2-Aryl-3-(6-trifluoromethoxy)benzo[d]thiazole-based thiazolidinone hybrids as potential anti-infective agents: Synthesis, biological evaluation and molecular docking studies.

The search for new antimicrobial agents is greater than ever due to the perpetual threat of multidrug resistance in known pathogens and the relentless emergence of new infections. In this manuscript, ten thiazole-based thiazolidinone hybrids bearing a 6-trifluoromethoxy substituent on the benzothiazole core were synthesized and evaluated against a panel of four bacterial strains Salmonella typhimurium, Staphylococcus aureus, Escherichia coli and Listeria monocytogenes and three resistant strains Pseudomonas aeruginosa, E. coli and MRSA. The evaluation of minimum bactericidal and minimum inhibitory concentrations was accomplished by microdilution assay. As reference compounds ampicillin and streptomycin were employed. All compounds displayed antibacterial efficiencies with MBCs/MICs at 0.25-1 mg/mL and 0.12-1 mg/mL respectively while ampicillin displayed MBCs/MICs at 0.15-0.3 mg/mL and at 0.1-0.2 mg/mL respectively. MICs/MBC of streptomycin varied from 0.05 to 0.15 mg/mL and from 0.1 to 0.3 mg/mL respectively. The best overall effect was observed for compound h4, while compound h1 exhibited the highest effective action against E. coli (MIC/MBC 0.12/0.25 mg/ml) among all tested compounds.

Synthesis of novel 1,2-diarylpyrazolidin-3-one-based compounds and their evaluation as broad spectrum antibacterial agents.

There is a continuous need to develop new antibacterial agents with non-traditional mechanisms to combat the nonstop emerging resistance to most of the antibiotics used in clinical settings. We identified novel pyrazolidinone derivatives as antibacterial hits in an in-house library screening and synthesized several derivatives in order to improve the potency and increase the polarity of the discovered hit compounds. The oxime derivative 24 exhibited promising antibacterial activity against E. coli TolC, B. subtilis and S. aureus with MIC values of 4, 10 and 20 µg/mL, respectively. The new lead compound 24 was found to exhibit a weak dual inhibitory activity against both the E. coli MurA and MurB enzymes with IC50 values of 88.1 and 79.5 µM, respectively, which could partially explain its antibacterial effect. A comparison with the previously reported, structurally related pyrazolidinediones suggested that the oxime functionality at position 4 enhanced the activity against MurA and recovered the activity against the MurB enzyme. Compound 24 can serve as a lead for further development of novel and safe antibiotics with potential broad spectrum activity.

Novel bis(pyrazole-benzofuran) hybrids possessing piperazine linker: Synthesis of potent bacterial biofilm and MurB inhibitors.

Novel 1,4-bis[(2-(3-(dimethylamino)-1-oxoprop-2-en-1-yl)benzofuran-5-yl)methyl]piperazine was prepared and used as a key synthon for the this study. Therefore, 1,3-dipolar cycloaddition of this synthon with the appropriate hydrazonyl chlorides afforded a new series of bis(1,3,4-trisubstituted pyrazoles), linked via piperazine moiety. Furthermore, it reacted with hydrazine hydrate and phenyl hydrazine individually to afford the corresponding 1,4-bis[(2-(1H-pyrazolyl)benzofuran-5-yl)methyl]piperazines. Different bacterial strains and cell lines were selected to study the in-vitro antibacterial and cytotoxic activities for the new derivatives. 1,4-Bis[((2-(3-acetyl-1-(4-nitrophenyl)-1H-pyrazole-4-yl)carbonyl)benzofuran-5-yl)methyl]piperazine 5e showed the best antibacterial efficacies with MIC/MBC values of 1.2/1.2, 1.2/2.4 and 1.2/2.4 µM against each of E. coli, S. aureus and S. mutans strains, respectively. In addition, the inhibitory activity of some new bis(pyrazoles) as MRSA and VRE inhibitors were studied. Compound 5e gave the best inhibitory activity with MIC/MBC values of 18.1/36.2, 9.0/18.1 and 18.1/18.1 µM, respectively, against MRSA (ATCC:33591 and ATCC:43300) and VRE (ATCC:51575) bacterial strains, respectively. Compound 5e showed more effective biofilm inhibition activities than the reference Ciprofloxacin. It showed IC50 values of 3.0 ± 0.05, 3.2 ± 0.08 and 3.3 ± 0.07 µM against S. aureus, S. mutans and E. coli strains, respectively. Furthermore, experimental study showed excellent inhibitory activities of 1,4-bis[((2-(3-substituted-1-aryl-1H-pyrazole-4-yl)carbonyl)benzofuran-5-yl)methyl]piperazine derivatives, attached to p-NO2 or p-Cl groups, against MurB enzyme. Compound 5e gave the best MurB inhibitory activity with IC50 value of 3.1 µM. The in-silico study was performed to predict the capability of new derivatives as potential inhibitors of MurB enzyme.

Mechanism of repression of 11ß-hydroxysteroid dehydrogenase type 1 by growth hormone in 3T3-L1 adipocytes.

11ß-Hydroxysteroid dehydrogenase type 1 (11ß-HSD1) is an NADPH-dependent reductase that converts cortisone to cortisol in adipose tissue. We previously reported that GH and IGF-I decrease 11ß-HSD1 activity and mRNA levels in adipocytes. Hexose-6-phosphate dehydrogenase (H6PDH) is involved in the production of NADPH, which is a coenzyme for 11ß-HSD1. The aim of the present study was to clarify further the mechanism of repression of 11ß-HSD1 activity by GH using linsitinib, an IGF-I receptor inhibitor. The suppression of 11ß-HSD1 mRNA by IGF-I was attenuated in the presence of 1 µM linsitinib (17.2% vs. 53.3% of basal level, P<0.05). 11ß-HSD1 mRNA levels in cells treated with GH in the presence of 1 µM linsitinib were not different from those in absence of linsitinib (35.9% vs. 33.9%). The increase in IGF-I mRNA levels with GH and 1 µM linsitinib was not different from that in the absence of linsitinib (359% vs. 347%). H6PDH mRNA levels were significantly decreased in cells treated with IGF-I for 8 and 24 h (55.6% and 33.7%, P<0.05). In the presence of 1 µM linsitinib, there was no repression of H6PDH mRNA (111.4%). H6PDH mRNA levels were significantly decreased in cells treated with GH in the absence of linsitinib for 24 h (55.9%, P<0.05), but not for 8 h (89.5%). The presence of 1 µM linsitinib also prevented repression of H6PDH mRNA by GH over 24 h (107.8%). These results suggest that GH directly represses 11ß-HSD1 mRNA rather than acting via the IGF-I receptor, and that GH represses H6PDH through locally produced IGF-I.

Hexose-6-phosphate dehydrogenase blockade reverses prostate cancer drug resistance in xenograft models by glucocorticoid inactivation.

Prostate cancer resistance to next-generation hormonal treatment with enzalutamide is a major problem and eventuates into disease lethality. Biologically active glucocorticoids that stimulate glucocorticoid receptor (GR) have an 11ß-OH moiety, and resistant tumors exhibit loss of 11ß-HSD2, the oxidative (11ß-OH → 11-keto) enzyme that normally inactivates glucocorticoids, allowing elevated tumor glucocorticoids to drive resistance by stimulating GR. Here, we show that up-regulation of hexose-6-phosphate dehydrogenase (H6PD) protein occurs in prostate cancer tissues of men treated with enzalutamide, human-derived cell lines, and patient-derived prostate tissues treated ex vivo with enzalutamide. Genetically silencing H6PD blocks NADPH generation, which inhibits the usual reductive directionality of 11ß-HSD1, to effectively replace 11ß-HSD2 function in human-derived cell line models, suppress the concentration of biologically active glucocorticoids in prostate cancer, and reverse enzalutamide resistance in mouse xenograft models. Similarly, pharmacologic blockade of H6PD with rucaparib normalizes tumor glucocorticoid metabolism in human cell lines and reinstates responsiveness to enzalutamide in mouse xenograft models. Our data show that blockade of H6PD, which is essential for glucocorticoid synthesis in humans, normalizes glucocorticoid metabolism and reverses enzalutamide resistance in mouse xenograft models. We credential H6PD as a pharmacologic vulnerability for treatment of next-generation androgen receptor antagonist-resistant prostate cancer by depleting tumor glucocorticoids.

Inhibition of the GDP-d-Mannose Dehydrogenase from Pseudomonas aeruginosa Using Targeted Sugar Nucleotide Probes.

Sufferers of cystic fibrosis are at extremely high risk for contracting chronic lung infections. Over their lifetime, one bacterial strain in particular, Pseudomonas aeruginosa, becomes the dominant pathogen. Bacterial strains incur loss-of-function mutations in the mucA gene that lead to a mucoid conversion, resulting in copious secretion of the exopolysaccharide alginate. Strategies that stop the production of alginate in mucoid Pseudomonas aeruginosa infections are therefore of paramount importance. To aid in this, a series of sugar nucleotide tools to probe an enzyme critical to alginate biosynthesis, guanosine diphosphate mannose dehydrogenase (GMD), have been developed. GMD catalyzes the irreversible formation of the alginate building block, guanosine diphosphate mannuronic acid. Using a chemoenzymatic strategy, we accessed a series of modified sugar nucleotides, identifying a C6-amide derivative of guanosine diphosphate mannose as a micromolar inhibitor of GMD. This discovery provides a framework for wider inhibition strategies against GMD to be developed.

Unraveling the mechanism of l-gulonate-3-dehydrogenase inhibition by ascorbic acid: Insights from molecular modeling.

l-Gulonate dehydrogenase (GuDH) is a crucial enzyme in the non-phosphorylated sugar metabolism or glucuronate-xylulose (GX) pathway. Some naturally occurring compounds inhibit GuDH. Ascorbic acid is one of such inhibitors for GuDH. However, the exact mechanism by which ascorbic acid inhibits GuDH is still unknown. In this study, we try to investigate GuDH inhibition using computational approaches by generating a model for buffalo GuDH. We used this model to perform blind dockings of ascorbic acid to GuDH. Some docked conformations of ascorbic acid bind near Asp39 and have steric clashes with crystal structure conformation of NADH. To assess the dynamic stability of the GuDH-ascorbic acid complex, we performed six molecular dynamics simulations for GuDH, three each in its free form and in complex with ascorbic acid for 50 ns, to obtain 300 ns of trajectories in total. During the simulations, ascorbic acid interacted with several residues nearby Asp39. As Asp39 is an important residue for NADH binding and specificity, the interaction of ascorbic acid near Asp39 hinders further NADH binding and ultimately affects the enzymatic functioning of GuDH. In this study, we analyze these interactions between ascorbic acid and GuDH. Our analysis reveals novel details on the mechanism of GuDH inhibition by ascorbic acid.

In silico identification, synthesis and biological evaluation of novel tetrazole inhibitors of MurB.

In the context of antibacterial drug discovery resurgence, novel therapeutic targets and new compounds with alternative mechanisms of action are of paramount importance. We focused on UDP-N-acetylenolpyruvylglucosamine reductase (i.e. MurB), an underexploited target enzyme that is involved in early steps of bacterial peptidoglycan biosynthesis. On the basis of the recently reported crystal structure of MurB in complex with NADP+ , a pharmacophore model was generated and used in a virtual screening campaign with combined structure-based and ligand-based approaches. To explore chemical space around hit compounds, further similarity search and organic synthesis were employed to obtain several compounds with micromolar IC50 values on MurB. The best inhibitors in the reported series of 5-substituted tetrazol-2-yl acetamides were compounds 13, 26 and 30 with IC50 values of 34, 28 and 25 µm, respectively. None of the reported compounds possessed in vitro antimicrobial activity against Staphylococcus aureus and Escherichia coli.

Pyrazolidine-3,5-diones and 5-hydroxy-1H-pyrazol-3(2H)-ones, inhibitors of UDP-N-acetylenolpyruvyl glucosamine reductase.

A series of pyrazolidine-3,5-dione and 5-hydroxy-1H-pyrazol-3(2H)-one inhibitors of Escherichia coli UDP-N-acetylenolpyruvyl glucosamine reductase (MurB) has been prepared. The 5-hydroxy-1H-pyrazol-3(2H)-ones show low micromolar IC(50) values versus E. coli MurB and submicromolar minimal inhibitory concentrations (MIC) against Staphylococcus aureus GC 1131, Enterococcus faecalis GC 2242, Streptococcus pneumoniae GC 1894, and E. coli GC 4560 imp, a strain with increased outer membrane permeability. None of these compounds show antimicrobial activity against Candida albicans, a marker of eukaryotic toxicity. Moreover, these compounds inhibit peptidoglycan biosynthesis, as assessed by measuring the amount of soluble peptidoglycan produced by Streptococcus epidermidis upon incubation with compounds. A partial least squares projection to latent structures analysis shows that improving MurB potency and MIC values correlate with increasing lipophilicity of the C-4 substituent of the 5-hydroxy-1H-pyrazol-3(2H)-one core. Docking studies using FLO and PharmDock produced several binding orientations for these molecules in the MurB active site.

Purification and properties of NAD+-dependent maltose dehydrogenase produced by alkalophilic Corynebacterium sp. No. 93-1.

NAD+-dependent maltose dehydrogenase was purified about 250-fold from the cell free extract of an alkalophilic Corynebacterium sp. No. 93-1. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis, sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis and ultracentrifugation. The molecular weight of the enzyme was determined to be 40 000 +/- 2000 by gel filtration and SDS-polyacrylamide gel electrophoresis. The enzyme appeared to be a single peptide chain. The isoelectric point was pH 4.50. The optimal pH was 10.2. The enzyme was stable over the range of pH 6 to 10. NAD+-dependent maltose dehydrogenase showed very wide substrate specificity on monosaccharides, disaccharides and trisaccharides. Among these substrates, maltose was the most reactive. Also, the enzyme showed oxidative activity on maltotetraose and maltopentaose. The Km values at pH 10 were 2.1 mM for maltose and 0.15 mM for NAD+. It was conjectured that the primary product of this reaction was maltono-delta-lactone and its was hydrolyzed non-enzymatically to maltobionic acid. p-Chloromercuribenzoic acid, Hg2+ and Ag2+ completely inhibited the activity, and HADH also showed competitive inhibition on the activity.

Effect of salts on D-glycerate dehydrogenase kinetic behavior.

Bovine liver D-glycerate dehydrogenase (D-glycerate:NAD (NADP) oxidoreductase, EC 1.1.1.29) adapts its kinetic behaviour to a sequential mechanism. The presence of NaCl causes an appreciable variation in the Km and V values. relative to the both substrates in the hydroxypyruvate/D-glycerate dehydrogenase/NADH system, which does not happen in the D-glycerate/D-glycerate dehydrogenase/NAD system. The former system is inhibited by high concentrations of NaCl and activated by low salt concentrations. The hydroxypyruvate concentration causing substrate inhibition increases as the concentration of NaCl increases; excess NADH inhibition is independent of the salt concentration. The variation of the initial rates of both systems, in the presence of chlorides having monovalent and divalent cations, or sodium halides, Na2SO4 and NaNO3 (at constant ionic strength) suggests that the anions have a specific action on the enzyme. An increase in the NaCl concentration causes a displacement of the optimum D-glycerate dehydrogenase pH (with hydroxypyruvate and NADH as substrates) towards the acid area. The enzyme stability, at varying pH, varies with the salt concentration.

Diethyl pyrocarbonate inactivation of human placental aldehyde reductase II.

Diethyl pyrocarbonate inactivated aldehyde reductase II (L-gulonate:NADP+ 6-oxidoreductase, EC 1.1.1.19) from human placenta. A concentration of 0.5-1.0 mM diethyl pyrocarbonate caused 40-65% loss of activity. The inactivation of the enzyme by diethyl pyrocarbonate was reversed by hydroxylamine and was accompanied by a large change in the absorbance of the protein at 242 nm, but not at 278 nm, indicating that only the histidine residues were modified. NADPH, but not glucuronate afforded significant protection to the enzyme from inactivation by diethyl pyrocarbonate. With 0.2-1.0 mM diethyl pyrocarbonate, 4-5 histidine residues were modified with a pseudo-first-order rate process. A double log plot of the fraction of the unmodified residues indicates that only one functional histidine residue is essential for the catalytic activity of aldehyde reductase II.

A conformational change in phosphoglycerate dehydrogenase induced by a shift in pH.

The fluorescence of NADH bound to phosphoglycerate dehydrogenase (3-phosphoglycerate: NAD+ oxidoreductase, EC 1.1.1.95) decreased by 42% between pH 8.5 and 7.0 Serine, an allosteric inhibitor, quenched the fluorescence of enzyme-bound NADH by 29% at pH 8.5, but not at all at pH 7.0. The kinetics of the fluorescence change which occurred when the pH of an enzyme-NADH solution was rapidly shifted from 8.5 to 7.0 was measured using stopped-flow fluorimetry. The kinetics were first order, with a rate constant of 2.83 s-1. This rate constant was similar in magnitude to the rate constants for fluorescence quenching at pH 8.5 by saturating concentrations of serine and glycine, another allosteric inhibitor (Dubrow, R. and Pizer, L.I. (1977) J. Biol. Chem. 252, 1527-1538). These results indicate that the conformation of phosphoglycerate dehydrogenase at pH 7.0 is similar to, but not identical with, the serine-induced conformation at pH 8.5.

Induced versus pre-existing asymmetry models for the half-of-the-sites reactivity effect in bovine liver uridine diphosphoglucose dehydrogenase.

Half-of-the-sites reactivity of the catalytic site thiol groups of UDPglucose dehydrogenase (UDPglucose:NAD+ 6-oxidoreductase, EC 1.1.1.22) can be ascribed either to the induction of conformational asymmetry following derivatization of one half of the subunits or to intrinsic conformational differences in the subunits of the native enzyme. If the half-sites reactivity behavior is due to induction effects, the magnitude of the induction could be expected to depend on the nature of the covalent modification. On the other hand, if the half-sites reactivity behavior is due to pre-existing asymmetry and there is no communication between catalytic centers, the properties of unmodified sub-units should be independent of the nature of the covalent derivative introduced on the modified subunits. According to the induced asymmetry hypothesis, the catalytic activity of half-sites modified enzyme might be different for different covalent modifications, whereas for the rigid pre-existing asymmetry hypothesis the catalytic activity of half-sites modified enzyme should be the same regardless of the modifying group. During the course of catalytic site thiol group modification by a number of thiol specific reagents, the loss of enzyme activity was equivalent to the degree of modification for most of the reagents employed. However, with iodoacetate and 5-(iodoacetamidoethyl)aminonaphthalene-1-sulfonic acid, half-sites modification of UDPglucose dehydrogenase reduced catalytic activity by 58 and 78%, respectively, of the initial activity. These observations are consistent with a model in which there is communication between catalytic sites. Electron microscopy shows that the six subunits of UDPglucose dehydrogenase are arranged as a hexagonal planar ensemble.

Structural and functional characterization of rabbit and human L-gulonate 3-dehydrogenase.

L-Gulonate 3-dehydrogenase (GDH) catalyzes the NAD(+)-linked dehydrogenation of L-gulonate into dehydro-L-gulonate in the uronate cycle. In this study, we isolated the enzyme and its cDNA from rabbit liver, and found that the cDNA is identical to that for rabbit lens lambda-crystallin except for lacking a codon for Glu(309). The same cDNA species, but not the lambda-crystallin cDNA with the codon for Glu(309), was detected in the lens, which showed the highest GDH activity among rabbit tissues. In addition, recombinant human lambda-crystallin that lacks Glu(309) displays enzymatic properties similar to rabbit GDH. These data indicate that GDH is recruited as lambda-crystallin without gene duplication. An outstanding feature of GDH is modulation of its activity by low concentrations of P(i), which decreases the catalytic efficiency in a dose dependent manner. P(i) also protects the enzyme against both thermal and urea denaturation. Kinetic analysis suggests that P(i) binds to both the free enzyme and its NAD(H)-complex in the sequential ordered mechanism. Furthermore, we examined the roles of Asp(36), Ser(124), His(145), Glu(157 )and Asn(196) in the catalytic function of rabbit GDH by site-directed mutagenesis. The D36R mutation leads to a switch in favor of NADP(H) specificity, suggesting an important role of Asp(36) in the coenzyme specificity. The S124A mutation decreases the catalytic efficiency 500-fold, and the H145Q, N196Q and N195D mutations result in inactive enzyme forms, although the E157Q mutation produces no large kinetic alteration. Thus, Ser(124), His(145) and Asn(196) may be critical for the catalytic function of GDH.

Genistein inhibits glucocorticoid amplification in adipose tissue by suppression of 11ß-hydroxysteroid dehydrogenase type 1.

Excess glucocorticoids promote visceral obesity, hyperlipidemia, and insulin resistance. The main regulator of intracellular glucocorticoid levels is 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1), which converts inactive glucocorticoids into bioactive forms such as cortisol in humans and corticosterone in rodents. Hexose-6-phosphate dehydrogenase (H6PD), which is colocalized with 11ß-HSD1 in the intralumen of the endoplasmic reticulum, supplies a crucial coenzyme, NADPH, for full reductase activity of 11ß-HSD1. Therefore, it is possible that inhibition of 11ß-HSD1 will become a considerable medical treatment for metabolic diseases including obesity and diabetes. Genistein, a soy isoflavone, has received attention for its therapeutic potential for obesity, diabetes, and cardiovascular disease, and has been proposed as a promising compound for the treatment of metabolic disorders. However, the mechanisms underlying the pleiotropic anti-obesity effects of genistein have not been fully clarified. Here, we demonstrate that genistein was able to inhibit 11ß-HSD1 and H6PD activities within 10 or 20min, in dose- and time-dependent manners. Inhibition of 11ß-HSD2 activity was not observed in rat kidney microsomes. The inhibition was not reversed by two estrogen receptor antagonists, tamoxifen and ICI182,780. A kinetic study revealed that genistein acted as a non-competitive inhibitor of 11ß-HSD1, and its apparent Km value for 11-dehydrocorticosterone was 0.5µM. Genistein also acted as a non-competitive inhibitor of H6PD, and its apparent Km values for G6P and NADP were 0.9 and 3.3µM, respectively. These results suggest that genistein may exert its inhibitory effect by interacting with these enzymes.

The relationship between effector binding and inhibition of activity in D-3-phosphoglycerate dehydrogenase.

The binding of L-serine to phosphoglycerate dehydrogenase from Escherichia coli displays elements of both positive and negative cooperativity. At pH 7.5, approximately 2 mol of serine are bound per mole of tetrameric enzyme. A substantial degree of positive cooperativity is seen for the binding of the second ligand, but the binding of the third and fourth ligand display substantial negative cooperativity. The data indicate a state of approximately 50% inhibition when only one serine is bound and approximately 80-90% inhibition when two serines are bound. This is consistent with the tethered domain hypothesis that has been presented previously. Comparison of the data derived directly from binding stoichiometry to the binding constants determined from the best fit to the Adair equation, produce a close agreement, and reinforce the general validity of the derived binding constants. The data also support the conclusion that the positive cooperativity between the binding to the first and second site involves binding sites at opposite interfaces over 110 A apart. Thus, an order of binding can be envisioned where the binding of the first ligand initiates a conformational transition that allows the second ligand to bind with much higher affinity at the opposite interface. This is followed by the third ligand, which binds with lesser affinity to one of the two already occupied interfaces, and in so doing, completes a global conformational transition that produces maximum inhibition of activity and an even lower affinity for the fourth ligand, excluding it completely. Thus, maximal inhibition is accomplished with less than maximal occupancy of effector sites through a mechanism that displays strong elements of both positive and negative cooperativity.

Evidence for glucose-6-phosphate transport in rat liver microsomes.

The existence of glucose-6-phosphate transport across the liver microsomal membrane is still controversial. In this paper, we show that S3483, a chlorogenic acid derivative known to inhibit glucose-6-phosphatase in intact microsomes, caused the intravesicular accumulation of glucose-6-phosphate when the latter was produced by glucose-6-phosphatase from glucose and carbamoyl-phosphate. S3483 also inhibited the conversion of glucose-6-phosphate to 6-phosphogluconate occurring inside microsomes in the presence of electron acceptors (NADP or metyrapone). These data indicate that liver microsomal membranes contain a reversible glucose-6-phosphate transporter, which furnishes substrate not only to glucose-6-phosphatase, but also to hexose-6-phosphate dehydrogenase.

Spiro[fluoreneisothiazolidin]one dioxides: new aldose reductase and L-hexonate dehydrogenase inhibitors.

The first examples of spiro[fluorene-9,4'- and -9,5'-isothiazolidin]one dioxides (1 and 2) were synthesized and screened for activity as aldose reductase and L-hexonate dehydrogenase inhibitors. Compared to compounds 1, and 9,5'-compounds 2, synthesized from fluorene-9-sulfonamides by alkylation at C(9) with ethyl bromoacetate followed by cyclization, were more active, but relatively nonselective, inhibitors of aldose reductase and L-hexonate dehydrogenase, with IC50 values for in vitro inhibition of both enzymes on the order of 10(-7)-10(-8) M. However, the isomeric 9,4'-compounds 1, prepared by alkylation of fluorene-9-carboxylic acid esters with bromo- or iodomethanesulfonamide followed by cyclization, were more selective inhibitors of L-hexonate dehydrogenase with IC50 values of about 10(-6) M.