[Acyclic nucleoside phosphonates as potential antineoplastic agents]. / Acyklické nukleosidfosfonáty jako potenciální antineoplastika.

Recently, Gilead Sciences (Foster City, CA, USA) presented a potential cytostatic drug GS-9219. It is a novel lipophilic prodrug of cyprPMEDAP, in vivo releasing the active compound PMEG in a two-step process. GS-9219 has shown a substantial therapeutic potential in treatment of spontaneous non-Hodgkin's lymphoma in dogs and its utilization in the human medicine is prospective. Hence, cyprPMEDAP represents a key intermediate in the intracellular activation of GS-9219. Both acyclic nucleoside phosphonates PMEG and cyprPMEDAP, serving as the basis for development of GS-9219, were discovered and their mechanism of action was investigated in detail at the Institute of Organic Chemistry and Biochemistry, Academy of Sciences of the Czech Republic. The biological studies using the rat lymphoma were carried out at the First Faculty of Medicine, Charles University.

Experimental therapy with 9-[2-(phosphonomethoxy)ethyl]-2,6-diaminopurine (PMEDAP): origin of resistance.

The role of MRP4 and MRP5 transporters in the acyclic nucleoside phosphonate PMEDAP efflux was studied in vitro (CCRF-CEM cells) and in vivo (spontaneous transplantable T-cell lymphoma of SD/Cub inbred rats). The increased resistance against the cytostatic agent PMEDAP during longterm treatment was found to be associated with overexpression of MRP4 and MRP5 genes. The course of both gene activation differs significantly. While the MRP5 function is important in the onset of PMEDAP resistance, the intensity of the relative MRP4 gene expression increases rather continuously. Our data indicate cooperative acting of both MRP4 and MRP5 genes during the PMEDAP resistance development.

Synthesis and cytostatic activity of substituted 6-phenylpurine bases and nucleosides: application of the Suzuki-Miyaura cross-coupling reactions of 6-chloropurine derivatives with phenylboronic acids.

The Suzuki-Miyaura reaction of protected 6-chloropurine and 2-amino-6-chloropurine bases and nucleosides with substituted phenylboronic acids led to the corresponding protected 6-(substituted phenyl)purine derivatives 6-9. Their deprotection yielded a series of substituted 6-phenylpurine bases and nucleosides 10-13. Significant cytostatic activity (IC(50) 0.25-20 micromol/L) in CCRF-CEM, HeLa, and L1210 cell lines was found for several 6-(4-X-substituted phenyl)purine ribonucleosides 12 (X = H, F, Cl, and OR), while the 6-phenylpurine and 2-amino-6-phenylpurine bases 10 and 11, as well as 2-amino-6-phenylpurine ribosides 13, were entirely inactive against these cell lines.

9-[2-(Phosphonomethoxy)ethyl]adenine diphosphate (PMEApp) as a substrate toward replicative DNA polymerases alpha, delta, epsilon, and epsilon*.

The diphosphoryl derivative of the acyclic nucleotide phosphonate analog 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA), found previously to weakly inhibit DNA pol delta/proliferating cell nuclear antigen, was studied as a substrate for pol alpha, delta, epsilon, and epsilon*. A comparison of the Vmax and Km for this derivative (PMEApp) and dATP demonstrated that the relative efficiency of the incorporation of this analog into the DNA chain is decreasing in the following order: pol delta approximately equal to pol epsilon approximately equal to pol epsilon* > pol alpha. Under the reaction conditions, this incorporation amounted to 4.4 to 0.7% of dAMP molecules. Similar Km values for PMEApp and dATP in pol epsilon and pol epsilon* catalyzed reactions revealed that proteolysis of the enzyme probably does not affect the dNTP binding site. The DNA polymerases tested were inhibited by the reaction product (PMEA terminated DNA chain) with similar Ki/Km ratios (pol alpha 0.2; pol delta, 0.1; pol epsilon 0.05; and pol epsilon*, 0.06). The associated 3'-5'-exonuclease activity of pol delta, epsilon, and epsilon* was able to excise PMEA from the 3'-OH end of DNA with a rate one order of magnitude lower than that of the dAMP residue.

Interaction of guanine phosphonomethoxyalkyl derivatives with GMP kinase isoenzymes.

Substrate activity and inhibitory potency of guanine phosphonomethoxyalkyl derivatives towards GMP kinase isoenzymes from L1210 cells were studied. 9-[2-(Phosphonomethoxy)ethyl]guanine (PMEG) and the (R)- and (S)-enantiomers of both 9-[3-hydroxy-2-(phosphonomethoxy)propyl]guanine (HPMPG) and 9-[2-(phosphonomethoxy)propyl]guanine (PMPG) were phosphorylated to the first step. Kinetic data showed that (R)-PMPG was a good substrate with a relative phosphorylation efficacy of 12% compared with the natural substrate GMP, whereas PMEG was a poor substrate with a relative phosphorylation efficacy of 1.1%. The structurally related 2,6-diaminopurine analogues 9-[2-(phosphonomethoxy)ethyl]-2, 6-diaminopurine (PMEDAP) and (R)- and (S)-9-[2-(phosphonomethoxy)propyl]-2,6-diaminopurine (PMPDAP) were not phosphorylated by any of the GMP kinase isoenzymes tested. The inhibitory activities of the individual compounds on GMP kinase isoenzymes decreased in the following order: (S)-HPMPG > (R)-PMPG > PMEG > (R)-HPMPG > (S)-PMPG > PMEDAP = (R)-PMPDAP = (S)-PMPDAP; each compound exerted a different type of inhibition.

2-Methylpropyl ester of 3-(adenin-9-yl)-2-hydroxypropanoic acid. Mechanism of antiviral action in vaccinia virus-infected L929 cells.

Alkyl esters of (RS)-3-(adenin-9-yl)-2-hydroxypropanoic acid (AHPA) were shown recently to be broad spectrum antiviral agents (De Clercq E and Holy A, J Med Chem 28: 282-287, 1985). It was postulated that these alkyl esters function as prodrugs by undergoing hydrolysis catalyzed by cellular esters to AHPA, a known inhibitor of S-adenosyl-L-homocysteine (AdoHcy) hydrolase. In this study, we describe the metabolic fate of the 2-methylpropyl ester of AHPA (AHPA-iBu) in murine L929 cells. When AHPA-iBu was included in the culture medium, it was taken up rapidly by murine L929 cells. The uptake was time- and concentration-dependent, resulting in the intracellular accumulation of the free acid, AHPA. Treatment with AHPA-iBu caused inhibition of cellular AdoHcy hydrolase in both a time- and a dose-dependent manner. Complete inhibition of the enzyme was achieved after a 1-hr incubation in culture medium containing 50 microM AHPA-iBu. The inhibition of the enzyme caused cellular accumulation of AdoHcy and a significant increase in the ratio of AdoHcy/S-adenosyl-L-methionine (AdoMet). Partial recovery of the AdoHcy hydrolase activity in L929 cells treated with 50 microM AHPA-iBu was observed after 24 hr. This recovery of enzyme activity was paralleled by a significant decrease in the cellular levels of AdoHcy and the ratio of AdoHcy/AdoMet. AHPA-iBu also exerted an inhibition (IC50 = 0.17 microM) of vaccinia virus plaque formation in monolayers of L929 cells. A 1 microM concentration of AHPA-iBu, which caused 80% inhibition of plaque formation, produced a 17-fold increase in AdoHcy content in drug-treated, virus-infected cells versus non-drug-treated, virus-infected cells and a 15% undermethylation of the poly(A)+ RNA. These data show that AHPA-iBu is a prodrug for AHPA which inhibits cellular AdoHcy hydrolase. The inhibition of this enzyme elevates cellular levels of AdoHcy, creating an unfavorable environment which suppresses replication of vaccinia virus.

Phosphorylation of 9-(2-phosphonomethoxyethyl)adenine and 9-(S)-(3-hydroxy-2-phosphonomethoxypropyl)adenine by AMP(dAMP) kinase from L1210 cells.

Acyclic nucleotide analogues 9-(2-phosphonomethoxyethyl)adenine (PMEA) and 9-(S)-(3-hydroxy-2-phosphonomethoxypropyl)adenine ((S)-HPMPA) which display potent antiviral activity are transformed in the cells to their mono- and disphosphoryl derivatives. We purified from mouse L1210 cells the enzyme that in two steps phosphorylates PMEA and (S)-HPMPA to their diphosphoryl derivatives and found that it co-purifies with AMP(dAMP) kinase activity; the best substrates of this enzyme were AMP, ADP and dAMP. Other nucleoside 5'-triphosphates or creatine phosphate could not be substituted for ATP as a phosphate donor. Our results also indicated that at least one other enzyme (creatine kinase) is capable of transforming the monophosphoryl derivatives of the studied compounds to their respective diphosphates.

Acyclic nucleotide analogues suppress growth and induce apoptosis in human leukemia cell lines.

Acyclic nucleotide analogues perturb DNA replication by terminating the growing DNA chain. The analogues selected for testing on human leukemia cell lines, namely 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA), 9-[2-(phosphonomethoxy)ethyl]-2,6-diaminopurine (PMEDAP), and 9-[2-(phosphonomethoxy)ethyl]guanine (PMEG) exhibited growth-inhibiting activity at low concentrations, and apoptosis-inducing activity at high concentrations. A common feature was a reduction of the proportion of G1 cell cycle phase. Activities of the analogues increased in the order PMEA

Inhibition of avian myeloblastosis virus reverse transcriptase by diphosphates of acyclic phosphonylmethyl nucleotide analogues.

Diphosphates of N-(2-phosphonylmethoxyethyl) derivatives of heterocyclic bases were studied in the endogenous oligo(dT)12-18 primed reaction of reverse transcriptase from detergent-disrupted AMV(MAV) retrovirions. These diphosphates (analogues of nucleotide 5'-triphosphates) exhibited an inhibitory activity towards reverse transcriptase. This inhibitory activity was dependent on the character of the heterocyclic base and decreased in the order: 2-aminoadenine greater than adenine greater than guanine much greater than cytosine much greater than thymine greater than uracil. The 2-aminoadenine derivative was more potent than either AZT-TP or ddTTP, while PMEApp had approximately the same potency as the two reference compounds (IC50 approximately 1 microM at 20 microM competing substrate). This finding is consistent with the antiviral activity of the parent nucleotide analogues against retroviruses (including HIV).

Properties of a 9-(2-phosphonylmethoxyethyl)adenine (PMEA)-resistant herpes simplex virus type 1 virus mutant.

After repeated passages of herpes simplex type 1 (HSV-1) KOS virus in the presence of 9-(2-phosphonylmethoxyethyl)adenine (PMEA) a mutant denoted PMEAr HSV-1 was isolated which grew well in the presence of 50-100 micrograms.ml-1 of the drug. PMEAr HSV-1 was still sensitive to the related phosphonate analogue (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl)adenine (HPMPA). In fact, it was more susceptible to the action of HPMPA than the original virus. PMEAr HSV-1 also retained sensitivity to 5-bromo-2'-deoxyuridine and other, viral thymidine kinase-dependent substances such as (E)-5-(2-bromovinyl)-2'-deoxyuridine. However, PMEAr HSV-1 was much less sensitive to acyclovir, 1-(beta-D-arabinofuranosyl)cytosine and 1-(beta-D-arabinofuranosyl)thymine than the parental KOS virus.

Inhibition of herpes simplex virus DNA polymerase by diphosphates of acyclic phosphonylmethoxyalkyl nucleotide analogues.

The inhibition of HSV-1 DNA polymerase and HeLa DNA polymerases alpha and beta by diphosphoryl derivatives of acyclic phosphonylmethoxyalkyl nucleotide analogues was studied and compared with the inhibition by ACV-TP, araCTP, ddTTP and AZT-TP. In the series of phosphonylmethoxyethyl (PME-) derivatives of heterocyclic bases, the inhibitory effect of their diphosphates on HSV-1 DNA polymerase decreased in the order 2-amino-PMEApp (Ki = 0.03 microM) much greater than PMEGpp greater than PMEApp greater than PMETpp much greater than PMECpp much greater than n8z7PMEApp greater than PMEUpp. The diphosphate derivative of the antiherpes agent (S)-9-(3-hydroxy-2-phosphonylmethoxypropyl) adenine (HPMPA) proved to be a relatively weak inhibitor of HSV-1 DNA polymerase (Ki = 1.4 microM). The inhibitors could be divided into three groups: (a) the diphosphoryl derivatives of acyclic nucleotide analogues (PME-type and HPMPA) and ACV-TP specifically inhibit HSV-1 DNA polymerase and DNA polymerase alpha and do not significantly inhibit DNA polymerase beta; (b) AZT-TP and ddTTP are effective only against DNA polymerase beta, and (c) araCTP inhibits all three enzymes. When dATP was omitted from the reaction mixture, the addition of HPMPApp stimulated DNA synthesis by HSV-1 DNA polymerase indicating that HPMPApp is an alternative substrate for in vitro DNA synthesis catalyzed by this enzyme.

Phosphonylmethyl ethers of acyclic nucleoside analogues: inhibitors of HSV-1 induced ribonucleotide reductase.

Diphosphates of N-(S)-(3-hydroxy-2-phosphonylmethoxypropyl) and N-(2-phosphonylmethoxyethyl) derivatives of purine and pyrimidine heterocyclic bases inhibit HSV-1 encoded ribonucleotide reductase. Of the compounds studied, the most efficient inhibitors of CDP reduction (at 5.1 mumols.l-1) by the HSV-1-encoded enzyme are HPMPApp (IC50 = 0.9 mumols.l-1) and PMEApp (IC50 = 8 mumol.l-1). PMEApp does not inhibit the enzyme isolated from the mutant HSV-1 KOS strain PMEAr which is resistant to PMEA at a concentration of 100 micrograms/ml. The enzyme isolated from the PMEA-resistant virus strain is also insensitive to inhibitory effects of hydroxyurea and HPMPApp. Thus, the inhibitory potency of HPMPApp and PMEApp toward HSV-1 encoded ribonucleotide reductase might be connected with the anti-HSV activity of HPMPA and PMEA.

Acyclic nucleotide analogues: synthesis, antiviral activity and inhibitory effects on some cellular and virus-encoded enzymes in vitro.

Several N-(S)-(3-hydroxy-2-phosphonylmethoxypropyl) (HPMP) and N-(2-phosphonylmethoxyethyl) (PME) derivatives of purine bases (adenine, guanine, 2-aminoadenine, 3-deazaadenine) and cytosine inhibit the growth of various DNA viruses. PME-derivatives (PMEA, PMEG and PMEDAP) are also active against retroviruses. Both types of nucleotide analogues undergo phosphorylation by cellular nucleotide kinases to their mono- and diphosphates. The phosphorylation with crude extracts of L-1210 cells is potentiated by an ATP-regenerating system. HPMPA is phosphorylated faster than PMEA with or without the ATP-regenerating system. The HPMP and PME analogues inhibit several virus-encoded target enzymes and their cellular counterparts: (1) HSV-1 DNA polymerase is inhibited by the diphosphates of the PME series; the virus-encoded enzyme is more sensitive than HeLa DNA pol alpha and beta. PMEApp terminates the growing DNA chain; it specifically replaces dATP. HPMPApp also acts as an alternative substrate of dATP, but, in contrast with PMEApp, it permits limited chain growth. (2) Diphosphates of both series inhibit HSV-1 ribonucleotide reductase; the greatest inhibition of CDP reduction to dCDP is exhibited by HPMPApp and PMEApp. The enzyme isolated from a PMEA-resistant HSV-1 mutant proved less sensitive to PMEApp, hydroxyurea and HPMPApp. (3) Diphosphates of PME derivatives efficiently inhibit AMV(MAV) reverse transcriptase. (4) The purine HPMP and PME analogues and, even more so, their monophosphate derivatives inhibit purine nucleoside phosphorylase from L-1210 cells.

Hypomethylation of CNG targets induced with dihydroxypropyladenine is rapidly reversed in the course of mitotic cell division in tobacco.

We followed the mitotic transmission of an experimentally induced hypomethylated state of several tobacco repetitive sequences in callus culture and plants. The initial hypomethylation was induced by a hypomethylation drug, dihydroxypropyladenine (DHPA), the competitive inhibitor of cellular S-adenosylhomocysteine hydrolase, which is known to preferentially inhibit methylation at CNG and non-symmetrical motifs while having a negligible effect on methylation at CG motifs. The deprivation of this drug resulted in an almost immediate remethylation of cytosines at CNG motifs ( MspI and EcoRII sites) leading us to conclude that, the hypomethylation effect of dihydroxypropyladenine is rather transient and differs from that of 5-azacytidine which often induces heritable changes in methylation patterns. The results suggest that de novo methylation of CNG motifs is a rapid and meiotically independent process on DNA sequences with pre-existing CG methylation.

Antitumour activity of N6-substituted PMEDAP derivatives against T-cell lymphoma.

The antitumour activity of four N6-substituted PMEDAP derivatives, Me2NEt-PMEDAP, allyl-PMEDAP, Me2-PMEDAP and cypr-PMEDAP, selected on the basis of their in vitro cytostatic activity, was studied in an in vivo model of haematological malignancy of inbred Sprague-Dawley rats. These compounds are believed to serve as the prodrugs of another (phosphonomethoxy)ethyl derivative, PMEG (9-[2-phosphonomethoxy) ethyl] guanine. We compared their toxicity and ability to inhibit tumour development in two different dosage regimes with those of their parent compound PMEDAP, as well with PMEG. The study confirmed the anticancer efficacy of the parental compound PMEDAP. Unlike PMEDAP, its N6-mono- and disubstituted congeners Me2NEt-PMEDAP, allyl-PMEDAP and Me2-PMEDAP were less potent or exhibited the same antineoplastic effect as PMEDAP. cypr-PMEDAP significantly decreased the survival of lymphoma-bearing rats due to high toxicity, which was approximately the same as that of PMEG. Therefore, these acyclic nucleoside phosphonates substituted at the 6-position of 2,6-diaminopurine ring do not seem to be promising drugs for the treatment of haematological malignancies.

Anticancer effect of PMEDAP--monitoring of apoptosis.

Antitumor effect of N-9-[2-(phosphonomethoxy) ethyl]-2,6-diaminopurine (PMEDAP) was studied in an in vivo model of s.c. transplanted Sprague-Dawley (SD/cub) rat T-cell lymphomas. Three individual SD/cub neoplasias (SD10/96, SD14/97, SD1/90) of different phenotypes were used. During the treatment, survival of the rats, increase of lymphoma mass, and DNA fragmentation detected by APO/BRDU kit, as well as Bcl2 and p53 protein expression, were followed. The study gives evidence of the positive therapeutic effect of PMEDAP in two of the three tested lymphomas, SD10/96 and SD14/97. Slowly growing SD1/90 lymphoma differs from the others in a uniform karyotype with trisomy of chromosome 11, CD4- immunophenotype, heterogeneous cellular morphology and constitutive expression of p53 protein found in some neoplastic cells. Thus, the diverse anticancer efficacy of PMEDAP treatment among SD/cub lymphomas could be associated with the different phenotypes of individual neoplasias.

Antitumour activity of a combined treatment with PMEDAP and docetaxel in the Prague inbred Sprague-Dawley/cub rat strain bearing T-cell lymphoma.

Antitumour efficiency of combined therapy with N-9-[2-(phosphonomethoxy)ethyl]-2,6-diaminopurine (PMEDAP) and docetaxel (DTX) was studied in an in vivo model of s.c. transplanted Sprague-Dawley (SD/Cub) rat T-cell lymphoma (phenotype SD10/96). The effect of the combined treatment of DTX with PMEDAP was significantly higher than that of DTX or PMEDAP alone. The s.c. administration of DXT into the vicinity of growing lymphoma together with i.p. administration of PMEDAP was found to be the most efficient combination. In this case, two out of four rats did not develop any lymphoma and remained alive. An irregular expression of Bcl2 protein was found in untreated and treated lymphomas, while the expression of protein p53 as well as MDM2 was not observed. All three types of the above-mentioned treatments (PMEDAP, DXT, DXT+PMEDAP) increased significantly the number of p21-positive cells, compared with untreated tumours.

9-[2-(phosphonomethoxy)ethyl]-2,6-diaminopurine (PMEDAP)--a potential drug against hematological malignancies--induces apoptosis.

Antitumor activity of the acyclic nucleotide analogs PMEDAP, PMEA, and PMEG was studied on a model of a spontaneous T-cell lymphoma in inbred SD/cub rats. Significant therapeutic effects were recorded after a treatment with 16 daily doses of PMEDAP at 5 mg/kg applied to the vicinity of the growing lymphoma. Identical administration of PMEA, or PMEG at a daily dose of 0.1 mg/kg did not affect the survival of lymphoma-bearing animals compared with untreated controls. A decrease in the lymphoma weight during PMEDAP administration was accompanied by the suppression of mitotic activity in neoplastic cells and increased chromatin condensation as witnessed by karyological examinations. Electron-microscopy showed the morphology of apoptotic cells (shrunken cells with condensed chromatin, apoptotic bodies) in lymphoma cell suspensions. An increase of nuclear DNA fragmentation was found during PMEDAP administration compared with spontaneous DNA fragmentation of untreated control lymphomas. These results indicate that PMEDAP application induces apoptosis in in vivo growing lymphomas. The antitumor effect of PMEDAP lasts only during the administration of the drug. After its cessation progression of neoplasia was reestablished.

Synthesis of 4- and 5-amino-1-(2-deoxy-D-erythro-pentofuranosyl)imidazole nucleosides by chemical and biotransformation methods.

(2-Deoxy-D-erythro-pentofuranosyl)imidazole nucleosides have been synthesised by glycosylation of the sodium salt of ethyl 5-aminoimidazole-4-carboxylate with 2-deoxy-3,5-di-O-p-toluoyl-alpha-D-erythro-pentofuranosyl chloride. Glycosylation of ethyl 5-aminoimidazole-4-carboxylate with 2-deoxy-beta-D-erythro-pentofuranose was also achieved enzymically using E. coli, immobilised by ionotropic gelation in an alginate gel, with 2'-deoxyuridine as glycosyl donor. 5-Amino-1-(2-deoxy-beta-D-erythro-pentofuranosyl)imidazole-4-carboxylic acid 5'-phosphate and 4-amino-1-(2-deoxy-beta-D-erythro-pentofuranosyl)imidazole-5-carboxylic acid 5'-phosphate were synthesised by phosphorylation of the respective nucleosides and examined as inhibitors of phosphoribosylaminoimidazole carboxylase (EC 4.1.1.21) and phosphoribosylaminoimidazolesuccinocarboxamide synthetase (EC 6.3.2.6), which are involved in the de novo biosynthesis of purine nucleotides.

The effect of purine phosphonomethoxyalkyl derivatives on DNA synthesis in CHO Chinese hamster cells.

The inhibition of incorporation of 3H-thymidine and the changes of the rate of nascent DNA chain elongation were investigated in CHO Chinese hamster cells treated with (S)-(3-hydroxy-2-phosphonomethoxypropyl) (HPMP) and N-(2-phosphonomethoxyethyl) (PME) derivatives of adenine (A), guanine (G) and 2,6-diaminopurine (DAP). No direct correlation was observed in PME and HPMP derivatives between cytotoxicity, inhibition of 3H-thymidine incorporation and inhibition of nascent DNA chain elongation. The highest cytotoxicity and inhibition of DNA synthesis were caused by PMEG. The limited extent of inhibition of DNA elongation was encountered in the case of HPMPG and HPMPA. With PMEA, weak inhibition of elongation of DNA was observed only after a prolonged exposure (6 h). None of the investigated drugs induced DNA breaks.