Strongly fluorescent indolizine-based coumarin analogs

Abstract

It is now possible to prepare 2-oxo-2H-pyrano[2,3-b]indolizine-3-carboxylates with an ordered arrangement of various substituents directly from pyridinium salts and diethyl 2-(ethoxymethylene)malonate, allowing for refined control of their photophysical properties. Facile entry into some previously unknown derivatives is disclosed to demonstrate the potential of this method. The use of substituted picolinium salts, as well as further functionalization of the pyrrole ring, permitted easy introduction of new moieties upon the dye, which enabled fine-tuning of the photophysical properties. The obtained dyes possess absorption and emission spectrum in the blue–green region and fluorescence quantum yields reaching 92%. The parent 2-oxo-pyrano[2,3-b]indolizine-3-carboxylate turned out to be an electron-deficient system with a low-lying LUMO, an electronic transition energy of 2.7 eV and possessing a large oscillator strength. Almost complete overlap of the HOMO and LUMO in the 2-oxo-pyrano[2,3-b]indolizine core is responsible for the large fluorescence quantum yields for almost all prepared derivatives. The reason for maintaining the large emission intensity in polar solvents is that the increase in the dipole moment is accompanied with a significant change in its orientation in space. Fluorescence imaging studies have proven that 2-oxo-pyrano[2,3-b]indolizines penetrate the membrane of living cells. A positively charged analog was synthesized and used to stain intracellular organelles in the H9c2 cell line. This compound did not penetrate the cell membrane, however after permeabilization, it specifically stained the nucleus.

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Introduction

Pyrano[2,3-b]indolizin-2-ones are a unique class of heterocycles that, contrary to their analogues pyrano[3,2-b]indol-2-ones^1–12 and pyrano[2,3-b]indol-2-ones,^13–22 have been greatly ignored after they were synthesized by Kakehi et al. in the 80s^23–25 (together with their isomers pyrano[3,2-a]indolizin-2-ones)^25,26 (Fig. 1). However, their distinct structure, composed of a fused tricyclic system, in which a pyran ring is fused to an indolizine moiety, offers a wide range of opportunities for the design and development of novel compounds, exhibiting immense potential for applications in various fields, ranging from medicinal chemistry to materials science. Structurally, pyrano[2,3-b]indolizin-2-ones can be considered analogues of coumarins^27–34 and benzocoumarins.^35–42

Fig. 1 Pyrano-indol-2-ones and pyrano-indolizin-2-ones.

The synthesis of pyrano[2,3-b]indolizines was investigated by Kakehi and co-workers mainly from 1980 to 1998, driven by the need to establish an efficient and reliable methodology. Numerous synthetic routes have been explored, involving the use of pyridinium salts as starting materials and the formation of various intermediates, e.g. 3-bis(methylthio)methylene-2,3-dihydroindolizin-2-ones,²³ 2,3-dihydroindolizin-2-ones,²⁴ 3-vinylindolizines²⁵ as well as the hydrolysis of pyrano[2,3-b]indolizine-2-imines.⁴³ These synthetic efforts have resulted in the successful preparation of a diverse array of pyrano[2,3-b]indolizine derivatives, expanding the chemical space available for further exploration and application.

In the context of search for novel fluorescent platforms we resolved to explore 2-oxo-pyrano[2,3-b]indolizine-3-carboxylates because of the easily available starting materials and the strong fluorescence emission claimed by the authors in their paper, but not sufficiently explored.²⁴ The Kakehi's synthesis (Scheme 1) consisted of four steps: (1) quaternization of 2,6-dimethylpyridine with ethyl 2-bromoacetate; (2) Dieckmann condensation to form the 5-methylindolizin-2-ol intermediate; (3) Friedel–Crafts reaction of the intermediate with diethyl 2-(ethoxymethylene)malonate; (4) final intramolecular cyclization via transesterification with the formation of the lactone ring and additional ethanol elimination. It is noteworthy that the last three steps occur in one pot. However, the drawback of this methodology is that a long reaction time (7 days) is required, and the yield is relatively poor (38%). By solving the synthetic drawbacks and shedding light on the properties of this class of compounds, we aimed to provide a comprehensive understanding of this intriguing heterocyclic system, thus paving the way for further research and development of novel compounds with enhanced properties and biological application.

Scheme 1 Synthesis of ethyl 6-methyl-2-oxo-pyrano[2,3-b]indolizine-3-carboxylate.

Results and discussion

Synthesis

Optimization. Our investigation started with the synthesis of the dye 2a (Scheme 2), and we used this reaction as a model for the optimization studies, Table 1. First, we attempted to decrease the reaction time by changing the solvent to DMF (entry 2), but the expected product was not detected; instead, 2i was obtained. Then we tried NaOEt as base (entry 3), but the product was observed only in trace amounts. At this point, before proceeding to test alternative bases, we hypothesized that sequential addition of reagents, allowing first the formation of the 5-methylindolizin-2-ol intermediate (Scheme 2) and then the following steps, could be the key to increasing the yield. Different bases were evaluated (entries 4–6) and we eventually found that the use of Cs₂CO₃ along with sequential addition of the reagents turned out to be pivotal to obtain a yield of 50% (entry 6). Interestingly, Cs₂CO₃ turned out to be the best base to promote the cyclization. This result can be partly attributed to the increased basicity strength versus that of K₂CO₃. And it is in line with some recently developed synthetic approaches.^44,45–47

Scheme 2 Scope of 2-oxo-pyrano[2,3-b]indolizine-3-carboxylate. ^a Obtained by simultaneous addition of malonate.

Table 1 Conditions for the optimization of 6-methyl-2-oxo-pyrano[2,3-b]indolizine-3-carboxylate

Entry	Base	Solvent	T/°C	Time/h	Yield %
a Kakehi's methodology. b Simultaneous addition of malonate. c Postponed addition of malonate.
1^a	K2CO3	EtOH	RT	7 days	38
2^b	K2CO3	DMF	RT	18 h	15% of 2i
3^b	NaOEt	EtOH	RT	18 h	Traces
4^c	KOH	EtOH	RT	18 h	30
5^c	LiOH	EtOH	RT	18 h	15
6^c	Cs2CO3	EtOH	RT	18 h	50

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Scope. Having established the optimal reaction conditions (Cs₂CO₃, EtOH, RT, 18 h), the substrate scope was evaluated. As shown in Scheme 2, different ‘formal’ picolinium salts, prepared according to literature procedure (1a–f)^48–51 and used as such, reacted with Cs₂CO₃ to form the indolizin-2(3H)-one intermediate that, after addition of diethyl 2-(ethoxymethylene) malonate, successfully gave pyranoindolizines possessing a hydrogen or an alkyl substituent in position R⁵ (2a, 2b, 2c) in 5–50% yield.

Consistent with what was observed by Kakehi and co-workers in 1986, the reaction with 2,3-cyclopentenopyridinium salts did not lead to the indolizine product,⁵² while the reaction with a 5,6,7,8-tetrahydroquinolinium salt led to the expected product 2d in 30% yield. 2-Methylquinolinium and 1-methylisoquinolinium salts also reacted nicely and gave π-expanded indolizine products with a free R⁵ position (2e, 2f) in 30 and 28% yield, respectively, while 3-methylisoquinolin-2-ium salt did not react as expected.

Additionally, α-picoline derivatives (1g and 1h) bearing EWG groups (2-Py and CO₂Et) attached to the methylene group (R⁵) furnished the target product (2g, 2h) in 44% and 5% yield respectively. It is noteworthy that in those cases, due to the highly acidic character of the proton at the α-position, the quaternization of the pyridine with ethyl 2-bromoacetate led to a 3-substituted indolizin-2-ol directly, even in the absence of base. In such cases the base was added later, only to promote the last two steps: nucleophilic substitution and transesterification.

Meanwhile, the reaction from a 4-bromopyridinium salt and diethyl 2-(ethoxymethylene)malonate did not produce the expected product, probably due to a less reactive indolizin-2-ol intermediate. Similarly, the reaction with a 4-dimethylaminopyridinium salt did not give the target product, in this case probably due to the difficulty in the formation of the indolizine intermediate caused by the effect of the electron-donating group, which turned the proton in the α-position less acidic, thus the reagent is less prone to cyclization.

Functionalization. The structure of indolizine 2a brings about exciting possibilities to create π-expanded, polarized derivatives. Among a few possibilities, the presence of a highly electron-rich carbon β to the nitrogen was appealing to exploit (Scheme 3). To explore these possibilities, three additional derivatives (3aa, 3ab, 3ac) were obtained by direct arylation. As expected, bromoarenes with EWG in the para position, such as CN or NO₂, were more reactive in the direct arylation and the products were obtained in good yields (60% and 50%). In contrast, the bromoarene bearing an EDG, such as tBu, gave the product 3ac in poor yield (17%).

Scheme 3 Synthesis of 3aa, 3ab, 3ac and 4a from 2a, and synthesis of 6a from 4a.

The Vilsmeier–Haack formylation also successfully occurred at the electron-rich position, resulting in the formation of 4a in 83% yield.

To probe the potential of this class of dyes, Knoevenagel condensation was carried out between 4a and 1,4-dimethylpyridin-1-ium iodide (5) (Scheme 3) forming a new styryl^53–55 dye 6a, which was obtained in 37% yield and later evaluated for cell staining (vide infra).

Considering the principles of green chemistry,⁵⁶ the reactions leading to compounds 2a–2i and 4a are considered to have moderate atom economy (AE) efficiency, in the range of 40–60%. Meanwhile, direct arylation products (3aa–3ac) and Knoevenagel condensation were obtained with AE efficiency above 80%, which is considered optimal according to the above principles. For all reactions, the reaction mass efficiency is very low, less than 10%, mainly due to the significant impact of the solvent in the calculation.

Photophysical properties

UV-Vis absorption and emission spectra were recorded in nonpolar toluene (dielectric constant ε = 2.38), moderately polar DCM (ε = 8.93), and polar DMSO (ε = 46.45) for all dyes, except 6a, which was measured only in DCM and DMSO and will be discussed separately and correlated to its precursor 4a.

A comparison between the photophysical parameters for 2-oxo-pyrano[2,3-b]indolizine-3-carboxylates and those available for similar structures (Fig. 2) such as 11-benzyl-7-hydroxyisochromeno[4,3-b]indol-5-one⁹ (Φ_fl = 42% in DCM, with λ_abs = 382 and λ_em 496) or methyl 2-oxo-4-(pyrrolidin-1-yl)-2,5-dihydropyrano[3,2-b]indole-3-carboxylate⁵⁷ (λ_abs = 335 in EtOH) reveals a significant bathochromic shift for absorption, while emission and fluorescence quantum yield (Φ_fl) remain in the same range.

Fig. 2 Indoles structures used as reference to discuss photophysical properties of synthesized dyes.

The collected data of compounds in DCM are presented in Table 2 and the spectra of selected dyes are compared in Fig. 4, while the remaining data for the other solvents are available in Table S5 in ESI.† Absorption maxima (λ_abs) of the dyes obtained are located within 420–470 nm in all solvents, while the emission maxima (λ_em) falls between 440 and 520 nm for almost all of them and is more bathochromically shifted in polar solvents (solvatochromism). It is worth noting that compound 3ab represents an exception to this trend in both DCM and DMSO, showing two emission bands overlapping, one at the ‘expected’ wavelength (491 nm) and another one around 642 nm. Excitation spectra were recorded to prove that both signals come from the same molecule and are not due to impurities in the sample. This property can be explained by hypothesizing that this structure, constituting of a strong donor, 2-oxo-pirano[2,3-b]indolizine, and a strong acceptor, nitrophenyl, can undergo internal charge transfer (ICT) in polar solvents and generate two possible excited states according to the geometry and consequently allow for two possible emitting paths.

Table 2 Photophysical data of synthesized 2-oxo-pyrano[2,3-b]indolizine derivatives in DCM

Cmp	Solv	λ abs [nm]	λ em [nm]	Δ [cm^−1]	ε [M^−1 cm^−1]	Φ fl
Fluorescence quantum yields measured with Coumarin 153 in EtOH as reference (Φfl = 0.55).a Fluorescence quantum yield measured with Rhodamine 6G in EtOH as reference (Φfl = 0.95).
2a	DCM	446	464	900	30 500	0.71
2b	DCM	449	470	1000	16 300	0.77
2c	DCM	468	494	1100	22 900	0.61
2d	DCM	467	499	1400	24 600	0.64
2e	DCM	456	470	700	39 700	0.73
2f	DCM	436	458	1100	39 700	0.33
2g	DCM	466	500	1500	17 600	0.92
2h	DCM	433	452	1000	29 200	0.81
2i	DCM	446	485	1800	21 000	0.25
3aa	DCM	453	490	1700	22 928	0.64
3ab	DCM	452	491/642	6500	27 700	0.03
3ac	DCM	457	514	2400	19 600	0.65
4a	DCM	426	448	1200	31 800	0.77
6a	DCM	509	575	2300	24 400	0.44

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The fluorescence quantum yields (Φ_fl) are in most cases on the same level regardless of the solvent and they vary among the studied 2-oxopyrano[3,2-b]indolizines from 0.92 for dye 2g in DCM to being barely detectable for 3ab in DCM and DMSO due to the preferred non-emissive decay (Fig. 3). Exceptions to this trend are 2c, 2d, 2g that show lower fluorescence in toluene; and 3ab, which shows a reverse trend by having the highest Φ_fl in the less polar solvent. The comparison of the photophysical properties of 2a, 2b and 2c (Fig. 4A), which vary their position of a methyl group (or is absent), reveals that this alkyl group at position R⁵ shifts both absorption and emission bathochromically (2cvs.2b) by about 20 nm, while the same group at position R¹ slightly shifts both signals hypsochromically (2avs.2b).

Fig. 3 Comparison of Fluorescence quantum yield in different solvents for representative compounds 2b, 2g and 3ab.

Fig. 4 Absorption and emission spectra of some representative 2-oxo-pyrano[2,3-b]indolizine-3-carboxylate derivatives in DCM. (A) Normalized absorption (solid line) and normalized emission (dashed line) of 2a, 2b, 2c (green, black, pink) (B) normalized absorption (solid line) and normalized emission (dashed line) of 2c, 3aa, 3ac and 4a (pink, brown, yellow and light blue) (C) normalized absorption (solid line) and normalized emission (dashed line) of 2b, 2e and 2f (black, purple, red) (D) normalized absorption (solid line) and normalized emission (dashed line) of 4a and 6a (light blue, orange).

Moreover, different electronic character of the substituent in R⁵ (Fig. 4B) influences both λ_abs and λ_em. If CHO is present (4a), λ_abs = 426 nm and λ_em = 448 nm, while if Me is present (2c), λ_abs = 468 nm and λ_em = 494. The presence of a π-extended system, as in 3aa and 3ac, slightly blue shifts the absorption. Additionally, having a tBu group in the para position (3ac) red shifts the emission, consequently increasing the Stokes shift (2400 cm⁻¹). Interestingly, the effect of the π-expansion of the heterocyclic core strongly depends on the position of the fused benzene ring. In the case of dye 2f a blue shift is observed accompanied with a decrease of the Φ_fl (0.33 vs. 0.77) compared to 2b (Fig. 4C).

The addition of a methylpyridinium conjugated through an alkene to the 2-oxo-pirano[2,3-b]indolizine system (6a) allowed for an increased dipole moment that translates to a bathochromic shift of 83 nm for λ_abs and almost 130 nm for λ_em when compared to 4a (Fig. 4D). Even though a decrease in the fluorescence quantum yield (0.44 vs. 0.77) was observed comparing 6a and 4a, the emission is still high suggesting that, in comparison to 3ab, the decay through a radiative channel is favoured.

Fluorescence kinetic studies were performed for selected compounds: 2a representing “typical” 2-oxo-pyrano[2,3-b]indolizine, 3ab exhibiting dual fluorescence and salt 6a. In all solvents fluorescence of dyes 2a and 6a decays exponentially (Fig. S11†) with decay times, τ_F, about 3.5 and 2.0 ns respectively (Table S6†). For both compounds τ_F changes slightly with solvent's polarity (represented by dielectric constant, ε) and the change is parallel to that of Φ_fl, – for 2a both τ_F and Φ_fl grow with increasing ε whereas for 6a they decrease (Table S6†). Consequently, the radiative rate constant k_r = Φ_fl/τ_F of the S₁ → S₀ transition remains constant and amounts to 1.3 × 10⁸ s⁻¹ and 2.1 × 10⁸ s⁻¹ respectively (Table S6†). Such high value is typical for the fully allowed ππ* transitions. For dye 3ab kinetic traces are complex. In non-polar toluene fluorescence decays exponentially (Fig. S12a†) with characteristic time 2.62 ns (Table S7†). Radiative rate is 8.8 × 10⁷ s⁻¹, much less than values obtained for dyes 2a and 6a. For polar solvents where dual fluorescence from LE and CT states is observed emission decays are non-exponential and depend on wavelength of observation. For DCM the high energy band of the LE state with maximum at 487 nm two components are clearly visible in the decay trace (Fig. S12b†), and deconvolution yields 20 and 356 ps decay times. Intensity of emission originating from low energy CT band with maximum at 700 nm grows at early times and reaches maximum much later than the LE profile (Fig. S12b†). This points to precursor (LE) – successor (CT) scheme and suggests that that initial population of the CT state is null. Indeed, deconvolution reveals grow time of 55 ps and decay time of 356 ps (Table S7†). Negative amplitude of the grow time is comparable to the (positive) amplitude of the decay time what confirms that CT state is not directly excited and is populated from the LE state. This provides experimental proof for the TICT concept hypothesised below. Long component of the LE profile has same decay time as decay time of CT trace, pointing to thermalization of both states. However, grow time of CT is a bit longer than the short decay time of LE so one may expect presence of a transition state or a slow relaxation to emissive CT structure, so the simple two states model used commonly for TICT can be not adequate. In more polar DMSO kinetic profiles are faster (Fig. S12c†) and the LE decay time decreases down to 15 ps (Table S7†) pointing to the increase of the intramolecular CT rate constant. Formation of TICT enhances also non-radiative relaxation, hence the Φ_fl decreases by two orders of magnitude.

Theoretical analysis

The DFT and TDDFT O3LYP/6-31G(d,p) calculations were performed to corroborate the experimental data. The choice of the O3LYP functional allows for the best reconstruction of the spectral data in comparison to our attempts using other functionals (Table S1†). Calculations included the optimization of molecular structures in their ground (S₀) and lowest electronic excited (S₁) states along with the determination of the corresponding absorption and fluorescence energies. Corresponding data for the molecules in the three solvents, toluene, DCM and DMSO, are collected in Table S2.† The calculated dipole moments for the S₀ and S₁ states are summarized in Table S3.† The two highest occupied (HOMO's) and two lowest unoccupied molecular orbital (LUMO's) energy levels are shown in the diagram in Fig. S1,† while the values of HOMO and LUMO energies are given and compared in Table S4.† More detailed results of calculations are in the ESI.†

Dye 2b lacking any substituents can be considered a model chromophore for this family of heterocycles (Scheme 2). This is a molecule with planar geometry in both S₀, and S₁, states. The electronic transition between them has a π–π* character and essentially it is described by a single HOMO/LUMO electronic configuration. A diagram of the calculated electronic states of heterocycle 2b with a short analysis is presented in ESI, Fig. S2.†

According to the computational results, the geometry of 2b in the excited state undergoes relatively small changes. This result is consistent with the small Stokes shift observed experimentally. Small changes in geometry are manifested by small Franck–Condon factors (Fig. S3†) which well reproduce the experimental spectrum (Fig. 1). The small values of the calculated spin–orbit coupling elements (H_so) between the excited S₁ state and the energetically close triplet states (Fig. S2†) together with the small Franck–Condon factors are indicative of relatively small rate constants of non-radiative processes,^58,59 which in the experiment (Table 2) is visible as significant values of Φ_fl achieved for dye 2b.

The calculation results show also, that 2b is a molecule with a large dipole moment, increasing in the excited state (9.6 D for S₀ state and 12 D in S₁ – Table S3†). In this light, slight red-shifts of its electronic spectra (both observed, Table 2 and Table S5† and calculated, Table S2†) as the solvent polarity increases may seem surprisingly small. This phenomenon, has also been observed in the case of various other molecules,^60,61 and can be explained with the use of classical theory of the solvent effect,^60,61 and is related to the fact that dipole moments are vector quantities (and not scalars). When the increase in the dipole moment in the S₁ state (relative to the S₀ state) is accompanied by a change of orientation in space, the solvent effect is smaller than expected based on values alone (see Fig. S4†).

Fig. S2† shows the shapes of the HOMO and LUMO of molecule 2b, providing high oscillator strength for the transition between the S₀ and S₁ states. They will constitute a reference orbital in the presentation of results for other 2-oxopyrano[3,2-b]indolizines.

The substitution of molecule 2b causes changes in the energy of the molecular orbitals (Fig. S1†), including changes in the spacing between them (Table S4†). This is reflected by the shifts of the electronic spectra, since the electronic transition has an initial and final state, the magnitude of this shift depends on the energy changes in both states.

In Fig. 5, correlation between the absorption S₀ → S₁ energy and the energy difference of the HOMO and LUMO for the studied 2-oxopyrano[2,3-b]indolizines is presented. This correlation demonstrates that transitions between the S₀ and S₁ states for all molecules, as in parent dye 2b, are described by a single HOMO/LUMO electronic configuration. At the same time, two groups of molecules can be distinguished, the first with moderate changes in the HOMO/LUMO energy gaps and excitation energies (in relation to 2b), and the other group of two molecules with large changes in these quantities.

Fig. 5 Correlation between the absorption energy and the energy difference of HOMO and LUMO orbitals (numerical data in Tables S2 and S4†). The point corresponding to molecule 2b is marked with a square. Two distant points (for lower energies, i.e. with red shift in comparison to 2b) correspond to molecules 3ab and 6a. These two molecules also differ from the others in large values and changes of the dipole moment between S₀ and S₁ states (Table S3†).

Fig. 6 (part of the diagram in Fig. S1†) illustrates the variation of the E(HOMO)–E(LUMO) gap and shows the shapes of these orbitals for four representatives of the studied family of 2-oxopyrano[2,3-b]indolizines: 2b – as a model chromophore, 3ac – a dye with observed red-shift in the absorption and fluorescence spectra (relative to the spectra of 2b), 4a – with a blue-shift of the spectra and dye 6a – as a molecule with large red-shift of electronic spectra (see also Fig. 4).

Fig. 6 Diagram of orbital energies: 2b as the model heterocycle and its three derivatives, 3ac, 4a and 6a in DMSO. The numbers in brackets indicate the size of the HOMO/LUMO energy gaps. In the case of 6a two LUMOs are present in the considered energy region and in contrary to previous cases lowest of them is localised on substituent.

Based on Fig. 6, it can be concluded that the effect of substitution of molecule 2b produces two qualitatively different effects depending on the electron affinity of the substituent. In the case of compounds 3ac and 4a, this is a modification effect, i.e. their HOMO and LUMO are the modified (to a larger or smaller extent) HOMO and LUMO of molecule 2b, but this is no longer the case of the LUMO of molecule 6a. Although the HOMO of 6a is still a modified HOMO of molecule 2b, the LUMO is the orbital located on the substituent (while the orbital located on core 2b lies above it, as shown in Fig. 6).

In addition to the diagram shown on Fig. 6, two other diagrams are shown in the ESI (Fig. S5 and S6†): HOMO/LUMO energy changes from methylation of the dye 2b in different positions (2avs. 2c) and with π-extension (2evs. 2f) of 2b. Methylation of 2b at the R⁵ position causes the largest change in HOMO energy leading to a red shift of the 2c spectra.

In contrast to the substitution with a single bond, the π-expansion of 2b becomes a problem not only at the level of the HOMOs, but also at the level of the (so far untouched) LUMO of molecule 2b.

Therefore, the reason for the large red shift of the 6a (and 3ab) spectra is a qualitative change of LUMO from localized on 2b to localized on the substituent. The S₀ → S₁ transition becomes, instead of the modified 2b excitation, intramolecular charge-transfer between the core and the substituent.

Both orbitals are extended into the attached ring. The energy effect depends on the geometry in which the expansion is carried out (E_LUMO(2e) < E_LUMO(2f)).

The calculation results for excited states indicate that most of the considered molecules retain features characteristic of parent dye 2b. The fluorescence energies of the molecules optimized in the excited state are not very far from the absorption energies, and these transitions are described by high oscillator strengths (Table S1†). Although these results are in agreement with the experiments, (small Stokes shift, large emission efficiencies) these facts may seem surprising at first. This is because in these dyes there is a convenient relaxation path, which is a rotation around a single bond connecting the substituents with the heterocyclic core 2b. We can expect two effects to play a role: a difference between the dihedral angles corresponding to the energy minimum in the ground and excited states, as well as the formation of rotational conformers. Differences between geometries optimized in the ground and excited states do indeed exist. For example, in systems containing a phenyl substituent at position R⁵, such as 3aa, in the ground state, the aromatic ring makes an angle of 41.3° with the 2b plane, while in the optimized excited state it makes an angle of 30.7°; a similar rotation occurs in 3ac, from 35.9° to 29.5°. However, they do not have a major impact on the spectroscopic properties because the LUMO is not extended to the substituent (Fig. 6). The exceptions are two molecules: 3ab and 6a, because their LUMOs are located on the substituent (Fig. 6, S7 and S8†). In both cases, the dark rotational conformers were optimised in the excited state (Table S1†). These conformers are characterised by zero oscillator strength for S₁ → S₀ transition at the perpendicular arrangement of the planes of the heterocyclic core and the substituent ring. The difference between 6a and 3ab is that the dark form of 3ab is the only form optimized in the excited state in polar DMSO. This result is consistent with experimental: while 6a in this solvent emits with significant efficiency, the emission yield of 3ab is extremely low. This means that the relaxation of 6a to the dark state is slower than the decay via the radiative channel or inhibited by a barrier due to a more complex link between substituent and the heterocyclic core.

In turn, the bright form of 3ab is experimentally observed and optimised in calculations for the S₁ state, in a non-polar medium only. Fig. S8† shows a diagram of electronic states of isolated molecule 3ab with both optimised forms of the excited state. This is a diagram typical of TICT systems.⁶²

Overall, it is worth to acknowledge the consistency between experimental and theoretical results. The ratio of radiative rate constant of 2a and 6a, 1.3/2.1 = 0.62 is very similar to the ratio oscillator strength calculated for them, 0.64 : 1.06 (Tables S1 and S7†). Also, the radiative rate obtained experimentally for 3ab is two/three times lower than for 2a/6a, exactly like the oscillator strength calculated for 3ab and 2a/6a (Tables S1 and S7†). For 3ab the theoretically predicted TICT mechanism is fully confirmed by fluorescence kinetic study. These as well as other correspondence between theory and experiment supports confidence for the HOMO and LUMO overlap statements.

Imaging studies

To prove the potential applicability of 2-oxo-pyrano[2,3-b]indolizines as a fluorescent probes, incubation of rat embryonic cardiomyoblast-derived cells H9c2 with selected dyes 2g, 2c, 4a and 6a was performed. The results showed that, neutral dyes from this family (i.e.2g, 2c and 4a) penetrate the living cell membrane and, as they are uncharged, accumulate nonspecifically in a wide range of cellular structures (Fig. S13†). In the case of dye 6a which is a quaternary pyridinium salt, imaging experiments did not lead to accumulation of the dye intracellularly (Fig. 7A). To promote the access to the cytosol, H9c2 were permeabilized with digitonin (100 μg ml⁻¹). Addition of digitonin to the incubation medium resulted in permeabilization of the cell membrane and the successful penetration of the dye into the cytosol and cell organelles (Fig. 7B). It turned out that under these conditions, compound 6a, as seen in the presented image (Fig. 7B), the dye was accumulated in the structure of the nucleus and the nuclear membrane. In order to compare the dye 6a with the classical cell nuclear stain, co-staining with Hoechst 33342 was performed (Fig. 7C). It appears that the fluorescence of these two dyes overlaps except for some intranuclear structures, which are more strongly stained by pyranoindolizine 6a (Fig. 7C and D). Additional intranuclear structures more strongly stained by the dye 6a may prove useful in distinguishing specific cell nuclear structures.

Fig. 7 Intracellular localization of compound 6a in H9c2 cells detected using confocal fluorescence microscopy. (A) Image of the cells incubated with dye 6a in a non-permeabilized cell membrane. Lower diagram structure of the dye. (B) Fluorescence of cells with digitonin (100 μg ml⁻¹) permeabilized membrane. (C) Fluorescence of permeabilized cell co-incubated with 0.5 μM Hoechst 33342 recorded with a 405 nm excitation wavelength and emission ranges from 420–520 nm. (D) Overlay of B and C. Lower pictures represents zoom upper square marked cells for B, C and D. The fluorescence of 6a was recorded with a 473 nm excitation wavelength and emission ranges from 510–610 nm.

Conclusions

In conclusion, the synthesis of pyrano[2,3-b]indolizin-2-ones from picolinium salts and diethyl 2-(ethoxymethylene) malonate under basic conditions could be optimized to reach 50% yields for the one-pot procedure. The presence of an electron-rich pyrrolic position can be harnessed to open the avenue towards various novel derivatives. The presence of different substituents on the starting picoline as well as post-functionalization allowed for detailed studies on their impact on the photophysical properties in polar and non-polar solvents. It was found that the fluorescence intensity is large for all studied pyrano[2,3-b]indolizin-2-ones regardless of the substitution pattern which originates from the almost complete overlap of the HOMO and LUMO. Combined experimental and computational study enabled to conclude that pyrano[2,3-b]indolizin-2-one core can be considered as an electron-acceptor and in most of the derivatives examined in this work, the consequence of π-expansion was the extension of the HOMO and the modification of the electronic transition energy in the range of 0.2 eV. We discovered that a simultaneous increase in the dipole moment and change of its orientation in space is the reason for maintaining the large emission intensity as well as the small Stokes shift and solvatochromism, in polar solvents. Studies on the staining of the H9c2 cell line with exemplary 2-oxo-pyrano[2,3-b]indolizines has proven that uncharged dyes easily penetrate the living cells’ membrane. On the other hand styryl derivative 6a does not, it specifically stains structures associated with the cell nucleus of permeabilized cells. All reported findings may serve as the guide enabling modulation of this class of molecules towards the design of improved dyes for cell imaging applications.

Experimental section

General synthetic information

All reagents and solvents were purchased from commercial sources and used as received. Reactions with moisture and oxygen sensitive compounds were performed under an argon atmosphere. The progress of reactions was monitored by thin-layer chromatography (TLC), performed on aluminum foil plates, covered with silica gel 60 F₂₅₄ (Merck). Pure products were achieved by means of column chromatography with silica gel 60 (230–400 mesh). The identity and purity of final products were established by ¹H and ¹³C NMR spectrometry as well as by MS-spectrometry (APCI-MS or ESI-MS). All reported ¹H NMR and ¹³C NMR spectra were recorded on Varian AM 500 MHz, or Varian AM 600 MHz spectrometers. Chemical shifts (ppm) were determined with TMS as internal reference; J values are given in Hz. All melting points were measured with EZ-Melt apparatus.

Synthesis of precursors for pyrano[2,3-b]indolizin-2-ones

Pyridinium salts 1a,⁴⁸1b,⁴⁹1c,⁵⁰1d,⁴⁸1e,⁵¹1f,⁵⁰ and bis(2-pyridyl)methane 0g ⁶³ were synthesized according to previously published procedures, and spectroscopic data are consistent.

1-(2-Ethoxy-2-oxoethyl)-2,6-dimethylpyridin-1-ium bromide (1a). ¹H NMR (600 MHz, DMSO-d₆) δ 8.46 (t, J = 7.9 Hz, 1H), 7.99 (d, J = 7.9 Hz, 2H), 5.62 (s, 2H), 4.26 (q, J = 7.1 Hz, 2H), 2.78 (s, 6H), 1.25 (t, J = 7.1 Hz, 3H).

1-(2-Ethoxy-2-oxoethyl)-2-methylpyridin-1-ium bromide (1b). ¹H NMR (600 MHz, DMSO-d₆) δ 9.01 (dd, J = 6.2, 1.3 Hz, 1H), 8.61 (td, J = 7.8, 1.5 Hz, 1H), 8.15 (d, J = 7.9 Hz, 1H), 8.08 (ddd, J = 7.7, 6.0, 1.5 Hz, 1H), 5.73 (s, 2H), 4.26 (q, J = 7.1 Hz, 2H), 2.77 (s, 3H), 1.26 (t, J = 7.1 Hz, 3H).

1-(2-Ethoxy-2-oxoethyl)-2-ethylpyridin-1-ium bromide (1c). ¹H NMR (600 MHz, DMSO-d₆) δ 9.07 (d, J = 5.4, Hz, 1H), 8.65 (td, J = 7.9, 1.5 Hz, 1H), 8.15 (d, J = 7.9 Hz, 1H), 8.11–8.07 (m, 1H), 5.78 (s, 2H), 4.23 (q, J = 7.1 Hz, 2H), 3.05 (q, J = 7.4 Hz, 2H), 1.28 (t, J = 7.4 Hz, 3H), 1.24 (t, J = 7.1 Hz, 3H).

1-(2-Ethoxy-2-oxoethyl)-5,6,7,8-tetrahydroquinolin-1-ium bromide (1d). ¹H NMR (500 MHz, DMSO-d₆) δ 8.90 (d, J = 6.1 Hz, 1H), 8.42 (d, J = 7.9 Hz, 1H), 7.97 (dd, J = 7.9, 6.2 Hz, 1H), 5.70 (s, 2H), 4.23 (q, J = 7.1 Hz, 2H), 3.00–2.97 (m, 4H), 1.90–1.84 (m, 2H), 1.76–1.70 (m, 2H), 1.23 (t, J = 7.1 Hz, 3H).

1-(2-Ethoxy-2-oxoethyl)-2-methylquinolin-1-ium bromide (1e). ¹H NMR (600 MHz, DMSO-d₆) δ 9.31 (d, J = 8.5 Hz, 1H), 8.48–8.50 (m, 2H), 8.30–8.23 (m, 2H), 8.03 (t, J = 7.5 Hz, 1H), 6.12 (s, 2H), 4.28 (q, J = 7.1 Hz, 2H), 3.12 (s, 3H), 1.27 (t, J = 7.1 Hz, 3H).

2-(2-Ethoxy-2-oxoethyl)-1-methylisoquinolin-2-ium bromide (1f). ¹H NMR (500 MHz, DMSO-d₆) δ 8.80 (d, J = 8.8, 1H), 8.70 (d, J = 6.9 Hz, 1H), 8.49 (d, J = 6.9 Hz, 1H), 8.33 (d, J = 8.1 Hz, 1H), 8.27 (dd, J = 7.5, 7.5 Hz, 1H), 8.08 (ddd, J = 8.4, 6.8, 1.3 Hz, 1H), 5.91 (s, 2H), 4.25 (q, J = 7.1 Hz, 2H), 3.21 (s, 3H), 1.25 (t, J = 7.1 Hz, 3H).

1-(Pyridin-2-yl)indolizin-2-ol (1g). In a round bottom flask, bis(2-pyridyl)methane (1.70 g, 10 mmol) was mixed with ethyl 2-bromoacetate (1.11 mL, 10 mmol) in ethanol and heated at 60 °C overnight. The pyridinium salt was not possible to isolate since the reaction spontaneously proceeded to the cyclization. After confirming the formation of the indolizine on TLC with Ehrlich's reagent, the solvent was evaporated, and the crude was used for the next reaction.

Ethyl 2-oxo-2,3-dihydroindolizine-1-carboxylate (1h). In a round bottom flask, ethyl 2-(pyridyn-2-yl)acetate (1.61 mL, 10 mmol) was mixed with ethyl 2-bromoacetate (1.11 mL, 10 mmol) in ethanol and heated at 60 °C overnight. The pyridinium salt was not possible to isolate since the reaction spontaneously proceeded to the cyclization. After confirming the formation of the indolizine on TLC with Ehrlich's reagent, solvent was evaporated, and the crude was used for the next reaction.

Di(pyridin-2-yl)methane (0g). ¹H NMR (600 MHz, DMSO-d₆) δ 8.45 (ddd, J = 4.9, 1.9, 0.9 Hz, 2H), 7.64 (td, J = 7.6, 1.9 Hz, 2H), 7.26 (dt, J = 7.9, 1.2 Hz, 2H), 7.16 (ddd, J = 7.7, 4.9, 1.2 Hz, 2H), 4.23 (s, 2H).

General procedure for the synthesis of 2-oxo-2H-pyrano[2,3-b]indolizine-3-carboxylates (condition A)

In a 20 mL Schlenk flask, pyridinium salt (6 mmol, 1 equiv.) and Cs₂CO₃ (6 mmol, 1 equiv.) were added and dissolved in 5 mL of ethanol. After 1–2 hours a color change in the mixture was observed and fluorescence, typical for indoles, was visible when irradiated with UV lamp. Diethyl 2-(ethoxymethylene) malonate (6 mmol, 1 equiv.) was added, and the reaction mixture was stirred overnight at room temperature. After completion, the mixture was extracted with DCM, and the combined organic layers were dried over Na₂SO₄ and concentrated under reduced pressure. The crude product was purified by column chromatography (silica, AcOEt/hexane 1 : 1).

Ethyl 6-methyl-2-oxo-2H-pyrano[2,3-b]indolizine-3-carboxylate (2a). According to the general procedure (condition A), 1-(2-ethoxy-2-oxoethyl)-2,6-dimethylpyridin-1-ium bromide (500 mg, 1.82 mmol), Cs₂CO₃ (594 mg, 1.82 mmol) and diethyl 2-(ethoxymethylene)malonate (0.369 mL, 1.82 mmol) were reacted affording 2a (250 mg, 50%) as yellow/orange crystals. R_f = 0.48 (SiO₂, AcOEt/hexane, 3 : 1); m.p. 189.0 °C. ¹H NMR (500 MHz, CDCl₃) δ 8.97 (s, 1H), 7.46 (d, J = 8.8 Hz, 1H), 7.25 (dd, J = 8.8, 7.0 Hz, 1H), 6.77 (d, J = 7.0 Hz, 1H), 6.43 (s, 1H), 4.41 (q, J = 7.1 Hz, 2H), 2.89 (s, 3H), 1.41 (t, J = 7.1 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 165.8, 158.3, 153.7, 142.1, 138.4, 138.1, 126.9, 117.0, 114.5, 112.7, 100.8, 89.0, 61.2, 22.0, 14.5 ppm. HRMS (ESI): m/z calcd for C₁₅H₁₃NO₄Na, 294.0742 [M + Na]⁺; found, 294.0741.

Ethyl 2-oxo-2H-pyrano[2,3-b]indolizine-3-carboxylate (2b). According to the general procedure (condition A), 1-(2-ethoxy-2-oxoethyl)-2-methylpyridin-1-ium bromide (0.500 mg, 2.77 mmol), Cs₂CO₃ (904 mg, 2.77 mmol) and diethyl 2-(ethoxymethylene)malonate (0.561 mL, 2.77 mmol) were reacted affording 2b (20 mg, 5%) as yellow crystals. R_f = 0.32 (SiO₂, AcOEt/hexane, 3 : 1); m.p. 199.5 °C. ¹H NMR (500 MHz, CDCl₃) δ 8.81 (s, 1H), 8.27 (d, J = 6.9 Hz, 1H), 7.50 (d, J = 9.0 Hz, 1H), 7.26–7.20 (m, 1H), 6.90 (t, J = 6.9 Hz, 1H), 6.33 (s, 1H), 4.41 (q, J = 7.1 Hz, 2H), 1.41 (t, J = 7.1 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 165.1, 158.8, 152.9, 140.5, 133.7, 126.3, 124.6, 119.4, 113.0, 111.2, 101.9, 88.2, 61.3, 14.5. HRMS (ESI): m/z calcd for C₁₄H₁₁NO₄Na, 280.0586 [M + Na]⁺; found, 280.0583.

Ethyl 10-methyl-2-oxo-2H-pyrano[2,3-b]indolizine-3-carboxylate (2c). According to the general procedure (condition A), 1-(2-ethoxy-2-oxoethyl)-2-ethylpyridin-1-ium bromide (0.200 mg, 1.03 mmol), Cs₂CO₃ (335 mg, 1.03 mmol) and diethyl 2-(ethoxymethylene)malonate (0.208 mL, 1.03 mmol) were reacted affording 2c (100 mg, 35%) as orange crystals. R_f = 0.54 (SiO₂, AcOEt/hexane, 3 : 1); m.p. 215.8 °C. ¹H NMR (500 MHz, CDCl₃) δ 8.76 (s, 1H), 8.23 (dt, J = 6.9, 1.1 Hz, 1H), 7.45 (dt, J = 9.0, 1.2 Hz, 1H), 7.21 (ddd, J = 9.0, 6.8, 1.1 Hz, 1H), 6.86 (td, J = 6.9, 1.2 Hz, 1H), 4.40 (q, J = 7.1 Hz, 2H), 2.30 (s, 3H), 1.41 (t, J = 7.1 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 165.3, 159.2, 150.7, 139.7, 133.1, 125.6, 124.5, 117.5, 112.6, 110.8, 100.8, 96.6, 61.1, 14.5, 6.2 ppm. HRMS (ESI): m/z calcd for C₁₅H₁₃NO₄Na, 294.0742 [M + Na]⁺; found, 294.0739.

Ethyl 8-oxo-5,6-dihydro-4H,8H-pyrano[2′,3′:4,5]pyrrolo[3,2,1-ij]quinoline-9-carboxylate (2d). According to the general procedure (condition A), 1-(2-ethoxy-2-oxoethyl)-5,6,7,8-tetrahydroquinolin-1-ium bromide (250 mg, 1.13 mmol), Cs₂CO₃ (370 mg, 1.13 mmol) and diethyl 2 (ethoxymethylene)malonate (0.229 mL, 1.13 mmol) were reacted affording 2d (110 mg, 30%) as orange/red crystals. R_f = 0.48 (SiO₂, AcOEt/hexane, 3 : 1); m.p. 244.1 °C. ¹H NMR (600 MHz, CDCl₃) δ 8.76 (s, 1H), 8.05 (dd, J = 6.9, 0.8 Hz, 1H), 6.90 (dd, J = 6.9, 0.8 Hz, 1H), 6.79 (t, J = 6.8 Hz, 1H), 4.40 (q, J = 7.1 Hz, 2H), 2.93–2.86 (m, 4H), 2.03 (p, J = 6.1 Hz, 2H), 1.41 (t, J = 7.1 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 165.4, 159.3, 148.0, 139.5, 133.6, 130.8, 121.8, 121.5, 113.4, 111.5, 99.9, 98.3, 61.1, 27.4, 22.0, 19.8, 14.5 ppm. HRMS (ESI): m/z calcd for C₁₇H₁₅NO₄Na, 320.0899 [M + Na]⁺; found, 320.0902.

Ethyl 9-oxo-9H-pyrano[2′,3′:4,5]pyrrolo[1,2-a]quinoline-10-carboxylate (2e). According to the general procedure (condition A), 1-(2-ethoxy-2-oxoethyl)-2-methylquinolin-1-ium bromide (200 mg, 0.645 mmol), Cs₂CO₃ (210 mg, 0.645 mmol) and diethyl 2-(ethoxymethylene)malonate (0.130 mL, 0.645 mmol) were reacted affording 2e (60 mg, 30%) as yellow crystals. R_f = 0.56 (SiO₂, AcOEt/hexane, 2 : 1); m.p. not measured, decomposed around 280 °C. ¹H NMR (500 MHz, CDCl₃) δ 9.36 (s, 1H), 8.32 (d, J = 8.5 Hz, 1H), 7.82 (dd, J = 7.9, 1.3 Hz, 1H), 7.77–7.72 (m, 1H), 7.54–7.48 (m, 2H), 7.41 (d, J = 9.2 Hz, 1H), 6.48 (s, 1H), 4.46 (q, J = 7.1 Hz, 2H), 1.45 (t, J = 7.1 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 165.5, 157.9, 154.3, 140.0, 138.0, 134.6, 130.6, 130.1, 127.8, 125.1, 124.3, 117.8, 115.6, 114.2, 103.5, 91.7, 61.6, 14.5 ppm. HRMS (APCI): m/z calcd for C₁₈H₁₃NO₄, 307.0845 [M−]; found, 307.0847.

Ethyl 10-oxo-10H-pyrano[2′,3′:4,5]pyrrolo[2,1-a]isoquinoline-9-carboxylate (2f). According to the general procedure (condition A), 2-(2-ethoxy-2-oxoethyl)-1-methylisoquinolin-2-ium bromide (250 mg, 0.806 mmol), Cs₂CO₃ (263 mg, 0.806 mmol) and diethyl 2-(ethoxymethylene)malonate (0.163 mL, 0.806 mmol) were reacted affording 2f (70 mg, 28%) as yellow crystals. R_f = 0.64 (SiO₂, AcOEt/hexane, 3 : 1); m.p. 261.2 °C. ¹H NMR (600 MHz, CDCl₃) δ 8.79 (d, J = 0.8 Hz, 1H), 8.11–8.08 (m, 1H), 8.01 (d, J = 7.1 Hz, 1H), 7.74–7.69 (m, 1H), 7.65–7.58 (m, 2H), 7.11 (d, J = 7.2 Hz, 1H), 6.84 (s, 1H), 4.41 (q, J = 7.1 Hz, 2H), 1.41 (t, J = 7.1 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 165.0, 158.8, 152.9, 138.6, 134.0, 129.6, 129.2, 128.7, 127.6, 124.3, 121.0, 113.2, 112.5, 103.3, 88.8, 61.4, 14.4 ppm. HRMS (ESI): m/z calcd for C₁₈H₁₃NO₄Na, 330.0742 [M + Na]⁺; found, 330.0746.

Ethyl 2-oxo-10-(pyridin-2-yl)-2H-pyrano[2,3-b]indolizine-3-carboxylate (2g). According to the general procedure (condition A), 1-(pyridin-2-yl)indolizin-2(3H)-one (250 mg, 1.19 mmol), Cs₂CO₃ (387 mg, 1.19 mmol) and diethyl 2-(ethoxymethylene)malonate (0.240 mL, 1.19 mmol) were reacted affording 2g (177 mg, 44%) as yellow/orange crystals. R_f = 0.37 (SiO₂, AcOEt/hexane, 2 : 1); m.p. 252.6 °C. ¹H NMR (500 MHz, CDCl₃) δ 9.02 (d, J = 9.1 Hz, 1H), 8.83 (s, 1H), 8.67–8.64 (m, 1H), 8.33 (d, J = 6.8 Hz, 1H), 8.23 (d, J = 8.1 Hz, 1H), 7.78 (td, J = 7.8, 1.9 Hz, 1H), 7.41 (ddd, J = 9.1, 6.9, 1.2 Hz, 1H), 7.16 (ddd, J = 7.5, 4.8, 1.1 Hz, 1H), 7.03 (td, J = 6.9, 1.3 Hz, 1H), 4.42 (q, J = 7.1 Hz, 2H), 1.43 (t, J = 7.1 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 164.8, 158.4, 151.4, 150.4, 148.9, 139.5, 136.7, 133.4, 127.9, 124.3, 122.6, 122.1, 120.9, 114.3, 111.1, 101.7, 101.2, 61.3, 14.5. HRMS (ESI): m/z calcd for C₁₉H₁₅N₂O₄, 335.1032 [M + H]⁺; found, 335.1031.

Diethyl 2-oxo-2H-pyrano[2,3-b]indolizine-3,10-dicarboxylate (2h). According to the general procedure (condition A), ethyl 2-oxo-2,3-dihydroindolizine-1-carboxylate generated in situ (404 mg, 2.00 mmol), Cs₂CO₃ (652 mg, 2.00 mmol) and diethyl 2 (ethoxymethylene)malonate (0.404 mL, 2.00 mmol) were reacted affording 2h (35 mg, 5%) as yellow crystals. R_f = 0.43 (SiO₂, AcOEt/hexane, 3 : 1); m.p. 227.9 °C. ¹H NMR (500 MHz, CDCl₃) δ 8.82 (s, 1H), 8.43 (d, J = 8.8 Hz, 1H), 8.37 (d, J = 6.8 Hz, 1H), 7.50 (t, J = 8.8 Hz, 1H), 7.13 (t, J = 6.8 Hz, 1H), 4.42 (q, J = 24.5 Hz, 2H), 4.40 (q, J = 24.5 Hz, 2H), 1.43 (t, J = 21.3, Hz, 3H), 1.40 (t, J = 21.3, Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 164.5, 162.4, 157.6, 152.3, 140.8, 134.0, 129.1, 124.6, 120.4, 115.1, 110.8, 104.2, 93.8, 61.5, 60.5, 14.5, 14.4 ppm. HRMS (ESI): m/z calcd for C₁₇H₁₅NO₆Na, 352.0797 [M + Na]⁺; found, 352.0798.

General procedure for the synthesis of 2-oxo-2H-pyrano[2,3-b]indolizine-3-carboxylates (condition B)

In a 20 mL Schlenk flask, pyridinium salt (6 mmol, 1 equiv.), potassium carbonate (6 mmol, 1 equiv.) and diethyl 2-(ethoxymethylene)malonate (6 mmol, 1 equiv.) were added and dissolved in 10 mL of DMF. The reaction mixture changed color and was stirred for 24 hours at room temperature. After completion, the mixture was extracted with DCM, and the combined organic layers were dried over Na₂SO₄ and concentrated under reduced pressure. The crude product was purified by column chromatography (silica, AcOEt/hexane 1 : 1).

Diethyl 2-((3-(ethoxycarbonyl)-6-methyl-2-oxo-2H-pyrano[2,3-b]indolizin-10-yl)methylene)malonate (2i). According to the general procedure (condition B), 1-(2-ethoxy-2-oxoethyl)-2,6-dimethylpyridin-1-ium bromide (450 mg, 2.3 mmol), potassium carbonate (320 mg, 2.3 mmol) and diethyl 2-(ethoxymethylene)malonate (0.467 mL, 2.3 mmol) were reacted affording 2i (100 mg, 15%) as yellow/orange crystals. R_f = 0.45 (SiO₂, AcOEt/hexane, 3 : 1); m.p. 210.5 °C. ¹H NMR (600 MHz, CDCl₃) δ 8.92 (s, 1H), 7.77 (s, 1H), 7.65 (d, J = 8.6 Hz, 1H), 7.36 (dd, J = 8.9, 7.1 Hz, 1H), 6.85 (d, J = 7.1 Hz, 1H), 4.53 (q, J = 7.1 Hz, 2H), 4.35 (q, J = 7.1 Hz, 2H), 4.25 (q, J = 7.2 Hz, 2H), 2.86 (s, 3H), 1.34 (t, J = 7.2 Hz, 3H), 1.32 (t, J = 7.2 Hz, 3H), 1.28 (t, J = 7.2 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 166.7, 165.1, 165.1, 156.5, 151.5, 141.6, 138.9, 138.1, 128.9, 128.7, 124.7, 116.4, 115.1, 113.3, 102.9, 96.9, 62.4, 61.6, 61.5, 22.1, 14.4, 14.2, 14.0 ppm. HRMS (APCI): m/z calcd for C₂₃H₂₂NO₈, 442.1345 [M − H]⁻; found, 440.1350.

General procedure for direct arylation

A Schlenk flask flushed with argon, was charged with ethyl 6-methyl-2-oxo-2H-pyrano[2,3-b]indolizine-3-carboxylate (0.55 mmol, 1 equiv.), aryl bromide (0.66 mmol, 1.2 equiv.), potassium acetate (1.11 mmol, 2 equiv.), NMP (3 mL) and PdCl₂(PPh₃)₂ (0.027 mmol, 0.05 equiv.). The solution was stirred at 100 °C for 10 min. Afterwards, water (1.11 mmol, 2 equiv.) was added, and the heating was continued at the same temperature overnight. Then, the mixture was cooled to room temperature and extracted with DCM. The combined organic layers were dried over Na₂SO₄ and concentrated under reduced pressure. The crude product was purified by column chromatography (silica, AcOEt/hexane 1 : 1).

Ethyl 10-(4-cyanophenyl)-6-methyl-2-oxo-2H-pyrano[2,3-b]indolizine-3-carboxylate (3aa). According to the general procedure for direct arylation, ethyl 6-methyl-2-oxo-2H-pyrano[2,3-b]indolizine-3-carboxylate (0.55 mmol, 1 equiv.), 4-bromobenzonitrile (0.66 mmol, 1.2 equiv.), potassium acetate (1.11 mmol, 2 equiv.), NMP (3 mL), PdCl₂(PPh₃)₂ (0.027 mmol, 0.05 equiv.), and water (1.11 mmol, 2 equiv.) were made to react affording 3aa (120 mg, 60%) as greenish yellow crystals. R_f = 0.45 (SiO₂, AcOEt/hexane, 3 : 1); m.p. 306.1 °C. ¹H NMR (600 MHz, CDCl₃) δ 9.06 (s, 1H), 7.84–7.81 (m, 2H), 7.80–7.77 (m, 3H), 7.38 (dd, J = 8.9, 7.0 Hz, 1H), 6.89 (d, J = 7.0 Hz, 1H), 4.42 (q, J = 7.1 Hz, 2H), 2.96 (s, 3H), 1.42 (t, J = 7.1 Hz, 3H). ¹³C NMR (151 MHz, CDCl₃) δ 165.3, 157.4, 150.5, 139.4, 138.9, 138.3, 135.1, 132.7, 129.0, 128.0, 118.9, 115.6, 115.4, 112.5, 110.4, 102.3, 101.4, 61.5, 22.3, 14.4 ppm. HRMS (APCI): m/z calcd for C₂₂H₁₅N₂O₄, 371.1032 [M − H]⁻; found, 371.1036.

Ethyl 6-methyl-10-(4-nitrophenyl)-2-oxo-2H-pyrano[2,3-b]indolizine-3-carboxylate (3ab). According to the general procedure for direct arylation, ethyl 6-methyl-2-oxo-2H-pyrano[2,3-b]indolizine-3-carboxylate (0.55 mmol, 1 equiv.), 1-bromo-4-nitrobenzene (0.66 mmol, 1.2 equiv.), potassium acetate (1.11 mmol, 2 equiv.), NMP (3 mL), PdCl₂(PPh₃)₂ (0.027 mmol, 0.05 equiv.), and water (1.11 mmol, 2 equiv.) were made to react affording 3ab (25 mg, 50%) as orange crystals. R_f = 0.45 (SiO₂, AcOEt/hexane, 3 : 1); m.p. 284.3 °C. ¹H NMR (500 MHz, CDCl₃) δ 9.07 (s, 1H), 8.36 (d, J = 8.8 Hz, 2H), 7.89 (d, J = 8.8 Hz, 2H), 7.83 (d, J = 8.9 Hz, 1H), 7.40 (dd, J = 9.0, 7.0 Hz, 1H), 6.91 (d, J = 7.1 Hz, 1H), 4.43 (q, J = 7.1 Hz, 2H), 2.97 (s, 3H), 1.42 (t, J = 7.1 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 165.3, 157.3, 150.7, 146.3, 139.4, 139.0, 138.3, 137.2, 129.0, 128.2, 124.3, 115.8, 115.5, 112.5, 102.5, 101.1, 61.5, 22.3, 14.4 ppm. HRMS (APCI): m/z calcd for C₂₁H₁₅N₂O₆, 391.0930 [M − H]⁻; found, 391.0932.

Ethyl 10-(4-(tert-butyl)phenyl)-6-methyl-2-oxo-2H-pyrano[2,3-b]indolizine-3-carboxylate (3ac). According to the general procedure for direct arylation, ethyl 6-methyl-2-oxo-2H-pyrano[2,3-b]indolizine-3-carboxylate (0.20 mmol, 1 equiv.), 1-bromo-4-(tert-butyl)benzene (0.26 mmol, 1.2 equiv.), potassium acetate (0.40 mmol, 2 equiv.), NMP (3 mL), PdCl₂(PPh₃)₂ (0.010 mmol, 0.05 equiv.), and water (0.40 mmol, 2 equiv.) were made to react affording 3ac (14 mg, 17%) as orange/brown crystals. R_f = 0.56 (SiO₂, AcOEt/hexane, 2 : 1); m.p. 241.6 °C. ¹H NMR (600 MHz, CD₂Cl₂) δ 8.93 (s, 1H), 7.71 (d, J = 8.7 Hz, 1H), 7.57–7.51 (m, 2H), 7.51–7.46 (m, 2H), 7.24 (dd, J = 8.9, 6.9 Hz, 1H), 6.76 (d, J = 7.0 Hz, 1H), 4.27 (q, J = 7.1 Hz, 2H), 2.84 (s, 3H), 1.32–1.29 (m, 12H). ¹³C NMR (126 MHz, CDCl₃) δ 165.7, 158.1, 150.3, 140.1, 138.5, 137.9, 128.9, 128.5, 127.0, 126.8, 125.9, 116.1, 114.9, 112.5, 103.3, 101.1, 61.2, 34.7, 31.3, 22.2, 14.5 ppm. HRMS (APCI): m/z calcd for C₂₅H₂₆NO₄, 404.1862 [M + H]⁺; found, 404.1861.

Procedure for the synthesis of styryl dye

Ethyl 10-formyl-6-methyl-2-oxo-2H-pyrano[2,3-b]indolizine-3-carboxylate (4a). In a round bottom flask, POCl₃ (0.280 mL, 3 mmol) was added to dry DMF (0.310 mL, 4 mmol), at 0 °C. The ice bath was removed and the solution was stirred for 30 min. A solution of ethyl 6-methyl-2-oxo-2H-pyrano[2,3-b]indolizine-3-carboxylate (0.250 g, 0.92 mmol) in dry DCE (5 mL) was added and the mixture was stirred at 85 °C for 16 hours. After cooling to room temperature, an aqueous solution of saturated sodium acetate (3 mL) was added and heated again for 1 hour at 100 °C, then the reaction mixture was cooled to room temperature and the water-organic layer was extracted with DCM. The combined organic layers were dried over Na₂SO₄ and concentrated under reduced pressure. The crude product was purified by column chromatography (silica, AcOEt/hexane 1 : 1) affording 4a (230 mg, 83%) as yellow crystals. R_f = 0.40 (SiO₂, AcOEt/hexane, 3 : 1); m.p. 241.4 °C. ¹H NMR (500 MHz, CDCl₃) δ 10.29 (s, 1H), 9.03 (s, 1H), 8.54 (d, J = 8.8 Hz, 1H), 7.60 (dd, J = 8.8, 7.2 Hz, 1H), 7.05 (d, J = 7.2 Hz, 1H), 4.43 (q, J = 7.1 Hz, 2H), 2.99 (s, 3H), 1.43 (t, J = 7.1 Hz, 3H). ¹³C NMR (126 MHz, CDCl₃) δ 181.8, 164.8, 156.6, 155.8, 140.6, 139.1, 139.0, 131.5, 118.5, 118.0, 112.2, 103.7, 102.3, 61.7, 22.1, 14.4. ppm. HRMS (APCI): m/z calcd for C₁₆H₁₂NO₅, 298.0714 [M − H]⁻; found, 298.0715.

4-Methylpyridinium iodide (5). Synthesis was conducted according to literature.⁶⁴¹H NMR (500 MHz, DMSO-d₆) δ 8.81 (d, J = 6.4 Hz, 2H), 7.94 (d, J = 6.2 Hz, 2H), 4.26 (s, 3H), 2.58 (s, 3H).

(E)-4-(2-(3-(Methoxycarbonyl)-6-methyl-2-oxo-2H-pyrano[2,3-b]indolizin-10-yl)vinyl)-1-methylpyridin-1-ium iodide (6a). In a round bottom flask, 4-methylpyridinium iodide (18 mg, 0.17 mmol), ethyl 10-formyl-6-methyl-2-oxo-2H-pyrano[2,3-b]indolizine-3-carboxylate (51 mg, 0.17 mmol) and piperidine (0.017 mL) were dissolved in MeOH (4 mL) and refluxed overnight. After cooling down to room temperature, a precipitate is formed, filtered and washed with MeOH to afford ethyl 10-formyl-6-methyl-2-oxo-2H-pyrano[2,3-b]indolizine-3-carboxylate as red crystals 6a (25 mg, 37%). M.p. 250.7 °C. ¹H NMR (500 MHz, DMSO-d₆) δ 8.94 (s, 1H), 8.78 (d, J = 6.3 Hz, 2H), 8.34–8.19 (m, 4H), 7.68 (t, J = 7.9 Hz, 1H), 7.31 (d, J = 16.0 Hz, 1H), 7.20 (d, J = 7.1 Hz, 1H), 4.23 (s, 3H), 3.81 (s, 3H), 2.95 (s, 3H). ¹³C NMR (126 MHz, DMSO-d₆) δ 164.9, 156.7, 153.4, 151.7, 145.0, 141.4, 141.2, 139.5, 130.5, 129.0, 123.0, 121.2, 117.4, 115.6, 113.8, 101.1, 99.2, 52.5, 47.0, 21.5 ppm. HRMS (ESI): m/z calcd for C₂₂H₁₉N₂O₄, 375.1345.1501 [M⁺]; found, 375.1348.

Photophysical measurements

HPLC grade solvents were purchased from Sigma-Aldrich and used as obtained. For optical studies, solutions of molecules at low concentrations were used to avoid dimerization or reabsorption effects. All absorption and fluorescence spectra were taken at room temperature (21 °C). A Shimadzu UV/VIS-NIR spectrophotometer, model UV-3600i Plus, was used for absorption spectra measurement. Fluorescence spectra were recorded with an FS5 spectrofluorometer from Edinburgh Instruments. Fluorescence quantum yields (Φ_fl) of molecules in solvents at 21 °C were determined using Coumarin 153 in EtOH (Φ_fl = 0.38) and Rhodamine 6 g in EtOH (Φ_fl = 0.95) as references. Solutions of low absorbance (A < 0.1) were used to avoid reabsorption or concentration quenching. Refractive index correction for solvents have been performed in the calculations of quantum yields. Molar absorption coefficient, ε, was calculated from the absorbance, A, of a solution of the given molar concentration, c, in a cuvette of length, l, according to Lambert Beer formula:

A = c ε l

Fluorescence kinetics studies were performed using the time correlated single photon counting technique. A mode-locked Coherent Mira-HP picosecond laser pumped by a Verdi 18 laser was used for excitation. The fundamental pulses of the Mira laser were up-converted to 387.5 nm. The temporal width of the excitation pulses was ∼280 fs and the instrument response function (IRF) about 40 ps. Fluorescence was dispersed with a 0.25 m Jarrell-Ash monochromator and detected with a HMP-100-07 hybrid detector coupled to an SPC-150 PC module, (Becker&Hickl GmbH). Fluorescence decays were analyzed with deconvolution software using a nonlinear least squares procedure with the Marquardt algorithm. A standard χ² test and Durbin–Watson (DW) parameter along with residual and autocorrelation function plots were used to assess the quality of a fit. The estimated accuracy for the determination of decay time was below 10 ps.

Computational details

The calculations were performed using the Gaussian 16 program.⁶⁵ The solvent effect was introduced in PCM procedure. The SOC elements were also calculated using the Orca program.⁶⁶

Biological studies

The rat embryonic cardiomyoblast-derived H9c2 cells were loaded with the fluorophore at the final concentration of 500 nM in DMEM medium supplemented with 10% foetal bovine serum, 2 mM glutamine, 100 U ml⁻¹ penicillin, and 100 μg ml⁻¹ streptomycin, and incubated at 37 °C in a humidified atmosphere containing 5% CO₂ for 15 to 60 minutes. After that, the incubation medium was replaced with FluoroBrite™ DMEM. The measurements were performed with the use of an Olympus IX83 confocal microscope with the silicone objective 60× UPLSAPO 60XS. Registered data were transferred to the ImageJ and analysed for presentation.

Author contributions

Conceptualization: D. T. G. and J. S. A. B.; investigation: J. S. A. B., A. W., I. D.; supervision: D. T. G., A. S.; visualization: J. S. A. B., A. W., I. D.; writing – original draft: J. S. A. B., A. W., I. D.; writing – review and editing: D. T. G., J. S. A. B.

All the authors discussed the results and commented on the manuscript.

Data availability

The data supporting this article have been included as part of the ESI.†

Conflicts of interest

There are no conflicts to declare.

Acknowledgements

This work was supported by the Polish National Science Center, Poland (grants OPUS 2020/37/B/ST4/00017 and HARMONIA 2018/30/M/ST5/00460). This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowka-Curie grant agreement no 101007804. The work was financially supported by the Foundation for Polish Science (TEAM POIR.04.04.00-00-3CF4/16-00). Calculations were performed at the Interdisciplinary Center for Mathematical and Computational Modeling (ICM) University of Warsaw under computational allocation G95-1734. We thank Joseph Milton for amending the manuscript.

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Footnote

† Electronic supplementary information (ESI) available: NMR, UV/vis, fluorescence spectra and computational details. See DOI: https://doi.org/10.1039/d4qo01216j

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