Synthesis and Structure Elucidation of 5-Aminomethinimino-3-methyl-4-isoxazolecarboxylic Acid Phenylamides and Their Immunological Activity

A series of 5-aminomethinimino-3-methyl-4-isoxazolecarboxylic acid phenylamides 4 has been prepared by condensation of 5-amino-3-methyl-4-isoxazolecarboxylic acid phenylamides 1 with trichloroacetic aldehyde. Alcoholysis of trichloro derivatives 2 gave 5-alkoxymethine derivatives 3 which, on reaction with an appropriate amine, formed the corresponding compounds 4 . The compounds obtained were evaluated for their immunological activity. The properties of three compounds, described in this report, permitted inhibition of the immune response in all possible ways: diminishing both types of immune response ( 4d ), humoral immune response ( 4a ), or cellular immune response ( 4c ). Preparation 4d is comparable in its effectiveness to CsA, so it may be potentially used as an agent for prolongation of the function of transplanted organs. Two other compounds may potentially be used in cases where only one type the immune response is required for combating pathogen invasion.     

Immunological activity of new heterocyclic amides of 5-amino-3-methylisoxazole-4-carboxylic acid

In the present study, some new amides of 5-amino-3-methylisoxazole-4-carboxylic acid were obtained. All new structures possessed markedly different groups of electron acceptor character, different spatial structure and they contained nitrogen heteroatom, enabling formation of salts and, at the same time, higher biological availability. They were examined for immunomodulating activity in comparison with cyclosporine A (CsA). We investigated effects of the compounds on the lipopolysaccharide (LPS)-induced production of tumor necrosis factor-alpha (TNF-alpha) and interleukin-6 (IL-6) by human peripheral blood cells. Some compounds exhibited suppressory action which corresponded with increasing electronoacceptor nature of the amide substituent. Two compounds, characterized by flat aromatic rings, demonstrated quite different properties. Much higher activity was expressed by compounds which contained -NH group, the group which conditioned immunostimulatory activity in other compounds described previously.     

A new class of isoxazole derivatives: the M 1-9 series of compounds with immunotropic activity

The isoxazole derivatives are interesting objects for synthesis in the search for various sorts of biological activity. Looking for more active immunomodulators we synthesized a series of 5-amino-3-methyl-4-isoxazolecarboxylic acid semicarbazides and thiosemicarbazides in the reaction of 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide with isocyanates and isothiocyanates. The biological effect of these compounds on the proliferative response of human mononuclear peripheral blood cells to phytohemagglutinin A (PHA) was described.     

Immunomodulating action and structure-activity relationships of substituted phenylamides of 5-amino-3-methylisoxazole-4-carboxylic acid.

A series of 5-amino-3-methylisoxazole-4-carboxylic acid amides has been prepared by condensation of 5-amino-3-methylisoxazole-4-carboxylic acid with ethyl chloroformate. The resulting mixed anhydride undergoes condensation with appropriate phenylamides to form the corresponding amides 6-16. The compounds obtained were evaluated for their immunological activities in cultures of human peripheral blood mononuclear cells (PMBC). We found that the activities of the compounds in the proliferation test and in the lipopolysaccharide (LPS)-induced cytokine production in PBMC cultures were differential. The stimulatory or inhibitory effects depended strongly on the origin and location of substituents in the phenyl ring which is described in the discussion and was supported by QSAR studies.     

Structure/activity investigations of 5-substituted 3-methylisoxazole[5, 4-d]1, 2, 3-triazin-4-one derivatives

The series of 5-substituted 3-methylisoxazole[5, 4-d]1, 2, 3-triazin-4-one derivatives was obtained by diazotization of 5-amino-3-methylisoxazol-4-carboxylic acid hydrazide. The immunological activity of these compounds was investigated experimentally in several in vitro and in vivo assays in mice and human models. In the next step, quantum-chemical investigations were performed using density functional theory with the B3LYP hybrid exchange-correlation energy functional and 6-31G(d, p) basis set. The Polarizable Continuum (SCRF/PCM) solvent model was also taken into account in order to show solvent influence on electron density and electrostatic potential around the exemplary molecules. Correlations between molecular structure and biological properties were found using a stepwise selection of scales for the multiple linear regression (MLR).     

Immunosuppressive activity of an isoxazolo[5,4-e]triazepine--compound RM33. I. Effects on the humoral and cellular immune response in mice

The aim of this investigation was to evaluate immunotropic properties of a new isoxazolotriazepine (compound RM33) in mice. We found that RM33 significantly inhibited induction of the humoral immune response to sheep red blood cells (SRBC), given 2 h prior to immunization. The development of the cellular immune response (delayed type hypersensitivity--DTH) to SRBC and to ovalbumin (OVA) was also suppressed, as well as the effector phase of the DTH to OVA. The compound was also effective when administered per os. The suppressive effects of RM33 on the immune response were comparable to those of cyclosporine A(CsA). We also showed that RM33 inhibited DTH to OVA if admixed with the sensitizing dose of an antigen and complete Freund's adjuvant (cFa) suggesting that the compound may affect initial events of antigen presentation. Such a hypothesis was supported by finding that RM33 significantly inhibited foot pad edema elicited by administration of cFa. The effects of RM33 on the activities of cytokines relevant to development and control of the immune response and inflammation such as: tumor necrosis factor alpha (TNF-alpha), interleukin 6 and 10 (IL-6 and IL-10) were also studied. The compound markedly (by 63%) inhibited lipopolysaccharide (LPS)-induced TNF-alpha serum level whereas IL-6 activity was lowered to a lesser extent (by 17%). The inducible IL-10 level in the splenocyte cultures was not affected at all. In summary, the presented results revealed immunosuppressive properties of RM33, which could be associated with its selective interference with co-stimulatory signals provided by adjuvant at initiation of the immune response.     

Synthesis and immunological activity of new 5-amino-3-methyl 4-amido and 4-ureilene isoxazole derivatives.

5-Amino-3-methylisoxazole-4-carboxylic acid amides and ureilenes have been synthesized from 5-amino-3-methylisoxazole-4-carbonyl azide. The compounds were investigated for potential immunotropic activity in several immunological tests. The most interesting suppressory activities in the humoral and cellular immune response were compared to activities of analogous compounds previously described as immunostimulatory.     

Reactions of 5-amino-3-methylisoxazole-4-carboxylic acid hydrazide with carbonyl compounds: immunological activity and QSAR studies of products

A series of 4-imino derivatives of the 5-amino-3-methylisoxazole-4-carboxylic acid hydrazide and 5-amino-3-methylisoxazole[5,4-d]-6,7-dihydropyrimidine has been prepared by condensation of 5-amino-3-methylisoxazole-4-carboxylic acid hydrazide with carbonyl compounds. The resulting products were evaluated for their immunological activities in the models of the humoral and cellular immune responses of mice in vivo and concanavalin A (Con A) and lipopolysaccharide (LPS)-induced splenocyte proliferation. In addition, effects on polyclonal antibody production by human peripheral blood cells in culture were investigated. For all studied compounds we carried out quantum chemical calculations at ab initio B3LYP 6-31G(d, p) level. The stimulatory or inhibitory effects depended strongly on the origin and location of substitunets, which is described in the conclusions and was supported by QSAR studies.     

In vitro immunomodulatory effects of 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide on the cellular immune response

The aim of this study was to determine the immunomodulatory activity of 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide in vitro. This compound was used for the synthesis of a series of 5-amino-3-methyl-4-isoxazolecarboxylic acid semicarbazides and thiosemicarbazides with documented immunotropic activity. The performed measurements assessed the cytotoxic effect of 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide on the murine macrophages (cell line J774E.1) and lymphoblasts (cell line D10.G4.1), the influence of this compound on the proliferation of murine lymphocytes isolated from peripheral lymphatic organs and murine peritoneal macrophages stimulated with mitogens (concanavalin A(ConA), lipopolysaccharide (LPS), phytohemagglutinin A (PHA)). Moreover, the production of tumor necrosis factor (TNF)-α and interleukin (IL)-1β by the murine peritoneal macrophages stimulated with LPS from Escherichia coli was assessed. It was found that 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide displayed no cytotoxic effects in the murine J774E.1 and D10.G4.1 cell lines in a wide range of concentrations (0.5–200?μg/ml). Furthermore, the compound stimulated proliferation of lymphocytes isolated from the spleen and mesenteric lymph nodes when used alone and in combination with mitogens (ConA and PHA). This effect was stronger in the nonstimulated cells, and it followed a dose–response relationship. The same phenomenon was observed for the proliferation of the murine peritoneal macrophages. The investigated hydrazide, at the highest used concentration of 150?μg/ml, increased the LPS-induced production of IL-1β and did not affect the level of TNF-α. These results confirmed the immunomodulatory properties of 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide and indicated that this compound could be useful in further research aimed at finding novel functional drugs.