Design, Synthesis, and Biological Evaluation of a Series of Oxazolone Carboxamides as a Novel Class of Acid Ceramidase Inhibitors

Acid ceramidase (AC) is a cysteine hydrolase that plays a crucial role in the metabolism of lysosomal ceramides, important members of the sphingolipid family, a diversified class of bioactive molecules that mediate many biological processes ranging from cell structural integrity, signaling, and cell proliferation to cell death. In the effort to expand the structural diversity of the existing collection of AC inhibitors, a novel class of substituted oxazol-2-one-3-carboxamides were designed and synthesized. Herein, we present the chemical optimization of our initial hits, 2-oxo-4-phenyl-N-(4-phenylbutyl)oxazole-3-carboxamide 8a and 2-oxo-5-phenyl-N-(4-phenylbutyl)oxazole-3-carboxamide 12a, which resulted in the identification of 5-[4-fluoro-2-(1-methyl-4-piperidyl)phenyl]-2-oxo-N-pentyl-oxazole-3-carboxamide 32b as a potent AC inhibitor with optimal physicochemical and metabolic properties, showing target engagement in human neuroblastoma SH-SY5Y cells and a desirable pharmacokinetic profile in mice, following intravenous and oral administration. 32b enriches the arsenal of promising lead compounds that may therefore act as useful pharmacological tools for investigating the potential therapeutic effects of AC inhibition in relevant sphingolipid-mediated disorders.


■ INTRODUCTION
Ceramides (Cer) and their metabolites are members of the sphingolipid (SL) family that play important roles as integral components of the eukaryotic cell membranes and signaling molecules in apoptosis, cell growth, differentiation, senescence, diabetes, insulin resistance, inflammation, neurodegenerative disorders, and atherosclerosis. 1−4 The proper regulation of ceramide biosynthesis and metabolism is controlled by a complex, highly compartmentalized, and interconnected network of enzymatic pathways essential to maintaining the cellular homeostasis and development. 1 Although a detailed analysis of this complex network is beyond the scope of this paper, a simplified representation of the ceramide metabolism is shown in Figure 1. Cer are the centerpiece of the SL metabolism, produced in response to stressful stimuli via two major pathways: the de novo pathway from serine and palmitoyl-CoA in the endoplasmic reticulum and the salvage pathway from the recycling of sphingosine (So), bypassing the formation of dihydroceramide. The generated Cer can be transported to distinct cellular compartments and further modified to more complex SLs, for example, glycosylated to hexosylceramides (HexCer) and, in turn, to more complex glycosylceramides; metabolized into sphingomyelin (SM) and ceramide-1phosphate (Cer-1P); and catabolized to produce So, which is further phosphorylated to sphingosine-1-phosphate (So-1P). Cellular Cer can also be generated through catabolic pathways in distinct sub-cellular compartments. In the lysosomes, for example, SM and HexCer (glucosylceramide and galactosylceramide) participate in distinct degradative pathways that contribute to the formation of lysosomal Cer. In the same compartment, HexCer can be hydrolyzed through distinct pathways to generate the corresponding lysosomal glycosylsphingosines. 5 Evidence to date suggests that imbalances in this complex network because of an altered expression and/or regulation of SL-modifying enzymes can lead to dysregulated cell signaling responses that contribute to the initiation and progression of several SL-related disorders. 3,4 During the past years, aided by the impressive advances of the modern biological and analytical technologies, the scientific community has focused much attention on improving the understanding of the functional roles of some basic components of this metabolic network, under physiological and pathological conditions. 6 Ceramidases (CDases) have attracted particular attention as key SL-metabolizing enzymes that regulate the levels and functions of different bioactive lipids, especially, Cer and So. 7 Thus far, five human CDases (hCDase) have been identified, which can be characterized by their different optimal pH for catalytic activity and localization in cells: acid ceramidase (AC), neutral ceramidase (NC), alkaline ceramidase 1 (ACER1), alkaline ceramidase 2 (ACER2), and alkaline ceramidase 3 (ACER3). 7,8 Because of differences in tissue distribution and expression level, cellular localization, optimum pH, and substrate specificity, these CDases appear to play distinct physiological roles in cellular responses. 8 The overexpression of NC has been implicated in colon carcinogenesis 9 and, therefore, it has emerged as a potential new therapeutic target for cancer therapy. 10 Recent reports have shown the implication of ACER1 in keratinocyte differentiation 11,12 and that of ACER2 in programmed cell death in response to DNA damage. 13 ACER3 has been reported to control both cell proliferation and apoptosis 14 and to be involved in motor coordinationassociated Purkinje cell degeneration. 15,16 Despite these fundamental studies on the functional roles of NC and ACER1-3 in certain biological processes, further investigations are still on going to better clarify their implications in human diseases. 8 By contrast, a growing body of evidence describes the important role of AC in the development and progression of different human pathological conditions, suggesting human AC (hAC) as a potential target for promising therapeutic applications. hAC (also known as N-acylsphingosine amidohydrolase-1, ASAH-1) is a lysosomal cysteine amidase that, at an optimal pH of 4.5, hydrolyzes Cer into So and fatty acids ( Figure  1). 17,18 Because the phosphorylation of So is the only pathway for the formation of So-1P, cellular So-1P is highly dependent on the availability of So; hence, hAC is a critical enzyme in regulating not only the hydrolysis of Cer but also the generation of both So and So-1P in cells. Cer and So-1P have opposing effects in the control of cell fate. 19 While Cer favor cell-cycle arrest 20 and apoptosis, 21,22 So-1P promotes angiogenesis, cell survival, and proliferation. 23−26 Hence, the altered Cer/So balance determines the shifting of cell fate toward apoptosis and proliferation, respectively, and contributes to the pathogenesis of some human diseases. For example, various common diseases, including inflammation, pain, and several pulmonary disorders, have been associated with aberrant hAC activities. 27 hAC is also deficient in two rare inherited disorders: spinal muscular atrophy with myoclonic epilepsy and Farber's disease. 28 By contrast, collected evidence has shown that hAC is abnormally expressed in various types of human cancer, for example, prostate, 29 melanoma, 30 head and neck, 31 colon, 32 and glioblastoma. 33 It has been observed that the overexpression of hAC renders the cells more resistant to pharmacological induction of apoptosis. 29,34 Therefore, the inhibition of hAC has been proposed as a potential strategy to enhance the therapeutic efficacy of standard antineoplastic agents and radiation. 34,35 Relevant evidence has shown that Alzheimer's disease (AD) brains exhibit elevated level and activity of hAC, suggesting a potential role of AC in controlling neuronal apoptosis and in the molecular mechanism of AD. 36 Notably, recent reports are proposing the role of hAC inhibition as an emerging strategy for treating some types of rare inherited metabolic disorders called lysosomal storage diseases (LSDs), 37−39 in particular, some severe neuropathic conditions related to Gaucher's 40 disease (GD) and Krabbe's 41 disease (KD). GD and KD are caused by the defective functions of some specific lysosomal proteins, acid β-glucocerebrosidase (GCase, β-glucosyl ceramidase) for GD and β-galactocerebrosidase (GALCase, β-galactosyl ceramidase) for KD. In GD patients, recent evidence suggests an active role of hAC in the catabolism of the lysosomal glucosylceramide, which is responsible for the accumulation of toxic glucosylsphingosine ( Figure 1). 42 In KD patients, deficiency of GALCase activity results in the buildup of the galactosylceramide and the galactosylsphingosine (psychosine) in nervous tissues, especially in the brain. Notably, a recent report suggests that genetic ablation or pharmacological inhibition of AC could eliminate the accumulation of the neurotoxic psychosine and prolong the life span of the KD mouse model. 43 There are no approved treatments for neuropathic GD and KD; targeting the inhibition of hAC may provide an innovative approach for treating these severe diseases. Although many efforts in the past decade have been made to identify new classes of hAC inhibitors, to date, these activities have resulted in limited success and a very limited number of suitable candidates for in vivo experiments are currently available. In a recent study, Gebai and co-workers reported the crystal structure analysis of mammalian AC (PDB code: 5U7Z), 44 which may assist future structure-guided drug discovery programs. First generation hAC inhibitors were designed on the basis of substrate (Cer)-based structures, for example, N-oleoylethanolamine (OEA, median inhibitory concentration (IC 50 ) ∼ 500 μM, 45 Figure 2). Despite being the first Cer-mimicking inhibitor to be described, the ability of OEA to inhibit hAC was not always reproducible. 45−47 Further representative examples are D-erithro-2-(N-myristoylamino)-1phenyl-1-propanol (D-e-MAPP, IC 50 > 500 μM in HL-60 cell lysates 45 and IC 50 = 500 μM in HaCaT cell lysates, 48 Figure 2) and its more water soluble derivative N-NMAPPD (B13, IC 50 ∼ 10 μM in HaCaT cell lysates, 48 Figure 2). Efforts to ameliorate these Cer-mimicking molecules led to several structurally varied analogues of B13, as compounds DP24a (IC 50 = 1.287 μM, 49 Figure 2) and the potent irreversible AC inhibitor SABRAC (IC 50 = 0.052 μM, 50,51 Figure 2). By contrast, the quinolinonebased compounds, Ceranib-1 and its optimized analogue Ceranib-2, represent the first class of non-Cer-mimicking inhibitors of hCDase identified by Draper and co-workers by screening a chemical library (hCDase IC 50 = 55 μM and 28 μM in SKOV3 cells, respectively, 52 Figure 2). In another study by Yildiz-Oze and co-workers, Ceranib-2 was found to inhibit hAC activity by 44% at 25 μM in H460 cells. 53 More recently, Cho and co-workers reported the identification of the hit compound [1,1′-biphenyl]-4-yl-2-(4-guanidinophenyl)acetate (E2, IC 50 = 52 μM, 54 Figure 2) from 68 guanidine-based derivatives tested for the discovery of new antiangiogenic inhibitors and determined the role of hAC as the E2-binding protein. Although a comparative analysis of the AC inhibitory activities of these different molecules is limited by the fact that the reported pharmacological data have been collected using different assay conditions and protein sources (Table S1), 45−50,52−54 overall, these AC inhibitors are characterized by low inhibitory potency (as those with IC 50 values in the μM range) 45−49,52−54 and poor drug-likeness (as those with, e.g., long lipophilic carbon chains). 45−50 A significant breakthrough was made by Realini and coworkers with the identification of carmofur [rat AC (rAC) IC 50 = 29 nM, 55 Figure 2 and Table S1] and some close uracil analogues, for example, compounds 1a−d, as nanomolar inhibitors of AC activity (1a, rAC IC 50 = 67 nM; 55 1b, rAC IC 50 = 12 nM; 55 1c, rAC IC 50 = 16 nM 56 and hAC IC 50 = 7.7 nM; 30 and 1d, hAC IC 50 = 12.8 nM, 30 Figure 2 and Table S1). Despite being potent AC inhibitors with some potential applications as chemo-sensitizing agents, the uracil derivatives showed low chemical and metabolic stability. Successively, using a ligand-based virtual screening approach, with carmofur as the template, Diamanti and co-workers identified a new class of potent hAC inhibitors, exemplified by the pyrazole carboxamide 2 (IC 50 = 14 nM, 57 Figure 2 and Table S1). However, these molecules exhibited low metabolic stability (2, mouse plasma half-life, t 1/2 = 9 min), 57 limiting their therapeutic potential. Through a systematic computational investigation, Ortega and co-workers reported the identification of benzimidazole derivatives 3a−d (3a, IC 50 = 2.5 nM; 3b, IC 50 = 13.9 nM; 3c, IC 50 = 22.5 nM; and 3d, IC 50 = 14.8 nM, 58 Figure 2 and Table  S1) with promising AC inhibitory activity in different melanoma cell lines. 58 A screening campaign of a small compound library was exploited by Pizzirani and co-workers resulting in the identification of the benzoxazolone (abbreviated as BOA, hereafter) carboxamide series, exemplified by the initial hit 4a (IC 50 = 64 nM, 59 Figure 2 and Table S1). 59 Preliminary studies led to the more advanced and systematically active analogues 4b (IC 50 = 79 nM, 59 Figure 2 and Table S1) and 4c (IC 50 = 33 nM, 60 Figure 2 and Table S1). 59,60 Although these molecules showed potent inhibitory effects on hAC activity, they generally suffered from low aqueous solubility and moderate metabolic stability, which impede their further development as oral drugs. During recent years, we directed the scope of our research work to solve these limitations. As part of our continued efforts in the optimization of the BOA carboxamide series, we recently reported the discovery of the piperidine 4d (IC 50 = 166 nM, 61 Figure 2 and Table S1) as a lead compound with good oral bioavailability, excellent brain penetration, and target engagement in two animal models of neuropathic GD and KD. 61 As an extension of this work while adopting a different strategy, we started an exploratory drug discovery program directed to the search for a novel class of hAC inhibitors with optimal drug-like properties, suitable for investigational studies in cellular and in vivo model systems. In the present study, we describe our strategies for the design and synthesis of a novel chemotype of hAC inhibitors (general structure, compound 5, Figure 3). The disruption of the molecular planarity of the fused bicyclic BOA moiety resulted in the identification of two initial hits, 2-oxo-4phenyl-N-(4-phenylbutyl)oxazole-3-carboxamide 8a and 2-oxo-5-phenyl-N-(4-phenylbutyl)oxazole-3-carboxamide 12a ( Figure  3). Herein, we present the structure−activity relationship (SAR) exploration of this novel series of substituted oxazol-2-one-3carboxamides and the chemical optimization which resulted in the identification of 5-[4-fluoro-2-(1-methyl-4-piperidyl)phenyl]-2-oxo-N-pentyl-oxazole-3-carboxamide 32b as a potent

■ CHEMISTRY
The synthetic routes for the preparation of all target compounds are described in Schemes 1−6. We introduced different substituents at the C(4)-and C(5)-positions of the 2-oxazolone core scaffold by exploring the synthetic pathways depicted in Schemes 1 and 2. The substituted 4-phenyl-oxazol-2-one derivatives 7a−c were obtained starting from the corresponding α-hydroxy ketones 6a−c, through the condensation reaction with potassium cyanate and in situ intramolecular cyclization under acidic conditions (Scheme 1). 62 A series of substituted 5phenyl-and 5-heteroaryl-oxazol-2-one derivatives 11a−q were synthesized starting from the commercially available α-bromo ketones 9a−q by condensation with 2,4-thiazolidinedione (TZD), followed by intramolecular cyclization of the intermediates 10a−q under basic conditions (LiOH or t-BuOK) (Scheme 2). 63 We introduced the carboxamide functionalities using standard conditions, by reacting intermediates 7a−c or 11a−q with the corresponding commercially available isocyanates, as in the synthesis of 8a−d (Scheme 1) or 12a−b, g−w (Scheme 3). Alternatively, the isocyanates were generated in situ, through the activation of the corresponding amines by reaction with Boc 2 O in the presence of 4-(dimethylamino)-pyridine (DMAP) 64 (12c−d, Scheme 3) or by reaction with triphosgene in the presence of N,Ndiisopropylethylamine (DIPEA) or Et 3 N 65 (12e−f, Scheme 3). The N-methylated analogue 13a and the carbamate 13b were prepared upon the activation of 11a with triphosgene in the presence of DIPEA, followed by the addition of N-methyl-4phenylbutylamine and 4-phenyl-1-butanol, respectively (Scheme 3). On the other hand, 11a was converted to the corresponding amide 13c by reaction with the corresponding freshly prepared 6-phenylhexanoic chloride. The oxazolidin-2one analogues 15a−b were prepared starting from the commercially available chiral (4S)-14a-and (4R)-14b-phenyloxazolidin-2-ones by carboxamide formation under standard conditions (Scheme 4A). A similar synthetic strategy was adopted for the preparation of analogues 18a−b upon the formation of the enantiomers (5S)-17a-and (5R)-17b-phenyloxazolidin-2-ones starting from the enantiomerically pure 2amino-1-phenylethanols 16a−b via 1,1′-carbonyldiimidazole (CDI)-mediated intramolecular cyclization (Scheme 4B). Similar procedures were exploited for the preparation of the targeted oxazolone carboxamides 25c−f and 32a−c, bearing a 4methylpiperidine moiety at the C(3′)-and C(2′)-positions of the phenyl ring, respectively (Schemes 5 and 6). The methyl ketones 22a−b and 28 were prepared in two steps, starting from the corresponding bromophenyls 20a−b and 26, using Pdcatalyzed cross-coupling reactions, in the presence of the commercially available boronic pinacol ester 19, followed by hydrogenation in EtOH at 60°C in the presence of 10% Pd/C and cyclohexene (as in the synthesis of 22a−b), or using Pd(OH) 2 and ammonium formate in MeOH at reflux (as in the synthesis of 28). The resulting methyl ketones 22a−b and 28 were transformed into the corresponding α-bromo ketones 22c−d and 29, through a slightly modified reported procedure, 66 consisting of an in situ addition of Nbromosuccinimide (NBS) to the corresponding silyl enol ethers in the presence of Et 3 N at a controlled low temperature (Schemes 5 and 6). The α-bromo ketones 22c−d and 29 were directly reacted with TZD to afford the corresponding intermediates 23a−b and 30 and then converted, through an intramolecular cyclization, to the 2-oxazolones 24a−b and 31a, respectively, as described above. Standard reaction conditions were exploited to convert the piperidines 24b and 31a to the corresponding 4-methylpiperidines 24d and 31c, which involved N-Boc removal and reductive amination in the derivative 25a, obtained by reacting 24a with 4-phenylbutyl isocyanate (Scheme 5). Finally, the carboxamide functionality of the targeted compounds 25d−f and 32a−c was introduced using standard reaction conditions, as described above.

■ RESULTS AND DISCUSSION
A common characteristic of some classes of known AC inhibitors is the presence of a cysteine (Cys)-targeting warhead as the α-bromo acetyl moiety or the urea-like functionality that can undergo a chemical reaction with the thiol group of the catalytic Cys143 of hAC to produce a covalent bond, 44 as reported for 2-bromoacetamide SABRAC 51 and the carboxamides 3a−b 58 and 4a 59 ( Figure 2). This evidence has been supported by recent studies, reported by Dementiev and co-workers, on the crystal structure analysis of carmofur covalently bound to Cys143 at a 2.7 Å resolution. 67 While potent and, in certain cases, systemically active, for example, analogues 4b− c, 59,60 these potent hAC inhibitors share two characteristics that hamper their applications as oral drugs. First, the chemical warheads which, on the one hand, are responsible for the covalent binding mechanisms of these inhibitors and, on the other hand, can contribute to the poor chemical and plasma stability of these molecules (e.g., carmofur, 1a−d and 2); 56,57 second, the hydrophobic linear side-chains, although funda- mental for target recognition and some degree of specificity, negatively affect the drug-like properties of these molecules (e.g., SABRAC and 4a). 51,59 Thus, there is a strong need for novel and optimized hAC inhibitors. In this respect, our continued efforts dedicated to the chemical optimization of the BOA carboxamide series, exemplified by 4a−c, 59,60 have recently led to the identification of the lead 4d as a potent and orally bioavailable hAC inhibitor with excellent brain penetration in mice and target engagement in two animal models of LSDs ( Figure 2). 61 As part of our more exploratory research program, our medicinal chemistry strategies were also focused on expanding the chemical diversity of the existing hAC inhibitors for the identification of new chemotypes with optimal physicochemical and metabolic properties suitable for cellular and in vivo studies. In this regard, by the disruption of the molecular planarity of the fused bicyclic aromatic BOA system, we designed a series of compounds with the general structure 5 and synthesized a few initial molecules, for example, the 2-oxo-4-phenyl-N-(4phenylbutyl)oxazole-3-carboxamide 8a and 2-oxo-5-phenyl-N-(4-phenylbutyl)oxazole-3-carboxamide 12a ( Figure 3). In particular, we were interested in studying the substitutedoxazolone ring system as a potential and attractive strategy for the BOA bioisosteric replacement. In addition, we envisaged that the insertion of this relatively unexplored heterocycle system, by reducing the molecular planarity of the core scaffold and, therefore, varying the nature of the leaving group at the reactive electrophilic functionality, might be a valuable strategy for the subsequent optimization of our targeted molecules. 68 By contrast, in order to somehow preserve the hAC recognition, we initially designed scaffolds that bear a lipophilic group on the lateral chain of the urea-like functionality, as the butyl phenyl group of 8a and 12a, already described in other series of known inhibitors (e.g., 3a−b and 4a−c, Figure 1) to be suitable for chemical optimization. Compounds 8a and 12a were screened against hAC using a fluorogenic assay and were able to inhibit the enzymatic activity with IC 50 values equal to 0.007 and 0.090 μM, respectively (Table 1). These initial results encouraged us to start a preliminary SAR exploration around these new scaffolds in the three main Regions A, B, and C, as depicted in Figure 4, with the objective of identifying the pharmacophore necessary for target inhibition.
In this regard, to first validate our initial hits, we prepared a set of representative analogues around the 4-phenyl-oxazol-2-one (4-POA) and 5-phenyl-oxazol-2-one (5-POA) carboxamide compounds 8a and 12a (Table 1). Interestingly, in the 4-POA carboxamide series, although a slight drop in potency was detected with the removal of the terminal aromatic ring, as in the n-pentyl analogue 8b (hAC IC 50 = 0.025 μM), the insertion of a Cl atom at the para phenyl position was tolerated, with compound 8d (hAC IC 50 = 0.005 μM) being equipotent to the parent 8a (hAC IC 50 = 0.007 μM) ( Table 1). In contrast, the insertion of a methyl group on the C(5)-position of the oxazolone ring, as in the di-substituted analogue 8c, resulted in a 6-fold loss of potency (hAC IC 50 = 0.042 μM). A slightly different trend was observed in the 5-POA carboxamide series; the removal of the terminal phenyl ring, as in the n-pentyl analogue 12b (hAC IC 50 = 0.039 μM), resulted in a weak improvement in potency compared to the parent compound 12a (hAC IC 50 = 0.090 μM). Both the insertion of a Cl atom at the para phenyl position, as in 12i, and the insertion of a methyl group at the C(4)-position of the oxazolone ring, as in 12h, afforded analogues (hAC IC 50 = 0.083 and 0.069 μM, respectively) with similar potency compared to 12a (Table 1).
These encouraging preliminary results confirmed that both the 4-and 5-(POA) carboxamide series were promising scaffolds and warranted further exploration. Nevertheless, a head-to-head comparison of the two hit series directed future investigations toward the 5-POA carboxamide series. Specifically, although being very potent hAC inhibitors, the 4-POA carboxamide series suffered from significantly poorer chemical stability compared to the 5-POA carboxamide series, as measured by performing the stability assay in aqueous media [8a, t 1/2 = 10 min, in phosphate buffered saline (PBS), pH 7.4; 12a, t 1/2 = >12 h, in PBS, pH 7.4].
First, we demonstrated the importance of the reactive carboxamide functionality of 12a because the corresponding unsubstituted analogue 11a was not active against hAC at the concentrations tested (Scheme 3 and Figure 5A), suggesting that inhibition by 12a could occur through covalent AC modification. Preliminary kinetic studies on hAC-enriched lysates showed that 12a causes a concentration-dependent reduction in the maximal catalytic velocity of AC (V max ) without  influencing the Michaelis−Menten constant (K M ) ( Figure 5B and Table S2) supporting irreversible binding. In addition, the replacement of the N−H of the urea-like functionality of 12a with a N−Me (13a), with an oxygen (13b) or with a methylene (13c) were detrimental to activity (Scheme 3), as these analogues were not active against hAC at the concentrations tested (1 and 10 μM). Moreover, we demonstrated that the presence of the oxazol-2-one ring of 12a was essential to maintaining inhibitory potency, since the corresponding chiral 1,3-oxazolidinone carboxamide analogues 18a−b were not active against hAC at the concentrations tested (1 and 10 μM) (Scheme 3). A similar outcome was also observed with the chiral 1,3-oxazolidinone carboxamide analogues of the more potent 8a, compounds 15a−b (Scheme 3). 57 Therefore, based on these results, we continued with a more focused SAR exploration, by targeting additional analogues bearing small linear and branched alkyl substituents on the sidechain at the N-terminal urea moiety of 12a series (Region C, Figure 4). Replacement of one methylene unit with an oxygen in the n-pentyl chain of 12b (as ethers 12c−d) significantly affected the inhibitory potency; specifically, 12c showed an hAC IC 50 = 2.04 μM, while a complete loss in potency was observed for 12d (Table 1). These results suggested that the lipophilic side-chains at region C were very likely occupying a hydrophobic channel of the enzyme. Our SAR exploration continued with the insertion of branched alkyl groups, the i-butyl 12e inhibited AC with an IC 50 of 1.70 μM, while the s-butyl analogue 12f was not active up to 10 μM. No improvement was also observed with the aryl urea 12g.
In parallel, our medicinal chemistry efforts were also focused on the exploration of region A by introducing different substituents on the phenyl ring of the 12a series. Besides, the p-Cl atom of 12i, both electron-withdrawing (F) and electrondonating (OCH 3 ) groups at different positions of the phenyl ring were tolerated, resulting in compounds, such as 12j (p-F) and 12k−m (p-, m-and o-OCH 3 ), with hAC IC 50 values in the submicromolar ranges (Table 1). In addition, the di-substituted derivative 12n (p-F, m-OCH 3 ) was almost equipotent (hAC IC 50 = 0.080 μM) to 12a.
A comparison of several of these analogues in terms of aqueous kinetic solubility (PBS, pH 7.4) and in vitro metabolism (t 1/2 in mouse plasma and mouse liver microsomes) underlined some important differences (Table 3), which informed the next steps of the SAR exploration. In general, these compounds showed poor aqueous solubility, which was not ameliorated by the removal of the terminal lipophilic phenyl group (e.g., npentyl analogue 12b), or by the introduction of more polar  a groups, such as an oxygen atom on the lateral chain (12c) or heteroaryl rings at the C(5)-position of the oxazol-2-one moiety (e.g., the pyridines 12o−q and the pyrazine 12r) ( Table 3). On the contrary, more pronounced differences were observed by comparing the mouse plasma and mouse liver microsomal stability properties of these analogues. In general, except for the parent 12a and the heteroaryls 12o and 12q−r, the selected compounds showed good mouse plasma stability. In contrast, poor mouse microsomal stabilities were observed within the phenyl derivatives, except when a F-atom was inserted in the phenyl ring, for example, p-F analogues 12j (m-liver microsomal t 1/2 = 60 min) and 12n (m-liver microsomal t 1/2 > 60 min, 74% compound remaining at 1 h). An improvement of the mouse microsomal stability was also detected with the i-butyl analogue 12e (m-liver microsomal t 1/2 = 50 min). A similar effect of the Fatom was shown by some nitrogen containing heteroaryl analogues, bearing an "aza"-group in the phenyl ring, for example, the 4-pyridyl 12q (m-liver microsomal t 1/2 > 60 min, 75% compound remaining at 1 h) and the pyrazine 12r (m-liver microsomal t 1/2 = 60 min) ( Table 3). Based on these results, we continued the SAR exploration by modifying the scaffold with the insertion of some potential solubilizing groups. We specifically focused our attention on regions A and B due to the fact that, as mentioned above, our SAR study suggested region C to be more involved in lipophilic interactions with hAC (Table 1). In this respect, our preliminary exploration of this series showed that the insertion of an hydrophilic group, such as the N-methyl-piperidine ring at the C(5)-position of the oxazol-2-one moiety (12y, Table 1), although detrimental for the inhibitory potency (hAC IC 50 = 1.40 μM), was essential for significantly improving the aqueous solubility (12y, kinetic solubility = 248 μM). As a consequence, we decided to investigate the effect of inserting the N-methylpiperidine group directly at the C(3′)-position of the phenyl ring of the 5-POA carboxamide series by preparing analogues 25c and 25d (Table 4). Although a similar decrease in potency (hAC IC 50 = 0.153 and 0.341 μM, respectively) was observed compared to the corresponding parent compounds 12a and 12j, we were pleased to notice that, overall, these structural modifications were tolerated. Moreover, a similar trend was observed when the N-methyl-piperidine ring was moved to the C(2′)-position of the phenyl ring, with 32a (hAC IC 50 = 0.337 μM) being equipotent to 25d. Notably, we observed that these targeted compounds showed moderately improved solubility (kinetic solubility > 30 μM, Table 5) compared to the parent compounds 12a and 12j.
These results prompted us to continue a more focused SAR study on the lateral chains of 25d and 32a scaffolds, selecting the optimal moieties previously identified in the exploration of region C (Tables 1 and 3) and, hence, synthetizing the corresponding n-pentyl analogues 25e and 32b and the i-butyl analogues 25f and 32c. Although, a complete loss in potency was observed with both i-butyl analogues 25f and 32c, the n-pentyl derivatives 25e and 32b gave unexpected results. While 25e showed a hAC IC 50 of 1.9 μM, surprisingly, compound 32b inhibited hAC with an IC 50 equal to 0.129 μM. In addition, compound 32b, bearing both the N-methyl piperidine ring and the small linear alkyl chain, showed a high solubility value (kinetic solubility > 250 μM) (Table 5). Finally, the most promising compounds were evaluated for in vitro metabolism (Table 5). In general, we were pleased to observe that the selected compounds exhibited good mouse plasma stabilities with t 1/2 values ≥2 h. On the other hand, the poor mouse liver microsomal stability observed for 25c was ameliorated by the insertion of a F-atom at the para-position of the phenyl ring (25d, m-liver microsomal t 1/2 > 60 min, 70% compound remaining at 1 h). A similar trend was observed with the other fluorinated analogues, compound 32a−b. Furthermore, additional in vitro metabolism studies were performed on 32b which showed acceptable h-plasma (t 1/2 = 40 min) and good h-liver microsomal stability (t 1/2 > 60 min, 80% compound remaining at 1 h).
Based on its inhibitory potency and good overall drug-like properties, compound 32b was selected for further biological and pharmacological characterizations. As previously anticipated for the initial hit 12a and based on our previous work with    Journal of Medicinal Chemistry pubs.acs.org/jmc Article 4d, 61 the mechanism of inhibition of 32b occurs through covalent hAC modification. This was further supported by the corresponding analogue 31c, lacking the reactive urea-like functionality of 32b, which was not active against hAC ( Figure  8A). Based on these considerations, we prioritized the selectivity evaluation of 32b against human N-acylethanolamine acid amidase (hNAAA), a lysosomal cysteine amidase that shares 33−34% sequence identity and a very similar reactive site with hAC. 69 Notably, 32b showed no effect at up to 125 μM against hNAAA under our assay conditions. In an effort to gain insights into the structural bases of these biological results, we then performed molecular modeling and docking studies using the Xray crystal structures of hAC (PDB code: 6MHM) 67 and hNAAA (PDB code: 6DXX). 69 The protein (hAC)−ligand (targeted compound) binding site was prepared by adding hydrogen atoms, optimizing hydrogen bonds, and verifying the protonation states of His, Gln, and Asn. The energy minimization was carried out using a default constraint of 0 Figure 8B. The docking pose suggests the alkyl sidechain (n-pentyl) of 32b sharing the same binding mode of the nhexyl chain of carmofur ( Figure 2) within the hAC binding pocket, 67 where lipophilic residues, such as Phe163, Tyr137, Leu211, and Meth161, are localized. In addition, hydrogen bonding interaction stabilizes the transition state around the catalytic Cys143 residue between the side-chain carboxyl group of Glu225 (2.77 Å) and the hydroxy group (tetrahedral intermediate) on the alpha carbon of the reaction center. The ligand pose itself is stabilized by a cation−pi interaction between the protonated N-methyl amino group of the piperidine ring and Trp395 and a second edge to face the pi−pi interaction between the fluorophenyl group of the ligand and Phe165 (4.56 and 3.43  Analysis of 32b in the anti-target hNAAA suggests that the binding site of the two enzymes share over 90% similarity in their amino acid composition. However, when compound 32b is docked into hNAAA, 69 the resultant equivalent pose has a very low score. Careful examination of the docking poses (modeling details in Experimental Section) show that there is a critical difference between hAC and hNAAA around residue 182 in the binding site. While hAC has Leu at position 182, the equivalent position is occupied by much larger Trp181 in hNAAA. The npentyl side-chain on the compound 32b easily fits in the wider pocket of hAC ( Figure 9A), but it causes steric clashes, as seen in the Figure 9B, in the case of hNAAA because of the much bulkier Trp181 side-chain. We believe this to be one of key reasons for selectivity observed in 32b that might be further exploited in future drug design programs.
Because of the overall property profile of 32b, this compound was selected for additional pharmacological studies with the aim of testing its ability to inhibit hAC in intact cells. In particular, we examined the effects of compound 32b treatment using human neuroblastoma SH-SY5Y cells, which are a well-characterized and widely used in vitro cell model. 70,71 Notably, Kyriakou and co-workers recently established and characterized a stable AC knockdown human neuroblastoma SH-SY5Y cell line, as a human in vitro cell model for studying the effects of AC deficiency. 72 Human neuroblastoma SH-SY5Y cells were incubated in the presence of 32b at different concentrations for 3 h (1, 2.5, 5, and 10 μM, Figure 6) and in the presence of 32b (10 μM) at different incubation times (1, 3, and 6 h, Figure  7). hAC activity was measured and SL levels were identified and quantified with a liquid chromatography/mass spectrometry (LC/MS)-based activity assay, as previously described. 55,56,61 We indeed demonstrated that 32b is effectively able to engage hAC in the complex cellular environment under our experimental conditions, causing the expected changes in the cellular levels of SL. Treatment of SH-SY5Y cell cultures with 32b caused a concentration ( Figure 6A) and time-dependent reduction of hAC activity ( Figure 7A). After 3 h incubation, we observed an intracellular accumulation of various Cer species, including Cer (d18:0/16:0) and Cer (d18:1/16:0) ( Figure  6B,C), and a corresponding decrease in So levels in a concentration-dependent manner ( Figure 6D). Conversely, no major variations were observed in the levels of SM (d18:1/16:0) ( Figure 6E) and HexCer (d18:1/16:0) ( Figure 6F). The effect of 32b (10 μM) on the inhibition of hAC activity and the intracellular SL levels is reported in Figure 7A−F. Specifically, we observed that 32b inhibits hAC in SH-SY5Y cells leading to an increased Cer (d18:0/16:0) and Cer (d18:1/16:0) ( Figure   7B,C) and decreased So levels ( Figure 7D), which persisted up to 6 h. No major variations were observed in the levels of SM (d18:1/16:0) ( Figure 7E) and HexCer (d18:1/16:0) ( Figure  7F).
Finally, we then took 32b for pharmacokinetic (PK) studies in C57BL/6 mice, following intravenous (i.v.) and oral administration (p.o. ). The relevant PK parameters are reported in Table 6. Values of plasma clearance (Cl p ), volume of distribution (V dss ), and plasma elimination half-life (t 1/2 ) were calculated after i.v. administration of 32b at 3 mg/kg. Cl p was relatively low (72 mL/min/kg) with acceptable plasma t 1/2 (119 min) and high V dss (12426 mL/kg), indicating that 32b is well distributed out of the circulating mouse plasma compartment. Compound 32b is an orally bioavailable hAC inhibitor at 10 mg/ kg (oral bioavailability, F = 40%) and is rapidly adsorbed in the plasma compartment (t max = 30 min), with a maximal plasma concentration (C max ) of 278 ng/mL and acceptable plasma t 1/2 (147 min). Moreover, 32b shows significant exposures in mouse plasma, after both i.v. and p.o. doses (AUC = 31978 and 42525 min × ng/mL, respectively).
Taking into account its overall profile, 32b was selected for further development studies aimed to elucidate the potential therapeutic applications of AC inhibition in cellular and in vivo model systems of relevant SL-mediated disorders, which will be described elsewhere in due course.

■ CONCLUSIONS
Although hAC inhibition has been the focus of intense discovery in the last decade, only a very limited number of valuable candidates for in vivo experiments are available. The scope of this work was directed to solve this limitation. The design and synthesis of a series of substituted oxazol-2-one-3-carboxamide derivatives were presented, resulting in the identification of two initial hits, 8a and 12a as a novel and versatile class of hAC inhibitors. Preliminary results of the hit expansion around these new scaffolds in the three main Regions A, B, and C contributed to the definition of the pharmacophore necessary for target inhibition and directed the strategies for chemical optimization. Our medicinal chemistry efforts around the most promising 5substituted oxazol-2-one-3-carboxamide series led to the identification of 5-[4-fluoro-2-(1-methyl-4-piperidyl)phenyl]-2-oxo-N-pentyl-oxazole-3-carboxamide (32b) as an optimized hAC inhibitor, structurally distinct from previous reported inhibitors, with good drug-like properties. Furthermore, 32b showed target engagement in human neuroblastoma SH-SY5Y cells and desirable PK properties in mice, with good F % and significant exposures in plasma, after intravenous and oral administrations. Compound 32b is a valuable lead that increases the arsenal of suitable hAC-targeting molecules, which can directly probe the link of hAC function to distinct physiological processes and investigate how the inhibition of its activity can provide health benefits under severe pathological conditions. The identification of novel hAC-modulating compounds, targeting active Cys143 with optimal drug-like properties, remains a challenging task and can be only achieved by a critical and balanced modulation of different parameters, whose objectives often clash during the chemical optimization process. Utilizing the recently reported crystal structures, we were able to dock our covalent inhibitors into the reactive site highlighting the basis for selectivity observed with 32b for hAC compared to hNAAA. In addition, the modeling that was undertaken can guide future optimization of this lead series accelerating the field ■ EXPERIMENTAL SECTION Chemicals, Materials, and Methods. Solvents and reagents were obtained from commercial suppliers and were used without further purification. Automated column chromatography purifications were done using a Teledyne ISCO apparatus (CombiFlash Rf) with prepacked silica gel or neutral alumina columns of different sizes (from 4 g until 120 g). Mixtures of increasing polarity of Cy and EtOAc or dichloromethane (DCM) and MeOH were used as eluents. Thin-layer chromatography (TLC) analyses were performed using Supelco silica gel on TLC Al foils 0.2 mm with a fluorescence indicator 254 nm. NMR experiments of all the intermediates and final compounds 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-gradient coil. Spectra were acquired at 300 K using DMSO-d 6 or CDCl 3 as solvents. Chemical shifts for 1 H and 13 C spectra were recorded in parts per million (ppm) using the residual nondeuterated solvent as the internal standard (for DMSO-d 6 Table S3. 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. Accurate mass measurements were performed on a Synapt G2 Quadrupole-ToF Instrument (Waters, USA), equipped with an ESI ion source; the compounds were diluted to 50 μM in CH 3 CN/H 2 O and analyzed. Leucine Enkephalin (2 ng/mL) was used as the lock mass reference compound for spectra recalibration. All final compounds displayed ≥95% purity as determined by NMR and UPLC/MS analysis.
General Procedure for the Synthesis of 4-Substitutedoxazol-2-ones (Procedure A). To a mixture of the appropriate αhydroxy ketone (1.0 equiv) and KNCO (2.0 equiv) in i-PrOH (0.2 M) was added dropwise AcOH (2.0 equiv) with stirring. The resulting suspension was heated at 70°C for 3 h, then poured into an ice/H 2 O bath, and extracted with DCM or EtOAc. The organic phase was dried over Na 2 SO 4 and concentrated under reduced pressure. The crude was purified by column chromatography (SiO 2 ), eluting with Cy/EtOAc or used as crude in the next step without further purification.
Step 1: to a stirred solution of the appropriate α-bromoketone (1.0 equiv) and TZD (1.2 equiv) in DMF (0.1−1 M) was added K 2 CO 3 (1.5 equiv). The resulting mixture was stirred at rt for 1−2 h and then poured into an ice/H 2 O bath. In some cases, the resulting solid was filtered off and washed with H 2 O. In other cases, the aq phase was extracted with EtOAc, and the organic layer was washed with 5% aq LiCl and brine and dried over Na 2 SO 4 . After evaporation of the solvent, the crude was purified by flash chromatography (SiO 2 ) eluting with Cy/EtOAc or used as a crude in the next step without further purification, as indicated in each case.
Step General Procedure for Palladium-Catalyzed Cross-Coupling Reaction (Procedure E). To a solution of the appropriate phenyl bromide (1.0 equiv) in dry 1,4-dioxane (0.1 M, previously degassed under a nitrogen atmosphere), 19 (1.1 equiv) was added followed by the addition of Pd(PPh 3 ) 4 (0.05 equiv) and Na 2 CO 3 (2.2 equiv, 2 M aq solution). The suspension was stirred at reflux on, cooled to rt, and then diluted with EtOAc and filtered through a pad of celite. The filtrate was concentrated under reduced pressure, diluted with EtOAc, washed with sat. aq NH 4 Cl solution and brine, and dried over Na 2 SO 4 . After evaporation of the solvent, the crude was purified by flash chromatography (SiO 2 ), eluting with Cy/EtOAc, as indicated in each case.
General Procedure for Catalytic Hydrogenation Reaction Then, the reaction mixture was poured into a saturated aqueous NaHCO 3 solution and extracted with EtOAc. The organic phase was washed with brine and dried over Na 2 SO 4 . After evaporation of the solvent, the crude was purified by flash chromatography (SiO 2 ) eluting with DCM/MeOH, or used as a crude, in the next step without further purification, as indicated in each case.
Synthesis of (5R)-(+)-2-Oxo-5-phenyl-N-(4-phenylbutyl)oxazolidine-3-carboxamide (18b). Compound 18b was prepared according to general procedure D (method A) using 17b (0.109 g, 0.67 mmol), DMAP (0.084 g, 0.74 mmol), and 4-phenylbutyl isocyanate (0.130 g, 0.74 mmol) in pyridine. The crude was purified by column chromatography (SiO 2 ), eluting with Cy/EtOAc (9:1), to afford 18b as colorless oil (0.180 g, 79%). 1  ■ MOLECULAR MODELING AND DOCKING STUDIES Covalent Docking. To carry out the covalent docking, we used docking modules available in a Schrodinger platform: CovDoc in Schrodinger 2018-4 version. These covalent docking tools require that the ligand set must be a series of compounds all of which should react with the receptor at the same site and by the same mechanism. The first step is the regular noncovalent docking of the ligands having the potential to form a covalent bond with the reactive residue of the receptor. Then, the program allows for the different conformations of the side-chain of the reactive residue, which is temporarily mutated to alanine at this stage. The reason for this temporary mutation is only to avoid the bias toward the particular ligand conformation if the side-chain of the reactive residue is present. After the noncovalent docking, the original side-chain of the reactive residue is replaced with the cysteine and the covalent bond is formed. The ligands where the covalent bond lengths between the reactive center of the ligand and the reactive residue of the receptor are longer compared to the standard chemical bonds are discarded. After the bond formation between the potential covalent ligand and the receptor, the complex is minimized using the Prime module of Schrodinger suite. Finally, the docked poses of the covalently linked ligands are to be visualized and rank ordered by energy and the docked score. The reaction site of ligands was determined using a SMART2 search pattern implemented in the covalent docking program. After determining the reactive sites from both the ligands and the receptor, a 12 A3 grid box was generated making sure that Cys143 would be in that grid box. The docked poses and the energetics were analyzed and 43 covalent inhibitors were showing good docking poses with lower complex energies and having docking scores ≥6. 0. Some of the ligands lacked the proper SMARTS for the reaction and were rejected from the calculations. Docking calculations for hNAAA were carried out under identical conditions using the X-ray structure (PDB code: 6DXX). Compound 32b did not natively dock into this receptor using the standard CovDock protocol. To understand the binding mode in greater details, each step of the docking run was analyzed and the raw unrefined poses were examined. It was clear that Trp181 was occluding binding of the n-pentyl sidechain of compound 32b leading to a docking score >10,000 (indicate no binding affinity to NAAA). The compound was thus modeled using a flexible ligand overlay using the AC docking pose for the purpose of generating a figure.

■ IN VITRO PHARMACOLOGICAL ASSAY
In Vitro hAC Fluorescent Assay. Cell Culture Conditions and Preparation of the hAC-Enriched Lysate. HEK293 cells stably expressing hAC (HEK293-hAC) were generated in our laboratory using a protocol, as previously described. 30 Briefly, hAC (variant 1 coding sequence, NM_177924) cDNA was purchased from Open Biosystems (clone ID 3923451) and subcloned in the mammalian expression vector pCDNA3.1, containing the neomycin resistance gene. HEK293 cells were transfected with the hAC-pCDNA3.1 construct using a JetPEI reagent (Polyplus Transfection, Illkirch-Graffenstaden, France) and following the manufacturer's instructions. A stable cell line was generated by selection with G418 (1 mg/mL), and cell clones were derived by limited dilution plating. HEK293-hAC were grown in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS), 1% glutamine, 1 mM sodium pyruvate, and 500 μg/mL G418. Cells were harvested and pellets were stored at −80°C until lysosomal-enriched lysate preparation. Cells were suspended in 20 mM Tris HCl (pH 7.5) with 0.32 M sucrose, sonicated, and centrifuged at 800 × g for 30 min at 4°C. Supernatants were then centrifuged at 12 000 × g for 30 min at 4°C. Pellets were re-suspended in PBS (pH 7.4) and subjected to three freeze−thaw cycles at −80°C. The suspension was finally centrifuged at 105 000 × g for 1 h at 4°C , and protein concentration was measured in the supernatant with a bicinchoninic acid based protein assay. This hACenriched preparation allowed us to further optimize the enzymatic assay and to use small amounts of lysate (2 μg/ well) at the 5 μM substrate (Rbm14-12) around its K M (K M = 5.0 μM).
Fluorogenic hAC Assay. The assay was performed in Optiplate 96-wells black plates, with each reaction well containing a mixture of 25 mM NaOAc buffer (pH 4.5) and a fixed amount of protein (2 μg) in a volume of 85 μL. After 10 min of pre-incubation with the test compounds (diluted 20× from DMSO stock solutions at different concentrations), the fluorogenic probe was added (diluted 40× from EtOH stock solution, final concentration 5 μM). After 3 h incubation at 37°C , reactions were stopped with 50 μL of MeOH and 100 μL of a 2.5 mg/mL NaIO 4 fresh solution in 100 mM glycine/NaOH buffer (pH 10.6 7.4) and subjected to three freeze−thaw cycles at −80°C. The suspension was finally ultracentrifuged at 105 000 × g for 1 h at 4°C, supernatants were collected, protein concentration was measured, and samples aliquoted and stored at −80°C until use. Chemical Stability Assay. Chemical stability of the selected compounds was evaluated under physiological pH conditions (0.01 M PBS, pH 7.4) for up to 8 h. The buffer was added with 10% of CH 3 CN. Stock solutions of each compound (10 mM) were freshly prepared in CH 3 CN. Each compound was incubated at a final concentration of 1 μM in pre-heated buffer (37°C). The sample solutions were divided into aliquots in glass vials (preheated at 37°C) for each time point. The samples were maintained at 37°C in an UPLC/MS autosampler during the study (no shaking). A reference solution of each compound (final concentration: 1 μM) in preheated CH 3 CN was prepared from the stock solutions and maintained at 37°C in the UPLC/MS autosampler during the study. For each time point, the samples were analyzed directly by LC/MS without any further sample preparation. The samples were analyzed by integrating the corresponding MRM peak areas. The relative compound concentration was calculated by dividing the peak area at each time point by the peak area at t = 0 min. The reference solution was analyzed at the beginning (t = 0 min) and at the end of the study (t = 8 h). The apparent half-life (t 1/2 ) of the disappearance of the compound was calculated using the best fitting equation by GraphPad Prism (GraphPad Software, Inc., USA). The analyses were performed on a Waters Acquity UPLC/MS triple quadrupole detection (TQD) system consisting of TQD MS equipped with an ESI interface and a PDA detector. The analyses were run on an Acquity UPLC BEH C18 1.7 μm 2.1 × 50 mm column with a VanGuard BEH C18 1.7 μm pre-column at 40°C. For each compound, the appropriate mobile phase was chosen. ESI was applied in positive mode. The values are the mean of at least two independent experiments performed in two technical replicates.
In Vitro Plasma Stability Study. Freshly prepared 10 mM CH 3 CN stock solution of the test compound was diluted 50-fold with DMSO/H 2 O (1:1) and incubated at 37°C for 2 h with mouse/human plasma added 5% DMSO (preheated at 37°C for 10 min). The final concentration was 2 μM. At each time point (0,5,15,30,60, and 120 min), 50 μL of the incubation mixture was diluted with 200 μL of cold CH 3 CN spiked with 200 nM of the internal standard, followed by centrifugation at 3300 × g for 20 min. The supernatant was further diluted with H 2 O (1:1) for analysis. The concentration of the test compound was quantified by LC/MS−MS on a Waters Acquity UPLC/MS TQD system consisting of TQD MS equipped with an ESI interface. The analyses were run on an Acquity UPLC BEH C18 (50 × 2.1 mm ID, particle size 1.7 μm) with a VanGuard BEH C18 pre-column (5 × 2.1 mm ID, particle size 1.7 μm) at 40°C. For each compound, the appropriate mobile phase was chosen. ESI was applied in positive mode. The response factors, calculated on the basis of the internal standard peak area, were plotted over time. When possible, response versus time profiles were fitted with Prism (GraphPad Software, Inc., USA) to estimate compounds t 1/2 in the plasma. The values are the mean of at least two independent experiments performed in two technical replicates.
In Vitro Microsomal Stability Study. Freshly prepared 10 mM CH 3 CN stock solution of the test compound was preincubated at 37°C for 15 min with mouse/human liver microsomes added 0.1 M Tris-HCl buffer (pH 7.4). The final concentration was 4.6 μM. After pre-incubation, the cofactors (NADPH, G6P, G6PDH, and MgCl 2 pre-dissolved in 0.1 M Tris-HCl) were added to the incubation mixture and the incubation was continued at 37°C for 1 h. At each time point (0,5,15,30, and 60 min), 30 μL of the incubation mixture was diluted with 200 μL of cold CH 3 CN spiked with 200 nM of the internal standard, followed by centrifugation at 3300g for 15 min. The supernatant was further diluted with H 2 O (1:1) for analysis. The concentration of the test compound was quantified by LC/MS−MS on a Waters Acquity UPLC/MS TQD system consisting of TQD MS equipped with an ESI interface. The analyses were run on an Acquity UPLC BEH C18 (50 × 2.1 mm ID, particle size 1.7 μm) with a VanGuard BEH C18 pre-column (5 × 2.1 mm ID, particle size 1.7 μm) at 40°C. For each compound, the appropriate mobile phase was chosen. ESI was applied in positive mode. The percentage of the test compound remaining at each time point relative to t = 0 was calculated. The t 1/2 were determined by a one-phase decay equation using a nonlinear regression of the compound concentration versus time. The values are the mean of at least two independent experiments performed in two technical replicates.

■ ANIMAL MODELS
In Vivo PK Study. C57 B6/J male mice, 8 weeks old (22−24 g), were used (Charles River, Calco). All procedures were performed in accordance with the Ethical Guidelines of European Communities Council (Directive 2010/63/EU of 22 September 2010) and accepted by the Italian Ministry of Health. All efforts were made to minimize animal suffering and to use the minimal number of animals required to produce reliable results, according to the "3Rs rules". Animals were group-housed in ventilated cages and had free access to food and water. They were maintained under a 12 h light/dark cycle (lights on at 8:00 am) at a controlled temperature (21 ± 1°C) and relative humidity (55 ± 10%). 32b was administrated intravenously (i.v.) at 3 mg/kg/5 mL, vehicle: PEG400/Tween 80/saline solution (10/10/80% in volume, respectively) via tail vein injection and via oral administration (p.o.) at 10 mg/kg/10 mL, vehicle: PEG400/Tween 80/saline solution (10/10/80% in volume, respectively) by an oral gavage. Sample collection. Samples were collected at 5 min, 15 min, 30 min, 1 h, 2 h, and 4 h (i.v. administration) and at 15 min, 30 min, 1 h, 2 h, 4 h, and 6 h (p.o. administration). N = 3 animals per dose time point were treated. Plasma samples were centrifuged at 21,100g for 15 min at 4°C. An aliquot of each sample was extracted (1:3) with cold CH 3 CN containing 200 nM of an appropriate internal standard being a close analogue of compound 32b. A calibration curve was prepared in blank mouse plasma over a 1 nM−10 μM range. Three quality controls were prepared by spiking compound 32b in the blank mouse plasma to 20, 200, and 2000 nM as final concentrations. The calibrators and quality controls were extracted (1:3) with the same extraction solution as the plasma samples. The plasma samples, the calibrators, and quality controls were centrifuged at 3270 × g for 15 min at 4°C. The supernatants were further diluted (1:1) with H 2 O and analyzed by LC/MS−MS on a Waters Acquity UPLC/MS TQD system consisting of TQD) MS equipped with an ESI interface and a PDA detector. ESI was applied in positive mode. Compounddependent parameters such as MRM transitions and collision energy were developed for compound 32b and the internal standard. The analyses were run on an Acquity UPLC BEH C18 (50 × 2.1 mm ID, particle size 1.7 μm) with a VanGuard BEH C18 pre-column (5 × 2.1 mm ID, particle size 1.7