key: cord-0784642-ew6kqsfr authors: Sun, Yuanpei; Zhang, Ning; Wang, Jian; Guo, Yu; Sun, Bo; Liu, Wei; Zhou, Honggang; Yang, Cheng title: Synthesis and Biological Evaluation of Quinolinone Compounds as SARS CoV 3CL(pro) Inhibitors date: 2013-07-19 journal: Chin J Chem DOI: 10.1002/cjoc.201300392 sha: 5b9e3206b85267b146c128de277dae479c0dd22e doc_id: 784642 cord_uid: ew6kqsfr SARS CoV 3CL(pro) is known to be a promising target for development of therapeutic agents against the severe acute respiratory syndrome (SARS). A quinolinone compound 1 was selected via virtual screening, and it was synthetized and tested for enzymatic inhibition in vitro. Compound 1 showed potent inhibitory activity (IC(50)=0.44 µmol/L) toward SARS CoV 3CL(pro). Further work on a series of quinolinone derivatives resulted in the discovery of the most potent compound 23, inhibiting SARS CoV 3CL(pro) with an IC(50) of 36.86 nmol/L. The structure‐activity relationships were also discussed. Severe acute respiratory syndrome (SARS), caused by a novel coronavirus (SARS CoV), is a highly infective upper respiratory tract disease. [1] [2] [3] [4] Due to the high mortality rate (up to 10%) SARS quickly became a global threat in 2003. [5, 6] Although about a year later from its first discovery the initial outbreak of the virus was stymied, the reemergence of SARS is quite possible and the development of new anti-SARS drugs is particularly important. [7, 8] The SARS CoV is a positive-sense single-stranded RNA virus. Its genome is comprised of about 29700 nucleotides, most of them (over 21000 nucleotides) belongs to the replicase gene, which encodes two overlapping polyproteins, pp1a (486 kDa) and pp1ab (790 kDa). The SARS CoV 3CL pro , a ~33 kDa cysteine protease, releases functional polypeptides from pp1a and pp1ab. Without these polypeptides the viral replication and transcription will be disabled. As the integral role of SARS CoV 3CL pro in SARS CoV lifecycle, it is an ideal target for drug discovery. [9] [10] [11] [12] Previously, a large number of SARS CoV 3CL pro inhibitors have been reported and their scaffolds are diverse. Yang et al. [6] developed a potent SARS CoV 3CL protease inhibitor (TG-0205221, Ki＝50 nmol/L, Figure 1 ). Sydnes et al. [9] synthetized a series of glutamic acid and glutamine peptides possessing a trifluoromethyl ketone group with effective inhibitory activity. Zhou et al. [11] have reported 1-alkylisatin-5-carboxamide (IC 50 ＝0.37 μmol/L, Figure 1 ) and its analogues to be highly potent SARS CoV 3CL pro inhibitors. We identified novel SARS CoV 3CL pro inhibitors among a database containing more than 600000 compounds by virtual screening. Compound 1 (Figure 2) was selected, synthetized and tested against SARS CoV 3CL pro in vitro. 1 showed potent inhibitory activity (IC 50 ＝0.44 μmol/L) toward SARS CoV 3CL pro . 23 compounds were synthesized and tested against SARS CoV 3CL pro in vitro. Biological evaluation results of the title compounds are listed in Table 1 . As shown in Table 1 , replacement of the thiophene moiety (compound 1) with benzene ring (compound 2) leads to the decrease of the inhibitory activity, especially when the benzene ring was substituted. It is worth , it was about 9-fold higher potency than compound 11. The nitrogen, oxygen, and sulfur atoms are colored blue, red, and orange, respectively. Hydrogen bonds are displayed as blue dashed lines. Figure 3 showed docking model of compounds 1 and 23, from which we can conclude that compound 1 binds competitively and strongly to the active site, and the quinolinone portion was recognized by S1 pocket. Moreover, the thiophene moiety extends into S1' site with hydrophobic interactions. The S1' pocket is not quite large, this may explain why compounds 14 and 15 , with long carbon chain between the aromatic ring and ester carbonyl, show lower potency than compound 2. Unlike compound 1, methylation of the N-H of the quinolinone brings a different binding interaction, the thiophene moiety of compound 23 occupies S2 pocket, and the methyl group fills into the crack of S1 pocket closely. As S1' pocket is much nearer the surface of the protein than S2 pocket, the new binding model makes compound 23 bind more tightly than compound 1 with the protein. This may partly explain why compound 23 showed more potent inhibitory effect compared with compound 1. Molecular insight into the two different binding modes of compound 1 and 23 was shown in Figure 4 , the oxygen at C-2 of quinolinone moiety of compound 1 makes a H-bond with the NH of His 163 (1.78 Å), and the NH of quinolinone forms a H-bond with Glu 166 (2.26 Å). The ester carbonyl oxygen forms a hydrogen bond with the NH of Gly 143 (2.26 Å). To compound 23, the hydro- gen bond of the amide carbonyl oxygen with the NH of His 163 (2.36 Å) was also conserved as well. But there is no hydrogen bond with Glu 166, because of the methylation. The ester carbonyl oxygen of compound 23 makes hydrogen bonds with the active site residues, His 41 (3.36 Å) and Cys 145 (2.23 Å) . This is the most difference between the two binding modes. The active site of SARS CoV 3CL pro contains a catalytic dyad constituted by Cys 145 and His 41, which functions as the common nucleophile in the proteolytic process. [11] That means that although the methylation disturbed the hydrogen bond of N-H of quinolinone, the distance between ester carbonyl oxygen and the S-H group of Cys 145 and N-H group of His 41 might be changed favorably resulting in dramatic inhibitory potency. In addition, the carbonyl of compound 23 may potentially react with the Cys 145 residue. This suppose remains to be further studied. In conclusion, twenty-three quinolinone compounds were synthesized and tested in vitro as inhibitors against SARS CoV 3CL pro . Compound 1, selected via virtual screening, was proved to have a potent inhibitory activity (IC 50 ＝ 0.44 μmol/L). Following optimization of 4-quinolinone ester derivatives resulted in the discovery of compound 23 with the most powerful potency (IC 50 ＝36.86 nmol/L). The present work demonstrated that 4-quinolinone ester analogs could be used as new leads for future Anti-SARS drugs discovery. Further structural optimization and in vivo activities about quinolinone compounds are well under way. Unless otherwise noted, all chemicals and solvents were commercially available and treated with standard methods before use. Melting points were determined using an SGW-X4B digital melting point apparatus and uncorrected. SHANGHAI SANPONT GF254 plates were used as analytical TLC. 1 H and 13 C NMR spectra were recorded on a 400 MHz Bruker Avance DPX spectrometers and using DMSO as solvent. All 13 C NMR spectra were recorded proton-decoupled. Chemical shifts for 1 H and 13 C spectra are quoted in ppm downfield from TMS. Coupling constants are referred to as J values in hertz. ESI mass spectra were acquired using a Bruker ESQUIRELCTM ESI ion trap spectrometer. FT-IR spectra were recorded at room temperature in the region of 4000-400 cm −1 with a Perkin-Elmer spectrum 65 FT-IR spectrometer using KBr pellets. Elemental analyses of carbon, hydrgen and nitrogen were obtained with an Elementar Vario MICRO cube Elemental Analyser. To a suspension of 4-hydroxy-2-quinolinone or Compound 5 was prepared according to general procedure by using 4-hydroxy-2-quinolinone and 4-ethylbenzoyl chloride, obtained a white solid in 96% yield. m.p. 233 -234 ℃. 1 Compound 6 was prepared according to general procedure by using 4-hydroxy-2-quinolinone and 4-tert-butylbenzoyl chloride, obtained a white solid in 95% yield. m.p. 263-264 ℃. 1 4-(6-Chloronicotinoyl)oxy-quinol-2-one (9) Compound 9 was prepared according to general procedure by using 4-hydroxy-2-quinolinone and 6-chloronicotinoyl chlorid, obtained a white solid in 97% yield. The sequence of SARS-CoV 3CL pro cloned into the pGEX-6p-1 vector was transformed into E. coli BL21 (DE3) cells. The recombinant protein with GST-tag was purified by GST-glutathione affinity chromatography and the ion exchange column. Eventually purified protein was of high purity (＞95%) as judged by SDS-PAGE analysis and the concentration is 0.5 μmol/L, and the buffer is 50 mmol/L Tris-HCl, pH 7.3, 1 mmol/L EDTA. The substrate synthesized in Shanghai Biological Engineering Company is dissolved in DMSO, with 0.8 mmol/L liquid storage for used. The SARS-CoV 3CL pro inhibition assays were conducted via fluorescence resonance energy transfer (FRET). The natural substrate amino acid sequence (AVLQ. SGFRKK) of SARS-CoV 3CL pro ends with MCA fluorescent group and Dnp fluorescence quenching group. The system of screening is as follows ( Table 2 ): The settled concentrations of proteins, compounds and substrate were preheated at 37 ℃ and oscillated. Excitation/emission light is 320/405 nm, test was carried out every 3 s for 60 times. Drawing curves, the maximum value of the negative control curve slope is V 0 , and the largest compound curve slope is V 1 . The inhibition ratio can be defined (1−V 1 /V 0 ). And the IC 50 value was calculated by the following equation: The crystal structure of SARS-CoV 3CL pro (code: 3SN8), obtained from The Protein Data Bank as a complex bound with inhibitors, was used in this docking study. And the docking studies were carried out in the Discovery Studio 3.0. To prepare the protein for docking, water molecules and inhibitors were removed and hydrogen atoms were added by using Prepare Protein Structure module, from the protein. (Cinnamoyl)oxy-quinol-2-one (16) Compound 16 was prepared according to general procedure by using 4-hydroxy-2-quinolinone and cinnamoyl chloride 93 (s, 1H); 13 C NMR (100 MHz 2-Oxo-1,2-dihydroquinolin-4-yl phenyl carbonate (17) Compound 17 was prepared according to general procedure by using 4-hydroxy-2-quinolinone and phenyl chloroformate, obtained a white solid in 91% yield Benzyl 2-oxo-1,2-dihydroquinolin-4-yl carbonate (18) Compound 18 was prepared according to general procedure by using 4-hydroxy-2-quinolinone and benzyl chloroformate, obtained a white solid in 92% yield (Propionyl)oxy-quinol-2-one (19) Compound 19 was prepared according to general procedure by using 4-hydroxy-2-quinolinone and propionyl chloride, obtained a white solid in 92% yield IR (KBr) ν: 3436, 1761, 1674, 1134, 752 cm −1 ; ESI-MS m/z: 218.02 ([M＋H ＋ ]). Anal. calcd for C 12 H 11 NO 3 : C 66.35, H 5.10, N 6.45; found C 66.54, H 5.32, N 6.74. Methyl 2-oxo-1,2-dihydroquinolin-4-yl succinate (20) Compound 20 was prepared according to general procedure by using 4-hydroxy-2-quinolinone and methyl 4-chloro-4-oxobutanoate Isobutyl 2-oxo-1,2-dihydroquinolin-4-yl carbonate (21) Compound 21 was prepared according to general procedure by using 4-hydroxy-2-quinolinone and isobutyl chloroformate, obtained a white solid in 94% yield cm −1 ; ESI-MS m/z: 262.33 ([M＋H ＋ ]). Anal. calcd for C 14 H 15 NO 4 : C 64.36, H 5.79, N 5.36; found C 64.12, H 5.89, N 5.08. 1-Methyl-4-(3-methoxybenzoyl)oxy-quinol-2-one (22) Compound 22 was prepared according to general procedure by using4-hydroxy-1-methyl-2-quinolinone and 3-methoxybenzol chloride This work was supported by Tianjin SME Technology Innovation Fund (No. 11ZXCXSY03500) and Na- tional Biomedical Special Project of International Innovation Park (No. 11ZCKFSY06800).