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

Abstract

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.

1. 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].

2. 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.

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.

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. Reactions of 2-methyl-3-nitropyridines with thiols.

Substrate	R1	n	R2	Product, Isolated Yield (%)
2a	NO2	0	PhCH2	5a, 70
3b	Br	1	PhCH2	5b, 96
3b	Br	1	4-Cl-C6H4	5c, 95
2b	Br	0	PhCH2	5d, 65
2c	CF3	0	PhCH2	5e, 60
3c	CF3	1	PhCH2	5f, 52

. Upon heating the reactants in DMF in the presence of K₂CO₃, the selective formation of 3-R²S-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. ¹H NMR spectra of the crude products generally contain an additional set of signals corresponding to the 5-R²S-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. Reactions of 2-(arylvinyl)-3-nitropyridines with thiols.

Substrate	R1	Ar	n	R2	Ratio 5/6 a	Product, Isolated Yield (%)
4a	NO2	4-Cl-C6H4	0	PhCH2	3:1	5g, 56 6g, 18
4a	NO2	4-Cl-C6H4	0	i-C4H9	2:1	5h, 62 6h, 31
4a	NO2	4-Cl-C6H4	0	2-furylmethyl	>20:1	5i, 56
4a	NO2	4-Cl-C6H4	0	4-Cl-C6H4	>20:1	5j, 67
4b	NO2	4-Me2N-C6H4	0	4-Cl-C6H4	>20:1	5k, 83
4b	NO2	4-Me2N-C6H4	0	i-C4H9	8:1	5l, 84
4b	NO2	4-Me2N-C6H4	0	PhCH2	10:1	5m, 88
4d	NO2	1-(4-fluorophenyl)pyrazol-4-yl	0	PhCH2	10:1	5n, 89
4d	NO2	1-(4-fluorophenyl)pyrazol-4-yl	0	4-Cl-C6H4	>20:1	5o, 93
4j	Br	4-Cl-C6H4	0	PhCH2	>20:1	5p, 60
4f	Br	4-Me2N-C6H4	1	PhCH2	>20:1	5q, 67

^a Determined from ¹H NMR spectrum of crude product.

.

Structures of compounds 5 and 6 were confirmed by NMR, HRMS, X-ray and elemental analysis. ¹H-¹H 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.

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. Photophysical properties of some synthesized compounds.

Compound	λmax nm	ε1 dm3 mol−1 cm−1	λmax nm	ε2 dm3 mol−1 cm−1
4a	-	-	368	46,400
4b	302	13,500	509	42,800
4c	263	16,200	397	34,900
4e	267	28,700	332	24,700
4i	-	-	326	39,000
4k	266	14,600	429	32,800
5g	296	16,000	384	20,300
5m	305	13,600	476	31,400
5n	266	18,100	403	25,900
5p	302	25,200	344	19,000
6g	267	15,000	337	31,400

). 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) 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.

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 Figure 9 and Figure 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.

3. 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.

4. Materials and Methods

4.1. 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). ¹H and ¹³C 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-d₆ or CDCl₃. 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 ¹³C NMR spectra of products due to insufficient solubility in common organic solvents (compounds 4d, 5,i,j,o and 6j).

4.2. 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 CHCl₃, evaporated and 50% H₂SO₄ (30 mL) was added to the residue. The mixture was stirred for 6 h at 120 °C, cooled, neutralized with Na₂CO₃ to pH 8 and extracted with CHCl₃. The organic phase was dried over Na₂SO₄, evaporated and the residue was purified via column chromatography (SiO₂/CHCl₃).

2-Methyl-3,5-dinitropyridin (2a) [19], brown oil; yield 62%; ¹H NMR (300 MHz, CDCl₃): δ 9.55 (d, 1H, J = 2.1 Hz), 9.06 (d, 1H, J = 2.1 Hz), 3.03 (s, 3H).

5-Bromo-2-methyl-3-nitropyridin (2b) [20], yellowish oil; yield 90%; 1H NMR (300 MHz, CDCl₃): δ 8.80 (d, 1H, J = 2.1 Hz), 8.44 (d, 1H, J = 2.1 Hz), 2.84 (s, 3H).

2-Methyl-3-nitro-5-trifluoromethylpyridine (2c), yellowish oil; yield 81%; 1H NMR (300 MHz, CDCl₃): δ 9.00 (s, 1H), 8.54 (s, 1H), 2.97 (s, 3H).

4.3. 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 CH₂Cl₂ (30 mL), a freshly prepared complex urea/H₂O₂ (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 Na₂S₂O₃ (50 mL) was added and the organic phase was separated. An aqueous layer was additionally extracted with CH₂Cl₂ and combined organic solutions were washed with the saturated solution of NaHCO₃, dried over Na₂SO₄ and evaporated. The residue was recrystallized from aqueous EtOH.

5-Bromo-2-methyl-3-nitropyridine N-oxide (3b), pale-yellow solid; yield 47%; mp 104–105 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.60 (s, 1H), 7.86 (s, 1H), 2.67 (s, 3H, CH₃). ¹³C NMR (125 MHz, CDCl₃): δ 148.5, 145.5, 143.5, 122.9, 116.4, 13.8. HRMS (ESI, m/z): calcd for C₆H₅BrN₂O₃ [M + H]⁺: 232.9556; found: 232.9563.

2-Methyl-3-nitro-5-trifluoromethylpyridine N-oxide (3c), pale-yellow solid; yield 65%; mp 82–83 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.69 (s, 1H), 7.89 (s, 1H), 2.75 (s, 3H). ¹³C NMR (75 MHz, CDCl₃): δ 149.9, 148.8, 139.5, 127.1 (q, ²J_CF = 36 Hz), 121.1 (q, ¹J_CF = 274 Hz), 116.1, 14.3. HRMS (ESI, m/z): calcd for C₇H₅F₃N₂O₃ [M + H]⁺: 223.0325; found: 223.0331.

4.4. 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.

(E)-2-[(4-Chlorophenyl)vinyl]-3,5-dinitropyridine (4a), yellow solid; yield 78%; mp 204–205 °C; ¹H NMR (300 MHz, CDCl₃): δ 9.58 (s, 1H), 9.05 (s, 1H), 8.30 (d, 1H, J = 15.3 Hz), 7.83 (d, 1H, J = 15.3 Hz), 7.64 (d, 2H, J = 8.4 Hz), 7.45 (d, 2H, J = 8.4 Hz). ¹³C NMR (150 MHz, DMSO-d₆): δ 152.0, 147.5, 143.2, 141.9, 140.3, 135.0, 134.0, 130.2, 129.2, 129.1, 121.6. HRMS (ESI, m/z): calcd for C₁₃H₈ClN₃O₄ [M + H]⁺: 306.0276; found: 306.0283.

(E)-4-(2-(3,5-Dinitropyridin-2-yl)vinyl)-N,N-dimethylaniline (4b), dark-violet solid; yield 91%; mp 260 °C (dec.); ¹H NMR (300 MHz, CDCl₃): δ 9.41 (d, 1H, J = 2.1 Hz), 8.93 (d, 1H, J = 2.1 Hz), 8.28 (d, 1H, J = 15.0 Hz), 7.64 (d, 1H, J = 15.0 Hz), 7.57 (d, 2H, J = 8.8 Hz), 6.77 (d, 2H, J = 8.8 Hz), 3.06 (s, 6H). ¹³C NMR (125 MHz, CDCl₃): δ 154.8, 152.4, 147.3, 146.4, 139.5, 131.1, 128.9, 123.2, 114.4, 111.9, 40.1. HRMS (ESI, m/z): calcd for C₁₅H₁₄N₄O₄ [M + H]⁺: 315.1088; found: 315.1085.

(E)-5-Bromo-2-(4-dimethylaminophenyl)vinyl-3-nitropyridine N-oxide (4c), red-brown solid; yield 73%; mp 217 °C (dec.); ¹H NMR (300 MHz, CDCl₃): δ 8.55 (d, 1H, J = 15.9 Hz), 8.49 (s, 1H), 7.64 (s, 1H), 7.49 (d, 2H, J = 8.7 Hz), 7.00 (d, 1H, J = 15.6 Hz), 6.69 (d, 2H, J = 8.7 Hz), 3.05 (s, 6H). ¹³C NMR (75 MHz, CDCl₃): δ 151.8, 147.2, 143.9, 142.8, 141.2, 129.9, 123.8, 123.0, 113.0, 111.9, 107.1, 40.1. HRMS (ESI, m/z): calcd for C₁₅H₁₄BrNO₃ [M + H]: 364.0291; found: 364.0292.

(E)-2-(2-(1-(4-Fluorophenyl)-1H-pyrazol-4-yl)vinyl)-3,5-dinitropyridine (4d), brown solid; yield 62%; mp 255 °C; ¹H NMR (300 MHz, CDCl₃): δ 9.53 (d, 1H, J = 2.1 Hz), 9.03 (d, 1H, J = 2.1 Hz), 8.30 (d,1H, J = 15.3 Hz), 8.19 (s, 1H), 8.06 (s, 1H), 7.70 (m, 3H), 7.20 (m, 2H). HRMS (ESI, m/z): calcd for C₁₆H₁₀FN₅O₄ [M + H]⁺: 356.0790; found: 356.0789.

(E)-2-(4-Chlorophenyl)vinyl-3-nitro-5-trifluoromethylpyridine N-oxide (4e), yellow solid; yield 81%; mp 161–162 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.65 (d,1H, J = 15.9 Hz), 8.64 (s, 1H), 7.73 (s, 1H), 7.52 (d, 2H, J = 8.4 Hz), 7.39 (d, 2H, J = 8.4 Hz), 7.17 (d, 1H, J = 15.9 Hz). ¹³C NMR (75 MHz, CDCl₃): δ 147.9, 143.6, 142.3, 140.4, 136.7, 134.1, 129.4, 129.3, 125.5 (q, ²J_CF = 36 Hz), 121.1 (q, ¹J_CF = 273 Hz), 116.0 (d, ³J_CF = 3.2 Hz), 112.8. HRMS (ESI, m/z): calcd for C₁₄H₈ClF₃N₂O₃ [M + H]⁺: 345.0248; found: 345.0239.

(E)-5-Bromo-2-(4-chlorophenyl)vinyl-3-nitropyridine N-oxide (4f), yellow solid; yield 69%; mp 173–174 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.45 (s, 1H), 8.30 (d, 1H, J = 16.1 Hz), 7.60 (s, 1H), 7.41 (d, 2H, J = 8.4 Hz), 7.29 (d, 2H, J = 8.4 Hz), 7.02 (d, 1H, J = 16.1 Hz). ¹³C NMR (125 MHz, CDCl₃): δ 147.8, 144.1, 140.3, 140.2, 136.1, 134.4, 129.2, 129.1, 122.7, 115.7, 113.1. HRMS (ESI, m/z): calcd for C₁₃H₈BrClN₂O₃ [M + H]⁺: 354.9480; found: 354.9472.

(E)-2-(4-Dimethylaminophenyl)vinyl-3-nitro-5-trifluoromethylpyridine N-oxide (4g), red-brown solid; yield 71%; mp 198-199 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.85 (d, 1H, J = 15.6 Hz), 8.58 (s, 1H), 7.68 (s, 1H), 7.52 (d, 2H, J = 8.4 Hz), 7.10 (d, 1H, J = 15.6 Hz), 6.69 (d, 2H, J = 8.4 Hz), 3.06 (s, 6H). ¹³C NMR (125 MHz, CDCl₃): δ 152.2, 146.8, 145.1, 144.4, 140.1, 130.5, 123.4, 122.7 (q, ²J_CF = 36 Hz), 121.4 (q, ¹J_CF = 273 Hz), 116.3 (q, ³J_CF = 3.6 Hz), 111.9, 106.6, 40.0. HRMS (ESI, m/z): calcd for C₁₆H₁₄F₃N₃O₃ [M + H]⁺: 354.1060; found: 354.1060.

4.5. General Procedure for the Synthesis of Compounds 4h–k

To a solution of the corresponding pyridine N-oxide (0.5 mmol), CH₂Cl₂ (10 mL) was added PCl₃ (0.13 mL) and the mixture was stirred under reflux for 2 h. After cooling, the reaction was washed with a saturated NaHCO₃ solution, dried over Na₂SO₄ and evaporated.

(E)-4-(2-(5-Bromo-3-nitropyridin-2-yl)vinyl)-N,N-dimethylaniline (4h), dark-brown solid; yield 89%; mp 202–203 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.75 (s, 1H), 8.34 (s, 1H), 8.05 (d, 1H, J = 15.3 Hz), 7.55 (d, 1H, J = 15.6), 7.53 (d, 2H, J = 8.4), 6.71 (d, 2H, J = 8.4 Hz), 3.04 (s, 6H). ¹³C NMR (75 MHz, CDCl₃): δ 153.8, 151.3, 148.8, 145.5, 142.8, 140.5, 135.0, 129.8, 115.5, 115.5, 112.3, 40.4, 29.7. HRMS (ESI, m/z): calcd for C₁₅H₁₄BrN₃O₂ [M + H]⁺: 348.0342; found: 348.0342.

(E)-2-(4-Chlorophenyl)vinyl-3-nitro-5-trifluoromethylpyridine (4i), yellow solid; yield 89%; mp 138 °C; ¹H NMR (300 MHz, CDCl₃): δ 9.02 (s, 1H), 8.50 (s, 1H), 8.16 (d, 1H, J = 15.6 Hz), 8.77 (d, 1H, J = 15.6 Hz), 7.59 (d, 2H, J = 8.4 Hz), 7.41 (d, 2H, J = 8.4 Hz). ¹³C NMR (75 MHz, CDCl₃): δ 152.0, 149.2 (q, ³J_CF = 3.6 Hz), 143.0, 140.8, 136.1, 133.9, 130.7 (q, ³J_CF = 3.6 Hz), 129.4, 129.3, 124.9 (q, ²J_CF = 35 Hz), 120.6. HRMS (ESI, m/z): calcd for C₁₄H₈ClF₃N₂O₂ [M + H]⁺: 329.0299; found: 329.0311.

(E)-5-Bromo-2-(4-chlorostyryl)-3-nitropyridine (4j), yellow solid; yield 94%; mp 184–185 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.83 (d, 1H, J = 1.8 Hz), 8.40 (d, 1H, J = 1.8 Hz), 8.02 (d, 1H, J = 15.6 Hz), 7.68 (d, 1H, J = 15.6 Hz), 7.56 (d, 2H, J = 8.4 Hz), 7.38 (d, 2H, J = 8.4 Hz). ¹³C NMR (75 MHz, CDCl₃): δ 154.0, 147.4, 143.7, 138.4, 135.5, 135.1, 134.2, 129.2, 129.1, 124.9, 117.6. HRMS (ESI, m/z): calcd for C₁₃H₈BrClN₂O₂ [M + H]⁺: 338.9530; found: 338.9533.

(E)-N,N-Dimethyl-4-(2-(3-nitro-5-trifluoromethylpyridin-2-yl)vinyl)aniline (4k), brown solid; yield 82%; mp 150–151 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.40 (s, 1H), 8.36 (s, 1H), 8.12 (d, 1H, J = 15.3 Hz), 7.56 (d, 1H, J = 15.3 Hz), 7.49 (d, 2H, J = 8.7 Hz), 6.66 (d, 2H, J = 8.7 Hz), 2.99 (s, 6H). ¹³C NMR (75 MHz, CDCl₃): δ 153.4, 151.9, 149.1 (q, ³J_CF = 4 Hz), 143.3, 141.8, 130.8 (q, ³J_CF = 4 Hz), 130.3, 122.8 (q, ¹J_CF = 271 Hz), 123.6, 122.9 (q, ²J_CF = 34 Hz), 115.0, 112.1, 40.2. HRMS (ESI, m/z): calcd for C₁₆H₁₄F₃N₃O₂ [M + H]⁺: 338.1111; found: 338.1120.

4.6. General Procedure for the Synthesis of Compounds 5 and 6

To a solution of compound 2, 3 or 4 (1 mmol) in anhydrous DMF (5 mL), thiol (1 mmol) and K₂CO₃ (0.138 g, 1 mmol) were added. The reaction mixture was stirred for 1-2 h at 60 °C, poured into H₂O (50 mL), acidified with conc. HCl to pH 3 and extracted with CHCl₃ (3 × 20 mL). The organic phase was dried over Na₂SO₄, evaporated and the residue was purified via column chromatography (SiO₂/CHCl₃) or recrystallized from EtOH.

3-(Benzylsulfanyl)-2-methyl-5-nitropyridine (5a), beige solid; yield 70%; mp 79–80 °C; ¹H NMR (300 MHz, CDCl₃): δ 9.06 (d, 1H, J = 2.1 Hz), 7.35 (m, 5H), 4.24 (s, 2H, CH₂), 2.65 (s, 3H, CH₃). ¹³C NMR (125 MHz, CDCl₃): δ 162.7, 142.8, 140.1, 134.9, 134.8, 128.9, 128.8, 128.0, 127.8, 37.1, 23.4. HRMS (ESI, m/z): calcd for C₁₃H₁₂N₂O₂S [M + H]⁺: 261.0692; found: 261.0699.

3-(Benzylsulfanyl)-5-bromo-2-methylpyridine N-oxide (5b), grey needles; yield 96%; mp 106–107 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.25 (s, 1H), 7.26-7.32 (m, 5H), 7.24 (s, 1H), 4.14 (s, 2H, SCH₂), 2.49 (s, 3H, CH₃). ¹³C NMR (75 MHz, CDCl₃): δ 147.3, 137.8, 136.8, 134.9, 128.9, 128.1, 127.7, 116.3, 38.5, 14.5. HRMS (ESI, m/z): calcd for C₁₃H₁₂BrNOS [M + H]⁺: 309.9896; found: 309.9896.

5-Bromo-3-(4-chlorophenylsulfanyl)-2-methylpyridine N-oxide (5c), brown solid; yield 95%; mp 131–132 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.27 (s, 1H), 7.44 (d, 2H, J = 8.4 Hz), 7.38 (d, 2H, J = 8.4 Hz), 6.89 (s, 1H), 2.56 (s, 3H). ¹³C NMR (75 MHz, CDCl₃): δ 146.7, 138.1, 137.3, 136.0, 134.8, 130.4, 129.0, 127.7, 116.6, 14.6. HRMS (ESI, m/z): calcd for C₁₂H₉BrClNOS [M + H]⁺: 329.9350; found: 329.9349.

3-(Benzylsulfanyl)-5-bromo-2-methylpyridine (5d), pale-yellow crystals; yield 65%; mp 55 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.33 (s, 1H), 7.58 (s, 1H), 7.31 (m, 5H), 4.09 (s, 2H, SCH₂), 2.48 (s, 3H, CH₃). ¹³C NMR (75 MHz, CDCl₃): δ 155.5, 146.5, 137.3, 135.6, 134.4, 128.9, 128.8, 127.8, 117.9, 37.8, 22.5. HRMS (ESI, m/z): calcd for C₁₃H₁₂BrNS [M + H]⁺: 293.9947; found: 293.9956.

3-(Benzylsulfanyl)-2-methyl-5-(trifluoromethyl)pyridine (5e), yellow oil; yield 60%; ¹H NMR (300 MHz, CDCl₃): δ 8.53 (s, 1H), 7.64 (s, 1H), 7.31 (s, 5H), 4.14 (s, 2H, SCH₂), 2.61 (s, 3H, CH₃). ¹³C NMR (75 MHz, CDCl₃): δ 161.0, 142.3 (q, ³J_CF = 4 Hz), 135.5, 133.4, 131.7 (q, ³J_CF = 3.5 Hz), 128.9, 128.8, 127.8, 124.7 (q, ²J_CF = 34 Hz), 123.5 (q, ¹J_CF = 273 Hz), 37.5, 23.2. HRMS (ESI, m/z): calcd for C₁₄H₁₂F₃NS [M + H]⁺: 284.0715; found: 284.0719.

3-(Benzylsulfanyl)-2-methyl-5-(trifluoromethyl)pyridine N-oxide (5f), pale-yellow crystals; yield 52%; mp 85–86 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.33 (s, 1H), 7.29 (m, 5H), 7.22 (s, 1H), 4.15 (s, 2H, SCH₂), 2.57 (s, 3H, CH₃). ¹³C NMR (75 MHz, CDCl₃): δ 151.8, 137.3, 134.7, 134.1, 128.9, 128.9, 128.1, 126.6 (q, ²J_CF = 35 Hz), 121.9 (q, ¹J_CF = 273 Hz), 120.1, 38.4, 15.0. HRMS (ESI, m/z): calcd for C₁₄H₁₂F₃NOS [M + H]⁺: 300.0664; found: 300.0662.

(E)-3-(Benzylsulfanyl)-2-((4-chlorophenyl)vinyl)-5-nitropyridine (5g), yellow crystals; yield 56%; mp 146–147 °C; ¹H NMR (300 MHz, CDCl₃): δ 9.17 (d, 1H, J = 2.1 Hz), 8.28 (d, 1H, J = 2.1 Hz), 7.92 (d, 1H, J = 15.6 Hz), 7.57 (d, 1H, J = 15.6 Hz), 7.53 (d, 2H, J = 8.7 Hz), 7.39 (d, 2H, J = 8.7 Hz), 7.30 (m, 5H), 4.18 (s, 2H, SCH₂). ¹³C NMR (75 MHz, CDCl₃): δ 159.0, 142.2, 142.1, 138.1, 135.5, 135.2, 134.5, 132.4, 132.3, 129.2, 129.2, 129.0, 128.9, 128.1, 122.8, 38.7. HRMS (ESI, m/z): calcd for C₂₀H₁₅ClN₂O₂S [M + H]⁺: 383.0616; found: 383.0603.

(E)-5-(Benzylsulfanyl)-2-((4-chlorophenyl)vinyl)-3-nitropyridine (6g), yellow crystals; yield 18%; mp 183–184 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.63 (d, 1H, J = 1.8 Hz), 8.06 (d, 1H, J = 2.1 Hz), 7.97 (d, 1H, J = 15.6 Hz), 7.67 (d, 1H, J = 15.6 Hz), 7.56 (d, 2H, J = 8.7 Hz), 7.39 (d, 2H, J = 8.7 Hz), 7.33 (m, 5H), 4.22 (s, 2H, SCH₂). HRMS (ESI, m/z): calcd for C₂₀H₁₅ClN₂O₂S [M + H]⁺: 383.0616; found: 383.0621.

2-[(E)-(4-Chlorophenyl)vinyl]-3-(isobutylsulfanyl)-5-nitropyridine (5h), yellow crystals; yield 62%; mp 139–140 °C; ¹H NMR (300 MHz, CDCl₃): δ 9.16 (d, 1H, J = 2.4 Hz), 8.31 (d, 1H, J = 2.1 Hz), 7.96 (d, 1H, J = 15.3 Hz), 7.63 (d, 1H, J = 15.3 Hz), 7.60 (d, 2H, J = 8.4 Hz), 7.41 (d, 2H, J = 8.4 Hz), 2.92 (d, 2H, J = 6.9 Hz), 1.99 (m, 1H), 1.14 (d, 6H, J = 6.6 Hz). ¹³C NMR (75 MHz, CDCl₃): δ 158.0, 147.0, 142.4, 141.0, 137.9, 135.4, 134.5, 134.3, 129.9, 122.8, 42.3, 28.1, 22.2. HRMS (ESI, m/z): calcd for C₁₇H₁₇ClN₂O₂S [M + H]⁺: 349.0772; found: 349.0783.

2-[(E)-(4-Chlorophenyl)vinyl]-5-(isobutylsulfanyl)-3-nitropyridine (6h), yellow crystals; yield 31%; mp 106 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.68 (d, 1H, J = 1.8 Hz), 8.09 (d, 1H, J = 1.8 Hz), 7.97 (d, 1H, J = 15.6 Hz), 7.68 (d, 1H, J = 15.6 Hz), 7.56 (d, 2H, J = 8.4 Hz), 7.38 (d, 2H, J = 8.4 Hz), 2.92 (d, 2H, J = 6.9 Hz), 1.96 (m, 1H), 1.10 (d, 6H, J = 6.6 Hz). ¹³C NMR (75 MHz, CDCl₃): δ 152.0, 145.4, 143.9, 136.8, 135.1, 134.9, 134.6, 130.9, 129.1, 129.0, 121.5, 42.1, 28.3, 22.0. HRMS (ESI, m/z): calcd for C₁₇H₁₇ClN₂O₂S [M + H]⁺: 349.0772; found: 349.0764.

2-[(E)-2-(4-Chlorophenyl)vinyl]-3-[(2-furylmethyl)sulfanyl]-5-nitropyridine (5i), yellow crystals; yield 56%; mp 156 °C; ¹H NMR (300 MHz, CDCl₃): δ 9.24 (d, 1H, J = 1.8 Hz), 8.42 (d, 1H, J = 2.1 Hz), 7.95 (d, 1H, J = 15.6 Hz), 7.65 (d, 1H, J = 15.6 Hz), 7.58 (d, 2H, J = 8.4 Hz), 7.40 (d, 2H, J = 8.4 Hz), 7.33 (s, 1H), 6.26 (s, 1H), 6.14 (d, 1H, J = 3.0 Hz), 4.18 (s, 2H). HRMS (ESI, m/z): calcd for C₁₈H₁₃ClN₂O₂S [M + H]⁺: 373.0408; found: 373.0419.

3-[(4-Chlorophenyl)sulfanyl]-2-[(E)-2-(4-chlorophenyl)vinyl]-5-nitropyridine (5j), yellow solid; yield 67%; mp 172 °C; ¹H NMR (300 MHz, DMSO-d₆): δ 9.26 (s, 1H), 8.22 (s, 1H), 7.98 (d, 1H, J = 15.6 Hz), 7.74 (d, 2H, J = 8.4 Hz), 7.66 (d, 1H, J = 15.6 Hz), 7.52 (br.s, 4H), 7.49 (d, 2H, J = 8.4 Hz). HRMS (ESI, m/z): calcd for C₁₉H₁₂Cl₂N₂O₂S [M + H]⁺: 403.0069; found: 403.0057.

(E)-4-(2-(3-((4-Chlorophenyl)sulfanyl)-5-nitropyridin-2-yl)vinyl)-N,N-dimethylaniline (5k), dark-red solid; yield 83%; mp 175 °C; ¹H NMR (300 MHz, DMSO-d₆): δ 9.21 (s, 1H), 8.20 (s, 1H), 7.96 (d, 1H, J = 15.6 Hz), 7.50 (m, 6H), 7.36 (d, 1H, J = 15.6 Hz), 6.74 (d, 2H, J = 8.4 Hz), 2.99 (s, 6H). ¹³C NMR (125 MHz, CDCl₃): δ 160.1, 151.6, 143.1, 141.3, 141.2, 134.9, 133.4, 133.2, 130.9, 130.1, 129.9, 123.7, 117.0, 111.9, 40.1. HRMS (ESI, m/z): calcd for C₂₁H₁₈ClN₃O₂S [M + H]⁺: 412.0881; found: 412.0870.

(E)-4-(2-(3-(Isobutylsulfanyl)-5-nitropyridin-2-yl)vinyl)-N,N-dimethylaniline (5l), dark-red solid; yield 84%; mp 165–166 °C; ¹H NMR (300 MHz, CDCl₃): δ 9.14 (d, 1H, J = 2.4 Hz), 8.27 (d, 1H, J = 2.1 Hz), 8.01 (d, 1H, J = 15.3 Hz), 7.58 (d, 2H, J = 8.7 Hz), 7.46 (d, 1H, J = 15.3 Hz), 6.74 (d, 2H, J = 8.7 Hz), 3.06 (s, 6H), 2.89 (d, 2H, J = 6.6 Hz), 1.96 (m, 1H), 1.13 (d, 6H, J = 6.6 Hz). ¹³C NMR (75 MHz, CDCl₃): δ 159.5, 151.5, 141.4, 141.3, 140.2, 132.5, 130.0, 129.8, 124.1, 117.4, 112.0, 43.0, 40.2, 28.1, 22.2. HRMS (ESI, m/z): calcd for C₁₉H₂₃N₃O₂S [M + H]⁺: 358.1584; found: 358.1583.

(E)-4-(2-(3-(Benzylsulfanyl)-5-nitropyridin-2-yl)vinyl)-N,N-dimethylaniline (5m), dark-red solid; yield 88%; mp 188 °C; ¹H NMR (300 MHz, CDCl₃): δ 9.13 (d, 1H, J = 2.1 Hz), 8.20 (d, 1H, J = 1.8 Hz), 7.98 (d, 1H, J = 15.3 Hz), 7.54 (d, 2H, J = 8.7 Hz), 7.43 (d, 1H, J = 15.3 Hz), 7.30 (m, 5H), 6.70 (d, 2H, J = 8.7 Hz), 4.16 (s, 2H), 3.05 (s, 6H). ¹³C NMR (75 MHz, CDCl₃): δ 160.4, 151.5, 142.3, 141.0, 140.5, 135.5, 132.2, 130.6, 129.9, 129.0, 128.8, 127.9, 124.0, 117.4, 112.0, 40.2, 38.5. HRMS (ESI, m/z): calcd for C₂₂H₂₁N₃O₂S [M + H]⁺: 392.1427; found: 392.1417.

(E)-3-(Benzylsulfanyl)-2-(2-(1-(4-fluorophenyl)-1H-pyrazol-4-yl)vinyl)-5-nitropyridine (5n), orange solid; yield 89%; mp 145–146 °C; ¹H NMR (300 MHz, CDCl₃): δ 9.15 (d, 1H, J = 2.1 Hz), 8.26 (d, 1H, J = 2.1 Hz), 8.05 (s, 1H), 7.98 (s, 1H), 7.92 (d, 1H, J = 15.3 Hz), 7.67 (m, 2H), 7.40 (d, 1H, J = 15.3 Hz), 7.30 (m, 5H), 7.18 (m, 2H), 4.18 (s, 2H). ¹³C NMR (75 MHz, CDCl₃): δ 161.4 (d, ¹J_CF = 247 Hz), 159.3, 142.1, 141.8, 140.3, 136.0 (d, ⁴J_CF = 3 Hz), 135.3, 132.2, 131.5, 129.3, 129.0, 128.9, 128.0, 126.4, 121.9, 121.4, 121.1 (d, ³J_CF = 8.5 Hz), 116.5 (d, ²J_CF = 23 Hz), 38.6. HRMS (ESI, m/z): calcd for C₂₃H₁₇FN₄O₂S [M + H]⁺: 433.1129; found: 433.1123.

(E)-3-((4-Chlorophenyl)sulfanyl)-2-(2-(1-(4-fluorophenyl)-1H-pyrazol-4-yl)vinyl)-5-nitropyridine (5o), orange solid; yield 93%; mp 219 °C; ¹H NMR (300 MHz, DMSO-d₆): δ 9.24 (s, 1H), 8.99 (s, 1H), 8.22 (s, 1H), 8.18 (s, 1H), 7.80 (m, 4H), 7.55 (s, 4H), 7.40 (m, 3H). HRMS (ESI, m/z): calcd for C₂₂H₁₄ClFN₄O₂S [M + H]⁺: 453.0583; found: 453.0573.

(E)-3-(Benzylsulfanyl)-5-bromo-2-[(4-chlorophenyl)vinyl]pyridine (5p), beige solid, yield 60%; mp 142–143 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.48 (s, 1H), 7.69–7.73 (m, 2H), 7.48–7.56 (m, 3H), 7.25-7.36 (m, 7H), 4.08 (s, 2H). ¹³C NMR (125 MHz, CDCl₃): δ 153.2, 148.4, 141.1, 135.9, 135.1, 134.3, 133.6, 132.5, 128.9, 128.8, 128.7, 128.6, 127.7, 123.7, 118.3, 39.4. HRMS (ESI, m/z): calcd for C₂₀H₁₅BrClNS [M + H]⁺: 415.9869; found: 415.9870.

(E)-3-(Benylsulfanyl)-5-bromo-2-[(4-(dimethylamino)phenyl)vinyl]pyridine N-oxide (5q), dark-red solid; yield 67%; mp 177 °C; ¹H NMR (300 MHz, CDCl₃): δ 8.49 (d, 1H, J = 15.9 Hz), 8.23 (s, 1H), 7.51 (d, 2H, J = 8.7 Hz), 7.28 (m, 7H), 6.71 (d, 2H, J = 8.7 Hz), 4.14 (s, 2H), 3.03 (s, 6H, NCH₃). ¹³C NMR (75 MHz, CDCl₃): δ 151.2, 144.9, 140.5, 139.0, 135.9, 135.0, 129.2, 129.1, 128.8, 128.4, 127.9, 127.8, 124.8, 114.1, 112.1, 40.3, 39.0. HRMS (ESI, m/z): calcd for C₂₂H₂₁BrN₂OS [M + H]⁺: 441.0631; found: 441.0635.

4.7. X-ray Crystallographic Data and Refinement Details

X-ray diffraction data for 5h were collected at 100 K on a Bruker Quest D8 diffractometer (Karlsruhe, Germany) equipped with a Photon III area detector, using graphite-monochromatized Mo K_α-radiation and a shutterless φ- and ω-scan technique. The intensity data were integrated by the SAINT program (version 8.04A) [21] and were semi-empirically corrected for absorption and decay using SADABS (2016/2) [22]. X-ray diffraction data for 4a, 4i and 5l were collected at 100 K on a four-circle Rigaku Synergy-S diffractometer (Wroclaw, Poland) equipped with a HyPix6000HE area detector, using monochromatized Cu K_α-radiation and a shutterless ω-scan technique. The intensity data were integrated and corrected for absorption and decay by the CrysAlisPro program [23]. All structures were solved by direct methods using SHELXT [24] and refined by the full-matrix least-squares method on F² using SHELXL-2018 [25]. Positions of all atoms were found from the electron density difference map. Atoms were refined with individual anisotropic (non-hydrogen atoms) or isotropic (hydrogen atoms) displacement parameters. The Mercury program [26] was used for molecular graphics. Crystal data, data collection and structure refinement details are summarized in Supplementary Materials Table S1. The structures were deposited at the Cambridge Crystallographic Data Center with the reference CCDC numbers 2191010–2191013; they also contain the supplementary crystallographic data. These data can be obtained free of charge from the CCDC via http://www.ccdc.cam.ac.uk/data_request/cif, accessed on 30 August 2022.