ABSTRACT
Novel 2,3-diarylindoles bearing an amine substituent at the indole 5- and 6-positions have been synthesized and evaluated as anticoccidial agents in both in vitro and in vivo assays. Both subnanomolar in vitro activity and broad spectrum in vivo potency were detected for several compounds, particularly compound 27.
Subject(s)
Coccidiostats/chemical synthesis , Indoles/chemical synthesis , Animals , Coccidiosis/drug therapy , Coccidiosis/enzymology , Coccidiosis/parasitology , Coccidiostats/pharmacology , Cyclic GMP-Dependent Protein Kinases/antagonists & inhibitors , Eimeria tenella/drug effects , Eimeria tenella/enzymology , Eimeria tenella/growth & development , Indoles/pharmacology , Poultry/parasitology , Pyridines/chemical synthesisABSTRACT
Novel 5,6-diarylimidazo[2,1-b][1,3]thiazoles bearing an amine substituent at the imidazothiazole 2-position have been synthesized and evaluated as anticoccidial agents in both in vitro and in vivo assays. Both subnanomolar in vitro activity and broad spectrum in vivo potency were detected for several compounds, particularly compound 10.
Subject(s)
Coccidiostats/chemical synthesis , Coccidiostats/pharmacology , Imidazoles/chemical synthesis , Imidazoles/pharmacology , Thiazoles/chemical synthesis , Thiazoles/pharmacology , Animals , Coccidiostats/chemistry , Combinatorial Chemistry Techniques , Cyclic GMP-Dependent Protein Kinases/antagonists & inhibitors , Eimeria/drug effects , Imidazoles/chemistry , Molecular Structure , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/chemistry , Protein Kinase Inhibitors/pharmacology , Thiazoles/chemistryABSTRACT
Diaryl-(4-piperidinyl)-pyrrole derivatives bearing cyclic amine substituents have been synthesized and evaluated as anticoccidial agents. Improvements in potency of Et-PKG inhibition, such as azetidine derivative 3a, and broad spectrum anticoccidial activities in feed, such as morpholine derivative 8c, have been achieved.
Subject(s)
Coccidiosis/drug therapy , Coccidiostats/pharmacology , Eimeria/drug effects , Pyrroles/pharmacology , Animals , Chickens , Coccidiostats/chemical synthesis , Cyclic GMP-Dependent Protein Kinases/antagonists & inhibitors , Eimeria/enzymology , Female , Molecular Structure , Oocysts/drug effects , Oocysts/physiology , Protein Kinase Inhibitors/pharmacology , Pyrroles/chemical synthesis , Pyrroles/chemistry , Structure-Activity RelationshipABSTRACT
Coccidiosis is the major cause of morbidity and mortality in the poultry industry. Protozoan parasites of the genus Eimeria invade the intestinal lining of the avian host causing tissue pathology, poor weight gain, and in some cases mortality. Resistance to current anticoccidials has prompted the search for new therapeutic agents with potent in vitro and in vivo activity against Eimeria. Recently, we reported the synthesis and biological activity of potent imidazo[1,2-a]pyridine anticoccidial agents. Antiparasitic activity is due to inhibition of a parasite specific cGMP-dependent protein kinase (PKG). In this study, we report the synthesis and anticoccidial activity of a second set of such compounds, focusing on derivatization of the amine side chain at the imidazopyridine 7-position. From this series, several compounds showed subnanomolar in vitro activity and commercial levels of in vivo activity. However, the potential genotoxicity of these compounds precludes them from further development.
Subject(s)
Coccidiostats/chemical synthesis , Coccidiostats/pharmacology , Pyridines/chemical synthesis , Pyridines/pharmacology , Animals , Cyclic GMP-Dependent Protein Kinases/antagonists & inhibitors , Eimeria/drug effects , Magnetic Resonance Spectroscopy , Protein Kinase Inhibitors/chemical synthesis , Protein Kinase Inhibitors/pharmacology , Spectrometry, Mass, Electrospray IonizationABSTRACT
Triptolide is a naturally occurring diterpene triepoxide whose anti-inflammatory effects correlate with transcriptional inhibition of various cytokines. Despite its use in herbal medicine for thousands of years, the cellular target and mode of action of this drug are unknown. [3H]-triptolide was prepared and a filtration assay designed to measure binding to cells and cellular extracts. Triptolide bound specifically and irreversibly to a single, 90 kDa protein in nuclear extracts from stimulated and non-stimulated monocytic and epithelial cell lines. Thiol reactivity of one or more of the epoxides on triptolide was necessary for the covalent binding, since thiol oxidizing agents dithiodipyridine and diamide, and the thiol alkylating agent N-ethylmaleimide all reduced the binding of [3H]-triptolide to nuclear extract. Neither glutathione nor the pro-oxidant tert-butylhydroperoxide affected the binding of [3H]-triptolide to the nuclear protein, ruling out a general oxidant effect. The number of epoxide moieties correlated with the ability to compete with radiolabeled triptolide for binding to the nuclear extract and with the potency of inhibition of TNFalpha secretion from monocytes, IL-2 secretion from Jurkat cells, and with inhibition of RNA synthesis. The correlation between the structure-activity relationship and observed binding suggests that identification of the triptolide binding protein could provide insight into the cellular mode of action of this anti-inflammatory natural product.
Subject(s)
Cytokines/biosynthesis , Diterpenes/metabolism , Epoxy Compounds/metabolism , Nuclear Proteins/metabolism , Phenanthrenes/metabolism , Apoptosis , Cell Line , Cytokines/immunology , Diterpenes/chemistry , Epoxy Compounds/chemistry , Humans , NF-kappa B/metabolism , Phenanthrenes/chemistryABSTRACT
Diaryl imidazo[1,2-a]pyridine derivatives, such as 6a and 7i, have been synthesized and found to be potent inhibitors of parasite PKG activity. The most potent compounds are the 7-isopropylaminomethyl analog 6a and 2-isopropylamino analog 7i. These compounds are also fully active in in vivo assay as anticoccidial agents at 25 ppm in feed.
Subject(s)
Coccidiosis/drug therapy , Coccidiostats/pharmacology , Imidazoles/chemistry , Protein Kinase Inhibitors/pharmacology , Pyridines/chemistry , Pyridines/pharmacology , Animals , Chemistry, Pharmaceutical/methods , Coccidiostats/chemistry , Cyclic GMP-Dependent Protein Kinases/metabolism , Drug Design , Eimeria tenella , Models, Chemical , Protein Kinase Inhibitors/chemistry , Structure-Activity RelationshipABSTRACT
Coccidiosis is the major cause of morbidity and mortality in the poultry industry. Protozoan parasites of the genus Eimeria invade the intestinal lining of the avian host causing tissue pathology, poor weight gain, and in some cases mortality. Resistance to current anticoccidials has prompted the search for new therapeutic agents with potent in vitro and in vivo activity against Eimeria. Antiparasitic activity is due to inhibition of a parasite specific cGMP-dependent protein kinase (PKG). In this study, we present the synthesis and biological activity of imidazo[1,2-a]pyridine anticoccidial agents. From this series, several compounds showed subnanomolar in vitro activity and commercial levels of in vivo activity. However, the potential genotoxicity of these compounds precludes them from further development.
Subject(s)
Coccidiostats/chemical synthesis , Coccidiostats/pharmacology , Eimeria/drug effects , Imidazoles/chemical synthesis , Imidazoles/pharmacology , Pyridines/chemical synthesis , Pyridines/pharmacology , Animals , Biological Availability , Coccidiostats/chemistry , Cyclic GMP-Dependent Protein Kinases/antagonists & inhibitors , Cyclic GMP-Dependent Protein Kinases/metabolism , Eimeria/physiology , Imidazoles/chemistry , Inhibitory Concentration 50 , Pyridines/chemistryABSTRACT
Compounds 10a-10d and 10i are very potent inhibitors of Eimeria tenella cGMP-dependent protein kinase (0.081-0.32 nM) and are very efficacious antiparasitic agents in vivo when administered to chickens at 12.5-25 ppm levels in the feed.
Subject(s)
Coccidiostats/chemical synthesis , Coccidiostats/pharmacology , Imidazoles/chemical synthesis , Imidazoles/pharmacology , Pyridines/chemistry , Pyridines/chemical synthesis , Pyridines/pharmacology , Animals , Coccidiostats/chemistry , Eimeria/drug effects , Imidazoles/chemistry , Molecular Structure , Structure-Activity RelationshipABSTRACT
Parasite cGMP-dependent protein kinase (PKG) is one of the validated biochemical targets for the treatment of coccidiosis. We screened our library of natural product extracts for inhibitors of parasite PKG for the discovery of anticoccidial leads. Terferol (1) and three new terphenyls (2, 3, and 4) were isolated using bioassay-guided fractionation of the microbial extract of a Phoma sp. by a high-throughput two-step isolation method employing LH-20 and reversed-phase HPLC. These compounds inhibited parasite PKG with IC(50) values in the range 0.9-5.8 microM.
Subject(s)
Ascomycota/chemistry , Coccidiosis/drug therapy , Cyclic GMP-Dependent Protein Kinases/antagonists & inhibitors , Eimeria tenella/drug effects , Enzyme Inhibitors , Terphenyl Compounds , Animals , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/isolation & purification , Enzyme Inhibitors/pharmacology , France , Molecular Structure , Terphenyl Compounds/chemistry , Terphenyl Compounds/isolation & purification , Terphenyl Compounds/pharmacologyABSTRACT
2-(4-Fluorophenyl)-3-(4-pyridinyl)-5-substituted pyrroles were prepared and evaluated as anticoccidial agents in both in vitro and in vivo assays. Among the compounds evaluated, the dimethylamine-substituted pyrrole 19a is the most potent inhibitor of Eimeria tenella PKG (cGMP-dependent protein kinase). Further SAR studies on the side chain of the 2-pyrrolidine nitrogen did not enhance in vivo anticoccidial activity.
Subject(s)
Coccidiostats/chemical synthesis , Coccidiostats/pharmacology , Pyrroles/chemical synthesis , Pyrroles/pharmacology , Animals , Coccidiostats/chemistry , Cyclic GMP-Dependent Protein Kinases/antagonists & inhibitors , Eimeria tenella/drug effects , Eimeria tenella/enzymology , Enzyme Inhibitors/chemical synthesis , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Pyrroles/chemistry , Structure-Activity RelationshipABSTRACT
Compounds 10a (IC50 110 pM) and 21 (IC50 40 pM) are the most potent inhibitors of Eimeria tenella cGMP-dependent protein kinase activity reported to date and are efficacious in the in vivo antiparasitic assay when administered to chickens at 12.5 and 6.25 ppm levels in the feed. However, both compounds are positive in the Ames microbial mutagenesis assay which precludes them from further development as antiprotozoal agents in the absence of negative lifetime rodent carcinogenicity studies.
Subject(s)
Antiprotozoal Agents/chemical synthesis , Cyclic GMP-Dependent Protein Kinases/antagonists & inhibitors , Eimeria tenella/drug effects , Enzyme Inhibitors/chemical synthesis , Imidazoles/chemical synthesis , Pyridines/chemical synthesis , Animal Feed , Animals , Antiprotozoal Agents/chemistry , Antiprotozoal Agents/pharmacology , Chickens , Coccidiosis/drug therapy , Eimeria tenella/enzymology , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Imidazoles/chemistry , Imidazoles/pharmacology , Male , Molecular Structure , Mutagenicity Tests , Oocysts/drug effects , Parasitic Sensitivity Tests , Pyridines/chemistry , Pyridines/pharmacology , Rats , Rats, Sprague-Dawley , Stereoisomerism , Structure-Activity RelationshipABSTRACT
Diaryl-(4-piperidinyl)-pyrrole derivatives bearing hydroxylated N-alkyl substituents have been synthesized and evaluated as anticoccidial agents. High potency in Et-PKG inhibition and broad-spectrum anticoccidial activities have been observed on compounds, such as 4b and 5h, which are fully efficacious in vivo at 50 ppm in feed.
Subject(s)
Coccidiostats/chemistry , Coccidiostats/pharmacology , Pyrroles/chemistry , Pyrroles/pharmacology , Cyclic GMP-Dependent Protein Kinases/antagonists & inhibitors , Hydroxylation , Structure-Activity RelationshipABSTRACT
Several analogs of 2,3-diaryl pyrroles were synthesized and evaluated as inhibitors of Eimeria tenella cGMP-dependent protein kinase and in in vivo anticoccidial assays. A 4-fluorophenyl group enhances both in vitro and in vivo activities. The most potent analogs are the 5-(N-methyl, N-ethyl, and N-methylazetidine methyl) piperidyl derivatives 12, 23, and 34. These compounds have a broad spectrum of activity. Based on the in vivo efficacy and cost of synthesis, the N-ethyl analog 23 was chosen as a novel anticoccidial agent for a field trial.
Subject(s)
Coccidiostats/chemical synthesis , Cyclic GMP-Dependent Protein Kinases/antagonists & inhibitors , Protozoan Proteins/antagonists & inhibitors , Pyrroles/chemical synthesis , Animals , Biological Availability , Chickens , Coccidiosis/drug therapy , Coccidiostats/pharmacokinetics , Coccidiostats/pharmacology , Eimeria , Half-Life , Inhibitory Concentration 50 , Pyrroles/pharmacokinetics , Pyrroles/pharmacology , Structure-Activity RelationshipABSTRACT
Parasite cGMP-dependent protein kinase (PKG) has been recently validated as a biochemical target for the treatment of coccidiosis. To discover new anticoccidial leads, we have screened our library of natural product extracts for inhibitors of parasite PKG. Bioassay-guided fractionation of the microbial extracts has led to the discovery of tenellones A (2) and B (3), two new highly substituted benzophenones. The isolation, structure, and activity of these compounds are described.
Subject(s)
Benzophenones/isolation & purification , Enzyme Inhibitors/isolation & purification , Fungi/chemistry , Animals , Benzophenones/chemistry , Benzophenones/pharmacology , Cyclic GMP-Dependent Protein Kinases , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Molecular Structure , Plants, Medicinal/chemistry , Spain , Toxoplasma/metabolismABSTRACT
The trisubstituted pyrrole 4-[2-(4-fluorophenyl)-5-(1-methylpiperidine-4-yl)-1H-pyrrol-3-yl]pyridine (Compound 1) inhibits the growth of Eimeria spp. both in vitro and in vivo. The molecular target of Compound 1 was identified as cGMP-dependent protein kinase (PKG) using a tritiated analogue to purify a approximately 120-kDa protein from lysates of Eimeria tenella. This represents the first example of a protozoal PKG. Cloning of PKG from several Apicomplexan parasites has identified a parasite signature sequence of nearly 300 amino acids that is not found in mammalian or Drosophila PKG and which contains an additional, third cGMP-binding site. Nucleotide cofactor regulation of parasite PKG is remarkably different from mammalian enzymes. The activity of both native and recombinant E. tenella PKG is stimulated 1000-fold by cGMP, with significant cooperativity. Two isoforms of the parasite enzyme are expressed from a single copy gene. NH(2)-terminal sequence of the soluble isoform of PKG is consistent with alternative translation initiation within the open reading frame of the enzyme. A larger, membrane-associated isoform corresponds to the deduced full-length protein sequence. Compound 1 is a potent inhibitor of both soluble and membrane-associated isoforms of native PKG, as well as recombinant enzyme, with an IC(50) of <1 nm.
