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

Article abstract of DOI:10.1021/jm200201k

Vitamin K₂ 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.

Full text of DOI:10.1021/jm200201k

BRIEF ARTICLE
pubs.acs.org/jmc
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
Yoshitomo Suhara,† Masato Watanabe,§ Sayaka Motoyoshi,^ Kimie Nakagawa,§ Akimori Wada,||
Kazuyoshi Takeda,‡ Kazuhiko Takahashi,† Hiroaki Tokiwa,^ and Toshio Okano*,§
†Laboratory of Environmental Sciences, and ‡Department of Medicinal Chemistry, Yokohama College of Pharmacy,
601 Matano-cho, Totsuka-ku, Yokohama 245-0066, Japan
§Department of Hygienic Sciences, and Department of Organic Chemistry for Life Science, Kobe Pharmaceutical University,
4-19-1, Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan
^Department of Chemistry, Faculty of Science, Rikkyo University, 3-34-1 Nishi-Ikebukuro, Toshima-ku, Tokyo 171-8501, Japan
S Supporting Information
b
ABSTRACT: Vitamin K2 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.
’ INTRODUCTION
vitamin K homologues. We predicted that the structure of the side
chain would play an important role in biological activities, since the
structural differences of the homologues are confined to only the
isoprene units. To evaluate the effects of the side chains of vitamin
K, we investigated SXR-mediated transcriptional activity of new
vitamin K analogues in comparison with vitamin K homologues.
We considered that the introduction of dual side chains at the C-2
and C-3 positions of vitamin K homologues (Figure 2) would
significantly increased activity. Indeed, some of the synthetic
analogues may have biological activity that exceeds that of the
natural homologues. In addition, if the new compounds prove to
be useful substrates, they may contribute to future investigations of
the mechanism that converts vitamin K analogues into MK-4 and
the compounds may clarify other biological actions of vitamin K.
In this study, we report the synthesis of new vitamin K analogues
and the results of SXR-mediated gene transcription.
Vitamin K serves as an essential cofactor of the γ-carboxylase,
an enzyme that transforms glutamic acid residues within specific
substrate proteins into γ-carboxyglutamic acid (Gla) residues, and
is therefore involved in the activation of the cascade of proteins for
blood coagulation.1 Recent investigations indicate that vitamin K
is also required for the synthesis of other calcium-binding proteins
such as bone Gla protein (osteocalcin), matrix Gla protein, protein
S, and gas6.2ꢀ4 The two molecular forms of “natural” vitamin K
can be distinguished by the differences of the alkyl side chains at
the 3-position of the common 2-methyl-1,4-naphthoquinone group
(Figure 1). Plant-derived vitamin K1 (1) (phylloquinone, PK)
contains the phytyl group as the side chain, while the bacterial-
derived vitamin K2 (menaquinone-n, MK-n) (2(MK-1), 3(MK-2),
4 (MK-3), and 5 (MK-4)) carries a polyisoprenyl side chain.
Vitamin K3 (6) (menadione, 2-methyl-1,4-naphthoquinone) has
been defined as a “synthetic” vitamin K analogue that lacks the
side chain at the C-3 position.
’ CHEMISTRY: SYNTHESIS OF VITAMIN K ANALOGUES
Menaquinone-4 (MK-4), one of the menaquinone homolo-
gues, showed additional biological activities related to gene tran-
scription through the steroid and xenobiotic receptor (SXR).5ꢀ10
MK-4 also protected oligodendrocyte precursors and immature
fetal cortical neurons from oxidative injury, independent of the
vitamin K-dependent γ-carboxylative reaction.11 There is con-
sistent evidence that the MK-4 is produced by conversion from
other dietary vitamin K homologues.12 We recently confirmed
that dietary PK was converted into MK-4 and then was accumu-
lated in various tissues in a high concentration.13
This background information prompted us to focus on the
SXR-mediated transcriptional activity and the biological roles of
the side chains of vitamin K analogues. No studies have examined
the biological activities of structure-modified vitamin
K analogues even though many reports have investigated natural
Vitamin K homologues 1ꢀ5 were a generous gift of Eisai Co.,
Ltd. We first planned to synthesize the requisite vitamin K
analogues through coupling of 1,4-naphthoquinone (12) and the
desired side chain moiety as outlined in Scheme 1. For the synthesis
of the analogues, we chose phyltol (14), prenol (15), geraniol (16),
farnesol (17), geranylgeraniol (18), and 12 as starting materials,
and the coupling reactions with the naphthoquinone part were
carried using our reported method.14 12 was vigorously stirred with
a 10% sodium hydrosulfite solution (aq) and Et2O to form
hydroquinone 13, to which 2 equiv of isoprene units 14ꢀ18 were
successively coupled in the presence of a catalytic amount of
BF3 Et2O. The resulting hydroquinone derivatives were oxidized
3
Received: February 22, 2011
Published: May 27, 2011
r
2011 American Chemical Society
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Journal of Medicinal Chemistry
BRIEF ARTICLE
to the respective quinones under atmospheric conditions. Thus,
the corresponding “double side chain” vitamin K analogues (PK-W
and MK-n-W) (7 (PK-W), 8 (MK-1-W), 9 (MK-2-W), 10 (MK-3-
W), and 11 (MK-4-W)) were obtained in 37ꢀ57% yields. When
the side chain was longer than geranylfarnesol, the chemical yield
was not satisfactory. Presumably, the bulkiness of the side chain
moiety affected the reactivity with 12.
pro luciferase reporter vector and with pRL-CMV using Lipo-
fectamine in the case of the CYP3A4 promoter assay. The SXR-
GAL4 assay measured SXR ligand-binding domain (LBD) driven
transcription, while the CYP3A4 promoter assay evaluated SXR/
RXR heterodimer-driven transcription.
All compounds were tested in the SXR-GAL4 assay at a fixed
concentration of 5 ꢁ 10ꢀ6 M. Among natural vitamin K homo-
logues, the transcriptional activity as an indicator of binding to
SXR increased in the order of 2 < 3 < 4 and then slightly decreased
in the case of 5. Among the double side chain analogues, 8 and 9
exhibited the highest activity. 10, 11, 1, and 7 had almost no effect.
The analogues had similar effects on the transcriptional activi-
ty of CYP3A4 promoter as shown in Figure 4. 3 and 4 followed
by 5 exhibited the most potent activity among the vitamin
K homologues. 8 and 9 were the most effective vitamin K
analogues.
’ RESULTS
Evaluation of SXR-Mediated Transcriptional Activity. All
the synthetic ligands and the vitamin K homologues were tested
in assays that measured the SXR-mediated transcriptional activ-
ity. Luciferase-based assays of SXR-GAL4 and CYP3A4 promoter
were carried out with HepG2 human hepatocellular carcinoma
cells. SXR is well-known to control the gene expression of
CYP3A4. The cells were transfected with pM-SXR and pGVP2-
GAL2 luciferase reporter vector and with pRL-CMV using
Lipofectamine15 in the case of the SXR-GAL4 assay. The cells were
also treated with pcDNA3.1-FLAG-SXR and pGL4.10-CYP3A4-
These findings suggest that the menaquinones 3ꢀ5 might be
favorable ligands for SXR while 8 and 9 may be the most
favorable of the double side chain analogues. The data also
indicate that the side chain length at the C-2 and C-3 positions in
vitamin K analogues affected the binding affinity to SXR, with
side chains of geranyl units as optimum size.
Docking Studies. We performed in silico binding analysis of
MK-n and MK-n-W to the SXR-LBD17 based on the ASEDock
method using the MOE (Molecular Operating Environment)
program package (see Computational Details). The docking
results (Figure 5A) clearly show that 9 has the highest docking
score. In the stable docking pose of 9 (Figure 5B), the side chain
part and the naphthalene moiety of 9 stably interact with Ser247
and His407. On the other hand, 10 has a low docking score, since
the strain of the molecule was large enough. The low docking
score for 11 suggests that the formation of a stable complex with
the ligand-binding pocket (LBP) of SXR would be difficult.
Figure 1. Structures of vitamin K homologues phylloquinone (1),
menaquinones 2ꢀ5, and menadione (6).
’ DISCUSSION
We describe the synthesis and the pharmacological profile
related to SXR-mediated transcriptional activities of new vitamin
K analogues in which the same side chain moiety was symmetrically
introduced at both the 2 and 3 positions of 1,4-naphthoquinone.
SXR is a master gene orchestrating the expression of a large
family of genes involved in uptake, metabolism, and disposal of a
number of endo- and xenobiotics, including drugs, bile acids,
steroid hormones, and metabolic intermediates in mammalian
cells. Following ligand binding, SXR forms a heterodimer with
the retinoid X receptor (RXR) that binds to SXR response
elements (SXREs) located in the 50-flanking regions of SXR
target genes, resulting in their transcriptional activation.
Figure 2. Structures of vitamin K homologues and our synthesized
“double side chain” analogues.
Scheme 1. Synthesis of Vitamin K Analogues That Have
Double Side Chain Parta
To determine if vitamin K analogues have a role in regulating
SXR, we evaluated the transcriptional activity of the new analogues
and vitamin K homologues by twoluciferase-basedassay methods.
The one-hybrid luciferase assay with SXR-GAL4 permitted us to
examine only the general effects of ligand-binding affinity to SXR in
the transcription and excluded any influence of heterodimerization
withRXRandbindingtoSXREs. Thesecondluciferase-basedassay
including SXRE with CYP3A4 promoter allowed us to examine the
influences of heterodimerization with RXR and binding to SXREs.
With respect to ligand binding (Figure 3), the activity of natural
vitamin K2 homologues 3ꢀ5 increased in a manner dependent on
the length of the side chain moiety, being greatest with 4, bearing a
farnesyl unit, having the greatest effect. Among all the double side
chain analogues, 8 and 9 showed the most potent activity.
a Reagents and conditions: (a) 10% Na2S2O4 aq, ether, quantitative; (b)
BF3 OEt2, dioxane/ethyl acetate, 37ꢀ57%.
3
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Figure 3. Transcriptional activity with a one-hybrid luciferase assay
with SXR-GAL4. HepG2 cells were treated with each compound at
5.0 ꢁ 10ꢀ6 M. DMSO or EtOH indicates control cells that were treated
with the solvent, without ligands. RIF indicates cells treated with
rifampicin, a known ligand for SXR.16 The histogram data are expressed
as the mean obtained from three independent experiments; the error
bars indicate the SD. Significant difference between control group and
each compounds group is as follows: ///, p < 0.001 (by Dunnett’s test).
Significant differences between the MK-n group and the MK-n-W group
are as follows: ###, p < 0.001; ##, p < 0.01 (by Student’s t test).
Figure 5. SXR-LBD docking analysis based on ASEDock of MOE: (A)
the docking score (U-dock) of the MK-n-W/SXR complex; (B) the
docking pose of the 9/SXR-LBD complex. The LBP of the SXR-LBD is
shown in white. 9 and key residues (Ser247 and His407) are shown in
stick model.
activity of 3 and 4 corresponds to that of 8 and 9, respectively,
the side chain length affected the transcriptional activity. The size
of the cavity of the LBP might be in the range of the size of 9
because the activity of 10 was remarkably decreased. On the
other hand, in a comparison of 5 with 1, their side chain lengths
were almost the same; however, the transcriptional activities
were quite different. From these findings, we considered that
the spatial configuration of the side chain part was important.
Consequently, the binding affinity to SXR depended on the
length of the side chain part and the fitting of the quinone part
of vitamin K analogues to the LBP.
Currently, as an effective treatment for osteoporosis and
prevention of fractures, vitamin K including MK-4 is frequently
prescribed for osteoporosis in Japan.18,19 The daily dosage of
MK-4 for osteoporotic patients is 45 mg, affording a body
concentration that corresponds to 10ꢀ6 M, and the tissue levels
are much higher (>10ꢀ5 M).20,21 These concentrations are well
correlated with those used in our in vitro transcriptional assay.
Recent studies have demonstrated that SXR also up-regulated
the expression of osteoblastic markers and the factors related to
bone formation such as a small leucine-rich proteoglycan named
tsukushi, an extracellular matrix protein matrilin-2, CD14
antigen,9 and Msx2 gene.22 Therefore, strong SXR agonists
based on vitamin K analogues may be a source for entirely new
therapeutic drugs for osteoporosis in both aspects of transcrip-
tional activity and coenzyme activity of γ-carboxylative action.
Figure 4. Transcriptional activity with luciferase assay including SXRE
with CYP3A4 promoter. HepG2 cells were treated with each compound
at 5.0 ꢁ 10ꢀ6 M. DMSO or EtOH indicates control cells that were
treated with the solvent, without ligands. RIF indicates cells treated with
rifampicin, a known ligand for SXR.16 The histogram data indicate the
mean obtained from three independent experiments; the error bars
indicate the SD. Significant differences between control group and each
compounds group are as follows: ///, p < 0.001; /, p < 0.05 (by
Dunnett’s test). Significant differences between the MK-n group and the
MK-n-W group are as follows: ###, p < 0.001; ##, p < 0.01 (by Student’s t
test).
Similar tendencies were noted in the assay that evaluated
heterodimerization and binding to SXREs (Figure 4). Com-
pounds 8 and 9 effectively increased the expression of a well-
characterized SXR target gene, CYP3A4, in a human hepatocyte
cell line, compared with other analogues. 3 and 4 showed strong
activitycomparabletothatoftheestablishedSXR ligandrifampicin.
To explore the docking state of 8 and 9 exhibited potent
transcriptional activities, we examined molecular modeling of 9
with the LBD of SXR. The docking study showed hydrophilic
interactions between the oxygen atom of 9 and His407 and
Ser247, a part of the constituent amino acids of SXR (Figure 5).
Of the menaquinones, 3ꢀ5 may be the most favorable ligands
for SXR, and in the same way, 8 and 9 may be the most favorable
of the double side chain analogues. Since the transcriptional
’ CONCLUSION
We report for the first time the synthesis of new vitamin
K2 analogues that have additional side chains introduced at the
1,4-naphthoquinone, and we demonstrate their effects on the
induction of transcription. Compound 9 exhibited the most
potent activity among the double side chain analogues in se-
parate assays that evaluated ligand binding and the role of
heterodimerization in the induction of transcription, and this
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Journal of Medicinal Chemistry
BRIEF ARTICLE
compound was as effective as the known substrate rifampicin.
These compounds would be useful not only in the development
of new biologically active agents but also in the evaluation of
still unknown biological roles of vitamin K.
silica gel (n-hexane/AcOEt = 20:1), giving 10 (86 mg, 48%) as a yellow
1
oil: H NMR (500 MHz, CDCl3) δ 8.10ꢀ8.08 (2H, m), 7.70ꢀ7.69
(2H, m), 5.12ꢀ5.03 (6H, m), 3.39 (4H, d, J = 7.0 Hz), 2.09ꢀ1.91 (16H,
m), 1.80 (6H, s), 1.67 (6H, s), 1.57 (12H, s); 13C NMR (125 MHz,
CDCl3) δ 185.1, 146.1, 137.4, 135.2, 133.3, 132.2, 131.2, 126.2,
124.3, 123.9, 119.8, 39.8, 39.7, 26.7, 26.5, 25.9, 25.7, 17.6, 16.5, 16.0;
ESI-HRMS (M þ Hþ) m/z calcd for C40H55O2 567.4196. Found
567.4197.
’ EXPERIMENTAL SECTION
High-resolution ESI-MS spectra were obtained with a Micromass
Q-TOF mass spectrometer. 1H NMR spectra were recorded at
500 MHz, and 13C NMR spectra were recorded at 125 MHz using
CDCl3 as a solvent unless otherwise specified. Chemical shifts are given
in parts per million (δ) using tetramethylsilane (TMS) as the internal
standard. Column chromatography was carried out on silica gel 60
(70ꢀ230 mesh), and preparative thin layer chromatography (TLC) was
run on silica gel 60F254. Unless otherwise noted, all reagents were
purchased from commercial suppliers. Purity of compounds was con-
firmed by a HPLC system.
Synthesis of 7. To a solution of 1,4-naphthoquinone (12) (150 mg,
948 μmol) in ether (20 mL) was added a 10% Na2S2O4 aqueous solution
(20 mL). The mixture was stirred vigorously at 30 ꢀC for 1 h under
argon. After the yellow ether layer turned colorless, the mixture was
extracted with AcOEt (50 mL ꢁ 3). The combined organic layer was
washed with brine (50 mL ꢁ 3), dried over MgSO4, and concentrated to
afford crude hydroquinone 13. The residue was immediately dissolved
in AcOEt (2 mL) and dioxane (2 mL). Then phytol (14) (845 mg, 2.85
mmol) and boron trifluoride ether complex (200 μL) were added. The
mixture was stirred at 70 ꢀC for 3 h under argon and cooled to room
temperature. The mixture was poured into iceꢀwater and extracted with
AcOEt (50 mL ꢁ 3). The combined organic layer was washed with water
(50 mL) and brine (50 mL), dried over MgSO4, and concentrated. The
residue was purified by preparative TLC on silica gel (n-hexane/AcOEt =
20:1) to afford 7 (373 mg, 55%) as a yellow oil: 1H NMR (500 MHz,
CDCl3) δ 8.09ꢀ8.06 (2H, m), 7.69ꢀ7.67 (2H, m), 5.01 (2H, t, J = 7.0
Hz), 3.37 (4H, d, J = 7.0 Hz), 1.95ꢀ1.92 (4H, m), 1.77 (6H, s),
1.55ꢀ1.49 (2H, m), 1.36ꢀ0.99 (36H, m), 0.88ꢀ0.81 (total 24H, m);
13C NMR (125 MHz, CDCl3) δ 185.1, 146.1, 137.8, 133.3, 132.2, 126.2,
119.5, 40.1, 39.4, 37.5, 37.4, 37.3, 32.8, 32.7, 29.7, 28.0, 26.0, 25.3, 24.8,
24.5, 23.5, 22.7, 22.6, 19.7, 16.4; ESI-HRMS (M þ Hþ) m/z calcd for
C50H83O2 715.6388. Found 715.6391.
Synthesis of 11. Similar to the synthesis of 7 from 12, the crude
product 11, which was obtained from 12 (50 mg, 316 μmol), geranyl-
geraniol (276 mg, 949 μmol), and boron trifluoride ether complex
(70 μL) in AcOEt (1 mL) and dioxane (1 mL), was purified by prepa-
rative TLC on silica gel (n-hexane/AcOEt = 20:1), giving 11 (89 mg,
40%) as a yellow oil: 1H NMR (500 MHz, CDCl3) δ 8.08ꢀ8.06 (2H,
m), 7.70ꢀ7.67 (2H, m), 5.09ꢀ5.01 (8H, m), 3.37 (4H, d, J = 7.0 Hz),
2.09ꢀ1.91(24H, m), 1.79(6H, s), 1.67(6H, s), 1.59(6H, s), 1.56(12H, s);
13C NMR (125 MHz, CDCl3) δ 185.1, 146.1, 137.5, 135.2, 134.9, 133.3,
132.2, 131.2, 126.2, 124.4, 124.2, 123.9, 119.7, 39.8, 39.7, 26.8, 26.7, 26.5,
25.9, 25.7, 17.7, 16.5, 16.0; ESI-HRMS (M þ Hþ) m/z calcd for
C50H71O2 703.5449. Found 703.5445.
Analytical Method Used To Determine Purity of the
Compounds. HPLC was conducted with a Shimadzu HPLC system
(Shimadzu, Kyoto, Japan) consisting of a binary pump (LC-20AD liquid
chromatography), an automatic solvent degasser (DGU-20A3 degasser),
and a manual injector. Separations were carried out using a reversed-phase
C18 analytical column (COSMOSIL 5C18-AR-II; 4.6 mm i.d. ꢁ 250 mm)
(Nakalai Tesque, Kyoto, Japan) with a solvent system consisting of an
isocratic solvent. The solvent contained either methanol, ethanol, or water
was delivered at 1.0 mL/min. This mobile phase was passed through the
column at 1.0 mL/min. The column was maintained at 40 ꢀC with a
CTO-20A column oven. Vitamin K analogues were detected at 258 nm
with an SPD-M20A diode array detector. The HPLC system was
controlled by a CBM-20A system controller (Shimadzu). The purity
of the each compound was calculated from a surface integral of detected
peaks. Those data satisfied more than 95% purity.23
Cloning and Construction of cDNAs. Human steroid and
xenobiotic receptor (SXR) expression vector (pcDNA3.1-FLAG-SXR)
were generated by PCR using human genomic DNA as templates and
inserted in-frame into pcDNA3.1(þ) vector (Invitrogen) at EcoRI and
XhoI sites. The CYP3A4 luciferase reporter plasmid pGL3-CYP3A4pro
was constructed in our laboratory. The CYP3A4 promoter, from base
pairs ꢀ362 to þ53, and a distal enhancer module of CYP3A4 promoter,
from base pairs ꢀ7876 to ꢀ7208, were generated by polymerase chain
reaction (PCR) using the DNA template isolated from the human
genome DNA. These products were then subcloned into the pGL4.10
vector, a promoter-less luciferase reporter vector at NheI and BamHI
sites. Finally, pGL4.10-CYP3A4pro, CYP3A4 luciferase reporter plas-
mid, containing ꢀ7876 to ꢀ7208 and ꢀ362 to þ53 bp of the CYP3A4
proximal promoter was prepared.15
Synthesis of 8. Similar to the synthesis of 7 from 12, the crude
product 8, which was obtained from 12 (50 mg, 316 μmol), prenol (82
mg, 949 μmol), and boron trifluoride ether complex (70 μL) in AcOEt
(1 mL) and dioxane (1 mL), was purified by preparative TLC on silica
gel (n-hexane/AcOEt = 20:1), giving 8 (34 mg, 37%) as a yellow oil: 1H
NMR (500 MHz, CDCl3) δ 8.09ꢀ8.05 (2H, m), 7.70ꢀ7.67 (2H, m),
5.03ꢀ5.00 (2H, m), 3.36 (4H, d, J = 7.0 Hz), 1.79 (6H, s), 1.69 (6H, s);
13C NMR (125 MHz, CDCl3) δ 185.2, 150.0, 133.8, 133.3, 132.2, 126.2,
119.9, 26.1, 25.8, 18.1; ESI-HRMS (M þ Hþ) m/z calcd for C20H23O2
295.1693. Found 295.1693.
Synthesis of 9. Similar to the synthesis of 7 from 12, the crude
product 9, which was obtained from 12 (50 mg, 316 μmol), geraniol
(146 mg, 949 μmol), and boron trifluoride ether complex (70 μL) in
AcOEt (1 mL) and dioxane (1 mL), was purified by preparative TLC on
silica gel (n-hexane/AcOEt = 20:1), giving 9 (78 mg, 57%) as a yellow
oil: 1H NMR (500 MHz, CDCl3) δ 8.10ꢀ8.06 (2H, m), 7.70ꢀ7.67 (2H,
m),5.05ꢀ5.00 (4H, m), 3.37(4H, d, J=7.0 Hz), 2.08ꢀ1.96 (8H, m), 1.78
(6H, s), 1.63 (6H, s), 1.56 (6H, s); 13C NMR (125 MHz, CDCl3) δ 185.1,
146.1, 137.4, 133.3, 132.2, 131.5, 126.2, 124.1, 119.8, 39.7, 26.6, 25.9, 25.7,
17.7, 16.4; ESI-HRMS (M þ Hþ) m/z calcd for C30H39O2 431.2945.
Found 431.2945.
Luciferase Assay. Human hepatoma cell line HepG2 cells were
maintained in Eagle’s modified medium (Nakalai Tesque) supplemen-
ted with 1% penicillin, 1% streptomycin, and 10% fetal calf serum (FCS)
(Gibco BRL). Luciferase assay was performed using HepG2 cells (2 ꢁ
105 cells/well in six-well plates) transfected with 0.25 μg of pcDNA3.1-
FLAG-SXR, 0.25 μg of pGL4.10-CYP3A4pro luciferase reporter vector
(Toyo Ink. Co. Ltd.), and 0.1 μg of pRL-CMV (Promega) using
Lipofectamine (Invitrogen). At 24 h after transfection, cells were treated
with rifampicin (Nakalai Tesque, Kyoto, Japan), vitamin K compounds,
or vehicle (ethanol) for 48 h in fresh medium, and luciferase activities
were determined by a Lumat LB9507 luminometer (Berthold Tech-
nologies) using the dual-luciferase assay system (Toyo Ink.). Firefly
luciferase activity was normalized to Renilla luciferase, which was used as
a transfection control. The experiments were repeated three times with
similar results.
Synthesis of 10. Similar to the synthesis of 7 from 12, the crude
product 10, which was obtained from 12 (50 mg, 316 μmol), farnesol
(211 mg, 949 μmol), and boron trifluoride ether complex (70 μL) in
AcOEt (1 mL) and dioxane (1 mL), was purified by preparative TLC on
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Computational Details. We built the three-dimensional structure of
the SXR-LBD/MK-n-W complexes from chain A of the crystal structure of
the hSXR-LBD/SR12813 complex (PDB accession code 1NRL17). All
hydrogen atoms and partially present atoms of Glu270, Arg303, and Gln695
were added on the basis of chain B by using the Sybyl 7.3 molecular modeling
system. Our docking analyses of MK-n-WtotheSXR were carriedout using
the MOE24 by the following procedure: (i) 500 trial conformations for
each MK-n-W were picked up; (ii) the positions of all hydrogen atoms in
hSXR-LBD were refined by conjugate gradient method; (iii) each MK-n-
W was docked to the hSXR-LBD using the ASEDock method. Calcula-
tions were performed on a Dell Precision T3500 workstation.
(6) 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 accumu-
lation in osteoblastic cells. J. Biol. Chem. 2006, 281, 16927–16934.
(7) Igarashi, M.; Yogiashi, Y.; Mihara, M.; Takada, I.; Kitagawa, H.;
Kato, S. Vitamin K induces osteoblast differentiation through pregnane
X receptor-mediated transcriptional control of the Msx2 gene. Mol. Cell.
Biol. 2007, 27, 7947–7954.
(8) Ichikawa, T.; Horie-Inoue, K.; Ikeda, K.; Blumberg, B.; Inoue, S.
Vitamin K2 induces phosphorylation of protein kinase A and expression of
novel target genes in osteoblastic cells. J. Mol. Endocrinol. 2007, 39, 239–247.
(9) Horie-Inoue, K.; Inoue, S. Steroid and xenobiotic receptor
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’ ASSOCIATED CONTENT
(10) Azuma, K.; Urano, T.; Ouchi, Y.; Inoue, S. VitaminK2 suppresses
proliferation and motility of hepatocellular carcinoma cells by activating
steroid and xenobiotic receptor. Endocr. J. 2009, 56, 843–849.
(11) Li, J.; Lin, J. C.; Wang, H.; Peterson, J. W.; Furie, B. C.; Furie, B.;
Booth, S. L.; Volpe, J. J.; Rosenberg, P. A. Novel role of vitamin K in
preventing oxidative injury to developing oligodendrocytes and neurons.
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Supporting Information. 1H and 13C NMR spectra of
S
b
7ꢀ11 and HPLC data. This material is available free of charge via
the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
(12) Yamamoto, R.; Komai, M.; Kojima, K.; Furukawa, Y.; Kimura,
S. Menaquinone-4 accumulation in various tissues after an oral admin-
istration of phylloquinone in Wistar rats. J. Nutr. Sci. Vitaminol. (Tokyo)
1997, 43, 133–143.
Corresponding Author
*Phone: þ81-78-441-7564. Fax: þ81-78-441-7565. E-mail:
t-okano@kobepharma-u.ac.jp.
(13) Okano, T.; Shimomura, Y.; Yamane, M.; Suhara, Y.; Kamao, M.;
Sugiura, M.; Nakagawa, K. Conversion of phylloquinone (vitamin K1)
into menaquinone-4 (vitamin K2) in mice: two possible routes for
menaquinone-4 accumulation in cerebra of mice. J. Biol. Chem. 2008,
283, 11270–11279.
(14) Suhara, Y.; Wada, A.; Okano, T. Elucidation of the mechanism
producing menaquinone-4 in osteoblastic cells. Bioorg. Med. Chem. Lett.
2009, 19, 1054–1057.
(15) Tirona, R. G.; Lee, W.; Leake, B. F.; Lan, L. B.; Cline, C. B.;
Lamba, V.; Parviz, F.; Duncan, S. A.; Inoue, Y.; Gonzalez, F. J.; Schuetz,
E. G.; Kim, R. B. The orphan nuclear receptor HNF4alpha determines
PXR- and CAR-mediated xenobiotic induction of CYP3A4. Nat. Med.
2003, 9, 220–224.
(16) Takeshita, A.; Taguchi, M.; Koibuchi, N.; Ozawa, Y. Putative
role of the orphan nuclear receptor SXR (steroid and xenobiotic
receptor) in the mechanism of CYP3A4 inhibition by xenobiotics.
J. Biol. Chem. 2002, 277, 32453–32458.
(17) Watkins, R. E.; Davis-Searles, P. R.; Lambert, M. H.; Redinbo,
M. R. Coactivator binding promotes the specific interaction between
ligand and the pregnane X receptor. J. Mol. Biol. 2003, 331, 815–828.
(18) Shiraki, M.; Shiraki, Y.; Aoki, C.; Miura, M. Vitamin K2
(menatetrenone) effectively prevents fractures and sustains lumbar bone
mineral density in osteoporosis. J. Bone Miner. Res. 2000, 15, 515–521.
(19) Booth, S. L.; Tucker, K. L.; Chen, H.; Hannan, M. T.; Gagnon,
D. R.; Cupples, L. A.; Wilson, P. W.; Ordovas, J.; Schaefer, E. J.; Dawson-
Hughes, B.; Kiel, D. P. Dietary vitamin K intakes are associated with hip
fracture but not with bone mineral density in elderly men and women.
Am. J. Clin. Nutr. 2000, 71, 1201–1208.
’ ACKNOWLEDGMENT
We are grateful to Dr. Atsuko Takeuchi and Dr. Chisato Tode
for spectroscopic measurements at Kobe Pharmaceutical Univer-
sity. We thank Dr. Seiichi Kobayashi, Ryoka Systems Inc. for
technical advice about MOE. We also thank Eisai Co., Ltd. for
generouslyprovidingthe intermediates ofthe vitaminK analogues.
Y.S. acknowledges Suzuken Memorial Foundation. This study was
supported in part by a Grant-in-Aid for Scientific Research
“KAKENHI” (C) (Grant No. 20590113 to Y.S.) from the Japan
Society for the Promotion of Science.
’ ABBREVIATIONS USED
Gla, γ-carboxyglutamic acid; gas6, growth arrest specific 6;PK,
phylloquinone; MK-n, menaquinone-n; SXR, steroid and xenobio-
tic receptor; PXR, pregnane X receptor; PAR, proteinase-activated
receptor; CYP3A4, cytochrome P450 3A4;MGP, matrix Gla
protein; RXR, retinoid X receptor; SXRE, SXR response element;
MOE, Molecular Operating Environment;ESI-HRMS, electro-
spray ionization high-resolution mass spectrometry; TMS, tetra-
methylsilane;TLC, thin layer chromatography; PCR, polymerase
chain reaction;PDB, Protein Data Bank
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