Novel (4-piperidin-1-yl)-phenyl sulfonamides as potent and selective human beta(3) agonists.

A series of novel (4-piperidin-1-yl)-phenyl sulfonamides was prepared and evaluated for their biological activity on the human beta(3)-adrenergic receptor (AR). Replacement of the 3,4-dihydroxyl group of the catechol moiety with 4-hydroxyl-3-methyl sulfonamide on the left-hand side of the compounds resulted in a number of potent full agonists at the beta(3) receptor. Modification of the right-hand side of the compounds by incorporation of a free carboxylic acid resulted in a few potent human beta(3) agonists with low affinities for beta(1)- and beta(2)-ARs. N-Alkyl substitution on the 4-piperidin-1-yl-phenylamine further increased the beta(3) potency while maintaining the selectivity. For example, sulfonamide 48 is a potent full beta(3) agonist (EC(50)=0.004 microM, IA=1.0) with > 500-fold selectivity over beta(1)- and beta(2)-ARs.

New oxadiazolidinedione derivatives as potent and selective human beta3 agonists.

As part of our investigation into the development of potent and selective human beta3 agonists, a series of thiazolidinedione analogues was prepared and evaluated for their biological activity on the human beta3-adrenergic receptor. The oxadiazolidinedione derivative 17 was found to be the most potent and selective compound in this study, with an EC50 value of 0.02 microM at the beta3 receptor, 259-fold selectivity over the beta1 receptor, and 745-fold selectivity over the beta2 receptor.

2,4-Thiazolidinediones as potent and selective human beta3 agonists.

Methylsulfonamide substituted 2,4-thiazolidinedione 22c is a potent (EC50=0.01 microM, IA=1.19) and selective (more than 110-fold over beta1 and beta2 agonist activity) beta3 agonist. This compound has also been proven to be active and selective in an in vivo mode.

Antihyperglycemic activity of new 1,2,4-oxadiazolidine-3,5-diones.

A series of 1,2,4-oxadiazolidine-3,5-diones was synthesized and evaluated as oral antihyperglycemic agents in the obese insulin resistant db/db and ob/ob mouse - the two models for Type 2 diabetes mellitus. The majority of the prepared methoxy- and ethoxy-linked oxazole 1,2,4-oxadiazolidine-3,5-diones normalized plasma glucose levels at the 100 mg kg(-1) oral dose in the db/db diabetic mouse model, and several amongst them reduced the glucose levels at the 20 mg kg(-1) oral dose. The most potent compounds in the db/db mouse model were also active in the ob/ob mouse model normalizing the plasma glucose levels at the 20 mg kg(-1) oral dose. The trifluoromethoxy analog 32 was the most active compound of the series, reducing significantly the plasma glucose levels at the 5 mg kg(-1) oral dose. Oxadiazole-tailed 1,2,4-oxadiazolidine-3,5-diones were also active in both the db/db and ob/ob diabetic mouse models normalizing plasma glucose levels at the 100 mg kg(-1) oral dose.

Evaluation of the antiviral activity of kaempferol and its glycosides against human cytomegalovirus.

The antiviral activity of seven flavonoids, belonging to the kaempferol series, was evaluated against human cytomegalovirus (HCMV) by a rapid method of detection of the immediate-early (IE) antigen, induced by the virus in infected cells. Flavonoids bearing acyl substituents were found to be the most active compounds.

Molecular modeling of the aldose reductase-inhibitor complex based on the X-ray crystal structure and studies with single-site-directed mutants.

Aldose reductase (AR) has been implicated in the etiology of the secondary complications of diabetes. This enzyme catalyzes the reduction of glucose to sorbitol using nicotinamide adenine dinucleotide phosphate as an essential cofactor. AR has been localized at the sites of tissue damage, and inhibitors of this enzyme prevent the development of neuropathy, nephropathy, retinopathy, and cataract formation in animal models of diabetes. The crystal structure of AR complexed with zopolrestat, a potent inhibitor of AR, has been described.(1) We have generated a model of the AR-inhibitor complex based on the reported Calpha coordinates of the protein and results of a structure-activity relationship study using four structurally distinct classes of inhibitors, recombinant human AR, and four single-site-directed mutants of this enzyme. The effects of the site-directed mutations on residues within the active site of the enzyme were evaluated by average interaction energy calculations and by calculations of carbon atom surface area changes. These values correlated well with the IC(50) values for zopolrestat with the wild-type and mutant enzymes, validating the model. On the basis of the zopolrestat-binding model, we have proposed binding models for 10 other AR inhibitors. Our models have enabled us to gain a qualitative understanding of the binding domains of the enzyme and how different inhibitors impact the size and shape of the binding site.

New azolidinediones as inhibitors of protein tyrosine phosphatase 1B with antihyperglycemic properties.

Insulin resistance in the liver and peripheral tissues together with a pancreatic cell defect are the common causes of type 2 diabetes. It is now appreciated that insulin resistance can result from a defect in the insulin receptor signaling system, at a site post binding of insulin to its receptor. Protein tyrosine phosphatases (PTPases) have been shown to be negative regulators of the insulin receptor. Inhibiton of PTPases may be an effective method in the treatment of type 2 diabetes. A series of azolidinediones has been prepared as protein tyrosine phosphatase 1B (PTP1B) inhibitors. Several compounds were potent inhibitors against the recombinant rat and human PTP1B enzymes with submicromolar IC(50) values. Elongated spacers between the azolidinedione moiety and the central aromatic portion of the molecule as well as hydrophobic groups at the vicinity of this aromatic region were very important to the inhibitory activity. Oxadiazolidinediones 87 and 88 and the corresponding acetic acid analogues 119 and 120 were the best h-PTP1B inhibitors with IC(50) values in the range of 0.12-0.3 microM. Several compounds normalized plasma glucose and insulin levels in the ob/ob and db/db diabetic mouse models.

Noradrenaline, dopamine, serotonin: different effects of psychological stress on brain biogenic amines in mice and rats.

The effect of restraint stress on central neurotransmission was evaluated in mice and rats. Noradrenaline (NA), dopamine (DA) and serotonin (5-HT) levels and their primary metabolites were measured in discrete brain regions following exposure to stress. Mice and rats demonstrated a similar response to stress in some brain regions. Both species responded to stress with lower NA and 5-HT in the locus coeruleus compared to non-stressed controls. Dopaminergic activity, assessed by DA turnover, was elevated in the hypothalamus. While DA turnover was suppressed in the amygdala, 5-HT turnover was similarly elevated in both species. In most cases, however, there were differences in biogenic neurotransmission between mice and rats in response to stress. In particular, NA levels were suppressed by stress in the dorsal cortex of mice, but in the rats NA levels were decreased in the hypothalamus. While stress produced lower DA levels in the hypothalamus, DA levels demonstrated a marked increase in the amygdala of mice. Stress was also associated with a decrease in DA levels in the rat striatum and with an increase of DA turnover in the locus coeruleus of mice. On the other hand, 5-HT was suppressed in the mouse striatum and in the rat hypothalamus and amygdala, while 5-HT turnover was markedly decreased in the hippocampus and dorsal cortex of rats alone. In conclusion, the changes in the central neurotransmission which are evoked by stress appear to be species-specific in most cases, a fact which may trigger discrete alterations in homeostatic mechanisms.

Antiproliferative and anticarcinogenic effects of an aqueous preparation of Abies alba and Viscum album se abies, on a L-1210 malignant cell line and tumor-bearing Wistar rats.

Extracts of plants have been widely tested for possible anticarcinogenic properties. In the present study a traditional remedy, consisting of an aqueous extract of mixed parts of the tree Abies alba and its mistletoe Viscum album se abies was tested on benzo(alpha)pyrene(BaP)-induced tumors in Wistar rats and on the L-1210 malignant cell line. Two main groups of male Wistar rats subcutaneously injected by 10 mg of BaP, a dose inducing 100% carcinogenesis, a control group (C-G, 15 rats) and a treatment group(TR-G, 18 rats), were used for the study. Five animals bearing BaP-induced tumors were also tested (TR-1-G). Animals of the TR-G were orally administered with the aqueous extract at doses of 50 ml/kg b.w, from the day of BaP injection and of the TR-1-G, from the 120th day of injection, till death. L-1210 malignant cells in cultivation, were administered with a powder obtained by condensation and lyophilization of the extract, at various concentrations and cytotoxicity was measured by the microculture tetrazolium assay. Autopsy of the rats, revealed metastasis in the lungs of the animals of all groups and the tumors developed were histologically identified as leiomyosarcomas. The results indicated that the extract of the above plants possess anticarcinogenic effects, documented by: a) its antiproliferative effects on L-1210 cells (IC50 = 49.6 +/- 1.4 micrograms/ml), b) the significant prolongation of life and reduction of tumor growth rate of the animals of the TR-G in comparison to the C-G, c) the inhibition by 16.6% of tumor induction in the TR-G and d) the prolongation of life and the necrotic effects of the extract on the tumors of the animals in the TR-1-G. The antiproliferative effects of the Abies alba and Viscum album se abies extract may be due to the lectins and thionins contained in Viscum album, as well as to the monoterpenes contained in Abies alba. Soft tissue tumors sensitive to the extract, are widespread among human organs, even in larynx, and are usually resistant to chemotherapy.

Novel benzofuran and benzothiophene biphenyls as inhibitors of protein tyrosine phosphatase 1B with antihyperglycemic properties.

Insulin resistance in the liver and peripheral tissues, together with a pancreatic cell defect, are the common causes of Type 2 diabetes. It is now appreciated that insulin resistance can result from a defect in the insulin receptor signaling system, at a site post binding of insulin to its receptor. Protein tyrosine phosphatases (PTPases) have been shown to be negative regulators of the insulin receptor. Inhibition of PTPases may be an effective method in the treatment of Type 2 diabetes. We have identified two novel series of benzofuran/benzothiophene biphenyl oxo-acetic acids and sulfonyl-salicylic acids as potent inhibitors of PTP1B with good oral antihyperglycemic activity. To assist in the design of these inhibitors, crystallographic studies have attempted to identify enzyme inhibitor interactions. Resolution of crystal complexes has suggested that the inhibitors bind to the enzyme active site and are held in place through hydrogen bonding and van der Waals interactions formed within two hydrophobic pockets. In the oxo-acetic acid series, hydrophobic substitutents at position-2 of the benzofuran/benzothiophene biphenyl framework interacted with Phe182 of the catalytic site and were very critical to the intrinsic activity of the molecule. The hydrophobic region of the catalytic-site pocket was exploited and taken advantage by hydrophobic substituents at either the alpha-carbon or the ortho aromatic positions of the oxo-acetic acid moiety. Similar ortho aromatic substitutions on the salicylic acid-type inhibitors had no effect, primarily due to the different orientation of these inhibitors in the catalytic site. The most active inhibitors of both series inhibited recombinant human PTP1B with phosphotyrosyl dodecapeptide TRDI(P)YETD(P)Y(P)YRK as the source of the substrate with IC(50) values in the range of 20-50 nM. Compound 68 was one of the most active compounds in vivo, normalizing plasma glucose levels at the 25 mg/kg dose (po) and the 1 mg/kg dose (ip). Compound 68 was also selective against several other PTPases.

Prodrugs of CL316243: a selective beta3-adrenergic receptor agonist for treating obesity and diabetes.

CL316243 is a highly selective and potent beta3-adrenergic receptor agonist, and has been shown in rodent models to be an effective agent for treating obesity and Type II diabetes. To improve the oral absorption and pharmacokinetic profiles of CL316243, a number of prodrugs have been synthesized and evaluated. Several ester-type prodrugs show significant improvements in oral bioavailability in both rodent and primate models.

The effect of sclareol on growth and cell cycle progression of human leukemic cell lines.

Sclareol, a labdane-type diterpene, was tested for cytotoxic effect against a panel of established human leukemic cell lines. The compound showed an IC50 lower than 20 microg/ml in most cell lines tested, while it was higher for resting peripheral blood mononuclear leukocytes (PBML). Furthermore, the compound was tested for cytostatic activity against four of the leukemic cell lines used. At a concentration of 20 microg/ml the compound showed a significant cytostatic effect as soon as 4 h after continuous incubation against two from B and two from T lineage cell lines. The morphology and the kind of death induced from sclareol in three cell lines, was also investigated. The effect of sclareol on the cell cycle progression of two cell lines, using flow cytometry, was examined. The results show that sclareol kills cell lines, through the process of apoptosis. The appearance of the apoptotic signs is time and dose dependent. From the flow cytometry experiments, a delay of the cell population on G0/1 seems to take place. This is the first report, that a labdane type diterpene kills tumor cells via a phase specific mechanism which induces apoptosis.

Beneficial effects of a vanadium complex with cysteine, administered at low doses on benzo(alpha)pyrene-induced leiomyosarcomas in Wistar rats.

BACKGROUND: Vanadium is a potent environmental and body metal, possessing remarkable antitumor and antidiabetic properties. Vanadium salts and complexes have been widely investigated for their anticarcinogenic properties in experimental carcinogenesis. In the present study the antitumor effects of a new vanadium complex with cysteine in relation to identical doses of vanadyl sulfate and cysteine, in tumor bearing rats are investigated. MATERIALS AND METHODS: Male wistar rats were injected with benzo(alpha)pyrene and divided into four groups of 21 rats each. Control group was treated only with BaP. The first group(TR-1) was treated by vanadyl sulfate per os at daily doses of 0.5 mg of V/kg b.w per day. The second (TR-2) by cysteine at doses of 4.5 mg/kg b.w per day and the third group (TR-3), by the complex V(III)-cysteine at daily doses of V 0.5 mg/kg b.w (containing cysteine at concentrations of 4.5 mg/b.w). Treatment was started when tumors were developed (evidenced from a palbable mass at the site of Bap injection) and went on till death. Toxicological tests were performed in 27 rats divided into a control group and two test groups; T-1 administered with vanadyl sulfate at daily doses of 18.5 mg V/kg b.w and T-2 group with V(III)-cysteine complex at daily doses of 18.5 V/kg b.w, for 9 weeks. Mean survival time, death rate, tumor growth rate, the carcinogenic potency of BaP, and the anticarcinogenic potency in relation to histological findings in each treatment group were calculated in each group in order to evaluate the antitumor effects of the substances used. RESULTS: Vanadyl sulfate, cysteine and V(III)-cysteine exerted antitumor effects on leiomyosarcoma bearing Wistar rats. However, V(III)-complex exerted much more potent effects than the other treatments, significantly prolonging mean survival time, retarding tumor growth rate and decreasing the carcinogenic potency of BaP in the TR-3 group, in comparison to the control and the TR-1 and TR-2 groups. Moreover V(III)-cysteine complex resulted in complete remission of 4 (19.7%) of the tumor bearing rats. Blood, urine, biochemical routine tests as well as autopsy did not reveal any toxic effects either of vanadyl sulafate or V(III)-cysteine complex. CONCLUSIONS: Vanadyl sulfate, cysteine and V(III)-cysteine complex exerted antitumor effects in tumor bearing rats. The V(III)-cysteine complex, however, exerts much more potent effects, as evident from the results of the present study. These beneficial effects of the above complex, in combination with its low toxicity provide evidence suggest its possible application in the treatment of human malignant diseases.

Probing the inhibitor-binding site of aldose reductase with site-directed mutagenesis.

Aldose reductase (AR) has been implicated in the etiology of the secondary complications of diabetes, and enzyme inhibitors have been proposed as therapeutic agents. While effectively preventing the development of diabetic complications in animals, results from clinical studies of AR inhibitors have been disappointing, possibly due to poor potency in man. To assist in the design of more potent and specific inhibitors, crystallographic studies have attempted to identify enzyme-inhibitor interactions. Resolution of crystal complexes has suggested that the inhibitors bind to the enzyme active site and are held in place through hydrogen bonding and van der Waals interactions formed within two hydrophobic pockets. To confirm and extend these findings we quantified inhibitor activity with single, site-directed, mutant, human AR enzymes in which the apolar active-site residues tryptophan 20, -79, -111 and phenylalanine 115 were replaced with alanine or tyrosine, decreasing the potential for van der Waals interactions. Consistent with molecular models, the inhibitory activity of Tolrestat, Sorbinil and Zopolrestat decreased 800-2000-fold when tested with the mutant enzyme in which Trp20 was replaced with alanine. Further, alanine substitution for Trp111 decreased Zopolrestat's activity 400-fold, while mutations to Trp79 and Phe115 had little effect on the activity of any of the inhibitors. The alanine mutation at Trp111 had no effect on Tolrestat's activity but decreased the activity of Sorbinil by about 1000-fold. These latter effects were unanticipated based on the number of non-bonded interactions between the inhibitors, Tolrestat and Sorbinil, and Trp20 and Trp111 that have been identified in the crystal structures. In spite of these unexpected findings, our results are consistent with the hypothesis that AR inhibitors occupy the enzyme active site and that hydrophobic interactions between the enzyme and inhibitor contribute to inhibitor binding stability.

Is epinephrine-induced platelet aggregation autoregulated by its metabolic degradation products in vivo?

BACKGROUND: Catecholamines play an important role in platelet activation and aggregation, epinephrine being the most potent one. Catecholamines are substantially increased during stress, exercise or smoking and could result in clinically important platelet activation if their action was not rapidly regulated. In the present study the possible fast regulation of epinephrine-induced platelet aggregation by its metabolic degradation products is investigated. MATERIALS AND METHODS: Human platelet rich plasma (hPRP) and washed rabbit platelets(wRP) were used for the study. The platelets irreversible aggregation induced by epinephrine and ADP were monitored by an aggregometer prior to and after the addition of the catecholamines degradation products metanephrine, 3-methoxy-4-hydroxy-phenyl-glycole-aldehyde (MHPGA),3-methoxy-4-hydroxy-phenyl-mandelic acid (VMA) 3,4-dihydroxy phenyl glycole (DHPG),3,4-Dihydroxy-phenyl-glycole-aldehyde (DHPGA) and the trimethoxy-phenyl-methyl-piperazine(TMP), a known free radical scavenger and calcium antagonist. Linoleic acid-lipoxygenase reaction, in vitro was monitored in the presence and absence of VMA. RESULTS: Metabolic degradation products possessing a methoxy group at position 3 of the phenolic ring markedly inhibited epinephrine and ADP-induced platelet aggregation at microM concentrations. The most potent inhibitor of both agonists was metahephrine, followed by MHPGA and VMA. TMZ also inhibited platelet aggregation at concentrations similar to VMA. Dihydroxy-phenyl compounds failed to induce any inhibition. None of the substances tested induced any aggregatory effect even at high concentrations (1 mM). VMA significantly inhibited linoleic acid-lipoxygenase reaction at 0.1 microM. CONCLUSIONS: Results indicate that catecholamines' degradation products possessing methoxy (-OCH3) groups can rapidly inhibit in vitro and ex vivo epinephrine-induced platelet aggregation. The inhibitory effects of methoxy phenolic derivatives on epinephrine-induced platelet aggregation may possibly be attributed to their free radical scavenging properties. There is substantial evidence to conclude that an internal rapid autoregulation of epinephrine-induced platelet aggregation, caused by its metabolic degradation products, takes place in vivo.

Cytotoxic activity of labdane type diterpenes against human leukemic cell lines in vitro.

Nine labdane type diterpenes isolated from the plant Cistus creticus subsp. creticus and from the resin "Ladano" which is excreted on the surface of the leaves and stems of this plant, were examined for their in vitro cytotoxic activity against 14 human leukemic cell lines. Compound 1, (13E)-labd-13-ene-8 alpha,15-diol, exhibited cytotoxic activity against 13 of the cell lines tested, while compound 2, (13E)-labd-7,13-dienol, was active only against HL60 cells. Further compound 1 was examined for its effect on the uptake of [3H]-thymidine as a marker of DNA synthesis.

Comparison of the therapeutic effects of two vanadium complexes administered at low dose on benzo[a]pyrene-induced malignant tumors in rats.

The antitumor effects of low dose administration of the vanadium(III) complexes with L-cysteine (complex 1) and N-(2-mercaptopropionyl)-glycine (complex 2) were compared on benzo[a]pyrene (BaP)-induced tumors in Wistar rats. Male Wistar rats, injected with 10.0 mg of BaP, were divided into one control (C-G) and two treatment (TR-G) groups of 17 animals each. Animals of the first treatment group were administered complex 2 (TR-2 group) and those of the second group were administered complex 1 (TR-1 group) at doses of 100 microg of vanadium per os daily, starting from the day a palpable tumor was developed till their death. BaP injection induced a 100% tumor (leiomyosarcomas) development in the animals of all groups. Administration of complex 1 to the animals resulted in a significant prolongation of the mean survival time, a complete remission of 17.6% of the tumors developed, a significant reduction of the carcinogenic potency (CP) of BaP and of the tumor growth rate (TGR) in TR-1 group animals, compared to the control and the TR-2 group. In marked contrast, complex 2 failed at the doses administered to exert any significant modulation of the above mentioned parameters. Results indicate that at low (100 micro/day) concentrations of vanadium, complex 1 exerts a significant anticarcinogenic effect on experimentally-induced leiomyosarcomas in rats, whereas complex 2 has no effect when administered at the same low concentrations of vanadium.

Azole phenoxy hydroxyureas as selective and orally active inhibitors of 5-lipoxygenase.

Azole phenoxy hydroxyureas are a new class of 5-lipoxygenase (5-LO) inhibitors. Structure-activity relationship studies have demonstrated that electronegative substituents on the 2-phenyl portion of the oxazole tail increased the ex vivo potency of these inhibitors. Similar substitutions on the thiazole analogs had only minor contribution to the ex vivo activity. The trifluoromethyl-substituted oxazole 24 was the best compound of the oxazole series in both the ex vivo (6 h pretreated rats) and in vivo (3 h pretreated rats) RPAR assay with ED50 values of approximately 1 and 3.6 mg/kg, respectively, but was weakly active in the allergic guinea pig assay. Oxazole 50 was equally active in both the RPAR and guinea pig in vivo models and was similar to zileuton. The unsubstituted thiazole 52 was the best compound of the thiazole series, by inhibiting the leukotriene B4 biosynthesis in the RPAR assay (3 h pretreated rats) by 99%, at an oral dose of 10 mg/kg, and the bronchoconstriction in the allergic guinea pig by 50%, at an intravenous dose of 10 mg/kg. Oxazole 24 demonstrated high and selective 5-LO inhibitory activity in the in vitro assays, with IC50 values ranging from 0.08 microM in mouse macrophages to 0.8 microM in human peripheral monocytes to 1.2 microM in human whole blood. This activity was selective for 5-LO, as concentrations up to 15 microM in mouse macrophages did not affect prostaglandin formation. Oxazole 59 was the most active inhibitor in the human monocyte assay with an IC50 value of 7 nM.

Novel spirosuccinimide aldose reductase inhibitors derived from isoquinoline-1,3-diones: 2-[(4-bromo-2-fluorophenyl)methyl]-6- fluorospiro[isoquinoline-4(1H),3'-pyrrolidine]-1,2',3,5'(2H)-tetrone and congeners. 1.

The high concentrations of plasma glucose formed during diabetic hyperglycemia rapidly translate into high levels of glucose in tissues where glucose uptake is independent of insulin. In these tissues that include the lens, retina, nerve, and kidney, this excess glucose enters the sorbitol (polyol) pathway. The first enzyme in this pathway, aldose reductase, reduces glucose to sorbitol. The diabetes-induced increased flux of glucose through the polyol pathway is believed to play an important role in the development of certain chronic complications of diabetes mellitus. Compounds that inhibit aldose reductase activity and block the flux of glucose through the polyol pathway prevent the development of neuropathy and nephropathy in diabetic animals and interrupt the progression of neuropathy in diabetic patients. Here we describe the preparation and characterization of novel aldose reductase inhibitors. These spiro[isoquinoline-4(1H),3'-pyrrolidine]-1,2',3,5'-(2H)-tetrones, based on the isoquinoline-1,3-dione framework, were evaluated in vitro for their ability to inhibit glyceraldehyde reduction, using a partially purified bovine lens aldose reductase preparation, and in vivo for their ability to inhibit galactitol accumulation in the lens and sciatic nerve of galactose-fed rats. Substitution at the N-2 position of the isoquinoline-1,3-dione framework with diverse structural substituents (i.e., aralkyl, benzothiazolylmethyl, methyl) produced several excellent series of ARIs. Optimization of these new series of spirosuccinimides through structure-activity relationship (SAR) studies, including analogy from other drug series (ponalrestat, zopolrestat), led to the design of the clinical candidate 2-[(4-bromo-2-fluorophenyl)methyl]-6-fluorospiro[isoquinoline-4(1H ),3'- pyrrolidine]-1,2',3,5'(2H)-tetrone (41). Compound 41 exhibited exceptional oral potency in two animal models of diabetic complications, the 14-day galactose-fed and streptozocin-induced diabetic rats, with ED50 values for the sciatic nerve of 0.1 and 0.09 mg/kg/day, respectively. Both enantiomeric forms of 41 exhibited similar inhibitory activity in both in vitro and in vivo assays possibly due to their rapid interconversion. In an ex vivo experiment, the pharmacodynamic effect of 41 in the plasma of rats and dogs, after a single dose, appeared to be comparable to that of tolrestat.

N-substituted spirosuccinimide, spiropyridazine, spiroazetidine, and acetic acid aldose reductase inhibitors derived from isoquinoline-1,3-diones. 2.

The isoquinoline-1,3-dione framework featured in our clinical candidate (1) and its congener was used as the template in the design of several new series of aldose reductase inhibitors (ARIs). These series included N'-substituted spirosuccinimide, spiropyridazine, spiroazetidine, and acetic acid analogues. Compounds within these series were evaluated in vitro for their ability to inhibit glyceraldehyde reduction by bovine lens aldose reductase and in vivo by their ability to inhibit galactitol accumulation in the lens and sciatic nerve of galactose-fed rats. The N'-amino- and N'-alkyl-substituted spiro[isoquinoline-4(1H),3'-pyrrolidine]-1,2',3,5'(2H)- tetrones 6 exhibited high oral potency, even though they were devoid of any intrinsic activity for the aldose reductase enzyme. Similar results were observed for the closely related spiropyridazines 8. Both of these groups are also considered to be prodrugs since they exhibited good oral potency, even though they were devoid of any intrinsic activity for the aldose reductase enzyme. In contrast, the isoquinoline-1,3-dione acetic acids 9 exhibited very high intrinsic activity for the aldose reductase enzyme, although minimal or no in vivo activity. The absence of in vivo activity for some of these compounds may be due to poor tissue penetration. In support of this suggestion, the more lipophilic acetyl alkyl carbamate derivatives of these isoquinoline-1,3-dione acetic acids, exhibited enhanced oral potency. The spiroazetidines 7 exhibited good activity for the aldoe reductase enzyme in both the in vitro and in vivo assays. The findings of this study demonstrate the utility of the isoquinoline-1,3-dione framework, as a versatile template for the design of divese series of potent ARIs.