Design, Synthesis and Biological Evaluation of Isoxazole-Based CK1 Inhibitors Modified with Chiral Pyrrolidine Scaffolds

Abstract

In this study, we report on the modification of a 3,4-diaryl-isoxazole-based CK1 inhibitor with chiral pyrrolidine scaffolds to develop potent and selective CK1 inhibitors. The pharmacophore of the lead structure was extended towards the ribose pocket of the adenosine triphosphate (ATP) binding site driven by structure-based drug design. For an upscale compatible multigram synthesis of the functionalized pyrrolidine scaffolds, we used a chiral pool synthetic route starting from methionine. Biological evaluation of key compounds in kinase and cellular assays revealed significant effects of the scaffolds towards activity and selectivity, however, the absolute configuration of the chiral moieties only exhibited a limited effect on inhibitory activity. X-ray crystallographic analysis of ligand-CK1δ complexes confirmed the expected binding mode of the 3,4-diaryl-isoxazole inhibitors. Surprisingly, the original compounds underwent spontaneous Pictet-Spengler cyclization with traces of formaldehyde during the co-crystallization process to form highly potent new ligands. Our data suggests chiral “ribose-like” pyrrolidine scaffolds have interesting potential for modifications of pharmacologically active compounds.

1. Introduction

To date, many drug discovery programs have been based on commercially or readily available building blocks and reagents to enable rapid synthesis of large compound libraries destined for various screening campaigns [1]. This approach often resulted in collections of large, “flat”, achiral and low-diverse molecules [2,3]. In this context, the “quality” of compounds and building blocks in terms of complexity, carbon bond saturation, chirality or natural product-likeness has been intensively discussed in the medicinal chemistry community [4].

In the field of protein kinases, most small molecule kinase inhibitors (SMKI) target the adenosine triphosphate (ATP) binding site in the catalytic cleft of the kinase (Figure 1A). Depending on their particular binding mode, several different types of inhibitors have been categorized [5]. Typical ATP-competitive kinase inhibitors (type I inhibitors) generally consist of a central nitrogen-containing heterocycle that is appended with mainly hydrophobic, rigid and achiral side chains. This design originates from Traxler’s pharmacophore model that defines distinct subsites within the ATP binding pocket, which can be sampled to develop selective inhibitors (Figure 1B) [6].

Usually the central heterocycle binds to the “adenine region” of the ATP binding site and undergoes hydrogen bonding to the so-called hinge region of the kinase, whereas the appended side chains of the heterocycle facilitate binding interactions within the “hydrophobic pocket I” and the solvent-exposed “hydrophobic region II” [5,6]. This approach inevitably gives rise to lipophilic, “flat” compounds that are often found to come with poor pharmakokinetic ADME parameters at the later stages of drug development [8].

In contrast, the more polar regions of the ATP binding site (ribose pocket and phosphate-binding region) have been exploited less frequently. This may be because binding to these hydrophilic water-exposed areas does not necessarily translate into an increased binding enthalpy of an inhibitor. Therefore, these regions are often only used to implement water-solubilizing groups (e.g., functionalized tetrahydropyran (1), pyrrolidine (2) or piperidine moieties) that enhance pharmacokinetic parameters, such as oral bioavailability (Figure 2) [9,10,11,12,13]. However, residues that comprise the ribose pocket have also been successfully targeted through stereochemically defined interactions, to create highly potent and selective inhibitors [14,15,16,17,18]. Examples include Pfizer’s interleukin-1 receptor-associated kinase 4 (IRAK4) inhibitor PF06650833 (3) [18], which is equipped with a chiral pyrrolidone scaffold, Merck’s carboribose-functionalized diaminopyrimidine derivative 4 [17] and a series of p38 mitogen-activated protein kinase (MAPK) inhibitors (e.g., 5) developed by Laufer and co-workers [16]. A special case is the well-known microbial alkaloid staurosporine (6) that targets the ribose pocket with a chiral “glycosyl” subunit (cf. Figure 2). This moiety is reported to contribute to the high potency, but also to the very high kinase promiscuity of staurosporine (6) [19,20,21,22,23]. Interestingly, modification of the sugar-like moiety of several closely related indolocarbazole analogs resulted in enhanced protein kinase selectivity profiles (e.g., 7) [24,25,26]. Taken together, these examples illustrate the potential of a stereochemical approach to developing potent and selective kinase inhibitors [19,27,28].

In 2009, our group reported isoxazole 8 (Scheme 1) as a nanomolar inhibitor of the protein kinase CK1δ [IC₅₀ (CK1δ) = 0.033 µM] [29]. CK1δ is a Ser/Thr-specific protein kinase and is one of seven mammalian CK1 isoforms (α, β, γ1, γ2, γ3, δ and ε) within the CK1 family. All CK1 isoforms share a highly conserved kinase domain [30] and phosphorylate a high number of substrates, including the tumor suppressor protein p53 [30,31]. Based on the wide spectrum of substrate proteins CK1 family members play an important role in the diverse signaling pathways that are involved in cell division, apoptosis, membrane transport, immune response and inflammation, spindle and centrosome-associated processes, DNA damage-related signal transduction and circadian rhythm. Deregulation and dysfunction of CK1 isoforms are associated with proliferative disorders, such as cancer [32]. Moreover, mutations, as well as disorders of CK1 expression and activity, have been observed in neurodegenerative diseases, such as Alzheimer’s or Parkinson’s disease, as well as sleeping disorders [32,33].

In the present study, we have modified diaryl-isoxazole 8 with functionalized enantiopure pyrrolidine scaffolds (Scheme 1) to promote selective binding interactions in the more hydrophilic areas of the ATP binding pocket. These kinds of pyrrolidines scaffolds have been frequently used in various medicinal chemistry projects, including the development of glycosidase inhibitors, azanucleosides and antiviral agents [34,35,36,37,38]. Especially hydroxy-functionalized pyrrolidines are remarkable scaffolds, as they can be regarded as sugar analogues with the furanose ring oxygen substituted by a nitrogen atom. Due to their structural and chemical similarity these “iminosugars” are effective carbohydrate mimics and are ideal scaffolds to address carbohydrate-related targets, including nucleoside or nucleotide-binding enzymes. In line with this notion, “iminosugars” provide access to polar active sites and thus to a chemical space that is usually inaccessible to Lipinski-compliant molecules [34].

2. Results and Discussion

2.1. Molecular Modelling

To understand how the lead compound 8 could be elaborated to exploit residues in the ribose pocket, compound 8 was docked into a CK1δ-PF670462 co-crystal structure (pdb: 3UZP). According to the predicted binding mode the amidopyridinyl moiety occupies the adenine region of the ATP binding site and forms a bidentate interaction with the backbone NH and carbonyl oxygen of Leu-85 (Figure 3). The isoxazole ring is predicted to pack between the sidechains of Ile-23 and Ile-148, and to form a hydrogen bond between the ring nitrogen atom and a structural water. The p-fluorophenyl substituent packs tightly in the hydrophobic pocket I, formed between the sidechains of Lys-38, Met-80 and the gate keeper residue Met-82. This characteristic “teardrop”-shaped [39] binding mode agrees with other related 4,5-diarylimidazoles that have been crystallized with CK1δ, as well as p38α MAPK [40]. Regarding isoxazole 8 this pharmacophore is extended towards the solvent-exposed hydrophobic region II by a cinnamic acid moiety. This configuration is projecting the aryl system to the solvent exposed area but leaving space for further interactions within the actual binding pocket.

To extend the pharmacophore of lead compound 8 towards the ribose pocket we replaced the susceptible double bond with a five-membered pyrrole ring to mimic and to rigidify the E-configured cinnamic acid moiety. Furthermore, the pyrrole nitrogen was used as a handle to attach the chiral scaffolds (Scheme 1). Docking of the designed ligands into CK1δ revealed that the key pharmacophore interactions described above are maintained. Additionally, the chiral scaffolds are predicted to pack between Ile-15 and Asp-91 and to crosslink the β1b strand of the N-terminal lobe and the αB helix of the C-terminal lobe with H-bonds (Figure 4). Asp-91 is one of three amino acid residues of CK1δ (beside Asp-132 and Ser-88) that are deemed to be involved in ribose binding of ATP [42,43]. While compound 29d is predicted to form H-bonds between its hydroxyl groups and the carboxyl group of Asp-91 (Figure 4A), enantiomer 29e is predicted to bind to Asp-91 with the NH-group of the pyrrolidine ring (Figure 4B). Moreover, H-bonding to the N-terminal carbonyl group of Ile-15 is predicted for both enantiomers. However, a clear stereochemical preference was not observed by scoring function [GScore −14.1 (29d) vs −13.8 (29e)].

2.2. Chemistry

The synthesis of our envisaged inhibitors began with the preparation of the two enantiomerically pure iminoribitol derivatives (−)-20 and (+)-20 (Scheme 2). Whilst (+)-20 was solely prepared from d-methionine its enantiomer (−)-20 was either synthesized from l-methionine or, particularly at larger scale, from the available 2,3-O-isopropylidene-l-lyxono-1,4-lactone, following published procedures [44,45,46,47,48,49]. Starting from the amino acids, d- and l-methionine were firstly converted into the corresponding tert-Butyloxycarbonyl (Boc) protected derivatives [50]. Upon reduction of the carboxylic acid functions [51] and subsequent tert-butyldiphenylsilyl (TBDPS)-protection of the primary alcohols (S)-10 and (R)-10 [52] sulfides (S)-11 and (R)-11 were oxidized to the sulfoxides (S)-12 and (R)-12 with meta-chloroperoxybenzoic acid (m-CPBA) [53,54]. Thermal syn-elimination [52] of sulfoxides (S)-12 and (R)-12 yielded the vinylglycinol intermediates (S)-14 and (R)-14 which underwent N-allylation to (S)-15 and (R)-15 when reacted with sodium hydride and allyl bromide. The allylation reaction, however, turned out to be capricious as higher yields were only achieved by repeated addition of the same amounts of sodium hydride and allyl bromide. Attempts to deploy potassium tert-butoxide as a base did not lead to any satisfactory product formation in our hands and other reaction conditions were reported to be unsuccessful [55,56]. The intramolecular cross-metathesis reaction of the N-allylvinylglycinol precursors (S)-15 and (R)-15 using benzylidene-bis(tricyclohexylphosphino)dichlororuthenium (Grubbs 1^st-generation catalyst) then yielded the 3,4-dehydroprolinol intermediates (S)-16 and (R)-16 [55,56,57]. The optical purity of the vinylglycinol intermediates (S)-14 and (R)-14 and the 3,4-dehydroprolinol intermediates (S)-16 and (R)-16 was analyzed by asymmetric high-performance liquid chromatography (HPLC) and determined to be > 99%. Employing established protocols (S)-16 and (R)-16 were then dihydroxylated with catalytic osmium tetroxide in the presence of 4-methylmorpholine N-oxide (NMO) and the so formed diols (−)-17 and (+)-17 were subsequently acetal protected with 2,2-dimethoxypropane (DMP) in the presence of catalytic amounts of para-toluenesulfonic acid (p-TSOH) to afford the fully protected iminoribitol derivatives (−)-18 and (+)-18 [57,58]. Finally, the silyl protecting groups were cleaved with tetra-n-butylammonium fluoride (TBAF) and the resulting primary alcohols (−)-19 and (+)-19 were activated as their mesylates (−)-20 and (+)-20 for coupling with the pyrrole building blocks 24a–c (Scheme 3) [47,48,49].

The required pyrrole building blocks 24a,b (Scheme 3) were prepared from commercially available ethyl 4-bromopyrrole-2-carboxylate (21) in two steps following procedures published by Handy et al. [40,59,60,61]. Previous studies showed that the use of unprotected pyrrole 21 in the envisaged Suzuki-Miyaura coupling results in extensive dehalogenation [61]. Accordingly, pyrrole 21 was initially N-Boc protected and subjected to palladium-catalyzed coupling with boronic acids 23a and 23b to give the desired pyrrole building blocks 24a and 24b. Now, mesylates (−)-20 and (+)-20, and pyrrole building blocks 24a,b were joint together in the presence of cesium carbonate at 80 °C to yield compounds 25d–f (Scheme 3) [62]. The enantiomeric purity of intermediates 25d and 25e was analyzed by asymmetric HPLC and determined to be >99%. In addition, the three-dimensional structure of 25d was confirmed by X-ray crystallographic analysis (Figure S1). To synthesize inhibitor 29c without aryl substituent attached to the pyrrole ring 1H-pyrrole-2-carboxylate 24c [63] was coupled with mesylate (−)-20 employing the same methodology as before to afford compound 25c. Furthermore, we aimed to prepare inhibitors 28a,b in which the iminosugar unit is replaced by a methyl group. For this purpose, pyrrole building blocks 24a,b were methylated under standard reaction conditions to give compounds 25a,b.

Following saponification of the esters 25a–f the isoxazole building block 30 [34] was coupled stepwise to the corresponding carboxylic acids 26a–f via their N-hydroxybenzotriazole (HOBt)-activated esters 27a–f (Scheme 4) [64]. Thus, coupling products 28a–f were obtained in yields of up to 67%. Finally, cleavage of the protecting groups under acidic reaction conditions furnished 29c–f.

2.3. Biological Evaluation

Compounds 28a–c and 29d–f were initially screened in in-vitro kinase assays at a concentration of 10 µM for their ability to inhibit CK1δ and CK1ε. Compounds that showed only low residual activity at this concentration were taken forward to measure IC₅₀ values (

Table 1. Activity and IC₅₀ values of compounds 28a,b and 29c–f in CK1δ and CK1ε kinase assays. Results are presented as mean ± SD from experiments performed in triplicate (n = 3).

#	R1	R2	CK1δ		CK1ε
			Residual Activity [%] at 10 µM ± SD	IC50 [µM] ± SD	Residual Activity [%] at 10 µM ± SD	IC50 [µM] ± SD
28a		Me	48 ± 7	n.d.	70 ± 21	n.d.
28b		Me	48 ± 4	n.d.	47 ± 11	n.d.
29c	H		7.7 ± 0.2	0.45 ± 0.07	18 ± 1	1.5 ± 0.3
29d			2.9 ± 0.3	0.038 ± 0.003	12 ± 2	0.065 ± 0.012
29e			3.4 ± 0.7	0.047 ± 0.004	14 ± 3	0.063 ± 0.009
29f			2.8 ± 0.5	0.037 ± 0.004	9.7 ± 1.6	0.1 ± 0.014

). In accordance with our molecular modelling studies 29d and 29e showed high activity in the CK1δ and CK1ε in-vitro kinase assays with IC₅₀ values in the nanomolar range. Interestingly, no preference was observed between chiral moieties of 29d and 29e, while replacement of the pyrrolidine scaffolds by a methyl (28a and 28b) ablated activity against CK1δ and CK1ε. Omitting the aryl moiety (29c) also resulted in a considerable decrease in activity relative to 29d and 29e in accordance with previously reported CK1δ inhibitors [40]. Taken together, both the aryl moiety and the pyrrolidine scaffold seem to contribute to the binding affinity. On the one hand, interactions between the pyrrolidine scaffold and the active site might could play an important role in determining inhibitor engagement with the active site. On the other hand, the molecular configuration of the scaffolds (29d vs 29e) does not seem to contribute to affinity and CK1-isoform selectivity.

In addition to the in-vitro kinase assays, all compounds were tested in cell viability assays against tumor cell lines. Since inhibition of CK1δ prolongs the survival of SV40 T-Ag/mutant CK1δ bitransgenic mice [65] and overexpression of CK1δ correlates with reduced survival rates of colorectal and breast cancer patients [66], we chose HT-29 and MCF-7 cells for biological testing [67,68]. These two cell lines are characterized on a molecular level in detail, exhibiting alterations in WNT and p53 signaling pathways, both being regulated by CK1 isoforms [30,69]. All compounds were initially screened at 5 µM, 10 µM and 20 µM, respectively (

Table 2. EC₅₀ values and cell viability of MCF-7 and HT-29 cells after treatment with compounds 28a,b and 29c–f. Results are presented as mean ± SD from experiments performed in triplicate (n = 3).

	MCF-7				HT-29
#	Cell Viability [%]			EC50 [µM]	Cell Viability [%]			EC50 [µM]
	5 µM	10 µM	20 µM		5 µM	10 µM	20 µM
28a	103 ± 16	88 ± 9	54 ± 5	n.d.	111 ± 19	98 ± 22	78 ± 10	n.d.
28b	89 ± 13	84 ± 11	79 ± 12	n.d.	94 ± 23	59 ± 14	52 ± 10	n.d.
29c	90 ± 10	81 ± 5	n.d.	39 ± 12	80 ± 16	78 ± 11	n.d.	24 ± 6
29d	100 ± 16	76 ± 19	5.6 ± 1.5	10 ± 2	76 ± 13	51 ± 8	14 ± 2	8.0 ± 1.7
29e	90 ± 27	58 ± 18	8.0 ± 3.4	10 ± 2	75 ± 13	42 ± 10	11 ± 3	8.8 ± 0.8
29f	87 ± 5	33 ± 7	3.9 ± 0.2	9.6 ± 0.8	50 ± 10	25 ± 5	9.4 ± 3.0	5.4 ± 0.4

). EC₅₀ values were determined for compounds that showed a clear reduction in cell viability. There is a trend between the EC₅₀ data for both cell lines and enzyme inhibitory data against CK1, with compounds 29d–f exhibiting a modest effect on both, HT-29 and MCF-7 cell viability. These results are consistent with previous findings that inhibitory effects of CK1 specific inhibitors are dependent on the cellular background [29,70,71,72]. Especially alterations in WNT and p53 signaling influence the effects of CK1 specific inhibitors [30,73]. The modest inhibitory effect on cancer cell viability may have several reasons: (i) The hydrophilic character of the pyrrolidine scaffolds might reduce their cellular uptake; (ii) efflux systems could contribute to a decreased availability of the inhibitors, (iii) compounds could be partly metabolized (e.g., the hydroxy groups of the pyrrolidine scaffolds) and, (iv) since these inhibitors are ATP competitive inhibitors and cellular ATP concentrations are much higher than the ATP concentration used in the in-vitro assays, higher compound concentrations are necessary to inhibit CK1δ within cells.

Next, we screened effective inhibitor 29d in a panel of 320 kinases at a concentration of 1 µM to examine its selectivity (Figure 5). Apart from CK1δ (residual activity = 1%) and CK1ε (residual activity = 4%) 29d hits only four other kinases, namely CK1α (residual activity = 16%), JNK2 (40%), JNK3 (15%), p38α (17%) thus resulting in an excellent selectivity score of 0.02 (number of kinases with residual activity < 50%/total number of tested kinases).

2.4. X-ray Analysis of ligand-CK1δ Complexes

In the next step we sought to elucidate structural details of the binding of our chiral inhibitors via X-ray crystallography. For this, a C-terminally truncated version of CK1δ (1-294, tCK1δ) together with 29d and 29e, respectively, were used in co-crystallization trials. Crystals suitable for diffraction analysis grew in presence of both compounds in crystallization conditions containing 0.1 M MES (pH = 5.5), 10% polyethylengylcol (PEG) 4000 and 0.2 M Li₂SO₄. These crystals diffracted to a resolution limit of 1.8 Å and belong to the monoclinic crystal system. In each asymmetric unit, two tCK1 molecules were present with an isoxazole-based ligand bound to each ATP binding pocket (Figure 6).

Surprisingly, the ligands in both protein-ligand complexes proved to be different from the actual target compounds submitted to co-crystallization. Since all analytical data for the originally employed inhibitors was consistent with the given structures, we postulate that the incorporation of an additional methylene group occurred via spontaneous Pictet-Spengler cyclization [74,75,76] during crystallization. In line with this notion, PEG, which has been used in our soaking solutions, is known to generate formaldehyde traces that have been shown in the past to affect the derivatization of both, proteins and ligands [77,78,79]. To further corroborate this hypothesis, we examined whether simple exposure of 29d/e to formaldehyde would give rise to the same Pictet-Spengler products 31a/b (Scheme 5). Indeed, when compounds 29d/e were treated with formaldehyde at room temperature 31a/b were formed readily and the obtained spectroscopic data was consistent with the predicted Pictet-Spengler products. In addition, aqueous solutions of 29d and 29e with and without PEG were analyzed by liquid chromatography-mass spectrometry (LC-MS) over a period of 7 days. Only in those solutions that had PEG present significant product formation of 31a and 31b was observed indicating that the degradation of PEG may have been the likely source of formaldehyde.

Following the discovery of new ligands as artefacts in the X-ray complexes, both these Pictet-Spengler products 31a and 31b were re-synthesized and subsequently assessed for their biological activities (

Table 3. Biological activity of compound 31a and 31b in in-vitro kinase assays (CK1δ and CK1ε) and cellular assays (MCF-7- and HT-29). Results are presented as mean ± SD from experiments performed in triplicate (n = 3).

#	R	IC50 (CK1δ) [µM] ± SD	IC50 (CK1ε) [µM] ± SD	EC50 (MCF-7) [µM] ± SD	EC50 (HT-29) [µM] ± SD
31a		0.011 ± 0.001	0.056 ± 0.005	1.3 ± 0.2	1.1 ± 0.1
31b		0.024 ± 0.004	0.196 ± 0.026	1.9 ± 0.3	1.4 ± 0.2

). In the in-vitro kinase assays both compounds exhibited nanomolar activity against CK1δ and CK1ε with a five-fold preference for CK1δ over CK1ε. The cellular assays revealed 31a and 31b to be more potent than the originally designed compounds 29d and 29e with EC₅₀ values below 2 µM. The higher cellular potency of both compounds is in a way surprising, because in contrast to compounds 29d and 29e the chiral moieties of the Pictet-Spengler products 31a and 31b are too far away to interact with residues in the ATP binding site (Figure 6). Further studies will need to elucidate compound’s kinase selectivity, and also their specificity in terms of addressing other targets. However, our results suggest that the chiral pyrrolidine scaffold attached to kinase inhibitors (and beyond) can be suitable for medicinal chemistry applications.

3. Materials and Methods

3.1. Molecular Modelling

Molecular modelling was performed on a DELL Precision T3610 four core workstation using Schrödinger Maestro, version 10.4, 2015, Schrödinger, LLC, New York, NY, USA. The protein crystal structure of CK1δ was used from the RCSB protein data bank www.rcsb.org (pdb 3UZP [41]) and prepared with the Protein Preparation Wizard 2015-4 (Epik version 2.4, Schrödinger, LLC, 2015; Impact version 5.9, Schrödinger, LLC, 2015; Prime version 3.2, Schrödinger LLC, 2015) regarding assignment of bond orders, addition of hydrogen atoms, identification of disulfide bonds and conversion of artificial selenomethionines to methionines (default settings) [80]. Designed ligands were minimized with MacroModel, version 11.0, Schrödinger, LLC, 2015 using an OPLS2005 force field. Ligand docking and receptor grid generation were performed with Glide, version 6.9, Schrödinger, LLC, 2015 using the standard protocol [81,82,83].

3.2. Chemistry

3.2.1. General Experimental Procedures

Melting points were determined either on a Stuart Melting Point (SMP3) apparatus and are uncorrected, or by differential scanning calorimetry (DSC) on a Mettler Toledo DSC1 instrument at a heating rate of 10 K·min⁻¹. Proton (¹H) and carbon (¹³C) NMR-spectra were recorded on Bruker Avance (III)-500 or Avance (I)-300 spectrometers. ¹⁹F NMR spectra were recorded at 470 MHz and are reported unreferenced. Chemical shifts are reported in ppm relative to Me₄Si (TMS, δ 0), or residual solvent peaks as an internal standard set to δ 7.26 and 77.00 (CDCl₃), or δ 3.34 and 49.05 (MeOD), or δ 2.50 and 39.43 (d₆-DMSO). NMR data is reported as follows: Chemical shift in ppm, multiplicity (ap = apparent, s = singlet, d = doublet, t = triplet, q = quartet, sp = septet, br = broad, dd = doublet of doublets, td = triplet of doublets, dt = doublet of triplets, m = multiplet), coupling constant in Hz, integration.

Electrospray ionization (ESI) mass spectrometry (MS) experiments were performed on a QTOF Premier mass spectrometer (Micromass, UK) under normal conditions. Sodium formate solution was used as a calibrant for high resolution mass spectra (HRMS) measurements. Elemental microanalyses were performed at The Campbell Microanalytical Laboratory, Department of Chemistry at the University of Otago on a Carlo-Erba EA 1108 elemental analyzer. Specific optical rotations were acquired on a Rudolph Autopol^® IV Automatic polarimeter at ambient temperature (20 °C), unless otherwise stated, λ = 589 nm and concentration (g/100 mL) in the solvent indicated, using a cell of 100 mm path length.

All reactions were monitored by thin layer chromatography (TLC) using 0.2 µm silica gel (Merck Kieselgel 60 F₂₅₄) precoated aluminium plates, using UV light, ammonium molybdate, ninhydrin or potassium permanganate staining solution to visualize. Flash column chromatography was performed on Davisil^® silica gel (60, particle size 0.040–0.063 mm), or using Reveleris^® silica or C-18 reversed phase flash cartridges on a Grace Reveleris^® automated flash system with continuous gradient facility. Solvents for reactions and chromatography were analytical grade and were used as supplied unless otherwise stated. 1-(tert-Butyl) 2-Ethyl 4-bromo-1H-pyrrole-1,2-dicarboxylate (22) [59], tert-Butyl (S)- and (R)-[1-hydroxy-4-(methylthio)butan-2-yl]carbamate [(S)-10 and (R)-10] [50,51] methyl 1H-pyrrole-2-carboxylate (24c) [63] and 4-[3-(4-fluorophenyl)-5-isopropylisoxazol-4-yl]pyridin-2-amine (30) [34] were prepared following literature procedures.

Chiral and achiral high-performance liquid chromatography (HPLC) analyses were performed on an Agilent 1100 Quaternary pump HPLC system (Waldbronn, Germany) with a diode array detector (200–400 nm), employing columns as indicated in the supporting information. Injection volumes were typically 10 μL (1–2 mg·mL⁻¹) and data was processed with Agilent Cerity System software.

LC-MS was performed with a Bruker Esquire ~LC ion trap mass spectrometer (Bremen, Germany) in the positive ion mode (dry gas 9 L/min, nebulizer 35 psi, drying temperature 350 °C) after chromatographic separation using an Agilent 1100 HPLC system (Waldbronn, Germany) with a RP-8 column (Agilent Eclipse XDB-C8, 150 × 4.6 mm, 5 μm) and a 0.1% acetic acid/acetonitrile gradient.

3.2.2. Syntheses

(S)-tert-Butyl 1-(tert-butyldiphenylsilyloxy)-4-(methylthio)-butan-2-ylcarbamate [(S)-11]. To a solution of alcohol (S)-10 (17.4 g, 73.9 mmol) in dry DMF (110 mL) was added imidazole (12.6 g, 185 mmol) and TBDPSCl (21.2 mL, 81.5 mmol), and the resulting reaction mixture was stirred at room temperature overnight. Water (500 mL) was added and the mixture extracted with ethyl acetate (3×). The combined organic layers were washed with water (2×) and brine, dried over MgSO₄ and concentrated. The crude product was purified by flash column chromatography (silica gel, ethyl acetate/petroleum ether 1:19, 1:9 and 1:6) to yield the desired product (S)-11 quantitatively as a colorless oil. ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{23}$ = −11.7 (c 1.20, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 7.66–7.61 (m, 4H), 7.45–7.34 (m, 6H), 4.76–4.64 (m, 1H), 3.81–3.65 (m, 2H), 3.61 (dd, 10.0, 3.0 Hz, 1H), 2.53–2.42 (m, 2H), 2.07 (s, 3H), 1.90–1.72 (m, 2H), 1.44 (s, 9H), 1.07 (s, 9H); ¹³C NMR (125 MHz, CDCl₃) δ 155.53, 135.58, 135.56, 133.26, 133.20, 129.82, 129.79, 127.77, 79.22, 65.62, 51.38, 31.75, 30.77, 28.41, 26.91, 19.32, 15.52. HRMS (ES+) m/z calcd for C₂₆H₃₉NO₃SSiNa⁺ 496.2312, found 496.2307.

tert-Butyl {(2S)-1-[(tert-butyldiphenylsilyl)oxy]-4-(methylsulfinyl)butan-2-yl} carbamate [(S)-12]. To a solution of (S)-11 (1.03 g, 2.17 mmol) in dichloromethane (46 mL) was added a solution of m-CPBA (50–55%; 750 mg, 2.17 mmol) in dichloromethane (4.5 mL) dropwise at −20 °C. After being stirred for 1 h at this temperature the reaction mixture was allowed to warm to 0 °C and was quenched with saturated sodium carbonate solution. The organic layer was separated, washed another two times with saturated sodium carbonate solution, dried over MgSO₄ and concentrated. The residue was purified by flash column chromatography (silica gel, ethyl acetate/petroleum ether 3:7 and 1:0) to give 737 mg (69%) of the sulfoxide (S)-12 as a light-yellow heavy oil that solidified on standing. In addition, 211 mg (19%) of the sulfone (S)-13 were isolated as a colorless solid.

tert-Butyl {(2S)-1-[(tert-butyldiphenylsilyl)oxy]-4-(methylsulfinyl)butan-2-yl}carbamate [(S)-12]. ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ = −9.3 (c 0.55, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 7.65–7.61 (m, 4H), 7.46–7.36 (m, 6H), 4.86–4.71 (m, 1H, NH) 3.85–3.61 (m, 3H), 2.78–2.64 (m, 2H), 2.54 (s, 1.5H), 2.53 (s, 1.5H), 2.07–1.90 (m, 2H), 1.44 (s, 9H), 1.07 (s, 9H); ¹³C NMR (125 MHz, CDCl₃) δ 155.72, 155.66, 135.55, 135.53, 133.00, 132.93, 129.91, 129.89, 127.83, 79.52, 65.85, 65.81, 51.70, 51.40, 51.01, 38.70, 38.65, 28.36, 26.90, 25.73, 25.22, 19.28; HRMS (ES+) m/z calcd for C₂₆H₃₉NO₄SSiNa⁺ 512.2261, found 512.2261.

tert-Butyl (S)-{1-[(tert-butyldiphenylsilyl)oxy]-4-(methylsulfonyl)butan-2-yl}carbamate [(S)-13]. m.p.: 95 °C; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ = −6.3 (c 1.115, CHCl₃); ¹H NMR (500 MHz, CDCl₃) δ 7.65–7.60 (m, 4H), 7.47–7.37 (m, 6H), 4.75 (d, 7.4 Hz, 1H), 3.80–3.67 overlapping signals (m, 1H and 3.71, dd, 10.4, 4.1 Hz, 1H), 3.63 (dd, 10.3; 3.8 Hz, 1H), 3.10–2.99 (m, 2H), 2.88 (s, 3H), 2.11–1.97 (m, 2H), 1.44 (s, 9H), 1.08 (s, 9H); ¹³C NMR (125 MHz, CDCl₃) δ 155.64, 135.57, 135.53, 132.87, 132.78, 130.00, 129.96, 127.90, 127.87, 79.78, 65.71, 51.99, 50.87, 40.69, 28.34, 26.90, 25.19, 19.27; HRMS (ES+) m/z calcd for C₂₆H₃₉NO₅SSiNa⁺ 528.2210, found 528.2202.

tert-Butyl (S)-{1-[(tert-butyldiphenylsilyl)oxy]but-3-en-2-yl}carbamate [(S)-14]. A mixture of sulfoxide (S)-12 (29.9 g, 61.1 mmol) and calcium carbonate (14.7 g, 147 mmol) in 1,2-dichlorobenzene (165 mL) was heated under reflux for 7 h (TLC analysis). After being cooled to room temperature the mixture was filtered through a pad of celite and the celite was additionally washed with ethyl acetate. The filtrate was concentrated to small volume and the residue was purified by flash column chromatography to afford 18.4 g (71%) of vinylglycinol (S)-14 as a light-yellow oil. ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{22}$ = −29.5 (c 1.18, CHCl₃), Ref. [56]: ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{25}$ = −27.7 (c 1.02, CHCl₃); ¹H NMR (300 MHz, CHCl₃) δ 7.67–7.61 (m, 4H), 7.46–7.33 (m, 6H), 5.84 (ddd, 17.2, 10.4, 5.4 Hz, 1H), 5.22 (dt, 17.3, 1.4 Hz, 1H), 5.16 (dt, 10.5, 1.4 Hz, 1H), 4.86–4.73 (m, 1H), 4.30–4.16 (m, 1H), 3.74 (dd, 10.1, 4.4 Hz, 1H), 3.64 (dd, 10.1, 4.6 Hz, 1H), 1.45 (s, 9H), 1.06 (s, 9H); ¹³C NMR (75 MHz, CHCl₃) δ 155.42, 136.48, 135.62, 135.56, 133.30, 133.19, 129.77, 127.72, 115.63, 79.35, 66.05, 54.41, 28.41, 26.84, 19.31; HRMS (ESI) m/z calcd for C₂₅H₃₅NO₃SiNa⁺ 448.2278, found 488.2589.

tert-Butyl (S)-allyl{1-[(tert-butyldiphenylsilyl)oxy]but-3-en-2-yl}carbamate [(S)-15]. To a solution of vinylglycinol (S)-14 (18.3 g, 43.0 mmol) and allyl bromide (6.78 mL, 78.4 mmol) in dry DMF (140 mL) was added sodium hydride (60%, in mineral oil; 2.35 g, 58.8 mmol) portionwise at room temperature followed by a catalytic amount of tetra-n-butyl ammonium iodide. The ice bath was removed, and the reaction was stirred at room temperature overnight. Another portion of allyl bromide (6.78 mL, 78.4 mmol) and sodium hydride (60%, in mineral oil; 2.35 g, 58.8 mmol) was added at room temperature and the mixture was again stirred overnight. After a third addition of allyl bromide (3.00 mL, 34.7 mmol) and sodium hydride (60%, in mineral oil; 1.00 g, 25.0 mmol) and stirring at room temperature overnight water was added carefully and the mixture was extracted with ethyl acetate (3×). The organic phases were combined, washed with water (2×) and brine, dried over MgSO₄ and concentrated. The obtained crude residue was purified by flash column chromatography (silica gel, ethyl acetate/petroleum ether 1:30) to yield 15.8 g (79%) of N-allylvinylglycinol (S)-15 as a colorless oil. ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{23}$ = +0.82 (c 0.90, CHCl₃). ¹H NMR (500 MHz, d₆-DMSO, rotamers) δ 7.64–7.59 (m, 4H), 7.50–7.41 (m, 6H), 5.87–5.74 (m, 2H), 5.15 (dt, 10.6, 1.4 Hz, 1H), 5.13–5.06 (overlapping signals: m, 1H and 5.10, dt, 17.4, 1.4 Hz, 1H), 5.05–5.01 (m, 1H), 4.70–4.55 (m, 0.5H), 4.36–4.20 (m, 0.4H), 3.87–3.64 (overlapping signals: 3.81, dd, 10.2, 8.3 Hz, 1H and m, 3H), 1.37 (s_br, 9H), 0.99 (s, 9H); ¹³C NMR (125 MHz, d₆-DMSO, rotamers) δ 154.48, 135.98, 135.52, 134.93, 134.46, 132.75, 129.79, 127.74, 117.28, 115.69, 115.27, 78.69, 63.81, 63.32, 60.69, 59.29, 47.71, 46.84, 27.90, 26.42, 18.63; ¹H NMR (500 MHz, d₆-DMSO, 100 °C) δ 7.65–7.61 (m, 4H), 7.48–7.39 (m, 6H), 5.87 (ddd, 17.3, 10.8, 6.4 Hz, 1H), 5.80 (ddt, 17.2, 10.5, 5.7 Hz, 1H), 5.17–5.07 (overlapping signals: 5.15, dt, 10.6, 1.5 Hz, 1H and, m, 2H), 5.02 (ddd, 10.3, 3.0, 1.5 Hz, 1H), 4.47–4.39 (m, 1H), 3.88 (dd, 10.3, 7.6 Hz, 1H), 3.85–3.77 (overlapping signals: 3.82, ddt, 16.1, 5.6, 1.4 Hz, 1H and 3.79, dd, 10.3, 6.1 Hz, 1H), 3.74 (ddt, 16.1, 5.8, 1.4 Hz, 1H), 1.38 (s, 9H), 1.03 (s, 9H), ¹³C NMR (125 MHz, d₆-DMSO, 100 °C) δ 154.04, 135.34, 134.52, 134.47, 132.71, 129.15, 127.12, 116.41, 114.87, 78.38, 63.62, 59.94, 47.19, 27.52, 26.12, 18.20; HRMS (ESI) m/z calcd for C₂₈H₃₉NO₃SiNa⁺ 488.2591, found 488.2589; Anal. calcd for C₂₈H₃₉NO₃Si: C, 72.21; H, 8.44; N, 3.01. Found C, 72.43; H, 8.69; N, 3.03.

tert-Butyl (S)-2-{[(tert-butyldiphenylsilyl)oxy]methyl}-2,5-dihydro-1H-pyrrole-1-carboxylate [(S)-16]. A solution of N-allylvinylglycinol (S)-15 (4.32 g, 9.28 mmol) in dry DCM (40 mL) was treated with Grubbs 1st-generation catalyst (40.0 mg, 0.049 mmol) employing the procedure described by Brackmann et al. [55] to afford 3.91 g (96%) of 3,4-dehydroprolinol (S)-16 as a colorless oil. ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{23}$ = −116 (c 0.95, CHCl₃), Ref. [58]: ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ = −90.1 (c 1.06, CHCl₃), Ref. [57] ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{23}$ = −24.6 (c 1.00, CHCl₃); ¹H NMR (500 MHz, CHCl₃; mixture of rotamers) δ 7.66–7.61 (m, 4H), 7.44–7.32 (m, 6H), 5.93–5.78 (m, 2H), 4.66–4.61 (m, 0.4H), 4.55–4.49 (m, 0.6H), 4.25 (dd, 15.4, 1.5 Hz, 0.6H), 4.17 (dd, 15.4, 1.2 Hz, 0.4H), 4.09 (d, 5.3 Hz, 0.6H), 4.06 (d, 5.3 Hz, 0.4H), 3.99 (dd, 9.8, 5.0 Hz, 0.4H), 3.88 (dd, 9.5, 3.1 Hz, 0.6H), 3.82 (dd, 9.8, 2.1 Hz, 0.4H), 3.67 (dd, 9.4, 6.5 Hz, 0.6H), 1.48 (s, 3.4H), 1.35 (s, 5.2H), 1.03 (s, 5H), 1.02 (s, 4H); ¹³C NMR (125 MHz, CHCl₃; mixture of rotamers) δ 154.13, 154.08, 135.55, 133.90, 133.80, 133.68, 133.57, 129.64, 129.52, 128.85, 128.75, 127.67, 127.59, 126.29, 79.46, 79.17, 65.57, 65.42, 65.14, 63.74, 54.27, 53.96, 28.57, 28.45, 26.78, 19.35, 19.28; HRMS (ESI) m/z calcd for C₂₆H₃₅NO₃SiNa⁺ 460.2278, found 460.2289.

tert-Butyl (2R,3R,4S)-2-{[(tert-butyldiphenylsilyl)oxy]methyl}-3,4-dihydroxypyrrolidine-1-carboxylate [(-)-17]. Following the procedure described by Murruzzu and Riera [57] 3,4-dehydroprolinol (S)-16 (461 mg, 1.05 mmol) was dissolved in a 3:1 mixture of acetone (14 mL) and water (4.7 mL) and reacted with osmium tetroxide (7 mg, 0.028 mmol) in the presence of N-methylmorpholine N-oxide (325 mg, 2.40 mmol) at room temperature overnight. The crude reaction product was purified by automated column chromatography (silica gel, ethyl acetate/petroleum ether 2–50%) to afford 477 mg (96%) of (−)-17 as a colorless oil. ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ = −27.3 (c 0.59, MeOH), Ref. [58]: ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ = −30.5 (c 1.01, MeOH); ¹H NMR (500 MHz, CD₃OD; mixture of rotamers) δ 7.68–7.61 (m, 4H), 7.47–7.36 (m, 6H), 4.41–4.34 (m, 1.6H), 4.30 (t, 3.8 Hz, 0.4H), 4.07 (dd, 10.5, 3.7, Hz, 0.4H), 3.88 (dd, 10.5, 4.3 Hz, 0.6H), 3.77 (dd, 10.5, 2.0 Hz, 0.6H), 3.73 (dd, 10.5, 1.4 Hz, 0.4H), 3.70–3.63 (m, 1H), 3.55 (dd, 11.1, 6.1 Hz, 0.4H), 3.51 (dd, 11.2, 6.2 Hz, 0.6H), 3.44–3.38 (m, 1H), 1.48 (s, 4H), 1.29 (s, 5H), 1.05 (s, 5H), 1.04 (s, 4H); ¹³C NMR (125 MHz, CD₃OD; mixture of rotamers) δ 156.53, 156.49, 136.71, 136.66, 134.57, 134.46, 134.36, 131.05, 130.98, 130.95, 128.99, 128.95, 128.92, 128.86, 81.25, 80.97, 74.99, 74.50, 71.33, 70.84, 66.35, 66.08, 63.81, 62.94, 52.81, 52.17, 28.89, 28.71, 27.39, 20.14, 20.10; HRMS (ESI) m/z calcd for C₂₆H₃₇NO₅SiNa⁺ 494.2333, found 494.2345.

tert-Butyl(3aR,4R,6aS)-4-{[(tert-butyldiphenylsilyl)oxy]methyl}-2,2-dimethyltetrahydro-5H-[1,3]dioxolo[4,5-c]pyrrole-5-carboxylate [(−)-18]. To a solution of compound (−)-17 (334 mg, 0.708 mmol) and 2,2-dimethoxypropane (0.20 mL, 1.63 mmol) in acetone (17 mL) was added a catalytic amount of p-toluenesulfonic acid monohydrate (26.6 mg, 0.140 mmol). After being stirred at room temperature overnight saturated bicarbonate solution (10 mL) and water are added, and the mixture was extracted with ethyl acetate (3×). The combined organic phases washed with brine, dried over MgSO₄ and concentrated. The residue was purified by automated column chromatography (silica gel, ethyl acetate/petroleum ether 2–5%) to yield 328 mg (91%) of (−)-18 as a colorless oil that solidified on standing. ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ = −47.9 (c 0.85, CHCl₃), Ref. [57]: ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{23}$ = −36.1 (c 1.05, CHCl₃); ¹H NMR (500 MHz, CHCl₃; mixture of rotamers) δ 7.66–7.56 (m, 4H), 7.46–7.34 (6H), 4.84–4.76 (overlapping signals: m, 1H and 4.78, d, 6.0 Hz, 0.6H), 4.74 (d, 6.1 Hz, 0.4H), 4.14–4.11 (m, 0.4H), 4.03–3.98 (m, 1H), 3.84 (dd, 12.4, 0.9 Hz, 0.6H), 3.77 (dd, 10.5, 3.7 Hz, 0.6H), 3.73 (dd, 12.3, 0.9 Hz, 0.4H), 3.70–3.63 (m, 2H), 1.49 (s, 4H), 1.47 (s, 1.6H), 1.46 (s, 1.4H), 1.37 (s, 5H), 1.35 (s, 1.6H), 1.33 (s, 1.4H), 1.05 (s, 4H), 1.04 (s, 5H); ¹³C NMR (125 MHz, CHCl₃; mixture of rotamers) δ 154.25, 154.06, 135.56, 135.45, 132.99, 132.94, 132.80, 132.70, 129.94, 129.89, 129.87, 129.80, 127.89, 127.84, 127.78, 111.47, 83.31, 82.66, 79.99, 79.67, 79.61, 79.20, 65.00, 64.68, 64.62, 64.42, 54.23, 53.54, 28.52, 28.42, 27.10, 26.90, 26.83, 25.14, 19.16, 19.08; HRMS (ESI) m/z calcd for C₂₉H₄₁NO₅SiNa⁺ 534.2646, found 534.2654.

tert-Butyl(3aR,4R,6aS)-4-(hydroxymethyl)-2,2-dimethyltetrahydro-5H-[1,3]dioxolo[4,5-c]pyrrole-5-carboxylate [(−)-19]. To a solution of (−)-18 (218 mg, 0.426 mmol) in THF (3 mL) was added a 1 M solution of tetra-n-butylammonium fluoride in THF (0.7 mL, 0.70 mmol) at room temperature. After being stirred overnight water was added and the resulting mixture extracted with ethyl acetate (3×). The organic phases were combined, washed with brine, dried over MgSO₄ and concentrated. The crude product was purified by automated column chromatography (silica gel, ethyl acetate/petroleum ether 2–20%) to give 100 mg (86%) of the desired alcohol (−)-19 as a colorless oil that solidified on standing. ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ = −47.6 (c 0.555, CHCl₃), Ref. [57]: ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{23}$ = −30.3 (c 0.3, CHCl₃); ¹H NMR (500 MHz, CH₃OD; mixture of rotamers) δ 4.78–4.73 (m, 1H), 4.72–4.68 (m, 1H), 3.98 (t, 3.7 Hz, 0.5H), 3.93 (t, 4.0 Hz, 0.5H), 3.69 (d, 11.8 Hz, 0.5H), 3.67 (d, 11.8 Hz, 0.5H), 3.64–3.59 (m, 2H), 3.50 (dd, 12.5 Hz, 5.1 Hz, 0.5H), 3.46 (dd, 12.5 Hz, 5.2 Hz, 0.5H), 1.46 (s, 9H), 1.40 (s, 3H), 1.30 (s, 3H); ¹³C NMR (125 MHz, CD₃OD; mixture of rotamers) δ 156.41, 156.26, 112.57, 84.29, 83.57, 81.36, 81.29, 81.00, 80.23, 66.93, 66.44, 62.77, 62.34, 54.52, 53.89, 28.75, 27.35, 25.10; HRMS (ESI) m/z calcd for C₁₃H₂₃NO₅Na⁺ 296.1468, found 296.1480.

tert-Butyl(3aR,4R,6aS)-2,2-dimethyl-4-{[(methylsulfonyl)oxy]methyl}tetrahydro-5H-[1,3]dioxo-lo[4,5-c]pyrrole-5-carboxylate [(−)-20]. To a solution of (−)-19 (6.98 g, 25.5 mmol) in dry DCM (160 mL) and triethylamine (10.7 mL, 76.7 mmol) was added methanesulfonyl chloride (3.0 mL, 38.8 mmol) dropwise at 0 °C. After being stirred for 30 min the reaction was poured into half concentrated bicarbonate solution. The organic phase was separated, and the aqueous layer extracted with ethyl acetate another three times. The organic phases were combined, washed with brine, dried over MgSO₄ and concentrated. The recovered crude product was used in the next reaction step without further purification. An analytical sample was purified by automated column chromatography (silica gel, ethyl acetate/petroleum ether 2–50%) to yield (−)-20 as a colorless oil. ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ = −50.8 (c 0.62, MeOH); ¹H NMR (500 MHz, CD₃OD; mixture of rotamers) δ 4.81–4.77 (m, 1H), 4.72 (ap d, 6.1 Hz, 1H), 4.41 (dd, 10.4, 4.5 Hz, 0.5H), 4.35 (dd, 10.4, 5.0 Hz, 0.5H), 4.31 (ap dd, 10.4, 3.6 Hz, 1H), 4.20–4.14 (m, 1H), 3.74 (d, 12.6 Hz, 0.5H), 3.70 (d, 12.7 Hz, 0.5H), 3.51 (dd, 12.7, 5.1 Hz, 0.5H), 3.47 (dd, 12.7, 5.1 Hz, 0.5H), 3.10 (s, 1.5H), 3.09 (s, 1.5H), 1.48 (s, 4.5H), 1.47 (s, 4.5H), 1.42 (s, 3H), 1.31 (s, 3H); ¹³C NMR (125 MHz, CD₃OD; mixture of rotamers) δ 156.19, 155.81, 113.06, 83.86, 83.08, 82.08, 81.83, 80.76, 80.00, 70.00, 69.81, 64.45, 64.03, 54.24, 53.46, 37.37, 37.27, 28.67, 27.30, 25.05; ¹H and ¹³C NMR data recorded in CDCl₃ was consistent with that reported in reference [48,49]. HRMS (ESI) m/z calcd for C₁₄H₂₅NO₇SNa⁺ 374.1244, found 374.1244.

tert-Butyl (R)-{1-[(tert-butyldiphenylsilyl)oxy]-4-(methylthio)butan-2-yl}carbamate [(R)-11]. Using the same procedure as for the preparation of compound (S)-11 alcohol (R)-10 (35.0 g, 149 mmol) was dissolved in dry DMF (250 mL) and reacted with TBDPSCl (46.4 mL, 179 mmol) in the presence of imidazole (25.3 g, 372 mmol) to yield 58.2 g (83%) of (R)-11 as a colorless oil. ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{21}$ = +6.8 (c 1.075, CHCl₃); All spectroscopic data was consistent with its enantiomer (S)-11. HRMS (ES+) m/z calcd for C₂₆H₃₉NO₃SSiNa⁺ 496.2318, found 496.2312.

tert-Butyl {(2R)-1-[(tert-butyldiphenylsilyl)oxy]-4-(methylsulfinyl)butan-2-yl}carbamate [(R)-12]. According to the protocol outlined for the preparation of (S)-12 sulfide (R)-11 (58.0 g, 122 mmol) dissolved in dichloromethane (1700 mL) was oxidized by adding dropwise a solution of m-CPBA (50–55%; 20 g, 61.2 mmol) in dichloromethane (160 mL) (dried over MgSO₄) at −40 °C to give 29.8 g (50%) of the desired sulfoxide (R)-12 as a colorless oil that solidified on standing. In addition, 1.77 g (3%) of the sulfone (R)-13 were isolated from the reaction as a colorless solid that also solidified on standing along with 26.1 g (45%) of starting material (R)-11.

tert-Butyl {(2R)-1-[(tert-butyldiphenylsilyl)oxy]-4-(methylsulfinyl)butan-2-yl}carbamate [(R)-12]. ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{21}$ = +9.4 (c 1.075, CHCl₃); All spectroscopic data was consistent with its enantiomer (S)-12. HRMS (ES+) m/z calcd for C₂₆H₃₉NO₄SSiNa⁺ 512.2261, found 512.2274.

tert-Butyl (R)-{1-[(tert-butyldiphenylsilyl)oxy]-4-(methylsulfonyl)butan-2-yl}carbamate [(R)-13]. ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{21}$ = +6.0 (c 1.14, CHCl₃); All spectroscopic data was consistent with its enantiomer (S)-13. HRMS (ES+) m/z calcd for C₂₆H₃₉NO₅SSiNa⁺ 528.2210, obsd 528.2209; Anal. calcd for C₂₆H₃₉NO₅SSi: C, 61.75; H, 7.77; N, 2.77. Found C, 62.02; H, 7.96; N, 2.68.

tert-Butyl (R)-{1-[(tert-butyldiphenylsilyl)oxy]but-3-en-2-yl}carbamate [(R)-14]. Employing the same procedure as for the preparation of (S)-14 a mixture of sulfoxide (R)-12 (29.6 g, 60.4 mmol) and calcium carbonate (14.5 g, 145 mmol) in 1,2-dichlorobenzene (165 mL) was heated at 200 °C for 7 h to yield 16.8 g (65%) of vinylglycinol (R)-14 as a light-yellow oil. ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{22}$ = +27.0 (c 1.03, CHCl₃), Ref. [84]: ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{23}$ = +25.4 (c 1.7, CHCl₃); The spectroscopic data was consistent with that for its enantiomer (S)-14. HRMS (ESI) m/z calcd for C₂₅H₃₅NO₃SiNa⁺ 448.2278, found 448.2275.

tert-Butyl (R)-allyl{1-[(tert-butyldiphenylsilyl)oxy]but-3-en-2-yl}carbamate [(R)-15]. Using the same procedure as for the preparation of N-allylvinylglycinol (S)-15 vinylglycinol (R)-14 (16.4 g, 38.5 mmol) dissolved in dry DMF (125 mL) was reacted with allyl bromide (6.67 mL, 77.1 mmol; second addition after ca. 20 h: 7.0 mL, 80.9 mmol; third addition after another ca. 20 h: 7.0 mL, 80.9 mmol) and sodium hydride (60% in mineral oil, 2.31 g, 57.8 mmol; second addition after ca. 20 h: 2.38 g, 59.5 mmol; third addition after another 20 h: 1.19 g, 29.8 mmol) in the presence of a catalytic amount of n-Bu₄NI to afford 12.7 g (71%) of N-allylvinylglycinol (R)-15 as a colorless oil. In addition, 3.86 g of mixed starting material and product were ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{23}$ = −0.76 (c 1.07, CHCl₃). The spectroscopic data was consistent with that reported for its enantiomer (S)-15. HRMS (ESI) m/z calcd for C₂₈H₃₉NO₃SiNa⁺ 488.2591, found 488.2596; Anal. calcd for C₂₈H₃₉NO₃Si: C, 72.21; H, 8.44; N, 3.01. Found C, 72.30; H, 8.52; N, 3.07.

tert-Butyl (R)-2-{[(tert-butyldiphenylsilyl)oxy]methyl}-2,5-dihydro-1H-pyrrole-1-carboxylate [(R)-16]. Following the method described by Brackmann et al. [55] N-allylvinylglycinol (R)-15 (9.80 g, 21.0 mmol) was dissolved in dry DCM (94 mL) and treated with Grubbs 1^st-generation catalyst (250 mg, 0.304 mmol) for 24 h at room temperature to yield 9.04 g (98%) of 3,4-dehydroprolinol (R)-16 as a colorless oil. ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{23}$ = +115 (c 1.38, CHCl₃). The spectroscopic data was consistent with those reported for its enantiomer (S)-16. HRMS (ES+) m/z calcd for C₂₆H₃₅NO₃SiNa⁺ 460.2274, found 460.2281.

tert-Butyl (2S,3S,4R)-2-{[(tert-butyldiphenylsilyl)oxy]methyl}-3,4-dihydroxypyrrolidine-1-carboxylate [(+)-17]. Deploying the procedure described by Murruzzu and Riera [57] compound (R)-16 (8.85 g, 20.2 mmol) was dissolved in a 10:1 mixture of acetone (360 mL) and water (36 mL) and treated with osmium tetroxide (59.9 mg, 0.236 mmol) in the presence of N-methylmorpholine N-oxide (6.01 g, 44.5 mmol) at room temperature overnight to yield 8.61 g (90%) of (+)-17 as a colorless oil. ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ = +28.6 (c 0.545, MeOH); All spectroscopic data was consistent with the data described for its enantiomer (−)-17. HRMS (ESI) m/z calcd for C₂₆H₃₇NO₅SiNa⁺ 494.2333, found 494.2344.

tert-Butyl(3aS,4S,6aR)-4-{[(tert-butyldiphenylsilyl)oxy]methyl}-2,2-dimethyltetrahydro-5H-[1,3]dioxolo[4,5-c]pyrrole-5-carboxylate [(+)-18]. Using the same procedure as for the preparation of its enantiomer (−)-18 compound (+)-17 (8.50 g, 18.0 mmol) and 2,2-dimethoxypropane (4.43 mL, 36.0 mmol) in acetone (430 mL) was treated with a catalytic amount of p-toluenesulfonic acid monohydrate (189 mg, 0.994 mmol) to give 8.76 g (95%) of (+)-18 as a colorless oil that solidified on standing. ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ = +46.1 (c 0.92, CHCl₃); All spectroscopic data was consistent with that of its enantiomer (−)-18. HRMS (ESI) m/z calcd for C₂₉H₄₁NO₅SiNa⁺ 534.2646, found 534.2652; Anal. calcd for C₂₉H₄₁NO₅: C, 68.07; H, 8.08; N, 2.74. Found C, 68.33; H, 8.20; N, 2.78.

tert-Butyl(3aS,4S,6aR)-4-(hydroxymethyl)-2,2-dimethyltetrahydro-5H-[1,3]dioxolo[4,5-c]pyrrole-5-carboxylate [(+)-19]. Following the same protocol as outlined for the preparation of its enantiomer (−)-19 the fully protected pyrrolidine (+)-18 (8.67 g, 16.9 mmol) was dissolved in THF (85 mL) and treated with a 1 M solution of tetra-n-butylammonium fluoride in THF (25 mL, 25 mmol) at room temperature overnight to yield 4.65 g (quant.) of (+)-19 as a colorless oil that solidified on standing. ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ = +46.7 (c 0.505, CHCl₃), Ref. [85]: ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{28}$ = +29 (c 1.0, CHCl₃), Ref. [86]: ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{24}$ = +29.4 (c 1.04, CHCl₃); All spectroscopic data was consistent with its enantiomer (−)-19. HRMS (ESI) m/z calcd for C₁₃H₂₃NO₅Na⁺ 296.1468, found 296.1468; Anal. calcd for C₁₃H₂₃NO₅; C, 57.13; H, 8.48; N, 5.12. Found C, 57.16; H, 8.53; N, 5.11.

tert-Butyl(3aS,4S,6aR)-2,2-dimethyl-4-{[(methylsulfonyl)oxy]methyl}tetrahydro-5H-[1,3]dioxolo[4,5-c]pyrrole-5-carboxylate [(+)-20]. Deploying the same protocol as described for the preparation of compound (−)-20 alcohol (+)-19 (2.51 g, 9.18 mmol) was dissolved in dry DCM (40 mL) and reacted with methanesulfonyl chloride (1.1 mL, 14.2 mmol) in the presence of triethylamine (3.9 mL, 28.0 mmol) to yield 3.21 g (quant.) of (+)-20 as a colorless oil upon purification by automated column chromatography (silica gel, ethyl acetate/petroleum ether 2–50%). ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ = +51.5 (c 0.565, MeOH); All spectroscopic data was consistent with that reported for its enantiomer (−)-20. HRMS (ESI) m/z calcd for C₁₄H₂₅NO₇SNa⁺ 374.1244, found 374.1242; Anal. calcd for C₁₄H₂₅NO₇S: C, 47.85; H, 7.17; N, 3.99; S, 9.12. Found C, 47.55; H, 7.21; N, 3.98; S, 8.95.

Ethyl 4-(4-methoxyphenyl)-1H-pyrrole-2-carboxylate (24a). To a solution of 1-(tert-butyl) 2-ethyl 4-bromo-1H-pyrrole-1,2-dicarboxylate 22 [59] (141 mg, 0.443 mmol) and 4-methoxyphenylboronic acid 23a (95%; 213 mg, 1.33 mmol) in DMF (7.5 mL) was added palladium-tetrakis(triphenylphosphine) (29 mg, 0.025 mmol) and a 2 M aqueous solution of sodium carbonate (2.25 mL) and the resulting reaction mixture was heated at 110 °C overnight. After being cooled to room temperature water was added and the mixture was extracted with ethyl acetate (3×). The combined organic phases were washed with water (2×) and brine, dried over MgSO₄ and concentrated. The crude product was purified by automated flash column chromatography (silica gel, ethyl acetate/petroleum ether 2–20%) to afford 88 mg (81%) of 24a as a colorless solid. m.p. 136–138 °C (DSC); ¹H NMR (500 MHz, CDCl₃) δ 9.22 (s_br, 1H), 7.44 (AA’XX’, J_AX = 8.8 Hz, 2H), 7.15–7.12 (m, 2H), 6.90 (AA’XX’, J_AX = 8.8 Hz, 2H), 4.34 (q, 7.2 Hz, 2H), 3.82 (s, 3H), 1.38 (t, 7.2 Hz, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 161.18, 158.31, 127.35, 126.64, 126.45, 123.60, 118.73, 114.22, 112.18, 60.43, 55.32, 14.45; HRMS (ESI) m/z calcd for C₁₄H₁₅NO₃Na⁺ 268.0944, found 268.0947.

Ethyl 4-(4-methoxyphenyl)-1-methyl-1H-pyrrole-2-carboxylate (25a). To a solution of 24a (727 mg, 2.96 mmol) in dry DMF (10 mL) was added sodium hydride (60% in mineral oil; 150 mg, 3.75 mmol) at 0 °C. After 20 min iodomethane (0.25 mL, 4.02 mmol) was introduced dropwise, the ice bath was removed after a further 15 min and the reaction was stirred for another 2.5 h. Subsequently, water was added, and the aqueous mixture was extracted with ethyl acetate (3×). The combined organic layers were washed with water (2×) and brine, dried over MgSO₄ and concentrated. The crude product was purified by automated flash column chromatography (silica gel, ethyl acetate/petroleum ether 2–20%) to yield 540 mg (70%) of 25a as a colorless oil that solidified on standing. ¹H NMR, (500 MHz, CDCl₃) δ 7.41 (AA’XX’, J_AX = 8.9 Hz, 2H), 7.15 (d, 2.1 Hz, 1H), 6.98 (d, 2.1 Hz, 1H), 6.89 (AA’XX’, J_AX = 8.9 Hz, 2H), 4.30 (q, 7.1 Hz, 2H), 3.95 (s, 3H), 3.81 (s, 3H), 1.37 (t, 7.2 Hz, 3H); ¹³C NMR, (125 MHz, CDCl₃) δ 161.34, 158.18, 127.38, 126.25, 125.57, 123.82, 123.31, 114.50, 114.21, 59.89, 55.33, 36.89, 14.48; HRMS (ESI) m/z calcd for C₁₅H₁₇NO₃Na⁺ 282.1101, found 282.1105; Anal. calcd for C₁₅H₁₇NO₃: C, 69.48; H, 6.61; N, 5.40. Found C, 69.45; H, 6.73; N, 5.43.

4-(4-Methoxyphenyl)-1-methyl-1H-pyrrole-2-carboxylic acid (26a). To solution of 25a (479 mg, 1.85 mmol) in a 1:1 mixture of THF (10 mL) and methanol (10 mL) was added an 8 M aqueous solution of potassium hydroxide (3.5 mL) and the resulting reaction mixture was heated at 50 °C overnight. After having evaporated the solvents water was added and the resulting mixture was acidified to pH 1 by slowly adding a 2 M aqueous solution of hydrochloric acid (to pH 3-4 by slowly adding a 1 M aqueous solution of hydrochloric acid in the case of O-isopropylidene-protected compounds: 26c–f). The precipitate was extracted with ethyl acetate (3×) and the combined organic phases were washed with brine, dried over MgSO₄ and concentrated to small volume which was then treated with a small amount of petroleum ether. The ochre crystalline solid was filtered off and washed with a 3:7 mixture of ethyl acetate and petroleum ether. The collected solid was dried under high vacuum to give 344 mg (81%) of 26a. m.p. 203–204 °C (DSC); ¹H NMR, (500 MHz, d₆-DMSO) δ 12.22 (s_br, 1H), 7.47 (AA’XX’, J_AX = 8.8 Hz, 2H), 7.42 (d, 2.0 Hz, 1H), 7.09 (d, 2.1 Hz, 1H), 6.90 (AA’XX’, J_AX = 8.8 Hz, 2H), 3.86 (s, 3H), 3.75 (s, 3H); ¹³C NMR, (125 MHz, d₆-DMSO) δ 161.86, 157.46, 126.89, 126.06, 125.60, 123.14, 122.37, 114.08, 113.59, 54.95, 36.33; HRMS (ESI) m/z calcd for C₁₃H₁₃NO₃Na⁺ 254.0788, found 254.0794; Anal. calcd for C₁₃H₁₃NO₃: C, 67.52; H, 5.67; N, 6.06. Found C, 67.51; H, 5.59; N, 6.21.

1H-Benzo[d][1,2,3]triazol-1-yl 4-(4-methoxyphenyl)-1-methyl-1H-pyrrole-2-carboxylate (27a). To a solution of 26a (278 mg, 1.20 mmol) and triethylamine (0.51 mL, 3.66 mmol) in dry DMF (10 mL) was added HBTU (559 mg, 1.47 mmol) in one portion at room temperature. After being stirred overnight water was added and the resulting mixture was extracted with ethyl acetate (3×). The combined organic phases were washed with water (2×) and brine, dried over MgSO₄ and concentrated. The crude reaction product was purified by automated flash column chromatography (silica gel, ethyl acetate/petroleum ether 2–50%) to yield 403 mg of 27a (96%) as a colorless foam. ¹H NMR (500 MHz, CDCl₃) δ 8.09 (dt, 8.5 Hz, 0.8 Hz, 1H), 7.63 (d, 2.0 Hz, 1H), 7.55 (ddd, 8.3, 6.6, 0.9 Hz, 1H), 7.51 (dt, 8.3, 1.0 Hz, 1H), 7.47 (AA’XX’, J_AX = 8.8 Hz, 2H), 7.43 (ddd, 8.3, 6.7, 1.4 Hz, 1H), 7.28 (d, 1.9 Hz, 1H), 6.95 (AA’XX’, J_AX = 8.8 Hz, 2H), 3.98 (s, 3H), 3.84, (s, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 158.79, 156.79, 143.57, 129.54, 129.15, 128.62, 126.55, 126.09, 125.77, 124.72, 120.54, 117.82, 116.83, 114.43, 108.49, 55.38, 36.97; HRMS (ESI) m/z calcd for C₁₉H₁₆N₄O₃Na⁺ 371.1115, found 371.1114. Anal. calcd for C₁₉H₁₆N₄O₃: C, 65.51; H, 4.63; N, 16.08. Found C, 65.54; H, 4.50; N, 16.02.

N-{4-[3-(4-Fluorophenyl)-5-isopropylisoxazol-4-yl]pyridin-2-yl}-4-(4-methoxyphenyl)-1-methyl-1H-pyrrole-2-carboxamide (28a). To a solution of isoxazole 30 (313 mg, 1.05 mmol) in dry DMF (10 mL) was added sodium hydride (60% in mineral oil; 39.7 mg, 0.993 mmol) in one portion at room temperature. After 30 min activated carboxylic acid 27a (195 mg, 0.560 mmol) was introduced in one portion and the resulting reaction was stirred for 48 h. Water was added and the mixture was extracted with ethyl acetate (3×). The combined organic phases were then washed with water (2×) and brine, dried over MgSO₄ and concentrated. The crude product was purified by automated flash column chromatography (silica gel, ethyl acetate/petroleum ether 2–40%) to afford 191 mg (67%) of 28a. Additional recrystallization from ethyl acetate/petroleum ether yielded 100 mg of pure 28a as a colorless solid. m.p. 157–158 °C (DSC); ¹H NMR, (500 MHz, CDCl₃) δ 8.42 (s_br, 1H), 8.27–8.23 (m, 2H), 7.43 (AA’BB’X, J_AB = 8.9, J_AF = 5.4 Hz, 2H), 7.40 (AA’XX’, J_AX = 8.8, 2H), 7.06–7.01 (overlapping signals: 7.03, AA’BB’X, J_AB = J_BF = 8.7 Hz, 2H and 7.02, d, 1.9 Hz, 1H), 7.00 (d, 1.8 Hz, 1H), 6.91 (AA’XX’, J_AX = 8.8 Hz, 2H), 6.74 (dd, 5.2, 1.5 Hz, 1H), 3.99 (s, 3H), 3.83 (s, 3H), 3.25 (sp, 7.0 Hz, 1H), 1.39 (d, 7.0 Hz, 6H); ¹⁹F NMR (470 MHz, CDCl₃) δ −111.12; ¹³C NMR, (125 MHz, CDCl₃) δ 175.49, 164.58/162.60 (d, 248.2 Hz), 160.04, 159.51, 158.39, 152.41, 148.03, 141.33, 130.50/130.43 (d, 8.2 Hz), 126.96, 126.27, 125.94, 125.55, 124.78, 123.98, 120.46, 115.88/115.71 (d, 21.7 Hz), 114.43, 114.35, 112.14, 110.45, 55.36, 37.05, 26.66, 21.00; HRMS (ESI) m/z calcd for C₃₀H₂₇FN₄O₃H⁺ 511.2140, found 511.2150; Anal. calcd for C₃₀H₂₇FN₄O₃: C, 70.57; H, 5.33; N, 10.97. Found C, 70.60; H, 5.41; N, 11.03.

Ethyl 4-(2,4-dimethoxyphenyl)-1H-pyrrole-2-carboxylate (24b). Applying the same method as for the preparation of 24a a mixture of 1-(tert-butyl) 2-ethyl 4-bromo-1H-pyrrole-1,2-dicarboxylate 22 (1.19 g, 3.74 mmol), 2,4-dimethoxyphenylboronic acid 23b (95%; 2.18 g, 11.4 mmol), palladium-tetrakis(triphenylphosphine) (225 mg, 0.195 mmol), and a 2 M aqueous solution of sodium carbonate (19 mL) in DMF (63 mL) was heated at 110 °C overnight to yield 892 mg (87%) of 24b as a colorless solid. M.p. 124–125 °C (DSC); ¹H NMR (500 MHz, CDCl₃) δ 9.13 (s_br, 1H), 7.44–7.41 (m, 1H), 7.38 (dd, 2.9, 1.6 Hz, 1H), 7.23 (dd, 2.7, 1.7 Hz, 1H), 6.54–6.51 (m, 2H), 4.34 (q, 7.1 Hz, 2H), 3.87 (s, 3H), 3.83 (s, 3H), 1.37 (t, 7.1 Hz, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 161.25, 159.32, 157.18, 128.48, 122.49, 122.41, 121.87, 116.44, 113.84, 104.74, 99.02, 60.28, 55.39, 14.47; HRMS (ESI) m/z calcd for C₁₅H₁₇NO₄Na⁺ 298.1050, found 298.1049.

Ethyl 4-(2,4-dimethoxyphenyl)-1-methyl-1H-pyrrole-2-carboxylate (25b). Employing the same procedure as described for the preparation of 25a pyrrole 24b (790 mg, 2.87 mmol) dissolved in dry DMF (10 mL) was treated with sodium hydride (60% in mineral oil; 140 mg, 3.50 mmol) and iodomethane (0.23 mL, 3.70 mmol) to yield 758 mg (91%) of 25b as a colorless oil. ¹H NMR (500 MHz, CDCl₃) δ 7.42–7.38 (m, 1H), 7.24–7.21 (m, 2H), 6.54–6.49 (m, 2H), 4.30 (q, 7.1 Hz, 2H), 3.94 (s, 3H), 3.87 (s, 3H), 3.82 (s, 3H), 1.36 (t, 7.1 Hz, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 161.47, 159.19, 157.11, 128.77, 128.32, 122.25, 119.63, 116.48, 116.20, 104.75, 98.99, 59.76, 55.41, 36.82, 14.51; HRMS (ESI) m/z calcd for C₁₆H₁₉NO₄Na⁺ 312.1206, found 312.1205; Anal. calcd for C₁₆H₁₉NO₄: C, 66.42; H, 6.62; N, 4.84. Found C, 66.59; H, 6.53; N, 4.90.

4-(2,4-Dimethoxyphenyl)-1-methyl-1H-pyrrole-2-carboxylic acid (26b). Following the procedure described for the preparation of 26a ethyl ester 25b (522 mg, 1.80 mmol) dissolved in a 1:1 mixture of THF and methanol (10 mL/10 mL) was treated with an 8 M aqueous potassium hydroxide solution (2.5 mL) to afford 294 mg (62%) of 26b as a crystalline, ochre solid. m.p. 175–176 °C (DSC); ¹H NMR (500 MHz, d₆-DMSO) δ 7.42 (d, 8.5 Hz, 1H), 7.41 (d, 2.0 Hz, 1H), 7.13 (d, 2.0 Hz, 1H), 6.60 (d, 2.5 Hz, 1H), 6.53 (dd, 8.5, 2.5 Hz, 1H), 3.86 (s, 3H), 3.84 (s, 3H), 3.77 (s, 3H); ¹³C NMR (125 MHz, d₆-DMSO) δ 161.96, 158.57, 156.60, 128.46, 127.55, 121.99, 118.68, 115.59, 105.18, 98.77, 55.28, 55.08, 36.22; HRMS (ESI) m/z calcd for C₁₄H₁₅NO₄Na⁺ 284.0893, found 284.0898; Anal. calcd for C₁₄H₁₅NO₄: C, 64.36; H, 5.79; N, 5.36. Found C, 64.46; H, 5.68; N, 5.38.

1H-Benzo[d][1,2,3]triazol-1-yl 4-(2,4-dimethoxyphenyl)-1-methyl-1H-pyrrole-2-carboxylate (27b). Carboxylic acid 26b (225 mg, 0.861 mmol) in dry DMF (8 mL) was reacted with HBTU (398 mg, 1.05 mmol) in the presence of triethylamine (0.40 mL, 2.87 mmol) according to the procedure outlined for the preparation of compound 27a to give 293 mg (90%) of 27b as a colorless foam. ¹H NMR (500 MHz, CDCl₃) δ 8.09 (d, 8.5 Hz, 1H), 7.73 (d, 1.8 Hz, 1H), 7.57–7.49 (m, 3H), 7.47–7.40 (m, 2H), 6.58–6.55 (m, 2H), 3.97 (s, 3H), 3.91 (s, 3H), 3.85 (s, 3H); ¹³C NMR (125 MHz, CDCl₃) δ 159.86, 157.26, 156.87, 143.57, 132.64, 129.21, 128.57, 128.52, 124.68, 121.69, 120.49, 119.67, 115.76, 115.22, 108.56, 104.96, 99.08, 55.48, 36.86; HRMS (ESI) m/z calcd for C₂₀H₁₈N₄O₄Na⁺ 401.1220, found 401.1229.

4-(2,4-Dimethoxyphenyl)-N-{4-[3-(4-fluorophenyl)-5-isopropylisoxazol-4-yl]pyridin-2-yl}-1-methyl-1H-pyrrole-2-carboxamide (28b). Deploying the same procedure as for the preparation of compound 28a isoxazole 30 (304 mg, 1.02 mmol) was dissolved in dry DMF (5 mL), deprotonated with sodium hydride (60% in mineral oil; 36 mg, 0.900 mmol) and subsequently reacted with a solution of 27b (212 mg, 0.560 mmol) in dry DMF (6 mL) to yield 172 mg (57%) of 28b as a colorless foam. The removal of a minor impurity required additional automated flash column chromatography on reversed phase C-18 silica gel (water/acetonitrile 10–90%). ¹H NMR (500 MHz, CDCl₃) δ 8.51 (s_br, 1H), 8.28 (s, 1H), 8.24 (d, 5.1 Hz, 1H), 7.43 (AA’BB’X, J_AB = 8.5, J_AF = 5.4 Hz, 2H), 7.38–7.34 (m, 1H), 7.23 (s, 1H), 7.14 (s, 1H), 7.03 (AA’BB’X, J_AB = J_BF = 8.6 Hz, 2H), 6.73 (d, 5.1, Hz, 1H), 6.56–6.51 (m, 2H), 3.99 (s, 3H), 3.89 (s, 3H), 3.83 (s, 3H), 3.26 (sp, 7.0 Hz, 1H), 1.39 (d, 7.0 Hz, 6H); ¹⁹F NMR (470 MHz, CDCl₃) δ −111.12; ¹³C NMR (125 MHz, CDCl₃) δ 175.46, 164.57/162.59 (d, 248 Hz), 160.03, 159.68, 159.42, 157.16, 152.58, 148.07, 141.23, 130.50/130.43 (d, 8.2 Hz), 128.89, 128.26, 124.80, 124.52, 120.36, 119.84, 116.05, 115.87/115.69 (d, 21.7 Hz), 114.46, 112.43, 112.18, 104.83, 99.08, 55.44, 36.99, 26.65, 20.99; HRMS (ESI) m/z calcd for C₃₁H₂₉FN₄O₄H⁺ 541.2246, found 541.2251. Anal. calcd for C₃₁H₂₉FN₄O₄·0.2(CHCl₃): C, 66.39; H, 5.21; N, 9.93. Found C, 66.35; H, 5.22; N, 9.88.

tert-Butyl(3aR,4R,6aS)-4-{[2-(methoxycarbonyl)-1H-pyrrol-1-yl]methyl}-2,2-dimethyltetrahydro-5H-[1,3]dioxolo[4,5-c]pyrrole-5-carboxylate (25c) To a solution of methyl 1H-pyrrole-2-carboxylate 24c [63] (325 mg, 2.60 mmol) and crude mesylate (−)-20 (1.14 g, 3.24 mmol) in dry DMF (28 mL) was added cesium carbonate (2.22 g, 6.81 mmol) and a catalytic amount of tetra n-butylammonium iodide, and the resulting mixture was heated at 80 °C overnight. After being cooled to room temperature a 10:1 mixture of water and brine was added, and the aqueous mixture was extracted with ethyl acetate (3×). The combined organic phases were washed with a 10:1 mixture of water and brine (2×), and brine, dried over MgSO₄ and concentrated. Finally, the crude reaction product was purified by automated flash column chromatography (silica gel, ethyl acetate/petroleum ether 2–40%) to yield 909 mg (92%) of 25c as a colorless oil that solidified on standing. ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ = +4.42 (c 0.77, CHCl₃); ¹H NMR (500 MHz, CDCl₃, mixture of rotamers) δ 6.95 (dd, 3.9, 1.5 Hz, 1H); 6.88 (s, 0.4H), 6.75-6.72 (m, 0.6H), 6.19–6.16 (m, 0.4H), 6.15 (t, 3.0 Hz, 0.6H), 4.66–4.51 (m, 3H), 4.49 (dd, 13.9, 5.2 Hz, 0.4H), 4.45–4.38 (m, 1H), 4.13 (dd, 13.7, 8.2 Hz, 0.6H), 3.96 (d, 13.1 Hz, 0.6H), 3.80 (s, 3H), 3.68 (d, 12.9 Hz, 0.4H), 3.26 (dd, 13.1, 5.0 Hz, 0.6H), 3.17 (dd, 12.8, 5.0 Hz, 0.4H), 1.46 (s, 3.4H), 1.41 (s, 3H), 1.34 (s, 5.6H), 1.27 (s, 2H), 1.25 (s, 1H); ¹³C NMR (125 MHz, CDCl₃, mixture of rotamers) δ 161.99, 161.80, 154.27, 129.33, 129.05, 122.00, 121.90, 118.61, 118.48, 111.83, 111.71, 109.22, 108.92, 82.39, 81.51, 80.05, 79.97, 79.32, 78.90, 64.56, 64.02, 52.19, 51.13, 51.06, 47.49, 46.32, 28.41, 28.11, 27.02, 26.93, 25.10, 25.06; HRMS (ESI) m/z calcd for C₁₉H₂₈N₂O₆Na⁺ 403.1840, found 403.1844. Anal. calcd for C₁₉H₂₈N₂O₆: C, 59.99; H, 7.42; N, 7.36. Found C, 60.07; H, 7.52; N, 7.39.

1-{[(3aR,4R,6aS)-5-(tert-Butoxycarbonyl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyrrol-4-yl]methyl}-1H-pyrrole-2-carboxylic acid (26c). According to the procedure described for the preparation of carboxylic acid 26a methyl ester 25c (752 mg, 1.98 mmol) dissolved in a 2:1 mixture of THF (12 mL) and MeOH (6 mL) was saponified with an aqueous 8 M potassium hydroxide solution 82.5 mL). The crude product was subsequently purified by automated flash column chromatography (silica gel, ethyl acetate/petroleum ether 2–70%) to give 606 mg (84%) of the desired carboxylic acid 26c as a colorless oil that solidified on standing. ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ = +13.2 (c 0.50, CHCl₃); ¹H NMR (500 MHz, CDCl₃, mixture of rotamers) δ 7.13–7.10 (m, 1H), 6.95 (s, 0.4H), 6.80–6.77 (s, 0.6H), 6.24–6.21 (m, 0.4H), 6.21–6.18 (m, 0.6H), 4.68–4.59 (m, 2H), 4.56–4.48 (m, 1.4H), 4.48–4.40 (m, 1H), 4.11 (dd, 13.7, 8.6 Hz, 0.6H), 3.99 (d, 13.2 Hz, 0.6H), 3.70 (d, 12.9 Hz, 0.4H), 3.27 (dd, 13.2, 4.9 Hz, 0.6H), 3.16 (dd, 12.8, 4.9 Hz, 0.4H), 1.46 (s, 3.4H), 1.41 (s, 3H), 1.34 (s, 5.6H), 1.29 (s, 2H), 1.26 (s, 1H); ¹³C NMR (125 MHz, CDCl₃, mixture of rotamers) δ 165.39, 165.23, 154.33, 130.71, 130.42, 121.13, 120.98, 120.79, 120.72, 111.92, 111.80, 109.71, 109.41, 82.35, 81.51, 80.20, 80.14, 79.31, 78.90, 64.48, 63.99, 52.23, 51.03, 47.67, 46.49, 28.39, 28.12, 27.01, 26.92, 25.06; HRMS (ESI) m/z calcd for C₁₈H₂₆N₂O₆Na⁺ 389.1683, found 389.1687. Anal. calcd for C₁₈H₂₆N₂O₆: C, 59.00; H, 7.15; N, 7.65. Found C, 59.22; H, 7.19; N, 7.60.

tert-Butyl(3aR,4R,6aS)-4-{[2-{[(1H-benzo[d][1,2,3]triazol-1-yl)oxy]carbonyl}-1H-pyrrol-1-yl]methyl}-2,2-dimethyltetrahydro-5H-[1,3]dioxolo[4,5-c]pyrrole-5-carboxylate (27c). Employing the same protocol as for the preparation of 27a carboxylic acid 26c (492 mg, 1.34 mmol) was dissolved in dry DMF (15 mL) and subsequently activated with HBTU (625 mg, 1.65 mmol) in the presence of triethylamine (0.57 mL, 4.09 mmol) to afford 648 mg (quant.) of 27c as a colorless foam. ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ = +31.4 (c 0.805, CHCl₃); ¹H NMR (500 MHz, CDCl₃, mixture of rotamers) δ 8.11–8.06 (m, 1H), 7.59–7.47 (m, 3H), 7.46–7.41 (m, 1H), 7.21 (s, 0.4H), 7.06–7.03 (s, 0.6H), 6.42–6.37 (m, 1H), 4.62 (t, 5.0 Hz, 0.6H), 4.54 (t, 4.8 Hz, 0.4H), 4.51–4.37 (m, 3.4H), 4.18 (dd, 13.5, 9.3 Hz, 0.6H), 4.03 (d, 13.4 Hz, 0.6H), 3.74 (d, 13.1 Hz, 0.4H), 3.22–3.13 (overlapping signals: 3.19, dd, 13.1, 4.5 Hz, 0.6H and m, 0.4H), 1.45 (s, 4H), 1.40 (s, 1H), 1.39 (s, 2H), 1.34 (s, 5H), 1.26 (s, 3H); ¹³C NMR (125 MHz, CDCl₃, mixture of rotamers) δ 157.23, 157.05, 154.32, 143.54, 133.20, 132.73, 129.21, 128.72, 128.59, 124.75, 122.37, 122.18, 120.56, 120.43, 116.27, 116.08, 112.07, 111.00, 110.67, 108.71, 108.49, 82.35, 81.53, 80.41, 80.28, 79.39, 78.98, 64.90, 64.02, 51.93, 50.84, 48.05, 47.09, 28.33, 28.14, 26.91, 26.83, 24.99, 24.92; HRMS (ESI) m/z calcd for C₂₄H₂₉N₅O₆Na⁺ 506.2010, found 506.2012.

tert-Butyl(3aR,4R,6aS)-4-{[2-({4-[3-(4-fluorophenyl)-5-isopropylisoxazol-4-yl]pyridin-2-yl}carbamoyl)-1H-pyrrol-1-yl]methyl}-2,2-dimethyltetrahydro-5H-[1,3]dioxolo[4,5-c]pyrrole-5-carboxylate (28c). Using the same procedure as for the preparation of compound 28a isoxazole 30 (376 mg, 1.26 mmol) dissolved in dry DMF (5 mL) was firstly deprotonated with sodium hydride (60% in mineral oil; 45.0 mg, 1.13 mmol) and subsequently reacted with a solution of compound 27c (339 mg, 0.701 mmol) in dry DMF (5 mL) at room temperature over a period of 48 h to afford 228 mg (50%) of the desired coupling product 28c as a colorless foam. ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ = +46.8 (c 0.66, acetone); ¹H NMR (500 MHz, CDCl₃, mixture of rotamers) δ 8.37 (s, 0.4H), 8.34 (s, 0.6H), 8.25–8.21 (m, 2H), 7.44 (AA’BB’X, J_AB = 8.6, J_AF = 5.4 Hz, 2H), 7.04 (AA’BB’X, J_AB = J_BF = 8.6 Hz, 2H), 6.92 (s, 0.4H), 6.84–6.78 (m, 1.6H), 6.76–6.72 (m, 1H), 6.24–6.20 (m, 1H), 4.70–4.54 (m, 3H), 4.47–4.41 (m, 1.4H), 4.26 (dd, 13.6, 8.1 Hz, 0.6H), 3.94 (d, 13.1 Hz, 0.6H), 3.71 (d, 12.9 Hz, 0.4H), 3.31–3.19 (overlapping signals: m, 1H and 3.26, sp, 7.0 Hz, 1H), 1.44 (s, 4H), 1.40 (s, 3H), 1.40 (d, 6.9 Hz, 6H), 1.34 (s, 5H), 1.26 (s, 2H), 1.22 (s, 1H); ¹⁹F NMR (470 MHz, CDCl₃, mixture of rotamers) δ −110.96, −111.01; ¹³C NMR (125 MHz, CDCl₃, mixture of rotamers) δ 175.48, 164.59/162.61 (d, 248.3 Hz), 160.06, 159.78, 159.52, 154.34, 154.23, 152.34, 152.28, 148.20, 141.09, 130.55/130.48 (d, 8.2 Hz), 129.27, 129.11, 124.79/124.76 (d, 3.4 Hz), 124.71, 120.43, 115.89/115.72 (d, 21.6 Hz), 114.46, 114.41, 114.03, 113.95, 112.18, 111.79, 111.69, 109.19, 109.01, 82.35, 81.53, 80.06, 79.94, 79.48, 78.94, 64.75, 64.01, 52.10, 51.09, 47.42, 46.41, 28.37, 28.19, 26.97, 26.92, 26.66, 25.04, 21.00; HRMS (ESI) m/z calcd for C₃₅H₄₀FN₅O₆H⁺ 646.3035, found 646.3044.

(2R,3R,4S)-2-{[2-({4-[3-(4-Fluorophenyl)-5-isopropylisoxazol-4-yl]pyridin-2-yl}carbamoyl)-1H-pyrrol-1-yl]methyl}-3,4-dihydroxypyrrolidin-1-ium trifluoroacetate (29c). Trifluoroacetic acid (4.75 mL) and water (0.50 mL) were added to compound 28c (163 mg, 0.252 mmol) and the resulting mixture was stirred for 3 h at 0 °C. After complete deprotection as indicated by TLC analysis the reaction was concentrated and the residue purified by automated column chromatography (silica gel, methanol/methylene chloride 0–10%) to yield 131 mg (84%) of 29c as a colorless foam. ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ = −4.5 (c 0.29, MeOH); ¹H NMR (500 MHz, CD₃OD) δ 8.35 (dd, 5.2, 0.7 Hz, 1H), 8.08 (dd, ~1.3, ~0.6 Hz, 1H), 7.45 (AA’BB’X, J_AB = 8.9, J_AF = 5.3 Hz, 2H), 7.16 (dd, 2.6, 1.7 Hz, 1H), 7.13 (AA’BB’X, J_AB = J_BF = 8.9 Hz, 2H), 7.10 (dd, 4.1, 1.6 Hz, 1H), 6.95 (dd, 5.2, 1.5, Hz, 1H), 6.30 (dd, 4.1, 2.6 Hz, 1H), 4.64 (d, 6.4 Hz, 2H), 4.20 (td, 4.1, 2.1 Hz, 1H), 4.02 (dd, 8.4, 4.1 Hz, 1H), 3.77 (dt, ~6.5, ~8.2 Hz, 1H), 3.40 (dd, 12.4, 4.2 Hz, 1H), 3.28 (sp, 7.0 Hz, 1H), 3.14 (dd, 12.4, 2.1 Hz, 1H), 1.38 (d, 7.0 Hz, 6H); ¹⁹F NMR (470 MHz, CD₃OD) δ −77.10, −112.81; ¹³C NMR (125 MHz, CD₃OD) δ 176.92, 166.15/164.17 (d, 247.1 Hz), 162.97, 161.49, 153.76, 149.81, 142.26, 131.83/131.76 (d, 8.3 Hz), 131.06, 126.52, 126.19, 122.22 117.21, 117.08, 116.96/116.79 (d, 22.1 Hz), 113.62, 110.55, 75.09, 71.13, 63.96, 51.24, 48.77, 27.96, 21.25; HRMS (ESI) m/z calcd for C₂₇H₂₈FN₅O₄H⁺ 506.2198, found 506.2203; Anal. calcd for C₂₇H₂₈FN₅O₄·1.3(CF₃COOH): C, 54.38; H, 4.52; N, 10.71. Found C, 54.28; H, 4.77; N, 10.85.

tert-Butyl(3aR,4R,6aS)-4-{[2-(ethoxycarbonyl)-4-(4-methoxyphenyl)-1H-pyrrol-1-yl]methyl}-2,2-dimethyltetrahydro-5H-[1,3]dioxolo[4,5-c]pyrrole-5-carboxylate (25d). Using the same procedure as for the preparation of compound 25c mesylate (−)-20 (2.65 g, 7.54 mmol) was dissolved in dry DMF (40 mL) and reacted with pyrrole 24a (1.5 g, 6.12 mmol) in the presence of cesium carbonate (4.38 g, 13.4 mmol) and a catalytic amount of tetrabutylammonium iodide at 80 °C overnight to give 2.19 g (72%) of 25d as a colorless foam. A crystalline sample (rosettes of fine transparent needles) for X-ray analysis was obtained by crystallization from a mixture of ethyl acetate and 2-propanol. m.p. 136–137 °C (DSC); ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ = +14.5 (c 0.725, acetone); ¹H NMR (500 MHz, CDCl₃, mixture of rotamers) δ 7.40 (AA’XX’, J_AX = 8.7 Hz, 0.8H), 7.38 (AA’XX’, J_AX = 8.7 Hz, 1.2H), 7.18–7.15 (m, 1H), 7.10 (d, 1.7 Hz, 0.4H), 6.92 (d, 1.7 Hz, 0.6H), 6.91–6.86 (overlapping AA’XX’ spin systems, J_AX = 8.7 Hz, 2H), 4.72–4.60 (m, 2H), 4.60–4.54 (m, 1H), 4.51–4.41 (m, 1.4H), 4.30 (q, 7.1 Hz, 2H), 4.14 (dd, 13.6, 8.5 Hz, 0.6H), 3.99 (d, 13.2 Hz, 0.6H), 3.82 (s, 3H), 3.69 (d, 13.0 Hz, 0.4H), 3.33 (dd, 13.1, 4.9 Hz, 0.6H), 3.21 (dd, 12.8, 5.1 Hz, 0.4H), 1.47 (s, 4H), 1.41 (s, 3H), 1.38/1.36 (overlapping tripletts, 7.0 Hz, 3H), 1.30 (s, 5H), 1.28 (s, 2H), 1.25 (s, 1H); ¹³C NMR (125 MHz, CDCl₃, mixture of rotamers) δ 161.59, 161.35, 158.32, 154.37, 154.27, 127.03, 126.42, 126.32, 125.37, 124.98, 124.87, 124.81, 122.90, 122.73, 115.33, 115.28, 114.21, 111.87, 111.71, 82.45, 81.48, 80.16, 80.00, 79.31, 78.93, 64.69, 64.11, 60.07, 55.33, 52.29, 51.04, 47.68, 46.27, 28.43, 28.09, 27.01, 26.92, 25.04, 14.47; HRMS (ESI) m/z calcd for C₂₇H₃₆N₂O₇Na⁺ 523.2415, found 523.2411; Anal. calcd for C₂₇H₃₆N₂O₇: C, 64.78; H, 7.25; N, 5.60. Found C, 64.78; H, 7.33; N, 5.54.

1-{[(3aR,4R,6aS)-5-(tert-Butoxycarbonyl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyrrol-4-yl]methyl}-4-(4-methoxyphenyl)-1H-pyrrole-2-carboxylic acid (26d). Deploying the same protocol as described for the preparation of compound 26a ethyl ester 25d (1.96 g, 3.92 mmol) dissolved in 2:1 mixture of THF (20 mL) and methanol (10 mL) was treated with a 4 M aqueous solution of sodium hydroxide (7.5 mL) at 50 °C overnight to yield 1.73 g (94%) of crude 26d. An analytical sample of 26d was obtained by recrystallization from methanol as a colorless and crystalline solid. m.p. 197–199 °C (DSC); ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ = +17.8 (c 0.695, acetone); ¹H NMR (500 MHz, CDCl₃, mixture of rotamers) δ 7.43–7.37 (overlapping AA’XX’ spin systems, J_AX = 8.7 Hz, 2H), 7.32 (d, 2.0 Hz, 1H), 7.18 (s, 0.4H), 6.99 (d, 1.3 Hz, 0.6H), 6.90 (AA’XX’, J_AX = 8.6 Hz, 2H), 4.73–4.55 (m, 3H), 4.51–4.45 (m, 1.4H), 4.13 (dd, 13.7, 8.8 Hz, 0.6H), 4.04 (d, 13.2 Hz, 0.6H), 3.82 (s, 3H), 3.72 (d, 12.8 Hz, 0.4H), 3.35 (dd, 13.2, 4.8 Hz, 0.6H), 3.22 (dd, 12.8, 5.0 Hz, 0.4H), 1.47 (s, 4H), 1.42 (s, 3H), 1.31/1.30 (overlapping singlets, 7H), 1.26 (s, 1H); ¹³C NMR (125 MHz, CDCl₃, mixture of rotamers) δ 165.17, 164.96, 158.46, 154.35, 126.84, 126.65, 126.47, 126.36, 125.51, 125.34, 121.65, 121.49, 117.34, 114.26, 111.95, 111.84, 82.40, 81.48, 80.30, 80.19, 79.31, 78.93, 64.63, 64.03, 55.32, 52.27, 51.00, 47.83, 46.47, 28.39, 28.07, 26.99/26.89, 25.04; HRMS (ESI) m/z calcd for C₂₅H₃₂N₂O₇H⁺ 473.2282, found 473.2281; Anal. calcd for C₂₅H₃₂N₂O₇: C, 63.55; H, 6.83; N, 5.93. Found C, 63.58; H, 6.88; N, 5.89.

tert-Butyl(3aR,4R,6aS)-4-[(2-{[(1H-benzo[d][1,2,3]triazol-1-yl)oxy]carbonyl}-4-(4-methoxyphen-yl)-1H-pyrrol-1-yl)methyl]-2,2-dimethyltetrahydro-5H-[1,3]dioxolo[4,5-c]pyrrole-5-carboxylate (27d). Applying the same procedure as for the preparation of compound 27a carboxylic acid 26d (1.05 g, 2.22 mmol) dissolved in dry DMF (30 mL) was reacted with HBTU (1.01 g, 2.66 mmol) in the presence of triethylamine (0.93 mL, 6.67 mmol) to yield 1.28 g (98%) of 27d as a colorless foam; ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ = +41.6 (c 0.700, CHCl₃); ¹H NMR (500 MHz, CDCl₃, mixture of rotamers) δ 8.12–8.07 (m, 1H), 7.67 (d, 1.6 Hz, 1H), 7.64–7.51 (m, 2H), 7.49–7.41 (m, 3.4H), 7.24 (m, 0.6H), 6.94 (AA’XX’, J_AX = 8.7 Hz, 2H), 4.67–4.62 (m, 0.6H), 4.61–4.51 (m, 1.2H), 4.51–4.39 (m, 2.6H), 4.22 (dd, 14.8, 11.5 Hz, 0.6H), 4.07 (d, 13.4 Hz, 0.6H), 3.85 (s, 3H), 3.75 (d, 13.7 Hz, 0.4H), 3.27–3.17 (m, 1H), 1.45 (s, 3H), 1.41 (s, 1H), 1.39 (s, 2H), 1.30 (s, 6H), 1.26 (s, 3H); ¹³C NMR (125 MHz, CDCl₃, mixture of rotamers) δ 158.91, 157.05, 154.43, 154.36, 143.55, 129.49, 129.23, 129.15, 128.78, 128.68, 126.92, 126.67, 125.78, 124.79, 120.59, 120.43, 118.64, 118.52, 116.77, 116.53, 114.47, 112.12, 108.77, 108.47, 82.41, 81.50, 80.50, 80.34, 79.36, 79.03, 65.04, 64.03, 55.37, 51.99, 50.83, 48.22, 47.03, 28.35, 28.09, 26.91, 26.84, 24.94; HRMS (ESI) m/z calcd for C₃₁H₃₅N₅O₇Na⁺ 612.2429, found 612.2429.

tert-Butyl(3aR,4R,6aS)-4-{[2-({4-[3-(4-fluorophenyl)-5-isopropylisoxazol-4-yl]pyridin-2-yl}carba-moyl)-4-(4-methoxyphenyl)-1H-pyrrol-1-yl]methyl}-2,2-dimethyltetrahydro-5H-[1,3]dioxolo[4,5-c]pyrrole-5-carboxylate (28d). Following the procedure as described for the preparation of compound 28a isoxazole 30 (266 mg, 0.895 mmol) dissolved in dry DMF (5 mL) was deprotonated with sodium hydride (60% in mineral oil; 32 mg, 0.80 mmol). Subsequently, a solution of compound 27d (309 mg, 0.524 mmol) in dry DMF (5 mL) was added dropwise via cannula and the reaction was stirred at room temperature for 48 h to obtain 220 mg (56%) of 28d as a colorless foam. ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ = +36.8 (c 0.595, acetone); ¹H NMR (500 MHz, CDCl₃, mixture of rotamers) δ 8.49 (s, 0.4H), 8.45 (s, 0.6H), 8.27-8.22 (m, 2H), 7.45 (AA’BB’X, J_AB = 8.5, J_AF = 5.4 Hz, 2H), 7.42–7.36 (m, 2H), 7.14 (s, 0.4H), 7.08–7.02 (m, 3H), 6.99 (s, 0.6H), 6.92 (AA’XX’, J_AX = 8.7 Hz, 2H), 6.78–6.73 (m, 1H), 4.77–4.66 (m, 1.4H), 4.64–4.56 (m, 1.6H), 4.52–4.44 (m, 1H), 4.39 (dd, 13.6, 5.6 Hz, 0.4H), 4.29 (dd, 13.6, 8.4 Hz, 0.6H), 3.98 (d, 13.1 Hz, 0.6H), 3.83 (s, 3H), 3.73 (d, 12.8 Hz, 0.4H), 3.32 (dd, 13.1, 4.8 Hz, 0.6H), 3.30–3.22 (overlapping signals: m, 0.4H; 3.27, sp, 6.9 Hz, 1H), 1.44 (s, 4H), 1.43–1.39 (overlapping signals: 1.41, s, 3H; 1.41, d, 6.9 Hz, 6H), 1.31 (s, 5H), 1.27 (s, 2H), 1.23 (s, 1H); ¹⁹F NMR (470 MHz, CDCl₃) δ −110.91, −110.96; ¹³C NMR (125 MHz, CDCl₃, mixture of rotamers) δ 175.54, 164.61/162.62 (d, 248.4 Hz), 160.07, 159.66, 159.41, 158.53, 154.39, 154.25, 152.29, 152.22, 148.06, 141.25, 130.56/130.50 (d, 7.8 Hz), 126.66, 126.62, 126.41, 126.36, 125.43, 125.31, 125.25, 125.11, 125.04, 124.77, 124.74, 120.46, 115.91/115.74 (d, 21.6 Hz), 114.49, 114.46, 114.37, 114.35, 112.15, 111.85, 111.73, 111.03, 82.41, 81.50, 80.18, 80.00, 79.48, 79.00, 64.87, 64.04, 55.36, 52.17, 51.06, 47.56, 46.41, 28.39, 28.15, 26.98, 26.92, 26.67, 25.05, 21.01; HRMS (ESI) m/z calcd for C₄₂H₄₆FN₅O₇H⁺ 752.3454, found 752.3461. Anal. calcd for C₄₂H₄₆FN₅O₇·0.1(CHCl₃): C, 66.20; H, 6.08; N, 9.17. Found C, 66.18; H, 6.09; N, 9.13.

(2R,3R,4S)-2-{[2-({4-[3-(4-Fluorophenyl)-5-isopropylisoxazol-4-yl]pyridin-2-yl}carbamoyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl]methyl}-3,4-dihydroxypyrrolidin-1-ium trifluoroacetate (29d). Employing the same method as for the preparation of 29c compound 28d (161 mg, 0.214 mmol) was treated with a mixture of trifluoroacetic acid (4.75 mL) and water (0.5 mL) at 0 °C for 2.5 h to yield 119 mg (77%) of 29d as a yellow semisolid. Pure 29d was obtained by subsequent recrystallization from ethyl acetate and petroleum ether. m.p. 191–193 °C (DSC); ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ = −2.4 (c 0.655, MeOH); ¹H NMR (500 MHz, CD₃OD) δ 8.38 (d, 5.3 Hz, 1H), 8.09–8.07 (m, 1H), 7.52 (AA’XX’, J_AX = 8.8 Hz, 2H), 7.49–7.44 (overlapping signals: 7.48, d, 1.7 Hz, 1H and 7.46, AA’BB’X, J_AB = 8.9, J_AF = 5.3 Hz, 2H), 7.42 (d, 1.9 Hz, 1H), 7.14 (AA’BB’X, J_AB = J_BF = 8.9 Hz, 2H), 6.99 (dd, 5.1, 1.5 Hz, 1H), 6.94 (AA’XX’, J_AX = 8.8 Hz, 2H), 4.73 (dd, 15.2, 8.8 Hz, 1H), 4.68 (dd, 15.2, 3.8 Hz, 1H), 4.25 (dt, 3.8, 1.6 Hz, 1H), 4.12 (dd, 8.8, 3.9 Hz, 1H), 3.91 (td, 8.8, 3.8 Hz, 1H), 3.81 (s, 3H), 3.48 (dd, 12.5, 3.9 Hz, 1H), 3.28 (sp, 7.0 Hz, 1H), 3.24 (dd, 12.5, 1.7 Hz, 1H), 1.38 (d, 7.0 Hz, 6H); ¹⁹F NMR (470 MHz, CD₃OD) δ −76.90, −112.83; ¹³C NMR (125 MHz, CD₃OD) δ 176.93, 166.15/164.18 (d, 247.3 Hz), 162.99, 161.47, 160.20, 153.67, 149.90, 142.32, 131.83/131.76 (d, 8.5 Hz), 127.90, 127.40, 127.11, 126.21/126.18 (d, 3.0 Hz), 122.40, 117.20, 116.98/116.80 (d, 22.1 Hz), 115.37, 114.13, 113.61, 74.82, 70.83, 63.72, 55.81, 51.13, 48.70, 27.98, 21.25; HRMS (ESI) m/z calcd for C₃₄H₃₄FN₅O₅H⁺ 612.2617, found 612.2616. Anal. calcd for C₃₄H₃₄FN₅O₅⁺·CF₃COOH: C, 59.58; H, 4.86; N, 9.65. Found C, 59.49; H, 4.88; N, 9.50.

tert-Butyl(3aS,4S,6aR)-4-{[2-(ethoxycarbonyl)-4-(4-methoxyphenyl)-1H-pyrrol-1-yl]methyl}-2,2-dimethyltetrahydro-5H-[1,3]dioxolo[4,5-c]pyrrole-5-carboxylate (25e). Using the same procedure as for the preparation of compound 25c crude mesylate (+)-20 (1.22 g, 3.47 mmol) dissolved in dry DMF (20 mL) was reacted with pyrrole 24a (712 mg, 2.90 mmol), cesium carbonate (2.08 g, 6.38 mmol) and a catalytic amount of tetra-n-butylammonium iodide to afford 1.12 g (77%) of 25e as a colorless foam. ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ = −15.4 (c 0.695, acetone). All spectroscopic data was consistent with that reported for its enantiomer 25d. HRMS (ESI) m/z calcd for C₂₇H₃₆N₂O₇Na⁺ 523.2415, found 523.2411; Anal. calcd for C₂₇H₃₆N₂O₇: C, 64.78; H, 7.25; N, 5.60. Found C, 64.61; H, 7.37; N, 5.59.

1-{[(3aS,4S,6aR)-5-(tert-Butoxycarbonyl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyrrol-4-yl]methyl}-4-(4-methoxyphenyl)-1H-pyrrole-2-carboxylic acid (26e). Applying the same protocol as for the preparation of compound 26a ethyl ester 25e (1.01 g, 2.02 mmol) was dissolved in a 2:1 mixture of THF and methanol (12 mL/6 mL) and treated with an aqueous solution of potassium hydroxide (8 M, 3.75 mL) at 50 °C overnight to afford 983 mg (quant.) of crude 26e as a light-yellow foam which was subsequently deployed in the next reaction step without further purification. An analytical sample was obtained by recrystallization from methanol. m.p. 196–199 °C (DSC). ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ = −17.7 (c 0.515, acetone). All spectroscopic data was consistent with the data reported for its enantiomer 26d. HRMS (ESI) m/z calcd for C₂₅H₃₂N₂O₇Na⁺ 495.2102, found 495.2102; Anal. calcd for C₂₅H₃₂N₂O₇: C, 63.55; H, 6.83; N, 5.93. Found C, 63.47; H, 6.89; N, 5.91.

tert-Butyl(3aS,4S,6aR)-4-{[2-{[(1H-benzo[d][1,2,3]triazol-1-yl)oxy]carbonyl}-4-(4-methoxyphenyl)-1H-pyrrol-1-yl]methyl}-2,2-dimethyltetrahydro-5H-[1,3]dioxolo[4,5-c]pyrrole-5-carboxylate (27e). Using the same method as described for the preparation of compound 27a crude carboxylic acid 26e (916 mg, 1.94 mmol) dissolved in dry DMF (20 mL) was reacted with HBTU (894 mg, 2.36 mmol) in the presence of triethylamine (0.83 mL, 5.96 mmol) to yield 1.05 g (92%) of 27e as a colorless foam. ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ = −39.7 (c 0.635, CHCl₃); All spectroscopic data was consistent with the data described for its enantiomer 27d. HRMS (ESI) m/z calcd for C₃₁H₃₅N₅O₇Na⁺ 612.2429, found 612.2437.

tert-Butyl(3aS,4S,6aR)-4-{[2-({4-[3-(4-fluorophenyl)-5-isopropylisoxazol-4-yl]pyridin-2-yl}carbamoyl)-4-(4-methoxyphenyl)-1H-pyrrol-1-yl]methyl}-2,2-dimethyltetrahydro-5H-[1,3]di-oxolo[4,5-c]pyrrole-5-carboxylate (28e). Following the procedure outlined for the preparation of compound 28a isoxazole 30 (474 mg, 1.59 mmol) dissolved in dry DMF (14 mL) was deprotonated with sodium hydride (60% in mineral oil; 75 mg, 1.88 mmol) and compound 27e (500 mg, 0.848 mmol) was subsequently introduced in one portion at room temperature to yield 335 mg (53%) of 28e as a light-yellow resin. ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ = −32.5 (c 0.76, acetone). All spectroscopic data of 28e was consistent with that reported for its enantiomer 28d. HRMS (ESI) m/z calcd for C₄₂H₄₆FN₅O₇Na⁺ 774.3273, found 774.3269. Anal. calcd for C₄₂H₄₆FN₅O₇: C, 67.10; H, 6.17; N, 9.31. Found C, 67.45; H, 6.53; N, 8.92.

(2S,3S,4R)-2-{[2-({4-[3-(4-Fluorophenyl)-5-isopropylisoxazol-4-yl]pyridin-2-yl}carbamoyl)-3-(4-methoxyphenyl)-1H-pyrrol-1-yl]methyl}-3,4-dihydroxypyrrolidin-1-ium trifluoroacetate (29e). Employing the same procedure as outlined for the preparation of 29c compound 28e (234 mg, 0.311 mmol) was treated with a mixture of TFA (6.8 mL) and water (0.76 mL) at 0 °C for 3 h and the crude product was purified by automated flash column chromatography (silica gel, methanol/dichloromethane 0–10%) to give 179 mg (79%) of 29e as a light-yellow resin. Subsequent recrystallization from ethyl acetate/petroleum ether provided 145 mg of pure 29e as a colorless solid. m.p. 188–191 °C (DSC); ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ = +2.6 (c 0.53, MeOH). All spectroscopic data was consistent with the data reported for its enantiomer 29d. HRMS (ESI) m/z calcd for C₃₄H₃₄FN₅O₅H⁺ 612.2617, found 612.2620. Anal. calcd for C₃₄H₃₄FN₅O₅⁺·CF₃COOH: C, 59.58; H, 4.86; N, 9.65. Found C, 59.71; H, 4.98; N, 9.68.

tert-Butyl(3aR,4R,6aS)-4-{[4-(2,4-dimethoxyphenyl)-2-(ethoxycarbonyl)-1H-pyrrol-1-yl]methyl}-2,2-dimethyltetrahydro-5H-[1,3]dioxolo[4,5-c]pyrrole-5-carboxylate (25f). Employing the same procedure as for the preparation of compound 25c crude mesylate (−)-20 (2.58 g, 7.34 mmol) dissolved in dry DMF (50 mL) was reacted with pyrrole 24b (1.68 g, 6.10 mmol) and cesium carbonate (4.48 g, 13.8 mmol) to afford 1.86 g (57%) of 25f as a colorless oil that solidified on standing. ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ = +13.2 (c 1.105, acetone); ¹H NMR (500 MHz, CDCl₃, mixture of rotamers) δ 7.42–7.37 (m, 1H), 7.32 (d, 1.6 Hz, 0.4H), 7.25 (d, 1.7 Hz, 0.4H), 7.24 (d, 1.7 Hz, 0.6H), 7.21 (d, 1.7 Hz, 0.6H), 6.53–6.49 (m, 2H), 4.67 (dd, 13.9, 7.8 Hz, 0.4H), 4.64–4.57 (m, 2.2H), 4.55 (dd, 14.0, 4.7 Hz, 0.4H), 4.51 (t, 5.2 Hz, 0.4H), 4.47–4.41 (m, 1H), 4.32–4.24 (overlapping signals: 4.29, q, 7.0 Hz, 2H and m, 0.6H), 3.91 (d, 13.0 Hz, 0.6H), 3.86 (s, 1H), 3.85 (s, 2H), 3.82 (s, 3H), 3.66 (d, 13.2 Hz, 0.4H), 3.33 (dd, 13.1, 4.2 Hz, 0.6H), 3.25 (dd, 12.7, 5.3 Hz, 0.4H), 1.47 (s, 3H), 1.41 (s, 3H), 1.39–1.33 (overlapping signals: m, 3H and 1.34, s, 6H), 1.27 (s, 2H), 1.24 (s, 1H); ¹³C NMR (125 MHz, CDCl₃, mixture of rotamers) δ 161.70, 161.52, 159.31, 157.18, 154.39, 154.14, 128.62, 128.35, 128.26, 121.67, 120.85, 120.60, 117.08, 116.84, 116.13, 111.75, 111.58, 104.73, 98.93, 82.42, 81.40, 80.03, 79.91, 79.30, 78.78, 64.61, 63.92, 59.94, 55.41, 55.37, 55.29, 52.39, 51.27, 47.47, 46.21, 28.44, 28.12, 27.06, 26.98, 25.08, 14.50; HRMS (ESI) m/z calcd for C₂₈H₃₈N₂O₈Na⁺ 553.2520, found 553.2529.

1-{[(3aR,4R,6aS)-5-(tert-Butoxycarbonyl)-2,2-dimethyltetrahydro-4H-[1,3]dioxolo[4,5-c]pyrrol-4-yl]methyl}-4-(2,4-dimethoxyphenyl)-1H-pyrrole-2-carboxylic acid (26f). Applying the same procedure as reported for the preparation of carboxylic acid 25a ethyl ester 25f (1.21 g, 2.28 mmol) dissolved in a 2:1 mixture of THF (12.5 mL) and methanol (6.5 mL) was treated with a 4 M aqueous sodium hydroxide solution (4.4 mL) at 50 °C overnight to obtain 1.05 g (92%) of 26f as a light-yellow solid. An analytical sample of 26f was obtained as colorless crystals by re-crystallization from ethyl acetate/petroleum ether. m.p. 180–181 °C (DSC); ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ = +12.6 (c 0.840, acetone); ¹H NMR (500 MHz, CDCl₃, mixture of rotamers) δ 7.43–7.37 (m, 2H), 7.27–7.25 (m, 1H), 6.54–6.50 (m, 2H), 4.71–4.51 (m, 3.4H), 4.51–4.44 (m, 1H), 4.25 (dd, 13.7, 7.8 Hz, 0.6H), 3.96 (d, 13.0 Hz, 0.6H), 3.87 (s, 1H), 3.86 (s, 2H), 3.83 (s, 3H), 3.70 (d, 13.1 Hz, 0.4H), 3.34 (dd, 13.0, 4.9 Hz, 0.6H), 3.25 (dd, 12.7, 5.2 Hz, 0.4H), 1.47 (s, 3H), 1.42 (s, 3H), 1.35 (s, 6H), 1.29 (s, 2H), 1.26 (s, 1H); ¹³C NMR (125 MHz, CDCl₃, mixture of rotamers) δ 165.50, 165.32, 159.49, 157.21, 154.40, 154.20, 130.07, 129.81, 128.38, 128.32, 121.48, 121.22, 120.43, 119.26, 119.10, 115.78, 111.87, 111.71, 104.78, 98.97, 82.40, 81.41, 80.18, 80.09, 79.33, 78.82, 64.55, 63.89, 55.43, 55.38, 55.31, 52.40, 51.25, 47.67, 46.40, 28.43, 28.13, 27.05, 26.98, 25.11; HRMS (ESI) m/z calcd for C₂₆H₃₄N₂O₈Na⁺ 525.2207, found 525.2220. Anal. calcd for C₂₆H₃₄N₂O₈: C, 62.14; H, 6.82; N, 5.57. Found C, 62.13; H, 6.96; N, 5.48.

tert-Butyl(3aR,4R,6aS)-4-{[2-{[(1H-benzo[d][1,2,3]triazol-1-yl)oxy]carbonyl}-4-(2,4-dimethoxyphenyl)-1H-pyrrol-1-yl]methyl}-2,2-dimethyltetrahydro-5H-[1,3]dioxolo[4,5-c]pyrrole-5-carboxylate (27f). Deploying the same procedure as for the preparation of compound 27a carboxylic acid 26f (317 mg, 0.631 mmol) dissolved in dry DMF (6 mL) was reacted with HBTU (304 mg, 0.802 mmol) in the presence of triethylamine (0.3 mL, 2.15 mmol) to give 335 mg (86%) of 27f as a colorless resin. ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ = +34.6 (c 0.735, CHCl₃); ¹H NMR (500 MHz, CDCl₃, mixture of rotamers) δ 8.11–8.06 (m, 1H), 7.78–7.75 (m, 1H), 7.65–7.49 (m, 3H), 7.47–7.41 (m, 2H), 6.58–6.54 (m, 2H), 4.61 (t, 5.0 Hz, 0.6H), 4.57 (dd, 12.3, 6.1 Hz, 0.4H), 4.54–4.39 (m, 3.4H), 4.27 (dd, 15.41, 10.4 Hz, 0.6H), 4.01 (d, 13.3 Hz, 0.6H), 3.91 (s, 1H), 3.90 (s, 2H), 3.85 (s, 3H), 3.72 (d, 12.8 Hz, 0.4H), 3.27–3.19 (overlapping signals: m, 0.4H and 3.24, dd, 13.2, 4.7 Hz, 0.6H), 1.45 (s, 3H), 1.41 (s, 1H), 1.40 (s, 2H), 1.31 (s, 6H), 1.26 (s, 3H); ¹³C NMR (125 MHz, CDCl₃, mixture of rotamers) δ 159.91, 157.27, 157.10, 154.35, 154.22, 143.49, 132.53, 132.07, 129.23, 129.16, 128.67, 128.56, 128.52, 128.40, 124.70, 122.87, 122.60, 120.54, 120.49, 120.33, 120.26, 115.47, 115.40, 114.90, 114.86, 111.99, 111.92, 108.79, 108.48, 104.91, 98.99, 82.36, 81.36, 80.32, 80.19, 79.28, 78.90, 64.91, 63.78, 55.41, 55.33, 52.05, 50.92, 47.98, 46.84, 28.31, 28.01, 26.91, 26.83, 24.93; HRMS (ESI) m/z calcd for C₃₂H₃₇N₅O₈Na⁺ 642.2534, found 642.2530.

tert-Butyl(3aR,4R,6aS)-4-{[4-(2,4-dimethoxyphenyl)-2-({4-[3-(4-fluorophenyl)-5-isopropylisoxa-zol-4-yl]pyridin-2-yl}carbamoyl)-1H-pyrrol-1-yl]methyl}-2,2-dimethyltetrahydro-5H-[1,3]dioxolo [4,5-c]pyrrole-5-carboxylate (28f). Following the same method as described for the preparation of compound 28a isoxazole 30 (196 mg, 0.659 mmol) dissolved in dry DMF (5 mL) was initially deprotonated with sodium hydride (60% in mineral oil; 24.7 mg, 0.618 mmol) which was then followed by introducing dropwise a solution of compound 27f (230 mg, 0.371 mmol) in dry DMF (5 mL) via cannula to give 103 mg (36%) of 28f as a colorless foam. ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ = +26.2 (c 0.775, acetone); ¹H NMR (500 MHz, CDCl₃, mixture of rotamers) δ 8.49 (s_br, 0.4H), 8.46 (s_br, 0.6H), 8.28-8.23 (m, 2H), 7.45 (AA’BB’X, J_AB = 8.4, J_AF = 5.5 Hz, 2H), 7.39–7.35 (m, 1H), 7.34 (s, 0.4H), 7.23 (s, 0.6H), 7.18–7.14 (m, 1H), 7.05 (AA’BB’X, J_AB = J_BF = 8.6 Hz, 2H), 6.74 (t, 4.5 Hz, 1H), 6.56–6.52 (m, 2H), 4.74–4.55 (m, 3H), 4.53–4.43 (m, 1.4H), 4.37 (dd, 13.4, 7.6 Hz, 0.6H), 3.92 (d, 13.1 Hz, 0.6H), 3.89 (s, 1H), 3.88 (s, 2H), 3.84 (s, 3H), 3.71 (d, 12.9 Hz, 0.4H), 3.36–3.22 (overlapping signals: 3.33, dd, 13.1, 4.9 Hz, 0.6H; m, 0.4H and 3.27, sp, 7.0 Hz, 1H), 1.45 (s, 3.5H), 1.43-1.39 (overlapping signals, 9H), 1.34 (s, 5.5H), 1.27 (s, 2H), 1.22 (s, 1H); ¹⁹F NMR (470 MHz, CDCl₃, mixture of rotamers) δ −110.94, −111.00; ¹³C NMR (125 MHz, CDCl₃, mixture of rotamers) δ 175.50, 164.60/162.62 (d, 247.8 Hz), 160.08, 159.81, 159.56, 157.25, 154.42, 154.17, 152.43, 152.38, 148.17, 141.11, 130.56/130.49 (d, 8.2 Hz), 128.40, 128.25, 128.20, 124.79, 124.78, 124.17, 124.13, 121.08, 120.91, 120.39, 115.90/115.73 (d, 21.7 Hz), 114.47, 112.98, 112.80, 112.20, 111.76, 111.60, 104.83, 99.05, 82.40, 81.44, 80.09, 79.92, 79.49, 78.87, 64.78, 63.90, 55.45, 55.34, 52.27, 51.26, 47.42, 46.26, 28.41, 28.15, 27.02, 26.98, 26.66, 25.09, 21.01; HRMS (ESI) m/z calcd for C₄₃H₄₈FN₅O₈Na⁺ 804.3379, found 804.3383.

(2R,3R,4S)-2-{[3-(2,4-Dimethoxyphenyl)-2-({4-[3-(4-fluorophenyl)-5-isopropylisoxazol-4-yl]pyridin-2-yl}carbamoyl)-1H-pyrrol-1-yl]methyl}-3,4-dihydroxypyrrolidin-1-ium trifluoroacetate (29f). Compound 28f (110 mg, 0.141 mmol) was treated with a 9:1 mixture of trifluoroacetate (3.6 mL) and water (0.4 mL) at 0 °C for 2.5 h applying the same protocol as for the preparation of compound 29c to yield 68.4 mg (64%) of 29f as a light-yellow foam. ${\left[\mathsf{\alpha }\right]}_{\mathrm{D}}^{20}$ = −1.14 (c 0.875, MeOH); ¹H NMR (500 MHz, CD₃OD) δ 8.37 (d, 3.3 Hz, 1H), 8.07 (s, 1H), 7.63 (d, 1.7 Hz, 1H), 7.52 (d, 1.8 Hz, 1H), 7.49 (d, 8.6 Hz, 1H), 7.46 (AA’BB’X, J_AB = 8.9, J_AF = 5.3 Hz, 2H), 7.14 (AA’BB’X, J_AB = J_BF = 8.8 Hz, 2H), 7.00 (d, 4.6 Hz, 1H), 6.61 (d, 2.4 Hz, 1H), 6.57 (dd, 8.5, 2.4 Hz, 1H), 4.72 (d, 6.3 Hz, 2H), 4.26 (td, 3.8, 1.6 Hz, 1H), 4.13 (dd, 8.8, 3.9 Hz, 1H), 3.96–3.88 (overlapping signals: m, 1H and 3.90, s, 3H), 3.82 (s, 3H), 3.49 (dd, 12.5, 3.9 Hz, 1H), 3.33–3.22 (overlapping signals: 3.29, sp, 7.0 Hz, 1H and 3.24, dd, 12.6, 1.7 Hz, 1H), 1.39 (d, 7.0 Hz, 6H); ¹⁹F NMR (470 MHz, CD₃OD) δ −77.01, −112.77; ¹³C NMR (125 MHz, CD₃OD) δ 177.05, 166.17/164.19 (d, 257.6 Hz), 163.13, 161.47, 161.33, 158.83, 153.53, 149.15, 142.83, 131.86/131.79 (d, 8.3 Hz), 130.25, 129.25, 126.14, 125.69, 123.30, 122.30, 117.22, 117.00, 116.83, 116.60, 116.15, 113.51, 106.38, 99.87, 74.81, 70.80, 63.74, 55.91, 51.11, 48.69, 27.98, 21.25; HRMS (ESI) m/z calcd for C₃₅H₃₆FN₅O₆H⁺ 642.2722, found 642.2735. Anal. calcd for C₃₅H₃₆FN₅O₆·2.3(CF₃COOH): C, 52.62; H, 4.27; N, 7.75. Found C, 52.61; H, 4.44; N, 7.73.

(1R,2S,10aR)-N-{4-[3-(4-Fluorophenyl)-5-isopropylisoxazol-4-yl]pyridin-2-yl}-1,2-dihydroxy-6-(4-methoxyphenyl)-2,3,10,10a-tetrahydro-1H,5H-dipyrrolo[1,2-a:1’,2’-d]pyrazine-8-carboxamide (31a). To a solution of 29d (20 mg, 0.043 mmol) in methanol (2 mL) were added 2 drops of formalin (formaldehyde solution, 37% in water). After being stirred at room temperature for 3 h the reaction was concentrated, and the crude product purified by automated column chromatography (silica gel, methanol/dichloromethane 0–10%) to afford 9 mg of 31a as a colorless solid. ¹H NMR (400 MHz, d_6-DMSO) δ 10.39 (s, 1H, NH), 8.36 (d, 4.6 Hz, 1H), 8.10 (s, 1H), 7.57 (s, 1H), 7.46 (AA’BB’X, J_AB = 8.9, J_AF = 5.5 Hz, 2H), 7.30 (AA’XX’, J_AX = 8.7 Hz, 2H), 7.27 (AA’BB’X, J_AB = J_BF = 8.9 Hz, 2H), 6.98 (AA’XX’, J_AX = 8.8 Hz, 2H), 6.89 (dd, 5.1, 1.5 Hz, 1H), 4.95–4.84 (overlapping signals: 4.93, dd, 13.0, 3.8 Hz, 1H; 4.93, s_br, 1H, OH), 4.84 (s_br, 1H, OH), 4.10 (d, 14.7 Hz, 1H), 4.09–4.03 (m, 1H), 3.80–3.74 (m, 1H; 3.77, s, 3H), 3.66 (m, 1H), 3.58 (d, 14.5 Hz, 1H), 3.47 (dd, 9.7, 6.4 Hz, 1H), 3.22 (sp, 7.0 Hz, 1H), 2.60–2.54 (m, 1H), 2.29–2.25 (m, 1H), 1.32 (d, 6.9 Hz, 6H); ¹⁹F NMR (376 MHz, d_6-DMSO) δ −111.04; ¹³C NMR (125 MHz, d_6-DMSO) δ 174.80, 164.0/161.6 (d, 247.3 Hz), 159.80, 159.58, 157.36, 152.97, 148.30, 139.42, 130.45/130.36 (d, 8.6 Hz), 130.1, 127.74, 127.23, 124.56/124.54 (d, 3.2 Hz), 122.83, 119.87, 118.34, 116.03/115.81 (d, 21.9 Hz), 114.49, 114.20, 114.08, 112.13, 73.99, 67.99, 63.13, 61.21, 55.00, 50.64 (br), 49.79 (br), 26.00, 20.60; HRMS (ESI) m/z calcd for C₃₅H₃₄FN₅O₅H⁺ 624.2617, found 624.2613.

(1S,2R,10aS)-N-{4-[3-(4-Fluorophenyl)-5-isopropylisoxazol-4-yl]pyridin-2-yl}-1,2-dihydroxy-6-(4-methoxyphenyl)-2,3,10,10a-tetrahydro-1H,5H-dipyrrolo[1,2-a:1’,2’-d]pyrazine-8-carboxamide (31b). Employing the same procedure as for the preparation of 31a compound 29e (31 mg, 0.043 mmol) was dissolved in methanol (2 mL) and treated with 2 drops of formalin (formaldehyde solution, 37% in water) at room temperature overnight to afford 30 mg of 31b as a light-yellow solid. All spectroscopic data of 31b was consistent with that recorded for 31a. HRMS (ESI) m/z calcd for C₃₅H₃₄FN₅O₅H⁺ 624.2617, found 624.2615.

3.3. Plasmids, Overexpression and Purification of Glutathion S-Transferase Fusion CK1δ TV1 Protein

Human CK1δ transcription variant 1 (CK1δ TV1_NM_001893.4) has been amplified from the cDNA obtained from HT1080 cell line, using CK1δ_forward-primer: 5′-GGA TCC ATG GAG CTG AGA GTC GGG AAC AG-3′ and CK1δ_reverse primer: 5′-GGA TCC TCA TCG GTG CAC GAC AGA CTG A-3′. The CK1δ DNA construct was cloned into pSC-A cloning vector (Agilent technologies, Munich, Germany) with BamHI enzyme, to generate plasmids pGEX6-P3-GST-CK1δ TV1 (FP1417). Overexpression and purification of GST-CK1δ TV1 protein has been performed as described before [87].

3.4. Kinase Assays

In vitro kinase assays have been performed to both screen, as well as to calculate IC₅₀ values of the compounds for CK1δ and ε. Each reaction was carried out using 2 μCi ³²P-γ-ATP in kinase buffer containing 25 mM Tris-HCl (pH = 7.5), 10 mM MgCl₂, 100 µM EDTA, and 10 µM ATP. Potential inhibitor compounds were used in a concentration of 10 µM for screening assays and in a dilution series ranging from 10 μM to 5 nM final reaction concentration, which was prepared by serial dilution in DMSO, in order to calculate the IC₅₀ values. Recombinant human CK1δ transcription variant 1 (expressed and purified as GST fusion protein as described earlier) and human GST-CK1ε (PV3500; Invitrogen) were used as sources of enzyme, while α-casein (C6780; Sigma-Aldrich) was used as substrate. Kinase reactions were incubated for 30 min at 30 °C and then stopped with loading dye (50 mM Tris-HCl (pH = 6.8), 5% (v/v) β-mercaptoethanol (MSH), 10% (v/v) glycerol, 2% (w/v) sodium dodecyl sulfate (SDS), 0.1% (w/v) bromphenol blue). Subsequently, reactions were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and phosphorylated protein bands were visualized on dried gels by autoradiography. The phosphorylated substrate protein bands were excised, and phosphorylation was quantified by Cherenkov counting. Dose-response analyses and calculation of IC₅₀ values were carried out using GraphPad Prism 6 statistical software (San Diego, CA, USA).

3.5. Cell Culture

3.5.1. Cell Lines

The human colon adenocarcinoma cell line HT-29 was grown in McCoy’s 5A medium, whereas MCF7 cells were grown in DMEM medium. All media were supplemented with 10% fetal calf serum (FCS), 100 units/mL penicillin, 100 µg/mL streptomycin and 2 mM glutamine. All cells were grown at 37 °C in a humidified 5% carbon dioxide atmosphere.

3.5.2. Cellular Assays and EC₅₀ Determination

In order to analyze the effects of selected inhibitors on the cell viability of HT-29 and MCF7 cells, MTT assays were performed. 1 × 10⁴ cells/well were seeded in 96-well cell culture plates. After 24 h cells were treated with increasing concentrations of the indicated inhibitor compounds, with untreated and DMSO-treated cells as control. After an incubation period of 48 h, 10 µL of MTT solution (5 mg/mL in PBS) were added and cells were incubated for 4 h. MTT-containing medium was carefully removed and 100 µL acidic isopropanol (0.04 n HCl in isopropanol) per well were added. To dissolve formazan crystals, plates were shaken for 30 min on an orbital shaker in the dark. Finally, dissolved crystals were measured spectrophotometrically at 570 nm. All experiments were performed in triplicate with four technical replicates per assay. Results were normalized considering the mean optical density value of control wells as 100%. GraphPad Prism 6 software (San Diego, CA, USA) was used to calculate EC₅₀ values.

3.6. Macromolecular Crystallography

For structural studies, His₆-tagged tCK1δ was recombinantly expressed in BL21 (DE3) TaKaRa 2 Escherichia coli cells (Clontech) and afterwards purified by immobilized metal affinity chromatography as described previously [40].

For co-crystallization of tCK1δ with 29d and 29e, protein stock solution (10 mg∙mL⁻¹) was mixed 30:1 with 10 mM of the respective compounds (solubilized in DMSO) and incubated for 30 min at room temperature. Sitting drop crystallization trials were set up at room temperature with drop ratios of 3 µL protein/inhibitor solution to 2 µL precipitant solution.

Crystals appeared after three to seven days in drops containing 0.1 M MES (pH = 5.5), 10% (w/v) PEG 4000 and 0.2 M lithium sulfate. For data collection, these crystals were cryo-protected by swiping them though reservoir solution supplemented with 25% (v/v) glycerol and subsequently flash frozen. Diffraction data were collected at beamline P13 at the PETRA/EMBL Hamburg, German Synchrotron Research Centre (DESY) campus, Hamburg, Germany (tCK1δ with 29d) and at beamline X06DA at the Swiss Light Source, Paul-Scherrer- Institute, Villigen, Switzerland. XDS [87] was used for data processing and the structures were solved by molecular replacement using the program PHASER [88,89] with a truncated crystal structure of CK1δ (pdb 4TWC [72]) as a search model. Between iterative cycles of refinement using phenix.refine [90] missing loops, as well as 31a/31b were manually built with Coot [91]. Restraints of 31a and 31b were calculated using phenix.elbow [92].

4. Conclusions

By using isoxazole-based CK1δ inhibitor 8 as model compound we implemented chiral pyrrolidine scaffolds to investigate specific interactions within the ATP binding pocket of this kinase. A synthetic approach leading to stereochemically defined compounds (29d–f) was established. Biological evaluation proved the compounds to be quite potent in in vitro CK1 kinase assays, but revealed only a minor impact of the different stereoisomers towards affinity differences and CK1 isoform-selectivity. However, selectivity profiling in a panel of 320 kinases showed compound 29d to be relatively specific for CK1. Compounds 29d–f showed also modest effects on HT-29 and MCF-7 cell lines. Surprisingly, X-ray crystallographic data revealed new cyclized compounds within the analyzed protein-ligand complexes, which were supposedly produced by spontaneous Pictet-Spengler cyclization implementing one equivalent formaldehyde during sample soaking procedures. In fact, this hypothesis could be confirmed by reacting the original compounds 29d/e with formaldehyde to yield the new ligands 31a/b. Taken together, functionalized pyrrolidines are expedient chiral scaffolds in medicinal chemistry and may have a potential for the development of potent and selective kinase inhibitors targeting the ribose pocket of the ATP binding site.