2,2^′-[2,4-Bis(4-chlorophenyl)cyclobutane-1,3-diyl]bis(8-bromo-6-chloro-3-nitroimidazo[1,2-a]pyridine)

Abstract

In the context of our ongoing studies on 3-nitroimidazo[1,2-a]pyridine derivatives as potent antileishmanial compounds, we isolated a new unexpected compound from the spontaneous cycloaddition of N-[2-(8-bromo-6-chloro-3-nitroimidazo[1,2-a]pyridin-2-yl)-1-(4-chlorophenyl)ethyl]-4-methylbenzenesulfonamide. The molecular structure was fully characterized by using ¹H and ¹³C NMR, X-ray crystallography, and HRMS.

1. Introduction

The World Health Organization (WHO) defines Neglected Tropical Diseases (NTDs) as a heterogeneous group of infections that are predominantly found in developing countries, particularly in tropical and subtropical environments [1]. These diseases have historically attracted little interest from the pharmaceutical industry. Among these NTDs, leishmaniasis is a parasitic disease transmitted by female phlebotomine sandfly as a vector caused by several species of flagellate protozoa of the genus Leishmania. It is estimated that 1 million new cases of leishmaniasis occur in over 100 countries worldwide each year. The most lethal form of the disease, visceral leishmaniasis (VL), is responsible for over 30,000 deaths annually [2].

The absence of a vaccine for humans against leishmaniasis restricts the range of drugs that can be employed for treatment. Pentavalent antimonials, such as meglumine antimoniate (Glucantime^®) and sodium stibogluconate (Pentostam^®), are still employed as inexpensive treatments in East Africa. However, the emergence of resistance, particularly in India, and the significant cardiotoxic, nephrotoxic, and hepatotoxic effects of these drugs considerably limit their use. Although amphotericin B (Fungizone^®) is effective, it can cause serious side effects, such as nephrotoxicity. The liposomal form of amphotericin B (Ambisome^®) has reduced renal toxicity, but is expensive and requires parenteral administration and cold chain compliance. There is only one oral medication available to treat VL, miltefosine (IMPAVIDO^®), which is well tolerated overall, but has teratogenic effects that limit its use [3,4].

In the course of our research into antileishmanial derivatives, our team identified a first hit compound (A) in the 3-nitroimidazo[1,2-a]pyridine series, substituted by a chlorine atom at position 6, a phenylsulfonyl group at position 2, and a 4-chlorophenylthioether group at position 8 (Figure 1) [5]. Subsequently, in vitro pharmacokinetic and physicochemical properties were improved, while maintaining the antileishmanial activity, by modulating the phenyl ring at position 2 with a gem-trifluoropropyl chain at position 2, and a pyridin-4-yl group at position 8, resulting in the identification of a second hit compound (B), the 6-chloro-3-nitro-8-(pyridin-4-yl)-2-[(3,3,3-trifluoropropylsulfonyl)methyl]imidazo[1,2-a]pyridine (Figure 1) [6].

To further explore the antileishmanial structure-activity relationship, we investigate the modulation at position 2 of the 3-nitroimidazo[1,2-a]pyridine ring without the introduction of a sulfone group. To this end, the tetrakis(dimethylamino)ethylene (TDAE) methodology previously described in our laboratory for the 3-nitroimidazo[1,2-a]pyridine scaffold was employed [7]. This allowed us to generate nucleophilic carbanion at position 2, which was subsequently employed in reactions with a range of electrophiles, including N-tosylbenzylimines [8]. A new series of compounds modulated at position 2 were synthesized. The initial in vitro results were promising, demonstrating encouraging antileishmanial activity.

With the aim to obtain analogs structurally similar to known hits, we set out to modulate the substituent at position 8 of the 3-nitroimidazo[1,2-a]pyridine ring using a Suzuki–Miyaura cross-coupling reaction. However, this reaction did not yield the expected product and an unexpected by-product was observed. Further tests allowed us to optimize the reaction and the structure of this by-product was determined.

2. Results

The N-[2-(8-bromo-6-chloro-3-nitroimidazo[1,2-a]pyridin-2-yl)-1-(4-chlorophenyl)ethyl]-4-methylbenzenesulfonamide intermediate (3) was obtained via a three-step synthesis developed in our laboratory (Scheme 1) [9]. Intermediate (1) was synthesized by the cyclocondensation of 1,3-dichloroacetone with commercially available 3-bromo-5-chloropyridin-2-amine in refluxing ethanol, followed by a selective nitration reaction at position 3, giving the intermediate (2). Subsequently, a TDAE methodology previously described by our team [10] and adapted (solvent and time), was employed to generate a stable 2-methyl carbanion, which was then reacted with (E)-N-(2-chlorobenzylidene)-4-methylbenzenesulfonamide [8,11] to give intermediate (3).

An attempt was made to modulate the position 8 of intermediate (3) by a Suzuki–Miyaura cross-coupling reaction, using the conditions previously developed in our laboratory [6]. However, the reaction did not yield the anticipated product, but instead produced an unexpected by-product (4) (Scheme 2).

To ascertain the source of this by-product (4), additional experiments were conducted using solely K₂CO₃, the base employed in the initial Suzuki–Miyaura cross-coupling reaction. These tests confirmed that using only K₂CO₃ with reflux heating resulted in the formation of compound (4) in moderate yield (23%) (Scheme 3).

Compound (4) was isolated and rapidly characterized by spectroscopic analysis (¹H and ¹³C NMR, HRMS, and X-ray crystallography) allowing its identification as 2,2^′-[2,4-bis(4-chlorophenyl)cyclobutane-1,3-diyl]bis(8-bromo-6-chloro-3-nitroimidazo[1,2-a]pyridine) (4) (Figure 2).

3. Discussion

The elucidation of the structure of compound (4) led to the formulation of a hypothesis regarding the mechanism of its formation. It is postulated that the use of a base such as K₂CO₃ led to an elimination, forming an alkene intermediate. This intermediate would have undergone a spontaneous cycloaddition, forming a central cyclobutane ring. A potential mechanism involves a spontaneous [2+2] cycloaddition (Scheme 4).

Nevertheless, this theory raises the question of the energy required for this reaction. One potential source is light, which could facilitate a [2+2] photocycloaddition, inducing a photodimerization. This process is well documented with analogous systems [13,14,15], such as stilbene [16,17,18]. However, while photocycloaddition has already been described for an imidazo[1,2-a]pyridine ring [19], it occurred under conditions of strong light irradiation (Scheme 5). In our case, the reaction was only subjected to ambient room light, which raises questions about the spontaneity of this cycloaddition.

The scope and mechanism of this reaction will be investigated and documented in due time.

4. Materials and Methods

4.1. General Information

Reagents were purchased from Sigma-Aldrich (Saint-Louis, MO, USA), Fischer Scientific (Pittsburgh, PA, USA) or Fluorochem (Hadfield, UK) and used without further purification. Reaction monitoring was performed using aluminium TLC plates (5 cm × 10 cm) coated with silica gel Xtra SIL G UV254 ALUGRAM® (Macherey-Nagel, Düren, Nordrhein-Westfalen, Germany) in an appropriate eluent. Visualization was performed with ultraviolet light (254 nm). Melting points were determined on a Stuart SMP3 melting point apparatus (Barloworld, Sandton, South Africa) and were uncorrected. HRMS spectra (ESI) were recorded on a SYNAPT G2 HDMS (Waters, Milford, MA, USA) at the Faculté des Sciences de Saint-Jérôme (Marseille, France). Single-Crystal X-Ray Diffraction was recorded on a SuperNova Dual Source Diffractometer (Agilent Technologies, Santa Carla, CA, USA) with Rigaku Oxford Diffraction (Rigaku, Tokyo, Japan) at the Faculté des Sciences de Saint-Jérôme (Marseille, France). NMR spectra were recorded on a Bruker Avance NEO 400 MHz NanoBay spectrometer (Bruker, Billerica, MA, USA) at the Faculté de Pharmacie of Marseille (France). (¹H NMR: reference CDCl₃ $\delta$ = 7.26 ppm and ¹³C NMR: reference CDCl₃ $\delta$ = 77.16 ppm). The following adsorbent was used for column chromatography: silica gel 60 (Merck KGaA, Darmstadt, Germany, particle size 0.063–0.200 mm, 70–230 mesh ASTM). Flash-chromatography was performed with a puriFlash^® 5.020 (Interchim, Montluçon, France), using a silica column (IR-50SI) with a size adapted to the crude sample load. The data were processed with InterSoft X. The purity determination of synthesized compounds was checked by LC/MS analyses, which were realized at the Faculté de Pharmacie of Marseille with a Thermo Scientific Vanquish^® (Dionex Softron GmbH, Part of Thermo Fisher Scientific, Germering, Germany) coupled using a single quadrupole mass spectrometer Thermo MSQ Plus^®. The LC/MS data were processed with Chromeleon 7. The RP-HPLC column is a Thermo Hypersil Gold^® 50 × 2.1 mm (C18 bounded), with particles of a diameter of 1.9 mm. The volume of the sample injected into the column was 5 µL. Chromatographic analysis, total duration of 10 min, was on the gradient of the following solvents: t = 0 min, methanol/water 5:95; 0 < t < 7 min, linear increase in the proportion of methanol to a methanol/water ratio of 100:0; 5 < t < 7 min, methanol/water 100:0; t = 7 min, return to a methanol/water ratio of 5:95; 7 < t < 10 min, methanol/water 5:95. The water and methanol used was buffered with 0.1 % formic acid. The flow rate of the mobile phase was 0.4 mL/min. The retention times (t_R) of the molecules analyzed were indicated in min.

4.2. Preparation of N-(2-(8-bromo-6-chloro-3-nitroimidazo[1,2-a]pyridin-2-yl)-1-(4-chlorophenyl)ethyl)-4-methylbenzenesulfonamide (3)

To a solution of 8-bromo-6-chloro-2-chloromethyl-3-nitroimidazo[1,2-a]pyridine (2) (200 mg, 0.62 mmol, 1 equiv) in 2-methyltetrahydrofuran (20 mL) cooled by an acetone bath at −20 °C, (E)-N-(4-chlorobenzylidene)-4-methylbenzenesulfonamide [11] (364 mg, 1.24 mmol, 2 equiv) was added. Then, TDAE (144 $\mathsf{\mu }$L, 0.62 mmol, 1 equiv) was added under an N₂ atmosphere, and the reaction mixture was stirred at −20 °C for 30 min. The TLC monitoring reaction was performed using DCM as eluent. The mixture was slowly poured into an ice-water mixture, extracted three times with dichloromethane, dried over anhydrous Na₂SO₄, filtered, and evaporated. Compound (3) was obtained after purification by flash-chromatography on silica-gel (eluent: dichloromethane/methanol gradient from 100/0 to 99/1 v/v) as a beige solid in 30 % yield (107 mg). mp 190 °C. ¹H NMR (400 MHz, CDCl₃) $\delta$: 9.26 (d, J = 1.7 Hz, 1H), 7.92 (d, J = 1.8 Hz, 1H), 7.41 (d, J = 8.5 Hz, 2H), 7.35 (d, J = 7.9 Hz, 2H), 7.31 (d, J = 8.5 Hz, 2H), 6.85 (d, J = 7.9 Hz, 2H), 5.69 (d, J = 6.9 Hz, 1H), 4.93–4.83 (m, 1H), 3.59 (dd, J = 14.1, 3.8 Hz, 1H), 3.40 (dd, J = 14.1, 11.0 Hz, 1H), 2.27 (s, 3H). (Figure S1). ¹³C NMR (101 MHz, CDCl₃) $\delta$: 149.3, 143.0, 141.0, 139.7, 137.4, 134.0, 133.8, 131.0, 129.0 (2C), 128.9 (2C), 127.8 (2C), 126.5 (2C), 124.8, 124.8, 112.8, 56.0, 37.7, 21.6. (Figure S2). LC/MS ESI + t_R 7.097 min, (m/z) [M + H]⁺ 583.32/585.27/587.34, HPLC Purity > 92%. HRMS (+ESI): 606.9405 [M + Na]⁺. Calcd for C₂₂H₁₇BrCl₂N₄O₄SNa: 606.9401.

4.3. Preparation of 2,2^′-[2,4-bis(4-chlorophenyl)cyclobutane-1,3-diyl]bis(8-bromo-6-chloro-3-nitroimidazo[1,2-a]pyridine) (4)

To a solution of N-(2-(8-bromo-6-chloro-3-nitroimidazo[1,2-a]pyridin-2-yl)-1-(4-chlorophenyl)ethyl)-4-methylbenzenesulfonamide (3) (1 g, 1.71 mmol, 1 equiv) in tetrahydrofuran (50 mL), K₂CO₃ (473 mg, 3.42 mmol, 2 equiv) was added. The reaction mixture was stirred for 24 h at 80 °C. The TLC monitoring reaction was performed using DCM/cyclohexane (1:1) as eluent. The mixture was slowly poured into an ice-water mixture. The mixture was extracted three times with ethyl acetate, dried over anhydrous Na₂SO₄, filtered, and evaporated. Compound (4) was obtained after purification by flash-chromatography on silica-gel (eluent: cyclohexane/dichloromethane gradient from 1/0 to 3/7 v/v) as a white solid in 23 % yield (160 mg). mp 287 °C. ¹H NMR (400 MHz, CDCl₃) $\delta$: 9.31 (d, J = 1.8 Hz, 2H), 7.82 (d, J = 1.8 Hz, 2H), 7.25–7.21 (m, 4H), 7.09–7.04 (m, 4H), 5.53–5.45 (m, 2H), 5.28–5.20 (m, 2H). (Figure S3). ¹³C NMR (101 MHz, CDCl₃) $\delta$: 152.6 (2C), 141.2 (2C), 137.8 (2C), 133.9 (2C), 132.7 (2C), 130.6 (2C), 129.6 (4C), 128.4 (4C), 124.8 (2C), 124.4 (2C), 112.9 (2C), 43.5 (2C), 43.4 (2C). (Figure S4). HRMS (+ESI): 826.8381 [M + H]⁺. Calcd for C₃₀H₁₇Br₂Cl₄N₆O₄: 826.8382.

4.4. Single-Crystal X-Ray Diffraction Determination

Crystal Data for compound (4): C₃₀H₁₆Br₂Cl₄N₆O (M = 826.09 g/mol): monoclinic, space group P 21/c, a = 13.4616(4) $\mathring{A}$, b = 6.5681(2) $\mathring{A}$, c = 18.3415(8) $\mathring{A}$, $\alpha$ = 90 °, $\beta$ = 108.653 (4) °, $\gamma$ = 90 °, V = 1536.52 (10) $\mathring{A}$³, Z = 2, T = 295 K, $\mu$(MoK$\alpha$) = 3.035 mm^-1, Dcalc = 1.786 g/cm³, 4360 reflections measured, 4124 unique. The final R1 values were 0.0468 (I > 2$\sigma$(I)) and 0.0919 (all data). The goodness of fit on F² was 1.032. (Figure S5). Crystallographic data (excluding structure factors) for compound (4) have been deposited with the Cambridge Crystallographic Data Centre (CCDC) under the number [CCDC-PUFQEQ-2391174] [12].