Discovery of a potent and selective human AC2 inhibitor based on 7-deazapurine analogues of adefovir.

Adefovir based acyclic nucleoside phosphonates were previously shown to modulate bacterial and, to a certain extent, human adenylate cyclases (mACs). In this work, a series of 24 novel 7-substituted 7-deazaadefovir analogues were synthesized in the form of prodrugs. Twelve analogues were single-digit micromolar inhibitors of Bordetella pertussis adenylate cyclase toxin with no cytotoxicity to J774A.1 macrophages. In HEK293 cell-based assays, compound 14 was identified as a potent (IC50 = 4.45 µM), non-toxic, and selective mAC2 inhibitor (vs. mAC1 and mAC5). Such a compound represents a valuable addition to a limited number of small-molecule probes to study the biological functions of individual endogenous mAC isoforms.

Stereo-Defined Acyclic Nucleoside Phosphonates are Selective and Potent Inhibitors of Parasite 6-Oxopurine Phosphoribosyltransferases.

Pathogens such as Plasmodium and Trypanosoma spp. are unable to synthesize purine nucleobases. They rely on the salvage of these purines and their nucleosides from the host cell to synthesize the purine nucleotides required for DNA/RNA production. The key enzymes in this pathway are purine phosphoribosyltransferases (PRTs). Here, we synthesized 16 novel acyclic nucleoside phosphonates, 12 with a chiral center at C-2', and eight bearing a second chiral center at C-6'. Of these, bisphosphonate (S,S)-48 is the most potent inhibitor of the Plasmodium falciparum and P. vivax 6-oxopurine PRTs and the most potent inhibitor of two Trypanosoma brucei (Tbr) 6-oxopurine PRTs yet discovered, with Ki values as low as 2 nM. Crystal structures of (S,S)-48 in complex with human and Tbr 6-oxopurine PRTs show that the inhibitor binds to the enzymes in different conformations, providing an explanation for its potency and selectivity (i.e., 35-fold in favor of the parasite enzymes).

Halogen-Dance-Based Synthesis of Phosphonomethoxyethyl (PME) Substituted 2-Aminothiazoles as Potent Inhibitors of Bacterial Adenylate Cyclases.

A series of acyclic nucleoside phosphonates (ANPs) was designed as inhibitors of bacterial adenylate cyclases (ACs), where adenine was replaced with 2-amino-4-arylthiazoles. The target compounds were prepared using the halogen dance reaction. Final AC inhibitors were evaluated in cell-based assays (prodrugs) and cell-free assays (phosphono diphosphates). Novel ANPs were potent inhibitors of adenylate cyclase toxin (ACT) from Bordetella pertussis and edema factor (EF) from Bacillus anthracis, with substantial selectivity over mammalian enzymes AC1, AC2, and AC5. Six of the new ANPs were more potent or equipotent ACT inhibitors (IC50 =9-18 nM), and one of them was more potent EF inhibitor (IC50 =12 nM), compared to adefovir diphosphate (PMEApp) with IC50 =18 nM for ACT and IC50 =36 nM for EF. Thus, these compounds represent the most potent ACT/EF inhibitors based on ANPs reported to date. The potency of the phosphonodiamidates to inhibit ACT activity in J774A.1 macrophage cells was somewhat weaker, where the most potent derivative had IC50 =490 nM compared to IC50 =150 nM of the analogous adefovir phosphonodiamidate. The results suggest that more efficient type of phosphonate prodrugs would be desirable to increase concentrations of the ANP-based active species in the cells in order to proceed with the development of ANPs as potential antitoxin therapeutics.

Acyclic nucleoside phosphonates with 2-aminothiazole base as inhibitors of bacterial and mammalian adenylate cyclases.

A series of novel acyclic nucleoside phosphonates (ANPs) was synthesized as potential adenylate cyclase inhibitors, where the adenine nucleobase of adefovir (PMEA) was replaced with a 5-substituted 2-aminothiazole moiety. The design was based on the structure of MB05032, a potent and selective inhibitor of fructose 1,6-bisphosphatase and a good mimic of adenosine monophosphate (AMP). From the series of eighteen novel ANPs, which were prepared as phosphoroamidate prodrugs, fourteen compounds were potent (single digit micromolar or submicromolar) inhibitors of Bordetella pertussis adenylate cyclase toxin (ACT), mostly without observed cytotoxicity in J774A.1 macrophage cells. Selected phosphono diphosphates (nucleoside triphosphate analogues) were potent inhibitors of ACT (IC50 as low as 37 nM) and B. anthracis edema factor (IC50 as low as 235 nM) in enzymatic assays. Furthermore, several ANPs were found to be selective mammalian AC1 inhibitors in HEK293 cell-based assays (although with some associated cytotoxicity) and one compound exhibited selective inhibition of mammalian AC2 (only 12% of remaining adenylate cyclase activity) but no observed cytotoxicity. The mammalian AC1 inhibitors may represent potential leads in development of agents for treatment of human inflammatory and neuropathic pain.

Xanthine-based acyclic nucleoside phosphonates with potent antiviral activity against varicella-zoster virus and human cytomegalovirus.

While noncanonic xanthine nucleotides XMP/dXMP play an important role in balancing and maintaining intracellular purine nucleotide pool as well as in potential mutagenesis, surprisingly, acyclic nucleoside phosphonates bearing a xanthine nucleobase have not been studied so far for their antiviral properties. Herein, we report the synthesis of a series of xanthine-based acyclic nucleoside phosphonates and evaluation of their activity against a wide range of DNA and RNA viruses. Two acyclic nucleoside phosphonates within the series, namely 9-[2-(phosphonomethoxy)ethyl]xanthine (PMEX) and 9-[3-hydroxy-2-(phosphonomethoxy)propyl]xanthine (HPMPX), were shown to possess activity against several human herpesviruses. The most potent compound was PMEX, a xanthine analogue of adefovir (PMEA). PMEX exhibited a single digit µM activity against VZV (EC50 = 2.6 µM, TK+ Oka strain) and HCMV (EC50 = 8.5 µM, Davis strain), while its hexadecyloxypropyl monoester derivative was active against HSV-1 and HSV-2 (EC50 values between 1.8 and 4.0 µM). In contrast to acyclovir, PMEX remained active against the TK- VZV 07-1 strain with EC50 = 4.58 µM. PMEX was suggested to act as an inhibitor of viral DNA polymerase and represents the first reported xanthine-based acyclic nucleoside phosphonate with potent antiviral properties.

Nucleobase Modified Adefovir (PMEA) Analogues as Potent and Selective Inhibitors of Adenylate Cyclases from Bordetella pertussis and Bacillus anthracis.

A series of 13 acyclic nucleoside phosphonates (ANPs) as bisamidate prodrugs was prepared. Five compounds were found to be non-cytotoxic and selective inhibitors of Bordetella pertussis adenylate cyclase toxin (ACT) in J774A.1 macrophage cell-based assays. The 8-aza-7-deazapurine derivative of adefovir (PMEA) was found to be the most potent ACT inhibitor in the series (IC50 =16 nm) with substantial selectivity over mammalian adenylate cyclases (mACs). AC inhibitory properties of the most potent analogues were confirmed by direct evaluation of the corresponding phosphonodiphosphates in cell-free assays and were found to be potent inhibitors of both ACT and edema factor (EF) from Bacillus anthracis (IC50 values ranging from 0.5 to 21 nm). Moreover, 7-halo-7-deazapurine analogues of PMEA were discovered to be potent and selective mammalian AC1 inhibitors (no inhibition of AC2 and AC5) with IC50 values ranging from 4.1 to 5.6 µm in HEK293 cell-based assays.

Crystal structures and inhibition of Trypanosoma brucei hypoxanthine-guanine phosphoribosyltransferase.

Human African Trypanosomiasis (HAT) is a life-threatening infectious disease caused by the protozoan parasite, Trypanosoma brucei (Tbr). Due to the debilitating side effects of the current therapeutics and the emergence of resistance to these drugs, new medications for this disease need to be developed. One potential new drug target is 6-oxopurine phosphoribosyltransferase (PRT), an enzyme central to the purine salvage pathway and whose activity is critical for the production of the nucleotides (GMP and IMP) required for DNA/RNA synthesis within this protozoan parasite. Here, the first crystal structures of this enzyme have been determined, these in complex with GMP and IMP and with three acyclic nucleoside phosphonate (ANP) inhibitors. The Ki values for GMP and IMP are 30.5 µM and 77 µM, respectively. Two of the ANPs have Ki values considerably lower than for the nucleotides, 2.3 µM (with guanine as base) and 15.8 µM (with hypoxanthine as base). The crystal structures show that when two of the ANPs bind, they induce an unusual conformation change to the loop where the reaction product, pyrophosphate, is expected to bind. This and other structural differences between the Tbr and human enzymes suggest selective inhibitors for the Tbr enzyme can be designed.

Bisamidate Prodrugs of 2-Substituted 9-[2-(Phosphonomethoxy)ethyl]adenine (PMEA, adefovir) as Selective Inhibitors of Adenylate Cyclase Toxin from Bordetella pertussis.

Novel small-molecule agents to treat Bordetella pertussis infections are highly desirable, as pertussis (whooping cough) remains a serious health threat worldwide. In this study, a series of 2-substituted derivatives of 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA, adefovir), in their isopropyl ester bis(L-phenylalanine) prodrug form, were designed and synthesized as potent inhibitors of adenylate cyclase toxin (ACT) isolated from B. pertussis. The series consists of PMEA analogues bearing either a linear or branched aliphatic chain or a heteroatom at the C2 position of the purine moiety. Compounds with a small C2 substituent showed high potency against ACT without cytotoxic effects as well as good selectivity over human adenylate cyclase isoforms AC1, AC2, and AC5. The most potent ACT inhibitor was found to be the bisamidate prodrug of the 2-fluoro PMEA derivative (IC50 =0.145 µM). Although the bisamidate prodrugs reported herein exhibit overall lower activity than the bis(pivaloyloxymethyl) prodrug (adefovir dipivoxil), their toxicity and plasma stability profiles are superior. Furthermore, the bisamidate prodrug was shown to be more stable in plasma than in macrophage homogenate, indicating that the free phosphonate can be effectively distributed to target tissues, such as the lungs. Thus, ACT inhibitors based on acyclic nucleoside phosphonates may represent a new strategy to treat whooping cough.

Amidate prodrugs of 9-[2-(phosphonomethoxy)ethyl]adenine as inhibitors of adenylate cyclase toxin from Bordetella pertussis.

Adenylate cyclase toxin (ACT) is the key virulence factor of Bordetella pertussis that facilitates its invasion into the mammalian body. 9-[2-(Phosphonomethoxy)ethyl]adenine diphosphate (PMEApp), the active metabolite of the antiviral drug bis(POM)PMEA (adefovir dipivoxil), has been shown to inhibit ACT. The objective of this study was to evaluate six novel amidate prodrugs of PMEA, both phenyloxy phosphonamidates and phosphonodiamidates, for their ability to inhibit ACT activity in the J774A.1 macrophage cell line. The two phenyloxy phosphonamidate prodrugs exhibited greater inhibitory activity (50% inhibitory concentration [IC50] = 22 and 46 nM) than the phosphonodiamidates (IC50 = 84 to 3,960 nM). The inhibitory activity of the prodrugs correlated with their lipophilicity and the degree of their hydrolysis into free PMEA in J774A.1 cells. Although the prodrugs did not inhibit ACT as effectively as bis(POM)PMEA (IC50 = 6 nM), they were significantly less cytotoxic. Moreover, they all reduced apoptotic effects of ACT and prevented an ACT-induced elevation of intracellular [Ca(2+)]i. The amidate prodrugs were less susceptible to degradation in Caco-2 cells compared to bis(POM)PMEA, while they exerted good transepithelial permeability in this assay. As a consequence, a large amount of intact amidate prodrug is expected to be available to target macrophages in vivo. This feature makes nontoxic amidate prodrugs attractive candidates for further investigation as novel antimicrobial agents.

Interactions with selected drug renal transporters and transporter-mediated cytotoxicity in antiviral agents from the group of acyclic nucleoside phosphonates.

Members of acyclic nucleoside phosphonates (ANPs) possess antiviral and antiproliferative activities. However, several clinically important ANPs may cause renal injury, most likely due to their active accumulation in the renal tubular cells. The goal of this study was to investigate in vitro relationships between the affinity of several structurally related potent ANPs to selected human transporters and their cytotoxicity. SLC (solute carrier family) transporters (hOAT1, hOCT2, hCNT2, hCNT3) and ABC (ATP-binding cassette) transporters (MDR1, BCRP), which are typically expressed in the kidney, were included in the study. The transport and toxic parameters of the tested compounds were compared to those of two clinically approved ANPs, adefovir and tenofovir. Transport studies with transiently transfected cells were used as the main method in the experiments. Most of the ANPs studied showed the potency to interact with hOAT1. GS-9191, a double prodrug of PMEG, displayed an affinity for hOAT1 comparable with that of adefovir and tenofovir. No significant interaction of the tested ANPs with hOCT2, hCNT2 and hCNT3 was observed. Only GS-9191 was found to be a strong inhibitor for both MDR1 and BCRP. PMEO-DAPy showed the potency to interact with MDR1. Most of the tested substances caused a significant decrease in cellular viability in the cells transfected with hOAT1. Only with the exclusion of GS-9191, a relatively lipophilic compound, did the in vitro cytotoxicity of the ANPs closely correspond to their potential to interact with hOAT1. The increased cytotoxicity of the studied ANPs found in OAT1 transfected cells was effectively reduced by OAT inhibitors probenecid and quercetin. The higher cytotoxicity of the compounds with affinity to hOAT1 proved in the inhibitory experiments evidences that ANPs are not only inhibitors but also substrates of hOAT1. Any clear relationship between the potency of ANPs to inhibit the studied efflux transporters and their cytotoxicity was not demonstrated. In conclusion, the study documented that among the studied transporters hOAT1 seems to be the decisive determinant for renal handling in most of the tested ANPs. This transporter may also play an important role in the mechanism of their potential cytotoxic effects. These facts are in good accordance with previous findings in the clinically used ANPs.

Synthesis and biological evaluation of guanidino analogues of roscovitine.

A series of 2,9-substituted 6-guanidinopurines, structurally related to the cyclin-dependent kinase (CDK) inhibitors olomoucine and roscovitine, has been synthesized and characterized. A new copper-catalyzed method for the synthesis of 2-substituted 6-guanidino-9-isopropylpurines under mild reaction conditions has been developed. All prepared compounds were screened for their CDK1 and CDK2 inhibitory activities, cytotoxicity and antiproliferative effects in the breast cancer-derived cell line MCF7. The most active derivative 16g possessed an identical side chain in the C2 position to roscovitine; this compound displayed approximately five fold higher inhibitory activity towards CDK2/cyclin E and more than ten fold increase in cytotoxicity in MCF7 cells. Interestingly and in contrast to previously described findings, (S)-6-guanidinopurine derivatives were generally more active than their (R)-counterparts. Kinase selectivity profiling of (R)- and (S)-enantiomers 16e and 16g, respectively, revealed that introduction of a guanidino group at the C6 position of the purine moiety decreased selectivity towards protein kinases compared to roscovitine. Nevertheless, increased inhibitory activity and decreased selectivity offer a good starting point for further development of new protein kinase inhibitors.

Synthesis of 9-phosphonoalkyl and 9-phosphonoalkoxyalkyl purines: evaluation of their ability to act as inhibitors of Plasmodium falciparum, Plasmodium vivax and human hypoxanthine-guanine-(xanthine) phosphoribosyltransferases.

The purine salvage enzyme, hypoxanthine-guanine-(xanthine) phosphoribosyltransferase [HG(X)PRT], catalyses the synthesis of the purine nucleoside monophosphates, IMP, GMP or XMP essential for DNA/RNA production. In protozoan parasites, such as Plasmodium, this is the only route available for their synthesis as they lack the de novo pathway which is present in human cells. Acyclic nucleoside phosphonates (ANPs), analogs of the purine nucleoside monophosphates, have been found to inhibit Plasmodium falciparum (Pf) HGXPRT and Plasmodium vivax (Pv) HGPRT with K(i) values as low as 100 nM. They arrest parasitemia in cell based assays with IC(50) values of the order of 1-10 µM. ANPs with phosphonoalkyl and phosphonoalkoxyalkyl moieties linking the purine base and phosphonate group were designed and synthesised to evaluate the influence of this linker on the potency and/or selectivity of the ANPs for the human and malarial enzymes. This data shows that variability in the linker, as well as the positioning of the oxygen in this linker, influences binding. The human enzyme binds the ANPs with K(i) values of 0.5 µM when the number of atoms in the linker was 5 or 6 atoms. However, the parasite enzymes have little affinity for such long chains unless oxygen is included in the three-position. In comparison, all three enzymes have little affinity for ANPs where the number of atoms linking the base and the phosphonate group is of the order of 2-3 atoms. The chemical nature of the purine base also effects the K(i) values. This data shows that both the linker and the purine base play an important role in the binding of the ANPs to these three enzymes.

Plasmodium vivax hypoxanthine-guanine phosphoribosyltransferase: a target for anti-malarial chemotherapy.

The malarial parasite, Plasmodium vivax (Pv), causes a serious infectious disease found primarily in Asia and the Americas. For protozoan parasites, 6-oxopurine phosphoribosyltransferases (PRTases) provide the only metabolic pathway to synthesize the purine nucleoside monophosphates essential for DNA/RNA production. We have purified the recombinant Pv 6-oxopurine (PRTase) and compared its properties with the human and Pf enzymes. The Pv enzyme uses hypoxanthine and guanine with similar catalytic efficiency to the Pf enzyme but xanthine is not a substrate, hence we identify this enzyme as PvHGPRT. Mass spectrometry suggests that PvHGPRT contains bound magnesium ions that are removed by EDTA resulting in loss of activity. However, the addition of Mg(2+) restores activity. Acyclic nucleoside phosphonates (ANPs) are good inhibitors of PvHGPRT having K(i) values as low as 3 microM. These compounds can form the basis for the design of new drugs aimed at combating malaria caused by Pv.

Synthesis of guanidino analogues of PMPDAP and their immunobiological activity.

A series of novel 9-, 7- and 3-substituted 2- or 6-guanidinopurines as analogues of potent antiviral and immunobiologically active compound enantiomers of PMPDAP was synthesized and evaluated for their biological activity. Compounds containing the combination of guanidino and amino group at the purine moiety enhanced the interferon-gamma-triggered NO production in murine macrophages and stimulated the secretion of cytokines and chemokines in both murine macrophages and human peripheral blood mononuclear cells. The most active compounds are 27 and 54. None of the compounds tested exhibited any significant cytostatic effect or antiviral effect.

Determination of acid-base dissociation constants of amino- and guanidinopurine nucleotide analogs and related compounds by capillary zone electrophoresis.

CZE has been applied for determination of acid-base dissociation constants (pKa) of ionogenic groups of newly synthesized amino- and (amino)guanidinopurine nucleotide analogs, such as acyclic nucleoside phosphonate, acyclic nucleoside phosphonate diesters and other related compounds. These compounds bear characteristic pharmacophores contained in various important biologically active substances, such as cytostatics and antivirals. The pKa values of ionogenic groups of the above compounds were determined by nonlinear regression analysis of the experimentally measured pH dependence of their effective electrophoretic mobilities. The effective mobilities were measured by CZE performed in series of BGEs in a broad pH range (3.50-11.25), at constant ionic strength (25 mM) and temperature (25 degrees C). pKa values were determined for the protonated guanidinyl group in (amino)guanidino 9-alkylpurines and in (amino)guanidinopurine nucleotide analogs, such as acyclic nucleoside phosphonates and acyclic nucleoside phosphonate diesters, for phosphonic acid to the second dissociation degree (-2) in acyclic nucleoside phosphonates of amino and (amino)guanidino 9-alkylpurines, and for protonated nitrogen in position 1 (N1) of purine moiety in acyclic nucleoside phosphonates of amino 9-alkylpurines. Thermodynamic pKa of protonated guanidinyl group was estimated to be in the range of 7.75-10.32, pKa of phosphonic acid to the second dissociation degree achieved values of 6.64-7.46, and pKa of protonated nitrogen in position 1 of purine was in the range of 4.13-4.89, depending on the structure of the analyzed compounds.

Synthesis of 9-alkyl and 9-heteroalkyl substituted 2-amino-6-guanidinopurines and their influence on the NO-production in macrophages.

9-Alkyl and 9-heteroalkyl substituted derivatives of the 2-amino-6-guanidinopurine were synthesized by alkylation of 2-amino-6-chloropurine and subsequent guanidinolysis. The activity of the thus prepared compounds on murine macrophages was examined. Compounds 4a, 4b, and 4d inhibit the LPS+IFN-gamma-induced NO production in murine macrophages while compound 4h stimulates this production.

A neoceptor approach to unraveling microscopic interactions between the human A2A adenosine receptor and its agonists.

Strategically mutated neoceptors, e.g., with anionic residues in TMs 3 and 7 intended for pairing with positively charged amine-modified nucleosides, were derived from the antiinflammatory A(2A) adenosine receptor (AR). Adenosine derivatives functionalized at the 5', 2, and N(6) positions were synthesized. The T88D mutation selectively enhanced the binding of the chain-length-optimized 5'-(2-aminoethyl)uronamide but not 5'-(2-hydroxyethyl)uronamide, suggesting a critical role of the positively charged amine. Combination of this modification with the N(6)-(2-methylbenzyl) group enhanced affinity at the Q89D- and N181D- but not the T88D-A(2A)AR. Amino groups placed near the 2- or N(6)-position only slightly affected the binding to mutant receptors. The 5'-hydrazide MRS3412 was 670- and 161-fold enhanced, in binding and functionally, respectively, at the Q89D-A(2A)AR compared to the wild-type. Thus, we identified and modeled pairs of A(2A)AR-derived neoceptor-neoligand, which are pharmacologically orthogonal with respect to the native species.