N -(2-Oxo-3-oxetanyl)carbamic acid esters as N-acylethanolamine acid amidase inhibitors: Synthesis and structure-activity and structure-property relationships

Article abstract of DOI:10.1021/jm300349j

The β-lactone ring of N-(2-oxo-3-oxetanyl)amides, a class of N-acylethanolamine acid amidase (NAAA) inhibitors endowed with anti-inflammatory properties, is responsible for both NAAA inhibition and low compound stability. Here, we investigate the structure-activity and structure-property relationships for a set of known and new β-lactone derivatives, focusing on the new class of N-(2-oxo-3-oxetanyl)carbamates. Replacement of the amide group with a carbamate one led to different stereoselectivity for NAAA inhibition and higher intrinsic stability, because of the reduced level of intramolecular attack at the lactone ring. The introduction of a syn methyl at the β-position of the lactone further improved chemical stability. A tert-butyl substituent in the side chain reduced the reactivity with bovine serum albumin. (2S,3R)-2-Methyl-4-oxo-3-oxetanylcarbamic acid 5-phenylpentyl ester (27, URB913/ARN077) inhibited NAAA with good in vitro potency (IC₅₀ = 127 nM) and showed improved stability. It is rapidly cleaved in plasma, which supports its use for topical applications.

Full text of DOI:10.1021/jm300349j

Article
pubs.acs.org/jmc
N-(2-Oxo-3-oxetanyl)carbamic Acid Esters as N-Acylethanolamine
Acid Amidase Inhibitors: Synthesis and Structure−Activity and
Structure−Property Relationships
Andrea Duranti,† Andrea Tontini,† Francesca Antonietti,† Federica Vacondio,‡ Alessandro Fioni,‡
Claudia Silva,‡ Alessio Lodola,‡ Silvia Rivara,‡ Carlos Solorzano,§ Daniele Piomelli,§,∥ Giorgio Tarzia,†
and Marco Mor*,‡
†Dipartimento di Scienze Biomolecolari, Universita
̀
degli Studi di Urbino “Carlo Bo”, Piazza del Rinascimento 6, I-61029 Urbino, Italy
‡Dipartimento Farmaceutico, Universita
̀
degli Studi di Parma, Viale G. P. Usberti 27/A, I-43124 Parma, Italy
§Department of Pharmacology, University of California, Irvine, 360 MSRII, California 92697-4625, United States
∥Department of Drug Discovery and Development, Italian Institute of Technology, via Morego 30, I-16163 Genova, Italy
ABSTRACT: The β-lactone ring of N-(2-oxo-3-oxetanyl)amides, a class of N-acylethanolamine acid amidase (NAAA) inhibitors
endowed with anti-inflammatory properties, is responsible for both NAAA inhibition and low compound stability. Here, we
investigate the structure−activity and structure−property relationships for a set of known and new β-lactone derivatives, focusing
on the new class of N-(2-oxo-3-oxetanyl)carbamates. Replacement of the amide group with a carbamate one led to different
stereoselectivity for NAAA inhibition and higher intrinsic stability, because of the reduced level of intramolecular attack at the
lactone ring. The introduction of a syn methyl at the β-position of the lactone further improved chemical stability. A tert-butyl
substituent in the side chain reduced the reactivity with bovine serum albumin. (2S,3R)-2-Methyl-4-oxo-3-oxetanylcarbamic acid
5-phenylpentyl ester (27, URB913/ARN077) inhibited NAAA with good in vitro potency (IC50 = 127 nM) and showed
improved stability. It is rapidly cleaved in plasma, which supports its use for topical applications.
amide hydrolase (FAAH),17 an intracellular membrane-bound
INTRODUCTION
Fatty acid ethanolamides (FAEs) make up a family of bioactive
■
protein belonging to the amidase signature (AS) family,
characterized by an optimal activity at pH 9.0.18 On the
mediators that have stimulated pharmaceutical interest because
other hand, PEA is primarily hydrolyzed by N-acylethanolamine
compounds blocking their deactivating hydrolysis offer a new
acid amidase (NAAA),19 which is not related to FAAH or other
members of the AS family, is localized in the lysosomes, and
shows an optimal activity at pH 5.0.20 NAAA is an N-terminal
nucleophile hydrolase (Ntn) and belongs to the choloylglycine
hydrolase family of hydrolases,21 which are characterized by the
ability to cleave nonpeptide amide bonds. Like other Ntn
enzymes, NAAA is converted by self-catalyzed proteolysis into
a shorter active form upon incubation at acidic pH.22
Processing of NAAA renders a cysteine residue (Cys131 in
rat NAAA and Cys126 in human NAAA) the N-terminal amino
acid. Site-directed mutagenesis23,24 and mass spectrometry25
studies demonstrated that this N-terminal cysteine plays a
pivotal role in both catalytic activity and proteolytic processing.
This is consistent with a catalytic mechanism involving the
exchange of a proton between the sulfhydryl and the amino
groups of the terminal cysteine and a nucleophile attack on the
amide carbonyl of the substrate, as supported by quantum
strategy for the treatment of pain and inflammation.1−3 An
endogenous FAE that has attracted considerable attention is
palmitoylethanolamide (PEA),4,5 which has been found to
modulate pain and inflammation by engaging peroxisome
proliferator-activated receptor type α.6,7 In particular, PEA has
been shown to inhibit peripheral inflammation and mast cell
degranulation8 and to exert antinociceptive effects in rodent
models of acute and chronic pain.9 PEA has also been found to
suppress pain behaviors induced by tissue injury, nerve damage,
or inflammation in mice.10
Along with PEA, other FAEs having neuromodulatory
functions have been identified. These include the endogenous
cannabinoid agonist N-arachidonoylethanolamine (ananda-
mide, AEA)11 and the feeding regulator oleoylethanola-
mide.12,13 FAEs share similar anabolic and catabolic pathways,
with their levels finely controlled by enzymes responsible for
synthesis and degradation.14 FAEs are produced by the action
of a selective phospholipase D, which catalyzes the cleavage of
the membrane precursor N-acylphosphatidylethanolamine.15,16
The hydrolysis of anandamide is mostly attributed to fatty acid
Received: March 13, 2012
Published: April 19, 2012
© 2012 American Chemical Society
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Table 1. Stabilities of Compounds 1−9 in Buffers and in the Presence of Thiols
mechanics/molecular mechanics (QM/MM) simulations for
cysteine Ntn hydrolases.26
with the acylation of the catalytic Cys131. Among the
synthesized N-(2-oxo-3-oxetanyl)amides, compound 7 (Table
1) had an IC50 of 115 nM and was effective at reducing the level
of carrageenan-induced leukocyte infiltration in vivo.33 The α-
amino-β-lactone moiety, although essential for the inhibitory
activity of N-(2-oxo-3-oxetanyl)amides, is responsible for the
low chemical stability of these compounds. Indeed, compounds
incorporating an α-amino-β-lactone portion promptly react
with bionucleophiles34 and are readily hydrolyzed in aqueous
media.35,36 As a consequence, their use in pharmacological
studies remains restricted to topical administration and requires
caution in their handling and storage.
In this work, we investigated the mechanism of degradation
of NAAA inhibitors with an α-amino-β-lactone scaffold,
including the known N-(2-oxo-3-oxetanyl)amides, and ex-
panded the series to new N-(2-oxo-3-oxetanyl)carbamic acid
esters, which were designed and synthesized to explore the
effect of the side chain on inhibitory activity and chemical
stability. SARs and structure−property relationships (SPRs)
demonstrated that the introduction of an α-carbamic acid ester
side chain and a β-methyl group on the β-lactone ring resulted
in potent NAAA inhibitors with enhanced chemical stability.
Recently, the level of interest in NAAA as a pharmaceutically
relevant target has increased, because of the observation that
NAAA inhibitors locally administered to inflamed tissues,
where the biosynthesis of PEA is downregulated,7 restore the
physiological levels of PEA.24 While several classes of potent
and selective FAAH inhibitors have been discovered and
employed to study the central27,28 and peripheral29 role of
FAAH, only few NAAA inhibitors have been reported.24,30−32
To further investigate the usefulness of NAAA inhibitors in
acute and chronic inflammation, the discovery of new potent
and selective compounds and the optimization of known
chemical classes are both needed.
In this context, our research group has recently identified a
class of β-lactone-based NAAA inhibitors, exemplified by (S)-
N-2-oxo-3-oxetanyl-3-phenylpropanamide [(S)-OOPP, 1
(Table 1)], which weakens responses to inflammatory stimuli
by elevating PEA levels in vitro and in vivo,24 and described the
structure−activity relationships (SARs) of a series of N-(2-oxo-
3-oxetanyl)amides.33 These compounds inhibit NAAA with a
rapid, noncompetitive, and reversible mechanism, consistent
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a
CHEMISTRY
Scheme 2
■
Compounds 1−9 were obtained as reported elsewhere.33 10a
was prepared by a literature procedure.36 N-Benzyloxycarbonyl-
L-serine (N-Cbz-L-serine, 11a) was purchased from Lancaster.
The β-lactone 10b37 and the cyclobutane derivative 1338
were synthesized starting from benzyl chloroformate (12)
(Scheme 1). 12 was treated with cyclobutylamine, in the case of
a
Scheme 1
a
Reagents and conditions: (a) PPh3, DMAD, THF, −78 °C for 20
min, room temperature for 2.5 h; (b) CF3COOH, p-TsOH, 0 °C for
10−15 min; (c) C6H5CH2NCO, Et3N, THF, 0 °C for 30 min, room
temperature for 3 h; (d) CbzCl, NaHCO3, H2O, THF, room
temperature for 1 h (21a,b), or Boc2O, NaHCO3, H2O, MeOH, room
temperature fir 36 h (22a−d); (e) BOP, Et3N, CH2Cl2, 0 °C for 1 h,
room temperature for 2 h (23a,b), or BOP, Et3N, CH2Cl2, room
temperature for 3 h (24a−d).
a
Reagents and conditions: (a) D-serine, NaHCO3, H2O, THF, room
temperature for 1 h; (b) PPh3, DMAD, THF, −78 °C for 3 h, room
temperature for 3 h; (c) c-C4H7NH2, NaHCO3, H2O, room
temperature for 3 h; (d) 1,2-bis(trimethylsilyloxy)cyclobutene, HCl/
Et2O, 0 °C and reflux for 4 h.
a
Scheme 3
13, or D-serine, in aqueous sodium bicarbonate. In the latter
case, the resulting intermediate N-Cbz-D-serine (11b) was
cyclized to 10b by means of a modified Mitsunobu reaction,
employing dimethyl azodicarboxylate (DMAD) and dry
triphenylphosphine.36
a
Cyclobutanone derivative 1536 was synthesized by a reaction
between benzyl carbamate (14) and 1,2-bis(trimethylsilyloxy)-
cyclobutene39 in ethereal hydrogen chloride (Scheme 1).36
Ureas 19a and 19b were obtained by reacting benzyl
isocyanate with (S)- and (R)-2-oxo-3-oxetanylammonium
toluene-4-sulfonate (18a40 and 18b41), respectively, in turn
synthesized by deprotection of β-lactone derivatives 17a37 and
17b,42 respectively, with dry trifluoroacetic acid in the presence
of dry p-toluenesulfonic acid (Scheme 2).40,41 Intermediates
17a and 17b were prepared starting from N-tert-butylox-
ycarbonyl-L-serine (N-Boc-L-serine, 16a) and N-Boc-D-serine
(16b), respectively, employing the same method used for 10b
(Scheme 2).37
Reagents and conditions: (a) (Cl3CO)2CO, pyridine, toluene, 0 °C
for 2 h, room temperature for 90 h; (b) 20b, NaHCO3, H2O, THF, (n-
Bu)4NBr, room temperature for 18 h; (c) HBTU, Et3N, CH2Cl2, 0 °C
for 3 h, room temperature for 6 h.
literature procedure;52 the latter was treated with 20b and
tetrabutylammonium bromide in aqueous sodium bicarbonate
to afford 26, which was cyclized by means of the 2-(1H-
benzotriazol-1-yl)-1,1,3,3-tetramethylaminium hexafluorophos-
phate (HBTU) carboxylic acid activating agent to give 27.
RESULTS AND DISCUSSION
■
The synthesis of threonine β-lactones 23a,43 23b, 24a,44
24b,33 24c, and 24d is illustrated in Scheme 2. The amino
groups of L-, D-, L-allo-, and D-allo-threonine (20a−d,
respectively) were protected with 12 or di-tert-butyl dicar-
bonate (Boc2O) in aqueous sodium bicarbonate to give 21a,45
21b,46 22a,47 22b,48 22c,49and 22d,50 respectively, employing a
slightly modified literature procedure.51 Cyclization to β-
lactones 23a,b and 24a−d was accomplished by treating the
parent compounds 21a,b and 22a−d, respectively, with
benzotriazol-1-yloxytris(dimethylamino)phosphonium hexa-
fluorophosphate (BOP reagent) by means of a literature
procedure.36
Chemical Stability of N-(2-Oxo-3-oxetanyl)amides.
The chemical stabilities of the previously published N-(2-oxo-
3-oxetanyl)amides 1−8 and compound 9 are summarized in
Table 1. Those compounds revealed high susceptibility to
chemical hydrolysis in buffered solution both at pH 7.4
(physiological pH) and at pH 5.0 (pH for the in vitro rat
NAAA inhibitory assay). Both aliphatic (1) and aromatic (2−7)
N-(2-oxo-3-oxetanyl)amides have half-lives (t1/2) of ≤18 min at
pH 7.4 and ≤28 min at pH 5.0. The presence of dithiothreitol
(DTT) (3 mM) in the pH 5.0 buffer did not significantly affect
half-life values, indicating that the β-lactones do not
preferentially react with thiols and that hydrolysis is the main
factor for their instability under the assay conditions. This result
is also relevant for the interpretation of the in vitro tests, as
DTT was added to NAAA samples to maintain the catalytic
activity of the enzyme over time.
D-Threonine β-lactone 27 was synthesized as reported in
Scheme 3. Commercially available 5-phenylpentanol (25) was
reacted with bis(trichloromethyl) carbonate in the presence of
pyridine to give 5-phenylpentyl chloroformate according to a
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The introduction of different electron-withdrawing (3 or 5)
or electron-donating (4 or 6) substituents at conjugated
positions did not significantly modify the stability of arylamides
2−7, which showed similar half-lives. Conversely, the
introduction of a syn methyl group at position 4 (compound
8) led to a significant improvement in half-life, which was
approximately 5-fold longer.
An investigation, performed by HPLC−ultraviolet (UV)−
mass spectrometry (MS) analysis, of degradation products of
amides 1−8 showed, at pH 7.4 and 5.0, the formation of N-
acylated L-serines, which resulted from the hydrolytic opening
of the β-lactone ring (data not shown). An important difference
in stability was observed between amides 1−7 and alkyl lactone
9, which was significantly more stable with half-lives passing
from 8.2−18.0 to 93.5 min at pH 7.4 and from 18.1−28.4 to
114.6 min at pH 5.0. Thus, the amide group of the side chain
plays a role in the chemical degradation of the lactone ring.
Taking compound 2 as the representative of compounds 1−
8, we conducted HPLC−UV−MS analysis at pH 7.4 and 5.0 of
its hydrolysis time course and that of formation of the resulting
products (Figure 1).
serine on the basis of its retention time and that of one
authentic sample.
Figure 1 shows the time course and degradation products of
2 at pH 5.0. At time ≤2 h, 2 was converted rapidly into N-(2-
naphthoyl)-L-serine [product A (Figure 2)] and more slowly
into O-(2-naphthoyl)-L-serine [product B (Figure 2)]. At 2 h,
no starting compound or 2-naphthoic acid [product C (Figure
2)] was detected, and approximately equal amounts of A and B
were present, accounting together for ∼60% of the total mass
balance. At early time points, a peak with an m/z value
corresponding to that of compound 2 ([M − H]− = 240.1) was
also observed, corresponding to an additional degradation
product [D (Figure 2)]. At longer time points, the slow
decrease in the level of N-(2-naphthoyl)-L-serine (product A)
was paralleled by a corresponding increase in the level of 2-
naphthoic acid. After 3 days, the sum of molar concentrations
for compounds A−C, measured through calibration curves with
pure standards, was more than 90% of the starting
concentration of compound 2, and the peak of the additional
degradation product (D) disappeared, probably because it
converted into compound A or B. We hypothesized a
degradation scheme for 2 involving an active role for the
amide group, following what had been already proposed for the
degradation of serine lactones in a different context (Figure
2).53 In particular, a nucleophilic attack from the amide
carbonyl to the β-methylene group of N-(2-oxo-3-oxetanyl)-
amides would give the intermediate 5-dihydro-2-(2-naphtha-
lenyl)-4-oxazolecarboxylic acid, having an m/z value consistent
with product D, which would quickly be hydrolyzed to O-
acylated amino acid B. At the same time, water could directly
attack the lactone carbonyl of 2, giving N-acylated amino acid
A. In fact, the time courses of starting lactone 2 and products A
and B (Figure 1) suggest two simultaneous pathways: one
involving hydrolysis of the lactone to give N-(2-naphthoyl)-L-
serine (A) and the second yielding O-(2-naphthoyl)-L-serine
(B) through the formation of oxazoline−carboxylic acid
intermediate D, which could not be isolated or quantified but
showed intense peaks in chromatograms at short time points
(Figure 3). This interpretation is further supported by the
experimental observation that N-(2-naphthoyl)-L-serine did not
directly convert to O-(2-naphthoyl)-L-serine but was slowly
hydrolyzed to 2-naphthoic acid when maintained at pH 5.0 for
3 days. O-(2-Naphthoyl)-L-serine was found to be stable at pH
5.0, whereas at pH 7.4, it was converted to N-(2-naphthoyl)-L-
serine (t1/2 ∼ 280 min).
Figure 1. Time courses of the degradation products of compound 2 at
◇
▲
●
pH 5.0: ( ) compound 2, ( ) N-(2-naphthoyl)-L-serine, ( ) O-(2-
▼
naphthoyl)-L-serine, and ( ) 2-naphthoic acid.
N-(2-Naphthoyl)-L-serine was detected at pH 7.4, and its
identity was confirmed by its m/z value ([M − H]− = 258.1)
and superposition of its HPLC peak with that of a standard
sample. The hydrolysis of the amide side chain was significantly
slower, as measurable amounts of 2-naphthoic acid were
detected after only 24 h. At pH 5.0, a degradation product not
observed at pH 7.4 was detected. The compound showed a
retention time on a C18 RP column shorter than that of N-(2-
naphthoyl)-L-serine and had an m/z value corresponding to the
addition of a water molecule to compound 2 ([M + H]+ =
260.1). The compound was identified as O-(2-naphthoyl)-L-
As the only substitution that improved the chemical stability
of the N-(2-oxo-3-oxetanyl)amides (i.e., the introduction of the
methyl group at the β-position of the lactone) had shown a
detrimental effect on rat NAAA inhibitory potency,33 our
attempt to optimize both potency and stability focused on the
Figure 2. Possible routes of formation of O- and N-(2-naphthoyl)-L-serine from compound 2.
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Figure 3. HPLC−MS chromatogram of compound 2 and its degradation products at pH 5.0 and 60 min, resulting from merging of selected ion
chromatograms at m/z 240.1, 258.1, and 260.1. For O-(2-naphthoyl)-L-serine (B, [M + H]+ = 260.09), tR = 2.67 min. For N-(2-naphthoyl)-L-serine
(A, [M − H]− = 258.09), tR = 4.40 min. For degradation product D ([M − H]− = 240.08), tR = 6.78 min. For compound 2 ([M − H]− = 240.08), tR
= 7.40 min.
class of N-(2-oxo-3-oxetanyl)carbamic acid esters (Table 2),
assuming that the lower nucleophilicity of the carbamate
carbonyl would weaken the negative effect of the side chain on
lactone stability. This hypothesis was based on quantum
mechanical calculations for the internal nucleophilic attack on
the β-carbon of the lactone ring by the side chain acyl oxygen,
simulated for the two model compounds N-(2-oxo-3-oxetanyl)-
acetamide and N-(2-oxo-3-oxetanyl)carbamic acid ester.
Potential energy paths, calculated at the density functional
theory (DFT) level (see Experimental Section for details),
revealed that the carbamate has a higher potential energy
barrier for oxazoline formation, probably because of the lower
nucleophilicity of the carbamate carbonyl, compared to that of
the carboxamide (Figure 4). The lower susceptibility of the
carbamic acid ester to undergo internal cleavage was also
confirmed by chemical stability data (e.g., carbamic acid esters
10a and 23b), which showed a significantly higher stability with
respect to the corresponding amides 1 and 8, the half-life going
at pH 7.4 from 18.0−73.8 to 26.7−107.7 min and at pH 5.0
from 23.1−134.6 to 39.2−185.1 min. To improve the potency,
we started a SAR exploration of N-(2-oxo-3-oxetanyl)carbamic
acid esters. SPR analysis was also conducted to investigate the
stereoelectronic requirements of the substituents at the α- and
β-positions of the α-amino-β-lactone and the role of the size
and shape of the carbamic acid ester side chain.
SAR of N-(2-Oxo-3-oxetanyl)carbamic Acid Esters. In
our previous SAR exploration of N-(2-oxo-3-oxetanyl)amides,
enantiomers with the (S) configuration at position 3 led to a
10-fold increase in rat NAAA inhibitory potency, compared to
that of (R) compounds. This trend is reversed in the present N-
(2-oxo-3-oxetanyl)carbamic acid esters, as the (R) analogue
(10b) became more potent than its (S) enantiomer (10a), with
IC50 values of 0.70 and 2.96 μM, respectively. As observed with
N-(2-oxo-3-oxetanyl)amides, the essential role played in rat
NAAA inhibitory potency by the intact β-lactone ring was
confirmed by the lack of potency of open derivatives 11a and
11b, the cyclobutane, and cyclobutanone derivatives 13 and 15
(IC50 > 100 μM). Replacing the carbamate group with a urea,
as in derivatives 19a and 19b, resulted in a marked decrease in
potency (IC50 values of 15 and 49 μM, respectively). Two
further structural modifications were attempted. First, we
introduced a tert-butyl substituent onto the carbamate group.
This substitution was tolerated for NAAA inhibition but caused
a lack of stereoselectivity, with IC50 values of 0.58 and 0.50 μM
for (S) (17a) and (R) (17b) enantiomers, respectively. The
second modification was the introduction of a methyl group at
the β-position of the ring. Within the class of N-(2-oxo-3-
oxetanyl)amides, syn methyl derivatives showed reduced
inhibitory potency (IC50 values of >100 μM for 8 and 100
μM for its enantiomer),33 whereas the corresponding anti
methyl derivatives were too unstable to be prepared or isolated
(unpublished results); however, methyl substitution could be
positively related to stability [e.g., compound 8 (Table 1 and
related results)]. The first of the two benzyl derivatives 23a and
23b, formally related to L- and D-threonine, respectively, was
slightly (10 times) more potent than the second. In the case of
the tert-butyl carbamates (24a−d), it was possible to synthesize
and test the four enantiomers corresponding to the relative
configurations of the two substituents on the β-lactone ring.
Among them, those having the Cβ of the α-amino-β-lactone
ring in the (S) configuration (24b and 24c) were endowed with
good inhibitory potency (IC50 values of 0.22 and 0.19 μM,
respectively), while their enantiomers were inactive (for 24a,
IC50 > 80 μM) or significantly less potent (for 24d, IC50 = 4.9
μM). The role of the lipophilicity of the carbamic acid ester
side chain was explored with the introduction of a 5-
phenylpentyl substituent (27). This compound showed a very
good potency with respect to rat NAAA inhibition, with an IC50
of 127 nM, which demonstrates the positive influence of
lipophilicity on rat NAAA inhibition.
Chemical Stability of N-(2-Oxo-3-oxetanyl)carbamic
Acid Esters. The decision to move from serine- to threonine-
based carbamic acid esters was conducive to chemical stability.
For example, half-lives of threonine lactone 23b were on
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Table 2. Inhibitory Potencies (IC50) of Compounds 10a,b,
11a,b, 13, 15, 17a,b, 19a,b, 23a,b, 24a−d, and 27 on Rat
NAAA Activity
Figure 4. DTF potential energy surfaces for the nucleophilic attack of
the side chain carbonyl oxygen at Cβ of the β-lactone ring for two
model compounds. Energies were calculated in the gas phase at the
B3LYP/6-31+G(d) level of theory. The black curve is calculated for a
carbamate derivative (X = OCH3), and the gray curve refers to the
same process for an amide derivative (X = CH3). The increasing
distances between the oxygen and Cβ atoms in the oxetane ring are
reported on the abscissa as the reaction coordinate. The inset structure
represents the transition state structure, as calculated for the carbamate
derivative.
Table 3. In Vitro Chemical Stabilities of Compounds 10a,
17a, 19a, 23b, 24a−c, and 27
t1/2 (min)
compd
pH 7.4
pH 5.0
pH 5.0 with DTT
10a
17a
19a
23b
24a
24b
24c
27
26.7 2.0
33.7 3.6
3.3 1.2
39.2 1.8
43.4 3.9
29.7 2.7
185.1 0.7
217.0 9.4
241.2 1.4
93.8 0.8
194.2 3.0
30.5 2.9
39.0 1.4
25.1 4.0
127.2 1.6
149.2 3.4
146.7 4.7
80.4 0.2
134.4 1.5
107.7 2.2
143.4 1.1
121.6 1.5
80.7 1.6
110.3 3.4
observed for all other threonine-based derivatives 24a−c and
27. The introduction of a bulky tert-butyl substituent onto the
carbamate side chain had no additive effect on chemical
stability; within this subset of derivatives, syn 24b was more
stable than the anti derivative (24c). Other threonine lactones,
characterized by the syn arrangement, showed half-lives of >100
min at pH 7.4 and 5.0.
Biological Stability. The N-(2-oxo-3-oxetanyl)amides and
N-(2-oxo-3-oxetanyl)carbamic acid esters were also assayed for
their biological stability in the presence of a prototypical off-
target protein, bovine serum albumin (BSA), the most
abundant protein in plasma, which is also endowed with
esterase activity,54,55 and 80% (v/v) rat plasma; this is a well-
established in vitro model for hydrolytic metabolism.56 Results
are reported in Table 4.
In the presence of 20 mg/mL BSA, half-lives of all
compounds significantly decreased with respect to chemical
stability assays. Threonine-based derivatives were in general
more stable than their serine-based analogues, which confirmed
the important role of methyl substitution of the β-lactone
system in chemical and BSA-catalyzed hydrolysis. For example,
1 had a half-life of 7.0 min in the presence of BSA, which
doubled for threonine-based amide 8 (t1/2 = 16 min). In the set
of carbamic acid esters, the very low stability of 10a (t1/2 ∼ 1
min) was improved in analogues 23b (t1/2 ∼ 5 min) and 27
average >4-fold longer than that of its homologue serine-based
10a (Table 3). A similar lengthening of half-lives was also
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Table 4. In Vitro Biological Stabilities for Compounds 1−9,
10a, 17a, 19a, 23b, 24a−c, and 27
Table 5. Plasma Stability of Compound 24b in the Presence
of Enzymatic Inhibitors
compd
20 mg/mL BSA t1/2 (min)
80% (v/v) rat plasma t1/2 (s)
t1/2 (min)
1
7.0 0.6
<1
<10
inhibitor
50% (v/v) rat plasma
50% (v/v) human plasma
2
<10
none
0.53 0.20
0.34 0.18
3.29 1.70
4.19 0.90
7.13 1.70
2.99 0.54
31.99 2.60
4.70 1.80
13.75 3.00
11.50 2.80
3
3.2 0.7
<1
75 20
EDTA
BNPP
eserine
PMSF
4
29
8
5
2.7 0.6
5.8 1.0
3.3 0.8
16.0 1.2
15.1 1.8
1.2 0.7
19.6 1.5
4.6 1.1
5.1 0.8
120.7 1.1
129.1 7.0
87.7 2.3
11.4 2.4
<10
6
65 30
32 15
<10
7
8
plasma, the cleavage of 24b seems to be mainly dependent on
the combined action of carboxylesterase and cholinesterase. In
fact, while EDTA did not change the stability of the compound
(t1/2 = 0.53 min vs 0.34 min in the presence of EDTA), BNPP,
PMSF, and eserine markedly increased the half-life of 24b. In
human plasma, which does not contain carboxylesterases,65
inhibition of calcium-dependent esterases by EDTA led to a 10-
fold increase in the half-life, a value higher than that observed
with other inhibitors. Human plasma paraoxonase had already
been identified as the main factor responsible for the quick
plasma hydrolysis of a set of anti-inflammatory glucocorticoid γ-
lactones.66 In that case, a potential application of these
compounds as “soft drugs” for the treatment of asthma was
postulated,67 based on the nonubiquitous distribution of
paraoxonase, and in particular on its absence from the lung
tissue.61 Soft drugs exert their therapeutic action in the target
tissue and are then converted into inactive metabolites upon
reaching the systemic circulation. In the literature, the soft drug
strategy has been successfully exploited for topically applied
drugs. With regard to the field of anti-inflammatory cortico-
steroids, attention was paid to carboxylic (thio)ester derivatives
and the development of related plasma labile soft drugs67,68 or
“antedrugs”.69,70 In the dermatologic field, boron-containing
type 4 phosphodiesterase inhibitors, developed for topical
treatment of psoriasis and atopic dermatitis, have been
successfully derivatized by incorporation of a cleavable ester
group, thus weakening their systemic side effects.71 With regard
to our set of N-(2-oxo-3-oxetanyl)carbamic acid esters, low
plasma stability may be turned to an advantage for topical
applications. PEA is an endogenous component of the human
epidermis and is generated from phospholipids in the stratum
granulosum. Topical application of a PEA-containing product
had been shown to significantly inhibit the development of UV
light-induced erythema and thymine dimer formation in normal
human skin.72 Topical application of plasma unstable NAAA
inhibitors could therefore improve the anti-inflammatory effects
of PEA, whereas the systemic instability of the compounds
would restrict NAAA inhibition to the site of administration,
thus weakening their potential systemic side effects.
9
14
9
10a
17a
19a
23b
24a
24b
24c
27
<10
<10
<10
<10
13
6
5
11
<10
<10
(t1/2 ∼ 11 min). Nevertheless, the most relevant stabilization
over BSA-catalyzed cleavage was obtained by the introduction
of the bulky tert-butyl substituent on the carbamic acid ester
side chain. The substitution of the benzyl (as in 10a) with a
tert-butyl group (as in 17a) had a marked effect on stability,
with the half-life increasing from ∼1 to ∼20 min. The effect was
more pronounced in the case of threonine-based tert-butyl
carbamic acid esters 24a−c. In fact, they showed half-lives, in
the presence of BSA, comparable to those observed in pH 7.4
buffered solution, indicating no significant BSA-catalyzed
cleavage and chemical stability as the limiting factor for these
compounds. With compound 24b, for example, it was possible
to maintain a good inhibitory potency on rat NAAA (IC50
0.22 μM) and half-lives of ∼2 h in the case of both chemical
and BSA-catalyzed hydrolysis.
=
The majority of the compounds were rapidly cleaved in the
presence of 80% (v/v) rat plasma, with half-lives shorter than
1−2 min. To obtain preliminary indications of which types of
plasma hydrolases might be responsible for the rapid
degradation of the molecules, 24b was chosen as a
representative of its class and subjected to a panel of stability
assays in rat and human plasma in the presence of different
enzymatic inhibitors.
Rat and human plasma contain different types of esterases,57
including carboxylesterases, cholinesterases, and paraoxonase.
Paraoxonase58−60 is a lipoprotein-associated esterase that
hydrolyzes organophosphorous compounds but is not inhibited
by them and is responsible for the cleavage of several arylesters
and aromatic and aliphatic lactones; it requires calcium ions as a
cofactor for activity and stability and is consequently inactivated
by ethylenediaminetetraacetic acid (EDTA).61,62 The esterase
inhibitors bis-4-nitrophenylphosphate (BNPP),63 phenylmetha-
nesulfonyl fluoride (PMSF),64 and eserine were employed for
the inhibition of carboxylesterases and cholinesterases, while
EDTA was employed for the inhibition of calcium-dependent
paraoxonase.62
CONCLUSIONS
■
A series of N-(2-oxo-3-oxetanyl)carbamic acid esters has been
synthesized as rat NAAA inhibitors, and SARs and SPRs were
studied to discover novel inhibitors endowed with an improved
potency and stability profile. Figure 5 summarizes the SARs for
the β-lactone carbamate NAAA inhibitors described here. They
confirmed the importance of the β-lactone ring for rat NAAA
inhibition and the positive role of the carbamate group in the
side chain. The lipophilicity of the side chain was also positively
correlated with NAAA inhibitory potency. The shift from
serine- to threonine-based carbamic acid esters was tolerated in
terms of NAAA inhibitory potency and led to an important
Table 5 reports the stability of compound 24b in rat and
human plasma with different esterase inhibitors. In rat plasma,
the cleavage of the compound was more rapid than in human
plasma. This could depend on the higher esterase activity of the
latter, as well as differences in enzyme composition.65 In rat
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Figure 5. Summary of SARs for β-lactone carbamate inhibitors of NAAA.
Quest (Italy) FlashEA 1112 elemental analyzer for C, H, and N.
Analyses were within 0.4% of the theoretical values. All tested
compounds were >95% pure as determined by elemental analysis.
Rat plasma was obtained from male Wistar rats (250−300 g)
(Charles River Laboratories, Milan, Italy). Animals were housed,
handled, and cared for according to European Community Council
Directive 86 (609) EEC, and the experimental protocol was conducted
in compliance with Italian regulations (DL 116/92) and with local
ethical committee guidelines for animal research. Pooled plasma was
obtained by cardiac puncture, collected into heparinized tubes,
centrifuged (1900g and 4 °C for 10 min) using an ALC refrigerated
centrifuge (ALC srl, Cologno Monzese, Italy), and stored at −70 °C
until it was used. BNPP, BSA, EDTA, eserine salicylate, human plasma,
and PMSF were obtained from Sigma (Sigma-Aldrich srl, Milan, Italy).
Synthesis of (R)-2-Oxo-3-oxetanylcarbamic Acid Benzyl Ester
(10b).37 The compound was prepared by means of a two-step
procedure reported in the literature:36 white solid; mp 133−134 °C
improvement in the chemical stability profile, even if the
preferred stereochemical configuration of the two substituents
showed a complex and not yet fully defined dependence on the
alkyl substitution of the carbamate side chain. The introduction
of a bulky tert-butyl substituent into the side chain made the
compounds less reactive toward BSA.
In particular, structural modulation led us to discover
(2S,3R)-2-methyl-4-oxo-3-oxetanylcarbamic acid 5-phenylpen-
tyl ester (27), which (i) is a potent in vitro rat NAAA inhibitor
(IC50 = 127 nM), (ii) has an improved chemical stability
profile, (iii) has a reduced propensity to react with BSA, and
(iv) is rapidly cleaved by plasma hydrolases. Compound 27,
also termed URB913 and ARN077, is the first potent
representative of a new class of NAAA inhibitors sharing a
carbamic acid ester side chain, which has been expanded for
further SAR investigation and potency improvement and
reported elsewhere.73 27 may be a useful new tool for
investigating the role of NAAA in inflammation and analgesia
and a possible soft drug candidate for topical use.
(CHCl3/n-hexane) [lit.36 133−134 °C (CHCl3/n-hexane)]; [α]20
=
D
+31 (c 0.5, CH3CN) [lit.36 [α]26D = +34.2 (c 0.5, CH3CN)]; ee >98%
(Chiral HPLC, AD-H; flow, 1 mL/min; λmax, 220 nm; eluent, 9:1 n-
hexane/i-PrOH; tR = 17.9 min); MS (EI) m/z 221 (M+), 91 (100); 1H
NMR (CDCl3) δ 4.45−4.48 (m, 2H), 5.15 (m, 3H), 5.51 (br s, 1H),
7.37 (m, 5H); 13C NMR (CDCl3) δ 59.6, 65.3, 67.8, 128.4, 128.6,
128.7, 135.4, 155.2, 168.7; IR (Nujol) νmax 3365, 1847, 1831, 1684,
1532 cm−1. Anal. (C11H11NO4) C, H, N.
EXPERIMENTAL SECTION
■
Chemicals, Materials, and Methods. All reagents were
purchased from Sigma-Aldrich, Lancaster, Novabiochem, or Acros at
the highest quality commercially available. Solvents were RP grade
unless otherwise indicated. Dry tetrahydrofuran was distilled over
sodium and benzophenone. Dry dimethylformamide, dichlorome-
thane, and triethylamine were used as supplied. Petroleum ether refers
to alkanes with boiling points of 40−60 °C. Melting points were
Synthesis of Cyclobutylcarbamic Acid Benzyl Ester (13).38 To a
stirred solution of NaHCO3 (0.504 g, 6.0 mmol) in H2O (7.0 mL)
were added c-C4H7NH2 (0.213 g, 0.260 mL, 3.0 mmol) and, dropwise,
12 (0.512 g, 0.430 mL, 3.0 mmol). The mixture was stirred at room
temperature for 3 h, diluted with H2O (10 mL), and extracted with
EtOAc. The combined organic phases were washed with brine, dried
(Na2SO4), and concentrated. Purification of the residue by flash
column chromatography (8:2 cyclohexane/EtOAc) and recrystalliza-
tion gave 13 as white needles: 67% yield (0.410 g); mp 75−76 °C
(Et2O/petroleum ether); MS (EI) m/z 205 (M+), 91 (100); 1H NMR
(CDCl3) δ 1.61−1.96 (m, 4H), 2.26−2.41 (m, 2H), 4.08−4.24 (m,
1H), 4.89 (br s, 1H), 5.09 (s, 2H), 7.36 (m, 5H); 13C NMR (CDCl3)
δ 14.7, 31.4, 46.2, 66.5, 128.1, 128.5, 136.6, 155.2; IR (neat) νmax 3317,
2977, 2945, 1682, 1539 cm−1. Anal. (C12H15NO2) C, H, N.
determined on a Buchi B-540 capillary melting point apparatus. The
̈
structures of the unknown compounds were unambiguously assessed
1
by MS, H NMR, 13C NMR, and IR. MS (EI) spectra were recorded
with a Fisons Trio 1000 (70 eV) spectrometer; only molecular ions
(M+) and base peaks are given. ESI-MS spectra were recorded with a
Waters Micromass ZQ spectrometer in a positive mode using a
nebulizing nitrogen gas at 400 L/min and a temperature of 250 °C, a
cone flow of 40 mL/min, a capillary of 3.5 kV, and a cone voltage of 60
V; only molecular ions in positive or negative ion mode ([M + H]+ or
Synthesis of (R,S)-2-Oxocyclobutylcarbamic Acid Benzyl Ester
(15).36 The compound was prepared following a literature
procedure:36 colorless oil; MS (EI) m/z 220 [(M + 1)+], 91 (100);
1H NMR (CDCl3) δ 1.94−2.14 (m, 1H), 2.38−2.56 (m, 1H), 2.87−
1
[M − H]−, respectively) are given. H NMR and 13C NMR spectra
were recorded on Bruker AC 200 and 50 spectrometers, respectively,
and analyzed using WIN-NMR. Chemical shifts were measured by
using the central peak of the solvent. IR spectra were recorded on a
Nicolet Atavar 360 FT spectrometer. Optical rotations were measured
on a Perkin-Elmer 241 digital polarimeter using a sodium lamp (589
nm) as the light source; concentrations are expressed as grams per 100
mL, and the cell length was 1 dm. Enantiomeric excess (ee) values
were determined by direct HPLC analysis with a Shimadzu
spectrometer: pump LC-10AS, UV detector SPD-10A, integrator C-
R6A, and the chiral column Chiralpak AD-H (0.46 cm × 25 cm) using
a combination of n-hexane and 2-propanol as the eluent. Column
chromatography purifications were performed under “flash” conditions
using Merck 230−400 mesh silica gel. TLC was conducted on Merck
silica gel 60 F254 plates, which were visualized by being exposed to
ultraviolet light and to an aqueous solution of ceric ammonium
molybdate. Reactions involving generation or consumption of β-
lactone were conveniently followed by TLC, using bromocresol green
spray (0.04% in EtOH, made blue by addition of NaOH) followed by
heating of the plate to detect the β-lactone as a yellow spot against a
blue background. The final compounds were analyzed on a Thermo-
2.96 (m, 2H), 4.82−4.95 (m, 1H), 5.11 (s, 2H), 5.28 (br s, 1H), 7.36
(m, 5H); 13C NMR (CDCl3) δ 19.8, 41.6, 65.2, 67.1, 128.2, 128.3,
128.6, 136.0, 155.4, 205.3; IR (neat) νmax 3356, 3033, 2961, 1792,
1701, 1522 cm−1. Anal. (C12H13NO3) C, H, N.
(S)-(2-Oxo-3-oxetanyl)carbamic Acid tert-Butyl Ester (17a).37 For
the white solid, the yield, mp, [α]20D, and MS (EI) are given in ref 33.
1H NMR and IR data are given in ref 37. Anal. (C8H13NO4) C, H, N.
(R)-(2-Oxo-3-oxetanyl)carbamic Acid tert-Butyl Ester (17b).42 For
1
the white solid, the yield, mp, [α]20D, MS (EI), H NMR, and IR are
given in ref 33. Anal. (C8H13NO4) C, H, N.
Synthesis of (S)-1-Methyl-3-(2-oxo-3-oxetanyl)urea (19a). To a
stirred mixture of 18a (0.130 g, 0.5 mmol) in dry THF (4 mL), at 0 °C
under a N2 atmosphere, were added Et3N (0.056 g, 0.085 mL, 0.55
mmol) and C6H5CH2NCO (0.073 g, 0.068 mL, 0.55 mmol). The
mixture was stirred at 0 °C for 0.5 h and at room temperature for 3 h
and then concentrated. Purification of the residue by column
chromatography (4:6 to 2:8 cyclohexane/EtOAc) and recrystallization
gave 19a as white crystals: 54% yield (0.060 g); mp 188−190 °C,
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decomposition with color change starting from 130 °C, sealed capillary
mmol) and Et3N (0.425 g, 0.585 mL, 4.2 mmol) in dry CH2Cl2 (20
mL), at 0 °C and under a N2 atmosphere, was added HBTU (0.796 g,
2.1 mmol). The mixture was stirred at 0 °C for 3 h and at room
temperature for 6 h and then concentrated. Purification of the residue
by flash column chromatography (7:3 cyclohexane/EtOAc) and
trituration gave 27 as an off-white solid: 7% yield (0.030 g); mp
80−83 °C (n-hexane); [α]20D = −68.2 [c 0.11, (CH3)CO(CH3)]; MS
tube [(CH3)CO(CH3)/petroleum ether]; [α]20 = −12 (c 0.5,
D
1
MeOH); MS (EI) m/z 220 (M+), 91 (100); H NMR (DMSO-d6) δ
4.20 (d, 2H, J = 6 Hz), 4.31−4.36 (m, 2H), 5.07−5.16 (m, 1H), 6.80−
6.92 (m, 2H), 7.21−7.32 (m, 5H); 13C NMR (DMSO-d6) δ 43.3, 59.2,
66.6, 127.1, 127.5, 128.7, 140.8, 157.4, 172.1; IR (Nujol) νmax 3421,
3295, 1845, 1815, 1651, 1545 cm−1. Anal. (C11H12N2O3) C, H, N.
Synthesis of (R)-1-Methyl-3-(2-oxo-3-oxetanyl)urea (19b). The
same protocol applied to 18b, apart from the eluent for column
chromatography (3:7 cyclohexane/EtOAc), gave 19b as white crystals:
42% yield (0.060 g); mp 183 °C, decomposition with color change
starting from 125 °C, sealed capillary tube [(CH3)CO(CH3)/
1
(EI) m/z 292 [(M + 1)+], 91 (100); H NMR [(CD3)CO(CD3)] δ
1.34−1.47 (m, 5H), 1.57−1.69 (m, 4H), 2.62 (t, 2H, J = 7.5 Hz),
4.00−4.10 (m, 2H), 4.85−4.97 (m, 1H), 5.49−5.56 (dd, 1H, J1 = 4.6
Hz, J2 = 6.3 Hz), 7.16−7.28 (m, 6H); 13C NMR [(CD3)CO(CD3)] δ
14.2, 25.3, 28.7, 31.1, 35.5, 60.4, 65.0, 74.4, 125.6, 128.2, 128.3, 142.4,
155.8, 169.2; IR (Nujol) νmax 3452, 3330, 1846, 1825, 1694, 1655,
1541 cm−1. Anal. (C16H21NO4) C, H, N.
petroleum ether]; [α]20 +10 (c 0.5, MeOH); MS (EI) m/z 220
D
(M+), 91 (100); 1H NMR (DMSO-d6) δ 4.21 (d, 2H, J = 6 Hz), 4.29−
4.37 (m, 2H), 5.08−5.17 (m, 1H), 6.78−6.87 (m, 2H), 7.17−7.32 (m,
5H); 13C NMR (DMSO-d6) δ 43.3, 59.2, 66.6, 127.1, 127.5, 128.7,
140.8, 157.4, 172.1; IR (Nujol) νmax 3421, 3295, 1845, 1815, 1651,
1545 cm−1. Anal. (C11H12N2O3) C, H, N.
(S)-2-Oxo-3-oxetanylammonium Toluene-4-sulfonate (18a).40
1
For the white solid, the yield, mp, and H NMR are given in ref 40.
(R)-2-Oxo-3-oxetanylammonium Toluene-4-sulfonate (18b).41
1
For the white solid, the H NMR is given in ref 41.
General Procedure for the Synthesis of Threonine β-Lactones
23a,b and 24a−d. To a stirred mixture of the opportune N-Cbz- and
N-Boc-threonine (21a,b and 22a−d) (3.65 mmol) in dry CH2Cl2 (75
mL), at 0 °C and under a N2 atmosphere, were added Et3N (1.1 g,
1.53 mL, 10.95 mmol) and BOP reagent (1.946 g, 4.4 mmol). The
mixture was stirred at 0 °C for 1 h and at room temperature for 2 h
(23a,b) or at room temperature for 3 h (24a−d) and then
concentrated. Purification of the residue by column chromatography
(8:2 cyclohexane/EtOAc for 23a, 7:3 cyclohexane/EtOAc for 23b and
24c,d, and 1:1 cyclohexane/EtOAc for 24a,b) and recrystallization
gave 23a,b and 24a−d.
General Procedure for the Synthesis of Threonine Derivatives
21a,b and 22a−d. To a stirred mixture of the opportune threonine
20a−d (1 g, 8.4 mmol) and NaHCO3 (1.76 g, 21 mmol, 21a,b; 1,092
g, 13 mmol, 22a−d) in H2O (17 mL) and THF (8.5 mL) (in the case
of 21a,b) or MeOH (17 mL) (in the case of 22a−d) was added 12
dropwise over 0.5 h (1.58 g, 1.32 mL, 9.24 mmol) (21a,b) or Boc2O
(2.684 g, 12.3 mmol) (22a−d). The mixture was stirred at room
temperature (1 h for 21a,b; 14 h for 22a−d), concentrated,
supplemented with Et2O, acidified with 2 N HCl, and extracted with
EtOAc. The combined organic layers were washed with brine, dried
(Na2SO4), and concentrated to give 21a,b and 22a−d.
(2R,3S)-2-Methyl-4-oxo-3-oxetanylcarbamic Acid Benzyl Ester
(2S,3R)-2-Benzyloxycarbonylamino-3-hydroxybutyric Acid
(23a):43 white needles; 50% yield (0.429 g); mp 120−122 °C
(21a):45 54% yield (1.13 g); [α]20 −2.2 (c 1.6, MeOH); HPLC/
D
(CHCl3/n-hexane); [α]20 +11.0 [c 0.29, (CH3)CO(CH3)]; MS (EI)
ESI(+) m/z 254 (M+), 271 (M + NH4); H NMR (CDCl3) δ 1.09−
1
D
m/z 235 (M+), 91 (100); 1H NMR (DMSO-d6) δ 1.33 (d, 3H, J = 6.3
Hz), 4.85 (m, 1H), 5.01−5.14 (ABq, 2H), 5.44 (dd, 1H, J1 = 9.4 Hz, J2
= 6.1 Hz), 7.27−7.43 (m, 5H), 8.36 (d, 1H, J = 9.4 Hz); 13C NMR
(CDCl3) δ 15.1, 60.4, 67.9, 74.7, 128.3, 128.6, 128.7, 135.5, 155.3,
168.7; IR (Nujol) νmax 3284, 1813, 1695, 1553 cm−1. Anal.
(C12H13NO4) C, H, N.
1.27 (m, 3H), 4.32−4.41 (m, 2H), 5.03−5.16 (m, 2H), 6.03 (br d,
1H), 6.25 (br s, 2H), 7.32 (m, 5H); IR (neat) νmax 3338, 2974, 1716,
1531 cm−1.
(2R,3S)-2-Benzyloxycarbonylamino-3-hydroxybutyric Acid
(21b):46 76% yield (1.67 g); [α]20 +2.5 (c 1.9, MeOH); HPLC/
D
1
ESI(+) m/z 254 (M+), 271 (M + NH4); H NMR (CDCl3) δ 1.09−
1.27 (m, 3H), 4.32−4.41 (m, 2H), 5.03−5.16 (m, 2H), 6.03 (br d,
1H), 6.25 (br s, 2H), 7.32 (m, 5H); IR (neat) νmax 3338, 2974, 1716,
1531 cm−1.
(2S,3R)-2-Methyl-4-oxo-3-oxetanylcarbamic Acid Benzyl Ester
(23b): white solid; 55% yield (0.472 g); mp 120−121 °C (CHCl3/
n-hexane); [α]20 −9.5 [c 0.2, (CH3)CO(CH3)]; MS (EI) m/z 235
D
(M+), 91 (100); 1H NMR (DMSO-d6) δ 1.33 (d, 3H, J = 6.4 Hz), 4.85
(m, 1H), 5.01−5.14 (ABq, 2H), 5.44 (dd, 1H, J = 9.4, 6.1 Hz), 7.27−
7.43 (m, 5H), 8.36 (d, 1H, J = 9.4 Hz); 13C NMR (CDCl3) δ 15.1,
60.4, 67.9, 74.7, 128.3, 128.6, 128.7, 135.5, 155.3, 168.7; IR (Nujol)
νmax 3284, 1813, 1695, 1553 cm−1. Anal. (C12H13NO4) C, H, N.
(2R,3S)-2-Methyl-4-oxo-3-oxetanylcarbamic Acid tert-Butyl Ester
(2S,3R)-2-tert-Butoxycarbonylamino-3-hydroxybutyric Acid
(22a).47 The yield, MS (EI), and H NMR are given in ref 33.
1
(2R,3S)-2-tert-Butoxycarbonylamino-3-hydroxybutyric Acid
(22b).48 The yield, MS (EI), and H NMR are given in ref 33.
1
(2R,3R)-allo-2-tert-Butoxycarbonylamino-3-hydroxybutyric Acid
(22c):49 78% yield (1.44 g); 1H NMR (CDCl3) δ 1.29 (m, 3H),
1.47 (s, 9H), 4.08−4.38 (m, 2H), 5.54 (br d, 1H), 5.84 (br s, 2H).
(2S,3S)-allo-2-tert-Butoxycarbonylamino-3-hydroxybutyric Acid
(22d):50 95% yield (1.75 g); 1H NMR (CDCl3) δ 1.28 (d, 3H),
1.46 (s, 9H), 4.08−4.38 (m, 2H), 5.60 (br s, 1H), 6.55 (br d, 2H).
S y n t h e s i s o f ( 2 R , 3 S ) - 3 - H y d r o x y - 2 - ( 5 -
phenylpentyloxycarbonylamino)butyric Acid (26). To a stirred
suspension of NaHCO3 (0.622 g, 7.4 mmol) in THF (1.6 mL) and
H2O (3.3 mL) was carefully added 20b (0.353 g, 2.96 mmol). After
gas evolution, 5-phenylpentyl chloroformate (0.740 g, 3.26 mmol),
previously prepared as reported in the literature,52 was added slowly
followed by (n-Bu)4NBr (0.035 g, 0.92 mmol). The mixture was
stirred at room temperature for 18 h, diluted with H2O, and washed
with Et2O. The aqueous phase was acidified to pH 2 with 2 N HCl and
extracted with EtOAc. The combined organic phases were washed
with brine, dried (Na2SO4), filtered, and concentrated to give 26 as a
colorless oil: 48% yield (0.440 g); HPLC/ESI m/z 310 (M+), 308
(24a).44 For the off-white solid, the yield, mp, [α]20D, MS (EI), H
1
NMR, and IR are given in ref 33: 13C NMR (CDCl3) δ 15.1, 28.2,
60.1, 74.9, 81.3, 154.4, 169.2. Anal. (C9H15NO4) C, H, N. Anal.
(C9H15NO4) C, H, N.
(2S,3R)-2-Methyl-4-oxo-3-oxetanylcarbamic Acid tert-Butyl Ester
(24b).33 For the white solid, the yield, mp, [α]20D, MS (EI), 1H NMR,
and IR are given in ref 33: 13C NMR (CDCl3) δ 15.1, 28.2, 60.1, 74.9,
81.3, 154.4, 169.2. Anal. (C9H15NO4) C, H, N.
(2S,3S)-allo-2-Methyl-4-oxo-3-oxetanylcarbamic Acid tert-Butyl
Ester (24c): white crystal; 76% yield (0.566 g); mp 119−121 °C
(CHCl3/n-hexane); [α]20D −81.7 (c 0.45, MeOH); MS (EI) m/z 202
1
(M+), 146 (100); H NMR (CDCl3) δ 1.47 (s, 9H), 1.61 (d, 3H, J =
6.1 Hz), 4.59 (m, 1H), 4.69−4.80 (m, 1H), 5.14 (br s, 1H); 13C NMR
(CDCl3) δ 18.9, 28.2, 64.3, 76.9, 81.4, 154.4, 168.1; IR (Nujol) νmax
3357, 1828, 1689, 1534 cm−1. Anal. (C9H15NO4) C, H, N.
(2R,3R)-allo-2-Methyl-4-oxo-3-oxetanylcarbamic Acid tert-Butyl
1
(M−); H NMR [(CD3)CO(CD3)] δ 1.14−1.24 (m, 5H), 1.36−1.48
Ester (24d): white crystal; 75% yield (0.558 g); mp 119−121 °C
(CHCl3/n-hexane); [α]20 +81.8 (c 0.4, MeOH); MS (EI) m/z 202
(m, 2H), 1.58−1.72 (m, 4H), 2.62 (t, 2H, J = 7.6 Hz), 4.05 (q, 2H, J =
7.1 Hz), 4.14−4.50 (m, 2H), 5.92 (br d, 1H), 7.12−7.31 (m, 5H); 13C
NMR [(CD3)CO(CD3)] δ 19.7, 25.3, 28.8, 31.1, 35.5, 59.2, 64.4, 67.0,
125.6, 128.2, 128.3, 142.5, 156.8, 171.6; IR (Nujol) νmax 3347, 1716,
1530 cm−1.
D
1
(M+), 146 (100); H NMR (CDCl3) δ 1.47 (s, 9H), 1.61 (d, 3H, J =
6.2 Hz), 4.58−4.61 (m, 1H), 4.69−4.80 (m, 1H), 5.14 (br s, 1H); IR
(Nujol) νmax 3356, 1828, 1690, 1540 cm−1; 13C NMR (CDCl3) δ 18.9,
28.2, 64.3, 76.9, 81.4, 154.4, 168.1. Anal. (C9H15NO4) C, H, N.
Synthesis of (2S,3R)-2-Methyl-4-oxo-3-oxetanylcarbamic Acid 5-
Phenylpentyl Ester (27). To a stirred solution of 26 (0.430 g, 1.4
In Vitro Chemical Stability. Chemical stability was investigated at a
fixed ionic strength (μ = 0.15 M) under physiological [0.01 M
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Article
phosphate-buffered saline (PBS) (pH 7.4)] and acidic [0.01 M
phosphate buffer (pH 5.0)] pH conditions, and in the presence of a 3
mM solution of the thiol nucleophile DTT in 0.01 M phosphate buffer
(pH 5.0), as previously reported.74 Stock solutions of compounds were
prepared in DMSO, and each sample was incubated at a final
concentration of 1−100 μM in a prewarmed (37 °C) buffer solution;
the final DMSO concentration in the samples was kept at 1%. The
samples were maintained at 37 °C in a temperature-controlled shaking
water bath (60 rpm). At various time points, 100 μL aliquots were
removed and analyzed by HPLC. Apparent half-lives (t1/2) for the
disappearance of test compounds were calculated from the pseudo-
first-order rate constants obtained by linear regression of plots of
log[compound] versus time and are listed in Tables 1 and 3 as mean
values of three experiments along with their standard deviations.
In Vitro Rat Plasma Stability. Rat plasma stability was investigated
as previously reported.74 Briefly, plasma was quickly thawed and
diluted to 80% (v/v) with PBS (pH 7.4) to buffer the solution pH,
which was checked during the course of the experiments. Pooled rat
plasma (400 μL) was incubated with PBS buffer (95 μL, pH 7.4) and a
compound stock solution (5 μL) in DMSO (final DMSO
concentration in samples of 1%; final compound concentrations of
5−100 μM). Samples were maintained at 37 °C in a temperature-
controlled shaking water bath (60 rpm) throughout the experiments.
At regular time points, aliquots (50 μL) were withdrawn,
supplemented with 2 volumes of CH3CN, centrifuged at 8000g for
potential (FP), 100 V; entrance potential (EP), 10 V. In all cases,
higher voltages led to compound in-source fragmentation. The ion
source temperature was set at 400 °C. A Supelcosil C18-DB column
(150 mm × 4.6 mm, 5 μm) (Supelco) was employed; the flow rate was
kept at 1 mL/min. Mobile phases consisted of various percentages of
acetonitrile and 10 mM ammonium acetate (pH 7.0). Data were
acquired by employing Analyst version 1.4 (AB/Sciex).
Pharmacology. NAAA Assay. Recombinant NAAA, espressed as
described previously,33 was incubated at 37 °C for 30 min in 0.2 mL of
sodium hydrogen phosphate buffer (50 mM, pH 5.0) containing 0.1%
Triton X-100, 3 mM dithiothreitol (DTT), and 50 mM
heptadecenoylethanolamide as the substrate. The reaction was
terminated by the addition of 0.2 mL of cold methanol containing 1
nmol of heptadecanoic acid (NuCheck Prep, Elysian, MN). Samples
were analyzed by liquid chromatography and mass spectrometry.
Heptadecenoic acid and heptadecanoic acid were eluted on an XDB
Eclipse C18 column isocratically at 2.2 mL/min for 1 min with a
solvent mixture of 95% methanol and 5% water, both containing
0.25% acetic acid and 5 mM ammonium acetate. The column
temperature was 50 °C. ESI was conducted in the negative mode. The
capillary voltage was 4 kV and the fragmentor voltage 100 V. N2 was
used as the drying gas at a flow rate of 13 L/min and 350 °C. The
nebulizer pressure was set at 60 psi. [M − H]− = 267 in the selected
ion monitoring mode. Calibration curves were generated using
commercial heptadecenoic acid (NuCheck Prep).
Quantum Mechanical Simulations of Reaction Kinetics.
Three-dimensional structures of the model compounds (S)-4,5-
dihydro-2-methyl-4-oxazolecarboxylic acid and (S)-4,5-dihydro-2-
methoxy-4-oxazolecarboxylic acid were built in their zwitterionic
forms (dihydro-4-oxazoliumcarboxylates) using Maestro,75 and their
geometry was optimized in the gas phase by means of DFT76
calculation as implemented in Jaguar.77 The B3LYP hybrid func-
tional78 was applied in combination with the 6-31+G(d) basis set,
which includes polarization and diffuse function for non-hydrogen
atoms. Default convergence criteria were applied during all the
calculations performed. Starting from the corresponding minimal-
energy structures, we explored the potential energy surfaces for the
internal nucleophilic substitution by gradually shortening (i.e., in 0.1 Å
steps) the distance between one of the oxygens of the 4-carboxylate
group and the methylene carbon at position 5 of the 4,5-dihydroxazole
scaffold. Then, at each step of the reaction coordinate, the geometry of
the system was optimized and its energy calculated at the B3LYP/6-
31+G(d) level of theory. The O−C distance was the only restraint
applied during the simulations, while other degrees of freedom were
allowed to change during the optimization step. The reaction
coordinate here employed yielded the two β-lactone derivatives,
namely, (S)-N-(2-oxo-3-oxetanyl)acetamide and (S)-(2-oxo-3-
oxetanyl)carbamic acid methyl ester, respectively, as products of the
simulated reaction. Approximate transition state (TS) structures
(corresponding to the points with the highest energy along the
path) were also identified. The geometry and the energetics of these
TSs were characterized more precisely by performing a linear
synchronous transit search in Jaguar. Finally, stationary points were
analyzed by the vibrational frequency calculations. The vibrational
motion associated with the negative frequency, characteristic of
transition states, resulted in the motion going toward products, in two
opposite directions.
5 min at 4 °C, and analyzed by RP-HPLC. Apparent half-lives (t1/2
)
for the disappearance of test compounds were calculated from the
pseudo-first-order rate constants obtained by linear regression of plots
of log[compound] versus time; apparent half-life values listed in Table
4 as the mean values of three experiments along with standard
deviations.
In Vitro Stability in the Presence of BSA. A 20 mg/mL solution of
bovine serum albumin in PBS (pH 7.4) was employed for stability
assays at 37 °C. Test compounds were incubated at final
concentrations of 5−100 μM with a final DMSO percentage of 1%
(v/v). Samples were processed as reported above for rat plasma
stability experiments.
In Vitro Rat and Human Plasma Stability in the Presence of
Selected Inhibitors. Rat and human plasma stability experiments in the
presence of selected inhibitors (BNPP, eserine, PMSF, and EDTA)
took place at 37 °C in the presence of 50% (v/v) plasma, because of
the very rapid hydrolysis of the test compounds under the previously
reported experimental conditions [80% (v/v) plasma at 37 °C].
Briefly, rat and human plasma were preincubated in the presence of
selected inhibitors or vehicle according to literature conditions.65 After
preincubation, compound 24b was added, and at regular time points,
aliquots of the sample (50 μL) were withdrawn, processed, and
analyzed as reported for in vitro rat plasma stability assays.
Conditions for HPLC−UV−MS Analysis. The degradation of
compounds 1−9, 10a, 19a, and 23b was monitored by RP-HPLC
and UV by employing a Shimadzu gradient system (Shimadzu Corp.,
Kyoto, Japan) consisting of two Shimadzu LC-10ADvp solvent
delivery modules, a 20 μL Rheodyne sample injector (Rheodyne
LLC, Rohnert Park, CA), and an SPD-10Avp UV−vis detector,
equipped with a reversed-phase C18 column [LC-18-DB, 5 μm, 150
mm × 4.6 mm (inside diameter) (Supelco, Bellefonte, PA)]. The
HPLC system was interfaced with PeakSimple version 2.83 for data
acquisition. Mobile phases consisted of various percentages of CH3CN
and 0.1% (v/v) formic acid delivered at a flow rate of 1 mL/min. Each
compound was monitored at its relative absorbance maximum for UV
detection. Compounds 17a, 24a−c, and 27 were monitored with an
API150EX single-quadrupole HPLC−MS system (AB/Sciex, Toronto,
ON) equipped with an atmospheric pressure chemical ionization
(APCI) ion source working in positive and negative ion mode and
coupled to an Agilent 1100 series HPLC system (Agilent
Technologies, Waldbronn, Germany) that consisted of a G1312A
binary pump, a G1379A degasser, and a 10 μL Rheodyne sample
injector. Compound-dependent parameters were optimized by flow
injection analysis (FIA) and ramping of the potentials. The final
settings were as follows: declustering potential (DP), 3.0 V; focusing
AUTHOR INFORMATION
■
Corresponding Author
*Phone: +39-0521-905059. Fax: +39-0521-905006. E-mail:
marco.mor@unipr.it.
Notes
The authors declare no competing financial interest.
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Article
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ACKNOWLEDGMENTS
This work was supported by MIUR (Ministero dell’Istruzione,
■
dell’Universita
Urbino “Carlo Bo”.
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e della Ricerca) and Universities of Parma and
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DEDICATION
■
This article is dedicated to the memory of Andrea Tontini, who
sadly died after the manuscript had been submitted. Andrea, a
valuable scientist and a great friend, joined the research group
in Urbino in 1991 and was a pillar of our teamwork. His sudden
death leaves a professional void in our research groups and
tremendous grief in our hearts.
ABBREVIATIONS USED
■
AEA, N-arachidonoylethanolamine; AS, amidase signature;
BNPP, bis-4-nitrophenylphosphate; BOP, benzotriazol-1-
yloxytris(dimethylamino)phosphonium hexafluorophosphate;
DFT, density functional theory; FAE, fatty acid ethanolamide;
HBTU, 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethylaminium
hexafluorophosphate; HPLC, high-performance liquid chroma-
tography; NAAA, N-acylethanolamine acid amidase; Ntn, N-
terminal nucleophile; PEA, palmitoylethanolamide; PMSF,
phenylmethanesulfonyl fluoride; PBS, phosphate-buffered
saline; QM/MM, quantum mechanics/molecular mechanics;
RP, reverse phase; SPR, structure−property relationship.
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