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 identied eight compounds inhibiting each target, with binding anity scores ranging from − 4,370 to -2,748 kcal/mol along with their toxicological, ADME, and drug-like properties. The SARS-CoV-2 Hatay strain (SARS-CoV-2, Hatay, 2020), isolated by Duran, was used in the study. The SARS-CoV-2 strain was propagated in the Vero E6 cell line (African green monkey kidney cells), obtained from the American Type Culture Collection (ATCC C1008). After seven days of incubation at 37°C in an atmosphere of 5% CO 2, the supernatant was harvested. Vero E6 cell line was used for viral isolation. Cell cultures were maintained with 10% fetal calf serum in RPMI-1640 medium containing 10 mM HEPES, 4 IM glutamine, and 100 IU/ml penicillin/streptomycin. Incubation of cells was carried out at 37 ºC in an incubator with 95% air and 5% CO 2 . Cell density was adjusted to 1x10 5 cells per ml. Experiments were performed by adding cell maintenance medium to 10% of the culture dish in culture dishes. Incubation of cell cultures continued for 96 hours. Cells were removed from the culture vessels with 0.25% trypsinization solution and collected in 50 ml centrifuge tubes by centrifugation at 1250 rpm for 10 minutes at the end of incubation. Cell number and viability were determined by hemocytometer with 1% trypan blue dye prepared in 0.9% NaCl. conrmed both by determining the presence of cytopathological effect and by the Real-Time PCR method. The formation of CPE was detected on the 4th day in the Vero cell line. PCR method, the presence or increase of viral replication was described as an increased number of viral copies. Viral RNA extraction was performed using a commercial kit under the commercial kit procedure. In the studies, the SYBR-Green RT-qPCR method was performed in accordance with the QuantiTect SYBR-Green PCR kit (Qiagen) test procedure. The PCR cycle starts with an initial denaturation at 95°C for 15 minutes, followed by three steps of 45 cycles of 94°C for 15 seconds, annealing at 60°C for 30 seconds and elongation at 72°C for 30 seconds, and a nal extension temperature of 72°C for 10 minutes. The Human β-actin gene (QuantiTect Primer Assays, Qiagen) was used as the reference gene. All reactions were tested in triplicate at different intervals.


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. 1,2 General treatment includes nutritional supplements, immune boosters, and some alternative medications in folk medicine. 3 Various 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. 4,5 Previous 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. Today, some of the available protease and RNA polymerase enzyme inhibitor drugs are being redesigned to treat SARS-CoV-2. [6][7][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 The synthesis of the second group of compounds (13)(14)(15)(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)(14)(15)(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 It 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 As 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.  Tyr505, Tyr495, Tyr453, Phe497, Phe390, Ala387 Glu288, Asp289, Glu290 Asp38, Glu37, Glu406 The replication mechanism of SARS-CoV-2 is mainly governed by RNA-dependent RNA polymerase (RdRp), the nsps 12, 8, and 7 complexes. 29 This 6M71 PDB encoded RdRp protein is mainly extracted from SARS-CoV2. 30 It has been reported that Ser759, Asp760, Asp761, Asp618, residues located in the catalytic region of the protein, are required for replication. 30 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). 32 The 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. 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.
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.

Viral propagation
The SARS-CoV-2 Hatay strain (SARS-CoV-2, Hatay, 2020), isolated by Professor Dr. Nizami Duran, was used in the study. The SARS-CoV-2 strain was propagated in the Vero E6 cell line (African green monkey kidney cells), obtained from the American Type Culture Collection (ATCC C1008). After seven days of incubation at 37°C in an atmosphere of 5% CO 2, the supernatant was harvested.

Titration of the Virus
Stock viral strains (maintained at -80 ºC) were rapidly dissolved in 37 ºC water (in a bain-marie) and were re-activated in cell lines (Vero E6 cell line).
The cells were then exploded by "freezing and thawing". Cells were collected from the culture dish and centrifuged at 4000 rpm for 20 minutes, and the supernatant virus was collected as a stock solution. Like in our previous study in which TCID 50 of the virus was calculated 34 ; in the present study, three different titers of SARS-CoV-2 (1, 10, and 100 TCID 50 ) were chosen to be studied.

3-(4,5-Dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide (MTT) Method
This method is based on the principle of the cleavage of the MTT tetrazolium ring in active mitochondria. In this method, cultured live cells can be detected colorimetrically and quantitatively. The MTT method, rst described by Mosmann and later developed, is a practical method used to determine cell viability. 35 MTT is a substance that is actively absorbed into cells and is reduced to colored water-insoluble form due to activity in mitochondria. The MTT reduction feature of cells is considered a measure of cell viability. The density of the dye obtained as a result of MTT analysis correlates with the number of live cells. This method was used to detect the presence of living cells to demonstrate the effectiveness of the chemical compounds at different concentrations against SARS-CoV-2. As viral replication would decrease the live-cell ratio, a detected decrease in cell viability was indicated as increased viral reproduction.
In the MTT assay, the decrease in cell viability was associated with increased viral replication, whereas high cell viability was considered as an indicator of antiviral activity. For this purpose, seawater at different concentrations was added to the SARS-CoV-2 inoculated cell lines and was incubated for 96 hours in an incubator with 5% carbon dioxide at 37 ºC. 10 µl of MTT was added to each well, and the plates were incubated for 4 hours under the same conditions. Absorbance measurements were made at 570 nm on a spectrophotometer.

Real-Time PCR
SARS-CoV-2 replication in cell culture was veri ed by the Real-Time PCR method. Compared to the control group using the Real-Time PCR method, the presence or increase of viral replication was described as an increased number of viral copies. Viral RNA extraction was performed using a commercial kit under the commercial kit procedure. In the studies, the SYBR-Green RT-qPCR method was performed in accordance with the QuantiTect SYBR-Green PCR kit (Qiagen) test procedure. The PCR cycle starts with an initial denaturation at 95°C for 15 minutes, followed by three steps of 45 cycles of 94°C for 15 seconds, annealing at 60°C for 30 seconds and elongation at 72°C for 30 seconds, and a nal extension temperature of 72°C for 10 minutes. The Human β-actin gene (QuantiTect Primer Assays, Qiagen) was used as the reference gene. All reactions were tested in triplicate at different intervals. 36

Cycle Threshold Value
In isolation, "Ct" values provide a relative measure of the viral quantity in the sample. Different known quantities of standards were included in the same study and tested in parallel with viral cultures. Thus, the "Ct" value could potentially provide some measure of viral copies. 37,38 An 8 to 10-fold dilutions series was prepared from the culture ltrate for SARS-CoV-2. Five replicates of each dilution were tested in parallel. All replicates were detected at dilutions of 10 − 1 to 10 − 5 . 39

Molecular docking studies
Molecular modeling studies were conducted using Maestro 11.8 (Schrödinger, LLC, New York, NY) software. Possible ionization states and tautomers of the ligands were determined by LigPrep (Schrödinger, LLC, New York, NY). Then, all ligands were optimized using the conjugate gradient method with force eld parameters MacroModel and OPLS3 (Schrödinger, LLC, New York, NY). 40 After the proteins were downloaded from the RCSB Protein Database (www.rcsb.org) 41 , this version of Protein Preparation Wizard was used for docking studies (Epik, Prime, Impact: Schrödinger, LLC, New York, NY). 42 In this process, unwanted structures in proteins were removed, hydrogen atoms and missing side-chain atoms were added, partial charges and bonds were assigned, ionization states, hydrogen, and disul de bonds were formed. Active regions of proteins were determined for docking, and grid maps of proteins were created using Maestro's receptor grid generation panel. Ligands were docked on these maps 100 times in extra precision mode using Glide (Schrödinger, LLC, NY) software. The poses obtained from docking were examined, and appropriate docking scores were determined, as shown in Table 1. 43 Figures Figure 1 Preparation of uoro/tri uoromethyl-substituted trimethoxy chalcones (6-9).

Figure 5
The effect of compounds 13 and 15 at three different concentrations on SARS-CoV-2 replication compared to standard drugs.