**1. 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 K3 (menadione), a vitamin K homologue that was considered as a synthetic vitamin K, has antitumor activity [2–5], as does vitamin K2 (menaquinone) [6, 7]. Among the homologues of vitamin K2, 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

© 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2017 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.

Based on the finding that Cpd5 inhibits Cdc25 and exerts antitumor activities, various menadione derivatives have been developed as candidate antitumor compounds. Bis(2‐ hydroxyethylthio)naphthoquinone derivative NSC 95397 (**Figure 3**) showed potent Cdc25‐ inhibitory activity and inhibited proliferation of several cancer cell lines with greater potency than that of Cpd 5 [20]. Hydroxylated NSC 95397 derivatives exhibited enhanced Cdc25‐ inhibitory 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 phosphor‐ ylation 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 IC50 value for HepB3 growth inhibition. Structure‐activity relationship study indicated that the biphenyl

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structure of PM‐20 is essential for activity (**Figure 3**) [24].

**Figure 3.** Structures of Cpd 5 derivatives bearing a 2‐hydroxyethylthio moiety.

such as **7** showed angiogenesis‐inhibitory activity (**Figure 4**) [28].

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

**Figure 1.** Structures of vitamin K homologues.

#### **2. 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 growth‐ regulatory phosphatase Cdc25 by arylating a cysteine residue in the catalytic site, causing cell‐ cycle arrest [16–19].

**Figure 2.** Compounds tested in the initial work on development of Cpd5.

Based on the finding that Cpd5 inhibits Cdc25 and exerts antitumor activities, various menadione derivatives have been developed as candidate antitumor compounds. Bis(2‐ hydroxyethylthio)naphthoquinone derivative NSC 95397 (**Figure 3**) showed potent Cdc25‐ inhibitory activity and inhibited proliferation of several cancer cell lines with greater potency than that of Cpd 5 [20]. Hydroxylated NSC 95397 derivatives exhibited enhanced Cdc25‐ inhibitory 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 phosphor‐ ylation 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 IC50 value for HepB3 growth inhibition. Structure‐activity relationship study indicated that the biphenyl structure of PM‐20 is essential for activity (**Figure 3**) [24].

**Figure 3.** Structures of Cpd 5 derivatives bearing a 2‐hydroxyethylthio moiety.

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

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 growth‐ regulatory phosphatase Cdc25 by arylating a cysteine residue in the catalytic site, causing cell‐

K activities.

240 Vitamin K2 - Vital for Health and Wellbeing

**Figure 1.** Structures of vitamin K homologues.

cycle arrest [16–19].

**2. Menadione derivatives as antitumor agents**

**Figure 2.** Compounds tested in the initial work on development of Cpd5.

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].

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‐d7), and alkyl derivative **12** were inactive. Removal of methyl groups also reduced the activity, but demethylated compounds **13**–**16** still

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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

Substitution at the terminal of the side chain of menaquinones significantly affects SXR ligand potency. Hydroxylated derivatives MK‐2‐ω‐OH, MK‐3‐ω‐OH, and MK‐4‐ω‐OH showed little activity in the SXR‐GAL4 one hybrid assay system, whereas compounds **17** and **18** bearing a

MK‐3‐W, MK‐4‐W, and PK‐W showed little activity (**Figure 7**) [35].

retained significant activity [34].

**Figure 6.** Compounds used in SAR study of SXR.

**Figure 7.** Structures of double side chain vitamin K analogs.

**Figure 4.** Examples of side chain‐modified Cpd 5 derivatives.

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]. Structur‐ ally 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 α‐ lapachone or β‐lapachone), in contrast to 2‐methyl‐1,4‐naphthoquinone.

**Figure 5.** Some vitamin K–related naphthoquinone derivatives with antitumor activity.
