C-2,8,14,20-tetra(propyl)-5,11,17,23-tetrakis(N–(piperidine)methyl)calix[4]resorcinarene

Abstract

Calix[4]resorcinarenes are polyhydroxylated macrocyclic compounds with four units of resorcinol. These compounds can be derivatized through modifications at the upper rim, allowing reactivity with secondary amines to produce Mannich base derivatives via Mannich-type aminomethylation reactions. In this paper, we report the reaction of C-tetra(propyl)calix[4]resorcinarene with piperidine in acetonitrile. The aminomethylated compound C-2,8,14,20-tetra(propyl)-5,11,17,23-tetrakis(N–(piperidine)methyl)calix[4]resorcinarene was obtained with a 52% yield, with an exact mass of 1044.6994 u and a mass error of 7.6 ppm. The reaction progress and product formation were monitored by RP-HPLC, and the compound was characterized using LC ESI-TOF/MS, one- and two-dimensional ¹H and ¹³C NMR, and FTIR spectroscopy. Chromatographic and spectroscopy data are presented and discussed.

1. Introduction

The Mannich reaction is a powerful tool in the field of organic chemistry for the formation and incorporation of C-C bonds in different molecules. This amino alkylation is a three-component nucleophilic condensation reaction between an acidic hydrogen compound, a non-enolizable aldehyde, e.g., formaldehyde, and a secondary amine or amide. The condensation produces a Mannich base, in which a hydrogen atom is replaced by an aminomethyl group [1,2,3]. In calix[4]resorcinarenes, polyhydroxylated macrocyclic molecules synthesized from resorcinol and aldehyde, which contain aromatic active hydrogens in the ortho position on the upper rim, can be replaced by the addition of an aminomethyl derivative in order to obtain the corresponding Mannich base. This process involves, as a first step, the reaction between amine and a methylation agent, e.g., formaldehyde, generating the hemiaminal intermediate, a subsequent dehydration, and the formation of an iminium cation. The second step involves a reaction in which the aromatic phenolic ring, rich in electrons in the calix[4]resorcinarene, attacks the iminium cation via an electrophilic aromatic substitution (S_EAr). In this type of substitution, the electron-rich aromatic ring, in the presence of an electrophile, forms a cationic intermediate, which recovers its aromaticity by the loss of a hydrogen atom in position 2. Therefore, the corresponding Mannich base is formed in the ortho position by replacing aromatic hydrogen (Figure 1) [2,4,5]. When primary amines are used, the product reaction is generally a benzoxazine, due to a second aminomethylation step and the intramolecular nucleophilic attack of a phenolic hydroxyl group on the iminium intermediate. In some cases, the Mannich base has been obtained using primary amines, depending on the pH and the solvent used [6].

Mannich bases have been obtained through Mannich-type aminomethylation reactions using calix[4]resorcinarenes and secondary amines as precursors in ethanolic solvent mixtures. An example of this process is the reaction of resorcinarenes such as tetra(nonyl)calix[4]resorcinarene or tetra(4-hydroxyphenyl)calix[4]resorcinarene which can be functionalized with L-proline [7,8,9].

Therefore, in this investigation, we present the synthesis of the incorporation of a heterocyclic piperidine amine into C-tetra(propyl)calix[4]resorcinarene by a Mannich aminomethylation reaction, obtaining a highly symmetrical 2-substituted cyclotetramer Mannich base, using acetonitrile (ACN), an aprotic polar solvent. RP-HPLC, LC-MS, and spectroscopy analyses are presented and discussed.

2. Results and Discussion

In the present study, we particularly wanted to see if the use of a polar aprotic solvent as an alternative to protic solvents such as ethanol, or more common mixtures such as EtOH:H₂O or EtOH:C₆H₆, commonly used for aminomethylation reactions [10], has any effect on the substitution, causing, for example, partial substitution at position 2 of the phenolic rings, no Mannich base formation, or an effect on the conformational properties of the product(s), taking as a starting point the symmetric C-tetra(propyl)calix[4]resorcinarene (1), which is stabilized in the crown conformation by the formation of intramolecular hydrogen bonds [6,11].

The synthesis of the precursor C-tetra(propyl)calix[4]resorcinarene (1) was performed following a previously reported procedure (see Section 3.2) [12]. The IR spectrum data of 1 show bands corresponding to functional groups in the resorcinarene precursor, notably the phenolic O–H stretching vibrations. ¹H and ¹³C NMR spectra show only seven proton and seven carbon signals. The limited number of signals observed in both the aromatic (downfield) and aliphatic (upfield) regions indicates that the majority of the carbon nuclei are chemically equivalent. Consequently, compound 1 is probably highly symmetrical, corresponding to C_4v point group symmetry, which corresponds to an rccc-crown conformation. In other conformations, a higher number of signals could be observed because of the spatial arrangement of the resorcinol units and the orientation of the substituents, resulting in a higher number of non-equivalent atoms. For example, in the C-tetra(methyl)calix[4]resorinarene rccc-crown (C_4v), when compared to the boat (C_2v) and chair (C_2h) conformers, a higher number of ¹H-NMR signals is observed in the corresponding spectrum, suggesting conformational differences in each case [13].

After obtaining precursor 1, the upper rim was functionalized by Mannich-type aminomethylation reaction using piperidine as the precursor amine. Since piperidine is a secondary cyclic amine, the reaction at position two in the aromatic units of calix[4]resorcinarene should correspond to a Mannich base. The reaction, using acetonitrile as a solvent, occurred at room temperature, in a short time and it was not necessary to use catalysts or reflux conditions.

The solid product (2) was analyzed by RP-HPLC at 280 nm (Figure 2a,b). A comparison of the chromatographic profile of calix[4]resorcinarene precursor 1 (Figure 2a) and the precipitated pink solid compound 2 showed a shorter retention time in RP-HPLC, suggesting the higher hydrophilicity of compound 2 than that of precursor 1, due to the presence of nitrogen atoms in the aminomethylated compound. The LC ESI-TOF/MS isotopic pattern of species [M+H]¹⁺ is shown in Figure 2c.

The resulting product showed a mass isotopic pattern with a [M+H]⁺ signal of m/z = 1045.6994. The experimental mass (Exp_mass) was calculated to be 1044.6994 u, which was compared with the theoretical mass—M: 1044.6915 u—with an error of 7.6 ppm, confirming the successful formation of the tetra-aminomethylated product between 1 and piperidine. Multiple charged ions were observed at m/z = 523.3533, corresponding to [M+2H]²⁺, and m/z = 349.2382, corresponding to [M+3H]³⁺. These ions result from the protonation of the nitrogen atoms in the ESI system, which is expected due to the presence of amines in the upper ring, as depicted in the structure in Figure 3.

The IR spectroscopic data are reported in Section 3.3, and the 1D and 2D NMR data are presented in

Table 1. Two-dimensional NMR correlation for Mannich base compound 2.

Structure Compound	C atom	δ (ppm)	Correlation (δ, ppm)
			HSQC (1H-13C)	HMBC (1H-13C)	COSY (1H-1H)
	1	122.0	7.11 (H1)	33.1 (C9); 107.0 (C4); 124.0 (C2); 151.0 (C3)	-
	2	124.0	-		-
	3	151.0	-		-
	4	107.0	-		-
	5	53.7	2.05 (H5); 2.97 (H5′)		-
	6	55.9	3.74 (H6)	53.7 (C5); 107.0 (C4); 151.0 (C3)	-
	7	25.6	1.64 (H7)	-	-
	8	23.9	1.76 (H8)	-	-
	9	33.1	4.32 (H9)	21.3 (C11); 35.5 (C10) 122.0 (C1); 124.0 (C2); 151.0 (C3)	2.19 (H10)
	10	35.5	2.19 (H10)	14.2 (C12); 21.3 (C11); 124.0 (C2)	4.32 (H9)
	11	21.3	1.33 (H11)	14.2 (C12); 33.1 (C9)	2.19 (H10)
	12	14.2	0.98 (H12)	21.3 (C11); 35.5 (C10)	1.33 (H11)

. The bands observed in FTIR are evidence of some characteristic functional groups of the aminomethylated compound 2. Bands were observed that correspond to the stretches of phenolic hydroxyl groups (-OH, 3200 cm⁻¹), aliphatic carbons and a methine bridge on the lower edge (-CH₂, -CH, 2930 and 2856 cm⁻¹), and the bonds of the tri-substituted cyclic amine derived from functionalization with piperidine (C-N, 1111 cm⁻¹), in the ortho position in the phenolic rings. Other intense bands corresponded to the C=C stretches of the aromatic rings (1609 and 1450 cm⁻¹).

As shown in Table 1, the ¹H NMR spectrum shows nine signals corresponding to the spin–spin couplings of the protons and the disappearance of one of the two signals in the aromatic range, confirming the substitution in the ortho position, and the ¹³C NMR spectrum exhibits twelve signals corresponding to the chemically differentiated carbons. The signal at 151.0 ppm is very weak but is identified through the vicinal coupling between H₁ and C₃, which is linked to the hydroxyl group in the ¹H-¹³C HMBC spectrum. The two-dimensional ¹H-¹³C HSQC spectrum shows nine carbons with hydrogen, as shown for the proposed structure. Note that the chemical shifts of C₅ and C₆ (δ = 53.7 and 55.9 ppm) and the respective hydrogen ones are within the ranges of aliphatic C-N reported in the literature [14]. In addition, the HMBC experiment showed long-distance heteronuclear correlations (²J, ³J, and ⁴J), establishing connectivity between the aromatic proton H₁ (7.11 ppm) and the aromatic carbons C₂ (124.0 ppm), C₃ (151.0 ppm), and C₄ (107.0 ppm), and the methine carbon C₉ (33.1 ppm). The connectivity between H₆ (3.74 ppm) of the piperidine ring, the methylene carbon C₅ (53.7 ppm), the ortho-substituted aromatic carbon C₄ (107.0 ppm), produced by the Mannich-type aminomethylation reaction, and C₃ was confirmed. This corresponds to the molecular structure presented and confirms the obtainment of the Mannich base 2.

The HSQC experiment showed that the geminal protons H₅ (2.05 ppm) and H_5′ (2.97 ppm) at the C₅ methylene bridge are diastereotopic. This difference in their chemical shifts can be explained by the two methylene bridge hydrogens being in different environments, i.e., axial and equatorial types, and consequently, they are not equivalent [15]. The ¹H-¹H COSY couplings are as expected for the alkyl propyl chain of the starting resorcinarene lower edge. The fact that there is the minimum number of equivalent signals in the ¹H and ¹³C spectra and no duplications for the same carbon or hydrogen indicates that the molecule is symmetric, tetra-substituted, and maintains the rccc-crown conformation of the precursor resorcinarene 1.

The adopted methodology did not use basic catalysts, as was carried out in other reported methodologies [8], and the use of an aprotic polar solvent did not affect the stabilization of the intermediates, so the reaction was carried out with good yields (52%) and worked in an open atmosphere, in contrast to methodologies that require the use of a N₂ atmosphere for cyclic amines with alkyl calix[4]resorcinarenes [12]. The synthesis was very clean, since no additional purification processes were required. To the best of our knowledge, this is the first time that this derivative has been obtained and characterized.

3. Materials and Methods

3.1. General Experimental Information

All the reagents and solvents used in the synthesis were of analytical grade and were used without further purification. Resorcinol, butyraldehyde, trifluoroacetic acid (TFA), acetonitrile (ACN), and piperidine were purchased from Sigma-Aldrich (St. Louis, MO, USA). CDCl₃ was obtained from Merck (Darmstadt, Germany). FTIR-ATR analysis was performed using a Thermo Nicolet iS5 FTIR equipped with an ATR iD7 accessory, and the absorption was recorded in cm⁻¹. The nuclear magnetic resonance spectrum of ¹H NMR was obtained at 400 MHz and that of ¹³C NMR at 101 MHz on a Bruker Avance 400 instrument, using CDCl₃ as a sample solvent. Chemical shifts (δ) are reported in ppm, using the solvent residual signal.

3.2. General Procedure for Synthesis of C-tetra(propyl)calix[4]resorcinarene

Resorcinol (4 mmol) was dissolved in 10 mL of ethanol and 4 mL of 37% hydrochloric acid with stirring. Separately, butanal (4 mmol) was dissolved in 6.5 mL of ethanol and added dropwise to the reaction under stirring. The mixture was refluxed for 6 h, and a clear yellow solution was obtained. The addition of 50 mL of Type I water induced the formation of a yellow precipitate. The yellow precipitate was separated by filtration, washed three times with water, and dried under reduced pressure. The product was characterized using RP-HPLC, ¹H NMR, ¹³C NMR, and ATR-FTIR.

C-2,8,14,20-tetra(propyl)calix[4]resorcinarene (1) was obtained as a pale orange solid with 92% yield. FTIR (ATR/cm⁻¹): 3270 ν(O–H), 2954–2867 ν(C–H), 1200–1100 ν(C–O) ¹H NMR, DMSO-d₆, (δ, ppm): 0.89 (t, J = 7.3 Hz, 12H, CH₃), 1.19 (m, J = 7.4 Hz, 8H, CH₂), 2.08 (q, 8H, CH₂), 4.22 (t, 4H, CH), 6.14 (s, 4H, CH aromatic), 7.24 (s, 4H, CH aromatic), 8.92 (s, 4H, OH); ¹³C NMR, DMSO-d₆, (δ, ppm): 151.4 (C of -OH), 124.8 (aromatic C), 123.0 (aromatic C), 102.1 (aromatic C), and 35.5 (methine C), 32.4, 20.5, 13.6 (aliphatic propyl chain C).

3.3. General Procedure for the Synthesis of C-2,8,14,20-tetra(propyl)-5,11,17,23-tetrakis(N-(piperidin)methyl)calix[4]resorcinarene

C-tetra(propyl)calix[4]resorcinarene (1 mmol) was dissolved in 5 mL of ACN, and 400 μL (10 mmol) of formaldehyde was added. A solution of 2 mL of piperidine (6 mmol) in ACN was added dropwise. The reaction mixture was stirred constantly at room temperature for 6 h. The precipitate was separated by filtration and dried under vacuum. The final product was analyzed and characterized via RP-HPLC, LC ESI-TOF/MS, ¹H NMR (400 MHz, CDCl₃, 297 K), ¹³C NMR (101 MHz, CDCl₃, 297 K), ¹H-¹H COSY (CDCl₃, 298 K), ¹H-¹³C HMBC (CDCl₃, 297 K), ¹H-¹³C HSQC (CDCl₃, 297 K), and ATR-FTIR.

C-2,8,14,20-tetra(propyl)-5,11,17,23-tetrakis(N-(piperidin)methyl)calix[4]resorcinarene (2) was obtained as a pale pink solid with a 52% yield. FTIR (ATR/cm⁻¹): 3200 ν(O-H), 2930, 2856 cm⁻¹ ν(C-H aliphatic), 1609, 1450 cm⁻¹ ν(C=C Ar), 1111 ν(C-N). ¹H NMR, CDCl₃, (δ, ppm): 0.98 (t, J = 7.3 Hz, 12H, CH₃), 1.35 (m, J = 7.4 Hz, 2H, CH₂), 1.62 (broad s, 8H, CH₂ of piperidine), 1.75 (broad s, 8H, CH₂ of piperidine), 2.05 (broad s, 4H, geminal hydrogen, CH₂), 2.17 (broad s, J = 8.0 Hz, 8H, CH₂), 2.97 (broad s, 4H, geminal hydrogen, CH₂), 3.74 (broad s, 8H, CH₂ of piperidine), 4.34 (t, J = 7.9 Hz, 4H, CH methine hydrogen), 7.11 (s, 4H, aromatic CH); ¹³C NMR, CDCl₃, (δ, ppm): 124.0 (aromatic C), 122.0 (aromatic C), 107.0 (ortho-substituted aromatic carbon relative to the hydroxyls), 55.9 (C-N heterocyclic piperidine), 53.7 (C-N, aminomethylation), 35.5 (CH₂, aliphatic), 33.1 (CH, methine), 25.6, 23.9 (C, heterocyclic piperidine), 21.3 (CH₂, aliphatic), 14.2 (CH₃, aliphatic). The ESI-TOF/MS spectrum showed a signal at m/z = 1045.6994 corresponding to [M+H]⁺, m/z = 523.3533 corresponding to [M+2H]²⁺, and m/z = 349.2382 corresponding to [M+3H]³⁺. Theoretical mass: 1044.6915 u; experimental mass (Exp_mass): 1044.6994 u; Δm: 7.6 ppm.

3.4. Reverse-Phase High-Performance Liquid Chromatography (RP-HPLC) Analysis

RP-HPLC analyses were performed on a Hitachi Primade 1410 with a DAD detector set to 280 nm. The mobile phase was ACN-TFA 0.05% (B) in H₂O-TFA 0.05% (A). The gradient elution was 20/20/100/100/20/20% B at 0/1/18/21/21.1/24 min, and the flow rate was 1.0 mL/min, with a Phenomenex Luna C8 column (100 × 4.6 mm, 5 μm, 100 Å).

3.5. LC-MS Methodologies

Compound analysis was performed using an Impact II LC-MS (Q-TOF) (Bruker Daltonik; Bremen, Germany) equipped with an electrospray ionization (ESI) source, operating in the positive mode. The chromatographic conditions were as follows: Intensity Solo C18 column (2.1 × 100 mm, 1.8 μm) (Bruker Daltonik; Bremen, Germany), temperature of 40 °C, and flow rate of 0.250 mL/min. The mobile phase consisted of water (A) and acetonitrile (B), each containing 0.1% formic acid. The gradient program was as follows: 5/5/95/95/5/5% B in 0/1/11/13/13.1/15 min. The ESI source conditions were as follows: endplate displacement 500 V, capillary at 4500 V, nebulizer at 1.8 bar, dry gas (nitrogen) 8.0 L/min, dry gas temperature 220 °C. Auto MS/MS scanning mode was used with a mass range of 20–2000 m/z, spectral rate of 6 Hz, and a collision energy of 5.0 eV.