23c the cis substituents (methyl or tert-
butyl) hindered the water molecule from approaching the car-
bonyl carbon of the lactone ring. This is particularly evident for
23c, where the presence of the tert-butyl group led to an Ea of
42.2 kcalmolꢀ1, almost 9 kcalmolꢀ1 higher than the Ea for the
same nucleophilic attack occurring from the anti side. This
means that compound 23c has a lower probability of being
Scheme 4. Representation of the hydrolysis mechanism of b-lactones. The
nucleophilic attack and protonation steps are concerted (R=reactants,
TS=transition State, P=products).
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hydrolyzed, as one of the two possible ways of attack is re-
markably disfavored. The Ea difference between the syn and
anti attack was 4 kcalmolꢀ1 also in the case of the cis deriva-
tive 6, carrying a methyl instead of a tert-butyl group, and was
almost zero for the two trans derivatives (24c and 38d). In ad-
dition, although comparison among different compounds
could be questionable, syn attack on compound 23c showed
the highest Ea among all the reactions simulated here (Table 3).
These DFT-based calculations help explain the half-life data ob-
tained at pH 7.4 for these compounds (Table 1), pointing to
23c as the compound with the highest stability in water
among the whole series of lactones reported herein.
than analogues belonging to the amide series. In addition, we
found that bulky substituents at the b-position of the b-lac-
tone ring have a negative effect on NAAA inhibitory potency.
We next focused our attention on the hydrolytic stability of
this class of compounds both under physiological (pH 7.4) and
acidic (pH 5.0) conditions, and in plasma. Indeed, it is known
that b-lactones have intrinsically low chemical stability due to
their propensity toward hydrolysis into the corresponding b-
hydroxy acids. We measured half-life values of several of the
present lactones, and laid down initial SSRs for these com-
pounds. We first noticed that molecules of the amide series
were generally less stable than carbamates. Second, in the car-
bamate series, the stability was related to the size of the alkyl
substituent and relative stereochemistry at the b-position. This
was particularly clear for compound 23c, carrying a b-tert-
butyl group in cis-configuration with respect to the chain at
the a-position. Finally, DFT calculations showed that in the
case of cis derivatives with a bulky substituent, one of the two
possible pathways of attack by a water molecule was energeti-
cally disfavored, providing a possible explanation for the im-
proved stability of 23c over other derivatives. Most of the a-
amino-b-lactones were unstable in rat plasma. However, a few
of them showed measurable half-life values on the order of
a few minutes. This may be due to the mutual contribution of
two effects, that is, increased b-substitution and amino group
functionalization.
The influence of the b-alkyl substitution on hydrolytic de-
composition for trans-carbamate analogues again became evi-
dent when studies were conducted at pH 5.0. In fact, along
with an expected higher stability of these b-lactones at acidic
pH relative to pH 7.4, high amounts of parent compound were
observed for derivatives displaying either the cis or trans con-
figuration (Table 1). This pH-dependency of hydrolytic stability
on b-substitution was also recognizable to some extent for cis-
amide derivatives 27a–c, 29a–c and 35g,h (Table 2).
Rat plasma stability
b-Substituted a-amino-b-lactone derivatives were also tested
for their stability in rat plasma (80% v/v; Tables 1 and 2). Most
cis- and trans-carbamate analogues, regardless of b-substitu-
tion and side chain modification, showed low half-life values
(t1/2 <5 min) due to extensive degradation of the parent com-
pounds. As observed for chemical stability, a bulky substituent
at the b-position also contributed to improve plasma stability,
with compounds 24c and 26c having half-lives >5 min (5.5
and 6.5 min, respectively). An analogous tert-butyl substitution
effect was also highlighted for cis-amide 27c, which showed
a t1/2 of 8.3 min, as a sole compound in this subset.
In summary, we have reported new SARs around a-amino-b-
lactones as NAAA inhibitors, along with an investigation on
the stability of these compounds in buffer and in plasma. This
information may be valuable in the discovery of better NAAA
inhibitors, which might help to validate NAAA as a therapeutic
target and serve as starting points for the development of
novel anti-inflammatory agents.
Although all the other cis-amides featuring a 6-phenylhexyl
side chain were found to be quite unstable (t1/2 <5 min), cis-
benzamide analogues 29a,c and 35h showed improved
plasma stability with significant half-life values (t1/2 =6.3–
10.3 min) for this class of compounds. The reason for this im-
provement in stability of compounds having a 4-benzyloxy-
phenyl chain is unclear at the moment and needs further in-
vestigation to be properly elucidated.
Experimental Section
Chemistry
Materials and methods: All of the commercially available reagents
and solvents were used as purchased from vendors without further
purification. Dry solvents (THF, Et2O, CH2Cl2, DMF, DMSO, MeOH)
were purchased from Sigma–Aldrich. Optical rotations were mea-
sured on a Rudolf Research Analytical Autopol II Automatic polar-
imeter using a sodium lamp (l 589 nm) as the light source; con-
centrations are expressed in g(100 mL)ꢀ1 using CHCl3 as a solvent
and a 1 dm cell. Melting points were determined on a Bꢁchi M-560
melting point apparatus. Automated column chromatography puri-
fications were done using a Teledyne ISCO apparatus (CombiFlash
Rf) with pre-packed silica gel columns of various sizes (4–120 g).
Mixtures of increasing polarity of cyclohexane and ethyl acetate
(EtOAc) or cyclohexane and methyl tert-butyl ether (MTBE) were
used as eluents. Hydrogenation reactions were performed using H-
Cube continuous hydrogenation equipment (SS-reaction line ver-
sion), employing disposable catalyst cartridges (CatCart) preloaded
with the required heterogeneous catalyst. Microwave heating was
performed using Explorer-48 positions instrument (CEM). NMR ex-
periments were run on a Bruker Avance III 400 system (400.13 MHz
Conclusions
Herein we report on a novel series of b-lactone inhibitors of
NAAA. NAAA is a promising target for the treatment of pain
and inflammation, but few classes of inhibitors of this enzyme
have been identified thus far. Among them, a-amino-b-lac-
tones have emerged as potential candidates in this respect, as
they can covalently and reversibly bind the catalytic cysteine
residue of NAAA. In this study, we described SARs of two series
of b-lactones: the carbamic acid ester and the amide series,
which differed in the nature of the chain at the a-position and
the aliphatic substituent at the b-position. We showed that de-
rivatives of the carbamate series are generally more potent
1
for H, and 100.62 MHz for 13C), equipped with a BBI probe and Z-
gradients. Spectra were acquired at 300 K, using deuterated di-
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1
methyl sulfoxide ([D6]DMSO) or deuterated chloroform (CDCl3) as
solvents. UPLC–MS analyses were run on a Waters Acquity UPLC–
MS system consisting of a single quadrupole detector (SQD) mass
spectrometer equipped with an electrospray ionization (ESI) inter-
face and a photodiode array (PDA) detector. The PDA range was
l 210–400 nm. Analyses were performed on an Acquity UPLC BEH
C18 column (100ꢂ2.1 mm ID, particle size: 1.7 mm) with a VanGuard
BEH C18 pre-column (5ꢂ2.1 mm ID, particle size: 1.7 mm). Mobile
phase was 10 mm NH4OAc in H2O at pH 5 adjusted with CH3COOH
(A) and 10 mm NH4OAc in CH3CN/H2O (95:5) at pH 5.0. ESI in posi-
tive and negative mode was applied. Accurate mass measurement
(HRMS) was performed on a Synapt G2 Quadrupole-ToF Instrument
(Waters, USA), equipped with an ESI ion source. All tested com-
pounds (6, 7, 23a–c–30a–c, 35g,h and 38d,e) showed ꢂ95%
purity by NMR and UPLC–MS analysis. Compounds 6,[19c,28b] 7,[28b]
35g,[28a] 38d,[28b] and activated alcohols 41–44[28b] were synthesized
by following published procedures; the synthesis of compounds
35h and 38e are described in the Supporting Information.
(0.125 g, 38%) as a colorless oil: H NMR (400 MHz, [D6]DMSO): d=
0.93 (s, 9H), 1.34 (m, 2H), 1.53–1.65 (m, 4H), 2.57 (t, J=7.8 Hz, 2H),
4.02 (q, J=6.2 Hz, 2H), 4.36 (d, J=6.3 Hz, 1H), 5.56 (dd, J=6.3,
8.5 Hz, 1H), 7.07–7.34 (m, 5H), 8.38 ppm (d, J=8.5 Hz, 1H);
13C NMR (100 MHz, [D6]DMSO): d=25.3 (3C), 25.4, 28.9, 31.1, 32.9,
35.5, 58.9, 65.1, 84.2, 126.1, 128.7 (4C), 142.5, 156.2, 169.8 ppm; MS
(ESI+) m/z (%): 351 (100) [M+NH4]+; (ESIꢀ) m/z (%): 332 (100)
[MꢀH]ꢀ; HRMS-ESI: m/z [M+H]+ calcd for C19H28NO4: 334.2018,
found: 334.2001.
(4-Phenylphenyl)methyl-N-[(2S*,3R*)-2-ethyl-4-oxo-oxetan-3-yl]-
carbamate (25a). The reaction was carried out using 16a (0.150 g,
0.44 mmol), dry CH2Cl2 (20 mL), Et3N (0.183 mL, 1.31 mmol), and
TBTU (0.168 g, 0.52 mmol) to give, after purification, pure 25a
(0.054 g, 38%) as a white solid: mp: 128.8–129.28C; 1H NMR
(400 MHz, [D6]DMSO): d=0.88 (t, J=7.4 Hz, 3H), 1.56–1.88 (m, 2H),
4.61 (dt, J=6.0, 8.1 Hz, 1H), 5.11 (d, J=12.9 Hz, 1H), 5.15 (d, J=
12.9 Hz, 1H), 5.49 (dd, J=6.0, 9.4 Hz, 1H), 7.29–7.57 (m, 5H), 7.60–
7.78 (m, 4H), 8.41 ppm (d, J=9.4 Hz, 1H); 13C NMR (75 MHz,
[D6]DMSO): d=8.9, 21.9, 59.6, 65.9, 79.1, 126.7 (2C), 126.7 (2C),
127.5, 128.5 (2C), 128.9 (2C), 135.7, 139.7, 139.9, 155.6, 169.8 ppm;
MS (ESI+) m/z (%): 343 (100) [M+NH4]+; (ESIꢀ) m/z (%): 324 (100)
[MꢀH]ꢀ; HRMS-ESI: m/z [M+NH4]+ calcd for C19H23N2O4: 343.1658,
found: 343.1660.
General procedure for the synthesis of cis- and trans-a-amino-b-
lactone carbamic acid ester derivatives (23a–c–26a–c;
Scheme 1).
Preparation of b-lactones 23a–c–26a–c. To a stirred mixture of
carbamates 15a–c–18a–c (1.0 equiv) in dry CH2Cl2, under nitrogen
atmosphere, at 08C, were added Et3N (3.0 equiv) and subsequently
TBTU (1.2 equiv). The mixture was left stirring at 08C for 1 h and at
room temperature for 15 h. Upon full conversion of the starting
material, the organics were removed under reduced pressure, and
the resulting crude product absorbed over silica gel and purified
by column chromatography, eluting with cyclohexane/EtOAc (from
90:10 to 60:40) to afford final products 23a–c–26a–c.
(4-Phenylphenyl)methyl-N-[(2S*,3R*)-2-isopropyl-4-oxo-oxetan-3-
yl]carbamate (25b). The reaction was carried out using 16b
(0.193 g, 0.54 mmol), dry CH2Cl2 (25.0 mL), Et3N (0.225 mL,
1.65 mmol), and TBTU (0.21 g, 0.65 mmol) to give, after purification,
pure 25b (0.041 g, 22%) as a white solid: mp: 149.7–150.78C;
1H NMR (400 MHz, [D6]DMSO): d=0.76 (d, J=6.6 Hz, 3H), 0.95 (d,
J=6.5 Hz, 3H), 1.93–2.13 (m, 1H), 4.25 (dd, J=5.9, 10.9 Hz, 1H),
5.14 (s, 2H), 5.48 (dd, J=5.9, 9.5 Hz, 1H), 7.34–7.52 (m, 5H), 7.61–
7.70 (m, 4H), 8.46 ppm (d, J=9.5 Hz, 1H); 13C NMR (100 MHz,
[D6]DMSO): d=16.8, 18.5, 27.4, 59.3, 65.9, 82.4, 126.8 (2C), 126.8
(2C), 127.7, 128.6 (2C), 129.1 (2C), 135.8, 139.7, 140.0, 155.7,
169.8 ppm; MS (ESI+) m/z (%): 357 (100) [M+NH4]+; (ESIꢀ) m/z
(%): 338 (100) [MꢀH]ꢀ; HRMS-ESI: m/z [M+NH4]+ calcd for
C20H25N2O5: 357.1814, found: 357.1832.
5-Phenylpentyl-N-[(2S*,3R*)-2-ethyl-4-oxo-oxetan-3-yl]carbamate
(23a). The reaction was carried out with 15a (0.070 g, 0.22 mmol),
dry CH2Cl2 (10 mL), Et3N (0.091 mL, 0.66 mmol), and TBTU (0.083 g,
0.26 mmol) to give, after purification, pure 23a (0.043 g, 65%) as
1
a white solid: mp: 63.8–64.78C; H NMR (400 MHz, [D6]DMSO): d=
0.89 (t, J=7.4 Hz, 3H), 1.27–1.41 (m, 2H), 1.45–1.64 (m, 4H), 1.64–
1.95 (m, 2H), 2.58 (t, J=7.7 Hz, 2H), 4.01 (t, J=6.5 Hz, 2H), 4.60 (dt,
J=6.0, 8.0 Hz, 1H), 5.45 (dd, J=6.0, 9.4 Hz, 1H), 7.05–7.38 (m, 5H),
8.23 ppm (d, J=9.4 Hz, 1H); 13C NMR (100 MHz, [D6]DMSO): d=8.9,
21.9, 24.9, 28.3, 30.6, 35.1, 59.5, 64.6, 79.1, 125.6, 128.2 (2C), 128.2
(2C), 142.1, 155.8, 169.9 ppm; MS (ESI+) m/z (%): 323 (100) [M+
NH4]+; (ESIꢀ) m/z (%): 304 (100) [MꢀH]ꢀ; HRMS-ESI: m/z [M+H]+
calcd for C17H24NO4: 306.1705, found: 306.1691.
(4-Phenylphenyl)methyl-N-[(2S*,3R*)-2-tert-butyl-4-oxo-oxetan-3-
yl]carbamate (25c). The reaction was carried using 16c (0.18 g,
0.48 mmol), dry CH2Cl2 (22 mL), Et3N (0.203 mL, 1.45 mmol), and
TBTU (0.186 g, 0.6 mmol) to give, after purification, pure 25c
(0.017 g, 10%, over two steps) as a white solid: mp: 83.3–87.78C;
1H NMR (400 MHz, [D6]DMSO): d=0.93 (s, 9H), 4.38 (d, J=6.2 Hz,
1H), 5.15 (d, J=2.8 Hz, 2H), 5.61 (dd, J=6.2, 8.5 Hz, 1H), 7.23–7.81
(m, 9H), 8.58 ppm (d, J=8.5 Hz, 1H); 13C NMR (100 MHz,
[D6]DMSO): d=24.9 (3C), 32.6, 58.7, 66.1, 83.9, 126.8 (4C), 127.7,
128.6 (2C), 129.1 (2C), 135.9, 139.9, 140.1, 155.8, 169.5 ppm; MS
(ESI+) m/z (%): 371(100) [M+NH4]+; (ESIꢀ) m/z (%): 352 (100)
[MꢀH]ꢀ; HRMS-ESI: m/z [M+NH4]+ calcd for C21H27N2O4: 371.1971,
found: 371.1960.
5-Phenylpentyl-N-[(2S*,3R*)-2-isopropyl-4-oxo-oxetan-3-yl]carba-
mate (23b). The reaction was carried out using 15b (0.14 g,
0.43 mmol), dry CH2Cl2 (20 mL), Et3N (0.18 mL, 1.30 mmol), and
TBTU (0.17 g, 0.55 mmol) to give, after purification, pure 23b
(0.052 g, 40%) as
a
white solid: mp: 82.6–83.18C; 1H NMR
(400 MHz, [D6]DMSO): d=0.75 (d, J=6.5 Hz, 3H), 0.96 (d, J=6.5 Hz,
3H), 1.25–1.39 (m, 2H), 1.52–1.65 (m, 4H), 2.03 (dt, J=6.5, 10.8 Hz,
1H), 2.57 (t, J=7.7 Hz, 2H), 4.01 (t, J=6.7 Hz, 2H), 4.24 (dd, J=5.9,
10.8 Hz, 1H), 5.44 (dd, J=5.9, 9.5 Hz, 1H), 7.13–7.34 (m, 5H),
8.27 ppm (d, J=9.5 Hz, 1H); 13C NMR (100 MHz, [D6]DMSO): d=
17.1, 18.8, 25.4, 27.7, 28.8, 31.1, 35.5, 59.6, 65.1, 82.7, 126.1, 128.7
(4C), 142.5, 156.2, 170.3 ppm; MS (ESI+) m/z (%): 337 (100) [M+
NH4]+; (ESIꢀ) m/z (%): 318 (100) [MꢀH]ꢀ; HRMS-ESI: m/z [M+
NH4]+ calcd for C18H29N2O4: 337.2127, found: 337.2143.
5-Phenylpentyl-N-[(2R*,3R*)-2-ethyl-4-oxo-oxetan-3-yl]carbamate
(24a). The reaction was carried out using 17a (0.155 g, 0.48 mmol)
as a 8:2 threo/erythro diastereomeric mixture, dry CH2Cl2 (15 mL),
Et3N (0.200 mL, 1.44 mmol), and TBTU (0.185 g, 0.58 mmol) to give,
after purification, 24a (0.75 g, 57%) as a pure trans diastereomer,
as a white solid: mp: 64.1–64.88C; 1H NMR (400 MHz, [D6]DMSO):
d=0.90 (t, J=7.4 Hz, 3H), 1.26–1.38 (m, 2H), 1.58 (m, 4H), 1.67–
1.90 (m, 2H), 2.57 (t, J=7.6 Hz, 2H), 3.98 (t, J=6.6 Hz, 2H), 4.51 (td,
J=4.3, 6.8 Hz, 1H), 4.69 (dd, J=4.3, 8.1 Hz, 1H), 7.09–7.32 (m, 5H),
8.06 ppm (d, J=8.1 Hz, 1H); 13C NMR (100 MHz, [D6]DMSO): d=8.7,
24.9, 25.2, 28.3, 30.6, 35.0, 62.0, 64.6, 79.6, 125.6, 128.2 (2C), 128.2
5-Phenylpentyl-N-[(2S*,3R*)-2-tert-butyl-4-oxo-oxetan-3-yl]carba-
mate (23c). The reaction was carried out using 15c (0.346 g,
0.98 mmol), dry CH2Cl2 (44 mL), Et3N (0.41 mL, 2.95 mmol), and
TBTU (0.38 g, 1.20 mmol) to give, after purification, pure 23c
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(2C), 142.1, 155.5, 169.1 ppm; MS (ESI+) m/z (%): 323 (100) [M+
NH4]+; (ESIꢀ) m/z: 304 (100) [MꢀH]ꢀ; HRMS-ESI: m/z [M+NH4]+
calcd for C17H27N2O4: 323.1970, found: 323.1971.
HRMS-ESI: m/z [M+NH4]+ calcd for C20H25N2O4: 357.1814, found:
357.1825.
(4-Phenylphenyl)methyl-N-[(2R*,3R*)-2-tert-butyl-4-oxo-oxetan-
3-yl]carbamate (26c). The reaction was carried out using 18c
(0.203 g, 0.55 mmol) as a 9:1 threo/erythro diastereomeric mixture,
dry CH2Cl2 (17 mL), Et3N (0.230 mL, 1.65 mmol), and TBTU (0.210 g,
0.66 mmol) to give, after purification, 26c (0.093 g, 48%) as a pure
trans diastereomer, as a white solid: mp: 147.1–147.88C; 1H NMR
(400 MHz, [D6]DMSO): d=0.95 (s, 9H), 4.37 (d, J=4.6 Hz, 1H), 4.83
(dd, J=4.6, 8.2 Hz, 1H), 5.14 (s, 2H), 7.32–7.56 (m, 5H), 7.60–7.75
(m, 4H), 8.25 ppm (d, J=8.2 Hz, 1H); 13C NMR (100 MHz,
[D6]DMSO): d=24.4 (3C), 32.4, 58.6, 66.4, 85.6, 127.1 (2C), 127.2
(2C), 128.0, 129.1 (2C), 129.4 (2C), 136.0, 140.6, 140.4, 155.8,
169.1 ppm; (ESI+) m/z (%): 371 (100) [M+NH4]+; (ESIꢀ) m/z (%):
352 (100) [MꢀH]ꢀ; HRMS-ESI: m/z [M+NH4]+calcd for C21H27N2O4:
371.1971, found: 371.1971.
5-Phenylpentyl-N-[(2R*,3R*)-2-isopropyl-4-oxo-oxetan-3-yl]carba-
mate (24b). The reaction was carried out using 17b (0.076 g,
0.24 mmol) as a 8:2 threo/erythro diastereomeric mixture, dry
CH2Cl2 (7.5 mL), Et3N (0.100 mL, 0.72 mmol), and TBTU (0.091 g,
0.29 mmol) to give, after purification, 24b (0.405 g, 56%) as a pure
trans diastereomer, as a white solid: mp: 63.7–64.68C; 1H NMR
(400 MHz, [D6]DMSO): d=0.87 (d, J=6.6 Hz, 3H), 0.95 (d, J=6.6 Hz,
3H), 1.27–1.43 (m, 2H), 1.49–1.69 (m, 4H), 1.87–2.08 (m, 1H), 2.57
(t, J=7.7 Hz, 2H), 3.99 (t, J=6.6 Hz, 2H), 4.22 (dd, J=4.4, 9.1 Hz,
1H), 4.73 (dd, J=4.4, 8.2 Hz, 1H), 7.10–7.35 (m, 5H), 8.06 ppm (d,
J=8.2 Hz, 1H); 13C NMR (100 MHz, [D6]DMSO): d=16.3, 17.9, 24.9,
28.3, 30.6, 30.7, 35.0, 60.9, 64.6, 83.2, 125.6, 128.2 (2C), 128.2 (2C),
142.1, 155.5, 168.9 ppm; MS (ESI+) m/z (%): 320 (100) [M+NH4]+;
(ESIꢀ) m/z (%): 318 (100) [MꢀH]ꢀ; HRMS-ESI: m/z [M+H]+ calcd
for C18H26NO4: 320.1862, found: 320.1869.
General procedure for the synthesis of cis- and trans-a-amino b-
lactone amide derivatives (27a–c–30a–c; Scheme 1).
5-Phenylpentyl-N-[(2R*,3R*)-2-tert-butyl-4-oxo-oxetan-3-yl]carba-
mate (24c). The reaction was carried out using 17c (0.325 g,
0.92 mmol) as a 9:1 threo/erythro diastereomeric mixture, dry
CH2Cl2 (29 mL), Et3N (0.387 mL, 2.77 mmol), and TBTU (0.356 g,
1.11 mmol) to give, after purification, 24c (0.165 g, 54%) as a pure
trans diastereomer, as a white solid: mp: 59.7–62.38C; 1H NMR
(400 MHz, [D6]DMSO): d=0.94 (s, 9H), 1.29–1.39 (m, 2H), 1.52–1.65
(m, 4H), 2.58 (t, J=7.6 Hz, 2H), 4.01 (t, J=6.6 Hz, 2H), 4.33 (d, J=
4.7 Hz, 1H), 4.76 (dd, J=4.7, 8.2 Hz, 1H), 7.11–7.36 (m, 5H),
8.05 ppm (d, J=8.2 Hz, 1H); 13C NMR (75 MHz, [D6]DMSO): d=
24.37 (3C), 25.47, 28.78, 31.07, 32.34, 35.53, 58.52, 65.14, 85.55,
126.10, 128.68 (2C), 128.72 (2C), 142.56, 155.99, 169.18 ppm; MS
(ESI+) m/z (%): 351 (100) [M+NH4]+; (ESIꢀ) m/z (%): 332 (100)
[MꢀH]ꢀ; HRMS-ESI: m/z [M+H]+ calcd for C19H28NO4: 334.2018,
found: 334.2004.
Preparation of b-lactone amides 27a–c–28a–c. In a pear flask,
under nitrogen atmosphere, at room temperature, to a stirred mix-
ture of 7-phenylheptanoic acid (1.1 equiv) in dry CH2Cl2 was added
TBTU (1.1 equiv). The reaction mixture was left stirring at room
temperature for 15 min. Subsequently was added a solution of
21a–c–22a–c (1.0 equiv) and Et3N (2.2 equiv) in dry CH2Cl2. The re-
action mixture was left stirring at room temperature for 16 h. The
solvent was rotary evaporated and the crude product was purified
by column chromatography, using a Teledyne ISCO apparatus, elut-
ing with cyclohexane/EtOAc (from 100:0 to 70:30) to afford com-
pounds 27a–c–28a–c.
N-[(2S*,3R*)-2-Ethyl-4-oxo-oxetan-3-yl]-7-phenylheptanamide
(27a). The reaction was carried out using 21a (0.095 g,
0.33 mmol), Et3N (0.097 mL, 0.73 mmol) in dry CH2Cl2 (4.5 mL), and
7-phenylheptanoic acid (0.075 mL, 0.37 mmol), TBTU (0.117 g,
0.37 mmol) in dry CH2Cl2 (4.5 mL) to give, after purification, pure
27a (0.045 g, 44%) as a white solid: mp: 70.1–72.68C; 1H NMR
(400 MHz, [D6]DMSO): d=0.87 (t, J=7.4 Hz, 3H), 1.29 (p, J=3.6 Hz,
4H), 1.44–1.68 (m, 5H), 1.67–1.82 (m, 1H), 2.16 (t, J=7.3 Hz, 2H),
2.54–2.63 (m, 2H), 4.59 (dt, J=6.0, 7.9 Hz, 1H), 5.59 (dd, J=6.0,
8.8 Hz, 1H), 7.12–7.23 (m, 3H), 7.24–7.33 (m, 2H), 8.77 ppm (d, J=
8.8 Hz, 1H); 13C NMR (100 MHz, [D6]DMSO): d=9.4, 22.6, 25.4, 28.8
(2C), 31.3, 35.3, 35.5, 58.1, 79.3, 126.1, 128.6 (4C), 142.7, 170.6,
172.9 ppm; MS (ESI+) m/z (%): 304 (100) [M+H]+; (ESIꢀ) m/z (%):
302 (100) [MꢀH]ꢀ; HRMS-ESI: m/z [M+H]+ calcd for C18H26NO3:
304.1913, found: 304.1930.
(4-Phenylphenyl)methyl-N-[(2R*,3R*)-2-ethyl-4-oxo-oxetan-3-yl]-
carbamate (26a). The reaction was carried out using 18a (0.193 g,
0.56 mmol) as a 8:2 threo/erythro diastereomeric mixture, dry
CH2Cl2 (17.5 mL), Et3N (0.235 mL, 1.69 mmol), and TBTU (0.217 g,
0.67 mmol) to give, after purification, 26a (0.089 g, 49%) as a pure
trans diastereomer, as a white solid: mp: 131.6–133.28C; 1H NMR
(400 MHz, [D6]DMSO): d=0.92 (t, J=7.4 Hz, 3H), 1.68–1.94 (m, 2H),
4.49–4.60 (m, 1H), 4.76 (dd, J=4.3, 8.1 Hz, 1H), 5.12 (s, 2H), 7.33–
7.42 (m, 1H), 7.42–7.53 (m, 4H), 7.62–7.76 (m, 4H), 8.25 ppm (d, J=
8.1 Hz, 1H); 13C NMR (100 MHz, [D6]DMSO): d=8.7, 25.2, 62.0, 65.8,
79.6, 126.6 (2C), 126.7 (2C), 127.5, 128.6 (2C), 128.9 (2C), 135.5,
139.6, 139.9, 155.3, 168.9 ppm; MS (ESI+) m/z (%): 343 (100) [M+
NH4]+; (ESIꢀ) m/z (%): 324 (100) [MꢀH]ꢀ; HRMS-ESI: m/z [M+
NH4]+calcd for C19H23N2O4: 342.1658, found: 343.1662.
N-[(2S*,3R*)-2-Isopropyl-4-oxo-oxetan-3-yl]-7-phenylheptana-
mide (27b). The reaction was carried out using 21b (0.100 g,
0.33 mmol), Et3N (0.097 mL, 0.73 mmol) in dry CH2Cl2 (4.5 mL), and
7-phenylheptanoic acid (0.075 mL, 0.37 mmol), TBTU (0.117 g,
0.37 mmol) in dry CH2Cl2 (4.5 mL) to give, after purification, pure
27b (0.032 g, 30%) as a white solid: mp: 91.8–92.88C; 1H NMR
(400 MHz, [D6]DMSO): d=0.72 (d, J=6.6 Hz, 3H), 0.95 (d, J=6.6 Hz,
3H), 1.18–1.38 (m, 4H), 1.40–1.64 (m, 4H), 1.98 (dp, J=6.6, 10.7 Hz,
1H), 2.15 (t, J=7.4 Hz, 2H), 2.53–2.61 (m, 2H), 4.23 (dd, J=5.9,
10.7 Hz, 1H), 5.58 (dd, J=5.9, 9.0 Hz, 1H), 7.08–7.34 (m, 5H),
8.81 ppm (d, J=9.0 Hz, 1H); 13C NMR (100 MHz, [D6]DMSO): d=
17.2, 18.7, 25.4, 27.9, 28.7, 28.8, 31.3, 35.4, 35.5, 57.6, 82.5, 126.1,
128.6 (4C), 142.7, 170.6, 172.8 ppm; MS (ESI+) m/z (%): 318 (100)
[M+H]+; (ESI) m/z: 316 (100) [MꢀH]ꢀ; HRMS-ESI: m/z [M+H]+
calcd for C19H28NO3: 318.2069, found: 318.2083.
(4-Phenylphenyl)methyl-N-[(2R*,3R*)-2-isopropyl-4-oxo-oxetan-
3-yl]carbamate (26b). The reaction was carried out using 18b
(0.059 g, 0.17 mmol) as a 8:2 threo/erythro diastereomeric mixture,
dry CH2Cl2 (5.3 mL), Et3N (0.070 mL, 0.51 mmol), and TBTU (0.065 g,
0.20 mmol) to give, after purification, 26b (0.010 g, 17%) as a pure
trans diastereomer, as a white solid: mp: 127.5–129.58C; 1H NMR
(400 MHz, [D6]DMSO): d=0.89 (d, J=6.7 Hz, 3H), 0.97 (d, J=6.7 Hz,
3H), 1.81–2.12 (m, 1H), 4.26 (dd, J=4.4, 9.1 Hz, 1H), 4.80 (dd, J=
4.4, 8.2 Hz, 1H), 5.12 (s, 2H), 7.29–7.54 (m, 5H), 7.63–7.77 (m, 4H),
8.26 ppm (d, J=8.2 Hz, 1H); 13C NMR (100 MHz, [D6]DMSO): d=
16.3, 17.9, 30.6, 61.0, 65.9, 83.2, 126.7 (2C), 126.7 (2C), 127.5, 128.6
(2C), 128.9 (2C), 135.5, 139.7, 139.9, 155.3, 168.7 ppm; MS (ESI+)
m/z (%): 357 (100) [M+NH4]+; (ESI) m/z (%): 338 (100) [MꢀH]ꢀ;
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N-[(2S*,3R*)-2-tert-Butyl-4-oxo-oxetan-3-yl]-7-phenylheptana-
mide (27c). The reaction was carried out using 21c (0.100 g,
0.32 mmol), Et3N (0.093 mL, 0.70 mmol) in dry CH2Cl2 (4.5 mL), and
7-phenylheptanoic acid (74 mL, 0.36 mmol), TBTU (0.116 g,
0.36 mmol) in dry CH2Cl2 (4.5 mL) to give, after purification, pure
Et3N (2.2 equiv) and the reaction stirred for 15 min. Subsequently,
at 08C, under nitrogen atmosphere was slowly added a solution of
4-benzyloxybenzoyl chloride (1.1 equiv) in dry CH2Cl2, and the reac-
tion stirred at 08C for 2 h. Upon full conversion of starting material,
the crude mixture was concentrated under reduced pressure, and
the crude residue purified by column chromatography, using a Tele-
dyne ISCO apparatus, eluting with cyclohexane/EtOAc (from 100:0
to 70:30) to afford compounds 29a–c–30a–c.
1
27c (0.040 g, 33%) as a colorless oil: H NMR (400 MHz, [D6]DMSO):
d=0.91 (s, 9H), 1.21–1.33 (m, 4H), 1.42–1.59 (m, 4H), 2.09–2.28 (m,
2H), 2.55 (t, J=7.7 Hz, 2H), 4.37 (d, J=6.2 Hz, 1H), 5.73 (dd, J=6.2,
8.1 Hz, 1H), 7.08–7.54 (m, 5H), 8.90 ppm (d, J=8.1 Hz, 1H);
13C NMR (100 MHz, [D6]DMSO): d=25.2 (3C), 25.4, 28.7, 28.9, 31.4,
32.9, 35.1, 35.5, 56.8, 84.2, 126.1, 128.6 (4C), 142.7, 170.6,
172.9 ppm; MS (ESI+) m/z (%): 332 (100) [M+H]+; (ESIꢀ) m/z: 330
(100) [MꢀH]ꢀ; HRMS-ESI: m/z [M+H]+ calcd for C20H30NO3:
332.2226, found: 332.2218.
N-[(2S*,3R*)-2-Ethyl-4-oxo-oxetan-3-yl]-4-benzyloxybenzamide
(29a). The reaction was carried out using 21a (0.087 g, 0.30 mmol)
in dry CH2Cl2 (4.2 mL), Et3N (0.088 mL, 0.67 mmol), and 4-benzylox-
ybenzoyl chloride (0.082, 0.33 mmol) in dry CH2Cl2 (4.2 mL), to give,
after purification, 29a (0.012 g, 12%) as a white solid: mp: 141.0–
1
144.28C; H NMR (400 MHz, [D6]DMSO): d=0.88 (t, J=7.4 Hz, 3H),
N-[(2R*,3R*)-2-Ethyl-4-oxo-oxetan-3-yl]-7-phenylheptanamide
(28a). The reaction was carried out using 22a (0.110 g, 0.38 mmol)
1.64–1.77 (m, 1H), 1.79–1.93 (m, 1H), 4.68 (dt, J=5.8, 8.0 Hz, 1H),
5.20 (s, 2H), 5.78 (dd, J=5.8, 8.5 Hz, 1H), 7.13 (d, J=8.8 Hz, 2H),
7.32–7.38 (m, 1H), 7.39–7.45 (m, 2H), 7.44–7.51 (m, 2H), 7.88 (d,
J=8.8 Hz, 2H), 9.21 ppm (d, J=8.5 Hz, 1H); 13C NMR (100 MHz,
[D6]DMSO): d=9.4, 22.5, 58.6, 69.9, 79.6, 115.0, 115.1, 125.7, 128.2
(2C), 128.4, 128.9 (2C), 129.9, 131.8, 137.1, 161.7, 166.3, 170.4 ppm;
MS (ESI+) m/z (%): 226 (100) [M+H]+; (ESIꢀ) m/z (%): 224
[MꢀH]ꢀ; HRMS-ESI: m/z [M+H]+ calcd for C19H20NO4: 326.1392,
found: 326.1400.
as
a 8:2 trans/cis diastereomeric mixture, Et3N (0.117 mL,
0.84 mmol) in dry CH2Cl2 (5.3 mL), and 7-phenylheptanoic acid
(86 mL, 0.42 mmol), TBTU (0.135 g, 0.42 mmol) in dry CH2Cl2
(5.3 mL) to give, after purification, 28a (0.040 g, 34%) as a pure
1
trans diastereomer, as a white solid: H NMR (400 MHz, [D6]DMSO):
d=0.91 (t, J=7.4 Hz, 3H), 1.21–1.39 (m, 4H), 1.46–1.64 (m, 4H),
1.79 (m, 2H), 2.14 (t, J=7.4 Hz, 2H), 2.57 (m, 2H), 4.53 (m, 1H),
4.75 (dd, J=4.3, 7.7 Hz, 1H), 7.14–7.22 (m, 3H), 7.28 (m, 2H),
8.65 ppm (d, J=7.7 Hz, 1H); 13C NMR (100 MHz, [D6]DMSO): d=9.2,
25.3, 25.8, 28.7, 28.8, 31.4, 35.2, 35.5, 61.3, 79.6, 126.1, 128.7 (2C),
128.7 (2C), 142.7, 169.5, 173.2 ppm; MS (ESI+) m/z (%): 304 (100)
[M+H]+; (ESIꢀ) m/z (%): 302 (100) [MꢀH]ꢀ.
N-[(2S*,3R*)-2-Isopropyl-4-oxo-oxetan-3-yl]-4-benzyloxybenza-
mide (29b). The reaction was carried out using 21b (0.100 g,
0.33 mmol) in dry CH2Cl2 (4.5 mL), Et3N (0.097 mL, 0.73 mmol), and
4-benzyloxybenzoyl chloride (0.090 g, 0.37 mmol) in dry CH2Cl2
(4.5 mL), to give, after purification, pure 29b (0.016 g, 13%) as
a white solid: mp: 148.0–149.08C; 1H NMR (400 MHz, [D6]DMSO):
d=0.71 (d, J=6.6 Hz, 3H), 0.98 (d, J=6.6 Hz, 3H), 2.07–2.26 (m,
1H), 4.31 (dd, J=5.9, 10.8 Hz, 1H), 5.19 (s, 2H), 5.78 (dd, J=5.9,
8.7 Hz, 1H), 7.12 (d, J=8.5 Hz, 2H), 7.29–7.59 (m, 5H), 7.87 ppm (d,
J=8.5 Hz, 2H), 9.26 (d, J=8.7 Hz, 1H); 13C NMR (100 MHz,
[D6]DMSO): d=16.8, 18.5, 27.6, 57.8, 69.6, 82.5, 114.8 (2C), 125.4,
127.9 (2C), 128.2, 128.6 (2C), 129.6 (2C), 136.8, 161.4, 165.9,
170.2 ppm; MS (ESI+) m/z (%): 340 (100) [M+H]+; (ESIꢀ) m/z (%):
338 (100) [MꢀH]ꢀ; HRMS-ESI: m/z [M+H]+ calcd for C20H22NO4:
340.1549, found: 340.1552.
N-[(2R*,3R*)-2-Isopropyl-4-oxo-oxetan-3-yl]-7-phenylheptana-
mide (28b). The reaction was carried out using 22b (0.100 g,
0.41 mmol) as
a 8:2 trans/cis diastereomeric mixture, Et3N
(0.125 mL, 0.90 mmol) in dry CH2Cl2 (5.6 mL), and 7-phenylheptano-
ic acid (0.092 g, 0.45 mmol), TBTU (0.144 g, 0.45 mmol) in dry
CH2Cl2 (5.6 mL) to give, after purification, 28b (0.06 g, 46%) as
a pure trans diastereomer, as a white solid: 1H NMR (400 MHz,
[D6]DMSO): d=0.87 (d, J=6.6 Hz, 3H), 0.96 (d, J=6.6 Hz, 3H), 1.28
(m, 4H), 1.40–1.65 (m, 4H), 1.89–2.04 (m, 1H), 2.14 (t, J=7.3 Hz,
2H), 2.53–2.61 (m, 2H), 4.24 (dd, J=4.3, 9.1 Hz, 1H), 4.80 (dd, J=
4.3, 7.8 Hz, 1H), 7.11–7.34 (m, 5H), 8.66 ppm (d, J=7.8 Hz, 1H);
13C NMR (100 MHz, [D6]DMSO): d=16.3, 17.9, 24.8, 28.2, 28.3, 30.7,
30.8, 34.7, 35.0, 59.7, 82.6, 125.6, 128.2 (4C), 142.2, 168.8,
172.7 ppm; MS (ESI+) m/z: 316 [M+H]+; (ESIꢀ) m/z: 318 [MꢀH]ꢀ.
N-[(2S*,3R*)-2-tert-Butyl-4-oxo-oxetan-3-yl]-4-benzyloxybenza-
mide (29c). The reaction was carried out using 21c (0.100 g,
0.32 mmol) in dry CH2Cl2 (4.5 mL), Et3N (0.93 mL, 0.70 mmol), and
4-benzyloxybenzoyl chloride (0.086 g, 0.35 mmol) in dry CH2Cl2
(4.5 mL) to give, after purification, pure 29c (0.019 g, 17%) as
a white solid: mp: 146.7–148.98C; 1H NMR (400 MHz, [D6]DMSO):
d=0.95 (s, 9H), 4.46 (d, J=6.2 Hz, 1H), 5.19 (s, 2H), 5.95 (dd, J=
6.2, 7.6 Hz, 1H), 7.00–7.16 (m, 2H), 7.29–7.52 (m, 5H), 7.84–8.01 (m,
2H), 9.34 ppm (d, J=7.6 Hz, 1H); 13C NMR (100 MHz, [D6]DMSO):
d=25.6 (3C), 33.1, 57.8, 70.0, 84.5, 115.1 (2C), 125.8, 128.4 (2C),
128.6, 129,1 (2C), 130.2 (2C), 137.2, 161.8, 166.4, 170.0 ppm; MS
(ESI+) m/z (%): 354 (100) [M+H]+; (ESIꢀ) m/z (%): 352 (100)
[MꢀH]ꢀ; HRMS-ESI: m/z [M+H]+ calcd for C21H24NO4: 354.1705,
found: 354.1718.
N-[(2R*,3R*)-2-tert-Butyl-4-oxo-oxetan-3-yl]-7-phenylheptana-
mide (28c). The reaction was carried out using 22c (0.075 g,
0.23 mmol) as
a 9:1 trans/cis diastereomeric mixture, Et3N
(0.068 mL, 0.52 mmol) in dry CH2Cl2 (3.2 mL), and 7-phenylheptano-
ic acid (0.052 g, 0.26 mmol), TBTU (0.084 g, 0.26 mmol) in dry
CH2Cl2 (3.2 mL) to give, after purification, 28c (0.035 g, 46%) as
a pure trans diastereomer, as a white solid: mp: 89.0–91.28C;
1H NMR (400 MHz, [D6]DMSO): d=0.92 (s, 9H), 1.24–1.32 (m, 4H),
1.45–1.60 (m, 4H), 2.13 (t, J=7.3 Hz, 2H), 2.55 (t, J=6.8, 2H), 4.32
(d, J=4.6 Hz, 1H), 4.84 (dd, J=4.6, 7.8 Hz, 1H), 7.13–7.19 (m, 3H),
7.24–7.29 (m, 2H), 8.62 ppm (d, J=7.8 Hz, 1H); 13C NMR (100 MHz,
[D6]DMSO): d=23.9 (3C), 24.9, 28.2, 28.3, 30.8, 31.8, 34.7, 35.1, 56.7,
84.6, 125.6, 128.2 (2C), 128.2 (2C), 142.3, 168.7, 172.8 ppm; MS
(ESI+) m/z (%): 332 (100) [M+H]+; (ESIꢀ) m/z (%): 330 (100)
[MꢀH]ꢀ; HRMS-ESI: m/z [M+H]+ calcd for C21H24NO4: 355.1783,
found: 355.1772.
N-[(2R*,3R*)-2-Ethyl-4-oxo-oxetan-3-yl]-4-benzyloxybenzamide
(30a). The reaction was carried out using 22a (0.150 g, 0.52 mmol)
as a 8:2 trans/cis diastereomeric mixture, dry CH2Cl2 (7.3 mL), Et3N
(0.159 mL, 1.14 mmol), and 4-benzyloxybenzoyl chloride (0.141 g,
0.57 mmol) in dry CH2Cl2 (7.3 mL) to give, after purification, 30a
(0.010 g, 8.5%) as a pure trans diastereomer, as a white solid:
1H NMR (400 MHz, [D6]DMSO): d=0.95 (t, J=7.4 Hz, 3H), 1.75–1.98
(m, 2H), 4.69 (td, J=4.3, 6.9 Hz, 1H), 4.92 (dd, J=4.3, 7.6 Hz, 1H),
5.20 (s, 2H), 7.05–7.17 (m, 2H), 7.34–7.48 (m, 5H), 7.90 (m, 2H),
Preparation of b-lactone amides 29a–c–30a–c. In a round-bot-
tomed flask, at 08C, under nitrogen atmosphere, to a stirred mix-
ture of 21a–c–22a–c (1.0 equiv) in dry CH2Cl2 was added dropwise
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9.18 ppm (d, J=7.6 Hz, 1H); 13C NMR (100 MHz, [D6]DMSO): d=9.3,
25.9, 61.8, 69.9, 79.6, 115.1 (2C), 125.5, 128.3 (2C), 128.4, 128.9 (2C),
129.8, 131.8, 137.0, 161.7, 167.4, 169.4 ppm; MS (ESI+) m/z (%):
226 (100) [MꢀH]+; (ESIꢀ) m/z (%): 224 (100) [MꢀH]ꢀ.
(0.01m phosphate buffer, pH 5.0) conditions for up to 24 h. Both
buffers were added with 10% CH3CN. Stock solutions of each com-
pound (10 mm) were prepared freshly in DMSO. Each compound
was incubated at a final concentration of 1.0 mm (1% DMSO) in
both preheated buffers (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 autosam-
pler during the study (no shaking). A reference solution of each
compound (final concentration: 1.0 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 (no shaking). The analyses were performed on
a Waters Acquity UPLC–MS TQD system consisting of a triple quad-
rupole detector (TQD) mass spectrometer equipped with an ESI in-
terface and a PDA detector. The 24 h time-course analysis for 29b,
30b, 25b, and 26b and their corresponding rearrangement/hy-
drolysis products (Supporting Information, figures S1–S4) was car-
ried out by UPLC–UV on the same instrument described above. A
calibration curve in the 0.2–50 mm concentration range was pre-
pared for each parent compound by serial dilution in CH3CN (R2 >
0.999 for all compounds). The concentration of the rearranged and
hydrolyzed products of each parent was then calculated on the
corresponding calibration curve, assuming no changes in the
molar absorptivity (e) values. To test this assumption, each parent
was fully hydrolyzed with 1.0m NaOH solution, and the concentra-
tion of the corresponding product was calculated on the parent
compound calibration curve. The measured concentration matched
the expected 30 mm value. The analyses were performed on an
Acquity UPLC BEH C18 1.7 mm 1.0ꢂ100 mm column, kept 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.: 10% B, 0.5–
4.0 min.: 10!100% B, 4.0–5.0 min.: 100% B, 5.0–5.1 min.: 100!
N-[(2R*,3R*)-2-Isopropyl-4-oxo-oxetan-3-yl]-4-benzyloxybenza-
mide (30b). The reaction was carried out using 22b (0.100 g,
0.33 mmol) as a 8:2 trans/cis diastereomeric mixture, dry CH2Cl2
(4.5 mL), Et3N (0.097 mL, 0.73 mmol) and 4-benzyloxybenzoyl chlo-
ride (0.090 g, 0.37 mmol) in dry CH2Cl2 (5.0 mL) to give, after purifi-
cation, 30b (0.021 g, 19%) as a pure trans diastereomer, as a white
solid: mp: 119.0–121.08C; 1H NMR (400 MHz, [D6]DMSO): d=0.92
(d, J=6.7 Hz, 3H), 0.99 (d, J=6.7 Hz, 3H), 1.95–2.09 (m, 1H), 4.39
(dd, J=4.4, 9.1 Hz, 1H), 4.95 (dd, J=4.4, 7.7 Hz, 1H), 5.19 (s, 2H),
7.09–7.17 (m, 2H), 7.31–7.51 (m, 5H), 7.81–7.90 (m, 2H), 9.19 ppm
(d, J=7.7 Hz, 1H); 13C NMR (100 MHz, [D6]DMSO): d=16.4, 17.9,
30.8, 60.2, 69.4, 82.7, 114.6 (2C), 124.9, 127.7 (2C), 127.9, 128.4 (2C),
129.3 (2C), 136.5, 161.2, 165.7, 168.8 ppm; MS (ESI+) m/z (%): 340
(100) [M+H]+; (ESIꢀ) m/z (%): 338 (100) [MꢀH]ꢀ; HRMS-ESI: m/z
[M+H]+ calcd for C20H22NO4: 340.1549, found: 340.1551.
N-[(2R*,3R*)-2-tert-Butyl-4-oxo-oxetan-3-yl]-4-benzyloxybenza-
mide (30c). The reaction was carried out using 22c (0.050 g,
0.16 mmol) as a 9:1 trans/cis diastereomeric mixture, dry CH2Cl2
(2.5 mL), Et3N (0.049 mL, 0.35 mmol) and 4-benzyloxybenzoyl chlo-
ride (0.042 g, 0.17 mmol) in dry CH2Cl2 (2.5 mL), to give, after purifi-
cation, 30c (0.03 g, 56%) as a pure trans diastereomer, as a white
solid: mp: 145.2–146.78C; 1H NMR (400 MHz, [D6]DMSO): d=0.97
(s, 9H), 4.48 (d, J=4.6 Hz, 1H), 4.99 (dd, J=4.6, 7.7 Hz, 1H), 5.19 (s,
2H), 7.13 (d, J=8.8 Hz, 2H), 7.29–7.53 (m, 5H), 7.84 (d, J=8.8 Hz,
2H), 9.17 ppm (d, J=7.7 Hz, 1H); 13C NMR (75 MHz, [D6]DMSO): d=
23.93 (3C), 31.92, 57.11, 69.42, 84.57, 99.50, 114.63 (2C), 127.77 (2C),
127.96, 128.45 (2C), 129.28 (2C), 136.54, 161.26, 165.68,
10% B, 5.1–6.5 min: 10% B at a flow rate of 0.1 mLminꢀ1
.
168.67 ppm; MS (ESI) m/z (%): 354 (100) [M+Na]+ HRMS-ESI: m/z
;
[M+H]+ calcd for C20H30NO3: 333.2304, found: 333.2312.
In vitro rat plasma stability:[19c] Compounds were diluted in rat
plasma added with 20% PBS (pH 7.4) and 5% DMSO to aid in solu-
bilization. Plasma was preheated at 378C (30 min). The final com-
pound concentration was 3.0 mm. At time points (immediately after
dilution, 1, 5, 15 and 30 min) a 40 mL aliquot of the incubation so-
lution was diluted in 150 mL cold CH3CN spiked with Warfarin
(200 nm) as internal standard. After vortexing for 30 s, the solution
was centrifuged for 15 min at 3500 g, and the supernatant trans-
ferred for LC–MS analysis. A reference 30 min incubation in PBS
(pH 7.4) added with 5% DMSO was also prepared for each com-
pound. Samples were analyzed on a UPLC-Xevo triple quadrupole
system (Waters Inc., Milford, USA).
Pharmacology
Fluorogenic hNAAA assay: HEK293 cells stably transfected with the
human NAAA coding sequence cloned from a human spleen cDNA
library were used as enzyme source. Recombinant HEK-hNAAA pel-
lets were resuspended in homogenizing buffer, and sonicated.
Samples were centrifuged at 800 g for 15 min at 48C, and the re-
sultant supernatants were then ultracentrifuged at 12000 g for
30 min at 48C. The pellets were resuspended in PBS pH 7.4 on ice
and subjected to a freeze/thaw cycle at ꢀ808C. The suspension
was finally centrifuged at 105000 g for 1 h at 48C. Protein concen-
tration was measured, and aliquots of samples were stored at
ꢀ808C until use. The assay was run in Optiplate 96-well black
plates, in a total reaction volume of 200 mL. NAAA protein prepara-
tion (4.0 mg) was pre-incubated for 10 min with various concentra-
tions of test compounds or vehicle control (5% DMSO) in 100 mm
citrate/phosphate buffer (pH 4.5) containing 3.0 mm DTT, 0,1%
Triton X-100, 0.05% BSA, 150 mm NaCl. N-(4-methyl-2-oxochro-
men-7-yl)hexadecanamide (PAMCA)[14c] was used as substrate
(5.0 mm), and the reaction was held for 30 min at 378C. The sam-
ples were then read in a PerkinElmer Envision plate reader using
an excitation wavelength of 360 nm and emission at 460 nm. IC50
values were calculated by nonlinear regression analysis of log[con-
centration]/inhibition curves using GraphPad Prism 5 (GraphPad
Software Inc., San Diego, CA, USA) applying a standard slope curve
fit.
Computational methods
The quantum chemical calculations were performed with the Gaus-
sian 09 (G09) program suite.[41] For all stationary points, both ge-
ometry and analytical frequency calculations were carried out at
the DFT level of theory by using the B3LYP functional. The em-
ployed basis set was Pople’s 6-31+ +G(d,p).[42] All the reported cal-
culations were performed in polarizable conductor calculation
model (CPCM),[43] and the chosen solvent was water. To mimic hy-
drolysis of the a-amino b-lactone ring, we used a water molecule
as nucleophile, assisted by a second one. We were able to identify
the transition states (TSs) for all the four compounds and for both
the two different attacks, and to decipher the nature of the reac-
tion mechanism. In total, eight reactions were studied. The struc-
tures of the identified TSs were further investigated: the diagonal-
ized mass-weighted Hessian matrix showed only one negative ei-
genvalue, revealing a first-order saddle point. To confirm the reac-
In vitro chemical stability: Chemical stability of selected compounds
was evaluated under physiological (0.01m PBS, pH 7.4) and acidic
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tion path, we performed intrinsic reaction coordinate (IRC)[44] calcu-
lations in order to follow the reaction starting from the TS reaching
the reactants and the products. Finally the end points of the IRC
pathway were optimized, and the nature of these points was inves-
tigated, confirming that they are energy minima in the potential
energy surface.
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The authors thank Luisa Mengatto (IIT) for hNAAA screenings, Dr.
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Received: October 18, 2013
Published online on January 8, 2014
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