Potent α-amino-β-lactam carbamic acid ester as NAAA inhibitors. Synthesis and structure-activity relationship (SAR) studies.

4-Cyclohexylbutyl-N-[(S)-2-oxoazetidin-3-yl]carbamate (3b) is a potent, selective and systemically active inhibitor of intracellular NAAA activity, which produces profound anti-inflammatory effects in animal models. In the present work, we describe structure-activity relationship (SAR) studies on 3-aminoazetidin-2-one derivatives, which have led to the identification of 3b, and expand these studies to elucidate the principal structural and stereochemical features needed to achieve effective NAAA inhibition. Investigations on the influence of the substitution at the β-position of the 2-oxo-3-azetidinyl ring as well as on the effect of size and shape of the carbamic acid ester side chain led to the discovery of 3ak, a novel inhibitor of human NAAA that shows an improved physicochemical and drug-like profile relative to 3b. This favourable profile, along with the structural diversity of the carbamic acid chain of 3b, identify this compound as a promising new tool to investigate the potential of NAAA inhibitors as therapeutic agents for the treatment of pain and inflammation.


Introduction
Identifying novel molecular targets for the treatment of pain and inflammation is of pivotal therapeutic importance and remains a major challenge for researchers in industry and academia [1]. Since its discovery in 2005 [2], the intracellular cysteine amidase Nacylethanolamine acid amidase (NAAA) has attracted increasing attention as a potential anti-inflammatory target, due to its essential role in the regulation of lipid-amide signalling at peroxisome proliferator-activated receptor-a (PPAR-a) in macrophages and other host-defence cells [2e8].
NAAA belongs to the N-terminal nucleophile (Ntn) family of enzymes and catalyses the deactivating cleavage of saturated and monounsaturated fatty acid ethanolamides (FAEs) into the corresponding free fatty acids and ethanolamine [3,4,9,10], thus interrupting their peroxisome proliferator-activated receptorea (PPARa) mediated actions. NAAA is primarily localized to macrophages [11] and B-lymphocytes [8] and, like other Ntn enzymes, is activated by auto-proteolysis, which occurs at acidic pH and generates a catalytically competent form of the enzyme [12]. The FAEs are a class of bioactive lipids [13e15]that contribute to the control of numerous physiological functions, including pain and inflammation [16e19]. Among these lipids, NAAA preferentially degrades palmitoylethanolamide (PEA) and oleoylethanolamide (OEA) [2e4]. PEA is produced by many mammalian cells [20], and has been shown to inhibit peripheral inflammation and mast cell degranulation [21,22] and to exhibit antinociceptive properties in rat and mouse models of acute and chronic pain [23e25]. In addition, evidence suggests that PEA may attenuate skin inflammation and neuropathic pain in humans [26e28]. These effects are mainly dependent on the ability of PEA to activate PPARea [19,29e31]. Recent studies have shown that sustaining endogenous PEA levels in vivo by protection from NAAA-catalysed degradation attenuates lung inflammation [8] and hyperalgesic and allodynic states elicited in mice and rats by local inflammation or nerve damage [6,32].
However, a-amino-b-lactone derivatives have limited chemical and plasma stability [36,40,41], due to the hydrolytic cleavage of the b-lactone ring, which restricts their in vivo use to topical applications [6,35]. To overcome the limited stability of this class of inhibitors, we investigated compounds in which an a-amino-blactam (3-aminoazetidin-2-one) group replaces the b-lactone ring. These compounds retain key structural features of the b-lactone series, and showed good human-NAAA (h-NAAA) inhibitory activity and favourable physicochemical properties for systemic administration [42]. In particular, N-[(S)-2-oxoazetidin-3-yl]nonanamide (2, Fig. 1) displayed an acceptable inhibitory potency (IC 50 ¼ 0.34 mM) against h-NAAA, good stability in buffer (PBS at pH 7.4 and 5.0) and in mouse and rat plasma, and good oral bioavailability after single oral administration in rats [42].
This prompted us to investigate further the structural and stereochemical features of this new series of derivatives, leading after a modification on the carbamic acid side chain of 3a to the discovery of 4-cyclohexylbutyl-N-[(S)-2-oxoazetidin-3-yl]carbamate (ARN726, 3b, Fig. 1), as a novel and potent NAAA inhibitor [8]. Compound 3b inhibited NAAA activity both in vitro and in vivo as demonstrated by a competitive activity-based protein profiling (ABPP) study with a structurally similar chemical probe [44]. Consistent with those findings, 3b exerted marked antiinflammatory effects in mouse models of inflammation and suppressed LPS-stimulated inflammatory reactions in human macrophages [8].
In the present work, we describe the structureeactivity relationship (SAR) work that has led to the identification of b-lactam 3b and the elucidation of key structural and stereochemical features needed to achieve h-NAAA inhibition with this class of compounds.
In particular, we investigated the influence of the substitution at bposition of the 2-oxo-3-azetidinyl ring as well as the effect of the size and shape of the carbamic acid ester side chain. We also outline the selectivity, stability, and solubility of the best among these novel NAAA inhibitors, which might aid further experimental investigations of the roles of NAAA.

Chemistry
The synthesis of differently substituted b-lactam carbamic acid esters was efficiently accomplished in an enantio-and diasteroselective fashion by a general strategy consisting of coupling an appropriate 3-amino-b-lactam core with a desired activated alcohol. Initially, a series of b-lactam carbamates bearing a 4cyclohexylbutyl side chain (3be10b) was synthesized as reported in Scheme 1.
Mesylation of 26 gave compound 30, which was cyclized under basic conditions (K 2 CO 3 ) to afford the desired 2-azetidinone (34) in high yields (73%, over 2 steps). Selective N-methoxy deprotection was carried out using a fresh solution of samarium iodide, leading to the formation of the b-methyl b-lactam 38 [49]. Cbzdeprotection of 38 and in situ trapping of the resulting 2-methyl-3-aminoazetidin-4-one with p-toluensulfonic acid gave the tosylate salt 16 in quantitative yield (Scheme 2).
Tosylate salts 17e19 were stereoselectively obtained using the same procedure, starting either from the commercially available N- Cbz-D-threonine or the synthesized L-and D-N-Cbz-allo-threonine (24 and 25), respectively (Scheme 2) (see Supporting Information).
Alcohols 11k,o,q were obtained in moderate to good yields starting from the corresponding aldehydes (47e49) via Wittig olefination with a benzyl-protected phosphonium salt, followed by a one-pot reduction/deprotection reaction, using Pd(OH) 2 in the H-Cube flow reactor (Scheme 5). A similar synthetic strategy was used for compound 11m, where cyclopentanecarbaldehyde was reacted with 2-(1,3-dioxolan-2-yl)-ethyl-(triphenyl)-phosphonium bromide to give olefin 53 (7:93 E/Z ratio). Reduction of the derivative 53, followed by dioxolane cleavage, afforded aldehyde 55 which upon reduction with NaBH 4 furnished the desired compound (Scheme 5). The synthesis of alcohols 11x,ag was accomplished using minor modifications of a protocol reported by Fu and co-workers (Scheme 6) [59].
Suzuki coupling between bromocyclopentane and 4methoxycarbonyl-phenyl boronic acid, using NiI 2 as a catalyst, furnished isopropyl ester 56, which was directly reduced with LiAlH 4 to give alcohol 11x. 4-Phenyl-tetrahydropyranyl derivative 57, obtained via Suzuki reaction between phenyl boronic acid and 4-chlorotetrahydropyran, was regioselectively acylated via a Frie-deleCrafts reaction [60] and then subjected to LiAlH 4 reduction to give compound 11ag. O-Alkylation of ethyl 4-hydroxybenzoate with 1-bromopropane led to derivative 59, which upon reduction of the ethyl ester under standard conditions gave the alcohol 11ai. Finally, alcohol 11am was obtained in a reasonable yield (27%) after a three steps sequence, which consisted in the formation of the oxazole ring starting from ethyl 4-acetylbenzoate followed by LiAlH 4 reduction of the ethyl ester (Scheme 6) [61,62].

Results and discussion
In a previous study on NAAA inhibitors we reported that aamino-b-lactam (3-aminoazetidin-2-one) amide derivatives display a set of pharmacological and physicochemical properties that render them suitable for systemic administration, overcoming the limited hydrolytic and plasma stability of a-amino-b-lactonebased NAAA inhibitors, such as ARN077 (1, Fig. 1) [42].
While the increase in potency observed with compound 3c is in accordance with the SAR observed in a-amino-b-lactone carbamate derivatives, where long alkyl chains are preferred over short ones [38], the detrimental effect of a 5-phenylpentyl or a p-biphenylmethyl group differentiates the SAR of b-lactam carbamate derivatives from that of the corresponding b-lactone series. We then explored the replacement of the long unsubstituted alkyl chain with a short, u-cycloalkyl-substituted chain, a moiety that led to potent NAAA inhibitors in the a-amino-b-lactone series [37,38]. The introduction of a 4-cyclohexylbutyl group yielded compound 3b, which allowed recovering double-digit nanomolar potency, while reducing the length of the alkyl chain. This compound was preferred over 3c for conducting further studies because available data on a-amino-b-lactone and a-amino-b-lactam derivatives showed that molecules bearing a long alkyl chain have lower selectivity for NAAA versus acid ceramidase, a cysteine amidase showing 33e34% identity and 70% similarity to NAAA and catalyses the hydrolysis of ceramide, a pro-inflammatory lipid messenger [63,64]. Compound 3b was shown to block NAAA covalently via Sacylation of the catalytic cysteine residue in in vitro experiments, to suppress lung inflammation in mice via a mechanism that requires PEA-and OEA-mediated PPARea activation, and to prevent endotoxin-induced inflammatory responses in human macrophages [8].
Encouraged by these results, we undertook a SAR study in order to elucidate the stereochemical and structural requirements necessary for NAAA inhibition by compound 3b and a series of analogues. We explored both the influence of the b-substitution on the b-lactam ring and the effect of modifications on the carbamic side chain, taking into account structural changes such as the introduction of heteroatoms, the length of the side-chain, and the insertion of conformationally constrained moieties.
A small set of representative compounds was also evaluated for selectivity, and physicochemical and drug-like properties, such as solubility in buffer, and mouse and rat plasma and liver microsomal stability ( Table 5).
As a first step in our study, we modified the a-amino b-lactam scaffold, while maintaining fixed the 4-cyclohexylbutyl carbamate side chain. We explored the role of the stereochemistry at a-position and the effect of mono/di-substitution at b-position of the azetidinone ring.
A strong stereo-recognition at the NAAA active-site was clearly proved with compound 4b, the (R)-enantiomer of 3b, which showed a 40-fold drop in activity (IC 50 ¼ 3.11 mM, Table 2) compared to its stereoisomer, in agreement with our previous observation with the 3-aminoazetidin-2-one amide derivative 2 and its enantiomer [42]. The importance of the (S)-configuration at a-position for inhibition of NAAA was also confirmed in vivo, as 4b showed no significant anti-inflammatory effect in mouse models in  contrast to its enantiomer 3b [8].
Then, as a-amino b-lactones bearing a methyl group in b-position of the lactone ring showed increased NAAA inhibition compared to the corresponding unsubstituted derivatives [35,38], we turned our attention to such modification on the b-lactam core.
This first set of modifications of the 3-azetidinone core showed that the configuration of the two stereogenic centers and the substitution at b-position play a key role in the modulation of the potency of these analogues.
Taking into account these results and our previous findings, showing that the b-lactam core is mandatory for the activity [42], we continued the SAR expansion on b-unsubstituted b-lactam carbamic acid esters. In particular, we decided to synthesize novel analogues of compound 3b to investigate the effects of the length and conformational flexibility of the aliphatic chain as well as of the insertion of heteroatoms (Table 3).
On the basis of the results of the SAR studies on the b-lactone carbamate [37,38] and b-lactam amide [42] classes, we learned that NAAA inhibition could be affected by varying the length of the aliphatic side chain. A limited exploration of this structural feature was carried out by decreasing (3f) or increasing (3g) the linear chain of carbamate 3b by a methylene unit. Unexpectedly, both compounds turned out to be slightly more potent (3f: Table 3) than 3b, differently to what we observed in the b-lactone carbamate series [37,38].
Next, we tested whether the replacement of a carbon atom in the side chain with a heteroatom might be beneficial for both inhibitory activity and physicochemical properties. Disappointingly, the insertion of an oxygen atom in b-(3h), a-(3i) or 4'-position (3j) of the cyclohexyl ring was not tolerated, as all the compounds showed only micromolar activity against NAAA (3h: IC 50 ¼ 1.28 mM; 3i: IC 50 ¼ 2.67 mM, 3j: IC 50 ¼ 9.51 mM, Table 3 Switching the 4-cyclohexylbutyl chain of 3b into a more conformationally rigid 4'-butylcyclohexanol-derived carbamate, as in analogue 3l, resulted only in a limited reduction of NAAA inhibition (IC 50 ¼ 0.32 mM).  To further evaluate the role of the terminal cyclohexyl ring, we replaced this group with either a smaller cyclopentyl residue (3m) or a planar phenyl ring (3n). Whereas a slight improvement in activity was observed for b-lactam 3m (IC 50 ¼ 0.033 mM), the phenyl substituted derivative displayed a decrease in potency of ca.
15-fold (3n: IC 50 ¼ 1.11 mM). To test whether substituents on the terminal phenyl ring could recover potency, we synthesized compounds 3oeq featuring moieties with different stereo-electronic properties. Although the 4-ethyl (3o) and 4-methoxy (3p) substituted phenyl derivatives turned out to be more active than the corresponding unsubstituted analogue 3n (IC 50 ¼ 0.23 mM and IC 50 ¼ 0.52 mM, respectively), their activity was still 3-to 8-fold lower than that of the cyclohexyl substituted carbamate 3b. Interestingly, the insertion of a 4-trifluoromethyl group (3q) was well tolerated, showing similar activity to 3b (IC 50 ¼ 0.073 mM). This recovery of activity for compound 3q could be explained by the more lipophilic nature of the trifluoromethyl group, which possibly   Table 3).
Overall, the results reported in Table 3 indicated a clear preference for lipophilic, linear side chains, and highlighted a limited possibility to capture further interactions by introducing heteroatoms. These outcomes further support the idea that lipophilic moieties, which resemble the enzyme natural substrate, are preferentially accommodated in the NAAA active site.
To obtain additional insights into the SAR of this novel chemical class, we evaluated the influence of the flexibility of the aliphatic side chain on NAAA inhibition. We explored the possibility to fix the 4-cycloexylbutyl moiety of 3b in a more rigid conformation and then we examined a series of less conformationally flexible analogues (Table 4).
First, we replaced the 4-cyclohexylbutyl moiety with a metacyclohexyl substituted benzyl carbamate, as in b-lactam 3u. This modification turned out to be detrimental for NAAA inhibition (IC 50 ¼ 1.72 mM), leading to a 25-fold drop in potency compared to 3b. To assess the effect of regiochemistry for this type of substitution, ortho-(3v) and para-(3w) regioisomers were synthesized and tested. While compound 3v displayed inhibitory activity only in the high micromolar range (IC 50 ¼ 12.83 mM), the para-cyclohexyl benzyl carbamate 3w exhibited a slight increase in potency (IC 50 ¼ 0.041 mM) with respect to the more flexible analogue 3b.
The finding that a para-substituted benzyl group retained a higher NAAA inhibitory activity compared to the corresponding ortho-and meta-isomers prompted us to focus our subsequent efforts on the synthesis of para-substituted benzyl carbamates. We evaluated the role of the ring size and type, the influence of linear alkyl substituents, and the combination of these changes with a simultaneous introduction of heteroatoms (Table 4). While a para-cyclopentyl ring (3x) showed an IC 50 value comparable to the para-cyclohexyl analogue (IC 50 ¼ 0.071 mM), the replacement of the cyclohexyl with a planar phenyl ring, as in 3e, resulted in a ca. 13-fold drop in activity (IC 50 ¼ 0.51 mM), as previously reported (Table 1) and in agreement with the analogous reduction in NAAA inhibition observed with b-lactam 3n versus 3b (Table 3). These outcomes clearly indicate that the presence of a terminal unsubstituted phenyl ring in the carbamic side chain is only marginally tolerated due to either shape or conformational reasons.
The replacement of the para-cyclohexyl moiety of 3w with short linear alkyl substituents (3yeab, Table 4) led to a progressive increase in inhibitory activity by extending the length of the alkyl chain from 1 to 4 methylene units (IC 50 ¼ 1.04 mM-0.023 mM). In particular the n-butyl substituted b-lactam 3ab displayed the highest potency (IC 50 ¼ 0.023 mM) within this small set of analogues. The replacement of the n-butyl moiety with a bulkier tbutyl group was not well tolerated, leading to a ca.13-fold drop in potency for b-lactam 3ac (IC 50 ¼ 0.31 mM) with respect to 3ab.
These observations are consistent with our previously findings on NAAA inhibitors from different chemical classes [34,38,42], in which derivatives with linear alkyl side-chains exhibited in general good to high potency.
Then, we turned our attention to carbamates possessing fused rings, such as the bicyclic tetralinyl-(3ad) and indanyl-derived (3ae) analogues. These compounds showed quite good potency (IC 50 ¼ 0.14 mM and 0.13 mM, respectively), although being slightly less active than the corresponding ring-opened derivatives 3ab and 3aa (3-to 6-fold drop in potency).
To further map the region of the enzyme occupied by the carbamate moiety and the adjacent carbon atom, we evaluated the effect of the substitution of the benzylic position in the potent blactam 3w. The introduction of a methyl group close to the carbamic function, as in 3af, turned out to be detrimental for potency (IC 50 ¼ 4.47 mM). Although compound 3af is a mixture of diastereoisomers, which could have a substantial difference in potency [38], the observed drastic drop in activity for 3af with respect to 3w appears to indicate a limited space in this region of the enzyme active site, at least for this type of derivatives.
After the identification of conformationally constrained derivatives with good activity, we explored the replacement of one or more carbon atoms of the carbamic side chain with an oxygen in the attempt to further improve potency and modulate physicochemical properties. Unfortunately, in analogy with the results obtained with the linear aliphatic derivatives (3hej , Table 3), the introduction of an oxygen atom in the side chain negatively affected potency. Although compounds 3ag and 3ah showed a 34-to 50fold reduction in NAAA inhibition (3ag: IC 50 ¼ 1.37 mM; 3ah: Table 5 Inhibitory potencies (IC 50 ) on h-NAAA and h-AC, % inhibitory activity (%Inhib.) on h-FAAH, mouse and rat plasma and microsomal stability (t ½ ), and solubility of compounds 3b,k,w,ab,ai,ak.
Notably, even if an evident loss in potency was observed for benzyl-1,3-dioxolane carbamate 3aj (IC 50 ¼ 2.19 mM), compared to compound 3ae, the insertion of a gem-difluoro moiety on the acetal carbon atom (3ak) restored NAAA inhibition in the low nanomolar range (IC 50 ¼ 0.085 mM). As seen for b-lactam 3k, we speculate that the increased lipophilicity of the gem-difluoromethylene group [65] may allow establishing additional interactions within the NAAA active site, thus increasing potency, which are somehow lost with the sole introduction of polar atoms. The replacement of the para-cyclohexyl moiety in compound 3w with a 3-pyridyl (3al, IC 50 ¼ 8.1 mM) or a 4-oxazolyl (3am, IC 50 ¼ 1.88 mM) group confirmed the negative effect of hetero-aryl substitution at least in this region, as already seen for para-phenyl derivative 3e.
A selection of structurally diverse b-lactam carbamic acid esters (3b,k,w,ab,ai,ak) identified in our SAR investigation was then tested for selectivity against human Fatty Acid Amide Hydrolase (h-FAAH), a member of the serine hydrolase family which can cleave FAEs [66], and human Acid Ceramidase (h-AC) [63]. The selectivity of the selected compounds versus h-AC was evaluated using a UPLC-MS-based assay [64], in order to measure IC 50 values under similar experimental conditions ( Table 5).
As previously reported for rat AC [8], compound 3b showed a high selectivity also versus h-AC (ca. 230-fold), having an activity only in the medium micromolar range (IC 50 ¼ 6.27 mM). This poor affinity towards h-AC was also maintained by its close gemdifluoro-analogue 3k, which displayed an IC 50 of 10.49 mM.
Disappointingly, the introduction of a conformationally constrained benzylic group on the carbamate moiety, favourable for h-NAAA inhibition, led to a good activity on h-AC. As a result, compounds 3w,ab,ai revealed a modest h-NAAA versus h-AC selectivity ratio (<130-fold, Table 5). Remarkably, the difluoro-benzodioxol substituted analogue 3ak was only a medium micromolar h-AC inhibitor (IC 50 ¼ 8.09 mM, Table 5), therefore recovering a high selectivity (>370-fold).
At the tested concentration (10 mM), none of the selected blactam carbamates inhibited h-FAAH in a significant manner ( Table 5).
The physicochemical and drug-like profiles of b-lactams 3b,k,w,ab,ai,ak were examined next (Table 5), evaluating their kinetic solubility in buffer (PBS, pH 7.4) and their plasma and metabolic stability in both mouse and rat plasma and liver microsomes, respectively. The carbamate derivatives 3b, was fairly soluble (144 mM), but showed a low metabolic (t ½ < 5 min in both species) and plasma stability (t ½ ¼ 12 min in rat; t ½ ¼ 41 min in mouse) [8].
The gem-di-fluoro substitution on the cyclohexyl ring, as for 3k, significantly improved the metabolic stability in mouse microsomes (t ½ ¼ 34 min) compared to 3b. This outcome would suggest the terminal cyclohexyl moiety in the side chain of 3b as a possible soft spot for oxidative metabolism in this species.
The substitution at the para position of the benzylic carbamic acid ester chain, as for compounds 3w and 3ab, positively affected the rat plasma stability (t ½ ¼ 104 min and >120 min, respectively), but negatively influenced the solubility (51 mM and 83 mM) and did not have any substantial effect on mouse plasma and on mouse and rat liver microsomal stability, which remained still quite low in both species (t ½ < 5 min). Reasonably, these data indicated an overall positive contribution of the benzylic substitution adjacent to the carbamic moiety on rat plasma stability, therefore reducing a possible hydrolytic cleavage of such electrophilic compounds in this species. The introduction of an oxygen atom in the linear alkyl chain of 3ab improved the solubility of the resulting compound, 3ai (>250 mM), maintained a good plasma stability in rat (t ½ >120 min), but did not ameliorate the high oxidative metabolism in both species.
The results reported above reasonably indicate that a carbamic acid side chain bearing an adjacent, substituted benzylic moiety could help improving plasma stability (especially in rat, 3k,ab vs. 3b), whereas the introduction of a gem-difluoro group in specific positions on the side chain could positively modulate the microsomal stability (3k vs. 3b). These outcomes were somehow confirmed for b-lactam 3ak, which altogether encompassed these structural requirements. In fact, to our gratification, this novel analogue showed excellent solubility (>250 mM) and good stability properties in rat (plasma t ½ > 120 min; micrososomal t ½ ¼ 40 min) coupled with a good activity and selectivity profile ( Table 5).

Conclusions
In the present work, we outline the key structural modifications that led to the identification of 4-cyclohexylbutyl-N-[(S)-2oxoazetidin-3-yl]carbamate (3b), as a potent, selective and systemically active inhibitor of intracellular NAAA activity. Furthermore, we expanded our previous SAR investigation on 3aminoazetidin-2-one derivative NAAA inhibitors. We structurally modified compound 3b with the aim of defining the relevant chemical features needed to achieve both good NAAA inhibition and suitable physicochemical and drug-like properties. We investigated primarily the effect of the size and shape of the carbamic acid ester side chains, as well as the influence of the substitution at b-position of the 2-oxo-3-azetidinyl ring. The studies demonstrated the importance of the configuration of the stereogenic center at position 3 and the detrimental effect on potency of b-substitution on the b-lactam ring. While the modifications of the linear lipophilic chain, in terms of the insertion of heteroatoms or substitution of the terminal ring, resulted in no or only minor beneficial effects on NAAA inhibition, the replacement of the 4-cyclohexylbutyl chain of compound 3b with substituted benzylic moieties turned out to be important to modulate the potency and the physicochemical profile. Thorough SAR explorations of the substitution patterns on the benzylic side-chains (site, type and shape of modifications, and insertion of heteroatoms) led to the discovery of 3ak, a novel human NAAA inhibitor with a good activity and an improved stability profile with respect to 3b. The structural diversity of the carbamic acid chain and the good physicochemical properties render compound 3ak a promising tool, which might help to further investigate the potential of NAAA inhibitors as new therapeutic agents for the treatment of pain and inflammatory conditions. The pharmacological profile of 3ak is currently under evaluation, and data will be reported in due course.

Chemistry
All the commercial 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 Sig-maeAldrich. Automated column chromatography purifications were done using a Teledyne ISCO apparatus (CombiFlash ® R f ) with pre-packed silica gel columns of different sizes (from 4 g up to 120 g). Mixtures of increasing polarity of cyclohexane and ethyl acetate (EtOAc), cyclohexane and methyl tert-butyl ether (MTBE) or dichloromethane and methanol (MeOH) were used as eluents. Hydrogenation reactions were performed using HeCube ® continuous hydrogenation equipment (SSereaction line version), employing disposable catalyst cartridges (CatCart ® ) preloaded with the required heterogeneous catalyst. Microwave heating was performed using Explorer ® e48 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 Zgradients. Spectra were acquired at 300 K, using deuterated dimethylsulfoxide (DMSOed 6 ) or deuterated chloroform (CDCl 3 ) as solvents. UPLC/MS analyses were run on a Waters ACQUITY UPLC/ MS system consisting of a SQD (single quadrupole detector) mass spectrometer equipped with an electrospray ionization interface and a photodiode array detector. The PDA range was 210e400 nm. Analyses were performed on an ACQUITY UPLC BEH C 18 column (100 Â 2.1mmID, particle size 1.7 mm) with a VanGuard BEH C 18 precolumn (5 Â 2.1 mmID, particle size 1.7 mm). Mobile phase was 10 mM NH 4 OAc in H 2 O at pH 5 adjusted with CH 3 COOH (A) and 10 mM NH 4 OAc in CH 3 CNeH 2 O (95:5) at pH 5.0. Electrospray ionization in positive and negative mode was applied. ESI was applied in positive and negative mode. Purifications by preparative HPLC/MS were run on a Waters Autopurification system consisting of a 3100 single quadrupole mass spectrometer equipped with an Electrospray Ionization interface and a 2998 Photodiode Array Detector. HPLC system included a 2747 sample manager, 2545 binary gradient module, system fluidic organizer and 515 HPLC pump. PDA range was 210e400 nm. Purifications were performed on a XBridge™ Prep C 18 OBD column (100 Â 19 mmID, particle size 5 mm) with a XBridge™ Prep C 18 (10 Â 19 mmID, particle size 5 mm) guard cartridge. Mobile phase was 10 mM NH 4 OAc in MeCNeH 2 O (95:5) at pH 5. Electrospray ionization in positive and negative mode was used. Accurate mass measurement (HMRS) was performed on a Synapt G2 quadrupole-Tof instrument (Waters, USA), equipped with an ESI ion source. All tested compounds (3beam, 4be10b) showed ! 95% purity by NMR and UPLC/MS analysis. Off-white solid. Experimental procedure and 1 H NMR are according to literature [42].