Design, synthesis and evaluation of thieno[3,2-d]pyrimidine derivatives as novel potent CDK7 inhibitors.

The targeting of cyclin-dependent kinase 7 (CDK7) has become a highly desirable therapeutic approach in the field of oncology due to its dual role in regulating essential biological processes, encompassing cell cycle progression and transcriptional control. We have previously identified a highly selective thieno[3,2-d]pyrimidine-based CDK7 inhibitor with demonstrated efficacy and safety in animal model. In this study, we sought to optimize the thieno[3,2-d]pyrimidine core to discover a novel series of CDK7 inhibitors with improved potency and pharmacokinetic (PK) properties. Through extensive structure-activity relationship (SAR) studies, compound 20 has emerged as the lead candidate due to its potent inhibitory activity against CDK7 and remarkable efficacy on MDA-MB-453 cells, a representative triple negative breast cancer (TNBC) cell line. Furthermore, 20 has demonstrated favorable oral bioavailability and exhibited highly desirable pharmacokinetic (PK) properties, making it a promising lead candidate for further structural optimization.

Design, synthesis and evaluation of novel thieno[2,3d]pyrimidine derivatives as potent and specific RIPK2 inhibitors.

In human cells, receptor-interacting protein kinase 2 (RIPK2) is mainly known to mediate downstream enzymatic cascades from the nucleotide-binding oligomerization domain-containing receptors 1 and 2 (NOD1/2), which are regulators of pro-inflammatory signaling. Thus, the targeted inhibition of RIPK2 has been proposed as a pharmacological strategy for the treatment of a variety of pathologies, in particular inflammatory and autoimmune diseases. In this work, we designed and developed novel thieno[2,3d]pyrimidine derivatives, in order to explore their activity and selectivity as RIPK2 inhibitors. Primary in vitro evaluations of the new molecules against purified RIPKs (RIPK1-4) demonstrated outstanding inhibitory potency and selectivity for the enzyme RIPK2. Moreover, investigations for efficacy against the RIPK2-NOD1/2 signaling pathways, conducted in living cells, showed their potency could be tuned towards a low nanomolar range. This could be achieved by solely varying the substitutions at position 6 of the thieno[2,3d]pyrimidine scaffold. A subset of lead inhibitors were ultimately evaluated for selectivity against 58 human kinases other than RIPKs, displaying great specificities. We therefore obtained new inhibitors that might serve as starting point for the preparation of targeted tools, which could be useful to gain a better understanding of biological roles and clinical potential of RIPK2.

Synthesis, spectral, DFT calculation, antimicrobial, antioxidant, DNA/BSA binding and molecular docking studies of bio-pharmacologically active pyrimidine appended Cu(II) and Zn(II) complexes.

A new pyrimidine derivative Schiff base (HL) [HL = 2-((4-amino-6-chloropyrimidin-2-ylimino)methyl)-4-nitrophenol] has been synthesized using 2,6-diamino-4-chloropyrimidine and 5-nitrosalicylaldehyde. Transition metal complexes of Cu(II) and Zn(II) complexes [CuL(OAc)] (1), [ZnL(OAc)] (2) are prepared with HL/metal(II) acetate with molar ratio of 1:1. The Schiff base (HL) and the complexes 1 and 2 are evaluated by UV-Visible, 1H-NMR, FT-IR, EI-MS and ESR spectral techniques. Complexes 1 and 2 are confirmed as square planar geometry. Electrochemical studies of the complexes 1 and 2 are used to analyse the quasi reversible process. Density Functional Theory (DFT) using the B3LYP/6-31++G(d,p) level basis set was used to get the optimised geometry and non-linear optical properties. The complexes 1 and 2 are good antimicrobial agents than Schiff base (HL). The interactions of the HL and complexes 1 and 2 with Calf Thymus (CT) DNA are investigated by electronic absorption methods and viscosity measurements. Various molecular spectroscopy techniques, such as UV absorption and fluorescence, were used to explore the mechanism of interaction between the BSA and the ligand HL and complexes 1 & 2 under physiological settings. Complexes 1 and 2 are act as potential antioxidants than free Schiff base (HL) by DPPH radical scavenging assay. Furthermore, the purpose of the molecular docking studies was to better understand how metal complexes interact with biomolecules (CT-DNA and BSA). From these biological analyses, complex 1 acts as good intercalator with CT DNA & BSA and potent antioxidant with DPPH radical than complex 2.Communicated by Ramaswamy H. Sarma.

Synthesis and Evaluation of 2-Amine-4-oxyphosaniline Pyrimidine Derivatives as EGFR L858R/T790M/C797S Mutant Inhibitors.

Epidermal growth factor receptor (EGFR) C797S mutation leads to Osimertinib drug resistance by disturbing the covalent biding of Michael acceptor group to the Cys797 residue in the ATP biding cleft. In this manuscript, a class of 2-amine-4-oxyphosaniline pyrimidine derivatives were designed, synthesized and evaluated as new noncovalent reversible EGFR inhibitors against L858R/T790M/C797S (CTL) triple mutant. The kinases inhibitiory activity evaluation showed that four compounds exhibited significant inhibitory activities against CTL (IC50 < 30 nM). In particularly, the most promising compound 7a showed excellent enzymatic inhibitory activity against CTL with IC50 value of 9.9 nM, which was more potent than control compound Osimertinib. Moreover, cell proliferation assays indicated that 7a effectively inhibited H1975-EGFR L858R/T790M/C797S with IC50 value of 0.33 µM. Furthermore, compound 7a displayed good metabolic stabilities in human, rat and mouse liver microsomes, and the putative biding mode of compound 7a with ATP was revealed by molecular docking study. These findings strongly indicated that compound 7a was a promising L858R/T790M/C797S mutant EGFR inhibitor.

Prediction of Anti-proliferation Effect of [1,2,3]Triazolo[4,5-d]pyrimidine Derivatives by Random Forest and Mix-Kernel Function SVM with PSO.

In order to predict the anti-gastric cancer effect of [1,2,3]triazolo[4,5-d]pyrimidine derivatives (1,2,3-TPD), quantitative structure-activity relationship (QSAR) studies were performed. Based on five descriptors selected from descriptors pool, four QSAR models were established by heuristic method (HM), random forest (RF), support vector machine with radial basis kernel function (RBF-SVM), and mix-kernel function support vector machine (MIX-SVM) including radial basis kernel and polynomial kernel function. Furthermore, the model built by RF explained the importance of the descriptors selected by HM. Compared with RBF-SVM, the MIX-SVM enhanced the generalization and learning ability of the constructed model simultaneously and the multi parameters optimization problem in this method was also solved by particle swarm optimization (PSO) algorithm with very low complexity and fast convergence. Besides, leave-one-out cross validation (LOO-CV) was adopted to test the robustness of the models and Q2 was used to describe the results. And the MIX-SVM model showed the best prediction ability and strongest model robustness: R2 = 0.927, Q2 = 0.916, mean square error (MSE) = 0.027 for the training set and R2 = 0.946, Q2 = 0.913, MSE = 0.023 for the test set. This study reveals five key descriptors of 1,2,3-TPD and will provide help to screen out efficient and novel drugs in the future.

Naked-eye chemosensor with high absolute fluorescence quantum yield for selective detection of Cu(II) and cell imaging.

The regulation of copper in the human body is important for the prevention of several diseases. Therefore, a rhodamine B-based chemosensor 1 that demonstrates substantial affinity and selectivity was synthesized for the fluorescence imaging of copper(II). In the presence of Cu2+, the chemosensor underwent a color change from colorless to amaranth that was visible to the naked eye, and the fluorescence intensity did not change when excess EDTA was added to the solution. Furthermore, strong fluorescence was observed at 575 nm. The limit of detection was determined as 12.1 nM. The absolute fluorescence quantum yield was as high as 77 % and the stoichiometry between 1 and Cu2+ was determined to be 1:1 using a job plot. An analytical method was developed and successfully used to evaluate the sensor's ability for the fluorescence imaging of Cu2+ in HeLa tumor cells.

Antiaging Effect of 4-N-Furfurylcytosine in Yeast Model Manifests through Enhancement of Mitochondrial Activity and ROS Reduction.

Small compounds are a large group of chemicals characterized by various biological properties. Some of them also have antiaging potential, which is mainly attributed to their antioxidant activity. In this study, we examined the antiaging effect of 4-N-Furfurylcytosine (FC), a cytosine derivative belonging to a group of small compounds, on budding yeast Saccharomyces cerevisiae. We chose this yeast model as it is known to contain multiple conserved genes and mechanisms identical to that of humans and has been proven to be successful in aging research. The chronological lifespan assay performed in the study revealed that FC improved the viability of yeast cells in a concentration-dependent manner. Furthermore, enhanced mitochondrial activity, together with reduced intracellular ROS level, was observed in FC-treated yeast cells. The gene expression analysis confirmed that FC treatment resulted in the restriction of the TORC1 signaling pathway. These results indicate that FC has antiaging properties.

A novel 1-((3-(2-toluyl)-4,5-dihydroisoxazol-5-yl)methyl)-4-(trifluoromethyl)pyrimidin-2(1H)-one activates intrinsic mitochondria-dependent pathway and decreases angiogenesis in PC-3 cells.

Prostate cancer is among the most common cancer diagnoses in men, and the best treatment for patients with metastatic disease in advanced stages is still unclear. Previously, we have demonstrated that the three 1-(3-(aryl-4,5-dihydroisoxazol-5-yl)methyl)-4-trihalomethyl-1H-pyrimidin-2- ones derivatives (8a, 8e and 9c) present important cytotoxicity and selectivity for tumoral cells. Considering that various cytotoxic drugs have been assessed in patients with prostate cancer, but few drugs show survival advantage, we decided to study these three compounds (8a, 8e and 9c) in prostate cancer cells, androgen receptor (AR)-positive 22Rv-1 and AR-negative PC-3 cells. We obtained the half maximal inhibitory concentration (IC50) of 8a, 8e and 9c in prostate cancer cells and based on high selectivity of 9c to PC-3 cells, we determined the mechanism of this compound to induce cell death through different methods. We show here that 9c compound induces cell cycle arrest in G2/M, increasing the levels of reactive oxygen species and DNA damage, and triggers DNA damage response by ataxia-telangiectasia mutated (ATM) and histone H2AX phosphorylation induction. The compound also led PC-3 to lipid peroxidation and mitochondrial depolarization which triggered the activation of intrinsic pathway, confirmed by increase of cleaved caspase-9 and 3. In this work we also show the ability of 9c in reducing vascular endothelial growth factor expression (VEGF) and inhibiting topoisomerase I enzyme, therefore indicating a potential new molecule to be further investigated for prostate cancer management.

The Pyrazolo[3,4-d]pyrimidine Derivative, SCO-201, Reverses Multidrug Resistance Mediated by ABCG2/BCRP.

ATP-binding cassette (ABC) transporters, such as breast cancer resistance protein (BCRP), are key players in resistance to multiple anti-cancer drugs, leading to cancer treatment failure and cancer-related death. Currently, there are no clinically approved drugs for reversal of cancer drug resistance caused by ABC transporters. This study investigated if a novel drug candidate, SCO-201, could inhibit BCRP and reverse BCRP-mediated drug resistance. We applied in vitro cell viability assays in SN-38 (7-Ethyl-10-hydroxycamptothecin)-resistant colon cancer cells and in non-cancer cells with ectopic expression of BCRP. SCO-201 reversed resistance to SN-38 (active metabolite of irinotecan) in both model systems. Dye efflux assays, bidirectional transport assays, and ATPase assays demonstrated that SCO-201 inhibits BCRP. In silico interaction analyses supported the ATPase assay data and suggest that SCO-201 competes with SN-38 for the BCRP drug-binding site. To analyze for inhibition of other transporters or cytochrome P450 (CYP) enzymes, we performed enzyme and transporter assays by in vitro drug metabolism and pharmacokinetics studies, which demonstrated that SCO-201 selectively inhibited BCRP and neither inhibited nor induced CYPs. We conclude that SCO-201 is a specific, potent, and potentially non-toxic drug candidate for the reversal of BCRP-mediated resistance in cancer cells.

[1,2,4]Triazolo[1,5-a]pyrimidine derivative (Mol-5) is a new NS5-RdRp inhibitor of DENV2 proliferation and DENV2-induced inflammation.

Dengue fever is an acute infectious disease caused by dengue virus (DENV) and transmitted by Aedes mosquitoes. There is no effective vaccine or antiviral drug available to date to prevent or treat dengue disease. Recently, RNA-dependent RNA polymerase (RdRp), a class of polymerases involved in the synthesis of complementary RNA strands using single-stranded RNA, has been proposed as a promising drug target. Hence, we screened new molecules against DENV RdRp using our previously constructed virtual screening method. Mol-5, [1,2,4]triazolo[1,5-a]pyrimidine derivative, was screened out from an antiviral compound library (~8000 molecules). Using biophysical methods, we confirmed the direct interactions between mol-5 and purified DENV RdRp protein. In luciferase assay, mol-5 inhibited NS5-RdRp activity with an IC50 value of 1.28 ± 0.2 µM. In the cell-based cytopathic effect (CPE) assay, mol-5 inhibited DENV2 infectivity with an EC50 value of 4.5 ± 0.08 µM. Mol-5 also potently inhibited DENV2 RNA replication as observed in immunofluorescence assay and qRT-PCR. Both the viral structural (E) and non-structural (NS1) proteins of DENV2 were dose-dependently decreased by treatment with mol-5 (2.5-10 µM). Mol-5 treatment suppressed DENV2-induced inflammation in host cells, but had no direct effect on host defense (JAK/STAT-signaling pathway). These results demonstrate that mol-5 could be a novel RdRp inhibitor amenable for further research and development.

Discovery of a new class of valosine containing protein (VCP/P97) inhibitors for the treatment of non-small cell lung cancer.

Valosine containing protein (VCP/p97) is a member of the AAA ATPase family involved in several essential cellular functions and plays an important role in the ubiquitin-mediated degradation of misfolded proteins. P97 has a significant role in maintaining the cellular protein homeostasis for tumor cell growth and survival and has been found overexpressed in many tumor types. No new molecule entities based on p97 target were approved in clinic. Herein, a series of novel pyrimidine structures as p97 inhibitors were designed and synthesized. After enzymatic evaluations, structure-activity relationships (SAR) were discussed in detailed. Among the screened compounds, derivative 35 showed excellent enzymatic inhibitory activity (IC50, 36 nM). The cellular inhibition results showed that compound 35 had good antiproliferative activity against the non-small cell lung cancer A549 cells (IC50, 1.61 µM). Liver microsome stability showed that the half-life of compound 35 in human liver microsome was 42.3 min, which was more stable than the control CB-5083 (25.8 min). The in vivo pharmacokinetic results showed that the elimination phase half-lives of compound 35 were 4.57 h for ig and 3.64 h for iv, respectively and the oral bioavailability was only 4.5%. These results indicated that compound 35 could be effective for intravenous treatment of non-small cell lung cancer.

Synthesis of some novel pyrimidine, thiophene, coumarin, pyridine and pyrrole derivatives and their biological evaluation as analgesic, antipyretic and anti-inflammatory agents

Pyrimidine derivative 3 was afforded through the reaction of compound (1) with 5-ureidohydantion (2). Product 3 underwent a cyclization to produce fused pyrimidine derivative 7, although the latter product 7 was synthesized through one step via the reaction of compound (1) with 5-ureidohydantion (2) using another catalyst. Compound 3 was oriented to react with cyclic ketones 8a,b in the presence of elemental sulfur, salicylaldehyde (10), aryldiazonium chlorides 12a,b and ω-bromo-4-methoxy- acetophenone (14), which afforded, fused thiophene derivatives 9a,b, coumarin derivative 11, arylhdrazono derivatives 13a,b and 4-methoxyphenyl butenyl derivative 15, respectively. The latter product 15 was reacted with either potassium cyanide (16a) or potassium thiocyanide (16b) to form cyano and thiocyano derivatives 17a,b, respectively. Compound 17a underwent further cyclization to afford pyridopyrimidine derivative 19. Compound 15 was reacted with either hydrazine (20a) or phenylhydrazine (20b) to produce hydrazo derivatives 21a,b and these products were cyclize to produce pyrrole derivatives 23a,b. Finally, 5-ureidohydantion (2) was reacted with compounds 24a,b,c to afford pyrimidine derivatives 25a,b,c. The structures of the synthesized compounds were confirmed using IR, 1H NMR, 13C NMR and mass spectrometry techniques. Compounds 11 and 19 have promising as analgesic and antipyretic activities

Crystal structure of 2-chloro-5-(3-hy-droxy-3-methyl-but-1-yn-1-yl)pyrimidine.

In the title compound, C9H9ClN2O, the ethynyl-pyrimidine moiety displays an almost planar geometry. In the crystal, mol-ecules are linked by O-H⋯N and C-Hpyrimidine⋯O hydrogen bonds, forming a three-dimensional supra-molecular architecture.

Differentially activated Src kinase in chemo-naïve human primary osteosarcoma cells and effects of a Src kinase inhibitor.

The therapeutic treatment of osteosarcoma (OS), a rare malignant teenage cancer of the skeletal system, still represents a great challenge as patient survival after conventional protocol chemotherapy treatment has not improved in the last four decades leaving poor patient prognoses. Therefore, many efforts have been done to find increasingly reliable OS cell models and to identify "druggable" targets in OS, in order to identify novel effective therapeutic approaches and treatment strategies. In this contest, the more successful use of patient-derived cell cultures in respect to human commercial lines and findings of Src kinase deregulation in cancer, prompted us to study for the first time the activation state of Src and the potential activity of our Src inhibitor SI-83 in a number of chemo-naïve patient-derived primary OS cells. We here demonstrate that Src is hyperactivated in OS cells in respect to the nonmalignant counterpart and that SI-83 is able to strongly decrease cell viability, proliferation, Src416 phosphorylation, and cell migration. © 2017 BioFactors, 43(6):801-811, 2017.

Biologically Active Cu(II), Co(II), Ni(II) and Zn(II) Complexes of Pyrimidine Derivative Schiff Base: DNA Binding, Antioxidant, Antibacterial and In Vitro Anticancer Studies.

A new pyrimidine derivative Schiff base ligand (HL) [HL = 2-(4,6-dimethylpyrimidin-2-ylimino)methyl)-4-nitrophenol] and its metal(II) complexes [CuL2] (1), [CoL2] (2), [NiL2] (3) and [ZnL2] (4) have been synthesized and characterized by several spectral techniques. The square planar geometry of the complexes 1-4 confirmed by UV-Visible, ESR and EI-mass spectral techniques. DNA binding study of the complexes 1-4 with Calf Thymus (CT) DNA using absorption spectral titration at different pH (4.0, 7.0 & 10.0) have been scrutinized that the complexes 1-4 bound by groove binding mode with significant binding constant values (K b  = 5.61 × 105 M-1 (1), 2.60 × 105 M-1 (2), 2.48 × 105 M-1 (3) and 6.98 × 104 M-1 (4) at pH = 10.0. Binding nature of the complexes 1-4 with CT DNA has further confirmed by emission, viscometry and cyclic voltammetry which also recommended that complexes 1-4 bound with CT DNA. The complexes 1-4 possessed effective scavenging effect during the DPPH and SOD radical scavenging method. The antibacterial activity of the complexes 1-4 was vetted against several bacterial strains and the results shows that the ligand (HL) and complexes 1-4 are more active in Bascillus subtilis. The anticancer activity of the complexes 1-4 was evaluated against Human Breast Cancer Cells (MCF-7), Human Cervical Cancer Cells (HeLa), Human Laryngeal Epithelial Carcinoma (HEp2) and Normal Human Dermal Fibroblast (NHDF) by MTT assay, which revealed that complexes 1 & 2 have modest activity against the cancer cell lines than ligand (HL) and complexes 3 & 4.

Spectroscopic analysis on the binding interaction of biologically active pyrimidine derivative with bovine serum albumin.

A biologically active antibacterial reagent, 2-amino-6-hydroxy-4-(4-N, N-dimethylaminophenyl)-pyrimidine-5-carbonitrile (AHDMAPPC), was synthesized. It was employed to investigate the binding interaction with the bovine serum albumin (BSA) in detail using different spectroscopic methods. It exhibited antibacterial activity against Escherichia coli and Staphylococcus aureus which are common food poisoning bacteria. The experimental results showed that the fluorescence quenching of model carrier protein BSA by AHDMAPPC was due to static quenching. The site binding constants and number of binding sites (n≈1) were determined at three different temperatures based on fluorescence quenching results. The thermodynamic parameters, enthalpy change (ΔH), free energy (ΔG) and entropy change (ΔS) for the reaction were calculated to be 15.15 kJ/mol, -36.11 kJ/mol and 51.26 J/mol K according to van't Hoff equation, respectively. The results indicated that the reaction was an endothermic and spontaneous process, and hydrophobic interactions played a major role in the binding between drug and BSA. The distance between donor and acceptor is 2.79 nm according to Förster's theory. The alterations of the BSA secondary structure in the presence of AHDMAPPC were confirmed by UV-visible, synchronous fluorescence, circular dichroism (CD) and three-dimensional fluorescence spectra. All these results indicated that AHDMAPPC can bind to BSA and be effectively transported and eliminated in the body. It can be a useful guideline for further drug design.

Crystal structure of methyl 4-(2-fluoro-phenyl)-6-methyl-2-sulfanylidene-1,2,3,4-tetra-hydro-pyrimidine-5-carb-oxy-late.

In the title compound, C13H13FN2O2S, the pyrimidine ring adopts a twist-boat conformation with the MeCN and methine-C atoms displaced by 0.0938 (6) and 0.2739 (3) Å, respectively, from the mean plane through the other four atoms of the ring. The 2-fluoro-benzene ring is positioned axially and forms a dihedral angle of 89.13 (4)° with the mean plane through the pyrimidine ring. The crystal structure features N-H⋯O, N-H⋯S and C-H⋯O hydrogen bonds that link mol-ecules into supra-molecular chains along the b axis. These chains are linked into a layer parallel to (10-1) by C-H⋯π inter-actions; layers stack with no specific inter-actions between them.

Crystal structure of ethyl 2-acetyl-3,7-dimethyl-5-(thio-phen-2-yl)-5H-thia-zolo[3,2-a]pyrimidine-6-carboxyl-ate.

In the title compound, C17H18N2O3S2, the pyrimidine ring adopts a shallow sofa conformation, with the C atom bearing the axially-oriented thio-phene ring as the flap [deviation = 0.439 (3) Å]. The plane of the thio-phene ring lies almost normal to the pyrimidine ring, making a dihedral angle of 79.36 (19)°. In the crystal, pairs of very weak C-H⋯O hydrogen bonds link the mol-ecules related by twofold rotation axes, forming R 2 (2)(18) rings, which are in turn linked by another C-H⋯O inter-action, forming chains of rings along [010]. In addition, weak C-H⋯π(thio-phene) inter-actions link the chains into layers parallel to [001] and π-π inter-actions with a centroid-centroid distance of 3.772 (10) Šconnect these layers into a three-dimensional network.

Crystal structure of ethyl 6-(2-fluoro-phen-yl)-4-hy-droxy-2-sulfanyl-idene-4-tri-fluoro-meth-yl-1,3-diazinane-5-carboxyl-ate.

In the title compound, C14H14F4N2O3S, the central di-hydro-pyrimidine ring adopts a sofa conformation with the C atom bearing the 2-fluoro-benzene ring displaced by 0.596 (3) Šfrom the other five atoms. The 2-fluoro-benzene ring is positioned axially and bis-ects the pyrimidine ring with a dihedral angle of 70.92 (8)°. The mol-ecular conformation is stabilized by an intra-molecular O-H⋯O hydrogen bond, generating an S(6) ring. The crystal structure features C-H⋯F, N-H⋯S and N-H⋯O hydrogen bonds, which link the mol-ecules into a three-dimensional network.

Research progress of synthesis and pharmacological activity of pyrazolo3, 4-d pyrimidine derivatives / 中国药学杂志

Due to the extensive pharmacological effects, pyrazolo[3, 4-d] pyrimidine derivatives have aroused great attention. There are a variety of ways in their synthesis, including using pyrazole ring, pyrimidine ring and other materials as parent ring. The compounds have strong bactericidal, anti-inflammatory, anti-cancer and anti-virus activities, etc. In this paper, the synthesis and pharmacological activities of pyrazolo[3, 4-d] pyrimidine derivatives taking the synthesis as the main line are summarized.