Recent advances in the piperazine based antiviral agents: A remarkable heterocycle for antiviral research

3.1 Anti-HIV activity of piperazine derivatives

Piperazine derivatives demonstrate a variety of antiviral activities (Fig. 2). Idoxuridine was the first antiviral drug licensed in 1963 and after that ninety (90) antiviral drugs have been approved which comprise 13 different functional groups (De Clercq and Li, 2016). HIV is a retrovirus which persists all over the map and is communicated by tainted blood transfusion, unprotected coupling and hypodermic syringes. In 2021, there were 38.4 million victims of HIV with 1.5 million new patients https://www.unaids.org/en/resources/fact-sheet. Presently, there is no licensed vaccine for HIV prevention. Anti-HIV agents reduce the dissemination of HIV infection and improve the health of HIV infected individuals (Cohen et al., 2011). Current HIV treatments comprise a combination of therapeutics that target viral reverse transcriptase, protease enzymes and viral entry. However, the existing therapeutic agents are associated with major side effects and HIV can develop resistance against this agent. Therefore, there is an immediate demand for the addition of novel antiviral agents with a distinct mode of action. In 2020, Jin et al. designed novel series of non-nucleoside HIV-1 reverse transcriptase inhibitors comprising biphenyl-substituted diaryltriazine core. The 4-cyanobiphenyl and 4-cyano aniline group are present in all derivatives (Jin et al., 2020). The synthesized compounds exhibited significant activity against both the wild type (WT) HIV-1 and a panel of HIV-1 mutant strains. However, the piperazine-based derivatives (10) and (11) (Fig. 3) were found to have excellent anti-HIV-1 IIIB wild-type activities with EC₅₀ values of 0.0033 ± 0.6 µM and 0.005 ± 1.0 µM, respectively with reference to nevirapine (EC₅₀ = 0.0045 ± 2 µM), efavirenz (EC₅₀ = 0.001 ± 0.3 µM) as standard drugs. Compounds (12) and (13) also presented prominent activity against WT HIV-1 reverse transcriptase with IC₅₀ values of 0.242 ± 0.020 µM and 0.368 ± 0.021 µM, respectively. In derivatives 10 and 11, the 4-cyanobiphenyl group is attached to triazine ring through the nitrogen bridge. In the case of derivatives 12 and 13, the 4-cyanobiphenyl group is connected to the triazine ring through the oxygen bridge. Sun et al. designed, synthesized and studied the HIV-1 capsid inhibition activity of novel benzenesulfonamide-containing phenylalanine derivatives. Derivatives having 2-piperazinone scaffold exhibited prominent anti-HIV activity. The phenylalanine derivative (14) having 2-piperazinone moiety was found to have 5.78-fold better anti-HIV activity (EC₅₀ = 0.09 ± 0.03 μM) in comparison to the reported standard PF-74 (EC₅₀ = 0.52 ± 0.18 μM). Derivative 14 presented a selectivity index>383.36. Derivative 14 carries a p-amino-substituted benzenesulfonyl group. Compound 14 also showed beneficial pharmacokinetic properties such as oral bioavailability and were found non-toxic till a dose of 1000 mg/kg (Sun et al., 2020). Esposito and co-researchers explored dihydroxyindole-2-carboxylic acid derivatives as blockers of reverse transcriptase associated ribonuclease (RNase) and HIV-1 integrase. Among all synthesized compounds, the bis-propyl-piperazine linker containing symmetric compound (15) exhibited significant inhibition (IC₅₀ = 7.0 ± 0.1 μM) of HIV-1 integrase (IN) activity. The dioxole analogue of derivative 15 was found to be inactive (IC₅₀ > 100 μM) (Esposito et al., 2020). Huang and his team discovered potent diarylpyrimidine-type derivatives in which heterocyclic rings such as piperazine, piperidine and morpholine were introduced. The HIV-1 inhibitory activity as a non-nucleoside reverse transcriptase inhibitor (NNRTI) against wild-type and E138K mutant virus was carried out. These derivatives presented prominent potency towards WT HIV-1 strain upon evaluation by anti-HIV assay. However, the piperazine containing compound (16) was found as the most agent (EC₅₀ = 0.0035 µM) with prominent selectivity index (SI < 48774). Most importantly the compound (16) also demonstrated excellent activity against resistant virus strain E138K with an EC₅₀ value of 0.0075 µM. Compound 16 also exhibited better water solubility (30.92 ± 0.02 µM) which makes it a strong candidate for the future drug (Huang et al., 2019). In 2018, Jin et al. synthesized a series of substituted piperazine-1-yl-pyrimidine derivatives as HIV-1 non-nucleoside inhibitor. The antiviral activity was evaluated as reverse transcriptase inhibitors in MT-4 cells. The results revealed inhibitory activities of 17 (EC₅₀ = 31.5 µM) and 18 (EC₅₀ = 3.36 µM) analogs against wild-type strain. In molecular docking studies of compound 17 with binding pocket of non-nucleoside reverse transcriptase (NNIBP), the biphenyl group interacted with aromatic rich pocket. The pyrimidine ring was involved in hydrogen bond formation with NH group of Lys10. The nitro group is also involved in hydrogen bond interactions. The piperazine ring increased the flexibility and lipophilicity of these derivatives (Jin et al., 2018).

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Fig. 2

Antiviral activities of piperazine derivatives.

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Fig. 3

Anti-HIV agents having piperazine core.

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In 2017, Zhou et al. carried out the structural modification of a novel viral infectivity factor (Vif) antagonist and reported that piperazine derivative 19 (Fig. 4) has good anti-HIV activity (EC₅₀ = 8.24 µM) by inhibiting Vifin non-permissive H9 cells. But derivative 19 was found more cytotoxic (CC₅₀ = 30.6 µM) in this series and showed less selectivity index (3.71) (Zhou et al., 2017). In 2015, Rivero-Buceta studied the anti-HIV activity of different compounds and also the mechanism of anti-viral activity. These derivatives contain 2,4,6-triethylbenzene as a central scaffold. Studies reported that piperazine containing compound (20) demonstrated the HIV-1 integrase inhibitory and gp120 binding capacity in the moderate range having EC₅₀ value>10 µM. The piperazine ring served as a linker and 2,3,4-trihydroxybenzoyl group is attached at position 1,3,5 of the triethylbenzene through the piperazine linker group (Rivero-Buceta et al., 2015). Zhao et al. discovered the novel N-aryl piperazine tetrahydroquinoline derivatives as chemokine receptor (CXCR4) inhibitors. Compounds (21) and (22) emerged as the most potent derivatives against HIV1 with the IC₅₀ values of 0.02 µM in HIV-1 repression assay. These derivatives contain benzoyl group and phenyl sulfonyl group attached with piperazine ring (Zhao et al., 2015). Ashok and co-workers synthesized and evaluated the β-carboline derivatives against HIV-1 viral strain. These compounds consist of central pyridoindole scaffold and different derivatives were synthesized by altering the phenyl substituent of piperazine ring. Among the reported analogues, the compound (23) demonstrated significant activity (EC₅₀ = 0.53 µM) as an inhibitor of syncytia formation and decent selectivity index (SI = 483). Compounds (24), (25) and (26) exhibited moderate activities presenting EC₅₀ values of 3.8 µM, 3.8 µM and 2.8 µM and selectivity index values > 105, >105 and 3.85 respectively. Compound (23) also showed inhibition of p24 antigen expression in acute HIV-1_IIIB infected cell line presenting an EC₅₀ value of 1.1 µM (Ashok et al., 2015). Derivatives in this series followed the Lipinski’s rule of five with minor violations (Lipinski, 2004).

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Fig. 4

Piperazine derivatives as anti-HIV agents.

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Chen et al. synthesized piperazine-based triazine derivatives (27), (28), (29) and (30) (Fig. 5) and also evaluated them against wild-type and resistant strains of HIV-1 (K103N/Y181C). Derivative (29) presented EC₅₀ values of 0.030 ± 0.0035 µM and ≥ 1.2 µM against wild-type and K103N/Y181C strains of HIV-1 in the MT-4 cell line (Chen et al., 2015). Hu et al. used fragment linking technique to design and synthesize novel 2-methylpiperazine analogues as CCR5 antagonist. Promising antiviral activities were observed for piperazine containing derivatives (31–34) having IC₅₀ values of 0.0472 µM, 0.146 µM, 0.0314 µM and 0.0751 µM, respectively with reference to maraviroc (IC₅₀ = 0.0055 µM). Derivative 31 carries 4-cyanobenzyl group while 32–34 carry 4-fluorobenzyl group attached at the position 4 of the piperazine group. Substitution of 4-fluorobenzyl by methyl benzyl or simple benzyl group produced less active derivatives. These derivatives in the concentration of 10 μM exhibited no cytotoxicity upon evaluation by CCK-8 assay (Hu et al., 2015). Tuyishime and coworkers used virtual blaze screening procedure to analyze million available compounds. Fifty derivatives were scrutinized for anti-HIV potential by using viral inhibition assays. The compound (35) appeared as the most potent against HIV-1_YU-2 displaying an IC₅₀ value of 13.1 ± 1.7 µM. The compound (35) contains a piperazine ring connected to acenaphthene group. The replacement of piperazine with di-pyrrolidine produced weakly active derivatives. Then, the acenaphthene scaffold was modified to produce more active derivatives (Tuyishime et al., 2014). In 2013, T. Liu et al. designed and synthesized new piperazine derivatives by using fragment linking and bioisosteric principles. Among different evaluated compounds, compound (36) was found the most potent anti-HIV-1 inhibitor (IC₅₀ = 0.44 µM) while other piperazine containing derivatives showed inferior activities. Derivative 36 also showed CC5 antagonistic activity having an IC₅₀ value of 6.29 µM (T. Liu et al., 2013).

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Fig. 5

Piperazine derived compounds as anti-HIV agents.

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Serrao et al. discovered the new 5-carbonyl-1H-imidazole-4-carboxamide analogues as HIV-1 inhibitors. Various aromatic rings and amines were introduced at positions 4 and 5. Piperazine containing derivatives (37 and 38) (Fig. 6) showed excellent activities towards the inhibition of integrase lens epithelium-derived growth factor (IN-LEDGF/p75) and HIV-1 with IC₅₀ values>20 µM (Serrao et al., 2013). Yeung et al. synthesized of indole-7-carboxamides and evaluated their HIV-1 inhibition capabilities. The piperazine containing indole derivatives were found as potent antiviral agents. The analogues (39–43) displayed promising in vitro antiviral activities with the EC₅₀ values of 5.2 × 10^-4 µM, 2.9 × 10^-4 µM, 2 × 10^-5 µM, 5.8 × 10^-6 µM, and 4 × 10^-5 µM, respectively. Derivatives 39–41 emerged as most promising because they were less toxic with CC₅₀ values>300 µM. While derivatives (42) and (43) are more toxic presenting CC₅₀ values of 29 µM and 9.2 µM, respectively. Derivative 39 exhibited better oral bioavailability and human liver microsomal stability (Yeung et al., 2013). Zentner et al. reported the piperazine derivatives as HIV-1 matrix (MA) protein inhibitors. The compound (44) came out as the most active inhibitor of HIV-1 MA with IC₅₀ values in the range of 7.5 to 15.6 µM against different subtypes. This compound competes with phosphatidylinosityl 4,5-biphosphate (PI[4,5]P2) for MA protein binding. The compound 44 interacted with HIV-I matrix protein presenting a Kd value of 22.6 ± 3.1 μM (Zentner et al., 2013). In 2012, Ali et al. reported the synthesis of benzamide analogues and also evaluated HIV-1 inhibition studies. Upon evaluation of antiviral activities, the piperazine containing analogue (45) showed prominent antiviral potential against H9 and MT4 cell lines having IC₅₀ values of 3.9 µM and 3.7 µM, respectively and presented CC₅₀ value of 70 µM. Compound 45 carries a piperazine ring attached with ring B through a carbonyl group. Derivatives having thioether linkage between ring A and B showed better activity. Similarly the amide or sulfonamide group was found effective between ring B and C (Ali et al., 2012).

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Fig. 6

Piperazine derivatives as anti-HIV agents.

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Dong et al. designed and synthesized novel 1,4-disubstituted piperidine/piperazine analogues and also evaluated antiviral activities against HIV-1 Bal (R5) in the CEMX174 5.25 M7 cells. Most of the synthesized derivatives demonstrated potent anti-HIV-1 activities showing IC₅₀ values. The piperazine hydrochloride analog (46) (Fig. 7) exhibited promising anti-HIV-1 activity (IC₅₀ = 0.00617 µM) and lower cytotoxicity (CC₅₀ < 100 µM). The compound (46) has also much higher solubility (25 mg/mL at 25 °C) and shorter plasma half-life (0.76 h) than the reference drug TAK-220 hydrochloride (2 mg/mL at 25 °C). The derivative (47) also showed excellent (IC₅₀ = 0.00185 ± 0.17 µM) anti-HIV-1 activity than the reference TAK-220 but showed higher cytotoxicity (CC₅₀ = 30.47 ± 7.99 µM) which obstruct it as a potential drug candidate. Derivatives 46 and 47 carry 4-fluorobenzyl and 4-trifluoromethyl benzyl fragments attached with piperazine. Replacement of benzyl group by benzoyl produced less active derivatives (Dong et al., 2012). Solyev et al. synthesized novel 5′-carbamate prodrugs of 3′-azido-3′-deoxythymidine (AZT) and also evaluated their anti-HIV properties. The piperazine-based analogue (48) of AZT showed moderate viral activity in MT-4 cell culture with an EC₅₀ value of 20 µM. This prodrug was stable and showed>24 h half-life (Solyev et al., 2012). Venkatraj et al. synthesized triazine dimers as anti-HIV agents. Alkylidenediamine, arylendiamine and piperazine act as linkers in compounds (49) and (50). Piperazine containing triazine dimers (49) and (50) have shown moderate antiviral activities against HIV-1 in TZM-b1 cells with EC₅₀ values of 6.07 µM and 3.19 µM respectively in comparison to standard compound TMC120 (EC₅₀ = 0.002 μM). Derivative 49 is a heterodimer in which triazine monomers are carrying different group (Venkatraj et al., 2012). Tabarrini et al. synthesized a series of naphthyridone derivatives as anti-HIV agents. The piperazine containing analogue (51) showed a prominent anti-HIV (EC₅₀ > 0.08 µM) activity and good selectivity (SI ≥ 3707). Compound 51 carries a benzothiazole piperazine scaffold attached at position #7 of 1,6-naphthyridone scaffold. Therefore, shifting the position of naphthyridone nitrogen from 8 to 6 produced highly active derivatives (Tabarrini et al., 2011). Tabarrini et al. presented the synthesis of quinolones derivatives having vinyl and amino group at position #1. The compound (52) emerged as the most potent derivative against HIV-1 (EC₅₀ = 2.30 ± 0.30 µM, SI 〈1 2 9) and HIV-2 (EC₅₀ = 2.84 ± 1.82 µM, SI 〈1 0 4) and exhibited excellent selectivity. Derivative 52 carries a benzothiazole piperazine scaffold attached at position #7 of quinolone ring. The compound 52 bears an amino group at position #1 and the substitution of amino group by vinyl group produced active derivatives. But derivatives having the vinyl group showed more toxicities (Tabarrini et al., 2010). Xu et al. discovered the anti-HIV piperazinone phenylalanine derivatives. The compound 53 was found to be the most promising antiviral small molecule acting against HIV CA protein through in vitro approach. The compound 53 showed EC₅₀ value of 5.89 ± 2.03 µM, CC₅₀ value of 16.36 ± 3.38 µM and selectivity index ratio of 2.78. Molecular docking simulations acknowledged that the compound 53 had 20% probability of showing H-bond interactions with key residue Glu71 (Xu et al., 2022).

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Fig. 7

Anti-HIV agents possessing basic skeleton of piperazine.

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Orally bioavailable, CCR5 antagonists as HIV-1 inhibitors were discovered by Tagat et al. (2001). The compound 54 exhibited modest binding data for CCR5-RANTES with Ki value of 0.007 µM, and CCR5 binding towards muscarinic receptor with Ki value of 0.25 µM. The compound 54 inhibited the entry and replication of HIV-1 with IC₅₀ value of 0.001 µM. McCombie et al. (2003) reported the pharmacokinetic profile of piperazine-based CCR5 antagonists as HIV-1 inhibitors by inserting some modifications in the compound 54 reported by Tagat et al. The unsymmetrical nicotinamide-N-oxide moiety in compound 54 was replaced with symmetrical isonicotinamides as well as 4,6-dimethyl pyrimidine-5-carboxamides. The newly made compound 55 showed the CCR5 binding data with Ki value of 0.003 µM, and binding affinity of CCR5 towards muscurainic receptor was observed to be 0.456 µM. IC₅₀ value noted while entrying into the cell was 0.005 µM. Sriram et al. (2005) described the anti-HIV effect of some biologically active lamivudine prodrugs, The study reported that among the series of compounds, compound 56 displayed the better anti-HIV potential with EC₅₀ value; 1.11 ± 0.52 µM, CC₅₀ value; 123 ± 14.3 µM with selectivity index of 110. In 2009, Wang et al. reported a drug candidate that demonstrated the antiviral activity in HIV-1- infected subjects. The compound 57 containing azaindole moiety displayed the better results than others. The inhibitory studies carried out to evaluate the anti-HIV-1 activity for compound 57 were EC₅₀, CC₅₀ and IC₅₀ with 0.88 ± 0.46 µM, > 300 µM, and > 40 µM values, respectively (Wang et al., 2009). Jiang et al. (2023) discovered diarylpyrimidine derivatives bearining piperazine as potent HIV-1 non-nucleoside reverese transcriptase inhibitors. The compound 58 showed the most potent antiviral activity against HIV-1 by showing EC₅₀ value of 0.0014 ± 0.00019 µM and CC₅₀ value 10.15 ± 1.82 µM. (In silico studies are depited in Table 1).

The structure–activity relationship (SAR) of the studies reported here in context of piperazine-based anti-HIV derivatives, piperazine containing indole derivatives (39–43) showed the best anti-HIV activity among the other groups attached with the piperazine-moiety displaying EC₅₀ in the range of 5.8 × 10^-6 µM to 5.2 × 10^-4 µM. Other moieties which exhibited the excellent anti-HIV activity (EC₅₀) includes; diarylpyrimidine derivatives (16, 58), biphenyl-substituted diaryltriazine (10), benzenesulfonamide-containing phenylalanine (14), β-carboline derivatives (23), and azaindole moiety (57) in the range of 0.0014 µM to 0.88 µM.

3.2 Anti-hepatitis B virus activity of piperazine derivatives

Hepatitis B virus (HBV) is a double-stranded DNA enveloped virus which infects>250 million people worldwide. About 900,000 deaths are reported annually due to hepatitis B virus infection (Razavi-Shearer et al., 2018). The higher death rate is due to the complications of cirrhosis, hepatocellular carcinoma and end-stage liver disease associated with HBV (Trépo et al., 2014). Interferon (IFN)-α,adefovir dipivoxil [bis(POM)PMEA] and lamivudine (3TC) are formally approved as the treatment of hepatitis B infection. However, the treatment does not completely cure the virus and virus reappears in patients after the withdrawal of drug (Ghany, 2017). Side effects and development of resistance necessitate the discovery of novel anti-HBV agents (De Clercq, 2004). In 2020, Li et al. synthesized a series of amide-containing α-hydroxytropolones as potential compounds for HBV drug development. The compound 59 (Fig. 8) was the most active (EC₅₀ = 0.33 ± 0.13 µM) against HBV replication among the piperazine derivatives. The compound 59 showed higher cytotoxicity (CC₅₀ = 4.5 ± 1.4 µM) and lower selectivity index (SI = 13.7) which makes it a poor candidate towards drug development. However, the piperazine containing compound 60 was also a potent compound (EC₅₀ = 0.46 ± 0.22 µM) against hepatitis B virus replication based on cytotoxicity value (CC₅₀ < 30 µM) and selectivity index (SI < 80) (Li et al., 2020). Gerasi et al. designed and synthesized novel substituted imidazo[4,5-b]pyridines and evaluated their anti-HBV activities. HBV infectious system was used to determine the anti-HBV activity of the synthesized derivatives at the level of HBV rcDNA secretion. Monochlorosubstituted imidazo[4,5-b]pyridine derivatives presented better activity as compared to dichlorosubstituted derivatives. The piperazine containing derivative 61 (EC₅₀ = 25.31 ± 1.45 μM) expressed excellent anti-HBV activity but presented low selectivity (SI < 7.9) (Gerasi et al., 2020). In 2018, Ren et al. reported the morpholine and piperazine-based novel analogues as HBV capsid inhibitor. The piperazine containing compound 62 was found to have low anti-HBV activity (EC₅₀ < 16.4 µM) as compared to morpholine analogues which showed better anti-HBV activities (Ren et al., 2018). In 2014, Xu et al. screened benzimidazole derivatives and identified novel piperazine containing benzimidazole 63 as a potent antiviral agent. Derivative 63 demonstrated prominent inhibition of hepatitis B virus (EC₅₀ = 0.6 µM) and HBsAg secretion (EC₅₀ = 1.5 µM) accompanied by less toxicity (CC₅₀ = 24.5 µM). Furthermore, it does not activate cellular stress reaction or host protein secretion and therefore it might be hypothesized that it is an excellent compound for drug development (Xu et al., 2014). In 2011, P. Wang et al. synthesized substituted aryl propenamide analogues and also separated the Z and E isomers. The Z isomer was found to have potent antiviral activity against HBV. The piperazine containing derivative 64 has less anti-HBV activity with an EC₉₀ value of ˃10 µM as compared to the piperidine and pyrrolidine derivatives (P. Wang et al., 2011). Kiruthika et al. reported a novel piperazine-based derivative 65 against tenofovir resistant hepatitis B virus that targets the hepatitis B surface antigen (HBsAg) using computational and in vitro approaches. The compound 65 was screened virtually and predicted binding free energy against HBsAg using MMGBSA analysis, and the score found was −50.01 kcal/mol. The compound 65 shows hydrogen bond interaction with Cys76 and Ala45. The in vitro analysis was performed on the Huh7 cells and the IC₅₀ value was found to be 11.39 µM against tenofovir-resistant hepatitis B virus (Kiruthika et al., 2021). Tan et al. (2006) presented 2-benzenesulfonylalkyl-5-substituted-sulfanyl-[1,3,4]-oxadiazoles as potential anti-hepatitis B virus agents. Among the reported series of anti-HBV agents, compound 66 was seen to inhibit the expression of HIV antigens HBsAg and HBeAg. The compound 66 displayed the EC₅₀ value of 1.63 µM which showed the greater reduction in virion production compared to that of nucleoside analog 3TC showing EC₅₀ value of 2.96 µM.

Among the above-mentioned piperazine-based anti-HBV derivatives, amide containing α-hydroxytropolone derivatives (59) demonstrated the best anti-HBV activity among the other groups attached with the piperazine-moiety, with EC₅₀ values ranging from 0.33 µM to 0.46 µM. Other molecules with excellent anti-HBV activity (EC₅₀) include chalcone (65), oxadiazole (66), and pyrimidine derivative (62) in the range of 0.6 µM to < 16.4 µM.

3.3 Anti-hepatitis C virus activity of piperazine derivatives

Hepatitis C virus (HCV) is a single-stranded RNA virus and infection due to HCV results in chronic liver disease leading to liver fibrosis, cirrhosis and hepatocellular carcinoma. According to some estimates, there are 71 million patients of HCV all over the world (Younossi, 2017). Inspite of the availability of direct-acting antiviral agents (DAAs), the HCV can develop resistance (Li and Chung, 2019). Consequently, there is an instant demand for new anti-HCV drugs. In 2020, Hu et al. reported the mechanism of action of chlorcyclizine (CCZ) against hepatitis C virus. Chlorcyclizine 67 (Fig. 9) is an effective hepatitis C virus (HCV) entry blocker which blocks HCV E1 with EC₅₀ values of 0.024 µM and 0.020 µM for (S)-CCZ and (R)-CCZ isomers, respectively. In molecular docking studies, chlorcyclizine formed hydrophobic interactions with HCV E1 fusion loop involving phenyl and chlorophenyl groups of chlorcyclizine. The chlorcyclizine analogue 68 was found to be more potent (EC₅₀ = 0.002 µM) than chlorcyclizine (Hu et al., 2020). Jiang et al. synthesized benzonitrile analogues as HCV inhibitors. The activity was evaluated by measuring the HCV-RNA level in the Huh7.5 cell line by using the qRT-PCR method. The piperazine containing derivative 69 was found as the most prominent HCV inhibitor presenting an EC₅₀ value of 0.022 µM and selectivity index of 600.The alkylation of the amino group and halogen substitution at the para-position are beneficial for the activity of these derivatives. Further evaluation of the mode of action showed that this compound acts at the point of viral entry into the host and was found specific against HCV (Jiang et al., 2020). Makino et al. discovered piperazine containing anti-HCV agent 70 while investigating the derivatives of cyclosporine. Derivative 70 presented an EC₅₀ value of 0.22 µM but were found less active as compared to the carbamoyl derivatives in this series. But compound 70 presented less immunosuppressive activity as compared to other members in this series (Makino et al., 2020). In 2018, Rolt et al. synthesized and disclosed the in vitro anti-HCV activity of analogues of an antihistamine chlorcyclizine. The derivative 71 exhibited significant anti-HCV activity (EC₅₀ = 0.0023 µM) with low toxicity (CC₅₀ = 19.8 µM) and large selectivity index (SI = 8609) in Huh7.5.1 cell line. Derivative 71 carries an ethyl group at the nitrogen of piperazine and it was found more active as compared to methyl, propyl, isopropyl and cyclobutyl containing derivatives. Authors concluded that this drug can be included in some directly acting antiviral agents (Rolt et al., 2018). In 2017, Bassetto et al. synthesized series of novel piperazine-based analogues and also evaluated in vitro antiviral activity against HCV replication in Huh5-2 cell line. Derivatives were synthesized by modifications of aromatic substituents, sulfonamide groups, piperazine ring and alkyl chain. Compounds 72, 73 were found to have promising inhibition of NS3 helicase unwinding activity having EC₅₀ values of 1.2 µM, and 1.3 ± 0.777 µM, respectively with reference to VX-950 (EC₅₀ = 0.8 ± 0.2 μM), primuline and aurintri-carboxylic acid. The central piperazine nucleus, two sulfonamides groups and at least one propyl chain were found essential for the activity. Derivative 73 inhibited NS3 helicase enzyme activity having a Kd value of 21 ± 13. The in silico study of a single compound 74 was reported (Bassetto et al., 2017). (In silico studies are depited in Table 1).

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Fig. 8

Piperazine nucleus as pharmacophoric unit to fight against HIV infections.

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In 2016, Vausselin et al. identified the novel piperazine containing derivatives of 2-amino benzimidazole as antiviral agents. The compound 75 (Fig. 10) inhibited HCV infection at IC₅₀ of 1.39 ± 0.69 µM and also showed a greater therapeutic index (TI = 18). Derivative 75 was found specific against HCV and inhibited HCV at various stages of its life cycle (Vausselin et al., 2016). In 2015, Jiang et al. reported the synthesis and antiviral activity of novel N-phenylacetophenone and N-phenyl-benzamide derivatives against HCV and EV71A strains. The piperazine was added as a substituent in one derivative but compound 76 did not display good antiviral activity (IC₅₀ > 168.36 µM) against EV71 (strain SZ98) (Jiang et al., 2015). Magri et al. synthesized the amidino-urea derivatives of an antiviral drug moroxydine. Derivatives having aryl piperazine group exhibited prominent activity. The presence of a p-chlorophenyl group at the N1 of amidine was also beneficial for activity. The novel amidino-urea derivatives 77 and 78 exhibited excellent HCV inhibition activity having EC₅₀ values of 1.30 µM and 1.69 µM in the fresh addition assay (FA). Derivatives 77 and 78 presented EC₅₀ values of 11.2 µM and 10.4 µM in the standard assay with reference to ribavirin (EC₅₀ = 30.4 µM) and telaprevir (EC₅₀ = 0.45 µM) as standard drugs. The derivatives 77 and 78 showed better activity than the reference drug ribavirin which has poor activity (EC₅₀ = 33.5 µM) and high cytotoxicity (CC₅₀ = 30.5 µM). In the FA, drugs were added to the cell medium at 1/10th of the concentration taken initially after every 24 h post-infection (Magri et al., 2015). In 2014, Rynearson et al. synthesized 2-aminobenzoxazole derivatives with different substituents at position #6 and #7. Upon evaluation of their antiviral activities as inhibitors of HCV internal ribosome entry site, the piperazine derived benzoxazole 79, 80 and 81 derivatives were found inactive (Rynearson et al., 2014). In 2010, Najda-Bernatowicz et al. reported the synthesis of novel tropolone derivatives attached with cyclic amines like piperidine, piperazine and pyrrolidine. The antiviral activity was studied by specific subgenomic inhibition of HCV replication. The piperazine containing analogue 82 was found to be the most active (IC₅₀ = 3.2 µM) in helicase inhibition assay. The compound 82 carries methyl piperazine group at positions 3, 5 and 7 of tropolone scaffold. The derivative 82 inhibited RNA amplification with EC₅₀ value of 46.9 µM but presented minimum cytotoxicity (CC₅₀ > 1000 μM) resulting in a better selectivity index (SI > 21.3). The 3,5,7-tri[(4′-methylpiperazin-1′-yl)methyl]tropolone 82 is the most favourable tropolone derivative which can be used as a lead to design new anti-HCV compound in future (Najda-Bernatowicz et al., 2010). (In silico studies are depited in Table 1).

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Fig. 9

Piperazine-based anti-HBV agents.

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Among the above-mentioned piperazine-based anti-HCV derivatives, chlorocyclizine derivatives (67–68, 71) showed the best anti-HCV activity among the other groups attached with the piperazine-moiety, with EC₅₀ values ranging from 0.0020 µM to 0.02 µM. In the EC₅₀ range of 0.2 µM to 1.69 µM, amidino-urea containing derivatives (77), phenyl substituted derivatives (70), benzonitrile analogues (69), sulphonamides (72), and derivatives of benzonitrile (70) also exhibit excellent anti-HCV activity.

3.4 Anti-herpesvirus activity of piperazine derivatives

Herpes simplex virus (HSV) infections are endemic and extensive. Although anti-HSV therapy is efficacious, the appearance of drug-resistant species and drug toxicity disrupt the treatment (Rechenchoski et al., 2017). The development of resistance against already available nucleoside analogues has also been reported (Jiang et al., 2016). The process of finding new drugs is very necessary to compete with resistant strains. In 2010, Aytemir and Özçelik synthesized mannich bases derivatives of chlorokojic acid as antimicrobial agents. The antiviral potential of the synthesized compounds was evaluated against Herpes simplex virus Type-1 (HSV-1). Among the synthesized compounds, compound 83 (Fig. 11) showed anti-herpes virus activity with less potency (Aytemir and Özçelik, 2010).

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Fig. 10

Piperazine derivatives having anti-HCV activity.

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3.5 Anti-coxsackie B virus and anti-coxsackie A activity of piperazine derivatives

Coxsackie B virus (CVB) is a prevalent human enterovirus which causes frequent systemic inflammatory disease. It can cause infection of cardiac muscles and pancreas leading to type I diabetes mellitus. Currently, no vaccine or antiviral agent is accredited for the treatment or avoidance of CVB infection (Sin et al., 2017; Song et al., 2019). In 2020, Volobueva et al. synthesized novel derivatives of anti-enteroviral drug pleconaril by modifying the propyl linker group. The antiviral activity was explored against Coxsackievirus B3 (CVB3) Nancy. The piperazine containing derivatives 84 and 85 exhibited better in vitro anti-CVB3 activity presenting IC₅₀ values of 9.1 ± 0.7 μM and 3.9 ± 0.3 μM, respectively as compared to pleconaril (IC₅₀ = 18.4 ± 1.5 μM). Derivative 85 was found more cytotoxic (CC₅₀ = 203.8 ± 14.7 μM, SI = 52) as compared to derivative 84 (CC₅₀ = 1867.1 ± 90.4 μM SI = 205) as evaluated by MTT 96-well plate assay (Volobueva et al., 2020). In 2017, Hao et al. synthesized N-6 piperazine substituted adenosine analogues and also evaluated their antiviral activities against CVB3 by using MTT assay in HEp-2 cells. A number of the piperazine-based compounds exhibited better antiviral activity as compared to the reference drug. Compound 85 showed promising activity (IC₅₀ = 5.1 ± 2.3 μM) and selectivity index (SI = 41) against CVB3 as compared to the reference drug ribavirin (IC₅₀ = 36.8 ± 9.6 μM, SI = 29.1) (Hao et al., 2017). In 2013, Zhang et al. identified novel piperazine derivatives having antiviral potential against coxsackie virus A16 (CVA16) and human enterovirus (EV71). The piperazine containing compound 86 revealed potent activity against EV71 and CVA16 having IC₅₀ values of 4.3 µM and 1.2 ± 0.5 µM accompanied by low cytotoxicity (TC₅₀ = 43.1 ± 4.7 μM). Derivative 86 carries thiazole ring directly attached with piperazine. Derivative 87 shows moderate activity against EV71 and CVA16 having IC₅₀ values of 4.9 ± 0.2 µM. In docking studies with EV71 capsid binding pocket, derivative 86 showed hydrophobic interactions with Ileu111, Val192, Met230 and Phe233. Derivative 86 also exhibited π-π interaction with Phe137 and Phe155 (Zhang et al., 2013).

According to the structure–activity relationship (SAR) of the studies discussed here regarding piperazine-based anti-coxsackie B virus hybrids, pleconaril derivatives (84, 85) and alkyl substitution on piperazine rings (86, 87) demonstrated the best anti-coxsackie B virus activity among the other groups attached with the piperazine-moiety, with IC₅₀ values ranging from 3.9 µM to 9.1 µM.

3.6 Anti-influenza virus activity of piperazine derivatives

Influenza remains a considerable hazard to public health despite years of surveillance and treatment. The influenza vaccine is also available but mutation of the influenza virus can reduce efficacy (Petrova and Russell, 2018). In 2020, Zhang et al. designed and synthesized a novel series of 2,4-disubstituted quinazolines and checked in vitro anti-influenza A virus effectiveness. The piperazine-based analogue 88 (Fig. 12) showed medium activity against influenza A virus (IAV) presenting an IC₅₀ value of 11.47 ± 0.54 µM and CC₅₀ value>100 µM. However, derivative 88 showed no violation of the Lipinski’s rule of five and showed drug-likeness properties (Zhang et al., 2020). In 2016, Arns et al. reported the novel spirothiazamenthane having a unique chemical structure as an obstructer of the influenza A-M2 proton channel. The piperazine containing derivative 89 inhibited the WT (wild type) virus having an EC₅₀ value of 2.1 µM by using plaque reduction assay in MDCK cells (Arns et al., 2016). In 2015, Bottini et al. reported the synthesis of an existing anti-influenza compound. The starting dimethoxy piperazinyl quinazolinamine compound 90 presented in vitro anti-influenza A virus activity having an IC₅₀ value of 549 µM. The derivative 91 represents the tenfold better anti-influenza A virus profile displaying an IC₅₀ value of 44.18 ± 11.28 µM as compared to the starting compound. The derivative 91 carries a propionyl group attached at the nitrogen of piperazine (Bottini et al., 2015). Tănase et al. synthesized novel analogues of N6-substituted adenine and pyrimidine. The antiviral activity against influenza virus was evaluated. The piperazine containing compounds 92 (EC₅₀ = 12 ± 1 µM) and 93 (EC₅₀ = 25 ± 2 µM) demonstrated moderate activities but better selectivity (SI = 73, 39) values. The presence of bulky substituents at position #6 of the nucleoside was necessary for optimum antiviral activity (Tănase et al., 2015). In 2014, Pagano et al. synthesized various benzofurazan derivatives and examined the activity against influenza A virus H1N1. The piperazine containing derivative 94 exhibited moderate activity (IC₅₀ = 10 µM) towards influenza virus strain and was found to be more cytotoxic (CC₅₀ = 20 µM) as compared to other derivatives. The ligand efficiency (LE) of compound 94 was found to be 0.22 kcal/mol (Pagano et al., 2014). Wang et al. reported antiviral piperazine containing products obtained via optimization on the JNJ4796 as novel anti-influenza A virus agents. The R-enantiomer of compound 95 exhibited excellent in vitro activity against IAV H1N1 with an IC₅₀ value of 0.27 µM than the reference drugs Ribavirin and Oseltamivir with IC₅₀ values 4.28 µM and 3.44 ± 0.84 µM, repectively. It is worth mentioning to report that it showed lower cytotoxicity (CC₅₀ > 200 µM) and higher selectivity index (SI > 6666) than the reference drugs Ribavirin and Oseltamivir (Wang et al., 2021). Winyakul et al. synthesized 2,5-diketopiperazine derivatives and evaluated them as potential anti-influenza (H5N2) agents through in vitro and in silico approaches. The compound 96 displayed the best binding energy value of −102.25 kcal/mol, and hydrogen bond interactions with Arg371 when compared to the reference drug Oseltamivir carboxylate (-93.75 kcal/mol). The cytotoxicity activities were performed on the Rhesus monkey kidney epithelial cells (LL-MK2 cell lines) showing IC₅₀ values of 287.65 µM, indicated the negligible cytotoxicity compared to the normal cells (Winyakul et al., 2022). The in silico studies are described in Table 1.

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Fig. 11

Piperazine derivatives having anti-HCV activity.

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In the analysis of the structure–activity relationship (SAR) regarding piperazine-based anti-influenza virus compounds, it was observed that the benzofuran derivative (95) demonstrated the most potent anti-influenza virus activity compared to other groups attached to the piperazine-moiety, with an impressive IC₅₀ value of 0.27 µM. Additionally, benzofurazan derivatives (94) and 2,4-disubstituted quinazolines (88) also exhibited noteworthy anti-influenza virus activity within the IC₅₀ range of 10 µM to 11.47 µM.

3.7 Anti-human cytomegalovirus activity of piperazine derivatives

Human cytomegalovirus is a ubiquitous DNA herpes virus that is common worldwide and is the chief cause of morbidity and death in immunocompromised patients (Davis et al., 2017). In 2013, Massari et al. synthesized piperazine containing derivatives of naphthyridone and evaluated their anti-human cytomegalovirus (HCMV) activity by using plaque reduction assay and anti-HIV activity by MTT assay. The piperazine containing compound 97 (Fig. 13) was the most significant anti-HCMV and anti-HIV agent with the EC₅₀ values of 0.9 ± 0.2 µM and 1.3 ± 0.02 µM, respectively. The cytotoxic concentration (CC₅₀) values of derivative 97 were 126.5 ± 27.8 µM and 10 ± 1.86 µM respectively against these viruses. The lower cytotoxicity of 97 against HCMV is responsible for its potency. However, the anti-HCMV activity of another derivative 98 was greater (EC₅₀ = 0.19 ± 0.04 µM) than 97 but its cytotoxicity (CC₅₀ = 15.3 ± 3.4 µM) was higher which make it less selective. The 4-(2-pyridinyl)piperazine was found as the best substituent at position #6. The replacement of the terminal pyridine ring by quinolone ring produced more cytotoxic compounds (Massari et al., 2013). In concern for structure–activity relationship, pyrimidine and benzothiazole substitution on the piperazine ring in addition to the cyclopropanyl group on the nitrogen of 1,4-dihydroquinolinoic acid in (97, 98) exhibited the best antiviral activity than the phenyl or alkyl substitution on the piperazine and quinolinoic acid’s nitrogen, respectively.

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Fig. 12

Piperazine-based anti-HSV and anti-CVB agents.

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3.8 Anti-human rhinovirus (HRV3) activity of piperazine derivatives

Rhinovirus (RV) is an RNA virus and is the most endemic contributor of upper respiratory tract infections in human beings (Vandini et al.,2019). In 2017, Da Costa et al. developed a novel series of 4,5-dimethoxybenzyl analogues and studied their activity against rhinovirus (RV). The piperazine-based derivative 99 exhibited little activity against rhinovirus RV-B14 with an EC₅₀ value of 18.10 ± 0.34 µM (Da Costa et al., 2017). Q. Zhang et al. designed, synthesized and evaluated 2,2-dimethyl-1,3-dioxolane derivatives for the development of novel anti-rhinoviral medicines. The piperazine containing compound 100 revealed excellent anti-rhinoviral activity with an IC₅₀ value of 2.50 ± 0.7 µM versus HRV-3C protease. Docking studies of derivative 100 with HRV-3C protease crystal structure showed hydrogen bond interactions involving oxygens of 1,3-dioxolone (His40, Gly145), hydrogen and oxygen of amide (Val162, Gly164) group. The thiazole ring formed π stacking interaction with His40 (Q. Zhang et al., 2017). H. Wang et al. synthesized a novel series of chloro-pyridazine piperazines as anti-HRV3 agents. The compound 101 demonstrated meaningful anti-HRV activity with an EC₅₀ value of < 0.0032 µM and was found to be most selective in this series. Molecular docking studies of compound 101 with HRV-3 viral protein-1 (VP1) showed that benzene ring interacts with methylene group of Tyr152. The ethoxy group formed hydrogen bonding with the amino group of Ser175. The N1 and N3 of pyridazine are involved in hydrogen bonding with the phenolic hydroxyl group of Tyr197 (H. Wang et al., 2011).

Among piperazine-based anti-human rhinovirus derivatives, it was observed that chloro-pyridazine derivatives (1 0 1) exhibited the most potent anti-human rhinovirus activity among the various groups attached to the piperazine moiety, boasting an impressive EC₅₀ value of < 0.0032 µM. Moreover, 4,5-dimethoxybenzyl analogues (99) and 2,2-dimethyl-1,3-dioxolane derivatives (1 0 0) also demonstrated remarkable anti-human rhinovirus activity.(Fig. 14)

3.9 Anti-respiratory syncytial virus activity of piperazine derivatives

Respiratory syncytial virus (RSV) is a vital and familiar respiratory pathogen for which no vaccine is available albeit>50 years of efforts. RSV is the primary cause of acute lower respiratory infections in children (Obando-Paceco et al., 2018). In 2020, Ferla et al. discovered a family of sixteen new dithiocarbamate analogues in which benzothiazole and piperazine ring were incorporated. The antiviral activity was explored against RSV. The compound 102 (Fig. 15) was found as the best compound in the series against RSV-A (EC₅₀ = 4.4 ± 1.1 μM) and RSV-B (EC₅₀ = 1.3 ± 0.2 μM) in cytopathic effect inhibition assay. Compound 102 showed less toxicity to normal Hep2 cell line having CC₅₀ value>100 μM. In derivative 102 phenyl ring is directly connected with the piperazine ring (Ferla et al., 2020). In 2015, Cancellieri et al. used structure-based drug design to synthesize novel zinc ejecting compounds in which dithiocarbamate was used as linker group. Aromatic substituent was added on one side of the carbamate group and alicyclic amine was added on the other side. Upon evaluation of their in vitro antiviral activity, compound 103 was found to be the most active against RSV having an IC₅₀ value of 6 µM. The presence of benzyl piperazine fragment produced more active compounds in this series. Molecular docking investigations with M2-1 protein showed that thiazole ring interacted with His14 and benzyl piperazine formed hydrophobic interaction with Lys19. The dithiocarbamoyl interacted with the zinc ion of protein (Cancellieri et al., 2015).

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Fig. 13

Piperazine-based anti-influenza active agents.

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In the context of the studies discussed here, the structures of compounds were focused to explore the structure–activity relationship (SAR) of piperazine-based anti-respiratory syncytial virus derivatives. It was found that dithiocarbamates (102, 103) exhibited the most potent anti-HBV activity among the compounds studied.

3.10 Anti-bovine viral diarrhea activity of piperazine derivatives

Bovine viral diarrhoea virus (BVDV) is notably infective pathogen from Flaviviridae family and is globally distributed. BVDV is responsible for serious economic damages in the cattle industry (Riitho et al., 2020). In 2018, Loddo et al. synthesized novel 9-aminoacridine-based derivatives and evaluated their anti-BVDV activity against both RNA and DNA-based viruses. The 4-(2′-hydroxyethyl) piperazine containing compounds 104 was the most potent derivative against BVDV presenting an EC₅₀ value of 0.8 µM. Compound 104 also showed prominent in vitro inhibition (IC₅₀ = 0.62 µM) of viral RNA dependent RNA polymerase (RdRp) enzyme. The presence of the amino group between piperazine and acridine nucleus was important for the activity of these derivatives (Loddo et al., 2018). Shiryaev et al. designed and synthesized cage compounds as inhibitors of BVDV. The piperazine-based cage compound 105 was found to have moderate antiviral activity against BVDV (IC₅₀ = 1.69 µM) but showed low toxicity with CC₅₀ values above 358.17 µM. In this compound, piperazine ring is condensed with adamantane fragment (Shiryaev et al., 2018). In 2016, Ibrahim et al. reported the preparation of piperazine containing Mannich bases of 7-hydroxycoumarins and studied the antiviral activities against HIV-1, BVDV, yellow fever virus (YFV), Reovirus (Reo-1), CVB-5, poliovirus (Sb-1), RSV and vesicular stomatitis virus (VSV). The piperazine containing compounds 106 and 107 endowed notable activities against BVDV with EC₅₀ values of 0.3 ± 0.01 µM and 1.0 µM as compared to EFV and other standard compounds. These compounds were specific for the BVDV because they showed no activity against other viruses. These derivatives demonstrated significant selectivity indices and less toxicity which makes them strong candidates towards the drug development. Derivatives 106 and 107 contain p-nitrobenzoyl group and hydroxyl group at position #7 respectively. In these derivatives, piperazine was found to be most suitable linker because the replacement of piperazine by piperidine and other alkyl amines produced less active derivatives (Ibrahim et al., 2016).

Among the various groups attached to the piperazine moiety, piperazine-containing 7-hydroxycoumarin derivatives (1 0 6) exhibited the most potent anti-bovine viral diarrhoea virus activity, with an EC₅₀ value of 0.3 µM. Additionally, other moieties that showed excellent anti-bovine viral diarrhoea virus activity include the adamantine fragment (1 0 5) and 9-aminoacridine-based derivatives (1 0 4), displaying IC₅₀ value ranging from 0.62 µM to 1.69 µM.(Fig. 15)

3.11 Anti-human norovirus activity of piperazine derivatives

Human norovirus (HNV) is one of the most leading etiological agents which causes acute gastroenteritis worldwide and therefore poses a substantial risk to human health. The development of a vaccine for norovirus faces challenges of short-lived immunity and antigenic unpredictability (Lucero et al., 2018; Nordgren and Svensson, 2019). In 2019, Giancotti and co-workers identified new antiviral scaffolds for HNV by using computer-assisted techniques on the viral polymerase. The piperazine containing hybrid analogues 108 and 109 (Fig. 16) produced the most amazing antiviral activity with EC₅₀ values of 6.1 ± 3.9 µM and 10.9 ± 11.1 µM respectively in HNV G1 assay (Giancotti et al., 2019). Harmalkar et al. carried out the design and synthesis of novel non-nucleoside vinyl-stilbene derivatives as potent inhibitors of norovirus. Amide derivatives of vinyl stilbene presented prominent activities and piperazine-derived amide 110 emerged as the most effective with an EC₅₀ value of 2.43 µM. Compound 110 also exhibited better selectivity index (>41.2) and less toxicity (CC₅₀ > 100 μM). The presence of the vinyl group was necessary for the activity because the removal of the vinyl group or replacement by ethyl produced less active derivatives. The promising anti-HNV activity of 110 may lead it towards the development of novel noroviral inhibitor (Harmalkar et al., 2019).

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Fig. 14

Piperazine-based anti-human cytomegalovirus and anti-human rhinovirus agents.

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The studies presented here demonstrate a structure–activity relationship (SAR) in the context of derivatives of piperazine that are directed against the human norovirus. The piperazine-containing non-nucleoside vinyl-stilbene derivatives (1 1 0) with an EC₅₀ value of 2.43 µM showed the strongest anti-human norovirus activity among the various groups attached to the piperazine moiety. The carbamothioyl benzamides (108, 109) with EC₅₀ values ranging from 1.6 µM to 10.9 µM also demonstrated excellent anti-human norovirus activity.

3.12 Anti-adenoviral activity of piperazine derivatives

Several human diseases are associated with adenovirus which includes conjunctivitis, gastroenteritis, myocarditis, pneumonia and hepatitis and to-date no antiviral agent is licensed to treat adenovirus. Adenovirus infections are considered as self-limiting in healthy individuals but individuals with impaired immune functions have encountered severe infections (Echavarría, 2008; Lenaerts et al., 2008; Ali et al., 2016). Here, we have reported potential compounds to control adenovirus replication. In 2020, Mazzotta et al. optimized piperazine-derived urea analogues for anti-adenoviral activity and reported a library of sixty-seven compounds by the refining procedure of piperazine-based privileged scaffolds. Structural modifications were carried out by replacement of urea by thiourea, the acyl group was substituted by substituted acetyl group and the piperazine ring was modified by 2,6-dimethyl piperazine. The biological evaluations of antiviral activity and cytotoxicity identified twelve derivatives that showed>80% inhibition of human adenovirus (HAdV) infections at low micromolar (µM) and nanomolar (nM) quantities accompanied by low cytotoxicity. Furthermore, eight out of twelve studied derivatives also blocked DNA replication of human cytomegalovirus (HCMV) at low micromolar concentrations. Finally, a total of seven compounds 111–117 were identified for their>90% HAdV inhibition in the plaque reduction analysis and with CC₅₀>100 µM. As the compound 114 significantly inhibited Phi29 DNA polymerase activity, therefore, it is suggesting that preferential targets are HAdV and CMV DNA polymerases (Mazzotta et al., 2020). In 2016 Sánchez-Céspedes et al. disclosed the synthesis, biological evaluation and SAR studies of novel phenylpiperazine derivatives. The compounds 118, 119, 120, 121 and 122 were found to have significant inhibition of DNA replication against human adenovirus (HAdV5) presenting IC₅₀ values of 3.4 ± 1.0 µM, 2.1 ± 0.1 µM, 2.5 ± 1.2 µM, 4.7 ± 0.1 µM, and 2.5 µM respectively. In these derivatives, piperazine is linked to benzofuran scaffold and substituted phenyl ring at both nitrogens (Sáanchez-Céspedes et al., 2016). In 2014, Sáanchez-Céspedes et al. prepared piperazine-2-one derivatives and also investigated the inhibition of adenosine replication. The trisubstituted piperazine-2-one analogue presented significant antiviral activity with very little cytotoxicity. The compound 123 selectively inhibited DNA replication of Ad5 and Ad16 adenovirus displaying IC₅₀ values of 1.3 ± 0.18 µM and 0.8 ± 0.74 µM, respectively. The studies on the mode of action showed that compound 123 did not interfere with the entry of the virus inside the host but inhibited the approach of attacking viral genome at the exact point in the nucleus. The compound 123 emerges as a potential drug towards the discovery of new antiviral agents (Sáanchez-Céspedes et al., 2014).

Among the various groups attached to the piperazine moiety, the derivative known as piperazin-2-one (1 2 3) displayed the most potent anti-adenovirus activity, with an EC₅₀ value of 1.3 µM. Furthermore, other derivatives, namely piperazin-2-ones (118–122), demonstrated remarkable anti-adenovirus activity, with EC₅₀ values ranging from 2.1 µM to 4.7 µM.(Fig. 16)

3.13 Anti-tobacco mosaic virus activity of piperazine derivatives

Tobacco mosaic virus (TMV) infects all tobacco species and many other plants worldwide and causes considerable loss of crops (R. Liu et al., 2013; W. Zhang et al., 2017). In this review, we identified piperazine containing favourable compounds having anti-TMV activities. In 2020, Nagalakshmamma et al. synthesized novel pyrimidine and piperazine containing derivatives by introducing urea/thiourea moieties and evaluated their inhibitory activity against TMV. Derivatives 124, 125 and 126 (Fig. 17) have exhibited high inclination towards TMV-coat proteins, TMV-helicase and were found as the most potent antiviral compounds versus TMV at 5.0 mg/mL concentration. The in vitro inhibition rates of 124, 125 and 126 were 68.3 ± 1.8%, 67.1 ± 2.2% and 65.1 ± 1.7% and found comparable to the standard drug ningnanmycin (72.3 ± 2.1%). In molecular docking studies, derivative 124 showed MM/GBSA score of −56.063 kcal/mol and presented hydrogen bonding interaction of its amino group and oxygen with Met245 and Ile275 of TMV-helicase enzyme (Nagalakshmamma et al., 2020). Febuxostat is non-purine analogue and is effective in the treatment of hyperuricemia and gout (Bruce, 2006). In 2013, Reddy et al. attached urea and thiourea analogues of piperazine with febuxostat and also evaluated them against TMV and other microbes. Derivatives 127 and 128 have shown significant in vivo anti-TMV activities with the inactivation rate of 85.72 ± 0.33% and 85.93 ± 0.54%, respectively as compared to standard drug ninganamycin (88.45 ± 0.68%). These derivatives contain 4-nitrophenyl and 3-bromophenyl attached with piperazine through amide and thioamide groups, respectively (Reddy et al., 2013). Yuan et al. synthesized ferulic acids containing piperazine moiety as potent inhibitor of mosaic viruses. The compound 129 exhibited outstanding antiviral activity against tobacco mosaic virus (TMV) and cucumber mosaic virus (CMV). The experiment was performed on the upper leaves of Nicotiana tobaccum and showed remarkable antiviral activity with EC₅₀ values of 189.0 µM and 401.7 µM against TMV and CMV, respectively compared to the reference drugs ningnanmycin (387.0, 519.3 µM) and ribavirin (542.1, 721.5 µM) (Yuan et al., 2022). The in silico studies are mentioned in Table 1. A number of trifluoromethylpyridine piperazine derivatives were prepared and tested for their anti-CMV activities, with compound 130 showing the most impressive anti-CMV activities; specifically, the EC₅₀ of compound 130 for curative, protective, and inactivation activities against CMV viruses was found to be 169.1, 95.0, and 18.1 µM, respectively, which were better than those of the commercial product ningnanmycin (Zhang et al., 2023).(Fig. 17)

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Fig. 15

Piperazine-based anti-RSV and anti-BVDV active agents.

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Fig. 16

Piperazine-based human anti-human norovirus and anti-human adenovirus agents.

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Fig. 17

Piperazine-based anti-tobacco mosaic virus, anti-cucumber mosaic virus, and anti-avian paramyxovirus agents.

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Fig. 18

Piperazine-based anti-SARS-CoV-2 compounds.

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Fig. 19

Piperazine-based anti-Nipah virus and anti-ZIKV agents.

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Studies on piperazine-based derivatives that target the TMV have found a good structure–activity relationship (SAR). Ferulic acid with a piperazine moiety attached (1 2 9) demonstrated the strongest anti-TMV activity among the various groups, with an 89.9% inactivation rate. Other derivatives, such as urea and thiourea analogues (124–128), also showed excellent anti–TMV activity, with inactivation effect rates ranging from 65 to 85 percent.

3.14 Anti-avian paramyxovirus activity of piperazine derivatives

Economically significance of avian paramyxoviruses has been reported around the world because huge mortality and morbidity rate is associated with them (Gogoi et al., 2017). Huge economic losses are reported from all over the world due to the devastating outbreaks of viruses particularly affecting the poultry industry and impose serious threats to chicken (Munir et al., 2012; Pedersen et al., 2013; Al-ilbadi, 2019). In 2018, Balaraman et al. synthesized novel benzamide derivatives by replacing the glutamic acid part of pemetrexed with different amines. The biological activity was evaluated against paramyxovirus (PMV) by plaque reduction assay. The piperazine containing derivative 131 has exhibited moderate plaque reduction (47%) at the minimal test concentration of 1 mM as compared to ribavirin (60% at 0.3 mM) (Balaraman et al., 2018). In 2017, Selvakumar et al. synthesized piperazine and morpholine derived compounds with N-aroyl/benzyl substitutions and screened their anti-viral activities against avian paramyxovirus (APMV-1). The compound 132 having carbonyl functionality appeared as the most potent derivative against avian paramyxovirus (APMV-1) (28% inhibition) as compared to reference drug ribavirin (32% inhibition) in plaque reduction assay (Selvakumar et al., 2017). In another research paper in 2018, Selvakumar et al. carried out the synthesis and anti-viral activity of piperazine and morpholine substituted derivatives against avian paramyxo virus (APMV-1). Among all the compounds piperazine substituted derivative 133 demonstrated higher antiviral activity (84% inhibition) in plaque reduction assay as compared to the reference drug ribavirin (32% inhibition) (Selvakumar et al., 2018).

The investigations focused on piperazine-based derivatives that specifically target the avian paramyxovirus, and these studies demonstrate a noteworthy relationship between the compound's structure and its activity. Among the various groups attached to the piperazine moiety, tetrahydroquinazoline (1 3 3) displayed the most potent anti-avian paramyxovirus activity, with inactivation rate of 84%. Furthermore,piperazine containing benzamide (1 3 1), demonstrated anti-avain paramyxovirus activity, with inactivation effect rate of 47%.(Fig. 17)

3.15 Anti-SARS-CoV-2 activity of piperazine derivatives

The ongoing COVID-19 pandemic has resulted in a global panic because of its continual evolution and recurring spikes. This serious malignancy is caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) (Rafiq et al., 2023b). Alaaeldin et al. investigated the in vitro antiviral activity of 7-(4-(N-substituted-carbamoyl-methyl)piperazin-1-yl)-chalcone 134 supported by molecular docking against SARS-CoV-2 main protease. The experiment was carried out using Vero cells which have an IC₅₀ value of 0.6 ± 0.05 μM, EC₅₀ value of 0.00393 µM, and an energy score of −8.9 kcal/mol, while ciprofloxacin was used as a reference drug displaying an IC₅₀ value of 5.13 ± 0.19 µM and an energy score of −7.0 kcal/mol (Alaaeldin et al., 2022). Omar et al. synthesized some novel piperazine compounds and evaluated them for their best antiviral activity against the SARS-CoV-2 protease enzyme through a virtual screening approach. Among these derivatives, the piperazine hybrids 135 and 136 showed the best docking scores towards the SARS-CoV-2 protease enzyme, i.e., −6.87 kcal/mol and −6.81 kcal/mol, respectively. The piperazine hybrid 135 showed hydrogen bond interactions with Thr26, Gln189, Asn142, and Gly143, while the compound 136 showed hydrogen bond interactions with His163 and hydrophobic interactions with His41 and Thr25 (Omar et al., 2021). Kumar et al. synthesized a novel piperazine-based compound 137 and tested it in vitro and in silico against the SARS-CoV-2 non-structural protein NSP15. The molecular docking simulations revealed the antiviral behavior of compound 137 via MM/GBSA analysis (ΔG-Bind), glide energy and potential energy values of −55.62 kcal/mol, 43.96 kcal/mol, and −84.37 kcal/mol, respectively. The viral entry assay showed that (2S,3S)-3-amino-1-(4-(4-(tert-butyl)benzyl)piperazin-1-yl)-4-phenylbutan-2-ol 137 demonstrated good inhibitory activity with an IC₅₀ value of 4.97 µM when compared to the reference drug ivermectin which had an IC₅₀ value of 7.28 µM. The spread assay indicated that compound 137 showed an IC₅₀ value of 8.46 µM compared to the ivermectin IC₅₀ value, i.e., 5.53 µM (Kumar et al., 2021). Antiviral activity of hydroxyethylamine analogs was investigated by Gupta et al. against SARS-CoV-2 main protease (3CL^pro) using computational and in vitro approaches. The piperazine-based compound 138 was synthesized, and its antiviral behavior was checked using the MM/GBSA score of −52.14 kcal/mol using an in vitro spread assay performed on Vero E6 cells that exhibited an IC₅₀ value of 12.44 µM against SARS-CoV-2 3CL^pro (Gupta et al., 2021). Gao et al. identified piperazine-based polyphenols as potent inhibitors of SARS-CoV-2 RdRp. The compound 139 demonstrated the best binding energy of −7.196 kcal/mol than remdesivir (-6.5 kcal/mol) to RdRp of SARS-CoV-2. The results revealed the confirmation of in silico analysis, i.e., the compound 139 demonstrated an IC₅₀ value of 9.2 ± 1.1 µM. It 139 blocked the active site by chelating with Mg⁺² thereby inhibiting the replication function of RdRp (Gao et al., 2022a). Gao et al. discovered nonpeptidic piperazine derivatives as SARS-CoV-2 main protease inhibitors. Among 30 synthesized compounds, compound 140 showed the significant enzymatic inhibitory activity of M^pro i.e., IC₅₀ = 0.18 µM and possessed the best antiviral activity against SARS-CoV-2 with an EC₅₀ value of 2.64 µM similar to that of remdesivir (EC₅₀ = 2.27 µM). The crystallographic studies revealed the covalent binding of the compound with the active site of M^pro (Gao et al., 2022b). Gao et al. discovered 1,2,4-trisubstituted piperazine derivatives as SARS-CoV-2 main protease inhibitors. Among synthesized compounds, compound 141 displayed the significant enzymatic inhibitory activity of M^pro i.e., IC₅₀ = 0.4 µM and demonstrated the excellent antiviral activity against SARS-CoV-2 with an EC₅₀ value of 1.1 µM. The study revealed that the compound 141 showed non-covalent, non-peptidic interactions with low cytotoxicity and high target specificity (Gao et al., 2022c).

Among piperazine-based anti-SARS-CoV-2 derivatives, chalcone (1 3 4) demonstrated the best anti-SARS-CoV-2 activity as compared to the other groups attached with the piperazine-moiety, with EC₅₀ value 0.0039 µM and IC₅₀ value 0.6 µM. On the other hand, 1,2,4-trisubstituted piperazine derivatives (1 4 1) exhibited the remarkable anti-SARS-CoV-2 activity with EC₅₀ value of 1.1 µM and IC₅₀ value of 0.4 µM. Other molecules with excellent anti-SARS-CoV-2 activity (IC₅₀) include phenylbutan-2-ol (1 3 7), hydroxyethylamine analogs (1 3 8), piperazine-based polyphenols (1 3 9), and nonpeptidic piperazine derivatives (1 4 0) in the range of 0.18 µM to 12.44 µM.(Fig. 18)

3.16 Anti-Nipah virus and anti-Zika virus activity of piperazine derivatives

Lipin et al. unraveled some piperazine-based derivatives of favipiravir for the treatment of Nipah virus through in silico approach. The compound 142 was observed to be the most potent compound against Nipah virus with docking score of −8.909 kcal/mol and glide energy value of −55.229 kcal/mol. The hydrogen bond residues were seen with Gln530, Ala532, and Ile580 and showed hydrophobic interactions with Val507, Pro488, Ala532, Cys240, Tyr581, Ala558, Ile580, Pro590, Ile588, Ile217, and Cys216. The predicted IC₅₀ value (pIC₅₀) was found out to be 4.86 µM, endorsing the docking studies, and predicted using a web server. (Lipin et al., 2021). In the context of the structure–activity relationship described by Lipin et al. (2021), piperazine-based derivatives of furans with phenyl groups that have electron-donating substituents (1 4 2) showed a stronger affinity to bind with the Nipah virus target than the compounds with electron-withdrawing substituents on the phenyl ring attached to the furan ring that is directly bonded to the piperazine. del Rosario García-Lozano et al. (2023) reported piperazine-derived compounds as inhibitors against Zika virus. Two lead compounds, 143 and 144, with promising broad-spectrum activity against ZIKV (IC₅₀ 6.6 μM and 1.9 μM respectively) were identified with a good security profile. Regarding structure activity relationship, piperazine ring possessing chalcone moiety (143, 144) exhibited better activity against ZIKV than the piperazine-based indole derivatives which are found to be inactive.(Fig. 19)