Synthesis of Novel Synthetic Vitamin K Analogues Prepared by Introduction of a Heteroatom and a Phenyl Group That Induce Highly Selective Neuronal Differentiation of Neuronal Progenitor Cells

Article abstract of DOI:10.1021/acs.jmedchem.6b01717

We synthesized novel vitamin K₂ analogues that incorporated a heteroatom and an aromatic ring in the side chain and evaluated their effect on the selective differentiation of neuronal progenitor cells into neurons in vitro. The results showed that a menaquinone-2 analogue bearing a p-fluoroaniline had the most potent activity, which was more than twice as great as the control. In addition, the neuronal selectivity was more than 3 times greater than the control.

Full text of DOI:10.1021/acs.jmedchem.6b01717

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Brief Article
Synthesis of Novel Synthetic Vitamin K Analogues Prepared by
Introduction of a Heteroatom and a Phenyl Group that Induce Highly
Selective Neuronal Differentiation of Neuronal Progenitor Cells
Kimito Kimura, Yoshihisa Hirota, Shigefumi Kuwahara, Atsuko Takeuchi,
Chisato Tode, Akimori Wada, Naomi Osakabe, and Yoshitomo Suhara
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01717 • Publication Date (Web): 22 Feb 2017
Downloaded from http://pubs.acs.org on March 1, 2017
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Journal of Medicinal Chemistry
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Synthesis of Novel Synthetic Vitamin K Analogues Prepared by
Introduction of a Heteroatom and a Phenyl Group that Induce
Highly Selective Neuronal Differentiation of Neuronal Progenitor
Cells
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†
‡
§
||
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Kimito Kimura, Yoshihisa Hirota, Shigefumi Kuwahara, Atsuko Takeuchi, Chisato Tode, Akimori
*
, †
⊥
#
Wada, Naomi Osakabe, and Yoshitomo Suhara
†Laboratory of Organic Synthesis and Medicinal Chemistry, Department of Bioscience and Engineering, College of Systems
Engineering and Science, Shibaura Institute of Technology, 307 Fukasaku, Minuma-ku, Saitama 337-8570, Japan
‡Laboratory of Biochemistry, Department of Bioscience and Engineering, College of Systems Engineering and Science, Shi-
baura Institute of Technology, 307 Fukasaku, Minuma-ku, Saitama 337-8570, Japan
§Laboratory of Applied Bioorganic Chemistry, Graduate School of Agricultural Science, Tohoku University, 1-1 Tsutsumi-
dori-Amamiyamachi, Aoba-ku, Sendai, 981-8555, Japan
||Analytical Laboratory, Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higashinada-ku, Kobe 658-8558,
Japan
⊥
Department of Life Science for Organic Chemistry, Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higash-
inada-ku, Kobe 658-8558, Japan
#
Food and Nutrition Laboratory, Department of Bioscience and Engineering, College of Systems Engineering and Science,
Shibaura Institute of Technology, 307 Fukasaku, Minuma-ku, Saitama 337-8570, Japan
KEYWORDS. Vitamin K, menaquinone-4, differentiation, neuronal cells, neural progenitor cells
2
ABSTRACT: We synthesized novel vitamin K analogues that incorporated a heteroatom and an aromatic ring in the side chain
and evaluated their effect on the selective differentiation of neuronal progenitor cells into neurons in vitro. The result showed that a
menaquinone-2 analogue bearing a p-fluoroaniline had the most potent activity, which was more than twice as great as the control.
In
addition,
the
neuronal
selectivity
was
more
than
three
times
greater
than
the
control.
INTRODUCTION
differentiation and maturation for establishing safe and practi-
cal cell therapies. Several natural products have been identi-
fied which directly affected differentiation of NPCs into neu-
rons, astrocytes, and oligodendrocytes, however, the com-
Neural progenitor cells (NPCs) are located in several region
of the human adult brain such as the subventricular zone and
the dentate gyrus of the hippocampus. NPCs can contribute to
neurogenesis in adulthood because they are capable of differ-
4
pounds demonstrated no selectivity or only weak activity.
1
entiating into neurons, astrocytes, and oligodendrocytes.
We previously reported that menaquinone-4, one of the vit-
amin K homologues, was biosynthesized and was used in the
Therefore, NPCs would have possible applications for devel-
opment of stem-cell-based therapies for neurodegenerative
5
brain. There are two natural forms of vitamin K homologues,
2
diseases. It have been reported that the grafting of NPCs im-
plant-derived vitamin K
terium-derived vitamin K
ing MK-2 (3), MK-3 (4), and MK-4 (5) (Figure 1). In contrast,
vitamin K (menadione) (6) is an artificial vitamin K. We have
1
(phylloquinone, PK) (1) and the bac-
proves neurological deficits in neurodegenerative diseases
2
(2) (menaquinone-n, MK-n) includ-
such as Parkinson’s disease, ischemic stroke, and spinal cord
3
injury. However, the induction of new neuronal cells in the
3
damaged brain is relatively low and might be nonfunctional.
Otherwise, the transplantation of neural stem cells was ex-
pected to repair lesions since they could differentiate into neu-
rons. But transplanted cells also had difficulties of differentia-
tion, maturation, and integration into host neural networks. We
have explored a new strategy to repair damaged brain tissues.
We focused on “organic compounds” which promote NPCs
recently revealed that MK-4 had the selective ability to cause
differentiation of NPCs, derived from mouse cerebrum, into
neuronal cells. Vitamin K homologues also have been report-
ed to play a role in preventing oxidative injury to developing
oligodendrocytes and neurons. Based on this findings, we have
synthesized new vitamin K analogues modified at the ω-
terminal group and several analogues with introduction of
6
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aromatic groups exhibited potent differentiation activity com-
pared to natural vitamin K. The activity of the compound was
tives. Thus, the individual desired vitamin K analogues of
7
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6
aniline derivatives 7-9 and 13-15 were obtained in 52-78%
yield. The phenol derivatives 10-12 and 16-18 were obtained
in 43-82 % yield.
double that of the control and it was also neuronal selective.
This finding showed modification of the side chain of vitamin
K could increase the differentiation activity. However, much
more potent activity is necessary for application as therapeutic
agents. If the analogues exhibited strong differentiation activi-
ty in neuronal cells, they may be applicable to elucidate the
mechanism of vitamin K in the brain. In this study, we synthe-
sized new analogues by introduction of a heteroatom as well
as an aromatic ring in the molecule with the expectation of
interaction with target proteins related to differentiation. Here,
we report the synthesis and structure-activity relationship of
Scheme 1. Synthesis of Vitamin K Analogues 7- 18
0
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2
novel vitamin K analogues that selectively induced neuronal
differentiation of multipotent neural progenitor cells.
1
Figure 1. Structure of vitamin K homologues: vitamin K (1),
vitamin K
(6).
2
(2) (MK-2 (3), MK-3 (4), and MK-4 (5)), and vitamin
K
3
aReagents and conditions: (a) 10% Na
2 2 4 2
S O aq., Et O, quant.;
(b) BF
30%; (d) MnO2, 76-78%; (e) aniline derivative, AcOH,
NaBH(OAc) , 52- 78%; (f) phenol derivative, Ph P, DIAD, 43-
2%.
3 2 2
•Et O, 32-34%; (c) SeO , salicylic acid, 70% TBHP, 5-
RESULTS AND DISCUSSION
The requisite vitamin K analogues containing aromatic
3
3
2
8
rings and heteroatoms at the ω-terminal position were obtained
by the synthetic method as shown in Scheme 1. Compounds
were prepared by introducing a nitrogen atom or an oxygen
atom as well as phenyl groups. At first, we planned the syn-
thesis of the side-chain by introduction of the nitrogen atom
using aniline derivatives, followed by a coupling reaction with
vitamin K by Friedel-Crafts alkylation. However, the cou-
3
pling reaction did not proceed at all. Presumably, the nitrogen
atom incorporated into the side chain interrupted the progress
We investigated the differentiation-inducing activity of the
vitamin K analogues using NPCs, which were prepared from
embryonic mouse brain. The general method for preparation
7
was as follows as we previously reported : (i) Neural stem
cells were dissociated from embryonic day 14 mouse cere-
brum and cultured according to the method previously de-
1
0
scribed. (ii) After the cells were seeded and cultured for 24 h,
they were treated with 1 µM of the vitamin K analogues every
second day for 4 days. We confirmed the differentiation of the
NPCs by an immunofluorescence staining method. The specif-
ic antigen microtubule-associated protein 2 (Map2) was ex-
pressed on the surface of neuronal cells that had successfully
differentiated from progenitor cells, and the sample emitted
red fluorescence. On the other hand, the expression of glial
fibrillary acidic protein (Gfap), indicating differentiation to the
astrocyte, was detected as green emission. The differentiation
could be evaluated from the resulting fluorescence with fluo-
rescence microscopy (Figure 2). In the ethanol control, we
observed the differentiation of NPCs into only neuronal cells.
On the other hand, natural menaquinone 5 promoted the dif-
ferentiation of NPCs into both neuronal cells and astrocytes.
The analogues 9 (with a nitrogen substituent in the side chain)
and 17 (with a phenol substituent) promoted the selective dif-
ferentiation of NPCs into neuronal cells since the differentiat-
ed cells mostly exhibited red fluorescence, but no green fluo-
rescence.
of the reaction with vitamin K
the order of the synthetic coupling reactions between the side
chain and vitamin K . We first synthesized vitamin K homo-
3
. Therefore, we reconsidered
3
logues 3 and 4 by a coupling reaction with geraniol (19) or
farnesol (20) and a hydroquinone according to a previously
8
reported method. After 6 was reduced to the hydroquinone
derivative with a 10% sodium hydrosulfite (aq) solution in
diethyl ether, the isoprene units 19 or 20 were successively
coupled with the hydroquinone in the presence of a catalytic
3 2
amount of BF •Et O to give 3 and 4 in 34% and 32% yield,
respectively. Then, the hydroxyl group was introduced to the
carbon atom of ω-terminal side chain in 3 and 4 to give alco-
hols 21 and 22 in 30% and 5% yield, respectively. Since the
side chain length of 4 was longer than that of 3, the selectivity
of introduction of the oxygen at the ω-position of 4 did not
proceed very well. The alcohols 21 and 22 were oxidized with
2
MnO to obtain aldehydes 23 and 24 in 78% and 76% yield,
9
respectively. Introduction of the nitrogen atom into the side
chain of vitamin K was provided by reductive amination reac-
tion between 23 or 24 and aniline derivatives. Incorporation of
an oxygen atom in the side chain was successfully carried out
with Mitsunobu reaction between 21 or 22 and phenol deriva-
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Journal of Medicinal Chemistry
control
5
9
17
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6
(
B)
2.0
*
*
1
.5
Figure 2. Vitamin K derivatives induce neuronal differentiation
of NPCs isolated from an embryonic mouse cerebrum. The cells
were treated with the indicated compounds 5, 9 and 17 at 1 μM.
After 48 h, cells were immunostained with markers for neurons
(Map2, red) and astrocytes (Gfap, green), and also with DAPI
(blue) for nuclei. Mature neurons and a very small amount of
astrocytes were respectively observed with Map2 (red) and Gfap
1
.0
**
*
0.5
.0
0
EtOH
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18
0
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2
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9
0
2
Figure 3. Differentiation-inducing activity of vitamin K ana-
logues from neuronal progenitor cells into neuronal cells as de-
(green) after treatment with MK-4 (5), a natural menaquinone. On
termined by quantitation of mRNA synthesis for Map2 and β-
the other hand, the synthesized analogues 9 (bearing a nitrogen)
and 17 (bearing a phenol) induced only neuronal differentiation.
actin (A), Gfap and β-actin (B) using PCR methodology. The
relative ratios were calculated for Map2/β-actin (A) and Gfap/β-
actin (B), respectively, and then the data were normalized with
respect to the EtOH control, which was expressed as 1.0. Cells
2
were treated with the indicated vitamin K analogues as well as
natural menaquinones 3-5 and menadione 6 at 1.0 ×10 M. Com-
pound 7- 12 were MK-2 analogues and compound 13- 18 were
MK-3 analogues. The histogram data are expressed as the means
obtained from three independent experiments; the error bars indi-
cate the SD. Significant difference: ***, p < 0.001, **, p < 0.005,
Then we quantitated the mRNA of Map2, Gfap and β-actin
of the cells using real-time PCR methodology to measure the
differentiation activity (Figure 3(A) (B)). The samples treated
with EtOH as untreated controls and with menaquinones 3-5
as positive controls were used for the purpose of comparison
with our compounds. In Figure 3 (A), the data were derived
-6
from calculation of the ratio of the mRNA levels of Map2/β-
*
, p < 0.01, between EtOH and compounds (by Dunnett’s t-test);
actin. The ratio obtained for the untreated control (EtOH) was
expressed as 1.0 and the values for the analogues were ex-
pressed with respect to the control value. Most of the ana-
logues effectively increased the expression of the mRNA level
of Map2. This result suggests that most compounds enhanced
the differentiation-inducing activity toward neuronal cells
compared to controls. Among natural menaquinones, 5
showed the highest activity that was more than twice the re-
sponse of the control in this experiment. Interestingly, the
MK-2 analogue 9, modified with the p-fluoroaniline at the ω-
terminal position of 3, showed the most potent activity among
the synthetic analogues. It was also more than three times as
effective as the EtOH control and displayed almost the same
activity as 5. There were no significant differences between 5
and 9 by Student’s t-test. While, the activity of the MK-2 ana-
logue 7 was significantly higher than that of the EtOH control.
Among the phenol derivatives, compound 16 and compound
There are no significant differences between 5 and 9 (by Student’s
t-test) in (A).
To evaluate selective differentiation activity of vitamin K
analogues toward neuronal cells, we also calculated the rela-
tive ratio of the mRNA of Map2/Gfap from the data of Figure
3
(A) and (B) as shown in Figure 4. From this result, 9 and 17
showed significantly higher ratios compared to control
EtOH). Thus, these compounds promoted selective differenti-
(
ation into neuronal cells rather than into astrocyte cell. Over-
all, MK-2 analogues bearing an aniline group and MK-3 ana-
logues possessing a phenol tended to exhibit highly selective
differentiation activity toward neuronal cells. Specifically, 9
showed the highest effects for both selectivity and activity
among the compounds. The selectivity was much higher than
2
that observed with natural vitamin K . As compared to the
1
7 showed higher activity, but with significantly lower effects
natural menaquinone 5, compound 9 selectively favored the
production of neuronal cells, as confirmed by Student’s t-test
(Figure 4).
than 7 and 9 (Figure 3(A)). On the other hand, Figure 3 (B)
was obtained from calculation of the mRNA level of Gfap/β-
actin same as Figure 3 (A). This graph indicated that 5 signifi-
cantly increased expression of the mRNA level both of Map2
and Gfap, while compounds 7, 9, 16, and 17 promoted only
Map2.
##
4
.0
***
***
3.0
*
2
.0
.0
1
(A)
4
.0
.0
.0
0.0
***
EtOH
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18
***
3
2
***
**
*
Figure 4. Relative ratios of differentiation-inducing activity of
vitamin K analogues to convert neuronal progenitor cells into
either neuronal cells or astrocyte cells, as determined by calcula-
tion of the relative ratios of Map2/Gfap from the data of Figure 3
(A) and (B). The data were normalized with respect to that ob-
tained with the EtOH control, which was expressed as 1.0. Cells
2
1.0
0.0
EtOH
3
4
5
6
7
8
9
10 11 12 13 14 15 16 17 18
2
were treated with the indicated vitamin K analogues 7- 18 as well
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Page 4 of 7
as natural menaquinones 3-5 and menadione 6 at 1.0 ×10-6 M.
7.17-7.11 (2H, m), 6.69-6.54 (3H, m), 5.36-5.32 (1H, m),
5.03-4.99 (1H, m), 3.74 (1H, br s), 3.57 (2H, s), 3.36 (2H, d, J
6.8 Hz), 2.18 (3H, s), 2.15-2.09 (2H, m), 2.05-1.99 (2H, m),
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Compound 7- 12 were MK-2 analogues, on the other hand, com-
pound 13- 18 were MK-3 analogues. The histogram data are ex-
pressed as the means obtained from three independent experi-
ments; the error bars indicate the SD. Significant differences:
=
13
1.78 (3H, s), 1.63 (3H, s). C NMR (100 MHz, CDCl
3
); δ
185.6, 184.6, 148.6, 146.2, 143.5, 137.3, 133.5, 133.4, 132.6,
*
(
**, p < 0.001, *, p < 0.01, between EtOH and the compounds
by Dunnett’s t-test); #, p < 0.005 between 5 and 9 (by Student’s
t-test).
1
1
32.24, 132.22, 129.2, 126.4, 126.3, 125.8, 119.5, 117.2,
12.9, 51.8, 39.4, 26.3, 26.1, 16.5, 14.8, 12.8. HRMS
+
([M+H] ) m/z calcd for C27
H30NO
2
400.2277; found 400.2279.
The effects of the vitamin K on NPCs differentiation has not
been investigated until now. Recently, it was reported that
2-((2E,6E)-3,7-Dimethyl-8-(p-tolylamino)octa-2,6-dien-1-
yl)-3-methylnaphthalene-1,4-dione (8). Similar to the syn-
thesis of 7 from 23, the crude product 8, which was obtained
from reaction of 23 (31 mg, 96 µmol), p-toluidine (12 mg, 115
µmol), acetic acid (7 µL, 115 µmol), MS4A, and sodium tri-
vitamin E isomer δ-tocopherol enhanced the efficiency of neu-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
ral stem cell differentiation via the L-type calcium channel.
The side chain part of δ-tocopherol and vitamin K resemble
each other, suggesting that the mechanism of differentiation
might also be mediated through a calcium channel.
2 2
acetoxyborohydride (41 mg, 192 µmol) in CH Cl (2 mL), was
purified by silica gel column chromatography (n-hexane/ ethyl
1
acetate= 10: 1), giving 8 (23 mg, 58%) as a brown oil. H
NMR (400 MHz, CDCl
m), 6.97-6.92 (2H, m), 6.55-6.48 (2H, m), 5.35-5.32 (1H, m),
.02-4.99 (1H, m), 3.55 (2H, s), 3.36 (2H, d, J = 7.2 Hz), 2.21
3H, s), 2.18 (3H, s), 2.14-2.09 (2H, m), 2.04-1.98 (2H, m),
3
); δ 8.09-8.07 (2H, m), 7.69-7.67 (2H,
CONCLUSION
5
(
1
In conclusion, we successfully introduced nitrogen or oxy-
gen atoms into the side chain of vitamin K derivatives to pro-
duce new analogues. We found that some of these compounds
had selective and potent activity toward the differential of
neuronal progenitor cells into mature neuronal cells. We clari-
fied that significant differences in differentiation activity could
be obtained by altering the functional group and the heteroa-
tom of the side chain. A more detailed examination is under-
way to clarify the mechanism of action of these vitamin K
analogues. If the mechanism and the target protein are clari-
fied, it may be possible to design and synthesize much more
potent compounds based on our findings.
13
3
.78 (3H, s), 1.63 (3H, s). C NMR (100 MHz, CDCl ); δ
185.5, 184.6, 146.3, 146.2, 143.5, 137.3, 133.5, 133.4, 132.8,
132.2, 129.7, 126.4, 126.3, 125.7, 119.5, 113.1, 52.2, 39.5,
+
26.3, 26.1, 20.4, 16.5, 14.8, 12.8. HRMS ([M+H] ) m/z calcd
2
for C28H32NO 414.2433; found 414.2432.
2-((2E,6E)-8-((4-Fluorophenyl)amino)-3,7-dimethylocta-
2,6-dien-1-yl)-3-methylnaphthalene-1,4-dione (9). Similar to
the synthesis of 7 from 23, the crude product 9, which was
obtained from reaction of 23 (92 mg, 285 µmol), p-
fluoroaniline (33 µL, 342 µmol), acetic acid (20 µL, 342
µmol), MS4A, and sodium triacetoxyborohydride (121 mg,
1
74 µmol) in CH
2 2
Cl (3 mL), was purified by silica gel col-
EXPERIMENTAL SECTION
umn chromatography (n-hexane/ ethyl acetate= 10: 1), giving
1
9
8
6
(
2
3
(93 mg, 78%) as a brown oil. H NMR (400 MHz, CDCl ); δ
High-resolution ESI-MS (ESI-HRMS) was performed with
1
.07-8.05 (2H, m), 7.68-7.66 (2H, m), 6.88-6.79 (2H, m),
.55-6.46 (2H, m), 5.34-5.30 (1H, m), 5.02-4.99 (1H, m), 3.66
1H, br s), 3.52 (2H, s), 3.35 (2H, d, J = 6.8 Hz), 2.17 (3H, s),
a Micro-mass Q-TOF mass spectrometer. H NMR spectra
1
3
were recorded at 400 MHz and C NMR spectra were record-
ed at 100 MHz using CDCl as a solvent unless otherwise
3
.14-2.09 (2H, m), 2.02-1.99 (2H, m), 1.78 (3H, s), 1.62 (3H,
specified. Chemical shifts are given in parts per million (δ)
using tetramethylsilane (TMS) as the internal standard. Col-
umn chromatography was carried out on silica gel 60 (70-230
mesh). The nuclei were stained with DAPI. Cells were fixed
with 10% formaldehyde solution, viewed under a fluorescent
microscope and photographed. Unless otherwise noted, all
reagents were purchased from commercial suppliers. We con-
firmed that the purities of the compounds 7-18 attained more
that 95% as determined by HPLC (high pressure liquid chro-
matography).
13
s). C NMR (100 MHz, CDCl
3
); δ 185.5, 184.6, 156.8, 154.5,
46.1, 144.9, 143.4, 137.2, 133.45, 133.40, 132.5, 132.20,
32.18, 126.4, 126.3, 125.9, 119.5, 115.6, 115.4, 113.7, 113.6,
1
1
+
52.4, 39.4, 26.2, 26.1, 16.5, 14.8, 12.8. HRMS ([M+H] ) m/z
calcd for C27 418.2182; found 418.2182.
-((2E,6E)-3,7-Dimethyl-8-phenoxyocta-2,6-dien-1-yl)-3-
H29FNO
2
2
methylnaphthalene-1,4-dione (10). To a solution of MK-2 ω-
alcohol 21 (24 mg, 110 µmol) in THF (2 mL) was added phe-
nol (31 mg, 330 µmol) and triphenylphosphine (87 mg, 330
µmol) at 0ºC under argon. After stirring for 30 min at the same
temperature, diisopropyl azodicarboxylate (67 mg, 330 µmol)
in THF (1 mL) was added dropwise to the mixture. The reac-
tion mixture was stirred for 1h at room temperature, then di-
luted with ethyl acetate (50 mL). The mixture was washed
2
-((2E,6E)-3,7-Dimethyl-8-(phenylamino)octa-2,6-dien-
-yl)-3-methylnaphthalene-1,4-dione (7). To a solution of
MK-2 ω-aldehyde 23 (51 mg, 158 µmol) in CH Cl (2 mL)
was added aniline (18 mg, 190 µmol) and acetic acid (11 mg,
90 µmol), and the mixture was stirred in the presence of mo-
1
2
2
1
with brine (50 mL), dried over MgSO
residue was purified with silica gel column chromatography
n-hexane/ ethyl acetate= 30: 1) to afford 10 (23 mg, 78%) as
4
, and concentrated. The
lecular sieves 4A (MS4A) at room temperature for 30 min
under argon. After the mixture was cooled to 0 °C, sodium
triacetoxyborohydride (67 mg, 316 µmol) was added to the
solution. The mixture was stirred at room temperature for 6 h,
then diluted with ethyl acetate (50 mL). The solution was
(
1
a yellow oil. H NMR (400 MHz, CDCl
m), 7.69-7.67 (2H, m), 7.26-7.22 (2H, m), 6.93-6.84 (3H, m),
.49-5.45 (1H, m), 5.05-5.02 (1H, m), 4.31 (2H, s), 3.37 (2H,
d, J = 6.8 Hz), 2.19 (3H, s), 2.19-2.14 (2H, m), 2.06-2.02 (2H,
3
); δ 8.09-8.06 (2H,
5
washed with 5% NaHCO
3
aq. (50 mL) and brine (50 mL),
dried over MgSO , and concentrated. The residue was purified
4
13
m), 1.80 (3H, s), 1.70 (3H, s). C NMR (100 MHz, CDCl
85.5, 184.6, 159.0, 146.1, 143.5, 137.2, 133.45, 133.40,
132.22, 132.19, 131.3, 129.4, 128.4, 126.4, 126.3, 120.7,
3
); δ
by silica gel column chromatography (n-hexane/ ethyl ace-
1
1
tate= 10: 1) to afford 7 (36 mg, 57%) as a brown oil. H NMR
(
3
400 MHz, CDCl ); δ 8.09-8.06 (2H, m), 7.69-7.67 (2H, m),
ACS Paragon Plus Environment

Page 5 of 7
Journal of Medicinal Chemistry
1
19.6, 114.9, 73.9, 39.2, 26.2, 26.1, 16.5, 14.0, 12.8. HRMS
toluidine (15 mg, 138 µmol), acetic acid (8 µL, 138 µmol),
MS4A, and sodium triacetoxyborohydride (49 mg, 230 µmol)
+
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
([M+H] ) m/z calcd for C27
29 3
H O 401.2117; found 401.2114.
in CH
2 2
Cl (2 mL), was purified by silica gel column chroma-
2
-((2E,6E)-3,7-Dimethyl-8-(m-tolyloxy)octa-2,6-dien-1-
tography (n-hexane/ ethyl acetate= 10: 1), giving 14 (38 mg,
yl)-3-methylnaphthalene-1,4-dione (11). Similar to the syn-
thesis of 10 from 21, the crude product 11, which was ob-
tained from the reaction of a mixture of 21 (61 mg, 188 µmol),
m-cresol (61 mg, 564 µmol), and triphenylphosphine (148 mg,
564 µmol) in THF (4 mL), and diisopropyl azodicarboxylate
(114 mg, 564 µmol) in THF (2 mL), was purified by silica gel
column chromatography (n-hexane/ ethyl acetate= 40: 1), giv-
1
68%) as a brown oil. H NMR (400 MHz, CDCl
3
); δ 8.09-8.06
(2H, m), 7.70-7.67 (2H, m), 6.95 (2H, d, J = 7.6 Hz), 6.53-
6
.50 (2H, m), 5.35-5.31 (1H, m), 5.05-4.99 (2H, m), 3.57 (2H,
s), 3.37 (2H, d, J = 6.8 Hz), 2.21 (3H, s), 2.18 (3H, s), 2.08-
1
3
1
.91 (8H, m), 1.80 (3H, s), 1.62 (3H, s), 1.55 (3H, s).
C
3
NMR (100 MHz, CDCl ); δ 185.6, 184.6, 146.4, 146.2, 143.4,
137.6, 135.0, 133.5, 133.4, 132.5, 132.24, 132.20, 129.7,
1
ing 11 (60 mg, 77%) as a yellow oil. H NMR (400 MHz,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
26.4, 126.3, 126.1, 124.2, 119.2, 113.0, 52.3, 39.8, 39.4,
CDCl
1H, m), 6.76-6.65 (3H, m), 5.48-5.44 (1H, m), 5.05-5.02 (1H,
m), 4.29 (2H, s), 3.37 (2H, d, J = 6.8 Hz), 2.30 (3H, s), 2.19
3
); δ 8.08-8.06 (2H, m), 7.69-7.65 (2H, m), 7.15-7.10
6.52, 26.45, 26.1, 20.5, 16.5, 16.1, 14.8, 12.8. HRMS
(
+
([M+H] ) m/z calcd for C33
H40NO
2
482.3059; found 482.3057.
(3H, s), 2.19-2.14 (2H, m), 2.06-2.02 (2H, m), 1.80 (3H, s),
2-((2E,6E,10E)-12-((4-Fluorophenyl)amino)-3,7,11-
1
3
1
1
1
1
.70 (3H, s). C NMR (100 MHz, CDCl
3
); δ 185.5, 184.6,
trimethyldodeca-2,6,10-trien-1-yl)-3-methylnaphthalene-
1,4-dione (15). Similar to the synthesis of 7 from 23, the crude
product 15, which was obtained from 24 (54 mg, 138 µmol),
p-fluoroaniline (16 µL, 166 µmol), acetic acid (10 µL, 166
µmol), MS4A, and sodium triacetoxyborohydride (59 mg, 276
59.0, 146.1, 143.5, 139.4, 137.2, 133.45, 133.40, 132.23,
32.21, 131.4, 129.1, 128.3, 126.4, 126.3, 121.5, 119.6, 115.8,
11.7, 73.9, 39.2, 26.3, 26.1, 21.6, 16.5, 14.0, 12.8. HRMS
+
([M+Na] ) m/z calcd for C28
H30NaO
3
437.2093; found
4
37.2092.
µmol) in CH
2 2
Cl (2 mL), was purified by silica gel column
chromatography (n-hexane/ ethyl acetate= 10: 1), giving 15
2
-((2E,6E)-3,7-Dimethyl-8-(4-nitrophenoxy)octa-2,6-
1
3
(45 mg, 67%) as a brown oil. H NMR (400 MHz, CDCl ); δ
dien-1-yl)-3-methylnaphthalene-1,4-dione (12). Similar to
the synthesis of 10 from 21, the crude product 12, which was
obtained from the mixture of 21 (39 mg, 120 µmol), p-
nitrophenol (50 mg, 360 µmol), and triphenylphosphine (94
mg, 360 µmol) in THF (4 mL), and diisopropyl azodicarbox-
ylate (73 mg, 360 µmol) in THF (1.5 mL), was purified by
8.09-8.06 (2H, m), 7.69-7.66 (2H, m), 6.85 (2H, t, J = 8.4 Hz),
6.54-6.49 (2H, m), 5.33 (1H, t, J = 6.9 Hz), 5.04-4.99 (2H, m),
3
.66 (1H, br s), 3.56 (2H, s), 3.37 (2H, d, J = 6.9 Hz), 2.18
(
(
1
3H, s), 2.08-1.91 (8H, m), 1.79 (3H, s), 1.61 (3H, s), 1.55
1
3
3H, s). C NMR (100 MHz, CDCl
54.5, 146.2, 144.9, 143.4, 137.5, 134.9, 133.5, 133.4, 132.22,
132.19, 132.15, 126.4, 126.3, 124.2, 119.2, 115.7, 115.4,
3
); δ 185.6, 184.6, 156.8,
silica gel column chromatography (n-hexane/ ethyl acetate=
1
1
0: 1), giving 12 (44 mg, 82%) as a yellow oil. H NMR (400
1
1
4
13.7, 113.6, 52.5, 39.8, 39.4, 26.5, 26.4, 26.1, 16.5, 16.1,
MHz, CDCl
.66 (2H, m), 6.92-6.88 (2H, m), 5.51-5.47 (1H, m), 5.05-5.02
1H, m), 4.40 (2H, s), 3.37 (2H, d, J = 6.8 Hz), 2.21-2.16 (2H,
m), 2.19 (3H, s), 2.07-2.04 (2H, m), 1.81 (3H, s), 1.70 (3H, s).
3
); δ 8.20-8.14 (2H, m), 8.08-8.05 (2H, m), 7.71-
+
4.7, 12.8. HRMS ([M+H] ) m/z calcd for C32
86.2756; found 486.2808.
H37FNO
2
7
(
2-Methyl-3-((2E,6E,10E)-3,7,11-trimethyl-12-
phenoxydodeca-2,6,10-trien-1-yl)naphthalene-1,4-dione
1
3
C NMR (100 MHz, CDCl
3
); δ 185.5, 184.6, 164.0, 146.1,
1
1
43.5, 141.4, 136.8, 133.5, 133.4, 132.2, 130.0, 129.6, 126.34,
(16). Similar to the synthesis of 10 from 21, the crude product
16, which was obtained from the mixture of MK-3 ω-alcohol
22 (41 mg, 104 µmol), phenol (29 mg, 312 µmol), and tri-
phenylphosphine (82 mg, 312 µmol) in THF (3 mL), and
diisopropyl azodicarboxylate (63 mg, 312 µmol) in THF (1.5
mL), was purified by silica gel column chromatography (n-
26.31, 125.9, 119.9, 114.8, 74.6, 39.1, 26.1, 16.4, 13.9, 12.8.
+
HRMS ([M+Na] ) m/z calcd for C27
found 468.1787.
5
H27NNaO 468.1787;
2
-Methyl-3-((2E,6E,10E)-3,7,11-trimethyl-12-
(phenylamino)dodeca-2,6,10-trien-1-yl)naphthalene-1,4-
hexane/ ethyl acetate= 40: 1), giving 16 (26 mg, 53%) as a
dione (13). Similar to the synthesis of 7 from 23, the crude
product 13, which was obtained from MK-3 ω-aldehyde 24
1
yellow oil. H NMR (400 MHz, CDCl
3
); δ 8.09-8.06 (2H, m),
7.69-7.67 (2H, m), 7.27-7.23 (2H, m), 6.93-6.89 (3H, m), 5.47
(1H, dd, J = 6.0, 6.8 Hz), 5.07-5.00 (2H, m), 4.35 (2H, s), 3.37
(
53 mg, 136 µmol), aniline (14 µL, 150 µmol), acetic acid (10
µL, 150 µmol), MS4A, and sodium triacetoxyborohydride (50
mg, 236 µmol) in CH Cl (2 mL), was purified by silica gel
(
2H, d, J = 6.8 Hz), 2.18 (3H, s), 2.14-1.95 (8H, m), 1.79 (3H,
2
2
1
3
s), 1.69 (3H, s), 1.57 (3H, s). C NMR (100 MHz, CDCl
3
); δ
column chromatography (n-hexane/ ethyl acetate= 10: 1), giv-
ing 13 (37 mg, 58%) as a brown oil. H NMR (400 MHz,
CDCl
1
185.5, 184.6, 159.1, 146.2, 143.4, 137.5, 134.8, 133.44,
1
1
1
33.38, 132.25, 132.22, 131.0, 129.4, 128.8, 126.4, 126.3,
24.3, 120.7, 119.2, 114.9, 74.0, 39.7, 39.2, 26.5, 26.4, 26.1,
6.5, 16.1, 14.0, 12.8. HRMS ([M+Na] ) m/z calcd for
3
); δ 8.09-8.06 (2H, m), 7.69-7.67 (2H, m), 7.16-7.12
(2H, m), 6.68-6.58 (3H, m), 5.36-5.33 (1H, m), 5.02 (2H, dd, J
+
=
6
7.2, 14.4 Hz), 3.77 (1H, br s), 3.60 (2H, s), 3.37 (2H, d, J =
C
32
H36NaO
3
491.2562; found 491.2562.
.8 Hz), 2.18 (3H, s), 2.09-1.91 (8H, m), 1.79 (3H, s), 1.62
1
3
(3H, s), 1.55 (3H, s). C NMR (100 MHz, CDCl
3
); δ 185.6,
2-Methyl-3-((2E,6E,10E)-3,7,11-trimethyl-12-(m-
1
1
1
1
4
84.6, 148.6, 146.2, 143.4, 137.5, 134.9, 133.5, 133.4, 132.3,
32.23, 132.20, 129.2, 126.4, 126.3, 126.2, 124.2, 119.2,
tolyloxy)dodeca-2,6,10-trien-1-yl)naphthalene-1,4-dione
(17). Similar to the synthesis of 10 from 21, the crude product
17, which was obtained from the mixture of 22 (59 mg, 150
µmol), m-cresol (49 mg, 450 µmol), and triphenylphosphine
(118 mg, 450 µmol) in THF (5 mL), and diisopropyl azodicar-
boxylate (91 mg, 450 µmol) in THF (1 mL), was purified by
17.2, 112.9, 51.9, 39.8, 39.4, 26.51, 26.45, 26.1, 16.5, 16.1,
+
4.8, 12.8. HRMS ([M+H] ) m/z calcd for C32
68.2903; found 468.2906.
H38NO
2
2
-Methyl-3-((2E,6E,10E)-3,7,11-trimethyl-12-(p-
tolylamino)dodeca-2,6,10-trien-1-yl)naphthalene-1,4-dione
14). Similar to the synthesis of 7 from 23, the crude product
4, which was obtained from 24 (45 mg, 115 µmol), p-
silica gel column chromatography (n-hexane/ ethyl acetate=
1
4
0: 1), giving 17 (31 mg, 43%) as a yellow oil. H NMR (400
(
1
3
MHz, CDCl ); δ 8.09-8.06 (2H, m), 7.69-7.67 (2H, m), 7.15-
ACS Paragon Plus Environment

Journal of Medicinal Chemistry
Page 6 of 7
7
.11 (1H, m), 6.75-6.69 (3H, m), 5.46 (1H, t, J = 6.8 Hz),
were cultured for 4 days while changing the medium every 2
days at 37 °C in the presence of 5% CO . The medium was
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
5.07-5.00 (2H, m), 4.33 (2H, s), 3.37 (2H, d, J = 6.8 Hz), 2.31
(3H, s), 2.19 (3H, s), 2.14-1.95 (8H, m), 1.79 (3H, s), 1.69
(3H, s), 1.57 (3H, s). C NMR (100 MHz, CDCl
184.6, 159.1, 146.2, 143.4, 139.4, 137.6, 134.8, 133.44,
1
1
2
2
removed, the cells were washed with PBS(-), PBS(-) contain-
ing 4% paraformaldehyde was added, and the cells were al-
lowed to stand for 20 minutes at room temperature to fix the
cells. The cells were then washed 3 times with PBS(-), and
then the cells were covered with PBS(-) containing 0.2% Tri-
ton X-100 and left at room temperature for 5 minutes. Then,
10% goat serum - containing PBS anti-Map 2 antibody was
added dropwise as a primary antibody and allowed to react
overnight at 4 °C. After washing three times with PBS(-),
CF594 dye was added dropwise as a secondary antibody, the
reaction was allowed to proceed for 1 hour at room tempera-
ture in a container, and washing was carried out three times
with PBS(-) and then the same procedure was carried out us-
ing anti Gfap antibody as the primary antibody and CF488A
dye as the secondary antibody. The cells were washed three
times with PBS(-) and then sealed with an encapsulant con-
taining DAPI. The spectra for DAPI (excitation wavelength
360 nm, fluorescence wavelength 460 nm), CF488A (excita-
tion wavelength 490 nm, fluorescence wavelength 515 nm)
and CF594 (excitation wavelength 593 nm, fluorescence
wavelength 614 nm) were observed.
1
3
3
); δ 185.6,
33.38, 132.3, 132.2, 131.1, 129.1, 128.7, 126.4, 126.3, 124.3,
21.5, 119.2, 115.8, 111.7, 74.0, 39.7, 39.2, 26.5, 26.4, 26.1,
+
1.6, 16.5, 16.1, 14.0, 12.8. HRMS ([M+Na] ) m/z calcd for
C
33
H
38NaO
3
505.2719; found 505.2722.
2-Methyl-3-((2E,6E,10E)-3,7,11-trimethyl-12-(4-
nitrophenoxy)dodeca-2,6,10-trien-1-yl)naphthalene-1,4-
dione (18). Similar to the synthesis of 10 from 21, the crude
product 18, which was obtained from the mixture of 22 (46
mg, 117 µmol), p-nitrophenol (49 mg, 351 µmol), and tri-
phenylphosphine (92 mg, 351 µmol) in THF (4 mL), and
diisopropyl azodicarboxylate (71 mg, 351 µmol) in THF (1.5
mL), was purified by silica gel column chromatography (n-
hexane/ ethyl acetate= 10: 1), giving 18 (38 mg, 63%) as a
yellow oil. H NMR (400 MHz, CDCl
8
5
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
3
); δ 8.18-8.15 (2H, m),
.09-8.06 (2H, m), 7.70-7.68 (2H, m), 6.96-6.93 (2H, m),
.51-5.48 (1H, m), 5.07-5.00 (2H, m), 4.45 (2H, s), 3.37 (2H,
d, J = 6.8 Hz), 2.18 (3H, s), 2.16-1.96 (8H, m), 1.79 (3H, s),
1.69 (3H, s), 1.57 (3H, s). C NMR (100 MHz, CDCl ); δ
3
1
3
185.5, 184.6, 164.1, 146.2, 143.4, 141.4, 137.4, 134.5, 133.44,
133.39, 132.24, 132.21, 130.0, 129.8, 126.4, 126.3, 125.9,
1
1
5
Evaluation of differentiation-inducing activity of vitamin
K analogues. After the cells were seeded and cultured for 24
h, they were treated with 1 μM of vitamin K analogues as well
as control (ethanol) for 4 days. Then the cells were collected
and their mRNA were extracted. The mRNA level of Map2,
Gfap, and β-actin were quantitated with real-time PCR meth-
od. The differentiation activity of vitamin K analogues toward
neuronal cells or astrocyte cells was calculated from the ratios
of mRNA levels of Map2/β-actin or Gfap/β-actin. Further-
more, selective differentiation activity of vitamin K analogues
toward neuronal cells was calculated from the relative ratio of
the mRNA of Map2/Gfap. The data were normalized with
24.5, 119.3, 114.8, 74.8, 39.7, 39.0, 26.5, 26.3, 26.1, 16.5,
+
6.0, 13.8, 12.8. HRMS ([M+H] ) m/z calcd for C32
14.2593; found 514.2591.
H36NO
5
Ethics statement. All animal experimental protocols were
performed in accordance with the Guidelines for Animal Ex-
periments at Shibaura Institute of Technology and were ap-
proved by the Animal Research and Ethics Committee of Shi-
baura Institute of Technology, Saitama, Japan. All surgical
procedures were performed under sodium pentobarbital anes-
thesia, and all efforts were made to minimize suffering.
respect to the EtOH control which was reported as 1.0. Theβ-
actin was used for a house-keeping gene.
Isolation of mouse embryonic cerebral-derived neural
stem cells. C57BL/6J female mice at 14 days of pregnancy
were sacrificed by cervical dislocation, the uterus was taken
out, and fetuses were removed from the uterus. The fetuses
were decapitated, the scalp and parietal bone were stripped in
L-15 medium, and the cerebrum was removed. Under stereo-
scopic microscope observation, the meninges and blood were
completely removed and the cerebrum was washed 5 times
with L-15 medium. To 9 mL of L-15 medium was added 1 mL
of 2.5 g / L trypsin solution and 100 μL of DNase I solution,
and the cerebrum was added and gently stirred at 37 °C for 20
to 25 minutes. DMEM (low glucose) medium containing 10%
FCS was added to the trypsin and DNase I-treated cerebrum,
the cerebrum was loosened by pipetting, and the cells were
dispersed and centrifuged. The supernatant was discarded,
DMEM (low glucose) containing 10% FCS medium was add-
ed to loosen the cells, and then centrifuged. After this opera-
tion was again performed, the cells were suspended in L-
DMEM medium containing 10% FCS. A plate-coated glass
ASSOCIATED CONTENT
Supporting Information
1H NMR and 13C NMR spectra of 7 - 18; HPLC data of 7 -
18 used in biological assays. This material is available free of
charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*
For Y.S.: phone, +81-48-720-6043; fax, +81-48-720-6011;
E-mail, suhara@shibaura-it.ac.jp
Notes
The authors declare no competing financial interest.
6
ACKNOWLEDGEMENTS
bottom culture dish was seeded at 3×10 cells / dish and cul-
tured at 37 °C for 1 day in the presence of 5% CO .
2
Y.S. acknowledges The Naito Foundation. This study was sup-
ported in part by a Grant-in-aid for Scientific Research
KAKENHI (16K08325 to Y.S.) from the Japan Society for Pro-
motion of Science.
Evaluation of cerebral nerve cell differentiation by im-
munostaining. The medium was removed from the isolated
neural stem cells and replaced with 10% FCS containing
DMEM (low glucose) medium supplemented with vitamin K
derivative to 10 M. For control samples, ethanol was used so
that the ethanol content in the medium was 0.1%. The cells
ABBREVIATIONS
NPCs, Neural progenitor cells; Map2, microtubule-
associated protein 2; Gfap, glial fibrillary acidic protein
-6
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Page 7 of 7
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