Discovery of Novel α,ß-Unsaturated Amide Derivatives as Candidate Antifungals to Overcome Fungal Resistance.

In our previous study, coumarin-containing CYP51 inhibitor A32 demonstrated potent antiresistance activity. However, compound A32 demonstrated unsatisfied metabolic stability, necessitating modifications to overcome these limitations. In this study, α,ß-unsaturated amides were used to replace the unstable coumarin ring, which increased metabolic stability by four times while maintaining antifungal activity, including activity against resistant strains. Subsequently, the sterol composition analysis and morphological observation experiments indicated that the target of these novel compounds is lanosterol 14α-demethylase (CYP51). Meanwhile, biofilm growth was inhibited and resistance genes (ERG11, CDR1, CDR2, and MDR1) expression was downregulated to find out how the antiresistance works. Importantly, compound C07 demonstrated the capacity to stimulate reactive oxygen species, thus displaying potent fungicidal activity. Moreover, C07 exhibited encouraging effectiveness in vivo following intraperitoneal administration. Additionally, the most potent compound C07 showed satisfactory pharmacokinetic properties and low toxicity. These α,ß-unsaturated amide derivatives, particularly C07, are potential candidates for treating azole-resistant candidiasis.

Listen to what the animals say: a systematic review and meta-analysis of sterol 14-demethylase inhibitor efficacy for in vivo models of Trypanosoma cruzi infection.

Sterol 14-demethylase (CYP51) inhibitors, encompassing new chemical entities and repurposed drugs, have emerged as promising candidates for Chagas disease treatment, based on preclinical studies reporting anti-Trypanosoma cruzi activity. Triazoles like ravuconazole (RAV) and posaconazole (POS) progressed to clinical trials. Unexpectedly, their efficacy was transient in chronic Chagas disease patients, and their activity was not superior to benznidazole (BZ) treatment. This paper aims to summarize evidence on the global activity of CYP51 inhibitors against T. cruzi by applying systematic review strategies, risk of bias assessment, and meta-analysis from in vivo studies. PubMed and Embase databases were searched for original articles, obtaining fifty-six relevant papers meeting inclusion criteria. Characteristics of animal models, parasite strain, treatment schemes, and cure rates were extracted. Primary outcomes such as maximum parasitaemia values, survival, and parasitological cure were recorded for meta-analysis, when possible. The risk of bias was uncertain in most studies. Animals treated with itraconazole, RAV, or POS survived significantly longer than the infected non-treated groups (RR = 4.85 [3.62, 6.49], P < 0.00001), and they showed no differences with animals treated with positive control drugs (RR = 1.01 [0.98, 1.04], P = 0.54). Furthermore, the overall analysis showed that RAV or POS was not likely to achieve parasitological cure when compared with BZ or NFX treatment (OD = 0.49 [0.31, 0.77], P = 0.002). This systematic review contributes to understanding why the azoles had failed in clinical trials and, more importantly, how to improve the animal models of T. cruzi infection by filling the gaps between basic, translational, and clinical research.

Design, Synthesis, and Activity Evaluation of Novel Benzazole Bifunctional Antifungal Inhibitors with an LDH Carrier.

Fungal infections maintain a close relation with the body's immune function. In this study, three series of benzazole compounds were designed as dual-target (PD-L1/CYP51) inhibitors using the skeleton splicing approach; their molecular structures were synthesized and evaluated accordingly. Among them, the compounds 9a-2, 12a-2, and 12b-1 exhibited potent antifungal activity and dual-target inhibition ability. Especially, the compound 12a-2 simultaneously exerted excellent bifunctional effects of fungal inhibition and immune activation. Moreover, a layered double hydroxide (LDH) carrier was also successfully constructed based on an infection microenvironment to improve the bioavailability and the targeting of compound 12a-2. This significantly accelerated the recovery process of deep and shallow fungal infections. In conclusion, this study expanded the development horizon of antifungal drugs and provided a novel drug delivery route for treating fungal infections.

Novel Pyrido[4,3-d]pyrimidine Derivatives as Potential Sterol 14α-Demethylase Inhibitors: Design, Synthesis, Inhibitory Activity, and Molecular Modeling.

Thirty-four new pyrido[4,3-d]pyrimidine analogs were designed, synthesized, and characterized. The crystal structures for compounds 2c and 4f were measured by means of X-ray diffraction of single crystals. The bioassay results showed that most target compounds exhibited good fungicidal activities against Pyricularia oryzae, Rhizoctonia cerealis, Sclerotinia sclerotiorum, Botrytis cinerea, and Penicillium italicum at 16 µg/mL. Compounds 2l, 2m, 4f, and 4g possessed better fungicidal activities than the commercial fungicide epoxiconazole against B. cinerea. Their half maximal effective concentration (EC50) values were 0.191, 0.487, 0.369, 0.586, and 0.670 µg/mL, respectively. Furthermore, the inhibitory activities of the bioactive compounds were determined against sterol 14α-demethylase (CYP51). The results displayed that they had prominent activities. Compounds 2l, 2m, 4f, and 4g also showed better inhibitory activities than epoxiconazole against CYP51. Their half maximal inhibitory concentration (IC50) values were 0.219, 0.602, 0.422, 0.726, and 0.802 µg/mL, respectively. The results of molecular dynamics (MD) simulations exhibited that compounds 2l and 4f possessed a stronger affinity to CYP51 than epoxiconazole.

Design, Synthesis, Bioactive Evaluation, and Molecular Dynamics Simulation of Novel 4H-Pyrano[3,2-c]pyridine Analogues as Potential Sterol 14α-Demethylase (CYP51) Inhibitors.

To discover potential sterol 14α-demethylase (CYP51) inhibitors, thirty-four unreported 4H-pyrano[3,2-c]pyridine derivatives were designed and synthesized. The assay results indicated that most compounds displayed significant fungicidal activity against Sclerotinia sclerotiorum, Colletotrichum lagenarium, Botrytis cinerea, Penicillium digitatum, and Fusarium oxysporum at 16 µg/mL. The half maximal effective concentration (EC50) values of compounds 7a, 7b, and 7f against B. cinerea were 0.326, 0.530, and 0.610, respectively. Namely, they had better antifungal activity than epoxiconazole (EC50 = 0.670 µg/mL). Meanwhile, their half maximal inhibitory concentration (IC50) values against CYP51 were 0.377, 0.611, and 0.748 µg/mL, respectively, representing that they also possessed better inhibitory activities than epoxiconazole (IC50 = 0.802 µg/mL). The fluorescent quenching tests of proteins showed that 7a and 7b had similar quenching patterns to epoxiconazole. The molecular dynamics simulations indicated that the binding free energy of 7a and epoxiconazole to CYP51 was -35.4 and -27.6 kcal/mol, respectively.

Construction and Activity Evaluation of Novel Bifunctional Inhibitors and a COF Carrier Based on a Fungal Infection Microenvironment.

Faced with increasingly serious fungal infections and drug resistance issues, three different series of novel dual-target (programmed death ligand 1/14 α-demethylase) compounds were constructed through the fragment combination pathway in the study. Their chemical structures were synthesized, characterized, and evaluated. Among them, preferred compounds 10c-1, 17b-1, and 18b-2 could efficiently exert their antifungal and antidrug-resistant fungal ability through blocking ergosterol biosynthesis, inducing the upregulation of reactive oxygen species level, and triggering apoptosis. Especially, compound 18b-2 exhibited the synergistic function of fungal inhibition and immune activation. Moreover, the covalent organic framework carrier was also generated based on the acidic microenvironment of fungal infection to improve the bioavailability and targeting of preferred compounds; this finally accelerated the body's recovery rate.

Identification of Potent and Selective Inhibitors of Acanthamoeba: Structural Insights into Sterol 14α-Demethylase as a Key Drug Target.

Acanthamoeba are free-living pathogenic protozoa that cause blinding keratitis, disseminated infection, and granulomatous amebic encephalitis, which is generally fatal. The development of efficient and safe drugs is a critical unmet need. Acanthamoeba sterol 14α-demethylase (CYP51) is an essential enzyme of the sterol biosynthetic pathway. Repurposing antifungal azoles for amoebic infections has been reported, but their inhibitory effects on Acanthamoeba CYP51 enzymatic activity have not been studied. Here, we report catalytic properties, inhibition, and structural characterization of CYP51 from Acanthamoeba castellanii. The enzyme displays a 100-fold substrate preference for obtusifoliol over lanosterol, supporting the plant-like cycloartenol-based pathway in the pathogen. The strongest inhibition was observed with voriconazole (1 h IC50 0.45 µM), VT1598 (0.25 µM), and VT1161 (0.20 µM). The crystal structures of A. castellanii CYP51 with bound VT1161 (2.24 Å) and without an inhibitor (1.95 Å), presented here, can be used in the development of azole-based scaffolds to achieve optimal amoebicidal effectiveness.

Antifungal and Antiparasitic Activities of Metallocene-Containing Fluconazole Derivatives.

The search for new anti-infectives based on metal complexes is gaining momentum. Among the different options taken by researchers, the one involving the use of organometallic complexes is probably the most successful one with a compound, namely, ferroquine, already in clinical trials against malaria. In this study, we describe the preparation and in-depth characterization of 10 new (organometallic) derivatives of the approved antifungal drug fluconazole. Our rationale is that the sterol 14α-demethylase is an enzyme part of the ergosterol biosynthesis route in Trypanosoma and is similar to the one in pathogenic fungi. To demonstrate our postulate, docking experiments to assess the binding of our compounds with the enzyme were also performed. Our compounds were then tested on a range of fungal strains and parasitic organisms, including the protozoan parasite Trypanosoma cruzi (T. cruzi) responsible for Chagas disease, an endemic disease in Latin America that ranks among some of the most prevalent parasitic diseases worldwide. Of high interest, the two most potent compounds of the study on T. cruzi that contain a ferrocene or cobaltocenium were found to be harmless for an invertebrate animal model, namely, Caenorhabditis elegans (C. elegans), without affecting motility, viability, or development.

Miconazole-like Scaffold is a Promising Lead for Naegleria fowleri-Specific CYP51 Inhibitors.

Developing drugs for brain infection by Naegleria fowleri is an unmet medical need. We used a combination of cheminformatics, target-, and phenotypic-based drug discovery methods to identify inhibitors that target an essential N. fowleri enzyme, sterol 14-demethylase (NfCYP51). A total of 124 compounds preselected in silico were tested against N. fowleri. Nine primary hits with EC50 ≤ 10 µM were phenotypically identified. Cocrystallization with NfCYP51 focused attention on one primary hit, miconazole-like compound 2a. The S-enantiomer of 2a produced a 1.74 Å cocrystal structure. A set of analogues was then synthesized and evaluated to confirm the superiority of the S-configuration over the R-configuration and the advantage of an ether linkage over an ester linkage. The two compounds, S-8b and S-9b, had an improved EC50 and KD compared to 2a. Importantly, both were readily taken up into the brain. The brain-to-plasma distribution coefficient of S-9b was 1.02 ± 0.12, suggesting further evaluation as a lead for primary amoebic meningoencephalitis.

Strategies of targeting CYP51 for IFIs therapy: Emerging prospects, opportunities and challenges.

CYP51, a monooxygenase associated with the sterol synthesis pathway, is responsible for the catalysis of the 14-methyl hydroxylation reaction of lanosterol precursors. This enzyme is widely present in microorganisms, plants, and mammals. In mammals, CYP51 plays a role in cholesterol production, oligodendrocyte formation, oocyte maturation, and spermatogenesis. In fungal cells, CYP51 is an enzyme that synthesizes membrane sterols. By inhibiting fungal CYP51, ergosterol synthesis can be inhibited and ergosterol membrane fluidity is altered, resulting in fungal cell apoptosis. Thus, targeting CYP51 is a reliable antifungal strategy with important implications for the treatment of invasive fungal infections (IFIs). Many CYP51 inhibitors have been approved by the FDA for clinical treatment. However, several limitations of CYP51 inhibitors remain to be resolved, including fungal resistance, hepatotoxicity, and drug-drug interactions. New broad-spectrum, anti-resistant, highly selective CYP51 inhibitors are expected to be developed to enhance clinical efficacy and minimize adverse effects. Herein, we summarize the structural features and biological functions of CYP51 and emphatically analyze the structure-activity relationship (SAR) and therapeutic potential of different chemical types of small-molecule CYP51 inhibitors. We also discuss the latest progress of novel strategies, providing insights into new drugs targeting CYP51 for clinical practice.

New pyrrole derivatives as DNA gyrase and 14α-demethylase inhibitors: Design, synthesis, antimicrobial evaluation, and molecular docking.

An efficient one-pot reaction utilizing readily available chemical reagents was used to prepare novel 2-amino-1,5-diaryl-1H-pyrrole-3-carbonitrile derivatives and the structures of these compounds were validated by spectroscopic data and elemental analyses. All the synthetic compounds were evaluated for their antimicrobial activities (MZI assay). The tested compounds proved high activities on Staphylococcus aureus (Gram-positive bacteria) and Candida albicans (Pathogenic fungi). However, they did not show any activity on Escherichia coli (Gram-negative bacteria). The most effective compounds in MZI assay 7c, 9a, 9b, 11a, and 11b were selected to determine their MIC on S. aureus and C. albicans. Furthermore, DNA gyrase and 14-α demethylase inhibitory assays were performed to study the inhibitory activities of 7c, 9a, 9b, 11a, and 11b. The results illustrated that compound 9b was the most DNA gyrase inhibitor (IC50 of 0.0236 ± 0.45 µM, which was 1.3- fold higher than gentamicin reference IC50 values of 0.0323 ± 0.81 µM). In addition, compound 9b demonstrated the highest 14-α demethylase inhibitory effect with IC50 of 0.0013 ± 0.02 µM, compared to ketoconazole (IC50 of 0.0008 ± 0.03 µM) and fluconazole (IC50 of 0.00073 ± 0.01 µM), as antifungal reference drugs. Lastly, docking studies were performed to rationalize the dual inhibitory activities of the highly active compounds on both DNA gyrase and 14-α demethylase enzymes.

Quinoline-imidazole/benzimidazole derivatives as dual-/multi-targeting hybrids inhibitors with anticancer and antimicrobial activity.

Two new classes of hybrid quinoline-imidazole/benzimidazole derivatives (the hybrid QIBS salts and QIBC cycloadducts) were designed and synthesized to evaluate their anticancer and antimicrobial activity. The strategy adopted for synthesis is straight and efficient, in four steps: N-acylation, N-alkylation, quaternization and a Huisgen 3 + 2 cycloaddition. The in vitro single-dose anticancer assay of forty six hybrid quinoline-benzimidazole compounds reveal that one QIBS salt (11h), has an excellent quasi nonselective activity against all type of cancer cell with an excellent PGI in the area of 90-100% and very good lethality. Three others quinoline-imidazole/benzimidazole hybrids (8h, 12h, 12f) has an excellent selective activity against some cancer cell lines: breast cancer MDA-MB-468 and Leukemia HL-60 TB). The five-dose assay screening confirms that compound 11h possesses excellent anti-proliferative activity, with GI50 in the range of nano-molar, against some cancer cell lines: Leukemia HL-60 TB, Leukemia K-526, Leukemia RPMI-8226, Breast cancer MDA-MB-468, Lung cancer HOP-92 and Ovarian cancer IGROV1. The antibacterial assay indicates that three hybrid QIBS salts (12f, 12c, 12d) have an excellent activity against Gram-negative bacteria E. coli (superior to control Gentamicin) while against Gram-positive bacteria S. aureus only one compound 8i (R2 = -CF3) exhibits a significant activity (superior to control Gentamicin). The MIC assay indicates that two other compounds (11h, 12h) are biologically active to a very low concentration, in the range of nano-molar. We believe that all these excellent assets related to anticancer and antibacterial activities, make from our hybrid quinoline-imidazole/benzimidazole compounds bearing a phenyl group (R2 = -C6H5) in the para (4)-position of the benzoyl moiety a good candidate for future drug developing.

Design, Synthesis, and Biological Evaluation of Dual-Target COX-2/CYP51 Inhibitors for the Treatment of Fungal Infectious Diseases.

The design of novel dual-target (COX-2/CYP51) inhibitors was proposed in the study, and three series of compounds were constructed though the pathway of skeleton screening and combination; their molecular structures were synthesized and evaluated. Most of the compounds exhibited significant antifungal ability. Among them, potential compounds (10a-2, 16b-3) with excellent antifungal and anti-drug-resistant fungal ability (MIC50, 0.125-2.0 µg/mL) were selected for the subsequent mechanistic study. On the one hand, these compounds could block the ergosterol biosynthesis pathway by inhibiting CYP51 and influence the internal physiological function of fungal cells, which included the increase of the ROS level, the anomaly of ΔΨm, and the emergence of an apoptotic state. On the other hand, these compounds also effectively showed COX-2 inhibition ability, eliminated the inflammatory reaction of the infected region, and activated the body's immune function. In summary, this study not only provided a novel antifungal drug design pathway but also discovered excellent target compounds.

Design, Synthesis, and Evaluation of New Sugar-Substituted Imidazole Derivatives as Selective c-MYC Transcription Repressors Targeting the Promoter G-Quadruplex.

c-MYC is a key driver of tumorigenesis. Repressing the transcription of c-MYC by stabilizing the G-quadruplex (G4) structure with small molecules is a potential strategy for cancer therapy. Herein, we designed and synthesized 49 new derivatives by introducing carbohydrates to our previously developed c-MYC G4 ligand 1. Among these compounds, 19a coupled with a d-glucose 1,2-orthoester displayed better c-MYC G4 binding, stabilization, and protein binding disruption abilities than 1. Our further evaluation indicated that 19a blocked c-MYC transcription by targeting the promoter G4, leading to c-MYC-dependent cancer cell death in triple-negative breast cancer cell MDA-MB-231. Also, 19a significantly inhibited tumor growth in the MDA-MB-231 mouse xenograft model accompanied by c-MYC downregulation. Notably, the safety of 19a was dramatically improved compared to 1. Our findings indicated that 19a could become a promising anticancer candidate, which suggested that introducing carbohydrates to improve the G4-targeting and antitumor activity is a feasible option.

Design and preparation of nanoarchitectonics of LDH/polymer composite with particular morphology as catalyst for green synthesis of imidazole derivatives.

This paper was designed and prepared a new nanoarchitectonics of LDH/polymer composite with specific morphology. For this purpose, CTAB surfactant was used to control the morphology of layered double hydroxide (LDH) and to prepare LDH/polymer nanocomposites (LDH-APS-PEI-DTPA). The polymer was synthesized using diethylenetriaminepentaacetic acid (DTPA), polyethylenimine and used with LDH to form a nanocomposite with high thermal stability. Subsequently, the prepared nanocomposite was identified using FTIR, EDX, TGA, XRD, FESEM, and BET techniques. In addition, the prepared LDH-APS-PEI-DTPA nanocomposite was used as a heterogeneous and recyclable catalyst for the synthesis of imidazole derivatives under green conditions. The results showed that the LDH-APS-PEI-DTPA nanocomposite benefit from suitable morphology, simple preparation, high catalytic activity, and high surface area. Also, the proposed LDH-APS-PEI-DTPA heterogeneous catalyst showed high stability and reusability for five consecutive runs which was consistent with the principles of green chemistry.

In silico Prediction and Pharmacokinetic Studies on Glucosinolates as a Potential Drug and Key Inhibitor Molecule for Lanosterol-14α- demethylase: A Fungal Membrane Biosynthesis Enzyme.

BACKGROUND: Glucosinolates (ß-thioglucoside-N-hydroxysulfates) are a water-soluble organic anion with sulfur- and nitrogen-containing glycosides which are found in abundance in Cruciferous plants. Ergosterol (ERG13) lanosterol-14α-demethylase protein has been targeted for inhibition studies as a key regulator enzyme of fungal membrane biosynthesis. OBJECTIVES: To understand the molecular mechanism of inhibition of Ergosterol (ERG13) lanosterol- 14α-demethylase by various phytochemicals from brassicales, i.e., glucosinolates and their potential role as putative drug molecules. METHODS: In this study, in silico analyses were performed to predict the molecular basis of various glucosinolates as a potential inhibitor of lanosterol-14α-demethylase protein, which is a key regulator of fungal membrane biosynthesis and its pharmacodynamics and toxicity profile. 3d structures of various glucosinolates were retrieved from PubChem, and the target protein, lanosterol-14α-demethylase (Pdb ID- 4lxj), was retrieved from the RCSB protein data bank. Molecular docking and interactions were carried out using the PyRx software using the AutoDOCK toolbar with default parameters. Dru- LiTo, ORISIS web servers were used to predict various drug likeliness predictions and Lipinski's Rule of 5, whereas admetSAR was used for prediction of toxicity, and PASS Program was used to study the antifungal and antimicrobial properties of these compounds. RESULTS: This study shows that among the different compounds screened, gluconasturtiin, Glucotropaeolin, and Indolylmethyl-Glucosinolate showed the highest binding energies of -8.7 kcal/mol, -8.5 kcal/mol, and -8.3 kcal/mol with the lanosterol-14α-demethylase, respectively. Further all the compounds follow the Lipinski's rule as well as they are found to be non-carcinogenic and non-cytotoxic in nature. These compounds also show antifungal properties. CONCLUSION: This study thus reveals that various glucosinolates interact with the ERG13 enzyme at various amino acid positions, which behaves as a catalytic site, thus indicates the probable mechanism of inactivation, and subsequently, these can be used as potential drug molecules. In vitro studies can be taken to further examine the utility of these compounds as antifungal agents.

Discovery of novel Thieno[2,3-d]imidazole derivatives as agonists of human STING for antitumor immunotherapy using systemic administration.

The activation of stimulator of interferon genes (STING) signaling pathways plays an important role in the innate immune response. Although several STING agonists have been developed recently, the majority of clinical CDN STING agonists are administered by intratumoral (IT) injection. Therefore, there remains a need to develop diverse non-CDN small-molecule STING agonists with systemic administration. Herein, by using a scaffold hopping strategy, we designed a series of thieno [2,3-d]imidazole derivatives as novel STING agonists. Further structure-activity relationship study and optimization led to the discovery of compound 45 as a highly potent human STING agonist with an EC50 value of 1.2 nM. Compound 45 was found to bind to multiple human STING isoforms and accordingly activated the downstream TBK1/IRF3 and NF-κB signaling pathways in the reporter cells bearing with different STING isoforms. The activation on STING signaling pathway was abolished in the STING knock-out cells, indicating that it is a specific STING agonist. Compound 45 significantly inhibited the tumor growth in allograft 4T1 and CT26 tumor models by systemic administration, and more significantly, 45 was able to induce tumor regression in CT26 tumor model without inducing weight loss, suggesting that compound 45 is a highly promising candidate worthy for further development.

Discovery of 5-methyl-1H-benzo[d]imidazole derivatives as novel P2X3 Receptor antagonists.

Drug discovery programs targeting P2X3 receptors (P2X3R), an extracellular adenosine 5'-triphosphate (ATP) gated cation channel family, have been actively investigated for several CNS-related diseases. The current unmet need in the field of P2X3R targeted drugs is to avoid a side effect, the loss of taste, that could be reduced by increase of the P2X3R selectivity vs P2X2/3R. In this study, 5-methyl-1H-benzo[d]imidazole derivatives were designed and synthesized from the analysis of key pharmacophores of current antagonists. In the structure-activity relationship study, the most potent compounds 17a-b was discovered as potent P2X3R antagonists with IC50 values of 145 and 206 nM, and selectivity index of 60 and 41, respectively. In addition, 17a-b showed the not-competitive antagonism, but poor binding score in the docking study at the known allosteric binding site of Gefapixant binding site, indicating that another allosteric binding site might be existing for the novel P2X3R antagonists.

Discovery of benzyloxyphenyl- and phenethylphenyl-imidazole derivatives as a new class of ante-drug type boosters.

Although cytochrome P450 3A4 (CYP3A4) inhibitors are used as boosters to increase drug absorption, the inhibition of CYP3A4 activity may affect the metabolism of other co-administered drugs. Therefore, we screened for and developed a new class of boosters to improve the oral availability of drugs. We identified benzyloxyphenyl imidazole and phenethylphenyl imidazole derivatives as new types of CYP3A4 inhibitors. Among the compounds synthesized, an ester 5c was found to inhibit CYP activity and the compound 5c was gradually converted to an inactive metabolite 5d under physiological conditions, indicating that the ester 5c may represent a novel ante-drug type booster.

Mulberrin inhibits Botrytis cinerea for strawberry storage by interfering with the bioactivity of 14α-demethylase (CYP51).

Currently, chemical agents hold great promise in preventing and combating Botrytis cinerea. However, the antifungal mechanism of some agents for B. cinerea remains rather vague, imposing restrictions on the research and development of novel antifungal inhibitors. In this work, we discovered that mulberrin (MBN), a natural compound from the root bark of Ramulus Mori, with an IC50 of 1.38 µM together, demonstrated marked anti-14α-demethylase (CYP51) activity through high throughput virtual screening and in vitro bioactivity assay. The computational biology results demonstrated that MBN and its derivatives were bound to the catalytic activity region of CYP51, but only MBN could form a strong π-cation interaction with the Fe ion of heme in CYP51 via the 2-methylpent-2-ene moiety at atom C9. MBN had a stronger binding free energy than the other three compounds with CYP51, implying that the 2-methylpent-2-ene moiety at atom C9 is a critical pharmacophore for CYP51 inhibitors. Subsequently, through an antifungal test, MBN demonstrated excellent anti-B. cinerea activity by inhibiting CYP51 activity. The EC50 values of MBN toward hyphal growth and spore germination in B. cinerea were 17.27 and 9.56 µg mL-1, respectively. The bioactivity loss of CYP51 by direct interaction with MBN induced the increase of cell membrane permeability, membrane destruction, and cell death. Meanwhile, in the B. cinerea infection model, MBN significantly prolonged the preservation of strawberries by preventing B. cinerea from infecting strawberries and could be used as a potential natural preserving agent for storing fruits.