NEW TOOLS IN SYNTHESIS
`
`SYNLETT
`
`233
`
`Pro-Nucleotides - Recent Advances in the Design of Efficient Tools for the Delivery of
`Biologically Active Nucleoside Monophosphates
`C. Meier*
`Institut für Organische Chemie, Julius-Maximilians-Universität Würzburg, Am Hubland, 97074 Würzburg, Germany
`Tel. +49-931-8885326; Fax +49-931-8884606; e-mail: meier@chemie.uni-wuerzburg.de
`Received 10 September 1997
`Dedicated to Professor Gernot Boche on the occasion of his 60th birthday
`
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`
`two nucleotide kinases)(8,9). Finally, the resistance of the human
`immunodeficiency
`virus
`to
`the
`clinically
`used
`antiviral
`dideoxynucleoside AZT 2 (Zidovudine, Retrovir®)(10) is on the one
`hand directly related to multiple point mutations within the HIV-1
`reverse transcriptase gene of the virus but may on the other hand be also
`due to lower susceptibility of resistant target cells to the drug related
`with a decreased activity or inability of the enzyme thymidine kinase to
`phosphorylate AZT 2
`to
`the dideoxynucleoside monophosphate
`AZTMP 9(11).
`
`Consequently, direct administration of the nucleotides d4TMP 4,
`ddAMP 8 and AZTMP 9 should bypass these limiting steps and hence
`has advantages for the biological activity. Unfortunately, because of the
`high polarity of the nucleoside monophosphates (nucleotides), these
`compounds are not able to easily penetrate cellular membranes or the
`blood-brain barrier. However, the phosphate moiety offers a suitable
`site to attach degradable lipophilic carrier residues. As a result, one
`effort to improve the therapeutic potential of nucleoside analogues is the
`delivery of the corresponding nucleotide from neutral, membrane-
`permeable prodrugs (Pro-Nucleotide Approach; Figure 2)(12).
`
`Abstract: A summary of the most recent advances to the design of pro-
`nucleotides will be presented. Approaches that have been designed to be
`activated by enzymes such as carboxyesterases [bis(POM)-, bis(POC)-,
`bis(SATE)-, bis(AB) phosphotriesters and the arylphosphoramidates] or
`by reductases [bis(SDTE) approach] will be discussed as well as the
`amino acids phosphoramidate diester concept with its still unknown
`delivery mechanism and the cycloSal approach that releases the
`nucleotides by an induced tandem reaction.
`Nucleoside analogues, e.g. 2',3'-dideoxy-2',3'-didehydrothymidine 1
`(d4T), 3'-azido-2',3'-dideoxythymidine 2
`(AZT) or 5-fluoro-2'-
`deoxyuridine 3 (5-FdU), are structurally different as compared to the
`corresponding natural DNA or RNA nucleosides with regard to
`modification of the glycon as well as the aglycon residue. Due to this
`modified structure, these compounds are widely used as antiviral or
`antitumor drugs in chemotherapy (Figure 1)(1). Since the discovery of
`AZT 2 as the first nucleoside drug for the treatment of AIDS,
`considerable efforts have been made to develop new nucleoside
`analogues that would be more active, less toxic inhibitors of the HIV-1
`reverse transcriptase (RT)(2). The general mode of action of nucleoside
`analogues is the inhibition of the HIV-1 RT by acting as competitive
`inhibitors or as DNA chain terminators. To act as DNA chain
`termination agents or RT inhibitors, intracellular conversion of the
`nucleoside analogues into their 5'-mono-, 5'-di- or 5'-triphosphates is a
`prerequisite after cell penetration(3). The enormous disparity in anti-
`HIV activity that is evident for a large number of dideoxynucleoside
`analogues belies their apparent structural similarity. Due to these
`structural differences as compared
`to natural nucleosides
`the
`metabolization to the corresponding dideoxynucleoside triphosphates is
`often inefficient and consequently the therapeutic efficacy is sometimes
`limited(4). For example,
`in
`the case of
`the anti-HIV active
`dideoxynucleoside analogue d4T 1 (Stavudine, Zerit®)(5) the first
`phosphorylation to d4T 5'-monophosphate 4 catalyzed by thymidine
`kinase (TK) is the rate-limiting step in human cells(6). More striking,
`however is 2',3'-dideoxyuridine triphosphate (ddUTP) which is one of
`the most powerful and selective inhibitors of HIV reverse transcriptase
`(Ki = 0.05 μM) while the parent nucleoside 2',3'-dideoxyuridine 5 (ddU)
`is virtually ineffective at blocking HIV infection in cultured cells.
`Biochemical and pharmacological studies in three different human T
`cell lines (CEM, ATH8 and Molt-4) showed that ddU 5 itself was not
`anabolized to the 5'-monophosphate, most apparently because it was a
`poor substrate for cellular nucleoside kinases because of
`the
`considerable substrate specificity of these enzymes(7). In contrast, in a
`few cases the limited efficacy is also due to a catabolic enzymatic
`reaction. For example, 2',3'-dideoxyadenosine 6 (ddA) is rapidly
`intracellularly deaminated to ddI 7 by adenosine deaminase (ADA)(8,9).
`As a consequence, ddI 7 has to be converted into its ultimate bioactive
`metabolite ddATP via ddAMP 8 by five enzymatic steps (5'-
`nucleotidase, adenylosuccinate synthase, adenylosuccinate lyase and
`
`1
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`234
`
`C. Meier
`
`SYNLETT
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`group attached to the phosphorus are known to be toxic suicide
`inhibitors of acetylcholinesterase(15). The anti-acetylcholinesterase
`activity of phosphorus derivatives is an inverse function of the pKa of
`the leaving group on the phosphorus atom and parallels the rate of
`spontaneous hydrolysis by P-O bond cleavage(16). In order to
`circumvent the possible problem of anti-cholinesterase activity, neutral
`phosphate ester prodrugs should be designed to undergo heterolytic
`cleavage of the C-O bond rather than the P-O bond of the ester.
`Many strategies have been developed to achieve this goal. As a general
`motive, uncharged nucleotide triesters are used as membrane-permeable
`nucleotide precursors(12). The major differences of these approaches are
`the delivery mechanisms to the nucleotides. First attempts have been
`made with simple dialkyl phosphotriesters. These compounds generally
`belong to the class of bipartate prodrug systems. After a first, sometimes
`selective hydrolysis of the phosphotriester via a nucleophilic reaction at
`the phosphorus center, the resulting phosphodiester is often extremely
`stable against a further chemical hydrolysis due to the charge at the
`phosphate which prevents a second nucleophilic reaction(17). Even if the
`subsequent hydrolysis is possible, one should take the pseudorotation
`phenomenon into account that excludes a selective delivery of the
`nucleotide(18). As a consequence, almost all approaches based on
`chemical hydrolysis reported so far were unable to deliver the
`nucleotide selectively except the cycloSal approach that will be
`discussed later. For this reason, the newer pro-nucleotide approaches are
`based on the concept of a tripartate prodrug system(14) and are based on
`the general idea of a selective chemical or enzymatic reaction within the
`masking group which leads to a second, spontaneous successive
`reaction yielding the charged phosphate ester. These approaches utilize
`and exploit the differences in reducing potentials, enzyme activity, and
`pH value. The concepts working with enzymatic trigger processes
`(bis(POM)-, bis(POC)-, bis(DTE)-, bis(SATE)-, bis(AB)- and the
`arylphosphoramidate concept) as well as the cycloSal approach based
`on a pH-driven degradation have demonstrated
`the successful
`intracellular delivery of free nucleotides from lipophilic precursors.
`It should be added, that it is not the intention of the author to give an
`entire overview of the pro-nucleotide field. The current review
`highlights some approaches that have been designed to deliver the
`nucleotide selectively by a special mechanism. With this selection, the
`author wants to point out that designing a delivery mechanism and
`bypassing a certain limiting metabolization the inherent biological
`potential of an already known nucleoside analogue could be used to a
`higher extent and consequently, the improvement in antiviral activity
`could be, in many cases, better than synthesizing new potential
`nucleoside analogues. Furthermore, this review is restricted to the
`delivery mechanism and so the synthesis of the compounds may be
`gleaned from the original literature.
`
`Chris Meier, born 1962 in Berlin, was trained in Chemistry at the
`University of Marburg. He passed his Diploma and Ph.D. thesis in the
`group of Prof. G. Boche in Organic Chemistry. Then he moved as post-
`doc fellow to the Pasteur-Institute in Paris where he started his work in
`nucleoside and oligonucleotide chemistry. In 1991 he joined the group
`of Prof. J.W. Engels at the University of Frankfurt/Main starting his
`Habilitation which he finished in July '96 and in May '97 he was
`appointed as an associate professor at the University of Würzburg.
`
`So, a lipophilic phosphotriester may penetrate into the target cell where
`first partial and at the end complete hydrolysis delivers the nucleotide.
`A suitable nucleotide prodrug has to fulfill two requirements: i) it has to
`be lipophilic enough for passive diffusion of the membrane and blood-
`brain barrier; ii) furthermore, it should be able to deliver the nucleoside
`hydrolytically or enzymatically leaving a non-toxic masking group(13).
`In principle, two different concepts for prodrug design are known:
`bipartate and tripartate prodrugs. In the former concept the drug is
`modified by a one-component masking group. In this form the drug is
`biologically inactive. After a simple cleavage of the mask, the active
`drug is liberated (Figure 3). In the latter concept, the drug is modified by
`a two-component masking group. Again, the drug is biologically
`inactive in this bound form. The mechanism of liberation involves a first
`chemical or enzymatic reaction under cleavage of part I of the masking
`moiety. The drug is still inactive but the effect of this first reaction is an
`activation of the remaining masking group II with the consequence of a
`spontaneous successive cleaving reaction releasing the now bioactive
`drug (Figure 3)(14).
`
`In the case of a nucleotide prodrug one should take into account that
`under physiological conditions two negatively charged phosphate
`oxygen's have to be masked in order to obtain a neutral, lipophilic
`phosphate ester. Consequently, not only one masking group is necessary
`but two. So, the efficient intracellular delivery of nucleotides from a
`prodrug requires the existence of a specific delivery mechanism or
`different rates of conversion of the prodrug to the drug intracellularly
`versus extracellularly. One comment with respect to toxic side events
`should be given. Neutral phosphorus derivatives with a good leaving
`
`Biographical Sketch
`
`2
`
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`

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`March 1998
`
`Pro-Nucleotides - Recent Advances in the Design of Efficient Tools
`
`235
`
`Bis(POM)- and Bis(POC)-Nucleotides
`The first approach reported by D. Farquhar et al. towards a new class of
`tripartate pro-nucleotides are
`the bis(pivaloyloxymethyl)- [POM]
`phosphotriesters of general type 10 (shown exemplified as the ddU
`derivative)(19,20). This approach utilizes a carboxyesterase-catalyzed
`cleavage of the pivaloyl ester within the POM-masking group to yield
`reactive hydroxymethyl phosphotriester 11 which
`the highly
`subsequently eliminates spontaneously formaldehyde to give the
`mono(POM) phosphodiester 12. The carboxyesterase which is used for
`this activation process may be more prevalent inside the cells. To obtain
`the free nucleotide, this enzymatic activation has to be repeated via 13
`or, alternatively, a phosphodiesterase cleaves the phosphodiester 12
`directly to yield the nucleotide (Figure 4).
`
`been shown that the bis(POM) phosphotriesters 10 were chemically
`unstable and highly susceptible to serum-mediated hydrolysis, factors
`which limit their potential utility for intracellular drug delivery(22,24a).
`A modification of the bis(POM)-approach and a way to overcome the
`limitations
`observed
`with
`these
`compounds
`are
`bis(isopropyloxycarbonyloxymethyl)-
`[bis(POC)]
`nucleotides
`of
`general type 14 (shown as the PMPA derivative) that have been
`published very recently by A. Fridland et al.(26) and L. Naesens et
`al.(27). This modification uses a carbonate diester within the masking
`group. The mechanism of action is again a carboxyesterase-catalyzed
`cleavage of the isopropyl ester to give isopropanol and in the shown
`example the intermediate phosphonate diester 15 that fragmentates via
`16 into carbon dioxide and formaldehyde and finally to the mono(POC)
`phosphonate ester 17. The mono(POC) ester 17 is subsequently
`degraded to yield PMPA or generally the nucleotide after a second
`carboxyesterase activation or by phosphodiesterase cleavage (Figure 5).
`
`This approach has been applied successfully to the delivery of 2',3'-
`bis(POM)-ddUMP(21).
`dideoxyuridine monophosphate
`from
`Furthermore, this concept has been used for the delivery of the anti-HIV
`drug AZTMP(22),
`the
`anti-herpes
`and
`anti-HIV
`drug
`phosphonomethoxyethyladenine (PMEA)(23) and for the antitumor
`active drug 5-fluoro-2'-deoxyuridine monophosphate (5-FdUMP)(24). In
`the case of the phosphonate analogue PMEA a highly improved
`bioavailability was obtained. Moreover, the bis(POM) derivatives of
`(R)-9-(2-phosphonomethoxypropyl)adenine
`(PMPA) and
`(R)-9-(2-
`phosphonomethoxypropyl)diaminopurine (PMPDAP) were found to be
`9- to 23-fold more active than the parental compounds(23). However, the
`cytotoxicity of the bis(POM) analogues was also increased by virtually
`similar degrees. Nevertheless, studies with radiolabelled bis(POM)-
`PMEA showed a considerable increase (100-fold) of the cellular uptake
`of PMEA by using the phosphonate diester(23). In an independent study,
`ten different PMEA prodrugs tested in vivo for oral bioavailability in
`cynomolgus monkeys. From the results of this study, bis(POM)-PMEA
`was selected for human clinical trails. In these clinical trails, the oral
`bioavailability of bis(POM)-PMEA from a single 500 mg dose was
`greater than 40% in fed subjects(25). The in vitro antitumor tests of the
`bis(POM)-5-FdUMP triester demonstrated high growth inhibition in
`cell lines that were resistant to the usually used drug 5-fluorouracil.
`Furthermore, this triester derivative showed in vivo bioactivity after
`intraperitonal application against P 388 leukemia in mice. Beside the
`antitumor activity, these results verified also the penetration of the pro-
`nucleotide through cellular membranes(24). A drawback of this approach
`is the requirement of a second identical activation reaction because the
`intermediate phosphodiester is a significantly poorer substrate for the
`activating carboxyesterases. Moreover, the delivery of one molecule of
`the nucleotides results in the liberation of two equivalents of potentially
`toxic formaldehyde and pivalinic acid (Figure 4). Additionally, it has
`
`In contrast to the bis(POM)-approach, the bis(POC) modification avoids
`the formation of two equivalents of pivalinic acid that accumulate in the
`cells and potentially cause toxicity. The bis(POC)-approach was applied
`to the anti-HIV drug PMPA in order to increase the low bioavailability
`of
`this phosphonate derivative (Figure 5). Bis(POC)-PMPA
`is
`chemically stable at low pH and has shown 30% bioavailability in dogs
`with minimal toxicity in repeat 5-day dosage of 60 mg/kg/day. The anti-
`HIV activity of bis(POC)-PMPA 14 in human peripheral blood
`lymphocytes and in dentritic T-cell coculture system was 35- and 16-
`fold, respectively, higher than that of PMPA. Bis(POC)-PMPA 14 was
`non-toxic at concentrations that completely suppressed viral replication.
`Studies of the metabolism of [3H]-bis(POC)-PMPA showed that is was
`readily taken into the human cells, hydrolyzed to PMPA, and
`phosphorylated to the mono- and diphosphate derivatives. These results
`show that bis(POC)-PMPA 14 is a membrane-permeable form of PMPA
`and shows promise as a drug for the treatment of HIV infections(26).
`
`Bis(SDTE)- and Bis(SATE)-Nucleotides
`Two further approaches that are also based on enzymatic activation
`have been reported by J.-L. Imbach and G. Gosselin(28). They designed
`the bis(S-[2-hydroxyethylsulfidyl]-2-thioethyl)- [bis(SDTE)] 18 and the
`bis(S-acyl-2-thioethyl)- [bis(SATE)] nucleotides 19. The former
`concept was constructed to take advantage of the greater reducing
`potential within the cells to liberate the nucleotide into the cytosol. After
`
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`236
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`C. Meier
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`SYNLETT
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`stability of the bis(SATE) phosphotriesters could be adjusted by varying
`the thio ester residue: the lowest stability was obtained for thio esters of
`acetic acid whereas with increasing alkyl residue of the carboxylic acid
`the stability was increased. The optimized compounds are bis(SATE)
`compounds bearing two S-pivaloyl-2-thioethyl side chains as shown in
`Figure 6 because the highly lipophilic and sterically demanding t-butyl
`residue protects the masking group from too rapid cleavage as compared
`to the S-acetyl or S-i-propyl counterparts(28d,30a,31). Again, J.-L. Imbach
`et al. demonstrated an increased stability of the bis(SATE) derivatives
`of AZTMP against degradation in culture medium as compared to
`cellular extracts(34). The bis(SATE) approach has been successfully
`applied to the thymidine kinase-bypass (TK-bypass) of d4T 1(35)
`(shown in Figure 6) and AZT 2(34), the adenosine deaminase-bypass
`(ADA-bypass) of the anti-HIV active nucleoside ddA 6(36) as well as for
`an improvement of the bioavailability of the anti-herpes and anti-HIV
`drug PMEA. Furthermore, the corresponding bis(SATE)-ddAMP
`triesters exhibited a higher stability than the dideoxynucleoside ddA 6
`against acid-catalyzed depurination(36a). It should be mentioned that the
`bis(SATE)-AZTMP derivatives demonstrated a 10-fold decrease in
`activity from the wild-type CEM/O cell line to the mutant TK- CEM cell
`line(34). This result is puzzling but has also been observed by others. On
`the other hand, J. Cinatl, Jr. et al.(11a) have shown that the bis(SATE)-
`AZTMP derivatives retained the biological activity of AZT 2 in AZT
`resistant Molt4/8 cells. Especially bis(S-pivaloyl-2-thioethyl)-AZTMP
`showed comparable cytotoxic and antiviral activity in sensitive and
`resistant cells. With these experiments, J. Cinatl et al. demonstrated that
`Molt4/8rAZT cells exert resistance to the anti-HIV activity of the drug
`mainly owing to the lack of AZT phosphorylation and that resistance
`may be bypassed by using AZT monophosphate prodrugs. However, as
`for the bis(POM) phosphotriesters 10 reported by D. Farquhar, the
`intermediate mono(SATE) phosphonate esters 21 and 23, respectively,
`are significantly lower substrates for the activating carboxyesterases due
`to the negative charge at the phosphonate residue in the vicinity of the
`enzyme cleavage site. Before hydrolysis takes place, the bis(SDTE) as
`well as the bis(SATE) phosphotriesters may serve as lipophilic
`precursors for a passive membrane permeation.
`J.-L. Imbach et al. presented the comparison of the bis(SATE)-approach
`to the isomeric S,S'-bis(O-acyl-2-oxyethyl) phosphorodithiolates(37).
`Surprisingly, these derivatives proved as active as the original
`bis(SATE) triesters with the nucleosides d4T 1 and ddA 6, although the
`delivery mechanism could not be the same. Further work is in progress
`to study the cleavage mechanism. Recently, the same group extended
`their prodrug design to bis(S-glycopyranosyl-2-thioethyl) [bis(SGTE)]
`nucleoside phosphotriesters(38). These compounds were designed to
`deliver the nucleotide by activation of glycosidases. However, no
`antiviral data have yet been published.
`
`Bis(AB)-Nucleotides
`One possibility to avoid the close vicinity of the negative charge of the
`intermediate phosphodiester and the enzyme cleavage site has been
`developed independently by S. Freeman et al.(39) and A. Glazier(40).
`The common motive of their approaches is to separate the phosphate
`group and the carboxyesterase cleaving site by a rigid spacer.
`Consequently,
`they are using bis(4'-acyloxybenzyl)-
`[bis(AB)]
`nucleotides 24 and 25, respectively (shown as their AZTMP derivatives)
`instead of the O-acyloxymethyl or the S-acyl-2-thioethyl residue,
`respectively. As Freeman calculated a separation of about 4 Å, which
`she states is enough to avoid electrostatic disturbance of the anionic
`phosphate and the carboxyester residue(41). The degradation mechanism
`of the bis(AB) nucleotides of type 24 is the following: the enzyme
`cleaves the ester moiety in the 4'-position of the aromatic ring to give
`
`the reductase cleavage of the disulfide bond in 18 with formation of
`thioethanol and thioethyl phosphotriester 20, this compound eliminates
`spontaneously episulfide to yield the intermediate phosphodiester 21.
`As in the case of the bis(POM)- (10) and the bis(POC) phosphotriester
`14, the bis(SDTE) derivative 18 requires a second identical enzyme-
`catalyzed activation step (Figure 6). The initial idea of this concept was,
`that the "soft" thiol-nucleophile in 20 will attack the "soft" electrophilic
`α-carbon atom rather than the "hard" phosphorus atom in the
`elimination reaction avoiding possible pseudorotation processes(18)
`because all reactions take place within the masking group without
`involving the phosphate ester moiety. This concept belongs also to the
`class of tripartate prodrugs. It could be shown that the bis(SDTE)
`triester of the nucleotides ddUMP and AZTMP 9 were cleaved 30-fold
`faster by reductases in cell extracts than in culture medium(28c).
`Furthermore, the bis(SDTE) approach has been applied to the
`nucleoside 5-FdU 3(29) and to the phosphonate nucleotide PMEA(30).
`Whereas an improvement in antiviral activity most probably due to an
`enhanced bioavailability for the bis(SDTE)-PMEA derivative 18
`compared with PMEA was found, the bis(SDTE)-5-FdUMP failed to
`show better antitumor potency as compared to the free nucleoside(29).
`The major limitation of this approach is the restricted chemically
`stability and high susceptibility to serum-mediated hydrolysis (Figure
`6).
`
`In contrast to the bis(SDTE) approach, the bis(SATE) approach is
`similar to the bis(POM) concept with respect to the activation process.
`Again, carboxyesterases are used to trigger the nucleotide delivery.
`Here, the enzyme cleaves a thioester in 19 to yield a carboxylic acid (as
`in the bis(POM) approach pivalinic acid) as well as the above
`mentioned thioethyl phosphonate diester 22 which undergoes a
`fragmentation to episulfide and the phosphonate monoester 23 (Figure
`6). Thus, the spontaneous, second step is identical in the bis(SDTE) and
`the bis(SATE) concepts. Nevertheless, again two independent activation
`steps are required to yield the free nucleotide. With these two
`approaches, two equivalents of episulfide, which has been shown to be
`chronically and acutely toxic in mice and rats, are released besides the
`nucleotide(31). The toxicity and moreover also mutagenicity has also
`been observed in vitro(32). J.-L. Imbach and J.-P. Sommadoussi have
`shown that the introduction of the SDTE and the SATE group do not
`induce additional cytotoxicity of the phosphotriesters as compared to
`the parent nucleosides (e.g. AZT) in human myeloid colony-forming
`cells (human bone marrow cells)(33). It should be mentioned that the
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`benzyl)methylphosphonate. As expected, the final products after
`enzymatic deesterification were methylphosphonic acid, acetic acid and
`trans-4'-hydroxycinnamic acid 34. Without addition of esterase no
`detectable decomposition was noted. Secondly, Glazier demonstrated
`convincingly, that after two hours of incubation with porcine liver
`esterase a nucleoside phosphotriester bearing the anti-herpes drug
`acyclovir monophosphate (ACVMP) has nearly completely been
`converted to methanol, acetic acid, trans-4'-hydroxycinnamic acid 34 as
`well as ACVMP as sole reaction products(45). This concept has been
`applied to the delivery of AZTMP(40) and ACVMP(45). However, a
`limitation of this approach may be the very short degradation half-lives
`of the compounds (t1/2 = 16 min). Furthermore, the bis(AB) derivatives
`reported by A. Glazier et al. were highly lipophilic (log Pa value = 1.07
`to 4.18; depending on the carboxyacid ester residue) making systemic
`applications problematic(45). Nevertheless,
`the bis(AB)-ACVMP
`derivatives showed promising in vivo activity against Herpes virus type-
`1 infections without toxic side effects using concentrations up to 100
`mg/kg body weight(45). Furthermore, no mutagenicity was found for the
`prodrugs in the Ames test with or without microsomal activation(45).
`However, the compounds showed only a limited improvement in
`antiviral activity in vitro(45). As acyclovir, the prodrugs showed no
`toxicity in vitro (CD50 >100 μM). Moreover, bis(AB) esters of PMEA
`were prepared and were found to be more potent in vitro than PMEA
`itself(40).
`
`Nucleoside Arylphosphoramidates
`triggered
`Another novel class of a membrane-soluble enzyme
`nucleotide delivery concepts are the phosphoramidate derivatives of
`type 35 recently reported by C. McGuigan et al.(46). The basic structure
`of these compounds (shown with the anti-HIV drug d4T 1) is depicted
`in Figure 8. In contrast to the previously mentioned phosphotriester
`approaches, in this case the structural motive involves a phosphate
`moiety that is linked to the nucleoside, a phenyl group and, through a
`phosphoramidate linkage, also to the methyl ester of α-amino acid ester.
`This concept has been applied to the wide variety of nucleotides active
`against several viruses: d4TMP(47), AZTMP(48), 3'-thiacytidine
`monophosphate (3TCMP)(49), ddAMP(50) and 2',3'-dideoxy-2',3'-
`didehydroadenosine monophosphate (d4AMP)(50,51).
`In in vitro antiviral tests, the d4T containing phosphoramidate was
`found to be slightly active as the parent nucleoside in wild-type CEM/O
`and MT-4 cells. More importantly and in contrast to d4T 1, the d4TMP
`phosphoramidate completely retained the biological activity in mutant
`thymidine kinase-deficient CEM/TK- cells(47) and suppresses HIV-1
`infection in natural peripheral blood lymphocytes and freshly isolated
`monocyte/macrophages. This result proves that d4TMP 4 was delivered
`intracellularly making the observed activity of the compound almost
`entirely independent of thymidine kinase and consequently these
`compounds serve as efficient tools for the TK-bypass. The d4T-
`phosphoramidate was found to be equally active against HIV-1 and
`HIV-2 replication, and has also proved inhibitory to other retroviruses
`including
`simian
`immunodeficiency virus
`(SIV)
`and
`feline
`immunodeficiency virus (FIV), Visna virus and Moloney murine
`sarcoma virus in cell culture(52). An interesting structure-activity
`relationship (SAR) has been found with these compounds: first, the
`presence of an α-amino acid ester is essentially important for the
`biological activity. In contrast to α-amino acid bearing compounds,
`simple alkyl amine derivatives exhibited a complete loss of antiviral
`activity(53). The same was observed for derivatives bearing β-amino
`acid side groups. Among the natural α-amino acids L-alanine was found
`to be the most effective(54) while the enantiomeric D-alanine showed a
`30-fold decrease in bioactivity(55). This interesting SAR points to an
`
`the 4'-hydroxybenzyl phosphotriester 26. Again, a subsequent
`spontaneous fragmentation of 26 is induced by this enzymatic reaction
`the formation of phosphodiester 27 and
`resulting
`in
`the 4'-
`hydroxybenzyl cation 28 which
`is oxidized
`to yield
`the 4'-
`quinonemethide 29 or
`is quenched by water
`to yield 4'-
`hydroxybenzylalcohol 30. Diester 27 could then be degraded after a
`second esterase activation or a phosphodiester cleavage to yield the
`nucleotide AZTMP 9 (Figure 7). The mechanism of delivery has been
`studied using methylphosphonate as model compound(42). Obviously,
`these compounds are also tripartate prodrugs.
`
`S. Freeman et al. applied their approach to the delivery of AZTMP(39,41)
`and obtained in vitro antiviral activity against HIV-1 and SIV that was
`comparable to AZT 2. Unfortunately, she has not tested the compounds
`in thymidine kinase-deficient cells which could prove the direct
`intracellular delivery of AZTMP 9. Recently, S. Freeman applied their
`bis(AB) approach to the intracellular delivery of foscarnet (PFA) from
`various precursors(43). A possible limitation of the approach is the
`generation of the reactive benzylcation 28 in close vicinity of the active
`site of the enzyme because such cations are known to be potential
`alkylating agents that could react with side chains of amino acids(44).
`This possible problem was faced by the approach reported by A. Glazier
`using the bis(AB) derivatives 25 (Figure 7)(45). Although the basic
`principle of the nucleotide delivery from 25 is essentially the same
`(carboxyesterase-mediated activation to give 32 via 31) as from triesters
`24, the difference to the Freeman concept is the introduction of a
`methylmethoxycarbonyl group in the benzylic position of the AB
`residue. The rationale for this substitution at the benzylic position is the
`possibility of a fast elimination reaction via
`the fleeting 4'-
`quinonemethide 33 due to the electron-withdrawing methoxycarbonyl
`group that facilitates proton abstraction by decreasing the intrinsic
`barrier to proton removal. The transition state for proton removal is
`further stabilized by resonance delocalisation into the aromatic ring. The
`adjacent carbonyl group may also facilitate proton tautomerisation by
`acting by intramolecular general base catalysis. Firstly, this concept has
`bis(4'-acetoxy-α-methylmethoxycarbonyl-
`been
`proven
`with
`
`5
`
`

`

`238
`
`C. Meier
`
`SYNLETT
`
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`
`J. Balzarini et al. showed that the corresponding ddA 6 and d4A
`derivatives (shown in Figure 8) markedly increased the antiviral
`potency of the parent nucleosides in different cell lines(50). The most
`obvious interpretation of these data is the efficient delivery of the
`appropriate nucleotides ddAMP 8 and d4AMP, respectively, which
`leads to the ADA-bypass. It should be added that for the ddA and the
`d4A phosphoramidate derivatives an increase in the selectivity index
`compared to the free nucleosides was observed. Furthermore, both
`adenosine analogues were efficient as 3TC in inhibiting hepatitis-B
`virus replication in hepatocytes. As a consequence, these derivatives
`rank among the most potent HIV and HBV inhibitors reported so far in
`cell culture.
`
`Amino Acid Phosphoramidate Diesters
`Recently, C. Wagner et al. published an approach using
`phosphoramidates 39 bearing aromatic amino acids that are still
`negatively charged(58). The approach has been applied
`to
`the
`nucleosides d4T 1(58), AZT 2(59), 5-FdU 3(60), and 3'-fluoro-3'-
`deoxythymidine (FLT)(58) (Figure 9). In few cases the reported
`phosphoramidates exhibited greater potency than the parent nucleosides
`in vitro. As amino acid L-tryptophan was found to be the best choice: in
`vitro, the L-tryptophan-AZT phosphoramidate demonstrated a 8-fold
`increase in antiviral activity compared to free AZT 2 against HIV-1
`replication in peripheral blood mononuclear cells without toxicity(59).
`Furthermore, cells treated with the active AZT diester contained 4-fold
`more phosphorylated AZT than those treated only with AZT(59). Again,
`this high biological activity could not be transferred to the antitumor-
`active nucleoside 5-FdU 3. Evaluation of the corresponding L-
`tryptophan-5-FdU phosphoramidate demonstrated a pronounced
`decrease in antitumor activity of the prodrug compared to free 5-FdU in
`several cell lines. Although some of the activity was gained back by
`using permeabilized cells, even in these cells the activity was still lower
`than the activity of the parent nucleoside 3 in intact cells. However,
`incubation of cell-free extracts of CEM cells with radiolabelled L-
`tryptophan-5-FdU phosphoramidate resulted in the rapid production of
`5-FdUMP and a lag in the generation of 5-FdU 3(60). It should be added
`that this approach is the only reported case that successfully uses
`phosphodiesters as a prodrug concept. It has been shown that due to the
`remaining charge, the compounds are still considerably water soluble
`and furthermore, the compounds are indefinitely stable in human blood.
`The high stability was also obser