New Chalcone Derivatives as Effective Anti-SARS-CoV-2 Agents

Flavonoids and related compounds, such as quercetin-based antiviral drug Gene-Eden-VIR/Novirin, inhibit the protease of severe acute respiratory syndrome coronavirus (SARS-CoV-2). The alkylated chalcones isolated from Angelica keiskei inhibit SARS-CoV proteases. Hydroxychloroquine and Favipiravir have been used in many countries since the beginning of the pandemic with the thought that they may have antiviral activity against SARS CoV-2. In this study, we aimed to compare the anti-SARS CoV-2 activities of both newly synthesized chalcone derivatives and these two drugs.The current study aimed to determine the potent antiviral activity of newly synthesized chalcone derivatives against SARS-CoV-2 by calculating the RT-PCR cycling threshold (Ct) values. Antiviral activities of the compounds varied due to being dose dependent. Compound 6, 7, 9 and 16 were highly effective against SARS-CoV-2 at concentrations of 1.60 µg/mL. Structure-based virtual screening was carried out against the most important druggable SARS-CoV-2 targets, viral RNA-dependent RNA polymerase (RdRp), to identify putative inhibitors that could facilitate the development of potential anti-COVID-19 drug candidates. Computational analyses identi�ed eight compounds inhibiting each target, with binding a�nity scores ranging from − 4,370 to -2,748 kcal/mol along with their toxicological, ADME, and drug-like properties.


Introduction
Coronavirus disease-2019 (COVID-19) is a highly contagious disease caused by a novel virus identi ed as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).To date, there is no speci c effective antiviral drug against SARS-CoV-2.The pandemic has adversely affected the global healthcare system.Treatment options for COVID-19 can be categorized into four: general treatment, coronavirus-speci c treatment, antiviral treatment, and other methods. 1,2General treatment includes nutritional supplements, immune boosters, and some alternative medications in folk medicine. 3rious treatment strategies based on the use of available drugs have been tried against SARS-CoV-2 globally, but no effective treatment protocol has been found.There are many drugs, such as chlorokin/hydroxychloroquine, favipiravir, ribavirin, remdesivir or combinations of these drugs, which are found to be effective against SARS-CoV-2. 4 Although some studies initially reported that one or more of these drugs were effective against SARS-CoV-2 in some countries, later some scienti c studies reported that these drugs did not have the expected level of effectiveness against SARS-CoV-2. 4,5evious treatments targeting SARS-CoV and MERS-CoV may accelerate the development of the COVID-19 treatment due to their structural and genomic similarities.Antiviral effective therapies act by arresting viral replication stages and the inactivation or inhibition of the enzymes or steps involved in these stages.One of the best characterized and conserved drug targets of coronaviruses is integrase, the main protease proteins.Inhibiting the activity of this enzyme would block viral replication, for which protease inhibitors are used.One of the most important targets that can inhibit viral replication against SARS-CoV-2 is the inactivation of the RNA-dependent RNA polymerase enzyme.Inactivation of this enzyme prevents virus replication.[8][9] In cases where vaccines against SARS-CoV-2 may be insu cient, humanity's most important weapon would be an effective antiviral drug.For this reason, it is crucial to produce an effective antiviral drug against this highly contagious and deadly virus.Small modi cations to the inhibitory ligand can signi cantly induce conformational changes in the molecules by changing the protein's binding modes and interactions to the active site and thus their activity.
In this context, some compounds that inhibit viral RNA polymerases in the literature were examined to nd a scaffold, which would be active against COVID-19.The scaffold was determined to show the best activity against COVID-19.Various modi cations were made to chalcone structure to design a newer, more active compound and reach the target molecules.A target molecule that could be effective against COVID-19 was designed.
Chalcone derivatives are the building blocks of pharmaceutical raw materials that have long been used to treat various diseases, and medicinal plants are structural analogs of the avonoids they carry.Flavonoids and related compounds, such as quercetin-based antiviral drug Gene-Eden-VIR/Novirin, inhibit the protease of SARS-CoV-2. 10 Chalcones also exhibit inhibitory activity against tobacco mosaic virus 11 , HIV 12 , herpes simplex virus 13 , and dengue virus. 14It also exhibits antioxidant 15 , antimicrobial 16 , antimycobacterial 17 and antileishmanial 18 properties.The alkylated chalcones isolated from Angelica keiskei inhibit SARS-CoV proteases. 19e current study aimed to determine the inhibitory activities of new eight chalcone derivatives against SARS-CoV-2 by calculating the SARS-CoV-2 RT-PCR cycling threshold (Ct) values.Moreover, the toxicological, ADME and drug-like properties of the compounds were also determined by in silico method.

Results
The synthesis of the target molecules (6-9) was accomplished in one step using the Claisen-Schmidt condensation reaction, as illustrated in Fig. 1.The corresponding chalcones were obtained in high purity, and good yields by treating 2,4,6-trimethoxy benzaldehyde (1) with uoro and tri uoromethyl substituted acetophenone derivatives (2-5) in an aqueous alkali medium.LiOH was the preferred alkaline base in the series.The reason for that nucleophilic aromatic substitution products is also obtained in reactions carried out in methanol and in a strongly basic medium, especially if the aromatic benzene ring contains uorine.As a result of the uorine atom's substitution by the methoxy group, methoxy chalcone structures arise as a by-product.To prevent the result from affecting the reaction e ciency, LiOH was used as an alkaline base.Tetrahydrofuran/water mixture (THF: H 2 O) was determined as the reaction solvent. 20he synthesis of the second group of compounds (13-16) was accomplished in two steps using bromination of compound 10 followed by the Claisen-Schmidt condensation reaction, as shown in Fig. 2. By treating 2,4,6-trimethoxyacetophenone (10) with LiBr and ammonium cerium (IV) nitrate in CH 3 CN, the corresponding compound 11 was obtained in high yield.Subsequently, target compounds (13-16) were obtained in high purity and good yield by the Claisen-Schmidt reaction of compound 11 with the corresponding benzaldehydes in an aqueous alkali medium (2-4, and 12).
Antiviral effects of all newly synthesized compounds against SARS-CoV-2 were assessed comparatively with standard drugs, such as favipiravir and hydroxychloroquine.The highest non-toxic concentrations of standard drugs and the compounds on Vero cells were selected for antiviral e cacy studies.The antiviral effects of three different concentrations of chalcone derivatives have been investigated.
In the study, viral growth in the Vero E6 cell line was detected by cytopathological effects (CPE) in the cells from the fourth day of incubation, as shown in Fig. 3. Activity studies in cell culture were performed after 96 hours of incubation.The characteristic CPE of SARS-CoV-2 was not detected in cell cultures in which viral reproduction was inhibited.Morphological analysis was performed by recording the CPE rate in the cells, especially the presence/absence of CPE.
The "Ct" values give a relative measure of the viral copy number in the sample compared to the curves using the standards.Different known quantities of standards were included in the same study and tested in parallel with viral cultures.Therefore, the "Ct" value was evaluated as the potential viral (SARS-CoV-2) copy number.In this study, the inhibitory activities of new chalcone derivatives against SARS-CoV-2 were determined by calculating the SARS-CoV-2 RT-PCR "Ct" values. 21,22 has been reported that low Ct values (high viral load) can be considered an indicator of severe disease and high contagiousness.In contrast, high Ct values (low viral load) indicate a decrease in both the risk of infection and contagiousness. 23 shown in Fig. 4, the effectiveness of compounds 6-9, 14, and 16 on SARS-CoV-2 at three different concentrations (0.40, 0.80, and 1.60 µg/mL) was compared with that of the standard drugs and the virus control group in terms of "Ct" values.When the effectiveness of standard drugs was compared with the virus control group (no drugs or chemicals included), no signi cantly high antiviral activities were found.It appears that there is no mention of the antiviral activity of both favipiravir and hydroxychloroquine.
The antiviral activities of compounds 6-9, 14, and 16 at high concentrations (1.6 and 0.8 µg/mL) were statistically signi cantly higher when compared to the virus control group and the standard drugs.No statistically signi cant differences were found when the antiviral activity of compound 8 at a concentration of 0.4 µg/mL was compared with the standard drugs and virus control group.The antiviral e cacy of compound 8 was lower than that of the compounds.
There was also a dose-dependent change in the antiviral e cacy of the compounds.While the highest e cacy was determined at a concentration of 1.60 µg/mL, it was evinced that antiviral activity decreased at a concentration of 0.8 and 0.4 µg/mL.Comparing the antiviral activities of the compounds at different concentrations; Compound 6 > 7 > 16 > 9 > 14 > 8 at 1.60 µg/mL; compound 7 > 6 > 9 > 16 > 14 > 8 at 0.80 µg/mL; 7 > 6 = 9 > 16 = 14 > 8 at 0.40 µg/mL ranking has been determined.After evaluating the results, it was found that the antiviral activities of 6, 7, 9 and 16 of the rst group compounds were remarkable.Hence it was noted that compounds 6, 7, 9, 14, and 16 exhibited statistically signi cantly higher antiviral activities than compound 8 against SARS-CoV-2 at all concentrations.
The antiviral effects of the second group of compounds (13 and 15) at three different concentrations (2.0, 1.0, 0.5 µg/mL) have been examined.As shown in Fig. 5, there was no signi cant difference between the 2.0 and 1.0 µg/mL concentrations of compound 13 against SARS-CoV-2.Compound 13 was found to exhibit a dose-dependent antiviral activity.However, at a concentration of 0.5 µg/mL, antiviral e cacy was not different from standard drugs and the virus control group.While the antiviral activity of compound 13 at a concentration of 1 µg/mL was not different from that of favipiravir, it was found to be statistically signi cantly higher than the effectiveness of hydroxychloroquine in 2 µg/mL.Also, the antiviral e cacy of compound 15 was statistically signi cantly higher than the effectiveness of standard drugs at all three tested concentrations.It was determined that the antiviral e cacy of compound 15 at a concentration of 2 µg/mL against SARS-CoV-2 was not signi cantly different from that of a concentration of 1 µg/mL.It was also noted that the compound exhibited a dose-dependent antiviral activity like other compounds.
In this study, eight new chalcone derivative compounds, expected to be effective against SARS-CoV2, have been synthesized.Molecular docking studies examine the binding of these compounds to target proteins and their interactions with essential residues in the proteins' active regions at the atomic level.Also, the structure-activity relationships (SAR) in this group of compounds were analyzed by comparing docking scores with in vitro results, as shown in Table 1.

Ser494
The replication mechanism of SARS-CoV-2 is mainly governed by RNA-dependent RNA polymerase (RdRp), the nsps 12, 8, and 7 complexes. 29This 6M71 PDB encoded RdRp protein is mainly extracted from SARS-CoV2. 30It has been reported that Ser759, Asp760, Asp761, Asp618, residues located in the catalytic region of the protein, are required for replication. 30,31The docking scores of the compounds varied between − 4,340 and − 3,266 kcal/mol.The docking scores of favipiravir and hydroxychloroquine were − 2,658 and − 2,215 kcal/mol, respectively.As a result of docking studies, compound 6 made a hydrogen bond with Ser814; a charged (negative) interaction with Asp760, Asp761, Asp618, and its binding a nity was determined at -4,370 kcal/mol, as shown in Fig. 6.
Viral RNAs were converted into various polyproteins with the effect of M pro of SARS-CoV2.Moreover, 6YB7 PDB encoded protein was used as a target protein in docking studies.The docking scores of the compounds ranged from − 3,953 to -2,748 kcal/mol, whereas the docking scores of favipiravir and hydroxychloroquine were − 2,271 and − 2,048 kcal/mol, respectively.It has been reported that Lys5, Ala285, Leu286, Glu288, Asp289, Glu290, located in the catalytic region of the protein, were responsible for the disruption of M pro . 28Compound 6 with the highest activity and docking score of -3,953 kcal/mol made hydrogen bond with Lys5; hydrophobic interaction with Leu286; and a charged (negative) interaction with lu288, Asp289, Glu290, as shown in Fig. 7.
The spike (S) protein, one of the target proteins for SARS-CoV2, gives the virus its typical shape, contains a speci c binding site that allows it to attach to the host cell.It attacks angiotensin-converting enzyme 2 (ACE-2) receptors in the host cell.6LZG PDB encoded protein was used as the target protein.The residues in the protein's catalytic region have been reported as Arg403, Tyr453, Gly496, Gln498, Asn501, Gly502, Tyr503, Tyr505.The docking scores of the compounds ranged from − 4.127 to -2.772 kcal/mol.The docking scores of favipiravir and hydroxychloroquine were − 2.555 and − 2.198 kcal/mol, respectively.In addition to having a high docking score of -4.127 kcal/mol, compound 6 made a hydrogen bond with Arg403 and Gly496, as shown in Fig. 6.
Docking studies conducted for different target proteins are in harmony with biological activity studies.All of the synthesized compounds are found to be more active than favipiravir and hydroxychloroquine.Compound 6 (Ct value: 32), the compound with the best activity against SARS-CoV2, also has the highest docking scores in all target proteins.The Ct values of favipiravir and hydroxychloroquine are calculated as 18 and 14, respectively.
Although the most active compound concentration of compound 6 on which it is effective is 4.78 µM, favipiravir and hydroxychloroquine are effective at 95.47, 59.54 µM, respectively.These results show that the e cacy of compound 6 against SARS-CoV2 is higher than that of favipiravir and hydroxychloroquine.Molecular docking studies support these activity results.
Besides bioactivity studies and molecular docking studies, the physicochemical and ADMET properties of the compounds are critical in terms of druglikelihood.Many compounds with signi cant biological activity do not make it to clinical trials due to their unsuitable physicochemical and toxicological properties.These properties of the compounds are described in QikProp (Maestro, Schrodinger 11.8), Datawarrior v4.07.02.and pre-ADMET software (https://preadmet.bmdrc.kr). 32The compounds' toxicological evaluation shows that none of them has the potential to be carcinogenic or mutagenic.Also, the compounds exhibit medium-level ability to inhibit the hERG potassium channel and CYP450.For ADMET properties, it has been shown that the properties of all compounds are suitable.In addition, all of the synthesized compounds comply with Lipinski's rule of ve.Details of the physicochemical and ADMET properties of the compounds are given in Table 3 and Table 4, respectively.In the study, the antiviral activities of new chalcone derivatives were evaluated by calculating their "Ct" values compared to that of the standard drugs and virus control group.In addition, the morphological appearance of the cells in each group and the viability rates of the collected cells were also evaluated.Morphological evaluation by microscopy and cell viability can also be considered a veri cation of "Ct" values.In cultures with a low "Ct" value (high viral load), intense CPE was detected in cells, and low cell viability was interpreted as an indicator of high viral reproduction.In cultures with a high "Ct" value (low viral load), the absence of CPE or the presence of minimal CPE in cells and high cell viability rates were interpreted as a low viral titer.Finally, in the experiments, it was indicated that compound 15 and all other compounds, especially the compounds 6, 7, and 9 showed remarkable antiviral activity against SARS-CoV-2.
Unfortunately, currently, there is no effective drug against SARS-CoV-2.For most people, the disease may cause mild illness; however, it can cause severe morbidity and even mortality in some people.Although there is no clear explanation for this situation, it is reported to be closely associated with host immunity.
The present study found that the newly synthesized chalcone derivatives showed a signi cant level of antiviral activity.The results indicated that the antiviral activities of the synthesized compounds were dose-dependent.Also, Compound 6, 15, and 16 have been found to possess potent activity against SARS-CoV-2 at the concentrations of 1.60, 2.0, and 1.60 µg/mL, respectively.
Chalcone derivatives were also analyzed in silico for their pharmacokinetic properties and were validated as having drug-like nature.This library was considered for tackling the modern-day issue of SARS-CoV-2 and further tested against homology modeled M pro , RdRp, and S proteins.Considering the potential e cacy of these newly synthesized compounds against SARS-CoV-2, we believe that these molecules may be potentially effective drug candidates.However, further studies are needed to test the effectiveness of these molecules by in-vivo tests.

Chemistry experimental procedure
General.All reagents used were commercially available unless otherwise speci ed and all solvents were distilled before use.Melting points were measured with Gallenkamp melting point devices.IR Spectra: PerkinElmer Spectrum One FT-IR spectrometer. 1 H-and 13 C-NMR Spectra: Varian 400 and Bruker 400 spectrometers.Elemental analysis results were obtained on a Leco CHNS-932 instrument.

Biological part
The effect of compounds 13 and 15 at three different concentrations on SARS-CoV-2 replication compared to standard drugs.
Interaction of compound 6 with the active site of 6M71.
Interaction of compound 6 with the active site of 6YB7.
mol MW: molecular weight of molecule.SASA: total solvent accessible surface area in square angstroms using a probe with a 1.4 Å radius.FOSA: hydrophobic component of SASA.FISA: hydrophilic component of SASA.PISA: Pi (carbon and attached hydrogen) component of SASA.WPSA: weakly polar component of the SASA (halogen, P and S).Total solvent-accessible volume in cubic angstroms using a probe with a 1.4 Å radius Mol Vol: Total solvent-accessible volume in cubic angstroms using a probe with a 1.4 Å radius.Donor HB: Estimated number of hydrogen bonds that would be donated by the solute to water molecule in an aqueous solution.accpt HB: number of hydrogen bonds that would be accepted by the solute from water molecule in an aqueous solution.LogP: Predicted octanol/water partition coe cient.PSA: polar surface area.Discussion COVID-19 is a highly contagious viral disease caused by coronavirus SARS-CoV-2, belonging to the beta coronavirus family.Currently, there is no effective speci c antiviral treatment option for SARS-CoV-2.However, several studies for using existing antivirals in isolation or combination and developing new therapeutic drugs to treat COVID-19 are underway.More extensive studies are needed to determine the reliability and effectiveness of these new drug candidates and to guide clinical decisions.Today, COVID-19 is a global health problem worldwide and is a serious and potential threat to human health.It is crucial to develop new protection strategies against SARS-CoV-2 and discover new active molecules against this virus.Studies are conducted on different structures; among them, one is chalcone scaffold.Chalcones are bioactive scaffolds of great medicinal interest due to their various pharmacological and biological activities to provide a valuable scaffold for discovering new drug leads.The current study aimed to determine the inhibitory activities of new chalcone derivatives against SARS-CoV-2 by calculating the "Ct" values of SARS-CoV-2 by RT-PCR method.

Figures Figure 1
Figures

Figure 2 Preparation
Figure 2

Table 1
Docking scores in kcal/mol for molecules in target proteins.The primary targets of drugs developed against SARS-CoV2 are speci ed as M pro , RdRp, and spike proteins in the literature.In silico studies have investigated the SARS-CoV2 inhibition potential of newly synthesized compounds using the target proteins.
28In this context, 6M71 PDB encoded protein (SARS-CoV-2 RNA-dependent RNA polymerase in complex with cofactors) was used for RNA-dependent RNA polymerase, 6YB7 PDB encoded protein (SARS-CoV-2 main protease with unliganded active sites) for M pro , and 6LZG PDB encoded protein (Structure of novel coronavirus spike receptor-binding domain complexed with its receptor ACE2) was used for spike protein.The interactions of synthesized compounds and reference molecules with target proteins are given in Table2.

Table 2
Interactions of compounds with active sites of the target proteins.