Structure-based screening and biological validation of the anti-thrombotic drug-dicoumarol as a novel and potent PPARγ-modulating ligand.

PPARγ full agonists, thiazolidinediones (TZDs), have been known as a class of most effective drugs for the treatment of type 2 diabetes mellitus (T2DM). However, recently their therapeutic benefits have been compromised by several undesirable side effects. In this study, a host-based repurposing strategy and in combination with comprehensive biological evaluations were synergistically employed to seek for potent PPARγ ligands, which led to the identification of an anti-thrombotic drug, dicoumarol (Dic), as the novel and safer selectively PPARγ modulator (SPPARγM) with advantages over current TZD drugs. The results in vitro showed that Dic had a potent binding affinity and weakly agonistic activity for PPARγ and its downstream key genes. Moreover, in diabetic model, it significantly reduced blood glucose without leading to the weight gain of both body and main organ tissues. Further mechanistic investigations revealed that Dic possessed such desired pharmacological properties mainly through effectively inhibiting the phosphorylation of PPARγ-Ser273 and selectively regulating the expressions of insulin-sensitive and resistance genes. Finally, the docking studies on the analysis of the potent binding mode of Dic with PPARγ revealed a remarkable difference on interaction region compared with other developed PPARγ agonists, which not only gave a proof of concept for the abovementioned mechanism but also provided the molecular basis for the discrimination of Dic from other PPARγ ligands, especially TZD drugs. Taken together, our findings suggested that Dic could serve as a new and promising candidate with good therapeutic index for treating T2DM, especially for those T2DM patients with thrombosis.

Isolation and In Silico SARS-CoV-2 Main Protease Inhibition Potential of Jusan Coumarin, a New Dicoumarin from Artemisia glauca.

A new dicoumarin, jusan coumarin, (1), has been isolated from Artemisia glauca aerial parts. The chemical structure of jusan coumarin was estimated, by 1D, 2D NMR as well as HR-Ms spectroscopic methods, to be 7-hydroxy-6-methoxy-3-[(2-oxo-2H-chromen-6-yl)oxy]-2H-chromen-2-one. As the first time to be introduced in nature, its potential against SARS-CoV-2 has been estimated using various in silico methods. Molecular similarity and fingerprints experiments have been utilized for 1 against nine co-crystallized ligands of COVID-19 vital proteins. The results declared a great similarity between Jusan Coumarin and X77, the ligand of COVID-19 main protease (PDB ID: 6W63), Mpro. To authenticate the obtained outputs, a DFT experiment was achieved to confirm the similarity of X77 and 1. Consequently, 1 was docked against Mpro. The results clarified that 1 bonded in a correct way inside Mpro active site, with a binding energy of -18.45 kcal/mol. Furthermore, the ADMET and toxicity profiles of 1 were evaluated and showed the safety of 1 and its likeness to be a drug. Finally, to confirm the binding and understand the thermodynamic characters between 1 and Mpro, several molecular dynamics (MD) simulations studies have been administered. Additionally, the known coumarin derivative, 7-isopentenyloxycoumarin (2), has been isolated as well as ß-sitosterol (3).

A pharmacological review of dicoumarol: An old natural anticoagulant agent.

Dicoumarol is an oral anticoagulant agent prescribed in clinical for decades. It is a natural hydroxycoumarin discovered from the spoilage of Melilotus officinalis (L.) Pall and is originally discovered as a rodenticide. Due to its structural similarity to that of vitamin K, it significantly inhibits vitamin K epoxide reductase and acts as a vitamin K antagonist. Dicoumarol is mainly used as an anticoagulant to prevent thrombogenesis and to cure vascular thrombosis. Other biological activities besides anticoagulants such as anticancer, antimicrobial, antiviral, etc., have also been documented. The side effects of dicoumarol raise safety concerns for clinical application. In this review, the physicochemical property, the pharmacological activities, the side effects, and the pharmacokinetics of dicoumarol were summarized, aiming to provide a whole picture of the "old" anticoagulant.

Characterization of the Particle Size and Polydispersity of Dicumarol Using Solid-State NMR Spectroscopy.

A variety of particle sizes of a model compound, dicumarol, were prepared and characterized in order to investigate the correlation between particle size and solid-state NMR (SSNMR) proton spin-lattice relaxation (1H T1) times. Conventional laser diffraction and scanning electron microscopy were used as particle size measurement techniques and showed crystalline dicumarol samples with sizes ranging from tens of micrometers to a few micrometers. Dicumarol samples were prepared using both bottom-up and top-down particle size control approaches, via antisolvent microprecipitation and cryogrinding. It was observed that smaller particles of dicumarol generally had shorter 1H T1 times than larger ones. Additionally, cryomilled particles had the shortest 1H T1 times encountered (8 s). SSNMR 1H T1 times of all the samples were measured and showed as-received dicumarol to have a T1 of 1500 s, whereas the 1H T1 times of the precipitated samples ranged from 20 to 80 s, with no apparent change in the physical form of dicumarol. Physical mixtures of different sized particles were also analyzed to determine the effect of sample inhomogeneity on 1H T1 values. Mixtures of cryoground and as-received dicumarol were clearly inhomogeneous as they did not fit well to a one-component relaxation model, but could be fit much better to a two-component model with both fast-and slow-relaxing regimes. Results indicate that samples of crystalline dicumarol containing two significantly different particle size populations could be deconvoluted solely based on their differences in 1H T1 times. Relative populations of each particle size regime could also be approximated using two-component fitting models. Using NMR theory on spin diffusion as a reference, and taking into account the presence of crystal defects, a model for the correlation between the particle size of dicumarol and its 1H T1 time was proposed.

Identification of novel ROS inducer by merging the fragments of piperlongumine and dicoumarol.

A series of novel ROS inducers were designed by merging the fragments of piperlongumine and dicoumarol. Most of these derivatives showed potent in vitro activity against three cancer cell lines and good selectivity towards normal lung cells. The most potent and selective compound 3e was proven to exhibit obvious ROS elevation and excellent in vivo antitumor activity with suppressed tumor growth by 48.46% at the dose of 5mg/kg. Supported by these investigation, these findings encourage further investigation around this interesting antitumor chemotype.

Synthesis, antibacterial activities, and theoretical studies of dicoumarols.

Four dicoumarols (DC, 2-PyDC, 3-PyDC and 4-PyDC) were synthesized and characterized via IR, (1)H NMR, HRMS, and single crystal X-ray crystallography. Two classical intramolecular O-H···O hydrogen bonds (HBs) stabilized their structures. The total HB energies in DC, 2-PyDC, 3-PyDC and 4-PyDC were calculated with the density functional theory (DFT) [B3LYP/6-31G*] method. The in vitro antibacterial activity of DC, 2-PyDC, 3-PyDC and 4-PyDC against Staphylococcus aureus (S. aureus ATCC 29213), methicillin-resistant S. aureus (MRSA XJ 75302), vancomycin-intermediate S. aureus (Mu50 ATCC 700699), and USA 300 (Los Angeles County clone, LAC) was evaluated by observing the minimum inhibitory concentration and time-kill curves. The results showed that among all the compounds, 2-PyDC exhibited the most potent antibacterial activity.

O-tert-butyldiphenylsilyl coumarin and dicoumarol: a case toward selective sensing of F- ions in organic and aqueous environments.

O-tert-Butyldiphenylsilyl coumarin 1 and 2 dicoumarol have been synthesized and their anion binding properties have been examined in organic and aqueous organic solvents. Compound 1 senses F(-) selectively over the other anions examined in CHCl3 by exhibiting a greater increase in emission. In contrast, compound 2 shows similar selectivity in CHCl3 giving ratiometric change in emission as well as color. In addition, both 1 and 2 are capable of detecting F(-) in water ensuing the cleavage of Si-O bonds. They also show cell permeability and demonstrate their abilities to detect F(-) in a living system.

Structure-activity relationships of 3,3'-phenylmethylene-bis-4-hydroxycoumarins: selective and potent inhibitors of gram-positive bacteria.

Dicoumarols and coumarin derivatives have shown a variety of pharmaceutical activities and have been found to be potent inhibitor for the NAD(P)H-dependent flavoproteins. In this report, dicoumarol and its derivatives containing the substituted benzene ring at the methylenebis position were synthesized and evaluated for their antibacterial activity against gram-positive bacteria: Staphylococcus aureus and Bacillus subtilis, and gram-negative bacteria: Escherichia coli and Klebsiella sp. The results showed that the synthesized dicoumarols affect cell growth but are selective against gram-positive over gram-negative bacterial cells. However, for most derivatives, the substitution of steric bulky benzene group on the methylenebis position appears to decrease in the efficacy of antibacterial effect. This finding is roughly described by the predicted poorer docked structure of the derivatives to a homology model of S. aureus flavoprotein. 3D-QSAR study highlighted structural features around the substituted benzene ring of dicoumarols as the antibacterial activity. CoMFA and CoMSIA contour maps support the idea that steric repulsion at the para position could diminish the antibacterial activity. The results of this study provide a better understanding of the molecular basis for the antibacterial activity of dicoumarols.

Synthesis and structure-activity relationships of novel amino/nitro substituted 3-arylcoumarins as antibacterial agents.

A new series of amino/nitro-substituted 3-arylcoumarins were synthesized and their antibacterial activity against clinical isolates of Staphylococcus aureus (Gram-positive) and Escherichia coli (Gram-negative) was evaluated. Some of these molecules exhibited antibacterial activity against S. aureus comparable to the standards used (oxolinic acid and ampicillin). The preliminary structure-activity relationship (SAR) study showed that the antibacterial activity against S. aureus depends on the position of the 3-arylcoumarin substitution pattern. With the aim of finding the structural features for the antibacterial activity and selectivity, in the present manuscript different positions of nitro, methyl, methoxy, amino and bromo substituents on the 3-arylcoumarin scaffold were reported.

A far-red fluorescent probe for sensing laccase in fungi and its application in developing an effective biocatalyst for the biosynthesis of antituberculous dicoumarin.

A far-red fluorescent probe has been developed for sensing fungal laccase. The probe was used to determine that Rhizopus oryzae had a high level endogenous laccase amongst 24 fungal strains. The Rhizopus oryzae was then used as a biocatalyst for the preparation of dicoumarin resulting in significant inhibition of Mycobacterium tuberculosis H37Ra.

NAD(P)H quinone oxidoreductase (NQO1): an enzyme which needs just enough mobility, in just the right places.

NAD(P)H quinone oxidoreductase 1 (NQO1) catalyses the two electron reduction of quinones and a wide range of other organic compounds. Its physiological role is believed to be partly the reduction of free radical load in cells and the detoxification of xenobiotics. It also has non-enzymatic functions stabilising a number of cellular regulators including p53. Functionally, NQO1 is a homodimer with two active sites formed from residues from both polypeptide chains. Catalysis proceeds via a substituted enzyme mechanism involving a tightly bound FAD cofactor. Dicoumarol and some structurally related compounds act as competitive inhibitors of NQO1. There is some evidence for negative cooperativity in quinine oxidoreductases which is most likely to be mediated at least in part by alterations to the mobility of the protein. Human NQO1 is implicated in cancer. It is often over-expressed in cancer cells and as such is considered as a possible drug target. Interestingly, a common polymorphic form of human NQO1, p.P187S, is associated with an increased risk of several forms of cancer. This variant has much lower activity than the wild-type, primarily due to its substantially reduced affinity for FAD which results from lower stability. This lower stability results from inappropriate mobility of key parts of the protein. Thus, NQO1 relies on correct mobility for normal function, but inappropriate mobility results in dysfunction and may cause disease.

Cancer-associated variants of human NQO1: impacts on inhibitor binding and cooperativity.

Human NAD(P)H quinone oxidoreductase (DT-diaphorase, NQO1) exhibits negative cooperativity towards its potent inhibitor, dicoumarol. Here, we addressed the hypothesis that the effects of the two cancer-associated polymorphisms (p.R139W and p.P187S) may be partly mediated by their effects on inhibitor binding and negative cooperativity. Dicoumarol stabilized both variants and bound with much higher affinity for p.R139W than p.P187S. Both variants exhibited negative cooperativity towards dicoumarol; in both cases, the Hill coefficient (h) was approximately 0.5 and similar to that observed with the wild-type protein. NQO1 was also inhibited by resveratrol and by nicotinamide. Inhibition of NQO1 by resveratrol was approximately 10,000-fold less strong than that observed with the structurally similar enzyme, NRH quinine oxidoreductase 2 (NQO2). The enzyme exhibited non-cooperative behaviour towards nicotinamide, whereas resveratrol induced modest negative cooperativity (h = 0.85). Nicotinamide stabilized wild-type NQO1 and p.R139W towards thermal denaturation but had no detectable effect on p.P187S. Resveratrol destabilized the wild-type enzyme and both cancer-associated variants. Our data suggest that neither polymorphism exerts its effect by changing the enzyme's ability to exhibit negative cooperativity towards inhibitors. However, it does demonstrate that resveratrol can inhibit NQO1 in addition to this compound's well-documented effects on NQO2. The implications of these findings for molecular pathology are discussed.

Atom- and bond-based 2D TOMOCOMD-CARDD approach and ligand-based virtual screening for the drug discovery of new tyrosinase inhibitors.

Two-dimensional atom- and bond-based TOMOCOMD-CARDD descriptors and linear discriminant analysis (LDA) are used in this report to perform a quantitative structure-activity relationship (QSAR) study of tyrosinase-inhibitory activity. A database of inhibitors of the enzyme is collected for this study, within 246 highly dissimilar molecules presenting antityrosinase activity. In total, 7 discriminant functions are obtained by using the whole set of atom- and bond-based 2D indices. All the LDA-based QSAR models show accuracies above 90% in the training set and values of the Matthews correlation coefficient (C) varying from 0.85 to 0.90. The external validation set shows globally good classifications between 89% and 91% and C values ranging from 0.75 to 0.81. Finally, QSAR models are used in the selection/identification of the 20 new dicoumarins subset to search for tyrosinase inhibitory activity. Theoretical and experimental results show good correspondence between one another. It is important to remark that most compounds in this series exhibit a more potent inhibitory activity against the mushroom tyrosinase enzyme than the reference compound, Kojic acid (IC(50) = 16.67 muM), resulting in a novel nucleus base (lead) with antityrosinase activity, and this could serve as a starting point for the drug discovery of novel tyrosinase inhibitor lead compounds. ( Journal of Biomolecular Screening 2008:1014-1024).

Synthesis, structure, antimicrobial and antioxidant investigations of dicoumarol and related compounds.

Different substituted 3,3'-arylidenebis-4-hydroxycoumarins (1-7) and tetrakis-4-hydroxycoumarin derivative 8 are the final products when 4-hydroxycoumarin and aromatic aldehydes containing different groups in ortho, meta or para positions condense in boiling ethanol or acetic acid. Upon heating 3,3'-arylidenebis-4-hydroxycoumarins, and tetrakis-4-hydroxycoumarin derivative in anhydride acetic acid, the epoxydicoumarins (9-16) were formed. From a study of nuclear magnetic resonance and infrared spectra, intramolecularly hydrogen-bonded structures are proposed for the dicoumarols (1-8). A possible relationship between such hydrogen-bonded structures and the antimicrobial and the antioxidant activities of compounds 1-8 is suggested.

Coumarin-based inhibitors of human NAD(P)H:quinone oxidoreductase-1. Identification, structure-activity, off-target effects and in vitro human pancreatic cancer toxicity.

The enzyme human NAD(P)H quinone oxidoreductase-1 (NQO1), which is overexpressed in several types of tumor cell, is considered a design target for cancer therapeutics. We identify new coumarin-based competitive inhibitors of NQO1, one of which is nanomolar. Using computational docking and molecular dynamics, we obtain insights into the structural basis of inhibition. Selected inhibitors were then assessed for off-target effects associated with dicoumarol and were found to have differing effects on superoxide formation and mitochondrial respiration. A comparison of NQO1 inhibition and off-target effects for dicoumarol and its derivatives suggests that the ability of dicoumarol to kill cancer cells is independent of NQO1 inhibition, that cellular superoxide production by dicoumarol does not seem linked to NQO1 inhibition but may be related to mitochondrial decoupling, and that superoxide does not appear to be a major determinant of cytotoxicity. Implications are discussed for NQO1 inhibition as an anticancer drug design target and superoxide generation as the dicoumarol-mediated mechanism of cytotoxicity.

Syntheses, characterization and fluorescent properties of two triethylene-glycol dicoumarin-3-carboxylates.

Two novel triethylene-glycol dicoumarin-3-carboxylates were synthesized and characterized by element analysis, (1)H NMR, FT-IR and UV-vis absorption spectra. The photoluminescent behaviors of triethylene-glycol dibenzo[5,6]coumarin-3-carboxylate doped in PMMA were discussed. These compounds exhibit strong blue emission under ultraviolet light excitation. Compared to coumarin-3-triethylene glycol diester, the absorption and emission spectra of benzo[5,6]coumarin-3-triethylene glycol diester was bathochromically shifted due to the larger conjugation of benzene moiety. These derivatives have potential possible to explore organic electroluminescent materials.

Dicoumarol: A Drug which Hits at Least Two Very Different Targets in Vitamin K Metabolism.

Dicoumarol, a symmetrical biscoumarin can be considered as the "parent" of the widely used anticoagulant drug, warfarin. The discovery of dicoumarol's bioactive properties resulted from an investigation into a mysterious cattle disease in the 1940s. It was then developed as a pharmaceutical, but was superseded in the 1950s by warfarin. Both dicoumarol and warfarin antagonise the blood clotting process through inhibition of vitamin K epoxide reductase (VKOR). This blocks the recycling of vitamin K and prevents the γ-carboxylation of glutamate residues in clotting factors. VKOR is an integral membrane protein and our understanding of the molecular mechanism of action of dicoumarol and warfarin is hampered by the lack of a three dimensional structure. There is consequent controversy about the membrane topology of VKOR, the location of the binding site for coumarin inhibitors and the mechanism of inhibition by these compounds. Dicoumarol (and warfarin) also inhibit a second enzyme, NAD(P)H quinone oxidoreductase 1 (NQO1). This soluble, cytoplasmic enzyme may also play a minor role in the recycling of vitamin K. However, its main cellular roles as an enzyme appear to be detoxification and the prevention of the build-up of reactive oxygen species. NQO1 is well characterised biochemically and structurally. Consequently, structure-based drug design has identified NQO1 inhibitors which have potential for the development of anti-cancer drugs. Many of these compounds are structurally related to dicoumarol and some have reduced "off target" effects. Therefore, it is possible that dicoumarol will become the "parent" of a second group of drugs.

Development of pyridine dicoumarols as potent anti HIV-1 leads, targeting HIV-1 associated topoisomeraseIIß kinase.

AIM: A structural study of a series of pyridine dicoumarol derivatives with potential activity against a novel Topoisomerase IIß kinase which was identified in the HIV-1 viral lysate, compounds were designed and synthesized based on a 3D-QSAR study. MATERIALS & METHODS: Based on QSAR model we have designed and synthesized a series of pyridine dicoumarol derivatives and characterized by spectral studies, all the molecules are biologically evaluated by kinase assay, cytotoxicity assay, ELISA and PCR method. RESULT: We demonstrated the achievement of water soluble disodium pyridine dicoumarate derivatives showing high anti-HIV-1 activity (IC50 <25 nM) which provides a crucial point for further development of pyridine dicoumarol series as HIV-1-associated topoisomerase IIß kinase inhibitors for clinical application against AIDS. CONCLUSION: A new class of anti-HIV-1 lead compounds have been designed and tested. Further studies would result in development of  novel and potential drugs.

Studies on the nitroreductase prodrug-activating system. Crystal structures of complexes with the inhibitor dicoumarol and dinitrobenzamide prodrugs and of the enzyme active form.

The E. coli nitroreductase enzyme (NTR) has been widely used in suicide gene therapy (GDEPT and ADEPT) applications as a activating enzyme for nitroaromatic prodrugs of the dinitrobenzamide class. NTR has been previously shown to be a homodimeric enzyme with two active sites. We present here the crystal structures of the reduced form of NTR and its complexes with the inhibitor dicoumarol and three dinitrobenzamide prodrugs. Comparison of the structures of the oxidized and reduced forms of the native enzyme shows that the principal structural changes occur in the FMN cofactor and indicate that the enzyme itself is a relatively rigid structure that primarily provides a rigid structural framework on which hydride transfer occurs. The aziridinyldinitrobenzamide prodrug CB 1954 binds in nonidentical ways in both of the two active sites of the homodimeric enzyme, employing both hydrophobic and (in active site B) a direct H-bond contact to the side chain of Lys14. In active site A the 2-nitro group stacks above the FMN, and in active site B the 4-nitro group does, explaining why reduction of either nitro group is observed. In contrast, the larger mustard group of the dinitrobenzamide mustard compound SN 23862 forces the prodrug to bind at both active sites with only the 2-nitro group able to participate in hydride transfer from the FMN, explaining why only the 2-hydroxylamine reduction product is observed. In each site, the nitro groups of the prodrug make direct H-bond contacts with the enzyme; in active Site A the 2-nitro to Ser40 and the 4-nitro to Asn71, while in active Site B the 2-nitro contacts the main chain nitrogen of Thr41 and the 4-nitro group the Lys14 side chain. The related amide-substituted mustard SN 27217 binds in a broadly similar fashion, but with the larger amide group substituent able to reach and contact the side chain of Arg107, further restricting the prodrug conformations in the binding site. The inhibitor dicoumarol appears to bind primarily by pi-stacking interactions and hydrophobic contacts, with no conformational changes in the enzyme. One of the hydroxycoumarin subunits stacks above the plane of the FMN via pi-overlap with the isoalloxazine ring, penetrating deep into the groove, with the other less well-defined. These studies suggest guidelines for further prodrug design. Steric bulk (e.g., mustard rather than aziridine) on the ring can limit the possible binding orientations, and the reducible nitro group must be located para to the mustard. Substitution on the carboxamide side chain still allows the prodrugs to bind, but also limits their orientation in the binding site. Finally, modulating substrate specificity by alteration of the structure of the enzyme rather than the prodrug might usefully focus on modifying the Phe124 residue and those surrounding it.

Further characterization of reversal of signs of induced cotton effects of dicumarol derivatives-alpha 1-acid glycoprotein systems by protriptyline.

The interaction of dicumarol derivatives and protriptyline with respect to the binding to alpha 1-acid glycoprotein (AGP) has been investigated by circular dichroism (CD), equilibrium dialysis and ultrafiltration. Investigation of the induced CD spectra of dicumarol derivatives bound to AGP indicated that the conformations of these compounds were different when bound to AGP. Though all the dicumarol derivatives, protriptyline and AGP formed a ternary complex, interaction modes were different, depending upon the substituent groups at position 3 of the dicumarol molecule. On the basis of the protriptyline effect on the CD spectra of all dicumarol derivatives bound to AGP, the compounds were classified in the following way: (1) Dicumarol, ethylidenebis 4-hydroxycoumarin and propylidenebis 4-hydroxycoumarin caused reversal of the sign of ellipticity. This interaction was explained by cooperative binding. (2) Butylidenebis 4-hydroxycoumarin and pentylidenebis 4-hydroxycoumarin generated new band and disappeared ellipticity of the original Cotton effect. This interaction was also explained by the cooperative binding mode. (3) Ethylbiscoumacetate which generated the CD band similar to that of dicumarol in the absence of protriptyline, reversed the sign of the CD spectrum only at 325 nm. The interaction was anticooperative in nature. (4) Benzylidenebis 4-hydroxycoumarin represented type four which had no change in the CD spectrum by the addition of protriptyline. This interaction was explained by the two-state model accompanying the conformational change of AGP. These results suggested that all compounds, except for benzylidenebis 4-hydroxycoumarin, induced negative Cotton effects at 325 nm by taking the same asymmetrical perturbation by the addition of protriptyline and the interaction was carried out according to model 2. An attempt to study the interaction mechanism of two or more drugs with regard to the binding to protein using these models is thought to help in understanding drug-protein interactions.