HPLC-UV Method Development and Validation to Monitor Difluprednate Synthesis.

During the synthesis of active pharmaceutical ingredients (APIs) there is a need for the development and validation of a simple and rapid high performance liquid chromatography (HPLC) method for the determination and quantification of the synthesized product and related by-products. An HPLC method gives a better understanding of how a synthesis is proceeding. A rapid and easy to use HPLC-UV (ultraviolet) method for the determination of difluprednate and monitoring of impurities generated during synthesis was developed and validated. A Shimadzu VP Series HPLC equipped with a LabSolutions software and UV detector set at 240 nm was used for analysis. The mobile phase consisted of phosphate buffer (pH 6) and acetonitrile 50:50 (v/v) and was eluted at a flow rate of 1.2 mL/min. Separation took place on a reversed-phase Kinetex C18 column (150 × 4.60 mm; 5 µm i.d.). Column temperature was set at 40°C. The developed method was found to have good linearity and acceptable accuracy and precision. The developed method may be effectively applied to determine products and by-products formed during synthetic reactions of steroids and to calculate the yield of the products obtained during each step of the synthesis.

Novel fluorinated antiinflammatory steroid with reduced side effects: methyl 9 alpha-fluoroprednisolone-16-carboxylate.

In an effort to test the hypothesis that 9 alpha-fluorination of a steroidal antedrug would enhance receptor binding affinity and local antiinflammatory activity, without concomitantly increasing adverse systemic effects, a fluorinated analog, 10, of methyl 11 beta, 21-dihydroxy- 3,20-dioxo-1,4-pregnadiene-16 alpha-carboxylate (DP16CM, 1) was synthesized and evaluated. In the acute rat croton oil-induced ear edema bioassay, 10 was found to be twice as potent as 1. This increase in topical potency was consistent with enhanced binding affinity of 10, relative to 1. The IC50 values for displacement of [3H]dexamethasone from glucocorticoid receptors of rat hepatoma tissue culture cells were 0.16, 1.2, and 0.03 microM for 10, 1, and prednisolone, respectively. Following multiple topical ID50 applications of predniosolone, 1, and its new fluorinated analog, 10, in the rat subacute croton oil-induced ear edema bioassay, only prednisolone exhibited significant untoward effects, such as reduction in relative thymus and adrenal weights, plasma corticosterone levels, and normal body weight gain. Thus, while fluroination of 1 enhanced its topical potency, there was not a concomitant increase in untoward systemic effects. This lack of adverse systemic effects is ostensibly due to the presence of the metabolically labile 16-carboxylate ester moiety.