Deoxy sugar analogues of triciribine: correlation of antiviral and antiproliferative activity with intracellular phosphorylation.

Triciribine (TCN) and triciribine monophosphate (TCN-P) have antiviral and antineoplastic activity at low micromolar or submicromolar concentrations. In an effort to improve and better understand this activity, we have conducted a structure-activity relationship study to explore requirements for the number of hydroxyl groups on the ribosyl moiety for biological activity. 2'-Deoxytriciribine (2'-dTCN), 3'-deoxytriciribine (3'-dTCN), 2', 3'-epoxytriciribine (2',3'-epoxyTCN), 2',3'-dideoxy-2', 3'-didehydrotriciribine (2',3'-d4TCN), and 2',3'-dideoxytriciribine (2',3'-ddTCN) were synthesized and evaluated for activity against human immunodeficiency virus (HIV-1), herpes simplex virus type 1 (HSV-1), and human cytomegalovirus (HCMV). Antiproliferative activity of the compounds also was tested in murine L1210 cells and three human tumor cell lines. All compounds were either less active than TCN and TCN-P or inactive at the highest concentration tested (100 microM) in both antiviral and antiproliferative assays. Reverse-phase HPLC of extracts from uninfected cells treated with the deoxytriciribine analogues only detected the conversion of 3'-dTCN and 2',3'-ddTCN to their respective monophosphates. Therefore, either the deoxytriciribine analogues were not transported across the cell membrane or, more likely, they were not substrates for a nucleoside kinase or phosphotransferase. We have concluded that the hydroxyl groups on the ribosyl ring system of TCN and TCN-P must be intact in order to obtain significant antiviral and antineoplastic activity.

6-N-Acyltriciribine analogues: structure-activity relationship between acyl carbon chain length and activity against HIV-1.

Triciribine (TCN) and triciribine-5'-monophosphate (TCN-P) are active against HIV-1 at submicromolar concentrations. In an effort to improve and better understand this activity, we have conducted a structure-activity relationship study to explore the tolerance of TCN to structural modifications at the 6-position. A number of 6-N-acyltriciribine analogues were synthesized and evaluated for antiviral activity and cytotoxicity. The cytotoxicity of these compounds was minimal in three human cell lines (KB, CEM-SS cells, and human foreskin fibroblasts (HFF)). The compounds were marginally active or inactive against herpes simplex virus type 1 (HSV-1) and human cytomegalovirus (HCMV). In contrast, most of the compounds exhibited moderate to high activity against human immunodeficiency virus type 1 (HIV-1), IC(50)'s = 0.03 to 1 microM. This structure-activity relationship study identified the N-heptanoyl group as having the optimal carbon chain length. This compound was as active against HIV-1 as TCN and TCN-P. Reverse phase HPLC of extracts from uninfected cells treated with 6-N-acyltriciribines detected sufficient TCN-P to account for anti-HIV activity thereby suggesting a prodrug effect. Studies in an adenosine kinase deficient cell line showed that the 6-N-acyl derivative was not phosphorylated directly but first was metabolized to triciribine which then was converted to TCN-P.

Acyclic sugar analogs of triciribine: lack of antiviral and antiproliferative activity correlate with low intracellular phosphorylation.

Triciribine and triciribine monophosphate have antiviral and antiproliferative activity at low or submicromolar concentrations. In an effort to improve and better understand this activity, we have synthesized a series of acyclic analogs and evaluated them for activity against select viruses and cancer cell lines. We conclude that the rigid ribosyl ring system of triciribine must be intact in order to be phosphorylated and to obtain significant antiviral and antiproliferative activity.

Phosphorylation of triciribine is necessary for activity against HIV type 1.

Triciribine (TCN) is a tricyclic nucleoside with known antineoplastic and antiviral activity. It is a potent and selective inhibitor of HIV-1 and HIV-2, including strains known to be resistant to AZT or TIBO. TCN is phosphorylated to its 5'-monophosphate (TCN-P) by intracellular adenosine kinase (AK), but is not converted to di- or triphosphates. We now report that 5'-phosphorylation is requisite for the activity of TCN against HIV-1. CEM cells incubated with TCN at concentrations ranging from 0.1 to 330 microM gave intracellular TCN-P concentrations from 27 to 775 microM, respectively. There was no difference in the amount of intracellular TCN-P detected in uninfected compared with HIV-1-infected CEM cells. The antiviral effect of TCN against HIV-1 was strongly antagonized by the AK inhibitor 5-iodotubercidin (ITu). In contrast, TCN and ITu only exhibited additive cytotoxicity. The 5'-deoxy analog of TCN, which cannot be phosphorylated, had no antiviral effect against HIV-1 at a concentration more than 100 times higher than the IC50 of TCN. Similarly, TCN was not active against HIV-1 in an AK-deficient cell line (AA-2) at concentrations shown to inhibit the virus by >95% in CEM cells. Consistent with its AK-deficient phenotype, this cell line phosphorylated TCN to only 3% of the extent observed in CEM cells. We conclude that TCN must be phosphorylated to TCN-P for activity against HIV-1.

Synthesis of 2,4-disubstituted thiazoles and selenazoles as potential antitumor and antifilarial agents: 1. Methyl 4-(isothiocyanatomethyl)thiazole-2-carbamates, -selenazole-2- carbamates, and related derivatives.

Methyl 4-(isothiocyanatomethyl)thiazole-2-carbamate and methyl 4-(isothiocyanatomethyl)selenazole-2-carbamate have been prepared via chemical transformations involving 2-amino-4-(chloromethyl)thiazole (1) and 2-amino-4-(chloromethyl)selenazole (2), respectively, as starting materials. The homoanalog, methyl 4-(2-isothiocyanatoethyl)thiazole-2-carbamate, was prepared from (2-aminothiazol-4-yl)acetic acid. All compounds prepared were evaluated for their ability to inhibit leukemia L1210 cell proliferation. Methyl 4-(isothiocyanatomethyl)thiazole-2-carbamate (7) was the most active compound in this screen, inhibiting the growth of L1210 leukemic cells with an IC50 = 3.2 microM. Mitotic blocking appears to be its primary mechanism of cytotoxic activity. Compound 7 also was the only compound which demonstrated significant in vivo antifilarial activity against the adult worms of Acanthocheilonema viteae in experimentally infected jirds. This compound was inactive against Brugia pahangi at a dosage of 100 mg/kg x 5 days.

Dual mechanisms of inhibition of DNA synthesis by triciribine.

The inhibition of DNA synthesis in triciribine (TCN)-treated L1210 cells was shown to involve two mechanisms, with different concentration dependence. (a) Initiation of new replicons and possibly of Okazaki fragments was inhibited when the cells were treated with 0.1 microM TCN. The inhibition of replicon initiation was shown by the rate of alkaline elution of [3H]DNA from 15-min-[3H]thymidine-labeled cells being slower if the cells had been pretreated with TCN, indicating that the average size of actively replicating DNA strands was increased. (b) At 1 microM TCN elongation of previously initiated DNA chains was also inhibited. This conclusion was suggested by the decrease in the rate of alkaline elution of [3H]DNA, during postlabeling incubation, being less if TCN was included in the medium. The mechanism of inhibition of DNA synthesis by TCN was shown not to involve DNA strand breakage or cross-linking, inhibition of polyamine biosynthesis, inhibition of purine de novo biosynthesis, inhibition of DNA polymerase alpha or DNA primase, or inhibition of ligation of Okazaki fragments. The effects of TCN on the incorporation of [3H]thymidine into Okazaki fragments and higher molecular weight DNA suggested the possibilities of inhibition of Okazaki fragment initiation and/or DNA polymerase delta.

Low nanogram detection of nucleotides using fast atom bombardment-mass spectrometry.

The effect of trimethylsilyl (TMS) derivatization on detection limits of mononucleotides in fast atom bombardment-mass spectrometry (FAB-MS) was examined. FAB-MS methods were developed to optimize sensitivity using adenosine 5'-monophosphate as a model compound and then applied to reference standards of two clinically important nucleotides: tricyclic nucleoside-5'-monophosphate (TCNMP) and 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP). The detection limit for the TMS derivative of TCNMP was 2.5-5 ng/microliters and less than 2.5 ng/microliters for FdUMP as its TMS derivative. This is greater than two orders of magnitude more sensitive than the FAB-MS analysis of the corresponding free compounds. These low detection limits for the TMS derivatives were obtained using a narrow scan range, signal averaging, detection in the negative ion mode, and 3-nitrobenzyl alcohol as the matrix. Hydrolysis of one or more of the labile TMS groups did occur, with the extent of hydrolysis being greatest in the more protic matrices.