Pronucleotides:Toward the InVivo
`Delivery of Antiviral and
`Anticancer Nucleotides
`
`CarstonR.Wagner,VidhyaV.Iyer,EdwardJ.McIntee
`
`Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota,
`Minneapolis, MN 55455
`
`!
`
`Abstract: To overcome the many hurdles preventing the use of antiviral and anticancer
`nucleosides as therapeutics, the development of a prodrug methodology (i.e., pronucleotide) for
`the in vivo delivery of nucleotides has been proposed as a solution. The ideal pronucleotide should
`be non-toxic, stable in plasma and blood, capable of being i.v. and/or orally dosed, and
`intracellularly convertible to the corresponding nucleotide. Although this goal has yet to be
`achieved, many clever and imaginative pronucleotide approaches have been developed, which are
`likely to be important pharmacological tools. This review will discuss the major advances and
`future directions of the emerging field of antiviral and anticancer pronucleotide design and
`development. (cid:223) 2000 John Wiley & Sons, Inc. Med Res Rev, 20, No. 6, 417–451, 2000
`
`Key words: prodrug; pronucleotide; nucleotide; antiviral; anticancer
`
`1. INTRODUCTION
`
`Nucleosides and nucleotides have demonstrated wide-spread utility as antiviral and anti-cancer
`therapeutics.1,2 Natural endogenous nucleosides must be phosphorylated to the corresponding 50-
`triphosphates (TP) to be incorporated into the DNA strand being synthesized in the cell. The first
`phosphorylation step leading to the formation of nucleoside 50-monophosphate (MP), is commonly
`catalyzed by a nucleoside kinase encoded by the host cell or the virus infecting the host cell (Fig. 1).3
`Conversion of nucleoside-MPs to the corresponding 50-diphosphates (DP) and triphosphates is
`carried out by nucleoside, nucleotidyl, and nucleoside diphosphate kinases, respectively. Thus,
`cellular kinases and virally-encoded kinases play a vital role in the metabolism and replication of
`cells and viruses.
`Based on the metabolite–antimetabolite approach, nucleoside analogs such as 20,30-
`dideoxynucleosides (ddNs) have been developed as competitors of natural 20-deoxynucleoside
`50-triphosphates (dNTPs). Typically, modifications at the 20 or the 30carbon atoms of the glycone
`(sugar) moiety of nuelcosides are introduced. By virtue of their resemblance to the natural 20-
`deoxynucleosides, ddNs are phosphorylated to the corresponding 50-triphosphates and incorporated
`
`Correspondence to: Carston R.Wagner
`
`(cid:223) 2000 John Wiley & Sons, Inc.
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`Figure1. Intracellular metabolism of nucleosides.3
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`into a growing DNA strand by a DNA polymerase, resulting in chain termination. Therefore, ddNs
`are in essence prodrugs, since they must be phosphorylated intracellularly in order to be biologically
`active.
`Therapies involving long-term administration of ddNs such as 30-azido-2030-dideoxythymidine
`(AZT) have been reported to lead to decreased activity of the first phosphorylating enzyme,
`thymidine kinase and thus, resistance.4 This type of resistance is observed not only in host tissues of
`the patients undergoing ddN therapy, but also in viruses. This resistance mechanism renders the
`ddNs less effective since their activation is hindered at the first phosphorylation step. In addition, for
`antiviral acyclic nucleosides such as acyclovir and penciclovir, the dependence of the nucleosides
`on activation to the triphosphates by virally encoded thymidine kinase, limits their spectrum of
`antiviral activity to those viruses such as herpes simplex virus (HSV) and Varicella zoster virus
`(VZV) which encode their own thymidine kinase.5 Thus, viruses such as hepatitis B virus (HBV),
`which do not encode their own nucleoside kinase, do not fall within the purview of activity of these
`antiviral nucleosides. Moreover, ddNs such as AZT, are associated with myelosuppressive side-
`effects, such as anemia and neutropenia.6 Toxic side-effects have been widely reported to lead to the
`discontinuation of ddN therapy. Certain other ddNs such as 20,30-dideoxyuridine (ddU), are poor
`substrates for thymidine kinase or other cellular kinases, and are, therefore, not converted to the
`corresponding triphosphates.7,8
`In principle, administration of the 50-phosphates would aid in overcoming the drawbacks of
`ddN therapy posed by resistance mechanisms and inherent biological differences. However, because
`phosphates are strongly acidic and thus negatively charged at physiological pH (pH(cid:136) 7.4), they are
`too hydrophilic to penetrate the lipid-rich cell membranes. In addition, blood and cell surface
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`phosphohydrolases (acid and alkaline phosphatases, 50-nucleotidases)
`phosphates to the corresponding nucleosides.
`In order to overcome the poor cell penetration of nucleoside 50-phosphates, Montgomery9
`proposed that ‘‘this difficulty might be overcome if one could prepare an ester of a nucleotide which
`could penetrate the cell wall and then be metabolized to the nucleotide itself.’’ Consequently,
`various prodrug or ‘‘pronucleotide’’8 approaches have been devised and investigated. In general, the
`goal of these approaches has been to promote passive diffusion through cell membranes and
`increase the bioavailability of phosphorylated nucleosides. This approach of derivatization has been
`applied using various protecting groups for the phosphate moiety. Ideally, the attempts have been
`designed to achieve stability in the extracellular medium and rapid intracellular hydrolysis to release
`the phosphate. In most cases, to be biologically active, the phosphate has to be activated to the
`diphosphate and triphosphate. This review will discuss what we believe to be the major advances
`and future directions of the emerging field of antiviral and anticancer pronucleotide design and
`development.
`
`rapidly convert
`
`the
`
`2. ALKYL AND ARYL PHOSPHATE
`DERIVATIVES
`
`A. AlkylandArylPhosphodiesters
`
`A simple solution to the delivery of nucleotide monophosphates to cells is the use of alkyl phosphate
`esters. For example, aryl phosphodiesters of 6-mercaptopurine (6-MP) riboside were prepared in
`order to deliver 6-MP to 6-MP resistant neoplasms.9 The attempt was unsuccessful, probably due to
`poor cell penetration.10
`Alkyl and aryl phosphodiesters of b-D-arabinofuranosylcytosine (Ara-C), 20,30-didehydro-30-
`deoxyadenosine (d4A), 20,30-didehydro-30-deoxycytosine (d4C), and ddU have also been
`synthesized (Fig. 2). It had been reported that of a series of lipophilic 50-(alkyl phosphate) esters
`of Ara-C, only the Ara-C 50-(glyceryl phosphate) possessed cytotoxicity comparable to that of Ara-
`C-MP (CC50(cid:136) 0.35 and 0.65 mM, respectively) towards L1210 leukemia cells.11 The glyceryl
`phosphate ester also demonstrated in vivo activity against a kinase-deficient P388 murine
`leukemia.12 These results suggested that the development of b-hydroxyalkyl phosphate esters and
`other hydroxyalkyl (steroids) substitutions would be a worthwhile endeavor.11 Hong and co-
`workers13 reported that alkyl esters of both Ara-C-MP and Ara-C-DP demonstrated lower growth
`inhibitory activity than steroidal esters of Ara-C and Ara-C-DP. Nevertheless, the Ara-C-DP
`steroidal esters were found to possess lower antitumor activity than Ara-C-MP and Ara-C-DP alkyl
`esters against L1210 lymphoid leukemia in mice in vivo.13 A decrease in anti-leukemic activity was
`observed with increasing alkyl chain length towards cultured L1210 leukemic cells. However, Ara-
`C-MP alkyl esters containing alkyl groups of 16–20 carbon atom length were reported to be orally
`active prodrugs of Ara-C.14
`Mullah and co-workers15 reported that 50-phenyl- and 50-methyl-phosphate diesters of d4A and
`d4C demonstrated in vitro anti-HIV activity and cytotoxicity comparable to the corresponding
`parent nucleosides (Fig. 2). The compounds were cleaved under test conditions and released the
`parent nucleosides or the nucleoside 50-monophoshates in medium containing 10% serum.15
`Synthesis of ionophore–nucleotide conjugates of 15 crown ether-AZT and 15 crown ether-ddU 50-
`phosphate diesters has also been reported (Fig. 2).16 After association with a metal cation, these
`conjugates likely form a lipophilic ion-pair which could diffuse through a bilayer membrane. Once
`inside the membrane, the aryl phosphate ester linkage of the conjugate could be cleaved to release
`the nucleoside 50-monophosphate. Although AZT was found to be approximately 14-fold more
`potent than the AZT–ionophore conjugate, the ddU–ionophore conjugate had approximately 11
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`Figure2. Alkyl and aryl phosphodiesters.9,11,15,16,18
`
`times the activity of ddU against HIV-1 infected CEM-SS cells, suggesting nucleoside 50-
`monophosphate delivery.16
`
`B. SteroidPhosphodiesters
`
`Based on the synergistic effect observed between certain drugs and the steroid prednisolone against
`human lymphoid leukemias and lymphomas, nucleotide–steroid conjugates were developed and
`evaluated for
`their anti-cancer effects. Ara-C-MP-prednisolone and Ara-C-MP-prednisone
`conjugates had greater anti-cancer effects against L1210 lymphoid leukemia in female mice than
`Ara-C alone or in combination with the individual steroid components.17 Ara-C-MP conjugates of
`corticosterone, cortexolone, 6a-methylprednisolone, and 11-deoxycorticosterone were also found to
`increase the life-span of mice with L1210 leukemia to a greater extent than Ara-C18 (Fig. 2) The
`conjugates were also found to be resistant to human liver cytidine deaminase and alkaline
`phosphatase, but sensitive to phosphodiesterase I, 50-nucleotidase, and acid phosphatase. They were
`shown to hydrolyze slowly in blood (t1/2(cid:136) 24–48 hr) and were able to cross the blood–brain barrier.
`These nucleotide–steriod conjugates did not deliver Ara-C-MP to the cells, as was evident from
`their lack of activity against a kinase-deficient L1210 leukemia resistant to Ara-C. However, many
`of
`the conjugates did show superior potency, compared to Ara-C, upon intraperitoneal
`administration against L1210 leukemia implanted intracerebrally.19,20 Other examples of steroid
`conjugates include the 3-fluorodeoxyuridine (FUdR)-MP-7b-hydroxycholestrol conjugate, which
`was found to possess in vitro cytotoxic activity against EL-4 murine leukemia cells and in vivo
`antitumor activity in mice with Krebs II ascitic carcinoma.21 The AZT-MP-3b(7b-hydroxychole-
`sterol) conjugate also demonstrated in vitro anti-HIV activity.22
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`C. AlkylandArylPhosphotriesters
`
`In general, alkyl and aryl phosphodiesters have proven to be unsuitable for the delivery of nucleo-
`tides, presumably due to their inherent polarity and sensitivity to phosphodiesterase. Consequently,
`simple alkyl and aryl phosphotriesters of nucleosides have been developed in order to increase the
`lipophilicity of the phosphate by the neutralization of negative charges of the phosphate moiety.9 The
`increased lipophilicity was postulated to overcome the inactivity of phosphodiesters by promoting
`passive diffusion of the phosphotriesters into the cells (Fig. 3). Literature reports have documented
`the synthesis and biological evaluation of alkyl and aryl phosphotriesters of AZT, Ara-C, dideoxy-
`cytidine (ddC), b-D-arabinofuranosyladenine (Ara-A), 20,30-didehydrodideoxythymidine (d4T), 30-
`acylthymidine and netivudine (1-(b-D-arabinofuranosy)-5-prop-1-ynyluracil) and 6-MP. In general,
`the phosphotriesters were found to be less potent than the corresponding parent nucleosides.9,10,23–34
`Ara-A and Ara-C are inactivated by the deamination catalyzed by adenosine deaminase and
`cytidine deaminase, respectively. However, the simple alkyl phosphotriesters of Ara-A and Ara-C
`were reported to display resistance to the action of deaminases, phosphodiesterase I, and lipase
`(Fig. 3).24,25 These phosphotriesters were found to be hydrolyzed slowly by alkaline phosphatase
`
`Figure3. Nucleoside alkyl and aryl phosphotriesters.24^26,28,29,33,34,38
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`and were stable at 37(cid:14)C, pH 7.4 for up to one week.25 In addition, because of their greater
`lipophilicity, they were shown to penetrate cell membranes. Unfortunately, they displayed no
`antiviral activity or cytotoxicity up to 100 mg/ml, although they were found to inhibit [3H]thymidine
`incorporation into the DNA of the mammalian epithelial cell line, CNCM 1221.24,25
`In an attempt to overcome the stability of simple alkyl phosphotriesters and induce the
`decomposition of these phosphotriesters, bis(2,2,2-trichloroalkyl) phosphates of thymidine and ddC
`were synthesized (Fig. 3).26 While the thymidine derivatives demonstrated modest anti-HIV activity,
`none of the analogs demonstrated antiviral potency comparable to that of the parent nucleosides.26
`Mixed alkyl monohaloalkyl phosphates of Ara-C were found to be more potent than simple alkyl
`phosphotriesters at inhibiting [3H]thymidine incorporation into DNA, with increasing potency
`correlating with increased lipophilicity (Fig. 3). Similarly, bis(haloalkyl) phosphotriesters of Ara-C
`were more potent than alkyl monohaloalkyl phosphotriesters of Ara-C.28
`Mixed aryl haloalkyl and diaryl phosphotriesters of AZT were synthesized and tested in human
`T-cell C8166 and the thymidine kinase-deficient JM cells for anti-HIV-1 activity. Except for the
`di(p-nitrophenyl) phosphate, none of the phosphotriesters were found to have greater anti-HIV-1
`potency than the nucleoside and were only marginally more potent than AZT in JM cells.29 The
`diaryl phosphates of AZT were found to have comparable or greater anti-HIV-2 activity compared to
`AZT in CEM/0, MT4, and the thymidine kinase-deficient CEM/TK(cid:255) cell lines, thus demonstrating
`their ability to not only penetrate the cell membranes, but also deliver
`the nucleoside
`monophosphates into the cells.32 With the exception of 30-acetyl, 30-ethyl, and 30-mesyl thymidine,
`bis(2,2,2-trihaloalkyl), simple alkyl, and mixed alkyl haloalkyl phosphates of thymidine analog
`antiviral nucleosides (i.e., d4T, and AZT) were found to be less potent against HIV-1 in C8166 cells
`than the parent nucleosides.27,30,31,35,36 Glycolate and lactate analogs of d4T were also examined,
`but were not found to be particularly active against HIV-1 or HIV-2 in the human T-lymphocyte
`CEM/0 and CEM/TK(cid:255) cell lines (Fig. 3).34 Similarly, alkyl, haloakyl, and aryl phosphotriesters
`of netivudine were synthesized and evaluated for anti-HCMV, HSV-2, VZV, influenza A, and
`HIV-1 activities (Fig. 3).34 All
`the phosphotriesters were found to be inactive at noncyto-
`toxic concentrations except for the 50-(4-nitrophenyl)phosphate which inhibited influenza A viral
`replication (EC50(cid:136) 2.3 mM).33
`
`D. CyclicAlkylPhosphotriesters
`
`Literature reports have demonstrated that while 5-membered cyclic phosphates are highly acid and
`alkali labile, 6-membered and 7-membered cyclic phosphates are stable under these conditions.37 A
`series of cyclic phosphates of FUdR and thymidine were synthesized and evaluated for cytotoxicity
`towards murine leukemia L1210 cells.38 A 50 0,50 0-difluoro-10 0,30 0-dioxa-20 0-phosphacyclohex-20 0-
`yl-20 0-oxide-50-cyclophosphate derivative of FUdR was found to be nearly as cytotoxic as FUdR
`toward L1210 cells (ID50(cid:136) 0.003 g/ml and 0.001 mg/ml, respectively) (Fig. 3). However, despite its
`
`lack of inhibitory activity towards cell-free thymidylate synthetase, the cytotoxicity of the cyclic
`phosphate of FUdR toward L1210 cells was reversed by the addition of exogenous thymidine
`(ID50(cid:136) 5 mg/ml). The comparable activity of the cyclic phosphate in the thymidine kinase-deficient
`L1210/BdUrd cell line to that of FUdR and FUdR-MP (ID50(cid:136) 2.42(cid:6) 0.4 mg/ml, 1.75(cid:6) 0.4 mg/ml,
`and 2.43(cid:6) 0.43 mg/ml, respectively) suggested that the compound was hydrolyzed extracellularly to
`FUdR, before cellular uptake.38
`
`E. Cyclosaligenyl(CycloSal)Phosphotriesters
`
`The cyclosaligenyl (cycloSal) phosphotriester approach has been investigated as a potential vehicle
`for the delivery of monophosphates of d4T, FUdR, acyclovir (ACV), penciclovir (PCV),
`T-penciclovir (T-PCV), dideoxyadenosine (ddA), d4A, dideoxyinosine (ddI), didehydro-dideox-
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`Figure4. Nucleoside CycloSal phosphotriesters.39^44,46
`
`yinosine (d4I), 20-fluoro-ara-20,30-dideoxyadenosine (F-ara-ddA), 20-fluoro-ara-20,30-dideoxya-
`denosine (F-ara-ddI), 20-fluoro-ribo-20,30dideoxyinosine (F-ribo-ddA), and 20-fluoro-ribo-20,30-
`dideoxyinosine (F-ribo-ddI) derivatives.39 –43 The cycloSal approach was designed to release the
`monophosphate selectively by a controlled, chemical tandem hydrolysis mechanism. The cycloSal-
`nucleotide conjugate incorporates unique phosphate ester bonds into the same molecule. The
`mechanism of generation of the nucleoside 50-monophosphate from the nucleoside cycloSal
`phosphotriester is described in Fig. 4 and is initiated by selective hydrolysis of the phenolic ester
`bond.39 Subsequently, spontaneous cleavage of the benzylic phosphate ester bond releases the
`nucleoside 50-monophosphate and the salicyl alcohol.39 Studies of the hydrolysis of the 5-chloro
`18O water
`cycloSal-d4T-MP and 5-methoxy cycloSal-d4T-MP conducted in the presence of H2
`revealed that 18O was incorporated into both the salicyl alcohol and monophosphate products,
`confirming chemical release of the monophosphate. However, the results of these experiments were
`unable to determine if the reaction proceeds through a benzyl carbocation or quinone methide
`intermediate.44 Although quinone methide has been shown to preferentially alkylate guanines in
`duplex DNA, the ability of the cycloSal derivatives to alkylate DNA has not been examined.45
`Because these compounds are prepared and evaluated as a diastereomeric mixture, the potential
`for the disastereomers to exhibit different levels of antiviral and anticancer activity has been
`evaluated for pronucleotides of d4T and ddA. Interestingly, Meier and co-workers42,44 have
`observed a difference of 3- to 80-fold in antiviral potency between the isolated diastereomers. An
`explanation for this preference has not been reported.
`The stabilities of cycloSal conjugates of FUdR were found to be greater in Tris buffer, pH 6.9
`(t1/2(cid:136) 6.8–59.2 hr) than in phosphate buffer, pH 7.29 (t1/2(cid:136) 0.9–10.6 hr) or in borate buffer, pH 8.9
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`(t1/2(cid:136) 0.4–2.1 hr).39 Besides decreased stability associated with increasing buffer pH,
`the
`conjugates also showed comparable or greater stability (t1/2(cid:136) 1.5–11.1 hr) in RPMI medium, pH
`7.4 than in RPMI containing 10% fetal calf serum (t1/2(cid:136) 2.2–10.7 hr).39 Increased stability was
`found to be associated with the decreased potency of the cycloSal phosphotriesters of FUdR.39
`In general, the cycloSal phosphotriesters were found to be more potent against HIV-1 in CEM/
`TK(cid:255) cells than the corresponding parent nucleosides. The cycloSal phosphotriesters of T-PCV, ddA,
`ddI, d4A, F-ribo-ddI, F-ribo-ddA, F-ara-ddI, and F-ara-ddA in CEM/0 cells demonstrated greater
`anti-HIV-1 potency, while those of d4T, ACV, PCV, and d4I had comparable or lower anti-HIV-1
`potency than the corresponding parent nucleosides.40 –43 However, the cycloSal phosphotriesters of
`FUdR displayed lower cytotoxicity than FUdR towards FM3A/0 and FM3A/TK(cid:255) cells (murine
`mammary carcinoma cells).39 CycloSal phosphotriesters have been used as mechanistic tools for
`understanding the effect of nucleoside structure on biological activity. For example, of the two
`20-fluoro analogs of ddA, F-ara-ddA and F-ribo-ddA, which were developed to overcome the
`susceptibility of ddA to deamination by adenosine deaminase (ADA) as well as acid-catalyzed
`glycosyl cleavage, F-ara-ddA proved to be more potent against HIV-1 and HIV-2 than F-ribo-
`ddA.43 It has been postulated that the conformations of the sugar rings and thus the activity of the
`two F-ddA isomers are strongly influenced by the orientation of 20-F.46,47 The cycloSal
`pronucleotide approach was applied to F-ara-ddA and F-ribo-ddA to determine if metabolic
`blockade at the monophosphorylation step is responsible for the difference in potency.43 The
`cycloSal approach did indeed appear to improve the anti-HIV activity of F-ribo-ddA by
`circumventing the metabolic barrier of hindered phosphorylation to the monophosphate due to
`the C-30-endo-conformation of the tetrahydrofuran ring.43
`Studies of the in vivo antiviral or anticancer potency of cycloSal phosphotriesters, as well as,
`toxicity have not been reported. In vivo toxicity studies have also not been reported. Salicyl alcohol
`(2-hydroxybenzyl alcohol) has been shown to be mild anesthetic.48 It is not known if similar in vivo
`biological activity is observed for substituted salicyl alcohols.
`
`3. BIOLABILE PROTECTING GROUPS FOR
`NUCLEOSIDE PHOSPHOTRIESTERS
`
`Although neutral lipophilic alkyl and aryl phosphotriesters are easily taken up by cells, in general,
`they were poorly hydrolyzed due to their stability. While phosphodiesters formed from
`phosphotriesters can be enzymatically converted to the nucleoside monophosphates, an endogenous
`eukaryotic phosphotriesterase activity has yet to be reported. In order to retain the advantages of
`nucleoside phosphotriesters with regard to their improved cellular uptake and still induce the
`hydrolysis of the phosphotriesters, a variety of biolabile moieties have been evaluated as potential
`protecting goups of the nucleoside monophosphate.
`
`A. S-Acyl-2-Thioethyl(SATE) Approach
`
`The SATE approach was first described by Imbach and co-workers and has been applied to the
`development of bis(SATE) phosphotriester analogs of ddU, ddA, ddI, AZT, 20,30-dideoxy-30-
`oxyadenosine (isoddA), d4T, and ACV.8,49 –52 The design and synthesis of closely related (iso)SATE
`analogs of D4T and ddA, monoSATE aryl phosphotriesters of AZT, S-pivaloyl-4-thiobutyl
`(tBuSATB) analogs of AZT and ddA and S-glycopyranosidyl (SGTE) analogs of AZT have also
`been reported (Fig. 5).53–56 The general decomposition pathway of these biolabile phosphotriesters
`to yield the nucleoside 50-monophosphates via the formation of a phosphodiester is described in
`Fig. 6. The hydrolysis of the bis(SATE) phosphotriester is initiated by the carboxyesterase-mediated
`hydrolysis of
`the thioester moiety of one of
`the SATE groups,
`to form the unstable
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`Figure5. Pronucleotide approaches closely related to the SATE approach.8,49,50,51,53^56
`
`Figure6. Mechanism of decomposition of SATE pronucleotides.49
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`O-2-mercaptoethylphosphotriester.49 The thiol generated, which is a soft nucleophile, attacks the
`soft electrophilic methylene carbon atom,
`releasing ethylene sulfide and the monoSATE
`phosphodiester.49 This phosphodiester
`is likely subject
`to hydrolysis by an intracellular
`phosphodiesterase to generate the nucleoside 50-monophosphate and S-acyl-thioethanol. Alter-
`natively, a second carboxyesterase-mediated thioester hydrolysis/ethylene sulfide step ensues to
`generate the nucleoside 50-monophosphate.49
`Another possible mechanism of hydrolysis of the bis(SATE) phosphotriester has also been
`proposed to occur via the direct nucleophilic attack of water on the phosphorus atom to release the
`S-acyl-thioethanol.49 The variation of the half-lives of the bis(SATE) phosphotriesters of ACV
`ranged from 2 min in mouse serum, to 5.5 hr in CEM cell extract, to 14 hr in human serum,
`suggesting that an esterase-mediated activation was more plausible than chemical hydrolysis.57
`Although not mutagenic by the Ames test, the potential for electrophilic ethylene sulfide or its
`metabolites released from SATE pronucleotides to react with nucleophilic groups on biological
`macromolecules has not been investigated.58
`The enhanced anti-HIV-1 activity of most of the SATE nucleoside phosphotriester analogs over
`that of the parent nucleosides, has been widely documented in CEM-SS, MT-4 (human T-
`lymphocytes), peripheral blood monocytes (PBMs), macrophages and CEM/TK(cid:255) cells, demon-
`strating that nucleoside 50-monophosphate
`can be
`successfully delivered into these
`cells.8,50,52,54,56,57,59 –61 Bis(SATE) d4T phosphotriesters demonstrated higher anti-HIV-1 potency
`than d4T in MT-4, CEM-SS, PBM, and CEM/TK(cid:255) cells.62 Only the AZT bis(SATE) phosphotriester
`analogs were found to be less potent than AZT in CEM-SS and MT-4 cells against HIV-1, but had
`superior anti-HIV-1 potency in CEM/TK(cid:255) cells, suggesting intracellular delivery of AZT-MP.8,49,61
`The closely related analogs—tBu(iso)SATE (O-pivaloyl-2-oxyethyl) thio-phosphotriesters of
`ddA and d4T, SGTE phosphotriester of AZT, the monoSATE aryl phosphotriesters of AZT, and
`tBuSATB phosphotriesters of AZT and ddA—were found to possess greater anti-HIV activity than
`the corresponding parent nucleosides, but were less active than the corresponding bis(SATE) pho-
`sphotriesters.53 –56 Some of the additional advantages of the bis(SATE) phosphotriester approach
`included not only the delivery of ddA-MP into the cells by the ddA bis(SATE) phosphotriester, but
`also resistance to adenosine deaminase (ADA). Deamination by ADA has been found to severely
`reduce the efficacy of ddA.8,51,63 In addition, unlike ACV, ACV bis(SATE) phosphotriesters were
`found to possess a broader antiviral spectrum of activity than ACV as was evident from their anti-
`hepatitis B viral activity.8,57,64,65 Moreover, AZT bis(SATE) phosphotriesters were able to
`overcome cellular resistance of MOLT4/8rAZR250 cells to AZT, because AZT-MP was delivered.59
`In vivo studies with duck HBV (DHBV) found that ACV bis(SATE) was somewhat more efficient
`than ACV when given over a short period of time orally or intraperitoneally.64 Thus, the bis(SATE)
`phosphotriester approach has proven to be a potentially effective way for both the in vitro and in vivo
`intracellular delivery of nucleoside 50-monophosphates.
`
`B. Dithioethyl(DTE)Approach
`
`The synthesis and biological evaluation of bis(DTE) (dithioethyl) phosphotriesters has been
`reported for AZT and ddU.49,50,59,61 The decomposition of the bis(DTE) analogs is described in
`Fig. 7 and is postulated to be initiated by a reductase-mediated reductive cleavage of the disulfide
`bond.8 As a result of
`the reductive cleavage,
`the unstable O-2-mercaptoethyl monoDTE
`phosphotriester
`is formed, followed by the release of ethylene sulfide or thioethanol by
`intramolecular nucleophilic displacement.8 The monoDTE phosphodiester can undergo hydrolysis
`mediated by phosphodiesterase or undergo a second reductase-mediated disulfide cleavage, to
`generate the nucleoside 50-monophosphate.8 As noted for the SATE approach, in vivo toxicity
`associated with the generation of ethylene sulfide from the DTE derivatives has not been
`reported.
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`Figure7. Mechanism of decomposition of dethioethyl (DTE) pronucleotides.8,51
`
`Like the bis(SATE) analogs, bis(DTE) phosphotriester analogs of AZT were found to possess
`lower anti-HIV-1 potency than AZT in CEM-SS, MT-4, and PBM cells, but did demonstrate greater
`anti-HIV-1 potency than AZT in CEM/TK(cid:255) cells.8,49,61 However, the bis(DTE) phosphotriesters of
`ddU were consistently more active against HIV-1 in CEM-SS, PBM, and CEM/TK(cid:255) cells than the
`parent nucleoside.50,61
`In general, bis(SATE) phosphotriesters of anti-HIV-1 nucleosides
`consistently displayed greater potency than the corresponding bis(DTE) phosphotriesters.8,49,50,61
`
`C. Pivaloyloxymethyl(POM)Approach
`
`The pivaloyloxymethyl (POM) phosphoester approach has been utilized for the development of
`bis(POM) phosphotriesters of ddU, FUdR, AZT and other nucleoside analogs7,8,49,61,66 –68 (Fig. 8).
`Like bis(DTE) phosphotriesters, the bis(POM) phosphotriesters of AZT were found to be less potent
`than AZT towards HIV-1 in CEM-SS, MT-4, and PBMs, but more active in CEM/TK(cid:255) cells.8,49,61,69
`Farquhar and co-workers7,69 demonstrated that unlike ddU or AZT, the incubation of CEM/TK(cid:255)
`cells with bis(POM) esters of ddU and AZT resulted in increased intracellular levels of ddU and
`AZT monophosphate, diphosphate, and triphosphate. The bis(POM) phosphotriesters displayed
`varying degrees of antiviral and cytotoxic activities relative to the corresponding bis(SATE) and
`bis(DTE) derivatives.8,49,61
`The mechanism of decomposition of bis(POM) derivatives to the nucleoside 50-monophosphate
`is described in Fig. 8, and like the bis(SATE) approach, relies on carboxyesterase-mediated
`hydrolysis to initiate release of the nucleoside 50-monophosphate.70 The unstable O-2-hydroxyethyl
`phosphotriester forms, then undergoes intramolecular nucleophilic displacement to generate the
`monoPOM phosphodiester and formaldehyde. Similar to the bis(SATE) approach, conversion of the
`phosphodiester to the nucleoside 50-monophosphate is likely rate limiting and carried out by a
`phosphodiesterase or esterase.70 The generation of electrophilic formaldehyde, as a result of the
`hydrolysis of the bis(POM) phosphotriester, could be a source of long term cellular toxicity arising
`from the interaction of formaldehyde with cellular nucleophiles such as proteins, DNA or RNA.
`The POM derivative of a cyclic phosphate of FUdR was moderately stable in aqueous buffers
`over a pH range of 1–7.4 (t1/2 > 30 hr), but was degraded to FUdR-MP in a concentration-dependent
`manner by carboxylate esterase (Fig. 8).68 The POM ester of FUdR cyclic phosphate was also found
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`Figure8. Pivaloyloxymethyl (POM) pronucleotide approach.68,70,71
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`to be as potent a growth inhibitor as 5-fluorouracil toward Chinese hamster ovary (CHO) cells
`(IC50(cid:136) 5(cid:2) 10(cid:255)6 M). In addition, FUdR monophosphate pronucleotide was found to increase the
`life spans of P388 leukemia bearing mice upon intraperitoneal administration for five consecutive
`days, and was active against a CHO variant cell line and a P388 mutant cell line, both of which were
`resistant to 5-fluorouracil.68 Taken together, this data suggested that the POM phosphate diester
`intracellularly delivered FUdR-MP.
`Like the AZT-bis(POM) phosphotriester, the bis(POM) FUdR phosphotriester was stable at
`pH 1–4 (t1/2 > 100 hr) and at pH 7.4 (t1/2(cid:136) 40.2 hr) but was hydrolyzed quickly in 0.05 M NaOH
`(t1/2 < 2 min).71 FUdR was found to be only 5-fold more cytotoxic (IC50(cid:136) 1(cid:2) 10(cid:255)6 M) towards
`CHO cells than bis(POM) 30-acetyl FUdR (IC50(cid:136) 5(cid:2) 10(cid:255)6 M), which had been synthesized as a
`member of a series of bis(acyloxymethyl) phosphotriesters of 30-acetyl thymidine.71 The bis(POM)
`phosphotriester of ddU was also found to generate the ddU-TP from ddU-MP, which had been
`delivered to MT-4 cells.69 The utility of the approach was evident from the successful decrease in
`the cytopathic effects and virus production of HIV-1 by CEM/TK(cid:255) cells.7
`
`D. Para-Acyloxybenzyl(PAOB)Approach
`
`In an extension of the p-acyloxymethyl approach, the p-acyloxybenzyl approach has also been used
`to develop phosphotriesters of antiviral agents. Among the antiviral agents, bis(PAOB)
`phosphotriesters of AZT, bis(AZT) PAOB phosphotriester homodimers of AZT, and PAOB
`phosphotriester heterodimers of AZT and ddI have been studied (Fig. 9).72 –74 The advantage of the
`p-acyloxybenzyl approach is that upon ester hydrolysis, formation of the quinone methide results in
`cleavage of the C–O bond, thus liberating a phosphate hydroxyl group.46 Similar to the SATE or
`POM approaches, a phosphodiester is generated as an intermediate.
`The mechanism of decomposition of the p-acyloxybenzyl phosphotriesters is described in
`Fig. 9.72,73 Analogous to the bis(POM) phosphotriester approach, the PAOB approach relies on
`esterase-mediated hydrolysis to initiate the activation of the phosphotriesters. Upon formation of the
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`Figure9. Bis(PAOB) pronucleotide approach.74
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`unstable p-hydroxybenzyl phosphotriester, generation of the p-hydroxybenzyl carbonium ion is
`followed by the formation of the mono(PAOB) phosphodiester. The phosphodiester in turn,
`undergoes esterase-mediated hydrolysis to release the nucleoside 50-monophosphate, an acylate
`anion and a p-hydroxybenzyl carbonium ion. The disadvantage of this approach was postulated to
`be the reactivity of the p-hydroxybenzyl carbonium ion, which may react with either DNA, protein,
`g