Synthesis, characterization, molecular docking, pharmacokinetics, and molecular dynamics of new bis-thiazoles based on bis-thiosemicarbazone as anti-coxsackievirus (2024)

Viral infections can range in severity from mild to life-threatening; some, including HIV, Hepatitis B, and Hepatitis C, are extremely serious problems for global public health¹. Coxsackieviruses B (Cox B) are known as RNA viruses that belong to the family Picornaviridae. They are transmitted viathe fecal-oral route, and upon infection, fever, headache, sore throat, gastrointestinal distress, extreme fatigue, as well as chest and muscle pain, occur². More than 90% of the cases don’t develop life-threatening symptoms, but on reaching the pancreas, liver, myocardium, and meninges viathe bloodstream, they can cause pancreatitis, hepatitis, aseptic meningitis, and myocarditis³. Due to a lack of anti-Cox B drugs⁴, antiviral activity of different plant extracts and synthetic compounds was tested as shown to affect the virus, as were the anti-cox B activity of Rheum palmatum⁵, quinoxaline derivatives⁶, and ginsenosides⁷. Focusing on the heterocyclic compounds, we noted that thiazole-containing compounds showed several pharmaceutical applications^8,9,10,11. It is spread in natural products like vitamins (thiamine), peptides¹², epothilone¹³, chlorophyll¹⁴, and alkaloids¹⁵. A long range of biological activities are attributed to the presence of a thiazole ring in the molecules as analgesic¹⁶, antioxidant¹⁷, antiallergic¹⁸, antibacterial¹⁹, anti-inflammatory²⁰, anticancer²¹, antimalarial²², and antihypertensive²³. The Food and Drug Administration (FDA) approved more than eighteen drugs containing thiazole moieties, with the Ritonavir drug containing a bis-thiazole moiety in its skeleton, as illustrated in Fig.1a. Our recently published review article²⁴ on the patents antiviral activity of thiazole ring revealed that more than 141 compounds having thiazole ring showed antiviral activity as anti-influenza (H1N1 and H3N2), anti-coronaviruses, anti-HCV, anti-HIV, and anti-HSV-I.

In addition, several studies^25,26,27 demonstrated that compounds with more than one thiazole ring unit have good biological activities. Numerous patents in Fig.1b list bis-thiazole compounds as effective antiviral agents²⁸. From all the above data and in continuation of our research field in the synthesis of bioactive heterocyclic compounds^{29,30,31,32,33,34,35,36} herein, we synthesized a series of bis-thiazole derivatives to investigate their antiviral activity and mechanism of action against Cox B. In addition, the molecular docking, pharmacokinetics ADMET Analysis, and MD stimulation were studied and discussed.

Chemistry

The condensation reaction of 2-(2,5-dimethoxy-benzylidene)indan-1,3-dione 1³⁷ with two mole equivalents of thiosemicarbazide 2 in ethanol with drops of HCl gave bis-thiosemicarbazone 3 (Scheme 1). The structure of bis-thiosemicarbazone derivative 3was established based on their ¹H NMR, ¹³C NMR, and IR spectra. As for instance, ¹H NMR spectrum of bis-thiosemicarbazone derivative 3 revealed six singlet signals at δ = 3.67, 3.73 (two OCH₃), 8.05, 8.15, 11.38 (three NH), and 8.35 (= CH), in addition to multiple signals in the aromatic region for seven aromatic protons at δ = 6.89–7.60 ppm. The three NH signals in ¹H NMR of compound 3 are attributed to the non-equivalent two protons of NH₂ due to one of these two protons forming an H-bond with the C = N as illustrated in Figure S1in the supplementary data Section²⁴. In addition, the ¹³C NMR spectrum exhibited a signal at 178.3 ppm, which was ascribed to the carbons of the two thioxo groups having overlapping. The mass of compound 3 was equivalent to the computed values.

Bis-thiosemicarbazone derivative 3 was used as a basic compound for the building of bis-thiazole derivatives. So firstly compound 3 (one mole equivalent) was reacted smoothly with hydrazonoyl chloride 4a-d (two mole equivalents) in dioxane/Et₃N (the progress of the reactions monitored with TLC). Such reaction proceeded through the formation of two non-isolable intermediates 5 & 6, followed by the elimination of two water molecules to afford the target bis-thiazole derivatives 7a-d (Scheme 2). ¹H NMR of all bis-thiazole derivatives 7a-d revealed the characteristic singlet signals of 2CH₃, 2OCH₃, =CH, and 2NH protons at δ = 2.27–2.56, 3.71–3.80, 7.41–8.14, and 8.67–8.75 ppm in addition to the other aromatic and aliphatic protons for each derivative. All IR spectra have an absorption band for the NH at ν = 3434 –3394cm⁻¹. ¹³C NMR showed the two methoxy group carbons in derivatives 7a-d as two signals at roughly 56.0 and 56.8 ppm. Additionally, the products 7a-d’s molecular weight measurement was found in the anticipated range.

In a similar way and under the same reaction conditions, bis-thiosemicarbazone derivative 3 was reacted with α-haloketones 8a, b, 10a-c, and 12 to afford another series of various bis-thiazole derivatives 9a, b, 11a-c, and 13, respectively. The structures of all bis-thiazole derivatives 9a, b, 11a-c and 13 were assured based on their spectroscopic data, For example, the IR spectrum of derivative 9b showed the characteristic absorption bands for C = O and C = N at ν = 1723 and 1638cm⁻¹, respectively. While in its ¹H NMR spectrum, a doublet signal was observed for 2CH₃at 1.12 ppm, a quartet signal for two CH-thiazole at 3.97 ppm, and 2NH protons at δ = 11.22 ppm. The signal detected in the ¹³C NMR spectra at 16.7 ppm was attributed to the overlapping carbons of the two methyl groups. Additionally, Compound 11a’s IR spectrum, revealed the presence of a distinctive absorption band at 3439cm⁻¹, which is linked to the NH group. At 1635 and 1574cm⁻¹, respectively, two medium bands formed that corresponded to the symmetric and asymmetric stretching vibrations of C = N. The creation of compound 11a was confirmed by the development of a single band for C = O stretching vibrations approximately 1702cm⁻¹, and the presence of a band for the NH group. Furthermore, the target compounds’ effective synthesis was confirmed by the ¹H NMR spectra. Accordingly, the two singlet peaks of methyl protons at δ 1.78 and 2.30 ppm with six protons integration for each signal and the existence of a singlet signal for NH at δ 11.18 confirmed the synthesis of compound 11a. While ¹³C NMR provided a signal for the carbonyl carbon at 168.2 ppm (overlapped). For compound 13, the NH group was detected in the IR spectrum at 3436cm⁻¹. While ¹H NMR provided multiple signals at 6.94–8.42 ppm for both CH-thiazole with the aromatic protons integrated for twenty protons, and a downfield signal at 11.63 ppm for the two imino protons. Otherwise, MS generated the molecular ion peak corresponding to the confirmed molecular weights of target 13.

In vitro antiviral activity

Cytotoxicity of tested compounds

Cytotoxicity assay Table1 shows the safe concentrations of compounds that can be applied to Vero cells without causing any morphological changes after 24h. Results showed that compounds have different safety effects on cells. Some were highly toxic at low concentration like 9a and 9b; others showed higher safety when applied to cells like 1, 3, 7d, and 13. Other compounds were dense and coloured that made masking to the cell sheet, and so it was hard to examine at higher concentrations as compounds 7a, 7b, 7c, 11a, 11c, and 11b, and accordingly suitable concentrations for each compound were used in the plaque reduction assay.

Antiviral activity of some compounds against Cox B virus

Plaque reduction assay was used to test the antiviral activity of chemical compounds under test at safe concentrations suitable for each compound. Results in Table2 showed that compound 7a caused high viral inhibition at tested concentrations.

Mechanism of action of active compounds

For an active compound having antiviral activity, three possible mechanisms by which it can affect viral infectivity (a) direct effect on the viral particle that we call virucidal; (b) effect on the replicating cycle of the virus, i.e., any of the steps that occur inside the cell supporting viral replication starting from uncoating till assembly and release; (c) effect on the binding site on the cells and thus preventing the virus from being able to enter the target cell and cause infection. On studying the mechanism of action of the most active bis-thiazole derivative 7a (Table3; Fig.2), we found that compound 7a had combined activity between adsorption and replication as it showed higher % inhibition on applying experiment steps that support testing those two mechanisms. Results showed that compound 7a has a double action, inhibiting both viral adsorption and replication. This means that compound 7a has the ability to bind to cell receptors of the virus and so prevent viral entry to the target cells, causing an effect on adsorption and also a high % reduction on testing the effect of the compound on replication showed that compound 7a caused inhibition to one of the important steps needed by the virus to complete its replicating cycle inside the cell.

On the other hand, the tested compound was found to have no direct effect on the viral particle itself, which means that the compound whether didn’t bind with outer surface of the virus or change its configuration and so turned it to be non-infectious.

Computational analysis

Molecular docking

Molecular docking is commonly used to predict the alignment of small molecule medicinal drugs with their protein targets, as well as the small molecule’s affinity and activity. Docking is an important tool in rational drug design. Given the biological and pharmacological significance of docking investigations, great effort has been made to improve the algorithms for docking prediction³⁸. The utility of docking in evaluating the interactions between synthesized compounds and protein receptors. It offers valuable insights into their binding modes and potential biological activities^39,40,41,42.

Docking and molecular interaction of synthesized compounds

Molecular docking was used to study the binding relationships between the generated compounds and protein targets linked to Coxsackievirus crucial protein receptors. The purpose of this analysis was to provide insight into the compounds’ effectiveness. Table4; Fig.3 show the findings of the docking investigations that investigated the binding affinities between the compounds and three Coxsackievirus vital protein receptors. Among affinity of compounds with these three proteins, compounds 7a and 7c have the highest binding energy with three Coxsackievirus vital protein receptors and comparing with Pleconaril (an antiviral medication, primarily acts against Coxsackievirus B by inhibiting the replication of the virus. Through a search in the Drug Bank Database using Pleconaril, the protein target identified was Capsid protein VP0. This protein plays a crucial role in immature procapsids, being cleaved into capsid proteins. It enables the capsid to remain inactive until the maturation process occurs) as a positive reference drug.

Docking and interaction studies with Coxsackievirus Adenovirus receptor (CAR)

The CAR receptor is the essential protease of adenovirus and, as such, represents a promising target for the treatment of ocular and other adenoviral infections. Based on docking outcomes, the protease exhibits a significant affinity towards 7a, 7c, 11b, and 11c, showcasing binding energies of −6.80, −6.80, −6.50, and − 6.30kcal/mol, respectively. These substances establish hydrogen bonds with key residues like Ser75, Asn130, Ser60, and Asp68, while also engaging in hydrophobic interactions such as (Pi-alkyl) with Leu73, Val70, Leu58, Tyr80, Val67, (Pi-cation) with Glu56, Asp68, Lys132, Asp54, Glu56, Asp68, and Lys121, (Pi-sigma) with Tyr80, (C-hydrogen bond) with Gln119 and Asp54, (Pi-sulfur) with Lys121, (unfavourable-bump) with Lys121, and (Pi-Pi-stacked) with Tyr80. Overall, these results indicate that 7a and 7c show the most promise among the compounds and merit further investigation as potential coxsackievirus adenovirus receptors. (Fig.4 and Table S1in the supplementary data section). Our findings are similar to those of⁴³, in which docking was employed to evaluate the inhibitory interaction between a molecule and adenovirus receptor protein.

Docking and interaction studies with 3C-protease from coxsackievirus

The 3C protease derived from coxsackievirus functions as a critical viral protease enzyme pivotal in the viral replication process. This cysteine protease is responsible for cleaving viral polyprotein precursors, leading to the production of essential viral proteins crucial for viral replication. Through docking analyses, it is evident that the compound exhibits a robust affinity for 7a, 7c, 11b, and 11c, with binding energies of −9.60, −9.30, −9.50, and − 9.40kcal/mol, respectively. These compounds establish hydrogen bonds with Gly164, Gly145, Gln146, Gly147, and Val162, while also engaging in hydrophobic interactions such as (Pi-alkyl) with Leu127, Phe170, Pro38, Lys108, Ile104, Val116, Ile114, His161, Leu102, Tyr138, Ala144, His40, Phe25, Phe140, (halogen) with Glu24, (Pi-Pi T shaped) with His40, (Pi-sulfur) with His161, His40, Phe25, and Cys147, (Pi-cation) with Gly147, Gly147, His40 and Glu71, (carbon-H-bond) with Gly169 and Glu71,(Pi-sigma) with Gly163. The amino acids Gly164, Gln146, Gly147, and Leu127 within the catalytic site appear to augment the binding affinity of the compounds significantly. In summary, these observations indicate that 7a demonstrates substantial potential as an inhibitor of the 3C protease from coxsackievirus. (Fig.5and Table S2 in the supplementary data section). These results were similar to⁴⁰ in which protease was used as a therapeutic protein target for drug development.

Docking with 3Dpol RNA-dependent RNA polymerase (RdRp) of Coxsackievirus

RdRp is a pivotal enzyme crucial for viral replication, facilitating the synthesis of RNA strands, a fundamental process for viral reproduction within host cells. Docking analyses indicate that compounds 7a, 7c, 11b, and 11c exhibit the highest affinity, with binding energies of −10.90, −9.80, −9.50, and − 9.40kcal/mol, respectively. These compounds establish hydrogen bonds with Ser295, Gly290, Leu175, Lys61, Gly293, Thr294, Gly290, Ala109, Arg188, Thr114, and Lys127, while also engaging in hydrophobic interactions such as (Pi-alkyl) with Arg174, Leu107, Ile398, Leu418, Leu421, Ile176, Lys172, Lys61, Ala178, Ile176, His199, Leu107, Leu110, and Tyr195, (Pi-Pi stacked) with His199, (carbon-hydrogen bond) with Ser289, Asp329, Ser401, (Pi-sigma) with Arg174 and Met393, (Pi-anion) with Glu227, Asp330, Tyr195, Asp329, Glu108, Asp111, Asp238. The amino acids Thr294, Arg188, Thr294, and Thr114 in the catalytic site appeared to enhance the binding affinity of the compounds. Overall, the amino acids Thr294, Arg188, Thr294, and Thr114 within the catalytic site appear to significantly enhance the binding affinity of these compounds. In conclusion, these results suggest that 7a shows promise as a potential candidate for further investigation as an inhibitor of the RdRp of Coxsackievirus. (Fig.6and Table S3 in the supplementary data section). These results were similar to that of⁴⁴ where they used RdRp as a viral protein receptor for explanations of antiviral activities of the compound through molecular docking analysis.

In silico pharmacokinetics ADMET prediction of synthesized compounds

Based on the results of molecular docking for compounds 7a, 7c, 11b, and 11c, the most promising candidates with the highest affinity concerning ADME and toxicity risks have been pinpointed. Initially, the physicochemical characteristics of the tested compounds are detailed in Table5; Fig.7. All relevant criteria were meticulously scrutinized. Consequently, all compounds exhibited a molecular weight exceeding 500 and possessed an adequate number of rotatable bonds (5–11 RBs), a pivotal factor for substantial structural flexibility. The count of hydrogen bond acceptors (HBA) and donors (HBD) was also determined for the four compounds, revealing that each compound featured fewer than 10 HBA and less than 5 HBD, indicating a favorable HBA-HBD equilibrium and enhanced potential for oral bioavailability. Moreover, the optimum reference TPSA were obtained as (20–160), therefore, our compounds displayed relatively high TPSA values, predominantly falling within the optimal range of 99–152 for efficient gut absorption and oral bioavailability. Subsequently, the lipophilicity and water solubility of compounds 7a, 7c, 11b, and 11c were evaluated. The results indicated poor water solubility across all active compounds, with Log S values ranging from − 9.894 to −6.352, signifying limited water solubility. Pharmacokinetic tests were then conducted, revealing high theoretical bioavailability for the compounds, and positioning them as promising drug candidates. However, moderate intestinal absorption was observed for all compounds, alongside the potential to interact with other drugs by inhibiting CYP2C9, CYP2C19, and CYP1A2 enzymes. Moreover, the drug-likeness of the compound was assessed through Lipinski, Golden Triangle, and Pfizer rules. All four compounds satisfied the drug-likeness requirements specified by the Pfizer Rule, suggesting promising physicochemical characteristics for drug development. Notably, all compounds, with the exception of 7a, adhered to the Lipinski criteria, while none met the criteria set by the Golden Triangle rule. Additionally, the distribution of compounds, including Plasma Protein Binding (PPB), was assessed, revealing high levels of protein binding exceeding 99%, indicative of a low therapeutic index and minimal unbound plasma fraction. Furthermore, the Blood-Brain Barrier (BBB) penetration analysis suggested that all compounds were BBB-, incapable of crossing the blood-brain barrier. Also, according to computational assessments, compounds 7b and 11b seem to be relatively safe and non-toxic. Conversely, compounds 7a and 11c exhibit mutagenic and tumorigenic effects, as outlined in Table6.

Molecular dynamics simulation (MDS)

Based on the docking of the three coxsackievirus vital receptors with a 7a and 11b compound, dynamic simulations were performed to investigate the behavior and stability of the complex at the atomic level. Firstly, the MDS of CAR complexes with 7a and 11b were performed to assess the stability and dynamics of the CAR complexes. The Root Mean Square Deviation (RMSD) was used to evaluate the stability of the protein structures. The results depicted in Fig.8a indicate that both CAR-7a and CAR-11b remained stable within the ranges of (0.20–0.25nm) and (0.25–0.30nm), respectively, and exhibited stabilization after 10 and 15 ns. Furthermore, Root Mean Square Fluctuation (RMSF) was conducted to evaluate the flexibility of amino acid residues throughout the simulation. The majority of residues displayed minimal fluctuations (0.1–0.45nm), signifying a state of relative stability (Fig.8b). Moreover, Radius of gyration (Rg) analysis was performed to assess the general conformation of the protein complexes. Figure8c illustrates Rg values for the CAR-7a and CAR-11b complexes falling within the range of 1.50 to 1.55nm (Fig.8c). The Rg values provide insights into the compactness or expansion of the protein structures during the simulation. Next, Surface Area of Solvent Accessible (SASA) analysis was conducted to understand the protein folding dynamics and stability. SASA values for the CAR-7a and CAR-11bcomplexes varied from 80 to 105nm² (Fig.8d). Furthermore, the intramolecular and intermolecular hydrogen bonds were analyzed to assess the stability of the complexes. The complexes formed a range of 120–140 intramolecular hydrogen bonds (Fig.8e). Also, it formed (1–12 bonds) intermolecular interactions bonds (Fig.8f). Our MD simulations were consistent with⁴³, who also performed MD simulations to validate interaction and stability of adenovirus receptor complexes. Secondly, MDs were conducted on the 3C-protease complex with 7a and 11b. The results presented in Fig.9a demonstrate that the RMSD values of the 3C protease with 7a and 11b remained consistent within the intervals of (0.20–0.25) and (0.20–0.30) nm respectively, stabilizing after 10 and 30 ns. Additionally, RMSF analysis was performed as illustrated in Fig.9b. The most residues exhibited minor fluctuations, typically ranging from 0.1 to 0.5nm. Figure9c exhibits the Rg values of 3C-protease with (7a and 11b), varying between (1.80–190nm). Furthermore, Fig.9d depicts the SASA values for 3C-protease with (7a and 11b) falling within the range of 140–150nm². Also, Fig.9e illustrate the intramolecular hydrogen bonds in 3C-protease with (7a and 11b), ranging from 220 to 245 bonds. Regarding intermolecular hydrogen bonds, 3C-protease with (7a and 11b) formed intermolecular hydrogen bonds (1–10) bonds (Fig.9f). Finally, MDs were performed to assess the stability of the RdRp complex with 7a and 11b. As illustrated in Fig.10a, the RdRp-7a and RdRp-11b complexes demonstrated stability, with an RMSD ranging between 0.20 and 0.35nm, achieving stability after 10 ns. RMSF analysis in Fig.10b revealed minor variations, typically between 0.1 and 0.50nm. Additionally, the Rg values for RdRp-7a fluctuated between 2.30 and 2.40nm, as shown in Fig.10c. Furthermore, Fig.10d shows the SASA values for RdRp-7a and RdRp-11b ranged from 220 to 235nm². Lastly, Fig.(10e and 10f) showcased the intramolecular and intermolecular hydrogen bonds formed by the RdRp complex with 7a and 11b. Initially, the complexes exhibited a range of 300–350 intramolecular hydrogen bonds. Compounds 7a and 11bformed intermolecular hydrogen bonds in the range of (1–8) bonds. Our molecular dynamics were comparable to those performed by⁴⁵, who used MD simulations to validate the contact and stability of receptor complexes.

Chemistry

General methods

All reagents were purchased at the highest available purity from Sigma-Aldrich and were used without further purification. Melting points were measured on a Gallenkamp melting point apparatus. IR spectra were applied on Shimadzu FT-IR 8101 PC infrared spectrophotometers (Shimadzu, Tokyo, Japan) using KBr disks. ¹H NMR spectra were run at 400MHz, and ¹³C NMR spectra were run at 100MHz in deuterated dimethyl sulfoxide (DMSO-d₆). Chemical shifts are given in parts per million and are related to that of the solvent. Mass spectra were recorded on a Bruker Daltonics spectrometer. Elemental analyses were recorded on an Elementar-Vario EL (Germany) automatic analyzer. Analytical thin-layer chromatography (TLC) was performed using silica gel 60 F254 glass plates, which was used to track each chemical reaction’s development and verify each derivative’s purity. Compound spots were visualized by UV light (254nm).

Synthesis of bis-thiosemicarbazone derivative 3

2,2’-((1E,3Z)−2-((E)−2,5-dimethoxybenzylidene)−1H-indene-1,3(2H)-diylidene)bis-(hydrazine-1-carbothioamide) (3).

In a 50 mL round Q.F. flask we added 0.01mol of 2-(2,5-dimethoxy-benzylidene)-indan-1,3-dione 1 (2.94g) with 0.02 moles of thiosemicarbazide 2 (≈ 2g) in abs. ethanol (40 mL). The mixture was heated under reflux to dissolve the thiosemicarbazide. After that, 2 mL of conc. HCl was added and the reflux was completed to 2h. Following cooling of the solution, compound 3 was obtained as yellow crystals by filtering and crystallizing the solid bis-thiosemicarbazone derivative 3 from ethanol/dioxane.

Yellow crystal, mp. 231–233°C. IR (ν): 3429, 3315, 3174 (NH₂, NH), 3010 (sp² CH₂), 2939 (sp³ CH) 1598(C = N), 1526, 1494, 1430, 1362, 1262, 1078cm⁻¹. ¹H NMR (DMSO-d₆) 3.67 (s, 3H, OCH₃), 3.73 (s, 3H, OCH₃), 6.88–6.94 (m, 5H, Ar-H), 7.61 (d, J= 8.5Hz, 2H, Ar-H), 8.10 (s, 2H, NH) 8.15 (s, 2H, NH) 8.34 (s, 1H, = CH) 11.38 (s, 2H, NH). ¹³C NMR 56.1 (OCH₃), 56.7 (OCH₃), 110.5, 113.6, 117.9, 123.3, 138.4, 138.6, 152.8, 153.8, 178.3 (two carbon overlapped). HR-ESI-MS 441.2516 [M + H]⁺. Anal. Calcd. (Found) for: C₂₀H₂₀N₆O₂S₂ (440.54): C, 54.53 (54.42); H, 4.58 (4.43); N, 19.08 (19.01)%.

General method for synthesis of bis-thiazole derivatives 7a-d, 9a, b, 11a-c and 13

A mixture of bis-thiazole 3 (10 mmol) with the appropriate hydrazonyl chloride (20 mmol) 4a-d or α-haloketones 8a, b, 10a-c or 12 and triethylamine (few drops) in dioxane (20 mL) was refluxed till all of the starting materials have disappeared (monitored by TLC). The solvent was evaporated and the solid formed was filtered off and recrystallized from dioxane/ethanol mixture to give compounds 7a-d, 9a, b, 11a-c and 13.

2,2’-(((1E,3E)−2-(2,5-dimethoxybenzylidene)−1H-indene-1,3(2H)-diylidene)bis(hydrazin-1-yl-2-ylidene))bis(4-methyl-5-((E)-phenyldiazenyl)thiazole) (7a).

Brown solid, mp. 123–125°C. IR (ν): 3394 (NH), 1602 (C = N), 1541, 1491, 1356, 1253, 1221, 1171, 1039cm⁻¹. ¹H NMR (DMSO-d₆) 2.59 (s, 6H, 2CH₃) 3.71 (s, 3H, OCH₃), 3.80 (s, 3H, OCH₃) 7.01–7.44 (m, 17H, Ar-H), 7.47 (s, 1H, =CH), 8.77 (s, 2H, 2NH). ¹³C NMR (DMSO-d₆) 17.6 (2CH_3, two methyl overlapped), 56.0 (OCH₃), 56.9 (OCH₃) 103.3 110.2, 113.7, 116.7, 126.4, 129.5, 132.1, 134.8, 136.4, 145.7, 148.3, 152.7, 154.0, 168.9. HR-ESI-MS 725.1879 [M + H]⁺. Anal. Calcd. (Found) for: C₃₈H₃₂N₁₀O₂S₂ (724.86): C, 62.97(62.86); H, 4.45 (4.39); N, 19.32 (19.29)%.

2,2’-(((1E,3E)−2-(2,5-dimethoxybenzylidene)−1H-indene-1,3(2H)-diylidene)bis(hydrazin-1-yl-2-ylidene))bis(4-methyl-5-((E)-p-tolyldiazenyl)thiazole) (7b).

Dark brown solid, mp. 132–135°C. IR (ν): 3394 (NH), 2919 (sp³CH), 1608 (C = N), 1539, 1492, 1423, 1220, 1170, 1039cm⁻¹. ¹H NMR (DMSO-d₆) 2.21 (s, 6H, 2CH₃), 2.27 (s, 6H, 2CH₃), 3.76 (s, 3H, OCH₃), 3.80 (s, 3H, OCH₃), 7.05–7.51 (m, 16H, ArH and = CH), 8.96 (s, 2H, 2NH). ¹³C NMR (DMSO-d₆) 17.6 (2CH_3, two methyl overlapped), 55.9 (OCH₃), 56.7 (OCH₃) 102.9, 103.6 109.8, 113.7, 116.4, 123.9, 129.5, 132.1, 134.8, 136.8, 152.0, 153.8, 158.6, 162.8, 168.5, 168.7.

HR-ESI-MS 753.8992 [M + H]⁺. Anal. Calcd. (Found) for: C₄₀H₃₆N₁₀O₂S₂ (752.91): C, 63.81 (63.66); H, 4.82 (4.71); N, 18.60 (18.53)%.

2,2’-(((1E,3E)−2-(2,5-dimethoxybenzylidene)−1H-indene-1,3(2H)-diylidene)bis(hydrazin-1-yl-2-ylidene))bis(5-((E)-(4-chlorophenyl)diazenyl)−4-methylthiazole) (7c).

Brown solid, mp. 119–121°C. IR (ν): 3420 (NH), 2919 (sp³ CH), 1602 (C = N), 1545, 1488, 1422, 1249, 1167, 1087cm⁻¹. ¹H NMR (DMSO-d₆) 2.56 (s, 6H, 2CH₃) 3.78 (s, 3H, OCH₃), 3.82 (s, 3H, OCH₃), 7.06–7.46 (m, 15H, ArH and = CH), 8.76 (s, 2H, 2NH). ¹³C NMR (DMSO-d₆) 16.7 (2CH_3, two methyl overlapped), 56.0 (OCH₃), 57.4 (OCH₃) 102.8, 103.7, 109.8, 112.0, 113.7, 116.4, 123.4, 126.9, 133.4, 136.1, 139.1, 150.4, 153.1, 157.0, 160.1, 170.1. HR-ESI-MS 794.2627 [M + H]⁺. Anal. Calcd. (Found) for: C₃₈H₃₀Cl₂N₁₀O₂S₂ (793.75): C, 57.50 (57.45); H, 3.81 (3.69); N, 17.65 (17.52)%.

2,2’-(((1E,3E)−2-(2,5-dimethoxybenzylidene)−1H-indene-1,3(2H)-diylidene)bis(hydrazin-1-yl-2-ylidene))bis(4-methyl-5-((E)-(4-nitrophenyl)diazenyl)thiazole) (7d).

Dark brown solid, mp. 181–183°C. IR (ν): 3434 (NH), 2920 (sp³ CH), 1595 (C = N), 1545, 1491, 1428, 1327, 1255, 1217, 1157, 1106, 1020cm⁻¹. ¹H NMR (DMSO-d₆) 2.51 (s, 6H, 2CH₃), 3.74 (s, 3H, OCH₃), 3.78 (s, 3H, OCH₃), 7.05–8.14 (m, 16H, ArH and = CH), 8.75 (s, 2H, 2NH).

¹³C NMR (DMSO-d₆) 13.1 (2CH_3, two methyl overlapped), 56.0 (OCH₃), 56.8 (OCH₃) 104.5, 109.8, 110.6, 114.2, 116.4, 117.6, 123.3, 126.0, 128.2, 129.1, 135.2, 136.9, 138.6, 150.4, 152.2, 153.1, 154.0, 168.4. HR-ESI-MS 815.2184 [M + H]⁺. Anal. Calcd. (Found) for: C₃₈H₃₀N₁₂O₆S₂ (814.85): C, 56.01 (55.93); H, 3.71 (3.59); N, 20.63 (20.58)%.

2,2’-(((1E,3E)−2-(2,5-dimethoxybenzylidene)−1H-indene-1,3(2H)-diylidene)bis(hydrazin-1-yl-2-ylidene))bis(thiazol-4(5H)-one) (9a).

Yellow solid, mp. 130–132°C. IR (ν): 3424 (NH), 2955 (sp³ CH), 1743 (C = O), 1616 (C = N), 1531, 1497, 1464, 1408, 1348, 1264 1173, 1036. ¹H NMR (DMSO-d₆) 3.74 (s, 3H, OCH₃), 3.80 (s, 3H, OCH₃), 4.59 (s, 4H, 2CH₂), 6.98–7.49 (m, 7H, Ar-H), 8.74 (s, 1H, = CH), 11.34 (s, 2H, NH). ¹³C NMR (DMSO-d₆) 29.8 (2CH_2, two methylene overlapped.), 56.5 (OCH₃), 57.7 (OCH₃) 114.6, 119.9, 120.7, 122.5, 123.0, 124.2, 129.1, 131.2, 133.4, 136.5, 137.8, 148.3, 150.0, 153.1, 154.0, 159.7, 185.0. HR-ESI-MS 521.1413 [M + H]⁺. Anal. Calcd. (Found) for: C₂₄H₂₀N₆O₄S₂ (520.58): C, 55.37 (55.28); H, 3.87 (3.74); N, 16.14 (16.04)%.

2,2’-(((1E,3E)−2-(2,5-dimethoxybenzylidene)−1H-indene-1,3(2H)-diylidene)bis(hydrazin-1-yl-2-ylidene))bis(5-methylthiazol-4(5H)-one) (9b).

Buff solid, mp. 198–200°C. IR (ν): 3424 (NH), 2939 (sp³ CH), 1723 (C = O), 1638 (C = N), 1564, 1495, 1455, 1370, 1328, 1252, 1644cm⁻¹. ¹H NMR (DMSO-d₆) 1.12 (d, J = 5.4Hz, 6H, 2CH₃), 3.72 (s, 3H, OCH₃), 3.77 (s, 3H, OCH₃), 3.97 (q, J= 6.8Hz, 2H, 2CH), 7.01–7.31 (m, 7H, Ar-H), 8.52 (s, 1H, = CH), 11.22 (s, 2H, NH). ¹³C NMR (DMSO-d₆) 16.7 (2CH_3, two methyl overlapped), 30.2, 57.7 (OCH₃), 58.6 (OCH₃) 112.0, 112.9, 115.1, 118.5, 121.2, 123.0, 124.2, 125.1, 129.1, 129.9, 130.8, 135.6, 136.9, 148.3, 154.0, 155.7, 160.5, 183.2. HR-ESI-MS 549.1424 [M + H]⁺. Anal. Calcd. (Found) for: C₂₆H₂₄N₆O₄S₂ (548.64): C, 56.92 (56.83); H, 4.41 (4.35); N, 15.32 (15.29)%.

1,1’-((((1E,3E)−2-(2,5-dimethoxybenzylidene)−1H-indene-1,3(2H)-diylidene)bis(hydrazin-1-yl-2-ylidene))bis(4-methylthiazole-2,5-diyl))bis(ethan-1-one) (11a).

Creamy (Off-White) solid, mp. 218 –210°C. IR (ν): 3439 (br. NH), 3152 (sp²CH), 3000, 2919 (SP³CH), 1702 (C = O), 1635 (C = N), 1574, 1497, 1425, 1361, 1261, 1165, 1133, 1098cm⁻¹. ¹H NMR (DMSO-d₆) 1.78 (s, 6H, 2CH₃), 2.30 (s, 6H, 2CH₃), 3.70 (s, 3H, OCH₃), 3.75 (s, 3H, OCH₃) 6.91–7.29 (m, 7H, Ar-H), 8.22 (s, 1H, = CH), 11.18 (s, 2H, NH). ¹³C NMR (DMSO-d₆) 17.7 (2CH_3, two methyl overlapped), 23 (CH₃), 56.6 (OCH₃), 57.3 (OCH₃) 103.2, 103.5, 109.1, 111.2, 113.3, 115.2, 116.4, 119.0, 122.5, 123.8, 126.9, 136.2, 155.6, 156.9, 163.6, 168.2. HR-ESI-MS 601.1593 [M + H]⁺. Anal. Calcd. (Found) for: C₃₀H₂₈N₆O₄S₂ (600.71): C, 59.98 (59.82); H, 4.70 (4.68); N, 13.99 (13.84)%.

Diethyl 2,2’-(((1E,3E)−2-(2,5-dimethoxybenzylidene)−1H-indene-1,3(2H)-diylidene)bis-(hydrazin-1-yl-2-ylidene))bis(4-methylthiazole-5-carboxylate) (11b).

Yellow solid, mp. 115–117°C. IR (ν): 3428 (NH), 3074 (sp² CH), 2922 (sp³ CH), 1698 (C = O), 1621(C = N), 1578, 1495, 1427, 1372, 1313, 1275, 1169, 1045cm⁻¹. ¹H NMR (DMSO-d₆) 1.19 (t, J = 6.3Hz, 6H, 2CH₃), 2.06 (s, 6H, 2CH₃), 3.70 (s, 3H, OCH₃), 3.75 (s, 3H, OCH₃), 4.38 (q, J = 7.9Hz, 4H, 2CH₂), 6.34–7.23 (m, 7H,, Ar-H), 8.23 (s, 1H, = CH), 11.26 (s, 2H, NH). ¹³C NMR (DMSO-d₆) 11.8 (CH₃) 17.9 (CH₃), 55.6 (OCH₃), 56.5 (OCH₃), 59.5 (CH₂), 103.3, 103.7, 109.4, 111.5, 113.3, 115.1, 116.4, 119.4, 112.1, 123.8, 126.9, 136.9, 155.7, 156.6, 163.6, 168.4 (C = O). HR-ESI-MS 661.2856 [M + H]⁺. Anal. Calcd. (Found) for: C₃₂H₃₂N₆O₆S₂ (660.76): C, 58.17 (58.06); H, 4.88 (4.68); N, 12.72 (12.63)%.

2,2’-(((1E,3E)−2-(2,5-dimethoxybenzylidene)−1H-indene-1,3(2H)-diylidene)bis(hydrazin-1-yl-2-ylidene))bis(4-methylthiazole) (11c).

Brown solid, mp. 178–180°C. IR (ν): 3439 (NH), 3078 (sp²CH), 1579 (C = N), 1494, 1428, 1373, 1274, 1216, 1167, 1137cm⁻¹. ¹H NMR (DMSO-d₆) 2.12 (s, 6H, 2CH₃), 3.75 (s, 3H, OCH₃), 3.81 (s, 3H, OCH₃), 6.34 (s, 2H, thiazole-H), 6.91–7.24 (m, 7H, Ar-H), 8.27 (s, 1H, = CH), 11.20 (s, 2H, NH). ¹³C NMR (DMSO-d₆) 17.6 (CH₃), 55.1, 56.5 (2OCH₃), 102.8, 105.4, 109.4, 109.8, 113.3, 116.7, 121.6, 122.9, 124.2, 127.7, 130.0, 136.4, 147.0, 151.8, 153.5, 158.3, 168.9. HR-ESI-MS 517.1412 [M + H]⁺. Anal. Calcd. (Found) for: C₂₆H₂₄N₆O₂S₂ (516.64): C, 60.44 (60.31); H, 4.68 (4.59); N, 16.27 (16.18)%.

2,2’-(((1E,3E)−2-(2,5-dimethoxybenzylidene)−1H-indene-1,3(2H)-diylidene)bis(hydrazin-1-yl-2-ylidene))bis(4-phenylthiazole) (13).

Brown solid, mp. 193–195°C. IR (ν): 3430(NH), 2922(sp³ CH), 1534, 1277, 1218, 1044cm⁻¹. ¹H NMR (DMSO-d₆) 3.76 (s, 3H, OCH₃), 3.81 (s, 3H, OCH₃), 6.94–8.42 (m, 20H, Ar-H, =CH and thiazole-H), 11.63 (s, 2H, 2NH). ¹³C NMR (DMSO-d₆) 56.0, 57.4 (2OCH₃), 102.8, 104.5, 109.8, 111.1, 111.5, 113.3, 116.4, 118.1, 123.3, 126.4, 128.2, 129.4, 130.0, 133.9, 135.2, 136.9, 139.1, 150.9, 151.3, 152.2, 152.7, 153.5, 168.4, 178.0. HR-ESI-MS 641.2004 [M + H]⁺. Anal. Calcd. (Found) for: C₃₆H₂₈N₆O₂S₂ (640.78): C, 67.48 (67.36); H, 4.40 (4.29); N, 13.12 (13.09)%.

Biological activity

Compounds:10mg were dissolved in 1 mL (10% DMSO and 90% deionized water), sterilized by 1% antibiotic–antimycotic mixture and stored in −20.

Cells:Vero cell line was used. The cells were propagated in DMEM medium (Lonza, USA) supplemented with 10% fetal bovine serum (Gibco, USA), 1% antibiotic–antimycotic mixture (Lonza, USA).

Virus:Cox B virus was propagated and titrated to give final count of 0.9 × 10⁵ viral particle/mL.

Cytotoxicity assay

Cytotoxic effect of compounds was measured by inoculation different doses of compounds on tested cells then examining them microscopically after 24h to determine any morphological changes that appeared on cells as a result of being subjected to the compounds⁴⁶. Vero cells were seeded onto 96-well plates, which were then incubated for the whole night. After a 24-hour incubation period at 37˚C in a humidified incubator with 5% CO₂, compounds were injected at concentrations of 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100µg /100 µl. Any morphological changes were monitored under a microscope.

Plaque reduction assay.

This test was done according to Tebas et al.⁴⁷, In a 12-well plate Vero cells (10⁵ cells/mL) were cultivated for 1day at 37^oC. The Cox B virus was mixed with the safe concentration of the compound and incubated for 1h at 37^oC before being added to the cells. Following the removal of the growth media, the cells were injected with virus-extract combinations (100 mL/well) and then rinsed with PBS (Phosphate Buffer Saline). Following a one-hour contact period for virus adsorption, the cell monolayer was supplemented with one milliliter of Dulbecco’s Modified Eagles Media (DMEM) containing 2% agarose. The plates were then allowed to solidify and incubated at 37°C until the development of viral plaques. Formalin (10%) was added for 2h then plates were stained with crystal violet. Control wells were included where untreated virus was incubated with Vero cells and finally plaques were counted and percentage reduction in plaques formation in comparison to control wells was recorded as following:

% inhibition = (viral count(untreated) - viral count(treated))/viral count(untreated)X100.

Mechanism of virus inhibition

For a compound having an antiviral effect, this might be due to the effect on viral replication, the effect on viral adsorption, or the direct effect on the viral particle.

Viral replication⁴⁸ Assay was carried out in a 6-well plate where Vero cells were cultivated (10⁵ cells/ml) for 1day at 37^oC. The cells were directly inoculated with the virus. After an hour of incubation, unadsorbed viral particles were eliminated by repeatedly washing the cells with PBS.

The compound was applied at different concentrations, and after 1h contact time, 2 mL of DMEM medium supplemented with 2% agarose were added to the cell monolayer. Plates were left to solidify then incubated till the appearance of plaques.

Viral adsorption⁴⁹ Vero cells were cultivated in a 6-well plate (10⁵ cells/mL) for 1day at 37^oC. The compound was applied at different concentrations in 200 mL medium without supplements and coincubated with the cells for 2h at 4^oC. The virus was coincubated with the pretreatment cells for one hour after the unadsorbed extract was eliminated by washing the cells three times in PBS. Next, 2 mL of DMEM supplemented with 2% agarose were added. The plates were left to solidify and incubated till the appearance of plaques.

Virucidal⁵⁰ the assay was conducted in a 6-well plate with Vero cells that were cultured for two days at 37°C (10⁵ cells/mL). A volume of 200 mL serum-free DMEM containing 1.5 × 10² PFU from Cox B was added to the concentration of the compound resulting in viral inhibition, after 1h incubation, The mixture was diluted three times using serum-free media, which leaves almost no extract but still permits the existence of virus particles to develop on Vero cells. 100 mL of each dilution was then introduced to the Vero cell monolayer. The DMEM overlayer was added to the cell monolayer after a one-hour contact period. The plates were left to solidify then incubated till the appearance of plaques.

Computational methods

Molecular docking simulation

To investigate the antiviral ability of the promising compounds, Cox B virus protein receptors were obtained from the Protein Data Bank, as listed in Table (7). The crystal structures of the target receptors were preprocessed by removing water molecules, ions, and existing ligands using PyMOL software. Remarkably, one of the reference antiviral drugs used as a positive ligand for docking with the chosen COX B structure was Pleconaril (see Fig.11). Subsequently, hydrogen atoms were added to the receptor molecule using Autodock Vina and saved in a pdbqt format. Moreover, each compound was minimized and converted to a mol2 format using Open Babel⁵¹. Ligand-centered maps were generated using the AutoGrid program. Additionally, the 2-D bond interactions between the target and ligands were analyzed using the Discovery Studio 4.5 program.

In-silico pharmacokinetics ADME and toxicity prediction

The physicochemical parameters and ADMET of compounds were calculated using the ADMETlab 2.0⁵⁵.

Molecular dynamics (MD) simulation

Molecular dynamics (MD) simulation is widely used to explain protein-ligand complexes’ binding interactions and binding affinities. In this study, MD simulations were performed using GROMACS 2018 software to further verify the rationality and reliability of the docking results. The topology of the protein was constructed using the CHARMM36 force field parameters. Also, the topology of compounds was generated using the Geoff server. After coordinating position restrictions were placed on ligands. NVT and NPT equilibrium were performed for 1000 ps in 300K under a 1.0bar atmosphere. After the MD simulations, Root Mean Square Deviation (RMSD), Root Mean Square Fluctuation (RMSF), and radius of gyration (Rg) were calculated⁵⁶.