Synthetic and computational efforts towards the development of peptidomimetics and small-molecule SARS-CoV 3CLpro inhibitors.

Severe Acute Respiratory Syndrome (SARS) is a severe febrile respiratory disease caused by the beta genus of human coronavirus, known as SARS-CoV. Last year, 2019-n-CoV (COVID-19) was a global threat for everyone caused by the outbreak of SARS-CoV-2. 3CLpro, chymotrypsin-like protease, is a major cysteine protease that substantially contributes throughout the viral life cycle of SARS-CoV and SARS-CoV-2. It is a prospective target for the development of SARS-CoV inhibitors by applying a repurposing strategy. This review focuses on a detailed overview of the chemical synthesis and computational chemistry perspectives of peptidomimetic inhibitors (PIs) and small-molecule inhibitors (SMIs) targeting viral proteinase discovered from 2004 to 2020. The PIs and SMIs are one of the primary therapeutic inventions for SARS-CoV. The journey of different analogues towards the evolution of SARS-CoV 3CLpro inhibitors and complete synthetic preparation of nineteen derivatives of PIs and ten derivatives of SMIs and their computational chemistry perspectives were reviewed. From each class of derivatives, we have identified and highlighted the most compelling PIs and SMIs for SARS-CoV 3CLpro. The protein-ligand interaction of 29 inhibitors were also studied that involved with the 3CLpro inhibition, and the frequent amino acid residues of the protease were also analyzed that are responsible for the interactions with the inhibitors. This work will provide an initiative to encourage further research for the development of effective and drug-like 3CLpro inhibitors against coronaviruses in the near future.

Introduction

In November 2002, Severe Acute Respiratory Syndrome (SARS) was first identified in Guangdong Province of Southern China and immediately within a few months was considered to be a global threat because of its highly contagious and deadly nature. From November 2002 to 2003, it spread rapidly over 29 countries. Total 8439 cases were reported globally till 3rd July 2003, and among which 812 patients died1, 2. The etiological agent solely responsible for this SARS was found to be a novel human coronavirus called SARS-CoV. Some changes in the phylogenetic characters and gene sequence of SARS-CoV distinguish it from other previously discovered3, 4 SARS is characterized by fever, and after a few days, it is followed by shortness of breath and dry nonproductive cough, headache, dyspnea, lower respiratory tract infection, lymphopenia, and diarrhoea.

Coronaviruses are enveloped, positive-stranded RNA virus and belong to order Nidovirales, family Coronaviridae, sub-family Coronavirinae genus Coronavirus. Genus is of four types alpha, beta, gamma, and delta Coronavirus. The beta coronavirus with “b” lineage is mainly responsible for SARS-CoV disease3, 6. The schematic representation of the taxonomy is described in Figure 1 . Coronaviruses are zoonotic viruses, primarily originated from non-humans (animal reservoirs, especially bats and avian species) and then transmitted to humans. The seven coronaviruses known to infect humans include HCoV-229E, HCoV-OC43, HCoV-NL63, HCoV-HKU1, SARS-CoV, MERS-CoV, and recently SARS-CoV-2. Amongst these seven SARS-CoV, Middle East Respiratory Syndrome Coronavirus (MERS-CoV) and SARS-CoV-2 are the most infectious and fatal. MERS-CoV was first reported in June 2012 in Saudi Arabia and caused severe respiratory problems. By the end of August 2015, 1511 cases were reported with 574 deaths (fatality rate – 38%)8, 9. The disease caused by SARS-CoV-2, also known as coronavirus disease 19 (COVID-19), as the first case was reported in Wuhan, China, in December 2019. COVID-19 has been declared a worldwide pandemic by World Health Organization (WHO) on March 11, 2020. Till the mid of August 2020, 21.2 million cases were reported, including 76,100 deaths10, 11. Even though the death rate is lower than SARS-CoV, the infection rate is quite high, so SARS-CoV-2 is considered a severe medical emergency. In recent times, several pharmaceutical companies successfully developed vaccines to treat COVID-19, but no specific anti-viral drugs have been discovered. Chymotrypsin-like cysteine protease (3CLpro) is the major protease involved in viral replication of SARS-CoV, and it has also been found that the sequence similarity between SARS-CoV and SARS-CoV-2 is 96%5, 8. By targeting this protease enzyme, many researchers have designed and developed potential inhibitors against coronaviruses. The potential SARS-CoV 3CLpro inhibitors have a particular parent moiety that can be modified structurally to develop a new effective drug. The most advanced pre-clinical therapeutic drugs for SARS-CoV-2 3CLpro are PF-00835231, PF-07304814, GC-376, and PF-07321332 (Figure 2 )12, 13. Based on the identical sequence similarity between SARS-CoV and SARS-CoV-2, the inventors of PF-0083523 have recently developed a new covalent inhibitor for treating COVID-19. Another potent and selective SMI against SARS-CoV-2 3CLpro is ALG-097111 which was evaluated in the COVID-19 infected Syrian Hamster Model for antiviral activity.

Figure 1

Taxonomic classification of SARS-CoV.

Figure 2

Structure of PF-00835231, PF-07304814, GC-376, PF-07321332, Nafamostat, and VR23.

To combat the current outbreak of COVID-19, the scientific community is trying to develop effective treatment within a feasible time. Many well-established strategies are taken by the research organizations to eradicate the COVID-19 pandemic16, 17. In the field of drug discovery and development, repurposing of existing drugs is one of the organized and economic strategies to fight against novel diseases, like novel coronavirus. The bioinformatics and pharmacoinformatic approaches have played a massive role in repurposing the existing drugs. By this strategy, many scientific workers have generated and identified numerous newer inhibitors against target diseases. In the battle of COVID-19, the anti-SARS-CoV compounds are mostly explored and repurposed for screening the best-fit inhibitors. With the help of computational chemistry, recently, Bhowmik and their collaborators identified Nafamostat and VR23 as promising candidates for treating COVID-19 by targeting SARS-CoV-2 3CLpro (Figure 2). The pharmacodynamic study revealed that the Nafamostat and VR23 are promising for drug-likeness but further pre-clinical/clinical investigation studies are needed to be procured for understanding the efficacy and potency of the compounds.

Apart from the pharmacoinformatic approaches, chemical synthesis is an integral part of drug discovery and development; to synthesize the novel molecules for the target diseases. Since now, many organic chemists have explored and discovered the different types of novel inhibitors targeting the SARS-CoV 3CLpro, such as peptidomimetic inhibitors (PIs), small-molecule inhibitors (SMIs), and metal conjugate inhibitors. Researchers can't evaluate the in vitro or in vivo efficacy and potency of inhibitors without the chemical synthesis of designed inhibitors. So, wet lab chemical synthesis has always a great interest for researchers in the pharmaceutical industry and scientific field to develop novel chemical entities as a drug.

Considering the global pandemic of COVID-19, this review focuses on the chemical synthesis and computational chemistry perspectives of SARS-CoV 3CLpro inhibitors, specially PIs and SMIs, which has been discovered by applying chemical synthesis, in vitro biological assay, and computational chemistry from 2004 to 2020.

The journey of different analogues towards development of SARS-CoV 3CLpro inhibitors is provided in Figure 3 . Here, we have classified the SARS-CoV 3CLpro inhibitors into two categories: 1) Peptidomimetic inhibitors (PIs), and 2) Small- molecule inhibitors (SMIs). The PIs consist of different analogues, including: Keto-Glutamine analogues, Anilide analogues, Peptidomimetic α,β-unsaturated esters, Glutamic acid and glutamine peptides, Peptidomimetic (TG-0205221), Phthalhydrazide peptide analogues, Cinanserin analogues, Trifluoromethyl ketone containing peptides, Trifluoromethyl, benzothiazolyl or thiazolyl ketone containing peptidomimetics, Michael type of peptidomimetics, Cysteine protease inhibitors, Potent dipeptide type of peptidomimetics, Novel dipeptide type of peptidomimetics, Nitrile based peptidomimetics, Tripeptide type of peptidomimetics, Peptidomimetics containing cinnamoyl warhead, Peptidomimetics containing decahydroisoquinolin moiety, Ketone-based covalent inhibitors, and α‐Ketoamide derivatives. The SMIs are classified into: isatin derivatives, Chloropyridyl ester derivatives, pyrazolone derivatives, pyrimidine derivatives, macrocyclic inhibitors, 5-sulphonyl isatin derivatives, pyrazolone derivatives, serine derivatives, phenylisoserine derivatives, and Octahydroisochromene derivatives.

Figure 3

Journey of different analogues in the discovery of SARS-CoV 3CLpro inhibitors.

We have also identified the most promising anti-SARS compounds from each class of derivatives and highlighted their structural features and binding information. This work, chemical synthesis, and computational chemistry aspect of SARS-CoV 3CLpro inhibitors will provide an initiative to encourage further research to the organic chemist and medicinal chemists for the design and synthesis of new 3CLpro inhibitors effective against coronaviruses in coming years.

SARS-CoV 3CLpro and its structural features:

Coronaviruses are positive polarity family of polyadenylated single-stranded RNA. In several animal species, even human beings, this virus can cause acute and chronic pulmonary and nervous system diseases. Coronavirus virions feature the largest viral genomes ranging from 27 to 31 kb long found to date. The genome is helically encapsidated by nucleocapsid (N) proteins. Virus cores are formed by the matrix proteins that surround the filamentous nucleocapsid. The virus membrane includes various spike-like protuberances called spike (S) proteins, a type of glycoprotein I along with membrane protein (M) that covers the membrane, and a hydrophobic protein called envelope protein (E) that surrounds the entire virus structure. These proteins make a crown-like appearance, hence the justification of the name Corona, which is derived from a Latin word, while seen in an electron microscope. S proteins act as a protein that binds with the receptor of the host and is a fusion of the viral and cellular membranes54, 55. The schematic figure is given in Figure 4 .

Figure 4

Structure of SARS-CoV and its proteins (S, M, E, and N).

The SARS-CoV genome consists of two open reading frames bound by a ribosomal frameshift that encodes two overlapping polyproteins, pp1a (approximately 450 kDa) and pp1ab (approximately 750 kDa), that helps to moderate the mechanism of viral replication and transcription. Complex proteolytic processing is involved in the maturation of coronavirus that controls viral gene expression and replication. Some viruses from the same family make use of three proteases in the proteolytic process. In comparison, it is known to encode only two proteases in the case of SARS-CoV. Other than 3CLpro, it also has papain-like cysteine protease (PLpro). 3CLpro, also known as the Main Protease (Mpro), plays a crucial role in the mechanism of viral replication and infection, allowing this macromolecule a prime candidate for antiviral therapy (PDB ID: 1UJ1)6, 57.

Yang et al solved the X-ray crystallographic structure of hexapeptidylchlromethyl ketone (CMK) inhibitor, which is bound to Mpro at various pH values. It was stated that with the two promoters referred to as “A” and “B” are focused almost perpendicular to each other, SARS-CoV 3CLpro acts as a dimer. As shown in the schematic diagram (Figure 5 ), the SARS-CoV Mpro crystal structure, analogous to other 3CLpro crystals, contains three domains. Domain I have 8–101 residues, and Domain II has 102–184 residues. Domain I and II contain β-sheets that form the framework of chymotrypsin, while domain III, having 201–306 residues, consists mainly of alpha-helices. The Cysteine-Histidine (Cys-His) catalytic dyad is present in SARS-CoV Mpro, and between domain I and domain II in a cleft, the binding site for the inhibitor is present. The inhibitor showing in Figure 6 , having binding subsite S1 specificity of a coronavirus protease in protomer A bestows complete specificity for the enzyme residue of the P1-Gln substrate. Squeezed between domain II and domain III of the parent monomer and domain II of the other monomer, each N-terminus residue (N-finger) plays an essential part in the dimerization process and development of the Mpro active site. The Mpro SARS-CoV dimer is intensely active, while the monomer is somewhat inactive. The study revealed that the pH-dependent triggering switch is a unique feature of SARS-CoV 3CLpro, resulting in significant, cooperative activities of the Glu-166, Phe-140, Leu-141, and Tyr-118 side chains and the dimer N terminus of the other protomer. A physiological reason for the lower P2 specificity of the SARS enzyme than those of other coronaviruses is due to the unusual binding mode of the substrate-analogue inhibitor. To facilitate the design and screening of inhibitors with anti-SARS action, these structures provide important structural details on dimerization and substrate binding sites6,58, .

Figure 5

Schematic diagram demonstrating the essential role of the N-finger in both dimerization and preservation of the enzyme's active form.

Figure 6

Structure of SARS-CoV 3CLpro inhibitor.

Chemical synthesis of SARS-CoV 3CLpro inhibitors:

Peptidomimetic inhibitors (PIs):

Therapeutic interactions between two proteins or protein–protein interactions (PPIs) have been essential in drug design in recent times against various diseases, including metabolic disorders, cancer, neurodegenerative disorders, and several other illnesses. It also has a significant role in various biological processes, including cell proliferation, signal transduction, and apoptosis. Peptidomimetics are molecules whose basic elements in 3D space resemble a standard peptide or protein, which maintain the capability to interact and create the same therapeutic effect with the biological target. Peptidomimetics are intended to overcome some of the complications associated with a native peptide, including proteolysis that affects the duration or half-life of the protein and low bioavailability. Other such properties may also be significantly improved, which includes receptor specificity and efficacy. Thus, in drug development, peptidomimetics have enormous potential. The peptidomimetic design method begins with developing the relationships between structure and activity, also known as SAR that can describe the biological effect61, 62.

There are various instances of PPI-targeting peptides and peptidomimetics that's been in clinical trials. For example, the first potent and specific p53 re-activator, an important tumor suppressor protein that binds and acts as an inhibitor of two p53 suppressor proteins, namely MDM2 and MDMX, is Aileron (ALRN-6924). p53 is one of the most favoured targets of antineoplastic drugs activated by its vital role in stopping multiple cancers from initiating and advancing. KAI Pharmaceuticals developed a small peptide named KAI-9803, which directly acts as dPKC inhibitor with its intracellular receptor, RACK. During reperfusion, dPKC activation initiates molecular pathways for cell death, which gradually leads to inflammation and injury during a stroke in the heart or brain. In phase II clinical trial to determine safety and effectiveness, KAI-9803 was evaluated for its efficacy in acute myocardial infarction patients.

The chemical synthesis of PIs under Keto-Glutamine analogues, Anilide analogues, Peptidomimetic α,β-unsaturated esters, Glutamic acid and glutamine peptides, Peptidomimetic (TG-0205221), Phthalhydrazide peptide analogues, Cinanserin analogues, Trifluoromethyl ketone containing peptides, Trifluoromethyl, benzothiazolyl or thiazolyl ketone containing peptidomimetics, Michael type of peptidomimetics, Cysteine protease inhibitors, Potent dipeptide type of peptidomimetics, Novel dipeptide type of peptidomimetics, Nitrile based peptidomimetics, Tripeptide type of peptidomimetics, Peptidomimetics containing cinnamoyl warhead, Peptidomimetics containing decahydroisoquinolin moiety, Ketone-based covalent inhibitors, and α‐Ketoamide derivatives; are discussed below:

Keto-Glutamine analogues:

In 2004, Jain and co-researchers synthesized a series of keto-glutamine analogues targeting the SARS-CoV 3CLpro. The synthesized compounds have been evaluated against SARS-CoV 3CLpro by using fluorometric assays and found the most potent inhibitors with IC50 values ranging from 0.60 to 70 μM. The compounds have been designed based on their previously synthesized molecule previously evaluated against human rhinovirus-14 (HRV-14) and hepatitis A virus (HAV) targeting on picornaviral 3C protease enzyme. The reported keto-glutamine analogues 1 and 2 of HAV 3C proteinase inhibitors with IC50 values are shown in Figure 7 . Jain et al designed the three synthesis scheme ( Scheme 1 : 1A, 1B, and 1C) by modification at P2, P3, and P4 position with amino acids of previously reported compound.

Figure 7

Keto-glutamine peptides as a potent inhibitor of HAV 3C-pro.

Scheme 1

Synthesis of Keto-Glutamine analogues. Scheme 1, Scheme 1A: Synthesis of N,N-dimethylglutamine analogues; Scheme 1, Scheme 1B: Synthesis of cyclic glutamine analogues with keto-phthalhydrazide moiety;Scheme 1, Scheme 1C: Synthesis of cyclic glutamine analogues without keto-phthalhydrazide moiety.

The synthetic route of a tetrapeptide, Scheme 1 A initiated according to the previously reported literature65, 66, 67 to produced 4a-c. After that, it reacts with trifluoracetic acid for removal of the Boc group. Simultaneously, tripeptide Ac-Val-Thr(OBn)-Leu-OH was added in the reaction medium of DMF to generate tetrapeptide 5a-b. In Scheme 1 A, the 5a and 5b compounds were reacted with respective reagents under specific reaction conditions to generate compounds 5c and 5d as shown in Scheme 1 B, the 9a and 9b compounds were reacted with appropriate reagents under specific reaction conditions to get another 9c and 9d compounds67, 68. Another compound 11 was synthesized without the keto-phthalhydrazide group to analyze the effect of tetrapeptide moiety in the compound 9a-d, as shown in Scheme 1 C. In-silico studies of the inhibitor 9b revealed that the pyrrolidine-2-one of S1 site interacted with His163 and nitro group of 5-nitro-2,3-dihydrophthalazine-1,4-dione of S2 site interacted with Asn142 residue of the protease (Figure 8 ). From all synthesized molecules, 9b has been found the most potent reversible inhibitors against SARS-CoV 3CLpro with IC50 value 0.6 µM (Figure 8).

Figure 8

Structure of peptidomimetic SARS-CoV 3CLpro inhibitors (9b, 14a, 38c, 53, 69, 83a, cinanserin, 108a, 113c, 137,149a, 162b, 172a, 180a, 204 214o, 226d, 247, 256 and 289s).

Anilide analogues:

A multiple series of peptide anilides were developed by Shie and co-workers in 2005 based on antihelmintic drug (Niclosamide) and evaluated the anti-SARS activity targeting the 3CLpro enzyme. The anilide analogues were derived from 2-chloro-4-nitroaniline, l-phenylalanine, and 4-(dimethylamino)benzoic acid. Based on 2-chloro-4-nitroanilide containing dipeptide (Scheme 2, Scheme 2 A), Shie et al synthesized various anilide analogues, such as tripeptide anilide, tetrapeptide anilide, dimeric anilide, and ketomethylene anilide (Scheme 2, Scheme 2 B and 2C).

Scheme 2

Synthesis of anilide analogues. Scheme 2, Scheme 2A: Synthesis of di-peptide anilides analogues; Scheme 2, Scheme 2B: Synthesis of tripeptide and tetrapeptide anilides analogues;Scheme 2, Scheme 2C: Synthesis of tripeptide ketomethylene anilide analogues.

The di-peptide anilides analogues 14a-e were synthesized by following previously reported synthetic strategy. First, 12 was reacted with Boc-l-phenylalanine to produced 13 and then, 13 was again treated with substituted carboxylic acid to produce the di-peptides 14a-e via coupling reaction (Scheme 2, Scheme 2 A). The intermediate compound 13 was taken further to synthesize the tripeptide 15a-x and tetrapeptide anilides 16a-x analogues via coupling with appropriate peptide (Scheme 2, Scheme 2 B). The synthesis of tripeptide ketomethylene anilide analogues 22a-p was synthesized via multistep organic reaction. First, intermediate 19 was prepared by adding two starting compounds, 17, 18, and then 19 was again reacted with HCl to remove Boc protection and substituted with respective R1COCl to form compound 20. The compound 20 further on coupling with another 2-chloro-4-nitroaniline derivative 21 in the presence of Palladium-tetrakis(triphenylphosphine) form tripeptide anilide analogues 22a-p.

All the synthesized molecules were screened via biological assay with fluorogenic tetrapeptide substrate, and the molecular docking study with PDB ID (1UK4) showed the binding affinity towards SARS-CoV 3CLpro enzyme. Under anilide peptide analogues, the most potent structure is 14a (shown in Figure 7 ), which acts as a competitive inhibitor of SARS-CoV 3CLpro with IC50 value 0.06 µM and Ki value 0.03 µM. Computer-modelling of the inhibitor 14a binding with SARS-CoV 3CLpro found that the benzyl group of S1 site was occupying Cys145, Ser144, His163, and Phe140 or the pocket formed by Thr25, His41, Cys44, Thr45, and Ala46. The 2-chloro-4 nitro benzyl group comprising the S2 site was found to interact with Ala46, while the chlorine atom is within 3 Å of Cys145 and His41. The dimethylamino benzyl of S3 site was found to be fitted into the pockets formed by Gln189-Gln192 and Met165-Pro68 residues ( Figure 8 ) .

Peptidomimetic α,β-unsaturated esters:

In 2005, Shie and co-researchers synthesized a series of peptidomimetics containing α,β-unsaturated esters targeting the 3CLpro for SARS-CoV. Researchers have synthesized a compound AG7088 and its related tripeptide α,β unsaturated ester, and ketomethylene isosteres (Scheme 3 ) and evaluated their inhibitory properties against SARS-CoV 3CLpro enzyme. These unsaturated peptidomimetic esters and ketomethylene isosteres showed moderate inhibition against the same enzyme. AG7088, previously reported as a potent PIs active against HRV-3Clpro that contain an α,β-unsaturated ester side chain . Based on this evidence, it has been thought that AG7088 could be a potent inhibitor of SARS-CoV 3CLpro but, it turned out to have no inhibition even in the concentration of 100 µM in an enzymatic assay. Although the related α,β unsaturated ester and ketomethylene isosteres were showed moderate inhibitory activity against SARS-CoV 3CLpro enzyme with IC50 value ranging from 11 to 39 µM.

Scheme 3

Synthesis of peptidomimetics (AG7088) containing α,β-unsaturated ester warhead. Scheme 3, Scheme 3A: Synthesis of intermediate 27; Scheme 3, Scheme 3B: Synthesis of intermediate 30;Scheme 3, Scheme 3C: Synthesis of peptidomimetics (AG7088) via coupling of 27 and 30 intermediates.

The synthesis of PIs (AG7088) containing α,β-unsaturated ester warhead and related compounds were synthesized based on previously developed literature25, 73. The synthesis strategy was divided into three parts: Scheme 3, Scheme 3 A, for the synthesis of intermediate 27; Scheme 3, Scheme 3 B, for the synthesis of intermediate 30; and Scheme 3, Scheme 3 C, for the synthesis of PIs (AG7088) via coupling of 27 and 30 intermediates.

Based on molecular modelling study, four different series (1, 2, 3, and 4) of AG7088 were designed and established. The series 1, 2, 3, and 4 were synthesized by changing in the three locations of the AG7088 (Scheme 3, Scheme 3 C). From that above series, it was found that the series 4 compounds were most promising for SARS-CoV 3CLpro inhibition. The set of Phe-Phe dipeptides containing conjugated amido moieties at the N-terminals were designed with the help of software. The series 4 compounds 38a-e were synthesized by using previously reported methods. The main starting material for the synthesis of 38a-e is Phe-Phe dipeptidyl α,β-unsaturated ester (Scheme 3, Scheme 3 C). Amidation of N-Boc-phenylalanine by 4-amino-5-phenyl-2-pentanoate and subsequent removal of Boc group with the help of 1,4-dioxane and HCl produces compound Phe-Phe dipeptides α,β-unsaturated ester. Then, with compound Phe-Phe dipeptides α,ß-unsaturated ester, several α,β-unsaturated carboxylic acid were reacted to produce a series of analogues (Scheme 3, Scheme 3 C). Before isolating the compounds, they have done an enzymatic assay and found that the compound 38c was the most potent inhibitor against SARS-CoV 3CLpro enzyme (Figure 8). Molecular modelling study reveals that, out of four series of compounds, the compounds of series 3 and 4 showed better inhibitory results than the compounds of 1 and 2 series against SARS-CoV 3CLpro enzyme. The in-silico study of compound 38c indicates that the (dimethylamino)cinnamyl group of S1 site was occupied by Glu166, Gln189, and Gln192 residues via hydrogen bonding ( Figure 8 ) .

Glutamic acid and glutamine peptides:

It has been established that glutamic acid and glutamine peptides with trifluoromethyl ketone groups show good inhibitory activity against the 3CLpro of SARS-CoV. Sydnes and co-workers synthesized a new series of tri- and tetra-glutamic acid and glutamine peptide and evaluated against 3CLpro inhibitory activity using a fluorescence-based peptide cleavage assay. Many research groups suggested that the trifluoromethyl ketone (CF3-ketone) moiety containing molecules play an important role in protease enzyme inhibition and an important group for fluorescence. Due to this background information, they thought that the trifluoromethyl ketone (CF3-ketone) could play a key role in SARS-CoV 3CLpro inhibition. Based on substrate binding recognition, the synthetic strategy of CF3-ketone containing glutamic acid and glutamine peptides has been designed. Four peptides were synthesized and biologically evaluated (enzyme inhibition assay) against SARS-CoV 3CLpro using fluorogenic substrate (Scheme 4 : Series 4A, 4B, and 4C). Although the related four peptides showed moderate inhibitory activity against the SARS-CoV 3CLpro enzyme, the most potential CF3-ketone containing glutamic acid and glutamine peptide was 53 with Ki value 116.1 ± 13.6 µM (Figure 8).

Scheme 4

Synthesis of CF3-ketone containing glutamic acid and glutamine peptides. Scheme 4, Scheme 4A: Synthesis of intermediate 45 (trifluoromethyl-b-amino alcohol); Scheme 4, Scheme 4B: Synthesis of intermediate 4z (glutamic acid and glutamine peptides with a CF3-ketone unit); Scheme 4, Scheme 4C: Synthesis of target compound (glutamic acid and glutamine peptides with a CF3-ketone unit) by coupling reaction.

The Synthesis of CF3-ketone containing glutamic acid and glutamine peptides can be accomplished by starting with compound 39. The synthesis of target compounds were divided into three parts: Scheme 4, Scheme 4 A, synthesis of intermediate 45 (trifluoromethyl- β-amino alcohol), Scheme 4, Scheme 4 B, synthesis of intermediate 48 (glutamic acid and glutamine peptides with a CF3-ketone unit), and Scheme 4, Scheme 4 C, Synthesis of the target compound (glutamic acid and glutamine peptides with a CF3-ketone unit) by coupling reaction. First, compound 39 was treated with paraformaldehyde and para-toluene sulfonic acid in a toluene mixture to produce oxazolidinone acid 40. The formation of oxazolidinone acid 40 was developed by Luesch et al . Then, compound 40 was transferred into 41, and subsequently, compound 41 was also converted into 42 under specific reaction conditions (Scheme 4, Scheme 4 A). The compound 42 was formed in two forms of stereoisomers, but the diastereomer form of 42 was further reacted with TBAF to produce 43. Then, the compound 42 and 43 were also transferred into compound 44 by reacting with sodium borohydride and methanol. Finally, by hydrogenation using Pd/C, compound 44 was transferred into final product 45 (Scheme 4, Scheme 4 A). The second part of synthesis is focused on peptide formation. The target compound 48 was synthesized based on published literature76, 77. At first, compound 46 was treated with H2 and Pd/C for the hydrogenation process that produced compound 47. Further, it was coupled with Cbz-l-Ala-OSu under basic medium to produce target peptide 48 (Scheme 4, Scheme 4 B). In the third part of the synthesis, the starting compound 45 was synthesized as shown in Scheme 4, Scheme 4 A. The compound 45 was reacted with different synthesized peptides such as: 46, 48, and 49 to get respective product 50, 51, and 52. Then, compounds 50, 51, and 52 reacted with TFA for removal of tert-butyl group to form compound 53, 54, and 55. In the final step, the last forming compounds 53, 54, and 55 were converted into respective compounds 56, 57, and 58 under suitable reaction conditions (Scheme 4, Scheme 4 C).

Peptidomimetic (TG-0205221):

TG-0205221 is a synthetic compound designed and synthesized by the scientists Syaulan Yang and their research group in 2006. The compound was compared with one natural peptide substrate and it was found that the natural compound showed moderate inhibition against the 3Cpro enzyme but a lower inhibitory activity against 3CLpro of SARS-CoV. Also, from the crystal structure of the drug-receptor complex, it was established that TG-0205221 binds firmly with the 3CLpro enzyme with extensive hydrophobic contact and involves ten hydrophobic bonds and one covalent bond. The compound TG-0205221 (69) was synthesized by a multi-step organic reaction showed in Scheme 5 and evaluated the inhibitory activity against SARS-CoV 3CLpro by using 229E and MRC-5 cells. The cytopathic effect was also performed with infected SARS-CoV Vero E6 cells. TG-0205221, 69 showed exceptional activity against SARS-CoV with Ki value 0.053 µM shown in Figure 8 .

Scheme 5

Synthesis of inhibitor TG-0205221 (69).

The synthesis of TG-0205221 (69) can be accomplished by a multi-step organic reaction. First, the l-Glutamic acid 59 was reacted with trimethylsilyl chloride and methanol as a solvent for 15 h and continued the reaction. After the desired time, the amine group was protected with Boc by adding Di-tert-butyl decarbonate and tri-ethylamine; and formed the compound 60. The compound 60 was again treated with strong base Lithium bis(trimethylsilyl)amide and bromo acetonitrile to get the compound 61. The intermediate compound 62 was formed by the hydrogenation of compound 61 and further reacted with base triethylamine to form cyclization product 63. Next, Boc protection of compound 63 was removed by adding the HCl at room temperature. After that, the addition of Boc-β-cyclohexyl-Ala-OH to the compound 64 produces the compound 65 and further deprotection of Boc produces the compound 66. In the next step, the Cbz-Thr(tBu)–OH was reacted with compound 66 to form compound 67. In the final step, the ester group of compound 67 was transferred to the alcohol by reacting with lithium hydride and forming compound 68. Further, reaction with triethylamine produced the final compound 69 (Scheme 5).

An in-silico study detected the binding interaction of compound 69 with 3CLpro. The result showed that the pyrrolidine-2-one of the S1 site was interacting with His163, Glu166, Phe140, and Asn142 residue. The amino acid residues, such as His164, Cys145, and Gly143, also interacted with the S2 site of aldehyde moiety. Similarly, in the N of -NHCbz and O of acetamide S3 site, the Glu166 amino acid residue interacted. The S4 site comprising amide residue was found to interact with Gln189 residue and cyclohexane moiety of S5 site interacted with His41. The trimethyl group of S6 site interacted with Met165, Leu167, Pro168, Asp187, Met49, Ala191, Arg188, and Thr190 residue (Figure 8).

Phthalhydrazide peptide analogues:

Zhang et al designed and synthesized a series of tetrapeptide containing phthalhydrazide ketones, pyridinyl esters, and their different analogues. They have designed the molecules based on a previous literature report on potent SARS-CoV 3CLpro inhibitor and screened the three compounds, such as keto-glutamine analogues, AG7088 , and aromatic ester; for the development of new potent SARS-CoV 3CLpro inhibitor molecule. They synthesized the designed peptides molecules via Scheme 6 : 6A, 6B, and 6C. The pyridinyl esters and their different analogues were also synthesized by two methods (method A and method B), shown in Scheme 6 E, Scheme 6 F, Scheme 6 G, and Scheme 6 H. All synthesized molecules were evaluated as SARS-CoV 3CLpro inhibitors by enzyme assay (FRET assay). The assay result revealed that some pyridinyl esters were a potent inhibitor against SARS-CoV 3CLpro with IC50 value ranging from 50 to 65 nM. Among them, the most potent inhibitor is 83a (IC50 value- 0.05 µM) (Figure 8).

Scheme 6

Phthalhydrazide peptide analogues. Scheme 6, Scheme 6A: Synthesis of tetrapeptide 73, Scheme 6, Scheme 6B: Synthesis of tetrapeptide 77 and 78;Scheme 6, Scheme 6C: Synthesis of tetrapeptide 82;Scheme 6, Scheme 6D: Synthesis of a series of 3-Chloropyridinyl Esters by Method A or B; Scheme 6, Scheme 6E: Synthesis of 3-Chloro-5-furan-(2-ylmethoxy)pyridine (85); Scheme 6, Scheme 6F: Synthesis of Furan-2-yl Nicotinate (87); Scheme 6, Scheme 6G: Synthesis of and N-(Pyridin-3-yl)thiophene-2-sulfonamide (89);Scheme 6, Scheme 6H: Synthesis of 2-(Pyridin-3-yl)-1-(thiophen-2-yl)ethanone (92);Scheme 6, Scheme 6I: Synthesis of 2-(5-Bromopyridin-3-yl)-1-(furan-2-yl)ethanone (95); Scheme 6, Scheme 6J: Synthesis of Thiazole-4-carbaldehyde (97), Scheme 6, Scheme 6K: Synthesis of 5-(4 Chlorophenyl)furan-2-carbaldehyde (99).

The synthesis of tetrapeptide 73 started via bromination of Boc-l-phenylalanine 70 in the presence of Ethyl chloroformate and tri-ethylamine. The brominated product 71, then reacted with Phthalhydrazide using DMF as the solvent and formed a new substituent product 72. In the last step of the reaction, compound 72 was treated with analogue of peptide molecule Ac-Val-Thr(OBn)-Leu-OH under suitable reaction conditions to produce the final tetrapeptide compound 73 (Scheme 6, Scheme 6 A). The other two tetrapeptides, 77 and 78 were also synthesized by changing the carboxylic part of compound 74. At first, the Boc-protected cyclic glutamic acid 74 was reacted with N,O-Dimethylhydroxylamine hydrochloride and other reactants, formed compound 75. Then, compound 75 was substituted with 2-Thienylmagnesium bromide under Isopropylmagnesium chloride solution to form compound 76. From this substituted product 76, the two tetrapeptides 77 and 78 were formed by reacting with different peptides (Scheme 6, Scheme 6 B). Based on Scheme 6, Scheme 6 B, they have synthesized another tetrapeptide 82, where the starting compound is Boc-protected cyclic glutamic acid ester 79. In the same fashion, the intermediates compounds were synthesized by reacting with peptide molecule, and treatment of lithium hydroxide leads to the compound 81. In the last step, the 5-Chloro-3-pyridinol was treated with compound 81 to produce the tetrapeptide compound 82. To explore the effect of Chloropyridinyl Esters in tetrapeptides, they have designed two methods (Method A and B) for synthesizing a series of tetrapeptides containing 3-Chloropyridinyl Esters group (Scheme 6, Scheme 6 D). The 3-Chloro-5-furan-(2-ylmethoxy)pyridine 85 was formed by reacting compound 84, 3-Chloro pyridinol and triphenylphosphine (Scheme 6, Scheme 6 E). The Furan-2-yl Nicotinate 87 was synthesized by the reaction of compound 86, thionyl chloride, and 2-Furanone (Scheme 6, Scheme 6 F). Another N-(Pyridin-3-yl)thiophene-2-sulfonamide 89 was also synthesized by treating compound 88 with 3-Aminopyridine (Scheme 6, Scheme 6 G). The synthesis of 2-(Pyridin-3-yl)-1-(thiophen-2-yl)ethanone 92 and 2-(5-Bromopyridin-3-yl)-1-(furan-2-yl)ethanone 95 are shown in Scheme 6 H and 6I. To explore the effect of aldehyde group, they synthesized the thiazole-4-carbaldehyde 97 and 5-(4 Chlorophenyl)furan-2-carbaldehyde 99 (Scheme 6, Scheme 6 J, and 6 K).

The result obtained from the in-silico study of compound 83a showed that the pyridine ring in the S1 site of the compound interacted with the Phe140, Leu141, Asn142, Glu166, His172, His163 amino acid residue and that of the keto group of S2 site interacted with the Ser144, Gly143, Cys145 amino acid residue of the protease (Figure 8).

Cinanserin analogues:

Cinanserin is a 5-HT receptor antagonist which was discovered in the 1960 s and clinically used in the treatment of neurological disorder in 1973 s. In 2005, Chen et al identified the cinanserin as an inhibitor of SARS-CoV 3CLpro, which was virtually screened from the structural database of existing drugs (more than 8000) and identified the best-fitted molecule to the target (SARS-CoV 3CLpro) through docking study. The virtual screening has identified the top 10 potential molecules that can act as anti-SARS agents and cinanserin is one of them. To confirmed the anti-SARS activity, the cinanserin was further evaluated in HCoV-229E tissue culture assays. It showed that the cinanserin has strong anti-SARS activity (IC50 ranging from 19 to 34 µM) via inhibiting the 3CLpro.

Based on the above research findings, the same research group designed and synthesized a series of cinanserin analogues (Figure 8) for SARS-CoV 3CLpro inhibition by collaborating the work with another research group and published the potential cinanserin analogues in 2008. All synthesized compounds were evaluated against SARS-CoV 3CLpro by FRET assay and showed that some of the compounds are potent and selective inhibitors with IC50 values ranging from 1.06 to 43.7 µM. The most potential cinanserin analogue is shown in Figure 8 (108a, IC50 value- 1.06 µM) and molecular interaction data showed that, the compound 108a binds tightly with 3CLpro enzyme subsite than cinanserin.

The cinanserin analogues were designed and synthesized via three series; series I, series II, and series III. In series I (Scheme 7 ), the compound 100 was reacted with various alkyl halides in the presence of isopropyl alcohol to produce the respective substituted product 101a-n. Again, these products are treated with substituted acyl chloride in the reaction medium and produce the final substituted product 102a-n (Scheme 7, Series I). Similarly, using another substituted starting material 103a-b, they synthesized 104a-b and 105a-b (Scheme 7 , Series II). In series III, they reacted the substituted compounds 107a-b with previous starting material 100 and by simple condensation process, the final compound was formed 108a-b (Scheme 7, Series III).

Scheme 7

Synthesis of a cinanserin analogues (Series I, Series II, and Series III).

The hydrophobic interaction atom pairs between the compound 108a and the enzyme were detected by using the LIGPLOT and it was evident that the benzyl group of S1, S4, S5 positions interacts with Thr25, Leu27, Gly143 (S1); Gln189, Met165, Cys145, Glu166 (S2); and HIs163, Phe140 (S3) respectively. The unsaturated alkyl chain of S2 and S3 sites was found interacting with His41 and Met49 amino acid residues (Figure 8).

Trifluoromethyl ketone containing peptides:

In 2008, Shao and co-workers designed and synthesized a series of trifluoromethyl ketones (TFMKs) targeting the 3CLpro for SARS-CoV. The P1- P4 sites of the TFMKs plays a vital role in covalent binding with the enzyme, like that of the substrate. They designed the compounds such that incorporation of benzyl group at P1 site and long-chain alkyl/varying amino acid at P2-P4 site significantly increased the binding ability. The four steps involved in the synthesis of the compounds is shown in Scheme 8 . All the compounds were subjected to in vitro assay, like enzyme and fluorescent substrate peptide assay against SARS-CoV 3CLpro. Amongst all the compounds, the most potent inhibitory activity was shown by compounds 113a, 113b, 113c with IC50 values 15 µM, 20 µM, and 10 µM, respectively. The compound 113c also showed a time-dependent decrease in SARS-CoV 3CLpro enzyme activity with respect to inhibitor concentration when incubated with 3CLpro for 4hrs (Figure 8).

Scheme 8

Synthesis of trifluoromethyl ketone containing peptides.

The synthesis of trifluoromethyl ketone containing peptides can be accomplished in four steps. In the first step, the substituted compound 109a was reacted with sodium nitrate in the presence of DMF and formed the intermediate compound 110a-c. Then, the compound 110a-c reacted with Trifluoroacetaldehyde ethyl hemiacetal under reaction conditions to form compound 111a-e and hydrogenation as well as reaction with N-protected amino acids transform the compound into 112a-g. In the last step, reduction of compound 112a-g leads to final compound 113a-h, as trifluoromethyl ketone containing peptides (Scheme 8).

From the computational studies, the interaction between inhibitor 113c and enzyme was studied. The interaction result showed that the benzyl group of S1 site interacting with Met165, Met49, His41, and Cbz-Ala of S2 site interacted with Gln189 amino acid residue. The cbz group of S3 site and alanine of S4 site interacting with amino acid residues, such as Pro168 (S3) and Glu166 (S4). Similarly, amino acid His163 and Phe140 were associated with leucine of S5 site; and keto group of S6 was also found to interact with amino acid residues Gly143 and Cys145 (Figure 8).

Trifluoromethyl, benzothiazolyl or thiazolyl ketone containing peptidomimetic inhibitors:

In 2006, Cai and co-workers reported moderate activity of trifluoromethyl ketone containing peptides against SARS-CoV 3CLpro. It was observed that the moderate inhibitory activity of the compounds was due to cyclization of the compounds, which decreases its interaction with the active site of the SARS-CoV 3CLpro. To increase the inhibitory activity of trifluoromethyl ketone containing peptides, Regnier T and co-workers in 2009 designed and synthesized a new trifluoromethyl series benzothiazolyl or thiazolyl ketone-containing peptidic compounds. The two approaches utilized to enhance the inhibitory activity were - Firstly, they modified the side chains of amino acid residues at the P1, P2, and P3 sites keeping the trifluoromethyl ketone moiety intact. Secondly, they replaced the trifluoromethyl moiety with electron-withdrawing groups such as thiazolyl and benzothiazolyl group. The synthetic route for target compounds are shown in Scheme 9 : 9A, 9B, 9C, and 9D. All the synthesized compounds were subjected to in vitro protease inhibition assay to check their 3CLpro inhibitory activity. Compounds 137 showed good inhibitory activity with Ki values 2.20 ± 0.8 (Figure 8) .

Scheme 9

Synthesis of trifluoromethyl, benzothiazolyl or thiazolyl ketone containing peptidomimetic inhibitors. Scheme 9, Scheme 9A: synthesis of CF3-ketone containing glutamic acid with Cbz protection, Scheme 9, Scheme 9B: Synthesis of peptides with thiazolyl and benzothiazolyl groups, Scheme 9, Scheme 9C: synthesis of pyrrolidone containing peptides, and Scheme 9, Scheme 9D: synthesis of dithiazolyl containing peptide inhibitor 137.

The synthetic strategy of target compounds were divided into four parts, first part: synthesis of CF3-ketone containing glutamic acid with Cbz protection(Scheme 9, Scheme 9 A), second part: replacing the trifluoromethyl moiety with electron-withdrawing groups such as thiazolyl and benzothiazolyl groups (Scheme 9, Scheme 9 B), third part: synthesis of pyrrolidone containing peptides (Scheme 9, Scheme 9 C), and fourth part: synthesis of dithiazolyl containing peptides (Scheme 9, Scheme 9 D).

The synthesis of target compounds 119a-e were started from N-Cbz-l-glutamine 114 and converted to the compound 115 under specific reaction conditions. Then, compound 115 was reacted with different amines to form the following compounds 116a-d and further converted to the compound 117a-d by using trifluoromethyltrimethylsilane. The compound 118a-d was prepared by a one-pot reaction from 117a-d under appropriate reaction conditions. In the final step, specific peptide fragments26, 71 were reacted with previous compound 118a-d and produced the final compounds 119a-e (Scheme 9, Scheme 9 A).

The compounds 125a-f were synthesized in four steps. In the first step, the compound 120 was converted to amides 121 and then reacted with thiazole or benzothiazole in the presence of n-Butyllithium to get compounds 122a-b. In the next step, treatment with formic acid compounds 122a-b produces the compounds 123a-b and further converted into 124a-e by using substituted amines under appropriate reaction conditions. In the last step, the final compounds 125a-f were formed by reacting the compounds 124a-e with peptide molecule under appropriate reaction conditions (Scheme 9, Scheme 9 B).

The compound 132 was synthesized following the previously published literature via multi-step process. In the first step, the compound 127 was prepared from previous literature methods by using third staring compound 126. Then, the compound 127 was converted to the 128 by using lithium hydroxide and further converted to 129 under reaction conditions. After that, the compound was treated with thiazole and n-Butyllithium to get compound 130. The reaction of trifluoracetic acid and compound 130 converts into compound 131 and further converted to the final product 132 by reacting with Cbz-Val-Leu-OH under reaction conditions (Scheme 9, Scheme 9 C).

Similarly, dithiazolyl compound 137 was prepared from Cbz-Val-Leu-OH 133 and converted to the compound 134 by reacting with n-hydroxy succinimide. Then, compound 134 reacted with glutamic acid to form the compound 135 and converted to the double-weinreb amide 136. In the final step, the compound 136 reacted with thiazole and n-Butyllithium to get the final compound 137 (Scheme 9, Scheme 9 D).

The binding interaction of compound 137 with the enzyme as observed in the in silico study showed that the keto group in the S1 site interacted with Gly143, Ser144, Cys145, the thiazole ring in the S2 site interacted with His41, keto group present in the 2nd position of pyrrolidine ring in the S3 site interacted with His163. Further, the Val-Leu and NH group of pyrrolidine comprise the S4 site found to interact with Gln189, Gln166, and that of amino group in S5 interacts with His164 (Figure 8).

Michael type of peptidomimetic inhibitors:

Akaji et al reported the design, synthesis and evaluation of peptide aldehyde as an inhibitor of SARS 3CLpro. The compounds were designed based on an initially reported pentapeptide aldehyde inhibitor (IC50 – 37 µM), and the optimum side-chain structure was determined by comparing inhibitory activity with Michael type inhibitors. The synthetic outline and associated reaction conditions are depicted in Scheme 10 : 10A, 10B, and 10C, respectively.

Scheme 10

Synthesis of Michael type of peptidomimetic inhibitors. Scheme 10, Scheme 10A: synthesis of Michael type inhibitor; Scheme 10, Scheme 10B: synthesis of peptide aldehyde 149a-f;Scheme 10, Scheme 10C: optimization and synthesis of potent peptide aldehyde inhibitor 156.

Protease inhibition assay was carried out on R188I mutant protease by simultaneous addition of both inhibitor and substrate to determine IC50 value. The result of the experiment suggested that the peptide aldehyde is a competitive inhibitor of SARS 3CLpro because there was no evidence in the formation of a stable covalent bond between inhibitor and protease.

In literature, structural optimization following X-ray crystallographic analysis of inhibitor-protease complex gives a potent tetrapeptide aldehyde with IC50 value 98 nM. The result demonstrated that peptide aldehyde could be a more effective inhibitor of SARS 3CLpro than Michael-type inhibitors.

The synthesis of Michael type inhibitor (Scheme 10, Scheme 10 A) was started from Boc-l-Glu-OBn (1 3 8). Coupling of the side chain carboxyl group with dimethylamine in BOP presence as a coupling reagent produces side-chain amide, simultaneous reduction with LiAlH4 gave alcohol upon Swern oxidation afforded aldehyde 139 . Horner − Wadsworth − Emmons olefination of aldehyde 139 produced Nα -protected amino acid ester 140. Cleavage of the protected group by TFA and following coupling with tetrapeptide yield compound 141. For the preparation of 145, aldehyde 139 was converted to 142 by Wittig reaction and following an acid treatment. The Horner − Wadsworth − Emmons olefination and coupling with tetrapeptide converted 142 to 145. To synthesize the analogue 146, 142 was further subjected to Wittig reaction and an acid treatment as above. The following Horner − Wadsworth − Emmons olefination and coupling with the tetrapeptide yielded 146 .

Peptide aldehyde 149(a-f) was synthesized by using conventional Weinreb amide resin (Scheme 10, Scheme 10 B). The conventional Fmoc-based solid-phase synthesis was used to introduce amino acid residues. After peptide chain construction, the resin was reduced with LiAlH4 to obtain desired compounds.

The synthesis of structurally optimized and potent peptide aldehyde is given in Scheme 10, Scheme 10 C. The synthesis was started with commercially available 9,10-decene-1-ol 150. Which on reaction with mCPBA and following hydrolysis obtained triol linker 151. Under appropriate reaction conditions, 151 was reacted with Fmoc-His (Trt)al to afford corresponding acetal 152. The acetal alcohol on oxidation by Jones reagent yielded carboxylic acid, which in coupling reaction produced amide resin 153. The amide resin on treatment with TFA converted to peptide acetal amide 154. After dissolving in AcOH treated with ethane thiol in BF3-Et2O, and after quenching with H2O obtained thioacetal peptide 155. The thioacetal on treatment with NBS yields final peptide aldehyde 156 .

The binding interaction of compound 149a with the 3CLpro was detected from computational modeling. The result showed the interaction of pyrazole in the S1 site of the compound with His163, Phe140, Leu141, Glu166 amino acid residues of the protease. Leucine in the S2 interacted with Met45, Met165, His41. Further, Valine in S3, Alanine in S4, and aldehyde group in S5 site interacted with Glu166, Thr190, Cys145 amino acid residue, respectively (Figure 8).

Tripeptide type cysteine protease inhibitors:

Prior et al designed and synthesized few transition state inhibitors in 2013. They designed these inhibitors of 3CLpro such that it contains a tripeptidyl group and warheads that include aldehyde, bisulfite adduct, a-ketoamide, or an alpha-hydroxy phosphonate transition state mimic. The glutamine surrogate at the P1 site is essential for recognition because of its compatibility with the substrate specificity for the targeted enzyme. The presence of leucine and arylalanine at the P2 and P3 site enhances the recognition and cell penetrability, respectively. They synthesized the tripeptidyl aldehyde from the starting material glutamine surrogate in five sequential steps, as shown in Scheme 11 : 11A and 11B . The FRET based assay and enzyme-based assay were conducted to evaluate the inhibitory activity of these compounds against SARS-CoV 3CLpro85, 86. The IC50 values obtained proved that compounds have moderate to good inhibitory activity. The most potent compound is shown in Figure 8 (162b, IC50 value- 0.23 ± 0.1 µM).

Scheme 11

Synthesis of tripeptide type cysteine protease inhibitors. Scheme 11, Scheme 11A: synthesis of tripeptide compounds 162a-e with aldehyde side chain; Scheme 11, Scheme 11B: synthesis of α -Ketoamide tripeptide cysteine protease inhibitor 163.

Synthesis of tripeptide cysteine protease inhibitors is divided into two parts: synthesis of tripeptide compounds 162a-e with aldehyde side chain (Scheme 11, Scheme 11 A) and second part involve the synthesis of α -Ketoamide tripeptide cysteine protease inhibitors (Scheme 11, Scheme 11 B). In the synthesis of tripeptide compounds 162a-e, the starting material 158 was synthesized based on previously published literature. First, the compound 158 was reacted with N-Boc-l-leucine 157 in the presence of EDCl to form compound 159. After that, reaction with different N-Cbz-amino acids 160a-e and previous compound 159 produces the tripeptide compounds 161a-e and further treatment with sodium borohydride and methanol converted to the final compounds 162a-e (Scheme 11, Scheme 11 A). In the second part of the synthesis, the starting material was 162b, which reacts with different conditions and reagents to transforms the final compounds 163 (Scheme 11, Scheme 11 B).

Potent dipeptide type of peptidomimetic inhibitors:

To develop a low molecular weight compound with 3CLpro inhibitory activity, Thanigaimalai et al designed a series of dipeptide-type compounds based on a previously reported lead compound C-terminal benzothiazolyl ketone containing dipeptide. Initial docking study involving binding interaction between the lead compound and 3CLpro, encouraged the replacement of P3 N-(3-methoxyphenyl) glycine with various rigid heterocyclic P3 moieties in the lead compound . The synthesis of the target compounds (172a-r) was achieved by assembling two key moieties: peptidics, 167 and C-terminal benzothiazole derivative, 171. Synthetic outline along with associated reaction conditions depicted in Scheme 12 : 12A and 12B. The inhibition constant value (Ki) of the compounds against 3CLpro were determined by accessing kinetic parameters in a fluorometric protease inhibition assay using constant substrate concentration. The IC50 value of only some potent inhibitor determined based on cleavage activity of R188I SARS-CoV 3CLpro on a synthetic peptide (H-TSAVLQSGFRK-NH2). The Ki/IC50 value was reported as a mean of three independent experiments. The SAR study revealed some potent inhibitors having Ki/IC50 value in the submicromolar to nanomolar range. The best potent inhibitor is compound 172a with Ki value, 0.006 µM (6 nM) and IC50 value, and 0.74 µM (Figure 8). Ki value of some compounds was also in good agreement with the binding affinity observed in the isothermal titration calorimetry (ITC) study.

Scheme 12

Synthesis of dipeptide cysteine protease inhibitors. Scheme 12, Scheme 12A: synthesis of N-protected amino acids 167a-r; Scheme 12, Scheme 12B: synthesis of dipeptide cysteine protease inhibitor 172a-r.

The synthesis of dipeptide cysteine protease inhibitors is divided into two parts: the first part involves the synthesis of peptide fragments 167a-r with carboxylic acid side chain (Scheme 12, Scheme 12 A) and the second part involves the synthesis of dipeptide cysteine protease inhibitor 172a-r (Scheme 12, Scheme 12 B). The peptide fragments 167a-r were synthesized via starting with compound 164 which reacted with different substituted carboxylic acid 165a-r under reaction conditions and formed the compounds 166a-r. Further, the compounds 166a-r reacted with trifluoroacetic acid to convert into final compounds 167a-r (Scheme 12, Scheme 12 A) . In the synthesis of dipeptide cysteine protease inhibitor 172a-r, the main starting compound is 168 and the compound was converted to the 169 by using the method given in published literature68, 87. Then, the compound 169 was further reacted with N,O-Dimethylhydroxylamine hydrochloride and formed the Weinreb amide 170. The compound 170 converted to compound 171 by coupling with benzothiazole in the presence of n-Butylamine. Lastly, the reaction of trifluoroacetic acid and previously synthesized compounds 167a-r produces the final dipeptide compounds 172a-r .

The –NH of the indole group along with the carbonyl moiety and the –NH group of the pyrolidin-2-one moiety comprises the S1 site, and this site is found to interact with the Glu166 residue. The –NH group of S2 and S3 interacted with Gln189 and His164 residues, respectively. The carbonyl moiety of S4 position interacted with Ser144 and the ketone group of the pyrolidin-2-one moiety was found to have an interaction with His163 residue of the protease (Figure 8).

Novel dipeptide type peptidomimetic inhibitors:

In search of novel low molecular weight SARS-CoV 3CLpro inhibitors Thanigaimalai et al designed some dipeptidic compounds based on their previously reported peptide lead compound Z-Val-Leu-Ala(-pyrrolidone-3-yl)-2-benzothiazole (Ki = 4.1 nM) by replacing P3 valine unit with varieties of substituents keeping P1 and P1 fixed. Synthesis of the designed dipeptide was achieved through a coupling reaction involving two intermediates, and N-protected amino acid esters 175a-f and a γ-lactam benzothiazole 179, those were synthesized in separate steps. The steps of synthesis depicted in Scheme 13 : 13A and 13B. The biological activity of the compounds was determined following a fluorometric protease inhibition assay against 3CLpro. Kinetic parameters were accessed at constant substrate concentration and inhibitor concentration varied to report Ki values. IC50 values were determined based on cleavage activity of SARS-CoV 3CL mutant protease (R188I) on a synthetic peptide substrate (H-Thr-Ser-Ala-Val-Leu-Gln-Ser-Gly-Phe-Arg-Lys-NH2). SAR study revealed two potential inhibitor 180a (Ki = 0.39 µM, IC50 = 10.0 µM) and 181b (Ki = 0.33 µM, IC50 = 14.0 µM). Further, a molecular docking study was also performed to understand the binding interaction of 180a with 3CLpro (Figure 8). The minimized energies of 181 m, obtained from the docking study, were −40.67 and −35.52 kcal/mol.

Scheme 13

Synthesis of dipeptide cysteine protease inhibitors. Scheme 13, Scheme 13A: synthesis of N-protected amino acids 175a-f; Scheme 13, Scheme 13B: synthesis of dipeptide cysteine protease inhibitor 180a-h, 181a-n, and 182a-d.

The synthesis of dipeptide cysteine protease inhibitors is divided into two parts, first part involves the synthesis of peptide fragments 175a-f with carboxylic acid side chain (Scheme 13, Scheme 13 A) and the second part involve the synthesis of dipeptide cysteine protease inhibitor 180a-h, 181a-n, and 182a-d (Scheme 13, Scheme 13 B). The peptide fragments 175a-f have been synthesized via compound 173a-f which reacted with different substituted carboxylic acid or benzyloxycarbonyl chloride compounds conditions and forms the compounds 174a-f. Further, the compounds 174a-f reacted with trifluoroacetic acid or lithium hydroxide under reaction conditions to convert into the final compounds 175a-f (Scheme 13, Scheme 13 A). In the synthesis of dipeptide cysteine protease inhibitor 180a-h, 181a-n, and 182a-d, the main starting compound is 176 and the compound was converted to the 177 by using published literature26, 31, 68, 37. Then, the compound 177 was further reacted with N,O-Dimethylhydroxylamine hydrochloride and formed the Weinreb amide 178. The compound 178 converted to the compound 179 by coupling with benzothiazole or 5-arylated thiazole in the presence of n-Butylamine or LDA. Lastly, reaction with trifluoroacetic acid and previously synthesized compounds 175a-f produces the final dipeptide compounds 180a-h, 181a-n, and 182a-d (Scheme 13, Scheme 13 B)26, 31, 68, 37 .

In silico studies revealed that the –NH group of the methoxy aniline, the ketone group and the –NH of pyrolidin-2-one comprising the S1 is found to interact with the Glu166 residue. The –NH group of S2 and S3 interacted with Gln189 and His164 residues, respectively. The carbonyl moiety of S4 position interacted with Ser144 and Cys145. In contrast, the pyrolidin-2-one moiety was found to interact with His163 residue of the protease (Figure 8).

Nitrile based peptidomimetic inhibitors:

In 2013, Wong and co-workers developed a four type of nitrile based broad-spectrum PIs for SARS-CoV 3CLpro. The four molecules were designed and synthesized with different N-terminal protective groups, such as, Boc (tert-butyloxycarbonyl), Cbz (carboxybenzyl), Mic (5-methylisoxazole-3-carboxyl) and different peptide length (Scheme 14 ). All four PIs 201, 202, 203, and 204 were evaluated by in vitro enzymatic assay against SARS-CoV 3CLpro and the four inhibitors showed potent inhibitory activity against the SARS-CoV 3CLpro with IC50 values ranging from 4.6 to 49 μM and among the compounds, 204 showed the best inhibitory activity against the SARS-CoV 3CLpro (IC50 = 4.6 μM) (Figure 8). Due to the best inhibitory effects towards SARS-CoV 3CLpro, the PIs 204 was taken for further study for broad-spectrum inhibition against different human coronavirus strains, such as 229E, NL63, OC43, HKU1 and infectious bronchitis virus. The study revealed that the inhibitor 204 has broad-spectrum activity and effective against SARS-CoV 3CLpro, with IC50 values ranging from 1.3 to 3.7 μM.

Scheme 14

Synthesis of nitrile-based peptidomimetic inhibitors.

The synthesis of nitrile based PIs was focused on: synthesis of different small peptide compounds containing Boc (tert-butyloxycarbonyl), Cbz (carboxybenzyl), and Mic (5-methylisoxazole-3-carboxyl) 195, 196, 199, and 200; and joined with peptide 187 to get final PIs 201, 202, 203 and 204. First, the synthesis of 187 starts via coupling of compound 183 and Gln(Trt)–OH in N,N’-dicyclohexylcarbodiimide N-hydroxysuccinimide; to form the compound 184 and reacts with ammonia, which converts the compound 184 to compound 185. Next, the primary amine transfer to the nitrile group via treatment with trifluoroacetic anhydride and formed the compound 186 and further deprotection of Boc can convert to the final peptide 187 (Scheme 14). Like previous compound 187 synthesis, the rest 195, 196, 199, and 200 intermediate peptides were formed via different reaction conditions shown in Scheme 14. These intermediate peptides were formed via reacting with different starting material, compound 188 and 192. In the last step, compound 187 was reacted with different previously synthesized peptides 195, 196, 199, and 200 to form the target compounds 201, 202, 203, and 204; via coupling the two peptides in mixed anhydride conditions.

The cyano group of the S1 site was found to interact with the Glu166, Phe140 residues. The amide group of S2 interacted with Cys145. Leu167 was found to interact with the dimethyl group of S3, the amide groups of S4 forms interaction with Thr190, Gln189 and the S5 site comprising the benzyl group interacted with Pro168 of the protease (Figure 8).

Tripeptide type of peptidomimetic inhibitors:

With an aim to develop novel peptidomimetics as inhibitors of SARS-CoV 3CLpro, Kanno et al designed a series of tripeptide-type compounds containing 4,5-dimethyl thiazole, 5-methyl thiazole, bezothiazole, and a series of 5-arylated thiazole at P1′ position, other substituents at P2 and P4 positions were also rigorously optimized to achieve the best fit for protease inhibition activity. The initial molecular modelling study involving a thiazolyl ketone-containing tripeptidic lead compound (of previous research work on SARS-CoV by the authors) and 3CLpro demonstrated a spacious requirement warhead group rather than a small thiazole in P1′ position, encourage to introduce those large groups. Synthetic outline of designed tripeptide-type compounds and associated reaction conditions depicted in Scheme 15 . The protease inhibitory activity of synthesized compounds evaluated using R188I SARS-Cov 3CLpro (SARS-Cov mutant protease) activity on a substrate (H-TSAVLQSGFRK-NH2). A fluorogenic substrate (Dabcyl-KTSAVLQSGFRKME-Edans) was also used for initial rate measurements. IC50 and/or Ki values were reported as the mean of three independent experiments. The study results revealed some compounds with benzothiazolyl moiety exhibit IC50 or Ki value in submicromolar to nanomolar range with two potential compound 214i (Ki = 4.1 nM) and 214p (Ki = 3.1 nM). The docking study of a potential compound, 214o of the series, showed strong binding interaction (Minimization energy: −43.65 kcal/mol) with protease enzyme than previously reported lead compound (Minimization energy: −37.56 kcal/mol). Docking structure was elucidated by X-ray crystallography (PDB ID: 1 WOF, Ki = 10.7 µM) (Figure 8).

Scheme 15

Synthesis of tripeptide type of peptidomimetic inhibitors. Scheme 15, Scheme 15A: synthesis of dipeptidic key fragment 209; Scheme 15, Scheme 15B: synthesis of tripeptide inhibitor 214a-u, 215a-d, and 216a-i; and Scheme 15, Scheme 15C: synthesis of tripeptide inhibitor-containing imidazole moiety 221a-c.

The synthesis outline of tripeptide type of PIs divided into three parts; the first part involves the synthesis of dipeptide key fragment 209 that has been synthesized with the help of peptide chemistry (Scheme 15, Scheme 15 A), the second part involves the synthesis of tripeptide inhibitor 214a-u, 215a-d, and 216a-i via synthesizing the key intermediate 213 (Scheme 15, Scheme 15 B), and third part involve the synthesis of tripeptide inhibitor-containing imidazole moiety 221a-c via synthesizing the key intermediate 220 (Scheme 15, Scheme 15 C).

The dipeptide key fragment 209 has been synthesized via starting with compound Wang-resin which reacted with different substituted Fmoc-amino acids and coupling reagent diisopropyl-carbodiimide under reaction conditions and formed the compound 206. To remove the F-moc group, the compound 206 has been reacted further with 20% piperidine in DMF and treated with Fmoc-valine to produce compound 207. Next, the compound 207 has been converted to compound 208 by reacting with different acid chlorides or acyl chlorides and 20% piperidine in DMF and further transferred to target fragment 209 by treating with trifluoroacetic acid under specific reaction conditions (Scheme 15, Scheme 15 A).

In the synthesis of tripeptide inhibitors 214a-u, 215a-d, and 216a-i, the main starting compound is 210 and the compound was converted to 211 by following the published literature method68, 87. Then, the compound 211 has been further reacted with N,O-Dimethylhydroxylamine hydrochloride under reaction conditions and formed the Weinreb amide 212. The Weinreb amide 212 has been further converted to the compound 213 by coupling with substituted benzothiazole or 5-arylated thiazole in the presence of n-Butylamine or LDA. Lastly, reaction with trifluoroacetic acid and previously synthesized compound 209 produces the final tripeptide compounds 214a-u, 215a-d, and 216a-i ( Scheme 15, Scheme 15 B)68, 87.

The synthesis of tripeptide inhibitor-containing imidazole moiety 221a-c has been started via commercially available compound 217. First, the compound 217 has been reacted with N,O-Dimethylhydroxylamine hydrochloride under reaction conditions and formed the respective Weinreb amide 218. Then, the Weinreb amide 218 has been treated with thiazole in the presence of n-Butylamine to produce compound 219 and further converted to compound 220 by reacting with tosyl chloride and triethylamine. Lastly, reaction with trifluoroacetic acid and previously synthesized compound 209 produces the final tripeptide inhibitors 221a-c (Scheme 15, Scheme 15 C) .

The dimethyl aniline group of the S1 site was found to interact with the Ala191 residue. The –NH group and the ketone of S2 interlinked with Glu166. Glu189, His41 residues were found to interact with the amide group of S3, the benzothiazole groups of S4 formed interaction with Cys145, Ser144 and Gly143 and the S5 site comprising the dimethyl group interacted with His163 of the protease (Figure 8).

Peptidomimetic containing cinnamoyl warhead:

In 2017, Vathan Kumar and co-workers have identified and evaluated six peptidomimetics as SARS-CoV 3CLpro inhibitors. They developed peptidomimetics with some modifications based on previous literature. The designed inhibitors were synthesized using previously published literature that was used in the synthesis of 3CLpro inhibitors for enterovirus. All the synthesized compounds were tested in vitro against SARS-CoV 3CLpro as well as MERS-CoV by using previously reported fluorescence assay. The biological evaluation data have reported some potent inhibitors against SARS-CoV 3CLpro, and compound 226d is one of them with an IC50 value of 0.2 ± 0.07 µM (Figure 8).

The synthesis of PIs containing cinnamoyl warhead and the related compound was synthesized based on previously developed literature. The synthesis strategy was divided into two parts: Scheme 16, Scheme 16 A, for the synthesis of intermediate 25; Scheme 16, Scheme 16 B, for the synthesis of target compounds 226a-d. At first, in the synthesis of intermediate compound 25, l-glutamic acid was used as a starting material and similar synthetic protocols were used which describe in Scheme 16, Scheme 16 A. Further, the intermediate compound 25 was coupled with Cbz-l-Phe under suitable reaction conditions to produce target peptide 222 and under reduction with Dess-martin periodinane and lithium hydride, the compound 222 converted into 223 (Scheme 16). In the final step, the respective compounds 222, 223, and 224 were reacted with substituted cinnamoyl derivatives 225a-d under specific reaction conditions to get desire compounds 226a-d (Scheme 16).

Scheme 16

Synthesis of peptidomimetics containing Cinnamoyl warhead. Scheme 16, Scheme 16A: synthesis of intermediate 25; Scheme 16, Scheme 16B: synthesis of target compounds 226a-d.

Peptidomimetic containing decahydroisoquinolin moiety:

With an aim to develop SARS-CoV 3CLpro inhibitor, Ohnishi K et al designed some compounds containing decahydroisoquinolin moiety with an additional substituent at non-prime site and warheads based on previous C-terminal aldehyde peptide inhibitor. Initially designed compound showed moderate activity, and the analysis of X-ray crystallographic structure of inhibitor in complexation with R188I SARS 3CLpro displayed the absence of interactions covering the P3 to P4 regions of substrate-based inhibitor, which led to the incorporation of a substituent at the non-prime site on the 4-position carbon of decahydroisoquinolin moiety. The synthetic outline and associated reaction condition for the compounds are depicted in Scheme 17 . The compound 247 (IC50 = 26 µM) exhibited clear but moderate inhibition potency on R188I SARS-CoV 3CLpro (Figure 8). The result of the study suggested increased interaction between protease and inhibitor 247 after incorporation of the substituent at the non-prime site. In addition, it is revealed from the study that the thioacetal warhead in association with decahydroisoquinolin moiety could be an efficient strategy for the design of potent SARS 3CLpro inhibitor.

Scheme 17

Synthesis of peptidomimetics containing Decahydroisoquinolin moiety. Scheme 17, Scheme 17A: synthesis of precursor molecule 233;Scheme 17, Scheme 17B: synthesis of precursor molecule Scheme 17, Scheme 17C: synthesis of intermediate compound 237;Scheme 17, Scheme 17D: synthesis of intermediate compound 242a, 242b, and 243; and Scheme 17, Scheme 17E: synthesis of target compounds including 247.

The synthesis of PIs containing decahydroisoquinolin moiety and the related compound was performed based on the retrosynthetic route. From the retrosynthetic analysis, it was revealed that a diol compound 227 is the starting compound for the synthesis of targeted derivatives. Hence, the synthesis of precursor molecule 233 is started from compound 227. The synthesis of decahydroisoquinolin derivatives has been divided into five parts, the first part has focused on the synthesis of precursor molecule 233 (Scheme 17, Scheme 17 A); the second part has focused on the synthesis of precursor molecule (Scheme 17, Scheme 17 B); the third part has focused on the synthesis of intermediate compound 237 (Scheme 17, Scheme 17 C); the fourth part has focused on the synthesis of intermediate compound 242a, 242b, and 243 (Scheme 17, Scheme 17 D); and the fifth part has focused on the synthesis of target compounds including 247 (Scheme 17, Scheme 17 E). Synthetic route of precursor compound 233 for the Pd(II) catalyzed cyclization given in Scheme 17, Scheme 17 A. Benzyl protection of one alcohol group and oxidation of other alcohol group to the aldehyde of diol 227, and then Wittig reaction deliver compound 228. Protection of a 1,2 diol with TBDMS group, obtained via asymmetric dihydroxylation of compound 228, yielded an alcohol 229 as a mixture of epimer. Amine 230 with Boc-protection was obtained via Mitsunobu reaction and catalytic hydrogenation of 229 in the presence of (Boc)2O. Mitsunobu reaction followed by reduction changed the alcohol group of 230 to amine group, which on acylation by p-bromobenzoic acid gave amide 231. Oxidation of primary alcohol group of 231 to an aldehyde, followed by Wittig reaction gave ether 232. The precursor compound 233 is obtained by reduction of 232 with DIBALH. After that with different amine configuration was achieved via synthetic Scheme 17, Scheme 17 B. The olefin presents at the terminal of 228 is transferred to epi-1,2-diol via asymmetric dihydroxylation by the use of different ligand that was used for 229. Protection of primary alcohol group with TBDMS yield as 8:1 diastereo mixture. Then the mixture in appropriate reaction conditions gave 234 as a single diastereomer, in addition to the required diastereomixture. To yield a single diastereomeric configuration of epi-13, the diol 234 was used. The compound 234 in a sequence of reaction gave compound 236. Then a single diastereomer was obtained via the desired sequence of reactions. Diastereoselective cyclization catalyzed by Pd(II) was then investigated (Scheme 17, Scheme 17 C). Bis-acetonitrile palladium chloride catalyzed cyclization of precursor 233 gave decahydroisoquinolin compound 237. Non-prime site substituents were introduced using compound 237. The preparation of P1 site fragment (242a-b) and nonprime site substituents covering P3 to P4 (2 4 3), to be incorporated on the decahydroisoquinolin moiety, depicted in Scheme 17, Scheme 17 D. For the synthesis of P1 site residue, an easily accessible Fmoc-His(Trt)–OH (2 4 0) was transferred to Weinreb amide, and reduction of the amide with DIBALH produced an aldehyde 241. The resulting compound 241 was then transformed to α, β-unsaturated ester or thioacetal, and under appropriate reaction conditions yield 242a or 242b. EDC/DMAP mediated coupling and additional debenzylation by catalytic hydrogenation of H-Gly-OBn and Ac-Thr-OH gave P3 to P4 fragment 243. Ultimately, the synthesized P1 site residue and P3 to P4 site substituent were introduced into the cyclic compound 237 (Scheme 17, Scheme 17 E). Boc group of 237 was detached using HCl, and then coupling of amine with 243 in presence of BOP yielded 244. Oxidation of terminal olefin followed by reductive coupling with 242b, the aldehyde product of 244 gave 245b. Then 245b was converted to 246b via elimination of the Trt group of imidazole in Histidine. To assess the influence of warheads on inhibitory potency, P1 residue 242a and H-His(Trt)-N(OMe)Me were similarly coupled with 244 to yield 246a and 246c. Treatment 246b with NBS changed the thioacetal to an aldehyde group and after purification of the product via preparative HPLC gave compound 247 .

Ketone-based covalent inhibitors:

In continuation of research efforts to develop novel molecule as a SARS-CoV 3CLpro inhibitor, Robert L. Hoffman et al pursued the design of ketone-based reversible and irreversible inhibitors based on previous Michael acceptor by changing warhead moiety and retaining P1 lactam, while P2 and P3 sites were randomly optimized. The synthesis of the designed compound and associated reaction condition depicted in Scheme 18 ; 18A, 18B, and 18C. The biological experiment was carried out following SARS-CoV-1 protease FRET assay and analysis, based on the proteolytic activity of 3CLpro on fluorogenic peptide substrate following sequence DABCY-LKTSAVLQ-SGFRKME-EDANS. Viral inhibition assay of the compounds was also carried out on SARS-CoV-1 and hCoV 229E. Two classes of potential inhibitors of SARS-CoV-1 have been developed viz. acyloxymethylketones and hydroxyethyl ketones. The biological evaluation data have reported some potent inhibitors against SARS-CoV 3CLpro under acyloxymethylketone derivatives, and compound 256 is one of them with an IC50 value of 0.017 ± 2 µM (Figure 8). Solving crystal structure of the ligand protein complex by X-ray crystallography analysis led to the identification of potent inhibitor PF-00835231 (IC50 = 4 ± 0.3 nM) with additional viral inhibition potential. Further, preclinical investigation of PF-00835231 revealed desirable pharmacokinetic property, which warrants the development of PF-00835231 as a drug candidate against COVID-19 patient.

Scheme 18

Synthesis of Ketone-based covalent inhibitors. Scheme 18, Scheme 18A: synthesis halomethylketone (HMK) intermediates 251, 252, 253, and 254; Scheme 18, Scheme 18B: Synthesis of chloromethylketone (CMK) intermediate 255; and Scheme 18, Scheme 18C: synthesis of target compounds including 274 and 275.

The halomethylketone (HMK) intermediates were obtained via a two-step synthetic procedure as given in Scheme 18, Scheme 18 A. Aminoester 248 on saponification yielded amino acid 249. The Reaction of acid 249 with isobutylchloroformate and trimethylamine produced mixed anhydride, which after reaction with diazomethane provide 250. Diazo ketone 250 was reacted with HCl for concurrent deprotection of nitrogen and transformation to the intermediate chloromethylketone (CMK) 253. On the other hand, reaction of 250 with stoichiometric amounts of 48% HBr in dichloromethane gave 254. Chloromethylketone (CMK) intermediate can be prepared differently by the treatment of 248 with too much LDA to produce 251. Conversion of 249 to a Weinreb amide, followed by reaction with Grignard of benzylchloromethyl ether produced oxymethylketone intermediate 252.

Substituted methyl ketone derivatives was achieved via generation of the fully elaborated chloromethylketone (CMK) intermediate 255 (Scheme 18, Scheme 18 B). Compound 253 in a series of reaction produce 255 in moderate yields without epimerization. Reaction of 255 with numerous carboxylic acids in a medium containing CsF gave acyloxymethylketones 256–271. The other derivatives were also obtained by following above synthetic procedure via substitution with appropriate functionalities.

The derivatives containing Hydroxymethylketone (HMK) functionality were obtained via two complementary processes depicted in Scheme 18, Scheme 18 C. Oxymethylketone intermediate 252 via a series of reactions yielded benzyl ether derivatives 274 in moderate yields. Moreover, by using above synthetic procedures along with the substitution of appropriate functionalities yielded other inhibitors.

The result obtained from the in silico study of compound 256 showed that the pyridine ring in the S1 site of the compound interacted with the Cys145 amino acid residue and that of the carbonyl group of S2 site interacted with the Cys145, Gly143, amino acid residues of the protease (Figure 8).

α‐Ketoamides derivatives:

Zhang et al in their research work, developed broad-spectrum antivirals against enterovirus, alpha coronavirus, and beta coronavirus. Because the protease of these virus shares a common active site architecture and an unique specificity for substrate recognition, they pursued a structure-based design of peptidomimetic α-ketoamides near equipotent against the three virus genera. The proteases targeted in this study specifically cleave peptide following glutamine residue in the P1 position, they have incorporated 5-membered γ-lactam ring (a derivative of glutamine) in P1 position, which causes ten-fold increase in potency of the inhibitors. Synthetic efforts were, therefore, aimed at optimizing the substituents at the P1′, P2, and P3 positions of the α-ketoamides. Chemical synthesis of α-ketoamides started with Boc protected l-glutamic acid dimethyl ester, synthetic outline and associated reaction conditions are depicted in Scheme 19 : 19A, 19B, and 19C . Synthesized compounds were tested against the recombinant viral proteases, viral replicons, and virus-infected cell cultures; most of them were non-toxic to the cell. The SAR study revealed that optimization at P2 substituent is important for achieving near equipotency against alpha coronavirus, beta coronavirus and enterovirus. Among the compounds, the best near equipotent compounds were 289u (P2 = cyclopentylmethyl) and 289r (P2 = cyclohexylmethyl). These compounds achieved the best fit between the different requirements for P2 substituent. However, in terms of viral protease inhibition activity, compound 289 s (IC50 = 0.24 μM) and 289n (IC50 = 0.33 μM) showed the best inhibition activity towards SARS-CoV 3CLpro (Figure 8). This study also demonstrated the fact that as there is a high similarity between the main protease of SARS-CoV and novel beta CoV (SARS-CoV-2/Wuhan/2019). All results reported here for the inhibition of SARS-CoV will most likely also apply to the new virus as well.

Scheme 19

Synthesis of α‐Ketoamides as peptidomimetic inhibitors. Scheme 19, Scheme 19A: synthesis of key lactam fragment 279; Scheme 19, Scheme 19B: synthesis of amino acids intermediates 283a-u, and Scheme 19, Scheme 19C: synthesis of α‐Ketoamides derivatives 289a-u by addition of two intermediates 279 and 283a-u.

The synthesis process of α‐Ketoamides derivatives has been divided into three parts, the first part involves the synthesis of key lactam fragment 279 (Scheme 19, Scheme 19 A), the second part involves the synthesis of amino acids intermediates 283a-u (Scheme 19, Scheme 19 B), and third part involves the addition of two synthesized intermediate compounds 279 and 283a-u; to produce the target compounds 289a-u (Scheme 19, Scheme 19 C) .

The key lactam fragment 279 has been synthesized via three steps which started with the N-Boc glutamic acid dimethyl ester 276 and reacted with bromoacetonitrile under specific reaction conditions to form compound 277. In the next step, the intermediate 277 has undergone a hydrogenation reaction and formed a cyclization product 278. In the last step, the cyclization product 278 has been reacted with trifluoroacetic acid to remove the Boc protection and transferred into final lactam fragment 279 (Scheme 19, Scheme 19 A).

Similarly, the second key amino acid intermediates 283a-u has been synthesized in two steps, which started with the substituted acyl chloride 280 and α-amino acid methyl ester 281. These two starting compounds have reacted with trifluoroacetic acid to produce compounds 282a-u and under alkaline hydrolysis, the compounds 282a-u have transferred into target amino acid intermediates 283a-u (Scheme 19, Scheme 19 B).

The synthesis of α‐Ketoamides derivatives has been started via the addition of two synthesized intermediates 279 and 283a-u. Under specific reaction conditions, these intermediates have transferred into compounds 284a-u. In the next step, the compounds 230a-u have undergoes a reduction reaction to form compounds 231a-u by reducing agent sodium borohydride and further reaction with Dess − Martin periodinane 285a-u has converted into 286a-u. In the next step, the compounds 286a-u has transferred into 287a-u by nucleophilic addition of isocyanides in acidic medium. The nucleophilic product 287a-u has converted into 288a-u by removing the acetyl group. In the last step, the alcohol group of 287a-u has converted to keto group by oxidation process with Dess − Martin periodinane and formed the α‐Ketoamides 289a-u (Scheme 19, Scheme 19 C).

In silico studies between inhibitor 289 s and 3CLpro revealed the interaction of cyclopropane of S1 site with amino acid residues such as: Gln189, Arg188, Asp187, Met49, and Met165 (Figure 8).

Small-molecule inhibitors (SMIs):

The chemical synthesis of SMIs under isatin derivatives, Chloropyridyl ester derivatives, pyrazolone derivatives, pyrimidine derivatives, macrocyclic inhibitors, 5-sulphonyl isatin derivatives, pyrazolone derivatives, serine derivatives, phenylisoserine derivatives, and Octahydroisochromene derivatives; are discussed below:

Isatin derivatives:

N-Substituted isatin derivatives were synthesized and evaluated in vitro against SARS-CoV 3CLpro by Li-Rung Chen and co-researchers. A series of isatin derivatives has been prepared from the reaction of isatin and various bromides (BrR4) in the presence of a strong base via a single-step process. The in vitro SARS-CoV 3CL protease assays have been conducted via FRET analysis for biological evaluation. The assay result showed that some compounds were potent and selective inhibitors with IC50 values ranging from 0.95 to 17.50 μM against SARS-CoV 3CLpro. The organic synthesis of isatin derivatives 291a-z is shown in Scheme 20 and among the synthesized compound, the most potent compound is 291a (IC50 = 0.95 μM), as shown in Figure 941.

Scheme 20

Synthesis of N-Substituted isatin derivatives.

The N-Substituted isatin derivatives 291a-z have been synthesized via a single-step process, which started with substituted isatin 290. Compound 236 reacted with different bromides in the presence of a strong base to obtain target compounds 291a-z ( Scheme 20 ). In compound 291a, the benzothiophene of the S1 site interacted with His164, the carbonyl groups of isatin of S2 site interacted with His41, Cys145, Gly143, and Iodine group of isatin of S3 site interacted with Phe140 amino acid residues of protease as detected in the in silico study (Figure 9 ).

Figure 9

Structure of small-molecule SARS-CoV 3CLpro inhibitors (291a, 294a, 300c, 306c, 316a, 328a, 337a, 348, 356, and 357b).

Chloropyridyl ester derivatives:

Chloropyridyl ester is a class of heteroaromatic organic molecule that was used previously in the development of SARS-CoV 3CLpro inhibitors. The first chloropyridyl ester-containing SARS-CoV 3CLpro inhibitor was reported by the Jianmin Zhang and their co-workers in 2007. Based on the previous history of Chloropyridyl ester as an antiviral potency, in 2008, Ghosh et al developed a series of SMIs containing Chloropyridyl ester and indole moiety, for SARS-CoV 3CLpro. Similarly, they have used another potent SARS-CoV 3CLpro inhibitor (i.e. Benzotriazole ester derivative) for designing the target molecule, which was developed by the Wong research group in 2006. They synthesized the designed SMIs via Scheme 21 . All the synthesized compounds were tested against SARS-CoV 3CLpro by using previously reported FRET enzyme assay as well as SARS-CoV cell-based assay. The assay result revealed that some Chloropyridyl esters were a potent inhibitor against SARS-CoV 3CLpro and among them, the most potent inhibitor is 294a (IC50 = 0.03 ± 0.01 µM, EC50 = 6.9 ± 0.9 µM) (Figure 9 ) .

Scheme 21

Synthesis of Chloropyridyl ester derivatives.Scheme 21, Scheme 21A: Synthesis of Chloropyridyl ester derivatives 294a-f; Scheme 21, Scheme 21B: conversion of indole compounds 294a-b to acetyl products 295a-b.

The Chloropyridyl ester derivatives 294a-f have been synthesized via a single step. Various carboxylic acid derivatives 292a-f were coupled with 5-chloro-3-pyridinol 293 in presence of DCC and DIMAP under appropriate reaction conditions and produces Chloropyridyl ester derivatives 294a-f (Scheme 21, Scheme 21 A). After synthesis of 294a-f, a few of the indole compounds 294a-b were converted to acetyl products 295a-b via acetylation reaction (Scheme 21, Scheme 21 B). They have also incorporated substituted phenyl sulphonyl group to the indole by direct sulfonamidation reaction but it has been found that the sulphonyl-indole Chloropyridyl products were not active in the anti-viral activity.

The binding interaction of compound 294a obtained by Gold dock with the enzyme as observed in the in silico study showed that the carbonyl group in the S2 site interacted with Gly143, Ser144, Cys145, the nitrogen of the chloropyridinyl in the S1 site is in close proximity with His163 residue of the macromolecule (Figure 9).

Pyrazolone Derivatives:

A major class of heterocyclic compounds that exist in many drugs and synthetic products are pyrazolone derivatives. Such molecules has excellent analgesic, antituberculosis, antifungal, antimicrobial, anti-inflammatory, antioxidant and anti-tumor functions. The pyrazolone structure plays an important role because of its easy preparation and rich biological activity and represents a fascinating template for combinatorial and medicinal chemistry. In 2010, Po-Huang Liang et al have reported a series of pyrazolone compounds having inhibitory activity against SARS-CoV 3CLpro. The compounds have been designed based on reported pyrazole containing drugs (like, Edaravone, a medicine that is effective for myocardial ischemia), synthesized and tested for anti SARS-CoV 3CLpro inhibitory activity by in vitro protease inhibition assay using fluorogenic substrate peptide in which several compounds exhibited potent inhibitions towards 3CLpro (Scheme 22 ). The most potent inhibitor is 300c with IC50 value 8.4 µM (Figure 9). Incidentally, one of the inhibitors also showed better effectiveness against Coxsackievirus B3 3C protease. They have also determined the cytotoxicity of the synthesized compounds using the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay and found that, none of them were cytotoxic at 200 µM.

Scheme 22

Synthesis of pyrazolone derivatives.

The pyrazolone derivatives 300a-u have been synthesized via two steps. The reaction started with substituted phenylhydrazine hydrochloride 296 and ethyl benzoylacetate 297 . These two starting materials have reacted in the presence of triethylamine to produce intermediate compounds 298 and further reaction with substituted aldehyde under desired reaction conditions, the compounds 298 has converted to the target compounds 300a-u (Scheme 22). The computational studies of compound 300c showed that the phenyl ring of the S1 site interacted with Met-49, Arg-188, and Gln-189; the 4-carboxy benzyl of the S2 site interacted with Gln-192 residues of protease as detected in Figure 9 .

Pyrimidine Derivatives:

In 2010, Ramajayam and co-workers developed a series of 2-(benzylthio)-6-oxo-4-phenyl-1,6-dihydropyrimidine derivatives as SARS-CoV 3CLpro inhibitors. They have been identified various heterocycles as a novel anti-SARS agent against SARS-CoV 3CLpro from high throughput screening. They have designed the compounds from the data of complete pharmacophore modelling, and synthesized some potential pyrimidine derivatives in two steps (Scheme 23 ) and tested in vitro against SARS-CoV 3CLpro, by using previously reported fluorescence assay. The biological evaluation data have reported some potent inhibitors against SARS-CoV 3CLpro, and compound 306c is one of them with IC50 value 6.1 ± 1.1 µM (Figure 9).

Scheme 23

Synthesis of pyrimidine derivatives.

The pyrimidine derivatives 306a-n has been synthesized via two steps. The reaction started with substituted benzaldehyde 301, ethyl cyanoacetate 302, and thiourea 303; all three compounds have reacted and converted into compounds 306a-n . In the next step, the compounds 304a-n were transferred to the target pyrimidine derivatives 306a-n by reacting with various halides 305 under appropriate reaction conditions (Scheme 23). In compound 306c, the para-nitro benzyl group of S1 site interacted with Gly-143 and Cys-145; the 4-chloro of the phenyl ring of S2 site interacted with Met-49 and Gln-189 amino acid residues of protease as detected in the in silico study (Figure 9).

Macrocyclic inhibitors:

Macrocycle is a large ring-shaped cyclic molecule with exciting features to interact with the intracellular proteins and were used as a potential drug candidate for many pharmacologically important targets through the inhibition of intracellular protein–protein complexes. Several compatible strategies were already developed over the past decade to synthesize and screen the macrocycle libraries for binding to pharmacologically important targets. Due to its important chemical features, such as higher affinity, higher metabolic stability, better membrane permeability, and selectivity towards proteins, macrocyclic inhibitors have attracted considerable attention to researchers in drug discovery. The first macrocyclic inhibitor (316a) for SARS-CoV 3CLpro has been designed and synthesized by Sivakoteswara and their research group in 2013. The macrocyclic inhibitor (316a) have been evaluated in vitro against different families of virus for anti-viral activity, such as picornaviridae, coronaviridae, and caliciviridae; which showed potent inhibition against 3CLpro of enterovirus (CVB3 Nancy strain), moderate inhibition against 3CLpro of norovirus and weak inhibition against 3CLpro of SARS-CoV with IC50 value 15.5 µM (Figure 9). The macrocyclic inhibitor (316a) has been synthesized via the macrocyclization process (Scheme 24, Scheme 24 C) by joining the previously synthesized intermediate compounds 309 and 312 through multi-step reactions (Scheme 24 : 24A and 24B).

Scheme 24

Synthesis of macrocyclic inhibitors. Scheme 24, Scheme 24A: synthesis of key acid fragment 309; Scheme 24, Scheme 24B: synthesis of azide intermediates 312, and Scheme 24, Scheme 24C: synthesis of macrocyclic inhibitors 316a-e by addition of two key intermediates 309 and 312.

The synthesis process of macrocyclic inhibitors 316a-e has been divided into three parts. The first part involves the synthesis of key acid fragment 309 (Scheme 24, Scheme 24 A), the second part involves the synthesis of acyclic intermediates 312 (Scheme 24, Scheme 24 B), and the third part the addition of two synthesized intermediate compounds 309 and 312; to produce the target compounds 316a-e (Scheme 24, Scheme 24 C) .

The key acid fragment 309 has been synthesized in two-steps by using previously published literature100, 101, 102, 103, which started with the Boc-propargyl-Gly-OH 307 and reacted with l-Leucine methyl ester hydrochloride under specific reaction conditions to form dipeptidyl ester 308. In the final step, the dipeptidyl ester 308 has been reacted with dry HCl under reaction conditions to remove the Boc protection (N-terminal) and further reaction with carbobenzoxy chloride transferred to the final cbz-protected acid fragment 309 (Scheme 24, Scheme 24 A).

Similarly, the second key azide intermediates 312 has been synthesized via two-step as described in previously published literature, which started with the Boc-Glu-ome 310 and NH2(CH2)N3 (n = 3). These two compounds have been coupled via EDCl mediated reaction and converted to compound 311. Further treatment with HCl in dioxane, the compound 311 has converted to final azide intermediate 312 (Scheme 24, Scheme 24 B).

The synthesis of macrocyclic inhibitors 316a-e has been started via the addition of two synthesized intermediates 309 and 312 and by coupling reaction conditions, the intermediates have transferred into acyclic compound 313. In the next step, the compound 313 has undergoes a cyclization reaction by applying click chemistry in the presence of Cu(I)Br/DBU and converted to cyclized product 314. The compound 314 further reacted with lithium borohydride in presence of THF to produce corresponding alcohol 315 and by oxidation of compound 315 with Dess–Martin periodinane produced the final macrocyclic compounds 316a-e (Scheme 24, Scheme 24 C)45, 104 .

5-sulphonyl isatin derivatives:

The substitution of a carboxamide group with a series of substituted sulfonamide moieties have been explored by Liu and co-workers in 2014 to enhance the inhibitory effect towards SARS-CoV 3CLpro. They have designed the target compounds based on previously reported noncovalent SARS-CoV 3CLpro inhibitors (N-substituted 5-carboxamide-isatin derivatives), which Zhou and co-workers developed in 2006. A series of designed compounds have been synthesized via multistep organic reaction (Scheme 25, Scheme 25 B) and screened by in vitro protease assay using fluorogenic substrate peptide. From the assay result, it has been found that several compounds have potent inhibitory activity against the SARS-CoV 3CLpro and among all the compounds, 328a showed the best inhibitory activity against the SARS-CoV 3CLpro with IC50 = 1.04 ± 0.01 μM (Figure 9).

Scheme 25

Synthesis of small-molecule inhibitors containing isatin moiety. Scheme 25, Scheme 25A: synthesis of isatin compounds 321 and 322; Scheme 25, Scheme 25B: synthesis a series of 5-sulphonyl isatin derivatives 328a-m.

The synthesis of isatin derivatives has been divided into two parts, the first part has focused on the synthesis of two compounds 321 and 322 (Scheme 25, Scheme 25 A); the second part has focused on the synthesis of series of 5-sulphonyl isatin derivatives 328a-m (Scheme 25, Scheme 25 B). The target compound 321 and 322 have been synthesized by reacting two corresponding starting material 317 and 318. Under reaction with hydroxylamine hydrochloride and chloral hydrate, the compounds 317 and 318 have converted to corresponding compounds 319 and 320. Lastly, reaction with concentrated sulfuric acid converts the compounds 319 and 320 to target isatin compounds 321 and 322 (Scheme 25, Scheme 25 A).

In the second part of the synthesis, a series of 5-sulphonyl isatin derivatives 328a-m has been prepared by referring to published literature107, 108. First, the isatin molecule 323 has been treated with chlorosulfonic acid that produces two compounds, 324 and 325. In the next step, both the compounds 324 and 325 have been isolated and reacted with a secondary amine under reaction condition and converted to 326. The compound 326 has directly converted to 327a-m by the treatment of secondary amine with high yields. In the last step, the compounds 327a-m have been alkylated with various bromides or iodides in the presence of sodium hydride to produce 5-sulphonyl isatin derivatives 328a-m ( Scheme 25, Scheme 25 B) .

The S1 site composed of the piperidine and the sulfonyl moiety was observed to fit in the hydrophobic pocket formed by Met49, Met165, Gln189, Arg188 residues, and the S2, which is in close proximity with the Cys145. The benzyl group present in S3 is near Phe140, Glu166, Leu141 residues. The two ketone groups and the nitrogen in S4 were also found to interact with Gly143, Asn142, Ser144 residues of the 3CLpro (Figure 9).

Pyrazolone derivatives:

Kumar et al have synthesized furfural-based pyrazolone derivatives and screened against SARS-CoV as well as MERS-CoV 3CLpro inhibitors as broad-spectrum antiviral agents. Neuraminidase (NA), is one of the key targets for the release of the viral particle from the cell surface. They have recently reported a new antiviral neuraminidase inhibitor for both N1 and N2 via cell-based antiviral assay. These NA inhibitors have some structural similarity with their previously reported SARS-CoV 3CLpro inhibitors. Due to these background data, they have screened and synthesized these NA analogues for SARS-CoV and MERS-CoV 3CLpro (Scheme 26 : 26A, 26B, 26C, and 26D). The biological activity showed that molecules have low micromolar inhibitory activity and could inhibit NA and 3CLpro enzymes as a dual target antiviral agent. Among all pyrazolone compounds, 337a has shown the best inhibitory activity against the SARS-CoV 3CLpro with IC50 = 5.8 ± 1.5 μM (Figure 9).

Scheme 26

Synthesis of pyrazolone derivatives. Scheme 26, Scheme 26A: synthesis of substituted furfural aldehyde fragment 332; Scheme 26, Scheme 26B: synthesis a series of pyrazolones derivatives 337a-s; Scheme 26, Scheme 26C: synthesis of compound 340; and Scheme 26, Scheme 26D: synthesis of compound 344.

The substituted furfural aldehyde fragment 332 has been synthesized by using published literature methods. At first, the diazonium salts 330 were synthesized by starting compound 329 and sodium nitrate under specific reaction conditions. Lastly, the diazonium salts 330 has been converted to final substituted furfural aldehyde fragment 332 by reacting with furfural and Copper (II) chloride (Scheme 26, Scheme 26 A).

In the second part of the synthesis, a series of pyrazolone derivatives 337a-s was prepared using a multistep organic reaction. To synthesize intermediate compounds 335a-f, the substituted hydrazines 333 and b-ketoesters 334 has been reacted in the presence of AcOH under reflux condition. Then, the intermediate compounds 335a-f have been converted to pyrazolone derivatives 337a-s by reacting with previously synthesized 336 (Scheme 26, Scheme 26 B) .

Similarly, another pyrazolone inhibitor 340 has been synthesized by one-step process via reaction of compounds 338 and 339; to explore the effect of aldehyde substitution (Scheme 26, Scheme 26 C). In the last part, another pyrazolone inhibitor 344 has been synthesized by a two-step process via the reaction of para-hydroxy benzaldehyde 283 and benzyl bromide under appropriate reaction conditions to produce compound 342. Further, compound 342 has been converted to final compound 344 by reacting with compound 343 under appropriate reaction conditions (Scheme 26, Scheme 26 D).

The S1 site composed of the chlorobenzoic acid moiety was observed to interact with Gly143, His163, Ser144, and Cys145 residues and the trimethyl group of S2 fit in the hydrophobic pocket formed by Met49 and Gln189. The carbonyl group of pyrazolone derivative present in the S3 was found to interact with the His41 residue of the 3CLpro (Figure 9).

Serine derivatives:

Konno et al has been aimed to design non-peptidyl SARS-CoV 3CLpro inhibitors based on previous lead compounds, tetrapeptide aldehyde Ac-Thr-Val-Cha-His-H and Bai’s inhibitors of SARS 3CLpro. They have designed a series of functionalized serine derivatives focusing on P1, P2, P4 site interaction of peptide aldehyde inhibitor with 3CLpro. The compounds have been generated through virtual screening on GOLD software with additional docking simulation. The synthetic outline and associated reaction conditions depicted in Scheme 27 , associated functionalities to prepare serine derivatives are given in Table 1, Table 2, Table 3 of their research article. The biological activity of the compounds has been evaluated using cleavage activity of SARS-3CL mutant protease, R188I, on a synthetic decapeptide with the S01 cleavage sequence as the substrate. In parallel with biological study, a cytotoxicity study of serine derivatives has also been performed on Hela cells. The SAR study, in combination with docking simulation, revealed two potential inhibitors (SK23) and (SK69), with d-configuration on serine template, have high protease selectivity. The potential compounds of this study practically do not have any cytotoxicity. The most potential inhibitor under serine derivatives is 348 (SK23) with IC50 = 30 µM (Figure 9).

Scheme 27

Synthesis of a series of serine derivatives.

Table 1

Summary of protein-inhibitor interaction for SARS-CoV 3CLpro.

Sl. No.	Compound no.	Derivatives	IC50 / Ki (µM)	Docking Study		Ref.
				PDB ID	Interaction (amino acid residues)Hydrogen = H Hydrophobic = Hb int.Covalent = (C)Van der waals int.
1	14a	Anilide analogues	0.06	1UK4	H = Ala46Pockets involved = Cys145, Ser144, His163, Phe140OrThr25, His41, Cys44, Thr45.	24
2	38c	Peptidomimetic α,β unsaturated esters	1	1c145a	H = Glu166, Gln189, Gln192	25
3	53	Glutamic acid and glutamine peptides	Ki = 116.1	N.A.	N.A.	26
4	69	Peptidomimetic (TG-0205221)	0.6	1Z1I	H = Gln189, His163, His164, Glu166, Cys145, Gly143, Phe140.C = Cys145Hb int. = Asn142, His41, Met165, Leu167, Pro168, Asp187, Met49, Ala191, Arg188, Thr190	27
5	83a	Phthalhydrazide peptide analogues	0.05	2A5K	H = His163, Ser144, Gly143, Cys145.Van der waals int. = Phe140, Glu166, Leu141, His172, Asn142,Hb int. = Cys145 (with the furan ring)	28
6	108a	Cinanserin analogues	1.06	1UJ1	Hb int. = Thr25, Leu27, Gly143, His41, Met49, Gln189, Met165, Cys145, Glu166, HIs163, Phe140	29
7	113c	Trifluoromethyl ketone containing peptides	10	1UK4	C = Cys145.H = Gly143, Glu166, Gln189.Hb int. = Met165, Met49, His163, Phe140, Pro168	30
8	137	Trifluoromethyl, benzothiazolyl or thiazolyl ketone containing peptidomimetics	Ki = 2.20 ± 0.8	1WOF	C = Cys145. H = His41, Ser144, Gly143, His163, Gln189, Glu166, His164.	31
9	149a	Michael type of peptidomimetics	5.7	3AW0	H = His163, Glu166, Thr190.	32
10	162b	Cysteine protease inhibitors	0.23 ± 0.1	N.A.	N.A.	33
11	172a	Potent dipeptide type of peptidomimetics	0.74	1WOF	H = Glu166, Gln189, His164, Ser144, His163	34
12	180a	Novel dipeptide type of peptidomimetics	10	1WOF	H = Glu166, Gln189, His164, Ser144, His163	35
13	204	Nitrile based peptidomimetics	4.6	3VB5	C = Cys145.H = Glu166, Phe140, Gln189, Thr190.Hb int. = Pro168	36
14	214o	Tripeptide type of peptidomimetics	0.65	1WOF	H = His41, Glu166, His163, Ser144, Cys145.Hb int. = Ala191	37
15	226d	Peptidomimetic containing Cinnamoyl warhead	0.2 ± 0.07	N.A.	N.A.	38
16	247	Peptidomimetic containing Decahydroisoquinolin moiety	26	4TWW	N.A.	39
17	256	Ketone-based covalent inhibitors	0.017 ± 2	6XHN	H = Gly143,Cys145C = Cys145	14
18	289s	α‐Ketoamides derivatives	0.24 ± 0.08	2BX4	H = Gln189, Phe140, Glu166, Gly143, Ser144, His41.Hb int. = Met49, Met165	40
19	291a	Isatin derivatives	0.95	1UK4	H = Gly143, Cys145, His41.Hb int. = His164, Phe140, Met165	41
20	294a	Chloropyridyl ester derivatives	0.03 ± 0.01	2HOB	H = Cys145, Ser144, Gly143	42
21	300c	Pyrazolone Derivatives	8.4	1UK4	H = Gly143, Gln192, Glu166.Hb int. = Met49, Arg188, Gln189	43
22	306c	Pyrimidines Derivatives	6.1 ± 1.1	1UK4	H = Gly143, Glu166, Cys145.Hb int. = Met49, Gln189	44
23	316a	Macrocyclic inhibitors	15.5	2ZU5	N.A.	45
24	328a	5-sulphonyl isatin derivatives	1.04 ± 0.01	1UK4	H = Gly143, Asn142, Ser144Hb int. = Met49, Met165, Gln189, Arg188, Cys145, Phe140, Glu166, Leu141	46
25	337a	Pyrazolone derivatives	5.8 ± 1.5	2ALV	H = His41, Ser144, His163, Cys145, Gly143,Hb int. = Met49, Gln189	47
26	348	Serine derivatives	30	3AW1	H = Cys145, Gln189.	48
27	356	Phenylisoserine derivatives	43	3AW1	H = Gln189, His41, Cys145, His164.	49
28	357b	Octahydroisochromene derivatives	95	4TWW	N.A.	50

N.A. = Not available.

Table 2

List of amino acid residues for SARS-CoV 3CLpro.

Function in SARS-CoV 3CLpro	Amino acid residues
Catalytic dyad	His41, Cys145
Ligand binding	Cys145, Glu166, Gln189, His163, Gly143, Ser144, Phe140, His41, Met49, His164, Met165, Asn142, Gln192, Pro168, Arg188, Thr190, Ala191, Leu141, Thr25, Ala46, Leu167, Asp187, His172, Leu27.

Table 3

Summary of cell-based assay for SARS-CoV 3CLpro.

Sl. No.	Compound no.	Derivatives	Cell-based assay		Ref.
			EC50 (µM)	Cell line
1	38c	Peptidomimetic α,β unsaturated esters	0.18	Vero E6	25
2	69	Peptidomimetic (TG-0205221)	0.6	Vero E6	27
3	256	Ketone-based covalent inhibitors	5 ± 1.4	Vero 76	14
4	289s	α‐Ketoamides derivatives	18.4 ± 6.7	Vero E6	40
5	294a	Chloropyridiyl ester derivatives	6.9 ± 0.9	Vero E6	42

A series of serine derivatives 348a-o, 349b-o, and 350a-c has been prepared using a three-step organic reaction. The synthesis has been started via a coupling reaction of Fmoc-Ser(tBu)–OH 345 and various amines (like cyclohexylamine, piperidine, morpholine, benzylamine, and cyclohexyl methylamine) under reaction conditions and transferred to the intermediate compounds 346a-e. Using different coupling reaction conditions (A, B, and C) and deprotection of t-butyl group, the compounds 288a-e have been converted to the 347a-o. In the last step, different acylation reaction conditions have been employed to convert the compounds 347a-o to the final serine derivatives 348a-o, 349b-o, and 350a-c (Scheme 27).

Results obtained from in silico study of compound 348 showed that the carbonyl group in the S1 site and amino group in S2 interacted with Cys145 and Gln189 amino acid residues of the enzyme, respectively (Figure 9).

Phenylisoserine derivatives:

Hiroyuki Konno, has designed and synthesized phenylisoserine derivatives from reported molecule tetrapeptide aldehyde (IC50 = 98 nM) and SK23 (IC50 = 30 µM) for SARS-CoV 3CLpro inhibition by the combination of SAR and docking simulation study. The molecular design of phenylisoserine derivatives has been started using three steps: serine to isoserine backbone, induction of the functionality at the P2 position, and SAR study. The inhibitory activity has been determined using a synthetic decapeptide as a substrate against SARS 3CL R188I mutant protease. The phenylisoserine containing inhibitors 356, 357a-e, 360, and 363 have been synthesized via multistep organic reaction as shown in Scheme 28 : 28A, 28B, and 28C. Amongst these synthesized inhibitors, the compound 356 has shown better inhibition against SARS-CoV 3CLpro with IC50 value 43 µM (Figure 9).

Scheme 28

Synthesis of phenylisoserine derivatives. Scheme 28, Scheme 28A: synthesis a series of phenylisoserine derivatives 356 and 357a-e; Scheme 28, Scheme 28B: synthesis of phenylisoserine containing inhibitor 360; Scheme 28, Scheme 28C: synthesis of phenylisoserine containing inhibitor 363.

The synthetic preparation of phenylisoserine derivatives 356 and 357a-e have been started via protection of three groups of the starting compound 351 amine, carboxylic acid, and hydroxyl and converted to compound 352. Next, the methyl ester of compound 352 has been saponified under suitable reaction conditions and further coupled with cyclohexylamine to prepare amide compound 353. Treatment with hydrogenation and cinnamic acid under reaction condition, the amide compound 353 has converted to cinnamoyl compound 354. The MOM group in compound 354 has been removed by treatment with trifluoroacetic acid to produce intermediate alcohol compound 355. From the compound 355, the phenylisoserine derivatives 356 and 357a-e have been prepared using appropriate reagents (Scheme 28, Scheme 28 A).

Similarly, two other phenylisoserine derivatives 360 and 363 have also been prepared. The synthesis of compound 360 started from compound 358 and converted to compound 359 by treatment with benzyl bromide as protection towards hydroxyl group, saponification, and introduction of cyclohexylamine under reaction conditions. In the last step, the compound 359 has transferred to the target compound 360 by hydrogenolysis process and coupling with cinnamic acid (Scheme 28, Scheme 28 B). The phenylisoserine derivatives 363 have also been prepared in two steps from the previous synthesized intermediate 361 (Scheme 28, Scheme 28 A) as a starting material. The MOM group in compound 361 has been removed by treatment with trifluoroacetic acid and reacted with cinnamic acid to produce compound 362. Further hydrogenolysis process and coupling with cinnamic acid have converted the compound 362 to target compound 363 ( Scheme 28, Scheme 28 C).

The binding interaction of compound 356 with the protease revealed the interaction of the ester group in S1 site of the compound with the amino acid residues Gln189, His41, Cys145 and that of keto in the S2 site interacted with His164 of the protease (Figure 9).

Octahydroisochromene derivatives:

Shin-ichiro Yoshizawa et al, conducted a study to design and synthesize novel nonpeptidyl inhibitors of SARS-CoV 3CLpro containing octahydrochromene scaffold based on previous peptide aldehyde inhibitor and nonpeptide inhibitor having decahydroisoquinolin moiety. However, in the previous work the effect of substituent on decahydroisoquinolin ring was not fully elaborated. In this study the effect of four substituents on 1-position of the octahydrochromene scaffold was rigorously analyzed. A novel hydrophobic core, octahydroisochromene scaffold has been introduced for interaction with the S2 pocket of the protease enzyme and substituents at 1-position was incorporated to form additional interactions.

Octahydroisochromene scaffold was built via Sharpless–Katsuki asymmetric epoxidation (SKAE) and Sharpless asymmetric dihydroxylation (SAD). The P1 site His-al and the substituent at 1-position was incorporated through subsequent reductive amination reactions. The synthetic outline and associated reaction condition for the construction of compounds are depicted in Scheme 29 . The inhibitory potency of the compounds (375a–d) with single diastereomeric configuration clearly indicates that a particular stereo-isomer of the octahydroisochromene moiety, (1S, 3S) 375b (IC50 = 95 µM), guides the P1 site imidazole, substituent at the 1-position and the warhead aldehyde of the fused ring to their proper pockets in the 3CL protease (Figure 9).

Scheme 29

Synthesis of Octahydroisochromene derivatives. Scheme 29, Scheme 29A: synthesis of key intermediate 369; Scheme 29, Scheme 29B: conversion of key intermediate 369 into the final product 372a-d; Scheme 29, Scheme 29C: synthetic route for the preparation of 375a, 375c and 375d.

The synthesis of SMIs containing octahydroisochromene moiety and the related compound was performed based on the retrosynthetic route. From the retrosynthetic analysis it was revealed that a diol compound 364 is the starting compound for the synthesis of targeted derivatives. Hence, the synthesis of precursor molecule 369 is started from compound 364. The key intermediate 369 was synthesized starting with compound 364 following the route shown in Scheme 29, Scheme 29 A. Protection of one hydroxyl group with TBDPSCl (tert-butyl diphenylsilyl chloride) and oxidation of another alcohol group via Ley-Griffith oxidation reaction using TPAP (tetrapropyl ammonium perruthenate) of diol 8, produced the corresponding aldehyde. The aldehyde without purification after reacting with (methoxymethyl) triphenylphosphonium chloride yielded 365. The reaction between 365 and 10-camphorsulfonic acid produced corresponding aldehyde. The isolated aldehyde after reaction with methyl triphenylphosphonium chloride gave terminal olefin. Removal of TBDPS group in the product upon treatment with TBAF (tetra-n-butylammonium fluoride) produced the intermediate 366. Oxidation of the primary alcohol in 366 via a Ley–Griffith oxidation reaction and homologation of the corresponding aldehyde through Horner–Wadsworth–Emmons (HWE) reaction using ethyl 2-(diethoxyphosphoryl) acetate produced 367.

Reduction of ethyl ester in 367 with DIBAL-H and stereo-specific oxidation of corresponding allylic alcohol via a SKAE reaction utilizing TBHP (tert-butyl hydroperoxide) mediated by (S,S)-diethyltartrate ((–)-DET) and titanium tetraisopropoxide (TTIP) gave 368 as a single diastereomer. The terminal olefin in 368 was oxidized via a Sharpless asymmetric dihydroxylation reaction under desired conditions. The production of the anticipated diol was confirmed by 1H NMR spectroscopy with the concurrent formation of a small proportion of the cyclic product 369 produced via the nucleophilic addition of the resultant alcohol. Then, desired cyclized product 369 was obtained via treatment of the mixture with p-toluenesulfonic acid without further purification.

The key intermediate 369 was transferred into the final product following the synthetic route displayed in Scheme 29, Scheme 29 B. Oxidative cleavage using sodium periodate followed by reductive amination of the corresponding aldehyde with four distinct amines by using NaBH3CN leads to aminated products. Methylation of aminated products by paraformaldehyde gave 370a-d. Oxidation of primary alcohol in the products and followed by reductive condensation of associated aldehyde with H-His-N(OMe)Me leads to Weinreb amides (371a–d). Reduction of each amide to aldehyde by treatment of DIBAL-H and followed by purification via LC/MS gave the required aldehyde products (372a–d). The synthetic route for the preparation (1-S, 3-R) 375a is depicted in Scheme 29, Scheme 29 C (i). The allylic alcohol produced after reducing the ester 367 was oxidized via Sharpless-Katsuki asymmetric epoxidation reaction in presence of (–)-DET as the chiral ligand to yield a single diastereoisomer 368. Oxidation of terminal olefin in 368 by Sharpless asymmetric dihydroxylation using a chiral ligand, (DHQ)2Pyr gave a primary product 369a .

Oxidative breakdown of the 1,2-diol in 369a and followed by reductive amination reaction using n-butyl amine in presence of paraformaldehyde were performed as illustrated for compound 370a. Finally coupling reaction with H-His-N(OMe)Me and following reduction by DIBAL-H was conducted as mentioned for compound 372a (Scheme 29, Scheme 29 C-i) yield 375a as the primary product. The diastereoisomer Compound 375b with the (1-S, 3-S) configuration was synthesized by using a combination of (DHQD)2Pyr and (–)-DET, those were employed in the SKAE reaction and the Sharpless asymmetric dihydroxylation reaction, respectively. Diastereoisomers, 375c and 375d, were synthesized with the (1-R, 3-R) and (1-R, 3-S) configurations, respectively; this was accomplished by using synthetic outline displayed in Scheme 29, Scheme 29 C-ii. To create the (1-R, 3-R) configuration in 13c, a combination of (+)-DET and (DHQ)2Pyr was utilized, those were used in the SKAE reaction (producing a single diastereoisomer) and the SAD reaction, respectively. In a similar way the (1-R, 3-S) configuration in 369d build using (+)-DET and (DHQD)2Pyr as ligand. Both the product then transferred to 375c and 375d following the given procedure.

Computational chemistry aspects:

Computational chemistry and pharmaceutical chemistry plays a tremendous role in drug discovery and development from the last decade. Nowadays, discovering lead molecules or identifying potential drug candidates for various diseases is one of the challenging scenarios for researchers. To overcome this challenge, the research groups are focusing on using pharmacoinformatic tools to minimize the time, cost, manpower, and errors for developing the potential drugs. By using the above tools, searching thousands of molecules from various databases and simultaneous identification of the best fit molecules for target protein becomes less tedious. The interactive information derived between proteins (macromolecule) and ligands can help the chemist to design and synthesize the most potent inhibitors against SARS-CoV by analyzing the SAR113, 114. Here, we have summarized the computational chemistry aspects of SARS-CoV 3CLpro inhibitors, including the various SARS-CoV 3CL proteases (PDB IDs), types of bond between the ligands and various amino acids of the protease, frequency of amino acid residues involved in the interaction (Table 1).

For virus replication and infection cycles, Mpro enzyme is critical, allowing it a perfect tool for the development of antiviral drugs. The binding site of 3CLpro comprises a catalytic dyad where a cysteine residue (Cys145) behaves as a nucleophile and a histidine residue (His41) serves either as general acid or base4, 115. The list of residues of amino acids that serve a vital part in ligand binding with the SARS-CoV 3CLpro are shown in Table 2. As found from various literature surveys amino acid residues and their percentage frequency are Cys145 (13.2%), Glu166 (11.1%), Gln189 (10.4%), His163 (8.3%), Gly143 (9%), Ser144 (6.3%), Phe140 (5.6%), His41 (5.6%), Met49 (4.9%), His164 (4.2%), Met165 (4.2%), Asn142 (2.8%), Gln192 (2.1%), Pro168 (2.1%), Thr190 (2.1%), Arg188 (1.4%), Ala191 (1.4%), Leu141 (1.4%), Thr25 (0.7%), Ala46 (0.7%), Leu167 (0.7%), Asp187 (0.7%), His172 (0.7%), Leu27 (0.7%) plays a vital role for the various interactions which includes hydrogen-bonding (55.9%), covalent (3.4%), hydrophobic (37.2%) and van der waals interactions (3.4%) in Figure 10, Figure 10 A. It was observed that the 11 frequent amino acid residues are mainly responsible for the interactions, which include Cys145, Glu166, Gln189, His163, Gly143, Ser144, Phe140, His41, Met49, His164, Met165 (Figure 10, Figure 10 B).

Figure 10

Amino acid residues predominantly involved in protein–ligand interactions. 10A. Amino acid residues occurrence in percentage. 10B. This figure represents the 11 most crucial amino acids responsible for interaction with the ligands present in the SARS-CoV 3CLpro (PDB ID: 1UK4) with 5-mer peptide of inhibitor (10C).

Cell-based assay:

Cell-based therapies can revolutionize contribution to currently unfulfilled healthcare problems, hence making effective manufacturing critical. Before any of this could become a possibility, numerous barriers must be solved, as well as a deeper understanding of the specific requirements for cell-based technologies. Vero E6, Vero 76, and other commonly known cell lines may be utilized for virus replication in vitro with evident cytopathic effects. The cytopathic effect of several viruses is known to be mediated by virus-induced apoptosis or cell necrosis. The 'Vero' cells that are used, are extracted from kidney epithelial cells of African green monkey. Vero E6 cells have been widely employed in SARS-CoV research in cell culture-based infection models since 2003, owing to their ability to sustain viral replication to high titers, due to their higher expression of the ACE-2 receptor. Translation cell-based assays for SARS-CoV had given proper insight into the mechanism of action of the potential inhibitors that are may be helpful for the drug discovery process in the near future117, 118. Table 3 shows the activities of cell-based assays of various inhibitors that are used in this review.

Conclusion & future perspective:

3CLpro is an important drug target for SARS-CoV, as virus-encoded protease involves the processing of the two viral polyproteins and responsible for the viral replication and infection process. This review deals with the chemical synthesis and medicinal chemistry perspectives of PIs and SMIs targeting SARS-CoV 3CLpro, which has been chemically synthesized and biologically screened as newer derivatives from 2004 to 2020 for therapeutic inventions of SARS-CoV. In this overview, we have also identified the most potent compounds from each derivative and highlighted their structural features and binding modes.

In the current scenario, the warhead-based strategy plays a high impact in the medicinal chemistry for the development of SMIs and PIs. Most of the PIs and SMIs for SARS-CoV 3CLpro have been designed by applying a warhead-based strategy, but few inhibitors have shown better inhibition and potency. Most of the inhibitor has been evaluated via in vitro SARS-CoV 3CL protease assays using FRET analysis. The majority of PIs have not been studied further for in-vivo evaluation due to their less optimal physicochemical characteristics and undesirable frameworks.

In the development of potential compounds against SARS-CoV 3CLpro, it has been observed that the first synthetic peptidomimetic compounds are under keto-glutamine analogue. It has been designed and synthesized in 2004 from previously reported anti-viral compounds like hepatitis virus 3C and human rhinovirus 3C inhibitors (Figure 6). Compound 9b is an example of a peptidomimetic inhibitor-containing cyclic keto-glutamine moiety that showed better inhibition against SARS-CoV 3CLpro with IC50 value 0.60 µM. Comparing the molecular docking study, inhibitor 9b is the only compound under SARS-CoV 3CLpro inhibitors studied with human rhinovirus-14 (PDB ID: 1CQQ).

From the keto-glutamine analogues other peptidomimetic inhibitors have been explored during this timeframe for the development of potential drug candidates, such as peptidomimetic containing α,β-unsaturated esters, anilide analogues, glutamic acid–glutamine peptides, and phthalhydrazide peptide; to improve the biological efficacy and potency against SARS-CoV 3CLpro. The chemical synthesis of many SMIs is also discussed in this review. The majority of inhibitors showed micromolar potency against SARS-CoV 3CLpro. The most promising inhibitors under SMIs are 291a, 294a, 300c, 306c, 316a, 328a, 337a, 348, 356, and 357b.

From the computational chemistry and protein interaction information’s data of SARS-CoV 3CLpro inhibitors, it may be suggested that peptidomimetics (covalent and non-covalent inhibitors) and SMIs exhibited specific and effective active-site inhibition. The protein-drug interaction of inhibitors 14a, 38c, 69, 83a, 108a, 113c, 137, 149a, 172a, 180a, 204, 214o, 256, 289 s, 291a, 294a, 300c, 306c, 328a, 337a, 348, and 356 has been also analyzed with different PDB IDs, except inhibitors 53, 162b, 226d, 247, 316a, and 357b (Table 1).

3CLpro inhibitors have been extensively studied, enabling the production of a percentage frequency graph that displays the most critical amino acid residues detected for interaction with the ligands. From this, it was verified that these compounds interact via hydrogen-bonding (55.9%), covalent (3.4%), hydrophobic (37.2%), and van der waals interactions (3.4%), where the Cys145 residue is the most frequent to exhibit hydrogen bond formation. His41-Cys145 is a part of a catalytic dyad. Met165 residue exhibits the most frequent hydrophobic interacting sites. Cys145 is the amino acid residue for which the covalent interaction is responsible. The 11 frequent amino acid residues responsible for the interactions includes Cys145, Glu166, Gln189, His163, Gly143, Ser144, Phe140, His41, Met49, His164, and Met165 that are discussed earlier in the medicinal chemistry perspective section and will be crucial for drug development process in future.

However, some of the peptidomimetics showed less inhibition activity against the SARS-CoV 3CLpro and poor absorption, distribution, metabolism, and excretion parameters to be a drug-like molecule. Peptidomimetic has few drawbacks over the SMIs, like high molecular weight, poor pharmacokinetic properties and pharmacodynamic property. To develop potential 3CLpro inhibitors against coronavirus, the strategy should be focused on developing low-molecular weight peptidomimetic inhibitors and its lead optimization for improvement of pharmacokinetic and pharmacodynamic properties. This project may soon emerge as a pioneer in the discovery of SARS-CoV 3CLpro inhibitors to succeed in the fight against coronavirus.

Declaration of Competing Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.