Recent Advances in the Medicinal Chemistry of Vitamin K Derivatives: An Overview (2000–2021)

*Shinya Fujii, Yoshitomo Suhara and Hiroyuki Kagechika*

## **Abstract**

In recent decades, many physiological and pharmacological functions of vitamin K other than its role as the cofactor of γ-glutamyl carboxylase (GGCX) have been identified, and consequently, many vitamin K derivatives and related congeners, including putative metabolites, have been designed and synthesized. Their biological activities include antitumor activity, anti-inflammatory activity, neuroprotective effects, neural differentiation-inducing activity, and modulating potency toward the nuclear steroid and xenobiotic receptor (SXR). These activities make vitamin K and its derivatives attractive candidates for drug discovery. In this chapter, an overview of recent advances in the medicinal chemistry of vitamin K, focusing especially on SXR modulation, neural differentiation, and antitumor activities, was provided.

**Keywords:** metabolites, synthetic analogs; neural differentiation, nuclear receptor, steroid and xenobiotic receptor, antitumor, phthalazine-1,4-dione

## **1. Introduction**

Vitamin K is the term used to describe derivatives of naphthoquinones **1**–**4** [1, 2]. It was originally identified as a specific cofactor for γ-glutamyl carboxylase (GGCX), which catalyzes the formation of γ-carboxyglutamyl (Gla) residues in vitamin K-dependent proteins. Since then, various other biological activities of vitamin K have been reported. For example, antitumor activity of vitamin K3 (**4**: menadione: 2-methyl-1,4-naphthoquinone) was reported in the 1980s, [3–6] and antitumor activity of vitamin K2 (**2**: menaquinone-n; MK-n) was also found in the 1990s [7, 8]. Among the homologs of vitamin K, menaquinone-4 (**3**, MK-4), which contains four isoprene units in the side chain, has been most intensively investigated [9–11]. MK-4 binds to human pregnane X nuclear receptor (PXR), which is also called the steroid and xenobiotic receptor (SXR), and regulates transcription of osteoblastic genes [12, 13]. MK-4 and its derivatives also have roles in neural differentiation, as well as neuroprotective effects [14]. In addition, MK-4 exhibits anti-inflammatory activity by suppressing the NF-κB pathway [15], exerts an inhibitory effect on

arteriosclerosis [16], and shows growth-inhibitory activity toward hepatocellular carcinoma (HCC) cells [17, 18]. Thus, vitamin K and its derivatives are attractive candidates for drug discovery. The following sections provide a review and perspective of the medicinal chemistry of vitamin K derivatives mainly developed from the beginning of this century (**Figure 1**).

## **2. Structure–activity relationship of vitamin K analogs for transcriptional activity through nuclear receptor SXR**

There are two kinds of natural vitamin K homologs, phylloquinone (PK) (**1**) and menaquinones (MK-n) (**2**). Since the discovery of vitamin K, research has mainly focused on its role in the blood coagulation system. However, it was recently revealed that MK-4 (**3**) binds to the steroid and xenobiotic receptor (SXR, pregnane X receptor in mice: PXR), a member of the nuclear receptor superfamily, and exhibits agonist activity [19]. The mechanism has been reported to be as follows; first, **3** binds to SXR and forms a heterodimer with retinoid X receptor (RXR). Then, the heterodimer binds to SXR-responsive elements on DNA and gene expression of a drug-metabolizing enzyme, CYP3A4, is induced [12]. The menaquinone **3** also induces gene expression of proteins involved in osteogenesis through binding to SXR [13]. Other menaquinones showed similar effects, but **3** was the most potent. Interestingly, **1** did not have such an effect.

Following describes several vitamin K derivatives synthesized for structure–activity relationship studies of SXR agonists. Focusing on the double bonds and methyl groups in the side chain of MK-4, compounds **5**–**14**, in which the double bonds in the isoprene side chain were progressively saturated or methyl groups were deleted, were synthesized (**Figure 2**) [20]. In this study, the deuterium-labeled compounds were

*Recent Advances in the Medicinal Chemistry of Vitamin K Derivatives: An Overview (2000–2021) DOI: http://dx.doi.org/10.5772/intechopen.101667*

**Figure 2.** *Structure of vitamin K analogs* **5–14.**

employed since these compounds enable us to investigate the conversion rate of the analogs to MK-4. The SXR-mediated transcriptional activity of each compound was evaluated in two ways, using SXR-GAL4 and CYP3A4 promoters. The results showed that as the number of double bonds was decreased, the transcriptional activity also significantly decreased [20]. This tendency was particularly pronounced with the CYP3A4 promoter assay. Deletion of methyl groups on the side chain also decreased the transcriptional activity. These results indicate that both the methyl groups and double bonds of the side chain of MK-4 are important for SXR-mediated transcriptional activity (**Figure 2**).

Since the isoprene structure of the side chain of menaquinones is important for the activity, vitamin K derivatives **15**–**19**, in which an isoprene side chain is symmetrically introduced into the naphthoquinone part, were next synthesized (**Figure 3**) [21]. The transcriptional activity of these analogs peaked at compound **17**, which contains two side chains of MK-2, and then remarkably decreased with increasing length of the side chains [20–22]. These results indicate that the transcriptional activity of vitamin K derivatives is greatly affected by the length and bulk of the side chains (**Figure 3**).

Then, in order to investigate how the transcriptional activity changes depending on the polarity of the side chain, vitamin K analogs introduced hydrophilic or hydrophobic functional groups at the end of the side chain; namely, compounds **20**–**22** with a hydroxyl group as a hydrophilic functional group and compounds **23** and **24** with a phenyl group as a hydrophobic functional group, were synthesized (**Figure 2**) [23]. The transcriptional activity of compounds **20**–**22** was decreased; on the other hand, that of compounds **23** and **24** was markedly increased. In particular, compound **23**, an analog of MK-3 bearing a phenyl group at the end of the side chain, showed comparable activity to that of rifampicin, a known SXR ligand. Computational analysis of the binding states of the vitamin K derivative **23** with the ligand-binding site of SXR using the MOE (Molecular Operating Environment)-integrated computational

**Figure 3.** *Vitamin K analogs 15–24.*

chemistry system indicated that the oxygen atoms of the quinone part of **23** form hydrogen bonds with His407 and Ser247 of SXR [23]. In view of their potent activity, MK-3 and MK-4 were expected to show similar interactions. Thus, the SXR-mediated transcriptional activity of vitamin K requires an appropriate length and bulk of the isoprene side chain, and the side chain structure also has a significant influence [20, 21, 23].

## **3. Neuronal differentiation-inducing activity of vitamin K and its analogs**

MK-4 is present at relatively high concentrations in the brain, though its physiological role remains unclear. As one of the biological action in the brain, it has been reported that it protects neurons against oxidative stress [14, 24–26]. It is also known that neural stem cells differentiate into neuronal progenitors and glial progenitors, and then, neuronal progenitors differentiate into neurons, while glial progenitors differentiate into astrocytes and oligodendrocytes [27]. Recently, it has been found that menaquinones selectively induce the differentiation of neural progenitors into neurons, although their potency was not high [28]. This activity differed depending on the repeat structure of the isoprene side chain of the menaquinones. Therefore, if this activity can be increased by derivatization of vitamin K, it might be possible to regulate differentiation using safe and small molecule inducers of neural differentiation. Thus, new vitamin K derivatives that would induce differentiation of neural stem cells into neurons were explored.

Considering the lipophilic environment of the brain, vitamin K analogs bearing various hydrophobic functional groups such as benzene or naphthalene in the side chain were designed and synthesized (**Figure 4**). The compounds were evaluated for neuronal differentiation-inducing activity toward stem cells derived from mouse fetal cerebrum. After the compounds were added to the cells and the cells were cultured, the expression levels of Map2 and Gfap, which are expressed specifically in neurons and astrocytes, were quantified by real-time PCR. Interestingly, most synthesized compounds showed a significant increase in the induction of neuronal differentiation compared with the control. In particular, derivative **26b**, in which an *m*-tolyl group was introduced at the end of the side chain of MK-3, exhibited the highest activity,

*Recent Advances in the Medicinal Chemistry of Vitamin K Derivatives: An Overview (2000–2021) DOI: http://dx.doi.org/10.5772/intechopen.101667*

#### **Figure 4.**

*Vitamin K analogs: (a) an aromatic substituent was introduced at the -terminal side chain. (b) A tert-butyl group or methyl groups were introduced at the -terminal side chain.*

and its differentiation-inducing effect was about twice that of the control. Based on the ratio of expression levels of Map2 and Gfap, compound **26b** selectively induces differentiation to neurons [28].

Then, compounds **30ab–35ab**, in which heteroatoms were incorporated and substituents such as fluorine and methyl groups were introduced into the phenyl group at the end of the side chain are reported (**Figure 3**). The results of biological evaluation showed that the fluorine-containing compounds enhanced the selectivity of neuronal differentiation. In order to investigate the effect of alkyl groups on the differentiation-inducing activity of compound **26b**, compounds **36–47**, in which several *t*-butyl and methyl groups were introduced into the phenyl group at the end of the side chain, were synthesized. Interestingly, it was clarified that derivatives **36** and **38**, in which two methyl groups were introduced at the 2,3- and 3,4-positions, respectively, of the phenyl group at the end of the MK-2 side chain, inhibited the differentiation of MK-2 into neurons (**Figure 4**) [29, 30].

Thus, the introduction of a hydrophobic functional group at the end of the side chain can enhance the differentiation-inducing activity of vitamin K from neural stem cells to neurons. It is known that natural products such as neuropathiazol, epolactaene, and retinoic acid (retinoid) induce neuronal differentiation. All of these compounds have double bonds or phenyl groups in their side chains, similar to the active vitamin K derivatives synthesized in this study [31–33]. Based on these findings, it might be possible to obtain compounds that have more potent neuronal differentiation activity. At present, the mechanism by which vitamin K derivatives induce neuronal differentiation is unknown. If the proteins upon which vitamin K acts were identified, this would be helpful for rational design of more potent compounds.

As described above, the biological activity of vitamin K is greatly affected by differences in the side chain structure. In addition to vitamin K, many other fatsoluble vitamins, such as vitamins A, D, and E, also have alkyl side chains containing double bonds. This may suggest that there is an optimal side chain structure for each target biological activity, because the specific action of each vitamin differs depending on the alkyl side chain structure. Further investigation of the structure–activity relationships of the side chains and the naphthoquinone part is needed (**Figure 4**).

## **4. Antitumor activity of vitamin K derivatives**

#### **4.1 Menadione-based Cdc25 inhibitors**

Antitumor activity is one of the most interesting features of vitamin K and its derivatives. Among synthetic compounds, a series of menadione-based alkylthio naphthoquinone derivatives including 2-hydroxyethylthio-3-methyl-1,4-naphthoquinone (Cpd 5; compound 5, NSC 672121: **48**) are representative examples of vitamin K derivatives with potent antitumor activity. Carr and coworkers designed and synthesized naphthoquinone derivatives bearing an alkyl, alkoxy, or alkylthio group, and evaluated their growth-inhibitory effect toward human hepatoma cell line HepB3. Almost all of the tested compounds, as well as menadione, exhibited significant growth-inhibitory activity toward HepB3 cells, and among the compounds, Cpd 5 exhibited the most potent activity [34]. Further studies revealed that Cpd 5 irreversibly inhibits growth-regulatory phosphatase Cdc25 by arylating the cysteine residue of the catalytic site, causing cell cycle arrest [35–37]. Based on the structure and the mode of action of Cpd 5, various compounds bearing 2-hydroxyethylthio group(s) have been developed as candidate antitumor agents. For example, bis(2-hydroxyethylthio)naphthoquinone derivative NSC 95397 (**49**) shows potent inhibitory activity toward Cdc25 phosphatase and was found to inhibit proliferation of several cancer cell lines [38]. Hydroxylated NSC 95397 derivatives (**50**, **51**) and a fluorinated Cpd 5 derivative (**52**) also exhibited more potent activity than the parent Cpd 5 [39, 40]. A maleimide moiety instead of naphthoquinone could also function as the arylating functionality, and a maleimide derivative PM-20 (**53**) exerted potent growth-inhibitory activity toward HepB3 cells [41].

In addition to the aryl moiety, modification of the sulfide side chain was also investigated. Garbay and coworkers developed carboxylic acid derivatives such as compounds **54**, **56**, and **57**. The carboxy functionality therein was introduced based on the consideration that the carboxylic acid moiety would interact with arginine residues in the catalytic site of Cdc25B, and indeed, these compounds exhibited potent Cdc25B3-inhibitory activity. Though the cytotoxic activity of these carboxylic acid derivatives, especially dicarboxylic acid **57**, was low, benzyl ester derivatives such as **55** and **58**, which could be considered as prodrugs, exhibited enhanced cell growthinhibitory activity [42, 43]. Suzuki and coworkers investigated the structure–activity relationship of the alkylthio moiety using a series of oxygen-containing derivatives such as **59**–**61** and found that the methoxy derivative **59** exhibited cytotoxic activity with selectivity toward neuroblastoma cell lines, whereas the parent menadione and Cpd 5 exhibited cytotoxicity toward both neuroblastoma cells and normal cell lines [44].

Because 1,4-naphthoquinone structure as well as quinolinedione structure is considered a promising scaffold for Cdc25 inhibitors, several naphthoquinone-based

## *Recent Advances in the Medicinal Chemistry of Vitamin K Derivatives: An Overview (2000–2021) DOI: http://dx.doi.org/10.5772/intechopen.101667*

Cdc25 inhibitors other than Cpd 5 derivatives have been also reported as candidate antitumor agents. Quinolinedione derivatives NSC663284 (**62**) and JUN-1111 (**63**) inhibit Cdc25 function, and the corresponding naphthoquinone derivative **64** also inhibits Cdc25B3 [45, 46]. Recently, Quinn and coworkers synthesized a series of naphthoquinone derivatives and examined their Cdc25-inhibitory activity as well as their binding affinity toward mitogen-activated protein kinase kinase 7 (MKK7). Most derivatives bearing alkylthio group(s) showed both Cdc25-inhibitory activity and MKK7-binding affinity, and NSC95397 (**49**), as well as compounds **65** and **66**, showed marked potency. They also found that compound **67** was a selective inhibitor of Cdc25A/B versus MKK7, whereas compound **68** was a selective inhibitor of MKK7 versus Cdc25A/B [47]. Cdc25 is a promising therapeutic candidate for not only HCC, but also other cancers including triple-negative breast cancer [48]. Development of vitamin K-based Cdc25 inhibitors could provide novel options for cancer chemotherapy (**Figures 5** and **6**).

## **4.2 Anti-hepatocellular carcinoma activity of menaquinone derivatives**

The inhibitory effect of menaquinones on tumor progression and the molecular mechanism involved have been intensively investigated [7, 49], and there is continuing interest in the use of menaquinones for the chemoprevention of hepatocellular carcinoma (HCC) due to their safety. Though several clinical studies have suggested a preventive effect of menaquinone against HCC recurrence [50, 51], the efficacy of menaquinones in suppressing HCC was not confirmed in a large-scale clinical

**Figure 5.** *Structures of Cpd 5 and related derivatives bearing an alkylthio moiety.*

**Figure 6.** *Examples of naphthoquinone- and quinolinedione-based Cdc25 inhibitors.*

study [52]. Therefore, further study of the anti-HCC activity of menaquinones and derivatives is needed. In order to investigate the anti-HCC activity of menaquinones, we focused on carboxylated derivatives, which include isolated and putative metabolites of menaquinones. In the case of MK-4, one of the most interesting vitamin K homologs because of its multifunctional properties, ω-carboxyl homologs of MK-4 (MK-4-ω-COOH: **69**), K acid I (**74**), K acid II (**76**), and their glucuronides, has been identified as metabolites [53–56]. It is considered that MK-4 is metabolized to MK-4 ω-COOH (**69**) by initial ω-oxidation, followed by sequential β-oxidation to afford intermediary carboxylic acids. We focused on the structural similarity between the ω-carboxyl homologs of menaquinones and acyclic retinoid (ACR, peretinoin; **77**), namely a hydrophobic isoprene chain and a carboxyl moiety. ACR, a chemopreventive agent currently under clinical investigation, selectively inhibits HCC cell growth, but also has a limited effect on normal hepatocytes.

Although several synthetic methods for oxidized vitamin K derivatives including K acid I (**74**) and K acid II (**76**) have been reported [57–61], there has been no systematic synthesis of ω-carboxyl menaquinone derivatives. Fujii and coworkers developed a method for systematic preparation of ω-carboxyl menaquinone derivatives using 1,4-dimethoxynaphthalene derivatives instead of reactive 1,4-naphthoquinones as synthetic intermediates [62]. By using the synthesized compounds as standards, McDonald and coworkers newly identified the presence of MK-1-ω-COOH (**75**) in human urine as a vitamin K metabolite (**Figures 7** and **8**) [63].

Then, the proliferation-inhibitory activity of ω-carboxyl menaquinone derivatives **70**, **71**, **73**, and **74** toward JHH7 human HCC cells were examined. All the tested carboxylic acid derivatives, including the known vitamin K metabolite K acid I (**74**), exhibited significant proliferation-inhibitory activity toward JHH7 cells, whereas the parent MK-4 had no effect on proliferation. Among the tested compounds, α,β-unsaturated carboxyl derivatives, that is, **71** and **73**, exhibited potent activity. Therefore, the activity profile of the potent compound MK-2-ω-COOH (**73**) was next investigated in detail. Compound **73** inhibited the proliferation of human HCC cell line HepG2, as well as JHH7, but had no significant effect on the proliferation of normal hepatocytes. These results suggested that the growth-inhibitory activity of **73** is cancer-selective. Since it was reported that menaquinone binds to Bak and induces apoptosis of HeLa cells, we next investigated the involvement of Bak in the growthinhibitory activity of **73**. However, a loss-of-function experiment using siRNA revealed that the observed effect of **73** was not mediated by Bak. As for ACR, it was

*Recent Advances in the Medicinal Chemistry of Vitamin K Derivatives: An Overview (2000–2021) DOI: http://dx.doi.org/10.5772/intechopen.101667*

**Figure 7.** *Putative catabolic pathway of MK-4 based on the identified metabolites.*

**Figure 8.** *Structure of acyclic retinoid (ACR).*

revealed that a caspase- and transglutaminase-dependent pathway is associated with ACR-induced apoptosis in HCC [64]. Cell growth inhibition caused by compound **73** was reversed by caspase inhibitor ZVAD and transglutaminase inhibitor cystamine, and combined treatment with ZVAD and cystamine almost completely blocked the cell death of JHH7 induced by compound **73**. These results suggested that the proliferation-inhibitory activity of the carboxylated menaquinone derivatives on HCC cells occurs at least partially via caspase- and transglutaminase-dependent pathways [65].

Fujii and coworkers have also developed a different type of candidate anti-HCC agents based on the structure of menaquinones. Specifically, a series of compounds with a phthalazine-1,4-dione core, instead of 1,4-naphthoquinone in the parent menaquinones, and a prenyl substituent corresponding in length to that of MK-1 to MK-4 (**79**–**82**), were designed. The corresponding ω-carboxylated compounds **83**–**86** were also synthesized. Phthalazine-1,4-dione is a heterocycle bearing two carbonyl groups, like 1,4-naphthoquinone, enabling us to probe the role of the naphthoquinone moiety in the antitumor effect of the parent menaquinone derivatives. Biological evaluation revealed that the compounds bearing an intact isoprene chain, such as geranyl

#### **Figure 9.**

*Structure of menaquinone-based phthalazine-1,4-dione derivatives* **79***–***86***.*

derivative **80**, exhibited potent anti-proliferative activity toward JHH7 cells, and the growth-inhibitory effects on normal hepatocytes were smaller than those on JHH7 cells. On the other hand, phthalazine-1,4-dione derivatives bearing a ω-carboxylated side chain were mostly inactive toward JHH7, in contrast to the corresponding naphthoquinone derivatives [66]. The SAR of phthalazine-1,4-dione derivatives was different from that of naphthoquinone derivatives. Further investigation of the mechanism of the anti-proliferative effect of phthalazine-1,4-dione derivatives might provide an improved understanding of the possibilities for chemoprevention of HCC (**Figure 9**).

## **5. Conclusion**

Vitamin K derivatives are attractive lead compounds for drug discovery. In this chapter, three topics in the medicinal chemistry of vitamin K, namely, SXR modulation, neural differentiation, and antitumor effect, were covered. Structure–activity relationship study of menaquinone-based SXR ligands has provided detailed information on the SXR-ligand recognition profile, contributing to the further development of novel SXR modulators. Neuronal differentiation-inducing compounds would be useful as chemical tools to probe signaling pathways that control neuronal specification, and also as candidate therapeutic agents for the treatment of neural diseases. The antitumor activity of vitamin K and its derivatives is also of great interest. Various studies have revealed that Cdc25 is an important target of the antitumor effect of naphthoquinone derivatives, including Cpd 5 and related compounds, and caspase- and transglutaminase-dependent pathways are also potential targets of vitamin K-based anti-HCC agents. Further investigation of the mechanism of the anti-proliferative effect of menaquinone derivatives might lead to agents for the chemoprevention of HCC.

*Recent Advances in the Medicinal Chemistry of Vitamin K Derivatives: An Overview (2000–2021) DOI: http://dx.doi.org/10.5772/intechopen.101667*

## **Author details**

Shinya Fujii1 , Yoshitomo Suhara<sup>2</sup> and Hiroyuki Kagechika1 \*

1 Institute of Biomaterials and Bioengineering, Tokyo Medical and Dental University, Tokyo, Japan

2 Faculty of Bioscience and Engineering, College of Systems Engineering and Science, Shibaura Institute of Technology, Saitama, Japan

\*Address all correspondence to: kage.chem@tmd.ac.jp

© 2022 The Author(s). Licensee IntechOpen. 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.

## **References**

[1] Litwack G, editor. Vitamin K, Vitamins and Hormones. Vol. 78. New York: Elsevier Academic Press; 2008

[2] Gordeladze JO, editor. Vitamin K2 - Vital for Health and Wellbeing. Rijeka: IntechOpen; 2017

[3] Prasad KN, Edwards-Prasad J, Sakamoto A. Vitamin K3 (menadione) inhibits the growth of mammalian tumor cells in culture. Life Sciences. 1981;**29**:1387-1392

[4] Ngo EO, Sun T-P, Chang J-Y, Wang C-C, Chi K-H, Cheng A-L, et al. Menadione-induced DNA damage in a human tumor cell line. Biochemical Pharmacology. 1991;**42**:1961-1968

[5] Wu FY-H, Chang N-T, Chen WJ, Juan C-C. Vitamin K3-induced cell cycle arrest and apoptotic cell death are accompanied by altered expression of c-fos and c-myc in nasopharyngeal carcinoma cells. Oncogene. 1993;**8**: 2237-2244

[6] Juan C-C, Wu FY-H. Vitamin K3 inhibits growth of human hepatoma HepG2 cells by decreasing activities of both p34-Cdc2 kinase and phosphatase. Biochemical and Biophysical Research Communications. 1993;**190**:907-913

[7] Lamson DW, Plaza SM. The anticancer effects of vitamin K. Alternative Medicine Review. 2003;**8**:303-318

[8] Wu FYH, Liao WC, Chang HM. Comparison of antitumor activity of vitamins K1, K2 and K3 on human tumor cells by two (MTT and SRB) cell viability assays. Life Sciences. 1993;**52**:1797-1804

[9] Miyazawa K, Yaguchi M, Funato K, Gotoh A, Kawanishi Y, Nishizawa Y, et al. Apoptosis/differentiation-inducing effects of vitamin K2 on HL-60 cells: Dichotomous nature of vitamin K2 in leukemia cells. Leukemia. 2001;**15**: 1111-1117

[10] Shibayama-Imazu T, Sakairi S, Watanabe A, Aiuchi T, Nakajo S, Nakaya K. Vitamin K2 selectively induced apoptosis in ovarian TYK-nu and pancreatic MIA PaCa-2 cells out of eight solid tumor cell lines through a mechanism different from geranylgeraniol. Journal of Cancer Research and Clinical Oncology. 2003;**129**:1-11

[11] Yoshida T, Miyazawa K, Kasuga I, Yokoyama T, Minemura K, Ustumi K, et al. Apoptosis induction of vitamin K2 in lung carcinoma cell lines: the possibility of vitamin K2 therapy for lung cancer. International Journal of Oncology. 2003;**23**:627-632

[12] Tabb MM, Sun A, Zhou C, Grün F, Errandi J, Romero K, et al. Vitamin K2 regulation of bone homeostasis is mediated by the steroid and xenobiotic receptor SXR. The Journal of Biological Chemistry. 2003;**278**:43919-43927

[13] Ichikawa T, Horie-Inoue K, Ikeda K, Blumberg B, Inoue S. Steroid and xenobiotic receptor SXR mediates vitamin K2-activated transcription of extracellular matrix-related genes and collagen accumulation in osteoblastic cells. The Journal of Biological Chemistry. 2006;**281**:16927-16934

[14] Li J, Lin JC, Wang H, Peterson JW, Furie BC, Furie B, et al. Novel role of vitamin k in preventing oxidative injury to developing oligodendrocytes and neurons. The Journal of Neuroscience. 2003;**23**:5816-5826

*Recent Advances in the Medicinal Chemistry of Vitamin K Derivatives: An Overview (2000–2021) DOI: http://dx.doi.org/10.5772/intechopen.101667*

[15] Ohsaki Y, Shirakawa H, Miura A, Giriwono PE, Sato S, Ohashi A, et al. Vitamin K suppresses the lipopolysaccharide-induced expression of inflammatory cytokines in cultured macrophage-like cells via the inhibition of the activation of nuclear factor kB through the repression of IKKα/β phosphorylation. The Journal of Nutritional Biochemistry. 2010;**21**: 1120-1126

[16] Spronk HMH, Soute BAM, Schurgers LJ, Thijssen HHW, De Mey JGR, Vermeer C. Tissue-specific utilization of menaquinone-4 results in the prevention of arterial calcification in warfarin-treated rats. Journal of Vascular Research. 2003;**40**:531-537

[17] Otsuka M, Kato N, Shao RX, Hoshida Y, Ijichi H, Koike Y, et al. Vitamin K2 inhibits the growth and invasiveness of hepatocellular carcinoma cells via protein kinase A activation. Hepatology. 2004;**40**:243-251

[18] Habu D, Shiomi S, Tamori A, Takeda T, Tanaka T, Kubo S, et al. Role of vitamin K2 in the development of hepatocellular carcinoma in women with viral cirrhosis of the liver. JAMA. 2004;**292**:358-361

[19] Azuma K, Ouchi Y, Inoue S. Vitamin K: novel molecular mechanisms of action and its roles in osteoporosis. Geriatrics & Gerontology International. 2014;**14**:1-7

[20] Suhara Y, Hanada N, Okitsu T, Sakai M, Watanabe M, Nakagawa K, et al. Structure−activity relationship of novel menaquinone-4 analogues: modification of the side chain affects their biological activities. Journal of Medicinal Chemistry. 2012;**55**:1553-1558

[21] Suhara Y, Watanabe M, Motoyoshi S, Nakagawa K, Wada A, Takeda K, et al.

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. Journal of Medicinal Chemistry. 2011;**54**:4918-4922

[22] Hirota Y, Tsugawa N, Nakagawa K, Suhara Y, Tanaka K, Uchino Y, et al. Menadione (vitamin K3) is a catabolic product of oral phylloquinone (vitamin K1) in the intestine and a circulating precursor of tissue menaquinone-4 (vitamin K2) in rats. The Journal of Biological Chemistry. 2013;**288**: 33071-33080

[23] Suhara Y, Watanabe M, Nakagawa K, Wada A, Ito Y, Takeda K, et al. Synthesis of novel vitamin K2 analogues with modification at the ω-terminal position and their biological evaluation as potent steroid and xenobiotic receptor (SXR) agonists. Journal of Medicinal Chemistry. 2011;**54**:4269-4273

[24] Josey BJ, Inks ES, Wen X, Chou CJ. Structure-activity relationship study of vitamin K derivatives yields highly potent neuroprotective agents. Journal of Medicinal Chemistry. 2013;**56**:1007-1022

[25] Moghadam BF, Fereidoni M. Neuroprotective effect of menaquinone-4(MK-4) on transient global cerebral ischemia/reperfusion injury in rat. PLoS One. 2020;**15**:e0229769

[26] Nakayama T, Asami S, Ono S, Miura M, Hayasaka M, Yoshida Y, et al. Effect of cell differentiation for neuroblastoma by vitamin K analogs. Japanese Journal of Clinical Oncology. 2009;**39**:251-259

[27] Eriksson PS, Perlieva E, Bjork-Eriksson T, Alborn AM, Nordborg C, Peterson DA, et al. Neurogenesis in the adult human hippocampus. Nature Medicine. 1998;**4**:1313-1317

[28] Suhara Y, Hirota Y, Hanada N, Nishina S, Eguchi S, Sakane R, et al. Synthetic small molecules derived from natural vitamin K homologues that induce selective neuronal deffirentiation of neuronal progenitor cells. Journal of Medicinal Chemistry. 2015;**58**: 7088-7092

[29] Kimura K, Hirota Y, Kuwahara S, Takeuchi A, Tode C, Wada A, et al. 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. Journal of Medicinal Chemistry. 2017;**60**:2591-2596

[30] Sakane R, Kimura K, Hirota Y, Ishizawa M, Takagi Y, Wada A, et al. Synthesis of novel vitamin K derivatives with alkylated phenyl groups introduced at the ω-terminal side chain and evaluation of their neural differentiation activities. Bioorganic & Medicinal Chemistry Letters. 2017;**27**:4881-4884

[31] Warashina M, Min KH, Kuwabara T, Huynh A, Gage FH, Schultz PG, et al. A synthetic small molecule that induces neuronal differentiation of adult hippocampal neural progenitor cells. Angewandte Chemie (International Ed. in English). 2006;**45**:591-593

[32] Kakeya H, Takahashi I, Okada G, Isono K, Osada H. Epolactaene, a novel neuritogenic compound in human neuroblastoma cells, produced by a marine fungus. Journal of Antibiotics (Tokyo). 1995;**48**:733-735

[33] Yu S, Levi L, Siegel R, Noy N. Retinoic acid induces neurogenesis by activating both retinoic acid receptors (RARs) and peroxisome proliferatoractivated receptor β/δ (PPARβ/δ). The Journal of Biological Chemistry. 2012;**287**:42195-42205

[34] Nishikawa Y, Carr BI, Wang M, Kar S, Finn F, Dowd P, et al. Growth inhibition of hepatoma cells induced by vitamin K and its analogs. The Journal of Biological Chemistry. 1995;**270**: 28304-28310

[35] Ni R, Nishikawa Y, Carr BI. Cell growth inhibition by a novel vitamin K is associated with induction of protein tyrosine phosphorylation. The Journal of Biological Chemistry. 1998;**273**: 9906-9911

[36] Nishikawa Y, Wang Z, Kerns J, Wilcox CS, Carr BI. Inhibition of hepatoma cell growth in vitro by arylating and non-arylating K vitamin analogs. Significance of protein tyrosine phosphatase inhibition. The Journal of Biological Chemistry. 1999;**274**: 34803-34810

[37] Tamura K, Southwick EC, Kerns J, Rosi K, Carr BI, Wilcox C, et al. Cdc25 inhibition and cell cycle arrest by a synthetic thioalkyl vitamin K analogue. Cancer Research. 2000;**60**:1317-1325

[38] Lazo JS, Nemoto K, Pestell KE, Cooley K, Southwick EC, Mitchell DA, et al. Identification of a potent and selective pharmacophore for Cdc25 dual specificity phosphatase inhibitors. Molecular Pharmacology. 2002;**61**: 720-728

[39] Peyregne VP, Kar S, Ham SW, Wang M, Wang Z, Carr BI. Novel hydroxyl naphthoquinones with potent Cdc25 antagonizing and growth inhibitory properties. Molecular Cancer Therapeutics. 2005;**4**:595-602

[40] Kar S, Wang M, Ham SW, Carr BI. Fluorinated Cpd 5, a pure arylating K-vitamin derivative, inhibits human hepatoma cell growth by inhibiting Cdc25 and activating MAPK.

*Recent Advances in the Medicinal Chemistry of Vitamin K Derivatives: An Overview (2000–2021) DOI: http://dx.doi.org/10.5772/intechopen.101667*

Biochemical Pharmacology. 2006;**72**:1217-1227

[41] Kar S, Wang M, Yao W, Michejda CJ, Carr BI. PM-20, a novel inhibitor of Cdc25A, induces extracellular signalregulated kinase 1/2 phosphorylation and inhibits hepatocellular carcinoma growth in vitro and in vivo. Molecular Cancer Therapeutics. 2006;**5**:1511-1519

[42] Brun M-P, Braud E, Angotti D, Mondésert O, Quaranta M, Montes M, et al. Design, synthesis, and biological evaluation of novel naphthoquinone derivatives with CDC25 phosphatase inhibitory activity. Bioorganic & Medicinal Chemistry. 2005;**13**: 4871-4879

[43] Braud E, Goddard M-L, Kolb S, Brun M-P, Mondésert O, Quaranta M, et al. Novel naphthoquinone and quinolinedione inhibitors of CDC25 phosphatase activity with antiproliferative properties. Bioorganic & Medicinal Chemistry. 2008;**16**: 9040-9049

[44] Kitano T, Yoda H, Tabata K, Miura M, Toriyama M, Motohashi S, et al. Vitamin K3 analogs induce selective tumor cytotoxicity in neuroblastoma. Biological & Pharmaceutical Bulletin. 2012;**35**:617-623

[45] Contour-Galcera MO, Sidhu A, Prévost G, Bigg D, Ducommun B. What's new on CDC25 phosphatase inhibitors. Pharmacology & Therapeutics. 2007;**115**:1-12

[46] Lavecchia A, Di Giovanni C, Novellino E. CDC25 Phosphatase inhibitors: an update. Mini-Reviews Med Chem. 2012;**12**:62-73

[47] Schepetkin IA, Karpenko AS, Khlebnikov A, Shibinska MO, Levandovskiy IA, Kirpotina LN, et al. Synthesis, anticancer activity, and molecular modeling of 1,4-naphthoquinones that inhibit MKK7 and Cdc25. European Journal of Medicinal Chemistry. 2019;**183**:111719

[48] Liu JC, Granieri L, Shrestha M, Wang D-Y, Vorobieva I, Rubie EA, et al. Identification of CDC25 as a common therapeutic target for triple-negative breast cancer. Cell Reports. 2018;**23**: 112-126

[49] Mizuta T, Ozaki I. Hepatocellular carcinoma and vitamin K. Vitamins Hormones. 2008;**78**(Vitamin K): 435-442

[50] Mizuta T, Ozaki I, Egichi Y, Yasutake T, Kawazoe S, Fujimoto K, et al. The effect of menatetrenone, a vitamin K2 analog, on disease recurrence and survival in patients with hepatocellular carcinoma after curative treatment: a pilot study. Cancer. 2006;**106**:867-872

[51] Kakizaki S, Sohara N, Sato K, Suzuki H, Yanagisawa M, Nakajima H, et al. Preventive effects of vitamin K on recurrent disease in patients with hepatocellular carcinoma arising from hepatitis C viral infection. Journal of Gastroenterology and Hepatology. 2007;**22**:518-522

[52] Yoshida H, Shiratori Y, Kudo M, Shiina S, Mizuta T, Kojiro M, et al. Effect of vitamin K2 on the recurrence of hepatocellular carcinoma. Hepatology. 2011;**54**:532-540

[53] Wiss O, Gloor U. Absorption, distribution, storage, and metabolites of vitamins K and related quinones. Vitamins and Hormones. 1966;**24**: 575-586

[54] Watanabe M, Toyoda M, Imada I, Morimoto H. Ubiquinone and related compounds. XXVI. The urinary

metabolites of phylloquinone and alpha-tocopherol. Chemical & Pharmaceutical Bulletin. 1974;**22** 176-182

[55] McBurney A, Shearer MJ, Barkhan P. Preparative isolation and characterization of the urinary aglycones of vitamin K1 (phylloquinone) in man. Biochemical Medicine. 1980;**24**:250-267

[56] Tadano K, Yuzuriha T, Sato T, Fujita T, Shimada K, Hashimoto K, et al. Identification of menaquinone-4 metabolites in the rat. Journal of Pharmacobio-Dynamics. 1989;**12**: 640-645

[57] Watanabe M, Kawada M, Nishikawa M, Imada I, Morimoto H. Ubiquinone and related compounds. XXVII. Synthesis of urinary metabolites of phylloquinone and α-tocopherol. Chemical & Pharmaceutical Bulletin. 1974;**22**:566-575

[58] Watanabe M, Okamoto K, Imada I, Morimoto H. Ubiquinone and related compounds. XXXI. Synthesis of urinary metabolites of ubiquinone, phylloquinone, α-tocopherol and related compounds. Chemical & Pharmaceutical Bulletin. 1978;**26**:774-783

[59] Teitelbaum AM, Scian M, Nelson WL, Rettie AE. Efficient syntheses of vitamin K chain-shortened acid metabolites. Synthesis. 2105;**47**: 944-948

[60] Okamoto K, Watanabe M, Kawada M, Goto G, Ashida Y, Oda K, et al. Synthesis of quinones having carboxy- and hydroxyl-alkyl side chains, and their effects on rat-liver lysosomal membrane. Chemical & Pharmaceutical Bulletin. 1982;**30**:2797-2819

[61] Terao S, Shiraishi M, Kato K, Ohkawa S, Ashida Y, Maki Y. Quinones. Part 2. General synthetic routes to quinone derivatives with modified polyprenyl side chains and the inhibitory effects of these quinones on the generation of the slow reacting substance of anaphylaxis (SRS-A). Journal of the Chemical Society, Perkin Transactions. 1982;**1**:2909-2920

[62] Fujii S, Shimizu A, Takeda N, Oguchi K, Katsurai T, Shirakawa H, et al. Systematic synthesis and antiinflammatory activity of ω-carboxylated menaquinone derivatives – Investigation on identified and putative vitamin K metabolites. Bioorganic & Medicinal Chemistry. 2015;**23**:2344-2352

[63] McDonald MG, Yeung CK, Teitelbaum AM, Johnson AL, Fujii S, Kagechika H, et al. A new LC-MS assay for the quantitative analysis of vitamin K metabolites in human urine. Journal of Lipid Research. 2019;**60**:892-899

[64] Karasawa S, Azuma M, Kasama T, Sakamoto S, Kabe Y, Imai T, et al. Vitamin K2 covalently binds to Bak and induces Bak-mediated apoptosis. Molecular Pharmacology. 2013;**83**: 613-620

[65] Qin X-Y, Fujii S, Shimizu A, Kagechika H, Kojima S. Carboxylic derivatives of vitamin K2 inhibit hepatocellular carcinoma cell growth through caspase/transglutaminaserelated signaling pathways. J Nutrit Sci Vitaminol. 2015;**61**:285-290

[66] Fujii S, Miura T, Oikawa T, Qin X-Y, Kojima S, Kagechika H. Design, synthesis and antitumor activity of phthalazine-1,4-dione-based menaquinone analogs. Bioorganic & Medicinal Chemistry Letters. 2021;**43**:128065

## **Chapter 2**
