Microwave-Assisted Reaction of 2 , 3-Dichloronaphthoquinone with Aminopyridines

The aminonaphthoquinone scaffold is part of the structure of numerous natural and synthetic products associated with cytotoxic, antibacterial, antimalarial, and antifungal activities. Furthermore, many heterocyclic aminoquinones have been reported to show antitumor activity. A classical route to obtain 2-amino-1,4-naphthoquinones is by reaction of 1,4-naphthoquinones with amines followed by air oxidation or by nucleophilic addition-elimination on 2-halo-1,4-naphthoquinones. However, the generality of the methods is limited and most of them require long reaction times. In our continuous effort towards the synthesis of heterocyclic quinones with useful biological activities, we were interested in the preparation of 2-pyridylamino3-chloro-1,4-naphthoquinones because their potential as antitumor agents was unexplored. Calandra and Adams, and Truit and co-workers. have reported that reaction of 2,3-dichloro-1,4-naphthoquinone (1) with 2-aminopyridine (2) under reflux of ethanol gives 2-(2-pyridylamino)-3-chloro1,4-naphthoquinone (3), which affords 1,4-naphthoquinone derivative 4 upon treatment with acetic acid. However, Mosby and Boyle found that the reaction of 1 and 2 under reflux of ethanol in the presence of sodium bicarbonate leads to 1,2-naphthoquinone derivative 5 (46%) and not to 2-pyridylamino-1,4-naphthoquinone 3 neither p-quinone 4 (Scheme 1). Considering the importance of the pyridylamino1,4-naphthoquinone moiety as pharmacophoric scaffold, we became interested in the application of microwave irradiation to study the reaction of 2,3-dichloro-1,4naphthoquinone (1) with different aminopyridines. It is noteworthy that microwave techniques have been demonstrated as a valuable tool in accelerating the rate and yield of organic reactions in general.


Introduction
The aminonaphthoquinone scaffold is part of the structure of numerous natural and synthetic products associated with cytotoxic, antibacterial, antimalarial, and antifungal activities. 1 Furthermore, many heterocyclic aminoquinones have been reported to show antitumor activity. 2 classical route to obtain 2-amino-1,4-naphthoquinones is by reaction of 1,4-naphthoquinones with amines followed by air oxidation or by nucleophilic addition-elimination on 2-halo-1,4-naphthoquinones. 3,4However, the generality of the methods is limited and most of them require long reaction times.In our continuous effort towards the synthesis of heterocyclic quinones with useful biological activities, 5 we were interested in the preparation of 2-pyridylamino-3-chloro-1,4-naphthoquinones because their potential as antitumor agents was unexplored.Calandra and Adams, 6 and Truit and co-workers. 7have reported that reaction of 2,3-dichloro-1,4-naphthoquinone (1) with 2-aminopyridine (2) under reflux of ethanol gives 2-(2-pyridylamino)-3-chloro-1,4-naphthoquinone (3), which affords 1,4-naphthoquinone derivative 4 upon treatment with acetic acid.However, Mosby and Boyle found that the reaction of 1 and 2 under reflux of ethanol in the presence of sodium bicarbonate leads to 1,2-naphthoquinone derivative 5 (46%) and not to 2-pyridylamino-1,4-naphthoquinone 3 neither p-quinone 4 (Scheme 1). 8onsidering the importance of the pyridylamino-1,4-naphthoquinone moiety as pharmacophoric scaffold, we became interested in the application of microwave irradiation to study the reaction of 2,3-dichloro-1,4naphthoquinone (1) with different aminopyridines.It is noteworthy that microwave techniques have been demonstrated as a valuable tool in accelerating the rate and yield of organic reactions in general. 9

Results and Discussion
We first performed the reaction of dichloronaphthoquinone 1 with 2-aminopyridine (2) employing conventional heating.Thus, treatment of quinone 1 with one equivalent of 2 in refluxing ethanol for 15 hours gave o-quinone 5 in 45% yield.The melting point and 1 HNMR spectra of compound 5 were in agreement with literature values, 10 and thus Mosby and Boyle results were confirmed. 8The microwave-assisted condensation reaction of 1 and 2 in ethanol, afforded o-quinone 5 in 57% yield on irradiation in a 10 mL microwave vial at 90 ºC for 60 minutes (Table 1, entry 1).Therefore, a dramatic reduction of reaction time as well as a yield increase was obtained using microwave irradiation.
The synthesis of pyridinylaminonaphthoquinone 11, as a model system, was studied as well.Amination reaction of 2-bromo-1,4-naphthoquinone (10) with 2-aminopyridine (2) under refluxing ethanol was unsuccessful, and starting materials were recovered.After this result, we decided to study this reaction using a palladium catalyst. 11It was found that Pd 2 (dba) 3 catalyst with rac-BINAP as ligand and Cs 2 CO 3 as base is a better choice for the coupling reaction between bromonaphthoquinone 10 and 2-aminopyridine (2).This coupling reaction was performed at 150 ºC in toluene for 12 hours, to give 35% yield of compound 11, but under microwave irradiation the reaction time was 15 minutes and the yield was improved to 61% (Table 1, entry 4).
In the present study the in vitro cytotoxicity of the synthesized naphthoquinone derivatives against MCF-7 breast cancer cell line was evaluated using the Sulforhodamine B (Sigma) assay described by Skehan et al. 12 The IC 50 values for compounds 5, 8, 9, 11, and daunorubicin as the reference drug, are shown in Table 2. On the basis of these IC 50 values, compound 5 was the most active although less potent than daunorubicin.The results are consistent with the concept that a planar conformation for a tricyclic structural pattern is a requirement for antitumor activity. 13heme 1.Previous thermal studies on the reaction of 1 with 2. In conclusion, we have shown that use of microwave irradiation diminished the reaction times and improved the yields substantially in all these reactions (Table 1).The pyridoaminonaphthoquinone derivatives prepared in this study can be a good starting point for further chemical modification in the search for novel anticancer drugs.

Experimental
Reactions were carried out with a single mode cavity Discover Microwave apparatus.Microwave experiments were performed in a 10 mL sealed tube.Melting points were determined with a Meltemp apparatus and are uncorrected.IR spectra were obtained on a Bruker Model Vector 22 spectrophotometer. 1 H NMR spectra were recorded on a Bruker ACP-200 and AM-400 instruments.Chemical shifts are expressed in parts per million downfield to tetramethylsilane (TMS, d scale) and coupling constants (J) are reported in hertz. 13C NMR spectra were recorded in CDCl 3 at 50 and 100 MHz.Column chromatography was performed on silica gel Merck 60 (70-230 mesh).Elemental analyses were performed on a Fison EA 1108 CHNS-O analyzer.Accurate MS measurements were determined using a Thermo Finnigan MAT 95XP spectrometer at the Facultad Ciencias Químicas y Farmacéuticas, Universidad de Chile.

Conventional heating
A solution of dichloronaphthoquinone 1 (2.0 g, 8.8 mmol) and the appropriate aminopyridine (0.94 g, 10 mmol) in ethanol (5 mL) was refluxed under nitrogen for 15 h.The solution was concentrated under reduced pressure and the residue partitioned between water (20 mL) and ethyl acetate (20 mL).The aqueous phase was extracted with ethyl acetate (2×20 mL) and the combined organic phases dried over MgSO 4 and the solvent evaporated.The residue was purified by column chromatography (AcOEt/CHCl 3 ; 2:8).