Synthesis, Crystal Structure and Anti-Leukemic Activity of (E)-Pyrrolo[1,2-a]quinoxalin-4-yl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one

Abstract

(E)-Pyrrolo[1,2-a]quinoxalin-4-yl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one was designed then synthesized using a multi-step pathway starting from commercially available 2-nitroaniline. Structure characterization of this original substituted pyrrolo[1,2-a]quinoxaline compound was achieved by using FT-IR, ¹H-NMR, ¹³C-NMR, X-Ray and HRMS spectral analysis. This new pyrroloquinoxaline shows interesting cytotoxic potential against different human leukemia cell lines (MV4-11, K562, MOLM14 and Jurkat cells).

1. Introduction

Among heterocyclic nitrogen derivatives that have attracted attention due to their broad profile of biological activities, the pyrrolo[1,2-a]quinoxaline moiety has shown wide interest for its various biological properties. [1,2]. Thus, many pyrrolo[1,2-a]quinoxaline compounds have been designed, synthesized and described as antipsychotic agents [3], antiviral agents [4], adenosine receptor modulators [5], antituberculosis agents [6,7], antiparasitic [8,9,10,11,12,13,14,15] and anticancer compounds [16,17,18,19,20,21,22]. However, despite the recent and promising development of targeted therapies, there is still a need to discover new cytotoxic molecules for the treatment of human cancers.

We previously published a series of new pyrrolo[1,2-a]quinoxaline derivatives endowed with promising pharmacological activity towards human leukemia cells [16,17,18,19,20,21]. In this context, and as an extension of our research work on the development and assessment of new antiproliferative heterocyclic derivatives, we decided to further modulate and substitute our heterocyclic pyrrolo[1,2-a]quinoxaline pharmacophore. DYT-1, a substituted quinoline, was recently screened from a chemical library and identified as a hit compound active against several leukemia [23,24]. Consequently, taking into account the experience of our lab in the domain of the synthesis of new bioactive nitrogen heterocyclic compounds based on this pyrrolo[1,2-a]quinoxaline heterocyclic scaffold, we report herein the design, synthesis and structural identification of (E)-pyrrolo[1,2-a]quinoxalin-4-yl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (JG1679) that could be considered a new structural bioisostere analogue of the previously described bioactive quinoline DYT-1 compound (Figure 1). This novel substituted pyrrolo[1,2-a]quinoxaline derivative JG1679 is then tested against four leukemia cell lines, namely, MV4-11, K562, Jurkat and MOLM14.

2. Results and Discussion

2.1. Synthesis of (E)-Pyrrolo[1,2-a]quinoxalin-4-yl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (JG1679)

The preparation of (E)-pyrrolo[1,2-a]quinoxalin-4-yl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (JG1679) has been accomplished in six steps starting from the commercially available 2-nitroaniline 1 according to the sequence depicted in Scheme 1. The Clauson-Kaas reaction of commercially available 2-nitroaniline 1 with 2,5-dimethoxytetrahydrofuran (DMTHF) in refluxing acetic acid under microwave conditions gave phenylpyrrole 2, which was then reduced using a NaBH₄-CuSO₄ treatment to provide 1-(2-aminophenyl)pyrrole 3. The reaction of acetyl chloride with 3 led to acetamide 4. 4-methylpyrrolo[1,2-a]quinoxaline 5 was prepared via the cyclisation of this amide 4 in refluxing phosphorus oxychloride according to the Bischler–Napieralski reaction. Then, we synthesized pyrrolo[1,2-a]quinoxaline-4-carboxaldehyde 6 via the oxidation of 5 with SeO₂ in refluxing dioxane. Finally, aldehyde 6 reacted with 3′,4′,5′-trimethoxyacetophenone under the presence of an aqueous NaOH solution, resulting in the synthesis of compound JG1679 as a single E isomer [23]. Thus, a coupling constant value of 15.15 Hz was in favor of a trans configuration in the double bond. The structure of this original synthesized pyrroloquinoxaline derivative JG1679 was then corroborated by FTIR, ¹H/¹³C-NMR, X-ray and ESI-MS experiments (see Supplementary Materials, Figures S1–S4). The 3D structural determination of this new substituted pyrrolo[1,2-a]quinoxaline JG1679 was established by X-ray crystallography (Figure 2) [25], and the confirmation of the structure being in the solid state was anticipated on the basis of NMR analysis.

2.2. Cytotoxic Activity

The cytotoxic activity of this new (E)-pyrrolo[1,2-a]quinoxalin-4-yl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one JG1679 was evaluated against the following human leukemia K562, MV4-11, Jurkat and MOLM14 cell lines with the MTS assay [16,17] (

Table 1. In vitro activity of compounds JG1679 and DYT-1 on K562, MV4-11, Jurkat, MOLM14 and HEK293 cells.

Compound	IC50 Values (μM) (a)
	K562	MV4-11	Jurkat	MOLM14	HEK293
JG1679	>50	1.7 ± 0.3	3.0 ± 0.5	>2	31.2 ± 0.3
DYT-1	6.2 ± 0.5 [23,24]	n.d. (b)	7.9 ± 0.4 [23,24]	n.d. (b)	n.d. (b)

^a The IC₅₀ (µM) values correspond to the mean ± standard deviation from 3 independent experiments. ^b n.d.: not done.

). The IC₅₀ values of quinoline DYT-1, used as reference a drug, are reported in Table 1 as previously described [23,24]. Against the human myeloid leukemia cell line K562, our new pyrroloquinoxaline derivative JG1679 displayed none antiproliferative activity (IC₅₀ > 50 μM), while this new compound inhibited the growth of MV4-11 human myeloid leukemia cells with an IC₅₀ value of 1.7 µM. Moreover, the IC₅₀ of JG1679 was noticed to be superior to 2 μM against the human MOLM14 acute myeloid leukemia cell line. Against the T-acute lymphoblastic leukemia Jurkat cell line, the biological result of compound JG1679 exhibited potent cytotoxicity with an IC₅₀ value of 3.0 μM.

In addition, (E)-pyrrolo[1,2-a]quinoxalin-4-yl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one JG1679 was also tested against human epithelial cell line HEK293 to evaluate its cytotoxicity on normal cells. Thus, compound JG1679 showed a low cytotoxicity against this HEK293 cell line (IC₅₀ = 31.2 μM). Index of selectivity (IS) was defined as the ratio of the IC₅₀ value on normal cell line HEK293 to the IC₅₀ value on the different leukemia cell lines. Derivative JG1679 showed a promising selectivity towards the MV4-11 cell line (IS = 18.35) and also towards the lymphoblastic leukemia Jurkat cell line with a calculated index of selectivity of 10.4.

3. Materials and Methods

Commercial reagents were used as received without additional purification. Melting points were determined with an SM-LUX-POL Leitz hot-stage microscope (Leitz GMBH, Midland, ON, USA) and are uncorrected. IR spectra were recorded on a NICOLET 380FT-IR spectrophotometer (Thermo Electron Scientific Instruments LLC, Madison, WI, USA). NMR spectra were recorded with tetramethylsilane as an internal standard using a BRUKER AVANCE 300 spectrometer (Bruker BioSpin, Wissembourg, France). Splitting patterns were reported as follows: s = singlet; bs = broad singlet; d = doublet; t = triplet; q = quartet; dd = double doublet; ddd = double double doublet; dt = double triplet; m = multiplet. 2D-NMR experiments were used for resonance assignments. Analytical TLC was carried out on 0.25 precoated silica gel plates (POLYGRAM SIL G/UV254) and the visualization of compounds after UV light irradiation. Silica gel 60 (70–230 mesh) was used for column chromatography. High-resolution mass spectra (electrospray in positive mode, ESI⁺) were recorded on a Waters Q-TOF Ultima apparatus (Bruker Daltonics, Bremen, Germany) [13,14].

3.1. (E)-Pyrrolo[1,2-a]quinoxalin-4-yl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one (JG1679)

To a solution of pyrrolo[1,2-a]quinoxaline-4-carboxaldehyde 5 (1.7 mmol) and 3′,4′,5′-trimethoxyacetophenone (1.7 mmol) in ethanol (7 mL) at 0 °C, a 40% aqueous solution of NaOH (0.8 mL) was added dropwise and stirred at this temperature for 30 min. The reaction mixture was then stirred at room temperature for 4 h, the formed solid was filtered, washed with water and then ethanol and dried to give the crude product. Column chromatography of this precipitate on silica gel using ethyl acetate-cyclohexane (3/7) as an eluent gave the pure product JG1679 (27%). Yellow crystals, m.p. 196–198 °C; IR (KBr) 1656 (CO), 1607 (C=N). ¹H-NMR (δ, ppm, CDCl₃, 300 MHz): 8.38 (d, 1H, J = 15.15 Hz, HC=), 8.18 (d, 1H, J = 15.15 Hz, =CH), 8.07 (dd, 1H, J = 8.05 and 1.30 Hz, H-9′), 8.03 (dd, 1H, J = 2.80 and 1.10 Hz, H-1′), 7.92 (dd, 1H, J = 8.05 and 1.30 Hz, H-6′), 7.59 (ddd, 1H, J = 8.05, 7.60 and 1.30 Hz, H-8′), 7.47 (ddd, 1H, J = 8.05, 7.60 and 1.30 Hz, H-7′), 7.44 (s, 2H, 2 CH_phenyl), 7.20 (dd, 1H, J = 4.05 and 1.10 Hz, H-3′), 6.99 (dd, 1H, J = 4.05 and 2.80 Hz, H-2′), 4.00 (s, 6H, 2OCH₃), 3.98 (s, 3H, OCH₃). ¹³C-NMR (δ, ppm, CDCl₃, 75 MHz): 188.7 (C=O), 153.2 (C-3_phenyl and C-5_phenyl), 147.8 (C-4′), 143.1 (C-4_phenyl), 137.9 (HC=), 135.9 (C-5a), 133.0 (C-7′), 130.4 (C-8′), 128.3 (C-6′), 128.0 (C-9′), 127.6 (C-3a), 126.4 (C-1_phenyl), 125.5 (C-9a), 114.8 (=CH), 114.3 (C-1′), 113.8 (C-2′), 106.5 (C-2_phenyl and C-6_phenyl), 106.2 (C-3′), 61.0 (OCH₃), 56.5 (2OCH₃). HR-MS m/z [M+H]⁺ Calcd for C₂₃H₂₁N₂O₄: 389.1501, Found: 389.1498.

3.2. X-ray Data

The structure of compound JG1679 was established using X-ray crystallography (Figure 2). The yellow single crystal of JG1679 was obtained by slow evaporation from a methanol/chloroform solution (v/v: 15/85): monoclinic, space group C c, a = 30.4488(6) Å, b = 4.8817(1) Å, c = 14.3011(3) Å, α = 90°, β = 109.613(1)°, γ = 90°, V = 2002.41(7)ų, Z = 4, δ(calcd) = 1.408 Mg.m⁻³, FW = 424.44 for C₂₃H₂₀N₂O₄.2H₂O, F(000) = 896.0. Full crystallographic results were deposited at the Cambridge Crystallographic Data Centre (CCDC-2237246), UK, as shown in the Supplementary X-ray Crystallographic Data [25]. The data were corrected for Lorentz and polarization effects and for empirical absorption correction [26]. The structure was solved by direct methods Shelx 2013 [26] and refined using Shelx 2013 [27] suite of programs.

3.3. Cell Culture

All leukemic cell lines (MV4-11, Jurkat, MOLM-14, K562) were cultured in Roswell Park Memorial Institute (RPMI) 1640 1X Glutamax (Gibco™, Thermo Fisher Scientific, Villebon-sur-yvette, France), supplemented with 10% fetal calf serum (FCS), 50 U/mL penicillin and 50 µg/mL streptomycin (Gibco™, Thermo Fisher Scientific, Villebon-sur-yvette, France). Cells were cultured with vehicle or the different compounds dissolved in dimethyl sulfoxide (0.1%) at 37 °C in a fully humidified atmosphere (37 °C, 5% CO₂) in a CO₂-regulated incubator (Jouan, Saint-Nazaire, France).

3.4. Cytotoxic Activity

Cell proliferation assays were performed using MTS tetrazolium (Cell Titer96 Aqueous; Promega, Charbonnières-les-Bains, France) on the human leukemic cell lines K562, MV4-11, Jurkat and MOLM14 as previously described by our team [16,17,21]. Cells (10⁵) were plated in quadruplicate into microtiter-plate wells in 100 µL of culture medium with various doses of compounds (1; 5; 10; 20; 50 µM). After 3 h of incubation of 20 µL of MTS, plates were read in a microplate autoreader (Imark Biorad, Marnes-la-Coquette, France) at a wavelength of 490 nm. The MTT proliferation test on the epithelial cell line HEK293 was performed as previously described [28]. The 50% inhibiting concentrations (IC₅₀) were determined by linear regression analysis.

4. Conclusions

By taking into account our previous research using the valuable and biological active pyrrolo[1,2-a]quinoxaline scaffold, we synthesized here a new (E)-pyrrolo[1,2-a]quinoxalin-4-yl)-1-(3,4,5-trimethoxyphenyl)prop-2-en-1-one JG1679 and then evaluated its antileukemic activity on different human leukemic cell lines, such as MV4-11, K562, Jurkat and MOLM14. This novel pyrrolo[1,2-a]quinoxaline JG1679 exhibited promising antileukemia properties on the MV4-11 and Jurkat human leukemia cell lines, meaning it could become an interesting candidate for further pharmacomodulations and biological investigations.