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The synthesis of the title compounds was carried out by reacting dicarboxylic acid chlorides with oximes in the presence of excess triethylamine. Disubstituted malonyl chlorides gave 2-alkenyl-4,4-dialkyl-3,5-isoxazolidinediones (8a-f) and 2,2'-ethylidene-bis[4,4-dialkyl-3,5-isoxazolidinedione]s (9a-f). Compounds 9 were formed from 8 and its N-unsubstituted 3,5-isoxazolidinedione decomposition product. Phthaloyl chlorides reacted with acetone oxime to yield 3-(1-methylethenyl)-1H-2,3-benzoxazine-1,4(3H)-diones (16a-e). Products 16 spontaneously decomposed to give N-unsubstituted 1H-2,3-benzoxazine-1,4(3H)-diones (17a-e) at rates that were dependent on temperature and solvent. Kinetic studies showed that two of the compounds decomposed by zero-order kinetics under neutral conditions. Butanedioyl chloride did not produce a cyclic product.

Bioactivity-directed fractionation of an extract of the leaves of Alvaradoa haitiensis, using the KB (human oral epidermoid carcinoma) cell line, led to the isolation and identification of 10 new anthracenone C-glycosides, alvaradoins E-N (1-10), along with the known compound chrysophanol (11). The cytotoxicity of all compounds was evaluated, and preliminary structure-activity relationships are suggested. The most potent compounds in the in vitro assays (1 and 2) were evaluated in vivo versus the P388 (murine lymphocytic leukemia) model, and alvaradoin E (1) showed antileukemic activity (125% T/C) at a dose of 0.2 mg kg-1 per injection when administered intraperitoneally.

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-S3 suspended uterine carcinoma screens with ED(50) values 0.3 to 12 microM. 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 K(i) values of 5.1 to 63 microM. 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., (Copyright 2001 Wiley-Liss, Inc.)

Inosine 5'-monophosphate dehydrogenase (IMPDH), the rate-limiting enzyme in the de novo pathway for synthesis of guanine nucleotides, is essential for normal cell proliferation and function. New derivatives of the 1,5-diazabicyclo[3.1.0]hexane-2,4-diones were synthesized and examined for antiproliferative effects and selective inhibition of human IMPDH type II activity. The 3,3-disubstituted-6,6-pentamethylene-1,5-diazabicyclo[3.1.0]hexane-2,4-diones proved to be effective antiproliferative agents in tumor cell lines derived from murine and human leukemias, lymphomas, uterine carcinoma, glioma, and breast effusion with ED50 values (concentration of compound that inhibits 50% of cell growth) ranging from 3.3 to 16 microM. The agents acted as antimetabolites suppressing de novo purine biosynthesis at the key regulatory enzyme IMPDH, resulting in the specific suppression of dGTP pool levels by 19 to 64% and DNA synthesis by 39 to 68%. The derivatives were specific inhibitors of IMPDH type II activity as opposed to type I, acting in a competitive manner with respect to inosine 5'-monophosphate, K(i) values of 44.2 to 62 microM. In addition, effects of agents on Tmolt4 cell growth and DNA synthesis could be reversed by coincubation with guanosine. Unlike mycophenolic acid and tiazofurin, the 6,6-pentamethylene-1,5-diazabicyclo[3.1.0]hexane-2,4-diones specifically targeted type II IMPDH, where activity is increased in replicating or neoplastic cells, and did not suppress type I activity, where expression is relatively unaffected by cell proliferation or transformation. Agents were not inhibitors of normal human lung fibroblast cell growth, WI-38, most likely due to the observed isoform selectivity.

3-Ethoxycarbonyl-5-phenyl-1, 3a, 4, 5, 6, 6a-hexahydropyrrolo[3,4-c]pyrazole-4, 6-dione, 2, 2, 6, 6-tetraethyl-1H, 5H-pyrazole[1, 2-a]pyrazole-1, 3, 5, 7-[2H, 6H]-tetraone and 6-ethoxycarbonyl-3-phenyl-3-azabicyclo[3.1.0] hexane-2, 4-dione demonstrated potent cytotoxic activity in the human Tmolt3, Tmolt4 and HL-60 leukemia screens, HuT-78 lymphoma and HeLa suspended uterine carcinoma cell lines. Most notable was the finding that these compounds were significantly more active than the standard cytotoxic agents examined in the MCF-7 breast (ED50 0.2-1.0 microg/ml) and U87MG glioma (ED50 1.3-2. 6 microg/ml) tumor screens. The agents inhibited Tmolt4 leukemia DNA and RVA syntheses after 60 min at 100 microM Multiple enzymes involved with nucleic acid metabolism appeared to be targeted including inhibition of RNA polymerases, ribonucleotide reductase and nucleoside kinase activities, however, inhibition of de novo purine synthesis at the key regulatory enzyme IMP dehydrogenase appeared to be the primary target. The predominant IMPDH isoform (Type II) detected in a number of human cancers, such as leukemias, ovarian and breast, was inhibited by the compounds yielding IC50 values in the microM range. Furthermore, inhibition of IMP dehydrogenase activity led to the selective depletion of dGTP pool levels by two of the compounds. The DNA molecule was not a target of the agents since no alkylation of the bases, cross-linking of the DNA strands or intercalation between base pairs occurred. Yet, the compounds did cause DNA fragmentation after incubating at 100 microuM for 24 h which was consistent with the observed decrease in ct-DNA viscosity.

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.

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 microM, less than half the values of the IC50 (microM) for the inhibition of Type II IMPDH. The IC50 microM 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 3,5-pyrazolidinediones proved to be potent cytotoxic agents against the growth of a number of murine and human tumor cell lines, e.g. human THP-I monocytic leukemia, Hut-78 lymphoma, MCF-7 breast effusion, A549 lung carcinoma, U87MG glioma, Hela uterine and A431 epidermoid skin cancer. In human Tmolt4 cell leukemia, the agents substantially suppressed DNA and RNA syntheses after 60 min at 100 microM. The de novo purine biosynthetic pathway appeared to be the major target of the agents with the inhibition of both PRPP-amido transferase and IMP dehydrogenase (IMPDH) activities. Suppression of IMPDH activity was due to the inhibition of both the Type I and II isoforms through an uncompetitive mechanism; however, the Type II isoform was preferentially inhibited at lower concentrations of compounds tested (>50-150 microM). Therefore IMPDH Type II activity, which predominates in cancer cells, was selectively inhibited over the Type I isoform (208-312 microM). The activities of other enzymes examined were inhibited which added to the overall suppression of DNA synthesis, i.e., ribonucleotide reductase, dihydrofolate reductase and nucleoside kinases. The agents caused Tmolt4 DNA strand scission but the DNA molecule itself did not appear to be a target of the compounds since there was no induced cross-linking of the DNA, intercalation between base pairs or alkylation of the DNA bases.

The 1,2,4-triazolidine-3,5-diones, 1-(1-(3-methylphenyl)ethylidineamino)-4,4-diethyl-3,5-azetidinediones, and 4,4-disubstituted-3,5-pyrazolidinediones proved to be potent competitive inhibitors of human Tmolt4 leukemia Type II IMP dehydrogenase [IMPDH] activity, an enzyme isoform which is induced in highly proliferating cells. On the other hand, the 3,5-isoxazolidinediones were shown to be uncompetitive inhibitors of Type II IMPDH activity. The correlation between inhibition of Type II IMPDH activity with the agents' ability to suppress DNA and purine syntheses in these Tmolt4 leukemia cell was positive. Type I IMPDH (i.e., the isoform that is present in normal cells) was not inhibited by these compounds suggesting that these agents would be less toxic to normal cells and have selective inhibition towards proliferating cells.

2-Etheny1-2,3-dihydrophthalazine-1,4-diones were successfully synthesized and proved to be effective cytotoxic agents against the growth of suspended murine and human leukemias and lymphomas. Selected compounds were also active in human HeLa uterine carcinoma, suspended effusion breast MCF-7 and glioma HS683 screens. These agents suppressed P388 lymphocytic leukemia DNA synthesis after 60 min at 100 microM. Their target appeared to be the de novo synthesis pathway with significant inhibition of the activities of both regulatory enzymes of the pathway, i.e. PRPP-amide transferase and IMP dehydrogenase resulting in a reduction in the d[NTP] pool levels for DNA incorporation. The compounds did not affect de novo pyrimidine synthesis and its regulatory enzymes. Very minor reduction by the agents was noted for the nucleoside kinases and the DNA and RNA polymerase activities within 60 min. DNA was not a target of the agents in that there was no alkylation of the nucleotide bases, intercalation between base pairs or cross-linking of the DNA strands; however, the agents did cause P388 DNA strand scission after 24 h at 100 microM.

The 1-(1-phenylalkylideneamino)-2,4-azetidinediones are potent cytotoxic agents against the growth of human and murine leukemias, lymphoma, and suspended HeLa uterine carcinoma. In cell lines cultured from solid human tumors, the agents were more selective with only a few agents demonstrating significant activity against the growth of HCT-8 ileum adenocarcinoma, Saos-2 osteosarcoma, KB nasopharynx, MCF-7 breast effusion, and ovary 1-A9 carcinoma A mode of action study in murine L1210 lymphoid leukemia cells showed that the agents inhibited DNA and RNA syntheses after 60 min. The compounds were potent inhibitors of the de novo purine synthesis suppressing the activity of both regulatory enzymes of the pathway, i.e., PRPP-amido transferase and IMP dehydrogenase. In addition, the agents reduced the activity of ribonucleotide reductase, dihydrofolate reductase, RNA polymerases, and thymidine kinases as well as the reduction of d[NTP] pools. All of these effects would contribute to the overall reduction of DNA and RNA syntheses. The DNA molecule itself was not a target for the agents in that alkylation of nucleoide bases, intercalation between base pairs, and cross-linking of DNA strands did not occur.

Inosine 5'-monophosphate dehydrogenase (IMPDH) is the rate-limiting enzyme in de novo purine biosynthesis. IMPDH activity results from expression of two isoforms. Type I is constitutively expressed and predominates in normal resting cells, while Type II is selectively up-regulated in neoplastic and replicating cells. Inhibitors of IMPDH activity selectively targeting the Type II isoform have great potential as cancer chemotherapeutic agents. For this study, an expression system was developed which yields 35-50 mg of soluble, purified recombinant Type I and II protein from 1 L of bacteria. In addition, three 1,5-diazabicyclo[3.1.0]hexane-2,4-diones were synthesized and shown to act as specific inhibitors of human recombinant Type II IMPDH. The agents are competitive inhibitors with respect to the endogenous substrate IMP and K(i) values range from 5 to 44 microM but were inactive as inhibitors of the Type I isoform at concentrations ranging from 0.5 to 500 microM. IC(50) values for recombinant Type II inhibition were determined and compared to IC(50) values obtained from Molt-4 cell extracts of IMPDH. Cytotoxicity assays revealed that the compounds inhibited Molt-4 leukemia growth with ED(50) values of 3.2-7.6 microM. Computational docking studies predict that the compounds bind to IMPDH in the IMP-binding site, although interactions with residues differ from those previously determined to interact with bound IMP. While all residues predicted to interact directly with the bound compounds are conserved in the Type I and Type II isoforms, sequence divergence within a helix adjacent to the active site may contribute to the observed selectivity for the human Type II isoform. These compounds represent the first class of selective IMPDH Type II inhibitors which may serve as lead compounds for the development of isoform-selective cancer chemotherapy.

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 microM after 60 min. Multiple enzyme sites in nucleic acid metabolism were affected by the compounds, i.e. DNA polymerase alpha, 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.

The aliphatic dicarboxylic acid surprisingly afforded potent cytotoxicity and in vivo antineoplastic activity. The agents were active against the growth of a variety of leukemias, lymphomas, and suspended HeLa uterine carcinoma. Suppression of growth of cell lines derived from human solid cancers, e.g. SW-480 colon adenocarcinoma, lung MB- 9812, glioma HS-683, and rat osteosarcoma UMR-106 was observed. A mode of action study in L1210 lymphoid leukemia demonstrated that DNA and RNA syntheses were inhibited at multiple sites including ribonucleoside reductase, purine de novo synthesis at PRPP-amidotransferase and IMP dehydrogenase and nucleic acid kinases. These studies could not exclude the possibility that the agents also interacted with the DNA molecule itself interfering with the utilization of the template.

1,3,5-Triazabicyclo[3.1.0]hexane-2,4-diones proved to be potent antineoplastic and cytotoxic agents in murine and human cancer cells. In L1210 lymphoid leukemia cells DNA synthesis was significantly suppressed over 60 min by the agents from 25 to 100 microM. DNA synthesis was blocked at multiple sites including DNA polymerase alpha, ribonucleoside reductase, dihydrofolate reductase, PRPP-amido transferase, and nucleoside kinases which would be additive overall in suppressing DNA synthesis. The DNA molecule itself did not appear to be at target of the agents since no alkylation of nucleotide bases, intercalation between base-pairs or cross-linking of strands occurred after 24 h incubation at 100 microM. Nevertheless, L1210 DNA fragmentation did occur after 24 h incubation at 100 microM which is usually associated with tumor cell apoptosis.

The 3,5-isoxazolidinediones and 2-isoxazolin-5-ones demonstrated potent cytotoxicity against the growth of human Tmolt3 T cell leukemia, murine P388 and L1210 leukemias, as well as human HeLa-S3 uterine carcinoma and glioma tumor cell growth. The specificity of the 3,5-isoxazolidinedione and 2-isoxazoline-5-one derivatives as cytotoxic agents varied with the histological type of tumor cell. Selected compounds were active against solid HeLa uterine. KB nasopharynx, skin A431, SW-480 adenocarcinoma, osteosarcoma and glioma growth. Selected compounds demonstrated in vivo antineoplastic activity against Ehrlich ascites carcinoma growth. In L-1210 leukemia cells, the agents blocked DNA and protein synthesis at 25, 50 and 100 microM over 60 min. The agents were effective in reducing rate limiting enzymes in the de novo purine and pyrimidine pathways. In addition they suppressed dihydrofolate reductase and ribonucleoside reductase activities with moderate inhibition of DNA and RNA polymerase activities. DNA itself was not a target of the agents.

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.

A series of monoacyl and diacyl 1,2,4-triazolidine-3,5-diones substituted at position 4 with either a phenyl or a tert-butyl group was prepared. Both acetyl and benzoyl groups were utilized as the acyl substituents. The diacylated compounds containing one or two acetyl groups were somewhat unstable to moisture. The acylated compounds were studied by (1)H, (13)C and (15)N NMR spectroscopy and X-ray crystallography to determine if they were acylated on nitrogen or ring carbonyl oxygen. The results indicated that the acylations occurred on nitrogen. The NMR spectra and molecular modeling computations were used to assign conformations to several of the diacylated compounds.

The 1-acyl- and 1,2-diacyl-4,4-diethyl-3,5-pyrazolinediones proved to be cytotoxic against the growth of a number of cell lines, including murine and human leukemias. HeLa suspended carcinoma, colon adencarcinoma SW480, KB nasopharynx and glioma tumors. Selected compounds were also active in the human lung bronchogenic MB-9812, and osteosarcoma TE418 screens. These derivatives were active in vivo in the Ehrlich ascites carcinoma screen in CF-1 mice at 8 mg/kg/day I.P. The mode of action in Tmol3 leukemia cells showed that the compounds reduced de novo synthesis of purines and pyrimidines and inhibited dihydrofolate reductase and ribonucleoside reductase activities. The DNA molecule was not a target although limited DNA strand scission may be possible.

1-Acyl and 1,2-diacyl 1,2,4-triazolidine-3,5-diones proved to be potent anti-inflammatory agents in rodents at 8 or 20 mg/kg. They were effective against induced edema, pleurisy, and septic shock. Furthermore, these agents were potent in blocking the writhing reflex suggesting that they should be effective against local pain generated by inflammatory processes. These compounds were not lysosomal hydrolytic enzyme or proteolytic enzyme inhibitors in mouse liver, macrophages or human leukocytes. However, the agents were potent inhibitors of prostaglandin and leukotriene de novo synthesis and have potential in acting as free radical scavengers.

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 CF1 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 CF1 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.

A series of 4-substituted 1,2-diacyl-1,2,4-triazolidine-3,5-diones were synthesized and shown to be hypolipidemic in rodents; serum cholesterol and triglyceride levels were significantly reduced following intraperitoneal and oral dosing at 20 mg/kg/day. The hypolipidemic activity of the triazolidine-3,5-diones was improved when R1 was either a phenyl or a butyl group. Tissue lipid levels were reduced in the liver, aorta, and small intestine, while fecal lipids, e.g. cholesterol, were increased after 14 days. Very low density lipid cholesterol levels were reduced but high density lipid cholesterol levels were significantly increased. It appears that the mode of action of the 1,2-diacyl-1,2,4-triazolidine-3,5-diones is by the inhibition of the de novo rate limiting enzyme for lipid synthesis. Enzyme activities suppressed by the agents included ATP-dependent citrate lyase, HMG CoA reductase, acyl CoA cholesterol acyl transferase, acetyl CoA carboxylase, sn-glycerol-3-phosphate aryl transferase, phosphatidylate phosphohydrolase, and cholesterol-7 alpha-hydroxylase.

1-Acetyl-4-phenyl-1,2,4-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. APTD's 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.

A series of 4-substituted 1-acyl-1,2,4-triazolidine-3,5-diones demonstrated potent activity in CF1 mice when administered intraperitoneally at 20 mg/kg/day, lowering both serum cholesterol and triglyceride levels significantly. The 4-(4-chlorophenyl)-substituted compounds demonstrated better hypolipidemic activity in rodents than 4-methoxy-, 4-nitro-, and 4-t-butylphenyl substitutions. Aryl and alkyl substitutions rather than benzoyl substitutions at position 4 demonstrated good hypocholesterolemic activity. Selected compounds were examined for the mode of action in rats in which serum cholesterol and triglyceride levels were reduced after administration of 20 mg/kg/day orally; tissue lipids were reduced after 14 days of administration, and bile and fecal lipids were increased by 44-250%. Serum lipoprotein levels were also modulated by the agents, with cholesterol levels in very low density lipoprotein and low density lipoprotein fractions being reduced by 2-57%. Cholesterol levels in the high density lipoprotein fraction were elevated by 94-341%. Activities of mouse hepatic enzymes were suppressed by the agents in a manner that suggested that the compounds interfere with de novo synthesis of lipids.

Significant hypolipidemic activity was demonstrated by 6-ethoxycarbonyl-1-3-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-triazine-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 7 alpha-hydroxylase, sn-glycerol-3-phosphate acyltransferase, phosphatidylate phosphohydrolase and heparin-induced lipoprotein lipase, involved in de 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.

A series of substituted 1-acyl- and 1,2-diacyl-3,5-pyrazolidinediones were found to have hypolipidemic properties lowering both serum cholesterol and triglyceride levels in rodents. For optimal activity of the pyrazolidinediones, both nitrogen atoms of the ring needed to be substituted preferentially with MeCO groups. This compound lowered rat VLDL but did not elevate HDL cholesterol content.

1-Acyl- and 1,2-diacyl-1,2,4-triazolidine-3,5-diones were found to be potent cytotoxic agents in murine and human cancer cell lines, e.g. L1210, P388, Tmolt3, colon adenocarcinoma, Hela cells and glioma. In vivo activity was demonstrated at 8 mg/kg/day against Ehrlich ascites carcinoma growth. In L1210 cells, 1-acetyl-4-phenyl-1,2,4-triazolidine-3,5-dione, 41, reduced DNA synthesis significantly with moderate reduction in RNA synthesis. Enzyme sites in L1210 cells which were markedly affected were m- and r-RNA polymerase, PRPP amidotransferase, IMP dehydrogenase, dihydrofolate reductase, thymidine, TMP and TDP kinases. Kinetic studies suggest the inhibition of rate limiting enzymes in the purine pathway by 41 was responsible for its cytotoxicity. Acute toxicity studies in mice indicated 41 was safe for therapeutic use at 20, 50, and 100 mg/ky/day.

2-(3,4,5-Trimethoxybenzoyl)-4,4-diethyl-3,5-isoxazolidione (TDI) and 1-acetyl-4-phenyl-1,2,4-triazolidine-3,5-dione (APTD) are two chemically related derivatives which have demonstrated potent hypolipidemic activity in mice at 20 mg/kg/day I.P. for 16 days. The purpose of this study is to correlate in vivo effects of TDI and APTD on rat serum lipoprotein lipids and apoprotein levels as well as plasma clearance and tissue uptake with effects of the agents on tissue cultured cells' LDL and HDL receptor binding, internalization and degradation. This study also correlates in vivo effects of TDI and APTD with endogenous enzyme activities regulated by these high affinity receptors. In rats at 20 mg/kg/day orally serum cholesterol, triglyceride, and VLDL-cholesterol levels were effectively reduced while HDL cholesterol levels were significantly elevated with both agents. These compounds in human hepatocytes lowered LDL receptor binding and degradation, whereas HDL receptor binding and degradation were elevated in human hepatocytes, rat small intestinal epithelium cells, human BG fibroblasts, rat aorta cells and mouse macrophages. These drugs inhibited HMG CoA reductase and sn-glycerol-3-phosphate acyl transferase activities, findings consistent with the observed in vivo reductions in serum cholesterol and triglyceride levels. Both drugs reduced activity of acyl CoA:cholesterol acyl transferase and accelerated activity of neutral cholesterol hydrolase in liver and aorta cells. This modulation by the drugs should reduce disposition of cholesterol esters in these tissues especially aorta wall; this effect was indeed observed in vivo. In the presence of TDI and APTD, HDL uptake of intracellular cholesterol from fibroblasts was accelerated. This was consistent with results from in vivo rat studies showing that HDL clearance was faster after treatment while clearance of LDL slowed. Tissue uptake of HDL and LDL after drug treatment was reduced for the major organs; however the liver accumulation was elevated. The accelerated uptake in the liver was probably due to the observed higher levels of Apo-E and Apo-AI in HDL after drug treatment. Increased excretion of cholesterol from the liver to the bile after drug treatment indicated that the reserve cholesterol transport system by HDL was accelerated by the agents in vivo.

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.

Small molecular weight aliphatic dicarboxylic acids, i.e. dimethylmalonic acid, diethylmalonic acid and maleic acid, afford greater than 35% reduction in serum cholesterol and triglycerides levels in CF1 mice at 20 mg/kg/day, i.p. Furthermore, these agents lowered greater than 40% serum cholesterol levels in rat after oral administration at 20 mg/kg/day. Dimethylmalonic and diethylmalonic acids lowered rat serum triglyceride levels by at least 23%. Rat tissue lipids, e.g. liver, small intestinal mucosa and aorta wall, were reduced in concentration and fecal lipids were elevated by dimethyl- and diethylmalonic acids. Rat serum lipoproteins after 14 days of treatment demonstrated reduction of VLDL and LDL cholesterol levels with elevated HDL cholesterol levels by dimethylmalonic and maleic acids. The agents also inhibited de novo hepatic enzyme activities, specifically mitochondrial citrate exchange, acetyl-CoA synthetase, ATP-dependent citrate lyase, acyl-CoA:cholesterol acyltransferase, cholesterol-7 alpha-hydroxyase, sn-glycerol-3-phosphate acyltransferase and phosphatidate phosphohydrolase, which would result in the reduction of de novo synthesis of fatty acids, cholesterol and triglycerides.

A series of benzo- and dibenzohydroxamic acids were shown to have potent hypolipidemic activity in mice and rats lowering both serum cholesterol and triglyceride levels at 20 mg/kg/day. Selected derivatives lowered tissue lipids, i.e. liver, small intestine and aorta, and accelerated fecal lipid excretion in rats. The VLDL and LDL cholesterol content was reduced and HDL cholesterol was significantly elevated after 14 days administration, orally. The agents were not HMG CoA reductase inhibitors; however, other lipid regulator enzyme activities were inhibited, e.g. acyl CoA cholesterol acyl transferase, ATP-dependent citrate lyase and acetyl CoA synthetase. The triglyceride levels were probably reduced due to the derivatives inhibiting the enzymatic activities of sn-glycerol-3-phosphate acyl transferase and phosphatidylate phosphohydrolase.