Preparation of 6-Substituted Quinoxaline JSP-1 Inhibitors by Microwave Accelerated Nucleophilic Substitution

A small library of 6-aminoquinoxalines has been prepared by nucleophilic substitution of 6-fluoroquinoxaline with amines and nitrogen-containing heterocycles under computer-controlled microwave irradiation. Some compounds were found to be potent inhibitors of JNK Stimulatory Phosphatase-1 (JSP-1) in an in vitro biological assay.

Through a high-throughput screening of our sample collection, an aminoquinoxaline derivative I (Figure 1) was found to show inhibitory activity towards JNK Stimulatory Phosphatase-1 (JSP-1) in an in vitro biological assay [14]. JSP-1, also known as VHX/MKPX [15] or JKAP [16], is a special member of the so-called Dual-Specificity Protein Phosphatases (DSPs) family [17], a class of protein tyrosine phosphatases (PTPs), which are a family of intracellular enzymes that dephosphorylate proteins with phosphate on serine, threonine and/or tyrosine, and play important regulative roles in cellular signal transduction. Unlike other DSPs, which directly inactivate mitogen-activated protein kinases (MAPK), JSP-1 is a selective activator of the MAPK Jun NH 2 -terminal kinase (JNK). The JNK signal transduction pathway is implicated in many pathological conditions, including cancer, diabetes and neurodegenerative diseases [18][19], therefore, JSP-1 might be a novel potential therapeutic target for treating diseases associated with dysfunctional JNK signaling. According to the literature 6-aminoquinoxalines could be prepared by condensation of corresponding ortho-phenylenediamines with 1,2-dioxo compounds [20][21] or by nucleophilic substitution of haloquinoxalines [22][23][24][25][26]. Since appropriately substituted ortho-phenylenediamines are not always readily available, the former route is not very useful for the construction of 6-aminoquinoxaline libraries and consequently, the latter one was considered a more attractive option to synthesize the target compounds. However, the nucleophilic substitution reaction, which affords 26-88% yields of the target compounds after reactions at high temperature for 3-22 hours, usually requires an electron-withdrawing substituent such as a fluorine atom or nitro group at the ortho-position of the 6-halo substituted quinoxalines [22][23][24][25]. There is one report claiming that 2,3-dimethyl-6chloroquinoxaline or methyl-6-fluoroquinoxaline reacted with methylamine in sealed tube for 16-26 hours to produce the expected product in 63-80% yield [26].
Recently, microwave-enhanced organic reactions have become quite attractive in organic synthesis [27][28][29][30][31][32][33]. The main advantages of this protocol are the potential to improve the yields and dramatically shorten the reaction times. Herein, we would like to report a microwave-assisted synthetic method to synthesize 6-aminoquinoxalines using 6-fluoroquinoxalines and amines or nitrogen-containing heterocycles in a single-mode microwave synthesizer.

Chemistry
The synthetic route used is outlined in Scheme 1. 4-Fluoro-2-nitroaniline was reduced by catalytic hydrogenation in the presence of 10% Pd/C to afford 4-fluoro-o-phenylenediamine (1) [34]. Condensation of compound 1 with 1,2-dioxo compounds gave the 6-fluoroquinoxalines 2a-d. As a model reaction to optimize the reaction conditions for preparing compounds 3-22, the synthesis of 6-(1-pyrrolidinyl)-quinoxaline (3) was examined in some detail. First, 1 equivalent of 6-fluoroquinoxaline (2a) was refluxed with 2 equivalents of pyrrolidine in the presence of 2 equivalents of potassium carbonate in dimethyl sulfoxide (DMSO). After 30 min, the resulting complex reaction mixture was checked by HPLC, which showed 21% of unreacted starting material 2a and 16% of the expected product 3 ( Table 1, entry 1). When the reflux time was prolonged to 3 hours (Table 1, entry 2), the disappearance of the starting material 2a was confirmed by HPLC, but the yield of substituted product 3 was only slightly increased to 22% and more side products were formed. To improve the nucleophilic substitution reaction, microwave irradiation was then employed, and the optimum reaction conditions for this reaction were investigated. As the results presented in Table 1 show, when a mixture of 1 equivalent of 6-fluoroquinoxaline (2a), 2 equivalents of pyrrolidine and 2 equivalents of potassium carbonate in N,N-dimethylformamide (DMF) was exposed to microwave irradiation at 120 °C for 30 min (Table 1, entry 3), the yield of substituted product 3 was not improved and considerable starting material 2a was detected. When the reaction temperature was raised to 200 °C and the solvent was changed from DMF to N-methyl pyrrolidone (NMP) or DMSO, a moderate yield improvement was observed for the reaction in NMP (Table 1, entry 4), but the reaction was complete within 30 min in DMSO to give the best yield (93%) ( Table 1, entry 5). Based on these reactions DMSO was selected as the solvent for subsequent work. The effect of the base, sodium hydroxide or 1,8-diazabicyclo [5,4,0]undec-7-ene (DBU), was also investigated, but no yield improvements were observed (Table 1, entries 6-7) and more side products were detected in both cases. When the amount of pyrrolidine used was reduceded to one equivalent, the yield also decreased ( Table  1, entry 8). Distinctly, the concentration of nucleophile played an important role in the outcome of the reactions. The yield was not improved regardless of whether the reaction temperature was raised or decreased. It was observed that the reaction was incomplete at temperatures lower than 200 °C ( Table  1, entry 9) and more by-products were formed at higher temperature ( Table 1, entry 10). Shortening the reaction time also resulted in a decrease in the yield ( Table 1, entry 11).
Following the results described above, it is suggested that the optimal reaction conditions involve a mixture of one equivalent of 6-fluoroquinoxaline (2a), two equivalents of pyrrolidine and two equivalents of potassium carbonate in DMSO exposed to microwave irradiation at 200°C for 30 min and that microwave irradiation might be a pivotal driving force to accelerate the nucleophilic substitution.
To extend the utility of the reaction, the above method was employed to synthesize other 6-aminoquinoxalines, and the reactions of substituted 6-fluoroquinoxalines and various amines were investigated ( Table 2). It could be shown that the reaction yields depended on the character of the nucleophilic reagents used. When nitrogen-containing aromatic heterocycles were employed as nucleophiles, excellent yields (88-97%) were obtained. When aliphatic amines were reacted with 6-fluoroquinoxalines, both nucleophilic and steric effects influenced the yields and reaction rates. No good reaction ocurred with primary amines, except in the case of 4-methoxybenzylamine, but when secondary amines were used, moderate to good yields were obtained. Changing from pyrrolidine to six-membered cyclic secondary amines, a decrease in yields was noted, which may be attributed to steric hindrance. Meanwhile, an effect of the substituents on the quinoxaline ring was also observed. 2,3-Difuryl substituted 6-fluoroquinoxaline reacted with cyclic secondary amines to give excellent yields of product within 30 min following the general procedure, but the reaction times had to be prolonged to complete the reaction of 2,3-dimethyl-or 2,3-di-p-tolyl-substituted 6-fluoroquinoxaline with amines.

Biological activity
All the synthesized compounds were assayed in vitro for their inhibiting activities towards JSP-1, and a number of these 2,3-diaryl substituted quinoxalines exhibited potent inhibitory activity, with compound 16 displaying the most potent activity.

Conclusions
In summary, an efficient method was developed to prepare 6-aminoquinoxalines in moderate to excellent yields within 5-60 min under microwave irradiation, whereas with standard heating this reaction could not be completed even after prolonged reaction times and a complex mixture of byproducts was formed. To extend the broad applicability of this procedure, this microwave assisted nucleophilic substitution was employed to construct a small parallel library of 6-substituted quinoxalines, which exhibited JSP-1 inhibiting activities in an in vitro enzymatic assay.

General
The reagents were purchased from Lancaster (Morecambe, England), Aldrich (St. Louis, MO, USA), Acros (Geel, Belgium) and Shanghai Chemical Reagent Company (Shanghai, China), and were used without further purification. The single-mode microwave synthesizer employed for this work was an Initiator from Biotage (Uppsala, Sweden), which is equipped with an internal probe that monitors reaction temperature and pressure, and maintains the desired temperature by computer control. Reactions were conducted in the 5 mL sealed vials. Analytical thin-layer chromatography was performed on HSGF 254 plates (150-200 µm thickness; Yantai Huiyou Company, Yantai, Shandong, China). Preparative thin-layer chromatography was carried out on HSGF 254 plates (400-500 µm thickness; Yantai Huiyou Company, Yantai, Shandong, China). Column chromatography was performed using 200-300 mesh silica gels (Qingdao Haiyang Chemical Company, Qingdao, Shandong, China). Yields were not optimized. Melting points were recorded in a capillary tube on a SGW X-4 melting point apparatus without correction. 1 H-NMR was recorded in CDCl 3 on a Varian AMX-300 (300 MHz) NMR spectrometer using tetramethylsilane as an internal standard. Chemical shifts were reported in parts per million (ppm, δ). Proton coupling patterns were described as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), and broad (br). Mass spectra (MS) were measured by the electric ionization (EI) method with a Finnigan MAT-95 instrument (Finnigan, Santa Clara, CA, USA).

General procedure for the preparation of 6-fluoroquinoxalines 2a-d: Preparation of 6-fluoroquinoxaline (2a)
4-Fluoro-2-nitroaniline (2.0 g, 0.013 mol), dissolved in ethanol (80 mL), was hydrogenated in the presence of 10% Pd/C (800 mg) under 1 atm H 2 at room temperature. When the hydrogen uptake was complete the catalyst was removed by filtration and a mixture of the filtrate and 40% glyoxal in water (1.5 mL, 0.013 mol) was heated at reflux for 24 hours. The solvent was evaporated under vacuum to give the crude product, which was further purified by flash column chromatography to give the desired compound 2a Pyrrolidine (112 µL, 97 mg, 1.36 mmol) was added in a 5 mL microwave vial containing 6-fluoroquinoxaline (100 mg, 0.68 mmol) and K 2 CO 3 (188 mg, 1.36 mmol) in DMSO (1.5 mL). The vial was sealed by a crimped cap and was placed in a Biotage microwave apparatus. The vessel was heated for 30 min at 200 o C. After completion of the reaction, the reaction mixture was cooled and the mixture was poured into ice-water (50 mL). The mixture was extracted three times with dichloromethane and the combined organic layers were washed with brine, dried over anhydrous sodium sulfate and filtered. The solvent was evaporated under vacuum, and the resulting crude product was purified by preparative thin-layer chromatography to give 125 mg of the title compound 3 as a yellow solid, yield 93%; mp: 77-79°C; 1 (16)

JSP-1 Inhibition Activities Assay
The JSP-1 activity was determined at room temperature by monitoring the hydrolysis of 3-omethylfluorescein phosphate (OMFP). In a typical assay a mixture (100 µL) containing 50 mM Bis·Tris, pH 6.5, 1 mM EDTA, 1 mM DTT, 10 µM OMFP and 30 nM purified recombinant GSTfusion human JSP-1 was used. The enzyme activity was continuously monitored with an excitation 485 nm/emission 535 nm filter set for 3 min and the initial rate of the hydrolysis was determined using the linear region of the enzymatic reaction kinetic curve. IC 50 values were calculated from the nonlinear curve fitting of percent inhibition (% inhibition) vs. inhibitor concentration [I] by using the equation: % Inhibition = 100/{1+(IC 50 /[I])k}, where k is the Hill coefficient.