molecules

Review Synthetic Compounds with 2-Amino-1,3,4-Thiadiazole Moiety Against Viral Infections

Georgeta Serban

Pharmaceutical Chemistry Department, Faculty of Medicine and Pharmacy, University of Oradea, 29 Nicolae Jiga, 410028 Oradea, Romania; [email protected]; Tel.: +4-0756-276-377



Academic Editor: Antonio Carta
 Received: 29 January 2020; Accepted: 18 February 2020; Published: 19 February 2020

Abstract: Viral infections have resulted in millions of victims in human history. Although great efforts have been made to find effective medication, there are still no drugs that truly cure viral infections. There are currently approximately 90 drugs approved for the treatment of human viral infections. As resistance toward available antiviral drugs has become a global threat to health, there is an intrinsic need to identify new scaffolds that are useful in discovering innovative, less toxic and highly active antiviral agents. 1,3,4-Thiadiazole derivatives have been extensively studied due to their pharmacological profile, physicochemical and pharmacokinetic properties. This review provides an overview of the various synthetic compounds containing the 2-amino-1,3,4-thiadiazole moiety that has been evaluated for antiviral activity against several viral strains and could be considered possible prototypes for the development of new antiviral drugs.

Keywords: 2-amino-1,3,4-thiadiazole; viral infections; antiviral agents; drug resistance; inhibitory concentration; mechanism of action

1. Human Viral Infections Viruses are the smallest among all self-replicating organisms and yet they are the etiological agents of many difficult to treat diseases in human populations [1]. There are broad types of human infections caused by viruses, such as respiratory infections (common cold, Influenza), digestive infections (viral gastroenteritis), central nervous system infections (viral meningitis, viral encephalitis), skin or mucosal infections (herpes, measles, mumps, smallpox and rubella), hepatic infections (hepatitis A, B, C, E), blood infections (acquired immunodeficiency syndrome) and hemorrhagic fever (yellow fever, Ebola hemorrhagic fever). Viruses are the most abundant and diverse biological entities on Earth and this is the reason for the high incidence of viral infections [2]. In addition, some viruses are etiological agents in the development of human tumors, particularly cervical cancer and hepatic cancer [3]. The main method and most cost-effective strategy for preventing viral infections is through vaccination, which is meant to prevent outbreaks by increasing immunity [4]. Vaccines for the prevention of several common acute viral infections, such as polio, rubella, measles, mumps, Influenza, yellow fever, encephalitis, rabies, smallpox and hepatitis B were developed during the 20th century and are available on a large scale [1,4]. Efforts to develop safe and effective vaccines against viruses that cause chronic infections, such as human immunodeficiency virus or hepatitis C virus did not give the expected results [1,4,5]. For many viral infections, only symptomatic treatment is indicated, while it is expected the immune system to fight off the virus. However, there are high-virulence viruses that cause serious viral infections where antiviral treatment is essential for patient survival. Although great efforts have been made to find effective medication, there are still no drugs that truly cure viral infections. Moreover,

Molecules 2020, 25, 942; doi:10.3390/molecules25040942 www.mdpi.com/journal/molecules Molecules 2020, 25, 942 2 of 22 due to the ability of viruses to undergo rapid mutations, the mechanisms involved in developing resistance to antiviral drugs are activated in most cases [1,3]. As resistance toward antiviral drugs is becoming a global health threat, there is an intrinsic need to identify new scaffolds that are useful in discovering innovative, less toxic and highly active antiviral agents [3,6,7].

2. Nitrogen-Containing Heterocycles and Thiadiazole Ring in Biology and Medicinal Chemistry Nitrogen-containing heterocycles are widely distributed in nature and are essential in vegetal and animal metabolism. They are found in nucleic acids, vitamins, antibiotics, alkaloids, etc. [8–14]. In addition, nitrogen-containing heterocycles are important targets for medicinal chemistry, as they are found in more than half of the commercially available drugs and can also act as versatile intermediates in the synthesis of complex products that exhibit outstanding biological activities [15]. Most of the nitrogen-containing heterocyclic compounds exhibit better biological activity than non-nitrogen compounds [12,16,17]. Currently, there are approximately 90 drugs approved for use in the treatment of nine human viral infections caused by human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV), herpes simplex virus (HSV), Influenza virus, human cytomegalovirus (HCMV), varicella-zoster virus (VZV), respiratory syncytial virus (RSV) and human papillomavirus [18]. Most of these drugs (e.g., acyclovir, cidofovir, idoxuridine, nevirapine, pleconaril, ribavirin, etc.) are nitrogen heterocycle molecules [3,19]. Five-membered aromatic systems with three heteroatoms at symmetrical positions, such as the 1,3,4-thiadiazole ring, have been extensively studied due to their pharmacological profile and physicochemical and pharmacokinetic properties. 1,3,4-Thiadiazole derivatives are known as compounds having significant and diverse biological activities such as antibacterial, antifungal, antitubercular [20], analgesic and anti-inflammatory [21,22], antidepressant and anxiolytic [23], kinesin inhibitors [24], etc. The 1,3,4-thiadiazole ring is also found in several medicines such as acetazolamide, methazolamide, cefazolin, cefazedone, sulfamethizole or megazol [20,25–27]. The 1,3,4-thiadiazole nucleus provides the compounds with high lipophilicity and the ability to form mesoionic systems associated with discrete regions of positive and negative charges. These distinct features allow mesoionic compounds to efficiently cross cellular membranes, leading to good oral absorption, bioavailability and strong unique interactions with biological molecules (e.g., DNA, proteins, etc.) thus increasing the potential of 1,3,4-thiadiazole derivatives to exhibit biological activities [20,27–29]. The thiadiazole ring is a bioisostere of the pyrimidine and pyridazine rings. The pyrimidine nucleus is commonly found in naturally occurring compounds of biochemical importance, as well as in drugs (e.g., pyrimidine nucleosides and nucleotides, nucleic acids, antiviral drugs), while the pyridazine nucleus is a component of some pharmacologically active compounds [20,30–32]. The thiadiazole ring also acts as a bioisostere of the thiazole ring, and therefore thiadiazole derivatives can act in the same way as third- and fourth-generation antibacterial cephalosporins. This is an additional feature that highlights the high potential of this ring system in medicinal chemistry [20,28]. Many researchers suppose there is a connection between the biological potential of 1,3,4-thiadiazoles and the strongly aromatic character of the 1,3,4-thiadiazole ring, as well as the presence of the toxophoric =N-C-S- linkage group. Furthermore, the high in vivo stability and low toxicity for higher organisms are also attributed to the 1,3,4-thiadiazole ring [20,33,34]. In addition, the amine derivatives of 1,3,4-thiadiazole are also studied. 2-Amino-1,3,4-thiadiazole moiety and its derivatives are known for their antitumor, antitrypanosomal and uricogenic properties [20,25,27,35–38]. 2-Amino-1,3,4-thiadiazole derivatives are currently being synthesized in many laboratories, and in previous papers, we described several 2-amino-1,3,4-thiadiazoles that exhibit antibacterial, antifungal, antitubercular [20,39–44], and antiparasitic activities [27,38]. The purpose of this paper is to present some small molecules possessing the 2-amino-1,3,4-thiadiazole moiety that have shown antiviral activity. Molecules 2020, 25, 942 3 of 22

3. The Activity of 2-Amino-1,3,4-Thiadiazole Derivatives Against Human Viral Pathogens

3.1. Human Immunodeficiency Virus (HIV) About 37 million people infected with human immunodeficiency virus (HIV) (Retroviridae family) were reported in 2016 [45], with the highest incidence of infection in sub-Saharan Africa. In the Third World, HIV infection coupled with tropical diseases, malaria and tuberculosis causes a high level of mortality. Due to sexual transmission, acquired immunodeficiency syndrome (AIDS) affects many young workers and therefore the disease has not only a social impact, but also a significant economic impact in these regions [3]. In the past two and half decades, different organic compounds have been developed as drug candidates for the treatment of AIDS targeting one or more stages of the virus life cycle such as absorption, fusion, entry, un-coating, reverse transcription, integration, transcription and maturation [46]. HIV-1 reverse transcriptase (RT) is an essential enzyme that converts single-stranded RNA from the viral genome into double-stranded DNA before its integration into host DNA [47]. Since RT is a key enzyme in the life cycle of HIV-1, some HIV-1 RT inhibitors with nucleoside or non-nucleoside structures are currently used in AIDS treatment [48]. Nucleoside reverse transcriptase inhibitors (NRTIs) such as zidovudine, didanosine, zalcitabine, stavudine, lamivudine, abacavir, tenofovir or emtricitabine interact competitively with the catalytic site of the RT, while the non-nucleoside reverse transcriptase inhibitors (NNRTIs)—nevirapine, delavirdine, efavirenz, etc.—follow an allosteric interaction with a site adjacent to the NRTI binding site, the non-nucleoside inhibitor binding site (NNBS) [47]. Due to their high selectivity and low cytotoxicity, NNRTIs have gained an increasingly important role in HIV infection therapy [49]. Five drugs in the class of NNRTIs have been approved for the treatment of HIV infection: nevirapine, delavirdine and efavirenz as the first generation drugs and etravirine (Intelence tablets, Janssen Therapeutics Company, 2008) and rilpivirine (Edurant tablets, Janssen Therapeutics Company, 2011) as the next-generation NNRTIs [46,50–52]. Doravirine (MK-1439A) is a new NNRTI developed by Merck Company that completed two 48-week studies in 2017 [53]. Doravirine demonstrated antiretroviral activity and immunological effects similar to efavirenz with significantly fewer central nervous system adverse events [54]. In January 2018, US Food and Drug Administration (FDA) accepted for review two new drug applications (NDAs) for doravirine, as a once-daily tablet in combination with other antiretroviral agents and as a once-daily fixed-dose combination single tablet of doravirine with lamivudine and tenofovir disoproxil fumarate [55]. On 30 August 2018, FDA approved both applications of doravirine for AIDS treatment as Pifeltro tablets (doravirine 100 mg) and Delstrigo tablets (doravirine 100 mg, lamivudine 300 mg and tenofovir disoproxil fumarate 300 mg) [56,57]. Several NNRTIs (e.g., fosdevirine, lersivirine) underwent clinical development programs but were discontinued due to unfavorable pharmacokinetic, efficacy and/or safety factors [58–60]. Approximately 17 million patients have access to antiretroviral therapy capable of controlling viremia and reducing mortality [61]. However, long-term treatment with antiretroviral agents can lead to drug resistance due to rapid mutations in the viral genome resulting in RT mutations and HIV chemotherapy failure [62]. These concerns have attracted a particular focus on research into new antiretroviral drugs that address the limitations of currently available agents for the treatment of HIV infection [46,59]. As a bioisostere of pyrimidine, a nucleoside component of nucleic acids, the thiadiazole ring can impart antiviral activity [28]. Studies concerning the antiviral activity of 1,3,4-thiadiazole derivatives often gave compounds with moderate or lower in vitro anti-HIV-1 and anti-HIV-2 activity than the reference drugs [63,64]. A relevant example is the chiral 2-substituted 5-(4-chlorophenylamino)-1,3,4-thiadiazoles 1–5 synthesized by Akhtar et al. by acidic cyclodehydration of the corresponding thiosemicarbazides [65]. Molecules 2020, 25, 942 4 of 22 Molecules 2020, 25, 942 4 of 22 Molecules 2020, 25, 942 4 of 22 Molecules 2020, 25, 942 4 of 22 H C Cl H3C N N Cl H3C H N N Cl 3 HN N N S NH S N S N S NH O S O S HN O O O R H O R R R = CH(CH3)2 (1); CH2CH(CH3)2 (2); CH(CH3)CH2CH3 (3) R = CH(CH3)2 (1); CH2CH(CH3)2 (2); CH(CH3)CH2CH3 (3) R = CH(CH3)2 (1); CH2CH(CH3)2 (2); CH(CH3)CH2CH3 (3) CH(OH)CH3 (4); CH2CH2SCH3 (5) CH(OH)CH3 (4); CH2CH2SCH3 (5) CH(OH)CH3 (4); CH2CH2SCH3 (5) In vitro HIV inhibitory activity using human T-lymphocyte (MT-4) cells gave moderate or low In vitrovitro HIV inhibitory inhibitory activity activity using using human human T-lymphocyte T-lymphocyte (MT-4) (MT-4) cells cells gave gave moderate moderate or low or half-maximalIn vitro HIV effectiv inhibitorye concentration activity using (EC50 human) values T-lymphocyte in comparison (MT-4) to efavirenz cells gave (EC moderate50 value of or 0.003 low lowhalf-maximal half-maximal effectiv effectivee concentration concentration (EC50 (EC) values50) values in comparison in comparison to efavirenz to efavirenz (EC50 (ECvalue50 valueof 0.003 of μhalf-maximalg/mL). The best effectiv resultse concentration were obtained (EC for50 )derivatives values in comparison1 and 5 (Figure to efavirenz1) with moderate (EC50 value EC 50of values. 0.003 0.003μg/mL).µg /ThemL). best The results best results were obtained were obtained for derivatives for derivatives 1 and 51 (Figureand 5 (Figure 1) with1) moderate with moderate EC50 values. EC 50 Thus,μg/mL). compound The best results1 showed were EC obtained50 > 14 forμg/mL derivatives against 1 HIV-1 and 5 (Figure(strain IIIB)1) with and moderate EC50 > 12.4EC50 values.μg/mL values.Thus, compound Thus, compound 1 showed1 showed EC50 EC> 1450 >μg/mL14 µg /mLagainst against HIV-1 HIV-1 (strain (strain IIIB) IIIB) and and ECEC5050 >> 12.412.4 μµg/mLg/mL againstThus, compound HIV-2 (strain 1 showed ROD), whileEC50 > the 14 compoundμg/mL against 5 showed HIV-1 EC (strain50 > 12.6 IIIB) μg/mL and ECagainst50 > 12.4 HIV-1 μg/mL and against HIV-2 HIV-2 (strain (strain ROD), ROD), while while the the compound compound 5 5showedshowed EC EC50 >50 12.6> 12.6μg/mLµg /againstmL against HIV-1 HIV-1 and ECagainst50 > 12.5HIV-2 μ g/mL(strain against ROD), whileHIV-2. the Low compound values for5 showed cytotoxicity EC50 > concentration12.6 μg/mL against 50% (compound HIV-1 and andEC50 EC> 5012.5> 12.5μg/mLµg/mL againstagainst HIV-2. HIV-2. Low Low values values for for cytotoxicity cytotoxicity concentration concentration 50% (compound(compound concentrationEC50 > 12.5 μ thatg/mL reduces against the HIV-2. viability Low of movaluesck-infected for cytotoxicity MT-4 cells concentrationby 50%), namely 50% the (compound CC50 value concentration thatthat reducesreduces the the viability viability of of mock-infected mock-infected MT-4 MT-4 cells cells by by 50%), 50%), namely namely the the CC CC50 value50 value of ofconcentration 14.0 ± 1.2 μ g/mLthat reduces for compound the viability 1 and of 13.3 mo ck-infected± 0.8 μg/mL MT-4 for compound cells by 50%), 5, respectively, namely the resultedCC50 value in 14.0of 14.01.2 ± 1.2µg /μmLg/mL for for compound compound1 and 1 and 13.3 13.30.8 ± 0.8µg /μmLg/mL for compoundfor compound5, respectively, 5, respectively, resulted resulted in low in lowof 14.0 ±selectivity ± 1.2 μg/mL index for SI ≤compound 1 (SI = CC 501 /ECand50 ).13.3± Other ± 0.8 derivatives μg/mL for showed compound lower 5, activityrespectively, with ECresulted50 within in selectivitylow selectivity index index SI 1SI (SI ≤ 1= (SICC =50 CC/EC5050/EC).50 Other). Other derivatives derivatives showed showed lower lower activity activity with with EC50 ECwithin50 within the thelow rangeselectivity of 47.4–125 index≤ SI ≤μ 1g/mL. (SI = CCHowever,50/EC50). Otherchemical derivatives modifications showed on lower this activityscaffold with might EC 50lead within to rangethe range of 47.4–125 of 47.4–125µg/mL. μg/mL. However, However, chemical chemical modifications modifications on this sca onffold this might scaffold lead might to compounds lead to compoundsthe range of with 47.4–125 enhanced μg/mL. activity However, as NNRTIs chemical [65]. modifications on this scaffold might lead to withcompounds enhanced with activity enhanced as NNRTIs activity [ 65as]. NNRTIs [65]. compounds with enhanced activity as NNRTIs [65]. H C Cl H3C N N Cl H3C H N N Cl 3 HN N N S NH S N S N S NH O S O S HN O O O R H O R R

butyl groups (2, 3) butyl groups (2, 3) Substitution with bulky groups CH(CH3)2 (1) or CH2CH2SCH3 (5) butyl groups (2, 3) Substitution with bulky groups CH(CH3)2 (1) or CH2CH2SCH3 (5) decreasedSubstitution antiviral with bulky activity groups CH(CH ) (1) or CH CH SCH (5) CH(OH)CH3 (4); decreased antiviral activity groups beneficial3 2 for antiviral2 2 activity3 CH(OH)CH (4); decreased antiviral activity CH(OH)CH3 (4); groups beneficial for antiviral activity 3 groups beneficial for antiviral activity Figure 1. Influence of substituents of 1,3,4-thiadiazole derivatives 1–5 on anti-HIV activity. Figure 1. InfluenceInfluence of of substituents substituents of of 1,3,4-thiadiazole 1,3,4-thiadiazole derivatives 1–5 on anti-HIV activity. Figure 1. Influence of substituents of 1,3,4-thiadiazole derivatives 1–5 on anti-HIV activity. Some 1,3,4-thiadiazole derivatives obtained by Hamad et al. [66] from amino acid analogs were Some 1,3,4-thiadiazole 1,3,4-thiadiazole derivatives derivatives obtained obtained by by Hamad Hamad et al. et [66] al. [from66] from amino amino acid analogs acid analogs were screenedSome for 1,3,4-thiadiazole anti-HIV-1 (strain derivatives IIIB) and obtained anti-HIV-2 by Hamad (strain et ROD) al. [66] acti fromvity amino by the acid inhibition analogs of were the werescreened screened for anti-HIV-1 for anti-HIV-1 (strain (strain IIIB) and IIIB) anti-HIV-2 and anti-HIV-2 (strain (strain ROD) ROD)activity activity by the byinhibition the inhibition of the virus-inducedscreened for anti-HIV-1 cytopathic (strain effect IIIB in) andhuman anti-HIV-2 MT-4 (straincells basedROD) action vity3-(4,5-dimethylthiazol-2-yl) by the inhibition of the ofvirus-induced the virus-induced cytopathic cytopathic effect effinect human in human MT-4 MT-4 cells cells based based on on 3-(4,5-dimethylthiazol-2-yl)3-(4,5-dimethylthiazol-2-yl) -2,5-diphenylvirus-induced tetrazoliumcytopathic effectbromide in human(MTT) assay.MT-4 cells2-(Naphthalen-2-yloxy)- based on 3-(4,5-dimethylthiazol-2-yl)N-((5-(phenylamino) -2,5-diphenyl-2,5-diphenyl tetrazoliumtetrazolium bromidebromide (MTT)(MTT) assay.assay. 2-(Naphthalen-2-yloxy)-2-(Naphthalen-2-yloxy)-N-((5-(phenylamino) -1,3,4-thiadiazol-2-yl)methyl)acetamide-2,5-diphenyl tetrazolium bromide (MTT) 6 showed assay. in vitro 2-(Naphthalen-2-yloxy)- inhibitory activity withN -((5-(phenylamino)EC50 values of 0.96 -1,3,4-thiadiazol-2-yl)methyl)acetamide-1,3,4-thiadiazol-2-yl)methyl)acetamide 66 showedshowed inin vitro vitro inhibitoryinhibitory activity activity with with EC50 EC values50 values of 0.96 of μ-1,3,4-thiadiazol-2-yl)methyl)acetamideg/mL (HIV-1 strain IIIB) and 2.92 μg/mL 6 showed (HIV-2 instrain vitro ROD), inhibitory respectively, activity withbut low EC 50selectivity values of (SI0.96 < 0.96μg/mLµg /(HIV-1mL (HIV-1 strain strain IIIB) IIIB)and 2.92 and μ 2.92g/mLµg /(HIV-2mL (HIV-2 strain strain ROD), ROD), respectively, respectively, but low but selectivity low selectivity (SI < 1).μg/mL Structure–activity (HIV-1 strain IIIB) relationship and 2.92 μ(SAR)g/mL (HIV-2studies strain have ROD), suggested respectively, that the but substitutionlow selectivity of (SIthe < (1).SI

Molecules 2020, 25, 942 5 of 22

A new class of HIV-1 NNRTIs, N-aryl-2-arylthioacetamides, has been identified in the last years [67–70]. Studies on the crystalline structure of the RT-NNRTI complex suggested that NNRTIs haveMolecules a common 2020, 25, 942 mode of action and interact with a hydrophobic pocket. N-aryl-2-arylthioacetamides5 of 22 adopted a butterfly-like conformation in which the arylthio moiety and the phenyl ring mimic the adopted a butterfly-like conformation in which the arylthio moiety and the phenyl ring mimic the butterfly wings. SAR studies showed that the arylthio moiety strongly influenced the antiviral activity, butterfly wings. SAR studies showed that the arylthio moiety strongly influenced the antiviral leading to different results depending on the steric/electronic properties of the groups [47]. Based on activity, leading to different results depending on the steric/electronic properties of the groups [47]. these findings, Xiaohe et al. synthesized 2-(5-amino-1,3,4-thiadiazol-2-ylthio)-N-(aryl) acetamide Based onMolecules these 2020 findings,, 25, 942 Xiaohe et al. synthesized 2-(5-amino-1,3,4-thiadiazol-2-ylthio)-5 of 22N -(aryl) derivatives 7–10 as new NNRTIs [47]. acetamide derivatives 7–10 as new NNRTIs [47]. adopted a butterfly-like conformation in which the arylthio moiety and the phenyl ring mimic the N N butterfly wings. SAR studies showed that the arylthioH moiety strongly influenced the antiviral activity, leading to different results depending on the steric/electronicN X properties of the groups [47]. H2N S S Based on these findings, Xiaohe et al. synthesized 2-(5-amino-1,3,4-thiadiazol-2-ylthio)-R N-(aryl) O X acetamide derivatives 7–10 as new NNRTIs [47]. N N 7-10 H N X H N S S 2 R Although they exhibited less anti-HIV-1 activityO X compared to standard drug zidovudine Although they exhibited less anti-HIV-1 activity compared to standard drug zidovudine (half-maximal inhibitory concentration IC50 = 0.016 μM), these compounds showed significant (half-maximal inhibitory concentration IC50 =7-100.016 µM), these compounds showed significant anti-HIV-1 activity at micromolar concentrations (IC50 within the range of 7.50–20.83 μM) (Table 1). It anti-HIV-1 activity at micromolar concentrations (IC within the range of 7.50–20.83 µM) (Table1). has been observedAlthough that they the exhibited 2-amino-1,3,4-thiadiazole less anti-HIV-1 activity50 moiety compared may to be standard a good drug group zidovudine for anti-HIV-1 It has been observed that the 2-amino-1,3,4-thiadiazole moiety may be a good group for anti-HIV-1 activity (half-maximalby providing inhibitory promising concentration antiviral agentsIC50 = 0.016. Moreover, μM), these the compoundselectronic propertiesshowed significant of the N-aryl activity by providing promising antiviral agents. Moreover, the electronic properties of the N-aryl group influencedanti-HIV-1 activity antiviral at micromolar potency. concentrations The introduc (IC50tion within of theelectron-withdra range of 7.50–20.83wing μM) (Tablegroups, 1). Itsuch as group influencedhas been observed antiviral that potency. the 2-amino-1,3,4-thiadiazole The introduction of moiety electron-withdrawing may be a good group groups, for anti-HIV-1 such as fluorine fluorine or trifluoromethyl on phenyl ring (derivatives 8 and 9), enhanced antiviral activity or trifluoromethylactivity by providing on phenyl promising ring (derivatives antiviral agents8 and. Moreover,9), enhanced the electronic antiviral properties activity of comparedthe N-aryl to the compared to the unsubstituted phenyl derivative 7. SAR studies suggested that the steric/electronic unsubstitutedgroup influenced phenyl derivative antiviral potency.7. SAR The studies introduc suggestedtion of thatelectron-withdra the steric/electronicwing groups, properties such as of the propertiesfluorine of theor trifluoromethylN-phenyl substituents on phenyl ringinfluenced (derivatives the 8antiretroviral and 9), enhanced activity antiviral more activity than their N-phenyl substituents influenced the antiretroviral activity more than their positions. In addition, positions.compared In addition, to the unsubstituted the nature phenyl of the derivative N-aryl 7ring. SAR influenced studies suggested the an thattiviral the steric/electronic potency as can be the nature of the N-aryl ring influenced the antiviral potency as can be observed for derivative 10 with observedproperties for derivative of the N10-phenyl with asubstituents pyrimidyl influenced ring which the wasantiretroviral the most activity active morecompound than their (Figure 2) a pyrimidyl ring which was the most active compound (Figure2)[47]. [47]. positions. In addition, the nature of the N-aryl ring influenced the antiviral potency as can be observed for derivative 10 with a pyrimidyl ring which was the most active compound (Figure 2)

[47]. Table 1. Structural details and IC50 values of compounds 7–10 (adapted from [47]). Table 1. Structural details and IC50 values of compounds 7–10 (adapted from [47]).

Table 1. Structural details andX IC50 values of Rcompounds 7–10 (adapted IC50 ( µfromM) [47]). X R IC50 (μM) 7 CHX R H IC50 (μM) 20.83 1.17 7 CH H 20.83 ± 1.17 ± 8 7 CHCH H 2,5-F220.83 ± 1.17 16.10 0.24 8 CH 2,5-F2 16.10 ± 0.24 ± 9 8 CHCH 2,5-F 3,5-(CF2 3 16.10)2 ± 0.24 14.93 0.84 9 CH 3,5-(CF3)2 14.93 ± 0.84 ± 10 9 CHN 3,5-(CF 4,6-(OCH3)2 14.933)2 ± 0.84 7.50 1.06 3 2 ± 10 10 N N 4,6-(OCH4,6-(OCH3))2 7.507.50 ± 1.06± 1.06 N NN N H H N N H N H2NS SS S 2 RR substitutionsubstitution with with F atoms F atoms (8, 9) (8, 9) O O enhancesenhances antiviral antiviral activity activity

X = CH unsubstitutet phenyl decreases activity (7) X = CH unsubstitutet phenyl decreases activity (7) N N H N N N X H2N S S H R NO XX H N S S 2 R 7-10O X X = N heterocyclic ring increases activity 7-10 N N HX = N heterocyclic ring increases activity N N H N S S 2 R introduction of CH O group (10) N N O N 3 H strengthens antiviral activity N N H N S S Figure 2. The2 influence of substituents of 1,3,4-thiadiazoleR introduction derivatives of7 –CH10 onO group anti-HIV (10) activity. Figure 2. The influence ofO substituentsN of 1,3,4-thiadiazole derivatives 7–10 on anti-HIV3 activity. strengthens antiviral activity

Figure 2. The influence of substituents of 1,3,4-thiadiazole derivatives 7–10 on anti-HIV activity.

Molecules 2020, 25, 942 6 of 22

Molecules 2020, 25, 942 6 of 22 MoleculesMolecular 2020, 25, 942 modeling studies for the pyrimidyl derivative 10 showed the formation of two potential6 of 22 Molecular modeling studies for the pyrimidyl derivative 10 showed the formation of two intermolecular hydrogen bonds involving a nitrogen atom and the amino group of the thiadiazole ring potential intermolecular hydrogen bonds involving a nitrogen atom and the amino group of the and aminoacidsMolecular modeling from NNBS studies of RT. for In addition,the pyrimidyl the electron-deficient derivative 10 showed pyrimidine the formation ring of the of ligand two thiadiazole ring and aminoacids from NNBS of RT. In addition, the electron-deficient pyrimidine establishespotential intermolecularπ–π interactions hydrogen with the bonds electron-rich involvin benzeneg a nitrogen rings of atom RT. Despite and the the amino docking group simulation of the ring of the ligand establishes π–π interactions with the electron-rich benzene rings of RT. Despite the results,thiadiazole the inhibitoryring and aminoacids activity of compoundfrom NNBS10 ofagainst RT. In addition, HIV-1 (strain the electron-deficient IIIB) replication in pyrimidine MT-4 cell docking simulation results, the inhibitory activity of compound 10 against HIV-1 (strain IIIB) culturering of the was ligand lower establishes than that of π zidovudine.–π interactions However, with the given electron-rich the butterfly-like benzene orientation rings of RT. as Despite a necessary the replication in MT-4 cell culture was lower than that of zidovudine. However, given the butterfly-like structuraldocking simulation condition forresults, antiretroviral the inhibitory activity, theseactivity results of compound reveal the promising10 against inhibitory HIV-1 (strain potential IIIB) of orientation as a necessary structural condition for antiretroviral activity, these results reveal the thisreplication scaffold in [47 MT-4]. cell culture was lower than that of zidovudine. However, given the butterfly-like promising inhibitory potential of this scaffold [47]. orientationThe series as a of necessary 5-(pyridin-2-ylmethyl)-1,3,4-thiadiazol-2-amine structural condition for antiretroviral derivatives activity, these11–16 resultswere synthesized reveal the The series of 5-(pyridin-2-ylmethyl)-1,3,4-thiadiazol-2-amine derivatives 11–16 were bypromising intramolecular inhibitory cyclization potential of of the this corresponding scaffold [47]. hydrazinecarbothioamides under acidic conditions synthesized by intramolecular cyclization of the corresponding hydrazinecarbothioamides under and theThe activity series against of 5-(pyridin-2-ylmethyl)-1,3,4-thiadiazol-2-amine HIV-1 was tested on MT-4 cells by MTT assay using derivatives efavirenz as11 a–16 standard were acidic conditions and the activity against HIV-1 was tested on MT-4 cells by MTT assay using drugsynthesized [64]. by intramolecular cyclization of the corresponding hydrazinecarbothioamides under acidicefavirenz conditions as a standard and the drug activity [64]. against HIV-1 was tested on MT-4 cells by MTT assay using efavirenz as a standard drug [64]. N N Ar N N N N S H Ar N S N 11-16 H Except for derivative 11 which showed low11-16 activity (EC50 values of 47 μM), the activity of the Except for derivative 11 which showed low activity (EC50 values of 47 µM), the activity of the otherExcept compounds for derivative was even 11 lower which (EC showed50 values low > 70activity μM) proving(EC50 values that substitutionof 47 μM), the with activity halogens of the or other compounds was even lower (EC50 values > 70 µM) proving that substitution with halogens or halogenoalkyl groups at C3 and/or C4 of phenyl ring led to a decrease or loss of activity (Figure 3). halogenoalkylother compounds groups was ateven C3 lower and/or (EC C450 of values phenyl > 70 ring μM) led proving to a decrease that substitution or loss of activity with halogens (Figure3 or). Although pyridine derivatives did not exhibit selective anti-HIV-1 activity, the fact that compound Althoughhalogenoalkyl pyridine groups derivatives at C3 and/or did not C4 exhibitof phenyl selective ring led anti-HIV-1 to a decrease activity, or theloss fact of activity that compound (Figure 3).11 11 showed some antiviral activity may encourage further research on this structure. Subsequent showedAlthough some pyridine antiviral derivatives activity may did encouragenot exhibit further selective research anti-HIV-1 on this activity, structure. the Subsequentfact that compound chemical chemical modifications by substitutions on aromatic rings with different groups may lead to modifications11 showed some by substitutionsantiviral activity on aromatic may encourage rings with fu dirtherfferent research groups on may this lead structure. to compounds Subsequent with compounds with improved activity [64]. improvedchemical modifications activity [64]. by substitutions on aromatic rings with different groups may lead to compounds with improved activity [64]. CH3 substitution with methyl beneficial for activity

Ar = CH3 substitution with methyl beneficial for activity Cl Ar = 11 Cl 11 halogen or halogenoalkyl groups at C3 μ decreasehalogen or the halogenoalkyl activity, EC50 groups values at > C370 M CF3 Br decrease the activity, EC values > 70 μM 12 13 50 CF3 Br 12 13 F Cl introduction of halogen at C3 and/or C4 Cl F μ introductionloss of activity, of halogen EC50 values at C3 >100 and/or M C4 Cl Cl Cl μ loss of activity, EC50 values >100 M 14 Cl 15 Cl 16 Cl 14 15 16 Figure 3. The influence of substituents of 1,3,4-thiadiazole derivatives 11–16 on anti-HIV activity. Figure 3. TheThe influence influence of substituents of 1,3,4-thiadiazole derivatives 11–16 on anti-HIV activity. The invasion of the central nervous system (CNS) by the HIV-1 virus frequently causes brain The invasion of the central nervous system (CNS) by the HIV-1 virus frequently causes brain inflammationThe invasion and of progressive the central nervousneurological system diseases, (CNS) bywhich the HIV-1are commonly virus frequently referred causes to as brainHIV inflammation and progressive neurological diseases, which are commonly referred to as HIV associated inflammationassociated neurocognitive and progressive disorders neurological (HAND) diseases,[71]. HAND which affects are 7%–15%commonly of AIDSreferred patients to as and HIV is neurocognitive disorders (HAND) [71]. HAND affects 7%–15% of AIDS patients and is characterized associatedcharacterized neurocognitive by neuronal disordersdysfunction (HAND) including [71]. synaptic HAND damage,affects 7%–15% neuronal of degenerationAIDS patients and and cell is by neuronal dysfunction including synaptic damage, neuronal degeneration and cell dropout [72]. characterizeddropout [72]. Cellsby neuronal involved dysfunction in HAND includingpathogenes synapticis are macrophages damage, neuronal and microglia, degeneration which and are cellthe Cells involved in HAND pathogenesis are macrophages and microglia, which are the main targets dropoutmain targets [72]. ofCells HIV-1 involved infection in HANDin the brain. pathogenes When isinfected are macrophages with HIV-1, and macrophages microglia, whichand microglia are the of HIV-1 infection in the brain. When infected with HIV-1, macrophages and microglia increase the mainincrease targets the ofproduction HIV-1 infection and releasein the brain. of several When infectedsoluble withneurotoxic HIV-1, factors,macrophages such andas glutamate, microglia production and release of several soluble neurotoxic factors, such as glutamate, inducing neuronal increaseinducing theneuronal production damage and [71]. release of several soluble neurotoxic factors, such as glutamate, damage [71]. inducingGlutamine neuronal is damagethe most [71]. abundant amino acid in the human body and is involved in more Glutamine is the most abundant amino acid in the human body and is involved in more metabolic metabolicGlutamine processes is the than most any abundant other aminoamino acid.acid inGlutamine the human is producedbody and fromis involved glutamate in more and processes than any other amino acid. Glutamine is produced from glutamate and ammonia by the metabolicammonia byprocesses the enzyme than glutamine any other synthetase amino acid. [73,74]. Glutamine The conversion is produced of glutamine from glutamateto glutamate and is ammoniacatalyzed byby thethe enzymemitochondrial glutamine enzyme, synthetase glutaminase [73,74]. [72,75]. The conversion The glutamine/glutamate of glutamine to glutamate cycle in the is catalyzedhuman body by the plays mitochondrial several important enzyme, metabolicglutaminase functions. [72,75]. TheThus, glutamine/glutamate glutamine and glutamate cycle in theare human body plays several important metabolic functions. Thus, glutamine and glutamate are

Molecules 2020, 25, 942 7 of 22

Moleculesenzyme 2020 glutamine, 25, 942 synthetase [73,74]. The conversion of glutamine to glutamate is catalyzed7 of by22 the mitochondrial enzyme, glutaminase [72,75]. The glutamine/glutamate cycle in the human body playsprecursors several to importantthe biosynthesis metabolic of proteins, functions. neurot Thus,ransmitters, glutamine nucleotides, and glutamate nucleic are precursors acids and toother the importantbiosynthesis biological of proteins, molecules. neurotransmitters, The glutamine/glut nucleotides,amate nucleic cycle acidsis the and substrate other importantfor the synthesis biological of ureamolecules. in the Theliver, glutamine genesis of/glutamate ammonia cycle in the is kidn the substrateeys and for for hepatic the synthesis and renal of urea gluconeogenesis. in the liver, genesis The glutamine/glutamateof ammonia in the kidneys exchange and regulates for hepatic the andacid-base renal balance gluconeogenesis. in kidneys, The acts glutamine as an oxidative/glutamate fuel forexchange the intestines regulates and the cells acid-base of the balance immune in kidneys,system and acts asprovides an oxidative the transport fuel for the of intestinesnitrogen andbetween cells organsof the immune [74–76]. systemThe existence and provides of a glutamine/glutam the transport ofate nitrogen cycle in between CNS was organs confirmed [74–76 in]. Thethe last existence years [76,77].of a glutamine Phosphate-activated/glutamate cycle mitochondrial in CNS was confirmed glutaminase in the is lastthe yearspredominant [76,77]. Phosphate-activated enzyme that uses glutaminemitochondrial in glutaminasethe brain. Glutamin is the predominante is present enzyme in the that extracellula uses glutaminer fluid in theof brain.the brain Glutamine at high is presentconcentrations in the extracellular and provides fluid an of abundant the brain atsubstrate high concentrations for glutaminase and provides[72,75]. Therefore, an abundant it has substrate been hypothesizedfor glutaminase that [72 ,75mitochondrial]. Therefore, itglutaminase has been hypothesized activation thatis mitochondrialresponsible for glutaminase the high activationlevels of glutamateis responsible in forthe thebrains high of levels HIV-1 of glutamateinfected pati in theents brains [72]. ofWhile HIV-1 glutamate infected patientsmediates [72 different]. While physiologicglutamate mediates processes, diff erentelevated physiologic extracellular processes, concentrations elevated extracellular of glutamat concentrationse can induce of glutamateneuronal damagecan induce (e.g., neuronal dementia, damage brain (e.g., atrophy) dementia, [71,72]. brain atrophy) [71,72]. Some glutaminase inhibitors (e.g., 6-diazo-5-oxo-L-norleucine,-norleucine, etc.) etc.) were were studied studied inin vitro vitro for their ability to preventprevent thethe generationgeneration ofof glutamateglutamate byby HIV-1HIV-1 infectedinfected macrophages.macrophages. The results support the hypothesis that glutaminase mediates glutamate generation in HIV-infected human macrophages. When When glutaminase glutaminase was was inhibited inhibited by by various inhibitors, HIV-induced glutamate production decreased and the neuronal damage waswas diminisheddiminished [[71,72,78].71,72,78]. Furthermore, for some glutaminase inhibitors, a non-competitive mechanis mechanismm of inhibition has been described [[78].78]. These findingsfindings support glutaminase as a potential comp componentonent of the HAND process and can provide a new therapeutic targettarget for for the the treatment treatment of neurocognitive of neurocognitive disorders disorders associated associated with HIV with infection HIV [infection71,72,78]. [71,72,78].In connection In withconnection these results, with th a largeese results, number a oflarge glutaminase number inhibitorsof glutaminase having ainhibitors bis-thiadiazole having (17 a) bis-thiadiazoleand a pyridazine-thiadiazole (17) and a pyridazine-thiadiazole (18) skeleton (Figure4), ( respectively,18) skeleton were (Figure synthesized 4), respectively, as a method were of synthesizedtreating or preventing as a method multiple of treating viral or infections, preventing including multiple infections viral infections, with retroviruses including infections [75]. with retroviruses [75]. O O N N N N N N N N X Y X N R R N S N HN S H S Y Y R1 R2 Y R1 R2

17 18

X = CH2SCH2, CH2CH2, CH2CH2CH2, CH2, CH2S, SCH2, CH2NHCH2, CH=CH

Y = H or CH2O(CO)R'

R1, R2 = H, alkyl, alkoxy, hydroxy R = alkyl, hydroxyalkyl, aminoalkyl, acylaminoalkyl, alkenyl, alkoxy, aryl, arylalkyl, aryloxyalkyl, heteroaryl, heteroarylalkyl, heteroaryloxy, heteroaryloxyalkyl, etc. R' = H, alkyl, alkoxy, aminoalkyl, alkylaminoalkyl, heterocyclylalkyl, arylalkyl or heterocyclylalkoxy

Figure 4. The general formula of glutaminase inhibitors 17 and 18. Figure 4. The general formula of glutaminase inhibitors 17 and 18. Multiple experiments were performed in order to study the biological profile of the compounds. Multiple experiments were performed in order to study the biological profile of the Some of the synthesized derivatives are prodrugs, which under physiologic conditions (in vivo), are compounds. Some of the synthesized derivatives are prodrugs, which under physiologic conditions converted into the therapeutically active parent compound. Studies have also been conducted to obtain (in vivo), are converted into the therapeutically active parent compound. Studies have also been pharmaceutical preparations suitable for use in human patients comprising any of the synthesized conducted to obtain pharmaceutical preparations suitable for use in human patients comprising any derivatives and one or more pharmaceutically acceptable excipients. In addition, the authors assume of the synthesized derivatives and one or more pharmaceutically acceptable excipients. In addition, that the derivatives may be used alone or in combination with known antiviral drugs. Studies on the authors assume that the derivatives may be used alone or in combination with known antiviral drugs. Studies on kidney-type glutaminase inhibition showed good results for many derivatives such as compound 19 with an IC50 value of 0.24 μM and its deuterium derivative 20 with an IC50 value of 0.54 μM [75].

Molecules 2020, 25, 942 8 of 22 kidney-type glutaminase inhibition showed good results for many derivatives such as compound 19 with anMolecules IC50 2020value, 25, of942 0.24 µM and its deuterium derivative 20 with an IC50 value of 0.54 µM[8 of75 22].

N N OCF3 N N D D OCF3 N N H2N S N O H2N S N O Molecules 2020, 25, 942 D D 8 of 22 N N H H N N OCF3 N N D D OCF3 19N 20 N H2N S N O H2N S N O D D 3.2. Human Cytomegalovirus (HCMV)N N 3.2. Human Cytomegalovirus (HCMV)H H HumanHuman cytomegalovirus cytomegalovirus19 (HCMV, (HCMV,Herpesviridae Herpesviridaefamily) family) is ais ubiquitous a ubiquitous 20 deoxyribonucleic deoxyribonucleic acid acid virus virus that infects people of all ages [79,80]. HCMV infection can be acquired through horizontal and that infects people of all ages [79,80]. HCMV infection can be acquired through horizontal and vertical vertical transmission. HCMV spreads from infected people through direct contact with body fluids transmission.3.2. Human HCMV Cytomegalovirus spreads from(HCMV) infected people through direct contact with body fluids that carry that carry the virus, such as urine, saliva, cervicovaginal secretions, sperm and breast milk. Vertical the virus,transmissionHuman such as cytomegalovirus urine,through saliva, organ cervicovaginal (HCMV,transplantation, Herpesviridae secretions, from family)mother sperm isto a and ubiquitouschild breast or transmission milk. deoxyribonucleic Vertical via transmission blood acid throughvirustransfusion, organ that infects transplantation, is also people possible of all from[79,80].ages [79,80]. mother Blood HCMV totests child indicateinfection or transmission thatcan be60%–90% acquired via of through blood the adult transfusion, horizontal population and is also possibleverticalexperienced [79,80 transmission.]. BloodHCMV tests infection HCMV indicate spreadsat some that 60%–90%fromtime infectedduring of thetheir people adult life through [81]. population Although direct experienced contact most of with these HCMVbody infections fluids infection at somethatare timeasymptomatic, carry during the virus, their certain such life as patient [81 urine,]. Although grou saliva,ps suchcervic most asovaginal ofbabies these that secretions, infections are infe ctedsperm are before asymptomatic, and birthbreast and milk. children certain Vertical patientor groupstransmissionadults such with as babiesweakened through that immuneorgan are infected transplantation, systems before due to birth fromdiseas and motheres or children medications to child or adults or(e.g., transmission withHIV-infected weakened via patients, blood immune transfusion, is also possible [79,80]. Blood tests indicate that 60%–90% of the adult population systemsorgan due transplant to diseases recipients) or medications can develop (e.g., severe HIV-infected illnesses that patients, require medical organ transplanttreatment [79]. recipients) HCMV can experienced HCMV infection at some time during their life [81]. Although most of these infections developis able severe to remain illnesses latent that in requireseveral cells medical of the treatment human body [79]. for HCMV a long is time able and to remaincan be reactivated latent in several if arethe asymptomatic,person develops certain immune patient system grou suppressionps such as babies [79,82]. that are infected before birth and children or cells of the human body for a long time and can be reactivated if the person develops immune system adultsThe with first-line weakened drugs immune recommended systems due for to the diseas treaestment or medications of HCMV (e.g., infection HIV-infected are intravenous patients, suppression [79,82]. organganciclovir transplant or orally recipients) administered can develop valganciclovir severe illnesses [83]. that Although require tolerabilitymedical treatment of ganciclovir [79]. HCMV and Theisvalganciclovir able first-line to remain drugs is latentacceptable, recommended in several hematological cells for of the the treatment orhuman neurological body of HCMV for sidea long infection effects time canand are canoccur. intravenous be reactivatedNeutropenia, ganciclovir if or orallythethrombocytopenia person administered develops and valganciclovir immune anemia system are th [ 83suppressione main]. Although toxic [79,82].effects tolerability that limit of therapy ganciclovir with these and drugs. valganciclovir Serum is acceptable,creatinineThe hematological first-line levels may drugs orincrease neurologicalrecommended during sideganciclovi for etheffects trear cantherapy,tment occur. ofwhich Neutropenia,HCMV requires infection monitoring thrombocytopenia are intravenous of renal and anemiaganciclovirfunction are the [84]. mainor orallyEncephalopathy toxic administered effects thatis thevalganciclovir limit neurotoxic therapy [83]. witheffect Although theseof ganciclovir drugs. tolerability Serum and valganciclovirof creatinine ganciclovir levels [85].and may increasevalganciclovirFoscarnet during is gancicloviralso is aacceptable, very effectiv therapy, hematologicale anti-HCMV which requires ordrug, neurological monitoring and cidofovir side of is renaleffects a broad-spectrum function can occur. [84]. antiviralNeutropenia, Encephalopathy with thrombocytopenia and anemia are the main toxic effects that limit therapy with these drugs. Serum is thegood neurotoxic activity against effect ofHCMV. ganciclovir Both drugs and cause valganciclovir a high level [of85 ].nephrotoxicity Foscarnet isthat also limits a verytreatment effective creatinine levels may increase during ganciclovir therapy, which requires monitoring of renal anti-HCMV[83]. drug, and cidofovir is a broad-spectrum antiviral with good activity against HCMV. Both functionNovel [84]. 2-amino-1,3,4-thiadiazole Encephalopathy is the derivatives neurotoxic with effect antiviral of ganciclovir activity against and valganciclovir HCMV have been[85]. drugs cause a high level of nephrotoxicity that limits treatment [83]. Foscarnetpatented [86].is also A largea very number effectiv ofe anti-HCMV 472 synthesized drug, compounds and cidofovir were is atested broad-spectrum in an HCMV antiviral polymerase with Novel 2-amino-1,3,4-thiadiazole derivatives with antiviral activity against HCMV have been goodassay activityat a concentration against HCMV. of 25 Both μM. drugs The degreecause a ofhigh enzyme level ofinhibition nephrotoxicity ranged that from limits 20% treatment to 100%. patented[83].Among [86 ].the A most large active number compounds, of 472 synthesized four derivatives compounds exhibited a were 100% tested inhibition in an rate, HCMV 29 derivatives polymerase assayshowed at aNovel concentration an 2-amino-1,3,4-thiadiazole inhibition ofrate 25 ofµM. 90.1%–99.9% The derivatives degree and ofwith 16 enzyme antiviralderivatives inhibition activity showed against ranged an HCMVinhibition from have 20% rate been to of 100%. Amongpatented80.3%–89.9%. the most [86]. active ThreeA large compounds,structural number ofseries 472 four standsynthesized derivatives out among compounds exhibited the most were aactive 100% tested 1,3,4-thiadiazole inhibition in an HCMV rate, polymerasederivatives: 29 derivatives showedassay1,3-dioxo-1,3-dihydro-2-benzofuran-5-carboxamide an at inhibitiona concentration rate of of 25 90.1%–99.9% μM. The degree and of 16enzymederivatives derivatives inhibition such showedas ranged 21–30 ,froman 9-octadecenamide inhibition 20% to 100%. rate of Among the most active compounds, four derivatives exhibited a 100% inhibition rate, 29 derivatives 80.3%–89.9%.derivatives Three such as structural 31–38 and series 2-ethoxy-1-naphthamide stand out among thederivatives most active such as 1,3,4-thiadiazole 39–42 (Tables 2, 3 derivatives: and 4). showed an inhibition rate of 90.1%–99.9% and 16 derivatives showed an inhibition rate of 1,3-dioxo-1,3-dihydro-2-benzofuran-5-carboxamideMost of the derivatives belong to the 1,3-dioxo-1,3-dihydro-2-benzofuran-5-carboxamide derivatives such as 21–30, 9-octadecenamide series. At 80.3%–89.9%. Three structural series stand out among the most active 1,3,4-thiadiazole derivatives: derivativesthe same such time, as the31 most–38 andactive 2-ethoxy-1-naphthamide compounds belong to this derivativesseries, so it can such be asconcluded39–42 (Tablesthat the2 –4). 1,3-dioxo-1,3-dihydro-2-benzofuran-5-carboxamide derivativesmoiety is asuch good as scaffold21–30, 9-octadecenamide for anti-HCMV Most of the derivatives belong to the 1,3-dioxo-1,3-dihydro-2-benzofuran-5-carboxamide series. derivativesactivity [86]. such as 31–38 and 2-ethoxy-1-naphthamide derivatives such as 39–42 (Tables 2, 3 and 4). At the same time, the most active compounds belong to this series, so it can be concluded that Most of the derivatives belong to the 1,3-dioxo-1,3-dihydro-2-benzofuran-5-carboxamideN Nseries. At the 1,3-dioxo-1,3-dihydro-2-benzofuran-5-carboxamidethe sameO time, the most active compounds belong to this moiety series, is so a it good can be sca concludedffold for that anti-HCMV theR S N 1,3-dioxo-1,3-dihydro-2-benzofuran-5-carboxamideN N N Omoiety is a good scaffold HNfor anti-HCMVH activity [O86]. N activity [86]. HN R S R N S N O O N H H N N O H O OC2H5 R S N N N N O HN H O N 21-30HN R 31-38 39-42 S R N S N O O N H H O H OC2H5

21-30 31-38 39-42

Molecules 2020, 25, 942 9 of 22 MoleculesMolecules 20202020,, 2525,, 942942 99 ofof 2222 MoleculesMolecules 2020 2020, 25,, 25942, 942 9 of9 22of 22 MoleculesMolecules 2020 20202020, 25,, 2525, 942,, 942942 9 9of9 of of22 2222 MoleculesMolecules 2020 2020, 25, ,942 25, 942 9 of 922 of 22 MoleculesMolecules 2020 2020, 25,, 94225, 942 9 of 922 of 22 Table 2.MoleculesStructural 2020, 25 details, 942 and human cytomegalovirus (HCMV) polymerase inhibition values of9 of 22 TableTableTable 2. 2.Structural2. StructuralStructural details detailsdetails and andand human humanhuman cytomegalovirus cytomegaloviruscytomegalovirus (HCMV) (HCMV)(HCMV) polymerase polymerasepolymerase inhibition inhibitioninhibition values valuesvalues of ofof compoundsTable21 2.– 30Structural(adapted details from and [86]). human cytomegalovirus (HCMV) polymerase inhibition values of TableTablecompounds 2. 2. Structural Structural 21– 30details details (adapted and and fromhuman human [86]). cytomegalovirus cytomegalovirus (HCMV) (HCMV) polymerase polymerase inhibition inhibition values values of of TablecompoundsTableTablecompounds 2. 2. Structural 2.Structural Structural 21 –2130– 30(adapteddetails details (adapted details and andfrom and fromhuman human [86]). human [86]). cytomegalovirus cytomegalovirus cytomegalovirus (HCMV) (HCMV) (HCMV) polymerase polymerase polymerase inhibition inhibition inhibition values values values of of of compoundsTablecompoundsTablecompounds 2. Structural2. 21Structural 21–30 –2130 (adapted– 30(adapteddetails details(adapted andfrom andfrom fromhuman [86]).human [86]). [86]). cytomegaloviruscytomegalovirus (HCMV)(HCMV) polymerase polymerase inhibition inhibition values values of of compoundscompoundscompoundsTable 21 21– 3021–2.30 –R(adaptedStructural30 (adapted (adapted fromdetails from from Inhibition[86]). [86]).and [86]). human (%) cytomegalovirus (HCMV) Rpolymerase inhibition Inhibition values (%) of compounds compounds 21– 2130– 30 R(adapted(adapted (adapted R R fromfrom from [86]).[86]). Inhibition[86]). Inhibition Inhibition (%) (%)(%) R R R Inhibition Inhibition Inhibition (%) (%)(%) Molecules compounds 2020, 25 R, R942 R 21 –30 (adapted Inhibition Inhibition from Inhibition [86]). (%) (%) (%) R R R Inhibition Inhibition Inhibition (%) (%)9 of (%) 22 R R R R Inhibition Inhibition Inhibition Inhibition (%) (%) (%) R R R R Inhibition Inhibition Inhibition Inhibition (%) (%) (%) (%) R R Inhibition Inhibition (%) (%) R R Inhibition Inhibition (%) (%) Molecules 2020, 25, 942 9 of 22 21Molecules 2020, 25(CH, 942(CH(CH 22))101022CH))1010CHCH33 33 100100100100 26 9 of 22 99.8 21 Table 2.(CH Structural2)10CH details3 and human100 cytomegalovirus 26 (HCMV) polymerase inhibition values 99.8 of Molecules 21Molecules 21 2020 2020,(CH 25, (CH25, 942, 9422 )102 )CH10CH3 3 100100 26 26 9 of9 of22 22 99.8 99.8 21 (CH(CH2)2)10210CHCH10 33 3 100100 26 99.8 21 21 compounds (CH(CH2)10 21CH)–30CH3 (adapted from [86]).100100 26 26 99.899.8 2121 compounds(CH(CH2))10 CH21(CH–303 (adapted2)10CH3 from [86]).100 100 2626 99.8 99.8 21 21Table 2. Structural details and human cytomegalovirus (HCMV)26 26 polymerase inhibition values of 99.899.8 21 Table 2.21 Structural details and human cytomegalovirus (HCMV)26 26 polymerase inhibition values of 99.899.8 Moleculescompounds 2020, 25, 942 21–30 (adapted R from [86]). Inhibition (%) R 9 Inhibitionof 22 (%) Molecules 2020Table, 25 , 2.942 Structural details and human cytomegalovirus (HCMV) polymerase inhibition values 9 of of 22 compoundsTable 2. Structural 21–30 (adapted details andfrom human [86]). cytomegalovirus (HCMV) polymerase inhibition values of 22 compounds 21– R30 (adapted from Inhibition [86]). 100100 (%)100100 27 R Inhibition (%) 99.8 22 22 22compounds 21–30 (adapted from [86]). 100 27 27 27 S 99.8 99.899.8 22 R Inhibition100100100 (%) 27 R InhibitionS S (%) 99.8 2222 Table22 2. Structural details R and human Inhibition cytomegalovirus100 100(%) 100 (HCMV)2727 polymerase R inhibitionS Inhibition valuesS of (%) 99.8 99.8 99.8 2222 Table22 2. 22Structural R(CHdetails 2)10 andCH human3 Inhibition cytomegalovirus100 (%) 100 (HCMV)2727 27 polymerase27 R inhibition InhibitionSS values (%)of 99.899.899.899.8 22 compounds 21 21–30 (adapted from [86]). 27 26 S SS 99.899.8 2 10 3 S compounds 21(CH–30 (adapted) CH from [86]). 100 21 (CH2)10CH3 100 26 99.8

21 (CH R 2)10CH3 Inhibition100 (%) 100100 26 R Inhibition99.8 (%) 23 23 21 (CH R2 )10CHN3 Inhibition100100 (%)100100 100 26 28 28 R Inhibition99.8 (%) 99.7 99.7 23 23 21 N N 100100 26 28 28 99.8NH 99.7 99.7 2323 23 NHNH 100100100 100 100 28 28 28 NHNH 99.7 99.7 99.7 2323 23 NNHH N 100 100 2828 28 NH NH 99.799.799.7 23 23 22 NH NH 100 28 2728 NHSNH NH 99.899.7 99.7 23 22 (CH2)10CHHNH3 H H 100 27 28 SNH NH 99.8 NH 99.7 21 (CH2)10HCHHHH3 100100 26 S NH99.8 2122 HHNH 100 2627 S 99.899.8 22 HHNN N H 100 27 99.8 22 HH HN 27 S S 99.8 NHN N N N 99.999.999.999.9 100 24 24 24 24 N 10099.9 99.9100 99.9 29 29 29 29 29 99.6 99.6 99.6 99.699.6 24 23 23 N N 100100 99.9 99.9 28 2829 99.7 99.7 99.6 24 24 22 24 H 100 99.999.9 27 29 29 29 S NH 99.8NH 99.699.6 99.6 242224 23 NH H 10099.9 2728 2929 99.899.7 99.699.6 24 23 H N 100 28 29 S NH 99.7 99.6 24 23 N 28 29 NH Cl 99.7Cl 99.6 H HH NH Cl Cl HN H N Cl N 99.8 Cl Cl NHH 10099.9 99.899.899.8 Cl Cl 232525 24 NN 100 99.899.92899.8 29 30 3030 99.7Cl99.6 99.5 99.599.5 2523 2425 NN F F 99.999.8 99.8 28 3029 30 NH Cl99.7 Cl 99.699.5 99.5 2525 25 2524 24 H F F F 99.899.899.899.8 29 30 30 3029 30 NH 99.6Cl Cl 99.699.599.5 99.5 99.5 2525 25 H F F F 99.999.999.8 3030 30 Cl 99.599.599.5 25 2424 H F F 2929 30 Cl99.6Cl99.6 99.5 25 F 30 Cl Cl Cl 99.5 Table 3. StructuralHN detailsF and HCMV polymerase inhibition valuesCl of compoundsClCl 31–38 (adapted TableTable 3.3. StructuralStructuralN detailsdetails andand HCMVHCMV99.8 polymerasepolymerase inhibitioninhibition valuesvalues ofof compounds compounds 3131––3838 (adapted(adapted TableTable 25 3. TableStructural3. Structural 3. Structural details details detailsand and HCMV99.9 andHCMV HCMV polymerase polymerase polymerase30 inhibition inhibition inhibition valuesCl values Clvalues of compoundsofof compounds 99.5 31 31 –3138– –38 38(adapted (adapted (adapted TableTable24from 3. 3. [86]).Structural Structural details detailsF and and HCMV HCMV99.999.8 polymerase polymerase99.829 inhibition inhibition values valuesCl of of compounds compounds99.6 31 31–38–38 (adapted (adapted TablefromTable24Tablefromfrom25 3.[86]). 3. 25 Structural[86]). [86]). from3.Structural Structural [86]). details details details and and and HCMV HCMV HCMV99.8 polymerase polymerase polymerase2930 inhibition inhibition30 inhibition values values values of of compounds ofcompounds99.6 compounds99.5 31 31– 383199.5–38 –(adapted38 (adapted (adapted TableTable 3.from Structural3.25 Structural [86]). details detailsF and and HCMV FHCMV 99.8 polymerase polymerase 30 inhibitioninhibition values valuesCl of of compounds compoundsCl99.5 3131–38–38 (adapted(adapted(adapted fromfromfrom [86]). 25 [86]).[86]). R F Inhibition30 (%) Cl R 99.5 Inhibition (%) fromfrom [86]). [86]). F ClCl fromfrom [ 86 [86]).Table ]). 3. Structural Rdetails R R and R HCMV polymerase Inhibition Inhibition Inhibition Inhibition inhibition (%) (%)(%) values (%) of compounds Cl R 31 R R R–38 (adapted Inhibition Inhibition Inhibition Inhibition (%) (%) (%)(%) Tablefrom [86]).3. Structural details R and HCMV99.8 polymerase Inhibition inhibition (%) values of compounds 31 R–38 (adapted Inhibition (%) 25 TableTable 3. Structural3. Structural R R R details details and HCMV and99.8 HCMV Inhibition polymerase Inhibition Inhibition polymerase30 inhibition (%) (%)(%) inhibition values of valuescompounds R of R R compounds 3199.5–38 (adapted Inhibition 31 Inhibition Inhibition– 38 (adapted (%) (%)(%) 25fromTable [86]). 3. Structural R details RF and HCMV polymerase Inhibition Inhibition30 inhibition (%) (%) values of compoundsClN R 31 R99.5–38 (adapted Inhibition Inhibition (%) (%) from [86]). RR F Inhibition Inhibition100 (%) (%) R Inhibition Inhibition (%) (%) 31from from from [86]). [86]). [86]). N R Inhibition (%) 100 35 R Cl N InhibitionN (%) 97.7 31 R Inhibition (%)100100 100 R 35 N InhibitionN (%) 97.7 31 H N 100 35 N 97.7 31Table 31 3. StructuralN detailsNN R and R HCMV polymerase Inhibition Inhibition100 inhibition100 (%) (%)values 35 of 35compounds R NN 31 R– Inhibition38 (adapted Inhibition(%) 97.7 97.7 (%) Table31 3. Structural details RN andH HCMV polymerase Inhibition inhibition(%)100100 values of 35compounds R N N31 Inhibition–38 (adapted (%) 97.7 3131 NNHCHHH 100 3535 N CHN 3 97.797.7 313131 from31 [86]). NNH3 100 100 353535 35 N CH 97.797.797.797.7 31 from 31 [86]). HNHN CH3 100 35 35 N CH 397.7 97.7 31 NNHHCHCH3 35 CH CH33 97.7 31 HCHNH 3 3 10099.9 35 N N CH3 97.7 H R CHN3 Inhibition100 (%) 100 N R CHN CH Inhibition3 (%) 32 31 CHCHH RN3 3 Inhibition (%) 99.935 36 RCH InhibitionCH3 N3 97.7 (%) 97.0 31 31 NCHCHNCHNNCH33N 35 35 3 CH 3CH3 397.7 97.7 32 CHH3 3 99.999.9 H 363C 3N 97.0 CHCHHN3 99.9 CH3 CHN3N 32 32 NNN 33 H 99.9 3636 H C N 97.097.0 32 NNCH 99.999.9 99.9 N 36N CH3 3N 97.0 32 32 NNCH3 3 100 99.999.9 36 CH363 HNCN 97.0 97.0 323232 31 N CH3 10099.999.9 99.9 35 3636N36 N H3CH33NCCHN3 97.7 97.097.097.0 3232 32 32 H3C NNN N 3 99.9 36 H36C36 36 H 3NC 97.0 97.097.097.0 32 31 NHN 99.999.9 35 363 NHH3C3C 97.7 97.0 32 H3C NNN 36 H3C N HHC3HC3C 97.0 32 H 99 99.936 H 3C N 97.0 33 H C CHNN3 37H3CH C 3 H3C N 92.6 H323HC 33CCNNNNNN 99 H3CCH36 97.0 H333 C CHNN3 3 37 92.6 HHCHC3CNNNNNNN 99 H3C H 3HC33HCNNCNNNNN 99.999 99 99 N 3233 33H 3CHH3CNN3CNNNN 99.999 3699 37 37 97.092.6 92.6 3332 33 H 3 CH3NNNO S 9999 36 N37 37 97.0 92.6 92.6 33 3333 3333 2 2 9999 9999 99 37 H373C 37 37 92.6 92.692.692.6 92.6 33 33 H3CH2NONN2S 99 99 37H 3C37 37 92.6 92.6 33 3333 99 37 37 37 92.6 92.6 92.6 33 H 2NO2S 98.3 99 37 92.6 34 HH3CHHNOHNO2NNNONOSS2SS 98.3 38 91.8 H33C2 H2 NN2NO22 2S 98.3 37 92.6 34 H H3NO34HNOH NO22SNOSS22S 99 38 38 91.8 91.8 33 2H22HNO2 2NO222S S 9998.3 98.337 92.6 34 H2NO22S 2 98.398.3 38 91.8 3434 3334 34 H2NO2SH NO S 98.398.398.3 98.337 38 38 38 38 92.6 91.891.8 91.891.8 343434 H2NO2S 98.398.3 3838 38 91.891.8 91.8 3434 Table 4. Structural details and HCMV polymerase98.398.3 inhibition 3838 values of compounds 39–42 (adapted91.8 91.8 3434 34 Table H Table24.NO Structural2S 4. Structural details and details HCMV and polymerase HCMV98.3 polymeraseinhibition98.3 values38 inhibition38 38 of compounds values 39 of–42 compounds (adapted 3991.8–91.84291.8 (adapted 34 from [86]).H2NO 2S 98.3 38 91.8 Table34 4. Structural details and HCMV polymerase 98.3 inhibition values38 of compounds 39–42 (adapted 91.8 34from [86]).from [86]). 98.3 38 91.8 TableTablefromTableTable [86]). 4.4. 4. Structural Structural StructuralStructural details details detailsdetails and and HCMV andand HCMV HCMVHCMV polymerase polymerase polymerasepolymerase inhibition inhibition values inhibition inhibitionvalues of of compounds compounds valuesvalues 39 ofof 39– 42 compounds–compounds42 (adapted (adapted 3939––4242 (adapted(adapted Table34Table 4. Structural4. Structural R details details and and HCMV Inhibition HCMV polymerase polymerase (%)38 inhibition inhibition values values R of compoundsof91.8 compounds Inhibition 39 –3942– 42(adapted(%) (adapted TableTableTable 4.from Structural4.from 4. Structural [86]). Structural [86]). details Rdetails details and and and HCMV HCMV InhibitionHCMV polymerasepolymerase polymerase (%) inhibition inhibition R values values values of ofof compounds Inhibitioncompoundscompounds (%) 39 39–42–42 (adapted (adapted(adapted TablefromTableTablefromfrom 4.[86]). 4. Structural[86]). [86]). 4.Structural Structural details details details R and and and HCMV HCMV HCMV polymerase polymerase Inhibition polymerase inhibition (%) inhibition inhibition values values values of of compounds of Rcompounds compounds 39 39– Inhibition4239–42 –(adapted42 (adapted (adapted (%) Tablefrom 4. TableStructural [86]). 4. Structural R details details and HCMV Inhibitionand HCMV polymerase (%) polymerase inhibition inhibition R values values of of compounds Inhibition compoundsO (%) 3939 ––4242 (adapted(adapted(adapted fromfromfromfrom [Table86 [86]).]). [86]).[86]). 4. Structural details R and HCMV Inhibition polymerase (%) inhibition values of compounds R O 39–42 Inhibition (adapted (%) fromTablefrom [86]). from 4.[86]). Structural [86]). Rdetails and HCMV Inhibition polymerase (%) inhibition values of R compounds O 39 Inhibition–42 (adaptedO (%) from from [86]). [86]).from [86]). R R R Inhibition93.1 Inhibition Inhibition93.1 (%) (%)(%) R R R Inhibition Inhibition Inhibition (%) (%)(%) 39from 39 [86]). RSO NH 93.1 Inhibition 93.141 (%) 41 OO R 83.9 83.9 Inhibition (%) 39 R R SO2 2NH2 2 Inhibition Inhibition (%) 41(%) R R O 83.9 Inhibition Inhibition (%) (%) 39 R R RSO NH Inhibition Inhibition93.1 Inhibition93.1 (%) (%) (%) 41 O R R R Inhibition Inhibition Inhibition83.9 (%) (%) (%) 39 R R H R2 2 SO Inhibition2 InhibitionNH2 Inhibition (%) (%) 41 (%) R R R Inhibition 83.9 (%)OO Inhibition Inhibition (%) (%) 39 R H SO NH Inhibition Inhibition (%) (%) 41 R N O R Inhibition83.9O (%) O Inhibition (%) HNSO NH2 2 ON N 2 H2 93.1 OO N H 92.9 93.193.1 N O OOOO 3939 H N 93.1 41 41 N O N O 83.983.9 39 39 40 N SO2NH2 93.193.192.9 93.1 92.993.1 42 41 41 N O 83.1O 83.983.9 394039 N SOSO2NH2NH2 2 93.193.193.193.1 41 42 41 83.9O O 83.1 83.9 3939 40 39 SO NHSO2 NH 2 93.1 92.9 93.1 92.942 4141 41 O 83.1 O 83.983.983.9 39 3939 40 SOSO2 2NH22NHSO 2 2NH 92.9 41 41 4142 83.9OOO 83.983.183.9 39 4040 SOSOHHNHSO2SONH NH 22 NH 2 4242 41 O 83.1OO 83.1 O 83.9 39 HSO2 2NH2 22 2 41 N O 83.9 H SONH2NH2 N N O HHHN N2H 2 N N NHH HN 92.9 N NN NNHN N 92.9 92.992.9 NN N 404040 N N 92.9 92.942 4242 N 83.1 83.183.1 40 40 40 N 92.992.992.9 92.9 42 42 42 N 83.183.1 83.1 40 40 40 40 92.992.9 42 42 42 42 83.1 83.183.183.1 4040 40 92.9 4242 42 83.183.183.1 40 42 83.1

Molecules 2020, 25, 942 10 of 22

Molecules 2020, 25, 942 10 of 22 Other compounds also showed good inhibitory activity. These derivatives contain a five- to six-memberedOther compounds saturated also heterocyclic showed good moiety, inhibitory such as imidazolidinyl,activity. These derivatives tetrahydrofuryl, contain piperidinyl, a five- to morpholinyl,six-membered thiomorpholinyl saturated heterocyclic or 5- to 10-memberedmoiety, such aromaticas imidazolidinyl, or unsaturated tetrahydrofuryl, heterocyclic moietypiperidinyl, such asmorpholinyl, furyl, pyrrolyl, thiomorpholinyl pyridyl, benzothiazolyl, or 5- to 10-membere etc. (e.g., derivativesd aromatic43 or,44 unsaturated)[86]. heterocyclic moiety such as furyl, pyrrolyl, pyridyl, benzothiazolyl, etc. (e.g., derivatives 43,44) [86]. H N

NO H 2 H N O S S N NO HN O 2 HN O N N N N O

43 (83.3% inhibition) 44 (81.6% inhibition)

WhileWhile thethe synthesizedsynthesized derivativesderivatives have shown inhibitoryinhibitory activity against HCMV polymerase, theirtheir antiviral activity cannot cannot be be limited limited to to a aspecific specific mechanism mechanism of of action. action. These These compounds compounds may may be beactive active against against cytomegalovirus cytomegalovirus by HCMV by HCMV polymerase polymerase inhibition inhibition or by orothe byr mechanisms other mechanisms of action. of action.In addition, In addition, during duringthe experiments, the experiments, many of many these of compounds these compounds also showed also showed activity activity against against other otherherpes herpes viruses, viruses, such as such varicella-zoster as varicella-zoster virus virus(VZV), (VZV), Epstein–Barr Epstein–Barr virus virus(EBV), (EBV), herpes herpes simplex simplex virus virus(HSV), (HSV), and andhuman human herpesvirus herpesvirus type type 8 (HHV-8). 8 (HHV-8). Pharmaceutical Pharmaceutical compositions compositions containing containing suchsuch compoundscompounds oror their their pharmaceutically pharmaceutically acceptable acceptable salts salts useful useful as antiviral as antiviral agents agents have also have been also studied. been Studiesstudied. have Studies been have conducted been conducted for the administration for the administration of pharmaceutical of pharmaceutical preparations preparations by parenteral, by topical,parenteral, oral topical, or rectal oral route, or rectal depending route, on depending the purpose on ofthe their purpose use to of treat their internal use to ortreat external internal viral or infectionsexternal viral [86]. infections [86].

3.3.3.3. Respiratory Viruses AcuteAcute respiratoryrespiratory infectionsinfections are aa majormajor globalglobal healthhealth problemproblem responsibleresponsible forfor aboutabout 3.93.9 millionmillion deathsdeaths worldwideworldwide each each year year [87 ,88[87,].88]. These These infections infections are of are the topof fivethe causestop five of mortalitycauses of worldwide mortality andworldwide the leading and the cause leading of mortality cause of among mortality children among under children five yearsunder of five age years in many of age developing in many countriesdeveloping [87 countries,89]. Acute [87,89]. respiratory Acute respiratory infections infections are most are often most caused often caused by viruses. by viruses. Over 200Over viral 200 serotypesviral serotypes are associated are associated with human with human respiratory respir diseasesatory diseases [90] including [90] including Influenza Influenza A and Influenza A and BInfluenza virus, respiratory B virus, respiratory syncytial virussyncytial (RSV), virus parainfluenza (RSV), parainfluenza virus (PIV), virus human (PIV), adenovirus human adenovirus (HAdV), human(HAdV), coronavirus human coronavirus (HCoV), human (HCoV), rhinovirus human rhinov (HRV),irus human (HRV), metapneumovirus human metapneumovirus (HMPV) and (HMPV) human bocavirusand human (HBoV). bocavirus In addition, (HBoV). two In human addition, polyomaviruses two human (HPyV),polyomaviruses KIPyV and (HPyV), WUPyV, KIPyV have beenand detectedWUPyV, in have patients been with detected respiratory in patients infections with [ 91respiratory]. These infections infections aff ect[91]. all These age groups, infections but nearlyaffect all severeage groups, episodes but occur nearly in childrenall severe under episodes five years, occur the in elderly children and under immunocompromised five years, the elderly individuals and (e.g.,immunocompromised HIV-infected patients) individuals [87,89]. (e.g., In adults, HIV-infected viral respiratory patients) infections [87,89]. In are adults, the cause viral of respiratory 30%–50% ofinfections pneumonia are the cases, cause 80% of of30%–50% asthma of complications pneumonia cases, and 20%–60% 80% of asthma of chronic complications obstructive and pulmonary 20%–60% diseaseof chronic exacerbations obstructive [87 ].pulmonary Consequently, disease common exac viralerbations respiratory [87]. infections Conseq causeuently, a greatercommon economic viral burdenrespiratory than infections many other cause clinical a greater conditions economic in terms burd of medicalen than many expenses other and clinical productivity conditions losses in [ 87terms,92]. Theof medical World Healthexpenses Organization and productivity has supported losses [87, the92]. monitoring The World of acute Health respiratory Organization diseases has worldwidesupported sincethe monitoring 1977 [91]. of acute respiratory diseases worldwide since 1977 [91]. TheThe InfluenzaInfluenza virusvirus belongsbelongs toto thethe OrthomyxoviridaeOrthomyxoviridae familyfamily andand causescauses respiratoryrespiratory infectionsinfections inin aboutabout 20%20% ofof thethe globalglobal populationpopulation everyevery year.year. TheThe 1918 fluflu pandemic was caused by InfluenzaInfluenza A subtypesubtype H1N1H1N1 andand killedkilled 5050 millionmillion peoplepeople aroundaround thethe worldworld [[93].93]. TheThe AsianAsian InfluenzaInfluenza caused by InfluenzaInfluenza AA subtypesubtype H2N2H2N2 occurredoccurred inin 19571957 andand thethe HongHong KongKong InfluenzaInfluenza causedcaused byby InfluenzaInfluenza AA subtypesubtype H3N2H3N2 took took place place in in 1968 1968 and and made made far fewerfar fewer victims victims than thethan 1918 the Spanish1918 Spanish flu. About flu. 70About people 70 diedpeople in Asiadied inin 2004–2005Asia in 2004–2005 due to the due H5N1 to the strain H5N1 ofavian strain flu of [93avian]. The flu 2009 [93]. flu The pandemic 2009 flu (swinepandemic flu) was(swine the flu) second was pandemic the second involving pandemic a strain involving of Influenza a strain Avirus. of Influenza It was classified A virus. as ItInfluenza was classified A H1N1 as 2009Influenza and the A geneticH1N1 2009 material and originatedthe genetic from material three or diiginatedfferent species: from three human, different avian andspecies: swine human, [7,94]. avian and swine [7,94]. The chemotherapy or prophylaxis of Influenza infections comprises agents blocking the Influenza A virus M2 proton-selective ion channel (amantadine, rimantadine) and neuraminidase inhibitors (zanamivir, oseltamivir, laninamivir, peramivir) [1,93]. Both classes can

Molecules 2020, 25, 942 11 of 22

The chemotherapy or prophylaxis of Influenza infections comprises agents blocking the Influenza A virus M2 proton-selective ion channel (amantadine, rimantadine) and neuraminidase inhibitors (zanamivir, oseltamivir, laninamivir, peramivir) [1,93]. Both classes can induce virus resistance and Molecules 2020, 25, 942 11 of 22 therefore there is an urgent need to develop new antiviral agents with novel mechanisms of action. An alternativeinduce virus concept resistance has and recently therefore emerged there is and an urgent it is based need on to thedevelop idea new of designing antiviral agents new molecules with targetingnovel host mechanisms cell factors of action. that are An hijacked alternative by concept the virus has during recently its emerged replication. and it Host-targeting is based on the antiviralsidea are anof alternativedesigning new strategy molecules for addressing targeting host host cell structures factors that involved are hijacked in the by virus the lifevirus cycle. during This its type of inhibitorsreplication. could Host-targeting exhibit a significantlyantivirals are greateran alternative barrier strategy for selecting for addressing drug-resistant host structures viruses and, in addition,involved display in the virus broad-spectrum life cycle. This antiviral type of inhibitors activity whencould exhibit interacting a significantly with a cellular greater targetbarrier common for to severalselecting viruses. drug-resistant The host viruses factor-directed and, in addition, antiviral display therapy broad-spectrum is recently studied. antiviral This activity is increasingly when interacting with a cellular target common to several viruses. The host factor-directed antiviral recognized as a relevant approach to combat viral resistance and provides broad-spectrum antiviral therapy is recently studied. This is increasingly recognized as a relevant approach to combat viral agentsresistance [95,96]. and provides broad-spectrum antiviral agents [95,96]. ManyMany studies studies are are currently currently being being developeddeveloped to to find find new new Influenza Influenza inhibitors. inhibitors. Tatar Tatar et al. et al. synthesizedsynthesized 2-phenylamino-1,3,4-thiadiazole 2-phenylamino-1,3,4-thiadiazole derivativesderivatives 4545––4848 [49]. [49 ].

SCH3 SCH3 SCH3 O S N N N N N N N H2N HN N H S S H S N N N H H R H

45 46 47 R = 4-CN 48 R = 3-CF3,4-Cl

TheThe antiviral antiviral activity activity against against some some respiratoryrespiratory viruses viruses such such as asInfluenza Influenza A H1N1, A H1N1, Influenza Influenza A A H3N2, Influenza B, Parainfluenza-3, RSV, Reovirus-1and Feline Coronavirus was investigated and H3N2, Influenza B, Parainfluenza-3, RSV, Reovirus-1and Feline Coronavirus was investigated and the the results are summarized in Table 5. No activity was observed at the highest concentration tested results are summarized in Table5. No activity was observed at the highest concentration tested or at or at subtoxic concentration against Influenza B and RSV [49]. subtoxic concentration against Influenza B and RSV [49]. Table 5. Antiviral evaluation and in vitro cytotoxicity of compounds 45–48 (adapted from [49]). Table 5. Antiviral evaluation and in vitro cytotoxicity of compounds 45–48 (adapted from [49]). Influenza A Influenza A Para Reovirus-1 Feline InfluenzaH1N1 A InfluenzaH3N2 A Influenza-3 CoronavirusFeline Para Influenza-3 Reovirus-1 H1N1 H3N2 Coronavirus EC50 CC50 EC50 CC50 EC50 MCC EC50 MCC EC50 CC50 EC(μ50M) CC(μ50M) EC(μ50M) CC(μ50M) EC(μ50M) MCC(μM) EC(μ50M) MCC(μM) EC(μM)50 CC(μM)50 (µM) (µM) (µM) (µM) (µM) (µM) (µM) (µM) (µM) (µM) 45 42 > 100 31.4 > 100 > 20 ≥ 20 > 20 ≥ 20 > 100 > 100 45 42 > 100 31.4 > 100 > 20 20 > 20 20 > 100 > 100 46 - 23 - 23 > 100 ≥ >100 > 100 ≥ > 100 > 100 > 100 46 - 23 - 23 > 100 >100 > 100 > 100 > 100 > 100 47 -- 79 79 - - 79 79 > >20 20 100 100 > >20 20 100 100 > > 100100 > > 100100 48 -- 2.7 2.7 - - 2.7 2.7 > >4 4 20 20 > >4 4 20 20 > > 44 11 11 Oseltamivir Oseltamivir 4.7 > 100 9 > 100 ------carboxylate 4.7 > 100 9 > 100 ------carboxylateRibavirine 8 > 100 8.1 > 100 50 > 250 > 250 > 250 - - AmantadineRibavirine 127 8 > >500 100 1.7 8.1 > >500 100 - 50 > - 250 > -250 > -250 - - - - Amantadine 127 > 500 MCC: 1.7 minimum > 500 cytotoxic - concentration. - - - - - MCC: minimum cytotoxic concentration. The inThe vitro antiviralin assay showedvitroN-{3-(methylthio)-1-[5-(phenylamino)-1,3,4-thiadiazol-2-yl]propyl} antiviral assay showed benzamideN-{3-(methylthio)-1-[5-(phenylamino)-1,3,4-thiadiazol-2-yl]propyl}benzamide45 as an Influenza A H3N2 virus subtype inhibitor. With an EC 4550 asvalue an Influenza of 31.4 µ AM, the derivativeH3N2 virus45 was subtype the mostinhibitor. potent With among an EC50 the value tested of 31.4 compounds μM, the derivative and moderate 45 was activethe most compared potent to standardamong drug the oseltamivir,tested compounds but a and promising moderate sca activeffold forcompared future to developments. standard drug Derivativesoseltamivir, but47 anda 48 exhibitedpromising activity scaffold against for Parainfluenza-3future developments. and Reovirus-1Derivatives and47 and probably 48 exhibited the thiourea activity moiety against favors Parainfluenza-3 and Reovirus-1 and probably the thiourea moiety favors antiviral activity on these antiviral activity on these strains (Figure5)[49]. strains (Figure 5) [49].

Molecules 2020, 25, 942 12 of 22 Molecules 2020, 25, 942 12 of 22 Molecules 2020, 25, 942 12 of 22

SCH3 SCH3 SCH3 SCH3 SCH3 SCH3 O S O S N N NN N N N N NN N N N N H2HN N HNHN N N H 2 H H S S SS H S S N N NN N N H H HH RR H H

amineamine group group ((4646)) amideamide group group (45) (45) loss of activity loss of activity thioureathiourea group group (47 (,47 48,) 48) beneficial against Influenza A H3N2 beneficial against Parainfluenza-3 beneficial against Influenza A H3N2 beneficial against Parainfluenza-3 and Reovirus-1 and Reovirus-1 FigureFigure 5. The 5. The influence influence of substituentsof substituents of of 1,3,4-thiadiazole 1,3,4-thiadiazole derivatives derivatives 4545–48– 48on onanti-respiratory anti-respiratory Figure 5. The influence of substituents of 1,3,4-thiadiazole derivatives 45–48 on anti-respiratory viruses’viruses’ activity. activity. viruses’ activity. 3.4. Hepatitis3.4. Hepatitis Viruses Viruses 3.4. Hepatitis Viruses ViralViral hepatitis hepatitis is a is liver a liver inflammation inflammation responsible responsible for about about 171,000 171,000 deaths deaths every every year year in the in the EuropeanViralEuropean Region.hepatitis Region. Patients is aPatients liver may inflammationmay have have an an acute acute responsible form form as as a a recent forrecent about infection, infection, 171,000 with deaths relatively every rapid rapid year onset onset in the or or a chronic form. There are five main hepatitis viruses (HAV, HBV, HCV, HDV and HEV) with Europeana chronic form.Region. There Patients are five may main have hepatitis an acute viruses form as (HAV, a recent HBV, infection, HCV, HDV with and relatively HEV) with rapid diff onseterent different ways of transmission and different impact on human health [97]. While HAV or HEV waysor a chronic of transmission form. There and are diff fiveerent main impact hepatiti on humans viruses health (HAV, [97 ].HBV, While HCV, HAV HDV or HEV and infectionHEV) with is infection is usually mild, with most people recovering quickly and completely, infection with HBV, different ways of transmission and different impact on human health [97]. While HAV or HEV usuallyHCV mild, or HDV with often most leads people to recoveringchronic infections quickly and and progressive completely, liver infection damage with the HBV, development HCV or HDV infectionoftenof leads cirrhosis is usually to chronic and mild, liver infections with cancer most [97]. and people progressiveThere recoveringare about liver 15quickly damage million and withpeople completely, the living development with infection chronic ofwith HBV cirrhosis HBV, HCVand liver infectionor HDV cancer andoften [about97 leads]. There14 to million chronic are with about infections HCV 15 millioninfection and progressive peoplein the European living liver with Region damage chronic [97]. with Safe HBV the and infectiondevelopment effective and ofabout cirrhosisvaccines 14 million and for the withliver prevention HCVcancer infection of[97]. HBV There infection in the are European have about been 15 available Region million [since97 people]. the Safe 1990 living ands. These e ffwithective vaccines chronic vaccines also HBV for infectionthe preventionprovide and protectionabout of HBV 14 frommillion infection HDV with infection. have HCV been Unfortunately,infection available in sincethe the EuropeanHCV the vaccine 1990s. Region has These not [97]. vaccinesyet beenSafe developedand also effective provide protectionvaccines[97,98]. for from theMany prevention HDV patients infection. infectedof HBV Unfortunately, withinfection HBV haveare the ad beults HCVen availableborn vaccine before since has the not thehepatitis yet 1990 been s.B Thesevaccine developed vaccines became [97 also,98]. provideManyavailable patients protection in infected the from 1990s. with HDV In HBVthese infection. arecases, adults drug Unfortunately, born treatmen beforet is the thethe hepatitisHCVonly option vaccine B vaccine [98]. has Several not became yet nucleoside been available developed and in the non-nucleoside derivatives with anti-HBV (e.g., adefovir, entecavir, lamivudine, telbivudine, 1990s.[97,98]. In Many these patients cases, drug infected treatment with isHBV the onlyare ad optionults born [98]. Severalbefore the nucleoside hepatitis and B vaccine non-nucleoside became availabletenofovir) in the or1990s. anti-HCV In these activity cases, (e.g., drug boceprevir treatmen,t isgrazoprevir, the only option elbasvir, [98]. ledipasvir, Several nucleosidesofosbuvir, and derivativestelaprevir) with are anti-HBV in use [3,99] (e.g., and adefovir, chronic infections entecavir, with lamivudine, HBV and HCV telbivudine, can be currently tenofovir) controlled or anti-HCV or non-nucleoside derivatives with anti-HBV (e.g., adefovir, entecavir, lamivudine, telbivudine, activityeven (e.g., cured. boceprevir, Due to the grazoprevir, costs of antiviral elbasvir, drugs ledipasvir, for chronic sofosbuvir, hepatitis, access telaprevir) to treatment are in useis a [major3,99] and tenofovir)chronicobstacle infections or inanti-HCV many with countries HBV activity andand HCVfinding(e.g., canboceprevir new, be less currently expensive, grazoprevir, controlled antiviral elbasvir, drugs or even is a cured.ledipasvir,necessity Due [97]. tosofosbuvir, the costs telaprevir)of antiviral1,2-Dihydro-4,6-dimethyl-2-oxo-1-[(5-(phenylamino)-1,3,4-thiadiazol-2-yl)methyl]pyridine-3-ca are drugs in use for [3,99] chronic and hepatitis, chronic accessinfections to treatment with HBV is and a major HCV obstacle can be currently in manycountries controlled and or evenfinding cured.rbonitrile new, Due less 49 expensivetowas the prepared costs antiviral of fromantiviral drugsthe drugscorresponding is a necessity for chronic phehylthiosemicarbazide [97 ].hepatitis, access to treatmentby cyclization is a inmajor obstacle1,2-Dihydro-4,6-dimethyl-2-oxo-1-[(5-(phenylamino)-1,3,4-thiadiazol-2-yl)methyl]pyridine-3-sulfuric in many acid [6].countries The derivative and finding 49 was new, tested less for expensiveantiviral activity antiviral against drugs the hepatitisis a necessity B virus [97]. (HBV) carbonitrile1,2-Dihydro-4,6-dimethyl-2-oxo-1-[(5-(phenylamino)-1,3,4-thiadiazol-2-yl)methyl]pyridine-3-causing HepG2.2.2.1549 was prepared cell line, from a human the correspondinghepatoblastoma phehylthiosemicarbazidecell line that produces HBV viral by cyclization particles. in rbonitrilesulfuricCytotoxicity acid 49 [6was]. Thewas prepared derivative also tested from49 by wasthe the tested correspondingcell viabil for antivirality method phehylthiosemicarbazide activity (MTT against assay). the Preliminary hepatitis by cyclization Bscreening virus (HBV) in indicated high inhibitory activity against HBV with an IC50 value of 0.3 μM, low cytotoxicity (CC50 sulfuricusing HepG2.2.2.15 acid [6]. The cellderivative line, a 49 human was tested hepatoblastoma for antiviral cell activity line thatagainst produces the hepatitis HBV viralB virus particles. (HBV) using valueHepG2.2.2.15 of 333.3 μ M)cell and line, a selectivity a human index hepatoblastoma SI of 1111 compared cell line to the that standard produces drug HBV lamivudine viral particles. (IC50 Cytotoxicityvalue of was 0.1 alsoμM). testedFrom bythese the results, cell viability it can methodbe conclu (MTTded assay).that pyridine-2-one Preliminary may screening be a indicatedgood Cytotoxicity was also tested by the cell viability method (MTT assay). Preliminary screening high inhibitorysubstituent activityon the 1,3,4-thiadiazole against HBV ring, with and an ICthese50 value two moieties of 0.3 µtogetherM, low make cytotoxicity a promising (CC scaffold50 value of indicated high inhibitory activity against HBV with an IC50 value of 0.3 μM, low cytotoxicity (CC50 333.3 µinM) promoting and a selectivity anti-HBV indexactivity SI [6]. of 1111 compared to the standard drug lamivudine (IC50 value of value0.1µM). of From333.3 μ theseM) and results, a selectivity it can be index concluded SI of 1111 that Ncompared pyridine-2-one to the standard may be adrug good lamivudine substituent (IC on50 O N value of 0.1μM). From these results, it can be concluded that pyridine-2-one may be a good the 1,3,4-thiadiazole ring, and these two moietiesN togetherS make a promising scaffold in promoting substituent on the 1,3,4-thiadiazole ring, and these two Nmoieties together make a promising scaffold anti-HBV activity [6]. NC CH3 H in promoting anti-HBV activity [6]. H3C N N O 49 N S N NC CH3 H

H3C 49

Molecules 2020, 25, 942 13 of 22 Molecules 2020, 25, 942 13 of 22

3.5.Molecules Miscellaneous 2020, 25, 942Viruses 13 of 22 3.5. Miscellaneous Viruses Sindbis fever, a less common human viral disease, is caused by a mosquito-borne virus called Sindbis fever, a less common human viral disease, is caused by a mosquito-borne virus called Sindbis3.5. Miscellaneous virus (Togaviridae Viruses family). Despite the wide distribution of Sindbis virus, symptomatic infections Sindbis virus (Togaviridae family). Despite the wide distribution of Sindbis virus, symptomatic in humans have been reported in only a few limited geographical areas such as northern Europe infectionsSindbis in fever,humans a less have common been reported human inviral only disease, a few limitedis caused geographical by a mosquito-borne areas such virus as northern called (Finland, Sweden and Russia), South Africa, Australia and China [100]. 1,3,4-Thiadiazole derivatives SindbisEurope virus(Finland, (Togaviridae Sweden andfamily). Russia), Despite South the Africa, wide Australiadistribution and of China Sindbis [100]. virus, 1,3,4-Thiadiazole symptomatic 50–55 were tested for antiviral activity against several viruses [101]. infectionsderivatives in 50 humans–55 were have tested been for reported antiviral in activity only a againstfew limited several geographical viruses [101]. areas such as northern Europe (Finland, Sweden and Russia), South Africa, AustraliaH and China [100]. 1,3,4-Thiadiazole N N derivatives 50–55 were tested for antiviral activity againstN severalR viruses [101]. S H N N N R F SOH

F F OH

R = CH3 (50); CH2CH=CHF 2 (51); C6H5 (52) C6H4CH3(p) (53); C6H4OCH3(p) (54); C6H11 (55) R = CH3 (50); CH2CH=CH2 (51); C6H5 (52) The derivatives 50 (methyl) and 51 (allyl) showed antiviral activity against herpes simplex C H CH (p) (53); C H OCH (p) (54); C H (55) virus-1The TK-KOS derivatives and50 herpes(methyl) simplex6 and4 513 virus-1(allyl) showedTK-KOS6 4 antiviral ACV,3 Sindbis activity6 11 virus, against Coxsackie herpes simplex virus virus-1B4 and TK-KOSPuntoThe Toro andderivatives virus herpes at simplexa 50concentration (methyl) virus-1 and of TK-KOS 1651 μ(allyl)g/mL. ACV, showedThe Sindbis highest antiviral virus, antiviral Coxsackie activity activity virusagainst was B4 exhibited herpes and Punto simplex against Toro virusvirus-1Sindbis at TK-KOS avirus concentration by and derivative herpes of 16 50µsimplexg /atmL. a concentration The virus-1 highest TK-KOS antiviral of 9.6 ACV, μ activityg/mL. Sindbis It was seems virus, exhibited that Coxsackie the against size virus Sindbisof the B4 amino virus and byPuntogroup derivative Toro substituent virus50 at at a influenceda concentration concentration the ofan of 9.6tiviral 16µ μgg/mL./ mL.activity. It The seems highestWhile that derivatives theantiviral size of activity the50 aminoand was 51 group exhibitedwith substituentsmall against alkyl influencedSindbisgroups showedvirus the by antiviral antiviralderivative activity. activity, 50 at While acompounds concentration derivatives with 50of bulky and9.6 μ51 g/mL.aromaticwith smallIt seems (52 alkyl, 53 that and groups the 54 )size or showed cycloalkyl of the antiviral amino (55) activity,groupgroups substituent were compounds not capable influenced with bulkyof vira the aromaticl inhibition antiviral (52 (Figure,activity.53 and 6) 54While [101].) or cycloalkyl derivatives (55 )50 groups and 51 were with not small capable alkyl of viralgroups inhibition showed (Figureantiviral6)[ activity,101]. compounds with bulky aromatic (52, 53 and 54) or cycloalkyl (55) CH increases antiviral activity (50) groups were not capable of viral inhibition (Figure3 6) [101]. H CH CH=CH beneficial for antiviral activity (51) N N CH3 increases2 antiviral2 activity (50) N R S H CH CH=CH beneficial for antiviral activity (51) N N 2 2 N R exchange with bulky groups F SOH exchange with bulky groupsCH3 F F OH 52 53 loss of activity 50-55 CH3 F 52 53 loss of activity 50-55 OCH3

54 55 OCH3 Figure 6. The influence of substituents of 1,3,4-thiadiazole derivatives 50–55 on viral inhibition. 54 55 2-Phenylamino-1,3,4-thiadiazoleFigure 6. TheThe influence influence of substituents derivatives of 1,3,4-thiadiazole 45–48 were derivatives also screened 50–55 on against viral inhibition. herpes simplex HSV-1 and HSV-2, herpes simplex virus-1 TK-KOS ACV, Sindbis virus, Coxsackie virus B4 and Punto2-Phenylamino-1,3,4-thiadiazole Toro virus (Table 6) [49]. While derivatives derivatives amide 45 4545and––4848 amine werewere 46also did screened not exhibit against antiviral herpes activity simplex at HSV-1subtoxic andand concentrations, HSV-2,HSV-2, herpes herpes in simplex simplexvitro tests virus-1 virus-1 showed TK-KOS TK-KOS antiviral ACV, ACV, Sindbisactivi Sindbisty virus,for thioureavirus, Coxsackie Coxsackie derivatives virus B4virus and47 B4and Punto and 48, ToroPuntohighlighting virus Toro (Table virus what6 )[(Table other49]. studies6) While [49]. amide haveWhile reported45 amideand amine 45that and derivatives46 aminedid not 46 bearing exhibitdid not the antiviral exhibit -NH-CS-NH- antiviral activity atgroupactivity subtoxic have at concentrations,subtoxicdemonstrated concentrations, antiviralin vitro tests activity. in vitro showed Derivativetests antiviral showed 48 activity exhibitedantiviral for activiactivity thioureaty foragainst derivatives thiourea different 47derivativesand strains48, highlighting of47 HSV and and48, whathighlightingboth otherderivatives studies what 47 other have and reported48studies showed have that activity reported derivatives against that bearing derivativesSindbis the virus, -NH-CS-NH- bearing Coxsackie the group-NH-CS-NH- virus have B4 and demonstrated group Punto haveToro antiviraldemonstratedvirus (Figure activity. 7)antiviral [49]. Derivative activity.48 exhibited Derivative activity 48 exhibited against activity different against strains different of HSV and strains both of derivatives HSV and 47bothand derivatives48 showed 47 activity and 48 against showed Sindbis activity virus, against Coxsackie Sindbis virus virus, B4 Coxsackie and Punto virus Toro virusB4 and (Figure Punto7)[ Toro49]. virus (Figure 7) [49].

Molecules 2020, 25, 942 14 of 22

MoleculesTable2020, 25 6., 942Antiviral evaluation and in vitro cytotoxicity of compounds 45–48 (adapted from [49]). 14 of 22 Molecules 2020, 25, 942 HSV-1 Sindbis 14 of 22 Coxsackie Punto Toro HSV-1 (TK-KOS virus Table 6. Antiviral evaluationHSV-2 and in vitro cytotoxicity of compounds 45–48 (adaptedvirus B4 from [49]). virus Table 6. Antiviral evaluation and in vitro ACV)cytotoxicity of compounds 45–48 (adapted from [49]). HSV-1 EC50 MCC EC50 MCC EC50 MCC ECSindbis50 MCC CoxsackieEC50 MCCPuntoEC Toro50 MCC HSV-1 HSV-2 (TK-KOSHSV-1 Sindbis Virus CoxsackieVirus B4 PuntoVirus Toro (μM) (μHSV-1M) (μM) (μM) (μM) (TK-KOSACV) (μM) (μvirusM) (μM) (μM) (μM) (μM) (μM) HSV-2 virus B4 virus 45 > 20 EC50≥ 20MCC > 20EC 50 ≥ 20MCC >EC 2050ACV) MCC≥ 20 EC > 5020 MCC≥ 20 EC50 > 20MCC ≥ 20EC 50 >MCC 20 ≥ 20 46 > 100 (µECM) >50 100 (MCC µM) > 100(ECµ M)50 > 100MCC(µM) > (EC100µM)50 MCC >(µ 100M) (ECµ >M) 50100 MCC(µM) > 100 EC( µM)50 > 100MCC(µ M) > 100EC(µM)50 >MCC (100µM) > 100 45 (>μ20M) (μ20M) (>μM)20 (μM)20 (>μM)20 (μM)20 (μ>M)20 (μM)20 (μ>M)20 (μM)20 (μ>M)20 (μM)20 47 > 100 > 100≥ > 100 > 100≥ > 100 >≥ 100 > 20 ≥ 100 > 20≥ 100 > ≥20 100 48 4645 > 20 >>100 20 100 >≥100 20> 20> > 10020 100>≥ 100 20 >> >20 10020 >≥ 100 10020 > > 10020 > 4 >≥ 20100 20 > 20100 > 4≥> 20100 20 > 20100 >≥> 204100 20 4746 >> 100100 > > 100 > 100 > 100 > 100100 > 100100 >> 10020 > 100100 >> 10020 > 100 > >10020 > 100 100 Acyclovir 0.9 > 250 0.4 > 250 > 250 > 250 ------4847 > 10020 > 100 100 >> 10020 > 100 100 >> 10020 > 100100 >> 204 100 20 >> 204 100 20 > >204 100 20 Acyclovir 0.9 > 250 0.4 > 250 > 250 > 250 ------Ribavirine 48 - > 20 - 100 - > 20 - 100 > - 20 100 - > > 4250 20 > 250 > 4 > 250 20 > 250 > 4 112 20 > 250 Ribavirine ------> 250 > 250 > 250 > 250 112 > 250 Acyclovir 0.9 > 250 0.4MCC: > minimum250 > 250 cytotoxic > 250 concentration. ------Ribavirine - - - MCC: -minimum - cytotoxic - concentration. > 250 > 250 > 250 > 250 112 > 250

SCH3 MCC: minimumSCH 3cytotoxic concentration. SCH3

O SCH3 SCH3 S SCH3 N N N N O N H N N HNS N N N 2 N N H S N S N H N S N N H2N N HN N N H S S H S H N HN R N H 45H 46H R 47 RH = 4-CN 45 46 47 R = 4-CN 48 R = 3-CF3,4-Cl amine group 48 R = 3-CF3,4-Cl amine group amide group amide group lossloss of of activityactivity thiourea group no activity at subtoxic concentrations thiourea group no activity at subtoxic concentrations beneficialbeneficial against against HSV, HSV, Sindbis Sindbis virus, virus, CoxsackieCoxsackie virus, virus, Punto Punto Toro virusToro virus Figure 7. The influence of substituents of 1,3,4-thiadiazole derivatives 45–48 on antiviral activity. Figure Figure7. The 7.influence The influence of substituents of substituents of of 1,3,4-thiadiazole 1,3,4-thiadiazole derivatives derivatives 45– 4845 on–48 antiviral on antiviral activity. activity. 2-Amino-5-(2-sulphamoylphenyl)-1,3,4-thiadiazole2-Amino-5-(2-sulphamoylphenyl)-1,3,4-thiadiazole 5656 reducedreduced the thereplication replication of some of DNA some DNA viruses2-Amino-5-(2-sulphamoylphenyl)-1,3,4-thiadiazoleviruses such as such adenovirus as adenovirus Ad17 Ad17 and and herpes herpes simplexsimplex HSV-1 HSV-1 56 andreduced and RNA RNA virusesthe viruses replication such suchas Poliovirus asof Poliovirussome 1, DNA 1, Echovirusviruses Echovirussuch 2 and as adenovirus Coxsackie2 and Coxsackie virus Ad17 virus B4 and at B4 concentrationsherpes at concentrations simplex ranging HSV-1 ranging fromand from RNA 20 20 toto 100viruses100 µμgg/mL/mL such [102,103]. [102 as, 103Poliovirus ].InIn vitro 1, experimentsEchovirusvitro 2 experimentsand were Coxsackie performed were virusperf usingormed B4 samples atusing concentrations samples of 106 humanof 10 ranging6 human aneuploid aneuploidfrom HEp-220 toHEp-2 100 cells cellsμg/mL that that were [102,103].were infected In infected with 10 infectious units per cell. Derivative 56 was6 highly active against all viral strains, withvitro 10experiments infectious unitswere per perf cell.ormed Derivative using samples56 was highly of 10 activehuman against aneuploid all viral HEp-2 strains, cells significantly that were significantly reducing viral replication at a concentration of 50 μg/mL. The best inhibition was infected with 10 infectious units per cell. Derivativeµ 56 was highly active against all viral strains, reducingrecorded viral replication against Echovirus at a concentration 2 virions that were of 50completelyg/mL. inhibited The best at inhibitiona concentration was of recorded 20 μg/mL against Echovirussignificantly(Table 2 virions 7).reducing Regarding that viral were the mechanism completelyreplication of inhibitedataction, a concentration the atauthors a concentration assume of 50that μ ofcompoundg/mL. 20 µg The/mL 56 may (Tablebest actinhibition7 ).on Regarding the was therecorded mechanismviral against structural of Echovirus action, proteins the authorspreventing2 virions assume thatthe assembly were that completely compound of virus particles 56inhibitedmay [102]. act at on a theconcentration viral structural of 20 proteins μg/mL preventing(Table 7). Regarding theDerivatives assembly the of mechanism ofcompound virus particles 56 of were action, [ 102prepared. ].the authors Methyl assume derivative that 57 compound and allyl derivative 56 may act58 on the viral Derivativesstructuralreduced theproteins of replication compound preventing of RNA56 were viruses the prepared. assembly (Poliovirus Methyl of 1 virus and derivativeCoxsackie particles virus 57[102].and B4) at allyl concentrations derivative of58 50reduced and 100 μg/mL, while ethyl derivative 59 was completely inactive against all viral strains (Table 7). the replicationDerivatives of of RNA compound viruses (Poliovirus56 were prepared. 1 and Coxsackie Methyl derivative virus B4) at57 concentrations and allyl derivative of 50 and 58 These results suggest the importance of the side chain for antiviral activity (Figure 8) [102]. reducedµ the replication of RNA viruses (Poliovirus 1 and Coxsackie virus B4) at concentrations of 50 100 g/mL, while ethyl derivative 59 was completelyN N inactive against all viral strains (Table7). Theseand 100 results μg/mL, suggest while the ethyl importance derivative of 59 the was side completely chain forR antiviral inactive activityagainst (Figureall viral8)[ strains102]. (Table 7). S N These results suggest the importance of the side chain Hfor antiviral activity (Figure 8) [102]. SON 2NHN 2 R R = H (56); CH3 (57); CH2SCH=CHN 2 (58); C2H5 (59) H SO2NH2

R = H (56); CH3 (57); CH2CH=CH2 (58); C2H5 (59)

Molecules 20202020,, 2525,, 942942 1515 ofof 2222

Molecules 2020, 25, 942 15 of 22 Table 7. Antiviral evaluation and in vitrotro cytotoxicitycytotoxicity ofof compoundscompounds 56–59 (adapted(adapted fromfrom [102]).[102]).

Table 7. Antiviral evaluation and in vitro cytotoxicity of compounds 56–59 (adaptedCoxsackie from [102]). Ad17 HSV-1 Poliovirus 1 Echovirus 2 Concn virus B4 MNC Concn Ad17 HSV-1 Poliovirus Echovirus Coxsackievirus virus B4 MNC Concn (Cells(Cells (Cells(Cells (Cells(Cells (Cells(Cells MNC ((μg/mL) (Cells (Cells 1 (Cells 2 (Cells B4 (Cells(Cells(Cells ((μg/mL) (µg/mL)Number) Number) Number) Number) (µg/mL) Number) Number) Number) Number) Number)Number) 8 6 9 9 8 blank 588 10 3 6610 3 10 99 2 10 99 3 10 88 blank 5 × 10 × 3 × 10× 3 × 10 × 2 × 10 × 3 × 10 20 20 7 × 10766 106 6 × 6105510 5 44 × 10666 0 0 6 105 6 × 1055 20 7 × 10 × 6 × 10× 4 × 10 0 × 6 × 10 56 50 4 102 5 104 2 103 0 2 103 900 56 50 4 × 1022× 5 5 ×× 1010×44 2 2 ×× 101033 0 0 × 2 2 ×× 101033 900 100 2 102 3 104 1 103 0 2 103 22× ×44 × 33 × 33 100 50 2 × 106 108 3 3 ×× 31010 10 6 1 12 ×× 10105 - 0 0 8 104 2 2 ×× 1010 57 × × × × 800 50 100 6 × 10688 108 3 3 ×× 210106610 6 2 28 ×× 1010455 - - - 2 104 8 8 ×× 101044 57 × 8 × 6 × 4 × 4 800 57 50 48 10 2 610 3 10 4 - 2 10 4 800 58 100 6 × 10 8× 2 2 ×× 1010×6 8 8 ×× 10104 - - × 2 2 ×× 10104 800 100 5 108 3 106 2 104 - 2 104 88× ×66 × 44 × 44 50 50 4 × 104 108 2 × 210 10 6 3 33 ×× 10109 2 109 - - 3 108 2 2 ×× 1010 58 59 × × × × × 1300 800 100 100 5 × 10588 108 3 3 ×× 310106610 6 2 23 ×× 1010944 2 109 - - 3 108 2 2 ×× 101044 × × × × × 50 4 Concn:× 1088 concentration;2 × 1066 MNC: maximum3 × 1099 noncytotoxic2 × concentration. 1099 3 × 1088 59 1300 100 5 × 1088 3 3 ×× 101066 3 3 ×× 101099 2 2 ×× 101099 3 3 ×× 101088 Concn: concentration; MNC: maximum noncytotoxic concentration.

introductionintroduction ofof ethylethyl groupgroup ((59)) lossloss ofof activityactivity N N N N R N N S N S NH2 N N H 2 CH22CH33 SO NH SO NH S N 22 22 SO22NH22 H SO22NH22

CH (57) or CH CH=CH (58) CH33 (57) or CH22CH=CH22 (58) freefree amineamine groupgroup ((56)) lossloss ofof activityactivity againstagainst DNADNA virusesviruses increasesincreases antiviralantiviral activityactivity beneficial for activity against RNA viruses Figure 8. The influence of substituents of 1,3,4-thiadiazole derivatives 56–59 on antiviral activity. Figure 8. The influence of substituents of 1,3,4-thiadiazole1,3,4-thiadiazole derivativesderivatives 56–59 onon antiviralantiviral activity.activity. Cui et al. synthesized several pyrrolyl-1,3,4-thiadiazoles with general formula 60 (Figure9). The compoundsCui et al. synthesized showed antiviral several activitypyrrolyl-1,3,4-thiadiazoles against some viruses with of the generalFlaviviridae formulafamily 60 (Figure(Figure such as 9).9). West TheThe compoundsNile virus and showed dengue antiviral virus [25 activity,104]. against some viruses of the Flaviviridae familyfamily suchsuch asas WestWest Nile virus and dengue virus [25,104]. X N N

Y N S N H R

O O Cl

Cl 60

X = O, S, NH; Y = OH, SH; R = H, OH, CN, halo, alkyl, alkoxy Figure 9. TheThe general general formula of derivatives 60.

The use ofof non-nucleosidenon-nucleoside derivatives as antiviralantiviral chemotherapeuticchemotherapeutic agents has stimulatedstimulated extensive research research into into the the synthesis synthesis of of compounds compounds of of this this class. class. However, However, many many antiviral antiviral drugs drugs are nucleosideare nucleoside analogs analogs that that act actby bysuppressing suppressing the the synthesis synthesis of ofviral viral DNA DNA or or RNA RNA which which leads leads to inhibitioninhibition of of virus virus replication replication or or cell division.division. Research Research has has been carried out to find find new nucleoside antiviral agents in which the natural nucleobases have been replaced by heterocyclic rings, as can be seen in derivatives 61–64 [105].

Molecules 2020, 25, 942 16 of 22 Molecules 2020, 25, 942 16 of 22 antiviralMolecules 2020 agents, 25, 942 in which the natural nucleobases have been replaced by heterocyclic rings, as can16 of be 22 seenantiviral in derivatives agents in which 61–64 the[105]. natural nucleobases have been replaced by heterocyclic rings, as can be seen in derivatives 61–64 [105]. R' R R' N N R N N S HN S S HN S 61-64 61-64 The antiviral activity was evaluated in vitro against viral strains parasitizing Chenopodium The antiviral activity was evaluated in vitro against viral strains parasitizing Chenopodium amaranticolorThe antiviral. The activityability ofwas derivatives evaluated 61in –vitro64 to against control viral the strainsviral infectionparasitizing of Chenopodium amaranticolor. The ability of derivatives 61–64 to control the viral infection of Chenopodium amaranticolor amaranticolor . leavesThe abilitywas studied of derivatives at two concentratio 61–64 to controlns: 1000 the ppm viral and infection 100 ppm. of Generally, Chenopodium the leaves was studied at two concentrations: 1000 ppm and 100 ppm. Generally, the compounds compoundsamaranticolor showed leaves goodwas studiedrates of viralat two infection concentratio controlns: at 1000 ppm.ppm Theand best 100 resultsppm. wereGenerally, observed the showed good rates of viral infection control at 1000 ppm. The best results were observed for the forcompounds the derivatives showed bearinggood rates th eof D-xylobutyl viral infection group cont rol(compound at 1000 ppm. 62 —82%The best control results and were derivative observed derivatives bearing the D-xylobutyl group (compound 62—82% control and derivative 64—76% 64for—76% the derivatives control). The bearing substituent the D-xylobutyl on the aryl ringgroup did (compound not significantly 62—82% influence control biological and derivative activity, control). The substituent on the aryl ring did not significantly influence biological activity, although although64—76% control).the compounds The substituent 61 and on 62 the having aryl ring a methoxy did not significantlygroup were influence slightly biologicalmore active activity, than the compounds 61 and 62 having a methoxy group were slightly more active than derivatives 63 and derivativesalthough the 63 compoundsand 64 bearing 61 anda methyl 62 having group a (Tablemethoxy 8, Figuregroup 10).were The slightly study moremay beactive useful than in 64 bearing a methyl group (Table8, Figure 10). The study may be useful in obtaining new pesticides obtainingderivatives new 63 pesticidesand 64 bearing for agriculture a methyl [105]. group (Table 8, Figure 10). The study may be useful in obtainingfor agriculture new pesticides [105]. for agriculture [105]. Table 8. Structural details and antiviral activity of compounds 61–64 (adapted from [105]). Table 8. Structural details and antiviral activity of compounds 61–64 (adapted from [105]). Table 8. Structural details and antiviral activity of compounds 61–64 (adapted from [105]). Viral infection control (%) R’ R Viral Infection Control (%) R’ R Viral1000 infection ppm control 100 ppm (%) R’ R 1000 ppm 100 ppm 61 4-CH3O D-glucopentyl 100069 ppm 100 26ppm 61 4-CH O D-glucopentyl 69 26 6261 4-CH33O D-glucopentylD-xylobutyl 8269 3526 62 4-CH3O D-xylobutyl 82 35 6362 4-CH2-CH3O3 D-glucopentylD-xylobutyl 5982 2235 63 2-CH3 D-glucopentyl 59 22 6463 2-CH3 D-glucopentylD-xylobutyl 7659 3022 64 2-CH3 D-xylobutyl 76 30 VirazoleVirazole64 2-CH 3 D-xylobutyl 100 76 100100 30 100 Virazole 100 100 D-xylobutyl (62, 64) D-xylobutylincreases activity (62, 64) R' R increases activity R' N N R N N S D-glucopentyl (61, 63) (61, 62) CH O group 3 HN S S D-glucopentyl (61, 63) (61, 62) CH3O group HN S moderate activity strenghten the activity moderate activity strenghten the activity 61-64 61-64

CH3 group (63, 64) CH3 group (63, 64) moderate antiviral activity moderate antiviral activity Figure 10. Influence of substituents of 1,3,4-thiadiazole derivatives 61–64 on antiviral activity. Figure 10. Influence of substituents of 1,3,4-thiadiazole derivatives 61–64 on antiviral activity. Figure 10. Influence of substituents of 1,3,4-thiadiazole derivatives 61–64 on antiviral activity. 4.4. Conclusions Conclusions 4. Conclusions TheThe research focused on 1,3,4-thiadiazole derivatives indicates indicates a a broad broad spectrum of pharmacologicalThe research activities activities focused associated associated on 1,3,4-thiadiazole with with good good physicochemical physicochemical derivatives indicates and and pharmacokinetic pharmacokinetic a broad spectrum properties. properties. of Thispharmacological articlearticle presents presents activities a literaturea literature associated review review ofwith 2-amino-1,3,4-thiadiazole of good 2-amino-1,3,4-thiadiazole physicochemical derivatives and pharmacokineticderivatives that have that been properties.have evaluated been evaluatedThisfor antiviral article for activitypresents antiviral against a literature severalactivity review viral against strains. of 2-amino-1,3,4-thiadiazole several In addition viral to thestrains. 2-amino-1,3,4-thiadiazole derivatives In addition that have to moiety, beenthe 2-amino-1,3,4-thiadiazoleevaluatedantiviral activity for isantiviral also dependent moiety,activity on antiviral theagainst nature activity ofseveral the substituents,is alsoviral dependent andstrains. structure–activity Inon theaddition nature studies toof havethe substituents,2-amino-1,3,4-thiadiazoleshown the most and e ffistructure–activitycient substituentsmoiety, antiviralstudies for antiviral have activity shown activity is thealso in most each dependent class.efficient Based substituentson onthe the nature literature for antiviral of data,the activitysubstituents,the 2-amino-1,3,4-thiadiazole in each and class. structure–activity Based scaon fftheold studies literature may be have considered data, shown the athe2-amino-1,3,4-thiadiazole possible most efficient pharmacophore substituents groupscaffold for that antiviralmay can be be consideredactivityincorporated in eacha possible into class. the structure pharmacophoreBased on of the known literature group compounds that data, ca nthe to be enhance2-amino-1,3,4-thiadiazole incorporated antiviral into activity the structure and scaffold contributes of mayknown be to compoundsconsideredthe search and a to possible developmentenhance pharmacophore antiviral of new activity medicines group and that ascont an caributes alternativen be incorporated to the to thesearch treatment into and the development ofstructure viral infections. of ofknown new medicinescompounds as toan enhance alternative antiviral to the treatmentactivity and of viralcont ributesinfections. to the search and development of new medicinesFunding: This as an research alternative received to nothe external treatment funding. of viral infections. Conflicts of Interest: The author declares no conflicts of interest.

Molecules 2020, 25, 942 17 of 22

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