¹9F nuclear magnetic resonance screening of glucokinase activators.

Human hexokinase enzyme IV (EC 2.7.1.1) catalyzes the phosphorylation of glucose and regulates the level of glucose. This enzyme exhibits strong positive cooperativity due to an allosteric transition between an inactive form and a closed active form. This form can be stabilized by activators and, thus, can increase its turnover by a kinetic memory effect characterized by a slow decay to the inactive state. The structural details of this kinetic allostery are known. Several synthetic activators have been reported. We present a preliminary nuclear magnetic resonance (NMR) screening of a chemical library in search of molecules with some affinity for glucokinase (GK). The library, composed of eight molecules with known activity as well as molecules that display no interaction, has been tested using the FAXS (fluorine chemical shift anisotropy and exchange for screening) method, based on monitoring the R2 relaxation of the (19)F spin. To ensure a valid interaction measurement, the enzyme was placed in the presence of glucose and magnesium. The binding signal of one known fluorinated ligand was measured by determining the displacement of the known ligand. This simple measure of the (19)F signal intensity after an 80-ms spin echo correlates nicely with the EC50, opening a route for NMR screening of GK activators.

Synthesis of 9-O-substituted derivatives of 9-hydroxy-5, 6-dimethyl-6H-pyrido[4,3-b]carbazole-1-carboxylic acid (2-(dimethylamino)ethyl)amide and their 10- and 11-methyl analogues with improved antitumor activity.

Analogues of the antitumor drug S 16020-2 modified at the 9, 10, or 11 position were synthesized and evaluated in vitro and in vivo on the P388 leukemia and B16 melanoma models. Starting from 9-methoxy-5, 11-dimethyl-6H-pyrido[4,3-b]carbazole-1-carboxylic acid ethyl ester, the 11-CH3 analogue of 9-hydroxy-5,6-dimethyl-6H-pyrido[4, 3-b]carbazole-1-carboxylic (2-(dimethylamino)ethyl)amide (1), compound 4, was synthesized using a four-step sequence, whereas its 10-CH3 analogue 5 was prepared using a two-step pathway, starting from compound 1. Finally starting from the 9-OH compounds 1, 4, and 5, a series of variously 9-O-substituted derivatives were synthesized. In these series, the most active compounds resulted from esterification of the 9-OH group with various aliphatic diacids, which led to 9-O-CO-( )-COOH derivatives of 1, 4, and 5. For these compounds, the number of long-term surviving mice obtained at the optimal dose were 60-100% in the ip/iv P388 leukemia and 10-35% in the ip/ip B16 melanoma, corresponding to an improved therapeutic index with respect to 1 and 4. This high antitumor activity, with curative examples in both models, was not due to a higher cytotoxicity since these compounds were equally or slightly less potent in vitro than 1 and 4. The most active compounds were thus selected for further in vivo evaluation.