HHS Public Access
`Author manuscript
`Expert Opin Drug Deliv. Author manuscript; available in PMC 2016 November 21.
`Published in final edited form as:
`Expert Opin Drug Deliv. 2009 April ; 6(4): 405–420. doi:10.1517/17425240902824808.
`
`Page 1
`
`Prodrug approaches to improving the oral absorption of antiviral
`nucleotide analogues
`
`Larryn W Peterson1 and Charles E McKenna†,2
`1PhD candidate, University of Southern California, Department of Chemistry, Los Angeles, CA
`90089-0744, USA
`2Professor of Chemistry, University of Southern California, Department of Chemistry, Los
`Angeles, CA 90089-0744, USA
`
`Abstract
`Nucleotide analogues have been well accepted as therapeutic agents active against a number of
`viruses. However, their use as antiviral agents is limited by the need for phosphorylation by
`endogenous enzymes, and if the analogue is orally administered, by low bioavailability due to the
`presence of an ionizable diacid group. To circumvent these limitations, a number of prodrug
`approaches have been proposed. The ideal prodrug achieves delivery of a parent drug by
`attachment of a non-toxic moiety that is stable during transport and delivery, but is readily cleaved
`to release the parent drug once at the target. Here, a brief overview of several promising prodrug
`strategies currently under development is given.
`
`Keywords
`antiviral agents; oral bioavailability; prodrugs; pronucleotides
`
`1. Introduction
`Of the approximately 40 antiviral drugs formally licensed for use, half are nucleoside or
`nucleotide analogues [1]. Nucleoside drugs per se must usually be phosphorylated to the 5(cid:5321)-
`mono-, 5(cid:5321)-di-, and finally, 5(cid:5321)-triphosphate by intracellular or viral kinases [2] in order to
`inhibit their therapeutic targets. This requirement limits efficacy, as phosphorylation to the
`monophosphate by endogenous kinases is slow and typically is the rate-limiting step in
`human cells [3,4].
`
`The administration of a nucleoside drug as its monophosphate (NMP) is a well-known
`approach to overcoming this obstacle [3,5]. However, this entails a penalty in the form of
`decreased membrane permeability. Nucleotide analogues contain an ionizable –O-P(O)
`(OH)2 group that exists chiefly as a dianion at physiological pH, resulting in low oral
`bioavailability [5]. In addition, if a NMP succeeds in crossing the intestinal membrane, it
`
`†Author for correspondence: Tel: +1 213 740 7007; Fax: +1 213 740 0930; mckenna@usc.edu.
`Declaration of interest
`LP and CM are co-inventors on a patent related to a portion of the work discussed in this review.
`
`Gilead 2014
`I-MAK v. Gilead
`IPR2018-00125
`
`Author Manuscript
`
`Author Manuscript
`
`Author Manuscript
`
`Author Manuscript
`
`

`

`Peterson and McKenna
`
`Page 2
`
`then becomes a potential substrate for phosphohydrolases (phosphatases and 5(cid:5321)-
`nucleotidases), which remove the phosphate group [6]. The use of a nucleoside phosphonate
`–CH2-P(O)(OH)2 circumvents dephosphorylation, but decreased transport remains an
`obstacle.
`
`Formulation strategies [7–11] to overcome these limitations are beyond the scope of this
`short review, which has as its focus an alternative approach: prodrug modification of
`nucleotide drugs. Promoieties can be attached at a number of positions on an NMP or
`nucleotide analogue [12,13]. However, the introduction of promoieties at the phosphorus (–
`[O,CH2]-P(O)(X)(Y) where X,Y = OR, OR(cid:5321), NHR(cid:5322)) directly addresses the problem of
`blocking P-OH ionization in vivo. The attachment of a well-designed promoiety increases
`delivery of the drug to its target, provided that its biochemical and physical properties –
`including lipophilicity, site-specificity and chemical stability – are conducive to this end
`[5,13,14].
`
`A prodrug must be stable under delivery conditions [3,5], but it must be capable of
`conversion to its active parent drug in vivo [5], at a rate consistent with pharmacological
`efficacy. The prodrug and metabolized promoiety/promoieties should have low acute and
`chronic toxicity [5]. Control of these and other crucial properties, such as aqueous solubility
`and lipophilicity, remains a key challenge in the development of an effective prodrug.
`
`Esterification with pivaloyloxymethyl (POM), p-acyloxybenzyl (PAOB), or isopropyloxy–
`carbonyloxymethyl (POC) groups has been reviewed extensively [3,5,6,13,15,16] and will
`not be addressed here. Also, of recent interest, but omitted from this discussion is the
`approach of Hostetler et al. to improve the oral bioavailability of certain antiviral
`phosphonate drugs by esterification with an ether lipid ester that mimics the natural lipid
`lysophosphatidylcholine, thus potentially delivering the prodrug within the cell intact
`[4,17,18]. Our review will examine the prodrug approaches represented by the structures in
`Figure 1.
`
`2. Phosphoramidate ‘ProTide’ approach
`McGuigan has introduced prodrugs (‘ProTides’) based on an amino acid ester promoiety,
`attached to the drug (as a aryl monophosphate or phosphonate) via a P-N bond, applying this
`approach to: 4(cid:5321)-azidouridine [19], 4(cid:5321)-azidoadenosine [20], 2(cid:5321),3(cid:5321)-dideoxyuridine (ddU) [21],
`carbocyclic L-d4A (L-Cd4A) [22], stauvidine (d4T) [23], 9-[2-(phosphonomethoxy)
`ethyl]adenine (PMEA) [24], 3(cid:5321)-azidothymidine (AZT) [25], abacavir (ABC) [26] and
`tenofovir (PMPA, 9-[(R)-2-(phosphonomethoxy)propyl]adenine) [27].
`
`The original approach involved preparation of simple alkyloxy phosphoramidates (Figure
`2A(1)), but has evolved into aryloxy phosphoramide pronucleotides with distinct structure–
`activity relationship detail [28]. Phosphorodiamidates (Figure 2A(2)) were also prepared, but
`no biological benefit versus the phosphoramidates was observed and synthetic yields were
`lower [28]. Interestingly, analogues linked through an oxygen resulted in a significant
`decrease in antiviral activity [29], possibly because the nucleoside monophosphate was not
`released from the diester intermediate [28]. Diaryl pronucleotides (Figure 2A(3)) were not
`
`Expert Opin Drug Deliv. Author manuscript; available in PMC 2016 November 21.
`
`Author Manuscript
`
`Author Manuscript
`
`Author Manuscript
`
`Author Manuscript
`
`

`

`Peterson and McKenna
`
`Page 3
`
`active in kinase-deficient cells [30], due to poor intracellular delivery of the NMP or
`possibly chemical instability of the diaryl masking groups. Overall, aryloxy
`phosphoramidates (Figure 2A(4)) appear to hold the most promise for delivery of the
`therapeutic agent.
`
`Aryloxy phosphoramidates were designed to release the NMP intracellularly via both
`chemical and enzymatic mechanisms (Figure 2B). The first step in the activation process is
`cleavage of the amino acid ester by a carboxyesterase [28] to afford 6 (Figure 2B), although
`a thorough investigation by Venkatachalam and coworkers found that activation by a lipase
`or protease is also possible and that these enzymes have different specificities for the
`substituent on the aryl group, the amino acid and the stereochemistry at the phosphorus [31].
`Subsequent nucleophilic attack at the phosphorus by the carboxyl group releases the aryloxy
`group, forming a transient cyclic diester, which is hydrolyzed to form the amino acyl
`metabolite (AAM, Figure 2B(7)) [28]. In the final step, the amino acid moiety is cleaved by
`a phosphoramidase to release the nucleoside monophosphate (Figure 2B(8)) and an amino
`acid [28].
`
`McGuigan and coworkers have thoroughly studied the aryloxy phosphoramidates of d4T and
`have been able to gain extensive structure–activity relationship insight. In general, the
`methyl, ethyl and benzyl esters lead to potent activity, while bulkier esters (t-butyl and
`isopropyl) are significantly less active than the methyl ester [32], most likely due to the
`increased stability to enzymatic hydrolysis [20]. A quantitative structure–activity
`relationship (QSAR) study on the variation of amino acid esters further described the most
`potent esters as those with considerable lipophilicity slightly removed from the ester bond
`[33]. In a separate report, it was found that the conversion of AAM to NMP was inhibited
`when benzyl alcohol was released [34]. This inhibition was not observed when ethanol or
`methanol was released [34].
`
`Although there are some exceptions depending on the nature of the drug used, the most
`successful pronucleotides contain L-alanine as the amino acid [34,35]. Exchange of L-
`alanine for either glycine or L-leucine reduced the antiviral activity 70- and 13-fold,
`respectively [36]. When L-valine was used, the antiviral activity was reduced 147-fold, and
`furthermore, 100% of the intact prodrug was recovered when it was exposed to pig liver
`carboxyesterases [34]. When D-alanine was substituted for L-alanine, the potency was
`decreased 35-fold [34]. The exact reason for the preference for alanine remains unknown.
`When the achiral amino acid analogue (cid:867),(cid:867)-dimethylglycine was prepared, the antiviral
`activity was only reduced three-fold [36], which illustrates the fact that natural amino acids
`are not essential for activity. However, when such amino acids were used, a preference for
`(cid:867)-amino acids was observed [34]. The (cid:868)-amino acid phosphoramidates showed efficient
`ester cleavage, but no phenyl loss was detected, and the AAM was not observed [34]. This
`suggests a possible entropy barrier that increases with chain length.
`
`Studies to determine the optimal aryloxy group were also performed [37]. The greatest
`activity was achieved when the aryl group had a p-Cl substituent, and generally for aryl
`groups that function as mildly electron-withdrawing, lipophilic substituents [37]. The
`potential for toxicity of the released phenol was not discussed. A naphthyl group was also
`
`Expert Opin Drug Deliv. Author manuscript; available in PMC 2016 November 21.
`
`Author Manuscript
`
`Author Manuscript
`
`Author Manuscript
`
`Author Manuscript
`
`

`

`Peterson and McKenna
`
`Page 4
`
`reported to be an effective aryl moiety for delivering anticancer agents [38], and this activity
`is most likely transferable to antiviral agents.
`
`To obtain successful intracellular delivery of NMP, the pronucleotides need to be resistant to
`hydrolysis during the absorption and distribution process. The chemical stability of the
`pronucleotides was studied, and all exhibited satisfying stability over the range of pHs
`studied (2.0 – 7.4) [39]. The aryloxy phosphoramidates were significantly less resistant to
`decomposition in plasma or cell extracts, indicative of the need for enzymatic activation
`[39]. After overnight exposure to pig liver carboxyesterases, CEM cell extract, human serum
`and mouse serum, AAM was formed from a majority of the L-amino acid-containing
`pronucleotides [34]. When carbocyclic adenosine phosphoramidates were evaluated in
`intestinal and liver S9 homogenates, some of the most antivirally potent analogues exhibited
`complete decomposition over 1 h in intestinal homogenate [22], but isopropyl and t-butyl
`esters on the amino acid increased intestinal stability [22]. Similarly, D-alanine and glycine
`exhibited the highest intestinal stability [22], which highlights the complexity in obtaining a
`structure–activity relationship. Although the usage of this aryloxy phosphoramidate prodrug
`approach with nucleotide analogues containing a phosphonate may be more difficult due to
`decreased chemical stability [40], an example of successful application to tenofovir has been
`described [27].
`
`The pharmacokinetics and oral bioavailability of aryloxy phosphoramidates, specifically
`abacavir phosphoramidates, were examined [35]. When the abacavir methyl alaninyl–
`phosphoramidate was administered intravenously, the pronucleotide was rapidly cleared
`from the plasma with a half-life of 7 min [35]. Similar results were observed following oral
`administration [35]. However, the major metabolite observed was the AAM [35]. Total
`exposure to the pronucleotide and its active metabolites was reported to approach that
`estimated for a similar dose of the parent drug, abacavir, resulting in an overall
`bioavailability of 50% [35]. The epithelial permeability of a series of d4T aryloxy
`phosphoramidates was evaluated in Caco-2 and MDCK monolayers [41]. The
`pronucleotides exhibited relatively low permeability, which may be partially explained by
`their susceptibility to first-pass metabolism in the intestinal epithelial cells and by being
`substrates of P-gp [41]. In general, this work exemplifies the difficulty in delivering the
`NMP to the target while avoiding significant metabolism during absorption and distribution.
`To obtain optimal antiviral activity of each pronucleotide, the fine tuning of each element
`(amino acid, ester, and aryl moiety) is required.
`
`3. Monoester prodrugs
`Amino acid phosphoramidate monoesters designed to release the NMP after a single
`activation by an endogenous phosphoramidase have been described by Wagner, who has
`applied this approach to AZT [42,43] and ddA [44], as well as anticancer drugs [45].
`
`After the delivery of AZT monophosphate by a glycoslyated carrier attached through lysine
`was reported [46], Wagner and coworkers proposed that NMP could be efficiently delivered
`by non-polar amino acid phosphoramidate monoesters and that the aryl group was not
`necessary. Furthermore, these phosphoramidate monoesters were stable in cell culture and
`
`Expert Opin Drug Deliv. Author manuscript; available in PMC 2016 November 21.
`
`Author Manuscript
`
`Author Manuscript
`
`Author Manuscript
`
`Author Manuscript
`
`

`

`Peterson and McKenna
`
`Page 5
`
`rat and human plasma [42]. A series of these compounds were synthesized containing an
`amino acid (tryptophan methyl ester [Figure 2C(9)] or phenylalanine methyl ester [Figure
`2C(11)]) connected via a P-N bond to the NMP with the other P-OH left as a free acid or
`esterified to a simple alkyl group [47]. The tryptophan monoester (Figure 2C(9)) exhibited
`the best antiviral activity, with an eight-fold increase over AZT with no cytotoxicity
`observed at the levels tested [47]. Further studies have been done to investigate the activation
`pathway of these pronucleotides and optimize their structures.
`
`The effect of changing the amino acid was studied in peripheral blood mononuclear cells
`(PBMC) [42]. The best antiviral activity was obtained with the L-alanine methyl ester [42],
`consistent with McGuigan’s ProTides. Furthermore, enhanced activity was observed with
`the L-tryptophan derivative (Figure 2C(9)) compared with the L-phenylalanine (Figure
`2C(11)), L-valine and L-leucine derivatives [42]. When evaluated in CEM cells, the L-
`alanine and L-phenylalanine derivatives exhibited antiviral activity comparable to AZT [42].
`This suggests that a simple structure–activity relationship does not exist. In order to avoid
`the polar carboxylate formed after interaction of the pronucleotides with carboxyesterases,
`the amino acid methyl ester was substituted by a methyl amide [42]. The authors reported
`that this exchange had little effect on the antiviral activity of the tryptophan derivatives,
`while the phenylalanine methyl amide derivatives exhibited increased potency [42].
`However, the methyl amide derivatives exhibited greater in vitro and in vivo stability [48].
`The antiviral activity did not exhibit a strong dependence on the amino acid stereochemistry
`[42], but the inclusion of the D-isomer versus the L-isomer led to decreased volumes of
`distribution [48]. Overall, the L-tryptophan methyl amide derivative (Figure 2C(10)) was
`selected for further studies.
`
`To better understand the differences in potency, Wagner and coworkers investigated the
`ability of the pronucleotide to deliver NMP intracellularly [42]. The antiviral activity is
`strongly related to the intracellular levels of nucleoside triphosphate. In both PBMCs and
`CEM cells, AZT was able to produce higher levels of AZT triphosphate than the
`pronucleotides [42]. However, when evaluated in CEM cells, the intracellular levels of the
`tryptophan methyl ester (Figure 2C(9)) and phenylalanine methyl ester (Figure 2C(11))
`pronucleotides did not plateau [49]. Therefore, the differences in potency may be derived
`from the ability of a phosphoramidase to cleave the P-N bond and release the NMP.
`
`The oral bioavailability, disposition and stability of the AZT phosphoramidate monoesters
`were evaluated in rats [50]. The phosphoramidate monoesters were stable in tissue
`homogenates, intestinal contents, and rat and human plasma [48,50]. The tryptophan methyl
`amide derivative (Figure 2C(10)) exhibited the best pharmacokinetic parameters. However,
`in simulated gastric fluids at pH 2.0, the pronucleotide exhibited a significantly reduced
`half-life of 5 h, but greater stability as the pH increased [50]. These results are consistent
`with greater chemical hydrolysis of P-N bonds at lower pH [51]. The pronucleotide was not
`detected in plasma or urine, which was confirmed in an in situ single pass perfusion study
`where little or no absorption of the pronucleotide in the 120 min perfusion period was
`detected [50]. AZT was observed in plasma and urine samples accounting for 29.5% of the
`dose, while 54.3% of the dose was recovered 4 h post-dosing (intravenously) as intact
`pronucleotide in the bile [50]. These results offer some possible explanations for the zero
`
`Expert Opin Drug Deliv. Author manuscript; available in PMC 2016 November 21.
`
`Author Manuscript
`
`Author Manuscript
`
`Author Manuscript
`
`Author Manuscript
`
`

`

`Peterson and McKenna
`
`Page 6
`
`oral bioavailability of the pronucleotide. Not only will the pronucleotide exist as a charged
`species at physiological pH, but pre-systemic hydrolysis of the P-N bond would lead to a
`dianionic monophosphate. This fact, combined with its molecular weight, makes biliary
`excretion difficult to avoid [50].
`
`The phosphoramidate monoester pronucleotide approach was applied to AZT to create a
`pronucleotide with increased antiviral activity and decreased cytotoxicity. Unfortunately,
`decomposition of the phosphoramidate monoester in simulated gastric fluid was observed,
`and the pronucleotide exhibited little or no bioavailability when evaluated in rats.
`
`4. SATE pronucleotides
`S-Acyl-2-thioethyl (SATE) protecting groups for nucleotide drugs have been introduced for
`the delivery of a number of NMP including d4T [52], PMEA [53], elvucitabine ((cid:868)-L-FD4C)
`[54], acyclovir [55,56], AZT [57–59] and cytarabine (Ara-C) [60].
`
`The SATE approach utilizes both enzymatic and chemical mechanisms to activate the
`pronucleotide and release the NMP (Figure 3A) [61]. Removal of the S-acyl-2-thioethyl
`protecting group and release of the monophosphate is initiated by esterase-mediated
`hydrolysis of the acyl group, which produces a reactive thiol group [61]. Nucleophilic attack
`on the (cid:867)-carbon results in a reactive 2-mercaptoethyl ester that decomposes spontaneously
`to release the diester (Figure 3A(14)) and ethylene sulfide (episulfide) [61]. If the second
`ester is also a SATE group, the process is then repeated, resulting in release of the NMP
`[61]. It has been proposed that if SATE mixed pronucleotides are used, the aryl group in
`SATE phosphotriesters is cleaved by a type 1 phosphodiesterase and a phosphoramidase
`cleaves the protecting group bound via a P-N bond [61]. However, the cleavage of the aryl
`ester or phosphoramidate could be concomitant with cleavage of the SATE group [61].
`
`Although the release of the NMP is essential, a further point to consider is promoiety
`toxicity. In the activation process, carboxylic acids and episulfide were released [61]. The
`body can metabolize the carboxylic acids, and no cytotoxicity was reported for the episulfide
`[61]. When the SATE promoiety and its metabolites were evaluated in various cell lines, no
`additional cytotoxicity was observed [61]. In vivo toxicity studies in cynomolgus monkeys
`showed neither clinical symptoms nor behavior problems indicative of toxicity [61].
`
`In all cases, application of the bis(SATE) phosphotriester (Figure 3B(15)) approach has led
`to increased in vitro antiviral activity compared to the parent nucleoside and efficient
`delivery of the NMP. Bis(MeSATE) d4T monophosphate was 10- to 17-fold more potent
`than d4T in wild-type CEM cells and PBMCs, and furthermore, antiviral activity was
`retained in thymidine kinase-deficient cells [52]. A slight increase in cytotoxicity was also
`observed for the pronucleotides [52]. Administration of the bis(tBuSATE) Ara-C
`phosphotriester resulted in significant antiviral activity in a resistant cell line due to lack of
`the appropriate kinase [60].
`
`Stability and transport across a Caco-2 monolayer was used to determine the optimal R1
`group (Figure 3B) [53]. Transport of the pronucleotides across Caco-2 monolayers resulted
`in intact pronucleotide in the basolateral compartment only when the bis(tBuSATE)
`
`Expert Opin Drug Deliv. Author manuscript; available in PMC 2016 November 21.
`
`Author Manuscript
`
`Author Manuscript
`
`Author Manuscript
`
`Author Manuscript
`
`

`

`Peterson and McKenna
`
`Page 7
`
`phosphotriester was evaluated, whereas no intact pronucleotide or metabolites were
`observed for the bis(MeSATE) phosphotriester [53]. Although very low amounts of the
`intact pronucleotide were detected, the greatest uptake in the Caco-2 monolayer was
`observed for the bis(tBuSATE) phosphotriester, which could be due to the increased
`enzymatic stability or lipophilicity of the pivaloyl group [53]. The bis(tBuSATE)
`phosphotriester was extensively metabolized to the monoester during transport in vivo,
`resulting in no intact pronucleotide observed in the plasma [62]. Some bis(tBuSATE)
`phosphotriesters exhibit poor aqueous solubility and had to be administered with 5%
`dimethyl sulfoxide (DMSO), which affected the esterase activity and significantly increased
`the half-lifes of the pronucleotides [62].
`
`In an attempt to obtain more favorable stability and bioavailability, a SATE group bearing a
`functionalized acyl moiety was investigated (Figure 3B(16)) [57,59,62]. The addition of one
`hydroxyl group led to antiviral activity comparable to the nucleoside and increased activity
`compared to the bis(tBuSATE) phosphotriester [62]. The addition of a hydroxyl group to the
`bis(tBuSATE) promoiety decreased the hydrolysis rate and increased the half-life and
`aqueous solubility of the pronucleotide [62]. Furthermore, intact pronucleotide was observed
`when the bis(hydroxyl-tBuSATE) phosphotriester was evaluated in a Caco-2 monolayer
`[62].
`
`Several variations of the SATE pronucleotide approach have been described. Initial attempts
`at the intracellular delivery of NMP with SATE esters involved the use of two SATE groups
`[52,55,60,62]. Although these bis(SATE) pronucleotides (Figure 3B(15 and 16)) exhibited
`increased antiviral activity and decreased cytotoxicity, the removal of the second SATE
`group proved difficult and proceeded much more slowly due to the negative charge at the
`phosphate [61]. Therefore, Gosselin and Imbach evaluated two different kinds of SATE
`mixed esters: aryl SATE phosphotriesters (Figure 3B(17)) [64,65] and SATE
`phosphoramidate diesters (Figure 3B(18)) [64,67].
`
`A phenyl (tBuSATE) mixed phosphotriester was synthesized, but the NMP was not released
`from the prodrug when studied in cell extract [63]. Several derivatives of L-tyrosine SATE
`phosphotriesters (Figure 3B(17)) were investigated for stability and antiviral activity [64,65].
`Since the presence of the free carboxylic acid on the tyrosine residue resulted in decreased
`antiviral activity, most likely due to decreased membrane permeability, the moiety was
`modified to contain polar, but not anionic, functionalities [66]. The pronucleotide with the
`shortest half-life exhibited the best antiviral activity [64]. The resulting mixed SATE
`phosphotriesters exhibited enhanced antiviral activity in CEM kinase-deficient cells,
`illustrating the successful delivery of the NMP intracellularly [64].
`
`Another solution to the slow activation of the bis(SATE) phosphotriesters was the SATE
`phosphoramidate diesters (Figure 3B(18)) modeled after the phosphoramidates of McGuigan
`and Wagner. Initial studies with various alkylamines illustrated that the rate limiting
`hydrolysis of the phosphoramidate was dependent on the basicity and bulk of the amine
`[65,67]. However, when the pKa of the amine was appropriate (approx. 5 – 11.2), the
`phosphoramidate diesters effectively delivered NMP intracellularly [67]. Phosphoramidate
`diesters of AZT were as potent as AZT in wild-type CEM cells and retained significant
`
`Expert Opin Drug Deliv. Author manuscript; available in PMC 2016 November 21.
`
`Author Manuscript
`
`Author Manuscript
`
`Author Manuscript
`
`Author Manuscript
`
`

`

`Peterson and McKenna
`
`Page 8
`
`antiviral activity in kinase-deficient cells [58]. Interestingly, the isopropylamino derivative
`exhibited the greatest potency, which illustrates the flexibility in the amine moiety and
`demonstrates that an (cid:867)-amino acid is not a structural requirement [58].
`
`Périgaud and coworkers have recently investigated the use of aryl SATE phosphotriesters
`[57,59]. Building on the ability of (hydroxyl-tBu)SATE to increase the solubility and
`stability of the prodrug, functionalization of the acyl moiety with polar groups was applied
`to the phenyl SATE mixed phosphotriester analogues. Additionally, an analogue with a
`valine replacing the acyl group was studied, but the compound was unstable in cell extract
`and did not maintain antiviral activity in a thymidine kinase (TK) deficient cell line [57,59].
`Introduction of one hydroxyl group on the acyl moiety resulted in greater stability in cell
`extracts compared to the tBuSATE analogue [57]. The derivative containing two hydroxyl
`groups showed decreased enzymatic stability compared to the (hydroxyl-tBu)SATE
`analogue, but it resulted in a significant loss in activity in a TK-deficient cell line [57]. The
`monohydroxylated prodrug showed anti-HIV activity in the micromolar range comparable to
`the tBuSATE analogue, and the solubility of the pronucleotide was greatly increased [57],
`which demonstrates the necessity of obtaining optimized pharmacokinetic properties to
`achieve effective prodrugs.
`
`Application of the SATE pronucleotide approach to numerous nucleoside monophosphates
`and nucleotide analogues has resulted in increased antiviral activity in vitro. However, when
`evaluated for transport across Caco-2 monolayers and for bioavailability, no intact prodrug
`was observed illustrating premature hydrolysis. As pointed out by Gosselin, Imbach, and
`Périgaud, these facts illustrate the necessary balance that needs to be achieved between
`lipophilicity, solubility and enzymatic stability [61].
`
`5. CycloSal prodrugs
`Meier et al. have shown that salicyl alcohol is an effective bifunctional masking unit for
`nucleotides, that is cleaved by a pH-dependent mechanism to deliver the active drug [68].
`They have illustrated the utility of this approach using various nucleoside and nucleotide
`analogues including acyclovir [69], 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA,
`adefovir) [70], 2(cid:5321),3(cid:5321)-dideoxyadenosine (ddA) [71], 2(cid:5321),3(cid:5321)-dideoxy-2(cid:5321),3(cid:5321)-didehydroadenosine
`(d4A) [71], 5-[(E)-2-bromovinyl]-2(cid:5321)-deoxyuridine (BVdU or brivudin) [72,73], 2(cid:5321),3(cid:5321)-
`dideoxy-2(cid:5321),3(cid:5321)-didehydrothymidine (d4T) [74–79] and carbocyclic 3(cid:5321)-azidothymidine
`analogues [80].
`
`Their original goal in creating these pronucleotides was to find a masking unit that would
`deliver the nucleotide analogue exclusively by a chemical mechanism. Initial attempts using
`bis(alkyl), bis(phenyl), or bis(benzyl) nucleotide triesters proved unsuccessful at releasing
`the NMP by a purely chemical mechanism [6]. The charge formed once one ester was
`cleaved led to a stable compound resistant to further chemical hydrolysis [16]. However,
`Meier and coworkers found that they could successfully mask the phosphate with phenyl and
`benzyl esters of salicyl alcohol, while the nucleoside was attached by esterification via the
`5(cid:5321)-hydroxyl group (Figure 4A(19)) [81]. These esters are distinct enough to achieve
`differentiated chemical hydrolysis independent of any enzymatic activity [82]. This principle
`
`Expert Opin Drug Deliv. Author manuscript; available in PMC 2016 November 21.
`
`Author Manuscript
`
`Author Manuscript
`
`Author Manuscript
`
`Author Manuscript
`
`

`

`Peterson and McKenna
`
`Page 9
`
`was validated by comparing the half-lifes of the triesters in phosphate buffer at pH 7.3 and
`cell extracts with fetal calf serum, which showed similar half-lifes for the triesters in both
`media [83].
`
`The successful cycloSal pronucleotide releases the nucleotide (Figure 4B(23)) and the
`salicyl alcohol (Figure 4B(24)) intracellularly [81]. The nucleotide is released through a
`cascade, which is initiated by cleavage of the phenyl ester via nucleophilic attack on the
`phosphorus atom by hydroxide to form the diester (Figure 4B(22)) (step a1). The ring, now
`activated by the strong electron-donating hydroxyl group, allows for cleavage of the benzyl
`ester to yield the nucleotide analogue (Figure 4B(23)) and salicyl alcohol (Figure 4B(24))
`via a SN1-type reaction at the benzyl position (step a2). It is also possible that hydrolysis of
`the benzyl ester can occur to first produce a charged intermediate (Figure 4B(25)) (step b1),
`which then reacts with water to yield 26 (Figure 2B) (step b2). Further hydrolysis of 26
`(Figure 2B) does not occur, thus preventing the release of the nucleotide analogue. However,
`in hydrolysis studies, the major products were the NMP and salicyl alcohol. When evaluated
`for cytotoxicity in mice, salicyl alcohol showed no toxicity [82].
`
`As the cycloSal pronucleotides were designed to release the active drug via a chemical
`cascade mechanism, the stability and hydrolysis pathways of these pronucleotides can be
`fine-tuned by varying the substituents on the aromatic ring. Acceptor substituents in the 5-
`or 6-position decrease stability, while donor substituents at the 3- or 5-position increase the
`stability of the triesters (Figure 4A(19)) [82]. Bulky substituents (tert-butyl groups) at the 3-
`and/or 5-position increase the amount of the phenyl phosphate diester (Figure 4B(25))
`observed. When substitution was made at the benzyl position, the half-life decreased
`drastically compared to the unsubstituted analogue and the major product was the diester
`(Figure 4B(25)) in hydrolysis studies [82]. However, the addition of a donor substituent at
`the 6-position caused the major hydrolysis product to be the desired diester (Figure 4B(22))
`[82].
`
`Although there is a benefit – lack of dependence on enzyme expression differences in
`tissues, individuals and species – to the use of a pronucleotide activated by a chemical
`mechanism, the possibility of extracellular release of the active drug or efflux of the
`pronucleotide, due to the establishment of a concentration equilibrium across the cell
`membrane, cannot be ignored. To remedy these potential problems, Meier et al. designed a
`way to trap the pronucleotide inside the cell [77,79,84]. In theory, the attachment of a moiety
`to the aromatic ring that can be enzymatically activated to release a more polar group will
`prevent penetration of the cellular membrane by the compound and trap the pronucleotide
`[75]. The initial attempts included the use of an esterase to release an alcohol or carboxylic
`acid [85]. The released alcohol group was not polar enough to prevent efflux of the
`pronucleotide, and when a two, three, or four carbon linker was used to attach a methyl or
`benzyl ester to the aromatic ring, the compounds did not show good esterase affinity [78].
`The feasibility of acetoxymethyl (AM) and pivaloyloxymethyl (POM) esters as enzyme-
`cleavable triggers was demonstrated by their significantly decreased stability in cell extracts
`versus plasma, illustrating selective activation of these compounds intracellularly [73].
`Attempts at intracellular trapping of the pronucleotide with an amino acid ester trigger
`moiety resulted in a large differential between the buffer and cell extract stability and
`
`Expert Opin Drug Deliv. Author manuscript; available in PMC 2016 November 21.
`
`Author Manuscript
`
`Author Manuscript
`
`Author Manuscript
`
`Author Manuscript
`
`

`

`Peterson and McKenna
`
`Page 10
`
`sustained antiviral activity in kinase-deficient cells [84]. Although a strongly polar group
`was required to trap the pronucleotide in the cell, Meier and coworkers found