key: cord-0889715-esogd26s
authors: Konno, Sho; Thanigaimalai, Pillaiyar; Yamamoto, Takehito; Nakada, Kiyohiko; Kakiuchi, Rie; Takayama, Kentaro; Yamazaki, Yuri; Yakushiji, Fumika; Akaji, Kenichi; Kiso, Yoshiaki; Kawasaki, Yuko; Chen, Shen-En; Freire, Ernesto; Hayashi, Yoshio
title: Design and synthesis of new tripeptide-type SARS-CoV 3CL protease inhibitors containing an electrophilic arylketone moiety
date: 2013-01-15
journal: Bioorg Med Chem
DOI: 10.1016/j.bmc.2012.11.017
sha: 43a01f3a7935d879533de01ab397fb4e5ae641fe
doc_id: 889715
cord_uid: esogd26s

We describe here the design, synthesis and biological evaluation of a series of molecules toward the development of novel peptidomimetic inhibitors of SARS-CoV 3CL(pro). A docking study involving binding between the initial lead compound 1 and the SARS-CoV 3CL(pro) motivated the replacement of a thiazole with a benzothiazole unit as a warhead moiety at the P1′ site. This modification led to the identification of more potent derivatives, including 2i, 2k, 2m, 2o, and 2p, with IC(50) or K(i) values in the submicromolar to nanomolar range. In particular, compounds 2i and 2p exhibited the most potent inhibitory activities, with K(i) values of 4.1 and 3.1 nM, respectively. The peptidomimetic compounds identified through this process are attractive leads for the development of potential therapeutic agents against SARS. The structural requirements of the peptidomimetics with potent inhibitory activities against SARS-CoV 3CL(pro) may be summarized as follows: (i) the presence of a benzothiazole warhead at the S1′-position; (ii) hydrogen bonding capabilities at the cyclic lactam of the S1-site; (iii) appropriate stereochemistry and hydrophobic moiety size at the S2-site and (iv) a unique folding conformation assumed by the phenoxyacetyl moiety at the S4-site.

Severe acute respiratory syndrome (SARS), a typical form of pneumonia, was first reported in southern China in November 2002. SARS rapidly spread to 32 countries, creating panic among the public and in the World Health Organization (WHO). [1] [2] [3] The initial outbreak of SARS included more than 8000 individuals diagnosed with the disease. Of these, 774 lives were claimed. 4 SARS is characterized by a high fever (>38°C), malaise, headache, rigor and a non-productive cough. 5 The causative agent of SARS has been identified as a novel human coronavirus (SARS-CoV) 6 that encodes several viral proteases that proteolyze polyproteins to yield functional proteins. One such highly conserved cysteine protease is the main protease (M pro ), also known as the dimeric chymotrypsin-like protease (3CL pro ). 7-9 3CL pro mediates the majority of proteolytic processes involved in producing two large viral polyproteins, replicases pb1a and pb1b. [7] [8] [9] The active site of SARS-CoV 3CL pro contains Cys145 and His41, which together constitute a catalytic dyad in which the cysteine moiety functions as a common nucleophile in the proteolytic process. 9, 10 Since SARS-CoV 3CL pro plays an important role in the virus life cycle, it has been recognized as a key target for the discovery of anti-SARS agents. Numerous SARS-CoV 3CL pro inhibitors have been identified through chemical library screenings [11] [12] [13] [14] [15] [16] or rational design approaches based on the active site properties. [17] [18] [19] [20] These protease inhibitors include C2-symmetric peptidomimetics, 11 3-quinolinecarboxylic acid derivatives, 12 bifunctional arylboronic acids, 13 keto-glutamine derivatives, 14 isatin derivatives, 15 thiophene-2-carboxylates, 16 zinc conjugated compounds, 17 cinanserin, 18 calmodulin, 19 benzotriazoles, 20 a,b-unsaturated acids, 21 anilide, 22 and glutamic acid and glutamine peptides possessing a trifluoromethyl ketone group. 23, 24 In our ongoing effort to develop anti-SARS agents, we previously identified a series of Z-Val-Leu-Ala(pyrrolidone-3-yl)-2-thiazoles as SARS-CoV 3CL pro inhibitors. 25 Among these compounds, compound 1 (Fig. 1 ) was identified as a potent lead compound with a K i value of 2.20 lM. 25 Primary structure-activity relationship (SAR) studies of 1 have revealed that the presence of a rigid cyclic amide (P1-site) and the electron-withdrawing chemical warhead thiazolyl-2-ketone (P1 0 -moiety) are very important for the inhibitory activity. In the present study, we performed a molecular modeling study involving 1 and the 3CL pro . The study revealed the presence of a space in the Table 2 . The imidazole-type compounds 4 were prepared as indicated in Scheme 3. Another key imidazole thiazole 17 was prepared from the commercially available Boc-His(Tos)-OH 14 using the same reactions and conditions as indicated for the preparation of the c-lactam thiazoles 13. The tosyl-protected 17 was successfully converted to the imidazole-type analogs 4 via deprotection and coupling chemistry, as described for the preparation of 2, 3 and 5.

The abilities of the synthetic compounds to inhibit the protease activity of SARS-CoV 3CL pro were evaluated. Briefly, IC 50 value of each inhibitor was assessed from the apparent decrease of a substrate (H-TSAVLQSGFRK-NH 2 ) by the digestion with R188I SARS 3CL pro as described previously. 28, 29 Cleavage reaction was monitored analytical HPLC and the cleavage rates were calculated from the decrease of the substrate peak area. Determining the kinetic parameters at a constant substrate concentration and different inhibitor concentrations assessed K i values. The initial rate measurements were determined as previously described using a fluorescence-based peptide cleavage assay with a fluorogenic substrate, Dabcyl-KTSAVLQSGFRKME-Edans. 24, 30 The rate of substrate cleavage was detected by the increase in fluorescent over time (see Section 6.7). Tables 1-3 report the IC 50 and/or K i values as the mean of 3 independent experiments.

In our previous report, inhibitor 1 was found to be a moderate SARS-CoV 3CL pro inhibitor with an IC 50 value of 9.5 lM and a K i value of 2.20 lM. In a first attempt to investigate the effects of the N-terminal substituents (P4-moiety) on the activity profile of 1 (Table 1) , the inhibitory activities of a series of aromatic (2a & 2b) and aliphatic (2c & 2d) carbamates were evaluated. None of these compounds showed inhibitory potency comparable to 1.

The same result was obtained for the acyl derivatives, such as 2e and 2f. Interestingly, the derivative 2g (IC 50 = 6.8 lM), a phenoxymethyl carbonyl, displayed a slightly higher activity than 1. Thus, the presence of the phenoxymethyl carbonyl at the N-terminal position appeared to enhance the activity of 1.

The molecular modeling study involving 1 and the 3CL pro enzyme ( Fig. 1 ) provided insight and understanding into the binding of the inhibitor to the active site of the enzyme (See Section 4 and Fig. 2) . The introduction of a spacious warhead group (P1 0 ) was suggested as a means for increasing the inhibitory activity; therefore, we modified inhibitor 1 to include larger units, such as 4,5dimethylthiazole, 5-methylthiazole, benzothiazole and a series of 5-arylated thiazoles at the P1 0 position.

The compound bearing 4,5-dimehtylthiazole, 2h, displayed a lower inhibitory activity (IC 50 = 22 lM) than 1. Compounds 2i and 2j bearing benzothiazole exhibited four-to fivefold higher activities (2i; IC 50 = 1.7 lM, 2j; IC 50 = 2.3 lM) than 1 and 10-to 13-fold higher activities than 2h. Notably, 2i exhibited very potent inhibition with a K i value of 4.1 nM. This finding strongly suggested that the benzothiazole unit in 1 was a suitable chemical warhead group for occupying the S1 0 -site. In the subsequent studies, inhibitors 2i and 2j were advanced as lead compounds for further optimization.

A series of electron-donating or electron-withdrawing substituents were introduced onto the phenyl ring of the P4-moiety of 2j. Indeed, compounds with an electron donating substituent such as methoxy, hydroxyl, or N,N 0 -dimethylamino at the o-, p-or m-positions (2k, 2l or 2m: p-, o-or m-methoxy, respectively; The most promising inhibitor was the m-N,N 0 -dimethylamino derivative (2p), with a K i value of 3.1 nM; however, analogs with an electron-withdrawing substituent (2q: p-carboxyl or 2r: pnitro) displayed relatively low potencies. These results strongly suggested that an electron-donating substituent on the phenyl ring of the P4-moiety was important and favorable to enhance inhibitory activity against 3CL pro . Isosteric replacement around the P4-scaffold in the context of 2j yielded notable differences from the inhibitory activities of 2j. Replacement of phenoxy (2j) with pyridinyloxy (2s) did not hamper the inhibitory potency. Interestingly, the potency was recovered upon phenylamino substitution (2t: IC 50 = 1.5 lM). The chain length between the P4-carbonyl and the phenyl group contributed significantly to the inhibitor potency, as indicated by a decreased in the activity of the analog 2u (IC 50 = 7.5 lM).

A variety of substituent groups were introduced into the P2moiety (3a-d; Table 2 ). Initially, the stereochemistry of P2 and a size-appropriate amino acid residue at the S2-position in 2i were tested. Compound 3a, in which L-leucine was replaced with D-leucine, dramatically reduced the inhibitory activity. Replacement with bulky hydrophobic side chains, such as cyclohexylmethyl (3b) and benzyl (3c) resulted in solubility issue in the enzyme assay, and replacement with polar glutamic acid (3d) did not increase the inhibitory potency. L-leucine, therefore, appeared to provide a suitable stereochemistry and appropriate size for the P2-moiety to fit into the S2-pocket. This result also well correlated with the docking study.

The hydrogen-bonding property of the pyrrolidone structure at the P1-side chain of 1 was studied with an imidazole moiety. As shown in Table 2 , none of these compounds (4a-c) exhibited notable inhibitory activity against SARS-CoV 3CL pro . Thus, the cyclic amide (c-lactam) at the P1 site was crucial for potent inhibitory activity.

The P1 0 -moiety was next examined by varying the 5-substituted thiazoles (5a-i; Table 3 ). The inhibitory activities (K i values) of compounds 5a-i are listed in Table 3 . Analog 5a (K i = 60 nM) showed 15-times lower activity than 2i; however, the other 5-arylated thiazoles (5b-i) generally exhibited very low inhibitory activities compared to 5a, although the K i values of some inhibitors, including 5b, 5g-i, were in the submicromolar range. These studies confirmed that the benzothiazole unit was appropriate as a warhead group for the P1 0 -moiety.

Previously, we demonstrated that the inhibitory activity of a compound containing a thiazole residue for insertion into the S1 0 -pocket was not time-dependent, suggesting a reversible strong binding interaction with the protease. 25, 27 Here, we examined the molecular docking of the potent active compound, 2o, in comparison with the lead compound 1 and a structurally similar ligand, 31 the docking structure of which has been elucidated by x-ray crystallography (PDB ID: 1WOF, K i = 10.7 lM) 31 (Fig. 2) . Several minimization processes were performed using the MMFF94X force field to model the solvation environment surrounding the inhibitor. A molecular simulation was subsequently performed. Figure  2A shows that the 2o moieties P1-P3 (yellow stick) interacted with the same region of the enzyme as the lead 1. Interestingly, the benzothiazole unit occupied the S1 0 -pocket more tightly than the smaller thiazole moiety. The minimized energies for 2o and 1 obtained from the docking study were À43.65 and À37.56 kcal/mol, respectively. 32 Additionally, the 4-N,N 0 -dimethylaminophenoxy acetyl group adopted a unique folding conformation by forming new hydrophobic interactions with a-carbon of Ala 191 at the P4 site, as shown in Figure 2B .

We describe here the design, synthesis and biological evaluation of a series of tripeptide-type SARS-CoV 3CL pro inhibitors in a SAR study. A docking study of the initial lead compound 1 bound to the SARS-CoV 3CL pro provided a better understanding of the inhibitor-active site structure and interactions. These studies led to the development of several potent inhibitors, including 2i, 2k, 2m, 2o, and 2p with IC 50 or K i values in the submicromolar to nanomolar range. Compounds 2i and 2p exhibited the most potent inhibitory activity, with K i values of 4.1 and 3.1 nM, respectively. These results clearly indicated that the peptidomimetics were promising inhibitors for the development of potential therapeutic agents against SARS. The structural requirements of the peptidomimetics displaying potent inhibitory activity against SARS-CoV 3CL pro could be summarized as follows: (i) a benzothiazole unit was effectively accommodated as a chemical warhead by the S1 0pocket; (ii) hydrogen-bonding capabilities at the cyclic lactam at the S1-position; (iii) appropriate stereochemistry and a size-appropriate L-leucine moiety in the S2-hydrophobic pocket and (iv) a unique folding conformation assumed by the phenoxyacetyl moiety at the S4-site.

6. Experimental

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 60F 254 pre-coated plates. Preparative HPLC was performed using a C18 reverse-phase column (19 Â 100 mm; Sun-Fire Prep C18 OBD™, 5 lm) with a binary solvent system: a linear gradient of CH 3 CN in 0.1% aqueous TFA at a flow rate of 6 mL/min, detected at UV 254 and 222 nm. All solvents used for HPLC were HPLC-grade. 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 BRU-KER 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 LCMS-2010EV (SHIMADZU).

6.2. General solid-phase synthetic procedure for the preparation of the dipeptides (9) To the Wang resin (1.0 mmol) in DMF (5 mL) was added Fmocamino acid (3 equiv), DIC (3 equiv) and DMAP (0.25 equiv), and the mixture was stirred for 3 h. The resin was filtered, washed with DMF and CH 2 Cl 2 and dried under vacuum to yield 6. After removal of the Fmoc-group using 20% piperidine in DMF over 20 min, the next appropriate amino acid was coupled to the resin using the coupling agents DIC (3 equiv) and HOBt (3 equiv) by solid-phase synthesis techniques. The resulting intermediate 7 was then treated with 20% piperidine in DMF for 20 min to remove the Fmocgroup and subsequently reacted with the corresponding carboxylic acid (3 equiv) by the DIC-HOBt method or acid chloride (3 equiv) in the presence of Et 3 N to produce 8. Finally, the resin was treated with TFA/H 2 O (10:1, 2 mL), and the mixture was filtered after 1 h. The filtrate was removed under high vacuum to give the desired dipeptides 9, which were used directly in the next step without further purification or analysis. 25 Compound 11 was prepared through sequential reactions from the well-known intermediate 10, as reported previously. 27 To a solution of the acid 11 (5.0 g, 12.0 mmol in DMF, 80 mL) was added EDCÁHCl (1.85 g, 13.44 mmol), HOBt (1.56 g, 13.44 mmol) and N,O-dimethylhydroxylamine (1.31 g, 13.44 mol) at ambient temperature. The solution was cooled to 0°C and TEA (1.87 mL, 13.44 mmol) was added slowly. After 2 h, the DMF was evaporated and the resulting residue was dissolved in ethyl acetate (100 mL). The organic phase was subsequently washed with 5% citric acid (50 mL), 5% NaHCO 3 (50 mL) and brine (50 mL). This organic layer was then dried over Na 2 SO 4 and concentrated under reduced pressure to yield the Weinreb amide derivative 12, which was purified by column chromatography (EtOAc/MeOH = 9.5:0.5).

To a solution of thiazole (1.373 g, 10.0 mmol) in THF at À78°C was added n-BuLi (2.0 mol in THF, 1.67 mL) dropwise over 15 min. After 1 h stirring, the Weinreb amide 12 (0.640 g, 2.0 mmol) in THF was slowly added dropwise over 20 min and the solution was stirred for 3 h. 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 ethyl acetate. This solution was washed with water and brine, and then dried over Na 2 SO 4 . The organic layer was concentrated under reduced pressure and the resulting residue was subjected to flash chromatography (EtOAc/ MeOH = 9:1) to obtain the pure compound 13a.

The data for the compound 12 & 13a has been reported in a previous article. 25 Compounds 13b-d were prepared from 12 according to the procedure described for the synthesis of 13a. 

Compound 13c 25 was prepared from 12 using a procedure similar to that described for the preparation of 13a. The data for the compound 13c has been reported in a previous article. 25 

To a cooled solution of the commercially available 5-phenylthiazole (4.78 mmol) in dry THF at À78°C, a solution of LDA (6.2 mmol) was slowly added. After 1 h, a pre-cooled solution of Weinreb amide 12 in dry THF was slowly added and the reaction was stirred at À78°C for 2 h. The solution was allowed to warm to room temperature, was quenched by the addition of water (35 mL), and was extracted with ethyl acetate (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 = 3:7) to furnish 13e. 55% yield from 12; brown solid; 1 Compounds 13f-j were prepared from 12 according to the procedure described for the synthesis of 13e. To a solution of 16 (1.6 mmol) in THF (20 mL) at 0°C was added tosyl chloride (1.5 equiv). After 10 min, Et 3 N (1.5 equiv) was added slowly and allowed to stir for 3 h at the same condition. The solvent was evaporated and the resulting residue was dissolved in ethyl acetate (50 mL). This organic phase was washed with 5% citric acid (25 mL), 5% NaHCO 3 (25 mL) and brine (25 mL). This layer was then dried over Na 2 SO 4 and concentrated under reduced pressure to yield 17, which was purified by column chromatography (n-hexane/EtOAc = 5:5 

To a solution of 13a (2 mmol) in CH 2 Cl 2 (2 mL) at 0°C was added TFA/H 2 O (10 mL) and the solution was stirred for 1 h. After evaporating the solvent under reduced pressure, the corresponding deprotected lactam residue (3 mmol) was coupled to the dipeptidic 9 (1.1 equiv) using the coupling agent HBTU (1.1 equiv) and HOBt (1.1 equiv) in the presence of diisopropylethylamine (DIPEA, 1.1 equiv) in DMF (3 mL) at 0°C. The reaction mixture was allowed to stir for 2-3 h under ambient conditions. The solvent was then evaporated under high vacuum, and the residue was dissolved in ethyl acetate (50 mL). The organic layer was washed with 5% citric acid (25 mL), 5% NaHCO 3 (25 mL) and brine (25 mL) . This solution was dried over Na 2 SO 4 , filtered and evaporated under reduced pressure to give a compound 2a.

Compounds 2f-u were prepared from 13a-c with 9 using a procedure similar to that described for the synthesis of 2a. Compounds 2a-u were purified by reverse phase HPLC. 25°C. The initial rate measurements were determined as previously described using a fluorescence-based peptide cleavage assay with a commercially available fluorogenic substrate, Dabcyl-KTSAVLQSGFRKME-Edans (Genesis Biotech). 24, 31 The change in fluorescence intensity was monitored in a Cary Eclipse fluorescence spectrophotometer (Varian) with 355 and 538 nm excitation and emission wavelengths, respectively. Substrate was dissolved in deionized water, and inhibitors were dissolved in DMSO. The experiments were performed in a buffer containing10 mM sodium phosphate, 10 mM sodium chloride, 1 mM EDTA, 1 mM TCEP and 2% DMSO (pH 7.4). While varying inhibitor concentrations (0-200 lM), the reaction was initiated by adding protease (final concentration 100 nM) to a solution of substrate at final concentration of 10 lM to a total volume of 120 lL in a microcuvette. The data from these assays were analyzed by using the non-linear regression analysis software Origin 7.

HRMS (ESI): m/z

methoxyphenoxy)acetamido)-3-methylbutanamido

18% yield from 13c

Hz, 1H), 8.11 (d, J = 8.0 Hz, 1H), 7.99 (d, J = 8.5 Hz, 1H), 7.67-7.57 (m, 2H), 7.02 (d, J = 7.2 Hz, 3H), 6.92-6.86 (m, 1H)

14% yield from 13c

Hz, 2H), 6.73 (s, 1H), 5.71 (dd

21% yield from 13c

Hz, 1H), 7.66-7.56 (m, 2H), 6.84 (d, J = 9.1 Hz, 2H), 6.72 (d, J = 9.0 Hz, 2H), 5.72 (dd, J = 3.4, 11.5 Hz, 1H)

18% yield from 13c

m, 12H); 13 C NMR (125 MHz, CD 3 OD

J = 8.0 Hz, 1H), 7.99-7.97 (d, J = 8.0 Hz, 1H), 7.61-7.58 (m, 2H), 7.43-7.40 (m, 1H)

HRMS (ESI): m/z calcd for C 35 H 48 N 6 O 6 S [M+H] + 679.3278, found 679

-yl)amino)-3-methyl-1-oxobutan-2-yl)amino)-2-oxoethoxy)benzoic acid (2q) 19% yield from 13c; white powder

Benzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)-4-methyl-2-((S)-3-methyl-2-(2-(4-nitrophenoxy)acetamido)butanamido)pentanamide (2r) 12% yield from 13c

Hz, 1H), 8.26-8.20 (m, 3H), 8.12 (d, J = 7.6 Hz, 1H), 7.67-7.58 (m, 2H), 7.15 (d, J = 9

Benzo[d]thiazol-2-yl)-1-oxo-3-((S)-2-oxopyrrolidin-3-yl)propan-2-yl)-4-methyl-2-((S)-3-methyl-2-(2-(pyridin-3-yloxy)acetamido)butanamido)pentanamide (2s) 33% yield from 13c

Hz, 1H), 8.21 (d, J = 7.4 Hz, 1H), 8.11 (d, J = 7.0 Hz, 1H), 7.98 (dd, J = 2.8, 8.7 Hz, 1H), 7.79 (dd, J = 5.3, 8.7 Hz, 1H)

HRMS (ESI): m/z calcd for C 32 H 41 N 6 O 6 S [M+H] + 637.2808, found 637

-(phenylamino)acetamido)butanamido)pentanamide (2t) 17% yield from 13c; slightly yellow powder; 1 H NMR (500 MHz, CD 3 OD) d 8.24 (d, J = 7.2 Hz, 1H), 8.20 (d, J = 7

HRMS (ESI)

3-methylbutanamido)-4-methylpentanamide (2u) 22% yield from 13c

-oxopyrrolidin-3-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (3a) 22% yield from 13c; White powder; 1 H NMR (500 MHz, DMSOd 6 ) d 8.21 (d, J = 8.4 Hz, 1H), 8.11 (d

-oxopyrrolidin-3-yl)propan-2-yl)amino)-3-cyclohexyl-1-oxopropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (3b) 26% yield from 13c

HRMS (ESI)

-oxopyrrolidin-3-yl)propan-2-yl)amino)-1-oxo-3-phenylpropan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (3c) 28% yield from 13c; white powder; 1 H NMR (500 MHz, DMSOd 6 ) d 8.27 (d, J = 8.4 Hz, 1H), 8.16 (d

HRMS (ESI): m/z

(benzyloxy)carb onyl)amino)-3-methylbutanamido)-5-oxopentanoicacid (3d) 22% yield from 13c; white powder; 1 H NMR (500 MHz, DMSOd 6 ) d 8.25 (d, J = 8.4 Hz, 1H), 8.23 (d

HRMS (ESI): m/z

1-oxo-1-(thiazol-2-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (4a) 36% yield from 17; colorless solid; 1 H NMR (300 MHz, DMSOd 6 ) d 8.81 (d, J = 9

HRMS (ESI): m/z

phenoxyacetamido)butanamido)pentanamide (4b) 43% yield from 17; colorless solid; 1 H NMR (300 MHz, DMSOd 6 ) d 8.89 (d, J = 4.65 Hz, 1H), 8.70-8.69 (m, 1H), 8.28 (br s, 1H), 8.15 (t, J = 6

HRMS (ESI): m/z

1-oxo-1-(thiazol-2-yl)propan-2-yl)amino)-4-methyl-1-oxopentan-2-yl)amino)-3-methyl-1-oxobutan-2-yl)carbamate (4c) 38% yield from 17

HRMS (ESI): m/z

(2S)-1-oxo-3-(2-oxopyrrolidin-3-yl)-1-(5-phenylthiazol-2-yl)propan-2-yl)amino)pentan-2-yl)amino)-1-oxobutan-2-yl)carbamate (5a) 33% yield from 13e

36-3.29 (m, 2H), 2.61-2.55 (m, 2H)

-phenylthiazol-2-yl)propan-2-yl)pentanamide (5b) 45% yield from 13e; colorless solid; 1 H NMR (400 MHz

HRMS (ESI)

p-tolyl)thiazol-2-yl)propan-2-yl)pentanamide (5c) 47% yield from 13f; colorless solid; 1 H NMR (400 MHz, CDCl 3 ) d 8.12 (s, 1H

(2S)-2-((S)-2-(2-(4-Methoxyphenoxy)acetamido)-3-methylbutanamido)-N-((2S)-1-(5-(4-methoxyphenyl)thiazol-2-yl)-1-oxo-3-(2-oxopyrrolidin-3-yl)propan-2-yl)-4-methylpentanamide (5d) 40% yield from 13g; yellow solid; 1 H NMR (400 MHz, CDCl 3 ) d 8.09 (s, 1H

-Chlorophenyl)thiazol-2-yl)-1-oxo-3-(2-oxopyrrolidin-3-yl)propan-2-yl)-2-((S)-2-(2-(4-methoxyphenoxy)acetamido)-3-methylbutanamido

67 (s, 3H), 3.30-3.19 (m, 2H

-Methoxyphenoxy)acetamido)-3-methylbutanamido)-4-methyl-N-((2S)-1-(5-(naphthalen-2-yl)thiazol-2-yl)-1-oxo-3-(2-oxopyrrolidin-3-yl)propan-2-yl)pentanamide (5f) 42% yield from 13i

1H), 7.91-7.87 (m, 3H), 7.71-7.69 (t, J = 8.4 Hz, 1H), 7.60-7.53 (m, 1H), 7.36-7.33 (m, 1H), 6.92-6.83 (m, 4H), 7.70-5.67 (m, 1H

-methoxyphenyl)thiazol-2-yl)-1-oxo-3-(2-oxopyrrolidin-3-yl)propan-2-yl)-4-methylpentanamide (5g) 39% yield from 13j; colorless solid; 1 H NMR (400 MHz, CDCl 3 ) d 8.14 (s, 1H), 8.08 (br s, 1H, NH

Dimethylamino)phenoxy)acetamido)-3-methylbutanamido)-4-methyl-N-((2S)-1-oxo-3-(2-oxopyrrolidin-3-yl)-1-(5-phenylthiazol-2-yl)propan-2-yl)pentanamide (5h) 36% yield from 13e

45 (s, 1H), 6.38-6.36 (d, J = 8.0 Hz, 1H), 5.68-5.63 (m, 1H

Dimethylamino)phenoxy)acetamido)-3-methylbutanamido)-4-methyl-N-((2S)-1-(5-methylthiazol-2-yl)-1-oxo-3-(2-oxopyrrolidin-3-yl)propan-2-yl)pentanamide (5i) 35% yield from 13d

Communicable Disease Surveillance & Response

The values quoted as minimized docking energies are non-bonded energy between the protease and inhibitors

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) 23Á01104 and a Grant-in-aid for Scientific Research 23659059.