Synthesis of Naphthoquinone Derivatives: Menaquinones, Lipoquinones and Other Vitamin K Derivatives.

Menaquinones are a class of isoprenoid molecules that have important roles in human biology and bacterial electron transport, and multiple methods have been developed for their synthesis. These compounds consist of a methylnaphthoquinone (MK) unit and an isoprene side chain, such as found in vitamin K1 (phylloquinone), K2, and other lipoquinones. The most common naturally occurring menaquinones contain multiple isoprene units and are very hydrophobic, rendering it difficult to evaluate the biological activity of these compounds in aqueous assays. One way to overcome this challenge has been the application of truncated MK-derivatives for their moderate solubility in water. The synthesis of such derivatives has been dominated by Friedel-Crafts alkylation with BF3∙OEt2. This attractive method occurs over two steps from commercially available starting materials, but it generally produces low yields and a mixture of isomers. In this review, we summarize reported syntheses of both truncated and naturally occurring MK-derivatives that encompass five different synthetic strategies: Nucleophilic ring methods, metal-mediated reactions, electrophilic ring methods, pericyclic reactions, and homologation and side chain extensions. The advantages and disadvantages of each method are discussed, identifying methods with a focus on high yields, regioselectivity, and stereochemistry leading to a detailed overview of the reported chemistry available for preparation of these compounds.

Daylight-Induced Antibacterial and Antiviral Nanofibrous Membranes Containing Vitamin K Derivatives for Personal Protective Equipment.

During the development of antibacterial and antiviral materials for personal protective equipment (PPE), daylight active functional polymeric materials containing vitamin K compounds (VKs) and impacts of polymer structures to the functions were investigated. As examples, hydrophobic polyacrylonitrile (PAN) and hydrophilic poly(vinyl alcohol-co-ethylene) (PVA-co-PE) polymers were directly blended with three VK compounds and electrospun into VK-containing nanofibrous membranes (VNFMs). The prepared VNFMs exhibited robust photoactivity in generating reactive oxygen species (ROS) under both daylight (D65, 300-800 nm) and ultraviolet A (UVA, 365 nm) irradiation, resulting in high antimicrobial and antiviral efficiency (>99.9%) within a short exposure time (<90 min). Interestingly, the PVA-co-PE/VK3 VNFM showed higher ROS production rates and better biocidal functions than those of the PAN/VK3 VNFM under the same photoirradiation conditions, indicating that PVA-co-PE is a better matrix polymer material for these functions. Moreover, the prepared PVA-co-PE/VK3 VNFM maintains its powerful microbicidal function even after five times of repeated exposures to bacteria and viruses, showing the stability and reusability of the antimicrobial materials. The fabrication of photoinduced antimicrobial VNFMs may provide new insights into the development of non-toxic and reusable photoinduced antimicrobial materials that could be applied in personal protective equipment with improved biological protections.

Vitamin K Analogs Influence the Growth and Virulence Potential of Enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) causes serious foodborne disease worldwide. It produces the very potent Shiga toxin 2 (Stx2). The Stx2-encoding genes are located on a prophage, and production of the toxin is linked to the synthesis of Stx phages. There is, currently, no good treatment for EHEC infections, as antibiotics may trigger lytic cycle activation of the phages and increased Stx production. This study addresses how four analogs of vitamin K, phylloquinone (K1), menaquinone (K2), menadione (K3), and menadione sodium bisulfite (MSB), influence growth, Stx2-converting phage synthesis, and Stx2 production by the EHEC O157:H7 strain EDL933. Menadione and MSB conferred a concentration-dependent negative effect on bacterial growth, while phylloquinone or menaquinone had little and no effect on bacterial growth, respectively. All four vitamin K analogs affected Stx2 phage production negatively in uninduced cultures and in cultures induced with either hydrogen peroxide (H2O2), ciprofloxacin, or mitomycin C. Menadione and MSB reduced Stx2 production in cultures induced with either H2O2 or ciprofloxacin. MSB also had a negative effect on Stx2 production in two other EHEC isolates tested. Phylloquinone and menaquinone had, on the other hand, variable and concentration-dependent effects on Stx2 production. MSB, which conferred the strongest inhibitory effect on both Stx2 phage and Stx2 production, improved the growth of EHEC in the presence of H2O2 and ciprofloxacin, which could be explained by the reduced uptake of ciprofloxacin into the bacterial cell. Together, the data suggest that vitamin K analogs have a growth- and potential virulence-reducing effect on EHEC, which could be of therapeutic interest.IMPORTANCE Enterohemorrhagic E. coli (EHEC) can cause serious illness and deaths in humans by producing toxins that can severely damage our intestines and kidneys. There is currently no optimal treatment for EHEC infections, as antibiotics can worsen disease development. Consequently, the need for new treatment options is urgent. Environmental factors in our intestines can affect the virulence of EHEC and help our bodies fight EHEC infections. The ruminant intestine, the main reservoir for EHEC, contains high levels of vitamin K, but the levels are variable in humans. This study shows that vitamin K analogs can inhibit the growth of EHEC and/or production of its main virulence factor, the Shiga toxin. They may also inhibit the spreading of the Shiga toxin encoding bacteriophage. Our findings indicate that vitamin K analogs have the potential to suppress the development of serious disease caused by EHEC.

Vitamin K for kidney transplant organ recipients: investigating vessel stiffness (ViKTORIES): study rationale and protocol of a randomised controlled trial.

BACKGROUND: Renal transplant recipients (RTRs) exhibit increased vascular stiffness and calcification; these parameters are associated with increased cardiovascular risk. Activity of endogenous calcification inhibitors such as matrix gla protein (MGP) is dependent on vitamin K. RTRs commonly have subclinical vitamin K deficiency. The Vitamin K in kidney Transplant Organ Recipients: Investigating vEssel Stiffness (ViKTORIES) study assesses whether vitamin K supplementation reduces vascular stiffness and calcification in a diverse population of RTR. METHODS AND ANALYSIS: ViKTORIES (ISRCTN22012044) is a single-centre, phase II, parallel-group, randomised, double-blind, placebo-controlled trial of the effect of vitamin K supplementation in 90 prevalent RTR. Participants are eligible if they have a functioning renal transplant for >1 year. Those on warfarin, with atrial fibrillation, estimated glomerular filtration rate <15 mL/min/1.73 m2 or contraindications to MRI are excluded. Treatment is with vitamin K (menadiol diphosphate) 5 mg three times per week for 1 year or matching placebo. All participants have primary and secondary endpoint measures at 0 and 12 months. The primary endpoint is ascending aortic distensibility on cardiac MR imaging. Secondary endpoints include vascular calcification (coronary artery calcium score by CT), cardiac structure and function on MR, carotid-femoral pulse wave velocity, serum uncarboxylated MGP, transplant function, proteinuria and quality of life. The study is powered to detect 1.0×10-3 mm Hg-1 improvement in ascending aortic distensibility in the vitamin K group relative to placebo at 12 months. Analyses will be conducted as between-group differences at 12 months by intention to treat. DISCUSSION: This trial may identify a novel, inexpensive and low-risk treatment to improve surrogate markers of cardiovascular risk in RTR.

Discovery of the First Vitamin K Analogue as a Potential Treatment of Pharmacoresistant Seizures.

Despite the availability of more than 25 antiseizure drugs on the market, approximately 30% of patients with epilepsy still suffer from seizures. Thus, the epilepsy therapy market has a great need for a breakthrough drug that will aid pharmacoresistant patients. In our previous study, we discovered a vitamin K analogue, 2h, which displayed modest antiseizure activity in zebrafish and mouse seizure models. However, there are limitations to this compound due to its pharmacokinetic profile. In this study, we develop a new series of vitamin K analogues by modifying the structure of 2h. Among these, compound 3d shows full protection in a rodent pharmacoresistant seizure model with limited rotarod motor toxicity and favorable pharmacokinetic properties. Furthermore, the brain/plasma concentration ratio of 3d indicates its excellent permeability into the brain. The resulting data shows that 3d can be further developed as a potential antiseizure drug in the clinic.

New Aspects of Vitamin K Research with Synthetic Ligands: Transcriptional Activity via SXR and Neural Differentiation Activity.

Vitamin K is classified into three homologs depending on the side-chain structure, with 2-methyl-1,4-naphthoqumone as the basic skeleton. These homologs are vitamin K1 (phylloquinone: PK), derived from plants with a phythyl side chain; vitamin K2 (menaquinone-n: MK-n), derived from intestinal bacteria with an isoprene side chain; and vitamin K3 (menadione: MD), a synthetic product without a side chain. Vitamin K homologs have physiological effects, including in blood coagulation and in osteogenic activity via γ-glutamyl carboxylase and are used clinically. Recent studies have revealed that vitamin K homologs are converted to MK-4 by the UbiA prenyltransferase domain-containing protein 1 (UBIAD1) in vivo and accumulate in all tissues. Although vitamin K is considered to have important physiological effects, its precise activities and mechanisms largely remain unclear. Recent research on vitamin K has suggested various new roles, such as transcriptional activity as an agonist of steroid and xenobiotic nuclear receptor and differentiation-inducing activity in neural stem cells. In this review, we describe synthetic ligands based on vitamin K and exhibit that the strength of biological activity can be controlled by modification of the side chain part.

Menaquinone-4 Suppresses Lipopolysaccharide-Induced Inflammation in MG6 Mouse Microglia-Derived Cells by Inhibiting the NF-κB Signaling Pathway.

The overactivation of microglia is known to trigger inflammatory reactions in the central nervous system, which ultimately induce neuroinflammatory disorders including Alzheimer's disease. However, increasing evidence has shown that menaquinone-4 (MK-4), a subtype of vitamin K2, can attenuate inflammation in the peripheral system. Whereas it was also observed at high levels within the brain, its function in this organ has not been well characterized. Therefore, we investigated the effect of MK-4 on microglial activation and clarified the underlying mechanism. Mouse microglia-derived MG6 cells were exposed to lipopolysaccharide (LPS) either with or without MK-4 pretreatment. Cell responses with respect to inflammatory cytokines (Il-1ß, Tnf-α, and Il-6) were measured by qRT-PCR. We further analyzed the phosphorylation of TAK1, IKKα/ß, and p65 of the NF-κB subunit by Western blotting. We observed that in LPS-induced MG6 cells, MK-4 dose-dependently suppressed the upregulation of inflammatory cytokines at the mRNA level. It also significantly decreased the phosphorylation of p65, but did not affect that TAK1 and IKKα/ß. Furthermore, the nuclear translocation of NF-κB in LPS-induced MG6 cells was inhibited by MK-4. These results indicate that MK-4 attenuates microglial inflammation by inhibiting NF-κB signaling.

Combination of Double-Mediator System with Large-Scale Integration-Based Amperometric Devices for Detecting NAD(P)H:quinone Oxidoreductase 1 Activity of Cancer Cell Aggregates.

NAD(P)H:quinone oxidoreductase 1 (NQO1) is a key enzyme providing cytoprotection from quinone species. In addition, it is expressed at high levels in many human tumors, such as breast cancer. Therefore, it is considered to be a potential target in cancer treatment. In order to detect intracellular NQO1 activity in MCF-7 aggregates as a cancer model, we present, in this study, a double-mediator system combined with large-scale integration (LSI)-based amperometric devices. This LSI device contained 20 × 20 Pt working electrodes with a 250 µm pitch for electrochemical imaging. In the detection system, menadione (MD) and [Fe(CN)6]3- were used. Since MD can diffuse into cells due to its hydrophobicity, it is reduced into menadiol by intracellular NQO1. The menadiol diffuses out of the cells and reduces [Fe(CN)6]3- of a hydrophilic mediator into [Fe(CN)6]4-. The accumulated [Fe(CN)6]4- outside the cells is electrochemically detected at 0.5 V in the LSI device. Using this strategy, the intracellular NQO1 activity of MCF-7 aggregates was successfully detected. The effect of rotenone, which is an inhibitor for Complex I, on NQO1 activity was also investigated. In addition, NQO1 and respiration activities were simultaneously imaged using the detection system that was further combined with electrochemicolor imaging. Thus, the double-mediator system was proven to be useful for evaluating intracellular redox activity of cell aggregates.

Inhibition of Polymorphic Property of Naphthoquinonepigment Derived from Vitamin K.

Organicpigment, 2-Methyl-3-(4-fluorophenylthio)-1,4-naphthalenedione (2), that is a new Vitamin K derivative bearing a fluorine group, did not show any polymorphism disregard of three different solvents. For comparison, its naphthoquinone analogue with a bromine group (1) showed clearly polymorphism susceptible to these solvents.

Synthesis of novel vitamin K derivatives with alkylated phenyl groups introduced at the ω-terminal side chain and evaluation of their neural differentiation activities.

Vitamin K is an essential cofactor of γ-glutamylcarboxylase as related to blood coagulation and bone formation. Menaquinone-4, one of the vitamin K homologues, is biosynthesized in the body and has various biological activities such as being a ligand for steroid and xenobiotic receptors, protection of neuronal cells from oxidative stress, and so on. From this background, we focused on the role of menaquinone in the differentiation activity of progenitor cells into neuronal cells and we synthesized novel vitamin K derivatives with modification of the ω-terminal side chain. We report here new vitamin K analogues, which introduced an alkylated phenyl group at the ω-terminal side chain. These compounds exhibited potent differentiation activity as compared to control.

Synthesis of Novel Synthetic Vitamin K Analogues Prepared by Introduction of a Heteroatom and a Phenyl Group That Induce Highly Selective Neuronal Differentiation of Neuronal Progenitor Cells.

We synthesized novel vitamin K2 analogues that incorporated a heteroatom and an aromatic ring in the side chain and evaluated their effect on the selective differentiation of neuronal progenitor cells into neurons in vitro. The results showed that a menaquinone-2 analogue bearing a p-fluoroaniline had the most potent activity, which was more than twice as great as the control. In addition, the neuronal selectivity was more than 3 times greater than the control.

Synthesis of 2-methyl-1,4-naphthoquinones with higher gamma-glutamyl carboxylase activity than MK-4 both in vitro and in vivo.

Vitamin K is the collective term for compounds that share a 2-methyl-1,4-naphthoquinone ring, but differ in the side-chain at the 3-position. We synthesized novel 2-methyl-1,4-naphthoquinone derivatives with different side chain length at the 3-position. Derivatives with C-14 and C-16 tails showed the highest in vitro bioactivity resulting in 2.5 and 2-fold higher carboxylated osteocalcin synthesis in MG63 cells than menaquinone-4 (MK-4, form of vitamin K2). Longer side chain lengths resulted in lower bioactivity. The in vivo vitamin K activity of the C-14 tail derivative was further tested in WKY rats receiving a vitamin K-deficient diet that resulted in a 40% decrease of prothrombin activity. The C-14 tail derivative was able to counteract the effects on vitamin K deficiency induced by the diet and resulted in the complete restoration of prothrombin activity. Compared to naturally occurring forms of vitamin K, synthetic vitamin K derivatives may have higher bioactivity and different pharmacological characteristics that are more favorable for use as supplements or in clinical settings.

The anticonvulsant action of the galanin receptor agonist NAX-5055 involves modulation of both excitatory- and inhibitory neurotransmission.

The endogenous neuropeptide galanin is ubiquitously expressed throughout the mammalian brain. Through the galanin receptors GalR1-3, galanin has been demonstrated to modulate both glutamatergic and GABAergic neurotransmission, and this appears to be important in epilepsy and seizure activity. Accordingly, galanin analogues are likely to provide a new approach to seizure management. However, since peptides are generally poor candidates for therapeutic agents due to their poor metabolic stability and low brain bioavailability, a search for alternative strategies for the development of galanin-based anti-convulsant drugs was prompted. Based on this, a rationally designed GalR1 preferring galanin analogue, NAX-5055, was synthesized. This compound demonstrates anti-convulsant actions in several animal models of epilepsy. However, the alterations at the cellular level leading to this anti-convulsant action of NAX-5055 are not known. Here we investigate the action of NAX-5055 at the cellular level by determining its effects on excitatory and inhibitory neurotransmission, i.e. vesicular release of glutamate and GABA, respectively, in cerebellar, neocortical and hippocampal preparations. In addition, its effects on cell viability and neurotransmitter transporter capacity were examined to evaluate potential cell toxicity mediated by NAX-5055. It was found that vesicular release of glutamate was reduced concentration-dependently by NAX-5055 in the range from 0.1 to 1000 nM. Moreover, exposure to 1 µM NAX-5055 led to a reduction in the extracellular level of glutamate and an elevation of the extracellular level of GABA. Altogether these findings may at least partly explain the anti-convulsant effect of NAX-5055 observed in vivo.

Synthesis of Vitamin K and Related Naphthoquinones via Demethoxycarbonylative Annulations and a Retro-Wittig Rearrangement.

Anionic annulations of 3-nucleofugal phthalides with α-alkyl(aryl)acrylates involving a demethoxycarbonylation provide a succinct synthesis of vitamin K and related naphthoquinones. Also reported is a new cascade reaction stemming from a Cope-retro-Wittig rearrangement. This cascade leads to direct formation of 1-hydroxy-4-prenyloxynaphthalene-2-carboxylates from the corresponding α-prenyl acrylate acceptors.

A novel salting-out assisted extraction coupled with HPLC- fluorescence detection for trace determination of vitamin K homologues in human plasma.

Recently, new physiological roles of vitamin K homologues have been established in the treatment of rheumatoid arthritis, osteoporosis, hepatocellular carcinoma and leukemia. However, relatively high plasma protein binding, low plasma concentrations and occurrences of interfering lipids make accurate determination of vitamin K homologues a challenging task. Therefore, a sensitive and reliable salting-out assisted liquid/liquid extraction (SALLE) method coupled with HPLC-Fluorescence detection was designed for efficient extraction and quantification of trace levels of vitamin K homologues in human plasma. The investigated vitamin K homologues were phylloquinone (PK, vitamin K1), menaquinone-4 (MK-4) and menaquinone-7 (MK-7). The method employed a new efficient fluorescence derivatization reaction using ethanolic solution of stannous chloride in acidic solution to generate highly fluorescent naphthohydroquinone derivatives. Correlation coefficients were more than 0.998 in the concentration ranges of 0.3-100 ng mL(-1) with detection limits of 0.1-0.17 ng mL(-1) in human plasma. The developed HPLC-FL system was successfully applied for sensitive determination of vitamin K homologues in plasma of healthy volunteers. The developed method may provide a valuable tool in the pharmacoinformatic studies concerning the roles of vitamin K homologues.

The inhibitory effect of vitamin K on RANKL-induced osteoclast differentiation and bone resorption.

To further understand the correlation between vitamin K and bone metabolism, the effects of vitamins K1, menaquinone-4 (MK-4), and menaquinone-7 (MK-7) on RANKL-induced osteoclast differentiation and bone resorption were comparatively investigated. Vitamin K2 groups (MK-4 and MK-7) were found to significantly inhibit RANKL-medicated osteoclast cell formation of bone marrow macrophages (BMMs) in a dose-dependent manner, without any evidence of cytotoxicity. The mRNA expression of specific osteoclast differentiation markers, such as c-Fos, NFATc1, OSCAR, and TRAP, as well as NFATc1 protein expression and TRAP activity in RANKL-treated BMMs were inhibited by vitamin K2, although MK-4 exhibited a significantly greater efficiency compared to MK-7. In contrast, the same dose of vitamin K1 had no inhibitory effect on RANKL-induced osteoclast cell formation, but increased the expression of major osteoclastogenic genes. Interestingly, vitamins K1, MK-4 and MK-7 all strongly inhibited osteoclastic bone resorption (p < 0.01) in a dose dependent manner. These results suggest that vitamins K1, MK-4 and MK-7 have anti-osteoporotic properties, while their regulation effects on osteoclastogenesis are somewhat different.

Multiple signals modulate the activity of the complex sensor kinase TodS.

The reason for the existence of complex sensor kinases is little understood but thought to lie in the capacity to respond to multiple signals. The complex, seven-domain sensor kinase TodS controls in concert with the TodT response regulator the expression of the toluene dioxygenase pathway in Pseudomonas putida F1 and DOT-T1E. We have previously shown that some aromatic hydrocarbons stimulate TodS activity whereas others behave as antagonists. We show here that TodS responds in addition to the oxidative agent menadione. Menadione but no other oxidative agent tested inhibited TodS activity in vitro and reduced PtodX expression in vivo. The menadione signal is incorporated by a cysteine-dependent mechanism. The mutation of the sole conserved cysteine of TodS (C320) rendered the protein insensitive to menadione. We evaluated the mutual opposing effects of toluene and menadione on TodS autophosphorylation. In the presence of toluene, menadione reduced TodS activity whereas toluene did not stimulate activity in the presence of menadione. It was shown by others that menadione increases expression of glucose metabolism genes. The opposing effects of menadione on glucose and toluene metabolism may be partially responsible for the interwoven regulation of both catabolic pathways. This work provides mechanistic detail on how complex sensor kinases integrate different types of signal molecules.

Vitamin K catabolite inhibition of ovariectomy-induced bone loss: structure-activity relationship considerations.

SCOPE: The potential benefit of vitamin K as a therapeutic in osteoporosis is controversial and the vitamin K regimen being used clinically (45 mg/day) employs doses that are many times higher than required to ensure maximal gamma-carboxylation of the vitamin K-dependent bone proteins. We therefore tested the hypothesis that vitamin K catabolites, 5-carbon (CAN5C) and 7-carbon carboxylic acid (CAN7C) aliphatic side-chain derivatives of the naphthoquinone moiety exert an osteotrophic role consistent with the treatment of osteoporosis. METHODS AND RESULTS: Osteoblast-like MG63 cell cultures were challenged with lipopolysaccharide and the levels of interleukin-6, an osteoclastogenic cytokine, measured with and without catabolites; low concentrations of CAN7C significantly inhibited interleukin-6 release, but CAN5C did not. In models of bone loss induced by ovariectomy or sciatic neurectomy in C57BL/6 mice, we found that the rarer CAN7C catabolite markedly restricted ovariectomy-induced bone loss and possibly limited sciatic neurectomy-induced bone loss. CAN7C activity depends on a free carboxylic acid and its particular side-chain structure. CONCLUSION: These in vivo data indicate for the first time that the clinical utility of vitamin K for osteoporosis may reside in an unusual catabolite.

Rapid, high performance method for the determination of vitamin K(1), menaquinone-4 and vitamin K(1) 2,3-epoxide in human serum and plasma using liquid chromatography-hybrid quadrupole linear ion trap mass spectrometry.

Unlike the other fat-soluble vitamins, vitamin K circulates in the human bloodstream at very low levels because of a low intake in the diet. Mammals have developed an efficient recycling system, known as vitamin K-epoxide cycle, which involve quinone, hydroquinone and epoxide forms of the vitamin. Phylloquinone (K(1)) is the main homologue, while menaquinone-4 (MK-4) is both a member of the vitamin K(2) family and metabolite of K(1) in extra-hepatic tissues. Notwithstanding the recent advances, many aspects of the complex vitamin K physiology still remain to be investigated. Therefore, there is a critical need to develop more reliable analytical methods for determining the vitamin K and its metabolites in biological fluids and tissues. Nevertheless, relatively low concentrations, unavailability of some authentic standards and occurrence of interfering lipids make this a challenging task. The method proposed in the present paper can directly and accurately estimate K(1), K(1) 2,3-epoxide (K(1)O), and MK-4 in human serum and plasma at concentrations in the ng/L-µg/L range, using labelled internal standards and a quadrupole linear ion trap instrument operated in multiple reaction monitoring (MRM) mode. High sensitivity was achieved by removing signal "endogenous suppressors" and making the composition of the non-aqueous mobile phase suitable to support the positive atmospheric pressure chemical ionization of the analytes. An excellent selectivity resulted from the combination of some factors: the MRM acquisition, the adoption of an identification point system, an extraction optimized to remove most of the lipids and a tandem-C18 column-system necessary to separate isobaric interferences from analytes. The method was validated according to the Food and Drug Administration (FDA) guidelines and its accuracy was assessed by analysing 9 samples from the Vitamin K External Quality Assessment Scheme (KEQAS). Its feasibility in evaluating vitamin K status in human serum was also tested by monitoring a group of six healthy subjects and a group of six patients under oral anticoagulant therapy (OAT). Warfarinised patients did not show deficiency of K1 but levels comparable with those of healthy people and an accumulation of K1O up to 3.760µg/L. MK-4 was not detected in either of the two groups.