Synthesis and biological activity of an acyclic analogue of 5,6,7,8-tetrahydrofolic acid, N-[4-[[3-(2,4-diamino-1,6-dihydro-6-oxo-5- pyrimidinyl)propyl]amino]-benzoyl]-L-glutamic acid.

The synthesis and biological evaluation of N-[4-[[3-(2,4-diamino-1,6-dihydro-6-oxo-5-pyrimidinyl)propyl]amino]- benzoyl]-L-glutamic acid (1) (5-DACTHF, 543U76), an acyclic analogue of 5,6,7,8-tetrahydrofolic acid (THFA), are described. The key intermediate, hemiaminal 8, was prepared in four stages from 3-chloropropionaldehyde diethyl acetal. Reaction of 8 with dimethyl N-(4-aminobenzoyl)-L-glutamate gave the 2,4-bis(acetylamino) derivative 11, which was hydrolyzed with 1 N sodium hydroxide to give 1; the glycine analogue 16 was prepared in a similar manner. The N-methyl analogue 2 and N-formyl analogue 3 were prepared from 11 and 1, respectively. Compounds 1-3 inhibited growth of Detroit 98 and L cells in cell culture, with IC50s ranging from 2 to 0.018 microM. Cell culture toxicity reversal studies and enzyme inhibition tests showed that 1 was cytotoxic but not by the mechanism of the dihydrofolate reductase inhibitor aminopterin. Compound 1 and its polyglutamylated homologues inhibited glycinamide ribonucleotide transformylase (GAR-TFase) and aminoimidazole ribonucleotide transformylase (AICAR-TFase), the folate-dependent enzymes in de novo purine biosynthesis; and 1 was an effective substrate for mammalian folyl-polyglutamate synthetase. The compound inhibited (IC50 = 20 nM) the conversion of [14C]formate to [14C]-formylglycinamide ribonucleotide by MOLT-4 cells in culture. These data suggest that the site of action of 1 is inhibition of purine de novo biosynthesis. Moderate activity was observed against P388 leukemia in vivo.

Imidazo[4,5-c]pyridines (3-deazapurines) and their nucleosides as immunosuppressive and antiinflammatory agents.

A variety of imidazo[4,5-c]pyridines (3-deazapurines) were synthesized. With use of these aglycons as pentosyl acceptors, the corresponding ribonucleosides and 2'-deoxyribonucleosides were prepared by an enzymatic method involving transfer of the pentosyl moiety from appropriate pyrimidine nucleosides. With most of the imidazo[4,5-c]pyridines, the products obtained from the enzyme-catalyzed reactions were pentosylated exclusively in the 1-position. However, some 3-pentosylation occurred with aglycons that had H or N3 in the 4-position. In addition to the 2'-deoxy congener of the ribonucleoside of 4-amino-1H-imidazo[4,5-c]pyridine, the 5'-deoxy and 2',5'-dideoxy congeners were synthesized. All of the aglycons and their nucleosides were tested for toxicity to mammalian cells in culture. None were markedly cytotoxic. These compounds were also evaluated for their ability to inhibit lymphocyte-mediated cytolysis in vitro. 3-Deazaadenosine (23) and its 2'-deoxy congener (38) were the most potent inhibitors (ED50 = 20 microM). In addition to these two in vitro tests, in vivo inhibition of the inflammatory response in the rat carregeenan pleurisy model was determined. 3-Deazaadenosine (23) was the most potent compound (ED50 = 3 mg/kg) in this in vivo test.

Biological action of inosine analogs in Leishmania and Trypanosoma spp.

Previous investigations have suggested that inosine analogs would be good models for the development of chemotherapeutic agents active against pathogenic hemoflagellates. We have systematically modified the five-membered heterocyclic ring of six inosine analogs and tested them for their antiprotozoal activities and toxicity to a mammalian cell line. All six analogs were very active against the three protozoan pathogens Leishmania donovani, Trypanosoma cruzi, and Trypanosoma gambiense. Two of the six, 9-deazainosine and allopurinol ribonucleoside, had very little toxicity for mouse L cells and offer promise as potential chemotherapeutic agents.

3'-Amino-2',3'-dideoxyribonucleosides of some pyrimidines: synthesis and biological activities.

3'-Amino-2',3'-dideoxyribonucleosides of thymine, uracil, and 5-iodouracil (1-3) were synthesized from the corresponding 2'-deoxyribonucleosides via the threo-3'-chloro and the erythro-3'-azido derivatives. Corresponding aminonucleosides of 5-bromouracil, 5-chlorouracil, and 5-fluorouracil (4-6) were synthesized enzymatically with 3'-amino-2',3'-dideoxythymidine as the aminopentosyl donor and thymidine phosphorylase (EC 2.4.2.4) as the catalyst. 3'-Amino-2',3'-dideoxycytidine (7) was synthesized by amination of the 3'-azido precursor of 3'-amino-2',3'-dideoxyuridine. The biological activity of 3'-amino-2',3'-dideoxy-5-fluorouridine (6) was notable among this group of aminonucleosides. It had an ED50 of 10 microM against adenovirus and was not appreciably cytotoxic to mammalian cells in culture. It also had activity against some Gram-positive bacteria but not against a variety of Gram-negative bacteria. The other aminonucleosides (1-5 and 7) lacked or exhibited weak antiviral and antibacterial activities. The only compounds in this group that were appreciably toxic to mammalian cells in culture were the thymidine and deoxycytidine analogues (1 and 7).

Pyrazolo[3,4-d]pyrimidine ribonucleosides as anticoccidials. 2. Synthesis and activity of some nucleosides of 4-(alkylamino)-1H-pyrazolo[3, 4-d]pyrimidines.

A series of 4-(alkylamino)-1-beta-D-ribofuranosyl-1H-pyrazolo[3, 4-d]pyrimidines was synthesized by enzymatic and chemical methods. On the basis of the previous finding that 4-(alkylthio)-1-beta-D-ribofuranosyl-1H-pyrazolo[3,4-d]pyrimidines were effective anticoccidial agents, this series was examined for efficacy against Eimera tenella in chicks. The most active anticoccidial agent in the present study was the 4-cyclopentylamino derivative (8), which cleared chicks of the parasite at 200 ppm in the diet. Some members of this series were toxic to embryonic chick liver cells, mouse cells, and human cells in vitro. The 4-diethylamino derivative (16), which was not toxic in vitro, appeared to be toxic in chicks.