Synthesis and Structure − Activity Relationship (SAR) of 2 ‑ Methyl-4-oxo-3-oxetanylcarbamic Acid Esters, a Class of Potent N ‑ Acylethanolamine Acid Amidase (NAAA) Inhibitors

: N -Acylethanolamine acid amidase (NAAA) is a lysosomal cysteine hydrolase involved in the degradation of saturated and monounsaturated fatty acid ethanolamides (FAEs), a family of endogenous lipid agonists of peroxisome proliferator-activated receptor- α , which include oleoylethano-lamide (OEA) and palmitoylethanolamide (PEA). The β lactone derivatives ( S )- N -(2-oxo-3-oxetanyl)-3-phenylpropionamide ( 2 ) and ( S )- N -(2-oxo-3-oxetanyl)-biphenyl-4-carboxamide ( 3 ) inhibit NAAA, prevent FAE hydrolysis in activated in ﬂ ammatory cells, and reduce tissue reactions to pro-in ﬂ ammatory stimuli. Recently, our group disclosed ARN077 ( 4 ), a potent NAAA inhibitor that is active in vivo by topical administration in rodent models of hyperalgesia and allodynia. In the present study, we investigated the structure − activity relationship (SAR) of threonine-derived β -lactone analogues of compound 4 . The main results of this work were an enhancement of the inhibitory potency of β -lactone carbamate derivatives for NAAA and the identi ﬁ cation of (4-phenylphenyl)-methyl- N -[(2 S ,3 R )-2-methyl-4-oxo-oxetan-3-yl]carbamate ( 14q ) as the ﬁ rst single-digit nanomolar inhibitor of intracellular NAAA activity (IC 50 = 7 nM on both rat NAAA and human NAAA). procedure (0.10 g, mmol), (0.06 mmol), the isomeric of 8i 9i (0.294 g, 1.02 mmol). puri preparative HPLC-MS give 14i as a white solid: yield, 41% (0.045 g); ] an isocratic step at 90% for 10 min, and a reconditioning to 3% of B. Mass spectrometry parameters were as follows: spray, 1.8 kV; cone, 25 V. Data-dependent acquisition of tandem mass spectra was activated for doubly charged ions in the m / z 300 − 800 range. A linear ramp of the collision energy from 15 to 35 eV was applied to the precursor ion to collect tandem mass spectra. Gluco ﬁ brinopeptide (500 nM) infused at 500 nL/min was used as lockspray mass. MS/MS data were analyzed using the Biolynx software embedded in the MassLynx software suite. MassLynx and Protein Lynx softwares (Waters) were used for the interpretation of LC-MS data.


■ INTRODUCTION
The ethanolamides of long-chain fatty acids, or fatty acid ethanolamides (FAEs), are a class of bioactive lipids that serve important signaling functions in both plants and animals. 1 Polyunsaturated FAEs such as arachidonoylethanolamide (anandamide) are endogenous agonists for G protein-coupled cannabinoid receptors and participate in the control of stresscoping responses and pain initiation. 2 On the other hand, monounsaturated and saturated FAEs, such as oleoylethanolamide (OEA) and palmitoylethanolamide (PEA), regulate energy balance, pain, and inflammation primarily by engaging peroxisome proliferator-activated receptor-α (PPAR-α), a member of the nuclear receptor superfamily. 3−6 The pharmacology of PEA has been extensively investigated. 7 The compound inhibits peripheral inflammation and mast cell degranulation 8,9 and exerts profound antinociceptive effects in rat and mouse models of acute and chronic pain. 3,9−11 Moreover, it suppresses pain behaviors induced, in mice, by tissue injury, nerve damage, or inflammation. 12 These properties are dependent on PPAR-α activation, because they are absent in PPAR-α-deficient mice and blocked by PPAR-α antagonists. 12,13 The possibility that PEA might attenuate skin inflammation and neuropathic pain in humans heightens the translational value of the results outlined above. 14,15 Tissue levels of bioactive FAEs are regulated at both the synthesis and degradation levels. 1 These compounds are generated by the action of a selective phospholipase D, which catalyzes the cleavage of the membrane precursor, Nacylphosphatidylethanolamine (NAPE), 16 and are deactivated by either of two intracellular lipid amidases: fatty acid amide hydrolase (FAAH) and N-acylethanolamine acid amidase (NAAA). 17−19 NAAA preferentially hydrolyzes PEA and OEA over anandamide, whereas FAAH displays an opposite substrate selectivity. 18,20 Whereas several classes of FAAH inhibitors have been reported in the literature, only a few NAAA inhibitors have been identified so far. 21−28 NAAA is a cysteine hydrolase that belongs to the N-terminal nucleophile (Ntn) family of enzymes 18,19 and bears a significant degree of sequence homology with the choloylglycine hydro-lases, which are characterized by the ability to cleave nonpeptide amide bonds. 29 Like other Ntn enzymes, NAAA is activated by autoproteolysis, which occurs at acidic pH and generates a catalytically competent form of the enzyme. 30 Sitedirected mutagenesis experiments have unequivocally identified Cys131 (in mice) and Cys126 (in humans) as the catalytic residues responsible for both autoproteolysis and FAE hydrolysis. 23,31−33 Compounds containing chemical groups susceptible to nucleophilic attack, such as a β-lactone moiety, are known to inhibit enzymes that contain a catalytic cysteine. 34 Within this class of compounds, (S)-2-oxo-3-oxetanyl-carbamic acid benzyl ester (1, Figure 1), an inhibitor of a viral cysteine hydrolase, 35 was found to inhibit NAAA activity with micromolar potency. 24 Replacement of the carbamic acid benzyl ester function of 1 with a 3-phenylpropionamide moiety led to (S)-N-(2-oxo-3oxetanyl)-3-phenylpropionamide [2, (S)-OOPP, Figure 1], a relatively potent NAAA inhibitor (IC 50 = 0.42 μM). 24 A pharmacological characterization of 2 showed that this compound prevents FAE hydrolysis in activated inflammatory cells and dampens tissue reactions to various pro-inflammatory stimuli. 24 In addition, compound 2 does not inhibit FAAH or other lipid hydrolases, such as monoacylglycerol lipase and diacylglycerol lipase type-α, a selectivity profile that allows its use as a pharmacological probe. Structure−activity relationship (SAR) studies of serine-derived 2-oxo-3-oxetanyl amides confirmed the key role of the β-lactone ring for NAAA inhibition and identified lipophilic side chains of the carboxamide moiety with optimal size and shape for potent enzyme inhibition. This work led to the identification of the NAAA inhibitor 3 (Figure 1; IC 50 = 0.115 μM), which prevented carrageenan-induced decrease of FAE levels in vivo with a potency that paralleled its ability to inhibit NAAA in vitro. 25 A first investigation of threonine-derived β-lactone derivatives, which have enhanced chemical stability compared to their serine-derived analogues, 28,35,36 led to the discovery of ARN077 (4, Figure 1), a potent NAAA inhibitor 28,37 that is active in vivo by topical administration in rodent models of hyperalgesia and allodynia caused by inflammation or nerve damage.
In the present study, we describe a further expansion of the SAR on threonine-derived β-lactones. We synthesized and tested a series of 2-methyl-4-oxo-3-oxetanylcarbamic acid esters to investigate the influence on NAAA inhibition of the size and shape of the carbamic acid ester side chain.
The preparation of compounds 6−9 was accomplished as reported in Scheme 2. Chloroformate 6b was prepared by reacting 3-phenylpropan-1-ol (5b) with triphosgene in the presence of pyridine. Imidazole 1-carboxylate 7c was obtained in a straightforward manner by reaction of the corresponding alcohol 5c with 1,1′-carbonyldiimidazole (CDI). 38 Compound 7c was not isolated and used in a one-pot procedure in the subsequent step of the synthetic pathway.
Synthesis problems encountered during the preparation of chloroformates 6 and the observed poor reactivity of imidazole 1-carboxylates 7c with either D-threonine (10a) or the tosylate salt 13 (Scheme 1) prompted us to search for alternative carbonic acid derivatives such as pyridyl carbonates deriving from di-2-pyridyl carbonate (DPC), to be reacted with compounds 10a−d or 13. DPC is reported to activate primary, secondary, and tertiary alcohols as alkyl 2-pyridyl carbonates and, as described in the literature, the pyridyl moiety makes these mixed carbonates highly reactive species toward amino acids. 39,40 Alcohols 5a,d,f,g,i−t were therefore reacted with DPC in the presence of triethylamine to give an isomeric mixture of 2pyridyl carbonates 8a,d,f,g,i−t and 2-oxopyridine-1-carboxylates 9a,d,f,g,i−t (Scheme 2, path c). 41,42 The mixture of isomers was used as such in the following synthesis step, as the limited stability of both compounds to chromatographic purification prevented the isolation of the pure products.
When procedure c was applied, a slight excess (1.5 equiv) of the isomeric mixtures containing 8 and 9 was required, leading to intermediates 15 in higher yields (36−97%) compared to that observed in the reaction with 13.
Compounds 19−21, respectively the enantiomer and the epimers of compound 4, were synthesized starting from the commercially available L-threonine 10b, L-allo-threonine 10c, and D-allo-threonine 10d. The amino acids were first reacted with the isomeric mixture of 8t and 9t to give the corresponding α-substituted β-hydroxycarboxylic acids 16−18 and subsequently cyclized to provide the desired analogues 19−21 (Scheme 5).  The diastereoisomeric mixture 14n was separated by chiral HPLC to afford the single diastereoisomers 22 and 23 as pure enantiomers (see Table 2). Compound 22 was also synthesized following the same procedure applied to compound 14n, starting from the commercially available optically pure (S)nonan-2-ol, and used as a reference to assign the correct absolute configuration of diastereoisomer 23 after chiral HPLC purification.

Scheme 4. Synthesis of 2-Methyl-4-oxo-3-oxetanylcarbamic Acid Esters via β-Hydroxycarboxylic
The synthesis of compound 29 (Scheme 6) was carried out starting from the commercially available product 24, which was N-methylated using methyl iodide and sodium hydride to afford intermediate 25. The benzyl group was then removed by catalytic hydrogenation and the obtained N-methyl-N-Boc-Dthreonine (26) was converted into the corresponding tosylate salt 27. Reaction of 27 with the isomeric mixture containing the 2-pyridyl carbonate 8t and the 2-oxopyridine-1-carboxylate 9t afforded the 5-phenylpentyl derivative 28, which was finally cyclized to give the desired compound 29.
The syntheses of alcohols 5j and 5l were accomplished according to literature procedures as reported in Scheme 7. The commercially available phenethyl alcohol 5a was reacted with chloroacetic acid, in the presence of sodium hydride, to afford 2-phenylethoxyacetic acid (30), which upon reduction with lithium aluminum hydride furnished the desired alcohol 5j. 43 Alcohol 5l was synthesized by addition of methylmagnesium bromide to 5-phenylpentanoic acid methyl ester, which was prepared in situ by treatment of the commercially available compound 31 with BF 3 −methanol complex. 44 Alcohols 5m,o were prepared in a two-step procedure starting from the commercially available alcohol 5t (Scheme 7). Swern oxidation to the aldehyde 32 followed by addition of methyl-lithium or isopropylmagnesium bromide furnished the desired compounds.
Alcohols 5p,r were prepared as reported in Scheme 8. The compound 5p was obtained via a Suzuki cross-coupling reaction between the commercially available 3-formylphenylboronic acid (34) and bromobenzene (33)  nium bromide to afford compound 37. 45 Removal of the acetal group under acidic conditions and subsequent reduction of ketone 38 furnished the alcohol 39, which was submitted to catalytic hydrogenation to afford 5r.

■ RESULTS AND DISCUSSION
Previous pharmacological studies by our group have identified the 2-methyl-4-oxo-3-oxetanylcarbamic acid ester derivative 4 (ARN077) as a potent NAAA inhibitor. 46 In vitro experiments have shown that compound 4 inhibits rat NAAA through a mechanism that is rapid, noncompetitive, and fully reversible after overnight dialysis. 46 Moreover, in rodent models of hyperalgesia and allodynia caused by inflammation or nerve damage, 4 administered by the topical route partially normalized FAE levels in the skin and sciatic nerve, which were reduced by inflammation and surgical ligation, respectively, and attenuated nociception through a mechanism that required PPAR-α activation. 46 We identified compound 4 starting from the serine-derived 2-oxo-3-oxetanylamide, (S)-OOPP (2), which inhibits NAAA with a median inhibitory concentration (IC 50 ) of 0.42 μM. 24  The (S)-configuration at position 3 in the β-lactone ring of 2 is essential for inhibitory potency, as indicated by the substantially lower activity of its enantiomer, (R)-OOPP (IC 50 = 6.0 μM). 24 The benzyl carbamate 1 was less potent than 2 at inhibiting NAAA (IC 50 = 2.96 μM), 28 whereas its enantiomer, (R)-2-oxo-3-oxetanylbenzyl carbamate, showed potency comparable to that of 2 (IC 50 = 0.70 μM). 28 This indicated an opposite stereochemical preference of NAAA at position 3 of the βlactone ring in the carbamic acid ester series relative to the amide series. The introduction of a methyl group with (S)stereochemistry at the β-position of the β-lactone ring of (R)-2oxo-3-oxetanylbenzyl carbamate led to the corresponding threonine-derived β-lactone analogue that, although slightly less potent (IC 50 = 1.0 μM), 28 showed an increased chemical stability with respect to serine-derived β-lactone analogues. 28,35,36 These results prompted us to focus our attention on carbamic acid ester derivatives bearing a methyl substitution at the β-position of the β-lactone ring.

Journal of Medicinal Chemistry
2-Methyl-4-oxo-3-oxetanylcarbamic acid esters were tested for their ability to inhibit the hydrolysis of 10-cis-heptadecenoylethanolamide (an unnatural FAE) by either native NAAA prepared from rat lungs (r-NAAA) or recombinant human NAAA heterologously expressed in HEK293 cells (h-NAAA). IC 50 values are reported in Tables 1−3 (r-NAAA) and Table 4 (h-NAAA). The pharmacological potencies of several test compounds slightly varied between r-NAAA and h-NAAA. We attribute such variations to differences in primary sequence and enzyme preparation.
As a first step in our study, we synthesized a small series of 2methyl-4-oxo-3-oxetanylcarbamic acid esters where the length of the aliphatic chain of the phenylalkyl alcohol was progressively increased from two to six methylene units (14a−c, 4, 14d). Compound 4 emerged as the most potent NAAA inhibitor (IC 50 = 0.05 μM) within this small series. The potency and the promising in vivo efficacy of 4 prompted us to investigate in greater detail the structural determinants for activity of this compound.
To test the role of the phenyl ring, this moiety was replaced either with an unsubstituted aliphatic chain (14e−h) or with a cyclohexyl residue (14k). Removal of the phenyl ring led to a >10-fold drop in potency (14e, IC 50 = 0.76 μM). However, potency was recovered by increasing the length of the aliphatic chain, with compounds bearing an n-heptyl (14g, IC 50 = 0.04 μM) or an n-octyl (14h, IC 50 = 0.03 μM) residue showing IC 50 values comparable to that of compound 4. Replacement of the phenyl ring with a cyclohexyl moiety, as in compound 14k, led to a significant improvement in NAAA inhibitory potency (IC 50 = 0.013 μM). The replacement of a carbon atom with an oxygen in the aliphatic chain of 4 (14i,j, Table 1) was tolerated only at a specific position, as shown by the different potencies of compounds 14i (IC 50 = 0.05 μM) and 14j (IC 50 = 0.14 μM).
To evaluate the influence of the shape of the phenylalkyl chain, we synthesized compounds 14l−n ( Table 2), which derive from secondary and tertiary alcohols bearing branched aliphatic chains. Introduction of a gem-dimethyl (14l) moiety close to the carbamic function was detrimental for potency, indicating a limited space in the region of the enzyme occupied by the carbamic function and the adjacent carbon atom. A single methyl group, however, appeared to be well accommodated in this region because compound 14m (IC 50 = 0.029 μM), although a mixture of diastereoisomers, displayed a slight increase in potency compared to compound 4. The introduction of the more sterically demanding isopropyl group (14o) decreased the inhibitory potency relative to 4.
To determine whether there is any stereochemical preference for a substituent at the carbon atom in the α-position to the carbamic function, we synthesized compounds 14n, as a diastereomeric mixture, and compound 22, from optically pure (S)-1-methyloctanol. Compound 14n (IC 50 = 0.06 μM) displayed a slightly decreased potency compared to the unsubstituted derivative 14h (IC 50 = 0.03 μM). The optically pure (S)-1-methyloctyl diastereoisomer 22 inhibited NAAA with IC 50 = 0.016 μM, a value comparable to that of the unsubstituted compound 14h, whereas the (R)-diastereoisomer 23 (IC 50 = 0.27 μM), obtained by chiral HPLC purification of 14n, was 10 times less potent than 14h. These results indicate a stereorecognition among the isomers and, specifically, a preference for the (S)-configuration at the carbon atom in position α to the carbamate function, at least for compounds bearing a 1-methyloctyl side chain. 47 The influence of the flexibility of the aliphatic chain on NAAA inhibition was studied by preparing the series of analogues 14p−s ( Table 2). Compounds bearing a 4benzylcyclohexyl (14r, IC 50 = 0.31 μM) or a p-benzyloxyphenyl (14s, IC 50 = 0.29 μM) moiety were less potent than compound 4, indicating that some flexibility is required in close proximity to the carbamate function. Of particular interest were the results obtained with the biphenylmethyl derivatives (14p,q). The p-biphenylmethyl compound 14q turned out to be a very potent NAAA inhibitor, showing an IC 50 = 0.007 μM. On the contrary, the m-biphenylmethyl analogue 14p did not effectively inhibit NAAA (IC 50 = 23 μM). These results show a clear preference for a linear moiety in the carbamic acid ester for optimal enzyme recognition.
The role of the carbamic function was also investigated by preparing compound 29 (Table 2), a derivative in which the carbamate nitrogen is methylated. Highlighting the importance of the N−H group, the compound showed no inhibitory activity on NAAA.
To investigate the effect of the stereochemistry at positions 2 and 3 of the β-lactone ring, we synthesized compound 19, the enantiomer of compound 4, and the epimers 20 and 21 (Table  3). A marked drop in inhibitory potency (70-fold) was observed with compound 19, whereas the (2S,3S) epimer 20 (IC 50 = 0.48 μM) showed a 10-fold decrease in potency compared to 4. By contrast, the (2R,3R) epimer 21 turned out to be a potent NAAA inhibitor (IC 50 = 0.02 μM). These findings show that the configuration of the two stereogenic centers is important for potency. Whereas the relative importance of the configuration at position 2 versus 3 remains to be fully explored, our data support a primary role for position 3.
To search for a rational explanation of the different potencies of compounds 4 and 19−21, we carried out density functional theory (DFT)-based calculations to mimic the nucleophilic attack of the sulfur atom onto the carbonyl group of the βlactone ring, the first step of the covalent inhibition mechanism hypothesized for the present series of compounds. 33,46 In our simulations, the four compounds were truncated and modeled as methyl carbamate, and the sulfur atom was provided by cysteamine (see Computational Methods under Experimental Section). Starting from the noncovalent complex, we performed a scan on the potential energy surface, using as reaction coordinate the distance between the sulfur atom of the cysteamine molecule and the carbon atom of the carbonyl group of the β-lactone ring. These calculations allowed us to identify the transition state (TS) for all four compounds under investigation and to determine the activation energy related to the nucleophilic attack carried by the sulfur atom. In Scheme 9 is reported a possible NAAA inhibition mechanism by βlactones. Going from the reactants (R) through the transition state (TS) to the product (P), the sulfur atom attacks the carbonyl group of the β-lactone ring, and the protonated amino terminal group donates the proton to the oxygen atom. The nucleophilic attack and the protonation steps are concerted.
In Figure 2, we report the potential energy plotted versus the reaction coordinate, whereas in Figure 3, we show the geometry of the four TSs. The activation energy (E a ) was calculated as the energy difference between TS and R. The E a values for compounds 4 and 21 were 13.0 and 13.3 kcal/mol, whereas the E a values for the nucleophilic attack to 19 and 20 were 18.9 and 17.0 kcal/mol. These calculations suggest that compounds 4 and 21 might be more prone to react with NAAA's nucleophilic cysteine, and therefore more potent, than compounds 19 and 20. This possibility is supported by the experimental IC 50 values obtained with these compounds (Table 3), which show that 4 and 21 are up to 2 orders of magnitude more potent than 19 and 20, respectively. The TSs structures (Figure 3) demonstrated that the presence of the carbamic substituent on the lactone ring allowed the formation of a transient intramolecular interactionbetween the carbonylic oxygen of the β-lactone and the hydrogen atom of the carbamateonly when the absolute configuration of the carbamate-carrying carbon was (R). Conversely, such a stabilizing interaction could not be established when the configuration of the same atom was (S). Moreover, in the reactants showing (S)-configuration, we observed an intermolecular stabilizing interaction between the sulfur atom of the cysteamine and the hydrogen atom of the carbamate. This interaction lowered the reactant energy, with a further increase of E a for 19 and 20 (see the Supporting Information). We conclude that the different potencies between (R)-and (S)-configurations of the carbon carrying the carbamate group may be due to both intra-and intermolecular interactions, responsible for markedly different  In the case of 4 and 21, the stabilization of the TS might be due to an intramolecular interaction between the oxygen atom of the lactone carbonyl and the hydrogen atom of the carbamate. The mechanism was a concerted asynchronous type, where the nucleophilic attack and the protonation of the carbonyl oxygen occur simultaneously. All distances are in Å. activation energies for the sulfur nucleophilic attack. However, we cannot exclude that compounds 4 and 19−21 might also bind to NAAA in different ways, and therefore, inhibitor− NAAA interactions at the Michaelis complex can also modulate their potency.
Selected 2-methyl-4-oxo-3-oxetanylcarbamic acid esters were tested in a functional assay using h-NAAA (Table 4). A common trend between relative potencies at human and rat NAAA was found. Potencies were usually 2−7-fold greater on human NAAA than on rat NAAA, with a particularly pronounced difference observed with compound 4. Compound 14q, the p-biphenylmethyl carbamate derivative, proved to be equally active on the two enzyme orthologues, showing very high potency on both h-NAAA (IC 50 = 0.007 μM) and r-NAAA (IC 50 = 0.007 μM).
The same compounds were also tested for selectivity against rat brain FAAH (r-FAAH), which can also cleave FAEs, 48 and rat lung acid ceramidase (r-AC), a cysteine amidase that exhibits 33% amino acid identity with r-NAAA (Table 4). 18 At the concentration tested (10 μM), the compounds had little or no effect on the activity of these enzymes, demonstrating a good selectivity toward NAAA.
Finally, we used high-resolution liquid chromatography− mass spectrometry (LC-MS) to probe the mechanism through which 14q, the most potent among the inhibitors described here, interacts with h-NAAA. We examined whether 14q might form a covalent adduct with the N-terminal cysteine of the active form of h-NAAA (see the Supporting Information), as previously shown for 4. 37 Consistent with our hypothesis, incubation of h-NAAA with 14q, followed by trypsin digestion, revealed the presence of a covalent adduct of 14q with the Nterminal peptide of h-NAAA (CTSIVAQDSR) (Figure 4). On the basis of the high-resolution MS/MS analysis of the modified peptide, we conclude that 14q inhibits h-NAAA through S-acylation of catalytic Cys126.

■ CONCLUSIONS
The present work expanded our previous SAR studies on threonine-derived 2-methyl-4-oxo-3-oxetanylcarbamic acid esters as NAAA inhibitors. Those studies had led to the discovery of 5-phenylpentyl-N-[(2S,3R)-2-methyl-4-oxo-oxetan-3-yl]carbamate 4, a potent inhibitor of intracellular NAAA activity. We modified compound 4 with the aim of clarifying the relevant structural determinants for NAAA inhibition by threonine-derived β-lactones. Our new investigations highlight the importance of the configuration of the stereogenic centers at positions 2 and 3 of compound 4 for potency. Although the relative importance of the configuration at position 2 versus 3 remains to be fully explored, our data support a primary role for position 3.
As previously observed with the serine-derived N-(2-oxo-3oxetanyl)amides, a linear lipophilic chain attached to the carbamic acid was important to achieve high potency. An unsubstituted carbamate nitrogen turned out to be mandatory for enzyme inhibition, whereas monosubstitution at the carbon atom in α-position to the carbamate oxygen with a small substituent, such as a methyl group, could be beneficial for potency, depending on the substituent configuration. Finally, the replacement of the phenylpentyl chain of compound 4 with a p-biphenylmethyl moiety led to the discovery of compound 14q, the first single-digit nanomolar inhibitor of both human and rat NAAA. This molecule represents a promising probe that may help characterize the functional roles of NAAA and assess the therapeutic potential of NAAA inhibitors as novel anti-inflammatory and analgesic agents.
■ EXPERIMENTAL SECTION a. Chemistry. Chemicals, Materials, and Methods. All of the commercially available reagents and solvents were used as purchased from vendors without further purification. Dry solvents (THF, Et 2 O, CH 2 Cl 2 , DMF, DMSO, MeOH) were purchased from Sigma-Aldrich. 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 3 as a solvent and a 1 dm cell. Automated column chromatography purifications were done using a Teledyne ISCO apparatus (CombiFlash R f ) with prepacked silica gel columns of different sizes (from 4 to 120 g). Mixtures of increasing polarity of cyclohexane and ethyl acetate (EtOAc) or cyclohexane and methyl tert-butyl ether (MTBE) were used as eluents. Hydrogenation reactions were performed using H-Cube continuous hydrogenation equipment (SS-reaction line version), employing disposable catalyst cartridges (CatCart) preloaded with the required heterogeneous catalyst. Microwave heating was performed using an Explorer-48 positions instrument (CEM). NMR experiments were run on a Bruker Avance III 400 system (400.13 MHz for 1 H and 100.62 MHz for 13 C), equipped with a BBI probe and Z-gradients. Spectra were acquired at 300 K, using deuterated dimethyl sulfoxide (DMSO-d 6 ) or deuterated chloroform (CDCl 3 ) as solvents. UPLC-MS analyses were run on a Waters ACQUITY UPLC-MS system consisting of a SQD (singlequadrupole 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 HSS T3 C 18 column (50 × 2.1 mm i.d., particle size = 1.8 μm) with a VanGuard HSS T3 C 18 precolumn (5 × 2.1 mm i.d., particle size = 1.8 μm). Mobile phase was either 10 mM NH 4 OAc in H 2 O at pH 5 adjusted with AcOH (A) or 10 mM NH 4 OAc in MeCN/H 2 O (95:5) at pH 5. Electrospray 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. The HPLC system included a 2747 sample manager, 2545 binary gradient module, system fluidic organizer, and 515 HPLC pump. PDA range was 210−400 nm. Purifications were performed on an XBridge Prep C 18 OBD column (100 × 19 mm i.d., particle size = 5 μm) with an XBridge Prep C 18 (10 × 19 mm i.d., particle size = 5 μm) guard cartridge. The mobile phase was 10 mM NH 4 OAc in MeCN/H 2 O (95:5) at pH 5. Electrospray ionization in positive and negative mode was used. Analyses by chiral HPLC were run on a Waters Alliance HPLC instrument consisting of Figure 4. Tandem mass spectrum of peptide CTSIVAQDSR covalently modified by 14q. The backbone fragment ion series perfectly matches the primary sequence of the peptide, clearly indicating that the mass increase (+311 Da) is carried by the Nterminal cysteine residue. A very intense tropylium ion (167.08 m/z) and a side-chain loss of the biphenyl group and CO 2 (1180.58 m/z) are also clearly visible in the spectrum. an e2695 separation module and a 2998 photodiode array detector. PDA range was 210−400 nm. Analyses were performed isocratically on a Daicel ChiralPak AD column (250 × 4.6 mm i.d., particle size = 10 μm). Mobile phase was 0.1% TFA heptane/2-propanol (75:25). Separations by preparative chiral HPLC were run on a Waters Alliance HPLC instrument consisting of a 1525 binary HPLC pump, Waters Fraction Collector III, and a 2998 photodiode array detector. UV detection was at 240 nm. Purifications were performed isocratically on a Daicel ChiralPak AD column (250 × 10 mm i.d., particle size = 10 μm). Mobile phase was 0.1% TFA heptane/2-propanol (75:25). All tested compounds (4, 14a−u, 19−22, and 29) showed ≥95% purity by NMR and UPLC-MS analysis.
Synthesis of 5-Phenylpentyl-[(2S,3R)-2-methyl-4-oxooxetan-3-yl]carbamate (4). 4 was obtained as an off-white solid. Experimental procedure and NMR are according to the literature. 28,37 General Procedure I for the Synthesis of Carbamates 14b,e,h (Scheme 4). Preparation of β-Hydroxycarboxylic Acids 15b,e,h (Step 1). To a suspension of NaHCO 3 (2.5 equiv) in THF and H 2 O was added D-threonine (1.0 equiv). The suitable chloroformate 6b,e,h (1.1 equiv) was slowly added, followed by a catalytic amount of (n-Bu) 4 NBr. After stirring at room temperature for 18 h, the mixture was diluted with H 2 O and washed with Et 2 O. The aqueous layer was acidified with 2 M HCl solution and extracted with EtOAc. The combined organic layers were dried over Na 2 SO 4 , filtered, and concentrated to give 15b,e,h, which were used in the next step without further purification.
Preparation of Carbamates 14b,e,h (Step 2). To a solution of βhydroxycarboxylic acid was added 15b,e,h (1.0 equiv) in dry CH 2 Cl 2 , Et 3 N (3.0 equiv) under argon. After cooling at 0°C, HBTU (1.5 equiv) or PyBOP (1.3 equiv) was added, and the mixture was stirred at 0°C for 3 h and then at room temperature for 16 h. The obtained solid was filtered off and the solvent removed under vacuum. The crude was purified by typical silica gel column chromatography, eluting with cyclohexane/EtOAc (from 80:20 to 30:70) to give compounds 14b,e,h.   Synthesis of Carbamate 14c (Scheme 4). (2R,3S)-3-Hydroxy-2-(4-phenylbutoxycarbonylamino)butanoic acid (15c). To a stirred mixture of 4-phenylbutan-1-ol (5c) (5.0 mL, 32.8 mmol, 1.0 equiv) in dry DMF (70 mL) under argon atmosphere was added CDI (10.6 g, 65.5 mmol, 2.0 equiv). After the mixture had stirred at room temperature for 2 h, D-threonine (3.90 g, 32.8 mmol, 1.0 equiv) dissolved in H 2 O (70 mL) and Et 3 N (6.8 mL, 49.1 mmol, 1.5 equiv) were added. The mixture was heated at 50°C for 16 h and then allowed to cool. Water was added and the mixture washed with Et 2 O (three times). The aqueous phase was acidified with 2 M HCl solution and then extracted with EtOAc (three times). The collected organic phases were dried over Na 2 SO 4 and filtered and the solvent removed under vacuum. The crude was purified through silica gel column chromatography eluting with cyclohexane/EtOAc (20:80) + 1% CH 3 COOH to afford 15c as a pale yellow oil: yield, 54% (5.
Preparation of Carbamates 14i,l,m,p,q,s (Step 2). To a stirred mixture of 13 (1.0 equiv) in dry CH 2 Cl 2 (1.0 mL) under nitrogen atmosphere was added dropwise DIPEA (1.0 equiv). Subsequently, the isomeric mixture of 8i,l,m,p,q,s and 9i,l,m,p,q,s (3.0 equiv) dissolved in dry CH 2 Cl 2 (2.0 mL) was added. The mixture was stirred at room temperature for 15 h, concentrated to dryness, and purified by column chromatography eluting with cyclohexane/MTBE (from 100:0 to 70:30). Compounds 14i,l,m,p,q,s were further purified by preparative HPLC-MS. General Procedure III for the Synthesis of Carbamates 14a,d,f,g,j,k,n,o,r and 19−22 (Schemes 4 and 5). Preparation of Activated Alcohols as Alkyl-2-pyridyl Carbonates 8a,d,f,g,j,k,n,o,r,t and Alkyl-2-oxopyridine-1-carboxylates 9a,d,f,g,j,k,n,o,r,t (Step 1). To a stirred mixture of the suitable alcohol 5a,d,f,g,j,k,n,o,r,t (1.0 equiv) in dry CH 2 Cl 2 , and under nitrogen atmosphere were added DMAP (0.1 equiv) and DPC (1.2 equiv). The reaction mixture was left to react at room temperature for 15 h, then diluted with CH 2 Cl 2 and washed with a saturated NH 4 Cl solution and, subsequently, with a saturated NaHCO 3 solution (three times). The combined organic layers were dried over Na 2 SO 4 , filtered, and concentrated to give a mixture (ratio 1.6:1) of alkyl-2-pyridyl carbonate 8a,d,f,g,j,k,n,o,r,t and alkyl-2-oxopyridine 1-carboxylate 9a,d,f,g,j,k,n,o,r,t. The mixture of isomers was not separated and used in the next step without any further purification.

Journal of Medicinal Chemistry
Preparation of β-Hydroxycarboxylic Acids 15a,d,f,g,j,k,n,o,r and 16−18 (Step 2). To a stirred mixture of D-or L-or D-allo-or L-allothreonine (1.0 equiv) and NaHCO 3 (1.5 equiv) in H 2 O (3.5 mL) was added the isomeric mixture containing the pyridyl carbonate 8a,d,f,g,j,k,n,o,r,t and the 2-oxopyridine-1-carboxylate 9a,d,f,g,j,k,n,o,r,t (1.5 equiv) in THF (3.5 mL). After 15 h at room temperature, the crude was evaporated and subsequently extracted with Et 2 O (3 × 5 mL). The aqueous layer was acidified with 2 M HCl solution to pH 2−3 and subsequently extracted with EtOAc (3 × 10 mL). The combined organic layers were dried over Na 2 SO 4 , filtered, and concentrated to give the threonine derivatives 15a,d,f,g,j,k,n,o,r and 16−18, which were used in the next step without further purification.