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 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.

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.

Synthesis of new vitamin K analogues as steroid and xenobiotic receptor (SXR) agonists: insights into the biological role of the side chain part of vitamin K.

Vitamin K(2) has been demonstrated to induce gene expression related to bone formation through a nuclear steroid and xenobiotic receptor (SXR). We synthesized new vitamin K analogues with the same isoprene side chains symmetrically introduced at the 2 and 3 positions of 1,4-naphthoquinone and evaluated the transcriptional activity of the target gene. The transcriptional activity was related to the length of the side chain which allowed optimal interaction with ligand-binding domain of SXR.

Synthesis and protein conjugation studies of vitamin K analogues.

Two vitamin K analogues bearing a carboxylic acid side chain (9a and its deuterated analogue 9b) were each synthesised in six steps from commercially available menadione. Analogue 9b was conjugated to lysozyme and bovine serum albumin (BSA) using EDCI/HOBT and by prior formation of its activated succinimidyl ester 11. Quantification of the thus formed conjugates by ESMS and LC-MS revealed that the number of equivalents of the analogue used in the couplings systematically controls the number of analogues that conjugate to the protein.

A convenient and asymmetric protocol for the synthesis of natural products containing chiral alkyl chains via Zr-catalyzed asymmetric carboalumination of alkenes. Synthesis of phytol and vitamins E and K.

[reaction: see text]. A convenient and asymmetric protocol for the synthesis of chiral oligoisoprenoids is described. Typically, a C14 vitamin E side chain 5 was synthesized in 47% yield over four steps. Isomeric purity of 5 was upgraded to >99% R at C-2 and 97% R at C-6 by the statistical formation of stereoisomeric p-phenylenebisurethanes and their diastereomeric separation. In addition, phytol and vitamin K were synthesized in 21% and 28% overall yields, respectively, over five steps from 1.

Prodrugs for systemic bioreductive activation-independent delivery of phyllohydroquinone, an active form of phylloquinone (vitamin K1). 1: Preparation and in vitro evaluation.

With the aim of overcoming the delivery problems (water-solubility and bioreductive activation problems) of phyllohydroquinone (PKH), an active form of phylloquinone (PK, vitamin K1), the N,N-dimethylglycine esters of phyllohydroquinone (1-mono, 1; 4-mono, 2; and 1,4-bis, 3) have been synthesized and assessed in vitro as a prodrug for the systemic bioreductive activation-independent delivery of PKH. The hydrochloride salts of the esters were found to be quite soluble in water. Hydrolysis of the esters in rat liver S9 fraction, rat plasma and phosphate buffer, pH 7.4, at 37 degrees C, was kinetically studied in the presence and absence of an esterase inhibitor. The hydrolysis was catalyzed by esterases located in rat liver and rat plasma and quantitatively yielded PKH. The enzymatic cleavage and the vitamin K-dependent carboxylation activity of the esters in the rat liver microsome preparation at pH 7.2 and 25 degrees C were studied. The regeneration of PKH from the esters was catalyzed by carboxylesterases located in the rat liver microsome, and the order was as follows: 1 > 3 > 2. The carboxylation was stimulated by selected ester 1 in the absence of dithiothreitol, an activator of the vitamin K cycle. The carboxylation activity of 1 was strongly inhibited in the presence of eserine, a carboxylesterase inhibitor. Compound 1 could also stimulate carboxylase under warfarin-poisoning conditions, where the vitamin K cycle was strongly inhibited. These results indicated that these highly water-soluble and liver-esterase hydrolyzable ester derivatives of PKH are potential candidates for parenteral prodrugs which can thus achieve the systemic bioreductive activation-independent delivery of PKH.

Vitamin K prodrugs: 1. Synthesis of amino acid esters of menahydroquinone-4 and enzymatic reconversion to an active form.

The efficacy and toxicity of vitamin K depends on the pathway and the extent of enzymatic reductive activation to vitamin K hydroquinone, which is an essential cofactor for the synthesis of clotting factors. Parenteral use of vitamin K is impaired by its water insolubility. With the aim to improve delivery problems associated with menahydroquinone-4 (MKH, 2), an active form of menaquinone-4, N,N-dimethylglycine esters of 2 (1-mono, 4-mono, and 1,4-bis) were synthesized and assessed as potential water-soluble prodrugs for parenteral use. The esters can deliver the hydroquinone to its active site without a quinone reductive activation step. The hydrochloride salts of the esters were found to be quite soluble in water. The hydrolysis of the esters in 20% rat liver homogenate 9000 x g supernatant, rat plasma and phosphate buffer, pH 7.4, at 37 degrees C was kinetically studied in the presence and absence of an esterase inhibitor. The hydrolysis was catalyzed by esterases located in the rat liver and rat plasma and quantitatively yielded 2. These results suggest that esterification of 2 with N,N-dimethylglycine is a promising way for obtaining water-soluble prodrug forms of 2. Based on the high susceptibility to liver esterase, the esters are potential prodrugs for achieving the site-specific delivery of 2.

Synthesis of fluoro- and hydroxy-derivatives of vitamin K as substrates or inhibitors of the liver microsomal vitamin K-dependent carboxylase.

The rat liver microsomal vitamin K-dependent carboxylase catalyzes the carboxylation of glutamyl to gamma-carboxyglutamyl residues in the presence of reduced vitamin K, O2 and CO2. The specificity of the enzyme for the vitamin substrate has been probed by the synthesis of a series of fluoro- hydroxy- and methoxy-analogs. 2-Fluoro-methyl-3-phytyl-1,4-naphthoquinone and 2-methyl-3-(1'-fluorodecyl)-1,4-naphthoquinone were synthesized but found to be unstable under enzyme assay conditions. The reduced (naphthohydroquinone) forms of 2-hydroxy-methyl-3-phytyl-1,4-naphthoquinone, 2-methoxymethyl-3-phytyl-1,4-naphthoquinone and 2-methyl-3-(1'-hydroxy-decyl)-1,4-naphthoquinone were inactive as substrates, but inhibitors of the enzyme. The two hydroxy analogs were shown to be low Ki (less than 10 microM) inhibitors of the reduced 2-methyl-3-phytyl-1,4-naphthoquinone-dependent activity of the enzyme. The oxidized forms of these compounds did not inhibit the enzyme and they had no activity as in vivo anticoagulants.

The toxicity of menadione (2-methyl-1,4-naphthoquinone) and two thioether conjugates studied with isolated renal epithelial cells.

Menadione (2-methyl-1,4-naphthoquinone) was used as a model compound to test the hypothesis that thioether conjugates of quinones can be toxic to tissues associated with their elimination through a mechanism involving oxidative stress. Unlike menadione, the glutathione (2-methyl-3-(glutathion-S-yl)-1,4-naphthoquinone; MGNQ) and N-acetyl-L-cysteine (2-methyl-3-(N-acetylcysteine-S-yl)-1,4-naphthoquinone; M(NAC)NQ) thioether conjugates were not able to arylate protein thiols but were still able to redox cycle with cytochrome c reductase/NADH and rat kidney microsomes and mitochondria. Interestingly, menadione and M(NAC)NQ were equally toxic to isolated rat renal epithelial cells (IREC) while MGNQ was nontoxic. The toxicity of both menadione and M(NAC)NQ was preceded by a rapid depletion of soluble thiols and was associated with a depletion of soluble thiols and was associated with a depletion of protein thiols. Treatment of IREC with the glutathione reductase inhibitor, 1,3-bis(2-chloroethyl)-1-nitrosourea, potentiated the thiol depletion and toxicity observed with menadione and M(NAC)NQ indicating the involvement of oxidative stress in this model of renal cell toxicity. The lack of MGNQ toxicity can be attributed to an intramolecular cyclization reaction which destroys the quinone nucleus and therefore eliminates its ability to redox cycle. These findings have important implications with regard to our understanding of the toxic potential of quinone thioether conjugates and of quinone toxicity in general.

Menadiol diphosphate, a new substrate for non-specific alkaline phosphatase in histochemistry and immunohistochemistry.

Menadiol diphosphate was introduced as a new substrate for nonspecific alkaline phosphatase, following a search for new and less expensive substrates, which give a more sensitive response and are easily synthesized in the laboratory. Menadiol released by phosphatase action can be assayed by its reduction of tetrazolium salts, or it can be coupled with diazonium salts; alternatively, the phosphate can be trapped by metal ions. The synthesis and purification of menadiol diphosphate are described, and it was shown to be sufficiently stable for qualitative and semiquantitative histochemistry, as well as for the immunohistochemistry of enzymes and cytoskeletal proteins with nonspecific alkaline phosphatase as the enzyme label. For qualitative as well as semiquantitative histochemistry and immunohistochemistry, the best results were obtained by applying the method with nitro-blue tetrazolium (NBT) to acetone-chloroform pretreated cryostat sections. Tetranitro-blue tetrazolium (TNBT), benzothiazolylphthalhydrazidyl tetrazolium (BSPT) and various diazonium salts were less suitable. Fast Blue BB and VB produced satisfactory results. Ce3+ ions and the DAB-Ni-H2O2 procedure yielded better results than Ca2+ ions in the Co-(NH4)2S visualization method. The NBT method with menadiol diphosphate is superior to existing methods employing azo, azoindoxyl or tetrazolium salts and to metal precipitation methods. The Ce3+ technique and the NBT/menadiol diphosphate method give similar results, and appear to be of equal value. In qualitative histochemistry and immunohistochemistry the NBT/menadiol diphosphate method resulted in higher quantities of precisely localized stain. Semiquantitative histochemistry with minimal incubation revealed more favorable kinetics for the menadiol diphosphate method, especially when using NBT.

Synthesis of trifluoromethyl analogs of vitamin K as substrates for the liver microsomal vitamin K-dependent carboxylase.

The rat liver microsomal vitamin K-dependent carboxylase catalyzes the carboxylation of glutamyl to gamma-carboxyglutamyl residues in the presence of reduced vitamin K, O2 and CO2. The specificity of the enzyme for the vitamin substrate has been probed by the synthesis of the trifluoromethyl analogs of menaquinone-2 (2-methyl-3-geranyl-1,4-naphthoquinone) and phylloquinone (2-methyl-3-phytyl-1,4-naphthoquinone). The reduced (naphthohydroquinone) forms of the trifluoromethyl analogs of the natural vitamins had no substrate activity but were competitive inhibitors of the reaction with a Ki in the same range as the Km of the normal substrate. The oxidized form of the trifluoromethyl analogs of vitamin K also caused inhibition by a mechanism that could not be established. Under the incubation conditions utilized, fluorine was lost from the trifluoromethyl group by a process that was dithiothreitol and high pH dependent.

Synthesis of menaquinone-2 derivatives as substrates for the liver microsomal vitamin K-dependent carboxylase.

The rat liver microsomal vitamin K-dependent carboxylase catalyzes the carboxylation of glutamyl to gamma-carboxyglutamyl residues in the presence of reduced vitamin K, O2 and CO2. The specificity of the enzyme for the vitamin substrate has been probed by the synthesis of a number of menaquinone-2 (2-methyl-3-geranyl-1,4-naphthoquinone) derivatives. The 2-des-methyl and 2-ethyl-MK-2 derivatives had very low activity as substrates. The 6- or 7-methyl-MK-2 derivatives and (6,7)-chloro-MK-2 were relatively high Vmax substrates with Km values increased over that seen for K-2. The 5- or 8-methyl-MK-2 derivatives were low Vmax substrates but also demonstrated low Km values. Although these observations suggested that 5-methyl-MK-2 might be a competitive inhibitor of the carboxylation reaction, it was not an effective inhibitor of either phylloquinone or 6-methyl-MK-2-dependent carboxylation.

Radioactive derivatives of 2-methyl-1, 4-naphthoquinol bis (diphosphate salt) as anti-cancer drugs with high LET radiations.

From the results of the alpha-particle track autoradiography, the alkaline phosphatase studies, the biodistribution investigations and the therapeutic experiments with the transplanted Franks and Hemmings (1978) adenocarcinoma of the rectum in mice, an important finding is the striking selectivity of the uptake of the compound 6-211 At-astato-2-methyl-1, 4-naphthoquinol bis (diphosphate salt), after intraperitoneal injection into the tumour cell nuclei and in many cases into the nuclei of tumour stem cells, together with negligible uptake into normal colon and narrow. The compound, 6-211 At-astato-2-methyl-1, 4-naphthoquinol bis (diphosphate salt) has been found to cure about two-thirds of the transplanted adenocarcinoma of the rectum in mice after a single intra-peritoneal injection of 3-4 microCi. This approach can be regarded as a form of metabolically-directed drug targetting on to a tumour product which is an enzyme, an alkaline phosphatase isozyme, present in the cells of some tumours. The compound is being studied further from the point of view of possible human therapeutic applications.