This protocol utilizes a recyclable and modified method in producing spirocyclic compounds. The MTT assay helps answer the question pertaining to the cytotoxicity of these novel molecules. This technique producers novel modifiable molecules using a regenerating starting material.
The MTT assay provides relatively quick, and easy to analyze biological results Begin by performing solid phase synthesis of spirocyclic hetero cycles six and seven, as previously demonstrated. Prepare 20 milliliters of a five milligrams per milliliter MTT solution using sterile PBS as the diluent. Filter and store the solution at minus 20 degrees Celsius.
Then, prepare a one-to-one dilution of the MTT solution in DMEM. Prepare one milliliter each of stock solutions in 1.5 milliliter micro centrifuge tubes of test compounds in DMSO. Store them at minus 20 degrees Celsius.
Prepare 200 microliters per dose of the working solutions of test compounds by diluting stock concentrations. One to 1000 in serum free medium. In the tissue culturehood seeds, COS seven cells in complete medium onto flat bottom tissue culture treated 96 well plates at a concentration of 4, 000 cells per 200 microliters per well.
Using a multi-channel pipeter. Incubate the cells for 24 hours at 37 degrees Celsius in an atmosphere containing 5%carbon dioxide. On the next day, aspirate the supernatant from the wells using a glass pasture pipette attached to a vacuum pump.
Dose the cells in triplicate with the test compounds using the previously prepared working solutions. Then return the cells to the incubator. After 24 hours, aspirate the supernatant from the wells and add 200 microliters of MTT solution to each well.
Incubate the plate for four hours. Gently aspirate the supernatant from the wells without disturbing the purple formazane crystals. Add 200 microliters of DMSO to each well to dissolve the crystals.
Then incubate the plate at room temperature for 15 minutes. Measure absorbance for each well at 590 or 600 nanometers using a 96 well plate reader. Use wells with no cells as background and average the absorbance value.
Spirocyclic gox seams six and seven were synthesized using a modified protocol. The progress of the reaction was monitored by detecting the disappearance of the alpha beta unsaturated Esther using infrared spectroscopy. The MTT assay was used for determining cell viability.
Cisplatin, which is known to induce cell death at high concentrations, served as a positive control. As expected, the higher the concentration of cisplatin the lower is the cell viability. The MTT assay was then used to test the spirocyclic compounds six and seven and furfurylamine.
Furfurylamine and spirocyclic gox seam six showed similar cytotoxicity. However, the toxicity of spirocyclic compound seven was noticeably greater than that of furfurylamine and six. When attempting this protocol, keep in mind that N-alkylation performed using bulky acyl halides was found to reduce the rate of beta elimination thus reducing the product yield.
