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Medicinal Chemistry of Vitamin K Derivatives and Metabolites

Vitamin K acts as a cofactor for γ‐glutamyl carboxylase. Recently, various biological activities of vitamin K have been reported. Anti‐proliferative activities of vitamin K, especially in vitamin K3, are well known. In addition, various physiological and pharmacological functions of vitamin K2, such as transcription modulators as nuclear steroid and xenobiotic receptor (SXR) ligands and anti‐inflammatory effects, have been revealed in the past decade. Characterization of vitamin K metabolites is also important for clinical application of vitamin K and its derivatives. In this chapter, recent progress on the medicinal chemistry of vitamin K derivatives and metabolites is discussed.


Introduction
Vitamin K is a specific cofactor for γ-glutamyl carboxylase (GGCX), which catalyzes formation of γ-carboxyglutamyl (Gla) residues in vitamin K-dependent proteins (Figure 1) [1]. Various other biological activities of vitamin K and its derivatives have also been reported. For example, vitamin K 3 (menadione), a vitamin K homologue that was considered as a synthetic vitamin K, has antitumor activity [2][3][4][5], as does vitamin K 2 (menaquinone) [6,7]. Among the homologues of vitamin K 2 , menaquinone-4 (MK-4), which contains four isoprene units, has been intensively investigated. It binds to nuclear receptor human pregnane X receptor (PXR), which is also called steroid and xenobiotic receptor (SXR), and regulates transcription of osteoblastic genes [8,9]. It also exhibits anti-inflammatory activity by suppressing the NF-kB pathway [10], and has an inhibitory effect on arteriosclerosis [11]. It binds 17β-hydroxysteroid dehydrogenase 4 and modulates estrogen metabolism [12]. Further, it enhances testosterone production [13,14], and shows growth-inhibitory activity toward hepatocellular carcinoma (HCC) cells [6,7]. These biological activities of vitamin K and its analogues are attractive targets of drug discovery, and the activities of vitamin K metabolites have also attracted much interest. A great many natural and synthetic biologically active 1,4-naphthoquinone derivatives (i.e., vitamin K derivatives) have been reported. In this chapter, we will focus on three medicinal-chemistry studies of vitamin K activities.

Menadione derivatives as antitumor agents
The antitumor activity of thioether derivatives is one of the most intensively investigated fields in the medicinal chemistry of menadione derivatives. Several series of naphthoquinone derivatives and benzoquinone derivatives bearing an alkyl, alkoxy, or alkylthio group as a side chain have been synthesized and biologically evaluated by assay of growth-inhibitory activity toward human hepatoma cell line HepB3. Almost all of the tested compounds, as well as the parent menadione, exhibited significant inhibitory activity, and the alkylthio derivatives were more potent than the corresponding alkyl and alkoxy derivatives. Among these compounds, a 2-hydroxyethylthio derivative Cpd 5 (compound 5; NSC 672121) exhibited the most potent activity (Figure 2) [15]. Subsequent studies revealed that Cpd 5 irreversibly inhibits growthregulatory phosphatase Cdc25 by arylating a cysteine residue in the catalytic site, causing cellcycle arrest [16][17][18][19]. Based on the finding that Cpd5 inhibits Cdc25 and exerts antitumor activities, various menadione derivatives have been developed as candidate antitumor compounds. Bis(2hydroxyethylthio)naphthoquinone derivative NSC 95397 (Figure 3) showed potent Cdc25inhibitory activity and inhibited proliferation of several cancer cell lines with greater potency than that of Cpd 5 [20]. Hydroxylated NSC 95397 derivatives exhibited enhanced Cdc25inhibitory activity and inhibited growth of several cancer cell lines [21]. Fluorinated Cpd 5 was three times more potent than Cpd-5 itself in Hep3B growth inhibition and induced phosphorylation of ERK1/2, JNK1/2 and p38 in HepB3 cells [22]. Calculations suggested that fluorinated Cpd 5 cannot generate reactive oxygen species because of its modified redox profile, and therefore, the compound appears to function as a pure arylating agent [23]. Modification of the core structure afforded a maleimide derivative PM-20 with a submicromolar IC 50 value for HepB3 growth inhibition. Structure-activity relationship study indicated that the biphenyl structure of PM-20 is essential for activity (Figure 3) [24]. Modification of the hydroxyethyl side chain of Cpd-5 and NSC 95397 was also investigated. Carboxylic acid derivatives such as compounds 1, 3, and 4 ( Figure 4) were designed to interact with arginine residues in the catalytic site of Cdc25B, and indeed, they exhibited potent Cdc25B3-inhibitory activity [25,26]. Though the cytotoxic activities of these carboxylic acid derivatives, especially dicarboxylic acid 4, were low, prodrug-type benzyl ester derivatives exhibited enhanced growth-inhibitory activity toward HeLa cells. It was also found that Cpd 5 derivatives bearing a modified terminal, such as 6, showed selective cytotoxicity toward neuroblastoma cell lines, whereas the parent menadione and Cpd 5 exhibited cytotoxicity toward both neuroblastoma cells and normal cell lines [27]. Aminoalkylmenadione derivatives such as 7 showed angiogenesis-inhibitory activity (Figure 4) [28].  A natural product, plumbagin (5-hydroxymenadione, Figure 5), shows anticancer and antiproliferative activities [29]. It suppresses the NF-kB activation pathway by modulating p65 and IkBα kinase activation to potentiate cytokine-and drug-induced apoptosis [30]. Structurally related naphthoquinone derivatives juglone and 1,4-naphthoquinone exerted similar TNFα-induced NF-kB inhibitory activities, whereas menadione did not [30]. Another natural product, lapachol, which has a hydroxyl group instead of the methyl group of MK-1, has anticancer activity [31]. A synthetic analogue 8 bearing two isoprene units also exerted antitumor activity ( Figure 5) [32], and various biologically active lapachol derivatives have been developed [33]. The 2-hydroxy-1,4-naphthoquinone structure has distinct chemistry; for example, it has the characteristics of 1,2-naphthoquinone (e.g., lapachol can cyclize to form αlapachone or β-lapachone), in contrast to 2-methyl-1,4-naphthoquinone.

Structure-activity relationship of MK-4 derivatives as nuclear SXR ligands
In the early twenty-first century, it was found that MK-4 binds a nuclear receptor, steroid, and xenobiotic receptor (SXR), which is a human homologue of pregnane X receptor (PXR), and Vitamin K2 -Vital for Health and Wellbeing 242 regulates transcription of osteoblastic genes [8,9]. Structure-activity relationships of MK-4 as an SXR ligand were intensively investigated by Suhara et al., using deuterated derivatives (Figure 6). Saturation of double bond(s) in the side chain significantly reduced the SXR agonistic activity. Triene derivative 9 bearing a 6,7-saturated side chain exerted only moderate activity, and diene 10, monoene 11 (phylloquinone-d 7 ), and alkyl derivative 12 were inactive. Removal of methyl groups also reduced the activity, but demethylated compounds 13-16 still retained significant activity [34]. The length of the side chain is important for the SXR activity of menaquinones. MK-1 bearing one prenyl group showed little ligand potency, while MK-2, MK-3, and MK-4 were more active. In the SXR-GAL4 one hybrid assay system, MK-3 was the most potent compound, and MK-2 and MK-4 showed somewhat lower activity. In the assay system using SXRE, MK-2, and MK-3 were the most potent compounds [35]. "Double side chain" vitamin K analogues bearing the same side chains at the 2-position and 3-position of the naphthoquinone ring were also designed and synthesized. MK-1-W and MK-2-W were as potent as MK-3 and MK-4, whereas MK-3-W, MK-4-W, and PK-W showed little activity (Figure 7) [35]. terminal phenyl group exhibited more potent activity than the parent menaquinones (Figure 8). Compounds 17 and 18 also exhibited potent activity in the SXRE assay system [36]. Thus, a suitable hydrophobic side chain is essential for SXR activity of menaquinones.
Interestingly, Suhara et al. also found that menaquinone derivatives bearing a terminal hydrophobic substituent have the ability to induce selective neuronal differentiation of neuronal progenitor cells. The most potent compound 19 was twice as effective as the EtOH control, based on quantitation of Map2 mRNA (Figure 8) [37].

Synthesis and biological activity of menaquinone metabolites
The biological activities of metabolites of vitamin K are also important. MK-4 is one of the most interesting vitamin K homologues because of its multifunctional properties, and ω-carboxyl homologues of MK-4 (MK-4-ω-COOH), K acid I, K acid II and their glucuronides have been identified as metabolites [38][39][40][41][42]. It is considered that MK-4 is initially metabolized to MK-4ω-COOH by ω-oxidation, followed by β-oxidation to afford intermediary carboxylic acids (Figure 9) [43]. These carboxylic acids can be categorized into two groups; MK-n-ω-COOH derivatives bearing a α,β-unsaturated carboxy group and MK-n-(ω-2)-COOH derivatives bearing a γ,δ-unsaturated carboxy group. Chemical synthesis of these metabolites is essential for evaluation of their properties, and several synthetic routes have been reported.
Vitamin K2 -Vital for Health and Wellbeing 244

Synthesis of menaquinone metabolites
The MK-4 metabolites K acid I and K acid II are also metabolites of phylloquinone (vitamin K 1 ). Several chemical syntheses of K acid I and K acid II have been reported. Watanabe et al. synthesized K acid I by direct addition of a carboxy side chain to the naphthoquinone framework using BF 3 etherate [44]. A route involving a malonyl derivative and decarboxylation was also investigated (Figure 10) [45]. They also synthesized K acid II. Addition of a side chain moiety by Friedel-Crafts acylation, followed by Clemmensen reduction, afforded naphthylcarboxylic acid, and oxidation of the naphthol moiety using Fremy's salt gave K acid II. Direct alkylation of naphthoquinone using peroxide also afforded K acid II (Figure 11) [44].

Vitamin K2 -Vital for Health and Wellbeing 246
Teitelbaum et al. synthesized K acid I and K acid II by oxidation of MK-2 and MK-1, respectively. They prepared intermediary MK-n using a menadione-cyclopentadiene adduct as the same starting material (Figure 12) [46].

Biological activities of menaquinone metabolites
These menaquinone carboxylic acid derivatives and related quinone carboxylic acids, including ubiquinone derivatives and tocopheryl derivatives, show lysosomal membrane-stabilizing activity [45,47]. Appropriate hydrophobicity of the side chain appears to be essential for this activity. Some of these compounds also exert inhibitory effects on the generation of the slowreacting substance of anaphylaxis [48]. MK-4 has various biological activities, such as anti-inflammatory activity and antitumor activity, and these activities of the menaquinone metabolites were also investigated. All tested menaquinone metabolites inhibited LPS-induced production of proinflammatory cytokines in RAW264.7 cells [50]. It is suggested that naphthoquinone structure is essential for the antiinflammatory activity of menaquinone derivatives. Regarding antitumor activity, several carboxylic acids, such as MK-2-ω-COOH, significantly inhibited proliferation of JHH7 and HepG2 hepatocellular carcinoma cell lines. On the other hand, MK-2-ω-COOH did not inhibit proliferation of normal hepatic cells. Anti-proliferative activity may be associated with caspase/transglutaminase-related pathways [53].
The ω-alcohols and ω-aldehydes showed apoptosis-inducing activity toward human leukemia cell line HL-60 and human osteosarcoma cell line MG-63. The ω-aldehydes were more potent than the corresponding ω-alcohols [51,52]. The vitamin K potency of MK-4-ω-OH, that is, its coenzyme activity for GGCX, was also evaluated. MK-4-ω-OH showed a larger V max /K m value than that of intact MK-4, indicating that MK-4-ω-OH has greater coenzyme activity than MK-4 [52].

Future perspective
Vitamin Ks are attractive lead compounds for drug discovery. One of the most promising applications is as candidate antitumor agents, though the mechanism of action of Cpd 5 could be different from that of intact vitamin Ks. In addition, bone homeostasis and neural effects are also possible targets of vitamin K derivatives. Vitamin K may also be used as a food supplement, and therefore, characterization of its metabolites is important. It is noteworthy that some menaquinone metabolites have characteristic activities distinct from those of intact vitamin K 2 . Though a clinical study of MK-4 as an agent to prevent recurrence of hepatocellular carcinoma was terminated [54], the metabolites and their analogs still represent potential drug candidates.