Rational design and synthesis of organic compounds for pharmaceutical purposes: from anticancer studies to nucleic acids delivery

Part A. Design and synthesis of anthraquinone-based Smo-inhibitors for the treatment of Hedgehog-dependent tumors The Hedgehog (Hh) signaling is one of the main oncogenic molecular pathway, since its aberrant activation causes or promotes the tumorigenesis mechanisms of various human tumors, which include basal cell carcinoma (BCC), medulloblastoma (MB), acute myeloid leukemia (AML), and rhabdomyosarcoma. For this reason, the components of the pathway (ie Ptch, Smo, Sufu, Gli1, and Gli2) represent attractive druggable target for anti-cancer therapy. The development of Smo antagonists, which can block the Hh pathway upstream, has received a lot of attention from the pharmaceutical companies. However, different side effects as well as the onset of mutations affecting the Smo receptor limit the use of some antagonists currently approved by the FDA. As a consequence, drug discovery of Smo antagonists has been recently focused on new and different chemotypes; in this scenario, a benzylphthalate derivative, Anta XV, has been identified by Novartis as a potent inhibitor of Smo. Given the availability of the crystallographic structure of the Smo receptor in complex with Anta XV (PDB ID: 4QIM), the design of new benzylphthalazine derivatives appears to be a promising path in the development of the candidate optimal drug. For this purpose, natural products have been a unique source of medicinal remedies since ancient times and, among the various classes of natural products, anthraquinones exhibit a wide variability of biological effects. From previous studies, synthetic C-alkylated emodin derivatives in position 7 have shown promising antiproliferative, apoptotic and DNA binding activity in tumor cell lines, such as HepG2 (human hepatocellular carcinoma cell line) and MDA-MB-231 (human breast cancer cell line). In particular, the presence of a piperazine cycle and its proven functionality highlighted an increase in antiproliferative activity. Based on the presence of a hyperactivation of Hh pathway in tumor cell lines and that known Smo inhibitors (i.e. Taladegib, Anta XV and its optimized derivatives, including NVP-LEQ506) also present a piperazine nucleus or 4-aminopiperidine in the Smo-targeting bullet, a small-focused library of anthraquinone-based compounds has been designed by combining the core of rhein with the Smo-targeting bullet of such known inhibitors. Our attention was particularly focused in finding an optimal hit compound as a starting point for further optimization and, noteworthy, molecular docking studies and simulations aided in the design of novel Smo antagonists endowed with a natural core. Thanks to a combination of visual inspection of binding modes and analyses of docking score, few derivatives were selected for organic synthesis and biological studies. The first series of RHs derivatives was synthesized and its luciferase activity was evaluated in vitro, using NIH3T3 Shh-Light II cells. Among the tested compounds, RH6 showed inhibitory activity with an IC50 of 5.698 μM and was subjected to further biological and cheminformatic analyses, which validated that it is a robust inhibitor of Hedgehog signaling by targeting Smo. Thus, on the bases of these findings, the small-focused library was further enlarged by designing and synthesizing a new series of RH6-analogues with the aim to investigate and study a structure-activity relationship. In conclusion, these results suggest that combining a natural core with analogues of the Smo-targeting bullet may broaden the spectrum of Smo-inhibitors design and facilitate the investigation of new highly effective small molecules Reference publication: Quaglio, D.; Infante, P.; Cammarone, S.; Lamelza, L.; Conenna, M.; Ghirga, F.; Adabbo, G.; Pisano, L.; Di Marcotullio, L.; Botta, B.; Mori, M. Exploring the Potential of Anthraquinone-Based Hybrids for Identifying a Novel Generation of Antagonists for the Smoothened Receptor in HH-Dependent Tumour. Chemistry - A European Journal, 2023, e202302237. DOI: https://doi.org/10.1002/chem.202302237 Part B. A cost-effective route to lipid ALC-0315: a key component of the Pfizer-BioNTech COVID-19 vaccine In November 2019, a new RNA Coronavirus (SARS-CoV-2) responsible for severe acute respiratory syndrome was identified in China and it was defined as COVID-19 (Coronavirus disease 2019). The World Health Organization (WHO) declared this new viral disease as a global public health emergency on January 30, 2020, and subsequently declared global pandemic status on March 11, 2020. The first COVID-19 vaccines to be authorized, namely BNT162b2 (Pfizer-BioNTech, Comirnaty®) and mRNA-1273 (Moderna, Spikevax®), are based on mRNA technology and they are both encapsulated into lipid nanoparticles (LNPs) in order to efficiently deliver nucleic acid material into organism that would otherwise be degraded in vivo by nucleases. The compound known as ALC-0315 is a key lipid component of the Pfizer-BioNTech COVID-19 vaccine and a highly sought-after lipid for nucleic acid therapeutics research. LNPs formulations of mRNA and siRNA including ALC-0315 and congeners are also important research resources in the developing field of nucleic acid therapies. As a result, research laboratories all over the world are very enthusiastic in ALC-0315. Accordingly, the need of finding the best route for the synthesis of this ionizable cationic lipid, that is about the 50% of the total composition of LNPs-mRNA, has considerably increased overtime. Indeed, the aim of this research, which was carried out in the laboratory of Prof. Dr. Marco Ciufolini at The University of British Columbia (Vancouver, Canada), was to devise a safe, faster, and cheaper synthetic route to lipid ALC-0315: the ionizable cationic lipid of Comirnaty® (Pfizer-BioNTech). Our research involved a study in reductive amination procedure, describing a synthetic route that more than doubles the overall yield relative to the published one. Relative to the public-domain route, the synthetic approaches described in this thesis demonstrated key advantages as: i) the performance of the oxidation without any hazardous Cr(VI) reagents; ii) unnecessary early-stage chromatography of intermediates; iii) employment of cleaner reactions which translated into easier chromatographic purification of final products and much higher yielding. In conclusion, this achieves significant efficiencies in terms of solvents, chromatographic supports and operator time. In addition, ALC-0315 analogues can also be created from diol 11b, making it easier the exploration of the structure-activity relationship of these remarkable lipids. It is worthy of note that this research led to a Very Important Paper (VIP) in Chemistry – A European Journal (© 2022 Wiley-VCH GmbH). Reference publication: Saadati, F.; Cammarone, S.; Ciufolini, M. A. A Route to Lipid ALC-0315: a Key Component of a COVID-19 mRNA Vaccine. Chemistry - A European Journal, 2022, 28 (48), e202200906. DOI: https://doi.org/10.1002/chem.202200906