A Practical Synthesis of Near-Infrared Benzannulated Xanthenoid Dyes

Jin Li and Ruwei Wei contributed equally.

Abstract

Near-infrared dyes are sought after for their potential in biomedical applications. Benzoxanthene dyes with a non-oxygen bridging atom are expected to exhibit longer absorption and emission wavelengths than their oxygen-containing counterparts, yet their synthesis remains unaddressed. Herein, we report the first syntheses of non-oxygen-bridged benzoxanthenes starting from a 7-bromo-2-aminonaphthalene derivative, and discuss their photophysical properties as well as their potential for in vivo imaging.

The development of long-wavelength dyes has been propelled by serendipity, color complementarity, and high-technology applications.[1] [2] [3] [4] Notable examples include IR photography in the 1930s, optoelectronics in the 1980s, and information storage media at the turn of the century. More recently, the deep tissue-penetrating ability of long-wavelength light, compared to that of visible light, has attracted interest from the biomedical community.[5–7] Dyes absorbing and emitting beyond ca. 650 nm, the cut-off point of biological background absorption, present valuable opportunities for practical applications. Hence, the development of dyes that are spectrally active beyond 650 nm is an important synthetic task.

The classes of long-wavelength dyes are rather limited.[8] [9] [10] [11] Cyanine dyes are archetypal long-wavelength dyes, and their absorption maxima can be fine-tuned over a large spectral range depending on the nature of the electron push–pull headgroups and the length of the conjugated polymethine backbone.[12] A problem with cyanine-type dyes is the structural freedom of the polymethine chain, accounting for their poor fluorescence brightness and poor resistance toward photobleaching.[13] Long-wavelength dyes with a rigid backbone are therefore sought-after. Efforts to develop long-wavelength dyes via judicious molecular engineering of other classic visible fluorochromes are ongoing and these endeavors have proved fruitful.[14] [15] [16] [17] [18]

Extensive efforts have been devoted toward the development of rigid long-wavelength dyes based on the BODIPY scaffold.[19] In comparison, benzannulation of the xanthene scaffold has received little attention. Xanthene is a rigid and superior fluorophore. The absorption maximum of xanthene dyes has been red-shifted by a number of approaches, e.g., the use of headgroups based on julolidine[20] or dihydroquinoline,[21] and substitution of the meso-methine carbon with an electron-withdrawing trifluoromethyl[22] or cyano[23] group. Extension of the conjugation between the push–pull headgroups is also a robust method to modulate the absorption maximum. Lee et al. first synthesized the [c]-type bisbenzannulated fluorescein in 1989 by acid-promoted fusing of 1,6-naphthadiol with phthalic anhydride at high temperature.[23] In 2005, Strongin harnessed the ortho-lithiation ability of a methoxy group, successfully activated the less nucleophilic sites for nucleophilic attack, and allowed for the preparation of a number of previously unattainable benzannulated xanthene scaffolds.[24] From 1,6-dimethoxynaphthalene, 1,8-dimethoxynaphthalene and 2,7-dimethoxynaphthalene, Strongin successfully obtained another five possible [a]-, [b]- and [c]-type monobenzannulated xanthene dyes.[25] [26] This chemistry was adopted by the groups of Tsubaki, Yamada and Ahn to further prepare a number of interesting benzocoumarins and bisbenzoxanthene dyes.[27] Another line of research has exemplified that replacement of the central bridging oxygen atom of xanthene dyes with a less electron-donating atom, e.g., C, Si, P, S or Se, is a robust approach to develop longer-wavelength-absorbing/emitting xanthenoid dyes. In fact, the first carbo-rhodamine was synthesized in 1963 by Barker (Figure [1]).[28] Further efforts in this research direction were inspired by the seminal work of Xiao, Qian and co-workers,[29] who in 2008 prepared the first silicon-bridged rhodamine dyes, which have been utilized extensively for demanding microscopic applications.[18] [30] In the following decade, xanthenoids with P, S, Se, Pb, Sn and Bi bridging groups were reported.[31–43] Such modifications led to significant spectral red-shifting and these compounds therefore became viable additions to the toolboxes of synthetic dye chemists. We have successfully prepared bis-/tetra-benzannulated xanthenoid dyes, e.g., EC5[33,44,45], ESi5[34], and EC7[46]. However, to date, the synthesis of mono-benzannulated xanthenoid dyes with a non-oxygen bridging atom remains an unaddressed challenge (Figure [1]).[23]

Herein, we report the synthesis of three monobenzannulated xanthenoid dyes possessing a carbon-, silicon- or phosphorous-based bridging group, respectively, i.e., EC4, ESi4, and EP4. We have also evaluated their spectral properties in various solvents as well as their photo- and chemostabilities. In addition, we showcase their potential applications with proof-of-concept fluorescence bioimaging studies in cells and in live mice.[44]

The synthesis of benzannulated dyes is often limited by the availability of appropriately substituted naphthalene derivatives. Here, 7-bromo-2-naphthol (1) was selected as a suitable starting material for preparing the scaffolds of EC4, ESi4 and EP4. Compound 1 is commercially available in large quantities at an affordable price. Since the scaffolds of EC4, ESi4 and EP4 bear an amino group as its push–pull headgroup, the hydroxy group of 1 should be converted into an amino group. Therefore, it could be triflated and then converted into an amino group via Buckwald–Hartwig amination. The advantage of this method is the convenient installation of easily substituted amino groups. However, we chose the Bucherer reaction to convert the hydroxy group of 1 into an amino group, since it did not require the use of an expensive transition-metal complex for catalysis. A downside was that the reaction had to be run in a pressure vessel with H₂O heated up to 145 °C. Under these conditions, a significant amount of the naphthol existed in its ketone form, which condensed with ammonia and then regained its aromatic scaffold via epimerization. A high yield (94%) of 2 was obtained and the product was collected by suction filtration and could be used for further transformation without additional purification (Scheme [1]). We initially dimethylated the NH₂ group of 2 with MeI to afford 7-bromo-N,N-dimethylnaphthalene-2-amine in a high yield. However, its subsequent Vilsmeier–Haack formylation did not occur at the desired C-6 position, but at C-1. For this reason, we attempted to prepare the julolidine version of 2 by reaction with 1-bromo-3-chloropropane. To our surprise, compound 3 was obtained in a very high yield of 81%. The chloropropyl group of 3 is compatible with organolithium chemistry and therefore we utilized 3 as the key intermediate for synthesis of the EC4/ESi4/EP4 dyes. The Vilsmeier–Haack formylation of 3 yielded aldehyde 4 [45] regioselectively, with a modest yield of 39%. Attempts to optimize the yield of this step by applying alternative formylation conditions were not successful. Nucleophilic attack of the formyl group of 4 with a 2-methylphenyl Grignard reagent yielded the carbinol 5 in a near quantitative yield.[46] Next, a mixture of 5 and 3-bromojulolidine (6) was heated at 70 °C in AcOH containing a trace of H₂SO₄; this reaction proceeded smoothly to give dibromo-substituted triarylmethane 7 in 73% yield.[47] In the presence of s-BuLi (2.2 equiv.), the two bromo atoms of 7 were exchanged to generate the corresponding bis-lithium reagent, the solution of which was subsequently transferred (via syringe) into a THF solution of methyl 2-phenoxybenzoate. Following work-up, the crude CH₂Cl₂ extract of the carbinol intermediate was acidified with MeSO₃H and heated at reflux for 30 minutes to effect closure of the spiro ring (Scheme [1]). Finally, after work-up and isolation, the crude intermediate 8 was oxidized with chloranil at room temperature to give the desired product EC4 in an overall yield of 25% from 7.[48]

Similarly, the products ESi4 [49] and EP4 [50] were prepared using dichlorodimethylsilane and dichlorophenylphosphine, respectively, in reactions with the bis-lithiated species derived from 7, followed by a chloranil-mediated oxidation of the triarylmethane intermediates (Scheme [1] and Figure S1 in the Supporting Information). The UV-Vis absorption and emission spectra of EC4, ESi4 and EP4 in CH₂Cl₂ along with their photophysical properties in different solvents are shown in Figure [2].

Next, the photo- and chemostabilities of EC4 were evaluated. Thus, solutions of EC4 and indocyanine green (ICG) in DMSO were irradiated with a 75 W halogen lamp and their absorption spectra were acquired frequently. Over a period of 60 minutes, the absorbance of EC4 decreased by 10.4% while that of ICG decreased by 62.3% (Figure [3]). The central methine carbon of xanthene dyes is electrophilic, and biological thiols have been reported to attack the methine of carbo-rhodamine. Ma et al. have also reported that ONOO^– can attack the methine of xanthene. Therefore, we tested the reactivity of EC4 toward various thiols and oxidative species. We found that biorelevantly high concentrations of three thiols, i.e., cysteine up to 2 mM, glutathione (GSH) up to 10 mM, and H₂S up to 2 mM, did not induce any spectral changes for EC4 (10 μM) in CH₃CN/H₂O (1:1, v/v). As for reactive species, H₂O₂ was totally unreactive toward EC4. However, ClO^– (up to 50 μM) and ONOO^– (up to 200 μM) induced a decrease of the solution absorbance of EC4 by 19% and 31%, respectively. This suggested that EC4 should be used with caution under oxidative-stress-impacted biological substrates (Figure [3]). The photo- and chemostabilities of ESi4 and EP4 were also tested. Their photostabilities were found to be equally high, similarly to that of EC4 (see Figures S8–S11 in in the Supporting Information). However, their chemostabilities toward nucleophiles, in particular ONOO^–, were inferior to those of EC4, presumably due to the electron-withdrawing ability of the Si/P-based bridging groups.

We also studied the photosensitization ability of EC4. When a solution of 1,3-diphenylisobenzofuran (DPBF) (a common indicator for oxidative species) was irradiated in the absence of EC4 at 418 nm for 10 minutes, a decrease of the solution absorption by 24.1% was observed (Figure [4]). In the presence of EC4, the decrease was 65.5%, suggesting that an oxidative species was generated upon photo-irradiation of EC4.

An important field of application for deep-red fluorophores is cell-based bioimaging. Currently, there are few robust dyes for this spectral region. For this reason, commercial fluorescence microscopes do not typically offer a matching laser line for excitation. Based on its higher stability, EC4 was selected for proof-of-concept cell imaging studies (Figure [5]). Therefore, off-peak excitation with a 647 nm laser of a confocal microscope was used. The emission beyond 663 nm was collected through a 663 nm long-pass filter. The emission beyond 800 nm could not be collected due to the diminishing detection quantum efficiency of the applied photomultiplier tube (PMT). Initially, EC4 exhibited minimal cytotoxicity toward HeLa cells up to 10 μM upon incubation for 24 hours. Next, HeLa cells were incubated with EC4 (5 μM) and MitoTracker (100 nM) for 15 minutes, and then rinsed and imaged. The images of the green channel for MitoTracker and the red channel for EC4 exhibited high degrees of colocalization (P = 0.94). This experiment showcased the potential of EC4 for cell imaging, notwithstanding the fact that the experiment setup could be further optimized toward the spectral properties of EC4.

We also tested the potentials of the prepared compounds for in vivo imaging. ESi4 was selected as a representative example for this experiment (Figure [6]). We injected ESi4 (0.5 mg/mL, PBS buffer, pH = 7.4) into BALB/c mice through the tail vein for in vivo imaging. Illumination was accomplished with a 755 nm laser (140 mW/cm²) and imaging utilized 900 nm or 1000 nm long-pass filters. Due to the high fluorescence intensity of ESi4, a short exposure time (ET) of 10 ms was sufficient. The fluorescent signals mainly originated from the liver and the blood vessels. Gauss fitting of mouse abdominal vascular signals yielded higher signal-to-noise ratios under the 1000 nm long-pass filter compared to the 900 nm long-pass filter (Figures [6]C and 6D). This experiment showcases the potential of ESi4 for in vivo imaging.

In conclusion, we have accomplished the syntheses of EC4, ESi4 and EP4, which have excellent photostability and chemical stability. To demonstrate the application potential of these benzannulated xanthenoid dyes in vivo and in vitro, HeLa cells were imaged utilizing EC4 and BALB/c mice were imaged in vivo by employing ESi4. Favorable imaging results were obtained in each case, indicating the potential of these dyes for ex vivo applications.

Conflict of Interest

The authors declare no conflict of interest.

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• 47 Compound 4 DMF (1.64 mL, 21 mmol, 1.2 equiv.) was mixed with 1,2-dichloroethane (10 mL) and cooled to 0 °C before dropwise addition of POCl₃ (1.98 mL, 21 mmol, 1.2 equiv.) and stirring for 30 min. The resulting mixture was added to a solution of compound 3 (3.5 g, 10 mmol, 1 equiv.) in anhydrous CH₂Cl₂ (10 mL) at 75 °C , and the obtained mixture was stirred for 3 h. The reaction was allowed to cool to the R.T. and quenched by adding saturated Na₂CO₃ solution. The aqueous layer was extracted with CH₂Cl₂ (3 × 20 mL), and the combined organic layers were dried over Na₂SO₄, filtered and concentrated in vacuo. The crude extract was purified by column chromatography (EtOAc/PE = 1:50 to 1:25, v/v) to give 4 (2.2 g, 39%) as a yellow solid. Mp 106.9–108.0 °C. ¹H NMR (400 MHz, CDCl₃): δ = 10.35 (s, 1 H), 8.24 (s, 1 H), 7.89 (s, 1 H), 7.66 (d, J = 9.2 Hz, 1 H), 7.11 (d, J = 9.2 Hz, 1 H), 3.69–3.56 (m, 4 H), 3.47–3.35 (m, 2 H), 2.98 (t, J = 6.5 Hz, 2 H), 2.11 (dd, J = 12.6, 5.2 Hz, 4 H). ¹³C NMR (101 MHz, CDCl₃): δ = 191.76, 146.02, 137.14, 132.32, 130.07, 125.98, 125.59, 124.30, 122.17, 115.65, 111.19, 49.59, 49.00, 42.50, 30.09, 23.44, 21.45. HRMS (ESI): m/z [M]⁺ calcd for C₁₇H₁₇BrClNO: 336.0255; found 336.0254.
• 48 Compound 5 Compound 4 (1 g, 2.8 mmol, 1 equiv.) was dissolved in anhydrous THF (20 mL) under an argon atmosphere and cooled to 0 °C before o-tolylmagnesium bromide (5.45 mL, 5.6 mmol, 2 equiv.) was added dropwise. The reaction was quenched after 30 min. Then the reaction was quenched with saturated NH4Cl aqueous solution (50 mL), and extracted with EtOAc (3 × 20 mL). The organic layer was combined, dried over anhydrous Na₂SO₄, filtered and evaporated to a yellow residue. The crude product was purified by column chromatography (PE/CH₂Cl₂ = 6:1, v/v) to yield 5 (1.18 g, 98%) as a white solid. Mp 101.0–102.4 °C. ¹1H NMR (400 MHz, CDCl₃) δ 7.94 (s, 1H), 7.53 (s, 1H), 7.45 (d, J = 9.1 Hz, 1H), 7.31 (d, J = 7.4 Hz, 1H), 7.18 (dd, J = 15.7, 7.4 Hz, 3H), 7.04 (d, J = 9.1 Hz, 1H), 6.32 (s, 1H), 3.61 (t, J = 6.1 Hz, 2H), 3.51 (td, J = 6.8, 3.2 Hz, 2H), 3.31 (dd, J = 6.2, 4.8 Hz, 2H), 2.95 (t, J = 6.5 Hz, 2H), 2.36 (s, 1H), 2.29 (s, 3H), 2.10 – 2.01 (m, 4H). ¹³C NMR (101 MHz, CDCl₃) δ 143.57, 140.40, 136.10, 134.19, 133.95, 130.44, 128.16, 127.66, 127.61, 126.49, 126.05, 125.51, 125.36, 122.38, 115.52, 111.65, 72.17, 49.48, 49.22, 42.82, 30.15, 23.63, 21.75, 19.24. HRMS (ESI): m/z [M + H]⁺ calcd for C₂₄H₂₆BrClNO: 458.0881; found: 458.0882.
• 49 Compound 7 Compound 5 (2 g, 2.25 mmol, 1 equiv.), 8-bromo-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline (6) (0.68 g, 2.7 mmol, 1.2 equiv.) and H₂SO₄ (0.5 mL) (catalytic quantity) were dissolved in AcOH (20 mL) and the resulting mixture was stirred at 70 °C for 8 h. The reaction was allowed to cool to R.T. and quenched by adding saturated NaHCO₃ solution (30 mL). The aqueous layer was extracted with CH₂Cl₂ (3 × 20 mL), and the combined organic layers were dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was purified by column chromatography (PE/CH₂Cl₂ = 8:1, v/v) to give 7 (2 g, 73%) as a white solid. Mp 156–157 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.98 (s, 1H), 7.39 (d, J = 9.1 Hz, 1H), 7.17 – 7.11 (m, 2H), 7.09 – 6.98 (m, 3H), 6.73 (d, J = 7.6 Hz, 1H), 6.22 (s, 1H), 6.16 (s, 1H), 3.63 (t, J = 6.1 Hz, 2H), 3.53 (t, J = 6.9 Hz, 2H), 3.36 – 3.29 (m, 2H), 3.10 (s, 4H), 2.98 (t, J = 6.5 Hz, 2H), 2.80 (t, J = 6.6 Hz, 2H), 2.55 (t, J = 6.1 Hz, 2H), 2.21 (s, 3H), 2.07 (dt, J = 12.8, 6.3 Hz, 4H), 2.04 – 1.90 (m, 4H). ¹³C NMR (101 MHz, CDCl₃): δ = 143.17, 143.15, 141.64, 137.19, 135.52, 133.31, 130.28, 130.19, 129.09, 129.00, 128.91, 127.37, 126.69, 126.30, 125.62, 125.56, 125.49, 125.49, 121.56, 120.17, 115.25, 111.81, 52.95, 50.14, 49.61, 49.54, 49.29, 42.89, 30.23, 29.63, 27.73, 23.74, 22.33, 22.00, 21.89, 19.78. HRMS (ESI): m/z [M + H]⁺ calcd for C₃₆H₃₈Br₂ClN₂: 691.1085; found: 691.1091.
• 50 EC4 Compound 7 (R¹ = H, R² = Me) (0.2 g, 0.29 mmol, 1 equiv.) was dissolved in anhydrous THF (10 mL) under an argon atmosphere and cooled to –78 °C before sec-BuLi (0.49 mL, 1.3 M in hexane, 0.64 mmol, 2.2 equiv.) was added dropwise. After stirring for 30 min, the mixture was added to a solution of methyl 2-phenoxybenzoate (0.13 g, 0.58 mmol, 2 equiv.) in THF (10 mL), and stirring was continued at 0 °C for 30 min. The reaction was quenched by adding saturated NH₄Cl aqueous solution (50 mL), and extracted with EtOAc (3 × 20 mL). The organic layer was combined, dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo The crude residue from concentrating the organic extract was dissolved in CH₂Cl₂ (20 mL) containing MeSO₃H (1 mL). After 2 h, chloranil (0.1 g) was added and the mixture was stirred for 4 h. The aqueous layer was extracted with CH₂Cl₂ (3 × 20 mL), and the combined organic layers were dried over Na₂SO₄, filtered and concentrated in vacuo The residue was purified by column chromatography (CH₂Cl₂/MeOH = 10:1, v/v) to give EC4 (50 mg, 25%) as a green solid. Mp 209.7–209.9 °C. ¹H NMR (400 MHz, CDCl3) δ 7.55 (d, J = 7.3 Hz, 1H), 7.49 (d, J = 7.4 Hz, 2H), 7.40 (s, 1H), 7.30 (dd, J = 10.5, 6.8 Hz, 5H), 7.23 (s, 1H), 6.92 (d, J = 8.7 Hz, 3H), 6.88 (s, 1H), 6.78 (d, J = 7.2 Hz, 1H), 6.73 (d, J = 7.9 Hz, 1H), 3.87 (s, 2H), 3.62 (s, 2H), 3.55 (t, J = 6.1 Hz, 4H), 3.38 – 3.30 (m, 2H), 2.69 (t, J = 5.6 Hz, 2H), 2.60 (t, J = 6.4 Hz, 2H), 2.21 (s, 3H), 2.14 (dd, J = 15.9, 8.2 Hz, 2H), 2.01 (dd, J = 12.9, 6.5 Hz, 4H), 1.98 – 1.93 (m, 2H), 1.50 (d, J = 4.9 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃): δ = 161.55, 156.10, 149.55, 148.64, 148.61, 147.38, 147.24, 136.07, 134.98, 134.76, 130.83, 130.67, 129.49, 128.99, 127.69, 127.38, 126.83, 124.83, 124.53, 117.13, 117.04, 115.48, 112.58, 53.89, 52.56, 48.99, 47.54, 42.26, 30.00, 27.45, 25.28, 22.76, 21.18, 20.22, 19.79, 19.54. HRMS (ESI): m/z [M]⁺ calcd for C₄₉H₄₄ClN₂O: 711.3137; found: 711.3132.
• 51 ESi4 Compound 7 (R¹ = H, R² = Me) (0.2 g, 0.29 mmol, 1 equiv.) was dissolved in anhydrous THF (10 mL) under an argon atmosphere and cooled to –78 °C before sec-BuLi (0.49 mL, 1.3 M in hexane, 0.64 mmol, 2.2 equiv.) was added dropwise. After stirring for 30 min, the resulting mixture was added to a solution of dichlorodimethylsilane (0.05 mL, 0.44 mmol, 1.5 equiv.) in THF (10 mL) and the obtained mixture was stirred at 0 °C for 30 min. The reaction was quenched by adding saturated NH₄Cl aqueous solution and extracted with EtOAc (3 × 20 mL). The organic layer was combined, dried over anhydrous Na₂SO₄, filtered and evaporated to a yellow residue. The residue from the organic extract was dissolved in CH₂Cl₂ (20 mL) containing chloranil (0.1 g) and the mixture was stirred for 4 h. The aqueous layer was extracted with CH₂Cl₂ (3 × 20 mL), and the combined organic layers were dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was purified by column chromatography (CH₂Cl₂/MeOH = 10:1, v/v) to give ESi4 (100 mg, 58%) as a green solid. Mp 241.5–241.9 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.97 (s, 1H), 7.47 (t, J = 7.4 Hz, 1H), 7.37 (d, J = 7.7 Hz, 2H), 7.33 (d, J = 9.2 Hz, 2H), 7.12 (d, J = 7.6 Hz, 1H), 7.05 (d, J = 9.2 Hz, 1H), 6.80 (s, 1H), 3.99 (d, J = 17.2 Hz, 4H), 3.71 – 3.58 (m, 4H), 3.53 – 3.47 (m, 2H), 3.11 (dd, J = 13.2, 6.4 Hz, 4H), 2.57 (s, 2H), 2.23 (s, 2H), 2.13 (dd, J = 12.7, 5.9 Hz, 4H), 2.03 (s, 3H), 2.03 – 1.97 (m, 2H), 0.70 (s, 3H), 0.67 (s, 3H). ¹³NMR (151 MHz, CDCl3) δ 165.69, 153.32, 147.51, 143.76, 140.14, 139.17, 138.95, 136.09, 135.66, 134.47, 133.94, 131.70, 130.29, 129.62, 129.54, 128.77, 126.47, 125.96, 125.71, 115.51, 113.32, 53.62, 52.94, 49.84, 49.15, 42.35, 31.44, 29.99, 28.70, 27.43, 23.27, 20.90, 20.44, 19.58, 0.24, 0.21.
• 52 EP4 Compound 7 (R¹/R² = Me) (0.2 g, 0.29 mmol, 1 equiv.) was dissolved in anhydrous THF (10 mL) under an argon atmosphere and cooled to –78 °C before sec-BuLi (0.49 mL, 1.3 M in hexane, 0.64 mmol, 2.2 equiv.) was added dropwise. After stirring for 30 min, the resulting mixture was added to a solution of dichlorophenylphosphine (0.17 g, 0.7 mmol, 2 equiv.) in THF (10 mL) and the obtained mixture was stirred at 0 °C for 30 min. The reaction was quenched by adding saturated NH₄Cl aqueous solution and extracted with EtOAc (3 × 20 mL). The organic layer was combined, dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo. The organic residue was dissolved in CH₂Cl₂ (20 mL) containing chloranil (0.1 g) and the mixture was stirred for 4 h. The aqueous layer was extracted with CH₂Cl₂ (3 × 20 mL), and the combined organic layers were dried over Na₂SO₄, filtered and concentrated in vacuo The residue was purified by column chromatography (CH₂Cl₂/MeOH = 10:1, v/v) to give EP4 (51 mg, 21%) as a green solid. Mp 248.8–249.5 °C. ¹H NMR (600 MHz, CDCl₃) δ 8.26 (d, J = 16.0 Hz, 1H), 7.72 (dd, J = 12.6, 7.5 Hz, 2H), 7.53 (d, J = 6.9 Hz, 1H), 7.49 (d, J = 5.2 Hz, 2H), 7.43 – 7.37 (m, 2H), 7.25 (d, J = 7.2 Hz, 3H), 7.10 (d, J = 9.3 Hz, 1H), 6.76 (d, J = 5.0 Hz, 1H), 4.30 (d, J = 12.4 Hz, 1H), 4.07 (d, J = 12.9 Hz, 1H), 3.83 (s, 2H), 3.76 (d, J = 15.0 Hz, 1H), 3.67 (d, J = 7.1 Hz, 2H), 3.61 (t, J = 5.8 Hz, 2H), 3.49 (dd, J = 10.6, 5.2 Hz, 2H), 3.09 (dd, J = 19.3, 9.6 Hz, 1H), 2.97 – 2.90 (m, 1H), 2.80 – 2.68 (m, 2H), 2.60 (d, J = 8.6 Hz, 1H), 2.10 (s, 3H), 2.10 – 2.02 (m, 8H), 2.02 (s, 3H). ¹³C NMR (151 MHz, CDCl3) δ 160.64, 154.11, 148.82, 137.30, 137.24, 136.67, 136.43, 136.13, 135.94, 135.78, 134.35, 134.26, 133.87, 133.16, 132.49, 130.05, 129.98, 129.39, 129.31, 128.72, 128.13, 128.00, 127.82, 126.99, 125.80, 116.70, 115.69, 54.16, 52.94, 49.98, 49.22, 42.23, 29.90, 29.70, 27.38, 25.67, 25.62, 23.17, 22.62, 20.91, 19.93. ³¹P NMR (243 MHz, CDCl₃): δ = 8.07. HRMS (ESI): m/z [M]⁺ calcd for C₄₃H₄₃ClN₂OP: 669.2796; found: 669.2797. The exact mass calculated with the ChemDraw for EP4 is 669.2797.
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Corresponding Author

Publication History

Received: 31 March 2023

Accepted after revision: 22 May 2023

Article published online:
07 August 2023

© 2023. Thieme. All rights reserved

Georg Thieme Verlag KG
Rüdigerstraße 14, 70469 Stuttgart, Germany

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• 47 Compound 4 DMF (1.64 mL, 21 mmol, 1.2 equiv.) was mixed with 1,2-dichloroethane (10 mL) and cooled to 0 °C before dropwise addition of POCl₃ (1.98 mL, 21 mmol, 1.2 equiv.) and stirring for 30 min. The resulting mixture was added to a solution of compound 3 (3.5 g, 10 mmol, 1 equiv.) in anhydrous CH₂Cl₂ (10 mL) at 75 °C , and the obtained mixture was stirred for 3 h. The reaction was allowed to cool to the R.T. and quenched by adding saturated Na₂CO₃ solution. The aqueous layer was extracted with CH₂Cl₂ (3 × 20 mL), and the combined organic layers were dried over Na₂SO₄, filtered and concentrated in vacuo. The crude extract was purified by column chromatography (EtOAc/PE = 1:50 to 1:25, v/v) to give 4 (2.2 g, 39%) as a yellow solid. Mp 106.9–108.0 °C. ¹H NMR (400 MHz, CDCl₃): δ = 10.35 (s, 1 H), 8.24 (s, 1 H), 7.89 (s, 1 H), 7.66 (d, J = 9.2 Hz, 1 H), 7.11 (d, J = 9.2 Hz, 1 H), 3.69–3.56 (m, 4 H), 3.47–3.35 (m, 2 H), 2.98 (t, J = 6.5 Hz, 2 H), 2.11 (dd, J = 12.6, 5.2 Hz, 4 H). ¹³C NMR (101 MHz, CDCl₃): δ = 191.76, 146.02, 137.14, 132.32, 130.07, 125.98, 125.59, 124.30, 122.17, 115.65, 111.19, 49.59, 49.00, 42.50, 30.09, 23.44, 21.45. HRMS (ESI): m/z [M]⁺ calcd for C₁₇H₁₇BrClNO: 336.0255; found 336.0254.
• 48 Compound 5 Compound 4 (1 g, 2.8 mmol, 1 equiv.) was dissolved in anhydrous THF (20 mL) under an argon atmosphere and cooled to 0 °C before o-tolylmagnesium bromide (5.45 mL, 5.6 mmol, 2 equiv.) was added dropwise. The reaction was quenched after 30 min. Then the reaction was quenched with saturated NH4Cl aqueous solution (50 mL), and extracted with EtOAc (3 × 20 mL). The organic layer was combined, dried over anhydrous Na₂SO₄, filtered and evaporated to a yellow residue. The crude product was purified by column chromatography (PE/CH₂Cl₂ = 6:1, v/v) to yield 5 (1.18 g, 98%) as a white solid. Mp 101.0–102.4 °C. ¹1H NMR (400 MHz, CDCl₃) δ 7.94 (s, 1H), 7.53 (s, 1H), 7.45 (d, J = 9.1 Hz, 1H), 7.31 (d, J = 7.4 Hz, 1H), 7.18 (dd, J = 15.7, 7.4 Hz, 3H), 7.04 (d, J = 9.1 Hz, 1H), 6.32 (s, 1H), 3.61 (t, J = 6.1 Hz, 2H), 3.51 (td, J = 6.8, 3.2 Hz, 2H), 3.31 (dd, J = 6.2, 4.8 Hz, 2H), 2.95 (t, J = 6.5 Hz, 2H), 2.36 (s, 1H), 2.29 (s, 3H), 2.10 – 2.01 (m, 4H). ¹³C NMR (101 MHz, CDCl₃) δ 143.57, 140.40, 136.10, 134.19, 133.95, 130.44, 128.16, 127.66, 127.61, 126.49, 126.05, 125.51, 125.36, 122.38, 115.52, 111.65, 72.17, 49.48, 49.22, 42.82, 30.15, 23.63, 21.75, 19.24. HRMS (ESI): m/z [M + H]⁺ calcd for C₂₄H₂₆BrClNO: 458.0881; found: 458.0882.
• 49 Compound 7 Compound 5 (2 g, 2.25 mmol, 1 equiv.), 8-bromo-2,3,6,7-tetrahydro-1H,5H-pyrido[3,2,1-ij]quinoline (6) (0.68 g, 2.7 mmol, 1.2 equiv.) and H₂SO₄ (0.5 mL) (catalytic quantity) were dissolved in AcOH (20 mL) and the resulting mixture was stirred at 70 °C for 8 h. The reaction was allowed to cool to R.T. and quenched by adding saturated NaHCO₃ solution (30 mL). The aqueous layer was extracted with CH₂Cl₂ (3 × 20 mL), and the combined organic layers were dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was purified by column chromatography (PE/CH₂Cl₂ = 8:1, v/v) to give 7 (2 g, 73%) as a white solid. Mp 156–157 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.98 (s, 1H), 7.39 (d, J = 9.1 Hz, 1H), 7.17 – 7.11 (m, 2H), 7.09 – 6.98 (m, 3H), 6.73 (d, J = 7.6 Hz, 1H), 6.22 (s, 1H), 6.16 (s, 1H), 3.63 (t, J = 6.1 Hz, 2H), 3.53 (t, J = 6.9 Hz, 2H), 3.36 – 3.29 (m, 2H), 3.10 (s, 4H), 2.98 (t, J = 6.5 Hz, 2H), 2.80 (t, J = 6.6 Hz, 2H), 2.55 (t, J = 6.1 Hz, 2H), 2.21 (s, 3H), 2.07 (dt, J = 12.8, 6.3 Hz, 4H), 2.04 – 1.90 (m, 4H). ¹³C NMR (101 MHz, CDCl₃): δ = 143.17, 143.15, 141.64, 137.19, 135.52, 133.31, 130.28, 130.19, 129.09, 129.00, 128.91, 127.37, 126.69, 126.30, 125.62, 125.56, 125.49, 125.49, 121.56, 120.17, 115.25, 111.81, 52.95, 50.14, 49.61, 49.54, 49.29, 42.89, 30.23, 29.63, 27.73, 23.74, 22.33, 22.00, 21.89, 19.78. HRMS (ESI): m/z [M + H]⁺ calcd for C₃₆H₃₈Br₂ClN₂: 691.1085; found: 691.1091.
• 50 EC4 Compound 7 (R¹ = H, R² = Me) (0.2 g, 0.29 mmol, 1 equiv.) was dissolved in anhydrous THF (10 mL) under an argon atmosphere and cooled to –78 °C before sec-BuLi (0.49 mL, 1.3 M in hexane, 0.64 mmol, 2.2 equiv.) was added dropwise. After stirring for 30 min, the mixture was added to a solution of methyl 2-phenoxybenzoate (0.13 g, 0.58 mmol, 2 equiv.) in THF (10 mL), and stirring was continued at 0 °C for 30 min. The reaction was quenched by adding saturated NH₄Cl aqueous solution (50 mL), and extracted with EtOAc (3 × 20 mL). The organic layer was combined, dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo The crude residue from concentrating the organic extract was dissolved in CH₂Cl₂ (20 mL) containing MeSO₃H (1 mL). After 2 h, chloranil (0.1 g) was added and the mixture was stirred for 4 h. The aqueous layer was extracted with CH₂Cl₂ (3 × 20 mL), and the combined organic layers were dried over Na₂SO₄, filtered and concentrated in vacuo The residue was purified by column chromatography (CH₂Cl₂/MeOH = 10:1, v/v) to give EC4 (50 mg, 25%) as a green solid. Mp 209.7–209.9 °C. ¹H NMR (400 MHz, CDCl3) δ 7.55 (d, J = 7.3 Hz, 1H), 7.49 (d, J = 7.4 Hz, 2H), 7.40 (s, 1H), 7.30 (dd, J = 10.5, 6.8 Hz, 5H), 7.23 (s, 1H), 6.92 (d, J = 8.7 Hz, 3H), 6.88 (s, 1H), 6.78 (d, J = 7.2 Hz, 1H), 6.73 (d, J = 7.9 Hz, 1H), 3.87 (s, 2H), 3.62 (s, 2H), 3.55 (t, J = 6.1 Hz, 4H), 3.38 – 3.30 (m, 2H), 2.69 (t, J = 5.6 Hz, 2H), 2.60 (t, J = 6.4 Hz, 2H), 2.21 (s, 3H), 2.14 (dd, J = 15.9, 8.2 Hz, 2H), 2.01 (dd, J = 12.9, 6.5 Hz, 4H), 1.98 – 1.93 (m, 2H), 1.50 (d, J = 4.9 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃): δ = 161.55, 156.10, 149.55, 148.64, 148.61, 147.38, 147.24, 136.07, 134.98, 134.76, 130.83, 130.67, 129.49, 128.99, 127.69, 127.38, 126.83, 124.83, 124.53, 117.13, 117.04, 115.48, 112.58, 53.89, 52.56, 48.99, 47.54, 42.26, 30.00, 27.45, 25.28, 22.76, 21.18, 20.22, 19.79, 19.54. HRMS (ESI): m/z [M]⁺ calcd for C₄₉H₄₄ClN₂O: 711.3137; found: 711.3132.
• 51 ESi4 Compound 7 (R¹ = H, R² = Me) (0.2 g, 0.29 mmol, 1 equiv.) was dissolved in anhydrous THF (10 mL) under an argon atmosphere and cooled to –78 °C before sec-BuLi (0.49 mL, 1.3 M in hexane, 0.64 mmol, 2.2 equiv.) was added dropwise. After stirring for 30 min, the resulting mixture was added to a solution of dichlorodimethylsilane (0.05 mL, 0.44 mmol, 1.5 equiv.) in THF (10 mL) and the obtained mixture was stirred at 0 °C for 30 min. The reaction was quenched by adding saturated NH₄Cl aqueous solution and extracted with EtOAc (3 × 20 mL). The organic layer was combined, dried over anhydrous Na₂SO₄, filtered and evaporated to a yellow residue. The residue from the organic extract was dissolved in CH₂Cl₂ (20 mL) containing chloranil (0.1 g) and the mixture was stirred for 4 h. The aqueous layer was extracted with CH₂Cl₂ (3 × 20 mL), and the combined organic layers were dried over Na₂SO₄, filtered and concentrated in vacuo. The residue was purified by column chromatography (CH₂Cl₂/MeOH = 10:1, v/v) to give ESi4 (100 mg, 58%) as a green solid. Mp 241.5–241.9 °C. ¹H NMR (400 MHz, CDCl₃) δ 7.97 (s, 1H), 7.47 (t, J = 7.4 Hz, 1H), 7.37 (d, J = 7.7 Hz, 2H), 7.33 (d, J = 9.2 Hz, 2H), 7.12 (d, J = 7.6 Hz, 1H), 7.05 (d, J = 9.2 Hz, 1H), 6.80 (s, 1H), 3.99 (d, J = 17.2 Hz, 4H), 3.71 – 3.58 (m, 4H), 3.53 – 3.47 (m, 2H), 3.11 (dd, J = 13.2, 6.4 Hz, 4H), 2.57 (s, 2H), 2.23 (s, 2H), 2.13 (dd, J = 12.7, 5.9 Hz, 4H), 2.03 (s, 3H), 2.03 – 1.97 (m, 2H), 0.70 (s, 3H), 0.67 (s, 3H). ¹³NMR (151 MHz, CDCl3) δ 165.69, 153.32, 147.51, 143.76, 140.14, 139.17, 138.95, 136.09, 135.66, 134.47, 133.94, 131.70, 130.29, 129.62, 129.54, 128.77, 126.47, 125.96, 125.71, 115.51, 113.32, 53.62, 52.94, 49.84, 49.15, 42.35, 31.44, 29.99, 28.70, 27.43, 23.27, 20.90, 20.44, 19.58, 0.24, 0.21.
• 52 EP4 Compound 7 (R¹/R² = Me) (0.2 g, 0.29 mmol, 1 equiv.) was dissolved in anhydrous THF (10 mL) under an argon atmosphere and cooled to –78 °C before sec-BuLi (0.49 mL, 1.3 M in hexane, 0.64 mmol, 2.2 equiv.) was added dropwise. After stirring for 30 min, the resulting mixture was added to a solution of dichlorophenylphosphine (0.17 g, 0.7 mmol, 2 equiv.) in THF (10 mL) and the obtained mixture was stirred at 0 °C for 30 min. The reaction was quenched by adding saturated NH₄Cl aqueous solution and extracted with EtOAc (3 × 20 mL). The organic layer was combined, dried over anhydrous Na₂SO₄, filtered and concentrated in vacuo. The organic residue was dissolved in CH₂Cl₂ (20 mL) containing chloranil (0.1 g) and the mixture was stirred for 4 h. The aqueous layer was extracted with CH₂Cl₂ (3 × 20 mL), and the combined organic layers were dried over Na₂SO₄, filtered and concentrated in vacuo The residue was purified by column chromatography (CH₂Cl₂/MeOH = 10:1, v/v) to give EP4 (51 mg, 21%) as a green solid. Mp 248.8–249.5 °C. ¹H NMR (600 MHz, CDCl₃) δ 8.26 (d, J = 16.0 Hz, 1H), 7.72 (dd, J = 12.6, 7.5 Hz, 2H), 7.53 (d, J = 6.9 Hz, 1H), 7.49 (d, J = 5.2 Hz, 2H), 7.43 – 7.37 (m, 2H), 7.25 (d, J = 7.2 Hz, 3H), 7.10 (d, J = 9.3 Hz, 1H), 6.76 (d, J = 5.0 Hz, 1H), 4.30 (d, J = 12.4 Hz, 1H), 4.07 (d, J = 12.9 Hz, 1H), 3.83 (s, 2H), 3.76 (d, J = 15.0 Hz, 1H), 3.67 (d, J = 7.1 Hz, 2H), 3.61 (t, J = 5.8 Hz, 2H), 3.49 (dd, J = 10.6, 5.2 Hz, 2H), 3.09 (dd, J = 19.3, 9.6 Hz, 1H), 2.97 – 2.90 (m, 1H), 2.80 – 2.68 (m, 2H), 2.60 (d, J = 8.6 Hz, 1H), 2.10 (s, 3H), 2.10 – 2.02 (m, 8H), 2.02 (s, 3H). ¹³C NMR (151 MHz, CDCl3) δ 160.64, 154.11, 148.82, 137.30, 137.24, 136.67, 136.43, 136.13, 135.94, 135.78, 134.35, 134.26, 133.87, 133.16, 132.49, 130.05, 129.98, 129.39, 129.31, 128.72, 128.13, 128.00, 127.82, 126.99, 125.80, 116.70, 115.69, 54.16, 52.94, 49.98, 49.22, 42.23, 29.90, 29.70, 27.38, 25.67, 25.62, 23.17, 22.62, 20.91, 19.93. ³¹P NMR (243 MHz, CDCl₃): δ = 8.07. HRMS (ESI): m/z [M]⁺ calcd for C₄₃H₄₃ClN₂OP: 669.2796; found: 669.2797. The exact mass calculated with the ChemDraw for EP4 is 669.2797.
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