Robert A. Izydore

Synthesis and cytotoxic action of 1-oxoalkyl and 1,2-dioxoalkyl-1,2,4-triazolidine-3,5-diones in murine and human tissue cultured cells.

1‐Oxoalkyl and 1,2‐dioxoalkyl‐1,2,4‐triazolidine‐3,5‐diones proved to be potent antineoplastic agents in mouse tumors and potent cytotoxic agents particularly against the growth of suspended tumor cells. The compounds with shorter substituents in position 1 or positions 1 and 2 afforded the better activity. In L1210 lymphoid leukemia cells DNA, RNA, and protein syntheses were inhibited at 100 μM after 60 min. Multiple enzyme sites in nucleic acid metabolism were affected by the compounds, i.e. DNA polymerase α, PRPP‐amido transferase, dihydrofolate reductase, thymidylate synthetase, and nucleoside kinases. These effects of the agents are probably additive in bringing about inhibition of DNA synthesis and cell death.

Investigation of 3,5-Isoxazolidinediones as Hypolipidemic Agents in Rodents

A series of 2-benzoyl-4,4-dialkyl-3,5-isoxazolidinediones proved to have potent hypolipidemic activity, lowering both serum cholesterol and triglyceride levels at 10 or 20 mg/kg/day, IP and orally in rodents. 2-(3,4,5-Trimethoxybenzoyl)-4,4-diethyl-3,5-isoxazolidinedione (4) afforded the best hypolipidemic activity lowering normolipidemic CFl mouse serum cholesterol levels 49% and serum triglyceride levels 34% at 20 mg/kg/day, IP. Compound 4 was selected as a typical derivative of the chemical class for further detailed studies. Serum cholesterol levels in normolipidemic Sprague Dawley male rats were reduced 45% after 8 weeks at 10 and 20 mg/kg/day of compound, orally. Serum triglyceride levels were reduced 38–49% at 10 and 20 mg/kg/day, orally. In vitro liver enzyme activities studies in normolipidemic CFl mice showed the compound inhibited mitochondrial citrate exchange, acetyl CoA synthetase, HMG CoA reductase, acyl CoA cholesterol acyl transferase, acetyl CoA carboxylase, sn-glycerol-3-phosphate acyl transferase, phosphatidylate phosphohydrolase and heparin-induced lipoprotein lipase activities with increases in the activities of cholesterol ester hydrolase and ATP-dependent citrate lyase. Similar enzyme activities were inhibited in vivo except HMG CoA reductase activity was not inhibited in rat liver or small intestinal mucosa after 8 weeks drug administration. Cholesterol levels were reduced in tissues after 8 weeks administration of compound 4 in normolipidemic rats. Bile cholesterol and triglyceride levels were elevated after two weeks administration to rats at 20 mg/kg/day. Serum lipoprotein levels in normolipidemic and hyperlipidemic rats showed the cholesterol levels in VLDL and LDL fractions after 4, 6 and 8 weeks at 10 and 20 mg/kg/day were reduced whereas HDL-cholesterol levels were significantly elevated. Studies demonstrated that 3H-cholesterol and 14C-palmitic acid incorporation into lipids of the lipoprotein fraction was reduced by the drug but 32P-incorporation was generally elevated. The agent demonstrated no observable toxicity in rats after 8 weeks administration, orally. The acute toxicity study in normolipidemic mice at 20, 40 and 100 mg/ kg/day, IP, demonstrated no observable harmful effects of the drug.

Implications of selective type II IMP dehydrogenase (IMPDH) inhibition by the 6-ethoxycarbonyl-3,3-disubstituted-1,5-diazabicyclo[3.1.0]hexane-2,4-diones on tumor cell death

It was shown previously that three 1,5-diazabicyclo[3.1.0]hexane-2,4-diones selectively inhibited human Type II IMP dehydrogenase (IMPDH) from Tmolt4 cell leukemia [Barnes et al., Biochemistry 2000;39:13641-50]. The agents acted as competitive inhibitors of this isoform, yet when tested against human Type I at concentrations ranging from 0.5 to 500 microM, Type I was not inhibited. This study focuses on the antineoplastic activity and cellular effects of one of these agents and two new derivatives containing ethoxycarbonyl substitution at position C6. Agents were studied for antiproliferative activity in human Tmolt4 leukemia (EC(50) 3.3 to 9.2 microM) and alterations in the levels of enzymes involved with cellular metabolism, including DNA and RNA syntheses due to IMPDH inhibition. Results reported here demonstrate that 6-ethoxycarbonyl-3,3-disubstituted-1,5-diazabicyclo[3.1.0]hexane-2,4-diones are effective inhibitors of DNA synthesis (30-66% inhibition) due to reductions in dGTP pool levels. Collectively, the three agents proved to be selective inhibitors of human IMPDH Type II activity (K(i) 11-33 microM), leading to cytotoxicity in a number of suspended and solid tumor lines, notably MCF-7 (EC(50) 0.7 to 6.0 microM). In addition, negative cytotoxic actions of these agents on WI-38 cell growth, a normal rapidly growing human line, suggest that specific targeting of Type II IMPDH would help to eliminate cell killing in lines where Type I predominates. Furthermore, effects of agents on DNA synthesis and cell death could be reversed by the addition of exogenous guanosine to the medium. Results from in vitro studies suggest that the 6-ethoxycarbonyl-3,3-disubstituted-1,5-diazabicyclo[3.1.0]hexane-2,4-diones may be used as effective isozyme-selective chemotherapeutic agents.

Mechanism of action of the antitumor agents 6‐benzoyl‐3,3‐disubstituted‐1,5‐diazabicyclo[3.1.0]hexane‐2,4‐diones: Potent inhibitors of human type II inosine 5′‐monophosphate dehydrogenase

The observation that expression of the IMPDH gene is tightly linked with cellular proliferation and transformation has led to an interest in developing inhibitors that deplete intracellular guanine nucleotide pools. IMPDH exists as 2 isoforms, one of which is induced in tumor cells, type II and thus has led to new interest in this target for the design of isoform‐selective anticancer chemotherapeutic agents. Several classes of IMPDH inhibitor are now in use or under development; however, only the 1,5‐diazabicyclo[3.1.0]hexane‐2,4‐diones show selectivity for the type II isoform. In the current study, we further evaluated chemical modification of this class to determine the necessary components for selective type II IMPDH inhibition. The 6‐benzoyl‐3,3‐disubstituted‐1,5‐diazabicyclo[3.1.0]hexane‐2,4‐diones were effective cytotoxic agents in human leukemias, lymphomas, breast, glioma and HeLa‐S 3 suspended uterine carcinoma screens with ED50 values 0.3 to 12 μM. The agents acted as antimetabolites by inhibiting de novo purine biosynthesis at the key regulatory enzyme IMPDH, resulting in suppression of DNA synthesis and dGTP pool levels within 60 min. Furthermore, the derivatives were specific for the type II isoform as opposed to type I, acting in a competitive manner with Ki values of 5.1 to 63 μM. Addition of the 6‐benzoyl moiety to the bicyclic parent ring structure afforded the most potent agent in the novel class of 1,5‐diazabicyclo[3.1.0]hexane‐2,4‐diones that selectively inhibits type II IMPDH activity.

Potential aldose reductase inhibitors: 1,2,4-triazolidine-3,5-diones and 2-(3,4,5-trimethoxybenzoyl)-4,4-diethyl-3,5-isoxazolidinedione .

1,2,4-Triazolidine-3,5-diones and the 3,5-isoxazolidinedione were, observed to be, potent inhibitors of rat lens aldose reductase activity. In vivo in streptozotocin-diabetic rats, selected agents at 20 mg/kg/day, orally for 21 days reduced significantly the sorbitol levels of rbc, lens and sciatic nerves, suggesting that these derivatives may have some usefulness to treat clinical complications of diabetes mellitus.

Specific inhibition of Type II inosine monophosphate dehydrogenase activity of Tmolt4 T cell human leukaemia cells by 3‐methoxy and di‐benzohydroxamic acids, maleic hydrazide and malonic acids

Small‐molecular‐weight benzohydroxamic and malonic acids and maleic hydrazide proved to be potent inhibitors of the activity of human Tmolt4 leukaemia Type II IMP (inosine monophosphate) dehydrogenase (IMPDH) activity. They were competitive inhibitors with respect to IMPDH demonstrating Ki values in the range 2.57–41.3 μm, less than half the values of the IC50 (μm) for the inhibition of Type II IMPDH. The IC50 μm values positively correlated with the ability of each compound to inhibit crude IMPDH activity, de‐novo purine and DNA syntheses and growth of the T leukaemia cell line. Compounds were not inhibitors of Type I IMPDH. Type I IMPDH predominates in normal resting cells compared with Type II which is found in rapidly proliferating cells. Discovery of agents which would selectivity target IMPDH found in proliferating cells should eliminate any antineoplastic therapeutic toxic effects in normal cells of the body.

The hypolipidemic effects of 1-acetyl-4-phenyl-1,2,4-triazolidine-3,5-dione in rodents.

Abstractl-Acetyl-4-phenyl-l ,2,4-triazolidine,5-dione (APTD), a potent hypolipidemic agent, lowered both serum cholesterol and triglyceride levels in normo- and hyperlipidemic rats at 10 or 20 mg/kg/day. The agent effectively lowered VLDL-cholesterol (VLDL-C) and LDL-C content and raised HDL-C content in normal and hyperlipidemic rats treated from 4 to 8 weeks. Similar effects on the incorporation of cholesterol into the lipoprotein fractions were observed after drug treatment. Tissue lipids, e.g. cholesterol, were lowered, whereas fecal cholesterol levels were increased. APTDs primary targets were acyl CoA cholesterol acyl transferase (ACAT) for cholesterol ester synthesis and sn-glycerol-3-phosphate acyl transferase (GPAT) and phosphatidylate phosphohydrolase (PPH) for triglyceride synthesis.

1,2-diacetyl-4-phenyl-1,2,4-triazolidine-3,5-dione.

The title crystal, C12H11N3O4, contains three essentially identical molecules in the asymmetric unit. The triazole rings in the three independent molecules are planar within 0.014 A. The N atom containing the phenyl substituent is trigonal, the sum of the three bond angles being 359.5 degrees. The other two N atoms are slightly pyramidal, the sum of the three bond angles being 346 degrees. The two acetyl groups are twisted out of the plane of the triazole ring by an average of 33 degrees and are trans to each other.

Disposition of the Pharmacologically Active Compound [1-14C-Acetyl]-1-Acetyl-4-Phenyl-1,2,4-Triazolidine-3,5-Dione in CF1 Mice

Summary1-Acetyl-4-phenyl-1,2,4-triazolidine-3,5-dione (APTD) has hypolipidaemic, anti-inflammatory, analgesic, antineoplastic, and aldose reductase inhibitory activities in animals. Disposition studies using pooled plasma and urine samples showed that [1-14C-acetyl]-1-acetyl-4-phenyl-1,2,4-triazolidine-3,5-dione (14C-APTD) had a maximum half-life of 20 hours. Urinary excretion accounted for less than 3% of the radioactivity elimination, while faecal excretion may account for up to 45% of the total elimination. In a 96-hour tissue distribution study, there was no sequestering of 14C-APTD in any of the organs. 14C-APTD demonstrated significant aqueous partitioning, and almost no binding to bovine serum albumin. In L1210 tumour cells, 14C-APTD was bound to DNA and RNA, and there was no binding to intracellular protein. 14C-APTD underwent significant metabolism in mice. One metabolite excreted in urine was identified; two other possible metabolites were proposed.

Hypolipidemic activity of 6-alkoxycarbonyl-3-aryl-1,3,5- triazabicyclo[3.1.0]hexane-2,4-diones and 2-alkoxycarbonyl-5-aryl-1,3,5-triazine-4,6(1H,5H)-diones in rodents.

Significant hypolipidemic activity was demonstrated by 6-ethoxycarbonyl-3-phenyl-1,3,5-triazabicyclo[3.1.0]hexane-2,4-dione, 2-ethoxycarbonyl-5-phenyl-1,3,5-triazine-4,6(1H,5H)-dione and 2-ethoxycarbonyl-5-(4-chlorophenyl)-1,3,5-trizine-4,6(1H,5H)-dione in rodents at 20 mg/kg/day. These agents lowered serum cholesterol and triglyceride levels by approximately 40% in mice after 16 d. Tissue lipids in rat liver, small intestinal mucosa, aortic wall and feces were reduced by treatment with the agents. Very low density lipoprotein (VLDL) and low density lipoprotein (LDL) cholesterol levels were reduced in the rat; high density lipoprotein (HDL) cholesterol levels were elevated after 14 d of treatment. The activities of regulatory enzymes,e.g., acetyl-CoA synthetase, acyl-CoA:cholesterol acyltransferase, cholesterol 7α-hydroxylase,sn-glycerol-3-phosphate acyltransferase, phosphatidylate phosphohydrolase and heparin-induced lipoprotein lipase, involved inde novo synthesis of hepatic lipids were affected by the agents. The new compounds may represent another class of potentially useful hypolipidemic agents for the treatment of atherosclerosis since HDL cholesterol levels were increased and VLDL and LDL cholesterol levels were lowered by some of the agents.