key: cord-0834080-3mqoexcr
authors: Liu, Pei; Liu, Hongbo; Sun, Qi; Liang, Hao; Li, Chunmei; Deng, Xiaobing; Liu, Ying; Lai, Luhua
title: Potent inhibitors of SARS-CoV-2 3C-like protease derived from N-substituted isatin compounds
date: 2020-08-01
journal: Eur J Med Chem
DOI: 10.1016/j.ejmech.2020.112702
sha: e672db0dbc349b3c1a450d0643d5359ba20a0ea3
doc_id: 834080
cord_uid: 3mqoexcr

SARS-CoV-2 3C-like protease is the main protease of SARS-CoV-2 and has been considered as one of the key targets for drug discovery against COVID-19. We identified several N-substituted isatin compounds as potent SARS-CoV-2 3C-like protease inhibitors. The three most potent compounds inhibit SARS-CoV-2 3C-like protease with IC(50)’s of 45 nM, 47 nM and 53 nM, respectively. Our study indicates that N-substituted isatin compounds have the potential to be developed as broad-spectrum anti-coronavirus drugs.

The coronavirus infectious disease 2019 (COVID-19) is a newly emerged infectious disease caused by a novel coronavirus, SARS-CoV-2. [1, 2] COVID-19 has been recognized as a global threat as it rapidly spreads and breaks out in many countries, causing significant health and economic impact. The SARS-CoV-2 is a positive-strand RNA virus that uses a complex set of enzymes to replicate its RNA genome. [3, 4] Among these, the 3C-like protease (3CL pro ), also known as the main protease (M pro ), is essential for processing the viral polyproteins that are translated from the viral RNA. [5] The active site of 3CL pro contains Cys145 and His41 to constitute a catalytic dyad, in which cysteine functions as the common nucleophile in the proteolytic process. [6, 7] The catalytic domain of 3CL pro in CoVs is highly conserved. [8] Hence, 3CL pro has been considered as an attractive drug target for broad-spectrum anticoronavirus therapy. [4] A variety of 3CL pro inhibitors have been reported in the literature over the past decade. [4, [9] [10] [11] [12] To date, several potential SARS-CoV-2 3CL pro inhibitors have been reported from compound library screening, [8] rational design [8, 13] , [14] and testing of ingredients from traditional Chinese medicine. [15, 16] The chemical structures of the experimentally identified SARS-CoV-2 3CL pro inhibi-tors are diverse, including α-ketoamide analogues [13] , peptidomimetics compounds [8, 14] , baicalein and its derivatives [15, 16] and several repurposed approved drugs and drug candidates [8] . However, only a few candidates have high inhibition activity against SARS-CoV-2 3CL pro and no effective therapy has been developed so far.

Previously, we reported a series of N-substituted 5-carboxamide-isatin compounds as inhibitors of SARS CoV 3CL pro . [12] The best compound showed a sub-micromolar IC 50 against SARS-CoV 3CL pro . [12] Apparently, the isatin scaffold with derivatization may also provide a good starting point for SARS CoV-2 3CL pro inhibitor development, because the two proteases share high sequence identity and the same active site. In order to verify whether isatin compounds can inhibit SARS-CoV-2 3CL pro , we selected a series of isatin compounds from an in-house synthetic compound library, synthesized a few new compounds, tested their inhibitory effects against SARS-CoV-2 3CL pro , and analyzed their structure-activity relationship (SAR).

The synthetic route used to prepare the test compounds 1-28 is shown in Scheme 1. Compound 26 was resynthesized by a simple and effective synthetic route within three steps. First, 2-((4carbamoylphenyl)amino)-2-oxoacetimidic acid (I-26) was obtained by reaction of 4-aminobenzamide, hydroxylamine hydrochloride, and chloral hydrate. Then, I-26 was converted into 2,3-dioxoindoline-5carboxamide (II-26) by treatment with concentrated sulfuric acid at 90 °C. Last, 26 was obtained by reaction of 2,3-dioxoindoline-5-carboxamide (II-26) with 2-(bromomethyl)naphthalene.

The synthetic route used to prepare compound 29 is shown in Scheme 2, which is different from other compounds. We started from 2-(4-aminophenyl)acetic acid using the general Sandmeyer synthetic route. [12, 17] After got 2-(2,3-dioxoindolin-5-yl)acetic acid (II-29), additional carboxyl protection to change to methyl 2-(2,3-dioxoindolin-5-yl)acetate (III-29) with methyl group, followed by the introduction of naphthyl group at N1 position was subjected to afford methyl 2-(1-(naphthalen-2-ylmethyl)-2,3dioxoindolin-5-yl)acetate (IV-29) and de-protection step to afford 2-(1-(naphthalen-2-ylmethyl)-2,3dioxoindolin-5-yl)acetic acid (V-29). Then through activating the carboxyl group using phosphorus oxychloride, 29 was obtained by reaction of the activated V-29 and ammonium hydroxide. Compounds 1-25 were collected from our synthesized in-house library. [12] Compounds 27, 28 and 29 are new compounds that have not been reported before. The structures of the newly synthesized compounds 26, 27, 28 and 29 and the potent compound 23 were confirmed by 1 H NMR, 13 C NMR and HRMS. 

The inhibition activity was measured following the previously published procedure using a synthetic peptide-pNA as substrate (Table 1 ). [12, 18] We used Tideglusib as a positive control for the enzyme assay. Our measured IC 50 value (1.91 µM) is in consistent with the reported one (1.55 µM, Fig. S1 ). 8 Among all the 29 compounds that we tested, 12 showed inhibition activity over 50 % at 50 µM. Hydrophobic groups at the R 1 position are required to insure the inhibitory effect. The carboxamide group at Fig. S1 ). Substitution to other groups at R 2 position like halogen (9), carboxylic acid N-hydroxysuccinimide ester (17) and ethanamide group (29) inactivate or decrease the activity of the compounds. The SARs of these compounds against SARS-CoV-2 3CL pro is similar to that found for SARS-CoV 3CL pro , [12] further indicating that the substrate binding pocket in 3CL pro of CoVs is highly conserved and other classes of reported SARS-CoV or other CoVs 3CL pro inhibitors may also be effective against SARS-CoV-2 3CL pro . Fig. S1 . c Inhibition less than 25% at 50 µM. 

To examine the potential binding mode of these compounds, we built the complex model structure of 26 and SARS-CoV-2 3CL pro using an induce-fit docking procedure. As shown in Fig. 2 , 26 fits snugly into the substrate-binding pocket. The carboxamide at C-5 makes H-bonds with the side-chain carboxyl groups of Asn142 and Glu166, and the oxygens at C-2 and C-3 form H-bonds with the main-chain amino group of Cys145. The naphthyl ring fits into the hydrophobic groove formed by Met49 and Met165

and forms π-π stacking interaction with His41. Combing the information from structure-activity relationship and docking analysis, we conclude that:

(1) The carboxamide group at C-5 is essential for inhibition activity. Even adding one carbon between the carboxamide group and the isatin ring (29) 

In conclusion, we have tested the inhibition activity of 29 N-substituted isatin derivatives against SARS-CoV-2 3CL pro . The most potent compound, 26, demonstrated an IC 50 of 45 nM against SARS-CoV-2 3CL pro , which is among the most potent SARS-CoV-2 3CL pro inhibitors known so far. We found that isatin compounds with a carboxamide substitution at the C-5 position and aromatic substitution at N-1 position are strong inhibitors of SARS-CoV-2 3CL pro . As these compounds also inhibit SARS-CoV 3CL pro , other known SARS-CoV 3CL pro inhibitors can be further tested for their inhibition activity against SARS-CoV-2 3CL pro . The active compounds have relatively high cytotoxicity that hinder quantitatively measurement of their anti-SARS-CoV-2 activity (as shown in Fig. S2 ). Further optimization to reduce the cytotoxicity and increase the cellular activity is necessary to develop this series of compounds as broad-spectrum anti-coronavirus drugs.

Melting points were obtained using an X4 apparatus and are uncorrected. Yields refer to isolated products. 1 12.31 g (85%) of product was obtained. The crude product is sufficiently pure for the next step. After stirring for 3h, the reaction was quenched with 20ml water. The mixture was extracted with EtOAc and the organic layer was washed with brine, dried over Na 2 SO 4 , filtered, and concentrated. 1.46 g (70%) of product is obtained. The crude product is sufficiently pure for the next step.

(1 g, 4.6 mmol), 2-(Bromomethyl)naphthalene (1.17 g, 5.3mmol) and K 2 CO 3 (1.63 g, 11.8 mmol) in 100 mL of acetonitrile was heating to reflux for 10 h. After the reaction was completed monitored by TLC.

The solution was evaporated and purified by column chromatography. 

The crystal structure of SARS-CoV-2 3CL pro (PDB ID 6LU7) was used for molecular docking [8] . The The full-length gene encoding SARS-CoV-2 3CL pro was synthesized for Escherichia coli (E. coil) expression (Hienzyme Biotech). The expression and purification of SARS-CoV-2 3CL pro were carried out using the reported protocol [15] .

A colorimetric substrate Thr-Ser-Ala-Val-Leu-Gln-pNA (GL Biochemistry Ltd) and assay buffer (40 mM PBS, 100 mM NaCl, 1 mM EDTA, 0.1% Triton 100, pH 7.3) was used for the inhibition assay. To evaluate the effects of compounds on SARS-CoV-2 3CL protease activity, compounds were first preincubated with enzyme samples in the assay buffer for 30 min at room temperature. And then 10 µl of 2 mM substrate was added into the above system to the final concentration of 200 µM to initiate the reaction. Increase in absorbance at 390 nm was recorded for 20 min at an interval of 30s with a kinetics mode program using a plate reader (Synergy, Biotek). IC 50 values were fitted with Hill1 function.

Corresponding Author # These authors contribute equally.

*Y.L.: e-mail, liuying@pku.edu.cn.

*L.L.: e-mail, lhlai@pku.edu.cn.

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3C-like proteinase from SARS coronavirus catalyzes substrate hydrolysis by a general base mechanism

CoV-2 3C-like protease is a key target for drug discovery against COVID-19

substituted isatin compounds are potent SARS-CoV-2 3C-like protease inhibitors. • Three compounds showed ten nanomolar level inhibitory potency. • The most potent compound 26 demonstrates an IC50 of 45 nM

This work was supported in part by the National Natural Science Foundation of China (21633001,21877003), and the Ministry of Science and Technology of China (2016YFA0502303).

The authors declare no competing financial interest.

COVID-19, CoV infectious disease; 3CL pro , 3C-like protease; TLC, thin layer chromatography.

The authors declare that they have no conflicts of interest to this work.