2024 年 72 巻 5 号 p. 518-523
We have developed a series of 2-monoaryl-5-diarylmethylene analogs of the green fluorescent protein chromophore to study their viscosity-induced emission (VIE) properties. The analogs were synthesized by a condensation with methyl imidate and N-(diarylmethylene)glycinate. Among the analogs, the N-methylpyrrol-2-yl-substituted analog 1h induced the most remarkable VIE behavior in triglyceride and lipid bilayers probably due to the high π-electron-rich property of the pyrrole ring. The pyrrole substituent in imidazolone analogs can be expected to become a common template for introducing VIE behavior.
p-Hydroxybenzylideneimidazolone (HO-BDI) is a common chromophore structure found in many fluorescent proteins such as green fluorescent protein (GFP),1) Dronpa protein,2,3) and Kaede protein,4,5) which are frequently used in fluorescence imaging6) (Fig. 1, upper left). The benzylideneimidazolone (BDI) structure is also an attractive target for many chemists because of its unique photochemical and photophysical properties. Owing to the rapid nonradiative decay caused by intramolecular bond rotations such as a Z/E-photoisomerization and a twist of the benzylidene single bond, HO-BDI emits fluorescence when conjugated with the β-barrel tertiary structure of proteins but not in the free state.7–9) Therefore, HO-BDI can be applied to ON/OFF switchable fluorescence systems by controlling the bond rotations. To exploit this property of the BDI structure, various synthetic BDI analogs have been developed to date.10,11) In particular, extending the conjugation of BDI by installing an aromatic substituent at the 2-position of the imidazolone moiety is a well-established strategy because it leads to a bathochromic shift of fluorescence like in the red Kaede chromophore.5) In most reported cases, the conjugated substituent at the 2-position is a simple phenyl group12–14) or, more recently, 2-arylvinyl groups, which have been installed in red Kaede-type analogs15–17) (Fig. 1, upper middle). In contrast, other aromatics including heterocycles are less explored.
Our group has developed a series of BDI analogs, namely, (diarylmethylene)imidazolones (DAINs), which contain a diarylmethylene moiety instead of the benzylidene18–20) (Fig. 1, upper middle-right). In such studies, we reported a 2-biaryl-conjugated DAIN (bar-DAIN) in which the nonradiative decay caused by the intramolecular bond rotations was suppressed under viscous or aggregated conditions, resulting in both aggregation-induced emission (AIE)21) and viscosity-induced emission (VIE)22) with different color patterns.20) Notably, we found that the aromatic substituent at the 2-position of imidazolone in bar-DAIN strongly affected the VIE property. Thus, we expected that the molecular size of bar-DAIN could be reduced by attaching an appropriate monoaromatic on the 2-position of imidazolone. Such modifications might facilitate the application of DAINs as fluorescent probes by preventing steric repulsion with target molecules or suppressing aggregation. For this purpose, we designed novel 2-monoaryl-DAIN analogs 1a–1h, and 1hN (mar-DAINs 1a–1h, and 1hN) bearing various monoaromatic rings, including π-electron-rich heterocycles, at the 2-position (Fig. 1, lower). As a control, cyclohexyl analog 1′ was also prepared. Herein, we report the synthesis and VIE properties of mar-DAINs 1a–1h, and 1hN.
The syntheses of 1a–1h, 1hN, and 1′ were performed by coupling18,19) imidates 3a–3h, and 3′, which were obtained from the corresponding amides 2a–2h, and 2′ via methylation, and imino-glycinate 4p or 4n under weak acidic conditions (Chart 1). In the case of 1e, thioimidate 6e was used as the coupling partner because the O-methylation reaction resulted in quite low yield.
Reagents and conditions: (a) MeOTf, CH2Cl2, room temperature (r.t.); (b) 4p or 4n, AcOH, toluene, reflux, 11–32% from 2a–2d, 2f–2h, 2′ or 5e; (c) Lawesson’s reagent, toluene, 90 °C, 86%; (d) MeI, t-BuOK, THF, r.t.
The maximum absorption wavelengths (λmax) of 1a–1h, 1hN, and 1′ in various solvents, namely, cyclohexane (CyH), ethyl acetate (EtOAc), acetonitrile (MeCN), and methanol (MeOH) are shown in Fig. 2, Supplementary Table S1, and Supplementary Fig. S1. The introduction of the aromatic ring at the 2-position of the imidazolone moiety led to a bathochromic shift of λmax compared with cyclohexyl analog 1′, which was expected considering the extended conjugation system. Furthermore, the incorporation of an electron-donating or electron-withdrawing group, or substitution within the π-electron-rich heterocycle, resulted in a notable bathochromic shift of λmax compared to the phenyl analog 1a, with the exception of the substitution with the 3-furyl group (1e). Interestingly, the solvatochromism23) of mar-DAIN was suppressed by introducing the aromatic ring (e.g., +14 nm shift from CyH to MeOH for 1′ and +0.5 nm shift from CyH to MeOH for 1a). Compared to the solvatochromism exhibited by 1′ and 1a, that of 1h was less suppressed despite having an aromatic ring at the 2-position (+7 nm shift from CyH to MeOH). The positive effect of 1h was further extended when using acetic acid (AcOH) as a more polar solvent, (+9.5 nm shift from CyH to AcOH, Supplementary Table S1). This result indicates that the charge transfer in 1h upon excitation is larger than that of other compounds of the 1 series, which is probably because of the stronger π-electron-rich property of pyrrole. As shown in Fig. 3, this property was supported by the highest occupied molecular orbital (HOMO) and lowest unoccupied molecular orbital (LUMO) of 1h, in which charge transfer seems to occur effectively via the push–pull mechanism from pyrrole to imidazolone (green arrow).24)
Optimization and energy calculation were performed at the B3LYP/6-31G* and B3LYP/6-311 + G(2df, 2p) levels of theory, respectively, using the Spartan 18 software. The charge transfer in 1h is indicated by a green arrow.
The emission spectra of 1a–1h, 1hN, and 1′ in EtOAc as a low-viscosity solvent and in glycerol tri-n-octanoate as a high-viscosity solvent are shown in Figs. 4A and B, respectively. In EtOAc, the solutions of all compounds were almost nonemissive (Fig. 4A), whereas visible emissions were observed in the glycerol tri-n-octanoate solutions of several analogs (Fig. 4B). The introduction of aromatic rings, in particular π-electron-rich rings, seemed to increase the emission (1b, 1f–1h). Among them, 1h showed a noticeable VIE (λem = 539 nm); the fluorescence quantum yield (FQY) of 1h was approximately 0.01 (in glycerol tri-n-octanoate). Notably, the emission intensity of 1h was higher than that of the structurally larger bar-DAIN analog 720) (Fig. 4C). Considering that the methyl group in the pyrrole moiety might more strongly affect the VIE than the electron-donating effect of the pyrrole-nitrogen atom, the structurally similar methyl furan analog 1f was examined. The VIE of 1f was found to increase compared with that of 1d but was lower than that of 1h, indicating that the strongly π-electron-rich nature of pyrrole is a crucial factor for the VIE. Replacing the diphenyl moiety with a dinaphtyl moiety increased the emission intensity in glycerol tri-n-octanoate by approximately 1.4 times (1hN: λem = 538 nm). The FQY of 1hN estimated through absorbance correction was approximately 1.1 times that of 1h.25) To verify the VIE of 1h, the emission intensity was evaluated in glycerol–MeOH mixtures at different ratios (Figs. 4D, E). Notably, the emission intensity increased by approximately 72 times from 0% glycerol to 100% glycerol. In addition, the Förster–Hoffmann plot was linear (Fig. 4E), which supports the viscosity dependence of the emission.26–28)
[A] Fluorescence spectra of 1a–1h, 1hN, and 1′ in EtOAc (1.0 × 10−4 M). λex for 1a–1h, 1hN, and 1′ (nm): 386, 390, 394, 401, 386, 405, 408, 406, 423, and 357, respectively. [B] Fluorescence spectra of 1a–1h, 1hN, and 1′ in glycerol tri-n-octanoate (1.0 × 10−4 M). λex for 1a–1h, 1hN, and 1′ (nm): 391, 396, 400, 406, 388, 407, 411, 409, 425, and 358, respectively. FQY of 1h: approx. 0.01. [C] Fluorescence spectra of 1h and bar-DAIN 7 in glycerol tri-n-octanoate (1.0 × 10−4 M). [D] Fluorescence spectra of 1h in glycerol (GL)–MeOH (1.0 × 10−4 M). λex for 0–100% glycerol (nm): 410, 412, 414, 417, 419, and 420, respectively. FQY of 1h in GL: approx. 0.04. [E] Förster–Hoffmann plot of 1h in GL–MeOH.
To evaluate the applicability of 1h as a VIE-based imaging tool, the emission in lipid bilayers was evaluated using 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) liposomes. As shown in Fig. 5A, the addition of 1h to a DMPC liposome aqueous solution immediately led to a notable increase in the emission intensity, changing visually from dark to green fluorescence (λem = 539 nm). By contrast, using 1a instead of 1h did not increase the emission intensity. These results indicate that pyrrole is an effective substituent for promoting the VIE behavior. Interestingly, in the case of 1hN, the fluorescence color gradually changed from yellow (λem = 555 nm) to green (λem = 542 nm) after the addition of the DMPC liposome probably because of the environmental change from water to the lipid bilayers causing a change from AIE to VIE.
[A] Fluorescence spectra of 1a, 1h, and 1hN in DMPC liposomes (mar-DAIN: 1.0 × 10−5 M, phospholipid: 4.0 × 10−4 M). λex for 1a, 1h, and 1hN (nm): 391, 409, and 426, respectively. FQY of 1hN: approx. 0.02 (in water). [B] Fluorescence spectra of 1h in DOPC, DMPC, and DSPC liposomes (1h: 1.0 × 10−5 M, phospholipid: 4.0 × 10−4 M). λex for 1h (nm): 409.
We then examined the VIE in lipid bilayers using other phospholipids, i.e., 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), which exhibit different phase transition temperatures (Tm: −20, 23, and 55 °C for DOPC, DMPC, and DSPC, respectively) (Fig. 5B). Although 1h was incorporated in the lipid bilayers in all cases within 10 min, producing emissions, the intensity seemed to depend on Tm. Thus, the DMPC bilayers, which exhibit a Tm close to room temperature, afforded the highest intensity probably because it showed the appropriate viscosity for incorporating 1h and suppressing its molecular motion. In the case of DOPC, the molecular motion of 1h would not be sufficiently suppressed due to its low Tm (i.e., soft bilayers), whereas the incorporation of 1h would be difficult in the DSPC bilayers due to its high Tm (i.e., rigid bilayers).
The reason for the highest VIE for the pyrrole analog 1h is unclear since a comparison of the LUMOs of 1a and 1h around the C=C double bond (Fig. 3) shows that the pyrrole ring is unlikely to exert a direct effect on excited-state C=C double bond rotation (ESDBR), which is known as a major nonradiative decay pathway in BDI analogs.29) Currently, we presume that the pyrrole–imidazolone single bond possesses a partial double bond character and will not easily rotate under viscous conditions, possibly indirectly suppressing the nonradiative decay pathway via (for example) suppression of a twisted intramolecular charge transfer mechanism30) and/or suppression of volume-conserving conformational changes during ESDBR.31) As shown in Fig. 3, the π-electron-rich property of pyrrole leads to a large amount of charge transfer at the excited state, endowing the pyrrole–imidazolone single bond with a certain double-bond character (Fig. 6).
We synthesized a series of 2-monoaryl-DAIN analogs to study their VIE properties. The N-methylpyrrol-2-yl-substituted analog induced the most remarkable VIE behavior in triglyceride and lipid bilayers probably due to its high π-electron-rich property. Therefore, the pyrrole substituent in imidazolone analogs such as DAIN and BDI can be expected to become a common template for introducing VIE behavior, thus leading to the development of new fluorescence imaging tools.
1H- and 13C-NMR spectra were recorded on a JNM-ECZ400S spectrometer at 400 and 100 MHz, respectively, and the chemical shifts are reported in parts per million (ppm) relative to tetramethylsilane or the solvent signal. Mass spectra were recorded using a JEOL JMS-T100LP mass spectrometer in positive electrospray ionization (ESI) mode. UV–vis spectra were obtained on a JASCO V-730BIO spectrophotometer. Fluorescence spectra were recorded using a JASCO FP-8300 spectrofluorometer, and FQYs were measured using an integrating sphere (JASCO ILF-835) and a quantum yield calculation program (JASCO Spectra Manager series). Melting points (Mp) values were measured using a Yanaco melting-point apparatus. The obtained Mp values were uncorrected. Column chromatography was performed using Fuji Silysia PSQ 100 silica gel. Amides 2a–2h, and 2′ were commercially available or synthesized from the corresponding acid chloride or carboxylic acid according to literature procedures.32,33) DAIN 1a was synthesized following our previously reported method.19)
General Procedures for the Synthesis of DAIN Compounds 1b–1h, 1hN, and 1′: Synthesis of 5-(Diphenylmethylene)-2-(4-methoxyphenyl)-3-methyl-3,5-dihydro-4H-imidazol-4-one (1b)Methyl trifluoromethanesulfonate (MeOTf) (397 µL, 3.63 mmol) was added to a stirred solution of amide 2b (400 mg, 2.42 mmol) in CH2Cl2 (14 mL) at room temperature. The mixture was stirred at room temperature for 1 d. Triethylamine (Et3N) (1.18 mL, 8.48 mmol) was added at ice-water temperature, and the mixture was stirred for 30 min at room temperature. The solvent was removed under reduced pressure, and the residue was passed through a short pad of silica gel (hexane/EtOAc = 4/1 containing 3% Et3N) to obtain crude methyl imidate 3b (254 mg), which was subjected to the next reaction without further purification because of its instability to moisture. A mixture of crude 3b (200 mg, approx. 1.12 mmol), N-(diphenylmethylene)glycine ethyl ester (4p; 1.49 g, 5.58 mmol), and AcOH (128 µL, 2.23 mmol) in toluene (5.6 mL) was stirred for 1 d under reflux. The mixture was diluted with EtOAc and washed with 5% aqueous HCl. The acidic aqueous layer was re-extracted with EtOAc after neutralization with a saturated solution of NaHCO3. The combined organic layers were washed with brine, dried over Na2SO4, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane/EtOAc = 3/1) and recrystallized from CH2Cl2/hexane to afford 1b as a yellow solid (225 mg, 32% over two steps). Mp: 189–190 °C. 1H-NMR (400 MHz, CDCl3) δ: 7.81 (2H, d, J = 8.7 Hz), 7.70–7.67 (2H, m), 7.45–7.42 (3H, m), 7.38–7.34 (5H, m), 7.01 (2H, d, J = 8.7 Hz), 3.88 (3H, s), 3.29 (3H, s). 13C-NMR (100 MHz, CDCl3) δ: 169.8, 162.0, 160.5, 146.2, 139.2, 138.2, 136.5, 132.7, 130.4, 130.2, 129.3, 128.8, 128.0, 127.7, 121.7, 114.2, 55.4, 29.0. HR-MS (ESI) Calcd for C24H20N2O2Na [M + Na]+: 391.1417. Found 391.1410.
2-(4-Acetylphenyl)-5-(diphenylmethylene)-3-methyl-3,5-dihydro-4H-imidazol-4-one (1c)Amide 2c (300 mg, 1.69 mmol), MeOTf (278 µL, 2.54 mmol), Et3N (888 µL, 6.37 mmol), and CH2Cl2 (11 mL) were subjected to the methylation conditions described for the synthesis of 3b to afford crude imidate 3c (158 mg) after passing the residue through a short pad of silica gel using hexane/EtOAc (4/1, containing 3% Et3N) as the eluent. Then, 1c (147 mg, 23% over two steps) was obtained as a yellow solid using 3c (158 mg, 0.83 mmol), 4p (1.11 g, 4.14 mmol), AcOH (95 µL, 1.65 mmol), toluene (4.4 mL), hexane/EtOAc (2/1) as the eluent for the silica gel column, and CH2Cl2/hexane as the solvent for recrystallization as described for 1b. Mp: 184–185.5 °C. 1H-NMR (400 MHz, CDCl3) δ: 8.09 (2H, d, J = 8.7 Hz), 7.93 (2H, d, J = 8.7 Hz), 7.67–7.65 (2H, m), 7.47–7.45 (3H, m), 7.39–7.36 (5H, m), 3.29 (3H, s), 2.66 (3H, s). 13C-NMR (100 MHz, CDCl3) δ: 197.3, 169.4, 159.7, 148.9, 139.0, 138.7, 137.8, 136.3, 133.6, 132.9, 130.5, 129.8, 129.3, 128.8, 128.6, 128.0, 127.8, 28.9, 26.8. HR-MS (ESI) Calcd for C25H20N2O2Na [M + Na]+: 403.1417. Found 403.1410.
5-(Diphenylmethylene)-2-(furan-2-yl)-3-methyl-3,5-dihydro-4H-imidazol-4-one (1d)Similarly, amide 2d (400 mg, 3.20 mmol), MeOTf (525 µL, 4.80 mmol), Et3N (1.57 mL, 11.2 mmol), CH2Cl2 (18 mL), and hexane/EtOAc (5/1, containing 3% Et3N) as the eluent for the silica gel pad were used for the methylation to obtain crude imidate 3d (210 mg). Subsequently, 3d (210 mg, 1.51 mmol), 4p (2.02 g, 7.56 mmol), AcOH (173 µL, 3.02 mmol), toluene (7.0 mL), hexane/EtOAc (3/1) as the eluent for the silica gel column, and EtOAc/hexane as the solvent for recrystallization were used for the synthesis of 1d, which was obtained as a yellow solid (128 mg, 12% over two steps). Mp: 187–188 °C. 1H-NMR (400 MHz, CDCl3) δ: 7.67–7.64 (3H, m), 7.45–7.42 (3H, m), 7.37–7.32 (5H, m), 7.30 (1H, d, J = 3.7 Hz), 6.62 (1H, dd, J = 3.7, 1.8 Hz), 3.44 (3H, s). 13C-NMR (100 MHz, CDCl3) δ: 168.8, 151.2, 146.8, 145.7, 144.6, 139.2, 138.1, 136.3, 132.8, 130.4, 129.5, 128.9, 128.0, 127.7, 115.7, 112.4, 28.3. HR-MS (ESI) Calcd for C21H16N2O2Na [M + Na]+: 351.1104. Found 351.1097.
5-(Diphenylmethylene)-3-methyl-2-(3-methylfuran-2-yl)-3,5-dihydro-4H-imidazol-4-one (1f)Amide 2f (100 mg, 0.72 mmol), MeOTf (236 µL, 2.16 mmol), Et3N (450 µL, 3.23 mmol), CH2Cl2 (3.1 mL), and hexane/EtOAc (10/1, containing 3% Et3N) as the eluent for the silica gel pad were used for the methylation to afford crude imidate 3f (40 mg), which required 2 d. Then, 3f (40 mg, 0.26 mmol), 4p (349 mg, 1.31 mmol), AcOH (30 µL, 0.52 mmol), toluene (1.4 mL), and hexane/EtOAc (5/1) as the eluent for the silica gel column were used for the synthesis of 1f, which was obtained as a yellow solid (28 mg, 11% over two steps). In this case, the re-extraction of the acidic aqueous layer and recrystallization processes were skipped. Mp: 172–173 °C. 1H-NMR (400 MHz, CDCl3) δ: 7.72–7.70 (2H, m), 7.52 (1H, d, J = 1.8 Hz), 7.44–7.43 (3H, m), 7.36–7.33 (5H, m), 6.48 (1H, d, J = 1.8 Hz), 3.43 (3H, s), 2.52 (3H, s). 13C-NMR (100 MHz, CDCl3) δ: 168.9, 152.1, 145.1, 144.1, 140.2, 139.3, 138.1, 136.6, 132.7, 130.4, 129.5, 129.2, 128.7, 128.0, 127.5, 115.8, 28.6, 12.1. HR-MS (ESI) Calcd for C22H18N2O2Na [M + Na]+: 365.1261. Found 365.1253.
5-(Diphenylmethylene)-3-methyl-2-(thiophen-2-yl)-3,5-dihydro-4H-imidazol-4-one (1g)Amide 2g (400 mg, 2.83 mmol), MeOTf (465 µL, 4.25 mmol), Et3N (1.39 mL, 9.97 mmol), CH2Cl2 (16 mL), and hexane/EtOAc (5/1, containing 3% Et3N) as the eluent for the silica gel pad were used for the methylation to obtain crude imidate 3g (196 mg). Subsequently, 3g (139 mg, 0.90 mmol), 4p (1.20 g, 4.48 mmol), AcOH (103 µL, 1.79 mmol), toluene (4.5 mL), hexane/EtOAc (3/1) as the eluent for the silica gel column, and EtOAc/hexane as the solvent for recrystallization were used to obtain 1g as a yellow solid (88 mg, 13% over two steps). Mp: 164–165 °C. 1H-NMR (400 MHz, CDCl3) δ: 7.73 (1H, dd, J = 3.7, 0.9 Hz), 7.71–7.69 (2H, m), 7.58 (1H, dd, J = 5.0, 0.9 Hz), 7.45–7.42 (3H, m), 7.38–7.32 (5H, m), 7.19 (1H, dd, J = 5.0, 3.7 Hz), 3.44 (3H, s). 13C-NMR (100 MHz, CDCl3) δ: 169.1, 154.9, 146.3, 139.1, 138.1, 136.2, 132.9, 132.7, 130.8, 130.4, 129.6, 129.5, 128.9, 128.2, 128.0, 127.7, 28.4. HR-MS (ESI) Calcd for C21H16N2OSNa [M + Na]+: 367.0876. Found 367.0871.
5-(Diphenylmethylene)-3-methyl-2-(1-methyl-1H-pyrrol-2-yl)-3,5-dihydro-4H-imidazol-4-one (1h)Amide 2h (100 mg, 0.72 mmol), MeOTf (119 µL, 1.09 mmol), Et3N (303 µL, 2.17 mmol), CH2Cl2 (3.5 mL), and hexane/EtOAc (4/1, containing 3% Et3N) as the eluent for the silica gel pad were used for the methylation to obtain crude imidate 3h (53 mg). Subsequently, 3h (53 mg, 0.35 mmol), 4p (511 mg, 1.91 mmol), AcOH (44 µL, 0.77 mmol), toluene (2.0 mL), hexane/EtOAc (4/1) as the eluent for the silica gel column, and EtOAc/hexane as the solvent for recrystallization were used to obtain 1h as a yellow solid (51 mg, 21% over two steps). The re-extraction of the acidic aqueous layer was not necessary in this case. Mp: 194–195 °C. 1H-NMR (400 MHz, CDCl3) δ: 7.66–7.64 (2H, m), 7.44–7.41 (3H, m), 7.36–7.33 (5H, m), 6.89 (1H, t like, J = 2.1 Hz), 6.80 (1H, dd, J = 4.1, 1.4 Hz), 6.25 (1H, dd, J = 4.1, 2.7 Hz), 4.05 (3H, s), 3.36 (3H, s). 13C-NMR (100 MHz, CDCl3) δ: 169.0, 153.6, 144.2, 139.6, 138.2, 136.3, 132.3, 130.5, 129.5, 129.0, 128.7, 128.0, 127.5, 121.0, 115.6, 108.7, 37.9, 29.0. HR-MS (ESI) Calcd for C22H19N3ONa [M + Na]+: 364.1420. Found 364.1414.
5-(Di(naphthalen-2-yl)methylene)-3-methyl-2-(1-methyl-1H-pyrrol-2-yl)-3,5-dihydro-4H-imidazol-4-one (1hN)Amide 2h (300 mg, 2.17 mmol), MeOTf (357 µL, 3.26 mmol), Et3N (909 µL, 6.52 mmol), CH2Cl2 (11 mL), and hexane/EtOAc (4/1, containing 3% Et3N) as the eluent for the silica gel pad were used for the methylation to obtain crude imidate 3h (139 mg). Subsequently, 3h (52 mg, 0.34 mmol), 4n (691 mg, 1.88 mmol), AcOH (86 µL, 1.50 mmol), toluene (2.0 mL), hexane/CH2Cl2 (1/4) as the eluent for the silica gel column, and CH2Cl2/hexane as the solvent for recrystallization were used to obtain 1hN as an orange solid (90 mg, 25% over two steps). The re-extraction of the acidic aqueous layer was not necessary in this case. Mp: 209–210 °C. 1H-NMR (400 MHz, CDCl3) δ: 8.10 (1H, s), 7.92–7.76 (7H, m), 7.71 (1H, d, J = 8.2 Hz), 7.55–7.44 (5H, m), 6.89 (1H, t like, J = 1.8 Hz), 6.83 (1H, dd, J = 4.0, 1.6 Hz), 6.26 (1H, dd, J = 4.0, 2.7 Hz), 4.09 (3H, s), 3.38 (3H, s). 13C-NMR (100 MHz, CDCl3) δ: 169.0, 153.6, 144.1, 137.5, 136.9, 135.8, 133.5, 133.4, 133.2, 133.0, 132.7, 130.4, 129.7, 129.5, 128.7, 128.6, 128.5, 127.8, 127.5, 127.4, 127.0, 126.7, 126.5, 126.1, 126.0, 121.0, 115.7, 108.7, 38.0, 29.1. HR-MS (ESI) Calcd for C30H23N3ONa [M + Na]+: 464.1733. Found 464.1728.
2-Cyclohexyl-5-(diphenylmethylene)-3-methyl-3,5-dihydro-4H-imidazol-4-one (1′)Amide 2′ (500 mg, 3.54 mmol), MeOTf (581 µL, 5.31 mmol), Et3N (1.48 mL, 10.6 mmol), CH2Cl2 (18 mL), and hexane/EtOAc (2/1, containing 3% Et3N) as the eluent for the silica gel pad were used for the methylation to afford crude imidate 3′ (312 mg). Using 3′ (200 mg, 1.29 mmol), 4p (1.72 g, 6.44 mmol), AcOH (158 µL, 2.76 mmol), toluene (6.4 mL), hexane/EtOAc (4/1) as the eluent for the silica gel column, and cyclohexane as the solvent for recrystallization, 1′ was obtained as a yellow solid (159 mg, 20% over two steps). Mp: 171.5–173 °C. 1H-NMR (400 MHz, CDCl3) δ: 7.69–7.67 (2H, m), 7.42–7.41 (3H, m), 7.34–7.27 (5H, m), 3.09 (3H, s), 2.55–2.49 (1H, m), 1.97–1.88 (4H, m), 1.77–1.63 (3H, m), 1.43–1.30 (3H, m). 13C-NMR (100 MHz, CDCl3) δ: 169.5, 166.9, 144.9, 139.0, 138.2, 136.2, 132.9, 130.2, 129.2, 128.5, 128.0, 127.6, 37.3, 29.6, 26.3, 25.8, 25.7. HR-MS (ESI) Calcd for C23H25N2O [M + H]+: 345.1961. Found 345.1972.
Alternative Synthesis of 5-(Diphenylmethylene)-2-(furan-3-yl)-3-methyl-3,5-dihydro-4H-imidazol-4-one (1e)The Lawesson’s reagent (292 mg, 0.72 mmol) was added to a stirred solution of amide 2e (129 mg, 1.03 mmol) in toluene (10 mL) at room temperature, and the resulting mixture was stirred at 90 °C for 3 h. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography (hexane/EtOAc = 4/1) to afford 5e as a white solid (125 mg, 86%). Potassium tert-butoxide (104 mg, 0.93 mmol) was added to a stirred solution of thioamide 5e (100 mg, 0.71 mmol) in THF (7.0 mL) at ice-water temperature. The mixture was stirred at the same temperature for 5 min. Iodomethane (52 µL, 0.84 mmol) was added to the mixture at ice-water temperature, and the mixture was stirred for 1 h at room temperature. After diluting the mixture with EtOAc, the organic layer was washed with water and brine, dried over Na2SO4, and concentrated under reduced pressure to afford 6e (75 mg), which was subjected to the next reaction without further purification. Specifically, a mixture of crude 6e (75 mg, approx. 0.48 mmol), 4p (650 mg, 2.43 mmol), and AcOH (55 µL, 0.96 mmol) in toluene (2.0 mL) was stirred for 1 d under reflux. The mixture was diluted with EtOAc and washed with 5% aqueous HCl, water, a saturated solution of NaHCO3, and brine. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by silica gel column chromatography (hexane/EtOAc = 3/1) to afford 1e as a yellow solid (27 mg, 12% over two steps). 5e: 1H-NMR (400 MHz, CDCl3) δ: 8.04 (1H, t-like s), 7.44–7.55 (1H, br s), 7.42 (1H, t, J = 1.7 Hz), 6.65 (1H, dd, J = 1.7, 0.9 Hz), 3.31 (3H, d, J = 4.6 Hz). 13C-NMR (100 MHz, CDCl3) δ: 190.5, 144.6, 144.0, 129.2, 107.1, 32.6. HR-MS (ESI) Calcd for C6H8NOS [M + H]+: 142.0321. Found 142.0292. 1e: Mp: 207.5–208.5 °C. 1H-NMR (400 MHz, CDCl3) δ: 8.04 (1H, t-like s), 7.68–7.66 (2H, m), 7.54 (1H, t, J = 1.6 Hz), 7.44–7.42 (3H, m), 7.37–7.31 (5H, m), 6.99 (1H, d, J = 1.4 Hz), 3.32 (3H, s). 13C-NMR (100 MHz, CDCl3) δ: 169.0, 154.5, 146.5, 143.9, 143.6, 139.1, 138.0, 136.3, 132.8, 130.3, 129.4, 128.9, 128.0, 127.7, 117.2, 109.9, 27.8. HR-MS (ESI) Calcd for C21H16N2O2Na [M + Na]+: 351.1104. Found 351.1088.
Evaluation of the Photophysical PropertiesThe fluorescence spectra were measured under excitation at λmax. The spectra recorded in glycerol tri-n-octanoate were corrected by subtracting the background emission from the triglycerides. The liposome solutions were prepared using a commonly used method, i.e., thin-film hydration and sonication method.34,35) The fluorescence spectra in the lipid bilayers were measured after adding a dimethylsulfoxide (DMSO) solution of mar-DAIN to the liposome solution (final concentration, 1a, 1h, and 1hN: 1.0 × 10−5 M, phospholipid: 4.0 × 10−4 M in water containing 0.1% DMSO).
This work was supported by JSPS KAKENHI Grant Number: 23K06040.
The authors declare no conflict of interest.
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