Nucleoside Phosphate-Conjugates Come of Age: Catalytic Transformation, Polymerase Recognition and Antiviral Properties.# Elisabetta Groaz* and Piet Herdewijn

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Send Orders for Reprints to [email protected] Current Medicinal Chemistry, Year, Volume 1

Medicinal Chemistry, Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, 3000 Leuven, Belgium

Abstract: Over the past few decades, different types of nucleoside phosphate-conjugates have been under extensive investigation due to their favorable molecular lability with interesting catalytic hydrolysis mechanisms, recognition as polymerase substrates, and especially for their development as antiviral/anticancer protide therapeutics. The antiviral conjugates such as nucleoside phosphoesters and phosphoramidates that were discovered and developed in the initial years have been well reviewed by the pioneers in the field. In the present review, we will discuss the basic chemical and biological principles behind consideration of some representative structural classes. We will also summarize the chemical and biological properties of some of the more recent analogues that were synthesized and evaluated in our laboratory and by others. This includes new principles for their application as direct substrates of polymerases, nucleobase-dependent catalytic and antiviral activity, and a plausible ‘prodrug of a prodrug’ strategy for tissue/organ-specific targeted drug delivery. Keywords: Prodrug – Delivery – Stability – Polymerase - Phosphoramidates

INTRODUCTION profile of these molecules through the “tuning” of the nature of the existing ligands and the pursuit of new protecting The ability of unnatural nucleoside-based therapeutics to moieties relying on chemical rather than enzymatic induce inhibition of uncontrolled cell proliferation or viral dependant activation mechanisms. Further attention has been replication hinges upon stepwise phosphorylation into their paid to the search for alternative nucleotide analogues that active species, inhibition or binding of intracellular enzymes could bypass multiple anabolic steps or skip over the entire (polymerases, ribonucleotide reductase), incorporation into a conventional kinase-mediated activation cascade.[7] The growing DNA/RNA strand, and subsequent chain archetype prodrug candidate should in fact possess the termination.[1] As most modified nucleoside substrates are ability to act as a direct substrate of the target polymerase. very poor substrates for natural kinases, their phosphorylated Apart from their therapeutic value, such strategies might metabolites are delivered to the cell, typically in the forms of prevent severe side effects and toxicity, which in some lipophilic precursors.[2] In addition to overcome instability instances were observed due to the accumulation of the related to phosphatases and poor cell-penetration associated nucleoside monophosphates.[8-10] with negatively charged nucleotides, the use of pronucleotides can turn inactive nucleosides into potent Exhaustive information on biological activities and mode of compounds, generate broader-spectrum antivirals and in action of nucleoside phosphate prodrugs has been reviewed some cases circumvent drug resistance.[3-6] in a series of authoritative articles.[1, 11-15] A comprehensive treatment of all their preparative methods The current availability of effective prodrug systems for the was also summarized in a recent review article.[16] Herein uptake of nucleoside phosphates is primarily based on we will discuss those aspects that concern our contribution to monophosphate conjugates (cycloSal, ProTides, SATE this topic and relevant examples from the recent literature approaches), as the first kinase-mediated step is generally from other groups. We will focus on the application of considered to be rate-limiting to nucleoside bioactivity. prodrugs as direct substrates of polymerases, nucleobase- However, premature and/or site unspecific removal of the dependent catalytic and antiviral activity together with a masking groups, substrate specificity of cellular enzymes discussion of a possible ‘prodrug of a prodrug’ strategy for involved in distal phosphorylation steps (NDPKs, creatine or site-specific targeted drug delivery. 3-phosphoglycerate kinases) and polymerase recognition remain relevant issues that need to be addressed to improve #Dedicated to Prof. Robert Vince on his 75th birthday. therapeutic activity. *Address correspondence to Dr. Elisabetta Groaz, Rega Institute for Medical Research, KU Leuven, Minderbroedersstraat 10, 3000 Leuven, In this respect, on-going rational drug design has been Belgium principally devoted to further refining the physicochemical E-mail: [email protected]

RECENT DEVELOPMENTS IN PRONUCLEOTIDES DESIGN: TOWARDS AN ANABOLIC DISENTANGLE? Antiviral prodrug approaches based on the conjugation of a nucleoside monophosphate to a α-amino acid moiety through a 5’-phosphoramidic bond are well documented in the literature. McGuigan and co-workers[17] and Wagner and co-workers,[18] have both made major contributions to the field. They have summarized the state of the art for aryloxy amino acid phosphoramidates diesters (ProTides) and amidate monoesters (aaNPs), respectively, in their reviews. Extensive mechanistic studies provided evidence for the nucleoside phosphoramidate deprotection to be initiated at the ester moiety by the action of a carboxyesterase-type enzyme or cathepsin A (Scheme 1). The resulting carboxylate 2 spontaneously undergoes an intramolecular rearrangement and displacement of the phenoxy group to form a five-membered cyclic anhydride intermediate 3, which, by reacting with a molecule of water gives a phosphoramidate diester 4. In a subsequent stage, the successful release of the nucleotidic moiety into the cell Scheme 2. Molecular structure and mode of action of amino relies on enzyme-catalysed selective P-N bond cleavage. acid phosphoramidate nucleoside acting as triphosphate mimics (IDA = iminodiacetic acid, IDP = iminodipropionic acid). Remarkably, since the amino acid amidate moiety has proven able to mimic the natural pyrophosphate as leaving group, it seems more likely that prodrugs derived from such substrates can be directly processed by the polymerase, without the need of any extra metabolic steps common to other approaches. In view of the potential advantages associated with the structural features discussed above, a variety of aspartic acid based phenoxy phosphoramidate diesters of stavudine (d4T)[28] and 2’-C-methyl containing nucleosides[29] (Asp-ProTides) were synthesized and tested Scheme 1. Intracellular activation of classical nucleoside for potential anti-HIV-1 and HCV inhibitory activity. In phosphoramidates (ProTides) nearly all reports on ProTides, L-alanine is most commonly used and has proved to be the best amino acid motif in terms In line with this theme, our group disclosed that of antiviral activity of the corresponding phosphoramidate. deoxynucleotide phosphoramidate monoester derivatives of This observation matched dramatically greater rates of L-aspartic acid (L-Asp) and L-hystidine (L-His) undergo alanine ester hydrolysis in the first step of the activation enzymatic incorporation into a growing DNA chain in the pathway (Scheme 1) when compared to branched presence of a DNA polymerase as HIV-1 Reverse counterparts (leucine, valine, isoleucine).[30] Interestingly, Transcriptase (RT), while still retaining the requisite the use of aspartic acid diesters in place of methyl alanine canonical base-pairing (Scheme 2).[19-21] Substrate design was marked in some cases by a significant improvement of was driven by both geometric and electronic considerations, activity, particularly when bulkier and more lipophilic alkyl with the aim of achieving a correct alignment of the α- groups were present as ester residues (n-butyl, amyl and phosphorus atom needed for the nucleotidyl transfer process isoamyl esters).[29] Although cytotoxicity occurred in the in the enzyme active site as well as coordination by catalytic low micromolar range, selectivity index values remained metal ions. The validity of this concept was illustrated favorable. through a systematic structure-substrate relationship study, designed to establish the influence of structural and chelating Further, metabolism studies demonstrated that in the properties of other modified or unnatural amino acids for the presence of liver esterases the hydrolysis of the β-carboxyl induction of HIV-1 RT-catalyzed DNA synthesis.[22-27] ester in the aspartate moiety preceded the cleavage of the ester at the α-position. Those events were followed by aryl displacement via neighboring group assistance of the α- carboxylic group and the other typical steps needed to unmask the protecting groups. The nucleoside monophosphate is then released into the cell. Thus, it was envisaged that the β-carboxylic ester functionality could be Short Running Title of the Article Journal Name, 2014, Vol. 0, No. 0 3 employed for guiding target-tissue drug delivery and that Parallels may therefore be drawn between the second Asp-ProTides could thus be considered as ‘prodrugs of generation DiPPro approach developed by Meier and our prodrugs’. Hypothetically, conjugation of the amino acid Asp-ProTide methodology. Although conceptually diverse, residue at the β-side chain with a biomolecule could facilitate they both attempt to combine different structural elements in hydrolysis in a specific tissue, where the β-carboxylic acid a single conjugate that could thereby address simultaneously containing Asp-Protide would then undergo different objectives. bioactivation.[29]

In spite of the broad scope offered forth by nucleoside di- and tri-phosphate prodrugs ability to deliver the appropriate REACTIVITY AND CHEMICAL DEGRADATION analogues to interact with polymerases, their utilization PATHS OF AMINO ACID NUCLEOSIDE PHOSPHORAMIDATES remains underexplored as an alternative delivery methodology as compared to monophosphates. This reflects The unravelling of each chemical/enzymatic step involved in an increased sensitivity of the pyrophosphate anhydride bond the process of prodrug release of the nucleoside phosphate towards nucleophilic attack upon diminution of the negative into the cell can therefore provide valuable and predictive charges. For instance, modifications of the β- or - knowledge for tailoring analogues with superior phosphorus atom based on diglycerides[31, 32] and physicochemical properties and therapeutic potency. cyclosaligenyl groups[33] (cycloSal) failed to deliver NDP However, the overall biological activity of a prodrug is owing to the preferential liberation of NMP. Analogues dictated not only by the successful access to the bearing liphophilic acyl chains were stable to P-βP corresponding active metabolite, but also by its inherent hydrolytic cleavage in aqueous buffer, but underwent capacity to withstand undesired metabolic reactions. For decomposition in biological media.[34] Of particular interest example, amino acid nucleoside phosphoramidates should be in this context are the recent findings by the Meier’s group sufficiently robust to undergo selective P-N bond cleavage challenging this knowledge. By introducing para- while resisting the unnecessary release of the nucleoside by (acyloxybenzyl) biolabile moieties of the β-phosphate means of P-O bond cleavage. For a prodrug strategy based moiety, it was possible to overcome the instability issues and on aaNPs acting as direct inhibitors/substrates of a viral the ineffective delivery associated with nucleoside polymerase to be successful, such compounds should remain diphosphates.[33, 35, 36] Such protecting groups convey to stable to both P-O and P-N bond cleavage before reaching the corresponding prodrugs (DiPPro) high chemical stability the enzyme’s pocket. Additionally, the free carboxylic at physiological pH, allowing for esterase-catalyzed or pH- groups present in the amino acid moiety, critical for dependent selective hydrolysis of the acyl ester group to polymerase recognition and binding,[20] could, under certain proceed faster than degradation of the pyrophosphate unit in conditions, induce unwanted chemical degradation reactions. cell extracts. This event triggers the 1,4-elimination of the benzyl group attached to phosphate function leading to a In view of these observations, recent studies[38, 39] were monosubstituted species that undergoes the same sequence a devoted to identify the elements that rule the selectivity for second time releasing NDP. P-N/P-O bond hydrolysis. Determination of the chemical stability of those compounds at high temperatures is also important in view of the potential use as substrates for thermostable polymerases. As a result, the structural determination of amino acid phosphoramidates in the gas- phase was investigated by electrospray ionization ESI-MS in both positive and negative ion modes and tandem mass spectrometry. The resulting fragmentation patterns provided important information for these studies.[40] Lonnberg et al. produced a first understanding of the solvolytic stability of amino acid phosphoramidate diesters Scheme 3. Mode of action of mixed nucleoside diphosphate by following the course of the hydrolytic breakdown of prodrugs. thymidine 5’-O-phenyl phosphoramidate conjugate with L- The introduction of bulkier substituents onto the acyl moiety alanine (L-Ala) methyl ester 11 at the temperature of 90 led to increased chemical stability, with a concurrent marked ºC.[38, 41] A sequence of degradation events has been reduction in enzymatic substrate conversion to the desired proposed in order to account for the observed distribution of NDP product. This resulted in higher amounts of the NMP reaction products summarized in Scheme 4 over a wide formed by chemical hydrolysis. Based upon this knowledge, range of pH values. the authors developed a second generation of mixed diphosphate prodrugs of the type 8 (Scheme 3) combining alkyl residues with different lengths at the β-phosphate in order to achieve the optimal compromise between lipophilicity and fast activation, thus minimizing NMP formation.[37] 4 Journal Name, 2014, Vol. 0, No. 0 Principal Author Last Name et al.

endocyclic oxygen atom.[43, 46] Direct displacement of the phenoxide moiety by a hydroxide ion is a minor side reaction and most likely follows an SN2-type mechanism to give 15.[47-51]

Scheme 4. Accumulation of products upon acid and base- catalysed hydrolysis of amino acid amidate ProTides. The key information to emerge from these studies was the identification of competing pathways accounting for the disappearance of the starting compound under acid-catalysed conditions. The first involves the nucleophilic displacement Scheme 6. Dominant base-catalysed hydrolytic pathway for of the alanyl ester moiety to afford thymidine 5’- 14. phenylphosphate 12 as a result of P-N bond cleavage. A comprehensive mechanistic study reported by our group Concurrently, the hydrolysed carboxylic function might also provided important new insight into the principal structural be involved in an intramolecular nucleophilic attack on the factors that determine the thermal and chemical stability of phosphorus. This would lead to either the nucleoside 5’- aaNPs of type 7.[39, 52, 53] A comparison of the kinetics of monophosphate 13, following displacement of phenol and hydrolysis under a set of conditions showed that the reaction decomposition of cyclic phosphoramidate intermediate 22, occurred at a faster rate in acidic solutions (pH 5-7) and at or alternatively, to thymidine via the formation of an higher temperatures (70 ºC), exhibiting a first-order intermediate of type 23 (Scheme 5), rather than by dependence on both phosphoramidate[41, 54, 55] and dephosphorylation of 16.[42] Notably, the corresponding hydronium ion[41, 54, 56] concentrations. As shown in monoester analogue of 11 was found to undergo exclusively Scheme 7 for L-Asp-NP, the presence of differentially P-N bond rupture, in analogy with the behavior of related protonated forms is conceivable in solution. Although compounds.[43-45] protonation at the amide nitrogen is thought to be favored over protonation at the phosphoryl oxygen,[56-60] a O O H a H a tautomeric equilibrium most likely exists between the N- and PhO P ONu PhO P ONu e O OPh eO ONu H O+ NH 3 NH a O P ONu a O P OPh O-protonated species.[43, 54] The hydrolysis of the P-N e e O -MeOH O NH e NH e Me Me O O bond is believed to operate by an SN2 concerted mechanism. OMe OH Me Me Engagement of the most stable N-protonated zwitterionic 11 16 17 18 species 26 and a molecule of water in an associative-type transition state, would then lead to the final departure of the path b PhOH path b' Nu aspartyl moiety. O O O O P ONu O P OPh Similarly, the mechanism involved in the P-O cleavage has Nu HO P O O NH O NH been rationalized with the nucleophilic attack of the α- O- Me Me carboxyl oxyanion at the phosphorus of the neutral 13 19 20 phosphate species 24, although another possible pathway Scheme 5. Postulated intermediates involved in the acid- interprets the reaction as proceeding through the catalysed intramolecular degradation of 14 (a = apical, e = monoanionic phosphate 25. The five-membered trigonal equatorial). bipyramidal phosphorane intermediate is thought to reflect the preference of the incoming oxygen nucleophile to adopt Under basic conditions compound 14 (Scheme 4) an apical position, thus forcing the nitrogen atom into an accumulated as the major product with no further equatorial position and the nucleoside moiety in the degradation observed. The mechanism involved in the remaining apical position. Proton transfer to the 5’-oxygen dominant reaction pathway, as shown in Scheme 6, has been followed by departure of the nucleoside gives a cyclic rationalized to occur with the saponification of the alanyl phosphoramidate, which, upon hydrolysis, further rearranges methyl ester, followed by the intramolecular attack of the to form the amino acid and organic phosphate products. resulting carboxylate on the phosphorus atom. This would lead to the formation of the cyclic transition state 22 followed by loss of the better leaving group - the phenoxide ion rather than the 5’-oxyanion of the nucleoside. The resulting cyclic anhydride is selectively hydrolysed at the P- OPh bond owing to the apical position occupied by the Short Running Title of the Article Journal Name, 2014, Vol. 0, No. 0 5

THE ULTIMATE CHALLENGING ENDEAVOR IN PRODRUG THERAPY: TARGETED DRUG DELIVERY In spite of the encouraging progress that has been achieved in the last few years in this area of drug discovery, especially in terms of enhanced oral bioavailability and pharmacological potency, general effective methods for site specific targeting and the release of therapeutically relevant nucleosides/tides are still lacking. Directing drugs to specific tissues, organs or cellular compartments would be beneficial Scheme 7. Mechanisms for P/O versus P/N acid-catalyzed not only to reduce off-target adverse side effects but also to hydrolysis of L-Asp-dAMP. lower the effective dose required. For the most part, Examination of the kinetic and theoretical data collected for nucleoside prodrug molecules that to date have been L-Asp-dAMP and related analogues revealed some proposed to overcome this problem were specifically interesting reactivity trends. The concomitant presence of a designed so as to be selectively activated by a particular α- and β-carboxylic group in L-Asp-dAMP appeared to enzyme which expression is restricted to a specific tissue or induce an accelerating effect on phosphoramidate hydrolysis to exploit differences in the redox environment of cellular (pH 6) in comparison with its glycine (α-COOH) and β- spaces. alanine (β-COOH) counterparts, with a relative reactivity In particular, HepDirect analogues are liver-selective cyclic order Gly < β-Ala < Asp. When only a β-carboxyl group was prodrugs derived from 1-aryl substituted 1,3-propanyl present, selective P-N bond cleavage was observed, phosphate (or phosphonate) esters. They represent the most otherwise both P-N and P-O bond cleavage pathways were prominent development within this context,[61] as found to be operative, although to different extents. The demonstrated by their advancement to human clinical trials extra group at the β-position further enhanced the for the treatment of hepatitis B virus (HBV) infections, destabilizing effect of the α-carboxyl group on the P-O bond. hepatocellular carcinoma and hypercholesterolemia.[62-64] This outcome has been ascribed to an increased entropic They are characterized by a remarkable resistance to contribution gained from the formation of a hydrogen bond enzymatic activity in plasma and most tissues, with the between the 3’-OH of the sugar and the aspartyl residue (NH exception of the liver, where they undergo oxidative and/or β-carboxyl group), which might force the α-carboxyl cleavage at the benzylic carbon atom by a liver specific moiety to adopt the required position for attack on the enzyme, namely cytochrome P450 (CYP3A). The phosphorus atom.[53] biologically active compound is then released from the ring- With respect to the impact of the nature of the nucleobase on opened ketophosphate intermediate 32 by elimination of an the reactivity, the estimation of hydrolysis rates following unsaturated aryl ketone by-product 33 (Scheme 8). Although the replacement of adenine in L-Asp-dAMP with different a detoxification process involving the enzyme glutathione S- deaza purines demonstrated a kinetic acceleration of P-O transferase is operative to help in the removal of 33 in the cleavage with the following order 3-deazaadenine < adenine hepatocytes, nevertheless its high reactivity may constitute a < 7-deazaadenine < 1-deazaadenine. The compound Asp-1- significant concern in terms of unpredicted toxicity. deazadAMP was found to be exceptionally prone to P-O bond hydrolysis even at low temperatures. The higher reactivity rates exhibited by 1-deaza and 7-deaza analogues compared to adenine were explained by a possible stabilizing role associated with the presence of a protonated N3 atom on the transition state involved in the intramolecular P-O bond decomposition pathway of the phosphoramidate species. Unlike the adenine-containing phosphoramidate, a favorable hydrogen-bonding interaction can occur owing to the rare Scheme 8. CYP450-catalysed liver activation of HepDirect syn-type conformation of the deaza nucleobases.[39] prodrugs. These findings open attractive possibilities for prodrug Another novel approach follows a similar logic taking design since data on the hydrolysis reaction preferences (P-N advantage of the reducing nature of the cytosolic vs. P-O) can be used as a predictive tool. For instance, it is environment, which represents one of the major cellular reasonable to assume that 3-deaza phosphoramidate pools of glutathione (GSH).[65] Substituted 1,2-dithiane analogues would be more successful in delivering nucleoside phosphate conjugates of type 35 undergo bioactivation in the monophosphates. This is due to their proclivity to P-N bond cytosol by a “reduction-release” mechanism, in response to rupture when compared with 1- or 7-deaza compounds, the high transmembrane GSH/GSSG redox potential which would instead afford the corresponding nucleosides difference between intra- and extracellular spaces. GSH- through chemical degradation. promoted reduction of the disulphide bond is followed by 6 Journal Name, 2014, Vol. 0, No. 0 Principal Author Last Name et al. cyclodeesterification with release of a cyclic phosphodiester predictions for the requirements that must be fulfilled but 36 and formation of a transient thiirane derivative, which, that could be experimentally probed to further expand this upon cyclization, is converted to the stable concept. tetrahydrothiophene species 39. Further enzymatic processing can then generate the monophosphate. CONCLUSION Several new principles for the design of new nucleoside conjugates as potential antivirals have been proposed over the last years, some of which have been described in this review. One major breakthrough would be the availability of nucleoside conjugates that are direct substrate for polymerases, circumventing the necessity for intracellular activation of the modified nucleosides by the kinases. Another interesting approach is the use of ProTides in which the masking group is removed in vivo by a chemical, rather than an enzymatic mechanism. In the event that the base Scheme 9. Cytosol-specific reductive bioactivation of moiety of the nucleoside is involved in prodrug degradation, cyclic-disulfide containing prodrugs. the approach will become base dependent. This would make the prodrug approach less reliant on the presence of Strategies based on the conjugation of therapeutics with degrading enzymes, and thus, more predictable. Tissue carrier molecules able to recognize tissue surface targeting could also be achieved by synthesizing ‘prodrugs antigens[66, 67] or disease-associated biomarkers, as well as liposome encapsulation[68, 69] and the use of diverse of prodrugs’, in which one entity is responsible for targeting nanocarriers may constitute valuable tools to ensure high a particular tissue and the other entity for transmembrane local accumulation of the parent drug.[70] Nevertheless, they transport and intracellular activation. None of these are not often associated with an increased therapeutic index, approaches however, has reached the level of clinical primarily due to inadequate exchange across the vascular research, but they may prove to inspire chemists to design endothelial barrier and slow rates of payloads release relative new conjugates and further study their chemical and to drug clearance from the system.[71] At the present time, biological properties. the development of drug conjugates as delivery method has also been delayed by concerns related to production costs, unpredictable immune responses and therapeutic ACKNOWLEDGEMENTS formulation. 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