Synthetic Small Molecules Derived from Natural Vitamin K

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Synthetic Small Molecules Derived from Natural Vitamin K Homologues that Induce Selective Neuronal Differentiation of Neuronal Progenitor Cells Yoshitomo Suhara,*,† Yoshihisa Hirota,‡,#,∇ Norika Hanada,§,# Shun Nishina,∥ Sachiko Eguchi,∥ Rie Sakane,† Kimie Nakagawa,‡,# Akimori Wada,⊥ Kazuhiko Takahashi,∥ Hiroaki Tokiwa,§ and Toshio Okano*,‡

Downloaded by UNIV OF NEW SOUTH WALES on August 31, 2015 | http://pubs.acs.org Publication Date (Web): August 31, 2015 | doi: 10.1021/acs.jmedchem.5b00999

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Department of Bioscience and Engineering, College of Systems Engineering and Science, Shibaura Institute of Technology, 307 Fukasaku, Minuma-ku, Saitama 337-8570, Japan ‡ Department of Hygienic Sciences, Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan § Department of Chemistry, Faculty of Science, Rikkyo University, 3-34-1 Nishi-Ikebukuro, Toshima-ku, Tokyo 171-8501, Japan ∥ Laboratory of Environmental Sciences, Yokohama College of Pharmacy, 601 Matano-cho, Totsuka-ku, Yokohama 245-0066, Japan ⊥ Department of Organic Chemistry for Life Science, Kobe Pharmaceutical University, 4-19-1 Motoyamakita-machi, Higashinada-ku, Kobe 658-8558, Japan S Supporting Information *

ABSTRACT: We synthesized new vitamin K2 analogues with ω-terminal modifications of the side chain and evaluated their selective differentiation of neuronal progenitor cells into neurons in vitro. The result of the assay showed that the menaquinone-3 analogue modified with the m-methylphenyl group had the most potent activity, which was twice as great as the control. This finding indicated that it is possible to obtain much more potent compounds with modification of the structure of vitamin K2.

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at the ω-terminal group and we examine their ability to induce differentiation activity in the transformation of progenitor cells into neural cells. Natural vitamin K includes two molecular forms of homologues, vitamin K1 (1) and vitamin K2 (2−5) (Figure 1).6 On the other hand, vitamin K3 (6), which lacks a side chain, is an artificial vitamin K. We recently found that 5 existed in brain at high concentrations7 and had the selective ability to differentiate NPCs, derived from mouse cerebrum, into neuronal cells. However, the activities of these compounds were too weak to use for lengthy studies. Vitamin K homologues have been reported to play a role in preventing

INTRODUCTION Neural progenitor cells (NPCs) have been identified in several regions of human adult brain, including the subventricular zone and the dentate gyrus of the hippocampus. The NPCs have the ability to differentiate into neurons, astrocytes, and oligodendrocytes; therefore, they can contribute to neurogenesis in adulthood.1 This finding suggests that the NPCs may have possible applications to development of stem-cell-based therapies for neurodegenerative diseases.2 However, selective methods for control of differentiation pathways of NPCs have not yet been identified. Retinoic acid, leukemia inhibitory factor, and insulin-like growth factor-1 are directly involved in differentiation of NPCs into neurons, astrocytes, and oligodendrocytes, however, they have no selectivity or display poor in vitro activity.3−5 Furthermore, few endogenous molecules that can control the fate of stem cells have been discovered so far. Small molecules that can directly lead to differentiation of adult NPCs could provide useful chemical tools to probe signaling pathways that control neuronal specification and could ultimately facilitate therapeutic application of NPCs. In the present report, we describe the synthesis and pharmacological profile of new vitamin K analogues modified © XXXX American Chemical Society

Figure 1. Structure of vitamin K homologues: phylloquinone (1), menaquinones 2−5, and menadione (6). Received: July 1, 2015

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DOI: 10.1021/acs.jmedchem.5b00999 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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oxidative injury to developing oligodendrocytes and neurons.8 We therefore designed new analogues that modified the chemical structure of vitamin K2 homologues, including 3 and 4, by introducing various hydrophobic groups at the ωposition of the side chain with the expectation that these compounds would be potent inducers of differentiation activity. If the analogues exhibited strong differentiation activity in neuronal cells, they may be applicable as therapeutic agents of neurodegenerative diseases such as Alzheimer’s disease. Consequently, the neural cells degenerated by the disease could be regenerated by the differentiation of stem cells or neuronal progenitor cells. This concept may be applicable for a new medical cure from the point of view of regenerative medical techniques in the future. Here, we report the synthesis and structure−activity relationship of novel vitamin K 2 analogues that selectively induce neuronal differentiation of multipotent neural progenitor cells.

embryonic mouse brain. The general method for preparation was as follows: (i) Neural stem cells were dissociated from embryonic day 14 mouse cerebrum and cultured according to the method previously described.12,13 (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. To investigate the activity of the side chain part of menaquinones, each 1 μM of geranylpyrophosphate (18), farnesylpyrophosphate (19), and geranylgeranylpyrophosphate (20) were also added to the cells. We confirmed the differentiation of the NPCs by an immunofluorescence staining method. If the specific antigen microtubule-associated protein 2 (Map2) was expressed on the surface of neuronal cells that had successfully differentiated rom progenitor cells, 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 confocal laser microscopy (Figure 2). Our analogues selectively differentiated NPCs to neuronal cells because the differentiated cells were mostly observed with red fluorescence. The green fluorescence was not observed in the cells throughout the duration of our study. However, we could not quantitate the fluorescence to compare the degree of differentiation among

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RESULTS AND DISCUSSION The requisite vitamin K2 analogues 7a,b−11a,b, containing aromatic groups introduced at the ω-terminal position were obtained by the synthetic method as shown in Scheme 1. Scheme 1. Synthesis of Vitamin K Analogues 7a,b−11a,ba

a

Reagents and conditions: (a) RMgBr, CuI, 65−75%; (b) Na2S2O4, Et2O, quant; (c) 13a,b−17a,b, BF3·Et2O, 55−65%.

Compounds 7a,b−11a,b were prepared by introducing the phenyl group, methylphenyl group, 1-naphthyl group, 2naphthyl group, and biphenyl group, respectively. The intermediates for the side chain part, compounds 12a and 12b, were synthesized according to our previously reported method.9,10 Conversion of the terminal THP group of 12a and 12b into the aromatic groups by Grignard reaction11 gave 13a,b−17a,b in good yields. Then the coupling reaction of 13a,b−17a, b with naphthoquinone ring 6 proceeded in two steps. After 6 was reduced to the hydroquinone derivative with a 10% sodium hydrosulfite (aq) solution in diethyl ether and then the isoprene unit, 13a,b−17a,b was successively coupled with the hydroquinone in the presence of a catalytic amount of BF3·Et2O. The resulting coupled compounds, obtained as hydroquinone derivatives, were immediately converted to quinones under atmospheric conditions. Thus, the individual desired vitamin K analogues 7a,b−11a,b were obtained in 55− 65% yield from the oxidation of the corresponding hydroquinones. We investigated the differentiation-inducing activity of the vitamin K analogues using NPCs which were prepared from

Figure 2. Vitamin K derivatives induce neuronal differentiation of NPCs isolated from an embryonic mouse cerebrum. The cells were treated with the indicated compounds 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 (green) after treatment with MK-4 (5). On the other hand, the synthesized analogues induced only neuronal differentiation. B



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high ratios compared to control (EtOH). This means that these compounds promoted selective differentiation into neuronal cells rather than into astrocyte cells. Specifically, 8b exhibited the highest effects for both selectivity and activity among the compounds. To analyze the structure−activity relationship between the differentiation-inducing activity and the modification at the side chain, we applied a QSAR analysis, part of the MOE suite, to our compounds, excluding 9a, 9b, and 11b. On the basis of the molecular mechanics method, we theoretically determined the optimized geometries of the compounds with the standard MMFF94x force field and obtained 32 kinds of VSA descriptors (SlogP_VSA (hydrophobicity, hydrophilicity), SMR_VSA (molar refraction), and PEOE_VSA (electronic characteristic)). Next, we derived five kinds of descriptors (PEOE_VSA-0, SMR_VSA4, SlogP_VSA4, SlogP_VSA5, and SlogP_VSA9) with AutoQSAR to extract the most appropriate descriptors. Computation of regression analysis between the measured values (Map2/β-actin) and their predictive values by calculation using the descriptors led to a regression equation of R2 = 0.78. As shown in Figure 5, 8b with four isoprene groups in the side


the cells. We therefore undertook the quantitation of the mRNA of Map2 and β-actin of the cells using real-time PCR methodology to measure differentiation activity into neuronal cells (Figure 3).

Figure 3. Differentiation-inducing activity of vitamin K2 analogues from neuronal progenitor cells into neuronal cells as determined by quantitation of mRNA synthesis for Map2 and β-actin using PCR methodology. Cells were treated with the indicated vitamin K2 analogues as well as natural menaquinones 3−5 at 1.0 × 10−6 M. The histogram data are expressed as the means obtained from three independent experiments; the error bars indicate the SD. Significant difference: ***, p < 0.001, between EtOH and compounds (by Dunnett’s t-test); #, p < 0.1 between 4 and 8b (by Student’s t-test).

For purposes of comparison, the sample treated with EtOH served as the baseline, untreated control, while the samples treated with menaquinones 3−5 served as positive controls. Most of the analogues effectively increased differentiationinducing activity compared to control. Menaquinones showed 1.5−1.7 times potency of control. Analogues 7a−9a and 11a showed almost the same activity as parent compound 3, while 10a did not exhibit any activity. On the other hand, analogues of 4 (8b) showed the most potent activity among the menaquinones. Interestingly, the compound 8b, modified with the m-methylphenyl group at the ω-terminal position of 4, exhibited potent activity, being twice as effective as the EtOH control. To evaluate selective differentiation activity of the vitamin K analogues toward neuronal cells, we also quantitated the mRNA of Gfap and β-actin of the cells using real-time PCR methodology to measure differentiation activity into astrocyte cells and calculated the relative ratio of Map2/Gfap as shown in Figure 4. From this result, 3, 4, and 8b showed significantly

Figure 5. Result of QSAR analysis of vitamin K analogues. The vertical axis and the horizontal axis respectively exhibited a measured activity and a calculated activity. The coefficient of correlation value (R2) of the regression equation was 0.78. This analysis showed 8b had the most potent activity corresponding to our experimental result.

chain and two m-methylphenyl moieties at the ω-terminal had the most potent activity. This result corresponded to our experimental results, therefore, it should be possible for us to predict the structure of more potent analogues based on vitamin K using this information. Our results indicated that the cell differentiation activity of the vitamin K derivatives depended on the structure of the side chain part and the lipid solubility of the functional group at the ω-terminal position. The ω-terminal m-methylphenyl group showed the most potent activity, while the naphthalenyl and biphenyl groups, i.e., “bulky” functional groups, did not increase or decrease differentiation activity. The QSAR analysis of the vitamin K derivatives suggested that introduction of bulky and highly lipid soluble substituents to the ω-terminal phenyl group of vitamin K would increase the differentiation activity.

Figure 4. Relative ratios of differentiation-inducing activity of vitamin K2 analogues to convert neuronal progenitor cells into either neuronal cells or astrocyte cells, as determined by quantitation of mRNA synthesis for Map2 and Gfap using PCR methodology. Cells were treated with the indicated vitamin K2 analogues as well as natural menaquinones 3−5 at 1.0 × 10−6 M. The histogram data are expressed as the means obtained from three independent experiments; the error bars indicate the SD. Significant difference: ***, p < 0.001, *, p < 0.01, between EtOH and the compounds (by Dunnett’s t-test).

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CONCLUSION In conclusion, we successfully obtained novel vitamin K derivatives based on natural MK-4 (5) and found that some of them had potent differentiation activity toward neuronal progenitor cells. To date, these derivatives are the only chemical compounds which have an ability to selectively affect C



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Hz), 2.16 (3H, s), 2.14−1.97 (4H, m), 1.77 (3H, s), 1.59 (3H, s). 13C NMR (125 MHz, CDCl3) δ 185.5, 184.6, 146.2, 143.4, 137.5, 136.2, 134.1, 133.9, 133.4, 132.6, 132.3, 132.2, 128.6, 127.0, 126.8, 126.4, 126.3, 126.4, 126.3, 125.5, 43.0, 39.6, 26.7, 26.1, 16.5, 12.8. HRMS (M+) m/z calcd for C31H30O2 434.2246; found 434.2243. 2-Methyl-3-((2E,6E,10E)-3,7,11-trimethyl-12-(naphthalene-1yl)dodeca-2,6,10-trien-1-yl)naphthalene-1,4-dione (9b). Similar to the synthesis of 8a from 6, the crude product 9b, which was obtained from 6 (150 mg, 871 μmol), 15b (366 mg, 1.05 mmol), and boron trifluoride ether complex (50 μL) in AcOEt (1 mL) and dioxane (1 mL), was purified by preparative TLC on silica gel (n-hexane/ AcOEt = 20:1), giving 9b (219 mg, 55%) as a yellow oil. 1H NMR (500 MHz, CDCl3) δ 8.07−8.02 (3H, m), 7.82−7.81 (1H, m), 7.71− 7.26 (7H, m), 5.14 (1H, t, J = 6.9 Hz), 5.00 (2H, t, J = 6.9 Hz), 3.71 (2H, s), 3.36 (2H, d, J = 6.9 Hz), 2.18 (3H, s), 2.08−1.91 (8H, m), 1.78 (3H, s), 1.58 (3H, s), 1.53 (3H, s). 13C NMR (125 MHz, CDCl3) δ 186.1, 185.2, 146.8, 144.0, 138.2, 136.9, 135.6, 134.5, 134.4, 134.0, 133.9, 132.8, 129.2, 127.6, 127.4, 127.3, 127.0, 126.8, 126.2, 126.1, 126.0, 125.0, 124.7, 119.7, 43.6, 40.3, 40.1, 27.3, 27.1, 26.7, 17.1, 16.6, 13.3. HRMS (M+) m/z calcd for C36H38O2 502.2872; found 502.2871. 2-((2E,6E)-3,7-Dimethyl-8-(naphthalen-2-yl)octa-2,6-dien-1yl)-3-methylnaphthalene-1,4-dione (10a). Similar to the synthesis of 8a from 6, the crude product 10a, which was obtained from 6 (150 mg, 871 μmol), 16a (294 mg, 1.05 mmol), and boron trifluoride ether complex (50 μL) in AcOEt (1 mL) and dioxane (1 mL), was purified by preparative TLC on silica gel (n-hexane/AcOEt = 20:1), giving 10a (220 mg, 58%) as a yellow oil. 1H NMR (500 MHz, CDCl3) δ 8.09− 8.05 (2H, m), 7.81−7.67 (5H, m), 7.60 (1H, s), 7.48−7.25 (3H, m), 5.24 (1H, t, J = 6.9 Hz), 5.04 (1H, t, J = 6.9 Hz), 3.40−3.37 (4H, m), 2.23−2.05 (4H, m), 2.18 (3H, s), 1.80 (3H, s), 1.52 (3H, s). 13C NMR (125 MHz, CDCl3) δ 186.1, 185.2, 146.8, 144.0, 138.6, 138.0, 135.1, 134.2, 134.0, 134.0, 132.8, 128.3, 128.1, 127.6, 127.0, 126.9, 126.5, 125.8, 120.0, 47.0, 40.3, 27.2, 26.7, 17.1, 16.5, 13.4. HRMS (M+) m/z calcd for C31H30O2 434.2246; found 434.2249. 2-Methyl-3-((2E,6E,10E)-3,7,11-trimethyl-12-(naphthalen-2yl)dodeca-2,6,10-trien-1-yl)naphthalene-1,4-dione (10b). Similar to the synthesis of 8a from 6, the crude product 10b, which was obtained from 6 (150 mg, 871 μmol), 16b (366 mg, 1.05 mmol), and boron trifluoride ether complex (50 μL) in AcOEt (1 mL) and dioxane (1 mL), was purified by preparative TLC on silica gel (n-hexane/ AcOEt = 20:1), giving 10b (241 mg, 55%) as a yellow oil. 1H NMR (500 MHz, CDCl3) δ 8.07−8.05 (2H, m), 7.78−7.67 (5H, m), 7.60 (1H, s), 7.48−7.25 (3H, m), 5.24 (1H, t, J = 6.9 Hz), 5.06 (1H, t, J = 6.9 Hz), 5.01 (1H, t, J = 6.9 Hz), 3.40 (2H, s), 3.36 (2H, d, J = 6.9 Hz), 2.18 (3H, s), 2.10−1.96 (8H, m), 1.80 (3H, s), 1.58 (3H, s), 1.52 (3H, s). 13C NMR (125 MHz, CDCl3) δ 185.6, 185.2, 146.8, 144.0, 138.7, 138.2, 135.7, 134.8, 134.2, 133.9, 132.8, 128.3, 128.2, 128.1, 127.8, 127.7, 127.3, 127.0, 126.9, 126.5, 125.8, 124.8, 119.8, 47.1, 40.4, 40.2, 27.3, 27.2, 26.7, 17.1, 16.7, 16.5, 13.4. HRMS (M+) m/z calcd for C36H38O2 502.2872; found 502.2871. 2-((2E,6E)-8-([1,1′-Biphenyl]-4-yl)-3,7-dimethylocta-2,6-dien1-yl)-3-methylnaphthalene-1,4-dione (11a). Similar to the synthesis of 8a from 6, the crude product 11a, which was obtained from 6 (150 mg, 871 μmol), 17a (321 mg, 1.05 mmol), and boron trifluoride ether complex (50 μL) in AcOEt (1 mL) and dioxane (1 mL), was purified by preparative TLC on silica gel (n-hexane/AcOEt = 20:1), giving 11a (237 mg, 59%) as a yellow oil. 1H NMR (500 MHz, CDCl3) δ 8.07−8.06 (2H, m), 7.74−7.18 (11H, m), 5.22 (1H, t, J = 6.9 Hz), 5.07 (1H, t, J = 6.9 Hz), 5.01 (1H, t, J = 6.9 Hz), 3.37 (2H, d, J = 6.9 Hz), 3.28 (2H, s), 2.18 (3H, s), 2.11−1.95 (8H, m), 1.80 (3H, s), 1.58 (3H, s), 1.52 (3H, s). 13C NMR (125 MHz, CDCl3) δ 185.6, 184.6, 146.19, 143.4, 137.5, 136.2, 134.1, 133.8, 133.4, 133.4, 132.6, 132.3, 132.2, 128.6, 127.0, 126.9, 126.8, 126.4, 126.3, 125.6, 125.5, 125.4, 124.4, 119.3, 42.9, 39.6, 26.7, 26.1, 16.5, 12.8. HRMS (M+) m/z calcd for C33H32O2 460.2402; found 460.2401. 2-((2E,6E,10E)-12-([1,1′-Biphenyl]-4-yl)-3,7,11-trimethyldodeca-2,6,10-trien-1-yl)-3-methylnaphthalene-1,4-dione (11b). Similar to the synthesis of 8a from 6, the crude product 11b, which was obtained from 6 (150 mg, 871 μmol), 17b (393 mg, 1.05 mmol), and boron trifluoride ether complex (50 μL) in AcOEt (1 mL) and

neuronal differentiation. We clarified that significant differences in differentiation activity could be obtained by altering the functional group of the side chain. A more detailed examination is underway to clarify the functional mechanism of the vitamin K analogues and to synthesize compounds which are much more potent.


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EXPERIMENTAL SECTION

High-resolution ESI-MS (ESI-HRMS) was performed with a Micromass Q-TOF mass spectrometer. 1H NMR spectra were recorded at 500 MHz and 13C NMR spectra were recorded at 125 MHz using CDCl3 as a solvent unless otherwise specified. Chemical shifts are given in parts per million (δ) using tetramethylsilane (TMS) as the internal standard. Column chromatography was carried out on silica gel 60 (70−230 mesh), and preparative thin layer chromatography (TLC) was run on silica gel 60F254. Unless otherwise noted, all reagents were purchased from commercial suppliers. We confirmed that the purities of the compounds 8a,b−11a,b were satisfied more than 95%. 2-((2E,6E)-3,7-Dimethyl-8-(m-tolyl)octa-2,6-dien-1-yl)-3methylnaphthalene-1,4-dione (8a). To a solution of menadion (6) (150 mg, 871 μmol) in ether (20 mL) was added a 10% Na2S2O4 aqueous solution (20 mL), and the mixture was stirred vigorously at 25 °C for 1 h under argon. After the yellow ether layer turned colorless, the mixture was extracted with AcOEt (50 mL × 3). The combined organic layer was washed with brine (50 mL × 3), dried over MgSO4, and concentrated to afford crude hydroquinone. The residue was immediately dissolved in AcOEt (1 mL) and dioxane (1 mL). Then the side chain part 14a (256 mg, 1.05 mmol) and boron trifluoride ether complex (50 μL) were added to the solution. The mixture was stirred at 70 °C for 3 h under argon and cooled to room temperature. The mixture was poured into ice−water and extracted with AcOEt (50 mL × 3). The combined 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 8a (226 mg, 65%) as a yellow oil. 1H NMR (500 MHz, CDCl3) δ 8.082−8.077 (2H, m), 7.698− 7.692 (2H, m), 7.12−7.09 (1H, m), 7.01−6.93 (3H, m), 5.17 (1H, t, J = 6.9 Hz), 5.04 (1H, t, J = 6.9 Hz), 3.38 (2H, d, J = 6.3 Hz), 3.18 (2H, s), 2.30 (3H, s), 2.19 (3H, s), 2.12−2.02 (4H, m), 1.80 (3H, s), 1.48 (3H, s). 13C NMR (125 MHz, CDCl3) δ 185.6, 184.7, 146.2, 143.5, 140.4, 137.8, 137.5, 134.7, 133.4, 129.7, 128.1, 126.7, 126.4, 126.3, 126.0, 119.3, 46.3, 39.7, 26.6, 26.1, 21.5, 16.5, 15.9, 12.8. HRMS (M+) m/z calcd for C28H30O2 398.2246; found 398.2248. 2-Methyl-3-((2E,6E,10E)-3,7,11-trimethyl-12-(m-tolyl)dodeca-2,6,10-trien-1-yl)naphthalene-1,4-dione (8b). Similar to the synthesis of 8a from 6, the crude product 8b, which was obtained from 6 (150 mg, 871 μmol), 14b (328 mg, 1.05 mmol), and boron trifluoride ether complex (50 μL) in AcOEt (1 mL) and dioxane (1 mL), was purified by preparative TLC on silica gel (n-hexane/AcOEt = 20:1), giving 8b (236 mg, 58%) as a yellow oil. 1H NMR (500 MHz, CDCl3) δ 8.08−8.07 (2H, m), 7.69−7.67 (2H, m), 7.17−7.12 (1H, m), 6.99−6.92 (3H, m), 5.18 (1H, t, J = 6.9 Hz), 5.06 (1H, t, J = 6.9 Hz), 5.01 (1H, t, J = 6.9 Hz), 3.38 (2H, d, J = 6.9 Hz), 3.20 (2H, s), 2.31 (3H, s), 2.19 (3H, s), 2.09−1.92 (8H, m), 1.79 (3H, s), 1.57 (3H, s), 1.48 (3H, s). 13C NMR (125 MHz, CDCl3) δ 186.1, 185.2, 146.8, 144.0, 141.0, 138.3, 138.2, 135.7, 135.0, 134.0, 132.8, 130.3, 128.7, 127.3, 127.0, 126.9, 126.5, 124.7, 119.8, 46.8, 40.4, 40.2, 27.3, 27.2, 26.7, 22.1, 17.1, 16.7, 16.4, 13.4. HRMS (M+) m/z calcd for C33H38O2 466.2872; found 466.2876. 2-((2E,6E)-3,7-Dimethyl-8-(naphthalen-1-yl)octa-2,6-dien-1yl)-3-methylnaphthalene-1,4-dione (9a). Similar to the synthesis of 8a from 6, the crude product 9a, which was obtained from 6 (150 mg, 871 μmol), 15a (294 mg, 1.05 mmol), and boron trifluoride ether complex (50 μL) in AcOEt (1 mL) and dioxane (1 mL), was purified by preparative TLC on silica gel (n-hexane/AcOEt = 20:1), giving 9a (227 mg, 60%) as a yellow oil. 1H NMR (500 MHz, CDCl3) δ 8.07− 8.05 (2H, m), 7.72−7.66 (5H, m), 7.46−7.34 (4H, m), 5.24 (1H, t, J = 6.9 Hz), 5.00 (1H, t, J = 6.9 Hz), 3.69 (2H, s), 3.34 (2H, d, J = 6.9 D




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dioxane (1 mL), was purified by preparative TLC on silica gel (nhexane/AcOEt = 20:1), giving 11b (258 mg, 56%) as a yellow oil. 1H NMR (500 MHz, CDCl3) δ8.07−8.06 (2H, m), 7.74−7.18 (11H, m), 5.22 (1H, t, J = 6.9 Hz), 5.07 (1H, t, J = 6.9 Hz), 5.01 (1H, t, J = 6.9 Hz), 3.37 (2H, d, J = 6.9 Hz), 3.28 (2H, s), 2.18 (3H, s), 2.11−1.95 (8H, m), 1.80 (3H, s), 1.58 (3H, s), 1.52 (3H, s). 13C NMR (125 MHz, CDCl3) δ 185.6, 184.6, 146.2, 143.4, 141.2, 139.7, 138.9, 137.6, 135.1, 134.2, 133.4, 132.2, 129.3, 128.8, 127.1, 127.0, 126.6, 126.4, 126.3, 124.2, 119.2, 45.9, 39.8, 39.7, 26.7, 26.6, 26.1, 16.5, 16.1, 15.9, 12.8. HRMS (M+) m/z calcd for C38H40O2 528.3028. Found 528.3022. Preparation of Neuronal Progenitor Cells from Mouse Cerebrum. Embryonic day 14 primary mouse neocortex neurons were prepared as follows. Cerebral cortices were dissected, minced, and dissociated with papain. The dissociated cells were plated onto 0.1% polyethylenimine-coated plates, at a density of 1.0−1.5 × 104 cells/cm2 for immunocytochemistry and 1.0 × 105 cells/cm2 for pulldown assays, and maintained in Neurobasal medium (Gibco-BRL) containing 2% B-27 supplement (Gibco-BRL) and 0.5 mM glutamine at 37 °C in a humidified 10% CO2 atmosphere for the periods indicated. The experimental protocols were approved by The Animal Care and Use Committee of the National Institute of Neuroscience. 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 method.

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ABBREVIATIONS USED NPCs, Neural progenitor cells; Map2, microtubule-associated protein 2; Gfap, glial fibrillary acidic protein; QSAR, quantitative structure−activity relationship

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REFERENCES

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.5b00999. 1 H and 13C NMR spectra of compounds 8a,b−11a,b, cells and cell culture, and incubation of cultured human cell lines with vitamin K analogues (PDF) Molecular formula strings (CSV)

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Brief Article

AUTHOR INFORMATION

Corresponding Authors

*For Y.S.: phone, +81-48-720-6043; fax, +81-48-720-6011; Email, [email protected]. *For T.O.: phone, +81-78-441-7563; fax, +81-78-441-7565; Email, [email protected]. Present Address ∇

For Y.H.: Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, 3500−3 Minamitamagaki-cho, Suzuka, Mie 513-8670, Japan. Author Contributions #

Y.H., NH., and K.N. contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are grateful to Dr. Seiichi Kobayashi, Ryoka Systems Inc. for technical advice about MOE. Y.S. acknowledges Takeda Science Foundation. This study was supported in part by a Grant-in-aid for Scientific Research KAKENHI (23590136 and 25460157 to Y.S. and H.T., respectively) from the Japan Society for Promotion of Science. H.T. acknowledges Rikkyo SFR project, 2014−2016, and MEXT Supported Program for the Strategic Research Foundation at Private Universities, 2013−2018. E


Synthetic Small Molecules Derived from Natural Vitamin K

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