Effect of Auxiliary Donors on 3,8-Phenothiazine Dyes for Dye-Sensitized Solar Cells.

Phenothiazines are one of the more common dye scaffolds for dye-sensitized solar cells. However, these sensitizers are exclusively based on a 3,7-substitution pattern. Herein, we have synthesized and characterized novel 3,8-substituted phenothiazine dyes in order to evaluate the effect of auxiliary donor groups on the performance of this new dye class. The power conversion efficiency increased by 7%-10% upon insertion of an auxiliary donor in position 8 of the phenothiazine, but the structure of the auxiliary donor (phenyl, naphthyl, pyrene) had a low impact when electrodes were stained with chenodeoxycholic acid (CDCA) additive. In the absence of CDCA, the highest power conversion efficiency was seen for the phenyl-based sensitizer attributed to a higher quality dye-monolayer. By comparing the novel dyes to their previously reported 3,7- analogues, only subtle differences were seen in photophysical, electrochemical, and performance measurements. The most notable difference between the two geometries is a lowering of the oxidation potentials of the 3,8-dyes by 40-50 mV compared to the 3,7-analogues. The best auxiliary donor for the 3,8-phenothiazine dyes was found to be pyrenyl, with the best device delivering a power conversion efficiency of 6.23% (99 mW cm-2, 10 eq. CDCA, JSC = 10.20 mA cm-2, VOC = 791 mV, and FF = 0.765).

Lipid accumulation inhibitory activities of novel isoxazole-based chenodeoxycholic acids: Design, synthesis and preliminary mechanism study.

In continuation of our drug discovery program on hyperlipidemia, a series of novel isoxazole-chenodeoxycholic acid hybrids were designed, synthesized and evaluated for their lipid-lowering effects. Preliminary screening of all the synthesized compounds was done by using a 3T3-L1 adipocyte model, in which the most active compound 16b could significantly reduce the lipid accumulation up to 30.5% at a nontoxic concentration 10 µM. Further mechanism studies revealed that 16b blocked lipid accumulation via activating FXR-SHP signaling pathway, efficiently down-regulated the expression of key lipogenesis regulator SREBP-1c.

Synthesis of 1ß-hydroxydeoxycholic acid in H-2 and unlabeled forms.

1ß-hydroxydeoxycholic acid in unlabeled and stable isotope labeled forms was required for use as a biomarker for Cytochrome P450 3A4/5 activity. A lengthy synthesis was undertaken to deliver the unlabeled compound and in the process, to develop a route to the deuterium labeled compound. The synthesis of the unlabeled compound was completed but in a very low yield. Concurrent with the synthetic approach, a biosynthetic route was pursued and this approach proved to be much more rapid and afforded the compound in both unlabeled and deuterium labeled forms in a 1-step oxidation from deoxycholic acid and [D4 ]deoxycholic acid, respectively.

Chemical Synthesis of Uncommon Natural Bile Acids: The 9α-Hydroxy Derivatives of Chenodeoxycholic and Lithocholic Acids.

The chemical synthesis of the 9α-hydroxy derivatives of chenodeoxycholic and lithocholic acids is reported. For initiating the synthesis of the 9α-hydroxy derivative of chenodeoxycholic acid, cholic acid was used; for the synthesis of the 9α-hydroxy derivative of lithocholic acid, deoxycholic acid was used. The principal reactions involved were (1) decarbonylation of conjugated 12-oxo-Δ(9(11))-derivatives using in situ generated monochloroalane (AlH2Cl) prepared from LiAlH4 and AlCl3, (2) epoxidation of the deoxygenated Δ(9(11))-enes using m-chloroperbenzoic acid catalyzed by 4,4'-thiobis-(6-tert-butyl-3-methylphenol), (3) subsequent Markovnikov 9α-hydroxylation of the Δ(9(11))-enes with AlH2Cl, and (4) selective oxidation of the primary hydroxyl group at C-24 in the resulting 3α,9α,24-triol and 3α,7α,9α,24-tetrol to the corresponding C-24 carboxylic acids using sodium chlorite (NaClO2) in the presence of a catalytic amount of 2,2,6,6-tetramethylpiperidine 1-oxyl free radical (TEMPO) and sodium hypochlorite (NaOCl). The (1)H- and (13)C-NMR spectra are reported. The 3α,7α,9α-trihydroxy-5ß-cholan-24-oic acid has been reported to be present in the bile of the Asian bear, and its 7-deoxy derivative is likely to be a bacterial metabolite. These bile acids are now available as authentic reference standards, permitting their identification in vertebrate bile acids.

Practical synthesis of chenodeoxycholic acid from phocaecholic acid.

In this study, we developed an effective method for the large-scale synthesis of chenodeoxycholic acid (CDCA) from phocaecholic acid (PhCA). A high total yield of up to 72 % was obtained via five steps including methyl esterification, Ts-protection, bromination, reduction, and hydrolysis. The structures of the intermediates were confirmed by 1H NMR (Nuclear Magnetic Resonance), 13C NMR, HRMS (High Resolution Mass Spectrometry), and IR (Infrared Spectroscopy) spectroscopies. This method offers a new and practical approach to the synthesizing of CDCA.

[Progress in the ligands and their complex structures of farnesoid X receptor].

Farnesoid X receptor (FXR) belongs to the nuclear receptor superfamily. It is highly related to the formation of metabolic syndrome and the glucose homeostasis, and therefore represents an important drug target against metabolic diseases and diabetes. In recent years, great progress has been made in the agonists, antagonists, and crystal structures of FXR. The diverse FXR ligands and their structure-activity relationship are reviewed in this article. The advances in the crystal structures of FXR in complex with different ligands are also introduced.

Development and in†vitro Profiling of Dual FXR/LTA4H Modulators.

Designed polypharmacology presents as an attractive strategy to increase therapeutic efficacy in multi-factorial diseases by a directed modulation of multiple involved targets with a single molecule. Such an approach appears particularly suitable in non-alcoholic steatohepatitis (NASH) which involves hepatic steatosis, inflammation and fibrosis as pathological hallmarks. Among various potential pharmacodynamic mechanisms, activation of the farnesoid X receptor (FXRa) and inhibition of leukotriene A4 hydrolase (LTA4Hi) hold promise to counteract NASH according to preclinical and clinical observations. We have developed dual FXR/LTA4H modulators as pharmacological tools, enabling evaluation of this polypharmacology concept to treat NASH and related pathologies. The optimized FXRa/LTA4Hi exhibits well-balanced dual activity on the intended targets with sub-micromolar potency and is highly selective over related nuclear receptors and enzymes rendering it suitable as tool to probe synergies of dual FXR/LTA4H targeting.

Structural requirements of the ASBT by 3D-QSAR analysis using aminopyridine conjugates of chenodeoxycholic acid.

The human apical sodium-dependent bile acid transporter (ASBT) is a validated drug target and can be employed to increase oral bioavailability of various drug conjugates. The aim of the present study was to investigate the chemical space around the 24-position of bile acids that influences both inhibition and uptake by the transporter. A series of 27 aminopyridine and aminophenol conjugates of glutamyl-chenodeoxycholate were synthesized and their ASBT inhibition and transport kinetics (parametrized as K(i), K(t), and J(max)) measured using stably transfected ASBT-MDCK cells. All conjugates were potent ASBT inhibitors. Monoanionic conjugates exhibited higher inhibition potency than neutral conjugates. However, neutral conjugates and chloro-substituted monoanionic conjugates were not substrates, or at least not apparent substrates. Kinetic analysis of substrates indicated that similar values for K(i) and K(t) implicate substrate binding to ASBT as the rate-limiting step. Using 3D-QSAR, four inhibition models and one transport efficiency model were developed. Steric fields dominated in CoMFA models, whereas hydrophobic fields dominated CoMSIA models. The inhibition models showed that a hydrophobic or bulky substitute on the 2 or 6 position of a 3-aminopyridine ring enhanced activity, while a hydrophobic group on the 5 position was detrimental. Overall, steric and hydrophobic features around the 24 position of the sterol nucleus strongly influenced bile acid conjugate interaction with ASBT. The relative location of the pyridine nitrogen and substituent groups also modulated binding.

Facial synthesis of key intermediate of obeticholic acid via Pd-catalyzed Kumada-Tamao-Corriu cross-coupling reaction.

Obeticholic acid (OCA) is used to treatment for Primary Biliary Cholangitis and other Famesoid X Receptor related diseases. Through the palladium catalyzed Kumada-Tamao-Corriu cross-coupling reaction, a novel and efficient method for synthesis of OCA with satisfied yield was successfully developed. The absolute configuration of the key intermediate was confirmed by Single-crystal X-ray Diffraction. It affords good strategy for large-scale synthesis of OCA.

Efficacy on anaplastic thyroid carcinoma of valproic acid alone or in combination with doxorubicin, a synthetic chenodeoxycholic acid derivative, or lactacystin.

The present study investigated the mechanism underlying the antitumor activity of the histone deacetylases inhibitor valproic acid (VPA), alone and in combination with doxorubicin, a synthetic chenodeoxycholic acid derivative (HS-1200), or the proteasome inhibitor lactacystin on cultured anaplastic thyroid carcinoma KAT-18 cells. Cell viability was evaluated by trypan-blue exclusion. Western blotting determined caspase and histone deacetylase activities and expression of poly(ADP)-ribose polymerase. Induction of apoptosis was identified by Hoechst staining, DNA electrophoresis, DNA hypoploidy and cell cycle phase analysis, and measurement of mitochondrial membrane potential. Subcellular translocation of apoptosis inducing factor and caspase-activated DNase after treatment was determined by confocal microscopy following immunofluorescent staining. VPA treatment increased apoptotic death of KAT-18 cells. VPA treatment was also associated with degradation of procaspase-3, procaspase-7, and poly(ADP)-ribose polymerase; induction of histone hyperacetylation; condensation of peripheral chromatin; decreased mitochondrial membrane potential and DNA content; and decreased translocation of apoptosis inducing factor and caspase-activated DNase. VPA in combination with doxorubicin, HS-1200, or lactacystin, applied at the highest concentrations that did not induce KAT-18 cell death, efficiently induced apoptosis in KAT-18 cells. The results suggest VPA combination therapy may represent an alternative therapeutic strategy for anaplastic thyroid carcinoma.

New lithocholic and chenodeoxycholic piperazinylcarboxamides with antiproliferative and pro-apoptotic effects on human cancer cell lines.

Six new synthetic bile acid derivatives were synthesized and tested in vitro against various human cancer cells (glioblastoma multiforme (GBM), multiple myeloma (KMS-11), and colonic carcinoma (HCT-116) cell lines. The best activity was obtained with compound IIIb on multiple myeloma cells (LD(50): 8.5+/-0.5 microM). This activity was associated with Mcl-1 and PARP-1 cleavage, inhibition of NFkappaB signaling, and DNA fragmentation, demonstrating an apoptotic cell death signaling pathway.

A facile synthesis of ursodeoxycholic acid and obeticholic acid from cholic acid.

A novel synthetic route of producing ursodeoxycholic acid (UDCA) and obeticholic acid (OCA) was developed through multiple reactions from cheap and readily-available cholic acid. The reaction conditions of the key elimination reaction of mesylate ester group were also investigated and optimized, including solvent, base and reaction temperature. In the straightforward synthetic route for preparation of UDCA and OCA, most of the reaction steps have high conversions with average yields of 94% and 92%, and overall yield up to 65% (7 steps) and 36% (11 steps) from cholic acid, respectively. This promising route offers economical and efficient strategies for potential large-scale production of UDCA and OCA.

Synthesis, characterization, and receptor interaction profiles of enantiomeric bile acids.

Bile acids are endogenous steroid detergents with receptor-mediated physiologic actions including activation of the G-protein coupled receptor TGR5 and gene regulation mediated by nuclear receptors. In this study, we report the first synthesis of enantiomeric lithocholic acid (ent-LCA, ent-1) and chenodeoxycholic acid (ent-CDCA, ent-2) via ent-testosterone (3). ent-1 was synthesized in 21 total steps in 4.2% yield, whereas ent-2 was obtained in 23 total steps in 0.8% yield. Critical micelle concentrations of the enantiomeric bile acids were found to be identical to their natural counterparts. Furthermore, enantiomeric bile acids were also tested for their ability to modulate bile acid activated proteins: farnesoid X receptor, vitamin D receptor, pregnane X receptor, and TGR5. Interestingly, ent-1 and ent-2 showed differential interactions with these proteins as compared to their corresponding natural bile acids. These data highlight the potential for using enantioselectivity as a way to distinguish between receptor and nonreceptor-mediated functions of natural bile acids.

Back door modulation of the farnesoid X receptor: design, synthesis, and biological evaluation of a series of side chain modified chenodeoxycholic acid derivatives.

Carbamate derivatives of bile acids were synthesized with the aim of systematically exploring the potential for farnesoid X receptor (FXR) modulation endowed with occupancy of the receptor's back door, localized between loops H1-H2 and H4-H5. Since it was previously shown that bile acids bind to FXR by projecting the carboxylic tail opposite the transactivation function 2 (AF-2, helix 12), functionalization of the side chain is not expected to interfere directly with the orientation of H12 but can result in a more indirect way of receptor modulation. The newly synthesized compounds were extensively characterized for their ability to modulate FXR function in a variety of assays, including the cell-free fluorescence resonance energy transfer (FRET) assay and the cell-based luciferase transactivation assay, and displayed a broad range of activity from full agonism to partial antagonism. Docking studies clearly indicate that the side chain of the new derivatives fits in a so far unexploited receptor cavity localized near the "back door" of FXR. We thus demonstrate the possibility of achieving a broad FXR modulation without directly affecting the H12 orientation.

First synthesis of 22-oxa-chenodeoxycholic acid analogue.

In this study, we report the first synthesis of 22-oxa-chenodeoxycholic acid analogue via androstenedione and progesterone, in 11 and 8 steps with overall yields of 6.4% and 12.7%, respectively. We anticipate this will help to facilitate the development of new drugs.

Synthesis, in vitro and in vivo small-animal SPECT evaluation of novel technetium labeled bile acid analogues to study (altered) hepatic transporter function.

INTRODUCTION: Hepatobiliary transport mechanisms are crucial for the excretion of substrate toxic compounds. Drugs can inhibit these transporters, which can lead to drug-drug interactions causing toxicity. Therefore, it is important to assess this early during the development of new drug candidates. The aim of the current study is the (radio)synthesis, in vitro and in vivo evaluation of a technetium labeled chenodeoxycholic and cholic acid analogue: [(99m)Tc]-DTPA-CDCA and [(99m)]Tc-DTPA-CA, respectively, as biomarker for disturbed transporter functionality. METHODS: [99mTc]-DTPA-CDCA([(99m)Tc]-3a) and [99mTc]-DTPA-CA ([(99m)Tc]-3b) were synthesized and evaluated in vitro and in vivo. Uptake of both tracers was investigated in NTCP, OCT1, OATP1B1, OATP1B3 transfected cell lines. Km and Vmax values were determined and compared to [(99m)Tc]-mebrofenin ([(99m)Tc]-MEB). Efflux was investigated by means of CTRL, MRP2 and BSEP transfected inside-out vesicles. Metabolite analysis was performed using pooled human liver S9. Wild type (n=3) and rifampicin treated (n=3) mice were intravenously injected with 37MBq of tracer. After dynamic small-animal SPECT and short CT acquisitions, time-activity curves of heart, liver, gallbladder and intestines were obtained. RESULTS: We demonstrated that OATP1B1 and OATP1B3 are the involved uptake transporters of both compounds. Both tracers show a higher affinity compared to [(99m)Tc]-MEB, but are in a similar range as endogenous bile acids for OATP1B1 and OATP1B3. [(99m)Tc]-3a shows higher affinities compared to [(99m)Tc]-3b. Vmax values were lower compared to [(99m)Tc]-MEB, but in the same range as endogenous bile acids. MRP2 was identified as efflux transporter. Less than 7% of both radiotracers was metabolized in the liver. In vitro results were confirmed by in vivo results. Uptake in the liver and efflux to gallbladder + intestines and urinary bladder of both tracers was observed. Transport was inhibited by rifampicin. CONCLUSION: The involved transporters were identified; both tracers are taken up in the hepatocytes by OATP1B1 andOATP1B3 with Km and Vmax values in the same range as endogenous bile acids and are secreted into bile canaliculi via MRP2. Dynamic small-animal SPECT imaging can be a useful noninvasive method of visualizing and quantifying hepatobiliary transporter functionality and disturbances thereof in vivo, which could predict drug pharmacokinetics.

Biliary excretion of chenodeoxycholyllysylrhodamine in Wistar rats: a possible role of a bile acid as a carrier for drugs.

The effect on biliary excretion of rhodamine after its conjugation to give chenodeoxycholyl-lysyl-rhodamine (cheno-lys-R) was studied in male Wistar rats. Following its intravenous injection via the jugular vein of animals cheno-lys-R was efficiently excreted into bile with a peak biliary excretion of 31.6 +/- 1.2% dose 5 min-1 and a cumulative biliary excretion of 96.4 +/- 2.0% in 30 min of the total dose administered. Unlike cheno-lys-R, rhodamine had a poor biliary excretion of 1.0 +/- 0.1% dose 5 min-1 and a cumulative biliary excretion of 3.3 +/- 0.6% in 30 min. Cheno-lys-R had a short plasma half-life (t1/2 alpha) of 4.0 +/- 0.5 min, whereas free rhodamine had a longer half life (t1/2 alpha) of 82.1 +/- 1.4 min. The plasma clearances of cheno-lys-R and rhodamine were 41.2 +/- 6.5 and 9.0 +/- 1.2 ml/min per kg, respectively. The data indicate that the cationic fluorescent xenobiotic, rhodamine, when conjugated to the bile salt analogue, greatly increased the biliary excretion of rhodamine and that cheno-lys acted as a carrier for hepatic uptake of rhodamine. Thus, an appropriate bile salt derivative may be used to target a drug to the liver.

6alpha-ethyl-chenodeoxycholic acid (6-ECDCA), a potent and selective FXR agonist endowed with anticholestatic activity.

A series of 6alpha-alkyl-substituted analogues of chenodeoxycholic acid (CDCA) were synthesized and evaluated as potential farnesoid X receptor (FXR) ligands. Among them, 6alpha-ethyl-chenodeoxycholic acid (6-ECDCA) was shown to be a very potent and selective FXR agonist (EC(50) = 99 nM) and to be endowed with anticholeretic activity in an in vivo rat model of cholestasis.

Facile synthesis, aggregation behavior, and cholesterol solubilization ability of avicholic acid.

[reaction: see text] Avicholic acid, a major constituent of the bile of several avian species, was synthesized in eight steps from readily available chenodeoxycholic acid in 9% overall yield using Breslow's remote functionalization strategy in a key step. Micelle formation and equilibrium cholesterol solubilization properties were studied for avicholate in aqueous solution.

Synthesis of 13C-labeled chenodeoxycholic, hyodeoxycholic, and ursodeoxycholic acids for the study of bile acid metabolism in liver disease.

In order to study the glycosidic conjugation of chenodeoxycholic, hyodeoxycholic, and ursodeoxycholic acids in patients with cholestasis after oral administration of pharmacological amounts of the respective bile acids avoiding the application of radioactive tracers we synthesized [24-13C]chenodeoxycholic, [24-13C]hyodeoxycholic, and [24-13C]ursodeoxycholic acids. The reaction intermediates of the bile acid syntheses were characterized by infrared spectroscopy. Purity was confirmed using thin-layer chromatography as well as gas chromatography-mass spectrometry. The 13C atom excess of approximately 90% of the synthesized bile acids was the same as the 13C atom excess of the sodium [13C]cyanide used for the labeling reaction confirming the successful synthesis. After oral administration of 0.5 g of [24-13C]ursodeoxycholic acid to a healthy volunteer, 13C label was detected in the nonamidated and glycine- or taurine conjugated glucosides and the N-acetylglucosaminide of ursodeoxycholic acid in urine. This establishes ursodeoxycholic acid as the first bile acid so far known to undergo both of the recently described glycosidic conjugation reactions in humans.