Progress in the development of β-lactams as N-Acylethanolamine Acid Amidase (NAAA) inhibitors: Synthesis and SAR study of new, potent N-O-substituted derivatives

Article abstract of DOI:10.1016/j.ejmech.2016.11.039

The anti-inflammatory effects resulting from raising the levels of palmitoylethanolamide (PEA), an endogenous bioactive lipid, led to envisage N-Acylethanolamine Acid Amidase (NAAA), the cysteine hydrolase mainly responsible for PEA degradation, as an att

Full text of DOI:10.1016/j.ejmech.2016.11.039

Accepted Manuscript
Progress in the development of β-lactams as N-Acylethanolamine Acid Amidase
(NAAA) inhibitors: Synthesis and SAR study of new, potent N-O-substituted
derivatives
R. Petracca, S. Ponzano, S.M. Bertozzi, O. Sasso, D. Piomelli, T. Bandiera, F.
Bertozzi
PII:
S0223-5234(16)30977-1
10.1016/j.ejmech.2016.11.039
EJMECH 9072
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 12 October 2016
Revised Date: 18 November 2016
Accepted Date: 19 November 2016
Please cite this article as: R. Petracca, S. Ponzano, S.M. Bertozzi, O. Sasso, D. Piomelli, T. Bandiera,
F. Bertozzi, Progress in the development of β-lactams as N-Acylethanolamine Acid Amidase (NAAA)
inhibitors: Synthesis and SAR study of new, potent N-O-substituted derivatives, European Journal of
Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.11.039.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Progress in the development of β-lactams as N-Acylethanolamine Acid
Amidase (NAAA) inhibitors: synthesis and SAR study of new, potent N-
O-substituted derivatives
†
†
†
†
†,ǁǁ
†
†,
Petracca, R.; Ponzano, S.; Bertozzi, S. M.; Sasso, O.; Piomelli, D.; Bandiera, T.; Bertozzi, F. *
†
Drug Discovery and Development, Istituto Italiano di Tecnologia, Via Morego 30, I-16163 Genova,
Italy;
ǁǁ
Departments of Anatomy and Neurobiology, Pharmacology and Biological Chemistry, University of
California, Irvine, USA, 92697-4625
1

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CORRESPONDING AUTHOR
For F.B.: phone, +39-010-71781235; e-mail, fabio.bertozzi@iit.it
AUTHOR CONTRIBUTIONS
R.P. and S.P. designed and synthesized compounds; S.M.B. performed chemical and plasma stability
tests; O.S. performed in vivo studies; D.P. ideated and coordinated the project; R.P., T.B. and F.B.
wrote the manuscript.
2

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GRAPHICAL ABSTRACT
3

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ABSTRACT
The anti-inflammatory effects resulting from raising the levels of palmitoylethanolamide (PEA), an
endogenous bioactive lipid, led to envisage N-Acylethanolamine Acid Amidase (NAAA), the cysteine
hydrolase mainly responsible for PEA degradation, as an attractive target for small molecule inhibitors.
Previous work in our group identified serine-derived β-lactams as potent and systemically active
inhibitors of NAAA activity. Aiming to expand the SAR study around this class of compounds, we
investigated the effect of the substitution on the endocyclic nitrogen by designing and synthesizing a
series of N-substituted β-lactams. The present work describes the synthesis of new N-O-alkyl and N-O-
aryl substituted β-lactams and reports the results of the structure activity relationship (SAR) study
leading to the discovery of a novel, single-digit nanomolar NAAA inhibitor (37). Compound 37 was
shown in vitro to inhibit human NAAA via S-acylation of the catalytic cysteine, and to display very
good selectivity vs. human Acid Ceramidase, a cysteine amidase structurally related to NAAA.
Preliminary in vivo studies showed that compound 37, administered topically, reduced paw edema and
heat hyperalgesia in a carrageenan-induced inflammation mouse model. The high in vitro potency of 37
as NAAA inhibitor, and its encouraging in vivo activity qualify this compound as a new tool for the
study of the role of NAAA in inflammatory and pain states.
KEYWORDS
N-Acylethanolamine Acid Amidase; cysteine hydrolase; N-O-substituted β-lactams; inhibitors;
Structure–Activity Relationships (SAR).
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ABBREVIATIONS USED
NAAA, N-acylethanolamine acid amidase; FAEs, fatty acid ethanolamides; OEA, oleoylethanolamide;
PEA, palmitoylethanolamide; FAAH, fatty acid amide hydrolase; AC, acid ceramidase; PPAR–α,
peroxisome proliferator-activated receptor–α; PAMCA, N-(4-methyl-2-oxo-chromen-7-yl)-
hexadecanamide; EDC HCl, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride; 2-DPC,
di-2-pyridyl carbonate; HOBt, 1-hydroxybenzotriazole; DEAD, diethyl azadicarboxylate; TCEP, tris(2-
carboxyethyl)phosphine;
TBTA,
tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl]amine;
THF,
tetrahydrofurane; DCM, dichloromethane; DIPEA, diisopropylethylamine; PPh
3
, triphenylphosphine.
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1
. INTRODUCTION
Palmitoylethanolamide (PEA), a member of the Fatty Acid Ethanolamides (FAEs) family, is an
endogenous bioactive lipid able to inhibit peripheral inflammation and mast cell degranulation by
activating the peroxisome proliferator-activated receptor-α (PPAR-α), [1,2] as well as to exert
antinociceptive effects in rat and mouse models of acute and chronic pain. [3,4,5] The anti-
inflammatory effects resulting from raising local levels of PEA led to envisage N-Acylethanolamine
Acid Amidase (NAAA), the lysosomal cysteine hydrolase mainly involved in PEA degradation, [6] as
an attractive target for small molecule inhibitors for the treatment of inflammatory and pain conditions.
[
7] N-Acylethanolamine Acid Amidase (NAAA) is activated by autoproteolysis at acidic pH generating
a catalytically competent subunit of the enzyme bearing a cysteine (Cys131 in mice, Cys126 in
humans) as the nucleophile residue responsible for FAEs hydrolysis. [8,9,10]
Compounds bearing electrophilic chemical functions, such as an α-amino β-lactone moiety, were
described in literature to covalently react with NAAA catalytic cysteine. [11,12,13,14,15] Among
them, the threonine-derived β-lactone
1 (ARN077, Figure 1), was reported to efficiently inhibit rat and
human NAAA in vitro activity (r-NAAA IC = 0.050 µM; h-NAAA IC = 0.007 µM), [16] and to
5
0
50
display in vivo activity by topical administration in rodent models of hyperalgesia and allodynia caused
by inflammation or nerve damage. [17] The administration of ARN077 attenuated, in a dose-dependent
manner, hyperalgesia and mechanical allodynia induced in mice by carrageenan injection or sciatic
nerve ligation, reversed the allodynia induced by ultraviolet B (UVB) radiation in rats, and normalized
FAE levels in inflamed mouse skin. [16]
Although potent towards our target enzyme, β-lactone inhibitors showed a limited plasma stability (t
½
<
1
0 sec), thus preventing their potential use as systemic drugs.[16,18,19] Aiming to further explore the
therapeutic utility of NAAA inhibitors, the replacement of the β-lactone core with a structurally similar
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β-lactam moiety was envisioned, leading to a first series of serine-derived β-lactam amides.[20]
Among the different amide analogues, the N-[(S)-2-oxoazetidin-3-yl]nonanamide (2, Figure 1) showed
good inhibitory potency in vitro against human NAAA (h-NAAA IC = 0.34 µM) and an acceptable
5
0
½
mouse plasma stability (t = 130 min). After intravenous and oral administration in rat, compound 2
exhibited a good oral bioavailability (F = 67 %), supporting the use of such class of compounds for
systemic administration.[20] Additionally, as demonstrated by a recently published SAR study, the
functionalization of the exocyclic amino group of the β-lactam scaffold as a carbamate afforded
compounds with potent NAAA inhibitory activity.[21] Notably, compound
3 (ARN0726, Figure 1)
showed a good inhibitory activity in vitro, on both rat and human NAAA (r-NAAA IC = 0.028 µM,
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0
h-NAAA IC50 = 0.073 µM) and was recently reported to exert anti-inflammatory effects in mouse
models of inflammation and suppress LPS-stimulated inflammatory reactions in human
macrophages.[22]
Figure 1.
Up to the present, no crystallographic structure of the target protein (NAAA) is available. Therefore,
although potent enzyme inhibitors have been identified, the precise interactions between those
molecules and the amino acidic residues of the catalytic binding site cannot be described in detail. In
order to collect additional information on the NAAA binding pocket, novel β-lactam derivatives
substituted on the endocyclic nitrogen (N1) were designed. The chemical growth of those compounds
on their left-hand side is expected to shed additional light on the stereo-electronic requirements of the
NAAA catalytic site. To this purpose, the extensively characterized ARN0726 was selected as starting
point for our SAR study. With the aim to maintain fixed the chemical functions essential for NAAA
inhibition (i.e. the β-lactam core and the exocyclic nitrogen derivatization as carbamate) as well as to
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further explore the available chemical space on the β-lactam scaffold, the functionalization of the
endocyclic nitrogen (N1) with different substituents was envisaged. To this purpose, a few synthetically
accessible N1-substituted derivatives were designed, synthesized and tested against h-NAAA. Notably,
among the possible chemical modifications on N1, the corresponding N-O-alkylation was clearly
envisaged as the most beneficial to retain significant h-NAAA inhibition (Table 1).
The aim of the present work was to investigate the effect of the N-O-substitution on h-NAAA
inhibitory activity as well as to evaluate the available space in the enzyme active site by designing and
synthesizing a novel class of N-O-substituted β-lactams. A conventional SAR study was performed to
uncover the chemical features beneficial for NAAA inhibition, leading to the discovery of new potent
β-lactam inhibitors that were further characterized also in term of stability, selectivity and in vivo
activity.
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2
. CHEMISTRY
A slight modification of the reported synthetic pathway to unprotected serine-derived β-lactams [21]
provided an efficient procedure to easily prepare the new N-substituted analogues. Based-catalyzed
nucleophilic substitutions using 3 (ARN0726) and the corresponding methyl iodide, acetyl chloride and
methyl-chloroformate afforded compounds 4, 5, and 6, respectively, in moderate yield (Scheme 1).
Scheme 1.
Concerning the N-O-methyl substituted serine- and threonine-derived stereoisomers, the final
compounds were obtained starting from the corresponding enantiomerically pure amino acid in a five-
step synthetic sequence, as already described in a previous work (see Supporting Information, Schemes
S1-3).[21]
The novel N-O-substituted α-amino β-methyl β-lactams were prepared by the enantioselective
synthetic pathway reported in Scheme 2. Except for the N-O-tert-butyl derivative 13 (see Supporting
Information, Scheme S4), all the N-O-alkyl and aryl derivatives were obtained by installation of the
corresponding substituent onto the endocyclic nitrogen of the common building block 18. Two
different series of N-O-substituted analogues were synthesized.
Starting from the enantiomerically pure Boc-L-threonine (14), the first step consisted in the conversion
of the free carboxylic acid into the benzyloxy amide derivative, followed by transformation of the
secondary alcohol into the corresponding mesylate (15). An intramolecular base-catalyzed (potassium
carbonate) cyclization reaction in acetone afforded the corresponding N-O-benzyl α-amino β-methyl β-
lactam core 16 with inversion of stereochemistry at C3. The insertion of the desired carbamic acid side
chain on the exocyclic amino group was accomplished as previously reported for the synthesis of other
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β-lactam derivatives.[21] First, compound 16 was Boc-deprotected and the obtained amine transformed
into the tosylate salt. The carbamoylation reaction was carried out with a mixture (1.6:1 isomeric ratio)
of 2-pyridyl carbonate (S181) and 2-oxopyridine-1-carboxylate (S182) derivatives, previously obtained
by activation of 4-cyclohexylbutan-1-ol with 2-DPC in the presence of catalytic DMAP (see
Supporting Information, Scheme S1),[21,23] to afford the 4-cyclohexylbutyl derivative 17. As last step,
the hydrogenolytic removal of the benzyl group of 17 afforded the N-hydroxy substituted β-lactam key
intermediate 18 in high yield. The yields of the single steps were generally good, varying from 50% in
the carbamoylation reaction to quantitative for the salt preparation.
The final step to achieve the N-O-substituted α-amino β-methyl β-lactam analogues started from 18
which was either submitted to a classic Mitsunobu reaction[24,25] with various alcohols to obtain the
N-O-alkyl derivatives (19-29), or to a copper catalyzed coupling reaction[26] with arylboronic acids to
afford the corresponding N-O-aryl β−lactams (30-39) (Scheme 2).
Scheme 2.
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3
. RESULTS and DISCUSSION
As the first step, in our study we investigated the possible substitutions on the endocyclic nitrogen (N1)
of β-lactam derivative ARN0726), compatible with the NAAA inhibitory activity of this chemical
3
(
class. We synthesized a small set of derivatives (Table 1) and tested their ability to inhibit the
hydrolysis of N-(4-methyl-2-oxo-chromen-7-yl)-hexadecanamide (PAMCA) by recombinant h-NAAA
heterologously expressed in HEK293 cells (See Supporting Info).[21]
Previous works on β-lactams carried out by our research group reported compound
3 as a potent,
double-digit nanomolar NAAA inhibitor with pronounced in vivo anti-inflammatory properties in
mouse models.[21,22] In this context, a strong stereorecognition by the target enzyme was highlighted,
being the (R)-enantiomer around 40-fold less active than the (S)-enantiomer in vitro and failing to show
anti-inflammatory activity in vivo.
Data reported in Table 1 showed that, while the insertion of a methyl (
methylcarbamoyl ( ) moiety resulted in decrease or loss of activity compared to ARN0726, the
substitution of the hydrogen on the endocyclic nitrogen with a methoxy group, as in compound
produced a significant improvement in the h-NAAA inhibitory potency (IC50 = 0.016 µM).
Surprisingly, the (R)-serine-derived compound , the enantiomer of , showed inhibitory potency in the
low nanomolar range (IC = 0.056 µM), leading to a loss of the stereorecognition previously observed
4), an acetyl (5) or a
6
7,
8
7
5
0
in similar derivatives.[21] Furthermore, based on previous findings, we asked whether the introduction
of a β-methyl substitution on the β-lactam ring would recover such stereorecognition. This
modification was earlier reported to restore the effect of stereochemistry on the activity of isomeric
threonine-derived analogues,[21] and also to beneficially affect the selectivity towards human acid
ceramidase (h-AC), a cysteine amidase that exhibits 33% amino acid identity with NAAA.[27,28] To
answer this question, the corresponding four stereoisomeric N-O-methyl substituted threonine-derived
1
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analogues (
9
-12) were synthesized. Interestingly, while stereoisomers 9-11 resulted to be poorly active
against h-NAAA (Table 1), the β-lactam 12, bearing the (2S,3S) configuration, retained a good potency
on the target enzyme (IC50 = 0.069 µM). Derivative 12 not only showed promising activity on h-
NAAA, but also a considerably high selectivity versus h-AC, displaying ca. 50% inhibition at 50 µM
(
data not reported).
Table 1.
The beneficial effect of the insertion of a methyl group in β-position both in terms of stereoselectivity
towards h-NAAA and increased selectivity versus h-AC, led us to identify carbamate 12 as the starting
point (hit compound) for a further structure-activity relationship (SAR) expansion of this class of N-O-
substituted β-lactam analogues.
Based on these findings, while maintaining fixed the (2S,3S)-α-amino β-methyl β-lactam core and the
4
-cyclohexylbutyl side chain, the effect on h-NAAA inhibition of the endocyclic nitrogen substitution
in terms of size and shape of the substituents was explored. Two different sets of N-O-alkyl and N-O-
aryl substituted α−amino β methyl β lactams were designed, synthesized and tested. Table 2 and
Table 3 report the outcomes of this investigation in terms of IC values.
−
−
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0
Taking into consideration the importance of N-O-substitution on NAAA inhibition, as demonstrated by
the modest inhibition (IC50 = 47.69 µM) of h-NAAA activity of the N-hydroxy derivative 18, the first
objective in our SAR exploration was the evaluation of O-alkyl substituents on the endocyclic nitrogen.
A first series of β
synthesized and tested (Table 2). First, the optimal length of a linear alkyl chain was evaluated (12
). Increasing the length from a simple methyl group (12, IC = 0.069 µM, Table 2) up to longer
−lactam derivatives bearing alkyl chains with different bulkiness and length were
,
19-
2
1
5
0
alkyl chains led to a progressive decrease in potency. Whereas a good inhibitory activity was retained
up to a C5 linear alkyl moiety (20, IC50 = 0.387 µM), a remarkable drop was observed for compound
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2
1
(IC50 = 2.23 µM) bearing a longer alkyl chain (C7). Notably, both the cyclohexyl (22, IC50 = 3.01
µM) and the tert-butyl substitutions (13, IC >50 µM) were detrimental for enzyme inhibitory activity,
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probably due to steric hindrance in close proximity of the catalytic active site. Based on these findings,
aiming to get additional structural information around the catalytic site, compound 20 was selected as a
starting point for the synthesis of a small set of derivatives (23-29) bearing different substitutions on
the alkyl chain. As shown in Table 2, the insertion of a terminal cyclohexyl (28) as well as of a phenyl
(
29) moiety on the C5 linear chain led to a decrease in potency (h-NAAA IC = 3.47 µM and 1.24 µM,
50
respectively), suggesting a limited space in this portion of enzyme catalytic site. Interestingly, the
derivatives 23-27, bearing an ether (23 24), a thioether (25), a methylsulfonyl (26), and a ketone (27
moiety respectively, displayed good inhibitory potencies with IC values ranging between 0.049 and
,
)
5
0
0
.187 µM. When the oxygen was moved from the 4 (compound 24) to the 3 position (compound 23) of
the alkyl chain, a slight drop in potency was observed (24: IC50 = 0.088 µM and 23: IC50 = 0.187 µM).
These results showed how the insertion of a heteroatom on the linear chain in the N-O-substituent could
lead to a significant modulation of the h-NAAA inhibitory activity. The introduction of a
methylsulfonyl group (26: IC50 = 0.049 µM) on the left hand side of the molecule led to an increase in
potency, suggesting the beneficial effect of a di-oxygenated substituent on NAAA inhibition.
Table 2.
We next turned our attention to the N-O-aryl β−lactams and investigated the role of the substituted
phenyl rings on NAAA inhibition (Table 3). In general, all N-O-aryl derivatives showed a very good
inhibitory potency, higher than that of the alkyl analogues, with IC s in the low nanomolar range. We
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0
speculated that the higher potency of these derivatives could be explained by an extended conjugation
of the phenoxy residue with the β-lactam amidic bond, making the carbonyl more prone to nucleophilic
attack by the catalytic cysteine of NAAA.
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The unsubstituted phenyl derivative 30 showed a three-fold improvement in IC50 compared to the
methyl derivative 12 (IC50 = 0.023 µM). A similar inhibitory potency (IC50 = 0.017-0.031 µM) was
also displayed by compounds 31-34, bearing electron-donating (31) or electron-withdrawing (32-34)
substituents on the phenyl ring. The apparent lack of correlation between the stereoelectronic properties
of the phenyl substitution and the inhibitory activity on the enzyme remains to be explained. Despite
the increased bulkiness, the para-phenyloxy (35) and para-phenyl (36) substitutions on the phenyl ring
demonstrated to be well tolerated by the enzyme (IC = 0.048 µM and 0.039 µM, respectively). At this
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point, considering the beneficial effect on activity of a methylsulfonyl substitution on the N-O-alkyl
substituted analogues, we explored such modification also on the aryl substituted β-lactam derivatives.
Noteworthy, a significant increase in potency with respect to phenyl compound (30) was observed with
the para-methylsulfonyl derivative 37 (IC = 0.006 µM), suggesting a possible electrostatic interaction
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of the two oxygen atoms with the amino acid residues of NAAA active site. To better investigate the
effect of this substitution on the phenyl ring, the meta- (38) and ortho- (39) methylsulfonyl derivatives
were also synthesized. As shown by the IC values (meta-: IC = 0.010 µM and ortho-:IC = 0.006
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0
50
50
µM) both substituted compounds resulted to be equally active to the para analogue 37, suggesting this
type of substitution to be beneficial for a potent NAAA inhibition regardless of the substituent position
on the phenyl ring.
In this context, we also synthesized and tested few N-O-aryl β-lactams bearing a substitution in meta-
or ortho position. As seen for methylsulfonyl substituted compounds (37-39), the position of the
substituent on the phenyl ring did not affect the compounds’ NAAA inhibitory activity (data not
reported).
Table 3.
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A selection of structurally relevant N-O-substituted α-amino β-methyl β-lactams (12, 26, 30, 37)
identified in our SAR study was also tested for selectivity against human Acid Ceramidase (h-AC),[27]
and human FAAH (h-FAAH), a serine hydrolase which can cleave FAEs[29] (Table 4).
The activity data on h-AC inhibition showed in the case of N-O-alkyl derivatives 12 and 26 a very high
selectivity (h-NAAA/h-AC > 1,000 fold), which drastically decreased when the endocyclic nitrogen
was substituted with a phenyloxy moiety (30), showing only a 13-fold difference between the activity
of the two cysteine hydrolases. The selectivity on h-NAAA was recovered when a methyl sulfonyl
group was inserted in para position (37, h-AC/h-NAAA = ca. 340 fold).
At the tested concentration (10 µM), none of the selected N-O-substituted β-lactam carbamates
inhibited h-FAAH in a significant manner (Table 4).
Table 4.
To investigate the physicochemical properties of this new β-lactam class, the same subset of N-O-
substituted α-amino β-methyl β-lactams was also evaluated for the chemical stability in buffer (PBS) at
pH 7.4 and for the mouse and rat plasma stability (Table 4).
The tested compounds showed an overall high chemical stability to hydrolytic cleavage, with half-life
of over 24 hours (12
, 26, 30: t > 1440 min). A lower but still acceptable half-life (t ) value was
½ ½
observed for the para-methylsulfonyl derivative 37 (t½= 727 min).
With respect to the plasma stability, all selected compounds displayed very low half-life (t < 5 min)
½
either in rat and mouse plasma, due to a fast hydrolytic cleavage.
In contrast with previously described β-lactam derivatives,[21] the low plasma stability displayed by N-
O-substituted analogues clearly prevents the potential use of this new class of NAAA inhibitors for
systemic administration. However, this limitation (i.e. low plasma stability) may be well turned into an
advantage when administering these compounds by topical applications, thus restricting NAAA
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inhibition to the site of administration and limiting their potential side effects. To assess the in vivo
activity of a representative compound of this series, we tested compound 37 by topical administration
in a mouse model of carrageenan-induced inflammation (see Supporting Information). After induction
of paw edema and heat hyperalgesia, a fresh suspension of 37 was given by intraplantar administration.
Figure 2.
A single dosing of 37 (0.5–50 µg/paw) was able to significantly attenuate both responses (Figure 2A,
and 2B), and when 37 was administered at the highest dose (50 µg/paw), the effect was still statistically
significant 24 h after treatment (Figure 2A, P<0.01).
The same compound 37 was also used as a tool to evaluate the mechanism of NAAA inhibition by this
new class of β-lactams. As previously shown for other β-lactam NAAA inhibitors,[16,21] high
resolution liquid chromatography mass spectrometry (HRLC/MS-MS) studies proved the formation of
a covalent thioester adduct with the N-terminal cysteine of the active form of h-NAAA (see Supporting
Information).
The covalent nature of NAAA inhibition by this class of compound was also confirmed by a
competitive Activity Based Protein Profiling study (ABPP) using ARN14686, a recently published
probe for NAAA. [30] We used the lysosomal enriched fraction from HEK-293 cells stably
overexpressing NAAA (NAAA HEK-293) as source of the target enzyme. This fraction was pre-
incubated with compound 37 (20 µM) for one hour and then a five-fold higher concentration of
ARN14686 was added after 15 minutes or 4 hours.
Figure 3.
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Subsequent click chemistry reaction with ARN14686 allowed the introduction of a fluorophore (i.e.,
rhodamine) to detect the catalytically active portion of the enzyme.
The SDS-PAGE analysis (Figure 3) clearly showed that β-lactam 37 was able to rapidly and fully
inhibit NAAA, as demonstrated by the absence of any fluorescent signals after 15 min incubation with
ARN14686. The same result was obtained after 4 hours incubation with ARN14686, thus indicating
that NAAA inhibition by compound 37 is irreversible, at least under this experimental conditions.
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4
. CONCLUSIONS
The present study expanded the previous SAR on serine- and threonine-derived β-lactams as NAAA
inhibitors by investigating the influence of the endocyclic nitrogen O-substitution on the activity. First,
we explored the effect of a β-methyl substitution on the β-lactam ring: as already seen with other
analogues, a strong recognition towards the (2S,3S) configuration (compound 12) was confirmed also
for this class. Then, N-O-alkyl and N-O-aryl substituted compounds were investigated, disclosing a
significant preference of the enzyme towards aryl and polar substituents. The expansion of the aryl
series, starting from the phenyl derivative 30, led us to compound 37, a single-digit nanomolar NAAA
inhibitor (h-NAAA IC50 = 0.006 µM). Surprisingly, similar IC50 values were observed for all para-
substituted phenyl compounds, indicating that the stereo-electronic properties of the substituent did not
affect NAAA inhibitory potency. Furthermore, the four-fold increased potency displayed by the
methylsulfonyl derivative 37 compared to compound 30, suggested the importance of oxygen-
containing substituents for the interaction with the enzyme. The results of our SAR study showed that
β-lactam NAAA inhibitors could be substituted at the endocyclic nitrogen with O-alkyl and O-aryl
groups, besides the exocyclic nitrogen of the α-amino group. This results in linearly extended
compounds where the cysteine warhead is no longer at one end of the molecule, as is the case in β-
lactones or β-lactams, like compounds
2 and 3. The enzyme appears therefore able to accommodate
relatively extended small molecules at the active site. The introduction of properly substituted O-aryl
moieties at the endocyclic nitrogen was particularly beneficial in terms of NAAA inhibitory activity,
and offered the possibility of modulating the substituent at the exocyclic nitrogen in order to access
further β-lactams with improved NAAA inhibitory activity and desired chemical functionalities.
Based on the interesting outcomes reported for the first NAAA activity-based probe,[29] N-O-
substituted β-lactams could also be exploited for the design of novel ABPs. This would allow the
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development of additional pharmacological tools for the in vitro and in vivo detection of the
catalytically active form of NAAA, thus contributing to improving our understanding of the enzyme’s
physiological roles.
A set of this new β-lactams was also evaluated for hydrolytic and plasma stability. While all selected
compounds showed a good chemical stability at pH 7.4 in PBS, upon incubation with rat and mouse
plasma they were rapidly hydrolyzed, suggesting their possible use as soft drugs for skin diseases. In
line with these data, a preliminary in vivo study with compound 37 in a carragenaan-induced
inflammation mouse model demonstrated the promising activity of such compound when administered
topically. This encouraging result lends support to its possible use as soft drug for skin diseases.
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5
. EXPERIMENTAL SECTION
5
5
.1 Chemistry
.1.1 Chemicals, materials and methods
All the commercial available reagents and solvents were used as purchased from vendors without
further purification. Dry solvents (THF, Et O, CH Cl , DMF, DMSO, MeOH) were purchased from
2
2
2
Sigma-Aldrich. Automated column chromatography purifications were performed using a Teledyne
ISCO apparatus (CombiFlash® Rf) with pre-packed silica gel columns of different sizes (from 4 g up
to 120 g). Mixtures of increasing polarity of cyclohexane (Cy) and ethyl acetate (EtOAc) or
cyclohexane and methyl tert-butyl ether (MTBE) were used as eluents. NMR experiments were run on
1
13
a Bruker Avance III 400 system (400.13 MHz for H, and 100.62 MHz for C), equipped with a BBI
probe and Z-gradients. Spectra were acquired at 300 K, using deuterated dimethylsulfoxide (DMSO-d6)
as solvent. UPLC/MS analyses were run on a Waters ACQUITY UPLC/MS system consisting of a
SQD (single quadrupole detector) mass spectrometer equipped with an electrospray ionization interface
and a photodiode array detector. The PDA range was 210-400 nm. Analyses were performed on an
ACQUITY UPLC BEH C column (100 x 2.1mmID, particle size 1.7 mm) with a VanGuard BEH C
18
18
precolumn (5 x 2.1 mmID, particle size 1.7 mm). Mobile phase was 10 mM NH
4
OAc in H
2
O at pH 5
adjusted with CH COOH (A) and 10 mM NH OAc in CH CN-H O (95:5) at pH 5.0. Electrospray
3
4
3
2
ionization in positive and negative mode was applied. 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 interface and a 2998 Photodiode Array Detector. HPLC
system included a 2747 sample manager, 2545 binary gradient module, system fluidic organizer and
5
15 HPLC pump. PDA range was 210-400 nm. Purifications were performed on a XBridge™ Prep C18
OBD column (100 x 19 mmID, particle size 5 mm) with a XBridge™Prep C18 (10 x 19 mmID, particle
2
0

ACCEPTED MANUSCRIPT
size 5 mm) guard cartridge. Mobile phase was 10 mM NH OAc in MeCN-H O (95:5) at pH 5.
4
2
Electrospray ionization in positive and negative mode was used. Optical rotations were measured on a
Rudolf Research Analytical Autopol II automatic polarimeter using a sodium lamp (589 nm) as the
light source; concentrations are expressed in g/100 mL using CHCl as a solvent and a 1 dm cell. All
3
tested compounds (4-12
,
18-39) showed ≥ 95% purity by NMR and UPLC/MS analysis.
-lactams (4-6).
ARN0726) are according to previously reported
5
.1.2 Synthesis of the N-substituted serine-derived
β
1
Experimental procedure and H-NMR relative to
literature.[21]
3 (
5
.1.2.1 4-Cyclohexylbutyl-(
Under nitrogen atmosphere, to a stirred solution of
Cs CO (0.026 g, 0.081 mmol) and methyl iodide (0.005 mL, 0.081 mmol) were subsequently added.
S
)-(1-methyl-2-oxoazetidin-3-yl)carbamate
(4).
3
(0.02 g, 0.074 mmol) in dry THF (1.5 mL),
2
3
After stirring 15 hours at 50 °C the solution was filtered over a celite pad, diluted with DCM and
evaporated to dryness. The crude product was absorbed over silica gel and purified by column
chromatography using a Teledyne ISCO apparatus, eluting with Cy/EtOAc (from 100:0 to 20:80) to
afford a crude product, which was submitted to a preparative HPLC/MS using a Waters
Autopurification system, affording the title compound (0.018 g, 15%) as a colorless oil. MS (ESI) m/z:
+
+
1
2
8
5
1
3
5
83.3 [M-H] , 300.3 [M-NH ] . [α]D20 (c= 0.1, CHCl ): +19.48. H NMR (DMSO-d ): δ 7.76 (d, J =
4 3 6
.65 Hz, 1H), 4.70-4.54 (m, 1H), 3.94 (td, J = 6.76, 1.6 Hz, 2H), 3.45 (t, J = 5.13 Hz, 1H), 3.12 (dd, J =
.13, 2.52 Hz, 1H), 2.72 (s, 3H), 1.72-1.56 (m, 5H), 1.50 (p, J = 6.76 Hz, 2H), 1.33-1.22 (m, 2H), 1.22-
1
3
6
.06 (m, 6H), 0.91-0.76 (m, 2H) ppm. C NMR (DMSO-d ): δ 171.1, 168.7, 65.2, 64.8, 47.6, 49.5,
7.0, 33.6, 29.6, 28.5, 26.6, 22.9 ppm.
.1.2.2 4-Cyclohexylbutyl-(
S
)-(1-acetyl-2-oxoazetidin-3-yl)carbamate
(0.05 g, 0.18 mmol) in dry DCM (3.0 mL),
pyridine (0.020 mL, 0.025 mmol) and acetyl chloride (0.018 mL, 0.025 mmol) were subsequently
(5).
Under nitrogen atmosphere, to a stirred solution of
3
2
1

ACCEPTED MANUSCRIPT
added. After stirring 15 hours at room temperature the solution was diluted with DCM (15 mL) and
3
washed with a saturated NaHCO aqueous solution (3 x 5.0 mL); the organic phase was dried over
NaSO , filtered and evaporated to dryness. The crude product was absorbed over silica gel and purified
4
by column chromatography using a Teledyne ISCO apparatus, eluting with Cy/EtOAc (from 100:0 to
+
7
0:30) to afford the title compound (0.01 g, 18%), as white solid. MS (ESI) m/z: 311.2 [M-H] , 328.3
+
-
1
[
M-NH ] , 309.2 [M-H] . [α]D20 (c= 0.03, CHCl ): -1.64. H NMR (DMSO-d ): δ 7.91 (d, J = 7.79 Hz,
4 3 6
1
6
1
2
5
H), 4.76 (td, J = 7.79, 3.80 Hz, 1H), 3.97 (t, J = 6.78 Hz, 2H), 3.75 (t, J = 6.88 Hz, 1H), 3.45 (dd, J =
.88, 3.80 Hz, 1H), 2.26 (s, 3H), 1.76-1.56 (m, 5H), 1.52 (p, J = 6.78 Hz, 2H), 1.35-1.20 (m, 2H), 1.21-
1
3
6
.05 (m, 6H), 0.93-0.75 (m, 2H) ppm. C NMR (DMSO-d ): δ 171.1, 168.7, 65.2, 58.6, 45.1, 37.0,
9.5, 29.3, 27.9, 26.8, 23.9, 21.7 ppm.
.1.2.3 Methyl-(
S)-3-(((4-cyclohexylbutoxy)carbonyl)amino)-2-oxoazetidine-1-carboxylate
(6).
Under nitrogen atmosphere, to a stirred solution of
3
(0.05 g, 0.18 mmol) in dry DCM (3.0 mL),
triethylamine (0.056 mL, 0.409 mmol) and methyl-carbonyl chloride (0.017 mL, 0.223 mmol) were
slowly added at 0 °C. After stirring 15 hours at room temperature (still starting material left) the
solution evaporated to dryness. The crude product was absorbed over silica gel and purified by column
chromatography using a Teledyne ISCO apparatus, eluting with Cy/EtOAc (from 100:0 to 40:60) to
afford a crude product, which was submitted to a preparative HPLC/MS using a Waters
Autopurification system, affording the title compound (0.037 g, 62%) as a colorless oil. MS (ESI) m/z:
+
+
1
3
8
27.3 [M-H] , 344.3 [M-NH ] . [α]D20 (c= 0.08, CHCl ): -4.62. H NMR (DMSO-d ): δ 7.88 (d, J =
4 3 6
.17 Hz, 1H), 4.79-4.70 (m, 1H), 3.96 (t, J = 6.76 Hz, 2H), 3.79 (t, J = 6.34 Hz, 1H), 3.73 (s, 3H), 3.53
(
(
dd, J = 3.81, 6.34 Hz, 1H), 1.72-1.56 (m, 5H), 1.51 (p, J = 6.76 Hz, 2H), 1.36-1.25 (m, 2H), 1.24-1.06
1
3
m, 6H), 0.91-0.76 (m, 2H). ppm. C NMR (DMSO-d ): δ 171.1, 168.7, 65.0, 57.4, 53.2, 46.1, 37.0,
6
3
3.4, 33.1, 33.2, 29.3, 26.4, 23.1 ppm.
.1.3 4-Cyclohexylbutyl- -[( )-1-methoxy-2-oxo-azetidin-3-yl]carbamate (7).
5
N
S
2
2

ACCEPTED MANUSCRIPT
Step 1 to 4. See Supporting Information.
Step 5. Under nitrogen atmosphere, a solution of S13 (0.082 g, 0.463 mmol) in dry CH
2
2
Cl (7.0 mL)
was treated with DIPEA (0.092 mL, 0.55 mmol). A second solution containing a mixture (ratio 1.6:1)
of 4-cyclohexylbutyl 2-pyridyl carbonate (S181) and 4-cyclohexylbutyl 2-oxopyridine-1-carboxylate
(
S182) (0.352 g, 1.27 mmol) in dry CH Cl (2.0 mL) was then added. The reaction was left to stir under
2 2
nitrogen atmosphere at room temperature for 15 hours. The solvent was evaporated and the crude
product was dissolved in EtOAc (10 mL), washed first with a saturated NH Cl aqueous solution (20.0
4
mL), subsequently with a saturated NaHCO (3x20 mL) and NaCl (20 mL) aqueous solutions. The
3
2 4
organic layer was dried over Na SO , filtered and evaporated to dryness. The crude was absorbed over
silica gel and purified by column chromatography using a Teledyne ISCO apparatus, eluting with
Cy/TBME (from 100:0 to 30:70) to afford the title compound (0.02 g, 31%), as a colorless oil. MS
+
+
1
(
(
(
(
ESI) m/z: 299.3 [M-H] , 316.3 [M-NH ] . [α]D20 (c= 0.1, CHCl ): -8.14. H NMR (DMSO-d ): δ 7.82
4 3 6
d, J = 8.35 Hz, 1H), 4.58-4.45 (m, 1H), 3.96 (t, J = 7.0 Hz, 2H), 3.78 (dd, J = 5.53, 4.15 Hz, 1H), 3.70
s, 3H), 3.48-3.40 (m, 1H), 1.73-1.57 (m, 5H), 1.52 (p, J = 6.86 Hz, 2H), 1.38-1.26 (m, 2H), 1.26-1.06
1
3
m, 6H), 0.93-0.78 (m, 2H) ppm. C NMR (DMSO-d ): δ 156.8, 154.3, 64.6, 62.6, 53.1, 50.5, 36.8,
6
3
3.4, 33.3, 29.5, 26.5, 26.3, 23.1 ppm.
.1.4 4-Cyclohexylbutyl- -[( )-1-methoxy-2-oxo-azetidin-3-yl]carbamate (8).
5
N
R
Step 1 to 3. See Supporting Information.
Step 4. To a refluxing (90 °C) slurry of powdered K CO (0.14 g, 1.02 mmol) in acetone (5.0 mL), a
2
3
solution of S20 (0.1 g, 0.254 mmol) in acetone (3.0 mL) was added. The resulting suspension was
stirred at 100 °C for 3 hours. Upon cooling, the thick slurry was filtered through celite and the collected
solid was extracted with EtOAc (20.0 mL). The organic layers were washed sequentially with a 1 N
HCl aqueous solution (10.0 mL), a saturated NaHCO solution (20.0 mL) and a saturated NaCl solution
3
(
2 4
10.0 mL), dried over Na SO , filtered and concentrated to dryness. The crude product was absorbed
2
3

ACCEPTED MANUSCRIPT
over silica gel and purified by column chromatography using a Teledyne ISCO apparatus, eluting with
Cy/TBME (from 100:0 to 70:30) to afford the title compound (0.042 g, 55%), as white solid. MS (ESI)
+
-
1
m/z: 299.2 [M-H] , 297.2 [M-H] . [α] (c= 0.1, CHCl ): -18.29. H NMR (DMSO-d ): δ 7.82 (d, J =
D20
3
6
8
3
6
3
5
.32 Hz, 1H), 4.57-4.44 (m, 1H), 3.96 (t, J = 6.72 Hz, 2H), 3.77 (dd, J = 4.18, 5.34 Hz, 1H), 3.70 (s,
H), 3.48-3.40 (m, 1H), 1.74-1.56 (m, 5H), 1.51 (p, J = 6.72 Hz, 2H), 1.37-1.25 (m, 2H), 1.25-1.07 (m,
1
3
6
H), 0.95-0.76 (m, 2H) ppm. C NMR (DMSO-d ): δ 156.8, 154.3, 64.3, 62.7, 53.2, 50.1, 37.8, 36.5,
3.3, 33.4, 28.8, 26.2, 22.5 ppm.
.1.5 4-Cyclohexylbutyl-N-[(2R,3S)-1-methoxy-2-methyl-4-oxo-azetidin-3-yl]carbamate (9).
Step 1. See Supporting Information.
Step 2. The reaction was carried out following the general procedure GP_S1 employing S14 (0.188 g,
0
0
.62 mmol), a mixture (1.6:1 ratio) of S181 and S182 (0.516 g, 1.86 mmol) and DIPEA (0.108 mL,
.62). Purification by column chromatography using a Teledyne ISCO apparatus, eluting with
Cy/EtOAc (from 100:0 to 60:40) afforded the title compound (0.11 g, 57%), as a white powder. MS
+
+
1
(
ESI) m/z: 313.1 [M-H] , 330.2 [M-NH
4
] . [α]D20 (c= 0.1, CHCl
3 6
): +44.11. H NMR (DMSO-d ): δ
7
.88 (d, J = 9.13 Hz, 1H), 4.69 (dd, J = 9.13, 5.10 Hz, 1H), 4.22-4.11 (m, 1H), 3.97 (td, J = 6.70, 1.63
Hz, 2H), 3.71 (s, 3H), 1.71-1.57 (m, 5H), 1.57-1.47 (m, 2H), 1.37-1.25 (m, 2H), 1.25-1.07 (m, 6H),
1
3
1
5
5
.14 (d, J = 6.08 Hz, 3H), 0.93-0.68 (m, 2H) ppm. C NMR (DMSO-d ): δ 161.7, 156.5, 64.6, 63.5,
6
8.1, 56.3, 37.2, 36.7, 33.0, 29.1, 26.4, 26.1, 22.8, 13.1 ppm.
.1.6 4-Cyclohexylbutyl-N-[(2S,3R)-1-methoxy-2-methyl-4-oxo-azetidin-3-yl]carbamate (10).
Step 1. See Supporting Information.
Step 2. The reaction was carried out following the general procedure GP_S1 employing S15 (0.188 g,
.62 mmol), a mixture of 4-cyclohexylbutyl 2-pyridyl carbonate (S18 ) and 4-cyclohexylbutyl 2-
oxopyridine-1-carboxylate (S18 ) (0.516 g, 1.86 mmol) and DIPEA (0.108 mL, 0.62). Purification by
column chromatography using a Teledyne ISCO apparatus, eluting with Cy/TBME (from 100:0 to
0
1
2
2
4

ACCEPTED MANUSCRIPT
+
5
3
9
3
0:50) afforded the title compound (0.093 g, 48%), as a colorless oil. MS (ESI) m/z: 313.2 [M-H] ,
+
-
1
4 3 6
30.2 [M-NH ] , 311.3 [M-H] . [α]D20 (c= 0.1, CHCl ): -53.21. H NMR (DMSO-d ): δ 7.88 (d, J =
.16 Hz, 1H), 4.70 (dd, J = 9.16, 5.11 Hz, 1H), 4.22-4.13 (m, 1H), 3.98 (td, J = 1.81, 6.73 Hz, 2H),
.72 (s, 3H), 1.76-1.57 (m, 5H), 1.52 (p, J = 6.73 Hz, 2H), 1.37-1.26 (m, 2H), 1.25-1.16 (m, 6H), 1.15
1
3
(
d, J = 6.21 Hz, 3H), 0.95-0.79 (m, 2H) ppm. C NMR (DMSO-d ): δ 161.4, 156.2, 64.3, 63.2, 57.8,
6
5
6.0, 36.9, 36.5, 32.8, 28.8, 26.2, 25.8, 22.5, 12.8 ppm.
.1.7 4-Cyclohexylbutyl- -[(2 ,3 )-1-methoxy-2-methyl-4-oxo-azetidin-3-yl]carbamate (11).
5
N
R
R
Step 1. See Supporting Information.
Step 2. The reaction was carried out following the general procedure GP_S1 employing S16 (0.193 g,
.64 mmol), a mixture of 4-cyclohexylbutyl 2-pyridyl carbonate (S181) and 4-cyclohexylbutyl 2-
0
oxopyridine-1-carboxylate (S182) (0.532 g, 1.92 mmol) and DIPEA (0.112 mL, 0.64). Purification by
column chromatography using a Teledyne ISCO apparatus, eluting with Cy/EtOAc (from 100:0 to
+
6
3
4
1
0:40) afforded the title compound (0.099 g, 50%), as a white powder. MS (ESI) m/z: 313.2 [M-H] ,
+
1
4 3 6
30.2 [M-NH ] . [α]D20 (c= 0.1, CHCl ): -10.81. H NMR (DMSO-d ): δ 7.82 (d, J = 8.07 Hz, 1H),
.06-4.00 (m, 1H), 3.95 (td, J = 6.66, 2.51 Hz, 2H), 3.90-3.83 (m, 1H), 3.72 (s, 3H), 1.72-1.55 (m, 5H),
.56-1.46 (m, 2H), 1.33 (d, J = 6.10 Hz, 3H), 1.31-1.25 (m, 2H), 1.25-1.08 (m, 6H), 0.93-0.77 (m, 2H)
1
3
ppm. C NMR (DMSO-d ): δ 161.6, 155.7, 64.3, 63.2, 59.8, 59.4, 37.0, 36.5, 32.8, 28.9, 26.2, 25.9,
6
2
2.6, 16.0 ppm.
5
.1.8 4-Cyclohexylbutyl-N-[(2S,3S)-1-methoxy-2-methyl-4-oxo-azetidin-3-yl]carbamate (12).
Step 1. See Supporting Information.
Step 2. The reaction was carried out following the general procedure GP_S1 employing S17 (0.290 g,
.96 mmol), a mixture of 4-cyclohexylbutyl 2-pyridyl carbonate (S181) and 4-cyclohexylbutyl 2-
0
oxopyridine-1-carboxylate (S182) (0.798 g, 2.88 mmol) and DIPEA (0.167 mL, 0.96). Purification by
column chromatography using a Teledyne ISCO apparatus, eluting with Cy/EtOAc (from 100:0 to
2
5

ACCEPTED MANUSCRIPT
+
6
3
4
1
6
3
5
0:40) afforded the title compound (0.17 g, 57%), as a colorless oil. MS (ESI) m/z: 313.4 [M-H] ,
+
1
4 3 6
30.2 [M-NH ] . [α]D20 (c= 0.1, CHCl ): +16.94. H NMR (DMSO-d ): δ 7.83 (d, J = 8.15 Hz, 1H),
.04 (dd, J = 8.15, 3.74 Hz, 1H), 4.00-3.93 (m, 2H), 3.87 (dt, J = 6.89, 3.74 Hz, 1H), 3.73 (s, 3H), 1.72-
.58 (m, 5H), 1.52 (p, J = 6.82 Hz, 2H), 1.34 (d, J = 6.89 Hz, 3H), 1.32-1.25 (m, 2H), 1.25-1.07 (m,
1
3
H), 0.92-0.78 (m, 2H).ppm. C NMR (DMSO-d ): δ 161.6, 155.7, 64.3, 63.1, 59.5, 59.3, 36.9, 36.5,
6
2.8, 28.8, 26.1, 25.8, 22.6, 16.0 ppm.
.1.9 4-Cyclohexylbutyl-((2S,3S)-1-(tert-butoxy)-2-methyl-4-oxoazetidin-3-yl)carbamate (13).
Step 1 to 4. See Supporting Information.
Step 5. 4-Cyclohexylbutyl-((2S,3S)-1-(tert-butoxy)-2-methyl-4-oxoazetidin-3-yl)carbamate (13).
Under nitrogen atmosphere, a solution of (2S,3S)-1-(tert-butoxy)-2-methyl-4-oxoazetidin-3-ammonium
tosylate salt S23 (0.130 g, 0.37 mmol) in dry CH Cl (3.0 mL) was treated with DIPEA (0.074 mL,
2
2
0
.45 mmol). A second solution containing a mixture (ratio 1.6:1) of 4-cyclohexylbutyl 2-pyridyl
carbonate (S181) and 4-cyclohexylbutyl 2-oxopyridine-1-carboxylate (S182) (0.418 g, 1.51 mmol) in
dry CH Cl (2.0 mL) was then added. The reaction was left to stir under nitrogen atmosphere at room
temperature for 15 hours. The solvent was evaporated and the crude product was dissolved in EtOAc
10 mL), washed first with a saturated NH Cl aqueous solution (20.0 mL), subsequently with a
2
2
(
4
saturated NaHCO (3x20 mL) and NaCl (20 mL) aqueous solutions. The organic layer was dried over
3
Na SO , filtered and evaporated to dryness. The crude was absorbed over silica gel and purified by
2
4
column chromatography using a Teledyne ISCO apparatus, eluting with Cy/TBME (from 100:0 to
0:70) to afford the title compound (0.077 g, 0.21 mmol, 57%), as colorless oil. MS (ESI) m/z: 355.2
3
+
+
-
1
[
(
M-H] , 372.3 [M-NH
d, J = 8.19 Hz, 1H), 4.04 (dd, J = 8.19, 2.18 Hz, 1H), 3.96 (td, J = 6.69, 2.65 Hz, 2H), 3.82-3.71 (m,
H), 1.72-1.57 (m, 5H), 1.52 (p, J = 6.69 Hz, 2H), 1.31-1.36 (m, 2H), 1.29 (d, J = 6.13 Hz, 3H), 1.24
4 3 6
] , 353.2 [M-H] . [α]D20 (c= 0.1, CHCl ): -259.6. H NMR (DMSO-d ): δ 7.89
1
2
6

ACCEPTED MANUSCRIPT
1
3
(
s, 9H), 1.23-1.05 (m, 6H), 0.95-0.76 (m, 2H) ppm. C NMR (DMSO-d ): δ 164.4, 155.9, 83.5, 64.4,
6
6
2.9, 60.3, 37.2, 36.7, 33.1, 29.1, 27.1, 26.4, 26.1, 22.8, 15.4.
.1.10 4-Cyclohexylbutyl-((2 ,3 )-1-hydroxy-2-methyl-4-oxoazetidin-3-yl)carbamate (18).
5
S S
Step 1. tert-Butyl-((2S,3R)-1-((benzyloxy)amino)-3-hydroxy-1-oxobutan-2-yl)carbamate.
To a solution of commercially available N-(tert-butoxycarbonyl)-L-threonine 14 (3.0 g, 13.7 mmol) in
THF (50 mL), O-benzylhydroxylamine as free base (1.85 g, 15.07 mmol) and HOBt (2.29 g, 15.07
mmol) were added. EDC HCl (2.89 g, 15.07 mmol) was added portion wise in 30 minutes and the
reaction was vigorously stirred for 5 hours at room temperature. After solvent evaporation the crude
mixture was extracted with EtOAc (20.0 mL) and washed with a saturated NaHCO
3 x 10 mL); the organic phase was dried over Na SO , filtered and the solvent removed under vacuum
to yield the title compound (3.37 g, 76%) as colorless oil, which was used in the next step without any
3
aqueous solution
(
2
4
+
- 1
further purification. MS (ESI) m/z: 325.2 [M-H] , 323.2 [M-H] . H NMR (DMSO-d ): δ 7.48-7.29 (m,
6
5
1
H), 6.34 (d, J = 8.52 Hz, 1H), 4.85-4.73 (m, 2H), 3.90-3.77 (m, 1H), 3.71 (dd, J = 8.52, 4.94 Hz, 1H),
.39 (s, 9H), 1.01 (d, J = 6.45 Hz, 3H) ppm.
Step
2.
(2R,3S)-4-((Benzyloxy)amino)-3-((tert-butoxycarbonyl)amino)-4-oxobutan-2-yl
methanesulfonate (15).
To a cooled (0 °C) solution of tert-butyl ((2S,3R)-1-((benzyloxy)amino)-3-hydroxy-1-oxobutan-2-
yl)carbamate (3.37 g, 10.39 mmol) in dry pyridine (41 mL), methanesulfonyl chloride (1.61 mL, 20.78
mmol) was added dropwise over 10 min. The reaction was left to stir under nitrogen atmosphere for 3
hours. The mixture was poured into 20 mL of iced water; the solution was then acidified with 2 N HCl
until reaching pH 4. The aqueous phase was extracted with EtOAc (20.0 mL) and the collected organic
layers were dried over Na SO , filtered and evaporated to dryness. The resulting crude product was
2
4
absorbed over silica gel and purified by column chromatography using a Teledyne ISCO apparatus,
eluting with Cy/EtOAc (from 100:0 to 0:100 for 10 minutes then only EtOAc) to afford the title
2
7

ACCEPTED MANUSCRIPT
+
+
compound (4.09 g, 10.5 mmol, 98%), as white solid. MS (ESI) m/z: 403.1 [M-H] , 420.1 [M-NH ] ,
4
-
1
4
4
01.1 [M-H] . H NMR (DMSO-d
6
): δ 7.25-7.49 (m, 5H), 7.09 (d, J = 9.47 Hz, 1H), 4.71-4.84 (m, 3H),
.08 (qd, J = 9.47, 6.32 Hz, 1H), 3.11 (s, 3H), 1.38 (s, 9H), 1.24 (d, J = 6.32 Hz, 3H), ppm.
Step 3. tert-Butyl-((2S,3S)-1-(benzyloxy)-2-methyl-4-oxoazetidin-3-yl)carbamate (16).
To a refluxing (90 °C) slurry of powdered K CO (4.35 g, 31.5 mmol) in acetone (160 mL), a solution
2
3
of 15 (4.09 g, 10.5 mmol) in acetone (80 mL) was added. The resulting suspension was stirred at 100
C for 3 hours. Upon cooling, the thick slurry was filtered through celite and the collected solid was
extracted with EtOAc (30.0 mL). The organic layers were washed sequentially with a 1 N HCl aqueous
solution (20.0 mL), a saturated NaHCO solution (20.0 mL) and a saturated NaCl solution (20.0 mL),
dried over Na SO , filtered and concentrated to dryness. The crude product was absorbed over silica
gel and purified by column chromatography using a Teledyne ISCO apparatus, eluting with Cy/TBME
°
3
2
4
(
[
(
from 100:0 to 70:30) to afford the title compound (2.72 g, 85%), as white solid. MS (ESI) m/z: 307.2
+
+ 1
M-H] , 324.2 [M-NH ] . H NMR (DMSO-d ): δ 7.58 (d, J = 8.25 Hz, 1H), 7.35-7.48 (m, 5H), 4.92
4
6
s, 2H), 3.99 (m, 1H), 3.70 (qd, J = 6.10, 2.08 Hz, 1H), 1.39 (s, 9H), 1.19 (d, J = 6.10 Hz, 3H) ppm.
Step 4. (2S,3S)-1-(Benzyloxy)-2-methyl-4-oxoazetidin-3-yl ammonium tosylate salt.
In a heart shaped flask purged with nitrogen, 16 (2.72 g, 8.87 mmol) was mixed with p-toluensulfonic
acid (1.60 g, 9.30 mmol) and the solid mixture was cooled to 0° C. Subsequently trifluoroacetic acid
(
36 mL) was added dropwise over 20 minutes and the reaction was left to stir at 0 °C for 15 minutes.
The solution was rotary evaporated maintaining the bath below 30 °C and the obtained oil was left
under high vacuum for 1 hour. Et O (20.0 mL) was added to the obtained crude and a white precipitate
2
was observed. After washing with Et O (5 x 10 mL) the title compound (3.35 g, 8.85 mmol,
2
1
quantitative) was recovered as white solid. H NMR (DMSO-d ): δ 8.70 (s, 3H), 7.38-7.54 (m, 7H),
6
7
3
.12 (d, J = 7.85 Hz, 2H), 4.99 (d, J = 2.64 Hz, 2H), 3.98-4.07 (m, 1H), 3.83-3.96 (m, 1H), 2.29 (s,
H), 1.26 (d, J = 6.10 Hz, 3H) ppm.
2
8