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`
`Review
`
`Antiviral Chemistry & Chemotherapy 2011; 22:23-49 (doi: 10.3851/IMP1797)
`
`Nucleotide prodrugs for HCV therapy
`
`Michael J Sofia'*
`
`'Pharmasset, Inc., Princeton, NJ, USA
`
`*Corresponding author e-mail: michael.sofia@pharmasset.com
`
`
`HCV infection is a significant worldwide health problem
`and is a major cause of hepatocellular carcinoma. The
`current standard of care, interferon and ribavirin, is only
`effective against a proportion of the patient population
`infected with HCV. To address the shortcomings of exist-
`ing therapy, the development of direct acting antiviral
`agents is under investigation. The HCV RNA dependent
`RNA polymeraseis an essential enzyme for viral replica-
`tion and is therefore a logical target against which to
`develop novel anti-HCV agents. Nucleosides have been
`shown to be effective as antiviral agents for other viral
`diseases and therefore, have been investigated as inhibi-
`tors of HCV replication. The development of prodrugs
`of nucleoside 5’-monophosphates has been pursued
`
`to address limitations associated with poor nucleoside
`phosphorylation. This is required to produce the nucle-
`oside 5’-triphosphate which is the anabolite that is the
`actual
`inhibitor of the polymerase enzyme. Prodrugs of
`nucleoside 5’-monophosphates have been developed that
`enable their delivery into cells and in vivo into the liver.
`The implementation of these prodrug strategies has ulti-
`mately led to the identification of several prodrugs of
`nucleoside 5’-monophosphates that are potent
`inhibi-
`tors of HCV replication in vitro. They have progressed into
`the clinic and the early data demonstrate greatly reduced
`viral load levels in HCV-infected patients. This review will
`survey the state of nucleotide prodrugs for the treatment
`of HCV.
`
`Introduction
`
`HCV is known to have infected approximately 180
`million individuals worldwide [1]. It is estimated that
`of those infected with HCV approximately 80% will
`develop chronic liver disease and a significant propor-
`tion of those infected will eventually developliver cir-
`rhosis and subsequently hepatocellular carcinoma [2].
`HCVis a single-stranded, positive sense RNA virus of
`the Flaviviridae. Six major viral genotypes with over
`100 viral subtypes have beenidentified for HCV. Geno-
`types la and 1b are the most prevalent genotypes in
`the western world with genotypes 2 and 3 comprising
`20-30% of this population. HCV genotypes 2-6 pre-
`dominate in the developing world [3,4]. Because HCV
`replicates in the cytoplasm ofinfected cells by a mem-
`brane associated replication complex and the virus has
`an RNA genome with no DNAintermediate during
`replication, no genomic templates are stably integrated
`into the host genome. Therefore, virological cures are
`possible for HCV patients. This is in contrast to other
`viruses such as HIV and HBV wheretheviral genomeis
`integrated into the host DNA anda virological cureis
`considered remote. However, for HCV-infected patients
`virological cures are madedifficult due to the high rate
`of HCVviral replication and by the high spontaneous
`
`©2011 International Medical Press 1359-6535 (print) 2040-2066 (online)
`
`mutation rate of the virus. This high mutationrate is a
`result of poor replication fidelity exhibited by the HCV
`polymerase and an apparentlack of proof reading[4].
`The current therapy for treating chronic HCV infec-
`tion consists of regular injections of o-interferon (IFN)
`with daily oral administration of ribavirin (RBV). This
`standard of care (SOC) regimen does notact by directly
`attacking the virus but functions by boosting the host
`immuneresponse. For genotype 1 patients regular IFN/
`RBV treatments for 48 weeksresult in only 40-50% of
`patients achievinga sustained virological response (SVR)
`indicative of a cure [5,6]. However, for genotype 2 and
`3 patients the SVRrates can beas high as 75%. It is also
`knownthat subpopulations which include individuals of
`African ancestry tend to respond less well to IFN/RBV
`treatments [7]. Recent genome-wideassociation studies
`have shownthat a single nucleotide polymorphism 3kb
`upstream of the IL28B genecorrelates with a significant
`difference in response to IFN therapy [8]. IL28B which
`encodesthe typeIII interferon IFN-A-3, is knownto be
`upregulated by IFNs and by RNAviralinfections.It has
`been shown that HCVpatients who harbour a TT or TC
`allele in their IL28B genetend to respondless well to IFN/
`RBVtreatment than do those having the CC genotype.
`
`Gilead 2005
`I-MAK v. Gilead
`IPR2018-00125
`
`

`

`MJ Sofia
`
`Figure 1. Model of HCV NS5B RNA complex
`
`
`
`Ribbon structure of the HCV NS5B RNA dependent RNA polymerase showing the
`Palm, Finger and Thumb domainstypical of a RNA polymerase.Also depicted is a
`bound template-primer strand of RNA andthe nucleotide bindingsite.
`
`Patients who choose to undergo IFN/RBV therapy face
`not only the possibility of not responding to treatment
`but also must contendwith the potential for multiple and
`sometimes serious side-effects that include influenza-like
`symptoms, fatigue, hemolytic anaemia and depression.
`The intolerable side effects can result in a high rate of
`drug discontinuations. Consequently, the modest cure
`rates and subpopulation differences combined with the
`side-effect profile for SOC have prompted an urgency to
`develop alternative novel, safe and effective therapies.
`As in the case of other viral diseases, the development
`of direct acting antivirals (DAAs) has become a focus.
`Development of small molecule agents to attack essen-
`tial viral proteins has the potential benefit of reducing
`toxicities and side effects associated with manipulating
`host functions and hopefully such DAA therapies will
`not have the intolerable side effects exhibited by cur-
`rent SOC.
`The push to identify small molecule DAAshasalso
`promptedthe discussion aroundthe possibility of elimi-
`nating or at least reducing the use of IFN/RBV from
`treatment regimens. Although clinical development of
`first generation DAAs has focused on combinations
`with SOC in the hope of both shortening duration of
`treatment and increasing the cure rate, the long-term
`desire is to completely eliminate the use of IFN/RBV
`from treatment regimens. Clearly this is an aspirational
`goal and a goalthat can only be achieved if an immune
`component of therapy is not absolutely required to
`eliminate those vestiges of undetectable virus
`[9].
`In addition, such a lofty goal can be realised if small
`
`24
`
`molecule DAAseither alone or more probably in com-
`bination can drive viral loads to undetectable limits and
`maintain those undetectable levels over the necessary
`course of therapy andafter cessation of treatment with-
`out having viral breakthrough resulting from the emer-
`genceofresistant virus. As has been shown with HIV
`highly active antiretroviral therapy (HAART), the HCV
`treatment paradigm will likely require combinations of
`anti-HCV agents [7]. The desire is to suppress virus as
`rapidly and completely as possible in order to give the
`body’s natural immunesystem the opportunity to clear
`residual virus and to hold back emergence of resistant
`virus. However, what will comprise those ideal com-
`binations of DAAsis yet to be determined and studies
`to clarify this question are under active discussion and
`investigation.
`HCVhasa 9.6 kb genomeof positive-stranded RNA.
`This genome encodes a precursor polyprotein thatis
`processed into 10 functional proteins: three structural
`proteins and seven non-structural proteins [10]. Sev-
`eral of the non-structural proteins have been the focus
`of intensive efforts to identify small molecule DAA
`agents as inhibitors of HCV replication. Of the seven
`non-structural proteins, molecules that inhibit the func-
`tions of the NS3/4 protease, NS4A, NS4B, NSSA and
`NSSB RNA dependent RNA polymerase (RdRp) have
`advanced to the clinic [11-15]. The most advanced
`agents are the NS3/4 protease inhibitors telaprevir and
`boceprevir. Each of these compounds has completed
`Phase III clinical
`investigation and both have been
`shownto be efficaciousin treating HCV infection when
`given in combination with SOC. However, each of these
`first generation protease inhibitors suffers from the lack
`of genotype coverage, undesired side effects, that may
`limit their usage, and the early emergence of resistant
`virus. It is therefore not surprising that even with the
`potential benefits of these first generation DAAs, ware-
`housingof patients by physicians occursin order to wait
`for approval of moreeffective and tolerable agents.
`The HCV RNA dependent RNA polymerase is an
`~68 kd protein that has the typical palm-finger-thumb
`structural motif found in many viral polymerases(Fig-
`ure 1) [16,17]. HCV polymeraseis an essential enzyme
`involved in RNA replication. Phylogenetic analysis
`shows a 65% homology of HCV RdRpacross genotypes
`and an 80% homologywithin a particular genotype [3].
`The HCV polymeraseactivesite is located in the palm
`domain where the conserved aspartic acid residue-con-
`taining GDD motifis located [18]. This conserved GDD
`motif is commonto viral polymerases in general [18].
`Through a divalent metal ion (Mg** or Mn**) the GDD
`motif functions to coordinate the bindingof the ribonu-
`cleoside triphosphate. The HCV polymerase catalyzes
`the addition of a single ribonucleoside triphosphate
`monomerto the 3’-end of the growing RNAchain by the
`
`©2011 International Medical Press
`
`

`

`formation of a 3’,5’-phosphodiester linkage. To accom-
`plish this process, the polymerase must simultaneously
`bind a template RNAstrand, a primer RNAstrand and
`a ribonucleoside triphosphate monomer[19,20]. There-
`fore, investigation of nucleoside analoguesis a rational
`choice for the development of inhibitors of the HCV
`NSS5B polymerase.
`Ofall the DAAsunderclinical investigation, nucleo-
`side/nucleotide NS5B polymerase inhibitors hold the
`promise of pan-genotype coverage and a high barrier
`to developmentofresistant virus. As in the case of HIV
`infection where nucleosides have becomethe backbone
`of therapy (for example, TRUVADA® and Combivir®),
`HCVnucleosides/nucleotides are positioned to assume
`a similar role. To date, only nucleosides/nucleotides
`have demonstrated broad genotype coverage both in
`the laboratory and in humanclinical studies [21]. In
`addition, to date, no pre-emergentresistant virus has
`been detected in clinical studies [22]. It is for these
`reasons that nucleosides/nucleotides are positioned to
`play a prominentrole in developing HCV treatment
`paradigms.
`The HCV polymerase has been shown to be a
`uniquely selective polymeraseas it relates to the devel-
`opment of nucleoside/nucleotide inhibitors. Over
`the last 10 years only two broad classes of nucleo-
`sides have emerged as inhibitors of this polymerase
`[23-25]. These include the 2’-methyl and the 4’-azido
`classes (Figure 2). However,
`these classes and sub-
`groups within these classes have clearly differentiated
`themselves in preclinical and clinical studies. This
`differentiation is exemplified in their viral selectivity,
`viral resistance, overall safety andclinical efficacy pro-
`files. Resistance associated with the 2’-methyl class of
`nucleosides is associated with the $282T amino acid
`alteration located in the finger domain of the HCV
`polymerase [26-29]. This mutation has been shown to
`be difficult to raise in vitro and has not been detected
`as a pre-existing mutation in clinical
`isolates [22].
`Similarly, for the 4’-azido class, the S96T amino acid
`alteration has been identified in vitro but has not been
`observedin the clinic [27].
`Implementation of a prodrug strategy has played
`a prominent role in the development of a number of
`nucleosides and nucleotides for the treatment of HCV
`infection [23,30]. These prodrugs have been developed
`to overcome, not only, bioavailability and stability
`issues but also to address key anabolism limitations
`important to nucleoside activation. Simple ester prod-
`rugs of both the 2’-methylcytidine, 2’-c-fluoro-2’-B-C-
`methylcytidine and 4’-azidocytidine nucleosides were
`developed to overcome both bioavailability issues and
`to curb undesirable metabolism [21,31,32]. Prodrugs of
`the phosphate group of nucleoside 5’-monophosphates
`were developed to address not only bioavailability
`
`Antiviral Chemistry & Chemotherapy 22.1
`
`Nucleotide prodrugs for HCV therapy
`
`Figure 2. Two major classes of nucleosides that are known to
`be inhibitors of HCV polymerase.
`
`2’-Methyl class
`
`4’-Azido class
`
`HO
`
`2
`
`BASE
`
`HO
`
`=X
`
`Xe OH, F
`
`BASE
`
`Ho“
`
`\ 9
`
`N3*
`HO
`
`=X
`
`X = OH, F
`
`These classes include the 2’-methyl and the 4’-azido ribosides.
`
`issues but also poor in vitro and in vivo conver-
`sion of the parent nucleoside to the active nucleoside
`5’-triphosphate [33]. Because nucleosides must be con-
`verted to their 5’-triphospates to be active as inhibitors
`of the HCV polymerase, they need to undergo a series
`of phosphorylation steps catalyzed by three separate
`kinases (Figure 3). These kinases convert the nucleoside
`first to the monophosphate, then to the diphosphate
`and finally to the active triphosphate. However, it is
`not uncommonthat in the phosphorylation cascade, a
`nucleoside or its corresponding mono- or diphosphate
`is a poor substrate for one of the kinases. In particular,
`it is the first kinase in the phosphorylation cascade that
`is generally the most substrate selective. Therefore, it
`is not unusual that bypassingthefirst kinase results in
`achieving high levels of the active triphosphate. Because
`nucleoside monophosphates are enzymatically dephos-
`phorylated and negatively charged, they do notreadily
`enter cells and therefore are not desirable as drug can-
`didates. To overcomethe limitations of administering
`a nucleoside monophosphate-containing agent, pro-
`drugs of the 5’-monophosphate nucleoside have been
`employed. Prodrugs of nucleoside monophosphates
`have been known for many years and a number of
`phosphate prodrug strategies have been developed to
`address the need to deliver a 5’-monophosphate nucleo-
`side into the cell [33-35]. However, there have been few
`examples where a nucleotide prodrug has been shown
`to deliver the corresponding 5’-monophosphate in vivo
`to the desired site of action [33]. Often the prodrug
`moiety decomposes prior to achieving its objective
`because of either chemical or enzymatic instability in
`the gastrointestinal tract and/or plasma.
`The development of a nucleoside phosphate prod-
`rug useful for the treatment of HCV faces several chal-
`lenges. The nucleoside phosphate prodrug must have
`sufficient chemical stability to be formulated for oral
`
`25
`
`

`

`M$ Sofia
`
`Figure 3. Nucleoside kinase activation pathway
`
`BASE
`[BASE
`ONo.
`[BASE|
`o.
`HO
`
`
`™ od _Kinase2aKinase 1 ad
`be X
`\ bx
`\__
`Y
`HO
`OY
`
`oo
`
`
`
`HO
`
`|BASE]
`_Kinase33
`= Phosphate
`
`Nucteoside kinase activation pathwayresulting in the nucleoside triphosphate whichis the active substrate for a polymerase allowing incorporation of the nucleoside
`or nucleoside analogueinto the growing RNA chain andthusresulting in inhibition of virus replication. Examples of kinases 1, 2, and 3 include deoxycytidine kinase
`(dCk), nucleoside monophosphate kinase (YMPK) and nucleoside disphosphate kinase (NDPK), respectively,
`
`administration. It must be stable to conditions of the
`gastrointestinal tract such that the prodrug reaches the
`site of absorption intact. The prodrug must have good
`absorption properties and must not undergo apprecia-
`ble enzymatic degradation during the absorption phase.
`Once absorbed, the prodrug needsto havesufficient sta-
`bility in the bloodin order to reach the target organ: for
`example theliver in the case of HCV. The prodrug must
`then be transported into hepatocytes andrelease the free
`5’-monophosphate nucleoside which can subsequently
`be converted to the active triphosphate derivative. Since
`HCVis a disease of the liver, and the liver is the first
`organ the prodrug encounters after absorption, HCVis
`an ideal disease for which to develop a targeted nucle-
`otide prodrug strategy. Consequently, several of these
`nucleoside monophosphate prodrugs have advanced
`to the clinic and have demonstrated proof of concept
`for treating HCV. Here, the application of phosphate
`prodrugs in the development of nucleotide inhibitors
`of HCV andthe status of nucleotide prodrugs under
`investigation for the treatment of HCV infection will
`be reviewed.
`
`Nucleotide phosphoramidates
`Nucleotide phosphoramidates were first disclosed
`by McGuigan et al. [36] as a prodrug strategy to
`deliver a nucleoside 5’-monophosphateforthe treat-
`ment of HIV and cancer. The structure of a nucle-
`otide phosphoramidatetypically consists of a nucleo-
`side 5’-monophosphate where the phosphate group
`is masked by appending an aryloxy group (usually a
`phenol) and an o-amino acid ester (Figure 4); how-
`ever, other related constructs have also appeared. The
`
`26
`
`phosphate groupis ultimately revealed by a sequence
`of enzymatic and chemical steps that requires either
`carboxyesterase or cathepsin A to cleave the terminal
`amino acid ester, intramolecular displacement of the
`phosphate phenoland then enyzmatic cleavage of the
`amino acid moiety by a phosphoramidaseorhistidine
`triad nucleotide-binding protein 1 (HINT 1) [37-39].
`It
`is believed that
`the phosphoramidate prodrug
`construct
`increases lipophilicity of the nucleoside
`5’-monophosphate and therefore increases cellular
`permeability and ultimately intracellular nucleotide
`concentrations. Since the phosphoramidate prodrug
`moiety contains a chiral phosphorus centre,
`issues
`arise with regard to development of a compoundthat
`consists of a mixture of isomers with implications
`arising from differential activity of each of the iso-
`mers, pharmacokinetics and manufacturing optimi-
`zation, etc. In addition, the typical phosphoramidate
`contains a phenolic substituent that is released during
`metabolism to the free monophosphate. Successful
`development also considers the metabolic release of
`this phenolic substituent. Selective examples employ-
`ing the phosphoramidate strategy to achieve kinase
`bypass for nucleoside inhibitors of HIV reverse tran-
`scriptase (RT) inhibitors, for example, ddA, d4T and
`d4A, showed that in vitro whole cell enhancement
`in potency could be achieved [40-42]. Although the
`phosphoramidate strategy was explored extensively to
`deliver nucleotides for the treatment of HIV and colon
`cancer [33,36], proof of concept in the clinic has yet
`to be reported. However, the phosphoramidate prod-
`rug approach has provento be a valuable strategy in
`the development of HCV nucleotide therapy.
`
`©2011 International Medical Press
`
`

`

`Figure 4. The phosphoramidate prodrug decomposition pathwaythatresults in the release of the nucleoside 5'-monophosphate
`
`Nucleotide prodrugs for HCV therapy
`
`Oo
`
`Ro
`
`OQ
`
`Ra
`
`Carboxyesterase \ R
`\ G
`¢ oe) “se
`fHoe)
`Ry
`9
`Cathepsin A
`H
`id
`Ar
`Ar
`
`
`
`Nucleotide phosphoramidate
`
`oO
`
`Spontaneous
`
`alae\-4
`
`A
`
`oO
`
`
`
`HINT-1
`
`h
`;
`(Phosphoramidase)
`
`4
`HO-F=0
`OH
`
`Ro
`
`a
`HN=F-o
`O
`
`|H
`
`oO
`
`G
`H
`
`2’-C-Methyl ribonucleotide phosphoramidates
`The 2’-C-methylcytidine nucleoside, NM107 (1; Fig-
`ure 5), was shown to be an inhibitor of HCV in cell
`culture (50% effective concentration [EC,,] =1.23 UM)
`and its triphosphate (3) was demonstrated to be a
`potent inhibitor of the HCV polymerase enzyme (50%
`inhibitory concentration [IC,,] =0.09-0.18 4M)acting
`as a nonobligate chain terminator [43]. NM107 also
`showed broad antiviral activity against not only HCV
`but also bovine virus diarrhoea virus (BVDV), yellow
`fever virus, dengue virus and West Nile virus [31]. To
`overcome bioavailability issues the 3’-valinate ester
`prodrug, NM283(valopicitabine; 2) [31,44], was taken
`into clinical development. In a Phase I monotherapy
`study, NM283 demonstrated proof of conceptdeliver-
`ing an ~1.2 log, [U/mlreductionin viral load at a dose
`of 800 mg twice daily given over 14 days. Unfortu-
`nately, NM283 was discontinued because of significant
`gastrointestinal toxicity in Phase II studies [24,30].
`Subsequent work on the development of the 2’-C-
`methyl class of nucleosides focused on 5’-phosphate
`nucleotide prodrugs. It was observed that the 2’-C-
`methylcytidine triphosphate (3) was highly active as
`an inhibitor of the NSSB polymerase, yet the parent
`
`Antiviral Chemistry & Chemotherapy 22.1
`
`nucleoside NM107 (1) was only modestly activein the
`whole cell based replicon assay. Studies had shown that
`NM107-triphosphate (3) formation was inefficient,
`particularly because of poor conversion of the nucleo-
`side to its monophosphate by 2’-deoxycytidine kinase
`[45]. Consequently, to circumvent this phosphoryla-
`tion problem and potentially improve the therapeutic
`index by increasing nucleoside triphosphate levels in
`the liver, a phosphoramidate prodrug approach was
`investigated [45]. This effort lead to the identification
`of phosphoramidate derivative 4 (Figure 5; EC,,<0.5
`uM) showing substantial increases in potency rela-
`tive to NM283[45]. The activity of compound 4 cor-
`related with the levels of triphosphate produced in
`humanhepatocytes and these levels were shown to be
`muchhigher than that seen with NM283 (2). In vivo
`studies assessing liver nucleoside triphosphate levels
`after oral administration in hamsters showedlowtri-
`phosphate concentrations only twofold higher than
`obtained with NM283. Since substantial
`liver tri-
`phosphatelevels were seen after subcutaneous admin-
`istration iz vivo and the compounds were shown to
`be stable in simulated gastric fluid, it was concluded
`that low oral bioavailability or metabolic degradation
`
`27
`
`

`

`M3 Sofia
`
`Figure 5. 2’-C-Methylcytidine nucleosides and nucleotide phosphoramidate prodrug inhibitors of HCV replication
`
`7X
`|
`vrN
`—
`HO
`O NY
`HO OA
`0
`QO
`NH2
`C
`.
`-
`OH
`ge
`O
`
`HO=OH |
`
`NH»
`
`NH»
`
`O
`
`NM107
`EC,,=1.23 uM
`1
`
`NM283
`2
`
`NH»
`
`oO
`O
`HO—P—o—P—o—P—o-*
`l
`l
`OH
`OH
`OH
`
`.0. Ny
`VV ~~
`toes
`
`F
`
`HO
`
`OH
`
`IC,,=0.09-0.18 uM
`3
`
`NHz
`
`NH2
`
`EC,=0.22 uM, CC,,=7 uM
`4
`
`EC,,=0.24 uM
`5
`
`opcing
`
`OH
`
`oO
`
`HO
`
`OH
`
`NTP (human hepatocytes) AUC,,,,=190 uMeh
`
`7
`
`sat
`
`0
`
`HO
`
`OH
`
`EC,=8.2 uM, CC,,=>100 1M
`NTP (human hepatocytes) AUC.
`‘o-ay= 1720 uMeh
`6
`
`
`
`AUC,area under the curve; CC,,, 50% cytotoxic concentration; EC,,, 50%effective concentration; IC,,, 50% inhibitory concentration.
`
`28
`
`©2011 International Medical Press
`
`

`

`Nucleotide prodrugs for HCV therapy
`
`in the intestine was the reason for the lack of oral
`efficacy [45].
`Anotherseries of 2’-C-methylcytidine phosphorami-
`date prodrugs having an acyloxyethylamino phospho-
`ramidate promoiety was studied (Figure 5) [46]. The
`phosphoramidate prodrug 5 provided up to a 30-fold
`improvement in HCV replicon potency over NM283
`(2), and it was also shown that this activity correlated
`to levels of nucleoside triphosphate in rat and human
`hepatocytes. However, when administeredorally to rats
`these phosphoramidates demonstrated no improvement
`in production of triphosphate in rat liver relative to
`NM283(2). These results put into question the oral
`bioavailability and conversion of these prodrugs to the
`nucleoside triphosphatein theliver.
`Phosphoramidate monoesters of 2’-C-methylcytidine
`were also explored (Figure 5) [45]. In this case the ami-
`date moiety was either an o-amino acid (6) or an acy-
`loxyethylamino substituent (7). Although phosphorami-
`date monoester 6 was shownto haveinferior replicon
`potencyrelative to its phenolic ester counterpart (<200-
`fold) it showed higher levels of triphosphate formation
`in human hepatocytes. Formation of nucleoside triphos-
`phate levels were observed in hepatocytes of various spe-
`cies for both 6 and 7 but the phosphoramidate monoester
`
`6 was shownto besuperiorto 7. In bothcases,liver levels
`of nucleoside triphosphate 3 were achieved in vivo after
`subcutaneous administration but not after oral adminis-
`tration suggesting a lack of oral bioavailability.
`Other 2’-C-methyl nucleosides containing purine
`bases were also investigated as inhibitors of HCV,
`including the 7-deaza-2’-C-methyladenosine derivative
`MK0608(8) which showed potentinhibition of HCV
`replication in vitro (EC,,=0.25 4M) and demonstrated
`SVR in an HCV-infected chimpanzee animal model
`[47,48]. Although MK0608 (8) was never progressed
`into clinical development, it did demonstrate the poten-
`tial of purine nucleosides as inhibitors of HCV. Sub-
`sequently, phosphoramidate prodrugs of 2’-C-methyl
`purine analogues werestudied to determineif improve-
`ment in potency could be achieved by kinase bypass.
`Although application of the phosphoramidate prodrug
`strategy was not successfulfor the adenosine derivative
`11 (Figure 6), its application to the guanosine analogue
`12 resulted in an 84-fold increasein activity in the rep-
`licon assay relative to the parent nucleoside (Figure 7)
`[49]. This result was rationalized by the observation
`that the guanosine triphosphate (13; IC,,=0.13 MM)
`was more potent as an inhibitor of the HCV polymer-
`ase than the adenosine triphosphate (10; IC,,=1.9
`
`
`
`Figure 6. 2’-C-Methyladenosine nucleoside and nucleotide phosphoramidate prodrug inhibitors of HCV replication
`
`EC,=0.25 uM, CC,,=>50 uM
`8
`
`EC,,=0.3 uM, CC,,=>50 uM
`9
`
`HO
`
`OH
`
`NH2
`
`NH»
`i
`hs
`“ Xk
`9
`O
`OQ
`N
`N
`if
`i
`i
`U1
`neeas y=
`
`OH
`
`OH
`
`OH
`
`1
`
`al
`
`-
`
`HO
`
`OH
`
`IC,,=1.9 uM, EC,,=0.3 uM
`10
`
`Eto
`
`oO
`
`CHs
`
`a
`
`¢ jn
`Ayttoe
`TON i, Vy n=
`
`oe HO
`=.
`
`OH
`
`EC,,=0.25 pM, CC,,=>50 uM
`14
`
`
`
`CC,,, 50% cytotoxic concentration; EC,,, 50% effective concentration; IC,,, 50% inhibitory concentration.
`
`Antiviral Chemistry & Chemotherapy 22.1
`
`29
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`

`

`MJ Sofia
`
`Figure 7. 2’-C-Methylguanosine nucleoside and nucleotide phosphoramidate prodrug inhibitors of HCV replication
`
`a
`
`O°
`N
`Cw
`NN
`at 2
`
`Lo.
`
`8
`e
`HO—P—O—P—o-P—o-*%
`
`OH
`
`OH
`
`OH
`
`0I
`
`O N
`
`H
`
`NC
`
`J
`iVey,
`
`HO~
`
`O
`
`HO
`
`OH
`
`EC,,=3.5 uM
`
`HO
`
`OH
`
`IC,,=0.13 uM
`13
`
`x
`0.
`
`CH
`
`pe,
`
`|
`
`;
`
`oc
`“Nn
`Ph
`=
`
`N
`
`¢ /
`
`N
`
`H
`
`f
`,
`4
`Oo
`Oo
`“Tt HNO"Ne
`co" °
`
`14 R.=CH,CH,Ph, R,=Ala EC,,=0.08 1M, CC,,=>100 uM
`15 R.=0-CIPh, R,=Val EC,,=0.43 uM, CC,,=>100 uM
`
`INX-08189
`
`EC,,=0.010 uM, CC,,=7 uM
`16
`
`IDX184
`
`EC,,=0.4 uM, CC,,>100 uM
`
`
`
`CC,,, 50% cytotoxic concentration; EC,,, 50% effective concentration; IC, 50% inhibitory concentration.
`
`uM), yet in the whole cell replicon assay the adenosine
`analogue (9; EC,,=0.3 4M; Figure 6) was more potent
`than the guanosine analogue (12; EC,,=3.5 1M;Figure
`7). Therefore, this finding coupled with the observa-
`tion that low levels of triphosphate were detected in
`cells for the guanosine nucleoside relative to that for
`the adenosine nucleoside hinted at poor phosphoryla-
`tion in the case of the guanosine nucleoside. The devel-
`opmentof the 2’-C-methylguanosine phosphoramidate
`derivative was further explored by systematically eval-
`uating structural modifications to the phosphorami-
`date moiety in an effort to achieve increased HCVrep-
`licon potency, plasma stability across multiple species,
`and appropriaterelative stability in intestinal andliver
`$9 preparations. Ultimately,
`these studies were able
`to identify 2’-C-methylguanosine phosphoramidate
`
`30
`
`prodrugs 14 and 15 (Figure 7) [50]. These prodrugs
`contained benzylor alkyl L-alanine and t-valine amino
`acid ester moieties and a naphthyl phosphate ester and
`exhibited a 10-30-fold enhancement in HCV repli-
`con potency with acceptable plasma andintestinal $9
`stability suitable for progression into in vivo studies.
`However, when mice were dosed orally in order to
`assessliver levels of the nucleoside triphosphate 13, the
`2’-C-methylguanosine phosphoramidates 14 and 15
`(Figure 7) produced substantial liver levels of triphos-
`phate but these levels were notsignificantly improved
`over that observed when mice were dosed with the par-
`ent guanosine nucleoside 12.
`Further investigation of the 2’-C-methylguanosine
`phosphoramidateseries led to the evaluation of substi-
`tution at the C-6-position of the guanosine base with the
`
`©2011 International Medical Press
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`

`

`Nucleotide prodrugs for HCV therapy
`
`reported to date. In the whole cell HCV replicon assay
`intention of increasing lipophilicity and thus improving
`IDX184 (17) was shownto be a potentinhibitor of HCV
`cellular uptakerelative to the natural guanosine deriva-
`replication (EC,,=0.4 1M; CC,,>100 mM)and was also
`tive 12. This led to the identification of the C-6-O-methy!
`active in the genotype 2a JFH1 replicon (EC,,=0.6—11
`derivative INX-08189 (16; Figure 7) containing both a
`LM)[59]. At a concentration of 2.5 1M it cleared HCV
`neopentyl ester on the L-alanyl amino acid moiety and
`replicon RNA after 14 days of treatment. Like other
`a naphthyl ester on phosphorus of the phosphorami-
`2’-C-methyl nucleosides it was shown to select for the
`date {51]. In INX-08189 (16), in addition to metabolic
`NSSB $282T mutation in the HCVreplicon. In combi-
`conversion of the phosphoramidate pro-moiety to the
`nation with the protease inhibitor IDX320, IFN-o, or
`5’-monophosphate, the 6-O-methy] group of the purine
`RBV, IDX184 exhibited an additive or synergistic profile
`base is metabolized to the guanine base. INX-08189
`[60]. When administered orally to cynomolgus monkeys,
`(16) demonstrated exceptional potency in the HCV 1b
`IDX184 (17) produced high live triphosphate levels
`replicon assay (EC,,=0.01 uM; CC,,=7 UM), was active
`relative to oral administration of the parent nucleoside
`against genotype 1a and 2 replicons and produced sub-
`with what appears to be high hepatic extraction. Sub-
`stantial levels of the guanosine triphosphate 13 in pri-
`sequent studies in a HCV-1-infected chimpanzee model
`mary human hepatocytes over 48 h. The known 2’-C-
`showed that oral administration of IDX184 (17) at 10
`methyl nucleoside $282T mutant replicon was shown
`mg/kg over 3 days produced a medianviral load decline
`to be moderately resistant (3- to 10-fold) to INX-08189
`of approximately -2.3 log,, at day 3 and 4 [61]. Conse-
`(16) [52]. No difference in HCV replicon potency was
`quently, IDX184 (17) was progressed into the clinic and
`observed for each of the individual diastereoisomers of
`in a PhaseIa single ascending dose study was shown to
`INX-08189 (16), thus INX-08189 was advanced into
`be generally safe and well tolerated at oral doses from 5
`clinical development as a mixture of isomers at the
`mg to 100 mg [56]. PK assessment supportedalivertar-
`phosphoruscentre of the prodrug.
`geting mechanism for the compound.In a Phase Ib mon-
`In a single-ascending-dose Phase Ia study in healthy
`otherapy study in HCV genotype-1-infected patients,
`volunteers administered doses ranging from 3 mg to
`IDX184 (17) was administered at doses from 25 to 100
`100 mg, INX-08189 (16) was shown to be generally
`mg once a day for 3 days. Day 4 viral load assessment
`well tolerated at all doses with no drug-related serious
`showedthatat the highest dose of 100 mg,a -0.74 log,,
`adverse events and pharmacokinetics (PK) supporting
`IU/ml reduction in viral load was observed [62]. Fol-
`once daily oral dosing [53,54]. INX-08189 was pro-
`lowing the positive Phase I clinical results, a Phase II
`gressed into a Phase Ib study in treatment-naive geno-
`study wasinitiated in which IDX184 (17), at doses from
`type 1 HCV patients dosed once daily at either 9 mg
`50-200 mg, was combined with pegylated IFN and RBV
`or 25 mg. Antiviral activity was observed with a mean
`for 14 days in treatment-naive HCV genotype i patients
`HCV RNAreduction of -0.71 and -1.03 log,, TU/ml,
`[63]. At day 14 viral load reductionsof-2.7 to -4.1 log,,
`respectively [54,55]. These early human data demon-
`TU/ml were achieved with no adverse events attributed
`strated clinical proof of concept for INX-08189 and
`to IDX184 (17) and with no detection of resistant virus
`consequently, the compound is continuing to undergo
`[64]. However, in an attempt to evaluate the efficacy
`clinical evaluation.
`of a DAA combination of IDX184 (17) with the NS3
`Another application of phosphoramidate prodrug
`protease inhibitor IDX320, three serious adverse events
`technology for
`delivering a
`2’-C-methylguanosine
`were reported in a drug—druginteraction study in healthy
`5’-monophosphate is exemplified by IDX184 (17; Figure
`volunteers and consequently, the programmewasplaced
`7) [56]. In this case the phosphoramidate prodrug moi-
`onclinical hold in September 2010, awaiting resolution
`ety utilized a benzyl! aminein place of an amino acid for
`of the cause of the adverse events [65,66]. In February
`the amine substituent and an S-acety]-2-thioethyl moiety
`2011,
`the US Food and Drug Administration (FDA)
`(SATE)as the phosphate ester substituent in place of the
`removed full clinical hold for IDX184 (17) owing to
`more commonaryl group. The SATE group is a known
`evidence that the toxicity waslikely caused by IDX320,
`phosphate ester prodrug construct [33,57,58]. Mecha-
`and the programme wasplaced onpartial clinical hold.
`nism for release of the desired 2’-C-methylguanosine
`Initiation of a Phase [lb 12-weektrial of IDX184 (17)in
`5’-monophosphateis believed to involve both CYP450-
`combination with pegylated IFN and RBVis