Activity, pharmacokinetics, and tissue distribution of 5-amino-N-tert-butyl-2-(methylsulfanyl)-4-(3-(nicotinamido)phenyl)thieno[2,3-d]pyrimidine-6-carboxamide (TP03), an allosteric agonist of the luteinizing hormone receptor

Gonadotropin preparations with luteinizing hormone (LH) activity are widely used to stimulate ovulation, but they have a number of side effects. Low-molecular allosteric LH receptor agonists, including our developed compound TP03, may be an alternative. TP03 was obtained with the acylation of 5-amino-4-(3-aminophenyl)-N-(tert-butyl)-2-(methylthio)thieno[2,3-d]pyrimidine-6-carboxamide, and, according to high-resolution mass spectrometry, this MW was 515.1304 (the calculated MW for [M+Na⁺] was 515.1294). Being a hydrophobic compound, TP03 is able to penetrate the transmembrane tunnel of the LH receptor, interacting with the transmembrane allosteric site. The aim of this study was to investigate the effect of TP03 on ovarian steroidogenesis and ovulation in immature female rats, as well as to investigate its pharmacokinetics in blood plasma and its distribution in ovaries and liver. Immature female rats stimulated with Follimag were administered TP03 (15 mg/kg, i.p. in DMSO), after which the levels of estradiol and progesterone in blood and the number of preovulatory follicles and corpora lutea in ovaries were assessed for 24 h. Using reversed-phase HPLC with tandem mass spectrometry, TP03 levels were measured in blood plasma, ovaries and liver. TP03 increased blood progesterone levels and, after 16–24 hours, led to the formation of corpora lutea in ovaries. The maximum plasma concentration of TP03 was 3530 ng/mL, and the time to reach maximum concentration was 15 min, indicating rapid absorption of TP03. The elimination constant, characterizing the rate of TP03 elimination, was 0.24 h^-1, and the half-life of TP03 was 2.94 h. Thus, TP03 is a long-lived compound in the bloodstream. Rapid accumulation of TP03 in ovaries and liver was demonstrated 1 hour after administration. Thus, the high efficacy of TP03 as an ovulation inducer is largely due to its rapid absorption in ovaries and long half-life in the bloodstream. The study was supported by the RSF (No 19-75-20122).