Synthesis of 6-Methyluracilpentylviologen Resorcinarene Cavitand

Abstract

Resorcinarenes, as macrocyclic compounds, are widely used to recognize substrates and create supramolecular assemblies. Their bowl-like form organizes functional groups at the upper and lower rims, which has a substantial impact on the molecular recognition of various substrates. As a result, resorcinarenes make good drug nanocarrier candidates. This paper presents the synthesis of a new resorcinarene cavitand functionalized along the upper rim with methyluracil and viologen fragments for its potential use in drug delivery. Methyluracils and viologens are well-known receptor-targeted compounds capable of facilitating the vector transfer of drugs and increasing the effectiveness of their action on cells. The paper describes the synthesis of resorcinarene modified with methyluracil and viologen groups along with its structure determined by ¹H-, ¹³C-NMR and IR spectroscopy.

1. Introduction

One of the emerging areas in medicinal chemistry is the development of vector nanocarriers for drug delivery [1,2]. The main feature of nanocarriers is their ability to encapsulate drugs for delivery to biotargets [3]. For this purpose, amphiphilic nanocarriers are being developed to increase the circulation time of drugs in the body, improve their penetration through tissues, and to accumulate in damaged areas [4,5].

There are several well-known types of macrocyclic compounds used as a platform for the synthesis of vector nanosystems [6,7,8,9]. The actively researched representatives in this field are resorcinarenes. Based on these macrocycles, various nanocontainers and nanocarriers have been created due to unique properties such as the presence of an aromatic cavity, the formation of “guest–host” complexes, as well as the possibility of modifying the upper and lower rims [10,11]. The use of specific functional groups makes it possible to change some properties, such as solubility in water, bioavailability, biological activity, and the ability to self-assemble [12].

Nanocarriers with viologen fragments have shown promise in early investigations for medication delivery. Dendrimers with viologens were shown to have an antiviral effect against the human immunodeficiency virus (HIV) [13]. These dendrimers function as polycationic ligands for the CXCR4 chemokine receptor and are capable of delivering genes while being simultaneously antagonistic to CXCR4 [14,15]. However, viologens cytotoxicity, interaction with the donor pieces of the cells, and formation of reactive oxygen species are their drawbacks. The introduction of additional receptor-directed groups into the nanocarrier base in addition to viologens could solve the toxicity problem.

Uracils have a wide spectrum of biological activity, and they are employed to produce drugs with different pharmacological effects [16,17]. Derivatives of uracil have anti-inflammatory, antiviral, and antioxidant properties and are used to treat herpes, hepatitis B and C, HIV, and other viruses. In vivo tests have demonstrated that 6-methyluracil derivatives can cross the blood–brain barrier and inhibit acetylcholinesterase in brain tissues [18]. Nanocarriers based on these compounds may also have anti-tumor activity and exhibit a synergistic effect with preparations.

The synthesis of resorcinarene cavitand with viologen and methyluracil groups on the upper rim is described in this study (UVCA-C4OH). Data from NMR, IR spectroscopy, and elemental analysis have all been used to confirm the structures of UVCA-C4OH and its intermediates. Future redox-controlled nanocarriers will be made from the resulting macrocycle. We are currently working on this.

2. Results

The synthesis of UVCA-C4OH was carried out as described in Scheme 1. First, bromomethylresorcinarene cavitand (BrCA-C4OAc) was synthesized starting from 3,4-dihydro-2H-pyran and 2-methylresorcine [19,20,21]. Simultaneously, an uracil-viologen derivative (UV) was obtained by reacting 3,(5-brompentyl)-6-methyluracil with 4,4’-bipyridine in DMF. Then, by reacting UV with BrCA-C4OAc, followed by acidic hydrolysis to remove acetate groups, UVCA-C4OH was created with a total yield of 45% (Scheme 1).

To obtain the UV compound, 3-(5-brompentyl)-6-methyluracil dissolved in DMF was added dropwise to a 4,4′-bipyridine solution at 80 °C and then it was mixed for two days. Then, the solvent was removed, and the remainder was washed with methylene chloride to eliminate the residual amount of bipyridine. The yield of the UV compound was 86.3%. The structure of the resulting compound was confirmed by ¹H and ¹³C-NMR spectra, in which all the expected signals of bipyridine, methyluracil, and pentyl tail fragment atoms were observed (see Supplementary Materials (SM), Figures S1 and S2). A strong band of valence vibrations of the C-N+ bond at 812 cm⁻¹ is fixed in the IR spectrum, confirming the formation of the pyridinium ion (Figure S3 in SM).

The UV compound was attached to the upper rim of the cavitand BrCA-C4OAc through the free nitrogen. The reaction was carried out in DMF at 80 °C for 24 h. Then DMF was removed and the hydrolysis with HCl was carried out in order to replace acetyl groups on the lower rim with hydroxyls. The unreacted reagents were removed by dialysis, which was carried out three times for 30 min. The yield of UVCA-C4OH was 62.8%.

In the ¹H-NMR spectrum, the proton peak of the methylene bridge between the cavitand and viologen is fixed at 5.87 ppm, while the proton signals of methylene bridges connecting resorcinol rings appear at 5.63 and 6.39 ppm (Figure S4 in SM). The proton signals of the UV groups are shifted to the low-field compared with the initial UV compound. The signals in the range of 1.30–2.50 ppm and at 3.55, 3.82 ppm were assigned to the hydroxybutylic groups of the lower rim and pentyl linkers. The structure of the UVCA-C4OH was also confirmed by the ¹³C-NMR spectrum, in which the signals of all carbon atoms are fixed (Figure S4 in SM). The signals were assigned using 2D-NMR spectroscopy (HSQC, COSY, HMBC, Figures S5–S7 in SM). The IR spectrum shows the broadened O-H and N-H stretching bands at 3393 cm⁻¹ and a vibration band of the C-N+ bond at 826 cm⁻¹ (Figure S3 in SM).

3. Materials and Methods

NMR spectra were recorded on a Bruker Avance 600 MHz spectrometer. IR spectra were recorded using a Vector-27 FTIR spectrometer (Bruker, Germany) in the 400–4000 cm⁻¹ range. The samples were prepared as KBr pellets. The elemental analysis was carried out on a CHNS analyzer Vario Macro cube (Elementar Analysensysteme GmbH, Langenselbold, Germany). The samples were weighed on Sartorius Cubis II (Goettingen, Germany) microbalance in tin capsules. VarioMacro Software V4.0.11 (Langenselbold, Germany) was used to perform quantitative measurements and evaluate the data received.

BrCA-C4OAc was synthesized as described in [19,20,21] from 2-methylresorcinol, 3,4-dihydro-2H-pyran, bromochloromethane, acetic anhydride, and N-bromosuccinimide. 3-bromopentyl-6-methyluracil was synthesized as described in [22]. Commercially available 4,4′-bypyridine (Alfa Aesar, 98%) was used without purification.

3.1. Synthesis of UV

3-(5-Bromopentyl)-6-methyluracil (0.810 g, 2.9 mmol) in 20 mL of DMF was added dropwise to a solution of 4,4′-bipyridine (1.811 g, 11.6 mmol) in 20 mL of DMF at 80 °C. Then, the mixture was heated at 80 °C for 48 h and DMF was removed at reduced pressure. The resulting oil was washed with methylene chloride to remove the residual amount of bipyridine. The precipitate was separated by centrifugation and dried to yield UV as a brown solid. The yield was 1.08 g (2.5 mmol, 85.3%). ¹H-NMR δ_H (600 MHz, D₂O): 8.91 (2H, d, J = 6.4 Hz, C-H_viologen), 8.74 (2H, d, J = 6.3 Hz, C-H_viologen), 8.37 (2H, d, J = 6.4 Hz, C-H_viologen), 7.87 (2H, d, J = 6.4 Hz, C-H_viologen), 5.58 (1H, s, CH_uracil), 4.63 (2H, t, J = 7.2 Hz, CH₂CH₂N⁺), 3.78 (2H, t, J = 7.2 Hz, CH₂CH₂N), 2.07 (2H + 3H, m CH₂CH₂N⁺ and CH₃), 1.61 (2H, p, J = 7.5 Hz, CH₂CH₂N), 1.32 (2H, p, J = 7.9 Hz, CH₂CH₂CH₂). ¹³C-NMR δ_C (600 MHz, D₂O): 166.3, 153.9, 153.6, 153.1, 150.2, 144.9, 142.7, 126.1, 122.6, 99.3, 61.5, 40.1, 30.0, 26.2, 22.5, 17.8. IR (KBr, cm⁻¹): 3400 br (N-H), 3140–2800 m (C-H), 1706 s (C=O), 1640 s (C=C), 1665 s (C-N), 1599, 1544 s (C-C_Ar), 812 m (C-N⁺). Anal. Calcd. for C₂₀H₂₃N₄O₂⁺ × Br⁻, %: C 55.69; H 5.37; N 12.99; Br 18.53. Found C 55.14; H 5.44; N 13.28; Br 18.85.

3.2. Synthesis of UVCA-C4OAc

1.08 g (2.5 mmol) of UV and 0.854 g (0.63 mmol) of BrCA-C4OAc were dissolved in 10 mL of DMF, and the mixture was stirred in an argon atmosphere at 80 °C for 24 h. Then, DMF was removed at reduced pressure to give an oily brown substance.

3.3. Synthesis of UVCA-C4OH

The oil obtained in the previous step was dissolved in 20 mL of ethanol, and 2 mL of the concentrated hydrochloric acid was added. The mixture was stirred at 80 °C in an argon atmosphere overnight. The solvent was removed at reduced pressure and the resulting solid was treated with acetonitrile. The solid was then dissolved in 5 mL of water, and the resulting solution underwent three dialyses for 30 min each (versus 800 mL of water). After the solvent was removed at reduced pressure, the remaining material was treated with acetonitrile and dried to produce UVCA-C4OH as a brown solid. The yield was 1.015 g (0.40 mmol, 62.8%). ¹H-NMR δ_H (600 MHz, D₂O): 9.08 (16H, m, C-H_viologen), 8.51 (16H, m, C-H_viologen), 7.79 (4H, s, H_Ar), 6.39 (4H, m, OCH), 5.87 (8H, s, CH₂), 5.64 (4H + 4H, m, CH_uracil and OCH), 3.82 (8H, m, CH₂CH₂N), 3.55 (8H, m, CH₂OH), 2.47 (8H, m, CH₂), 2.13 (12H + 8H, m, CH₂ and CH₃), 1.64 (8H + 8H, m, CH₂CH₂N and CH₂CH₂OH), 1.38 (8H + 8H, m, CH₂CH₂CH₂ and CH₂CH₂CH). ¹³C-NMR δ_C (600 MHz, D₂O): 166.4, 153.6, 153.5, 153.1, 150.6, 149.9, 145.8, 145.6, 139.2, 127.1, 126.1, 124.7, 119.9, 100.4, 99.3, 62.1, 61.7, 55.2, 40.1, 37.4, 31.2, 30.1, 28.9, 26.2, 23.6, 22.7, 17.9. IR (KBr, cm⁻¹): 3393 br (O-H), 3118–2859 m (C-H), 1711 s (C=O), 1636 s (C=C, C-N), 1557, 1505 s (C-C_Ar), 826 w (C-N⁺). Anal. Calcd. for C₁₃₂H₁₅₂N₁₆O₂₀⁸⁺ × 8Cl⁻, %: C 61.78; H 5.97; N 8.73; Cl 11.05. Found C 61.39; H 6.23; N 8.95; Cl 10.78.

4. Conclusions

In this work, 6-methyluracilpentylviologen resorcinarene cavitand was synthesized for potential application as a nanocarrier for drug delivery. The cavitand and intermediate products are characterized by a set of physicochemical methods (¹H-NMR, ¹³C-NMR, and IR spectroscopy).