Structure-activity relationships in the conversion of vitamin K analogues into menaquinone-4. Substrates essential to the synthesis of menaquinone-4 in cultured human cell lines

Article abstract of DOI:10.1016/j.bmc.2010.03.035

To reveal an essential biological role of menaquinone-4, we have clarified that dietary PK was converted to menaquinone-4 (MK-4) in animal tissues using deuterated vitamin K analogues. However, the kinds of analogue converted into MK-4 have not been elucidated. In this study, we examined structure-activity relationships in the conversion of several vitamin K analogues, with a substituted side chain, into MK-4 using cultured human cell lines. The results differed with the side chain of the analogues, that is, (1) the length of the isoprene unit and (2) the number of double bonds in the side chain. These findings would be useful for clarifying the mechanism of conversion of other vitamin K homologs into MK-4 as well as related enzymes.

Full text of DOI:10.1016/j.bmc.2010.03.035

Bioorganic & Medicinal Chemistry 18 (2010) 3116–3124
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Structure–activity relationships in the conversion of vitamin K analogues
into menaquinone-4. Substrates essential to the synthesis of menaquinone-4
in cultured human cell lines
Yoshitomo Suhara a, , Akimori Wada b, Yoji Tachibana c, Masato Watanabe a, Kanae Nakamura a,
Kimie Nakagawa a, Toshio Okano a,
*
a Department of Hygienic Sciences, Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan
b Department of Organic Chemistry for Life Science, Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan
c Fine Chemical Research Laboratory, Nisshin Flour Milling Co., Ltd, 5-3-1 Tsurugaoka, Fujimino City, Saitama 356-8511, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
To reveal an essential biological role of menaquinone-4, we have clarified that dietary PK was converted
to menaquinone-4 (MK-4) in animal tissues using deuterated vitamin K analogues. However, the kinds of
analogue converted into MK-4 have not been elucidated. In this study, we examined structure–activity
relationships in the conversion of several vitamin K analogues, with a substituted side chain, into
MK-4 using cultured human cell lines. The results differed with the side chain of the analogues, that
is, (1) the length of the isoprene unit and (2) the number of double bonds in the side chain. These findings
would be useful for clarifying the mechanism of conversion of other vitamin K homologs into MK-4 as
well as related enzymes.
Received 19 February 2010
Revised 15 March 2010
Accepted 16 March 2010
Available online 19 March 2010
Keywords:
Vitamin K
Menaquinone-4
Conversion
Ó 2010 Elsevier Ltd. All rights reserved.
Structure–activity relationships
Analogues
Deuterated compound
1. Introduction
reaction.11 These findings suggest that MK-4 is the preferred form
among vitamin K homologs and plays an important role in the
Vitamin K is a fat-soluble vitamin and a well-known cofactor for
-glutamyl carboxylase (GGCX), an enzyme that converts specific
function of the brain. However, it is not known why this conversion
occurs because MK-4 has long been thought to be only a cofactor
c
glutamic acid residues in several proteins to
c-carboxyglutamic
for the vitamin K-dependent c-carboxylative reaction. As a first
acid (Gla) residues related to blood coagulation1 and bone forma-
tion in the vitamin K cycle.2–4 It has two major homologs, the
plant-derived vitamin K1 (A) (phylloquinone: PK) and the bacte-
rium-derived vitamin K2 (B) (menaquinone-n: MK-n) (Fig. 1).5
Among these homologs, MK-4 has attracted attention because of
its interesting biological activities. For example, MK-4 showed
additional biological actions related to gene transcription through
the steroid and xenobiotic receptor (SXR),6 suppression of cancer
cell proliferation,7–9 and so on. Recently we confirmed that MK-4
was converted from dietary PK, and then accumulated in various
tissues in mice.10 Notably, certain tissues with a high lipid content,
such as brain tissue, contain high concentrations of MK-4. In a
study of the brain in vitro, MK-4 was shown to prevent oxidative
injury to oligodendrocyte precursors and immature fetal cortical
step toward clarifying the biological importance of MK-4, we
investigated the kinds of vitamin K derivatives easily converted
to MK-4 in vitro. No report has focused on the side chains of vita-
min K analogues, and examined structure–activity relationships for
conversion to MK-4.12–14 The development of new analogues with
high rates of conversion to MK-4 would help to elucidate the
mechanism of conversion as well as the enzymes involved. Fur-
thermore, it can be expected to lead to new drugs once the biolog-
ical importance of MK-4 is clarified. We report here a method of
O
O
O
H
n
H
3
neurons, independent of the vitamin K-dependent c-carboxylative
O
* Corresponding author. Tel.: +81 78 441 7563; fax: +81 78 441 7565.
E-mail address: t-okano@kobepharma-u.ac.jp (T. Okano).
Present address: Laboratory of Environmental Sciences, Yokohama College of
Pharmacy, Yokohama 245-0066, Japan.
A
B
Figure 1. Structure of vitamin K homologs: phylloquinone (A) and menaquinones
(B).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.03.035

Y. Suhara et al. / Bioorg. Med. Chem. 18 (2010) 3116–3124
3117
synthesizing vitamin K analogues and results of conversion to MK-
4 using cultured human cell lines.
Ltd. The deuterated MK-4 analogue 7 and its side-chain moiety
were prepared as reported.15 Meanwhile, the naphthoquinone
moiety used was a commercially available ‘menadione-d8 (K3-d8)’
(13).15 After the reduction of K3-d8 (13) with sodium hydrosulfite
in Et2O and water to form the hydroquinone 14, several side-chain
moieties were successively coupled with 14 in the presence of a
catalytic amount of BF3ꢀEt2O before the hydroquinone was oxi-
dized to the quinone 13 under atmospheric conditions. Thus,
MK-2-d7 (1)–MK-7-d7 (6), MK-4-d12 (7), and PK-d7 (10) were all
obtained in good yields. We did not use ‘MK-1-d7’, supposedly con-
sisting of prenol and K3-d8, because it was difficult to obtain and
the chemical yield was not satisfactory with our method.
The vitamin K analogues (8)–(11), in which the number of
double bonds in the side chain of MK-4 was decreased, are
shown in Figure 2. The synthesis of the 60,70-dihydro analogue
(6,7-DH-MK-4-d7 (8)) is shown in Scheme 2. We chose farnesol
(15) as a starting material to produce the side chain of the ana-
logue. After the 2,3-double bond nearest to the hydroxyl group
had been saturated by selective catalytic reduction, the hydroxyl
group of 16 was converted into the iodide 17 through methane
sulfonate in good yield in two steps. The introduction of a
carbonyl group into 17 under basic conditions in two steps, fol-
2. Results and discussion
2.1. Synthesis of vitamin K analogues
We synthesized several analogues of deuterated vitamin K to
examine the conversion to MK-4 in vitro. In this study, we focused
on the side-chain moiety of vitamin K and synthesized two general
groups of compounds. Each compound was modified in the side
chain as follows, (1) the length of the isoprene unit was changed
and (2) the number of double bonds in the side chain was de-
creased, as shown in Figure 2. MK-4 was focused on because it
potentially plays an important role in the living body. Deuterated
menaquinones were synthesized because they can be distin-
guished from the native vitamin K homologs originally contained
in cells and tissues with LC–APCI–MS/MS.
The deuterated menaquinones in which the length of the iso-
prene unit was changed MK-2-d7 (1) to MK-7-d7 (6), MK-4-d12
(7), and PK-d7 (10) (Fig. 2). Compound 7 contains deuterium in
the side chain as well as menadione for observing its conversion
to MK-4-d7. As shown in Scheme 1, the analogues 1–6 and 10 were
obtained from isoprene side-chain moieties such as prenol, gera-
niol, farnesol, geranylgeraniol, geranylfarnesol, farnesylfarnesol,
geranylgeranylfarnesol, and phytol, kindly provided by Eisai Co.,
lowed by
a Horner–Wadsworth–Emmons reaction, gave the
methyl ester 19 in 75% yield. Reduction of 19 with Vitride
reagent16 gave 6,7-dihydrogeranylgeraniol (20) in 57% yield. Fi-
nally, 6,7-DH-MK-4-d7 (8) was obtained from the alcohol 20
and K3-d8 (13) as mentioned in 43% yield.
D
O
D
O
1: n= 1
2: n= 2
3: n= 3
4: n= 4
5: n= 5
6: n= 6
D
D
CD3
CD3
D
D
CD3
CD3
D2
C
H
n
D
D
O
O
CD3
D
D
O
O
7
9
D
D
D
D
D
D
O
O
D
D
O
O
8
D
D
CD3
D
D
CD3
D
D
O
O
D
O
10
11
D
D
CD3
O
D
O
12
Figure 2. The newly synthesized deuterated vitamin K analogues with different alkyl side chains.
D
D
D
D
OH
OH
O
O
1: (55% yield)
2: (57% yield)
3: (43% yield)
4: (40% yield)
5: (38% yield)
6: (35% yield)
7: (55% yield)
10: (45% yield)
D
D
CD3
CD3
D
D
a
b
D
D
13: K3-d8
14: hydroquinone-d8
Scheme 1. Synthesis of vitamin K-d7 analogues. Reagents and conditions: (a) 10% Na2S2O4 aqueous, Et2O; (b) isoprenyl alcohols, BF3ꢀEt2O, EtOAc/dioxane (1:1).

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Y. Suhara et al. / Bioorg. Med. Chem. 18 (2010) 3116–3124
b, c
a
I
HO
HO
17
15
16
g
f
d, e
H3CO
O
O
18
19
h
HO
8
20
Scheme 2. Synthesis of vitamin K analogues. Reagents and conditions: (a) 2% PtO2, H2 (quant.); (b) CH3SO2Cl, Et3N; (c) NaI (73% in two steps); (d) CH3COCH2COOC2H5, 28%
MeONa, THF; (e) KOH (70% in two steps); (f) PO(OC2H5)2CH2COOC2H5, 28% MeONa (81%); (g) Vitride reagent (90%); (h) 13, BF3ꢀEt2O, EtOAc/dioxane (1:1) (43%).
Scheme 3 shows the synthesis of 60,70,100,110-tetrahydro-MK-4
(6,7,10,11-TH-MK-4-d7 (9)). After the hydroxyl group of b-citronel-
lol (21) was converted to the iodide 22 in 87% yield in two steps,
the introduction of a carbonyl group into the molecule was fol-
lowed by conversion of the ketone 23 into the methyl ester 24 with
a Horner–Wadsworth–Emmons reaction in the same manner as
above. 24 was reduced to the alcohol 25 with Vitride reagent in
92% yield, then the 2,3-double bond nearest the hydroxyl group
of 25 was reduced by selective catalytic reduction with monitoring
using TLC to give 26 in quantitative yield. With the same reaction
leading from 21 to 26, 6,7,10,11-tetrahydrogeranylgeraniol (30)
was obtained in good yield. The synthesis of 6,7,10,11-TH-MK-4-
d7 (9) was carried out with the side chain of 30 and K3-d8 (13) in
43% yield. The 60,70,100,110,140,150-hexahydro-MK-4 was identical
compound with PK-d7 (10). The vitamin K analogue 11, in which
all double bonds of the side chain are saturated, was obtained by
hydrogenation as described in the Section 4. ‘20,30-Dihydrophyllo-
quinone (2,3-DH-PK-d7 (11))’ is frequently used in the manufactur-
ing of foods because of its stability.17
Besides these vitamin K analogues, we synthesized menaquin-
one-4-epoxide-d7 (MK-4-epoxide-d7) (12) as shown in Figure 2 to
detect turnover of the vitamin K cycle in cells. The method used
was reported previously.18
Regarding the two general groups of compounds, we investi-
gated the conversion of analogues to MK-4-d7 as well as MK-4-
epoxide-d7, one of the metabolites of the vitamin K cycle, with
LC–APCI–MS/MS as reported,13 then elucidated the effect of substi-
tution of the side chain moiety on conversion to MK-4 and turn-
over of the vitamin K cycle.
2.2. Evaluation of the rate of conversion of vitamin K
derivatives into MK-4
The conversion of vitamin K analogues into MK-4 was examined
in vitro using human osteosarcoma-derived MG-63 cells. First, the
synthesized compounds were added at 1 lM, and the cells incu-
bated at 37 °C for 24 h. After reactants were collected, the vitamin
K compounds contained in cells were extracted with a n-hexane
solvent. The amount of converted MK-4-d7 in each sample was
measured by LC–APCI–MS/MS.
The results for each vitamin K analogue added to MG-63 cells
are shown in Figures 3 and 4. Figure 3 shows the results for the
compounds in which the isoprene unit of the side chain was chan-
ged as well as K3-d8. The vertical and horizontal axes in the graph
show the concentration of MK-4-d7 and MK-4-epoxide-d7 con-
verted from deuterated vitamin K analogues with the vitamin K cy-
cle, and the kinds of vitamin K derivatives, respectively. The rate of
conversion to MK-4 increased as the side chain became shorter.
Consequently, MK-2 was the best substrate for conversion to
MK-4 among menaquinones in MG-63 cells. The amount of K3-d8
obtained was largest, however, K3 was reported to have serious
toxicity.19 The conversion might depend on the amount taken up
into cells or ease with which the side chain of vitamin K analogues
was cleaved. Interestingly, a deuterated MK-4-epoxide, generated
a, b
c, d
e
HO
I
O
23
HO
22
21
g
f
HO
H3CO
O
26
25
24
j, k
l
h, i
I
O
28
27
n
m
HO
H3CO
9
O
30
29
Scheme 3. Synthesis of vitamin K analogues. Reagents and conditions: (a) CH3SO2Cl, Et3N; (b) NaI (87% in two steps); (c) CH3COCH2COOC2H5, 28% MeONa, THF; (d) KOH (99%
in two steps); (e) PO(OC2H5)2CH2COOC2H5, 28% MeONa (91%); (f) Vitride reagent (92%); (g) 2% PtO2, H2, (quant.); (h) CH3SO2Cl, Et3N; (i) NaI (94% in two steps); (j)
CH3COCH2COOC2H5, 28% MeONa, THF; (k) KOH (quant.); (l) PO(OC2H5)2CH2COOC2H5, 28% MeONa (63%); (m) Vitride reagent (57%); (n) 13, BF3ꢀEt2O, EtOAc/dioxane (1:1)
(43%).

Y. Suhara et al. / Bioorg. Med. Chem. 18 (2010) 3116–3124
3119
Figure 3. The conversion of deuterated vitamin K analogues (1)–(7) as well as 13, in which an isoprene unit of the side chain was changed, to MK-4-d7 (3) in MG-63 cells. The
vertical and horizontal axes show the concentrations of MK-4-d7 (3) ( ) and MK-4-epoxide-d7 (12) ( ), and ligands of vitamin K derivatives, respectively. Each analogue was
incubated at 1 lM with MG-63 cells for 24 h. The resulting vitamin K analogues were extracted from each reactant, and concentrations of MK-4-d7 (3) and MK-4-epoxide-d7
(12) were measured with LC–APCI–MS/MS as described in Section 4.
Figure 4. The conversion of deuterated vitamin K analogues (8)–(11) as well as 7, some double bonds of which were hydrogenated, to MK-4-d7 (3) in MG-63 cells. The vertical
and horizontal axes show the concentrations of MK-4-d7 (3) ( ) and MK-4-epoxide-d7 (12) ( ), and ligands of vitamin K derivatives, respectively. Experimental conditions
were similar to those in Figure 3.
in the vitamin K cycle, was also detected in cells. This finding
indicates that the converted MK-4-d7 was utilized in the vitamin
K cycle in cells.
the mevalonate pathway, with an enzymatic reaction to produce
MK-4.
Regarding the derivatives in which some double bonds were
hydrogenated in the side chain, the rate of conversion to MK-4
was proportional to the number of double bonds as shown in
Figure 4. Notably, 2,3-dihydro-phylloquinone (2,3-DH-PK) (11), a
hydrogenated form of PK produced during the hydrogenation of
PK-rich plant oils,17 was not converted into MK-4 in our ‘in vitro’
experiment. MK-4-epoxide-d7 was also generated and utilized in
cells the same as in Figure 3. The total amount of MK-4-d7 and
MK-4-epoxide-d7 obtained from MK-d12 (7) was largest, therefore,
MK-4-d12 might be much easier to convert to MK-4 than other
hydrogenated analogues. This finding suggests the double bonds
of the side chain to be important for conversion to MK-4.
Similar results were obtained with human hepatocarcinoma
HepG2 cells. This means that the conversion to MK-4 occurred in
sequence regardless of cell species. The mechanism of conversion
has been reported by us20 and another group.21 We can speculate
further based on the present results. Briefly, once absorbed by cells,
the analogues would be transferred to intracellular compartments,
then the side chain would be cleaved and the resulting naphtho-
quinone derivative would be bound to GGPP, biosynthesized in
3. Conclusions
In this study, we synthesized various vitamin K derivatives, and
examined their conversion to MK-4 in cells. We clarified that the
rate of conversion differed significantly with the side chain, there-
fore, the structure of the side chain part of vitamin K is important
for the conversion to MK-4. These findings should help to reveal
the system converting vitamin K to MK-4. Moreover, it may be pos-
sible to greatly improve the rate of conversion to MK-4 by modify-
ing the side chain. A more detailed examination is underway to
clarify the physiological significance of MK-4 and the precise
mechanism of conversion.
4. Experimental
4.1. Synthesis
4.1.1. General
The synthesis of and the data on deuterated vitamin K homo-
logs are shown in Schemes 1–3. 1H NMR spectra were recorded

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Y. Suhara et al. / Bioorg. Med. Chem. 18 (2010) 3116–3124
on a Varian VXR-500 spectrometer at 500 MHz and 13C-NMR spec-
tra were recorded at 125 MHz using deuterized chloroform (CDCl3)
(Merck, Germany). Chemical shifts are given in ppm (d) using tet-
ramethylsilane (TMS) as an internal standard. Mass spectra were
recorded on a JMS SX-102A. Preparative thin layer chromatography
(TLC) was carried out on Silica Gel 60 F254 (Merck). Unless other-
wise noted, all reagents were purchased from commercial suppli-
ers and used as received.
4.1.5. Menaquinone-5-d7 (MK-5-d7) (4)
Similar to the synthesis of 1 from 13, a crude product (4), which
was obtained from 13 (50 mg, 278
l
mol), geranylfarnesol (111 mg,
L) in AcOEt
310 mol), and boron trifluoride ether complex (30
l
l
(1 mL) and dioxane (1 mL), was purified by preparative TLC on sil-
ica gel (n-hexane/AcOEt = 20:1), giving 4 (58 mg, 40%) as a yellow
oil: 1H NMR (500 MHz, CDCl3) d 1.56 (6H, s), 1.587 (3H, s), 1.593
(3H, s), 1.67 (3H, s), 1.79 (3H, s), 1.98–2.09 (16H, m), 3.37 (2H, d,
J = 6.5 Hz), 5.01–5.11 (5H, m); 13C NMR (125 MHz, CDCl3) d
15.96, 16.01, 16.4, 17.7, 25.7, 26.0, 26.5, 26.6, 26.8, 39.7, 119.1,
123.8, 123.9, 124.2, 124.3, 124.4, 131.2, 132.1, 134.8, 134.9,
135.2, 137.5, 143.2, 146.2, 184.5, 185.5; D NMR (500 MHz, CHCl3)
d 2.13 (3D, s), 7.70 (2D, s), 8.10 (2D, s): EI-LRMS m/z 519 (M+).
EI-HRMS calcd for C36H41D7O2: 519.4091. Found: 519.4100.
4.1.2. Menaquinone-2-d7 (MK-2-d7) (1)
To a solution of menadion-d8 (K3-d8) (13) (150 mg, 833 lmol) in
ether (20 mL) was added a 10% Na2S2O4 aqueous solution (20 mL),
and the mixture stirred vigorously at 30 °C for 1 h under argon.
After the yellow ether layer turned colorless, the mixture was ex-
tracted with AcOEt (50 mL ꢁ 3). The combined organic layer was
washed with brine (50 mL ꢁ 3), dried over MgSO4, and concen-
trated to afford crude hydroquinone (14). The residue was immedi-
ately dissolved in AcOEt (1 mL) and dioxane (1 mL), then, geraniol
4.1.6. Menaquinone-6-d7 (MK-6-d7) (5)
Similar to the synthesis of 1 from 13, a crude product (5), which
was obtained from 13 (50 mg, 278
l
mol), farnesylfarnesol
(132 mg, 310 mol), and boron trifluoride ether complex (30
l
lL)
(139 mg, 900 lmol) and boron trifluoride ether complex (50 lL)
in AcOEt (1 mL) and dioxane (1 mL), was purified by preparative
TLC on silica gel (n-hexane/AcOEt = 20:1), giving 5 (61 mg, 38%)
as a yellow oil: 1H NMR (500 MHz, CDCl3) d 1.56 (6H, s), 1.591
(6H, s), 1.595 (3H, s), 1.68 (3H, s), 1.79 (3H, s), 1.91–2.09 (20H,
m), 3.37 (2H, d, J = 7.0 Hz), 5.00–5.12 (6H, m); 13C NMR
(125 MHz, CDCl3) d 15.99, 16.01, 16.02, 16.04, 16.4, 17.7, 25.7,
26.0, 26.5, 26.7, 26.8, 39.7, 119.1, 123.9, 124.2, 124.3, 124.4,
131.2, 132.1, 134.9, 135.2, 137.6, 143.2, 146.2, 184.5, 185.5; D
NMR (500 MHz, CHCl3) d 2.13 (3D, s), 7.71 (2D, s), 8.10 (2D, s):
EI-LRMS m/z 587 (M+). EI-HRMS calcd for C41H49D7O2 587.4717.
Found 587.4723.
were added. The mixture was stirred at 70 °C for 3 h under argon,
and cooled to room temperature. The reaction mixture was poured
into ice-water, and extracted with AcOEt (50 mL ꢁ 3). The com-
bined organic layer was washed with water (100 mL) and brine
(100 mL), dried over MgSO4, and concentrated. The residue was
purified by preparative TLC on silica gel (n-hexane/AcOEt = 20:1)
to afford 1 (145 mg, 55%) as a yellow oil: 1H NMR (500 MHz, CDCl3)
d 1.57 (3H, s), 1.63 (3H, s), 1.80 (3H, s), 2.00 (2H, t, J = 7.5 Hz), 2.06
(2H, t, J = 7.5 Hz), 3.38 (2H, d, J = 7.0 Hz), 5.01–5.16 (2H, m); 13C
NMR (125 MHz, CDCl3) d 16.4, 17.7, 25.6, 26.0, 26.5, 39.7, 119.2,
124.0, 131.5, 132.2, 137.5, 143.3, 146.3, 184.6, 185.5; D NMR
(500 MHz, CHCl3) d 2.14 (3D, s), 7.72 (2D, s), 8.11 (2D, s): EI-LRMS
m/z 315 (M+). EI-HRMS calcd for C21H17D7O2: 315.2214. Found:
315.2221.
4.1.7. Menaquinone-7-d7 (MK-7-d7) (6)
Similar to the synthesis of 1 from 13, a crude product (6), which
was obtained from 13 (50 mg, 278
l
mol), farnesylfarnesol
(153 mg, 310 mol), and boron trifluoride ether complex (30
l
lL)
in AcOEt (1 mL) and dioxane (1 mL), was purified by preparative
TLC on silica gel (n-hexane/AcOEt = 20:1), giving 6 (64 mg, 35%)
as a yellow oil: 1H NMR (500 MHz, CDCl3) d 1.56 (6H, s), 1.59
(12H, s), 1.67 (3H, s), 1.79 (3H, s), 1.91–2.07 (24H, m), 3.37 (2H,
d, J = 7.0 Hz), 5.01–5.12 (7H, m); 13C NMR (125 MHz, CDCl3) d
16.01, 16.03, 16.4, 17.7, 25.7, 26.0, 26.5, 26.7, 26.8, 39.7, 119.1,
123.9, 124.2, 124.3, 124.4, 131.2, 132.1, 134.9, 135.2, 137.6,
143.3, 146.2, 184.5, 185.5; D NMR (500 MHz, CHCl3) d 2.14 (3D,
s), 7.70 (2D, s), 8.10 (2D, s): EI-LRMS m/z 656 (M+). EI-HRMS calcd
for C46H57D7O2: 655.5343. Found: 655.5352.
4.1.3. Menaquinone-3-d7 (MK-3-d7) (2)
Similar to the synthesis of 1 from 13, a crude product (2), which
was obtained from 14 (50 mg, 278
l
mol), farnesol (69 mg,
L) in AcOEt
310 mol), and boron trifluoride ether complex (30
l
l
(1 mL) and dioxane (1 mL), was purified by preparative TLC on sil-
ica gel (n-hexane/AcOEt = 20:1), giving 2 (61 mg, 57%) as a yellow
oil: 1H NMR (500 MHz, CDCl3) d 1.56 (6H, s), 1.65 (3H, s), 1.80 (3H,
s), 1.91 (2H, t, J = 7.5 Hz), 1.99 (4H, t, J = 8.0 Hz), 2.06 (2H, t,
J = 8.0 Hz), 3.37 (2H, d, J = 6.5 Hz), 5.01–5.06 (3H, m); 13C NMR
(125 MHz, CDCl3) d 16.0, 16.4, 17.6, 25.7, 26.0, 26.5, 26.7, 39.67,
39.71, 119.1, 123.9, 124.3, 131.2, 132.1, 135.2, 137.5, 143.3,
146.2, 184.5, 185.5; D NMR (500 MHz, CHCl3) d 2.14 (3D, s), 7.72
(2D, s), 8.11 (2D, s): EI-LRMS m/z 383 (M+). EI-HRMS calcd for
C26H25D7O2: 383.2840. Found: 383.2837.
4.1.8. Menaquinone-4-d12 (MK-4-d12) (7)
Similar to the synthesis of 1 from 13, a crude product (7), which
was obtained from 13 (50 mg, 278
l
mol), geranylgeraniol-d5
(91 mg, 310
l
mol)6, and boron trifluoride ether complex (30
lL)
in AcOEt (1 mL) and dioxane (1 mL), was purified by preparative
TLC on silica gel (n-hexane/AcOEt = 20:1), giving 7 (70 mg, 55%)
as a yellow oil: 1H NMR (500 MHz, CDCl3) d 1.56 (3H, s), 1.59
(3H, s), 1.67 (3H, s), 1.79 (3H, s), 1.91–2.07 (10H, m), 3.37 (2H, d,
J = 6.5 Hz), 5.00–5.10 (4H, m); 13C NMR (125 MHz, CDCl3) d 15.9,
16.4, 17.7, 25.7, 26.0, 26.6, 26.8, 39.57, 39.61, 39.7, 119.1, 123.8,
124.2, 124.4, 131.2, 132.12, 132.15, 134.9, 135.2, 137.6, 143.3,
146.2, 184.5, 185.5; D NMR (500 MHz, CHCl3) d 1.51 (3D, s), 2.02
(2D, s), 2.14 (3D, s), 7.72 (2D, s), 8.11 (2D, s): EI-LRMS m/z 456
(M+). EI-HRMS calcd for C31H28D12O2: 456.3780. Found: 456.3777.
4.1.4. Menaquinone-4-d7 (MK-4-d7) (3)
Similar to the synthesis of 1 from 13, a crude product (3),
which was obtained from 13 (50 mg, 278
l
mol), geranylgeraniol
L)
(90 mg, 310 mol), and boron trifluoride ether complex (30
l
l
in AcOEt (1 mL) and dioxane (1 mL), was purified by preparative
TLC on silica gel (n-hexane/AcOEt = 20:1), giving 3 (54 mg, 43%)
as a yellow oil: 1H NMR (500 MHz, CDCl3) d 1.557 (3H, s),
1.561 (3H, s), 1.59 (3H, s), 1.67 (3H, s), 1.79 (3H, s), 1.91–2.07
(12H, m), 3.37 (2H, d, J = 7.0 Hz), 5.00–5.11 (4H, m); 13C NMR
(125 MHz, CDCl3) d 15.9, 16.0, 16.4, 17.7, 25.7, 26.0, 26.5, 26.6,
26.7, 39.6, 39.69, 39.71, 119.1, 123.9, 124.1, 124.4, 131.2,
132.09, 132.12, 134.9, 135.2, 135.5, 143.2, 146.2, 184.5, 185.5;
D NMR (500 MHz, CHCl3) d 2.14 (3D, s), 7.71 (2D, s), 8.10 (2D,
s): EI-LRMS m/z 451 (M+). EI-HRMS calcd for C31H33D7O2:
451.3460. Found: 451.3466.
4.1.9. 60,70-Dihydro-menaquinone-4-d7 (6,7-DH-MK-4) (8)
Similar to the synthesis of 1 from 13, a crude product (8), which
was obtained from 13 (50 mg, 278
and boron trifluoride ether complex (30
dioxane (1 mL), was purified by preparative TLC on silica gel
lmol), 20 (90 mg, 310
lmol),
lL) in AcOEt (1 mL) and

Y. Suhara et al. / Bioorg. Med. Chem. 18 (2010) 3116–3124
3121
(n-hexane/AcOEt = 20:1), giving 8 (54 mg, 43%) as a yellow oil: 1H
NMR (500 MHz, CDCl3) d 0.83 (3H, d, J = 6.5 Hz), 0.84–1.48 (6H, m),
1.57 (3H, s), 1.59 (3H, s), 1.60–1.61 (1H, m), 1.67 (3H, s), 1.78 (3H,
s), 1.90–2.07 (8H, m), 3.35 (2H, d, J = 7.0 Hz), 4.99–5.13 (3H, m); 13C
NMR (125 MHz, CDCl3) d 15.9, 16.3, 17.7, 19.6, 25.2, 25.4, 25.7,
26.0, 26.7, 32.2, 36.5, 37.0, 39.7, 40.0, 119.1, 124.4, 124.8, 131.2,
132.08, 132.12, 134.6, 135.2, 137.9, 143.2, 146.3, 184.5, 185.5; D
NMR (500 MHz, CHCl3) d 2.14 (3D, s), 7.72 (2D, s), 8.11 (2D, s):
EI-LRMS m/z 453 (M+). EI-HRMS calcd for C31H35D7O2: 453.3622.
Found: 453.3610.
and the organic layer was combined. The ether layer was washed
with brine (10 mL), dried over MgSO4, and concentrated in vacuo.
The residue was purified by preparative TLC on silica gel (n-hex-
ane/AcOEt = 20:1) to afford 12 (41 mg, 92%) as a pale yellow oil:
1H NMR (500 MHz, CDCl3) d 1.56 (3H, s), 1.57 (3H, s), 1.59 (3H,
s), 1.67 (3H, s), 1.76 (3H, s), 1.91–2.09 (12H, m), 2.43 (1H, dd,
J = 7.0, 15.0 Hz), 3.25 (1H, dd, J = 7.0, 15.0 Hz), 5.05–5.14 (4H, m);
13C NMR (125 MHz, CDCl3) d 16.0, 16.1, 16.6, 17.7, 25.6, 25.7,
26.5, 26.6, 26.8, 39.68, 39.73, 39.79, 67.5, 116.7, 123.8, 124.2,
124.4, 131.3, 131.9, 132.2, 134.9, 135.3, 139.1, 192.1, 193.1; D
NMR (500 MHz, CHCl3) d 1.73 (3D, s), 7.77 (2D, s), 8.03 (2D, s):
EI-LRMS m/z 467 (M+). EI-HRMS calcd for C31H33D7O3: 467.3414.
Found: 467.3422.
4.1.10. 60,70-100,110-Tetrahydro-menaquinone-4-d7 (6,7,10,
11-TH-MK-4-d7) (9)
Similar to the synthesis of 1 from 13, a crude product (9), which
was obtained from 13 (50 mg, 278
l
mol), 30 (90 mg, 310
l
mol),
4.1.14. (E)-3,7,11-Trimethyldodeca-6,10-dien-1-ol (16)
and boron trifluoride ether complex (30
l
L) in AcOEt (1 mL) and
Farnesol (15) (5.0 g, 22.5 mmol) was hydrogenated with PtO2
(100 mg) in AcOEt (100 mL) for 40 min. The completion of the
hydrogenation was checked by 1H NMR spectroscopy with part
of the reaction mixture. The reaction mixture was filtrated and
evaporated in vacuo. The residue was purified by flash column
chromatography on silica gel (n-hexane/AcOEt = 10:1) to give 16
(5.0 g, quantitative yield) as a colorless oil: 1H NMR (500 MHz,
CDCl3) d 0.91 (3H, d, J = 6.5 Hz), 1.18–1.43 (5H, m), 1.56–1.65
(1H, m), 1.60 (6H, s), 1.68 (3H, s), 1.95–2.09 (6H, m), 3.64–3.73
(2H, m), 5.07–5.12 (2H, m); 13C NMR (125 MHz, CDCl3) d 15.9,
17.7, 19.5, 25.3, 25.7, 26.7, 29.2, 37.2, 39.7, 39.9, 61.2, 124.4,
124.6, 131.3, 134.9: EI-LRMS m/z 224 (M+). EI-HRMS calcd for
C15H28O: 224.2139. Found: 224.2140.
dioxane (1 mL), was purified by preparative TLC on silica gel
(n-hexane/AcOEt = 20:1), giving 9 (54 mg, 43%) as a yellow oil:
1H NMR (500 MHz, CDCl3) d 0.81 (3H, d, J = 7.0 Hz), 0.86 (3H, d,
J = 7.0 Hz), 1.00–1.41 (14H, m), 1.59 (3H, s), 1.67 (3H, s), 1.78
(3H, s), 1.91–1.97 (4H, m), 3.37 (2H, d, J = 7.0 Hz), 4.99–5.11 (2H,
m); 13C NMR (125 MHz, CDCl3) d 16.2, 17.6, 19.6, 22.6, 22.7, 24.4,
25.2, 25.4, 25.6, 25.7, 26.0, 28.0, 32.2, 36.6, 37.3, 39.3, 40.0,
118.8, 125.1, 130.9, 132.08, 132.12, 137.9, 143.2, 146.3, 184.5,
185.5; D NMR (500 MHz, CHCl3) d 2.14 (3D, s), 7.72 (2D, s), 8.11
(2D, s): EI-LRMS m/z 455 (M+). EI-HRMS calcd for C31H37D7O2:
455.3781. Found: 455.3788.
4.1.11. Phylloquinone-d7 (PK-d7) (10)
Similar to the synthesis of 1 from 13, a crude product (10),
4.1.15. (E)-12-Iodo-2,6,10-trimethyldodeca-2,6-diene (17)
To a stirred solution of 16 (2.0 g, 8.9 mmol) in dichloromethane
(23.5 mL) was added methanesulfonyl chloride (1.2 g, 10.5 mmol)
and Et3N (1.2 g, 11.9 mmol) at 0–5 °C, and the resulting mixture
was stirred for 30 min. The mixture was washed with a saturated
aqueous NaHCO3 solution and brine, dried over MgSO4, filtered,
and concentrated. Acetone (20 mL) and NaI (5.0 g, 33.3 mmol)
were added to the residue and the mixture was refluxed for
1.5 h. The reaction mixture was quenched with water, extracted
with AcOEt, and subjected to a standard work up. The residue
was purified by column chromatography on silica gel (n-hexane/
AcOEt = 10:1) to afford 17 (2.18 g, 73% yield) as a colorless oil:
1H NMR (500 MHz, CDCl3) d 0.88 (3H, d, J = 6.5 Hz), 1.15–1.49
(4H, m), 1.56–1.65 (1H, m), 1.60 (6H, s), 1.68 (3H, s), 1.85–2.09
(6H, m), 3.14–3.27 (2H, m), 5.08–5.12 (2H, m); 13C NMR
(125 MHz, CDCl3) d 16.0, 17.7, 18.7, 25.2, 25.7, 26.7, 33.5, 36.3,
39.7, 40.9, 124.30, 124.35, 131.3, 135.1: EI-LRMS m/z 334 (M+).
EI-HRMS calcd for C15H27I: 334.1156. Found: 334.1167.
which was obtained from 13 (50 mg, 278
lmol), phytol (92 mg,
310 mol), and boron trifluoride ether complex (30
l
lL) in AcOEt
(1 mL) and dioxane (1 mL), was purified by preparative TLC on sil-
ica gel (n-hexane/AcOEt = 20:1), giving 10 (57 mg, 45%) as a yellow
oil: 1H NMR (500 MHz, CDCl3) d0.79–0.85 (total 12H, m), 0.99–1.34
(18H, m), 1.49 (1H, m), 1.76 (3H, s), 1.92 (2H, m), 3.35 (2H, d,
J = 7.0 Hz), 4.98 (1H, dt, J = 1.0, 7.0 Hz); 13C NMR (125 MHz, CDCl3)
d 16.3, 19.70, 19.71, 22.6, 22.7, 24.4, 24.8, 25.3, 26.0, 28.0, 32.6,
32.8, 36.6, 37.3, 37.36, 37.39, 39.4, 40.0, 118.8, 119.3, 132.1,
137.9, 138.3, 143.2, 184.6, 185.5; D NMR (500 MHz, CHCl3) d 2.14
(3D, s), 7.71 (2D, s), 8.10 (2D, s): EI-LRMS m/z 457 (M+), 193. EI-
HRMS calcd for C31H39D7O2: 457.3935. Found: 457.3935.
4.1.12. 20,30-Dihydro-phylloquinone-d7 (2,3-DH-PK-d7) (11)
To a solution of 10 (20 mg, 44 lmol) in AcOEt (10 mL) was
added a catalytic amount of PtO2 (2 mg), and the mixture stirred
vigorously under a hydrogen atmosphere for 12 h at room temper-
ature. After filtration through Celite, the filtrate was concentrated.
The residue was purified by preparative TLC on silica gel (n-hex-
ane/AcOEt = 20:1) to give 11 (20 mg, quantitative yield) as a yellow
oil: 1H NMR (500 MHz, CDCl3) d 0.84–0.87 (12H, m), 0.98 (3H, dd,
J = 1.5, 11.5 Hz), 1.05–1.38 (22H, m), 1.48–1.55 (2H, m), 2.56–2.68
(2H, m); 13C NMR (125 MHz, CDCl3) d 19.47, 19.53, 19.7, 19.8, 22.6,
22.7, 24.4, 24.5, 24.8, 28.0, 32.8, 33.4, 35.7, 35.8, 37.0, 37.1, 37.3,
37.4, 37.5, 39.4, 132.1, 132.2, 142.8, 148.1, 184.7, 185.4; D NMR
(500 MHz, CHCl3) d 2.14 (3D, s), 7.72 (2D, s), 8.11 (2D, s): EI-LRMS
m/z 459 (M+). EI-HRMS calcd for C31H41D7O2: 459.4092. Found:
459.4090.
4.1.16. (E)-6,10,14-Trimethylpentadeca-9,13-dien-2-one (18)
To a solution of 17 (2.18 g, 6.5 mmol) in THF (20 mL) were
added a 28% sodium methoxide in methanol solution (3.3 mL)
and CH3COCH2COOC2H5 (4.5 g, 34.6 mmol), and then refluxed for
20 h. After addition of water to the mixture and extraction with
AcOEt, the organic layer was dried and concentrated. The residue
was dissolved in isopropyl alcohol (4.5 mL) and a 50% aqueous
KOH solution (4.0 mL), then heated at 85 °C and subjected to a
standard work up. The residue was purified by column chromatog-
raphy on silica gel (n-hexane/AcOEt = 10:1) to afford 18 (1.2 g, 70%
yield) as a colorless oil: 1H NMR (500 MHz, CDCl3) d 0.88 (3H, d,
J = 6.5 Hz), 1.10–1.16 (2H, m), 1.25–1.43 (3H, m), 1.52–1.62 (2H,
m), 1.60 (6H, s), 1.68 (3H, s), 1.93–2.07 (6H, m), 2.13 (3H, s), 2.40
(2H, t, J = 7.5 Hz), 5.08–5.11 (2H, m); 13C NMR (125 MHz, CDCl3)
d 16.0, 17.6, 19.4, 21.4, 25.4, 25.7, 26.7, 29.8, 32.2, 36.4, 36.9,
39.7, 44.1, 124.4, 124.7, 131.2, 134.7, 209.2: EI-LRMS m/z 264
(M+). EI-HRMS calcd for C18H32O: 264.2453. Found: 264.2469.
4.1.13. Menaquinone-4-epoxide-d7 (MK-4-epoxide-d7) (12)
A mixture of 43 mg (95 lmol) of MK-4-d7 (3) and 100 lL of 30%
hydrogen peroxide solution in 2.5 mL of ethanol was combined
with an aqueous solution of 100 mg of sodium carbonate in
0.3 mL of H2O and heated at 75 °C for 1 h. The mixture was poured
into 10 mL of H2O and extracted with three 10 mL portions of
ether. The reaction mixture was extracted with ether (3 ꢁ 10 mL)

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Y. Suhara et al. / Bioorg. Med. Chem. 18 (2010) 3116–3124
4.1.17. (2E,10E)-Methyl 3,7,11,15-tetramethylhexadeca-2,10,
14-trienoate (19)
flux for 12 h. After cooling to room temperature, the mixture was
poured into water. The reaction mixture was extracted with AcOEt
(100 mL ꢁ 3), and the combined organic layer was washed with
brine (100 mL), dried over MgSO4, and concentrated. The residue
was dissolved in isopropyl ether (10 mL), added to a 50% KOH solu-
tion (12 mL), and stirred at 85 °C for 3 h. After addition of water to
the solution, the mixture was extracted with AcOEt (100 mL ꢁ 3),
washed with brine (100 mL), dried over MgSO4, and concentrated.
The residue was purified by flash column chromatography on silica
gel (n-hexane/AcOEt = 6:1) to give 23 (5.1 g, 99% yield) as a color-
less oil: 1H NMR (500 MHz, CDCl3) d 0.87 (3H, d, J = 6.5 Hz), 1.08–
1.17 (2H, m), 1.25–1.36 (2H, m), 1.37–1.43 (1H, m), 1.52–1.63
(2H, m), 1.60 (3H, s), 1.68 (3H, s), 1.92–2.00 (2H, m), 2.13 (3H, s),
2.40 (2H, t, J = 7.0 Hz), 5.07–5.10 (1H, m); 13C NMR (125 MHz,
CDCl3) d 19.4, 21.3, 25.4, 25.7, 29.8, 30.9, 32.2, 36.4, 36.9, 44.1,
124.8, 131.0, 209.2: EI-LRMS m/z 196 (M+). EI-HRMS calcd for
C13H24O: 196.1826. Found: 196.1832.
To a solution of 18 (0.9 g, 3.4 mmol) in n-hexane (6.0 mL) were
added PO(OCH2CH3)2CH2COOC2H5 (1.3 g, 5.8 mmol) and a 28% so-
dium methoxide in methanol solution (1.35 g), and the mixture
was stirred for 2 h at room temperature. Following the addition
of water to the reaction mixture followed by extraction with n-
hexane, the organic layer was washed with 60% methanol, dried
over MgSO4, and concentrated. The residue was purified by flash
column chromatography on silica gel (n-hexane/AcOEt = 95:5) to
give 19 (0.89 g, 81% yield) as a colorless oil: 1H NMR (500 MHz,
CDCl3) d 0.87 (3H, d, J = 6.5 Hz), 1.08–1.18 (2H, m), 1.25–1.33
(3H, m), 1.40–1.44 (2H, m), 1.60 (6H, s), 1.68 (3H, s), 1.93–2.13
(8H, m), 2.15 (3H, s), 3.67 (3H, s), 5.08–5.11 (2H, m), 5.65–5.67
(1H, m); 13C NMR (125 MHz, CDCl3) d 15.9, 17.7, 18.7, 19.5, 24.8,
25.4, 25.7, 26.7, 32.2, 36.4, 37.0, 39.7, 41.2, 50.7, 115.0, 124.4,
124.7, 131.2, 134.7, 160.7, 167.3: EI-LRMS m/z 320 (M+). EI-HRMS
calcd for C21H36O2: 320.2713. Found: 320.2693.
4.1.21. (E)-Methyl 3,7,11-trimethyldodeca-2,10-dienoate (24)
To a solution of 23 (5.1 g, 26.0 mmol) in n-hexane (50 mL) were
added PO(OC2H5)2CH2COOC2H5 (9.0 g, 40.1 mmol) and 28% sodium
methoxide in methanol (10.0 g), and the mixture was stirred for
2 h at room temperature. Addition of water to the reaction mixture
was followed by extraction with n-hexane (100 mL ꢁ 3), and the
organic layer was washed with 60% methanol (100 mL), dried over
MgSO4, and concentrated. The residue was purified by flash col-
umn chromatography on silica gel (n-hexane/AcOEt = 95:5) to give
24 (6.0 g, 91% yield) as a colorless oil: 1H NMR (500 MHz, CDCl3) d
0.87 (3H, d, J = 6.5 Hz), 1.10–1.51 (7H, m), 1.60 (3H, s), 1.68 (3H, s),
1.90–2.00 (2H, m), 2.10–2.13 (2H, m), 2.16 (3H, s), 3.68 (3H, s), 5.10
(1H, m), 5.66 (1H, s); 13C NMR (125 MHz, CDCl3) d 19.4, 22.6, 24.8,
25.5, 25.7, 31.6, 32.2, 36.4, 36.9, 41.2, 50.7, 115.0, 124.9, 131.1,
160.7, 167.3: EI-LRMS m/z 252 (M+). EI-HRMS calcd for C16H28O2:
252.2088. Found: 252.2092.
4.1.18. (2E,10E)-3,7,11,15-Tetramethylhexadeca-2,10,14-trien-
1-ol (20)
To a solution of 19 (0.89 g, 2.8 mmol) in n-hexane (10 mL) was
added Vitride reagent (1.2 g), and stirred for 2 h. After the addition
of acetone (1 mL) and 50% acetic acid (10 mL), the mixture was
dehydrated with a Dean–Stark apparatus. The reaction mixture
was washed with water, saturated aqueous NaHCO3, and brine,
and the organic layer was dried over MgSO4, filtered, and concen-
trated. The residue was purified by flash column chromatography
on silica gel (n-hexane/AcOEt = 10:1) to give 20 (0.72 g, 90% yield)
as a colorless oil: 1H NMR (500 MHz, CDCl3) d 0.87 (3H, d,
J = 6.5 Hz), 1.08–1.18 (3H, m), 1.25–1.44 (5H, m), 1.60 (6H, s),
1.67 (3H, s), 1.68 (3H, s), 1.93–2.08 (8H, m), 4.15–4.16 (2H, m),
5.08–5.12 (2H, m), 5.39–5.42 (1H, m); 13C NMR (125 MHz, CDCl3)
d 15.9, 16.2, 17.7, 19.6, 25.1, 25.4, 25.7, 26.7, 32.3, 36.6, 37.0,
39.7, 39.9, 59.4, 123.1, 124.4, 124.9, 131.2, 134.6, 140.3: EI-LRMS
m/z (M+) 292. EI-HRMS calcd for C20H36O: 292.2765. Found:
292.2760.
4.1.22. (E)-3,7,11-Trimethyldodeca-2,10-dien-1-ol (25)
To a solution of 24 (6.0 g, 23.7 mmol) in n-hexane (60 mL) was
added Vitride reagent (10 g), and the mixture stirred for 2 h. After
the addition of acetone (2 mL) and 50% acetic acid (10 mL), the
mixture was dehydrated with the Dean–Stark apparatus. The reac-
tion mixture was washed with water, saturated aqueous NaHCO3,
and brine, and the organic layer was dried over MgSO4, filtered,
and concentrated. The residue was purified by flash column chro-
matography on silica gel (n-hexane/AcOEt = 10:1) to give 25 (5.3 g,
92% yield) as a colorless oil: 1H NMR (500 MHz, CDCl3) d 0.86 (3H,
d, J = 6.5 Hz), 1.06–1.16 (2H, m), 1.25–1.43 (6H, m), 1.60 (3H, s),
1.67 (3H, s), 1.68 (3H, s), 1.93–2.05 (4H, m), 4.14 (2H, d,
J = 5.5 Hz), 5.08–5.11 (1H, m), 5.39–5.42 (1H, m); 13C NMR
(125 MHz, CDCl3) d 16.1, 17.6, 19.5, 25.1, 25.5, 25.7, 32.2, 36.6,
37.0, 39.8, 59.4, 123.1, 125.0, 131.0: EI-LRMS m/z 224 (M+). EI-
HRMS calcd for C15H28O: 224.2140. Found: 224.2152.
4.1.19. 8-Iodo-2,6-dimethyloct-2-ene (22)
To a solution of b-citronellol (21) (8.5 g, 54.4 mmol) and meth-
anesulfonyl chloride (7.5 g, 65.4 mmol) in CH2Cl2 (100 mL) was
added triethylamine (10 g, 169 mmol) dropwise at 0 °C under ar-
gon, and the mixture stirred at room temperature for 2 h. After
extraction with AcOEt (50 mL ꢁ 3), the combined organic layer
was washed with saturated sodium bicarbonate aqueous solution
(100 mL) and then brine (100 mL), dried over MgSO4, and concen-
trated to afford a crude product. The residue was dissolved in ace-
tone (100 mL), and sodium iodide (16.5 g, 110 mmol) was added.
The mixture was heated to reflux for 3 h under argon. After the
reaction mixture had cooled to room temperature, it was poured
into ice-water, and extracted with AcOEt (50 mL ꢁ 3). The com-
bined organic layer was washed with brine (100 mL), dried over
MgSO4, and concentrated. The residue was purified by flash col-
umn chromatography on silica gel (n-hexane/AcOEt = 6:1) to give
22 (12.5 g, 87% yield) as a colorless oil: 1H NMR (500 MHz, CDCl3)
d 0.89 (3H, d, J = 6.5 Hz), 1.14–1.21 (1H, m), 1.27–1.38 (2H, m),
1.53–1.71 (2H, m), 1.61 (3H, s), 1.68 (3H, s), 1.85–2.01 (2H, m),
3.14–3.19 (1H, m), 3.22–3.27 (1H, m), 5.09 (1H, t, J = 7.0 Hz); 13C
NMR (125 MHz, CDCl3) d 5.1, 17.7, 18.6, 25.3, 25.7, 33.5, 36.3,
40.9, 124.4, 131.4: EI-LRMS m/z 266 (M+). EI-HRMS calcd for
C10H19I: 266.0530. Found: 266.0543.
4.1.23. 3,7,11-Trimethyldodec-10-en-1-ol (26)
Compound 25 (5.2 g, 23.2 mmol) was hydrogenated with PtO2
(100 mg) in AcOEt (100 mL) for 40 min. Completion of the hydro-
genation was checked by 1H NMR spectroscopy with part of the
reaction mixture. The reaction mixture was filtrated and evapo-
rated in vacuo. The residue was purified by flash column chroma-
tography on silica gel (n-hexane/AcOEt = 10:1) to give 26 (5.2 g,
quantitative yield) as a colorless oil: 1H NMR (500 MHz, CDCl3) d
0.85 (3H, d, J = 6.5 Hz), 0.90 (3H, d, J = 6.5 Hz), 1.07–1.40 (11H,
m), 1.50–1.63 (2H, m), 1.60 (3H, s), 1.68 (3H, s), 1.92–2.02 (2H,
m), 3.64–3.72 (2H, m), 5.08–5.12 (1H, m); 13C NMR (125 MHz,
CDCl3) d 17.6, 22.6, 22.7, 24.3, 24.8, 25.6, 25.7, 28.0, 39.3, 39.96,
40.0, 61.2, 125.0, 130.9: EI-LRMS m/z 226 (M+). EI-HRMS calcd for
C15H30O: 226.2295. Found: 226.2310.
4.1.20. 6,10-Dimethylundec-9-en-2-one (23)
To a solution of 22 (7.0 g, 26.3 mmol) in THF (70 mL) were
added a 28% sodium methoxide methanol solution (10 g) and ethyl
acetoacetate (15 g, 115 mmol), and the mixture was heated to re-

Y. Suhara et al. / Bioorg. Med. Chem. 18 (2010) 3116–3124
3123
4.1.24. 12-Iodo-2,6,10-trimethyldodec-2-ene (27)
(trans), s), 1.74 (0.3H (cis), s), 1.94–2.06 (4H, m), 4.12–4.22 (2H,
m), 5.09–5.12 (1H, m), 5.40–5.43 (1H, m); 13C NMR (125 MHz,
CDCl3) d 16.1, 17.6, 19.6, 22.6, 24.4, 25.1, 25.7, 28.0, 30.9, 32.7,
37.1, 39.4, 39.9, 59.4, 123.1, 125.1 130.9, 140.3: EI-LRMS m/z 294
(M+). EI-HRMS calcd for C20H38O: 294.2923. Found: 294.2922.
To a stirred solution of 26 (5.2 g, 23.2 mmol) in dichlorometh-
ane (50 mL) were added methanesulfonyl chloride (3.2 g,
27.9 mmol) and Et3N (3.5 g, 34.6 mmol) at 0–5 °C, and the resulting
mixture was stirred for 30 min. The mixture was washed with a
saturated aqueous NaHCO3 solution and brine, dried over MgSO4,
filtered, and concentrated. Acetone (100 mL) and NaI (9.0 g,
60.0 mmol) were added to the residue and the mixture was re-
fluxed for 1.5 h. The reaction mixture was quenched with water,
extracted with AcOEt, and subjected to a standard work up. The
residue was purified by column chromatography on silica gel
(n-hexane/AcOEt = 10:1) to afford 27 (7.3 g, 94% yield) as a color-
less oil: 1H NMR (500 MHz, CDCl3) d 0.86 (3H, d, J = 3.0 Hz), 0.87
(3H, d, J = 3.0 Hz), 1.07–1.32 (10H, m), 1.50–1.67 (2H, m), 1.61
(3H, s), 1.69 (3H, s), 1.85–2.00 (2H, m), 3.14–3.19 (1H, m), 3.23–
3.28 (1H, m), 5.09–5.12 (1H, m); 13C NMR (125 MHz, CDCl3) d
5.4, 17.6, 18.7, 18.8, 22.6, 24.8, 25.6, 25.7, 28.0, 34.0, 36.5, 39.3,
41.0, 125.0, 131.0: EI-LRMS m/z 336 (M+). EI-HRMS calcd for
C15H29I: 336.1312. Found: 336.1322.
4.2. Cells and cell culture
The human hepatoblastoma cell line (HepG2) and human oste-
osarcoma cell line (MG-63) were obtained from Riken cell bank
(Tsukuba Science City, Japan). Minimum essential medium
(MEM), Dulbecco’s modified Eagle’s medium (DMEM) (low-
glucose type), Dulbecco’s phosphate-buffered saline (PBS), penicil-
lin–streptomycin (10,000 U/mL penicillin), sodium pyruvate
(100 mM), and MEM non-essential amino acid solution (10 mM)
were all purchased from Gibco (Grand Island, NY). Trypsin (2.5%
solution) and trypsin-EDTA (2.5% trypsin, 1 mM EDTA-solution)
were purchased from Nacalai Tesque (Kyoto, Japan). Fetal Bovine
Serum (FBS) was purchased from Termo Trace (Melbourne, Austra-
lia). HepG2 was maintained in MEM supplemented with 1 mM so-
4.1.25. 6,10,14-Trimethylpentadec-13-en-2-one (28)
dium pyruvate, 100 lM MEM non-essential amino acid solution,
To a solution of 27 (5.8 g, 17.2 mmol) in THF (60 mL) were
added 28% sodium methoxide in methanol (7 mL) and CH3COCH2-
COOC2H5 (12 g, 92.2 mmol), and the mixture was refluxed for 20 h.
After addition of water to the mixture and extraction with AcOEt,
the organic layer was dried and concentrated. The residue was dis-
solved in isopropyl alcohol and a 50% aqueous KOH solution,
heated at 85 °C, and subjected to a standard work up. The residue
was purified by column chromatography on silica gel (n-hexane/
AcOEt = 10:1) to afford 28 (6.0 g, quantitative yield) as a colorless
oil: 1H NMR (500 MHz, CDCl3) d 0.85 (3H, d, J = 4.5 Hz), 0.86 (3H,
d, J = 4.5 Hz), 1.06–1.42 (14H, m), 1.60 (3H, s), 1.68 (3H, s), 1.88–
2.01 (2H, m), 2.13 (3H, s), 2.40 (2H, t, J = 7.0 Hz), 5.09–5.12 (1H,
m); 13C NMR (125 MHz, CDCl3) d 17.6, 19.5, 21.4, 22.6, 24.4, 25.6,
28.0, 29.8, 32.4, 36.5, 37.2, 39.4, 44.1, 125.1, 130.9, 209.4: EI-LRMS
m/z 266 (M+). EI-HRMS calcd for C18H34O: 266.2610. Found:
266.2622.
10% heat-inactivated FBS, and 1% penicillin–streptomycin. MG-63
was maintained in DMEM supplemented with 10% heat-inacti-
vated FBS and 1% penicillin–streptomycin. Cells were cultured in
T-75 flasks (BD Falcon, MA) at 37 °C in a humidified 5% CO2
atmosphere.
4.3. Incubation of cultured human cell lines with vitamin K
analogues
To compare the conversion to MK-4-d7, and utilization of MK-4-
d7 in cultured human cell lines, concentrations of vitamin K metab-
olites in cells were measured after the addition of the analogues.
The experimental medium consisted of culture medium containing
1
l
M of each analogue. In the case of HepG2 cells, 8 ꢁ 105 cells/
well were seeded onto 6-well plates on the first day, and the cul-
ture medium was changed every 2 days. Six days later, when cells
were confluent, the medium was replaced with the experimental
medium. In the case of MG-63 cells, 4 ꢁ 105 cells/well were seeded
onto six-well plates on the first day. When cells were confluent
3 days later, the medium was changed to the experimental med-
ium. After cells were incubated in the presence of each analogue
for 24 h, they were washed with cold PBS three times and then
refrigerated at ꢂ30 °C. After being warmed to room temperature,
4.1.26. (E)-Methyl 3,7,11,15-tetramethylhexadeca-2,14-dienoate
(29)
To a solution of 28 (6.0 g, 22.5 mmol) in n-hexane (40 mL) were
added PO(OC2H5)2CH2COOC2H5 (9.8 g, 43.7 mmol) and 28% sodium
methoxide in methanol (8.5 g), and the mixture was stirred for 2 h
at room temperature. After quenching with water, and extraction
with n-hexane, the organic layer was washed with a 60% methanol
solution, and concentrated. The residue was purified by column
chromatography on silica gel (n-hexane/AcOEt = 10:1) to give 29
(4.6 g, 63% yield) as a colorless oil: 1H NMR (500 MHz, CDCl3) d
0.84–0.87 (6H, m), 1.06–1.52 (14H, m), 1.60 (3H, s), 1.68 (3H, s),
1.90–2.00 (2H, m), 2.10–2.13 (2H, m), 2.15 (3H, s), 3.69 (3H, s),
5.09–5.12 (1H, m), 5.65–5.67 (1H, m); 13C NMR (125 MHz, CDCl3)
d 17.6, 18.7, 19.6, 22.6, 24.4, 24.9, 25.7, 28.0, 32.6, 39.4, 41.2,
50.7, 115.0, 125.1, 131.0, 160.8, 167.3: EI-LRMS m/z 322 (M+). EI-
HRMS calcd for C21H38O2: 322.2872. Found: 322.2874.
cells were lysed in 1 mL of PBS. With this procedure, 10 lL of cell
lysate was analyzed for protein determination with a BCA protein
assay kit (Pierce, IL, USA).
4.4. Preparation of standard solutions
A stock solution (100 lg/mL) of each standard (MK-4-d7 (3) and
MK-4-epoxide-d7 (12)) as a reference was prepared in ethanol
according to the solubility of the solute and stored in the dark at
ꢂ30 °C prior to use. For the analytical curves, working solutions
of the standard mixture, ranging from 25 to 400 ng/mL, were pre-
pared by dilution of the stock solution with ethanol. The stock
4.1.27. (E)-3,7,11,15-Tetramethylhexadeca-2,14-dien-1-ol (30)
To a solution of 29 (4.6 g, 14.3 mmol) in n-hexane (50 mL) was
added Vitride reagent (6.1 g), and the mixture stirred for 1 h. After
the addition of acetone (1 mL) and 50% acetic acid (10 mL), the
mixture was dehydrated with the Dean–Stark apparatus. The reac-
tion mixture was washed with water, saturated aqueous NaHCO3,
and brine, and the organic layer was dried over MgSO4, filtered,
and concentrated. The residue was purified by flash column chro-
matography on silica gel (n-hexane/AcOEt = 10:1) to give 30 (3.7 g,
57% yield) as a colorless oil: 1H NMR (500 MHz, CDCl3) d 0.84–0.87
(6H, m), 1.05–1.41 (15H, m), 1.61 (3H, s), 1.67 (3H, s), 1.69 (2.7H
solution (10 lg/mL) of the MK-4 analogue containing heavy oxy-
gen (MK-4-18O)18,20 for the internal standard was prepared by dilu-
tion in ethanol and stored in the dark at ꢂ30 °C prior to use.
Dilution of this solution with ethanol gave working internal stan-
dard solutions of 3.6 and 100 ng/mL MK-4-18O, respectively.18,20
The 3.6 ng/mL mixture was used for the determination of vitamin
K homologs in cells and medium. An equal amount of the standard
solution (25–400 ng/mL) and internal standard solution (100 ng/
mL) gave the solution used for the standard curve. The final con-

3124
Y. Suhara et al. / Bioorg. Med. Chem. 18 (2010) 3116–3124
centration ranged from 12.5 to 200 ng/mL in the case of the stan-
dard and contained 50 ng/mL of internal standard.
and 200 ng/mL. The points were given by the calculated peak area
ratio of the standard and internal standard.
4.5. Sample preparation (extraction of vitamin K and its
metabolites from cells)
Acknowledgments
We are grateful to Dr. Makiko Sugiura, Dr. Atsuko Takeuchi, and
Dr. Chisato Tode for the spectroscopic measurements at Kobe Phar-
maceutical University. We also thank Eisai Co., Ltd for generously
providing the intermediate of the vitamin K analogues. Y.S.
acknowledges Takeda Science Foundation. This study was sup-
ported in part by a Grant-in-aid for Scientific Research <KAKENHI>
(C) (Grant number 18590112 to Y.S.) from the Japan Society for the
Promotion of Science.
The medium (1 mL) or cell lysate (PBS solution) (1 mL) in a
brown screw-capped Pyrex tube was supplemented with 1 mL of
3.6 ng/mL MK-4-18O in ethanol as an internal standard. The addi-
tion of extra ethanol (1 mL) to denature the protein and 3 mL of
n-hexane was followed by shaking for 5 min. The solution was cen-
trifuged at 3000 rpm for 5 min, and the upper layer was separated.
After the organic layer was evaporated under reduced pressure, the
dried sample was dissolved in 60
lL of ethanol and sonicated for
10 s. The solutions were transferred to microvials, capped, and
Supplementary data
placed in a SIL-10AD vp autosampler rack. Aliquots (30
automatically injected into the HPLC system.
lL) were
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.03.035.
4.6. Apparatus and HPLC conditions
References and notes
The HPLC analyses were conducted with a Shimadzu HPLC sys-
tem (Simadzu, Kyoto, Japan) consisting of a binary pump (LC-10AD
liquid chromatography), an automatic solvent degasser (DGU-14A
degasser), and an autosampler (SIL-10AD autoinjector). Separa-
tions were carried out using a reversed-phase C18 analytical col-
umn (Capcell PAK C18 UG120, 5 mm; 4.6 mm id ꢁ 250 mm)
(Shiseido, Tokyo, Japan) with a solvent system consisting of an iso-
cratic solvent A (0–25 min) and then a linear gradient from 0% to
20% ethanol (25–50 min). Solvent A contained methanol/0.1% ace-
tic acid aqueous (95:5, v/v) and 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 35 °C with a column oven (CTO-10AC
column oven). The HPLC system was controlled by a SCL-10A Sys-
tem Controller (Shimadzu). Acetic acid both functioned as an ion
pair reagent during reversed-phase HPLC and facilitated the forma-
tion of protonated vitamin K, [M+H]+, in the positive ion mode with
an APCI. The autosampler was maintained at 25 °C.
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A quantitative analysis was carried out using MS/MS-MRM (mul-
tiple reaction monitoring) of the precursor ion of vitamin K homo-
logs (m/z 451 (MK-4-d7 (3)), 468 (MK-4-epoxide-d7 (12)), and 449
(MK-4-18O),18,20 and their product ion (m/z 194 (d7-labeled vitamin
K analogues), 168 (d7-labeled vitamin K epoxide analogues) and 191
(
18O-labeled vitamin K analogues)) with a dwell time of 300 ms. Cal-
ibration, using internal standardization, was done by linear regres-
sion analysis using five different concentrations, 12.5, 25, 50, 100,