Development of Positive Allosteric Modulators of the Thyroid Stimulating Hormone Receptor Based on the Thieno[2,3-d]-Pyrimidine Structure and their Effect on Thyroid Status in Rats

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Abstract

Thyroid stimulating hormone (TSH) levels in autoimmune hypothyroidism and hypothyroidism caused by inactivating mutations in the TSH receptor are normal or elevated, which is due to increased thyroliberin (TRH)-stimulated TSH production in thyroid hormone (TH) deficiency. The main cause of hypothyroidism is a weakened thyroid response to TSH. This requires the development of approaches to increase the sensitivity of thyrocytes to TSH. One of them is the use of positive allosteric modulators (PAMs) of the TSH receptor, which enhance the effects of TSH on TH production. However, such PAMs are currently lacking. The aim of the work was to synthesize new thieno[2,3-d]-pyrimidine derivatives TPYox and TPYmp with PAM activity of the TSH receptor and to study their effects on basal and TRH-stimulated blood TH concentration and expression of genes involved in TH synthesis in the rat thyroid. When administered to rats, TPYox and TPYmp (20 mg/kg) had little effect on blood TH concentration and expression of TH synthesis genes, except for an increase in tT3 concentration and expression of the Dio2 gene encoding type 2 deiodinase when using TPYmp. At the same time, despite the absence of differences with the control, in the TPYox-treated group, the blood TH concentrations and the expression of the Tg, Tpo, Dio2, and Tshr genes decreased as compared to the TPYmp-treated rats, which we believe is due to the high reactivity of the oxirane cycle in TPYox and its inhibitory effect on some components of the thyroid system in the thyroid gland. Pretreatment of rats with TPYox and TPYmp preserved the stimulatory effects of TRH on TH concentration and thyroid gene expression, and in some cases significantly enhanced them. However, the dynamics and severity of the potentiating effect of TPYox and TPYmp differed. TPYox potentiated the stimulatory effects of TRH on blood tT4, fT3, and tT3 concentrations 1.5 h after TRH treatment, while TPYmp enhanced the stimulatory effects of TRH on fT3 and tT3 concentrations 3 h later, when the potentiating effect of TPYox had already disappeared. In the thyroid gland, TPYox enhanced TRH-induced Tpo gene expression, while TPYmp enhanced Dio2 and Nis gene expression. Based on the data obtained, it was concluded that the most promising drug for increasing the TSH receptor response to endogenous TSH is TPYmp, 5-amino-N-(tert-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 that we developed.

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K. V. Derkach

Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences

Author for correspondence.
Email: derkatch_k@list.ru
Russian Federation, Saint Petersburg

A. S. Pechalnova

Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences

Email: derkatch_k@list.ru
Russian Federation, Saint Petersburg

I. R. Nazarov

Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences

Email: derkatch_k@list.ru
Russian Federation, Saint Petersburg

E. A. Didenko

Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences; Saint Petersburg State University

Email: derkatch_k@list.ru
Russian Federation, Saint Petersburg; Saint Petersburg

V. N. Sorokoumov

Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences; Saint Petersburg State University

Email: derkatch_k@list.ru
Russian Federation, Saint Petersburg; Saint Petersburg

A. O. Shpakov

Sechenov Institute of Evolutionary Physiology and Biochemistry of the Russian Academy of Sciences

Email: derkatch_k@list.ru
Russian Federation, Saint Petersburg

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2. Fig. 1. Scheme for the synthesis of thieno[2,3-d]-pyrimidine derivatives of TPYox (a) and TPYmp (b). The designations in the figure are: 1 - 5-amino-N-(tert-butyl)-4-(4-iodophenyl)-2-(methylthio)thieno[2,3-d]pyrimidine-6-carboxamide; 2 - 2-[(prop-2-in-1-yloxy)methyl]oxirane; 3 - 5-amino-N-(tert-butyl)-2-(methylthio)-4-{4-[3-(oxiran-2-ylmethoxy)prop-1-in-1-yl]phenyl}thieno[2,3-d]pyrimidine-6-carboxamide (TPYox); 4 - 5-amino-4-(4-bromophenyl)-N-(tert-butyl)-2-(methylthio)thieno[2,3-d]pyrimidine-6-carboxamide; 5 - 1-morpholino-3-(pro-2-in-1-yloxy)propan-2-ol; 6 - 5-amino-N-(tert-butyl)-4-{4-[3-(2-hydroxy-3-morpholino-propoxy)pro-1-in-1-yl]phenyl}-2-(methylthio)thieno[2,3-d]pyrimidine-6-carboxamide (TPYmp). Abbreviations: PPh3, triphenylphosphine; Pd(PPh3)4, palladium tetrakis(triphenylphosphine); CuI, copper (I) iodide; DMF, dimethylformamide; THF, tetrahydrofuran; Et3N, triethylamine; RT, room temperature.

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3. Fig. 2. Basal and thyroliberin-stimulated TH concentrations in the blood of rats when they were treated with TPYox and TPYmp (w/w, 20 mg/kg) 2 and 3.5 h after TP treatment. (a) - Free thyroxine (fT4), pmol/l. (b) - Total thyroxine (tT4), nmol/l. (c) - Free triiodothyronine (fT3), pmol/l. (d) - Total triiodothyronine, (tT3), nmol/L. Initial TG concentrations are not given because there were no significant differences with both controls and between groups in this case. Groups were formed randomly from a pool of animals that were generally homogeneous in terms of blood TG concentrations. Differences with control (a) and with TRH group (b), as well as between TPYox and TPYmp groups (c) and TPYox + TRH and TPYmp + TRH groups (d) at one time point are statistically significant at p < 0.05. Values are presented as M ± SEM, in all groups n = 6.

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4. Fig. 3. Blood TTH concentrations in rats during their treatment with TPYox and TPYmp (w/w, 20 mg/kg). Blood TTH concentrations were assessed 3.5 h after TP treatment or 3 h after TRH administration. Differences with control (a) are statistically significant at p < 0.05. M ± SEM, in all groups n = 6.

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5. Fig. 4. Expression of thyroglobulin (Tg), thyroperoxidase (Tpo) and sodium iodide symporter (Nis) genes in the thyroid gland of rats during their treatment with TPYox and TPYmp (w/w, 20 mg/kg). Gene expression was assessed after the end of the experiments, 3.5 h after TP treatment or 3 h after TRH administration. Tg and Tpo gene expression data (RQ values) are non-normally distributed and presented as median and interquartile ranges (25%; 75%). Nis gene expression data are normally distributed and presented as M ± SEM. In all groups, n = 6. Differences with control (a) and with the TRH group (b) and between the TPYox and TPYmp groups (c) are statistically significant at p < 0.05.

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6. Fig. 5. Expression of genes of type 2 and 3 deiodinases (Dio2, Dio3) and TTG receptor (Tshr) in rat thyroid gland during their treatment with TPYox and TPYmp (w/w, 20 mg/kg). Gene expression was assessed after the end of the experiments, 3.5 h after TP treatment or 3 h after TRH administration. Dio3 gene expression data (RQ values) are non-normally distributed and presented as median and interquartile ranges (25%; 75%). Dio2 and Tshr gene expression data are normally distributed and presented as M ± SEM. In all groups, n = 6. Differences with control (a) are statistically significant at p < 0.05.

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