Abstract <p>Thyroid stimulating hormone (TSH) levels in hypothyroidism,both autoimmune and caused by inactivating mutations in the TSHreceptor gene, are either normal or elevated due to increased thyroliberin(TRH)-stimulated TSH production in thyroid hormone (TH) deficiency.Since the main cause of hypothyroidism is an attenuated thyroidresponse to TSH, it appears reasonable to develop approaches toincreasing the sensitivity of thyrocytes to TSH. One of such approachesimplies the use of TSH receptor positive allosteric modulators (PAMs),able to enhance TSH effects on TH production. However, such PAMsare currently unavailable. This work was aimed to synthesize thenew thieno[2,3-d]-pyrimidine-based derivatives, TPYox and TPYmp,sharing a TSH receptor PAM activity, and to study their effectson basal and TRH-stimulated blood TH levels, as well as the expressionof genes involved in TH synthesis, in the rat thyroid. When administeredto rats, TPYox and TPYmp (20 mg/kg) had little effect on blood THlevels and the expression of TH synthesis genes, except for an increasein tT3 concentration and the expression of the <i>Dio2</i> geneencoding type 2 deiodinase when using TPYmp. At the same time, despiteno differences versus controls, blood TH levels and the expressionof <i>Tg</i>, <i>Tpo</i>, <i>Dio2</i>, and <i>Tshr</i> genesin the TPYox-treated group decreased compared to TPYmp-treated rats,which we assume is due to a high reactivity of the oxirane cyclein the TPYox molecule and the inhibitory effect of this compoundon some components of the thyroid system. Rat pretreatment withTPYox and TPYmp preserved the stimulating effects of TRH on TH concentrationand thyroid gene expression, while significantly enhancing themin some cases. Meanwhile, the dynamics and severity of TPYox and TPYmppotentiating effects differed, namely TPYox potentiated TRH stimulatingeffects on blood tT4, fT3, and tT3 levels 1.5 h after TRH treatment,whereas TPYmp enhanced these effects on blood fT3 and tT3 levels3 h later, when the potentiating effect of TPYox had already fadedaway. In the thyroid gland, TPYox enhanced TRH-induced <i>Tpo</i> expression, while TPYmp enhanced <i>Dio2</i> and <i>Nis</i> expression. Thus,it was concluded that the most promising drug for increasing theTSH receptor response to endogenous TSH is TPYmp, 5-amino-N-(<i>tert</i>-butyl)-4-{4-[3-(2-hydroxy-3-morpholinopropoxy)pro-1-yn-1-yl]phenyl}-2-(methylthio)thieno[2,3-d]pyrimidine-6-carboxamide,the first functionally active PAM of the TSH receptor.</p>