Synthesis of 2-amino-1-methyl-6-phenylimidazo[4,5 -b ]pyridine (PhIP), a heterocyclic food mutagen

The synthesis of the heterocyclic food mutagen, 2-amino-1-methyl-6-phenylimidazo[4,5 - b ]pyridine (PhIP) 7 , is reported. PhIP 7 was synthesized in six steps from commercially available 2,3-diaminopyridine 1 in 6% overall yield. This route features a new ring closure reagent, N - dichloromethylene-p -toluenesulfonamide, to assemble the imidazo[4,5 -b ]pyridine ring system and uses a Suzuki coupling to introduce an aryl ring onto the heterocyclic core as the last synthetic step.


Introduction
Diet is one of the most significant environmental causes of cancer in humans. 1 A series of mutagenic and carcinogenic heterocyclic amines has been identified from cooked meat and fish. 2 These heterocyclic amines are believed to arise from the condensation amino acids, glucose and creatinine via the Maillard reaction during cooking at high temperatures. 3The most mass abundant of the heterocyclic amines is 2-amino-1-methyl-6-phenylimidazo [4,5-b]pyridine (PhIP) 7. 1 PhIP 7 forms covalent DNA adducts resulting in genetic mutations in animal and human models. 45][6] Chemical standards of these mutagens need to be made for biological assays to assess the risk associated with their consumption.In our continued effort to develop new methods for the syntheses of biologically important mutagens and carcinogens, we now report a new method for the synthesis of 7.
We were interested in developing a expedient and flexible synthesis of 7 that would be also amenable to making isotope-labeled and metabolites of 7.This synthesis features new ringclosure reagent, N-dichloromethylene-p-toluenesulfonamide, 7 that reacts with an aromatic vicinal diamine 3 that contains both primary and secondary amine groups to give the tosyl-protected Nalkyl imidazo [4,5-b]pyridine 4.This ring-closure reagent is more reactive than the analogous N-(bis-methylthio)methylene-p-toluenesulfonamide that reacts most readily with primary aliphatic diamines. 8The N-tosyl cyclic amine 4 can be deprotected in high yield by anhydrous HF 8 to give the 2-amino-1-methyl-imidazo [4,5-b]pyridine heterocyclic core 5.
Previous published routes for synthesis of 7 [9][10][11][12] were not amenable to facile derivatization of the aryl ring that will be important to make isotope-labeled 13 and metabolites of 7. The Suzuki coupling 14 stood out as an ideal method to introduce the aryl ring onto a brominated heterocycle in the last step of this synthesis.This flexible coupling reaction allows for the synthesis of 7 and a potentially large variety of aryl-substituted PhIP derivatives from commercially available arylboronic acids.

2-Amino-3-methylaminopyridine (3).
A dry 250-mL round bottom flask was charged with 50 mL anhydrous THF and N 3 -benzoyloxycarbonyl-2,3-diaminopyridine (4.81 g, 20 mmol).At 0 °C, 50 mL of 1 M LiAlH 4 in THF was added to the flask over 20 minutes.The reaction mixture was heated to reflux (65 °C) for 4 h.At 0 °C, the reaction was quenched with 10 mL deionized water followed by 100 mL 3 M HCl.The aqueous layer was extracted with 4 x 50 mL portions of diethyl ether.At 0 °C, the aqueous layer was made basic (pH = 12) with solid KOH.
The aqueous layer was extracted with 4 x 50 mL portions of THF/diethyl ether.The organic layer was dried over anhydrous MgSO 4 , filtered and the solvent was removed under vacuum (25 °C, 0.1 torr) to give 3.60 g (80%) of a brown solid; mp = 115-118 °C (lit. 13