Synthesis and evaluation of a (99m)Tc-MAMA-propyl-thymidine complex as a potential probe for in vivo visualization of tumor cell proliferation with SPECT.

INTRODUCTION: Cytosolic thymidine kinase (TK1) catalyzes phosphorylation of thymidine to its monophosphate. TK1 activity is closely related with DNA synthesis, and thymidine analogs derivatized with bulky carboranylalkyl groups at the N-3 position were reported to be good substrates for TK1. Accordingly, we have synthesized (99m)Tc-MAMA-propyl-thymidine and evaluated it as a potential tumor tracer. METHODS: The bis(S-trityl)-protected MAMA-propyl-thymidine precursor (3-N-[S-trityl-2-mercaptoethyl]-N-[N'-(S-trityl-2-mercaptoethyl)amidoacetyl]-aminopropyl-thymidine) was prepared in three steps, and its structure was confirmed with (1)H NMR and mass spectrometry. Deprotection of the thiols and labeling with (99m)Tc were done in a two-step, one-pot procedure, yielding (99m)Tc-MAMA-propyl-thymidine, which was analyzed with high-performance liquid chromatography, radio-LC-MS analysis (ESI+) and electrophoresis, and its log P was determined. The biodistribution in normal mice was evaluated, and its biodistribution in a radiation-induced fibrosarcoma (RIF) tumor mouse was compared with that of 3'-deoxy-3'-[(18)F] fluorothymidine [(18)F]FLT. RESULTS: (99m)Tc-MAMA-propyl-thymidine was obtained with a radiochemical yield of 70%. Electrophoresis indicated that the complex is uncharged, and its log P was 1.0. The molecular ion mass of the Tc complex was 589 Da, which is compatible with the hypothesized N(2)S(2)-oxotechnetium structure. Tissue distribution showed fast clearance from plasma primarily by the hepatobiliary pathway. Whole-body planar imaging after injection of (99m)Tc-MAMA-propyl-thymidine in an RIF tumor-bearing mouse showed high uptake in the liver and the intestines. No uptake was observed in the tumor, in contrast to the clear uptake observed for [(18)F] FLT visualized with muPET. CONCLUSIONS: Although it has been reported that TK1 accepts large substituents at the N-3 position of the thymine ring, the results of this study show that (99m)Tc-MAMA-propyl-thymidine cannot be used as a single photon emission computed tomography tumor tracer, probably because the (99m)Tc-MAMA ligand is too bulky to be tolerated by TK1.

Pronounced anti-proliferative activity and tumor cell selectivity of 5-alkyl-2-amino-3-methylcarboxylate thiophenes.

5-(2-(4-Methoxyphenyl)ethyl)-2-amino-3-methylcarboxylate thiophene (TR560) is the prototype drug of a recently discovered novel class of tumor-selective compounds that preferentially inhibit the proliferation of specific tumor cell types (e.g. leukemia/lymphoma). Here, we further increased tumor selectivity by simplification of the molecule through replacing the 4-methoxyphenyl moiety by an alkyl chain. Several 2-amino-3-methylcarboxylate thiophene derivatives containing at C-5 an alkyl group consisting of at least 6 (hexyl) to 9 (nonyl) carbon units showed pronounced anti-proliferative activity in the mid-nanomolar range with 500- to 1000-fold tumor cell selectivity. The compounds preferentially inhibited the proliferation of T-lymphoma CEM and Molt/4, prostate PC-3, kidney Caki-1 and hepatoma Huh-7 tumor cells, but were virtually inactive against other tumor cell lines including B-lymphoma Raji and cervix carcinoma HeLa cells. The novel prototype drug 3j (containing a 5-heptyl chain) elicited a cytotoxic, rather than cytostatic activity, already after 4 h of exposure. The unusual tumor selectivity could not be explained by a differential uptake (or efflux) of the drug by sensitive versus resistant tumor cells. Exposure of a fluorescent derivative of 3j revealed pronounced uptake of the drug in the cytoplasm, no visible appearance in the nucleus, and a predominant localization in the endoplasmic reticulum. These observations may be helpful to narrow down the intracellular localization and identification of the molecular target of the 5-substituted thiophene derivatives.

Guanine α-carboxy nucleoside phosphonate (G-α-CNP) shows a different inhibitory kinetic profile against the DNA polymerases of human immunodeficiency virus (HIV) and herpes viruses.

α-Carboxy nucleoside phosphonates (α-CNPs) are modified nucleotides that represent a novel class of nucleotide-competing reverse transcriptase (RT) inhibitors (NcRTIs). They were designed to act directly against HIV-1 RT without the need for prior activation (phosphorylation). In this respect, they differ from the nucleoside or nucleotide RTIs [N(t)RTIs] that require conversion to their triphosphate forms before being inhibitory to HIV-1 RT. The guanine derivative (G-α-CNP) has now been synthesized and investigated for the first time. The (L)-(+)-enantiomer of G-α-CNP directly and competitively inhibits HIV-1 RT by interacting with the substrate active site of the enzyme. The (D)-(-)-enantiomer proved inactive against HIV-1 RT. In contrast, the (+)- and (-)-enantiomers of G-α-CNP inhibited herpes (i.e. HSV-1, HCMV) DNA polymerases in a non- or uncompetitive manner, strongly indicating interaction of the (L)-(+)- and the (D)-(-)-G-α-CNPs at a location different from the polymerase substrate active site of the herpes enzymes. Such entirely different inhibition profile of viral polymerases is unprecedented for a single antiviral drug molecule. Moreover, within the class of α-CNPs, subtle differences in their sensitivity to mutant HIV-1 RT enzymes were observed depending on the nature of the nucleobase in the α-CNP molecules. The unique properties of the α-CNPs make this class of compounds, including G-α-CNP, direct acting inhibitors of multiple viral DNA polymerases.