Dihydroartemisinin (DHA), a first-line treatment for uncomplicated malaria, has demonstrated antitumor activity against a variety of human cancers, emphasizing its potential for repurposing as an anticancer agent. However, its short half-life and poor bioavailability hinder its application in cancer therapy. We previously demonstrated that the molecular hybridization of DHA with bile acids (BAs) enhances its anticancer activity by improving stability and reducing toxicity. Based on this rationale, here, we designed and synthesized a library of DHA-based hybrids through conjugation with ursodeoxycholic and chenodeoxycholic bile acids. Different conjugation sites and both cleavable and non-cleavable linkages were explored to enable a comprehensive structure-activity relationship analysis. The resulting BA-DHA hybrids were evaluated in vitro for their anticancer activity against HCT116 and RKO colorectal cancer cell lines. As a result of the synergistic effect of the linker type and conjugation site, the BA-DHA hybrids synthesized via click chemistry emerged as the most active compounds in both cell lines, displaying 2- to 20-fold higher activity than the parent DHA. Mechanistic investigations further revealed that the click-derived BA-DHA hybrids possess enhanced anticancer activity and antimetastatic potential, achieving comparable or even superior efficacy to the parent compound at markedly lower concentrations.

Ursodeoxycholic acid (UDCA) and ferulic acid (Fer) are recognized for their neuroprotective effects against oxidative stress, being antioxidant agents able to preserve cell mitochondrial functions. This makes them potentially useful for neuronal protection against neurodegenerative disorders. Consequently, a new prodrug (UDC-Fer-Me) was developed as an ester-conjugate of the methyl-derivative of ferulic acid (Fer-Me), which retains the antioxidant properties of Fer and UDCA. The prodrug was encapsulated in compritol-based solid lipid nanoparticles (SLNs) for its nasal administration and nose-to-brain delivery. HPLC measurements demonstrated that (i) UDC-Fer-Me is hydrolyzed in rat liver and brain homogenates, as well as following intravenous administration in rats, leading to the release of UDCA and Fer-Me; (ii) the prodrug is not detectable in the cerebrospinal fluid (CSF) after intravenous administration to rats; however, (iii) it permeates in vitro nasal mucosal cells, inducing its partial hydrolysis. UDC-Fer-Me also counteracted reactive oxygen species production induced by H2O2 on cultured neuronal cells. The UDC-Fer-Me-loaded SLNs showed a particle size distribution in the similar to 0.1 mu m order of magnitude and markedly improved the very slow dissolution rate of the prodrug in aqueous environments. Differential scanning calorimetry measurements suggested a good dispersion of the prodrug within the lipid matrix of SLNs; the drug loading was about 8% (61% entrapment efficiency). Following nasal administration of SLNs to rats (2 mg/kg dose), both the prodrug and Fer-Me were detected in the CSF at concentrations reaching up to 2 mu g/mL, consistent with drug delivery to the central nervous system following intranasal administration.

Delivery represents a major hurdle to the clinical advancement of oligonucleotide therapeutics for the treatment of disorders such as Duchenne muscular dystrophy (DMD). In this preliminary study, we explored the ability of 2′-O-methyl-phosphorothioate antisense oligonucleotides (ASOs) conjugated with lipophilic ursodeoxycholic acid (UDCA) to permeate across intestinal barriers in vitro by a co-culture system of non-contacting IEC-6 cells and DMD myotubes, either alone or encapsulated in exosomes. UDCA was used to enhance the lipophilicity and membrane permeability of ASOs, potentially improving oral bioavailability. Exosomes were employed due to their biocompatibility and ability to deliver therapeutic cargo across biological barriers. Exon skipping was evaluated in the DMD myotubes to reveal the targeting efficiency. Exosomes extracted from milk and wild-type myotubes loaded with 5′-UDC-3′Cy3-ASO and seeded directly on DMD myotubes appear able to fuse to myotubes and induce exon skipping, up to ~20%. Permeation studies using the co-culture system were performed with 5′-UDC-3′Cy3-ASO 51 alone or loaded in milk-derived exosomes. In this setting, only gymnotic delivery induced significant levels of exon skipping (almost 30%) implying a possible role of the intestinal cells in enhancing delivery of ASOs. These results warrant further investigations to elucidate the delivery of ASOs by gymnosis or exosomes.

The advantages of a treatment modality that combines two or more therapeutic agents with different mechanisms of action encourage the study of hybrid functional compounds for pharmacological applications. Molecular hybridization, resulting from a covalent combination of two or more pharmacophore units, has emerged as a promising approach to overcome several issues and has also been explored for the design of new drugs for COVID-19 treatment. In this review, we presented an overview of small-molecule hybrids from both natural products and synthetic sources reported in the literature to date with potential antiviral anti-SARS-CoV-2 activity.

Herein, we report the synthesis of a new hybrid compound based on a 2′-deoxyuridine nucleoside conjugated with a NO photo-donor moiety (dU-t-NO) via CuAAC click chemistry. Hybrid dU-t-NO, as well as two previously reported 2′-deoxyadenosine based hybrids (dAdo-S-NO and dAdo-t-NO), were evaluated for their cytotoxic and cytostatic activities in selected cancer cell lines. dAdo-S-NO and dAdo-t-NO hybrids displayed higher activity with respect to dU-t-NO. All hybrids showed effective release of NO in the micromolar range. The photochemical behavior of the newly reported hybrid, dU-t-NO, was studied in the RKO colon carcinoma cell line, whereas the dAdo-t-NO hybrid was tested in both colon carcinoma RKO and hepatocarcinoma Hep 3B2.1-7 cell lines to evaluate the potential effect of NO released upon irradiation on cell viability. A customized irradiation apparatus for in vitro experiments was also designed.

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,7β-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 7β-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.

Artemisinin is a natural compound extracted from Artemisia species belonging to the Asteraceae family. Currently, artemisinin and its derivatives are considered among the most significant small-molecule antimalarial drugs. Artemisinin and its derivatives have also been shown to possess selective anticancer properties, however, there are several limitations and gaps in knowledge that retard their repurposing as effective anticancer agents. Hybridization resulting from a covalent combination of artemisinin with one or more active pharmacophores has emerged as a promising approach to overcome several issues. The variety of hybridization partners allows improvement in artemisinin activity by tuning the ability of conjugated artemisinin to interact with various molecule targets involved in multiple biological pathways. This review highlights the current scenario of artemisinin-derived hybrids with potential anticancer activity. The synthetic approaches to achieve the corresponding hybrids and the structure–activity relationships are discussed to facilitate further rational design of more effective candidates.

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.

Hepatocellular carcinoma is the third most common cause of cancer-related death according to the International Agency for Research on Cancer. Dihydroartemisinin (DHA), an antimalarial drug, has been reported to exhibit anticancer activity but with a short half-life. We synthesized a series of bile acid–dihydroartemisinin hybrids to improve its stability and anticancer activity and demonstrated that an ursodeoxycholic–DHA (UDC-DHA) hybrid was 10-fold more potent than DHA against HepG2 hepatocellular carcinoma cells. The objectives of this study were to evaluate the anticancer activity and investigate the molecular mechanisms of UDCMe-Z-DHA, a hybrid of ursodeoxycholic acid methyl ester and DHA via a triazole linkage. We found that UDCMe-Z-DHA was even more potent than UDC-DHA in HepG2 cells with IC50 of 1 μM. Time course experiments and stability in medium determined by cell viability assay as well as HPLC-MS/MS analysis revealed that UDCMe-Z-DHA was more stable than DHA, which in part accounted for the increased anticancer activity. Mechanistic studies revealed that UDCMe-Z-DHA caused G0/G1 arrest and induced reactive oxygen species (ROS), mitochondrial membrane potential loss and autophagy, which may in turn lead to apoptosis. Compared to DHA, UDCMe-Z-DHA displayed much lower cytotoxicity toward normal cells. Thus, UDCMe-Z-DHA may be a potential drug candidate for hepatocellular carcinoma.

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.

Bio-based 5-hydroxymethylfurfural (HMF) is upgraded to the fully furan-based poly(hydroxymethylfuroate) (PHMF) through a two-step organocatalyzed process.

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.

The synthesis of a small library of NH-Boc- or NH-Fmoc-protected l-phenylalanines carrying methyl groups at positions 2 and 6 and diverse functionalities at position 4 has been achieved. The approach, which took advantage of a Pd-catalyzed directed C-H dimethylation of picolinamide derivatives, allowed the electronic and steric properties of the resulting amino acid derivatives to be altered by appending a variety of electron-withdrawing, electron-donating, or bulky groups.

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.

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.

The advantages of a treatment modality that combines two or more therapeutic agents in cancer therapy encourages the study of hybrid functional compounds for pharmacological applications. In light of this, we reviewed recent works on hybrid molecules based on bile acids. Due to their biological properties, as well as their different chemical/biochemical reactive moieties, bile acids can be considered very interesting starting molecules for conjugation with natural or synthetic bioactive molecules.

Oligonucleotides (ONs) are gaining increasing importance as a promising novel class of biopharmaceuticals. Thanks to their fundamental role in gene regulation, they can be used to develop custom-made drugs (also called N-to-1) able to act on the gene expression at pre-translational level. With recent approvals of ON-based therapeutics by the Food and Drug Administration (FDA), a growing demand for high-quality chemically modified ONs is emerging and their market is expected to impressively prosper in the near future. To satisfy this growing market demand, a scalable and economically sustainable ON production is needed. In this paper, the state of the art of the whole ON production process is illustrated with the aim of highlighting the most promising routes toward the auspicated market-size production. In particular, the most recent advancements in both the upstream stage, mainly based on solid-phase synthesis and recombinant technology, and the downstream one, focusing on chromatographic techniques, are reviewed. Since ON production is projected to expand to the large scale, automatized multicolumn countercurrent technologies will reasonably be required soon to replace the current ones based on batch single-column operations. This consideration is supported by a recent cutting-edge application of continuous chromatography for the ON purification.

Hepatocellular carcinoma (HCC) is the most common primary liver malignancy in adults and accounts for 85–90% of all primary liver cancer. Based on the estimation by the International Agency for Research on Cancer in 2018, liver cancer is the fourth leading cause of cancer death globally. Dihydroartemisinin (DHA), the main active metabolite of artemisinin derivatives, is a well-known drug for the treatment of malaria. Previous studies have demonstrated that DHA exhibits antitumor effects toward a variety of human cancers and has a potential for repurposing as an anticancer drug. However, its short half-life is a concern and may limit the application in cancer therapy. We have reported that UDC-DHA, a hybrid of bile acid ursodeoxycholic acid (UDCA) and DHA, is ∼12 times more potent than DHA against a HCC cell line HepG2. In this study, we found that UDC-DHA was also effective against another HCC cell line Huh-7 with an IC50 of 2.16 μM, which was 18.5-fold better than DHA with an IC50 of 39.96 μM. UDC-DHA was much more potent than the combination of DHA and UDCA at 1:1 molar ratio, suggesting that the covalent linkage rather than a synergism between UDCA and DHA is critical for enhancing DHA potency in HepG2 cells. Importantly, UDC-DHA was much less toxic to normal cells than DHA. UDC-DHA induced G0/G1 arrest and apoptosis. Both DHA and UDC-DHA significantly elevated cellular reactive oxygen species generation but with different magnitude and timing in HepG2 cells; whereas only DHA but not UDC-DHA induced reactive oxygen species in Huh-7 cells. Depolarization of mitochondrial membrane potential was detected in both HepG2 and Huh-7 cells and may contribute to the anticancer effect of DHA and UDC-DHA. Furthermore, UDC-DHA was much more stable than DHA based on activity assays and high performance liquid chromatography-MS/MS analysis. In conclusion, UDC-DHA and DHA may exert anticancer actions via similar mechanisms but a much lower concentration of UDC-DHA was required, which could be attributed to a better stability of UDC-DHA. Thus, UDC-DHA could be a better drug candidate than DHA against HCC and further investigation is warranted.

A series of hybrid compounds based on natural products-bile acids and dihydroartemisinin-were prepared by different synthetic methodologies and investigated for their in vitro biological activity against HL-60 leukemia and HepG2 hepatocellular carcinoma cell lines. Most of these hybrids presented significantly improved antiproliferative activities with respect to dihydroartemisinin and the parent bile acid. The two most potent hybrids of the series exhibited a 10.5- and 15.4-fold increase in cytotoxic activity respect to dihydroartemisinin alone in HL-60 and HepG2 cells, respectively. Strong evidence that an ursodeoxycholic acid hybrid induced apoptosis was obtained by flow cytometric analysis and western blot analysis.

A series of glycomimetics of UDP-GlcNAc, in which the β-phosphate has been replaced by either an alkyl chain or a triazolyl ring and the sugar moiety has been replaced by a pyrrolidine ring, has been synthesized by the application of different click-chemistry procedures. Their affinities for human O-GlcNAc transferase (hOGT) have been evaluated and studied both spectroscopically and computationally. The binding epitopes of the best ligands have been determined in solution by means of saturation transfer difference (STD) NMR spectroscopy. Experimental, spectroscopic, and computational results are in agreement, pointing out the essential role of the binding of β-phosphate. We have found that the loss of interactions from the β-phosphate can be counterbalanced by the presence of hydrophobic groups at a pyrroline ring acting as a surrogate of the carbohydrate unit. Two of the prepared glycomimetics show inhibition at a micromolar level.