Design and synthesis of anti-breast cancer agents from 4-piperazinylquinoline: A hybrid pharmacophore approach

A novel class of 4-piperazinylquinoline derivatives based on the isatin scaffold were designed by molecular hybridization approach and synthesized for biological evaluation. Subsequently, the compounds were examined for their cytotoxic effects on two human breast tumor cell lines, MDA-MB468 and MCF7, and two non-cancer breast epithelial cell lines, 184B5 and MCF10A. Although all compounds examined were quite effective on the breast cancer cell lines examined, the compound 4-bromo-1-[4-(7-chloro-quinolin-4-yl)-piperazin-1-ylmethyl]-1H-indole-2,3-dione (5b) and N¹-[4-(7-trifluoromethyl-quinolin-4-yl)]-piperazin-1-ylmethyl-4-chloro-1H-indole-2,3-dione-3-thiosemicarbazone (8a) emerged as the most active among this series. It appeared that both 5b and 8a caused apoptosis to MCF7 cancer cells, but not MCF10A non-cancer cells. Thus, 4-piperazinylquinoline linked isatin analog can serve as the prototype molecule for further development of a new class of anti-breast cancer agents.     

Design,synthesis and in vitro cytotoxicity evaluation of 5-(2-carboxyethenyl)isatin derivatives as anticancer agents

Forty four di- or trisubstituted novel isatin derivatives were designed and synthesized in 5–6 steps in 25–45% overall yields. Their structures were confirmed by ¹H NMR and ¹³C NMR as well as LC–MS. The anticancer activity of these new isatin derivatives against three human tumor cell lines, K562, HepG2 and HT-29, were evaluated by MTT assay in vitro. SAR studies suggested that the combination of 1-benzyl and 5-[trans-2-(methoxycarbonyl)ethen-1-yl] substitution greatly enhance their cytotoxic activity, whereas an intact carbonyl functionality on C-3 as present in the parent ring is required to such a potency. This study leads to the identification of two highly active molecules, compounds 2h (IC₅₀ = 3 nM) and 2k (IC₅₀ = 6 nM), against human leukemia K562 cells.     

Altered Circulating Levels of Retinol Binding Protein 4 and Transthyretin in Relation to Insulin Resistance,Obesity, and Glucose Intolerance in Asian Indians

Objective: Retinol binding protein 4 (RBP4) has been implicated in metabolic disorders including type 2 diabetes mellitus (T2DM), but few studies have looked at transthyretin (TTR) with which RBP4 is normally bound to in the circulation. We report on the systemic levels of RBP4 and TTR and their associations with insulin resistance, obesity, prediabetes, and T2DM in Asian Indians.Methods: Age-matched individuals with normal glucose tolerance (NGT, n = 90), impaired glucose tolerance (IGT, n = 70) and T2DM (n = 90) were recruited from the Chennai Urban Rural Epidemiology Study (CURES). Insulin resistance was estimated using the homeostasis model assessment of insulin resistance (HOMA-IR). RBP4 and TTR levels were measured by enzyme-linked immunosorbent assay (ELISA).Results: Circulatory RBP4 and TTR levels (in μg/mL) were highest in T2DM (RBP4: 13 ± 3.9, TTR: 832 ± 310) followed by IGT (RBP4: 10.5 ± 3.2; TTR: 720 ± 214) compared to NGT (RBP4: 8.7 ± 2.5; TTR: 551 ± 185; P<.001). Compared to nonobese NGT individuals, obese NGT, nonobese T2DM, and obese T2DM had higher RBP4 (8.1 vs. 10.6, 12.1, and 13.2 μg/mL, P<.01) and TTR levels (478 vs. 737, 777, and 900 μg/mL, P<.01). RBP4 but not TTR was significantly (P<.001) correlated with insulin resistance even among NGT subjects. In regression analysis, RBP4 and TTR showed significant associations with T2DM after adjusting for confounders (RBP4 odds ratio [OR]: 1.107, 95% confidence interval [CI]: 1.008–1.216; TTR OR: 1.342, 95% CI: 1.165–1.547).Conclusion: Circulatory levels of RBP4 and TTR showed a significant associations with glucose intolerance, obesity, T2DM and RBP4 additionally, with insulin resistance.Abbreviations: BMI = body mass index CI = confidence interval HDL = high-density lipoprotein IGT = impaired glucose tolerance LDL = low-density lipoprotein NGT = normal glucose tolerance OGTT = oral glucose tolerance test OR = odds ratio RBP4 = retinol binding protein 4 T2DM = type 2 diabetes mellitus TTR = transthyretin WC = waist circumference     

Synthesis of new fluorinated, 2-substituted 5-pyrrolidinylsulfonyl isatin derivatives as caspase-3 and caspase-7 inhibitors: Nonradioactive counterparts of putative PET-compatible apoptosis imaging agents

Downstream caspases-3 and -7 are essential to execute the programmed type I cell death (apoptosis). In order to better understand their role, specific inhibitors of these enzymes are required, which after radiolabeling can be applied to non-invasively visualize and monitor apoptotic pathways in vivo using Positron Emission Tomography (PET). Therefore, 2-methoxyethyl-, 2-methoxypropyl-, 2-ethoxymethyl-, 2-(2-fluoroethoxymethyl)-, and 2-(2,2,2-trifluoroethoxymethyl)pyrrolidinyl analogues of (S)-5-[1-(2-methoxymethylpyrrolidinyl)sulfonyl]isatin (2) were prepared and their in vitro binding affinities towards caspases-1, -3, -6 and -7 were evaluated and compared to that of the lead structure 2. While the inhibition potencies against caspases-1 and -6 were in the micromolar range, all synthesized compounds exhibited excellent and selective inhibition of caspases-3 and -7 in the nanomolar range up to IC₅₀ = 4.79 nM and 7.47 nM, respectively. These highly potent 2-substituted analogues of 2 might be developed as anti-apoptosis agents and some selected fluorinated inhibitors might be useful as potential PET radiotracers for apoptosis imaging after ¹⁸F-labeling.     

Novel Zn2+-binding Sites in Human Transthyretin: IMPLICATIONS FOR AMYLOIDOGENESIS AND RETINOL-BINDING PROTEIN RECOGNITION*

Human transthyretin (TTR) is a homotetrameric protein involved in several amyloidoses. Zn²⁺ enhances TTR aggregation in vitro, and is a component of ex vivo TTR amyloid fibrils. We report the first crystal structure of human TTR in complex with Zn²⁺ at pH 4.6–7.5. All four structures reveal three tetra-coordinated Zn²⁺-binding sites (ZBS 1–3) per monomer, plus a fourth site (ZBS 4) involving amino acid residues from a symmetry-related tetramer that is not visible in solution by NMR. Zn²⁺ binding perturbs loop E-α-helix-loop F, the region involved in holo-retinol-binding protein (holo-RBP) recognition, mainly at acidic pH; TTR affinity for holo-RBP decreases ∼5-fold in the presence of Zn²⁺. Interestingly, this same region is disrupted in the crystal structure of the amyloidogenic intermediate of TTR formed at acidic pH in the absence of Zn²⁺. HNCO and HNCA experiments performed in solution at pH 7.5 revealed that upon Zn²⁺ binding, although the α-helix persists, there are perturbations in the resonances of the residues that flank this region, suggesting an increase in structural flexibility. While stability of the monomer of TTR decreases in the presence of Zn²⁺, which is consistent with the tertiary structural perturbation provoked by Zn²⁺ binding, tetramer stability is only marginally affected by Zn²⁺. These data highlight structural and functional roles of Zn²⁺ in TTR-related amyloidoses, as well as in holo-RBP recognition and vitamin A homeostasis.     

Monoaryl derivatives as transthyretin fibril formation inhibitors: Design,synthesis, biological evaluation and structural analysis

Transthyretin (TTR) is a ß-sheet-rich homotetrameric protein that transports thyroxine (T4) and retinol both in plasma and in cerebrospinal fluid. TTR also interacts with amyloid-β, playing a protective role in Alzheimer’s disease. Dissociation of the native transthyretin (TTR) tetramer is widely accepted as the critical step in TTR amyloids fibrillogenesis, and is responsible for extracellular deposition of amyloid fibrils. Small molecules, able to bind in T4 binding sites and stabilize the TTR tetramer, are interesting tools to treat and prevent systemic ATTR amyloidosis. We report here the synthesis, in vitro evaluation and three-dimensional crystallographic analyses of new monoaryl-derivatives in complex with TTR. Of the derivatives reported here, the best inhibitor of TTR fibrillogenesis, 1d, exhibits an activity similar to diflunisal.     

Synthesis and evaluation of isatin analogs as caspase-3 inhibitors: introduction of a hydrophilic group increases potency in a whole cell assay

A series of isatin analogs containing a hydrophilic group, including a pyridine ring, ethylene glycol group, and a triazole ring, have been synthesized, and their inhibition potency for caspase-3 was measured both in vitro (i.e., recombinant enzyme) and in whole cells (HeLa cells). The analogs having a hydrophilic group, including 12, 13, 16, 38, and 40, have dramatically increased activity in vitro and in HeLa cells compared to the corresponding unsubstituted N-phenyl isatin analogs.     

Synthesis of 5- and 6-substituted 2-(4-dimethylaminophenyl)-1,3-benzoxazoles and their in vitro and in vivo evaluation as imaging agents for amyloid plaque

A series of novel 5- and 6-substituted 2-(4-dimethylaminophenyl)-1,3-benzoxazoles was synthesized and their potential as imaging probes for Alzheimer’s Disease (AD)-related amyloid plaque was evaluated in vitro and in vivo. In vitro binding affinities for Aβ1–40 peptide of several of these compounds were in the low-nanomolar range . The lowest K_i of 9.3 nM was found for N-(2-(4-(dimethylamino)phenyl)-1,3-benzoxazol-5-yl)-4-iodobenzamide (1e). Its ¹²³I-radiolabeled form ([¹²³I]1e) was subsequently prepared by iododestannylation of the corresponding tributylstannyl precursor and evaluated in vivo in a baboon model using SPECT imaging. Contrary to our expectations, 1e did not cross the blood–brain barrier (BBB) to any significant extent.     

Acetolysis of Leuconostoc mesenteroides NRRL B-1299 Dextran. Isolation and characterization of tetrasacharrides containing secondary linkages from the borate-insoluble fraction

Fractionation of the deacetylated acetolyzate of the borate-insoluble fraction of the dextran elaborated by Leuconostoc mesenteroides NRRL B-1299 gave five tetrasaccharide fractions, isolated after chromatography on charcoal—Celite, paper chromatography, and paper electrophoresis. Examination of partial acid hydrolyzates of the tetrasaccharide fractions and their corresponding alditols, the relation between the logarithm of their partition functions (α') and molecular size, and methylation studies, showed them to be (a) 2³-α-d-glucosyl-nigerotriose (1), (b) a mixture of 6-α-nigerotriosyl-d-glucose (2) and 6¹-α-d-glucosyl-nigerotriose (3) and/or 6²-α-d-glucosyl-nigerotriose (4), (c) a mixture of 2¹-α-nigerosyl-isomaltose (5) and 3²-α-isomaltosyl-kojibiose (6) and/or 6²-α-nigerosyl-kojibiose (7), (d) 2-α-nigerotriosyl-d-glucose (8) and (e) nigerotetraose (9).     

Three new compounds isolated from the bulbs of Fritillaria pallidiflora Schrenk and their anti-inflammatory activity

Three new compounds, 17β-cevanin-6-oxo-5α,20β-diol yibeinine (1), 2-(tetrahydro-5-(2-hydroxyphenyl)-2H-pyran-3-yl) phenol (2), 1,3-O-diferuloyl-2-methoxypropane diol (3), as well as four known compounds (4–7), have been isolated from the ethanol extract of dried bulbs of Fritillaria pallidiflora Schrenk. All structures were determined based on their spectroscopic data (1D and 2D NMR (including ¹H NMR, ¹³C NMR, ¹H-¹H COSY, HMBC, HSQC, HSQC-TOCSY, and NOESY experiments), and MS). Biological evaluation showed that compounds 1–4 inhibited the production of nitric oxide (NO) in LPS-stimulated RAW 264.7 cells with IC₅₀ values of 18.0, 38.7, 29.5, and 47.1 μM, respectively. These results indicated that compound 1 has potential anti-inflammatory activity.     

Synthesis and biological evaluation of some new mono Mannich bases with piperazines as possible anticancer agents and carbonic anhydrase inhibitors

New mono Mannich bases, (2-(4-hydroxy-3-((4-substituephenylpiperazin-1-yl)methyl)benzylidene)-2,3-dihydro-1H-inden-1-one), were prepared to evaluate their cytotoxic/anticancer properties and also their inhibitory effects on human carbonic anhydrase I and II isoenzymes (hCA I and II). Amine part was changed as [N-phenylpiperazine (1), N-benzylpiperazine (2), 1-(2-fluorophenyl)piperazine (3), 1-(4-fluorophenyl)piperazine (4), 1-(2-methoxyphenyl)piperazine (5)]. The structure of the synthesized compounds was characterized by ¹H NMR, ¹³C NMR and HRMS spectra. Cytotoxicity results of the series pointed out that the compound 4 had the highest tumor selectivity value (TS: 59.4) possibly by inducing necrotic cell death in series. Additionally, all compounds synthesized showed a good inhibition profile towards hCA I and II isoenzymes with the Ki values between 29.6 and 58.4 nM and 38.1–69.7 nM, respectively. These values were lower than the reference compound AZA. However, it seems that the compounds 4 and 2 can be considered as lead compounds of CA studies with the lowest Ki values in series for further designs.     

Cytotoxic palladium(II) complexes of 8-aminoquinoline derivatives and the interaction with human serum albumin

Palladium(II) complexes are potential antitumor metallodrugs for their chemical resemblance to platinum(II) complexes. Two palladium(II) complexes (1 and 2) in the formula of [PdLⁿCl] [L¹ = N-(tert-butoxycarbonyl)-l-methionine-N′-8-quinolylamide, L² = L-alanine-N′-8-quinolylamide] have been synthesized accordingly. The structures of the complexes were fully characterized by X-ray crystallography. The palladium(II) center in 1 is coordinated by two N atoms and an S atom from L¹ with one chloride anion as the leaving group; while that in 2 is coordinated by three N atoms from L² with one chloride anion as the leaving group. The interaction between complex 1 and human serum albumin (HSA) has been investigated using fluorescence and circular dichroism spectroscopies. The complex seems to react with HSA chiefly through hydrophobic and electrostatic interactions, and it does not alter the α-helical nature of HSA. The cytotoxicity of these complexes has been tested against the human cervical cancer (HeLa), human mammary cancer (MCF-7), and human lung cancer (A-549) cell lines. Complex 1 displays a cytotoxic activity comparable to that of cisplatin, but complex 2 is less active than cisplatin.     

Two new lignans with their biological activities in Portulaca oleracea L.

Two new lignans, identified as 6,7-dihydroxy-4-(4′’-hydroxy-3′’-methoxyphenyl)-2-naphthoic acid, named Oleralignan C (1), and 4-(3,4-dihydroxyphenyl)-6-hydroxy-7-methoxy-2-naphthoic acid, named Oleralignan D (2), were obtained from Portulaca oleracea L. The structures were determined by spectroscopic methods, including UHPLC-ESI-QTOFMS, ¹D and ²D NMR. Both Oleralignan C (1) and Oleralignan D (2) inhibited the inflammatory factors, IL-1β and TNF-α in RAW 264.7 cells induced by lipopolysaccharide (LPS). Both compounds also could clear 1,1-diphenyl-2-picryl-hydrazyl (DPPH) radicals indicating their antioxidant potential.     

Facile one-pot synthesis of novel dispirooxindole-pyrrolidine derivatives and their antimicrobial and anticancer activity against A549 human lung adenocarcinoma cancer cell line

Novel dispirooxindole-pyrrolidine derivatives have been synthesized through 1,3-dipolar cycloaddition of an azomethine ylide generated from isatin and sarcosine with the dipolarophile 3-(1H-indol-3-yl)-3-oxo-2-(2-oxoindolin-3-ylidene)propanenitrile, and also spiro compound of acenaphthenequinone obtained by the same optimized reaction condition. Synthesized compounds were evaluated for their antimicrobial activity and all the compounds shown significant activity. Anticancer activity was evaluated against A549 human lung adenocarcinoma cancer cell lines. Compounds 7b, 7g, 7i and 7r exhibit very good anticancer activity 62.96%, 62.03%, 67.67% and 60.22%, respectively, at the dose of 200 μg/mL and compound 7i shows IC₅₀ value in 50 μg/mL.     

Characterization of Oncorhynchus mykiss 5-hydroxyisourate hydrolase/transthyretin superfamily: Evolutionary and functional analyses

Teleosts have highly diverged genomes that resulted from whole genome duplication, which leads to an extensive diversity of paralogous genes. Transthyretin (TTR), an extracellular thyroid hormone (TH) binding protein, is thought to have evolved from an ancestral 5-hydroxyisourate hydrolase (HIUHase) by gene duplication at some stage of chordate evolution. To characterize the functions of proteins that arose from duplicated genes in teleosts, we investigated the phylogenetic relationship of teleost HIUHase and TTR aa sequences, the expression levels of Oncorhynchus mykiss HIUHase and TTR mRNA in various tissues and the biological activities of the O. mykiss re-HIUHase and re-TTR. Phylogenetic analysis of the teleost aa sequences revealed the presence of two HIUHase subfamilies, HIUHase 1 (which has an N-terminal peroxisomal targeting signal-2 [PTS2]) and HIUHase 2 (which does not have an N-terminal PTS2), and one TTR family. The tissue distributions of HIUHase 1 and TTR mRNA were similar in juvenile O. mykiss and the mRNA levels were highest in the liver. The O. mykiss re-HIUHase and re-TTR proteins were both 40–50 kDa homotetramers consisting of 14–15 kDa subunits, with 30% identity. HIUHase had 5-hydroxyisourate (5-HIU) hydrolysis activity with Zn^2 + sensitivity, whereas TTR had ligand binding activity with a preference for THs and several environmental chemicals, such as halogenated phenols. Our results suggest that O. mykiss HIUHase and TTR have diverged from a common ancestral HIHUase with no functional complementation.     

Facile synthesis of carbon-11-labeled sEH/PDE4 dual inhibitors as new potential PET agents for imaging of sEH/PDE4 enzymes in neuroinflammation

To develop PET tracers for imaging of neuroinflammation, new carbon-11-labeled sEH/PDE4 dual inhibitors have been synthesized. The reference standard N-(4-methoxy-2-(trifluoromethyl)benzyl)benzamide (1) and its corresponding desmethylated precursor N-(4-hydroxy-2-(trifluoromethyl)benzyl)benzamide (2) were synthesized from (4-methoxy-2-(trifluoromethyl)phenyl)methanamine and benzoic acid in one and two steps with 84% and 49% overall chemical yield, respectively. The standard N-(4-methoxy-2-(trifluoromethyl)benzyl)-1-propionylpiperidine-4-carboxamide (MPPA, 4) and its precursor N-(4-hydroxy-2-(trifluoromethyl)benzyl)-1-propionylpiperidine-4-carboxamide (5) were synthesized from methyl 4-piperidinecarboxylate, propionyl chloride and (4-methoxy-2-(trifluoromethyl)phenyl)methanamine in two and three steps with 62% and 34% overall chemical yield, respectively. The target tracers N-(4-[¹¹C]methoxy-2-(trifluoromethyl)benzyl)benzamide ([¹¹C]1) and N-(4-[¹¹C]methoxy-2-(trifluoromethyl)benzyl)-1-propionylpiperidine-4-carboxamide ([¹¹C]MPPA, [¹¹C]4) were prepared from their corresponding precursors 2 and 5 with [¹¹C]CH₃OTf through O-[¹¹C]methylation and isolated by HPLC combined with SPE in 25–35% radiochemical yield, based on [¹¹C]CO₂ and decay corrected to end of bombardment (EOB). The radiochemical purity was >99%, and the molar activity (A_M) at EOB was 370–740 GBq/μmol with a total synthesis time of 35–40-minutes from EOB.     

Studies on dehydro-l-ascorbic acid 2-arylhydrazone 3-oximes: Conversion into substituted triazoles and isoxazolines

l-threo-2,3-Hexodiulosono-1,4-lactone 2-(arylhydrazones) (2) were prepared by condensation of dehydro-l-ascorbic acid with various arylhydrazines. Reaction of 2 with hydroxylamine gave the 2-(arylhydrazone) 3-oximes (3). On boiling with acetic anhydride, 3 gave 2-aryl-4-(2,3-di-O-acetyl-l-threo-glycerol-l-yl)-1,2,3-triazole-5-carboxylic acid 5,4¹-lactones (4). On treatment of 4 with liquid ammonia, 2-aryl-4-(l-threo-glycerol-l-yl)-1,2,3-triazole-5-carboxamides (5) were obtained. Acetylation of 5 with acetic anhydride-pyridine gave the triacetates, and vigorous acetylation with boiling acetic anhydride gave the tetraacetyl derivatives. Periodate oxidation of 5 gave the 2-aryl-4-formyl-1,2,3-triazole-5-carboxamides (8), and, on reduction, 8 gave the 2-aryl-4-(hydroxymethyl)-1,2,3-triazole-5-carboxamides, characterized as the monoacetates and diacetates. Controlled reaction of 2 with sodium hydroxide, followed by neutralization, gave 3-(l-threo-glycerol-l-yl)-4,5-isoxazolinedione 4-(arylhydrazones), characterized by their triacetates. Reaction of 2 with HBr-HOAc gave 5-O-acetyl-6-bromo-6-deoxy-l-threo-2,3-hexodiulosono-1,4-lactone 2-(arylhydrazones); these were converted into 4-(2-O-acetyl-3-bromo-3-deoxy-l-threo-glycerol-l-yl)-2-aryl-1,2,3-triazole-5-carboxylic acid 5,4¹-lactones on treatment with acetic anhydride-pyridine.     

Design,synthesis, and biological evaluation of radioiodinated benzo[d]imidazole-quinoline derivatives for platelet-derived growth factor receptor β (PDGFRβ) imaging

Several malignant tumors and fibrotic diseases are associated with PDGFRβ overexpression and excessive signaling, making this receptor attractive for molecular targeting and imaging approaches. A series of benzo[d]imidazole-quinoline derivatives were designed and synthesized to develop radioiodinated compounds as PDGFRβ-specific imaging probes. The structure activity relationship (SAR) evaluation of the designed compounds was performed. Among them, 2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]-8-(piperazin-1-yl)quinoline (5a) and 4-{2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]quinolin-8-yl}morpholine (5d) exhibited a relatively high PDGFRβ-TK inhibitory potency, whereas iodinated 5a derivative 5-iodo-2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]-8-(piperazin-1-yl)quinoline (8) exhibited a superior inhibitory potency as PDGFRβ inhibitor than iodinated 5d derivative 4-{5-iodo-2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]quinolin-8-yl}morpholine (11). Furthermore, [¹²⁵I]8 and [¹²⁵I]11 were synthesized and evaluated for PDGFRβ radioligand ability, both in vitro and in vivo. Cellular uptake experiments showed that [¹²⁵I]8 had a higher uptake in BxPC3-luc cells as PDGFRβ-positive cells than [¹²⁵I]11. Incubation of [¹²⁵I]8 after pretreatment of PDGFRβ ligands significantly reduced the uptake of [¹²⁵I]8. In biodistribution experiments using tumor-bearing mice, [¹²⁵I]8 accumulation in the tumor 1?h postinjection was higher than that of the benzo[d]imidazol-quinoline derivative [¹²⁵I]IIQP, used in our previous research. These results indicate that [¹²⁵I]8 could be a promising PDGFRβ imaging agent. Although its clinical application requires further structural modifications, the results obtained in this research may be useful for the development of PDGFRβ-specific radioligands.     

S-sulfonation of transthyretin is an important trigger step in the formation of transthyretin-related amyloid fibril

Senile systemic amyloidosis and familial amyloid polyneuropathy are caused by oxidative deposition of conformationally altered transthyretin (TTR). We identified oxidative modification of the 10th cysteine of TTR through S-sulfonation in vitro. Based on mass spectrometric analysis, we determined the spectrophotometric, western blotting, and fluororescent microscopic properties of TTR incubated with and without cysteine-S-sulfonate in acidic (pH 4) and alkaline (pH 8) conditions at 37°. The absorption of the aggregated TTR molecules increased more with incubation time and the concentration of cysteine-S-sulfonate at pH 4 than at pH 8. The Congo red binding to the S-sulfonated TTR at pH 4 was saturated with an apparent Bmax of 2.01 mol per mole of the S-sulfonated TTR and apparent K_D of 7.75 × 10⁻⁶ M. On the other hand, the Bmax of cysteinyl TTR was 1.38, and its K_D was 3.52 × 10⁻⁶ M while the Bmax of reduced TTR was 0.86, and its K_D was 2.86 × 10⁻⁶ M. Moreover, we detected positive amyloid fibril staining using Thioflavin T and Congo red with the S-sulfonated TTR but not with untreated or reduced TTR by microscopic fluororescent analysis. After modification of TTR in vitro, oligomers resisted reduction and denaturation was irreversibly induced, and which contributed differences in the Western blotting patterns obtained with four anti-TTR antibodies. In conclusion, this study showed that the formation of S-sulfonation of TTR through oxidative modifications of the thiol residue on the 10th cysteine of TTR is an important trigger step in the formation of transthyretin-related amyloid fibril.     

The metabolism of [2-14C]indole in the rat

1. [2-¹⁴C]Indole has been synthesized from [¹⁴C]formate and o-toluidine via N[¹⁴C]-formyltoluidine. 2. When fed to rats, the ¹⁴C of [¹⁴C]indole (dose 70–80mg./kg. body wt.) is fairly rapidly excreted, and in 2 days an average of 81% appears in the urine, 11% in the faeces and 2·4% as carbon dioxide in the expired air. 3. Radioactivity is excreted in the urine as indoxyl sulphate (50% of the dose), indoxyl glucuronide (11%), oxindole (1·4%), isatin (5·8%), 5-hydroxyoxindole conjugates (3·1%), N-formylanthranilic acid (0·5%) and unchanged indole (0·07%). The faeces contain indoxyl sulphate (0·4% of the dose) and indole (0·2%), but the major metabolites have not been identified. 4. Fed to rats with biliary cannulae an average of 5·6% of a dose of [¹⁴C]indole (20–60mg./kg. body wt.) is excreted in the bile in 2 days. Radioactivity is present as indoxyl sulphate (0·8% dose) and 5-hydroxyoxindole conjugates (0·6%). 5. Rats further metabolize indoxyl into N-formylanthranilic acid and anthranilic acid, and oxindole into 5-hydroxyoxindole. 6. With rat-liver microsomes plus supernatant under aerobic conditions, indole gives indoxyl, oxindole, possibly isatin, N-formylanthranilic acid and anthranilic acid, but under anaerobic conditions gives only oxindole. Similarly, under aerobic conditions, oxindole gives 5-hydroxyoxindole, anthranilic acid and o-aminophenylacetic acid. 7. Indole is metabolized by two pathways, one via indoxyl to isatin, N-formylanthranilic acid and anthranilic acid, and the other via oxindole to 5-hydroxyoxindole and possibly to o-aminophenylacetic and anthranilic acid. 8. The following new compounds are described: 4-hydroxy-2-nitrophenylacetic acid, 3-, 4- and 5-benzyloxy-2-nitrophenylacetic acid, 5- and 7-hydroxyoxindole and 5-aminoacridine indoxyl sulphate.