Current literature highlights - June 2002

Selective Cdk4 Inhibitors: Cyclins and cyclin-dependent kinases (Cdks) play important roles in regulation of the cell cycle. In particular, D-type cyclins, which have been shown to be amplified or over-expressed in several tumour cells, associate with Cdk4/6 to activate their phosphorylation activity. Cyclin D-Cdk4/6 complexes phosphorylate the retinoblastoma protein (pRB) and regulate the cell cycle during G₁/S transition. Loss of function or deletion of p16^ink4a (endogenous Cdk4/6 specific inhibitor protein) frequently occurs in clinical cancer cells. Thus, selective Cdk4/6 inhibitors should be useful as a new class of cytostatic antitumour agents. In order to overcome the problem of selectivity for Cdk4/6 over the hundreds of homologous kinases in the superfamily, specific amino acid residues were identified around the ATP binding pocket of Cdk4 by comparing the amino acid sequences of 390 representative kinases. Subsequently, a chemical library was designed using this information about the locations of these amino acid residues (A novel approach for the development of selective Cdk4 inhibitors: Library design based on locations of Cdk4 specific amino acid residues, Honma, T. et. al., J. Med. Chem., 44, (2001), 4628-4640).     

Identification of novel Plasmodium falciparum PI4KB inhibitors as potential anti-malarial drugs: Homology modeling,molecular docking and molecular dynamics simulations

The current study was set to discover selective Plasmodium falciparum phosphatidylinositol-4-OH kinase type III beta (pfPI4KB) inhibitors as potential antimalarial agents using combined structure-based and ligand-based drug discovery approach. A comparative model of pfPI4KB was first constructed and validated using molecular docking techniques. Performance of Autodock4.2 and Vina4 software in predicting the inhibitor-PI4KB binding mode and energy was assessed based on two Test Sets: Test Set I contained five ligands with resolved crystal structures with PI4KB, while Test Set II considered eleven compounds with known IC50 value towards PI4KB. The outperformance of Autodock as compared to Vina was reported, giving a correlation coefficient (R²) value of 0.87 and 0.90 for Test Set I and Test Set II, respectively. Pharmacophore-based screening was then conducted to identify drug-like molecules from ZINC database with physicochemical similarity to two potent pfPI4KB inhibitors –namely cpa and cpb. For each query inhibitor, the best 1000 hits in terms of TanimotoCombo scores were selected and subjected to molecular docking and molecular dynamics (MD) calculations. Binding energy was then estimated using molecular mechanics–generalized Born surface area (MM-GBSA) approach over 50 ns MD simulations of the inhibitor-pfPI4KB complexes. According to the calculated MM-GBSA binding energies, ZINC78988474 and ZINC20564116 were identified as potent pfPI4KB inhibitors with binding energies better than those of cpa and cpb, with ΔG_binding ≥ −34.56 kcal/mol. The inhibitor-pfPI4KB interaction and stability were examined over 50 ns MD simulation; as well the selectivity of the identified inhibitors towards pfPI4KB over PI4KB was reported.     

JNK inhibitor SP600125 promotes the formation of polymerized tubulin, leading to G2/M phase arrest, endoreduplication, and delayed apoptosis

The JNK inhibitor SP600125 strongly inhibits cell proliferation in many human cancer cells by blocking cell-cycle progression and inducing apoptosis. Despite extensive study, the mechanism by which SP600125 inhibits mitosis-related effects in human leukemia cells remains unclear. We investigated the effects of SP600125 on the inhibition of cell proliferation and the cell cycle, and on microtubule dynamics in vivo and in vitro. Treatment of synchronized leukemia cells with varying concentrations of SP600125 results in significant G₂/M cell cycle arrest with elevated p21 levels, phosphorylation of histone H3 within 24 h, and endoreduplication with elevated Cdk2 protein levels after 48 h. SP600125 also induces significant abnormal microtubule dynamics in vivo. High concentrations of SP600125 (200 µM) were required to disorganize microtubule polymerization in vitro. Additionally, SP600125-induced delayed apoptosis and cell death was accompanied by significant poly ADP-ribose polymerase (PARP) cleavage and caspase-3 activity in the late phase (at 72 h). Endoreduplication showed a greater increase in ectopic Bcl-2-expressing U937 cells at 72 h than in wild-type U937 cells without delayed apoptosis. These results indicate that Bcl-2 suppresses apoptosis and SP600125-induced G₂/M arrest and endoreduplication. Therefore, we suggest that SP600125 induces mitotic arrest by inducing abnormal spindle microtubule dynamics.     

High-throughput virtual screening and preclinical analysis identifies CB-1, a novel potent dual B-Raf/c-Raf inhibitor,effective against wild and mutant variants of B-Raf expression in colorectal carcinoma

Paradoxical Raf activation via Raf dimerization is a major drawback of wild/mutant B-Raf inhibitors. Herein, we report that CB-1 a novel, potent B-Raf/c-Raf dual inhibitor, effective against colon cancer cells, irrespective of their genetic status. High-throughput virtual screening of the ChemBridge library against wild B-Raf (B-Raf^WT), mutant B-Raf (B-Raf^V600E), and c-Raf was performed using an automated protocol with the AutoDock-VINA. Caco-2 and HT-29 cells were used. Of the 23,365 compounds screened computationally, CB-1 showed the highest binding energy towards B-Raf^WT with a ΔG_binding score of ? 13.0 kcal/mol. The compound was also predicted to be effective against B-Raf^V600E and c-Raf molecules with ΔG_binding energies of ? 10.6 and ? 10.1 kcal/mol, respectively. The compound inhibited B-Raf^WT, B-Raf^V600E and c-Raf kinases with IC₅₀ values of 27.13, 51.70, and 40.23 nM, respectively. The GI₅₀ value of CB-1 was 247.9 nM in B-Raf^WT-expressing Caco-2 cells and 352.4 nM in B-RafV600E-expressing HT-29 cells. Dose-dependent increases in total apoptosis and G₁ cell cycle phase arrest was observed in CB-1-treated colon cancer cells. The compound decreased B-Raf expression in both wild and mutant colon cancer cells. CB-1, a novel, potent dual B-Raf/c-Raf inhibitor was effective against colon cancer cells bearing wild-type and mutant variants of B-Raf expression.

     

Imidazopyridazine Acetylcholinesterase Inhibitors Display Potent Anti-Proliferative Effects in the Human Neuroblastoma Cell-Line,IMR-32

Imidazo[1,2-b]pyridazine compounds are a new class of promising lead molecules to which we have incorporated polar nitro and amino moieties to increase the scope of their biological activity. Two of these substituted 3-nitro-6-amino-imidazo[1,2-b]pyridazine compounds (5c and 5h) showed potent acetylcholinesterase (AChE) inhibitory activity (IC₅₀ 40–50 nM), which we have previously reported. In this study, we wanted to test the biological efficacy of these compounds. Cytotoxicity assays showed that compound 5h mediated greater cell death with over 43% of cells dead at 100 μM and activation of caspase 3-mediated apoptosis. On the other hand, compound 5c mediated a dose-dependent decrease in cell proliferation. Both compounds showed cell cycle arrest in the G₀/G₁ phase and reduced cellular ATP levels leading to activation of adenosine monophosphate-activated protein kinase (AMPK) and enhanced mitochondrial oxidative stress. It has to be noted that all these effects were observed at doses beyond 10 μM, 200-fold above the IC₅₀ for AChE inhibition. Both compounds also inhibited bacterial lipopolysaccharide-mediated cyclooxygenase-2 and nitric oxide release in primary rat microglial cells. These results suggested that the substituted imidazo (1,2-b) pyridazine compounds, which have potent AChE inhibitory activity, were also capable of antiproliferative, anti-migratory, and anti-inflammatory effects at higher doses.     

Identification of novel HDAC8 selective inhibitors through ligand and structure based studies: Exploiting the acetate release channel differences among class I isoforms

Histone deacetylases (HDACs) are key regulators of gene expression and have emerged as crucial therapeutic targets for cancer. Among the HDACs, inhibition of HDAC8 enzyme has been reported to be a novel strategy in the treatment of female-specific cancers. Most of the HDAC inhibitors discovered so far inhibit multiple HDAC isoforms causing toxicities in the clinic thus limiting their potential. Therefore, the discovery of isoform-selective HDAC8 inhibitors is highly desirable. In the present study, a combination of ligand and structure based drug design tools were utilized to build a statistically significant pharmacophore based 3D QSAR model with statistical parameters R²: 0.9964, and Q²: 0.7154, from a series of 31 known HDAC8 inhibitors. Top 1000 hits obtained from Virtual screening of Phase database were subjected to docking studies against HDAC8. Top 100 hits obtained were redocked into HDAC Class I (HDAC 1,2,3) and Class II isoforms (HDAC 4, 6) and rescored with XP Glide Score. Based on fitness score, XP glide score and interacting amino acid residues, five HDAC8 inhibitors (1–5) were selected for in vitro studies. The HDAC8 activity assay followed by enzyme kinetics clearly indicated Compounds 1, 2 and 3 to be potent HDAC8 selective inhibitors with IC₅₀ of 126 pM, 112 nM, and 442 nM respectively. These compounds were cytotoxic to HeLa cells where HDAC8 is overexpressed but not to normal cells, HEK293. Also, they were able to induce apoptosis by modulating Bax/Bcl2, cleavage of PARP and release of Cytochrome C. Molecular Dynamics simulations observed most favorable interaction patterns and presented a rationale for the activities of the identified compounds. Selectivity against HDAC8 was due to exploitation of the architectural difference in the acetate release channel among class I HDAC isoforms.     

Dibenzofuran Derivatives Inspired from Cercosporamide as Dual Inhibitors of Pim and CLK1 Kinases

Pim kinases (proviral integration site for Moloney murine leukemia virus kinases) are overexpressed in various types of hematological malignancies and solid carcinomas, and promote cell proliferation and survival. Thus, Pim kinases are validated as targets for antitumor therapy. In this context, our combined efforts in natural product-inspired library generation and screening furnished very promising dibenzo[b,d]furan derivatives derived from cercosporamide. Among them, lead compound 44 was highlighted as a potent Pim-1/2 kinases inhibitor with an additional nanomolar IC₅₀ value against CLK1 (cdc2-like kinases 1) and displayed a low micromolar anticancer potency towards the MV4-11 (AML) cell line, expressing high endogenous levels of Pim-1/2 kinases. The design, synthesis, structure–activity relationship, and docking studies are reported herein and supported by enzyme, cellular assays, and Galleria mellonella larvae testing for acute toxicity.     

Prediction of new chromene-based inhibitors of tubulin using structure-based virtual screening and molecular dynamics simulation methods

Multidrug resistance (MDR) is one of the serious problems in cancer research that causes failure in chemotherapy. Chromene-based compounds have been proven to be the novel anti-MDR agents for inhibiting proliferation of tumor cells through tubulin polymerization inhibition of by binding at the colchicine binding site. In this study, we screened a chromene-based database of small molecules using physicochemical, ADMET properties and molecular docking to identify potential hit compounds. In order to validate our hit compounds, molecular dynamics simulations and related analysis were carried out and the results suggest that our hit compounds (PubChem CIDs: 16814409, 17594471, 57367244 and 69899719) can prove to be potential inhibitors of tubulin. The in silico results show that the present hits, like colchicine, effectively suppressed the dynamic instability of microtubules and induced microtubule-depolymerization and cell cycle arrest.     

Discovery of novel inhibitors for <Emphasis Type="Italic">Leishmania</Emphasis> nucleoside diphosphatase kinase (NDK) based on its structural and functional characterization

Nucleoside diphosphate kinases (NDKs) are ubiquitous enzymes that catalyze the transfer of the γ-phosphate moiety from an NTP donor to an NDP acceptor, crucial for maintaining the cellular level of nucleoside triphosphates (NTPs). The inability of trypanosomatids to synthesize purines de novo and their dependence on the salvage pathway makes NDK an attractive target to develop drugs for the diseases they cause. Here we report the discovery of novel inhibitors for Leishmania NDK based on the structural and functional characterization of purified recombinant NDK from Leishmania amazonensis. Recombinant LaNDK possesses auto-phosphorylation, phosphotransferase and kinase activities with Histidine 117 playing an essential role. LaNDK crystals were grown by hanging drop vapour diffusion method in a solution containing 18% PEG-MME 500, 100 mM Bis-Tris propane pH 6.0 and 50 mM MgCl₂. It belongs to the hexagonal space group P6₃22 with unit cell parameters a?=?b?=?115.18, c?=?62.18 Å and α?=?β?=?90°, γ?=?120°. The structure solved by molecular replacement methods was refined to crystallographic R-factor and R_free values of 22.54 and 26.52%, respectively. Molecular docking and dynamics simulation-based virtual screening identified putative binding compounds. Protein inhibition studies of selected hits identified five inhibitors effective at micromolar concentrations. One of the compounds showed ~45% inhibition of Leishmania promastigotes proliferation. Analysis of inhibitor-NDK complexes reveals the mode of their binding, facilitating design of new compounds for optimization of activities as drugs against leishmaniasis.     

Synthesis of 4‐Heteroaryl–Quinazoline Derivatives as Potential Anti‐breast Cancer Agents

4‐Heteroaryl or heteroalkyl–quinazoline derivatives were prepared as dual epidermal growth factor receptor (EGFR) and vascular endothelial growth factor receptor‐2 (VEGFR‐2) inhibitors. The new compounds were tested for their dual enzyme inhibition as well as their cytotoxic activity on MCF7 cell line. The results indicated that almost all the compounds showed moderate dual inhibition of both enzymes. Compound 3 (methyl piperidine‐4‐carboxylate derivative) showed the highest inhibitory activity against both enzymes with IC₅₀ 97.6 and 64.0 µM against EGFR and VEGFR‐2 kinases, respectively. Most of the test compounds showed potent to moderate antitumor activity on MCF7 cell line. Five compounds ( 3 , 9c , 11 , 13 , and 15b ) showed potent cytotoxic activity with IC₅₀ values between 10 and 17 µM .     

Discovery of a New Drug-like Series of OGT Inhibitors by Virtual Screening

O-GlcNAcylation is an essential post-translational modification installed by the enzyme O-β-N-acetyl-d-glucosaminyl transferase (OGT). Modulating this enzyme would be extremely valuable to better understand its role in the development of serious human pathologies, such as diabetes and cancer. However, the limited availability of potent and selective inhibitors hinders the validation of this potential therapeutic target. To explore new chemotypes that target the active site of OGT, we performed virtual screening of a large library of commercially available compounds with drug-like properties. We purchased samples of the most promising virtual hits and used enzyme assays to identify authentic leads. Structure-activity relationships of the best identified OGT inhibitor were explored by generating a small library of derivatives. Our best hit displays a novel uridine mimetic scaffold and inhibited the recombinant enzyme with an IC₅₀ value of 7 µM. The current hit represents an excellent starting point for designing and developing a new set of OGT inhibitors that may prove useful for exploring the biology of OGT.     

Revealing the Potential Application of EC-Synthetic Retinoid Analogues in Anticancer Therapy

(1) Background and Aim: All-trans retinoic acid (ATRA) induces differentiation and inhibits growth of many cancer cells. However, resistance develops rapidly prompting the urgent need for new synthetic and potent derivatives. EC19 and EC23 are two synthetic retinoids with potent stem cell neuro-differentiation activity. Here, these compounds were screened for their in vitro antiproliferative and cytotoxic activity using an array of different cancer cell lines. (2) Methods: MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, AV/PI (annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI)), cell cycle analysis, immunocytochemistry, gene expression analysis, Western blotting, measurement of glutamate and total antioxidant concentrations were recruited. (3) Results: HepG2, Caco-2, and MCF-7 were the most sensitive cell lines; HepG2 (ATRA; 36.2, EC19; 42.2 and EC23; 0.74 µM), Caco-2 (ATRA; 58.0, EC19; 10.8 and EC23; 14.7 µM) and MCF-7 (ATRA; 99.0, EC19; 9.4 and EC23; 5.56 µM). Caco-2 cells were selected for further biochemical investigations. Isobologram analysis revealed the combined synergistic effects with 5-fluorouracil with substantial reduction in IC₅₀. All retinoids induced apoptosis but EC19 had higher potency, with significant cell cycle arrest at subG₀-G₁, -S and G₂/M phases, than ATRA and EC23. Moreover, EC19 reduced cellular metastasis in a transwell invasion assay due to overexpression of E-cadherin, retinoic acid-induced 2 (RAI2) and Werner (WRN) genes. (4) Conclusion: The present study suggests that EC-synthetic retinoids, particularly EC19, can be effective, alone or in combinations, for potential anticancer activity to colorectal cancer. Further in vivo studies are recommended to pave the way for clinical applications.     

Discovery of a Potent GLUT Inhibitor from a Library of Rapafucins by Using 3D Microarrays

Glucose transporters play an essential role in cancer cell proliferation and survival and have been pursued as promising cancer drug targets. Using microarrays of a library of new macrocycles known as rapafucins, which were inspired by the natural product rapamycin, we screened for new inhibitors of GLUT1. We identified multiple hits from the rapafucin 3D microarray and confirmed one hit as a bona fide GLUT1 ligand, which we named rapaglutin A (RgA). We demonstrate that RgA is a potent inhibitor of GLUT1 as well as GLUT3 and GLUT4, with an IC₅₀ value of low nanomolar for GLUT1. RgA was found to inhibit glucose uptake, leading to a decrease in cellular ATP synthesis, activation of AMP‐dependent kinase, inhibition of mTOR signaling, and induction of cell‐cycle arrest and apoptosis in cancer cells. Moreover, RgA was capable of inhibiting tumor xenografts in vivo without obvious side effects. RgA could thus be a new chemical tool to study GLUT function and a promising lead for developing anticancer drugs.     

Furanone derivatives as new inhibitors of CDC7 kinase: development of structure activity relationship model using 3D QSAR,molecular docking,and in silico ADMET

Cell division cycle 7 (CDC7) is a serine/threonine kinase, which plays a vital role in the replication initiation of DNA synthesis. Overexpression of the CDC7 in various tumor growths and in cell proliferation makes it a promising target for treatment of cancers. To investigate the binding between the CDC7 and furanone inhibitors, and in order to design highly potent inhibitors, a three-dimensional quantitative structure activity relationship (3D-QSAR) with molecular docking was performed. The optimum CoMSIA model showed significant statistical quality on all validation methods with a determination coefficient (R²?=?0.945), bootstrapping R² mean (BS-R²?=?0.960), and leave-one-out cross-validation (Q²) coefficient of 0.545. The predictability of this model was evaluated by external validation using a test set of nine compounds with a predicted determination coefficient R²_test of 0.96, besides the mean absolute error (MAE) of the test set was 0.258 log units. The extracted contour maps were used to identify the important regions, where the modification was necessary to design a new molecule with improved activity. Furthermore, a good consistency between the molecular docking and contour maps strongly demonstrates that the molecular modeling is reliable. Based on those obtained results, we designed several new potent CDC7 inhibitors, and their inhibitory activities were validated by the molecular models. Additionally, those newly designed inhibitors showed promising results in the preliminary in silico ADMET evaluations.     

Fragment Linking and Optimization of Inhibitors of the Aspartic Protease Endothiapepsin: Fragment‐Based Drug Design Facilitated by Dynamic Combinatorial Chemistry

Fragment‐based drug design (FBDD) affords active compounds for biological targets. While there are numerous reports on FBDD by fragment growing/optimization, fragment linking has rarely been reported. Dynamic combinatorial chemistry (DCC) has become a powerful hit‐identification strategy for biological targets. We report the synergistic combination of fragment linking and DCC to identify inhibitors of the aspartic protease endothiapepsin. Based on X‐ray crystal structures of endothiapepsin in complex with fragments, we designed a library of bis‐acylhydrazones and used DCC to identify potent inhibitors. The most potent inhibitor exhibits an IC₅₀ value of 54 nm , which represents a 240‐fold improvement in potency compared to the parent hits. Subsequent X‐ray crystallography validated the predicted binding mode, thus demonstrating the efficiency of the combination of fragment linking and DCC as a hit‐identification strategy. This approach could be applied to a range of biological targets, and holds the potential to facilitate hit‐to‐lead optimization.     

Integrating pharmacophore mapping,virtual screening,density functional theory,molecular simulation towards the discovery of novel apolipoprotein (apoE ε4) inhibitors

AimAn integrated protocol of virtual screening involving molecular docking, pharmacophore probing, and simulations was established to identify small novel molecules targeting crucial residues involved in the variant apoE ε4 to mimic its behavior as apoE2 thereby eliminating the amyloid plaque accumulation and facilitating its clearance.Materials and MethodsAn excellent ligand-based and structure-based approach was made to identify common pharmacophoric features involving structure-based docking with respect to apoE ε4 leading to the development of apoE ε4 inhibitors possessing new scaffolds. An effort was made to design multiple-substituted triazine derivatives series bearing a novel scaffold. A structure-based pharmacophore mapping was developed to explore the binding sites of apoE ε4 which was taken into consideration. Subsequently, virtual screening, ADMET, DFT searches were at work to narrow down the proposed hits to be forwarded as a potential drug likes candidates. Further, the binding patterns of the best-proposed hits were studied and were forwarded for molecular dynamic simulations of 10 ns for its structural optimization.ResultsSelectivity profile for the most promising candidates was studied, revealing significantly C13 and C15 to be the most potent compounds. The proposed hits can be forwarded for further study against apoE ε4 involved in neurological disorder Alzheimer’s.     

Phloroglucinol Derivatives from Dryopteris crassirhizoma as Potent Xanthine Oxidase Inhibitors

Dryopteris crassirhizoma rhizomes are used as a traditional medicine in Asia. The EtOAc extract of these roots has shown potent xanthine oxidase (XO) inhibitory activity. However, the main phloroglucinols in D. crassirhizoma rhizomes have not been analyzed. Thus, we investigated the major constituents responsible for this effect. Bioassay-guided purification isolated four compounds: flavaspidic acid AP (1), flavaspidic acid AB (2), flavaspidic acid PB (3), and flavaspidic acid BB (4). Among these, 1 showed the most potent inhibitory activity with a half-maximal inhibitory concentration (IC₅₀) value of 6.3 µM, similar to that of allopurinol (IC₅₀ = 5.7 µM) and better than that of oxypurinol (IC₅₀ = 43.1 µM), which are XO inhibitors. A comparative activity screen indicated that the acetyl group at C3 and C3′ is crucial for XO inhibition. For example, 1 showed nearly 4-fold higher efficacy than 4 (IC₅₀ = 20.9 µM). Representative inhibitors (1–4) in the rhizomes of D. crassirhizoma showed reversible and noncompetitive inhibition toward XO. Furthermore, the potent inhibitors were shown to be present in high quantities in the rhizomes by a UPLC-QTOF-MS analysis. Therefore, the rhizomes of D. crassirhizoma could be used to develop nutraceuticals and medicines for the treatment of gout.     

Synthesis of 5-deoxy-5-phospho-d-ribonohydroxamic acid: a new competitive and selective inhibitor of type B ribose-5-phosphate isomerase from Mycobacterium tuberculosis

5-Deoxy-5-phospho-d-ribonohydroxamic acid, a mimic of the 1,2-cis-enediolate high-energy intermediate species of the allose-6-phosphate isomerase reaction, was obtained by a six-step synthesis from d-erythronolactone. In contrast to the known competitive ribose-5-phosphate isomerase (Rpi) inhibitors 4-deoxy-4-phospho-d-erythronohydroxamic acid, 4-deoxy-4-phospho-d-erythronate, and 4-deoxy-4-phosphonomethyl-d-erythronate, the new hydroxamic acid selectively inhibits Mycobacterium tuberculosis RpiB (K_i = 0.40 mM, K_m/K_i = 4.5) versus Spinacia oleracea RpiA, and hence appears as a promising lead for the design of potent species-specific inhibitors of the bacterial enzyme.     

Identification of Novel Antagonists Targeting Cannabinoid Receptor 2 Using a Multi-Step Virtual Screening Strategy

The endocannabinoid system plays an essential role in the regulation of analgesia and human immunity, and Cannabinoid Receptor 2 (CB2) has been proved to be an ideal target for the treatment of liver diseases and some cancers. In this study, we identified CB2 antagonists using a three-step “deep learning–pharmacophore–molecular docking” virtual screening approach. From the ChemDiv database (1,178,506 compounds), 15 hits were selected and tested by radioligand binding assays and cAMP functional assays. A total of 7 out of the 15 hits were found to exhibit binding affinities in the radioligand binding assays against CB2 receptor, with a pK_i of 5.15–6.66, among which five compounds showed antagonistic activities with pIC₅₀ of 5.25–6.93 in the cAMP functional assays. Among these hits, Compound 8 with the 4H-pyrido[1,2-a]pyrimidin-4-one scaffold showed the best binding affinity and antagonistic activity with a pK_i of 6.66 and pIC₅₀ of 6.93, respectively. The new scaffold could serve as a lead for further development of CB2 drugs. Additionally, we hope that the model in this study could be further utilized to identify more novel CB2 receptor antagonists, and the developed approach could also be used to design potent ligands for other therapeutic targets.