Virginija Jakubkiene

Anti-Inflammatory Activity of a Novel Family of Aryl Ureas Compounds in an Endotoxin-Induced Airway Epithelial Cell Injury Model

Background Despite our increased understanding of the mechanisms involved in acute lung injury (ALI) and the acute respiratory distress syndrome (ARDS), there is no specific pharmacological treatment of proven benefit. We used a novel screening methodology to examine potential anti-inflammatory effects of a small structure-focused library of synthetic carbamate and urea derivatives in a well established cell model of lipopolysaccharide (LPS)-induced ALI/ARDS. Methodology/Principal Findings After a pilot study to develop an in vitro LPS-induced airway epithelial cell injury model, a library of synthetic carbamate and urea derivates was screened against representative panels of human solid tumor cell lines and bacterial and fungal strains. Molecules that were non-cytotoxic and were inactive in terms of antiproliferative and antimicrobial activities were selected to study the effects on LPS-induced inflammatory response in an in vitro cell culture model using A549 human alveolar and BEAS-2B human bronchial cells. These cells were exposed for 18 h to LPS obtained from Escherichia coli, either alone or in combination with the test compounds. The LPS antagonists rhein and emodin were used as reference compounds. The most active compound (CKT0103) was selected as the lead compound and the impact of CKT0103 on pro-inflammatory IL-6 and IL-8 cytokine levels, expression of toll-like receptor-4 (TLR4) and nuclear factor kappa B inhibitor alpha (IκBα) was measured. CKT0103 significantly inhibited the synthesis and release of IL-6 and IL-8 induced by LPS. This suppression was associated with inhibition of TLR4 up-regulation and IκBα down-regulation. Immunocytochemical staining for TLR4 and IκBα supported these findings. Conclusions/Significance Using a novel screening methodology, we identified a compound – CKT0103 – with potent anti-inflammatory effects. These findings suggest that CKT0103 is a potential target for the treatment of the acute phase of sepsis and sepsis-induced ALI/ARDS.

Synthesis of 5-(6-Methyl-2,4-dioxo-1,2,3,4-tetrahydro-3-pyrimidinyl)-methyl-4-amino-1,2,4-triazole-3-thione and its Reactions with Polyfunctional Electrophiles

Summary. In the reaction of 5-(6-methyl-2,4-dioxo-1,2,3,4-tetrahydro-3-pyrimidinyl)-methyl-1,3,4-oxadiazole-2-thione with hydrazine hydrate, 5-(6-methyl-2,4-dioxo-1,2,3,4-tetrahydro-3-pyrimidinyl)-methyl-4-amino-1,2,4-triazole-3-thione was formed. The reactions of the latter with ethyl bromoacetate and chloroacetonitrile in the presence of triethylamine proceeded under formation of the corresponding S-alkylated derivatives, whereas from its reaction with ω-bromoacetophenone and ethyl 4-chloroacetoacetate triazolothiadiazines were obtained. Treatment of the title compound with ethyl 2-chloroacetoacetate led to the formation of 5-(6-methyl-2,4-dioxo-1,2,3,4-tetrahydro-3-pyrimidinyl)-methyl-4-N-acetylamino-(3-ethoxy-carbonylmethylthio)-1,2,4-triazole. Performing of the latter reaction without basic catalyst gave a triazolothiadiazine. Treatment of the S-alkylated derivatives with sodium methoxide resulted in triazolothiadiazines via a cyclocondensation reaction.

Synthesis of 1-(6-methyl-2,4-dioxo-1,2,3,4-tetrahydro-3-pyrimidinyl)acetyl-4-alkyl(aryl)thios emicarbazides and their heterocyclisation to 1,2,4-triazoles and 1,3,4-thiadiazoles

5-(6-Methyl-2,4-dioxo-1,2,3,4-tetrahydro-3-pyrimidinyl)methyl-1,3,4-oxadiazole-2-thione reacts with amines to give 1-(6-methyl-2,4-dioxo-1,2,3,4-tetrahydro-3-pyrimidinyl)acetyl-4-alkyl(aryl)thiosemicarbazides, which on treatment with base or acid undergo cyclisation to 4-alkyl-1,2,4-triazole-2-thiones or 4-amino-1,3,4-thiadiazoles, respectively.

Reactions of 5-(6-Methyl-2,4-dioxo-1,2,3,4-tetrahydro-3-pyrimidinyl)-methyl-1,3,4-oxadiazole-2-thione with Electrophiles

Summary. Treatment of 5-(6-methyl-2,4-dioxo-1,2,3,4-tetrahydro-3-pyrimidinyl)-methyl-1,3,4-oxadiazole-2-thione with haloalkanes yielded oxadiazole S-alkyl derivatives, whereas its reaction with formaldehyde and amines resulted in formation of oxadiazole N(3)-aminomethyl derivatives. The alkylation of 2-alkylsulfanyl-5-(6-methyl-2,4-dioxo-1,2,3,4-tetrahydro-3-pyrimidinyl)-methyl-1,3,4-oxadiazoles with methyl bromoacetate proceeded at the N(1)-position of pyrimidine to give 2-alkylsulfanyl-5-(1-methoxycarbonylmethyl-6-methyl-2,4-dioxo-1,2,3,4-tetrahydro-3-pyrimidinyl)-methyl-1,3,4-oxadiazoles, whereas aminomethylation, bromination, or nitration took place at position 5 of pyrimidine ring and afforded the corresponding 5-pyrimidine substituted derivatives.

Synthesis of novel derivatives of 5-(4,6-dimethyl-2-pyrimidinylsulfanyl)methyl-1,2,4-triazole-3-thione

Alkylation of 5-(4,6-dimethyl-2-pyrimidinylsulfanyl)methyl-1,2,4-triazole-3-thione (2) with various alkyl halides, 4-chlorophenacyl bromide, chloroacetic acid, and α-chloroacetanilide afforded S-substituted 1,2,4-triazoles (3–11). 3-Carboxymethylsulfanyl-5-(4,6-dimethyl-2-pyrimidinylsulfanyl)methyl-1,2,4-triazole (9), in the presence of acetic anhydride, was cyclized to 6-(4,6-dimethyl-2-pyrimidinylsulfanyl)methylthiazolo[3,2-b][1,2,4]triazol-3(2H)-one (12). The later condensed with aromatic aldehydes to give 6-(4,6-dimethyl-2-pyrimidinylsulfanyl)methyl-6-[(1-aryl)methylidene]thiazolo[3,2-b][1,2,4]triazol-3(2H)-ones (13, 14) and under treatment with aniline underwent ring-disclosure reaction to yield 3-(phenylcarbamoyl)methylsulfanyl-5-(4,6-dimethyl-2-pyrimidinylsulfanyl)methyl-1,2,4-triazole (11).

The first example of the Fischer–Hepp type rearrangement in pyrimidines

Summary A N-nitroso moiety can be used for the activation of chloropyrimidines toward a nucleophilic substitution reaction with amines. The subsequent treatment of the obtained products with aq H2SO4 can lead to either N-denitrosation to obtain 4,6-pyrimidinediamines or to a Fischer–Hepp type rearrangement to obtain 5-nitroso-4,6-pyrimidinediamines. It was found that the outcome of the reaction strongly depends on the structure of the pyrimidines. Activation of the pyrimidine ring by three groups with a positive mesomeric effect is crucial for the intramolecular nitroso group migration.

Transformations of methyl 6-methyl-2-methylsulfanyl-4-oxo-3,4-dihydro-3-pyrimidinylacetate under oxidative conditions

The oxidation of methyl 6-methyl-2-methylsulfanyl-4-oxo-3,4-dihydro-3-pyrimidinylacetate by reagents which oxidized the SMe group to SO2Me gave the products of the further transformation of the corresponding 2-methylsulfonyl-substituted ester obtained: methyl 5,5-dichloro-6-methoxy-6-methyl-2,4-dioxohexahydro-3-pyrimidinylacetate (using Cl2 in 70 or 50% MeOH), its mixture (about 1:10) with methyl 6-methyl-2,4-dioxo-1,2,3,4-tetrahydro-3-pyrimidinylacetate (Cl2 in 30% MeOH) or only to the latter compound (Cl2 in H2O, m-ClC6H4CO3H in CHCl3, H2O2 in MeOH). The reaction did not take place with NaOCl in DMF.

Synthesis and properties of (6-methyl-2-oxo-4-thioxo-1,2,3,4-tetrahydro-3-pyrimidinyl)acetic acid methyl ester

On interacting of the methyl ester of (6-methyl-2,4-dioxo-1,2,3,4-tetrahydro-3-pyrimidinyl)acetic acid with the Lawesson’s reagent the corresponding 4-thioxo derivative is synthesized. Its alkylation with methyl bromoacetate has been studied as has its interaction with N-nucleophiles, amines, and hydrazines.