Revisiting and Updating Cyanorona-20: The Selective SARS-CoV-2 Inhibitor

Evident inhibition/blockade of the viral RNA-dependent RNA polymerase (RdRp) of the newly-emerged fatal Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is considered one of the most promising and efficient approaches for developing highly potent remedies for Coronavirus Disease 2019 (COVID-19). However, almost all of the reported viral RdRp inhibitors (either repurposed or new antiviral drugs) lack specific selectivity against the novel coronaviral-2 RdRp and still at a beginning phase of advancement. In this complementary research study, the new pyrazine derivative cyanorona-20 was revisited with an update about its synthetic and toxicological data. This promising selective specific anti-COVID-19 compound is deemed to be the first distinctive derivative of favipiravir. Cyanorona-20, the unrivalled nucleoside/nucleotide analog, was designed, synthesized, characterized, computationally studied, and biologically evaluated for its anti-COVID-19/cytotoxic actions. The results of the biological assay displayed that cyanorona-20 surprisingly exhibited very high and largely significant anti-COVID-19 activities (anti-SARS-CoV-2 EC₅₀ = 0.45 μM), and, in addition, it could be also a very promising guide and lead compound for the design and synthesis of new anti-SARS-CoV-2 and anti-COVID-19 agents through structural modifications and further computational studies. Further appraisal for the improvement of cyanorona-20 medication, through performing deeper in vivo biological evaluations and extensive clinical trials, is a prerequisite requirement in the coming days. In this short communication paper, the comprehensive chemicobiological data and information about the "Corona Antidote", cyanorona-20 compound, were briefly and collectively revisited and the synthetic and pharmacotoxicological data were updated.

A novel coronavirus (2019-nCoV), officially known as Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2; figure 1), recently appeared in Wuhan (Wuhan City, Hubei Province, China) in the last days of 2019 [1]. In spite of strict and rigorous containment measures, the transmission of this mysterious fatal viral microbe is ongoing resulting in the spread of the virus characteristic infection or disease, Coronavirus Disease 2019 (COVID-19), with its major signs and symptoms concentrated in the respiratory system of human (i.e., characterized by pulmonary infection in humans) [1,2]. This outbreak of 2019‐nCoV infection in China has spread across the Earth planet [1,2]. The extreme efforts of multinational pharmaceutical companies, drug discovery research centers, and other related places have since focused on the search for effective medications and therapies able to destroy the virus, stop the viral replication, and/or counter the most severe and fatal effects of the disease [2,3]. No specific antiviral agents have been officially and internationally approved for the 100% successful treatment of COVID‐19 to date [3].

Figure 1: Representation of SARS-CoV-2 morphology and structure.

In the present status of the absence of any known effective anti-COVID-19 therapy and, in addition, because of the intolerable situation of this public-health emergency of the COVID-19 pandemic, many researchers have proposed the repurposing of the known potent antiinfluenza drug favipiravir (it is a purine nucleic acid analog, which was officially approved for medical use in Japan since 2014; figure 2) to fight the novel COVID-19 [4,5]. Preceding studies and investigations have identified the viral RNA-dependent RNA polymerase (RdRp) as a very potential drug target in COVID-19 therapy due to its highly significant and pivotal role in SARS-CoV-2 replication and transcription (i.e., in the virus life cycle), and, furthermore, this enzyme has a strategic feature and advantage of being absolutely absent in the coronavirus-uninfected human cells (i.e., viral RdRp is a drug target of extremely selective toxicity against SARS-CoV-2 particles) [5-7]. Favipiravir, as an antiviral agent, acts by selectively inhibiting viral RdRp (some other researches suggest that favipiravir, in addition of being a potent RdRp inhibitor, induces lethal RNA transversion mutations, thus producing a nonviable viral phenotype) [4,8]. Favipiravir is a prodrug that is metabolized to its active form, favipiravir-ribofuranosyl-5'-triphosphate (favipiravir-RTP), mainly by the human enzyme Hypoxanthine-Guanine Phosphoribosyltransferase (HGPRT) so as to stop the replication processes of the viral RNA genome (i.e., to inhibit the reproduction of the virus) [9]. However, many limitations have restricted the successful complete use of favipiravir as an efficient and potent anti-COVID-19 agent to date, e.g., reliable data regarding the in vitro SARS-CoV-2 inhibition are still unavailable [4,5]; broad documented data regarding the in vivo SARS-CoV-2 inhibition and efficacy in preclinical animal studies are still unavailable [5,10]; the few available animal experiments of favipiravir show the tendency and potential for teratogenic effects (there is a strong evidence that its use during pregnancy may cause harm to the fetus) [4]; favipiravir has not been shown to be effective in primary human airway cells [11]; lack of additional virus-toxic functional groups in favipiravir structure to augment its antiviral mechanism of action against the lethal and resistant SARS-CoV-2 [5,12]; lipophilic/hydrophilic properties of favipiravir are not extremely balanced to achieve maximal bioavailability (specially in lungs) in humans suffering from that fatal COVID-19 [13]; expected binding affinities of active favipiravir-RTP molecule (as a viral RdRp inhibitor) to SARS-CoV-2 RdRp enzyme protein are not that great [12]; all data concerning its clinical use in human (mainly in Japan) are still mysterious and not that clear [4,10,13]; favipiravir as an anti-COVID-19 has not been used that much outside Japan till now (just in very few countries like China and Italy) [4]; favipiravir as an anti-COVID-19 drug is almost used only locally (off-label use) in Japan as its medical use as anti-COVID-19 has not been officially and internationally approved till now (the drug has not been approved as anti-COVID-19 by the WHO and the FDA, and it has not any complete or overall international scientific and medical consensus till now) [4,13]; and many favipiravir published articles and papers have considerably irreproducible data and results [5,10].

Figure 2: Chemical structures of favipiravir (6-fluoro-3-hydroxypyrazine-2-carboxamide) and the newly-discovered target compound cyanorona-20 ((E)-N-(4-cyanobenzylidene)-6-fluoro-3-hydroxypyrazine-2-carboxamide).

All these disadvantages and limitations of favipiravir anti-COVID-19 usage together with some importantly-needed additional properties in the favipiravir chemical structure to be a highly effective potent anti-SARS-CoV-2 drug (e.g., inhibiting and acting on many or more than one SARS-CoV-2 target; augmenting all the other expected modes of action on all the other possible viral protein targets, i.e., other than the main enzyme SARS-CoV-2 RdRp; overcoming the possibly-emerged resistance against favipiravir from the newly-mutated/-created coronaviruses; enhancing the distribution and bioavailability of the drug in the human respiratory system/lungs and the other nearer and target systems/organs; and achieving faster onset of action against this detrimental virus) have motivated me to start searching for a well-designed alternative or derivative of favipiravir to block SARS-CoV-2 RdRp very efficiently. The major aim is to add moieties, to favipiravir chemical structure, that have both polar (or hydrophilic) and nonpolar (or lipophilic) parts, viral replication-inhibiting activities, structure-stabilizing properties (mainly through resonance), biological compatibility with the human body living systems, and the smallest possible molecular volume/weight to fulfill all the previously-mentioned required characteristics together with solving the previously-mentioned issues and problems associated with favipiravir use. After extensive computational molecular modeling studies (including compound libraries screening and SARS-CoV-2 RdRp docking studies) together with my hypothetical intellectual suggestions, (E)-N-(4-cyanobenzylidene)-6-fluoro-3-hydroxypyrazine-2-carboxamide (cyanorona-20; figure 2), a new derivative of favipiravir, has been selected to do this urgently-required mission to save our planet from this destroying novel single-stranded RNA virus, SARS-CoV-2 [14]. Herein, as a continuation of my previous work [14], in the next paragraphs, I briefly revisit almost all the comprehensive chemical, biological, pharmacological, and toxicological information of cyanorona-20, with new interesting and important updates about the optimization of the synthesis of the compound (i.e., concerning the enhanced/optimized synthesis of cyanorona-20) along with updated toxicological, cytotoxicological, and safety data.

Cyanorona-20 is the 4-cyanobenzylidene derivative of favipiravir at the amino group, and is expected to be a prodrug that is metabolized inside the human body to its logically-predicted active nucleotide triphosphate form, cyanorona-20-ribofuranosyl-5'-triphosphate (cyanorona-20-RTP). The structural features of this newly-synthesized compound are optimal, as the structure of cyanorona-20 molecule totally obeys Lipinski's rule of five (Ro5), has extremely high druglikeness score, and has almost all the structural requirements (most of them have been mentioned above) for a compound to be a highly effective and potent anti-COVID-19 agent (i.e., SARS-CoV-2 RdRp inhibitor) [15-17]. The mechanism of interaction of cyanorona-20-RTP with the large molecule of SARS-CoV-2 RdRp has not been fully elucidated, but I hypothesize that cyanorona-20 in its in vivo active form may act through, at least, six different and synergistic major modes of action (i.e., through a very potent anti-COVID-19 multiaction or multiple activity; figure 3) [9,11,18-21], as it may be misincorporated in a nascent SARS-CoV-2 RNA (thus preventing RNA strand elongation and viral proliferation), it may evade RNA proofreading by viral exoribonuclease (ExoN; thus causing a decrease in SARS-CoV-2 RNA production), it may competitively bind to conserved polymerase domains (thus preventing incorporation of mainly purine nucleotides required for SARS-CoV-2 RNA replication and transcription), it may cause the SARS-CoV-2 RdRp to pause, it may induce an irreversible chain termination in the growing SARS-CoV-2 RNA, or it may induce lethal mutagenesis (thus, mainly, making the virus less effective and reducing its titer "viral titer") during SARS-CoV-2 infection.

Figure 3: Hypothetical representation of cyanorona-20 prime mechanism of anti-COVID-19 action.

Cyanorona-20, to the best of my knowledge (until the date of submitting this discovery article for publication), is the first bioactive derivative of favipiravir and also the first selective and potent direct-acting anti-COVID-19 agent. Cyanorona-20 fulfills and satisfies all the needed requirements to be an ideal anti-COVID-19 drug (i.e., to be a better antiviral and anti-COVID-19 drug than its parent compound favipiravir). For example, cyanorona-20 molecule has a virus-toxic cyano group (it may be also called a SARS-CoV-2 RdRp-destabilizing moiety, as it chemically causes major steric clash with the SARS-CoV-2 RdRp molecule at some residues, this greatly helps in RdRp preliminary partial blockade and results in delayed chain termination in RNA synthesis which gives cyanorona-20 an extrapotency against the principal resistance mechanisms that might be emerged by SARS-CoV-2 against favipiravir and most other classical potent antiviral nucleos(t)ide analogs [21]) (a strong aliphatic polar group) and a resonance-stabilized benzene ring (in the benzylidene group) (a strong aromatic lipophilic moiety), both groups with a one-carbon-atom linker form a highly stable 4-cyanobenzylidene moiety (a SARS-CoV-2-toxic moiety which is extremely stabilized through strong resonance and inductive effects) which is not present in the parent favipiravir molecule, and adds an exceptional and excellent balanced lipophilic/hydrophilic properties along with electronic extrastability to the molecule (this makes the molecule more bioavailable and more biocompatible).

One of the interesting characteristics of cyanorona-20 structure is its clear ability to serve as a multizincophore (zinc ion carriers or zinc ionophores, e.g., chloroquine [22], hydroxychloroquine [22], quercetin [23], epigallocatechin gallate [23], and CoViTris2020/ChloViD2020 [24], transport extracellular Zn²⁺ ions across the hydrophobic cell membranes to enter the living cell, and have been studied mainly for their antiviral activities, as they have been shown to effectively inhibit the replication of various viruses in vitro [25]). Zn²⁺ inhibits coronavirus RdRp activity (i.e., inhibits coronaviral replication and transcription) in vitro (Zn²⁺ ion is the only known elemental cofactor and ligand present in the crystal structure of SARS-CoV-2 RdRp and, thus, it has an extremely important role in controlling/regulating the activity of this COVID-19 RNA-synthesizing enzymatic machine) and, therefore, zinc ionophores have been shown to successfully hinder the replication process of coronaviruses intracellularly in cell cultures [26-28]. Based on this fact, inhibiting SARS-CoV-2 RdRp and its associated replication processes is one of the best strategies to inhibit and kill SARS-CoV-2 through designing and synthesizing (or searching for) molecules that have good zincophoric properties, and as much the molecule has more zincophoric centers in its structure as higher its zincophoric and related SARS-CoV-2-killing activities are. Cyanorona-20 is supposed to have about six zincophoric moieties or centers (four active nitrogen atoms and two active oxygen atoms), making it an ideal candidate to act as a potent zincophore (a multizincophoric anti-COVID-19 compound).

Here, in this communication research paper (along with its adjusted Supporting Information file), the previous data for the design, synthesis, characterization, computational studies, and anti-COVID-19 biological activities of this novel compound, named cyanorona-20, are supported and updated with interesting new pharmacotoxicological studies (i.e., the updated discovery of cyanorona-20, the first bioactive derivative of favipiravir and the first selective/specific and potent anti-COVID-19 drug, is reported).

Synthesis, chemistry, and stability of cyanorona-20

Cyanorona-20 was successfully synthesized, as shown in figure 4, in very good yields from its parent favipiravir via direct condensation with 4-cyanobenzaldehyde (equimolar amounts) in the presence of the strong dehydrating agent glacial acetic acid (gla. AcOH) along with excess sodium acetate (NaOAc). The reaction could proceed either by conventional heating (with more than 85% yield) or under Microwave Irradiation (MWI) (with about 96% yield). The structure of cyanorona-20 was characterized, elucidated, and confirmed through spectroscopic analyses (IR, ¹H-NMR, 13C-NMR, and mass spectrometry) and microanalyses (elemental analysis for C, H, and N atoms). Spectral and elemental analyses of the sample of this product were in an ideal and full agreement with the proposed structure of cyanorona-20 as presented in the Supporting Information file.

Figure 4: Schematic representation of the optimized conventional and microwave-assisted synthetic pathways of cyanorona-20 from favipiravir.

Cyanorona-20, quite like its parent compound favipiravir, is expected to be a tautomeric molecule [29]. According to the computational simulations studies, in almost all cases the molecule favors the enol-like tautomeric structure (the predominant form), which is substantially much more stable in the aqueous media as compared to the keto-like tautomeric structure as shown in figure 5.

Figure 5: Schematic representation of the tautomeric forms of cyanorona-20 molecule in aqueous solutions.

The Schiff base-like structure of cyanorona-20 molecule rendered me to accurately and extensively investigate and examine the stability behavior of this new compound (specially in aqueous media similar to those of the human body) using all the possible analytical and physicochemical methods. The final results of aqueous dissolution testing were excellent and sufficient as they showed that only less than 5% (as a maximum) of total cyanorona-20 amount undergoing hydrolysis to minor products and impurities after the maximum period of 3 months, and less than 50% of this 5% amount (i.e., less than 2.5% of total cyanorona-20 amount) undergoing hydrolysis to the parent favipiravir during the same 3-month period (see the representative HPLC chart, Chart S4, in the Supporting Information file), thus proving the good and adequate practical stability of cyanorona-20.

Evaluation of the antiviral/anti-COVID-19 and toxic/cytotoxic activities of cyanorona-20

The results demonstrated in table 1 explicitly revealed the extremely higher and surprising anti-COVID-19 effectiveness of the new compound cyanorona-20 (the most potent anti-SARS-CoV-2 compound) relative to that of each of the reference drugs. Among the five tested compounds, four compounds (cyanorona-20, remdesivir, HCl-arbidol-H2O, and favipiravir, respectively) were found to inhibit SARS-CoV-2 replication in Vero E6 cells with EC₅₀ under 100 μM, while hydroxychloroquine sulfate was found to do that above 100 μM. Surprisingly, cyanorona-20 (EC₅₀ = 0.45 μM) was found to be about 209 and 45 times more potent than favipiravir (EC₅₀ = 94.09 μM) and remdesivir (EC₅₀ = 20.17 μM), respectively, in anti-SARS-CoV-2 activity (in vitro). According to the assay, cyanorona-20 is expected to have very high clinical selectivity index (SI; SI = CC₅₀/EC₅₀) and safety margin (CC₅₀ is much larger than 100 μM), while, on the other hand, hydroxychloroquine sulfate is expected to have very narrow clinical therapeutic index (EC₅₀ is just above 100 μM, CC₅₀ = 93.06 μM). Cyanorona-20 is also having amazingly very small values of the concentration that causes 100% inhibition of the SARS-CoV-2 cytopathic effects in vitro (cyanorona-20 has the best CPEIC₁₀₀ value, among all the five compounds tested, of 1.40 μM) and of the concentration that is required for 50% reduction in the number of SARS-CoV-2 RNA copies in vitro (cyanorona-20 has the best EC₅₀ value, among all the five compounds tested, of 0.48 μM).

The prediction of toxicities and adverse/side effects of any new potential medicine is a very critical issue of the drug discovery trip. Interestingly, modern virtual toxicity predictions have several favorable merits over the practical classic toxic dose determinations in animals, as they, e.g., provide more rapid data, reduce research costs, and decrease the number of diverse animal experiments. The data obtained from using the new ProTox-II Virtual Laboratory disclosed the high predicted biosafety of cyanorona-20 compound (over most of the reference drugs) [30]. The results showed that cyanorona-20 lies in the safe toxicity class 4 with a predicted lethal dose 50% (LD50) ≥ 600 mg/kg body weight. With a molecular weight of about 270.22 (which is approaching the mean molecular weight of dataset, 319.67), cyanorona-20 resides in the molecular weights region of the safest drug-like compounds in the dataset of this virtual laboratory (figure 6). The resulted data also predicted that cyanorona-20 is cytotoxicologically inactive (with a probability percentage of more than 81%). Interestingly, analysis of all diverse toxicity endpoints using the different ProTox-II toxicity model tests revealed that cyanorona-20 has high levels of safety in respect to the diverse organ toxicities and adverse outcomes pathways. These virtual toxicological predictions significantly support the previous results of the in vitro cytotoxicity evaluation for cyanorona-20 compound to efficiently act as a very potent safe anti-COVID-19 agent (i.e., an effective SARS-CoV-2 inhibitor).

Figure 6: Comparison of molecular weight (molweight) of cyanorona-20 with that of dataset compounds (using ProTox-II Virtual Laboratory methodology).

We have to put into consideration that the three nucleoside/nucleotide analogs, cyanorona-20 (guanine analog), favipiravir (guanine analog), and remdesivir (adenosine analog), mainly undergo prior intracellular metabolic activation into their triphosphate forms by the host cellular enzymes (chiefly nucleoside kinases), which may differ among several cell types, thus evaluation of the actions of nucleos(t)ide analogs in primary human airway epithelial cells would undoubtedly facilitate the interpretation of the results. The metabolic activation would surely add additional anti-COVID-19 activities to the three drugs, and it would also successfully increase the clinical effectiveness of the three drugs. Interestingly, the four reference drugs (favipiravir, remdesivir, HCl-arbidol-H2O, and hydroxychloroquine sulfate) are currently undergoing extensive clinical trials, as anti-SARS-CoV-2/anti-COVID-19 agents, worldwide. The very high value of CC₅₀ of cyanorona-20 indicates that cyanorona-20 would be expectedly well tolerated in the human body. The extremely minute value of anti-SARS-CoV-2 EC₅₀ and the very high value of mammalian cells CC₅₀ (i.e., the fantastically desirable and promising high value of SI) of cyanorona-20 indicate that this compound clearly favors the resistant RNA virus over DNA virus and mammalian cells, and this, in turn, expresses and proves the previously-expected selective specificity of this compound as anti-COVID-19 drug "Corona Antidote or Corona Killer" (see Introduction part). Using a combination formula (a mixture) of cyanorona-20 and remdesivir is a suggested option, as it may have an exceptional and astonishing combinational synergistic anti-COVID-19 effect in further assays in animals (in vivo) and clinical trials. Almost all the practical results obtained, here, in the antiviral anti-COVID-19 biological evaluation are compliant with the previous theoretical results extracted from the computational molecular and pharmacological predictions for the new compound cyanorona-20 and its four reference compounds.

Selective potent inhibition of the novel SARS-CoV-2 RdRp has been validated as the most effective viable approach for targeted COVID-19 therapy and, therefore, all my efforts in our laboratories in 2020 were focused on designing and creating new drugs (specially, new potent derivatives of old drugs repurposed against COVID-19) targeting and efficiently inhibiting this important active viral polymerase. These efforts led to the discovery of an extremely promising selective specific and potent SARS-CoV-2 RdRp inhibitor, cyanorona-20 ((E)-N-(4-cyanobenzylidene)-6-fluoro-3-hydroxypyrazine-2-carboxamide), which dramatically inhibited SARS-CoV-2 RdRp with EC₅₀ values of 0.45 and 0.48 μM (relatively and according to the used assay, cyanorona-20 is the most potent SARS-CoV-2 RdRp inhibitor till now), and interestingly presented about 209- and 45-fold anti-SARS-CoV-2 RdRp selectivity/potency more than favipiravir and remdesivir, respectively. On the other hand, the design and discovery of cyanorona-20 by the structural modification at the active amide moiety of the antiinfluenza favipiravir molecule opens the door for us to establish the first class of anti-COVID-19 agents (of the type "nucleoside analogs"), which will specifically comprise a series of pyrazine derivatives (beginning with the first two effective members, favipiravir and cyanorona-20) [31]. Prior extensive preparatory computational molecular calculations and studies showed that cyanorona-20 has the most ideal and the best balanced values of the pharmacokinetic and druglikeness descriptors (among the five evaluated drugs, cyanorona-20 and the four reference drugs, favipiravir, remdesivir, arbidol, and hydroxychloroquine) required to be an effectively potent anti-COVID-19 drug inside the human body [32,33]. Computational molecular modeling analysis of the best inhibitory binding mode of the expected active metabolite of cyanorona-20 inside the human cell, cyanorona-20-RTP, showed that the added 4-cyanobenzylidene moiety interestingly increases the blocking affinity and potency at active and/or allosteric sites of the SARS-CoV-2 RdRp (binding free energy = -10.50 kcal/mol) when compared to those of the active metabolite of its parent favipiravir inside the human cell (which lacks this 4-cyanobenzylidene moiety), favipiravir-RTP (binding free energy = -8.40 kcal/mol). Surprisingly, cyanorona-20 and its active metabolite surpassed the four moderately to highly potent reference drugs and their active metabolites, respectively, in the values of almost all compared theoretical and practical anti-COVID-19 parameters, scores, and activities. Cyanorona-20 will enter nowadays all the needed complementary in vivo/preclinical studies. If cyanorona-20 compound successfully passes the in vivo bioassays and preclinical/clinical biostudies with effectively significant results as anti-COVID-19 agent, a combination therapy (e.g., as an oral tablet or a nasal/oral prophylactic gel) with a second highly potent antiviral drug, such as remdesivir, will be a recommended possible choice for the COVID-19 treatment in the near future. In brief summary, in this new research paper, the first derivative of favipiravir, cyanorona-20 "Corona Killer or Coronacide", was discovered and reported as a fantastic hit molecule (it can also be considered as the first potent anticoronaviral hybrid molecule of both potent antivirals favipiravir and remdesivir) with selective specific and potent successful inhibition against SARS-CoV-2 RdRp and, thus, the first known promising under-investigation candidate drug for the treatment of COVID-19 was found out.

I hereby declare that I totally have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this new paper.

I gratefully thank and deeply acknowledge anyone who gave a hand to make this new discovery and research coming out to light.

The data that support the findings of this research study are available in the supplementary material of this article (Supporting Information file).

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