Coumarin Compounds Research Articles

THE PHENOLIC PROFILE OF RAPESEED HONEY ENRICHED WITH MELILOTUS ALBUS FLOWERS AND ITS INHIBITORY EFFECT ON RAT PLATELET AGGREGATION

Background. The Melilotus plants are highly valued in herbal medicine, recommended for the prevention and treatment of thrombotic vein inflammation and varicose veins. Their health-promoting properties primarily result from the presence of coumarin and its derivatives. The aim of the study was to determine whether honey enriched with dried Melilotus albus flowers has the ability to inhibit platelet aggregation. The inhibitory activity of rapeseed honey (control sample), rapeseed honey enriched with 1 % w/w of M. albus dried flowers and pure coumarin were evaluated against ADP (5 µМ) and collagen (2 µg/ml) in duced aggregation of rat platelets. Platelet aggregation was measured by the turbidimetric method. Moreo ver, the quantitative polyphenolic profile determined using HPLC-PDA and volatile compounds profiles obtained by GC-MS methods for rapeseed and M. albus enriched honey were compared to identify the chemical factors that limit platelet aggregation. Results and conclusions. A beneficial, dose-dependent effect of enriched honey on the inhibition of the aggregation process was demonstrated, and for the highest concentration used (20 %), the inhibition of aggregation induced by ADP and collagen amounted to 75 % and 90%, respectively. A weaker inhibitory effect was found for pure rapeseed honey at 20 % concentration – 40 % and 55 % of inhibition, respective ly and pure coumarin (0.5 mg/ml) – 44 % and 20 %, respectively. The results obtained demonstrated that rapeseed honey enriched with M. albus flowers possesses antiplatelet activity resulting from the synergistic effect of coumarin and other bioactive compounds occurred in dried M. albus flowers, including phe nolic and volatile components.. However, the mechanism of this action needs further study.

Osthole: A potential AMPK agonist that inhibits NLRP3 inflammasome activation by regulating mitochondrial homeostasis for combating rheumatoid arthritis

BackgroundOsthole (OST), a characteristic coumarin compound in Angelicae pubescentis radix (APR), has shown potent efficacy in the treatment of rheumatoid arthritis (RA), but its specific targets and potential mechanism are limited. PurposeThis study aimed to explore the potential targets and molecular mechanisms of OST against RA using computer-assisted techniques in combination with RA fibroblast-like synoviocytes (FLS) inflammation model and CIA rat model. MethodsNetwork pharmacology and molecular docking were applied to initially predict the potential targets of OST for the treatment of RA. Thereafter, TNFα was used to stimulate FLS to build an in vitro model of inflammation, combined with RNA-seq technology and molecular biology such as qPCR to investigate the anti-inflammatory effects and related mechanisms of OST. Finally, the anti-RA effect of OST was demonstrated by establishing a CIA rat model. ResultsThe network model results showed that the anti-RA effect of OST was mainly related to its anti-inflammatory effect, and AMPK was identified as a potential target for the potency of OST. In the TNFα-induced FLS cells, OST inhibited the secretion of FLS inflammatory factors, which was attributed to the ability of OST to activate AMPK to inhibit the activation of the NLRP3 inflammasome. Further, it was observed that the activation of AMPK by OST facilitated mitochondrial biogenesis, and corrected abnormal mitochondrial dynamics in FLS, which was favoured to the restoration of mitochondrial homeostasis, and further promoted the occurrence of apoptosis and the decrease of ROS in FLS. Consistent with in vivo studies, administration of OST significantly improved joint deformity and toe erythema, reduced arthritis index scores and inhibited synovial inflammation in CIA rats. ConclusionOur study proposed for the first time that AMPK, served as a potential target of OST, positively participated in the anti-RA therapeutic effect of OST. By regulating mitochondrial homeostasis and function, OST can effectively inhibit the activation of inflammasome and the secretion of inflammatory factors in vitro and in vivo, and finally achieve beneficial effects in the treatment of RA, which provides support and greater possibility to make further efforts on pharmacological research and clinical application of OST.

GlPS1 overexpression accumulates coumarin secondary metabolites in transgenic Arabidopsis.

The online version contains supplementary material available at 10.1007/s11240-022-02427-w.

Anticancer Properties and Clinical Trials of Coumarins: A Review

Cancer has evolved as one of the most common causes of mortality, worldwide. Though numerous chemotherapeutic treatments are available, their side effects such as cytotoxicity and drug resistance form a big problem during the cancer treatment. Recent studies of anticancer activities conducted on natural products isolated from plants, namely coumarin and related compounds, prove them to be a promising drug candidate in cancer treatments. Efforts made by the scientists to design and develop novel anti-cancer agents using coumarins as lead compounds and study their effectiveness using Structural Activity Relationship is worth appreciating. This review, therefore, focuses on the recent progress in the discovery of coumarin derivatives with potential antitumor activity. It also summarizes their structureactivity relationship, and mechanism of action studies

Pharmacological Activities and Pharmacokinetics of Glycycoumarin.

The online version contains supplementary material available at 10.1007/s43450-022-00342-x.

Evaluation of on-site testing methods with a novel 3-in-1 miniaturized spectroscopic device for cinnamon screening

“True cinnamon” is often fraudulently replaced by other varieties for economic reasons. In the powdered form, it is not possible to distinguish the varieties visually, but they differ in their sensory profile, in particular in the aromatic compound coumarin content which has also been deemed hepatotoxic in animal models. Molecular and analytical techniques exist which can be used for authentication but are expensive, time-consuming, and destructive. As an alternative, we tested three different miniaturized spectroscopic techniques namely, ultraviolet–visible (UV–Vis), near-infrared (NIR) and fluorescence (FLUO) to authenticate cinnamon samples. Out of the three, UV–Vis and NIR were superior to FLUO. The separation with UV–Vis and FLUO could be visually identified after pre-processing the spectral data and subsequently submitting it to principal component analysis (PCA). When chemometrics were applied a correct classification rate by variety of 89%, 90% and 89% for UV–Vis, NIR, and fluorescence spectroscopy, respectively, was observed. The usage of miniaturized spectrophotometers combined with PCA and classification algorithms was found promising to authenticate cinnamon.

Recovery of Naringin-Rich Flavonoid Extracts from Agroresidues with Anxiolytic- and Antidepressant-like Effects in Mice

Citrus paradisi species belong to the Rutaceae family, and it is commonly known as grapefruit. Grapefruit consumption involves a large amount of waste that goes to landfills and produces significant pollution affecting the human health. To examine this phenomenon, we designed an efficient chemical method that recovers naringin-rich flavonoid extracts from the fresh waste of grapefruits, by using the solvent impregnation resin method (SIR) with XAD-4 amberlite and either methanol or water as elution systems. Additionally, we focused on evaluating these extracts' anxiolytic- and antidepressant-like effects in behavioral predictive paradigms in mice. According to direct Principal Component Analysis (PCA) by NMR, and Direct Injection Electrospray Ionization-Mass Spectrometry (DIESI-MS), methanol extracts obtained after resin treatment were free of coumarin compounds and evinced had a high content of naringin. Poncirin, phenylalanine, chrysin 5,7-dimethyl ether, 5,7-dimethoxy-4'-hydroxyflavanone, 2,3-dihydro-2-(4-hydroxyphenyl)-5,6,7,8-tetramethoxy-4H-1-benzopyran-4-one, tetrahydrocurcumin, corchoionoside C, 6'-coumaroyl-1'-O-[2-(3,4-dihydroxyphenyl) ethyl]-β-D-glucopyranoside were also detected. Naringin-rich methanol extract caused a clear anxiolytic-like effect in the Elevated Plus Maze (EPM) and the Hole-Board (HBT) Tests, increasing oral doses of this extract did not produce a sedative effect. A single oral dose caused an antidepressant-like effect in the Tail Suspension Test (TST), while repeated administrations of the methanol extract elicited a robust antidepressant effect in the Forced Swimming Test (FST) in mice. Our evidence highlights the importance of bioprospecting studies of organic waste with therapeutic potentials, such as anxiety and depression disorders.

Natural products as sources of acetylcholinesterase inhibitors: Synthesis, biological activities, and molecular docking studies of osthole-based ester derivatives.

Osthole is a natural coumarin compound which isolated from Cnidium monnieri (L.) Cusson, has extensive pharmacological activities and could be used as a leading compound for drug research and development. In a continuous effort to develop new acetylcholinesterase inhibitors from natural products, eighteen osthole esters were designed, synthesized, and confirmed by 1H NMR, 13C NMR and HRMS. The anti-AChE activity of These derivatives was measured at a concentration of 1.0 mol/mL in vitro by Ellman's method, and the result showed that 4m and 4o had moderate inhibitory activities with 68.8% and 62.6%, respectively. Molecular docking study results further revealed AChE interacted optimally with docking poses 4m and 4o. Network pharmacology also predicted that compound 4m could be involved in Ras signaling pathway, which made it a potential therapeutic target of AD.

Photophysical and photochemical properties and comparison of tolyl and tosyl coumarin-bearing phthalocyanines

In this study, peripheral and non-peripherally substituted Zn(II) phthalocyanine complexes were synthesized from 7‑hydroxy-3-(p-tolyl)coumarin and 7‑hydroxy-3-(p-tosyl)coumarin compounds. The synthesized new compounds were characterized using elemental analysis, FT-IR, UV–Vis, Fluorescence 1HNMR spectroscopy and MALDI-TOF mass spectrometry. All the synthesized phthalocyanine complexes showed good solubility in organic solvents such as acetone, dichloromethane, chloroform, pyridine, and ethyl acetate. Fluorescent quenching behavior was investigated using 1,4-benzoquinone and potassium iodide as a quencher. The photophysical (fluorescent quantum yields and lifetimes) and photochemical (single oxygen and photodegradation quantum yields) properties of these new phthalocyanines were examined in dimethyl sulfoxide. Phthalocyanine complexes containing 7‑hydroxy-3-(p-tolyl)coumarin had higher singlet oxygen quantum yields than phthalocyanine complexes containing 7‑hydroxy-3-(p-tosyl)coumarin. Phthalocyanines to which coumarins are peripherally bound were more advantageous than their non-peripherally bound derivatives. As a result of their photophysical and photochemical properties, coumarin-phthalocyanine complexes containing tolyl-/tosyl- groups can be used as photosensitizing candidates in photodynamic therapy and can be developed with targeted modifications.

A Research Study in the Synthesis and the Applications of Coumarin Derivatives

Many organic and medicinal chemists are interested in the coumarin compounds due to their potential for use in pharmaceuticals and other industries. Coumarin compounds are a significant class of biologically active chemicals. Here, we outline the steps forproducing coumarin derivatives quickly and effectively from commercially accessible substrates via multicomponent condensation processes involving pyrone and benzene derivatives. These substances were examined for their potential anti-tumor effects, including their ability to block carbonic anhydrase, activate the cell apoptosis protein, and coumarin and its derivatives are the primary oral anticoagulants. Candida albicans, Aspergillus fumigatus, and Fusarium solani are three distinct fungus species that are resistant to the antifungal properties of synthetic coumarins. Long known are the coumarins' antibacterial due to its biological activities against some kinds of bacteria such as Zeitschrift für Naturforschung C, as well as their antifilarial, antiulcerogenic, anti-inflammatory, and antioxidant effects for its capacity to inhibit their growth activities. Coumarin has long been valued as a major raw element in the fragrance sector due to its distinct sweet aroma and stability. When mixed with organic essential oils like lavender, citrus, rosemary, and oak moss, it is used as an odour-enhancer to produce a long-lasting impact. It has been utilized as a bleaching or brightening agent in detergents when it revealed a remarkable brightening effect with excellent overall stability on fibers of synthetic origin. Materials such as liquid crystalline, organic/inorganic composites, as well as light harvesting, energy transferring compounds, and electro-optics materials are all employed as a dye in organic photo-redox catalysis and as potent photo-sensitizers reagent. These materials polymerize epoxy-silicones, under near-UV and visible light. Coumarin has also found useful as metal-free sensitizers for solar cells.

Pharmacological and Therapeutic Applications of Esculetin.

Esculetin is a coumarin compound, which belongs to the class of benzopyrone enriched in various plants such as Sonchus grandifolius, Aesculus turbinata, etc. Free radicals lead to the development of oxidative stress causing inflammation, arthritis, cancer, diabetes, fatty liver disease, etc. These further reduce the efficacy of anticancer drugs, activate inflammatory signaling pathways, degrade joints and cartilage, and disrupt the glycemic index and normal function of liver enzymes. For instance, the current treatment modalities used in arthritis such as non-steroidal anti-inflammatory drugs, disease-modifying anti-rheumatoid drugs, and lipoxygenase inhibitors present limited efficacy and adverse effects. Thus, there is a constant need to find newer and safer alternatives. Esculetin has an immense antioxidative potential thereby alleviating arthritis, diabetes, malignancies, and hepatic disorders. Structurally, esculetin contains two hydroxyl groups, which enhance its ability to function as an antioxidant by inhibiting oxidative stress in pathological conditions. Leukotriene B4 synthesis, NF-κB and MPAK pathway activation, and inflammatory cytokine production are the main causes of bone and joint deterioration in arthritis, whereas esculetin treatment reverses these factors and relieves the disease condition. In contrast, lipid peroxidation caused by upregulation of TGF-β-mediated expression and dysfunction of antioxidant enzymes is inhibited by esculetin therapy, thus reducing liver fibrosis by acting on the PI3K/FoxO1 pathway. Therefore, targeting NF-κB, pro-inflammatory cytokines, TGF-β and oxidative stress may be a therapeutic strategy to alleviate arthritis and liver fibrosis.

Novel Coumarin 7-Carboxamide/Sulfonamide Derivatives as Potential Fungicidal Agents: Design, Synthesis, and Biological Evaluation.

Coumarin compounds have a variety of biological activities such as anti-tumor, anti-coagulation, anti-HIV, anti-fungal, and insecticidal. Amide and sulfonamide compounds have been used as fungicides for half a century, and dozens of varieties have been developed so far. This study focused on the introduction of carboxamide and sulfonamide moieties in a coumarin core to discover novel derivatives. Based on this strategy, we synthesized two series of novel carboxamide and sulfonamide substituted coumarin derivatives, and their fungicidal activity was also investigated. Some designed compounds possessed potential activities against six phytopathogenic fungi in the primary assays, highlighted by compound 6r. Compound 6r exhibited stronger fungicidal activity against Botrytis cinerea (EC50 = 20.52 µg/mL) and will be the lead structure for further study.

Dihydroeugenol derivatives associated with blue LED light: A different form to face multidrug resistant (MDR) bacteria

Eugenol has already had its pharmacological properties elucidated in previous studies, including antibacterial and antifungal properties. Based on such information, this study aimed to evaluate the antibacterial and modulatory activity of coumarin compounds prepared from dihydroeugenol and to associate them with blue LED light for the same activity. For this study, five of the substances available: compound 1 (C1), 8-methoxy-2-oxo-6-propyl-2H-chromen-3-carboxylic acid, compound (C2), 3-(hydroxy(4-nitrophenyl)methyl)-8- methoxy-6-propyl-2H-chromen-2-one, compound 7 (C3), 8-hydroxy-3-(4-nitrobenzoyl)-6-propyl-2H-chromen-2-one, compound 8 (C4), 3-(4-aminobenzoyl)-8-methoxy-6-propyl-2H-chromen-2-one and Compound 9 (C5), 8-methoxy-3-(4-nitrobenzoyl)-6-propyl-2H-chromen-2-one 2-one. To determine the MIC, the broth microdilution technique was used. The products were evaluated for their potential to modulate the activity of antibiotics. Afterward, the plates were submitted to blue LED light for 20 min. When exposed to LED, C3 exhibited a decrease in MIC for SA ATCC and C5 for EC ATCC, with an average of 645.08 μg/mL for both cases. C2 and C4 exhibited synergism in a greater number of situations. However, C3 showed promising activity against S. aureus. C1 and C2 already acted better against E. coli, with the difference that C1 acted better against these bacteria when associated with LED. In general, the compounds studied here exhibited good antibacterial activity when associated with LED.

Scopoletin Induced Metabolomic Profile Disturbances in Zebrafish Embryos.

Scopoletin, a typical example of a coumarin compound, exists in several Artemisia species and other plant genera. However, the systemic metabolic effects induced by scopoletin remain unclear. In the present study, we evaluated the metabolic profiles in scopoletin-exposed zebrafish embryos using UHPLC-Q-Obitrap-HRMS combined with multivariate analysis. Compared with the control group, 33 metabolites in scopoletin group were significantly upregulated, while 27 metabolites were significantly downregulated. Importantly, scopoletin exposure affected metabolites mainly involved in phosphonate and phosphinate metabolism, vitamin B6 metabolism, histidine metabolism, sphingolipid metabolism, and folate biosynthesis. These results suggested that scopoletin exposure to zebrafish embryos exhibited marked metabolic disturbance. This study provides a perspective of metabolic impacts and the underlying mechanism associated with scopoletin exposure.

Multi-targeted molecular docking, pharmacokinetics, and drug-likeness evaluation of coumarin based compounds targeting proteins involved in development of COVID-19

COVID-19 is a progressing pandemic of coronavirus disease-2019, which had drowned the whole world in a deep sorrow sea. Uncountable deaths were extending the list of deaths every single day. The present research was aimed to study the multi-target interaction of coumarins against COVID-19 using molecular docking analysis. The structure of coumarin compounds was checked for ADME and Lipinski rule of five by using SwissADME, an online tool. SARS-CoV-2 proteins such as RdRp, PLpro, Mpro and spike protein were collected from the Protein Data Bank. The molecular docking study was performed in the PyRx tool, and the molecular interactions were visualised by Discovery Studio Visualizer. All the coumarin compounds used in the study were obeyed Lipinski’s rule of 5 without any violations. All the three designed derivatives of phenprocoumon, hymecromone, and psoralen were showed high binding affinity and prominent interactions with the drug target. The presence of –OH groups in the compound, His41, a catalytic dyad in Mpro, number of and the distance of hydrogen bond interactions with SARS-CoV-2 targets was accountable for the high binding attractions. The modified drug structures possess better binding efficacy towards at least three targets compared to their parent compounds. Further, molecular dynamic studies can be suggested to find the ligand–protein complex stability. The present study outcome reveals that the designed coumarins can be synthesised and examined as a potent inhibitory drug of SARS-CoV-2.

Colon cancer therapy with calcium phosphate nanoparticles loading bioactive compounds from Euphorbia lathyris: In vitro and in vivo assay

Amorphous calcium phosphate nanoparticles (ACP NPs) exhibit excellent biocompatibility and biodegradability properties. ACP NPs were functionalized with two coumarin compounds (esculetin and euphorbetin) extracted from Euphorbia lathyris seeds (BC-ACP NPs) showing high loading capacity (0.03% and 0.34% (w/w) for esculetin and euphorbetin, respectively) and adsorption efficiency (2.6% and 33.5%, respectively). BC-ACP NPs, no toxic to human blood cells, showed a more selective cytotoxicity against colorectal cancer (CRC) cells (T-84 cells) (IC50, 71.42 µg/ml) compared to non-tumor (CCD18) cells (IC50, 420.77 µg/ml). Both, the inhibition of carbonic anhydrase and autophagic cell death appeared to be involved in their action mechanism. Interestingly, in vivo treatment with BC-ACPs NPs using two different models of CRC induction showed a significant reduction in tumor volume (62%) and a significant decrease in the number and size of polyps. A poor development of tumor vasculature and invasion of normal tissue were also observed. Moreover, treatment increased the bacterial population of Akkermansia by restoring antioxidant systems in the colonic mucosa of mice. These results show a promising pathway to design innovative and more efficient therapies against CRC based on biomimetic calcium phosphate NPs loaded with natural products.

Intramolecular Coumarin-Dimer Containing Polyurethanes: Optical Tuning via Single- and Two-Photon Absorption Processes

The synthesis of an isomer-pure intramolecular coumarin dimer (ICD) in virtually unlimited amounts using a photo flow reactor is described. Based on this compound, polyurethanes with different ratios of hexamethylene diisocyanate and polytetrahydrofuran were synthesized and characterized. The reversibility of the photoinduced cycloaddition and cycloreversion reactions, one of the critical features, was compared for polymers prepared from closed ICDs and open ICDs. It was found that closed ICDs lead to a pre-orientation of the photoactive groups in the polymers, which results in a reduced loss in photoswitchable absorption. By varying the ICD content of the polymers, refractive indices ranging from 1.47 to 1.59 were achieved. With illumination, refractive index changes of Δn ≥ 0.02, as well as fluorescence changes, were achieved. Photoreactions induced by both single-photon absorption (SPA) and two-photon absorption (TPA) were studied. For the TPA-induced reaction, an autocatalytically sensitized mechanism was found. These novel polymers based on an intramolecular dimeric coumarin compound have great potential as optical materials with light-switchable properties, in particular, by TPA processes.

Osthole: An up-to-date review of its anticancer potential and mechanisms of action.

With its high incidence and mortality rates, cancer is one of the largest health problems worldwide. Investigating various cancer treatment options has been the focus of many domestic and international researchers, and significant progress has been made in the study of the anticancer effects of traditional Chinese medicines. Osthole, a coumarin compound extracted from Cnidium monnieri (L.) Cuss., has become a new research hotspot. There have been many reports on its anticancer effects, and recent studies have elucidated that its underlying mechanism of action mainly involves inhibiting cancer cell proliferation, inducing cancer cell apoptosis, inhibiting invasion and migration of cancer cells, inhibiting cancer angiogenesis, increasing sensitivity to chemotherapy drugs, and reversing multidrug resistance of cancer cells. This mini-review summarizes the research progress on the anticancer effects of osthole in recent years.

Penicillium growth inhibition, fruit decay reduction, and polymethoxyflavones and scoparone induction in satsuma mandarin irradiated with ultraviolet-A light-emitting diodes

In this study, we investigated the effects of ultraviolet (UV)-A light-emitting diodes (LEDs) on the inhibition of fungal spore germination, fruit decay, and accumulation of secondary metabolites, including phytoalexins, in satsuma mandarin. We examined the growth and development of blue (Penicillium italicum) and green (Penicillium digitatum) molds exposed to UV-A LED irradiation in vitro and found that UV-A LEDs with an emission peak of 375 nm and light intensity of 9.4 W m−2 significantly reduced the growth of both these fungi. In addition, wounded fruits inoculated with P. digitatum were irradiated with UV-A LEDs for 6 days, and we monitored the subsequent changes in green mold symptoms. The results indicated that low-intensity UV-A LED treatment significantly reduced green mold symptoms in fruit, being the effect most pronounced inhibitory on Penicillium sporulation. Furthermore, wounded fruits subjected to UV-A LED treatment were found to have significantly higher contents of the coumarin compound scoparone than fruits maintained in the dark. Contrastingly, scoparone was barely detectable in non-wounded fruits, irrespective of UV-A LED irradiation. The contents of nobiletin, tangeretin, and hesperidin were likewise found to be higher in wounded fruits treated with UV-A LED than in either non-wounded or non-irradiated fruits. These results indicate that treatment with low-intensity UV-A LED irradiation is sufficient to reduce mold symptom development, and accordingly could represent a promising safe approach to the control of post-harvest decay in citrus fruits. On the basis of our observations, we speculate that the biosynthesis of scoparone, polymethoxyflavones, and flavanones could be induced when the peel is injured and irradiated with UV-A contributing to a reduction in fruit decay.

Spectroscopic studies of 7,8-diacetoxy-4-methylcoumarin and 7,8-dipentynoyl-4-methylcoumarin binding with calreticulin.

Calreticulin (CRT) is a protein found mainly in the endoplasmic reticulum (ER) that maintains calcium levels and controls protein folding, but has recently been found at the cell surface, cytoplasm, and in the extracellular matrix. CRT participates in multiple physiological processes such as gene expression, the immune response, and cancer. Calreticulin has been shown to autoacetylate with the binding of preferred ligand 7,8-diacetoxy-4-methylcoumarin (DAMC). This project aims to develop a chemical biology approach to investigate importance of CRT acylating abilities on its nonendoplasmic reticulum functions by targeting the downstream substrates of CRT acetylation. Our goal was to use CRT to transfer a pentynoyl tag (using a novel ligand, DPeMC) to its substrates, which can then be used as a handle for protein identification. Molecular modelling using available data in the literature was used to approximate the binding interface between CRT and the acylation ligands. Molecular Operating Environment (MOE) software was used to perform sequence alignment, simulated annealing, positional refinement, and blind docking of acylated coumarins with the CRT model. Docking studies pointed to the P domain as the most thermodynamically and sterically favourable region for acylated coumarin binding with tryptophan residue 200 within the active site on CRT. Absorption and fluorescence spectra of all coumarin compounds in ethanol:PBS (1:9v/v) solution were investigated. Stern-Volmer quenching constant (KSV ), binding constant (K), and number of binding sites (n) of each coumarin compound with CRT was determined. Our studies demonstrated that acyl coumarin compounds bind to CRT using a dynamic quenching mechanism, bind to a single binding site on the P domain, and that the protein-ligand interaction is spontaneous and exothermic.