Preparation of (S)-HPMP derivatives (HPMPC, HPMPA, HPMPDAP, HPMP-5azaC) mostly utilizes the first approach: base catalysed nucleophilic opening of the oxirane ring in (S)-2-(trityloxymethyl)oxirane or (S)-glycidol butyrate with appropriate nucleobase (Figure 1). Thus created 3-O-substituted (S)-2,3-dihydroxypropyl derivatives are then treated with diisopropyl tosyloxymethanephosphonate and finally deprotected. Preparation of diisopropyl tosyloxymethanephosphonate consists in the treatment of diisopropyl phosphite with paraformaldehyde and triethylamine followed by tosylation (Holý, 1993). The alternative way is introduction of phosphonomethyl ether group using diisopropyl bromomethylphosphonate (Göbel et al., 1992; Holý, 2005; Oh and Hong, 2008).

Cidofovir has unique activity against practically all DNA viruses. Intracellularly, it is converted to cidofovir diphosphate which suppresses virus replication selectively by competitive inhibition of viral DNA polymerase. Incorporation of the drug disrupts further chain elongation. Under the brand name Vistide™, cidofovir was approved for the treatment of cytomegalovirus retinitis in AIDS patients (Plosker and Noble, 1999; Drugs.com, 2021). Off label it is used for the treatment of severe cases of papillomatoses (Gi et al., 2012), progressive multifocal leukoencephalopathy (Andrei et al., 2015), adenovirus infections (Neofytos et al., 2007) and poxvirus infections, e.g., recalcitrant molluscum contagiosum and orf in immunocompromised patients (Cundy, 1999; Andrei and Snoeck, 2010). In justified cases, it was exceptionally applied also to immunocompetent pediatric patients with very complicated adeno-, polyoma- or cytomegalovirus infections (Neant et al., 2018; Alcamo et al., 2020).

Application of cidofovir is possible only intravenously and its bioavailability is very low. More than 90% of its intravenous dose is excluded unchanged by kidney. Nephrotoxicity of the cidofovir can be partially overcome by the pre-treatment with the nephroprotective drug probenecid (Lacy et al., 1998; Wolf et al., 2003).

To improve pharmacokinetic properties of cidofovir, various prodrugs are being investigated. So far the most important prodrugs are alkoxyalkyl esters (Kern et al., 2002; Beadle et al., 2002; Ruiz, 2011). They can be prepared by alkylation of N,N,-dicyclohexyl-4-morpholinocarboxamidinium salt of cyclic cidofovir (cHPMPC) with appropriate alkoxyalkyl bromides (Method 1, Figure 2) (Beadle et al., 2002; Kern et al., 2002) or condensation of cHPMPC with alkoxyalcohols under Mitsunobu conditions (Method 2) (Ruiz, 2011). Thus formed esters of cyclic HPMPC are subsequently cleaved to corresponding monoesters by the heating with aqueous sodium hydroxide. The method is applicable also for various other phosphonates (not only in the HPMP series) (Hostetler, 2009). Hexadecyloxypropyl (HDP) ester of cidofovir (Brincidofovir, CMX001) was being developed by Chimerix, Inc. as a drug against cytomegalovirus and adenovirus infections in transplant recipients. Oral brincidofovir was investigated in the Phase III of clinical trials for CMV prophylaxis in stem cell transplants (Marty et al., 2019) but the trials failed due to the toxicity in gastrointestinal tract. Also two other Phase III trials for its use in preventing infection after kidney transplantation and some trials targeted to adenovirus were not successful (Marty et al., 2019; Clinical.trials.gov.; Brincidofovir, 2021). It is speculated that the compound tolerability may be improved by a change in the drug formulation, e.g., by intravenous application. The present plans with brincidofovir are targeted mostly to poxviruses, especially variola virus for the case of a smallpox outbreak. Finally, in June 2021, brincidofovir was approved by FDA as a drug for the treatment of smallpox and is marketed under the brand name Tembexa (FDA, 2021). While the smallpox is eradicated in nature, there is still possibility of variola virus abuse as a biological weapon or its accidental release (FDA, 2021). During Ebola outbreak in 2014 Chimerix also received an FDA approval for investigational applications of brincidofovir for the treatment of Ebola virus disease in patients and approval for the Phase II clinical trials. The trials were subsequently discontinued because of a lack of suitable subjects. Anyway, they highlighted the need to establish better practices for preclinical in vitro and animal screening of therapeutics for potentially emerging epidemic infectious diseases prior to their use in patients (Dunning et al., 2016).

Synthesis of acyclic nucleoside phosphonates of the PME series consists in the base catalysed condensation of a purine or pyrimidine base with a relevant synthetic precursor, i.e., compound containing the whole aliphatic part including a phosphonomethyl arrangement and an appropriate leaving group (mostly tosyl or halogene).

N-2-(Phosphonomethoxy)ethyl derivatives are synthesized from dialkyl (diethyl or diisopropyl) 2-(chloroethoxy)methylphosphonates. These precursors are accessible by the treatment of trialkyl phosphites with 2-(chloroethoxy)methyl chloride (Holý et al., 1999); Holý et al., 1989) The synthesis of PMEA (adefovir) according to this procedure is described in Figure 3. Analogous procedures were applied also to the preparation of the cytostatic guanine derivative PMEG and the series of N⁶-substituted PMEDAP, i.e., 9-[2-(phosphonomethoxy)ethyl]-2,6-diaminopurine including 9-[(2-phosphonomethoxy)ethyl]-N⁶-cyclopropyl-2,6-diaminopurine (cPrPMEDAP, the compound which amidate prodrug is used as a veterinary drug rabacfosadine). In these cases condensation of diisopropyl 2-(chloroethoxymethyl)phosphonate is performed with 2-amino-6-chloropurine and the resulting product was further transformed according to Figure 3 (Holý et al., 2001).

Alternative way to 2-(phosphonomethoxy)ethyl derivatives is the reaction of purine bases with dialkyl 2-hydroxyethylphosphonate under Mitsunobu conditions (Chen et al., 1996). Other methods utilize reaction of dialkyl tosyloxymethylphosphonate with an appropriate 9-(2-hydroxyethyl)purine derivative (Figure 4A) or reaction of an analogous purine 2-iodoethyl derivative with diethyl hydroxymethylphosphonate (Figure 4B on the example of adefovir). The advantage of the latter one is commercial availability of diethyl hydroxymethylphosphonate (Jones et al., 2019).

Adefovir is active against retroviruses and some types of DNA viruses (Naesens et al., 1994; Holý, 2003; De Clercq and Holý, 2005; De Clercq, 2007). Its activity against HIV and hepatitis B virus consists in the inhibitory effect towards reverse transcriptase.

Synthesis of adefovir prodrug (adefovir dipivoxil, Bis(POM)-PMEA) proceeds from adefovir by the action of chloromethyl pivalate (Figure 3) (Starrett et al., 1992).

Adefovir in the form of bis(pivaloyloxymethyl) ester was being developed originally as anti-HIV agent but the trials were finally discontinued due to the nephrotoxicity at the required daily therapeutic dose 120 mg (Kahn et al., 1999). On the other hand, the efficacy for HBV was found higher when the therapeutic effect was achieved with a ten times lower dose of 10 mg (Izzedine et al., 2004; Fontaine et al., 2005). It enabled development of the compound for the treatment of chronic hepatitis B in adults, especially for those with lamivudine resistance. In 2002, adefovir dipivoxil was approved by FDA under the trade name Hepsera^TM. Clinical practice shows that sometimes a higher dose of the drug would have been more efficacious but of course, it is ruled out due to the renal safety (Izzedine et al., 2004; Hézode et al., 2007). In rare cases, it is also reported that prolong treatment with adefovir even in a low dose of 10 mg can cause adefovir-induced osteomalacia, a metabolic bone disease that leads to softening of the bones. It is caused by hypophosphatemia as a result of renal tubular dysfunction (Kim et al., 2013; Park et al., 2018). Clinical use of adefovir dipivoxil started to grow down since the more effective tenofovir disoproxil fumarate and later on, tenofovir alafenamide were approved for HBV (2008 and 2016).

Alternative prodrug approaches for adefovir to improve adefovir pharmacokinetics were also investigated, e.g., bis(S-acyl-2-thioethyl esters (SATE-esters) (Benzaria et al., 1996) or substitutes aryl esters, e.g., bis(o-ethoxyphenyl) ester (Figure 3) (Shaw et al., 1997). The bis(tBu-SATE)PMEA has 50-times increased stability in human plasma compared to bis(POM)-PMEA and improved stability in human gastric fluid (Benzaria et al., 1996).

Extensive research was paid also to cyclosaligenyl (cycloSal) phosphonates, i.e., cyclic esters with variously substituted salicyl alcohol and cycloAmb phosphonates (cyclic ester amidates with 2-aminobenzyl alcohol). Investigation of anti-HIV-1 and anti-HIV-2 activity of various cycloSal-PMEA on cell cultures revealed increased antiviral activity compared to the parent PMEA and in parallel, decreased toxicity and decreased stability compared to other types of prodrugs [e.g., SATE or bis(amidates)]. cycloAmb-derivatives had generally increased stability and lower antiviral activity in comparison with cycloSal-PMEAs (Meier et al., 2005). (Figure 5)

An original prodrug technology are HepDirect^TM prodrugs. They were developed by Ligand Pharmaceuticals, Inc. with the aim to target adefovir directly to the liver due to specific activation by enzymes overexpressed in the liver tissue (Erion et al., 2004; Tillmann, 2007; Reddy et al., 2008). HepDirect prodrugs are cyclic 1-aryl-1,3-propanyl esters which are susceptible to oxidative cleavage of the ring by a cytochrome P450 isozyme (CYP3A4) (Erion et al., 2004). Chemical synthesis consists in condensation of adefovir with 1-(3-chlorophenyl)-1,3-propanediol. While only cis-(2R,4S)-isomer was found as appropriate prodrug, the synthesis includes stereoselective resolution of racemic intermediate 1-(3-chlorophenyl)-1,3-dihydroxypropane through diastereomeric menthone adducts (Figure 6) (Reddy et al., 2008).

Pradefovir was advanced to Phase II of clinical trials where a 12-fold improvement in the liver/kidney ratio over adefovir dipivoxil has been proven. Further investigation in United States was discontinued when the more effective tenofovir disoproxil fumarate became available. Nevertheless, pradefovir is still in clinical development for Hepatitis B in China. Promising results of their Phase 1b study have been recently published (Zhang et al., 2020).

Besides antiviral effects extensive research was targeted to antibacterial activity of PMEA prodrugs. Studies performed with Bis(POM)PMEA revealed that adefovir upon intracellular conversion into the active metabolite adefovir diphosphate has strong activity against adenylate cyclase toxin (ACT) from Bordetella pertussis, a causal agent of whooping cough, and both ACT and edema factor (EF) from Bacillus anthracis. Various symmetrical amino acid ester based bis(amidates) of PMEA have been developed in our Institute as less toxic and more stable alternatives to bis(POM)PMEA (Česnek et al., 2015; Břehová et al., 2016; Šmídková et al., 2014). The highest activity and optimal pharmacokinetic profile was achieved with bis(isopropyl phenylalanine) amidate promoiety. Besides, also various base modified PMEA analogues (aza/deaza purine derivatives) were synthesized. One of the most promising ACT inhibitors was found 8-aza-7-deazapurine analogue of PMEA in the form of bis(isopropyl phenylalanine) amidate with IC₅₀ value16 nM and substantial selectivity over mammalian adenylate cyclases (Česnek et al., 2018). Synthesis of symmetrical bis(amidates) can be performed from phosphonate diisopropyl esters which are deprotected first with bromotrimethylsilane, followed by condensation with appropriate amino acid ester in the presence of 2,2′-dithiopyridine (Aldrithiol). The reaction scheme is outlined in Figure 7. Extraordinary effect to ACT inhibitory potency has replacement of the adenine moiety with another heterocyclic base able to mimic adenine, namely 2-aminothiazole. These compounds are 5-aryl-4-PME-2-aminothiazoles, 4-aryl-5-PME-2-aminothiazoles and their bis(amidate)prodrugs and phosphono diphosphate analogues (Břehová et al., 2021; Česnek at al., 2022). The most potent inhibitor was diphosphate of 4-(4-(benzylcarbamoyl)phenyl-5-PME-2-aminothiazole (Figure 7). It is the most potent ANP-based inhibitor of ACT (IC₅₀ = 9 nM) and EF (IC₅₀ = 11.6 nM) known to date.

Several years ago, a considerable attention started to be paid to cytostatic 9-[2-(phosphonomethoxy)ethyl] derivatives derived from guanine, 2,6-diaminopurine and N⁶-substituted 2,6-diaminopurine (Figure 3). These compounds work via perturbing DNA replication by terminating the growing DNA chain and suppressing the cell growth (at low concentrations) while at higher concentrations they induce apoptotic activity. In cells, they are phosphorylated to diphosphates as active metabolites inhibiting the cell growth due to a potent inhibition of nuclear DNA polymerases. It results in inhibition of DNA synthesis and/or DNA repair (Kramata et al., 1996; Pisarev et al., 1997; Kramata et al., 1998). The most potent compound is PMEG, 9-[(2-phosphonomethoxy)ethyl]guanine; the activities generally increase in the order PMEA < PMEDAP < PMEG. Antitumor effects of PMEG was proven also in animal models in a Sprague–Dawley rat experimental model for T-cell lymphoblastic leukemia/lymphoma (Rose et al., 1990; Holý, 2003) but the utility of PMEG for clinical practice is very limited by its poor cellular permeability and toxicity (Kreider et al., 1990; Rose et al., 1990). A fundamental breakthrough was made by the N⁶-substitution of the 6-amino group in 2,6-diamino-9-[2-(phosphonomethoxy)ethyl]purine (PMEDAP) leading to numerous compounds with very promising activity (Holý et al., 2001). Finally, the cyclopropyl derivative, cPrPMEDAP was selected as the lead compound. Its antiproliferative effects were similar to PMEG in vitro, however, in vivo, toxicity of cPrPMEDAP was substantially reduced. In cell cultures, cPrPMEDAP is deaminated to PMEG and then converted to the PMEG-diphosphate—it is evident that cPrPMEDAP acts as an intracellular prodrug of PMEG (Compton et al., 1999; Hatse et al., 1999). The enzyme capable of this conversion was identified as N⁶-methyl-AMP/dAMP aminohydrolase (Schinkmanová et al., 2006; Schinkmanová et al., 2008).

Nevertheless, cPrPMEDAP, similarly as other ANPs, has a low cellular permeability making its direct clinical use practically impossible and transformation to an appropriate prodrug was necessary. Development of amino acid amidate prodrugs resulted finally in the development of compound GS-9219, the ethyl alaninate prodrug of cPrPMEDAP and concurrently a double prodrug of PMEG. The compound was designed as a cytotoxic agent preferentially targeting lymphoid cells. The compound is prepared from cPrPMEDAP by the action of L-alanine ethyl ester hydrochloride after activation of phosphonic acid residue with Aldrithiol and triphenylphosphine (Cheng et al., 2005; Jansa et al., 2011). The scheme of metabolic conversion of GS-9219 to PMEGpp is outlined in Figure 8. Preclinical trials with dogs with spontaneous non-Hodgkin’s lymphoma proved GS-9219 as generally well tolerated drug with significant antitumor activity warranting its further study in humans (Reiser et al., 2008).

Clinical trials of GS-9219 (Phase I/II) were started in July 2007. The compound was tested in adult patients with non-Hodgkin’s lymphoma, chronic lymphocytic leukemia and multiple myeloma in twenty leading experimental clinics in United States, Russia and Czech Republic. Despite expectations, 3 years later, in October 2010, the study on GS-9219 has been terminated due to the unacceptable safety profile of the compound (Clinical.trials.gov. GS-9219, 2014). Later on, it was licensed to the veterinary drug company VetDC where the compound was being developed as a veterinary drug against canine lymphoma (VDC-1101, rabacfosadine). In April 2017, it has been approved by FDA as a veterinary drug. The drug is currently marketed by Elanco Animal health under the trade name Tanovea^TM (Elanco, 2021). The whole story has been recently reviewed by De Clercq (De Clercq, 2018).

Syntheses of enantiomeric N-[2-(phosphonomethoxy)propyl derivatives are based on the condensation of a nucleobase with the appropriate chiral building block. The starting compound for the synthesis of (R)-PMPA (tenofovir) is (R)-2-[bis(2-propyl)phosphonomethoxy]propyl]-p-toluenesulfonate, i.e., “(R)-PMP building block”, the compound prepared easily from (R)-1-benzyloxy-2-propanol by multi-step process involving chloromethylation, Arbuzov reaction with triisopropyl phosphite, catalytic hydrogenation and final tosylation (Holý et al., 1995). Condensation of adenine with this precursor followed by deprotection of ester groups gives (R)-9-[(2-phosphonomethoxy)propyl]adenine [(R)- PMPA, tenofovir]. For clinical utilization this compound is transformed to the neutral prodrug, bis(isopropoxycarbonyloxymethyl) ester [tenofovir disoproxil, Bis(POC)- (R)-PMPA] and marketed in the form of fumarate salt under the trade name Viread^TM. The prodrug is synthesized from (R)-PMPA and chloromethyl isopropyl carbonate (Arimilli et al., 1997; Arimilli et al., 1999). The overall scheme of the synthesis of tenofovir and its bis(POC) prodrug is outlined in Figure 9.

Tenofovir is a typical antiretroviral agent acting as a nucleotide reverse transcriptase inhibitor (Srinivas and Fridland, 1998; Suo and Johnson, 1998; Cihlar et al., 2002). In 2001, its bis(isopropoxycarbonyloxymethyl) ester (tenofovir disoproxil fumarate, TDF) was approved for the treatment of HIV infections as one-component drug Viread^TM (Srinivas and Fridland, 1998; Lyseng-Williamson, 2005). Current trend in anti-HIV therapy is a combined antiretroviral therapy (cART) consisting in several anti-HIV drugs with different mechanism of action in all-in-one pill. Marketed combinations of TDF are: Truvada (TDF + emtricitabine), Atripla (TDF + emtricitabine + efavirenz), Complera (emtricitabine + rilpivirine) and Stribild (TDF + elvitegravir + cobicistat + emtricitabine) (Gilead, 2022; Deeks and Perry, 2010; De Clercq, E., 2019).

Tenofovir is the most effective, safe and best selling anti-HIV drug. Its special advantages are preventive effects in mother-to-child HIV transmission and relative safety for pregnant women and pediatric patients (Nachega, et al., 2017; Gibb et al., 2012; Siberry et al., 2012; Wang et al., 2013). In 2012, FDA approved Truvada (emtricitabine/tenofovir disoproxil fumarate) as the first drug effective to reduce the risk of HIV transmission to uninfected individuals. Extensive investigations are also carried out in the field of preventive effects of tenofovir containing microbicides. Several clinical studies including daily use of tenofovir vaginal gel or its combination with oral tenofovir are under way in several African countries. Despite an initial enthusiasm from the results of the Caprisa 004 study published in 2010, the following studies (VOICE and FACTS 001) failed due to a failure of the human factor: An adherence in all aspects of the study was too low to confirm effectiveness. The same result was obtained from the FEM-PrEP clinical trial designed to assess prevention of HIV infection with a daily dose of one pill of Truvada (Van Damme et al., 2012; Marrazzo et al., 2015).

Tenofovir has been also investigated as anti-hepatitis B agent and in 2008 approved by FDA for the treatment of chronic hepatitis B in adults and pediatric patients 12 years of age and older. Tenofovir disoproxil is more efficacious than previously approved adefovir dipivoxil, and so far there is no reported resistance (Yang et al., 2015; Tillmann and Samuel et al., 2019; de Fraga et al., 2020). Both ANPs (tenofovir and adefovir) act as chain terminators when their metabolites get incorporated into the viral DNA strands while they undergo replication by polymerases or reverse transcriptase. However, recent discoveries showed also an additional immunomodulatory mechanism. Both ANP drugs, (but not nucleoside analogues), induce interferon (IFN)-λ3 in the gastrointestinal tract. Pretreatment of peripheral blood mononuclear cells from HBV patients with these ANPs inhibited LPS-mediated interleukin (IL)-10 production (Murata et al., 2020).

The new prodrug form of tenofovir developed by Gilead Sciences is tenofovir alafenamide (TAF) (De Clercq, 2016; Lee et al., 2005; Birkus et al., 2017). It is preferentially taken up by the lymphatic tissue and, also by liver cells (Birkus et al., 2017). The compound is a typical representative of “ProTides” - aryloxy amino acid phosphoramidates, the technology developed originally by C. McGuigan. “ProTides” are widely used due to optimal pharmacokinetic properties and relatively easy synthesis (Pradere et al., 2014; Heidel and Dowd, 2019). Synthesis of TAF has been described in many patents, including variations of experimental conditions and separation of diastereoisomers (GS-7340 and GS-7339, Figure 10) (Chapman et al., 2001; Ramanathan, 2013). The large-scale synthesis and process for separation of diastereomers by simulated moving bed chromatography (SMBC) is described in ref. (Chapman et al., 2001). The desired isomer was found GS-7340 with anti-HIV-1 activity data about ten-fold higher than GS-7339. For clinical application, the compound is used in the form of hemifumarate (Liu, 2013). For HBV therapy, it is sold under the brand name Vemlidy. At present, TAF has largely replaced TDF in HIV treatments, primarily due to the significant difference in dosage—only 30 vs. 300 mgs, with lower incidence of adverse side effects as well as greatly increased levels of tenofovir inside the virally infected cells (De Clercq, 2016; Seley-Radtke and Yates, 2018; De Clercq, 2019). TAF is also used in various combinations with other antiretroviral drugs (Genvoya, Odefsey, Descovy, Symtuza).

During more than 30 years of ANP research at the IOCB, hundreds of structures with excellent biological activities have been synthesized. An integral part of this story was always a personality of Erik De Clercq, Antonin Holy´s friend and collaborator leading all antiviral investigations and also author of many ANP reviews (De Clercq, 2009; De Clercq, 2011; De Clercq, 2013; De Clercq, 2019). Promising structures deserving further investigation are discussed below (Figure 11).

a) FPMP derivatives, i.e., (3-Fluoro-2-phosphonomethoxy)propyl derivatives.

This group of purine ANPs inhibits selectively retroviruses (HIV-1, HIV-2) and also HBV, with no effect towards other DNA viruses. The adenine derivative, (S)-FPMPA has better parameters in vivo compared to PMEA (adefovir) (Balzarini et al., 1991; Jindřich et al., 1993). Anti-HIV activity was also found in both enantiomers of FPMPDAP and FPMPG and some N⁶-substituted derivatives of (R)-FPMPDAP (cyclopropyl, propyl, allyl) (Baszczyňski and Janeba, 2013).

In the pyrimidine series, no antiviral activity was found but both enantiomers of the thymine derivative 1-[3-fluoro-2-phosphonomethoxy)propyl]thymine (FPMPT) have inhibitory activity towards thymidine phosphorylase, the enzyme playing the key role in the angiogenesis in tumors (Folkman and Shing, 1992; Esteban-Gamboa et al., 2000; Pomeisl et al., 2005). A study carried out on human purine nucleoside phosphorylase (PNP) revealed that the monophosphates derived from both enantiomers of FPMPG are its potent inhibitors. PNP is a purine salvage pathway enzyme catalysing the phosphorolysis of guanosine, inosine and 2′-deoxyguanosine to the corresponding purine base and ribose-1-phosphate or 2′-deoxyribose-1-phosphate, respectively. Pharmacological aspect of PNP inhibition is connected with the treatment of human T-cell proliferative disorders (Votruba et al., 2010)¹⁰¹.

b) (R)-PMP-derivatives, i.e., (R)-2-(Phosphonomethoxy)propyl derivatives.

The most active antiretroviral compound of this series, 2,6-diaminopurine analogue of tenofovir, (R)-PMPDAP, has not been thus far sufficiently studied, despite the fact that it is 10-fold more potent against HIV-1 compared to tenofovir in vitro and in vivo (Balzarini et al., 1993; Balzarini et al., 1996). It is also strongly active against animal retroviruses, especially feline immunodeficiency virus. Although strong conclusions cannot still be done, some studies revealed slightly positive (Vahlenkamp et al., 1995; Taffin et al., 2015). It is clear that the activity of (R)-PMPDAP in vivo warrants further investigations including synthesis of prodrugs (Krečmerová et al., 2013).

c) “Open-ring” analogues are ANPs having PME-, PMP or HPMP-grouping attached to the position 6 of the 2,4-diaminopyrimidine ring via oxygen atom. They mimic 2,6-diaminopurine ANPs with an open imidazole ring. Their antiviral activity is essentially identical to that of their parent compounds, including the enantiomeric specificity. It means that (R)-HPMPO-DAPy is a typical anti-DNA virus agent (similarly as cidofovir and other HPMP derivatives) whereas PMEO-DAPy, (R)-PMPO-DAPy a 5-substituted PMEO-DAPy inhibit retroviruses and HBV (Balzarini et al., 2002; Holý et al., 2002; Hocková et al., 2003; De Clercq et al., 2005; Herman et al., 2010). The most effective antiretroviral compounds are PMEO-5-methyl-DAPy and other 5-substituted PMEO-DAPy derivatives (Hocková et al., 2003). Compound PMEO-DAPy is active not only against retroviruses but also against many DNA viruses, especially herpesviruses affecting immunocompromised patients including those with HIV/AIDS. For this purpose we studied PMEO-DAPy also in the form of various structural types of prodrugs (Krečmerová et al., 2017).

1-(S)-[3-hydroxy-2-(phosphonomethoxy)propyl]-5-azacytosine (HPMP-5-azaC) has been developed as less toxic and more effective alternative to cidofovir. It shows potent and selective activity against all DNA viruses. The activity is comparable to cidofovir concerning herpes viruses (HSV-1, HSV-2) and vaccinia virus, and 2 to 7 times more active against varicella zoster virus (VZV), human cytomegalovirus (HCMV), human herpesvirus type 6 (HHV-6) and adenovirus (Ad2), with generally lower cytotoxicity (Krečmerová et al., 2007a). The prodrug form, hexadecyloxyethyl ester of cyclic HPMP-5-azaC revealed extremely high values of anti-DNA virus activities including imposing selectivity indices on the order of thousands, e.g., 1,160 for herpes simplex virus (HSV-1), ≥5,800 for varicella zoster virus (VZV) or ≥24,600 for human cytomegalovirus (HCMV) (Krečmerová et al., 2007b). Unfortunately, HPMP-5-azaC has rather complicated metabolic profile due to instability of the 5-azacytosine ring and the compound was finally not advanced to clinical investigations (Dračínský et al., 2008; Naesens et al., 2008).

e) HPMPA, HPMPDAP and aza/deaza purine base analogues

(S)-Enantiomers of HPMPA, HPMPDAP and 3-deazaHPMPA are compounds strongly active against DNA viruses, especially herpesviruses but their therapeutic possibilities are so far underexplored. Some of them, e.g., HPMPDAP could be useful for veterinary medicine for their effects towards African swine fever virus (Gil-Fernandez et al., 1987; Holý, 2003). First experiments with HPMPDAP performed in mice were successful. Unfortunately, application of free HPMPDAP to pigs caused serious side effects—the compound was strongly toxic. More research concerning dosing and formulation must be done but it is clear that future investigations need to be performed with prodrug forms (Roels et al., 2013).

Special effort has been also paid to investigation of (S)-HPMPDAP and its prodrugs as anti-pox virus agents. The collaborative project of our institute with Rega Institute and Gilead Sciences had to find a potential drug candidate against poxviruses for the purpose of bioterrorist attack with variola virus (Krečmerová et al., 2010). Large series of cyclic and acyclic HPMPDAP esters were prepared: alkoxyalkyl, POM, 2,2,2-(trifluoro)ethyl, butylsalicylyl, and prodrugs based on peptidomimetics. The most potent prodrugs in vitro (tested on vaccinia virus) were the alkoxyalkyl ester derivatives with 50% effective concentrations 400- to 600-fold lower than those of the parent compound. Nevertheless, further in vivo experiments selected finally as the best candidate the acyclic POM ester (Figure 12).

Compounds deserving further investigation are also ANPs active against herpesviruses so far lacking any treatment (e.g., human herpesvirus type 6, HHV-6). HHV-6 takes part in pathogenesis of exanthema subitum (roseola), lymphoproliferative diseases, chronic fatigue syndrome and it is known also a co-factor of AIDS. It causes severe complications (e.g., encephalitis) in immunocompromised patients. Testing of ANPs for activity against HHV-6 selected 3-deaza-HPMPA and its cyclic form as compounds with the highest activity and selectivity (Reymen et al., 1995). In the series of 8-azapurine analogues, PME-8-azaguanine and (R)-PMP-8-azaguanine are compounds with interesting antiretroviral potency (Holý et al., 1996).

New ANP structures modified in the aliphatic side chain and/or in the base moiety are currently being developed in many laboratories. The successful example is besifovir, 9-[2-cyclopropyl-2-(phosphonomethoxy)ethyl]-2-aminopurine. Its prodrug form, besifovir disoproxil is investigated for the HBV treatment in the Phase III of clinical trials (Jung et al., 2020; Kim et al., 2020).

Replacement of a natural nucleobase with another heterocycle has been already mentioned in antibacterial PME-2-aminothiazoles (chapter 1.1.5). Another example is a series of HPMP and PME derivatives bearing a 1,2,4-thiadiazole base moiety which was intended as a cytosine mimic. In contrast to HPMPC, none of these compounds has antiviral activity but they were found potent inhibitors of cysteine dependent enzymes, such as Cathepsine K and glycogen synthase kinase 3β (Pomeislová et al., 2021).

Many side-chain modified ANPs are investigated as potent antiparasitic agents (see chapter 1.2.4.)

Prodrugs are pharmacologically inactive compounds which are transformed in vivo to active drugs via metabolic and/or chemical processes occurring in the body. They are developed to optimize pharmacological properties of parent drugs. It is estimated that around 10% of all marketed drugs are prodrugs and their share in the new drug development is continuously increasing (Najjar and Karaman, 2019).

Prodrugs developed for acyclic nucleoside phosphonates can be categorized to the following groups (Pradere et al., 2014; Heidel and Dowd, 2019):

- Symmetrical phosphonate diesters—Alkyl, aryl, acyloxyalkyl (pivaloyloxymethyl, POM), alkoxycarbonyloxyalkyl (isopropoxycarbonyloxymethyl, POC), S-acylthioalkyl (SATE) (Benzaria et al., 1991).

Besides improving antiviral activity, cellular uptake and toxicity profile, some types of ANP prodrugs are also reported with respect to influence the antiviral activity spectrum of parent compounds. In the HPMP series, it concerns octadecyloxyethyl (ODE) monoesters derived of HPMPA. (S)-HPMPA is active exclusively against most double-stranded DNA viruses but has no in vitro effect against RNA viruses and retroviruses (Holý, 2003; De Clercq and Holý, 2005). Its opposite enantiomer, (R)-HPMPA is completely inactive against all viruses. Despite these facts, HDP and ODE monoesters of (S)-HPMPA were reported as active compounds against HCV (positive sense single-stranded RNA virus). Interestingly, even a prodrug derived from R-enantiomer was active against HCV (Wyles et al., 2009). Moreover, alkoxyalkyl esters of (S)-HPMPA and its 3-O-methyl derivative, (S)-MPMPA are potent inhibitors of HIV-1 replication (EC₅₀ value of 7 nM for HDP ester of (S)-HPMPA) (Hostetler et al., 2006; Valiaeva et al., 2006). As expected, remarkable increase in antiviral activity against DNA viruses (HSV-1, HCMV, vaccinia, cowpox and ectromelia virus) was observed in ODE esters of (S)-HPMPA, (S)-HPMPG, (S)-HPMPDAP and its 6-cyclopropylamino analogue (S)-HPMP-cPrDAP. The two most active compounds against HSV-1 were ODE–(S)-HPMPA and ODE–(S)-HPMPC with subnanomolar EC₅₀ values in cell cultures (Valiaeva et al., 2009).

Another example of influencing antiviral activity spectrum via synthesis of prodrugs are both enantiomers of 3-fluoro-2-(phosphonomethoxy)propyl]adenine (FPMPA) and other FPMP derivatives. These ANPs are effective antihuman immunodeficiency virus (HIV) agents, but have no activity against a wide range of DNA viruses. The introduction of a diamyl aspartate amidate motif together with a phenyl ester moiety at the phosphorus (de facto a special kind of proTides) not only enhanced antiviral potency against HIV (by a factor up to 1,500), but also against HBV. Interestingly, some of the synthesized compounds exhibited activity against DNA viruses, namely herpes viruses (Luo et al., 2017).

Despite of the huge number of prepared structures and promising in vitro or in vivo studies, only a few of them have been proceeded into clinical trials or clinics (POM, POC, HDP, proTides). It can be attributed to the difficulty of achievement of the right prodrug properties balance, particularly suitable chemical stability at physiological pH, rate of conversion, safety with respect to formed by-products, avoidance of stereoisomers, physical properties (ability to crystallize, solubility), and ability to synthesize substantial quantities. Therefore, search for new structural types of prodrug promoiety is highly desirable.

One such promising strategy is the concept of amino acid prodrugs (Krečmerová, 2017). Single amino acid ester prodrugs were reported in Herdewijn´s group for (S)-HPMPA and its cyclic form (Luo et al., 2018).

Extensive research is paid to prodrugs where the phosphonic acid residue is esterified by a hydroxyl group of hydroxyl amino acids (serine, threonine, tyrosine) which can be used either as single amino acid or as a component of small peptides (dipeptides and tripeptides). To circumvent the problem of the low permeation of peptides through the cell membranes and enzymatic instability in the gastrointestinal tract, structural modifications to form peptidomimetics are the solution. These modifications consist in esterification of a carboxyl group or its transformation. This approach enables wide spectrum of “fine tuning” of pharmacokinetic properties. Additionally, introduction of D-configurated N-terminal amino acids to the dipeptide increases enzymatic stability of the prodrug and its uptake to plasma (Eriksson et al., 2008; Peterson et al., 2011). Extensive research finally showed the best pharmacokinetic profiles in single amino acid prodrugs, especially tyrosine derivatives (Krylov et al., 2013; Zakharova et al., 2011; Williams et al., 2011). Synthesis and structures of peptidomimetic and tyrosine based ANP prodrugs are depicted in Figure 13 on the example of cidofovir. Analogous prodrugs were prepared also from (S)-HPMPA (Zakharova et al., 2011). They have excellent antiviral activity against DNA viruses (HCMV, poxviruses, HSV-1) without any cytotoxicity. Activity data of the cyclic forms are not dependent on stereochemistry at the phosphorus atom (EC₅₀ for R_{P ≈}S_P). Oral bioavailability in mice was 8-10x higher than that of parent phosphonates (39% vs. <5%) while the tyrosine alkylamide esters had better stability than carboxylate ester derivatives (Zakharova et al., 2011; Krylov et al., 2013).

Significant results have been recently published using tyrosine in synthesis of ANP proTides whose phenyl group is replaced with a modified (S)-tyrosine moiety (Figure 13). This approach applied to tenofovir leads to compounds with substantially increased potency and selectivity index towards HIV as well as HBV compared to tenofovir alafenamide. It is caused by their high cellular uptake and rapid cleavage to the parent drug tenofovir in the target cells (Kalčic et al., 2021).

Various purine ANPs have remarkable antiprotozoal activity. The therapeutic potential for the treatment of African trypanosomiasis was identified originally in (S)-HPMPA and (S)-HPMPDAP. The compounds revealed inhibitory effects against Trypanosoma brucei brucei both in vitro and in vivo while inhibitory activity against Trypanosoma congolense was found in PMEDAP (Kaminsky et al., 1994; Kaminsky et al., 1996). Recent studies selected as the best inhibitors 6-oxopurine ANPs further modified in the side chain (branched nitrogen containing derivatives, bisphosphonates and C-1′-branched ANPs) (Doleželová et al., 2018; Teran, 2020; Doležalová et al., 2021; Kalčic et al., 2021).

Extensive research is currently paid to ANPs as antimalarial agents. The parasitic 6-oxopurine phosphoribosyltransferases (PRTase) have been shown to be drug targets to inhibit malarial parasites (Plasmodium falciparum, Plasmodium vivax). 6-Oxopurine PRTases are present in all organisms. For a drug candidate, high selectivity towards parasitic enzyme PfHGPRT (and low or no inhibition of the human HGPRT) is always required (De Jersey et al., 2011; Keough, et al., 2013a).

Effective inhibitors of Plasmodium HGXPRT are guanine and hypoxanthine ANPs with the following arrangements of the side chain:

a) 2-(Phosphonoethoxy)ethyl derivatives (PEE derivatives: PEEG, PEEHx) (De Jersey et al., 2011; Keough, et al., 2013a).

Besides above described guanine and hypoxanthine ANP inhibitors, there are also highly effective and selective “ANP-like“inhibitors with unnatural pyrrolidine base moiety (Keough et al., 2018). Purine metabolism was also identified as a ubiquitous factor in the physiology of various other pathogens including Mycobacterium tuberculosis, Mycobacterium smegmatis and other mycobacteria. The purine salvage enzyme HGPRT was found essential for Mycobacterium tuberculosis growth in vitro; however, its precise role in M. tuberculosis physiology is so far unclear. Membrane permeable prodrugs of HGPRT inhibitors arrest the growth of M. tuberculosis and represent potential new antituberculosis compounds (Knejzlík et al., 2020).