A new working hypothesis that there is a hitherto unrecognized binding site on the aldose reductase (AR) enzyme with strong affinity for benzothiazoles was pursued for the design of novel, potent aldose reductase inhibitors (ARIs). The first application of this hypothesis led to a novel series of 3,4-dihydro-4-oxo-3-(benzothiazolylmethyl)-1-phthalazineacetic+ + + acids. The parent of this series (207) was a potent inhibitor of AR from human placenta (IC50 = 1.9 x 10(-8) M) and was orally active in preventing sorbitol accumulation in rat sciatic nerve, in an acute test of diabetic complications (ED50 = 18.5 mg/kg). Optimization of this lead through medicinal chemical rationale, including analogy from other drug series, led to more potent congeners of 207 and culminated in the design of 3,4-dihydro-4-oxo-3-[[5-(trifluoromethyl)-2-benzothiazolyl] methyl]-1-phthalazineacetic acid (216, CP-73,850, zopolrestat). Zopolrestat was found to be more potent than 207, both in vitro and in vivo. Its IC50 against AR and ED50 in the acute test were 3.1 x 10(-9)M and 3.6 mg/kg, respectively. Its ED50s in reversing already elevated sorbitol accumulation in rat sciatic nerve, retina, and lens in a chronic test were 1.9, 17.6, and 18.4 mg/kg, respectively. It was well absorbed in diabetic patients, resulting in high blood level, showed a highly favorable plasma half-life (27.5 h), and is undergoing further clinical evaluation. An assortment of synthetic methods used for the construction of benzothiazoles, including an efficient synthesis of zopolrestat, is described. Structure-activity relationships in the new series are discussed.

Ethyl 1-benzyl-3-hydroxy-2(5H)-oxopyrrole-4-carboxylate (1, EBPC) is a potent and specific inhibitor of aldose reductase. It was greater than 4000X more potent in its inhibition of rat lens aldose reductase than the closely related rat or pig kidney aldehyde reductase, thus making it the most selective inhibitor of a NADPH-dependent carbonyl reductase identified to date. In agreement with this observation, it was found to be a highly potent inhibitor of aldose reductase from rat sciatic nerve with greater than 98% inhibition at 1 microM, but it was practically devoid of activity against aldehyde reductases from rat liver and brain. Inhibition of aldose reductase was mixed type for glyceraldehyde (Ki = 8.0 x 10(-8) M) and noncompetitive for NADPH (Ki = 1.70 x 10(-8) M). Its potential as an in vitro tool to quantitate monomeric aldo/keto reductase activities in crude tissue extracts is presented. Structure-activity relationships emerging from synthetic modifications of EBPC are discussed. Several modifications were found to be active in vitro against aldose reductase from human placenta and in vivo in a rat model of diabetic complications, but none was more potent than EBPC.

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Benzothiazole side chains featured in zopolrestat (1a) and its congeners were incorporated into oxophthalazineacetic acid replacements, including indazole, pyridazinone, and pyridopyridazinone with a pendant acetic acid moiety. Potent aldose reductase inhibition activity among resulting compounds is as widespread as it is in the earlier zopolrestat series, thus lending further support to our hypothesis that there is a binding site on the aldose reductase enzyme with strong affinity for benzothiazoles. Representative new compounds 1-[(5,7-difluoro-2-benzothiazolyl)-methyl]-1H-indazoleacetic acid (62), [6-[[5-(trifluoromethyl)benzothiazol-2-yl]methyl]-8-oxo- 6H-pyrido[2,3-d]-pyridazin-5-yl]acetic acid (70), 3,4-dihydro-4-oxo-5,6-dimethyl-3-[(5,7-difluorobenzothiazol-2-yl) methyl]-1-pyridazineacetic acid (79), and 3,4-dihydro-4-oxo-5,6-cyclohexano-3-[[5-(trifluoromethyl) benzothiazol-2-yl]-methyl]-1-pyridazineacetic acid (82) are potent aldose reductase inhibitors with IC50s of 30, 2.1, 5, and 52.2 nM, respectively. The best of these compounds, 79 and 82, also inhibit accumulation of sorbitol in rat sciatic nerve in a model of diabetic complications, when administered orally at 10 mg/kg. The inhibition values are 76 and 61%, respectively. In addition to benzothiazole, we have examined its surrogates effective in potentiating aldose reductase inhibition activity, including benzoxazole and aryl[1,2,4]oxadiazole. Structure-activity relationships emerging from this program are also discussed.

A pictorial map of the lactose synthase (galactosyl transferase) acceptor binding site has been formulated from this and published studies on substrate analogs and inhibitors. The basic requirements are a pyranose, thiopyranose or inositol ring structure and equatorial substituents (if any) at C-2, C-3, C-4, and C-5. The aglycone (at C-1) may be either alpha or beta-, but alpha- is somewhat preferred. In the absence of alpha-lactalbumin galactosyl transferase will accept long chain 2-N-acyl substituents on the glucosamine (GlcNH2) structure. An equatorial amino or N-acetyl substituent (e.g. mannosamine, N-acetylmannosamine) is also a suitable acceptor in the absence of alpha-lactalbumin since both N-acetylglucosamine and N-acetylmannosamine have complementary binding loci for the N-acyl moiety. The aglycone moiety must be equatorial (beta-configuration). However, upon alpha-lactalbumin binding the aglycone specificity allows for axial (alpha-configuration) as well as equatorial substituents. Furthermore, the 2-N-acyl substituent binding locus is blocked beyond a 2-N-hexanoyl group. It is suggested that alpha-lactalbumin binds to a hydrophobic site some distance from the C-2 group.