Short report: floxacrine analog WR 243251 inhibits hematin polymerization.

Floxacrine was a promising antimalarial compound that led to the identification of WR 243251. On the basis of their structures, we suspected that these compounds might be good inhibitors of hematin polymerization. Indeed, WR 243251 was as potent and floxacrine was only 2-fold less potent than chloroquine as inhibitors of this process. However, this hematin polymerization inhibition did not completely account for the increased antimalarial potency of WR 243251 versus chloroquine. The WR 243251 ketone hydrolysis product WR 243246 was without activity against hematin polymerization. These data also confirm that hematin polymerization inhibition can be quite sensitive to small changes in inhibitor structure.

Analysis of stereoelectronic properties of camptothecin analogues in relation to biological activity.

Camptothecin and four of its 10,11-methylenedioxy analogues were examined for their activity against the pathogenic protozoan Leishmania donovani in vitro. The methylenedioxy analogues were 36- to 180-fold more potent than the parent camptothecin, possessing IC50 values ranging from 160 to 32 nM against the parasite. Our finding that the methylenedioxy camptothecins possess greater activity than camptothecin, which is also the case for other cell types and for the generation of cleavable complex in the presence of DNA and purified mammalian topoisomerase I, prompted us to examine the molecular features of camptothecin and methylenedioxy camptothecin analogues. A delocalization of positive potential was observed in the methylenedioxy camptothecin analogues, which could increase the affinity of these molecules for DNA. In addition, geometrical and electronic differences between the E ring of camptothecin and its methylenedioxy analogues were noted. One or both of these factors may contribute to the superior biological activity of the methylenedioxy camptothecin analogues.

Molecular characterization of Plasmodium falciparum S-adenosylmethionine synthetase.

S-Adenosylmethionine (AdoMet) synthetase (SAMS: EC 2.5.1.6) catalyses the formation of AdoMet from methionine and ATP. We have cloned a gene for Plasmodium falciparum AdoMet synthetase (PfSAMS) (GenBank accession no. AF097923), consisting of 1209 base pairs with no introns. The gene encodes a polypeptide (PfSAMS) of 402 amino acids with a molecular mass of 44844 Da, and has an overall base composition of 67% A+T. PfSAMS is probably a single copy gene, and was mapped to chromosome 9. The PfSAMS protein is highly homologous to all other SAMS, including a conserved motif for the phosphate-binding P-loop, HGGGAFSGKD, and the signature hexapeptide, GAGDQG. All the active-site amino acids for the binding of ADP, P(i) and metal ions are similarly preserved, matching entirely those of human hepatic SAMS and Escherichia coli SAMS. Molecular modelling of PfSAMS guided by the X-ray crystal structure of E. coli SAMS indicates that PfSAMS binds ATP/Mg(2+) in a manner similar to that seen in the E. coli SAMS structure. However, the PfSAMS model shows that it can not form tetramers as does E. coli SAMS, and is probably a dimer instead. There was a differential sensitivity towards the inhibition by cycloleucine between the expressed PfSAMS and the human hepatic SAMS with K(i) values of 17 and 10 mM, respectively. Based on phylogenetic analysis using protein parsimony and neighbour-joining algorithms, the malarial PfSAMS is closely related to SAMS of other protozoans and plants.

Synthesis and biological evaluation of 4-chloro-3, 5-dinitrobenzotrifluoride analogues as antileishmanial agents.

Desnitro analogues of 4-chloro-3,5-dinitrobenzotrifluoride (chloralin) (2), an in vitro microtubule inhibitor of several Leishmania species, have been synthesized from 2-halo-5-(trifluoromethyl)benzenesulfonyl chlorides 4 and 5. The analogues exhibited moderate to excellent activity when tested against Leishmania donovani amastigotes in vitro. Two representative compounds, 7f and 8, were tested against the Khartoum strain of L. donovani in a hamster model using chloralin (2) and Glucantime (one of the current therapeutics of choice in the treatment of Leishmania) as standards, the results of which will be discussed herein.

Chagas' disease: a search for treatment and a question--should the disease be of military concern?

If military forces are required to operate in areas that are endemic for Chagas' disease, the occupation should be of critical concern. These areas, located in Central and South America, are many. The matter is of particular importance because no suitable drug exists to treat individuals who contract the disease. We examined 60 compounds of a chemical class, thiosemicarbazones, known to have some activity against the disease. The work was carried out using Trypanosoma cruzi-infected mice. Of the 60 potential drugs evaluated, 12 showed significant suppressive activity. One of these compounds was almost 50% greater than the reference drug used in the test system.

Simulation analysis of formycin 5'-monophosphate analog substrates in the ricin A-chain active site.

Ricin is an RNA N-glycosidase that hydrolyzes a single adenine base from a conserved loop of 28S ribosomal RNA, thus inactivating protein synthesis. Molecular-dynamics simulation methods are used to analyze the structural interactions and thermodynamics that govern the binding of formycin 5'-monophosphate (FMP) and several of its analogs to the active site of ricin A-chain. Simulations are carried out initiated from the X-ray crystal structure of the ricin-FMP complex with the ligand modeled as a dianion, monoanion and zwitterion. Relative changes in binding free energies are estimated for FMP analogs constructed from amino substitutions at the 2- and 2'-positions, and from hydroxyl substitution at the 2'-position.

The cytotoxicity of N-Pyridinyl and N-quinolinyl substituted derivatives of phthalimide and succinimide.

The N-pyridinyl and N-quinolinyl substituted derivatives of phthalimides and succinimides demonstrated cytotoxicity against the growth of a number of cultured cell lines. The substituted succinimides were more effective than the unsubstituted succinimide derivative in reducing cell growth. On the other hand, phthalimide demonstrated more potent cytotoxicity than its N-substituted derivatives. Three representative examples N-[2-pyridinyl-1-oxide) methyl] phthalimide 8, 1-[N-2-phthalimidoethyl]-3,4-dihydroiso-quinoline 12, and 1-[N-(2-(1,2,3,4-tetrahydro-2-quinolinyl)] ethylphthalimide 14 were shown to inhibit L1210 leukemia DNA synthesis whereas RNA synthesis was not inhibited at 25-100 uM. All three agents inhibited the activities of DNA polymerase alpha, PRPP-amido transferase, nucleoside kinases, and dihydrofolate reductase. The cellular pool levels of d[GTP], d[CTP], and d[TTP] were reduced after 60 minutes incubation at 100 uM. The DNA molecule itself was not a target of these agents.

Drugs that show protective effects from ricin toxicity in in vitro protein synthesis assays.

We used an in-vitro, inhibition of protein synthesis assay (PSI) to test a wide variety of drugs for possible therapeutic use against ricin, a toxic glycoprotein that causes death in animals by inhibiting protein synthesis. Selection of test drugs was based on possible interference with ricin activity at different stages of the toxic process. Most of the drugs tested had no effect on ricin-induced PSI, were toxic when tested alone, or enhanced the toxicity of ricin. The only ones showing protection were galactose, lactose, and several derivatives of these sugars, Brefeldin A (BFA), 3'-azido-3'-deoxythymidine (AZT), and a purine derivative (BM33203). THe sugar derivatives provided 50% protection against a PSI ED99 of ricin (0.1 micrograms/ml). Concentrations of BFA greater than 0.5 micro M caused about 50% PSI by itself, but blocked any further inhibitory effects of ricin. AZT, at optimum concentrations, reached a maximum protection level of about 40% in the presence of an ED99 dose of ricin, while the nucleoside derivative, BM33203 and AZT appeared to have an additive effect, showing up to 80% protection from an ED99 dose of ricin. Drugs showing protection in the PSI cell assay showed no protection from ricin in a cell-free translation assay used to determine if they would block ricin at the protein synthesis site.

Hypolipidemic activity of a series of N-pyridinyl and N-quinolinyl substituted derivatives of phthalimide and succinimides in rodents.

N-Pyridinyl- and N-quinolinyl-ethyl substituted succinimide and phthalimide derivatives demonstrated significant hypolipidemic activity in rodents lowering both serum cholesterol and triglyceride levels at 20 mg/kg.day i.p. The pyridinyl, quinolinyl and tetrahydro-2-quinolinyl substitution improved the hypolipidemic activity of the succinimide derivatives. Whereas select N-quinolinylethylphthalimide derivatives demonstrated less activity than phthalimide, itself, in lowering serum cholesterol and triglyceride levels, they did cause significant increases in HDL cholesterol and lower LDL cholesterol levels after 14 days of drug administration. Phthalimide actually caused reductions of HDL cholesterol levels in rats. These new agents with heterocyclic substitution of the N atom of phthalimide increased fecal excretion of lipids and suppressed in vitro hepatic rate limiting enzyme activities for de novo synthesis of cholesterol, fatty acids and triglycerides. Acute toxicity studies of the N-quinolinylethylphthalimide derivatives indicated that the agents are safe as potential therapeutic agents at doses necessary for hypolipidemic activity.

2-Acetylpyridine thiosemicarbazones. 12. Derivatives of 3-acetylisoquinoline as potential antimalarial agents.

A series of 3-acetylisoquinoline thiosemicarbazones and their related thiosemicarbazides was prepared for evaluation as potential antimalarial agents. The former were synthesized by the reaction of 3-acetylisoquinoline with methyl hydrazinecarbodithioate to give methyl 3-[1-(3-isoquinolinyl)ethylidene]hydrazinecarbodithioate, IV. Displacement of the S-methyl group of this intermediate by the requisite amines gave 3-acetylisoquinoline thiosemicarbazones, V. The corresponding thiosemicarbazides, in which the azomethine bond was reduced, were prepared by the reduction of IV with sodium borohydride to give methyl 3-[1-(3-isoquinolinyl)ethyl]hydrazinecarbodithioate, VI. Reaction of this dithioester with amines gave 1-[1-(3-isoquinolinyl)ethyl-3-thiosemicarbazides, VII. The antimalarial properties of series V and VII were evaluated in mice infected with Plasmodium berghei. Significant curative activity could be observed at doses as low as 40 mg/kg for 3 of 10 compounds in series V and at 160 mg/kg for 3 of 11 compounds in series VII.

Antibacterial properties of 2-acetylpyridine-1-oxide thiosemicarbazones.

We have investigated the in vitro antibacterial activity of 13 2-acetylpyridine-1-oxide thiosemicarbazones and 5 thiosemicarbazides against 80 clinically significant bacterial cultures, including 13 isolates with known antibiotic resistance. Of the thiosemicarbazones tested, 5 had minimal inhibitory concentrations (MICs) of 0.25 microgram/ml for Neisseria gonorrhoeae isolates; 1 of these had an MIC range of 0.25-0.5 microgram/ml for the Neisseria meningitidis cultures, and 2 had MICs of 2 and 2-4 micrograms/ml for Staphylococcus aureus and Streptococcus faecalis isolates, respectively. Two of the thiosemicarbazides had MICs of 0.25 microgram/ml for N. gonorrhoeae, whereas 2 others had MICs of 2-4 and 4-8 micrograms/ml for S. aureus and S. faecalis isolates, respectively. The test compounds were ineffective against the gram-negative enteric cultures and the Pseudomonas isolates.

2-Acetylpyridine thiosemicarbazones XI: 2-(alpha-Hydroxyacetyl)pyridine thiosemicarbazones as antimalarial and antibacterial agents.

A series of 2-(alpha-hydroxyacetyl)pyridine thiosemicarbazones was synthesized as potential antimalarial and antibacterial agents. Their synthesis was achieved by the condensation of N4-mono- or N4,N4-disubstituted thiosemicarbazides with 2-(alpha-hydroxyacetyl)pyridine. The latter was prepared by selective bromine oxidation of (2-pyridinyl)-1,2-ethanediol. The new compounds show potent inhibitory activity against penicillin-sensitive as well as penicillin-resistant Neisseria gonorrhoeae (MIC, 0.5-0.004 micrograms/mL), against Neisseria meningitidis (MIC, 0.5-0.032 micrograms/mL), and Staphylococcus aureus (MIC, 0.5-2 micrograms/mL). Good in vitro antimalarial effects against Plasmodium falciparum (Smith strain; ID50, 6.7-38 ng/mL) were observed in most of these new agents, but only 3 of 12 compounds exhibit moderate in vivo activity against Plasmodium berghei. These new agents appear to be less toxic to the host and more water soluble than the corresponding 2-acetylpyridine thiosemicarbazones.

2-Acetylpyridine thiosemicarbazones and Mycobacterium leprae.

Four 2-acetylpyridine thiosemicarbazones were tested in mice against Mycobacterium leprae by the kinetic method and found to be nearly inactive in a dosage of 0.05% in the diet. At the same dosage, thiacetazone, as a positive control, exhibited its expected activity.

2-Acetylpyridine thiosemicarbazones. 8. Derivatives of 1-acetylisoquinoline as potential antimalarial agents.

A series of 1-acetylisoquinoline thiosemicarbazones was prepared in order to evaluate their antimalarial properties. This was achieved by the reaction of 1-acetylisoquinoline with methyl hydrazinecarbodithioate to give methyl 3-[1-(1-isoquinolinyl)ethylidene]hydrazinecarbodithioate (II). Displacement of the S-methyl group from this intermediate by various primary and secondary amines afforded the desired 1-acetylisoquinoline thiosemicarbazones (III). Thiosemicarbazides in which the azomethine moiety of the latter was reduced could be prepared by the reaction of II with NaBH4 to give methyl 3-[1-(1-isoquinolinyl)ethyl]hydrazinecarbodithioate (VIII). Reaction of VII with the appropriate amine gave 1-[1-(1-isoquinolinyl)ethyl]thiosemicarbazides (IX). Evaluation of the antimalarial activity of series III and IX in mice infected with Plasmodium berghei indicated that cures were attainable at dose levels of 40-160 mg/kg.

2-Acetylpyridine thiosemicarbazones. 9. Derivatives of 2-acetylpyridine 1-oxide as potential antimalarial agents.

In view of the antimalarial activity in mice of 2-acetylpyridine thiosemicarbazones, a series of analogous 1-oxides was prepared for evaluation. Their synthesis was achieved by the reaction of 2-acetylpyridine 1-oxide with methyl hydrazinecarbodithioate to give methyl 3-[1-(2-pyridinyl 1-oxide)ethylidene]hydrazinecarbodithioate (II). Reaction of the latter intermediate with secondary amines afforded the desired 2-acetylpyridine 1-oxide thiosemicarbazones (III). Reduction of the azomethine linkage of II with NaBH4 gave methyl 3-[1-(2-pyridinyl 1-oxide)ethyl]-hydrazinecarbodithioate (IV) whose S-methyl group was then displaced by amines to give a 1-[1-(2-pyridinyl 1-oxide)ethyl]thiosemicarbazide, V. Antimalarial activity of III was evaluated against both Plasmodium berghei in the mouse and Plasmodium falciparum in an automated in vitro test system. In both cases, 2-acetylpyridine 1-oxide thiosemicarbazones were found to be less active than the corresponding de-1-oxide analogues. When compounds V were evaluated against Plasmodium berghei in the mouse, a diminution of activity was similarly seen in comparison to the analogues not bearing the 1-oxide moiety.

2-Acetylpyridine thiosemicarbazones. 10. 2-Propionyl-, 2-butyryl-, and 2-(2-methylpropionyl)pyridine thiosemicarbazones as potential antimalarial agents.

In view of the antimalarial properties observed for many 2-acetylpyridine thiosemicarbazones, a series of N4,N4-disubstituted thiosemicarbazones derived from 2-propionyl-, 2-butyryl-, and 2-(2-methylpropionyl)pyridine was prepared for evaluation against the malaria parasite, Plasmodium berghei, in the mouse. The thiosemicarbazones were made by the reaction of methyl hydrazinecarbodithioate with a 2-acylpyridine to give the intermediate methyl 3-[1-2-pyridinyl)alkylidene]hydrazinecarbodithioates. Reaction of the latter with 3-azabicyclo[3.2.2]nonane, 1-(2-pyridinyl)piperazine, or 1H-hexahydroazepine gave the requisite thiosemicarbazones. The three thiosemicarbazones derived from 3-azabicyclo[3.2.2]nonane were most effective, the greatest potency being exhibited by 3-azabicyclo-[3.2.2]nonane-3-thiocarboxylic acid 2-[1-(2-pyridinyl)butylidene]hydrazide (4) which cured 4/5 mice at a dose of 160 mg/kg. In contrast to the related thiosemicarbazones derived from 2-acetylpyridine, virtually no toxic effects were observed in the series described here.

2-Acetylpyridine thiosemicarbazones. 5. 1-[1-(2-Pyridyl)ethyl]-3-thiosemicarbazides as potential antimalarial agents.

Reduction of the azomethine bond of 2-acetylpyridine thio- and selenosemicarbazones with sodium borohydride readily afforded the corresponding thio- or selenosemicarbazides when they were N4,N4-disubstituted. This conversion failed, however, when the thio- or selenosemicarbazones were N4-substituted or unsubstituted. A more general route to the desired thio- or selenosemicarbazides consisted of reduction with sodium borohydride of methyl 3-[1-(2-pyridyl)ethylidene]hydrazinecarbodithioate to give the 2-pyridylethyl derivative. Displacement of methyl mercaptan from the thio ester moiety of the latter by amines produced 1-[1-(2-pyridyl)ethyl]-3-thiosemicarbazides. These compounds were somewhat more active as antimalarial agents in Plasmodium berghei infected mice than the corresponding thiosemicarbazones; however, the enhancement of activity was accompanied by an increase in toxicity. Compound 7, 3-azabicyclo[3.2.2]nonane-3-carbothioic acid 2-[1-(2-pyridyl)ethyl]hydrazide, is the most potent derivative of 2-acetylpyridine we have evaluated to date.

Thiosemicarbazones of 2-acetylpyridine, 2-acetylquinoline, 1- and 3-acetylisoquinoline and related compounds as inhibitors of clinically significant bacteria in vitro.

Antibacterial activity of 64 thiosemicarbazones of 2-acetylpyridine, 2-acetylquinoline, 1- and 3-acetylisoquinoline and related compounds was determined by testing clinical isolates of ten bacterial genera, including some which were antibiotic resistant. Minimal inhibitory concentrations (MICs) of 0.016 to 0.125 microgram/ml were obtained with 26% of the compounds for Neisseria meningitidis and 0.002 to 0.25 microgram/ml with 19% of the compounds for N. gonorrhoeae. Staphylococcus aureus was inhibited in the MIC range of 0.062 to 0.5 microgram/ml by 34% of the thiosemicarbazones, whereas 26% inhibited Streptococcus faecalis with an MIC range of 0.25 to 1.0 microgram/ml. Less antibacterial activity was shown toward the Gram-negative bacilli, i.e., Pseudomonas, Klebsiella-Enterobacter, Shigella, Escherichia coli, Serratia marcescens and Proteus.