Derivatization and Optimization of a LCMS-MS method to quantify selective estrogen receptor β agonists to determine their metabolic stability, protein binding, and pharmacokinetics.

Quantification of compounds without nitrogen atoms are challenging to measure by mass spectrometry due to a very low level of ionization. Therefore, we developed and validated a method of derivatization using 1,2- dimethylimidazole-5-sulfonyl-chloride (DMIS) to form the corresponding nitrogen-containing sulfonamide. Using this method, the sensitivity of detection was increased by more than 10-fold. We were able to carry out microsomal stability assays with compounds 1 and 2. Regarding phase 1, 55-75% of compound 1 was intact after 2 hours when employing mouse, human, rat, and dog liver S9 fractions. Compound 2 exhibited slightly higher stability. Regarding phase 2 conjugation (glucuronidation), only 25 % of compound 1 was detected after 2 hours using mouse, human, rat, and dog liver S9 fractions. Compound 2 was less stable under these conditions. It can be concluded that both compounds are rapidly glucuronidated in the presence of liver S9 fraction, where oxidation by P450 enzymes is relatively slow. We were also able to carry out pharmacokinetic studies with two compounds. To calculate the bioavailability, we utilized data available from oral and intravenous PK studies. Overall, we found a low bioavailability for both compounds in mice. These results were expected based on metabolic studies, that showed rapid glucuronidated in the presence of human and mouse liver S9 fraction. References: 1. Edward A Wetzel, Kylee J Marks, Alexandra A Gleason, Sandra Brown-Ford, Terry-Elinor Reid, Subhabrata Chaudhury, Sergey Lindeman, Daniel S Sem, William A Donaldson “Discovery of two novel (4-hydroxyphenyl) substituted polycyclic carbocycles as potent and selective estrogen receptor beta agonists” Bioorg Med Chem Lett, 2022, 73(1), 128906. 2. Cecilia Jalabert, Maria A Shock, Chunqi Ma, Taylor J Bootsma, Megan Q Liu, Kiran K Soma “Ultrasensitive quantification of multiple estrogens in songbird blood and microdissected brain by LC-MS/MS” Eneuro, 2022, 9(4). ENEURO.0037-22.2022.