Hydrogen-bonded cocrystals of the drug metformin: From molecular interactions analysis to supramolecular synthesis and characterization of Met-bis(DCA), a pharmaceutical cocrystal with enhanced anti-leukemic activity

N,N-dimethylbiguanide, metformin (Met) in the medical literature, is a widely used active pharmaceutical ingredient (API) of the biguanide class. Besides being the drug of first choice for oral therapy of type 2 diabetes, its anticancer activity is the subject of active research. Met can exist in three resonance-stabilized forms, i.e. as neutral molecule (Met), monoprotonated (MetH+) or diprotonated (MetH22+) cation, with dissociation constants in water typical of biguanides: pKa1~12.40; pKa2=2.96. We have investigated the crystal chemistry of Met in series of pharmaceutical cocrystals (PCC) prepared using various acids as coformers (CF), with particular focus on Generally Recognized as Safe (GRAS) compounds, nutraceuticals and APIs. By cocrystallization under different conditions we obtained 33 PCCs of a quality suitable for structural determination, 27 containing MetH+ and 6 MetH22+. The delocalized biguanide fragment is never found to be planar and the hydrogen bond (H-bond) is the dominating molecular interaction. Crystal packing analysis reveals that Met and CF molecules are linked by extended H-bond networks with a number of conserved patterns (dimers, rings, ribbons, sheets, etc). The H-bond strength is increasing with decreasing ΔpKa according to the PA/pKa equalization principle. All complexes formed by Met with acidic compounds are salts, as predictable from the thermodynamic acid-base constants. As a consequence of the high value of pKa1 (12.40), neutral Met is easily protonated at the iminic nitrogen even by weak acids, giving rise to 1:1 ionic adducts consisting of monoprotonated MetH+ and one anion, but since pKa2 is much smaller (2.96), only strong acids succeed in protonating the secondary amino nitrogen, forming 1:2 ionic adducts consisting of diprotonated MetH22+ and two anions. As successful application, we report the crystal engineering, supramolecular synthesis, crystal structure determination and in vitro biological activity testing of two new PCCs of Met with the antileukemic drug dichloroacetic acid (DCA, pKa=0.9) in 1:1 (1, MetH+∙DCA-) and 1:2 (2, MetH22+∙2DCA-) ratio, respectively. The activity of 1 resembles closely the 1:1 physical mixture of NaDCA and Met.HCl, while 2 displays a significantly higher activity and induces a synergistic apoptotic cell death on primary cells of human patients. To our best knowledge, 2 is the first PCC displaying a synergistic enhancement of the anticancer activity of two APIs.