PCSK9 inhibitors: a patent review 2018-2023.

INTRODUCTION: Proprotein convertase subtilisin/kexin 9 (PCSK9) plays a crucial role in breaking down the hepatic low-density lipoprotein receptor (LDLR), thereby influencing the levels of circulating low-density lipoprotein cholesterol (LDL-C). Consequently, inhibiting PCSK9 through suitable ligands has been established as a validated therapeutic strategy for combating hypercholesterolemia and cardiovascular diseases. AREA COVERED: Patent literature claiming novel compounds inhibiting PCSK9 disclosed from 2018 to June 2023 available in the espacenet database, which contains more than 150 million patent documents from over 100 patent-granting authorities worldwide. EXPERT OPINION: The undisputable beneficial influence of PCSK9 as a pharmacological target has prompted numerous private and public institutions to patent chemical frameworks as inhibitors of PCSK9. While several compounds have advanced to clinical trials for treating hypercholesterolemia, they have not completed these trials yet. These compounds must contend in a complex market where new, costly, and advanced drugs, such as monoclonal antibodies and siRNA, are prescribed instead of inexpensive and less potent statins.

Alkene carboamination/oxidative denitrogenation of 3-allyl-3-hydrazinylindolin-2-ones: one-pot entry to spirocyclopropyloxindoles.

A one-pot protocol, consisting of a Pd-catalysed carboamination reaction, followed by N-deprotection and oxidative denitrogenation, has been developed for the synthesis of diversely substituted spirocyclopropyloxindoles, in yields up to 73% and with diastereoselectivity close to 1 : 1. Readily accessible starting materials, mild reaction conditions, an easy to operate one-pot procedure and good functional group tolerance make this transformation a versatile tool for the synthesis of substituted spirocyclopropyloxindoles. This protocol successfully works on the gram-scale and allows access to both diastereoisomers separately. A plausible mechanism was proposed, and a series of post-transformations were performed on the obtained products, showing their remarkable synthetic versatility.

Computational Design, Synthesis, and Biological Evaluation of Diimidazole Analogues Endowed with Dual PCSK9/HMG-CoAR-Inhibiting Activity.

Proprotein convertase subtilisin/kexin 9 (PCSK9) is responsible for the degradation of the hepatic low-density lipoprotein receptor (LDLR), which regulates circulating cholesterol levels. Consequently, the PCSK9 inhibition is a valuable therapeutic approach for the treatment of hypercholesterolemia and cardiovascular diseases. In our studies, we discovered Rim13, a polyimidazole derivative reducing the protein-protein interaction between PCSK9 and LDLR with an IC50 of 1.6 µM. The computational design led to the optimization of the shape of the PCSK9/ligand complementarity, enabling the discovery of potent diimidazole derivatives. In fact, carrying out biological assays to fully characterize the cholesterol-lowering activity of the new analogues and using both biochemical and cellular techniques, compound Dim16 displayed improved PCSK9 inhibitory activity (IC50 0.9 nM). Interestingly, similar to other lupin-derived peptides and their synthetic analogues, some compounds in this series showed dual hypocholesterolemic activity since some of them complementarily inhibited the 3-hydroxy-3-methylglutaryl coenzyme A reductase.

Synthesis of SARS-CoV-2 Mpro inhibitors bearing a cinnamic ester warhead with in vitro activity against human coronaviruses.

COVID-19 now ranks among the most devastating global pandemics in history. The causative virus, SARS-CoV-2, is a new human coronavirus (hCoV) that spreads among humans and animals. Great efforts have been made to develop therapeutic agents to treat COVID-19, and among the available viral molecular targets, the cysteine protease SARS-CoV-2 Mpro is considered the most appealing one due to its essential role in viral replication. However, the inhibition of Mpro activity is an interesting challenge and several small molecules and peptidomimetics have been synthesized for this purpose. In this work, the Michael acceptor cinnamic ester was employed as an electrophilic warhead for the covalent inhibition of Mpro by endowing some peptidomimetic derivatives with such a functionality. Among the synthesized compounds, the indole-based inhibitors 17 and 18 efficiently impaired the in vitro replication of beta hCoV-OC-43 in the low micromolar range (EC50 = 9.14 µM and 10.1 µM, respectively). Moreover, the carbamate derivative 12 showed an antiviral activity of note (EC50 = 5.27 µM) against another hCoV, namely hCoV-229E, thus suggesting the potential applicability of such cinnamic pseudopeptides also against human alpha CoVs. Taken together, these results support the feasibility of considering the cinnamic framework for the development of new Mpro inhibitors endowed with antiviral activity against human coronaviruses.

Computational Design, Synthesis, and Biophysical Evaluation of ß-Amido Boronic Acids as SARS-CoV-2 Mpro Inhibitors.

The COVID-19 pandemic has given a strong impetus to the search for antivirals active on SARS-associated coronaviruses. Over these years, numerous vaccines have been developed and many of these are effective and clinically available. Similarly, small molecules and monoclonal antibodies have also been approved by the FDA and EMA for the treatment of SARS-CoV-2 infection in patients who could develop the severe form of COVID-19. Among the available therapeutic tools, the small molecule nirmatrelvir was approved in 2021. It is a drug capable of binding to the Mpro protease, an enzyme encoded by the viral genome and essential for viral intracellular replication. In this work, by virtual screening of a focused library of ß-amido boronic acids, we have designed and synthesized a focused library of compounds. All of them were biophysically tested by microscale thermophoresis, attaining encouraging results. Moreover, they also displayed Mpro protease inhibitory activity, as demonstrated by performing enzymatic assays. We are confident that this study will pave the way for the design of new drugs potentially useful for the treatment of SARS-CoV-2 viral infection.

Discovery of a Novel Trifluoromethyl Diazirine Inhibitor of SARS-CoV-2 Mpro.

SARS-CoV-2 Mpro is a chymotrypsin-like cysteine protease playing a relevant role during the replication and infectivity of SARS-CoV-2, the coronavirus responsible for COVID-19. The binding site of Mpro is characterized by the presence of a catalytic Cys145 which carries out the hydrolytic activity of the enzyme. As a consequence, several Mpro inhibitors have been proposed to date in order to fight the COVID-19 pandemic. In our work, we designed, synthesized and biologically evaluated MPD112, a novel inhibitor of SARS-CoV-2 Mpro bearing a trifluoromethyl diazirine moiety. MPD112 displayed in vitro inhibition activity against SARS-CoV-2 Mpro at a low micromolar level (IC50 = 4.1 µM) in a FRET-based assay. Moreover, an inhibition assay against PLpro revealed lack of inhibition, assuring the selectivity of the compound for the Mpro. Furthermore, the target compound MPD112 was docked within the binding site of the enzyme to predict the established intermolecular interactions in silico. MPD112 was subsequently tested on the HCT-8 cell line to evaluate its effect on human cells' viability, displaying good tolerability, demonstrating the promising biological compatibility and activity of a trifluoromethyl diazirine moiety in the design and development of SARS-CoV-2 Mpro binders.

Enantiopure ß-isocyano-boronic esters: synthesis and exploitation in isocyanide-based multicomponent reactions.

Various boron-containing isocyanides have been efficiently synthesized from the corresponding enantiopure ß-substituted ß-amino boronic acid pinacol esters, without need for protecting group interconversion, through a two-step, purification-free procedure. They were employed in a variety of isocyanide-based multicomponent reactions, proving to be reliable components for all of them and allowing the efficient synthesis of unprecedented, boron-containing peptidomimetics and heteroatom-rich small molecules, including biologically relevant cyclic boronates. Jointing together the ß-amido boronic acid moiety, deriving from the isocyanide component, with prominent pharmacophoric rings emerging from the multicomponent process, a successful application of the molecular hybridization concept could be realized.

Synthesis of potent and selective HDAC6 inhibitors led to unexpected opening of a quinazoline ring.

Histone deacetylase (HDAC) inhibitors are highly involved in the regulation of many pharmacological responses, which results in anti-inflammatory and anti-cancer effects. In the present work, chemoinformatic analyses were performed to obtain two potent and selective aminotriazoloquinazoline-based HDAC6 inhibitors. We unexpectedly obtained an aminotriazole from a water-driven ring opening of the triazoloquinazoline scaffold. Both compounds were evaluated as HDAC6 inhibitors, resulting in subnanomolar inhibitory activity and high selectivity with respect to class I HDAC1 and HDAC8. Importantly, the compounds were about 3- and 15-fold more potent compared to the reference compound trichostatin A. Additionally, the predicted binding modes were investigated with docking. Considering that the aminotriazole scaffold has never been embedded into the chemical structure of HDAC6 inhibitors, the present study suggests that both the aminotriazoloquinazoline and aminotriazole classes of compounds could be excellent starting points for further optimization of potential anticancer compounds, introducing such novel groups into a relevant and new area of investigation.

Unexpected chiral vicinal tetrasubstituted diamines via borylcopper-mediated homocoupling of isatin imines.

Addressing the asymmetric synthesis of oxindole-based α-aminoboronic acids, instead of the expected products we disclosed the efficient homocoupling of oxindole-based N-tert-butanesulfinyl imines, with the generation of chiral, quaternary 1,2-diamines in a mild and completely stereoselective way. The obtained 3,3'-bisoxindole derivatives were fully characterized by NMR and single-crystal X-ray diffraction analysis and proved to be single diastereoisomers and atropisomers. A plausible mechanism for the one-pot Cu(II)-catalyzed Bpin addition to the isatin-derived ketimine substrate and subsequent homocoupling is described.

Highly diastereoselective entry to chiral oxindole-based ß-amino boronic acids and spiro derivatives.

We here describe the first Cu-catalysed, diastereoselective 1,2-addition of 1,1-diborylmethane to chiral ketimines for the synthesis of quaternary stereocenters and spiro compounds. The method provides easy access to a range of chiral, highly functionalized compounds, namely oxindole-based ß,ß'-disubstituted ß-amino boronates, boron-containing peptidomimetics and six-, seven-membered spirocyclic hemiboronic esters. Such unprecedented compounds are mostly obtained in high yields and easily isolated as single diastereoisomers, paving the way to a more intense exploitation of boron-containing compounds in diversity-oriented chemistry and drug-discovery programs. Concerning stereochemistry, the application of Ellman's auxiliary strategy allows in principle to access both steric series of target compounds.

1,3-Dioxolan-4-Ones as Promising Monomers for Aliphatic Polyesters: Metal-Free, in Bulk Preparation of PLA.

The first example of solvent-free, organocatalyzed, polymerization of 1,3-dioxolan-4-ones, used as easily accessible monomers for the synthesis of polylactic acid (PLA), is described here. An optimization of reaction conditions was carried out, with p-toluensulfonic acid emerging as the most efficient Brønsted acid catalyst. The reactivity of the monomers in the tested conditions was studied following the monomer conversion by 1H NMR and the molecular weight growth by SEC analysis. A double activation polymerization mechanism was proposed, pointing out the key role of the acid catalyst. The formation of acetal bridges was demonstrated, to different extents depending on the nature of the aldehyde or ketone employed for lactic acid protection. The polymer shows complete retention of stereochemistry, as well as good thermal properties and good polydispersity, albeit modest molecular weight.

Ecdysteroid Derivatives that Reverse P-Glycoprotein-Mediated Drug Resistance.

The expression of multidrug resistance P-glycoprotein (P-gp) by cancer cells represents one of the major drawbacks to successful cancer therapy. Accordingly, the development of drugs that inhibit the activity of this transporter remains a major challenge in cancer drug discovery. In this context, several new ecdysteroid derivatives have been synthesized and evaluated as P-gp inhibitors. Two of them (compounds 9 and 14) were able to resensitize CEMVbl100 and LoVoDoxo resistant cell lines to vinblastine and doxorubicin, respectively. Indeed, both compounds 9 and 14 increased the cellular accumulation of rhodamine 123 in cells expressing P-gp and stimulated basal P-glycoprotein-ATPase activity at a 1 µM concentration, demonstrating their interference with the transport of other substrates in a competitive mode. Moreover, in a medulloblastoma cell line (DAOY), compounds 9 and 14 reduced the side population representing cancer stem cells, which are characterized by a high expression of ABC drug transporters. Further, in DAOY cells, the same two compounds synergized with cisplatin and vincristine, two drugs used commonly in the therapy of medulloblastoma. Molecular docking studies on the homology-modeled structure of the human P-glycoprotein provided a rationale for the biological results, validating the binding mode within the receptor site, in accordance with lipophilicity data and observed structure-activity relationship information. Altogether, the present results endorse these derivatives as promising P-gp inhibitors, and they may serve as candidates to reverse drug resistance in cancer cells.

Isonitrile-Based Multicomponent Synthesis of ß-Amino Boronic Acids as ß-Lactamase Inhibitors.

The application of various isonitrile-based multicomponent reactions to protected (2-oxoethyl)boronic acid (as the carbonyl component) is described. The Ugi reaction, both in the four components and in the four centers-three components versions, and the van Leusen reaction, proved effective at providing small libraries of MIDA-protected ß-aminoboronic acids. The corresponding free ß-aminoboronic acids, quantitatively recovered through basic mild deprotection, were found to be quite stable and were fully characterized, including by 11B-NMR spectroscopy. Single-crystal X-ray diffraction analysis, applied both to a MIDA-protected and a free ß-aminoboronic acid derivative, provided evidence for different conformations in the solid-state. Finally, the antimicrobial activities of selected compounds were evaluated by measuring their minimal inhibitory concentration (MIC) values, and the binding mode of the most promising derivative on OXA-23 class D ß-lactamase was predicted by a molecular modeling study.

Computationally Driven Structure Optimization, Synthesis, and Biological Evaluation of Imidazole-Based Proprotein Convertase Subtilisin/Kexin 9 (PCSK9) Inhibitors.

Proprotein convertase subtilisin/kexin 9 (PCSK9) is responsible for the degradation of the hepatic low-density lipoprotein receptor (LDLR), which in turn regulates the circulating low-density lipoprotein cholesterol (LDL-C) level. For this reason, the PCSK9 inhibition, by small molecules or peptides, is a validated therapeutic approach for fighting hypercholesterolemia and cardiovascular diseases. In this field, we have recently reported an imidazole-based peptidomimetic that has shown PCSK9 inhibitory activity in the micromolar range. Here, by applying advanced computational techniques, the binding mechanism of that imidazole peptidomimetic was predicted. Then, among a small set of poly-imidazole analogs, compounds showing the highest theoretical affinity were suitably synthesized, relying on a van Leusen reaction based multicomponent strategy. One compound (named RIm13) displayed a PCSK9 inhibitory activity 10-fold lower than the template compound, and, remarkably, at a concentration of 1 µM, it successfully prevented the LDLR degradation mediated by PCSK9 on HepG2 cells. As well as increasing the LDL uptake at the same concentration, RIm13 represents currently one of the most potent small molecules targeting the PCSK9/LDLR protein-protein interaction.

Exploitation of the Ugi-Joullié reaction in drug discovery and development.

Introduction: Multicomponent reactions are paramount in drug discovery for their ability to achieve high levels of diversity within the chemical space, generating complex structures from simple building blocks. Among them, the isocyanide-based Ugi-Joulliè reaction is particularly suited for the rapid synthesis of peptidomimetics and nitrogen-containing compounds. Areas covered: The latest achievements in drug discovery and synthetic chemistry regarding the application of the Ugi-Joulliè reaction in the field of natural compounds, peptidomimetics and small molecules, are reported in this article. All relevant literature was disclosed applying most common web-based literature searching tools, namely Web of Science, PubMed, SciFinder and Google Scholar. Expert opinion: The Ugi-Joulliè reaction represents an extremely versatile and simple synthetic methodology, useful for designing efficiently new molecular frameworks. Particularly relevant to drug discovery is the Ugi-Joulliè-based synthesis of conformationally constrained peptidomimetics and antibacterial depsipeptides. On the other hand, the many syntheses of new, nitrogen-containing heterocycles are not always followed by biological evaluation, losing opportunities for the disclosure of unprecedented lead compounds.

Allylation of isatin-derived N-Boc-hydrazones followed by Pd-catalyzed carboamination reaction: an entry to 3-spiro-pyrazolidyl-oxindoles.

The indium-mediated allylation of novel 3-(2-Boc-hydrazono)indolin-2-one derivatives, followed by a palladium-catalysed carboamination reaction, is described to afford unprecedented spirocyclic oxindoles in good yields. The method provides an efficient access to both cis and trans diastereoisomers of highly functionalized compounds, bearing an N-Boc, 5-substituted pyrazolidine ring at the C3-oxindole spiro junction. The versatility of the method is fully demonstrated starting from a series of substituted isatins and employing a variety of aryl halides in the key cyclization step.

Phytosterol and γ-Oryzanol Conjugates: Synthesis and Evaluation of their Antioxidant, Antiproliferative, and Anticholesterol Activities.

Fifteen new multifunctional conjugates were designed and synthesized by chemically linking the steroidal framework of natural occurring γ-oryzanol and γ-oryzanol-derived phytosterols to a wide range of bioactive natural compounds (fatty acids, phenolic acids, amino acids, lipoic acid, retinoic acid, curcumin, and resveratrol). Starting from γ-oryzanol, which is the main component of rice bran oil, this study was aimed at assessing if the conjugation strategy might enhance some γ-oryzanol bioactivities. The antioxidant activity was evaluated through three different mechanisms, namely, DPPH-scavenging activity, metal-chelating activity, and ß-carotene-bleaching inhibition. Measurement of the in vitro cell growth inhibitory effects on three different human cancer cellular lines was also carried out, and the potential hypocholesterolemic effect was studied. Compounds 10 and 15 displayed an improved antioxidant activity, with respect to that of γ-oryzanol. Compounds 2, 6, and 12 exerted an antiproliferative activity in the low micromolar range against HeLa and DAOY cells (GI50 < 10 µM). As for the claimed hypocholesterolemic effect of γ-oryzanol, none of the synthesized compounds inhibited the 3-hydroxy-3-methylglutaryl-coenzyme A reductase, a key enzyme in cholesterol biosynthesis.

Heteronanoparticles by Self-Assembly of Ecdysteroid and Doxorubicin Conjugates To Overcome Cancer Resistance.

Heteronanoparticles (H-NPs) consisting of conjugates characterized by a squalene tail linked to doxorubicin and ecdysteroid derivatives are presented. Biological evaluation on A2780ADR cell line confirms not only the maintenance of the activity of the parental drug but also the ability to overcome cancer resistance. The in vitro cell uptake was demonstrated, and the involvement of an endosomal-mediated pathway was suggested.

Sequential Multicomponent Strategy for the Diastereoselective Synthesis of Densely Functionalized Spirooxindole-Fused Thiazolidines.

We developed two Ugi-type three-component reactions of spirooxindole-fused 3-thiazolines, isocyanides, and either carboxylic acids or trimethylsilyl azide, to give highly functionalized spirooxindole-fused thiazolidines. Two diverse libraries were generated using practical and robust procedures affording the products in typically good yields. The obtained thiazolidines proved to be suitable substrates for further transformations. Notably, both the Ugi-Joullié and the azido-Ugi reactions resulted highly diastereoselective, affording predominantly the trans-configured products, as confirmed by X-ray crystallographic analysis.

One step access to oxindole-based ß-lactams through Ugi four-center three-component reaction.

A multicomponent Ugi reaction involving isatin, isocyanide and ß-amino acid components has been developed. The reactions proceeded smoothly to give ß-lactam-containing 3,3-disubstituted oxindoles in only one step and generally high yields. When chiral, non racemic, ß-amino acids were used, products were obtained as enantiomerically pure ß-lactams diastereoisomers, whose relative stereochemistry was determined by X-ray analysis. For one compound, a weak antibacterial activity has been preliminarily highlighted.