Synthesis and antilipidemic properties of cis-7-chloro-3a, 8b-dihydro-3a-methylfuro[3,4-b]benzofuran-3(1H)-one, a tricyclic clofibrate related lactone having a structural resemblance to mevalonolactone.

The synthesis for the title lactone 2, designed to be an antagonist of the enzyme HMG-CoA reductase (E.C.1.1.1.34), is described. Lactone 2, its synthetic tricyclic hemiacetal precursor 4, and clofibrate were investigated for their antilipidemic activity in 7-day treated normal and in Triton WR-1339 induced hyperlipidemic male Sprague-Dawley rats. After 7-day drug administration to normal rats, lactone 2 was less effective than clofibrate in lowering HMG-CoA reductase activity and serum cholesterol; however, unlike clofibrate, lactone 2 did not increase liver weight or liver-body weight ratio or lower serum triglycerides. Since hemiacetal 4 selectively influenced triglycerides in normal animals, lactone 2 and hemiacetal 4 appear to have differential hypolipidemic effects. In the Triton hyperlipidemic model 2 and 4 lowered elevated triglycerides; only 4 significantly reduced elevated cholesterol levels; but neither 2 nor 4 was as effective as clofibrate. Differences in the observed antilipidemic properties for clofibrate, 2, and 4 in the two animal models are discussed. On the basis of preliminary biological data described in this article it is concluded that tricyclic analogues 2 and 4 represent reasonable leads for the development of new antilipidemic agents.

Inhibition of cytosolic epoxide hydrolase by trans-3-phenylglycidols.

The inhibition of murine cytosolic epoxide hydrolase has been studied with both racemic and enantiomerically pure trans-3-phenylglycidols. These compounds are the first enantioselective, slow binding inhibitors of cytosolic epoxide hydrolase. The (2S,3S)-3-phenylglycidol enantiomer was always a better inhibitor than the (2R,3R)-enantiomer. When the I50 values of (2S,3S)- and (2R,3R)-3-(4-nitrophenyl)glycidol were compared, the (2S,3S)-enantiomer was at least a 750-fold better inhibitor (I50 = 1.6 microM) than the (2R,3R)-enantiomer (I50 = 1200 microM), and it was the most potent inhibitor tested in the 3-phenylglycidol series. If the hydroxyl group of the glycidol was masked or converted to another functionality, the potency of the inhibitor decreased and the (2S,3S)-enantiomer was not necessarily the better inhibitor. In addition, trans-3-phenylglycidols demonstrated slow binding inhibition of cytosolic epoxide hydrolase. Inhibitors without a hydroxyl group, or with a blocked hydroxyl group, were not slow binding inhibitors. These results suggested that the hydroxyl group was important in both enantioselectivity and time dependence of inhibition of cytosolic epoxide hydrolase by trans-3-phenylglycidols. The hydration pattern of (2S,3S)- and (2R,3R)-2,3-epoxy-3-(4- nitrophenyl)glycidol by cytosolic epoxide hydrolase also differed. When incorporation of [18O] from water catalyzed by cytosolic epoxide hydrolase was measured, the (2S,3S)-enantiomer gave 12% incorporation into the benzylic carbon and the (2R,3R)-enantiometer gave 40% incorporation into the benzylic carbon. Finally, trans-3-phenylglycidols were found to be poor inhibitors of microsomal epoxide hydrolase.

Photoaffinity labeling of methyl farnesoate epoxidase in cockroach corpora allata.

The last enzyme in the biosynthetic pathway to juvenile hormone III in the corpora allata of hemimetabolous insects is methyl farnesoate epoxidase, a cytochrome P450 monooxygenase. Assays with intact glands incubated in vitro and with gland homogenates have identified a series of 1,5-disubstituted imidazoles as potent inhibitors of the enzyme. We have designed, synthesized and tested two imidazoles, diazirine-Ice T and benzophenone-Ice T, in which a radiolabeled and photoactivatable diazirine or benzophenone group was introduced to label the hydrophobic substrate binding site of the enzyme. Our results show that these bifunctional compounds inhibit JH III synthesis by intact glands as well as methyl farnesoate epoxidation by gland homogenates. Moreover both compounds selectively label a protein of ca. 55 kDa in corpora allata of the cockroach, Diploptera punctata. These photoaffinity labels, which use an imidazole to coordinate to the heme iron and a photoreactive group to modify the hydrophobic substrate binding pocket, are specific and effective probes for the molecular analysis of methyl farnesoate epoxidase.

Comparative receptor surface analysis of agonists for tyramine receptor which inhibit sex-pheromone production in Plodia interpunctella.

The quantitative structure-activity relationship (QSAR) of a set of 29 agonists for tyramine (TA) receptor responsible for the inhibition of sex-pheromone production in Plodia interpunctella, was analyzed using comparative receptor surface analysis (CoRSA). Using the common steric and electrostatic features of the most active members of a series of compounds, CoRSA generated a virtual receptor model, represented as points on a surface complementary to the van der Waals or Wyvill steric surface of the aligned compounds. Three-dimensional energetics descriptors were calculated from receptor surface model (RSM)/ligand interaction and these three-dimensional descriptors were used in genetic partial least squares data analysis to generate a QSAR model, giving a 3D QSAR with r(2)=0.969 for calibration and CV- r(2)=0.635 for the leave-one-out cross validation.

Effects of pindolol on serum lipids, apolipoproteins, and lipoproteins in patients with mild to moderate essential hypertension.

The effects of 12 weeks' administration of the beta-blocker pindolol (5 mg twice daily) on serum lipids, apolipoproteins (apo), and lipoproteins were studied in 20 normolipidemic patients with mild to moderate essential hypertension (WHO I-II). Pindolol significantly increased both high-density lipoprotein cholesterol (HDL-C) and low-density lipoprotein cholesterol (LDL-C), while very-low-density lipoprotein cholesterol (VLDL-C) was significantly decreased. Apo A-II levels were increased significantly and the apo B/apo A-I ratio, which is one of the atherogenic indexes, was decreased significantly after pindolol therapy. Total cholesterol, HDL subfraction cholesterols, the LDL-C/HDL-C ratio, triglycerides, apo A-I, apo B, apo C-II, apo C-III, and apo E did not change significantly.

Three-dimensional pharmacophore hypotheses of octopamine receptor responsible for the inhibition of sex-pheromone production in Helicoverpa armigera.

Three-dimensional pharmacophore hypotheses were built on the basis of a set of nine octopamine (OA) agonists responsible for the inhibition of sex-pheromone production in Helicoverpa armigera. Of the 10 models generated by the program Catalyst/Hypo, hypotheses including hydrogen-bond acceptor (HBA), hydrophobic (Hp), and hydrophobic aliphatic (HpAl) features were considered important and predictive in evaluating OA agonists. An HBA and four hydrophobic features are the minimum components of an effective OA agonist-binding hypothesis, which resembles the results of binding activity to locust OAR3. Active agonists mapped well onto all of the features of the hypothesis, such as HBA, Hp, and HpAl features. On the other hand, inactive compounds lacking binding affinity were shown to be poorly capable of achieving an energetically favorable conformation shared by the active molecules in order to fit the 3D chemical feature pharmacophore models. Those hypotheses are considered useful in designing new leads for more active compounds. Further research on the comparison of models from agonists may help elucidate the mechanisms of OA receptor-ligand interactions.

Three-dimensional molecular field analyses of octopaminergic agonists and antagonists for the locust neuronal octopamine receptor class 3.

The quantitative structure-activity relationship (QSAR) of a set of 70 octopaminergic agonists and 20 antagonists against octopamine receptor class 3 (OAR3) in locust nervous tissue was analyzed by molecular field analysis (MFA). MFA of these compounds evaluated effectively the energy between a probe and a molecular model at a series of points defined by a rectangular grid. Contour surfaces for the molecular fields are presented. These results provide useful information in the characterization and differentiation of octopaminergic receptor types and subtypes.

Identification of novel inhibitors of calling and in vitro [14C]acetate incorporation by pheromone glands of Plodia interpunctella.

Some octopamine agonists were found to suppress in vitro biosynthesis of the calling pheromone of the Indian meal moth, Plodia interpunctella. Isolated pheromone-gland preparations incorporated sodium [14C]acetate at a linear rate for 3 h when incubated with the pheromone biosynthesis activating neuropeptide (PBAN). This incorporation was dependent on the dose of PBAN (up to 0.5 microM). Thin-layer chromatography of a pheromone-gland extract revealed quantitative incorporation of radioactivity into a product exhibiting the same mobility as (Z,E)-9,12-tetradecadienyl acetate, the main component of the calling pheromone of P interpunctella. Twenty-seven octopamine agonists were initially screened using a calling behaviour bioassay of female P interpunctella. Four derivatives with activity in the nanomolar range were identified which were, in order of decreasing pheromonostatic activity: 2-(2,6-diethylphenylimino)thiazolidine > 2-(2,6-diethylphenylimino)oxazolidine > 2-(2,6-dimethylphenylimino)thiazolidine > 2-(2-ethylphenylimino)oxazolidine. These compounds also showed in vitro inhibitory activity in intracellular de novo pheromone biosynthesis. The results of the present study indicate that these derivatives could provide useful information in the characterization and differentiation of octopaminergic receptor types and subtypes.

Quantitative structure-activity studies of octopaminergic agonists and antagonists against Locusta migratoria using similarity indexes.

The quantitative structure-activity relationship of 39 octopamine (OA) agonists and 12 antagonists against the thoracic nerve cord of the migratory locust, Locusta migratoria L. was analyzed using atom based rigid fit method or flexible fitting offered by PowerFit 1.0 from MicroSimulation. For OA agonists, the more similar to reference compound NC (24) the structure of test compound, the higher the activity, whereas for OA antagonists it was not the case. Antagonists may not interact with the same part of the membrane with which the agonists interact. Taken the part of the membrane with which the agonist interacts as the true receptor, the antagonist may well interact with an area surrounding the receptor including the ionophore.

Effect of tropine derivatives, antimuscarinic agents, on the growth of Bombyx mori larvae.

A number of atropine analogs were synthesized and their effects on larval growth of the silkworm, Bombyx mori, were investigated by both topical application and dietary administration. Among the tested compounds, 8-methyl-8-azabicyclo[3.2.1]octan-3 alpha-ol 2,2-diphenylpropionate (5), an antagonist of the muscarinic acetylcholine receptor in mammals, significantly prolonged the duration of the instar. When fed on compound 5 at 30 ppm, some of the larvae failed to molt. A 2,2-diphenylpropionate moiety was indispensable for this activity. Compound 5 had more potent activity than atropine which is known to inhibit PTTH release in vitro.

Docking study of enantiomeric fonofos oxon bound to the active site of Torpedo californica acetylcholinesterase.

Molecular interaction between enantiomeric fonofos oxon (O-ethyl S-phenyl ethylphosphonothiolate) and acetylcholinesterase (AChE) of Torpedo californica was evaluated by using the Cerius2 program. It was suggested that the difference in the inhibitory activity of the two enantiomers of fonofos oxon on AChE is due to the steric hindrance in binding to the AChE active site.

Synthesis and lateral root-inducing activity of novel N-substituted-2-piperidones with a 1,4-benzodioxan ring.

Novel N-substituted-2-piperidones with a 1,4-benzodioxan ring were prepared and evaluated for their activity to induce lateral roots in lettuce seedlings. Compounds were obtained by aldol condensation of the lithium enolate of N-substituted-2-piperidones with 1,4-benzodioxan-6-carbaldehyde. Of the series compounds tested, N-cinnamyl-3-[1-(1,4-benzodioxan-6-yl)-1-hydroxymethyl]-2-piperidone (2e) had the highest activity. In seedlings treated with 10 ppm of 2e, all of the primary roots formed lateral roots. Only erythro-2e showed lateral root-inducing activity, while threo-2e was inactive.

Nature of N-S bond cleavage of 2,3-dihydro-2,2-dimethyl-7-benzofuranyl (di-n-butylaminosulfenyl) (methyl)carbamate.

The cleavage of the N-S bond in 2,3-dihydro-2,2-dimethyl-7-benzofuranyl (di-n-butylaminosulfenyl) (methyl)carbamate was examined in different buffer solutions (hydrolysis), in buffer solution containing sulfhydryl reagents (thiolysis) and on thin-layer chromatographic plates. In buffer solution and on thin-layer plates, N-S bond cleavage readily occurred to give carbofuran as a major product, with minor amounts of bis-carbofuran-N,N'-disulfide and -trisulfide. The hydrolysis reaction in buffer proceeded with first-order kinetics. Significant amounts of an unknown polar compound were obtained in buffer solution and on thin-layer plates. In the presence of excess cysteine and glutathione at pH 7.0, thiolytic N-S bond cleavage occurred with first-order kinetics to give carbofuran as the sole identifiable product. At pH 5.0, three minor products were obtained along with carbofuran.

Root Growth-promoting Activities of N-Acyl-L-proline Derivatives.

A number of N-acyl-L-proline derivatives were synthesized and their biological activities were investigated by using lettuce (Lactuca sativa L. cv. Sacramento) seedling test. A wide variety of these compounds promoted root growth at 25°C both under light and in darkness. Of the compounds tested, N-(2-ftuorobenzoyl)-L-proline methyl ester (4) showed the highest activity and caused a 270% increase in the root elongation compared to the control. N-(2-Naphthoyl)-L-proline methyl ester (14) promoted the root growth, while N-(1-naphthoyl)-L-proline methyl ester inhibited it. L-Proline, benzoic acid, and 2-naphthoic acid had no significant effect on lettuce seedlings. Compounds 4 and 14, and N-(2-chlorobenzoyl)-L-proline methyl ester (7) reduced the inhibitory effect of 1 ppm ABA on the root growth, while the D-isomer of 4 was less activite than compound 4. Compounds 4, 7, and 14 did not show any rescue-activity for the complete inhibition of germination that was caused by treating 10 ppm of ABA.

Spectrophotometric substrates for cytosolic epoxide hydrolase.

In this study, we demonstrate the utility of a broad class of spectrophotometric substrates for the assay of cytosolic epoxide hydrolase purified from murine liver. These substrates, epoxy esters or carbonates, cyclize spontaneously upon or during hydrolysis of the epoxide functionality. The alcohol released by cyclization may then be assayed directly or by coupling to a second reaction. The alcohol produced, or its secondary reaction products, can be selected to give an absorption in the visible or near-uv range of the spectrum. This allows the synthesis of a wide variety of useful spectrophotometric substrates. 4-Nitrophenyl (2S,3S)-2,3-epoxy-3-phenylpropyl carbonate, at pH 6.4 and 25 degrees C, had a Vmax of 22 mumol min-1 mg-1 and a Km of 16 microM when assayed with a conventional spectrophotometer. When assayed under the same conditions with a 96-well plate reader, the measured Vmax was 15 mumol min-1 mg-1 and the Km was 6.6 microM. Some of these compounds were also found to be substrates for glutathione S-transferase, microsomal epoxide hydrolase, and porcine liver carboxylesterase. Indeed, 4-nitrophenyl 3,4-epoxy-3-phenylbutanoate was a 3.4-fold better substrate for porcine liver carboxylesterase than 4-nitrophenyl acetate when initial rates of hydrolysis were measured under the same conditions.

Three-dimensional quantitative structure-activity studies of octopaminergic agonists responsible for the inhibition of sex-pheromone production in Helicoverpa [correction of Hercoverpa] armigera.

The quantitative structure activity relationship (QSAR) of octopaminergic agonists responsible for the inhibition of sex-pheromone production in Helicoverpa [corrected] armigera, was analyzed using physicochemical parameters, molecular shape analysis (MSA), molecular field analysis (MFA), and receptor surface model (RSM), respectively. The dose-response studies were performed in vitro analyzing the effect of these compounds on intracellular cAMP production in the presence of pheromone biosynthesis activating neuropeptide (PBAN) at 1 pmol/intersegment. Six active derivatives were identified in the order of decreasing pheromonostatic activity: 2-(2,6-dimethylanilino)imidazolide (6) > 2-(2-methyl-4-chloroanilino)oxazolidine (1) > clonidine (5) > 2-(2,6-diethylanilino)thiazolidine (8) > 2-(3,5-dichlorobenzylamino)-2-oxazoline (4) > tolazoline (10) which were all active in the nanomolar range in inhibition of cAMP production by 1 pmol PBAN/intersegment. Four other compounds were less active having Ki in the micromolar range. An MSA was tried to obtain QSAR equation that incorporates spatial molecular similarity data of those compounds. MFA on the training set of those compounds evaluated effectively the energy between a probe and a molecular model at a series of points defined by a rectangular or spherical grid. An RSM was generated using some subset of the most active structures. Three-dimensional energetics descriptors were calculated from RSM/ligand interaction and these three-dimensional descriptors were used in QSAR analysis. These results indicate that these derivatives could provide useful information in the characterization and differentiation of octopaminergic receptor types and subtypes.

The interaction of cytosolic epoxide hydrolase with chiral epoxides.

1. The kinetic parameters of the cytosolic epoxide hydrolase were examined with two sets of spectrophotometric substrates. The (2S,3S)- and (2R,3R)-enantiomers of 4-nitrophenyl trans-2,3-epoxy-3-phenylpropyl carbonate had a KM of 33 and 68 microns and a Vmax of 16 and 27 mumol/min/mg, respectively. With the (2S,3S)- and (2R,3R)-enantiomers of 4-nitrophenyl trans-2,3-epoxy-3-(4-nitrophenyl)propyl carbonate, cytosolic epoxide hydrolase had a KM of 8.0 and 15 microM and a Vmax of 7.8 and 5.0 mumol/min/mg, respectively. 2. Glycidyl 4-nitrobenzoate had the lowest I50 of the compounds tested in the glycidyl 4-nitrobenzoate series (I50 = 140 microM). The I50 of the (2R)-enantiomer was 3.7-fold higher. The inhibitor with the lowest I50 in the glycidol series, and the lowest I50 of any compound tested, was (2S,3S)-3-(4-nitrophenyl)glycidol (I50 = 13.0 microM). It also showed the greatest difference in I50 between the enantiomers (330-fold). 3. All enantiomers of glycidyl 4-nitrobenzoates and trans-3-phenylglycidols gave differential inhibition of cytosolic epoxide hydrolase. However, neither the (S,S)-/(S)- or (R,R)-/(R)-enantiomer always had the lower I50. 4. Addition of one or more methyl groups to either enantiomer of glycidyl 4-nitrobenzoate resulted in increased I50. However, addition of a methyl group to C2 of either enantiomer of 3-phenylglycidol resulted in a decreased I50. Finally, when the hydroxyl group of trans-3-(4-nitrophenyl)glycidol was esterified the I50 of the (2S,3S)- but not the (2R,3R)-enantiomer increased.

Role of the carbohydrate moiety of a glucoamylase from Aspergillus awamori var. kawachi in the digestion of raw starch.

The digestion of raw starch by a glucoamylase (GA MU-H) from a mutant strain of Aspergillus awamori var. kawachi was closely correlated with mannoside chains O-linked to the Gp-I region (A470-V514), but not sugar chains N-linked to catalytic GAI' domain of GA MU-H. The partial replacement of mannose residues by glucose residues led to a significant decrease raw starch digestion. By the substitution of D2O for H2O in the reaction mixture, the raw starch digestion of GA MU-H decreased to 80% of that at 30 degrees C, although the rate of hydrolysis of soluble starch by and the ability to bind beta-cyclodextrin of GA MU-H were unchanged. Glycerol, known as an antichaotropic reagent, decreased the raw starch digestion of GA MU-H significantly. However, it did not have any effect on the enzymatic activity for soluble starch when soluble starch was the substrate. The efficient digestion of raw starch with raw starch-digesting glucoamylase needed the mannoside chains O-linked to the Gp-I region, which were suggested to contribute to digestion of raw starch through the interaction with water.

Effect of 1-(4-phenoxyphenoxypropyl)imidazole (KS-175) on larval growth in the silkworm Bombyx mori.

1-(4-Phenoxyphenoxypropyl)imidazole (KS-175), which has two types of characteristic moieties of insect growth regulators (IGRs), the phenoxyphenoxyalkyl group of juvenile hormone analogs (JHAs) and imidazole of 1,5-disubstituted imidazole such as KK-42, was tested for its biological activity on the silkworm, Bombyx mori. Penultimate (4th) instar larvae topically treated with KS-175 did not molt for more than 20 days. This activity was different from that reported for any IGRs. After the treatment, ecdysteroid levels in the hemolymph did not increase and the cells of the prothoracic gland had shrunk. When the treated penultimate larvae were fed an artificial diet supplemented with 20 ppm of 20-hydroxyecdysone, the larvae molted to the ultimate (5th) instar with a timing similar to that of control larvae fed a diet with or without 20-hydroxyecdysone. These results suggest that topical application of KS-175 irreversibly damages ecdysone biosynthesis in the prothoracic glands.

Enantioselective Synthesis of a (+)-(2R, 3R)-1,4-Benzodioxane-7-carbaldehyde Derivative, a Key Intermediate in the Total Synthesis of Haedoxan Analogs.

(+)-(2R,3R)-7-Formyl-6-methoxy-2-methoxymethyl-3- (3,4-methylenedioxyphenyl)-2,3-dihydro-1,4-benzodioxin (2), a key building block for the total synthesis of haedoxan A, was synthesized from (4R)-4-(phenylmethyl)-2-oxazolidinone (3) in ten steps with a 12% overall yield.