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Structure-activity relationships of talaumidin derivatives: Their neurite-outgrowth promotion in vitro and optic nerve regeneration in vivo
European Journal of Medicinal Chemistry 148 (2018) 86e94
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Research paper
Structure-activity relationships of talaumidin derivatives: Their neurite-outgrowth promotion in vitro and optic nerve regeneration in vivo Kenichi Harada a, *, Katsuyoshi Zaha a, Rina Bando a, Ryo Irimaziri a, Miwa Kubo a, Yoshiki Koriyama b, Yoshiyasu Fukuyama a, ** a b
Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Yamashiro-cho, Tokushima 770-8514, Japan Graduate School and Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, 3500-3 Minamitamagaki, Suzuka, Mie, 513-8670, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 December 2017 Received in revised form 5 February 2018 Accepted 6 February 2018 Available online 9 February 2018
(e)-Talaumidin (1), a 2,5-biaryl-3,4-dimethyltetrahydrofuran lignan, shows potent neurotrophic activities such as neurite-outgrowth promotion and neuroprotection. Previously, we found that (e)-(1S,2R,3S,4R)-stereoisomer 2 exhibited more significant activity than did the natural product talaumidin (1). However, the preparation of optically active (e)-2 requires a complicated synthetic route. To explore new neurotrophic compounds that can be obtained on a large scale, we established a short step synthetic route for talaumidin derivatives and synthesized fourteen analogues based on the structure of (e)-2. First, we synthesized a racemic compound of (e)-2 (2a) and assessed its neurotrophic activity. We found that the neurotrophic property of racemic 2a is similar in activity to that of (e)-2. Using the same synthetic methodology, several talaumidin derivatives were synthesized to optimize the oxy-functionality on aromatic rings. As a result, bis(methylenedioxybenzene) derivative 2b possessed the highest neurotrophic activity. Furthermore, examination of the structure-activity relationships of 2b revealed that the 2,5-diphenyl-tetrahydrofuran structure was an essential structure and that two methyl groups on THF ring could enhance neurotrophic activity. In addition, compounds 2a and 2b were found to induce mouse optic nerve regeneration in vivo. © 2018 Elsevier Masson SAS. All rights reserved.
Keywords: Talaumidin Neurotrophic activity Neurite-outgrowth promotion Optic nerve regeneration
1. Introduction Neurotrophic factors (NTFs) have been recognized to play important roles in the differentiation of nerve stem cells, neuriteoutgrowth, survival and regeneration of neurons [1e4]. The NTFs have been predicted to improve neurodegenerative diseases such as depression, glaucoma, and Alzheimer's diseases. However, their poor bioavailability and pharmacokinetics due to their polypeptidyl structures have made clinical trials ineffective [5]. Therefore, research on small neurotrophic molecules has garnered significant scientific attention. As part of our research in this area, we have continued to search for neurotrophic molecules from plants [6e10]. In the course of our studies on neurotrophic compounds, we
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (K. Harada), [email protected]. ac.jp (Y. Fukuyama). https://doi.org/10.1016/j.ejmech.2018.02.014 0223-5234/© 2018 Elsevier Masson SAS. All rights reserved.
isolated a tetrahydrofuran-type lignan, (e)-(2S,3S,4S,5S)-talaumidin (1) from Aristolochia arcuata Masters (Fig. 1). Remarkably, 1 shows not only significant neurite-outgrowth promotion in primary cultured rat cortical neurons and in NGF-differentiated PC12 cells but also exhibits protective effects against cell death induced by several insults [11,12]. Further work such as in vivo experiments and mechanistic analysis of neurotrophic activities is currently required to develop the therapeutic agents for neurodegenerative diseases. In addition to its neurotrophic activity, talaumidin has an interesting structure consisting of a 2,5-biaryl-3,4dimethyltetrahydrofuran skeleton with four continuous stereogenic centers. Previously, we synthesized both enantiomers and all diastereomers of natural product 1 to investigate stereochemistryactivity relationships [13e16]. As a result, (1S,2R,3S,4R)-stereoisomer (e)-2 bearing all-cis-substituents showed the most significant neurite-outgrowth promoting activity in NGF-differentiated PC12 cells. However, the preparation of optically active
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Fig. 1. Structures of ()-talaumidin (1), stereoisomers ()-2, and synthesized talaumidin derivatives.
talaumidin and isomer 2 suffered from high cost, long synthetic steps, and low total chemical yield. In this study, we established a new synthetic methodology for talaumidin derivatives in order to explore new neurotrophic compounds that can be obtained on a large scale. In addition, we discuss the structure-activity relationships between talaumidin derivatives and neurotrophic activities. Furthermore, we report their regenerative activity towards mouse optic nerves as a neurotrophic activity in vivo to increase the potential as a therapeutic agent for neurodegenerative disease. Because the central nerve system is not able to regenerate after injury, nerve regenerative activity is one of the most important roles as a neurotrophic factor [16]. 2. Results and discussion 2.1. Synthesis and neurite-outgrowth promoting activity of racemic compound 2a In a previous paper [17], it was indicated that there is little difference in neurite-outgrowth promoting activity between ()-talaumidin and its enantiomer, suggesting that racemic compound 2a has the same biological activity as that of the optically active compound. Therefore, this synthetic study began from the synthesis of 2a, aiming to establish a concise synthetic methodology for talaumidin derivatives. Generally, synthesis of a racemic compound is much easier than that of optically compound because asymmetric reactions, chiral starting materials, and chiral reagents
Scheme 1. Synthesis of racemic 2a.
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are not needed. We envisioned a short steps synthesis of 2a, as shown in Scheme 1. First, a successive Grignard reaction and DessMartin oxidation of commercially available 3 gave the ketone 4a in 99% yield over 2 steps. After a-bromination, the obtained bromide 5a was coupled to 6a in the presence of KHMDS in DMF to provide a 1,4-diketone 7a in good yield. Next, the 1,4-diketone was subjected to Paal-Knorr reaction to afford a tetra-substituted furan 8 in 87% yield. Finally, the synthesis of 2a was attained by hydrogenation in dilute solution (5 mmol/L). It should be noted that the usual reaction concentrations (10e100 mmol/L) induce overreactions such as hydrogenolysis of THF ring in any solvent. According to the previously reported experimental procedure [18], we compared the neurotrophic activity of 2a to the optically active (e)-talaumidin (1) and (1S,2R,3S,4R)-stereoisomer (e)-2 at 30 mmol/L17 (Fig. 2). As expected, 2a showed neurite-outgrowth promoting activity in NGF-differentiated PC12 cells at the same level as the optically active (e)-2, which was higher than that of talaumidin. Thus, no different activity was observed between the enantiomers and racemic compound. 2.2. Optimization of oxy-functional group on benzene rings Next, our attention was focused on optimizing oxy-functional groups on aromatic rings to enhance neurotrophic activity. To elucidate the correlation between functional groups and activity, we decided to synthesize symmetrical derivatives with the same functional group on both benzene rings. In accordance with the synthetic methodology of 2a, five tetrahydrofuran compounds 2bed, 2f and 2g were synthesized in 4 steps (Scheme 2). The coupling reaction of brominated 5aed with 4aed provided dimers 7bee. Paal-Knorr's furan synthesis of 7bee afforded the corresponding tetra-substituted furans 8bee. The methoxy 8c was converted to alcohol 8f using BBr3 in DCM, and 8e, with a benzyloxy group, was converted to 8g by hydrogenolysis. Finally, reduction of the obtained furans 8beg gave rise to tetrahydrofurans 2beg, respectively. All-cis-configuration of 2beg was determined by comparison of the chemical shifts and coupling constants with those of stereoisomers reported in our preceding paper [17]. As shown in Fig. 3, synthesized tetrahydrofuran derivatives were evaluated for their ability to induce neurite-outgrowth in NGF-differentiated PC12 cells at 30 mmol/L, together with their synthetic precursors 8ae8d, 8f and 8g. The degree of their effects on neurite-outgrowth promotion in PC12 cells was demonstrated by morphological observations and quantitative analysis of the neurite length extending from the cell bodies. Consequently, tetrahydrofuran compounds had higher activity than did furan compounds. For 40 -substituents, a specific difference was not
Fig. 2. Comparison of neurite length of NGF-differentiated PC12 cells promoted by (e)-1, (e)-2, and 2a at 30 mmol/L. Data are expressed as the mean as ±SE. *, P < 0.05; **, P < 0.01 compared with NGF by Dunnett's t-test.
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nerve [16] was adopted as an in vivo experiment. The optic nerve is part of the central nervous system that cannot regenerate spontaneously. The optic nerve has been used to examine the response of CNS neurons to injury because of its accessibility, anatomy, and functional importance [19]. The number of regenerating axons in injured mouse optic nerves was counted as described by Leon [20]. Notably, three compounds, 1, 2a and 2b, were found to induce the regeneration of the injured optic nerve, as shown in Fig. 5. In addition, compounds 2a and 2b induced more significant regeneration of nerves than did natural product (e)-1 as well as their neurite-outgrowth promotion in vitro at 30 mmol/L. Although the difference between 2a and 2b was hardly observed in this screening, the all-cis-substituted compounds 2a and 2b were found to show more potent activity than the all-trans-substituted (e)-1. Thus, stereochemistry of talaumidin derivatives was demonstrated to be important for not only neurite-outgrowth promotion in vitro but also for regeneration of the optic nerve in vivo. 3. Conclusion
Scheme 2. Synthesis of talaumidin derivatives with all cis configurations.
observed among methoxy-, methyl-, and hydroxy-derivatives 2cef or 8ce8f. Moreover, the 30 -methoxy group was found to show some enhancement of the activity between 2f and 2g, or 8f and 8g. Ultimately, 2b bearing the methylenedioxy group was found to exhibit higher activity than 2a. 2.3. Examination of structure-activity relationships of talaumidin derivatives Subsequently, we decided to investigate the structure-activity relationships for the neurite-outgrowth activity of talaumidin derivatives. To determine the roles of aromatic rings and methyl groups on tetrahydrofuran in 2b, we designed two compounds, mono-phenyl compound 9 and compound 10. The synthesis of 9 was started from dimethylmaleic anhydride 11 (Scheme 3). The Grignard reaction of 11 with 3,4-methylenedioxyphenyl magnesium bromide (12) provided a lactol 13 in good yield. After reduction with Et3SiH/TFA, the obtained lactone 14 was converted to the tri-substituted furan 15. Finally, the mono-phenyl derivative 9 was prepared by Pd-catalyzed hydrogenation. In addition, compound 10 was synthesized in two steps involving Suzuki-Miyaura reaction of 2,5-dibromofuran (16) with boronate 17, and hydrogenation of 18 (Scheme 4). Synthesized compounds 9 and 10 were assessed for their neurite-outgrowth promoting activity in NGF-differentiated PC12 cells and compared with 2b (Fig. 4). As a result, monophenyl compound 9 showed no activity, while compound 10 exhibited lower activity than did 2b. These results suggest that two phenyl groups on tetrahydrofuran are the crucial structural units, and two methyl groups serve to enhance the neurotrophic activity. 2.4. Evaluation of regeneration of mouse optic nerve in vivo Finally, we examined the neurotrophic activity of (e)-1, 2a, and 2b in vivo. In this study, the regenerative activity of injured optic
We established a concise synthetic methodology for talaumidin derivatives that could be used to prepare a variety of analogues in short steps and on a large scale. The synthesized racemic compound 2a showed neurite-outgrowth promoting activity in NGFdifferentiated PC12 cells at the same level as the optically active (e)-2, revealing that the relative configuration bearing all-cissubstituents is important for potent neurotrophic activities although the absolute configurations do not affect their activities. Using this synthetic methodology, fourteen compounds based on (e)-2 were prepared. Among them, compounds 2b with a methylenedioxy group on both benzene rings was found to exhibit the most significant neurite outgrowth promotion in NGFdifferentiated PC12 cells in vitro. In addition, 2a and 2b also induced regeneration of the mouse optic nerve in vivo, and their activity was higher than talaumidn's activity as well as the in vitro experiments. Furthermore, the structure-activity relationships of 2b showed that the two benzene rings were essential structures, and that the methyl groups on the THF ring could enhance the neurotrophic activity. This result suggests that modification of alkyl groups on the THF ring can enhance the neurotrophic activities. Further studies involving optimization of structure, detailed pharmacological experiments and mechanistic analysis are now underway. Our results demonstrate that talaumidin derivatives can be novel therapeutic agents for neurodegenerative diseases such as glaucoma and Alzheimer's diseases. 4. Experimental 4.1. General information All reagents, general solvents, and dry solvents were purchased from Tokyo Chemical Industry, Wako Pure Chemical Industries, and Sigma-Aldrich. IR spectra were recorded on JASCO FT-IR 410 infrared spectrophotometer. High resolution mass spectra were acquired on MStation JMS-700. 1H and 13C NMR spectra were obtained on Varian 200, 300, 400 MHz and JEOL 400 MHz and Bruker 500 MHz instruments in CDCl3 with TMS as internal standard and CD3OD. Silica gel column chromatography was carried out on Merck Silica gel 60 N (70e230 mesh). 4.1.1. (±)-4-{5-(benzo[d][1,3]dioxol-5-yl)-3,4dimethyltetrahydrofuran-2-yl}-2-methoxyphenol (2a) To a solution of 8 (200 mg, 467 mmol) in EtOH (93.4 mL) was added Pd(OH)2/C (40.0 mg). The mixture was stirred vigorously under hydrogen atmosphere at rt for 96 h. After being filtrated, the
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Fig. 3. Comparison of neurite length of NGF-differentiated PC12 cells promoted by furan compounds and tetrahydrofuran compounds at 30 mmol/L. Data are expressed as the mean as ±SE. *P < 0.05, **P < 0.01 compared with NGF by Dunnett's t-test.
Scheme 4. Synthesis of compound 10. Scheme 3. Synthesis of mono-phenyl compound 9.
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2972, 2892, 1503, 1490, 1254, 1237, 1039 cm1. HRMS (EI) m/z: calcd. for C20H20O5 [M]þ 340.1311, found 340.1300. 4.1.3. 2,5-bis(4-methoxyphenyl)-3,4-dimethyltetrahydrofuran (2c) Compound 2c was prepared in the same manner as compound 2a using tetra-substituted furan 8c (345 mg, 1.12 mmol). The product 2c (101 mg, 29%) was obtained as a colorless oil. 1H NMR (300 MHz, CDCl3) d 7.36 (4H, d, J ¼ 8.5 Hz), 6.91 (4H, d, J ¼ 8.5 Hz), 5.13 (2H, d, J ¼ 6.8 Hz), 3.83 (6H, s), 2.68e2.64 (2H, m), 0.58 (6H, d, J ¼ 6.9 Hz). 13C NMR (75 MHz, CDCl3) d 158.4, 132.7, 127.6, 113.4, 113.3, 82.7, 82.5, 55.2, 41.6, 11.9. IR (ATR) 2967, 2835, 1513, 1247 cm1. HRMS (EI) m/z: calcd. for C20H24O3 [M]þ 312.1725, found 312.1715.
Fig. 4. Comparison of neurite length of NGF-differentiated PC12 cells promoted between 2b and tetrahydrofuran compounds at 30 mmol/L. Data are expressed as the mean as ±SE. *P < 0.05, **P < 0.01 compared with NGF by Dunnett's t-test.
solution was concentrated in vacuo. The residue was purified by column chromatography (hexane:DCM ¼ 1:30) to afford 2a (108 mg, 68%) as a colorless oil. 1H NMR, 13C NMR, IR, and MS data of 2a were consistent with those of ()-2. 1H NMR (400 MHz, CDCl3) d 6.96 (1H, d, J ¼ 1.5 Hz), 6.94 (1H, d, J ¼ 1.4 Hz), 6.92 (1H, d, J ¼ 8.0 Hz), 6.88 (1H, dd, J ¼ 8.0, 1.4 Hz), 6.86 (1H, dd, J ¼ 8.1, 1.5 Hz), 6.81 (1H, d, J ¼ 8.1 Hz), 5.97 (2H, s), 5.09 (2H, d, J ¼ 6.4 Hz), 3.91 (3H, s), 2.61e2.68 (2H, m), 0.61 (3H, d, J ¼ 6.1 Hz), 0.59 (3H, d, J ¼ 6.1 Hz). 13 C NMR (100 MHz, CDCl3) d 147.4, 146.2, 144.3, 134.5, 132.4, 119.5, 119.3, 118.0, 114.0, 109.0, 107.9, 107.1, 100.9, 82.8, 82.7, 56.0, 41.5, 41.5, 11.8, 11.8. IR (ATR) 3472, 2969, 1516, 1236, 1038, 455 cm1. HRMS (EI) m/z: calcd for C20H22O5 [M]þ 342.1467, found 342.1476. 4.1.2. 5,5'-(3,4-dimethyltetrahydrofuran-2,5-diyl)bis(benzo[d][1,3] dioxole) (2b) Compound 2b was prepared in the same manner as compound 2a using tetra-substituted furan 8b (67.2 mg, 200 mmol). The product 2b (47.2 mg, 69%) was obtained as a colorless oil. 1H NMR (400 MHz, CDCl3) d 6.93 (2H, d, J ¼ 1.0 Hz), 6.86 (2H, dd, J ¼ 7.9, 1.0 Hz), 6.80 (2H, d, J ¼ 7.9 Hz), 5.96 (4H, s), 5.07 (2H, d, J ¼ 6.7 Hz), 2.70e2.57 (2H, m), 0.59 (6H, d, J ¼ 7.0 Hz). 13C NMR (75 MHz, CDCl3) d 147.4, 146.3, 134.4, 119.5, 107.9, 107.1, 100.9, 82.7, 41.5, 11.8. IR (ATR)
4.1.4. 3,4-Dimethyl-2,5-di-p-tolyltetrahydrofuran (2d) Compound 2d was prepared in the same manner as compound 2a using tetra-substituted furan 8d (20.3 mg, 73.6 mmol). The product 2d (10.2 mg, 49%) was obtained as a colorless oil. 1H NMR (200 MHz, CDCl3) d 7.32 (4H, d, J ¼ 7.9 Hz), 7.17 (4H, d, J ¼ 7.9 Hz), 5.16 (2H, d, J ¼ 6.6 Hz), 2.73e2.62 (2H, m), 2.36 (6H, s), 0.58 (6H, d, J ¼ 6.9 Hz). 13C NMR (75 MHz, CDCl3) d 137.5, 136.2, 128.6, 126.3, 82.8, 41.5, 21.2, 11.9. IR (ATR) 2972, 2919, 1514, 1015, 817 cm1. HRMS (EI) m/z: calcd. for C20H24O [M]þ 280.1827, found 280.1830. 4.1.5. 4,4'-(3,4-dimethyltetrahydrofuran-2,5-diyl)diphenol (2f) Compound 2f was prepared in the same manner as compound 2a using tetra-substituted furan 8f (50.0 mg, 179 mmol). The product 2f (18.8 mg, 37%) was obtained as a colorless oil. 1H NMR (300 MHz, CD3OD) d 7.23 (4H, d, J ¼ 8.5 Hz), 6.78 (4H, d, J ¼ 8.5 Hz), 5.07 (2H, d, J ¼ 6.5 Hz), 2.68e2.00 (2H, m), 0.56 (6H, d, J ¼ 7.0 Hz). 13 C NMR (75 MHz, CD3OD) d 154.7, 129.6, 125.9, 113.0, 81.5, 40.0, 9.5. IR (ATR) 3224, 1515 cm1. HRMS (EI) m/z: calcd. for C18H20O3 [M]þ 284.1412, found 284.1427. 4.1.6. 4,4'-(3,4-dimethyltetrahydrofuran-2,5-diyl)bis(2methoxyphenol) (2g) Compound 2g was prepared in the same manner as compound 2a using tetra-substituted furan 8g (107 mg, 205 mmol). The product 2g (36.4 mg, 52%) was obtained as a colorless oil. 1H NMR (400 MHz, CDCl3) d 6.98e6.88 (6H, m), 5.53 (2H, s), 5.12 (2H, d, J ¼ 6.6 Hz), 3.92 (6H, s), 2.70e2.62 (2H, m), 0.59 (6H, d, J ¼ 6.9 Hz). 13 C NMR (75 MHz, CDCl3) d 147.4, 146.3, 134.4, 119.5, 107.9, 107.1, 100.9, 82.8, 41.5, 11.8. IR (ATR) 3433, 3967, 1515, 1275, 1034 cm1. HRMS (EI) m/z: calcd. for C20H24O5 [M]þ 344.1624, found 344.1617.
Fig. 5. Talaumidin-related compounds induce mouse optic nerve regeneration in vivo at 30 mmol/L. Longitudinal sections of the adult mouse optic nerve showing GAP-43 positive axons extending over the injury site (red asterisks) after 2 weeks of optic nerve injury. (A) Vehicle control, Scale ¼ 200 mm. (B) Talaumidin. (C) 2a. (D) 2b. (E) Quantification of axonal regrowth at an indicated proximal point from the injury site (200 mm). **P < 0.01, *P < 0.05, vs injury (n ¼ 6). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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4.1.7. 1-(4-Benzyloxy-3-methoxyphenyl)propan-1-one (4a) To a solution of 4-benyloxy-3-methoxybenzaldehyde (3, 5.00 g, 20.6 mmol) in THF (206 mL) was added EtMgBr (30.0 mL, 1.0 M solution in THF) at 0 C. After being stirred for 16 h, the reaction was quenched with saturated NH4Cl. The aqueous layer was extracted with ether. The combined organic layers were washed with brine, dried over anhydrous MgSO4, and concentrated in vacuo. To the residue was added Dess-Martin periodinane (11.3 g, 30.1 mmol) in DCM (103 mL). After being stirred for 12 h, the mixture was added to excess ether and filtered through Celite, and the filtrate was concentrated in vacuo. The residue was purified by column chromatography (hexane:EtOAc ¼ 3:1) to afford 4a (5.51 g, 99%) as a colorless oil. 1H NMR (200 MHz, CDCl3) d 7.56e7.33 (7H, m), 6.89 (1H, d, J ¼ 8.4 Hz), 5.23 (2H, s), 3.94 (3H, s), 2.94 (2H, q, J ¼ 7.3 Hz), 1.21 (3H, t, J ¼ 7.3 Hz). 13C NMR (75 MHz, CDCl3) d 199.5, 152.1, 149.4, 136.3, 130.3, 128.6, 128.1, 127.1, 122.3, 112.1, 110.5, 70.7, 56.0, 31.3, 8.6. IR (ATR) 2972, 2935, 1671, 1584, 1510, 1453, 1416, 1340 cm1. HRMS (EI) m/z: calcd. for C17H18O3 [M]þ 270.1256, found 270.1252. 4.1.8. 1-(4-Benzyloxy-3-methoxyphenyl)-2-bromopropan-1-one (5a) To a solution of 4a (3.90 g, 14.4 mmol) in CHCl3 (100 mL) was added Br2 (741 mL, 14.4 mmol) in CHCl3 (45.0 mL) at 0 C. After being stirred overnight, saturated NaHCO3 solution was added and extracted with DCM. The organic layers were dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography (hexane:EtOAc ¼ 88:12) to afford 5a (3.93 g, 78%) as a colorless oil. 1H NMR (300 MHz, CDCl3) d 7.59 (1H, brs), 7.58 (1H, brd, J ¼ 8.2 Hz), 7.44e7.31 (5H, m), 6.90 (1H, d, J ¼ 8.2 Hz), 5.25 (1H, q, J ¼ 6.6 Hz), 5.23 (2H, s), 3.94 (3H, s), 1.87 (3H, d, J ¼ 6.6 Hz). 13C NMR (75 MHz, CDCl3) d 192.1, 152.9, 149.7, 136.1, 128.7, 128.2, 127.2, 123.2, 112.0, 111.5, 70.8, 56.1, 41.2, 20.4. IR (ATR) 2933, 2358, 1671, 1591, 1453, 1376, 1260 cm1. HRMS (EI) m/z: calcd. for C17H17O3Br [M]þ 348.0362, found 348.0356. 4.1.9. 1-(benzo[d][1,3]dioxol-5-yl)-2-bromopropan-1-one (5b) Compound 5b was prepared in the same manner as compound 5a using ketone 4b (9.00 g, 50.0 mmol). The product 5b (11.3 g, 87%) was obtained as a pale yellow oil. 1H NMR (300 MHz, CDCl3) d 7.64 (1H, dd, J ¼ 8.1, 1.8 Hz), 7.50 (1H, d, J ¼ 1.8 Hz), 6.88 (1H, d, J ¼ 8.1 Hz), 6.07 (2H, s), 5.22 (1H, q, J ¼ 6.6 Hz), 1.88 (3H, d, J ¼ 6.6 Hz). 13C NMR (75 MHz, CDCl3) d 191.6, 152.3, 148.4, 128.6, 125.3, 108.7, 108.1, 102.1, 41.3, 20.3. IR (ATR) 2953, 1683, 1443, 1245, 1039 cm1. HRMS (EI) m/z: calcd. for C10H9O3Br [M]þ 257.9867, found 257.9899. 4.1.10. 2-Bromo-1-(4-methoxyphenyl)propan-1-one (5c) Compound 5c was prepared in the same manner as compound 5a using ketone 4c (2.00 g, 12.2 mmol). The product 5c (2.45 g, 83%) was obtained as a pale yellow oil. 1H NMR (300 MHz, CDCl3) d 8.01 (2H, d, J ¼ 9.3 Hz), 6.95 (2H, d, J ¼ 9.3 Hz), 5.26 (1H, q, J ¼ 6.6 Hz), 3.88 (3H, s), 1.89 (3H, d, J ¼ 6.6 Hz). 13C NMR (75 MHz, CDCl3) d 191.9, 163.9, 131.3, 126.8, 113.9, 55.5, 41.4, 20.2. IR (ATR) 2933, 1674, 1597, 1238, 1157, 840 cm1. HRMS (EI) m/z: calcd. for C10H11O2Br [M]þ 241.9942, found 241.9950. 4.1.11. 2-Bromo-1-(p-tolyl)propan-1-one (5d) Compound 5d was prepared in the same manner as compound 5a using ketone 4d (2.01 mL, 13.5 mmol). The product 5d (2.05 g, 67%) was obtained as a pale yellow oil. 1H NMR (300 MHz, CDCl3) d 7.92 (2H, d, J ¼ 8.4 Hz), 7.28 (2H, d, J ¼ 8.4 Hz), 5.28 (1H, q, J ¼ 6.5 Hz), 2.43 (3H, s), 1.89 (3H, d, J ¼ 6.5 Hz). 13C NMR (75 MHz, CDCl3) d 193.1, 144.8, 131.5, 129.5, 129.1, 41.6, 21.8, 20.2. IR (ATR) 2973, 1667, 1440, 1245, 1037 cm1. HRMS (EI) m/z: calcd. for C10H11OBr [M]þ 225.9993, found 226.0007.
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4.1.12. 1-(benzo[d][1,3]dioxol-5-yl)-4-(4-benzyloxy-3methoxyphenyl)-2,3-dimethylbutane-1,4-dione (7a) To a solution of 6a (1.00 g, 5.58 mmol) in DMF (35.0 mL) was added KHMDS (13.4 mL, 0.5 M solution in toluene) at 45 C. After being stirred for 1 h, a solution of 5a (1.95 g, 5.58 mmol) in DMF (35.0 mL) was added to the reaction mixture at the same temperature. After being stirred overnight, the reaction was quenched with saturated NH4Cl. The aqueous layer was extracted with ether. The combined organic layers were washed with brine, dried over anhydrous MgSO4, and concentrated in vacuo. The residue was purified by column chromatography (hexane:ether ¼ 1:1) to afford 7a (2.15 g, 86%) as a pale yellow oil. 1H NMR (400 MHz, CDCl3) d 7.63 (1H, dd, J ¼ 8.4, 1.5 Hz), 7.61 (1H, dd, J ¼ 8.4, 1.8 Hz), 7.50 (1H, d, J ¼ 1.8 Hz), 7.44e7.32 (6H, m), 6.90 (1H, d, J ¼ 8.4 Hz), 6.86 (1H, d, J ¼ 8.4 Hz), 6.02 (2H, s), 5.23 (2H, s), 3.90 (3H, s), 3.90e3.85 (2H, m), 1.27 (3H, d, J ¼ 6.6 Hz), 1.26 (3H, d, J ¼ 6.6 Hz). 13C NMR (100 MHz, CDCl3) d 202.9, 202.5, 152.4, 151.7, 149.5, 148.2, 136.4, 131.0, 129.4, 128.7, 128.1, 127.2, 124.7, 122.9, 112.3, 111.1, 108.4, 108.0, 101.8, 70.8, 56.0, 43.5, 43.4, 16.0, 15.8. IR (ATR) 3018, 2974, 1665, 1594, 1505, 1454, 1246 cm1. HRMS (EI) m/z: calcd. for C27H26O6 [M]þ 446.1730, found 446.1717. 4.1.13. 1,4-bis(benzo[d][1,3]dioxol-5-yl)-2,3-dimethylbutane-1,4dione (7b) Compound 7b was prepared in the same manner as compound 7a using a-bromoketone 4b (100 mg, 389 mmol). The product 7b (66.6 mg, 48%) was obtained as a pale yellow oil. 1H NMR (300 MHz, CDCl3) d 7.63 (2H, dd, J ¼ 8.3, 1.8 Hz), 7.42 (2H, d, J ¼ 1.8 Hz), 6.86 (2H, d, J ¼ 8.3 Hz), 6.03 (4H, s), 3.84e3.78 (2H, m), 1.27 (6H, d, J ¼ 7.0 Hz). 13C NMR (75 MHz, CDCl3) d 202.4, 151.7, 148.2, 130.8, 124.7, 108.4, 108.0, 101.8, 43.5, 15.9. IR (ATR) 2956, 1667, 1440, 1244, 1038 cm1. HRMS (EI) m/z: calcd. for C20H18O6 [M]þ 354.1103, found 354.1099. 4.1.14. 1,4-bis(4-methoxyphenyl)-2,3-dimethylbutane-1,4-dione (7c) Compound 7c was prepared in the same manner as compound 7a using a-bromoketone 4c (2.05 g, 8.47 mmol). The product 7c (1.66 g, 60%) was obtained as a pale yellow oil. 1H NMR (200 MHz, CDCl3) d 7.97 (4H, d, J ¼ 8.7 Hz), 6.93 (4H, d, J ¼ 8.7 Hz), 3.95e3.90 (2H, m), 3.86 (6H, s), 1.28 (6H, d, J ¼ 6.7 Hz). 13C NMR (75 MHz, CDCl3) d 202.9, 163.4, 130.8, 129.1, 113.7, 55.5, 43.2, 15.8. IR (ATR) 2971, 1667, 1597, 1168, 968 cm1. HRMS (EI) m/z: calcd. for C20H22O4 [M]þ 326.1518, found 326.1508. 4.1.15. 2,3-Dimethyl-1,4-di-p-tolylbutane-1,4-dione (7d) Compound 7d was prepared in the same manner as compound 7a using a-bromoketone 4d (400 mg, 1.78 mmol). The product 7d (472 mg, 90%) was obtained as a pale yellow oil. 1H NMR (200 MHz, CDCl3) d 7.89 (4H, d, J ¼ 8.2 Hz), 7.26 (4H, d, J ¼ 8.2 Hz), 3.98e3.90 (2H, m), 2.41 (6H, s), 1.27 (6H, d, J ¼ 6.7 Hz). 13C NMR (75 MHz, CDCl3) d 203.9, 143.6, 133.6, 129.2, 128.6, 43.4, 21.6, 15.5. IR (ATR) 2963, 1666, 1541, 966 cm1. HRMS (EI) m/z: calcd. for C20H22O2 [M]þ 294.1620, found 294.1619. 4.1.16. 1,4-bis(4-benzyloxy-3-methoxyphenyl)-2,3-dimethylbutane1,4-dione (7e) Compound 7e was prepared in the same manner as compound 7a using a-bromoketone 4e (150 mg, 431 mmol). The product 7e (137 mg, 59%) was obtained as a pale yellow oil. 1H NMR (300 MHz, CDCl3) d 7.61 (2H, dd, J ¼ 8.7, 2.0 Hz), 7.49 (2H, d, J ¼ 2.0 Hz), 7.44e7.31 (10H, m), 6.90 (2H, d, J ¼ 8.7 Hz), 5.23 (4H, m), 3.97e3.87 (2H, m), 3.89 (6H, s), 1.27 (6H, d, J ¼ 6.5 Hz). 13C NMR (75 MHz, CDCl3) d 203.0, 152.3, 149.5, 136.4, 129.4, 128.7, 128.1, 127.2, 122.9, 112.2, 111.1, 70.8, 56.0, 43.2, 16.0. IR (ATR) 2972, 1666, 1593, 1511,
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1260 cm1. HRMS (EI) m/z: calcd. for C34H34O6 [M]þ 538.2355, found 538.2340. 4.1.17. 5-{5-(4-benzyloxy-3-methoxyphenyl)-3,4-dimethylfuran-2yl}benzo[d][1,3]dioxole (8) A solution of 7a (66.6 mg, 188 mmol) in DCM/benzene (500 mL/ 3.76 mL) containing p-TsOH$H2O (10.7 mg, 56.4 mmol) was refluxed under removal of water by a Dean-Stark apparatus for 4 h. The cooled solution was diluted with EtOAc, washed with saturated NaHCO3 solution, dried over anhydrous MgSO4, and concentrated in vacuo. The residue was purified by column chromatography (hexane:EtOAc ¼ 1:1) to afford 8 (55.2 mg, 87%) as a yellow oil. 1H NMR (400 MHz, CDCl3) d 7.45 (2H, d, J ¼ 7.0 Hz), 7.37 (2H, t, J ¼ 7.0 Hz), 7.31 (1H, t, J ¼ 7.0 Hz), 7.23 (1H, d, J ¼ 1.8 Hz), 7.17 (1H, d, J ¼ 1.5 Hz), 7.14 (1H, dd, J ¼ 8.1, 1.5 Hz), 7.13 (1H, dd, J ¼ 8.4, 1.8 Hz), 6.92 (1H, d, J ¼ 8.4 Hz), 6.87 (1H, d, J ¼ 8.1 Hz), 5.98 (2H, s), 5.19 (2H, s), 3.95 (3H, s), 2.19 (3H, s), 2.18 (3H, s). 13C NMR (100 MHz, CDCl3) d 149.7, 147.8, 147.2, 146.8, 146.8, 146.3, 137.1, 128.5, 127.8, 127.3, 126.2, 125.6, 119.4, 118.3, 117.9, 117.9, 114.1, 109.6, 108.5, 106.3, 101.2, 71.1, 56.1, 9.9, 9.9. IR (ATR) 2897, 1503, 1486, 1452, 1243 cm1. HRMS (CI) m/z: calcd. for C27H25O5 [MþH]þ 429.1702, found 429.1695. 4.1.18. 5,5'-(3,4-dimethylfuran-2,5-diyl)bis(benzo[d][1,3]dioxole) (8b) Compound 8b was prepared in the same manner as compound 8a using diketone 7b (66.6 mg, 188 mmol). The product 8b (55.2 mg, 87%) was obtained as a yellow oil. 1H NMR (300 MHz, CDCl3) d 7.16e7.13 (4H, m), 6.85 (2H, d, J ¼ 8.4 Hz), 5.99 (4H, s), 2.19 (6H, s). 13 C NMR (75 MHz, CDCl3) d 147.8, 146.7, 146.3, 126.2, 119.4, 117.9, 108.5, 106.2, 101.1, 9.9. IR (ATR) 1503, 1231, 904, 728 cm1. HRMS (CI) m/z: calcd. for C20H17O5 [MþH]þ 337.1076, found 337.1068. 4.1.19. 2,5-bis(4-methoxyphenyl)-3,4-dimethylfuran (8c) Compound 8c was prepared in the same manner as compound 8a using diketone 7c (1.65 g, 5.06 mmol). The product 8c (1.49 g, 96%) was obtained as a yellow oil. 1H NMR (200 MHz, CDCl3) d 7.61 (4H, d, J ¼ 8.8 Hz), 6.96 (4H, d, J ¼ 8.8 Hz), 3.85 (6H, s), 2.20 (6H, s). 13 C NMR (75 MHz, CDCl3) d 158.3, 146.8, 126.9, 125.0, 117.4, 114.0, 55.3, 9.9. IR (ATR) 2930, 1507, 1247, 1177, 836 cm1. HRMS (EI) m/z: calcd. for C20H20O3 [M]þ 308.1412, found 308.1435. 4.1.20. 3,4-Dimethyl-2,5-di-p-tolylfuran (8d) Compound 8d was prepared in the same manner as compound 8a using diketone 7d (460 mg, 1.56 mmol). The product 8d (374 mg, 87%) was obtained as a yellow oil. 1H NMR (200 MHz, CDCl3) d 7.58 (4H, d, J ¼ 8.2 Hz), 7.23 (4H, d, J ¼ 8.2 Hz), 2.38 (6H, s), 2.22 (6H, s). 13 C NMR (75 MHz, CDCl3) d 147.2, 136.3, 129.2, 145.5, 125.5, 118.4, 21.3, 9.9. IR (ATR) 2918, 1510, 1108, 822 cm1. HRMS (EI) m/z: calcd. for C20H20O [M]þ 276.1514, found 276.1509. 4.1.21. 2,5-bis(4-benzyloxy-3-methoxyphenyl)-3,4-dimethylfuran (8e) Compound 8e was prepared in the same manner as compound 8a using diketone 7e (31.7 mg, 58.9 mmol). The product 8e (15.4 mg, 50%) was obtained as a yellow oil. 1H NMR (300 MHz, CDCl3) d 7.46e7.30 (10H, m), 7.22 (2H, d, J ¼ 1.8 Hz), 7.13 (2H, dd, J ¼ 8.1, 1.8 Hz), 6.92 (2H, d, J ¼ 8.1 Hz), 5.19 (4H, s), 3.95 (6H, s), 2.19 (6H, s). 13 C NMR (50 MHz, CDCl3) d 149.7, 147.1, 146.9, 137.1, 128.6, 127.9, 127.3, 125.6, 118.3, 117.9, 114.1, 109.7, 71.1, 56.1, 9.9. IR (ATR) 1509, 1249, 1024 cm1. HRMS (EI) m/z: calcd. for C34H32O5 [M]þ 520.2250, found 520.2257. 4.1.22. 4,4'-(3,4-dimethylfuran-2,5-diyl)diphenol (8f) A solution of 8c (560 mg, 1.82 mmol) in DCM was added BBr3 (4.54 mL, 1.0 M solution in DCM) at 0 C. After being stirred for 13 h,
the reaction was quenched with saturated NaHCO3 solution was added and extracted with DCM. The organic layers were dried over MgSO4 and concentrated in vacuo. The residue was purified by column chromatography (hexane:EtOAc ¼ 2:1) to afford 8f (477 mg, 94%) as a yellow oil. 1H NMR (300 MHz, CD3OD) d 7.56 (4H, d, J ¼ 8.8 Hz), 6.89 (4H, d, J ¼ 8.8 Hz), 2.19 (6H, s). 13C NMR (75 MHz, CD3OD) d 185.2, 176.0, 155.9, 153.3, 145.9, 144.4, 37.9. IR (ATR) 3397, 1504, 830 cm1. HRMS (CI) m/z: calcd. for C18H16O3 [M]þ 280.1099, found 280.1100. 4.1.23. 4,4'-(3,4-dimethylfuran-2,5-diyl)bis(2-methoxyphenol) (8g) To a solution of 8e (5.4 mg, 10.4 mmol) in EtOH (1.90 mL) was added Pd(OH)2/C (1.8 mg). The mixture was stirred vigorously under hydrogen atmosphere at rt for 14 h. After being filtrated, the solution was concentrated in vacuo. The residue was purified by column chromatography (toluene:EtOAc ¼ 5:1) to afford 8g (1.1 mg, 33%) as a yellow oil. 1H NMR (300 MHz, CDCl3) d 7.18 (2H, d, J ¼ 2.0 Hz), 7.16 (2H, dd, J ¼ 8.7, 2.0 Hz), 6.97 (2H, d, J ¼ 8.7 Hz), 5.64 (2H, s), 3.96 (6H, s), 2.20 (6H, s). 13C NMR (50 MHz, CDCl3) d 147.0, 146.6, 144.7, 125.4, 124.6, 119.3, 114.5, 108.5, 56.0, 9.9. IR (ATR) 3428, 2935, 1510, 1257, 1118 cm1. HRMS (EI) m/z: calcd. for C20H20O5 [M]þ 340.1311, found 340.1340. 4.1.24. 5-(3,4-Dimethyltetrahydrofuran-2-yl)benzo[d][1,3]dioxole (9) To a solution of 15 (32.0 mg, 148 mmol) in EtOH (27.8 mL) was added Pd(OH)2/C (1.5 mg). The mixture was stirred vigorously under hydrogen atmosphere at rt for 17 h. After being filtrated, the solution was concentrated in vacuo. The residue was purified by column chromatography (hexane:EtOAc ¼ 11:1) to afford 9 (26.5 mg, 87%) as a colorless oil. 1H NMR (400 MHz, CDCl3) d 6.80e6.71 (3H, m), 5.93 (2H, s), 5.02 (1H, d, J ¼ 4.8 Hz), 4.06 (1H, t, J ¼ 8.0 Hz), 3.59 (1H, dd, J ¼ 10.0, 8.0 Hz), 2.68e2.58 (1H, m), 2.28e2.22 (1H, m), 1.00 (3H, d, J ¼ 7.0 Hz), 0.50 (3H, d, J ¼ 7.3 Hz). 13 C NMR (100 MHz, CDCl3) d 147.3, 146.1, 134.7, 119.0, 107.8, 106.8, 100.8, 84.6, 72.8, 41.8, 37.8, 12.5, 8.8. IR (ATR) 2965, 1504, 1490, 1443, 1039 cm1. HRMS (EI) m/z: calcd. for C13H16O3 [M]þ 220.1099, found 220.1090. 4.1.25. 2,5-bis(benzo[d][1,3]dioxol-5-yl)tetrahydrofuran (10) To a solution of 18 (9.4 mg, 30.5 mmol) in benzene (1.00 mL) was added Pd(OH)2/C (3.6 mg). The mixture was stirred vigorously under hydrogen atmosphere at rt for 11 h. After being filtrated, the solution was concentrated in vacuo. The residue was purified by preparative TLC (hexane:ether ¼ 5:1) to afford 2a (3.1 mg, 33%) as a colorless oil. 1H NMR (300 MHz, CDCl3) d 6.94 (2H, d, J ¼ 1.2 Hz), 6.86 (2H, dd, J ¼ 7.8, 1.2 Hz), 6.78 (2H, d, J ¼ 7.8 Hz), 5.96 (4H, s), 4.94e4.91 (2H, m), 2.37e2.33 (2H, m), 1.94e1.92 (2H, m). 13C NMR (75 MHz, CDCl3) d 147.7, 146.8, 136.8, 119.4, 108.0, 106.6, 100.9, 81.1, 34.4. IR (ATR) 2924, 1489, 1444, 1249, 1038 cm1. HRMS (EI) m/z: calcd. for C18H16O5 [M]þ 312.0998, found 312.0991. 4.1.26. 5-(benzo[d][1,3]dioxol-5-yl)-5-hydroxy-3,4-dimethylfuran2(5H)-one (13) To a solution of 2,3-dimethylmaleic anhydride (11, 3.03 g, 23.8 mmol) in THF (240 mL) was added 3,4-methylenedioxy phenyl magnesium bromide (48.1 mL, 0.5 M solution in THF) at 0 C. After being stirred for 3 h, the reaction was quenched with saturated NH4Cl. The aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous MgSO4, and concentrated in vacuo. The residue was purified by recrystallization (hexane/EtOAc) to afford 13 (5.90 g, quant.) as a colorless amorphous. 1H NMR (300 MHz, CDCl3) d 6.97 (1H, dd, J ¼ 8.1, 1.8 Hz), 6.89 (1H, d, J ¼ 1.8 Hz), 6.79 (1H, d, J ¼ 8.1 Hz), 5.98 (2H, s), 1.86 (6H, s). 13C NMR (75 MHz, CDCl3) d 173.4, 159.8, 148.3,
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147.9, 130.7, 123.7, 119.6, 108.2, 106.3, 106.0, 101.4, 10.7, 8.5. IR (ATR) 3334, 1739, 1504, 1491, 1246, 1038 cm1. HRMS (EI) m/z: calcd. for C13H12O5 [M]þ 248.0685, found 248.0685. 4.1.27. 5-(benzo[d][1,3]dioxol-5-yl)-3,4-dimethylfuran-2(5H)-one (14) To a solution of 13 (3.79 g, 15.3 mmol) in TFA (153 mL) was added Et3SiH (2.95 mL, 18.4 mmol) at rt. After being stirred for 1.5 h, the solution was concentrated in vacuo. The residue was purified by column chromatography (toluene:EtOAc ¼ 9:1) to afford 14 (3.55 g, qunat.) as a colorless oil. 1H NMR (300 MHz, CDCl3) d 6.81 (1H, d, J ¼ 8.0 Hz), 6.73 (1H, dd, J ¼ 8.0, 1.8 Hz), 6.59 (1H, d, J ¼ 1.8 Hz), 5.98 (2H, s), 5.51 (1H, brs), 1.90 (3H, brs), 1.81 (3H, brs). 13C NMR (75 MHz, CDCl3) d 174.6, 159.0, 148.4, 148.2, 128.5, 123.2, 121.3, 108.4, 106.6, 101.4, 84.9, 12.2, 8.6. IR (ATR) 2904, 1747, 1489, 1444, 1089, 1036 cm1. HRMS (EI) m/z: calcd. for C13H12O4 [M]þ 232.0736, found 232.0740. 4.1.28. 5-(3,4-Dimethylfuran-2-yl)benzo[d][1,3]dioxole (15) To a solution of 14 (1.05 g, 4.53 mmol) in DCM (4.74 mL) was added Dibal-H (43.1 mL, 1.0 M solution in DCM) at 78 C. After being stirred for 19 h, the reaction was concentrated in vacuo. the reaction was quenched with saturated 2 mol/L HCl was added and extracted with DCM. The organic layers were dried over MgSO4 and concentrated in vacuo. The residue was separated by column chromatography (hexane:DCM ¼ 9:1) to afford a crude compound 15 (520 mg, 56%) as a colorless oil. Because compound 15 was instable, the spectral data were not obtained.
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Statistical analyses were performed using Dunnett's t-test. 4.3. Evaluation of regeneration of mouse optic nerve 4.3.1. Animals All animals were maintained and handled in accordance with the Guiding Principles in the Care and Use of Animals and the Suzuka University of Medical Science's guidelines for animal experiments. Male C57BL/6 mice (9 weeks old; Japan SLC, Inc., Shizuoka, Japan) were reared in clear plastic cages and kept under a 12 h lightedark cycle at 23 C. Mice were anesthetized by intraperitoneal injection of sodium pentobarbital (30e40 mg/kg body weight). The optic nerve was crushed using forceps 1 mm posterior to the eyeball. The study was designed to minimize the number of experimental animals used and to minimize their pain and suffering. 4.3.2. Quantitation of optic nerve regeneration in vivo Optic nerves samples embedded in OCT compound were cut into longitudinal sections of 14 mm thickness. Regenerating optic axons were visualized by staining with mouse anti-GAP43 antibody (1∶250, CST Japan) followed by a fluorescently labeled secondary antibody and captured by fluorescent microscopy (BZ-9000, Keyence, Osaka, Japan). Axons were counted manually in at least eight sections per conditions (six mice in each treatment group) at 200 mm away from the injury site. The numbers of regenerating axons were counted as described by Leon et al. [20]. Acknowledgments
4.1.29. 2,5-bis(benzo[d][1,3]dioxol-5-yl)furan (18) To a solution of 17 (600 mg, 4.53 mmol) and H3PO4 (996 mg, 3.69 mmol) in 1,4-dioxane (4.74 mL) was added 16 (269 mg, 1.23 mmol) and PdCl2(dppf) (102 mg, 120 mmol) at rt. After being stirred for 11 h at 100 C, the reaction mixture was filtrated through Celite and concentrated in vacuo. The residue was purified by column chromatography (hexane:EtOAc ¼ 9:1) to afford 18 (72.9 mg, 19%) as a colorless oil. 1H NMR (300 MHz, CDCl3) d 7.24 (2H, dd, J ¼ 8.1, 1.8 Hz), 7.19 (2H, d, J ¼ 1.8 Hz), 6.85 (2H, d, J ¼ 8.1 Hz), 6.56 (2H, s), 6.00 (4H, s). 13C NMR (75 MHz, CDCl3) d 152.6, 148.0, 146.9, 125.3, 117.5, 108.7, 106.1, 104.4, 101.2. IR (ATR) 1479, 1251, 1221, 1034 cm1. HRMS (EI) m/z: calcd. for C18H12O5 [M]þ 308.0685, found 308.0691. 4.2. Evaluation of neurite-outgrowth promotion 4.2.1. PC12 cells PC (Pheochromocytoma) 12 cell line was purchased from JCRB Cell Bank. 4.2.2. Quantitation of neurite-outgrowth promotion PC12 cells were cultured in a 24-well plate at a density of 8 103 cells/cm2 in DMEM þ10% HS, 5% FBS, 100 IU/mL penicillin, and 100 mg/mL streptomycin at 37 C under a humidified atmosphere of 95% air and 5% CO2 for 24 h. The culture medium was then changed to DMEM þ2% HS, 1% FBS, 100 IU/mL penicillin, and 100 mg/mL streptomycin. At the same time, 30 mM concentration of test samples with 2 ng/mL NGF were added. The experiment was repeated in three wells. After incubation with samples for 4 days, the cultures were fixed with 4% paraformaldehyde/PBS and stained with methylene blue. Cell morphology was observed under a phase-contrast microscope, and neurite length was quantified. Five images were selected randomly under a microscope for each well, and five significantly differentiated cells were selected to measure the longest neurite length extending from a cell body for each picture. At least 100 cells were calculated for each test sample.
We thank Dr. Yasuko Okamoto for measuring MS spectra. This work was supported by a Grant from Tokushima Bunri University for Education Reform and Collaborative Research (No. TBU2015-2-1 and TBU2016-2-1). Appendix A. Supplementary data Supplementary data related to this article can be found at https://doi.org/10.1016/j.ejmech.2018.02.014. References [1] B.L. Irwin, K.K. Leonard, The Neuron: Cell Molecular Biology, third ed, Oxford University Press, New York, NY, 2002, pp. 410e412. [2] H.L. Weiner, D.J. Selkoe, Inflammation and therapeutic vaccination in CNS diseases, Nature 420 (2002) 879e884. [3] T. Finkel, N.J. Holbrook, Oxidants, oxidative stress and the biology or agents, Nature 408 (2000) 239e247. [4] R.H. Woodruff, R.J. Franklin, Growth factors and demyelination in the CNS, Histol. Histopathol. 12 (1997) 459e466. €ckman, G.M. Rose, B.J. Hoffer, M.A. Henry, R.T. Bartus, P. Friden, [5] C. Ba A.C. Granholm, Systematic administration of a nerve growth factor conjugate reverses age-related cognitive dysfunction and prevents cholinergic neuron atrophy, J. Neurosci. 16 (1996) 5437e5442. [6] J.M. Huang, R. Yokoyama, C.S. Yang, Y. Fukuyama, Merrilactone A, a novel neurotrophic sesquiterpene dilactone from Illicium merrillianum, Tetrahedron Lett. 41 (2000) 6111e6114. [7] R. Yokoyama, J.M. Huang, C.S. Yang, Y. Fukuyama, New seco-prezizaane-type sesquiterpenes, jiadifenin with neurotrophic activity and 1,2dehydroneomajucin from Illicium jiadifengpi, J. Nat. Prod. 65 (2002) 527e531. [8] M. Kubo, C. Okada, J.M. Huang, K. Harada, H. Hioki, Y. Fukuyama, Novel pentacyclic seco-prezizaane-type sesquiterpenoids with neurotrophic properties from Illicium jiadifengpi, Org. Lett. 11 (2009) 5190e5193. [9] M. Kubo, Y. Kishimoto, K. Harada, H. Hioki, Y. Fukuyama, NGF-potentiating vibsane-type diterpenoids from Viburnum sieboldii, Bioorg. Med. Chem. Lett 20 (2010) 2566e2571. [10] M. Kubo, M. Gima, K. Baba, M. Nakai, K. Harada, M. Suenaga, Y. Matsunaga, E. Kato, S. Hosoda, Y. Fukuyama, Novel neurotrophic phenylbutenoids from Indonesian ginger Bangle, Zingiber purpureum, Bioorg. Med. Chem. Lett 25 (2015) 1586e1591. [11] H. Zhai, T. Inoue, M. Moriyama, T. Esumi, Y. Mitsumoto, Y. Fukuyama, Neuroprotective effects of 2,5-diaryl-3,4-dimethyltetrahydrofuran neolignans,
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Biol. Pharm. Bull. 28 (2005) 289e293. [12] H. Zhai, M. Nakatsukasa, Y. Mitsunoto, Y. Fukuyama, Neurotrophic Effects of talaumidin, a neolignan from Aristolochia arcuate, in primary cultured rat cortical neurons, Planta Med. 70 (2004) 598e602. [13] Y. Fukuyama, K. Harada, T. Esumi, D. Hojyo, Y. Kujime, N. Kubo, M. Kubo, H. Hioki, Synthesis of ()-talaumidin, a neurotrophic 2,5-biaryl-3,4-dimethyltetrahydrofuran lignin, and its stereoisomers, Heterocycles 76 (2008) 551e567. [14] K. Harada, H. Horiuchi, K. Tanabe, R.G. Carter, M. Kubo, H. Hioki, Y. Fukuyama, Asymmetric synthesis of ()-chicanine using a highly regioselective intramolecular Mitsunobu reaction and revision of its absolute configuration, Tetrahedron Lett. 52 (2011) 3005e3008. [15] K. Harada, N. Kubo, K. Tanabe, M. Kubo, T. Esumi, H. Hioki, Y. Fukuyama, Asymmetric synthesis of (þ)-machillin F by unusual stereoselective Mitsunobu reaction, Heterocycles 82 (2011) 1127e1132.
[16] S. der Lima, Y. Koriyama, T. Kurimoto, J.T. Oliveira, Y. Yin, Y. Li, H.Y. Gilbert, M. Fagiolini, A.M.B. Martinez, L. Benowitz, Full-length axon regeneration in the adult mouse optic nerve and partial recovery of simple visual behaviors, Proc. Natl. Acad. Sci. Unit. States Am. 109 (2012) 9149e9154. [17] K. Harada, M. Kubo, H. Horiuchi, A. Ishii, T. Esumi, H. Hioki, Y. Fukuyama, Systematic asymmetric synthesis of all diastereomers of ()-talaumidin and their neurotrophic activity, J. Org. Chem. 80 (2015) 7076e7088. [18] W. Tang, H. Hioki, K. Harada, M. Kubo, Y. Fukuyama, Clerodane diterpenes with NGF-potentiating activity from Ptychopetalum olacoides, J. Nat. Prod. 71 (2008) 1760e1763. [19] L.I. Benowitz, Z. He, J.L. Goldber, Reaching the brain: Advances in optic nerve regeneration, Exp. Neurol. 287 (2017) 365e373. [20] S. Leon, Y. Yin, J. Nguyen, N. Irwin, L.I. Benowitz, Lens injury stimulates axon regeneration in the nature rat optic nerve, J. Neurosci. 20 (2000) 4615e4626.
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European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Structure-activity relationships of talaumidin derivatives: Their neurite-outgrowth promotion in vitro and optic nerve regeneration in vivo Kenichi Harada a, *, Katsuyoshi Zaha a, Rina Bando a, Ryo Irimaziri a, Miwa Kubo a, Yoshiki Koriyama b, Yoshiyasu Fukuyama a, ** a b
Faculty of Pharmaceutical Sciences, Tokushima Bunri University, 180 Yamashiro-cho, Tokushima 770-8514, Japan Graduate School and Faculty of Pharmaceutical Sciences, Suzuka University of Medical Science, 3500-3 Minamitamagaki, Suzuka, Mie, 513-8670, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 7 December 2017 Received in revised form 5 February 2018 Accepted 6 February 2018 Available online 9 February 2018
(e)-Talaumidin (1), a 2,5-biaryl-3,4-dimethyltetrahydrofuran lignan, shows potent neurotrophic activities such as neurite-outgrowth promotion and neuroprotection. Previously, we found that (e)-(1S,2R,3S,4R)-stereoisomer 2 exhibited more significant activity than did the natural product talaumidin (1). However, the preparation of optically active (e)-2 requires a complicated synthetic route. To explore new neurotrophic compounds that can be obtained on a large scale, we established a short step synthetic route for talaumidin derivatives and synthesized fourteen analogues based on the structure of (e)-2. First, we synthesized a racemic compound of (e)-2 (2a) and assessed its neurotrophic activity. We found that the neurotrophic property of racemic 2a is similar in activity to that of (e)-2. Using the same synthetic methodology, several talaumidin derivatives were synthesized to optimize the oxy-functionality on aromatic rings. As a result, bis(methylenedioxybenzene) derivative 2b possessed the highest neurotrophic activity. Furthermore, examination of the structure-activity relationships of 2b revealed that the 2,5-diphenyl-tetrahydrofuran structure was an essential structure and that two methyl groups on THF ring could enhance neurotrophic activity. In addition, compounds 2a and 2b were found to induce mouse optic nerve regeneration in vivo. © 2018 Elsevier Masson SAS. All rights reserved.
Keywords: Talaumidin Neurotrophic activity Neurite-outgrowth promotion Optic nerve regeneration
1. Introduction Neurotrophic factors (NTFs) have been recognized to play important roles in the differentiation of nerve stem cells, neuriteoutgrowth, survival and regeneration of neurons [1e4]. The NTFs have been predicted to improve neurodegenerative diseases such as depression, glaucoma, and Alzheimer's diseases. However, their poor bioavailability and pharmacokinetics due to their polypeptidyl structures have made clinical trials ineffective [5]. Therefore, research on small neurotrophic molecules has garnered significant scientific attention. As part of our research in this area, we have continued to search for neurotrophic molecules from plants [6e10]. In the course of our studies on neurotrophic compounds, we
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (K. Harada), [email protected]. ac.jp (Y. Fukuyama). https://doi.org/10.1016/j.ejmech.2018.02.014 0223-5234/© 2018 Elsevier Masson SAS. All rights reserved.
isolated a tetrahydrofuran-type lignan, (e)-(2S,3S,4S,5S)-talaumidin (1) from Aristolochia arcuata Masters (Fig. 1). Remarkably, 1 shows not only significant neurite-outgrowth promotion in primary cultured rat cortical neurons and in NGF-differentiated PC12 cells but also exhibits protective effects against cell death induced by several insults [11,12]. Further work such as in vivo experiments and mechanistic analysis of neurotrophic activities is currently required to develop the therapeutic agents for neurodegenerative diseases. In addition to its neurotrophic activity, talaumidin has an interesting structure consisting of a 2,5-biaryl-3,4dimethyltetrahydrofuran skeleton with four continuous stereogenic centers. Previously, we synthesized both enantiomers and all diastereomers of natural product 1 to investigate stereochemistryactivity relationships [13e16]. As a result, (1S,2R,3S,4R)-stereoisomer (e)-2 bearing all-cis-substituents showed the most significant neurite-outgrowth promoting activity in NGF-differentiated PC12 cells. However, the preparation of optically active
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Fig. 1. Structures of ()-talaumidin (1), stereoisomers ()-2, and synthesized talaumidin derivatives.
talaumidin and isomer 2 suffered from high cost, long synthetic steps, and low total chemical yield. In this study, we established a new synthetic methodology for talaumidin derivatives in order to explore new neurotrophic compounds that can be obtained on a large scale. In addition, we discuss the structure-activity relationships between talaumidin derivatives and neurotrophic activities. Furthermore, we report their regenerative activity towards mouse optic nerves as a neurotrophic activity in vivo to increase the potential as a therapeutic agent for neurodegenerative disease. Because the central nerve system is not able to regenerate after injury, nerve regenerative activity is one of the most important roles as a neurotrophic factor [16]. 2. Results and discussion 2.1. Synthesis and neurite-outgrowth promoting activity of racemic compound 2a In a previous paper [17], it was indicated that there is little difference in neurite-outgrowth promoting activity between ()-talaumidin and its enantiomer, suggesting that racemic compound 2a has the same biological activity as that of the optically active compound. Therefore, this synthetic study began from the synthesis of 2a, aiming to establish a concise synthetic methodology for talaumidin derivatives. Generally, synthesis of a racemic compound is much easier than that of optically compound because asymmetric reactions, chiral starting materials, and chiral reagents
Scheme 1. Synthesis of racemic 2a.
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are not needed. We envisioned a short steps synthesis of 2a, as shown in Scheme 1. First, a successive Grignard reaction and DessMartin oxidation of commercially available 3 gave the ketone 4a in 99% yield over 2 steps. After a-bromination, the obtained bromide 5a was coupled to 6a in the presence of KHMDS in DMF to provide a 1,4-diketone 7a in good yield. Next, the 1,4-diketone was subjected to Paal-Knorr reaction to afford a tetra-substituted furan 8 in 87% yield. Finally, the synthesis of 2a was attained by hydrogenation in dilute solution (5 mmol/L). It should be noted that the usual reaction concentrations (10e100 mmol/L) induce overreactions such as hydrogenolysis of THF ring in any solvent. According to the previously reported experimental procedure [18], we compared the neurotrophic activity of 2a to the optically active (e)-talaumidin (1) and (1S,2R,3S,4R)-stereoisomer (e)-2 at 30 mmol/L17 (Fig. 2). As expected, 2a showed neurite-outgrowth promoting activity in NGF-differentiated PC12 cells at the same level as the optically active (e)-2, which was higher than that of talaumidin. Thus, no different activity was observed between the enantiomers and racemic compound. 2.2. Optimization of oxy-functional group on benzene rings Next, our attention was focused on optimizing oxy-functional groups on aromatic rings to enhance neurotrophic activity. To elucidate the correlation between functional groups and activity, we decided to synthesize symmetrical derivatives with the same functional group on both benzene rings. In accordance with the synthetic methodology of 2a, five tetrahydrofuran compounds 2bed, 2f and 2g were synthesized in 4 steps (Scheme 2). The coupling reaction of brominated 5aed with 4aed provided dimers 7bee. Paal-Knorr's furan synthesis of 7bee afforded the corresponding tetra-substituted furans 8bee. The methoxy 8c was converted to alcohol 8f using BBr3 in DCM, and 8e, with a benzyloxy group, was converted to 8g by hydrogenolysis. Finally, reduction of the obtained furans 8beg gave rise to tetrahydrofurans 2beg, respectively. All-cis-configuration of 2beg was determined by comparison of the chemical shifts and coupling constants with those of stereoisomers reported in our preceding paper [17]. As shown in Fig. 3, synthesized tetrahydrofuran derivatives were evaluated for their ability to induce neurite-outgrowth in NGF-differentiated PC12 cells at 30 mmol/L, together with their synthetic precursors 8ae8d, 8f and 8g. The degree of their effects on neurite-outgrowth promotion in PC12 cells was demonstrated by morphological observations and quantitative analysis of the neurite length extending from the cell bodies. Consequently, tetrahydrofuran compounds had higher activity than did furan compounds. For 40 -substituents, a specific difference was not
Fig. 2. Comparison of neurite length of NGF-differentiated PC12 cells promoted by (e)-1, (e)-2, and 2a at 30 mmol/L. Data are expressed as the mean as ±SE. *, P < 0.05; **, P < 0.01 compared with NGF by Dunnett's t-test.
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nerve [16] was adopted as an in vivo experiment. The optic nerve is part of the central nervous system that cannot regenerate spontaneously. The optic nerve has been used to examine the response of CNS neurons to injury because of its accessibility, anatomy, and functional importance [19]. The number of regenerating axons in injured mouse optic nerves was counted as described by Leon [20]. Notably, three compounds, 1, 2a and 2b, were found to induce the regeneration of the injured optic nerve, as shown in Fig. 5. In addition, compounds 2a and 2b induced more significant regeneration of nerves than did natural product (e)-1 as well as their neurite-outgrowth promotion in vitro at 30 mmol/L. Although the difference between 2a and 2b was hardly observed in this screening, the all-cis-substituted compounds 2a and 2b were found to show more potent activity than the all-trans-substituted (e)-1. Thus, stereochemistry of talaumidin derivatives was demonstrated to be important for not only neurite-outgrowth promotion in vitro but also for regeneration of the optic nerve in vivo. 3. Conclusion
Scheme 2. Synthesis of talaumidin derivatives with all cis configurations.
observed among methoxy-, methyl-, and hydroxy-derivatives 2cef or 8ce8f. Moreover, the 30 -methoxy group was found to show some enhancement of the activity between 2f and 2g, or 8f and 8g. Ultimately, 2b bearing the methylenedioxy group was found to exhibit higher activity than 2a. 2.3. Examination of structure-activity relationships of talaumidin derivatives Subsequently, we decided to investigate the structure-activity relationships for the neurite-outgrowth activity of talaumidin derivatives. To determine the roles of aromatic rings and methyl groups on tetrahydrofuran in 2b, we designed two compounds, mono-phenyl compound 9 and compound 10. The synthesis of 9 was started from dimethylmaleic anhydride 11 (Scheme 3). The Grignard reaction of 11 with 3,4-methylenedioxyphenyl magnesium bromide (12) provided a lactol 13 in good yield. After reduction with Et3SiH/TFA, the obtained lactone 14 was converted to the tri-substituted furan 15. Finally, the mono-phenyl derivative 9 was prepared by Pd-catalyzed hydrogenation. In addition, compound 10 was synthesized in two steps involving Suzuki-Miyaura reaction of 2,5-dibromofuran (16) with boronate 17, and hydrogenation of 18 (Scheme 4). Synthesized compounds 9 and 10 were assessed for their neurite-outgrowth promoting activity in NGF-differentiated PC12 cells and compared with 2b (Fig. 4). As a result, monophenyl compound 9 showed no activity, while compound 10 exhibited lower activity than did 2b. These results suggest that two phenyl groups on tetrahydrofuran are the crucial structural units, and two methyl groups serve to enhance the neurotrophic activity. 2.4. Evaluation of regeneration of mouse optic nerve in vivo Finally, we examined the neurotrophic activity of (e)-1, 2a, and 2b in vivo. In this study, the regenerative activity of injured optic
We established a concise synthetic methodology for talaumidin derivatives that could be used to prepare a variety of analogues in short steps and on a large scale. The synthesized racemic compound 2a showed neurite-outgrowth promoting activity in NGFdifferentiated PC12 cells at the same level as the optically active (e)-2, revealing that the relative configuration bearing all-cissubstituents is important for potent neurotrophic activities although the absolute configurations do not affect their activities. Using this synthetic methodology, fourteen compounds based on (e)-2 were prepared. Among them, compounds 2b with a methylenedioxy group on both benzene rings was found to exhibit the most significant neurite outgrowth promotion in NGFdifferentiated PC12 cells in vitro. In addition, 2a and 2b also induced regeneration of the mouse optic nerve in vivo, and their activity was higher than talaumidn's activity as well as the in vitro experiments. Furthermore, the structure-activity relationships of 2b showed that the two benzene rings were essential structures, and that the methyl groups on the THF ring could enhance the neurotrophic activity. This result suggests that modification of alkyl groups on the THF ring can enhance the neurotrophic activities. Further studies involving optimization of structure, detailed pharmacological experiments and mechanistic analysis are now underway. Our results demonstrate that talaumidin derivatives can be novel therapeutic agents for neurodegenerative diseases such as glaucoma and Alzheimer's diseases. 4. Experimental 4.1. General information All reagents, general solvents, and dry solvents were purchased from Tokyo Chemical Industry, Wako Pure Chemical Industries, and Sigma-Aldrich. IR spectra were recorded on JASCO FT-IR 410 infrared spectrophotometer. High resolution mass spectra were acquired on MStation JMS-700. 1H and 13C NMR spectra were obtained on Varian 200, 300, 400 MHz and JEOL 400 MHz and Bruker 500 MHz instruments in CDCl3 with TMS as internal standard and CD3OD. Silica gel column chromatography was carried out on Merck Silica gel 60 N (70e230 mesh). 4.1.1. (±)-4-{5-(benzo[d][1,3]dioxol-5-yl)-3,4dimethyltetrahydrofuran-2-yl}-2-methoxyphenol (2a) To a solution of 8 (200 mg, 467 mmol) in EtOH (93.4 mL) was added Pd(OH)2/C (40.0 mg). The mixture was stirred vigorously under hydrogen atmosphere at rt for 96 h. After being filtrated, the
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Fig. 3. Comparison of neurite length of NGF-differentiated PC12 cells promoted by furan compounds and tetrahydrofuran compounds at 30 mmol/L. Data are expressed as the mean as ±SE. *P < 0.05, **P < 0.01 compared with NGF by Dunnett's t-test.
Scheme 4. Synthesis of compound 10. Scheme 3. Synthesis of mono-phenyl compound 9.
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2972, 2892, 1503, 1490, 1254, 1237, 1039 cm1. HRMS (EI) m/z: calcd. for C20H20O5 [M]þ 340.1311, found 340.1300. 4.1.3. 2,5-bis(4-methoxyphenyl)-3,4-dimethyltetrahydrofuran (2c) Compound 2c was prepared in the same manner as compound 2a using tetra-substituted furan 8c (345 mg, 1.12 mmol). The product 2c (101 mg, 29%) was obtained as a colorless oil. 1H NMR (300 MHz, CDCl3) d 7.36 (4H, d, J ¼ 8.5 Hz), 6.91 (4H, d, J ¼ 8.5 Hz), 5.13 (2H, d, J ¼ 6.8 Hz), 3.83 (6H, s), 2.68e2.64 (2H, m), 0.58 (6H, d, J ¼ 6.9 Hz). 13C NMR (75 MHz, CDCl3) d 158.4, 132.7, 127.6, 113.4, 113.3, 82.7, 82.5, 55.2, 41.6, 11.9. IR (ATR) 2967, 2835, 1513, 1247 cm1. HRMS (EI) m/z: calcd. for C20H24O3 [M]þ 312.1725, found 312.1715.
Fig. 4. Comparison of neurite length of NGF-differentiated PC12 cells promoted between 2b and tetrahydrofuran compounds at 30 mmol/L. Data are expressed as the mean as ±SE. *P < 0.05, **P < 0.01 compared with NGF by Dunnett's t-test.
solution was concentrated in vacuo. The residue was purified by column chromatography (hexane:DCM ¼ 1:30) to afford 2a (108 mg, 68%) as a colorless oil. 1H NMR, 13C NMR, IR, and MS data of 2a were consistent with those of ()-2. 1H NMR (400 MHz, CDCl3) d 6.96 (1H, d, J ¼ 1.5 Hz), 6.94 (1H, d, J ¼ 1.4 Hz), 6.92 (1H, d, J ¼ 8.0 Hz), 6.88 (1H, dd, J ¼ 8.0, 1.4 Hz), 6.86 (1H, dd, J ¼ 8.1, 1.5 Hz), 6.81 (1H, d, J ¼ 8.1 Hz), 5.97 (2H, s), 5.09 (2H, d, J ¼ 6.4 Hz), 3.91 (3H, s), 2.61e2.68 (2H, m), 0.61 (3H, d, J ¼ 6.1 Hz), 0.59 (3H, d, J ¼ 6.1 Hz). 13 C NMR (100 MHz, CDCl3) d 147.4, 146.2, 144.3, 134.5, 132.4, 119.5, 119.3, 118.0, 114.0, 109.0, 107.9, 107.1, 100.9, 82.8, 82.7, 56.0, 41.5, 41.5, 11.8, 11.8. IR (ATR) 3472, 2969, 1516, 1236, 1038, 455 cm1. HRMS (EI) m/z: calcd for C20H22O5 [M]þ 342.1467, found 342.1476. 4.1.2. 5,5'-(3,4-dimethyltetrahydrofuran-2,5-diyl)bis(benzo[d][1,3] dioxole) (2b) Compound 2b was prepared in the same manner as compound 2a using tetra-substituted furan 8b (67.2 mg, 200 mmol). The product 2b (47.2 mg, 69%) was obtained as a colorless oil. 1H NMR (400 MHz, CDCl3) d 6.93 (2H, d, J ¼ 1.0 Hz), 6.86 (2H, dd, J ¼ 7.9, 1.0 Hz), 6.80 (2H, d, J ¼ 7.9 Hz), 5.96 (4H, s), 5.07 (2H, d, J ¼ 6.7 Hz), 2.70e2.57 (2H, m), 0.59 (6H, d, J ¼ 7.0 Hz). 13C NMR (75 MHz, CDCl3) d 147.4, 146.3, 134.4, 119.5, 107.9, 107.1, 100.9, 82.7, 41.5, 11.8. IR (ATR)
4.1.4. 3,4-Dimethyl-2,5-di-p-tolyltetrahydrofuran (2d) Compound 2d was prepared in the same manner as compound 2a using tetra-substituted furan 8d (20.3 mg, 73.6 mmol). The product 2d (10.2 mg, 49%) was obtained as a colorless oil. 1H NMR (200 MHz, CDCl3) d 7.32 (4H, d, J ¼ 7.9 Hz), 7.17 (4H, d, J ¼ 7.9 Hz), 5.16 (2H, d, J ¼ 6.6 Hz), 2.73e2.62 (2H, m), 2.36 (6H, s), 0.58 (6H, d, J ¼ 6.9 Hz). 13C NMR (75 MHz, CDCl3) d 137.5, 136.2, 128.6, 126.3, 82.8, 41.5, 21.2, 11.9. IR (ATR) 2972, 2919, 1514, 1015, 817 cm1. HRMS (EI) m/z: calcd. for C20H24O [M]þ 280.1827, found 280.1830. 4.1.5. 4,4'-(3,4-dimethyltetrahydrofuran-2,5-diyl)diphenol (2f) Compound 2f was prepared in the same manner as compound 2a using tetra-substituted furan 8f (50.0 mg, 179 mmol). The product 2f (18.8 mg, 37%) was obtained as a colorless oil. 1H NMR (300 MHz, CD3OD) d 7.23 (4H, d, J ¼ 8.5 Hz), 6.78 (4H, d, J ¼ 8.5 Hz), 5.07 (2H, d, J ¼ 6.5 Hz), 2.68e2.00 (2H, m), 0.56 (6H, d, J ¼ 7.0 Hz). 13 C NMR (75 MHz, CD3OD) d 154.7, 129.6, 125.9, 113.0, 81.5, 40.0, 9.5. IR (ATR) 3224, 1515 cm1. HRMS (EI) m/z: calcd. for C18H20O3 [M]þ 284.1412, found 284.1427. 4.1.6. 4,4'-(3,4-dimethyltetrahydrofuran-2,5-diyl)bis(2methoxyphenol) (2g) Compound 2g was prepared in the same manner as compound 2a using tetra-substituted furan 8g (107 mg, 205 mmol). The product 2g (36.4 mg, 52%) was obtained as a colorless oil. 1H NMR (400 MHz, CDCl3) d 6.98e6.88 (6H, m), 5.53 (2H, s), 5.12 (2H, d, J ¼ 6.6 Hz), 3.92 (6H, s), 2.70e2.62 (2H, m), 0.59 (6H, d, J ¼ 6.9 Hz). 13 C NMR (75 MHz, CDCl3) d 147.4, 146.3, 134.4, 119.5, 107.9, 107.1, 100.9, 82.8, 41.5, 11.8. IR (ATR) 3433, 3967, 1515, 1275, 1034 cm1. HRMS (EI) m/z: calcd. for C20H24O5 [M]þ 344.1624, found 344.1617.
Fig. 5. Talaumidin-related compounds induce mouse optic nerve regeneration in vivo at 30 mmol/L. Longitudinal sections of the adult mouse optic nerve showing GAP-43 positive axons extending over the injury site (red asterisks) after 2 weeks of optic nerve injury. (A) Vehicle control, Scale ¼ 200 mm. (B) Talaumidin. (C) 2a. (D) 2b. (E) Quantification of axonal regrowth at an indicated proximal point from the injury site (200 mm). **P < 0.01, *P < 0.05, vs injury (n ¼ 6). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)
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4.1.7. 1-(4-Benzyloxy-3-methoxyphenyl)propan-1-one (4a) To a solution of 4-benyloxy-3-methoxybenzaldehyde (3, 5.00 g, 20.6 mmol) in THF (206 mL) was added EtMgBr (30.0 mL, 1.0 M solution in THF) at 0 C. After being stirred for 16 h, the reaction was quenched with saturated NH4Cl. The aqueous layer was extracted with ether. The combined organic layers were washed with brine, dried over anhydrous MgSO4, and concentrated in vacuo. To the residue was added Dess-Martin periodinane (11.3 g, 30.1 mmol) in DCM (103 mL). After being stirred for 12 h, the mixture was added to excess ether and filtered through Celite, and the filtrate was concentrated in vacuo. The residue was purified by column chromatography (hexane:EtOAc ¼ 3:1) to afford 4a (5.51 g, 99%) as a colorless oil. 1H NMR (200 MHz, CDCl3) d 7.56e7.33 (7H, m), 6.89 (1H, d, J ¼ 8.4 Hz), 5.23 (2H, s), 3.94 (3H, s), 2.94 (2H, q, J ¼ 7.3 Hz), 1.21 (3H, t, J ¼ 7.3 Hz). 13C NMR (75 MHz, CDCl3) d 199.5, 152.1, 149.4, 136.3, 130.3, 128.6, 128.1, 127.1, 122.3, 112.1, 110.5, 70.7, 56.0, 31.3, 8.6. IR (ATR) 2972, 2935, 1671, 1584, 1510, 1453, 1416, 1340 cm1. HRMS (EI) m/z: calcd. for C17H18O3 [M]þ 270.1256, found 270.1252. 4.1.8. 1-(4-Benzyloxy-3-methoxyphenyl)-2-bromopropan-1-one (5a) To a solution of 4a (3.90 g, 14.4 mmol) in CHCl3 (100 mL) was added Br2 (741 mL, 14.4 mmol) in CHCl3 (45.0 mL) at 0 C. After being stirred overnight, saturated NaHCO3 solution was added and extracted with DCM. The organic layers were dried over Na2SO4 and concentrated in vacuo. The residue was purified by column chromatography (hexane:EtOAc ¼ 88:12) to afford 5a (3.93 g, 78%) as a colorless oil. 1H NMR (300 MHz, CDCl3) d 7.59 (1H, brs), 7.58 (1H, brd, J ¼ 8.2 Hz), 7.44e7.31 (5H, m), 6.90 (1H, d, J ¼ 8.2 Hz), 5.25 (1H, q, J ¼ 6.6 Hz), 5.23 (2H, s), 3.94 (3H, s), 1.87 (3H, d, J ¼ 6.6 Hz). 13C NMR (75 MHz, CDCl3) d 192.1, 152.9, 149.7, 136.1, 128.7, 128.2, 127.2, 123.2, 112.0, 111.5, 70.8, 56.1, 41.2, 20.4. IR (ATR) 2933, 2358, 1671, 1591, 1453, 1376, 1260 cm1. HRMS (EI) m/z: calcd. for C17H17O3Br [M]þ 348.0362, found 348.0356. 4.1.9. 1-(benzo[d][1,3]dioxol-5-yl)-2-bromopropan-1-one (5b) Compound 5b was prepared in the same manner as compound 5a using ketone 4b (9.00 g, 50.0 mmol). The product 5b (11.3 g, 87%) was obtained as a pale yellow oil. 1H NMR (300 MHz, CDCl3) d 7.64 (1H, dd, J ¼ 8.1, 1.8 Hz), 7.50 (1H, d, J ¼ 1.8 Hz), 6.88 (1H, d, J ¼ 8.1 Hz), 6.07 (2H, s), 5.22 (1H, q, J ¼ 6.6 Hz), 1.88 (3H, d, J ¼ 6.6 Hz). 13C NMR (75 MHz, CDCl3) d 191.6, 152.3, 148.4, 128.6, 125.3, 108.7, 108.1, 102.1, 41.3, 20.3. IR (ATR) 2953, 1683, 1443, 1245, 1039 cm1. HRMS (EI) m/z: calcd. for C10H9O3Br [M]þ 257.9867, found 257.9899. 4.1.10. 2-Bromo-1-(4-methoxyphenyl)propan-1-one (5c) Compound 5c was prepared in the same manner as compound 5a using ketone 4c (2.00 g, 12.2 mmol). The product 5c (2.45 g, 83%) was obtained as a pale yellow oil. 1H NMR (300 MHz, CDCl3) d 8.01 (2H, d, J ¼ 9.3 Hz), 6.95 (2H, d, J ¼ 9.3 Hz), 5.26 (1H, q, J ¼ 6.6 Hz), 3.88 (3H, s), 1.89 (3H, d, J ¼ 6.6 Hz). 13C NMR (75 MHz, CDCl3) d 191.9, 163.9, 131.3, 126.8, 113.9, 55.5, 41.4, 20.2. IR (ATR) 2933, 1674, 1597, 1238, 1157, 840 cm1. HRMS (EI) m/z: calcd. for C10H11O2Br [M]þ 241.9942, found 241.9950. 4.1.11. 2-Bromo-1-(p-tolyl)propan-1-one (5d) Compound 5d was prepared in the same manner as compound 5a using ketone 4d (2.01 mL, 13.5 mmol). The product 5d (2.05 g, 67%) was obtained as a pale yellow oil. 1H NMR (300 MHz, CDCl3) d 7.92 (2H, d, J ¼ 8.4 Hz), 7.28 (2H, d, J ¼ 8.4 Hz), 5.28 (1H, q, J ¼ 6.5 Hz), 2.43 (3H, s), 1.89 (3H, d, J ¼ 6.5 Hz). 13C NMR (75 MHz, CDCl3) d 193.1, 144.8, 131.5, 129.5, 129.1, 41.6, 21.8, 20.2. IR (ATR) 2973, 1667, 1440, 1245, 1037 cm1. HRMS (EI) m/z: calcd. for C10H11OBr [M]þ 225.9993, found 226.0007.
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4.1.12. 1-(benzo[d][1,3]dioxol-5-yl)-4-(4-benzyloxy-3methoxyphenyl)-2,3-dimethylbutane-1,4-dione (7a) To a solution of 6a (1.00 g, 5.58 mmol) in DMF (35.0 mL) was added KHMDS (13.4 mL, 0.5 M solution in toluene) at 45 C. After being stirred for 1 h, a solution of 5a (1.95 g, 5.58 mmol) in DMF (35.0 mL) was added to the reaction mixture at the same temperature. After being stirred overnight, the reaction was quenched with saturated NH4Cl. The aqueous layer was extracted with ether. The combined organic layers were washed with brine, dried over anhydrous MgSO4, and concentrated in vacuo. The residue was purified by column chromatography (hexane:ether ¼ 1:1) to afford 7a (2.15 g, 86%) as a pale yellow oil. 1H NMR (400 MHz, CDCl3) d 7.63 (1H, dd, J ¼ 8.4, 1.5 Hz), 7.61 (1H, dd, J ¼ 8.4, 1.8 Hz), 7.50 (1H, d, J ¼ 1.8 Hz), 7.44e7.32 (6H, m), 6.90 (1H, d, J ¼ 8.4 Hz), 6.86 (1H, d, J ¼ 8.4 Hz), 6.02 (2H, s), 5.23 (2H, s), 3.90 (3H, s), 3.90e3.85 (2H, m), 1.27 (3H, d, J ¼ 6.6 Hz), 1.26 (3H, d, J ¼ 6.6 Hz). 13C NMR (100 MHz, CDCl3) d 202.9, 202.5, 152.4, 151.7, 149.5, 148.2, 136.4, 131.0, 129.4, 128.7, 128.1, 127.2, 124.7, 122.9, 112.3, 111.1, 108.4, 108.0, 101.8, 70.8, 56.0, 43.5, 43.4, 16.0, 15.8. IR (ATR) 3018, 2974, 1665, 1594, 1505, 1454, 1246 cm1. HRMS (EI) m/z: calcd. for C27H26O6 [M]þ 446.1730, found 446.1717. 4.1.13. 1,4-bis(benzo[d][1,3]dioxol-5-yl)-2,3-dimethylbutane-1,4dione (7b) Compound 7b was prepared in the same manner as compound 7a using a-bromoketone 4b (100 mg, 389 mmol). The product 7b (66.6 mg, 48%) was obtained as a pale yellow oil. 1H NMR (300 MHz, CDCl3) d 7.63 (2H, dd, J ¼ 8.3, 1.8 Hz), 7.42 (2H, d, J ¼ 1.8 Hz), 6.86 (2H, d, J ¼ 8.3 Hz), 6.03 (4H, s), 3.84e3.78 (2H, m), 1.27 (6H, d, J ¼ 7.0 Hz). 13C NMR (75 MHz, CDCl3) d 202.4, 151.7, 148.2, 130.8, 124.7, 108.4, 108.0, 101.8, 43.5, 15.9. IR (ATR) 2956, 1667, 1440, 1244, 1038 cm1. HRMS (EI) m/z: calcd. for C20H18O6 [M]þ 354.1103, found 354.1099. 4.1.14. 1,4-bis(4-methoxyphenyl)-2,3-dimethylbutane-1,4-dione (7c) Compound 7c was prepared in the same manner as compound 7a using a-bromoketone 4c (2.05 g, 8.47 mmol). The product 7c (1.66 g, 60%) was obtained as a pale yellow oil. 1H NMR (200 MHz, CDCl3) d 7.97 (4H, d, J ¼ 8.7 Hz), 6.93 (4H, d, J ¼ 8.7 Hz), 3.95e3.90 (2H, m), 3.86 (6H, s), 1.28 (6H, d, J ¼ 6.7 Hz). 13C NMR (75 MHz, CDCl3) d 202.9, 163.4, 130.8, 129.1, 113.7, 55.5, 43.2, 15.8. IR (ATR) 2971, 1667, 1597, 1168, 968 cm1. HRMS (EI) m/z: calcd. for C20H22O4 [M]þ 326.1518, found 326.1508. 4.1.15. 2,3-Dimethyl-1,4-di-p-tolylbutane-1,4-dione (7d) Compound 7d was prepared in the same manner as compound 7a using a-bromoketone 4d (400 mg, 1.78 mmol). The product 7d (472 mg, 90%) was obtained as a pale yellow oil. 1H NMR (200 MHz, CDCl3) d 7.89 (4H, d, J ¼ 8.2 Hz), 7.26 (4H, d, J ¼ 8.2 Hz), 3.98e3.90 (2H, m), 2.41 (6H, s), 1.27 (6H, d, J ¼ 6.7 Hz). 13C NMR (75 MHz, CDCl3) d 203.9, 143.6, 133.6, 129.2, 128.6, 43.4, 21.6, 15.5. IR (ATR) 2963, 1666, 1541, 966 cm1. HRMS (EI) m/z: calcd. for C20H22O2 [M]þ 294.1620, found 294.1619. 4.1.16. 1,4-bis(4-benzyloxy-3-methoxyphenyl)-2,3-dimethylbutane1,4-dione (7e) Compound 7e was prepared in the same manner as compound 7a using a-bromoketone 4e (150 mg, 431 mmol). The product 7e (137 mg, 59%) was obtained as a pale yellow oil. 1H NMR (300 MHz, CDCl3) d 7.61 (2H, dd, J ¼ 8.7, 2.0 Hz), 7.49 (2H, d, J ¼ 2.0 Hz), 7.44e7.31 (10H, m), 6.90 (2H, d, J ¼ 8.7 Hz), 5.23 (4H, m), 3.97e3.87 (2H, m), 3.89 (6H, s), 1.27 (6H, d, J ¼ 6.5 Hz). 13C NMR (75 MHz, CDCl3) d 203.0, 152.3, 149.5, 136.4, 129.4, 128.7, 128.1, 127.2, 122.9, 112.2, 111.1, 70.8, 56.0, 43.2, 16.0. IR (ATR) 2972, 1666, 1593, 1511,
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1260 cm1. HRMS (EI) m/z: calcd. for C34H34O6 [M]þ 538.2355, found 538.2340. 4.1.17. 5-{5-(4-benzyloxy-3-methoxyphenyl)-3,4-dimethylfuran-2yl}benzo[d][1,3]dioxole (8) A solution of 7a (66.6 mg, 188 mmol) in DCM/benzene (500 mL/ 3.76 mL) containing p-TsOH$H2O (10.7 mg, 56.4 mmol) was refluxed under removal of water by a Dean-Stark apparatus for 4 h. The cooled solution was diluted with EtOAc, washed with saturated NaHCO3 solution, dried over anhydrous MgSO4, and concentrated in vacuo. The residue was purified by column chromatography (hexane:EtOAc ¼ 1:1) to afford 8 (55.2 mg, 87%) as a yellow oil. 1H NMR (400 MHz, CDCl3) d 7.45 (2H, d, J ¼ 7.0 Hz), 7.37 (2H, t, J ¼ 7.0 Hz), 7.31 (1H, t, J ¼ 7.0 Hz), 7.23 (1H, d, J ¼ 1.8 Hz), 7.17 (1H, d, J ¼ 1.5 Hz), 7.14 (1H, dd, J ¼ 8.1, 1.5 Hz), 7.13 (1H, dd, J ¼ 8.4, 1.8 Hz), 6.92 (1H, d, J ¼ 8.4 Hz), 6.87 (1H, d, J ¼ 8.1 Hz), 5.98 (2H, s), 5.19 (2H, s), 3.95 (3H, s), 2.19 (3H, s), 2.18 (3H, s). 13C NMR (100 MHz, CDCl3) d 149.7, 147.8, 147.2, 146.8, 146.8, 146.3, 137.1, 128.5, 127.8, 127.3, 126.2, 125.6, 119.4, 118.3, 117.9, 117.9, 114.1, 109.6, 108.5, 106.3, 101.2, 71.1, 56.1, 9.9, 9.9. IR (ATR) 2897, 1503, 1486, 1452, 1243 cm1. HRMS (CI) m/z: calcd. for C27H25O5 [MþH]þ 429.1702, found 429.1695. 4.1.18. 5,5'-(3,4-dimethylfuran-2,5-diyl)bis(benzo[d][1,3]dioxole) (8b) Compound 8b was prepared in the same manner as compound 8a using diketone 7b (66.6 mg, 188 mmol). The product 8b (55.2 mg, 87%) was obtained as a yellow oil. 1H NMR (300 MHz, CDCl3) d 7.16e7.13 (4H, m), 6.85 (2H, d, J ¼ 8.4 Hz), 5.99 (4H, s), 2.19 (6H, s). 13 C NMR (75 MHz, CDCl3) d 147.8, 146.7, 146.3, 126.2, 119.4, 117.9, 108.5, 106.2, 101.1, 9.9. IR (ATR) 1503, 1231, 904, 728 cm1. HRMS (CI) m/z: calcd. for C20H17O5 [MþH]þ 337.1076, found 337.1068. 4.1.19. 2,5-bis(4-methoxyphenyl)-3,4-dimethylfuran (8c) Compound 8c was prepared in the same manner as compound 8a using diketone 7c (1.65 g, 5.06 mmol). The product 8c (1.49 g, 96%) was obtained as a yellow oil. 1H NMR (200 MHz, CDCl3) d 7.61 (4H, d, J ¼ 8.8 Hz), 6.96 (4H, d, J ¼ 8.8 Hz), 3.85 (6H, s), 2.20 (6H, s). 13 C NMR (75 MHz, CDCl3) d 158.3, 146.8, 126.9, 125.0, 117.4, 114.0, 55.3, 9.9. IR (ATR) 2930, 1507, 1247, 1177, 836 cm1. HRMS (EI) m/z: calcd. for C20H20O3 [M]þ 308.1412, found 308.1435. 4.1.20. 3,4-Dimethyl-2,5-di-p-tolylfuran (8d) Compound 8d was prepared in the same manner as compound 8a using diketone 7d (460 mg, 1.56 mmol). The product 8d (374 mg, 87%) was obtained as a yellow oil. 1H NMR (200 MHz, CDCl3) d 7.58 (4H, d, J ¼ 8.2 Hz), 7.23 (4H, d, J ¼ 8.2 Hz), 2.38 (6H, s), 2.22 (6H, s). 13 C NMR (75 MHz, CDCl3) d 147.2, 136.3, 129.2, 145.5, 125.5, 118.4, 21.3, 9.9. IR (ATR) 2918, 1510, 1108, 822 cm1. HRMS (EI) m/z: calcd. for C20H20O [M]þ 276.1514, found 276.1509. 4.1.21. 2,5-bis(4-benzyloxy-3-methoxyphenyl)-3,4-dimethylfuran (8e) Compound 8e was prepared in the same manner as compound 8a using diketone 7e (31.7 mg, 58.9 mmol). The product 8e (15.4 mg, 50%) was obtained as a yellow oil. 1H NMR (300 MHz, CDCl3) d 7.46e7.30 (10H, m), 7.22 (2H, d, J ¼ 1.8 Hz), 7.13 (2H, dd, J ¼ 8.1, 1.8 Hz), 6.92 (2H, d, J ¼ 8.1 Hz), 5.19 (4H, s), 3.95 (6H, s), 2.19 (6H, s). 13 C NMR (50 MHz, CDCl3) d 149.7, 147.1, 146.9, 137.1, 128.6, 127.9, 127.3, 125.6, 118.3, 117.9, 114.1, 109.7, 71.1, 56.1, 9.9. IR (ATR) 1509, 1249, 1024 cm1. HRMS (EI) m/z: calcd. for C34H32O5 [M]þ 520.2250, found 520.2257. 4.1.22. 4,4'-(3,4-dimethylfuran-2,5-diyl)diphenol (8f) A solution of 8c (560 mg, 1.82 mmol) in DCM was added BBr3 (4.54 mL, 1.0 M solution in DCM) at 0 C. After being stirred for 13 h,
the reaction was quenched with saturated NaHCO3 solution was added and extracted with DCM. The organic layers were dried over MgSO4 and concentrated in vacuo. The residue was purified by column chromatography (hexane:EtOAc ¼ 2:1) to afford 8f (477 mg, 94%) as a yellow oil. 1H NMR (300 MHz, CD3OD) d 7.56 (4H, d, J ¼ 8.8 Hz), 6.89 (4H, d, J ¼ 8.8 Hz), 2.19 (6H, s). 13C NMR (75 MHz, CD3OD) d 185.2, 176.0, 155.9, 153.3, 145.9, 144.4, 37.9. IR (ATR) 3397, 1504, 830 cm1. HRMS (CI) m/z: calcd. for C18H16O3 [M]þ 280.1099, found 280.1100. 4.1.23. 4,4'-(3,4-dimethylfuran-2,5-diyl)bis(2-methoxyphenol) (8g) To a solution of 8e (5.4 mg, 10.4 mmol) in EtOH (1.90 mL) was added Pd(OH)2/C (1.8 mg). The mixture was stirred vigorously under hydrogen atmosphere at rt for 14 h. After being filtrated, the solution was concentrated in vacuo. The residue was purified by column chromatography (toluene:EtOAc ¼ 5:1) to afford 8g (1.1 mg, 33%) as a yellow oil. 1H NMR (300 MHz, CDCl3) d 7.18 (2H, d, J ¼ 2.0 Hz), 7.16 (2H, dd, J ¼ 8.7, 2.0 Hz), 6.97 (2H, d, J ¼ 8.7 Hz), 5.64 (2H, s), 3.96 (6H, s), 2.20 (6H, s). 13C NMR (50 MHz, CDCl3) d 147.0, 146.6, 144.7, 125.4, 124.6, 119.3, 114.5, 108.5, 56.0, 9.9. IR (ATR) 3428, 2935, 1510, 1257, 1118 cm1. HRMS (EI) m/z: calcd. for C20H20O5 [M]þ 340.1311, found 340.1340. 4.1.24. 5-(3,4-Dimethyltetrahydrofuran-2-yl)benzo[d][1,3]dioxole (9) To a solution of 15 (32.0 mg, 148 mmol) in EtOH (27.8 mL) was added Pd(OH)2/C (1.5 mg). The mixture was stirred vigorously under hydrogen atmosphere at rt for 17 h. After being filtrated, the solution was concentrated in vacuo. The residue was purified by column chromatography (hexane:EtOAc ¼ 11:1) to afford 9 (26.5 mg, 87%) as a colorless oil. 1H NMR (400 MHz, CDCl3) d 6.80e6.71 (3H, m), 5.93 (2H, s), 5.02 (1H, d, J ¼ 4.8 Hz), 4.06 (1H, t, J ¼ 8.0 Hz), 3.59 (1H, dd, J ¼ 10.0, 8.0 Hz), 2.68e2.58 (1H, m), 2.28e2.22 (1H, m), 1.00 (3H, d, J ¼ 7.0 Hz), 0.50 (3H, d, J ¼ 7.3 Hz). 13 C NMR (100 MHz, CDCl3) d 147.3, 146.1, 134.7, 119.0, 107.8, 106.8, 100.8, 84.6, 72.8, 41.8, 37.8, 12.5, 8.8. IR (ATR) 2965, 1504, 1490, 1443, 1039 cm1. HRMS (EI) m/z: calcd. for C13H16O3 [M]þ 220.1099, found 220.1090. 4.1.25. 2,5-bis(benzo[d][1,3]dioxol-5-yl)tetrahydrofuran (10) To a solution of 18 (9.4 mg, 30.5 mmol) in benzene (1.00 mL) was added Pd(OH)2/C (3.6 mg). The mixture was stirred vigorously under hydrogen atmosphere at rt for 11 h. After being filtrated, the solution was concentrated in vacuo. The residue was purified by preparative TLC (hexane:ether ¼ 5:1) to afford 2a (3.1 mg, 33%) as a colorless oil. 1H NMR (300 MHz, CDCl3) d 6.94 (2H, d, J ¼ 1.2 Hz), 6.86 (2H, dd, J ¼ 7.8, 1.2 Hz), 6.78 (2H, d, J ¼ 7.8 Hz), 5.96 (4H, s), 4.94e4.91 (2H, m), 2.37e2.33 (2H, m), 1.94e1.92 (2H, m). 13C NMR (75 MHz, CDCl3) d 147.7, 146.8, 136.8, 119.4, 108.0, 106.6, 100.9, 81.1, 34.4. IR (ATR) 2924, 1489, 1444, 1249, 1038 cm1. HRMS (EI) m/z: calcd. for C18H16O5 [M]þ 312.0998, found 312.0991. 4.1.26. 5-(benzo[d][1,3]dioxol-5-yl)-5-hydroxy-3,4-dimethylfuran2(5H)-one (13) To a solution of 2,3-dimethylmaleic anhydride (11, 3.03 g, 23.8 mmol) in THF (240 mL) was added 3,4-methylenedioxy phenyl magnesium bromide (48.1 mL, 0.5 M solution in THF) at 0 C. After being stirred for 3 h, the reaction was quenched with saturated NH4Cl. The aqueous layer was extracted with EtOAc. The combined organic layers were washed with brine, dried over anhydrous MgSO4, and concentrated in vacuo. The residue was purified by recrystallization (hexane/EtOAc) to afford 13 (5.90 g, quant.) as a colorless amorphous. 1H NMR (300 MHz, CDCl3) d 6.97 (1H, dd, J ¼ 8.1, 1.8 Hz), 6.89 (1H, d, J ¼ 1.8 Hz), 6.79 (1H, d, J ¼ 8.1 Hz), 5.98 (2H, s), 1.86 (6H, s). 13C NMR (75 MHz, CDCl3) d 173.4, 159.8, 148.3,
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147.9, 130.7, 123.7, 119.6, 108.2, 106.3, 106.0, 101.4, 10.7, 8.5. IR (ATR) 3334, 1739, 1504, 1491, 1246, 1038 cm1. HRMS (EI) m/z: calcd. for C13H12O5 [M]þ 248.0685, found 248.0685. 4.1.27. 5-(benzo[d][1,3]dioxol-5-yl)-3,4-dimethylfuran-2(5H)-one (14) To a solution of 13 (3.79 g, 15.3 mmol) in TFA (153 mL) was added Et3SiH (2.95 mL, 18.4 mmol) at rt. After being stirred for 1.5 h, the solution was concentrated in vacuo. The residue was purified by column chromatography (toluene:EtOAc ¼ 9:1) to afford 14 (3.55 g, qunat.) as a colorless oil. 1H NMR (300 MHz, CDCl3) d 6.81 (1H, d, J ¼ 8.0 Hz), 6.73 (1H, dd, J ¼ 8.0, 1.8 Hz), 6.59 (1H, d, J ¼ 1.8 Hz), 5.98 (2H, s), 5.51 (1H, brs), 1.90 (3H, brs), 1.81 (3H, brs). 13C NMR (75 MHz, CDCl3) d 174.6, 159.0, 148.4, 148.2, 128.5, 123.2, 121.3, 108.4, 106.6, 101.4, 84.9, 12.2, 8.6. IR (ATR) 2904, 1747, 1489, 1444, 1089, 1036 cm1. HRMS (EI) m/z: calcd. for C13H12O4 [M]þ 232.0736, found 232.0740. 4.1.28. 5-(3,4-Dimethylfuran-2-yl)benzo[d][1,3]dioxole (15) To a solution of 14 (1.05 g, 4.53 mmol) in DCM (4.74 mL) was added Dibal-H (43.1 mL, 1.0 M solution in DCM) at 78 C. After being stirred for 19 h, the reaction was concentrated in vacuo. the reaction was quenched with saturated 2 mol/L HCl was added and extracted with DCM. The organic layers were dried over MgSO4 and concentrated in vacuo. The residue was separated by column chromatography (hexane:DCM ¼ 9:1) to afford a crude compound 15 (520 mg, 56%) as a colorless oil. Because compound 15 was instable, the spectral data were not obtained.
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Statistical analyses were performed using Dunnett's t-test. 4.3. Evaluation of regeneration of mouse optic nerve 4.3.1. Animals All animals were maintained and handled in accordance with the Guiding Principles in the Care and Use of Animals and the Suzuka University of Medical Science's guidelines for animal experiments. Male C57BL/6 mice (9 weeks old; Japan SLC, Inc., Shizuoka, Japan) were reared in clear plastic cages and kept under a 12 h lightedark cycle at 23 C. Mice were anesthetized by intraperitoneal injection of sodium pentobarbital (30e40 mg/kg body weight). The optic nerve was crushed using forceps 1 mm posterior to the eyeball. The study was designed to minimize the number of experimental animals used and to minimize their pain and suffering. 4.3.2. Quantitation of optic nerve regeneration in vivo Optic nerves samples embedded in OCT compound were cut into longitudinal sections of 14 mm thickness. Regenerating optic axons were visualized by staining with mouse anti-GAP43 antibody (1∶250, CST Japan) followed by a fluorescently labeled secondary antibody and captured by fluorescent microscopy (BZ-9000, Keyence, Osaka, Japan). Axons were counted manually in at least eight sections per conditions (six mice in each treatment group) at 200 mm away from the injury site. The numbers of regenerating axons were counted as described by Leon et al. [20]. Acknowledgments
4.1.29. 2,5-bis(benzo[d][1,3]dioxol-5-yl)furan (18) To a solution of 17 (600 mg, 4.53 mmol) and H3PO4 (996 mg, 3.69 mmol) in 1,4-dioxane (4.74 mL) was added 16 (269 mg, 1.23 mmol) and PdCl2(dppf) (102 mg, 120 mmol) at rt. After being stirred for 11 h at 100 C, the reaction mixture was filtrated through Celite and concentrated in vacuo. The residue was purified by column chromatography (hexane:EtOAc ¼ 9:1) to afford 18 (72.9 mg, 19%) as a colorless oil. 1H NMR (300 MHz, CDCl3) d 7.24 (2H, dd, J ¼ 8.1, 1.8 Hz), 7.19 (2H, d, J ¼ 1.8 Hz), 6.85 (2H, d, J ¼ 8.1 Hz), 6.56 (2H, s), 6.00 (4H, s). 13C NMR (75 MHz, CDCl3) d 152.6, 148.0, 146.9, 125.3, 117.5, 108.7, 106.1, 104.4, 101.2. IR (ATR) 1479, 1251, 1221, 1034 cm1. HRMS (EI) m/z: calcd. for C18H12O5 [M]þ 308.0685, found 308.0691. 4.2. Evaluation of neurite-outgrowth promotion 4.2.1. PC12 cells PC (Pheochromocytoma) 12 cell line was purchased from JCRB Cell Bank. 4.2.2. Quantitation of neurite-outgrowth promotion PC12 cells were cultured in a 24-well plate at a density of 8 103 cells/cm2 in DMEM þ10% HS, 5% FBS, 100 IU/mL penicillin, and 100 mg/mL streptomycin at 37 C under a humidified atmosphere of 95% air and 5% CO2 for 24 h. The culture medium was then changed to DMEM þ2% HS, 1% FBS, 100 IU/mL penicillin, and 100 mg/mL streptomycin. At the same time, 30 mM concentration of test samples with 2 ng/mL NGF were added. The experiment was repeated in three wells. After incubation with samples for 4 days, the cultures were fixed with 4% paraformaldehyde/PBS and stained with methylene blue. Cell morphology was observed under a phase-contrast microscope, and neurite length was quantified. Five images were selected randomly under a microscope for each well, and five significantly differentiated cells were selected to measure the longest neurite length extending from a cell body for each picture. At least 100 cells were calculated for each test sample.
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