New Smoothened ligands based on the purine scaffold as potential agents for treating pancreatic cancer.

Aberrant activation of the Hedgehog (Hh) signalling pathway has been associated with the development and progression of pancreatic cancer. For this reason, blockade of Hh pathway by inhibitors targeting the G protein-coupled receptor Smoothened (SMO) has been considered as a therapeutic target for the treatment of this cancer. In our previous work, we obtained a new SMO ligand based on a purine scaffold (compound I), which showed interesting antitumor activity in several cancer cell lines. In this work, we report the design and synthesis of 17 new purine derivatives, some of which showed high cytotoxic effect on Mia-PaCa-2 (Hh-dependent pancreatic cancer cell lines) and low toxicity on non-neoplastic HEK-293 cells compared with gemcitabine, such as 8f, 8g and 8h (IC50 = 4.56, 4.11 and 3.08 µM, respectively). Two of these purines also showed their ability to bind to SMO through NanoBRET assays (pKi = 5.17 for 8f and 5.01 for 8h), with higher affinities to compound I (pKi = 1.51). In addition, docking studies provided insight the purine substitution pattern is related to the affinity on SMO. Finally, studies of Hh inhibition for selected purines, using a transcriptional functional assay based on luciferase activity in NIH3T3 Shh-Light II cells, demonstrated that 8g reduced GLI activity with a IC50 = 6.4 µM as well as diminished the expression of Hh target genes in two specific Hh-dependent cell models, Med1 cells and Ptch1-/- mouse embryonic fibroblasts. Therefore, our results provide a platform for the design of SMO ligands that could be potential selective cytotoxic agents for the treatment of pancreatic cancer.

New Inhibitors of Bcr-Abl Based on 2,6,9-Trisubstituted Purine Scaffold Elicit Cytotoxicity in Chronic Myeloid Leukemia-Derived Cell Lines Sensitive and Resistant to TKIs.

Bcr-Abl is an oncoprotein with aberrant tyrosine kinase activity involved in the progression of chronic myeloid leukemia (CML) and has been targeted by inhibitors such as imatinib and nilotinib. However, despite their efficacy in the treatment of CML, a mechanism of resistance to these drugs associated with mutations in the kinase region has emerged. Therefore, in this work, we report the synthesis of 14 new 2,6,9-trisubstituted purines designed from our previous Bcr-Abl inhibitors. Here, we highlight 11b, which showed higher potency against Bcr-Abl (IC50 = 0.015 µM) than imatinib and nilotinib and exerted the most potent antiproliferative properties on three CML cells harboring the Bcr-Abl rearrangement (GI50 = 0.7-1.3 µM). In addition, these purines were able to inhibit the growth of KCL22 cell lines expressing Bcr-AblT315I, Bcr-AblE255K, and Bcr-AblY253H point mutants in micromolar concentrations. Imatinib and nilotinib were ineffective in inhibiting the growth of KCL22 cells expressing Bcr-AblT315I (GI50 > 20 µM) compared to 11b-f (GI50 = 6.4-11.5 µM). Molecular docking studies explained the structure-activity relationship of these purines in Bcr-AblWT and Bcr-AblT315I. Finally, cell cycle cytometry assays and immunodetection showed that 11b arrested the cells in G1 phase, and that 11b downregulated the protein levels downstream of Bcr-Abl in these cells.

Synthesis and Anti-Diabetic Activity of an 8-Purine Derivative as a Novel DPP-4 Inhibitor in Obese Diabetic Zücker Rats.

Type 2 diabetes mellitus (T2DM) is one of the world's principal metabolic diseases characterized by chronic hyperglycemia. The gut incretin hormones, glucagon-like peptide 1 (GLP-1) and gastric inhibitory polypeptide (GIP), which has been proposed as a new treatment for T2DM, are extensively metabolized by Dipeptidyl peptidase 4 (DPP-4). Inhibitors of DPP-4 block the degradation of GLP-1 and GIP and may increase their natural circulating levels, favoring glycemic control in T2DM. A novel and potent selective inhibitor of DPP-4 with an 8-purine derived structure (1) has been developed and tested in vitro and in vivo in Zücker obese diabetic fatty (ZDF) rats, an experimental model of the metabolic syndrome and T2DM to assess the inhibitory activity using vildagliptin as reference standard. ZDF rats were subdivided into three groups (n = 7/group), control (C-ZDF), and those treated with compound 1 (Compound1-ZDF) and with vildagliptin (V-ZDF), both at 10 mg/kg/d rat body weight, in their drinking water for 12 weeks, and a group of lean littermates (ZL) was used. ZDF rats developed DM (fasting hyperglycemia, 425 ± 14.8 mg/dL; chronic hyperglycemia, HbA1c 8.5 ± 0.4%), compared to ZL rats. Compound 1 and vildagliptin reduced sustained HbAl1c (14% and 10.6%, P < 0.05, respectively) and fasting hyperglycemia values (24% and 19%, P < 0.05, respectively) compared to C-ZDF group (P < 0.001). Compound 1 and vildagliptin have shown a potent activity with an IC50 value of 4.92 and 3.21 µM, respectively. These data demonstrate that oral compound 1 administration improves diabetes in ZDF rats by the inhibitory effect on DPP-4, and the potential to be a novel, efficient and tolerable approach for treating diabetes of obesity-related T2DM, in ZDF rats.

Design, Synthesis, and Biological Evaluation of Novel Purine Derivatives as Herbicide Safeners.

Mesosulfuron-methyl, an inhibitor of acetolactate synthase (ALS), has been extensively used in wheats. However, it can damage wheat (Triticum aestivum) and even lead to crop death. Herbicide safeners selectively shield crops from such damage without compromising weed control. To mitigate the phytotoxicity of mesosulfuron-methyl in crops, several purine derivatives were developed based on active substructure splicing. The synthesized title compounds underwent thorough characterization using infrared spectroscopy, 1H nuclear magnetic resonance (1H NMR), 13C nuclear magnetic resonance (13C NMR), and high-resolution mass spectrometry. We evaluated chlorophyll and glutathione contents as well as various enzyme activities to evaluate the safer activity of these compounds. Compounds III-3 and III-7 exhibited superior activity compared with the safener mefenpyr-diethyl. Molecular structure analysis, along with predictions of absorption, distribution, metabolism, excretion, and toxicity, indicated that compound III-7 shared pharmacokinetic traits with the commercial safener mefenpyr-diethyl. Molecular docking simulations revealed that compound III-7 competitively bound to the ALS active site with mesosulfuron-methyl, elucidating the protective mechanism of the safeners. Overall, this study highlights purine derivatives as potential candidates for novel safener development.

Discovery of 2,9-diaryl-6-carbamoylpurines as a novel class of antitubercular agents.

A series of novel 9-alkyl/aryl-2-aryl-6-carbamoylpurines were synthesized, and their activity against Mycobacterium tuberculosis strain H37Rv was assessed. The SAR analysis on the first set of derivatives, with an alkyl or aryl unit at N-9 and a phenolic unit at C-2, showed that the activity depends on the purine ring substituents at N-9 and C-2. A phenyl group at N-9 combined with a 3-hydroxyphenyl or 4-hydroxyphenyl at C-2 improve the activity. The most active compound of this set has a phenyl group at N-9 and a 4-hydroxyphenyl group at C-2, displaying an IC90 = 1.2 µg/mL and a selectivity index higher than 25.5. This compound served as a Hit to design the second set of derivatives. A phenyl group at N-9 was maintained, and the group at C-2 was diversified. The SAR analysis showed that the aryl unit at C-2 must have an oxygen or nitrogen atom bonded in the para position. A proton, a small alkyl or a substituted aryl group may also be bonded to the oxygen. The compound with the 4-methoxyphenyl group at C-2, 1Bd, exhibits the highest activity with an IC90 < 0.19 µg/mL. This compound is highly potent against M. tuberculosis strain H37Rv and non-toxic for VERO mammalian cells with an SI > 153.8. Compound 1Bd was also non-cytotoxic against primary macrophage cultures at IC90, 2xIC90, and 10xIC90 and significantly reduced the bacterial load in M. tuberculosis-infected macrophages at the same concentrations. Compound 1Bd showed a favorable pharmacokinetic profile when administered orally, with major lung and liver accumulation. In vivo antimycobacterial efficacy of 1Bd was tested at 25 mg/kg. At the tested regimen, a decrease in bacterial burden was observed in the liver. Optimization of the treatment regimen should be performed to fully potentiate the in vivo efficacy of our lead molecule, particularly in the lung, the main target organ of M. tuberculosis.

Recent Developments and Future Perspectives of Purine Derivatives as a Promising Scaffold in Drug Discovery.

Numerous purine-containing compounds have undergone extensive investigation for their medical efficacy across various diseases. The swift progress in purine-based medicinal chemistry has brought to light the therapeutic capabilities of purine-derived compounds in addressing challenging medical conditions. Defined by a heterocyclic ring comprising a pyrimidine ring linked with an imidazole ring, purine exhibits a diverse array of therapeutic attributes. This review systematically addresses the multifaceted potential of purine derivatives in combating various diseases, including their roles as anticancer agents, antiviral compounds (anti-herpes, anti-HIV, and anti-influenzae), autoimmune and anti-inflammatory agents, antihyperuricemic and anti-gout solutions, antimicrobial agents, antitubercular compounds, anti-leishmanial agents, and anticonvulsants. Emphasis is placed on the remarkable progress made in developing purine-based compounds, elucidating their significant target sites. The article provides a comprehensive exploration of developments in both natural and synthetic purines, offering insights into their role in managing a diverse range of illnesses. Additionally, the discussion delves into the structure-activity relationships and biological activities of the most promising purine molecules. The intriguing capabilities revealed by these purine-based scaffolds unequivocally position them at the forefront of drug candidate development. As such, this review holds potential significance for researchers actively involved in synthesizing purine-based drug candidates, providing a roadmap for the continued advancement of this promising field.

Nutrient intake, digestibility, and utilization in goats fed graded levels of hempseed cake finisher diets.

Globally, the price of soybean meal, the most common proteinaceous ingredient in livestock diets, has become highly expensive prompting a search for alternative ingredients. Hemp seed cake is a promising alternative but could be limited by its high neutral detergent fiber and ether extract contents which impede nutrient intake and digestibility. However, some ruminant species such as goats have superior ability to digest high fiber and ether extract diets. Thus, the current research evaluated nutrient intake and digestibility, rumen fermentation, and microbial protein synthesis of goats fed hempseed cake as a substitute for soybean meal in finisher diets. A total of 25 Kalahari Red castrates (27 ± 3 kg, 4-5 months old) were assigned to five dietary treatments (5 goats/ diet) in a completely randomized design. A maize-lucerne-based finishing diet was formulated with hempseed cake substituting soybean meal as the primary protein ingredient at 0, 25, 50, 75, or 100 g/kg dry matter. Ether extract intake exhibited a positive linear trend (P ≤ 0.05) while crude protein intake and microbial nitrogen supply exhibited a negative linear trend (P ≤ 0.05) with dietary inclusion of hempseed cake. However, feeding hempseed cake did not influence (P > 0.05) apparent nutrient digestibility, rumen fermentation parameters and nitrogen use efficiency. In conclusion, the substitution of soybean meal for hempseed cake decreased crude protein intake and microbial nitrogen supply in goat finisher diets without compromising nutrient digestibility and nitrogen use efficiency. The study recommends partial or full replacement of soybean meal with hempseed cake in goat finisher diets.

Estimating Microbial Protein Synthesis in the Rumen-Can 'Omics' Methods Provide New Insights into a Long-Standing Question?

Rumen microbial protein synthesis (MPS) provides at least half of the amino acids for the synthesis of milk and meat protein in ruminants. As such, it is fundamental to global food protein security. Estimating microbial protein is central to diet formulation, maximising nitrogen (N)-use efficiency and reducing N losses to the environment. Whilst factors influencing MPS are well established in vitro, techniques for in vivo estimates, including older techniques with cannulated animals and the more recent technique based on urinary purine derivative (UPD) excretion, are subject to large experimental errors. Consequently, models of MPS used in protein rationing are imprecise, resulting in wasted feed protein and unnecessary N losses to the environment. Newer 'omics' techniques are used to characterise microbial communities, their genes and resultant proteins and metabolites. An analysis of microbial communities and genes has recently been used successfully to model complex rumen-related traits, including feed conversion efficiency and methane emissions. Since microbial proteins are more directly related to microbial genes, we expect a strong relationship between rumen metataxonomics/metagenomics and MPS. The main aims of this review are to gauge the understanding of factors affecting MPS, including the use of the UPD technique, and explore whether omics-focused studies could improve the predictability of MPS, with a focus on beef cattle.

Evaluation of methodologies of urine collection to estimate microbial synthesis in bovines diets containing lipids or not / Avaliação de metodologias de coleta de urina para estimar a síntese microbiana em dietas de bovinos contendo ou não lipídios

This study investigated the effectiveness of the urine sample collection method in predicting the volume urinary and synthesis of microbial nitrogen. Eight fistulated steers were used with accessible rumens and kept in individual stalls. Their diets consisted of corn silage; corn silage + concentrate; corn silage + concentrate with addition of lipids in the form of soybean oil; and corn silage + concentrate with addition of lipids in the form of soybean grains. Estimates of microbial protein synthesis were obtained based on the urinary excretion of purine derivatives. There was no effect of diets on daily creatinine excretion (P>0.05). There were differences (P<0.05) between the urinary volume and microbial synthesis values determined by the total urine collection and those estimated from the urine spot samples and equations proposed by different authors. The estimation of microbial synthesis based on the urine excretion of purine derivatives should be performed from the total collection of the urine for a period of 24 hours.

Este estudo investigou a eficácia do método de coleta de amostras de urina sobre a predição do volume urinário e síntese de nitrogênio microbiano. Oito novilhos fistulados foram utilizados com rúmen acessível e mantidos em baias individuais. Suas dietas consistiram de silagem de milho; silagem de milho + concentrado; silagem de milho + concentrado com adição de lipídios na forma de óleo de soja; e silagem de milho + concentrado com adição de lipídios na forma de grãos de soja. Estimativas de síntese de proteína microbiana foram obtidas com base na excreção urinária de derivados de purina. Não houve efeito de dietas na excreção diária de creatinina (P> 0,05). Houve diferenças (P <0,05) entre o volume urinário e os valores de síntese microbiana determinados pela coleta total de urina e os estimados a partir das amostras de manchas de urina e equações propostas por diferentes autores. A estimativa da síntese microbiana baseada na excreção de urina dos derivados de purina deve ser realizada a partir da coleta total da urina por um período de 24 horas.