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

Article abstract of DOI:10.1016/j.bmcl.2016.11.073

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.

Full text of DOI:10.1016/j.bmcl.2016.11.073

Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis of 2-methyl-1,4-naphthoquinones with higher gamma-
glutamyl carboxylase activity than MK-4 both in vitro and in vivo
⇑
Cees Vermeer , Cynthia van ’t Hoofd, Marjo H.J. Knapen, Sofia Xanthoulea
R&D Group VitaK, Maastricht University, Oxfordlaan 70, 6229 EV Maastricht, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
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 vita-
min 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 vita-
min K derivatives may have higher bioactivity and different pharmacological characteristics that are
more favorable for use as supplements or in clinical settings.
Received 19 October 2016
Revised 22 November 2016
Accepted 23 November 2016
Available online xxxx
Keywords:
2
-Methyl-1,4-naphthoquinone
Vitamin K
Phylloquinone
Menaquinone
Osteocalcin
Ó 2016 Elsevier Ltd. All rights reserved.
Vitamin K is the common designation for compounds sharing a
-methyl-1,4-naphthoquinone ring, but differing in side-chain
a diversity of regulatory functions in important physiological
processes, including bone mineralization (osteocalcin (OC), synthe-
sized by osteoblasts) and vascular calcification (matrix Gla-protein,
2
1
structure at the 3-position. Natural vitamin K exists in different
forms: it is present in plants as vitamin K1 (phylloquinone, struc-
ture 1 in Fig. 1) and produced by bacteria as vitamin K2 (menaqui-
nones, MK-n, structure 2).2
9
,10
synthesized by vascular smooth muscle cells).
Chemical synthesis may yield entirely new compounds with
vitamin K activity and pharmacological properties different from
those of the natural homologues 1 and 2, that may in turn lead
to novel commercially interesting biologically active drugs. A lim-
ited number of papers have described the synthesis of vitamin K
analogues and their biological activities. Suhara et al. synthesized
–4
Phylloquinone has a phytyl side-chain containing four iso-
prenoid residues (one unsaturated) and is mainly found in green
leafy vegetables. Menaquinones have a side-chain containing mul-
tiple unsaturated isoprenoid residues (n), where the length of the
side-chain governs important physicochemical properties, includ-
two analogues with hydroxyl or phenyl groups at the
x-terminal
1
–5
6
11
ing lipophilicity
and kinetic constants for cofactor function.
of the side-chain. The same group also synthesized vitamin K
analogues with demethylation or reduction of the double bonds
of the side-chain of menaquinone-4 (MK-4) (structure 3 in
The main sources of menaquinones are meat (MK-4, structure 3),
fermented foods like cheese (MK-8 and MK-9) and fermented
soybeans known as natto (MK-7, structure 4). Synthetic forms of
vitamin K include menadione (structure 5 in Scheme 1), a water-
soluble compound lacking vitamin K activity by itself but which
1
2
Fig. 1). The authors concluded that more potent ligands may arise
if new analogues are constructed with the following structural fea-
tures: maintenance of the double bonds of the side-chain and sub-
stitution of one of the methyl groups with other functional groups.
In this study we provide proof-of-concept for a novel synthesis
route enabling researchers to create a broad spectrum of 2-methyl-
1,4-naphthoquione derivatives with high vitamin K activity. We
report the synthesis of several examples of such derivatives and
demonstrate their biological activity both in vitro and in vivo.
Structures of compounds referred below are indicated in
Schemes 1 and 2 and detailed synthesis steps are provided in
Supplementary data. Briefly, ethyl bromoacetate and pyridine
–
like phylloquinone – may be converted into active MK-4 in sev-
7
eral tissues.
Vitamin K drives the posttranslational carboxylation of gluta-
mate (Glu) residues into -carboxyglutamate (Gla), which are pre-
c
8
sent in the Gla-protein family, conferring them functionality.
Beyond their central role in blood coagulation, Gla-proteins have
⇑
Corresponding author.
E-mail address: c.vermeer@vitak.com (C. Vermeer).
http://dx.doi.org/10.1016/j.bmcl.2016.11.073
960-894X/Ó 2016 Elsevier Ltd. All rights reserved.
0
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2
C. Vermeer et al. / Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
amounts of cOC were produced, which is consistent with minimal
6
vitamin K activity. As previously found , menadione (compound 5
in Scheme 2) was not biologically active. In line with this, 6 and its
reduced form 7 showed no activity in the in vitro experiments. The
ethylester of 7, i.e. 8, exhibited almost no activity (data not shown).
However, as shown in Fig. 2, its oxidized form 9, and the carboxylic
acid forms 10–11 showed substantially reduced ucOC levels and
significantly increased cOC levels compared to MK-4 (compound
3). These data demonstrate that vitamin K bioactivity of com-
pounds 9–11 was higher than that of MK-4.
Longer side-chain derivatives may possess even higher vitamin
K activity, and therefore, we developed and tested compounds 12–
1
5 (depicted in Scheme 2) in an analogous follow-up experiment.
Levels of ucOC in supernatants of cells were lower than controls
where no vitamin K or vitamin K analogue was added), in all com-
(
pounds tested (3 and 12–15) (Fig. 3) with a stronger effect seen for
compounds 13 (C-16 tail) and 15 (C-14 tail) (lines of 3 and 15 are
superimposed in the graph). Comparably, compounds 13 and 15
resulted in the most efficient production of cOC, with concentra-
tions after 72 h that were approximately 2-fold higher for com-
pound 13 and 2.5-fold higher for compound 15, respectively,
compared to compound 3. Cells supplemented with 12 (C-17 tail)
and 14 (C-18 tail) produced minimal levels of cOC.
Fig. 1. Structures of natural vitamin K homologues. 1: phylloquinone (K1); 2:
menaquinones (K2, general structure); 3: menaquinone-4 (MK-4); 4: menaqui-
none-7 (MK-7).
served as starting materials providing an ylide. Next, the ylide
reacted with compound 5 to give compound 9. Subsequently, com-
pound 9 was further reduced with tetrabutylammonium iodide
producing compound 8. Then, the ester was hydrolyzed yielding
compound 11. Finally, compound 11 can react with any primary
amine. Re-oxidation can follow through standard treatment with
ceric ammonium nitrate. The structures of the final products were
verified by NMR spectroscopy (see Supplementary data).
These results indicate that while the shortest tail (compound
15, C-14) resulted in the highest potency with 2.5 times higher
activity than MK-4 (compound 3), more than 16 C-atoms appeared
to be detrimental to activity.
Based on these results, we selected compound 15 in order to
further characterize it in WKY rats. Two compound 15 enriched
diets were prepared: a high-dose diet containing 5
pound 15/g of food and a low-dose diet containing 0.5
l
l
g of com-
g of com-
The bioactivity of compounds 5–15 was defined by their capa-
pound 15/g of food. The low-dose was selected in order to provide
the minimal amount of vitamin K required for normal coagulation
activity in rats. At baseline (after one-week of vitamin K-deficient
diet (VKDD) feeding), four rats were sacrificed (t = 0; baseline,
Fig. 4). The remaining 20 rats were randomly assigned to either
receive the VKDD alone for 2 or 5 days (controls, n = 8), the low-
dose compound 15 diet for 5 days (n = 4) or the high-dose com-
pound 15 diet for either 2 or 5 days (n = 8). Rats were sacrificed
after two or five days to study short-term or long-term effects,
respectively. Body weights were determined before sacrifice and
bility to serve as agonists for the endoplasmic enzyme
c-glutamyl
carboxylase, and thus to promote the formation of -carboxy glu-
c
tamate residues in proteins belonging to the Gla-protein family.
We used the human osteosarcoma cell line MG63 and determined
the levels of uncarboxylated (ucOC) and carboxylated (cOC) osteo-
calcin in the culture media. No cytotoxicity, measured as cell via-
bility, of the vitamin K derivatives 5–15 was observed with
concentrations up to 500 nM (data not shown). In controls, where
no vitamin K or vitamin K analogue was added, no measurable
Scheme 1. Schematic representation of the steps for synthesis of the 2-methyl-1,4-naphthoquinone derivatives.
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C. Vermeer et al. / Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
3
Scheme 2. Structures of novel synthesized 2-methyl-1,4-naphthoquinone derivatives with different side-chains at the 3-position. The lower structure shows compound 15
the C-14 tailed derivative), while compounds 12, 13 and 14 refer to structures with a C-17, a C-16 and a C-18 tail, respectively.
(
Fig. 2. Effect of vitamin K derivatives on the synthesis of uncarboxylated (ucOC) and carboxylated (cOC) osteocalcin by human MG63 cells. In a first experiment, the effect of
compounds 3 (MK-4) and 9–11 on the synthesis of ucOC (left) and cOC (right) osteocalcin was studied. Experiments were performed in triplicates.
Fig. 3. The effect of compounds 3 and 12–15 on the production of uncarboxylated (ucOC) (left) and carboxylated (cOC) (right) osteocalcin was investigated. Experiments were
performed in triplicates.
no substantial differences were observed between the different
treatments (data not shown).
the low-dose compound 15 diet for 5 days, increased plasma pro-
thrombin levels back to normal. Therefore, even at the lowest dose
equaling the minimal dose of vitamin K necessary for normal blood
coagulation, 15 was able to quickly restore prothrombin activity.
Vitamin K bioactivity was determined by assessing circulating
levels of prothrombin, since vitamin K is known to promote pro-
thrombin biosynthesis is the rat model system.13 In male WKY rats,
plasma prothrombin levels decreased by approximately 40% after
one-week of VKDD feeding (Table 1; baseline). Supplementing
These results indicate that 15 may serve as a cofactor for
c-car-
boxylation of the vitamin K-dependent coagulation factors synthe-
sized in the liver. Administration of the high-dose compound 15
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4
C. Vermeer et al. / Bioorganic & Medicinal Chemistry Letters xxx (2016) xxx–xxx
active drug. The data presented here are, however, only proof-of-
principle and further studies are required to compare the activity
of the compound 15 or other vitamin K analogues to the natural
vitamin K homologues like vitamin K1, MK-4 and MK-7 in
in vitro and in vivo models.
Acknowledgements
The authors would like to thank Dr. Ketie Saralidze and Prof. Dr.
Leo H. Koole from the Department of Biomedical Engineering,
Maastricht University, The Netherlands, for assistance with synthe-
sis of the compounds. This study was supported by Nattopharma
(
Høvik, Norway), which was not involved in conducting the
research or in the preparation of the article.
A. Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2016.11.
Fig. 4. Study design of a rat study for the determination of the in vivo biological
activity of derivative 15.
0
73.
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methyl-1,4-naphthoquinone derivatives and demonstrate their
biological activity as carboxylase agonists. Most of the vitamin K
derivatives possessed vitamin K activity and this differed depend-
ing on the structural characteristics of the side-chain: while com-
pounds 13 (C-16 tail) and 15 (C-14 tail) showed strong
bioactivity, compounds 12 (C-17 tail) and 14 (C-18 tail) showed
only weak activity.
1
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Please cite this article in press as: Vermeer C., et al. Bioorg. Med. Chem. Lett. (2016), http://dx.doi.org/10.1016/j.bmcl.2016.11.073