Methyl 2-Amino-4-[1-(tert-butoxycarbonyl)azetidin-3-yl]-1,3-selenazole-5-carboxylate

Abstract

Methyl 2-amino-4-[1-(tert-butoxycarbonyl)azetidin-3-yl]-1,3-selenazole-5-carboxylate as a newly functionalized heterocyclic amino acid was obtained via [3+2] cycloaddition. The structure of the novel 1,3-selenazole was unequivocally confirmed by detailed ¹H, ¹³C, ¹⁵N, and ⁷⁷Se NMR spectroscopic experiments, HRMS and elemental analysis.

1. Introduction

Selenium is an essential bio-trace element that plays an important role in antioxidant selenoproteins for protection against oxidative stress in humans and other animal species [1]. Functionalized organoselenium compounds possess a wide range of biological activities and are present in many pharmacologically important substances [2]. Well-known representative bioactive molecules that contain selenium in their structure are potent the antiviral agent selenazofurin [3], histamine H2-agonist known as amselamine [4], as well as ebselen and its analogues exhibiting anti-inflammatory, antioxidant, and cytoprotective properties [5,6]. Moreover, the synthesis of new Se-containing β-lactams such as selenapenam, selenacepham and selenazepine as a potential antibacterial agents has been reported [7] (Figure 1).

On the other hand, the azetidine ring has been identified as a conformationally restricted component of important pharmacological molecules [8], including synthetic analogues of natural amino acids, such as γ-aminobutyric acid (GABA) [9]. The azetidine ring is also present in the molecular structure of the well-known antihypertensive drug azelnidipine (Figure 2) [10].

The aim of the present study was to extend our previous work on heterocyclic amino acids containing selenazole and azetidine cores [11,12,13,14], as it is still a new and potentially relevant field. In this work, a molecular system containing both selenazole and azetidine scaffolds was synthesized through Hantzsch cyclization of β-ketoester.

2. Results and Discussion

Selenazoles can be obtained using analogous synthetic strategies similar to the ones used for thiazoles. Among a variety of ring construction reactions, the most widely used approach is Hantzsch synthesis and its variations [15,16,17]. This methodology reliably leads to the formation of diverse heterocyclic rings in good yields.

The synthetic strategy for obtaining novel selenazole-azetidine building blocks is outlined in Scheme 1. The target compound was synthesized in four steps using commercially available N-Boc-protected azetidine-3-carboxylic acid 1. The synthetic sequence was started with preparation of β-ketoester 2 by its adduct methanolysis with Meldrum’s acid. In the following step, bromination of ester 2 was carried out in the presence of NBS in acetonitrile. The reaction afforded an α-bromocarbonyl compound 3, which was immediately used in the final step. The condensation of 3 with selenourea afforded the desired methyl 2-amino-4-[1-(tert-butoxycarbonyl)azetidin-3-yl]-1,3-selenazole-5-carboxylate 4.

The final structure of compound 4 was easily deduced after a detailed analysis of spectral data (Figures S1–S8). The ¹H-¹⁵N HSQC experiment revealed that the most downfield protons at 7.25 ppm belong to the NH₂ group, which resonates at −294.6 ppm. The multiplicity-edited ¹H-¹³C HSQC spectrum revealed a cross-peak correlating a methine proton at 4.67 ppm with the ¹³C signal at 28.1 ppm from the azetidine ring (Figure 3).

The connectivity of the 2-amino-1,3-selenazole-5-carboxylate moiety and the N-Boc-protected azetidine fragment could be confirmed based on long-range ¹H-¹³C and ¹H-¹⁵N correlations, obtained from gs-HMBC spectra of the aforementioned protons. The ¹H-¹⁵N HMBC experiment revealed a strong three-bond correlation between the 1,3-selenazole N-3 nitrogen, which resonated at −128.1 ppm, and the protons from the 2-amino functional group. In the case of 3-H from the azetidine ring system, it showed a strong correlation with the selenazole N-3, and additionally revealed data for azetidine N-1 at −309.0 ppm. The data from ¹H–¹³C HMBC and LR-HSQMBC experiments allowed an unambiguous assignment of the 1,3-selenazole ring system, as we were able to easily distinguish the carbonyl carbons from the N-Boc and carboxylate moieties. Lastly, the protonated azetidine carbon C-3 at 21.8 ppm showed a correlation with an adjacent selenazole quaternary carbon C-4 at 162.2 ppm in the 1,1-ADEQUATE spectrum. By a process of elimination, this allowed the assignment of the last selenazole C-2 signal, which resonated at 173.6 ppm. The ⁷⁷Se NMR spectra contained a singlet at 581.4 ppm. The observed ¹⁵N and ⁷⁷Se chemical shifts are consistent with data reported in the literature for 1,3-selenazoles possessing similar structures [18].

The optical properties of compound 4 were investigated by UV/vis spectroscopy. The electronic absorption spectra of compound 4 in tetrahydrofuran (THF) contained an intense absorption band at 314 nm. Fluorescence spectra displayed an emission maximum at 375 nm (Figure 4).

3. Materials and Methods

3.1. General

All chemicals and solvents were purchased from Sigma-Aldrich (St. Louis, MO, USA) and used without further purification unless otherwise specified. Prior to use, dichloromethane (DCM) was stored over molecular sieves (4 Å). Reaction progress was monitored by thin-layer chromatography (TLC) analysis on Macherey–Nagel™ ALUGRAM^® Xtra SIL G/UV₂₅₄ plates (Macherey−Nagel™, Düren, Germany). TLC plates were visualized with ultraviolet (UV) light (wavelength 254 nm). Compounds were purified by flash chromatography in a glass column (stationary phase—silica gel, high-purity grade 9385, pore size 60 Å, particle size: 230–400 mesh, Sigma-Aldrich, St. Louis, MO, USA). The ¹H, ¹³C and ¹⁵N-NMR spectra were recorded in chloroform-D (CDCl₃) at 25 °C on a Bruker Avance III 700 (700 MHz for ¹H, 176 MHz for ¹³C, 71 MHz for ¹⁵N) spectrometer equipped with a 5 mm TCI ¹H-¹³C/¹⁵N/D z-gradient cryoprobe (Bruker BioSpin AG, Fallanden, Switzerland). ⁷⁷Se NMR spectra (76.31 MHz, absolute referencing via Ξ ratio) were obtained on a Bruker Avance III 400 (Bruker BioSpin AG, Fallanden, Switzerland) instrument with a ‘directly’ detecting broadband observe probe (BBO). The chemical shifts, expressed in ppm, were relative to tetramethylsilane (TMS). The ¹⁵N NMR spectrum was referenced to neat, external nitromethane (coaxial capillary). The full and unambiguous assignments of the ¹H, ¹³C, ¹⁵N, and ⁷⁷Se-NMR resonances were achieved using standard Bruker software (TopSpin 3.5.6, Bruker BioSpin AG, Fallanden, Switzerland) and a combination of standard NMR spectroscopic techniques, such as distortionless enhancement by polarization transfer (DEPT), homonuclear correlation spectroscopy (COSY), gradient-selected heteronuclear single quantum coherence (gs-HSQC), gradient-selected heteronuclear multiple bond correlation (gs-HMBC), heteronuclear 2-bond correlation (H2BC), long-range heteronuclear single-quantum multiple-bond correlation (LR-HSQMBC) and 1,1-ADEQUATE experiments. The infrared (IR) spectra were recorded on a Bruker TENSOR 27 spectrometer (Bruker Optik GmbH, Ettlingen, Germany) using potassium bromide (KBr) pellets. The melting point was determined in open capillary tubes with a DigiMelt MPA160 apparatus (temperature gradient: 1 °C/min) and was uncorrected. The HRMS spectrum was obtained in electrospray ionization (ESI) mode on a Bruker MicrOTOF-Q III spectrometer (Bruker Daltonik GmbH, Bremen, Germany). Elemental analysis (C, H and N) was determined using a CE-440 Elemental Analyzer (Exeter Analytical Ltd, Coventry, UK), Model 440 CHN/O/S. The UV/vis spectrum was recorded using 10⁻⁴ M solution of the compound in THF on a Shimadzu 2600 UV/vis spectrometer (Shimadzu EUROPA GmbH, Duisburg, Germany). The fluorescence spectrum was recorded on a FL920 fluorescence spectrometer from Edinburgh Instruments (Edinburg Instruments Ltd, Livingston, UK).

3.2. Synthesis

1-Boc-azetidine-3-carboxylic acid 1 (3.267 g, 16.2 mmol), Meldrum‘s acid (2.811 g, 19.5 mmol), DMAP (2.969 g, 24.3 mmol) and EDC (3.738 g, 19.5 mmol) were dissolved in DCM (70 mL). The resulting mixture was stirred at room temperature for 18 h. The reaction mixture was diluted with an additional volume of DCM (50 mL) and washed with 10% KHSO₄ aqueous solution (3 × 50 mL) and brine (1 × 100 mL). The organic layer was separated, dried over anhydrous Na₂SO₄, filtered, and the solvent was evaporated under reduced pressure. The residue was dissolved in MeOH (70 mL) and stirred at 60 °C for 18 h. The solvent was evaporated in vacuo. The residue was purified by column chromatography on silica gel (n-hexane/ethyl acetate, gradient from 6:1 to 1:1 v/v). Obtained β-ketoester 2 (3.458 g, 13.4 mmol) was dissolved in ACN (100 mL) and NBS (3.588 g, 20.2 mmol) was added. The reaction mixture was stirred at room temperature for 2 h. After the reaction, the solvent was removed under reduced pressure. The residue was dissolved in EtOAc (50 mL) and filtered through a pad of silica. The filter pad was washed out with EtOAc (150 mL). The filtrate was concentrated in vacuo. The residue was dissolved in MeOH (40 mL) and selenourea (0.915 g, 7.4 mmol) was added. The reaction mixture was stirred at room temperature. After 2 h, the resulting mixture was added dropwise to 2% Na₂CO₃ aqueous solution (100 mL) while stirring. The precipitate was filtered and dissolved in DCM (100 mL). The solution was dried over anhydrous Na₂SO₄, filtered, and the solvent was removed in vacuo. The residue was purified by column chromatography on silica gel (n-hexane/ethyl acetate, gradient from 4:1 to 1:1 v/v) to afford compound 4. The obtained solid was recrystallized from hexane. Colorless crystals (683 mg) were obtained with an overall of yield 21%. R_f = 0.19 (n-hexane/ethyl acetate 2/1, v/v), m.p. 167−168 °C. IR (KBr) ν_max, cm⁻¹: 3388 (NH), 3293 (NH), 3175, 2975, 2955, 2890, 1680 (C=O), 1628 (C=O), 1505, 1415, 1284, 1139, 1067, 909, 772 and 757, 527. ¹H NMR (700 MHz, CDCl₃) δ, ppm: 1.47 (9H, s, 3 × CH₃), 3.75 (3H, s, OCH₃), 4.01 (2H, br s, 2 × CH_aH_b), 4.20−4.27 (2H, m, 2 × CH_aH_b), 4.67 (1H, tt, J = 8.7, 5.5 Hz, CH), 7.25 (2H, s, NH₂). ¹³C NMR (176 MHz, CDCl₃) δ, ppm: 28.1 (CH), 28.5 (3 × CH₃), 51.8 (OCH₃), 53.6 (CH₂), 55.1 (CH₂), 79.9 (C(CH₃)₃), 113.8 (Sel C-5), 156.6 (Boc C=O), 162.2 (Sel C-4), 164.0 COOCH₃), 173.6 (Sel C-2). ¹⁵N NMR (71 MHz, CDCl₃) δ, ppm: −309.0 (Az N-1), −294.6 (NH₂), −128.1 (Sel N-3). ⁷⁷Se NMR (76 MHz, CDCl₃) δ, ppm: 581.4. HRMS (ESI) for C₁₃H₁₉N₃NaO₄Se ([M + Na]⁺): found m/z 384.0434, calculated m/z 384.0434. Elem. An. for C₁₃H₁₉N₃O₄Se (%): found C, 43.87; H, 5.33; N, 11.22; calculated C, 43.34; H, 5.32; N, 11.66.

4. Conclusions

In this short note, we reported the synthesis and structure elucidation of methyl 2-amino-4-[1-(tert-butoxycarbonyl)azetidin-3-yl]-1,3-selenazole-5-carboxylate 4. This compound is a valuable building block for more complex molecular systems as well as for the development of DNA-encoded chemical libraries.