Advances and Perspectives in the Management of Varicella-Zoster Virus Infections

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Review Advances and Perspectives in the Management of Varicella-Zoster Virus Infections

Graciela Andrei * and Robert Snoeck

Laboratory of Virology and Chemotherapy, Rega Institute for Medical Research, KU Leuven, 3000 Leuven, Belgium; [email protected] * Correspondence: [email protected]; Tel.: +32-16-32-19-51

Abstract: Varicella-zoster virus (VZV), a common and ubiquitous human-restricted pathogen, causes a primary infection (varicella or chickenpox) followed by establishment of latency in sensory ganglia. The virus can reactivate, causing herpes zoster (HZ, shingles) and leading to signiﬁcant morbidity but rarely mortality, although in immunocompromised hosts, VZV can cause severe disseminated and occasionally fatal disease. We discuss VZV diseases and the decrease in their incidence due to the introduction of live-attenuated vaccines to prevent varicella or HZ. We also focus on acyclovir, valacyclovir, and famciclovir (FDA approved drugs to treat VZV infections), brivudine (used in some European countries) and amenamevir (a helicase-primase inhibitor, approved in Japan) that augur the beginning of a new era of anti-VZV therapy. Valnivudine hydrochloride (FV-100) and valomaciclovir stearate (in advanced stage of development) and several new molecules potentially good as anti-VZV candidates described during the last year are examined. We reﬂect on the role of antiviral agents in the treatment of VZV-associated diseases, as a large percentage of the at-risk

population is not immunized, and on the limitations of currently FDA-approved anti-VZV drugs. Their low efﬁcacy in controlling HZ pain and post-herpetic neuralgia development, and the need of

Citation: Andrei, G.; Snoeck, R. multiple dosing regimens requiring daily dose adaptation for patients with renal failure urges the Advances and Perspectives in the development of novel anti-VZV drugs. Management of Varicella-Zoster Virus Infections. Molecules 2021, 26, Keywords: varicella-zoster virus (VZV); chickenpox; HZ; shingles; nucleoside analogues; helicase- 1132. https://doi.org/10.3390/ primase inhibitors; valnivudine hydrochloride (FV-100); valomaciclovir stearate; amenamevir; anti- molecules26041132 VZV drugs

Academic Editors: Libor Grubhoffer and Masanori Baba 1. Introduction Received: 12 January 2021 Varicella-zoster virus (VZV), also known as human herpesvirus 3 (HHV-3) has a Accepted: 12 February 2021 Published: 20 February 2021 double-stranded DNA genome of 125 kb, encoding for approximately 71 open reading frames (ORFs). The herpesvirus family includes three subfamilies, α, β, and γ-

Publisher’s Note: MDPI stays neutral herpesvirinae. VZV together with herpes simplex virus 1 and 2 (HSV-1 and HSV-2) belong α with regard to jurisdictional claims in to the -herpesvirinae, characterized by establishment of latency in neurons. published maps and institutional afﬁl- VZV is the causative agent of chickenpox (varicella), a common infantile illness. iations. Like all herpesviruses, VZV undergoes a lifelong latent state following primary infection. During latency, the viral DNA persists in the dorsal root ganglia and cranial root ganglia. VZV reactivation produces skin lesions characteristic of herpes zoster (shingles), causing signiﬁcant morbidity, but rarely mortality [1]. Herpes zoster (HZ) is characterized by a localized rash in a unilateral, dermatomal distribution and is often associated with severe Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. neuropathic pain. This article is an open access article VZV is highly infectious and enters the body via the respiratory tract, followed by distributed under the terms and rapid spread from the pharyngeal lymphoid tissue to circulating T lymphocytes [2]. After conditions of the Creative Commons 10–21 days, the virus arrives at the skin, producing the typical vesicular rash characteristic Attribution (CC BY) license (https:// of varicella. In most individuals, VZV infection results in lifelong immunity. During VZV creativecommons.org/licenses/by/ reactivation, the virus is transported along microtubules within sensory axons to infect 4.0/). epithelial cells, usually without viremia. This gives a skin rash within the dermatome

Molecules 2021, 26, 1132. https://doi.org/10.3390/molecules26041132 https://www.mdpi.com/journal/molecules Molecules 2021, 26, 1132 2 of 34

innervated by a single sensory nerve [the trigeminal (cranial nerve), cervical and thoracic sensory nerves are the most common nerves involved in VZV reactivation]. The virus can also be transmitted by fomites from varicella and shingles skin lesions. Reactivation from latency occurs when there is a weakening in cell-mediated immunity (CMI) as a natural consequence of aging since VZV-speciﬁc T cells lose their ability to proliferate with age or with immune suppression. Risks factors for HZ include older age, CMI dysfunction, diabetes, female gender, genetic susceptibility, physical trauma, recent psychological stress, and white race [2]. The narrow host range (VZV infection is highly restricted to humans) coupled with the highly cell-associated nature of the virus, has hampered the development of a reliable animal model that mimics VZV diseases in human, thereby hindering VZV pathogenesis studies.

2. Clinical Characteristics of VZV Infections Primary VZV infection generally occurs in childhood and is usually mild but complications can occur in adults and in immunocompromised patients. Disseminated varicella infections with multi-organ failure have been reported in both immunocompetent [3–5] and immunocompromised patients [5–7]. Although VZV reactivation leading to HZ may occur at any age, the highest incidence is seen in the elderly and among immunocompromised individuals (HIV/AIDS individuals or patients suffering from malignancies, organ or hematopoietic stem cell transplant (HSCT) recipients or persons receiving high-dose corticosteroid therapy). Age-related impaired CMI facilitates VZV reactivation from latency. HZ affects up to 25% of human beings during their lifetime, with 50% of persons being aged 80 years or more [8]. HZ is rarely life threatening but it is associated with a number of acute syndromes, including a vesicular rash and pain. It can lead to prolonged pain, known as post-herpetic neuralgia (PHN), a debilitating condition, which can be very difficult to manage, mainly in the elderly where the disease tends to be more serious [9,10]. PHN is associated with a loss of physical function, encompassing fatigue, anorexia, weight loss, reduced mobility, physical inactivity, sleep disturbance (especially insomnia) resulting in a loss of social contact. A positive correlation between enhanced severity of pain and higher interference with daily activities was reported [9]. Ophthalmological manifestations of VZV may also occur. About 10–15% of HZ cases are subtyped as HZ ophthalmicus (HZO), occurring when the virus is reactivated in the ophthalmic division of the trigeminal nerve [11]. Approximately 50% of individuals with HZO develop ophthalmic complications [12]. Chronic ocular inflammation, loss of vision, and debilitating pain can be permanent sequelae of HZO. Similar to other α-herpesviruses, VZV is able to infect the central nervous system (CNS) to cause encephalitis, either as a complication of varicella or HZ, but also in the absence of rash. About 50% of VZV encephalitis cases occur in individuals with immunosuppressive conditions, in whom viral reactivation is more common. A high viral diversity and mixed infections with different clades in the cerebrospinal fluid (CSF) from patients with VZV encephalitis, which can be explained by viral reactivation from multiple neurons, may contribute to the pathogenesis of VZV encephalitis [13]. VZV infections among immunocompromised patients can be more severe, of longer duration than in immunocompetent individuals and may have unusual clinical presentation with multidermal involvement and hyperkeratotic skin lesions [14,15]. VZV can be associated with severe acute retinal necrosis (ARN), a disease with poor prognosis; typically occurring in immunocompetent individuals though it can also occur in immunocompromised patients [16,17]. Unlike VZV necrotizing retinitis, progressive outer retinal necrosis (PORN), a form of VZV chorioretinitis, is found mostly in severely compromised people though, exceptionally, some cases of PORN have been described in immunocompetent persons [18–21]. Treatment strategies for PORN are rather unsuccessful and the disease can evolve fast leading to blindness within a few days or weeks. Molecules 2021, 26, 1132 3 of 34

3. Vaccination Strategies and Post-Exposure Prophylaxis Varivax® (the live-attenuated vaccine for varicella from Merck Sharp & Dohme Corp., Table1), which is based on the VZV attenuated Oka strain (vaccine Oka, vOka), was licensed in United States in 1995 for children aged 12 to 18 months, leading to a substantial drop in the incidence of varicella. GlaxoSmithKline also developed and launched a live vOka preparation (Varilrix®), for immunization against VZV infections in 1998. To prevent mild breakthrough infections among vaccinated children following a single dose of vOka, a recommendation for a 2-dose schedule was given in 2006 to improve immunity to VZV. The live-attenuated heterogeneous vaccine preparation vOka is used routinely in many countries worldwide, which resulted in a signiﬁcant decline in the incidence of varicella disease, with a decrease risk of developing zoster [22]. The varicella vaccine has a very good safety proﬁle. Only less than 5% vaccine recipients can develop a papular or vesicular rash, usually at the site of infection within 6 weeks following vaccination. Vaccinated individuals have minimum risk for transmitting vOka to contact persons and transmission occurs only in the presence of a rash [23]. Generalized varicella-like rashes can develop rarely within 14 days after vaccination and are due to wild-type VZV incidentally acquired soon after vaccination. vOka also establishes latent infection in vaccine recipients and may very rarely reactivate to cause HZ. Vaccine-related HZ occurs less frequently and is less severe than wild-type VZV reactivation and always manifests as HZ of one dermatome. Like most live attenuated viral vaccines, the emergence of vaccine-wild type recombinant strains can occur. VZV frequently undergoes genetic recombination, and the vaccine strains have already been found in recombination events with the wild-type [24].

Table 1. Vaccines available against varicella-zoster virus. Comparative characteristics of the available varicella zoster and herpes zoster vaccines.

Varicella Vaccine Herpes Zoster Vaccine Name Varivax® & Varilrix® Zostavax® Shingrix® Year of FDA 1995 2006 2018 licensure Merck (Varivax®) Manufacturer Merck GSK GSK (Varilrix®) Inactivated; Recombinant Type Life-attenuated viral vaccine Life-attenuated viral vaccine subunit Vaccine components Oka strain (1350 PFU) Oka strain (19,400 PFU) VZV glycoprotein E (gE) • Routine 2-dose vaccination 1st dose at 12–15 months old 2nd dose at 4–6 years old Number of doses • 2nd dose catch-up vaccination ≥3 1 dose 2 doses (2–6 months apart) administered months after 1st dose for children aged <13 years of age • Adolescent & adults 2 doses 4 to 8 weeks apart Storage Freezer Freezer Refrigerator Diluent Sterile water Sterile water Adjuvant 0.65 mL vial for subcutaneous 0.5 mL vial for intramuscular Dose form 0.5 mL vial for intramuscular injection injection injection

•≥ children 12 months • FDA approved: ≥50 • FDA approved: ≥50 years old years old Recommended age • Adults without evidence of immunity to varicella • CDC recommendation: • CDC recommendation: ≥60 years old ≥60 years old Molecules 2021, 26, 1132 4 of 34

Table 1. Cont.

Varicella Vaccine Herpes Zoster Vaccine Name Varivax® & Varilrix® Zostavax® Shingrix® • Shingles prevention Age • Shingles prevention Age 50–59: 69% Age 60–69: 50–59: 97.2% Age 60–69: 64% Age 70–79: 41% Age About 98% protection in children and 96.6% Age 70–79: 91.3% ≥80: 18% efficacy Efficacy about 75% protection in teenagers and Age ≥80: 91.4% • PHN protection Age adults • PHN protection Age ≥ 60–69: 51% Age 70–79: 50: 91.2% Age ≥ 70: 64% Age ≥80: 41% 88.8% Overall: 51% Unknown; however, long-term efficacy studies have demonstrated continued Immunity duration Age-dependent Age-dependent protection up to 10 years after vaccination • People with a history of • Individuals with a severe allergic reaction history of severe allergic • Immunocompromised (primary or (e.g., anaphylaxis) to any reaction (e.g., acquired) patients component of the anaphylaxis) to any • Family history of congenital vaccine component of the • Immunocompromised vaccine or after a Contraindications immunodeficiency’s • Individuals with anaphylactic patients previous dose of reaction to any component of the • Individuals with family Shingrix. vaccine efficacy history of congenital • Pregnant and immunodeficiency’s breastfeeding women • Pregnant and • Persons who currently breastfeeding women have shingles Most commonly: Most commonly: • Pain, redness, and Most commonly: swelling at the • Fever • Pain, redness, swelling, injection site • Pain, redness, and swelling at the warmth or itching at the • Muscle pain Possible side effects injection site site of injection at the • Tiredness • Varicella-like rash (transmission of injection site • Headache varicella-virus can occur) • Headache • Shivering • Fever • Upset stomach

Two vaccines (Zostavax® and Shingrix®, Table1) are currently available to boost the CMI to VZV. Both vaccines proved to be safe and immunogenic and to reduce the incidence of HZ and PHN. The efﬁcacy of the life-attenuated HZ vaccine (Zostavax®) decreases with age at the time of vaccination and with time since vaccination. The vaccine efﬁcacy of recombinant HZ vaccine (Shingrix®) remains higher and appears to decline more slowly than that of the life-attenuated vaccine across all age groups. Both vaccines are cost-effective in individuals ≥50 years of age compared with no vaccination, especially among those 65–79 years of age and Shingrix appears to be more cost-effective than Zostavax®. Impor- tantly, a live attenuated vaccine, such as Zostavax®, cannot be given to immunosuppressed patients and this patient population is at high risk for developing HZ. In contrast, Shingrix can be given to persons with impaired immune conditions [25]. VariZIG® (Saol Therapeutics) is a VZV immune globulin preparation available for post-exposure prophylaxis of varicella in persons at high-risk for severe disease who lack immunity to VZV and who are ineligible for varicella vaccine. High-risk groups encompass immunocompromised persons (children and adults), newborns of mothers who developed varicella just before or shortly after delivery, premature babies, children younger than one year of age, adults without evidence of immunity and pregnant women. The time during Molecules 2021, 26, 1132 5 of 34

which a patient may receive VariZIG® after VZV exposure has been prolonged from 4 days to 10 days.

4. Treatment of VZV-Associated Diseases MoleculesMolecules 2021 ,2021 26, 1132, 26, 1132 5 of 355 of 35 Molecules 2021, 26, 1132 5 of 35 Safe and effective anti-VZV therapy considerably contributed to diminish the morbidity and mortality associated with varicella and HZ, in particular in immunocompromised populations. The drugs licensed for the treatment of VZV-associated disease in United 4. Treatment4. Treatment of VZV of VZV‐Associated‐Associated Diseases Diseases States include4. Treatment acyclovir of VZV (ACV),‐Associated its oral Diseases prodrug valacyclovir (VACV), and famciclovir Safe and effective anti‐VZV therapy considerably contributed to diminish the mor‐ (FCV),Safe the and oralSafe effective prodrug and effective anti of‐VZV penciclovir anti therapy‐VZV therapy (PCV) considerably (Tableconsiderably 2contributed). contributed to diminish to diminish the mor the‐ mor‐ biditybidity and and mortality mortality associated associated with with varicella varicella and and HZ, HZ, in particular in particular in immunocompro in immunocompro‐ ‐ Acyclovirbidity and [ACV, mortality 9-(2-hydroxyethoxymethyl)guanine, associated with varicella and HZ, Zovirax in particular®], a structuralin immunocompro analogue‐ misedmised populations. populations. The The drugs drugs licensed licensed for forthe thetreatment treatment of VZVof VZV‐associated‐associated disease disease in in of the naturalmised compoundpopulations. 2 The0-deoxyguanosine drugs licensed (Tablefor the2 ),treatment is a potent of VZV and‐ selectiveassociated inhibitor disease in of UnitedUnited States States include include acyclovir acyclovir (ACV), (ACV), its itsoral oral prodrug prodrug valacyclovir valacyclovir (VACV), (VACV), and and VZV, HSV-1,United HSV-2, States andinclude Epstein-Barr acyclovir virus(ACV), (EBV) its oral with prodrug modest activityvalacyclovir against (VACV), human and cy- famciclovirfamciclovirfamciclovir (FCV), (FCV), the (FCV),theoral oral prodrug the prodrug oral of prodrug penciclovir of penciclovir of penciclovir (PCV) (PCV) (Table (Table(PCV) 2). 2).(Table 2). tomegalovirus (HCMV). Acyclovir and its prodrug valacyclovir® ® (L-valyl ester of acyclovir) AcyclovirAcyclovir Acyclovir[ACV, [ACV, 9‐(2 [ACV,9‐hydroxyethoxymethyl)guanine,‐(2‐hydroxyethoxymethyl)guanine, 9‐(2‐hydroxyethoxymethyl)guanine, Zovirax Zovirax], Zoviraxa ],structural a structural®], a structuralana ‐ana‐ ana‐ are the gold standard for prophylaxis and therapy of HSV and VZV associated diseases. loguelogue of the oflogue thenatural natural of the compound natural compound compound 2′‐deoxyguanosine 2′‐deoxyguanosine 2′‐deoxyguanosine (Table (Table 2), is2),(Table a ispotent a 2),potent isand a potentand selective selective and in selective‐ in‐ in‐ hibitorAcyclovirhibitor of VZV, hibitorof VZV, is HSV converted of HSV ‐VZV,1, HSV‐1, HSV HSV‐ to2, ‐and its‐1,2, HSV and monophosphateEpstein ‐Epstein2, and‐Barr Epstein‐Barr virus virus‐ (ACV-MP)(EBV)Barr (EBV) virus with with (EBV) modest by modest the with activity viral modest activity thymidine against activity against against kinase human(TK),human cytomegalovirus whichhuman cytomegalovirus is furthercytomegalovirus (HCMV). phosphorylated (HCMV). Acyclovir(HCMV). Acyclovir by andAcyclovir cellular and its prodrugits andprodrug kinases its valacyclovir prodrug tovalacyclovir ACV-triphosphate valacyclovir (L‐valyl (L‐valyl ester (L ester‐ valyl (ACV-TP) ester of (Figureacyclovir)of acyclovir)1of). areacyclovir) ACV-TP, arethe thegold are thegold standard the active standard gold formfor standard prophylaxisfor of prophylaxis acyclovir, for prophylaxis and and is therapy a competitivetherapy and of therapyHSV of HSV and inhibitor of and VZV HSV VZV associ and with associ VZV‐ respect‐ associ to‐ atedtheated diseases. natural diseases.ated Acyclovir substratediseases. Acyclovir isAcyclovir dGTPconverted is converted (deoxyguanosine is toconverted its to monophosphate its monophosphate to its monophosphate triphosphate) (ACV (ACV‐MP)‐MP) and(ACV by the by it‐MP) canviralthe viralby be thymi the a thymi substrate viral‐ ‐ thymi for‐ dinethedine kinase viral kinasedine DNA(TK), kinase(TK), which polymerase which (TK), is further whichis further and phosphorylatedis then further phosphorylated be incorporatedphosphorylated by cellularby cellular to by thekinases cellular kinases 30-end to kinasesACV ofto aACV‐ synthesizedtriphos to‐triphos ACV‐ ‐triphos‐ DNA‐ phatemolecule.phate (ACV (ACVphate‐TP) The‐ TP) (Figure(ACV DNA (Figure‐TP) 1). polymerase-associated ACV(Figure 1). ACV‐TP, 1).‐ TP,the ACV activethe‐ TP,active form the form 3active of0-5 acyclovir,0 exonucleaseof form acyclovir, of is acyclovir, a competitiveis cannot a competitive is a excise competitive inhibitor inhibitor the 3 0 -terminalinhibitor with respect to the natural substrate dGTP (deoxyguanosine triphosphate) and it can be a ACV-TPwith respectwith residues, respect to the resultingnatural to the natural substrate in prevention substrate dGTP (deoxyguanosine dGTP of chain (deoxyguanosine elongation triphosphate) as triphosphate) ACV and lacks it can the and be 3 it0hydroxyl acan be a substratesubstrate for thefor theviral viral DNA DNA polymerase polymerase and and then then be incorporated be incorporated to the to the3′‐end 3′‐end of a of syn a ‐syn‐ group requiredsubstrate forfor DNAthe viral elongation. DNA polymerase Acyclovir and hasthen limited be incorporated oral bioavailability to the 3′‐end (15–30%)of a syn‐ thesizedthesized DNAthesized DNA molecule. molecule. DNA Themolecule. The DNA DNA polymeraseThe polymerase DNA polymerase‐associated‐◦associated‐ associated3′‐5 ′3exonuclease′‐5′exonuclease 3′‐5′exonuclease cannot cannot excise cannotexcise excise theand 3the′‐terminal restricted3′‐terminal ACV solubilityACV‐TP ‐residues,TP residues, in water resulting resulting (~0.2%, in prevention in 25preventionC), of requiring chain of chain elongation relatively elongation as ACV large as ACV lacks doses lacks and frequent administrationthe 3′‐terminal ACV to maintain‐TP residues, plasma resulting levels in ofprevention acyclovir of high chain enough elongation to achieveas ACV lacks viral the the3′hydroxyl 3′hydroxylthe 3 group′hydroxyl group required requiredgroup for required DNAfor DNA elongation. for elongation. DNA elongation. Acyclovir Acyclovir hasAcyclovir haslimited limited has oral limited oral bioavaila bioavaila oral‐ bioavaila‐ ‐ inhibition. The valine ester of acyclovir, valacyclovir (VACV, Valtrex, Zelitrex) (Table2 ) bilitybility (15–30%) (15–30%)bility and (15–30%) and restricted restricted and solubility restricted solubility in solubility water in water (~0.2%, in (~0.2%, water 25 °C),(~0.2%, 25 °C), requiring 25requiring °C), relatively requiring relatively large relatively large large dosesisdoses a and safe and frequentdoses and frequent and efﬁcacious administration frequent administration prodrug administration to maintain to (54% maintain to oral plasma maintain plasma bioavailability). levels plasma levels of acyclovir of levels acyclovir It of is high acyclovir rapidly high enough enough metabolizedhigh to enough to to to achieveyieldachieve viral acyclovirachieve viral inhibition. inhibition. andviral the inhibition.The essentialThe valine valine Theester amino estervaline of acyclovir, of acid ester acyclovir,L of-valine valacycloviracyclovir, valacyclovir [26] valacyclovir due (VACV, to (VACV, a carrier-mediated Valtrex, (VACV, Valtrex, Zeli Valtrex, Zeli‐ ‐ intesti- Zeli‐ trex)naltrex) (Table absorption, (Tabletrex) 2) is (Table2) a is viasafe a 2)safe theand is humanand aefficacious safe efficacious and intestinal efficaciousprodrug prodrug peptide (54% prodrug (54% oral transporter oral bioavailability).(54% bioavailability). oral hPEPT1,bioavailability). It is It followed rapidly is rapidly It is by rapidly rapid metabolized to yield acyclovir and the essential amino acid L‐valine [26] due to a carrier‐ conversionmetabolizedmetabolized to to acyclovir yield to acyclovir yield by acyclovir ester and hydrolysis the and essential the essential in amino the small acidamino L intestine.‐ valineacid L ‐[26]valine Several due [26] to adue clinical carrier to a ‐carrier studies‐ mediateddemonstratedmediated intestinalmediated intestinal that absorption, intestinal valacyclovirabsorption, absorption, via thevia has thehuman a viahuman safety the intestinal human intestinal proﬁle peptide intestinal comparable peptide transporter peptide transporter to transporter that hPEPT1, hPEPT1, of acyclovir fol hPEPT1,‐ fol‐ in fol HZ‐ lowedpatients.lowed by rapidbylowed Valacyclovir rapid conversion by conversion rapid conversion becameto acyclovir to acyclovir a to better by acyclovir esterby option ester hydrolysis by hydrolysis inester the hydrolysisin treatment the in thesmall small in intestine. ofthe VZVintestine. small infectionsSeveral intestine. Several Several since it clinical studies demonstrated that valacyclovir has a safety profile comparable to that of requiresclinicalclinical studies a less studies frequentdemonstrated demonstrated dosing that regimen valacyclovir that valacyclovir than has acyclovir, a safety has a profile contributingsafety profilecomparable comparable to increased to that toof patientthat of acycloviracyclovir in HZ in HZpatients. patients. Valacyclovir Valacyclovir became became a better a better option option in the in thetreatment treatment of VZV of VZV adherenceacyclovir to therapy. in HZ patients. Valacyclovir became a better option in the treatment of VZV infectionsinfections sinceinfections since it requires it since requires ita lessrequires a lessfrequent frequent a less dosing frequent dosing regimen dosingregimen than regimen than acyclovir, acyclovir, than contributingacyclovir, contributing contributing to to to increasedincreased patient patient adherence adherence to therapy. to therapy. Table 2. Currently licensed anti-herpesvirusincreased agents patient used adherence to treat to VZV therapy. infections. Anti-VZV drugs approved in United States,Table 2. Europe Currently and/or licensed Japan anti for‐herpesvirus treatment agents of VZV-associated used to treat VZV diseases infections. and drugs Anti‐VZV used drugs off label approved to treat in acyclovir-resistant United Table 2.Table Currently 2. Currently licensed licensed anti‐herpesvirus anti‐herpesvirus agents usedagents to usedtreat toVZV treat infections. VZV infections. Anti‐VZV Anti drugs‐VZV approved drugs approved in United in United States, Europe and/or Japan for treatment of VZV‐associated diseases and drugs used off label to treat acyclovir‐resistant infections.States, EuropeStates, Europeand/or Japanand/or for Japan treatment for treatment of VZV ‐ofassociated VZV‐associated diseases diseases and drugs and used drugs off used label off to labeltreat acyclovirto treat acyclovir‐resistant‐resistant infections. infections.infections. Mode of Drug Prodrug Mode of Action/Ap‐ Natural Analogue Action/Approval/IndicationMode ofMode Action/Ap of Action/Ap‐ ‐ DrugDrug Drug ProdrugProdrug Prodrug NaturalNatural AnalogueNatural Analogue Analogue proval/Indicationproval/Indicationproval/Indication I. Nucleoside analogues I. I.Nucleoside NucleosideI. Nucleoside analogues analogues analogues  • DNA Polymerase AcyclovirAcyclovir (ACV),Acyclovir (ACV), Zovirax (ACV), Zovirax Zovirax ValaciclovirValaciclovir Valaciclovir (VACV), (VACV), Val (VACV), ‐Val‐ DNA Val DNA‐ Polymerase Polymerase DNA Polymerase inhibi inhibi‐ 2‐ ′‐inhibiDeoxyguanosine2′‐Deoxyguanosine‐ 2′‐Deoxyguanosine (natural (natural (natural inhibitor—chain 9‐(29‐hydroxyethoxyme‐(2‐hydroxyethoxyme9‐(2‐hydroxyethoxyme‐ ‐ trex,trex,‐ Zelitrex Zelitrextrex, Zelitrex tor—chaintor—chain tor—chainterminator terminator terminator at the at thenucleoside) atnucleoside) the nucleoside) thyl)guanine L‐valine ester of acyclovir incorporationterminator site at the thyl)guaninethyl)guanine L‐valineL ‐estervaline of ester acyclovir of acyclovir incorporation incorporation site site O O incorporation site O N  FDA FDA approved approved HN N  HN N • FDAFDA approved approved HN  Treatment Treatment and and suppres suppres‐ ‐ H N N N N  2 H2N N N • TreatmentTreatment and and suppres‐ H2N N sionsion of VZV of VZV infections infections (1st (1st HO HO sionsuppression of VZV infections of VZV (1st O O HO O lineline treatment treatmentlineinfections fortreatment VZVfor VZV (1st‐asso for line‐asso‐ VZV‐ ‐asso‐ ciatedciated diseases) diseases)treatment for OH OH ciated diseases) OH VZV-associated diseases)

Molecules 2021, 26, 1132 6 of 34

Table 2. Cont. MoleculesMoleculesMolecules 20212021 2021,, 2626,, ,11321132 26, 1132 66 ofof 35635 of 35

Molecules 2021Molecules, 26, 1132 2021 , 26, 1132 Mode of 6 of 35 6 of 35 MoleculesDrug 2021,, 26,, 1132 Prodrug Natural Analogue 6 of 35 Molecules 2021, 26, 1132 Action/Approval/Indication 6 of 35

Molecules 2021, 26, 1132 6 of 35 PenciclovirPenciclovirPenciclovir (PCV),(PCV), (PCV), Denavir,Denavir, Denavir, FamciclovirFamciclovir Famciclovir (FAM),(FAM), (FAM), FamvirFamvir Famvir  DNADNA DNA• PolymerasePolymerase PolymeraseDNA Polymerase inhibiinhibi inhibi‐‐ ‐22′‐′‐DeoxyguanosineDeoxyguanosine2′‐Deoxyguanosine (natural(natural (natural Vectavir Diacetyl ester of 9‐(4‐hy‐ tor—chain terminator after nucleoside) VectavirPenciclovirVectavir Penciclovir (PCV), Denavir, (PCV), DiacetylDenavir,FamciclovirDiacetyl ester Famciclovir ester(FAM), of 9 of‐(4 9Famvir‐hy‐(4 (FAM),‐hy‐ tor—chain FamvirDNAtor—chain Polymerase terminatorinhibitor—chain DNA terminator Polymerase inhibi after after‐ nucleoside)2 inhibiinhibi′‐Deoxyguanosinenucleoside)‐ 2 ′‐′‐Deoxyguanosine (natural (natural PenciclovirPenciclovir (PCV), (PCV), Denavir, Denavir, Famciclovir Famciclovir (FAM), (FAM), Famvir Famvir  DNA  DNAPolymerase Polymerase inhibi inhibi‐ 2′‐‐Deoxyguanosine2′‐Deoxyguanosine (natural (natural 9Vectavir9‐‐(4(49‐‐hydroxy‐hydroxy(4‐hydroxy ‐‐33‐‐hydroxymehydroxyme‐3‐hydroxyme‐‐ droxyDiacetyldroxy‐ droxy‐‐33‐‐ hydroxymethylhydroxymethylester‐3‐hydroxymethyl of 9‐(4‐hy‐‐ ‐ incorporationincorporationtor—chainincorporation terminator andand and elongaelonga elonga after ‐‐ ‐ nucleoside) OO O PenciclovirVectavirVectavir (PCV), Denavir, FamciclovirDiacetylDiacetyl (FAM), ester esterFamvir ofof 9 9‐‐(4(4 ‐‐DNAhyhy‐‐ Polymerasetor—chaintor—chaintor—chain inhibi terminatorterminatorterminator‐ 2′‐Deoxyguanosine afterafter nucleoside)nucleoside) (natural Vectavir Diacetyl ester of 9‐(4‐hy‐ tor—chain terminator after nucleoside) N thyl‐but‐1‐yl)guanine but‐ 1‐yl)‐6‐deoxyguanine tion of severalincorporation bases and HN N N thyl9‐(4thyl‐buthydroxy‐‐but1‐yl)guanine‐1‐3yl)guanine‐hydroxyme ‐ butdroxy‐ but1‐‐yl)‐ 3‐1hydroxymethyl‐6yl)‐deoxyguanine‐6‐deoxyguanine‐ tion incorporationtion of several of several andbases bases elonga ‐ HN OHN O 99‐‐(4(4‐‐hydroxyhydroxy‐‐33‐‐hydroxymehydroxyme‐‐ droxydroxy‐‐33‐‐hydroxymethylhydroxymethyl‐‐ incorporationincorporationincorporation andand elongaelonga‐‐ O Vectavir9‐(4 ‐hydroxy‐3‐hydroxymeDiacetyl‐ droxy ester‐3‐hydroxymethyl of 9‐(4‐hy‐ ‐tor—chainincorporation terminatorelongation and after of elonga several nucleoside)‐ O  FDA approved N thyl ‐but ‐1‐yl)guanine but‐ 1‐yl)‐6‐deoxyguanine tion FDA ofFDA severalapproved approved bases H2N HNH NNN NN N N thylthyl‐but‐1‐yl)guanine but‐ 1‐yl)‐6‐deoxyguanine tiontion of several bases H2N 2 HN N 9‐(4‐hydroxythyl‐3‐‐buthydroxyme‐1‐yl)guanine‐ droxy ‐3 ‐hydroxymethylbut‐ 1‐yl)‐6‐deoxyguanine‐ incorporation tionbases and of several elonga bases‐ O HNN thyl‐but‐1‐yl)guanine but‐ 1‐yl)‐6‐deoxyguanine Treatmenttion of several of HZ bases HN  TreatmentFDA Treatment approved of HZ of HZ HOH N N N  FDA approved HO2 HO O N H N N N thyl‐but‐1‐ yl)guanine but‐ 1‐yl)‐6‐deoxyguanine tion of • severalFDAFDA bases approved approved O O H22N N N  FDA approved HN H2N N  H2N N Treatment Treatmentof HZ of HZ HO • Treatment Treatment of of HZ HZ O HO  FDA approved H N N NHO O Treatment of HZ 2 HO O O  OHOH OH Treatment of HZ HO O OH OH OH OH

Brivudine,Brivudine,Brivudine, Zostex,Zostex, Zostex, Zonavir,Zonavir, Zonavir, OrallyOrally OrallyOrally bioavailablebioavailable bioavailable bioavailable •• IncorporationIncorporation• Incorporation• Incorporation intointo into viralviral viral into 22 viral′‐′‐deoxythymidinedeoxythymidine2′‐deoxythymidine (natural(natural (natural ZerpexZerpex DNA DNA with with replicationDNA replication with‐in‐‐in‐ nucleoside)nucleoside) ZerpexBrivudine, Brivudine,Brivudine, Zostex, Zonavir, Zostex,Zostex, Zonavir,Zonavir,Orally bioavailable OrallyOrally bioavailablebioavailable DNA • Incorporation with• •replication IncorporationIncorporation into viral‐in‐ intointointonucleoside)2 ′‐viralviraldeoxythymidine 22 ′‐′‐′‐deoxythymidinedeoxythymidine (natural (natural(natural Brivudine, Zostex, Zonavir, Orally bioavailable • Incorporationreplication-incompetent into viral 2′‐deoxythymidineO (natural (Zerpex(EE))‐‐(55E‐‐(2)(2‐5 ‐‐bromovynil)‐bromovynil)(2Zerpex‐bromovynil) ‐‐22′‐′‐dede‐2′‐‐‐de‐ competentDNAcompetentcompetent with DNA virusvirusreplication virus withproductionproduction production replication‐in‐ nucleoside) ‐in‐ nucleoside) O O Zerpex DNA with replication‐inin‐ nucleoside)CHCH3 CH Brivudine,Zerpex Zostex, Zonavir, Orally bioavailable • IncorporationDNA withvirus replicationinto production viral ‐in‐2′‐deoxythymidinenucleoside)HN (natural3 3 oxyuridine (BVDU), bromo‐ • Approved in some Euro‐ HN OHN O oxyuridine(E)‐oxyuridine5‐(2‐bromovynil)( E(BVDU),)‐5 (BVDU),‐(2‐bromovynil) bromo‐2′‐ debromo‐‐ ‐‐2′‐′‐de‐ •competentApproved• Approved• competent virus in some in production some Eurovirus Euro‐ production ‐ O Zerpex (E)‐5‐(2‐bromovynil)‐2′‐de‐ DNA with competentreplicationApproved virus‐ in‐ some productionnucleoside) CHO3 (E)‐5‐(2‐bromovynil)‐2′‐de‐ competent virus production O N CH 3 vynildeoxyuridinevynildeoxyuridine pean pean countries countries O HN ON N HN CH33 vynildeoxyuridineoxyuridineoxyuridine (BVDU), bromo (BVDU),‐ bromo‐ pean• Approved countries• Approvedin some Euro in some‐ Euro‐ O HNCH3 oxyuridine (BVDU), bromo‐ •European Approved countries inin some EuroHO‐ HN (E)‐5‐oxyuridine(2‐bromovynil) (BVDU),‐2′‐de bromo‐ ‐ competent• Approved virus production in some Euro ‐ HO HO O • Treatment of HZ OO NO CH vynildeoxyuridine •pean Treatment• Treatmentcountries• of HZ of HZ 3 O N vynildeoxyuridinevynildeoxyuridine peanpeanTreatment countriescountries of HZ HN O N oxyuridinevynildeoxyuridine (BVDU), bromo ‐ • Approvedpean countries in some Euro‐ HO O HON O HO O • Treatment of HZ O •• TreatmentTreatment ofof HZHZ OHO N vynildeoxyuridine pean• countries Treatment of HZ OHOH OH O HO • Treatment of HZ O OH OH OH OH

II. Nucleotide analogue II.II. II.NucleotideNucleotide Nucleotide analogueanalogue analogue HPMPC, Cidofovir, Vis‐ Brincidofovir (BCV), • DNA Polymerase inhibi‐ 2′‐deoxycytidine 5′‐mono‐ HPMPC,II. HPMPC,Nucleotide Cidofovir,II.II. Cidofovir, NucleotideNucleotide analogue Vis ‐Vis‐ analogueanalogueBrincidofovirBrincidofovir (BCV), (BCV), • DNA• DNA• Polymerase PolymeraseDNA Polymerase inhibi inhibi‐ ‐2′‐deoxycytidine2′‐deoxycytidine 5′‐mono 5′‐mono‐ ‐ ®II. Nucleotide analogue tidetideHPMPC,tide®,, ((SS®)),‐ ‐ (1Cidofovir,1S‐‐(3)(3‐1‐‐hydroxy‐hydroxy(3‐hydroxy Vis‐‐22‐‐‐ ‐2‐ CMX001,BrincidofovirCMX001,CMX001, hexadecyloxyprohexadecyloxypro hexadecyloxypro (BCV), ‐‐ tor—chain•tor—chain‐ DNAtor—chain Polymerase terminatorterminatorinhibitor—chain terminator inhibi afterafter after‐ phosphatephosphate2 ′‐deoxycytidinephosphate (natural(natural (natural 5′‐ nucleomononucleo nucleo‐‐‐ ‐ II. NucleotideHPMPC,HPMPC, analogue Cidofovir,Cidofovir, VisVis‐‐ BrincidofovirBrincidofovir (BCV),(BCV), •• DNADNA PolymerasePolymerase inhibiinhibiinhibi‐‐ 22′‐′‐′‐deoxycytidinedeoxycytidine 55′‐′‐′‐monomono‐‐ phosphonylmethoxypro®HPMPC, Cidofovir, ‐Vis‐ pyl‐cidofovirBrincidofovir (HDP (BCV),‐CDV) incorporation• DNA Polymerase and elonga inhibi‐ tide)‐ 2 ′‐deoxycytidine 5′‐mono‐ phosphonylmethoxyprotidephosphonylmethoxypro, (S)‐1tide‐(3‐hydroxy®®, (S)‐1‐(3‐2‐‐‐hydroxy‐ pylCMX001,‐pyl‐cidofovir2‐ ‐cidofovir hexadecyloxyproCMX001, (HDP (HDP‐ CDV)hexadecyloxypro‐CDV) ‐ incorporation tor—chainincorporation‐ tor—chain terminator and and elonga terminatorelonga after ‐ ‐tide)phosphate tide)after phosphate (natural nucleo (natural‐ nucleo‐ HPMPC,® Cidofovir,tidetide ,, (S)‐ 1Vis‐(3‐‐hydroxyBrincidofovir‐2‐ CMX001, (BCV), hexadecyloxypro • DNA Polymerase‐ tor—chaintor—chain inhibi terminatorterminator‐ 2′‐deoxycytidine after phosphate 5′‐mono (natural‐ nucleo‐ pyl)cytosinetide , (S )(HPMPC)‐1‐(3‐hydroxy ‐2‐ CMX001, hexadecyloxyprotion‐ tor—chainof severalincorporation bases terminator and after phosphate (naturalNH nucleo‐ pyl)cytosinephosphonylmethoxypropyl)cytosine (HPMPC) (HPMPC) ‐ pyl ‐cidofovir (HDP‐CDV) tionincorporationtion of several of several andbases bases elonga ‐ tide) NH22 NH2 ® phosphonylmethoxypro NH2‐NH pyl‐cidofovir (HDP‐CDV) incorporationincorporation and elonga ‐ tide)tide) tide phosphonylmethoxypro, (S)‐1phosphonylmethoxypro‐(3‐hydroxy‐2‐ ‐CMX001, NHpyl2‐ ‐2cidofovir hexadecyloxypropyl‐cidofovir (HDP (HDP‐CDV)‐ tor—chain‐ CDV)incorporation incorporationterminator and after elonga and elongaphosphate‐ tide)‐ tide)(natural nucleo‐ NHNH2 NH2 elongation of several pyl)cytosine (HPMPC)2 tion of several bases N NHN2 pyl)cytosine (HPMPC) tion of several bases N NH 2 pyl)cytosine (HPMPC) N NH 2 tiontion of several bases NH22 phosphonylmethoxypro‐ pylN ‐cidofovirN NH(HDP2 ‐CDV) incorporationbases and elonga‐ tide) NH pyl)cytosineNH (HPMPC) NH22 • Cidofovir:tion of several off label bases use— 2 NN N2 NH2 NH2 • Cidofovir:• Cidofovir: off labeloff label use— use— O N N NH22 O ON N N pyl)cytosine (HPMPC)NH N tion of• several bases NH2 N 2 OO ONN N ONO O Cidofovir: off label N NH2 N treatment• Cidofovir:treatment of ACVoff of ACVlabel and/or use—and/or O NON N N N treatment of• Cidofovir:ACV and/or off label use— O O O N O NHN 2 N OPO(CH) O(CH ) CH •use—treatment Cidofovir: off labellabel of ACV use—HOHO HOO O N OPO(CHOPO(CH22)33O(CH2)3O(CH22)1515CH2)3315CH 3 P O N O N N O N O O N O • Cidofovir: off label use— P O P O O O O N O N O PFAtreatmentPFAPFA‐‐resistantresistant‐resistant of ACV VZVVZV VZV and/or infecinfec infec‐‐ ‐ HO HO O O N HO O OO N OH OH treatmenttreatment of ACV and/orHO O HO HO N O N O OPO(CHN OH O ) O(CH ) CH • Cidofovir:treatmentand/or off label PFA-resistant of use— ACV and/orHO HO O P O O N 2OPO(CH3 2 15 3 ) O(CH )treatmentCH of ACV and/or P O OHO NO P PO OO N OPO(CH22))33O(CHtions22))1515 CH 33 O O P O O HOHON HO OOPO(CH) O(CH )2 3CHtionsPFA2tions15‐ resistant3 PFA VZV‐resistant infec ‐VZV infec‐HO HO P O O HOHO HOO O OH 2 3 2 15 3 PFAVZV‐resistant infections VZV infecinfec‐ P OHO O HO O OH treatmentPFA‐ resistantof ACV and/or VZV infec ‐ HO O P OO OHON OH • Brincidofovir: herpesvirus HO OHO OH HO P O OPO(CHOH2)3O(CH2)15CH 3 •tions Brincidofovir:• Brincidofovir:• herpesvirus herpesvirus OH OH HO HO P O HO HO tionstionsBrincidofovir: P O HOP HOHOO HO PFA‐resistanttions VZV infec‐ HO O HO O HO HO OH development tions discontinued HO development• Brincidofovir:development•herpesvirus Brincidofovir: discontinued herpesvirusdiscontinued herpesvirus OH OH P HOO tions • Brincidofovir: herpesvirus OH HOHO HO • Brincidofovir: herpesvirus OH III.III.HO III. PyrophosphatePyrophosphate PyrophosphateHO analogueanalogue analogue developmentdevelopmentdevelopment discontinued discontinued • Brincidofovir:development herpesvirus discontinued OH Foscarnet,HO Foscavir • Directdevelopment DNAdiscontinued polymerase discontinued Foscarnet,III.Foscarnet, Pyrophosphate FoscavirIII. Foscavir Pyrophosphate analogue analogue • Direct• Direct DNA DNA polymerase polymerase III. PyrophosphateIII. Pyrophosphate analogue analogue development discontinued III. Foscarnet, Pyrophosphate OO FoscavirO analogue inhibitor•inhibitor Directinhibitor DNA withoutwithout without polymerase incorporaincorpora incorpora ‐‐ ‐ Foscarnet,Foscarnet,OO O FoscavirFoscavir •• DirectDirect DNADNA polymerasepolymerase III. Foscarnet,-Pyrophosphate Foscavir analogue • Direct DNA polymerase ++- O + - P C tion NaNa ONa PO CP C O tioninhibitortion • withoutinhibitorDirect incorpora DNA without polymerase‐ incorpora‐ O-- +- O O inhibitorinhibitor without incorporaincorpora‐ Foscarnet, -Foscavir+ O NaO+ + O • Direct DNA polymerase + - O- + -O Na+ Na inhibitor without incorpora‐ O OP NaNaCO Na+ -- O •tion• OffOff• Off labellabel label useuse ‐ use ‐ treatmenttreatment ‐ treatment ofof of Na Na++ -O P C tioninhibitor without O+ - Na - O + P C inhibitor withouttiontion incorpora‐ Na O- +OPO NaC -- + tion O - + O- Na++ acyclovir ‐resistant VZV in‐ Na O- -- Na+++ O Na acyclovir• Offacyclovir label‐resistant• incorporationuse ‐Offresistant ‐ labeltreatment VZV useVZV in ‐ ‐ of treatmentin ‐ of + - - OONaNa • Off labellabel use ‐ ‐ treatmenttreatment of Na O P C O Na+ tion • Off label use ‐ treatment of - fections• O Na+ fectionsacyclovirfections ‐resistantacyclovir Off label VZV‐ use—treatmentresistant in‐ VZV in‐ O- Na+ • Off labelacyclovir use ‐ treatment‐resistant of VZV inin‐ IV.IV. IV. NonNon Non‐‐nucleosidenucleoside‐nucleoside analogueanalogue analogue fectionsacyclovir of‐ acyclovir-resistantresistant VZV in‐ acyclovir‐resistantfectionsfections VZV in‐ IV.AmenamevirAmenamevir AmenamevirNon‐nucleoside {{NN‐‐ (2,6{(2,6N‐(2,6‐‐dime dimeanalogue‐dime‐‐ OrallyOrally‐ Orally bioavailablebioavailable bioavailable •• Helicase Helicase• Helicasefections‐‐primaseprimaseVZV ‐primase infections inhibitorinhibitor inhibitor IV.IV. NonNon‐‐nucleosidenucleoside analogueanalogue fections Amenamevirthylphenyl)thylphenyl)IV.thylphenyl) Non‐‐NN ‐{‐nucleosideN‐[2‐[2N‐(2,6‐‐[4‐[4[2‐‐(1,2,4‐(1,2,4[4dime‐(1,2,4 ‐‐analogue‐ Orally‐ bioavailable •• Approved HelicaseApproved• Approved‐primase onlyonly only inin inhibitor JapanJapan in Japan IV. Non‐nucleosideAmenamevirAmenamevir analogue {{NN‐‐(2,6(2,6 ‐‐dimedime‐‐ OrallyOrally bioavailablebioavailable •• Helicase Helicase‐‐primaseprimase inhibitorinhibitorinhibitor oxadiazolAmenamevir‐3‐yl)anilino] {N‐(2,6‐2‐ox‐dime‐ ‐ Orally bioavailable • Treatment• Helicase of‐ HZprimase inhibitor oxadiazolthylphenyl)oxadiazol‐3‐thylphenyl)thylphenyl)yl)anilino]‐N3‐‐yl)anilino][2‐[4‐(1,2,4‐‐2N‐ox‐‐[2‐2‐‐‐ox[4‐‐(1,2,4‐ •• Treatment Approved• Treatment• Approved ofonly HZ of in HZ Japan only inin Japan Amenamevirthylphenyl)thylphenyl) {N‐(2,6‐N‐[2‐dime‐‐[4N‐‐(1,2,4[2‐ ‐[4Orally‐‐(1,2,4 bioavailable‐ • Helicase• Approved‐•primase Approved only inhibitor in only Japan in Japan oxadiazoloethyl]oethyl]oethyl]‐‐1,11,1‐oxadiazol3‐‐1,1yl)anilino]dioxothianedioxothiane‐dioxothiane‐3‐‐yl)anilino]2‐‐44ox‐‐ ‐4‐ ‐2‐ox‐ • Treatment• Treatmentof HZ of HZ thylphenyl)oxadiazol‐N‐[2‐[4‐‐3(1,2,4‐yl)anilino]‐ ‐2‐ox‐ • Approved • Treatmentonly in Japan of HZ oethyl]oxadiazolcarboxamide}carboxamide}‐1,1carboxamide}‐dioxothiane‐3‐yl)anilino] ‐4‐‐2‐ox‐ •Treatment of HZ oxadiazol‐3‐oethyl]oethyl]yl)anilino]‐‐1,11,1‐‐dioxothiane2dioxothiane‐ox‐ ‐‐44‐‐ • Treatment of HZ oethyl]‐1,1‐dioxothiane‐4‐ carboxamide}carboxamide} oethyl]‐1,1‐carboxamide}dioxothianecarboxamide}‐4 ‐

carboxamide}

Molecules 2021, 26, 1132 6 of 35

Penciclovir (PCV), Denavir, Famciclovir (FAM), Famvir  DNA Polymerase inhibi‐ 2′‐Deoxyguanosine (natural Vectavir Diacetyl ester of 9‐(4‐hy‐ tor—chain terminator after nucleoside)

9‐(4‐hydroxy‐3‐hydroxyme‐ droxy‐3‐hydroxymethyl‐ incorporation and elonga‐ O

N thyl‐but‐1‐yl)guanine but‐ 1‐yl)‐6‐deoxyguanine tion of several bases HN

 FDA approved N H2N N  Treatment of HZ HO O

Brivudine, Zostex, Zonavir, Orally bioavailable • Incorporation into viral 2′‐deoxythymidine (natural Zerpex DNA with replication‐in‐ nucleoside) (E)‐5‐(2‐bromovynil)‐2′‐de‐ competent virus production O CH3 oxyuridine (BVDU), bromo‐ • Approved in some Euro‐ HN vynildeoxyuridine pean countries O N HO • Treatment of HZ O

II. Nucleotide analogue HPMPC, Cidofovir, Vis‐ Brincidofovir (BCV), • DNA Polymerase inhibi‐ 2′‐deoxycytidine 5′‐mono‐ tide®, (S)‐1‐(3‐hydroxy‐2‐ CMX001, hexadecyloxypro‐ tor—chain terminator after phosphate (natural nucleo‐ phosphonylmethoxypro‐ pyl‐cidofovir (HDP‐CDV) incorporation and elonga‐ tide)

pyl)cytosine (HPMPC) tion of several bases NH2 NH2 NH 2 N N • Cidofovir: off label use— N O N O N O treatment of ACV and/or O N O OPO(CH) O(CH ) CH HO 2 3 2 15 3 P O PFA‐resistant VZV infec‐ HO O HO O OH P O tions HO HO • Brincidofovir: herpesvirus OH HO Molecules 2021, 26, 1132 development discontinued 7 of 34 III. Pyrophosphate analogue Foscarnet, Foscavir • Direct DNA polymerase O O inhibitor without incorpora‐ - Table 2. Cont. Na+ O P C tion O- Na+ O- Na+ • Off label use ‐ treatment of Mode of Drug Prodrug acyclovir‐resistant VZV in‐ Natural Analogue Action/Approval/Indication fections IV.IV. Non-nucleoside Non‐nucleoside analogue analogue Amenamevir {N‐(2,6‐dime‐ OrallyOrally bioavailable bioavailable • Helicase• ‐primaseHelicase-primase inhibitor thylphenyl)‐N‐[2‐[4‐(1,2,4‐ • Approved inhibitoronly in Japan oxadiazol‐3‐yl)anilino]‐2‐ox‐ • Treatment• Approvedof HZ only in Japan oethyl]‐1,1‐dioxothiane‐4‐ • Treatment of HZ carboxamide}

Penciclovir [PCV,9-(4-hydroxy-3-hydroxymethyl-but-1-yl)guanine, Denavir®, Vectavir®],

a 20-deoxyguanosine analog, resembles acyclovir in chemical structure, mechanism of action and spectrum of antiviral activity (Table2 and Figure1)[ 27,28]. PCV-TP inhibits viral DNA polymerases through competition with 20-deoxyguanosine triphosphate and incorporation into the synthesized viral DNA. Unlike ACV-TP, PCV-TP has two hydroxyl groups on the acyclic chain and thus PCV-TP is not an obligate chain terminator and can be incorporated into the extended DNA chain. Penciclovir is very poorly absorbed when given orally and thus, famciclovir (FAM), the diacetylester of 6-deoxypenciclovir, was developed as the oral prodrug, which has an oral bioavailability of 77%. Famciclovir is rapidly and extensively absorbed and efficiently converted to penciclovir in two steps: (1) removal of the two acetyl groups (the first one by esterases in the intestinal wall and the second one on the liver), and (2) oxidation at the six position catalyzed by aldehyde oxidase that accounts for the conversion of 6-deoxypenciclovir to penciclovir. Famciclovir is well tolerated in patients and is effective against HSV-1 and HSV-2 (for both therapy and long-term suppression of recurrent infections) and against VZV (for treatment of HZ). Brivudine (BVDU), (E)-5-(2-bromovinyl)-20-deoxyuridine, bromovinyldeoxyuridine Zostex®, Zonavir®, Zerpex® (Table2) is licensed for the therapy of HZ in several countries in Europe. This thymidine analogue is a highly selective antiviral agent active against HSV-1 and VZV [29]. Several congeners of brivudine have been synthesized, including BVaraU (sorivudine), the arabinofuranosyl counterpart of brivudine. The selective anti- HSV-1 and anti-VZV activity of brivudine is dependent on a specific phosphorylation of the compound by the HSV-1 and VZV TK to its monophosphate (BVDU-MP) and diphosphate (BVDU-DP) (Figure2). Following conversion to the triphosphate form (BVDU-TP) by a nucleoside diphosphate (NDP) kinase, BVDU-TP competes with the natural substrate dTTP (deoxythymidine triphosphate) for the viral DNA polymerase, inhibiting the incorporation of dTTP into the viral DNA or, as an alternate substrate is incorporated leading to the formation of a structurally and functionally disabled viral DNA [29]. Approximately 90% is absorbed following oral administration of brivudine and about 70% of the oral dose is rapidly transformed to bromovinyluracil (BVU) during the first passage through the liver [30]. Brivudine is effective in the treatment of HZ, both the short-term (formation of new lesions) and long-term (prevention of PHN) effects, and is as efficient and/or convenient as the other anti-VZV drugs acyclovir, valacyclovir and famciclovir. A recent retrospective study compared efficiencies of valacyclovir, famciclovir and brivudine in terms of pain relief in HZ patients [31]. All three drugs were effective in treating pain in acute HZ with no significant difference between mild and moderate HZ patients. A significant reduction in intensity of pain was observed in several cases on day 3 (brivudine group), on day 7 (famciclovir group), and at 2–3 weeks (valacyclovir group). Significant side effects were not observed in any of the groups. Based on the results of this study, brivudine could be considered as the first choice to treat severe HZ cases considering that is administered once daily and can control pain earlier. Molecules 2021, 26, 1132 8 of 34 Molecules 2021, 26, 1132 8 of 35

Figure 1. ActivationActivation and and mechanism mechanism of of action of ( A)) acyclovir (ACV) (ACV) and penciclovir (PCV) (PCV) and ( (B)) cidofovir (CDV) (CDV) and foscarnet (PFA). Valacyclovir (VACV), the oral prodrug of ACV, has improved absorption compared to ACV because of foscarnet (PFA). Valacyclovir (VACV), the oral prodrug of ACV, has improved absorption compared to ACV because of a a stereo‐selective transporter in the human intestine by dipeptide transporters followed by rapid and efficient hydrolysis stereo-selective transporter in the human intestine by dipeptide transporters followed by rapid and efﬁcient hydrolysis to to ACV by estereases found in the gut lumen, intestinal wall and liver. Famciclovir (FAM), the oral prodrug of PCV, followsACV by first estereases deacetylation found inat the 3 gut and lumen, 4 positions intestinal of the wall acyclic and side liver. chain Famciclovir and then (FAM), oxidation the at oral the prodrug 6 position of of PCV, the follows purine ringﬁrst deacetylationyielding the active at the metabolite, 3 and 4 positions PCV. The of the conversion acyclic side to the chain monophosphate and then oxidation (MP) atforms the 6of position ACV and of thePCV purine is carried ring outyielding by the the viral active thymidine metabolite, kinase PCV. (TK). The The conversion cellular enzyme to the guanosine monophosphate monophosphate (MP) forms kinase of ACV or guanylate and PCV kinase is carried (GMP) out performsby the viral further thymidine phosphorylation kinase (TK). to Thethe diphosphate cellular enzyme (DP) guanosine forms. Following monophosphate conversion kinase to their or guanylatetriphosphate kinase (TP) (GMP)forms byperforms the cellular further nucleoside phosphorylation 5′‐diphosphate to the diphosphate (NDP) kinase, (DP) the forms. active Following metabolites conversion inhibit to viral their DNA triphosphate polymerases (TP) formsbecause by theythe cellular act as competitive nucleoside 5 inhibitors0-diphosphate of the (NDP) natural kinase, substrate the active (i.e., deoxyguanosine metabolites inhibit triphosphate, viral DNA polymerases dGTP) and/or because as alternative they act substratesas competitive when inhibitors incorporated of the into natural the growing substrate DNA (i.e., deoxyguanosinechain. Incorporation triphosphate, of ACV‐TP dGTP) into the and/or growing as alternative DNA chain substrates results in chain termination due to the lack of an OH at the 5′ position. PCV‐TP is not an obligate chain terminator due to the when incorporated into the growing DNA chain. Incorporation of ACV-TP into the growing DNA chain results in chain presence of an OH at the 5′ position and its incorporation results in slowdown of DNA chain elongation. The acyclic termination due to the lack of an OH at the 50 position. PCV-TP is not an obligate chain terminator due to the presence nucleotide analogue0 cidofovir (CDV) does not require activation by a virus‐encoded enzyme for activation as the molecule alreadyof an OH carries at the a 5phosphonateposition and bond. its incorporation In contrast to the results O‐P inlinkage slowdown (phosphate), of DNA the chain CH2 elongation.‐P‐bond (phosphonate) The acyclic is nucleotide resistant toanalogue phosphodiesterase cidofovir (CDV) and doesphosphatase not require hydrolysis. activation Therefore, by a virus-encoded acyclic nucleoside enzyme phosphonates for activation as(ANPs), the molecule such as already CDV, whichcarries mimic a phosphonate the nucleoside bond. monophosphates, In contrast to the can O-P bypass linkage the (phosphate), initial enzymatic the CH2-P-bond phosphorylation (phosphonate) by viral kinases. is resistant Similar to tophosphodiesterase a nucleoside monophosphate, and phosphatase a nucleoside hydrolysis. phosphonate Therefore, acyclicis further nucleoside phosphorylated phosphonates by cellular (ANPs), nucleotide such as kinases. CDV, which The conversionmimic the nucleosideof CDV to its monophosphates, active metabolite, can i.e., bypass CDV‐diphosphate the initial enzymatic (CDVpp) phosphorylation is performed by cellular by viral kinases kinases. [UMP/CMP Similar to kinasea nucleoside 1 (UMP/CMPK monophosphate,‐1) and 5′‐ adiphosphate nucleoside phosphonate(NDP) kinase]. is CDV further‐DP, phosphorylated recognized by the by viral cellular DNA nucleotide polymerase, kinases. will then The blockconversion DNA ofsynthesis CDV to by its acting active as metabolite, competitive i.e., inhibitor CDV-diphosphate with respect (CDVpp) of the natural is performed substrate by cellular dCTP or kinases as alternative [UMP/CMP sub‐ strate leading to incorporation into the growing DNA. Chain termination occurs when two consecutive CDVpp’s are in‐ kinase 1 (UMP/CMPK-1) and 50-diphosphate (NDP) kinase]. CDV-DP, recognized by the viral DNA polymerase, will then corporated. CDVp‐choline is regarded as an intracellular reservoir of CDVp and CDVpp. Foscarnet (PFA, phosphonofor‐ block DNA synthesis by acting as competitive inhibitor with respect of the natural substrate dCTP or as alternative substrate mic acid) does not require any activation by viral or cellular kinases and directly interacts with the viral DNA polymerase. PFAleading binds to incorporation to the pyrophosphate into the growing exchange DNA. site of Chain the viral termination DNA polymerase, occurs when blocking two consecutive the release CDVpp’s of pyrophosphate are incorporated. from theCDVp-choline terminal nucleoside is regarded triphosphate as an intracellular and thus, reservoir impeding of CDVp the formation and CDVpp. of the Foscarnet 3′‐5′‐phosphodiester (PFA, phosphonoformic linkage essential acid) does for viralnot require DNA elongation. any activation by viral or cellular kinases and directly interacts with the viral DNA polymerase. PFA binds to the pyrophosphate exchange site of the viral DNA polymerase, blocking the release of pyrophosphate from the terminal nucleoside triphosphate and thus, impeding the formation of the 30-50-phosphodiester linkage essential for viral DNA elongation.

Molecules 2021, 26, 1132 9 of 34 Molecules 2021, 26, 1132 9 of 35

Figure 2. Activation, mechanism of action and catabolism of brivudine (BVDU). The VZV thymidine kinase (TK) as well as HSV HSV-1‐1 TK, display both thymidine kinase and thymidylate (dTMP) kinase activities, responsible for the activation of BVDU to the monophosphate (BVDU‐MP) and diphosphate (BVDU‐DP) forms, respectively. The conversion of BVDU‐ BVDU to the monophosphate (BVDU-MP) and diphosphate (BVDU-DP) forms, respectively. The conversion of BVDU- DP to the active triphosphate metabolite (BVDU‐TP) is carried out by the cellular nucleoside 5′‐diphosphate (NDP) kinase. DP to the active triphosphate metabolite (BVDU-TP) is carried out by the cellular nucleoside 50-diphosphate (NDP) BVDU‐TP is recognized by DNA polymerases as an alternative substrate and is incorporated into the DNA molecule via internucleotidekinase. BVDU-TP linkages. is recognized Pyrimidine by nucleoside DNA polymerases analogues, as such an alternative as BVDU and substrate BVaraU, and can is incorporatedbe degraded by into pyrimidine the DNA catabolicmolecule enzymes via internucleotide (such as uridine linkages. phosphorylase Pyrimidine or nucleoside thymidine analogues, phosphorylase such as (TPase) BVDU leading and BVaraU, to their can free be base degraded metabo by‐ litespyrimidine without catabolic antiviral enzymes activity. (such BVDU as uridinecannot be phosphorylase administered or together thymidine with phosphorylase 5‐flurouracil (TPase) or its prodrug leading capecitabine to their free base be‐ causemetabolites BVU (the without product antiviral formed activity. following BVDU BVDU cannot degradation be administered by the together thymidine with phsophorylase), 5-flurouracil or its is prodruga potent capecitabine inhibitor of dihydropyrimidinebecause BVU (the product dehydrogenase. formed following This enzyme BVDU is degradationneeded for the by first the thymidinestep in the phsophorylase),catabolic pathway is a of potent pyrimidines inhibitor and of fordihydropyrimidine 5‐fluorouracil degradation dehydrogenase. and hence This co enzyme‐administration is needed of for 5‐ thefluorouracil first step and in the brivudine catabolic leads pathway to increased of pyrimidines exposure and to 5for‐fluorouracil. 5-fluorouracil degradation and hence co-administration of 5-fluorouracil and brivudine leads to increased exposure to 5-fluorouracil. One important limitation for the use of brivudine is the absolute contraindication of the combinationOne important of brivudine limitation with for the 5‐fluorouracil use of brivudine or its is oral the absoluteprodrug contraindicationcapecitabine since of BVU,the combination the degradation of brivudine product with of brivudine, 5-fluorouracil is a potent or its oralinhibitor prodrug of dihydropyrimidine capecitabine since dehydrogenase,BVU, the degradation the enzyme product responsible of brivudine, for the is afirst potent step inhibitorin the catabolic of dihydropyrimidine pathway of py‐ rimidinesdehydrogenase, (Figure the 2). Dihydropyrimidine enzyme responsible dehydrogenase for the first step is needed in the for catabolic 5‐fluorouracil pathway deg of‐ radationpyrimidines and, (Figure thereby2). concomitant Dihydropyrimidine administration dehydrogenase of 5‐fluorouracil is needed together for 5-fluorouracil with brivu‐ dinedegradation results and,in increased thereby concomitantexposure to administration5‐fluorouracil of(since 5-fluorouracil BVU hampers together 5‐fluorouracil with brivu- degradationdine results to in the increased inactive exposure 5‐fluoro‐ to5,6 5-fluorouracil‐dihydrouracil (since product, BVU significantly hampers 5-fluorouracil increasing 5‐ fluorouracildegradation half to the‐life) inactive [32]. Clinicians 5-fluoro-5,6-dihydrouracil should thus be aware product, of the life significantly‐threatening increasing and pos‐ sible5-fluorouracil fatal drug half-life)‐drug interaction [32]. Clinicians of brivudine should thusand becapecitabine/5 aware of the‐fluorouracil life-threatening [33–35]. and Sorivudine,possible fatal like drug-drug brivudine, interaction is metabolized of brivudine to BVU and and capecitabine/5-fluorouracil therefore, its administration [33 with–35]. 5Sorivudine,‐fluorouracil like is contraindicated. brivudine, is metabolized Sorivudine, to BVUlicensed and in therefore, Japan in its 1993 administration for the treatment with of5-fluorouracil HZ, was withdrawn is contraindicated. following Sorivudine,several deaths licensed related in to Japan the co in‐administration 1993 for the treatment with 5‐ fluorouracilof HZ, was [36,37]. withdrawn following several deaths related to the co-administration with 5-fluorouracilThe helicase [36,37‐primase]. inhibitor (HPIs) amenamevir {ASP2151, N‐(2,6‐dime‐ thylphenyl)The helicase-primase‐N‐[2‐[4‐(1,2,4‐ inhibitoroxadiazol (HPIs)‐3‐yl)anilino] amenamevir‐2‐oxoethyl] {ASP2151,‐1,1‐Ndioxothiane-(2,6-dimethylphenyl)-‐4‐carbox‐ amide}N-[2-[4-(1,2,4-oxadiazol-3-yl)anilino]-2-oxoethyl]-1,1-dioxothiane-4-carboxamide} (Table 2) was approved for treatment of HZ in Japan in September 2017 while (Table the2) phasewas approved I trial with for this treatment drug was of HZ halted in Japan in United in September States due 2017 to while safety the concerns. phase I trialThe withher‐ pesvirusthis drug helicase–primase was halted in United complex States (encompassing due to safety the concerns. helicase, The the herpesvirus primase and helicase– a cofac‐ torprimase protein complex with 1:1:1 (encompassing stoichiometry) the helicase, possesses the multiple primase andenzymatic a cofactor activities protein including with 1:1:1

Molecules 2021, 26, 1132 10 of 34

stoichiometry) possesses multiple enzymatic activities including DNA helicase, single- stranded DNA (ssDNA)-dependent ATPase and primase, all essential for viral DNA replication and viral growth. Agents targeting the helicase–primase complex represent a breakthrough in the development of anti-herpesvirus agents. The helicase–primase complex is well conserved among members of the herpesvirus family and genes encoding the VZV helicase subunit (ORF55), primase subunit (ORF6) and cofactor subunit (ORF52) share homology with, respectively, the HSV-1 UL5, UL52, and UL8 genes and with the HCMV genes UL105, UL70 and UL102. Amenamevir is an oxadiazole phenyl derivative with potent activity against both VZV and HSV [38], while the two other classes of HPIs, i.e., the thiazole urea derivative Pritelivir (AIC316, BAY 57-1293) and the 2-aminothiazolylphenyl type, BILS 179 BS, have a limited antiviral spectrum inhibiting only HSV-1 and HSV-2. HPIs are virus-specific, have low in vitro toxicity, inhibit both reference and clinical viral strains, and are orally bioavailable and effective in different mouse models of HSV infection. They have a completely different mechanism of action (direct inhibition of the helicase-primase complex) compared to the classical anti-herpesvirus agents (target the DNA polymerase) and do not need activation by the viral TK. Thus, HPIs are active against viral mutants with a defective TK (TK−). Furthermore, combination of HPI with nucleoside analogues showed a synergistic effect in vitro, pointing at combination therapy as a potential approach for treating severe conditions, such as encephalitis or infections in patients with immunosuppression [39,40]. To confirm that the anti-VZV activity of amenamevir was due to inhibition of the VZV helicase-primase complex, an amenamevir VZV mutant was selected and characterized [41]. Sequencing analysis of ORF55 (helicase gene) and ORF6 (primase gene) of this mutant indicated three amino acid changes from the parent strain: N336K in the helicase motif IV, one of the six well-conserved sequence motifs in ORF55, R446H in ORF55 and N939D in ORF6. Notably, this mutant showed a marked defect in viral replication. Astellas Pharma originally developed amenamevir for treatment of genital herpes and HZ. The efficacy of amenamevir 2× daily was comparable to acyclovir twice a day (BID) for 3 days using the primary endpoint ‘time to lesion healing’ in a phase II dose-finding study in patients with genital herpes (NCT00486200) [42]. Time to healing was shorten by 1–2 days in both treatment groups compared to the placebo group. The results of the clinical trial (NCT00487682), sponsored by Astellas Pharma, to investigate the efficacy and safety of three different doses of ASP2151 compared to valacyclovir in subjects with HZ and to determine the recommended clinical dose, have not yet been reported. Following a phase 1, randomized, double blind, multiple dose, multicenter study (NCT00870441) to compare the safety of amenamevir to valacyclovir and placebo in healthy male and female subject, Astellas Pharma halted the clinical development of amenamevir due to treatment-emergent serious adverse events. Maruho Co., Ltd. (Kyoto, Japan) resumed the development of amenamevir and conducted a randomized, double-blind, valacyclovir-controlled phase 3 study to evaluate the efficacy and safety of amenamevir in Japanese HZ patients that receive the drug within 72 h following the start of the rash. The study included 751 patients who were randomly distributed to be treated with either amenamevir 400 mg or 200 mg per os, 1× per day or valacyclovir 1000 mg 3× per day (3000 mg total daily dose) for 7 days (NCT01959841) [43]. The cessation proportion of development of new lesions at day 4, i.e., day 4 cessation proportion, was considered the primary efficacy end-point and these proportions were 81.1% (197/243), 69.6% (172/247) and 75.1% (184/245), respectively, for amenamevir 400 and 200 mg and valacyclovir, with non-inferiority of amenamevir 400 mg to valacyclovir confirmed by a closed testing procedure. Secondary end-points (days to cessation of new lesions formation, complete crusting, healing, pain resolution and virus disappearance) were not significantly different among the three treatment groups. Amenamevir (400 and 200 mg) and valacyclovir 3000 mg were well tolerated and the proportions of patients experiencing drug-related adverse events were 10.0% (25/249), 10.7% (27/252) and 12.0% (30/249), respectively. This study showed that amenamevir Molecules 2021, 26, 1132 11 of 34

400 mg is effective and well tolerated for treatment of HZ in immunocompetent Japanese patients, leading to amenamevir approval for this indication in Japan. Cidofovir, the ﬁrst FDA-approved acyclic nucleoside phosphonate (ANP) for intravenous treatment of HCMV retinitis in AIDS patients in 1996, is mostly used off-label for the intravenous or topical treatment of severe infections caused by various DNA viruses, including various herpesviruses other than HCMV, polyoma-, adeno-, pox- (molluscum contagiosum virus and orf virus) and human papillomaviruses (HPV). In the case of VZV, the drug is used off label for therapy of acyclovir and/or foscarnet resistant infections. Cid- ofovir has a phosphonate group bypassing the ﬁrst phosphorylation step by viral kinases. Cellular kinases convert the drug to the active diphosphate form (CDVpp), which acts as a competitive inhibitor of the viral DNA polymerase, causing slow down elongation and premature chain termination during viral DNA synthesis (Figure1). CDVpp inhibits viral DNA polymerases more potently than cellular DNA polymerases. The metabolites of the ANPs show an unusually long intracellular half-life, accounting for a long-lasting antiviral activity. The formation of CDVp-choline adduct serves as an intracellular reservoir for the mono-(CDVp) and diphosphonate (CDVpp), explaining the long-lasting activity of the drug. CDV has two important drawbacks that restrict its use in clinic. Due to its low oral bioavailability (<5%), the drug needs to be administered intravenously, normally once a week because of its long-lasting activity. CDV is also known for its dose dependent nephrotoxicity, which can be reduced by pre-hydration with at least 1 L of 0.9% saline solution intravenously before each CDV infusion and concomitant oral administration of probenecid. The pyrophosphate analogue foscarnet (PFA, Foscavir®), a direct inhibitor of viral DNA polymerases, is independent of activation by the viral TK (Figure1 and Table2). Hence, PFA is the therapy of choice for acyclovir-resistant (ACV-R) VZV infections due to mutations in the viral TK gene,

5. Medical Need for New Antiviral Agents to Manage VZV-Associated Diseases 5.1. Management of PHN and other Complications Current antiviral drugs available for HZ treatment significantly decrease the incidence of new lesion formation, accelerate healing, and shorten the duration of viral shedding thereby reducing the incidence, severity and duration of pain, and limiting neuron damage [44]. The effect on the resolution of pain is extremely important in the antiviral therapy of HZ. Pain associated with HZ can be measured in three ways: (i) pain at presentation (acute pain), quantified over the first 30 days; (ii) PHN (post-herpetic neuralgia), defined as “pain that has not resolved after 30 days of disease onset" or as “pain that persists after healing or pain 90 days after rash onset” and (iii) zoster-associated pain (ZAP), pain recorded from the time of acute zoster until its complete resolution [44]. Acyclovir, valacyclovir and famciclovir are approved worldwide for the treatment of HZ in both immunocompetent and immunocompromised patients, brivudine is available in some European countries, and amenamevir is licensed only in Japan. Valacyclovir proved superior to acyclovir according to ZAP analysis from different clinical studies [44]. Famciclovir and acyclovir were therapeutically equivalent in terms of healing rate and pain resolution in immunocompetent patients aged >50 years. Brivudine proved similar efficacy on pain and rash as well as a similar tolerability compared to famciclovir in a large multicenter study that enrolled patients with acute HZ aged ≥50 years [44]. However, existing antiviral therapies are not completely effective in avoiding PHN, most likely because antivirals should be started within 72 h of rash appearance. The delay between onset of symptoms and start of treatment is likely the major cause of reduced efficacy of antiviral therapy. Clinical trials of antiviral drugs for HZ have enrolled patients within 72 h from rash onset; however, no well-controlled clinical trials comparing early- onset therapy with later therapy (>72 h) have been performed. Therefore, antiviral agents with a higher potency may achieve a more rapid decrease in viral replication consequently reducing neural damage and both acute and chronic symptoms of HZ. In addition, currently Molecules 2021, 26, 1132 12 of 34

available antivirals require 3–5 times daily dosing regimens that need to be modified for patients with renal impairment. The medications used to treat the pain associated with PHN are only palliative, are often insufficient in terms of relief for the patients and do not provide a cure for HZ. Hence, drugs with superior anti-VZV activity, with the ability to prevent PHN, able to provide better pain relief, and with a more simplified dosing regimen are indeed needed. These drugs would also be very useful for the management of disseminated VZV primary infections and complications of VZV reactivation, including VZV vasculopathy, meningoradiculitis, cerebellitis, myelopathy, ocular disease, and zoster sine herpete (ZSH, radicular pain in the absence of skin rash).

5.2. Managing Rare but Signiﬁcant Side Effects Linked to the VZV Life Vaccine The varicella vaccine in infants diminishes the consequences of chickenpox in terms of both healthcare and economic burden, while the zoster vaccine protects immunocompetent adults from HZ and reduces disease severity in those who develop HZ. Adverse events were not seen in clinical trials performed with the VZV vaccines. However, rare but important side effects are being increasingly described most likely due to the increased administration of VZV life vaccines worldwide. A few cases of disseminated varicella infections due to the VZV vaccine strain were described in immunocompromised patients, requiring treatment with antiviral agents [45,46]. VZV vaccine can occasionally reactivate in healthy children and cause HZ [47,48] and in adults [49]. A disseminated VZV infection with CNS involvement directly following vaccine administration has been reported in a previously healthy elderly woman [50]. Herpes zoster and meningitis due to reactivation of the VZV vaccine virus has been described in an immunocompetent child [51]. Also, a case of vaccine-associated HZ ophtalmicus and encephalitis in an immunocompetent child requiring acyclovir therapy has been described [52]. Clinicians should also be aware that very rarely, the varicella vaccine can be transmitted and cause invasive disease as shown by a case report showing that the VZV vaccine was transmitted within a family from a child with shingles resulting in varicella meningitis in an immunocompetent adult [53]. Therefore, antiviral therapy will be needed for treatment of rare VZV diseases even after widespread implementation of vaccination programs.

5.3. Emergence of Drug-Resistant Viruses Treatment of VZV infections with acyclovir or valacyclovir in the immunocompetent hosts is not associated with emergence of. In the immunocompromised hosts, VZV infection tends to be severe and persistent, requiring prolonged therapy with antivirals and ACV-R mutants have been isolated after long-term treatment with acyclovir [54–58]. Persistent VZV and VZV-related complications occur more frequently among hematological patients and antiviral resistance was found in 27% of patients with persistent VZV, including patients that progressed to severe retinal or cerebral infection [59]. Besides, compartmentalization of ACV-R VZV has been reported with important implications for sampling in molecular diagnostics [60]. Notably, ACV-R VZV keratitis was reported in an immunocompetent patient [61], highlighting the need to determine the antiviral-resistance patterns of corneal VZV isolates from chronic keratitis even in immunocompetent patients, as the eye can be considered an immune privileged site. Resistance to acyclovir in VZV appears because of mutations in either the TK or the DNA polymerase genes. The most frequent mutants isolated both in cell culture and in the clinic are TK mutants [62,63]. Mutations associated with resistance to nucleoside analogues are distributed throughout the entire VZV TK gene, similarly to HSV-1 and HSV-2. How- ever, conserved regions, such as the ATP- and the nucleoside-binding sites, and the amino acid position 231 are considered as hot spots for drug-resistance mutations [56,57,64–66]. Several case reports documented the use of foscarnet as salvage therapy for ACV-R VZV infections in immunocompromised patients [67–69]. However, mutations in the VZV DNA Molecules 2021, 26, 1132 13 of 34

polymerase gene associated with PFA-R have also been described in immunocompromised patients [70–72]. The amino acid changes in the VZV DNA polymerase linked to PFA-R show generally cross-resistance to acyclovir. Remarkably, acyclovir and penciclovir were shown to select in vitro for different types of drug-resistant VZV genotypes: TK mutants under acyclovir pressure and DNA polymerase mutants under penciclovir selection [63,73]. This is different from HSV ﬁndings, where both drugs selected in vitro for TK mutants. Penciclovir remains active against some HSV-1 and VZV TK and DNA polymerase mutants that show ACV-R [74–76], indicating that the interactions between human α-herpesviruses TK and penciclovir or acyclovir, and also between the viral DNA polymerases and PCV- TP or ACV-TP are dissimilar, which may explains the differences between ACV-R and PCV-R VZV strains. Additionally, the frequency of VZV mutants was signiﬁcantly higher following acyclovir pressure than penciclovir exposure [77]. As cidofovir (CDV, HPMPC) (Table2) is a nucleotide analogue that is converted to the active form CDVpp, by cellular kinases, it is a therapeutic option for therapy of ACV-R, PCV-R and/or PFA-R resistant VZV infections [21,78,79]. Emergence of ACV-R in the course of a chronic infection caused by the Oka vaccine VZV strain was reported in an immunosuppressed child vaccinated prior a tumor diagnosis requiring intensive antitumor therapy [80]. Clinical ACV-R in this patient was linked to a TK mutation that responded to foscarnet therapy. ACV-R in the Oka vaccine strain was also described in a child with neuroblastoma [69], suggesting that the Oka vaccine strain can be linked to severe disease in the immunocompromised host following reactivation from latency, being necessary prolonged acyclovir therapy with the subsequent risk of emergence of drug-resistance. Hence, novel potent anti-herpesvirus agents with a target other than the viral DNA polymerase would be very useful to manage drug-resistance to the currently available antiviral agents.

6. Novel Anti-VZV Agents in Advanced Development The gold standard for VZV therapy remains acyclovir and its prodrug valacyclovir. Other nucleoside analogues such as penciclovir, its prodrug famciclovir and brivudine can also be used. These antiviral agents rely on the viral TK for their first phosphorylation and have as target the viral DNA polymerase. VZV mutants arising under pressure with these nucleoside analogues bearing mutations in the viral TK can be treated with foscarnet, a direct inhibitor of viral DNA polymerases. However, foscarnet can be associated with significant renal toxicity and cannot be used for VZV DNA polymerase mutants emerging under acyclovir as most of them show cross-resistance to foscarnet. As cidofovir is a nucleotide analogue that bypass the activation by the viral TK and usually DNA polymerase mutants that are resistant to acyclovir and/or foscarnet remain sensitive to cidofovir, this drug can be used for VZV infections refractory to acyclovir, penciclovir and/or foscarnet. Unfortunately, cidofovir can also cause renal toxicity. Amenamevir, an helicase-primase inhibitor, has only been approved in Japan and its development was discontinued in United States due to toxicity concerns. The drawbacks of the currently available treatments for VZV-associated diseases highpoint the necessity for new, safe and highly effective anti-VZV agents. Drugs able to inhibit virus growth by targeting different steps of the VZV replicative cycle will be very useful for the management of drug-resistance infections in the clinic as well as for limiting the probability of emergence of antiviral drug-resistance and could also form the base for combination therapy. Research should focus on the discovery and development of new anti-herpesvirus compounds having more potent activity than the currently available VZV antivirals. Be- sides, the search of new lead compounds able to block viral enzymes other than the viral DNA polymerase should be favored. In the last decade, only a few anti-VZV drugs were compared to valacyclovir in clinical trials to evaluate their efficacy in diminishing HZ associated pain and severity. Molecules 2021, 26, 1132 14 of 34

Molecules 2021, 26, 1132 14 of 35

6.1. Bicyclic Nucleoside Analogues (BCNAs) 6.1. InBicyclic 1999, Nucleoside the potent Analogues and selective (BCNAs) anti-VZV activity of some unusual bicyclic nucleoside analoguesIn 1999, (BCNAs) the potent was and reported selective by anti Mc‐ GuiganVZV activity and collaborators of some unusual [81]. bicyclic For BCNAs nucleo with‐ simpleside analogues alkyl side-chain (BCNAs) on was the reported bicyclic by base Mc ring, Guigan the optimaland collaborators carbon chain [81]. lengthFor BCNAs for anti- VZVwith activity simple alkyl ranked side between‐chain on 8the and bicyclic 10, with base the ring, 6-octyl-substituted the optimal carbon derivative chain length Cf-1368 for beinganti‐VZV the most activity active ranked and selectivebetween 8 compound and 10, with of thisthe series6‐octyl of‐substituted BCNAs. derivative Cf‐ 1368Although being the most BCNAs active are and structurally selective compound related to of BVDU, this series they of differ BCNAs. in their spectrum of antiviralAlthough activity BCNAs as theyare structurally exclusively related inhibit to VZV, BVDU, in contrastthey differ to in BVDU their spectrum that possesses of potentantiviral anti- activity HSV-1 as and they anti-VZV exclusively activity. inhibit AmongVZV, in BCNAscontrast withto BVDU an aromatic that possesses ring system po‐ (phenyl)tent anti in‐ HSV the side-chain,‐1 and anti the‐VZV n-pentylphenyl- activity. Among and BCNAs n-hexylphenyl-derivatives with an aromatic ring (Cf-1742 system and Cf-1743,(phenyl) respectively) in the side‐chain, (Figure the3) emergedn‐pentylphenyl as the‐ mostand potentn‐hexylphenyl compounds‐derivatives with 50% (Cf effective‐1742 and Cf‐1743, respectively) (Figure 3) emerged as the most potent compounds with 50% concentration (EC50) values as low as 0.0001–0.0005 µM against reference VZV strains effective concentration (EC50) values as low as 0.0001–0.0005 μM against reference VZV as well as clinical isolates in vitro [82,83]. A strong correlation between the length of the strains as well as clinical isolates in vitro [82,83]. A strong correlation between the length n-alkyl and n-alkylaryl moiety of the BCNAs and antiviral activity was observed [84]. Cf- of the n‐alkyl and n‐alkylaryl moiety of the BCNAs and antiviral activity was observed 1743 was also able to reduce the replication and spread of VZV in organotypic epithelial raft [84]. Cf‐1743 was also able to reduce the replication and spread of VZV in organotypic cultures as measured by morphological changes induced by the virus and quantiﬁcation epithelial raft cultures as measured by morphological changes induced by the virus and of the viral DNA load [85]. Similar to acyclovir and brivudine, the BCNAs were inactive quantification of the viral DNA load [85]. Similar to acyclovir and brivudine, the BCNAs − againstwere inactive TK-deﬁcient against (TK TK‐)deficient strains, pointing(TK−) strains, to a crucial pointing role to of a thecrucial VZV-encoded role of the TKVZV in‐ the activationencoded TK (phosphorylation) in the activation of(phosphorylation) the BCNAs. VZV of mutantthe BCNAs. strains VZV selected mutantin strains vitro underse‐ pressurelected in of vitro BCNAs under showed pressure mutations of BCNAs in showed the viral mutations TK gene in [63 the]. viral TK gene [63].

FigureFigure 3. 3.Anti-VZV Anti‐VZV drugs in in advanced advanced development. development. Chemical Chemical structures structures of CF‐1743 of CF-1743 and its prodrug and its valnivudine prodrug valnivudine hydro‐ chloride (FV‐100) and of omaciclovir (H2G) and its prodrug valomaciclovir stearate. hydrochloride (FV-100) and of omaciclovir (H2G) and its prodrug valomaciclovir stearate. BCNAs also strikingly differ from brivudine in their mechanism of activation (Figure BCNAs also strikingly differ from brivudine in their mechanism of activation 4) [86,87]. BCNAs are recognized by VZV TK as a substrate, but not by HSV‐1 TK, nor by (Figure4) [86,87]. BCNAs are recognized by VZV TK as a substrate, but not by HSV-1 TK, cytosolic TK‐1 or mitochondrial TK‐2 in kinetic studies with purified enzymes [84]. VZV nor by cytosolic TK-1 or mitochondrial TK-2 in kinetic studies with puriﬁed enzymes [84]. TK was demonstrated to phosphorylate the BCNAs not only to their corresponding 5′‐ VZV TK was demonstrated to phosphorylate the BCNAs not only to their corresponding mono‐ but also to their 5′‐diphosphate derivatives due to the intrinsic dTMP kinase activ‐ 50-mono- but also to their 50-diphosphate derivatives due to the intrinsic dTMP kinase ac- ity of the VZV TK. Thus, BCNAs are selectively phosphorylated to their 5′ diphosphates tivity of the VZV TK. Thus, BCNAs are selectively phosphorylated to their 50 diphosphates by the two successive enzyme activities of VZV TK (thymidine kinase and dTMP kinase). by the two successive enzyme activities of VZV TK (thymidine kinase and dTMP kinase). However, no clear-cut correlation between the antiviral potency of the compounds and

MoleculesMolecules2021 2021, 26,, 26 1132, 1132 15 of15 35 of 34

theirHowever, afﬁnity no for clear VZV‐cut TK correlation could be demonstrated,between the antiviral pointing potency to a different of the compounds structure/activity and their affinity for VZV TK could be demonstrated, pointing to a different structure/activity relationship of the eventual antiviral target of these compounds. In contrast to BVDU, relationship of the eventual antiviral target of these compounds. In contrast to BVDU, human NDP kinase was unable to convert BCNAs to their triphosphate derivatives (BCNA- human NDP kinase was unable to convert BCNAs to their triphosphate derivatives TP) [84], in agreement with studies reporting no traces of BCNA-TP in VZV infected cells. (BCNA‐TP) [84], in agreement with studies reporting no traces of BCNA‐TP in VZV in‐ These data clearly indicate that the mechanism of action of BCNAs differs from that of fected cells. These data clearly indicate that the mechanism of action of BCNAs differs BVDU and suggests that BCNAs exert their antiviral effects via their monophosphate or from that of BVDU and suggests that BCNAs exert their antiviral effects via their mono‐ diphosphate derivatives, virtually excluding the DNA polymerase as the molecular target phosphate or diphosphate derivatives, virtually excluding the DNA polymerase as the ofmolecular the BCNAs. target The of exact the BCNAs. molecular The target exact of molecular the BCNAs target could of the not BCNAs be identiﬁed could yetnot andbe is currentlyidentified under yet and investigation. is currently under investigation.

FigureFigure 4. Activation,4. Activation, mechanism mechanism of of action action and and catabolism catabolism of bicyclic nucleoside nucleoside analogues analogues (BCNAs). (BCNAs). VZV VZV TK TK converts converts BCNA’s to the mono‐ and diphosphate forms although whether there is conversion to the triphosphate form and which BCNA’s to the mono- and diphosphate forms although whether there is conversion to the triphosphate form and which is is the active metabolite and mechanism of action remain unclear to date. Striking differences between BCNAs and BVDU theexist active regarding metabolite their and catabolic mechanism pathways. of action In contrast remain to unclear BVDU, to human date. Striking TPases do differences not recognize between BCNA’s BCNAs as substrates and BVDU and exist regardingthe free their bases catabolic of BCNAs pathways. do not inhibit In contrast human to DPD BVDU, (dihydropyrimidine human TPases do dehydrogenases) not recognize BCNA’s and thereof, as substrates there is a and normal the free basesmetabolism of BCNAs of do Capecitabine/5 not inhibit human‐fluorouracyl. DPD (dihydropyrimidine Dashed grey arrows dehydrogenases) indicate lack of activation. and thereof, there is a normal metabolism of Capecitabine/5-fluorouracyl. Dashed grey arrows indicate lack of activation. BCNAs have also different catabolic pathways than BVDU and BVaraU (Figure 4), providingBCNAs BCNAs have alsowith differentsignificant catabolic advantages. pathways Pyrimidine than nucleoside BVDU and analogues BVaraU are (Figure sus‐4), providingceptible to BCNAs pyrimidine with catabolic significant enzymes advantages. (such Pyrimidineas uridine phosphorylase nucleoside analogues or thymidine are sus- ceptiblephosphorylase to pyrimidine and are catabolichydrolyzed enzymes to their free (such base as uridinemetabolites phosphorylase that lack antiviral or thymidine activ‐ phosphorylaseity. In contrast, and BCNAs are hydrolyzed were not recognized to their free as substrates base metabolites by human that thymidine lack antiviral phosphor activity.‐ Inylase contrast, and thereby BCNAs they were were not not recognized converted to as their substrates inactive byfree human base derivatives thymidine [88]. phospho- Fur‐ rylasethermore, and the thereby free bases they of were BCNAs not were converted not inhibitory to their to inactive human free dihydropyrimidine base derivatives de [‐88]. Furthermore,hydrogenase, the the free catabolic bases enzyme of BCNAs involved were not in the inhibitory degradation to human of pyrimidines dihydropyrimidine analogues. dehydrogenase, the catabolic enzyme involved in the degradation of pyrimidines analogues. As mentioned above, the BVU free base is inhibitory to dihydropyrimidine dehydrogenase, an enzyme necessary for the catabolic inactivation of the chemotherapeutic pyrimidine analogue 5-fluorouracil.

Molecules 2021, 26, 1132 16 of 34

The safety of the oral prodrug of Cf-1743, i.e., FV-100 (valnivudine hydrochloride), was evaluated in three randomized, double-blind, placebo-controlled clinical trials: (i) a single-ascending-dose study in 32 healthy subjects aged 18 to 55 years (100-, 200-, 400-, and 800-mg doses); (ii) a multiple-ascending-dose study including 48 subjects (18 to 55 years- old) that received 100 mg once daily (QD), 200 mg QD, 400 mg QD, 400 mg twice a day, and 800 mg QD for 7 days); (iii) a two-part study in subjects aged 65 years and older with a single 400-mg dose in 15 subjects and a 400-mg QD dosing regimen for 7 days in 12 subjects [89]. FV-100 was shown to be rapidly and extensively converted to CF-1743; CF-1743 renal excretion was very low; high-fat but not a low-fat meal reduced exposure to CF-1743; and the FV-100 pharmacokinetic profile was similar in elderly and younger subjects. All doses maintained mean drug plasma levels of the active form of FV-100 that exceeded the EC50 for approximately 24 h, supporting the potential for once-a-day dosing in phase II trials. FV-100 was well tolerated at all doses and no serious adverse events were reported. The safety of FV-100 and its efficacy in reducing pain associated with acute HZ compared to valacyclovir was evaluated in a prospective, multicenter, parallel-group, double-blind, randomized study [90]. Patients were ≥50 years old, had a diagnosis of HZ within 72 h of the formation of lesions and presented HZ-associated pain. They were randomized to receive a 7-day course of either FV-100 200 mg 1× per day (n = 117), FV-100 400 mg 1× per day (n = 116), or valacyclovir 1000 mg 3× per day (n = 117). Burden of illness (BOI), incidence and duration of clinically significant pain (CSP), pain scores, incidence and severity of PHN, and times to full lesion crusting and to lesion healing were used to evaluate efficacy. Safety was evaluated based on adverse event (AE)/ severe adverse events (SAE) profiles, changes in laboratory and vital signs values, and results of electrocardiograms. The BOI scores for pain through 30 days were 114.5 (200 mg FV-100), 110.3 (400 mg FV-100), and 118.0 (valacyclovir). The incidences of PHN at 90 days were 17.8%, 12.4%, and 20.2%, respectively, for FV-100 200 mg, FV-100 400 mg, and VACV. Adverse event and SAE profiles were similar in the two FV-100 and the valacyclovir groups. Although this trail missed the primary end-point (no statistically significant difference among the treatment groups for BOI at 30 days), a difference between FV-100 400 mg and valacyclovir groups was found in the 90 days data (14% reduction). These results point to a potential effect of FV-100 on subacute and chronic pain and demonstrate the potential utility of FV-100 as an antiviral for the treatment of shingles, diminishing both pain burden of the acute episode and incidence and severity of PHN. Reduced incidence of PHN, the most clinically meaningful endpoint, and reduced use of additional pain medication (opioids) for the management of PHN in the FV-100 arm vs. valacyclovir arm was seen. Compared to the standard of care valacyclovir, FV-100 offers secondary benefits over valacyclovir because FV-100 is once daily dosing vs. valacyclovir three-times dosing.

6.2. Carbocyclic Nucleoside Analogues: H2G (Omaciclovir) and Its Prodrug (Valomaciclovir) H2G, (R)-9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine, omaciclovir (Figure3), a carbocyclic nucleoside analogue, has potent activity against different herpesviruses, being especially active against VZV, HSV and EBV but lacking activity against HCMV [91]. The average EC50 for omacyclovir was 2.3 ± 0.8 µM, compared to 46.8 µM for acyclovir and 78.7 µM for penciclovir against 20 VZV clinical isolates, pointing to a superior activity of omaciclovir against this virus [92]. Omaciclovir has a mode of action similar to that of acyclovir, but with less selectivity as a substrate for TK and resistance to omaciclovir maps to the TK [93]. However, omaciclovir, in contrast to acyclovir, is not an obligate chain terminator, although incorporation of the triphosphate form (omaciclovir-TP) results in limited chain elongation. Omaciclovir-TP has a longer intracellular half-life than ACV-TP (providing dosing advantages over acyclovir) and is a potent inhibitor of VZV DNA polymerase though less active than ACV-TP. The anti- herpesvirus activity of omaciclovir can be markedly enhanced by the immunosuppressive agent mycophenolate mofetil [94]. Molecules 2021, 26, 1132 17 of 34

Since omaciclovir oral bioavailability is of 17% in cynomolgus monkeys, similar to that of acyclovir, the diester prodrug valomaciclovir stearate, EPB-348 [L-valine, (3R)-3[2- amino-1,6-dihydro-6-oxo-purin-9-yl]methyl]-4[(1-oxooctadecyl)oxo]butylester] (Figure3) was synthesized with an oral bioavailability of >70% in rats and monkeys, and >60% in humans [95]. Omaciclovir was licensed from the Swedish biotech company Medivir AB to Epiphany Biosciences for further development under the name EPB-348. A phase IIb trial enrolled 373 immunocompetent patients with acute HZ, randomized into three arms: 1 g 1× daily valomaciclovir stearate, 2 g once daily valomaciclovir stearate, and 1 g 3× per day valacyclovir [96]. Eighteen patients also received 3 g of valomaciclovir stearate once daily. The primary endpoint was non-inferiority in terms of time to complete crusting of the shingles rash for the 1× daily valomaciclovir stearate cohort compared to valacyclovir cohort. Once daily valomaciclovir stearate 2 g met the primary endpoint, being more convenient than 3× daily valacyclovir for the treatment of HZ and also equally safe. Valomaciclovir stearate proved non-inferior to VACV in the secondary endpoints (time to complete pain resolution, time to rash resolution and time to cessation of new lesion formation). Moreover, the highest valomaciclovir stearate dose (3 g 1× daily) demonstrated superiority to valacyclovir for the primary endpoint with no significant adverse event differences between valomaciclovir stearate and valacyclovir groups, being nausea the most common adverse event in all patient groups. Valomaciclovir stearate proved also effective against acute infectious mononucleosis due to primary EBV infection, for which there is no FDA-approved treatment. The findings of a randomized, placebo-controlled, double-blind trial of valomaciclovir stearate for infectious mononucleosis were reported in 2009 at a conference but data have not yet been published [97]. Omaciclovir produced a significant decrease in median EBV load in the oral compartment compared to placebo but no differences were found in clearance of EBV DNAemia, CD8:CD4 ratios, CD8 lymphocytosis, or CD8 responses to lytic and latent EBV tetramers in the valomaciclovir stearate vs. placebo group. On Epiphany Biosciences’ website, omaciclovir stearate is still listed as active but without information on further development.

6.3. Brincidofovir Brincidofovir (CMX001) (Table2) is an orally bioavailable lipid acyclic nucleoside phosphonate (ANP) with the same broad-spectrum activity against DNA viruses as its parent compound cidofovir. Because of the limitations associated with the use of cidofovir, the hexadecyloxypropyl (HDP) ester of cidofovir (CMX001, brincidofovir) (Table2) was synthesized. In alkoxyalkyl esters prodrugs, a natural fatty acid (lysophosphatidylcholine) molecule is used as carrier to facilitate drug absorption in the gastrointestinal tract [98,99]. Brincidofovir has improved uptake and absorption, an oral bioavailability in mice of 88–97% (compared to less than 5% for cidofovir), increased cell penetration (10–20 fold) and higher intracellular levels (100-fold) of CDV-DP than those reached with cidofovir, resulting in superior antiviral activity. Furthermore, brincidofovir is not nephrotoxic because, in contrast to cidofovir, it is not a substrate of the human organic anion transporter 1 enzyme present in the proximal renal tubular cells. Despite promising preclinical data with brincidofovir, the results of a phase III trial evaluating its efficacy in the prevention of HCMV disease in seropositive allogeneic hematopoietic stem cell transplant patients were disappointing (SUPPRESS trial, NCT01769170) [100]. The end-point of the study was prevention of clinical significant HCMV infections 24 weeks’ post-transplantation. Unfortunately, a higher viral infection rate was found in the brincidofovir arm than in the placebo group (22% vs. 11%) as well as a higher proportion of patients developing graft-versus-host-disease (GvHD) and digestive symptoms. It is unclear if the emergence of digestive symptoms was linked to drug toxicity or if the drug favored the onset of digestive GvHD, for which the patients had to start immunosuppression explaining the increase in clinically significant HCMV infections. Based on the results of this study, Chimerix suspended further trials Molecules 2021, 26, 1132 18 of 34

with oral brincidofovir for HCMV. In September 2019, SymBio Pharmaceuticals Limited (SymBio, Tokyo, Japan) announced an exclusive global license agreement with Chimerix Inc. for brincidofovir. Chimerix granted SymBio exclusive worldwide rights to develop, manufacture, and commercialize BCV in all human indications, except for the prevention and treatment of smallpox. Regarding the clinical data of brincidofovir against VZV, a retrospective study including 30 HSCT recipients, provided limited-evidence on the potential efﬁcacy of brincidofovir for prophylaxis of HSV and VZV in this group of patients [101]. The successful use of brincidofovir in management of disseminated acyclovir- and cidofovir-resistant VZV in a hematopoietic stem cell transplant patient with chronic GvHD who was intolerant to foscarnet has also been reported [102].

7. Candidate anti-VZV Drugs 7.1. Nucleoside/Nucleotide Analogues 7.1.1. Phenoxazine Derivatives Phenoxazine [1,3-diaza-2-oxophenoxazine] binds strongly to guanine in a duplex and improves TT-TT stacking interactions with adjacent bases. The phenoxazine scaffold is commonly employed to stabilize nucleic acid duplexes and fluorescent probes incorporating a phenoxazine component have been used for the study of nucleic acid structure, recognition, and metabolism. Phenoxazines are well-known in the field of medicinal chemistry because they have various biological activities, i.e., anticancer, antiviral, antidiabetic, antioxidant, anti-Alzheimer, anti-inflammatory, and antibiotic properties [103]. The antiviral activity against a panel of diverse viruses of newly synthesized phenoxazine- 0 based nucleoside derivatives has been recently reported [104]. 3-(2 -Deoxy-β-D-ribofuranosyl)- 1,3-diaza-2-ox-ophenoxazine (compound 7a) (Table3) proved to be a potent inhibitor of VZV replication with superior activity against wild-type than TK− strains (EC50 = 0.06 µM and 10 µM, respectively) [104]. Interestingly, compound 7a was not cytotoxic or cytostatic for HEL cells at 100 µM (the maximum concentration tested), resulting in selectivity indices (ratio CC50/EC50) of 1667 (reference Oka strain) and 10 (TK− 07-1). The mechanism of action of phenoxazines derivatives against VZV is unknown but previously described MoleculesMolecules 20212021,, 2626,, 11321132 phenoxazine derivatives with antiviral activity against HCMV, HSV-1 and HSV-2,1919 were ofof 3535

shown to directly inactive herpesviruses [105].

Table 3. Candidate anti-VZV drugs. Compounds described in the last years showing promising activity against VZV. TableTable 3.3. CandidateCandidate antianti‐‐VZVVZV drugsdrugs.. CompoundsCompounds describeddescribed inin thethe lastlast yearsyears showingshowing promisingpromising activityactivity againstagainst VZV.VZV.

EC50 EC50 EC50 EC50 Activity Against Other EC50 EC50 ActivityActivity against Against Other Human Other CompoundCompound TK+ TK− CC50 CC50 Compound CC50 Herpesviruses VZVTK+TK+ VZVVZVVZV TKTK−− VZVVZV HumanHuman HerpesvirusesHerpesviruses

Nucleoside/nucleotideNucleoside/nucleotideNucleoside/nucleotide analoguesanalogues

PhenoxazinePhenoxazine derivativesderivatives 0.060.06 μ μMM 1010 μ μMM >100>100 μ μMM NoNo activityactivity againstagainst HCMVHCMV

0.06 µM 10 µM >100 µM No activity against HCMV CompoundCompound 7a7a

22′′‐‐DeoxyriboseDeoxyribose emimycineemimycine nucleosidesnucleosides 0.990.99 μ μMM 6.916.91 μ μMM >412>412 μ μMM ActivityActivity againstagainst HSVHSV‐‐1,1, butbut Activitylowerlower against activityactivity HSV-1, againstagainst but lower TKTK− − activity against TK− HSV-1 and CompoundCompound 27a27a 0.99 µM 6.91 µM >412 µM HSVHSV‐‐11 andand againstagainst HSVHSV‐‐2.2. NoNo against HSV-2. No anti-HCMV activityantianti‐‐HCMVHCMV activityactivity

C5C5‐‐SubstitutedSubstituted‐‐(1,3(1,3‐‐diyne)diyne)‐‐22‐‐deoxyuridinesdeoxyuridines ~1~1 μ μMM >20>20 μ μMM 5555 μ μMM DecreasedDecreased activityactivity againstagainst HSVHSV‐‐11 TKTK−− andand HSVHSV‐‐2.2. ForFor compoundcompound 2626,, RR == pp‐‐ NoNo antianti‐‐HCMVHCMV activityactivity

(OCF3)C6H4– (OCF3)C6H4–

CarbocyclicCarbocyclic nucleosides:nucleosides: 0.110.11 μ μMM NoNo datadata >300>300 μ μMM ActivityActivity againstagainst HCMV,HCMV, nono 33‐‐SubstitutedSubstituted 33‐‐deazadeaza‐‐55′′‐‐noraristeromyinnoraristeromyin derivativesderivatives availableavailable datadata onon otherother herpesvirusesherpesviruses

InIn compoundcompound 44,, XX == BrBr

XanthineXanthine‐‐basedbased acyclicacyclic nucleosidenucleoside phosphonatesphosphonates 2.622.62 μ μMM 4.584.58 μ μMM 111111 μ μMM ActivityActivity againstagainst HSVHSV‐‐1,1, HSVHSV‐‐ PMEXPMEX 22 andand HCMVHCMV

CyclopentylCyclopentyl nucleosidenucleoside phosphonatesphosphonates 5.355.35 μ μMM 8.838.83 μ μMM >289>289 μ μMM ActivityActivity againstagainst HSVHSV‐‐1,1, HSVHSV‐‐ 22 andand HCMVHCMV CompoundCompound 2323

Nuceoside/nucleotideNuceoside/nucleotide prodrugsprodrugs

Molecules 2021, 26, 1132 19 of 35 Molecules 2021, 26, 1132 19 of 35 Molecules Molecules 2021 2021, 26, 26, 1132, 1132 1919 of of 35 35

Table 3. Candidate anti‐VZV drugs. Compounds described in the last years showing promising activity against VZV. Table 3. Candidate anti‐VZV drugs. Compounds described in the last years showing promising activity against VZV. TableTable 3. 3. Candidate Candidate anti anti‐VZV‐VZV drugs drugs. .Compounds Compounds described described in in the the last last years years showing showing promising promising activity activity against against VZV. VZV. EC50 EC50 Activity Against Other Compound EC50 EC50 CC50 Activity Against Other ECEC5050 ECEC5050 ActivityActivity Against Against Other Other Compound TK+ VZV TK− VZV CC50 Human Herpesviruses CompoundCompound TK+ VZV TK− VZV CCCC5050 Human Herpesviruses TK+TK+ VZV VZV TKTK−− VZV VZV HumanHuman Herpesviruses Herpesviruses Nucleoside/nucleotide analogues Nucleoside/nucleotide analogues Nucleoside/nucleotideNucleoside/nucleotide analogues analogues Phenoxazine derivatives 0.06 μM 10 μM >100 μM No activity against HCMV Phenoxazine derivatives 0.06 μM 10 μM >100 μM No activity against HCMV PhenoxazinePhenoxazine derivatives derivatives 0.060.06 μ μMM 1010 μ μMM >100>100 μ μMM NoNo activity activity against against HCMV HCMV

Compound 7a Compound 7a CompoundCompound 7a 7a Molecules 2021, 26, 1132 19 of 34

2′‐Deoxyribose emimycine nucleosides 0.99 μM 6.91 μM >412 μM Activity against HSV‐1, but 2′‐Deoxyribose emimycine nucleosides 0.990.99 μ μMM 6.916.91 μ μMM >412>412 μ μMM ActivityActivity againstagainst HSVHSV‐1,‐1, butbut 22′‐′Deoxyribose‐Deoxyribose emimycine emimycine nucleosides nucleosides 0.99 μM 6.91 μM >412 μM lowerActivity activity against againstHSV‐1, TKbut− Table 3. Cont. lower activity against TK− Compound 27a HSVlowerlower‐1 andactivityactivity against againstagainst HSV‐ 2. TK NoTK− − Compound 27a EC EC HSV‐1 and against HSV‐2. No CompoundCompound 27a 27a 50 50 ActivityHSVHSV‐1‐ against1 and and against against Other HSV HumanHSV‐2.‐2. No No Compound TK+ TK− CC anti‐HCMV activity 50 anti‐HCMVHerpesviruses activity VZV VZV antianti‐HCMV‐HCMV activity activity C5‐Substituted‐(1,3‐diyne)‐2‐deoxyuridines ~1 μM >20 μM 55 μM Decreased activity against C5C5‐Substituted‐Substituted‐(1,3‐(1,3‐diyne)‐diyne)‐2‐2‐deoxyuridines‐deoxyuridines ~1~1 μ μMM >20>20 μ μMM 5555 μ μMM DecreasedDecreased activityactivity againstagainst C5‐Substituted‐(1,3‐diyne) ‐2‐deoxyuridines ~1 μM >20 μM 55 μM HSVDecreased‐1 TK− andactivity HSV ‐2.against

HSVHSV‐1‐1 TK TK− and and HSV HSV‐2.‐2. For compound 26, R = p‐ DecreasedNoHSV anti‐1 activityTK‐HCMV−− and against activityHSV‐2. HSV-1 For compound 26, R = p‐ No anti‐HCMV activity ForFor compound compound 26 26, ,R R = = p p‐ ‐ ~1 µM >20 µM 55 µM TK−NoNoand anti anti HSV-2.No‐HCMV‐HCMV activity anti-HCMV activity (OCF3)C6H4– (OCF3)C6H4– activity (OCF(OCF3)C3)C6H6H4–4–

Carbocyclic nucleosides: 0.11 μM No data >300 μM Activity against HCMV, no CarbocyclicCarbocyclic nucleosides: nucleosides: 0.110.11 μ μMM NoNo datadata >300 >300 μ μMM ActivityActivity againstagainst HCMV,HCMV, nono 3Carbocyclic‐Substituted nucleosides: 3‐deaza‐5′‐ noraristeromyin derivatives 0.11 μM availableNo data >300 μM dataActivity on other against herpesviruses HCMV, no 3‐Substituted 3‐deaza‐5 ‐noraristeromyin derivatives available data on other herpesviruses 33‐Substituted‐Substituted 3 3‐deaza‐deaza‐5‐5′‐′noraristeromyin‐noraristeromyin derivatives derivatives availableavailable datadata on on other other herpesviruses herpesviruses No data In compound 4, X = Br Activity against HCMV, no data 0.11 µM avail- >300 µM In compound 4, X = Br on other herpesviruses InIn compound compound 4 4, ,X X = = Br Br able

Xanthine‐based acyclic nucleoside phosphonates 2.62 μM 4.58 μM 111 μM Activity against HSV‐1, HSV‐ Xanthine‐based acyclic nucleoside phosphonates 2.62 μM 4.58 μM 111 μM Activity against HSV‐1, HSV‐ XanthineXanthine‐based‐based acyclic acyclic nucleoside nucleosidePMEX phosphonates phosphonates 2.622.62 μ μMM 4.584.58 μ μMM 111111 μ μMM 2Activity Activityand HCMV against against HSV HSV‐1,‐1, HSV HSV‐ ‐ PMEX 2 and HCMV PMEXPMEX 22 and and HCMV HCMV Activity against HSV-1, HSV-2 2.62 µM 4.58 µM 111 µM and HCMV

Cyclopentyl nucleoside phosphonates 5.35 μM 8.83 μM >289 μM Activity against HSV‐1, HSV‐ Cyclopentyl nucleoside phosphonates 5.35 μM 8.83 μM >289 μM Activity against HSV‐1, HSV‐ CyclopentylCyclopentyl nucleoside nucleoside phosphonates phosphonates 5.355.35 μ μMM 8.838.83 μ μMM >289>289 μ μMM 2Activity Activityand HCMV against against HSV HSV‐1,‐1, HSV HSV‐ ‐ 2 and HCMV Compound 23 22 and and HCMV HCMV Compound 23 CompoundCompound 23 23 Activity against HSV-1, HSV-2 5.35 µM 8.83 µM >289 µM and HCMV

Nuceoside/nucleotide prodrugs MoleculesNuceoside/nucleotide 2021, 26, 1132 prodrugs 20 of 35 Nuceoside/nucleotideNuceoside/nucleotide prodrugs prodrugs

Nuceoside/nucleotide prodrugs (E)‐But‐2‐enyl nucleoside phosphonoamidates 0.39 μM 0.33 μM ≥83 μM Activity against HSV‐1, HSV‐ 2 and HCMV For compound 19, Activity against HSV-1, HSV-2 0.39 µM 0.33 µM ≥83 µM and HCMV R = Bn

R1 = CH3

ProdrugsProdrugs of of C5-substituted C5‐substituted pyrimidine pyrimidine acyclic acyclic nucleosides nucleosides Bis(POM) derivative of (E)-TbutP (compound 16) Bis(POM)Bis(POM) derivative derivative of of ( E(E)-5Br-UbutP)‐TbutP (compound (compound 16) 18) Bis(POM) derivative of (E)‐5Br‐UbutP (compound 18)

R = CH3 (E)‐compound 16 0.41 μM 0.19 μM 35 μM Activity against HSV‐1 & HSV‐2 and no anti‐HCMV activity

R = Br (E)‐ compound 18 20 μM 15 μM 35 μM Weak activity against HSV‐1, HSV‐2, no anti‐HCMV activity In compound 15, X = Me, 0.5 μM 0.09 μM 40 μM Activity against HSV‐1, HSV‐

R1 = Neopent and Ar = naphtol 2 and HCMV

In compound 21, X = Me, R1 = Neopent 0.47 μM 0.20 μM >100 μM Activity against HSV‐1, HSV‐ 2 and HCMV

Prodrugs of pyrimidine acyclic nucleoside analogues Parent compound PMEO‐DAPy 2.07 μM 4.72 μM 90.3 μM Activity against HSV‐1 & HSV‐2 but not against HCMV

Bis(POC)‐PMEO‐DAPy 0.32 μM 0.29 μM 15.1 μM Activity against HSV‐1 & (Compound 4) HSV‐2 and marginal activity against HCMV

MoleculesMoleculesMolecules 2021 20212021,, , 26 2626,, , 1132 11321132 202020 of ofof 35 3535

Molecules 2021, 26, 1132 20 of 35

Molecules(E)‐But 2021‐2‐,enyl 26, 1132 nucleoside phosphonoamidates 0.39 μM 0.33 μM ≥83 μM Activity against20 of 35HSV ‐1, HSV‐ (E(E)‐)But‐But‐2‐2‐enyl‐enyl nucleoside nucleoside phosphonoamidates phosphonoamidates 0.390.39 μ μMM 0.330.33 μ μMM ≥ ≥8383 μ μMM ActivityActivity against against HSV HSV‐1,‐1, HSV HSV‐‐ (E)‐But‐2‐enyl nucleoside phosphonoamidates 0.39 μM 0.33 μM ≥83 μM 2Activity22 and andand HCMV HCMVHCMV against HSV‐1, HSV‐ ForForFor compound compoundcompound 19 1919,, , (E)‐But‐2‐enyl nucleoside phosphonoamidates 0.39 μM 0.33 μM ≥83 μM Activity against2 and HSV HCMV‐1, HSV ‐ R = Bn Molecules 2021, 26, 1132 RForR = = Bn compoundBn 19, 2 and HCMV 20 of 34 R1 = CH3 ForR1R compoundR1 = = Bn= CH CH 3 3 19,

R = R1Bn = CH3 Prodrugs of C5‐substituted pyrimidine acyclic nucleosides ProdrugsProdrugs of of C5 C5‐substituted‐substitutedR1 = CH pyrimidine pyrimidine3 acyclic acyclic nucleosides nucleosidesTable 3. Cont. Bis(POM)ProdrugsBis(POM)Bis(POM) of derivative derivativederivative C5‐substituted of ofof ( (E(EE))‐)‐TbutP ‐TbutPpyrimidineTbutP (compound (compound(compound acyclic 16 1616nucleosides)) ) EC50 EC50 ProdrugsBis(POM)Bis(POM) of derivativeC5derivative‐substituted ofof ( (pyrimidineEE))‐‐5Br5Br‐‐UbutPUbutP acyclic (compound(compound nucleosides 1818 )) Activity against Other Human Bis(POM)Bis(POM) derivativederivative ofofCompound ((EE))‐‐5BrTbutP‐UbutP (compound (compound 16) 18) TK+ TK− CC50 Bis(POM) derivative of (E)‐TbutP (compound 16) Herpesviruses R = CH3 (E)‐compound 16 0.41 μM 0.19 μM 35 μM Activity against HSV‐1 & Bis(POM) derivative of (RER )= ‐= 5BrCH CH‐3UbutP 3( E(E)‐)compound‐compound (compound 16 16 18) VZV0.410.41 μ μMM VZV0.190.19 μ μMM 3535 μ μMM ActivityActivity againstagainst HSVHSV‐1‐1 && Bis(POM) derivative of (E)‐5Br‐UbutP (compound 18) HSV‐2 and no anti‐HCMV R = CH3 (E)‐compound 16 0.41 μM 0.19 μM 35 μM HSVActivityHSV‐2‐2 andandagainst nono antiantiHSV‐HCMV‐HCMV‐1 & R = CH3 (E)‐compound 16 0.41 μM 0.19 μM 35 μM Activity against HSV‐1 & activityHSVactivityactivity‐2 and no anti‐HCMV HSV‐2 Activityand no againstanti‐HCMV HSV-1 & HSV-2 0.41 µM 0.19 µM 35 µM activity andactivity no anti-HCMV activity

RRR = == Br BrBr ( (E(EE))‐ )‐ ‐ compoundcompoundcompound 18 1818 202020 μ μ μMMM 151515 μ μ μMMM 353535 μ μ μMMM WeakWeakWeak activity activityactivity against againstagainst HSV HSVHSV‐‐1,‐1,1, HSV‐2, no anti‐HCMV RR = = Br Br ( E(E)‐ )compound‐ compound 18 18 20 μM 20 μ15M μ M 15 μ35M μ M 35Weak μM activityWeakHSVWeakHSV activityagainst‐2,‐ 2,activity HSV againstnono ‐ against1, anti HSV-1,anti‐ HCMV‐HSVHCMV‐1, R = Br (E)- compound 18 20 µM 15 µM 35 µM HSV‐2, HSV-2,activityHSVnoactivityactivity no‐2,anti anti-HCMV ‐HCMVno anti activity‐HCMV InInIn compound compoundcompound 15 1515,, , X XX = == Me, Me,Me, 0.50.50.5 μ μ μMMM 0.090.090.09 μ μ μMMM 404040activity μ μ μMMM ActivityactivityActivityActivity against againstagainst HSV HSVHSV‐‐1,‐1,1, HSV HSVHSV‐‐‐ In Rcompound1 = Neopent 15, Xand = Me, Ar = naphtol 0.5 μM 0.09 μM 40 μM Activity against2 and HSV HCMV‐1, HSV ‐ RInR1 1=compound = Neopent Neopent and 15and, XAr Ar = =Me, = naphtol naphtol 0.5 μM 0.09 μM 40 μM 2Activity2 and and HCMV HCMV against HSV‐1, HSV‐ R1 = Neopent and Ar = naphtol 2 and HCMV 1 R = Neopent and Ar = naphtol Activity2 and against HCMV HSV-1, HSV-2 0.5 µM 0.09 µM 40 µM and HCMV

In compound 21, X = Me, R1 = Neopent 0.47 μM 0.20 μM >100 μM Activity against HSV‐1, HSV‐ In IncompoundIn compound compound 21, X21 21=, Me,,X X = = RMe, Me,1 = Neopent R R1 1= = Neopent Neopent 0.47 μ M0.47 0.47 μ0.20 μMM μ M 0.200.20>100 μ μMM μ M >100>100Activity μ μMM againstActivityActivity HSV against ‐against1, HSV ‐HSV HSV‐1,‐1, HSV HSV‐‐ 2 and HCMV In compound 21, X = Me, R1 = Neopent 0.47 μM 0.20 μM >1002 and μM HCMV 2Activity2 and and HCMV HCMV against HSV‐1, HSV‐

Activity2 and against HCMV HSV-1, HSV-2 0.47 µM 0.20 µM >100 µM and HCMV

Prodrugs of pyrimidine acyclic nucleoside analogues Prodrugs of pyrimidine acyclic nucleoside analogues ProdrugsProdrugsProdrugs of ofof pyrimidine pyrimidinepyrimidine acyclic acyclicacyclic nucleoside nucleosidenucleoside analogues analoguesanalogues Parent compound PMEO‐DAPy 2.07 μM 4.72 μM 90.3 μM Activity against HSV‐1 & Prodrugs of pyrimidine acyclicParentParentParent nucleoside compound compoundcompound analogues PMEO PMEOPMEO‐‐DAPy‐DAPyDAPy 2.072.072.07 μ μ μMMM 4.724.724.72 μ μ μMMM 90.390.390.3 μ μ μMMM ActivityActivityActivity againstagainstagainst HSVHSVHSV‐‐1‐11 &&& HSV‐2 butActivity not against against HCMV HSV-1 & HSV-2 2.07 µM 4.72 µM 90.3 µM HSV‐2 but not against HCMV Parent compound PMEO‐DAPy 2.07 μM 4.72 μM 90.3 μM butHSVActivity notHSV against‐2‐2 but but againstnot not HCMV against against HSV HCMV HCMV‐1 & Bis(POC)‐PMEO‐DAPy 0.32 μM 0.29 μM 15.1 μM Activity againstHSV‐ 2 butHSV not‐1 against& HCMV (CompoundBis(POC)Bis(POC)Bis(POC) 4‐)‐PMEO ‐PMEOPMEO‐‐DAPy‐DAPyDAPy 0.320.320.32 μ μ μMMM 0.290.290.29 μ μ μMMM 15.115.115.1HSV μ μ μM‐M2M and Activity ActivityActivitymarginal againstactivityagainstagainst HSVHSVHSV‐‐1‐11 &&& (CompoundBis(POC)(Compound(Compound‐PMEO 4 44)) ) ‐DAPy 0.32 μM 0.29 μM 15.1against μM HCMVHSVActivityHSVHSV ‐‐2‐22 andand andagainst marginalmarginalmarginal HSV activityactivityactivity‐1 & (Compound 4) ActivityagainstHSVagainstagainst‐ against2 HCMVand HCMVHCMV marginal HSV-1 & HSV-2activity 0.32 µM 0.29 µM 15.1 µM and marginal activity against HCMVagainst HCMV Molecules 2021,, 26,, 1132 21 of 35

Bis(aminoacid) 0.56 μM 1.69 μM 25.9 μM Activity against HSV‐1 & Phosphoramidate ActivityHSV‐2 against and HSV-1low & HSV-2activity 0.56 µM 1.69 µM 25.9 µM and low activity against HCMV (Compound 5) against HCMV

Parent compound PME‐5azaC 100 μM 40 μM >200 μM Marginal activity against

MarginalHSV‐1, activity HSV‐2 against& HCMV HSV-1, 100 µM 40 µM >200 µM HSV-2 & HCMV

0.32 μM 0.79 μM 79.2 μM Activity against HSV‐1, HSV‐ Mono‐octadecyloxyethyl‐ 2 and HCMV PME‐5‐azaC (Compound 17)

Diamyl aspartate amidate prodrugs of 3‐Fluoro‐2‐ (phosphonomethoxy)propyl acyclic nucleoside phosphonates Base adenine: (S) 22a, (R) 22b Base cytosine: (S) 23a, (R) 23b Base cytosine: (S) 25a, (R) 25b

22a 0.05 μM 0.057 μM 1.96 μM No selectivity against HCMV 23a 3.15 μM 1.32 μM 74.7 μM Activity and selectivity against HCMV 25a 0.59 μM 0.079 μM 27.8 μM Activity but low selectivity 25b 0.89 μM 0.11 μM 10.2 μM against HCMV (S)‐cHPMPA Phosphonamidates 0.00045 0.00054 12 μM Activity against HSV‐1, HSV‐2, μM μM and HCMV In (S)-Ile-cHPMPA (compound 16), R =

Non‐nucleoside analogues

Pyrazolo[1,5−c]1,3,5‐triazin‐4‐one (35B2) 0.75 μM 4.6 μM 152.5 μM Weak activity against HSV‐1, no anti‐HCMV activity

Molecules 2021,, 26,, 1132 21 of 35

MoleculesMolecules 2021 2021, ,26 26, ,1132 1132 2121 of of 35 35

Bis(aminoacid) 0.56 μM 1.69 μM 25.9 μM Activity against HSV‐1 & Phosphoramidate HSV‐2 and low activity Bis(aminoacid)Bis(aminoacid) 0.560.56 μ μMM 1.691.69 μ μMM 25.925.9 μ μMM ActivityActivity againstagainst HSVHSV‐‐11 && (Compound 5) against HCMV Molecules 2021, 26, 1132 PhosphoramidatePhosphoramidate HSVHSV‐‐22 andand lowlow activityactivity 21 of 34 (Compound(Compound 55)) againstagainst HCMVHCMV Parent compound PME‐5azaC 100 μM 40 μM >200 μM Marginal activity against

ParentParent compoundcompound PMEPME‐‐5azaC5azaC 100100Table μ μMM 3. Cont.4040 μ μMM >200>200 μ μMM MarginalMarginal HSVactivityactivity‐1, HSV againstagainst‐2 & HCMV HSV‐1, HSV ‐2 & HCMV EC EC HSV‐1, HSV ‐2 & HCMV 50 50 Activity against Other Human Compound TK+ TK− CC 50 Herpesviruses VZV VZV 0.32 μM 0.79 μM 79.2 μM Activity against HSV‐1, HSV‐ 0.320.32 μ μMM 0.790.79 μ μMM 79.279.2 μ μMM ActivityActivity againstagainst HSVHSV‐‐1,1, HSVHSV‐‐ Mono‐octadecyloxyethyl‐ 2 and HCMV MonoMono‐‐octadecyloxyethyloctadecyloxyethyl‐‐ 22 andand HCMVHCMVActivity against HSV-1, HSV-2 PME‐5‐azaC (Compound 17)0.32 µM 0.79 µM 79.2 µM PMEPME‐‐55‐‐azaCazaC (Compound(Compound 1717)) and HCMV

Diamyl aspartate amidate prodrugs of 3‐Fluoro‐2‐ DiamylDiamyl aspartateaspartate amidateamidate prodrugsprodrugs ofof 33‐‐FluoroFluoro‐‐22‐‐ (phosphonomethoxy)propyl acyclic nucleoside (phosphonomethoxy)propyl(phosphonomethoxy)propyl(phosphonomethoxy)propyl acyclicacyclic acyclic nucleosidenucleoside nucleoside phosphonatesphosphonatesphosphonates BaseBaseBase adenine:adenine: adenine: ((SS)) 22a22a (S,, ()(R R22a)) 22b22b, ( R ) 22b BaseBaseBase cytosine:cytosine: cytosine: ((SS)) 23a23a (S,, ()(R R23a)) 23b23b, ( R ) 23b BaseBaseBase cytosine:cytosine: cytosine: ((SS)) 25a25a (S,, ()(R R25a)) 25b25b, ( R ) 25b

0.057 22a 0.05 µM 1.96 µM No selectivity against HCMV 22a22a 0.050.05 μ μMM 0.0570.057µ μ μMM 1.961.96 μ μMM NoNo selectivityselectivity againstagainst HCMVHCMV 22a 0.05 μM M0.057 μM 1.96 μM No selectivity against HCMV 23a23a 3.153.15 μ μMM 1.321.32 μ μMM 74.774.7 μ μMM ActivityActivityActivity andand andselectivityselectivity selectivity against 23a23a 3.153.15µM μM 1.321.32µM μM 74.7 µ74.7M μM Activity and selectivity againstagainst HCMV HCMVHCMV against HCMV 25a25a 0.590.59 μ μMM 0.0790.0790.079 μ μMM 27.827.8 μ μMM ActivityActivity butbut lowlow selectivityselectivity 25a 0.59 µM 27.8 µM Activity but low selectivity 25a 0.59 μM µM0.079 μM 27.8 μM Activity but low selectivity 25b25b 0.890.89 μ μMM 0.110.11 μ μMM 10.210.2 μ μMM againstagainst against HCMVHCMV HCMV µ µ µ against HCMV (25b(S25bS))‐‐cHPMPAcHPMPA PhosphonamidatesPhosphonamidates 0.000450.000450.89 0.89M0.000540.00054 μM 0.11 0.1112M12 μ μMM 10.2 10.2ActivityMActivity μM against againstagainst HSV HSV ‐HCMV‐1,1, HSV HSV‐‐2, 2, (S)‐cHPMPA Phosphonamidates μμMM 0.00045μμMM 0.00054 12andand μM HCMVHCMV Activity against HSV‐1, HSV‐2, In (S)-Ile-cHPMPA In (S)-Ile-cHPMPA μM μM and HCMV (compound(compoundIn (S)-Ile-cHPMPA 1616),), RR == 0.00045 0.00054 Activity against HSV-1, HSV-2, (compound 16), R = 12 µM µM µM and HCMV

NonNon‐‐nucleosidenucleoside analoguesanalogues

Pyrazolo[1,5Pyrazolo[1,5Non‐nucleoside−−c]1,3,5c]1,3,5 analogues‐‐triazintriazin‐‐44‐‐oneone (35B2)(35B2) 0.750.75 μ μMM 4.64.6 μ μMM 152.5152.5 μ μMM WeakWeak activityactivity againstagainst HSVHSV‐‐1,1, Non-nucleosideNon‐nucleoside analogues analogues nono antianti‐‐HCMVHCMV activityactivity Pyrazolo[1,5−c]1,3,5‐triazin ‐4‐one (35B2) 0.75 μM 4.6 μM 152.5 μM Weak activity against HSV‐1, no anti‐HCMV activity Weak activity against HSV-1, no 0.75 µM 4.6 µM 152.5 µM anti-HCMV activity Molecules 2021, 26, 1132 22 of 35

[(5‐Chlorobenzo[b]thiophen‐3‐yl)methyl] [(4‐chloro‐ 16.9 μM 18.3 μM >100 μM phenyl)sulfonyl]amine (45B5) 16.9 µM 18.3 µM >100 µM NoNo data data available available on other on viruses other viruses

N‐(4‐Chlorophenyl)‐5‐nitro‐3‐thienylcarboxamide (133G4) 19.3 μM No data 537 μM Activity against HCMV

available

Indole‐based derivatives 2.6 μM 2.0 μM >100 μM No activity against HSV‐1, HSV‐2, HCMV

17a

Cephalotaxine esters

16.15 45.82 ng/mL ng/mL No data available on other (HT) (HT) No data viruses—Low selectivity for 9.96 74.71 VZV ng/mL ng/mL

(HTT) (HTT) HT HTT Hybrid molecules Dihydropyrimidinone/1,2,3‐triazole hybrid molecules

3.62 μM 7.85 μM >100 μM (8a) (8a) (8a) No anti‐HCMV activity 11.33 μM 10.79 μM >100 μM (7d) (7d) (7d)

8a 7d

7.1.2. 2′‐Deoxyribose Emimycin Nucleosides Emimycin (1,2‐dihydro‐2‐oxopyrazine 4‐oxide) is a pyrazine analogue structurally resembling uracil with known antibacterial properties. Emimycin, its 5‐substituted con‐ geners and the ribonucleoside derivatives are devoid of antiviral activity against RNA viruses. Interestingly, some of the 2′‐deoxyribosyl emimycin derivatives proved potent inhibitors of the replication of HSV and VZV but were completely devoid of activity

MoleculesMolecules 2021, 26 2021, 1132, 26 , 1132 22 of 3522 of 35

MoleculesMolecules 2021 2021, 26, ,26 1132, 1132 22 22of 35of 35

Molecules 2021, 26, 1132 22 of 35

[(5‐Chlorobenzo[b]thiophen[(5‐Chlorobenzo[b]thiophen‐3‐yl)methyl]‐3‐yl)methyl] [(4‐chloro [(4‐chloro‐ ‐ 16.9 μ16.9M μM18.3 μ18.3M μM>100 μ>100M μM Molecules 2021, 26, 1132 22 of 35 phenyl)sulfonyl]amine[(5 phenyl)sulfonyl]amine[(5‐Chlorobenzo[b]thiophen‐Chlorobenzo[b]thiophen (45B5) (45B5) ‐3‐‐yl)methyl]3 ‐yl)methyl] [(4 [(4‐chloro‐chloro‐ ‐ 16.916.9 μM μ M 18.318.3 μM μ M >100>100 μM μ M phenyl)sulfonyl]aminephenyl)sulfonyl]amine[(5‐Chlorobenzo[b]thiophen (45B5) (45B5) ‐ 3‐yl)methyl] [(4‐chloro‐ 16.9 μM 18.3 μM >100 μNoM dataNo dataavailable available on otheron other Molecules 2021, 26, 1132 22 of 34 phenyl)sulfonyl]amine (45B5) virusesNovirusesNo data data available available on on other other [(5‐Chlorobenzo[b]thiophen‐3‐yl)methyl] [(4‐chloro‐ 16.9 μM 18.3 μM >100 μM virusesvirusesNo data available on other phenyl)sulfonyl]amine (45B5) viruses Table 3. Cont. No data available on other N‐(4‐Chlorophenyl)N‐(4‐Chlorophenyl)‐5‐nitro‐5‐nitro3‐thienylcarboxamide‐3‐thienylcarboxamide (133G4) (133G4) 19.3 μ19.3M μMNo Nodata 537data μ M537 μMActivity Activity against against HCMV HCMV viruses EC EC N‐N(4‐‐(4Chlorophenyl)‐Chlorophenyl)‐5‐‐nitro5‐nitro‐3‐‐thienylcarboxamide3‐thienylcarboxamide (133G4) (133G4) 19.319.3 μ50M μ Mavailable NoavailableNo 50 datadata 537 537 μM μ M ActivityActivityActivity against against against HCMV Other HCMV Human Compound TK+ TK− CC 50 Herpesviruses N‐(4‐Chlorophenyl)‐5‐nitro‐3‐thienylcarboxamide (133G4) VZV19.3 μM availableVZVavailableNo data 537 μM Activity against HCMV available N‐(4‐Chlorophenyl)‐5‐nitro‐3‐thienylcarboxamide (133G4) 19.3 μM No data 537 μM Activity against HCMV

Noavailable data 19.3 µM avail- 537 µM Activity against HCMV

able IndoleIndole‐based‐based derivatives derivatives 2.6 μM2.6 μM2.0 μM2.0 μM>100 μ>100M μMNo activityNo activity against against HSV‐ 1,HSV ‐1, IndoleIndole‐based‐based derivatives derivatives 2.62.6 μM μ M 2.02.0 μM μ M >100>100 μM μ M NoHSVNo activity activity‐2,HSV HCMV against‐2, against HCMV HSV HSV ‐1,‐ 1, Indole‐based derivatives 2.6 μM 2.0 μM >100 μM NoHSV activityHSV‐2,‐ HCMV2, against HCMV HSV ‐1, HSV‐2, HCMV Indole‐based derivatives 2.6 μM 2.0 μM >100 μM No activity against HSV‐1, No activity against HSV-1, HSV-2, 17a 17a 2.6 µM 2.0 µM >100 µM HCMV HSV‐2, HCMV 17a17a Cephalotaxine esters Cephalotaxine esters17a CephalotaxineCephalotaxine esters esters Cephalotaxine17a esters 16.15 16.15 45.82 45.82 Cephalotaxine esters ng/mL16.15ng/mL16.15 ng/mL45.82ng/mL45.82 16.15 45.82 No dataNo dataavailable available on other on other Cephalotaxine esters ng/mL(HT) 16.15 ng/mL(HT)45.82 (HT)ng/mL ng/mL (HT)ng/mL ng/mL No dataNo data viruses—LowNoviruses—LowNo data data available selectivityavailable selectivity on on forother other for (HT)(HT)9.96ng/mL (HT)(HT)74.71ng/mL No data available on other 9.96 (HT)16.15 No data 74.71 (HT)45.82 9.96 NoNo data data 74.71 VZVviruses—Lowviruses—Low VZVviruses—LowNo data selectivityavailable selectivity selectivity foron for VZV otherfor ng/mL9.96ng/mL9.96 (HT) ng/mL74.71ng/mL74.71 (HT) ng/mLng/mL No datang/mL ng/mL VZVviruses—Low selectivity for VZVNo data available on other (HTT)ng/mL(HTT)(HTT)ng/mL 9.96 (HTT)(HTT)ng/mL(HTT)ng/mL 74.71 (HT) (HT) VZV No data viruses—Low selectivity for HT HTT (HTT) (HTT)ng/mL (HTT)(HTT)ng/mL HT HTHTT HTT 9.96 74.71 VZV Hybrid molecules (HTT) (HTT) HybridHTHTHybrid molecules molecules HTTHTT ng/mL ng/mL HybridDihydropyrimidinone/1,2,3HT molecules ‐triazoleHTT hybrid molecules Dihydropyrimidinone/1,2,3HybridDihydropyrimidinone/1,2,3-triazole molecules ‐triazole hybrid hybrid molecules molecules (HTT) (HTT) Dihydropyrimidinone/1,2,3Hybrid molecules ‐triazole hybrid molecules Dihydropyrimidinone/1,2,3HT ‐triazoleHTT hybrid molecules Dihydropyrimidinone/1,2,3‐triazole hybrid molecules Hybrid molecules 3.62 μ3.623.62Mµ μM 7.85M7.85 μ 7.85µMM μ >100M>100 μ>100µMM μ M Dihydropyrimidinone/1,2,3‐triazole hybrid molecules (8a) (8a) (8a) (8a)3.62 (3.628a μ) μ(M8a M)7.85 (7.858a μ) μ(M8a M)>100 (>1008a μ) μM MNo anti-HCMV activity 11.33 10.79 >100 µM No antiNo‐HCMV anti‐HCMV activity activity 11.33(µ8a μM(11.33()8a3.62 M7d) μ μ)10.79MµM( 8aM( μ 10.79()8a7.85 7dM) ) μ μ>100M(7dM( 8a μ) >100()8a>100 M) μ μMM NoNo anti anti‐HCMV‐HCMV activity activity (7d)11.33 (11.337d(8a μ) ) μ(M7d M)10.79 (10.797d(8a μ) ) μ(M7d M)>100 (>1007d(8a μ) ) μ M M 3.62 μM 7.85 μM >100 μM No anti‐HCMV activity (7d)11.33 μM(7d )10.79 μM(7d )>100 μM (7d(8a)) (7d(8a)) (7d(8a))

(7d) (7d) (7d) No anti‐HCMV activity 0 11.33 μM 10.79 μM >100 μM 8a 8a 7d 7d7.1.2. 2 -Deoxyribose Emimycin Nucleosides (7d) (7d) (7d) 8a 8a 7d7d Emimycin (1,2-dihydro-2-oxopyrazine 4-oxide) is a pyrazine analogue structurally 7.1.2. 2′‐Deoxyribose Emimycin Nucleosides 8a 7.1.2. 2resembling7d′‐Deoxyribose uracil Emimycin with known Nucleosides antibacterial properties. Emimycin, its 5-substituted con-

7.1.2.Emimycin7.1.2.geners 2Emimycin′‐ 2Deoxyribose′‐ andDeoxyribose (1,2 the‐ dihydro(1,2 ribonucleoside ‐Emimycindihydro Emimycin‐2‐oxopyrazine‐2 ‐Nucleosidesoxopyrazine derivativesNucleosides 4‐oxide) 4 are ‐oxide) devoidis a pyrazineis a of pyrazine antiviral analogue analogue activity structurally against structurally RNA viruses. Interestingly, some of the 20-deoxyribosyl emimycin derivatives proved potent 8a resemblingresembling7d7.1.2.Emimycin uracil2′‐Deoxyribose uracil with (1,2 ‐ withdihydroknown Emimycin known antibacterial‐2‐ oxopyrazine antibacterial Nucleosides properties. 4‐ oxide)properties. Emimycin,is a pyrazineEmimycin, its analogue5‐ substitutedits 5‐substituted structurally con‐ con ‐ inhibitorsEmimycin of the (1,2 replication‐dihydro of‐2 HSV‐oxopyrazine and VZV but4‐oxide) were completelyis a pyrazine devoid analogue of activity structurally against genersresemblinggeners and theand uracilribonucleoside the ribonucleosidewith known derivatives antibacterial derivatives are devoid properties.are devoid of antiviral Emimycin, of antiviral activity its activity 5 ‐againstsubstituted against RNA con RNA ‐ resemblingHCMVEmimycin [106 uracil]. The (1,2 2with0-deoxyribonucleosides‐dihydro known‐2 ‐antibacterialoxopyrazine with properties.4‐oxide) an emimycin is Emimycin,a pyrazine nucleobase itsanalogue 5‐ (structurallysubstituted structurally con sim-‐ viruses.genersviruses.7.1.2. Interestingly, and 2 ′‐Interestingly,Deoxyribose the ribonucleoside some Emimycinsome of the of derivatives2 the′‐ deoxyribosylNucleosides 2′‐deoxyribosyl are devoid emimycin emimycin of antiviral derivatives derivatives activity proved againstproved potent RNApotent genersilarresembling to uracil) and theuracil did ribonucleoside not with display known inhibitory derivativesantibacterial activity are properties. againstdevoid VZV, of Emimycin, antiviral HSV-1 and activity its HSV-2. 5‐substituted against In contrast, RNA con ‐ inhibitorsviruses.inhibitors ofEmimycin Interestingly, the of replication the (1,2replication ‐somedihydro of ofHSV of‐the2 ‐ oxopyrazineHSVand 2′‐deoxyribosyl VZVand VZVbut 4‐ oxide) werebut emimycin werecompletely is a completelypyrazine derivatives devoid analogue devoid provedof activity structurallyof potentactivity viruses.thegeners presence andInterestingly, the of 5-methylemimycin ribonucleoside some of the derivatives 2 (related′‐deoxyribosyl toare the devoid natural emimycin of thymidine antiviral derivatives activity nucleobase) proved against inpotent com-RNA inhibitorsinhibitorsresembling of ofthe uracil the replication replication with known of ofHSV antibacterialHSV and and VZV VZV properties. but but were were completelyEmimycin, completely itsdevoid devoid 5‐substituted of ofactivity activity con ‐ poundviruses.27a Interestingly,(Table3) conferred some of potent the 2 antiviral′‐deoxyribosyl activity emimycin against VZV derivatives TK+ ( EC proved50 = 0.99 potentµM) geners and the ribonucleoside derivatives are devoid of antiviral activity against RNA andinhibitors HSV-1 TK+of the (EC replication= 1.79 µ M),of HSV with and selectivity VZV indicesbut were (ratio completely CC /EC devoid) of 416 of and activity 230, 50 50 50 respectively.viruses. Interestingly, Compound some27a wasof the 7-fold 2′‐deoxyribosyl (VZV) and 30-fold emimycin (HSV-1) derivatives less active proved when testedpotent inhibitors of the replication of HSV and VZV but were completely devoid of activity against mutant TK–strains and was devoid of anti-HCMV activity. The 5-ethylemimycin 0 2 -deoxyribose congener 28a has a very similar proﬁle as its 5-methyl congener 27a, but its antiviral activity is less pronounced.

Molecules 2021, 26, 1132 23 of 34

7.1.3. C5-substituted-(1,3-diyne)-2-deoxyuridines C5-substituted pyrimidine nucleosides, in particular those in the 20-deoxyuridine series, constitute a unique class of compounds, playing an important role as components of nucleotide-derived tools for molecular genetics and as antiviral and anticancer agents. For instance, signiﬁcant efforts have been expended to develop C5-substituted analogues that can be incorporated into DNA by viral DNA polymerases, such as the 5-(2- substituted vinyl)-20-deoxyuridines, including brivudine. Novel C5-substituted-(1,3-diyne)- 20-deoxyuridines (with cyclopropyl, hydroxymethyl, methylcyclopentane, p-(substituted) phenyl and disubstituted-phenyl substituents) have been synthesized and evaluated against several herpesviruses [107]. The compound 5-[4-(4-triﬂuoromethoxyphenyl)buta- 1,3-diynyl]-2-deoxyuridine (26, Table3) emerged as the most potent inhibitor of this series against VZV with an EC50 of ∼1 µM and a CC50 of 55 µM. This compound had similar activity as acyclovir but it was less active than brivudine against the VZV TK+ YS and OKA strains and lost potency against TK− VZV strains (EC50 > 20µM). Compound 26 displayed moderate activity against HSV-1, HSV-2 and HSV-1 TK− with EC50 values of ~10–15 µM.

7.1.4. Carbocyclic Nucleosides: C-3 halo and 3-methyl substituted 50-nor-3-deazaaristeromycins To expand on the antiviral properties of the carbocyclic nucleoside analogue 50- noraristeromycin, 3-substituted 3-deaza-50-noraristeromyin derivatives (i.e., bromo, iodo, chloro, and methyl) were synthesized [108]. An extensive characterization of their antiviral activities showed compound 4 (the bromo congener, Table3) was most favorable towards the herpesviruses HCMV (EC50 = 1.7 µM), VZV (EC50 = 0.11 µM) without inhibiting cell growth at a concentration of 300 µM. This compound was also able to inhibit the replication of hepatitis B virus (HBV) and vaccinia virus but the activity against HSV was not reported.

7.1.5. Xanthine-Based Acyclic Nucleoside Phosphonates (ANPs) Noncanonic xanthine nucleotides XMP/dXMP play an important role in the balance and maintenance of intracellular purine nucleotide pool as well as in potential mutage- nesis. A series of ANPs bearing a xanthine nucleobase were synthesized and evaluated for their activity against a wide range of DNA and RNA viruses [109]. Within this series, two ANPs, i.e., 9-[2-(phosphonomethoxy)ethyl]xanthine (PMEX) and 9-[3-hydroxy-2- (phosphonomethoxy)-propyl]xanthine (HPMPX), showed activity against several human herpesviruses. PMEX (Table3), a xanthine analogue of adefovir (PMEA), emerged as the most important compound, exhibiting also activity against VZV (EC50 = 2.62 µM, TK+ Oka strain and EC50 = 4.58 µM, TK− 07-1 strain). The (S)HPMPX was less active with EC50 values of 22.7 µM and 17.1 µM, for respectively, TK+ and TK− VZV strains. The hexadecyloxypropyl monoester prodrug of PMEX (i.e., HDP-PMEX) proved 7- to 26-fold more active than the parent compound PMEX against VZV, although a concomitant increase in its cytostatic activity of 11-fold was also observed indicating successful increase in the cell uptake. The structures of HPMPX and HDP-PMEX are not reported here but can be found in the original study by Baszczynski and coworkers [109]. An increase in the EC50 of PMEX of the same magnitude as that measured for adefovir was found for well-characterized HSV-1 DNA polymerase mutant viruses indicating that the target of action of the active form of PMEX (i.e., PMEXpp) is the herpesvirus DNA polymerase. Furthermore, when the inhibitory activity of PMEXpp, was evaluated in an enzymatic assay against VZV DNA polymerase compared to cellular (α and β) DNA polymerases, PMEXpp proved inhibitory to VZV DNA polymerase when dGTP was used as competitive radiolabeled substrate with an IC50 = 7.4 µM without inhibition towards cellular DNA polymerases.

7.1.6. Cyclopentyl Nucleoside Phosphonates Both 20-hydroxy-30-deoxy- and 20-deoxy-30-hydroxycyclopentyl nucleoside phosphonates with the natural nucleobases adenine, thymine, cytosine and guanine were synthe- Molecules 2021, 26, 1132 24 of 34

sized and evaluated for activity against several herpesviruses [110]. The guanine containing analogues displayed antiviral activity; in particular, the 30-deoxy congener 23 (Table3) was active, with an EC50 of 5.35 µM against TK+ VZV strain and an EC50 of 8.83 µM against TK− VZV strain, while lacking cytotoxicity (CC50 > 289 µM). The application of phosphonodiamidate prodrug strategy did not result in a boost in antiviral activity.

7.1.7. (E)-but-2-enyl Nucleoside Phosphonoamidates A signiﬁcant amount of research and development on aryl phosphoramidate prodrugs has been carried out by McGuigan’s group. However, the development of aryl phosphonoamidates, in particular in the ﬁeld of ANPs, has been poorly investigated. Recently, the synthesis and antiviral evaluation of hitherto unknown (E)-but-2-enyl nucleosidephospho- noamidates using the cross-metathesis in water-under ultrasound irradiation has been reported by Agrofolio’s group [111]. The overall yield obtained was high, well above the previously data for the preparation of phosphonoamidates (15% vs. ~3%). Among the synthesized (E)-but-2-enylnucleoside phosphonoamidates, the thymine analogue 19 (Table3) proved to be the best prodrug tested against VZV with an EC50’s of 0.39 and 0.33 µM for TK+ and TK− strains, respectively, and a selectivity index ≥200. This innovative synthetic approach may open the way for the synthesis of new purine and pyrimidine (E)-but-2-enyl phosphonoamidates.

7.1.8. Prodrugs of C5-Substituted Pyrimidine Acyclic Nucleosides for Antiviral Therapy While C5-Substituted-Uracil ANPs were devoid of antiviral activity, the bis(POM) prodrug of (E)-TbutP (compound 16) (Table3) presented a potent anti-herpesvirus activity, namely, HSV-1, HSV-2 and VZV (EC50 = 2.5−6.1 µM for these three viruses) [112]. Interest- ingly, the compound showed a decreased inhibitory effect (EC50 = 9.2 µM) against the TK− HSV-1 strain, but an increase in activity against the VZV TK− strain with EC50 = 0.19 µM compared to EC50 = 0.41 µM for VZV TK+ strain. There was no activity against HCMV. The (E)-bis(POM)-5Br-UbutP congener 18 proved weakly inhibitory against HSV-1, HSV-2, and VZV with EC50’s = 16−33 µM, and of 15–20 µM for VZV) but not against HCMV and kept activity against TK− HSV-1 and VZV strains. The compounds showed no cytotoxic at 200 µM, but were slightly cytostatic at ~35 µM for HEL cells. In contrast with the (E) isomer of bis(POM)-TbutP and bis(POM)-UbutP, the corresponding (Z) isomers exhibited negligible or no antiviral activity. The phosphoro(no)amidate approach proved very successful both on nucleoside analogues (e.g., Sofosbuvir for hepatitis C virus) as well as on ANPs (e.g., Tenofovir alafenamide for HIV infection and HBV). Excellent results using the phosphoroamidate approach on adefovir were also obtained. In search for more potent and safe ANPs, the impact of the phosphonoamidate and phosphonodiamidate prodrugs on the antiviral activity of C5-pyrimidine acyclic nucleosides derivatives functionalized with but-2-enyl- chain was studied [113]. In the phosphonoamidate series, the most active compound 15 showed sub- micromolar activity against VZV (EC50 = 0.09–0.5 µM) and µM activity against HCMV and HSV. Notably, the phosphonodiamidate 21 showed excellent activity against wild type and TK− VZV strains (EC50 = 0.47 and 0.2 µM, respectively) and HCMV (EC50 = 3.5–7.2 µM) without cytostatic effect at the highest tested concentration (CC50 > 100).

7.1.9. Prodrugs of the Pyrimidine Acyclic Nucleoside Phosphonates PMEO-DAPy and PME-5-azaC Novel prodrugs of 2,4-diamino-6-[2-(phosphonomethoxy)ethoxy]pyrimidine (PMEO- DAPy) and 1-[2-(phosphonomethoxy)ethyl]-5-azacytosine (PME-5-azaC) prodrugs were synthesized with a pro-moiety consisting of carbonyloxymethyl esters (POM, POC), alkoxyalkyl esters, amino acid phosphoramidates and/or tyrosine (Table3)[ 114]. PMEO-DAPy be- longs to the class of open-ring ANPs, which are characterized by the phosphonomethoxy group containing an aliphatic part linked to the position 6 of 2,4-diaminopyrimidine via the oxygen atom. They are mimics of the appropriate 2,6-diaminopurine derivatives with an open imidazole ring. Interestingly, PMEO-DAPy has activity against retroviruses, HBV as Molecules 2021, 26, 1132 25 of 34

well as herpesviruses. The replication of herpesvirus was inhibited by the bis(POC) and the bis(amino acid) phosphoramidate prodrugs of PMEO-DAPy with lower EC50 values than those for the parent compound PMEO-DAPy though the selectivity against HSV and VZV was only slightly improved. Thus, the VZV EC50 values and CC50 values for PMEO-DAPy were respectively, of 2.07–4.72 µM and 90.3 µM vs. ~0.3 µM and 15.1 µM [Bis(POC), compound 4] and 0.56–1.69 µM and 25.9 µM [bis(amino acid) phosphoramidate, compound 5]. The PME-5-azaC mono-octadecyl ester (compound 17) was the most effective and selective PME-5-azaC prodrug when evaluated against HSV (EC50 of 0.15–0.35 µM), VZV (EC50’s of 0.32–0.79 µM) and HCMV (EC50’s of 0.24–1.12 µM) compared to a minimal anti-herpesvirus activity of the parent compound PME-5-azaC. Although the bis(hexadecylamido-L-tyrosyl) and the bis(POM) esters of PME-5-azaC proved to be very potent anti-herpesvirus compounds, their selectivity indices were reduced relative to the mono-octadecyl ester prodrug.

7.1.10. Diamyl Aspartate Amidate Prodrugs of 3-Fluoro-2-(phosphonomethoxy)propyl Acyclic Nucleoside Phosphonates Acyclic nucleosides bearing a 3-ﬂuoro-2-(phosphonomethoxy)propyl (FPMP) side chain have moderate anti-HIV efﬁcacy but are devoid of activity against DNA viruses. Two enantiomeric series of FPMP nucleosides bearing the four natural nucleobases was recently synthesized [115] (Table3). Conversion of the phosphonic acid functionality of FPMPs into a diamyl aspartate phenoxyamidate group resulted in a novel generation of compounds having considerably improved antiretroviral potency as well as a larger spectrum of antiviral activity including HBV and herpesviruses. The (S)-FPMPA amidate prodrug displayed interesting activity against HIV (in the low nanomolar range), HBV and VZV. The (S)-FPMPA amidate prodrug (compound 22a), one of the most active compounds, had 2000-fold better activity than the parent phosphonate against both TK+ and TK− VZV strains (EC50s in the 0.05 µM range). Compound 22a was also active against HCMV but lacked selectivity for HCMV (CC50 = 1.96 µM for HEL cells, which was lower than the HCMV EC50 values) though it proved selective for VZV. Compounds 25a and 25b (both enantiomers of the guanine containing amidate prodrugs) showed similar activity and selectivity against VZV (EC50’s in the 0.079−0.89 µM range) and HCMV (EC50’s in the 1.98−5.94 µM range), but compound 25b proved more active against HIV and HBV than its diasteromer 25a. Among the pyrimidine analogues, compound 23a (the cytosine containing (S)-Asp-prodrug) was the only congener displaying moderate activity against both VZV TK+ (EC50 = 3.15 µM) and TK− (EC50 = 1.32 µM) viruses and against HCMV [EC50 = 0.76 µM (AD-169 strain) and EC50 = 2.02 µM (Davis strain)].

7.1.11. Amidate Prodrugs of Cyclic 9-(S)-[3-Hydroxy-2-(phosphonomethoxy) propyl]adenine, cHPMPA The use of aryloxy monoamidates, a promising prodrug strategy for nucleoside phosphates and phosphonates developed the last years, was applied to HPMP-based compounds, resulting in the synthesis of phosphono amidate prodrugs of (S)-cHPMPA bearing different amino acid motifs [116]. All (S)-cHPMPA phosphonamidates prodrugs displayed broad-spectrum activity against herpesviruses with EC50 values in the low nanomolar range. Compound 8a (cHPMPA diamyl aspartate phosphonoamidate prodrug) (Table3) displayed good activity against HSV, VZV, and HCMV (EC 50 values in the 0.0009−0.027 µM range, being the EC50’s for VZV of ~0.0005 µM) while the (R)-counterpart 8b showed 24- to 180-fold lower potency. The (S)-Phe-cHPMPA (10), (S)-Glu-cHPMPA (13), (S)-Val-cHPMPA (14), (S)-Leu-cHPMPA (15), and (S)-Ile-cHPMPA (16) had also very potent anti-herpesvirus activity, with EC50 values of 0.00045−0.0043 µM for VZV. The structures of compounds 8b, 10, 13, 14 and 15 are not shown here but were reported by Luo and colleagues [116]. Among these prodrugs, compound 16 (Table3) inhibited VZV with EC50’s of 0.00045–0.00054 µM and emerged as the most selective one (selectivity indices’ of 20,000 and 1800, for VZV and HCMV, respectively). All prodrugs showed improved antiviral properties compared to the parent compound, which can be explained by their increased lipophilicity leading to enhanced cellular permeability. Additionally, HPMPA Molecules 2021, 26, 1132 26 of 34

prodrugs displayed more potent activity than the reference anti-herpesvirus drugs and were equally active against wild-type and TK− mutants of HSV and VZV. Furthermore, the leucine ester prodrug of (S)-cHPMPA as well as the phosphonobisamidate valine ester prodrug of (S)-HPMPA were shown to be stable in human plasma.

7.2. Non-Nucleoside Analogues 7.2.1. Pyrazolo[1,5-c]1,3,5-triazin-4-one Derivative A derivative of pyrazolo[1,5-c]1,3,5-triazin-4-one, coded as 35B2 (2-[(2,6-dichlorophenyl) methylthio]-3H-pyrazolo[1,5-c]1,3,5-triazin-4-one) (Table3) was identified from a library of 9600 random compounds as a novel anti-VZV compound screened by using a reporter cell line that produces luciferase upon infection with VZV [117]. The EC50 value for the vOka strain was 0.75 µM and the selective index was more than 200. The compound inhibited neither immediate-early gene expression nor viral DNA synthesis. Viral clones resistant to 35B2 had a mutation(s) in the amino acid sequence of the open reading frame 40 (ORF40), which encodes the major capsid protein, with most of the mutations located in the regions corresponding to the “floor” domain of the major capsid protein of HSV-1. Treatment with 35B2 altered the localization of major capsid protein in vOka-infected fibroblasts but not in fibroblasts infected with the drug-resistant clones. Normal major capsid protein localization in the 35B2-treated infected cells was restored by overexpression of the scaffold proteins. Using electron microscopic analysis, lack of capsid formation could be demonstrated in the 35B2-treated infected cells. These findings indicated a new class of anti-VZV agents targeting the herpesvirus major capsid proteins and inhibiting normal capsid formation.

7.2.2. 5-Chlorobenzo[b]thiophen Derivative From the screening of the 9600 compounds in the search of anti-VZV inhibitors, the compound [(5-chlorobenzo[b]thiophen-3-yl)methyl][(4-chlorophenyl)sulfonyl]amine, coded as 45B5 (Table3), was identiﬁed also as a new anti-VZV agent [ 118]. Its EC50 against VZV vOka in HEL was 16.9 µM and its CC50 was >100 µM. The EC50 values against the parental Oka strain, acyclovir-resistant strains and 35B2-resistant strains were similar to that against the vOka strain. Treatment of cells with 45B5 led to a weak but signiﬁcant decrease of viral DNA synthesis and IE62 expression. Several 45B5-resistant viral clones were isolated and characterized, having all of them at least one mutation in the ORF54 that encodes the portal protein. No effects on interaction between the portal and scaffold proteins were found, suggesting that 45B5 may inhibit nuclear delivery of viral DNA.

7.2.3. Thienylcarboxamide Derivative The screening of 9600 compounds also allowed the identiﬁcation of a thienylcarboxamide derivative, coded as 133G4 [N-(4-chlorophenyl)-5-nitro-3-thienylcarboxamide] (Table3), which was effective not only against VZV (EC 50 = 19.3 µM) but also against HCMV [119]. The compound displayed a selectivity of 27.8 and >35.5 µM against VZV and HCMV, respectively. It was concluded that 133G4 was able to inhibit the activation of early gene promoters by HCMV IE2 and VZV IE62 considering that: (i) in HCMV-infected cells, 133G4 reduced HCMV early and late genes expression but not HCMV immediately early (IE1/IE2) gene expression, (ii) in HCMV-infected cells, 133G4 inhibited the activation of various HCMV early gene promoters of transiently-transfected plasmids, and (iii) in tran- sient transfection assays, the compound reduced activation of HCMV (or VZV) early gene promoters by HCMV IE2 (or VZV IE62) in absence of other viral proteins. The inhibition of early gene activation was cell dependent as it was demonstrated in human and African green monkey cell lines but not in rodent cell lines, being the compound not effective against murine CMV. It was then hypothesized that 133G4 may target a cellular factor used commonly in activation of human herpesvirus promoters, however, the compound did not inhibit the recruitment of some cellular proteins that have been well-characterized as involved in VZV IE62-dependent viral gene activation to their binding sites, nor inhibited the direct interactions of VZV IE62 with cellular proteins. Molecules 2021, 26, 1132 27 of 34

7.2.4. Indole-Based Derivatives A library of indole-based derivatives was designed, synthesized and evaluated for antiviral activity against a broad spectrum of viruses [120]. The N-biphenylethyl acetamide derivative 17a (Table3) displayed signiﬁcant inhibitory activity against VZV replication, including TK+ and TK− VZV strains, pointing to a mechanism of action independent from the virus-encoded thymidine kinase. Compound 17a inhibited VZV plaque formation with EC50’s of 1.7–3.6 µM and a selectivity of 10–20 (ratio MCC/ EC50), being inactive against a variety of RNA and DNA viruses. A structure-activity relationship study showed that the substitution on the tryptamine moiety strongly inﬂuenced the compounds’ activity/toxicity. The concomitant presence of a biphenyl and methyl(phenyl) carboxy group at the amino group of tryptamine was required for anti-VZV activity.

7.2.5. Cephalotaxine Esters The alkaloid esters isolated from the genus Cephalotaxus, harringtonine (HT) and homoharringtonine (HHT) (Table3), exhibit antitumor activity. Indeed, a semisynthetic HHT, omacetaxine mepesuccinate, has been approved by the FDA for treatment of chronic myelogenous leukemia patients resistant or intolerant to tyrosine kinase inhibitors. In addition to their known antitumor activity, HT and HHT possess potent antiviral activity with HT inhibiting chikungunya virus replication through down-regulation of viral protein expression and HHT displaying activity against HBV and coronavirus. HT and HHT, but not their biologically inactive parental alkaloid cephalotaxine, signiﬁcantly hampered VZV replication of the recombinant VZV-pOka luciferase strain and a clinical isolate of VZV in vitro though the anti-VZV activity was lower for the clinical isolate than the reference strain [121]. HT and HHT reduced plaque formation with an estimated EC50 of 16.15 and 9.96 ng/mL, respectively. Their selectivity indices were low considering their CC50 values, i.e., 45.82 ng/mL (HT) and 74.71 ng/mL (HTT). The inhibition of VZV replication by HT and HHT was related to down-regulation of VZV lytic genes. Given that HT and HHT inhibit replication of both RNA and DNA viruses, antiviral activities could be due to effects on cellular rather than viral factor(s) required for viral replication. HT and HHT were suggested to interfere with signal transduction pathways and transcription factors involved in VZV lytic gene expression. Furthermore, HT and HHT were reported to block cell cycle progression at G1/S or G2/M transition and considering that cell cycle regulation affects herpesvirus lytic gene expression and replication, a plausible mechanism to explain the anti-VZV activity of HT and HHT may be through effects on cell cycle in infected cells [120]. This study supports the potential of HT and HHT as candidates for treatment of VZV-associated diseases.

7.3. Hybrid Molecules Dihydropyrimidinone/1,2,3-triazole Hybrid Molecules A feasible strategy for producing drug molecules with potent activity is to combine two bioactive molecules belonging to different therapeutic categories. For instance, 1,2,3- triazole-based heterocycles have been used for the generation of many medicinal scaffolds with inhibitory properties against several viruses, including VZV. The multi-functionalized pyrimidinone scaffold represents a class of heterocyclic compounds with known pharmacological efﬁciency, including antiviral activity. Therefore, the synthesis of novel hybrid molecules can be regarded as a way to increase activity and/or decreased toxicity of the molecules. Novel hybrid compounds have been synthesized by combining the structural features of dihydropyrimidinone and 1,2,3-triazole heterocycles. Some of the tested compounds showed valuable antiviral activities, with EC50 values ranging from 3.6 to 11.3 µM against TK+ and TK− VZV and without measurable cell-growth inhibition. Compound 8a (Table3) emerged as the most active derivative with antiviral activities against VZV at EC50 of 3.62 µM (TK+ strain) and 7.85 µM (TK− strain) and CC50 >100 µM, followed by compound 7d [EC50 of ~10–11 µM and CC50 >100 µM] [122]. None of the tested compounds exhibited measurable antiviral activity against HCMV. These ﬁndings suggest a Molecules 2021, 26, 1132 28 of 34

selectivity of dihydropyrimidinone/1,2,3-triazole hybrid molecules into the herpesviridae family against VZV.

8. Conclusions and Perspectives Despite the availability of a vaccine for the prevention of pediatric varicella in children and for the prevention of HZ in adults, there will continue to be a need for antiviral drugs. Some people in the elderly category are not able to mount a strong response to the vaccine. In immunocompromised persons, including patients with advanced HIV infection, varicella vaccination should be done exclusively with the subunit HZ vaccine as the life- attenuated vaccine is contraindicated for fear of disseminated vaccine-induced disease. On the other hand, the vaccine coverage is still quite limited and childhood immunization with lower coverage could theoretically shift the epidemiology of the disease leading to an increase in the number of severe cases in older children, teenagers and adults. Infections with VZV are a serious cause of morbidity and mortality among immunosuppressed patients and in the elderly as PHN (the major complication of HZ) can be very difficult to manage. Currently available antiviral agents can shorten the duration of HZ and can promote rash healing, though they are not quite effective in preventing PHN, most likely due to the delay between diagnosis and the start of antiviral treatment. New antiviral chemotherapeutics with a different mechanism of action are under development for the management of HZ, including valnivudine hydrochloride (FV-100), prodrug of the bicyclic nucleoside analogue Cf-1743, the helicase-primase inhibitor amenamevir (ASP2151) that got only approval in Japan because of toxicity issues, and the carbocyclic nucleoside analogue valomaciclovir stearate (EPB-348), the omaciclovir prodrug. Although the development of amenamevir has been halted due to problems of toxicity following a trial in United States, the results with valnivudine hydrochloride and valomaciclovir stearate indicated that these drugs are effective, well-tolerated, with once-daily therapy regimen in the treatment of HZ. Further studies are needed to prove an advantage of valnivudine hydrochloride (FV-100) and valomaciclovir stearate over the standard of care valacyclovir. The development of other helicase-primase inhibitors and new drugs with a different target against VZV would be of value to manage ACV-resistant strains. Furthermore, inhibition of multiple targets in the viral replication cycle has the potential for synergistic effects when used in combination therapy, which would be of particular importance for immunocompromised hosts because it may avoid the need for extended therapeutic regimens and the risk of developing antiviral resistance during prolonged monotherapy. A potential anti-VZV drug candidate needs to be at least as effective and safe as the gold standard of treatment for HZ, i.e., valacyclovir. It will need to have also some advantages over valacyclovir, such as longer intracellular half-life allowing once a day dosing, superior efficacy, independence of TK for activation and/or target another enzyme than the DNA polymerase. Antiviral agents will still have a role in the treatment of VZV-associated diseases. A large percentage of the HZ at-risk population does not receive the vaccine. The currently FDA-approved antiviral drugs, including acyclovir, valacyclovir and famciclovir have low efficacy in the control of pain for HZ patients and a significant proportion of these patients (~20–40%) develops PHN. These antivirals require multiple dosing regimens daily that need to be modified for patients with renal failure. Treatment with currently approved antiviral agents is recommended for VZV-associated infections in immunocompromised individuals, where VZV infections can be very severe. Among immunocompetent patients, antiviral therapy is recommended for adolescents and adults suffering from varicella and for patients (especially those older than 50 years) with HZ. Antiviral therapy will also be valuable to treat rare but significant side effects caused by the VZV vaccine. Although development of drug-resistance in considered much less common in VZV than in HSV, emergence of VZV drug-resistance in an emerging concern among immunosuppressed patients who have received prolonged antiviral therapy. Novel antiviral chemotherapeutics with different mode of action than the currently available anti-VZV drugs are required for the treatment of ACV-resistant strains in the immunocompromised host. Furthermore, Molecules 2021, 26, 1132 29 of 34

compounds with a better activity than the currently approved drugs will be extremely useful if they are able to shorten the time to complete crusting of the shingles rash, time to complete pain resolution and prevention of PHN. In the medical literature, cases of coronavirus disease 2019 (COVID-19) and HZ co- infections are being reported. An atypical bilateral acute retinal necrosis was diagnosed in a COVID-19 positive 75-year-old woman, who was immunosuppressed because of chemotherapy for a relapse of diffuse large cell B-cell lymphoma (DLBCL) [123]. Tartari and colleagues reported four cases of HZ in COVID-19 patients over 65 years old, three of them being admitted to ICU, requiring mechanical ventilation, and developing a necrotic HZ on the second branch of the trigeminal nerve [124]. The fourth patient, under immune suppressive therapy because of a cardiac transplant, showed a more indolent COVID-19 disease not requiring hospitalization and developed a classical HZ. Shors reported on a 49-year-old who developed HZ lesions 7 days following the emergence of COVID-19 symptoms and despite receiving valacyclovir 1 g 3× daily within 12 h of the initial eruption, the patient developed severe herpetic neuralgia [125]. A case of an immunocompetent middle-aged man presented with COVID-19 and 2 days later developed HZ. In another report, two cases of HZ reactivation preceding the emergence of respiratory symptoms in COVID-19 patients were demonstrated [126]. Furthermore, HZ may occur in asymptomatic COVID-19 patients, as found in a 39-year-old immunocompetent man who proved COVID- 19 positive but symptoms free and presented HZ with trigeminal neuropathy [127]. These studies suggest an association between COVID-19 and VZV reactivation [128]. Given the nature of VZV reactivation, most commonly linked to stress, advanced age, and impaired immune conditions, more co-infection cases are expected to follow, which will require not only management of COVID-19 but also of VZV disease. To date VZV reactivation in COVID-19 patients has been described mostly in immunocompetent individuals and it has been proposed that the acute COVID-19 disease, associated with physical and emotional stress, might be a trigger factor for the development of HZ. Covid-19-associated lymphopenia, occurring due to direct damage to the thymus and spleen or due to the induced cytokine storm may impair antiviral responses. Furthermore, corticosteroids are being used to manage COVID-19, which may also favor VZV reactivation. Therefore, reactivation of herpesviruses, in particular of VZV, could be an emerging complication of COVID-19. Considering the ongoing COVID-19 pandemic, the CDC recommends vaccination against shingles for individuals at high risk, in particular for patients ≥50 years old. Besides, more potent and selective anti-VZV agents would be important to treat the unusual presentations and complications of HZ in COVID-19 patients.

Author Contributions: Conceptualization, G.A.; writing—original draft preparation, G.A.; writing— review and editing, G.A. and R.S. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Conﬂicts of Interest: The authors declare no conﬂict of interest

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