Synthesis, physicochemical properties of allopurinol derivatives and their biological activity against Trypanosoma cruzi.

Chagas disease is caused by Trypanosoma cruzi (T. cruzi) leading to a huge number of infections and deaths per year, because in addition to many sufferers only having limited access to health services only an inefficient chemotherapy is available using drugs such as benznidazole and nifurtimox. Here, C6-alkyl (2a-c) and N1-acyl (3a-c) derivatives of Allopurinol (Allop, compound with activity against T. cruzi) were synthesized in good yields and their structures were unambiguously characterized. Only 2a, 2b and 3c showed inhibitory activity against the proliferative stages of the parasite when tested at 1 µg mL(-1) with the 3c derivative exhibiting an IC50 value similar to that of Allop and not being toxic for mammalian cells. Relevant pharmaceutical physicochemical properties (pKa, stability, solubility, lipophilicity) were also determined as well by using Lipinski's rule, polar surface area and molecular rigidity. Taken together, the results demonstrated that the studied derivatives had optimal properties for bioavailability and oral absorption. For the stability studies, Micellar Liquid Chromatography was used as the analytical method which was fully validated according to the FDA guidelines and shown to be a suitable, sensitive and simple method for routine analysis of these Allop derivatives.

Synthesis, antiprotozoal and cytotoxic activities of new N-(3,4-dimethyl-5-isoxazolyl)-1,2-naphthoquinone-4-amino derivatives.

Three derivatives of N-(3,4-dimethyl-5-isoxazolyl)-1,2-naphthoquinone-4-amino (1), a compound which exhibits significant activity against Trypanosoma cruzi and Plasmodium falciparum but with cytotoxicity toward murine L-6 cells, were synthesized with the aim of ameliorating its cytotoxicity. The in vitro antiprotozoal and cytotoxic activities of the synthesized compounds were evaluated against T. cruzi, Trypanosoma brucei rhodesiense, P. falciparum and murine L-6 cells. The hydroxymethyl (2) and the oxime (3) derivatives were active against T. cruzi, with IC50 values in a range comparable to those of 1 (IC50: 0.65 microg/ml) and benznidazole (IC50: 0.56 microg/ml) while the carboxymethyloxime (4) was inactive. Compounds 2 and 3 were cytotoxic toward L-6 cells, with IC50 values identical to that of 1 (IC50: 0.50 microg/ml). The results did not support the suggestion that 2 and 3 may be used as prodrugs of 1.

Human serum albumin binding of novel antiretroviral nucleoside derivatives of AZT.

The binding of novel nucleoside derivatives (2-7) to the Human Serum Albumin (HSA) was studied using zidovudine (AZT), as standard compound. The applicability of two different techniques to separate unbound drug from drug-protein complex was analyzed: the gel filtration and ultrafiltration methods. Ultrafiltration was found to be an adequate procedure for the separation of unbounded drug from the drug-protein complex. Incubation temperature ranging from 0 to 37 degrees C did not modify considerably the bound fractions. The same effects were observed as HSA concentration was modified. Binding assays of studied compounds to purified 1% (w/v) HSA at 0 degrees C, indicate that bound fraction of 2-7 ranges from 13 to 47%, exhibiting a higher affinity to HSA than AZT (12%), which would introduce some interesting improvements in their pharmacokinetic properties. In addition, by means of displacement studies using HSA site specific drugs such as diazepam and salicylate, it was determined that AZT binds to site I of the HSA molecule, by a mainly entropy driven process (DeltaS = 10.834 cal/mol degrees K), being these observations extensive to 2-7. Some structural basis to explain enhanced affinity of these novel derivatives was also established.

Solubility profiles of some isoxazolyl-naphthoquinone derivatives.

Water, ethanol and n-hexane solubility and pH-solubility behavior of a homologous series of isoxazolyl-naphthoquinone derivatives, which exhibit important biological activity, were studied. Their pK(a) values were determined spectrophotometrically as well as from their pH-solubility profiles. Also, the molar aqueous solubility of these compounds was estimated with the equations developed by Yalkowsky and Valvani using the data of their melting points and octanol/water partition coefficients.

Mechanism of antibacterial and degradation behavior of a chlorinated isoxazolylnaphthoquinone.

The chemical stability of 3-chloro-2-hydroxy-(3, 4-dimethyl-5-isoxazolyl)-1,4-naphthoquinon-4-imine (ClQ(1)), a new potential antimicrobial agent was analyzed at different pH values by first-derivative spectroscopy. The degradation of ClQ(1) followed a pseudo-first-order kinetics in aqueous media at different pH values. The interaction of antibiotics with respiratory chain of Staphylococcus aureus generates superoxide anion, an oxygen radical capable of producing damage to the bacteria. The performed assays have demonstrated that ClQ(1) presents higher activity and toxic oxidant generation at pH 5.0 than at pH 7.5. In addition, the antibacterial activity of other halogenated isoxazolylnaphthoquinones was also studied in different collection and clinical strains which presented the following decreasing activity, ClQ(1) > BrQ(1) > DClQ(1) whereas DBrQ(1) did not show inhibition properties. The antibacterial and stability properties evidenced by ClQ(1) are so important that must be taken into account when new alternative treatments against beta-lactamase-positive S. aureus strains are investigated.

Synthesis, lipophilicity and anti-HIV activity of a new brominated analog of zidovudine.

A novel cyclic bromine zidovudine analog, (-)-trans-(5S,6S)-5-bromo-6,5'- epoxy-5,6-dihydro-3'-azido-3'-deoxythymidine (2), and its diastereoisomer (+)-trans-(5R,6R)-(3) were synthesized and characterized by spectroscopic methods, obtaining 3 in very low yields. The major product 2 presents a selectivity index (CCID50/IC50) similar to zidovudine but 55.5 times with higher lipophilicity, which should increase the ability of 2 to cross the blood-brain barrier by a non facilitated diffusion mechanism.

Second-derivative UV spectrophotometric assay for determining the degradation kinetics of 3-bromo-N-bromo-N-(3,4-dimethyl-5-isoxazolyl-4-amine)-1,2-naphthoquinone.

The application of the second-derivative UV spectrophotometry for determining the stability of 3-bromo-N-bromo-N-(3,4-dimethyl-5-isoxazolyl-4-amine)-1,2-naphthoquinone in ethanolic solutions is described. The validity of this method was evaluated using synthetic mixtures of the intact drug and its degradation products and by statistical analysis of the calibration data. In order to verify the usefulness of this method for stability studies, recovery experiments by the standard addition method were also carried out.

Stability determination of 3-bromo-2-hydroxy-N-(3,4-dimethyl-5-isoxazolyl)- 1,4-naphthoquinon-4-imine in ethanol by first-derivative spectrophotometry.

The degradation kinetics of a new potential tripanocidal and antibacterial agent, 3-bromo-2-hydroxy-N-(3,4-dimethyl-5-isoxazolyl)-1,4- naphthoquinon-4-imine (2), in 95% ethanol, was investigated between 35 and 50 degrees C under room-light and light-protected conditions. The decomposition product was isolated and identified as 2-hydroxy-N-(3,4-dimethyl- 5-isoxazolyl)-1,4-naphthoquinon-4-imine (1). A simple, rapid, and stability-indicating method for the determination of 2 in the presence of 1 using "zero crossing" first-derivative spectrophotometry is reported. The validity of this method was proved using synthetic mixtures of the intact drug with its decomposition product and by statistical analysis of the calibration data. Pseudo-first-order constants for the degradation reaction of 2, obtained from linear plots of the residual concentration logarithms vs time, the calculated activation parameters Ea, delta H not equal to, and delta S not equal to were similar under room-light and light-protected conditions. The in vitro antibacterial activity of 2 was also evaluated.

Synthesis and some physiochemical properties of 2-hydroxy-N-(3,4-dimethyl-5-isoxazolyl)-1,4-naphthoquinone 4-imine derivatives.

Some derivatives of 2-hydroxy-N-(3,4-dimethyl-5-isoxazolyl)- 1,4-naphthoquinone 4-imine (3), a poorly soluble drug, were synthesized in an attempt to improve their physicochemical properties. The new compounds were characterized by spectroscopic methods including an iterative NMR method (the LAOCOON III program). The physicochemical properties such as solubility, relative lipophilicity (RM), and partition coefficients (Leo-Hansch fragmental system) were determined. Some derivatives were more lipophilic than 3 and one was water soluble. In vitro antibacterial activity was also reported for some derivatives.

Isoxazoles. VII: Hydrolysis of 4-methyl-5-isoxazolylnaphthoquinone derivatives in aqueous solutions.

The kinetics for the degradation of 2-(4-methyl-5-isoxazolylamine)-N-(4-methyl-5-isoxazolyl)-1,4 -naphthoquinone-4- imine (1) in solution were investigated at 70 degrees C and at a constant ionic strength of 0.5 over a pH range of 1.75 to 12.85. The degradation rates were determined by absorption and second-derivative UV spectrometry. Two degradation products were identified in acidic and neutral pHs; they are 4-N-(4-methyl-5-isoxazolyl)-1,2-naphthoquinone (2) and 2-methyl-cyanoacetamide (5), respectively. In alkaline pH, two degradation products, 2-hydroxy-N-(4-methyl-5-isoxazolyl)-1,4-naphthoquinone-4-imine (3) and 5-amino-4-methylisoxazole (4), were isolated. The pathway for degradation of 1 in acidic and neutral pH followed consecutive first-order kinetics since 2 undergoes hydrolysis giving 2-hydroxy-1,4-napthoquinone (6) and 2-methylcyanoacetamide (5). No appreciable buffer effect on the degradation of 1 and 2 was observed for any of the buffer species in this study. The pH-rate profiles exhibited specific acid and specific basic catalysis for 1 and specific acid catalysis for 2. The maximum stability for 1 and 2 occurred in the neutral pH region.

Isoxazoles V: chemical stability of diisoxazolylnaphthoquinone in aqueous solution.

The hydrolytic degradation of 2-(3,4-dimethyl-5-isoxazolylamine)-N-(3,4-dimethyl-5-isoxazolyl )-1,4- naphthoquinone-4-imine (1) was investigated over a wide range of pH values and at different temperatures. The degradation rates were determined by reversed-phase HPLC and were observed to follow pseudo-first-order kinetics with respect to the concentration of 1. The pH-rate profile was linear with slopes -1 and +1 in acid and alkaline pH, respectively, becoming pH independent in the region of maximum stability from pH 4.5 to 10.0. Neither primary salt effects nor buffer catalysis was observed due to the buffer species employed.