molecules

Review Isoxazole Derivatives as Regulators of Immune Functions

Michał Zimecki 1, Urszula B ˛achor 2 and Marcin M ˛aczy´nski 2,*

1 Laboratory of Immunobiology, Institute of Immunology and Experimental Therapy, Polish Academy of Sciences, Weigla 12, 53-114 Wroclaw, Poland; [email protected] 2 Department of Organic Chemistry, Faculty of Pharmacy, Wroclaw Medical University, Borowska 211a, 50-556 Wroclaw, Poland; [email protected] * Correspondence: [email protected]; Tel.: +48-717840344



Received: 4 October 2018; Accepted: 20 October 2018; Published: 22 October 2018


Abstract: In this review, we present reports on the immunoregulatory properties of isoxazole derivatives classified into several categories, such as immunosuppressive, anti-inflammatory, immunoregulatory, and immunostimulatory compounds. The compounds were tested in various models using resident cells from rodents and humans, cell lines, and experimental animal disease models corresponding to human clinical situations. Beneficial features of the described isoxazole derivatives include low toxicity and good bioactivity at low doses. In a majority of studies, the activities of investigated compounds were comparable or even higher than registered reference drugs. Whenever possible, a plausible mechanism of action of the investigated compounds and their potential therapeutic utility were proposed. Among the described compounds, particular attention was paid to the class of immune stimulators with a potential application in chemotherapy patients.

Keywords: isoxazoles; anti-inflammatory; immune suppression; immune stimulation

1. Introduction Compounds containing the isoxazole ring [1] serve as an important source of valuable drugs that are designed to treat infections and diseases of different etiologies. Syntheses, classification, mechanisms of action, and the therapeutic application of registered isoxazole derivatives, as well as these under preclinical investigations, were recently in detail described in several reviews [2–5]. Therefore, we wished to focus in this short review only on data regarding the immunoregulatory properties of compounds containing the isoxazole ring. Although a majority of isoxazole derivatives under interest display immunosuppressive activities, including anti-inflammatory ones, less is known about immunostimulatory compounds of potential therapeutic utility. Such a group of compounds, besides immunosuppressive molecules, was also synthesized, and their potential therapeutic usefulness evaluated by our research team. In this review, the results regarding immunoregulatory actions of selected isoxazole derivatives in various immunological settings in vitro and in vivo human and rodent models are described (Table1). In some investigations, the description of the immunoregulatory properties is accompanied with the suggested mechanism of action, and potential therapeutic applications are proposed.

Molecules 2018, 23, 2724; doi:10.3390/molecules23102724 www.mdpi.com/journal/molecules Molecules 2018, 23, 2724 2 of 17 Molecules 2018, 23, x 2 of 17 Molecules 2018, 23, x 2 of 17 Molecules 2018, 23, x 2 of 17 Molecules 2018, 23, x 2 of 17 Molecules 2018, 23, x 2 of 17 TableTable 1. Isoxazole 1. Isoxazole derivatives derivatives with potential with application potential in therapy. application in therapy. Table 1. Isoxazole derivatives with potential application in therapy. Table 1. Isoxazole derivatives with potential application in therapy. Compound Structure ActivityMolecular/Cellular Molecular/Cellular Mechanism of MechanismReference of Action Reference Number Compound Table Struc1. Isoxtureazole derivatives with potential applicationActivity in therapy. Table 1. Isoxazole derivatives with potential application in therapy. Molecular/CellularAction Mechanism of ReferenceNumber Compound Structure ImmunosuppressiveActivity actions Molecular/Cellular Mechanism of Reference Compound Structure Immunosuppressive actionsActi vity Molecular/CellularAction Mechanism of ReferenceNumber Compound Structure Activity Molecular/CellularAction Mechanism of ReferenceNumber Compound Structure3CH Immunosuppressive actionsActivi ty Action Number CH ImmunosuppressiveO actions Action Number 3CH Immunosuppressive actions 3 O N HWA-486/Leflunomide 5-methyl-N-[4- NH 3CH OImmunosuppressiveImmunosuppressive actions and anti- COX-2 inhibitor, non-cytotoxic HWA-486/Leflunomide 5-methyl-N-[4- O N Immunosuppressive and anti-inflammatory COX-2 inhibitor, non-cytotoxic inhibitor of HWA-486/Leflunomide 5-methyl-N-[4- NH 3CH Immunosuppressive and anti- COX-2 inhibitor, non-cytotoxic (trifluoromethyl)phenyl]-1,2-oxazole-4-HWA-486/Leflunomide 5-methyl-N-[4- O N Immunosuppressiveinflammatory regulation and anti- of COX-2inhibitor inhibitor, of dihydroorotate non-cytotoxic [6] [6] (trifluoromethyl)phenyl]-1,2-oxazole-4-carboxamide F NH N regulation of autoimmune lymphocytes dihydroorotate dehydrogenase (trifluoromethyl)phenyl]-1,2-oxazole-4-HWA-486/Leflunomide 5-methyl-N-[4- O Immunosuppressiveinflammatory regulation and anti- of COX-2inhibitor inhibitor, of dihydroorotate non-cytotoxic [6] (trifluoromethyl)phenyl]-1,2-oxazole-4-carboxamide F NH N inflammatoryautoimmune lymphocytesregulation of inhibitordehydrogenase of dihydroorotate [6] HWA-486/(trifluoromethyl)phenyl]-1,2-oxazole-4-Leflunomidecarboxamide 5-methyl- N-[4- F NH O Immunosuppressiveinflammatoryautoimmune lymphocytesregulation and anti- of COX-2inhibitor inhibitor,dehydrogenase of dihydroorotate non-cytotoxic [6] carboxamide F O autoimmune lymphocytes dehydrogenase (trifluoromethyl)phenyl]-1,2-oxazole-4- F F inflammatory regulation of inhibitor of dihydroorotate [6] carboxamide FF O autoimmune lymphocytes dehydrogenase F F 3CH carboxamide F O autoimmune lymphocytes dehydrogenase F 3CH F O CH O 3FCH O 3 F O 3CH O 3CH O ON CH O CH Parecoxib 3 ONHOS 3 ON Anti-inflammatory registered drug COX-2 inhibitor [7] 3CH OS N ParecoxibParecoxib ONH O O Anti-inflammatoryAnti-inflammatory registered registereddrug drugCOX-2 inhibitor COX-2 inhibitor[7] [7] Parecoxib 3CH NHOS N Anti-inflammatory registered drug COX-2 inhibitor [7] NH SO N Parecoxib SO Anti-inflammatory registered drug COX-2 inhibitor [7] Parecoxib NH O Anti-inflammatory registered drug COX-2 inhibitor [7] O

Inhibits the proliferative response of 8g/5-(4-amino-5-benzoyl-1,2-oxazol-3-yl)- O O Inhibits the proliferative response of 8g/5-(4-amino-5-benzoyl-1,2-oxazol-3-yl)- O N mouseInhibits splenocytes the proliferative to concanavalin response of A, N-[(pyridin-4-yl)methyl]-1,3,4-oxadiazole- O N N.D. [8] 8g/5-(4-amino-5-benzoyl-1,2-oxazol-3-yl)- O N 8g/5-(4-amino-5-benzoyl-1,2-oxazol-3-yl)-N- O mouseInhibitssuppressesInhibits splenocytes the proliferative thethe humoral proliferative to concanavalin response immune response of A, of mouse N8g/-[(pyridin-4-yl)methyl]-1,3,4-oxadiazole-5-(4-amino-5-benzoyl-1,2-oxazol-3-yl)- 2NH O N N mouse splenocytes to concanavalin A, N.D. [8] N2-carboxamide-[(pyridin-4-yl)methyl]-1,3,4-oxadiazole- O O N Inhibitssuppresses the proliferative the humoral response immune of N.D. [8] [(pyridin-4-yl)methyl]-1,3,4-oxadiazole- 2NH O N mouse splenocytessplenocytes to concanavalin to concanavalin A, A, N.D. [8] 8g/5-(4-amino-5-benzoyl-1,2-oxazol-3-yl)- N response 2-carboxamideN-[(pyridin-4-yl)methyl]-1,3,4-oxadiazole- O 2NH O N suppresses the humoral immune N.D. [8] 2-carboxamide N NH mouse splenocytesresponse to concanavalin A, 2-carboxamide NH N N O N suppressessuppresses the humoral the humoral immune immune response N-[(pyridin-4-yl)methyl]-1,3,4-oxadiazole- 2 NH response N.D. [8] 2-carboxamide N N O suppresses the humoral immune 2NH O NH response 2-carboxamide N N O O NH N N response O NH N O O O 10f/5-(5-amino-7-phenyl[1,2]oxazolo[4,5- N Stimulates mitogen-induced O N 10f/5-(5-amino-7-phenyl[1,2]oxazolo[4,5- N Stimulates mitogen-induced 10f/d]pyrimidin-3-yl)-5-(5-amino-7-phenyl[1,2]oxazolo[4,5-N-[(pyridin-3- O N proliferationStimulates of mitogen-induced mouse splenocytes, N.D. [8] NH N N 10f 10f/d]pyrimidin-3-yl)-5-(5-amino-7-phenyl[1,2]oxazolo[4,5-N-[(pyridin-3- 2 O N N proliferationStimulates of mitogen-induced mouse splenocytes, N.D. [8] /5-(5-amino-7-phenyl[1,2]oxazolo[4,5-d]d]pyrimidin-3-yl)-yl)methyl]-1,3,4-oxadiazole-2-carboxamN-[(pyridin-3- ide NH N N O proliferationsuppresses of mouse DTH splenocytes, N.D. [8] 10f/5-(5-amino-7-phenyl[1,2]oxazolo[4,5- 2 O N N StimulatesStimulates mitogen-induced mitogen-induced proliferation of d]pyrimidin-3-yl)-yl)methyl]-1,3,4-oxadiazole-2-carboxamN-[(pyridin-3- ide 2NH N N N proliferationsuppresses of mouse DTH splenocytes, N.D. [8] pyrimidin-3-yl)-yl)methyl]-1,3,4-oxadiazole-2-carboxamN-[(pyridin-3 ide N O NH N suppresses DTH N.D. [8] d]pyrimidin-3-yl)-N-[(pyridin-3- 2NH N N N O proliferationmouse of splenocytes,mouse splenocytes, suppresses DTH N.D. [8] NH N -yl)methyl]-1,3,4-oxadiazole-2-carboxamideyl)methyl]-1,3,4-oxadiazole-2-carboxamide NH N N O suppresses DTH 2 N O NH N yl)methyl]-1,3,4-oxadiazole-2-carboxamide N N O suppresses DTH OC HNH N N O2 5 O NH N OC2H5 OCO2H5 4d/5-(2- OCO2H5 O Inhibits the DTH and humoral hydroxyethyl)piperazinomethinimino-3-4d/5-(2- OC2H5 4d/5-(2- CH O immuneInhibits response the DTH to SRBC and humoral in vitro and N.D. [9] hydroxyethyl)piperazinomethinimino-3-methyl-4-isoxazolecarboxylic 4d/5-(2- acid 4-(4- 3 O NH Inhibits the DTH and humoral hydroxyethyl)piperazinomethinimino-3- 3CH O NH immuneInhibits response the DTH to SRBC and humoral in vitro and N.D. [9] methyl-4-isoxazolecarboxylic4d/5-(2- acid 4-(4- CH immune responsein tovivo SRBC in vitro and N.D. [9] hydroxyethyl)piperazinomethinimino-3-ethoxyphenyl)-amide 3 O NH Inhibits the DTH and humoral 4d/5-(2-hydroxyethyl)piperazinomethinimino-3methyl-4-isoxazolecarboxylic acid 4-(4- CH N N OH immune responsein tovivo SRBC in vitro and N.D. [9] hydroxyethyl)piperazinomethinimino-3-ethoxyphenyl)-amide 3 NNH Inhibitsin the vivo DTH and humoral immune methyl-4-isoxazolecarboxylic acid 4-(4- O OH -methyl-4-isoxazolecarboxylicethoxyphenyl)-amide acid 4- 3CH N NH N NH immune response to SRBC in vitro and N.D. N.D.[9] [9] methyl-4-isoxazolecarboxylic acid 4-(4- N N N OH in vivo ethoxyphenyl)-amide O N response to SRBC in vitro and in vivo (4-ethoxyphenyl)-amide N O N NH OH in vivo ethoxyphenyl)-amide N NH N O N OH N NH O Molecules 2018, 23, x NH 3 of 17

CH O 3 * Stimulation of caspase* Stimulation 9 expression of caspase 9 expression in N RM-33/3,5,7-trimethyl-5,6,7,8-tetrahydro- 3CH in thymocytes andthymocytes splenocytes andand splenocytes and Fas in RM-33/3,5,7-trimethyl-5,6,7,8-tetrahydro- NH InhibitsInhibits LPS-induced LPS-induced TNF-α TNF- and IL-6α and IL-6 activity 4H-[1,2]oxazolo[5,4- Fas in bone marrowbone cells marrow and cells and[10] splenocytes, [10] 4H-[1,2]oxazolo[5,4-e][1,2,4]triazepin-4-one activity inhibitsinhibits antibody antibody production production e][1,2,4]triazepin-4-one N splenocytes, inhibitioninhibition of ERK1 of and ERK1 and p38g in bone N O CH3 H p38g in bone marrow cells marrow cells OH 1020/ 5-{[(4- O 3CH Displays strong immunosuppressive hydroxyphenyl)methylidene]amino}-3- N action, inhibits polyclonal antibody N.D. [11] methyl[1,2]oxazolo[5,4-d]pyrimidin-4(5H)- N N production one O N

1025/5-(cyclohexylideneamino)-3- O Shows immunosuppressive property, 3CH methyl[1,2]oxazolo[5,4-d]pyrimidin-4(5H)- N lowers polyclonal antibody N.D. [11] one N production N O N O O NH M5/2-[(5-amino-3-methylisoxazol-4- CH 3 NH NH yl)carbonyl]-N-(4- Cl Inhibits antibody production in mice N.D. [12] chlorophenyl)hydrazinecarboxamide N NH O 2 N NH N 2 Inhibits the humoral immune RM56/5-(5-amino-3-methyl-1,2-oxazol-4- 3CH O response, the carrageenan reaction and N.D. [13] yl)-1,3,4-oxadiazol-2-amine proliferation of lymphocytes N NH O 2 O MO5/5-amino-3-methyl-N-[(4- CH 3 NH Inhibits the humoral immune response methylphenyl)methyl]-1,2-oxazole-4- Inhibitor of TNFα production [14] in vitro carboxamide N CH3 O NH2 O NH 2 Modulates the content of T cell subsets 3CH Upregulation of fractalkine 5-amino-3-methyl-1,2-oxazole-4- NH and B cells in lymphoid organs, and (CX3CL1) and IL-17F, and [15] carbohydrazide elevates the humoral immune downregulation of IL-10 and TLR4 N response in mice O NH2 OH Inhibits the mitogen-induced MM3/5-amino-N′-[2-(2,4- O N OH proliferation of PBMC and LPS- Strong increases in the expression of dihydroxyphenyl)ethylidene]-N,3- [16] 3CH N induced TNF α production in human caspases, Fas, and NF-kB1 dimethyl-1,2-oxazole-4-carbohydrazide CH3 blood cell culture N NH O 2 MoleculesMolecules2018, 232018, 2724, 23, x 3 of 17 3 of 17 Molecules 2018, 23, x 3 of 17 Molecules 2018, 23, x 3 of 17 Molecules 2018, 23, x O CH3 3 of 17 Molecules 2018, 23, x CH * Stimulation of caspase 9 expression 3 of 17 O N 3 * Stimulation of caspase 9 expression Molecules 2018, 23, x 3CH O CH3 in thymocytes and splenocytes and 3 of 17 MoleculesRM-33/ 20183,, 5,7-trimethyl-5,6,7,8-tetrahydro-23, x CH NCHNH InhibitsTable LPS-induced 1. Cont. TNF-α and IL-6 * Stimulation of caspase 9 expression 3 of 17 3 O N 3 *in Stimulation thymocytesFas in bone of and marrowcaspase splenocytes cells9 expression and and [10] RM-33/3,5,7-trimethyl-5,6,7,8-tetrahydro- 3CH CHNH Inhibits LPS-induced TNF-α and IL-6 in thymocytes and splenocytes and 4H-[1,2]oxazolo[5,4-e][1,2,4]triazepin-4-one O N 3 activity inhibits antibody production * Stimulation of caspase 9 expression RM-33/3,5,7-trimethyl-5,6,7,8-tetrahydro- 3CH N O CHNH3 Inhibits LPS-induced TNF-α and IL-6 splenocytes,in thymocytesFas in bone inhibition andmarrow splenocytes ofcells ERK1 and andand [10] 4H-[1,2]oxazolo[5,4-e][1,2,4]triazepin-4-one NN activity inhibits antibody production * StimulationFas in bone of marrowcaspase cells9 expression and [10] RM-33/Compound3,5,7-trimethyl-5,6,7,8-tetrahydro- 3CH N StructureO O CHNH3 CH3 Inhibits LPS-induced TNF- Activityα and IL-6 splenocytes,in thymocytes inhibition and Molecular/Cellular splenocytes of ERK1 andand Mechanism of Action Reference Number 4H-[1,2]oxazolo[5,4-e][1,2,4]triazepin-4-one CH HN activity inhibits antibody production * StimulationFasp38g in bonein boneof marrowcaspase marrow cells9 expression cells and [10] RM-33/3,5,7-trimethyl-5,6,7,8-tetrahydro- 3 N N NHCH3 Inhibits LPS-induced TNF-α and IL-6 splenocytes,in thymocytes inhibition and splenocytes of ERK1 andand 4H-[1,2]oxazolo[5,4-e][1,2,4]triazepin-4-one CH O NN activity inhibits antibody production Fasp38g in bonein bone marrow marrow cells cells and [10] RM-33/3,5,7-trimethyl-5,6,7,8-tetrahydro- 3 N O H NHCH3 OH Inhibits LPS-induced TNF-α and IL-6 splenocytes,in thymocytes inhibition and splenocytes of ERK1 andand 41020/RM-33/H-[1,2]o5-{[(4-3,5,7-trimethyl-5,6,7,8-tetrahydro-xazolo[5,4-e][1,2,4]triazepin-4-one O NH NHCH Inhibitsactivity LPS-inducedinhibits antibody TNF- productionα and IL-6 Fasp38g in bonein bone marrow marrow cells cells and [10] 1020/5-{[(4-hydroxyphenyl)methylidene]amino}-4H-[1,2]oxazolo[5,4-e][1,2,4]triazepin-4-one 3CH N O 3 OH activityDisplays inhibits strong antibodyimmunosuppressive production splenocytes,Fasp38g in bonein boneinhibition marrow marrow of cells ERK1 cells and and [10] 1020/5-{[(4- NO NH CH3 OH splenocytes, inhibition of ERK1 and 4hydrH-[1,2]oxaoxyphenyl)methylidene]amino}-3-zolo[5,4-e][1,2,4]triazepin-4-one 3CH O N activityDisplays inhibits strong antibody immunosuppressive production action,p38g in bone marrow cells 1020/5-{[(4- NO HN CH3 OH Displaysaction, inhibits strong polyclonalimmunosuppressive antibody splenocytes, inhibitionN.D. of ERK1 and [11] 3-methyl[1,2]oxazolo[5,4-hydroxyphenyl)methylidene]amino}-3- 3CH ON N Displays strong immunosuppressive p38g in bone marrow cells N.D. [11] 1020/methyl[1,2]oxazolo[5,4-5-{[(4- d]pyrimidin-4(5H)- N O NH CH3 OH inhibits polyclonal antibody production hydroxyphenyl)methylidene]amino}-3- 3CH ON action, inhibits polyclonal antibody N.D. [11] emphd]pyrimidin-4(51020/5 H)-one O N H Displays strongproduction immunosuppressive p38g in bone marrow cells methyl[1,2]oxazolo[5,4-one -{[(4- d]pyrimidin-4(5H)- NCH O N OH action, inhibits polyclonal antibody N.D. [11] hydroxyphenyl)methylidene]amino}-3- 3 ON N Displays strongproduction immunosuppressive 1020/methyl[1,2]oxazolo[5,4-5-{[(4- d]pyrimidin-4(5H)- NCH N OH action, inhibits polyclonal antibody N.D. [11] 1020/hydroxone 5-{[(4-yphenyl)methylidene]amino}-3- 3 O ON N Displays strongproduction immunosuppressive methyl[1,2]oxazolo[5,4-d]pyrimidin-4(5H)- NCH N action, inhibits polyclonal antibody N.D. [11] hydroxypheone nyl)methylidene]amino}-3- 3 O N N Displays strongproduction immunosuppressive methyl[1,2]oxazolo[5,4-hydroxyphenyl)methylidene]amino}-3-d]pyrimidin-4(5H)- N N action, inhibits polyclonal antibody N.D. [11] methyl[1,2]oxazolo[5,4-one d]pyrimidin-4(5H)- O N N production one N O N action, inhibits polyclonal antibody N.D. [11] methyl[1,2]oxazolo[5,4-1025/5-(cyclohexylideneamino)-3-d]pyrimidin-4(5H)- N N O Shows immunosuppressiveproduction property, one O 3CH N production one1025/methyl[1,2]oxazolo[5,4- 5-(cyclohexylideneamino)-3-d]pyrimidin-4(5H)- O O Showslowers immunosuppressive polyclonal antibody property, N.D. [11] 1025/5-(cyclohexylideneamino)-3-methyl[1,2]oxazolo1025/5-(cyclohexylideneamino)-3- 3CH N O N ShowsShows immunosuppressive immunosuppressive property, property, lowers methyl[1,2]oxazolo[5,4-d]pyrimidin-4(5H)- CH lowers polyclonal antibody N.D. N.D.[11] [11] 1025/one 5-(cyclohexylideneamino)-3- 3 O N N Shows immunosuppressiveproduction property, [5,4-methyl[1,2]oxazolo[5,4-d]pyrimidin-4(5H)-oned]pyrimidin-4(5H)- NCH lowerspolyclonal polyclonal antibody production N.D. [11] 1025/one 5-(cyclohexylideneamino)-3- 3 O N N Shows immunosuppressiveproduction property, methyl[1,2]oxazolo[5,4-d]pyrimidin-4(5H)- N3CH N N lowers polyclonal antibody N.D. [11] 1025/one 5-(cyclohexylideneamino)-3- O ON Shows immunosuppressiveproduction property, methyl[1,2]oxazolo[5,4-d]pyrimidin-4(5H)- 3NCH N lowers polyclonal antibody N.D. [11] 1025/one 5-(cyclohexylideneamino)-3- O O N Shows immunosuppressiveproduction property, methyl[1,2]oxazolo[5,4-d]pyrimidin-4(5H)- N3CH N ON N lowers polyclonal antibody N.D. [11] methyl[1,2]oxazolo[5,4-one d]pyrimidin-4(5H)- O O N lowers polyclonalproduction antibody N.D. [11] one N O NH ON N production oneM5/2-[(5-amino-3-methylisoxazol-4- ON N ON production 3CH O NHNH M5/2-[(5-amino-3-methylisoxazol-4- ON NHN NHO yl)carbonyl]-N-(4- CH O NH N Cl Inhibits antibody production in mice N.D. [12] M5 /2-[(5-amino-3-methylisoxazol-4- 3 O NH NHO yl)carbonyl]-N-(4- 3CH O NH N Inhibits antibody production in mice N.D. [12] M5/2-[(5-amino-3-methylisoxazol-4-yl)carbonyl]-M5chlorophenyl)hydrazinecarboxamide/2-[(5-amino-3-methylisoxazol-4- O NH NHO Cl yl)carbonyl]-N-(4- 3CH N O NH O Cl Inhibits Inhibitsantibody antibody production production in mice in miceN.D. N.D.[12] [12] M5chlorophenyl)hydrazinecarboxamide/2-[(5-amino-3-methylisoxazol-4- NH2NH NH N-(4-chlorophenyl)hydrazinecarboxamideyl)carbonyl]-N-(4- 3CH N O O NH Cl Inhibits antibody production in mice N.D. [12] M5chlorophenyl)hydrazinecarboxamide/2-[(5-amino-3-methylisoxazol-4- CH NH NH NH M5yl)carbonyl]-/2-[(5-amino-3-methylisoxazol-4-N-(4- 3 N O NH2 NNH Cl Inhibits antibody production in mice N.D. [12] chlorophenyl)hydrazinecarboxamide CH NH2 NH2 yl)carbonyl]-N-(4- 3 N O NH N NNH Cl Inhibits antibody production in mice N.D. [12] chyl)carbonyl]-lorophenyl)hydrazinecarboxamideN-(4- NH2 NH Inhibits antibody production in mice N.D. [12] chlorophenyl)hydrazinecarboxamide N O N N 2 Cl Inhibits the humoral immune N CH NH2 O NH2 chRM56loroph/5-(5-amino-3-methyl-1,2-oxazol-4-enyl)hydrazinecarboxamide O 3 NH N N 2 NH2 response,Inhibits the the carrageenan humoral immune reaction and N.D. [13] RM56/5-(5-amino-3-methyl-1,2-oxazol-4- N O 3CH N N O yl)-1,3,4-oxadiazol-2-amine NH2 NH Inhibits the humoral immune RM56/5-(5-amino-3-methyl-1,2-oxazol-4- O 3CH N O 2 response, the carrageenan reaction and N.D. [13] yl)-1,3,4-oxadiazol-2-amine N NH InhibitsproliferationInhibits the the humoral of humoral lymphocytes immune immune response, the RM56/5-(5-amino-3-methyl-1,2-oxazol-4-yl)-1,3,4-RM56/5-(5-amino-3-methyl-1,2-oxazol-4- 3CH N N N O 2 response, the carrageenan reaction and N.D. [13] yl)-1,3,4-oxadiazol-2-amine NH2 NH2 Inhibitsproliferation the humoral of lymphocytes immune RM56/5-(5-amino-3-methyl-1,2-oxazol-4- 3CH N O N O response,carrageenan the carrageenan reaction reaction and and proliferation of N.D. N.D.[13] [13] oxadiazol-2-amineyl)-1,3,4-oxadiazol-2-amine NH Inhibitsproliferation the humoral of lymphocytes immune RM56/5-(5-amino-3-methyl-1,2-oxazol-4- 3CH ON O O2 response,Inhibitsproliferation the the carrageenan humoral of lymphocytes immune reaction and N.D. [13] yl)-1,3,4-oxadiazol-2-amine CH NHO2 response, the carrageenanlymphocytes reaction and N.D. [13] RM56/5-(5-amino-3-methyl-1,2-oxazol-4- 3 ON O proliferation of lymphocytes yl)-1,3,4-oxadiazol-2-amine NH2 response, the carrageenan reaction and N.D. [13] MO5/5-amino-3-methyl-N-[(4- 3CH ON O NH proliferation of lymphocytes yl)-1,3,4-oxadiazol-2-amine NH2 Inhibits the humoral immune response MO5/5-amino-3-methyl-N-[(4- 3CH ON O NH proliferation of lymphocytes methylphenyl)methyl]-1,2-oxazole-4- NH2 Inhibitor of TNFα production [14] MO5/5-amino-3-methyl-N-[(4- 3CH ON O NH Inhibits the humoralin vitro immune response methylphenyl)methyl]-1,2-oxazole-4- NH2 CH Inhibits the humoral immune response Inhibitor of TNFα production [14] MO5/5-amino-3-methyl-carboxamide N-[(4- 3CH N O O NH 3 methylphenyl)methyl]-1,2-oxazole-4- NH CH Inhibits the humoralin vitro immune response Inhibitor of TNFα production [14] methylphenyl)methyl]-1,2-oxazole-4-MO5/5-amino-3-methyl-carboxamide N-[(4- 3CH N O O NH2 3 in vitro Inhibitor of TNFα production [14] MO5/5-amino-3-methyl-MO5/5-amino-3-methyl-carboxamideN-[(4-methylphenyl) N-[(4- CH NH CH3 Inhibits Inhibitsthe humoral the immune humoral response immune response methylphenyl)methyl]-1,2-oxazole-4- 3 N O NH2O in vitro Inhibitor of TNFα productionInhibitor of TNFα production [14] [14] MO5/5-amino-3-methyl-carboxamide N-[(4- 3CH NH2 NH CH3 Inhibits the humoral immune response methyl]-1,2-oxazole-4-carboxamidemethylphenyl)methyl]-1,2-oxazole-4- N O NHO 2 Modulates the contentin vitro of inT cell vitro subsets Inhibitor of TNFα production [14] carboxamide N CH NH2 NH CH3 Inhibits the humoral immune response methylphenyl)methyl]-1,2-oxazole-4- O 3 O NH 2 CH in vitro InhibitorUpregulation of TNF ofα fractalkineproduction [14] 5-amino-3-methyl-1,2-oxazole-4-carboxamide N NH2 NH 3 Modulatesand B cells the in contentlymphoid of Torgans, cell subsets and O 3CH O NH 2 CH Modulates the contentin vitro of T cell subsets Upregulation of fractalkine carboxamide N O CH NH2 NH 3 (CX3CL1) and IL-17F, and [15] 5-amino-3-methyl-1,2-oxazole-4-carbohydrazide 3 NH O NH 2 Modulatesandelevates B cells the inthe contentlymphoid humoral of Torgans,immune cell subsets and Upregulation of fractalkine 5-amino-3-methyl-1,2-oxazole-4- O CH 2 NH and B cells in lymphoid organs, and (CX3CL1) and IL-17F, and [15] carbohydrazide 3 N O NH 2 Modulateselevates the the content humoral of Timmune cell subsets downregulationUpregulation(CX3CL1) and of of IL-17F,IL-10 fractalkine and and TLR4 [15] 5-amino-3-methyl-1,2-oxazole-4- CH O NH NH2 and B cellsresponse in lymphoid in mice organs, and Upregulation of fractalkine carbohydrazide 3 N O NH2NH Modulateselevates the the content humoral of Timmune cell subsets downregulation(CX3CL1) and of IL-17F,IL-10 and and TLR4 [15] 5-amino-3-methyl-1,2-oxazole-4- 3CH N NHNH 2 ModulatesandModulates B cells responsethe in contentlymphoid the in content ofmice Torgans, cell of subsets Tand cell subsetsdownregulationUpregulation and B Upregulation of of IL-10 fractalkine and TLR4 of fractalkine (CX3CL1) and 5-amino-3-methyl-1,2-oxazole-4-carbohydrazide CH O OH2 andelevates B cellsresponse inthe lymphoid humoral in mice organs,immune and (CX3CL1) and IL-17F, and [15] 3 N NHNH2 downregulationUpregulation of of IL-10 fractalkine and TLR4 5-amino-3-methyl-1,2-oxazole-4-carbohydrazide5-amino-3-methyl-1,2-oxazole-4-carbohydrazide O OH andelevates Bcells cellsresponse in inthe lymphoid lymphoid humoral in mice organs,immune organs, and and elevates(CX3CL1) the andIL-17F, IL-17F, and and downregulation [15] of IL-10 and [15] carbohydrazide NH2 elevates the humoral immune downregulation of IL-10 and TLR4 N O OH Inhibitsresponse the mitogen-induced in mice (CX3CL1) and IL-17F, and [15] carbohydrazide N NH2 elevateshumoral the humoral immune immune response indownregulation mice of IL-10 and TLR4 TLR4 MM3/5-amino-N′-[2-(2,4- O O OH OH Inhibitsresponse the mitogen-induced in mice N ON NH2 proliferation of PBMC and LPS- Strongdownregulation increases inof theIL-10 expression and TLR4 of dihydroxyphenyl)ethylidene]-MM3/5-amino-N′-[2-(2,4- N,3- O NHOH OH Inhibitsresponse the mitogen-induced in mice [16] MM3/5-amino-N′-[2-(2,4- 3CH NON OH 2 inducedproliferation TNF α ofproduction PBMC and in LPS-human Strongcaspases, increases Fas, in andthe expression NF-kB1 of dihydroxyphenyl)ethylidene]-N,3- O N OH Inhibits the mitogen-induced [16] dimethyl-1,2-oxazole-4-carbohydrazideMM3/5-amino-N′-[2-(2,4- 3CH N OH proliferation of PBMC and LPS- Strong increases in the expression of dihydroxyphenyl)ethylidene]-N,3- O CH3 OH inducedInhibits TNF the α productionmitogen-induced in human caspases, Fas, and NF-kB1 [16] 3CH N N proliferationblood ofcell PBMC culture and LPS- Strong increases in the expression of dimethyl-1,2-oxazole-4-carbohydrazideMM3/5-amino-N′-[2-(2,4- O OH inducedInhibits TNF the α productionmitogen-induced in human caspases, Fas, and NF-kB1 dihydroxyphenyl)ethylidene]-N,3- CH N CHN 3 proliferation of PBMC and LPS- Strong increases in the expression of [16] dimethyl-1,2-oxazole-4-carbohydrazide MM3/5-amino-N′-[2-(2,4- 3 NHN2 OH Inhibitsblood the mitogen-inducedcell culture dihydroxyphenyl)ethylidene]-0 N,3- N O O CHN 3 induced TNF α production in human caspases, Fas, and NF-kB1 [16] MM3/5-amino-dimethyl-1,2-oxazole-4-carbohydrazideN MM3-[2-(2,4-dihydroxyphenyl)/5-amino-N′-[2-(2,4- 3CH NHN proliferationInhibitsblood the ofcell mitogen-induced PBMC culture and LPS- proliferationStrong increases of in the expression of dihydroxyphenyl)ethylidene]-N,3- N O O 2CH3 OH induced TNF α production in human caspases, Fas, and NF-kB1 [16] 3CH NHN N proliferationblood ofcell PBMC culture and LPS- Strong increases in Strongthe expression increases of in the expression of dimethyl-1,2-oxazole-4-carbohydrazidedihydroxyphenyl)ethylidene]-N,3- N O CH2 induced TNF α production in human α caspases, Fas, and NF-kB1 [16] ethylidene]-N,3-dimethyl-1,2-oxazole-4 3CH 3 PBMC and LPS-induced TNF production [16] dimethyl-1,2-oxazole-4-carbohydrazide NHN2 blood cell culture N O CH3 induced TNF α production in human caspases, Fas, and NF-kB1caspases, Fas, and NF-kB1 dimethyl-1,2-oxazole-4-carbohydrazide NH blood cell culture -carbohydrazide N O 2CH3 in human blood cell culture NH blood cell culture N O 2 NH O 2 Molecules 2018, 23, 2724 4 of 17

Table 1. Cont.

Molecules 2018Compound, 23, x Structure Activity Molecular/Cellular Mechanism4 of 17 Action Reference Number Molecules 2018, 23, x 4 of 17 Molecules 2018, 23, x Immune regulators 4 of 17 Molecules 2018, 23, x Immune regulators 4 of 17 Molecules 2018, 23, x 3CH Immune regulators 4 of 17 O 3CH Immune regulators Molecules 2018, 23, x O 4 of 17 HWA-486/Leflunomide 5-methyl-N-[4- 3CH N Immune regulatorsSuppresses T cell-dependent B-cell NH O HWA-486/Leflunomide 5-methyl-N-[ CH N Suppresses T cell-dependent B-cell responses, HWA-486(trifluoromethyl)phenyl]-1,2-oxazole-4-/Leflunomide 5-methyl-N-[4- NH 3 Immune regulatorsSuppressesresponses, T cell-dependent does not affect B-cellT- COX-2 inhibitor [17] ON COX-2 inhibitor [17] 4-(trifluoromethyl)phenyl]-1,2-oxazole-4-carboxamideHWA-486/Leflunomide 5-methyl-N-[4- F 3CH Suppressesdoes not T affect cell-dependent T-independent B-cell B-cell function (trifluoromethyl)phenyl]-1,2-oxazole-4- NH O O Immune regulatorsresponses, does not affect T- COX-2 inhibitor [17] HWA-486/Leflunomidecarboxamide 5-methyl- N-[4- F N Suppressesindependent T cell-dependent B-cell function B-cell (trifluoromethyl)phenyl]-1,2-oxazole-4- NHOCH responses, does not affect T- COX-2 inhibitor [17] carboxamide FF 3 N independent B-cell function HWA-486(trifluoromethyl)phenyl]-1,2-oxazole-4-/Leflunomide 5-methyl-N-[4- F NH O O Suppressesresponses, T cell-dependent does not affect B-cellT- COX-2 inhibitor [17] carboxamide F F independent B-cell function (trifluoromethyl)phenyl]-1,2-oxazole-4-HWA-486/Leflunomidecarboxamide 5-methyl- N-[4- F O O N Suppressesresponses,independent T cell-dependentdoes B-cell not functionaffect B-cellT- COX-2 inhibitor [17] FF NH NH2 Modulates T cell subset composition F O O independent B-cell function (trifluoromethyl)phenyl]-1,2-oxazole-4-carboxamide F 3CH NHNH2 responses, does not affect T- COX-2 inhibitor [17] 5-amino-3-methyl-1,2-oxazole-4- F F O ModulatesandModulates the levels T cell of T subsetB cell cells subset compositionin lymphoid composition Modulation and the of T and B cell content F CH O NH2 5-amino-3-methyl-1,2-oxazole-4-carboxamide F3 O NH Modulatesand independentthelevels levels T ofcell of B B subsetB-cell cells cells infunctionincomposition lymphoidlymphoid organs,Modulation and of T andModulation B cell content of T and B[18] cell content in carbohydrazide 3CH NHNH2 organs, and enhances anti-SRBC in lymphatic organs 5-amino-3-methyl-1,2-oxazole-4-carbohydrazide5-amino-3-methyl-1,2-oxazole-4- F N O andModulates the levels T cell of Bsubset cells incomposition lymphoid Modulation of T and B cell content [18] [18] carbohydrazide F3CH NHNHNH2 Modulateshumoralorgans,enhances immuneand T cell enhances subset anti-SRBCresponses anti-SRBCcomposition in humoral mice immunein lymphatic organs lymphatic organs[18] 5-amino-3-methyl-1,2-oxazole-4-carbohydrazide CH N O 2 andorgans, the levels and enhancesof B cells anti-SRBCin lymphoid Modulationin lymphatic of T and organs B cell content 3 O NHNH humoral immune responses in in mice mice [18] 5-amino-3-methyl-1,2-oxazole-4- N O S 2 NH2 Modulatesand the levels T cell of Bsubset cells incomposition lymphoid Modulation of T and B cell content carbohydrazide NH humoralorgans, immune and enhances responses anti-SRBC in mice in lymphatic organs [18] 3CH N O S NH2 5-amino-3-methyl-1,2-oxazole-4-carbohydrazide O NH NH andhumoralorgans,Induces the levels immuneand lymphopoiesis enhancesof B responsescells inanti-SRBC lymphoidand in the mice Modulationin lymphatic of T and organs B cell content 06K/ 2-(5-amino-3-methyl-1,2-oxazole-4- N O S 2 Increases percentage of CD8+ and [18] O NHNH NHNH Cl humoralgenerationInduces immune lymphopoiesis of regulatory responses and T-cells, in the mice 06K/ 2-(5-amino-3-methyl-1,2-oxazole-4-carbohydrazide 3CH O S 2 organs, and enhances anti-SRBC Increasesin lymphaticpercentage organs of CD8+ and carbonyl)-N-(4-chlorophenyl)hydrazine-1- O N NHNH NH Cl generationInducesInduces lymphopoiesis lymphopoiesisof regulatory and T-cells, and the the generationregulatory of CD4+CD25+Fox3+ T cells [19] 06K/ 2-(5-amino-3-methyl-1,2-oxazole-4- CH S stimulates the humoral immune Increases percentageIncreases of CD8+ percentageand of CD8+ and carbonyl)-N-(4-chlorophenyl)hydrazine-1- 3 O NH NH2 humoralInduces immune lymphopoiesis responses and in themice regulatory CD4+CD25+Fox3+ T cells [19] 06K/2-(5-amino-3-methyl-1,2-oxazole-4-carbonyl)-carbothioamide N- CH NHO NH Cl generationregulatory of regulatory T-cells, stimulates T-cells, the humoralin spleens and lymph nodes carbonyl)-06K/ 2-(5-amino-3-methyl-1,2-oxazole-4-N-(4-chlorophenyl)hydrazine-1- 3 O NH response,stimulates decreases the humoral the DTH immune reaction regulatoryIncreases CD4+CD25+Fox3+percentage of CD8+ T andcells [19] carbothioamide N NHNH2 SNH Cl Inducesgeneration lymphopoiesis of regulatory and T-cells, the in spleens andregulatory lymph nodes CD4+CD25+Fox3+ T cells in [19] (4-chlorophenyl)hydrazine-1-carbothioamidecarbonyl)-06K/ 2-(5-amino-3-methyl-1,2-oxazole-4-N-(4-chlorophenyl)hydrazine-1- 3CH O response,stimulatesimmune decreases the humoral response, the DTH immune decreasesreaction theregulatoryIncreases DTH percentageCD4+CD25+Fox3+ of CD8+ T and cells [19] carbothioamide N NHNH2 NH Cl generation of regulatory T-cells, in spleens and lymphspleens nodes and lymph nodes 3CH OO NH S response,stimulatesInduces decreases lymphopoiesis the humoral the DTH immuneand reaction the carbonyl)-06K/ 2-(5-amino-3-methyl-1,2-oxazole-4-N-(4-chlorophenyl)hydrazine-1-carbothioamide N NH2 reaction regulatoryIncreasesin spleens percentageCD4+CD25+Fox3+ and lymph of CD8+ nodes T and cells [19] O NH NHS Cl response,stimulatesgeneration decreases theof regulatory humoral the DTH immune T-cells, reaction carbothioamide 3CH N O NH2 NH in spleens and lymph nodes carbonyl)-01K/2-(5-amino-3-methyl-1,2-oxazole-4-N-(4-chlorophenyl)hydrazine-1- O S response, decreases the DTH reaction regulatoryRegulation CD4+CD25+Fox3+ of IL-1β and TNF- T cellsα [19] N O NHNH2 NH NH Regulatesstimulates proliferation the humoral of thymocytes,immune 01K/2-(5-amino-3-methyl-1,2-oxazole-4-carbothioamide 3CH O O NH S Regulationin spleens of and IL-1 lymphβ and nodes TNF-α carbonyl)-N-phenylhydrazine-1- NH NH response, decreases the DTH reaction production in peritoneal cell [20] 01K/ 2-(5-amino-3-methyl-1,2-oxazole-4- CH N NH2 S Regulatessplenocytes, proliferation and lymph of thymocytes,node cells Regulation of IL-1β and TNF-α carbonyl)-carbothioamideN-phenylhydrazine-1- 3 O O NHNH NH Regulates proliferation of thymocytes, productioncultures in peritoneal cell [20] 01K/ carbonyl)-2-(5-amino-3-methyl-1,2-oxazole-4-N-phenylhydrazine-1- 3CH O NH splenocytes, and lymph node cells Regulationproduction of in IL-1 peritonealβ and TNF- cell α [20] 01K/2-(5-amino-3-methyl-1,2-oxazole-4-carbonyl)-carbothioamide N- N NHNH SNH RegulatesRegulates proliferation proliferation of thymocytes, of thymocytes, culturesRegulation of IL-1β and TNF-α production 01K/ carbonyl)-2-(5-amino-3-methyl-1,2-oxazole-4-N-phenylhydrazine-1- 3CH 2 splenocytes, and lymph node cells Regulationproduction of in IL-1 peritonealβ and TNF- cellα [20] carbothioamide N OO NHNHNH NH Regulates proliferation of thymocytes, cultures [20] phenylhydrazine-1-carbothioamide 3CH 2 splenocytes,splenocytes, and lymph and node lymph cells node cells in peritoneal cell cultures 01K/ carbonyl)-2-(5-amino-3-methyl-1,2-oxazole-4-N-phenylhydrazine-1- O O Regulationproduction of in IL-1 peritonealβ and TNF- cellα [20] carbothioamide N NH2 splenocytes, and lymph node cells cultures CH O O NH NH Regulates proliferation of thymocytes, carbonyl)-MO5/5-amino-3-methyl-carbothioamideN-phenylhydrazine-1- N-[(4- 33CH N NHNH2 productioncultures in peritoneal cell [20] N OO NH Inhibitssplenocytes, the humoral and lymph immune node response cells methylphenyl)methyl]-1,2-oxazole-4-MO5/5-amino-3-methyl-carbothioamide N-[(4- 3CH NH2 Inhibition ofcultures TNFα production [14] CH O O Inhibits the humoral immune response MO5/5-amino-3-methyl-N-[(4- 3 NH CH in vitro methylphenyl)methyl]-1,2-oxazole-4-carboxamide NN O NH2 3 Inhibits the humoral immune response Inhibition of TNFα production [14] methylphenyl)methyl]-1,2-oxazole-4- MO5/5-amino-3-methyl-N-[(4- 3CH O NHNH in vitro Inhibition of TNFα production [14] carboxamide N O 2 CH3 Inhibits the humoral immune response methylphenyl)methyl]-1,2-oxazole-4- MO5/5-amino-3-methyl-N-[(4- 3CH O NHNH CH in vitro Inhibition of TNFα production [14] MO5/5-amino-3-methyl-carboxamideN-[(4-methylphenyl)methyl]- N O 2 3Anti-inflammatoryInhibits Inhibitsthe humoral in the vitro immune humoral response immune response methylphenyl)methyl]-1,2-oxazole-4- NH2 CH Inhibition of TNFα production α [14] MO5/5-amino-3-methyl-carboxamide N-[(4- 3CH N O NH 3 Inhibition of TNF production [14] 1,2-oxazole-4-carboxamide NH OC2H5 Anti-inflammatoryInhibits the humoral in vitro immune in vitro response carboxamide N O 2 CH3 methylphenyl)methyl]-1,2-oxazole-4- OC2H5 Anti-inflammatory Inhibition of TNFα production [14] O NH2 in vitro carboxamide N OC2H5 CH3 Anti-inflammatory NH 5-amino-N-(4-ethoxyphenyl)-3-methyl-1,2- O O 2 OC2H5 Anti-inflammatoryLowers carrageenan-induced paw O OC2H5 Anti-inflammatory N.D. [21] 5-amino-N-(4-ethoxyphenyl)-3-methyl-1,2- 3CH NH Anti-inflammatoryLowers carrageenan-induced paw oxazole-4-carboxamide O edema, exhibits antibacterial activity N.D. [21] 5-amino-N-(4-ethoxyphenyl)-3-methyl-1,2- 3CH NH OC H Lowers carrageenan-induced paw oxazole-4-carboxamide O 2 5 edema, exhibits antibacterial activity N.D. [21] 5-amino-Noxazole-4-carboxamide-(4-ethoxyphenyl)-3-methyl-1,2- 3CH NH edema,Lowers exhibits carrageenan-induced antibacterial activity paw N O N.D. [21] 5-amino-N-(4-ethoxyphenyl)-3-methyl-1,2- 3CH NHNH2 Lowers carrageenan-induced paw oxazole-4-carboxamide N O edema, exhibits antibacterial activity CH NH N.D. [21] 5-amino-N5-amino--(4-ethoxyphenyl)-3-methyl-1,2-oxazole-Noxazole-4-carboxamide-(4-ethoxyphenyl)-3-methyl-1,2- 3 N O O NH2 Cl edema,LowersLowers exhibits carrageenan-induced carrageenan-induced antibacterial activity paw paw edema, NH2 CH N O Cl N.D. N.D.[21] [21] 4-carboxamideoxazole-4-carboxamide 3 NHNH edema, exhibitsexhibits antibacterial antibacterial activity activity N O 2 Cl NH O O 2 Cl Shows anti-inflammatory activity in 5-benzamido-N-(4-chlorophenyl)-3- N O 3CH NHNH Cl theShows carrageenan-induced anti-inflammatory reaction, activity hasin N.D. [21] 5-benzamido-N-(4-chlorophenyl)-3- O 2 methyl-1,2-oxazole-4-carboxamide CH O O Shows anti-inflammatory activity in 5-benzamido-N-(4-chlorophenyl)-3- 3 NH Cl the carrageenan-inducedantibacterial properties reaction, has N.D. [21] methyl-1,2-oxazole-4-carboxamide CH O Shows anti-inflammatory activity in 5-benzamido-N-(4-chlorophenyl)-3- 3 N NHO the carrageenan-inducedantibacterial properties reaction, has N.D. [21] methyl-1,2-oxazole-4-carboxamide CH O NHO Shows anti-inflammatory activity in 5-benzamido-N-(4-chlorophenyl)-3- 3 N O NH the carrageenan-inducedantibacterial properties reaction, has N.D. [21] methyl-1,2-oxazole-4-carboxamide CH NHO 3 N O O NH theShows carrageenan-induced antibacterialanti-inflammatory properties reaction, activity hasin N.D. [21] methyl-1,2-oxazole-4-carboxamide NHO 5-benzamido-N-(4-chlorophenyl)-3- N O 3CH NHNH the carrageenan-inducedantibacterial properties reaction, has N.D. [21] N O methyl-1,2-oxazole-4-carboxamide NHO O antibacterial properties N NH O Molecules 2018, 23, x 4 of 17

Immune regulators 3CH O N HWA-486/Leflunomide 5-methyl-N-[4- NH Suppresses T cell-dependent B-cell (trifluoromethyl)phenyl]-1,2-oxazole-4- responses, does not affect T- COX-2 inhibitor [17] F carboxamide O independent B-cell function F F O NH2 Modulates T cell subset composition CH 5-amino-3-methyl-1,2-oxazole-4- 3 NH and the levels of B cells in lymphoid Modulation of T and B cell content [18] carbohydrazide organs, and enhances anti-SRBC in lymphatic organs N O NH2 humoral immune responses in mice S O NH Induces lymphopoiesis and the 06K/ 2-(5-amino-3-methyl-1,2-oxazole-4- Increases percentage of CD8+ and NH NH Cl generation of regulatory T-cells, carbonyl)-N-(4-chlorophenyl)hydrazine-1- 3CH regulatory CD4+CD25+Fox3+ T cells [19] stimulates the humoral immune carbothioamide in spleens and lymph nodes N NH2 response, decreases the DTH reaction O S O NH 01K/ 2-(5-amino-3-methyl-1,2-oxazole-4- Regulation of IL-1β and TNF-α NH NH Regulates proliferation of thymocytes, carbonyl)-N-phenylhydrazine-1- 3CH production in peritoneal cell [20] splenocytes, and lymph node cells carbothioamide cultures N NH2 O O MO5/5-amino-3-methyl-N-[(4- CH 3 NH Inhibits the humoral immune response methylphenyl)methyl]-1,2-oxazole-4- Inhibition of TNFα production [14] in vitro carboxamide N CH3 O NH2 Molecules 2018, 23, 2724 Anti-inflammatory 5 of 17 OC2H5

5-amino-N-(4-ethoxyphenyl)-3-methyl-1,2- O Lowers carrageenan-induced paw Table 1. Cont. N.D. [21] oxazole-4-carboxamide 3CH NH edema, exhibits antibacterial activity

N Compound StructureNH Activity Molecular/Cellular Mechanism of Action Reference Number O 2 Cl

O ShowsShows anti-inflammatory anti-inflammatory activity in activity in the 5-benzamido-N5-benzamido--(4-chlorophenyl)-3-methyl-1,2-oxazole-N-(4-chlorophenyl)-3- 3CH NH the carrageenan-inducedcarrageenan-induced reaction, reaction,has has N.D. N.D.[21] [21] methyl-1,2-oxazole-4-carboxamide4-carboxamide O antibacterialantibacterial properties properties N NH Molecules 2018, 23, x O 5 of 17 Molecules 2018, 23, x 5 of 17 Molecules 2018, 23, x 5 of 17 Molecules 2018, 23, x 5 of 17 Molecules 2018, 23, x 5 of 17 SuppressesSuppresses carrageenan-induced carrageenan-induced Suppression pleurisy, of TNFSuppression α, IL-1β, MIF, of TNF α, IL-1β, MIF, VGX-1027VGX-1027(S,R)-(3-phenyl-4,5-dihydro-1, (S,R)-(3-phenyl-4,5-dihydro-1,2- Suppresses carrageenan-induced Suppression of TNF α, IL-1β, MIF, VGX-1027 (S,R)-(3-phenyl-4,5-dihydro-1,2- O pleurisy, LPS-inducedLPS-induced lethality, lethality, and and typeinhibition of NFκB inhibitionand p38, and of NFκB and[22] p38, and [22] 2-oxazol-5-yl)aceticoxazol-5-yl)acetic acid acid O Suppresses carrageenan-induced Suppression of TNF α, IL-1β, MIF, VGX-1027 (S,R)-(3-phenyl-4,5-dihydro-1,2- pleurisy,Suppressestype II-collagen-induced LPS-induced carrageenan-induced lethality, arthritis and Suppressioninhibitionupregulation of of NF TNFofκ BERK andα, IL-1signaling p38,β, andMIF, [22] oxazol-5-yl)acetic acid HO O N pleurisy, LPS-inducedII-collagen-induced lethality, and arthritis inhibition of NFκB andupregulation p38, and of ERK[22] signaling VGX-1027 oxazol-5-yl)acetic(S,R)-(3-phenyl-4,5-dihydro-1,2- acid typeSuppresses II-collagen-induced carrageenan-induced arthritis Suppressionupregulation of ofTNF ERK α, signalingIL-1β, MIF, VGX-1027 (S,R)-(3-phenyl-4,5-dihydro-1,2- OH O O N pleurisy, LPS-induced lethality, and inhibition of NFκB and p38, and [22] oxazol-5-yl)acetic acid ONH type II-collagen-induced arthritis upregulation of ERK signaling OH O O N O pleurisy,type II-collagen-induced LPS-induced lethality, arthritis and inhibitionupregulation of NF ofκ ERKB and signaling p38, and [22] oxazol-5-yl)acetic acid OH ONHO N O O type II-collagen-induced arthritis upregulation of ERK signaling OH ONHN O N O ONH O ON O O N-(2-(Benzo[d][1,3]dioxol-5-yl)ethyl)-5- ONH O Inhibition of FAAH (fatty acid O N O N d O Protects against experimental colitis [23] -(2-(Benzo[N(biphen-4-yl)-isoxazole-3-carboxamide-(2-(Benzo[][1,3]dioxol-5-yl)ethyl)-5-(biphen-4-d][1,3]dioxol-5-yl)ethyl)-5- ON Inhibitionamide of FAAHhydrolase))Inhibition (fatty acid of FAAH (fatty acid amide O Protects Protectsagainst experimental against experimental colitis colitis [23] [23] N-(2-(Benzo[d][1,3]dioxol-5-yl)ethyl)-5- N Inhibition of FAAH (fatty acid yl)-isoxazole-3-carboxamide(biphen-4-yl)-isoxazole-3-carboxamideN-(2-(Benzo[d][1,3]dioxol-5-yl)ethyl)-5- O Protects against experimental colitis Inhibitionamide of hydrolase))FAAH (fatty acidhydrolase)) [23] N(biphen-4-yl)-isoxazole-3-carboxamide-(2-(Benzo[d][1,3]dioxol-5-yl)ethyl)-5- O Protects against experimental colitis Inhibitionamide of FAAHhydrolase)) (fatty acid [23] (biphen-4-yl)-isoxazole-3-carboxamide Protects against experimental colitis amide hydrolase)) [23] (biphen-4-yl)-isoxazole-3-carboxamide amide hydrolase)) OCH3 OCH3 H3CO OCH H C Inhibits tumor growth, peritoneal 2b/5-(3-methylthiophen-2-yl)-3-(3,4,5- H CO 3 3 Inhibitory activity toward 3 OCH Inhibits tumor growth, peritoneal 3 3CH angiogenesis, and ascite formation in [24] 2b /5-(3-methylthiophen-2-yl)-3-(3,4,5- H3CO Inhibits Inhibitstumor growth, tumor peritoneal growth, peritonealInhibitory activity toward trimethoxyphenyl)-1,2-oxazole H CO OCH3 3CH lipooxygenase (LOX) and COX-2 2b/5-(3-methylthiophen-2-yl)-3-(3,4,5-2b /5-(3-methylthiophen-2-yl)-3-(3,4,5- H33CO angiogenesis, and ascite formation in Inhibitory activityInhibitory toward activity toward[24] lipooxygenase trimethoxyphenyl)-1,2-oxazole CH InhibitsErlich carcinomatumor growth, mouse peritoneal model lipooxygenase (LOX) and COX-2 HH3COCO 3 angiogenesis,angiogenesis, and ascite and asciteformation formation in in Erlich [24] [24] 2b /5-(3-methylthiophen-2-yl)-3-(3,4,5- 3 N O S InhibitsErlich carcinoma tumor growth, mouse peritoneal model Inhibitory activity toward trimethoxyphenyl)-1,2-oxazoletrimethoxyphenyl)-1,2-oxazole H CO 3CH angiogenesis, and ascite formation in lipooxygenase (LOX) and COX-2(LOX) and COX-2[24] 2b /5-(3-methylthiophen-2-yl)-3-(3,4,5- 3 N S Erlich carcinomacarcinoma mouse mousemodel model Inhibitory activity toward trimethoxyphenyl)-1,2-oxazole H CO R O O angiogenesis, and ascite formation in lipooxygenase (LOX) and COX-2 [24] 39/4-(4-Chlorophenyl)-5-[4-(quinolin-2- 3 N S Erlich carcinoma mouse model trimethoxyphenyl)-1,2-oxazole H CO OR O lipooxygenase (LOX) and COX-2 3 N S Erlich carcinoma mouse model 39ylmethoxy)phenyl]isoxazol-3-carboxylic/4-(4-Chlorophenyl)-5-[4-(quinolin-2- OR O O 39/4-(4-Chlorophenyl)-5-[4-(quinolin-2- OR N O S ylmethoxy)phenyl]isoxazol-3-carboxylic O N Inhibitory activity against cellular 5- 39/4-(4-Chlorophenyl)-5-[4-(quinolin-2-acid 39 R=Quinolin-2-ylOR ylmethoxy)phenyl]isoxazol-3-carboxylic O N Anti-inflammatory agent InhibitoryLO (5-lipoxygenase) activity against product cellular 5- [25] 39/4-(4-Chlorophenyl)-5-[4-(quinolin-2-ylmethoxy) 39/4-(4-Chlorophenyl)-5-[4-(quinolin-2-acid 3940 R=Quinolin-2-yl R=Benzothiazol-2-yl 40ylmethoxy)phenyl]isoxazol-3-carboxylic/4-(4-Chlorophenyl)-5-[4-(benzothiazol- O Inhibitory activity against cellular 5- acid 39 R=Quinolin-2-yl N Anti-inflammatory agent LO (5-lipoxygenase) product [25] phenyl]isoxazol-3-carboxylic40ylmethoxy)phenyl]isoxazol-3-carboxylic/4-(4-Chlorophenyl)-5-[4-(benzothiazol- acid 40 R=Benzothiazol-2-yl O COOH Inhibitory activitysynthesis against cellular 5- 2-ylmethoxy)phenyl]acid 39 R=Quinolin-2-yl N Anti-inflammatory agent LO (5-lipoxygenase) product [25] 40/4-(4-Chlorophenyl)-5-[4-(benzothiazol- 40 R=Benzothiazol-2-yl COOH Inhibitory activitysynthesisInhibitory against cellular activity 5- against cellular 5-LO 40/4-(4-Chlorophenyl)-5-[4-(benzothiazol-22-ylmethoxy)phenyl]acid 39 R=Quinolin-2-yl N Anti-inflammatoryAnti-inflammatory agent agent LO (5-lipoxygenase)synthesis product [25] [25] 40/4-(4-Chlorophenyl)-5-[4-(benzothiazol-isoxazol-3-carboxylic acid 40 R=Benzothiazol-2-ylCl COOH Anti-inflammatory agent LO (5-lipoxygenase)(5-lipoxygenase) product product[25] synthesis -ylmethoxy)phenyl]2-ylmethoxy)phenyl] 40 R=Benzothiazol-2-yl COOH synthesis 40/4-(4-Chlorophenyl)-5-[4-(benzothiazol-isoxazol-3-carboxylic2-ylmethoxy)phenyl] acid O Cl isoxazol-3-carboxylic acid COOH synthesis isoxazol-3-carboxylic2-ylmethoxy)phenyl] acid O Cl isoxazol-3-carboxylic acid O Cl O O Blocks corticosteroid-insensitive lung isoxazol-3-carboxylic acid H3C O Cl O CH O NH Blocks corticosteroid-insensitive lung 3 O O inflammation and airway hyper- ISO-1/(S,R)-methyl [3-(4-oxocyclohexa-2,5- CH O NH Blocks corticosteroid-insensitive lung Inhibition of migration inhibitory 3 O inflammation and airway hyper- ISO-1/(S,R)-methyl [3-(4-oxocyclohexa-2,5- CH O NH Blocksresponsiveness) corticosteroid-insensitive suppresses lung lung Inhibition of migration inhibitory [26] dien-1-ylidene)-1,2-oxazolidin-5-yl]acetate 3 O inflammation and airway hyper- factor (MIF) function ISO-1/(S,R)-methyl [3-(4-oxocyclohexa-2,5- CH O NH Blocksresponsiveness) corticosteroid-insensitiveBlocks corticosteroid-insensitive suppresses lung lung Inhibition lung of migration inhibitory [26] dien-1-ylidene)-1,2-oxazolidin-5-yl]acetate 3 inflammationinflammation in andozone-exposed airway hyper- mice, factor (MIF) function ISO-1/(S,R)-methyl [3-(4-oxocyclohexa-2,5- NH inflammationinflammationresponsiveness) ininflammation ozone-exposedand suppresses airway hyper- andlung mice, airway Inhibition of migration inhibitory [26] ISO-1/(S,R)-methyl ISO-1dien-1-ylidene)-1,2-oxazolidin-5-yl]acetate/(S,R)-methyl [3-(4-oxocyclohexa-2, [3-(4-oxocyclohexa-2,5- responsiveness)in contrast to dexamethasone suppresses lung Inhibitionfactor of (MIF)migrationInhibition function inhibitory of migration inhibitory[26] factor dien-1-ylidene)-1,2-oxazolidin-5-yl]acetate inflammationresponsiveness)in contrasthyper-responsiveness) in to ozone-exposeddexamethasone suppresses lung mice, suppresses lungfactor (MIF) function [26] [26] 5-dien-1-ylidene)-1,2-oxazolidin-5-yl]acetatedien-1-ylidene)-1,2-oxazolidin-5-yl]acetate O inflammation in ozone-exposed mice, factor (MIF) function (MIF) function inflammationin contrast in to ozone-exposed dexamethasone mice, O HO in inflammationcontrast to dexamethasone in ozone-exposed mice, in O OH O in contrastcontrast to dexamethasone to dexamethasone OH O O O N HO O OH Cpd #15/(16S,17R)-30-bromo-6,9-difluoro- O O Inhibits eosinophilic infiltration in a OH N OH O Inhibits LPS-induced nitric oxide Cpd #15/(16S,17R)-30-bromo-6,9-difluoro- H O N Inhibits eosinophilic infiltration in a 11b,21-dihydroxy-40H-pregna-1,4- OH Br model of allergen-induced pulmonary Inhibits LPS-induced nitric oxide [27] Cpd #15/(16S,17R)-30-bromo-6,9-difluoro- O O Inhibits eosinophilic infiltration in a production 11b,21-dihydroxy-40H-pregna-1,4- H N Br model of allergen-induced pulmonary [27] dieno[16,17-d]isoxazole-3,20-dione OH O inflammation in rats Inhibits LPS-induced nitric oxide Cpd #15/(16S,17R)-30-bromo-6,9-difluoro- FHH N Inhibits eosinophilic infiltration in a production 11b,21-dihydroxy-40H-pregna-1,4- OH Br model of allergen-induced pulmonary Inhibits LPS-induced nitric oxide [27] Cpd dieno[16,17- #15/(16S,17R)-30-bromo-6,9-difluoro-d]isoxazole-3,20-dione O FH Inhibitsinflammation eosinophilic infiltration in rats in a production 11b,21-dihydroxy-40dieno[16,17-d]isoxazole-3,20-dioneH-pregna-1,4- H Br model of inflammationallergen-induced in rats pulmonary Inhibits LPS-induced nitric oxide [27] O FHH production 11b,21-dihydroxy-40H-pregna-1,4- F Br model of allergen-induced pulmonary [27] dieno[16,17-d]isoxazole-3,20-dione O FH inflammation in rats production F dieno[16,17-d]isoxazole-3,20-dione O FH inflammation in rats F O F F

Molecules 2018, 23, x 5 of 17

Suppresses carrageenan-induced Suppression of TNF α, IL-1β, MIF, VGX-1027 (S,R)-(3-phenyl-4,5-dihydro-1,2- O pleurisy, LPS-induced lethality, and inhibition of NFκB and p38, and [22] oxazol-5-yl)acetic acid type II-collagen-induced arthritis upregulation of ERK signaling OH N O ONH O

O N N-(2-(Benzo[d][1,3]dioxol-5-yl)ethyl)-5- O Inhibition of FAAH (fatty acid Protects against experimental colitis [23] (biphen-4-yl)-isoxazole-3-carboxamide amide hydrolase))

OCH3 H3CO Inhibits tumor growth, peritoneal 2b /5-(3-methylthiophen-2-yl)-3-(3,4,5- 3CH Inhibitory activity toward angiogenesis, and ascite formation in [24] trimethoxyphenyl)-1,2-oxazole lipooxygenase (LOX) and COX-2 H3CO Erlich carcinoma mouse model N O S OR 39/4-(4-Chlorophenyl)-5-[4-(quinolin-2- ylmethoxy)phenyl]isoxazol-3-carboxylic O Inhibitory activity against cellular 5- acid 39 R=Quinolin-2-yl N Anti-inflammatory agent LO (5-lipoxygenase) product [25] 40/4-(4-Chlorophenyl)-5-[4-(benzothiazol- 40 R=Benzothiazol-2-yl synthesis 2-ylmethoxy)phenyl] COOH

isoxazol-3-carboxylic acid Cl Molecules 2018, 23, 2724 O 6 of 17

O O 3CH Blocks corticosteroid-insensitive lung NH inflammation and airway hyper- ISO-1/(S,R)-methyl [3-(4-oxocyclohexa-2,5- Inhibition of migration inhibitory responsiveness) suppresses lung [26] dien-1-ylidene)-1,2-oxazolidin-5-yl]acetate Table 1. Cont. factor (MIF) function inflammation in ozone-exposed mice, in contrast to dexamethasone Compound Structure Activity Molecular/Cellular Mechanism of Action Reference Number O OH

O O Cpd N Cpd #15/(16S,17R)-30-bromo-6,9-difluoro- OH InhibitsInhibits eosinophilic eosinophilic infiltration infiltration in a in a model of #15/(16S,17R)-30-bromo-6,9-difluoro-11b,21- Inhibits LPS-inducedInhibits nitric oxide LPS-induced nitric oxide 11b,21-dihydroxy-40H-pregna-1,4- H Br modelallergen-induced of allergen-induced pulmonary pulmonary inflammation [27] [27] dihydroxy-40H-pregna-1,4-dieno[16,17-d] production production dieno[16,17-d]isoxazole-3,20-dione FH inflammation in ratsin rats isoxazole-3,20-dione Molecules 2018, 23, x O 6 of 17 Molecules 2018, 23, x 6 of 17 Molecules 2018, 23, x F 6 of 17 MoleculesMolecules 2018, 232018, x , 23, x 6 of 176 of 17

N O N N O N N 7a/N-(4-(isoxazol-5-yl)phenyl)-4,6- N NN N OO N Reduces carrageenan-induced mice N O N COX-2 inhibitor [28] 7a/N-(4-(isoxazol-5-yl)phenyl)-4,6-di(piperidin-1-yl)-di(piperidin7a/N-(4-(isoxazol-5-yl)phenyl)-4,6--1-yl)-1,3,5-triazin-2-amine N N pawReduces edemaReduces carrageenan-induced in comparison carrageenan-induced to celecoxib mice mice paw 7a7a//NN-(4-(4-(isoxazol-5-yl)phenyl)-4,6--(isoxazol-5-yl)phenyl)-4,6- NNNN N NN H ReducesReduces carrageenan-induced carrageenan-induced mice mice COX-2 inhibitor COX-2 inhibitor[28] [28] 1,3,5-triazin-2-aminedi(piperidin7a/N-1-yl)-1,3,5-triazin-2-amine-(4-(isoxazol-5-yl)phenyl)-4,6- N N paw edemaReducesedema in comparison carrageenan-induced in comparison to celecoxib to mice celecoxib COX-2COX-2 inhibitor inhibitor [28][28] di(piperidindi(piperidin-1-yl)-1,3,5-triazin-2-amine-1-yl)-1,3,5-triazin-2-amine NNN H pawpaw edema edema in in comparison comparison to to celecoxib celecoxib COX-2 inhibitor [28] di(piperidin-1-yl)-1,3,5-triazin-2-amine NNNNNN HH paw edema in comparison to celecoxib NNN H O H3CO NH CH O H3CO 3 O H CO O NH H3CO3 OCH CH O H CO 3 3 NH 3 Inhibits carrageenan-induced footpad CH NH MZO-2 /ethyl N-(4-{[(2,4- 3CH 3 N N NH OCH3 Suppression of LPS-induced TNFα, 3CH OCH Inhibits carrageenan-induced footpad MZO-2/ethyl O OCH33 inflammationInhibits carrageenan-induced and the contact footpad Suppression of LPS-induced TNFα, and MZO-2 /ethyl N-(4-{[(2,4- N N OCH3 InhibitsInhibits carrageenan-induced carrageenan-induced footpad footpad Suppression of LPS-induced TNFα, dimethoxyphenyl)methyl]carbamoyl}-3- OC H Inhibits carrageenan-induced footpadand the expression of caspases 3, 8, [29] MZO-2 MZO-2 /et/ethylhyl NN-(4-{[(2,4--(4-{[(2,4- NN O NN 2 5 sensitivityinflammation in mice andto oxazolone the contact when SuppressionSuppression of of LPS-induced LPS-induced TNF TNFαα, , N-(4-{[(2,4-dimethoxyphenyl)methyl]carbamoyl}-3-MZO-2 /ethyl N-(4-{[(2,4- 3CH N inflammation and the contact sensitivitySuppression in the of expressionLPS-induced of TNF caspasesα, 3, 8, and 9 in [29] dimethoxyphenyl)methyl]carbamoyl}-3- OO OC HN inflammationinflammation and and the the contact contact and the expression of caspases 3, 8, [29] methyl-1,2-oxazol-5-yl)ethanimidate O 2 5 and 9 in Jurkat cells dimethoxyphenyl)methyl]carbamoyl}-3-dimethoxyphenyl)methyl]carbamoyl}-3- CH OC H sensitivityinflammation in mice to oxazolone and the whencontact andand the the expression expression of of caspases caspases 3, 3, 8, 8, [29][29] methyl-1,2-oxazol-5-yl)ethanimidatedimethoxyphenyl)methyl]carbamoyl}-3- 3 OC2H2 55 miceapplied to oxazolone in ointment when applied in ointmentand the expression of caspasesJurkat 3, 8, cells [29] methyl-1,2-oxazol-5-yl)ethanimidate CH OC2H5 sensitivitysensitivity in in mice mice to to oxazolone oxazolone when when and 9 in Jurkat cells 3CH 3 sensitivity in mice to oxazolone when methyl-1,2-oxazol-5-yl)ethanimidatemethyl-1,2-oxazol-5-yl)ethanimidate 3CH applied in ointment andand 9 9 in in Jurkat Jurkat cells cells methyl-1,2-oxazol-5-yl)ethanimidate applied in ointment and 9 in Jurkat cells appliedapplied in ointment in ointment

N

N N CH Demonstrates analgesic activity and H NN 3 4-(2-(4-(1H-benzimidazol-2- N N N CH DemonstratesDemonstrates analgesic analgesicactivity and activity and H NH 3 protection in paw edema test, is 4-(2-(4-(1H-benzimidazol-2-yl)phenyl)hydrazono)-1- NN CHN 4-(2-(4-(1H-benzimidazol-2- N CH 3 DemonstratesDemonstrates analgesic analgesic activity activity and and yl)phenyl)hydrazono)-1-(4-chlorophenyl)- HH N 3 N.D. [30] NH CH3 protectionDemonstrates in paw analgesic edema activity test, isand comparable N.D. [30] 4-(2-(4-(14-(2-(4-(1HH--benzimidazol-2-benzimidazol-2- H NN N protection in paw edema test, is H H O N comparable with that of the reference (4-chlorophenyl)-3-methyl-1yl)phenyl)hydrazono)-1-(4-chlorophenyl)-4-(2-(4-(1-pyrazol-5(4H-benzimidazol-2-)-one NHNH N protectionprotection in in paw paw edema edema test, test, is is N.D. [30] yl)phenyl)hydrazono)-1-(4-chlorophenyl)-3-methyl-1H-pyrazol-5(4H)-one NHN NN withprotection that of thein paw reference edema drugtest, is Diclofenac N.D. [30] yl)phenyl)hydrazono)-1-(4-chlorophenyl)- O N comparabledrug with Diclofenac that of the reference N.D. [30] 3-methyl-1yl)phenyl)hydrazono)-1-(4-chlorophenyl)-H-pyrazol-5(4H)-one NN comparable with that of the reference N.D. [30] OO N comparable with that of the reference 3-methyl-13-methyl-1HH-pyrazol-5(4-pyrazol-5(4HH)-one)-one O comparabledrug Diclofenac with that of the reference 3-methyl-1H-pyrazol-5(4H)-one drug Diclofenac Cl drug Diclofenacdrug Diclofenac NO 2Cl Cl NO2 Cl NO Cl NO22 NO2 R N R Is analgesic and anti-inflammatory N SNO2 Isoxazole-mercaptobenzimidazole hybrids RR N.D. [31] NN Is analgesic and anti-inflammatory R SNON 2 Is analgesicactivity and in vivo anti-inflammatory activity Isoxazole-mercaptobenzimidazoleIsoxazole-mercaptobenzimidazole hybrids hybrids SNOO IsIs analgesic analgesic and and anti-inflammatory anti-inflammatory N.D. N.D.[31] [31] N SNO22 Is analgesic and anti-inflammatory Isoxazole-mercaptobenzimidazoleIsoxazole-mercaptobenzimidazole hybrids hybrids N SNO2 activity in vivoin vivo N.N.D.D. [[31]31] Isoxazole-mercaptobenzimidazole hybrids NN O activityactivity in in vivo vivo N.D. [31] ONO N activity in vivo NN O Ar N Ar ArAr Ar

O (3S,3aR,6aS)-5-benzyl-3-(1H-indol-3-yl)-2- O N Inhibition of LPS-induced TNF α H O Increases survival in a mouse model of (3S,3aR,6aS)-5-benzyl-3-(1H-indol-3-yl)-2- O N Inhibition of LPS-induced TNF α phenyl-hexahydro-2H-pyrrolo[3,4- NH H O and IL-6 production in macrophage [32] (3S,3aR,6aS)-5-benzyl-3-(1H-indol-3-yl)-2-(3S,3aR,6aS)-5-benzyl-3-(1H-indol-3-yl)-2-(3S,3aR,6aS)-5-benzyl-3-(1H-indol-3-yl)-2- NN O Increases survivalsepsis in a mouse model of InhibitionInhibition of of LPS-induced LPS-induced TNF TNF α α (3S,3aR,6aS)-5-benzyl-3-(1H-indol-3-yl)-2- HH N Increases survival in a mouse model of Inhibition of LPS-induced TNF α phenyl-hexahydro-2H-pyrrolo[3,4-d][1,2]oxazole-4,6-dione (9ª) NH H O Increases survival in a mouse model of and IL-6 productionTHP-1Inhibition cells in macrophage of LPS-induced [32] TNF α and IL-6 phenyl-hexahydro-2H-pyrrolo[3,4- H NO H Increases survivalsepsis in a mouse model ofand IL-6 production in macrophage [32] phenyl-hexahydro-2H-pyrrolo[3,4-phenyl-hexahydro-2H-pyrrolo[3,4-d][1,2]oxazole-4, NH NH OO Increases survival in a mouse modeland of IL-6 sepsis production in macrophage [32] [32] d][1,2]oxazole-4,6-dionephenyl-hexahydro-2H-pyrrolo[3,4- (9ª) NH O sepsissepsis and IL-6THP-1 production cells in macrophage [32] H NO H sepsis production in macrophage THP-1 cells 6-dionedd][1,2]oxazole-4,6-dione][1,2]oxazole-4,6-dione (9ª) (9ª) (9ª) H H THP-1THP-1 cells cells d][1,2]oxazole-4,6-dione (9ª) H NONOH THP-1 cells H NO H

Immunostimulatory OH OH Immunostimulatory ImmunostimulatoryImmunostimulatory OH P OH Immunostimulatory OOH O OH OH P OH OH OH PP O NO P HAB-439/(3-phenyl-4,5-dihydro-1,2- OO Stimulates DTH response to Salmonella OO O Inhibitor of aminopeptidase B [33] HAB-439/oxazol-5-yl)phosphonic(3-phenyl-4,5-dihydro-1,2- acid N O Stimulatestyphimurium DTH and response Listeria monocytogenes to Salmonella HAB-439/HAB-439/(3-phenyl-4,5-dihydro-1,2-(3-phenyl-4,5-dihydro-1,2- NN StimulatesStimulates DTH DTH response response to to Salmonella Salmonella Inhibitor of aminopeptidase B [33] oxazol-5-yl)phosphonicHAB-439/(3-phenyl-4,5-dihydro-1,2- acid N typhimuriumStimulates and DTHListeria response monocytogenes to Salmonella InhibitorInhibitor of of aminopeptidase aminopeptidase B B [33] [33] oxazol-5-yl)phosphonic acid typhimurium and Listeria monocytogenes Inhibitor of aminopeptidase B [33] oxazol-5-yl)phosphonicoxazol-5-yl)phosphonic acid acid typhimuriumtyphimurium and Listeria and Listeria monocytogenes monocytogenes Molecules 2018, 23, x 6 of 17

N O N 7a/N-(4-(isoxazol-5-yl)phenyl)-4,6- Reduces carrageenan-induced mice N N COX-2 inhibitor [28] di(piperidin-1-yl)-1,3,5-triazin-2-amine paw edema in comparison to celecoxib NNN H

O H3CO NH 3CH OCH3 Inhibits carrageenan-induced footpad MZO-2/ethyl N-(4-{[(2,4- N N Suppression of LPS-induced TNFα, O inflammation and the contact dimethoxyphenyl)methyl]carbamoyl}-3- and the expression of caspases 3, 8, [29] OC2H5 sensitivity in mice to oxazolone when methyl-1,2-oxazol-5-yl)ethanimidate 3CH and 9 in Jurkat cells applied in ointment

N

N CH Demonstrates analgesic activity and H 3 4-(2-(4-(1H-benzimidazol-2- N NH protection in paw edema test, is yl)phenyl)hydrazono)-1-(4-chlorophenyl)- N N.D. [30] N comparable with that of the reference 3-methyl-1H-pyrazol-5(4H)-one O drug Diclofenac

Cl NO2

R N Is analgesic and anti-inflammatory Isoxazole-mercaptobenzimidazole hybrids SNO2 N.D. [31] N activity in vivo O N

Ar Molecules 2018, 23, 2724 7 of 17

O (3S,3aR,6aS)-5-benzyl-3-(1H-indol-3-yl)-2- N Inhibition of LPS-induced TNF α H Increases survival in a mouse model of phenyl-hexahydro-2H-pyrrolo[3,4- and IL-6 production in macrophage [32] NH O sepsis d][1,2]oxazole-4,6-dione (9ª) Table 1. Cont. THP-1 cells H NO H Compound Structure Activity Molecular/Cellular Mechanism of Action Reference Number Immunostimulatory Immunostimulatory OH OH P O O HAB-439/(3-phenyl-4,5-dihydro-1,2-oxazol-5-HAB-439/(3-phenyl-4,5-dihydro-1,2- N StimulatesStimulates DTH response DTH to response Salmonella to Salmonella Inhibitor of aminopeptidaseInhibitor B of aminopeptidase [33] B [33] yl)phosphonicoxazol-5-yl)phosphonic acid acid typhimuriumtyphimurium and Listeriaand monocytogenesListeria monocytogenes Molecules 20182018,, 2323,, xx 77 ofof 1717 Molecules 2018, 23, x 7 of 17 Molecules 2018, 23, x 7 of 17 Molecules 2018, 23, x 7 of 17 Molecules 2018, 23, x CH StimulatesStimulates mitogen-inducedmitogen-induced 7 of 17 O CH33 Stimulates mitogen-induced O CH3 StimulatessplenocyteStimulates mitogen-inducedproliferation mitogen-induced and splenocyte NCH splenocyte proliferation and H3CH C O N 3 splenocyteStimulates proliferation mitogen-induced and RM-11/3,5-dimethyl-5,6-dihydro-4RM-11/3,5-dim/3,5-dimethyl-5,6-dihydro-4ethyl-5,6-dihydro-4H-[1,2]oxazoloH-- CH 3 N CH acceleratesacceleratesproliferationsplenocyte thethe proliferation restorationrestoration and accelerates ofandof thethe the restoration 3 O N NHNH3 Stimulates mitogen-induced N.D. [34,35] RM-11/3,5-dimethyl-5,6-dihydro-4H- 3CH O CH3 acceleratessplenocyte the proliferation restoration of and the N.D. N.D.[34,35] [34,35] [5,4-[1,2]oxazolo[5,4-[1,2]oxazolo[5,4-RM-11e][1,2,4]triazepin-4-one/3,5-dimethyl-5,6-dihydro-4ee][1,2,4]triazepin-4-one][1,2,4]triazepin-4-oneH- N NH accelerateshumoralhumoralof the andand the humoral cellularcellularrestoration immuneimmune and of cellular the immune N.D. [34,35] [1,2]oxazolo[5,4-e][1,2,4]triazepin-4-one 3CH NH humoralsplenocyte and proliferationcellular immune and N.D. [34,35] RM-11/3,5-dimethyl-5,6-dihydro-4H- CH NN N responseaccelerates in cyclophosphamide-treated the restoration of the [1,2]oxazolo[5,4-RM-11/3,5-dimethe][1,2,4]triazepin-4-oneyl-5,6-dihydro-4H- 3N NN NH responseaccelerateshumoralresponse in cyclophosphamide-treated and the in cellular cyclophosphamide-treatedrestoration immune of the mice N.D. [34,35] O NH response in cyclophosphamide-treated [1,2]oxazolo[5,4-e][1,2,4]triazepin-4-one N O N responsehumoral in cyclophosphamide-treated andmice cellular immune N.D. [34,35] [1,2]oxazolo[5,4-e][1,2,4]triazepin-4-one N N humoral andmice cellular immune O CH response in cyclophosphamide-treatedmice N OO CHN 33 mice O CHN3 response in cyclophosphamide-treatedmice H C OO NCH3 RecruitRecruitss CD19+ B and accelerates the 33CH mice Stimulation of LPS-induced IL-6 CH O N CH3 Recruits CD19+Recruits B and CD19+ accelerates B and the accelerates Stimulation the of LPS-induced IL-6 R-11/3,5-dimethyl-5,8-dihydro-4R-11/3,5-dimethyl-5,8-dihydro-4/3,5-dimethyl-5,8-dihydro-4H-[1,2]oxazoloH-- 3 N N Recruitdevelopmentdevelopments CD19+ of ofB andbothboth accelerates typestypes ofof thethe the Stimulation of LPS-inducedStimulation IL-6 of LPS-induced IL-6 3CH O CH3 R-11/3,5-dimethyl-5,8-dihydro-4H- N N Recruitdevelopmentdevelopments CD19+ of B both and of typesaccelerates both of types the the of theproductionproduction immuneStimulation inin of humanhuman LPS-induced wholewhole blood bloodIL-6 [36][36] [36] [5,4-[1,2]oxazolo[5,4-[1,2]oxazolo[5,4-e][1,2,4]triazepin-4-oneR-11/3,5-dimethyl-5,8-dihydro-4ee][1,2,4]triazepin-4-one][1,2,4]triazepin-4-oneH- 3CH N developmentimmuneimmune of responseresponse both types inin of the productionStimulation in humanofproduction LPS-induced whole inblood IL-6 human whole[36] blood cultures [1,2]oxazolo[5,4-e][1,2,4]triazepin-4-one CH N N Recruitresponseimmunes CD19+ Bresponse in and immunocompromised accelerates in the production mice inculturescultures human whole blood [36] R-11/3,5-dimethyl-5,8-dihydro-4H- 3N N N developmentimmunocompromised of both types mice of the Stimulation of LPS-induced IL-6 [1,2]oxazolo[5,4-R-11/3,5-dimethyl-5,8-dihydro-4e][1,2,4]triazepin-4-oneH- O N N developmentimmunocompromisedimmune of response both types in mice of the production incultures human whole blood [36] [1,2]oxazolo[5,4-e][1,2,4]triazepin-4-one N O NH (base) ( base) immunocompromisedimmune response micein production incultures human whole blood [36] N O HN (base) immunocompromised mice cultures [1,2]oxazolo[5,4-e][1,2,4]triazepin-4-one CH (base) immune response in N OO CHNH 33 (base) immunocompromised mice cultures O CH OO HN3 (base) immunocompromised mice H C O NNCH3 RecruitRecruitss CD4+ T cells and accelerates R-13/33,5-dimethyl-5,8-dihydro-4,5-dimethyl-5,8-dihydro-4H-- 33CH H (base) Recruits CD4+ T cells and accelerates CH O N CH3 R-13/3,5-dimethyl-5,8-dihydro-4H- 3 CH N NN Recruitthethe restorationrestorationRecruitss CD4+ TCD4+ of ofcells bothboth and T typestypes cells accelerates ofof and thetheaccelerates the R-13/3,5-dimethyl-5,8-dihydro-4[1,2]oxazolo[5,4-[1,2]oxazolo[5,4- R-13/3,5-dimethyl-5,8-dihydro-4ee][1,2,4]triazepin-4-one][1,2,4]triazepin-4-oneH-[1,2]oxazoloH- 3 O CHN 3 Recruitthe restorations CD4+ Tof cells both and types accelerates of the N.N.D.D. [[37]37] [1,2]oxazolo[5,4-e][1,2,4]triazepin-4-one 3CH N N the restorationrestorationimmune of response both of both types in types of the of the immune N.D. N.D.[37] [37] R-13/3,5-dimethyl-5,8-dihydro-4H- NN Recruitsimmune CD4+ T cellsresponse and acceleratesin [5,4-e][1,2,4]triazepin-4-one[1,2]oxazolo[5,4-hydrochloridehydrochloridee][1,2,4]triazepin-4-one hydrochloride 3CH NN N the restorationimmune responseof both types in of the N.D. [37] [1,2]oxazolo[5,4-R-13/3,5-dimethyl-5,8-dihydro-4hydrochloridee][1,2,4]triazepin-4-one H- N OO N immunocompromisedimmunocompromisedresponseimmune inresponse immunocompromised in micemice mice N.D. [37] hydrochloride N O NHH N + HCL+ (salt) theimmunocompromised restorationimmune of response both types micein of the [1,2]oxazolo[5,4-e][1,2,4]triazepin-4-one N O HN HCL (salt) immunocompromised mice N.D. [37] hydrochloride O + HCL+ HCL (salt) (salt) immune response in N O O NH + HCL (salt) immunocompromised mice hydrochloride O HN H3CH C O O NH + HCL (salt) immunocompromised mice M4/.2-(5-amino-3-methyl-1,2-oxazole-4- 3 HNH SS Enhances the mitogen-induced M4/.2-(5-amino-3-methyl-1,2-oxazole-4- 3CH O NH + HCL (salt) Enhances the mitogen-induced M4/.2-(5-amino-3-methyl-1,2-oxazole-4- CH NH S Enhances the mitogen-induced carbonyl)-carbonyl)- M4/.2-(5-amino-3-methyl-1,2-oxazole-4-N-(prop-2-en-1-yl)hydrazine-1--(prop-2-en-1-yl)hydrazine-1- 3 O NHNH S proliferativeproliferativeEnhances the responseresponse mitogen-induced ofof humanhuman N.N.D.D. [[12]12] 3CH N NH M4/.2-(5-amino-3-methyl-1,2-oxazole-4-carbonyl)-carbonyl)-M4/.2-(5-amino-3-methyl-1,2-oxazole-4-N-(prop-2-en-1-yl)hydrazine-1- N NHNH NHS proliferativeEnhancesEnhances the response the mitogen-induced mitogen-induced of human proliferative N.D. [12] carbothioamide 3NCH O NHNH22 NH CHCH2 PBMC carbonyl)-N-(prop-2-en-1-yl)hydrazine-1-carbothioamide O NH NH NHS 2 proliferative PBMCresponse of human N.D. N.D.[12] [12] N-(prop-2-en-1-yl)hydrazine-1-carbothioamideM4/.2-(5-amino-3-methyl-1,2-oxazole-4-carbothioamide N O 2 CH2 Enhances theresponsePBMC mitogen-induced of human PBMC carbonyl)-N-(prop-2-en-1-yl)hydrazine-1- NH2NH NH CHCHCH CH proliferative response of human N.D. [12] carbothioamide N O 2 CH22 PBMC carbonyl)-N-(prop-2-en-1-yl)hydrazine-1- NH2 NH CHCH CH proliferative response of human N.D. [12] carbothioamide N O O CH2 2 PBMC NHO NH CH2 O 2 NHCHNH22 carbothioamide O CH2 CH PBMC H C O NH2 2 33CH NHNHNHCH StimulatesStimulates mitogen-inducedmitogen-induced 5-amino-3-methyl-1,2-oxazole-4- CH O 2 CH2 Increase of LPS-induced induced IL- 5-amino-3-methyl-1,2-oxazole-4- 3 NH NH Stimulates mitogen-induced Increase of LPS-induced induced IL- 5-amino-3-methyl-1,2-oxazole-4- 3CH O NH 2 proliferationproliferationStimulates ofof splenocytes splenocytesmitogen-induced andand lymphlymph Increase of LPS-induced induced IL- [38][38] 5-amino-3-methyl-1,2-oxazole-4-carbohydrazide NH Increase1β production of LPS-induced by peritoneal induced cells IL- carbohydrazide 3CH NH 2 proliferationStimulates of splenocytes mitogen-induced and lymph 1β production by peritoneal cells [38] carbohydrazide NN proliferation ofnodenode splenocytes cellscells and lymph 1β production by peritoneal cells [38] β 5-amino-3-methyl-1,2-oxazole-4- 3CH NHNH StimulatesStimulates mitogen-induced mitogen-induced proliferationIncrease of of LPS-inducedIncrease induced of LPS-induced IL- induced IL-1 5-amino-3-methyl-1,2-oxazole-4-carbohydrazidecarbohydrazide N O NH22 proliferation ofnode splenocytes cells and lymph 1β production by peritoneal cells [38] [38] 5-amino-3-methyl-1,2-oxazole-4- N O NH node cells Increase of LPS-induced induced IL- carbohydrazide HOO O 2 proliferationsplenocytes of splenocytes and and lymph lymph node cells1β production by peritonealproduction cells by peritoneal[38] cells carbohydrazide N O OOH NH2 node cells 1β production by peritoneal cells OOH NH N O OOH 2 node cells HO NH OH O OOH 2 8e/8e/3-(4-methoxyphenyl)-4(3-hydroxy-4-3-(4-methoxyphenyl)-4(3-hydroxy-4- OH OH OOH Augments the proliferative response 8e/3-(4-methoxyphenyl)-4(3-hydroxy-4- O Augments the proliferative response carboxybenzoyl)-5-(3-chlorophenyl)-4,5-carboxybenzoyl)-5-(3-chlorophenyl)-4,5-8e/3-(4-methoxyphenyl)-4(3-hydroxy-4- OH O Augments the proliferative response IncreasedIncreased IL-2IL-2 secretionsecretion [39] [39] carboxybenzoyl)-5-(3-chlorophenyl)-4,5- H CO O Augmentsofof humanhuman theandand proliferative mousemouse lymphocyteslymphocytes response Increased IL-2 secretion [39] 8e/ 3-(4-methoxyphenyl)-4(3-hydroxy-4-dihydrodihydroisoxazoline H33CO OH O carboxybenzoyl)-5-(3-chlorophenyl)-4,5-dihydrodihydroisoxazoline H3CO ofAugments human and the mouseproliferative lymphocytes response Increased IL-2 secretion [39] 8e/3-(4-methoxyphenyl)-4(3-hydroxy-4-8e/ 3-(4-methoxyphenyl)-4(3-hydroxy-4-dihydrodihydroisoxazoline H CO O of human and mouse lymphocytes carboxybenzoyl)-5-(3-chlorophenyl)-4,5- 3 AugmentsAugments the proliferative the proliferative response responseIncreased of IL-2 secretion [39] dihydrodihydroisoxazoline H CO O of human and mouse lymphocytes carboxybenzoyl)-5-(3-chlorophenyl)-4,carboxybenzoyl)-5-(3-chlorophenyl)-4,5- 3 NO ClCl Increased IL-2 secretionIncreased IL-2 secretion[39] [39] dihydrodihydroisoxazoline H CO NO of human and mouse lymphocytes 3 NO Cl human and mouse lymphocytes 5-dihydrodihydroisoxazolinedihydrodihydroisoxazoline Cl ** RecentlyRecentlyNO established,established, unpuunpublished;blished; N.D.—notN.D.—not defined.defined. * RecentlyNO established,Cl unpublished; N.D.—not defined. * RecentlyNO established,Cl unpublished; N.D.—not defined. * Recently established, unpublished; N.D.—not defined. * Recently* Recently established, established, unpublished; unpublished;N.D.—not defined. N.D.—not defined.

Molecules 2018, 23, 2724 8 of 17

2. Results and Discussion

2.1. Immune Suppression Leflunomide and its active metabolite [6] are the most well-recognized immunosuppressive drugs, and they have found therapeutic applications if used alone or in combination with other drugs in rheumatoid arthritis [40] or transplantation [41]. As a prime representative of the isoxazole family, the drug is widely used as a reference drug to compare the potency of other investigated isoxazoles. Another example of a widely used isoxazole drug is Parecoxib, a pain reliever that acts as cyclooxygenase 2 (COX-2) inhibitor [7]. Its safety has been a subject of many multicenter trials, as summarized in a recent article [42], which could not definitely confirm the lack of its side effects reported earlier [43]. In investigations with rodents, a number of isoxazole derivatives was evaluated for their in vitro and in vivo immunosuppressive activities. In a study on the derivatives of isoxazole[4,5-d]pyrimidine [8], two compounds, 8g and 10f, inhibited the humoral immune response to sheep erythrocytes (SRBC) in vivo, but only one of them suppressed (10f) the delayed-type hypersensitivity response (DTH), showing that closely related compounds may differentially affect these two major types of immune response. Suppressive properties of 5-amino-3-methylisoxazole-4-carboxylic acid amides and ureilenes [44], and 5-aminomethinimino-3-methyl-4-isoxazolecarboxylic acid phenylamides [9] on the humoral and cellular immune response in mice were subsequently found. One of the compounds, 5-(2-hydroxyethyl)piperazinomethinimino-3-methyl-4-isoxazolecarboxylic acid 4-(4-ethoxyphenyl)-amide (4d)[9] exhibited an exceptionally strong level of activity, comparable to cyclosporine. In turn, a study on the immunosuppressive activities of 5-substituted 3-methylisoxazole[5,4-d]-1,2,3-triazin-4-one derivatives [45], was supplemented with quantum-chemical calculations using AM1, a semi-empirical method for geometry optimization, the estimation of descriptor values, and the localization of the HOMO and LUMO orbital. The structure/activity investigations were extended by the application of the B3LYP hybrid exchange-correlation energy functional and the 6-31G(d, p) basis set. [46] The Polarizable Continuum (SCRF/PCM) solvent model was also taken into account, in order to show the solvent influence on the electron density, and the electrostatic potential around the exemplary molecules. Correlations between the molecular structure and its biological properties were subsequently found by using a stepwise selection of scales for the multiple linear regression (MLR). The studies on an isoxazole[5,4-e]triazepine derivative (compound RM33) led us to a demonstration of the very potent immunosuppressive activities of the compound in mouse models [10,47]. The compound effectively suppressed both the humoral response to SRBC, and the cellular response to ovalbumin (OVA) when administered intraperitoneally or per os. The compound could also act locally by the inhibition of foot pad edema when admixed with Freund’s complete adjuvant. In addition, serum tumor necrosis factor alpha (TNF α) and interleukin 6 (IL-6) levels, induced by administration of lipopolysaccharide (LPS) were reduced, but not an inducible interleukin 10 (IL-10) concentration in splenocyte cultures. RM33 inhibited also foot pad edema that was induced by carrageenan in Wistar rats [48], accompanied by lower serum TNF α levels. The inhibition of the foot pad edema was correlated with adequate histological changes, such as a lesser level of damage to mast cells, and smaller macrophage infiltration. Very recent (unpublished data) indicate that the immunosuppressive action of RM33 may consist of its apoptotic action on thymocytes and splenocytes, associated with strong increases of caspase 9, p53, and NFκB1 and NFκB2 expression. In another investigation of our group, a series of 5-amino-3-methylisoxazole[5,4-d]pyrimidin-4-one derivatives were obtained by the reaction of N0-substituted derivatives of 5-amino-3-methyl-1,2-oxazole-4-carbohydrazide with ethyl orthoformate [11]. The compounds differentially inhibited in vivo cellular and humoral immune responses in mice and pokeweed mitogen (PWM)-induced polyclonal antibody production in a culture of human peripheral Molecules 2018, 23, 2724 9 of 17 blood mononuclear cells (PBMC). The derivatives displayed differential inhibitory activities in these models, depending on the character and location of the substituted groups. Also, a significant suppression of the in vivo humoral immune response to SRBC in mice was achieved upon low doses of 5-amino-3-methyl-4-isoxazolecarboxylic acid semicarbazides and thiosemicarbazides [12]. Other studies on new lead structures of the isoxazole system [13] revealed their strong immunosuppressive activities in the humoral immune response, the carrageenan model, and the proliferative response of lymphocytes. Density Functional Theory (DFT) was employed to shed light on the molecular properties of the investigated compounds. The geometrical parameters of the studied molecular structures were fully optimized at the B3LYP/6-311G (d, p) level. The atomic charge distribution, derived on the basis of the Mulliken population analysis, was correlated with the immunological activity of the compounds. The obtained relationships revealed that the isoxazole ring played an important role in the observed immunological activities. In a study on 5-amino-3-methyl-4 isoxazolecarboxylic acid benzylamides [14] a selected MO5 compound (5-amino-3-methyl-N-[(4-methylphenyl)methyl]-1,2-oxazole-4-carboxamide) inhibited the humoral immune response in vitro and stimulated the inductive phase of DTH in vivo, although it suppressed the eliciting phase of that response and the foot pad carrageenan reaction. Its action on phytohemagglutinin A (PHA)-induced PBMC proliferation, and TNF α production was also inhibitory. In a parallel study [15], effects of 5-amino-3-methyl-1,2-oxazole-4-carbohydrazide on selective gene expression in Caco-2 cells were studied, together with Leflunomide (5-methyl-N-[4-(trifluoromethyl)phenyl]-4-isoxazolecarboxamide), as a reference drug. It appeared that both compounds similarly regulated selected genes i.e., the upregulation of fractalkine (CX3CL1) and IL-17F, and the downregulation of IL-10 and toll-like receptor 4 (TLR4). Nevertheless, the expression of 12 genes was differently regulated by these compounds: interleukins (IL) IL-1B, IL-6, and a chemokine CCL22 were upregulated by the compound, but significantly suppressed by Leflunomide. IL-2 and IL-27 in turn were upregulated by Leflunomide and suppressed by the compound. The authors concluded that the studied compound has a potential for further modifications as an immunosuppressive agent, with applications that are different from that of Leflunomide. In a new series of N0-substituted derivatives of 5-amino-N,3-dimethyl-1,2-oxazole-4- carbohydrazide [16], a selected 5-amino-N0-[2-(2,4-dihydroxyphenyl)ethylidene]-N,3-dimethyl-1,2- oxazole-4-carbohydrazide (MM3 compound) lowered the PHA-induced proliferation of PBMC and LPS-induced TNF α production in human blood cell culture. In the model of Jurkat cells, MM3 elicited strong increases in the expression of caspases, Fas, and NF-κB1, indicating that a proapoptotic action may account for its immunosuppressive action in the studied models.

2.2. Inhibition of Inflammation Izoxazole derivatives exhibiting anti-inflammatory properties constitute a major group from this class of potential therapeutics. In an early study [21] the authors demonstrated strong anti-inflammatory and antibacterial activity of p-etoxyphenylamid and p-chlorophenylamid of 5-benzoylamino-3-methyl-4-isoxazolecarboxylic acid. The most potent activity of these compounds were attributed to the benzoyl group in position 5 of the isoxazole ring. HWA-486 (5-methyl-N-[4-(trifluoromethyl)phenyl]-1,2-oxazole-4-carboxamide), together with cyclophosphamide, a reference drug, was investigated in the model of adjuvant arthritis in Lewis rats [17]. The compound prevented the development of the disease and suppressed the mitogen-elicited proliferation of lymphocytes derived from adjuvant arthritis rats. In contrast to cyclophosphamide, the compound did not affect the proliferation of lymphocytes from healthy rats. Leflunomide and its active metabolite A771726 [49] suppressed the immune response by means of various mechanisms [50,51]. In a model of J774.2 macrophages treated with LPS, both agents were shown to inhibit PGE2 accumulation in cell cultures. Further, in a whole blood human cell culture stimulated with calcium ionophore, Leflunomide inhibited both COX-1 and COX-2 activity [50]. In vivo effects of the compound were restricted by experimental substrate supply, and probably by Molecules 2018, 23, 2724 10 of 17 plasma binding. Other important actions of A771726 include suppression of proinflammatory cytokine production, such as TNF α and IL-1 [51]. In an extensive study on in vitro, ex vivo, and in vivo immunopharmacological effects of VGX-1027 (S,R)-(3-phenyl-4,5-dihydro-1,2-oxazol-5-yl)acetic acid [22], the investigators evaluated the potential efficacy of the compound in the amelioration of acute and chronic immunoinflammatory diseases, based on its ability to inhibit LPS-induced cell signaling pathways. A lack of toxicity predisposes the compound for further preclinical investigations. A potential utility of new fatty acid amine hydrolase (FAAH) inhibitors based on 3- carboxamido-5-aryl-isoxazole scaffolds in protection against experimental colitis was recently also proposed [23]. A search for novel anticancer drug via inhibition of LOX and COX enzymes constitutes an important strategy, since the overproduction of leukotrienes and prostaglandins contributes to tumor growth by inducing formation of new blood vessels that support tumor cell growth. It was demonstrated [24] that one of the synthesized compounds, 5-(3-methylthiophen-2-yl)-3-(3,4,5-trimethoxyphenyl)-1,2-oxazole (2b) exhibited significant inhibitory activity toward lipooxygenase (LOX) and COX-2. 4,5-diarylisoxazol-3-carboxylic acids could also serve as leukotriene synthesis inhibitors [25], and thus they are potential anti-inflammatory drugs. Macrophage migration inhibitory factor (MIF) is an inflammatory cytokine that is associated with inflammatory disorders, as only cytokine is resistant to steroid action. Airway hyper-responsiveness is also relatively resistant to steroid therapy. A MIF antagonist (S,R)-methyl [3-(4-oxocyclohexa-2,5-dien-1-ylidene)-1,2-oxazolidin-5-yl]acetate (ISO-1)[26] proved to be efficient in the suppression of lung inflammation in ozone-exposed mice, in contrast to Dexamethasone. MIF is also implicated in development of autoimmunity, so that the therapeutic efficacy of ISO-1 was also evaluated in a model of glomerulonephritis in lupus-prone NZB/NZW F1 and MLR/lpr mice [52]. The results showed that ISO-1, like anti-MIF, inhibited the interaction between MIF and its receptor, and reduced the functional and histological parameters of glomerulonephritis, leukocyte recruitment, proinflammatory cytokine and chemokine expression. These data support the therapeutic value of the compound by the downregulation of MIF-dependent pathways of tissue damage in systemic lupus erythematosus. For the prevention of another type of autoimmunity, (S,R)-(3-phenyl-4,5-dihydro-1,2-oxazol-5-yl)acetic acid (VGX-1027) was tested in a model of NOD mice, with spontaneous or accelerated forms of diabetes induced either by injection of cyclophosphamide or by transfer of spleen cells from acutely diabetic syngeneic donors, representing a preclinical model of human type 1 diabetes mellitus [53]. The compound significantly diminished the cumulative incidence of diabetes and insulitis in this model. The animals receiving VGX-1027 exhibited reduced production of the proinflammatory mediators, such as TNF α, IL-1β, and MIF as well as inducible nitric-oxide synthase-mediated nitric oxide generation in both pancreatic islets and peripheral organs. The authors recommended VGX-1027 for further investigations as a prime candidate to treat this type of the autoimmune disease. To suppress pulmonary inflammation, a novel glucocorticoids series of (GCs), 6α,9α-di-fluoro 3-substituted C-16,17-isoxazolines was designed [27]. A selected Cpd #15 compound inhibited LPS-induced nitric oxide release in Raw264.7 mouse macrophages, and TNF α-induced IL-8 production in human airway smooth muscle cells with a potency higher than that of Dexamethasone. The authors concluded that the compound displayed a pharmacokinetic and pharmacodynamic profile that was suitable for topical treatment of conditions that are associated with lung inflammatory reactions. A series of new isoxazole derivatives of expected immunosuppressive activities was synthesized [29]. Following in vitro screening in the human cell models, the activity of a most active MZO-2 compound (ethyl N-(4-{[(2,4- dimethoxyphenyl)methyl]carbamoyl}-3-methyl-1,2-oxazol-5-yl)ethanimidate) in mouse in vivo models was evaluated. The compound, administered intraperitoneally, inhibited carrageenan-induced footpad edema, and the contact sensitivity in mice to oxazolone when applied in ointment, with a Molecules 2018, 23, 2724 11 of 17 potency that was comparable to tacrolimus (Protopic®). The compound inhibited also expression of caspases 3, 8, and 9 in Jurkat cells. Several new 3-methylisoxazol-5(4H)-one/2-hydroxy/mercapto-6-methylpyrimidin-4(5H)-one/3 -methyl-1-substituted-1H-pyrazol-5(4H)-one substituted benzimidazole derivatives [30], were synthesized. A most potent representative of this series, 4-(2-(4-(1H-benzimidazol-2-yl) phenyl)hydrazono)-1-(4-chlorophenyl)-3-methyl-1H-pyrazol-5(4H)-one demonstrated significant analgesic activity, protection in carrageenan-induced footpad edema, efficient antibacterial, and moderate antiviral activities. Among the triazines, novel series [28] were synthesized, including isoxazoles with COX-2 inhibitory activity. To evaluate their anti-inflammatory activities, the compounds were investigated by the paw thickness inhibition assay. The most potent compound in the group of isoxazole derivatives that reduced the carrageenan-induced mice paw edema in comparison to Celecoxib, was compound 7a (N-(4-(isoxazol-5-yl)phenyl)-4,6-di(piperidin-1-yl)-1,3,5-triazin-2-amine) (51% inhibition of paw edema). Most of the compounds were even more potent than the reference drugs, such as: Celecoxib, Diclofenac, and Indomethacin. Analgesic and anti-inflammatory properties were also found when investigating isoxazole–mercaptobenzimidazole hybrids [31]. Indolyl–isoxazolidines are another category of compounds with potent anti-inflammatory-analgesic activities [32]. A selected compound, 9a, significantly inhibited LPS-induced TNF α and IL-6 production in macrophage THP-1 cells. The compound also caused analgesia and showed anti-inflammatory effects in the carrageenan test, with a potency that was comparable to that of indomethacin. Compound 9a was also effective in the protection of LPS-induced death in mice, indicating its potential usefulness as an analgesic/anti-inflammatory drug.

2.3. Immunoregulation Following demonstration of the suppressive effects of HWA-486 (Leflunomide) compound in adjuvant-induced arthritis in rats using cyclophosphamide, prednisolone, and cyclosporine A as reference drugs [17], the studies were extended by using several in vivo and ex vivo methods that were aimed at the elucidation of differences between the mechanisms of the actions of HWA-486 and of reference immunosuppressors. The authors came to a conclusion [17] that the compound has inhibitory activity on T-cell-dependent B-cell immune responses, yet it does not affect T-cell-independent B-cells; thus T-cell responsiveness may not be affected. Therefore, these findings may explain the disease-modifying activity of HWA-486 in adjuvant-induced arthritic rats. Immunoregulatory actions were described in a series of reports on the derivatives of phenylamides of 5-amino-3-methyl-4-isoxazolecarboxylic acid [44]. The activities of the compounds in the proliferative response of PBMC to PHA, and in LPS-induced cytokine production in the PBMC cultures were differential. The stimulatory or inhibitory effects depended strongly on the origin and location of substituents in the phenyl ring, as supported by QSAR (Quantitative Structure–Activity Relationship) studies. Similar conclusions could be drawn from in vivo and in vitro investigations on 4-imino derivatives of the 5-amino-3-methyl-1,2-oxazole-4-carbohydrazide and 5-amino-3-methylisoxazole[5,4-d]-6,7-dihydropyrimidine in mouse models [54]. Immunoregulatory properties were also observed with derivatives of 5-amino-3-methyl-1,2-oxazole-4-carbohydrazide [18]. The compound exhibited modulatory effects on T-cell subsets and levels of B-cells in lymphoid organs, and enhanced anti-SRBC antibody production in mice. The authors suggest its potential use in the treatment of autoimmune diseases, infections, or as an adjuvant for boosting the efficacy of vaccines. The immunoregulatory properties of 2-(5-amino-3-methyl-1,2-oxazole-4-carbonyl)-N-(4- chlorophenyl)hydrazine-1-carbothioamide (06K compound) in mouse in vivo models were also investigated [19]. Interestingly, the actions of the compounds were differential, depending on a model used. The compound stimulated antibody production against SRBC, as measured by the numbers of antibody-forming cells, but the cellular immune response to Molecules 2018, 23, 2724 12 of 17

OVA was decreased. Further, the compound significantly diminished carrageenan-induced foot pad edema. Phenotypic studies revealed that the immunoregulatory properties of the compound involved the mobilization of lymphopoiesis, and the generation of regulatory T cells. In another report with the application of in vitro models [20] 2-(5-amino-3-methyl-1,2-oxazole-4-carbonyl)-N-phenylhydrazine-1-carbothioamide (01K), and 2-(5- amino-3-methyl-1,2-oxazole-4-carbonyl)-N-(4-chlorophenyl)hydrazine-1-carbothioamide (06K) exhibited regulatory activity in the proliferation tests using cells from the thymus, spleen, and lymph nodes, as well as in the effects of the compounds on IL-1β, and the production of TNF-α in peritoneal cell cultures. These actions were associated with the different influence of the compounds on signaling protein expression in immature T cell Jurkat cell lines. Immunosuppressive isoxazole Leflunomide and a stimulatory RM-11 compound (1,7-dimethyl-8-oxo-1,2H-isoxazole[5,4-e]triazepine) were applied as reference drugs. A study on the immunoregulatory properties of the selected 5-amino-3-methyl-4- isoxazolecarboxylic acid benzylamides [14], and its selected derivative MO5 (5-amino- 3-methyl-N-[(4-methylphenyl)methyl]-1,2-oxazole-4-carboxamide), showed that the compounds 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 in mice, and inhibited LPS-induced TNF α production in human whole blood culture, as well as PHA-induced proliferation of PMBC.

2.4. Immunostimulation Immunostimulatory actions of therapeutics are equally important as immunosuppressive ones, and they are invaluable in the restoration of immune functions in immunocompromised individuals that are subjected to antibiotics and chemotherapy. In a study with HAB-439 isoxazoline derivative the compound appeared to be an inhibitor of aminopeptidase [33]. HAB-439 stimulated the DTH response to Salmonella typhimurium and Listeria monocytogenes. It also restored a decreased DTH reaction that was caused by the ampicillin treatment of mice infected with L. monocytogenes. Several immunostimulatory isoxazole derivatives were synthesized, and their potential therapeutic utility was evaluated by our research team. RM-11 (3,5-dimethyl- 5,6-dihydro-4H-[1,2]oxazolo[5,4-e][1,2,4]triazepin-4-one) was found to potently stimulate both humoral and cellular immune response to SRBC in mice [34]. The compound also stimulated ConA-induced splenocyte proliferation. RM-11 showed no sign of toxicity when given to mice at a dose of 250 mg/kg body weight. The compound was subsequently investigated in a model of immunocompromised mice treated with cyclophosphamide (CP) [35]. In this model, the compound, given i.p. in repeatable doses, caused a rapid recovery of both antibody production against SRBC and DTH responses to OVA, in comparison with control mice. In addition, the compound accelerated the process of myelopoiesis as measured by the percentage of neutrophils and their precursors in the peripheral blood. The compound was also protective in the humoral immune response in vitro to SRBS suppressed by methotrexate. The phenotypic studies revealed that RM-11 preferentially increased the percentage of mature, single positive CD4+ and CD8+ T cells in the spleen of normal mice. Similar results were obtained with another T-cell tropic isoxazole derivative, 3,5-dimethyl-5,8-dihydro-4H-[1,2]oxazolo[5,4-e][1,2,4]triazepin-4-one hydrochloride (R 13), which also induced a significant increase of CD4+ T cells in the spleen, and in the lymph nodes of mice [37]. The compound significantly accelerated both the antibody production and the cellular immune response. Unlike RM-11, this compound decreased the content of myelocytic cells in the circulating blood, but increased the level of immature lymphocyte forms, indicating the preferential promotion of lymphopoiesis in CP-treated mice. In contrast to the two previously described compounds, 3,5-dimethyl-5,8-dihydro-4H-[1,2]oxazolo[5,4-e][1,2,4]triazepin-4-one (R-11) appeared to preferentially induce the recruitment of CD19+ B cells in normal mice [36]. Moreover, the development of both the humoral immune response to SRBC and DTH to OVA was also accelerated Molecules 2018, 23, 2724 13 of 17 in CP-immunocompromised mice by this compound. The compound also stimulated IL-6 production, elicited by LPS in human whole blood cultures. We postulate that the above described compounds may be invaluable in the restitution of the immune function of patients undergoing chemotherapy. At present, the only therapeutic that is accepted for this purpose is granulocyte colony stimulating factor (Filgrastim®)[55]. However, the use of this cytokine has serious limitations, since it is costly and temperature-sensitive. In addition, this cytokine predominantly promotes myelopoiesis, the process is spontaneously recovered quickly [56]. However, the main problem with the reconstitution of the immune cell compartments of the chemotherapy patients are T- and B-cell compartments, where recovery takes much longer [57]. Other stimulatory izoxazole derivatives included 7-amino-3,5-dimethylisoxazole[5,4-e][1,3,4] -triazepin-4-one, showing the stimulatory effect on Con A-induced mouse splenocyte proliferation and cytokine production by the P388D1 macrophage cell line [58] and 2-(5-amino- 3-methyl-1,2-oxazole-4-carbonyl)-N-(prop-2-en-1-yl)hydrazine-1-carbothioamide (compound M4)[12], which stimulated PHA-induced proliferation of human PBMC. The aim of another study was to determine the immunomodulatory activity of 5-amino-3-methyl-1,2-oxazole-4-carbohydrazide in vitro [38]. The compound was not cytotoxic against reference cell lines, up to a concentration of 200 µg/mL. The compound stimulated the mitogen-induced proliferation of lymphocytes isolated from spleens and mesenteric lymph nodes when they were used alone and in combination with mitogens, and it increased LPS-elicited IL-1β production in peritoneal cell culture. Interestingly, among salicylic acid derivatives containing an isoxazole ring, 3-(4- methoxyphenyl)-4-(3-hydroxy-4-carboxybenzoyl)-5-(3-chlorophenyl)-4,5-dihydroisoxazoline (8e), which demonstrated mitogenic activity towards human lymphocytes and mouse splenocytes, was found [39]. The ability of the compound to stimulate cell division was caused by increased IL-2 secretion. It seems that an advantage of this new mitogenic compound is that it should not bind to and inactivate sugar-containing biologically active proteins, in contrast to lectins (ConA, PHA). A property of binding to sugars in biologically active glycoproteins like lactoferrin [59] by protein mitogens (lectins) may hamper the interpretation of results deriving from models where mitogenic lectins are applied.

3. Conclusions The isoxazole derivatives described herein have differential activities and mechanisms of action, and it may be applied in such disease and immunological disorders as inflammation, infection, autoimmune disorders, and impaired immune responsiveness. It is worthy to underline that many of the compounds, presented in this review, have higher potency in comparison to relevant reference drugs; thus they are potential therapeutics and they may replace registered drugs. Alternatively, they may enrich the pool of existing drugs in the market. Keeping in mind that even registered drugs are not completely devoid of side-effects and toxicity, the search for new compounds with the possible highest selectivity of action and the lowest toxicity is highly justified.

Funding: This research was funded by Wroclaw Medical University, grant number STD090.17.012. Conflicts of Interest: The authors declare no conflict of interest.

References

1. Giomi, D.; Cordero, F.M.; Machetti, F. Comprehensive Heterocyclic Chemistry III; Katrizky, A.R., Ramsden, C.A., Scriven, E.F.V., Taylor, R.J.K., Eds.; Elsevier: Oxford, UK, 2008; pp. 365–486. 2. Barmade, M.A.; Murumkar, P.R.; Sharma, M.K.; Yadav, M.R. Medicinal chemistry perspective of fused isoxazole derivatives. Curr. Top. Med. Chem. 2016, 16, 2863–2883. [CrossRef][PubMed] 3. Uto, Y. 1, 2-Benzisoxazole: A privileged structure with a potential for polypharmacology. Curr. Pharm. Des. 2016, 22, 3201–3211. [CrossRef][PubMed] Molecules 2018, 23, 2724 14 of 17

4. Sysak, A.; Obmi´nska-Mrukowicz, B. Isoxazole ring as a useful scaffold in a search for new therapeutic agents. Eur. J. Med. Chem. 2017, 137, 292–309. [CrossRef][PubMed] 5. Zhu, J.; Mo, J.; Lin, H.Z.; Chen, Y.; Sun, H.P. The recent progress of isoxazole in medicinal chemistry. Bioorg. Med. Chem. 2018, 23, 3065–3075. [CrossRef][PubMed] 6. Herrmann, M.L.; Schleyerbach, R.; Kirschbaum, B.J. Leflunomide: An immunomodulatory drug for the treatment of rheumatoid arthritis and other autoimmune diseases. Immunopharmacology 2000, 47, 273–289. [CrossRef] 7. Satyanarayana, P.S.; Jain, N.K.; Singh, S.; Kulkarni, S.K. Effect of selective inhibition of cyclooxygenase-2 on lipopolysaccharide-induced hyperalgesia. Inflammopharmacology 2004, 12, 57–68. [CrossRef][PubMed] 8. Wagner, E.; Al-Kadasi, K.; Zimecki, M.; Sawka-Dobrowolska, W. Synthesis and pharmacological screening of derivatives of isoxazolo[4,5-d]pyrimidine. Eur. J. Med. Chem. 2008, 43, 2498–2504. [CrossRef][PubMed] 9. Ryng, S.; Macho´n,Z.; Wieczorek, Z.; Zimecki, M.; Głowiak, T. Synthesis and structure elucidation of 5-aminomethinimino-3-methyl-4-isoxazolecarboxylic acid phenylamides and their immunological activity. Arch. Pharm. 1997, 330, 319–326. [CrossRef] 10. Ryng, S.; Zimecki, M.; Maczy´nski,M.; Chodaczek, G.; Kocieba, M. Immunosuppressive activity of an isoxazolo[5,4-e]triazepine—Compound RM33. I. Effects on the humoral and cellular immune response in mice. Pharmacol. Rep. 2005, 57, 195–202. [PubMed] 11. Maczy´nski,M.; Zimecki, M.; Drozd-Szczygieł, E.; Ryng, S. The synthesis, physicochemical properties and immunological activity of 5-amino-3-methylisoxazolo[5,4-d]4-pyrimidinone derivatives. Cell. Mol. Biol. Lett. 2005, 10, 613–623. [PubMed] 12. Maczy´nski, M.; Zimecki, M.; Taraszkiewicz, M.; Ryng, S. Synthesis, immunological activity and computational study of 5-amino-3-methyl-4-isoxazolecarboxylic acid semicarbazides and thiosemicarbazides. Acta Pol. Pharm. 2008, 65, 543–549. [PubMed] 13. Maczy´nski,M.; Ryng, S.; Artym, J.; Kocieba, M.; Zimecki, M.; Brudnik, K.; Jodkowski, J.T. New lead structures in the isoxazole system: Relationship between quantum chemical parameters and immunological activity. Acta Pol. Pharm. 2014, 71, 71–83. [PubMed] 14. Maczy´nski,M.; Artym, J.; Kocieba, M.; Sochacka-Cwikła,´ A.; Drozd-Szczygieł, E.; Ryng, S.; Zimecki, M. Synthesis and immunoregulatory properties of selected 5-amino-3-methyl-4 isoxazolecarboxylic acid benzylamides. Acta Pol. Pharm. 2016, 73, 1201–1211. [PubMed] 15. Płoszaj, P.; Regiec, A.; Ryng, S.; Piwowar, A.; Kruzel, M.L. Influence of 5-amino-3-methyl- 4-isoxazolecarbohydrazide on selective gene expression in Caco-2 cultured cells. Immunopharmacol. Immunotoxicol. 2016, 38, 486–494. [CrossRef][PubMed] 16. M ˛aczy´nski,M.; Borska, S.; Mieszała, K.; Koci˛eba,M.; Zaczy´nska,E.; Kochanowska, I.; Zimecki, M. Synthesis, immunosuppressive properties, and mechanism of action of a new isoxazole derivative. Molecules 2018, 23, 1545. [CrossRef][PubMed] 17. Bartlett, R.R.; Schleyerbach, R. Immunopharmacological profile of a novel isoxazol derivative, HWA 486, with potential antirheumatic activity—I. Disease modifying action on adjuvant arthritis of the rat. Int. J. Immunopharmacol. 1985, 7, 7–18. [CrossRef] 18. Drynda, A.; Obmi´nska-Mrukowicz, B.; M ˛aczy´nski, M.; Ryng, S. The effect of 5-amino-3-methyl- 4-isoxazolecarboxylic acid hydrazide on lymphocyte subsets and humoral immune response in SRBC-immunized mice. Immunopharmacol. Immunotoxicol. 2015, 37, 148–157. [CrossRef][PubMed] 19. Drynda, A.; Obmi´nska-Mrukowicz, B.; Zaczy´nska, E.; Zimecki, M.; Ryng, S.; M ˛aczy´nski, M. Immunoregulatory effects of 4-(4-chlorophenyl)-1-(5-amino-3-methylisoxazole-4-carbonyl)-thiosemicarbazide (06K) in non-immunized and SRBC-immunized mice. J. Pharm. Pharmacol. 2016, 68, 1613–1620. [CrossRef] [PubMed] 20. Drynda, A.; Obmi´nska-Mrukowicz, B.; Zaczy´nska,E.; Zimecki, M.; Kochanowska, I.; Ryng, S.; M ˛aczy´nski,M. 5-Amino-3-methyl-4-isoxazolecarboxylic acid hydrazide derivatives with in vitro immunomodulatory activities. Chem. Biol. Drug Des. 2017, 89, 705–713. [CrossRef][PubMed] 21. Macho´n,Z.; Ryng, S. Synthesis and biological properties of 5-benzoylamino-3-methyl-4-isoxazolocarboxylic acid derivatives. Arch. Immunol. Ther. Exp. 1981, 29, 813–821. Molecules 2018, 23, 2724 15 of 17

22. Stojanovic, I.; Cuzzocrea, S.; Mangano, K.; Mazzon, E.; Miljkovic, D.; Wang, M.; Donia, M.; Al Abed, Y.; Kim, J.; Nicoletti, F.; et al. In vitro, ex vivo and in vivo immunopharmacological activities of the isoxazoline compound VGX-1027: Modulation of cytokine synthesis and prevention of both organ-specific and systemic autoimmune diseases in murine models. Clin. Immunol. 2007, 123, 311–323. [CrossRef][PubMed] 23. Andrzejak, V.; Muccioli, G.G.; Body-Malapel, M.; El Bakali, J.; Djouina, M.; Renault, N.; Chavatte, P.; Desreumaux, P.; Lambert, D.M.; Millet, R. New FAAH inhibitors based on 3-carboxamido-5-aryl-isoxazole scaffold that protect against experimental colitis. Bioorg. Med. Chem. 2011, 19, 3777–3786. [CrossRef] [PubMed] 24. Rakesh, K.S.; Jagadish, S.; Balaji, K.S.; Zameer, F.; Swaroop, T.R.; Mohan, C.D.; Jayarama, S.; Rangappa, K.S. 3,5-disubstituted isoxazole derivatives: Potential inhibitors of inflammation and Cancer. Inflammation 2016, 39, 269–280. [CrossRef][PubMed] 25. Banoglu, E.; Çeliko˘glu,E.; Völker, S.; Olgaç, A.; Gerstmeier, J.; Garscha, U.; Çalı¸skan,B.; Schubert, U.S.; Carotti, A.; Macchiarulo, A.; Werz, O. 4,5-diarylisoxazol-3-carboxylic acids: A new class of leukotriene biosynthesis inhibitors potentially targeting 5-lipoxygenase-activating protein (FLAP). Eur. J. Med. Chem. 2016, 4, 1–10. [CrossRef][PubMed] 26. Russell, K.E.; Chung, K.F.; Clarke, C.J.; Durham, A.L.; Mallia, P.; Footitt, J.; Johnston, S.L.; Barnes, P.J.; Hall, S.R.; Simpson, K.D.; et al. The MIF antagonist ISO-1 attenuates corticosteroid-insensitive inflammation and airways hyperresponsiveness in an ozone-induced model of COPD. PLoS ONE 2016, 11, 0146102. [CrossRef][PubMed] 27. Ghidini, E.; Capelli, A.M.; Carnini, C.; Cenacchi, V.; Marchini, G.; Virdis, A.; Italia, A.; Facchinetti, F. Discovery of a novel isoxazoline derivative of prednisolone endowed with a robust anti-inflammatory profile and suitable for topical pulmonary administration. Steroids 2015, 95, 88–95. [CrossRef][PubMed] 28. Elshemy, H.A.H.; Abdelall, E.K.A.; Azouz, A.A.; Moawad, A.; Ali, W.A.M.; Safwat, N.M. Synthesis, anti-inflammatory, cyclooxygenases inhibitions assays and histopathological study of poly-substituted 1,3,5-triazines: Confirmation of regiospecific pyrazole cyclization by HMBC. Eur. J. Med. Chem. 2017, 127, 10–21. [CrossRef][PubMed] 29. M ˛aczy´nski,M.; Artym, J.; Koci˛eba,M.; Kochanowska, I.; Ryng, S.; Zimecki, M. Anti-inflammatory properties of an isoxazole derivative—MZO-2. Pharmacol. Rep. 2016, 68, 894–902. [CrossRef][PubMed] 30. Chikkula Krishna, V.; Sundararajan, R. Analgesic, anti-inflammatory, and antimicrobial activities of novel isoxazole/pyrimidine/pyrazole substituted benzimidazole analogs. Med. Chem. Res. 2017, 26, 3026–3037. [CrossRef] 31. Kankala, S.; Kankala, R.K.; Gundepaka, P.; Thota, N.; Nerella, S.; Gangula, M.R.; Guguloth, H.; Kagga, M.; Vadde, R.; Vasam, C.S. Regioselective synthesis of isoxazole-mercaptobenzimidazole hybrids and their in vivo analgesic and anti-inflammatory activity studies. Bioorg. Med. Chem. Lett. 2013, 23, 1306–1309. [CrossRef][PubMed] 32. Singh, G.; Singh, G.; Bhatti, R.; Gupta, M.; Kumar, A.; Sharma, A.; Singh Ishar, M.P. Indolyl-isoxazolidines attenuate LPS-stimulated pro-inflammatory cytokines and increase survival in a mouse model of sepsis: Identification of potent lead. Eur. J. Med. Chem. 2018, 153, 56–64. [CrossRef][PubMed] 33. Dickneite, G.; Schwab, W.; Schorlemmer, H.U.; Gebert, U.; Sedlacek, H.H. Effect of the new immunostimulator HAB 439 on cell-mediated immunity against intracellular bacteria. Int. J. Immunopharmacol. 1991, 13, 541–548. [CrossRef] 34. Ryng, S.; Sonnenberg, Z.; Zimecki, M. RM-11, a new izoxasole derivative, is a potent stimulator of the humoral and cellular immune responses in mice. Arch. Immunol. Ther. Exp. 2000, 48, 127–131. 35. Zimecki, M.; Artym, J.; Ryng, S.; Obmi´nska-Mrukowicz, B. RM-11, an isoxazole derivative, accelerates restoration of the immune function in mice treated with cyclophosphamide. Pharmacol. Rep. 2008, 60, 183–189. [PubMed] 36. Zimecki, M.; Artym, J.; Koci˛eba,M.; Obmi´nska-Mrukowicz, B.; M ˛aczy´nski,M.; Ryng, S. Restoration of immune system function is accelerated in immunocompromised mice by the B-cell-tropic isoxazole R-11. Pharmacol. Rep. 2012, 64, 403–411. [CrossRef] 37. Zimecki, M.; Artym, J.; Koci˛eba,M.; Obmi´nska-Mrukowicz, B.; M ˛aczy´nski,M.; Ryng, S. Immune function in cyclophosphamide-treated mice is restored by the T-cell-tropic isoxazole derivative R-13. J. Immunotoxicol. 2015, 12, 322–329. [CrossRef][PubMed] Molecules 2018, 23, 2724 16 of 17

38. Drynda, A.; M ˛aczy´nski, M.; Ryng, S.; Obmi´nska-Mrukowicz, B. In vitro immunomodulatory effects of 5-amino-3-methyl-4-isoxazolecarboxylic acid hydrazide on the cellular immune response. Immunopharmacol. Immunotoxicol. 2014, 36, 150–157. [CrossRef][PubMed] 39. Puttaswamy, N.; Pavan Kumar, G.S.; Al-Ghorbani, M.; Vigneshwaran, V.; Prabhakar, B.T.; Khanum, S.A. Synthesis and biological evaluation of salicylic acid conjugated isoxazoline analogues on immune cell proliferation and angiogenesis. Eur. J. Med. Chem. 2016, 114, 153–161. [CrossRef][PubMed] 40. Kremer, J.M. Methotrexate and leflunomide: Biochemical basis for combination therapy in the treatment of rheumatoid arthritis. Semin. Arthritis Rheum. 1999, 29, 14–26. [CrossRef] 41. Furst, D.E. Innovative treatment approaches for rheumatoid arthritis. Cyclosporin, leflunomide and nitrogen mustard. Baillieres Clin. Rheumatol. 1995, 9, 711–729. [CrossRef] 42. Schug, S.A.; Parsons, B.; Li, C.; Xia, F. The safety profile of parecoxib for the treatment of postoperative pain: A pooled analysis of 28 randomized, double-blind, placebo-controlled clinical trials and a review of over 10 years of postauthorization data. J. Pain Res. 2017, 10, 2451–2459. [CrossRef][PubMed] 43. Zhang, J.; Ding, E.L.; Song, Y. Adverse effects of cyclooxygenase 2 inhibitors on renal and arrhythmia events meta-analysis of randomized trials. J. Am. Med. Assoc. 2006, 296, 1619–1632. [CrossRef][PubMed] 44. Ryng, S.; Macho´n, Z.; Wieczorek, Z.; Zimecki, M. Synthesis and immunological activity of new 5-amino-3-methyl 4-amido and 4-ureilene isoxazole derivatives. Pharmazie 1999, 54, 359–361. [CrossRef] [PubMed] 45. Maczy´nski,M.; Jezierska, A.; Zimecki, M.; Ryng, S. Synthesis, immunological activity and theoretical study of new 5-substituted 3-methylisoxazole[5,4-d] 1,2,3-triazin-4-one derivatives. Acta Pol. Pharm. 2003, 60, 147–150. [PubMed] 46. Jezierska, A.; Maczy´nski, M.; Koll, A.; Ryng, S. Structure/activity investigations of 5-substituted 3-methylisoxazole[5,4-d]1, 2, 3-triazin-4-one derivatives. Arch. Pharm. 2004, 337, 81–89. [CrossRef][PubMed] 47. Zimecki, M.; M ˛aczy´nski,M.; Artym, J.; Ryng, S. Closely related isoxazoles may exhibit opposite immunological activities. Acta Pol. Pharm. 2008, 65, 793–794. [PubMed] 48. Zimecki, M.; Ryng, S.; Maczy´nski, M.; Chodaczek, G.; Kocieba, M.; Kuryszko, J.; Kaleta, K. Immunosuppressory activity of an isoxazolo[5,4-e]triazepine-compound RM-33 II. Effects on the carrageenan-induced inflammation. Pharmacol. Rep. 2006, 58, 236–241. [PubMed] 49. Kalgutkar, A.S.; Nguyen, H.T.; Vaz, A.D.; Doan, A.; Dalvie, D.K.; McLeod, D.G.; Murray, J.C. In vitro metabolism studies on the isoxazole ring scission in the anti-inflammatory agent lefluonomide to its active alpha-cyanoenol metabolite A771726: Mechanistic similarities with the cytochrome P450-catalyzed dehydration of aldoximes. Drug Metab. Dispos. 2003, 31, 1240–1250. [CrossRef][PubMed] 50. Hamilton, L.C.; Vojnovic, I.; Warner, T.D. A771726, the active metabolite of leflunomide, directly inhibits the activity of cyclo-oxygenase-2 in vitro and in vivo in a substrate-sensitive manner. Br. J. Pharmacol. 1999, 127, 1589–1596. [CrossRef][PubMed] 51. Yao, H.W.; Li, J.; Chen, J.Q.; Xu, S.Y. A 771726, the active metabolite of leflunomide, inhibits TNF-alpha and IL-1 from Kupffer cells. Inflammation 2004, 28, 97–103. [CrossRef][PubMed] 52. Leng, L.; Chen, L.; Fan, J.; Greven, D.; Arjona, A.; Du, X.; Austin, D.; Kashgarian, M.; Yin, Z.; Huang, X.R.; et al. A small-molecule macrophage migration inhibitory factor antagonist protects against glomerulonephritis in lupus-prone NZB/NZW F1 and MRL/lpr mice. J. Immunol. 2011, 186, 527–538. [CrossRef][PubMed] 53. Stosic-Grujicic, S.; Cvetkovic, I.; Mangano, K.; Fresta, M.; Maksimovic-Ivanic, D.; Harhaji, L.; Popadic, D.; Momcilovic, M.; Miljkovic, D.; Kim, J.; et al. A potent immunomodulatory compound, (S,R)-3-Phenyl-4,5-dihydro-5-isoxazole acetic acid, prevents spontaneous and accelerated forms of autoimmune diabetes in NOD mice and inhibits the immunoinflammatory diabetes induced by multiple low doses of streptozotocin in CBA/H mice. J. Pharmacol. Exp. Ther. 2007, 320, 1038–1049. [PubMed] 54. Ryng, S.; Zimecki, M.; Fedorowicz, A.; Jezierska, A. Reactions of 5-amino-3-methylisoxazole-4-carboxylic acid hydrazide with carbonyl compounds: Immunological activity and QSAR studies of products. Arch. Pharm. 2001, 334, 71–78. [CrossRef] 55. Mitchell, S.; Li, X.; Woods, M.; Garcia, J.; Hebard-Massey, K.; Barron, R.; Samuel, M. Comparative effectiveness of granulocyte colony-stimulating factors to prevent febrile neutropenia and related complications in cancer patients in clinical practice: A systematic review. J. Oncol. Pharm. Pract. 2016, 22, 702–716. [CrossRef][PubMed] Molecules 2018, 23, 2724 17 of 17

56. Renwick, W.; Pettengell, R.; Green, M. Use of filgrastim and pegfilgrastim to support delivery of chemotherapy: Twenty years of clinical experience. BioDrugs 2009, 23, 175–186. [CrossRef][PubMed] 57. Talmadge, J.E.; Jackson, J.D.; Borgeson, C.D.; Perry, G.A. Differential recovery of polymorphonuclear neutrophils, B- and T-cell sub-populations in the thymus, bone marrow, spleen and blood of mice following split-dose polychemotherapy. Cancer Immunol. Immunother. 1994, 39, 59–67. [CrossRef][PubMed] 58. Drozd-Szczygieł, E.; Ryng, S.; Zimecki, M. 7-amino-3,5-dimethylisoxazole [5,4-e] [1,3,4]-triazepin-4-one- a potential new lead structure: Its structure, physicochemical properties and biological activity. Acta Pol. Pharm. 2004, 61, 84–85. [CrossRef][PubMed] 59. Zimecki, M.; Stepniak, D.; Szynol, A.; Kruzel, M.L. Lactoferrin regulates proliferative response of human peripheral blood mononuclear cells to phytohemagglutinin and mixed lymphocyte reaction. Arch. Immunol. Ther. Exp. 2001, 49, 147–154.

Sample Availability: Sample Availability: Not available.

© 2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).