Synthesis and Biological Investigation of Bile Acid-Paclitaxel Hybrids.

Chenodeoxycholic acid and ursodeoxycholic acid (CDCA and UDCA, respectively) have been conjugated with paclitaxel (PTX) anticancer drugs through a high-yield condensation reaction. Bile acid-PTX hybrids (BA-PTX) have been investigated for their pro-apoptotic activity towards a selection of cancer cell lines as well as healthy fibroblast cells. Chenodeoxycholic-PTX hybrid (CDC-PTX) displayed cytotoxicity and cytoselectivity similar to PTX, whereas ursodeoxycholic-PTX hybrid (UDC-PTX) displayed some anticancer activity only towards HCT116 colon carcinoma cells. Pacific Blue (PB) conjugated derivatives of CDC-PTX and UDC-PTX (CDC-PTX-PB and UDC-PTX-PB, respectively) were also prepared via a multistep synthesis for evaluating their ability to enter tumor cells. CDC-PTX-PB and UDC-PTX-PB flow cytometry clearly showed that both CDCA and UDCA conjugation to PTX improved its incoming into HCT116 cells, allowing the derivatives to enter the cells up to 99.9%, respect to 35% in the case of PTX. Mean fluorescence intensity analysis of cell populations treated with CDC-PTX-PB and UDC-PTX-PB also suggested that CDC-PTX-PB could have a greater ability to pass the plasmatic membrane than UDC-PTX-PB. Both hybrids showed significant lower toxicity with respect to PTX on the NIH-3T3 cell line.

Synthesis and Biological Activity of New Brassinosteroid Analogs of Type 24-Nor-5ß-Cholane and 23-Benzoate Function in the Side Chain.

Brassinosteroids are polyhydroxysteroids that are involved in different plants' biological functions, such as growth, development and resistance to biotic and external stresses. Because of its low abundance in plants, much effort has been dedicated to the synthesis and characterization of brassinosteroids analogs. Herein, we report the synthesis of brassinosteroid 24-nor-5ß-cholane type analogs with 23-benzoate function and 22,23-benzoate groups. The synthesis was accomplished with high reaction yields in a four-step synthesis route and using hyodeoxycholic acid as starting material. All synthesized analogs were tested using the rice lamina inclination test to assess their growth-promoting activity and compare it with those obtained for brassinolide, which was used as a positive control. The results indicate that the diasteroisomeric mixture of monobenzoylated derivatives exhibit the highest activity at the lowest tested concentrations (1 × 10-8 and 1 × 10-7 M), being even more active than brassinolide. Therefore, a simple synthetic procedure with high reaction yields that use a very accessible starting material provides brassinosteroid synthetic analogs with promising effects on plant growth. This exploratory study suggests that brassinosteroid analogs with similar chemical structures could be a good alternative to natural brassinosteroids.

Novel Derivatives of Deoxycholic Acid Bearing Linear Aliphatic Diamine and Aminoalcohol Moieties and their Cyclic Analogs at the C3 Position: Synthesis and Evaluation of Their In Vitro Antitumor Potential.

A series of novel deoxycholic acid (DCA) derivatives containing aliphatic diamine and aminoalcohol or morpholine moieties at the C3 position were synthesized by 3,26-epoxide ring-opening reactions. These compounds were investigated for their cytotoxicity in four human tumor cell lines and murine macrophages and for inhibitory activity against macrophage-mediated NO synthesis in vitro. Obtained data revealed that: (i) all amine-containing substituents significantly increased the cytotoxicity of the novel compounds (IC502-10 = 1.0-36.0 µM) in comparison with DCA (IC50DCA ≥ 82.9 µM); (ii) aminoalcohol moieties were more preferable than diamine moieties due to the fact they imparted better selectivity for tumor cells of the novel derivatives; (iii) the susceptibility of tested cell lines to derivatives diminished in the following order: HuTu-80 (duodenal carcinoma) ≈ HepG2 (hepatocarcinoma) > KB-3-1 (cervical carcinoma) > RAW264.7 (macrophages) > A549 (lung carcinoma); (iv) compounds 8 and 9, bearing aminoethanol and aminopropanol moieties, respectively, exhibited high cytotoxic selectivity indexes (SIHuTu-80 = 7.9 and 8.3, respectively) and good drug-likeness parameters; (v) the novel compounds do not display anti-NO activity. Mechanistic study revealed that compound 9 induces ROS-dependent cell death by activation of intrinsic caspase-dependent apoptosis and cytodestructive autophagy in HuTu-80 cells and vitamin D receptor can be considered as its primary target.

Synthesis of 2-Deoxybrassinosteroids Analogs with 24-nor, 22(S)-23-Dihydroxy-Type Side Chains from Hyodeoxycholic Acid.

Natural brassinosteroids are widespread in the plant kingdom and it is known that they play an important role in regulating plant growth. In this study, two new brassinosteroid analogs with shorter side chains but keeping the diol function were synthesized. Thus, the synthesis of 2-deoxybrassinosteroids analogs of the 3α-hydroxy-24-nor, 22,23-dihydroxy-5α-cholestane side chain type is described. The starting material is a derivative from hyodeoxycholic acid (4), which was obtained with an overall yield of 59% following a previously reported five step route. The side chain of this intermediate was modified by oxidative decarboxylation to get a terminal olefin at the C22-C23 position (compound 20) and subsequent dihydroxylation of the olefin. The resulting epimeric mixture of 21a, 21b was separated and the absolute configuration at the C22 carbon for the main product 21a was elucidated by single crystal X-ray diffraction analysis of the benzoylated derivative 22. Finally, lactonization of 21a through a Baeyer-Villiger oxidation of triacetylated derivative 23, using CF3CO3H/CHCl3 as oxidant system, leads to lactones 24 and 25 in 35% and 14% yields, respectively. Deacetylation of these compounds leads to 2-deoxybrassinosteroids 18 and 19 in 86% and 81% yields. Full structural characterization of all synthesized compounds was achieved using their 1D, 2D NMR, and HRMS data.

Novel derivatives of deoxycholic acid bearing aliphatic or cyclic diamine moieties at the C-3 position: Synthesis and evaluation of anti-proliferative activity.

A new library of deoxycholic acid derivatives bearing nitrogen-containing moieties at the C-3 position was synthesised from epoxy derivative 1 via an epoxide ring-opening reaction promoted by aliphatic or cyclic diamines and fully characterised by NMR and mass-spectroscopy. The synthesised compounds were screened for cytotoxicity against four human tumour cell lines. The results showed that some of the novel diamine-bearing derivatives displayed improved anti-proliferative activities over the parent compound DCA. Among them, a 1-methylpiperazine containing compound (6) showed promising activity and the highest selectivity against tumour cells of enterohepatic origin (HepG2: IC50=3.6µM, SI=9.0; HuTu-80: IC50=4.6µM, SI=6.9) and was identified as a lead molecule.

Discovery and Synthesis of Amino Acids Modified Deoxycholic Acid Derivatives and in Vitro Antiproliferative Evaluation.

A series of deoxycholic acid (DCA) derivatives bearing amino acid moiety has been synthesized and investigated for their potential antiproliferative activities. DCA derivative compounds were synthesized by a two or three step synthetic approach. Their bioactivities were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and Western blotting analysis on three tumor cell lines A549 (human lung cancer cell line), MCF-7 (human breast cancer cell line) and HeLa (human cervical carcinoma cell). The novel derivatives DCA3d, DCA5a, DCA5b, DCA5c, and DCA5d were found to be promising antiproliferative agents. Furthermore, DCA5b showed the greatest cytotoxic activity by induction of apoptosis. These compounds show potentiality for further optimization as antitumor drugs.

Micellization parameters (number average, aggregation number and critical micellar concentration) of bile salt 3 and 7 ethylidene derivatives: Role of the steroidal skeleton II.

BACKGROUND: Bile salts are steroidal biosurfactants. Micellar systems of bile salts are not only important for solubilization of cholesterol, but they also interact with certain drugs thus changing their bioavailability. METHODS: The number-average aggregation numbers (n¯) are determined using the Moroi-Matsuoka-Sugioka thermodynamic method. Critical micellar concentrations were determined by spectrofluorometric method using pyren and by surface tension measurements. RESULTS: Micelles of ethylidene derivatives possess the following values for n¯: 7-Eth-D (n¯=11 (50 mM)-n¯=14.8 (100 mM)); 12-Ox-7-Eth-L (n¯≈8.8, without concentration dependence) and 7,12-diOx-3-Eth-Ch (n¯≈2.9, without concentration dependence). In the planes n¯-ln k and ln CMC-ln k derivative 7-Eth-D is outlier in respect to hydrophobic linear congeneric groups. CONCLUSION: Gibbs energy of formation for 7-Eth-D anion micelles in addition to the Gibbs energy of hydrophobic interactions consists excess Gibbs energy (GE) from hydrogen bond formation between building blocks of micelles. Gibbs energy of formation for 7,12-diOx-3-Eth-Ch and 12-Ox-7-Eth-L anion micelle is determined by the Gibbs energy of hydrophobic interactions. Relative increase in hydrophobicity and aggregation number for ethylidene derivatives is larger when ethylidene group is introduced from the C7 lateral side of steroidal skeleton then it is when ethylidene group is on C3 carbon. GENERAL SIGNIFICANCE: Position of outlier towards hydrophobic congeneric groups from n¯-ln k and ln CMC-ln k planes indicates the existence of excess Gibbs energy (GE) which is not of hydrophobic nature (formation of hydrogen bonds). For the bile salt micelles to have GE (formation of secondary micelles) it is necessary that steroidal skeleton possesses C3-α-(e)-OH and C12-α-(a)-OH groups.

Synthesis and biological activity of novel deoxycholic acid derivatives.

We report the synthesis and biological activity of new semi-synthetic derivatives of naturally occurring deoxycholic acid (DCA) bearing 2-cyano-3-oxo-1-ene, 3-oxo-1(2)-ene or 3-oxo-4(5)-ene moieties in ring A and 12-oxo or 12-oxo-9(11)-ene moieties in ring C. Bioassays using murine macrophage-like cells and tumour cells show that the presence of the 9(11)-double bond associated with the increased polarity of ring A or with isoxazole ring joined to ring A, improves the ability of the compounds to inhibit cancer cell growth.

Synthesis and antiproliferative activity of C-homo-lactam derivatives of 7-deoxycholic acid.

Using deoxycholic acid as starting materials, a series of 12a-aza-C-homo-12-one 7-deoxycholic acid derivatives were synthesized The antiproliferative activity of the synthesized compounds against some carcinoma cell lines was investigated. The results showed that some 12-oxy-12a-aza-C-homo-7-deoxycholic acid derivatives displayed distinct cytotoxicity to HeLa (human cervical carcinoma) and Tu 686 (laryngocarcinoma) tumor cell lines. In particular, the IC50 values of the compounds 6 and 7 against Tu 686 cells are 16.7 and 19.8 µM/L respectively. The information obtained from the studies may be useful for the design of novel chemotherapeutic drugs.

Synthesis and quantitative structure-property relationships of side chain-modified hyodeoxycholic acid derivatives.

Bile acids have emerged as versatile signalling compounds of a complex network of nuclear and membrane receptors regulating various endocrine and paracrine functions. The elucidation of the interconnection between the biological pathways under the bile acid control and manifestations of hepatic and metabolic diseases have extended the scope of this class of steroids for in vivo investigations. In this framework, the design and synthesis of novel biliary derivatives able to modulate a specific receptor requires a deep understanding of both structure-activity and structure-property relationships of bile acids. In this paper, we report the preparation and the critical micellization concentration evaluation of a series of hyodeoxycholic acid derivatives characterized by a diverse side chain length and by the presence of a methyl group at the alpha position with respect to the terminal carboxylic acid moiety. The data collected are instrumental to extend on a quantitative basis, the knowledge of the current structure-property relationships of bile acids and will be fruitful, in combination with models of receptor activity, to design and prioritize the synthesis of novel pharmacokinetically suitable ligands useful in the validation of bile acid-responsive receptors as therapeutic targets.

New fluorescent bile acids: synthesis, chemical characterization, and disastereoselective uptake by Caco-2 cells of 3-deoxy 3-NBD-amino deoxycholic and ursodeoxycholic acid.

Deoxycholic acid (DCA), a secondary bile acid (BA), and ursodeoxycholic acid (UDCA), a tertiary BA, cause opposing effects in vivo and in cell suspensions. Fluorescent analogues of DCA and UDCA could help investigate important questions about their cellular interactions and distribution. We have prepared a set of isomeric 3α- and 3ß-amino analogues of UDCA and DCA and derivatised these with the discrete fluorophore, 4-nitrobenzo-2-oxa-1,3-diazol (NBD), forming the corresponding four fluorescent adducts. These absorb in the range 465-470 nm and fluoresce at approx. 535 nm. In order to determine the ability of the new fluorescent bile acids to mimic the parents, their uptake was studied using monolayers of Caco-2 cells, which are known to express multiple proteins of the organic anion-transporting peptide (OATP) subfamily of transporters. Cellular uptake was monitored over time at 4 and 37°C to distinguish between passive and active transport. All four BA analogues were taken up but in a strikingly stereo- and structure-specific manner, suggesting highly discriminatory interactions with transporter protein(s). The α-analogues of DCA and to a lesser extent UDCA were actively transported, whereas the ß-analogues were not. The active transport process was saturable, with Michaelis-Menten constants for 3α-NBD DCA (5) being K(m)=42.27±12.98 µM and V(max)=2.8 ± 0.4 nmol/(mg protein*min) and for 3α-NBD UDCA (3) K(m)=28.20 ± 7.45 µM and V(max)=1.8 ± 0.2 nmol/(mg protein*min). These fluorescent bile acids are promising agents for investigating questions of bile acid biology and for detection of bile acids and related organic anion transport processes.

Tandem facial amphiphiles for membrane protein stabilization.

We describe a new type of synthetic amphiphile that is intended to support biochemical characterization of intrinsic membrane proteins. Members of this new family displayed favorable behavior with four of five membrane proteins tested, and these amphiphiles formed relatively small micelles.

Conjugation of bile esters to cellulose by olefin cross-metathesis: A strategy for accessing complex polysaccharide structures.

Bile salts tend to form micelles in aqueous media and can thereby contribute to drug solubilization; they also exhibit crystallization inhibition properties that can stabilize supersaturated drug solutions. Herein, we explore conjugation of bile salts with polysaccharides to create new, amphiphilic polysaccharide derivatives with intriguing properties, portending broad utility in various applications. We introduce efficient conjugation of cholesterol (as a model steroid), lithocholic acid, and deoxycholic acid by mild, modular olefin cross-metathesis reactions. These small molecules were first modified with an acrylate group from the A-ring hydroxyl, then reacted with cellulose derivatives bearing olefin-terminated metathesis "handles". Successful conjugation of bile acids has demonstrated chemoselective cross-metathesis with complex, polyfunctional structures, and large multi-ring systems. It also enabled an efficient, general pathway for polysaccharide-bile salt conjugates, which promise synergy for applications such as amorphous solid dispersion (ASD).

Identify liver X receptor ß modulator building blocks by developing a fluorescence polarization-based competition assay.

The liver X receptors (LXRs) of the nuclear receptor family are promising therapeutic targets of multiple diseases like lipid disorders, chronic inflammation, as well as different human cancers. To date, no LXR agonists or antagonists can be used in clinics, emphasizing the importance for discovering new LXR modulators. Fragment-based lead discovery (FBLD) is powerful for designing new scaffolds and new mechanistic drugs, but fragment screening has not been applied to LXRs yet, which might be due to the lack of a specific fragment screening method against the dynamic and hydrophobic ligand binding domain (LBD) of LXRs. Herein, a series of fluorescent tracers were designed, synthesized and tested. The tracer based on hyodeoxycholic acid exhibited a good capability for competitively detecting the ligand binding of LXRß using a fluorescence polarization approach. Then, 1074 fragments were screened against the LBD of LXRß (LXRß-LBD), resulting in 27 binding hits. These fragment hits were further tested using the co-activator recruitment assay and reporter gene assay, and efforts in X-ray crystallography fortunately solved a co-crystal structure of LXRß-LBD with the fragment F3 (tert-butyl-7-amino-3,4-dihydroisoquinoline-2(1H)-carboxylate). The fluorescence-based fragment screening tool and the newly identified LXRß binding fragments provide the basis for developing novel LXRß modulators.

Novel hyaluronic acid coated hydrophobically modified chitosan polyelectrolyte complex for the delivery of doxorubicin.

The aim of this work was to examine the formation and properties of a novel polyelectrolyte complex of drug carrier system for the delivery of doxorubicin (DOX), which consists of hyaluronic acid (HA) coated hydrophobically modified chitosan (CS). Various batches of polyelectrolyte complexes with the molar ratio of deoxycholic acid (DCA) and chitosan (CS) of 0.1, 0.2, 0.3 were prepared, and were termed as CS-DCA10, CS-DCA20, and CS-DCA30 respectively. The samples were characterized by Fourier transform infrared (FTIR) spectroscopy, X-ray diffraction (XRD), Transmission electron microscopy (TEM), nuclear magnetic resonance hydrogen spectrum (1H NMR) and dynamic light scattering (DLS). Particle sizes of synthesized polyelectrolyte complex nanoparticles (PCNs) were found to be in the range of 280-310 nm, larger than those of uncoated nanoparticles (~150 nm). The PCNs have large zeta potentials (about 26 mV) which make them stable and no sizes' change was determined. DOX could be easily incorporated into the PCNs with encapsulation efficiency (56%) and kept a sustained release manner without burst effect when exposed to PBS (pH 7.4) at 37 °C. Overall, these findings confirmed the potential of these PCNs for drug carrier and prolonged and sustained delivery in the bloodstream.

Vascular targeted chitosan-derived nanoparticles as docetaxel carriers for gastric cancer therapy.

A gastric cancer angiogenesis marker peptide, GX1, is promising to be a desirable ligand for anti-angiogenesis targeted drug of gastric cancer treatment. In this study, GX1 was utilized to fabricate a multifunctional vascular targeting docetaxel (DCT)-loaded nanoparticle with N-deoxycholic acid glycol chitosan (DGC) as the carrier and GX1-PEG-deoxycholic acid (GPD) conjugate as the targeting ligand. The mean size of obtained GX1-DGC-DCT was 150.9 nm with a narrow size distribution and their shape was spherical with smooth surface texture. The in vitro drug release test revealed a sustained release manner and an acid pH could accelerate the release compared with the neutral pH. Furthermore, GX1-DGC-DCT showed stronger cytotoxicity against co-cultured gastric cancer cells and human umbilical vein endothelial cells (co-HUVEC) than DCT within 100 µM. In addition, GX1 efficiently enhanced the cellular uptake of nanoparticles in co-HUVEC cells as confirmed by confocal fluorescence scanning microscopy. Moreover, in vivo delivery of GX1-DGC-DCT was demonstrated to inhibit tumor growth in SGC791 tumor-bearing mice with tumor inhibition rate (TIR) of 67.05% and no weight loss of mice was observed. The anti-tumor effects were further confirmed by H&E and TUNEL analysis. Therefore, this new drug delivery system represents a potential strategy for gastric cancer therapy.

Synthesis and biological evaluation of bile acid dimers linked with 1,2,3-triazole and bis-beta-lactam.

We report herein the synthesis and biological evaluation of bile acid dimers linked through 1,2,3-triazole and bis-beta-lactam. The dimers were synthesized using 1,3-dipolar cycloaddition reaction of diazido bis-beta-lactams , and terminal alkynes derived from cholic acid/deoxycholic acid in the presence of Cu(i) catalyst (click chemistry). These novel molecules were evaluated in vitro for their antifungal and antibacterial activity. Most of the compounds exhibited significant antifungal as well as antibacterial activity against all the tested fungal and bacterial strains. Moreover, their in vitro cytotoxicities towards HEK-293 and MCF-7 cells were also established.

Bile acids: Lipase-catalyzed synthesis of new hyodeoxycholic acid derivatives.

In this work we present an efficient, environmentally friendly approach to the synthesis of a series of hyodeoxycholic acid derivatives applying Biocatalysis. Fifteen acetyl and ester derivatives, twelve of them new, were obtained through an enzymatic strategy in a fully regioselective way and in very good to excellent yield. In order to find the optimal reaction conditions, the influence of several parameters such as enzyme source, alcohol or acylating agent:substrate ratio, enzyme:substrate ratio, temperature and reaction solvent was considered. The excellent results obtained made this procedure very efficient, particularly considering the low amount of enzyme required. In addition, this methodology uses mild reaction conditions and has reduced environmental impact, making biocatalysis a suitable way to obtaining these bile acids derivatives.

Synthesis, Characterization, and Biodistribution of a Dinuclear Gadolinium Complex with Improved Properties as a Blood Pool MRI Agent.

A dinuclear gadolinium(III) chelate containing two moieties of diethylenetriaminepentaacetic acid (DTPA), covalently conjugated to an analogue of deoxycholic acid, was synthesized and thoroughly characterized. A full relaxometric analysis was carried out, consisting of 1) the acquisition of nuclear magnetic resonance dispersion (NMRD) profiles in various media; 2) the study of binding affinity to serum albumin; 3) the measurement of 17 O transverse relaxation rate versus temperature, and 4) a transmetallation assay. In vivo biodistribution MRI studies at 1 T and blood pharmacokinetics assays were carried out in comparison with Gd-DTPA (Magnevist) and gadocoletic acid trisodium salt (B22956/1), two well-known Gd complexes that share the same chelating cage and the same deoxycholic acid residue of the Gd complex investigated herein ((GdDTPA)2 -Chol). High affinity for plasma protein and, in particular, the availability of more than one binding site, allows the complex to reach a fairly high relaxivity value in plasma (∼20 mm-1 s-1 , 20 MHz, 310 K) as well as to show unexpectedly enhanced properties of blood pooling, with an elimination half-life in rats approximately seven times longer than that of B22956/1.

Novel steroid derivatives: synthesis, antileishmanial activity, mechanism of action, and in silico physicochemical and pharmacokinetics studies.

The search for new drugs for the treatment of leishmaniasis is an important strategy for improving the current therapeutic arsenal for the disease. There are several limitations to the available drugs including high toxicity, low efficacy, prolonged parenteral administration, and high costs. Steroids are a diverse group of compounds with various applications in pharmacology. However, the antileishmanial activity of this class of molecules has not yet been explored. Therefore, in the present study, we investigated the antileishmanial activity and cytotoxicity of novel steroids against murine macrophages with a focus on the derivatives of cholesterol (CD), cholic acid (CA), and deoxycholic acid (DA). Furthermore, the mechanism of action of the best compound was assessed, and in silico studies to evaluate the physicochemical and pharmacokinetic properties were also conducted. Among the sixteen derivatives, schiffbase2, CD2 and deoxycholic acid derivatives (DOCADs) were effective against promastigotes of Leishmania species. Despite their low toxicity to macrophages, the majority of DOCADs were active against intracellular amastigotes of L. amazonensis, and DOCAD5 exhibited the best biological effect against these parasitic stages (IC50 = 15.34 µM). Neither the CA derivatives (CAD) nor DA alone inhibited the intracellular parasites. Thus, the absence of hydroxyl in the C-7 position of the steroid nucleus, as well as the modification of the acid group in DOCADs were considered important for antileishmanial activity. The treatment of L. amazonensis promastigote forms with DOCAD5 induced biochemical changes such as depolarization of the mitochondrial membrane potential, increased ROS production and cell cycle arrest. No alterations in parasite plasma membrane integrity were observed. In silico physicochemical and pharmacokinetic studies suggest that DOCAD5 could be a good candidate for an oral drug. The data demonstrate the potential antileishmanial effect of certain steroid derivatives and encourage new in vivo studies.