Synthesis and antitumor activity of pyrrolo[2,3-d]pyrimidine antifolates with a bridge chain containing a nitrogen atom.

Novel pyrrolo[2,3-d]pyrimidine antifolates (1a, b and 2a, b) with a nitrogen atom in the bridge chain between the 2,4-diaminopyrrolo[2,3-d]pyrimidine and phenylene rings were designed and efficiently synthesized. These compounds exhibited more potent inhibitory activities than methotrexate (MTX) against the proliferation of human epidermoid carcinoma KB cells and human non-small cell lung carcinoma A549 cells despite their modest dihydrofolate reductase (DHFR)-inhibitory potency.

Novel pyrrolo[2,3-d]pyrimidine antifolates: synthesis and antitumor activities.

New antifolates, characterized by a 6-5 fused ring system, a pyrrolo[2,3-d]pyrimidine ring, and a trimethylene bridge at position 5 (12a,b and 13a,b) were designed and efficiently synthesized. The synthetic method included (1) construction of the key intermediary acyclic skeleton, 5-[4-(tert-butoxycarbonyl)phenyl]- 2-(dicyanomethyl)pentanoates (6a,b), (2) cyclization with guanidine, followed by reduction to the pyrrolo[2,3-d]pyrimidine derivatives (8a,b and 9a,b), and (3) subsequent glutamate coupling and saponification. These antifolates were more growth-inhibitory by about 1 order of magnitude than methotrexate (MTX) against KB human epidermoid carcinoma cells and A549 human nonsmall cell lung carcinoma cells in in vitro culture. Growth inhibitory IC50 values for N-[4-[3-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5- yl)propyl]benzoyl]-L-glutamic acid (12a) against KB and A549 were 0.27 and 4.5 ng/mL, while those for MTX were 5.0 and 35 ng/mL, respectively. Other members of this class of antifolates, 12b and 13a,b, showed good activities nearly equal to that of 12a.

The high content of natural suppressor serine tRNA in dystrophic mouse muscle.

In order to gain an insight into the pathogenesis of mouse muscular dystrophy, we investigated the natural suppressor serine tRNA. The natural suppressor seryl-tRNA was distinguished from the other seryl-tRNAs on the basis of its specific property of being converted into phosphoseryl-tRNA by a tRNA kinase. On a wet-weight basis, the content of total tRNA in dystrophic muscles was 47% of that in normal muscles. Although the serine-accepting activities of tRNA were similar in muscles of 3-month-old dystrophic and normal mice, the ratio of [32P]phosphoseryl-tRNA (suppressor tRNA) to the total serine tRNA was significantly enhanced in dystrophic muscles compared with that in normal muscles. This high content of suppressor tRNA in dystrophic muscles was further confirmed by dot-blot hybridization experiments with the DNA probes CGTAGTCGGCAGGAT and CGCCCGAAAGGTGGAA for major tRNA(IGASer) and suppressor tRNA respectively. At the early postnatal age of 3 weeks, when only a week had elapsed since the first manifestation of the dystrophic symptom (hindleg dragging), the ratio of suppressor tRNA to major tRNAs in dystrophic hindleg muscles was abnormally increased. Thereafter it decreased with age in normal mice but remained almost unchanged in dystrophic mice. Consequently, at 3 months old, it was 1.7 times higher in dystrophic than in normal mice. The suppressor tRNA is now accepted to play a role in the synthesis of glutathione peroxidase. The present study showed that the content of this enzyme was abnormally elevated in dystrophic mice. Previously we had demonstrated that the docosahexaenoic (C22:6) acid content in phospholipids was decreased, possibly resulting from the enhanced oxidative milieu caused by the dystrophic condition. Thus far, the findings suggest that an increase in the contents of suppressor tRNA and glutathione peroxidase in dystrophic muscle may have been secondarily induced by such a highly oxidative state in the dystrophic condition. However, it is difficult to exclude the possibility that the natural suppressor tRNA plays a primary role in the pathogenesis of muscular dystrophies.

Fatty acid composition of lipids in tongue and hindleg muscles of muscular dystrophic mice.

In order to understand the pathogenesis of mouse muscular dystrophy, fatty acid and lipid compositions in tongue and hindleg muscles of dystrophic mice were analyzed. The phospholipid contents in tongue and hindleg muscles were 71-73% and 23-24% of the total lipids, respectively. The content of triglyceride in tongue and hindleg muscles was 8% and 66% of the total lipids, respectively. There were no significant differences in the phospholipid content of the tongue or hindleg muscles between normal and dystrophic mice. However, analyses of the fatty acid composition in phospholipids showed that the content of 16:0 and 22:6 in the hindleg muscles of dystrophic mice decreased significantly, while the content of C-18 fatty acids (18:0, 18:1 and 18:2) increased. In addition, the fatty acid composition in phospholipids of tongues of dystrophic mice was identical to that of normal mice. This latter result supports the bone-muscle imbalance hypothesis for the pathogenesis of mouse muscular dystrophy.

The detection of natural opal suppressor seryl-tRNA in Escherichia coli by the dot blot hybridization and phosphorylation by a tRNA kinase [corrected] .

It was believed that there was no natural suppressor tRNA in Escherichia coli, however, it has been suggested that selC, relating to the synthesis of formate dehydrogenase of a selenoprotein [(1988) Nature 331, 723-725], codes for tRNA, even though the presence of tRNA has not yet been demonstrated. We detected the product of selC in the tRNA preparation of the E. coli MC 4100 strain by the dot blot hybridization method with a DNA probe (ACCGCTGGCGGC) corresponding to the extra arm of selC tRNA. Two hybridization peaks were found in the chromatographic pattern from Sephadex A50. The amount of tRNA was estimated to be about 0.03% of the total tRNA. Some tRNA [corrected] was phosphorylated by a tRNA kinase in E. coli B. These results suggest that the opal suppressor seryl-tRNA in E. coli should be converted to selenocysteyl-tRNA [corrected] and occurs in vertebrates as a general phenomenon.

The conversion of phosphoserine residues to selenocysteine residues on an opal suppressor tRNA and casein.

This study has been undertaken in order to elucidate the mechanisms of incorporation of Se into glutathione peroxidase (GSHPx), in which selenocysteine corresponds to the opal termination codon UGA on the mRNA. We studied the above mechanisms using an opal suppressor tRNA, prepared from bovine liver, and casein as a model protein for the GSHPx apo-enzyme which might contain phosphoserine. The results showed that opal suppressor tRNA did not accept selenocysteine (lower than 0.1 mmol/mol) under the standard conditions. A trace amount of phosphoseryl-tRNA was converted to selenocysteyl-tRNA by incubation with H2Se and some enzymes. Meanwhile, a number of phosphoserine residues in casein were converted to selenocysteine residues by incubation with H2Se and enzymes. These results suggest that opal suppressor tRNA plays a role in synthesizing GSHPx via co- and/or post-translational mechanisms.

Stronger affinity of reticulocyte release factor than natural suppressor tRNASer for the opal termination codon.

Animal natural suppressor tRNA did not affect the release reaction of reticulocyte release factor (RF) at the same concentration of tRNA (both estimated as being present at a similar level of 3-5 X 10(-8) M in vivo); even at a 10-fold greater concentration the tRNA did not prevent the release reaction with RF. In order to confirm this result, the Ka values were determined. The Ka value between RF and UGA was 1.26 X 10(6) M-1 and that between the suppressor tRNA and UGA amounted to 8 X 10(3) M-1. This result showed that RF had a 150-fold stronger affinity than suppressor tRNA for the opal termination codon. Incorporation of phosphoserine into phosphoprotein via phosphoseryl-tRNA was inhibited by addition of RF to the reaction mixture. These results suggest that animal natural suppressor tRNA in the normal state does not perform its suppressor function, except in special cases where mRNA has the context structure near the opal termination codon (UGA).

Selenocysteine on glutathione peroxidase may be converted from phosphoserine on the apo-enzyme synthesized with an opal suppressor phosphoseryl-tRNA.

There are two possible mechanisms (co- or post-translational) for incorporation of Se into glutathione peroxidase in which selenocysteine presents at the active site of the enzyme and corresponds to UGA on the mRNA. We studied the above mechanisms using opal suppressor tRNA in mammals. Opal suppressor tRNA did not accept any selenocysteine and phosphoseryl-tRNA did not change to selenocysteyl-tRNA. Meanwhile, phosphoprotein changed to a protein containing selenocysteine by the incubation with H2Se and some enzymes. From these results, we propose that phosphoserine on glutathione peroxidase (apo-enzyme), which is synthesized with phosphoseryl-tRNA, is converted to selenocysteine in the mature enzyme, by a posttranslational mechanism. Opal suppressor tRNA may play a role to synthesize the apo-enzyme of glutathione peroxidase.

Opal suppressor phosphoseryl-tRNA is not a substrate of phosphoserine aminotransferase.

A proposal of the role of animal opal suppressor phosphoseryl (Ps)-tRNA is that Ps-tRNA plays a role as an intermediate in the metabolic pathway from 3-phosphoglycerate to glycine. The labeled [32P]phospho[3H]seryl-tRNA was prepared and used as a substrate in the reaction of bovine brain Ps aminotransferase (EC 2.6.1.52) in the presence of alpha-ketoglutarate. By analysis of the reaction product, no amount of 3-phosphohydroxypyruvate was found, even though phosphohydroxypyruvate was noted in the control experiment by use of Ps and alpha-ketoglutarate. These results showed that Ps-tRNA was not a substrate of Ps aminotransferase.

Semisynthetic beta-lactam antibiotics. 8 Structure-activity relationships of alpha-sulfocephalosporins.

Synthesis and in vitro activity of a number of cephalosporins having alpha-sulfoacyl- or other acyl groups, e.g., alpha-carboxyacyl- and alpha-sulfoaminoacyl- at the 7-position and bearing a variety of heterocyclic thioether or pyridinium moieties at the 3-position are described.