## 5 Conclusions  
We have shown that TP4/2, a LHCGR allosteric agonist developed by us, stimulates T production in male Wistar rats during a five-day administration. With a single administration, TP4/2 is inferior in the steroidogenic effect to hCG, which is widely used in the clinic to stimulate steroidogenesis, but with a long-term use, on the third to fifth days after administration, the steroidogenic effect of TP4/2 is comparable to that of gonadotropin. The compound TP4/2 is effective not only in the treatment of young adult rats, but also in the treatment of both aging, 18-month-old, rats and diabetic rats with streptozotocin-induced T1 DM. When administered to the aging and diabetic rats, TP4/2 is as effective as hCG in normalizing T levels and improving testicular morphology and germ cell maturation. Unlike hCG, TP4/2 does not cause a decrease in the _Lhr_ gene expression, thereby preventing the resistance of Leydig cells to endogenous gonadotropins, and also does not cause hyperactivation of the gene expression of cholesterol-transporting StAR protein and some steroidogenic enzymes. A more moderate, but at the same time more stable stimulation of steroidogenesis, induced by TP4/2, demonstrates good prospects for its use of TP4/2 and, as one can assume, other LMW allosteric full agonists of LHCGR to compensate for androgen deficiency, both in certain physiological states, including aging, starvation and others, as well as in a number of diseases, which are characterized by an impairment of the hypothalamic-pituitary-gonad axis, including diabetes mellitus and metabolic syndrome. New results obtained in this study, as well as earlier data on the study of other TP derivatives [23] show that, unlike gonadotropins, the LMW allosteric agonists of LHCGR can be used for a long time, but not cause hyperactivation of both LH signaling system and steroidogenesis in Leydig cells. In addition, taking into account chaperone-like properties of TP derivatives in relation to LHCGR [37] and the absence of a decrease in the _Lhr_ gene expression in the case of long-term TP4/2 treatment demonstrated in our study, it can be assumed that the LMW allosteric agonists are able to enhance the effects of LH and hCG, and this will reduce the dose of clinically used gonadotropins, thus preventing some of their side effects. The following are available online at [[http://www.mdpi.com/1422-006/721/20/7493/s1](http://www.mdpi.com/1422-006/721/20/7493/s1)]([http://www.mdpi.com/1422-006/721/20/7493/s1](http://www.mdpi.com/1422-006/721/20/7493/s1)), Figure S1: The molecular mechanisms of luteinizing hormone/human chorionic gonadotropin-induced stimulation of testicular steroidogenesis in rat Leydig cells. Figure S2: The chemical structures of 5-amino-\(N\)-_tert_-butyl-4-(3-(1-methyl)pyrazole-4-carboxamidophenyl)-2-(methylthio)thien2[3,3-_d_]pyrimidine-6-carboxylic acid amide (TP4/2) (A) and the compound Org 43553 (B) selected as a standard for molecular docking into the LHCGR allosteric site. Figure S3: The experimental design and the studied groups of animals. Figure S4: The synthesis of 5-amino\(N\)-_tert_-butyl-4-(3-(1-methylpyrazole-4-carboxamido)phenyl)-2-(methylthio)thieno[2,3-4]pyrimidine-6-carboxylic acid amide (A) and the \({}^{1}\)H-NMR (B) and \({}^{13}\)C-NMR spectra (C) of this compound. Figure S5: Three-dimensional structures of full-length rat luteinizing hormone/chorionic gonadotropin (LHCGR) and thyroid-stimulating hormone (TSHR) receptors generated by homology modeling. Table S1: The basal and forskolin- and GppNHp-stimulated adenylyl cyclase activity in the testicular membranes isolated from the young adult, aging and diabetic rats. Table S2: The effect of TP4/2 on the basal and forskolin-, GppNHp- and TSH-stimulated adenylyl cyclase activity in the rat thyroidal membranes. Table S3: The basal and thyrotropin-releasing hormone (TRH)-stimulated blood levels of free (fT4) and total (fT4) thyroxine and total triiodynorine (tT3) in rats treated with TP4/2.