A novel and efficient one-pot synthesis of symmetrical diamide (bis-amidate) prodrugs of acyclic nucleoside phosphonates and evaluation of their biological activities.

A novel and efficient method for the one-pot synthesis of diamide (bis-amidate) prodrugs of acyclic nucleoside phosphonates, starting from free phosphonic acids or phosphonate diesters is reported. The approach from phosphonate diesters via their bis(trimethylsilyl) esters is highly convenient, eliminates isolation and tedious purification of the phosphonic acids, and affords the corresponding bis-amidates in excellent yields (83-98%) and purity. The methodology has been applied to the synthesis of the potent anticancer agent GS-9219, and symmetrical bis-amidates of other biologically active phosphonic acids. Anti-HIV, antiproliferative, and immunomodulatory activities of the compounds are discussed including the bis-amidate prodrugs 14 and 17 that exhibited anti-HIV activity at submicromolar concentrations with minimal cytotoxicity.

Inhibition of SAH-hydrolase activity during seed germination leads to deregulation of flowering genes and altered flower morphology in tobacco.

Developmental processes are closely connected to certain states of epigenetic information which, among others, rely on methylation of chromatin. S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) are key cofactors of enzymes catalyzing DNA and histone methylation. To study the consequences of altered SAH/SAM levels on plant development we applied 9-(S)-(2,3-dihydroxypropyl)-adenine (DHPA), an inhibitor of SAH-hydrolase, on tobacco seeds during a short phase of germination period (6 days). The transient drug treatment induced: (1) dosage-dependent global DNA hypomethylation mitotically transmitted to adult plants; (2) pleiotropic developmental defects including decreased apical dominance, altered leaf and flower symmetry, flower whorl malformations and reduced fertility; (3) dramatic upregulation of floral organ identity genes NTDEF, NTGLO and NAG1 in leaves. We conclude that temporal SAH-hydrolase inhibition deregulated floral genes expression probably via chromatin methylation changes. The data further show that plants might be particularly sensitive to accurate setting of SAH/SAM levels during critical developmental periods.

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.

Nitric oxide production in mouse and rat macrophages: a rapid and efficient assay for screening of drugs immunostimulatory effects in human cells.

Activation of inducible nitric oxide (NO) synthase (iNOS) and resulting high-output NO release is known to depend on the action of cytokines. We investigated in vitro production of NO by resident peritoneal macrophages from mice and rats, and secretion of cytokines by these cells as well as by human peripheral blood mononuclear cells (PBMC). The cells were cultured in the presence of a selected group of acyclic nucleoside phosphonates that have previously been shown to possess immunobiological potential. Several of the compounds enhanced production of NO in animal macrophages. This activity was associated with stimulatory effects on secretion of cytokines such as TNF-alpha in all mouse and rat macrophages and human PBMC, and IL-10 in mouse and human cells. Statistically highly significant correlation between the range of NO biosynthesis in rodent cells and extent of cytokine stimulation in human PBMC has been observed. It is suggested that the NO assay may be regarded as an efficient, economical and relatively reliable tool in primary screening for intrinsic immunostimulatory activity of compounds in human cell system, at least from the point of view of cytokine secretion.

Purine P1 receptor-dependent immunostimulatory effects of antiviral acyclic analogues of adenine and 2,6-diaminopurine.

Acyclic nucleoside phosphonates are widely recognised antivirals. The oral prodrugs of prototype compounds, e.g., 9-[2-(phosphonomethoxy)ethyl]adenine (PMEA; adefovir), and 9-(R)-[2-(phosphonomethoxy)propyl]adenine [(R)-PMPA; tenofovir] were approved by FDA for treatment of hepatitis B (Hepsera), and acquired immunodeficiency syndrome (AIDS) (Viread), respectively. A number of acyclic nucleoside phosphonates possess immunostimulatory activity. The present experiments demonstrate that activation of cytokine and chemokine secretion is mediated by adenosine receptors. Included in the study were 9-(R)-[2-(phosphonomethoxy)propyl]adenine [tenofovir], N(6)-cyclopentyl-(R)-9-[2-(phosphonomethoxy)propyl]-2,6-diaminopurine, N(6)-cyclopropyl-(R)-9-[2-(phosphonomethoxy)propyl]-2,6-diaminopurine, and N(6)-isobutyl-9-[2-(phosphonomethoxy)ethyl]-2,6-diaminopurine. All of them activate secretion of tumor necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10), "regulated on activation of normal T cell expressed and secreted" (RANTES/CCL5), and macrophage inflammatory protein-1alpha (MIP-1alpha/CCL3) in murine macrophages. With exception of MIP-1alpha, the effects were inhibited by antagonists of adenosine A(1), A(2B), and A(3) receptors (not by adenosine A(2A) receptor antagonist). The adenosine A(1) receptor antagonist inhibited TNF-alpha, IL-10, and RANTES, adenosine A(2B) receptor antagonist inhibited TNF-alpha and RANTES, and adenosine A(3) receptor antagonist inhibited IL-10 and RANTES. The suppression is due to decreased transcription of cytokine mRNA. It may be suggested that acyclic nucleoside phosphonates are nonspecific ligands for purine P(1) receptors.

Immunobiological activity of N-[2-(phosphonomethoxy)alkyl] derivatives of N6-substituted adenines, and 2,6-diaminopurines.

Acyclic nucleoside phosphonates are novel class of virostatics effective against replication of both DNA-viruses and retroviruses. We found recently, that in addition to the antimetabolic mode of action, some acyclic nucleoside phosphonates such as 9-[2-(phosphonomethoxy)propyl]adenine [(R)-PMPA; tenofovir], which is used in treatment of human immunodeficiency virus (HIV) infection, possess immunostimulatory and immunomodulatory activities known to interfere with replication of viruses. The present experiments analyzed immunobiological effects of more than 70 novel derivatives of acyclic nucleoside phosphonates. They comprise substitutions at the N6-amino function of adenine (A) or 2,6-diaminopurine (DAP) by monoalkyl, dialkyl, cycloalkyl, alkenyl, alkynyl or substituted alkyl group, and at the N9-side chain represented by (R)- or (S)-enantiomeric 9-[2-(phosphonomethoxy)ethyl] (PME) and 9-[2-(phosphonomethoxy)propyl] (PMP) moieties. Their biological effects were investigated in vitro using mouse resident peritoneal macrophages. A number of the compounds under scrutiny, mainly the N6-cycloalkyl derivatives of 9-[2-(phosphonomethoxy)ethyl]2,6-diaminopurine (PMEDAP) and (R)-enantiomeric 9-[2-(phosphonomethoxy)propyl]adenine [(R)-PMPDAP] stimulate secretion of cytokines [tumor necrosis factor-alpha (TNF-alpha), interleukin-10 (IL-10)] and chemokines ["regulated-upon-activation, normal T expressed and secreted" (RANTES), macrophage inflammatory protein-1alpha (MIP-1alpha)]. Moreover, they substantially augment production of nitric oxide (NO) triggered by interferon-gamma. The effects are produced in a dose-dependent fashion. The most potent derivatives, i.e. N6-isobutyl-PMEDAP, N6-cyclopentyl-PMEDAP, N6-cyclooctyl-PMEDAP, N6-dimethylaminoethyl-(R)-PMPDAP, N6-cyclopropyl-(R)-PMPDAP, and N6-cyclopentyl-(R)-PMPDAP are more effective than (R)-PMPA (tenofovir) itself. They exhibit immunostimulatory effects at concentrations as low as 1 to 5 microM. It is suggested that these compounds might be prospective candidates for antiviral therapeutic exploitation.