2-(2-(Benzylamino)-2-Oxoethyl)-1-Methyl-1H-Pyrrole-3-Carboxylic Acid

Abstract

Here, 2-(2-(Benzylamino)-2-oxoethyl)-1-methyl-1H-pyrrole-3-carboxylic acid was efficiently synthesised in good yield via an amide coupling reaction between 2-carboxymethyl-1-methyl-1H-pyrrole-3-carboxylic acid and benzylamine, employing TBTU as the coupling reagent and DIPEA as the base. The reaction was carried out in dichloromethane at room temperature. The compound was characterised by melting point determination, ¹H and ¹³C NMR, IR spectroscopy, and mass spectrometry. The combined analytical data confirm the target molecule’s successful synthesis and structural integrity.

1. Introduction

Pyrrole derivatives represent a prominent class of heterocyclic compounds widely utilised in the development of biologically active molecules. Their five-membered nitrogen-containing ring offers a versatile scaffold for designing compounds with diverse pharmacological properties. Numerous pyrrole-based compounds have demonstrated anticancer [1], antimicrobial [2,3], and antitubercular [4] properties. Their role in neuroprotection is exemplified by compounds that inhibit acetylcholinesterase or provide antioxidant activity, which is particularly relevant in Alzheimer’s and Parkinson’s diseases. [5]. Additionally, 2-(Carboxymethyl)-1-methyl-1H-pyrrole-3-carboxylic acid is a bifunctional pyrrole derivative featuring carboxymethyl and carboxylic acid substituents. The specific biological activities of 2-(carboxymethyl)-1-methyl-1H-pyrrole-3-carboxylic acid have not been extensively documented. However, the dual carboxylic functionalities in this compound offer versatile sites for chemical modifications, enabling the synthesis of amide or ester derivatives.

Here, 2-(Carboxymethyl)-1-methyl-1H-pyrrole-3-carboxylic acid was taken as a template for the design of new multifunctional ligands with psychotropic activity based on amide derivatives of long-chain arylpiperazines (LCAPs). However, the synthesis of amide derivatives from 2-(carboxymethyl)-1-methyl-1H-pyrrole-3-carboxylic acid presents specific challenges due to the presence of two carboxylic acid groups: one at the 3-position of the pyrrole ring and another at the 2-position linked by a methylene spacer. This dual functionality can lead to complications in selective amide bond formation, as both carboxylic acid groups are potential sites for reaction. One of the primary issues is achieving regioselectivity in amidation reactions.

The reaction conditions were selected based on literature precedent and our prior internal screening for amidation involving pyrrole carboxylic acids. We applied the protocol as previously described and successfully used by Monika Marcinkowska et al. [6]. Standard coupling reagents, such as carbodiimides DCC or EDC, may not effectively discriminate between the two carboxylic acid groups [7]. Benzylamine was chosen for coupling to facilitate our primary goal: to validate the site selectivity of the amidation reaction with 2-carboxymethyl-1-methyl-1H-pyrrole-3-carboxylic acid. Benzylamine provides a diagnostically advantageous aromatic moiety that is easily traceable in NMR, IR, and MS spectra.

Herein, we present the structural analysis of the amide coupling reaction product in order to determine which carboxylic acid group of 2-carboxymethyl-1-methyl-1H-pyrrole-3-carboxylic acid is more reactive under the applied conditions with benzylamine. The established procedure was utilised in the preparation of new multifunctional ligands with psychotropic activity based on amide derivatives of long-chain arylpiperazines (LCAPs) with 2-(carboxymethyl)-1-methyl-1H-pyrrole-3-carboxylic acid.

2. Results

The synthesis of 2-(2-(benzylamino)-2-oxoethyl)-1-methyl-1H-pyrrole-3-carboxylic acid was described (Figure 1). Briefly, 2-Carboxymethyl-1-methyl-1H-pyrrole-3-carboxylic acid (1 mmol) and o-benzotriazol-1-yl-N,N,N’,N’-tetramethyluronium tetrafluoroborate (TBTU) (2 mmol) were dissolved in dichloromethane (DCM). After that, N,N-diisopropylethylamine (DIPEA) (0.25 mmol) was added, and the reaction mixture was stirred for 15 min. The next step was to add benzylamine (1.2 mmol) and stir the reaction in a microwave for 5 min at 30 °C. Completion of the reaction was confirmed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) analysis. The final compound was purified by column chromatography and preparative RP-HPLC.

DIPEA is a commonly used non-nucleophilic base suitable for amide coupling. DCM was chosen as a standard aprotic solvent. Benzylamine was chosen because it provides a diagnostically advantageous aromatic moiety that is easily traceable in NMR, IR, and MS spectra. This feature is beneficial when attempting to identify substitution sites unambiguously, as seen in the NOESY and HMBC experiments. This facilitated our primary goal: to validate the site-selectivity of the amidation reaction involving 2-carboxymethyl-1-methyl-1H-pyrrole-3-carboxylic acid. A low reaction temperature (30 °C) was intended to maintain the replicability of reaction conditions in the microwave reactor.

The structure of the synthesised compound was verified through detailed spectral analysis, including ¹H-, ¹³C-, HMBC-, and NOESY-NMR, as well as IR spectroscopy and mass spectrometry. The collected data confirms the formation of the regioisomer, as presented in Figure 1.

The ¹H-NMR spectrum analysis confirms that the methyl group resonates at 3.60 ppm as a singlet. The aromatic protons from the benzyl group are distinctly identified by a multiplet 7.28–7.23 (m, 2H) and two overlapping doublets in the range of 7.21–7.16. Similarly, aromatic protons from the pyrrole ring were identified as two doublets at 6.61 (d, J = 3.0 Hz, 1H) and 6.46 (d, J = 3.0 Hz, 1H). In the ¹³C-NMR spectrum, distinct signals corresponding to the methyl group and the aromatic rings can be identified, respectively. The signal coming from the methyl group is observed at 30.05 ppm. Signals corresponding to the phenyl ring appear at 126.98, 128.19, and 138.52 ppm. Additionally, signals attributed to the pyrrole group are seen at 109.42, 113.18, 122.35, and 132.75 ppm.

An HMBC spectrum was recorded to unambiguously assign the two singlet signals, 4.33 and 3.99 ppm, to specific protons in the structure. This spectrum confirms that the 4.33 ppm signal originates from the CH₂ group protons adjacent to the benzyl group and the 3.99 ppm signal from the CH₂ group protons adjacent to the pyrrole ring. The HMBC spectrum alone was insufficient to determine which regioisomer was formed.

For this purpose, the NOESY-NMR and IR were recorded. The NOESY-NMR was focused on the signal 4.33 ppm from the ¹H-NMR spectrum. This signal is coupled with aromatic protons from the benzyl ring and protons from the CH₂ group adjacent to the pyrrole ring (3.99 ppm). In this spectrum, there is no observed coupling with protons from the methyl group and pyrrole. The infrared spectrum exhibits characteristic bands corresponding to the molecular structure. First, a characteristic N-H stretching band is observed at 3289 cm⁻¹. Next to it, broadband can be observed in the range of 2587–3065 cm⁻¹, corresponding to the O–H group from the carboxylic acid. Additionally, there is a signal at 1670 cm⁻¹, which confirms amide C=O stretching vibrations, and a signal at 1278 cm-1, corresponding to the C-O stretching band.

IR spectral analysis of the dicarboxylic acid substrate confirms two distinct carbonyl stretching vibrations at 1699 cm⁻¹ and 1652 cm⁻¹. The absorption band at 1652 cm⁻¹ is attributed to the carbonyl group conjugated with the aromatic pyrrole ring. In contrast, the band at 1699 cm⁻¹ corresponds to the non-conjugated carboxylic acid carbonyl group, which exhibits a typical higher-frequency C=O stretch. After the amidation reaction, the IR spectrum of the product showed two carbonyl stretching bands at 1670 cm⁻¹ and 1638 cm⁻¹. Comparing these values with the spectrum of the starting material (1699 cm⁻¹ for the non-conjugated and 1652 cm⁻¹ for the conjugated carboxylic acid), it can be inferred that the non-conjugated carboxyl group (initially at 1699 cm⁻¹) has undergone amidation, as a lower-frequency amide band has replaced its C=O absorption.

Mass spectrometry revealed a molecular ion peak at m/z 270.940, which matched the calculated mass. ¹H-, ¹³C-, HMBC-, and NOESY-NMR data confirm mono-substitution, with signals corresponding to a single amide and no evidence of a second. Collected spectra NOESY-NMR and IR allow us to formulate a conclusion and propose a structure of the final compound, as depicted in Figure 1. Together, these results demonstrate that amidation occurred selectively in the non-conjugated carboxyl group (Supplementary Materials).

3. Materials and Methods

All the starting materials were purchased from commercial suppliers and were used for synthesis without further purification. Analytical TLC was performed on Merck Kieselgel 60 F₂₅₄ (0.25 mm) precoated aluminium sheets (Merck, Darmstadt, Germany). Chromatograms were visualised using a 254 nm UV lamp. Column chromatography was performed using silica gel (particle size 0.063–0.200 mm; 70–230 Mesh ATM) purchased from Merck. Melting points (mp) were determined with a Melting Point M-560 apparatus (Büchi, Flawil, Switzerland) and are uncorrected. The UPLC-MS or UPLC-MS/MS analyses were run on a UPLC-MS/MS system comprising Waters ACQUITY UPLC (Waters Corporation, Milford, MA, USA) coupled with a Waters TQD mass spectrometer (electrospray ionisation mode, ESI with tandem quadrupole). An LC-4000 system (Jasco, Easton, MD, USA) equipped with a Phenomenex Luna C8 column (5 µm, 15 × 21.2 mm) was employed for reverse-phase purification. The mobile phase consisted of a CH₃CN/H₂O gradient containing 0.05% formic acid (HCOOH). The UPLC/MS purity of the compound was determined to be greater than 99%. ¹H NMR, ¹³C NMR, and high-resolution mass spectral measurements were obtained with an FT-NMR 500 MHz spectrometer (Joel Ltd., Akishima, Tokyo, Japan). Chemical shifts are reported in terms of δ values (ppm) relative to TMS δ = (¹H) as an internal standard. The J values are expressed in Hertz (Hz). The following abbreviations represent signal multiplicities: s (singlet), brs (broad singlet), d (doublet), dd (doublet of doublets), dt (doublet of triplets), t (triplet), td (triplet of doublets), q (quartet), m (multiplet). Infrared (IR) spectra were recorded using a Fourier-transform infrared (FT-IR) spectrometer, ThermoFisher Scientific Nicolet iS5 with ATR iD7 (Waltham, MA, USA), and are reported in wavenumbers (cm⁻¹).

Synthesis of 2-(2-(benzylamino)-2-oxoethyl)-1-methyl-1H-pyrrole-3-carboxylic acid.

A solution of 2-carboxymethyl-1-methyl-1H-pyrrole-3-carboxylic acid (100 mg, 0.54 mmol), TBTU (346.77 mg, 1.08 mmol), and DIPEA (23 µL, 0.13 mmol) in DCM (10 mL) was stirred at room temperature for 15 min. After that time, benzylamine (71 µL, 0.65 mmol) was added. The mixture was stirred for 5 min in the microwave (2.45 GHz frequency and 200 W of the microwave’s power) at 30 °C. The reaction was monitored by thin-layer chromatography (TLC) using a DCM:MeOH (9:1) solvent system, as well as high-performance liquid chromatography (HPLC). Purification was achieved using column chromatography with a DCM:MeOH (9:1) mixture and preparative high-performance liquid chromatography (HPLC). Conversion rate: 40%, yield: 14% pure product, white solid, m.p. 163.5–165.1 °C; IR (ATR): 3289, 3065, 3029, 2921, 2662, 2587, 1670, 1638, 1278, ¹H-NMR (500 MHz, METHANOL-D4) δ 7.28–7.23 (m, 2H), 7.21–7.16 (m, 3H), 6.61 (d, J = 3.0 Hz, 1H), 6.46 (d, J = 3.0 Hz, 1H), 4.33 (s, 2H), 3.99 (s, 2H), 3.60 (s, 3H).; ¹³C-NMR (126 MHz, METHANOL-D4) δ 170.78, 167.92, 138.52, 132.75, 128.19, 126.98, 122.35, 113.18, 109.42, 42.81, 33.06, 32.20, 17.05.