In silico study and in vitro antileishmanial and antitrypanosomal evaluation of azopyrazoles and azopyrimidines.

Hydrazones 1-6, azo-pyrazoles 7-9 and azo-pyrimidines 10-15 are compounds that exhibit antibacterial activity. The mode of action and structures of these derivatives have been previously confirmed as antibacterial. In this investigation, biological screening and molecular docking studies were performed for derivatives 1-15, with compounds 2, 7, 8, 14 and 15 yielding the best energy scores (from -20.7986 to -10.5302 kcal/mol). Drug-likeness and in silico ADME prediction for the most potent derivatives, 2, 7, 8, 14 and 15, were predicted (from 84.46 to 96.85%). The latter compounds showed good recorded physicochemical properties and pharmacokinetics. Compound 8 demonstrated the strongest inhibition, which was similar to the positive control (eflornithine) against Trypanosoma brucei brucei (WT), with an EC50 of 25.12 and 22.52µM, respectively. Moreover, compound 14 exhibited the best activity against Leishmania mexicana promastigotes and Leishmania major promastigotes (EC50 =46.85; 40.78µM, respectively).

RuBi-Ruxolitinib: A Photoreleasable Antitumor JAK Inhibitor.

We describe the synthesis and biological testing of ruthenium-bipyridine ruxolitinib (RuBiRuxo), a photoreleasable form of ruxolitinib, a JAK inhibitor used as an antitumoral agent in cutaneous T-cell lymphomas (CTCL). This novel caged compound is synthesized efficiently, is stable in aqueous solution at room temperature, and is photoreleased rapidly by visible light. Irradiation of RuBiRuxo reduces cell proliferation and induces apoptosis in a light- and time-dependent manner in a CTCL cell line. This effect is specific and is mediated by a decreased phosphorylation of STAT proteins. Our results demonstrate the potential of ruthenium-based photocompounds and light-based therapeutic approaches for the potential treatment of cutaneous lymphomas and other pathologies.

Design, synthesis and evaluation of a pyrazolo[3,4-d]pyrimidine derivative as a novel and potent TGFß1R1 inhibitor.

The transforming growth factor ß1 (TGFß1)/SMAD signaling pathway regulates many vital physiological processes. The development of potent inhibitors targeting activin receptor-like kinase 5 (ALK5) would provide potential treatment reagents for various diseases. A significant number of ALK5 inhibitors have been discovered, and they are currently undergoing clinical evaluation at various stages. However, the clinical demands were far from being met. In this study, we utilized an alternative conformation-similarity-based virtual screening (CSVS) combined with a fragment-based drug designing (FBDD) strategy to efficiently discover a potent and active hit with a novel chemical scaffold. After structural optimization in the principle of group replacement, compound 57 was identified as the most promising ALK5 inhibitor. Compound 57 demonstrated significant inhibitory effects against the TGF-ß1/SMAD signaling pathway. It could markedly attenuate the production of extracellular matrix (ECM) and deposition of collagen. Also, the lead compound showed adequate pharmacokinetic (PK) properties and good in vivo tolerance. Moreover, treatment with compound 57 in two different xerograph models showed significant inhibitory effects on the growth of pancreatic cancer cells. These results suggested that lead compound 57 refers as a promising ALK5 inhibitor both in vitro and in vivo, which merits further validation.

Unexpected Noncovalent Off-Target Activity of Clinical BTK Inhibitors Leads to Discovery of a Dual NUDT5/14 Antagonist.

Cofactor mimicry represents an attractive strategy for the development of enzyme inhibitors but can lead to off-target effects due to the evolutionary conservation of binding sites across the proteome. Here, we uncover the ADP-ribose (ADPr) hydrolase NUDT5 as an unexpected, noncovalent, off-target of clinical BTK inhibitors. Using a combination of biochemical, biophysical, and intact cell NanoBRET assays as well as X-ray crystallography, we confirm catalytic inhibition and cellular target engagement of NUDT5 and reveal an unusual binding mode that is independent of the reactive acrylamide warhead. Further investigation of the prototypical BTK inhibitor ibrutinib also revealed potent inhibition of the largely unstudied NUDIX hydrolase family member NUDT14. By exploring structure-activity relationships (SARs) around the core scaffold, we identify a potent, noncovalent, and cell-active dual NUDT5/14 inhibitor. Cocrystallization experiments yielded new insights into the NUDT14 hydrolase active site architecture and inhibitor binding, thus providing a basis for future chemical probe design.

Identification of CH2-linked quinolone-aminopyrimidine hybrids as potent anti-MRSA agents: Low resistance potential and lack of cross-resistance with fluoroquinolone antibiotics.

The structural optimization of B14, an antibacterial agent we previously obtained, has led to the discovery of a new class of CH2-linked quinolone-aminopyrimidine hybrids with potent anti-MRSA activities. Surprisingly, the hybrids lacking a C-6 fluoro atom at the quinolone nucleus showed equal or even stronger anti-MRSA activities than their corresponding 6-fluoro counterparts, despite the well-established structure-activity relationships (SARs) indicating that the 6-fluoro substituent enhances the antibacterial activity in conventional fluoroquinolone antibiotics. Moreover, these new hybrids, albeit structurally related to conventional fluoroquinolones, showed no cross-resistance with fluoroquinolone drugs. The most active compound, 15m, exhibited excellent activities with a MIC value of 0.39 µg/mL against both fluoroquinolone-sensitive strain USA500 and -resistant MRSA isolate Mu50. Further resistance development studies indicated MRSA is unlikely to acquire resistance against 15m. Moreover, 15m displayed favorable in vivo half-life and safety profiles. These findings suggest a rationale for further evolution of quinolone antibiotics with a high barrier to resistance.

Tetrahydropyridine LIMK inhibitors: Structure activity studies and biological characterization.

LIM Kinases, LIMK1 and LIMK2, have become promising targets for the development of inhibitors with potential application for the treatment of several major diseases. LIMKs play crucial roles in cytoskeleton remodeling as downstream effectors of small G proteins of the Rho-GTPase family, and as major regulators of cofilin, an actin depolymerizing factor. In this article we describe the conception, synthesis, and biological evaluation of novel tetrahydropyridine pyrrolopyrimidine LIMK inhibitors. Homology models were first constructed to better understand the binding mode of our preliminary compounds and to explain differences in biological activity. A library of over 60 products was generated and in vitro enzymatic activities were measured in the mid to low nanomolar range. The most promising derivatives were then evaluated in cell on cofilin phosphorylation inhibition which led to the identification of 52 which showed excellent selectivity for LIMKs in a kinase selectivity panel. We also demonstrated that 52 affected the cell cytoskeleton by disturbing actin filaments. Cell migration studies with this derivative using three different cell lines displayed a significant effect on cell motility. Finally, the crystal structure of the kinase domain of LIMK2 complexed with 52 was solved, greatly improving our understanding of the interaction between 52 and LIMK2 active site. The reported data represent a basis for the development of more efficient LIMK inhibitors for future in vivo preclinical validation.

Structure-Guided Discovery and Preclinical Assessment of Novel (Thiophen-3-yl)aminopyrimidine Derivatives as Potent ERK1/2 Inhibitors.

The RAS-RAF-MEK-ERK signaling cascade is abnormally activated in various tumors, playing a crucial role in mediating tumor progression. As the key component at the terminal stage of this cascade, ERK1/2 emerges as a potential antitumor target and offers a promising therapeutic strategy for tumors harboring BRAF or RAS mutations. Here, we identified 36c with a (thiophen-3-yl)aminopyrimidine scaffold as a potent ERK1/2 inhibitor through structure-guided optimization for hit 18. In preclinical studies, 36c showed powerful ERK1/2 inhibitory activities (ERK1/2 IC50 = 0.11/0.08 nM) and potent antitumor efficacy both in vitro and in vivo against triple-negative breast cancer and colorectal cancer models harboring BRAF and RAS mutations. 36c could directly inhibit ERK1/2, significantly block the phosphorylation expression of their downstream substrates p90RSK and c-Myc, and induce cell apoptosis and incomplete autophagy-related cell death. Taken together, this work provides a promising ERK1/2 lead compound for multiple tumor-treatment drug discovery.

Akt Inhibitor Advancements: From Capivasertib Approval to Covalent-Allosteric Promises.

Akt kinase is vital in cell growth, survival, metabolism, and migration. Dysregulation of Akt signaling is implicated in cancer and metabolic disorders. In the context of cancer, overactive Akt promotes cell survival and proliferation. This has spurred extensive research into developing Akt inhibitors as potential therapeutic agents to disrupt aberrant Akt signaling. Akt inhibitors are classified into three main types: ATP-competitive, allosteric, and covalent-allosteric inhibitors (CAAIs). ATP-competitive inhibitors compete with ATP for binding to Akt, allosteric inhibitors interact with the Pleckstrin homology (PH) domain, and covalent-allosteric inhibitors form covalent bonds, making them more potent and selective. Notably, capivasertib (AZD5363), a potent ATP-competitive Akt inhibitor, received FDA approval in November 2023 for use in combination with the estrogen receptor degrader fulvestrant to treat breast cancer. Challenges remain, including improving selectivity, identifying biomarkers to tailor treatments, and enhancing therapeutic efficacy while minimizing adverse effects. Particularly covalent-allosteric inhibitors hold promise for future more effective and personalized treatments.

Synthetic access to diverse thiazetidines via a one-pot microwave assisted telescopic approach and their interaction with biomolecules.

A one-pot microwave assisted telescopic approach is reported for the chemo-selective synthesis of substituted 1,3-thiazetidines using readily available 2-aminopyridines/pyrazines/pyrimidine, substituted isothiocyanates and 1,2-dihalomethanes. The procedure involves thiourea formation from 2-aminopyridines/pyrazines/pyrimidine with the substituted isothiocyanates followed by a base catalysed nucleophilic attack of the CS bond on the 1,2-dihalomethane. Subsequently, a cyclization reaction occurs to yield substituted 1,3-thiazetidines. These four membered strained ring systems are reported to possess broad substrate scope with high functional group tolerance. The above synthetic sequence for the formation of four membered heterocycles is proven to be a modular and straightforward approach. Further the mechanistic pathway for the formation of 1,3-thiazetidines was supported by computational evaluations and X-ray crystallography analyses. The relevance of these thiazetidines in biological applications is evaluated by studying their ability to bind bio-macromolecules like proteins and nucleic acids.

Synthesis of fluorescent 5-heteroarylpyrimidine-containing oligonucleotides via post-synthetic trifluoromethyl conversion.

Post-synthetic conversion of the trifluoromethyl group to a heteroaryl group at the C5 position of the pyrimidine base in DNA oligonucleotides was achieved. Specifically, the oligonucleotides containing 5-trifluoromethylpyrimidine bases were treated with o-phenylenediamines and o-aminothiophenols as nucleophiles to afford the corresponding 5-(benzimidazol-2-yl)- and 5-(benzothiazol-2-yl)-pyrimidine-modified bases. Furthermore, evaluation of the fluorescence properties of the obtained oligonucleotides revealed that among them the oligonucleotide containing 5-(5-methylbenzimidazol-2-yl)cytosine exhibited the highest fluorescence intensity. These results indicated that post-synthetic trifluoromethyl conversion, which is practical and operationally simple, is a powerful tool for exploring functional oligonucleotides.

Discovery of a Meisoindigo-Derived PROTAC as the ATM Degrader: Revolutionizing Colorectal Cancer Therapy via Synthetic Lethality with ATR Inhibitors.

Meisoindigo (Mei) has long been recognized in chronic myeloid leukemia (CML) treatment. To elucidate its molecular target and mechanisms, we embarked on designing and synthesizing a series of Mei-derived PROTACs. Through this endeavor, VHL-type PROTAC 9b was identified to be highly cytotoxic against SW620, SW480, and K562 cells. Employing DiaPASEF-based quantitative proteomic analysis, in combination with extensive validation assays, we unveiled that 9b potently and selectively degraded ATM across SW620 and SW480 cells in a ubiquitin-proteasome-dependent manner. 9b-induced selective ATM degradation prompted DNA damage response cascades, thereby leading to the cell cycle arrest and cell apoptosis. This pioneering discovery renders the advent of ATM degradation for anti-cancer therapy. Notably, 9b-induced ATM degradation synergistically enhanced the efficacy of ATR inhibitor AZD6738 both in vitro and in vivo. This work establishes the synthetic lethality-inducing properties of ATR inhibitors in the ATM-deficient context, thereby providing new avenues to innovative therapies for colorectal cancer.

Insights into the Overcoming EGFRDel19/T790M/C797S Mutation: A Perspective on the 2-Aryl-4-aminothienopyrimidine Backbone.

The epithelial growth factor receptor (EGFR) signaling pathway has been proposed to benefit non-small cell lung cancer (NSCLC) treatment. In this manuscript, we investigated the modification of 2-aryl-4-aminoquinazoline, the classical backbone of the fourth-generation EGFR inhibitors, in addition to obtaining a series of novel 2-aryl-4-aminothienopyrimidine derivatives (A1~A45), we also gained further understanding of the modification of this framework. Derivatives were tested for cytotoxicity against cancer cell lines (cervical cancer cell line Hela, lung cancer cell lines A549, H1975, and PC-9, Ba/F3-EGFRDel19/T790M/C797S cells, and human normal hepatocytes LO2) as well as for the derivative's inhibitory activity against EGFRWT, EGFRL858R/T790M, and EGFRDel19/T790M/C797S kinase inhibitory activities. The results showed that most of the target compounds showed moderate to excellent activity against one or more cancer cell lines. Among them, the antitumor activity (IC50) of the most promising A9 against A549 and H1975 cell lines was 0.77±0.08 µM, 6.90±0.83 µM, respectively. At concentration of 10 µM, A9 can be employed as the fourth-generation of EGFR inhibitors with the ability to overcome the C797S drug resistance since it can suppress EGFRDel19/T790M/C797S cells and kinase by 98.90 % and 85.88 %, respectively. Moreover, the tumor-bearing nude mice experiment further shows that A9 can significantly inhibit the growth of tumor in vivo, with the tumor inhibition rate (TIR) of 55.92 %, which was equivalent to the positive group. After that, from the result of HE staining experiment and blood biochemical analysis experiment, A9 show low toxicity and good safety, which is worthy of further research and development.

Rational design and synthesis of 2,4-dichloro-6-methyl pyrimidine derivatives as potential selective EGFRT790M/L858R inhibitors for the treatment of non-small cell lung cancer.

Many patients with non-small cell lung cancer (NSCLC) initially benefit from epidermal growth factor receptor (EGFR) targeted therapy. Unfortunately, varying degrees of resistance or side effects eventually develop. Overcoming and preventing the resistance and side effects of EGFR inhibitors has become a hot topic of research today. Based on the previous studies on AZD-9291, we designed and synthesized two series of 2,4-dichloro-6-methylpyrimidine derivatives, 19 compounds in total, as potential inhibitors of the EGFR kinase. The most promising compound, L-18, showed better inhibitory activity (81.9%) and selectivity against EGFRT790M/L858R kinase. In addition, L-18 showed strong antiproliferative activity against H1975 cells with an IC50 value of 0.65 ± 0.06 µM and no toxicity to normal cells (LO-2). L-18 was able to dose-dependently induce the apoptosis of H1975 cells and produced a cell-cycle-blocking effect, and it can also dose-dependently inhibit the migration and invasion of H1975 cells. L-18 also showed in vivo anticancer efficacy in H1975 cells xenograft mice. We also performed a series of in vivo and in vitro toxicological evaluations of compound L-18, which did not cause obvious injury in mice during administration. These results suggest that L-18 may be a promising drug candidate that warrants further investigation.

Synthesis, DFT calculations, and anti-proliferative evaluation of pyrimidine and selenadiazolopyrimidine derivatives as dual Topoisomerase II and HSP90 inhibitors.

Novel series of aminopyrimidines bearing a biologically active cyclohexenone 3a-f and oxo-selaneylidene moiety 4, besides selenadiazolopyrimidines (5a-e and 7), were synthesised using 5,6-diaminouracils as starting materials. Compound 3a exhibited strong anti-proliferative activity against three cell lines: HepG-2 (IC50 14.31 ± 0.83 µM), A-549 (IC50 30.74 ± 0.76 µM), and MCF-7 (IC50 27.14 ± 1.91 µM). Also, it was four times more selectively cytotoxic against WI-38 cell lines than doxorubicin. Furthermore, Topoisomerase II (IC50 4.48 ± 0.65 µM) and HSP90 (IC50 1.78 ± 0.11 µM) were both strongly inhibited in vitro by 3a. The cell cycle was halted at the G1-S phase, and total apoptotic cells were 65 times more than control Hep-G2 cells. Besides, it increased caspase-3 gene expression, triggering mitochondrial cell death. Molecular docking study indicated that it could bind to Topoisomerase II and HSP90 binding sites in an inhibitory mode. Its geometric properties were investigated using the density functional theory (DFT). Furthermore, compound 3a demonstrated in silico good oral bioavailability.

Design, Synthesis and Bioactivity of Novel Pyrimidine Sulfonate Esters Containing Thioether Moiety.

Pesticides play an important role in crop disease and pest control. However, their irrational use leads to the emergence of drug resistance. Therefore, it is necessary to search for new pesticide-lead compounds with new structures. We designed and synthesized 33 novel pyrimidine derivatives containing sulfonate groups and evaluated their antibacterial and insecticidal activities. Results: Most of the synthesized compounds showed good antibacterial activity against Xanthomonas oryzae pv. Oryzae (Xoo), Xanthomonas axonopodis pv. Citri (Xac), Pseudomonas syringae pv. actinidiae (Psa) and Ralstonia solanacearum (Rs), and certain insecticidal activity. A5, A31 and A33 showed strong antibacterial activity against Xoo, with EC50 values of 4.24, 6.77 and 9.35 µg/mL, respectively. Compounds A1, A3, A5 and A33 showed remarkable activity against Xac (EC50 was 79.02, 82.28, 70.80 and 44.11 µg/mL, respectively). In addition, A5 could significantly improve the defense enzyme (superoxide dismutase, peroxidase, phenylalanine ammonia-lyase and catalase) activity of plants against pathogens and thus improve the disease resistance of plants. Moreover, a few compounds also showed good insecticidal activity against Plutella xylostella and Myzus persicae. The results of this study provide insight into the development of new broad-spectrum pesticides.

Design, synthesis and anti-tumor activity studies of novel pyrido[3, 4-d]pyrimidine derivatives.

In order to find high-efficiency and low-toxic anti-tumor drugs, 29 pyrido[3,4-d]pyrimidine compounds were designed, synthesized and evaluated by MTT assay in vitro. The results presented that most of the compounds had good antitumor activities, among which compound 30 had the best anti-tumor activity on MGC803 cells (IC50 = 0.59 µM). Mechanistic studies exhibited that compound 30 inhibited migration of MGC803 and induced apoptosis. It was proved that compound 30 up-regulated expression of Bid and PARP, down-regulated expression of CycD1 by western blot experiments. This study indicated that compound 30 might be served as a lead agent for the treatment of human gastric cancers.

Human dihydrofolate reductase inhibition effect of 1-Phenylpyrazolo[3,4-d]pyrimidines: Synthesis, antitumor evaluation and molecular modeling study.

A new series of pyrazolo[3,4-d]pyrimidine analogues bearing different amino acid conjugates 10a-m were synthesized with the aim to evaluate their antitumor effect through simultaneous inhibition of human dihydrofolate reductase (hDHFR). All novel compounds were tested to screen their enzyme inhibition activity against (hDHFR) beside their in vitro cytotoxicity against six human MTX resistant cancer cell lines namely, human prostate cancer (PC-3), pancreatic human cancer cell lines (BxPC-3), colorectal carcinoma (HCT-116), human hepatocellular carcinoma (HepG-2), cervical carcinoma (HeLa), and mammary gland breast cancer (MCF-7), besides normal immortalized pancreatic cell line (HPDE). Compounds 10e, 10f, 10g inhibited DHFR at considerable low (IC50 < 1 µM) in comparison to MTX (IC50 = 5.61 µM) beside their characteristic cytotoxic effects on different resistant cancer cell lines. Flow cytometry was done for the most active candidate compound 10e against MCF-7 breast cancer cell line. The results illustrated that compound 10e induced apoptosis and arrested MCF-7 cell cycle in the G1/S phase. Western blot for visualization and quantification was used to confirm the capability of compound 10e to induce the expression of proapoptotic caspases and Bax proteins in MCF-7 breast cancer cell line beside its ability to reduce the expression of antiapoptotic Bcl-2 protein. Molecular modeling studies demonstrated that compound 10e elucidated binding energy of (S= - 8.4390 Kcal/mol) that exceed that of the normal ligand MTX (S= - 8.3951Kcal/mol) in addition to several favorable binding interactions with the active site residues.

Design, synthesis and activity evaluation of isopropylsulfonyl-substituted 2,4- diarylaminopyrimidine derivatives as FAK inhibitors for the potential treatment of pancreatic cancer.

A series of isopropylsulfonyl-substituted 2,4-diarylaminopyrimidine derivatives were designed and synthesized as FAK inhibitors to evaluate their biological activity against pancreatic cancer. One of the most promising compound, 9h, effectively interfered with FAK-mediated phosphorylation and suppressed the proliferation of human pancreatic cancer AsPC-1 cells with half maximal inhibitory concentration (IC50) values of 0.1165 nM and 0.1596 µM, respectively. In addition, 9h also exhibited relatively low toxicity against immortalized normal human liver L-02 cells, indicating its low hepatotoxicity at an equivalent dosage. Furthermore, the elucidation of the mechanism of action revealed that compound 9h effectively inhibited cell migration and inhibited the proliferation of AsPC-1 by blocking the cell cycle at the G2/M phase. Moreover, 9h also demonstrated efficacy in inhibiting tumor growth in a murine AsPC-1 cell xenograft model at the dosage of 10 mg/kg without losing noticeable body weight. All these findings provide important clues for the identification of potent FAK inhibitors.

6-Amino-2,4,5-trimethylpyridin-3-ol and 2-amino-4,6-dimethylpyrimidin-5-ol derivatives as selective fibroblast growth factor receptor 4 inhibitors: design, synthesis, molecular docking, and anti-hepatocellular carcinoma efficacy evaluation.

A novel series of aminotrimethylpyridinol and aminodimethylpyrimidinol derivatives were designed and synthesised for FGFR4 inhibitors. Structure-activity relationship on the FGFR4 inhibitory activity of the new compounds was clearly elucidated by an intensive molecular docking study. Anti-cancer activity of the compounds was evaluated using hepatocellular carcinoma (HCC) cell lines and a chick chorioallantoic membrane (CAM) tumour model. Compound 6O showed FGFR4 inhibitory activity over FGFR1 - 3. Compared to the positive control BLU9931, compound 6O exhibited at least 8 times higher FGFR4 selectivity. Strong anti-proliferative activity of compound 6O was observed against Hep3B, an HCC cell line which was a much more sensitive cell line to BLU9931. In vivo anti-tumour activity of compound 6O against Hep3B-xenografted CAM tumour model was almost similar to BLU9931. Overall, compound 6O, a novel derivative of aminodimethylpyrimidinol, was a selective FGFR4 kinase inhibitor blocking HCC tumour growth.

Design, synthesis and biological evaluation of a series of dianilinopyrimidines as EGFR inhibitors.

This paper described our efforts to develop dianilinopyrimidines as novel EGFR inhibitors. All the target compounds were tested for inhibitory effects against wild type EGFR (EGFRwt) and three tumour cells, including A549, PC-3, and HepG2. Some of the compounds performed well in antitumor activities. Especially, compound 4c 2-((2-((4-(3-fluorobenzamido)phenyl)amino)-5-(trifluoromethyl) pyrimidin-4-yl)amino)-N-methylthiophene-3-carboxamide showed higher anti-tumour activities than Gefitinib. The IC50 values of compound 4c against A549, PC-3, and HepG2. reached 0.56 µM, 2.46 µM, and 2.21 µM, respectively. In addition, further studies indicated that compound 4c could induce apoptosis against A549 cells and arrest A549 cells in the G2/M phase. Molecular docking studies showed that compound 4c could closely interact with EGFR. Generally, compound 4c was the potential for developing into an anti-tumour drug.