Absorption, Fluorescence, and Two-Photon Excitation Ability of 5-Phenylisolidolo[2,1-a]quinolines.

We report on the absorption, fluorescence, and two-photon excitation spectra of a series of 5-phenylisoindolo[2,1-a]quinoline dyes. Depending on the substituents, we observed increasing two-photon absorption cross sections, with values up to 56 GM@973 nm, which are similar to those of the enhanced green fluorescent protein and fluorescein, common fluorescent chromophores.

Introduction

Two-photon excitation is a phenomenon in which two photons are simultaneously absorbed by a molecule, causing excitation. Because visible fluorophores are used for bioimaging, near-infrared (NIR) laser light pulses are used to induce two-photon excitation processes. The excitation induced by NIR laser light offers superior penetration depth and is less invasive for living specimens. Furthermore, two-photon excitation is spatially localized at the focus of the excitation laser light because of nonlinear dependence on photon density, which allows for the acquisition of optically sectioned fluorescence images. Thus, out-of-focus photobleaching, photodamage, and attenuation of the excitation beam by out-of-focus absorption rarely occur. Therefore, two-photon excitation microscopy is currently the strongest methodology used to evaluate molecular and cellular mechanisms at some depths of an organ.[1]

Also, it became evident that in order for two-photon technology to realize its full potential, more diverse dye scaffolds are needed in the design and synthesis of more active dye molecules with the necessary solubility and photostability. To facilitate the design and synthesis of new active molecules, research was needed to establish well-defined structure/property relationships for a large number of organic structures. This required both the synthesis of highly pure, well-defined organic dyes with systematically varied molecular structures and the careful reproducible characterization of their two-photon excitation properties.

In 2013, we reported a new tandem process that combined a ruthenium-catalyzed ring-closing metathesis (RCM) reaction with intermolecular 1,3-dipolar cycloaddition to give isoindolo[2,1-a]quinoline,[2] a kind of indolizine,[2b−2g] core 2 (Scheme ). Among the novel dyes, dyes 2a–2c had fluorescence absorption in the region longer than 600 nm and the quantum yield of dye 2c was the highest among these three in chloroform (Scheme ).

Scheme 1

One-Pot Reaction of RCM and Intermolecular 1,3-Dipolar Cycloaddition to Give Isoindolo[2,1-a]quinoline

Scheme 2

Fluorescence Spectra of Compounds 2a–2c in Chloroform

This report describes the absorption, fluorescence, and two-photon excitation ability of several 5-phenylisoindolo[2,1-a]quinoline derivatives.

Results and Discussion

First, we decided to synthesize five new 5-phenylisoindolo[2,1-a]quinoline derivatives, as shown in Scheme , that is, 3-methyl form 2d, 3-fluoro form 2e, 3-methoxy form 2f, 3-phenyl form 2g, and derivative 2h, in which one more condensed ring is added, were successfully prepared by our own one-pot procedure, as explained in Scheme .

Scheme 3

Synthesis 5-Phenylisoindolo[2,1-a]quinoline Derivatives

The absorption and fluorescence spectra of these dyes are shown in Schemes and 5. In addition, Table summarizes the quantum yield and the maximum wavelength of these dyes in the absorption spectrum and the fluorescence spectrum. In the wavelength region longer than 380 nm of the absorption spectrum, although there was a slight difference in the maximum absorption wavelength (468–494 nm), no significant difference could be confirmed between these dyes. On the other hand, in the wavelength range shorter than 380 nm, each dye exhibited a characteristic waveform, and 2g and 2h had large absorption bands at 280 and 300 nm, respectively.

Scheme 4

Absorption Spectra of 2c–2h [50 μM in Dimethyl Sulfoxide (DMSO)]

Scheme 5

Fluorescence Spectra of 2c–2h (50 μM in DMSO)

Table 1

Quantum Yield and the Maximum Wavelength of 2c–2h (50 μM in DMSO)

dye	ϕ	λex (nm)	Fmax (nm)
2c	0.0029	475	594
2d	0.0043	477	595
2e	0.012	468	511
2f	0.0012	481	599
2g	0.0034	477	597
2h	0.0018	494	540

In the fluorescence spectrum, 2c and 2g showed almost the same waveform, but in the case of the other dyes, not only the waveform but also the maximum value in the NIR region greatly changed, depending on the substituent present in the scaffold.

Looking at the quantum yield, it was lower than 2c, 2f, and 2h, but it increased for the other three compounds (2d, 2e, and 2g). In particular, while the quantum yield of the methoxy form (2f) was about one-fourth of that of 2c, the quantum yield of the fluoro form (2b) was about three times as large as 2c. Furthermore, as a result of measuring the two-photon excitation spectrum of these compounds, dyes 2c and 2g were found to be two-photon excitation dyes (Scheme , 2c: σ973 nm = 56 GM, 2g: σ972 nm = 20 GM). Dyes 2c or 2g have almost twice brighter or comparable two-photon absorption (TPA) cross-sectional values, respectively, as typical fluorophores for two-photon excitation microscopy, fluorescein, and enhanced green fluorescent protein (EGFP)[3,4] and follow the magnitude of one-photon absorption.

Scheme 6

Two-Photon Excitation Spectra of 2c–2h (1 mM in DMSO) and Fluorescein (100 μM in pH 11 Buffer: DMSO = 1:9)

Conclusions

In summary, we synthesized five new 5-phenylisoindolo[2,1-a]quinoline derivatives by one-pot reaction of RCM and intermolecular 1,3-dipolar cycloaddition. We also measured the absorbance, fluorescence, and two-photon excitation spectra of these dyes and found that the 5-phenylisoindolo[2,1-a]quinoline derivative is a two-photon excited dye, even though their quantum yields are in the range of 0.0018–0.012,[5] whose TPA cross-section value is similar to EGFP and fluorescein, common fluorescent chromophores.

Experimental Section

Synthesis of Substituted 2-(1-Phenylvinyl)anilines

2-(1-Phenylvinyl)aniline derivatives S1a–e were synthesized based on the literature.[6,7]

Procedure A for the Synthesis of Substituted N-Methyl-2-(1-phenylvinyl)aniline (Reductive Amination)[8]

A methodology using zinc and aqueous formaldehyde was applied for the synthesis of S2a–c.

To a mixture of the 2-(1-phenylvinyl)aniline derivative (1.0 equiv), AcOH (8.0 equiv) and zinc dust (2.0 equiv) in dioxane (0.5 M) were added to 37% aqueous formaldehyde (1.5 equiv) and was stirred under nitrogen at room temperature. After 2 h, the mixture was quenched by the addition of saturated NaHCO3 solution and the organic compounds were then extracted with AcOEt. The combined organic layer was washed with brine, dried over Na2SO4, and concentrated in vacuo. The obtained residue was then subjected to column chromatography (neutral silica gel, hexane/AcOEt = 75:1).

N-Methyl-4-methyl-2-(1-phenylvinyl)aniline (S2a)

Following procedure A, S2a (557 mg, 2.49 mmol, 34%) was obtained as pale yellow oil from S1a (1.54 g, 7.36 mmol). 1H NMR (400 MHz, CDCl3): δ 7.37–7.26 (5H, m), 7.08 (1H, d, J = 8.3 Hz), 6.91 (1H, s), 6.58 (1H, d, J = 7.8 Hz), 5.81 (1H, d, J = 1.4 Hz), 5.32 (1H, d, J = 1.4 Hz), 3.56 (1H, s), 2.70 (3H. d, J = 5.0 Hz), 2.26 (3H, s); 13C NMR (100 MHz, CDCl3): δ 147.16, 144.58, 139.76, 131.12, 129.43, 128.62, 128.15, 127.45, 126.62, 125.82, 116.19, 110.25, 31.24, 20.47; HRMS (MALDI-TOF): calcd for C16H17N (M)+, 223.13530; found, 223.13555.

N-Methyl-4-fluoro-2-(1-phenylvinyl)aniline (S2b)

Following procedure A, S2b (670 mg, 2.95 mmol, 50%) was obtained as yellow oil from S1b (1.25 g, 5.86 mmol). 1H NMR (400 MHz, CDCl3): δ 7.36–7.29 (5H, m), 6.98 (1H, td, J = 8.8, 3.2 Hz), 6.84 (1H, dd, J = 9.2, 3.2 Hz), 6.56 (1H, dd, J = 8.7, 4.6 Hz), 5.84 (1H, d, J = 0.9 Hz), 5.34 (1H, d, J = 1.4 Hz), 3.54 (1H, s), 2.69 (3H, d, J = 5.5 Hz); 13C NMR (100 MHz, CDCl3): δ 155.31 (d, J = 230.0 Hz), 146.18, 143.22 (d, J = 1.9 Hz), 139.03, 128.75, 128.43, 128.29, 126.55, 117.2 (d, J = 22.9 Hz), 116.84, 115.03 (d, J = 21.0 Hz), 110.64 (d, J = 7.6 Hz), 31.42; 19F NMR (400 MHz, CDCl3): δ −127.32 (1F, m, J = 4.6 Hz); HRMS (MALDI-TOF): calcd for C15H14NF (M)+, 227.11043; found, 227.11048.

N-Methyl-4-methoxy-2-(1-phenylvinyl)aniline (S2c)

Following procedure A, S2c (225 mg, 0.94 mmol, 42%) was obtained as yellow oil from S1c (500 mg, 2.22 mmol). 1H NMR (300 MHz, CDCl3): δ 7.39–7.29 (5H, m), 6.88 (1H, dd, J = 8.7, 2.7 Hz), 6.75 (1H, d, J = 3.2 Hz), 6.62 (1H, d, J = 9.2 Hz), 5.84 (1H, d, J = 1.4 Hz), 5.34 (1H, d, J = 1.4 Hz), 3.77 (3H, s), 3.41 (1H, s), 2.69 (3H, s); 13C NMR (75 MHz, CDCl3): δ 151.49, 146.90, 141.24, 139.38, 128.67, 128.64, 128.25, 126.57, 116.84, 116.40, 114.10, 111.29, 55.97, 31.75; HRMS (MALDI-TOF): calcd for C16H17NO (M)+, 239.13055; found, 239.13047.

Procedure B for the Synthesis of Substituted N-Methyl-2-(1-phenylvinyl)aniline (Methylation)[9]

A methodology using MeOTf for the monomethylation of primary amines was applied for the synthesis of S2d–e.

To a solution of 2-(1-phenylvinyl)aniline derivative (1.0 equiv) in hexafluoro-2-propanol (1.0 M) was added MeOTf (1.5 equiv) and was stirred under nitrogen at room temperature for 1 h. The solution was then quenched by the addition of 2.0 N HCl (2 equiv), stirred for 5 min, and then neutralized with saturated NaHCO3. The organic compounds were extracted with AcOEt, and the combined organic layer was then washed with brine, dried over Na2SO4, and concentrated in vacuo. The obtained residue was then subjected to column chromatography (neutral silica gel, hexane/AcOEt = 75:1).

N-Methyl-4-phenyl-2-(1-phenylvinyl)aniline (S2d)

Following procedure B, S2d (728 mg, 2.55 mmol, 79%) was obtained as pale yellow oil from S1d (880 mg, 3.24 mmol). 1H NMR (400 MHz, CDCl3): δ 7.58–7.53 (3H, m), 7.42–7.23 (9H, m), 6.72 (1H, d, J = 8.7 Hz), 5.87 (1H, d, J = 1.8 Hz), 5.40 (1H, d, J = 1.4 Hz), 3.79 (1H, s), 2.76 (3H, d, J = 4.6 Hz); 13C NMR (100 MHz, CDCl3): δ 146.96, 146.19, 141.22, 139.48, 129.46, 129.13, 128.77, 128.70, 128.29, 127.57, 127.54, 126.65, 126.40, 126.16, 116.57, 110.25, 30.92; HRMS (MALDI-TOF): calcd for C21H19N (M)+, 285.15147; found, 285.15120.

N-Methyl-2-(1-phenylvinyl)naphthalen-1-amine (S2e)

Following procedure B, S2e (450 mg, 1.74 mmol, 71%) was obtained as brown oil from S1e (600 mg, 2.44 mmol). 1H NMR (400 MHz, CDCl3): δ 8.15 (1H, dd, J = 8.2, 1.9 Hz), 7.83 (1H, dd, J = 7.3, 2.3 Hz), 7.50–7.24 (9H, m), 5.92 (1H, d, J = 1.4 Hz), 5.36 (1H, d, J = 0.9 Hz), 3.73 (1H, s), 2.74 (3H, s); 13C NMR (100 MHz, CDCl3): δ 147.63, 144.48, 140.67, 134.72, 129.20, 128.59, 128.56, 128.48, 128.22, 127.87, 126.87, 125.87, 125.34, 124.36, 121.93, 116.32, 37.72; HRMS (MALDI-TOF): calcd for C19H17N (M)+, 259.13607; found, 259.13555.

Procedure C for the Synthesis of Substituted N-Allyl-N-methyl-2-(1-phenylvinyl)aniline

To a mixture of N-methyl-2-(1-phenylvinyl)aniline derivative (1.0 equiv) and K2CO3 (4.0 equiv) in a 2:1 ratio of EtOH/H2O (0.15 M) was added allyl bromide (3.0 equiv) and was stirred under nitrogen at 70 °C for 2 h. After cooling down to room temperature, the reaction was diluted with H2O. The organic compounds were extracted with AcOEt, and the combined organic layer was then washed with brine, dried over Na2SO4, and concentrated in vacuo. The obtained residue was then subjected to column chromatography (neutral silica gel, hexane/AcOEt = 150:1).

N-Allyl-N-methyl-4-methyl-2-(1-phenyl vinyl)aniline (1d)

Following procedure C, 1d (373 mg, 1.42 mmol, 79%) was obtained as colorless oil from S2a (400 mg, 1.79 mmol). 1H NMR (400 MHz, CDCl3): δ 7.26–7.22 (5H, m), 7.10 (2H, d, J = 5.9 Hz), 6.90 (1H, d, J = 8.7 Hz), 5.62 (1H, d, J = 1.4 Hz), 5.35 (1H, d, J = 1.4 Hz), 5.33–5.25 (1H, m), 5.94 (1H, d, J = 7.3 Hz), 4.91 (1H, s), 3.37 (2H, d, J = 6.4 Hz), 2.43 (3H, s), 2.32 (3H, s); 13C NMR (100 MHz, CDCl3): δ 150.44, 148.82, 141.39, 136.10, 135.91, 132.80, 131.33, 129.09, 127.87, 127.28, 126.53, 119.73, 116.50, 114.87, 58.73, 39.93, 20.76; HRMS (MALDI-TOF): calcd for C19H22N (M + H)+, 264.17453; found, 264.17468.

N-Allyl-N-methyl-4-fluoro-2-(1-phenyl vinyl)aniline (1e)

Following procedure C, 1e (528 mg, 1.98 mmol, 45%) was obtained as colorless oil from S2b (1.0 g, 4.4 mmol). 1H NMR (400 MHz, CDCl3): δ 7.26–7.23 (5H, m), 7.02–6.92 (3H, m), 5.64 (1H, d, J = 0.9 Hz), 5.36 (1H, d, J = 1.4 Hz), 5.30–5.23 (1H, m), 4.94 (1H, td, J = 3.0,1.4 Hz), 4.91 (1H, s), 3.33 (2H, d, J = 6.4 Hz), 2.40 (3H, s); 13C NMR (100 MHz, CDCl3): δ 158.35 (d, J = 240.0 Hz), 149.44, 147.41 (d, J = 1.9 Hz), 140.84, 138.08 (d, J = 7.6 Hz), 135.70, 127.97, 127.50, 126.42, 121.25 (d, J = 8.6 Hz), 118.50 (d, J = 23.0 Hz), 116.80, 115.63, 114.73 (d, J = 22.0 Hz), 58.89, 40.16; 19F NMR (400 MHz, CDCl3): δ −116.70 (1F, m, J = 2.3 Hz); HRMS (MALDI-TOF): calcd for C18H19NF (M + H)+, 268.14899; found, 268.14960.

N-Allyl-N-methyl-4-methoxy-2-(1-phenyl vinyl)aniline (1f)

Following procedure C, 1f (200 mg, 0.72 mmol, 78%) was obtained as colorless oil from S2c (220 mg, 0.92 mmol). 1H NMR (400 MHz, CDCl3): δ 7.26 (5H, m), 6.96 (1H, d, J = 8.3 Hz), 6.87 (1H, d, J = 2.3 Hz), 6.85 (1H, dd, J = 8.2, 3.2 Hz), 5.63 (1H, d, J = 1.4 Hz), 5.34 (1H, d, J = 1.4 Hz), 5.32–5.23 (1H, m), 4.95–4.89 (2H, m), 3.81 (3H, d, J = 2.3 Hz), 3.30 (2H, d, J = 6.0 Hz), 2.38 (3H, s); 13C NMR (100 MHz, CDCl3): δ 155.12, 150.17, 144.96, 141.39, 138.26, 136.22, 127.88, 127.27, 126.43, 121.38, 117.39, 116.46, 115.08, 113.43, 59.28, 55.65, 40.49; HRMS (MALDI-TOF): calcd for C19H21NO (M)+, 279.16154; found, 279.16177.

N-Allyl-N-methyl-4-phenyl-2-(1-phenyl vinyl)aniline (1g)

Following procedure C, 1g (373 mg, 1.42 mmol, 79%) was obtained as colorless oil from S2d (400 mg, 1.79 mmol). 1H NMR (400 MHz, CDCl3): δ 7.61 (1H, d, J = 0.9 Hz), 7.60 (1H, d, J = 5.1 Hz), 7.54 (1H, d, J = 2.7 Hz), 7.52 (1H, d, J = 2.3 Hz), 7.43 (1H, d, J = 2.3 Hz), 7.40 (1H, d, J = 7.8 Hz), 7.32–7.25 (6H, m), 7.04 (1H, d, J = 7.8 Hz), 5.69 (1H, d, J = 1.4 Hz), 5.46 (1H, d, J = 1.4 Hz), 5.38–5.31 (1H, m), 4.98 (1H, d, J = 5.5 Hz), 4.95 (1H, s), 3.49 (2H, d, J = 6.0 Hz), 2.53 (3H, s); 13C NMR (100 MHz, CDCl3): δ 150.50, 150.37, 140.92, 140.86, 135.72, 135.35, 134.19, 131.00, 128.83, 127.96, 127.49, 127.04, 126.81, 126.76, 126.61, 119.68, 116.78, 115.15, 58.29, 39.57; HRMS (MALDI-TOF): calcd for C24H24N (M + H)+, 326.18979; found, 326.19033.

N-Allyl-N-methyl-2-(1-phenylvinyl)naphthalen-1-amine (1h)

Following procedure C, 1h (328 mg, 1.10 mmol, 75%) was obtained as pale yellow oil from S2e (380 mg, 1.46 mmol). 1H NMR (400 MHz, CDCl3): δ 8.16 (1H, dd, J = 8.2, 1.6 Hz), 7.85 (1H, dd, J = 5.7, 2.5 Hz), 7.62 (1H, d, J = 8.2 Hz), 7.49–7.46 (2H, m), 7.32–7.26 (6H, m), 5.79 (1H, d, J = 1.4 Hz), 5.70–5.64 (1H, m), 5.27 (1H, d, J = 1.4 Hz), 5.10 (1H, dd, J = 17.0, 1.8 Hz), 5.01 (1H, dd, J = 10.3, 1.6 Hz), 3.53 (2H, broad peak), 2.57 (3H, s); 13C NMR (100 MHz, CDCl3): δ 150.09, 147.06, 141.97, 137.01, 136.21, 134.80, 132.64, 130.17, 128.34, 128.22, 127.73, 126.92, 125.79, 125.74, 125.12, 124.28, 116.22, 115.27, 59.33, 40.76; HRMS (MALDI-TOF): calcd for C22H22N (M + H)+, 300.17442; found, 300.17468.

Procedure D for the Synthesis of 5-Phenylisoindolo[2,1-a]quinoline Derivatives (2d–h)

To a 0.01 M solution of N-allyl-N-protected aniline derivative (1 equiv) in benzene was added the second-generation Grubbs catalyst (10 mol %) under the Ar atmosphere and the mixture was refluxed for 0.5 h. To the mixture was then added benzoquinone (10 equiv) and refluxed for an additional 1 h. The mixture was cooled down to room temperature and quenched with a saturated solution of NH4Cl. The solvent was partially removed by an evaporator and the organic compounds were extracted with AcOEt. The combined organic layer was then washed with brine, dried over Na2SO4, and concentrated under vacuum. The obtained residue was then subjected to column chromatography (first time neutral silica gel, toluene/AcOEt = 20:1 to 5:1, second time neutral flash silica gel, toluene/AcOEt = 20:1)

3-Methyl-5-phenylisoindolo[2,1-a]quinoline-7,10-dione (2d)

Following procedure D, 2d (60 mg, 0.178 mmol, 23%) was obtained as a red solid from 1d (200 mg, 0.76 mmol). 1H NMR (500 MHz, CDCl3): δ 8.33 (1H, s), 8.05 (1H, s), 7.94 (1H, d, J = 8.4 Hz), 7.59 (1H, s), 7.54–7.49 (6H, m), 6.79 (1H, d, J = 10.3 Hz), 6.71 (1H, d, J = 10.3 Hz), 2.44 (3H, s); 13C NMR (125 MHz, CDCl3): δ 183.58, 181.16, 161.97, 141.90, 140.53, 138.35, 136.24, 131.45, 129.66, 128.75, 128.56, 128.03, 127.49, 124.35, 123.68, 118.17, 115.36, 114.52, 113.83, 111.90, 21.46; HRMS (MALDI-TOF): calcd for C23H16NO2 (M + H)+, 338.11857; found, 338.11756; mp 276–279 °C (from EtOAc/n-hexane).

3-Fluoro-5-phenylisoindolo[2,1-a]quinoline-7,10-dione (2e)

Following procedure D, 2e (90 mg, 0.26 mmol, 35%) was obtained as a red solid from 1e (200 mg, 0.75 mmol). 1H NMR (300 MHz, CDCl3): δ 8.34 (1H, s), 8.14 (1H, s), 8.05 (1H, dd, J = 9.2, 4.6 Hz), 7.55–7.46 (7H, m), 6.82 (1H, d, J = 10.6 Hz), 6.75 (1H, d, J = 10.6 Hz); 19F NMR (400 MHz, CDCl3): δ −124.77 (1F, m, J = 5.4 Hz); 13C NMR (125 MHz, CDCl3): δ 183.39, 181.34, 160.51 (d, J = 245.0 Hz), 141.89, 139.76 (d, J = 3.6 Hz), 138.64, 137.52, 132.62, 129.59, 129.06, 128.98, 126.49, 126.43, 124.23, 119.43, 118.26 (d, J = 24.0 Hz), 117.53 (d, J = 9.5 Hz), 113.96, 113.78 (d, J = 7.0 Hz), 112.61; HRMS (MALDI-TOF): calcd for C22H13NO2F (M + H)+, 342.09206; found, 342.09248; mp 281–285 °C (from EtOAc/n-hexane).

3-Methoxy-5-phenylisoindolo[2,1-a]quinoline-7,10-dione (2f)

Following procedure D, 2f (99 mg, 0.28 mmol, 39%) was obtained as a red solid from 1f (200 mg, 0.72 mmol). 1H NMR (400 MHz, CDCl3): δ 8.28 (1H, s), 8.06 (1H, s), 7.96 (1H, d, J = 9.2 Hz), 7.54–7.51 (5H, m), 7.30 (1H, dd, J = 9.2, 2.8 Hz), 7.24 (1H, d, J = 2.7 Hz), 6.78 (1H, d, J = 10.3 Hz), 6.70 (1H, d, J = 10.3 Hz), 3.77 (3H, s) 13C NMR (125 MHz, CDCl3): δ 183.65, 181.21, 157.63, 141.91, 140.26, 138.38, 137.94, 132.46, 129.55, 128.87, 128.71, 127.14, 125.74, 123.71, 118.67, 118.65, 116.89, 113.68, 112.00, 110.29, 55.78; HRMS (MALDI-TOF): calcd for C23H16NO3 (M + H)+, 354.11216; found, 354.11247; mp 269–270 °C (from EtOAc/n-hexane).

3,5-Diphenylisoindolo[2,1-a]quinoline-7,10-dione (2g)

Following procedure D, 2g (107 mg, 0.27 mmol, 44%) was obtained as a red solid from 1g (200 mg, 0.61 mmol). 1H NMR (300 MHz, CDCl3): δ 8.37 (1H, s), 8.10 (2H, t, J = 4.3 Hz), 8.02 (1H, d, J = 1.9 Hz), 7.93 (1H, dd, J = 8.7, 1.9 Hz), 7.57–7.37 (10H, m), 6.79 (1H, d, J = 10.5 Hz), 6.71 (1H, d, J = 10.5 Hz); 13C NMR (125 MHz, CDCl3): δ 183.47, 181.16, 141.85, 140.67, 139.58, 139.23, 138.40, 137.75, 132.83, 131.82, 129.67, 129.18, 128.89, 128.73, 128.10, 127.29, 126.45, 124.71, 123.90, 118.53, 116.04, 113.90, 112.11; HRMS (MALDI-TOF): calcd for C28H18NO2 (M + H)+, 400.13250; found, 400.13321; mp 265–268 °C (from EtOAc/n-hexane).

7-Phenylbenzo[h]isoindolo[2,1-a]quinoline-9,12-dione (2h)

Following procedure D, 2h (125 mg, 0.33 mmol, 49%) was obtained as a red solid from 1h (200 mg, 0.67 mmol). 1H NMR (500 MHz, CDCl3): δ 9.13, (1H, s), 9.04 (1H, d, J = 8.4 Hz), 8.34, (1H, s), 8.03 (1H, dd, J = 7.8, 1.3 Hz), 7.83–7.72 (4H, m), 7.56–7.51 (5H, m), 6.84 (1H, d, J = 10.3 Hz), 6.76 (1H, d, J = 10.3 Hz); 13C NMR (125 MHz, CDCl3): δ 183.70, 180.72, 142.08, 140.85, 138.17, 138.07, 134.99, 134.30, 129.88, 129.80, 129.15, 128.77, 128.57, 127.98, 127.60, 126.87, 124.27, 124.05, 123.93, 123.15, 122.93, 119.04, 118.87, 110.66; HRMS (MALDI-TOF): calcd for C26H16NO2 (M + H)+, 374.11881; found, 374.11756; mp 255–259 °C (from EtOAc/n-hexane).

TPA Cross-Section Measurements

TPA cross-section spectra of dyes 2c, 2d, 2e, 2f, 2g, and 2h were estimated as reported previously by comparison to fluorescein as a standard with minor modifications.[3,10] Dyes 2c, 2d, 2e, 2f, 2g, and 2h were dissolved in DMSO to concentrations of 1 mM, and fluorescein was dissolved in pH 11 sodium phosphate buffer solution to a concentration of 0.1 mM. The fluorescence images of their solutions filled in glass-bottom dishes were measured by a two-photon excitation microscopy system equipped with a mode-locked titanium:sapphire laser light source (Mai tai DeepSee eHP, Spectra Physics), of which the wavelength is continuously tunable from 690 to 1040 nm, with a ca. 100 fs pulse width and 80 MHz repetition rate. The laser light beam was introduced into a spinning-disk confocal scanner (CSU-MP; Yokogawa Electric)[11,12] installed on an inverted microscope (Ti-E, Nikon) with a water-immersion objective lens (LWD Lambda S 40XC WI 40×/1.27 NA, Nikon), and it was focused on a specimen. Fluorescent lights acquired by the objective lens were reflected by a dichroic mirror (700 nm shortpass filter, Yokogawa Electric) and detected by an EM-CCD camera (iXon Ultra 897, Andor Technology, Belfast, UK). The wavelength range of the irradiation laser light pulses was from 750 to 1000 nm, with 10 nm intervals. All fluorescent images of dye solutions were measured under the same optical conditions, making it possible to correct the difference between our two-photon microscopy system and the referred system. To avoid fluorescence reabsorptions or two-photon excitation saturations, irradiated laser powers were adjusted to the range where fluorescent intensities were linear to the squared values of the excitation laser power values. Because fluorescent signal intensities were in linear relation to products of TPA cross-section values, quantum yield values, solution concentrations, and TPA cross-section values of the targeted dye were calculated backward to obtained fluorescent intensities and reported values of fluorescein. Finally, the calculated TPA cross-section values were plotted against the irradiated laser light wavelength.