key: cord-0864362-pgzogpa2
authors: Shao, Yi-Ming; Yang, Wen-Bin; Kuo, Tun-Hsun; Tsai, Keng-Chang; Lin, Chun-Hung; Yang, An-Suei; Liang, Po-Huang; Wong, Chi-Huey
title: Design, synthesis, and evaluation of trifluoromethyl ketones as inhibitors of SARS-CoV 3CL protease
date: 2008-04-15
journal: Bioorg Med Chem
DOI: 10.1016/j.bmc.2008.02.040
sha: c7f00c1af00cd27223dd8cb5020788e4e06e48cc
doc_id: 864362
cord_uid: pgzogpa2

A series of trifluoromethyl ketones as SARS-CoV 3CL protease inhibitors was developed. The inhibitors were synthesized in four steps from commercially available compounds. Three different amino acids were explored in the P1-position and in the P2–P4 positions varying amino acids and long alkyl chain were incorporated. All inhibitors were evaluated in an in vitro assay using purified enzyme and fluorogenic substrate peptide. One of the inhibitors showed a time-dependent inhibition, with a K(i) value of 0.3 μM after 4 h incubation.

Severe acute respiratory syndrome-associated coronavirus (SARS-CoV), identified to be the causative agent of this life-threatening epidemic, [1] [2] [3] [4] [5] leads to a respiratory disease with the symptoms including cough, high fever, chills, rigor, myalgia, headache, dizziness, and progressive radiographic changes of the chest and lymphopenia. The spread of this contagious disease in 2003 infected more than 8000 people with a high mortality. In total, there were 774 deaths reported around the world. During the life cycle of SARS-CoV, 3CL protease cleaves the polyprotein into individual polypeptides to provide all the essential proteins for viral replication and transcription. 6, 7 This enzyme is thus recognized as a primary target for the therapeutic intervention.

In contrast to the common serine proteases containing a Ser-His-Asp catalytic triad, SARS-CoV 3CL protease has a Cys-His catalytic dyad (Cys-145 and His-41), which is similar to porcine transmissible gastroenteritis virus main protease (Cys-144 and His-41) and human coronavirus 229E main protease (Cys-144 and His-41). 8 In addition, it cleaves the replicase polyprotein at no less than 11 conserved sites with canonical Leu-Gln#(Ser, Ala, Gly) sequences. 9 Taken together, this information provides good understanding to the design of potent inhibitors.

To date, a number of 3CL protease inhibitors have been prepared, including C 2 -symmetric diols, 10 bifunctional aryl boronic acids, 11 keto-glutamine analogs, 12 isatin derivatives, 13 a,b-unsaturated esters, 14 anilide, 15 and benzotriazole. 16 Here, we report the synthesis of trifluoromethyl ketones as inhibitors against SARS-CoV 3CL protease, and provide kinetic analysis and computer modeling to address the issue of covalent binding.

Trifluoromethyl ketones (TFMKs) are well known as the inhibitors of serine 17 and cysteine 18 proteases. Owing to the high electronegativity of fluorine, the carbonyl carbon of TFMK is a highly active electrophile. It is generally believed that hemiketal or hemithioketal is formed by the nucleophilic attack of the hydroxyl or thiol group at the active site when TFMKs are employed as the inhibitors against serine or cysteine proteases, respectively. Previous studies 19 indicated that TFMKs demonstrate a competitive slow, tight-binding inhibition against human leukocyte elastase. Recently, Zhang et al. 20a described N,N-dimethyl glutaminyl fluoromethyl ketones as 3CL protease inhibitors. One of these compounds was found to have low toxicity in mice, and another one was found to have an EC 50 value of 2.5 lM based on the cytopathic effect (CPE) inhibition assay. However, the in vitro inhibition has not been characterized in detail. Sydnes et al. 20b also reported the synthesis of glutamic acid and glutamine peptides with a CF 3 -ketone unit as 3CL protease inhibitors.

In order for the synthetic simplicity, we assumed that the benzyl group as the P1 site can mimic the Gln residue of the substrate. Scheme 1 shows the four-step synthesis of various N-protected trifluoromethyl ketones. The preparation of nitro alcohols 3 was carried out by C-C bond formation between nitroalkanes 2 and trifluoroacetaldehyde ethyl hemiacetal under the basic condition of catalytic potassium carbonate. The choice of nitroalkanes defines the P 1 group of the final inhibitor. For instance, 1-nitro-2-phenylethane 2a introduces a benzyl group at the P1 site. Subsequent reduction to amine alcohols was performed either by PtO 2 -or Raney nickel-catalyzed hydrogenation. The use of PtO 2 was avoided in the reduction of 3a because undesired saturation of the phenyl ring was observed. At this stage, the trifluoroamine alcohols were coupled with N-protected amino acids or long-chain acids by using HBTU and DIEA (or Et 3 N) to afford 4a-g. Final oxidation using Dess-Martin reagent generated the desired trifluoromethyl ketones 5a-h.

TFMKs 5a-h were evaluated to interfere with SARS-CoV 3CL protease activity according to the reported procedure 21 (Table 1 ). The activity of 5a, 5b, 5f, 5g, and 5h, having benzyl group as the side chain at the P1 site, supports the idea that the P2-P4 sites still have a significant contribution to the binding affinity though they are far from the active site. The best inhibitor 5h, containing the same residues as the reported substrate sequence at the P2, P3, and P4 sites, displayed a compet-

5a R = Bn, X = Cbz-Leu (86%) 5b R = Bn, X = Cbz-Phe (87%) 5c R = Me, X = Boc-Leu (86%) 5d R = H, X = Boc-γGlu(OtBu)-Ala (14%) 5e R = H, X = γGlu-Ala 5f R = Bn, X = CH 3 (CH 2 ) 8 CO-Leu (85%) 5g R = Bn, X = CH 3 (CH 2 ) 7 CO-Leu (70%) 5h R = Bn, X = Cbz-Ala-Val-Leu (67%) Bn CH 3 (CH 2 ) 8 CO-Leu 50 5g

Bn CH 3 (CH 2 ) 7 CO-Leu >50 5h

Bn Cbz-Ala-Val-Leu 10 itive inhibition against 3CL protease ( Fig. 1) . Moreover, in consistence with the previous reports of cathepsin B and human leukocyte elastase, 18b,19 prolonged incubation of 3CL protease with 5h exhibited a time-dependent decrease in enzyme activity as a function of the inhibitor concentration. The inhibitor was found to produce progressive tightening of inhibition, as shown by a 30-fold decrease in the K i value (from 8.8 to 0.3 lM) in 4 hr ( Table 2 and Fig. 2 ). As indicated by the NMR studies, the trifluoromethyl ketone moiety exists as an equilibrium mixture of ketone and hydrate forms. The timedependent tightening of inhibition is likely due to the slow formation of a covalent adduct through the nucleophilic attack of the thiol group on the carbonyl carbon.

Compound 5h and 3CL protease complex have been crystallized in our laboratory, but the X-ray crystallography experiments were nevertheless unsuccessful in structural refinement due to fragmented electron density maps. Alternatively, computational molecular modeling was used to construct a model for the acyl-enzyme complex. On the basis of the crystal structure of 3CL protease with a chloromethyl ketone (CMK) inhibitor, the analog of trifluoromethyl ketone, determined by Yang et al., 7 we first constructed the models for the four possible stereoisomers of the covalent adducts between the protein and compound 5h. All the models were constrained with a covalent link between the thiol group of Cys-145 and compound 5h, in consistent with the analog experimental complex structure by Yang et al. 7 In comparison with the analog experimental structure, only the (S,S,S,S) isomer of compound 5h with the R configuration of carbonyl carbon adjacent to CF 3 group agreed with the binding mode of the CMK inhibitor, in particular all four amino acid side chains of compound 5h fitted into the bind pockets of the 3CL protease active site. All the other three stereoisomers were ruled out because all these molecules were unable to bind to the active site under the covalent constraint. The computational model of the (S,S,S,S) isomer is different from the binding mode of the CMK-3CL protease complex structure in that the P1, P2, P4 side chains in compound 5h occupied S2, S1, and S4 sites, respectively, in 3CL protease (Fig. 3) . The binding mode discrepancies were expected consequences due to the difference between the amino acid side chains of the two inhibitor analogs. The proposed detailed covalent attacking mechanism was shown in Figure 4 . into a mixture of substrate (6 lM) and inhibitor 5h. Over the entire 120 min time window, the uninhibited enzyme displayed a linear progress curve, whereas the inhibited enzyme with a different concentration of inhibitor showed a time-dependent reduction of activity.

The substrate-based design and synthesis of trifluoromethyl ketones as SARS-CoV 3CL protease inhibitors have been reported. The most potent inhibitor 5h, which possesses the same moiety as the substrate on P1-P4 site, supported the covalent binding. Also, the time-dependent inhibition displayed by inhibitor 5h advanced our understanding of the interactions between the cysteine protease and the electrophilic compound, thereby furthering the discovery of cysteine protease inhibitors.

All reactions with air-and moisture-sensitive materials were performed in oven-dried glassware fitted with rub-ber septa or three-way T taps under a positive pressure of argon or nitrogen. Air-and moisture-sensitive liquids and solutions were transferred via syringe. Organic solutions were concentrated by rotary evaporation at 23-80°C (water-bath temperature). Column chromatography was performed employing Merck silica gel (60 Å pore size, 70-230 mesh ASTM). Analytical thin-layer chromatography (TLC) was performed using glass plates pre-coated with Merck silica gel (60 Å pore size) impregnated with a fluorescent indicator (254 nm). TLC plates were visualized by I 2 vapors, UV lamp, phosphomolybdic acid solution in ethanol, or 0.5% ninhydrin in ethanol followed by brief heating on a hot plate. Commercial solvents and reagents were used as received without further purification. They were purchased from Aldrich, ACROS, BACHEM, or other commercial sources. Compounds are characterized by nuclear magnetic resonance spectroscopy and high resolution mass spectroscopy. Proton nuclear magnetic resonance ( 1 H NMR) spectra and carbon nuclear magnetic resonance ( 

As described, 21, 22 the inhibitory effects of each compound on the enzymatic activities of 3CL protease were evaluated using purified enzyme and fluorogenic substrate peptide. The kinetic measurements were performed in 20 mM Bis-Tris (pH 7.0) at 25°C. The initial velocities of the inhibited reactions of 50 nM 3CL protease and 6 lM fluorogenic substrate were plotted against the different inhibitor concentrations to obtain the IC 50 by fitting with Eq. 1. K i measurement was performed at two fixed inhibitor concentrations of 1 · IC 50 and 2· IC 50 . Substrate concentrations ranged from 8 to 40 lM in a reaction mixture containing 50 nM 3CL protease. Lineweaver-Burk plots of kinetic data were fitted with the computer program KinetAsyst II (IntelliKinetics, State College, PA) by nonlinear regression to obtain the K i values of competitive inhibitors using Eq. 2

AðIÞ ¼ Að0Þ Â f1 À ½½I=ð½I þ IC 50 Þg ð1Þ

In Eq. 1, A(I) is the enzyme activity with inhibitor con- To a stirred solution of the above amine (385.9 mg, 1.76 mmol) and Cbz-Leu-OH (492 mg, 1.76 mmol) in dry DMF (15 mL) were added HBTU (1720 mg, 4.40 mmol) and Et 3 N (1227 lL, 8.80 mmol). The reaction mixture was stirred under N 2 at 23°C for 36 h. DMF was evaporated under reduced pressure, and the resulting brown oil was diluted with CH 2 Cl 2 and washed with 1 N HCl. The water layer was separated and extracted with CH 2 Cl 2 for three times. The organic layers were combined and washed with H 2 O for two times, dried over Na 2 SO 4 , filtered, and concentrated under reduced pressure. The residue was then purified by SiO 2 column chromatography (20% ! 25% EtOAc-hexanes) to give 4a as a yellow solid (574 mg, 70%). R f = 0.26 (hexanes/EtOAc 2:1); 1 H NMR (500 MHz, CD 3 OD): d = 7.34-7.18 (m, 10H; ArH), 5.14-5.05 (m, 2H; PhCH 2 O), 4.42-4.31 (m, 1H; CH(OH)CF 3 ), 4.14-3.89 (m, 2H; 2· CH a ), 3.12-2.77 (m, 2H; CH 2b(Phe) ), 1.63-1.00 (m, 3H; CH 2b(Leu) + CH c(Leu) ), 0.91-0.76 (m, 6H; 2· CH 3d(Leu) ); 13 

Compound 4b was prepared in a similar way to compound 4a, except Cbz-Phe-OH was used here in place of Cbz-Leu-OH. Compound 4b was isolated as a white solid (1240.7 mg, 66%). 175.8, 174.0, 173.2, 158.5, 139.0, 138.1, 130.5, 129.6,  129.5, 129.1, 128.9, 127.9, 67.8, 60.3, 53.1, 51.9 

]-4-phenyl-1,1,1trifluorobutan-2-one (5a). To a solution of 4a (57.6 mg, 0.12 mmol) in dry CH 2 Cl 2 (5 mL) was added the Dess-Martin reagent (15 wt% soln. in CH 2 Cl 2 , 769 lL, 0.37 mmol). TFA (28 lL, 0.37 mmol) was added and then the reaction mixture was stirred at 22°C for 3 h. The reaction was concentrated under reduced pressure and the remaining residue was treated with a mixture of EtOAc and saturated aqueous solutions of NaHCO 3 . The water layer was extracted with EtOAc, washed with brine, dried (Na 2 SO 4 ), filtered, and concentrated under vacuum. Purification (twice) by SiO 2 column chromatography (1st: 25% EtOAc-hexanes; 2nd: 10% EtOAc-CH 2 Cl 2 ) afforded 5a as a white solid (49.6 mg, 86%). 4.3.16. 3-(N-L L-cGlu-L L-Ala)-1,1,1-trifluoropropan-2-one (5e). Compound 5d (7.2 mg) was dissolved in TFA (5 mL) and stirred for 40.5 h. TFA was evaporated under reduced pressure to give 5e (TFA salt) as a white solid. 1 8 (C@O)-L L-Leu]}-4-phenyl-1,1, 1-trifluorobutan-2-one (5f). Compound 5f was prepared in a similar way to compound 5a, except the starting material used was 4e (85% yield). R f = 0.41 (hexanes/EtOAc 2:1); 1 H NMR (400 MHz, CDCl 3 ): d = 7.33-7.14 (m, 5H; ArH), 6.38-5.27 (m, 2H; 2· NH), 5.10-4.13 (m, 2H; 2· CH a ), 3.51-2.70 (m, 2H; CH 2b(Phe) ), 2.18-2.07 (m, 2H; CH 2b(Leu) ), 1.97-0.69 (m, 26H; CH 3 (CH 2 ) 8 C(@O)NH + CH c(Leu) + 2· CH 3d(Leu) ); 13 7 (C@O)-L L-Leu]}-4-phenyl-1,1, 1-trifluorobutan-2-one (5g). Compound 5g was prepared in a similar way to compound 5a, except the starting material used was 4f (70% yield). R f = 0.26 (hexanes/ EtOAc 2:1); 1 H NMR (400 MHz, CDCl 3 ): d = 7.34-7.14 (m, 5H; ArH), 7.10-5.65 (m, 2H; 2· NH), 5.36-4.95 (m, 1H; CH a ), 4.48-4.11 (m, 1H; CH a ), 3.30-2.79 (m, 2H; CH 2b(Phe) ), 2.20-2.07 (m, 2H; CH 2b(Leu) ), 1.74-0.71 (m, 24H; CH 3 (CH 2 ) 7 C(@O)NH + CH c(Leu) + 2· CH 3d(Leu) ); 13 

Benzyloxycarbonyl-L L-Ala-L L-Val-L L-Leu]}-4-phenyl-1,1,1-trifluorobutan-2-one (5h). Compound 5h was prepared in a similar way to compound 5a, except the starting material used was 4g (67% yield). 

The crystal structure of SARS-CoV 3CL protease in complex with a substrate-analog inhibitor (coded 1uk4) was obtained from The Protein Data Bank (PDB; http://www.rcsb.org/pdb/). We constructed four stereomeric compound 5h complex as hemithioketal (DISCOVERY STUDIO 1.7) to determine which isomer can form the protein-inhibitor adduct. GOLD 3.2 23,24 was used for the flexible docking of compound 5h into the enzyme to explore the wide range of its conformational flexibility. The atoms of the enzyme and compound 5h were assigned with Kollmann all-atom charges 25 with SYBYL 7.3 program. 26 To distinguish the four possible stereoisomers of enzyme-inhibitor complex, the carbonyl carbon adjacent to the CF 3 group of compound 5h was constrained to form a covalent bonding with the sulfur atom of Cys-145. Initial 1000 independent genetic algorithm running cycles were carried out with inhibitor torsion angles varying between À180 and 180 degree. The search efficiency was set up at 200% to ensure the most exhaustive search for docking conformation space. The docking processes were carried out in a 40-CPU (Intel Xeon(TM) CPU 3.00 GHz) Linux cluster. For each stereoisomer conformation, the resultant enzyme-inhibitor complex structures were ranked with the CHEMSCORE scoring 27 function to determine the top 10 hits.

SARS study group Lancet

Proc. Natl. Acad

This work is supported by National Science Council, Taiwan and Genomics Research Center, Academia Sinica. The SYBYL computation was conducted at the National Center for High Performance Computing, Taiwan. The DISCOVERY STUDIO 1.7 computation was conducted at the computational center of Academia Sinica.