Microwave-Assisted Synthesis of Fluorescent Pyrido[2,3-b]indolizines from Alkylpyridinium Salts and Enaminones

Pyridinium ylides are well recognized as dipoles for cycloaddition reactions. In its turn, the microwave-assisted interaction of N-(cyanomethyl)-2-alkylpyridinium salts with enaminones unexpectedly proceeds as a domino sequence of cycloisomerization and cyclocondensation reactions, instead of a 1,3-dipolar cycloaddition. The reaction takes place in the presence of sodium acetate as base and employs benign solvents. The optical properties of the resulting pyrido[2,3-b]indolizines were studied, showing green light emission with high fluorescence quantum yields.

In this work we discovered that the reactions of 2-alkyl-N-(cyanomethyl)pyridinium salts 1 with enaminones 2 proceed unexpectedly as a two-component domino sequence of cycloisomerization and cyclocondensation reactions, while cycloaddition processes were not observed (Scheme 1). Scheme 1. General representation of the current work.

Results
Based on the previously investigated reaction [31], we started optimization of the conditions with the use of sodium acetate as base and an iso-propanol/water mixture as solvent (Table 1, entries 1-6). Varying the ratio of the starting materials and the base, the target compound 3a was obtained with 50% yield (Table 1, entry 5). The use of other bases such as Et3N, DIPEA, or NH4OAc did not improve the yield (Table 1, entries 7-9). Inorganic bases did not ameliorate the process either (Table  1, entries 10 and 11). The variation of reaction time or temperature commonly led to diminished yields (Table 1, entries [12][13][14].

Results
Based on the previously investigated reaction [31], we started optimization of the conditions with the use of sodium acetate as base and an iso-propanol/water mixture as solvent (

Results
Based on the previously investigated reaction [31], we started optimization of the conditions with the use of sodium acetate as base and an iso-propanol/water mixture as solvent ( With the optimized conditions in hand, we went on to investigate the reaction scope. It turned out that the reaction of N-cyanomethyl-2,3-dimethylpyridinium salt with enaminone was more effective, and the target product 3b was isolated with 82% yield (Scheme 2). On the contrary, the interaction of 2,5-dimethylpyridinium salt with the enaminone delivered product 3c with 27% yield. When p-methylphenyl-substituted enaminone was used, the compounds 3d-f were isolated with 21-37% yield. Bromo-substituted enaminones could be also used with various pyridinium salts to give indolizines 3g-i with 19-62% yield. Pyridoindolizines 3j-l with a phenol moiety were prepared with 24-66% yield. Moreover, we were pleased to find the pyridyl-containing enaminones to work successfully, producing the corresponding compounds 3m-r with poor to moderate yields. It is worth noting that taking N-cyanomethyl-2,3-dimethylpyridinium bromide in a large excess increased the yield of the compound 3n from 33% to 85%. Unfortunately, increasing the loading of the pyridinium salts in other cases did not result in yield improvement. The use of aliphatic enaminones (R 3 = Me or Et) in the reactions with N-cyanomethyl-2,3-dimethylpyridinium bromide generated complex mixtures, and no target product could be isolated. As a rule, the use of 2,3-dimethylpyridiniums resulted in greater yields of the target pyridoindolizines 3. The scope of the enaminones included various aryl groups, even phenols and heterocycles, while the use of aliphatic enaminones was found to be a limitation of the method.
Molecules 2020, 25, x FOR PEER REVIEW 3 of 12 With the optimized conditions in hand, we went on to investigate the reaction scope. It turned out that the reaction of N-cyanomethyl-2,3-dimethylpyridinium salt with enaminone was more effective, and the target product 3b was isolated with 82% yield (Scheme 2). On the contrary, the interaction of 2,5-dimethylpyridinium salt with the enaminone delivered product 3c with 27% yield. When p-methylphenyl-substituted enaminone was used, the compounds 3d-f were isolated with 21-37% yield. Bromo-substituted enaminones could be also used with various pyridinium salts to give indolizines 3g-i with 19-62% yield. Pyridoindolizines 3j-l with a phenol moiety were prepared with 24-66% yield. Moreover, we were pleased to find the pyridyl-containing enaminones to work successfully, producing the corresponding compounds 3m-r with poor to moderate yields. It is worth noting that taking N-cyanomethyl-2,3-dimethylpyridinium bromide in a large excess increased the yield of the compound 3n from 33% to 85%. Unfortunately, increasing the loading of the pyridinium salts in other cases did not result in yield improvement. The use of aliphatic enaminones (R 3 = Me or Et) in the reactions with N-cyanomethyl-2,3-dimethylpyridinium bromide generated complex mixtures, and no target product could be isolated. As a rule, the use of 2,3-dimethylpyridiniums resulted in greater yields of the target pyridoindolizines 3. The scope of the enaminones included various aryl groups, even phenols and heterocycles, while the use of aliphatic enaminones was found to be a limitation of the method. Scheme 2. The scope of the reaction between N-cyanomethyl-2-methylpyridinium bromides and various enaminones a . a General conditions: a mixture of pyridinium salt 1 (0.591 mmol), enaminone 2 (0.394 mmol), and sodium acetate (0.197 mmol) in isopropyl alcohol (3 mL) and water (1 mL) was placed into the microwave reactor and irradiated at 150 °C for 30 min. b The reaction was performed on 1.182 mmol scale of N-cyanomethyl-2,3-dimethylpyridinium salt.

Scheme 2.
The scope of the reaction between N-cyanomethyl-2-methylpyridinium bromides and various enaminones a . a General conditions: a mixture of pyridinium salt 1 (0.591 mmol), enaminone 2 (0.394 mmol), and sodium acetate (0.197 mmol) in isopropyl alcohol (3 mL) and water (1 mL) was placed into the microwave reactor and irradiated at 150 • C for 30 min. b The reaction was performed on 1.182 mmol scale of N-cyanomethyl-2,3-dimethylpyridinium salt.
The structure of pyridoindolizine 3b was unambiguously determined by a single crystal X-ray diffraction study (Figure 1, CCDC 1922817).
Molecules 2020, 25, x FOR PEER REVIEW 4 of 12 The structure of pyridoindolizine 3b was unambiguously determined by a single crystal X-ray diffraction study (Figure 1, CCDC 1922817).  The optical properties of the synthesized compounds were evaluated and all the spectra were measured in toluene solutions ( Figure 2, Table 2, separate images are available in Supplementary Materials). Indolizines 3a-c, m-q exhibited absorption peak maxima at 403-420 nm. The emission peak maxima lay in the green region 505-528 nm, and the largest Stokes shift 4950 cm −1 was registered for compound 3b. The fluorescence quantum yields (FQYs) were determined using coumarin 153 as a standard [32]. The lowest FQY values of 55-63% were measured for 4-pyridyl-substituted pyridoindolizines 3m-o, while the phenyl-substituted pyridoindolizine 3b demonstrated the highest FQY, 82%. This optical behavior is in accordance with the literature. For instance, indolizines, condensed with isoindole [33], quinoline [34,35] or dihydropyrrole [36] cycles also emit in the blue to green region 410-556 nm with FQYs up to 77%. The reaction presumably starts with the intramolecular cyclization of a deprotonated α-methyl group on a nitrile moiety, eventually giving an aminoindolizine intermediate A [27] (Scheme 3). The interaction of the latter with enaminone produces an intermediate B. The cyclocondensation of B completes the reaction sequence, delivering pyridoindolizine 3a. The intermediate A is evidently a highly nucleophilic species, containing a π-extensive pyrrole fragment combined with an amino group, readily reacting with the present electrophiles. Unfortunately, our attempts to isolate this intermediate failed. Even experiments in the absence of the enaminone generated multicomponent mixtures, pointing out the possibility for A to interact with the starting salt 1a.
Molecules 2020, 25, x FOR PEER REVIEW 4 of 12 The structure of pyridoindolizine 3b was unambiguously determined by a single crystal X-ray diffraction study (Figure 1, CCDC 1922817).  The optical properties of the synthesized compounds were evaluated and all the spectra were measured in toluene solutions ( Figure 2, Table 2, separate images are available in Supplementary Materials). Indolizines 3a-c, m-q exhibited absorption peak maxima at 403-420 nm. The emission peak maxima lay in the green region 505-528 nm, and the largest Stokes shift 4950 cm −1 was registered for compound 3b. The fluorescence quantum yields (FQYs) were determined using coumarin 153 as a standard [32]. The lowest FQY values of 55-63% were measured for 4-pyridyl-substituted pyridoindolizines 3m-o, while the phenyl-substituted pyridoindolizine 3b demonstrated the highest FQY, 82%. This optical behavior is in accordance with the literature. For instance, indolizines, condensed with isoindole [33], quinoline [34,35] or dihydropyrrole [36] cycles also emit in the blue to green region 410-556 nm with FQYs up to 77%. The optical properties of the synthesized compounds were evaluated and all the spectra were measured in toluene solutions ( Figure 2, Table 2, separate images are available in Supplementary Materials). Indolizines 3a-c, m-q exhibited absorption peak maxima at 403-420 nm. The emission peak maxima lay in the green region 505-528 nm, and the largest Stokes shift 4950 cm −1 was registered for compound 3b. The fluorescence quantum yields (FQYs) were determined using coumarin 153 as a standard [32]. The lowest FQY values of 55-63% were measured for 4-pyridyl-substituted pyridoindolizines 3m-o, while the phenyl-substituted pyridoindolizine 3b demonstrated the highest FQY, 82%. This optical behavior is in accordance with the literature. For instance, indolizines, condensed with isoindole [33], quinoline [34,35] or dihydropyrrole [36] cycles also emit in the blue to green region 410-556 nm with FQYs up to 77%.
Molecules 2020, 25, x FOR PEER REVIEW 5 of 12 (a) (b) Figure 2. The absorbance (a) and emission (b) spectra of the above synthesized compounds in toluene.

General Information
Starting reagents were purchased from commercial sources and were used without any additional purification. Enaminones 2 were prepared according to the literature procedures [37]. Microwave reactions were conducted in a Monowave 300 Microwave Reactor (Anton Paar GmbH, Graz, Austria). Column chromatography was performed using silica gel (230-400 mesh) and mixtures in different proportions of ethyl acetate with hexane as the mobile phase. Melting points were determined on a SMP-10 apparatus (Barloworld Scientific Limited, Stone, UK). 1 H NMR spectra were recorded on a 400 MHz spectrometer (Bruker, 100 MHz for 13 C NMR) and referenced to the residual signals of the solvent (for 1 H and 13 C). Chemical shifts are reported in parts per million (δ/ppm). Coupling constants are reported in Hertz (J/Hz). The peak patterns are indicated as follows: s, singlet; d, doublet; t, triplet; q, quadruplet; m, multiplet; dd, doublet of doublets and td, triplet of doublets. Low resolution mass spectra were recorded with an LCMS-8040 triple quadrupole liquid chromatograph mass-spectrometer (Shimadzu corp., Tokyo, Japan). The reaction progress was monitored by TLC and the spots were visualized under UV light (254 or 365 nm). Elemental analysis was performed on a EuroVector EA-3000 instrument (EuroVector S.p.A., Milan, Italy).

General Information
Starting reagents were purchased from commercial sources and were used without any additional purification. Enaminones 2 were prepared according to the literature procedures [37]. Microwave reactions were conducted in a Monowave 300 Microwave Reactor (Anton Paar GmbH, Graz, Austria). Column chromatography was performed using silica gel (230-400 mesh) and mixtures in different proportions of ethyl acetate with hexane as the mobile phase. Melting points were determined on a SMP-10 apparatus (Barloworld Scientific Limited, Stone, UK). 1 H NMR spectra were recorded on a 400 MHz spectrometer (Bruker, 100 MHz for 13 C NMR) and referenced to the residual signals of the solvent (for 1 H and 13 C). Chemical shifts are reported in parts per million (δ/ppm). Coupling constants are reported in Hertz (J/Hz). The peak patterns are indicated as follows: s, singlet; d, doublet; t, triplet; q, quadruplet; m, multiplet; dd, doublet of doublets and td, triplet of doublets. Low resolution mass spectra were recorded with an LCMS-8040 triple quadrupole liquid chromatograph mass-spectrometer (Shimadzu corp., Tokyo, Japan). The reaction progress was monitored by TLC and the spots were visualized under UV light (254 or 365 nm). Elemental analysis was performed on a EuroVector EA-3000 instrument (EuroVector S.p.A., Milan, Italy).

General Procedure for the Synthesis of Salts 1a-c
Bromoacetonitrile (0.026 mol) was added to a stirred solution of corresponding pyridine (0.022 mol) in acetonitrile (10 mL). The reaction mixture was heated at reflux for 4 h. The precipitate was filtered, washed with acetonitrile, and dried in vacuum over P 2 O 5 .   13

General Procedure for the Synthesis of Enaminones 2
A mixture of dimethylformamide dimethylacetal (14.8 mmol) and methyl ketone (14.8 mmoL) was placed into the microwave reactor and irradiated at 150 • C for 15 min, then left to cool to room temperature. After cooling, the precipitate was filtered-off, washed twice with toluene and dried on air.

General Procedure for the Synthesis of Compounds 3a-r
Method A (for 3a-g, 3i, 3m-r): A mixture of pyridinium salt 1 (0.591 mmol), enaminone 2 (0.394 mmol), and sodium acetate (0.197 mmol) in isopropyl alcohol (3 mL) and water (1 mL) was placed into the microwave reactor and irradiated at 150 • C for 30 min. After cooling to room temperature, the solvent was then evaporated under reduced pressure. The products were isolated by column chromatography on silica gel, eluting with ethyl acetate-hexane mixture in different proportions.
Method B (for 3h, 3k): A mixture of pyridinium salt 1 (0.591 mmol), enaminone 2 (0.394 mmol), and sodium acetate (0.197 mmol) in isopropyl alcohol (3 mL) and water (1 mL) was placed into the microwave reactor and irradiated at 150 • C for 30 min and left to cool to room temperature. After cooling, the precipitate was filtered-off and washed with ethanol, water (2 times) and ethanol again, then dried in air. Method C (for 3j, 3l): A mixture of pyridinium salt 1 (0.591 mmol), enaminone 2 (0.394 mmol), and sodium acetate (0.197 mmol) in isopropyl alcohol (3 mL) and water (1 mL) was placed into the microwave reactor and irradiated at 150 • C for 30 min and left to cool to room temperature. The reaction mixture was diluted with water (70 mL) and extracted with DCM. The combined organic layer was dried over Na 2 SO 4 . After filtration, the solvent was evaporated under reduced pressure. The residue was recrystallized from the isopropyl alcohol-DCM 3-1 mixture. The precipitate was filtered-off and washed with isopropyl alcohol for 3 times, then dried in air.  13 13 13

Conclusions
In conclusion, we discovered a novel domino route to condensed indolizines-pyrido [2,3-b] indolizines, containing various aromatic or heteroaromatic moieties at C (2) and alkyl groups at C(7) or C (9). The route is based on the interaction of 2-alkyl-N-(cyanomethyl)pyridinium salts with enaminones. The synthesized compounds are effective fluorophores, emitting green light with FQYs up to 82%.