Facile approach to benzo[d]imidazole-pyrrolo[1,2-a]pyrazine hybrid structures through double cyclodehydration and aromatization and their unique optical properties with blue emission

A modular approach to polycyclic N-fused heteroaromatics is described. Acid-catalyzed reactions of various 1-(2-oxo-2-arylethyl)-1H-pyrrole-2-carbaldehydes with several o-phenylenediamines provided facile access to a number of new benzo[d]imidazole-pyrrolo[1,2-a]pyrazine hybrid structures through double cyclodehydration and aromatization. Optical characterization of the synthesized compounds revealed unique emission properties, with deep blue emission in the aggregated and solid states, and a dramatic substituent effect was observed. Fusion of an additional benzene ring into the benzo[4,5]imidazo[1,2-a]pyrrolo[2,1-c]pyrazine scaffold resulted in a remarkable increase in the intensity of blue fluorescence from the solution along with good cell permeability and negligible phototoxicity, indicating the potential for bioimaging applications.


Introduction
Novel organic uorophores are critical tools in biomedical research and play an important role in disease diagnosis and bioimaging applications. [1][2][3][4] Due to the detrimental aggregationcaused quenching (ACQ) effects of conventional uorophores, various approaches to achieve stable and efficient emission have been explored. 5,6 Along this line, aggregation-induced emission (AIE) and aggregation-induced emission enhancement (AIEE) have drawn much attention recently, as dramatic enhancements of emission in the aggregated or solid state have been observed. [7][8][9] Although AIE luminogens have shown the potential to prevent quenching at high concentrations or in the solid state, practical issues remain, such as solubility and cell permeability, which need to be overcome to facilitate practical applications, particularly in various bioassays. 6 Furthermore, the AIE effect has challenged our current understanding of the photophysical properties of photoluminescence. Therefore, novel uorophores with unique photophysical features are of great signicance in practical applications. Blue or deep-blue emissive materials are particularly valuable in organic lightemitting diodes (OLEDs), but good blue or deep-blue emitters (AIEgens) are still rare due to their wide energy gaps. [10][11][12][13] Among the approaches reported to date, combinatorial synthesis approaches with high content imaging screening systems have shown great advantages to effectively accelerate the development of novel emissive materials, since the context of the cellular system is unpredictable in terms of the permeability, photostability and cytotoxicity of uorophores. 14,15 Nitrogen-fused heterocycles are an important structural motif frequently found in a number of natural products, pharmaceuticals, and dyes. Among them, a number of N-fused 5,6-ring systems, such as imidazo [1,2-a]pyridine, 16 indolizine, 17 and pyrazolo [1,5-a] pyrimidine, 18 have received much attention, as shown in Fig. 1.
Accordingly, numerous synthetic methods for these scaffolds have been well established, including ring annulation and intermolecular cross-coupling. [19][20][21][22] Although a number of medicinal agents based on pyrrolo[1,2-a]pyrazine, another N-fused heterobicyclic system, have been disclosed in the literature, synthetic approaches to prepare this scaffold have been relatively underdeveloped, which prompted us to investigate the chemistry of pyrrolo [1,2-a]pyrazine. In this context, we have been involved in the design and synthesis of new chemical spaces based on the pyrrolo[1,2-a] pyrazine core (Scheme 1). As a means to decorate the basic skeleton via intermolecular cross-coupling reactions, we have studied Pdcatalyzed direct (hetero)arylation 23,24 and electrophilic acetylation/ formylation 25,26 of various pyrrolo[1,2-a]pyrazines (2), which were this method gave rise to the desired products with a methyl group at the C6 position, reactions with unsymmetrical o-phenylenediamines resulted in a mixture of regioisomers. Moreover, regioisomeric mixtures were obtained with arylpropargyl-substituted substrates as a result of incomplete isomerization. In contrast, our strategy could lead to the target skeleton with not only methyl but also various aryl moieties at the C6 site, demonstrating versatility of our method. Since an additional functional group can be introduced to the a position of the ketone of our substrate 5, we deemed that our approach to this skeleton would be more exible, affording a wide variety of derivatives with substituent(s) at the C5 and/or C6 site(s). Mechanistically, dehydrative condensation of o-phenylenediamine with the aldehyde of 5 would form dihydrobenzo [d] imidazole, which would further react with other carbonyl moieties in 5 to give rise to tetracyclic compound 8 aer air oxidation. Overall, we anticipated that a polycyclic heteroaromatic ring system would be constructed by a one-pot domino protocol consisting of double cyclodehydration and aromatization.
Furthermore, as we expected that the tetracyclic skeleton formed as a hybrid structure of benzo[d]imidazole and pyrrolo [1,2-a]pyrazine, 29-32 two versatile pharmacophores in medicinal chemistry, might exhibit intriguing photophysical properties, we decided to embark on the synthesis and optical characterization of this scaffold, which is described herein.

Design and synthesis
The reaction was optimized with 5a 33 and o-phenylenediamine (Table 1). Reactions in AcOH or AcOH/toluene (1 : 1) at 90 C gave 8a in 54-55% yields (entries 1 and 2). While the use of DBSA (dodecylbenzenesulfonic acid) or PTSA as a catalyst improved the yield of 8a (entries 3-5), reactions in the presence of a Lewis acid furnished the product at a yield of 32-55% (entries 6-8). Finally, we were pleased to nd that desired product 8a was obtained in 80% yield by initial treatment of 5a and o-phenylenediamine in TFA/DMSO at room temperature for 8 h followed by warming at 65 C for an additional 3 h (entry 9). 34 The reaction in a closed vial gave similar results as that in open air. Intermediate I was identied at room temperature, and subsequent heating facilitated dehydrative cyclization to furnish 8a.
As shown in Table 2, reactions of 5a with several o-phenylenediamines were performed under optimized conditions to furnish the corresponding 6-arylbenzo [4,5]imidazo[1,2-a]pyrrolo[2,1-c]pyrazines (8a-8g) in good to excellent yields. Notably, the use of unsymmetrical o-phenylenediamines such as 4-uoro-o-phenylenediamine and 4-chloro-o-phenylenediamine under these conditions gave rise to regioisomers 8c and 8e, respectively. The structure of 8c was unambiguously conrmed by X-ray crystallographic analysis (CCDC number: 1919367, Fig. 2 Table 3, exhibiting a wide range of functional group tolerances under these reaction conditions. DBSA/toluene was used in some cases where TFA/DMSO afforded low chemical yields (8n, 8p, 8v, 8x, and 8y). The basic tetracyclic hybrid skeleton 9 was also obtained, although conversion of 5g to 9 required a higher reaction temperature (Scheme 2). When 10, derived from indole-2carbaldehyde, was allowed to react with o-phenylenediamine, the corresponding hexacyclic 11 was obtained in quantitative yield. As noted in the Introduction, benzo[d]imidazole-pyrrolo [1,2-a]pyrazines 13a-b bearing two substituents at the C5 and C6 sites were constructed upon exposure of 12a-b to the optimal reaction conditions, demonstrating the benets of our protocol over the previous approach. In addition, further elaboration of the resulting 8a was conducted: N-alkylation of 8a with methyl iodide delivered the corresponding salt 14 in quantitative yield.

Optical properties and structure-property relationship analysis
We examined the optical properties of the synthesized compounds, as shown in Fig. 3 and Table S1. † The compounds exhibited absorption maxima at approximately 320-360 nm and emission Table 2 Synthesis of 8a-8h a,b a Aer a mixture of 5a (0.188 mmol) and o-phenylenediamine (0.244 mmol, 1.3 equiv.) in TFA (0.056 mmol, 0.3 equiv.)/DMSO (1 mL) was stirred at rt for 8 h, the reaction mixture was stirred at 65 C for an additional 3 h. b Isolated yield (%). This journal is © The Royal Society of Chemistry 2020

RSC Advances Paper
This journal is © The Royal Society of Chemistry 2020 RSC Adv., 2020, 10, 7265-7288 | 7273 Paper RSC Advances  Fig. 3 along with their absorption and emission spectra. In Group A, pyrrolo [1,2-a]pyrazines without an aromatic ring at the C6 position showed strong blue emission in solution (8ai), but they were not emissive in the solid state due to the ACQ effect. The addition of benzene or naphthalene groups at the C6 position decreased the emission intensity, but the emission maxima were red-shied (Table S1 †).
Further structure-property relationship analysis revealed that the uorescence intensity and emission maxima were signicantly affected by attaching different substituents to the R 1 and R 2 positions of the benzo [4,5]imidazo[1,2-a]pyrrolo[2,1c]pyrazine scaffold (Fig. S1a †). When attaching an electronwithdrawing halide to the R 1 position (8d, Fig. S1b †), the uorescence intensity increased, and a hypsochromic shi was observed relative to 8a (Fig. S1a †). However, by attaching an electron-donating -CH 3 group to R 1 , as shown for 8g (Fig. S1c †), the opposite effect occurred, that is, a bathochromic shi. 8m with 4-ClC 6 H 4 group at the C6 position (Fig. S1d †) exhibited a slight bathochromic shi ($10 nm) compared to 8a, while 8u with 4-MeOC 6 H 4 group at R 2 (Fig. S1e †) showed blue-shied emission with increased intensity. Overall, the electronic effect from the electron-withdrawing R 1 and the electrondonating substituent on the phenyl ring of R 2 was found to be important for enhancing the uorescence intensity in Group A.
Extension with an additional benzene ring in Group B, which includes 6-phenylnaphtho [ Fig. S2 †), remarkably enhanced the uorescence intensity compared to that from Group A.

Aggregation induced blue-shied emission from Group A
Interestingly, 8c, 8g, and 8i showed an unusual hyposochromic shi in water. By increasing the water content in THF, blue-shied emission at 362 nm was observed as the emission at 435 nm gradually decreased (Fig. 4a, b and S3 †). As shown in Fig. 4c and d, the absorption and emission spectra of 8g were insensitive to solvent polarity. Therefore, the observed hyposochromic shi in aqueous solutions was suggested to be more related to the aggregated state emission. Indeed, the intensity of deep-blue emission at 362 nm in 95% water in THF solution increased by 12-fold  compared to that in a pure THF solution (Fig. 4b, inset). The observed blue shi was interesting since a bathochromic shi in the aggregated state is more common. 35 With the addition of DMSO stock to water during sonication, moderately uniform nanoparticles were formed with a diameter of 198 nm (Fig. 5). Compared to the emission of 8g in DMSO, the emission of 8g as nanoparticles exhibited a signicant blue shi by approximately 80 nm.
Bioimaging application with highly uorescent 5BP series (Group B) 5BP derivatives with an additional aromatic ring exhibited strong uorescence intensity in solution, as shown in Table 4 Fig. S2a †) but with a weaker intensity than that of 8h.
The 5BP compounds were also tested for live cell imaging in HeLa cells using the Operetta high-content imaging system (Fig. 6). Extension with an additional aromatic ring in 8h, 8k, 8q, 8t, 8z, 8ab, 8ae, and 8ah showed bright blue uorescence in the Operetta high-content screening system, demonstrating good cell permeability and potential for bioimaging applications. Meanwhile, extension with an additional benzene fused to the pyrrole side (11) did not induce signicant cellular uorescence. The phototoxicities of 8a, 8h, 8z, and 11 were negligible in HeLa cells, as shown in Fig. S4. †

Solid state emission and XRD analysis
As organic optoelectronic materials work in the solid state, the optical properties of BP scaffolds in the solid state were investigated and are summarized in Table 5. 8h and 11 exhibited signicant red shis of approximately 100 nm in their solidstate emission compared to those in solution, whereas 8a and 8g showed blue shis in the solid state (Fig. 7).
Intrigued by different patterns of emission spectra induced by aggregation, we investigated the intermolecular interactions of 8c that contributed to the blue emission in the solid and nanoaggregated states. The geometry and packing arrangements were analyzed in crystal states using single-crystal X-ray diffraction (XRD) measurements (Fig. 8).
The single crystal of 8c was depicted as a monomer, two of which were paired in an anti-parallel alignment. Notably, the torsion angle between the phenyl group and benzo [4,5]imidazo [1,2-a]pyrrolo[2,1-c]pyrazine was 90.4 , suppressing the conjugation between the phenyl ring and ABCD ring (Fig. 8a). As shown in Fig. 8b and c, the molecules were arranged in an antiparallel mode and columnar stacked array with an intermolecular vertical distance of 3.58Å between two adjacent planes. Each assembled pair was separated from neighboring pairs with a contact distance of 5.09Å between the phenyl and pyrrole plane centroids (Fig. 8d). Between the antiparallel monomers, there were intermolecular CH/N interactions (CH/N bond: 2.56Å) with the phenyl ring and the nitrogen of the imidazole B ring (Fig. 8d), as shown by yellow lines. With the columnar stacked array, the intercolumnar contact between the imidazole (B ring) and uorobenzene (A ring) formed intermolecular CH/N interactions of 2.58Å, as shown by purple lines. Additionally, nonclassical hydrogen bonds of 8c (CH/F bond: 2.73Å) incorporated the intermolecular network, rigidifying the molecular conformation, which contributed to the aggregation-induced blue shi (Fig. 8d).
Generally, in the aggregated or solid state, the emission is red-shied by the geometry change in p-stacks from charge transfer or the formation of J-aggregates. 36,37 On the other hand, solid emission at shorter wavelengths can be induced by modifying the linkage position of substituents, reducing the conjugation effect, or twisting the conformation. 11 In the case of 8c, the observed blue-shied emission was ascribed to the weakened intermolecular p-p interactions due to the longer distance between two planes in the crystal packing. 38 Conformational twisting and spatial restraint also inhibited planarization. Overall, with increasing distance, the conjugation degree was reduced, which caused a blue shi in the solid and nanoparticle states of 8c.

Conclusions
In conclusion, a novel benzo[d]imidazole-pyrrolo[1,2-a]pyrazine hybrid system, benzo [4,5] was designed and synthesized via a cascade reaction consisting of double cyclodehydration and aromatization as part of our continued efforts to expand pyrrolo[1,2-a]pyrazine-based chemical space. A wide range of derivatives were readily accessed with high atom efficiency by this modular approach under mild reaction conditions. Optical characterization of the synthesized polycyclic N-fused aromatics revealed that the uorescence intensity and emission properties were signicantly affected by attaching different substituents to the R 1 and R 2 positions of the benzo [4,5]imidazo[1,2-a]pyrrolo[2,1-c]pyrazine scaffold. Among the synthesized compounds, 8c, 8g, and 8i considerably showed aggregation-induced blue-shied emission in the solid and nanoaggregated states, which would be valuable for organic lightemitting diode (OLED) applications. Fusion with an additional benzene ring into a benzo [4,5]imidazo[1,2-a]pyrrolo[2,1-c]pyrazine scaffold resulted in a remarkable increase in blue

General methods
Unless specied, all reagents and starting materials were purchased from commercial sources and used as received without purication. "Concentrated" refers to the removal of volatile solvents via distillation using a rotary evaporator. "Dried" refers to pouring onto, or passing through, anhydrous magnesium sulfate followed by ltration. Flash chromatography was performed using silica gel (230-400 mesh) with hexanes, ethyl acetate, and dichloromethane as the eluents. All reactions were monitored by thin-layer chromatography on 0.25 mm silica plates (F-254) visualized with UV light. Melting points were measured using a capillary melting point apparatus. 1 H and 13 C NMR spectra were recorded on a 400 MHz NMR spectrometer and were described as chemical shis, multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; m, multiplet), coupling constant in hertz (Hz), and number of protons. HRMS were measured with an electrospray ionization (ESI) and Q-TOF mass analyzer.     9,10-Dichloro-6-phenylbenzo [4,5]