Novel 1,2,5-Oxadiazole 2-Oxide Derivatives with Analgesic and Fetal Hemoglobin Induced Properties Designed As Drug Candidate to Treat Sickle Cell Disease Symptoms

The combination of multiple activities in the same structure using molecular modification is a powerful tool to discover more effective and safe compounds to treat sickle cell disease (SCD) symptoms. The only FDA approved drug for SCD, at present, is hydroxyurea (HU). The release of NO by HU is an important mechanism of its HbF-inducing properties. Thalidomide and some analogues are known to inhibit cytokines release, presenting important analgesic activity. Recently the drug has also been reported to induce gamma-globin expression. Our rational drug design used molecular hybridization between thalidomide and NO donor subunits, represented by 1,2,5-oxadiazole 2-oxide. The aim of this study was to evaluate the effects of eight novel hybrid compounds (3a-h) on NO donor activity, analgesic activity and γ-globin gene expression. Methods. 1. Detection of nitrite. A solution of the appropriate compound (20 μL) in DMSO was added to 2 mL of a mixture of 50 mM phosphate buffer (pH 7.4) and methanol (1:1, v:v), containing 5 mM of L-cysteine. The final concentration of the compound was 10⁻₄ M. After 1 h at 37 °C, 1 mL of the reaction mixture was treated with 250 μL of Griess reagent. After 10 min at room temperature, the absorbance was measured at 540 nm using a spectrophotometer. Standard sodium nitrite solutions (10–80 nmol/mL) were used to construct the calibration curve. The yields of nitrite are expressed as % NO₂^∓ (mol/mol). 2. Antinociceptive activity. Analgesic activity was determined in vivo with the acetic-acid-induced (0.6%, 0.1 mL/10 g) abdominal constriction test in mice. Swiss mice of both sexes (18–23 g) were used. The compounds were administered orally (100 μmol/kg) as a suspension in 5% arabic gum in saline (vehicle). Dypirone (100 μmol/kg) was used as the standard drug. Acetic acid solution was administered i.p. 1 h after the administration of the compounds. Ten minutes after the i.p. acetic acid injection, the number of constrictions per animal was recorded for 20 min. The control animals received an equal volume of vehicle. Antinociceptive activity was expressed as percentage inhibition of the constrictions compared with those in the vehicle-treated control group. The data were analyzed statistically with Student's t test at a significance level of P < 0.05. 3. Gamma-globin gene expression. Human K562 cells were maintained in DMEM with 10% FBS, Pen/Strep, in humidified air (5% CO_2, 37°C). Cells (1×10⁷cells/100mL) were incubated with compounds at different concentrations (5, 30, 60, and 100μM) for 24, 48, 72 and 96h. g-Globin gene expression was analyzed by qRT-PCR and quantified using the Gnorm program. Results are expressed as arbitrary units. Results. 1. Detection of nitrite: The quantification of the nitrite produced resulted from the oxidative reaction of NO, oxygen and water. All eight compounds were capable of inducing nitrite formation at concentrations of between 9.5% and 28%. Isosorbide dinitrate (DNS), used as the control, induced 11.7% nitrite formation. 2.Antinociceptive activity: Compounds 3c and 3d were the most active antinociceptive compounds, significantly reducing the acetic-acid-induced abdominal constrictions by 43% and 38%, respectively, while dypirone used as a control inhibited constrictions by 34%. 3. Gamma-globin gene expression: Compound 3c demonstrated inverse dose-response relationships, achieving the highest levels of γ-globin induction at 5 and 30 μM. Compound 3c achieved maximal γ-globin induction as early on as 48h after treatment ([48h; 5 μM]: 1,88±0.06 AU; control 0.78±0.1 AU; P<0.05). Testing of compound 3d is currently underway. Conclusions. Results demonstrate that molecular hybridization between a thalidomide derivative and nitric oxide donors may be an important tool to treat SCD symptoms. These compounds demonstrated NO donor and analgesic activity. Specifically, the compound 3c was capable of inducing gamma-globin expression. Testing of our lead compound 3c in a preclinical mouse model of SCD will be performed in order to evaluate its efficacy in the treatment of this hemoglobinopathy.

No relevant conflicts of interest to declare.