(E)-N-(4-Methoxyphenyl)-1-(4′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)methanimine

Abstract

The title compound (1) was obtained within a project aimed at synthesizing inhibitors of the human enzyme lactate dehydrogenase A (hLDHA). Compound 1 was synthesized by Suzuki cross-coupling of (E)-1-(4-bromophenyl)-N-(4-methoxyphenyl)methanimine (2a) or (E)-1-(4-iodophenyl)-N-(4-methoxyphenyl)methanimine (2b) with 4-(trifluoromethyl)phenylboronic acid (3) using Pd(PPh₃)₄ as a catalyst in 86% and 87% yields, respectively. Halo-imines 2a and 2b were previously obtained by condensation reaction between p-anisidine (4) and 4-bromo-benzaldehyde (5a) or 4-iodo-benzaldehyde (5b), respectively. The structure of compound 1 was established by 1D and 2D NMR spectroscopy, infrared and ultraviolet spectroscopies, and high-resolution mass spectrometry.

1. Introduction

The human lactate dehydrogenase (hLDH) enzyme is an oxidoreductase found in nearly all tissues. It catalyzes the reversible conversion of L-lactate/pyruvate, glyoxylate/oxalate and L-glycerate/hydroxypyruvate, with nicotinamide adenine dinucleotide (NAD⁺/NADH) as a cofactor (Scheme 1) [1,2].

hLDH is a tetrameric enzyme with known isoforms, formed by the association of two types of subunits, H (heart) and M (muscle). Specifically, hLDHA is a homotetramer composed of four M-type subunits that catalyze the conversion of pyruvate to lactate under anaerobic conditions, such as those found in skeletal muscles or hypoxic tumors [3,4,5,6]. Notably, it is the isoform most extensively studied in this context [7,8,9].

Lactate dehydrogenase activity is associated with various cardiovascular diseases, cancer and neurodegenerative disorders [1]. Dysregulation of LDH significantly contributes to cancer development by promoting the Warburg effect [10,11]. Targeting the LDH enzyme presents a promising therapeutic approach for those pathologies [1,7,8,9].

The synthesis of the title compound 1 is part of a project focused on preparing compounds with a quinoline skeleton (Scheme 2). The goal of that study will be the synthesis of a series of N-aryl aromatic imines, such as 1, via a previous condensation reaction of aromatic amines and benzaldehyde derivatives, followed by constructing the quinoline ring system through the Povarov reaction between those N-aryl aromatic imines and electron-rich dienophiles (Scheme 2), following standard methodologies [12].

The significant pharmacological properties of quinoline moieties have led to relevant applications in the field of bioorganic chemistry, and it is considered an important pharmacophore in medicinal chemistry [13]. In fact, many compounds containing the quinoline moiety show antibacterial, antifungal, antiviral, antineoplastic, anti-inflammatory and neuroprotective activities [14]. In addition, several hLDHA inhibitors with a quinoline core have been described and their potential applications reported [3,15,16,17], although none have been approved for clinical use yet (Figure 1).

2. Results and Discussion

Compound 1 was synthesized via a Suzuki–Miyaura coupling reaction between a halo-imine (2a or 2b) and 4-(trifluoromethyl)phenylboronic acid (3), catalyzed by tetrakis(triphenylphosphine)palladium(0) under basic conditions (Scheme 3). Optimal experimental conditions, as reported in the literature, involved stirring at 120 °C in the absence of oxygen [18]. Additionally, due to the sensitivity of the palladium catalyst to light, the reaction was conducted in the dark.

Previously, both (E)-1-(4-bromophenyl)-N-(4-methoxyphenyl)methanimine (2a) and (E)-1-(4-iodophenyl)-N-(4-methoxyphenyl)methanimine (2b) were synthesized through a condensation reaction between p-anisidine (4) and the corresponding halogenated benzaldehyde with bromine (5a) or iodine (5b), respectively, following common procedures (Scheme 3) [19].

The synthesis of compound 1 was performed using both halo-imines 2a and 2b to evaluate their reaction behavior in each case. When the brominated imine 2a was used, the reaction took 25 min and an 86% yield was achieved. On the other hand, using the iodinated imine 2b, the reaction was complete in just 10 min (87%). Although the yields were similar, the reaction was twice as fast as from the iodo-imine 2b. This result was expected, as the iodide ion is a better leaving group than the bromide ion due to iodine’s larger size and lower electronegativity, among other factors.

The ¹H NMR spectrum of compound 1 (see Figure S1 in Supplementary Materials), shows notable signals including singlets at 3.85 ppm and 8.54 ppm, corresponding to the methoxy group and the iminic methine, respectively. The remaining signals, associated with the aromatic rings, appear as multiplets. In the ¹³C NMR spectrum (see Figure S2 in Supplementary Materials), significant signals are observed for carbons located in the ortho (C-3‴ and C-5‴) and ipso (C-4‴) positions relative to the trifluoromethyl group, as well as the trifluorinated carbon (CF₃). These signals exhibit a quartet-splitting due to carbon–fluorine couplings (Figure 2). Specifically, the coupling constants are 272.5 Hz for the trifluorinated carbon, 32.9 Hz for the ipso carbon, and 3.7 Hz for the ortho carbon.

The mass spectrum of compound 1 (see Figure S6 in Supplementary Materials) shows the molecular ion peak at m/z 355.11804 with a relative abundance of 79%, consistent with a molecular formula of C₂₁H₁₆F₃NO. The base peak is observed at m/z 340.09454 (100%) and is attributed to the loss of a CH₃ fragment. A peak at m/z 285.08896 (12%) corresponds to the loss of a CF₃ fragment. Additionally, the loss of a C₇H₈NO fragment results in the appearance of a peak at m/z 233.05765 (5%). The IR spectrum of compound 1 (see Figure S7 in Supplementary Materials) allowed us to recognize the presence of the imino group (1616 cm⁻¹) and the aromatic rings (3034, 1574, 1504, 1464, 837, 818 cm⁻¹). The UV spectrum of compound 1 (see Figure S8 in Supplementary Materials) showed two absorbance bands at 289 and 350 nm.

3. Materials and Methods

3.1. General Experimental Methods

All solvents and reagents used in the synthesis were purchased and used as received, except N,N-dimethylformamide (DMF), which was previously dried, using standard methodologies [20]. We purchased 4-(trifluoromethyl)phenylboronic acid (3) from Boron Molecular (Boron Molecular Inc., Melbourne, Australia); tetrakis(triphenylphosphine)palladium(0) (Pd(PPh₃)₄) was purchased from Sigma-Aldrich (Merck KGaA, Darmstadt, Germany) and NaOH was purchased from Panreac (Panreac Química, Barcelona, España). All solvents (n-hexane (Hex), ethyl acetate (EtOAc), dichloromethane (DCM), ethanol (EtOH), DMF) were purchased from VWR (Avantor, Radnor, PA, USA).

Reactions were monitored by analytical thin-layer chromatography (TLC) on silica gel 60 F₂₅₄ precoated aluminum sheets (0.25 mm, Merck Chemicals, Darmsdadt, Germany) and spots were visualized using UV light (λ = 254 nm). The purification of the synthesized compound 1 was carried out by column chromatography (CC) using Silica gel 60 (particle size 0.040–0.063 mm) (Merck Chemicals, Darmsdadt, Germany). The melting point of compound 1 was recorded on an Electrothermal 9100 (Antylia Scientific, Vernon Hills, IL, USA) equipment.

¹H NMR and ¹³C NMR spectra of compound 1 (and compounds 2a and 2b) were measured on a Bruker Avance 400 spectrometer (Bruker Daltonik GmbH, Bremen, Germany) operating at 400 and 100 MHz for ¹H and ¹³C, respectively. Deuterated chloroform (CDCl₃) was used as a solvent. Chemical shifts (in ppm) were referenced to solvent peaks as internal reference. The coupling constants (J) are expressed in hertz (Hz). The coupling systems are described by using the following abbreviations: s, singlet; q, quartet; m, multiplet. The total assignment of ¹H and ¹³C signals was made using 2D NMR spectra such as COSY, HSQC, and HMBC.

The high-resolution mass spectrum of 1 (HRMS) was conducted on a Thermo Orbitrap Exploris GC 240 mass spectrometer (Thermo Fisher Scientific, Waltham, MA, USA), via a Direct Insertion Probe (DIP) and using an ionizing voltage of 70 eV (EI).

The IR spectrum of compound 1 was recorded on a FT-IR spectrometer Bruker Vertex 70 (Bruker Daltonik GmbH, Bremen, Germany) equipped with the attenuated total reflection (ATR) diamond accessory Bruker Platinum (Bruker Daltonik GmbH, Bremen, Germany).

The UV spectrum of compound 1 was recorded on a Varian Cary 4000 UV-Vis double beam spectrophotometer (Varian Inc., Palo Alto, CA, USA). A solution of 1 in EtOH at 1.13 μM was used for the measurement.

3.2. Synthesis of Compound 1

In a round-bottom flask, the imine 2a or 2b (1.03 mmol) and DMF (3 mL) were introduced, and the mixture was allowed to stir for a few minutes at room temperature under argon. Subsequently, a solution of 3 (407 mg, 2.14 mmol) in DMF (3 mL) was added dropwise, followed by the addition of a solution of Pd(PPh₃)₄ (36 mg, 0.03 mmol) in DMF (3 mL) in the dark. Finally, NaOH (169 mg, 4.67 mmol) dissolved in water (3 mL) was introduced and the reaction vessel was heated to 120 °C under stirring, in the absence of light. All solutions were previously purged with argon. The reaction progress was monitored by TLC (Hex:EtOAc 8:2). Once the reaction was complete (25 or 10 min starting from imine 2a or 2b, respectively), the resulting solution was cooled to room temperature, poured into water and extracted with DCM (3 × 15 mL). The combined organic layers were washed with water (2 × 15 mL) and brine (2 × 15 mL), and dried for over anh. Na₂SO₄ evaporated to dryness under reduced pressure. The resulting product was purified by column chromatography (CC) using mixtures of Hex:EtOAc. With an 85:15 solvent mixture, pure compound 1 was eluted and isolated as a yellowish solid, yielding 314 mg starting from bromo-imine 2a (86%) and 319 mg starting from iodo-imine 2b (87%).

• •(E)-N-(4-methoxyphenyl)-1-(4′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)methanimine (1). Melting point: 190.4–191.2 °C; ¹H NMR (400 MHz, CDCl₃) δ (ppm) 8.54 (s, 1H, H-1), 7.98–8.01 (m, 2H, H-2″, H-6″), 7.69–7.76 (m, 6H, H-3″, H-5″, H-2‴, H-3‴, H-5‴, H-6‴), 7.26–7.30 (m, 2H, H-2′, H-6′), 6.94–6.98 (m, 2H, H-3′, H-5′), 3.85 (s, 3H, OCH₃); ¹³C NMR (100 MHz, CDCl₃) δ (ppm) 158.6 (C-4′), 157.6 (C-1), 144.8 (C-1′), 144.0 (C-4″), 142.2 (C-1‴), 136.4 (C-1″), 130.0 (q, J = 32.9 Hz, C-4‴), 129.3 (C-2″, C-6″), 127.7 (C-3″, C-5″)*, 127.6 (C-2‴, C-6‴)*, 126.0 (q, J = 3.7 Hz, C-3‴, C-5‴), 124.4 (q, J = 272.5 Hz, CF₃), 122.4 (C-2′, C-6′), 114.6 (C-3′, C-5′), 55.7 (OCH₃); * interchangeable signals. HRMS (EI) (M⁺) calc. for C₂₁H₁₆F₃NO 355.11840, found 355.11804; m/z (%): 355.11804 (M⁺, 79), 340.09454 (M⁺–CH₃, 100), 311.09204 (8), 285.08896 (M⁺–CF₃, 12), 233.05765 (M⁺–C₇H₈NO, 5), 215.08588 (6), 167.55579 (4), 77.03868 (2). IR (cm⁻¹): 3034 (aromatic C–H), 2922, 2853 (C–H), 1616 (C=N), 1574, 1504, 1464 (aromatic C=C), 1124 (C–F)*, 1111 (C–O)*, 837, 818 (aromatic C–H, p-substituted); * interchangeable signals. UV (EtOH), λ_max (log ε): 289 (5.37), 350 (5.30) nm.

4. Conclusions

The novel compound (E)-N-(4-methoxyphenyl)-1-(4′-(trifluoromethyl)-[1,1′-biphenyl]-4-yl)methanimine (1) was successfully synthesized and characterized by IR, UV, and NMR spectroscopies and HRMS spectrometry. The synthesis was performed using two halo-imines, with bromine (2a) and iodine (2b), achieving high yields in both cases. Additionally, the iodide proved to be better leaving group than bromide, as the synthesis of 1 from 2b resulted in a shorter reaction time. The imino group of halo-imines 2a and 2b was stable in the experimental conditions. Compound 1 was isolated in pure form as a yellowish solid and would be used to synthesize potential hLDHA inhibitors with a quinoline moiety via the Povarov reaction.