Synthesis and Characterization of New Monosubstituted Pillar[5]arene with Terminal Carboxyl Group

Abstract

The subject of this article is a new monosubstituted pillar[5]arene with a terminal carboxylic group. Previously, it was shown that monosubstituted pillar[5]arene forms solid lipid nanoparticles with various morphologies depending on the nature of the terminal group. The present work is devoted to the synthesis of monosubstituted pillar[5]arene with an anionic carboxylic group for the further creation of drug–carrier systems based on them. The chemical structure of the product obtained was established based on ¹H and ¹³C NMR, IR spectroscopy, MALDI TOF mass spectrometry, and elemental analysis.

1. Introduction

The development of targeted delivery has led to the creation of new carrier systems capable of the targeted transport of drugs [1,2,3,4,5,6,7]. These include polymer compounds with active substances [8], polymer micelles [9], inorganic nanoparticles [10], liposomes [11], etc. Solid lipid nanoparticles [12], as another promising carrier option, are of particular interest to researchers, since they have such properties as a slower drug release rate, low toxicity, biodegradability, high encapsulation capacity for both hydrophobic and hydrophilic drugs, as well as the possibility of large-scale production due to the economical process of nanoparticle synthesis.

Previously, this type of associate was synthesized using amphiphilic thiacalix[4]arene as an example [13]. In addition, such particles were shown to be able to effectively interact with DNA; however, particles based on thiacalix[4]arene caused partial denaturation of the biopolymer [13,14]. The use of promising pillar[5]arenes for the synthesis of solid lipid nanoparticles is of particular interest [15,16,17,18]. In our previous works, the relationship between the structure of the synthesized macrocycle and the properties (size, stability, morphology) of the nanoparticles was demonstrated [17]. Thus, depending on the ability of the macrocycle to include/not include its “tail” in the cavity, spherical, spindle-shaped, and rod-shaped particles can be formed. The self-assembly strongly depends on the chemical nature of the “tail”. Such particles have been shown to be able to act as new nanoscale carriers for the targeted delivery of pharmaceutical agents or to track the localization of particles loaded with a pharmaceutical agent in the body [17].

The introduction of even one new functional group into the macrocycle leads to the appearance of new properties without significantly changing its ability to recognize guests with a macrocyclic cavity. Functionalization of the macrocyclic platform with amide fragments [19] containing a carbonyl group—a donor of unshared electron pairs, and a proton-donor NH fragment—is a promising approach to the creation of polyfunctional structures capable of binding both charged and neutral guest molecules of an organic and inorganic nature, as well as regulating the selectivity of interaction with biopolymers [17]. The introduction of two amide fragments and one carboxyl group into the structure of the substituent in the macrocycle will increase the affinity for the biosubstrate due to additional H-bonds.

The present work is devoted to the synthesis of monosubstituted pillar[5]arene with anionic carboxylic group and two amide fragments for the further creation of drug–carrier systems based on them.

2. Results and Discussion

We synthesized new monosubstituted pillar[5]arene containing terminal carboxyl and two amide groups linked by hexamethylene spacer (Scheme 1).

The synthesis of the initial monosubstituted pillar[5]arene 1, containing an ester fragment, was carried out using methods from the literature [19]. The synthesis of the monosubstituted pillar[5]arene 2, containing a terminal amine group, was carried out using methods from the literature [20]. We synthesized a new monosubstituted pillar[5]arene derivative containing diglycolic acid fragment 3 from the reaction of 2 with diglycolic anhydride. The latter was synthesized by us by means of dehydration of diglycolic acid with the action of acetic acid chloride in chloroform. Reactions with glycolic anhydride were carried out with a 4-fold excess of the reagent in tetrahydrofuran (THF) at the boiling point of the solvent (66 °C) (Scheme 1). The choice of THF as a solvent is determined by several factors: (1) polar aprotic solvents are used when carrying out acylation reactions; (2) the reagents have high solubility in THF; and (3) spontaneous opening of the glycolic anhydride ring does not occur in this solvent. After 40 h, compound 3 was isolated with 71% yield.

The structure of 3 were decisively assigned based on IR, ¹H and ¹³C NMR spectroscopy, MALDI TOF mass spectrometry, and elemental analysis (see Figures S1–S5). Previously, Wang et al. showed the formation of an intramolecular hydrogen bond NH∙∙∙OCH₂ and inclusion of an alkylamide fragment into the macrocyclic cavity by means of X-ray diffraction for compounds with a similar structure [20]. In our previous works, we showed that macrocycle 2 tends to form a self-inclusion complex in CDCl₃ as well as in DMSO because of this intramolecular NH⋯OCH₂ hydrogen bond [21]. A (6′-amino)hexamethyleneamide fragment was included in the macrocyclic cavity up to the fourth carbon atom, while the amino group remained outside the macrocyclic cavity. The same behavior was observed for compound 3 described in the current manuscript. There are significant upfield shifts (−2.11 and −1.77 ppm, respectively) of the methylene proton (H⁶ and H⁷) signals of the alkyl fragment of 3 in the ¹H NMR spectrum. H⁸ and H⁹ methylene proton signals were also observed in the upfield (−0.72 and −0.33 ppm, respectively). This fact unambiguously indicates inclusion of the alkyl substituent in the pillar[5]arene cavity (see Figure S1).

A narrow intense band at 3405 cm^–1 in the IR spectra (see Figure S4) of pillar[5]arene 3 also indicates the formation of a strong hydrogen bond between the NH protons and oxygen of the methoxyl fragment. We assumed that the presence of such a strong hydrogen bond is possible only for the self-inclusion complex characterized for compound 3.

The comparison of ¹H NMR spectra of compounds 2 and 3 also confirms the inclusion of a (6′-amino)hexamethyleneamide fragment in the macrocyclic cavity up to the fourth carbon atom (see Figure S6).

These facts unambiguously indicate inclusion of the alkyl substituent in the pillar[5]arene cavity [21].

3. Materials and Methods

General

¹H, ¹³C, and 2D NOESY NMR spectra were obtained on a Bruker Avance-400 spectrometer (Bruker Corp., Billerica, MA, USA) (¹³C{1H}–100 MHz and ¹H and 2D NOESY–400 MHz). The chemical shifts were determined against the signals of residual protons of a deuterated solvent (CDCl₃, DMSO-d₆). The concentrations of the compounds were equal to 3–5% by the weight in all the records. The FTIR ATR spectra were recorded on the Spectrum 400 FT-IR spectrometer (Perkin Elmer Inc, Waltham, MA, USA) with a Diamond KRS-5 attenuated total internal reflectance attachment (resolution 0.5 cm⁻¹, accumulation of 64 scans, recording time 16 s in the wavelength range 400–4000 cm⁻¹). Mass spectra were obtained on a Bruker Ultraflex III MALDI-TOF instrument (Bruker Daltonik GmbH, Bremen, Germany) with p-nitroaniline as the matrix. Elemental analysis was performed on Perkin–Elmer 2400 Series II instruments (Perkin Elmer, Waltham, MA, USA). Melting points were determined using a Boetius Block apparatus (VEB Kombinat Nagema, Radebeul, Germany).

Diglycolic acid and sodium chloride were purchased from Acros (USA) and used as received. Chemically pure organic solvents were purified by standard methods. All the aqueous solutions were prepared with Millipore-Q deionized water (>18.0 MW cm at 298 K).

4-(Ethoxycarbonylmethoxy)-8,14,18,23,26,28,31,32,35-nonamethoxypillar[5]arene (1) was synthesized according to the literature [21]. Yield: 1.77 g (76%). M.p.: 209 °C (208–210 °C).

4-[(N-{6′-aminohexyl}-amino)-carbonylmethoxy]-8,14,18,23,26,28,31,32,35-nonamethoxypillar[5]arene (2) was synthesized according to the literature [22]. Yield: 0.36 g (78%). M.p.: 218 °C.

4-(2-[6-(2-[Carbomethoxy]acetamido)hexylamino]-2-oxoethoxo)-8,14,18,23,26,28,31,32,35-nonamethoxy-pillar[5]arene (3). Macrocycle 2 (0.2 g, 0.2 mmol) was dissolved in 6 mL of tetrahydrofuran in a round-bottomed flask equipped with a magnetic stirrer. Then, 0.09 g (0.9 mmol) of diglycolic anhydride was added. The reaction mixture was refluxed for 40 h. The solvent was removed under reduced pressure at the end of the synthesis. The product was recrystallized from water.

Yield: 0.18 g (71%). Decomposition point = 150 °C. ¹H NMR (400 MHz, 298 K, DMSO-d₆): −1.65–(−1.18) (m, 4H, C(O)NHCH₂CH₂), 0.02–0.19 (m, 2H, C(O)NHCH₂CH₂CH₂), 1.13–1.51 (m, 4H, CH₂CH₂CH₂NH), 2.16–2.31 (m, 2H, CH₂CH₂CH₂NH), 3.58–3.80 (m, 37H, -OCH₃- and -CH₂-), 3.97 (s, 2H, -NH-C(O)-CH₂-O), 4.19 (s, 2H, -O-CH₂-C(O)OH), 4.38–4.73 (m, 2H, Ar-O-CH₂), 4.74–4.84 (m, 1H, -C(O)NH-), 6.60–6.98 (m, 10H, ArH). ¹H NMR (400 MHz, 298 K, CDCl₃): δ −2.23–(−1.97) (m, 2H, C(O)NHCH₂), −1.91–(−1.65) (m, 2H, C(O)NHCH₂CH₂), −0.92–(−0.52) (m, 2H, C(O)NHCH₂CH₂CH₂), −0.47–(−0.23) (m, 2H, CH₂CH₂CH₂NH), 2.34–2.50 (m, 4H, CH₂CH₂CH₂NH), 3.66–3.86 (m, 37H, -OCH₃- and -CH₂-), 4.24 (s, 2H, -NH-C(O)-CH₂-O), 4.29 (s, 2H, -O-CH₂-C(O)OH), 4.63 (s, 2H, Ar-O-CH₂), 5.40–5.54 (m, 1H, -C(O)NH-), 6.73–7.00 (m, 10H, ArH). ¹³C NMR (100 MHz, DMSO-d₆): δ 28.8, 28.9, 29.5, 35.5, 55.4, 55.5, 65.4, 67.2, 67.8, 70.2, 113.3, 113.9, 127.4, 127.5, 127.6, 149.2, 149.9, 150.0, 168.8, 171.3, 171.5. IR, ν/cm⁻¹: 3405 (NH), 2936 (OH), 2853, 2830, 1735, 1449, 1244 (COOH), 1636, 1535, 1207 (C(O)NH). MS (MALDI TOF) calc. [M+H⁺]⁺ m/z = 1009.5, [M+K⁺]⁺ m/z = 1047.4, found [M+H⁺]⁺ m/z = 1009.9, [M+K⁺]⁺ m/z = 1047.9. Found (%): C, 66.58, H, 6.90, N, 2.70. Calc. for C₅₆H₆₈N₂O₁₅. (%): C, 66.65, H, 6.79, N, 2.78.

4. Conclusions

Thus, we have demonstrated the synthesis of pillar[5]arene containing terminal carboxyl and two amide groups linked by a hexamethylene spacer with high yield. The chemical structure of the product obtained was established based on ¹H and ¹³C NMR, IR spectroscopy, MALDI TOF mass spectrometry, and elemental analysis.