Synthesis and Biological Evaluation of Benzothiazolyl-pyridine Hybrids as New Antiviral Agents against H5N1 Bird Flu and SARS-COV-2 Viruses

Abstract

A novel series of benzothiazolyl-pyridine hybrids 8a–h and 14a–e were produced from the reaction of enamine derivative 4 with each of the arylcyanoacetamides 5a–h and cyanoacetohydrazides 9a–e. The new products were characterized by spectral techniques (IR, ¹H NMR, ¹³C NMR, and MS). Biological evaluation of 8a–h and 14a–e in vitro against H5N1 and SARS-COV-2 viruses showed that several compounds had significant activity. Compounds 8f–h, which contain fluorine atoms, have better activity against H5N1 and anti-SARS-CoV-2 viruses than the other compounds included in this study. Compound 8h has a trifluoromethyl group at position-3 of the phenyl ring and exhibits a high activity against H5N1 virus with 93 and 60% inhibition at concentrations of 0.5 and 0.25 μmol/μL, respectively, among the tested compounds, and it also showed anti-SARS-CoV-2 virus with a half-maximum inhibition rate of 3.669 μM, among the remaining compounds. The mechanism of action of 8f–h, which is expected to be repurposed against COVID-19, was investigated. The results showed that the compounds have virucidal effects at different stages of the three mechanisms of action. Furthermore, compounds 8f–h were found to possess CoV-3CL protease inhibitory activities with IC₅₀ values of 544.6, 868.2, and 240.6 μg/mL, respectively, compared to IC₅₀ = 129.8 μg/mL of the standard drug lopinavir. Interestingly, compounds 8f–h also showed high inhibitory activity against the H5N1 virus as well as the SARS-CoV-2 virus. Moreover, compounds 8f–h fit admirably into the active site of the SARS-CoV-2 main protease (PDB ID: 6LU7) using the molecular docking Moe software 2015.10.

1. Introduction

Influenza, corona, and other related pneumotropic viruses cause illness and death by inducing an excessive inflammatory immune response in the host′s airways.

Influenza A viruses are classified into subtypes according to two proteins present on the virus surface. Hemagglutinin (HA), which promotes receptor binding and membrane fusion, and neuraminidase (NA), which helps in the release of viral progeny.¹ Subtypes of influenza A include H1N1, H2N2, H3N2, H5N1, H7N7, and H9N2, which can be isolated from humans, suggesting that human host constraints as a virus have been suggested.² There are two types of H5N1: highly pathogenic (HPAI), commonly referred to as “Bird flu” (HPAI), and low pathogenic. In 1997, there were few cases of H5N1 infection between chickens and humans in Hong Kong, but the number of infected people increased until outbreaks were reported in several Asian countries in 2004 and 2005, and these outbreaks were slow to subside. In 2006, the total number of confirmed human cases of H5N1 had amounted to 174, of which 94 were fatal.³ WHO confirmed 455 human death cases from 861 of total infected human cases since 2003–2020.⁴ A human pandemic with H5N1 virus could potentially be catastrophic due to an almost complete lack of antibody-mediated immunity to the H5 surface protein in most human populations and the virulence of this viral subtype. Data also indicate that the H5N1 strain is particularly harmful due to it having 50% mortality rate in humans and is more likely to be transferred among humans than seasonal influenzas, which have a higher mortality rate.

Currently, anti-influenza drugs (H5N1) are classified into two classes, as shown in Figure 1. The first class is the M2 proton channel inhibitors like amantadine and rimantadine.^5,6 The second class is the neuraminidase inhibitors like oseltamivir, peramivir, and zanamivir (Figure 1).^7,8

Figure 1.

Approved drugs used for avian influenza A.

Meanwhile, the severe acute respiratory syndrome coronavirus (SARS-CoV-2) spread rapidly, causing a pandemic coronavirus infection 2019 (COVID-19). Since the first outbreak in Wuhan, China, in December 2019, according to the WHO, more than 760 million people worldwide of COVID-19 cases have been confirmed, and 6.8 million people have died. To date, SARS-CoV-2 is the most interesting topic in coronavirus infections. There are many potential candidates for therapeutics against this type of virus, including chloroquine,⁹ hydroxychloroquine,¹⁰ remdesivir,¹¹ and lopinavir/ritonavir¹² (Figure 2). Various phytochemicals were also utilized against SARS-CoV-2 virus such as flavone and coumarin derivatives,¹³ theophyllines,¹⁴ and saikosaponins.¹⁵

Figure 2.

Structures of some drugs used as anti-COVID-19.

SARS-CoV-2 is composed of two overlapping polyproteins (pp1a and pp1ab) encoded in the RNA genome, whose cleavage is essential for replication and transcription processes.¹⁵ These cleavage processes are mediated by the major protease M^pro (3-chymotrypsin-like protease 3CLpro) and nonstructural viral proteins such as the papain-like protease PLpro.¹⁶⁻¹⁹ In this regard, inhibition of viral RNA synthesis is considered as one way to combat coronaviruses.²⁰

According to reports, influenza A and COVID-19 are both contagious respiratory diseases, although caused by different viruses. Influenza is caused by infection with the influenza virus, whereas COVID-19 is caused by infection with a coronavirus (SARS-CoV-2), which was first discovered in 2019. Avian influenza A (H5N1) and COVID-19 have similar symptoms including fever, muscle aches, headache, sneezing, and coughing.^21,22 However, despite similar disease symptoms, influenza viruses and COVID-19 contrast in terms of replication, immune stimulation, and overall fatality.²³ Moreover, some clinical and laboratory findings have been reported to be similar for both infection in patients with influenza and COVID-19 coinfection, making the diagnoses of these coinfected patients difficult. For suspected patients, detection methods specific for influenza virus and SARS-V-2 are recommended.²⁴

In addition, there are prominent antiviral drugs containing pyridine and dihydropyridine cores such as dorvirine, which is considered a nonnucleoside reverse transcriptase inhibitor and is used to treat HIV infection.²⁵ Tipranavir, which contains the trifluoromethyl group is used also as an anti-HIV drug and in combination with ritonavir used as a combination medicine.²⁶ Baloxavir marboxil drug is used as a treatment for influenza A and influenza Bird flu²⁷ (Figure 3). Compound I has been reported to have low toxicity with high activity against the H5N1 influenza²⁸ (Figure 3). Moreover, a natural product with 2-pyridine scaffold has been used as a therapeutic agent against the novel coronavirus SARS-CoV-2.²⁹

Figure 3.

Structures of antiviral market drugs containing pyridine and benzothiazole and our targets present in the current study.

Compounds containing the benzothiazole moiety demonstrated to exhibit antiviral activity.³⁰ For example, frentizole (benzothiazolyl urea derivative) II is used as an HSV and immunosuppressive agent,³¹ as shown in Figure 3. The bis-tetrahydrofuranyl-urethane analogue of benzothiazole III is used as an HIV as well as in phase-3-ongoing COVID-19 (Figure 3).³² It is worth noticing that joining two or more diverse heterocyclic rings into one structure may bring about new compounds with upgraded bioactivity.³³ As an example, pyridines with the benzothiazole moiety have enhanced antibacterial and antifungal activities.^34,35 To achieve this goal, new strategies for synthesizing pyridine-bearing benzothiazole moieties have been generated.³⁶⁻³⁹ As a continuation of our previous work on the synthesis of bioactive heterocyclic ring systems,⁴⁰⁻⁵⁴ we herein report that the cytotoxic activity of some novel arylpyridines incorporated benzothiazole moieties 8a–h against H5N1, SARS-COV-2 viruses, and their inhibition of SARS-COV-2 main protease (M^pro). Molecular docking studies of more potent main protease inhibitors were also performed to find a potential candidate as antiviral agent toward H5N1 and SARS-CoV-2 viruses.

2. Results and Discussion

2.1. Chemistry

The starting 2-cyanomethylbenzothiazole 3 was synthesized from the grinding of o-aminothiophenol 1 and malononitrile 2 using lemon juice as a natural acid catalyst at room temperature for 30 min as well as the conventional method,⁵⁵ as shown in Scheme 1.

Scheme 1. Synthesis of 2-Cyanomethylbenzothiazole 3.

Furthermore, the enamine derivative 4(56) was prepared from grinding compound 3 with N,N-dimethylformamide dimethyl acetal (DMF-DMA) at ambient temperature for 10 min, as depicted in Scheme 2.

Scheme 2. Synthesis of Enamine Derivative 4.

On the other hand, grinding of equimolar amounts of compound 4, N-aryl-2-cyanoacetamides 5a–h, and potassium hydroxide in a mortar at room temperature for 20 min gave the new N-substituted 2-pyridyl benzothiazoles 8a–h, as shown in Scheme 3 and Figure 6. Their essential examination, such as spectral data, confirmed the obtainable products 8a–h. For illustration, the ¹H NMR spectrum of compound 8b appeared with two singlet signals at δ = 3.75 and 3.86 ppm for the two methoxy protons, and another three doublets at δ = 7.66, 7.83, and 8.11 ppm with coupling constants J = 7.2, 8.4, and 7.2 Hz, respectively, due to the protons of aryl groups. Also, a peak at δ = 8.27 ppm for the pyridine proton showed up. The ¹³C NMR spectrum of compound 8b showed characteristic signals at 55.7 (OCH₃), 56.5 (OCH₃), 104.6 (CN), 149.5 (C–NH₂), 161.0 (CO), and 165.0 (C=N) ppm. In addition, other signals correspond to aryl carbons at 74.8, 76.8, 99.1, 120.1, 120.3, 120.5, 121.7, 122.9, 126.3, 144.4, and 155.0 ppm. The mass spectrum of compound 8b contains molecular ion peaks at m/z = 404 (M⁺, 100%), corresponding to the molecular formula C₂₁H₁₆N₄O₃S. The reaction mechanism was illustrated as the disposal of a dimethylamine molecule following Michael addition, and intramolecular cyclization was achieved by adding an amino proton to the cyano group, followed by tautomerization forming 2-pyridyl benzothiazoles 8a–h.

Scheme 3. Synthesis of 5-(Benzothiazolyl)-1-(aryl)-pyridine-2-ones 8a–h.

Figure 6.

CC₅₀ (50% cytotoxic concentration) of the tested N-pyridone-based triazoles in Vero-E6 cells and IC₅₀ (50% inhibitory concentration) against NRC-03-nhCoV virus in Vero-E6 cells (A and B). CC₅₀ and IC₅₀ were plotted for each tested compound using Graph Pad Prism and were calculated from the nonlinear regression curve-fit analysis, relative to the virus and cell controls. Compound 14b shows significant activity toward SARS-CoV-2 with IC₅₀ = 70.48 μM and SI = 14.897 among the tested compounds 14a–e.

Additionally, grinding compound 4 with 2-cyano-N′-(4-substituted benzylidene) acetohydrazides 9a–e in a mortar at room temperature for 20 min yielded compounds 14a–e, as shown in Scheme 4. A fundamental investigation analysis of compounds 14a–e was used to confirm the suggested structures. The IR spectrum of compound 14b showed absorption bands at 3398, 2206, and 1612 cm^–1 due to the NH, CN, and CO groups, respectively. The ¹H NMR spectrum of 14b showed two singlet signals at δ = 3.85 and 3.91 ppm due to the two methoxy protons and another two doublet signals at δ = 7.90 and 8.04 with a coupling constant of J = 7.6 Hz, which demonstrated the aryl protons. A singlet signal at δ = 8.47 ppm was of the pyridine CH proton. Also, ¹³C NMR affirmed the structure of compound 14b, where the characteristic peaks appeared at δ = 56.0 (OCH₃), 57.2 (OCH₃), 114.7 (CN), 153.4 (C=N), 160.3 (C=N), and 163.0 (CO) ppm. Moreover, other signals due to aryl carbons at 81.1, 101.1, 116.6, 117.1, 120.4, 121.5, 121.8, 122.2, 123.8, 126.3, 135.0, 135.2, 152.1, and 152.9 ppm were presented. The proposed reaction pathway included the addition of the active methylene group of 2-cyano-N′-(4-substituted benzylidene)acetohydrazides 9a–e to the double bond of the beginning compound 4 to form intermediate 10; Michael adduct with the consequent end of the dimethylamino group yielded intermediate 11. Tautomerism of intermediate 11 happens to make intermediate 12, via intramolecular cyclization, involving the bonding of the amino group to the C=N to give intermediate 13, followed by autoxidation yielding the pyridone hybrids 14a–e.

Scheme 4. Synthesis of Benzothiazolyltriazolo[1,5-a]pyridine Derivatives 14a–e.

2.2. Biological Activity

2.2.1. H5N1 Influenza Virus

The MTT assay was used to determine the safe dose of each of the synthesized benzothiazolyl-pyridine derivatives 8a–h and 14a–e. From a linear equation generated for each compound, IC₅₀ values are listed in Table 1. The antiviral activity of safe doses of each compound against H5N1 virus was investigated using a plaque reduction assay. The results demonstrated that compound 8h (contains the 3-fluoromethyl group) exhibited the highest activity against H5N1 influenza virus strain among the tested derivatives where inhibition appeared at 93 and 60% at concentrations equal to 0.5 and 0.25 μmol/μL, respectively, compared to ribavirin as a broad-spectrum antiviral standard drug of 100% inhibition at concentration 0.5 μmol/μL and 86% inhibition at 0.25 μmol/μL. The following compounds 8f (contains one fluoro group), 8g (contains two fluoro groups), and 14d (N,N-dimethyl groups) exhibited 88, 67, and 73% inhibition at 0.5 μmol/μL, respectively. Compound 14c with an amino group also showed 65% inhibition at 0.25 μmol/μL. However, compounds 8e, 14a, and 14e showed moderate antiviral activity against H5N1, with inhibition rates 47, 55, and 20% at concentration 0.25 μmol/μL, while 8a (17%) and 8b (7%) showed a very weak rate of inhibition at concentration 0.25 μmol/μL. Compound 8c, with methyl and chloro groups at 2- and 4-positions in the phenyl ring, respectively, showed no cytotoxic activity against H5N1 among all tested compounds at any concentration (Figure 4). Some reports revealed that heterocyclic compounds with substituent methyl or chloro groups compared to other functional groups showed weak or no antiviral activity.^57,58

Table 1. Antiviral Activities of All Synthesized Compounds.

compound no	concentration (μmol/μL)	initial viral count	viral count (PFU/mL)	inhibition (%)
8a	0.5	5.2 × 10–6	6 × 10–6	13
	0.25	5 × 10–6	6 × 10–6	17
8b	0.5	5.5 × 10–6	6 × 10–6	8
	0.25	5.6 × 10–6	6 × 10–6	7
8c	0.5	unable	6 × 10–6	0
	0.25	unable	6 × 10–6	0
8d	0.5	3 × 10–6	6 × 10–6	60
	0.25	4.7 × 10–6	6 × 10–6	22
8e	0.5	2.3 × 10–6	6 × 10–6	52
	0.25	3.2 × 10–6	6 × 10–6	47
8f	0.5	0.7 × 10–6	6 × 10–6	88
	0.25	1.5 × 10–6	6 × 10–6	69
8g	0.5	2 × 10–6	6 × 10–6	67
	0.25	2.4 × 10–6	6 × 10–6	56
8h	0.5	5 × 10–6	6 × 10–6	93
	0.25	2.5 × 10–6	6 × 10–6	60
14a	0.5	2.4 × 10–6	6 × 10–6	60
	0.25	2.7 × 10–6	6 × 10–6	55
14b	0.5	2 × 10–6	6 × 10–6	67
	0.25	2.4 × 10–6	6 × 10–6	60
14c	0.5	3 × 10–6	6 × 10–6	50
	0.25	2 × 10–6	6 × 10–6	65
14d	0.5	1.6 × 10–6	6 × 10–6	73
	0.25	1.6 × 10–6	6 × 10–6	73
14e	0.5	3.6 × 10–6	6 × 10–6	40
	0.25	4.8 × 10–6	6 × 10–6	20
ribavirin	0.5	5 × 10–6	6 × 10–6	100
	0.25	2.8 × 10–6	6 × 10–6	86

Figure 4.

Antiviral activities of all synthesized compounds against H5N1.

2.2.2. Detection of In Vitro Cytotoxicity

The colorimetric MTT assay was used to assess the cytotoxicity of compounds 8a–h and 14a–e on Vero-E6 cells. The results showed that CC₅₀ and safety index (SI) of compounds 8a–h ranged 1665–152 μM and 106.704–0.0487, respectively (Table 2, Figure 5). Compounds 14a–d, on the other hand, exhibited CC₅₀ and SI ranging 6276–143 μM and 14.897–0.0274, respectively (Table 2, Figure 6). The three best SARS-COV-2 inhibitors were 8f–g with IC₅₀ values of 10.52, 21.46, and 3.669 μM and SI values of 52.282, 115.971, and 106.704, respectively.

Table 2. Anti-SARS-CoV-2 Activities of Compounds 8a–h.

compound no.	R	aCC50 (Vero-E6) μM	bIC50 (NRC-03-nhCoV) μM	cSI
8a	4-OMeC6H4	1651	994.3	1.660
8b	2,4-(OMe)2C6H3	152	3120	0.0487
8c	2-Me-4-ClC6H3	1665	142.2	11.645
8d	2,4-Cl2C6H3	1593	1596	0.998
8e	4-BrC6H4	1279	73.84	17.321
8f	4-FC6H4	550	10.52	52.281
8g	2,3-FC6H3	425.8	21.46	115.971
8h	3-CF3C6H4	391.5	3.669	106.704

^aCC₅₀.

^bIC₅₀ values were derived from results of at least two independent experiments in Vero cells infected with SARS-CoV-2.

^cSI (Selective index) = CC₅₀/IC₅₀ for inhibitory SARS-CoV-2 infection.

Figure 5.

CC₅₀ (50% cytotoxic concentration) of the tested N-pyridones in Vero-E6 cells and IC₅₀ (50% inhibitory concentration) against the NRC-03-nhCoV virus in Vero-E6 cells (A and B). The CC₅₀ and IC₅₀ were plotted for each tested compound using Graph Pad Prism and were calculated from the nonlinear regression curve-fit analysis, relative to the virus and cell controls. The compounds 8a–h exhibited antiviral activity against the NRC-03-nhCoV virus in Vero-E6 cells with IC₅₀ ranging from 3.669 to 3120 μM. The most active compound is 8h with IC₅₀ = 3.669 μM and SI = 106.704 compared to the weakest compound 8b with IC₅₀ = 3120 μM and SI = 0.0487 in this series.

2.2.3. Anti-SARS-CoV-2 Activity

The inhibitory effects of compounds 8a–h and 14a–e on the SARS-COV-2 (NRC-03-nhCoV) virus in Vero-E6 cells were investigated. The results in Table 1 show that most of the compounds tested significantly inhibited the replication of the NRC-03-nhCoV virus in a dose-dependent profile. Compounds 8f–h demonstrated superior inhibitory activity compared to compounds 14a–e (Table 2 and Figure 5A and B). The most potent anti-SARS-COV-2 compound was 8f–h, with IC₅₀ values of 10.52, 21.46, and 3.669 μM, respectively. Compound 8a (R = 4-methoxy as the donating group in the benzene ring) showed an IC₅₀ value equal to 994.3 μM. Furthermore, insertion of another methoxy group at the 2-position of the benzene ring decreased the IC₅₀ by 3.13-fold in IC₅₀ as in the case of 8b (IC₅₀ = 3120 μM). The introduction of the withdrawing group such as the chlorine atom at the 4-position of the benzene ring improved the antiviral activity as in the case of 8c (IC₅₀ = 142.2 μM), while the insertion of another group of a chlorine atom at the 2-position of the benzene ring decreased the antiviral activity again as in 8d (IC₅₀ = 1596 μM). Changing the chlorine atom at position-4 of the benzene ring to a bromine atom increased the IC₅₀ by 1.92-fold compared to 8c (IC₅₀ = 73.84 μM). Furthermore, since fluorine-containing drugs have become one of the essential structural features in modern pharmaceuticals, the introduction of a fluorine atom in place of chlorine or bromine increases the antiviral activity, as in the case of compound 8f, which contains one fluorine atom at the 4-position of the benzene ring, decreased the IC₅₀ value to 10.520 μM and SI = 0.05228 as shown in Table 2. On the other hand, adding another fluorine atom at the 2-position of the benzene ring decreased the activity to IC₅₀ = 21.46 μM as in the case of 8g, but increasing the number of fluorine atoms as the trifluoromethyl group at the 3-position of the benzene ring (8h) resulted in a decrease of at least 2.86-fold in IC₅₀ value (3.669 μM) compared to 8f and 5.84-fold in IC₅₀ value compared to 8g, suggesting that the significance of the trifluoromethyl group (withdrawing group) is important for the SARS-COV-2 replication inhibitory activity in Vero-E6 cells.

In the second series, 14a–e, compound 14b (R = 2,5-dimethoxyphenyl) exhibited some activity against SARS-CoV-2 with IC₅₀ = 70.48 μM, while the other tested compounds in this series showed weak activity (Table 3 and Figure 6A and B).

Table 3. Anti-SARS-CoV-2 Activities of Compounds 14a–e.

compound no.	R1	R2	R3	R4	aCC50 (Vero-E6) μM	bIC50 (NRC-03-nhCoV) μM	cSI
14a	OMe	H	H	H	6276	4246	1.4781
14b	OMe	H	H	OMe	1050	70.48	14.897
14c	H	H	NH2	H	1914	269.4	7.104
14d	H	H	N(Me)2	H	143	5217	0.0274
14e	Cl	H	NO2	H	3725	1151	3.236

^aCC₅₀.

^bIC₅₀ values were derived from results of at least two independent experiments in Vero cells infected with SARS-CoV-2.

^cSI (Selective index) = CC₅₀/IC₅₀ for inhibitory SARS-CoV-2 infection.

2.2.4. Mechanism of Anti-SARS-CoV-2 Activity

The inhibition rates for different mechanisms of action (virus adsorption, replication, and virucidal) of the most active compounds 8f–h are shown in Table 4. The data depicted in this table show that compound 8f, which contains one fluorine atom, decreases in concentration to ¹/₁₀₀ and reduces virucidal inhibition by about 50%. However, adsorption and replication varied in both cases from 28.6 to 8.3%, and the concentration had no impact on inhibition. On the other hand, the adsorption rate of compound 8g decreases with decreasing concentration, but in viral replication, concentration does not affect the inhibition rate (60%, 60%, and 53%). With compound 8h, a precise effect on adsorption and viral replication appeared; both compounds 8g and 8h showed equally good results in virucidal properties, which were not affected by decreasing concentration. Thus, compound 8g showed 80, 76.7, and 73.3% virucidal efficacy at concentrations of 10, 1, and 0.1 μM, respectively, while compound 8h exhibited 83.3, 80, and 73.3% virucidal efficacy at concentrations of 10, 1, and 0.1 μM, respectively. Thus, in the three stages of compounds, 8f–h exhibited multiple inhibitory effects, but the potency of their activity was mainly virucidal effect (Table 4 and Figure 7).

Table 4. Mechanism of Action of Compounds 8f–h (Viral Titers).

compd no	conc (μM)	adsorption (PFU/mL)	viral inhibition (%)	viral replication (PFU/mL)	viral inhibition (%)	virucidal (PFU/mL)	viral inhibition (%)
8f	10	1 × 105	28.6	0.5 × 105	8.3	0.36 × 105	48
	1	1.2 × 105	26.5	0.5 × 105	0	0.32 × 105	40
	0.1	1.28 × 105	14.3	0.6 × 105	0	0.38 × 105	24
8g	10	1.0 × 105	33.3	0.6 × 105	60	0.3 × 105	80
	1	1.2 × 105	20	0.6 × 105	60	0.35 × 105	76.7
	0.1	1.4 × 105	7	0.7 × 105	53.3	0.4 × 105	73.3
8h	10	1.2 × 105	20	0.75 × 105	50	0.25 × 105	83.3
	1	1.2 × 105	20	0.85 × 105	43.3	0.3 × 105	80
	0.1	1.5 × 105	0	1.0 × 105	33.3	0.4 × 105	73.3
virus control		1.5 × 105		1.5 × 105		1.5 × 105

Figure 7.

Modes of action of compounds 8f–h. Viral Adsorption, replication, and virucidal mechanisms were studied for each compound at different concentrations using a plaque reduction assay. The three tested compounds act by the virucidal activity against SARS-CoV-2. The untreated virus was included in each mode of action for each compound as a virus control, and viral inhibition % for each mechanism for each tested compound calculated based on the percentage of virus reduction.

2.2.5. Inhibitory Activity against SARS-CoV-2 M^pro

The Protein Data Bank currently contains several potential target proteins isolated from coronaviruses (COVID-19). One of these crucial proteins, a protease, has been targeted by a panel of inhibitors with various chemical scaffolds, suggesting the potential for the creation of drugs against this challenging virus.^59,60 It is interesting to note that this enzyme and the comparable human proteases have completely different cleavage specificities, which makes it an excellent antiviral target with great selectivity and safety margins.⁶¹ It was interesting in view of these to investigate the potential protease inhibitory effect of compounds 8f–h against COVID-19. The inhibitory activities of compounds 8f–h against SARS-CoV-2 Mpro were evaluated according to previously published procedure.⁶² The results recorded in Table 3 indicated that the biological activity of these compounds depends on the nature of the substituent groups at a benzene ring and the number of fluorine atoms. Thus, the IC₅₀ values were 4.195 times higher for 4-fluoro, such as 8f, and 688 times higher for compound 8g, which contains 2,3-difluoro, compared to the standard drug lopinavir. On the other hand, compound 8h containing 3-trifluoromethyl showed 1.853-fold decrease compared to lopinavir. Thus, the 3-trifluoromethyl side chain showed higher inhibition against SARS-CoV-3CL protease (IC₅₀ = 240.6 μg/mL) than 4-fluoro (544.6 μg/mL) and 2,3-difluoro (868.2 μg/mL) as shown in Table 5 and Figure 8.

Table 5. Resulting Data of IC₅₀ of Compounds 8f–h and a Lopinavir against CoV-3CL Protease.

compound no.	IC50 (μg/mL)	SD±
8f	544.6	27.6
8g	868.2	44
8h	240.6	12.2
lopinavir	129.8	6.58

Figure 8.

IC₅₀ graph of compounds 8f–h against CoV-3CL protease and Lopinavir.

2.2.6. Molecular Docking Study

Additionally, compounds 8f–h were docked with the active site of N-[(5-methylisoxazol-3-yl)carbonyl)alanyl-l-valyl-N1-((1R,2Z)-4-(benzyloxy)-4-oxo-1-{[(3R)-2-oxopyrolidin-3-yl]-methyl}but-2-enyl)-l-leucinamide (PDB ID: 6LU7). The binding modes of the ligand (PDB ID: 6LU7) with compounds 8f–h were studied using molecular docking software 2015.10, and the results showed that the ligand formed four hydrogen bonds (HBs) with the active site of 6LU7 associated with a binding energy of −7.9074 kcal/mol and root-mean-square deviation (rmsd) equal to 1.6312; the first three HBs between the CO groups and Ans 142, Gly 143, and Glu 166 were with bond lengths of 3.08 3.2, and 3.18 Å, respectively. The interaction of HB between NH and Ans 143 had a bond length of 3.24 (Figures 9 and 10). The binding energy was −6.0086 kcal/mol, and the rmsd was 0.8430, indicating that compound 8f fully occupied the receptor domains. Compound 8f showed one hydrogen bond with a bond length of 3.22 Å between Gly 143 and the cyano group and another interaction between Glu 166 and arene-H of the thiophene ring (Figures 11 and 12). Compound 8g showed two arene–H bond interactions of the benzothiophene moiety, His 163 and Glu 166 with a binding energy of −5.6918 kcal/mol and a rmsd of 0.7399 (Figures 13 and 14). Additionally, compound 8h, which exhibited more potent anti-CoV-2 SARS activity, showed two hydrogen bond interactions with the active site of 6LU7, one between a cyano group and His 163 and the other between an amino group and Gln 189, with bond lengths of 3.09 and 3.10 Å associated with the bonging energy of −6.5953 kcal/mol and rmsd = 1.1970, in addition to another arene–H interaction between phenyl, which contains the trifluoromethyl group and Gly 143 (Figures 15 and 16). Furthermore, the standard drug lopinavir docked also with the active site 6LU7 with a binding energy of −7.1477 kcal/mol and rmsd equal to 1.0194. The results showed an HB interaction between Gln 189 and CO with a bond length of 3.03 Å, as well as two arene–phenyl interactions, the first an arene–arene interaction of His 41 and the second an arene-H with Pro 168 (Figures 17 and 18). These diverse binding modes of HBs formation affect the metabolism of the compounds, resulting in compounds 8a–h being considered SARS-CoV-2 inhibitors.

Figure 9.

Interaction in 2D of the active site of 6LU7.

Figure 10.

Interaction in 3D of the ligand active site of 6LU7 (in yellow color).

Figure 11.

Interaction in 2D of compound 8f with an active site of 6LU7.

Figure 12.

Interaction in 3D of compound 8f (in brown color) with the active site of 6LU7.

Figure 13.

Interaction in 2D of compound 8g with the active site of 6LU7.

Figure 14.

Interaction in 3D of compound 8g (in brown color) with an active site of 6LU7.

Figure 15.

Interaction in 2D of compound 8h with an active site of 6LU7.

Figure 16.

Interaction in 3D of compound 8h (in brown color) with the active site of 6LU7.

Figure 17.

Interaction in 2D of lopinavir with the active site of 6LU7.

Figure 18.

Interaction in 3D of lopinavir (in brown color) with the active site of 6LU7 (in yellow color).

3. Conclusions

In this study, a novel series of benzothiazolyl-pyridine hybrids were synthesized. The antiviral activity of these compounds was screened against H5N1, SARS-COV-2, and SARS-COV-2 viruses. The results showed that some of the synthesized compounds exhibited significant cytotoxic activity. Compounds 8f, 8g, and 14d showed high antiviral effects against H5N1. Compounds 8f, 8g and 8h, which contain fluoro groups, showed a good antiviral effect against SARS-COV-2. Furthermore, compound 8h, with a 3-trifluoromethyl side chain, showed higher SARS-CoV-3CL protease inhibition (IC₅₀ = 240.6 μg/mL) than 4-fluoro (544.6 μg/mL) and 2,3-difluoro (868.2 μg/mL) compared to lopinavir. These outcomes suggest that these compounds could be hopeful leads for producing more potent H1N5 and SARS-COV-2 inhibitors.

4. Experimental Section

4.1. Chemistry

All melting points were determined without correction using an Electrothermal (9100) apparatus. At Ain Shams, the ¹H and ¹³C NMR spectra were recorded on a Bruker Avance (III)-400 Spectrometer (400 and 100 MHz, respectively) in DMSO-d₆ by using Si(Me)₄. At 70 eV, mass spectra were collected on a Shimadzu GCMS-QP 1000 Ex mass spectrometer and were achieved at Cairo University’s Microanalyses Center. The IR spectra were done at Cairo University’s Microanalyses Center as KBr pellets on a PerkinElmer 1430 spectrophotometer. Elemental analyses were carried out at Cairo University’s Microanalyses Center using a Vario EL III Elemental CHNS analyzer. Biological evaluations of H5N1 and SARS-CoV-2 and mechanism of action of the newly synthesized compounds were performed at the Center of Scientific Excellence for Influenza Viruses, Environmental Research Division, National Research Center (NRC), Dokki, and Cairo 12622, Egypt.

4.1.1. Synthesis of 2-Cyanomethylbenzothiazole 3

At room temperature for 30 min, a mixture of o-aminothiophenol 1 (0.01 mol) and malononitrile 2 (0.01) was ground in a mortar with lemon juice (1 mL). The resulting solid was triturated with water, filtered, and recrystallized from methanol to yield pale yellow crystals 3, yield 87%; mp 100 °C,⁵⁵v_max/cm^–1 (KBr) 2225 (CN); ¹H NMR (DMSO-d₆) δ = 3.67 (s, 2H, CH₂), 7.51–7.53 (m, 2H, Ar), 8.02 (d, 1H, Ar), 8.18 (d, 1H, Ar); anal. calcd for C₉H₆N₂S: C, 62.05; H, 3.47; N, 16.08; S, 18.40. Found: C, 62.22; H, 3.67; N, 16.31; S, 18.21%.

4.1.2. Synthesis of 2-(Benzothiazolyl)-3-(dimethylamino)acrylonitrile 4

For 10 min, equimolar quantities of 2-cyanomethylbenzothiazole 3 and N,N-dimethylformamide dimethyl acetal (DMF-DMA) were ground in a mortar. The solid was triturated with water and recrystallized from ethanol to produce orange crystals with a melting point of 168 °C⁵⁶ and a 70% yield; ν_max/cm^–1 (KBr) 2220 (CN); ¹H NMR (DMSO-d₆) δ = 3.0 (s, 3H, CH₃), 3.4 (s, 3H, CH₃), 7.38–7.75 (m, 4H, Ar), 7.76 (s, 1H, CH); anal. calcd for C₁₂H₁₁N₃S: C, 62.86; H, 4.84; N, 18.33; S, 13.98. Found: C, 62.65; H, 4.63; N, 18.56; S, 13.80%.

4.1.3. Synthesis of Compounds 8a–h

In a mortar, equimolar amounts of compound 4 (0.01 mol) and N-aryl-2-cyanoacetamides 5a–h (0.01 mol) were mixed for 20 min at room temperature in the presence of potassium hydroxide (0.01 mol). The resulting solid was triturated with water, purified, and then recrystallized from DMF to yield the products.

4.1.3.1. 5-(Benzothiazolyl)-6-amino-1-(4-methoxyphenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile (8a)

Yellow crystals, yield 81%; mp 280 °C, v_max/cm^–1 (KBr) 3372 (NH₂) 2206 (CN), 1667 (CO); ¹H NMR (DMSO-d₆) δ = 3.85 (s, 3H, OCH₃), 7.16–7.50 (m, 6H, NH₂ and Ar); 7.85 (d, 2H, J = 8.8 Hz, Ar), 8.06 (d, 2H, J = 8.8 Hz, Ar), 8.40 (s, 1H, CH); ¹³C NMR (DMSO-d₆) δ = 55.9, 116.11 117.9, 121.7, 122.2, 125.4, 126.8, 127.1, 130.2, 132.1, 144.6, 152.4, 155.0, 160.0, 160.4, 166.51; m/z = 374 (M⁺, 100%), 359 (26.3%), 330 (5.8%), 252 (10.6%), 224 (12.8%), 197 (8.8%), 187 (6.8%), 123 (9.2%), 108 (27.0%), 77 (12.6%), 64 (10.4%); anal. calcd for C₂₀H₁₄N₄O₂S: C, 64.16; H, 3.77; N, 14.96; S, 8.56. Found: C, 64.34; H, 3.58; N, 14.74; S, 8.76%.

4.1.3.2. 5-(Benzothiazolyl)-6-amino-1-(2,4-dimethoxyphenyl)-2-oxo-1,2-dihydro-pyridine-3-carbonitrile (8b)

Orange crystals, yield 79%; mp 288 °C, v_max/cm^–1 (KBr) 3398 (NH₂), 2195 (CN), 1640 (CO); ¹H NMR (DMSO-d₆) δ = 3.75 (s, 3H, OCH₃), 3.86 (s, 3H, OCH₃), 6.50–6.64 (m, 1H, Ar), 7.15–7.18 (m, 3H, Ar and NH₂), 7.32–7.36 (m, 1H, Ar), 7.66 (d, 1H, J = 7.2 Hz, Ar), 7.83 (d, 1H, J = 8.4 Hz, Ar), 8.05 (s, 1H, Ar), 8.11 (d, 1H, J = 7.2 Hz, Ar), 8.27 (s, 1H, CH); ¹³C NMR (DMSO-d₆) δ = 55.7, 56.5, 74.8, 76.8, 99.1, 104.6, 120.1, 120.3, 120.5, 121.7, 122.9, 126.3, 144.4, 149.5, 155.0, 161.0, 165.0; m/z = 404 (M⁺, 100%), 387 (11.5%), 373 (70.0%), 333 (31.5%), 251 (4.4%), 224 (16.3%), 198 (15.6%), 138 (18.8%), 122 (23.8%), 95 (31.4%), 77 (39.7%), 69 (58.1%), 56 (96.1%); anal. calcd for C₂₁H₁₆N₄O₃S: C, 62.36; H, 3.99; N, 13.85; S, 7.93. Found: C, 62.55; H, 3.81; N, 13.62; S, 7.76%.

4.1.3.3. 5-(Benzothiazolyl)-6-amino-1-(4-chloro-2-methylphenyl)-2-oxo-1,2-dihydro-pyridine-3-carbonitrile (8c)

Yellow crystals, yield 86%; mp 292 °C, v_max/cm^–1 (KBr) 3370 (NH₂), 2208 (CN), 1666 (CO); ¹H NMR (DMSO-d₆) δ = 2.06 (s, 3H, CH₃), 7.39–7.53 (m, 6H, Ar and NH₂), 7.64 (s, 1H, Ar), 7.90 (d, 1H, J = 4.4 Hz, Ar), 8.0 (d, 1H, J = 6 Hz, Ar), 8.49 (s, 1H, CH); m/z 392 = (M⁺, 100%), 393 (M⁺ + 1, 34.2%), 375 (81.2%), 340 (15.0%), 312 (15.1%), 223 (12.0%), 197 (22.9%), 164 (10.4%), 125 (14.4%), 109 (18.2%), 89 (45.0%), 77 (29.8%), 63 (30.3%), 51 (17.6%); anal. calcd for C₂₀H₁₃ClN₄OS: C, 61.15; H, 3.34; Cl, 9.02; N, 14.26; S, 8.16. Found: C, 61.32; H, 3.17; N, 14.49; S, 8.35%.

4.1.3.4. 5-(Benzothiazolyl)-6-amino-1-(2,4-dichlorophenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile (8d)

Orange crystals, yield 92%; mp ≥ 300 °C, v_max/cm^–1 (KBr) 3378 (NH₂), 2196 (CN), 1644 (CO); ¹H NMR (DMSO-d₆) δ = 7.18–7.22 (m, 1H, Ar), 7.34–7.42 (m, 3H, Ar and NH₂), 7.65 (s, 1H, Ar), 7.69 (d, 1H, J = 6 Hz, 1H, Ar), 7.87 (d, 1H, J = 6.8 Hz, Ar), 8.26–8.28 (m, 2H, Ar), 8.35 (s, 1H, CH); ¹³C NMR (DMSO-d₆) δ = 74.10, 78.50, 120.6, 121.8, 123.2, 124.2, 126.4, 127.3, 128.2, 128.9, 133.2, 135.4, 144.5, 154.6, 164.2; m/z = 412 (M⁺, 48.5%), 413 (M⁺ + 1, 15%), 414 (M⁺ + 2, 31.6%), 392 (35.4%), 377 (69.4%), 224 (15.7%), 198 (15.1%), 124 (19.2%), 109 (36.1%), 97 (34.3%), 71 (45.9%), 69 (92.6%), 55 (100%); anal. calcd for C₁₉H₁₀Cl₂N₄OS: C, 55.22; H, 2.44; Cl, 17.16; N, 13.56; S, 7.76. Found: C, 55.42; H, 2.61; N, 13.79; S, 7.94%.

4.1.3.5. 5-(Benzothiazolyl)-6-amino-1-(4-bromophenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile (8e)

Beige crystals, yield 79%; mp ≥ 300 °C, v_max/cm^–1 (KBr) 3327 (NH₂), 2211 (CN), 1652 (CO); ¹H NMR (DMSO-d₆) δ = 7.19 (d, 1H, J = 7.6 Hz, Ar); 7.28 (d, 1H, J = 7.6 Hz, Ar); 7.38–7.69 (m, 8H, Ar and NH₂), 8.39 (s, 1H, CH); anal. calcd for C₁₉H₁₁BrN₄OS: C, 53.91; H, 2.62; Br, 18.88; N, 13.24; S, 7.57. Found: C, 53.72; H, 2.83; N, 13.47; S, 7.39%.

4.1.3.6. 5-(Benzothiazolyl)-6-amino-1-(4-fluorophenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile (8f)

Beige crystals, yield 75%; mp ≥ 300 °C, v_max/cm^–1 (KBr) 3356 (NH₂), 2208 (CN), 1666 (CO); ¹H NMR (DMSO-d₆) δ = 7.28–7.48 (m, 6H, Ar and NH₂); 7.87 (d, 1H, J = 7.6 Hz, Ar); 8.02–8.04 (m, 2H, Ar), 8.06 (d, 1H, J = 8 Hz, Ar), 8.43 (s, 1H, CH); m/z 362 = (M⁺, 100%), 318 (7.3%), 197 (8.8%), 146 (5.5%), 111 (18.0%), 95 (35.9%), 75 (20.5%), 69 (22.9%), 55 (6.7%); anal. calcd for C₁₉H₁₁FN₄OS: C, 62.97; H, 3.06; F, 5.24; N, 15.46; S, 8.85. Found: C, 62.77; H, 3.25; N, 15.21; S, 8.67%.

4.1.3.7. 5-(Benzothiazolyl)-6-amino-1-(2,3-difluorophenyl)-2-oxo-1,2-dihydropyridine-3-carbonitrile (8g)

Beige crystals, yield 71%; mp = 286 °C, v_max/cm^–1 (KBr) 3433 (NH₂), 2212 (CN), 1641 (CO); ¹H NMR (DMSO-d₆) δ = 7.26–7.46 (m, 7H, Ar and NH₂); 7.86 (d, 1H, J = 7.8 Hz, Ar); 8.02 (d, 1H, J = 7.8 Hz, Ar), 8.41 (s, 1H, CH); ¹³C NMR (DMSO-d₆) δ = 80.5, 100.4, 119.9, 121.0, 121.4, 121.7, 123.3, 125.8, 126.5, 134.3, 134.7, 151.3, 152.5, 156.8, 160.3, 162.6, 165.6; m/z 380 = (M⁺, 100%), 360 (50.7%), 332 (26.4%), 224 (9.2%), 197 (11.3%), 170 (6.76%), 146 (7.28%), 129 (12.16), 113 (18.29%), 96 (5.1%), 69 (23.3%); anal. calcd for C₁₉H₁₀F₂N₄OS: C, 60.0; H, 2.65; F, 9.99; N, 14.73; S, 8.43. Found: C, 60.19; H, 2.83; N, 14.49; S, 8.61%.

4.1.3.8. 5-(Benzothiazolyl)-6-amino-1-[3-(trifluoromethyl)phenyl]-2-oxo-1,2-dihydro-pyridine-3-carbonitrile (8h)

Beige crystals, yield 78%; mp ≥ 300 °C, v_max/cm^–1 (KBr) 3428 (NH₂), 2213 (CN), 1643 (CO); ¹H NMR (DMSO-d₆) δ = 7.27–7.31 (m, 4H, Ar); 7.41 (m, 2H, Ar); 7.86 (d, 2H, J = 8.1 Hz, Ar); 8.02 (d, 2H, J = 7.8 Hz, Ar); 8.41 (s, 1H, CH); ¹³C NMR (DMSO-d₆) δ = 80.5, 100.4, 120.0, 121.0, 121.7, 123.3, 125.9, 134.3, 134.7, 152.1, 152.5, 156.9, 161.0, 162.6, 165.6; anal. calcd for C₂₀H₁₁F₃N₄OS: C, 58.25; H, 2.69; F, 13.82; N, 13.59; S, 7.77. Found: C, 58.43; H, 2.50; N, 13.82; S, 7.94%.

4.1.4. Synthesis of Compounds 14a–e

Equimolar amounts of 4 (0.01 mol) and 2-cyano-N′-(4-substituted benzylidene) acetohydrazide 9a–e (0.01 mol) were ground in a mortar in the presence of potassium hydroxide for 20 min at room temperature. The solid so formed was triturated with water, filtered off, and then recrystallized from DMF.

4.1.4.1. 8-(Benzothiazolyl)-2-(2-methoxyphenyl)-5-oxo-3,5-dihydro[1,2,4]triazolo[1,5- a]-pyridine-6-carbonitrile (14a)

Reddish brown crystals, yield 74%; mp ≥ 300 °C, v_max/cm^–1 (KBr) 3431 (NH), 2195 (CN), 1609 (CO); ¹H NMR (DMSO-d₆) δ = 3.88 (s, 3H, OCH₃), 7.12 (t, 1H, J = 7.2 Hz, Ar), 7.32 (t, 1H, J = 7.6 Hz, Ar), 7.44–7.52 (m, 2H, Ar), 7.74 (d, 1H, J = 7.8 Hz, Ar), 7.90 (d, 1H, J = 8 Hz, Ar), 8.03 (m, 3H, Ar and NH), 8.47 (s, 1H, CH); ¹³C NMR (DMSO-d₆) δ = 56.3, 81.1, 101.11, 112.8, 117.7, 120.7, 121.5, 122.2, 123.8, 126.3, 131.4, 131.7, 134.9, 135.2, 152.0, 153.0, 157.5, 158.1, 160.5, 161.1; anal. calcd for C₂₁H₁₃N₅O₂S: C, 63.15; H, 3.28; N, 17.53; S, 8.03. Found: C, 63.34; H, 3.45; N, 17.75; S, 8.20%.

4.1.4.2. 8-(Benzothiazolyl)-2-(2,5-dimethoxyphenyl)-5-oxo-3,5-dihydro[1,2,4]triazolo[1,5-a]-pyridine-6-carbonitrile (14b)

Reddish brown crystals, yield 83%; mp ≥ 300 °C, v_max/cm^–1 (KBr) 3398 (NH), 2206 (CN), 1612 (CO); ¹H NMR (DMSO-d₆) δ = 3.85 (s, 3H, OCH₃), 3.91 (s, 3H, OCH₃), 7.06–7.15 (m, 2H, Ar), 7.30–7.44 (m, 3H, Ar and NH), 7.56 (s, 1H, Ar), 7.90 (d, 1H, J = 7.6 Hz, Ar), 8.07 (d, 1H, J = 7.6 Hz, Ar), 8.47 (s, 1H, CH); ¹³C NMR (DMSO-d₆) δ = 56.0, 57.2, 81.1, 101.1, 114.7, 116.6, 117.1, 120.4, 121.5, 121.8, 122.2, 123.8, 126.3, 135.0, 135.2, 152.1, 152.9, 153.4, 160.3, 163.0; anal. calcd for C₂₂H₁₅N₅O₃S: C, 61.53; H, 3.52; N, 16.31; S, 7.47. Found: C, 61.71; H, 3.69; N, 16.09; S, 7.29%.

4.1.4.3. 8-(Benzothiazolyl)-2-(4-aminophenyl)-5-oxo-3,5-dihydro[1,2,4]triazolo[1,5-a]-pyridine-6-carbonitrile (14c)

Dark brown crystals, yield 84%; mp ≥ 300 °C, v_max/cm^–1 (KBr) 3345 (NH₂), 3220 (NH), 2203 (CN), 1617 (CO); ¹H NMR (DMSO-d₆) δ = 6.71 (s, 2H, NH₂), 7.19–7.89 (m, 7H, Ar and NH), 7.97 (d, 2H, J = 8.4 Hz, Ar), 8.46 (s, 1H, CH); m/z = 384 (M⁺, 3.7%), 358 (15%), 305 (3.4%), 251 (5.8%), 223 (7.03%), 185 (3.7%), 146 (6.4%), 135 (25.49%), 106 (28.7%) 85 (32.1%) 55 (100%); anal. calcd for C₂₀H₁₂N₆OS: C, 62.49; H, 3.15; N, 21.86; S, 8.34. Found: C, 62.31; H, 3.34; N, 21.63; S, 8.17%.

4.1.4.4. 8-(Benzothiazolyl)-2-(4-(dimethylamino)phenyl)-5-oxo-3,5-dihydro[1,2,4]- triazolo[1,5-a]pyridine-6-carbonitrile (14d)

Black crystals, yield 71%; mp ≥ 300 °C, v_max/cm^–1 (KBr) 3384 (NH), 2195 (CN), 1605 (CO); ¹H NMR (DMSO-d₆) δ = 2.99 (s, 6H, CH₃), 6.85 (d, 1H, J = 8.8 Hz, Ar), 7.30 (t, 1H, J = 7.6 Hz, Ar), 7.44 (t, 1H, J = 8 Hz, Ar), 8.08–8.12 (m, 5H, Ar and NH), 8.15 (d, 1H, J = 8.4 Hz, Ar), 8.45 (s, 1H, CH); ¹³C NMR (DMSO-d₆) δ = 66.8, 81.2, 112.2, 121.4, 122.2, 123.8, 128.4, 151.8, 153.0, 158.7, 161.4; m/z 412 = (M⁺, 3.4%), 381 (4.5%), 305 (92.4%), 279 (16.6%), 223 (3.5%), 185 (4.09%), 149 (16.1%), 132 (34.08%), 97 (28.2%), 73 (54.3%), 69 (71.3%), 57 (100%), 55 (87.5%); anal. calcd for C₂₂H₁₆N₆OS: C, 64.06; H, 3.91; N, 20.38; S, 7.77. Found: C, 64.25; H, 3.73; N, 20.60; S, 7.59%.

4.1.4.5. 8-(Benzothiazolyl)-2-(2-chloro-4-nitrophenyl)-5-oxo-3,5-dihydro[1,2,4]triazolo1,5-a]pyridine-6-carbonitrile (14e)

Reddish brown crystals, yield 76%; mp ≥ 300 °C, v_max/cm^–1 (KBr) 3398 (NH), 2206 (CN), 1612 (CO); ¹H NMR (DMSO-d₆) δ = 6.80–7.35 (m, 3H, Ar), 7.45 (d, 1H, J = 8 Hz, Ar), 7.51–7.60 (m, 3H, Ar and NH), 8.15 (d, 1H, J = 8.4 Hz, Ar), 8.50 (s, 1H, CH); ¹³C NMR (DMSO-d₆) δ = 95.2, 102.4, 117.7, 119.6, 120.9, 122.8, 123.9, 125.8, 127.6, 128.8, 132.9, 137.0, 152.5, 156.9, 158.5, 159.9, 161.8; anal. calcd for C₂₀H₉ClN₆O₃S: C, 53.52; H, 2.02; Cl, 7.90; N, 18.72; S, 7.14. Found: C, 53.70; H, 2.19; N, 18.49; S, 7.35%.

4.2. Biological Section

4.2.1. Virus, cell, and MTT assay

At 37 °C, 5% CO₂, the virus and Vero-E6 cells were grown in Dulbecco’s Modified Eagle’s medium (DMEM) containing 10% FBS (Invitrogen) and 1% pen/strep antibiotic mixture. To yield virus stock, cells were distributed in tissue culture jars for 24 h before infection with CoV-19/Egypt/NRC-3/2020 isolates at a multiplicity of infection (MOI) of 0.1 infection medium (DMEM containing 2% FBS, 1% pen/strep, and 1% TPCK-treated trypsin). The infection medium comprising of the virus inoculum was enlarged 2 h later. To produce virus stock, cells were dispersed into tissue culture jars for 24 h earlier to infection with CoV-19/Egypt/NRC-3/2020 isolate at a MOI of 0.1 infection medium (DMEM including 2% FBS, 1% pen/strep, and 1% TPCK-treated trypsin). The virus inoculum-containing infection medium was ejected 2 h later and replaced with a fresh infection medium, incubated for 3 days. Cell supernatant was collected at the indicated time point and centrifuged for 5 min at 2500 rpm to remove small particulate cell flotsam and jetsam. The supernatant was transferred to a new 50 mL falcon tube, and the plaque infectivity assay was used to titrate it.

The following steps carried out the method.

• I.Stock solutions of the test compounds were prepared in 10% DMSO in DDH₂O and diluted further to working solutions with DMEM to determine the half-maximal cytotoxic concentration (CC₅₀). The extracts’ cytotoxic activity was determined in Vero-E6 cells using the MTT process, which was slightly modified.
• II.The cells were seeded in 96-well plates (100 l/well at a density of 3105 cells/mL) and incubated at 37 °C in 5% CO₂ for 24 h. Cells were treated in triplicates with different concentrations of the studied compounds after 24 h.
• III.The supernatant was discarded after 24 h, and cell monolayers were washed three times with phosphate-buffered saline (PBS). MTT solution (20 l of 5 mg/mL stock solution) was applied to each well and incubated at 37 °C for 4 h before medium aspiration.
• IV.Formazan crystals were dissolved in each well with 200 mL of acidified isopropanol (0.04 M HCl in isopropanol).
• V.Using a multiwell plate reader, the absorbance of formazan solutions was measured at max 540 nm with 620 nm as a reference wavelength.
• VI.The following equation was used to calculate the percentage of cytotoxicity relative to untreated cells. The concentration that exhibited 50% cytotoxicity was calculated using a plot of percent cytotoxicity versus sample concentration.⁶³

(Absorbance of cells without treatment – absorbance of cells with treatment) × 100 absorbance’s cells without treatment = % cytotoxicity

4.2.2. Plaque Infectivity Assay

The plaque infectivity assay was used to titrate hCoV-19/Egypt/NRC-03/2020 (NRC-03-nhCoV) (Accession Number on GSAID: EPI-ISL-430820) as previously mentioned. In a nutshell, the propagated virus was serially diluted 10-fold in a medium containing no FBS. To inoculate 80–90% confluent Vero-E6 cell monolayers, 100 L of each virus dilution was combined with 400 L of infection medium and used to inoculate 80–90% confluent Vero-E6 cell monolayers.

A control well was inoculated with 500 mL of serum-free medium in the same plate, then incubated for 1 h at 37 °C with 5% CO₂ to enable virus adsorption and rocked every 15 min to ensure uniform infection and prevent drying of the cells. The virus inoculum was discarded after 1 h. The cell monolayers were overlaid with 3 mL of DMEM plus 0.6% agarose containing 1 g/mL TPCK-treated trypsin, 10% FBS, one pen/strep, and the required concentration of the test drug. The plate was left at R.T. for 10 min to enable the agarose portion of the overlayer medium to solidify before being incubated at 37 °C under 5% CO₂. After 72 h, 1 mL of fixation solution (10% formalin) was applied to each well for 1 h for cell fixation and virus inactivation. The fixer was eventually discarded, and the plate wells were washed and dried. One mL of the staining solution (0.1% crystal violet) was applied to each well for 5 min to visualize the plaques, then the dye was discarded, and the plate wells were rinsed in water. In a violet (stained cells) background, viral plaques appeared as simple unstained spots (due to viral infection). The following equation was used to measure the virus titer

Plaque-forming unit (PFU) per mL = number of plaques multiplied by the virus’s inoculated volume multiplied by the virus’s dilution factor of 10.⁶⁴

4.2.3. Plaque Reduction Assay

The plaque reduction assay⁶⁵ was performed in a six-well plate with Vero-E6 cells (1.2 106 cells) cultivated for 24 h at 37 °C to determine the preliminary antiviral activity of the FDA-approved drugs. Before being applied to the cells, the NRC-03-nhCoV virus was diluted to give 102 plaque-forming units (PFU) per well, combined with the safe concentration of the tested compounds, and incubated for 1 h at 37 °C. The cells were inoculated with (100 mL/well) virus with the tested compounds after removing the growth medium from the cell culture plates. After 1 h of virus adsorption, the cell monolayers were given 3 mL of DMEM supplemented with the overlay medium containing specified concentrations of the tested compounds. The plates were allowed to solidify before incubating at 37 °C for 3 days to form viral plaques. After 1 h in cell fixing solution, the plates were stained with 0.1% crystal violet in distilled water. The untreated virus was incubated with Vero-E6 cells in control wells, and plaques were counted, and the percentage reduction in plaque formation was calculated.

4.2.4. Inhibitory Concentration 50 (IC₅₀)

Vero-E6 cells were distributed in each well of 96-well tissue culture plates and incubated overnight in a humidified 37 °C incubator under a 5% CO₂ environment. After that, the cell monolayers were washed in 1 PBS and exposed to virus adsorption for 1 h at room temperature. The monolayers were then overlaid with 50 μmol DMEM containing differing concentrations of the selected test compounds azithromycin, niclosamide, and nitazoxanide. The cells were fixed with 100 l of 4% paraformaldehyde for 20 min and stained with 0.1% crystal violet in distilled water for 15 min after being incubated at 37 °C in a 5% CO₂ incubator for 72 h. The optical density of the color was calculated at 570 nm using an Anthos Zenyth 200 rt plate reader after the crystal violet dye was dissolved in 100 mL of absolute methanol per well (Anthos Lab tec Instruments, Heerhugowaard, Netherlands). The compound’s IC₅₀ is described as the amount of the compound needed to reduce the virus-induced cytopathic effect (CPE) by 50% when compared to the virus control.⁶⁶

To test the effect of different virus concentrations on replication performance, confluent Vero-E6 cell monolayers were infected in triplicate with NRC-03-nhCoV at MOIs of 0.005, 0.001, and 0.001 at 37 °C. At 1 h postinfection (hpi), the inocula were removed, the cell monolayers were washed with 1 PBS, and infection media (1 DMEM supplemented with 1% Pen/Strep, 0.3% bovine serum albumin (BSA), and 2 g/mL TPCK-treated trypsin) was overlaid. At 48 h postinduction, the cell culture supernatants were collected. A plaque infectivity assay was used to assess the virus titer.

4.2.5. Viral Adsorption Mechanism

Zhang et al.⁶⁷ used a procedure with minor changes to investigate the viral adsorption process. Vero-E6 cells were grown for 24 h at 37 °C in a 6-well plate (105 cells per mL). Each medication was mixed into 200 L of medium without supplements and incubated with the cells for 2 h at 4 °C. The nonabsorbed drug was extracted from the inocula by washing the cells three times with a supplement-free medium. The pretreated cells were coincubated for 1 h with SARS-CoV-2 virus diluted to 104 PFU/well, and then 3 mL of DMEM supplemented with 2% agarose was added. Plates were allowed to solidify before being incubated at 37 °C to allow viral plaques to form. The plaques were fixed and stained as mentioned above to measure the percentage reduction in plaque formation compared to the control wells, which contained untreated Vero-E6 cells directly infected with NRC-03-nhCoV.

4.3.6. Viral Replication Mechanism

As previously mentioned,⁶⁸ the effect of the tested drug on viral replication was determined, which Vero-E6 cells were grown for 24 h at 37 °C in a 6-well plate (105 cells per mL). The virus was inoculated directly into the cells and incubated at 37 °C for 1 h. Three times with a supplement-free medium, washing the cells removed the nonadsorbed viral particles from the inocula. Crystal violet was used to stain cell monolayers after being fixed in a 10% formalin solution for 1 h, then Vero-E6 cells were incubated with the virus in the control wells. The number of plaques formed was counted, and the percentage reduction in plaque formation was compared to that in control wells.

4.3.7. Virucidal Mechanism

The virucidal mechanism was investigated using a protocol previously mentioned⁶⁹ Vero-E6 cells (105 cells/mL) were grown for 24 h at 37 °C in a 6-well plate, and 200 mL of serum-free DMEM containing SARS-CoV-2 was added to each sample with promising inhibition. The mixture was diluted 10-fold three times in a serum-free medium after 1 h of incubation, and viral particles could still develop on Vero-E6 cells. The Vero-E6 cell monolayer was then treated with 100 mL of each dilution. After 1 h of contact time, a DMEM overlayer was added to the cell monolayer. Plates were allowed to solidify before being incubated at 37 °C to allow viral plaques to form. The plaques were fixed and stained to measure the percentage reduction in the level of plaque formation, as mentioned above. This value was compared to that of control wells that contained virus-infected cells that had not been pretreated with the tested substance.

4.2.8. Molecular Modeling

SARS-CoV-2 main protease (Mopar) (PDB ID: 6LU7) X-ray crystal structure coordinate was discovered using PDB. The MOE 2015.10 software was used to perform the docking study.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsomega.3c01987.

• Experimental details: chemistry, biological evaluations, MTT assay, molecular docking, characterization data of all compounds, ¹H NMR, ¹³C NMR, IR, mass spectra, and IC₅₀ for tested compounds (PDF)

The authors declare no competing financial interest.