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The microbial, biofilm-based infections of chronic wounds are one of the major challenges of contemporary medicine. The use of topically administered antiseptic agents is essential to treat wound-infecting microorganisms. Due to observed microbial tolerance/resistance against specific clinically-used antiseptics, the search for new, efficient agents is of pivotal meaning. Therefore, in this work, 15 isoxazole derivatives were scrutinized against leading biofilm wound pathogens Staphylococcus aureus and Pseudomonas aeruginosa, and against Candida albicans fungus. For this purpose, the minimal inhibitory concentration, biofilm reduction in microtitrate plates, modified disk diffusion methods and antibiofilm dressing activity measurement methods were applied. Moreover, the cytotoxicity and cytocompatibility of derivatives was tested toward wound bed-forming cells, referred to as fibroblasts, using normative methods. Obtained results revealed that all isoxazole derivatives displayed antimicrobial activity and low cytotoxic effect, but antimicrobial activity of two derivatives, 2-(cyclohexylamino)-1-(5-nitrothiophen-2-yl)-2-oxoethyl 5-amino-3-methyl-1,2-oxazole-4-carboxylate (PUB9) and 2-(benzylamino)-1-(5-nitrothiophen-2-yl)-2-oxoethyl 5-amino-3-methyl-1,2-oxazole-4-carboxylate (PUB10), was noticeably higher compared to the other compounds analyzed, especially PUB9 with regard to Staphylococcus aureus, with a minimal inhibitory concentration more than x1000 lower compared to the remaining derivatives. The PUB9 and PUB10 derivatives were able to reduce more than 90% of biofilm-forming cells, regardless of the species, displaying at the same time none (PUB9) or moderate (PUB10) cytotoxicity against fibroblasts and high (PUB9) or moderate (PUB10) cytocompatibility against these wound cells. Therefore, taking into consideration the clinical demand for new antiseptic agents for non-healing wound treatment, PUB9 seems to be a promising candidate to be further tested in advanced animal models and later, if satisfactory results are obtained, in the clinical setting.

The hybrid peptides consisting of α and β-amino acids show great promise as peptidomimetics that can be used as therapeutic agents. Therefore, the development of new unnatural amino acids and the methods of their incorporation into the peptide chain is an important task. Here, we described our investigation of the possibility of 5-amino-3-methyl-isoxazole-4-carboxylic acid (AMIA) application in the solid phase peptide synthesis. This new unnatural β-amino acid, presenting various biological activities, was successfully coupled to a resin-bound peptide using different reaction conditions, including classical and ultrasonic agitated solid-phase synthesis. All the synthesized compounds were characterized by tandem mass spectrometry. The obtained results present the possibility of the application of this β-amino acid in the synthesis of a new class of bioactive peptides.

Cancer is a large group of diseases in which the rapid proliferation of abnormal cells generally leads to metastasis to surrounding tissues or more distant ones through the lymphatic and blood vessels, making it the second leading cause of death worldwide. The main challenge in designing a modern anticancer therapy is to develop selective compounds that exploit specific molecular targets. In this work, novel oxazolo[5,4

Herein we present a synthesis and characterization of a new and unique low-weight heterocyclic compound 5-amino-2-(5-amino-3-methyl-1,2-oxazol-4-yl)-3-methyl-2,3-dihydro-1,3,4-oxadiazol-2-ylium bromide with the unusual electron charge delocalization owing the local positive charge at the carbon atom of oxadiazole moiety. X-ray single crystal of C7H10N5O2·Br− showed the molecule crystalized in monoclinic, space group P21/c. Both five membered rings are planar and twisted forming the ring motif with the counter ion where H⋯Br interactions are one of the dominant. The presented compound is characterized by high ionization efficiency in ESI-MS mode and undergoes dissociation within oxadiazole moiety under ESI-MS/MS conditions even under low collision energies. The presented compound is an interesting example of heterocyclic stable carbocation which may serve as a new lead structure.

Cancer is a large group of diseases in which the rapid proliferation of abnormal cells generally leads to metastasis to surrounding tissues or more distant ones through the lymphatic and blood vessels, making it the second leading cause of death worldwide. The main challenge in designing a modern anticancer therapy is to develop selective compounds that exploit specific molecular targets. In this work, novel oxazolo[5,4-d]pyrimidine derivatives were designed, synthesized, and evaluated in vitro for their cytotoxic activity against a panel of four human cancer cell lines (lung carcinoma: A549, breast adenocarcinoma: MCF7, metastatic colon adenocarcinoma: LoVo, primary colon adenocarcinoma: HT29), along with their P-glycoprotein-inhibitory ability and pro-apoptotic activity. These oxazolo[5,4-d]pyrimidine derivatives, which are structurally similar to nucleic purine bases in general, are characterized by the presence of a pharmacologically favorable isoxazole substituent at position 2 and aliphatic amino chains at position 7 of the condensed heterocyclic system. In silico analysis of the obtained compounds identified their potent inhibitory activity towards human vascular endothelial growth factor receptor-2 (VEGFR-2). Molecular docking was performed to assess the binding mode of new derivatives to the VEGFR-2 active site. Then, their physicochemical, pharmacokinetic, and pharmacological properties (i.e., ADME—administration, distribution, metabolism, and excretion) were also predicted to assess their druglikeness. In particular, compound 3g (with a 3-(N,N-dimethylamino)propyl substituent) was found to be the most potent against the HT29 cell line, with a 50% cytotoxic concentration (CC50) of 58.4 µM, exceeding the activity of fluorouracil (CC50 = 381.2 μM) and equaling the activity of cisplatin (CC50 = 47.2 µM), while being less toxic to healthy human cells (such as normal human dermal fibroblasts (NHDFs)) than these reference drugs. The results suggest that compound 3g is a potentially promising candidate for the treatment of primary colorectal cancer.

Previous studies demonstrated strong anti-inflammatory properties of isoxazolo[5,4-e]-1,2,4-triazepine (RM33) in vivo. The aim of this investigation was to describe synthesis, determine physicochemical characteristics, evaluate biological activities in murine and human in vitro models, as well as to propose mechanism of action of the compound. The compound was devoid of cell toxicity up to 100 μg/mL against a reference A549 cell line. Likewise, RM33 did not induce apoptosis in these cells. The compound stimulated concanavalin A (ConA)-induced splenocyte proliferation but did not change the secondary humoral immune response in vitro to sheep erythrocytes. Nevertheless, a low suppressive effect was registered on lipopolysaccharide (LPS)-induced splenocyte proliferation and a stronger one on tumor necrosis factor alpha (TNFα) production by rat peritoneal cells. The analysis of signaling pathways elicited by RM33 in nonstimulated resident cells and cell lines revealed changes associated with cell activation. Most importantly, we demonstrated that RM33 enhanced production of cyclooxygenase 2 in LPS-stimulated splenocytes. Based on the previous and herein presented results, we conclude that RM33 is an efficient, nontoxic immune suppressor with prevailing anti-inflammatory action. Additionally, structural studies were carried out with the use of appropriate spectral techniques in order to unequivocally confirm the structure of the RM33 molecule. Unambiguous assignment of NMR chemical shifts of carbon atoms of RM33 was conducted thanks to full detailed analysis of 1H, 13C NMR spectra and their two-dimensional (2D) variants. Comparison between theoretically predicted chemical shifts and experimental ones was also carried out. Additionally, N-deuterated isotopologue of RM33 was synthesized to eliminate potentially disturbing frequencies (such as NH, NH2 deformation vibrations) in the carbonyl region of the IR (infrared) spectrum to confirm the presence of the carbonyl group.

Heterocyclic moieties, especially five and six-membered rings containing nitrogen, oxygen or sulfur atoms, are broadly distributed in nature. Among them, synthetic and natural alike are pharmacologically active compounds and have always been at the forefront of attention due to their pharmacological properties. Heterocycles can be divided into different groups based on the presence of characteristic structural motifs. The presence of β-amino acid and heterocyclic core in one compound is very interesting; additionally, it very often plays a vital role in their biological activity. Usually, such compounds are not considered to be chemicals containing a β-amino acid motif; however, considering them as this class of compounds may open new routes of their preparation and application as new drug precursors or even drugs. The possibility of their application as nonproteinogenic amino acid residues in peptide or peptide derivatives synthesis to prepare a new class of compounds is also promising. This review highlights the actual state of knowledge about β-amino acid moiety-containing heterocycles presenting antiviral, anti-inflammatory, antibacterial compounds, anaplastic lymphoma kinase (ALK) inhibitors, as well as agonist and antagonists of the receptors.

Thanks to the progress in oncology, pharmacological treatment of cancer is gaining in importance and in the near future anti-cancer chemotherapeutics are expected to be the main method of treatment for cancer diseases. What is more, the search for new anti-cancer compounds with the desired application properties is constantly underway. As a result of designed syntheses, we obtained some new N’-substituted 5-chloro-3-methylisothiazole-4-carboxylic acid hydrazide derivatives with anticancer activity. The structure of new compounds was determined by mass spectrometry (MS), elemental analysis, proton nuclear magnetic resonance spectroscopy (1H-NMR), carbon nuclear magnetic resonance spectroscopy (13C-NMR), 1H-13C NMR correlations and infrared spectroscopy (IR). Moreover, the structures of the compounds were confirmed by crystallographic examination. The antiproliferative MTT tests for 11 prepared compounds was conducted towards human biphenotypic B cell myelomonocytic leukemia MV4-11. SRB test was used to examine their potential anticancer activity towards human colon adenocarcinoma cell lines sensitive LoVo, resistant to doxorubicin LoVo/DX, breast adenocarcinoma MCF-7 and normal non-tumorigenic epithelial cell line derived from mammary gland MCF-10A. The most active compound was 5-chloro-3-methyl-N′-[(1E,2E)-(3-phenyloprop-2-en-1-ylidene]isothiazole-4-carbohydrazide, which showed the highest antiproliferative activity against all tested cell lines.

Thanks to the progress in oncology, pharmacological treatment of cancer is gaining in importance and in the near future anti-cancer chemotherapeutics are expected to be the main method of treatment for cancer diseases. What is more, the search for new anti-cancer compounds with the desired application properties is constantly underway. As a result of designed syntheses, we obtained some new N&rsquo, substituted 5-chloro-3-methylisothiazole-4-carboxylic acid hydrazide derivatives with anticancer activity. The structure of new compounds was determined by mass spectrometry (MS), elemental analysis, proton nuclear magnetic resonance spectroscopy (1H-NMR), carbon nuclear magnetic resonance spectroscopy (13C-NMR), 1H-13C NMR correlations and infrared spectroscopy (IR). Moreover, the structures of the compounds were confirmed by crystallographic examination. The antiproliferative MTT tests for 11 prepared compounds was conducted towards human biphenotypic B cell myelomonocytic leukemia MV4-11. SRB test was used to examine their potential anticancer activity towards human colon adenocarcinoma cell lines sensitive LoVo, resistant to doxorubicin LoVo/DX, breast adenocarcinoma MCF-7 and normal non-tumorigenic epithelial cell line derived from mammary gland MCF-10A. The most active compound was 5-chloro-3-methyl-N&prime, [(1E,2E)-(3-phenyloprop-2-en-1-ylidene]isothiazole-4-carbohydrazide, which showed the highest antiproliferative activity against all tested cell lines.

Here, the Conductor-like Polarizable Continuum Model (CPCM) was used as a less demanding substitute of Periodic Boundary Conditions (PBC) method to attempt to reliably simulate infrared spectra and some structural parameters of solid compound - 5-amino-3-methyl-4-isoxazolecarbohydrazide (HIX). The measured relative electric permittivity of HIX was used as a simplified equivalent of electric intensity generating by other molecules of 5-amino-3-methyl-4-isoxazolecarbohydrazide in crystal structure. The application of CPCM has resulted in better conformity of calculated molecular parameters with experimentally found. Theoretical geometry was compared with data obtained with X-ray crystallography. Comparison of harmonic approximation with anharmonic one, based on the six selected characteristic vibrations, shows that non-scaled harmonic wavenumbers, calculated with CPCM, well correspond to experimental spectra. Additionally, the results point out that anharmonic approximation appeared to be strong sensitive for input geometry and calculation parameters used, so the results are sometimes unreliable, especially for low wavenumbers. Also, the new feasible ways of the synthesis of the titled compound is presented, one of which is particularly easy and highly efficient.

Simple Summary Hematological malignancies are diseases involving the abnormal production of blood cells. The aim of the study is to collect comprehensive information on new drugs used in the treatment of blood cancers which have introduced into therapy in the last decade. The approved drugs were analyzed for their structures and their biological activity mechanisms. Abstract Hematological malignancies, also referred to as blood cancers, are a group of diseases involving abnormal cell growth and persisting in the blood, lymph nodes, or bone marrow. The development of new targeted therapies including small molecule inhibitors, monoclonal antibodies, bispecific T cell engagers, antibody-drug conjugates, recombinant immunotoxins, and, finally, Chimeric Antigen Receptor T (CAR-T) cells has improved the clinical outcomes for blood cancers. In this review, we summarized 52 drugs that were divided into small molecule and macromolecule agents, approved by the Food and Drug Administration (FDA) in the period between 2011 and 2021 for the treatment of hematological malignancies. Forty of them have also been approved by the European Medicines Agency (EMA). We analyzed the FDA-approved drugs by investigating both their structures and mechanisms of action. It should be emphasized that the number of targeted drugs was significantly higher (46 drugs) than chemotherapy agents (6 drugs). We highlight recent advances in the design of drugs that are used to treat hematological malignancies, which make them more effective and less toxic.

(1) Background: Melanoma is an aggressive neoplasm derived from melanocyte precursors with a high metastatic potential. Responses to chemotherapy and immunotherapy for melanoma remain weak, underlining the urgent need to develop new therapeutic strategies for the treatment of melanoma. (2) Methods: The viability of NHDF and A375 cell cultures after the administration of the tested isoxazole derivatives was assessed after 24-h and 48-h incubation periods with the test compounds in the MTT test. ROS and NO scavenging analyses, a glycoprotein-P activity analysis, a migration assay, a test of apoptosis, and a multiple-criteria decision analysis were also performed. (3) Results: All compounds that were tested resulted in a slower migration of melanoma neoplastic cells. The mechanism of the antitumor activity of the tested compounds was confirmed—i.e., the pro-apoptotic activity of the compounds in A375 cell cultures. Compound O7K qualified for further research. (4) Conclusions: All the tested compounds inhibited the formation of melanoma metastases and demonstrated the ability to reduce the risk of developing drug resistance in the tumor. The MCDA results showed that O7K showed the strongest antitumor activity.

Isoxazoles are an important class of compounds of potential therapeutic value. The aim of this study was to determine immunotropic effects of 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide derivatives on spontaneous and mitogen-induced lymphocyte proliferation in young and old mice, cytokine production by peritoneal cells as well as possible mechanism of action in a model of Jurkat cells. Three month and thirteen month old BALB/c mice were used as donors of the cells from a thymus, a spleen, mesenteric lymph nodes and a peritoneal cavity. Spontaneous and concanavalin A (ConA) or lipopolysaccharide (LPS)-induced cell proliferation was measured by MTT colorimetric method. IL-1β and TNF-α production induced by LPS in macrophage-enriched peritoneal cell cultures was measured by enzyme-linked immunoassay. 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide, 01K (4-phenyl-1-(5-amino-3-methylisoxazole-4-carbonyl)-thiosemicarbazide) and 06K (4-(4-chlorophenyl)-1-(5-amino-3-methylisoxazole-4-carbonyl)-thiosemicarbazide) exhibited regulatory activity in the proliferation tests. Prevailing stimulatory activity of the hydrazide and inhibitory activity of 01K and 06K was observed. Those effects were connected with different influence of the compounds on signaling proteins expression in Jurkat cells. The regulatory effects of the compounds on IL-1β production were more profound than those on TNF-α. Differences in the compound activity in young vs. old mice were mainly restricted to 01K. Immunosuppressive isoxazole leflunomide and a stimulatory RM-11 (1,7-dimethyl-8-oxo-1,2H-isoxazole[5,4-e] triazepine) were applied as reference drugs. This article is protected by copyright. All rights reserved.

The synthesis of a series of novel 7-aminooxazolo[5,4-d]pyrimidines 5, transformations during their synthesis and their physicochemical characteristics have been described. Complete detailed spectral analysis of the intermediates 2&ndash, 4, the N&prime, cyanooxazolylacetamidine by-products 7 and final compounds 5 has been carried out using MS, IR, 1D and 2D NMR spectroscopy. Theoretical research was carried out to explain the privileged formation of 7-aminooxazolo[5,4-d]pyrimidines in relation to the possibility of their isomer formation and the related thermodynamic aspects. Additionally, the single-crystal X-ray diffraction analysis for 5h was reported. Ten 7-aminooxazolo[5,4-d]pyrimidines 5 (SCM1&ndash, 10) were biologically tested in vitro to preliminarily evaluate their immunological, antiviral and anticancer activity. Compounds SCM5 and SCM9 showed the best immunoregulatory profile. The compounds displayed low-toxicity and strongly inhibited phytohemagglutinin A-induced proliferation of human peripheral blood lymphocytes and lipopolysaccharide-induced proliferation of mouse splenocytes. Compound SCM9 caused also a moderate suppression of tumor necrosis factor &alpha, (TNF-&alpha, ) production in a human whole blood culture. Of note, the compounds also inhibited the growth of selected tumor cell lines and inhibited replication of human herpes virus type-1 (HHV-1) virus in A-549 cell line. Molecular investigations showed that the compounds exerted differential changes in expression of signaling proteins in Jurkat and WEHI-231 cell lines. The activity of SCM5 is likely associated with elicitation of cell signaling pathways leading to cell apoptosis. The compounds may be of interest in terms of therapeutic utility as inhibitors of autoimmune disorders, virus replication and antitumor agents.

Reconstitution of the immune function in chemotherapy patients will lead to decreases in post-operative complications. A preliminary investigation showed that an isoxazole derivative R-13 (3,5-dimethyl-isoxazole[5,4-e]8H-triazepin-4-one) hydrochloride, given in a single oral dose to normal mice, induced significant increases in the content of CD4(+) cells in the spleens and lymph nodes. That observation prompted the authors to assess the immune reconstituting effects of R-13 in mice pre-treated with cyclophosphamide (CP). Mice were given intraperitoneally (IP) a sublethal dose of CP (200 mg/kg) and then R-13 (as 20 µg IP doses, every 3 days post-CP treatment). Control mice, not treated with CP, received R-13 or the vehicle (DMSO in appropriate dilution). Blood leukocyte and splenocyte numbers, blood cell type levels, splenocyte spontaneous and ConA-induced proliferation, and delayed-type hypersensitivity (DTH) to ovalbumin (OVA) were investigated on day 15 post-CP treatment and five R-13 doses. The humoral immune response (antibody-forming cell development to sheep erythrocytes) was measured 30 days post-CP treatment and 10 R-13 doses. In CP-treated mice, five dosings with R-13 led to increases in numbers of splenocytes and blood leukocytes, as well as in spontaneous and ConA-induced splenocyte proliferation, relative to levels in mice that received only CP 15 days earlier. Blood analysis revealed decreases in neutrophil and eosinophil contents and an increased appearance of lymphocyte immature forms in all mice that received the R-13. Both cell-mediated responses to OVA and humoral immune response to sheep erythrocytes in CP-treated hosts were restored. Based on the data here, it is concluded that R-13 may be of potential value for reconstitution of the immune function of chemotherapy patients.

Currently, the basic method of treatment of colon cancer is surgery. The range of anticancer drugs used in the treatment of colorectal cancer is small and is based mainly on systemic combination chemotherapy. As a result of the designed syntheses, we received new isothiazole derivatives with anticancer activity. The synthesized 5-hydrazino-3-methylisothiazole-4-carboxylic acid has never been obtained before. It is also a substrate for the synthesis of its innovative derivatives, i.e. compounds that are Schiff bases. The identification of the structure of new compounds was carried out using mass spectrometry (MS), proton nuclear magnetic resonance spectroscopy ( 1 H NMR), carbon nuclear magnetic resonance spectroscopy ( 13 C NMR) and infrared spectroscopy (IR). Potential antitumor activity was confirmed in antiproliferative MTT and SRB tests. The selected, most biologically active substances were characterized by high selectivity towards leukemia and colon cancer cell lines. They caused high inhibition of proliferation of human biphenotypic B cell myelomonocytic leukemia MV4-11 (13 compounds), human colon adenocarcinoma cell lines sensitive LoVo (8 compounds) and resistant to doxorubicin LoVo/DX (12 compounds). However, in the conducted studies, their activity against breast adenocarcinoma MCF-7 and normal non-tumorigenic epithelial cell line derived from mammary gland MCF-10A was substantially lower. The result of this work is claimed Polish patent application.

Objectives Immunoregulatory properties of 06K derivative (4-(4-chlorophenyl)-1-(5-amino-3-methylisoxazole-4-carbonyl)-thiosemicarbazide) in mouse in vivo models were investigated. Methods Several in vivo models were used: humoral and cellular immune response, carrageenan inflammatory reaction and determination of lymphocyte subsets in non-immunized mice. Key findings The compound administered before or after immunization with sheep erythrocytes (sheep red blood cell (SRBC)) elevated the number of plaque-forming cells (PFC), and this effect was stronger at lower doses. Although total haemagglutinin titres to SRBC decreased upon postimmunization treatment, IgG titre increased. In the model of delayed-type hypersensitivity (DTH) to ovalbumin (OVA), the compound, applied intraperitoneally before an eliciting dose of an antigen but not before immunization, inhibited the magnitude of a cutaneous reaction. Further, 06K significantly diminished carrageenan-induced foot pad inflammation when administered 1 h before carrageenan. The compound, administered intraperitoneally to naïve mice, elicited changes in weight, cell number in lymphoid organs and content of lymphocyte subsets, depending on the dose and number of applications. Phenotypic changes included increased turnover of thymocytes, changes in B-cell distribution in spleens and lymph nodes, increased percentage of CD8+ cells and regulatory CD4+CD25+ Foxp3+ T cells. Conclusions Immunoregulatory properties of 06K involve mobilization of lymphopoiesis and generation of regulatory T cells.

Background A series of new isoxazole derivatives of expected immunosuppressive activities was synthesized. Following in vitro screening in the human cell models, the activity of MZO-2 compound (ethyl N-{4-[(2,4-dimethoxybenzyl)carbamoyl]-3-methylisoxazol-5-yl}acetimidate) in mouse in vivo models was evaluated. Methods In vitro tests included evaluation of: peripheral blood mononuclear cells (PBMC) viability, phytohemagglutinin (PHA)-induced PBMC proliferation and lipopolysaccharide (LPS)-induced tumor necrosis factor α (TNF α) production in whole blood cell cultures. MZO-2 was studied in mice for its effects on: humoral immune response to sheep erythrocytes (SRBC), delayed type hypersensitivity (DTH) to ovalbumin (OVA), contact sensitivity to oxazolone and carrageenan-induced foot pad edema. In addition, the effect of MZO-2 on expression of caspases in Jurkat cells was determined. Results The studied compounds exhibited differential, dose-dependent effects to suppress PHA-induced PBMC proliferation and a weak property to suppress LPS-induced production of TNF α. MZO-2 had no effect on the induction phase of the humoral immune response to SRBC in vitro and in vivo , but moderately suppressed the induction phase of DTH to OVA. Its inhibitory effect on carrageenan-induced paw inflammation was potent. Likewise, MZO-2, applied in ointment, was very effective in reducing ear edema and number of lymphocytes in draining lymph nodes of mice sensitized to oxazolone, comparably to tacrolimus, the reference drug. The expression of caspases 3, 8 and 9 in Jurkat cells was inhibited by the compound. Conclusion MZO-2, applied systemically or locally, may serve as a potential drug for amelioration of inflammatory processes.

Restoration of impaired immune response in immunocompromised patients is a crucial problem. In this study we evaluated the efficacy of isoxazole R-11 in reconstitution of the immune response in immunosuppressed mice.Mice were given a sublethal dose (250 mg/kg b.w.) of cyclophosphamide (CP). The cellular immune response to ovalbumin (OVA) and the humoral immune response to sheep erythrocytes (SRBC) were generated. R-11 was administered at repetitive, intraperitoneal doses (20 μg/mouse) until determination of the immune responses: 7 and 15 doses on alternate days for cellular and humoral immune response, respectively. For phenotypic studies R-11 was given per os, at a single dose of 20 μg/mouse. The ability of R-11 to affect interleukin- 6 (IL-6) production was determined in the whole human blood cell culture.R-11 increased the content of CD19+ cells in the spleens and lymph nodes with a concomitant decrease of CD3+ and CD4+ cells. The compound significantly accelerated restoration of both cellular and humoral immune responses, elevated the numbers of circulating leukocytes and splenocytes and normalized the blood cell picture. Supplementary experiments showed that R-11 was not toxic with regard to human peripheral blood mononuclear cells (PBMC) and that it upregulated IL-6 production in blood cell culture stimulated with lipopolysaccharide (LPS).We demonstrated that R-11 is likely a B-cell tropic agent which can restore both cellular and humoral immune responses in immunocompromised mice and may have a potential to be applied in therapy of immunocompromised patients.

This work describes the synthesis of a new series of isoxazole derivatives, their immunosuppressive properties, and the mechanism of action of a representative compound. A new series of N&prime, substituted derivatives of 5-amino-N,3-dimethyl-1,2-oxazole-4-carbohydrazide (MM1&ndash, MM10) was synthesized in reaction of 5-amino-N,3-dimethyl-1,2-oxazole-4-carbohydrazide with relevant carbonyl compounds. The isoxazole derivatives were tested in several in vitro models using human cells. The compounds inhibited phytohemagglutinin A (PHA)-induced proliferation of peripheral blood mononuclear cells (PBMCs) to various degrees. The toxicity of the compounds with regard to a reference A549 cell line was also differential. 5-amino-N&prime, (2,4-dihydroxyphenyl)methylidene-N,3-dimethyl-1,2-oxazole-4-carbohydrazide (MM3) compound was selected for further investigation because of its lack of toxicity and because it had the strongest antiproliferative activity. The compound was shown to inhibit lipopolysaccharide (LPS)-induced tumor necrosis factor (TNF &alpha, ) production in human whole blood cell cultures. In the model of Jurkat cells, MM3 elicited strong increases in the expression of caspases, Fas, and NF-&kappa, B1, indicating that a proapoptotic action may account for its immunosuppressive action in the studied models.

5-Amino-3-methyl-4-isoxazolecarboxylic acid hydrazide is a non-cytotoxic synthetic isoxazole derivative with considerable immunomodulatory properties demonstrated in in vitro experiments. The aim of this study was to investigate the influence of this compound, depending on the dosage and schedule of treatment, on lymphocyte subsets in non-immunized mice and humoral immune response in SRBC (sheep red blood cells)-immunized mice. An analysis of lymphocyte subsets was carried out by flow cytometry, using specific monoclonal antibodies stained with fluorescein isothiocyanate (FITC) or phycoerythrin (PE). In the SRBC-immunized mice, the influence of the compound on the humoral response was determined, depending on the time of administration relative to the antigen. The number of plaque forming cells (PFC) was determined by a local hemolysis technique in an agar gel. Total and 2-mercaptoethanol resistant serum agglutination titers were defined by active hemagglutination test carried out on microplates. The investigated hydrazide was able to modulate the percentage and absolute number of T lymphocyte subsets in the thymus, and T and B lymphocytes in the peripheral lymphatic organs. It also enhanced humoral immune response in SRBC-immunized mice by increasing the number of cells producing hemolytic anti-SRBC antibodies (PFC) and by augmenting the level of total and 2-mercaptoethanol resistant hemagglutinin. The present study showed modulatory effects of 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide on lymphocyte subsets and humoral immune response in mice. This compound could be potentially useful for the treatment of autoimmune diseases, infections or as an adjuvant for boosting the efficacy of vaccines.

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.

The aim of the study was to characterize a series of isoxazole derivatives in several immunological tests in vitro and in vivo, in mouse and human models. The human model included measurement of: viability of peripheral blood mononuclear cells (PBMC), phytohemagglutinin A (PHA)-induced proliferation of PBMC, production of tumor necrosis factor a (TNF a) in whole blood cultures stimulated with lipopolysaccharide (LPS) and growth of SW-948 and L1210 tumor cell lines. Experiments in mice encompassed the following tests: secondary, humoral immune response splenocytes to sheep erythrocytes (SRBC) in vitiv, delayed type hypersensitivity (DTH) to ovalbumin (OVA) and carrageenan-induced foot edema. All compounds were non-toxic against PMBC and displayed differential, dose-dependent suppressive properties in the model of PHA- induced PMBC proliferation. They also exhibited differential, mostly inhibitory effects on TNF a production. The inhibitory actions on growth of tumor cell lines were moderate. M05 (5-amino-3-methyl-N-(4-methyl-benzyl)-4-isoxazolecarboxamide) was most suppressive in the proliferation and TNF a production tests, it was, therefore, selected for in vitro and in vivo studies in the mouse models. The compound inhibited the humoral immune response in vitro, stimulated the inductive phase of DTH in vivo, although it inhibited the eliciting phase of that response. The compound also inhibited the carrageenan skin reaction. M05 combines strong anti-proliferative and anti-inflammatory activities, it is therefore attractive for further studies in more advanced animal models as a potential therapeutic.