Structural and Synthetic Aspects of Small Ring Oxa- and Aza-Heterocyclic Ring Systems as Antiviral Activities

5.1.1. Anti-HIV Agent Darunavir

Kate et al. have demonstrated the synthesis of darunavir (Figure 6), which is considered to be a protease inhibitor [112] and used in low doses for the treatment of HIV. Raltegravir [113] and stavudine [114] are the other available drugs which also show similar properties in this particular domain.

Darunavir becomes stabilized inside the cavity of the enzyme by making a hydrogen bonding interaction through the coordination of the hydroxyl group, 4-amino phenyl and tetrahydrofuran ring system with the active site (Asp25′) or near to the active site (Asp29/29′, Asp30/30′) of the amino acid residues as present in HIV-1 PR (Figure 7) [115].

Key synthetic steps for Scheme 1 [116]: (a) ring opening of epoxide with primary amine; (b) N-protected 4-aminobenzenesulfonyl halide; (c) Boc deprotection; and (d) nucleophilic addition followed by separation of the product 1.

Reagents and conditions: (a) A mixture of (2S,3S)-1,2-Epoxy-3-(Boc-amino)–4-phenylbutane and isobutyl amine was heated at 65–75 °C and (b) N-acetyl sulphanilyl chloride was added at 5–15 °C to the pre-cooled mixture of (1S,2R)-(1-Benzyl-2-hydroxy-3-(isobutyl-amino) propyl) carbamic acid tert-butyl ester in N, N-dimethylacetamide. Then, triethyl amine was added to the reaction mixture at a temperature below 30 °C and (c) Boc deprotection was done by taking the corresponding carbamic acid tert-butyl ester in isopropyl alcohol at 25–35 °C; after that, aqueous sulphuric acid solution was added to the reaction mixture at 25–35 °C. Then, the amino- N-((2R,3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-isobutylbenzene sulfonamide sulphate salt was treated with potassium carbonate solution in water and 4-Amino-N-((2R,3S)-3-amino–2-hydroxy-4-phenylbutyl)-N–isobutylbenzenesulfonamide was obtained above in water. Then, to a stirred mixture of (d) potassium carbonate, isopropyl acetate and water, 4-amino-N-((2R,3S)-3-amino-2-hydroxy-4-phenylbutyl)-N-isobutyl benzene sulphonamide was added at 25–35 °C, and the reaction mixture was cooled to 15–25 °C; after that, (3R,3aS,6aR)-Hydroxyhexa hydrofuro [2,3-b] furanyl succinimidyl carbonate was added at 15–25 °C. After completion of the reaction, the crude reaction mixture was purified to obtain the final product 1.

Structure–Activity Relationship of Darunavir: The structure–activity relationship of darunavir analogues is shown in Table 1, in which the incorporation of thiazole and ethyl phosphonate subunit as R¹, along with benzo[d][1,3]dioxole moiety as R², increases its activity significantly [117]. Darunavir and its corresponding synthetic analogues show a distinctive mechanism of action, as characterized by dual functionality. It works as HIV-1 protease inhibitor and also hinders the dimerization process of the HIV-1 protease [118]. Mostly, the Darunavir class of compounds exhibits a binding affinity towards plasma proteins such as alpha-1-acid glycoprotein (AAG or AGP) [119]. The CheckMate^TM Mammalian Two-Hybrid System was utilized to establish a dual luciferase assay. This assay was employed to assess the susceptibility of HIV-1_LAI to a variety of drugs and evaluate the cytotoxic effects of the drugs.

Table 1.

SAR analysis of darunavir.

Compound	Substitution		Activity
	−R1	−R2	Inhibition Concentration (nM)	Inhibitor Constant (Ki)
			IC90 = 4.1	16 pM
			IC90 = 1.4	14 pM
			IC50 = 0.22	14 pM
			IC50 = 0.7	15 fM
			IC50 = 1	8 pM

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A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay was employed for determining drug susceptibility and cytotoxicity [120]. The chromogenic substrate Lys-Ala-Arg-Val-Nle-paranitro-Phe-Glu-Ala-Nle-amide was used to determine the kinetic parameters [121].

Mechanism of Action of Darunavir: Darunavir interacts with the protease enzyme of HIV-1 to prevent the dimerization and enhance the catalytic activity. As a result, the cleavage of the proteins is disturbed, and ultimately the replication of the virus is stopped, after the application of this drug to HIV-infected cells [122]. Generally, the interaction takes place with the primary chains of Asp-29 and Asp-30 amino acids present in the active site of the protease enzyme.

5.1.2. Anti-HIV Agent Fluoroquinolone-Isatin-Thiosemicarbazone Hybrids

The molecular-hybrid approach was introduced to understand the synergistic effects of the two compounds in order to generate a new structural entity with superior properties [123] for inhibiting the viral replication. Isatin derivatives have been shown to exhibit antiviral activity against a range of viruses, including HIV-1. Recently, hybrid fluoroquinolone-isatin derivatives have attracted attention due to their promising anti-HIV properties (Figure 8) [124].

Key synthetic steps for Scheme 2 [125]: (a) A solution of N-hydroxylamine in absolute ethanol was added to potassium hydroxide and carbon disulphide, and the mixture was stirred at 0−5 °C to form the corresponding potassium salt of dithiocarbamates; (b) hydrazine hydrate was added to the reaction mixture and stirred at 80 °C; after completion of the reaction, it was cooled to 0 °C to obtain the corresponding thiosemicarbazide; (c) to a hot dispersion of thiosemicarbazide in ethanol was added an equimolar aqueous solution of sodium acetate, and to this solution a further equimolar ethanolic solution of 5-F-isatin was added, and the mixture was stirred while being heated on a hot plate for 4−15 min. The resultant precipitate was filtered off and dried. The product was recrystallized from 95% ethanol; (d) the N-Mannich bases were further synthesized by condensing the acidic imino group of isatin derivatives with formaldehyde and secondary amine (4-ethyl-7-fluoro-1-oxo-6-(piperazin-1-yl)-1,4-dihydronaphthalene-2-carboxylic acid) by irradiating the reaction vessel in a microwave reactor for 3−15 min at 455 W, followed by purification to obtain the corresponding product 2.

Reagents and conditions: (a) CS₂, KOH, C₂H₅OH, 0−5 °C; (b) NH₂NH₂.H₂O, 80 °C, conc. HCl; (c) 5-F-Isatin, CH₃COONa; (d) 30% HCHO, 455 W, 3−15 min.

Mechanism of Action of Fluoroquinolone-isatin-thiosemicarbazone: Isatinyl thiosemicarbazone derivatives have been found to exhibit anti-HIV activity by hindering the viral protease enzyme’s function. The viral protease enzyme plays a pivotal role in the maturation of the HIV virion by interrupting its activity; as a result, the production of the corresponding infectious virions stops. Isatinyl thiosemicarbazone derivatives interact with the active site of the viral protease enzyme, and hence its activity is stopped [126,127].

5.1.3. Anti-HIV Agent Amprenavir

The drug amprenavir (Figure 9) is primarily used to treat HIV infections; it acts as a protease inhibitor. It binds to the active site of the enzyme and inhibits its activity. It prevents the cleavage of viral polyproteins, which leads to the development of immature non-infectious viruses [128]. Amprenavir’s hydroxyl group interacts with Asp25 and Asp25′ residues of the protein at the catalytic site. Along with that, there are stable H-bonding interactions between the hydroxyl group of the drug with the catalytic site of the aspartic acid side chains (Figure 10) [129].

Key synthetic steps for Scheme 3 [130]: (a) Formation of chalcone by the reaction of 2-phenylacetaldehyde and Ph₃PCHCO₂Et at 90 °C in toluene; (b) reduction of the ester by lithium aluminium hydride and AlCl₃ in diethyl ether; (c) chiral epoxidation; (d) epoxide ring opening, followed by (e) epoxide ring closing and further(f) ring opening; and formation of gem-diol derivatives which formed product 3 by reacting with (g) isobutyl amine and (h) N-hydroxysuccinimidyl carbonate of (S)-3-hydroxytetrahydrofuran.

Reagents and conditions: (a) Ph₃PCHCO₂Et, PhH, 90 °C; (b) LiAlH₄, AlCl₃ (30 mol %), Et₂O, 0 °C; (c) mCPBA, CH₂Cl₂, 0 °C; (d) Ti(OⁱPr)₄, TMSN₃, C₆H₆, 70 °C; (e) p-TsCl, Bu₂SnO (2 mol %), Et₃N, DMAP (10 mol %), CH₂Cl₂, 0 °C; (f) K₂CO₃, MeOH, 0 °C; (g) (S,S)-Co(salen)OAc (0.5 mol %), THF, H₂O (0.5 equiv), 25 °C; (h) (1) ⁱBuNH₂, ⁱPrOH, 50 °C, (2) PPh₃, H₂O, THF, 25 °C; (2) N-hydroxysuccinimidyl carbonate of (S)-3-hydroxytetrahydrofuran, Et₃N, CH₂Cl₂, 25 °C.

Structure–Activity Relationship of Amprenavir: It shows antiviral properties against the HIV-1 virus in an in vitro study against the C8166 cell line [131]. The efficacy of HIV-1 protease inhibitors was assessed using the fluorescence resonance energy transfer (FRET) technique. A specific protease substrate [Arg-Glu(EDANS)-Ser-Gln-Asn-Tyr-Pro-Ile-Val-Gln-Lys(DABCYL)-Arg], was employed. The determination of the inhibitor’s binding dissociation constant (K_i) involved fitting the initial velocity plot against inhibitor concentrations to the Morrison equation through non-linear regression analysis [132]. The EC₅₀ values were compared with those of amprenavir, resulting in the conclusion that the biaryl subunit with varying substituents showed lower efficiency. However, compounds that featured substituents of –NH₂ for R¹ and 3-pyridyl or 4-pyridyl for R² exhibited increased solubility and stronger enzyme inhibitory activity at a sub-nanomolar level. These compounds were found to be 2–10 times more active than amprenavir, as described in Table 2 [133].

Table 2.

SAR analysis of amprenavir.

Compound	Substitution		Activity
	−R1	−R2	EC50 (nM)	Inhibitor Constant (Ki) (nM)
			2.93	0.84
			4.29	5.8
			2.32	6.4
			6.89	5.8
			6.04	7.4
			3.63	5.2
			5.53	1.8
			4.30	1.9
			3.46	4.3
			9.2	1.6
			4.48	8.1
			3.08	3.5
			2.68	1.6
			2.25	3.8
			0.10	1.95
			0.10	0.33
			2.93	0.086

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Mechanism of Action of Amprenavir: Amprenavir binds to the active site of the protease and inhibits the activity of the enzyme. This inhibition prevents the cleavage of the gag-pol polyprotein. Amprenavir competes with the natural substrate of the viral protease enzyme, which is a precursor protein of the viral genome. This competition leads to the formation of a stable complex between the drug and the enzyme, preventing the enzyme from cleaving the protein. As a result, non-infectious viral particles are produced, leading to a reduction in the number of viral particles in the body [134]. In Table 3 other marketed anti-HIV drugs are listed with their mechanism of action, ways of use and side effects.

Some other synthesized compounds that show activity against HIV are given in Table 4.

Table 4.

Synthesized anti-HIV compounds.

Sl. No.	Antiviral Agent	Drug Target	Activity
1.		HIV [142]	Methylthiazole derivatives effectively inhibit HIV-1 entry into cells, demonstrating low micromolar potency (IC50 = 1.6 µM) and a favorable selectivity index (SI) in both single-cycle TZM-bl cell assays and multicycle MT-2 cell assays.
2.		HIV-1 and HIV-2 [143]	3″-alkenyl-substituted TSAO derivatives exhibit anti-HIV-1 and anti-HIV-2 activity at subtoxic concentrations against HIV-2 RT involved the RNA-dependent DNA polymerase assay with EC50(HIV-1MT-4) = 0.06 ± 0.03 µM and EC50(HIV-2MT-4) >10 µM.
3.		HIV [144]	4′-ethynyl-2-fluoro-2′-deoxyadenosine demonstrates exceptional potency in inhibiting HIV-1 replication in phytohemagglutinin-activated peripheral blood mononuclear cells with EC50(HIV-1NL4-3) = 50 pM.
4.		HIV-1 [145]	Deoxythreosyl phosphonate nucleosides exhibit potent anti-HIV-1 activity (EC50(HIV-1) = 2.53 μM) by binding effectively to the active site pocket of HIV-1 reverse transcriptase. These compounds show no cytotoxicity, even at high concentrations (CC50 > 316 µM). The incorporation kinetics of these compounds into DNA were studied using their diphosphate form as a substrate and HIV-1 reverse transcriptase as the catalyst.
5.		HIV-1 [146]	Flavopiridol, a cyclin-dependent kinase (CDK) inhibitor, blocks HIV-1 Tat transactivation and viral replication by inhibiting P-TEFb kinase activity. The cytotoxicity of Flavopiridol and its analogues was evaluated using an MTT-based cell viability assay, demonstrating their potential as anti-HIV-1 therapeutics with EC50 = 7.4 nM.

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5.2.1. Anti-HCV Agent Asunaprevir

Asunaprevir (Figure 11) is an orally efficacious NS3 protease inhibitor used for the treatment of hepatitis C virus infection. This tripeptidic acyl sulfonamide is an inhibitor of the enzyme NS3/4A, and is now in phase III clinical trials for the treatment of hepatitis C virus infection. The activity of asunaprevir showed a robust antiviral response in early clinical trials. Suzuki et al. have studied the antiviral activity and toxicological profile of asunaprevir [147]. It inhibits the activity of proteases by binding to the active site. Viral polyproteins cannot be cleaved by this inhibition, which produces undeveloped and non-infectious viral particles (Figure 12) [148].

Key synthetic steps for the Scheme 4 [149]: (a,b) (E)-3-(4-chlorophenyl)acrylic acid is cyclized, and the derivatization (c) reacts with N-Boc-3-(R)-hydroxy-L-proline via nucleophilic addition (d) by peptide coupling reaction. The synthesized fragment reacts with (1R,2S)-1-amino-N-(cyclopropylsulfonyl)-2-vinylcyclopropanecarboxamide (TsOH salt), followed by (e) deprotection of proline nitrogen. (f) The final product 4 was obtained by the peptide coupling reaction with N-Boc-t-butyl-L-glycine.

Reagents and conditions: (a) (i) DPPA, Et₃N, benzene, rt; (ii) Ph₂CH₂, reflux; (iii) NBS, MeCN, reflux. (b) (i) POCl₃, reflux; (ii) (1) n-Bu-Li, THF, −78 °C, (2) (i-PrO)₃B, −78 °C; (3) 50% H₂O₂, Na₂SO₃ −78 °C; (iii) MeOH, MeCN, TMSCHN₂, 0 °C–rt. (c) N-Boc-3-(R)-hydroxy-L-proline, t-BuOK, DMSO, 10 °C. (d) HATU, Hunig’s base i-Pr₂Net, (1R,2S)-1-amino-N-(cyclopropylsulfonyl)-2-vinylcyclopropanecarboxamide (TsOH salt), rt; (e) HCl (conc.), MeOH, reflux; (f) HATU, Hunig’s base, N-Boc-t-butyl-L-glycine, DCM, 0 °C–rt.

Structure–Activity Relationship of Asunaprevir: The structure-activity relationship studies show that the incorporation of a hydroxyl group at the R¹ position decreases its antiviral activity (Table 5). The isoquinoline series with methoxy and chlorinated analogues proved to be potent inhibitors of the NS3/4A protease (GT-1a NS3/4A enzyme) which extended to excellent inhibitory activity in the replicon at the R² substituent. The antiviral activity of the drug was evaluated through a two-part study. Initially, a single ascending dose (SAD) study was conducted in patients infected with genotype 1, followed by a subsequent multiple ascending dose (MAD) study [149].

Table 5.

SAR analysis of asunaprevir derivatives.

Compound	Substitution				Activity
	−R1	−R2	−R3	−R4	IC50 (nM)
					2
					247
					2
					4
					4
					2
					7
					1
					7
					2

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Mechanism of Action of Asunaprevir: Asunaprevir is highly active against HCV NS3 protease [150], which is responsible for processing the HCV polyprotein into individual viral proteins. The production of new viral proteins is prevented with the use of this drug compound, and as a result, the progression of HCV-related liver disease is reduced. It is typically used in combination with other antiviral drugs to enhance efficacy and minimize drug resistance [52].

5.2.2. Anti-HCV Agent 3-(1,2,4-oxadiazole)-quinolone

Studies have shown that 3-heterocyclic quinolones (Figure 13) can inhibit NS5B polymerase activity by binding to an allosteric site. This binding triggers a change in the protein’s structure, resulting in the inhibition of RNA replication. In vitro and in vivo studies have demonstrated the significant antiviral activity of these compounds against HCV and indicated their roles as promising lead candidates for further development [151].

Key synthetic steps for Scheme 5 [151]: (a) The nitrile moiety of quinolone is converted to the intermediate hydroxyamidine through reaction with hydroxyl amine; (b) the resulting hydroxyamidine intermediates are converted to a variety of 1,2,4-oxadiazole target compounds by reacting with appropriate carboxylic acids to obtain product 5.

Reagents and conditions: (a) CH₃CN, reflux (b) NH₂OH, DIEA, aq. EtOH; (c) HBTU, DIPEA, 200 °C, microwave.

Structure–Activity Relationship of 3-(1,2,4-oxadiazole)-quinolone derivatives: The activity (IC₅₀) against the NS5B polymerase enzyme (Table 6) was determined by scintillation proximity assays (SPA) [152]. For a high-throughput screening (HTS) campaign to identify inhibitors of NS5B polymerase, a scintillation proximity assay (SPA) format was employed. Scintillation proximity assays (SPAs) are an efficient technique that can be used to detect enzymes, receptors, radioimmunoassays and molecular interac-tions. This assay format allowed for the screening of compounds that effectively inhibited the enzymatic function of NS5B polymerase, specifically targeting the wild-type (genotype 1b) enzyme. The IC₅₀ values of the compounds were assessed against NS5B polymerase, and their efficacy was determined in using a cell-based viral replication surrogate assay called the replicon system [152]. R¹ and R² mainly stabilize the compounds in the hydrophobic pockets. The inhibition activity of these two functionalities present in the quinolone moiety is shown in Table 6 [153]. It is clearly found that the presence of the –F or –CF₃ group in either R¹ or R² is very much responsible for modulating the corresponding activity [153].

Table 6.

SAR analysis of 3-(1,2,4-oxadiazole)-quinolone derivatives.

Compound	Ubstitution		Activity
	−R1	−R2	IC50 (μM)
	4-Cl-C6H4	4-F4-C6H4	0.019
	4-Cl-C6H4	2,4-DiF-C6H3	0.075
	4-CF3-C6H4	C6H5	0.024
	2-F,4-CF3-C6H3	C6H5	0.014
	2-F,4-CF3-C6H3	4-F4-C6H4	0.015

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Mechanism of Action of 3-(1,2,4-oxadiazole)-quinolone derivatives: 3-(1,2,4-oxadiazole)-quinolone derivatives show promising inhibitory activity against HCV NS5B polymerase and NS3 protease. Cyclophilin, a protein present in the host cell of HCV, also is affected by the interaction of such heterocyclic compounds; consequently, the replication process of this virus becomes affected. By targeting multiple stages of the HCV life-cycle, these compounds can reduce the amount of virus and slow or stop the progression of HCV-related liver diseases [154].

5.2.3. Anti-HCV Agent Grazoprevir

Grazoprevir (Figure 14) is a potent inhibitor of RNA synthesis in HCV (due to the action of two different DAAs as NS5A and NS3/4A inhibitors), representing high genetic barriers to resistance. The mechanism of action and pharmacodynamic properties, as well as the pharmacokinetics, clinical uses, safety and efficacy of elbasvir/grazoprevir in managing a large variety of conditions, including cases in the presence of cirrhosis, co-infection with HIV and patients having inherited blood disorders, were nicely reviewed by Kassas et al. [155]. Sofosbuvir [156], ledipasvir [157] and telaprevir [158] are the reported anti-HCV drugs with similar synthetic procedures.

Key synthetic steps for Scheme 6 [159]: Grazoprevir was synthesized starting from the (a) cross coupling strategy of 2,3-dichloro-6-methoxyquinoxaline with substituted proline derivative to obtain the corresponding five membered heterocyclic core followed by (b) esterification of the (1R,2R)-2-(pent-4-yn-1-yl)cyclopropan-1-ol with (S)-2-amino-3,3-dimethylbutanoic acid and (c) metal catalyzed coupling reactions of the fragments, followed by (d) cyclization through intramolecular peptide coupling (e). The final product 7 was obtained by the peptide coupling with the allylic sulfonamide, as shown in Scheme 6.

Reagents and conditions: (a) DBU (1.05 equiv.), DMAc, 50 °C; (b) CDI, Hunig’s base, 95 °C, 2.5 h; (c) Pb(OAc)₂, P(t-Bu)₃BF₄, K₂CO₃, CPME/MeCN; (d) (1) Pd/C, H₂, IPAc/MeOH; (2) (i) PhSO₃H, (ii) HATU, NEt₃, MeCN; (e) DEC, pyridine, MeCN.

Structure–Activity Relationship of Grazoprevir: A group of linear HCV NS3/4A protease inhibitors was created by removing the macrocyclic linker found in grazoprevir. This allows for the exploration of diverse quinoxalines while conferring conformational flexibility. Inhibitors with small substituents at the 3-position (R¹) of quinoxaline were found to be effective in maintaining potency. The 3-chloroquinoxaline demonstrated outstanding potency against wild-type HCV NS3/4A protease. Replacing the cyclopropyl-sulfonamide with a more hydrophobic 1-methyl cyclopropyl-sulfonamide group (R²) generally enhances the potency of the resulting analogues. Similarly, substituting the tert-butyl group (R³) with a bulkier cyclopentyl moiety led to the development of compounds with improved potency (Table 7) [160]. The enzyme inhibition constants (K_i) were determined for the wild-type genotype 1a NS3/4A protease, as well as the resistant variants R155K and D168A. Additionally, a subset of compounds underwent testing to determine their cellular antiviral potencies (EC₅₀) using replicon-based antiviral assays. These assays, which assessed the efficacy of the compounds, were not only run against the wild-type HCV strain but also against the drug-resistant variants R155K, A156T, D168A, and D168V [161].

Table 7.

SAR Analysis of grazoprevir derivatives.

Compound	Substitution			Activity
	−R1	−R2	−R3	EC50 (nM)	Inhibitor Constant (Ki) (nM)
				24	19 ± 2.7
				6.6	7.8 ± 1.1
				6.3	6.1 ± 1.1
				10	9.2 ± 0.9
				4.5	7.1 ± 1.1
				3.1	3.9 ± 0.7

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Mechanism of Action of Grazoprevir: Grazoprevir is a potent and selective inhibitor of the NS3/4A protease enzyme in the hepatitis C virus (HCV). The NS3/4A protease enzyme plays a critical role in HCV replication by cleaving the HCV polyprotein into the individual functional proteins necessary for the virus to replicate and propagate. Grazoprevir’s mechanism of action has been extensively studied and documented. Grazoprevir effectively inhibited the NS3/4A protease enzyme by binding to the enzyme’s active site, thereby preventing the cleavage of the HCV polyprotein and inhibiting HCV replication [162]. There are other drugs available on the market for the treatment of HCV, such as boceprevir, sofosbuvir, etc. Their mechanisms of action, ways of use and side effects are given in Table 8.

Some other synthesized compounds that show activity against HCV are given in Table 9.

Table 9.

Synthesized anti-HCV compounds.

Sl. No.	Antiviral Agent	Drug Target	Activity
1.		Hepatitis C Virus (HCV) [167]	The fatty acid synthase inhibitors showed good antiviral activity in a cell-based HCV replicon assay and an acceptable selectivity index. Their observed cell permeability in an MDCK permeability assay supports these findings. The antiviral activities align with the biochemical inhibition (IC50 values > 30 μM) of Hfasn [168].
2.		HCV [169]	Imidazo[2,1-b]thiazole targets HCV NS4B, specifically the second amphipathic α helix (4BAH2). The effectiveness of the compound against Huh-7 cells was assessed, screening an EC50 value of 18 nM [170].
3.		HCV [171]	The efficacy of the compound against Huh-7 cells shows promising activity, which reflects in the EC50 value (16 nM).

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5.3.1. Anti-HBV Agent Lamivudine

Lamivudine (Figure 15) is a nucleoside reverse transcriptase inhibitor that inhibits the reverse transcriptase of the human hepatitis B virus (HBV). It is a safe medicine with minimal side effects and can be prescribed for pregnant women and children over five years of age [172].

Key synthetic steps for Scheme 7 [173,174]: Formation of mixture of diastereomer at 0 °C (b) separation of diastereomers is done by recrystallization, (c) diastereo pure compound is treated with methanolic K₂CO₃ to obtain product 6.

Reagents and conditions: (a) Reactants are mixed and cooled to 0 °C in MeOH; (b) separation of diastereomers; (c) MeOH, K₂CO₃.

Mechanism of Action of Lamivudine: Lamivudine is a nucleoside analogue that is used in the treatment of hepatitis B virus (HBV) infection. The mechanism of action of lamivudine is based on its ability to inhibit HBV reverse transcriptase, which is a critical enzyme for viral replication. Once inside the infected cell, lamivudine is phosphorylated by cellular enzymes into its active triphosphate form. This active form of lamivudine competes with the natural nucleotide building blocks for incorporation into the growing viral DNA chain. However, lamivudine lacks the 3′-OH group required for further chain extension, thereby resulting in the termination of viral DNA synthesis [175].

5.3.2. Anti-HBV Agent Entecavir

Entecavir (Figure 16) is a guanosine nucleoside analogue active against hepatitis B (HBV). It is highly efficient in preventing all stages of replication. Compared to the other Hepatitis B drugs, lamivudine and entecavir are more effective; the corresponding triphosphate binds with HBVpol with amino acid residues ARG A: 23, LYS A: 14, ASN A: 18 and ALA A: 68 and effectively inhibits its activity (Figure 17) [176,177].

Key synthetic steps for Scheme 8 [178]: (a) Protection of aliphatic alcohol; (b) activation of the terminal alkyne; (c,d) synthesis of the epoxide followed by (e) intramolecular cyclization; (f,g) protection and deprotection of the alcohols; (h) Mitsunobu reaction with 2-amino-6-chloropurine; followed by (i) acid treatment; and (j) saponification to obtain the Entecavir 28.

Reagents and conditions: (a) TBSCI (1.1 equiv.), imidazole, THF, rt; (b) K₂CO₃ cat., MeOH; (c) m-CPBA, CH₂Cl₂; (d) Ac₂O, NEt₃, DMAP cat., CH₂Cl₂; (e) Cp₂TiCl₂ 20 mol%, IrCl(CO)(PPh₃)₂ 10 mol%, Mn (2 equiv.), collidine, TMSCl, H₂ (4 bar), THF; (f) p-O₂NBzCl, NEt₃, CH₂Cl₂; (g) 5% (+)-CSA, MeOH; (h) 2-amino-6-chloropurine, DIAD, PPh₃, THF, −10 °C; (i) HCOOH, 50 °C; (j) MeONa, MeOH.

Structure–Activity Relationship of Entecavir: An extensive investigation of the structure–activity relationship (SAR) of entecavir and its analogues is shown in Table 10. It was discovered that these compounds are the most potent inhibitors of HBV replication, with the ability to effectively inhibit lamivudine-resistant HBV. These compounds are carbocyclic guanosine nucleoside analogues (R¹) and are highly effective when tested against HBV in HepG2.2.15 cells [179]. The plasma half-life of entecavir in rats and dogs is 4–9 h. It is metabolized by HepG2 cells to the corresponding mono-, di-, and triphosphates.

Table 10.

SAR analysis of entecavir derivatives.

Compound	Substitution		Activity
	−R1	−R2	IC50 (μM)
			0.03
			0.128
			>100

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Mechanism of Action of Entecavir: Entecavir is a nucleoside analogue that inhibits hepatitis B virus (HBV) DNA replication by interfering with the activity of viral polymerase, an enzyme essential for the virus to replicate its genetic material. In HBV-infected cells, it is phosphorylated into its active form, entecavir triphosphate, which competes with the natural substrate, deoxyguanosine triphosphate, for incorporation into the elongating viral DNA chain. The incorporation of entecavir triphosphate into the viral DNA chain leads to chain termination, preventing further extension of the viral DNA and ultimately inhibiting HBV replication. Entecavir’s mechanism of action has been extensively studied and has been shown to be highly effective in suppressing HBV replication and reducing liver damage. Due to its high potency and low risk of developing viral resistance, entecavir has become one of the preferred first-line treatments for chronic HBV infection [180,181].

5.3.3. Anti-HBV Agent Dehydro-Andrographolide and Andrographolide Derivatives

Dehydro-andrographolide and andrographolide compounds have demonstrated the ability to inhibit the replication of HBV DNA, with IC₅₀ values of 22.58 and 54.07 μM, respectively [172].

Key synthetic steps for Scheme 9 [172]: (a) Compounds are obtained with the help of esterification reaction with acids in the presence of 4-dimethylaminopyridine (DMAP) and N′,N′-dicyclohexylcarbodiimide (DCC) in anhydrous dichloromethane to obtain the product 35.

Reagents and conditions: (a) corresponding acids, DMAP, DCC, CH₂Cl₂, rt.

Structure–Activity Relationship of dehydro-Andrographolide: The SARs of the derivatives indicate that having a free hydroxyl group at C-2 can result in enhanced anti-HBV properties. Additionally, maintaining the double bond between C-8 and C-17, as well as the conjugated double bonds between C-11 and C-14, or C-12 and C-15, is crucial for preserving anti-HBV activity and decreasing cytotoxicity. To improve the anti-HBV activity, it is useful to incorporate the 3-methoxycinnamoyl, nicotinoyl, 2-furoyl, or 2-thenoyl groups shown in Table 11 [105]. The anti-HBV activity of the compounds derived from dehydro-andrographolide and andrographolide were investigated. Specifically, their ability to inhibit the secretion of HBsAg and HBeAg, as well as HBV DNA replication, was evaluated using HepG 2.2.15 cells in vitro. Tenofovir, a known antiviral agent, was used as the positive control in the study [182].

Table 11.

SAR analysis of dehydro-andrographolide and andrographolide derivatives.

Compound	Substitution	Activity
	−R	IC50 (μM)
		18
		1970
		207
		40
		2313
		19.2
		162
		210
		1137
		121
		880
		280
		565
		1317

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Mechanism of Action of dehydro-Andrographolide: Dehydro-andrographolide inhibits HBV replication by blocking the binding of the HBV core protein to viral RNA. It also activates the host immune response, which can help control HBV replication and clear infected cells [183]. Overall, dehydro-andrographolide has shown promising anti-HBV activity through its ability to inhibit viral replication and enhance the host immune response. However, further studies are needed to fully understand its mechanisms of action and potential clinical applications [184]. The details of other anti-HBV drugs, along with their mechanisms of action, are mentioned in Table 12.

Some other synthesized compounds with activity against HBV are tabulated below (Table 13).

Capsid assembly modulators (CpAMs) belong to a novel category of antiviral compounds that specifically target the core protein of the hepatitis B virus (HBV) to interfere with the assembly process. HepG2.2.15 cells are a type of human hepatoblastoma cells that have been genetically modified to stably express the hepatitis B virus (HBV). The compounds under investigation inhibit HBV replication by interfering with the assembly of the HBV capsid protein [188].

Table 13.

Synthesized anti-HBV compounds.

Sl. No.	Antiviral Agent	Drug Target	Activity
1.		HBV [189]	Capsid assembly modulators (CpAMs) are antiviral compounds that target the core protein of the hepatitis B virus (HBV) to disrupt assembly. In HepG2.2.15 cells, which express HBV, these compounds inhibit HBV replication by interfering with capsid protein assembly with EC50 = 511 nM.
2.		HBV [190]	The anti-HCV activities were tested in the Huh-Luc/neo cell line and cytotoxicity of the test compound was determined on both MT-2 cell lines with EC50 = 10 µM.

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Anti-RSV Agent Ribavirin

The antiviral property of this drug (Figure 18) was studied in 1972 [191]. This drug is presently used for the treatment of RSV. DeVincenzo et al. have studied the inhibitory activities of ribavirin against the RSV F-protein [192].

Key synthetic step for the Scheme 10 [193]: (a) Nucleophilic substitution in the presence of bio-catalyst, (b) amide formation in the presence of NH₃ to obtain product 10.

Reagents and conditions: (a) Purine nucleoside phosphorylase, buffer solution of pH = 7; (b) NH₃, MeOH.

Mechanism of Action of Ribavirin: Ribavirin is a broad-spectrum antiviral agent that has activity against a range of RNA and DNA viruses, including respiratory syncytial virus (RSV). The mechanism of action of ribavirin is not completely understood, but it is believed to involve several different mechanisms [194]. One proposed mechanism is that ribavirin interferes with the synthesis of viral RNA by inhibiting the activity of the viral RNA-dependent RNA polymerase. Another proposed mechanism is that ribavirin induces mutations in the viral genome, leading to non-functional or less-virulent viral particles. Additionally, ribavirin has been shown to stimulate the host’s immune response, which may contribute to its antiviral effects [195,196,197]. RSV-IGIV and palivizumab are the available drugs on the market for the treatment of RSV. Their mechanisms of action, ways of use and side effects are given in Table 14.

Some other synthesized compounds that show activity against RSV are given in Table 15.

Table 15.

Synthesized anti-RSV compounds.

Sl. No.	Antiviral Agent	Drug Target	Activity
1.		Respiratory syncytial virus [201]	The anti-RSV property of the compound tested with Hep2 cells with IC50 = 1.2 nM. The cell viability was measured using MTT reagents and cell cytotoxicity was evaluated through parallel assessments with plaque reduction assays.
2.		Respiratory syncytial virus [202]	The screening of the compounds against nonattenuated respiratory syncytial virus was done by high-throughput protocol with EC50 = 0.36–0.55 μM.

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5.5.1. Anti-HCMV Agent Iso-Valganciclovir Hydrochloride

Iso-Valganciclovir hydrochloride (Figure 19) is used for the treatment of cytomegalovirus (CMV). It is a type of nucleoside analogue and is the cutting-edge drug candidate against CMV [203].

Key synthetic step for Scheme 11 [204]: (a) Addition of reaction of 3-(chloromethoxy)prop-1-ene to o-benzyl guanine in the presence of a base, followed by oxidation; (b) addition reaction with S-2-azido-3-methylbutanoic acid and further reduction gives the final product 21, as shown in Scheme 11.

Reagents and conditions: (a) (i) NaH, DMF; (ii) KMnO₄, acetone; (ii) 10% Pd/C, MeOH; (iv) (S)-2-azido-3-methylbutanoic acid, DCC, DMSO; (b) 10% Pd/C, MeOH.

Mechanism of Action of Iso-Valganciclovir Hydrochloride: Iso-Valganciclovir hydrochloride is a prodrug of the antiviral agent ganciclovir, which is converted to its active form by hydrolysis of the valine ester in the liver and blood. The active form of ganciclovir works by inhibiting the viral DNA polymerase, which is essential for the replication of HCMV. By inhibiting the viral DNA polymerase, ganciclovir prevents the formation of new viral DNA chains, which ultimately inhibits HCMV replication [205].

5.5.2. Anti-HCMV Agent Ganciclovir

Ganciclovir (Figure 20) is a marketed drug for the treatment of HCMV; it acts as a DNA polymerase to inhibit synthesis of viral DNA [206].

Key synthetic steps for Scheme 12 [207]: (a) N-Acylation of the guanine then (d) reacts with 2-(acetoxymethoxy)propane-1,3-diyl diacetate to form N-(9-(((1,3-dihydroxypropan-2-yl)oxy)methyl)-6-oxo-6,9-dihydro-1H-purin-2-yl)acetamide (prepared from 4-(chloromethyl)-1,3-dioxolane) (e) by the deprotection of the amine and alcohol group; the final product 22 was thereby obtained.

Reagents and conditions: (a) Ac₂O/HOAc, 140 °C; (b) Ac₂O/HOAc/ZnCl₂, r.t.; (c) KOAc/DMF, 150 °C; (d) EtSO₃H, 165–170 °C; (e) 40% aq. MeNH₂, 75 °C.

Mechanism of Action of Ganciclovir: Ganciclovir is an antiviral drug used to treat HCMV infections. It stops viral DNA synthesis by acting as a chain terminator, which inhibits the elongation of the viral DNA strand. Ganciclovir triphosphate, its active form, is similar to guanosine and is selectively toxic to infected cells as it is preferentially incorporated into viral DNA, reducing viral replication and controlling infections [206].

5.5.3. Anti-HCMV Agent 1,2,4-Triazol-Quinoxalin Derivative

Another potential anti-HCMV agent is represented by quinoxaline derivatives, which have been found in recent research studies. Quinoxaline is a heterocyclic compound containing a benzene ring fused to a pyrazine ring, and its derivatives have diverse biological activities, including antiviral properties. These compounds have exhibited greater antiviral activity against HCMV compared to the standard drug ganciclovir [208]. The triazole and quinoxaline moieties in 1,2,4-triazoloquinoxaline (Figure 21) have been reported to exhibit antiviral activity against HCMV. The triazole moiety is a five-membered heterocyclic ring containing three nitrogen atoms, which has been reported to possess antiviral activity. The quinoxaline moiety is a bicyclic aromatic ring system that has also been reported to exhibit antiviral activity. Studies have shown that 1,2,4-triazoloquinoxaline derivatives can inhibit HCMV replication by targeting the viral DNA polymerase, which is a key enzyme involved in viral replication. These compounds have also been reported to have low cytotoxicity toward human cells, making them potentially useful as antiviral agents [209].

Key synthetic steps for Scheme 13 [210]: (a) The compound 2-(6,7-dimethyl-3-oxo-3,4-dihydroquinoxalin-2-yl)acetohydrazide was reacted with triethylorthoformate in ethanol to afford ethyl [(6,7-dimethyl-3-oxo-3,4- dihydroquinoxalin-2-yl)acetyl]hydrazonoformate; further, (b) treatment of the hydrazonoformate with 2-aminopyridine in acetic acid reflux afforded 6,7-dimethyl-3-{[4-(pyridin-2-yl)- 4H-1,2,4-triazol-3-yl]methyl}quinoxalin-2(1H)-one to obtain the product 19.

Reagents and conditions: (a) C₂H₅OH, rt; (b) CH₃COOH, reflux.

Mechanism of Action of 1,2,4-triazol-quinoxalin Derivatives: 1,2,4-triazol-quinoxalin derivatives have potential as antiviral agents against HCMV, but their exact mechanism of action is not fully understood. They may inhibit viral DNA replication and interfere with viral gene expression or virion assembly. Additionally, they may have immunomodulatory effects that enhance antiviral activity or inhibit immune evasion strategies against HCMV [211]. Valganciclovir is another available drug for the treatment of HCMV. The mechanism of action, ways of use and side effects of valganciclovir and ganciclovir are given in Table 16.

Some other synthesized compounds that show activity against HCMV are given in the Table 17.

Table 17.

Synthesized anti-HCMV compounds.

Sl. No.	Antiviral Agent	Drug Target	Activity
1.		HCMV [214]	Synthesized trichlorinated indole nucleosides were tested for activity against human cytomegalovirus (HCMV). Cytotoxicity was assessed using two methods: microscopic inspection of uninfected HFF cells [215] and crystal violet staining with spectrophotometric quantitation in KB cells [216]. The IC50 was found to be 0.23 µM.
2.		HCMV [217]	The compound was assayed for antiviral activity against HCMV (Davis, VR-807) cells in a cytotoxic assay. The EC50 value of 53.1 µM showed only moderate antiviral activity against HCMV.

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