2-Benzyl-6-carboxy-5,6,7,8-tetrahydroimidazo[1,2-a]pyrimidin-1-ium 2,2,2-trifluoroacetate

Abstract

Cyclic guanidines are valuable scaffolds for the design of compounds acting on GABAergic neurotransmission, owing to their ability to mimic the amino functionality of GABA as bioisosteres. With the aim to obtain a more potent and selective betaine/GABA transporter (BGT1) inhibitor, a basic hydrolysis of ethyl (E)-2-(acetylimino)-1-(3-phenylprop-2-yn-1-yl)hexahydropyrimidine-5-carboxylate was attempted. However, we isolated a byproduct, which was identified as the trifluoroacetate salt of 2-benzyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyrimidine-6-carboxylic acid. The structure was confirmed by NMR spectroscopy and LC-MS. Herein we report the preparation, characterization, and spectral data of this fused heterocyclic compound.

1. Introduction

Cyclic guanidines represent a versatile class of structural motifs frequently encountered in biologically active molecules [1]. They are found across diverse pharmacological areas, ranging from antimicrobials [2] to compounds for central nervous system targets [3]. Their resemblance to the side chain of arginine, together with their positive charge at physiological pH and conformational rigidity, allows them to engage in directional hydrogen bonding and electrostatic interactions with biological targets [4,5]. In nature, several cyclic guanidines carry a carboxylic acid functionality, reflecting their biosynthetic origin from arginine; examples include the non-proteinogenic amino acids enduracididine [6] and capreomicidine [7], which are also building blocks of bioactive cyclic peptides [3]. As guanidines can also serve as bioisosteres of amino groups, we previously reported guanidine-containing and cyclic amino acids as structural analogs of γ-aminobutyric acid (GABA), the primary inhibitory neurotransmitter in the brain [8]. Among these, 2-amino-1,4,5,6-tetrahydropyrimidine-5-carboxylic acid (ATPCA, Figure 1) emerged as a selective substrate of the betaine/GABA transporter (BGT1), an important regulator of GABA uptake.

Aiming at developing more potent and selective BGT1 inhibitors based on ATPCA, we explored the impact of endocyclic N-substitution [8]. During the synthesis of one of the designed analogs, 1-(3-phenylprop-2-yn-1-yl)-ATPCA, we unexpectedly isolated a byproduct instead. Herein, we report the synthesis and structural elucidation of this compound, identified as the TFA salt of 2-benzyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyrimidine-6-carboxylic acid (4). Notably, this scaffold features a functionalized 5,6,7,8-tetrahydroimidazo[1,2-a]pyrimidine nucleus, a reduced form of the imidazo[1,2-a]pyrimidine pharmacophore, bearing a carboxylic acid handle suitable for further functionalization in medicinal chemistry.

2. Results and Discussion

The starting compound 3 was synthesized by alkylation [8] of the easily accessible ethyl 2-(acetylimino)hexahydropyrimidine-5-carboxylate (2), in turn prepared in good yields from the commercially available 2-aminopyrimidine-5-carboxylic acid (1) by esterification [9], acetylation [8], and catalytic hydrogenation of the heterocycle [8] (Scheme 1).

Next, we attempted to deprotect 3 using strong basic hydrolytic conditions. The reaction was monitored by LC-MS, which showed conversion of 3 to a new major peak with an m/z of 258.1, coherent with the m/z ratio of the desired hydrolyzed product (m/z calculated for C₁₄H₁₅N₃O₂ [M + H]⁺: 258.12). We therefore purified the reaction mixture by preparative HPLC and isolated the newly formed product as a pure TFA salt (95% by analytical HPLC). However, ¹H- and ¹³C-NMR analysis of the purified compound clearly revealed that the isolated compound, even if with the same m/z ratio, was not the desired product: one new singlet (integration 1, H₁ in Figure 2A) was detected in the aromatic area (6.77 ppm), and the carbon signals typical of the alkyne moiety were absent (Figure 2B).

With this information, and knowing that the alkynyl-arenes can react with nucleophiles [10], we hypothesized that an intramolecular cyclization between the hexocyclic nitrogen and the alkyne had occurred during basic hydrolysis, forming the bicyclic byproduct 4 instead of the desired product. 2D-NMR analysis confirmed our hypothesis and allowed us to identify the compound as the trifluoroacetate salt of the 2-benzyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyrimidine-6-carboxylic acid (4). In detail, we identified by HSQC (Figure 3) the correlation between the new aromatic singlet H₁ and its corresponding carbon C₁, and we revealed by HMBC that the benzylic protons H₂ (3.80 ppm) were correlating with the aromatic carbons C₁, C₂, C₃, and C₄.

3. Materials and Methods

The starting ethyl 2-(acetylimino)hexahydropyrimidine-5-carboxylate (2) was prepared in good yields from the commercially available 2-aminopyrimidine-5-carboxylic acid 1 according to known procedures [8,9]. 3-phenylprop-2-yn-1-yl methanesulfonate was prepared from the commercial 3-phenylprop-2-yn-1-ol as previously reported [11]. Solvents and chemicals were purchased from commercial suppliers and used without additional purification. Anhydrous DMF was obtained from a Glass Contour Solvent System (SG Water, Nashua, NH, USA). Thin layer chromatography (TLC) was carried out on Merck silica gel 60 F254 aluminum plates (Darmstadt, Germany), and visualization was performed by UV light (254 nm). Flash column chromatography (FC) was performed using a CombiFlash Rf 300+ (Teledyne Isco, Lincoln, NE, USA) and RediSep Rf Silica columns (Teledyne Isco), with the eluents indicated in the procedures. ¹H-, ¹³C-NMR, HSQC, and HMBC spectra were recorded on a Bruker Avance III HD 600 MHz spectrometer (Billerica, MA, USA) equipped with a cryogenically cooled 5 mm dual probe optimized for ¹³C and ¹H at 300 K. Chemical shifts (δ) are reported in parts per million (ppm) downfield from TMS (δ = 0) using solvent resonance as the internal standard (dimethylsulfoxide-d₆, ¹H: 2.50 ppm, ¹³C: 39.52 ppm). Coupling constants (J) are given in Hertz (Hz). Multiplicities are reported. LC-MS mass spectra were obtained with an Agilent 6130 Mass Spectrometer instrument (Santa Clara, CA, USA) using electron spray ionization (ESI) coupled to an Agilent 1200 HPLC system (ESI-LC/MS) with a C18 reverse phase column (Zorbax Eclipse XBD-C18, 4.6 mm × 50 mm), autosampler, and diode array detector, using a linear gradient of the binary solvent system of buffer A (milliQ 58 H₂O:ACN:TFA, 95:5:0.1 v/v%) to buffer B (ACN:TFA, 100:0.1 v/v%) with a flow rate of 1 mL⁄min. High-resolution mass spectra (HRMS) were recorded on a QExactive Orbitrap mass spectrometer (Thermo Scientific, Bremen, Germany) equipped with an SMALDI5 ion source. The sample was analyzed in the positive ion mode using a peak from the DHB matrix for internal mass calibration. Analytical HPLC was carried out on an UltiMate HPLC system (Thermo Scientific, Waltham, MA, USA) consisting of an LPG-3400A pump (1 mL/min), a WPS-3000SL autosampler, and a DAD-3000D diode array detector using a Gemini-NX C18 column (4.6 × 250 mm, 3 μm, 110 Å); gradient elution 0 to 100% B (ACN:H₂O:TFA 90:10:0.1) in solvent A (H₂O:TFA 100:0.1) over 20 min. Analytical purity of compound 4 was ≥95%. Preparative HPLC was carried out on an UltiMate HPLC system (Thermo Scientific) consisting of an LPG-3400BX pump (20 mL/min), a Rheodyne 9725i injector, a 10 mL loop, and an MWD-3000SD detector (200; 247; 254; and 280 nm) using a Gemini-NX C18 column (21.2 × 250 mm, 5 μm, 110 Å). The gradient elution of the solvents A (H₂O:TFA 100:0.1) and B (ACN:H₂O:TFA 90:10:0.1) is described.

• Ethyl (E)-2-(acetylimino)-1-(3-phenylprop-2-yn-1-yl)hexahydropyrimidine-5-carboxylate (3). To a flame-dried two-neck flask, compound 2 (300 mg, 1.41 mmol, 1 eq) was added in dry DMF. Cs₂CO₃ (3 eq) was then added, and the suspension was stirred for 30 min under a nitrogen atmosphere. The alkylating agent 3-phenyl-2-propyn-1-yl methanesulfonate (350 mg, 1.7 mmol, 1.2 eq) was added, and the reaction mixture was stirred at 70 °C. After 48, the mixture was diluted with water in a 10-fold volume of the DMF, followed by 3 extractions with EtOAc. The combined organic layers were then washed with brine, dried over Na₂SO₄, filtered, and evaporated to dryness under vacuum. FC (Heptane/EtOAc) gave the pure compound 3 in 43% yield as a white solid. ¹H NMR (600 MHz, DMSO-d₆) δ 10.30 (t, J = 2.9 Hz, 1H), 7.49–7.42 (m, 2H), 7.42–7.34 (m, 3H), 4.73 (d, J = 17.5 Hz, 1H), 4.63 (d, J = 17.5 Hz, 1H), 4.10 (m, 2H), 3.67–3.60 (m, 2H), 3.56–3.51 (m, 1H), 3.49–3.44 (m, 1H), 3.19–3.12 (m, 1H), 1.90 (s, 3H), 1.16 (t, J = 7.1 Hz, 3H). ¹³C NMR (151 MHz, DMSO) δ 181.52, 170.42, 156.35, 131.48, 128.67, 128.65, 122.08, 85.16, 83.19, 60.74, 44.74, 39.20, 37.36, 35.74, 28.18, 13.94. LC-MS (ESI): m/z calculated for C₁₈H₂₁N₃O₃ [M + H]⁺: 328.2, found: 328.2, retention time: 3.240 min.
• 2-benzyl-6-carboxy-5,6,7,8-tetrahydroimidazo[1,2-a]pyrimidin-1-ium 2,2,2-trifluoroacetate (4). Compound 3 (65 mg, 0.2 mmol) was stirred in 7M KOH_aq at 60 °C for 3 h. Purification by preparative HPLC (20 min, 0% B to 80% B) provided the pure compound 4 (TFA salt) in 35% yield as an off-white solid. ¹H NMR (600 MHz, DMSO-d₆) δ 13.03 (broad s, 1H), 12.45 (broad s, 1H), 8.31 (t, J = 2.9 Hz, 1H), 7.36–7.31 (m, 2H), 7.27–7.22 (m, 3H), 6.77 (s, 1H), 4.05 (dd, J = 12.7, 5.7 Hz, 1H), 4.02 (dd, J = 12.7, 5.1 Hz, 1H), 3.80 (m, 2H), 3.56–3.49 (m, 2H), 3.25–3.19 (m, 1H). ¹³C NMR (151 MHz, DMSO-d₆) δ 171.63, 157.90 (q, J_CF = 30.3 Hz), 143.68, 137.49, 128.54, 128.48, 126.72, 125.67, 117.31 (q, J_CF = 300.9 Hz), 112.62, 43.24, 39.73, 35.37, 30.06. LC-MS (ESI): m/z calculated for C₁₄H₁₅N₃O₂ [M + H]⁺: 258.1, found: 258.1. Retention time: 2.744 min. HRMS (ESI): m/z calculated for C₁₄H₁₅N₃O₂ [M + H]⁺: 258.1238, found: 258.1242. Purity by analytical HPLC (210 nm): 95%.

4. Conclusions

In summary, the attempted basic hydrolysis of ethyl (E)-2-(acetylimino)-1-(3-phenylprop-2-yn-1-yl)hexahydropyrimidine-5-carboxylate (3) resulted in the unexpected intramolecular cyclization to afford 2-benzyl-5,6,7,8-tetrahydroimidazo[1,2-a]pyrimidine-6-carboxylic acid 4. Structural elucidation was carried out by ¹H-, ¹³C-, HSQC-, and HMBC-NMR spectroscopy, supported by MS data. The formation of 4 results from an intramolecular cyclization under strong basic conditions. To avoid it and favor the formation of 1-(3-phenylprop-2-yn-1-yl)-ATPCA instead, milder reaction conditions may be screened, including weaker or more diluted bases, lower temperature, shorter reaction times, or hydrolysis in acidic conditions. Unlike the symmetric ATPCA, compound 4 contains an asymmetric carbon. Future studies might investigate the use of asymmetric protecting groups as chiral auxiliaries [12] or asymmetric bases [13] to induce facial selectivity during cyclization and thereby access enantioenriched derivatives. This result highlights a facile entry point for functionalized tetrahydroimidazo[1,2-a]pyrimidines under simple reaction conditions and expands the accessible chemical space of this scaffold.