Nucleophilic Functionalization of 2-R-3-Nitropyridines as a Versatile Approach to Novel Fluorescent Molecules

A number of new 2-methyl- and 2-arylvinyl-3-nitropyridines were synthesized and their reactions with thiols were studied. It was found that 3-NO2 tends to be selectively substituted under the action of sulfur nucleophiles in the presence of another nucleofuge in position 5. Correlations between the substitution pattern and regioselectivity as well as photophysical properties were established. Some synthesized compounds possessed a large Stokes shift.


Introduction
Pyridine is an important heterocyclic motif and a part of various natural products. The pyridine ring system is incorporated into alkaloids, medicines (for example, omeprazole, lorlatinib, ivosidenib and many others), fungicides, herbicides and insecticides. Application of the pyridine derivatives as biologically active precursors and coordination complexes was reviewed recently [1]. Nitropyridines are of particular interest due to their biological significance [2][3][4][5]. In addition, some nitropyridines are considered to be promising energetic compounds [6][7][8][9][10] and efficient organic optical materials [11]. The introduction of the nitro group into the pyridine ring facilitates its functionalization in different ways. Recently, we investigated reactions of 3-R-5-nitropyridines with various types of nucleophiles [12]. It was found that in the case of anionic S-, N-and O-nucleophiles, the substitution of the nonactivated nitro group occurred while carbon nucleophiles underwent dearomatization of the pyridine ring with the formation of 1,2-or 1,4-addition products. As a result, a number of novel or hardly accessible pyridines and their dihydro derivatives were synthesized [12].
In this work we report on the synthesis, reactivity and photophysical properties of 2-methyl-and 2-(2-arylvinyl)-3-nitropyridines, as shown in Figure 1. 2-Alkenylpyridines are widely employed as precursors to pharmaceuticals (vorapaxar, axitinib, nifurpirinol) and other biologically active compounds [13]. In addition, 2-(2-arylvinyl)pyridines were proven to be the fluorescent molecules, with the fluorescence quantum yield showing a large dependence on the acidity of media [14].

Introduction
Pyridine is an important heterocyclic motif and a part of various natural products The pyridine ring system is incorporated into alkaloids, medicines (for example omeprazole, lorlatinib, ivosidenib and many others), fungicides, herbicides and insecticides. Application of the pyridine derivatives as biologically active precursors and coordination complexes was reviewed recently [1]. Nitropyridines are of particular interest due to their biological significance [2][3][4][5]. In addition, some nitropyridines are considered to be promising energetic compounds [6][7][8][9][10] and efficient organic optical materials [11] The introduction of the nitro group into the pyridine ring facilitates its functionalization in different ways. Recently, we investigated reactions of 3-R-5-nitropyridines with various types of nucleophiles [12]. It was found that in the case of anionic S-, N-and O-nucleophiles, the substitution of the non-activated nitro group occurred while carbon nucleophiles underwent dearomatization of the pyridine ring with the formation of 1,2 or 1,4-addition products. As a result, a number of novel or hardly accessible pyridines and their dihydro derivatives were synthesized [12].
In this work we report on the synthesis, reactivity and photophysical properties of 2-methyl-and 2-(2-arylvinyl)-3-nitropyridines, as shown in Figure 1. 2-Alkenylpyridines are widely employed as precursors to pharmaceuticals (vorapaxar, axitinib, nifurpirinol and other biologically active compounds [13]. In addition, 2-(2-arylvinyl)pyridines were proven to be the fluorescent molecules, with the fluorescence quantum yield showing a large dependence on the acidity of media [14].

Results and Discussion
2-Methyl-3-nitropyridines 2a-c were synthesized from the corresponding commercially available 2-chloro-3-nitropyridines 1a-c by the reaction with diethyl malonate, followed by acidic hydrolysis/decarboxylation, as shown in Scheme 1. The oxidation of compounds 2b,c with the hydrogen peroxide-urea complex gave N-oxides 3b,c in moderate yields.

Scheme 1. Synthesis of 2-methyl-3-nitropyridines.
We examined 2-methylpyridines 2 and 3 in reactions with aldehydes under piperidine catalysis. Our attempts to isolate condensation products of compounds 2b,c failed, whereas 2-methyl-3,5-dinitropyridine 2a and N-oxides 3b,c gave diarylethenes 4a-g in high yields, as shown in Scheme 2. It should be noted that compound 2a reacts several times faster than pyridine N-oxides 3b,c, indicating that para-NO2 is a more potent activating group for this reaction than the N-oxide moiety neighboring the 2-methyl group. The functional group tolerance, along with the relatively mild conditions and availability of aromatic aldehydes, makes this method a valid alternative for Pd-catalyzed coupling reactions [15][16][17][18]. Deoxygenation of pyridine N-oxides 4c,e-g with PCl3 allowed us to obtain four additional 2-ethenylpyridines 4h-k, which were inaccessible via direct condensation of 2-methylpyridines 2b,c with aldehydes, as shown in Scheme 2.  We examined 2-methylpyridines 2 and 3 in reactions with aldehydes under piperidine catalysis. Our attempts to isolate condensation products of compounds 2b,c failed, whereas 2-methyl-3,5-dinitropyridine 2a and N-oxides 3b,c gave diarylethenes 4a-g in high yields, as shown in Scheme 2. It should be noted that compound 2a reacts several times faster than pyridine N-oxides 3b,c, indicating that para-NO 2 is a more potent activating group for this reaction than the N-oxide moiety neighboring the 2-methyl group. The functional group tolerance, along with the relatively mild conditions and availability of aromatic aldehydes, makes this method a valid alternative for Pd-catalyzed coupling reactions [15][16][17][18]. Deoxygenation of pyridine N-oxides 4c,e-g with PCl 3 allowed us to obtain four additional 2-ethenylpyridines 4h-k, which were inaccessible via direct condensation of 2-methylpyridines 2b,c with aldehydes, as shown in Scheme 2.
We examined 2-methylpyridines 2 and 3 in reactions with aldehydes under piperidine catalysis. Our attempts to isolate condensation products of compounds 2b,c failed, whereas 2-methyl-3,5-dinitropyridine 2a and N-oxides 3b,c gave diarylethenes 4a-g in high yields, as shown in Scheme 2. It should be noted that compound 2a reacts several times faster than pyridine N-oxides 3b,c, indicating that para-NO2 is a more potent activating group for this reaction than the N-oxide moiety neighboring the 2-methyl group. The functional group tolerance, along with the relatively mild conditions and availability of aromatic aldehydes, makes this method a valid alternative for Pd-catalyzed coupling reactions [15][16][17][18]. Deoxygenation of pyridine N-oxides 4c,e-g with PCl3 allowed us to obtain four additional 2-ethenylpyridines 4h-k, which were inaccessible via direct condensation of 2-methylpyridines 2b,c with aldehydes, as shown in Scheme 2. The possibility of the substitution of the non-activated nitro group in pyridines was studied recently by our group [12]. In 3-nitro-5-Cl(Br)-pyridines, 3-NO2 was found to be more nucleofugal than halogen in position 5. The reactions of 2-methyl-3-nitropyridines 2 and 3 with thiols are summarized in Scheme 3 and Table 1. Upon heating the reactants in DMF in the presence of K2CO3, the selective formation of 3-R 2 S-products 5 was observed in all cases; however, the reaction of 2a with BnSH gave 5a with a trace amount of the isomer 6a.  Interestingly, diarylethenes 4 react with thiols under the same mild conditions, but with lower selectivity. 1 H NMR spectra of the crude products generally contain an additional set of signals corresponding to the 5-R 2 S-isomer, whereas the ratio of 5/6 varied from 2:1 to 20:1 depending on the substrate and thiol. Moreover, compounds 6g,h were isolated and fully characterized, but in all other cases we were unable to isolate isomers 6, as shown in Scheme 4 and Table 2. The possibility of the substitution of the non-activated nitro group in pyridines was studied recently by our group [12]. In 3-nitro-5-Cl(Br)-pyridines, 3-NO 2 was found to be more nucleofugal than halogen in position 5. The reactions of 2-methyl-3-nitropyridines 2 and 3 with thiols are summarized in Scheme 3 and Table 1. Upon heating the reactants in DMF in the presence of K 2 CO 3 , the selective formation of 3-R 2 S-products 5 was observed in all cases; however, the reaction of 2a with BnSH gave 5a with a trace amount of the isomer 6a. In all cases, only trans-diarylethenes are formed, which was confirmed by NMR spectroscopy: coupling constants of 15-16 Hz were observed for proton signals of the double bonds. In addition, X-ray analysis for compounds 4a,i was performed, undoubtedly proving our assumption, as shown in Figure 2. The possibility of the substitution of the non-activated nitro group in pyridines was studied recently by our group [12]. In 3-nitro-5-Cl(Br)-pyridines, 3-NO2 was found to be more nucleofugal than halogen in position 5. The reactions of 2-methyl-3-nitropyridines 2 and 3 with thiols are summarized in Scheme 3 and Table 1. Upon heating the reactants in DMF in the presence of K2CO3, the selective formation of 3-R 2 S-products 5 was observed in all cases; however, the reaction of 2a with BnSH gave 5a with a trace amount of the isomer 6a.  Interestingly, diarylethenes 4 react with thiols under the same mild conditions, but with lower selectivity. 1 H NMR spectra of the crude products generally contain an additional set of signals corresponding to the 5-R 2 S-isomer, whereas the ratio of 5/6 varied from 2:1 to 20:1 depending on the substrate and thiol. Moreover, compounds 6g,h were isolated and fully characterized, but in all other cases we were unable to isolate isomers 6, as shown in Scheme 4 and Table 2.  Interestingly, diarylethenes 4 react with thiols under the same mild conditions, but with lower selectivity. 1 H NMR spectra of the crude products generally contain an additional set of signals corresponding to the 5-R 2 S-isomer, whereas the ratio of 5/6 varied from 2:1 to 20:1 depending on the substrate and thiol. Moreover, compounds 6g,h were isolated and fully characterized, but in all other cases we were unable to isolate isomers 6, as shown in Scheme 4 and Table 2.
Structures of compounds 5 and 6 were confirmed by NMR, HRMS, X-ray and elemental analysis. 1 Н-1 Н NOESY spectra of compounds 5m and 5q revealed interactions of the spatially close protons of the double bond and benzyl substituent, as shown in Figure  3. The structures of 5h,l were determined by the X-ray diffraction single-crystal method, as shown in Figure 4.   Structures of compounds 5 and 6 were confirmed by NMR, HRMS, X-ray and elemental analysis. 1 H-1 H NOESY spectra of compounds 5m and 5q revealed interactions of the spatially close protons of the double bond and benzyl substituent, as shown in Figure 3. The structures of 5h,l were determined by the X-ray diffraction single-crystal method, as shown in Figure 4.
The above-mentioned results allow us to conclude that electron-releasing substituents in the aryl group and the bulky thiolate anion favor substitution at position 3: the best selectivity was observed for reactions of 4b with α-toluene thiol, whereas 4a with isobutyl mercaptan gave the lowest selectivity (2:1). Reactions with 4-chlorothiophenol afforded exclusively a 3-substituted product.   Structures of compounds 5 and 6 were confirmed by NMR, HRMS, X-ray and ele mental analysis. 1 Н-1 Н NOESY spectra of compounds 5m and 5q revealed interactions o the spatially close protons of the double bond and benzyl substituent, as shown in Figure  3. The structures of 5h,l were determined by the X-ray diffraction single-crystal method as shown in Figure 4.    Compounds with multiple conjugated double bonds, such as diarylethenes 4-6, can be expected to have strong absorbance in the UV and visible region; therefore, the photophysical properties of some representative compounds with various substitution patterns were studied. Indeed, it was found that all recorded UV-Vis spectra in the MeCN solution have a strong and distinctive absorption band in the 326-509 nm region accompanied by one or more weaker and non-informative bands around 260-300 nm ( Figure 5, Table 3). Notable exceptions are compounds 4a,i with only one dominant absorption maximum and compound 4e with a stronger shortwave band, which can be attributed to the N-oxide moiety. The above-mentioned results allow us to conclude that electron-releasing substituents in the aryl group and the bulky thiolate anion favor substitution at position 3: the best selectivity was observed for reactions of 4b with α-toluene thiol, whereas 4a with isobutyl mercaptan gave the lowest selectivity (2:1). Reactions with 4-chlorothiophenol afforded exclusively a 3-substituted product.
Compounds with multiple conjugated double bonds, such as diarylethenes 4-6, can be expected to have strong absorbance in the UV and visible region; therefore, the photophysical properties of some representative compounds with various substitution patterns were studied. Indeed, it was found that all recorded UV-Vis spectra in the MeCN solution have a strong and distinctive absorption band in the 326-509 nm region accompanied by one or more weaker and non-informative bands around 260-300 nm ( Figure 5, Table 3). Notable exceptions are compounds 4a,i with only one dominant absorption maximum and compound 4e with a stronger shortwave band, which can be attributed to the N-oxide moiety.  In the case of dinitro compounds 4a-c, the electron-releasing Me2N group in the phenyl ring leads to a considerable red shift of the absorption maximum (by 141 nm)   In the case of dinitro compounds 4a-c, the electron-releasing Me 2 N group in the phenyl ring leads to a considerable red shift of the absorption maximum (by 141 nm) with respect to the electron-withdrawing chlorine atom (compounds 4a and 4b, Figure 6). Compound 4b, with a strong electron-releasing 4-dimethylaminophenyl group, absorbs light in the visible region, whereas compounds 4a and 4c have their absorption maxima at the border between the visible and UV regions. This can be explained by the difference in the degree of charge transfer along the conjugation chain between the strongly withdrawing nitropyridine ring and the second electron-donating ring through the double bond. with respect to the electron-withdrawing chlorine atom (compounds 4a and 4b, Figure 6). Compound 4b, with a strong electron-releasing 4-dimethylaminophenyl group, absorbs light in the visible region, whereas compounds 4a and 4c have their absorption maxima at the border between the visible and UV regions. This can be explained by the difference in the degree of charge transfer along the conjugation chain between the strongly withdrawing nitropyridine ring and the second electron-donating ring through the double bond. On the other hand, the replacement of 5-NO2 with the CF3 group in a pyridine cycle caused a 42 nm blue shift for compounds 4a/4i and an 80 nm shift for 4-dimethylaminophenyl derivatives 4b/4k, as well as a small decrease in molar absorptivity (Figure 7). It can be concluded that substituents at the double bond as well as in position 5 can be independently altered to predictably fine-tune absorption spectra of these compounds. On the other hand, the replacement of 5-NO 2 with the CF 3 group in a pyridine cycle caused a 42 nm blue shift for compounds 4a/4i and an 80 nm shift for 4-dimethylaminophenyl derivatives 4b/4k, as well as a small decrease in molar absorptivity (Figure 7). It can be concluded that substituents at the double bond as well as in position 5 can be independently altered to predictably fine-tune absorption spectra of these compounds.
The study of isomeric substitution products of 3-and 5-NO 2 in compound 4a revealed an important dependence, as shown in Figure 8. Substitution of the nitro group at position 5 gave compound 6g, whose absorption spectrum generally resembles that of the parent compound and follows the same pattern described above for the 5-NO 2 /5-CF 3 pair. On the other hand, substitution of the nitro group at position 3 gave compound 5g, which is qualitatively different from both compounds. In this case, the absorption maximum shifts slightly towards the visible region, and the absorption spectrum itself acquires a more complex structure. From this we can conclude that the combination of 2-alkenyl and 3-alkylthio substituents leads to the appearance of a characteristic electronic structure. A similar pattern was observed for the substitution of 3-NO 2 in compound 4c, but not for compound 4b, which can be explained by the predominance of a strong charge transfer over the finer electronic structure. It should be noted that the alkylthio substituent does not significantly affect the photophysical properties of the obtained compounds. On the other hand, the replacement of 5-NO2 with the CF3 group in a pyridine cycle caused a 42 nm blue shift for compounds 4a/4i and an 80 nm shift for 4-dimethylaminophenyl derivatives 4b/4k, as well as a small decrease in molar absorptivity (Figure 7). It can be concluded that substituents at the double bond as well as in position 5 can be independently altered to predictably fine-tune absorption spectra of these compounds. The study of isomeric substitution products of 3-and 5-NO2 in compound 4a revealed an important dependence, as shown in Figure 8. Substitution of the nitro group at position 5 gave compound 6g, whose absorption spectrum generally resembles that of the parent compound and follows the same pattern described above for the 5-NO2/5-CF3 pair. On the other hand, substitution of the nitro group at position 3 gave compound 5g, which is qualitatively different from both compounds. In this case, the absorption maximum shifts slightly towards the visible region, and the absorption spectrum itself acquires a more complex structure. From this we can conclude that the combination of 2-alkenyl and 3-alkylthio substituents leads to the appearance of a characteristic electronic structure. A similar pattern was observed for the substitution of 3-NO2 in compound 4c, but not for compound 4b, which can be explained by the predominance of a strong charge transfer over the finer electronic structure. It should be noted that the alkylthio substituent does not significantly affect the photophysical properties of the obtained compounds. Compounds 5g,n showed fluorescence upon excitation by light with the wavelength equal to the λ max in the visible region, as shown in Figures 9 and 10. Compound 5g has an emission maximum at 538 nm and a Stokes shift of 154 nm, whereas for compound 5n, these values are 571 nm and 168 nm, respectively. The large values of Stokes shifts (150-170 nm) almost completely eliminate the overlap between the absorption and emission regions. In addition, the properties of these fluorescent molecules can be tuned by changing the substituent at the double bond.

Conclusions
In conclusion, a number of the previously unknown 2-methyl-and 2-arylvinyl-3-nitropyridines were synthesized. Their reactions with S-nucleophiles proceeded under mild conditions and were found to be regioselective with a predominance of 3-NO2 substitution; the influence of the substituents in positions 2 and 5 on the observed selectivity was revealed. The synthesized compounds showed promising tunable photophysical properties, such as large Stokes shifts. The reported synthetic approach can be considered as a convenient tool for rapid access to pyridine-based building blocks of various potential applications.

Conclusions
In conclusion, a number of the previously unknown 2-methyl-and 2-arylvinyl-3-nitropyridines were synthesized. Their reactions with S-nucleophiles pro ceeded under mild conditions and were found to be regioselective with a predominanc of 3-NO2 substitution; the influence of the substituents in positions 2 and 5 on the ob served selectivity was revealed. The synthesized compounds showed promising tunabl photophysical properties, such as large Stokes shifts. The reported synthetic approach can be considered as a convenient tool for rapid access to pyridine-based building block of various potential applications.

Conclusions
In conclusion, a number of the previously unknown 2-methyl-and 2-arylvinyl-3nitropyridines were synthesized. Their reactions with S-nucleophiles proceeded under mild conditions and were found to be regioselective with a predominance of 3-NO 2 substitution; the influence of the substituents in positions 2 and 5 on the observed selectivity was revealed. The synthesized compounds showed promising tunable photophysical properties, such as large Stokes shifts. The reported synthetic approach can be considered as a convenient tool for rapid access to pyridine-based building blocks of various potential applications.

General Information
All chemicals were of commercial grade and used directly without purification. Melting points were measured on a Stuart SMP20 apparatus (Stuart (Bibby Scientific), UK). 1 H and 13 C NMR spectra were recorded on a Bruker AM-300 (at 300.13 and 75.13 MHz, respectively, Bruker Biospin, Germany) or Bruker Avance DRX 500 (at 500 and 125 MHz, respectively, Bruker Biospin, Germany) in DMSO-d 6 or CDCl 3 . J values are given in Hz. HRMS spectra were recorded on a Bruker micrOTOF II mass spectrometer using ESI. UV-Vis absorption spectra were recorded in MeCN (2 × 10 −5 M) in standard 10 × 10 × 45 mm quartz cuvettes on a Cary 60 UV-Vis spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). Fluorescence spectra were recorded in MeCN (2 × 10 −6 M) in standard 10 × 10 × 45 mm quartz cuvettes on a Cary Eclipse fluorescence spectrophotometer (Agilent Technologies). All reactions were monitored by TLC analysis using ALUGRAM SIL G/UV254 plates, which were visualized with UV light. Compounds 1a-c were purchased from commercial suppliers. In some cases we were unable to record the 13 C NMR spectra of products due to insufficient solubility in common organic solvents (compounds 4d, 5,i,j,o and 6j).

General Procedure for the Synthesis of 2-Methyl-3-nitropyridines 2a-c
To a stirred suspension of NaH (60% in mineral oil, 0.80 g, 20 mmol) in anhydrous THF (30 mL), diethyl malonate (1.52 mL, 10 mmol) was added dropwise. The suspension was stirred for 15 min until hydrogen evolution ceased and a solution of the corresponding 2-chloropyridine 1 (10 mmol) in THF (20 mL) was added. The reaction mixture was stirred at r.t. (room temperature) for 6 h, poured in water (200 mL) and acidified with conc. HCl to pH 3. This was then extracted with CHCl 3 , evaporated and 50% H 2 SO 4 (30 mL) was added to the residue. The mixture was stirred for 6 h at 120 • C, cooled, neutralized with Na 2 CO 3 to pH 8 and extracted with CHCl 3 . The organic phase was dried over Na 2 SO 4 , evaporated and the residue was purified via column chromatography (SiO 2 /CHCl 3 ).

General Procedure for the Oxidation of 2-Methyl-3-nitropyridines 2b,c
To a solution of the corresponding 2-methyl-3-nitropyridine 2 (10 mmol) in CH 2 Cl 2 (30 mL), a freshly prepared complex urea/H 2 O 2 (1.88 g, 20 mmol) was added. The resulting suspension was cooled to 0 • C and trifluoroacetic anhydride (5 mL, 36 mol) was added dropwise. The reaction mixture was stirred for 30 min at 0 • C and 4 h at r.t. A saturated aqueous solution of Na 2 S 2 O 3 (50 mL) was added and the organic phase was separated. An aqueous layer was additionally extracted with CH 2 Cl 2 and combined organic solutions were washed with the saturated solution of NaHCO 3 , dried over Na 2 SO 4 and evaporated. The residue was recrystallized from aqueous EtOH.

General Procedure for the Synthesis of Compounds 4a-g
To a solution of the corresponding 2-methylpyridine 2 or 3 (5 mmol) in toluene (30 mL), aromatic aldehyde (5 mmol) and 50 µL of piperidine was added. The reaction mixture was stirred under reflux with a Dean-Stark adapter until water separation was completed. The solvent was evaporated and the residue was triturated with 20 mL of cold EtOH. The precipitate was filtered off and air-dried.  13