Synthesis of Pyridinium Moiety Containing Triazolyl Purines

Abstract

Pyridinium salts of 2-piperidinyl-6-triazolylpurine derivatives were obtained by the introduction of pyridinium moieties into the propane-1,3-diol fragment at the N(9) position of purine to enhance the solubility of 2-amino-6-triazolylpurine derivatives in water. Target structures were obtained using the tosylation of hydroxyl groups of 2-(6-(4-(4-methoxyphenyl)-1H-1,2,3-triazol-1-yl)-2-(piperidin-1-yl)-9H-purin-9-yl)propane-1,3-diol, the subsequent introduction of pyridine, and ion exchange. The compounds were characterized using ¹H- and ¹³C-NMR spectra, FTIR, UV–Vis, and HRMS data.

1. Introduction

Purine derivatives are extensively studied due to their biological activity [1]. Purines are one of the most common naturally occurring heterocycles and can easily undergo modifications at C2, C6, C8, N9, and N7 positions [2,3,4,5,6,7,8,9], leading to countless new compounds with new reactivities and possible applications in biochemistry. They can be used as adenosine receptor agonists and antagonists, inhibitors of phosphodiesterases, sulfotransferases, and Src tyrosine and P38α MAP kinase inhibitors [10,11].

Many purine derivatives also possess fluorescent properties and are widely used in studies of biological and biochemical processes in cells and nucleic acids [12,13,14] due to the fact that a purine moiety works as a building block in DNA and RNA syntheses. Such compounds should usually have low cytotoxicity and are soluble in water. Many derivatization approaches are used to increase the solubility of purine derivatives in aqueous media, for example, introducing an amine-containing moiety to the purine N9 position [15], making purine derivatives co-crystals with benzenetricarboxylic acids [16] or obtaining purine-based ionic liquids [17].

In addition, a variety of reported novel fluorescent purine derivatives are also exploited as pH sensors [18], photocatalysts [19] and metal ion sensors [20].

2-Piperidinyl-6-triazolylpurine derivatives are known as push–pull chromophores that possess photophysical properties [21,22,23]. In our study, we decided to enhance their solubility, especially in water, and synthesize pyridinium salts of 2-piperidinyl-6-triazolylpurine derivatives, and then examine how much the fluorescence is quenched upon introduction of pyridine moieties in the structure. There are a few examples in the literature that report that the introduction of pyridinium moieties in organic structures can increase the solubility [24,25] and also quench the fluorescence [26,27].

2. Results and Discussion

Starting material 1 was prepared according to the literature [21]. Then, two hydroxyl groups of propane-1,3-diol at the N9 position of purine derivative 1 were tosylated using TsCl, DMAP, and Et₃N in DCM, resulting in product 2 with a 76% yield (Scheme 1).

To make the triazolyl purine derivative’s pyridinium tosylate salt 3, the solution of compound 2 in pyridine was heated in the pressure vial at 120 °C for 1 h. After workup, compound 3 was acquired with a 72% yield with no need for further purification. Although pyridinium salt 3 was soluble in water, it still possessed bulky and aromatic tosylate anions that potentially could limit the solubility and impact fluorescence. Therefore, the tosylate anions were exchanged to chlorides using ion exchange resin, and product 4 was obtained in an 84% yield. Next, the solubility of compounds 1–4 in water was determined by using qNMR (1—0.21 mg/mL, 2—0.19 mg/mL, 3—57 mg/mL, 4—133 mg/mL).

Absorption and emission spectra were measured for compound 3 in H₂O, DMSO, and DCM (Figure 1) and for compound 4 in MeCN, MeOH, H₂O, DMSO, and DCM (Figure 2). Compound 3 exhibited absorption maxima at 359–362 nm and emission maxima at 428–565 nm (

Table 1. Photophysical properties of compounds 3 and 4 in various solvents.

Solvent	Compound 3			Compound 4
	λabs max, nm	λem max, nm	QY, %	λabs max, nm	λem max, nm	QY, %
MeCN	-	-	-	360	457	<0.5
MeOH	-	-	-	362	461	<0.5
H2O	362	457	<0.5	363	463	<0.5
DMSO	361	428	<0.5	362	454	<0.5
DCM	359	565	<0.5	360	452	<0.5

). Compound 4 exhibited absorption maxima at 360–363 nm and emission maxima at 452–463 nm (Table 1). Purine derivative 3 showed a blue shift in DCM and red shift in DMSO for emission spectra compared to derivative 4, probably due to the presence of tosylate ions (Figure 1 and Figure 2). The quantum yields for 10⁻⁴ M solutions of both compounds were below the detection range (<0.5%). In contrast, starting material 1 was reported to have a 98% QY in DMSO solution at 10⁻⁵ M concentration [21]. Thus, upon derivatization of propane-1,3-diol fragments at the purine N(9) position with pyridinium moieties, the solubility of compounds in water was greatly enhanced, but the fluorescence was practically quenched.

3. Materials and Method

NMR spectra were recorded on Bruker Avance 300 and Bruker Avance 500 spectrometers. ¹H-NMR spectra were recorded at 300 or 500 MHz with internal references from nondeuterated solvents (δ = 7.26 for CDCl₃, δ = 2.50 for DMSO-d₆) at 20° or 80 °C. ¹³C-NMR spectra were recorded at 75.5 or 125.7 MHz with internal references from nondeuterated solvents (δ = 77.16 for CDCl₃, δ = 39.52 for DMSO-d₆) at 20° or 80 °C. Coupling constants are reported in Hz, chemical shifts of signals are given in ppm, and standard abbreviations are used for multiplicity assignments. Fourier transform infrared (FTIR) spectra were recorded using a Thermo Scientific Nicolet™ iSTM50 (Thermo Fisher, Waltham, MA, USA) spectrometer in the Attenuated Total Reflectance (ATR) mode. Spectra were obtained over a range of wavenumbers from 400 to 4000 cm⁻¹, co-adding 64 scans at 4 cm⁻¹ resolution. Before every measurement, a background spectrum was taken and deducted from the sample spectrum. An Orbitrap Exploris 120 (Thermo Scientific, Waltham, MA, USA) mass spectrometer was used for high-resolution mass spectra (ESI). UV–Vis absorption spectra were recorded with a PerkinElmer Lambda 35 spectrometer. Emission spectra and quantum yields for solutions were recorded using a QuantaMaster 40 steady-state spectrofluorometer (Photon Technology International, Inc., Birmingham, NJ, USA) equipped with a 6-inch integrating sphere by LabSphere (North Sutton, NH, USA), using the software package provided by the manufacturer.

2-{6-[4-(4-Methoxyphenyl)-1H-1,2,3-triazol-1-yl]-2-[piperidin-1-yl]-9H-purin-9-yl}propane-1,3-diyl bis(4-methylbenzenesulfonate) 2. To a suspension of compound 1 (250 mg, 0.56 mmol, 1.0 eq.) and 4-dimethylaminopyridine (7 mg, 0.056 mmol, 0.1 eq.) in dry DCM (30 mL), Et₃N (0.19 mL, ρ = 0.73 g/cm³, 1.34 mmol, 2.4 eq.) was added, and the reaction mixture was cooled to 0 °C temperature. Tosyl chloride (253 mg, 1.34 mmol, 2.4 eq.) was added in small portions to the reaction mixture at 0 °C temperature, and the reaction was left stirring for 2 h and then overnight at room temperature. DCM was washed with water (2 × 30 mL) and brine (30 mL), dried over anhydrous Na₂SO₄, filtered and evaporated. Recrystallization from hot ethanol (15 mL) provided product 2 (322 mg, 76%) as a beige amorphous solid. ¹H-NMR (500 MHz, CDCl₃) δ (ppm): 9.01 (s, 1H, H-C(triazole)), 7.80 (d, 2H, ³J = 9.0 Hz, 2×H-C(Ar)), 7.53 (s, 1H, H-C(purine)), 7.45 (d, 4H, ³J = 8.2 Hz, 4×H-C(Ts)), 7.07 (d, 2H, ³J = 9.0 Hz, 2×H-C(Ar)), 7.03 (d, 4H, ³J = 8.2 Hz, 4×H-C(Ts)), 4.75 (tt, 1H, ³J = 8.0, 4.6 Hz, (-CH-)), 4.59 (dd, 2H, ²J = 10.7 Hz, ³J = 8.0 Hz, (-CH₂-)), 4.42 (dd, 2H, ²J = 10.7 Hz, ³J = 4.6 Hz, (-CH₂-)), 3.90 (s, 3H, (-CH₃)), 3.83–3.70 (m, 4H, 2×(-CH₂-)), 2.27 (s, 6H, 2×(-CH₃)), 1.74–1.68 (m, 2H, (-CH₂-)), 1.68–1.62 (m, 4H, 2×(-CH₂-)). ¹³C-NMR (125.7 MHz, CDCl₃) δ (ppm): 160.2, 158.3, 152.8, 147.6, 145.7, 144.5, 140.7, 131.4, 130.4, 129.9, 127.6, 124.7, 123.0, 122.5, 115.0, 65.2, 55.8, 54.5, 45.4, 26.0, 25.0, 21.8. IR (neat) ν (cm⁻¹): 2934, 2853, 1628, 1564, 1445, 1362, 1250, 1175, 1095, 1017, 977, 808, 785, 640, 554. HRMS (ESI): m/z calculated for [C₃₆H₃₈N₈O₇S₂ + H]⁺ 759.2378, found 759.2349.

1,1′-(2-{6-[4-(4-Methoxyphenyl)-1H-1,2,3-triazol-1-yl]-2-[piperidin-1-yl]-9H-purin-9-yl}propane-1,3-diyl)bis(pyridin-1-ium) di-4-methylbenzenesulfonate 3. In the pressure vial, compound 2 (120 mg, 0.158 mmol) in pyridine (7 mL) was heated at 120 °C temperature for 1 h. The reaction mixture was evaporated, then dissolved in DCM (20 mL) and washed with cold water (2 × 30 mL) and cold brine (30 mL). The organic phase was concentrated in a vacuum, giving product 3 (104 mg, 72%) as a yellow oil. ¹H-NMR (300 MHz, DMSO-d₆, 80 °C) δ (ppm): 9.22 (s, 1H, H-C(triazole)), 9.02 (d, 4H, ³J = 6.0 Hz, 4×H-C(Pyridine)), 8.58 (t, 2H, ³J = 7.7 Hz, 2×H-C(Pyridine)), 8.34 (s, 1H, H-C(purine)), 8.06 (d, 4H, ³J = 7.0 Hz, 4×H-C(Pyridine)), 7.92 (d, 2H, ³J = 8.7 Hz, 2×H-C(Ar)), 7.58 (d, 4H, ³J = 8.1 Hz, 4×H-C(Ts)), 7.12 (d, 4H, ³J = 8.1 Hz, 4×H-C(Ts)), 7.06 (d, 2H, ³J = 8.7 Hz, 2×H-C(Ar)), 6.05–5.90 (m, 1H, (-CH-)), 5.72–5.52 (m, 4H, 2×(-CH₂-)), 3.83 (s, 3H, (-CH₃)), 3.83–3.75 (m, 4H, 2×(-CH₂-)), 2.30 (s, 6H, 2×(-CH₃)), 1.74–1.53 (m, 6H, 3×(-CH₂-)). ¹³C-NMR (75.5 MHz, DMSO-d₆, 80 °C) δ (ppm): 159.4, 157.2, 155.8, 146.4, 146.3, 145.5, 144.9, 144.6, 141.5, 137.3, 128.0, 127.6, 126.9, 125.2, 121.9, 118.7, 114.5, 114.2, 59.4, 56.2, 55.0, 44.7, 24.9, 23.7, 20.3. IR (neat) ν (cm⁻¹): 3058, 2936, 1627, 1560, 1490, 1444, 1176, 1120, 1009, 910, 782, 680, 564. HRMS (ESI): m/z calculated for [C₃₂H₃₄N₁₀O]²⁺ 287.1453, found 287.1449.

1,1′-(2-{6-[4-(4-Methoxyphenyl)-1H-1,2,3-triazol-1-yl]-2-[piperidin-1-yl]-9H-purin-9-yl}propane-1,3-diyl)bis(pyridin-1-ium) dichloride 4. Compound 3 (52 mg, 0.057 mmol) was dissolved in water (0.3 mL) and flashed through wet Bio-Rex^® 9 ion exchange resin (2 mL), using water (5 mL) as an eluent. Water was evaporated, giving product 4 (31 mg, 84%) as a yellow oil. ¹H-NMR (500 MHz, DMSO-d₆, 80 °C) δ (ppm): 9.43 (d, 4H, ³J = 6.0 Hz, 4×H-C(Pyridine)), 9.22 (s, 1H, H-C(triazole)), 8.78 (s, 1H, H-C(purine)), 8.57 (t, 2H, ³J = 7.7 Hz, 2×H-C(Pyridine)), 8.08 (t, 4H, ³J = 7.0 Hz, 4×H-C(Pyridine)), 7.92 (d, 2H, ³J = 8.7 Hz, 2×H-C(Ar)), 7.06 (d, 2H, ³J = 8.7 Hz, 2×H-C(Ar)), 6.27 (tt, 1H, ³J = 10.4, 3.5 Hz, (-CH-)), 5.92 (dd, 2H, ²J = 13.7 Hz, ³J = 3.5 Hz, (-CH₂-)), 5.74 (dd, 2H, ²J = 13.7 Hz, ³J = 10.4 Hz, (-CH₂-)), 3.83 (s, 3H, (-CH₃)), 3.75 (t, 4H, ³J = 5.3 Hz, 2×(-CH₂-)), 1.71–1.64 (m, 2H, (-CH₂-)), 1.64–1.54 (m, 4H, 2×(-CH₂-)). ¹³C-NMR (125.7 MHz, DMSO-d₆, 80 °C) δ (ppm): 159.4, 157.1, 155.9, 146.29, 146.27, 145.0, 144.4, 142.0, 127.8, 126.8, 121.9, 118.7, 114.3, 114.2, 59.1, 56.8, 55.0, 44.6, 24.9, 23.7. IR (neat) ν (cm⁻¹): 3046, 2935, 2853, 1626, 1560, 1489, 1442, 1249, 1177, 1017, 910, 838, 782, 684. HRMS (ESI): m/z calculated for [C₃₂H₃₄N₁₀O]²⁺ 287.1453, found 287.1446.

4. Conclusions

1,1′-(2-{6-[4-(4-Methoxyphenyl)-1H-1,2,3-triazol-1-yl]-2-[piperidin-1-yl]-9H-purin-9-yl}propane-1,3-diyl)bis(pyridin-1-ium) di-4-methylbenzenesulfonate and 1,1′-(2-{6-[4-(4-methoxyphenyl)-1H-1,2,3-triazol-1-yl]-2-[piperidin-1-yl]-9H-purin-9-yl}propane-1,3-diyl)bis(pyridin-1-ium) dichloride salts were obtained in 72 and 84% yields by substitution of tosylates of propane-1,3-diol fragments at the N9 position of purine with pyridinium moieties. 2-Amino-6-triazolyl purine pyridinium conjugates were easily soluble in water, and this opens the possibility of using them in biological studies in the future. 1,1′-(2-{6-[4-(4-Methoxyphenyl)-1H-1,2,3-triazol-1-yl]-2-[piperidin-1-yl]-9H-purin-9-yl}propane-1,3-diyl)bis(pyridin-1-ium) dichloride salt showed decreased fluorescence intensity in MeCN, MeOH, H₂O, DMSO, and DCM and had a QY below 0.5% in comparison to its highly fluorescent analog 2-(6-(4-(4-methoxyphenyl)-1H-1,2,3-triazol-1-yl)-2-(piperidin-1-yl)-9H-purin-9-yl)propane-1,3-diol.