Fused pyrimidines as d-alanyl-d-alanine ligase inhibitors
A technology of amino and compound, which is applied in the direction of drug combination, medical preparations containing active ingredients, antibacterial drugs, etc., and can solve the problem of decreased sensitivity
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Embodiment 1
[0214] Example 1 - Synthesis of Ligase Inhibitors
[0215]
[0216] N7-methyl-N7-(1-naphthalen-1-yl-propyl)-pyrimido[4,5-d]pyrimidine-2,4,7-triamine
[0217] Compound 1 : In an ice-water bath, slowly add 13 ml of ethylmagnesium bromide in ether (3M) to a solution of 4.25 g (27.2 mmol) of naphthyl formaldehyde in 30 ml of anhydrous ether. The mixture was stirred at room temperature for 30 minutes, then quenched by the slow addition of 40 ml of 1N HCl solution. The organic layer was washed with water (20ml), saturated sodium bicarbonate (20ml×2), brine (20ml), and dried over anhydrous sodium sulfate. Evaporation of the organic solvent gave the crude product 1 , which was used directly in the next step of the reaction without further purification.
[0218] Compound 2 : the crude product 1 Dissolved in 30 ml of acetone and to the resulting mixture in an ice-water bath was slowly added Jones' reagent until brown color persisted. The solution was further stirred at r...
Embodiment 2
[0306] Example 2-D-Ala-D-Ala-ligase Ki assay
[0307] On the day of screening, the synthetic analog of Example 1 was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 100 mM using a vortex mixer and sonication if necessary to facilitate dissolution. The solution was kept at room temperature until the screening was complete.
[0308]On the day of screening, a 10 mM NADH (Sigma) stock solution was prepared by dissolving 32 μmol NADH in 3.2 ml double distilled water. Keep the NADH solution on ice. Contains 50 mM phosphoenolpyruvate (PEP; Sigma), 500 μM HERMES, 30 mM adenosine triphosphate (ATP; Sigma), 200 mM D-alanine (Sigma) and 4× core buffer (i.e. 400 mM Hepes, 40 mM MgCl and 40 mM Chloride Potassium) stock solutions were also kept on ice. A stock solution of pyruvate kinase / lactate dehydrogenase (PK / LDH) was also obtained from Sigma.
[0309] filter type
Embodiment 3
[0310] Example 3 - Determination of the Ki of analogs
[0311] For each group of tested compounds, two 96-well plates were used: an inhibitor plate and an enzyme plate. Test compounds correspond to rows A-G of the plate. Adenosine (Sigma) dissolved in DMSO was used as a control in row H of each plate.
[0312] Allow the enzyme solution to equilibrate at 25 °C.
[0313] Dilutions were prepared in the suppressor plate as follows: 50 [mu]l DMSO was added to each well of the suppressor plate, columns 1-11, rows A-G. Add 50 [mu]l DMSO to each well of columns 1-11, row H. Add 100 μl of 100 mM test solution to column 12, rows A-G (ie first compound in row A, second compound in row B, and so on). Add 100 μl of a 100 mM adenosine solution to column 12, row H.
[0314] 50 μl of the test solution was transferred from column 12 of each row to column 11 of the same row, and this solution was mixed with DMSO. Then transfer 50 μl of the solution from column 11 of each row into column...
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