The stimulating effect of thienopyrimidines structurally similar to Org 43553 on adenylate cyclase activity in the testes and on testosterone production in male rats

The functional activity of the luteinizing hormone (LH) receptor can be regulated not only by gonadotropins, but also by its low molecular weight agonists, which, in contrast to gonadotropins, bind to the allosteric site located in the transmembrane channel of the receptor. The most promising low molecular weight agonists are thienopyrimidine derivatives, which are structural analogs of the Org 43553 compound. The purpose of this work was to synthesize novel thienopyrimidine derivatives—5-amino-N-(tert-butyl)-4-(3-(2-methoxynicotinamido)-phenyl)-2-(methylthio)thieno[2,3-d]pyrimidine-6-carboxamide (TP-21), 4-((3-(5-amino-6-(tert-butylcarbamoyl)-2-(methylthio)thieno[2,3-d]pyrimidine-4-yl)phenyl)carbamoyl)pyridine 1-oxide (TP-22), and 5-amino-N-(tert-butyl)-4-(3-(2-chloroxynicotinamido)phenyl)-2-(methylthio)thieno[2,3-d]pyrimidine-6-carboxamide (TP-23)—and to investigate their effects on adenylate cyclase (AC) activity in rat testicular membranes in vitro and on testosterone levels in male rats following intratesticular or intraperitoneal administration in vivo. Compounds TP-21, TP-22, and TP-23 stimulated basal AC activity in rat testicular membranes with EC₅₀ values of 1556, 358, and 372 nM; their efficiency was ordered as follows: TP-23 > TP-21 ≈ TP-22. When thienopyrimidines (10^–4 M) were applied in combination with human chorionic gonadotropin (HCG, 10^–8 M), the AC-stimulating effect of HCG was maintained, and, at an HCG concentration of 10^–10 M, the effects of thienopyrimidines and HCG on AC activity were additive. Intratesticular administration of 10 mg/kg TP-21, TP-22, and TP-23 increased testosterone levels in male rats: in 5 h after treatment, its levels were 32.8, 36.4, and 76.9 nM, respectively, higher than in the control group. Following intraperitoneal administration, TP-21 and TP-22 had little effect on testosterone levels, while TP-23 induced a significant increase in testosterone levels (by 34.8 and 18.9 nM in comparison to control in 1 and 3 h, respectively). These data suggest that compound TP-23 is an active stimulator of testosterone synthesis and secretion and represents a promising basis for development of highly efficient LH receptor agonists.     

In vitro and in vivo studies of functional activity of new low molecular weight agonists of the luteinizing hormone receptor

The use of luteinizing hormone (LH) and its structural and functional homolog human chorionic gonadotropin (hCG) leads to hyperstimulation of LH-dependent signalling pathways and to desensitization of LH receptors. Therefore, the development of low molecular weight agonists of the LH receptor without these disadvantages has been carried out in recent years. These agonists, unlike gonadotropins, are active when administered orally. The greatest prospects are associated with the development of the thienopyrimidine derivatives structurally similar to compound Org 43553. The purpose of this work was the synthesis of new thienopyrimidine derivatives TP03 and TP04, the study of the regulation of LH-sensitive adenylyl cyclase signalling system in rat testes, and the evaluation of the ability of TP03 and TP04 to stimulate testosterone synthesis in male rats at different routes of administration. In the concentration range 10^–7–10^–3 M, TP03 and TP04 induced an increase of the basal adenylyl cyclase (AC) activity in rat testicular membranes with the EC₅₀ values of 390 and 759 nM, respectively. At a concentration of 10^–4 M, AC-stimulating effects of TP03 and TP04 were 213 and 122%, respectively, which indicates a higher activity of TP03 under in vitro conditions. At the same time, the AC-stimulating effect of TP03 was approximately 2.5-fold weaker than that of hCG (10^–8 M). Upon simultaneous administration of hCG and thienopyrimidine derivatives, their stimulating effects on the AC activity were additive due to the different localization of their binding sites in the LH receptor. Compound TP03 (25 mg/kg), when administered intraperitoneally to male rats, was more efficient than TP04: the maximal increase in the testosterone level (3 h after administration) was by 60% higher than that induced by TP04. TP03 increased the testosterone level after oral administration (50 mg/kg) as well, but the effect was weaker than in the case of the intraperitoneal injection. Orally administered TP04 exhibited a low activity. The results suggest that 5-amino-N-tert-butyl-2-(methylsulfonyl)-4-(3-(nicotinamide)phenyl) thieno[2,3-d]pyrimidine-6-carboxamide (TP03) can be used for the development of the drugs that stimulate steroidogenesis in Leydig cells.     

Specificity of heterotrimeric G protein regulation by human chorionic gonadotropin and low-molecular agonist of luteinizing hormone receptor

Luteinizing hormone (LH) and its homologue, human chorionic gonadotropin (hCG), are very important regulators of the reproductive system. These hormones stimulate various types of G proteins—primarily, G_s and G_q proteins—by binding to the specific LH-hCG receptor, which leads to the activation of adenylate cyclase (AC) and phospholipase C, respectively. It has been suggested that many side effects of LH and hCG are associated with low selectivity of their effect on G proteins. Low-molecular agonists of LH-hCG receptor developed on the basis of thienopyrimidine derivatives do not cause these side effects, and differences in the interaction with G proteins may be ones of the cause for this. To test this, a comparative study of the effect of hCG and synthesized by us thienopyrimidine derivative, 5-amino-N-tert-butyl-2-(methylsulfanyl)-4-(3-(nicotinamido)phenyl)thieno[2,3-d]pyrimidine-6-carboxamide (TP03) on the AC activity and GTP binding of G proteins in plasma membranes isolated from the rat ovaries and testes was performed. Cholera toxin (CT) and pertussis toxin (PT) were used to selectively switch off the signal transduction via G_s and G_i/o proteins, the peptide corresponding to the C-terminal segment 349–359 of the Gα_q subunit was used to suppress G_q-dependent cascades. It was shown that treatment of ovarian and testicular membranes with CT resulted in suppression of TP03 and hCG stimulatory effects on the AC activity, but in different ways influenced the GTP binding stimulation: it completely blocked the effect of 10^–6 M TP03 and reduced by 45–46% the effect of hCG (10^–8 M). Preincubation of membranes with the peptide 349–359 reduced the hCG stimulatory effect on GTP binding by 34 (ovaries) and 45% (testes), but did not affect the corresponding effect of 10^–6 M TP03. Preincubation with the peptide 349–359 also reduced the GTP stimulatory effect of 10^–4 M TP03, but to a small extent. The obtained data indicate that, in contrast to hCG, the targets of which in the ovaries and testes are G_s and G_q proteins, the action of TP03 is realized mainly via G_s proteins. Only at a concentration that exceeds EC₅₀ by two orders TP03 is capable to relatively weakly activate G_q proteins. The PT treatment of the membranes did not affect the effects of TP03 and hCG, which indicates the lack of their effective interaction with G_i/o proteins. Thus, the selectivity of activation of G_s-dependent cascades responsible for the synthesis and production of steroid hormones is a significant advantage of low-molecular agonists of LH-hCG receptor over gonadotropins.     

Synthesis and structure-activity relationships of thieno[2,3-d]pyrimidine-2,4-dione derivatives as potent GnRH receptor antagonists

The synthesis and SAR studies of thieno[2,3-d]pyrimidine-2,4-diones as human GnRH receptor antagonists to treat reproductive diseases are discussed. It was found that the 2-(2-pyridyl)ethyl group on the 5-aminomethyl functionality of the core structure was a key feature for good receptor binding activity. SAR study of the 6-(4-aminophenyl) group suggests that hydrophobic substituents were preferred. The best compound from this series had binding affinity (K(i)) of 0.4 nM to the human GnRH receptor.     

Discovery of novel thieno[2,3-d]pyrimidin-4-yl hydrazone-based inhibitors of Cyclin D1-CDK4: Synthesis,biological evaluation,and structure–activity relationships

The synthesis and evaluation of new analogues of thieno[2,3-d]pyrimidin-4-yl hydrazones are described. 2-Pyrdinecarboxaldehyde [6-(tert-butyl)thieno[2,3-d]pyrimidine-4-yl]hydrazone derivatives have been identified as cyclin-dependent kinase 4 (CDK4) inhibitors. The potency, selectivity profile, and structure–activity relationship of this series of compounds are discussed.     

Inhibition of adenylyl cyclase activity in rat corpora luteal tissue by glycopeptides of human chorionic gonadotropin and the alpha-subunit of human chorionic gonadotropin

Indirect evidence has indicated that the carbohydrate moieties of the glycoprotein hormones are involved in the activation of the receptor-adenylyl cyclase system of reproductive tissues. In the present study, we have isolated the glycopeptides (GP) from human chorionic gonadotropin (hCG), the alpha-subunit of hCG, fetuin, and bovine gamma-globulin (b gamma G). These along with a number of synthetic oligosaccharides were tested for their ability to inhibit adenylyl cyclase (AC). There was less than 0.001% cross-reactivity of the GP from hCG, hCG alpha, fetuin, and b gamma G when tested in a double-antibody hCG radioimmunoassay or rat corpora lutea radioreceptor assay. The GP of fetuin, b gamma G, and the synthetic oligosaccharides did not inhibit AC activity of 2000 g corpora lutea membranes when coincubated with 100 ng of hCG/mL (ED50). However, when the GP of hCG and hCG alpha were included with intact hCG, there was a dose-related inhibition. Inhibition of cyclase activity was enhanced when the hCG GP were desialylated. This occurred without a change in the lag time of hCG activation which was calculated to be 1-1.5 min. Changing the concentration of ATP and Mg2+ did not affect the inhibitory effects of the hCG alpha GP on hCG-stimulated AC activity. Inhibition by hCG GP followed uncompetitive kinetics. The inhibition by the GP of hCG seems to be restricted to the LH/hCG-stimulatable AC system because the same dosage of hCG GP which inhibited the rat luteal AC system did not have any effect on the rat hepatocyte AC system when coincubated with glucagon or on NaF-stimulated activity in luteal membranes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thienopyrimidine-based dual EGFR/ErbB-2 inhibitors

Two new series of potent and selective dual EGFR/ErbB-2 kinase inhibitors derived from novel thienopyrimidine cores have been identified. Isomeric thienopyrimidine cores were evaluated as isosteres for a 4-anilinoquinazoline core and several analogs containing the thieno[3,2-d]pyrimidine core showed anti-proliferative activity with IC₅₀ values less than 1 μM against human tumor cells in vitro.     

Synthesis of N-1β-D-Arabinofuranosyl and N-1-2′-Deoxy-β-D-erythro-pentofuranosyl Thieno [3,2-d] Pyrimidine Nucleosides

Abstract

Synthetic methods for 1-(β-D-arabinofuranosyl) and 1-(2-deoxy-β-D-erythro-pentofuranosyl)thieno[3,2-d]pyrimidine-2,4-diones from the orresponding 1-(β-D-ribofuranosyl) nucleoside have been developed in this report. These compounds were tested against HIV-1 in CEM cl 13 cell cultures, but none of them exhibited significant inhibitory activity against this virus.     

Pyrimidine-5-carbonitrile based potential anticancer agents as apoptosis inducers through PI3K/AKT axis inhibition in leukaemia K562

A novel series of 4-(4-Methoxyphenyl)-2-(methylthio)pyrimidine-5-carbonitrile was developed linked to an aromatic moiety via N-containing bridge and then evaluated for their cytotoxic activity against MCF-7 and K562 cell lines. Seven compounds exhibited the highest activity against both cell lines where compounds 4d and 7f were the most active against K562 cell line. Exploring their molecular mechanisms by enzyme inhibition assay on PI3Kδ/γ and AKT-1 showed that compound 7f was promising more than 4d with IC₅₀ = 6.99 ± 0.36, 4.01 ± 0.55, and 3.36 ± 0.17 uM, respectively. Also, flowcytometric analysis revealed that 7f caused cell cycle arrest at S-phase followed by caspase 3 dependent apoptosis induction. Mechanistically, compound 7f proved to modulate the expression of PI3K, p-PI3K, AKT, p-AKT, Cyclin D1, and NFΚβ. Furthermore, in-vivo toxicity study indicated good safety profile for 7f. These findings suggest that the trimethoxy derivative 7f has strong potential as a multi-acting inhibitor on PI3K/AKT axis targeting breast cancer and leukaemia.     

Synthesis and pharmacological studies of new pyrazole analogues of podophyllotoxin

The pyrazole analogues of podophyllotoxin were synthesized by the chalcone route. This route attracts the attention because of its simple operating conditions and easy availability of the chemicals. Initially, benzylideneacetophenones (chalcones) were prepared in high yields by Claisen-Schmidt reaction of acetophenones with 4-(methylthio)benzaldehyde. The cyclopropyl ketones were prepared in good yields by the reaction of chalcones with trimethylsulfoxonium iodide. Tetralones were prepared in good yields by the Friedel-Craft’s intramolecular cyclization reaction of cyclopropyle ketones in the presence of anhyd. stannic chloride and acetic anhydride. The tetralones on formylation to give substituted hydroxylmethylene tetralones. Condensation of substituted hydroxylmethylene tetralones with hydrazine hydrate afforded target compounds. The structures of the synthesized compounds were confirmed by IR, ¹H NMR and Mass spectral technique. The title compounds were screened for their antimitotic and antimicrobial activities. Among the synthesized compounds cyclopropyl ketones and pyrazole analogues of podophyllotoxin, compound 7-(methylthio)-5-(4-(methylthio)phenyl)-4,5-dihydro-² H-benzo[g]indazole is more active than 5-(4-(methylthio)phenyl)-4,5-dihydro-² H-benzo[g]indazole, 7-methyl-5-(4-(methylthio)phenyl)-4,5-dihydro-² H-benzo[g]indazole, 7-methoxy-5-(4-(methylthio)phenyl)-4,5-dihydro-² H-benzo[g]indazole and the key intermediate tetralones in 100, 200 and 400 ppm at 12, 18 and 24 h and also showed very good activity against screened bacteria and fungi compared to their standard.     

Synthetic strategy for increasing solubility of potential FLT3 inhibitor thieno[2,3-d]pyrimidine derivatives through structural modifications at the C2 and C6 positions

Acute myeloid leukemia (AML) is a clonal disorder of hematopoietic progenitor cell. In AML, a mutation in FLT3 is commonly occurs and is associated with poor prognosis. We have previously reported that thieno[2,3-d]pyrimidine derivative compound 1 exhibited better antiproliferative activity against MV4-11 cells which harbor mutant FLT3 than AC220, which is a well-known FLT3 inhibitor, and has good microsomal stability. However, compound 1 had poor solubility. We then carried out further structural modification at the C₂ and the C₆ positions of thieno[2,3-d]pyrimidine scaffold. Compound 13b, which possesses a thiazole moiety at the C₂ position, exhibited better antiproliferative activity than compound 1 and showed increased solubility and moderate microsomal stability. These results indicate that compound 13b could be a promising potential FLT inhibitor for AML chemotherapy.     

Synthesis of 1-(β-D-Ribofuranosyl)Thieno[3,2-d] Pyrimidine-2,4-Dione and 4-Substituted Derivatives

Abstract

Regiospecific ribosylation of the bis(trimethylsilyl) derivative of thieno[3,2-d]pyrimidine-2,4-dione in the presence of a Lewis acid followed by debenzoylation has afforded 1-(β-D-ribofuranosyl)thieno[3,2-d]pyrimidine-2,4-dione, a uridine analogue. The site of ribosylation and anomeric configuration of this N-nucleoside were established by NMR and UV. Thiation of the β-anomer was followed by treatment with methanolic ammonia to afford 4-amino-1-(β-D-ribofuranosyl)thieno [3,2-d]pyrimidin-2-one, a cytidine analogue.     

Synthesis of novel spiro[pyrazolo[4,3-d]pyrimidinones and spiro[benzo[4,5]thieno[2,3-d]pyrimidine-2,3′-indoline]-2′,4(3H)-diones and their evaluation for anticancer activity

An efficient and novel method for the preparation of spiro[pyrazolo[4,3-d]pyrimidin]-7′(1′H)-ones by the condensation of 4-amino-1-methyl-3-propylpyrazole-5-carboxamide with ketones under mild conditions using catalytic InCl₃ was reported. This method has been extended for the synthesis of novel spiro[benzo[4,5]thieno[2,3-d]pyrimidine-2,3′-indoline]-2′,4(3H)-dione which are having potential applications in medicinal chemistry. All the synthesized compounds were evaluated for their anti-proliferative properties in vitro against cancer cell lines and several compounds were found to be active. Further in vitro studies revealed that inhibition of sirtuins could be the possible mechanism of action of these molecules.     

Synthesis and antimicrobial activity of some new substituted pyrido[3′,2′:4,5]thieno[3,2-d]-pyrimidinone derivatives

A series of new 3-substituted-7-(2-chloro-6-ethoxypyridin-4-yl)-9-(2,4-dichlorophenyl)-2-methylpyrido[3′,2′:4,5]thieno[3,2-d]pyrimidin-4(3H)-one derivatives were synthesized as antimicrobial agents using 7-(2-chloro-6-ethoxypyridin-4-yl)-9-(2,4-dichlorophenyl)-2-methyl-4H-pyrido[3′,2′:4,5]thieno[3,2-d]-[1,3]oxazin-4-one as a starting compound. Its condensation with substituted aniline derivatives or phenyl hydrazine gave the corresponding N-substituted derivatives. Treatment of the starting compound with hydrazine hydrate afforded the corresponding N-amino derivative, which was reacted with substituted phenylisocyanate and phenylisothiocyanate derivatives to give the corresponding semicarbazides and thiosemicarbazide derivatives. All the newly synthesized compounds were evaluated for their antimicrobial activities in comparison to streptomycin and fusidic acid as positive controls. The structure assignments of the new compounds are based on chemical and spectroscopic evidence.     

Synthesis and binding properties of novel selective 5-HT3 receptor ligands

This work reports on the synthesis and affinities for the 5-HT(3) versus the 5-HT(4) receptor of new piperazinyl-substituted thienopyrimidine derivatives 20-45 with a view to identify potent and selective ligands for the 5-HT(3) receptor. Some of the new compounds show good affinity for the 5-HT(3) receptor and, notably, do not display any affinity for the 5-HT(4) receptor. 4-(4-Methyl-1-piperazinyl)-2-methylthio-6,7-dihydro-5H-cyclopenta[4,5]thieno[2,3-d]pyrimidine 31 exhibits the highest affinity for the 5-HT(3) receptor (Ki = 33 nM) and behaves as noncompetitive antagonist.     

Changes in sulfur compounds of Awamori during aging

Eleven sulfur compounds in awamori were identified, of which the main component was dimethyl sulfide (DMS). The ratio of DMS to the remaining other ten sulfur compounds (DMS/total sulfur compounds) was more than 50 per cent. Next in order by weight was 3-(methylthio) propyl acetate, them dimethyl trisulfide (DMTS). The total value of the 11 sulfur compounds ranged from 1777 to 6571 μg per l at 100% alcohol. Qualitative and quantitative differences in the sulfur component contents were found between unaged awamori and unaged whisky. The total value of the 11 sulfur compounds in awamori was about three times higher than those in whisky.In kame-aging, some components tended to decrease [DMS, DMTS, 3-(methylthio)-propanal, dihydro-2-methyl-3(2H)-thiophenone, ethyl-3-(methylthio)propanoate and 3-(methylthio)propyl acetate], while others showed no distinct change [dimethyl disulfide (DMDS), 2-thiphenecarboxaldehyde, 3-(methylthio)propanol, benzothiophene and benzothiazole]. During aging in non-porous containers, DMS, DMTS, 3-(methylthio) propanal and dihydro-2-methyl-3(2H)-thiophenone also tended to decrease, while the other seven components showed no distinct change. Thus sulfur compounds change more during aging in kame than in non-porous containers.     

Discovery and SAR of a novel series of Natriuretic Peptide Receptor-A (NPR-A) agonists

Novel thienopyrimidine compounds 2 and 3 were discovered from high-throughput screening as Natriuretic Peptide Receptor A (NPR-A) agonists. Scaffold hopping of a thienopyrimidine ring to a quinazoline ring, introduction of the basic functional group and optimization of the substituent on the 6-position of the benzene ring of quinazoline led to improved agonistic activity. We discovered compound 48, which showed potent agonistic activity for NPR-A with an EC₅₀ value of 0.073 μM, indicating 350-fold potency compared to the hit compound 3.     

Biochemical studies of toxic agents. The metabolism of iodomethane

1. The metabolism of iodomethane has been studied after the administration of the compound to rats by subcutaneous injection. 2. The urinary excretion of S-methylcysteine, methylmercapturic acid, methylthioacetic acid and N-(methylthioacetyl)glycine has been demonstrated by paper chromatography. 3. Methylmercapturic acid and N-(methylthioacetyl)glycine have been isolated from the urine of the dosed animals. 4. The methylthio compounds detected represented about 2% of the iodomethane administered.     

Tranylcypromine and 6-trifluoroethyl thienopyrimidine hybrid as LSD1 inhibitor

Tranylcypromine moiety extracted from LSD1 inhibitors and 6-trifluoroethyl thienopyrimidine moiety from menin-MLL1 PPI inhibitors were merged to give new chemotypes for medicinal chemistry study. Among 15 new compounds prepared in this work, some exhibited nanomolar LSD1 activity and good selectivity over MAO-A/B, low micromolar menin-MLL1 PPI inhibitory activity, as well as submicromolar MV4-11 antiprofilative activities. Intracellular LSD1 engagement of compounds with higher enzymatic and antiproliferative activities was confirmed by CD86 mRNA up-regulation experiments.     

Synthesis and pharmacology of pyrido[2,3-d]pyrimidinediones bearing polar substituents as adenosine receptor antagonists

Amino-substituted pyrido[2,3-d]pyrimidinediones have previously been found to bind to adenosine A1 and A2A receptors in micromolar concentrations. The present study was aimed at studying the structure-activity relationships of this class of compounds in more detail. Most of the investigated compounds were provided with polar substituents, such as ethoxycarbonyl groups and basic amino functions, in order to improve their water-solubility. The compounds were synthesized starting from 6-amino-1,3-dimethyluracil via different reaction sequences involving (cyano)acetylation, Vilsmeier formylation, or reaction with diethyl ethoxymethylenemalonate (EMME). The most potent and selective compound of the present series was 6-carbethoxy-1,2,3,4-tetrahydro-1,3-dimethyl-5-(2-naphthylmethyl)aminopyrido[2,3-d]pyrimidine-2,4-dione (11c) with a Ki value of 5 nM at rat and 25 nM at human A1 receptors. The compound was more than 60-fold selective versus A3 and more than 300-fold selective versus A2A receptors. It showed an over 300-fold improvement with respect to the lead compound. In GTPgammaS binding studies at membranes of Chinese hamster ovary cells recombinantly expressing the human adenosine A1 receptor, 11c behaved as an antagonist with inverse agonistic activity. A regioisomer of 11c, 6-carbethoxy-1,2,3,4-tetrahydro-1,3-dimethyl-7-(2- naphthylmethyl)aminopyrido[2,3-d]pyrimidine-2,4-dione (7a) in which the 2-naphthylmethylamino substituent at position 5 of 11c was moved to the 7-position, was a relatively potent (Ki=226 nM) and selective (>20-fold) A3 ligand. In the series of compounds lacking an electron-withdrawing ethoxycarbonyl or cyano substituent in the 6-position, compounds with high affinity for adenosine A2A receptors were identified, such as 1,2,3,4-tetrahydro-1,3-dimethyl-5-(1-naphthyl)aminopyrido[2,3-d]pyrimidine-2,4-dione 16b (Ki human A2A=81.3 nM, Ki human A1=153 nM, and Ki human A3>10,000 nM).