Synthesis of New Pyrrolo[1,2-a]quinoxaline Derivatives as Potential Inhibitors of Akt Kinase

Abstract

Akt kinases are attractive targets for small molecule drug discovery because of their key role in tumor cell survival/proliferation and their overexpression/activation in many human cancers. Recent efforts in the development and biological evaluation of small molecule inhibitors of Akt have led to the identification of novel Akt kinase inhibitors, based on a quinoxaline or pyrazinone scaffold. A series of new substituted pyrrolo[1,2-a]quinoxaline derivatives, structural analogues of these active quinoxaline or pyrazinone pharmacophores, was synthesized from various substituted 2-nitroanilines or 1,2-phenylenediamine via multistep heterocyclization process. These new compounds were tested for their in vitro ability to inhibit the proliferation of the human leukemic cell lines K562, U937 and HL60, and the breast cancer cell line MCF7. Three of these human cell lines (K562, U937 and MCF7) exhibited an active phosphorylated Akt form. The most promising active pyrroloquinoxalines were found to be 1a that inhibited K562 cell line proliferation with an IC₅₀ of 4.5 μM, and 1h that inhibited U937 and MCF7 cell lines with IC₅₀ of 5 and 8 μM, respectively. These two candidates exhibited more potent activities than the reference inhibitor A6730.

• Pyrrolo[1,2-a]quinoxaline
• Akt kinase
• inhibitor
• antiproliferative agents

Introduction

Protein kinase B (PKB), also known as Akt, is a serine/threonine kinase that has recently garnered a great deal of attention as a promising molecular target for cancer therapy due to its critical role as a regulator of the cell's apoptotic machinery 12345. Akt is comprised of three mammalian isoforms, namely Akt1, Akt2, and Akt3. Akt as a downstream target of PI-3 kinase can induce a variety of biological responses. Overexpression of Akt can result from inactivation of tumor suppressor PTEN and has been correlated with an increasing number of human cancers. Akt is also responsible for promoting survival signals that downregulate apoptotic pathways and contribute to cancer progression. Thus, Akt has a wide range of downstream targets that regulate tumor-associated cell processes such as cell growth, cell cycle progression, survival, migration, epithelial-mesenchymal transition and angiogenesis. Correlation between resistance to chemotherapy and Akt activation has also been observed in prostate cancer cell lines and in human tumors tissue [3]. Inhibition of Akt alone or in combination with other standard cancer chemotherapeutics results in increased programmed death of cancer cells, leading to decreased tumor growth and tumor resistance to chemotherapy [3]. However, the development of Akt inhibitors as small molecule therapeutics for the treatment of cancer has been hindered by a lack of Akt specific inhibitors (versus the AGC family of kinases) and isozyme selective (Akt1, Akt2, and Akt3) Akt inhibitors due to high sequence identity homology 12345. The 2,3-diphenylquinoxaline I was identified through a high throughput screening effort devoted to select compounds capable of inhibiting the three Akt isozymes (Figure 1) [6,7]. Further optimisation around this initial hit I resulted in the identification of more potent Akt1 (II)-, Akt2 (III)- and dual Akt1/2-selective (IV and V) kinase inhibitors 456868–10.

Figure 1 Structures of 2,3-diphenylquinoxaline I, pyrazinones II-III, quinoxaline IV and V (A6730), Akt kinase inhibitors.

We previously described a novel synthetic approach to pyrrolo[1,2-a]quinoxaline derivatives designed as interesting bioactive analogues of quinoline, quinoxaline or pyridine derivatives 11121314. They could be developed as new isosteres of quinoxalines I, IV-V and pyrazinones II-III. Hence, we reported here the synthesis of a series of pyrrolo[1,2-a]quinoxaline derivatives 1 (Figure 2), and the preliminary results of their in vitro ability to inhibit the proliferation of the human leukemic cell lines U937, K562 and HL60, and the breast cancer cell line MCF7. Three of these human cell lines (K562, U937 and MCF7) exhibited an active phosphorylated Akt form.

Figure 2 General structure of synthesized substituted pyrrolo[1,2-a]quinoxaline derivatives 1.

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. Compound A6730 was purchased from Sigma-Aldrich.

Synthesis of 4-(pyrrolo[1,2-a]quinoxalin-4-yl)benzaldehydes 6a-e

Method A: A mixture of the 4-chloropyrrolo[1,2-a]quinoxaline 5a-e [11,12,14] (5 mmol), the 4-formylphenylboronic 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 at 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. The crude residue was triturated in a mixture of diethyl ether-petroleum ether (1/3). The resulting precipitate was filtered, washed with diethyl ether-petroleum ether (1/3), then with ethanol, dried and crystallized from ethanol to give the pure product 6a-e. Method B: To a suspension of potassium 4-formylphenyltrifluoroborate (1.5 mmol), cesium carbonate (4.5 mmol), PdCl₂(dppf)·CH₂Cl₂ (0.15 mmol), and 4-chloropyrrolo[1,2-a]quinoxaline 5a-b (1.65 mmol), in THF (15 mL) was added water (1.5 mL) under a nitrogen atmosphere. The reaction mixture was stirred at reflux 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. The crude residue was triturated in a mixture of diethyl ether-petroleum ether (1/3). The resulting precipitate was filtered, washed with diethyl ether-petroleum ether (1/3), then with ethanol, dried and crystallized from ethanol to give the pure product 6a-b.

4-(Pyrrolo[1,2-a]quinoxalin-4-yl)benzaldehyde (6a)

Yield: 88% (method A), 34% (method B), yellow crystals, mp = 114°C; IR ν_max (KBr)/cm^{− 1} 1700 (CHO); ¹H NMR δ (300 MHz, CDCl₃) 10.16 (s, 1H, CHO), 8.21 (d, 2H, J 8.20 Hz, H-2 and H-6), 8.08 (d, 2H, J 8.20 Hz, H-3 and H-5), 8.08-8.04 (m, 2H, H-1′ and H-9′), 7.94 (dd, 1H, J 8.15 and 1.30 Hz, H-6′), 7.59 (ddd, 1H, J 8.15, 7.40 and 1.30 Hz, H-8′), 7.51 (ddd, 1H, J 8.15, 7.40 and 1.30 Hz, H-7′), 7.01 (dd, 1H, J 4.05 and 1.30 Hz, H-3′), 6.96 (dd, 1H, J 4.05 and 2.70 Hz, H-2′). Anal. Calcd. for C₁₈H₁₂N₂O: C, 79.39; H, 4.44; N, 10.29. Found: C, 79.55; H, 4.72; N, 10.08%.

4-(7-Methoxypyrrolo[1,2-a]quinoxalin-4-yl)benzaldehyde (6b)

Yield: 74% (method A), 31% (method B), yellow crystals, mp = 168°C; IR ν_max (KBr)/cm^{− 1} 1710 (CHO); ¹H NMR δ (300 MHz, CDCl₃) 10.15 (s, 1H, CHO), 8.21 (d, 2H, J 8.10 Hz, H-2 and H-6), 8.10 (d, 2H, J 8.10 Hz, H-3 and H-5), 8.06-8.03 (m, 1H, H-1′), 7.87 (d, 1H, J 9.10 Hz, H-9′), 7.62 (m, 1H, H-6′), 7.22 (dd, 1H, J 9.10 and 2.60 Hz, H-8′), 7.05-7.02 (m, 1H, H-3′), 6.96-6.92 (m, 1H, H-2′), 3.96 (s, 3H, CH₃O). Anal. Calcd. for C₁₉H₁₄N₂O₂: C, 75.48; H, 4.67; N, 9.27. Found: C, 75.32; H, 4.47; N, 9.44%.

4-(8-Methoxypyrrolo[1,2-a]quinoxalin-4-yl)benzaldehyde (6c)

Yield: 90%, yellow crystals, mp = 196°C; IR ν_max (KBr)/cm^{− 1} 1705 (CHO); ¹H NMR δ (300 MHz, CDCl₃) 10.13 (s, 1H, CHO), 8.18 (d, 2H, J 8.00 Hz, H-2 and H-6), 8.05 (d, 2H, J 8.00 Hz, H-3 and H-5), 7.99 (d, 1H, J 9.00 Hz, H-6′), 7.95-7.93 (m, 1H, H-1′), 7.30 (d, 1H, J 2.85 Hz, H-9′), 7.09 (dd, 1H, J 9.00 and 2.85 Hz, H-7′), 6.98-6.93 (m, 2H, H-3′ and H-2′), 3.99 (s, 3H, CH₃O). Anal. Calcd. for C₁₉H₁₄N₂O₂: C, 75.48; H, 4.67; N, 9.27. Found: C, 75.62; H, 4.80; N, 9.13%.

4-(8-Phenylpyrrolo[1,2-a]quinoxalin-4-yl)benzaldehyde (6d)

Yield: 80%, yellow crystals, mp = 176°C; IR ν_max (KBr)/cm^{− 1} 1700 (CHO); ¹H NMR δ (300 MHz, CDCl₃) 10.16 (s, 1H, CHO), 8.23 (d, 2H, J 7.93 Hz, H-2 and H-6), 8.17-8.08 (m, 3H, H-1′, H-6′ and H-9′), 7.78-7.75 (m, 3H, H-2″, H-6″ and H-7′), 7.55 (t, 2H, J 7.20 Hz, H-3″ and H-5″), 7.46 (t, 1H, J 7.20 Hz, H-4″), 7.06-7.03 (m, 1H, H-3′), 7.00-6.98 (m, 1H, H-2′). Anal. Calcd. for C₂₄H₁₆N₂O: C, 82.74; H, 4.63; N, 8.04. Found: C, 82.57; H, 4.69; N, 7.93%.

4-(7-Cyanopyrrolo[1,2-a]quinoxalin-4-yl)benzaldehyde (6e)

Yield: 55%, yellow crystals, mp = 310°C; IR ν_max (KBr)/cm^{− 1} 2240 (N = C), 1705 (CHO); ¹H NMR δ (300 MHz, CDCl₃) 10.16 (s, 1H, CHO), 8.38 (dd, 1H, J 2.80 and 1.10 Hz, H-1′), 8.20 (d, 2H, J 8.30 Hz, H-2 and H-6), 8.11-8.09 (m, 3H, H-3, H-5 and H-6′), 8 (d, 1H, J 8.5 Hz, H-9′), 7.81 (dd, 1H, J 8.5 and 1.80 Hz, H-8′), 7.10 Hz (dd, 1H, J 4.05 and 1.10 Hz, H-3′), 7.05 (dd, 1H, J 4.05 and 2.80 Hz, H-2′). Anal. Calcd. for C₁₉H₁₁N₃O: C, 76.75; H, 3.73; N, 14.13. Found: C, 76.89; H, 3.64; N, 14.27%.

Synthesis of 1,3-dihydro-1-{1-[4-(pyrrolo[1,2-a]quinoxalin-4-yl)benzyl]piperidin-4-yl}-2H-benzimidazol-2-one (1a-f), 1,3-dihydro-1-{1-[(4-phenylpyrrolo[1,2-a]quinoxalin-1-yl)methyl]piperidin-4-yl}-2H-benzimidazol-2-one (1h), and 1,3-dihydro-1-{1-[4-(4-phenylpyrrolo[1,2-a]quinoxalin-1-yl)benzyl]piperidin-4-yl}-2H-benzimidazol-2-one (1i)

The pH of a solution of the aldehyde 6a-e, 9, or 11 (2.5 mmol) and secondary amine (3.0 mmol) in 40 mL methanol was adjusted to 6 by the dropwise addition of acetic acid. Powered sodium cyanoborohydride (6.9 mmol) was then added, and the resultant mixture was refluxed for 5 h. After removal of the methanol by rotary evaporation, the residue was triturated in water and extracted with dichloromethane. The organic layer was washed with water, dried over magnesium sulfate and evaporated to dryness. Solids were crystallized from propan-2-ol, filtered, washed with diethyl ether and dried under reduced pressure to give the compounds 1a-f, 1h-i.

1,3-Dihydro-1-{1-[4-(pyrrolo[1,2-a]quinoxalin-4-yl)benzyl]piperidin-4-yl}-2H-benzimidazol-2-one (1a)

Yield: 45%, white crystals, mp = 215°C; IR ν_max (KBr)/cm^{− 1} 3360 (NH), 1685 (C = O); ¹H NMR δ (300 MHz, CDCl₃) 10.15 (s, 1H, NH), 8.08 (dd, 1H, J 7.95 and 1.45 Hz, H-9″), 8.03 (dd, 1H, J 2.75 and 1.40 Hz, H-1″), 8.01 (d, 2H, J 8.15 Hz, H-3′ and H-5′), 7.90 (dd, 1H, J 7.95 and 1.45 Hz, H-6″), 7.57 (d, 2H, J 8.15 Hz, H-2′ and H-6′), 7.51 (ddd, 1H, J 7.95, 7.30 and 1.45 Hz, H-8″), 7.48 (ddd, 1H, J 7.95, 7.30 and 1.45 Hz, H-7″), 7.35-7.32 (m, 1H, H benzimid.), 7.10-7.06 (m, 3H, H benzimid.), 7.03 (dd, 1H, J 4.05 and 1.40 Hz, H-3″), 6.93 (dd, 1H, J 4.05 and 2.75 Hz, H-2″), 4.44-4.41 (m, 1H, CH pip.), 3.69 (s, 2H, CH₂N), 3.13-3.09 (m, 2H, CH₂ pip.), 2.54-2.50 (m, 2H, CH₂ pip.), 2.27-2.23 (m, 2H, CH₂ pip.), 1.86-1.83 (m, 2H, CH₂ pip.). ¹³C NMR δ (75 MHz, CDCl₃) 155.0 (CO), 154.2 (C4″), 140.4 (C5a″), 137.2 (C9a″), 136.2 (C1′), 130.1 (C6″), 129.2 (C3a), 129.0 (C3′ and C5′), 128.5 (C7a), 127.9 (C2′ and C6′), 127.4 (C8″), 127.1 (C3a″), 125.3 (C4′), 125.2 (C7″), 121.0 (C6), 120.9 (C5), 114.5 (C9″), 113.9 (C3″), 113.6 (C1″), 109.8 (C4), 109.6 (C7), 108.6 (C2″), 62.6 (CH₂N), 53.1 (CH₂ pip.), 53.0 (CH₂ pip.), 50.7 (CH pip.), 29.3 (CH₂ pip.), 29.2 (CH₂ pip.). Anal. Calcd. for C₃₀H₂₇N₅O: C, 76.08; H, 5.75; N, 14.79. Found: C, 75.87; H, 5.86; N, 14.94%.

1,3-Dihydro-1-{1-[4-(7-methoxypyrrolo[1,2-a]quinoxalin-4-yl)benzyl]piperidin-4-yl}-2H-benzimidazol-2-one (1b)

Yield: 53%, pale-yellow crystals, mp = 110°C; IR ν_max (KBr)/cm^{− 1} 3365 (NH), 1680 (C = O); ¹H NMR δ (300 MHz, CDCl₃) 8.84 (s, 1H, NH), 8.01 (d, 2H, J 7.95 Hz, H-3′ and H-5′), 7.96 (dd, 1H, J 2.70 and 1.20 Hz, H-1″), 7.82 (d, 1H, J 9.05 Hz, H-9″), 7.60 (d, 2H, J 7.95 Hz, H-2′ and H-6′), 7.55 (d, 1H, J 2.80 Hz, H-6″), 7.35-7.32 (m, 1H, H benzimid.), 7.13 (dd, 1H, J 9.05 and 2.80 Hz, H-8″), 7.11-7.08 (m, 3H, H benzimid.), 7.03 (dd, 1H, J 4.05 and 1.20 Hz, H-3″), 6.89 (dd, 1H, J 4.05 and 2.70 Hz, H-2″), 4.45-4.41 (m, 1H, CH pip.), 3.94 (s, 3H, CH₃O), 3.70 (s, 2H, CH₂N), 3.13-3.10 (m, 2H, CH₂ pip.), 2.54-2.51 (m, 2H, CH₂ pip.), 2.27-2.24 (m, 2H, CH₂ pip.), 1.89-1.85 (m, 2H, CH₂ pip.). Anal. Calcd. for C₃₁H₂₉N₅O₂: C, 73.93; H, 5.80; N, 13.91. Found: C, 74.05; H, 5.89; N, 13.72%.

1,3-Dihydro-1-{1-[4-(8-methoxypyrrolo[1,2-a]quinoxalin-4-yl)benzyl]piperidin-4-yl}-2H-benzimidazol-2-one (1c)

Yield: 48%, pale-yellow crystals, mp = 156°C; IR ν_max (KBr)/cm^{− 1} 3355 (NH), 1680 (C = O); ¹H NMR δ (300 MHz, CDCl₃) ¹H NMR δ (300 MHz, CDCl₃) 10.27 (s, 1H, NH), 8.01 (d, 2H, J 8.00 Hz, H-3′ and H-5′), 7.98 (d, 1H, J 8.65 Hz, H-6″), 7.92 (dd, 1H, J 2.75 and 1.30 Hz, H-1″), 7.57 (d, 2H, J 8.00 Hz, H-2′ and H-6′), 7.37-7.33 (m, 2H, 2H benzimid.), 7.30 (d, 1H, J 2.85 Hz, H-9″), 7.11-7.06 (m, 3H, H-7″ and 2H benzimid.), 7.01 (dd, 1H, J 4.00 and 1.30 Hz, H-3″), 6.92 (dd, 1H, J 4.00 and 2.75 Hz, H-2″), 4.46-4.42 (m, 1H, CH pip.), 3.98 (s, 3H, CH₃O), 3.71 (s, 2H, CH₂N), 3.15-3.11 (m, 2H, CH₂ pip.), 2.54-2.51 (m, 2H, CH₂ pip.), 2.27-2.23 (m, 2H, CH₂ pip.), 1.88-1.83 (m, 2H, CH₂ pip.). Anal. Calcd. for C₃₁H₂₉N₅O₂: C, 73.93; H, 5.80; N, 13.91. Found: C, 74.05; H, 5.89; N, 13.72%.

1,3-Dihydro-1-{1-[4-(8-phenylpyrrolo[1,2-a]quinoxalin-4-yl)benzyl]piperidin-4-yl}-2H-benzimidazol-2-one (1d)

Yield: 61%, pale-yellow crystals, mp = 150°C; IR ν_max (KBr)/cm^{− 1} 3365 (NH), 1680 (C = O); ¹H NMR δ (300 MHz, CDCl₃) 9.17 (s, 1H, NH), 8.12 (d, 2H, J 8.00 Hz, H-3′ and H-5′), 8.09 (dd, 1H, J 2.75 and 1.25 Hz, H-1″), 8.05 (m, 2H, H-6″ and H-9″), 7.76-7.72 (m, 3H, 3H phényl), 7.61-7.57 (m, 4H, 2H phenyl, H-2′ and H-6′), 7.44 (d, 1H, J 8.40 Hz, H-7″), 7.35-7.31 (m, 1H, H benzimid.), 7.08 (m, 3H, H benzimid.), 7.03 (dd, 1H, J 4.00 and 1.25 Hz, H-3″), 6.95 (dd, 1H, J 4.00 and 2.75 Hz, H-2″), 4.46-4.42 (m, 1H, CH pip.), 3.74 (s, 2H, CH₂N), 3.14-3.10 (m, 2H, CH₂ pip.), 2.56-2.52 (m, 2H, CH₂ pip.), 2.28-2.24 (m, 2H, CH₂ pip.), 1.87-1.83 (m, 2H, CH₂ pip.). Anal. Calcd. for C₃₆H₃₁N₅O: C, 78.66; H, 5.68; N, 12.74 Found: C, 78.86; H, 5.49; N, 12.57%.

1,3-Dihydro-1-{1-[4-(7-cyanopyrrolo[1,2-a]quinoxalin-4-yl)benzyl]piperidin-4-yl}-2H-benzimidazol-2-one (1e)

Yield: 68%, pale-yellow crystals, mp = 152°C; IR ν_max (KBr)/cm^{− 1} 3370 (NH), 2230 (N = C), 1695 (C = O); ¹H NMR δ (300 MHz, CDCl₃) 9.57 (s, 1H, NH), 8.35 (d, 1H, J 1.75 Hz, H-6″), 8.06 (dd, 1H, J 2.90 and 1.05 Hz, H-1″), 8.02 (d, 2H, J 8.15 Hz, H-3′ and H-5′), 7.96 (d, 1H, J 8.60 Hz, H-9″), 7.76 (dd, 1H, J 8.60 and 1.75 Hz, H-8″), 7.57 (d, 2H, J 8.15 Hz, H-2′ and H-6′), 7.36-7.32 (m, 1H, H benzimid.), 7.14 (dd, 1H, J 4.00 and 1.05 Hz, H-3″), 7.12-7.09 (m, 3H, H benzimid.), 7.02 (dd, 1H, J 4.00 and 2.90 Hz, H-2″), 4.46-4.42 (m, 1H, CH pip.), 3.79 (s, 2H, CH₂N), 3.22-3.19 (m, 2H, CH₂ pip.), 2.58-2.56 (m, 2H, CH₂ pip.), 2.36-2.33 (m, 2H, CH₂ pip.), 1.89-1.86 (m, 2H, CH₂ pip.). Anal. Calcd. for C₃₁H₂₆N₆O: C, 74.68; H, 5.25; N, 16.86. Found: C, 74.81; H, 5.10; N, 17.03%.

1,3-Dihydro-1-{1-[4-(pyrrolo[1,2-a]quinoxalin-4-yl)benzyl]piperidin-4-yl}-5-chloro-2H-benzimidazol-2-one (1f)

Yield: 21%, white crystals, mp = 245°C; IR ν_max (KBr)/cm^{− 1} 3350 (NH), 1680 (C = O); ¹H NMR δ (300 MHz, CDCl₃) 10.22 (s, 1H, NH), 8.13 (d, 1H, J 8.10 Hz, H-9″), 8.11-8.04 (m, 3H, H-1″, H-3′ and H-5′), 7.91 (d, 1H, J 8.10 Hz, H-6″), 7.57-7.52 (m, 4H, H-2′, H-6′, H-7″ and H-8″), 7.17-7.15 (m, 1H, H benzimid.), 7.08-7.04 (m, 2H, H benzimid.), 6.96-6.93 (m, 2H, H-2″ and H-3″), 4.41-4.38 (m, 1H, CH pip.), 3.67 (s, 2H, CH₂N), 3.02-2.97 (m, 2H, CH₂ pip.), 2.42-2.33 (m, 2H, CH₂ pip.), 2.24-2.215 (m, 2H, CH₂ pip.), 1.81-1.71 (m, 2H, CH₂ pip.). Anal. Calcd. for C₃₀H₂₆ClN₅O: C, 70.93; H, 5.16; N, 13.79. Found: C, 71.13; H, 5.31; N, 14.01%.

1,3-Dihydro-1-{1-[(4-phenylpyrrolo[1,2-a]quinoxalin-1-yl)methyl]piperidin-4-yl}-2H-benzimidazol-2-one (1h)

Yield: 31%, white crystals, mp = 257°C; IR ν_max (KBr)/cm^{− 1} 3370 (NH), 1685 (C = O); ¹H NMR δ (300 MHz, CDCl₃) 9.44 (s, 1H, NH), 8.75 (dd, 1H, J 8.05 and 1.40 Hz, H-9″), 8.11 (dd, 1H, J 8.05 and 1.40 Hz, H-6′), 7.99-7.96 (m, 2H, H-2″ and H-6″), 7.63-7.54 (m, 5H, H-7′, H-8′, H-3″, H-4″ and H-5″), 7.15-7.03 (m, 4H, 4H benzimid.), 6.95 (d, 1H, J 3.85 Hz, H-2″), 6.80 (d, 1H, J 3.85 Hz, H-3″), 4.52-4.49 (m, 1H, CH pip.), 4.07 (s, 2H, CH₂N), 3.29-3.26 (m, 2H, CH₂ pip.), 2.52-2.36 (m, 4H, 2CH₂ pip.), 1.93-1.89 (m, 2H, CH₂ pip.). Anal. Calcd. for C₃₀H₂₇N₅O: C, 76.08; H, 5.75; N, 14.79. Found: C, 75.98; H, 5.55; N, 14.70%.

1,3-Dihydro-1-{1-[4-(4-phenylpyrrolo[1,2-a]quinoxalin-1-yl)benzyl]piperidin-4-yl}-2H-benzimidazol-2-one (1i)

Yield: 70%, pale-yellow crystals, mp = 165°C; IR ν_max (KBr)/cm^-1 3360 (NH), 1680 (C = O); ¹H NMR δ (300 MHz, CDCl₃) 9.20 (s, 1H, NH), 8.04 (d, 2H, J 8.15 Hz, H-3′ et H-5′), 7.99 (dd, 1H, J 7.95 and 1.30 Hz, H-9″), 7.56-7.47 (m, 8H, H-2′, H-6′, H-6″, H-7″, H-8″, H-2‴, H-4‴ and H-6‴), 7.37 (t, 2H, J 7.85 Hz, H-3‴ and H-5‴), 7.17-7.09 (m, 4H, 4H benzimid.), 7.06 (d, 1H, J 4.05 Hz, H-3″), 6.81 (d, 1H, J 4.05 Hz, H-2″), 4.48-4.45 (m, 1H, CH pip.), 3.74 (s, 2H, CH₂N), 3.20-3.16 (m, 2H, CH₂ pip.), 2.60-2.57 (m, 2H, CH₂ pip.), 2.34-2.31 (m, 2H, CH₂ pip.), 1.93-1.89 (m, 2H, CH₂ pip.). Anal. Calcd. for C₃₆H₃₁N₅O: C, 78.66; H, 5.68; N, 12.74. Found: C, 78.42; H, 5.45; N, 12.93%.

1,3-Dihydro-1-{1-[4-(7-(1H-tetrazol-5-yl)pyrrolo[1,2-a]quinoxalin-4-yl)benzyl]piperidin-4-yl}-2H-benzimidazol-2-one (1g)

A solution of 1e (1 mmol), sodium azide (5 mmol), and ammonium chloride (5 mmol) in DMF (8 mL) was heated to 110°C for 8 h. After the solution cooled to room temperature, water was added and the resultant solid filtered, washed with ethanol then with diethyl ether, and dried to give 1g. Yield: 73%, beige crystals, mp = 271°C; IR ν_max (KBr)/cm^{− 1} 3380 (NH), 1690 (C = O); ¹H NMR δ (300 MHz, DMSO-d₆) 10.92 (s, 1H, NH), 8.60 (dd, 1H, J 2.95 and 1.10 Hz, H-1″), 8.55 (d, 1H, J 1.95 Hz, H-6″), 8.47 (d, 1H, J 8.75 Hz, H-9″), 8.24 (dd, 1H, J 8.75 and 1.95 Hz, H-8″), 8.07 (d, 2H, J 7.90 Hz, H-3′ and H-5′), 7.66 (d, 2H, J 7.90 Hz, H-2′ and H-6′), 7.28-7.25 (m, 1H, H benzimid.), 7.10 (dd, 1H, J 3.95 and 1.10 Hz, H-3″), 7.03-6.99 (m, 4H, H benzimid. and H-2″), 4.36-4.33 (m, 1H, CH pip.), 4.03 (s, 2H, CH₂N), 3.32-3.28 (m, 2H, CH₂ pip.), 2.70-2.55 (m, 4H, 2CH₂ pip.), 1.82-1.77 (m, 2H, CH₂ pip.). Anal. Calcd. for C₃₁H₂₇N₉O: C, 68.74; H, 5.02; N, 23.28. Found: C, 68.90; H, 5.13; N, 23.07%.

Synthesis of 1,3-dihydro-1-{1-[4-(4-phenylpyrrolo[1,2-a]quinoxalin-2-yl)methyl]piperidin-4-yl}-2H-benzimidazol-2-one (1j) and 1,3-dihydro-1-{1-[α-cyano-4-(4-phenylpyrrolo[1,2-a]quinoxalin-2-yl)methyl]piperidin-4-yl}-2H-benzimidazol-2-one (1k)

The pH of a solution of the aldehyde 15 (2.5 mmol) and secondary amine (3.0 mmol) in 40 mL methanol was adjusted to 6 by the dropwise addition of acetic acid. Powered sodium cyanoborohydride (6.9 mmol) was then added and the resultant mixture was refluxed for 5 h. The resulting precipitate was filtered, washed with methanol then with diethyl ether and dried under reduced pressure to give 1k. After evaporation of the solvents, the second residue was triturated in water and extracted with dichloromethane. The organic layer was washed with water, dried over magnesium sulfate and evaporated to dryness. Solid was crystallized from propan-2-ol, filtered, washed with diethyl ether and dried under reduced pressure to give compound 1j.

1,3-Dihydro-1-{1-[4-(4-phenylpyrrolo[1,2-a]quinoxalin-2-yl)methyl]piperidin-4-yl}-2H-benzimidazol-2-one (1j)

Yield: 43%, white crystals, mp = 148°C; IR ν_max (KBr)/cm^{− 1} 3185 (NH), 1690 (C = O); ¹H NMR δ (300 MHz, CDCl₃) 10.41 (s, 1H, NH), 8.07 (dd, 1H, J 8.00 and 1.35 Hz, H-9′), 8.06-8.02 (m, 3H, H-1′, H-2″ and H-6″), 7.89 (dd, 1H, J 8.00 and 1.35 Hz, H-6′), 7.59-7.56 (m, 4H, H-3″, H-4″, H-5″ and H-8′), 7.48 (ddd, 1H, J 8.00, 7.45 and 1.35 Hz, H-7′), 7.33-7.30 (m, 1H, H benzimid.), 7.13-7.02 (m, 3H, 3H benzimid.), 7.00 (d, 1H, J 1.20 Hz, H-3′), 4.42-4.40 (m, 1H, CH pip.), 3.76 (s, 2H, CH₂N), 3.20-3.17 (m, 2H, CH₂ pip.), 2.55-2.52 (m, 2H, CH₂ pip.), 2.25-2.22 (m, 2H, CH₂ pip.), 1.86-1.83 (m, 2H, CH₂ pip.). Anal. Calcd. for C₃₀H₂₇N₅O: C, 76.08; H, 5.75; N, 14.79. Found: C, 76.28; H, 5.82; N, 14.83%.

1,3-Dihydro-1-{1-[α-cyano-4-(4-phenylpyrrolo[1,2-a]quinoxalin-2-yl)methyl]piperidin-4-yl}-2H-benzimidazol-2-one (1k)

Yield: 25%, white crystals, mp = 260°C; IR ν_max (KBr)/cm^{− 1} 3190 (NH), 2230 (N = C), 1695 (C = O); ¹H NMR δ (300 MHz, CDCl₃) 8.75 (s, 1H, NH), 8.19 (d, 1H, J 1.15 Hz, H-1′), 8.08 (dd, 1H, J 8.05 and 1.35 Hz, H-9′), 8.05-8.02 (m, 2H, H-2″ and H-6″), 7.96 (dd, 1H, J 8.05 and 1.35 Hz, H-6′), 7.62-7.58 (m, 4H, H-3″, H-4″, H-5″ and H-8′), 7.50 (ddd, 1H, J 8.05, 7.40 and 1.30 Hz, H-7′), 7.22-7.18 (m, 1H, H benzimid.), 7.12 (d, 1H, J 1.15 Hz, H-3′), 7.10-7.07 (m, 3H, H benzimid.), 5.15 (s, 1H, CHCN), 4.38-4.36 (m, 1H, CH pip.), 3.22-3.19 (m, 1H, CH₂ pip.), 2.98-2.95 (m, 1H, CH₂ pip.), 2.72-2.70 (m, 2H, CH₂ pip.), 2.44-2.41 (m, 2H, CH₂ pip.), 2.01-1.98 (m, 1H, CH₂ pip.), 1.86-1.83 (m, 1H, CH₂ pip.). ¹³C NMR δ (75 MHz, CDCl₃) 154.2 (CO), 153.4 (C-4′), 138.0 (C-5a′), 136.0 (C-9a′), 130.6 (C-1″), 130.1 (C-4″), 129.8 (C-6′), 129.1 (C-3″ and C-5″), 128.9 (C-2″ and C-6″), 128.8 (C-3a), 128.7 (C-7a), 126.8 (C-8′), 126.4 (C-3a′), 124.8 (C-7′), 123.0 (C-6), 121.0 (C-5), 120.8 (C-1′), 116.9 (N = C), 115.9 (C-9′), 115.6 (C-3′), 109.2 (C-4), 109.0 (C-2′), 108.0 (C-7), 55.4 (CH), 52.0 (CH₂ pip.), 50.4 (CH₂ pip.), 47.4 (CH pip.), 29.0 (CH₂ pip.), 28.7 (CH₂ pip.). Anal. Calcd. for C₃₁H₂₆N₆O: C, 74.68; H, 5.26; N, 16.86. Found: C, 74.41; H, 5.46; N, 17.04%.

Synthesis of 1-bromo-4-phenylpyrrolo[1,2-a]quinoxaline (10)

To a solution of 4-phenylpyrrolo[1,2-a]quinoxaline 8 (15 mmol) in CH₂Cl₂ (65 mL) was added a solution of N-bromosuccinimide (15 mmol) CH₂Cl₂ (30 mL). After the mixture was stirred at room temperature for 30 min, a 2% aqueous solution of NaOH (65 mL) was added and the mixture extracted with CH₂Cl₂ (3 × 35 mL). The combined organic extracts were dried over sodium sulfate and concentrated under reduced pressure. The residue was triturated in ethanol, filtered, washed with ethanol then with petroleum ether and dried, yielding 10. Yield: 82%, white crystals, mp = 159°C; ¹H NMR δ (300 MHz, CDCl₃) 9.35 (dd, 1H, J 7.95 and 1.75 Hz, H-9″), 8.07 (dd, 1H, J 7.95 and 1.75 Hz, H-6), 7.95-7.92 (m, 2H, H-2′ and H-6′), 7.57-7.54 (m, 5H, H-7, H-8, H-3′, H-4′ and H-5′), 6.97 (d, 1H, J 4.25 Hz, H-2), 6.92 (d, 1H, J 4.25 Hz, H-3). Anal. Calcd. for C₁₇H₁₁BrN₂: C, 63.18; H, 3.43; N, 8.67. Found: C, 63.09; H, 3.22; N, 8.95%.

4-(4-Phenylpyrrolo[1,2-a]quinoxalin-1-yl)benzaldehyde (11)

To suspension of 1-bromo-4-phenylpyrrolo[1,2-a]quinoxaline 10 (4.64 mmol) and Pd(PPh₃)₄ (0.232 mmol) in a mixture of toluene/EtOH (75/4.1 mL) under nitrogen were added K₂CO₃ (5.1 mmol) and 4-formylphenylboronic acid (5.1 mmol). The reaction mixture was refluxed for 24 h, and the cooled suspension was extracted with CH₂Cl₂ (3 × 80 mL). The organic layer was washed with a saturated solution of NaCl (70 mL), and the combined organic extracts were dried over sodium sulfate, filtered, and evaporated under reduced pressure. The residue was triturated in ethanol, filtered, washed with ethanol then with petroleum ether and dried to give 11. Yield: 84%, yellow crystals, mp = 215°C; IR ν_max (KBr)/cm^{− 1} 1705 (C = O); ¹H NMR δ (300 MHz, CDCl₃) 10.16 (s, 1H, CHO), 8.06 (d, 2H, J 8.20 Hz, H-2 and H-6), 8.06-8.02 (m, 2H, H-6′ and H-9′), 7.78 (d, 2H, J 8.20 Hz, H-3 and H-5), 7.62-7.58 (m, 2H, H-2″ and H-6″), 7.47-7.44 (m, 3H, H-7′, H-8′ and H-4″), 7.26-7.14 (m, 3H, H-2′, H-3″ and H-5″), 6.92 (d, 1H, J 3.85 Hz, H-3′). Anal. Calcd. for C₂₄H₁₆N₂O: C, 82.74; H, 4.63; N, 8.04. Found: C, 82.97; H, 4.57; N, 8.11%.

Pharmacology

Cell culture

The human leukemic cell lines U937, K562 and HL60, and the breast cancer cell line MCF7 were grown in RPMI 1640 medium (Life Technology, France) supplemented with 10% fetal calf serum (FCS), antibiotics (100 U/ml penicillin, 100 μg/mL streptomycin) and L-glutamin, at 37°C, 5% CO₂ in air. The toxicity of various molecules was also evaluated on non-activated, freshly isolated normal human peripheral blood mononuclear cells (PBMNC), as well as phytohemagglutinin (lymphoproliferative agent) (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).

Cytotoxicity test

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 washed twice in PBS (Phosphate Buffer Saline) and plated in quadruplicate into microtiter-plate wells in 100 μL culture media without or with our various compounds at increasing concentration (0, 1, 5, 10 and 20 μM). After 3 h of incubation with 20 μL MTS/well, the plates were read using an ELISA microplate reader (Thermo, Electrocorporation) 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 cell growth by 50% after a 3 days incubation period. The 50% inhibitory concentrations (IC₅₀) were determined by linear regression analysis, expressed in μM ± SD.

Results and discussion

Chemistry

The synthesis of the 1,3-dihydro-1-{1-[4-(pyrrolo[1,2-a]quinoxalin-4-yl)benzyl]piperidin-4-yl}-2H-benzimidazol-2-ones 1a-f has been accomplished in six or seven steps starting from 2-nitroaniline according to the sequence depicted in Schbeme 1. The Clauson-Kaas reaction of 2-nitroanilines with 2,5-dimethoxytetrahydrofuran (DMTHF) in acetic acid gave the pyrrolic derivatives 2a-e, which were reduced using a NaBH₄-CuSO₄ system to provide the attempted 1-(2-aminophenyl)pyrroles 3a-d [14] or using a SnCl₂, 2H₂O treatment to give 3e [15]. The reaction of 3a-e with triphosgene in toluene gave the lactams 4a-e, which were subsequently chlorodehydroxylated with phosphorous oxychloride, leading to the 4-chloroquinoxalines 5a-e [12,14]. 4-(Pyrrolo[1,2-a]quinoxalin-4-yl)benzaldehydes 6a-e were easily prepared in quite good yields (55-90%) by a direct Suzuki-Miyaura cross-coupling reaction of 4-chloropyrroloquinoxalines 5a-e with 4-formylphenylboronic acid performed in the presence of Pd(PPh₃)₄ as a catalyst, and in the presence of sodium carbonate used as the base (method A) [14,16,17]. The Suzuki-Miyaura-type reaction was then expanded to the use of potassium (E)-4-formylphenyltrifluoroborate 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 (method B) [14,18,19]. The aldehydes 6a-e were then engaged in a reductive amination with NaBH₃CN and 4-(2-ketobenzimidazolin-1-yl)piperidine or 4-(5-chloro-2-ketobenzimidazolin-1-yl)piperidine to give the pyrroloquinoxalines 1a-f [20]. The tetrazole derivative 1g was synthesized by reacting sodium azide with 1e [21].

Scheme 1 Synthesis of the 1,3-dihydro-1-{1-[4-(pyrrolo[1,2-a]quinoxalin-4-yl)benzyl]piperidin-4-yl}-2H-benzimidazol-2-ones 1a-g. Reagents and conditions: (i) DMTHF, AcOH, Δ; (ii) Method A: CuSO₄, NaBH₄, EtOH, RT for 3a-d; Method B: SnCl₂, 2H₂O, EtOH, Δ for 3e; (iii) CO(OCCl₃)₂, toluene, Δ; (iv) POCl₃, Δ; (v) Method A: OHC-C₆H₄-B(OH)₂, Pd[P(C₆H₅)₃]₄, Na₂CO₃, C₆H₆, EtOH, Δ; Method B: OHC-C₆H₄-BF₃K, PdCl₂(dppf)·CH₂Cl₂, Cs₂CO₃, THF-H₂O, Δ; (vi) 4-(2-ketobenzimidazolin-1-yl)piperidine, NaBH₃CN, MeOH, Δ; (vii) 1e, NaN₃, NH₄Cl, DMF, Δ.

The 4-phenylpyrrolo[1,2-a]quinoxaline 8 was prepared by cyclisation of the amide 7 in refluxing phosphorus oxychloride. Under Vilsmeier-Haack reaction conditions, formylation of 8 occurs selectively using a POCl₃/DMF complex at position 1 to give the 4-phenylpyrrolo[1,2-a]quinoxaline-1-carbaldehyde 9 [22]. Reaction of 8 and one equivalent of N-bromosuccinimide (NBS) afforded the 1-bromo-4-phenylpyrrolo[1,2-a]quinoxaline 10 as the sole reaction product. It was then followed by the Suzuki-type cross-coupling of 10 with 4-formylphenylboronic acid in order to introduce the benzaldehyde moiety in position 1 of this 4-phenylpyrrolo[1,2-a]quinoxaline skeleton to give 11 [23,24]. Reaction of 4-(2-ketobenzimidazolin-1-yl)piperidine with 9 and 11 and using sodium cyanoborohydride as reductive agent in methanol gave the amines 1h and 1i, respectively (Scheme 2).

Scheme 2 Synthesis of the 1,3-dihydro-1-{1-[4-(4-phenylpyrrolo[1,2-a]quinoxalin-1-yl)benzyl- or -methyl]piperidin-4-yl}-2H-benzimidazol-2-ones 1h-i. Reagents and conditions: (i) C₆H₅COCl, toluene, pyridine, Δ; (ii) POCl₃, Δ; (iii) POCl₃/DMF, DMF, Δ; (iv) 4-(2-ketobenzimidazolin-1-yl)piperidine, NaBH₃CN, MeOH, Δ; (v) NBS, CH₂Cl₂, RT; (vi) OHC-C₆H₄-B(OH)₂, Pd[P(C₆H₅)₃]₄, K₂CO₃, toluene, EtOH, Δ; (vii) 4-(2-ketobenzimidazolin-1-yl)piperidine, NaBH₃CN, MeOH, Δ.

The 2-(aminomethyl)-4-phenylpyrrolo[1,2-a]quinoxalines 1j-k, structural analogues of compound 1h in position 2 of the pyrrolo[1,2-a]quinoxaline moiety, were prepared according to the sequence as shown in Scheme 3. Reaction of commercially available phenylenediamine with 1-phenylpropan-1,2-dione in acetic acid gave the methylphenylquinoxaline 12. Treatment of compound 12 with ethyl bromopyruvate in refluxing ethanol led to ethyl 4-phenylpyrrolo[1,2-a]quinoxaline-2-carboxylate 13. Reduction of the ester group of 13 with LiAlH₄ in anhydrous tetrahydrofuran gave the alcohol 14, subsequently oxidized into the attempted aldehyde 15 using MnO₂ in refluxing chloroform [22]. The aldehyde 15 was then engaged in a reductive amination with NaBH₃CN and 4-(2-ketobenzimidazolin-1-yl)piperidine to give in majority (43%) the pyrroloquinoxaline 1j by applying the same experimental procedure as described in the synthesis of compounds 1a-f and 1h-i.

Scheme 3 Synthesis of the 1,3-dihydro-1-{1-[4-(4-phenylpyrrolo[1,2-a]quinoxalin-2-yl)methyl]piperidin-4-yl}-2H-benzimidazol-2-ones 1j-k. Reagents and conditions: (i) C₆H₅COCOCH₃, AcOH, Δ; (ii) BrCH₂COCOOC₂H₅, EtOH, Δ; (iii) LiAlH₄, THF, Δ; (iv) MnO₂, CHCl₃, Δ; (v) 4-(2-ketobenzimidazolin-1-yl)piperidine, NaBH₃CN, MeOH, Δ.

During this synthesis we also isolated a second amine 1k (25%) presenting a nitrile group on the methylene function. The mechanism of formation of this new compound is probably due to the nucleophilic addition of a cyanide ion (CN⁻ ), formed by hydrolysis of the cyanoborohydride in the reactional medium [25,26], on the iminium intermediate during the reductive amination.

The 3D spatial determinations of 1j and 1k were established by X-ray crystallography [27], and confirmed the structures in the solid state as anticipated on the basis of IR, ¹H and ¹³C NMR data (Figure 3). Moreover, 1k was found as a racemic (R)/(S) mixture as highlighted by the determined spatial group (C2/c).

Figure 3 The ORTEP drawing of 1,3-dihydro-1-{1-[4-(4-phenylpyrrolo[1,2-a]quinoxalin-2-yl)methyl]piperidin-4-yl}-2H-benzimidazol-2-ones 1j and 1k with thermal ellipsoids at 30% level.

Pharmacology

Cytotoxicity

All compounds 1a-k were tested on activated (PBMNC + PHA) human peripheral blood mononuclear cells (Table I) [14]. As expected, most of the pyrrolo[1,2-a]quinoxalines 1a-k showed significant level of cytotoxicity against lymphocytes with IC₅₀ ranging from 5 to > 50 μM. These preliminary results were used to determine their respective range of toxic concentration.

Table I.  In vitro activity of compounds 1a-k on U937, K562, HL60 and MCF7 cells, and cytotoxicity on human peripheral blood mononuclear cells PBMNC + PHA.

	IC50 values (μM)^a
Compound	U937	K562	HL60	MCF7	Cytotoxicity on activated human peripheral blood mononuclear cells (PBMNC) PBMNC+PHA
A6730	8 ± 0.2	8 ± 0.3	5.5 ± 0.2	>20	n.d.^b
1a	>20	4.5 ± 0.2	14 ± 0.4	20 ± 1	10 ± 0.5
1b	>20	>20	>20	>20	43 ± 2
1c	>20	10 ± 0.3	10 ± 0.3	>20	9 *** ± 0.4
1d	>20	16 ± 0.4	>20	>20	>50
1e	>20	>20	10 ± 0.4	>20	44 ± 2
1f	>20	9 ± 0.3	>20	>20	8 ± 0.4
1g	>20	>20	>20	>20	>50
1h	5 ± 0.1	>20	20 ± 1	8 ± 0.3	9 ± 0.4
1i	>20	>20	15.5 ± 0.4	>20	>50
1j	16 ± 0.3	8 ± 0.2	14 ± 0.3	17 ± 0.4	5 ± 0.1
1k	17 ± 0.3	17 ± 0.4	>20	>20	35 ± 3

^a The IC₅₀ (μM) values correspond to the mean+/ − standard deviation from 3 independent experiments.

^b n.d. = not determined.

Antiproliferative effect

Compounds 1a-k were assessed for their ability to inhibit the in vitro proliferation of the human leukemic cell lines U937, K562 and HL60, and the breast carcinoma line MCF7. Compound A6730 (Figure 1) was used in these tests as the reference standard drug. The results are summarized in Table I. The pyrrolo[1,2-a]quinoxalines 1h, 1j and 1k were found the most antiproliferative compounds on the growth of human myeloid U937 cell line with IC₅₀ from 5 to 17 μM. In particular, 1h displayed strong cytotoxic properties on U937 cell line with an IC₅₀ of 5 μM, and showed a better activity in comparison with the reference compound A6730 (IC₅₀ = 8 μM). Interestingly, these three pyrroloquinoxalines 1h, 1j and 1k were substituted by a methylpiperidinyl benzimidazolone moiety in position 1 or 2 and by a phenyl ring in position 4 of the pyrrolo[1,2-a]quinoxaline core. Moreover, the displacement of the methylpiperidinyl benzimidazolone substitutent from the position 1 (compound 1h) to position 2 (compound 1j) induced a slight decrease in the antiproliferative activity on U937 cell line (IC₅₀ 16 μM for 1j compared with 5 μM for 1h). All other compounds 1a-f derived from the incorporation of the benzylpiperidinyl benzimidazolone moiety, which was present in the reference compounds II-V, into the 4-position of the pyrroloquinoxaline ring were found inactive at 20 μM on the U937 cell line. It was also interesting to notice the difference of antiproliferative activities between the structural analogues 1i and 1h in which the replacement of the benzylpiperidinyl benzimidazolone moiety (compound 1i) with a methylpiperidinyl benzimidazolone group (compound 1h) was beneficial for inhibitory activity (IC₅₀ = 5 μM for 1h compared with > 20 μM for 1i). From a SAR point of view, these preliminary biological results on U937 cell line enlightened the importance of the substitution at C-1 position of the pyrroloquinoxaline scaffold by a methylpiperidinyl benzimidazolone group.

The antiproliferative potencies of these new derivatives 1a-k were also examined towards the human myeloid leukaemia cell lines K562 and HL60.

Among the eleven compounds tested for antiproliferative activities on K562 cell line, pyrroloquinoxaline 1a was found the most active compound with an IC₅₀ of 4.5 μM. In the same series, the substitution of the 7- or 8-position of the pyrrolo[1,2-a]quinoxaline moiety (compounds 1b-e and 1g) seems to slightly or totally decrease the activity in comparison with their unsubstituted derivative 1a with IC₅₀ ranging from 10 to >20 μM. However, as the substitution in position 8 led to slightly active compounds 1c-d (IC₅₀ = 10 and 16 μM, respectively), the substitution at position 7 of the pyrroloquinoxaline heterocycle by one methoxy, one cyano or one tetrazole group (compounds 1b, 1e and 1g) decreased once more the antiproliferative activity upon K562 cell line with IC₅₀ superior to 20 μM. On the other hand, introduction of a chlorine atom on the 5 position of the benzimidazolone moiety reduced the activity up to two times (i.e.; IC₅₀ = 9 μM for 1f compared to 4.5 μM for 1a). Introduction of the methyl- or benzylpiperidinyl benzimidazolone group in position 1 of the pyrroloquinoxaline skeleton (compounds 1h, 1i) led to a decrease in the activity, whereas introduction of this methylpiperidinyl benzimidazolone group in position 2 (compound 1j) provided a better antiproliferative activity (IC₅₀ = 8 μM). The presence of a cyano group on the methylene in position 2 (compound 1k) also decreased the activity (IC₅₀ = 17 μM).

Against the HL60 human acute promyeloid leukemia cell line, most of the tested compounds had only weak antiproliferative activity with IC₅₀ values from 10 to 20 μM. The most active derivatives on this HL60 line were the 8-methoxy and 7-cyano pyrroloquinoxalines 1c and 1e which, with an IC₅₀ value of 10 μM, were 2-fold less potent than A6730 (IC₅₀ = 5.5 μM). The results against the HL60 cell line do not enable us to determine precisely the structure-activity relationship in this series of compounds.

Against the MCF7 breast adenocarcinoma, none of the pyrroloquinoxalines 1a-g, 1i and 1k exhibited relevant cytotoxicity (IC₅₀ ≧ 20 μM). Nevertheless, the two compounds, bearing a methylpiperidinyl benzimidazolone group in position 1 and 2 of the tricyclic structure (compounds 1h and 1j), have shown significant antiproliferative activities (IC₅₀ = 8 and 17 μM, respectively).

Conclusion

In the present report, we described the synthesis of new pyrrolo[1,2-a]quinoxaline derivatives bearing the piperidin-4-yl-2H-benzimidazol-2-one moiety via Suzuki cross-coupling and reductive amination reactions and presented their antiproliferative activities on the human leukemic cell lines U937, K562 and HL60, and the breast cancer cell line MCF7. These results have been discussed in a preliminary SAR study. The first biological evaluation of our compounds showed antiproliferative activity against U937, K562 and MCF7 cell lines. The most promising active pyrroloquinoxalines were found to be 1a that inhibited K562 cell line proliferation with an IC₅₀ of 4.5 μM, and 1h that inhibited U937 and MCF7 cell lines with IC₅₀ of 5 and 8 μM, respectively. These two candidates exhibited more potent activities than the reference inhibitor A6730. None of these new synthesized compounds 1a-k had a significant effect on the HL60 cell line proliferation, which expressed an inactive Akt form, suggesting that these compounds exhibited a specificity for phosphorylated Akt form. Moreover, it would be now interesting to enlarge the biological evaluation of these two new pyrrolo[1,2-a]quinoxaline derivatives by studying the phosphorylation level of Akt by Western-Blot using (Ser473 or Thr308) phosphoAkt antibodies, as well as their isoenzyme selectivity. Finally, the new substituted pyrrolo[1,2-a]quinoxalines could open the way to new valuable medicinal chemistry scaffolding in the oncology domain.

Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.