Multi-gram Preparation of 7-Nitroquinoxalin-2-amine

Methodologies to obtain quinoxaline compounds regioselectively are rarely reported in literature, thus regioselective and multi-gram methodologies to obtain these derivatives are desirable to explore the entire potential of these scaffolds for academic and/or commercial application. A facile and multi-gram methodology is described to obtain compound 7-nitroquinoxalin-2-amine using o-phenylenediamine, a cheap and readily available reactant, as starting material in a five-step procedure in good yields and high purity without further purification such as crystallization or column chromatography.


Introduction
Quinoxalines or benzopyrazines are heterocyclic compounds that present designed properties for commercial and/or academic applications as dyes, drugs or pharmacological tools. 1,2Methodologies to obtain quinoxaline compounds regioselectively are rarely reported in literature, thus regioselective and multi-gram methodologies to obtain those derivatives are desirable to explore the entire potential of these scaffolds.
It is worth mentioning that 7-nitroquinoxalin-2-amine is an interesting functionalized scaffold, which can easily be a substrate for chemoselective functional group interconversion, aiming the further synthesis of bioactive compounds, containing pharmacophore groups linked to the nitrogen-substituents of positions 2 and 7 of quinoxaline nucleus.The N- (2-(2-phenylureido) quinoxalin-7-yl) acrylamides, designed in order to contain the structural requirements to inhibit epidermal growth factor receptors, are examples of structural pattern that can be obtained from 7-nitroquinoxalin-2-amine scaffold.
So far, synthesis of 7-nitroquinoxalin-2-amine was only described once in the literature by Wolf et al. 3 in 1949.Wolf's methodology uses 4-nitro-o-phenylenediamine as starting material and it is not regioselective.Separation of regioisomers was done by crystallization from the mixture of aryl chlorides using ligroin and benzene as solvents.Regioisomers are described regarding their melting point (mp) as A (higher melting point) and B (lower melting point).Besides, characterization of regioisomers 6-nitroquinoxalin-2-amine and 7-nitroquinoxalin-2-amine was not done, as scales and yields were also not described.

Results and Discussion
Our procedure (Scheme 1) uses o-phenylenediamine as starting material to prepare initially quinoxalin-2-ol (2) in good yields and different scales (0.3-10.0 g) as described by Kobayashi et al. 4 Next, 7-nitroquinoxalin-2-ol (3) was regioselectively obtained by a methodology using fuming nitric acid and glacial acetic acid at room temperature as described by Deng et al. 5 This step is scale sensitive, and attempts to increase or decrease scale (0.5 or 4.0 g) were unsuccessful.Nitration of quinoxaline-2-ol scaffold was confirmed by DEPT-135 (distortionless enhancement by polarization) spectra and melting point.Chemical shifts from 1 H and 13 C nuclear magnetic resonance (NMR) spectra of the obtained isomer are in accordance with values previously described for the 7-nitroquinoxalin-2-ol. 5 NOE (nuclear Overhauser effect) difference experiment also confirmed obtainment of regioisomer 3.By using phosphoryl chloride under refluxing conditions, we prepared the corresponding aryl chloride 2-chloro-7-nitroquinoxaline (4) in good yields by filtration after slowly addition of the reaction mixture into a mixture of ice and water. 6lthough the amination of 2-chloroquinoxaline is described as easily done, we could not reproduce chlorine substitution by an amino group at 7-nitroquinoxaline scaffold as previously described in literature. 3,7Ammonia in methanolic solution at high temperatures, through nucleophilic aromatic substitution in similar procedure as described by Wolf et al. 3 and by Gowenlock et al. 7 did not yield the desired product.Different methodologies were employed to prepare amidine compound such as: ammonium hydroxide with copper iodide under microwave irradiation, urotropine in ethanol under reflux, and Koródi reaction. 8However, none of these methodologies were effective.
Therefore, use of benzylamines was proposed to act as amino group carrier and then, in a second step, after a deprotection reaction, we could obtain the final compound. 9onsidering the nitro group and atom economy green chemistry principle, p-methoxybenzylamine (PMBAM) was chosen as amino carrier group, once benzylamine moiety removal by hydrogenation with paladium over carbon would lack chemioselectivity, also reducing the nitro group, and considering that 3,5-dimethoxybenzylamine has more methoxy groups in its structure than PMBAM.
Firstly, compound 5 was obtained by a methodology using DMF (dimethylformamide) as solvent. 10This methodology uses higher amounts of amine and the removal of DMF was a time demanding issue.To circumvent these issues, a more environment friendly methodology was employed using 1.05 equivalent of amine and 3 equivalent of triethylamine in ethanol under refluxing conditions, 11 see Table 1.Corresponding p-methoxybenzylamine derivative (5) was obtained after solvent removal and water washing by filtration in good yields (> 90%).

Conclusions
In summary, we described a methodology that is affordable for academic and/or commercial purposes by a five-step synthetic pathway through common synthetic procedures.Intermediates and the final product were obtained by filtration from water and needed no further purification procedures to yield the compounds in multigram scales (up to 10 g of starting material).Chemical characterization ( 1 H and 13 C NMR, ATR/FTIR (attenuated total reflection/Fourier Transform infrared secptroscopy) and MS (mass spectrometry)) and regioselective preparation of 7-nitroquinoxalin-2-amine are herein described for the first time.

Experimental
All commercially available reagents and solvents were used without further purification. 1H and 13 C NMR and DEPT-135 spectra were determined in DMSO-d 6 or D 2 O solutions using a Bruker AC-200 or a Bruker Avance 400 spectrometer.The chemical shifts are given in parts per million (ppm) from solvent residual peaks and the coupling constant values (J) are given in Hz.Signal multiplicities are represented by: s (singlet), d (doublet), dd (double doublet), t (triplet), m (multiplet) and br (broad signal).FTIR spectra were obtained using a Thermo Scientific Nicolet's Avatar iS10 spectrometer equipped with smart endurance diamond ATR unit for direct measurements.Mass spectra were obtained from a TLC-MS (thin layer chromatography) interface CAMAG in negative mode and from a Hewlette Packard HP 5973 mass selective detector (70 eV).Melting points were determined using a MP70 Mettler Toledo and are uncorrected.The purity of compounds was determined by high-performance liquid chromatography (HPLC, Merck Hitachi L-6200 intelligent pump, Merck Hitachi AS-2000 auto sampler, Merck Hitachi L-4250 UV-Vis detector) using a Zorbax ® Eclipse XDB C8 column (5 mm), employing a gradient of 0.01 M KH 2 PO 4 (pH 2.3) and methanol as solvent system with a flow rate of 1.5 mL min -1 and detection at 230 and 254 nm.