key: cord-0905419-2i6795ga authors: Thanigaimalai, Pillaiyar; Konno, Sho; Yamamoto, Takehito; Koiwai, Yuji; Taguchi, Akihiro; Takayama, Kentaro; Yakushiji, Fumika; Akaji, Kenichi; Kiso, Yoshiaki; Kawasaki, Yuko; Chen, Shen-En; Naser-Tavakolian, Aurash; Schön, Arne; Freire, Ernesto; Hayashi, Yoshio title: Design, synthesis, and biological evaluation of novel dipeptide-type SARS-CoV 3CL protease inhibitors: Structure–activity relationship study date: 2013-05-20 journal: Eur J Med Chem DOI: 10.1016/j.ejmech.2013.05.005 sha: ef47a59c6ef2eebf1474aa9270c2e666fcf6e084 doc_id: 905419 cord_uid: 2i6795ga This work describes the design, synthesis, and evaluation of low-molecular weight peptidic SARS-CoV 3CL protease inhibitors. The inhibitors were designed based on the potent tripeptidic Z-Val-Leu-Ala(pyrrolidone-3-yl)-2-benzothiazole (8; K(i) = 4.1 nM), in which the P3 valine unit was substituted with a variety of distinct moieties. The resulting series of dipeptide-type inhibitors displayed moderate to good inhibitory activities against 3CL(pro). In particular, compounds 26m and 26n exhibited good inhibitory activities with K(i) values of 0.39 and 0.33 μM, respectively. These low-molecular weight compounds are attractive leads for the further development of potent peptidomimetic inhibitors with pharmaceutical profiles. Docking studies were performed to model the binding interaction of the compound 26m with the SARS-CoV 3CL protease. The preliminary SAR study of the peptidomimetic compounds with potent inhibitory activities revealed several structural features that boosted the inhibitory activity: (i) a benzothiazole warhead at the S1′ position, (ii) a γ-lactam unit at the S1-position, (iii) an appropriately hydrophobic leucine moiety at the S2-position, and (iv) a hydrogen bond between the N-arylglycine unit and a backbone hydrogen bond donor at the S3-position. Since its first appearance in Southern China in late 2002, severe acute respiratory syndrome (SARS) has been recognized as a global threat [1, 2] . Its rapid and unexpected spread to 32 countries has affected more than 8000 individuals and caused nearly 800 (w10%) fatalities worldwide within a few months [1e3] . The causative SARS pathogen is a novel coronavirus, SARS-CoV [4, 5] . SARS-CoV is a positive-strand RNA virus with a genome sequence that is only moderately homologous to other known coronaviruses [6, 7] . SARS-CoV encodes a chymotrypsin-like protease (3CL pro ), also referred to as the main protease (M pro ), which plays a pivotal role in processing viral polyproteins and controlling replicase complex activity [8] . This enzyme is indispensable for viral replication and infection processes, making it an ideal target for the design of antiviral therapies. The 3CL pro active site contains a catalytic dyad in which a cysteine residue (Cys145) acts as a nucleophile and a histidine (His41) residue acts as a general acid or base [9, 10] . The SARS epidemic was successfully controlled in 2003; however, the potential reemergence of pandemic SARS-CoV continues to pose a risk, and new strains of SARS could potentially be more virulent than the strains that contributed to the 2003 outbreak. Since 2003, two additional human coronaviruses, NL63 and HKU1, have been identified in patients around the world [11, 12] . Recently, a new SARS-like virus, HCoV-EMC, was identified in at least two individuals, one of whom died [13] . Very recently, the first case of a fatal respiratory illness similar to the deadly SARS was confirmed in Britain [14] . The World Health Organization (WHO) has announced that it is closely monitoring the situation and is working to "ensure a high degree of preparedness, should the new virus be found to be sufficiently transmissible to cause a community outbreak". There is a significant need to develop anti-SARS agents that are capable of treating this potentially fatal respiratory illness. Several reports of crystalline forms of the SARS-CoV 3CL pro protein bound to hexapeptidyl chloromethyl ketone inhibitors have been reported [6, 9] , and numerous peptidic structures have been reported in the context of targeted antiviral drug design [15e22] . The reported protease inhibitors are generally peptidic in nature, often five to three residues in length, and bear a reactive warhead group at the C-terminus which forms an interaction with the protease catalytic Cys145 (Fig. 1, 1e9) . Two of these compounds (8 and 9) , recently described in a separate report from our group [22] , exhibited excellent potent inhibitory activities with K i values of 4.1 and 3.1 nM, respectively. These peptidic inhibitors provided valuable insight into the design constraints for this system and quickly led to the development of nonpeptidic small molecule inhibitors (Fig. 1, 10e17) [23e30]. These small molecular inhibitors generally showed moderate to good activities. Recently, we performed a structureeactivity relationship study based on the lead compound, Z-Val-Leu-Ala(pyrrolidone-3-yl)-2thiazole (7) [21] . This study led to the discovery of the potent compounds 8 and 9, with K i values in the low nanomolar range [22] . Extending our studies toward the development of new anti-SARS agents, we now report the design, synthesis, and evaluation of a series of low-molecular weight dipeptide-type compounds in which the P3 valine unit is removed from the previous lead Z-Val-Leu-Ala(pyrrolidone-3-yl)-2-benzothiazole compound (8, Fig. 1) . A preliminary SAR study led to the identification of inhibitors with moderate to good inhibitory activities. In particular, compounds 26m and 26n exhibited potent inhibitory activities with K i values of 0.39 and 0.33 mM, respectively. The binding interactions of 26m were predicted using molecular modeling studies. We describe the results of these extensive studies in detail, including the design, synthesis, molecular modeling, and biological evaluation of a series of SARS-CoV 3CL pro inhibitors. The synthesis of the title inhibitors was achieved through a coupling reaction involving two key fragments, as shown in Scheme 1. One of the key fragment intermediates (19) (20) , which were used directly in the subsequent step. The g-lactam-thiazoles (24) were synthesized using an approach similar to the syntheses reported previously [21, 22, 31] . Accordingly, the optically pure L-glutamic acid ester 21 was converted to the g-lactam ester 22 by treatment with bromoacetonitrile, followed by reduction with PtO 2 (5%) and cyclization. The resulting ester 22 was hydrolyzed in the presence of 4 M NaOH in methanol to yield the corresponding acid, which was coupled to N,O-dimethylhydroxylamine via the EDCeHOBt method to afford the Weinreb amide 23. The Weinreb amide 23 was then coupled to the appropriate thiazoles in the presence of n-butyl lithium (n-BuLi) or lithium diisopropylamide (LDA) at À78 C to afford the glactam-thiazoles 24. The Boc protecting group was then removed, and the resulting intermediate was subsequently reacted with the above-mentioned N-protected amino acids 20 in the presence of Obenzotriazole-N,N,N 0 ,N 0 -tetramethyluroniumhexafluoro phosphate (HBTU) and DIPEA in DMF to afford the title compounds 25e27. All compounds were purified by reverse phase HPLC and characterized by 1 H and 13 C NMR, and mass spectrometry analysis. The purity of each compound exceeded 90e95%. The compounds were subjected to a fluorometric protease inhibitory assay against SARS-CoV 3CL pro , as described previously [32, 33] . Briefly, the kinetic parameters were determined at a constant substrate concentration, and the inhibitor concentrations were varied to assess the K i values [22] . The IC 50 values were determined only for certain potent inhibitors, based on the apparent decrease in the substrate concentration (H-Thr-Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys-NH 2 ) upon digestion by R188I SARS 3CL pro , as described previously [19, 34] . The cleavage reaction was monitored by analytical HPLC, and the cleavage rates were calculated from the decrease in the substrate peak area. Tables 1e4 report the K i or IC 50 values as the mean of 3 independent experiments. In an effort to develop low-molecular weight peptidic SARS-CoV 3CL pro inhibitors, we designed a series of dipeptide-type inhibitors based on the previous potent lead compound 8 (see Fig. 1 ). In a first attempt, the P3 valine region was removed and the P4benzyloxycarbonyl (Cbz) in 8 was replaced with a series of small moieties with sizes similar to that of the P3 valine group in 8. The inhibitory activities of the resulting dipeptidic compounds bearing a valine mimic, isopentanoyl (25a; K i ¼ 5.9 mM) or tert-butoxy carbonyl (25b; K i ¼ 23 mM), were dramatically reduced compared to the activity of 8 (K i ¼ 0.0041 mM) [22] ; however, the introduction of Cbz (25c; K i and IC 50 ¼ 0.46 and 21.0 mM) as a P3 moiety resulted in a 12-fold or 50-fold activity increase for 25a or 25b, respectively, although the potency was reduced relative to the value for the tripeptidic lead 8. This result suggested that the Cbz group, which was introduced in place of the P3 scaffold in the dipeptidic 25c, conveyed appreciable activity; therefore, compound 25c could serve as a lead for further optimization steps. By retaining the P3 Cbz moiety in 25c, we examined the relevance of the leucine residue (or isobutyl unit) for P2 substrate selectivity in comparison with a variety of its congeners. Accordingly, a series of isosteres was introduced, including n-butyl (25d; K i ¼ 1.60 mM), isopropyl (25e; K i ¼ 1.71 mM), sec-butyl (25f; K i ¼ 29.0 mM), 3-methyl(thio)ethyl (25g; K i ¼ 9.40 mM), and benzyl (25h; K i ¼ 1.20 mM). The compounds bearing n-butyl (25d), isopropyl (25e), or benzyl (25h) groups exhibited reasonable inhibitory activities, although the potencies were reduced by a factor of 3e4 relative to the value for 25c. The inhibitory activity of the compound bearing sec-butyl (25f) or 3-methyl(thio)ethyl (25g) was severely reduced compared to the activity of 25c. Therefore, the leucine residue (or isobutyl group) was more selective and appropriate as a P2 group in 25c, providing enhanced inhibitory activity. The P3 moiety of the lead compound 25c was examined by introducing a wide variety of substituents, such as aryl (or heteroaryl) acetyls and propionyls, arylacrylyls, aryloxyacetyls, and N-arylglycyls. Initially, the aryl (or heteroaryl) acetyls and propionyls were introduced, including phenylacetyl (26a; K i ¼ 3.20 mM), 4-methoxyphenylacetyl (26b; K i and IC 50 ¼ 0.42 and 43 mM), 4-methoxyphenylpropionyl (26c; K i ¼ 0.62 mM), and pyridine-3-propionyl (26d; K i ¼ 7.4 mM). Among the compounds, a 4methoxyphenylacetyl derivative (26b) exhibited potent inhibitory activity; the phenylacetyl (26a) and pyridine-3-propionyl (26d) derivatives in particular displayed low inhibitory activities. Therefore, the 4-methoxyphenylacetyl moiety provided a good alternative to the P3 Cbz group in 25c. We next introduced the arylacrylyls, including cinnamoyl (26e; K i ¼ 0.69 mM), 4-methoxycinnamoyl (26f; K i ¼ 0.70 mM), and 3,4-dimethoxycinnamoyl (26g; K i ¼ 1.30 mM). These compounds showed moderate inhibitory activities relative to the lead 25c; thus, the acrylyls were not considered further as P3 moieties in the context of 25c. A previous report describing a SAR study of the tripeptidomimetic 7 (see Fig. 1 ) as a SARS inhibitor revealed that the introduction of an aryloxyacetyl at the N-terminal position (the P4 position) appeared to significantly enhance the inhibitory activity against SARS-CoV 3CL pro [22] . Therefore, we introduced a series of aryloxyacetyls, including phenoxyacetyl (26h; K i and IC 50 ¼ 0.56 and 24 mM), 4-methoxyphenoxyacetyl (26i; K i ¼ 1.56 mM), 4hydroxyphenoxyacetyl (26j; K i ¼ 8.4 mM), and 3-dimethyl aminophenoxyacetyl (26k; K i ¼ 0.84 mM) as P3 groups in place of the Cbz group in 25c. Compounds 26h and 26k exhibited comparable activities to 25c; however, the other derivatives (26i and 26j) displayed reduced inhibitory activities relative to the lead compound 25c. These results suggested that the introduction of an aryloxyacetyl P3 group in the dipeptidic 25c did not appreciably improve the inhibitory activity against 3CL pro . We also introduced several N-arylglycyls, including N-(4-methoxyphenyl)glycyl (26l; K i ¼ 3.20 mM), N-(3-methoxyphenyl)glycyl (26m; K i and IC 50 ¼ 0.39 and 10.0 mM), and N-(2-methoxyphenyl)glycyl (26n; K i and IC 50 ¼ 0.33 and 14.0 mM). The results of these studies revealed that compounds 26m and 26n displayed relatively potent inhibitory activities compared to the lead 25c. The compound bearing an N-(2-methoxyphenyl)glycyl (26n) was identified as the most potent inhibitor in the present study. This result suggested that the hydrogen bonding properties of the amino group on the N-arylglycyl moiety might have contributed to the improvement in activity (see Fig. 2B ). The P1 0 moiety was examined next by varying the 5-substituted thiazoles (27aed). The inhibitory activities of compounds 27ae d are illustrated in Table 3 . Inhibitor 27a exhibited an inhibitory activity comparable to that of 25c. The other 5-arlylated thiazoles (27bed) generally exhibited very low inhibitory activities compared to 25c. These studies confirmed that the benzothiazole unit was more suitable as a warhead group on the P1 0 moiety in 25c. The binding mode of compound 26m with 3CL pro was simulated using a molecular docking program as described previously [22] . We examined the molecular docking of the potent active compound 26m in comparison with docking of the tripeptidic 9 and a structurally similar ligand, the docking structure of which has been elucidated previously by X-ray crystallography (PDB ID: 1WOF, [35] . Several minimization processes were performed using the MMFF94X force field to model the solvation environment surrounding the inhibitor. A molecular simulation was then performed. As shown in Fig. 2A , the P1 0 eP2 moieties in 26m, in the tripeptidic compound 9, and in the original ligand interacted with the same region of the protease. Interestingly, the aminoacetyl group on the N-(3-methoxyphenyl)glycyl group in 26m mimicked the P3 valine moieties of the tripeptidomimetic 9 and the reported ligand. The interactions of compound 26m with the protein are described in detail in Fig. 2B . Particularly, a notable hydrogen bonding involved between the amino group (eNH) of P3 N-(3-methoxyphenyl)glycine with a backbone amino acid residue Glu166 of 3CL pro was observed to have relatively potent inhibition than other inhibitors presented in the study against SARS-CoV 3CL pro . The essential nature of this hydrogen bond will be explored in further optimization studies of dipeptide-type SARS-CoV 3CL pro inhibitors. In an effort to develop low-molecular weight peptidic anti-SARS agents, we designed, synthesized, and evaluated a series of dipeptide-type inhibitors in which the P3 valine unit in our potent tripeptidomimetic lead compound 8 was replaced with a number of distinct functionalities. In a preliminary study, the compound 25c, bearing a benzyloxycarbonyl (Cbz) P3 moiety, was identified as a lead compound. The P3 moiety in 25c was systematically modified with a view to improve the biological activity. This study led to the identification of a compound bearing an N-arylglycyl as a P3 moiety as having higher inhibitory activity due to the presence of a hydrogen bond with the backbone amino acid residue Glu166 of 3CL pro . Leucine and benzothiazole units were identified as appropriate P2 and P1 0 moieties, respectively, in 25c. Accordingly, compounds 26m and 26n were recognized as potent inhibitors in the present study. These potent dipeptidic inhibitors provide attractive leads, which are undergoing further structural modifications in an effort to improve the pharmaceutical profiles. These processes are underway and will be reported in the near future. Reagents and solvents were purchased from Wako Pure Chemical Ind., Ltd. (Osaka, Japan), and Aldrich Chemical Co. Inc. (Milwaukee, WI) and were used without further purification. Analytical thin-layer chromatography (TLC) was performed on Merck Silica Gel 60F254 pre-coated plates. Preparative HPLC was performed using a C18 reverse-phase column (19 Â 150 mm; Sun-Fire Prep C18 All other chemicals were of analytical grade or better. 1 H and 13 C NMR spectra were obtained using a JEOL 400 MHz spectrometer, a Varian Mercury 300 spectrometer (300 MHz), or a BRUKER AV600 spectrometer (600 MHz) with tetramethylsilane as an internal standard. High-resolution mass spectra (ESI or EI) were recorded on a micromass Q-Tof Ultima API or a JEOL JMS-GCmate BU-20 spectrometer. Mass spectra (ESI) were recorded on an LCMS-2010EV (SHIMADZU). 4.2.1. Synthetic procedures for the preparation of (S)-tert-butyl 2- To a solution of the commercially available L-leucine tert-butyl ester 18a (0.200 g, 0.89 mmol) in DMF (15 mL) was added an isovaleric acid (0.100 g, 0.98 mmol), HOBt$H 2 O (0.151 g, 0.98 mmol), and EDC$HCl (0.189 g, 0.98 mmol). The resulting solution was cooled to 0 C under ice bath conditions, and TEA was then added dropwise. After 5 min, the ice bath was removed and the mixture was allowed to stir for 2 h at ambient temperature. DMF was removed under high vacuum, and the resulting residue was dissolved in EtOAc (30 mL). The organic layer was washed with 5% citric acid (20 mL Â 2), 5% NaHCO 3 (20 mL Â 2), and brine (20 mL). The solution was dried over Na 2 SO 4 , filtered, and evaporated under reduced pressure to give compound 19a. The resulting crude compound was purified by silica gel column chromatography using hexaneeEtOAc as eluents. Yield 65%; white solid; 1 The intermediates 19heu were prepared from L-leucine tertbutyl ester 18a and various commercially available carboxylic acids according to the procedure described for the synthesis of 19a. To a solution of the commercially available L-leucine tert-butyl ester 18a (0.500 g, 2.20 mmol) in CH 2 Cl 2 (20 mL) was added benzyloxycarbonyl chloride (0.410 mL, 2.5 mmol). The resulting solution was cooled to 0 C and TEA (0.380 mL, 2.5 mmol) was then added dropwise. After 5 min, the ice bath was removed and the mixture was allowed to stir for 2 h at room temperature. The mixture was washed with H 2 O (20 mL) and brine (10 mL). The Yield 76% from L-leucine tert-butyl ester (18a); colorless oil; 1 Yield 60% from L-leucine tert-butyl ester (18a); light pink solid; Procedure A: To a solution of the corresponding tert-butyl ester 19 (0.110 g, 0.31 mmol) in CH 2 Cl 2 (2 mL) at 0 C was added TFA/H 2 O (10:1, 3 mL). After 5 min stirring, the reaction mixture was allowed to stir at room temperature for 1 h. The solvent was completely evaporated under reduced pressure to give the corresponding acids 20, which were used directly in the subsequent step without further characterization. Procedure B: To a solution of the corresponding methyl ester 19 (0.100 g, 0.35 mmol) in THF (3 mL) at room temperature was added LiOH in water (0.102 g, 2.45 mmol). After 3 h stirring, the solvent was completely evaporated under reduced pressure and the resulting residue was neutralized with 2 M HCl. The solution was extracted with EtOAc (20 mL Â 2), dried over Na 2 SO 4 , filtered and evaporated under reduced pressure to give the corresponding acids 20, which were directly used for next step without further characterizations. Compound 22 was prepared through sequential reactions from the well-known intermediate 21, as reported previously [20e 22, 31] . To a solution of 22 (3.27 g, 8.0 mmol) in methanol (5 mL) was added a 4 M NaOH solution (0.04 mol) at room temperature. The mixture was stirred for 4 h and the methanol was evaporated. The residue was neutralized with 2 M HCl and extracted with EtOAc (20 mL Â 4). The organic layer was washed with brine, dried over Na 2 SO 4 , and evaporated under reduced pressure to yield the corresponding acid. To a solution of the acid (12.0 mmol) in DMF (30 mL) were added EDC$HCl (0.540 g, 10.0 mmol), HOBt$H 2 O (0.330 g, 11.0 mmol), and N,O-dimethylhydroxylamine (0.213 g, 11.0 mmol) at ambient temperature. The solution was cooled to 0 C, and TEA (0.240 mL, 11.0 mmol) was added slowly. After 2 h, the DMF was evaporated under high vacuum, and the resulting residue was dissolved in EtOAc (100 mL). The resulting solution was subsequently washed with 5% citric acid (20 mL Â 2), 5% NaHCO 3 (20 mL Â 2), and brine (50 mL). The organic layer was then dried over Na 2 SO 4 and concentrated under reduced pressure to yield the Weinreb amide derivative 23, which was purified by column chromatography (EtOAc/MeOH ¼ 9.5:0.5). The data for compounds 22 and 23 were reported previously [20, 21] . To a solution of benzothiazole (1.37 g, 10.0 mmol) in THF at À78 C was added n-BuLi (2.0 M in THF, 1.67 mL) dropwise over 20 min. After 1 h stirring, the Weinreb amide 23 (0.064 g, 2.0 mmol) in THF was slowly added dropwise over 20 min and then the solution was stirred for 3 h at same temperature. The reaction was quenched with sat. NH 4 Cl and allowed to stir at 0 C for 20 min. The mixture was evaporated and dissolved in EtOAc. This solution was washed with water (100 mL) and brine (50 mL), and then dried over Na 2 SO 4 . After filtration, the organic layer was concentrated under reduced pressure and the resulting residue was subjected to flash chromatography (EtOAc/MeOH ¼ 9:1) to obtain the pure compound 24a. The data for the compound 24a has been reported in a previous article [21] . To a cooled solution of the commercially available 5phenylthiazole (0.175 g, 10 mmol) in dry THF at À78 C was slowly added a solution of LDA (1.0 mL, 14 mmol). After 1 h, a precooled solution containing the Weinreb amide 23 (0.329 g, 10 mmol) in anhydrous THF was slowly added, and the reaction was stirred at À78 C over 2 h. The solution was allowed to warm to room temperature, was quenched by the addition of water (35 mL), and then extracted with EtOAc (3 Â 50 mL). The combined organic layers were dried over anhydrous Na 2 SO 4 , filtered, and evaporated in vacuo. The crude mixture was then purified using flash chromatography (n-hexane/EtOAc ¼ 3:7) to furnish 24b. The data for the compound 24b were reported previously [22] . Compounds 24cee were prepared from 23 according to the procedure described for the synthesis of 24b. The data for the compound 24c has been reported in a previous article [22] . The data for the compound 24d has been reported in a previous article [22] . The data for the compound 24e has been reported in a previous article [22] . To a solution of 24a (0.200 g, 0.5 mmol) in CH 2 Cl 2 (3 mL) at 0 C was added TFA/H 2 O (10:1, 5 mL), and the solution was stirred for 1 h. After evaporating the solvent under reduced pressure, the corresponding deprotected lactam residue (0.100 g, 0.53 mmol) was coupled to the carboxylic acid 20a (0.136 g, 0.38 mmol) using the coupling agent HBTU (0.147 g, 0.38 mmol) in the presence of diisopropylethylamine (0.050 mL, 0.38 mmol) in DMF (3 mL) at 0 C. The reaction mixture was allowed to stir for 2e3 h under ambient conditions. The solvent was then evaporated under a high vacuum, and the residue was dissolved in EtOAc (50 mL). The organic layer was washed with 5% citric acid (20 mL Â 2), 5% NaHCO 3 (20 mL Â 2), and brine (25 mL). This solution was dried over Na 2 SO 4 , filtered, and evaporated under reduced pressure to give compound 25a. Yield 35%; light yellow solid; 1 Compounds 25beh were prepared from 24a with 20aeh using a procedure similar to that described for the synthesis of 25a. Yield 37% from 24a; colorless solid; 1 H NMR (400 MHz, Compounds 26aer were prepared from 24a with 20ieu using a procedure similar to that described for the synthesis of 25a. The crystal structure of the SARS-CoV 3CL protease in complex with a substrate analog inhibitor (coded 1WOF) [35] was obtained from the Protein Data Bank (PDB; http://www.rcsb.org/pdb/home/ home.do). Initially, a binding model of 26m with 3CL pro was simulated to form the basis of a comparison with our previous potent lead tripeptidomimetic 9 and a substrate analog, using molecular operating environment (MOE) software. Several minimization processes were performed using the MMFF94X force field to model the solvation environment surrounding the inhibitor. Structures having a relatively low binding free energy and a high number of cluster members were selected for the subsequent docking conformation optimization step. The minimized energies of 26m, obtained from the docking study, were À40.67 and À35.52 kcal/mol. -(dimethylamino)phenoxy) acetamido)-4-methylpentanamide (26k) Yield 35% from 24a )-1-(Benzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-oxopyrro lidin-3-yl)propan-2-yl Hz, 1H), 7.68e7.60 (m, 2H), 7.06 (t, J ¼ 8.4 Hz, 1H), 6.30e6.17 (m, 3H), 5.72e5.68 (m, 1H), 4.53e4.50 (m, 1H), 3.84e3.65 (m, 2H), 3.82 (s, 3H), 3.37e3.28 (m, 2H, merged with CD 3 OD), 2.72e2.55 (m, 1H), 2.54e2.29 (m, 1H), 2.28e1.83 (m, 3H), 1.62e1.41 (m, 3H), 0.90e0.83 (m, 6H). 13 C NMR (400 MHz -methoxyphenyl)amino) acetamido)-4-methylpentanamide (26m) Yield 41% from 24a; light yellow solid; 1 H NMR (400 MHz -methoxyphenyl)amino) acetamido)-4-methylpentanamide (26n) Yield 45% from 24a; light yellow solid S)-2-oxopyr rolidin-3-yl)-1-(5-phenylthiazol-2-yl)propan-2-yl)amino)pentan-2-yl)carbamate (27a Yield 55% from 24b J ¼ 7.20 Hz, 2H), 7.48e7.40 (m, 3H), 7.37e7.24 (m, 5H), 5.65e5.61 (m, 1H), 5.15e5.03 (m, 2H), 4.30e4.20 (m, 1H), 3.40e3.23 (m, 2H, merged with CD 3 OD), 2.75e2.65 (m, 1H), 2.64e2.06 (m, 2H), 2.05e1.80 (m, 2H), 1.79e1.60 (m, 1H), 1.59e 1.52 (m, 2H), 0.99e0.89 (m, 6H). 13 C NMR (400 MHz (S)-2-oxopyr rolidin-3-yl)-1-(5-(p-tolyl)thiazol-2-yl)propan-2-yl)amino)pentan-2-yl)carbamate (27b) Yield 51% from 24c 38 (s, 3H), 2.29e2.09 (m, 1H), 2.08e1.75 (m, 2H), 1.74e1.65 (m, 1H), 1.63e1.49 (m, 2H), 0.97e0.90 (m, 6H) 2-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)carbamate (27d) Yield 51% from 24e; light yellow solid; 1 H NMR (400 MHz This research was supported by Grants from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan, including a Grant-in-aid for Young scientist (Tokubetsu Kenkyuin Shorei-hi) 23$01104 and a Grant-in-aid for Scientific Research 23659059 and 23390029. E.F. acknowledges support from the National Institutes of Health (grant GM57144). Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.ejmech.2013.05.005.