1 Introduction

The chemical [1,2,3] or photochemical [4, 5] oxidation of 2-phenyl- or naphtyridinylindoles is a good way to obtain 4H-3,1-benzoxazin-4-ones. Benzoxazinone derivatives are an important class of N-heterocyclic compounds that find synthetic, pharmaceutical, and biological applications [6,7,8,9,10,11,12,13].

In a previous work we have shown that 2-substitued benzoxazinone can be easily formed upon photooxidation of 2-substitued indole derivative in acetonitrile in the presence of air. No additional photosensitizer is necessary in this reaction. The substrate itself acts as a photosensitizer: after UV excitation the energy is transferred to the triplet state of the chromophore and, next, to molecular oxygen with the formation of singlet oxygen and, finally, the product [5].

In this work we investigate the influence of protic and aprotic environment on the photooxidation process of two indole derivatives, 2-(pyrazin-2′-yl)-1H-indole (1) and 2,5-di(1H-indol-2′-yl)pyrazine (2) (Scheme 1) which leads to the formation of 4H-3,1-benzoxazin-4-ones derivatives.

Scheme 1.
scheme 1

Structures of 2-(pyrazin-2′-yl)-1H-indole (1) and 2,5-di(1H-indol-2′-yl)pyrazine (2)

Full size image

Photophysical and photochemical properties of bifunctional azaaromatic indole derivatives that possess both the proton donor and proton acceptor abilities can be changed by the environment. In protic solvents these compounds form hydrogen-bonded complexes, which can effectively diminish their photodestruction. To explain this effect, two mechanisms were proposed, each leading to an increased rate of depopulation of the excited singlet state: (i) fast excited state double proton transfer occurring in 1:1 cyclic complexes with water or alcohol and (ii) efficient S0 ← S1 internal conversion in 1:2 stoichiometry complexes. Both of these processes are faster than T1 ← S1 intersystem crossing and can therefore diminish the quantum yield of triplet state formation, as well as the singlet oxygen formation efficiency [14,15,16,17,18].

Previously we have demonstrated the photostabilizing abilities of alcohols for three azaaromatic chromophores: 2-(1H-indol-2′-yl)-[1,5]naphthyridine (3), 2-(1H-indol-2′-yl)-[1,8]naphthyridine (4) [17] and 12,13-dihydro-5H-indolo[3,2-c]acridine (5) [18] (Scheme 2).

Scheme 2.
scheme 2

Structures of 2-(1H-indol-2′-yl)-[1,5]naphthyridine (3), 2-(1H-indol-2′-yl)-[1,8]naphthyridine (4) and 12,13-dihydro-5H-indolo[3,2-c]acridine (5)

Full size image

In this report we investigate photostability of two indolepyrazines: 2-(pyrazin-2′-yl)-1H-indole (1) and 2,5-di(1H-indol-2′-yl)pyrazine (2). These molecules possess the same structural motif as 3–5, consisting of pyridine and pyrrole nitrogen atoms separated by three bonds, but the presence of pyrazine ring suggests that the electronic properties and the ability to form intra- and intermolecular hydrogen bonds may be different. We find that the quantum yields of photodegradation of 1 and 2 are comparable and the molecules are about ten times more photostable in the protic solvents than in aprotic ones. The photostabilization ability due to hydrogen bonding interactions is, however, significantly lower than that previously observed for compounds 3–5(30 to 200 times). The mechanism of the photoreaction involves singlet oxygen mediated oxidation of the indole ring of the studied chromophore with formation of benzoxazinone derivatives: 2-(pyrazin-2-yl)-4H-3,1-benzoxazin-4-one (12) and 2-[5-(1H-indol-2-yl)pyrazin-2-yl]-4H-3,1-benzoxazin-4-one (14) for molecules 1 and 2, respectively. The formation of the photoproducts is slower and less efficient in methanol because the quantum yield of singlet oxygen formation in this solvent is diminished by hydrogen bonding interactions with the chromophore.

2 Experimental

2.1 Synthesis of 1 and 2

The pyrazinyl indoles 1 and 2 were prepared from the corresponding phenylhydrazone derivatives 8 and 11 by application of the Fisher indole synthesis (Schemes 3 and 4).

Scheme 3.
scheme 3

Preparation of 2-(pyrazin-2′-yl)-1H-indole (1)

Full size image
Scheme 4.
scheme 4

Preparation of 2,5-di(1H-indol-2′-yl)pyrazine (2)

Full size image

NMR spectra were obtained on a General Electric QE-300 spectrometer at 300 MHz for 1H NMR and at 75 MHz for 13C NMR. Chemical shifts were reported in parts per million relative to the residual proton or carbon of the corresponding solvent. Melting points were measured on Thomas Hoover capillary melting point apparatus and are not corrected. Elemental analyses were carried out by QTI, P.O. Box 470, Whitehouse, NJ 08,888–0470. All chemicals are commercially available, and all solvents were reagent grade.

2.1.1 Phenylhydrazone of 2-acetylpyrazine (8)

A mixture of phenylhydrazine (6, 0.30 g, 2.8 mmol), 2-acetylpyrazine (7, 0.28 g, 2.3 mmol), and glacial AcOH (3 drops) in absolute EtOH (7 cm3) provided 8 as a yellow solid which was washed with hexane and filtered (0.46 g, 94%): mp 141–142 °C; 1H-NMR (DMSO-d6) \(\delta\) 8.36 (d, 1H, J = 1.5 Hz), 8.40 (d, 1H, J = 2.7 Hz), 8.33 (d, 1H, J = 2.4 Hz), 7.63 (bs, 1H), 7.26 (t, 2H, J = 7.2 Hz), 7.17 (t, 2H, J = 7.2 Hz), 6.89 (t, 1H, J = 7.2 Hz), 2.29 (s, 3H).

2.1.2 2-(pyrazin-2′-yl)-1H-indole (1)

A mixture of 8 (0.42 g, 2.0 mmol) and polyphosphoric acid (PPA 3.0 g) in p-xylene (10 cm3) was mechanically stirred at 110–120 °C for 2 h. After cooling to room temperature, p-xylene was decanted and the remaining solid was dissolved in water (100 cm3). The solution was made basic by adding 10% NaOH until pH 14. A dark green precipitate is obtained which was filtered and dried under vacuum. Chromatography on alumina, eluting with CH2Cl2 gave 1 (0.35 g, 80%) as a white solid which was recrystallized from CH2Cl2/hexane: mp 158–160 °C; 1H-NMR (CDCl3) \(\delta\) 9.44 (bs, 1H), 9.10 (s, 1H), 8.50 (t, J = 0.9 Hz, 1H), 8.41 (t, J = 1.5 Hz 1H), 7.44 (d, J = 7.8 Hz, 1H), 7.27 (m, 1H), 7.15 (m, 2H); 13C-NMR (CDCl3) \(\delta\) 146.2, 143.5, 142.2, 142.0, 136.9, 133.5, 128.9, 124.0, 121.6, 120.6, 111.4, 102.1; Anal. Calcd for C12H9N3: C, 73.85; H, 4.61; N, 21.54. Found: C, 73.57; H, 4.56; N, 21.40.

2.1.3 2, 5-Diacetylpyrazine (10) [19]

To a solution of pyrazine (3.0 g, 38 mmol) and acetaldehyde (8.3 g, 19 mmol) in glacial AcOH (45 cm3) and H2O (45 cm3) was added conc. H2SO4 (9 cm3). The resulting solution was cooled to 0 °C. In a clean beaker, FeSO4-7H2O (2.1 g, 75 mmol) was dissolved in 70% t-butylhydroperoxide (9.7 g) and H2O (30 cm3). The peroxide solution was slowly added over 5 min to the chilled pyrazine solution and the mixture was allowed to stand at room temperature for 1 h. The homogeneous solution was kept at 4 °C for 3 d. The resulting yellow crystals were collected, washed with H2O (2 × 20 cm3) and air dried to give 10 (2.2 g, 36%): mp 156–158 °C (lit. mp1 158 °C); 1HNMR (CDCl3) δ 9.25 (s, 2H), 2.76 (s, 6H).

2.1.4 Bis-phenylhydrazone of 2,5-diacetylpyrazine (11)

In the same manner as described for 1, the reaction of phenyl hydrazine (0.26 g, 2.4 mmol), 6 (0.20 g, 1.2 mmol), and glacial AcOH (0.5 cm3) in absolute EtOH (20 ml) provided 11 as a yellow solid (0.33 g, 80%): mp 234–236 °C; 1H-NMR (CDCl3) δ 9.31 (s, 1H), 7.66 (s, 1H), 7.33 (dd, J = 8.1 Hz, 2H), 7.24 (d, J = 7.5 Hz, 2H), 6.95 (dd, J = 6.9 Hz, 1H), 2.39 (s, 6H).

2.1.5 2,5-di(1H-indol-2′-yl)pyrazine (2)

In the manner described above for 8, a mixture of 11 (1.0 g, 2.9 mmol) and PPA (3.0 g) provided 2 as a green solid which was extracted using soxhlet extraction. Chromatography on alumina, eluting with CH2Cl2 gave 2 as a yellow solid (0.85 g, 95%): mp > 300 °C; 1HNMR (DMSO-d6) δ 11.86 (s, 2H), 9.27 (s, 2H), 7.60 (d, J = 8.1 Hz, 2H), 7.47 (d, J = 8.4 Hz, 2H), 7.36 (s, 2H), 7.16 (dd, J = 6.9 Hz, 2H), 7.03 (dd, J = 6.9 Hz, 2H); 13C (DMSO-d6) δ 144.1, 141.1, 138.0, 134.8, 128.7, 123.3, 121.2, 120.3, 112.5, 102.3.

2.2 Solvents

The following solvents (spectroscopic or LCMS grade) were used: n-hexane, acetonitrile, methanol, 1-butanol, and water (Aldrich or Merck). Prior to measurements the solvents were carefully examined and showed no traces of absorption or emission.

2.3 Absorption and fluorescence

Electronic absorption spectra were measured with a Shimadzu UV 2700 spectrophotometer. Stationary emission and fluorescence excitation spectra were obtained using an Edinburgh FS 5 spectrofluorometer. The spectra were corrected for the spectral sensitivity of the instrument. Fluorescence curves, recorded as a function of wavelength (λ), were subsequently multiplied by λ2 in order to convert counts per wavelength into counts per wavenumber. The quantum yield of fluorescence, Φf, was determined by a reference method, using 0.05 mol dm−3 quinine sulfate in sulfuric acid (Φf = 0.51) as the standard [20]. Fluorescence decay lifetimes were determined by time-resolved single photon counting (SPC) on a home-built SPC setup consisting of an IBH Nanoled emitting diode (297 nm, and pulse width < 750 ps), Becker&Hickl PMC 100-4 photomultiplier, Spectral Products Digikröm CM 110 monochromator, and PicoQuant TimeHarp 100 PC card. PicoQuant Fluofit software (version 3.3) was used to analyze the decay curves.

2.4 Singlet oxygen generation

The quantum yield of singlet oxygen generation, ΦΔ, was determined with a home-built setup. An Opotek Radiant 355 laser was used for excitation at 340 or 370 nm. The emission was detected using a BENTHAM DTMc300 double monochromator, equipped with a Hamamatsu H10330C-75 thermoelectrically cooled photomultiplier. In order to determine ΦΔ amplitudes of singlet oxygen phosphorescence decay curves were measured at 1275 nm, the emission maximum, photosensitized by the standard (signal amplitude A0) and by the compound under investigation (amplitude Ax). Both samples were compared using the same solvent (acetonitrile or methanol, depending on the experiment). ΦΔ = Φ0Δ × Ax × \((1-10^{{-{\text {OD}}}_0})\)/(A0 × (1–10−ODx)), where \(1-10^{{-{\text {OD}}}_0}\) and 1–10−ODx correspond to fractions of light absorbed by the phenalenone and the measured compound, respectively, at a given excitation wavelength. At room temperature the quantum yield of singlet oxygen generation for phenalenone is equal to 0.98 both in n-hexane and methanol [21].

2.5 Photodegradation

The photodegradation of the compounds was studied in the regime of 365 nm irradiation. Solutions of 1 and 2 (~ 5 × 10–5 mol dm−3) were prepared in n-hexane, acetonitrile, and methanol and then irradiated in a sealed quartz cuvette. High power (270 mW) 365 nm LED diode (Roithner Laser Technik, H2A1-H365-r4) was used for irradiation. The light intensity was determined by potassium ferrioxalate actinometry as 2.17 × 1016 quanta s−1 [22]. The photodegradation process was monitored by UV–Vis absorption measurements. The detailed procedure of the determination of the quantum yield of photodegradation for the studied compounds is described in Supporting Information.

2.6 LC–MS analysis

The photoproducts were analysed using Shimadzu Nexara X2 LC/MS/MS 8050 system with positive or negative ESI ionization methods. The samples dissolved in acetonitrile (concentration of about 5 × 10–5 mol dm−3) were irradiated by the 365 nm UV diode in a quartz cuvette and next transferred into UPLC vials and diluted 5 times with the solvent. The samples were subsequently injected into the LC/MS/MS system for the determination of the masses of the substrate and the photoproducts. Chromatographic separation was achieved with a 00D-4424-B0 Synergi™ 4 µm Fusion-RP 80 Å, LC column 100 × 2 mm (Phenomenex). The ESI–MS settings were as follows: capillary voltage 4000 V (positive mode) and 3000 V (negative mode), nebulizing gas flow 3.0 L/min, and drying gas flow 5 L/min at 300 °C. The mobile phase was composed of acetonitrile or methanol (90%) and water (10%). The flow rate was 0.4 mL/min with an isocratic elution. The injection volume was 2 μL. The column temperature was set at 40 °C. The mass-to-charge ratio (m/z) scan range was from 80 to 1000 (positive and negative). The data were processed using LabSolutions software (Shimadzu).

2.7 Quantum chemical calculations

Gaussian 09 software package was used [23]. All the calculations were performed by applying the density functional theory (DFT) method using B3LYP functional [24, 25] with 6−31+G(d,p) basis set. Vertical excitation energies and excited-state geometries were obtained by the time-dependent DFT (TDDFT) method. Vibrational frequency analysis was performed to predict zero-point vibrational energies (ZPVE) of different forms. The counterpoise technique [26, 27] was used to correct for the basis-set superposition error (BSSE).

3 Results and discussion

3.1 Photophysical data

3.1.1 Solvent effects on electronic absorption, fluorescence, and excitation spectra

1 can exist in two and 2 in three different rotameric forms (Fig. 1). The syn form of 1 is about 16 kJ mol−1 lower in energy than the anti conformer. For 2 the DFT calculations predict that the syn-anti and anti-anti forms have energies higher than the most stable syn-syn conformer by about 16 and 31 kJ mol−1, respectively.

Fig. 1
figure 1

Rotameric forms of 1 and 2 with the predicted relative energies

Full size image

Figure 2 shows the simulated electronic absorption spectra of the rotameric forms of 1 and 2.

Fig. 2
figure 2

Absorption spectra of the rotameric forms of 1 and 2 simulated using optimized ground-state geometries. The spectral envelopes were obtained by convolution of the respective stick spectra with a Gaussian function of 3000 cm−1 FWHM

Full size image

It is well known that solvent can influence a chromophore by electrostatic or hydrogen bonding interactions and change its spectral, photophysical, and photochemical characteristics. Therefore, the electronic absorption and fluorescence spectra of 1 and 2 have been measured in three solvents of different polarity and proticity: n-hexane (aprotic, nonpolar), acetonitrile (aprotic, polar), and methanol (protic, polar) (Fig. 3).

Fig. 3
figure 3

Normalized absorption and fluorescence spectra of 1 (top) and 2 (bottom) in n-hexane (full black lines), acetonitrile (dotted red lines) and methanol (dashed blue lines)

Full size image

Spectral and photophysical characteristics of the experimental absorption and emission spectra in different solvents are presented in Table 1.

Table 1 Spectral and photophysical parameters of 1 and 2 in n-hexane, acetonitrile, and methanol measured at room temperature
Full size table

The spectroscopic and photophysical data of 1 and 2 show a distinct change with the nature of the solvent (Table 1). The position of the lowest absorption band of 1 and 2 is almost the same in all solvents, but the maximum of the emission band is moving to the red with increasing polarity of the solvent. For molecule 1 the observed Stokes shifts are larger in all solvents than for molecule 2. Most probably this relates to the differences in dipole moments of these compounds in S0 and S1 states (compare Table S3 in the Supporting Information). The fluorescence quantum yields, Φf, are about one hundred (for 1) and ten times (for 2) higher in aprotic solvents (n-hexane and acetonitrile) than in methanol. In all solvents 2 exhibits higher values of Φf than observed for compounds 1 and 3–5 (see Table 1 and Table S1 in the Supporting Information for comparison). For this type of bifunctional indole derivatives the fluorescence quantum yields in n-hexane were reported to span the range from 0.99 for 2-(3΄-pyridyl)indole to 0.04 for 3,3΄-tetramethylene-2-(2΄-pyridyl)indole [28].

In nonpolar n-hexane the fluorescence decays are monoexponential and the lifetime values are 0.92 ns for 1 and 1.5 ns for 2. The quantum yields (Φf) and lifetimes (τ) obtained in this medium lead to large values of the radiative rate constants (Eq. (1)), 2.6 × 108 s−1 (1) and 4.1 × 108 s−1 (2). Radiative and radiationless rate constants were calculated using the Eqs. (1) and (2), respectively. The data are shown in the Table 1.

$$k_{{\text{f}}} = \Phi_{{\text{f}}} /\tau ,$$
(1)
$$k_{{{\text{nr}}}} = \left( {1 - \Phi_{{\text{f}}} } \right)/\tau .$$
(2)

The electronic transition dipole moments in fluorescence Mfl, calculated from Eq. (3) [29, 30] are 4.7 D and 6.5 D for 1 and 2, respectively.

$$k_{{\text{f}}} = \frac{{64\pi^{4} }}{3h}\left( {n\tilde{\nu }_{{{\text{fl}}}} } \right)^{3} \left| {\vec{M}_{{{\text{fl}}}} } \right|^{2} .$$
(3)

In this equation knr is the radiationless rate constant, h is the Planck constant, n is the refractive index of the solvent, and \(\tilde{\nu }_{{{\text{fl}}}}\) is the wavenumber of fluorescence maximum.Also the Mfl values (which are the best quantitative measure of the transition probability) are comparable to those of the electronic transition dipole moments Mabs (Eq. 4 [30]) related to the lowest 1(π,π*) ← S0 absorption band

$$\left| {\vec{M}_{{{\text{abs}}}} } \right|^{2} = \frac{3\ln 10hc}{{8\pi^{3} N_{{\text{A}}} n\tilde{\nu }_{{{\text{abs}}}} }}\int {\varepsilon (\tilde{\nu }){\text{d}}\tilde{\nu }} .$$
(4)

In this equation \(\varepsilon (\tilde{\nu })\) is the molar absorption coefficient, \(\tilde{\nu }_{{{\text{abs}}}}\) is the wavenumber of absorption maximum, NA is the Avogadro number, and c is the speed of light. The similarity of Mfl and Mabs values indicates a similar nature of the Franck–Condon excited states initially reached in absorption and the solvent-equilibrated fluorescent states. Most probably, the absorption and fluorescence in n-hexane correspond to the syn forms of 1 and 2. In this solvent monoexponential fluorescence decay was observed.

Compounds 1 and 2 in acetonitrile show biexponential fluorescence decay which, similarly to the case of 2-(2΄-pyridyl)pyrrole [31, 32] and 3 and 4 [17] in polar aprotic media is due most probably to the presence of both syn and anti rotameric forms. In acetonitrile both decay times are longer than the monoexponential decay time observed in n-hexane. The stabilization mechanism of the anti forms of 1 in in this polar solvent, is not obvious, because the syn and anti forms have predicted similar dipole moment values of equal 1.4 D and 0.9 D, respectively. In the case of 2 the calculations predict zero dipole moment values for centrosymmetric syn-syn and anti-anti forms and 2.2 D for the syn-anti structure. These data suggest that in a case of 2 the syn-anti conformer can be stabilized in polar acetonitrile; the fluorescence decay data show that in acetonitrile a few percent of fluorescence originate from the syn-anti form with longer decay time values (~ 8 ns).

In alcohols both compounds have very short decay times (< 0.2 ns), probably originating from the emission of hydrogen-bonded syn forms of the compounds. For 1:1 syn complexes of 4 with water [17] and 5 with methanol [18] similar short lifetimes were observed. Additionally, for 2 a longer lifetime (~ 0.5 ns) is observed in methanol, which can be assigned to hydrogenbonded syn-anti form of this molecule.

3.1.2 Complexation with methanol

The B3LYP/6–31+G(d,p) optimized structures of the most stable complexes of 1 and 2 with methanol of 1:1 are shown in Figs. 4 and 5, respectively. The stabilization energies of these complexes are listed in Table 2. These results indicate that the most stable structures of 1:1 stoichiometry are cyclic complexes of the syn rotamers, with proton donating and accepting units connected by a methanol link with two cooperative hydrogen-bonds. This type of complexes is predicted to be the most stable in the case of all molecules 1–5. The complexes of 1–3 have similar binding energies (ΔEC) about −27 kJ mol−1, whereas for 4 and 5 the stabilization energies are about 10 kJ mol−1 lower (Table S2 in the Supporting Information).

Fig. 4
figure 4

Optimized structures of 1:1 complexes of 1 with methanol

Full size image
Fig. 5
figure 5

Optimized structures of 1:1 complexes of 2 with methanol

Full size image
Table 2 Calculated binding energies of the ground state complexes of 1 and 2 with methanol
Full size table

To establish the stoichiometry of the ground state complexes of 1 with alcohols we performed the titration experiments with mixed solvents [14]. Upon adding small amounts of 1-butanol to n-hexane solutions of 1 the growth of the low-energy side intensities and isosbestic points in the absorption spectra are observed (Fig. 6). This shows the formation of equilibrium between two species in the ground state, which are assigned to the uncomplexed and complexed forms of 1.

Fig. 6
figure 6

Changes in absorption observed upon titrating n-hexane solutions of 1 with 1-butanol. The alcohol concentration varied from 0 to 0.036 mol dm−3 (A). Determination of the stoichiometry of 1-butanol solvates of 1 (B). The number of alcohol molecules in the complex, n, is given by the slope of the plot (Eqs. 5 and 6 in the text). Absorbance changes were monitored at 385 nm (26,000 cm−1), n = 1.04 ± 0.04, correlation coefficient r = 0.986

Full size image

For a reaction:

$$X + nY = XY_{n} ,$$
(5)

which involves one molecule of chromophore X and n molecules of alcohol Y the equilibrium constant, K1n, may be expressed as follows:

$$K_{1n} = \frac{{\left[ {XY_{n} } \right]}}{{\left[ X \right]\left[ Y \right]^{n} }}\;K_{1n} = \frac{{A - A_{0} }}{{\left( {A_{\infty } - A} \right)\left[ Q \right]^{n} }},$$
(6)

where A0 and A∞ are the absorbance values obtained when only the uncomplexed or complexed forms are present, respectively; A is the optical density measured at an alcohol concentration [Q]. The number of alcohol molecules, n, in the complex can be derived from the plot of ln[(A − A0)/(A∞ − A)] vs. ln[Q].

The absorption data yielded the 1:1 stoichiometry of the complexes of 1 for which the equilibrium constants is equal to 12 (± 5) dm3 mol−1. The obtained equilibrium constant is similar to these observed for 3 (14 (± 10) dm3 mol−1), but much smaller than the values derived for 4 and 5 (515 (± 20) dm3 mol−1 and 122 (± 10) dm3 mol−1, respectively) [17, 18]. This observation is in line with the results of DFT calculations, which predict a much higher binding energy for the cyclic doubly hydrogen-bonded syn complexes of 4 and 5 than for complexes of 1 and 3 (Table S2 in Supporting Information).

For molecule 2, an analogous experiment was also done, but because of poor solubility of the chromophore in n-hexane the obtained data are not quantitatively reliable and are not presented here.

3.1.3 Singlet oxygen quantum yield

The determined values of quantum yield of singlet oxygen generation are higher in acetonitrile than for methanol solution for all studied compounds 1–5 (Table 3). The largest difference is observed for compound 1. In this case the \({\Phi }_{\Delta }\) in alcohol is about 35 times lower than in acetonitrile. For compound 2 this difference is about ten times smaller than for 1.

Table 3 Comparison of the quantum yield of singlet oxygen formation \({\Phi }_{\Delta }\) determined in acetonitrile (ACN) and methanol (MeOH) for compounds 1–5
Full size table

3.2 Photochemistry

3.2.1 Photodegradation quantum yield

Photostability of 1 and 2 dissolved in n-hexane, acetonitrile, and methanol was studied by measuring the absorption intensity during continuous illumination at 365 nm by a high power LED. Figure 7 shows the spectral changes observed during the UV irradiation of 1 and 2 in acetonitrile. Upon irradiation the absorption bands of the indolepyrazines gradually disappear and new bands appear, belonging to the photoproduct. The appearance of clear isosbestic points in the absorption spectrum suggests that under irradiation the stoichiometry of the photoreaction is preserved. Most probably the direct substrate to product transformation occurs and only a one photoproduct is formed. A similar spectral behaviour was observed also for n-hexane and methanol solutions of these compounds.

Fig. 7
figure 7

Changes in absorption observed upon 365 nm irradiation of 1 and 2 in acetonitrile. The spectra recorded before and after irradiation are represented by thick black and red lines, respectively

Full size image

The photodegradation quantum yields, \({\Phi }_{\mathrm{pho}}\), determined for molecules 1 and 2 dissolved in n-hexane, acetonitrile, and methanol are listed in Table 4 and compared with the values obtained previously for chromophores 3–5. A detailed description of the procedures used for the determination of \({\Phi }_{\mathrm{pho}}\) values is given in the Supporting Information. The data show that alcohol acts for all compounds as a photostabilizer. The molecules 1 and 2 are, respectively, 20 and 10 times more photostable in methanol than in acetonitrile. These results are comparable with the data obtained for 3, but the stabilizing effect of hydrogen bonding interactions is smaller than observed for 4 and 5.

Table 4 The comparison of photodegradation quantum yield, \({\Phi }_{\mathrm{pho}}\), of the indole derivatives 1–5 determined in n-hexane, acetonitrile, and methanol solutions under 365 nm irradiation
Full size table

3.2.2 Photoproducts

To establish the nature of the photoproducts, solutions of 1 and 2 before and after 365 nm irradiation were analyzed using LC–MS. Figures S1 and S2 in Supporting Information show the ESI–MS (positive and negative) spectra of 1 before and after irradiation in acetonitrile and methanol, respectively. In both solvents, as a result of irradiation, the amount of ions of the substrate with m/z equal to 196.1 ([M + H]+) and 194.1 ([M − H]−) decreases and new ions belonging to photoproducts appear with m/z equal to 226.1 (positive) and 243.3 (negative). Based on our previous studies on 3 [5] and the existing literature data on singlet oxygen-mediated photooxidation of 2-phenylindole [4], we assign the ions with m/z = 226.1 to 2-(pyrazin-2-yl)-4H-3,1-benzoxazin-4-one (12) and the ions with m/z = 243.3 to the product of hydrolysis of 12, namely 2-[(pyrazine-2-carbonyl)amino]benzoic acid (13). The products of hydrolysis of 4H-3,1-benzoxazin-4-one derivatives with analogous structure were identified during the synthesis of benzoxazinones from 2-phenylindoles [2]. The LC–MS data obtained for 2 in acetonitrile show that upon UV irradiation the amount of positive ions with m/z = 311.1 decreases and a new ion appears with the m/z = 341.2 (see Figure S3 in Supporting Information). The new ion can be assigned to 2-[5-(1H-indol-2-yl)pyrazin-2-yl]-4H-3,1-benzoxazin-4-one (14). For 2 no negative MS signals which could be assigned to the substrate or product were observed. The schematic photooxidation pathways of 1 and 2 are presented in Schemes 5 and 6, respectively.

Scheme 5.
scheme 5

Proposed pathway for the formation of 2-(pyrazin-2-yl)-4H-3,1-benzoxazin-4-one (12) and 2-[(pyrazine-2-carbonyl)amino]benzoic acid (13) from 1

Full size image
Scheme 6.
scheme 6

Proposed pathway for the formation of 2-[5-(1H-indol-2-yl)pyrazin-2-yl]-4H-3,1-benzoxazin-4-one (14) from 2

Full size image

4 Summary

The photophysical properties of indolepyrazines: 2-(pyrazin-2′-yl)-1H-indole (1) and 2,5-di(1H-indol-2′-yl)pyrazine (2), similar to other 2-substitued indole derivatives studied previously, 2-(1H-indol-2′-yl)-[1,5]naphthyridine (3), 2-(1H-indol-2′-yl)-[1,8]naphthyridine (4), and 12,13-dihydro-5H-indolo[3,2-c]acridine (5) strongly depend on the nature of the solvent (its polarity and protic abilities). Fluorescence quantum yields drastically decrease (10 to 100 times) in methanol in comparison to the values observed in aprotic n-hexane and polar acetonitrile. The strong increase of photostability can be explained by formation of hydrogen-bonded complexes with alcohol, which leads to efficient nonradiative deactivation of the excited S1 state of the studied chromophores. The interactions with the protic solvent lead to less efficient intersystem crossing and therefore the values of the quantum yield of singlet oxygen formation in methanol are significantly lower (from 3 to 35 times) in comparison to the values measured in acetonitrile. Therefore, all the studied molecules 1–5 are more photostable in methanol than in acetonitrile or n-hexane. The photodegradation quantum yields of molecules 1–3 are, respectively, 20, 10, and 16 times lower in methanol than in acetonitrile. For chromophores 4 and 5 this difference is larger: 39- and 147-fold.

The explanation of the origin of the observed differences in photodegradation quantum yields of molecules 1–5 requires additional investigations. The photodegradation quantum yield (Φpho) is proportional to the quantum yield of triplet state formation (ΦT), the rate constant of photoreaction (kpho) and the triplet state lifetime (τT). These three factors need not correlate for different molecules. Therefore, it is extremely difficult to provide a general model for predicting relative photostability in a series of heteroazaaromatic compounds. Assuming that the quantum yield of singlet oxygen generation is a low limit value of the quantum yield of the triplet state formation, the photodegradation quantum yield can be correlated with the quantum yield of singlet oxygen formation. The observed lack of such correlation in the case of compounds 1–5 suggests that the triplet state lifetime is an additional important parameter to be involved in the qualitative characterization of the photodegradation quantum yield. In our previous publication we showed that the triplet states lifetime of molecule 5 differs significantly between nonpolar and polar solvents (from tens of ns in hexane to a few ns in acetonitrile) [18]. In order to assess the role of three different factors in the photostability, we plan to measure triplet lifetimes of molecules 1–4 in different solvents.

365 nm irradiation of 1 and 2 in acetonitrile or methanol at ambient temperature in the presence of air, similarly as was observed for 3 [5], leads to formation of the photoproduct with benzoxazinone structure. Starting from 1 leads to 2-(pyrazin-2-yl)-4H-3,1-benzoxazin-4-one (12). Additionally, a product of hydrolysis of 12, namely 2-[(pyrazine-2-carbonyl)amino]benzoic acid (13) was also observed.

In the case of 2 as a substrate, 2-[5-(1H-indol-2-yl)pyrazin-2-yl]-4H-3,1-benzoxazin-4-one (14) was identified as the photoproduct. The mechanism of photoreaction most likely involves two steps: (i) the singlet oxygen formation and (ii) its insertion into the indole ring of the studied compound. This photooxidation reaction does not require the use of any external photosensitizer. The studied indole derivatives act simultaneously as the substrate and the photosensitizer.