c-Met inhibitors have demonstrated encouraging efficacy in the treatment of non-small cell lung cancer. However, some of these drugs are conditionally approved and still face certain limitations and challenges, primarily...c-Met inhibitors have demonstrated encouraging efficacy in the treatment of non-small cell lung cancer. However, some of these drugs are conditionally approved and still face certain limitations and challenges, primarily manifested as poor blood-brain barrier penetration, off-target toxicity, drug resistance, and low oral bioavailability. These factors restrict their clinical efficacy and widespread application. To discover novel c-Met inhibitors with high potency, minimal toxic side effects, and the ability to penetrate the blood-brain barrier, we designed and synthesized 33 new pyrimidine derivatives using Tepotinib as the lead, employing bioisosterism and conformational restriction strategies. Their anti-tumor activities were evaluated in vitro and in vivo. Among these derivatives, the optimal compound 11g exhibited IC values of 4.01 nM and 3.50 nM against MHCC97H and EBC-1 cells, respectively. In the EBC-1 xenograft mouse model, at a dose of 4 mg/kg, 11g achieved a tumor growth inhibition (TGI) rate of 64.9%, which was significantly higher than that of Tepotinib (33.5%) at the same dose. Pathological evaluation further confirmed that 11g possessed improved safety and reduced toxic side effects. In addition, 11g displayed superior blood-brain barrier permeability and metabolic stability compared with the lead compound. Mechanistic studies demonstrated that 11g effectively inhibits tumor cell proliferation and migration by binding to the c-Met protein and induces cell apoptosis. In summary, as a novel and highly potent c-Met inhibitor, 11g shows promising potential for the treatment of NSCLC, particularly in the prevention and treatment of tumor brain metastasis.
Hybrid molecules, chemical entities with two or more pharmacophore domains, are widely investigated for their antiparasitic potential. First-in-class antimalarial hybrids were designed and tested against Plasmodium falci...Hybrid molecules, chemical entities with two or more pharmacophore domains, are widely investigated for their antiparasitic potential. First-in-class antimalarial hybrids were designed and tested against Plasmodium falciparum. Our multistep synthesis, initiated by a Mn(III)-mediated radical reaction, led to new hybrids containing both the pharmacophores of atovaquone (naphthoquinone) and artemisinin (endoperoxide) separated by a piperazine linker. With 2 or 3 asymmetric carbons, 4 to 8 theoretical configurational isomers were expected. For two of our 5 final products, chiral chromatography separated all isomers. 20 single isomers or stereoisomeric mixtures were obtained and tested against Plasmodium falciparum W2 and K1 chloroquine-resistant strains for their antiplasmodial activity and against HepG2 and CHO cells for their cytotoxicity. Antiplasmodial activity was better than those obtained for reference compound chloroquine for 15 compounds on PfW2 strain and 8 compounds on PfK1 strain. Hit compounds were identified among stereoisomer mixtures (12, ICPfW2 = 0.489 μM, ICPfK1 = 1.061 μM, CC HepG2 = 11.99 μM, CC CHO >125 μM) but also among several stereoisomers. These results suggest that hybrid compounds naphthoquinone/endoperoxide synthesized by adding two pharmacophores can lead to a synergistic effect.
Sainas S, Vigato C, Circosta P
… +19 more, Zonfrillo M, Servidei T, Alberti M, Rescigno M, Miggiano R, Gaidano V, Vitale N, Passoni A, Lanno A, Buccinnà B, Piccinini M, Lorenzati M, Gentile S, Sferrazza G, Sgambato A, Saglio G, Giorgis M, Boschi D, Lolli ML
Targeting cancer metabolism, particularly de novo nucleotide biosynthesis, has emerged as a promising and innovative therapeutic strategy for both hematologic and solid malignancies, including those of the Central Nervou...Targeting cancer metabolism, particularly de novo nucleotide biosynthesis, has emerged as a promising and innovative therapeutic strategy for both hematologic and solid malignancies, including those of the Central Nervous System (CNS). Glioblastoma cancer stem-like cells are especially vulnerable to pyrimidine synthesis inhibition, highlighting human dihydroorotate dehydrogenase (hDHODH), a rate-limiting enzyme in the de novo pathway, as a potential therapeutic target. MEDS433 is a best-in-class hDHODH inhibitor, that shows efficacy in vivo after oral administration but lacks efficient penetration of the blood-brain barrier (BBB), limiting its utility against CNS tumors. Its lipophilic analogue MEDS613 showed enhanced cellular potency, but its poor metabolic stability and rapid conversion to hydroxylated metabolites precluded its future clinical development. In this study, we aimed to design a novel BBB-permeable hDHODH inhibitor, capable of effectively targeting CNS-localized hDHODH. We began by identifying the metabolic soft spots present in the propyloxy side chain of MEDS613 using this information to develop a metabolically stable analogue, MEDS700 (compound 3, as named in the manuscript), that was shown to inhibit hDHODH in the low nanomolar range (IChDHODH 1.5 nM). Subsequently, MEDS700 was fully profiled, including detailed analysis of its crystallographic binding mode, pan-antitumor activities in cell-based assays and in vitro cytotoxicity on Peripheral Blood Mononuclear Cells (PBMC). An in vivo pharmacokinetic experiment demonstrated that MEDS700 was able to cross the blood-brain barrier, maintaining therapeutically relevant intracerebral concentrations for up to 24 h after oral administration. Our findings establish MEDS700 as a potent, safe, metabolically stable hDHODH inhibitor, indicating it as a promising candidate for the treatment of hard-to-reach brain tumors.
Oxidative stress is closely associated with aging and a wide range of major diseases, including cancers, neurodegenerative disorders, and inflammatory conditions. Antioxidant proteins therefore constitute important funct...Oxidative stress is closely associated with aging and a wide range of major diseases, including cancers, neurodegenerative disorders, and inflammatory conditions. Antioxidant proteins therefore constitute important functional biomolecules and potential therapeutic targets in drug discovery. Accurate and efficient identification of antioxidant proteins is thus of relevance for biomedical research and early-stage target exploration. To address the limitations of existing computational approaches, such as restricted dataset size, limited generalization ability, and simplistic feature representations, we constructed a high-quality benchmark dataset comprising 1144 antioxidant proteins and 2959 non-antioxidant proteins. On this basis, we propose SENET-AOP, an attention-based deep learning framework for antioxidant protein classification and target prioritization. The model integrates complementary semantic representations derived from two protein language models, ESM-2 and ProtT5, and adopts a dual-branch CNN-SENet architecture to capture local sequence patterns and global physicochemical properties, while adaptively recalibrating channel-wise feature importance. Experimental results demonstrate that SENET-AOP achieves an accuracy of 0.9360, a Matthews correlation coefficient of 0.8376, and an AUROC of 0.9721 under five-fold cross-validation. On the independent test set, the model attains an accuracy of 0.9367 and an AUROC of 0.9795, consistently outperforming other methods. Moreover, the proposed framework exhibits favorable interpretability. Collectively, SENET-AOP provides an effective and reliable tool for high-throughput identification and prioritization of antioxidant protein targets, supporting oxidative stress-related diseases research and medicinal chemistry-oriented drug discovery workflows. For user convenience, a freely accessible web server has been developed at: http://www.senetaop.com.cn/.
Kułaga D, Bosak N, Ptaszkiewicz M
… +14 more, Chrzan J, Staroń K, Marzec K, Drabczyk AK, Siemieńska I, Kot M, Latacz G, Karnafał J, Velázquez-Martínez CA, Malarz K, Mrozek-Wilczkiewicz A, Boguszewska-Czubara A, Greber K, Ciura K
Nur77, an orphan nuclear receptor, is involved in the development and progression of multiple tumors. In our previous study, we have shown that the protein level of Nur77 is elevated in colon tumors compared to adjacent...Nur77, an orphan nuclear receptor, is involved in the development and progression of multiple tumors. In our previous study, we have shown that the protein level of Nur77 is elevated in colon tumors compared to adjacent normal tissues, highlighting its potential as a promising target for colorectal cancer therapy. Significantly, we have identified BI1071 as a Nur77-targeting compound that induces apoptosis in colorectal cancer cells. Based on the scaffold of BI1071, by substituting the indole group of BI1071 with a pyrrolyl group on one side, we rationally designed and synthesized a series of novel BI1071 analogues named SIM-C-PhCFCl targeting Nur77, and the structure-activity relationship of these BI1071 derivatives was summarized. From this series of compounds, A6 exhibited the strongest binding affinity to Nur77 (K = 0.40 ± 0.05 μM) and the most potent anti-proliferative activity against HCT116 and MC38 colorectal tumor cell lines, with IC values of 0.53 ± 0.06 μM and 0.16 ± 0.007 μM, respectively. Interestingly, unlike BI1071, which triggers Nur77-dependent apoptosis, compound A6 suppressed colon cancer cell proliferation predominantly by inducing Nur77-dependent mitotic arrest. Collectively, our findings provide a foundation for further investigation and development of Nur77-targeting antimitotic molecules toward colorectal cancer therapy.
Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO) are rate-limiting enzymes of the kynurenine pathway and are closely associated with tumor immune evasion and neuropsychiatric disorders. The failu...Indoleamine 2,3-dioxygenase 1 (IDO1) and tryptophan 2,3-dioxygenase (TDO) are rate-limiting enzymes of the kynurenine pathway and are closely associated with tumor immune evasion and neuropsychiatric disorders. The failure of single-target IDO1 inhibition, exemplified by the phase III setback of epacadostat, has highlighted the limitations imposed by compensatory TDO activity and the heterogeneity of the tumor microenvironment. This review focuses on three generations of IDO1 inhibitors, selective TDO inhibitors, and emerging dual-target strategies, and provides a comprehensive overview of IDO1/TDO dual-target inhibitors. We summarize the research progress of β-carboline, indazole, indole, isoquinoline/alkaloid-derived, and other natural product-related scaffolds, with particular emphasis on their structure-activity relationships, binding modes, translational pharmacology, and preclinical efficacy in tumor and central nervous system models. Clinical-stage candidates have further demonstrated the translational potential of simultaneous IDO1/TDO blockade. Overall, this review offers new insights and strategic perspectives for therapeutic paradigms spanning cancer immunotherapy and neuropsychiatric intervention through modulation of the integrated immunity-metabolism-neurology axis.
Hexokinase 2 (HK2) promotes M1 macrophage polarization through aerobic glycolysis, so as to increase the release of inflammatory factors and to maintain the pro-inflammatory phenotype. HK2 inhibitors may regulate the phe...Hexokinase 2 (HK2) promotes M1 macrophage polarization through aerobic glycolysis, so as to increase the release of inflammatory factors and to maintain the pro-inflammatory phenotype. HK2 inhibitors may regulate the phenotype of immune cells and exert anti-inflammatory effects by modulating glycolysis. Building upon our prior discovery that indole-chalcone derivatives specifically bind to HK2 and inhibit its enzymatic activity, a focused library of novel indole-pyridine chalcone derivatives was rationally designed and facilely synthesized via Claisen-Schmidt condensation of corresponding indole-3-carboxaldehydes with 3-acetylpyridine. Systematic screening of HK2 enzyme inhibitory activity identified compound ZSW as a potent lead candidate, exhibiting significant HK2 inhibition with an IC value of 0.48 ± 0.13 μM. Both molecular docking and biotin probe pull-down experiments confirmed ZSW-HK2 binding. Further experiments indicated that ZSW alleviated inflammatory bowel disease primarily via two possible mechanisms: one is modulating macrophage phenotypes through glycolysis inhibition, and the other is inhibiting NF-κB nuclear translocation by preventing IκBα phosphorylation and degradation. Animal experiments demonstrated that ZSW improved the intestinal inflammatory state of IBD mice. This study not only lays the research foundation for HK2 as a target for the development of anti-IBD drugs, but also provides theoretical support for expanding the application of HK2 inhibitors in the development of anti-inflammatory drugs.
Pathogens can cause serious infections in humans. Over time, pathogens have developed resistance to antimicrobial treatments, which enables them to survive, continue multiplying, and thrive in the presence of antibiotics...Pathogens can cause serious infections in humans. Over time, pathogens have developed resistance to antimicrobial treatments, which enables them to survive, continue multiplying, and thrive in the presence of antibiotics. This rise in antimicrobial-resistant pathogens has led to significant loss of life and has detrimental effects on social and economic conditions. As a result, there is an urgent need to develop new antibiotics that act through diverse mechanisms to effectively tackle pathogens. Natural products exhibit a remarkable variety of scaffolds, characterized by complex structures and diverse biological activities. The contribution of natural products in the advancement of antibiotic discovery is significant. Among natural products, secondary metabolites such as polyenes are lipophilic or amphipathic compounds that easily permeate the membrane of pathogens and inhibit their growth. Notably, polyene-based compounds such as amphotericin, nystatin, and natamycin have been approved for the treatment of fungal diseases and have shown the least likelihood of developing resistance, which creates a ray of hope in combating antibiotic-resistant pathogens. This review summarizes recently identified polyene-based natural leads for the treatment of fungal, bacterial, protozoal, and viral infections. The discussed natural polyenes have immense potential for further development in clinical studies to treat human infections.
The α6β4 nAChR represents promising therapeutic target for neuropathic pain and substance use disorders, yet potent and selective antagonists remain limited. We systematically investigated the structure-activity relation...The α6β4 nAChR represents promising therapeutic target for neuropathic pain and substance use disorders, yet potent and selective antagonists remain limited. We systematically investigated the structure-activity relationship of α4/4-conotoxin LvIC0 at the human α6β4 nAChR using structure-guided design and electrophysiology. Four analogues-LvIC2 (A4S), LvIC10 (G10V), LvIC12 (G10Q), and LvIC13 (K11R)-exhibited substantially enhanced potency (IC = 2.4-4.5 nM) and improved selectivity. Combinatorial substitutions revealed non-additive effects, while loop elongation generated LvIC17 which retained most of the activity. Penicillamine substitution produced sequence-dependent stability outcomes. Receptor mutagenesis validated predicted interactions. In vivo, LvIC13 induced less motor impairment despite greater potency, suggesting target inhibition can be dissociated from side effects. These findings establish a framework for designing selective α6β4 antagonists and provide promising leads for treating neuropathic pain and addiction.
Although coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is no longer a major global pandemic, the disease is still present and may progress into severe respiratory s...Although coronavirus disease (COVID-19) caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is no longer a major global pandemic, the disease is still present and may progress into severe respiratory syndrome for many elder or immunocompromised individuals. The treatment options are very limited, and therefore improved therapies for COVID-19 are still needed. Herein, we described a novel class of covalent SARS-CoV-2 3C-like protease (3CL) inhibitors bearing a spirooxindole moiety. By minimizing the peptidomimetic properties of the original structure, we obtained a series of compounds exhibiting excellent potency and high metabolic stability in liver microsomes. This work culminated in the discovery of preclinical candidate CG-0988, which demonstrates excellent antiviral activity (EC50 = 4.0 nM against Omicron EG.5.1). Pharmacokinetic (PK) study of CG-0988 in rats and dogs exhibited favorable PK profiles, with oral availability of 59.2% (in rats) and 94.0% (in dogs), respectively. These findings indicate that CG-0988 is a promising 3CL inhibitor for oral administration, potentially without the need for a PK boosting agent.
The escalating crisis of antibiotic resistance poses a severe threat to global public health systems and food safety, which have substantially compromised the therapeutic efficacy of existing antibacterial agents. In thi...The escalating crisis of antibiotic resistance poses a severe threat to global public health systems and food safety, which have substantially compromised the therapeutic efficacy of existing antibacterial agents. In this study, a rational molecular hybridization strategy was employed to design and identify a novel amphiphilic small-molecule antibacterial candidate, H14d. In vitro studies demonstrated that H14d exhibits potent broad-spectrum antibacterial activity (MIC = 0.125-4 μg/mL), with MIC values of 0.25 μg/mL against Methicillin-resistant Staphylococcus aureus ATCC43300 (MRSA) and 2 μg/mL against Acinetobacter baumannii ATCC19606. In addition, H14d displayed rapid bactericidal kinetics, a low propensity for resistance development, favorable biosafety profiles, and an exceptional ability to significantly reduce mature bacterial biofilms. Mechanistic investigations revealed that H14d specifically binds to phosphatidylglycerol (PG), a key phospholipid component of bacterial cell membranes, while concurrently inhibiting the essential cell division protein FtsZ, thereby exerting antibacterial activity through a dual-target mode of action. Pharmacokinetic studies showed that H14d displayed stable elimination kinetics (t = 10.6-10.9 h) and a large apparent volume of distribution in mice. In vivo efficacy evaluations further demonstrated that H14d outperformed the frontline clinical antibiotic linezolid in a MRSA-infected Galleria mellonella model and multiple murine infection models. Collectively, this study provides valuable insights for the treatment of drug-resistant infections and offers a rational framework for the design and development of next-generation new antibacterial agents.
Targeting cancer stem cells (CSCs) has emerged as a promising strategy for cancer therapy and prevention. The human cytochrome P450 enzyme CYP4Z1 has been identified as a potential therapeutic target due to its role in p...Targeting cancer stem cells (CSCs) has emerged as a promising strategy for cancer therapy and prevention. The human cytochrome P450 enzyme CYP4Z1 has been identified as a potential therapeutic target due to its role in promoting breast cancer stemness. Aiming to develop potent and selective CYP4Z1 inhibitors, our strategy involved systematic structure-activity relationship (SAR) studies of the lead compound XD-2 (1-benzyl-1H-imidazo [4,5-c] pyridine), which led to its structural optimization. A series of derivatives were designed and synthesized to enhance drug-like properties, inhibitory activity, and selectivity. Among all the synthesized compounds, the preferred analog C8, which features an imidazo[4,5-c]pyridine core connected to a terminal butyl group via an amide-containing linker, exhibited the most potent CYP4Z1 inhibitory activity, with an IC value of 55.3 nM against CYP4Z1. Molecular docking studies revealed that the introduced side chain extended into the hydrophobic subpocket and the phenyl group established additional aromatic stacking interactions with Trp120. Subsequent in vitro and in vivo biological assessments confirmed that compound C8 potently diminished stemness marker expression, impeded spheroid formation, and attenuated both metastatic potential and tumor-initiating capacity in breast cancer cells. Collectively, these results underscore the promise of C8 as a leading candidate for advancing clinically viable CYP4Z1-targeted therapies in breast cancer.
The benzo[b]thiophene scaffold has emerged as a privileged chemotype in target-directed anticancer drug discovery because of its structural versatility and distinctive sulfur-centered interactions that support selective...The benzo[b]thiophene scaffold has emerged as a privileged chemotype in target-directed anticancer drug discovery because of its structural versatility and distinctive sulfur-centered interactions that support selective target binding. This review provides a focused, mechanism-based analysis of benzothiophene derivatives developed as anti-cancer agents, emphasizing compounds with experimentally validated molecular targets and confirmed cellular activity rather than nonspecific cytotoxins. Benzothiophenes have been designed to modulate diverse oncogenic targets, including receptor and non-receptor kinases, STAT3, estrogen receptor pathways, epigenetic regulators, metabolic enzymes, tubulin, and DNA-associated systems. Many derivatives show nanomolar potency, pathway-selective effects, in vivo anti-tumor activity, improved selectivity, and enhanced metabolic stability. Raloxifene remains the only benzothiophene-based drug approved for an oncological indication, highlighting both the scaffold's clinical promise and the translational barriers that have limited its broader therapeutic advancement. This review also examines key structure-activity relationships, binding determinants, and optimization strategies guiding benzothiophene-based anti-cancer therapeutics.
Antimicrobial resistance demands therapeutics that combine potent activity with low resistance potential. Ultrashort antimicrobial peptides (AMPs) are attractive due to their manufacturability and tunability, but activit...Antimicrobial resistance demands therapeutics that combine potent activity with low resistance potential. Ultrashort antimicrobial peptides (AMPs) are attractive due to their manufacturability and tunability, but activity is often lost upon sequence minimization because of conformational instability. Here, we establish a conformational engineering strategy based on backbone amide cyclization to restore ultrashort AMP function. Using a snake venom-derived cathelicidin template ZY4, we identified a minimal Trp/Lys/Arg-rich motif and generated the ultrashort cyclic peptide WKR-cyl, which exhibits enhanced antibacterial potency, membrane selectivity, and proteolytic stability compared with linear and disulfide-cyclized analogs. Mechanistically, amide cyclization stabilizes membrane-active conformations and increases hydrophobic driving forces, reducing the energetic barrier for membrane insertion. WKR-cyl preferentially targets peptidoglycan-rich bacterial surfaces, enabling rapid membrane permeabilization and bactericidal activity. WKR-cyl shows potent activity against MRSA, strong anti-biofilm activity, high plasma stability, and minimal resistance induction, and demonstrates therapeutic efficacy in murine MRSA pneumonia and skin infection models, supporting development of clinically translatable ultrashort anti-infective peptides.
The repeated outbreaks of coronavirus show how hard it is to keep antiviral effectiveness when the virus mutates. Coronaviruses depend on tightly regulated proteolytic processing mediated by the 3-chymotrypsin-like prote...The repeated outbreaks of coronavirus show how hard it is to keep antiviral effectiveness when the virus mutates. Coronaviruses depend on tightly regulated proteolytic processing mediated by the 3-chymotrypsin-like protease (3CL), making it a key antiviral target. Most current inhibitors engage the catalytic site, an approach that remains vulnerable to resistance driven by active-site mutations. Here, we investigate an alternative strategy based on allosteric regulation of 3CL by targeting a pocket surrounding residue Asn28, previously shown to influence enzymatic activity and dimer stability. Structure-based virtual screening identified novobiocin as a candidate ligand for this region, which lies adjacent to but distinct from the catalytic center. Biophysical experiments showed direct binding of novobiocin to 3CL in solution, with sub-micromolar affinity (K ∼ 3 × 10 M). Protease thermal stability and dimeric assembly were lowered by ligand binding. Enzymatic assays revealed a pronounced reduction in catalytic turnover with minimal effects on substrate binding, consistent with an allosteric mechanism of inhibition, and yielded IC values of ∼0.5 μM across independent assays. Molecular docking and simulation analyses supported stable binding at the Asn28-associated pocket and revealed localized changes in conformational dynamics. These findings show that novobiocin allosterically inhibits 3CL and identify the Asn28-associated pocket as a relevant target for developing inhibitors with improved resistance to viral evolution.
Hepatocyte nuclear factor 4 alpha (HNF4α) is a key nuclear receptor involved in liver function and metabolic regulation, making it a potential therapeutic target for lipid-metabolism disorders. Combining time-resolved fl...Hepatocyte nuclear factor 4 alpha (HNF4α) is a key nuclear receptor involved in liver function and metabolic regulation, making it a potential therapeutic target for lipid-metabolism disorders. Combining time-resolved fluorescence resonance energy transfer (TR-FRET) high-throughput screening with a diffusion-based structure-guided generative design workflow (DiffSBDD), we identified and prioritized a series of compounds with HNF4α agonist-like activity. Among them, (Z)-19 showed good HNF4α activation with an EC of 9.2 μM, outperforming the reference agonist N-trans-caffeoyltyramine (NCT; EC = 129.6 μM). Surface plasmon resonance (SPR) supported a direct interaction with HNF4α (K = 4.6 μM). In HepG2 cells, (Z)-19 reduced triglyceride and total cholesterol accumulation with minimal cytotoxicity and exhibited ferroptosis-protective activity, with a response profile differing from that of NCT. In a hyperlipidemia mouse model, (Z)-19 lowered plasma triglyceride and total cholesterol levels, indicating encouraging in vivo efficacy. Additionally, all-atom molecular dynamics (MD) simulations supported a possible binding mode characterized by persistent hydrogen-bonding and hydrophobic contacts within the ligand-binding domain, and yielded favorable binding-free-energy estimates consistent with the experimental potency ranking. Together, these results identify (Z)-19 as a promising starting point for further optimization and mechanistic investigation of compounds that modulate HNF4α.
Selective COX-2 inhibitors are associated with cardiovascular safety concerns, motivating continued exploration of alternative chemotypes. In this study, we investigated the COX-2 inhibitor activity of a library of alkyl...Selective COX-2 inhibitors are associated with cardiovascular safety concerns, motivating continued exploration of alternative chemotypes. In this study, we investigated the COX-2 inhibitor activity of a library of alkylaminophenols. This was achieved through single-concentration screens used to prioritize compounds with COX-2 inhibition and acceptable cellular tolerability in THP-1 cells, generation of analogs via positional analog scanning (PAS) and molecular docking studies to guide scaffold optimization. Novel compounds were then synthesized by a green Petasis borono-Mannich protocol. Preliminary structure activity relationships (SAR) and molecular descriptor analysis of our one-shot assays allowed us to observe that indoline and para substituted phenols derivatives showed the most promising inhibitory activity. Compound 35e demonstrated a 23-fold increase in COX-2 potency over its parent compound 35, while simultaneously increasing COX-1/COX-2 selectivity by a factor of 25. In LPS-stimulated THP-1 macrophages, 35e reduced IL-6 and TNF-α release in a concentration-dependent manner, with estimated IC values of approximately 35 μM and 42 μM, respectively. These concentrations remained below the cytotoxic IC in THP-1 cells (67.9 μM), indicating cytokine modulation under non-cytotoxic conditions. Although cellular activity required higher concentrations than direct enzymatic inhibition (COX-2 IC = 8.43 μM), these results support a measurable functional anti-inflammatory phenotype. This work establishes alkylaminophenols as an early-stage COX-2 inhibitory chemotype and identifies 35e as a starting point for further optimization.