Expanding The Scope Of Oligo-pyrrolinone-pyrrolidines As Protein-protein Interface Mimics

Abstract

Oligo-pyrrolinone-pyrrolidines (generic structure 1) have the potential to interfere with protein-protein interactions (PPIs), but to reduce this to practice it is necessary to be able to synthesize these structures with a variety of different side-chains corresponding to genetically encoded proteins. This paper describes expansion of the synthetic scope of 1, the difficulties encountered in this process, particularly issues with epimerization and slow coupling rates, and methods to overcome them. Finally, spectroscopic and physicochemical properties as well as proteolytic stabilities of molecules in this series were measured; these data highlight the suitability of oligo-pyrrolinone-pyrrolidines for the development of pharmacological probes or pharmaceutical leads.

INTRODUCTION

Minimalist mimics of secondary structures is a term we used to describe organic scaffolds that present amino acid side chains (usually three) in restricted conformations relative to peptides A. Typical examples of these include Hamilton’s helical systems beginning with terphenyls and now encompassing a range of variations from his group^1–5 and others,^6–12 and Hirchmann Smith turn and sheet mimics.^13–17 Some specific examples of recent minimalist mimics include Arora’s oxopiperazines B, Smith/Hirschmann pyrrolinones C,¹⁷, selected Bartlett’s aza-@tide structures D,^18,19, and our own oligo-(pyrrolinone-pyrrolidines) 1.²⁰

Several attributes shared by the generalized structures B - D and 1 enhance their value as minimalist mimics. First, they all must occupy more narrowly defined regions of conformational space than the peptides A because their backbones have fewer significant degrees of freedom (red arrows). Second, they are not comprised of repeating secondary amide units, enhancing the likelihood their derivatives will be proteolytically stable, cell permeable, and orally bioavailable,²¹ though each of these parameters is partly-determined by the side-chains. Their relative conformational rigidities,²² and the fact that they will not form polycationic ammonium species under physiological conditions, are also desirable features with respect to retarding proteolysis and promoting absorption. Third, water solubilities of compounds having these structures will tend to be promoted by their heteroatoms and heterocycles, relative to structures featuring mostly benzenoid rings. Fourth, each of these minimalist mimic scaffolds have side-chains arranged in 1, 4, 7, 10 ….. relationships (we colloquially refer to this feature of some minimalist mimics as side-chain periodicities). The fact that this is the same side-chain periodicity as peptides and proteins is almost certainly advantageous when attempting to mimic consecutive amino acid side chains. Finally, and related to this, all these structures presumably could be made from amino acids, and this may facilitate preparations of systems with more functionalized, less synthetically accessible, side-chains like those found in Arg, His, Trp, and Cys.

In practice, the fact that structures like B – D could be made from functionalized amino acids is very different to actually demonstrating the methods and conditions by which they can be prepared with these side-chains. Just as protocols in peptide synthesis have taken decades to develop and refine, syntheses of any of these systems with different combinations of side-chains corresponding to a diverse set of amino acids are difficult. Consequently, one key objective of this article is to describe syntheses of compounds 1 incorporating a variety of amino acid side-chains. We have not prepared a set of systems that shows all 20 naturally encoded amino acids can be incorporated, but have made enough to support an immediate need for further validating the concept of Exploring Key Orientations (EKO).²³ This technique features data mining to match preferred conformations of a minimalist mimic with PPI interface regions.

RESULTS AND DISSUSSION

Preparation Of Tetramic Acid Derivatives 2

The first of two routes to the requisite tetramic acid synthons involved BOC-protected amino acids as starting materials in condensation/cyclization processes involving Meldrum’s acid as shown in Scheme 1a.^24–27 This strategy has the advantage that many BOC-protected amino with appropriate side-chain protection are commercially available, and inexpensive relative to other protected amino acid derivatives. Moreover, the condensation/cyclization process is experimentally convenient because strictly anhydrous conditions are not required. However, this “Meldrum’s acid route” described above also has drawbacks. In our hands, the condensation procedure does not proceed to completion for some amino acids (eg Phe) unless excess carboxylate activating agent are used (eg 2.0 – 2.5 eq EDCI, even though some procedures have reported using less EDCI).²⁷ Consequently, a procedure was devised wherein an aqueous work up was used to remove excess EDCI and DMAP salts after first step of the process, and the crude material was used in the next transformation without further purification.

Scheme 1.

Two methods for preparing tetramic acid derivatives: a Meldrum’s acid approach. b Bestmann’s ylid^28–30 route.

The procedures described above were based on work by Tønder and co-workers^24–27 who made these products but exclusively using hydrophobic amino acids for which the purified products could be obtained via precipitation from ether. The current studies showed this approach was not universally applicable to functionalized side-chains, depending on the scale, the side-chain, and residual TFA levels in the crude material. Thus isolation of some tetramic acids (eg that from Trp) required chromatograpy, but several others (Met/Phe/Thr and Leu) were crystallized directly after rigorous removal of TFA. Overall, we find this “Meldrum’s acid route” to be the most useful entry point into tetramic acids with NH N-termini; it tends to be practical because many of the requisite BOC-protected amino acids starting materials are commercially available.

Scheme 1b shows an alternative route to tetramic acid derivatives based on cumulative literature, largely originating from Schobert’s laboratory,^29–33 regarding additions of (triphenylphosphoranylidine)ketene (the Bestmann ylid) to N-alkyl α-amino esters. These routes are conceptually different to those based on Meldrum’s acid insofar as the starting materials are C- rather than N-protected. Removal of Ph₃PO after the cyclization in method b can be problematic, particularly for > 5 g scale. When trying to overcome this difficulty, we found the N-PMB protected products tend to be ether soluble thus facilitating precipitation of Ph₃PO from concentrated ether solutions of the crude material at −20 °C. Samples obtained in this way could be subjected to column chromatography, even on >5 g scales. This approach is more practical, if less elegant, than ones based on supported variants of Bestmann’s ylid³³ that involve inconveniently large amounts of solid-phase reagent for large scale reactions.

Overall, the Bestmann’s ylid approach in Scheme 1b is preferred if N-capped tetramic acid derivatives are required. N-PMB tetramic derivatives are useful in the syntheses described below, and the starting materials can be prepared conveniently from amino acids via reductive amination with 4-methoxybenzaldehyde.

A possible complication for preparation of tetramic acids from aspartic acid derivatives is competing cyclization to give the six-membered ring product in competition with the desired material 2d′. To explore this possibility, the di-tert-butyl ester of aspartic acid was transformed as indicated in reaction 1, but only the five-membered ring compound 2d′ was observed. The corresponding reaction with glutamic acid was not attempted, but in that case the competitive processes would be between formation of a 5-membered ring and a 7-membered ring, the latter is less likely, so side-chain complications are predicted to be even less of a concern.

reaction 1

Table 1 shows all the tetramic acids isolated in the course of this work and indicates how the one-letter codes for side-chains in this paper correspond to those of the parent amino acids.

Table 1.

Preparation of Tetramic Acid Derivatives^a

compound	R1	yield (%)
2a		92
2d′		71
2f		75
2i		68
3i		78
2l		70
2m		64
2t′		82
2w		58

^aAll tetramic acids were synthesized from the corresponding L-amino acids except w which was made from the D-amino acid.

Preparation of pyrrolinone-pyrrolidine nucleophiles 2

Our preparation of the scaffold systems 1 begin with a Merck-procedure to decarboxylate trans-4-hydroxyproline that can afford more than 50 g of crystalline (R)-3-hydroxypyrrolidine without chromatography.³⁴ N-protection of (R)-3-hydroxypyrrolidine followed by nucleophilic displacement of its triflate-derivative (under conditions optimized to avoid elimination)³⁵ gave the amino esters 4 (Scheme 2). Variable amounts of epimerization occurred in these reactions but this is not a serious problem because all the esters 4 featured in this paper were obtained as single diastereomers via one crystallization from either ethanol or acetonitrile. The communication that precedes this work reported an X-ray analysis of 4f•HCl (after crystallization from ethanol) that proved it formed via a single inversion, ie without a neighboring group effect.²⁰ Here 4i•HCl (after crystallization from acetonitrile) was similarly shown to be the product of one inversion.

Scheme 2^a.

Syntheses Of The Target Systems 1. b One-pot Modification Of The Key Coupling Step.^a

A series of experiments were undertaken to elucidate which of the two chiral centers in structure 4 epimerized in the synthesis of this material. First a mixture that would be formed from “acyclic epimerization”, (S,S)-4f + (R,S)-4f, was made by reacting the Cbz-pyrrolidine 3-triflate precursor with (rac)-tert-butyl phenylalanine. Second, racemic Cbz-pyrrolidine 3-triflate was reacted with optically pure tert-butyl phenylalanine to form the isomers that would be formed from “ring epimerization”, ie (S,S)-4f + (R,S)-4f and (S,R)-4f + (R,R)-4f. After removal of the Cbz group, ¹³C NMR spectroscopy of these mixtures allowed (S,S)-4f and (R,S)-4f to be differentiated {and, of course, the enantiomers (R,R)-4f from (S,R)-4f}. Comparison of that ¹³C NMR data with analytical traces from a chiral HPLC column established that (R,S)-4f could be chromatographically distinguished from the other three stereoisomers that eluted at almost the same time under these conditions. Comparison of all this data with ¹³C NMR and chiral HPLC analyses of the partially epimerized mixture (step 1, Scheme 1) was sufficient to confirm that ring epimerization was the dominant process and acyclic epimerization did not occur to within the limits of detection in the ¹³C spectra that were accumulated (see Supporting Information).

Epimerization in the first step of Scheme 1 was found to be dependent upon the side-chains involved and the scale of the reaction. Loss of stereochemical integrity in this reaction could occur via minor pathways involving S_N1 mechanisms and/or a neighboring group effect from the carbamate. It was conspicuous in this study because the nucleophile is homochiral. Several papers in the literature feature reactions of N-carbamate-protected 3-hydroxy pyrrolidine derivatives with achiral nucleophiles;^36–43 partial racemization in these reactions would have been less noticeable.

Once crystallized, the hydrochlorides 4 are stable on the bench for at least several weeks. It is convenient that the products are hydrochlorides because this provides the one equivalent of acid that is required to activate Bestmann’s ylid^28–30 in the next step: cyclization to Cbz-protected forms of the pyrrolidines 5. Intermediates E are labeled with a letter and not a number, because they were subjected to hydrogenolysis “as is”. Epimerization can occur in cyclizations using Bestmann’s ylid, but we found it is negated in THF using relatively short reaction times (3 h). Dimerization of the ketene formed from Bestmann’s ylid after protonation competes with the desired cyclizations in these processes. To minimize this complication, the solid ylid was added portion-wise every 15 min until completion of the reaction (monitored via 1H NMR spectroscopy; see Supporting Information).

Condensation of the free pyrrolidine-NH of 5 with 5-substituted 2,4-pyrrolidinediones (tetramic acids) 2 gave the pyrrolinones-pyrrolidine-pyrrolinone systems 6. Unfortunately, the free pyrrolidines 5 seem to be basic enough to mediate self-epimerization, at rates dependent on the side-chains and the C-terminal cap but, in the worst cases, even during storage at −20 °C. For instance, the tert-butyl ester 5a (Me side-chain, O^tBu C-terminus) was isolated and characterized as one stereoisomer, then used immediately in the next step without compromising its stereochemical purity, whereas the corresponding methyl ester derivatives were far less stable (see below). Similarly, to illustrate dependence of epimerization on side-chains, storage of 5a at −20 °C for 4 d resulted in epimerization, but none was detected for the derivative from Ile (5i) under the same conditions. Overall, we conclude the tendencies for compounds 5 to epimerize are suppressed when the side-chain and/or the O-substituent are large, probably because this sterically disfavors formation of planar, extended enolates.

Pyrrolidines 5 tend to be protonated by adventitious protons upon standing. One attempt to crystallize the free base 5f using untreated glassware deposited crystals of the corresponding ammonium chloride (X-ray analysis). It is important to avoid such protonation events because the salts of 5 do not couple with tetramic acids to give the desired vinylogous ureas 6. For this reason we routinely purified the free-bases 5 immediately before the next coupling step via column chromatography using an eluent containing 1 % Et₃N. Epimerization may occur if the fractions are directly concentrated in that basic medium, but azeotropic removal with toluene prevents epimerization in the rotary evaporation process, because, we hypothesize, the mol fraction of Et₃N is not increased under these conditions.

It is to be anticipated that C-terminal tetramate-OMe units will be preferred when dealing with those PPI targets where the corresponding tert-butyl esters are too bulky and lipophilic to be accommodated. This consideration led us to investigate the methyl tetramate forms of 5; however, these were found to epimerize faster than the corresponding tert-butyl compounds. Consequently, a “one-pot” procedure was developed to transform intermediate E directly into the products 9 (methyl forms) and 6 (tert-butyl). In the event, the modification that was developed (Scheme 2b) facilitated these reactions without epimerization and made it easier to remove triphenylphosphine oxide from the products. Moreover, selective hydrogenolysis of the Cbz group in the presence of O-benzyl side-chain protection occurred in this process; that is an advantage in situations where downstream steps require side-chain O-benzyl protection.

Pyrrolinone-pyrrolidine-pyrrolinone-pyrrolidine-pyrrolinones 1

Optimized conditions for coupling of the pyrrolidine 5 with the tetramic acids 2 as described above worked well for several substrates, but coupling of the same pyrrolidines 5 with the larger electrophiles derived from deprotection of vinylogous carbamates 6 was significantly slower. We therefore investigated reaction of the nucleophiles with mesylates derived from the tetramic acid, with or without catalytic Lewis acids like Yb(OTf)₃⁴⁴ and ⁱPrOH/AcOH/molecular sieves,⁴⁵ but this did not improve the product yield. Qualitatively, we observed that reaction of the pyrrolidines 5 was faster in alcohol solvents, but MeOH caused methanolysis of the vinylogous urea bonds leading to byproducts, so a more hindered alcohol medium, ⁱPrOH, was used. Trimethylorthoformate was added to scavenge water in this reaction; it was more effective than molecular sieves or azeotropic removal. Better product yields were obtained with 1.5 eq of trimethylorthoformate, though use of this as a co-solvent was not helpful. No epimerization was observed when products 1 or 6 were formed under the conditions specified in Scheme 2a and, most particularly, using a slight excess of the C-terminal tetramate electrophile, whereas it was problematic in test reactions in which the pyrrolidine nucleophile was used in excess. Fortunately, protecting group requirements for functionalized nucleophiles 5 and electrophiles 2 do not place severe constraints on the reaction; for instance, the unprotected indole side-chain in 2w tolerated coupling with the nucleophiles 5. However, when the R² substituent is large, eg for Thr(OBn) or Ile, the desired reaction is relatively slow, and the product yield is diminished.

Properties of the pyrroline-pyrrolidine oligomers

Stabilities of the oligomers containing pyrroline-pyrrolidine were challenged under commonly encountered conditions to determine synthetic limitations, constraints for in vitro assays, and to reveal how the featured compounds might be changed in vivo. In the first instance, influence of aqueous acid was examined by HPLC for 6t’l in pH 7.4 phosphate buffer (100 mM) and at pH 4.5 (100 mM acetate buffer) and 25 °C. At pH 7.4 there was no change to 6t’l over 15 h (after which time the experiment was stopped, see Supporting Information), but at pH 4.5 this compound hydrolyzed with a half life of ~ 40 h⁻¹ (Figure 1a). Similarly, 6ll was completely stable in pH 7.8 10 mM Tris buffer at 55 °C for 60 h (after which time the experiment was stopped).

Figure 1.

a Compound 6t’l in pH 4.5 100 mM acetate buffer has a half-life of ca 40 h⁻¹ at 25 °C. b UV Absorbance Spectrum Of 1fla in 100 mM Acetate Buffer pH 4.5. c Fluorescence Spectrum Of The C-Deprotected Form Of 6fl.

Proteolytic stabilities were assessed by treating 1fla and 6ll with 63 μM Pronase (a mixture of proteases from Streptomyces griseus that is routinely used to hydrolyze peptides)⁴⁶ at pH 7.8 in 10 mM Tris buffer containing 5 mM CaCl₂, at 37 °C. No decomposition of either compound was observed by HPLC over a period of 60 h (after which time the experiment was stopped). Under the same conditions a control tetrapeptide GATV-DAP (GATV fluorescently labeled on the N-terminus with 4-dimethyolaminophthalimide) was completely hydrolyzed to smaller fragments within 2 h. Collectively, these data indicate the compounds 1 and 6 tend to be stable under neutral aqueous conditions at 37 °C, even in the presence of proteases, but they would be hydrolyzed at decreased pH values, such as in the stomach.

Figure 1 shows these compounds have a maximal UV absorbance at around 295 nm: it has an extinction coefficient of 3.88 × 10⁴ M⁻¹cm^−1. The products with an alkylated C-terminus have no significant fluorescence, but the deprotected forms like the C-deprotected form of 6fl (ie 6fl-OH; Φ = 0.006 in 100 mM acetate buffer, pH 4.5) fluoresce with a low quantum yield, but which might influence assays set to observe around 400 nm.

Two molecular features govern the bioavailability of structures 1; specifically, parameters of the: (i) scaffold unit; and, (ii) the side chains. Jorgenssen’s QikProp program^47,48 was used to simulate rates of permeation into Caco-2 cells (Table 2) in comparison with corresponding peptides (Table 3) to begin to understand these properties. Rates of >20 nm/sec⁻¹ are widely considered to be a good indicator of favorable oral bioavailability; standards for permeation into cells would be somewhat less than this.

Table 2.

Rates Of Permeation Into Caco-2 Cells Simulated Using QikProp

	R1	R2	R3	R	PCaco-2 (nm/s)
1	H	H	H	H	4
1	H	H	H	tBu	15
1	Me	Me	Me	H	6
1	Me	Me	Me	tBu	10
1	Phe	Leu	Me	H	15
1	Phe	Leu	Me	tBu	25
1	Leu	Me	Ile	H	12
1	Leu	Me	Ile	tBu	19
1	H	H	Arg	H	0.3
1	Lys	Me	Me	H	0.7
1	Lys	Me	Asp	H	0.03
1	Lys	Me	Asp	tBu	0.06
6	H	H	-	H	34
6	H	H	-	tBu	118
6	Me	Me	-	H	49
6	Me	Me	-	tBu	89
6	Leu	Me	-	H	74
6	Leu	Me	-	tBu	218
6	Leu	Lys	-	H	5
6	Leu	Lys	-	tBu	9

Table 3.

Rates Of Permeation Into Cells Simulated Via QikProp.

R1	R2	R3	PCaco-2 (nm/s)
H	H	H	3
Me	Me	Me	3
Me	H	Me	3
Leu	Me	Ile	33
Phe	Leu	Me	3

Table 2 lists simulated rates of permeation into Caco-2 cells for compounds based on the generic structures 1 and 6, and Table 3 gives the data for peptides with the corresponding side chains. This data covers six compounds 1 that were prepared in this work and six more that have not been prepared yet. Similarly, that same table covers four compounds 6 that were made, and four others that were not. None of the peptides are predicted to be bioavailable (PCaco < 11 nm/s). However, most of the PCaco data is above the 20 nm/s threshold for the smaller mimics 6, that have about the same molecular masses as the tripeptides. The only exceptions were for the most polar analogs, eg ones containing a Lys side-chain. Predicted Caco-2 data for the larger compounds 1 was found to be less, as expected from their larger sizes. Nevertheless, the least polar of these structures had pCaco values in an acceptable region (15 – 25 nm/s). Overall, we conclude that molecules based on 1 and 6 are reasonable candidates for experimental determinations of bioavailabilities, and they are probably much more cell-permeable than closely related peptides.

Conclusions

Several issues emerged when attempting to expand the scope of the procedures to obtain interface mimics 1. First, preparation of the requisite tetramic acids with several different side-chains is relatively easy. The first real problem encountered in the syntheses was “ring-epimerization” in the C-protected N-nucleophile syntheses corresponding to fragments 4. This issue was address by crystallizing those nucleophiles; this proved to be possible for every different side-chain R² or R³ examined here. The second issue relates to slow couplings of the nucleophiles 5 with electrophiles like 6; this was an issue that was improved, and steric constraints regarding the side-chains were elucidated. Furthermore, a one-pot procedure was developed for the first key coupling step in the syntheses of 6.

The target molecules 1 are stable at neutral pH levels around physiological, but have a half-life of about 40 h⁻¹ at pH 4.5. They are proteolytically stable, thermally resilient to 55 °C, and potentially cell permeable based on QikProp calculations. The molecular scaffolds 1 and 6 explored in this work have potentially much better cell permeability than the corresponding peptides.

EXPERIMENTAL SECTION

General Experimental Methods

All reactions were carried out under an inert atmosphere (nitrogen or argon where stated) with dry solvents under anhydrous conditions. Glassware for anhydrous reactions were dried in an oven at 140 °C for minimum 6 h prior to use. Dry solvents were obtained by passing the previously degassed solvents through activated alumina columns. Yields refer to chromatographically and spectroscopically (¹H-NMR) homogeneous materials, unless otherwise stated. Reagents were purchased at a high commercial quality (typically 97 % or higher) and used without further purification, unless otherwise stated. Analytical thin layer chromatography (TLC) was carried out on Merck silica gel plates with QF-254 indicator and visualized by UV, ceric ammonium molybdate, and/or potassium permanganate stains. Flash column chromatography was performed using silica gel 60 (Silicycle, 230–400 mesh) as per the Still protocol. ¹H and ¹³C spectra were recorded on a 300 MHz spectrometer and were calibrated using residual non-deuterated solvent as an internal reference (CDCl₃: ¹H-NMR = 7.26, ¹³C-NMR = 77.16, DMSO-d6: ¹³C-NMR = 39.52, CD₃OD: ¹H-NMR = 3.31, ¹³C-NMR = 49.00). The following abbreviations or combinations thereof were used to explain the multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, p = pentet, br = broad, dd = doublet of doublet, app = apparent. IR spectra were recorded using NaCl plates. Melting points were recorded on an automated melting point apparatus and are uncorrected. Optical rotations were obtained on a polarimeter at the D-line of sodium.

All the HPLC analyses were carried out with UV detection monitored at 254 nm. Analytical reversed phase HPLC analyses were performed with a 150 × 4.6 mm C-18 column using gradient conditions (10 – 90 % acetonitrile in water, flow rate = 0.75 mL/min). Chiralpak AD (250 × 4.6 mm ID) column was utilized for the chiral HPLC analysis (hexanes : isopropyl alcohol 85 : 15, flow rate = 1 mL/min).

QikProp 3.5 from Schrödinger (2012) was used to evaluate pharmaceutically relevant properties for compound 1, 6 and tripeptides with various side chains.

General Procedure for the Synthesis of N-PMB α-amino Ester Hydrochlorides

To a solution of the α-amino ester hydrochloride (40.0 mmol) in methanol (50 ml) at 0 °C was added triethylamine (5.6 mL, 40.0 mmol, 1.0 equiv), dropwise. p-Methoxybenzaldehyde (5.82 mL, 48.0 mmol, 1.2 equiv) was added and the reaction was allowed to proceed at 25 °C for 2 h. Sodium borohydride (3.02 g, 80.0 mmol, 2.0 equiv) was carefully added portion-wise at 0° C over 60 min. The reaction was stirred for 30 min at 25 °C and concentrated. The residue was partitioned between Et₂O (250 mL) and saturated NaHCO₃ (150 mL). The aqueous layer was extracted twice with Et₂O (75 mL) and the combined organic layers was washed with brine (75 mL), dried over MgSO₄ and filtered. The filtrate was cooled to 0 °C and treated with a 4M solution of HCl in dioxane (10 mL, dropwise addition) to precipitate the product, which was collected by filtration.

(S)-tert-Butyl 2-((4-methoxybenzyl)amino)propanoate (N-PMB Ala tert-butyl ester•HCl)

White solid, 10.4 g, 86 %; ¹H-NMR (300 MHz, CDCl₃) δ 7.47 (d, J = 8.4 Hz, 2H), 6.82 (d, J = 8.4 Hz, 2H), 4.05 (m, 2H), 3.69 (s, 3H), 3.45 (m, 1H), 1.59 (d, J = 7.2 Hz, 3H), 1.50 (s, 9H) ¹³C-NMR (75 MHz, CDCl₃) δ 168.2, 160.2, 132.1, 122.4, 114.3, 83.9, 55.2, 53.8, 48.6, 27.9, 15.7; IR (film, cm⁻¹) 1735, 1613, 1562, 1421, 1390, 1257, 1090; MS (ESI-TOF) m/z calcd for C₁₅H₂₃NO₃ (M+H)⁺ 266.18; found 266.19

(2S,3S)-tert-Butyl 2-((4-methoxybenzyl)amino)-3-methylpentanoate (N-PMB Ile tert-butyl ester•HCl)

White solid, 8.1 g, 79 %; [α]²⁰ +15.5 (c 1.0, MeOH); ¹H-NMR (300 MHz, CDCl₃) δ 10.52 (br s, 1H), 9.60 (br s, 1H), 7.58 (d, J = 8.7 Hz, 2H), 6.90 (d, J = 8.7 Hz, 2H), 4.32 (d, J = 13.2 Hz, 1H), 4.15 (d, J = 13.5 Hz, 1H), 3.77 (s, 3H), 3.38 (br s, 1H), 2.45-2.32 (m, 1H), 1.50 (s, 10H), 1.45-1.27 (m, 1H), 1.14 (d, J = 6.9 Hz, 3H), 0.87 (t, J = 7.4 Hz, 3H); ¹³C-NMR (75 MHz, CDCl₃) δ 165.9, 160.4, 132.5, 121.7, 114.4, 84.3, 61.7, 55.2, 49.7, 36.1, 28.1, 26.8, 15.0, 11.6; IR (film, cm⁻¹) 1732, 1614, 1557, 1421, 1393, 1257, 1092, 1031; MS (ESI-TOF) m/z calcd for C₁₈H₃₀NO₃ (M+H)⁺ 308.22; found 308.29

(S)-di-tert-Butyl 2-((4-methoxybenzyl)amino)succinate (N-PMB Asp(O^tBu) tert-butyl ester•HCl)

Light yellow solid, 8.1 g, 74 %; mp = 112.0–113.3 °C; ¹H-NMR (300 MHz, CDCl₃) δ 7.27 (dd, J₁ = 2.1 Hz, J₂ = 6.6 Hz, 2H), 6.87 (dd, J₁ = 2.1 Hz, J₂ = 6.6 Hz, 2H), 3.87-3.82 (m, 4H), 3.67 (d, J = 12.6 Hz, 1H), 3.51 (dd, J₁ = 5.7 Hz, J₂ = 6.9 Hz, 1H), 2.63 (dd, J₁ = 5.7 Hz, J₂ = 15.6 Hz, 1H), 2.54 (dd, J₁ = 6.9 Hz, J₂ = 15.6 Hz, 1H), 1.92 (b, 1H), 1.50 (s, 9H), 1.47 (s, 9H); ¹³C-NMR (75 MHz, CDCl₃) δ 173.0, 170.3, 158.7, 132.0, 129.5, 113.7, 81.3, 80.8, 57.8, 55.3, 51.4, 39.5, 28.1; IR (film, cm⁻¹) 3420(br), 2981, 1735, 1614, 1517, 1371, 1255, 1154; HRMS (ESI-TOF) m/z calcd for C₂₀H₃₁NO₅ (M+H)⁺ 366.2275; found 366.2274 (0.3 ppm)

General Procedure for the Cyclization of N-PMB α-amino Ester Hydrochlorides

To a stirred suspension of the PMB-protected α-amino ester hydrochloride (5.0 mmol) in anhydrous dioxane (0.25 M) under Argon was added Bestmann’s ylide (11.0 mmol, 2.2 equiv) in one portion at 25 °C. The reaction was heated at 100 °C under Argon for 12 h. Upon cooling, the reaction mixture was concentrated and re-suspended in 50 mL of ether. The suspension was stirred vigorously for 30 min and the solids were removed by filtration. The filtrate was concentrated to ~ 10 mL and placed at − 20 °C for 14 h following which the crystallized Ph₃PO by-product was removed by filtration. The filtrate was concentrated to obtain an oil which was purified by flash chromatography.

(S)-4-(tert-Butoxy)-1-(4-methoxybenzyl)-5-methyl-1H-pyrrol-2(5H)-one

Colorless oil, 1.30 g, 90 %; ¹H-NMR (300 MHz, CDCl₃) δ 7.15 (d, J = 8.4 Hz, 2H), 6.81 (d, J = 9.0 Hz, 2H), 5.03-4.96 (m, 2H), 3.96 (d, J = 15.0 Hz, 1H), 3.76 (s, 3H), 3.66 (q, J = 6.7 Hz, 1H), 1.39 (s, 9H), 1.18 (d, J = 6.9 Hz, 3H); ¹³C-NMR (75 MHz, CDCl₃) δ 172.2, 172.0, 158.8, 130.0, 129.2, 113.9, 94.9, 81.4, 56.6, 55.2, 42.3, 27.4, 16.0; MS (ESI-TOF) m/z calcd for C₁₇H₂₄NO₃ (M+H)⁺ 290.17; found 290.10

(S)-4-(tert-Butoxy)-5-((S)-sec-butyl)-1-(4-methoxybenzyl)-1H-pyrrol-2(5H)-one

Colorless oil, 1.23 g, 74 %; [α]²⁰ +11.8 (c 1.0, MeOH); ¹H-NMR (300 MHz, CDCl₃) δ 7.18 (d, J = 8.7 Hz, 2H), 6.87 (d, J = 9.0 Hz, 2H), 5.18-5.10 (m, 2H), 3.88 (d, J = 15.3 Hz, 1H), 3.82 (s, 3H), 3.72 (d, J = 3.0 Hz, 1H), 1.96-1.82 (m, 1H), 1.45 (s, 9H), 1.55-1.33 (m, 2H), 0.89 (t, J = 7.4 Hz, 3H), 0.77 (d, J = 6.9 Hz, 3H); ¹³C-NMR (75 MHz, CDCl₃) δ 172.9, 170.8, 158.8, 129.9, 129.3, 113.9, 96.5, 81.6, 63.4, 55.2, 42.3, 34.9, 27.5, 25.7, 12.9, 12.6; IR (film, cm⁻¹) 1682, 1514, 1372, 1329, 1246, 1171; MS (ESI-TOF) m/z calcd for C₂₀H₃₀NO₃ (M+H)⁺ 332.22; found 332.23

(S)-tert-Butyl 2-(3-(tert-butoxy)-1-(4-methoxybenzyl)-5-oxo-2,5-dihydro-1H-pyrrol-2-yl)acetate (2d′)

Colorless oil, 1.54 g, 75 %, ¹H-NMR (300 MHz, CDCl₃) δ 7.22 (dd, J₁ = 2.1 Hz, J₂ = 6.6 Hz, 2H), 6.86 (dd, J₁ = 2.1 Hz, J₂ = 6.6 Hz, 2H), 5.10 (s, 1H), 5.05 (d, J = 15.3 Hz), 4.07 (t, J = 5.1 Hz), 4.03 (d, J = 15.3 Hz), 3.81 (s, 3H), 2.62 (dd, J₁ = 5.1 Hz, J₂ = 15.3 Hz, 1H), 2.51 (dd, J₁ = 5.1 Hz, J₂ = 15.3 Hz, 1H), 1.45 (s, 18H); ¹³C-NMR (75 MHz, CDCl₃) δ 172.5, 170.2, 169.0, 158.9, 129.7, 129.3, 114.0, 95.7, 82.1, 81.3, 57.8, 55.2, 42.8, 36.6; IR (film, cm⁻¹) 2979, 2361, 1731, 1615, 1513, 1368, 1248, 1147; HRMS (ESI-TOF) m/z calcd for C₂₂H₃₁NO₅ (M+Na)⁺ 412.2094; found 412.2101 (1.7 ppm)

Procedure for the Syntheses of 3i

(S)-4-(tert-Butoxy)-5-((S)-sec-butyl)-1-(4-methoxybenzyl)-1H-pyrrol-2(5H)-one (1 mmol) was dissolved in dichloromethane (5 mL) and cooled to 0 °C. TFA (5 mL) was added and the reaction was allowed to warm up to room temperature. After stirring for 2h, toluene (25 mL) was added and the solution was concentrated. Residual TFA was azeotroped 2 times with toluene (25 mL × 2) and the residue was purified by flash chromatography (50 % ethyl acetate in dichloromethane) to afford (S)-5-((S)-sec-Butyl)-1-(4-methoxybenzyl)pyrrolidine-2,4-dione (3i) in 78 % yield.

Colorless oil, 215.4 mg, 78 %; ¹H-NMR (300 MHz, CDCl₃) δ 7.12 (d, J = 8.4 Hz, 2H), 6.80 (d, J = 9.0 Hz, 2H), 5.22 (d, J = 14.7 Hz, 1H), 3.82 (d, J = 14.7 Hz, 1H), 3.74 (s, 3H), 3.61 (d, J = 3.3 Hz, 1H), 2.92 (s, 2H), 1.90-1.78 (m, 1H), 1.49-1.33 (m, 2H), 0.86-0.73 (m, 6H); ¹³C-NMR (75 MHz, CDCl₃) δ 205.5, 170.4, 159.5, 129.8, 126.6, 114.3, 68.6, 55.2, 43.1, 42.4, 35.1, 25.1, 13.4, 12.0; IR (film, cm⁻¹) 1770, 1514, 1418, 1248, 1034; MS (ESI-TOF) m/z calcd for C₁₆H₂₂NO₃ (M+H)⁺ 276.15; found 276.19

General Procedure for the Syntheses of Tetramic Acids 2

A modified literature procedure was used. To a stirred solution of meldrum’s acid (476 mg, 3.3 mmol, 1.1 equiv) and DMAP (550 mg, 4.5 mmol, 1.5 equiv) at 0 °C in dichloromethane (30 mL) was added N-Boc-amino acid (3.0 mmol, 1.0 equiv) in one portion. EDCI (1.2 g, 7.2 mmol, 2.4 equiv) was added in one portion and the reaction mixture was stirred at 25 °C for 14 h. The yellow reaction mixture was transferred to a separatory funnel and diluted with EtOAc (80 mL) and washed with cold 5 % KHSO₄ (3 × 100 mL) and brine (75 mL). The organic layer was dried over MgSO₄ and filtered. The filtrate was refluxed for 30 min under N₂. Upon concentration, the residue was dissolved in dichloromethane (5 mL) and cooled to 0 °C. TFA (5 mL) was added and the reaction was stirred for 30 min. Toluene (25 mL) was added and the solution was concentrated. Residual TFA was azeotroped 2 times with toluene (25 mL ea) and then 5 mL ether was added. After stirring for 10 min, the solid product was collected by filtration.

(S)-5-((1H-Indol-3-yl)methyl)pyrrolidine-2,4-dione (2w)

White solid, 407.3 mg, 58 %; ¹H-NMR (300 MHz, CDCl₃) δ 8.41 (br s, 1H), 7.56 (d, J = 7.8 Hz, 1H), 7.38-7.32 (m, 1H), 7.26-7.18 (m, 1H), 7.17-7.10 (m, 1H), 7.25 (br s, 1H), 6.93 (d, J = 2.4 Hz, 1H), 4.23 (dd, J = 7.5, 3.3 Hz, 1H), 3.01 (dd, J = 14.9, 7.9 Hz, 1H), 2.89 (d, J = 22.2 Hz, 1H), 2.68 (dd, J = 22.2, 1.5 Hz, 1H); ¹³C-NMR (75 MHz, CDCl₃) δ 207.3, 171.4, 136.2, 126.9, 123.5, 122.5, 119.9, 118.6, 111.5, 109.2, 64.7, 40.9, 28.1; IR (film, cm⁻¹) 3400 (br), 2918, 1767, 1684, 1361, 1236, 1093, 745; HRMS (ESI-TOF) m/z calcd for C₁₃H₁₂N₂O₂ (M+Li)⁺ 235.1020; found 235.1028 (3.4 ppm)

(S)-5-((S)-sec-Butyl)pyrrolidine-2,4-dione (2i)

White solid, 316.4 mg, 68 %; ¹H-NMR (300 MHz, CDCl₃) δ 8.06 (br s, 1H), 3.90 (d, J = 3.6 Hz, 1H), 2.95 (m, 2H), 1.91-1.84 (m, 1H), 1.38-1.27 (m, 2H), 0.99 (d, J = 6.9 Hz, 3H), 0.89 (t, J = 7.2 Hz, 3H); ¹³C-NMR (75 MHz, CDCl₃) δ 207.7, 172.3, 69.1, 41.7, 37.7, 24.3, 15.3, 11.6; IR (film, cm⁻¹) 3200 (br), 2957, 1766, 1697, 1471, 1363, 1291; HRMS (ESI-TOF) m/z calcd for C₈H₁₃NO₂ (M+H)⁺ 156.1019; found 156.1018 (0.6 ppm)

General Procedure for the Syntheses of 4

To a solution of Cbz-protected pyrrolidin-3-ol (2.57 g, 11.6 mmol) in dry dichloromethane (15 mL) at −78 °C was added diisopropylethyl amine (2.2 mL, 12.8 mmol, 1.1 equiv), dropwise. Freshly distilled (P₂O₅) triflic anyhydride (2.0 mL, 12.1 mmol, 1.05 equiv) was added using a syringe pump at rate of 4 mL/hr ensuring that the bath temperature does not exceed −70 °C. The reaction mixture turned pink. On complete addition of triflic anyhydride, the reaction was stirred for 10 min. A solution of amino acid tert-butyl ester (17.4 mmol, 1.5 equiv) in dichloromethane (15 mL) was then added at a rate of 30 mL/hr. The reaction was stirred for 10 minutes at −78 °C, and allowed to warm to 25 °C. After 18 h, the reaction mixture was transferred to a separatory funnel and diluted with dichloromethane (125 mL). The organic layer was extracted with saturated sodium bicarbonate (2 × 150 mL) and brine (1 × 100 mL). The organic layer was dried over MgSO₄, filtered and concentrated. The residue was purified by column chromatography (SiO₂, 1:9 ethyl acetate/dichloromethane; cerric ammonium molybdate stain and UV for visualization) to afford the product as a mixture of diastereomers. To a solution of the mixture in dry ether (70 mL) at 0 °C was added HCl in dioxane (4M, 1.1 equiv) dropwise. Removal of the ether by filtration afforded a hydroscopic white solid. The solid was dissolved in dichloromethane (10 mL) and transferred to a 50 mL erlenmeyer flask. Hexanes (35 mL) was slowly added to form a second layer. The flask was placed at − 20 °C for 12 h to afford a white, non-hydroscopic solid. The resulting solid was collected by filtration and to afford the required pure single diastereomer.

(S)-di-tert-Butyl 2-(((S)-1-((benzyloxy)carbonyl)pyrrolidin-3-yl)amino)pentanedioate (4e′)

White crystals, crystallized from hot MeCN (~ 12 mL/g), 1.62 g after crystallization, 28 %; mp = 171–172 °C; ¹H-NMR (300 MHz, CDCl₃) δ 7.36-7.26 (m, 5H), 5.16-5.02 (m, 2H), 3.98-3.86 (m, 1H), 3.85-3.62 (m, 4H), 3.44-3.24 (m, 1H), 2.72-2.42 (m, 4H), 2.39-2.22 (m, 2H), 1.50 (s, 9H), 1.40 (s, 9H); ¹³C-NMR (75 MHz, CDCl₃) δ 170.8, 166.4, 154.4, 136.4, 128.5, 128.2, 128.09, 128.03, 127.9, 85.2, 81.1, 67.2 and 67.1, 59.2, 55.7 and 54.9, 47.5 and 47.1, 44.3 and 44.0, 30.7, 28.9, 28.1, 28.0, 25.4, 25.2; Note: Carbon spectra shows more than the expected number of peaks due to restricted rotation about the NH–C=O bond. This was confirmed by hydrogenolysis of the product (see Supporting Information); IR (film, cm⁻¹) 2978, 2628, 1714, 1697, 1417, 1153; HRMS (ESI-TOF) m/z calcd for C₂₅H₃₉N₂O₆ (M+H)⁺ 463.2808; found 463.2817 (1.9 ppm)

(S)-Benzyl 3-(((2S,3R)-3-(benzyloxy)-1-methoxy-1-oxobutan-2-yl)amino)pyrrolidine-1-carboxylate (4t′)

White crystals, crystallized from hot MeCN (~ 14 mL/g), 1.85 g after crystallization, 40 %; mp = 164–166 °C; ¹H-NMR (300 MHz, CDCl₃) δ 7.41-7.21 (m, 10 H), 5.16-4.98 (m, 2H), 4.72-4.56 (m, 2H), 4.50-4.39 (m, 1H), 4.01-3.90 (m, 1H), 3.88-3.78 (m, 2H), 3.78 (m, 3H), 3.70-3.59 (m, 1H), 3.34-3.19 (m, 1H), 2.63-2.40 (m, 1H), 2.40-2.27 (m, 1H), 1.38 (d, J = 6.3 Hz, 3H); ¹³C-NMR (75 MHz, CDCl₃) δ 166.4, 154.4, 137.3, 136.5, 128.51, 128.48, 128.28, 128.12, 128.05, 127.9, 73.2, 71.5, 67.0, 63.3, 57.3 and 56.7, 53.4, 48.1 and 47.6, 44.2 and 43.9, 28.5 and 27.7, 16.7; IR (film, cm⁻¹) 2953, 2886, 2636, 1747, 1213, 1123, 741; HRMS (ESI-TOF) m/z calcd for C₂₄H₃₁N₂O₅ (M+H)⁺ 427.2233; found 427.2219 (3.3 ppm)

General Procedure for the Syntheses of 5

The re-crystallized hydrochloride salt 4 (0.97 mmol) was suspended in dry THF (10 mL) and Bestmann’s ylid (re-crystallized from PhMe, 352 mg, 1.2 equiv) was added in one portion. The reaction mixture was heated to 75 °C under an Argon atmosphere. After 30 min, a second portion of Bestmann’s ylid (59 mg, 0.2 equiv) was added, and this process was repeated four additional times at 15 min intervals to complete the addition of 2.2 equiv of ylid. The reaction was monitored by NMR spectroscopy. After completion of reaction (~ 3 h), the solvent was evaporated. Upon cooling, the THF was removed in vacuo and the residue was loaded onto a short SiO₂ column. Elution with 5 % EtOAc/CH₂Cl₂ (to remove traces of unreacted starting material) followed by 50 % % EtOAc/CH₂Cl₂ afforded a mixture of the cyclized product and triphenylphosphine oxide. The mixture was directly utilized in the next step.

General Procedure for the Hydrogenolysis of the N-Cbz Group

To a solution of the substrate in methanol (0.1 M) under nitrogen was added 10 wt % Pd/C (0.1 equiv Pd). The reaction was placed under an atmosphere of hydrogen (1 atm, balloon) for 12 h and purged with N₂. The reaction mixture was filtered over a Celite pad and concentrated to afford the crude product. The crude product was purified by flash chromatography (SiO₂, 3 % MeOH/CH₂Cl₂ → 3 % MeOH/CH₂Cl₂ containing 1 % Et₃N) to afford the product.

tert-Butyl 3-((S)-3-(tert-butoxy)-5-oxo-1-((S)-pyrrolidin-3-yl)-2,5-dihydro-1H-pyrrol-2-yl)propanoate (5e′)

Colorless oil, 225.4 mg, 64 % over two steps; ¹H-NMR (300 MHz, CDCl₃) δ 5.23 (s, 1H), 4.22-4.10 (m, 1H), 3.95-3.89 (m, 1H), 3.63-3.51 (m, 3H), 3.50-3.36 (m, 1H), 2.50-2.29 (m, 1H), 2.12-1.94 (m, 5H), 1.44 (s, 9H), 1.41 (s, 9H) (Note: Complete removal of residual solvent (eg Et₃N, CH₂Cl₂) used in chromatography was not done to avoid epimerization. After characterization, the sample was immediately carried to the next step.); ¹³C-NMR (75 MHz, CDCl₃) δ 174.2, 171.9, 171.1, 96.7, 83.1, 81.0, 61.8, 51.9, 49.5, 45.8, 45.2, 30.1, 28.1, 27.4, 23.4; IR (film, cm⁻¹) 2980, 2935, 2870, 1728, 1682, 1614, 1369, 1339, 1258, 1167, 844, 808; HRMS (ESI-TOF) m/z calcd for C₁₉H₃₃N₂O₄ (M+H)⁺ 353.2440; found 353.2431 (2.6 ppm)

General Procedure for the Syntheses of 6

These compounds were prepared via the general procedure described previously.⁴⁹

(S)-4-(tert-Butoxy)-5-methyl-1-((S)-1-((S)-2-(2-(methylthio)ethyl)-5-oxo-2,5-dihydro-1H-pyrrol-3-yl)pyrrolidin-3-yl)-1H-pyrrol-2(5H)-one (6ma)

Pale yellow oil, 231.9 mg, 59 %; ¹H-NMR (300 MHz, CDCl₃) δ 6.37 (br s, 1H), 5.00 (app s, 1H), 4.56 (d, J = 1.2 Hz, 1H), 4.37-4.25 (m, 2H), 3.94-3.82 (m, 1H), 3.64-3.52 (m, 1H), 3.53-3.38 (m, 2H), 3.38-3.30 (m, 1H), 2.57 (t, J = 7.3 Hz, 2H), 2.53-2.40 (m, 1H), 2.28-2.13 (m, 2H), 2.11 (s, 3H), 1.90-1.75 (m, 1H), 1.45 (s, 9H), 1.35 (d, J = 6.6 Hz, 3H); ¹³C-NMR (75 MHz, CDCl₃) δ 176.2, 172.7, 172.1, 165.4, 95.5, 89.2, 81.9, 77.3, 57.9, 56.3, 51.7, 50.1, 47.6, 31.9, 29.9, 27.4, 18.0, 15.7; IR (film, cm⁻¹) 3214 (br), 2958, 2873, 1653, 1599, 1480, 1456, 1399, 1373, 1339, 1302, 1259, 1214, 1168, 1096, 880, 840, 781, 757; HRMS (ESI-TOF) m/z calcd for (M+H)⁺ C₂₀H₃₂N₃O₃S 394.2164; found 394.2175 (2.7 ppm)

(S)-4-(tert-Butoxy)-5-((S)-sec-butyl)-1-((S)-1-((S)-2-isobutyl-5-oxo-2,5-dihydro-1H-pyrrol-3-yl)pyrrolidin-3-yl)-1H-pyrrol-2(5H)-one (6li)

Pale yellow solid, 115.1 mg, 55 %; ¹H-NMR (300 MHz, CDCl₃) δ 5.70 (br s, 1H), 4.99 (s, 1H), 4.52 (d, J = 1.5 Hz, 1H), 4.14 (d, J = 9.6 Hz, 1H), 4.07-3.88 (m, 1H), 3.86 (d, J = 2.7 Hz, 1H), 3.83-3.69 (m, 1H), 3.46-3.15 (m, 3H), 2.74-2.52 (m, 1H), 1.87-1.47 (m, 5H), 1.42 (s, 9H), 1.40-1.35 (m, 1H), 1.0-0.89 (m, 6H), 0.75 (d, J = 6.9 Hz, 3H); ¹³C-NMR (75 MHz, CDCl₃) δ 176.2, 173.3, 170.4, 166.7, 97.4, 88.3, 82.0, 65.8, 55.7, 52.6, 49.6, 47.4, 42.2, 36.4, 27.4, 26.0, 25.9, 23.7, 21.4, 12.6, 12.4; HRMS (ESI-TOF) m/z calcd for C₂₄H₄₀N₃O₃ (M+H)⁺ 418.3070; found 418.3083 (3.1 ppm)

(S)-1-((S)-1-((R)-2-((R)-1-(Benzyloxy)ethyl)-5-oxo-2,5-dihydro-1H-pyrrol-3-yl)pyrrolidin-3-yl)-4-(tert-butoxy)-5-methyl-1H-pyrrol-2(5H)-one (6t’a)

White solid, 212.6 mg, 67 %; ¹H-NMR (300 MHz, CDCl₃) δ 7.37-7.19 (m, 5H), 6.29 (br s, 1H), 4.94 (s, 1H), 4.62-4.54 (m, 2H), 4.43 (d, J = 11.7 Hz, 1H), 4.39-4.26 (m, 1H), 4.24 (d, J = 1.5 Hz, 1H), 3.87-3.76 (m, 1H), 3.69-3.58 (m, 1H), 3.38-3.24 (m, 3H), 3.23-3.09 (m, 1H), 2.30-2.04 (m, 2H), 1.42 (s, 9H), 1.23 (d, J = 6.6 Hz, 3H), 1.17 (d, J = 6.3 Hz, 3H); ¹³C-NMR (75 MHz, CDCl₃) δ 176.7, 172.7, 172.0, 163.8, 138.1, 128.3, 127.8, 127.7, 95.2, 90.6, 81.8, 74.2, 71.1, 61.2, 57.3, 51.0, 50.9, 48.1, 29.0, 27.4, 18.2, 15.3; IR (film, cm⁻¹) 3250 (br), 2978, 2872, 1597, 1398, 1375, 1257, 1213, 1167, 1096, 735; MS (ESI-TOF) m/z calcd for C₂₆H₃₆N₃O₄ (M+H)⁺ 454.27; found 454.26

tert-Butyl 3-((S)-1-((S)-1-((S)-2-benzyl-5-oxo-2,5-dihydro-1H-pyrrol-3-yl)pyrrolidin-3-yl)-3-(tert-butoxy)-5-oxo-2,5-dihydro-1H-pyrrol-2-yl)propanoate (6fe′)

Colorless oil that crashed into a solid under vacuum, 134.0 mg, 64 %; ¹H-NMR (300 MHz, CDCl₃) δ 7.35-7.13 (m, 5H), 5.23 (br s, 1H), 5.03 (s, 1H), 4.52 (s, 1H), 4.32 (dd, J = 9.9, 3.0 Hz, 1H), .3.04–4.14 (m, 1H), 4.04-3.95 (m, 1H), 3.82-3.66 (m, 1H), 3.65-3.53 (m, 1H), 3.52-3.30 (m, 2H), 3.25 (dd, J = 13.5, 3.0 Hz, 1H), 2.66-2.41 (m, 2H), 2.30-2.16 (m, 1H), 2.15-1.95 (m, 4H), 1.44 (s, 9H), 1.43 (s, 9H); ¹³C-NMR (75 MHz, CDCl₃) δ 175.6, 173.3, 172.3, 170.1, 165.5, 136.8, 129.1, 128.8, 128.6, 127.1, 97.0, 88.8, 82.3, 80.9, 60.7, 58.4, 52.1, 49.9, 47.7, 39.4, 28.1, 27.4, 24.8; IR (film, cm⁻¹) 3250 (br), 2978, 2931, 2872, 1724, 1667, 1601, 1395, 1371, 1339, 1371, 1339, 1258, 1165, 1151, 702; HRMS (ESI-TOF) m/z calcd for C₃₀H₄₂N₃O₅ (M+H)⁺ 524.3124; found 524.3115 (1.8 ppm)

(S)-1-((S)-1-((S)-2-((1H-Indol-3-yl)methyl)-5-oxo-2,5-dihydro-1H-pyrrol-3-yl)pyrrolidin-3-yl)-4-(tert-butoxy)-5-isobutyl-1H-pyrrol-2(5H)-one (6wl)

Pale yellow solid, 127.4 mg, 52 %; ¹H-NMR (300 MHz, CDCl₃) δ 9.79 (s, 1H), 7.47 (d, J = 7.5 Hz, 1H), 7.36 (d, J = 7.8 Hz, 1H), 7.14 (t, J = 7.8 Hz, 1H), 7.06 (t, J = 7.2 Hz, 1H), 6.95 (d, J = 1.5 Hz, 1H), 5.35 (br s, 1H), 5.00 (s, 1H), 4.56 (s, 1H), 4.38-4.28 (m, 1H), 4.26-4.12 (m, 1H), 3.94-3.76 (m, 2H), 3.76-3.55 (m, 2H), 3.49-3.31 (m, 2H), 2.69 (dd, J = 14.7, 9.6 Hz, 1H), 2.59-2.41 (m, 1H), 2.28-2.13 (m, 1H), 1.91-1.74 (m, 1H), 1.69-1.56 (m, 2H), 1.44 (s, 9H), 0.94 (d, J = 0.9 Hz, 3H), 0.92 (d, J = 0.9 Hz, 3H); ¹³C-NMR (75 MHz, CDCl₃) δ 176.4, 173.4, 171.9, 166.2, 136.6, 126.9, 123.4, 121.9, 119.3, 118.0, 111.9, 110.4, 96.2, 88.9, 82.1, 61.4, 57.5, 52.4, 50.9, 47.8, 39.6, 30.2, 28.8, 27.4, 24.1, 24.0, 23.1; IR (film, cm⁻¹) 3414, 3246 (br), 2976, 2928, 2868, 1647, 1597, 1422, 1341, 1167, 908, 735; HRMS (ESI-TOF) m/z calcd for C₂₉H₃₉N₄O₃ (M+H)⁺ 491.3022; found 491.3035 (2.6 ppm)

General Procedure for the One-pot Syntheses of 6

To a solution of the tetramic acid substrate (1.3 mmol) and N-Cbz protected dimer (1.0 mmol) in ethanol (0.1 M) under nitrogen was added trimethylorthoformate (1.5 equiv) and 10 wt % Pd/C (0.2 equiv Pd). The reaction was stirred under an atmosphere of H₂ (1 atm, balloon) for 12 h. The reaction was purged with N₂ for a few minutes and filtered over a pad of Celite. The filtrate was concentrated to obtain the crude product, which was purified by flash chromatography (4–5 % MeOH/CH₂Cl₂) to afford the trimers as a white solid.

(S)-4-(tert-Butoxy)-5-isobutyl-1-((S)-1-((S)-2-isobutyl-5-oxo-2,5-dihydro-1H-pyrrol-3-yl)pyrrolidin-3-yl)-1H-pyrrol-2(5H)-one (6ll)

White solid, 157.7 mg, 54 %; ¹H-NMR (300 MHz, CDCl₃) δ 5.78 (br s, 1H), 4.97 (s, 1H), 4.51 (d, J = 1.5 Hz, 1H), 4.21-3.97 (m, 2H), 3.87 (dd, J = 6.3, 3.8 Hz, 1H), 3.79-3.64 (m, 1H), 3.46-3.33 (m, 2H), 3.32-3.14 (m, 1H), 2.65-2.43 (m, 1H), 2.23-2.06 (m, 2H), 1.87-1.67 (m, 2H), 1.67-1.53 (m, 3H), 1.42 (s, 9H), 0.99-0.83 (m, 12H); ¹³C-NMR (75 MHz, CDCl₃) δ 176.3, 173.3, 171.8, 166.8, 96.1, 88.3, 82.0, 61.3, 55.8, 52.5, 49.8, 47.4, 42.1, 39.7, 27.4, 25.8, 24.1, 23.9, 23.7, 23.0, 21.3; IR (film, cm⁻¹) 3211 (br), 2955, 1661, 1651, 1601, 1472, 1371, 1341, 1268, 1167, 731; HRMS (ESI-TOF) m/z calcd for C₂₄H₄₀N₃O₃ (M+H)⁺ 418.3070; found 418.3067 (0.6 ppm)

(S)-4-(tert-Butoxy)-5-isobutyl-1-((S)-1-((S)-2-methyl-5-oxo-2,5-dihydro-1H-pyrrol-3-yl)pyrrolidin-3-yl)-1H-pyrrol-2(5H)-one (6al)

White solid, 180.1 mg, 48 %; ¹H-NMR (300 MHz, CDCl₃) δ 5.72 (br s, 1H), 4.97 (s, 1H), 4.48 (d, J = 1.2 Hz, 1H), 4.20 (q, J = 6.7 Hz, 1H), 4.14-3.99 (m, 1H), 3.87 (dd, J = 6.4, 3.8 Hz, 1H), 3.77-3.62 (m, 1H), 3.51-3.33 (m, 2H), 3.33-3.16 (m, 1H), 2.62-2.43 (m, 1H), 2.19-2.06 (m, 1H), 1.87-1.71 (m, 1H), 1.65-1.53 (m, 2H), 1.42 (s, 9H), 1.35 (d, J = 6.6 Hz, 3H), 0.90 (d, J = 6.3 Hz, 6H); ¹³C-NMR (75 MHz, CDCl₃) δ 176.2, 173.3, 171.9, 167.3, 96.1, 87.7, 82.0, 61.2, 52.9, 52.5, 49.8, 47.5, 39.7, 27.7, 27.4, 24.1, 23.9, 23.0, 19.0; IR (film, cm⁻¹) 3304 (br), 2976, 2871, 1653, 1601, 1397, 1339, 1258, 1167, 731; HRMS (ESI-TOF) m/z calcd for C₂₁H₃₄N₃O₃ (M+H)⁺ 376.2600; found 376.2610 (2.6 ppm)

(5S)-1-((3S)-1-(2-((R)-1-(Benzyloxy)ethyl)-5-oxo-2,5-dihydro-1H-pyrrol-3-yl)pyrrolidin-3-yl)-4-(tert-butoxy)-5-isobutyl-1H-pyrrol-2(5H)-one (6t’l)

White solid, 131.2 mg, 53 %; ¹H-NMR (300 MHz, CDCl₃) δ 7.36-7.21 (m, 5H), 5.78 (br s, 1H), 4.98 (s, 1H), 4.64-4.55 (m, 2H), 4.44 (d, J = 11.7 Hz, 1H), 4.21 (d, J = 1.5 Hz, 1H), 4.19-4.07 (m, 1H), 3.83-3.74 (m, 1H), 3.52-3.27 (m, 3H), 3.24-3.11 (m, 1H), 2.41-2.25 (m, 1H), 2.14-2.05 (m, 1H), 1.99-1.86 (m, 1H), 1.82-1.69 (m, 1H), 1.63-1.51 (m, 2H), 1.44 (s, 9H), 1.19 (d, J = 6.3 Hz, 3H), 0.89 (d, J = 6.3 Hz, 6H); ¹³C-NMR (75 MHz, CDCl₃) δ 176.6, 173.3, 171.8, 163.8, 138.2, 128.3, 127.8, 127.6, 96.0, 90.3, 81.9, 73.8, 71.0, 61.3, 60.8, 51.9, 50.8, 48.1, 39.7, 28.1, 27.4, 24.0, 23.1, 15.7; IR (film, cm⁻¹) 3227 (br), 2976, 2868, 1599, 1339, 1256, 1167, 1098, 737; HRMS (ESI-TOF) m/z calcd for C₂₉H₄₂N₃O₄ (M+H)⁺ 496.3175; found 496.3171 (0.9 ppm)

tert-Butyl 3-((S)-3-(tert-butoxy)-1-((S)-1-((S)-2-isobutyl-5-oxo-2,5-dihydro-1H-pyrrol-3-yl)pyrrolidin-3-yl)-5-oxo-2,5-dihydro-1H-pyrrol-2-yl)propanoate (6le′)

White solid, 119.9 mg, 49 %; ¹H-NMR (300 MHz, CDCl₃) δ 5.71 (br s, 1H), 5.02 (s, 1H), 4.51 (s, 1H), 4.29-4.10 (m, 2H), 4.01-3.94 (m, 1H), 3.67-3.54 (m, 1H), 3.48-3.32 (m, 2H), 3.32-3.19 (m, 1H), 2.57-2.37 (m, 2H), 2.26-1.96 (m, 5H), 1.80-1.67 (m, 1H), 1.66-1.53 (m, 1H), 1.43 (s, 9H), 1.41 (s, 9H), 0.95 (d, J = 6.6 Hz, 3H), 0.92 (d, J = 6.6 Hz, 3H); ¹³C-NMR (75 MHz, CDCl₃) δ 176.2, 173.2, 172.2, 170.0, 166.7, 97.0, 88.4, 82.3, 80.8, 60.7, 55.7, 52.0, 49.7, 47.4, 42.1, 28.1, 27.4, 25.8, 24.7, 23.7, 21.4; IR (film, cm⁻¹) 3123 (br), 2976, 2871, 1724, 1670, 1600, 1395, 1369, 1341, 1258, 1167, 922, 887, 847, 731; HRMS (ESI-TOF) m/z calcd for C₂₇H₄₄N₃O₅ (M+H)⁺ 490.3281; found 490.3270 (2.2 ppm)

(S)-5-((R)-1-(Benzyloxy)ethyl)-4-methoxy-1-((S)-1-((S)-2-methyl-5-oxo-2,5-dihydro-1H-pyrrol-3-yl)pyrrolidin-3-yl)-1H-pyrrol-2(5H)-one (9at′)

White solid, 104.1 mg, 51 %; ¹H-NMR (300 MHz, CDCl₃) δ 7.35-7.24 (m, 5H), 5.68 (br s, 1H), 5.04 (s, 1H), 4.70 (d, J = 12.0 Hz, 1H), 4.50-4.41 (m, 2H), 4.11-3.96 (m, 3H), 3.78 (s, 3H), 3.78-3.70 (m, 1H), 3.67-3.55 (m, 1H), 3.33-3.21 (m, 2H), 3.21-3.08 (m, 1H), 2.42-2.25 (m, 1H), 2.01-1.88 (m, 1H), 1.30 (d, J = 6.6 Hz, 3H), 1.18 (d, J = 6.0 Hz, 3H); ¹³C-NMR (75 MHz, CDCl₃) δ 176.1, 175.0, 172.7, 167.2, 137.7, 128.6, 128.0, 127.7, 95.8, 87.7, 74.0, 71.1, 64.5, 58.3, 53.6, 52.8, 49.7, 47.3, 27.2, 19.0, 14.8; IR (film, cm⁻¹) 3260 (br), 2928, 2868, 1670, 1595, 1396, 1361, 1238, 1099, 995, 733, 696; HRMS (ESI-TOF) m/z calcd for C₂₃H₃₀N₃O₄ (M+H)⁺ 412.2236; found 412.2277 (3.7 ppm)

General Procedure for the Syntheses of 1

These compounds were prepared via the general procedure described previously.⁴⁹

(S)-1-((S)-1-((S)-2-Benzyl-5-oxo-2,5-dihydro-1H-pyrrol-3-yl)pyrrolidin-3-yl)-4-((S)-3-((S)-3-(tert-butoxy)-2-methyl-5-oxo-2,5-dihydro-1H-pyrrol-1-yl)pyrrolidin-1-yl)-5-isobutyl-1H-pyrrol-2(5H)-one (1fla)

White solid, 99.7 mg, 54 %; ¹H-NMR (300 MHz, CDCl₃) δ 7.32-7.14 (m, 5H), 5.11 (br s, 1H), 4.96 (s, 1H), 4.58 (s, 1H), 4.50 (s, 1H), 4.33-4.23 (m, 2H), 4.24-4.08 (m, 2H), 3.86 (q, J = 6.6 Hz, 1H), 3.63-3.20 (m, 8H), 2.66-2.38 (m, 4H), 2.26-2.08 (m, 2H), 1.77-1.59 (m, 3H), 1.42 (s, 9H), 1.32 (d, J = 6.6 Hz, 3H), 0.91 (d, J = 6.3 Hz, 3H), 0.86 (d, J = 6.0 Hz, 3H); ¹³C-NMR (75 MHz, CDCl₃) δ 175.5, 174.2, 172.7, 172.1, 165.5, 163.5, 137.0, 129.1, 128.8, 127.1, 95.5, 90.6, 88.6, 82.0, 60.2, 58.5, 57.8, 52.9, 51.7, 50.3, 49.9, 47.6, 47.5, 39.4, 38.2, 27.4, 24.1, 23.9, 23.1, 18.0; HRMS (MALDI-TOF) m/z calcd for C₃₆H₅₀N₅O₄ (M+H)⁺ 616.3857; found 616.3865 (1.3 ppm)

tert-Butyl 3-((S)-1-((S)-1-((S)-2-benzyl-1-((S)-1-((S)-2-methyl-5-oxo-2,5-dihydro-1H-pyrrol-3-yl)pyrrolidin-3-yl)-5-oxo-2,5-dihydro-1H-pyrrol-3-yl)pyrrolidin-3-yl)-3-(tert-butoxy)-5-oxo-2,5-dihydro-1H-pyrrol-2-yl)propanoate (1afe′)

Pale yellow solid, 86.6 mg, 42 %; ¹H-NMR (300 MHz, CDCl₃) δ 7.25-7.01 (m, 5H), 5.72 (br s, 1H), 5.00 (s, 1H), 4.41 (s, 1H), 4.38 (s, 1H), 4.32 (t, J = 4.5 Hz, 1H), 4.25-4.16 (s, 1H), 4.15-4.05 (s, 1H), 4.01-3.82 (m, 2H), 3.71-3.42 (m, 3H), 3.42-3.17 (m, 4H), 3.17-3.05 (m, 2H), 2.91 (dd, J = 14.3, 5.3 Hz, 1H), 2.55-2.29 (m, 2H), 2.25-2.13 (m, 1H), 2.12-1.95 (m, 5H), 1.42 (s, 9H), 1.40 (s, 9H), 1.31 (d, J = 6.3 Hz, 3H); ¹³C-NMR (75 MHz, CDCl₃) δ 176.2, 174.3, 173.3, 172.3, 170.1, 167.3, 163.5, 135.1, 129.3, 128.3, 127.1, 96.9, 91.1, 87.4, 82.4, 80.8, 61.2, 60.6, 53.4, 52.9, 52.0, 50.1, 50.0, 47.6, 47.4, 37.6, 28.1, 27.4, 24.7, 19.0; IR (film, cm⁻¹) 3123 (br), 2976, 2871, 1724, 1670, 1600, 1395, 1369, 1341, 1258, 1167, 922, 887, 847, 731; HRMS (MALDI-TOF) m/z calcd for C₃₉H₅₄N₅O₆ (M+H)⁺ 688.4067; found 688.4045 (3.2 ppm)

General Procedure for the Deprotection of tert-Butyl Group

(3′S,5S)-5-((S)-sec-Butyl)-1′-((S)-1-((S)-1-((S)-2-Isobutyl-5-oxo-2,5-dihydro-1H-pyrrol-3-yl)pyrrolidin-3-yl)-2-methyl-5-oxo-2,5-dihydro-1H-pyrrol-3-yl)-[1,3′-bipyrrolidine]-2,4-dione (8lai)

Off-white solid, 84.2 mg, 80 %; ¹H-NMR (300 MHz, CDCl₃) δ 6.01 (br s, 1H), 4.65-4.48 (m, 2H), 4.27-4.02 (m, 4H), 3.93 (d, J = 3.0 Hz, 1H), 3.77 (t, J = 9.5 Hz, 1H), 3.68-3.51 (m, 3H), 3.50-3.37 (m, 3H), 3.37-3.22 (m, 1H), 2.98 (s, 2H), 2.69-2.42 (m, 2H), 2.36-2.11 (m, 2H), 1.93-1.78 (m, 1H), 1.77-1.48 (m, 2H), 1.47-1.32 (m, 3H), 1.03-0.92 (m, 12 H), 0.89 (d, J = 6.6 Hz, 3H); ¹³C-NMR (75 MHz, CDCl₃) δ 205.1, 176.2, 173.2, 169.7, 167.1, 165.0, 89.7, 87.8, 71.4, 56.2, 55.9, 53.2, 52.2, 51.8, 50.1, 49.7, 47.4, 47.3, 43.3, 41.9, 37.5, 27.8, 25.8, 25.3, 23.7, 21.4, 18.6, 13.3, 12.1; HRMS (MALDI-TOF) m/z calcd for C₂₉H₄₄N₅O₄ (M+H)⁺ 526.3393; found 526.3381 (2.3 ppm)

General Procedure for the pH Stability Assay

Procedure

Stock solutions of trimer 6t’l (200 μM) in 100 mM acetate buffer, pH 4.5 (containing less than 5 % DMSO) and 100 mM phosphate buffer pH 7.4 (containing less than 5 % DMSO) were prepared and stored at 25 °C. Samples (25 μL) were injected into a RP-HPLC system (see general methods) at regular intervals.

General Procedure for the Heat Stability Assay

Procedure

A stock solution of 1fla in the protease assay buffer (pH 7.8) was prepared as described above and incubated at 55 °C. Samples (200 μL) were aliquoted periodically diluted with 200 μL MeCN and 600 μL protease assay buffer II and analysed by RP-HPLC (see general methods).

Protease Assay

Procedure

Two buffers were used in the protease stability assay. Buffer I consisted of 50 mM Tris, 10 mM CaCl₂, pH 7.8 in 50 % (v/v) glycerol. Buffer II consisted of 50 mM Tris, 10 mM CaCl₂, pH 7.8. Pronase (3.2 mg) from Streptomyces griseus (Aldrich) was dissolved in buffer I (1mL) to obtain a stock solution of 160 μM (assuming an average molecular weight of 20 kDa). Pentamer 1fla or trimer 6ll (2 mg) was dissolved in 50 μL DMSO and diluted with 500 μL buffer I and then buffer II to make up 2 mL of solution to obtain the sample stock solutions (1.6 mM 1fla, 2.4 mM 6ll). The stock solutions were filtered through a 0.2 μ membrane filter to remove particulate matter for HPLC analysis. To the respective sample stock solutions, 70 μL of pronase stock solution was added and gently mixed to obtain the reaction mixture, which was incubated at 37 °C.

The positive control was prepared was follows. Pronase stock solution (70 μM) was added to a solution of the the N-labeled tetrapeptide GATV-OH (3.8 mM, 2 mL), which was prepared similar to the sample stock solution above. The reaction mixture was incubated at 37 °C.

Samples (200 μL) were aliquoted periodically and quenched with 200 μL MeCN to halt the reaction, then diluted with 600 μL buffer II and analysed by RP-HPLC (see general methods). Under the described conditions, the control peptide completely decomposed in 2 h.

General Procedure for X-Ray Structure Determination

A microscope was used to identify a suitable colorless multi-faceted crystal with very well defined faces with dimensions (max, intermediate, and min) 0.05 mm × 0.03 mm × 0.01 mm from a representative sample of crystals of the same habit. The crystal mounted on a nylon loop was then placed in a cold nitrogen stream maintained at 110 K.

A X-ray diffractometer was employed for crystal screening, unit cell determination, and data collection. The goniometer was controlled using the FRAMBO software suite.^φ The sample was optically centered with the aid of a video camera such that no translations were observed as the crystal was rotated through all positions. The detector was set at 6.0 cm from the crystal sample (MWPC Hi-Star Detector, 512×512 pixel). The X-ray radiation employed was generated from a Cu sealed X-ray tube (K_α = 1.54184Å with a potential of 40 kV and a current of 40 mA) fitted with a graphite monochromator in the parallel mode (175 mm collimator with 0.5 mm mono-capillary optics).

The rotation exposure indicated acceptable crystal quality and the unit cell determination was undertaken. 2100 data frames were taken at widths of 0.5° with an exposure time of 10 seconds. Over 6000 reflections were centered and their positions were determined. These reflections were used in the auto-indexing procedure to determine the unit cell. A suitable cell was found and refined by nonlinear least squares and Bravais lattice procedures and reported here in Table 1. No super-cell or erroneous reflections were observed.

After careful examination of the unit cell, a standard data collection procedure was initiated. This procedure consists of collection of one hemisphere of data collected using omega scans, involving the collection 0.5° frames at fixed angles for φ, 2θ, and χ (2θ; = −28°, χ = 54.73°, 2θ; = −90°, χ = 54.73°), while varying omega. Addition data frames were collected to complete the data set. Each frame was exposed for 10 sec. The total data collection was performed for duration of approximately 24 hours at 110K. No significant intensity fluctuations of equivalent reflections were observed.

Data Reduction, Structure Solution, and Refinement

Integrated intensity information for each reflection was obtained by reduction of the data frames with the program SAINT.^* The integration method employed a three dimensional profiling algorithm and all data were corrected for Lorentz and polarization factors, as well as for crystal decay effects. Finally the data was merged and scaled to produce a suitable data set. The absorption correction program SADABS^** was employed to correct the data for absorption effects.

Systematic reflection conditions and statistical tests for the data suggested the space group P2₁. A solution was obtained readily using SHELXTL (SHELXS).^* All non-hydrogen atoms were refined with anisotropic thermal parameters. The Hydrogen atoms bound to carbon were placed in idealized positions [C–H = 0.96 Å, Uiso(H) = 1.2 × Uiso(C)]. The structure was refined (weighted least squares refinement on F²) to convergence. X-seed was employed for the final data presentation and structure plots.^**