Enzymatic Synthesis of New Acetoacetate-Ursodeoxycholic Acid Hybrids as Potential Therapeutic Agents and Useful Synthetic Scaffolds as Well.

Ursodeoxycholic acid (UDCA) and acetoacetate are natural compounds present in the human intestine and blood, respectively. A number of studies highlighted that besides their well-known primary biological roles, both compounds possess the ability to influence a variety of cellular processes involved in the etiology of various diseases. These reasons suggested the potential of acetoacetate-UDCA hybrids as possible therapeutic agents and prompted us to develop a synthetic strategy to selectively derivatize the hydroxyl groups of the bile acid with acetoacetyl moieties. 3α-acetoacetoxy UDCA was obtained (60% isolated yield) via the regioselective transesterification of methyl acetoacetate with UDCA promoted by the Candida antarctica lipase B (CAL-B). 3α,7ß-bis-acetoacetoxy UDCA was obtained instead by thermal condensation of methyl acetoacetate and UDCA (80% isolated yield). This bis-adduct was finally converted to the 7ß-acetoacetoxy UDCA (82% isolated yield) via CAL-B catalyzed regioselective alcoholysis of the ester group on the 3α position. In order to demonstrate the value of the above new hybrids as UDCA-based scaffolds, 3α-acetoacetoxy UDCA was subjected to multicomponent Biginelli reaction with benzaldehyde and urea to obtain the corresponding 4-phenyl-3,4-dihydropyrimidin-2-(1H)-one derivative in 65% isolated yield.

Dihydroartemisinin-Ursodeoxycholic Bile Acid Hybrids in the Fight against SARS-CoV-2.

Here, we propose the molecular hybridization of dihydroartemisinin (DHA) and ursodeoxycholic bile acid (UDCA), approved drugs, for the preparation of antiviral agents against SARS-CoV-2. DHA and UDCA were selected on the basis of their recently demonstrated in vitro activity against SARS-CoV-2. A selection of DHA-UDCA-based hybrids obtained by varying the nature of the linkage and the bile acid conjugation point as well as unconjugated DHA and UDCA were tested in vitro for cytotoxicity and anti-SARS-CoV-2 activity on Vero E6 and Calu-3 human lung cells. The hybrid DHA-t-UDCMe, obtained by conjugation via click chemistry on a gram scale, was identified as a potential candidate for SARS-CoV-2 infection treatment due to significant reduction of viral replication, possibly involving ACE2 downregulation, no cytotoxicity, and chemical stability.

Antisense Oligonucleotides Conjugated with Lipophilic Compounds: Synthesis and In Vitro Evaluation of Exon Skipping in Duchenne Muscular Dystrophy.

Our groups previously reported that conjugation at 3'-end with ursodeoxycholic acid (UDCA) significantly enhanced in vitro exon skipping properties of ASO 51 oligonucleotide targeting the human DMD exon 51. In this study, we designed a series of lipophilic conjugates of ASO 51, to explore the influence of the lipophilic moiety on exon skipping efficiency. To this end, three bile acids and two fatty acids have been derivatized and/or modified and conjugated to ASO 51 by automatized solid phase synthesis. We measured the melting temperature (Tm) of lipophilic conjugates to evaluate their ability to form a stable duplex with the target RNA. The exon skipping efficiency has been evaluated in myogenic cell lines first in presence of a transfection agent, then in gymnotic conditions on a selection of conjugated ASO 51. In the case of 5'-UDC-ASO 51, we also evaluated the influence of PS content on exon skipping efficiency; we found that it performed better exon skipping with full PS linkages. The more efficient compounds in terms of exon skipping were found to be 5'-UDC- and 5',3'-bis-UDC-ASO 51.

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 Exon-Skipping Properties of a 3'-Ursodeoxycholic Acid-Conjugated Oligonucleotide Targeting DMD Pre-mRNA: Pre-Synthetic versus Post-Synthetic Approach.

Steric blocking antisense oligonucleotides (ASO) are promising tools for splice modulation such as exon-skipping, although their therapeutic effect may be compromised by insufficient delivery. To address this issue, we investigated the synthesis of a 20-mer 2'-OMe PS oligonucleotide conjugated at 3'-end with ursodeoxycholic acid (UDCA) involved in the targeting of human DMD exon 51, by exploiting both a pre-synthetic and a solution phase approach. The two approaches have been compared. Both strategies successfully provided the desired ASO 51 3'-UDC in good yield and purity. It should be pointed out that the pre-synthetic approach insured better yields and proved to be more cost-effective. The exon skipping efficiency of the conjugated oligonucleotide was evaluated in myogenic cell lines and compared to that of unconjugated one: a better performance was determined for ASO 51 3'-UDC with an average 9.5-fold increase with respect to ASO 51.

Modified Nucleosides, Nucleotides and Nucleic Acids via Click Azide-Alkyne Cycloaddition for Pharmacological Applications.

The click azide = alkyne 1,3-dipolar cycloaddition (click chemistry) has become the approach of choice for bioconjugations in medicinal chemistry, providing facile reaction conditions amenable to both small and biological molecules. Many nucleoside analogs are known for their marked impact in cancer therapy and for the treatment of virus diseases and new targeted oligonucleotides have been developed for different purposes. The click chemistry allowing the tolerated union between units with a wide diversity of functional groups represents a robust means of designing new hybrid compounds with an extraordinary diversity of applications. This review provides an overview of the most recent works related to the use of click chemistry methodology in the field of nucleosides, nucleotides and nucleic acids for pharmacological applications.

Surface Phenomena Enhancing the Antibacterial and Osteogenic Ability of Nanocrystalline Hydroxyapatite, Activated by Multiple-Ion Doping.

The present work describes a novel nanocrystalline, multidoped hydroxyapatite featuring excellent eukaryotic versus prokaryotic cell selectivity, attested by excellent osteoinductive character and evaluated with human stem cells, and anti-infective ability, tested against different pathogens. Physicochemical analysis and transmission electron microscopy (TEM)/scanning STEM observations highlighted that such enhanced biological features are related to the lower crystallinity level and increased surface charge of hydroxyapatite, both induced by multiple-ion doping. Specifically, the lattice substitution of Ca2+ with Zn2+ promotes the segregation of Ca2+ and doping Mg2+ cations to a less-ordered surface layer, thus promoting dynamic ion absorption/release acting as bioactive signals for cells and exerting an antiproliferative effect on all tested pathogens. These findings open the design of new biodevices, combining regenerative ability and effective microbial inhibition without using any antibiotic drugs. This is extremely important to circumvent bacterial resistance to antibiotics, which is today considered as one of the biggest threats to global health.

A Graded Multifunctional Hybrid Scaffold with Superparamagnetic Ability for Periodontal Regeneration.

The regeneration of dental tissues is a still an unmet clinical need; in fact, no therapies have been completely successful in regenerating dental tissue complexes such as periodontium, which is also due to the lack of scaffolds that are able to guide and direct cell fate towards the reconstruction of different mineralized and non-mineralized dental tissues. In this respect, the present work develops a novel multifunctional hybrid scaffold recapitulating the different features of alveolar bone, periodontal ligament, and cementum by integrating the biomineralization process, and tape casting and electrospinning techniques. The scaffold is endowed with a superparamagnetic ability, thanks to the use of a biocompatible, bioactive superparamagnetic apatite phase, as a mineral component that is able to promote osteogenesis and to be activated by remote magnetic signals. The periodontal scaffold was obtained by engineering three different layers, recapitulating the relevant compositional and microstructural features of the target tissues, into a monolithic multifunctional graded device. Physico-chemical, morphological, and ultrastructural analyses, in association with preliminary in vitro investigations carried out with mesenchymal stem cells, confirm that the final scaffold exhibits a good mimicry of the periodontal tissue complex, with excellent cytocompatibility and cell viability, making it very promising for regenerative applications in dentistry.