Prolyl oligopeptidase inhibitors

Abstract

Provided is a compound of formula 1, wherein X is O or S and R is an electrophilic warhead group that comprises nitrile, a boronic ester or an a-ketoamide motif. Said compound is a prolyl oligopeptidase inhibitor that is useful in the treatment of Alzheimer's disease or Parkinson's disease and for inhibiting autophagy. (I)

Description

PROLYL OLIGOPEPTIDASE INHIBITORSCROSS-REFERENCE TO A RELATED APPLICATION

[0001] This disclosure claims priority from U.S. provisional application 63 / 754,025 filed February 5, 2025, which is incorporated herein by reference in its entirety.TECHNICAL FIELD

[0002] This disclosure relates to the field of prolyl oligopeptidase inhibitors, methods of making same and methods of using same.BACKGROUND OF THE ART

[0003] Prolyl oligopeptidase (POP, PREP) is a serine endoprotease of the S9 family which hydrolyzes peptides at the C-terminus of proline residues — and less commonly — alanine residues. This enzyme was first isolated from human uterine tissue over fifty years ago, it was then known as the post-proline cleaving enzyme. Its longstanding association with cognitive functions by neuropeptidase activity had been suggested through several earlier studies. The inhibitory potential was investigated using proline-containing peptides, of which the now known Z-Pro-Prolinal (Kj = 0.35 nm, see Formula A) was found to be highly potent. Follow-up studies inspired by this particular scaffold resulted in the fruition of covalent and non-covalent inhibitors such as JTP-4819 (Kj = 0.055 nm, see Formula B), KYP-2047 (Kj = 0.023 nm see Formula C), SUAM-1221 (Kj = 43.0 nm see Formula D), and S-17092 (Kj = 1.5 nm see Formula E).

[0004] Recent studies involving POP inhibitors were found to reduce levels of a-synuclein, a protein associated with Parkinson’s disease (PD). In addition to its effect on, and co-localization with a-synuclein, POP has been found to be co-localized with p-amyloid, another marker of Alzheimer’s disease (AD) and PD. Later studies showed that POP inhibitor (KYP-2047) could decrease levels of a-synuclein aggregation and promote clearance thereof and restore motor behavior in mouse models.

[0005] More recently, the underlying protein-protein interactions (PPIs) surrounding POP have been further elaborated, highlighting its regulatory role in autophagy and ROS production via interaction with protein phosphatase 2A (PP2A).

[0006] Accordingly, improvements in POP inhibitors are still desired, particularly it would be highly desired to be provided with an inhibitor that has improved efficacy and that is capable of crossing the blood brain barrier to treat AD and PD.SUMMARY

[0007] In one aspect, there is provided a compound of formula 1:wherein X is O or S, and R is an electrophilic covalent warhead group.

[0008] In some embodiments, R is a warhead group that comprises a nitrile, a boronic ester or an a-ketoamide motif.

[0009] In some embodiments, the compound is of formula 2:

[0010] wherein X is as defined above in formula 1.

[0011] In some embodiments, the compound is of formula 3:

[0012] wherein X is as defined in above in formula 1 and each R2is independently H or -O- CH3.

[0013] In some embodiments, the compound is selected from the group consisting of:

[0014] In one aspect, there is provided a pharmaceutical composition comprising a compound as defined herein and a pharmaceutically acceptable excipient.

[0015] In a further aspect, there is provided the compound as defined herein for the treatment of Alzheimer’s disease or Parkinson’s disease. In a further aspect, there is provided the compound as defined herein for inhibiting autophagy.

[0016] Many further features and combinations thereof concerning the present improvements will appear to those skilled in the art following a reading of the instant disclosure.DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1 is a schematic showing the docking pose in the POP active site of an inhibitor having a [5,5]-fused thiazolidine substituted by -NH-C(O)-O-Benzyl (see Formula F).

[0018] FIG. 2 is a schematic showing the docking pose in the POP active site of an inhibitor having a [5,5]-fused thiazolidine substituted by -O-Benzyl (see Formula 5).

[0019] FIG. 3 is a schematic showing the docking pose in the POP active site of an inhibitor having a [5,5]-fused oxazolidine substituted by -O-Benzyl (see Formula 8).

[0020] FIG. 4 is a schematic showing the crystal structure of compound 15a.

[0021] FIG. 5 is a schematic showing the crystal structure of compound 17b.

[0022] FIG. 6 is a graph showing the average enzyme activity in function of the log of the concentration of the inhibitor (compounds 15a, 15b, 31a, and 31b).

[0023] FIG. 7 is a graph showing the average enzyme activity in function of the log of the concentration of the inhibitor (compounds 17a, 17b and 25).

[0024] FIG. 8 is a bar graph selectivity profile of compound 31a against related serine proteases (fibroblast activation protein (FAP)) and dipeptidases (dipeptidyl peptidase (DPP)).DETAILED DESCRIPTION

[0025] It was previously thought that an optimal docking pose to inhibit POP enzyme can be achieved with a [5,6]-fused heterocycle scaffold. [5,5]-fused heterocycle-based inhibitors such as the compound of formula F were previously dismissed and abandoned because they were oxidized by cytochrome P450. The oxidation products led to subsequent isomerization into complex mixtures of metabolites with unknown pharmacodynamic properties. This was an undesirable result. However, it was surprisingly found that a modified [5,5]-fused thiazolidine ring bicyclic core exhibited additional steric and / or electronic constraints, to overcome the previously observed disadvantages with [5,5]-fused heterocycles.

[0026] The inhibitors (formulas A to E) are flexible molecules when it comes to the movement of the cyclic rings. In other words, because these compounds have at least two cyclic rings that are linked by a flexible bond, there can be many three-dimensional conformations for these compounds. For example, when considering formulas A and C which are some of the most potent POP inhibitors reported to date, one could see two potential sites of rigidification. If Cbz-Pro-Prolinal (Formula A) or KYP-2047 (Formula C) are taken as template, one could rigidity by means of a bicyclic core, either the left, or right, side of these flexible ligands. These approaches have already been done and compounds of formula F (Kj = 200-700 nm, human brain), formula G (Kj = 45 nm, human brain), formula H (Kj = 2.1), and formula I (Kj = 1.0) have been developed.

[0027] As such, rigidification of the left side has resulted in potent inhibitors demonstrating single digit nanomolar activity. Furthermore, the bicyclo-[4.3.0]-aromatic and aliphatic scaffolds (formulas G and I) that contained an additional proline ring saw retention of the 1 ,4-dicarbonyl motif, albeit on the righthand side. As a result, key hydrogen bonding interactions were maintained and both series were favorably accommodated in the POP active site. Unfortunately, studies on compound of formula F using liquid chromatography tandem mass spectrometry had shown oxidation of the sulfur atom by P450 enzymes in liver microsomes leading to reactive metabolites. One issue with compound G was that it had a low-medium clearance in human liver microsomes (HLM), and medium clearance in rat liver microsomes (RLM). Compounds H-l were tested in cellule, but did not show sufficient reduction in alpha-synuclein aggregation.

[0028] Compared to Formula F, it was presently found that [5,5]-fused heterocycle with a smaller substitution (-O-Benzyl instead of -NH-C(O)-O-Benzyl) provided a better fit into the catalytic pocket, a better inhibitory activity and an improved metabolic stability. Accordingly, there is presently provided a compound of formula 1 :

[0029] where X is O or S and R is a warhead group that forms a covalent bond with the nucleophilic residue Ser554 in the catalytic site of POP. In some embodiments, the warhead group comprises or is nitrile, a boronic ester, a boronic acid, an epoxide, a p-lactam, an oxetane substituted by a nucleophile (such as =0) or an a-ketoamide motif. It should be noted that the boronic ester becomes a boronic acid when exposed to aqueous medium.

[0030] The compound of formula 1 has been synthesized by attaching a pyrrolidine featuring a warhead (e.g. nitrile or boronate ester moiety) to the scaffold via peptide coupling with an appropriately substituted pyrrolidine ring.

[0031] Acyclic inhibitors have a higher level of clearance compared to the cyclic inhibitors, thus the present cyclic inhibitors have a higher metabolic stability in comparison. A preferred embodiment is when X is O because that eliminates sulfur oxidation by P450 enzymes leading to potential reactive metabolites.

[0032] It was presently found that electrophilic warhead groups such as the nitrile and boronic acid warheads have an increased residence time in the active site of the catalytic pocket compared to their non-covalent counterparts. Covalent warheads feature an additional binding mode which gives rise to a two-step binding process constituting a non-covalent enzyme inhibitor complex (El), followed by a covalent ligand-enzyme complex (E-l), that is responsible for this requisite feature. Without wishing to be bound by theory, it is believed that boronic acids have higher residence times due to their ability to mimic the natural conformation of a tetrahedral peptide intermediate compared to trigonal planar adopted nitriles.

[0033] In some embodiments, the compound of the present disclosure is selected from:

[0034] where X is O or S, and each R2 is independently H or -O-Me.

[0035] In some embodiments, the compound of the present disclosure is selected from:

[0036] Thanks to their activity as inhibitors of POP, the compounds of the present disclosure may be used in the treatment, prevention or alleviation of symptoms for conditions of POP hyperactivity (e.g. when POP activity is upregulated or when POP is overproduced). These conditions associated with POP activity can for example be AD and / or PD. Since POP is associated with autophagy, the present compounds may also be used to promote autophagy.

[0037] It should be noted that the carbon chiral center that links the -O-benzyl to the fused bicyclic ring in all the compounds contemplated herein (compounds 1-9 and 10a-10f) could be either stereoisomer (exo- or endo- to the ring). Indeed, both were synthesized (see Schemes 1 and 3 in the Example section). Therefore, the straight line can refer to either stereoisomer, a mixture thereof or a racemate. As an example, compound 1 could be compound 1a, compound 1b, a mixture thereof or a racemate thereof.

[0038] The Forecaster drug discovery platform including the docking program Fitted was used to design these intended molecules. To generate an inhibitor with promising protease activity, the molecule should satisfy three structural guidelines. The proline core which features the 1,4-dicarbonyl motif should be contained within the S1 and S2 specificity pockets, while a hydrophobic, often aromatic substituent, should align in the S3 specificity pocket. Thus, to improve the fit of the compound when considering the structure of Formula F, it was important to consider the length of the carboxybenzyl substituent group that extends out of the [5,5] bicyclic core. The docking studies were unsuccessful in predicting an appropriate docking pose with the carboxybenzyl group, which was satisfactorily improved by replacing this substituent with a benzyloxy group containing two less atoms. This subtle but important difference provided a pose that overlayed closely with the co-crystallized ligand (Figures 1 to 3). Figure 1 represents the [5,5]-fused thiazolidine inhibitor (formula F) with nitrile warhead overlayed with the co-crystallized ligand. Figures 2 and 3 represent the [5,5]-fused thiazolidine and oxazolidine inhibitors of the present disclosure in the POP active site.

[0039] Moreover, the acquired docking pose for the two inhibitors of Figures 2 and 3 suggested key hydrogen bonding interactions with Arg643 and Trp595 would be maintained. Hydrophobic interaction with Phe173 was also present which further highlighted the appeal of pursuing this particular set of molecules. The benzyloxy group with (S)-stereochemistry had a comparable fit in the active site relative to its (R)- diastereomer. The nitrile and boronate ester warheads requiring respective (S)- and (R)- stereochemistry adopted trigonal planar and tetrahedral intermediate conformations upon coordination by reactive nucleophilic residue Ser554 in the catalytic site. It should be noted that the presently used docking software is unable todistinguish between the inhibitory potential of different electrophilic groups within 1-2 orders of magnitude. Lastly, Tyr473 which is known to stabilize the tetrahedral intermediate by stabilization of charge via the oxyanion hole was present near the electrophilic warheads in the acquired docking pose. Taken together, by retaining the required non-covalent interactions in the presently proposed scaffold, it was envisioned that rigidifying the lefthand side of the bicyclic compound could be favorably accommodated into the active site. This rigidification strategy can also serve to improve metabolic stability and offer higher specificity over the corresponding acyclic analogues.

[0040] Following the simulation observations, the compounds were synthesized. All reagents were purchased from commercial suppliers and used without further purification unless otherwise stated. Thin layer chromatography (TLC) analysis was carried out on silica gel 60 F254 aluminium backed plates supplied by EMD Millipore Corporation and visualized by an ultraviolet lamp (A = 254 nm). Silica gel column chromatography was performed using SiliaFlash™ P60 Academic Silica Gel (40-63 pm). Nuclear magnetic resonance (NMR) spectra were collected on a Bruker™ Avance 400 MHz, 500 MHz or Qanuc 800 MHz instrument and all chemical shifts are reported as 5 values.1H NMR spectra were measured relative to the deuterated solvent and referenced to the residual solvent peak.13C NMR spectra were measured relative to the carbon signals of the deuterated solvent. Correlated spectroscopy (COSY), heteronuclear single quantum coherence (HSQC), and heteronuclear double bond coherence (HMBC) 2D correlation techniques were used for assignments in some cases. High resolution mass spectrometry was performed by Electrospray ionization (ESI) on a Bruker™ Maxis Impact API QqTOF or by ESI or atmospheric pressure chemical ionization (APCI) on a ThermoFisher Exactive Plus Orbitrap-API at McGill University. All tested compounds were at least 95% pure as determined by high performance liquid chromatography (HPLC) and / or NMR.

[0041] To synthesize the compounds of formula 1, first one needs to access the bicyclic core. The first step involved Sakurai-Hosomi conditions to install an allyl group onto ethyl glyoxylate using boron trifluoride as a Lewis-acid catalyst to generate 11.1 in good yields (see Scheme 1). The secondary alcohol was then benzylated under standard conditions (see Scheme 1) using benzyl bromide to provide 11.2 as a racemic mixture. It was observed that smaller reaction quantities (< 500 mg) often benefited from using silver oxide, whereas sodium hydride was used for larger-scale purposes. The olefin was then oxidatively cleaved, followed by reaction with L-cysteine methyl ester under buffered conditions. After aqueous workup, the reaction mixture was heated to reflux in toluene to complete the cyclization to afford 12a / b as a mixture of separablediastereomers. It is believed the bicyclic core is constructed from three steps of imine formation after the ozonolysis, followed by unfavorable 5-encfo-trig cyclization facilitated by the longer carbon-sulfur bond, then amidation by the transient secondary amine to displace the ethyl ester. The stereochemistry of the methine proton at the ring junction is directly influenced by the nature of the amino acid used, which often has a driving force to be exo- to the bicyclo[3.3.0] ring under thermodynamic conditions. It should be noted this protocol avoids the use of pyridine as required by the traditional Baldwin procedure. With the desired scaffold in hand, the methyl ester was hydrolyzed using lithium hydroxide in a biphasic mixture of tetrahydrofuran / water. Interestingly, no epimerization was observed for both diastereomers at carbonyl a-positions and also the ring junction. Carboxylic acids 13a / b were then coupled to either pyrrolidine or (S)-2-cyanopyrrolidine to furnish the non-covalent (14a / b) and covalent analogue containing the nitrile warhead (15a / b).In the case of the boronate ester, the carboxylic acid was first coupled to / V-hydroxy phthalimide via DCC-coupling. This route was favorable to optimize the costs associated with using 2-boroprolinepinanediol esters. Noteworthy, the coupling of activated ester 16a with a racemic mixture of 2-boroproline furnished only the desired (R)-diastereomer (17a), suggesting the topology of the molecule is influencing the chiral discrimination. Crystal structures obtained for 15a and 17b confirm the stereochemistry at the ring junction alongside the other chiral centres (Figures 4 and 5 respectively).Scheme 1.a) allyltrimethylsilane, BF3 ■ OEt2, 0 °C - rt, 3 h, 57%; b) BnBr, silver (I) oxide, diethyl ether, rt, 24 h, 40%; c) O3, DCM, -78 °C, 0.5 h then dimethyl sulfide, -78 °C - rt, overnight; ii) NaHCCh, L-cysteine methyl ester, EtOH / H2O, rt, 4 h; iii) toluene, 111 °C, 16 h, 30% (1:1 12a:12b); d) LiOH, THF / H2O, rt, 1 h, 86%; e) PivCI, DCM, DIPEA, 4A MS, pyrrolidine or (S)-2-cyanopyrrolidine tosylate, 0 °C - rt, 49 - 65%; f) N-hydroxyphthalimide, DCC, THF, 4A MS, 0 °C - rt, 2 h, 49%; g) (R)-2-pyrrolidineboronic acid pinanediol ester hydrochloride, DCM, DIPEA, 4A MS, 0 °C - rt, 1 h, 48 - 54%.

[0042] The required warhead for the a-ketoamide derivative (see Formula 4) was synthesized using the protocols as described in Scheme 2. Boc-L-proline was first coupled to commercially available stabilized ylide (triphenylphosphoranylidene)acetonitrile through 1 -Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) coupling to provide crude 18, which was subsequently oxidized to 19 via ozonolysis conditions. The methyl ester was hydrolyzed by use of lithium hydroxide to generate the a-keto acid 20, which undergone Hexafluorophosphate Azabenzotriazole Tetramethyl Uronium (HATLI) coupling with 3,4-dimethoxybenzylamine. Amide intermediate 21 was reduced to the corresponding a-hydroxyamide 22 prior to removal of the tert-butyloxycarbonyl-protecting group. It was found that this was necessary to avoid self-reactivity with the labile a-ketoamide motif.Scheme 2.a) EDC, DMAP, (triphenylphosphoranylidene)acetonitrile, DCM, 0°C- rt, 16 h; b) O3, DCM / MeOH, -78 °C, 40 min, 31% over 2 steps; c) LiOH, THF / H2O, rt, 3 h; d) HATU, DIPEA, 3,4-dimethoxybenzyl amine, DMF, rt, 16 h, 72% over 2 steps; e) NaBF , THF, rt, 0.5 h; f) TFA, DCM, rt, 1 h; g) 16a, DIPEA, DCM, 4 MS, O °C - rt, 30% over 3 steps; h) PDC, SiO2, DCM, rt, 2 h, 16%.

[0043] The generated pyrrolidine 23 was then coupled with activated ester 16a to provide precursor 24, which underwent oxidation with pyridinium dichromate in the presence of normalphase silica to furnish the desired a-ketoamide 25 in sufficient quantities (Scheme 2). Noteworthy, this same methodology was unable to afford the other diastereomer despite multiple attempts to couple the a-hydroxyamide warhead 23 with activated ester 16b.

[0044] An alternative route was required to construct the oxazolidine bicyclic system as a mixture of products was obtained with the initial methodology used to implement the fused thiazolidine ring. Use of L-serine methyl ester in place of L-cysteine methyl ester with either toluene or pyridine as a solvent and various time / temperature considerations were unsuccessful in affording the desired [3.3.0]-oxazolidine bicycle. A one-pot procedure was also investigated, but this also failed to furnish the requisite target molecule precursor. A strategy to overcome thislimitation was to pre-install the L-serine moiety to bias the mechanistic pathway to instead undergo 5-exo-trig cyclization via the hemiaminal intermediate (Scheme 3). Hence, intermediate 11 was hydrolyzed under standard conditions to provide the free acid. EDC activation of 26 and coupling with L-serine methyl ester in the presence of HOBt afforded the necessary acyclic system 27. It should be noted that while coupling with protected ferf-butyl serine methyl ester provides the acyclic precursor in higher yields, subsequent removal of the terf-butyl group under TFA conditions to reveal the primary alcohol were irreproducible. Hence, use of unprotected reactant provided 27 in higher purity, albeit in diminished yields. In a one-pot, three-step procedure, the olefin was oxidatively cleaved, followed by in-situ reduction and acid-catalyzed cyclization to satisfactorily afford the fused bicycle 28 as a mixture of inseparable diastereomers. After ester hydrolysis, carboxylic acid 29 was coupled with either pyrrolidine or (S)-2-cyanopyrrolidine to provide enantiopure non-covalent inhibitors 30a and 30b and covalent inhibitors 31a and 31b, which undergone biological testing alongside the sulfur inhibitors.Scheme 3.>a) allyltrimethylsilane, BF3■ OEt2, 0 °C - rt, 3 h, 57%; b) BnBr, silver (I) oxide, diethyl ether, rt, 24 h, 40%; c) THF, NaOH (aq), rt, overnight, 68% ; d) EDC, HOBt, DCM, NEfe, rt, 1 h, 50%; e) O3, DCM, -78 °C, 0.5 h then DMS, -78 °C - rt, overnight; ii) PhMe, 90 °C, 1 h; iii) PhMe, TFA, 50 °C, 2 h, 26% over 3 steps; f) THF, NaOH (aq), 0 °C, 1 h, quantitative; g) PivCI, DCM, DIPEA, 4 MS, pyrrolidine or (S)-2-cyanopyrrolidine tosylate, 0 °C - rt, 45-50%.Characterization of the compounds and detailed synthesis

[0045] Ethyl 2-hydroxypent-4-enoate (rac-11.1): A flame-dried 250 mL round bottom flask equipped with a magnetic stir bar was charged with ethyl glyoxylate (50% in toluene, 6.0 mL, 29.5 mmol), allyltrimethylsilane (9.6 mL, 60.4 mmol), anhydrous dichloromethane (150 mL) and 4 molecular sieves. The flask was flushed with argon then boron trifluoride diethyl etherate (7.2 mL, 58.3 mmol) was added dropwise at 0 °C. After stirring at room temperature for 3 h, the material was extracted against distilled water (50 mL) with dichloromethane (2 x 25 mL). The combined organic layers were washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The yellow oil was purified by column chromatography, eluting with a gradient of ethyl acetate in hexane (0:1 —> 1 :9) to afford the product as an off-white oil. Yield 57%. TLC (1:3 ethyl acetate in hexane): Rf= 0.43.1H NMR (500 MHz, CDCI3) 6 5.84-5.75 (m, 1H), 5.17-5.11 (m, 2H), 4.31-4.18 (m, 3H), 2.84 (d, J = 6.0 Hz, 1H), 2.59-2.53 (m, 1H), 2.46-2.39 (m, 1H), 1.28 (t, J= 7.2 Hz, 3H);13C NMR (100 MHz, CDCI3) 6 174.5, 132.6, 118.8, 70.1, 61.8, 38.8, 14.3 ppm.

[0046] Ethyl 2-(benzyloxy)pent-4-enoate (rac-11.2): In a 250 mL flame-dried round bottom flask equipped with a magnetic stir bar was dissolved the secondary alcohol (2500.0 mg, 17.3 mmol) in dry tetrahydrofuran (100 mL). Sodium hydride (60% in mineral oil, 1600.0 mg, 40.0 mmol) was added portionwise at 0 °C. After stirring for 30 minutes, benzyl bromide (2.1 mL, 17.7 mmol) and tetrabutylammonium iodide (640.0 mg, 1.73 mmol) were added on ice and the reaction was allowed to warm to room temperature and stirred for 24 hours. The excess sodium hydride was quenched with saturated ammonium chloride solution. The organic phase was separated and the contents were further extracted with dichloromethane (3 x 25 mL). The combined organic phases were washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The oil was purified by column chromatography, eluting with a gradient of ethyl acetate in hexane (0:100 — > 10:90) to afford a pale-yellow oil. Yield was 51%.

[0047] An alternative procedure can be used for smaller scale reactions using silver (I) oxide:

[0048] A flame-dried 50 mL round bottom flask equipped with a magnetic stir bar was charged with the secondary alcohol (225.0 mg, 1.97 mmol), diethyl ether (25 mL), and silver (I) oxide (913.0 mg, 3.94 mmol). The flask was flushed with argon then benzyl bromide (0.94 mL, 7.91 mmol) was added dropwise. The reaction was stirred at room temperature for 24 hours. The suspension was filtered under gravity. The oil was purified by column chromatography, elutingwith a gradient of ethyl acetate in hexane (0:100 — > 10:90) to afford a pale-yellow oil. Yield 40%. TLC (1:9 ethyl acetate in hexane): Rf= 0.41.1H NMR (500 MHz, CDCh) 6 7.39 - 7.28 (m, 5H), 5.88-5.80 (m, 1H), 5.18-5.08 (m, 2H), 4.72 (d, J= 11.8 Hz, 1H), 4.46 (d, J= 11.7 Hz, 1H), 4.27 - 4.17 (m, 2H), 4.00 (dd, J= 6.3, 6.3 Hz, 1H), 2.56 - 2.52 (m, 2H), 1.29 (t, J= 7.2 Hz, 3H) ppm;13C NMR (126 MHz, CDCh) 6 172.2, 137.6, 133.2, 128.5, 128.1, 128.0, 118.0, 77.9, 72.4, 61.0, 37.5, 14.4 ppm.

[0049] Methyl (3R,6S,7aS)-6-(benzyloxy)-5-oxohexahydropyrrolo[2,1-b]thiazole-3-carboxylate (12a-exo) and methyl (3R,6R,7aS)-6-(benzyloxy)-5-oxohexahydropyrrolo[2,1-b]thiazole-3-carboxylate (12b-enofo): A flame-dried 250 mL round bottom flask equipped with a magnetic stir bar was charged with the alkene (2072.0 mg, 8.84 mmol) and dichloromethane (90 mL) then cooled to -78 °C. Nitrogen was bubbled through the solution followed by ozone until a light blue solution persisted. The reaction was stirred for 10 minutes, at which point nitrogen was bubbled through the flask. Dimethyl sulfide (6.50 mL, 10 equiv.) was added and the reaction stirred at room temperature overnight under an atmosphere of argon. The solvent was concentrated in vacuo and the material was used without further purification. Cysteine methyl ester hydrochloride (1518.0 mg, 8.84 mmol) was dissolved in distilled water (24 mL) and ethanol (8 mL). Sodium bicarbonate (742.0 mg, 8.84 mmol) was added to this followed by the crude material in ethanol (16 mL). The reaction stirred at room temperature for 4 hours. The material was extracted with dichloromethane (3 x 25 mL) against distilled water (25 mL) and brine (25 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The resulting residue was dissolved in toluene (150 mL), heated to 111 °C and stirred overnight. The solvent was removed under reduced pressure and the material was purified by column chromatography, eluting with a gradient of ethyl acetate in hexane (5:95 — > 1:3) to afford the diastereomers as yellow oils. Yield 30% (1:1 12a:12b). TLC (1:1 ethyl acetate in hexane): Rf= 0.80 (12a-exo), 0.70 (12b-enofo).

[0050] 12a-exo:1H NMR (500 MHz, CDCh) 6 7.39 - 7.27 (m, 5H), 5.27 (dd, J = 6.7, 4.1 Hz, 1H), 5.06 (dd, J= 7.6, 4.7 Hz, 1H), 4.91 (d, J= 11.7 Hz, 1H), 4.71 (d, J= 11.7 Hz, 1H), 4.25 (dd, J= 7.9, 3.9 Hz, 1H), 3.78 (s, 3H), 3.45 - 3.38 (m, 2H), 2.54 (ddd, J= 14.3, 6.7, 4.0 Hz, 1H), 2.40 (ddd, J = 14.3, 7.9, 4.1 Hz, 1H) ppm.13C NMR (126 MHz, CDCh) 6 173.4, 170.0, 137.5, 128.6, 128.2, 128.1, 77.5, 72.2, 64.2, 57.5, 53.0, 36.8, 34.0 ppm. HRMS (ESI+) for CI5HI8O4NS (M + H), calcd: 308.09511, found 308.09427.

[0051] 12b-enofo:1H NMR (500 MHz, DCI3) 67.39 - 7.27 (m, 5H), 5.19 (dd, J = 6.9, 4.0 Hz, 1H), 5.01 - 4.98 (m, 2H), 4.75 (d, J = 11.9 Hz, 1H), 4.49 (dd, J = 9.6, 8.1 Hz, 1H), 3.75 (s, 3H), 3.38 - 3.33 (m, 2H), 2.90 (ddd, J = 12.8, 8.1, 6.3 Hz, 1H), 2.12 (ddd, J = 13.0, 9.6, 7.2 Hz, 1H) ppm.13C NMR (126 MHz, CDCh) 6 172.3, 169.8, 137.4, 128.6, 128.3, 128.1, 77.1, 72.6, 61.0, 57.5, 53.0, 37.4, 35.1 ppm. HRMS (ESI+) for CI5HI8O4NS (M + H), calcd: 308.09511, found 308.09427.

[0052] (3 / ?,6S,7aS)-6-(Benzyloxy)-5-oxohexahydropyrrolo[2,1-b]thiazole-3-carboxylic acid (13a-exo): In a 2-dram vial equipped with a magnetic stir bar was dissolved the methyl ester (90.0 mg, 0.29 mmol) in tetrahydrofuran (2 mL). A solution of lithium hydroxide (1 M, 0.29 mL, 0.29 mmol) was added and the reaction stirred at room temperature for 1 hour. The reaction was neutralized with 4 M HCI (pH ~ 2), diluted with distilled water (2 mL) and extracted with ethyl acetate (3 x 2 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to afford an oil. The material was used without further purification. Yield 86%.1H NMR (500 MHz, CDCh) 69.01 (bs, 1H), 7.38-7.28 (m, 5H), 5.29 (dd, J = 6.6, 4.7 Hz, 1 H), 5.00 (t, J = 6.6 Hz, 1 H), 4.89 (dd, J = 11.7 Hz, 1 H), 4.70 (d, J = 11.7 Hz, 1 H), 4.30 (dd, J = 7.8, 3.4 Hz, 1H), 3.52 - 3.46 (m, 2H), 2.56 (ddd, J = 14.3, 6.6, 3.3 Hz, 1H), 2.39 (ddd, J = 14.3, 7.7, 4.8 Hz, 1H) ppm;13C NMR (126 MHz, CDCh) 6 173.8, 172.7, 137.2, 128.6, 128.3, 128.2, 78.0, 72.4, 64.6, 57.5, 36.7, 34.2 ppm; HRMS (ESI+) for Ci4Hi4NNa2O4S (M + 2Na), calcd: 338.0433, found 338.0433.

[0053] (3 / ?,6 / ?,7aS)-6-(Benzyloxy)-5-oxohexahydropyrrolo[2,1-b]thiazole-3-carboxylic acid (13b-enofo): In a 2-dram vial equipped with a magnetic stir bar was dissolved the methyl ester (162.0 mg, 0.53 mmol) in tetrahydrofuran (2 mL). A solution of lithium hydroxide (1 M, 0.53 mL, 0.53 mmol) was added and the reaction stirred at room temperature for 30 minutes. The reaction was neutralized with 3 M HCI (pH ~ 1) and re-basified with saturated sodium bicarbonate (pH ~ 8). The organic impurities were extracted with ethyl acetate (2 x 2 mL). The solution was acidified (pH ~ 1) with 3 M HCI followed by extraction with ethyl acetate (3x2 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to afford an oil. The material was used without further purification. Yield 45%.1H NMR (500 MHz, CDCh) 69.75 (bs, 1H), 7.39 - 7.29 (m, 5H), 5.14 (dd, J= 7.2, 4.5 Hz, 1H), 4.99 - 4.96 (m, 2H), 4.80 - 4.72 (m, 1H), 4.57 - 4.50 (m, 1H), 3.48 - 3.36 (m, 2H), 2.92 - 2.87 (m, 1H), 2.18 - 2.10 (m, 1H) ppm.13C NMR (126 MHz, CDCh) 6 173.0, 171.3, 137.3, 128.7, 128.3, 128.2, 77.4, 72.8, 61.3, 57.6, 37.3, 35.1 ppm; HRMS (ESI+) for Ci4Hi4NNa2O4S (M + 2Na), calcd: 338.0433, found 338.0426.

[0054] (3 / ?,6S,7aS)-6-(Benzyloxy)-3-(pyrrolidine-1-carbonyl)tetrahydropyrrolo[2,1-b]thiazol-5(6H)-one (14a-exo): A 2-dram vial equipped with a magnetic stir bar was charged with the carboxylic acid (72.0 mg, 0.24 mmol), diisopropylethylamine (0.05 mL, 0.29 mmol), dichloromethane (1 mL) and 4A molecular sieves. The solution was cooled to 0 °C then pivaloyl chloride (30 pL, 0.24 mmol) was added dropwise. The reaction stirred for 30 minutes, at which point a solution of pyrrolidine in dichloromethane (1 M, 0.24 mL, 0.24 mmol) and DIPEA (0.05 mL, 0.29 mmol) were added. After stirring overnight at ambient temperature, the material was directly loaded into the column and purified by column chromatography, eluting with 1:1 ethyl acetate in hexane to afford an oil. Yield 49%. TLC (neat ethyl acetate): Rf = 0.61.1H NMR (500 MHz, CDCh) 5 7.41 - 7.28 (m, 5H), 5.24 (dd, J = 6.3, 3.4 Hz, 1H), 5.08 (dd, J = 8.1, 4.3 Hz, 1H), 4.91 (d, J = 11.9 Hz, 1 H), 4.73 (d, J = 11.9 Hz, 1 H), 4.29 (dd, J = 7.8, 6.0 Hz, 1 H), 3.84 - 3.79 (m, 1 H), 3.66 (dd, J = 10.8, 4.3 Hz, 1H), 3.59 - 3.54 (m, 1H), 3.51 - 3.45 (m, 2H), 3.30 (dd, J = 10.8, 8.0 Hz, 1H), 2.51 - 2.42 (m, 2H), 2.02 - 1.94 (m, 2H), 1.90 - 1.84 (m, 2H) ppm.13C NMR (126 MHz, CDCh) 6 174.3, 167.0, 137.5, 128.6, 128.2, 128.1, 76.6, 72.4, 64.2, 57.0, 46.9, 46.7, 36.4, 31.6, 26.4, 24.2 ppm. HRMS (ESI+) for Ci8H22N2O3NaS (M + Na), calcd: 369.1243, found 369.1239.

[0055] (3 / ?,6 / ?,7aS)-6-(Benzyloxy)-3-(pyrrolidine-1 -carbonyl)tetrahydropyrrolo[2,1 -b]thiazol-5(6H)-one (14b-enofo): A 2-dram vial equipped with a magnetic stir bar was charged with the carboxylic acid (71.0 mg, 0.24 mmol), diisopropylethylamine (0.05 mL, 0.29 mmol), dichloromethane (1 mL) and 4A molecular sieves. The solution was cooled to 0 °C then pivaloyl chloride (0.03 mL, 0.24 mmol) was added dropwise. The reaction stirred for 30 minutes, at which point a solution of pyrrolidine in dichloromethane (1 M, 0.24 mL, 0.24 mmol) and DIPEA (0.05 mL, 0.29 mmol) were added. After stirring overnight at ambient temperature, the material was directly loaded into the column and purified by column chromatography, eluting with neat ethyl acetate to afford an oil. Yield 65%. TLC (neat ethyl acetate): Rf= 0.27.1H NMR (500 MHz, CDCh) 67.39 -7.27 (m, 5H), 5.15 - 5.09 (m, 2H), 4.99 (d, J = 11.7 Hz, 1 H), 4.74 (d, J = 11.9 Hz, 1 H), 4.49 (t, J = 8.6 Hz, 1H), 3.86 (dt, J= 10.1, 6.9 Hz, 1H), 3.53 - 3.37 (m, 5H), 2.88 (ddd, J= 13.3, 8.5, 6.6 Hz, 1H), 2.15 (ddd, J= 13.1, 8.9, 6.4 Hz, 1H), 2.04- 1.94 (m, 2H), 1.91 - 1.83 (m, 2H) ppm.13C NMR (126 MHz, CDCh) 6 171.9, 167.5, 137.6, 128.6, 128.2, 128.1, 77.6, 72.6, 62.6, 56.2, 46.9, 46.6, 35.9, 35.4, 26.4, 24.2 ppm. HRMS (ESI+) for C18H23N2O3S (M + H), calcd: 347.1424, found 347.1418.

[0056] (S)-1-((3 / ?,6S,7aS)-6-(Benzyloxy)-5-oxohexahydropyrrolo[2,1-b]thiazole-3-carbonyl)-pyrrolidine-2-carbonitrile (15a-exo): A 2-dram vial equipped with a magnetic stir bar was charged with the carboxylic acid (50.0 mg, 0.17 mmol), diisopropylethylamine (0.03 mL, 0.17mmol), dichloromethane (2 mL) and 4A molecular sieves. The solution was cooled to 0 °C then pivaloyl chloride (21 pL, 0.17 mmol) was added dropwise. The reaction was stirred for 30 minutes, at which point a solution of (S)-2-cyanopyrrolidinium tosylate (46.0 mg, 0.17 mmol) and diisopropylethylamine (0.03 mL, 0.17 mmol) in dichloromethane (1 mL) was added. After stirring overnight at ambient temperature, the material was diluted with dichloromethane (10 mL) and washed with 0.1M HCI (10 mL). After further extraction with dichloromethane, the combined organic layers were washed with brine (25 mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude material was purified by column chromatography, eluting with 1:1 ethyl acetate in hexane to afford the product as a white solid. The material was further purified by dissolution in diethyl ether, followed by slow evaporation of the filtrate to afford white crystals. Yield 56%. TLC (neat ethyl acetate): Rf= 0.75.1H NMR (500 MHz, CDCI3) 67.39 - 7.28 (m, 5H), 5.16 (dd, J = 6.6, 3.2 Hz, 1H), 5.05 (dd, J = 8.0, 4.5 Hz, 1H), 4.89 (d, J = 11.7 Hz, 1H), 4.77 -4.71 (m, 2H), 4.29 (dd, J= 7.9, 5.6 Hz, 1H), 3.89 - 3.84 (m, 1H), 3.81 - 3.77 (m, 1H), 3.73 (dd, J = 11.1, 4.6 Hz, 1H), 3.36 - 3.32 (m, 1H), 2.53 -2.42 (m, 2H), 2.32 - 2.13 (m, 4H) ppm.13C NMR (126 MHz, CDCI3) 6 174.4, 167.6, 137.3, 128.6, 128.2, 128.1, 118.2, 76.6, 72.5, 64.2, 56.9, 47.2, 46.9, 36.2, 31.7, 30.0, 25.4 ppm. HRMS (ESI+) for Ci9H2iN3O3NaS (M + Na), calcd: 394.1196, found 394.1191.

[0057] (S)-1-((3R,6R,7aS)-6-(Benzyloxy)-5-oxohexahydropyrrolo[2,1-b]thiazole-3-carbonyl)pyrrolidine-2-carbonitrile (15b-enofo): A 2-dram vial equipped with a magnetic stir bar was charged with the carboxylic acid (50.0 mg, 0.17 mmol), diisopropylethylamine (0.03 mL, 0.17 mmol), dichloromethane (2 mL) and 4A molecular sieves. The solution was cooled to 0 °C then pivaloyl chloride (21 pL, 0.17 mmol) was added dropwise. The reaction was stirred for 30 minutes, at which point a solution of (S)-2-cyanopyrrolidinium tosylate (46.0 mg, 0.17 mmol) in dichloromethane (1 mL) and diisopropylethylamine (0.03 mL, 0.17 mmol) were added. After stirring overnight at ambient temperature, the material was directly loaded into the column and purified by column chromatography, eluting with 3:1 ethyl acetate in hexane to afford the product as a white solid. Spectroscopic data was obtained for a mixture of rotamers Yield 60%. TLC (neat ethyl acetate): Rf= 0.54.1H NMR (500 MHz, CDCI3) 5 7.40 - 7.28 (m, 5H), 5.30 (d, J = 6.1 Hz, 0.3H), 5.08 (t, J = 6.5 Hz, 0.7H), 5.04 - 5.00 (m, 1 H), 4.97 - 4.90 (m, 1 ,2H), 4.76 - 4.66 (m, 2H), 4.48 (t, J = 8.7 Hz, 0.7H), 3.97 - 3.90 (m, 1 H), 3.75 - 3.71 (m, 0.8H), 3.65 - 3.53 (m, 1 ,4H), 3.46 - 3.42 (m, 0.8H), 3.02 - 2.96 (m, 0.6H), 2.91 - 2.86 (m, 0.8H), 2.36 - 2.10 (m, 5H) ppm.13C NMR (126 MHz, CDCI3) 5 174.2, 172.1, 168.2, 166.3, 137.4, 128.6, 128.25, 128.20, 128.17, 119.2, 118.1, 77.5, 77.4, 73.1, 72.6, 62.7, 60.1, 59.1, 56.0, 48.0, 47.4, 47.1, 46.9, 39.1, 36.0, 35.3, 32.9,32.3, 29.9, 29.82, 25.5, 22.2 ppm. HRMS (ESI+) for Ci9H2iN3O3NaS (M + Na), calcd: 394.1196, found 394.1190.

[0058] 1,3-dioxoisoindolin-2-yl (3 / ?,6S,7aS)-6-(benzyloxy)-5-oxohexahydropyrrolo[2,1-b]thiazole-3-carboxylate (16a-exo): A 6-dram vial equipped with a magnetic stir bar was charged with the carboxylic acid (85.0 mg, 0.29 mmol), N-hydroxyphthalimide (47.0 mg, 0.29 mmol), tetrahydrofuran (6 mL) and 4A molecular sieves. The flask was submerged in an ice-bath then a solution of DCC (60.0 mg, 0.29 mmol) in tetrahydrofuran (2 mL) was added. After stirring at room temperature for 2 hours, the urea by-product was filtered through celite and the pad of celite was subsequently washed with additional tetrahydrofuran. The filtrate was concentrated in vacuo and the off-white solid was triturated with isopropanol. The solid was filtered under vacuum using a Hirsch funnel to afford the product as a white solid. Yield 49%.1H NMR (500 MHz, CDCI3) 5 7.92 - 7.89 (m, 2H), 7.83 - 7.80 (m, 2H), 7.39 - 7.28 (m, 5H), 5.46 (dd, J = 8.2, 4.2 Hz, 1 H), 5.36 (dd, J = 6.7, 3.7 Hz, 1 H), 4.93 (d, J = 11.8 Hz, 1 H), 4.72 (d, J = 11.8 Hz, 1 H), 4.29 (dd, J = 7.9, 4.3 Hz, 1 H), 3.68 (dd, J = 11.7, 4.2 Hz, 1 H), 3.60 (dd, J = 11.7, 8.2 Hz, 1 H), 2.59 (ddd, J = 14.3, 6.8, 4.3 Hz, 1H), 2.47 (ddd, J = 14.3, 7.9, 3.7 Hz, 1H) ppm.13C NMR (126 MHz, CDCI3) 5 173.8, 166.5, 161.4, 137.4, 135.1, 128.9, 128.6, 128.3, 128.2, 124.3, 76.8, 72.4, 64.3, 56.1, 37.0, 33.6 ppm. HRMS (ESI+) for C22Hi8N2O6NaS (M + Na), calcd: 461.0778, found 461.0789.

[0059] 1,3-Dioxoisoindolin-2-yl (3 / ?,6 / ?,7aS)-6-(benzyloxy)-5-oxohexahydropyrrolo[2,1-b]thiazole-3-carboxylate (16b-enofo): A 2-dram vial equipped with a magnetic stir bar was charged with the carboxylic acid (50.0 mg, 0.17 mmol), N-hydroxyphthalimide (28.0 mg, 0.17 mmol), tetrahydrofuran (4 mL) and 4A molecular sieves. The flask was submerged in an ice-bath then a solution of DCC (40.0 mg, 0.19 mmol) in tetrahydrofuran (1 mL) was added. After stirring at room temperature for 2 hours, the urea by-product was filtered through celite and the pad of celite was subsequently washed with additional tetrahydrofuran. The filtrate was concentrated in vacuo and the off-white solid was triturated with isopropanol (~4 mL). The solid was filtered under vacuum using a Hirsch funnel to afford the product as a white solid. Yield 46%.1H NMR (500 MHz, CDCI3) 5 7.90 - 7.89 (m, 2H), 7.82 - 7.80 (m, 2H), 7.40 - 7.29 (m, 5H), 5.62 (dd, J = 7.4, 3.2 Hz, 1H), 5.11 (dd, J= 6.6 Hz, 6.6 Hz, 1H), 5.02 (d, J= 11.9 Hz, 1H), 4.77 (d, J= 11.9 Hz, 1H), 4.52 (dd, J= 8.8 Hz, 8.8 Hz, 1H), 3.64 (dd, J= 11.5, 3.2 Hz, 1H), 3.50 (dd, J= 11.6, 7.5 Hz, 1H), 2.96 (ddd, J = 12.9, 8.2, 6.3 Hz, 1H), 2.18 (ddd, J = 13.1, 9.5, 6.9 Hz, 1H) ppm.13C NMR (126 MHz, CDCI3) 5 172.7, 166.2, 161.4, 137.4, 135.1, 128.9, 128.7, 128.3, 128.2, 124.3, 76.7, 72.8, 61.2, 56.0, 37.3, 35.2 ppm. HRMS (ESI+) for C22HI9N2O6S (M + H), calcd: 439.09583, found 439.09485.

[0060] (3 / ?,6S,7aS)-6-(Benzyloxy)-3-(( / ?)-2-((3aS,4S,6S,7a / ?)-3a,5,5-trimethylhexahydro- 4,6-methanobenzo[d][1 ,3,2]dioxaborol-2-yl)pyrrolidine-1 -carbonyl)tetrahydropyrrolo[2,1 -b]thiazol-5(6H)-one (17a-exo): A 2-dram vial equipped with a magnetic stir bar was charged with the activated ester (52.0 mg, 0.12 mmol), pyrrolidinium HCI salt (34.0 mg, 0.12 mmol), dichloromethane (3 mL) and 4A molecular sieves. The solution was cooled to 0 °C then DI PEA (21 pL, 0.12 mmol) was added. The reaction was allowed towarm to room temperature and stirred for 3 hours, at which point it was quenched with a saturated solution of ammonium chloride. The contents were extracted with dichloromethane. The combined organic layers were washed with saturated sodium bicarbonate solution, brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The crude residue was purified by column chromatography, eluting with 2:3 ethyl acetate in hexane to afford the product as an off-white oil. The compound was further purified by trituration with hexane followed by slow evaporation of the filtrate to afford white crystalline material. Yield 48%. TLC (1:1 ethyl acetate in hexane): Rf = 0.41.1H NMR (800 MHz, CDCI3) 6 7.37 - 7.33 (m, 4H), 7.30 - 7.28 (m, 1H), 5.21 (dd, J = 7.0, 2.4 Hz, 1H), 5.08 (dd, J = 8.1, 4.4 Hz, 1H), 4.91 (d, J= 11.9 Hz, 1H), 4.72 (d, J= 11.9 Hz, 1H), 4.30 - 4.27 (m, 2H), 3.95 -3.92 (m, 1H), 3.66 (dd, J= 10.9, 4.4 Hz, 1H), 3.59 -3.56 (m, 1H), 3.30 (dd, J= 10.8, 8.1 Hz, 1H), 3.16 (dd, J = 10.0, 7.3 Hz, 1H), 2.49 - 2.46 (m, 1H), 2.43 - 2.40 (m, 1H), 2.35 - 2.31 (m, 1H), 2.16 - 2.14 (m, 1H), 2.13 - 2.08 (m, 1H), 2.07 - 2.03 (m, 1H), 2.02 - 1.96 (m, 2H), 1.91 - 1.85 (m, 2H), 1.80 - 1.75 (m, 1H), 1.41 (s, 3H), 1.34 (d, J = 10.9 Hz, 1H), 1.28 (s, 3H), 0.84 (s, 3H) ppm.13C NMR (200 MHz, CDCI3) 6 174.4, 166.7, 137.5, 128.6, 128.2, 128.1, 86.1, 78.0, 76.4, 72.3, 64.2, 56.3, 51.4, 46.9, 45.1, 39.7, 38.4, 36.5, 35.7, 31.1, 28.7, 27.7, 27.30, 27.26, 26.3, 24.2 ppm. HRMS (ESI+) for C28H37BN2O5NaS (M + Na), calcd: 547.2408, found 547.2406.

[0061] (3R,6 / ?,7aS)-6-(Benzyloxy)-3-(( / ?)-2-((3aS,4S,6S,7a / ?)-3a,5,5-trimethylhexahydro- 4,6-methanobenzo[d][1 ,3,2]dioxaborol-2-yl)pyrrolidine-1 -carbonyl)tetrahydropyrrolo[2,1 -b]thiazol-5(6H)-one (17b-enofo): A 2-dram vial equipped with a magnetic stir bar was charged with the activated ester (54.0 mg, 0.12 mmol), pyrrolidinium HCI salt (40.0 mg, 0.14 mmol), dichloromethane (3 mL) and 4A molecular sieves. The solution was cooled to 0 °C then DI PEA (21 pL, 0.11 mmol) was added. The reaction was allowed towarm to room temperature and stirred for 1 hour, at which point it was quenched with a saturated solution of ammonium chloride. The contents were extracted with dichloromethane. The combined organic layers were washed with saturated sodium bicarbonate solution, brine, dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The crude residue was purified by column chromatography, eluting with 1:1 ethyl acetate in hexane to afford the product as an off-white oil. The compound was furtherpurified by trituration with hexane followed by slow evaporation of the filtrate to afford white crystalline material. Yield 54%. TLC (1:1 ethyl acetate in hexane): Rf = 0.26.1H NMR (500 MHz, CDCI3) 67.38-7.27 (m, 5H), 5.12 - 5.07 (m, 2H), 4.98 (d, J= 11.7 Hz, 1H), 4.72 (d, J = 11.7 Hz, 1 H), 4.45 (t, J = 8.6 Hz, 1 H), 4.28 (dd, J = 8.9, 2.2 Hz, 1 H), 4.01 - 3.96 (m, 1 H), 3.55 - 3.48 (m, 2H), 3.41 (dd, J= 11.1, 7.7 Hz, 1H), 3.17 (dd, J= 10.1, 7.2 Hz, 1H), 2.84 (ddd, J= 13.3, 8.5, 6.6 Hz, 1H), 2.35 - 2.29 (m, 1H), 2.17 - 1.97 (m, 6H), 1.90 - 1.73 (m, 3H), 1.39 (s, 3H), 1.32 (d, J = 10.8 Hz, 1H), 1.27 (s, 3H), 0.83 (s, 3H) ppm.13C NMR (126 MHz, CDCI3) 6 171.8, 167.2, 137.6, 128.6, 128.2, 128.1, 86.1, 78.0, 77.7, 72.5, 62.7, 55.4, 51.3, 46.9, 45.0, 39.7, 38.4, 36.0, 35.6, 34.9, 28.7, 27.7, 27.3, 27.2, 26.3, 24.2 ppm. HRMS (ESI+) for C28H37BN2O5NaS (M + Na), calcd: 547.2408, found 547.2395.

[0062] tert-Butyl (S)-2-(2-cyano-2-(triphenyl-l5-phosphaneylidene)acetyl)pyrrolidine-1 -carboxylate (18): Boc-L-proline (3.000 g, 13.9 mmol), EDC HCI (2.310 g, 14.9 mmol) and DMAP (0.170 g, 1.39 mmol) were dissolved in dichloromethane (30 mL). A solution of (Triphenylphosphoranylidene)acetonitrile (2.150 g, 30.0 mmol) in dichloromethane (70 mL) was added dropwise at 0 °C. The reaction was allowed to warm to room temperature then stirred for 16 hours. After dilution with distilled water (50 mL), the contents were extracted with dichloromethane (3 x 50 mL). The combined organic layers were washed with brine (50 mL), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The crude material was used without further purification.

[0063] tert-Butyl (S)-2-(2-methoxy-2-oxoacetyl)pyrrolidine-1 -carboxylate (19):Compound 18 (6.940 g, 13.9 mmol) was dissolved in 7:3 DCM:MeOH (90 mL) at -78 °C. Nitrogen was bubbled through the solution followed by ozone until a light blue solution persisted. The reaction was stirred for 40 minutes, at which point nitrogen was bubbled through the flask. The solution was allowed to warm to room temperature then concentrated in vacuo. The crude material was purified by column chromatography, eluting with a gradient of ethyl acetate in hexane (0:1 — > 15:85) to afford the product as a colorless oil. Spectroscopic data was obtained for a mixture of rotamers. Yield 31% (over2 steps).1H NMR (500 MHz, CDCI3) 64.88-4.86 (m, 0.4H), 4.77 -4.74 (m, 0.6H), 3.88 - 3.81 (m, 3H), 3.54 - 3.39 (m, 2H), 2.35 -2.22 (m, 1H), 2.06 - 1.85 (m, 3H), 1.45 - 1.30 (m, 9H) ppm. Spectroscopic data are consistent with previously reported literature.

[0064] (S)-2-(1-(tert-Butoxycarbonyl)pyrrolidin-2-yl)-2-oxoacetic acid (20): Compound 19 (100.0 mg, 0.43 mmol) was dissolved in THF (1 mL). A solution of 1M LiOH (0.43 mL, 0.43mmol) was added. The reaction was stirred at ambient temperature for 3 hours. After acidification with 1M HCI (pH ~ 1) and dilution with distilled water (5 mL), the contents were extracted with ethyl acetate (3x 5 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The crude material was used without further purification.

[0065] tert-Butyl (S)-2-(2-((3,4-dimethoxybenzyl)amino)-2-oxoacetyl)pyrrolidine-1-carboxylate (21): Compound 20 (283.0 mg, 1.17 mmol) was dissolved in DMF (6 mL). HATU (975.0 mg, 2.56 mmol), DIPEA (0.447 mL, 2.56 mmol) and 3,4-dimethoxybenzylamine (0.176 mL, 1.17 mmol) were added. The reaction was stirred at ambient temperature for 16 hours. After dilution with distilled water (10 mL), the contents were extracted with ethyl acetate (2 x 10 mL). The combined organic layers were washed with 1M HCI (10 mL), saturated sodium bicarbonate solution (10 mL) and brine (10 mL), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The crude material was purified by column chromatography, eluting with a gradient of ethyl acetate in hexane (0:1 — > 1:1) to afford the product as a yellow oil. Spectroscopic data was obtained for a mixture of rotamers. Yield 72% (over 2 steps). TLC (1:1 ethyl acetate in hexane): Rf= 0.81.1H NMR (500 MHz, CDCI3) 67.21 - 7.16 (m, 0.5H), 7.13 - 7.07 (m, 0.5H), 6.84 - 6.79 (m, 3H), 5.22 (dd, J = 9.1, 4.8 Hz, 0.5H), 5.14 (dd, J = 9.1, 5.3 Hz, 0.5H), 4.47 - 4.36 (m, 2H), 3.89-3.86 (m, 6H), 3.58 -3.41 (m, 2H), 2.41 - 2.32 (m, 1H), 2.00 - 1.81 (m, 3H), 1.43 (s, 4.5H), 1.32 (s, 4.5H) ppm.13C NMR (126 MHz, CDCI3) 6196.7, 196.4, 159.6, 159.3, 154.5, 153.4, 149.4, 149.3, 148.9, 148.8, 135.9, 129.5, 125.6, 120.54, 120.49, 111.41, 111.38, 111.36, 80.1, 80.0, 68.1, 60.9, 60.3, 56.1, 56.0, 56.0, 46.9, 46.64, 46.57, 43.3, 34.3, 30.4, 30.2, 29.3, 28.5, 28.3, 28.1, 25.7, 24.7, 23.8 ppm. HRMS (ESI+) forC2oH28N206Na (M + Na), calcd: 415.1840, found 415.1845.

[0066] tert-Butyl (S)-2-(2-((3,4-dimethoxybenzyl)amino)-1-hydroxy-2-oxoethyl)pyrrolidine-1 -carboxylate (22): Compound 21 (327.0 mg, 0.83 mmol) was dissolved in THF (6 mL). Sodium borohydride (47.3 mg, 1.25 mmol) was added and the reaction stirred at ambient temperature for 30 minutes. The reaction was quenched with distilled water (10 mL) then extracted with ethyl acetate (10 mL). The organic layer was washed with 1M HCI (2 x 10 mL) and brine (10 mL), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The crude material was used without further purification.

[0067] (S)- / V-(3,4-Dimethoxybenzyl)-2-hydroxy-2-(pyrrolidin-2-yl)acetamide (23):Compound 22 (233.0 mg, 0.59 mmol) was dissolved in dichloromethane (3 mL). TFA (0.068 mL, 0.89 mmol) was added. The reaction stirred at ambient temperature for 1 hour, then concentrated in vacuo. The crude material was used without further purification.

[0068] 2-((S)-1 -((3 / ?,6S,7aS)-6-(Benzyloxy)-5-oxohexahydropyrrolo[2,1 -b]thiazole-3-carbonyl)pyrrolidin-2-yl)-N-(3,4-dimethoxybenzyl)-2-hydroxyacetamide (24): A 6-dram vial was charged with the activated ester 16a (53.1 mg, 0.12 mmol), pyrrolidine 23 (35.6 mg, 0.12 mmol), 4 MS and dichloromethane (1 mL). DIPEA (0.021 mL, 0.12 mmol) was added at 0 °C. The reaction stirred at ambient temperature for 1 hour. After reaction quench with saturated ammonium chloride (5 mL), the contents were extracted with dichloromethane (3 x 5 mL). The combined organic layers were washed with saturated sodium bicarbonate solution (5 mL) and brine (5 mL), dried over anhydrous sodium sulfate, filtered and concentrated in vacuo. The crude material was purified by column chromatography, eluting with a gradient of ethyl acetate in hexane (1:1 — > 1:0) to afford the product as an off-white solid. Yield 30% (over 3 steps). TLC (neat ethyl acetate): Rf = 0.14.1H NMR (500 MHz, DMSO-de, variable temperature at 80 °C) 5 7.94 - 7.90 (m, 1H), 7.35 - 7.28 (m, 5H), 6.93 - 6.79 (m, 3H), 5.63 - 5.37 (m, 1H), 5.23 - 5.02 (m, 2H), 4.76 (d, J = 11.8 Hz, 1H), 4.66 (d, J = 12.0 Hz, 1H), 4.33 - 4.22 (m, 5H), 3.83 - 3.78 (m, 0.3H), 3.75 (s, 3H), 3.74 (s, 3H), 3.70 - 3.65 (m, 0.7H), 3.57 - 3.43 (m, 3H), 2.47 -2.41 (m, 2H), 1.91 - 1.75 (m, 4H) ppm.13C NMR (126 MHz, DMSO-d6, variable temperature at 80 °C) 5 172.0, 171.4, 167.0, 148.7, 147.9, 137.5, 131.9, 127.8, 127.3, 127.2, 119.6, 119.4, 112.3, 77.0, 70.7, 69.4, 64.2, 60.0, 56.3, 55.6, 55.5, 46.7, 41.4, 35.8, 30.9, 25.0, 23.2 ppm. HRMS (ESI+) for C29H35N3O7NaS (M + Na), calcd: 592.2088, found 592.2096.

[0069] 2-((S)-1 -((3 / ?,6S,7aS)-6-(Benzyloxy)-5-oxohexahydropyrrolo[2,1 -b]thiazole-3-carbonyl)pyrrolidin-2-yl)-N-(3,4-dimethoxybenzyl)-2-oxoacetamide (25): Compound 24 (21.0 mg, 0.037 mmol) was dissolved in dichloromethane (1 mL). SiO2(6.64 mg, 0.11 mmol) and PDC (13.9 mg, 0.037 mmol) were added. The reaction stirred at ambient temperature for 2 hours. The solution was filtered through Celite then concentrated in vacuo. The crude material was purified by column chromatography, eluting with a gradient of ethyl acetate in hexane (1 :1 — > 1 :0) to afford the product as an off-white oil. Yield 16%. TLC (neat ethyl acetate): Rf = 0.61.1H NMR (500 MHz, CDCI3) 57.38 - 7.27 (m, 5H), 7.09 - 7.05 (m, 1 H), 6.85 - 6.73 (m, 3H), 5.31 - 5.26 (m, 1 H), 5.22 - 5.17 (m, 1H), 5.13 - 5.07 (m, 1H), 4.97 - 4.87 (m, 1H), 4.74 - 4.70 (m, 1H), 4.40 - 4.37 (m, 2H), 4.31 - 4.26 (m, 1 H), 3.98 - 3.94 (m, 1 H), 3.88 (s, 3H), 3.87 (s, 3H), 3.82 - 3.74 (m, 1 H), 3.54 (dd, J = 11.0, 4.4 Hz, 1H), 3.32 (dd, J = 11.0, 8.1 Hz, 1H), 2.51 - 2.32 (m, 3H), 2.09 - 2.05 (m, 1H), 2.03 - 1.87 (m, 2H) ppm.13C NMR (126 MHz, CDCI3) 5 194.9, 174.2, 167.2, 159.4, 149.4, 148.9, 137.4, 129.4, 128.6, 128.2, 128.1, 120.5, 111.41, 111.38, 76.7, 72.4, 64.3, 61.4, 56.5, 56.1, 47.6, 43.4, 36.2, 31.9, 28.5, 25.6 ppm. HRMS (ESI+) for C29H33N3O7NaS (M + Na), calcd: 590.19314, found 590.19155.

[0070] 2-(Benzyloxy)pent-4-enoic acid (rac-26): General procedure for hydrolysis: In a an appropriately sized round bottom flask equipped with a magnetic stir bar was dissolved the ethyl ester (1 equiv.) in tetrahydrofuran (0.5 M). A solution of sodium hydroxide (1 M, 2 equiv.) was added and the reaction stirred at room temperature overnight. The organic impurities were extracted with ethyl acetate (25 mL). The aqueous phase was acidified with 1 M HCI (pH ~ 1) and extracted with dichloromethane (3 x 25 mL). The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to afford an off-white oil. The material was used without further purification. Yield 68%.1H NMR (500 MHz, CDCh) 6 7.39 -7.30 (m, 5H), 5.85 (ddt, J= 17.1, 10.1, 6.9 Hz, 1H), 5.20-5.13 (m, 2H), 4.74 (d, J= 11.6 Hz, 1H), 4.54 (d, J= 11.6 Hz, 1H), 4.08 (dd, J= 6.8, 5.1 Hz, 1H), 2.65 -2.54 (m, 2H) ppm.13C NMR (126 MHz, CDCh) 6 176.8, 137.0, 132.6, 128.7, 128.3, 128.2, 118.7, 77.4, 72.7, 37.0 ppm.

[0071] Methyl (2-(benzyloxy)pent-4-enoyl)-L-serinate (27a-exo and 27b-endo) A 250-mL round bottom flask equipped with a magnetic stir bar was charged with the carboxylic acid (1134.0 mg, 5.50 mmol), L-serine methyl ester hydrochloride (855.0 mg, 5.50 mmol), DCM (50 mL) and triethylamine (1.54 mL, 11.0 mmol). EDC hydrochloride (1054.0 mg, 5.50 mmol) and HOBt (842.0 mg, 5.50 mmol) were added and the reaction stirred at ambient temperature overnight under an atmosphere of argon. The contents were diluted with DCM (25 mL) then washed with 1M HCI (25 mL). After further extraction with dichloromethane (25 mL), the combined organic phases were washed with saturated sodium bicarbonate solution (25 mL) and brine (25 mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude material was purified by column chromatography, eluting with a gradient of ethyl acetate in hexane (1 :4 — > 1 : 1 ) to afford the product as a mixture of diastereomers which solidified upon storage in the freezer. Yield 50%. TLC (1:1 ethyl acetate in hexane): Rf= 0.32, 0.22.1H NMR (500 MHz, CDCh) 67.52 - 7.29 (m, 10H), 5.89 - 5.79 (m, 2H), 5.17 - 5.09 (m, 4H), 4.69 - 4.65 (m, 2H), 4.63 - 4.55 (m, 4H), 3.99 -3.94 (m, 4H), 3.90 - 3.83 (m, 2H), 3.79 (s, 3H), 3.77 (s, 3H), 2.63 - 2.57 (m, 2H), 2.55 - 2.47 (m, 2H), ppm.13C NMR (126 MHz, CDCh) 6 172.7, 172.6, 170.7, 170.6, 137.2, 137.1, 133.3, 133.2, 128.74, 128.71, 128.34, 128.32, 128.27, 118.3, 79.6, 79.5, 73.0, 72.9, 63.45, 63.39, 54.5, 54.4, 52.90, 52.87, 37.3, 37.2 ppm. HRMS (ESI+) for C16H22O5N (M + H), calcd: 308.14925, found 308.14844.

[0072] Noteworthy, a significant amount of white precipitate forms after addition of 1M HCI during aqueous workup, which satisfactorily is removed after the sodium bicarbonate wash. Alternatively, the basic wash can be performed first which contributes to a cleaner extraction.

[0073] Methyl (3S,7aS)-6-(benzyloxy)-5-oxohexahydropyrrolo[2,1-b]oxazole-3-carboxylate (28a-exo and 28b-enofo): A flame-dried 100 mL round bottom flask equipped with a magnetic stir bar was charged with the alkene (460.0 mg, 1.50 mmol) and anhydrous dichloromethane (25 mL) then cooled to -78 °C. Nitrogen was bubbled through the solution for 5 minutes, followed by ozone until a deep blue solution persisted. The reaction was stirred for 10 minutes, at which point nitrogen was bubbled through the flask. Dimethyl sulfide (0.55 mL, 7.44 mmol, 5 equiv.) was added and the reaction stirred at room temperature overnight under an atmosphere of argon. The reaction was concentrated in vacuo then re-suspended in toluene (20 mL). After stirring at 90 °C for 2 hours, the filtrate was transferred to a secondary 100 mL round bottom flask. TFA (0.08 mL, 1.04 mmol) was added and the reaction was heated to 50 °C and stirred for an additional 2 hours. The contents were concentrated then purified by column chromatography, eluting with a gradient of ethyl acetate in hexane (1:4 — > 1:1) to afford the product as a mixture of inseparable diastereomers. Yield 26% (1:1 19a: 19b). TLC (1:1 ethyl acetate in hexane) Rf= 0.55.1H NMR (500 MHz, CDCI3) 6 7.41 - 7.28 (m, 10H), 5.29 (dd, J = 5.8, 2.6 Hz, 1 H), 5.08 (dd, J = 5.9, 4.2 Hz, 1 H), 4.98 (d, J = 11.7 Hz, 1 H), 4.88 (d, J = 11.7 Hz, 1 H), 4.76 - 4.68 (m, 3H), 4.62 (dd, J = 8.2, 6.6 Hz, 1 H), 4.48 (dd, J = 8.8 Hz, 8.8 Hz, 1 H), 4.42 -4.36 (m, 2H), 4.22 (dd, J = 8.0, 3.1 Hz, 1H), 3.97 (ddd, J = 17.1, 8.9, 6.6 Hz, 2H), 3.79 (s, 3H), 3.76 (s, 3H), 2.82 (ddd, J= 13.6, 8.7, 6.0 Hz, 1H), 2.45 -2.40 (m, 1H), 2.35 -2.29 (m, 1H), 2.02 (ddd, J = 13.3, 8.9, 4.2 Hz, 1H) ppm.13C NMR (126 MHz, CDCI3) 6 176.1, 175.3, 170.3, 170.2, 137.39, 137.35, 128.63, 128.60, 128.28, 128.25, 128.15, 128.11, 91.5, 88.6, 77.7, 76.6, 72.6, 72.2, 70.5, 69.8, 55.6, 55.0, 52.93, 52.90, 34.9, 33.6 ppm. HRMS (ESI+) for CI5HI8O5N (M + H), calcd: 292.11795, found 292.11770.

[0074] Unfortunately, an additional adduct (with + CH2O mass) is present directly underneath the product that required careful separation during column chromatography. It was observed that any attempt to carry forward contaminated material often complicated the subsequent two steps.

[0075] (3S,6S,7aS)-6-(Benzyloxy)-5-oxohexahydropyrrolo[2,1-b]oxazole-3-carboxylic acid (29a-exo) and (3S,6R,7aS)-6-(benzyloxy)-5-oxohexahydropyrrolo[2,1-b]oxazole-3-carboxylic acid (29b-enofo): In a 6-dram vial equipped with a magnetic stir bar was dissolved the methyl ester (82.0 mg, 0.28 mmol) in tetrahydrofuran (2 mL). A solution of sodium hydroxide (1 M, 0.28 mL, 0.28 mmol) was added. The reaction was stirred at 0 °C for 1 hour. After acidification with 1M HCI (pH ~ 1) and further dilution with distilled water (5 mL), the contents were extracted with ethyl acetate (3 x 5 mL). The combined organic phases were dried over anhydrous sodiumsulfate, filtered, and concentrated in vacuo to afford the product as an off-white oil. The material was used without further purification.

[0076] (3S,6S,7aS)-6-(Benzyloxy)-3-(pyrrolidine-1 -carbonyl)tetrahydropyrrolo[2,1 -b]oxazol-5(6H)-one (30a-exo) and (3S,6 / ?,7aS)-6-(benzyloxy)-3-(pyrrolidine-1-carbonyl)tetrahydropyrrolo[2,1-b]oxazol-5(6H)-one (30b-enofo): A 6-dram vial equipped with a magnetic stir bar was charged with the carboxylic acid (100.0 mg, 0.36 mmol), DI PEA (0.06 mL, 0.36 mmol), dichloromethane (2 mL) and 4 molecular sieves. The solution was cooled to 0 °C then pivaloyl chloride (44 pL, 0.36 mmol) was added dropwise. The reaction was stirred for 30 minutes, at which point a solution of pyrrolidine (29 pL, 0.36 mmol) and DIPEA (0.06 mL, 0.36 mmol) in dichloromethane (1 mL) was added. After stirring overnight at ambient temperature, the material was diluted with dichloromethane (10 mL) and washed with 0.1 M HCI (10 mL). After further extraction with dichloromethane, the combined organic layers were washed with brine (25 mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude material was purified by column chromatography, eluting with a gradient of ethyl acetate in hexane (0:1 — > 1:0) to afford the product as separable diastereomers (clear oils). Yield 45% (1:1 30a:30b). TLC (neat ethyl acetate): Rf = 0.44 (30a-exo), 0.31 (30b-enofo).

[0077] 30a-exo:1H NMR (500 MHz, CDCI3) 67.37 - 7.28 (m, 5H), 5.29 (dd, J = 5.2, 2.4 Hz, 1H), 4.86 (d, J= 11.7 Hz, 1H), 4.71 -4.68 (m, 2H), 4.37 (dd, J= 8.4, 6.0 Hz, 1H), 4.23-4.16 (m, 2H), 3.94 - 3.89 (m, 1H), 3.59 - 3.44 (m, 3H), 2.41 - 2.32 (m, 2H), 2.04 - 1.95 (m, 2H), 1.91 -1.85 (m, 2H) ppm.13C NMR (126 MHz, CDCI3) 6 176.9, 167.0, 137.4, 128.6, 128.2, 128.1, 91.0, 77.1 , 72.2, 69.9, 55.4, 46.8, 46.7, 32.9, 26.4, 24.2 ppm. (ESI+) for Ci8H22N2NaO4 (M + Na), calcd: 353.14718, found 353.14641.

[0078] 30b-endo.1H NMR (500 MHz, CDCI3) 67.39 - 7.28 (m, 5H), 5.15 (dd, J= 6.0, 3.8 Hz, 1 H), 4.98 (d, J = 11.7 Hz, 1 H), 4.79 (dd, J = 6.6 Hz, 6.5 Hz, 1 H), 4.72 (d, J = 11.8 Hz, 1 H), 4.45 (dd, J = 8.6 Hz, 8.5 Hz, 1 H), 4.33 - 4.30 (m, 1 H), 4.22 (dd, J = 8.3, 6.5 Hz, 1 H), 3.97 - 3.92 (m, 1H), 3.53 - 3.41 (m, 3H), 2.81 (ddd, J = 13.6, 8.8, 5.9 Hz, 1H), 2.05 - 1.95 (m, 3H), 1.91 - 1.83 (m, 2H) ppm.13C NMR (126 MHz, CDCI3) 6 175.3, 167.3, 137.5, 128.6, 128.2, 128.1, 88.7, 76.8, 72.6, 70.0, 54.3, 46.8, 46.6, 34.7, 26.3, 24.2 ppm. (ESI+) for Ci8H22N2NaO4(M + Na), calcd: 353.14718, found 353.14636.

[0079] (S)-1-((3S,6S,7aS)-6-(Benzyloxy)-5-oxohexahydropyrrolo[2,1-b]oxazole-3-carbonyl)pyrrolidine-2-carbonitrile (31a-exo) and (S)-1-((3S,6 / ?,7aS)-6-(benzyloxy)-5-oxohexahydropyrrolo[2,1-b]oxazole-3-carbonyl)pyrrolidine-2-carbonitrile (31b-enofo): A 6-dram vial equipped with a magnetic stir bar was charged with the carboxylic acid (76.0 mg, 0.28 mmol), DIPEA (0.05 mL, 0.28 mmol), dichloromethane (1 mL) and 4 molecular sieves. The solution was cooled to 0 °C then pivaloyl chloride (34 pL, 0.28 mmol) was added dropwise. The reaction was stirred for 30 minutes, at which point a solution of (S)-2-cyanopyrrolidinium tosylate (75.0 mg, 0.28 mmol) and DIPEA (0.05 mL, 0.28 mmol) in dichloromethane (1 mL) was added. After stirring overnight at ambient temperature, the material was diluted with dichloromethane (10 mL) and washed with 0.1 M HCI (10 mL). After further extraction with dichloromethane, the combined organic layers were washed with brine (25 mL), dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude material was dried onto silica gel and purified by column chromatography, eluting with a gradient of ethyl acetate in hexane (0:1 — > 1:1) to afford the product as separable diastereomers (clear oils). Yield 50% (3:1 31a:31b). TLC (1:1 ethyl acetate in hexane): Rf = 0.28 (31a-exo), 0.19 (31b-enofo).

[0080] 31a-exo:1H NMR (500 MHz, CDCI3) 67.37 - 7.28 (m, 5H), 5.23 (dd, J = 5.6, 2.2 Hz, 1 H), 4.84 (d, J = 11.7 Hz, 1 H), 4.76 - 4.74 (m, 1 H), 4.70 (d, J = 11.8 Hz, 1 H), 4.68 - 4.65 (m, 1H), 4.46 - 4.43 (m, 1H), 4.23 - 4.20 (m, 1H), 3.99 - 3.93 (m, 1H), 3.84 -3.80 (m, 1H), 2.43 -2.18 (m, 6H) ppm.13C NMR (126 MHz, CDCI3) 6 177.2, 167.6, 137.2, 128.6, 128.2, 128.1, 118.2, 90.9, 76.9, 72.4, 69.6, 55.2, 47.2, 46.8, 32.8, 29.9, 25.4 ppm. (ESI+) for Ci9H2iN3NaO4(M + Na), calcd: 378.1424, found 378.1422.

[0081] 31b-enofo:1H NMR (500 MHz, CDCI3) 6 7.40 - 7.28 (m, 5H), 5.15 (d, J = 7.5 Hz, 0.2H), 5.12 (dd, J = 5.9 Hz, 3.9 Hz, 0.8H), 5.07 (dd, J = 6.0 Hz, 3.5 Hz, 0.2H), 5.00 (d, J = 11.8 Hz, 0.2H), 4.96 (d, J = 11.8 Hz, 0.8H), 4.79 - 4.70 (m, 2.8H), 4.62 - 4.58 (m, 0.2H), 4.54 (dd, J = 8.8 Hz, 4.9 Hz, 0.2H), 4.45 - 4.42 (m, 0.8H), 4.38 - 4.35 (m, 0.8H), 4.30 - 4.27 (m, 0.8H), 4.21 -4.18 (m, 0.2H), 4.03 - 3.98 (m, 0.8H), 3.75 - 3.71 (m, 0.8H), 3.64 - 3.53 (m, 0.4H), 2.90 - 2.80 (m, 1H), 2.36 - 2.18 (m, 4H), 2.09 - 2.02 (m, 1H) ppm.13C NMR (126 MHz, CDCI3) 6 175.6, 167.9, 137.3, 128.7, 128.21, 128.19, 118.1, 88.8, 76.7, 72.6, 69.8, 54.1, 47.1, 46.8, 34.7, 30.0, 25.4 ppm. Ci9H2iN3NaO4 (M + Na), calcd: 378.1424, found 378.1427.Biological testing

[0082] The inhibitory activities of the synthesized compounds were investigated using recombinant human POP (Table 1, Figures 6-7). The non-covalent inhibitors 14a and 14b were shown to display reduced activity (200 - 350 nM) compared to their covalent counterparts.Installation of the respective nitrile and boronate ester warheads reinforced the high inhibitory potential of these electrophilic groups (0.8 - 7.5 nM). a-ketoamide inhibitor 25 showed slightly higher activity than ZPP-control (formula A). Intermediate 16a which contained the activated ester was tested but deemed to be inactive. Inhibitors 14a / 15a / 17a in which the benzyloxy group is oriented up, display comparable reactivity to their respective diastereomeric counterparts (i.e. benzyloxy position possessing (R)-stereochemistry). Interestingly, the covalent oxygen analogues 31a and 31b demonstrated an order of magnitude less activity (i.e. 3.1 nM compared to 11 nM) with respect to the sulfur inhibitors possessing the same left-hand and right-hand motifs, (i.e. (R)- or (S)-benzyloxy and nitrile warhead). The endo- non-covalent analogue 30b was an order of magnitude less active than the exo- variant 30a. Compound 17a and 25 were determined to be the lead candidates in this series, displaying sub-nanomolar (0.4 - 0.8 nM) activity. The high inhibitory potential of these compounds demonstrates that a-ketoamides and boronate esters are highly compatible with the catalytic serine residue in POP and that rigidification of the LHS of ZPP-control is a successful option when designing POP inhibitors. POP in vitro assays to determine IC50 values were performed as described in Mariaule, Gaelle, et al. "3-Oxo-hexahydro-1 H-isoindole-4-carboxylic Acid as a Drug Chiral Bicyclic Scaffold: Structure-Based Design and Preparation of Conformationally Constrained Covalent and Noncovalent Prolyl Oligopeptidase Inhibitors." Journal of Medicinal Chemistry 59.9 (2016): 4221-4234.Table 1. Inhibitory activities of bicyclic inhibitors against POP>

[0083] Neuron Seeding Assay. The use of immune pluripotent stem cells (iPSCs) and iPSCs derived cells in this research was approved by the McGill University Research Ethics Board (IRB Study Number A03-M19-22A / eRAP 22-03-027). Human iPSC lines used herein are a familial PDpatient iPSC line with a triplication of the aSyn gene (SNCA) locus (SNCA Tri) and its isogenic control line generated by CRISPR-mediated deletion of two copies of SNCA gene (Isog Ctrl). SNCA Tri, and Isog Ctrl hiPSC cultures were maintained as feederfree cultures following the protocols described in Chen, Carol X-Q., et al. "A multistep workflow to evaluate newly generated iPSCs and their ability to generate different cell types." Methods and protocols 4.3 (2021): 50. The final differentiation of the dopaminergic (DA) neuronal progenitor cells (NPCs) into dopamine neurons (DNs) was achieved as described in Shtilbans, Alexander, et al. "Combination of tauroursodeoxycholic acid, co-enzyme Q10 and creatine demonstrates additive neuroprotective effects in in-vitro models of Parkinson’s disease." Frontiers in Neuroscience 18 (2024): 1492028. Pre-formed fibril (PFF) production and quercetin (QC) were performed as Bayati, Armin, et al. "Visualization of a-synuclein trafficking via nanogold labeling and electron microscopy." STAR protocols 4.1 (2023): 102113. DA NPCs were seeded into 96-well plates and maintained in DN maturation media for 2 weeks. After 2- weeks of neuronal maturation, DNs were treated with PFFs (at a final concentration of 500 nM or at concentrations indicated for different experiments) or the aSyn monomers (at the same concentrations as PFFs) and left 14 days in the presence of PFFs or the aSyn monomers. For the small-molecule compound tests, different compounds were added to DNs in addition to PFFs. After the PFF treatment, the DNs were fixed with PFA 4% and newly formed aSyn aggregates were visualized by IF staining with the phosphorylated aSyn antibody as described previously. Images were acquired in triplication on an automated microscope high content system (ThermoFisher Scientific Celllnsight CX7 Pro High-Content Screening Platform) using a 20X objective and analyzed with Neuronal profiling BioApplication (HCS Studio 5.0 Cell Analysis Software, ThermoFisher Scientific). Nuclei were identified in Channel 1 with Hoechst 33342 staining and were used as autofocus reference. Neurites were identified in Channel 2 with TLIBB3 staining through the nNeurite identification module and total neurite areas were set as Region of Interest (ROI). Phosphorylated a-synuclein (pS129-syn) spots (Channel 3) were identified within the ROI mask from Til BBS-positive nNeurite Channel 2. Total fluorescence intensity of pS129-syn spots per field was measured and normalized against total neurite length per field. The normalized total fluorescence intensity of pS129-syn sSpots Channel 3 (phosphorylated a-synuclein) was used as readout for a-synuclein aggregate formation.

[0084] Solubility Assay. Molecule solubility assays were performed in phosphate-buffered saline (1* phosphate buffered saline (PBS) pH 7.4) in triplicate. Incubations were carried out in 0.75 mL Thermo Matrix tubes fitted into a 96-well plate holder by mixing 500 pL of PBS with 10 pL of each compound (10 mg / mL stock in DMSO). Samples were incubated at 37 °C in a CorningLSE shaking incubator set at 130 rpm for 4 h, followed by centrifugation at 2000 x g for 20 min (Eppendorf 5804R). The supernatant was diluted (5 pL into 245 pL) with internal standard (IS) solution containing 50 nM loperamide in 40% acetonitrile, 10% methanol, and 50% water. A further 5-fold dilution was prepared by mixing 20 pL of the first dilution with 80 pL of IS solution.

[0085] LogD7.4 Determination. The partitioning of compounds between octanol and aqueous phases was evaluated to determine LogD in triplicate. Briefly, 10 pL of each compound (10 mg / mL in DMSO) was added to 0.75 mL tubes, followed by 240 pL of saturated octanol. Samples were agitated on a tube shaker (Rocking platform) at 700 rpm for 5 min and then centrifuged at 2000 rpm for 2 min. Subsequently, 250 pL of phosphate-buffered saline (1X PBS, pH 7.4) was added, and the tubes were shaken for 48 h at room temperature. After phase separation by centrifugation (2000 rpm, 5 min), 5 pL of the octanol phase was transferred into a 96-well injection plate containing 245 pL of internal standard (IS) solution (Dilution 1). A second dilution was prepared by mixing 20 pL of Dilution 1 with 80 pL of IS solution. The same procedure was repeated for the aqueous phase; to minimize octanol phase contamination, 100 pL of the aqueous phase was first transferred into a fresh plate before sampling.

[0086] Calibration Curve and Quantification. Calibration curves were prepared for each compound from 10 mg / mL stock solutions, yielding 13 concentration levels (2-750 pg / mL) plus a blank, generated by serial dilution in internal standard (IS) solution. Calibration points were selected for each compound according to sensitivity and expected concentration range. Samples and calibration standards were analyzed by liquid chromatography tandem mass spectrometry (LC-MS / MS), and concentrations were determined from the analyte-to-IS peak area ratio using quadratic regression. Solubility was expressed as the percent ratio of the calculated concentration to the nominal concentration. The distribution coefficient was calculated as the logarithm of the concentration ratio between the octanol and aqueous (PBS, pH 7.4) phases.

[0087] LC-MS / MS Analysis. Samples were analyzed using a Waters l-Class UPLC system coupled to a Xevo™ G2-XS QTof mass spectrometer with electrospray ionization (ESI) operated in positive ion mode. Chromatographic separation was performed on an ACQUITY Premier BEH C18 column (1.7 pm, 2.1 x 50 mm; Waters) maintained at 40 °C, with a 1 pL injection volume. The mobile phases consisted of water with 0.1% formic acid (A) and acetonitrile with 0.1% formic acid (B). The gradient was programmed as follows: 0-0.5 min, 90% A; 0.5-4.5 min, linear change to 50% A; 4.5-4.8 min, linear change to 10% A; 4.8-4.9 min, return to 90% A; and held until 6.0 min for re-equilibration. The flow rate was set to 0.4 mL / min. Mass spectrometric detection wascarried out over an m / z range of 50-1200, with MSE. Source parameters were as follows: capillary voltage, 1.5 kV; source temperature, 150 °C; desolvation temperature, 550 °C; cone gas, 80 L / h; and desolvation gas, 1000 L / h.

[0088] a-synuclein aggregation in neurons. The evaluation of the inhibitors’ protease activity revealed potencies that ranged from micromolar to subnanomolar. While these are not predictive of the effect on a-syn aggregation; relatively weaker inhibitors can be strong modulators of the PPIs and vice versa, at the very least inhibitors must bind strongly enough to POP. The present enzymatic assays do suggest that the inhibitors are bound to the catalytic site, which ultimately influence the conformational dynamics of prolyl oligopeptidase. Hence, the synthesized inhibitors were tested in dopaminergic neurons derived from pluripotent stem cells to probe this effect. To induce a Parkinson’s-like environment, two different cell-types (healthy neurons and SNCA-gene triplication) were allowed to mature over 2-weeks, then spiked with preformed fibrils to stimulate aggregation of a-syn monomers into insoluble oligomers. These primed neurons were then incubated with varied concentrations of the inhibitors and the change in aggregation was measured (Table 2). These inhibitors were tested in a head-head fashion with compound 7 as comparative as it had demonstrated reduction of a-synuclein aggregation in a dose-responsive manner.Table 2: Effect of inhibitors on reduction of a-synuclein aggregation.Neuron types #1: SNCA gene triplication (elevated a-Syn); #2: Control healthy iPSC-derived neurons.

[0089] It is important to note that complete inhibition of aggregation is not required, but rather, reduction of oligomers by a sufficient amount to then undergo macroautophagy in a complementary process. As the data suggests, a-synuclein aggregation was reduced in both cell types which consisted of healthy neurons and those expressing higher concentration of a-synuclein. The reduction was found to be as low and high as 20% and 70%, respectively. Interestingly, inhibitors which demonstrated sub-nanomolar IC50 values (compounds 17, 25) were found to be weaker modulators of the protein-protein interactions with respect to the non-covalent and nitrile derivatives of the fused-thiazolidine scaffold (compounds 14-15). Once again, this illustrates the partial disconnect that exists between the proteolytic activity and protein-protein interactions, and the difficulty in discovering potent modulators of the PPIs. Compound 15a which contained the nitrile warhead was found to reduce aggregation by 68% at concentrations as low as 367 nM in the SNCA triplication cell-line, while the non-covalent encfo-counterpart (compound 14b) showed 22% reduction at similar concentration. The fused-oxazolidine inhibitors (compounds 30-31) also demonstrated impressive reduction of aggregation, albeit at higher concentrations (micromolar). It is important to note that cell viability was unaffected by all tested inhibitors, which further reinforces the potential of targeting prolyl oligopeptidase as a therapeutic target.

[0090] Physicochemical properties. As mentioned earlier, the previous use of [3.3.0]-thiazolidine revealed the metabolic liability of this scaffold. It is proposed herein to address this issue by its substitution for a [3.3.0]-oxazolidine scaffold. In order to confirm the validity of this strategy, preliminary DMPK studies focused on solubility was performed, with logD and microsomal stability assessments (Tables 3 & 4). As shown, select compounds from both the thiazolidine and oxazolidine scaffold demonstrated high solubility at pH 7.4 in combination with favorable logD profiles. Unfortunately, while the steric constraints attributed onto the newly designed thiazolidine scaffold failed to improve clearance, the oxygen analogue remained metabolically stable over several hours. Thus, this observation reinforces the problematic nature of the sulfur as previously mentioned and its successful replacement whilst maintaining a-synuclein aggregation reduction properties. Other parameters such as solubility at different pHs, selectivity for POP over analogous serine proteases from the same S9 family, plasma stability, plasma protein binding, and Caco-2 permeability were measured for compound 31a (Table 5, Figure 8). The data shows that 31a is soluble across a broad pH range, has minimal metabolic turnover in both human and mouse plasma (ti / 2> 240 minutes), and remains in a primarily unbound form. The directionality assay also revealed high permeability in the apical-to-basolateral direction (Papp A — > B = 12.7 ± 0.6 x 106cm / s) with limited to no efflux, while the MDCK-MDR1 cell permeability results suggested minimal P-glycoprotein involvement and high CNS exposure.Table 3: Solubility at pH 7.4 and LogDy^assessment of selected compounds.n = 3 measurementsTable 4: Microsomal stability assessment of selected compounds.n = 2 measurementsTable 5: Properties of compound 31a.>

[0091] Microsomal Stability Assay. Incubations were performed in deep-well 96-well plates. Potassium phosphate buffer with 5 mM MgCh containing mouse or human liver microsomes (0.50 mg protein / mL) was pre-incubated separately with the compounds (2 pM) or positive control (loperamide, 1 pM) in a water bath at a temperature set at 37°C for 15 minutes (N=2). Reactions were initiated by adding NADPH (final concentration 1 mM) in all the wells. Reactions without NADPH were also incubated (final point only) to rule out non-NADPH metabolism or chemical instability in the incubation buffer. Reactions were terminated at 5 time points (0, 5, 15, 30 and 60min) by transferring 50 pL of incubation solution in a new 96 well plate and adding 125 pl of acetonitrile / methanol solvent mixture (80:20, v / v) containing an internal standard (labetalol, 0.5 pM). The plates were centrifuged at 3700 rpm for 10 minutes at4°C, and an aliquot of supernatant (50 pL) was diluted with two volumes of water containing 0.1% formic acid (100 pL) before analysis by LC-MS / MS. Reference samples were also prepared at concentrations between 0.002 and 10 pM in buffer containing microsomes, by adding the diluted solutions of the compound to the buffer containing microsomes quenched with 2.5 volume of solvent containing the internal standard (whereas the incubation To is obtained by quenching the microsomes containing the compound, then adding NADPH).

[0092] Plasma Protein Binding Assay. Dialysis membranes were soaked in Milli-Q™ water for one hour, then transferred into a 20:80 ethanol-water solution for at least 20 minutes. Before use, membranes were rinsed with Milli-Q™ water according to the recommendations of the instrument provider. For each test compound, premixes were prepared by adding 5 pL of the appropriate DMSO stock to 995 pL of matrix and incubating for 20 minutes at 37 °C. Human plasma, mouse plasma, and phosphate buffer were used as matrices. This procedure yielded final concentrations of 1 pM propranolol and 5 pM 31a, and 0.1 pM propranolol and 0.5 pM of each test compound in buffer. Following the pre-incubation, 150 pL of the spiked plasma premix were transferred into the upper compartments of the equilibrium dialysis plate, and 150 pL of phosphate buffer were added to the corresponding bottom compartments, in triplicate for each matrix and compound. The sealed HTDialysis plate was incubated for 5 hours at 37 °C under a 95 / 5 O2 / CO2atmosphere with gentle mixing at 50 rpm. At the end of the incubation, 50 pL were collected from both the plasma side and the buffer side for bioanalysis as described below. For each test compound, control samples consisting of compound in buffer were dialyzed against buffer at one-tenth of the plasma concentration in order to evaluate potential non-specific binding to the system. Non-dialyzed samples kept at 4 °C and at 37 °C were analyzed in parallel to assess overall recovery and potential instability during the incubation period.

[0093] Plasma Extraction. To 50 pL of plasma or buffer sample, 125 pL of protein precipitation solution were added. The precipitation solvent consisted of acetonitrile and methanol in an 80 / 20 volume ratio and contained loperamide at 25 nM as the internal standard. After mixing, the plate was centrifuged for 10 minutes at 3500 rpm and 4 °C. An aliquot of 50 pL of the supernatant was transferred into an HPLC plate and 100 pL of water containing 0.1 percent formic acid were added prior to LC-MS / MS analysis.

Claims

WHAT IS CLAIMED IS:

1. A compound of formula 1:wherein X is O or S and R is an electrophilic covalent warhead group.

2. The compound of claim 1 , wherein R is a warhead group that comprises nitrile, a boronic ester or an a-ketoamide motif.

3. The compound of claim 1 or 2, wherein the compound is of formula 2:wherein X is as defined in claim 1.

4. The compound of claim 1 or 2, wherein the compound is of formula 3:Wherein X is as defined in claim 1 and each R2 is independently H or -O-CH3.

5. The compound of claim 1 or 2, wherein the compound is selected from the group consisting of:

6. A pharmaceutical composition comprising a compound as defined in any one of claims 1 to 5 and a pharmaceutically acceptable excipient.

7. Use of the compound as defined in any one of claims 1 to 5, for the treatment of Alzheimer’s disease or Parkinson’s disease.

8. Use of the compound as defined in any one of claims 1 to 5, for the manufacture of a medicament for the treatment of Alzheimer’s disease or Parkinson’s disease.

9. Use of the compound as defined in any one of claims 1 to 5, for inhibiting autophagy.

10. Use of the compound as defined in any one of claims 1 to 5, for the manufacture of a medicament for inhibiting autophagy.

11. The compound as defined in any one of claims 1 to 5 for use in a method of treating Alzheimer’s disease or Parkinson’s disease.

12. The compound as defined in any one of claims 1 to 5, for manufacturing a medicament for the treatment of Alzheimer’s disease or Parkinson’s disease.

13. The compound as defined in any one of claims 1 to 5, for use in a method of inhibiting autophagy.

14. The compound as defined in any one of claims 1 to 5, for use in the manufacture of a medicament for inhibiting autophagy.

15. A method of treating Alzheimer’s disease or Parkinson’s disease in a subject in need thereof, the method comprising administering a therapeutically effective amount of the compound as defined in any one of claims 1 to 5 to the subject in need thereof.

16. A method of inhibiting autophagy in a subject in need thereof, the method comprising administering a therapeutically effective amount of the compound as defined in any one of claims 1 to 5 to the subject in need thereof.

17. A method of treating Alzheimer’s disease or Parkinson’s disease in a subject in need thereof, the method comprising administering a therapeutically effective amount of the pharmaceutical composition as defined in claim 6 to the subject in need thereof.

18. A method of inhibiting autophagy in a subject in need thereof, the method comprising administering a therapeutically effective amount of the pharmaceutical composition as defined in claim 6 to the subject in need thereof.

19. A method for treating a condition or disease where prolyl opligopeptidase (POP) is overexpressed and where a reduced POP activity is desired in a subject in need thereof, the method comprising administering a therapeutically effective amount of the composition as defined in claim 6 to the subject in need thereof.

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