Synthesis of new 4-(E)-alkenylpyrrolo[1,2-a]quinoxalines as antileishmanial agents by Suzuki-Miyaura cross-coupling reactions

Abstract

A series of new 4-(E)-alkenylpyrrolo[1,2-a]quinoxaline derivatives, structural analogues of alkaloid chimanine B, was synthesized in good yields using efficient palladium(0)-catalyzed Suzuki-Miyaura cross-coupling reactions. These new compounds were tested for in vitro antiparasitic activity upon Leishmania amazonensis and Leishmania infantum strains. Biological results showed activity against the promastigote forms of L. amazonensis and L. infantum with IC₅₀ ranging from 0.5 to 7 μM. From a Structure-Activity Relationships point of view, these pharmacological results mainly enlightened the importance of the 4-lateral C₆, C₇ or C₈ α-unsaturated trans-alkenyl chain of unsubstituted pyrrolo[1,2-a]quinoxaline moiety.

Keywords::

• 4-Alkenylpyrrolo[1,2-a]quinoxaline
• antileishmanial agents
• leishmania amazonensis
• leishmania infantum
• suzuki reaction

Introduction

Parasitic diseases cause enormous suffering in many parts of the world. The leishmaniasis is a complex of disease syndromes caused by at least 20 species of the protozoan parasite of the genus Leishmania 1-2. The disease is distributed worldwide, but mainly in the tropics and subtropics, with a prevalence of 12 million cases and an approximated incidence of 0.5 million cases of visceral (VL, or Kala-azar) and 1.5 million cases of cutaneous leishmaniasis (CL). The pentavalent antimonial compounds, sodium stibogluconate (Pentostam®) and meglumine antimoniate (Glucantime®), which have been the first-line treatments of leishmaniases for the last 50 years, are subjects to development of resistance. Amphotericin B (Fungizone®, AmBisome®) and pentamidine (Pentacarinat®), the parenteral alternatives to antimony, cause serious and irreversible toxic effects which preclude their use [3]. Newly introduced first orally active drug miltefosine is quite effective but shows teratogenic effects and cannot be used in the pregnant women [4]. As current treatments are not ideal because they possess one or more negative attributes, including toxicity, loss of effectiveness due to resistance, expense, and inconvenience 5-8, novel therapies to combat leishmaniasis are urgently needed. Amongst new drugs, natural products 9-10 and derivatives of quinoline alkaloids such as chimanine B and some 2-substituted quinoline derivatives isolated from Galipea longiflora (Figure 1) have been reported to possess activity against experimental animal infections 11-13.

Figure 1 Structures of chimanine B, previously synthesized 4-(E)-pentenylpyrrolo[1,2-a]quinoxalines and new 4-(E)-alkenylpyrrolo[1,2-a]quinoxalines 1a–m.

In the course of our work devoted to discover new compounds employed in the leishmaniasis chemotherapy, we previously identified a series of 4-substituted pyrrolo[1,2-a]quinoxaline derivatives designed as interesting bioactive isosteres of Galipea species quinoline alkaloids [14]. From these preliminary results, it appeared that 4-alkenylpyrrolo[1,2-a]quinoxalines endowed with lipophilic unsaturated carbon chain, such as the 4-(E)-pentenyl derivatives (Figure 1), could initiate new valuable antileishmanial chemistry scaffolding. Thus, taking into account our experience in the field of the synthesis of new bioactive heterocyclic compounds of the type pyrrolo[1,2-a]quinoxaline 15-17, we designed new 4-(E)-alkenylpyrrolo[1,2-a]quinoxalines correlated with a particular lipophilic behavior (Figure 1). In this paper, we present preliminary results concerning the synthesis of 1a–m via Suzuki-Miyaura cross-coupling reactions, and initial in vitro antileishmanial activity upon Leishmania amazonensis and Leishmania infantum.

Materials and methods

Chemistry

Instrumentation

Melting points were determined with an SM-LUX-POL Leitz hot-stage microscope and reported uncorrected. NMR spectra were recorded on a BRUKER AVANCE 300 spectrometer (300 MHz). Chemical shifts refer to tetramethylsilane which was used as an internal reference. Analytical TLC was carried out on 0.25 precoated silica gel plates (POLYGRAM SIL G/UV₂₅₄) with visualisation by irradiation with a UV lamp. Silica gel 60 (70–230 mesh) was used for column chromatography. Elemental analyses were conducted by CNRS, Vernaison, France.

Synthesis of 4-[(E)-Hex-1-enyl]pyrrolo[1,2-a]quinoxalines 1a–c

A mixture of the 4-chloropyrrolo[1,2-a]quinoxaline 5a–c [15,17] (3.4 mmol), the (E)-1-hexenylboronic acid pinacol ester (3.8 mmol) and Pd(PPh₃)₄ (0.10 mmol) in benzene (15 mL), and 4 M aqueous potassium hydroxide solution (2.4 mL) was stirred and heated under reflux under nitrogen for 5 h. It was then cooled, transferred to a separating funnel, and the reaction flask washed out with water (3 × 50 mL) and benzene (2 × 40 mL), the washings being added to the separating funnel. The organic layer was separated, washed with an aqueous saturated sodium hydrogen carbonate solution, then with a brine solution, dried over Na₂SO₄, and evaporated to dryness. Column chromatography of the residue on silica gel using diethyl ether-petroleum ether (1/3) as eluent gave the pure product 1a–c.

4-[(E)-Hex-1-enyl]pyrrolo[1,2-a]quinoxaline (1a)

Yield: 76%, yellow oil; ¹H NMR δ (300 MHz, CDCl₃) 7.96 (dd, J 8.80 and 2.20 Hz, 1H, H-9), 7.93 (dd, J 2.90 and 1.25 Hz, 1H, H-1), 7.83 (dd, J 8.80 and 2.20 Hz, 1H, H-6), 7.46 (m, 2H, H-7 and H-8), 7.24 (ddd, J 15.50 and 7.05 Hz, 1H, = CH), 7.02 (dd, J 3.95 and 1.25 Hz, 1H, H-3), 6.88 (dd, J 3.95 and 2.90 Hz, 1H, H-2), 6.85 (ddd, J 15.50 and 1.50 Hz, 1H, HC = ), 2.41 (m, 2H, CH₂), 1.62 (m, 2H, CH₂), 1.47 (m, 2H, CH₂), 0.98 (t, J 7.20 Hz, 3H, CH₃). Anal. Calcd. for C₁₇H₁₈N₂: C, 81.56; H, 7.25; N, 11.19. Found: C, 81.31; H, 7.39; N, 11.28%.

4-[(E)-Hex-1-enyl]-7-methoxypyrrolo[1,2-a]quinoxaline (1b)

Yield: 72%, yellow oil; ¹H NMR δ (300 MHz, CDCl₃) 7.85 (dd, J 2.60 and 1.20 Hz, 1H, H-1), 7.73 (d, J 8.95 Hz, 1H, H-9), 7.43 (d, J 2.75 Hz, 1H, H-6), 7.22 (ddd, J 15.50 and 7.05 Hz, 1H, = CH), 7.06 (dd, J 8.95 and 2.75 Hz, 1H, H-8), 6.98 (dd, J 4.05 and 1.20 Hz, 1H, H-3), 6.86 (dd, J 4.05 and 2.60 Hz, 1H, H-2), 6.82 (ddd, J 15.50 and 1.55 Hz, 1H, HC = ), 3.93 (s, 3H, CH₃O), 2.39 (m, 2H, CH₂), 1.61 (m, 2H, CH₂), 1.47 (m, 2H, CH₂), 0.98 (t, J 7.25 Hz, 3H, CH₃). Anal. Calcd. for C₁₈H₂₀N₂O: C, 77.11; H, 7.19; N, 9.99. Found: C, 76.94; H, 7.03; N, 10.06%.

4-[(E)-Hex-1-enyl]-8-methoxypyrrolo[1,2-a]quinoxaline (1c)

Yield: 63%, pale-yellow crystals, mp = 28°C; ¹H NMR δ (300 MHz, CDCl₃) 7.87 (d, J 8.90 Hz, 1H, H-6), 7.81 (dd, J 2.75 and 1.25 Hz, 1H, H-1), 7.27 (d, J 2.65 Hz, 1H, H-9), 7.15 (ddd, J 15.60 and 7.00 Hz, 1H, = CH), 7.02 (dd, J 8.90 and 2.65 Hz, 1H, H-7), 6.97 (dd, J 3.95 and 1.25 Hz, 1H, H-3), 6.87 (dd, J 3.95 and 2.75 Hz, 1H, H-2), 6.82 (ddd, J 15.60 and 1.50 Hz, 1H, HC = ), 3.95 (s, 3H, CH₃O), 2.39 (m, 2H, CH₂), 1.59 (m, 2H, CH₂), 1.49 (m, 2H, CH₂), 0.98 (t, J 7.20 Hz, 3H, CH₃). Anal. Calcd. for C₁₈H₂₀N₂O: C, 77.11; H, 7.19; N, 9.99. Found: C, 77.19; H, 7.35; N, 10.14%.

Synthesis of 4-[(E)-Hept-1-enyl]pyrrolo[1,2-a]quinoxalines 1d-f, 4-[(E)-Oct-1-enyl]pyrrolo[1,2-a]quinoxalines 1g–i and 4-[(E)-Non-1-enyl]pyrrolo[1,2-a]quinoxaline 1j–k

A mixture of the 4-chloropyrrolo[1,2-a]quinoxaline 5a–c [15,17] (5 mmol), the alkenylboronic acid (5.5 mmol) and Pd(PPh₃)₄ (0.15 mmol) in benzene (25 mL), ethanol (1.6 mL) and 2 M aqueous sodium carbonate solution (5.4 mL) was stirred and heated under reflux under nitrogen for 24 h. It was then cooled, transferred to a separating funnel, and the reaction flask washed out with water (3 × 50 mL) and dichloromethane (3 × 90 mL), the washings being added to the separating funnel. The organic layer was separated and the aqueous phase extracted with dichloromethane (2 × 100 mL). The combined organic extracts were then washed with water (3 × 130 mL), dried over Na₂SO₄, filtered and the filtrate evaporated under reduced pressure. Column chromatography of the residue on silica gel using diethyl ether-petroleum ether (1/3) as eluent gave the pure product 1d–k.

4-[(E)-Hept-1-enyl]pyrrolo[1,2-a]quinoxaline (1d)

Yield: 66%, yellow oil; ¹H NMR δ (300 MHz, CDCl₃) 7.95 (dd, J 8.85 and 2.15 Hz, 1H, H-9), 7.93 (dd, J 2.90 and 1.15 Hz, 1H, H-1), 7.83 (dd, J 8.85 and 2.15 Hz, 1H, H-6), 7.45 (m, 2H, H-7 and H-8), 7.26 (ddd, J 15.35 and 7.05 Hz, 1H, = CH), 7.02 (dd, J 3.95 and 1.15 Hz, 1H, H-3), 6.86 (dd, J 3.95 and 2.90 Hz, 1H, H-2), 6.86 (ddd, J 15.35 and 1.40 Hz, 1H, HC = ), 2.40 (m, 2H, CH₂), 1.60 (m, 2H, CH₂), 1.42 (m, 4H, 2CH₂), 0.94 (t, J 7.20 Hz, 3H, CH₃). Anal. Calcd. for C₁₈H₂₀N₂: C, 81.78; H, 7.62; N, 10.60. Found: C, 81.95; H, 7.55; N, 10.63%.

4-[(E)-Hept-1-enyl]-7-methoxypyrrolo[1,2-a]quinoxaline (1e)

Yield: 55%, pale-yellow crystals, mp = 41°C; ¹H NMR δ (300 MHz, CDCl₃) 7.85 (dd, J 2.65 and 1.25 Hz, 1H, H-1), 7.72 (d, J 9.00 Hz, 1H, H-9), 7.43 (d, J 2.80 Hz, 1H, H-6), 7.22 (ddd, J 15.55 and 7.05 Hz, 1H, = CH), 7.07 (dd, J 9.00 and 2.80 Hz, 1H, H-8), 6.99 (dd, J 4.05 and 1.25 Hz, 1H, H-3), 6.85 (dd, J 4.05 and 2.65 Hz, 1H, H-2), 6.84 (ddd, J 15.55 and 1.50 Hz, 1H, HC = ), 3.93 (s, 3H, CH₃O), 2.41 (m, 2H, CH₂), 1.61 (m, 2H, CH₂), 1.42 (m, 4H, 2CH₂), 0.94 (t, J 7.20 Hz, 3H, CH₃). Anal. Calcd. for C₁₉H₂₂N₂O: C, 77.52; H, 7.53; N, 9.52. Found: C, 77.72; H, 7.78; N, 9.49%.

4-[(E)-Hept-1-enyl]-8-methoxypyrrolo[1,2-a]quinoxaline (1f)

Yield: 59%, pale-yellow crystals, mp = 51°C; ¹H NMR δ (300 MHz, CDCl₃) 7.87 (d, J 8.95 Hz, 1H, H-6), 7.81 (dd, J 2.70 and 1.30 Hz, 1H, H-1), 7.23 (d, J 2.65 Hz, 1H, H-9), 7.15 (ddd, J 15.55 and 7.00 Hz, 1H, = CH), 7.03 (dd, J 8.95 and 2.65 Hz, 1H, H-7), 6.98 (dd, J 4.00 and 1.30 Hz, 1H, H-3), 6.87 (dd, J 4.00 and 2.70 Hz, 1H, H-2), 6.82 (ddd, J 15.55 and 1.50 Hz, 1H, HC = ), 3.95 (s, 3H, CH₃O), 2.38 (m, 2H, CH₂), 1.60 (m, 2H, CH₂), 1.40 (m, 4H, 2CH₂), 0.94 (t, J 7.25 Hz, 3H, CH₃). Anal. Calcd. for C₁₉H₂₂N₂O: C, 77.52; H, 7.53; N, 9.52. Found: C, 77.61; H, 7.70; N, 9.42%.

4-[(E)-Oct-1-enyl]pyrrolo[1,2-a]quinoxaline (1 g)

Yield: 52%, yellow oil; ¹H NMR δ (300 MHz, CDCl₃) 7.97 (dd, J 8.90 and 2.20 Hz, 1H, H-9), 7.93 (dd, J 2.70 and 1.30 Hz, 1H, H-1), 7.83 (dd, J 8.90 and 2.20 Hz, 1H, H-6), 7.45 (m, 2H, H-7 and H-8), 7.24 (ddd, J 15.50 and 7.00 Hz, 1H, = CH), 7.02 (dd, J 4.00 and 1.30 Hz, 1H, H-3), 6.88 (dd, J 4.00 and 2.70 Hz, 1H, H-2), 6.86 (ddd, J 15.50 and 1.50 Hz, 1H, HC = ), 2.41 (m, 2H, CH₂), 1.61 (m, 2H, CH₂), 1.43 (m, 2H, CH₂), 1.37 (m, 4H, 2CH₂), 0.93 (t, J 6.85 Hz, 3H, CH₃). Anal. Calcd. for C₁₉H₂₂N₂: C, 81.97; H, 7.96; N, 10.08. Found: C, 82.17; H, 8.05; N, 10.13%.

7-Methoxy-4-[(E)-oct-1-enyl]pyrrolo[1,2-a]quinoxaline (1 h)

Yield: 42%, pale-yellow crystals, mp = 72°C; ¹H NMR δ (300 MHz, CDCl₃) 7.86 (dd, J 2.60 and 1.20 Hz, 1H, H-1), 7.73 (d, J 8.95 Hz, 1H, H-9), 7.43 (d, J 2.80 Hz, 1H, H-6), 7.22 (ddd, J 15.50 and 7.05 Hz, 1H, = CH), 7.08 (dd, J 8.95 and 2.80 Hz, 1H, H-8), 6.99 (dd, J 4.05 and 1.20 Hz, 1H, H-3), 6.85 (dd, J 4.05 and 2.60 Hz, 1H, H-2), 6.84 (ddd, J 15.50 and 1.45 Hz, 1H, HC = ), 3.93 (s, 3H, CH₃O), 2.40 (m, 2H, CH₂), 1.61 (m, 2H, CH₂), 1.45 (m, 2H, CH₂), 1.35 (m, 4H, 2CH₂), 0.93 (t, J 6.80 Hz, 3H, CH₃). Anal. Calcd. for C₂₀H₂₄N₂O: C, 77.88; H, 7.84; N, 9.08. Found: C, 77.97; H, 7.68; N, 9.22%.

8-Methoxy-4-[(E)-oct-1-enyl]pyrrolo[1,2-a]quinoxaline (1i)

Yield: 57%, pale-yellow crystals, mp = 52°C; ¹H NMR δ (300 MHz, CDCl₃) 7.88 (d, J 8.95 Hz, 1H, H-6), 7.82 (dd, J 2.75 and 1.20 Hz, 1H, H-1), 7.25 (d, J 2.65 Hz, 1H, H-9), 7.15 (ddd, J 15.55 and 7.00 Hz, 1H, = CH), 7.04 (dd, J 8.95 and 2.65 Hz, 1H, H-7), 6.98 (dd, J 3.95 and 1.20 Hz, 1H, H-3), 6.88 (dd, J 3.95 and 2.75 Hz, 1H, H-2), 6.82 (ddd, J 15.55 and 1.40 Hz, 1H, HC = ), 3.96 (s, 3H, CH₃O), 2.38 (m, 2H, CH₂), 1.57 (m, 2H, CH₂), 1.46 (m, 2H, CH₂), 1.38 (m, 4H, 2CH₂), 0.93 (t, J 6.75 Hz, 3H, CH₃). Anal. Calcd. for C₂₀H₂₄N₂O: C, 77.88; H, 7.84; N, 9.08. Found: C, 77.80; H, 7.94; N, 9.14%.

4-[(E)-Non-1-enyl]pyrrolo[1,2-a]quinoxaline (1j)

Yield: 71%, yellow oil; ¹H NMR δ (300 MHz, CDCl₃) 7.95 (dd, J 8.90 and 2.25 Hz, 1H, H-9), 7.93 (dd, J 2.75 and 1.30 Hz, 1H, H-1), 7.83 (dd, J 8.90 and 2.25 Hz, 1H, H-6), 7.45 (m, 2H, H-7 and H-8), 7.23 (ddd, J 15.50 and 7.00 Hz, 1H, = CH), 7.02 (dd, J 4.00 and 1.30 Hz, 1H, H-3), 6.88 (dd, J 4.00 and 2.75 Hz, 1H, H-2), 6.85 (ddd, J 15.50 and 1.55 Hz, 1H, HC = ), 2.40 (m, 2H, CH₂), 1.61 (m, 2H, CH₂), 1.42 (m, 2H, CH₂), 1.37 (m, 6H, 3CH₂), 0.93 (t, J 6.85 Hz, 3H, CH₃). Anal. Calcd. for C₂₀H₂₄N₂: C, 82.15; H, 8.27; N, 9.58. Found: C, 82.32; H, 8.23; N, 9.67%.

7-Methoxy-4-[(E)-non-1-enyl]pyrrolo[1,2-a]quinoxaline (1k)

Yield: 56%, yellow oil; ¹H NMR δ (300 MHz, CDCl₃) 7.85 (dd, J 2.65 and 1.25 Hz, 1H, H-1), 7.72 (d, J 8.95 Hz, 1H, H-9), 7.42 (d, J 2.75 Hz, 1H, H-6), 7.22 (ddd, J 15.50 and 7.05 Hz, 1H, = CH), 7.07 (dd, J 8.95 and 2.75 Hz, 1H, H-8), 6.99 (dd, J 4.05 and 1.25 Hz, 1H, H-3), 6.85 (dd, J 4.05 and 2.65 Hz, 1H, H-2), 6.84 (ddd, J 15.50 and 1.45 Hz, 1H, HC = ), 3.92 (s, 3H, CH₃O), 2.41 (m, 2H, CH₂), 1.60 (m, 2H, CH₂), 1.41 (m, 2H, CH₂), 1.36 (m, 6H, 3CH₂), 0.90 (t, J 6.85 Hz, 3H, CH₃). Anal. Calcd. for C₂₁H₂₆N₂O: C, 78.22; H, 8.13; N, 8.69. Found: C, 78.45; H, 7.96; N, 8.77%.

Synthesis of 4-[(E)-dec-1-enyl]pyrrolo[1,2-a]quinoxaline (1 l) and 4-[(E)-dec-1-enyl]-7-methoxypyrrolo[1,2-a]quinoxaline (1m)

To a suspension of potassium (E)-decenyltrifluoroborate (1.5 mmol), cesium carbonate (4.5 mmol), PdCl₂(dppf)·CH₂Cl₂ (0.15 mmol), and 4-chloropyrrolo[1,2-a]quinoxaline 5a–b [15,17] (1.65 mmol), in THF (15 mL) was added water (1.5 mL) under a nitrogen atmosphere. The reaction mixture was stirred at reflux temperature for 18 h, then cooled to room temperature, diluted with water (25 mL), and extracted with diethyl ether. The combined organic extracts were washed with brine and then dried over Na₂SO₄, filtered and the filtrate evaporated under reduced pressure. Column chromatography of the residue on silica gel using diethyl ether-petroleum ether (1/3) as eluent gave the pure product 1l–m.

4-[(E)-Dec-1-enyl]pyrrolo[1,2-a]quinoxaline (1 l)

Yield: 66%, pale-yellow crystals, mp = 30°C;¹H NMR δ (300 MHz, CDCl₃) 7.96 (dd, J 8.85 and 2.20 Hz, 1H, H-9), 7.93 (dd, J 2.70 and 1.25 Hz, 1H, H-1), 7.84 (dd, J 8.85 and 2.20 Hz, 1H, H-6), 7.45 (m, 2H, H-7 and H-8), 7.23 (ddd, J 15.50 and 7.00 Hz, 1H, = CH), 7.02 (dd, J 4.05 and 1.25 Hz, 1H, H-3), 6.89 (dd, J 4.05 and 2.70 Hz, 1H, H-2), 6.85 (ddd, J 15.50 and 1.45 Hz, 1H, HC = ), 2.41 (m, 2H, CH₂), 1.59 (m, 2H, CH₂), 1.41 (m, 2H, CH₂), 1.36 (m, 8H, 4CH₂), 0.91 (t, J 6.75 Hz, 3H, CH₃). Anal. Calcd. for C₂₁H₂₆N₂: C, 82.31; H, 8.55; N, 9.14. Found: C, 82.17; H, 8.63; N, 9.07%.

4-[(E)-Dec-1-enyl]-7-methoxypyrrolo[1,2-a]quinoxaline (1m)

Yield: 69%, pale-yellow crystals, mp = 49°C; ¹H NMR δ (300 MHz, CDCl₃) 7.86 (dd, J 2.70 and 1.25 Hz, 1H, H-1), 7.74 (d, J 8.95 Hz, 1H, H-9), 7.44 (d, J 2.75 Hz, 1H, H-6), 7.21 (ddd, J 15.50 and 7.05 Hz, 1H, = CH), 7.08 (dd, J 8.95 and 2.75 Hz, 1H, H-8), 6.99 (dd, J 4.00 and 1.25 Hz, 1H, H-3), 6.85 (dd, J 4.00 and 2.70 Hz, 1H, H-2), 6.84 (ddd, J 15.50 and 1.45 Hz, 1H, HC = ), 3.93 (s, 3H, CH₃O), 2.40 (m, 2H, CH₂), 1.58 (m, 2H, CH₂), 1.40 (m, 2H, CH₂), 1.34 (m, 8H, 4CH₂), 0.89 (t, J 6.85 Hz, 3H, CH₃). Anal. Calcd. for C₂₂H₂₈N₂O: C, 78.53; H, 8.39; N, 8.32. Found: C, 78.58; H, 8.54; N, 8.22%.

Pharmacology

In vitro L. amazonensis and L. infantum Culture and Drug Assays

Promastigotes of the L. infantum (clone MCAN/GR/82/LEM497) and L. amazonensis (MHOM/BR/1987/BA) were maintained at 26°C in RPMI-1640 medium supplemented with 10% heat-inactivated fetal calf serum (FCS), 200 U/mL penicillin, 100 μg/mL streptomycin, sodium bicarbonate and non-essential amino acids (all from Gibco, Peisley, UK). At stationary growth phase parasites (10⁶/mL) were harvested, washed and incubated in culture media with our molecules. The viability of promastigotes was checked using the MTS tetrazolium colorimetric assay CellTiter 96 Aqueous (Promega, USA). The MTS cell proliferation assay is a colorimetric assay system, which measures the reduction of a tetrazolium component (MTS) into formazan produced by the mitochondria of viable cells. Cells were plated in triplicate into microtiter-plate wells in 100 μM culture media with our various compound at increasing concentration (0, 1, 5, 10 and 25 μM). After 3 h of incubation with 20 μL MTS/well, the samples were read using an ELISA plate reader at 490 nm wavelength. The amount of colour produced was directly proportional to the number of viable cells. The results are expressed as the concentrations inhibiting parasite growth by 50% (IC₅₀) after a 1-2 day incubation period.

Cytotoxicity test upon human cells

The toxicity of various molecules was evaluated on non-activated, freshly isolated normal human peripheral blood mononuclear cells (PBMNC), as well as phytohemagglutinin (PHA)-induced cells. PBMNC from healthy volunteers were obtained following centrifugation on Ficoll gradient. Cells were then incubated in medium alone or induced to enter cell cycle by the addition of PHA (5 μg/mL, Murex Biotech Limited, Dartford, UK). Our molecules were added as described under results. Following cell cultures during 3–4 days, cells were harvested, washed, and counted with trypan blue exclusion. In some experiments, the proliferation of PBMNC was checked using the MTS dye colorimetric method as described previously. The 50% inhibitory concentrations (IC₅₀) were determined by linear regression analysis, expressed in μM ± SD and the maximum tolerated concentration expressed in μM was evaluated for each compound.

Partition coefficients-Log D (pH 7.4)

In this study, the relative log D (pH 7.4) were assessed at pH 7.4 by the micro-HPLC method [14]. Determinations were performed with a chromatographic apparatus (Spectra Series, San Jose, USA) equipped with a model P1000XR pump and a model SCM 1000 vacuum membrane degasser, a model UV 150 ultraviolet detector (203 nm ≤ λ_max ≤ 253 nm) and a ChromJet data module integrator (ThermoFinnigan, San Jose, USA). The reversed phase column used, was an Equisorb (C.I.L. Cluzeau) C₈ (4.6 × 150 mm; 5 μm particle size) with a mobile phase consisting of acetonitrile – potassium dihydrogenophosphate (6.24 × 10^{− 2} M; pH = 3.5 adjusted with orthophosphoric acid) (65: 35, v/v). The compounds were partitioned between n-octanol (HPLC grade) and phosphate buffer (pH = 7.4). Octanol was presatured with the adequate phosphate buffer (2%), and conversely. An amount of 1 mg of each compound was dissolved in an adequate volume of methanol in order to achieve 1 mg/mL stock solutions. Then, an appropriate aliquot of these methanolic solutions was dissolved in buffer to obtain final concentration of 50 μg/mL. Under the above-described chromatographic conditions, 50 μL of aqueous phase was injected into the chromatograph, leading to the determination of a peak area before partitioning (W₀). In screw-capped tubes, 4000 μL of the aqueous phase (V_aq) was then added to 10 μL of n-octanol (V_oct). The mixture was shaken by mechanical rotation during 30 min, followed by centrifugation achieved at 3000 rpm during 15 min. An amount of 50 μL of the lower phase was injected into the chromatograph column. This led to the determination of a peak area after partitioning (W₁). For each compound, the log D value was calculated using the formula: log D = log [(W₀ – W₁)V_aq/W₁V_oct].

Results and discussion

Chemistry

All reported 4-(E)-alkenylpyrrolo[1,2-a]quinoxaline derivatives 1a–m were obtained from 1-(2-nitrophenyl)pyrroles 2a–c [15,17] (Scheme 1), prepared in acetic acid according to the Clauson-Kaas reaction, starting from 2-nitroanilines and 2,5-dimethoxytetrahydrofuran (DMTHF).

Scheme 1 Synthesis of 4-(E)-alkenylpyrrolo[1,2-a]quinoxaline derivatives 1a–m. Reagents and conditions: (i) DMTHF, AcOH, Δ; (ii) CuSO₄, NaBH₄, EtOH, RT; (iii) CO(OCCl₃)₂, toluene, Δ; (iv) POCl₃, Δ; (v) Method A: CH₃(CH₂)₃CH = CH-B(O-C(CH₃)₂-)₂, KOH; Pd[P(C₆H₅)₃]₄, C₆H₆, Δ; Method B: CH₃(CH₂)_4-6CH = CH-B(OH)₂, Pd[P(C₆H₅)₃]₄, Na₂CO₃, C₆H₆, EtOH, Δ.; Method C: CH₃(CH₂)₇CH = CH-BF₃K, PdCl₂(dppf)·CH₂Cl₂, Cs₂CO₃, THF-H₂O, Δ.

The resulting 1-(2-nitrophenyl)pyrroles intermediates 2a–c were subsequently reduced into the attempted 1-(2-aminophenyl)pyrroles 3a–c [15,17] using a sodium borohydride-copper(II) sulfate in ethanol at room temperature according to the conditions described by Yoo and Lee [18]. This NaBH₄-CuSO₄ system was found to be quite powerful in reducing the aromatic nitro groups with excellent yields (85–94%). Not commercialy available 5-methoxy-2-nitroaniline was prepared according to the literature 19-20. The reaction of 3a–c with triphosgene in toluene gave the lactams 4a–c, which were subsequently chlorodehydroxylated with phosphorous oxychloride, leading to the 4-chloroquinoxalines 5a–c [15,17]. 4-(E)-Hex-1-enylpyrrolo[1,2-a]quinoxalines 1a–c were easily prepared in quite good yields (63–76%) by a direct Suzuki-Miyaura cross-coupling reaction of 4-chloropyrroloquinoxalines 5a–c with (E)-1-hexenylboronic acid pinacol ester performed in the presence of Pd(PPh₃)₄ as a catalyst and a 4 M aqueous solution of potassium hydroxide solution 21-22. Then, the 4-(E)-alkenylpyrrolo[1,2-a]quinoxalines 1d–k were synthesized via the palladium-catalyzed cross-coupling reaction of various (E)-alkenylboronic acids with 5a–c in the presence of sodium carbonate used as the base 1423-24. The Suzuki-Miyaura-type reaction was then expanded to the use of potassium (E)-decenyltrifluoroborate and 4-chloropyrrolo[1,2-a]quinoxaline 5a–b as coupling partner by using PdCl₂(dppf)·CH₂Cl₂ as the catalyst, cesium carbonate as the base, and THF-H₂O as the solvent system 25-27. Thus, this new palladium-catalyzed cross-coupling reaction led to the expected 4-[(E)-dec-1-enyl]pyrrolo[1,2-a]quinoxalines 1l–m with an 66–69% yield (Table I). From NMR assignations, it appeared that 4-alkenyl derivatives 1a–m were afforded as single E isomers. Thus, their ¹H NMR spectra showed two characteristic chemical shifts of a (E)-alkenyl chain in the olefinic region [δ 7.26–7.15 ppm (1H, ddd, J = 15.60–15.35 and 7.05–7.00 Hz), and 6.86–6.82 ppm (1H, ddd, J = 15.60–15.35 and 1.55–1.40 Hz)]. A coupling constant value of 15.60–15.35 Hz was in favor of a (E)-configuration in the double bond.

Table I.  Physical properties of 4-(E)-alkenylpyrrolo[1,2-a]quinoxalines 1a–m.

Compound	log D7.4	mp (°C)	% Yield
1a	4.47	–	76
1b	4.70	–	72
1c	4.73	28	63
1d	4.90	–	66
1e	5.05	41	55
1f	5.10	51	59
1 g	5.05	–	52
1 h	4.77	72	42
1i	5.12	52	57
1j	5.05	–	71
1k	4.83	–	56
1 l	5.09	30	66
1m	4.98	49	69

Pharmacology

Antileishmanial activity

Compounds 1a–m were tested for their in vitro antileishmanial activity upon the L. amazonensis and L. infantum strains 28-29 with chimanine B as the reference and Amphotericin B as the reference standard drug (Table II). All the 4-(E)-alkenylpyrrolo[1,2-a]quinoxaline 1a–m were found active against the promastigote forms of L. amazonensis with IC₅₀ ranging from 0.5 to 7 μM.

Table II.  In vitro sensitivity of compounds 1a–m on L. amazonensis and L infantum strains, and cytotoxicity on human peripheral blood mononuclear cells PBMNC + PHA.

	IC50 values (μM)^a			Index of selectivity^b
	Leishmania strains		Cytotoxicity on activated human peripheral blood mononuclear cells (PBMNC)	L. amazonensis	L. infantum
Compound	L. amazonensis promastigotes	L. infantum promastigotes	PBMNC + PHA
Amphotericin B	1 ± 0.2	0.9 ± 0.1	96 ± 10	96	106.6
Chimanine B	5 ± 1	7 ± 2	42 ± 4	8.4	6
1a	0.5 ± 0.1	0.6 ± 0.1	7 ± 0.5	14	11.7
1b	2 ± 0.1	2.5 ± 0.1	5 ± 0.5	2.5	2
1c	2.5 ± 0.1	2.5 ± 0.1	5 ± 0.5	2	2
1d	1 ± 0.1	1.5 ± 0.2	7 ± 0.4	7	4.7
1e	2 ± 0.1	0.5 ± 0.1	5 ± 0.4	2.5	10
1f	2.5 ± 0.1	5 ± 0.2	10 ± 1	4	2
1 g	0.5 ± 0.1	0.6 ± 0.1	8 ± 0.5	16	13.3
1 h	2 ± 0.3	1.2 ± 0.1	8 ± 0.6	4	6.7
1i	7 ± 0.1	5 ± 0.1	20 ± 3	2.8	4
1j	3 ± 0.1	3 ± 0.1	7 ± 0.5	2.3	2.3
1k	2 ± 0.2	4 ± 0.1	5 ± 0.6	2.5	1.25
1 l	4 ± 0.4	3 ± 0.2	7 ± 0.4	1.75	2.3
1m	1 ± 0.2	4 ± 1	5 ± 0.4	5	1.25

^aIC₅₀ values were measured on the promastigotes of the Leishmania amazonensis (MHOM/BR/1987/BA) and Leishmania infantum (clone MCAN/GR/82/LEM497) strains. The IC₅₀ (μM) values correspond to the mean +/ − standard deviation from 3 independent experiments

^bIndex of selectivity (I.S.) was defined as the ratio between the IC₅₀ value on the PBMNC + PHA cells and the IC₅₀ value against the Leishmania amazonensis and Leishmania infantum strains.

The pyrrolo[1,2-a]quinoxalines 1a, 1d and 1 g, substituted at the 4-position with a (E)-hexenyl, a (E)-heptenyl or a (E)-octenyl chain, respectively presented the best activities of all investigated compounds against L. amazonensis with IC₅₀ of 0.5, 1 and 0.5 μM, respectively. In these same series, comparison of 1a, 1d and 1 g with their corresponding 7-methoxy substituted derivatives 1b, 1e and 1 h respectively, showed that the 7-methoxy substitution led to a slight decrease in the antileishmanial activity (IC₅₀ = 2 μM). On the other hand, the substitution at position 8 of the C₆, C₇ or C₈ 4-alkenylpyrrolo[1,2-a]quinoxaline heterocycle by one methoxy group (compounds 1c, 1f and 1i) decreased once more the antiparasitic activity upon the L. amazonensis strain with IC₅₀ values of 2.5 to 7 μM. Surprisingly, the 7-methoxysubstituted 4-(E)-nonenyl 1k and 4-(E)-decenyl derivatives 1m were more potent than their unsubstituted analogues 1j and 1 l (IC₅₀ = 2 and 1 compared with 3 and 4 μM for 1j and 1 l).

In the 7-methoxy substituted 4-alkenylpyrrolo[1,2-a]quinoxaline derivative series, the length of the trans α-unsaturated lateral chain at position 4 of the pyrroloquinoxaline moiety seemed not to be crucial as illustrated by results obtained for compounds 1b, 1e, 1 h, 1k and 1m (IC₅₀ = 1–2 μM) in comparison with those of their unsubstituted homologues 1a, 1d, 1 g, 1j and 1 l. From a SAR point of view, these preliminary pharmacological results mainly enlightened the importance of the 4-lateral C₆, C₇ or C₈ α-unsaturated trans-alkenyl chain in the unsubstituted pyrrolo[1,2-a]quinoxaline moiety, in our series.

A second pharmacological evaluation was achieved against the promastigote forms of L. infantum in an experimental visceral leishmaniasis model, with chimanine B as the reference (Table II). All tested compounds 1a–m showed almost the same level of activity as those observed upon the L. amazonensis strain with IC₅₀ ranging from 0.5 to 5 μM. It must be noticed that all these new 4-(E)-alkenyl pyrroloquinoxaline derivatives 1a–m were found more potent than chimanine B, the reference alkaloid (IC₅₀ = 7 μM). Moreover, three compounds 1a, 1e and 1 g showed an IC₅₀ < 1 μM. As a general rule, the introduction of a methoxy substituent in position C-7 or C-8 of the 4-alkenyl pyrrolo[1,2-a]quinoxaline skeleton seemed to slightly decrease the antileishmanial activity in comparison with their respective unsubstituted 4-alkenyl pyrrolo[1,2-a]quinoxaline homologues (i.e., 1a compared to 1b and 1c, 1d to 1f, 1 g to 1 h and 1i, 1j to 1k, and 1 l to 1m), with the exception of 1e, which showed a better antileishmanial activity than its unsubstituted analogue 1d (IC₅₀ = 0.5 μM versus 1.5 μM for 1d).

In summary, all compounds showed almost the same level of activity on L. amazonensis and on L. infantum promastigotes. Further pharmacological studies should be investigated to determine the intracellular target(s) of these new 4-(E)-alkenylpyrrolo[1,2-a]quinoxalines, and to clarify their action mechanism against Leishmania parasites.

Cytotoxicity

All compounds 1a–m were tested on activated (PBMNC + PHA) human peripheral blood mononuclear cells (Table II) [30]. As expected, most of the active pyrrolo[1,2-a]quinoxalines 1a–m showed significant level of cytotoxicity against monocytes (IC₅₀ = 5–20 μM). The different substituents introduced on the pyrrolo[1,2-a]quinoxaline moiety seemed to have less influence on the PBMNC + PHA IC₅₀ values than those observed for the antileishmanial activity. Index of selectivity (IS) was defined as the ratio of the IC₅₀ value on the human mononuclear cells to the IC₅₀ value on the L. amazonensis or L. infantum strains (promastigotes). This IS led to the identification of compound 1 g with IS of 16 on L. amazonensis, and 13.3 on L. infantum. On the other hand, the 4-(E)-hexenylpyrrolo[1,2-a]quinoxaline 1a is also remarkable with IS = 14 and 11.7, on both strains respectively. These two molecules would constitute suitable pharmacophores for the design of new candidates in forthcoming pharmacological investigations.

Lipophilicity

Preliminary pharmacological results could be discussed in terms of physicochemical behaviour through the partitioning theory, determined here by the distribution coefficient D, usually expressed as log D. Consequently, HPLC determination of log D at pH = 7.4, considered as the biological medium pH, for the thirteen bispyrrolo[1,2-a]quinoxalines 1a–m was achieved. All compounds were found very lipophilic with Log D values between 4.47 for 1a and 5.12 for 1i. A plot of antileishmanial IC₅₀ versus log D values was presented in Figure 2, permitting to classify compounds in various subsets. The less lipophilic compound (1a) is the most active (IC₅₀ = 0.5–0.6 μM) while the four more lipophilic ones (1j, 1 l, 1f and 1i) are found slightly less active (IC₅₀ = 2.5–7 μM) on both L. amazonensis or L. infantum strains. A more heterogenous behaviour was found for compounds with log D between 4.90 and 5.05, as for 1d, 1m and 1 g compounds which were are active on L. amazonensis. Such an observation could be noticed for 1 g and 1e with log D values of 5.05 from results observed on the L. infantum strain.

Figure 2 Log D / activity relationship for pyrrolo[1,2-a]quinoxalines 1a–m L. amazonensis and L. infantum strains.

These results indicated that the choice and the length of the α-unsaturated trans-alkenyl chain in position 4 of the pyrrolo[1,2-a]quinoxaline moiety could be correlated with a particular lipophilic behaviour for the antileishmanial activity observed for this new series.

Conclusion

In the present report, we described the synthesis of new 4-(E)-alkenyl pyrrolo[1,2-a]quinoxaline derivatives via Suzuki cross-coupling reactions and presented their leishmanial activity. The physico-chemical profile of new 4-(E)-alkenylpyrrolo[1,2-a]quinoxaline derivatives was studied through log D HPLC determination, achieved at physiological pH. These results have been discussed in terms of lipophilic behaviour in a preliminary SAR study. Nevertheless, as antiparasitic activity is generally related to the distribution of studied compounds to the intracellular target, it is not possible to establish pertinent and definitive correlation. These preliminary pharmacological results mainly enlightened the importance of the 4-lateral C₆, C₇ or C₈ α-unsaturated trans-alkenyl chain in the unsubstituted pyrrolo[1,2-a]quinoxaline moiety. Finally, with respect to our previously described 4-(E)-pentenylpyrrolo[1,2-a]quinoxalines, that showed IC₅₀ ranging from 2 to 7 μM on both L. amazonensis and L. infantum strains [14], these new pyrrolo[1,2-a]quinoxalines 1a–m endowed with a more lipophilic unsaturated carbon chain presented, in general, better antiparasitic activities, and could be further developed as potential antileishmanial drugs.