Protonation equilibria for the parent compound of three highly potent antivirals have been studied by 1H NMR spectroscopy.

Acyclic nucleoside phosphonates are of medical relevance and deserve detailed chemical characterization. We focus here on (S)‐9‐[3‐hydroxy‐2‐(phosphonomethoxy)propyl]adenine (HPMPA) and include for comparison 9‐[2‐(phosphonomethoxy)ethyl]adenine (PMEA), as well as the nucleobase‐free (phosphonomethoxy)ethane (PME) and (R)‐hydroxy‐2‐(phosphonomethoxy)propane (HPMP). The acidity constants of H3(HPMPA)+ were determined and compared with those of the related phosph(on)ate derivatives; they are also needed to understand the properties of the metal ion complexes. Given that in vivo nucleotides and their analogues participate in reactions typically as divalent metal ion (M2+) complexes, the stability constants of the M(H;HPMPA)+ and M(HPMPA) species with M2+ = Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, and Cd2+ were measured. Comparisons between the results for HPMPA2– and the previous data for PMEA2–, HPMP2– and PME2– revealed that for most M(HPMPA) complexes the enhanced stability (the enhancement relative to the stability of a simple phosphonate‐M2+ coordination), can solely be explained by the formation of 5‐membered chelates involving the ether oxygen. These chelates occur in equilibrium with simple ′open′ phosphonate‐M2+ species, the phosphonate group being the primary binding site. The only exceptions are the M(HPMPA) complexes of Ni2+, Cu2+, and Zn2+, which show an additional stability enhancement; in these instances not only the indicated 5‐membered chelates are formed, but M2+ coordinates in addition to N3 of the adenine residue forming a 7‐membered chelate ring. This observation regarding N3 is important because it emphasizes the metal ion affinity of this site (which is often ignored). Note that in the DNA double helix N3 is exposed to the solvent in the minor groove. The stability data for the monoprotonated M(H;HPMPA)+ complexes suggest that these carry H+ at the phosphonate group whereas M2+ is partly at the nucleobase and partly also at the phosphonate group. The ratios of these isomers depend on the metal ion involved, e.g., for Cu(H;HPMPA) the ratio of the isomers is about 1:1.

Acyclic nucleoside phosphonates (ANPs) are of medical relevance and deserve detailed chemical characterization. We focus here on 1-[2-(phosphonomethoxy)ethyl]cytosine (PMEC), (S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine (HPMPC), 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA), and (S)-9-[3-hydroxy-2-(phosphonomethoxy)propyl]adenine (HPMPA) and include for comparison the nucleobase-free (phosphonomethoxy)ethane (PME) and (R)-hydroxy-2-(phosphonomethoxy)propane (HPMP). The acidity constants of H-3(ANP)(+) and H-2(NP) (NP2-=nucleoside phosph(on)ate derivative) are needed to understand the properties of the ternary neutral Cu(Arm)(ANP/NP) [Arm=2,2-bipyridine (Bpy) or 1,10-phenanthroline (Phen)] and the monoprotonated Cu(Arm)(H;ANP)(+) complexes. The Cu(Arm)(ANP) species are considerably more stable than the corresponding Cu(Arm)(R-PO3), where R-PO32- represents a phosph(on)ate ligand with a non-coordinating group R. The observed stability enhancements are due to intramolecular stack formation (st) between the aromatic rings of Arm and the nucleobase residues and also to the formation of five-membered chelates involving the ether oxygen of the -CH(R)-O-CH2-PO32- residue (cl/O) (R=H or CH2-OH). In intramolecular equilibria, three structurally different Cu(Arm)(ANP) isomers occur; for example, of Cu(Phen)(HPMPA) about 5% exist as an open (op) Cu(Phen)(HPMPA)(op) isomer, 17% as Cu(Phen)(HPMPA)(cl/O), and 78% as Cu(Phen)(HPMPA)(st). In Cu(Arm)(ANP) the stacking tendency decreases in the order PMEA(2-)>HPMPA(2-)>PMEC2->HPMPC2-. In monoprotonated Cu(Arm)(H;ANP)(+) both H+ and Cu(Arm)(2+) are at PO32- undergoing similar intramolecular equilibria as indicated above.

The acyclic nucleoside phosphonate (S)-1-[3-hydroxy-2-(phosphonomethoxy)propyl]cytosine (HPMPC; Cidofovir) has been approved for clinical use in antiviral therapy. We determined the acidity constants of H2(HPMPC)±, as well as that of the nucleobase-free (hydroxy-2-(phosphonomethoxy)propane (H(HPMP)−) (I=0.1M, NaNO3; 25°C). Given that in vivo nucleotides and their analogues participate in reactions typically as metal ion (M2+) complexes, the stability constants of the M(H;HPMPC)+, M(HPMPC), and M(HPMP) complexes with M2+=Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, and Cd2+ were measured. Comparisons between results for HPMP2− and previous data for PME2− (CH3CH2OCH2PO32-; phosphonomethoxyethane) revealed the hydroxyl-group effect. The hydroxyl group stabilizes only complexes with the heavier alkaline earth metal ions (Ca2+, Sr2+, Ba2+). For all other complexes, the enhanced stability can solely be explained by the formation of 5-membered chelates involving the ether oxygen; these occur in equilibrium with simple 'open' phosphonate-M2+ species. The stability of the M(HPMPC) complexes is also higher than expected for a phosphonate-only coordination, indicating that chelates are formed, but comparison with the HPMP2− data shows that the cytosine base does not affect complex stability. Similar observations were made previously with related cytosine derivatives. The stability data for the monoprotonated M(H;HPMPC)+ complexes suggest that these carry H+ predominantly on the phosphonate group, and M2+ on the nucleobase.

The acyclic nucleoside phosphonate (ANP2−) 9-[2-(phosphonomethoxy)ethyl]guanine (PMEG) is anticancer and antivirally active. The acidity constants of the threefold protonated H3(PMEG)+ were determined by potentiometric pH titrations (aq. sol.; 25 °C; I = 0.1 M, NaNO3). Under the same conditions and by the same method, the stability constants of the binary Cu(H;PMEG)+ and Cu(PMEG) complexes as well as those of the ternary ones containing a heteroaromatic N ligand (Arm), that is, of Cu(Arm)(H;PMEG)+ and Cu(Arm)(PMEG), where Arm = 2,2′-bipyridine (Bpy) or 1,10-phenanthroline (Phen), were measured. The corresponding equilibrium constants, taken from our earlier work for the systems with 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA) and 9-[2-(phosphonomethoxy)ethyl]-2,6-diaminopurine (PMEDAP) as well as those for Cu(PME) and Cu(Arm)(PME), where PME2− = (phosphonomethoxy)ethane = (ethoxymethyl)phosphonate, were used for comparisons. These reveal that in the monoprotonated ternary Cu(Arm)(H;PE)+ complexes, the proton and Cu(Arm)2+ are at the phosphonate group; the ether oxygen of the –CH2–O–CH2–P(O)2−(OH) residue also participates to some extent in Cu(Arm)2+ coordination. Furthermore, the coordinated Cu(Arm)2+ forms a bridge with the purine moiety undergoing π–π stacking which is more pronounced with H·PMEDAP− than with H·PMEA−. Most intense is π stack formation (st) with the guanine residue of H·PMEG−; here the bridged form Cu(Arm)(H·PMEG)st+ occurs next to an open (op), unbridged (binary) stack, formulated as Cu(Arm)2+/(H·PMEG)op−. The unprotonated and neutral ternary Cu(Arm)(PE) complexes are considerably more stable than the corresponding Cu(Arm)(R-PO3) species, where R-PO32− represents a phosph(on)ate ligand with a group R that is unable to participate in any intramolecular interaction. The observed stability enhancements are mainly due to intramolecular stack formation (st) between the aromatic rings of Arm and the purine residue in the Cu(Arm)(PE) complexes and also, to a smaller extent, to the formation of five-membered chelates involving the ether oxygen of the –CH2–O–CH2–PO32− residue (cl/O) of the PE2− species. The quantitative analysis of the intramolecular equilibria reveals three structurally different Cu(Arm)(PE) isomers; e.g., of Cu(Phen)(PMEG) ca. 1.1% exist as Cu(Phen)(PMEG)op, 3.5% as Cu(Phen)(PMEG)cl/O, and 95% as Cu(Phen)(PMEG)st. Comparison of the various formation degrees reveals that within a given Cu(Arm)(PE) series the stacking tendency decreases in the order PMEG2− ⩾ PMEDAP2− > PMEA2−. Furthermore, stacking is more pronounced in the acyclic Cu(Arm)(PE) complexes compared with that in the Cu(Arm)(NMP) species, where NMP2− = corresponding parent (2′-deoxy)nucleoside 5′-monophosphate. Here is possibly one of the reasons for the biological activity of the ANPs. One is tempted to speculate that the pronounced stacking tendency of PMEG2−, together with a different H-bonding pattern, leads to enhanced binding in the active site of nucleic acid polymerases, thus being responsible for the pronounced anticancer and antiviral activity of PMEG.

Affinity capillary electrophoresis (ACE) has been applied to estimation of apparent binding constant of complexes of ( R , S )‐enantiomers of selected acyclic nucleoside phosphonates (ANPs) with chiral selector β‐cyclodextrin (βCD) in aqueous alkaline medium. The noncovalent interactions of five pairs of ( R , S )‐enantiomers of ANPs‐based antiviral drugs and their derivatives with βCD were investigated in the background electrolyte (BGE) composed of 35 or 50 mM sodium tetraborate, pH 10.0, and containing variable concentration (0–25 mM) of βCD. The apparent binding constants of the complexes of ( R , S )‐enantiomers of ANPs with βCD were estimated from the dependence of effective electrophoretic mobilities of ( R , S )‐enantiomers of ANPs (measured simultaneously by ACE at constant reference temperature 25°C inside the capillary) on the concentration of βCD in the BGE using different nonlinear and linear calculation methodologies. Nonlinear regression analysis provided more precise and accurate values of the binding constants and a higher correlation coefficient as compared to the regression analysis of the three linearized plots of the effective mobility dependence on βCD concentration in the BGE. The complexes of ( R , S )‐enantiomers of ANPs with βCD have been found to be relatively weak – their apparent binding constants determined by the nonlinear regression analysis were in the range 13.3–46.4 L/mol whereas the values from the linearized plots spanned the interval 12.3–55.2 L/mol.

Capillary electrophoresis (CE) and quantum mechanical density functional theory (DFT) were applied to the investigation of the acid-base and electromigration properties of important compounds: newly synthesized derivatives of 5-azacytosine - analogs of efficient antiviral drug cidofovir. These compounds exhibit a strong antiviral activity and they are considered as potential new antiviral agents. For their characterization and application, it is necessary to know their acid-base properties, particularly the acidity constants (pKa) of their ionogenic groups (the basic N3 atom of the triazine ring and the acidic phosphonic acid group in the alkyl chain). First, the mixed acidity constants (pKamix) of these ionogenic groups and the ionic mobilities of these compounds were determined by nonlinear regression analysis of the pH dependence of their effective electrophoretic mobilities. Effective mobilities were measured by CE in a series of background electrolytes in a wide pH range (2.0-10.5), at constant ionic strength (25mM) and constant temperature (25°C). Subsequently, the pKamix values were recalculated to thermodynamic pKa values using the Debye-Hückel theory. The thermodynamic pKa value of the NH+ moiety at the N3 atom of the triazine ring was found to be in the range 2.82-3.30, whereas the pKa of the hydrogenphosphonate group reached values from 7.19 to 7.47, depending on the structure of the analyzed compounds. These experimentally determined pKa values were in good agreement with those calculated by quantum mechanical DFT. In addition, DFT calculations revealed that from the four nitrogen atoms in the 5-azacytosine moiety, the N3 atom of the triazine ring is preferentially protonated. Effective charges of analyzed compounds ranged from zero or close-to-zero values at pH 2 to -2 elementary charges at pH≥9. Ionic mobilities were in the range (-16.7 to -19.1)×10-9m2V-1s-1 for univalent anions and in the interval (-26.9 to -30.3)×10-9m2V-1s-1 for divalent anions.

Based on the previous discovery of the inhibitory effect of the 5-substituted 2-amino-4,6-dichloropyrimidines on nitric oxide (NO) production in vitro, a series of novel pyrimidine derivatives, namely 4,6-dichloro-2-[(N,N-dimethylamino)methyleneamino]pyrimidines, 2,4-diamino-6-chloropyrimidines, and 2,4-diamino-6-(2-hydroxyethoxy)pyrimidines, were prepared bearing various substituents at the C-5 position on the pyrimidine, such as hydrogen, methyl, ethyl, propyl, isopropyl, propargyl, allyl, butyl, sec-butyl, phenyl, benzyl, and fluorine. The intrinsic biological potential of the prepared compounds was characterized by effects on the in vitro production of immune-activated NO in mouse peritoneal cells. All 5-substituted 4,6-dichloro-2-[(N,N-dimethylamino)methyleneamino]pyrimidines strongly inhibited NO production. The IC50s were <5 µM in most cases. The highest inhibitory activity was observed for the 5-sec-butyl analog (IC50 = 2.57 µM), the lowest one for 5-unsubtituted compound (IC50 = 11.49 µM). With the exception of the 5-fluoro-4,6-dichloro-2-[(N,N-dimethylamino)methyleneamino] derivative, all other compounds were devoid of cytotoxic effects. The hitherto obtained data suggest that the NO-inhibitory activity depends on the presence of the 2-amino-4,6-dichloropyrimidine scaffold.

A series of 5-substituted 2-amino-4,6-dihydroxypyrimidines were prepared by a modified condensation of the corresponding monosubstituted malonic acid diesters with guanidine in an excess of sodium ethoxide. The optimized procedure using Vilsmeier–Haack–Arnold reagent, followed by immediate deprotection of the (dimethylamino)methylene protecting groups, has been developed to convert the 2-amino-4,6-dihydroxypyrimidine analogs to novel 5-substituted 2-amino-4,6-dichloropyrimidines in high yields. Pilot screening for biological properties of the prepared compounds was done in mouse peritoneal cells using the in vitro nitric oxide (NO) assay. Irrespective of the substituent at the 5 position, 2-amino-4,6-dichloropyrimidines inhibited immune-activated NO production. The most effective was 5-fluoro-2-amino-4,6-dichloropyrimidine with an IC 50 of 2 µM (higher activity than the most potent reference compound) while the IC 50s of other derivatives were within the range of 9–36 µM. The 2-amino-4,6-dihydroxypyrimidine counterparts were devoid of any NO-inhibitory activity. The compounds had no suppressive effects on the viability of cells. The Mechanism of action remains to be elucidated.

The acidity constants of protonated 9-[2-(phosphonomethoxy)ethyl]-2-amino-6-dimethylaminopurine (H3(PME2A6DMAP)+) are considered, and the stability constants of the M(H;PME2A6DMAP)+ and M(PME2A6DMAP) complexes (M2+ = Mg2+, Ca2+, Sr2+, Ba2+, Mn2+, Co2+, Ni2+, Cu2+, Zn2+, or Cd2+) were measured by potentiometric pH titrations in aqueous solution (25 °C; I = 0.1 mol/L, NaNO3). In the M(H;PME2A6DMAP)+ species, H+ and M2+ (mainly outersphere) are at the phosphonate group; this is relevant for phosphoryl-diester bridges in nucleic acids because, in the present system, there is no indication for a M2+–purine binding. This contrasts, for example, with the complexes formed by 9-[2-(phosphonomethoxy)ethyl]adenine, M(H;PMEA)+, where M2+ is mainly situated at the adenine residue. Application of log [Formula: see text] vs. [Formula: see text] plots for simple phosph(on)ate ligands, R–PO32− (R being a residue that does not affect M2+ binding), proves that all M(PME2A6DMAP) complexes have larger stabilities than what would be expected for a M2+–phosphonate coordination. Comparisons with M(PME–R) complexes, where R is a noncoordinating residue of the (phosphonomethoxy)ethane chain, allow one to conclude that the increased stability is due to the formation of five-membered chelates involving the ether–oxygen of the –CH2–O–CH2–PO32− residue: the percentages of formation of these M(PME2A6DMAP)cl/O chelates, which occur in intramolecular equilibria, vary between 20% (Sr2+, Ba2+) and 50% (Zn2+, Cd2+), up to a maximum of 67% (Cu2+). Any M2+ interaction with N3 or N7 of the purine moiety, as in the parent M(PMEA) complexes, is suppressed by the (C2)NH2 and (C6)N(CH3)2 substituents. This observation, together with the previously determined stacking properties, offers an explanation why PME2A6DMAP2– has remarkable therapeutic effects.

Protozoan parasites from the Plasmodiidae family are the causative agents of malaria. Inhibition of hypoxanthine-guanine-(xanthine) phosphoribosyltransferase (HG(X)PRT) has been suggested as a target for development of new anti-malarial therapeutics. Acyclic nucleoside phosphonates (ANPs) are potent and selective inhibitors of plasmodial HG(X)PRTs. A new series of ANPs, based on the chemical structure and inhibitory activity of three ANPs, 2-(phosphonoethoxy)ethyl with either guanine or hypoxanthine as the base (PEEG and PEEHx) and 3-hydroxy-2-(phosphonomethoxy)propyl with guanine as the base (HPMPG), were prepared. These compounds are stereoisomers of 3-fluoro-(2-phosphonoethoxy)propyl (FPEPs) and 3-fluoro-(2-phosphonomethoxy)propyl (FPMPs) analogues. Both the (R)- and (S)-isomers of these fluorinated derivatives have higher Ki values (by 10- to 1000-fold) for human HGPRT and Plasmodium falciparum HGXPRT than the non-fluorinated ANPs. Possible explanations for these changes in affinity are proposed based on docking studies using the known crystal structures of human HGPRT in complex with PEEG.

Stability constants of the ternary Cu(Arm)(H;PMEC)(+) and Cu(Arm)(PMEC) complexes {PMEC2- = dianion of 1-[2-(phosphonomethoxy)ethyl]cytosine, Arm = 2,2'-bipyridine (Bpy) or 1,10-phenanthroline (Phen)} were measured by potentiometric pH titrations (aq. sol.; 25 degrees C; I = 0.1 M, NaNO3) and compared with those of Cu(Arm)(H;PMEA)(+) and Cu(Arm)(PMEA) {PMEA(2-) = dianion of 9-[2-(phosphonomethoxy)ethyl]adenine}, and related species. The basicity of the terminal phosphonate group is similar in PMEC2- and PMEA(2-). Stability-constant comparisons reveal, that in the monoprotonated ternary Cu(Arm)(H;PMEC)(+) complexes H+ is at the phosphonate group, that the ether oxygen atom of the -CH2-O-CH2-P(O)(2)(-)(OH) residue participates, next to the P(O)(2)(-)(OH) group, in Cu(Arm)(2+) coordination, and that pi-pi stacking between the aromatic rings of Cu(Arm)(2+) and the pyrimidine moiety is important. The Cu(Arm)(PMEC) complexes are considerably more stable than the corresponding Cu(Arm)(R-PO3) species, where R-PO32- is a phosph(on)ate with a group R unable to interact intramolecularly. The stability enhancements are mainly attributed to intramolecular stacks and, to a smaller extent, to the formation of five-membered chelates involving the ether oxygen atom of the -CH2-O-CH2-P(O)(3)(2-) residue of PMEC2-. Analysis of the intramolecular equilibria reveals that ca. 10% of the isomeric ternary complexes exist with Cu(Arm)(2+) solely coordinated to the phosphonate group, ca. 25% as a five-membered chelate involving the ether oxygen, and ca. 65% with an intramolecular pi-pi stack between the pyrimidine moiety of PMEC2- and the rings of Bpy or Phen. For a given Cu(Arm)(2+) the stacking intensity increases from PMEC2- to PMEA(2-). It seems feasible that the reduced stacking intensity of PMEC2-, together with a different hydrogen bonding pattern, leads to a different orientation of the cytosine residue (compared to the adenine moiety) in the active site of the nucleic acid polymerases, thus resulting in a reduced antiviral activity of PMEC compared to PMEA.

Human immunodeficiency virus (HIV) infection is often accompanied by infection with other pathogens, in particular herpes simplex virus type 2 (HSV-2). The resulting coinfection is involved in a vicious circle of mutual facilitations. Therefore, an important task is to develop a compound that is highly potent against both viruses to suppress their transmission and replication. Here, we report on the discovery of such a compound, designated PMEO-DAPym. We compared its properties with those of the structurally related and clinically used acyclic nucleoside phosphonates (ANPs) tenofovir and adefovir. We demonstrated the potent anti-HIV and -HSV activity of this drug in a diverse set of clinically relevant in vitro, ex vivo, and in vivo systems including (i) CD4⁺ T-lymphocyte (CEM) cell cultures, (ii) embryonic lung (HEL) cell cultures, (iii) organotypic epithelial raft cultures of primary human keratinocytes (PHKs), (iv) primary human monocyte/macrophage (M/M) cell cultures, (v) human ex vivo lymphoid tissue, and (vi) athymic nude mice. Upon conversion to its diphosphate metabolite, PMEO-DAPym markedly inhibits both HIV-1 reverse transcriptase (RT) and HSV DNA polymerase. However, in striking contrast to tenofovir and adefovir, it also acts as an efficient immunomodulator, inducing β-chemokines in PBMC cultures, in particular the CCR5 agonists MIP-1β, MIP-1α and RANTES but not the CXCR4 agonist SDF-1, without the need to be intracellularly metabolized. Such specific β-chemokine upregulation required new mRNA synthesis. The upregulation of β-chemokines was shown to be associated with a pronounced downmodulation of the HIV-1 coreceptor CCR5 which may result in prevention of HIV entry. PMEO-DAPym belongs conceptually to a new class of efficient multitargeted antivirals for concomitant dual-viral (HSV/HIV) infection therapy through inhibition of virus-specific pathways (i.e. the viral polymerases) and HIV transmission prevention through interference with host pathways (i.e. CCR5 receptor down regulation).

We designed and synthesised a series of new cationic lipids based on spermine linked to various hydrophobic anchors. These lipids could be potentially useful for the preparation of stable cationic liposomes intended for the construction of drug targeting systems applicable in the field of anticancer/antiviral therapy, vaccine carriers, and vectors for the gene therapy. Low in vitro toxicity was found for these compounds, especially for LD1, in several cell lines. The delivery of both a fluorescence marker (calcein) and antiviral drugs into cells has been achieved owing to a large extent of internalization of cationic liposomes (labelled by Lyssamine-Rhodamine PE or fluorescein-PE) as demonstrated by fluorescent microscopy and quantified by flow cytometry. The bovine herpes virus type 1 (BHV-1) virus infection in vitro model using MDBK cells was employed to study the effect of the established antiviral drug HPMPC (Cidofovir®) developed by Prof. A. Holý. Inhibition of BHV-1 virus replication was studied by quantitative RT-PCR and confirmed by both Hoffman modulation contrast microscopy and transmission electron microscopy. We found that in vitro antiviral activity of HPMPC was significantly improved by formulation in cationic liposomes, which decreased the viral replication by about 2 orders of magnitude.

The oxidation reactions of 5-aminopyrimidine derivatives in dimethyl sulphoxide (DMSO) were studied. The DMSO solutions of the studied compounds became deeply coloured within a few hours or days. The oxidation products can undergo further condensation reactions with the starting pyrimidines to yield bipyrimidines and/or pyrimidopteridines. The reaction mechanism of the oxidation-condensation reaction was also supported by reactions of the 5-aminopyrimidines with alloxan (2,4,5,6-tetraoxopyrimidine). DMSO is often used as the solvent in in vitro tests of biological activities, but it is also an oxidising agent and may react with solute molecules and significantly affect the quality of the generated biochemical data.

6-Oxopurine acyclic nucleoside phosphonates (ANPs) have been shown to be potent inhibitors of hypoxanthine-guanine-xanthine phosphoribosyltransferase (HGXPRT), a key enzyme of the purine salvage pathway in human malarial parasites. These compounds also exhibit antimalarial activity against parasites grown in culture. Here, a new series of ANPs, hypoxanthine and guanine 9-[2-hydroxy-3-(phosphonomethoxy)propyl] derivatives with different chemical substitutions in the 2'-position of the aliphatic chain were prepared and tested as inhibitors of Plasmodium falciparum (Pf) HGXPRT, Plasmodium vivax (Pv) HGPRT and human HGPRT. The attachment of an hydroxyl group to this position and the movement of the oxygen by one atom distal from N(9) in the purine ring compared with 2-(phosphonoethoxy)ethyl hypoxanthine (PEEHx) and 2-(phosphonoethoxy)ethyl guanine (PEEG) changes the affinity and selectivity for human HGPRT, PfHGXPRT and PvHGPRT. This is attributed to the differences in the three-dimensional structure of these inhibitors which affects their mode of binding. A novel observation is that these molecules are not always strictly competitive with 5-phospho-α-d-ribosyl-1-pyrophosphate. 9-[2-Hydroxy-3-(phosphonomethoxy)propyl]hypoxanthine (iso-HPMP-Hx) is a very weak inhibitor of human HGPRT but remains a good inhibitor of both the parasite enzymes with K(i) values of 2μM and 5μM for PfHGXPRT and PvHGPRT, respectively. The addition of pyrophosphate to the assay decreased the K(i) values for the parasite enzymes by sixfold. This suggests that the covalent attachment of a second group to the ANPs mimicking pyrophosphate and occupying its binding pocket could increase the affinity for these enzymes.