Study of Different Substituted Cyclic and Acyclic Benzylpronucleotides of d4T Relative to Their Hydrolytic Stability and Antiviral Activity

Abstract

CycloSal-d4TMP and two different bis(benzyl) phosphate triesters of the antivirally active nucleoside analog d4T were studied with regard to their chemical hydrolysis behavior at pH 7.3, in CEM/0 cell extracts, and their anti-HIV activity. In contrast to triesters 2–4, bis-(o-AB)-d4TMP 1 was found to be chemically exquisitely stable. All compounds led to the formation of d4TMP in cell extracts and all triesters achieved the TK-bypass.     

Second Generation of cycloSal‐Pronucleotides with Esterase‐Cleavable Sites: The ”Lock‐In”‐Concept

A conceptual extension of the cycloSal‐pronucleotide approach is presented. The characteristic feature of the new cycloSal‐derivatives of the anti‐HIV active nucleoside analogue d4T 1 is the incorporation of an enzymatically cleavable carboxylic ester moiety with the intention to trap the triesters inside cells (”lock‐in”‐concept). CycloSal‐triesters bearing different ester groups in the 3‐or 5‐position of the cycloSal‐moiety are described. Surprisingly, only acetyl‐and levulinyl esters are cleaved readily in CEM cell extracts while alkyl esters were found to be stable. Nevertheless, in in‐vitro anti‐HIV assays most of the compounds achieve the thymidine–kinase bypass, thus proving that they act at least as nucleotide delivery systems.     

Second generation of cycloSal-pronucleotides with esterase-cleavable sites: the "lock-in"-concept

A conceptual extension of the cycloSal-pronucleotide approach is presented. The characteristic feature of the new cycloSal-derivatives of the anti-HIV active nucleoside analogue d4T 1 is the incorporation of an enzymatically cleavable carboxylic ester moiety with the intention to trap the triesters inside cells ("lock-in"-concept). CycloSal-triesters bearing different ester groups in the 3-or 5-position of the cycloSal-moiety are described. Surprisingly, only acetyl-and levulinyl esters are cleaved readily in CEM cell extracts while alkyl esters were found to be stable. Nevertheless, in in-vitro anti-HIV assays most of the compounds achieve the thymidine-kinase bypass, thus proving that they act at least as nucleotide delivery systems.     

Pathway of thiamine pyrophosphate synthesis in Micrococcus denitrificans.

The pathway of thiamine pyrophosphate (TPP) biosynthesis, which is formed either from exogeneously added thiamine or from the pyrimidine and thiazole moieties of thiamine, in Micrococcus denitrificans was investigated. The following indirect evidence shows that thiamine pyrophosphokinase (EC 2.7.6.2) catalyzes the synthesis of TPP from thiamine: (i) [35S]thiamine incubated with cells of this microorganism was detected in the form of [35S]thiamine; (ii) thiamine gave a much faster rate of TPP synthesis than thiamine monophosphate (TMP) when determined with the extracts; and (iii) a partially purified preparation of the extracts can use thiamine, but not TMP, as the substrate. The activities of the four enzymes involved in TMP synthesis from pyrimidine and thiazole moieties of thiamine were detected in the extracts of M. denitrificans. The extracts contained a high activity of the phosphatase, probably specific for TMP. After M. denitrificans cells were grown on a minimal medium containing 3 mM adenosine, which causes derepression of de novo thiamine biosynthesis in Escherichia coli, the activities of the four enzymes involved with TMP synthesis, the TMP phosphatase, and the thiamine pyrophosphokinase were enhanced two- to threefold. These results indicate that TPP is synthesized directly from thiamine without forming TMP as an intermediate and that de novo synthesis of TPP from the pyrimidine and thiazole moieties involves the formation of TMP, followed by hydrolysis to thiamine, which is then converted to TPP directly. Thus, the pathway of TPP synthesis from TMP synthesized de novo in M. denitrificans is different from that found in E. coli, in which TMP synthesized de novo is converted directly to TPP without producing thiamine.     

Bis-ketol nucleoside triesters as prodrugs of the antiviral nucleoside triphosphate analogues of 3'-deoxythymidine and 3'-deoxy-2',3'-didehydrothymidine

Derivatives of 3'-deoxythymidine (ddT) and 3'-deoxy-2',3'-didehydrothymidine (d4T) were prepared in which the 5'-hydroxyl group of the nucleoside was esterified to a bis-ketol phosphate. The resulting phosphate triesters are postulated to be prodrugs of the corresponding 5'-mononucleotides, which are formed intracellularly by the hydrolysis of the two ketol ester groups. The triesters were tested for anti-HIV activity with the result that those derived from ddT showed enhanced antiviral activity when compared to the parent nucleoside.     

Effects of cycloSal-d4TMP derivatives in H9 cells with induced AZT resistance phenotype.

Cytotoxic and antiretroviral activity of cycloSal-d4TMP derivatives were tested in a new AZT-resistant H9 cell subline (H9rAZT250). The results showed, that cycloSal-d4TMP derivatives overcame resistance of HIV-1 to d4T in H9rAZT250 cells, which exert decreased thymidine kinase (TK) gene expression.     

d4TMP delivery from 7-substituted cycloSal-d4TMPs

Benzyl-substituted cycloSal-d4T monophosphates were prepared and evaluated for their ability to release d4TMP selectively. In contrast to previously reported derivatives, two of the new compounds release d4TMP as the sole product while two others gave the expected benzyl phosphate diesters. However, these diesters were surprisingly stable against degradation to release d4TMP.     

A th-1 Mutant of Arabidopsis thaliana Is Defective for a Thiamin-Phosphate-Synthesizing Enzyme: Thiamin Phosphate Pyrophosphorylase

We have examined the activity of the thiamin phosphate pyrophosphorylase in Arabidopsis thaliana wild type and in a mutant (th-1) which requires exogenous thiamin for growth. Mutant and wild-type plants grown in 1 × 10⁻⁷ molar thiamin were used for the examination of the production of thiamin and thiamin monophosphate (TMP) using 4-methyl-5-hydroxyethylthiazole phosphate and 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate as substrates. While the wild-type strain formed both thiamin and TMP, the th-1 mutant did not. When TMP was added to the extracts, the th-1 mutant, as well as wild type, produced thiamin. Accordingly, it was concluded that the th-1 mutant was defective in the activity of TMP pyrophosphorylase. Some of the characteristics of the enzyme from the wild-type plant were examined. The optimum temperature for the reaction is 45°C, and the K_m values for the substrates are 2.7 × 10⁻⁶ molar for 4-methyl-5-hydroxyethylthiazole phosphate and 1.8 × 10⁻⁶ molar for 2-methyl-4-amino-5-hydroxymethylpyrimidine pyrophosphate.     

Different mutations responsible for the elevated sister-chromatid exchange frequencies in Bloom syndrome and X-irradiated B-lymphoblastoid cell lines originating from acute leukemia

Cell hybridization and co-cultivation protocols have been used to determine whether the increased rates of sister-chromatid exchanges (SCEs) exhibited by Bloom syndrome (BS) and a human mutant cell line (CCRF-SB-T1), originating from an X-irradiated acute leukemia-derived B-lymphoblastoid cell line, have the same or different bases. Cell fusion of CCRF-SB-T1 with each of 4 different BS B-lymphoblastoid cell lines (LCLs), retaining a high-SCE character, exhibited low (normal level) numbers of SCEs, signifying complementation. Co-cultivation of CCRF-SB-T1 and BS B-LCLs also resulted in a significant reduction in SCE level, from 70 to 35, in BS cells, lowered the BrdU concentrations necessary for sister-chromatid differential staining (SCD) from 15 micrograms/ml (0.05 mM) to 2.0 micrograms/ml (0.01 mM) and resulted in a completely normal level of SCE in CCRF-SB-T1 cells. This strongly suggests that the defects in the 2 cell types are different. In the assay of cell extracts, the 4 BS cell lines appear to have lost thymidylate (TMP) synthetase activity (about 50% reduction from that of normal cells), whereas CCRF-SB-T1 cells show a 20% increase of TMP synthetase activity compared to normal cells.     

d4TMP Delivery from 7-Substituted cycloSal-d4TMPs

Abstract

Benzyl-substituted cycloSal-d4T monophosphates were prepared and evaluated for their ability to release d4TMP selectively. In contrast to previously reported derivatives, two of the new compounds release d4TMP as the sole product while two others gave the expected benzyl phosphate diesters. However, these diesters were surprisingly stable against degradation to release d4TMP.     

Benzyl-functionalized cycloSal-d4T monophosphates

Synthetic routes to benzyl-functionalized cycloSal-d4T monophosphates (7CH2X-cycloSal-d4TMP) have been developed. Their hydrolytic behavior in basic aqueous solution (pH = 7.3) was studied and their hydrolysis half-lives were determined. It turned out that two different degradation pathways are leading to different products: beside the formation of the expected d4TMP and a styrene type derivative, a phenyl-d4T-phosphodiester was obtained as well. The product distribution was specified.     

Synthesis and in vitro evaluation of S-acyl-3-thiopropyl prodrugs of Foscarnet

A new enzyme-labile group called S-acyl-3-thiopropyl group (SATP) has been synthesized from allylic esters of phosphonate. After demonstration of the enzyme-labile character of the SATP in cellular extracts, it has been introduced onto the phosphonate moiety of PFA (Foscarnet) to obtain potential lipophilic prodrugs. To ponder the lipophilicity of the triesters of PFA, esters of monomethylether of polyethyleneglycols and of thioglycerol were introduced on the PFA carboxylate moiety. The SATP groups were introduced in an attempt to deliver PFA after bioactivation inside the cells. The PFA prodrugs were evaluated in vitro for their activity against human immunodeficiency viruses (HIV-1 and HIV-2).     

Effects of cycloSal-D4TMP Derivatives in H9 Cells with Induced AZT Resistance Phenotype

Abstract

Cytotoxic and antiretroviral activity of cycloSal-d4TMP derivatives were tested in a new AZT-resistant H9 cell subline (H9^rAZT²⁵⁰). The results showed, that cycloSal-d4TMP derivatives overcame resistance of HIV-1 to d4T in H9^rAZT²⁵⁰ cells, which exert decreased thymidine kinase (TK) gene expression.     

BENZYL-FUNCTIONALIZED cycloSal-d4T MONOPHOSPHATES

Synthetic routes to benzyl-functionalized cycloSal-d4T monophosphates (7CH₂X-cycloSal-d4TMP) have been developed. Their hydrolytic behavior in basic aqueous solution (pH = 7.3) was studied and their hydrolysis half-lives were determined. It turned out that two different degradation pathways are leading to different products: beside the formation of the expected d4TMP and a styrene type derivative, a phenyl-d4T-phosphodiester was obtained as well. The product distribution was specified.     

Effect of thyroxine in vitro on the transmembrane potential of rat liver mitochondria

It has been shown in in vitro experiments that a certain latent period after the addition of thyroxine (T4) and triiodothyronine (T3) was necessary for the manifestation of their effects on transmembrane potential (TMP) of the rat liver mitochondria. The duration of the lag-period decreased upon an increase in the concentrations of these hormones, and T4 at a dose of 2.10(-4) M produced a fall in TMP immediately after its addition. The rate of TMP fall was in proportion with the concentrations of thyroid hormones introduced into the cell, with T3 30-40% more effective than T4. It was established that the action of I2 resembled that of thyroid hormones, namely, a fall in TMP, mitochondrial swelling, activation of transhydrogenase Kl was ineffective. It is suggested that the appearance of the lag-period upon the action of thyroid hormones might be explained by the period of time necessary for the formation of the active iodine forms, as well as by the formation of fatty acids (donators of H+) by mitochondrial phospholipases. All these factors lead to TMP fall resulting in decreased formation of sufficient ATP quantities in mitochondria.     

NOVEL CYCLOSAL NUCLEOTIDES WITH REDUCED INHIBITORY POTENCY TOWARD HUMAN BUTYRYLCHOLINESTERASE

Two novel cycloSal-d4T monophosphates (d4TMPs) with increased steric demand have been synthesized via a new synthetic route. While 3-cyclohexyl-cycloSal d4TMP did not show a significantly reduced inhibitory potency toward human butyrylcholinesterase, the opposite was the case for the second novel pronucleotide, bis-(cycloSal-d4TMP).     

Novel cyclosal nucleotides with reduced inhibitory potency toward human butyrylcholinesterase

Two novel cycloSal-d4T monophosphates (d4TMPs) with increased steric demand have been synthesized via a new synthetic route. While 3-cyclohexyl-cycloSal d4TMP did not show a significantly reduced inhibitory potency toward human butyrylcholinesterase, the opposite was the case for the second novel pronucleotide, bis-(cycloSal-d4TMP).     

New developments of the "lock-in" modified cycloSal-d4TMPs

New developments of the "lock-in" modified cycloSal-d4TMP are reported. Novel prodrugs with variations in the linker chain were introduced. The synthesis, hydrolysis properties in different media (PBS, CEM/0- and liver extracts) and the antiviral activities against HIV are shown.     

Tetramethylpyrazine Suppresses Transient Oxygen-Glucose Deprivation-Induced Connexin32 Expression and Cell Apoptosis via the ERK1/2 and p38 MAPK Pathway in Cultured Hippocampal Neurons

Tetramethylpyrazine (TMP) has been widely used in China as a drug for the treatment of various diseases. Recent studies have suggested that TMP has a protective effect on ischemic neuronal damage. However, the exact mechanism is still unclear. This study aims to investigate the mechanism of TMP mediated ischemic hippocampal neurons injury induced by oxygen-glucose deprivation (OGD). The effect of TMP on hippocampal neurons viability was detected by MTT assay, LDH release assay and apoptosis rate was measured by flow cytometry. TMP significantly suppressed neuron apoptosis in a concentration-dependent manner. TMP could significantly reduce the elevated levels of connexin32 (Cx32) induced by OGD. Knockdown of Cx32 by siRNA attenuated OGD injury. Moreover, our study showed that viability was increased in siRNA-Cx32-treated-neurons, and neuron apoptosis was suppressed by activating Bcl-2 expression and inhibiting Bax expression. Over expression of Cx32 could decrease neurons viability and increase LDH release. Furthermore, OGD increased phosphorylation of ERK1/2 and p38, whose inhibitors relieved the neuron injury and Cx32 up-regulation. Taken together, TMP can reverse the OGD-induced Cx32 expression and cell apoptosis via the ERK1/2 and p38 MAPK pathways.