1-Phenyl-8-[[4-(pyrrolo[1,2-a]quinoxalin-4-yl)phenyl]methyl]-1,3,8-triazaspiro[4.5]decan-4-one: Synthesis, Crystal Structure and Anti-Leukemic Activity

1-Phenyl-8-[[4-(pyrrolo[1,2-a]quinoxalin-4-yl)phenyl]methyl]-1,3,8-triazaspiro[4.5]decan-4-one has been successfully synthesized via a multi-step pathway starting from 2-nitroaniline. Structure characterization of this original pyrrolo[1,2-a]quinoxaline derivative was achieved by FT-IR, 1H-NMR, 13C-NMR, X-Ray and HRMS spectral analysis. This title compound shows interesting cytotoxic potential against several human leukemia cell lines (K562, HL60, and U937 cells).


Cytotoxic Activity
The cytotoxic activity of the 1-phenyl was evaluated against K562, U937, and HL60 cell lines with the MTS assay using compounds A6730 and JG454 as the reference standard drugs [14,15].
As listed in Table 1, the IC 50 values of [4.5]decan-4-one 7 was found in the same range as those observed for the reference drug A6730. Firstly, the antiproliferative potencies of this new derivative 7 was examined towards the human myeloid leukemia cell lines K562 and HL60. Against the human K562 chronic myeloid leukemia cell line, this substituted pyrroloquinoxaline 7 showed significant antiproliferative activity with an IC 50 of 3.5 µM, similar to that of our compound JG454 (IC 50 = 4.5 µM), but better than that of the reference compound A6730 (IC 50 = 17 µM). On the contrary, against the HL60 human acute myeloblastic leukemia cell line, our tested derivative was found less active than the reference quinoxaline A6730; i.e., IC 50 = 15 µM for 7 versus 5.5 µM for A6730. This activity for derivative 7 against HL60 was also found to be in the same order as the one observed for JG454 (IC 50 = 14 µM). Nevertheless, our pyrrolo[1,2-a]quinoxaline 7 was found to be inactive against the human myeloblastic U937 cell line (IC 50 >20 µM), whereas A6730 showed an antiproliferative activity of 8.0 µM against this leukemia cell line. Compound 7 was also tested on normal human peripheral blood mononuclear cells activated with phytohemagglutinin (PBMNC + PHA) to evaluate its cytotoxicity on normal cells (Table 1). Pyrroloquinoxaline 7 demonstrated lower level of cytotoxicity against T-lymphocytes with an IC 50 over 35.0 µM. This preliminary result was used to determine its range of toxic concentration. Index of selectivity (IS) was defined as the ratio of the IC 50 value on T-lymphocytes to the IC 50 value on the various leukemia cell lines. Compounds that demonstrated high selectivity (high index of selectivity) should offer a potential of safer therapy. In our case, we could note that our compound 7 showed an interesting selectivity towards K562 cell line (SI = 10).

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 an 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 have been 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 have been used for resonance assignments. Analytical TLC were carried out on 0.25 precoated silica gel plates (POLYGRAM SIL G/UV254) and 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].

X-ray Data
The structure of compound 7 was established by X-ray crystallography (Figure 1). The colorless single crystal of 7 was obtained by slow evaporation from a methanol/chloroform solution (v/v: 20/80): triclinic, space group P-1, a = 6.6189 (2) [20]. The data were corrected for Lorentz and polarization effects and for empirical absorption correction [21]. The structure was solved by direct methods Shelx 2013 [22] and refined using Shelx 2013 [22] suite of programs.

Cytotoxic Activity
The MTS proliferation tests on the human leukemic cell lines U937, K562, and HL60 were performed as previously described by our team [13,14,23].