The recent outbreaks of human coronaviruses: A medicinal chemistry perspective

Abstract Coronaviruses (CoVs) infect both humans and animals. In humans, CoVs can cause respiratory, kidney, heart, brain, and intestinal infections that can range from mild to lethal. Since the start of the 21st century, three β‐coronaviruses have crossed the species barrier to infect humans: severe‐acute respiratory syndrome (SARS)‐CoV‐1, Middle East respiratory syndrome (MERS)‐CoV, and SARS‐CoV‐2 (2019‐nCoV). These viruses are dangerous and can easily be transmitted from human to human. Therefore, the development of anticoronaviral therapies is urgently needed. However, to date, no approved vaccines or drugs against CoV infections are available. In this review, we focus on the medicinal chemistry efforts toward the development of antiviral agents against SARS‐CoV‐1, MERS‐CoV, SARS‐CoV‐2, targeting biochemical events important for viral replication and its life cycle. These targets include the spike glycoprotein and its host‐receptors for viral entry, proteases that are essential for cleaving polyproteins to produce functional proteins, and RNA‐dependent RNA polymerase for viral RNA replication.

The interruption of any stage of the viral life cycle can become an important therapeutic approach for treating CoV-related diseases. A recent SARS-CoV-2-human protein-protein interaction analysis showed that SARS-CoV-2 contains approximately 66 druggable proteins, each of which has several ligand binding sites. 25 The most interesting coronavirus proteins are the S glycoprotein, proteases M pro and PL pro , RdRP, and helicase. In this   36 It is worth highlighting that a similar strategy could work for the new SARS-CoV-2. The recently solved cryo-EM structure of SARS-CoV-2 in complex with human ACE2 can provide a structural rationale for the peptide design. 29 For viral entry, MERS-CoV uses its spike protein (S) to interact with the host-receptor DPP4, [37][38][39] also known as adenosine deaminase-complexing protein-2 or CD26. 37 MERS-CoV was also the first virus reported to use this particular path. 35,37 DPP4 is a type II transmembrane glycoprotein, that forms homodimers on the cell surface, and it is involved in the cleavage of dipeptides. 37,40 In humans, DPP4 is predominantly found on the bronchial epithelial and alveolar cells in the lower lungs. 40 A heptad repeat (HR) is a repeating structural pattern of seven amino acids. A crucial membrane fusion framework of SARS-CoV is the 6-helix-bundle (6-HB) that is formed by HR1 and HR2 of the viral S protein.
Enfuvirtide (T-20) is an FDA approved HR2 peptide and the first HIV fusion inhibitor. It has opened up new avenues toward identifying and developing peptides as viral entry inhibitors. Such molecules represent a promising strategy against enveloped viruses with class 1 fusion proteins such as Nipah virus, Hendra virus, Ebola virus, and other paramyxoviruses, simian immunodeficiency virus, feline immunodeficiency virus, and respiratory syncytial virus. [43][44][45][46] The HR regions of SARS-CoV-1 and SARS-CoV-2 S protein share a high degree of conservation, and such fusion inhibitors have potential applications in preventing SARS-CoV-2 entry.
Small molecule entry inhibitors, on the other hand, are reported to target the RBD. Compared to peptides, proteins, and biologics, small molecules have several advantages due to lower production costs, improved pharmacokinetics, stability, and dosage accuracy. Sarafianos et al. identified the oxazole-carboxamide derivative SSAA09E2 (1; Figure 4) as an entry inhibitor against SARS-CoV-1 by screening a chemical library composed of 3000 compounds. 47 This inhibitor directly blocks ACE2 recognition by interfering with the RBD with an EC 50 value of 3.1 µM and a 50% cytotoxic concentration (CC 50 ) value of greater than 100 µM, not affecting ACE2 expression levels. 48 Xu et al. 49 identified two small molecules, TGG (2; Figure 4) and luteolin (3; Figure 4), that can bind avidly to the SARS-CoV-1 S2 protein and inhibit its entry into Vero E6 cells (EC 50   and block viral entry into host cells. 51 Imatinib (6; Figure 4), an Abelson kinase inhibitor, could inhibit CoV S protein-induced fusion with an EC 50 value of 10 µM and showed no cytotoxic effects in Vero cells up to 100 µM concentration. 52,53 2.2 | Inhibitors targeting the cellular receptor The genetic code of SARS-CoV-2 shares noticeable similarities with SARS-CoV-1, which caused the SARS epidemic in 2002. 26,54 More importantly, both viruses have identical mechanisms of infection. SARS-CoV-1 uses the host's ACE2 as a portal to infect cells, which has high expression in the vascular endothelium 55 and the lung, particularly in type 2 alveolar epithelial cells. 56 SARS-CoV-2 shares 76% of its spike (S) protein with SARS-CoV-1. Despite a few amino acid differences in its RBD compared to the SARS-CoV-1 S protein, the SARS-CoV-2 S protein binds to ACE2 with even greater affinity 27 offering an explanation for its greater virulence and preference for the lung.
ACE, a highly glycosylated type I integral membrane protein, is an essential component of the reninangiotensin (Ang) system, which controls blood pressure homeostasis. Both ACE1 and ACE2 cleave Ang peptides.
However, they differ markedly: ACE1 cuts and converts the inactive decapeptide Ang I into the octapeptide Ang II by removing the dipeptide His-Leu. This Ang II induces vaso-and bronchoconstriction, increased vascular permeability, inflammation, and fibrosis, thus promoting acute respiratory distress syndrome (ARDS) and lung failure in patients infected with SARS-CoV-1 or SARS-CoV-2 57 ( Figure 5). Therefore, ACE-inhibitors (ACEis) and angiotensin II receptor blockers (ARBs) could block the disease-propagating effect of Ang II. [58][59][60] ACE2, on the other hand, is a zinc-containing metalloenzyme, and shares merely 42% of its amino acid sequence with ACE1. 61 It cleaves only one amino acid residue (Leu or Phe) from Ang I and Ang II, respectively, generating Ang (1-9) and Ang (1-7) (a vasodilator) ( Figure 5). Thus, ACE2 has been considered a potential therapeutic target for cardiovascular diseases.
Virtual screening combined with a molecular docking approach targeting the ACE2 catalytic site with around 140 000 compounds led to the identification of inhibitor N-(2-aminoethyl)-1 aziridine-ethanamine (7; Figure 6) with an IC 50 value of 57 µM and a K i of 459 µM. However, no information about the cytotoxicity of this compound is available so far. 62 Chloroquine (8; Figure 6) is a relatively safe, cheap, and effective medication for the treatment of malaria and amebiasis. Savarino et al. 63 reported its antiviral effects. At a molecular level, it increases late endosomal and lysosomal pH, resulting in impaired liberation of virions from endosomes or lysosomes. The virus is therefore unable to release its genetic material into the cell and replicate. 64,65 Furthermore, they hypothesize that chloroquine might block the production of proinflammatory cytokines (such as IL-6), thereby blocking the pathway that subsequently leads to ARDS. 63 Chloroquine is reasonably active in vitro against SARS-CoV-1, MERS-CoV, and SARS-CoV-2. It was found to inhibit SARS-CoV-2 with an EC 50 value of 5.47 µM in vitro. 66 Antiviral activity against SARS-CoV-1 was reported with an IC 50 of 8.8 μM in Vero cells, but it is unclear how this translates into activity in respiratory epithelial cells and in vivo. 67,68 Mechanistic studies of chloroquine for SARS-CoV-1 infection revealed that it could also weaken the interaction between the RBD of SARS-CoV-1 and ACE2 by interfering with terminal glycosylation of ACE2, thereby reducing its affinity to SARS-CoV-1 S. 69 During the SARS-CoV-2 pandemic, chloroquine has been recommended by Chinese, South Korean, and Italian health authorities for the experimental treatment of COVID-19, 70,71 despite contraindications for patients with heart disease or diabetes. 72 However, health experts and agencies like the US FDA and European Medicines Agency warned against broad uncontrolled use after reports of misuse of low-quality versions of chloroquine phosphate intended for fish.
Hydroxychloroquine (9; Figure 6) is being studied as an experimental treatment for COVID-19. 73 However, the benefits of treatment with this drug are unclear. 74 Hydroxychloroquine was found to inhibit SARS-CoV-2 with an EC 50 value of 0.74 µM in vitro. 66 Some studies imply synergistic effects of hydroxychloroquine and azithromycin. Azithromycin is active in vitro against Zika and Ebola virus 75,76 and can be used to guard against life-threatening bacterial superinfections when administered to patients suffering from viral infections. 77 A small study that compared hydroxychloroquine monotherapy and combination treatment with azithromycin found a significant advantage of the combination. While evaluating the efficacy of therapeutic intervention with hydroxychloroquine as monotherapy and its impact in combination with azithromycin, the number of patients testing negative in polymerase chain reaction (PCR) tests was substantially different in the two groups with 100% of patients cured (6 days post inclusion) in the combination arm of the study versus 57% in the monotherapy group. At the same time, 12% of patients in the control group receiving only standard care were cured. 78,79 The WHO declared on 18 March that chloroquine and its derivative hydroxychloroquine will be among the four medicines studied in the solidarity clinical trial 80 for the treatment of COVID-19. In April 2020, the US National Institutes of Health (NIH) also commenced a study with the drug for treating COVID-19 patients. 81 The recent clinical trial involving 96 032 patients with COVID-19 concluded that it was unable to confirm a benefit of hydroxychloroquine or chloroquine, when used alone or in combination with a macrolide such as  azithromycin (or clarithromycin). 82 The study actually reported decreased survival rates for patients treated with each of these drug regimens. Additionally, patients had an increased risk of developing ventricular arrhythmia under treatment. However, still more evidence is needed to adequately assess the drugs' risks or benefits for the treatment or prevention of COVID-19 (it is important to note that chloroquine and hydroxychloroquine are still considered safe treatment options in certain autoimmune diseases and malaria). Besides, the WHO announced the premature pause of its clinical trials using hydroxychloroquine as a safety precaution on 24 May 2020.
On a different note, it was found that ACE2 undergoes proteolytic shedding; releasing an enzymatic ectodomain during viral entry. 83 A disintegrin and metalloproteinase (ADAM), also known as TNF-α converting enzyme (TACE), assisted the shedding regulation of ACE2. Inhibition of this enzyme led to reduced shedding of ACE2.
GW280264X (10; Figure 6) was found to be a specific inhibitor of ADAM-induced shedding of ACE2 at 1 nM. 84 Two TACE inhibitors, TAPI-0 (11) and TAPI-2 (12; Figure 6), reduced ACE2 shedding, with IC 50 values of 100 and 200 nM, respectively. 83 MLN-4760 (13; Figure 6) inhibited the catalytic activity of ACE2 with an IC 50 of around 440 pM. 85 This is the most potent and selective small-molecule inhibitor against soluble human ACE2 described to date, thus making it a very promising candidate for SARS-CoV-2 interference. It binds to the active site zinc and emulates the transition state peptide. However, no antiviral data for this compound is available at this time.
The interference of a virus-host cell fusion, which is mediated by the viral S protein to its receptor ACE2 on host cells, may be a viable prevention strategy. Umifenovir (14; brand name Arbidol), a broad spectrum antiviral drug used against influenza, prevents viral entry by inhibiting virus-host cell fusion. 86 It is currently being investigated in a clinical trial for the treatment of SARS-CoV-2. 87,88 Do ACEis or ARBs amplify SARS-CoV-2 pathogenicity and aggravate the clinical course of COVID-19? After ACE2 was recognized as the SARS-CoV-2 receptor, 14,29 speculations emerged about potentially negative consequences F I G U R E 6 Inhibitors for SARS-CoV-1 and 2 targeting ACE2. ACE, angiotensin-converting enzyme; SARS-CoV, severe acute respiratory syndrome coronavirus of ACEi or ARB therapy in COVID-19 patients. This theory caused confusion in the public and alarmed patients taking these medicines. One report said that the expression of ACE2 was increased in patients with heart disease compared to healthy individuals. It was also insisted that ACE2 expression could be increased by taking ACEis and ARBs, 89 although there is no supporting report of this happening in the lungs.
In another report, it was suggested that patients suffering from high blood pressure receiving "ACE2increasing drugs" have a higher risk for severe COVID-19, since ACEis and ARBs could elevate levels of ACE2. 90 A joint declaration by the presidents of the HFSA/ACC/AHA on 17 March 2020, 91 followed by a similar statement of the European Medicines Agency, 92 clarified that there was no scientific basis for stopping ACEi or ARB therapy. [93][94][95] This was in accordance with the editors of the New England Journal of Medicine. 96 In case of SARS-CoV, the experimental data showed that such medications may be beneficial rather than damaging, which led to a new therapeutic approach for lung diseases. 97

| Proteolytic processing inhibitors
CoVs enter the host cells via both clathrin (endosomal) and nonclathrin pathways (nonendosomal); however, both pathways are dependent upon receptor binding. 98,99 The clathrin-mediated pathway involves the binding of CoV S protein to the host receptor followed by the internalization of vesicles that maturate to late endosomes. Acidification of the endosome promotes the H +dependent activation of cellular cathepsin L proteinase in late endosomes and lysosomes, which cleaves and activates the S protein, thus initiating viral fusion. Recent research shows that in addition to ACE2 SARS-CoV-2 can also use the host cell receptor CD147 to gain access into host cells. 100 Membrane fusion is also the crucial step for the CoV life cycle in the nonclathrin/endosomal route, in which host proteases such as cathepsin L, TMPRSS2, and TMPRSS11D (airway trypsin-like protease) cut the S protein at the S1/S2 cleavage site to activate the S protein for membrane fusion. 101 Interference with this process by targeting these proteases could become an attractive strategy for combating CoV infections. A recent study confirms the role of TMPRSS2 for the viral life cycle in SARS-CoV-2-infected VeroE6 cells. 5 Furin (a serine endoprotease) activates MERS-CoV to initiate the nonclathrin mediated membrane fusion event. 102 The neurotransmitter receptor blockers chlorpromazine (15), promethazine (16), and fluphenazine (17; Figure 7), were reported to inhibit MERS-CoV and SARS-CoV-1 most probably by impeding S protein-induced fusion. 103 Chlorpromazine, a clathrin-mediated viral entry inhibitor, was already described to inhibit human CoV-229E, hepatitis C virus, infectious bronchitis virus, as well as mouse hepatitis virus-2 (MHV2). 104 Recently, K11777 (19; Figure 8), a cysteine protease inhibitor, was shown in tissue cultures to inhibit SARS-CoV-1 and MERS-CoV replication in the subnanomolar range. 110,111 Future tissue culture and animal model studies should be conducted to clarify, whether its antiviral activity is mediated by targeting TMPRSS2.
Teicoplanin is a glycopeptide antibiotic used to prevent infections with Gram-positive bacteria like methicillinresistant Staphylococcus aureus and Enterococcus faecalis. It was found that teicoplanin inhibits the entry of PILLAIYAR ET AL.

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SARS-CoV-1, MERS-CoV, and Ebola virus by specifically targeting cathepsin L. 112 This knowledge has also been used to block the entry of new SARS-CoV-2 pseudoviruses with an IC 50 value of 1.66 µM. Therefore, teicoplanin could be considered a potential candidate for the treatment of COVID-19. 113

| Small-molecules as cathepsin L inhibitors
Human cathepsin L is a cysteine endopeptidase and plays a key role for infection efficiency by activation of the S protein into a fusogenic state to escape the late endosomes. Targeting this protease with small molecules could interfere with virus-cell entry and therefore be a possible intervention strategy for CoV infection. 114 Bates et al. II (residues 102-184), which are β-barrel domains that shape the chymotrypsin-like structure, while domain III (residues 201-306) is made up by α-helices. 124 The CoV M pro active site uses a catalytic dyad (Cys145-His41), in which cysteine acts as the nucleophile in the proteolysis while histidine behaves as general acid-base. The peptide substrate or inhibitor binds in a cleft between domains I and II. 125 As far as the development of new therapeutics against SARS-and MERS-CoV infection is concerned, efforts have mainly focused on protease inhibitors. These enzymes are highly attractive drug targets because they are so essential to the virus. Peptides, peptidomimetics, and even small molecules can inhibit them, which leads to markedly reduced viral transmission and pathogenicity. Although most of the reported molecules display only weak anti-CoV activity, several of studies elucidated structure-activity relationships that can be used to further improve their activity. 100,126-128

| Substrate-derived M pro inhibitors
To date, no approved drugs or vaccines are available for treating a coronavirus infection. In a race to identify chemotherapeutic options, various approaches, such as chemical synthesis, testing of natural products, and virtual screening of compound libraries, have been used. The systematic design of inhibitors of CoV M pro was essentially based on the enzyme's substrate. In general, a substrate can be transformed into a good inhibitor by modifying part of its sequence such that it binds to the catalytic cysteine in either a reversible or an irreversible manner. Peptide inhibitors are designed by attaching a reactive group (also known as warhead group) to peptides that mimic the natural substrate. The partial peptide substrate sequence for SARS-CoV-1 M pro is mentioned in Figure 10, indicating the specific subsite of each amino acid residue.

| Inhibitors with Michael acceptor as a warhead group
The disclosure of the first crystal structure of the SARS-CoV-1 M pro in complex with a peptidic inhibitor Cbz-Val-Asn-Ser-Thr-Leu-Gln-chloromethyl ketone (also known as hexapeptide chloromethyl ketone; 28) 125 provided clues for the substrate-based design. Although it is a substrate analog for the porcine transmissible gastroenteritis CoV (TGEV) M pro , it offers a structural explanation for the P1-Gln entering into the specific subsite S1 pocket and decreased P2-leucine specificity in the hydrophobic S2 site of SARS-CoV-1 M pro . Additionally, rupintrivir (29; AG7088), a peptidomimetic inhibitor of human rhinovirus 3C protease is oriented similar to inhibitor 28 in the binding pocket of TGEV M pro . 129 These two molecules became prototype compounds for the development of SARS-CoV-1 M pro inhibitors.
Compound 29 was only weakly active against SARS-CoV-1 M pro (IC 50 , 800 µM) also in cellular antiviral assays. 130 However, systematic structural modifications led to a series of analogs that show moderate to good activity. 131 For example, compound 30 ( Figure 11), in which the P1-lactam was replaced by a phenyl ring, showed moderate activity. Compound 31, in which the larger P2 p-fluorophenyl was replaced with a phenyl group, was even more effective. By taking 29 as a lead, Ghosh et al. designed new molecules mainly focusing on the replacement of the large P2 p-fluorobenzyl group. Two of the resulting structures with P2-benzyl (32) and prenyl (33) moieties showed decent inhibitory potencies at both enzymatic (K inact , 0.014 and 0.045 min −1 , respectively) and cell-based (IC 50 , 45 and 70 µM) assays. 132 Besides, no cytotoxicity was observed for these compounds up to 100 µM concentration. However, 32 and 33 were inactive at MERS-CoV M pro . 133 The same research group further modified the molecule with the introduction of P4 Boc-serine, to establish additional hydrogen bond interactions as described in compound 34 (IC 50 , 75 µM). Unfortunately, the activity of the resulting compound was not improved. Further modification of the isobutyl group in compound 34 to isoprenyl group in compound 35 displayed potent activity with K i = 3.6 µM ( Figure 11). 14 On the other hand, Yang et al. 134 reported a series of peptide inhibitors with a greater inhibitory potency. In general, they systematically changed the backbone of inhibitor 29. As a result, they were able to identify more specific residues for each subsite (compounds 36-38; Figure 12): At first, the P1-lactam ring was identified as a more specific moiety for the S1-site, forming multiple hydrogen-bond interactions with the enzyme as can be seen in the crystal structure (36); P2-leucine showed a fourfold increased inhibitory activity when compared to the P2-phenylalanine or -4-fluorophenylalanine (37). A lipophilic tert-butyl residue was recognized to be a better P3-moiety than the P3-valine (38). Finally, the replacement of P4-methylisoxazole with a benzyloxy group was the best option for activity enhancement (compare 29 vs 36). They all showed moderate to high antiviral activity against HCoV-229E in cell-based assays.
Shie et al. 131 reported another series of peptide inhibitors with comparatively reduced molecular weight to increase drug-like properties. These pseudo-C2-symmetric inhibitors consist of a Phe-Phe-dipeptidic α,β-unsaturated ester. One of these inhibitors (39) had an outstanding inhibitory activity with an EC 50 value of 0.52 µM (see Figure 12). Besides, it displayed remarkable antiviral activity with an EC 50 value of 0.18 µM. Structurally, the presence of 4-dimethylamine on the phenyl ring was found to be crucial for activity enhancement.
Another peptidic drug with a Michael acceptor was N3 (40), which was reported to inhibit SARS-CoV-1 3CL pro SARS-CoV-2 shares only 82% of its genome with its relative SARS-CoV-1. However, essential viral enzymes of both species show sequence similarities of greater than 90%. 137,139-142 SARS-CoV-2 3CL pro is highly similar to SARS-CoV-1 3CL pro , sharing 96% of its sequence. Therefore, one could expect that SARS-CoV-1 M pro inhibitors are active against SARS-CoV-2 M pro . Compound 40 was found to be active against SARS-CoV-2 M pro and its value of kobs/[I] for the COVID-19 virus M pro was determined to be 11 300 ± 880 M −1 ·s −1 . 143 Peptide N3 was cocrystalized with SARS-CoV-1 M pro at 2.1 Å resolution (see Figure 13). Its binding mode to SARS-CoV-2 M pro is highly similar to that of other CoV main proteases. Some key features include the Cys-His catalytic dyad and the substrate-binding pocket situated in a gap between domain I and II.
In general, inhibitors possessing a Michael acceptor group as a warhead moiety could form an irreversible (covalent) bond with the catalytic cysteine residue in the following manner ( Figure 14): First, the cysteine residue undergoes 1,4-addition at the inhibitor's Michael acceptor group (warhead). Rapid protonation of the α-carbanion from His-H + leads to the covalent bond formation between the warhead of the inhibitor and the cysteine residue.

| Inhibitors with aldehyde as a warhead group
Although the above-described inhibitors with 1,4-Michael acceptors (e.g., α,β-vinyl ethyl ester, -CH═CH-C (O)-OEt) showed enzymatic or cell-based in-vitro activities, they can be cleaved to their carboxylic acids by plasma esterases; for instance, AG7088 (29) was inactive in the plasma of rodents and rabbits. 144,145 Therefore, scientists explored different reactive groups that are stable in vivo. shown in Figure 16. Among them, 49 was moderately more active against SARS-CoV M pro when compared to 46. 148 The X-ray structure of M pro in complex with 49 revealed that the P2-decahydroisoquinoline moiety was fittingly placed in the S2-subsite, while the P1-imidazole moiety occupied the S1-subsite. With these key residues located appropriately in their respective pockets, the terminal functional group fits tightly into the active site.
This group further extended their study to find inhibitors that interact with S2 to S4 subsites. Taking  as shown in example 52. 149 The resulting 52 showed more than twofold increased M pro inhibitory activity compared to 49. This indicates that the additional interactions at S2-S4 sites enhance inhibitory activity.
Rather recently, the same research group explored the ability of octahydroisochromene to interact with the hydrophobic S2 pocket as an innovative P2-moiety. 150 To identify the best specific configuration, all possible diastereomers were evaluated. It was found that the molecule with (1S,3S)-octahydroisochromene 53-56 could secure the optimal position of the P1-imidazole as well as the aldehyde functional group at the active site.
Additionally, the N-butyl side chain attached to the 1-position of the fused ring system was recognized to be important for establishing hydrophobic interactions.
In 2018, Groutas et al. 151  Compound 63 was identified as SARS-CoV M pro inhibitor with an IC 50 value of 1.95 µM (Figure 19). 156 Taking 63 as a lead, aided by its X-ray structure in complex with SARS-CoV-1, HCoV-NL63, and coxsackievirus M pros , systematic structural modifications were investigated, focusing on the P2-moiety. As a result, the replacement of P2-phenyl with P2-cyclohexyl (64) was found to be the best substitution, while P2-cyclopentyl (65) showed similar potency against the enzyme SARS-CoV-1 M pro . In Huh7 cells, 64 also showed strong antiviral activity with an EC 50   at the surface of each protomer, between domains I and II. The thioketal that resulted from the nucleophilic Cys145 attacking the inhibitor, is stabilized by a H-bond from His41, whereas the amide oxygen of 67 accepts a H-bond from the main-chain amides of Gly143, Cys145, and in part, Ser144 that make up the cysteine protease's canonical oxyanion hole. 157 The P1 lactam moiety is deeply embedded in the S1 pocket where the lactam nitrogen donates a three-center H-bond to the main chain oxygen of the Phe140 and the carboxylate of Glu166. The carbonyl oxygen forms a H-bond to His163. The P2-cyclopropyl moiety fits into the S2 subsite. The P3-P2 pyridone moiety occupies the space normally filled by the substrate's main chain. The Boc group is not situated in the canonical S4 site, rather it is located near Pro168, which explains why the removal of the Boc group as in 68 weakened the inhibitory activity.

| Inhibitors with electrophilic ketone
It was envisioned that a fluorinated ketone moiety could be utilized as a warhead for targeting proteases, because it forms a thermodynamically stable hemiketal or hemithioketal after nucleophilic attack by Ser-OH or Cys-SH residues, which are present in the active sites of serine or cysteine proteases, respectively (see Figure 22). was exchanged for a variety of functional groups. 161 The study determined N-arylglycyl to be the optimal P3moiety. Compound 72 displayed the best inhibitory activity. Docking studies of 72 to the protease highlighted the amino hydrogen of the P3-N-phenyl glycyl forming a H-bond with backbone Glu166 of M pro , in addition to the best P2-leucine and P1'-benzthiazole moieties (see Figure 23A). Further structural optimization at the P3-N-arylglycyl moiety found the indole-2 carbonyl group to be one of the best P3-moeities, thus reaching inhibitors with low nanomolar potency, for example 73 (K i , 0.006 µM) against SARS-CoV-1 M pro . 162

| Small molecule inhibitors of M pro
Benzotriazole esters (88-91; Figure 26) were discovered as novel nonpeptidic irreversible inhibitors of SARS-CoV-1 M pro . 168  bound more strongly to SARS-CoV-1 M pro than to papain protease, while etacrynic acid ester 104 was more active at papain protease (K i , 3.2 µM) than at SARS-CoV-1 M pro (K i , 45.8 µM; Figure 28). SAR studies suggested that chloro substituents were necessary for protease inhibition. Docking studies of 105 to M pro revealed that it forms hydrogen bonds with Gln189, Glu166, Thr190, and Gln192 with its terminal amino group. The Michael system carbonyl group interacts with Gly143, and the reactive double bond remained next to the Cys145 sulfur.
They also observed moderate inhibitory activity against CVB3 3C pro . Structure-functionality analyses illustrated that the benzylidene ring next to pyrazolone C4 in addition to electron-withdrawing groups, favors inhibitory activity. Molecular modeling studies of 112 predicted that for its inhibitory function, the N1-phenyl residue in the M pro S1 site as well as the carboxyl benzylidene moiety in the S3 pocket are important. SAR studies focusing on the benzotriazole moiety of 129 were performed to improve activity. The replacement of this group with 4-phenyl-1,2,3-triazole (as in 130) was somewhat tolerated (IC 50 of 11 µM; Figure 32). To cut overall molecular weight of the inhibitors, P3-truncation was performed, which led to potent derivatives bind to M pro . Antiviral activity assays, using real-time reverse transcription-PCR, indicated that ebselen and inhibitor "N3" (40; Figure 12) had the strongest antiviral effects. Ebselen displayed an EC 50 value of 4.67 µM, and "N3" showed an EC 50 value of 16.77 µM in a plaque-reduction assay. Ebselen's IC 50 value for SARS-CoV-2 M pro was reported at 0.67 µM. The activity data of remaining compounds is summarized in Figure 35.
Ebselen has been studied for an array of diseases and has a very low toxicity. [191][192][193] Its safety has been demonstrated in clinical trials. 191,192,194 It can therefore be considered a promising molecule for the treatment or prevention of CoV infections.

| Coronavirus PL pro inhibitors
Along with the M pro , papain-like protease (PL pro ) also cleaves polyproteins which is an important process for viral replication. PL pro cleaves at the first three positions creating three nonstructural functional proteins (nsp1-nsp3).
In particular, nsp3 is central for the generation of the viral replication complex. The multifunctionality of PL pro in deubiquitinating, de-ISGylation (ISG: interferon-stimulated gene), 195,196 and in the evasion of the innate immune response make PL pro an attractive antiviral drug target.
PL pro is a cysteine protease and its active site contains a catalytic triad composing of Cys112-His273-Asp287.
Cys112 behaves as a nucleophile, and His273 is a general acid-base. Asp287 helps His273 to align perfectly, thus promoting His to deprotonate Cys-SH.
Next, the same group studied the SARs for compound 158 further. This led to the discovery of compound 165 with high PL pro inhibitory activity of SARS-CoV-1 (IC 50 , 0.32 µM) and antiviral activity (EC 50 , 9.1 µM) in Vero cells. 199 The mode of action of 165 was found to be a noncovalent, competitive inhibition of PL pro . Unlike the previous series, the stereochemistry at the α-methyl group did not make a significant difference in inhibition of Chou et al. 201 identified thiopurine (171) and 6-thioguanine (172) as SARS-CoV-1 PL pro inhibitors by the screening of a library containing 160 compounds. The thiocarbonyl group was important for PL pro inhibition.
However, the toxicity of these anticancer agents limits their therapeutic utility as anti-SARS agents. Disulfiram (151; Figure 35) was also reported as a SARS-CoV-1 PL pro inhibitor (IC 50 , 24.1 µM), 205 probably by reacting with the active site cysteine, thereby covalently modifying the enzyme target, as was reported for other targets.

| RdRP AND ITS INHIBITORS
The ability to produce new RNA copies from available template molecules is necessary for life on earth. RNA polymerases are therefore found in all living cells as well as many viruses. RdRP are essential enzymes to all RNA viruses, as they catalyze the synthesis of new RNA from a given RNA template. 206 Due to their importance for viral life cycles, and their high conservation among different RNA viruses, they have been attractive drug targets for antiviral therapy for a long time.
F I G U R E 37 Broad spectral PL pro inhibitors from different sources. PL pro , papain-like protease; SARS-CoV, severe acute respiratory syndrome coronavirus SARS-CoV-2 also uses an RdRP to replicate its genome within the host cell. Three nonstructural viral proteins (nsp) form its replication/transcription complex, with nsp12 forming the catalytic subunit. Bound to it are nsp7 and nsp8-accessory factors that facilitate template binding. [207][208][209] Their individual structures and that of the complex have been solved. [210][211][212][213][214] Interestingly, the only nsp that interacts directly with RNA seems to be nsp12, whereas nsp7 and nsp8 are needed to increase its efficiency. 210,215 RdRP is the target of inhibitors like remdesivir (178), galidesivir (179), ribavirin (180), favipiravir (181), and EIDD-2801 (182). These molecules have shown promise for the treatment of COVID-19 patients. 87,[216][217][218] (For structures and biological data see Figure 38) Remdesivir is a 1'-cyano-substitued adenine C-nucleoside analog prodrug. The prodrug strategy used is similar to that of the FDA-approved anti-hepatitis C drug sofosbuvir (183; see Figure 38). Upon its diffusion into cells, the phosphoramidate undergoes an intracellular conversion process that results in the formation of the triphosphate active metabolite (RTP). The triphosphate is recognized as adenine by viral RdRP, which causes heavy disruptions in RNA synthesis.
The exact molecular mechanism of remdesivir's action against SARS-CoV-2 has recently been elucidated by Yin et al. 219  Galidesivir is another C-nucleoside analog that resembles adenosine. However, the base is not linked to a ribose, but to an aza-sugar. Although it is recognized as adenosine by RdRP, its properties are different enough to cause a disruption in chain elongation. Galidesivir has been used in the treatment of Ebola and Marburg virus infections, and in vitro studies against SARS-and MERS-CoVs have suggested efficacy against CoVs. 221 Therefore, it is a likely future anti SARS-CoV-2 agent, and currently being studied in clinical trials. 222,223 Ribavirin is a nucleoside analog, which shows structural similarity to guanosine. But guanosine's 6-membered ring is only hinted at by the amide group. As such, it is incorporated by viral RdRPs, but interrupts RNA polymerization. 224 It is an approved drug in most countries and used against a variety of viral infections. Although its efficacy against SARS-CoV-2 has not been determined in large clinical trials, ribavirin has shown some promise in the treatment of COVID-19 patients. 225 Favipiravir (Avigan®) is an approved antiviral drug for the treatment of influenza in Japan and China. It is a pyrazinamide derivative that has shown some activity against a variety of RNA viruses. 226 Favipiravir inhibits viral RdRP via its similarity to guanine. After biotransformation into its active metabolite, favipiravir-ribofuranosyl-5'triphosphate, it is incorporated into newly synthesized RNA by RdRP, leading to premature chain termination 227 similar to remdesivir's mode of action. Favipiravir is currently being studied around the world as a treatment option against COVID-19.
Very recently, Sheahan et al. 228 reported the discovery of EIDD-1931 and its orally bioavailable prodrug EIDD-2801. These nucleoside analogs have shown remarkable potency against SARS-CoV-2 and other related CoVs in vitro and in vivo, with IC 50 values in the low nanomolar range, outperforming remdesivir 3-10-fold. The reason for this increased potency could be additional interactions with viral RdRP involving the N4-hydroxyl group of the cytidine ring. 219 The efficacy of EIDD-2801 in COVID-19 patients is being evaluated in a clinical trial. 229 Remdesivir and other potential RdRP inhibitors 230 are currently being studied in clinical trials around the world, but even though preliminary results appear promising, it is too early to assess their clinical value against COVID-19.  231 Several studies also described the combination of IFNs with antiviral drugs like ribavirin (180) or lopinavir-ritonavir for treating SARS. 232,233 In 2004, SARS patients in an open-label study had better clinical outcomes when treated with ribavirin in combination with lopinavir-ritonavir (400 and 100 mg, respectively) than the control group receiving only ribavirin. 227 A study in SARS patients found that viral replication could not be blocked at ribavirin concentrations achievable in human serum. 234 Nevertheless, the combination of ribavirin with IFN-β had a synergistic effect on the inhibition of SARS-CoV-1 replication. The effects of PEGylated IFN together with ribavirin against SARS-CoV-2 are being studied in clinical trials. 87 Nitazoxanide (178; Figure 39), a broad-spectrum antiparasitic drug, was reported to inhibit SARS-CoV-2 (EC 50 , 2.12 μM in Vero E6 cells). 215 It is also an IFN-inducing agent, and it is being studied for treating a wide range of infections.

| DRUGS REPOSITIONING APPROACH
The antiarrhythmic drug amiodarone (179, Figure 39) also inhibited SARS-CoV-1 replication in infected Vero cells. 235 The drug appears to alter the endocytotic pathway, thus inhibiting endosomal viral entry.
Glycyrrhizin inhibited viral replication in Vero cells with an EC 50 value of 300 mg/L, possibly by blocking viral entry as well. 232 As nitric oxide (NO) has been associated with antiviral activity, the NO donor, S-nitroso-N-acetylpenicillamine (180; Figure 39) was reported to inhibit SARS-CoV-1 replication in a dose-dependent manner. 236 In a search for potential antiviral agents against SARS-CoV-1, the screening of a library of 8000 approved drugs identified cinanserin (150; Figure 39), a serotonin antagonist, as a potential inhibitor of SARS-CoV-1 targeting its M pro with IC 50 value 4.0 µM. 189 A virtual screening and docking study identified the calmodulin antagonist calmidazolium as a SARS-CoV-1 M pro inhibitor (K i , 61 µM). 237 In 2014, Dyall et al. reported an array of pharmaceutical drugs with antiviral activity against MERS-CoV, and SARS-CoV-1 ( Table 2, chemical structure of all drugs were indicated in Figure S1). 121 The agents were grouped according to their modes of action. Hits inhibited both investigated CoVs.
In particular, the protein-processing inhibitors cycloheximide and anisomycin showed strong inhibitory activities against both CoVs. The HIV protease inhibitor lopinavir was more effective against SARS-CoV-1 than against MERS-CoV. The antidiarrheal agent loperamide showed moderate inhibitory activitiy against both CoVs.  Niclosamide (181; Figure 39), an anthelmintic drug, exhibited very potent antiviral activity against SARS-CoV-1 replication and stopped viral antigen synthesis at 1.56 μM concentrations. 238 It prevented the cytopathic effect of SARS-CoV-1 at low concentrations of 1 μM and halted SARS-CoV-1 replication with an EC 50 less than 0.1 μM in Vero E6 cells. 188  Famotidine has been proposed as a therapeutic against COVID-19, and a clinical trial is underway. 245 It is used to treat peptic ulcers and gastroesophageal reflux disease, among others. Cimetidine is a similar drug and has also been suggested as a treatment for COVID-19.
Dipyridamole was proposed as a treatment for COVID-19 as well, and a clinical study is being conducted. 246 It is a nucleoside transport and PDE3 inhibitor that prevents blood clot formation.
Sildenafil was proposed as treatment for COVID-19, and it is currently being investigated in a small trial. 247 It is a medication used to treat erectile dysfunction and pulmonary arterial hypertension.
Fenofibrate and bezafibrate have been suggested for the treatment of COVID-19. Fenofibrate is a blood lipidlowering medicine of the fibrate class. 248,249 Bezafibrate is a related lipid-lowering agent.
The HIV-protease inhibitor nelfinavir (183; Figure 39) strongly inhibited replication of SARS-CoV-1 in Vero cells with an EC 50 value of 0.048 µM. It was suggested to exert its effect at the post-entry step of SARS-CoV-1 infection. 250 Recently, Yamamoto et al reported that nelfinavir also potently inhibited replication of SARS-CoV-2 among nine other Anti-HIV drugs tested (IC 50 , 1.13 µM; CC 50 , 24.32 µM; SI = 21.52). 251 The measured serum concentrations of nelfinavir were 3-6 times higher than the reported EC 50

| Conclusions and future directions
The SARS-CoV-2 outbreak has caused worldwide disruption and was recently declared a global pandemic by the World Health Organization (WHO) owing to its rapid spread and high fatality rate. As there is no effective treatment to date, the number of infections continues to rise globally. This has led numerous research groups around the world to prioritize the identification and development of new therapeutics against COVID-19.
Although it is often considered the most promising method to prevent or contain future coronavirus outbreaks, an all-round anti-CoV vaccine is possibly a long way away. Small molecule drugs have the potential to be effective, rapidly produced, and widely available. Indeed, several small molecules have been investigated and advanced to clinical trials for the treatment of COVID-19, selected drug candidates are indicated in Table 3 (https://covid-19.heigit.org/clinical_trials.html).
As outlined in this review, inhibitors of important viral enzymes or structures, such as M pro , PL pro , or RdRP have displayed encouraging activity against various human-infecting CoVs. Since, both contagious viruses, SARS-CoV-1 and SARS-CoV-2, have a similar mechanism of infection; and both share the same human receptor, ACE2, for viral entry, for example-already developed inhibitors against the former could potentially be used to combat the latter. But despite the efficacy demonstrated by many inhibitors of SARS-CoV-1, no specific prophylactic or postexposure therapy is currently available.
The first step in the viral life cycle is the viral entry. It represents an attractive intervention point by blocking the RBD-ACE2 interaction or the virus-cell membrane fusion event. A large number of inhibitors, including peptides, antibodies, small-molecule compounds, and natural products have been identified to hamper viral entry.
Some of the peptides and antibodies displayed substantial anti-SARS activity and are therefore considered promising entry inhibitors with high potencies in the low micromolar range. Despite the apparent match of SARS-CoV-2 S and ACE2, other possible viral entry receptors should not be left unexplored. The glucose-regulated protein 78 (GRP-78, aka HSPA5), for instance, is employed as a coreceptor for entry by several viruses, including bat-CoVs and MERS-CoV, 260 and a study predicted that SARS-CoV-2 S might utilize this mechanism as well. 261 Elevated levels of GRP-78 in COVID-19 patients suggest a supplementary link. 262 Although as of yet unconfirmed, the development of therapeutics against additional targets like GRP-78 should receive due attention.
Viral proteases are another very important target for the development of antiviral therapies, as they are directly involved in the viral replication processes. Especially the M pro is one of the best-characterized viral targets, and numerous medicinal chemistry efforts have been already reported for the past outbreaks of SARS-1 and MERS. Main proteases are highly conserved among other CoVs, which allows the development of broad spectral antiviral agents. Moreover, no human protease analog to the M pro is known. Thus, drugs targeting M pro could be highly virus-selective and safe.
In light of the urgency of the current outbreak, repositioning of already approved drugs is becoming a popular approach due to the availability of toxicity and safety data. Drug repurposing has become fashionable, promising quick solutions to complicated questions. Old and, presumably, safe drugs are proclaimed miracle cures. The reality is a different one: Widely employed broad-spectrum antiviral drugs, such as (hydroxy)chloroquine, favipiravir, ribavirin, or umifenovir were reported to be effective against SARS-CoV-2, but could not convince in clinical trials yet. Clinicians are faced with an avalanche of contraindications and a myriad of case reports to choose the right drug. The drug repositioning strategy is, therefore, not a sound scientific path to a cure. At best, it can provide a basis for extensive future research in all related fields, including synthetic organic medicinal chemistry.
A new problem with the current COVID-19 outbreak is related to the spread of scientific information. When initial unfounded speculations about the alleged dangers of antihypertensive therapies with ACEis and ARBs were widely publicized in the media they caused great uncertainty among patients. Impetuous communications such as these can have serious consequences and should not be proclaimed carelessly. As it turns out, the benefits of continued antihypertensive therapy with these medicines in COVID-19 patients far outweigh their risks. There is even evidence of additional protective effects of ACEis and ARBs in this cohort, although the clinical relevance of this has yet to be investigated.
It is clear that governments and societies all over the world have been surprised by the recent coronavirus outbreak-as they were by the SARS outbreak in 2003 and the MERS epidemic in 2013. Human-infecting CoVs are on the rise, but quickly forgotten once life returns to normal. However, this problem will not disappear by itself, but likely increase in intensity. Viral spillover events are expected to increase in frequency as humans continue to invade new territories. We hope that, this time, the world will heed nature's warning to finance and conduct groundbreaking research on CoVs and their disease patterns. Only with a profound understanding of the viral life cycle and the affected human physiology we can prevent and control future outbreaks.