Design and synthesis of biologically active analogues of vitamin K2: Evaluation of their biological activities with cultured human cell lines

Article abstract of DOI:10.1016/j.bmc.2007.12.025

Novel ω-oxygenated vitamin K₂ analogues were efficiently synthesized and their biological activities were evaluated. Some were biologically active and the side-chain played an important role in γ-carboxylation and apoptosis-inducing activity. The results provide useful information on the structure-activity relationship of vitamin K₂ analogues for the development of new drugs.

Full text of DOI:10.1016/j.bmc.2007.12.025

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry 16 (2008) 3108–3117
Design and synthesis of biologically active analogues
of vitamin K2: Evaluation of their biological activities
with cultured human cell lines
Yoshitomo Suhara, Yoshihisa Hirota, Kimie Nakagawa, Maya Kamao,
Naoko Tsugawa and Toshio Okano*
Department of Hygienic Sciences, Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higashinada-ku,
Kobe 658-8558, Japan
Received 19 November 2007; revised 13 December 2007; accepted 13 December 2007
Available online 2 January 2008
Abstract—Novel x-oxygenated vitamin K2 analogues were efficiently synthesized and their biological activities were evaluated.
Some were biologically active and the side-chain played an important role in c-carboxylation and apoptosis-inducing activity.
The results provide useful information on the structure–activity relationship of vitamin K2 analogues for the development of
new drugs.
ꢀ 2007 Elsevier Ltd. All rights reserved.
O
O
O
O
1. Introduction
Vitamin K is an essential cofactor required for the post-
translational conversion of glutamic acid residues into c-
carboxyglutamic acid (Gla) via c-glutamylcarboxylase
within vitamin K-dependent proteins.1 As Gla-contain-
ing proteins, a number of blood coagulation factors
including coagulation factors II (prothrombin), VII,
IX, and X, a bone-specific protein such as osteocalcin,
and other proteins have been identified.2,3 The physio-
logical function and putative health roles of vitamin
K-dependent proteins include bone mineralization,4,5
inhibition of vascular calcification,6 cell cycle regulation,
and signal transduction.7
3
n-1
1
2
Figure 1. Structure of vitamin K homologues: phylloquinone (1) (PK)
and menaquinones (2) (MK-n).
aquinone-4 (MK-4) shows strong coagulation activities
in comparison with PK and other MK-n. Some groups
recently reported that MK-4 exhibits anti-arteriosclero-
sis activity8 and anti-tumor actions in various cancer
cells,9–11 and acts as an SXR-specific ligand for the reg-
ulation of gene transcription.12 MK-4 is accumulated in
various tissues at high concentrations after its biosyn-
thesis by conversion from PK and other MK-n in the
body.13 However, its biologically essential roles other
than as a coenzyme for c-glutamylcarboxylase are still
unknown.
There are two naturally occurring forms of vitamin K:
vitamin K1 (1) (phylloquinone: PK) and vitamin K2
(2) (menaquinone-n: MK-n) (Fig. 1). PK is synthesized
by plants and is present in large amounts in various
green vegetables, which are the major source of dietary
vitamin K. On the other hand, MK-n are of bacterial
origin and have a variable side-chain length of four to
thirteen isoprene units. Among these homologues, men-
The aim of our study is to investigate the structure–
activity relationship of vitamin K analogues. Various
analogues have been synthesized and their biological
activities investigated.14–17 We have especially focused
on modifications of the side-chain of vitamin K. Meta-
bolic pathways are typical examples of modifications.
In terms of the study of vitamin K homologues
Keywords: Vitamin K; Metabolite; Menaquinone-4; x-Oxidation; K
acid.
*
Corresponding author. Tel.: +81 78 441 7563; fax: +81 78 441
7565; e-mail: t-okano@kobepharma-u.ac.jp
0968-0896/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2007.12.025

Y. Suhara et al. / Bioorg. Med. Chem. 16 (2008) 3108–3117
3109
in vivo, major aglycone metabolites have been detected.
O
O
O
They are urinary metabolites as glucuronides of x-car-
boxylic acid (3) and (4),18,19 K acid I (also called
‘7C-aglycon’; 2-methyl-3-(50-carboxy-30- methyl-20-pen-
tenyl)-1, 4-naphthoquinone) (5), and K acid II (or
‘5C-aglycon’; 2-methyl-3-(30-30-carboxymethylpropyl)-
1, 4-naphthoquinone) (6)20 as shown in Figure 2. In this
catabolic pathway, 3 or 4 is formed by oxygen attacking
hepatic metabolites at the x-carbon of PK or MK-n,
then 5 and 6 are successively generated by a side-chain
shortening of b-oxidation.21 The resultant aglycone
metabolites are then conjugated with glucuronic acid
to facilitate their biliary and urinary elimination.22,23
Despite the metabolic study, biological activities of 3–
6 have hardly been evaluated so far. Studying the deriv-
atives in detail would provide insight into the biological
significance of vitamin K and valuable information for
the development of new drugs. In this study, we focused
on MK homologues and synthesized six kinds of ana-
logues because MK-4 has various biological activities.
But the known metabolites such as x-carboxylic acid
(4) were quite unstable and consequently unable to be
used for assays. Therefore, we synthesized new ana-
logues, into which was introduced a terminal x-hydro-
xyl or x-aldehyde group instead of x-carboxylic acid
in the side-chain moiety of MK (menaquinone-2, 3,
and 4) (7)–(12) as shown in Figure 3. We report here
the design, synthesis, and structure–activity relationship
of new vitamin K analogues based on metabolites.
CHO
OH
n-1
O
n-1
7: (n= 2)
8: (n= 3)
9: (n= 4)
10: (n= 2)
11: (n= 3)
12: (n= 4)
Figure 3. Chemical structures of our compounds, which are x-
hydroxyl analogues (7)–(9) and x-aldehyde analogues (10)–(12).
x-oxygenated 15c did not give a good yield under the
same conditions. A possible reason for the low chemical
yield is that selective reactivity of SeO2 was reduced due
to a longer alkyl chain and x-1-oxygenated and diol
derivatives were generated. The mixture was separated
by silica gel column chromatography to give the desired
15c in 40% yield. Protection of the hydroxyl group of
15a–c with 3, 4-dihydro-2H-pyran (DHP) and p-tolu-
enesulfonic acid (TsOH) in CH2Cl2 gave 16a–c, which
were protected by a tetrahydropyran (THP) group, in
excellent yield. Removal of the acetyl group in 16a–c
gave the corresponding alcohol 17a–c in 78–82%.
The naphthoquinone part 18 was prepared from 1, 4-
selective
diacetoxy-2-methylnaphthoquinone
with
hydrolysis of the acetyl group. Scheme 2 shows the
synthesis of x-oxygenated vitamin K analogues. 1-Acet-
oxy-2-methyl-4-naphthalenol (18)25 was used as a
naphthoquinone synthon for the coupling with side-
chain analogues. Treatment of the monoacetate 18 with
an alkyl side-chain alcohol 17a–c in the presence of bor-
on trifluoride etherate yielded 19a–c in 45–51% yield.
Although we first tried to obtain x-alcohol compounds
7–9 directly by alkaline hydrolysis, the chemical yield
was extremely low. Presumably, the quinone form of
19a–c was degraded under alkaline conditions or a
side-chain moiety reacted and polymerization occurred.
Therefore, we employed a 2-step synthesis by way of
dimethyl ether analogues 20a–c. In short, monoacetate
derivatives 19a–c were treated with an excess amount
of potassium hydride in anhydrous conditions. This
was followed by the addition of methyl iodide to give
20a–c. Finally, deprotection of the THP group of
naphthohydroquinone methyl ether 20a–c with Ce(N-
H4)2(NO3)6 in water gave the desired x-oxygenated
2. Results and discussion
We planned to synthesize the requisite analogues
through coupling of the naphthoquinone derivative
and side-chain moiety. The outlines are shown in
Schemes 1 and 2. For the synthesis of the side-chain,
we chose geraniol (13a), farnesol (13b), and geranylger-
aniol (13c) as the starting material (Scheme 1). The pri-
mary hydroxyl group of 13a–c was protected with an
acetyl group to give 14a–c in quantitative yield. The
selective terminal x-oxygenation of 14a and 14b with
SeO2, 70% t-BuOOH solution, and salicylic acid in
CH2Cl2 according to a reported method afforded the
x-oxygenated alcohols 15a and 15b with the x-aldehyde
analogue in good yield.24 The conversion of 14c to
O
O
O
O
COOH
3
3
O
O
O
O
1
3
COOH
COOH
O
O
O
O
5
6
COOH
n-1
n-1
4
2
Figure 2. Metabolic pathway of vitamin K homologues and structural formulas of phylloquinone (1), menaquinones (2), x-carboxylic acid (3) and
(4), K acid I (5), and K acid II (6).

3110
Y. Suhara et al. / Bioorg. Med. Chem. 16 (2008) 3108–3117
Scheme 1. Synthesis of side-chain moiety. Reagents and conditions: (a) pyridine, Ac2O, 92–95%; (b) SeO2, 70% t-BuOOH, salicylic acid, CH2Cl2, 40–
75%; (c) DHP, TsOH, CH2Cl2, 75–81%; (d) 1 N NaOH aq, MeOH, 78–82%.
Scheme 2. Synthesis of vitamin K analogues. Reagents and conditions: (a) 17a–c, BF3Et2O, EtOAc/dioxane (1:1), 45–51%; (b) KH, CH3I, THF, 60–
65%; (c) CAN, CH3CN–H2O, 71–76%; (d) PDC, CH2Cl2 35–55%.
vitamin K homologues 7–9 in good yield. These ana-
logues were further converted to x-aldehyde analogues
10–12 with PDC treatment in good yield. Thus, six kinds
of x-oxygenated analogues were prepared.
12 should be converted to 9 during the reduction proce-
dure for the conversion of quinone to hydroquinone in
this assay. The Km and Vmax values for MK-4 and x-
hydroxylated MK-4 (9) (both in their hydroquinone
form) were determined from the initial carboxylation
rates at 8 different vitamin concentrations and the data
are presented in Figure 4. We have also determined
the kinetic constants for the hydroquinone forms of
the vitamins and summarized the data in Table 1. The
Km for MK-4 was 6 times higher than that for MK-4
x-OH (9), whereas the Vmax value for MK-4 was also
1.3 times higher than that for MK-4 x-OH (9). The
Vmax/Km values were 5 times higher for 9 than for
MK-4. This result means that 9 showed strong activity
toward GGCX compared to MK-4. The hydrophobicity
as well as the terminal hydrophilicity for the side chain
of vitamin K might be related to the interaction with
the vitamin K-binding site of GGCX. There are reports
that vitamin K promotes the calcification of bone in
cooperation with vitamin D.31 Therefore, if potent vita-
To investigate the biological activities of those ana-
logues, we compared coenzyme activity for c-glutamyl-
carboxylase (GGCX), which is a classical role of
vitamin K homologues, between natural vitamin K
and our new analogues. This in vitro assay was con-
ducted using previously described methods.26–29 In
short, the amount of 14CO2 incorporated into the exog-
enous substrate peptide ‘FLEEL’ was measured in reac-
tion mixtures containing reduced vitamin K, mouse liver
microsome, and NaH14CO3. We investigated whether
the GGCX activity increases or not if functional groups
are introduced into the vitamin K molecule. We chose
MK-4 and x-hydroxylated MK-4 (9) because MK-4 is
now used as a therapeutic agent for osteoporosis in Ja-
pan.30 x-Aldehyde MK-4 (12) was not assayed because

Y. Suhara et al. / Bioorg. Med. Chem. 16 (2008) 3108–3117
3111
125
100
75
50
25
0
A
B
.
0.08
0.07
0.06
0.05
0.04
0.03
0.02
0.01
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
..
-0.5 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5 0.6
1/Reduced vitamin K analogue (µM/30 min)
0
50
100
150
200
250
Reduced vitamin K analogue (µM)
Figure 4. Kinetic analysis of vitamin K hydroquinone analogues. (A) The CO2 incorporation as a function of the concentration of MK-4
hydroquinone (——) and MK-4 x-OH hydroquinone (- - - - - -). (B) Double-reciprocal plots of v vs. [S] for MK-4 hydroquinone (——–) and MK-4
x-OH hydroquinone (- - - - - -).
Table 1. Comparison of the kinetic parameters of vitamin K analogues
ity toward HL-60 cells (human leukemia cells), and the
introduction of a x-hydroxyl group increased the activ-
ity in the case of MK-4 analogues.33 We further ex-
plored the possibility of using our compounds against
other cancer cells such as the osteosarcoma cell line
MG-63, and human hepatoma cell lines HepG2 and
Huh-7. Samples of analogues were added at 5 and
10 lM to cells. After 72 h, the cells were collected
and treated with propidium iodide, and then apoptosis-
inducing activity was assessed with a fluorescence-acti-
vated cell sorter (FACS) as previously reported.34 In
the case of MG-63 cells, the activity of most analogues
increased in a dose-dependent manner as shown in Fig-
ure 5. The activity increased in the order MK-2, MK-3,
and MK-4 analogues at 10 lM. The x-aldehyde ana-
logues are likely to be more active than the x-hydroxyl-
ated analogues in these cells. On the other hand, HepG2
and Huh-7 cells showed no significant apoptosis as re-
ported previously.32 Thus, the apoptosis-inducing activ-
Substrate
Km (lM) Vmax
Vmax/Km (pmol/
(pmol/30 min) 30 min/lM)
MK-4
MK-4 x-OH (9)
13.3
2.0
126.4
99.9
9.5
50.0
min K analogues, which have coenzyme activity, were
synthesized, they might be expected to have synergic ef-
fects on bone formation in the presence of vitamin D.
However, further experimentation is necessary to study
the biological activities involved.
We next evaluated the apoptosis-inducing activity of
these analogues in cancer cell lines. As MK-4 is expected
to become a therapeutic agent against hepatic cancer,
vitamin K analogues have been studied for use as anti-
cancer drugs.32 We have already reported that some
vitamin K analogues exhibited apoptosis-inducing activ-
Figure 5. Apoptosis-inducing activity of menaquinone derivatives in MG-63 cells. MG-63 cells were treated with vitamin K2 analogues at 10 lM for
3 days. Apoptosis-inducing activity was identified with flow cytometric analysis of DNA content. Columns, mean obtained from three independent
experiments; bars, SD. Significant difference: **p < 0.01, ***p < 0.001, compared with cells untreated with vitamin K2 analogues.

3112
Y. Suhara et al. / Bioorg. Med. Chem. 16 (2008) 3108–3117
ity of our analogues as well as natural vitamin K homo-
logues exhibited was selective of cancer cells. In addi-
(5.0 g, 17 mmol) and acetic anhydride (10 mL) in pyri-
dine (20 mL), was purified by flash column chromatog-
raphy on silica gel (hexane/ethyl acetate = 10/1) to give
14c (5.3 g, 92%) as a colorless oil: IR (CHCl3): 2969,
tion,
increasing
hydrophilicity
through
the
introduction of an aldehyde or hydroxyl group into
the side-chain moiety might enhance the apoptosis-
inducing activity. Therefore, the physiological activity
of the vitamin K changed with the length of the side-
chain and the functional group of the terminal alkyl
group.
2929, 1728, 1448, 1386, 1239, 1023 cmꢀ1 1H NMR
;
(500 MHz, CDCl3): d 1.55 (3H, s), 1.60 (6H, s), 1.68
(3H, s), 1.71 (3H, s), 1.96–2.14 (12H, m), 2.05 (3H, s),
4.59 (2H, d, J = 7.0 Hz), 5.08–5.12 (3H, m), 5.33–5.36
(1H, m); 13C NMR (125 MHz, CDCl3): d 15.90, 16.20,
16.5, 17.7, 21.0, 25.7, 26.2, 26.5, 26.8, 39.5, 39.6, 39.7,
61.4, 118.2, 123.6, 124.2, 124.4, 131.3, 135.0, 135.5,
142.3, 171.1; EI-LRMS MHz 332 (M+), 272. EI-HRMS
calcd for C22H36O2 332.2715. Found 332.2737.
In summary, our results indicated that vitamin K ana-
logues might have potential as biologically active com-
pounds and can provide information with which to
develop new drugs based on vitamin K.
3.4. (2E,6E)-8-Hydroxy-3,7-dimethylocta-2,6-dienyl ace-
tate (15a)
3. Experimental
To a suspension of selenium dioxide (283 mg, 2.5 mmol)
and salicylic acid (353 mg, 2.5 mmol) in CH2Cl2
(125 mL) was added 70% tert-butyl hydroperoxide
(10 mL) at room temperature, and the mixture was stirred
at room temperature for 10 min. After the mixture was
cooled to 0 ꢁC, geranyl acetate (14a) (5.0 g, 25 mmol) in
CH2Cl2 (5 mL) was added to it. The reaction mixture
was stirred at the same temperature for 5 min and then
continuously stirred at room temperature for 24 h. The
suspension was diluted with ethyl acetate, and successively
washed with 5% NaHCO3 aqueous solution, saturated
aqueous CuSO4, saturated aqueous Na2S2O3, water, and
brine. The organic layer was dried over anhydrous MgSO4
and concentrated in vacuo. The residue was chromato-
graphed on silica gel (hexane/ethyl acetate = 10/1) to give
15a (4.1 g, 75%) as a colorless oil: IR (CHCl3): 3468,
3.1. (E)-3,7-Dimethylocta-2,6-dienyl acetate (Geranyl
acetate) (14a)
To a suspension of geraniol (13a) (5.0 g, 32 mmol) in pyr-
idine (20 mL) was added acetic anhydride (10 mL) at 0 ꢁC
under argon, and the suspension was stirred for 12 h at
room temperature. The reaction mixture was diluted with
ethyl acetate, and successively washed with saturated
CuSO4 aqueous solution, water, and brine. The organic
layer was dried over anhydrous MgSO4 and concentrated
in vacuo. The residue was purified by flash column chro-
matography on silica gel (hexane/ethyl acetate = 10/1) to
afford 14a (6.0 g, 95%) as a colorless oil: IR (CHCl3):
2969, 2930, 1728, 1445, 1381, 1240, 1023 cmꢀ1 1H
;
NMR (500 MHz, CDCl3): d 1.60 (3 H, s), 1.68 (3H, s),
1.70 (3H, s), 2.03–2.12 (4H, m), 2.06 (3H, s), 4.59 (2H,
d, J = 7.0 Hz), 5.07–5.10 (1H, m), 5.33–5.36 (1H, m);
13C NMR (125 MHz, CDCl3): d 16.4, 17.6, 21.0, 25.6,
26.2, 39.5, 61.4, 118.2, 123.7, 131.8, 142.2, 171.1; EI-
LRMS MHz 196 (M+), 136. EI-HRMS calcd for
C12H20O2 196.1463. Found 196.1478.
2935, 1728, 1445, 1367, 1242, 1023 cmꢀ1 1H NMR
;
(500 MHz, CDCl3): d 1.67 (3H, s), 1.71 (3H, s), 2.06 (3H,
s), 2.08 (2H, dd, J = 7.0, 14.0 Hz), 2.18 (2H, dd, J = 7.0,
14.0 Hz), 3.99 (2H, s), 4.58 (2H, d, J = 7.5 Hz), 5.32–5.38
(2H, m); 13C NMR (125 MHz, CDCl3): d 13.6, 16.4,
21.0, 25.6, 39.0, 61.4, 68.8, 118.6, 125.2, 135.2, 141.7,
171.2; EI-LRMS MHz 212 (M+). EI-HRMS calcd for
C12H20O3 212.1412. Found 212.1435.
3.2. (2E,6E)-3,7,11-Trimethyldodeca-2,6,10-trienyl ace-
tate (Farnesyl acetate) (14b)
3.5. (2E,6E,10E)-12-Hydroxy-3,7,11-trimethyldodeca-
2,6,10-trienyl acetate (15b)
In a manner similar to that for the synthesis of 13a from
12a, a crude product, which was obtained from 13b
(5.0 g, 22 mmol) and acetic anhydride (10 mL) in pyridine
(20 mL), was purified by flash column chromatography
on silica gel (hexane/ethyl acetate = 10/1) to give 14b
(5.5 g, 93%) as a colorless oil: IR (CHCl3): 2969, 2928,
1728, 1447, 1382, 1241, 1023 cmꢀ1; 1H NMR (500 MHz,
CDCl3): d 1.60 (6H, s), 1.68 (3H, s), 1.71 (3H, s), 1.96-
2.13 (8H, m), 2.05 (3H, s), 4.59 (2H, d, J = 7.0 Hz),
5.08–5.11 (2H, m), 5.33–5.36 (1H, m); 13C NMR
(125 MHz, CDCl3): d 16.0, 16.4, 17.6, 21.0, 25.6, 26.1,
26.7, 39.5, 39.6, 61.4, 118.2, 123.6, 124.3, 131.3, 135.4,
142.2, 171.1; EI-LRMS MHz 264 (M+), 288. EI-HRMS
calcd for C17H28O2 264.2089. Found 264.2080.
In a manner similar to that for the synthesis of 15a from
14a, a crude product, which was obtained from 14b
(1.0 g, 3.8 mmol), selenium dioxide (42 mg, 0.38 mmol),
salicylic acid (52 mg, 0.38 mmol), and 70% tert-butyl
hydroperoxide (3 mL) in CH2Cl2 (30 mL), was purified
by flash column chromatography on silica gel (hexane/
ethyl acetate = 10/1) to give 15b (647 mg, 61%) as a col-
orless oil: IR (CHCl3): 3504, 2932, 1725, 1448, 1367,
1
1238, 1023 cmꢀ1; H NMR (500 MHz, CDCl3): d 1.56
(1H, s), 1.60 (3H, s), 1.67 (3H, s), 1.71 (3H, s), 2.00–
2.07 (4H, m), 2.06 (3H, s), 2.11–2.13 (4H, m), 3.99
(2H, s), 4.58 (2H, d, J = 7.0 Hz), 5.10–5.12 (1H, m),
5.33–5.36 (1H, m), 5.37–5.41 (1H, m); 13C NMR
(125 MHz, CDCl3): d 13.6, 15.9, 16.4, 21.0, 26.08,
26.12, 39.2, 39.4, 61.3, 68.9, 118.3, 123.8, 125.8, 134.7,
135.0, 142.1, 171.1; EI-LRMS MHz 280 (M+), 220.
EI-HRMS calcd for C17H28O3 280.2038. Found
280.2045.
3.3. (2E,6E,10E)-3,7,11,15-Tetramethylhexadeca-2,6,10,14-
tetraenyl acetate (Geranylgeranyl acetate) (14c)
In a manner similar to that for the synthesis of 14a from
13a, a crude product, which was obtained from 13c

Y. Suhara et al. / Bioorg. Med. Chem. 16 (2008) 3108–3117
3113
3.6. (2E,6E,10E,14E)-16-Hydroxy-3,7,11,15-tetrameth-
ylhexadeca-2,6,10,14-tetraenyl acetate (15c)
(1H, m), 5.33–5.36 (1H, m), 5.40–5.43 (1H, m); 13C
NMR (125 MHz, CDCl3): d 14.1, 16.0, 16.5, 19.6, 21.1,
25.5, 26.2, 26.4, 30.7, 39.3, 39.5, 61.4, 62.2, 73.0, 97.4,
118.3, 123.9, 127.8, 131.9, 135.2, 142.3, 171.1; EI-LRMS
MHz 364 (M+), 304. EI-HRMS calcd for C22H36O4
364.2614. Found 364.2627.
In a manner similar to that for the synthesis of 15a from
14a, a crude product, which was obtained from 14c
(1.4 g, 4.0 mmol), selenium dioxide (45 mg, 0.4 mmol),
salicylic acid (55 mg, 0.4 mmol), and 70% tert-butyl
hydroperoxide (3 mL) in CH2Cl2 (30 mL) was purified
by flash column chromatography on silica gel (hexane/
ethyl acetate = 10/1) to give 15c (587 mg, 40%) as a col-
orless oil: IR (CHCl3): 3504, 2927, 1727, 1448, 1383,
3.9. (2E,6E,10E,14E)-3,7,11-Trimethyl-15-((tetrahydro-
2H-pyran-2-yloxy)methyl)hexadeca-2,6,10,14-tetraenyl
acetate (16c)
1
1240, 1022 cmꢀ1; H NMR (500 MHz, CDCl3): d 1.60
In a manner similar to that for the synthesis of 16a from
15a, a crude product, which was obtained from 15c
(730 mg, 2.1 mmol), 3, 4-dihydro-2H-pyran (353 mg,
4.2 mmol), and a catalytic amount of p-toluenesulfonic
acid in CH2Cl2 (5 mL), was purified by flash column
chromatography on silica gel (hexane/ethyl acetate = 5/
1) to give 16c (681 mg, 75%) as a colorless oil: IR
(CHCl3): 3008, 2946, 2855, 1728, 1440, 1239,
(6H, s), 1.66 (3H, s), 1.71 (3H, s), 2.05 (3H, s), 1.99–
2.13 (12H, m), 3.99 (2H, br s), 4.58 (2H, d,
J = 7.5 Hz), 5.11 (2H, dd, J = 7.0, 12.5 Hz), 5.34 (1H,
t, J = 7.0 Hz), 5.39 (1H, t, J = 7.0 Hz); 13C NMR
(125 MHz, CDCl3): d 13.6, 15.91, 15.94, 16.4, 21.0,
26.1, 26.2, 26.5, 39.2, 39.4, 39.6, 61.3, 68.9, 118.2,
123.6, 124.4, 125.9, 134.5, 134.7, 135.3, 142.2, 171.1;
EI-LRMS MHz 348 (M+), 288. EI-HRMS calcd for
C22H36O3 348.2664. Found 348.2679.
1
1018 cmꢀ1; H NMR (500 MHz, CDCl3): d 1.60 (3H,
s), 1.66 (3H, s), 1.69 (3H, s), 1.71 (3H, s), 1.73–1.79
(2H, m), 1.83–1.89 (2H, m), 2.05 (3H, m), 3.50–3.54
(1H, m), 3.86–3.90 (2H, m), 4.09 (1H, d, J = 11.5 Hz),
4.59 (2H, d, J = 7.0 Hz), 4.60 (1H, d, J = 3.0 Hz), 5.10
(2H, dd, J = 7.0, 13.5 Hz), 5.34 (1H, dt, J = 1.5,
7.0 Hz), 5.42 (1H, dt, J = 1.5, 7.0 Hz); 13C NMR
(125 MHz, CDCl3): d 14.0, 15.9, 16.0, 16.4, 19.5, 21.0,
25.5, 26.4, 26.6, 30.6, 39.3, 39.5, 39.6, 61.4, 62.1, 72.9,
97.4, 118.2, 123.6, 124.4, 127.9, 131.8, 134.7, 135.4,
142.2, 171.1; EI-LRMS MHz 432 (M+). EI-HRMS
calcd for C27H44O4 432.324. Found 432.3262.
3.7. (2E,6E)-3-Methyl-7-((tetrahydro-2H-pyran-2-yloxy)-
methyl)octa-2,6-dienyl acetate (16a)
A catalytic amount of p-toluenesulfonic acid was added to
a cooled solution of 15a (500 mg, 2.4 mmol) and 3, 4-dihy-
dro-2H-pyran (396 mg, 4.7 mmol) in CH2Cl2 (5 mL) at
0 ꢁC under argon, and the mixture was stirred at room
temperature for 2 h. The reaction mixture was poured
into ice-water and extracted with ethyl acetate twice.
The organic layer was combined and washed with brine,
dried over anhydrous MgSO4, and concentrated in vacuo.
The residue was chromatographed on silica gel (hexane/
ethyl acetate = 5/1) to give 16a (566 mg, 81%) as a
colorless oil: IR (CHCl3): 3017, 2945, 2854, 1728, 1454,
1240, 1075 cmꢀ1; 1H NMR (500 MHz, CDCl3): d
1.51–162 (4 H, m), 166 (3H, s), 1.71 (3H, s), 1.81–1.86
(2H, m), 2.06 (3H, s), 2.09 (2H, t, J = 7.5 Hz), 2.17
(2H, t, J = 7.5 Hz), 3.49–3.53 (1H, m), 3.84 (1H, d,
J = 11.5 Hz), 3.86–3.90 (2H, m), 4.10 (1H, d,
J = 11.5 Hz), 4.58 (1H, d, J = 6.5 Hz), 4.60 (1H, d,
J = 6.5 Hz), 5.33–5.37 (1H, m), 5.39–5.43 (1H, m); 13C
NMR (125 MHz, CDCl3): d 14.0, 16.4, 19.5, 21.0, 25.5,
25.9, 30.7, 39.1, 61.3, 62.2, 73.0, 97.4, 118.4, 127.0,
132.4, 141.9, 171.1; EI-LRMS MHz 296 (M+). EI-HRMS
calcd for C17H28O4 296.1988. Found 296.1958.
3.10. (2E,6E)-3-Methyl-7-((tetrahydro-2H-pyran-2-ylox-
y)methyl)octa-2,6-dien-1-ol (17a)
To a solution of compound 16a (525 mg, 1.8 mmol) in
MeOH (10 mL) was added 1 N aqueous NaOH until the
pH reached 11–12 at room temperature, and the reaction
mixture was stirred at room temperature for 1 h. The solu-
tion was poured into ice-water and immediately extracted
withethyl acetate three times. The combined extracts were
successively washed with water and brine, dried over
anhydrous MgSO4, and concentrated in vacuo. The resi-
due was chromatographed on silica gel (hexane/ethyl ace-
tate = 5/1) to afford 17a (369 mg, 82%) as a colorless oil:
IR (CHCl3): 3448, 3009, 2945, 2856, 1667, 1454, 1231,
1
1118 cmꢀ1; H NMR (500 MHz, CDCl3): d 1.38 (1H, t,
J = 5.5 Hz), 1.51–1.63 (4H, m), 1.66 (3H, s), 1.67 (3H,
s), 1.69–1.75 (1H, m), 1.78–1.88 (1H, m), 2.08 (2H, t,
J = 7.5 Hz), 2.13–2.23 (2H, m), 3.49–3.53 (1H, m), 3.85
(1H, d, J = 11.0 Hz), 3.84–3.89 (1H, m), 4.09 (1H, d,
J = 11.0 Hz), 4.11–4.15 (2H, m), 4.61 (1H, t,
J = 3.5 Hz), 5.39–5.42 (2H, m); 13C NMR (125 MHz,
CDCl3): d 14.0, 16.1, 19.4, 25.5, 25.8, 30.6, 39.0, 59.4,
62.0, 72.9, 97.0, 123.9, 127.7, 132.1, 139.1; EI-LRMS
MHz 254 (M+). EI-HRMS calcd for C15H26O3
254.1882. Found 254.1862.
3.8. (2E,6E,10E)-3,7-Dimethyl-11-((tetrahydro-2H-pyr-
an-2-yloxy)methyl)dodeca-2,6,10-trienyl acetate (16b)
In a manner similar to that for the synthesis 16a from 15a,
a crude product, which was obtained from 15b (1.4 g,
0.50 mmol), 3, 4-dihydro-2H-pyran (840 mg, 1.0 mmol),
and catalytic amount of p-toluenesulfonic acid in CH2Cl2
(10 mL), was purified by flash column chromatography
on silica gel (hexane/ethyl acetate = 5/1) to give 16b
(1.47 g, 81%) as a colorless oil: IR (CHCl3): 3010, 2946,
2855, 1728, 1442, 1239, 1021 cmꢀ1; 1H NMR (500 MHz,
CDCl3): d 1.60 (3H, s), 1.66 (3H, s), 1.71 (3H, s), 1.51–
1.63 (4H, m), 1.80–1.87 (2H, m), 2.05 (3H, m), 2.00–2.14
(8H, m), 3.49–3.54 (1H, m), 3.83–3.90 (2H, m), 4.09
(1H, d, J = 12.0 Hz), 4.59 (3H, d, J = 6.5 Hz), 5.09–5.12
3.11. (2E,6E,10E)-3,7-Dimethyl-11-((tetrahydro-2H-pyr-
an-2-yloxy)methyl)dodeca-2,6,10-trien-1-ol (17b)
In a manner similar to that for the synthesis of 17a from
16a, a crude product, which was obtained from 16b

3114
Y. Suhara et al. / Bioorg. Med. Chem. 16 (2008) 3108–3117
(1.1 g, 3.0 mmol), 1 N aqueous NaOH in MeOH
(20 mL), was purified by flash column chromatography
on silica gel (hexane/ethyl acetate = 5/1) to give 17b
(777 mg, 80%) as a colorless oil: IR (CHCl3): 3455,
4.09 (1H, d, J = 12.0 Hz), 4.61 (1H, t, J = 3.5 Hz),
5.21–5.24 (1H, m), 5.36–5.39 (1H, m), 5.89 (1H, br s),
7.41–7.47 (2H, m), 7.63–7.64 (1H, m), 8.12–8.14 (1H,
m); 13C NMR (125 MHz, CDCl3): d 13.5, 14.0, 16.3,
19.3, 20.7, 25.5, 25.9, 26.4, 30.6, 39.2, 62.0, 72.8, 96.9,
119.9, 120.6, 121.7, 121.8, 124.1, 125.0, 126.1, 126.3,
127.5, 132.4, 138.4, 147.8, 169.7; EI-LRMS MHz 452
(M+). EI-HRMS calcd for C28H36O5 452.2563. Found
452.2560.
3010, 2944, 2855, 1667, 1454, 1231, 1118 cmꢀ1 1H
;
NMR (500 MHz, CDCl3): d 1.38 (1H, br s), 1.51–1.63
(4H, m), 1.60 (3H, s), 1.66 (3H, s), 1.68 (3H, s), 1.69–
1.75 (1H, m), 1.81–1.88 (1H, m), 2.00–2.06 (4H, m),
2.08–2.16 (4H, m), 3.48–3.53 (1H, m), 3.85 (1H, d,
J = 12.0 Hz), 3.85–3.91 (1H, m), 4.09 (1H, d,
J = 12.0 Hz), 4.15–4.17 (2H, m), 4.59–4.61 (1H, m),
5.10–5.13 (1H, m), 5.40–5.43 (2H, m); 13C NMR
(125 MHz, CDCl3): d 14.1, 16.0, 16.3, 19.6, 25.5,
26.26, 26.29, 30.7, 39.2, 39.5, 59.4, 62.2, 73.0, 97.4,
123.5, 124.1, 127.8, 131.9, 135.0, 139.6; EI-LRMS
MHz 322 (M+). EI-HRMS calcd for C20H34O3
322.2508. Found 322.2509.
3.14. 1-Hydroxy-3-methyl-2-((2E,6E,10E)-3,7-dimethyl-
11-((tetrahydro-2H-pyran-2-yloxy)methyl)dodeca-2,6,10-
trienyl)naphthalen-4-yl acetate (19b)
In a manner similar to that for the synthesis of 19a from
17a, a crude product, which was obtained from 17b
(328 mg, 1.0 mmol), a catalytic amount of boron tri-
fluoride ether complex, and compound 18 (200 mg,
0.93 mmol) in acetate/dioxane 1:1 (1 mL), was purified
by flash column chromatography on silica gel (hexane/
ethyl acetate = 5/1) to give 19b (246 mg, 51%) as a yel-
low oil: IR (CHCl3): 3691, 3008, 2929, 2856, 1762,
3.12. (2E,6E,10E,14E)-3,7,11-Trimethyl-15-((tetrahydro-
2H-pyran-2-yloxy)methyl)hexadeca-2,6,10,14-tetraen-1-
ol (17c)
In a manner similar to that for the synthesis of 17a from
16a, a crude product, which was obtained from 16c
(550 mg, 1.3 mmol), 1 N aqueous NaOH in MeOH
(5 mL), was purified by flash column chromatography
on silica gel (hexane/ethyl acetate = 5/1) to give 17c
(387 mg, 78%) as a colorless oil: IR (CHCl3): 3450,
1669, 1456, 1233, 1021 cmꢀ1 1H NMR (500 MHz,
;
CDCl3): d 1.59 (3H, s), 1.63 (3H, s), 1.85 (3H, s),
1.99–2.13 (8H, m), 2.24 (3H, s), 2.47 (3H, s), 3.49 (2H,
d, J = 7.0 Hz), 3.51 (1H, m), 3.82 (1H, d, J = 11.5 Hz),
3.85–3.90 (1H, m), 4.07 (1H, d, J = 11.5 Hz), 4.59 (1H,
m), 5.07 (1H, t, J = 7.5 Hz), 5.23 (1H, t, J = 7.5 Hz),
5.38 (1H, t, J = 7.0 Hz), 5.86 (1H, s), 7.38–7.45 (2H,
m), 7.62 (1H, d, J = 8.5 Hz), 8.10 (1H, d, J = 8.5 Hz);
13C NMR (125 MHz, CDCl3): d 13.5, 14.0, 14.1, 16.1,
16.4, 19.5, 20.6, 22.7, 25.5, 26.3, 26.4, 30.7, 31.6, 39.2,
39.6, 62.1, 73.0, 97.4, 120.0, 120.6, 121.3, 121.8, 123.8,
124.9, 126.1, 126.3, 127.9, 131.8, 135.4, 137.9, 138.8,
147.8, 169.7; EI-LRMS MHz 520 (M+). EI-HRMS
calcd for C33H44O5 520.3189. Found 520.3192.
3010, 2940, 2855, 1667, 1454, 1231, 1118 cmꢀ1 1H
;
NMR (500 MHz, CDCl3): d 1.27 (4H, m), 1.60 (3H,
s), 1.61 (3H, s), 1.66 (3H, s), 1.68 (3H, s), 1.97–2.14
(12H, m), 3.48–3.53 (1H, m), 3.83–3.91 (2H, m), 4.09
(1H, d, J = 12.0 Hz), 4.15 (2H, d, J = 7.0 Hz), 4.61
(1H, dd, J = 2.5, 7.0 Hz), 5.10–5.13 (2H, m), 5.40–5.43
(2H, m); 13C NMR (125 MHz, CDCl3): d 14.0, 16.0,
16.3, 19.5, 22.6, 25.5, 26.3, 26.6, 30.7, 31.6, 39.3, 39.5,
39.6, 59.4, 62.1, 73.0, 97.4, 123.4, 123.8, 124.4, 127.9,
131.8, 134.7, 135.3, 139.7; EI-LRMS MHz 390 (M+).
EI-HRMS calcd for C25H42O3 390.3134. Found
390.3152.
3.15. 1-Hydroxy-3-methyl-2-((2E,6E,10E,14E)-3,7,11-
trimethyl-15-((tetrahydro-2H-pyran-2-yloxy)methyl)-
hexadeca-2,6,10,14-tetraenyl)naphthalen-4-yl acetate
(19c)
3.13. 1-Hydroxy-3-methyl-2-((2E,6E)-3-methyl-7-((tetra-
hydro-2H-pyran-2-yloxy)methyl)octa-2,6-dienyl)naphtha-
len-4-yl acetate (19a)
In a manner similar to that for the synthesis of 19a from
17a, a crude product, which was obtained from 17c
(258 mg, 0.66 mmol), a catalytic amount of boron tri-
fluoride ether complex, and compound 18 (130 mg,
0.6 mmol) in acetate/dioxane 1:1 (0.8 mL), was purified
by flash column chromatography on silica gel (hexane/
ethyl acetate = 5/1) to give 19c (170 mg, 48%) as a yellow
oil: IR (CHCl3): 3690, 3503, 2987, 2942, 2856, 1732,
Compound 18 (205 mg, 0.95 mmol) was dissolved in
ethyl acetate/dioxane 1:1 (1 mL), and a catalytic amount
of boron trifluoride ether complex was added to the
solution. After the resulting mixture was stirred at
45 ꢁC for 5 min, compound 17a (330 mg, 1.0 mmol) in
dioxane (0.2 mL) was added to the mixture and contin-
uously stirred at the same temperature for 3 h. The reac-
tion mixture was cooled to room temperature, poured
into ice-water, and extracted with ethyl acetate three
times. The combined organic layer was washed with
water and brine, dried over anhydrous MgSO4, and con-
centrated in vacuo. The residue was chromatographed
on silica gel using hexane/ ethyl acetate 5:1 to give 19a
(193 mg, 45%) as a yellow oil: IR (CHCl3): 3690, 3006,
1601, 1445, 1250, 1046 cmꢀ1 1H NMR (500 MHz,
;
CDCl3): d 1.57 (3H, s), 1.51–1.71 (6H, m), 1.60 (3H,
s), 1.65 (3H, s), 1.87 (3H, s), 1.98–2.14 (12H, m), 2.26
(3H, s), 2.47 (3H, s), 3.41–3.51 (1H, m), 3.53 (2H, d,
J = 6.5 Hz), 3.83 (1H, d, J = 11.5 Hz), 3.85–3.90 (1H,
m), 4.08 (1H, d, J = 11.5 Hz), 4.59 (1H, t, J = 4.0 Hz),
5.06–5.13 (2H, m), 5.24–5.27 (1H, m), 5.39–5.42 (1H,
m), 5.76 (1H, s), 7.40–7.46 (2H, m), 7.62–7.64 (1H, m),
8.11–8.13 (1H, m); 13C NMR (125 MHz, CDCl3): d
13.7, 14.1, 16.0, 16.1, 16.4, 19.5, 20.6, 22.7, 25.5, 26.3,
26.41, 26.46, 26.59, 30.7, 39.3, 39.7, 62.2, 73.0, 97.4,
120.6, 121.2, 121.8, 123.5, 124.0, 124.4, 124.9, 126.06,
126.13, 126.3, 128.0, 131.8, 134.7, 135.8, 138.0, 139.3,
2937, 2854, 1758, 1601, 1456, 1232, 1020 cmꢀ1 1H
;
NMR (500 MHz, CDCl3): d 1.53–1.73 (6H, m), 1.65
(3H, s), 1.86 (3H, s), 2.12–2.18 (4H, m), 2.25 (3H, s),
2.47 (3H, s), 3.50–3.53 (3H, m), 3.85–3.96 (2H, m),

Y. Suhara et al. / Bioorg. Med. Chem. 16 (2008) 3108–3117
3115
148.0, 169.7; EI-LRMS MHz 588 (M+). EI-HRMS
calcd for C38H52O5 588.3815. Found 588.3831.
3.18. 2-((2E,6E,10E,14E)-16-(1,4-dimethoxy-2-methyl-
naphthalen-3-yl)-2,6,10,14-tetramethylhexadeca-2,6,10,14-
tetraenyloxy)-tetrahydro-2H-pyran (20c)
3.16. 2-((2E,6E)-8-(1,4-dimethoxy-2-methylnaphthalen-3-
yl)-2,6-dimethylocta-2,6-dienyloxy)-tetrahydro-2H-pyran
(20a)
In a manner similar to that for the synthesis of 20a from
19a, a crude product, which was obtained from 19c
(100 mg, 0.17 mmol), 30% potassium hydride (disper-
sion in mineral oil) (91 mg, 0.68 mmol), and methyl io-
dide (0.63 mL, 1.0 mmol), was purified by flash
column chromatography on silica gel (hexane/ethyl ace-
tate = 10/1 to 5/1) to give 20c (59 mg, 60%) as a pale yel-
low oil: IR (CHCl3): 3690, 2989, 2940, 2854, 1732, 1602,
To a stirred suspension of 30% potassium hydride (dis-
persion in mineral oil) (142 mg, 1.1 mmol) in THF
(8 mL) was added compound 19a (120 mg, 0.27 mmol)
in THF (2 mL) at 0 ꢁC under argon. The mixture was
warmed to room temperature and then stirred for
20 min. After the color of the suspension changed to
dark green, methyl iodide (1 mL, 1.6 mmol) was added,
and the reaction mixture was stirred at room tempera-
ture further for 12 h. Addition of saturated aqueous
NH4Cl to the mixture at 0 ꢁC was followed by extraction
with ether three times. The organic layer was dried over
anhydrous MgSO4 and concentrated dry in vacuo. The
residue was chromatographed on silica gel using hexane/
ethyl acetate 10:1 to 5:1 to give 20a (76 mg, 65%) as a
pale yellow oil: IR (CHCl3): 3691, 3009, 2944, 2853,
1456, 1353, 1250, 1046 cmꢀ1 1H NMR (500 MHz,
;
CDCl3): d 1.51–1.71 (6H, m), 1.56 (3H, s), 1.56 (3H,
s), 1.65 (3H, s), 1.83 (3H, s), 1.92–2.12 (12H, m), 2.37
(3H, s), 3.48–3.51 (1H, m), 3.56 (2H, d, J = 6.5 Hz),
3.83 (1H, d, J = 11.5 Hz), 3.84–3.92 (1H, m), 3.87 (3H,
s), 3.88 (3H, s), 4.09 (1H, d, J = 11.5 Hz), 4.59 (1H, t,
J = 4.0 Hz), 5.06–5.12 (3H, m), 5.39–5.41 (1H, m),
7.44–7.48 (2H, m), 8.03–8.07 (2H, m); 13C NMR
(125 MHz, CDCl3): d 12.3, 14.0, 15.95, 16.03, 16.4,
19.6, 25.5, 26.3, 26.4, 26.6, 26.7, 30.7, 39.3, 39.7, 61.3,
62.17, 62.19, 73.0, 97.4, 122.1, 122.3, 122.8, 124.1,
124.4, 125.3, 125.4, 126.9, 127.3, 127.5, 127.9, 130.9,
131.8, 134.7, 135.1, 135.8, 149.7, 150.1; EI-LRMS
MHz 574 (M+). EI-HRMS calcd for C38H54O4
574.4022. Found 574.4042.
1594, 1456, 1353, 1206, 1020 cmꢀ1
;
1H NMR
(500 MHz, CDCl3): d 1.48-1.60 (4H, m), 1.63 (3H, s),
1.65–1.73 (2H, m), 1.83 (3H, s), 2.05 (2H, t,
J = 8.0 Hz), 2.14 (2H, t, J = 8.0 Hz), 2.37 (3H, s), 3.45–
3.49 (1H, m), 3.56 (2H, d, J = 5.5 Hz), 3.79 (1H, d,
J = 11.5 Hz), 3.82–3.86 (1H, m), 3.87 (3H, s), 3.88 (3H,
s), 4.05 (1H, d, J = 11.5 Hz), 4.56 (1H, t, J = 3.0 Hz),
5.11–5.13 (1H, m), 5.37–5.40 (1H, m), 7.44–7.46 (2H,
m), 8.03–8.06 (2H, m); 13C NMR (125 MHz, CDCl3): d
12.4, 14.0, 16.4, 19.5, 25.5, 26.29, 26.32, 30.6, 39.3,
61.3, 62.1, 62.2, 72.9, 97.4, 122.1, 122.3, 125.3, 125.4,
126.9, 127.2, 127.5, 127.6, 130.8, 132.0, 135.5, 149.7,
150.1; EI-LRMS MHz 438 (M+). EI-HRMS calcd for
C28H38O4 438.2770. Found 438.2781.
3.19. 2-((2E,6E)-8-hydroxy-3,7-dimethylocta-2,6-dienyl)-
3-methylnaphthalene-1,4-dione (MK-2 x-OH) (7)
To a stirred suspension of di-ammonium cerium (IV) ni-
trate (158 mg, 0.29 mmol) in water (0.1 mL) was added
20a (42 mg, 96 lmol) in CH3CN/ ether 5:1 (0.6 mL).
After addition of extra CH3CN (0.3 mL), the mixture
was stirred for 20 min. The reaction mixture was poured
into water and extracted with ether three times. The
combined ether layer was washed with water and brine,
dried over anhydrous MgSO4, and concentrated in va-
cuo. The residue was purified by preparative TLC (hex-
ane/ethyl acetate = 3/1) to give 7 (22 mg, 72%) as a
yellow oil: IR (CHCl3): 3690, 3602, 2997, 2928, 1660,
1597, 1458, 1378, 1332, 1297, 973 cmꢀ1;1H NMR
(500 MHz, CDCl3): d 1.63 (3H, s), 1.79 (3H, s), 2.03
(2H, t, J = 7.5 Hz), 2.12 (2H, t, J = 7.5 Hz), 2.19 (3H,
s), 3.36 (2H, d, J = 6.5 Hz), 3.94 (2H, br s), 5.01 (1H,
t, J = 7.0 Hz), 5.32 (1H, t, J = 7.0 Hz), 7.68–7.70 (2H,
m), 8.07–8.09 (2H, m); 13C NMR (125 MHz, CDCl3):
d 12.7, 13.7, 16.3, 25.9, 26.1, 39.2, 68.9, 119.6, 125.6,
126.2, 126.3, 132.1, 133.3, 133.4, 135.0, 137.0, 143.4,
146.1, 184.6, 185.4; EI-LRMS MHz 324 (M+). EI-
HRMS calcd for C21H24O3 324.1725. Found 324.1748.
3.17. 2-((2E,6E,10E)-12-(1,4-dimethoxy-2-methylnaphth-
alen-3-yl)-2,6,10-trimethyldodeca-2,6,10-trienyloxy)-tet-
rahydro-2H-pyran (20b)
In a manner similar to that for the synthesis of 20a from
19a, a crude product, which was obtained from 19b
(110 mg, 0.21 mmol), 30% potassium hydride (disper-
sion in mineral oil) (113 mg, 0.85 mmol), and methyl io-
dide (0.78 mL, 1.3 mmol), was purified by flash column
chromatography on silica gel (hexane/ethyl acetate = 10/
1 to 5/1) to give 20b (66 mg, 62%) as a pale yellow oil: IR
(CHCl3): 3690, 2939, 2854, 1732, 1602, 1456, 1376, 1227,
1
1017 cmꢀ1; H NMR (500 MHz, CDCl3): d 1.49–1.71
(6H, m), 1.57 (3H, s), 1.62 (3H, s), 1.83 (3H, s), 1.95–
2.09 (8H, m), 2.37 (3H, s), 3.47–3.50 (1H, m), 3.56
(2H, d, J = 6.5 Hz), 3.78–3.85 (1H, m), 3.82 (1H, d,
J = 11.5 Hz), 3.87 (3H, s), 3.88 (3H, s), 4.07 (1H, d,
J = 11.5 Hz), 4.58 (1H, t, J = 3.5 Hz), 5.07–5.11 (2H,
m), 5.36–5.39 (1H, m), 7.44–7.47 (2H, m), 8.03–8.06
(2H, m); 13C NMR (125 MHz, CDCl3): d 12.4, 14.0,
16.0, 16.4, 19.5, 25.5, 26.34, 26.37, 26.6, 30.7, 39.3,
39.7, 61.3, 62.16, 62.19, 73.0, 97.4, 122.1, 122.2, 122.8,
124.3, 125.3, 125.4, 126.9, 127.3, 127.5, 127.8, 130.9,
131.8, 134.8, 135.7, 149.7, 150.1; EI-LRMS MHz 506
(M+). EI-HRMS calcd for C33H46O4 506.3396. Found
506.3401.
3.20. 2-((2E,6E,10E)-12-hydroxy-3,7,11-trimethyldo-
deca-2,6,10-trienyl)-3-methylnaphthalene-1,4-dione (MK-
3 x-OH) (8)
In a manner similar to that for the synthesis of 7 from
20a, a crude product, which was obtained from 20b
(45 mg, 88 lmol), di-ammonium cerium (IV) nitrate
(146 mg, 0.27 mmol) in water, and CH3CN/ ether 5:1
(0.6 mL), was purified by preparative TLC (hexane/ethyl
acetate = 3/1) to give 8 (26 mg, 76%) as a yellow oil: IR

3116
Y. Suhara et al. / Bioorg. Med. Chem. 16 (2008) 3108–3117
(CHCl3): 3677, 3620, 2979, 2926, 1657, 1430, 1332, 1229,
36 lmol) and pyridinium dichromate (27 mg, 71 lmol)
in CH2Cl2, was purified by preparative TLC (hexane/
ethyl acetate = 5/1) to give 11 (7.7 mg, 55%) as a yellow
oil: IR (CHCl3): 3016, 1682, 1660, 1598, 1435, 1332,
1
929 cmꢀ1; H NMR (500 MHz, CDCl3): d 1.57 (3H, s),
1.63 (3H, s), 1.79 (3H, s), 1.93–2.09 (8H, m), 2.17 (3H,
s), 3.67 (2H, d, J = 7.5 Hz), 3.97 (2H, br s), 5.00–5.07
(2H, m), 5.32–5.35 (1H, m), 7.68–7.70 (2H, m), 8.07–
8.09 (2H, m); 13C NMR (125 MHz, CDCl3): d 12.7,
13.6, 16.0, 16.4, 26.0, 26.2, 26.3, 39.2, 39.6, 69.0, 119.2,
124.1, 126.0, 126.2, 126.3, 132.1, 132.2, 133.28, 133.34,
134.7, 134.8, 137.4, 143.3, 146.1, 184.5, 185.5; EI-LRMS
MHz 392 (M+). EI-HRMS calcd for C26H32O3
392.2351. Found 392.2376.
1
1227 cmꢀ1; H NMR (500 MHz, CDCl3): d 1.59 (3H,
s), 1.70 (3H, s), 1.79 (3H, s), 2.00 (2H, t, J = 7.5 Hz),
2.06–2.11 (4H, m), 2.19 (3H, s), 2.39 (2H, dd, J = 7.5,
15.0 Hz), 3.37 (2H, d, J = 7.5 Hz), 5.02 (1H, dt,
J = 1.5, 7.5 Hz), 5.09 (1H, dt, J = 1.5, 7.5 Hz), 6.40
(1H, dt, J = 1.5, 7.5 Hz), 7.68–7.70 (2H, m), 8.07–8.09
(2H, m), 9.35 (1H, s); 13C NMR (125 MHz, CDCl3): d
9.19, 12.7, 15.9, 16.4, 26.1, 26.4, 27.4, 37.9, 39.5, 119.4,
125.2, 126.3, 126.3, 132.17, 132.21, 133.37, 133.41,
133.6, 137.3, 139.4, 143.4, 146.2, 154.4, 184.6, 185.5,
195.3; EI-LRMS MHz 390 (M+). EI-HRMS calcd for
C26H30O3 390.2195. Found 390.2201.
3.21. 2-((2E,6E,10E,14E)-16-hydroxy-3,7,11,15-tetram-
ethylhexadeca-2,6,10,14-tetraenyl)-3-methylnaphthalene-
1,4-dione (MK-4 x-OH) (9)
In a manner similar to that for the synthesis of 7 from
20a, a crude product, which was obtained from 20c
(41 mg, 71 lmol), di-ammonium cerium (IV) nitrate
(117 mg, 0.21 mmol) in water, and CH3CN/ether 5:1
(0.6 mL), was purified by preparative TLC (hexane/ethyl
acetate = 3/1) to give 9 (23 mg, 71%) as a yellow oil: IR
(CHCl3): 3607, 3605, 2979, 2926, 1658, 1430, 1332, 1229,
3.24. (2E,6E)-8-(1,4-dihydro-2-methyl-1,4-dioxonaphtha-
len-3-yl)-2,6-dimethylocta-2,6-dienal (MK-4 x-CHO)
(12)
In a manner similar to that for the synthesis of 10 from 7, a
crude product, which was obtained from 9 (10 mg,
22 lmol) and pyridinium dichromate (16 mg, 43 lmol)
in CH2Cl2, was purified by preparative TLC (hexane/
ethyl acetate = 5/1) to give 12 (5.1 mg, 51%) as a yellow
oil: IR (CHCl3): 2978, 2929, 2855, 1682, 1660, 1618,
1
973 cmꢀ1; H NMR (500 MHz, CDCl3): d 1.53 (3H, s),
1.56 (3H, s), 1.66 (3H, s), 1.79 (3H, s), 1.91–2.13 (12H,
m), 2.19 (3H, s), 3.37 (2H, d, J = 7.5 Hz), 3.99 (2H, br
s), 5.00–5.08 (3H, m), 5.38 (1H, dt, J = 1.5, 7.5 Hz),
7.68–7.70 (2H, m), 8.07–8.09 (2H, m); 13C NMR
(125 MHz, CDCl3): d 12.7, 13.7, 15.96, 16.0, 16.4,
26.0, 26.2, 26.5, 26.6, 39.3, 39.6, 39.7, 69.0, 119.1,
123.9, 124.5, 126.16, 126.22, 126.3, 132.2, 133.3, 133.4,
134.5, 135.1, 137.5, 143.4, 146.2, 184.5, 185.4; EI-LRMS
MHz 460 (M+). EI-HRMS calcd for C31H40O3
460.2977. Found 460.2965.
1597, 1440, 1331, 1227 cmꢀ1 1H NMR (500 MHz,
;
CDCl3): d 1.56 (3H, s), 1.59 (3H, s), 1.74 (3H, s), 1.79
(3H, s), 1.92 (2H, t, J = 7.0 Hz), 1.98–2.07 (6H, m), 2.14
(2H, t, J = 7.5 Hz), 2.19 (3H, s), 2.43 (2H, dd, J = 7.0,
14.5 Hz), 3.37 (2H, d, J = 7.5 Hz), 5.00-5.12 (3H, m),
6.45 (1H, dt, J = 1.5, 7.5 Hz), 7.68–7.70 (2H, m), 8.07–
8.09 (2H, m), 9.37 (1H, s); 13C NMR (125 MHz, CDCl3):
d 9.23, 12.7, 15.9, 16.4, 26.0, 26.5, 26.6, 27.4, 38.0, 39.5,
39.7, 119.2, 124.1, 125.6, 126.2, 126.3, 132.2, 133.3,
133.4, 135.0, 137.5, 139.4, 143.4, 146.2, 154.5, 184.6,
185.5, 195.3; EI-LRMS MHz 458 (M+). EI-HRMS calcd
for C31H38O3 458.2821. Found 458.2812.
3.22. (2E,6E,10E,14E)-16-(1,4-dihydro-2-methyl-1,4-
dioxonaphthalen-3-yl)-2,6,10,14-tetramethylhexadeca-
2,6,10,14-tetraenal (MK-2 x-CHO) (10)
To a solution of 7 (15 mg, 46 lmol) in CH2Cl2 (3 mL)
was added pyridinium dichromate (35 mg, 92 lmol) at
room temperature, and the mixture was stirred for
12 h. After the solution was concentrated, the residue
was purified by preparative TLC (hexane/ethyl acetate
5/1) to give 10 (5.2 mg, 35%) as a yellow oil: IR (CHCl3):
3.24.1. Carboxylase activity assays. Carboxylase activity
was assayed by previously described methods.26–29 The
amount of 14CO2 incorporated into exogenous substrates
was measured in reaction mixtures of 125 lL containing
substrate at the indicated concentration, 222 lM of re-
duced MK-4, 16 lM of the propeptide ProFIX19 which
contains the sequence AVFLDHENANKILNRPKRY,
1.4 mM NaH14CO3 (5 lCi), 25 mM MOPS (pH 7.0),
500 mM NaCl, 0.16% (w/v) phosphatidylcholine, 0.16%
(w/v) CHAPS, 8 mM DTT, and 0.8 M ammonium sul-
fate, unless stated otherwise. All of the assay components
except for the microsomal fraction were prepared as a
master mixture. The incorporation of 14CO2 into peptide
substrates for over 30 min was assayed in a scintillation
counter. Stimulation experiments with reduced MK-4
were performed at a constant concentration of the enzyme
sample and substrate (3.6 mM FLEEL) with increasing
concentrations of reduced MK-4, as indicated. All assays
were performed in quadruplicate.
2929, 2855, 1682, 1660, 1618, 1597, 1258 cmꢀ1 1H
;
NMR (500 MHz, CDCl3): d 1.72 (3H, s), 1.83 (3H, s),
2.18 (2H, dd, J = 7.5, 15.0 Hz), 2.19 (3H, s), 2.45 (2H,
dd, J = 7.5, 15.0 Hz), 3.39 (2H, d, J = 7.0 Hz), 5.09
(1H, dt, J = 1.5, 7.5 Hz), 6.41 (1H, dt, J = 1.5, 7.5 Hz),
7.69–7.71 (2H, m), 8.07–8.10 (2H, m), 9.33 (1H, s); 13C
NMR (125 MHz, CDCl3): d 9.2, 12.7, 16.3, 26.1, 27.3,
38.0, 120.5, 121.3, 126.27, 126.31, 132.1, 133.41,
133.44, 136.0, 143.5, 145.7, 153.8, 184.5, 185.4, 195.1;
EI-LRMS MHz 322 (M+). EI-HRMS calcd for
C21H22O3 322.1569. Found 322.1554.
3.23. (2E,6E,10E)-12-(1,4-dihydro-2-methyl-1,4-diox-
onaphthalen-3-yl)-2,6,10-trimethyldodeca-2,6,10-trienal
(MK-3 x-CHO) (11)
3.24.2. Evaluation of apoptosis-inducing activity of vita-
min K analogues. MG-63 cells were maintained in con-
tinuous culture in DMEM supplemented with 10%
In a manner similar to that for the synthesis of 10 from
7, a crude product, which was obtained from 8 (14 mg,

Y. Suhara et al. / Bioorg. Med. Chem. 16 (2008) 3108–3117
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Acknowledgments
We are grateful to Dr. Makiko Sugiura, Dr. Atsuko
Takeuchi, and Dr. Chisato Tode for the spectroscopic
measurements. This investigation was supported in parts
by The Mochida Memorial Foundation for Medical and
Pharmaceutical Research, and a Grant-in-Aid for Scien-
tific Research <KAKENHI> from the Japan Society for
the Promotion of Science.
Supplementary data
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