Exploring new horizons: design, synthesis, and comprehensive evaluation of novel 1ɑ,25-Dihydroxyvitamin D3 analogues for therapeutic applications
- Autores: Uxía Gómez Bouzó
- Directores de la Tesis: Yagamare Fall Diop (dir. tes.), Generosa Gómez Pacios (dir. tes.)
- Lectura: En la Universidade de Vigo ( España ) en 2024
- Idioma: inglés
- Tribunal Calificador de la Tesis: Miguel Ferrero Fuertes (presid.), María Joâo Matos (secret.), Verónica Isabel Dodero Bedaña (voc.)
- Programa de doctorado: Programa de Doctorado en Ciencia y Tecnología Química por la Universidad de Santiago de Compostela y la Universidad de Vigo
- Menciones:
- Internacional
- Calificación: Sobresaliente Cum Laude
- Materias:
- Enlaces
- Tesis en acceso abierto en: Investigo
- Resumen
This Doctoral Thesis is organized into three chapters: the first focuses on the design and synthesis of new vitamin D analogues; the second addresses their comprehensive evaluation through various studies; and the third, with a different approach, presents the development of new inverse agonists for the CB2 receptor, a result of one of the research internships.
Chapter I. Design and Synthesis of Vitamin D Analogues.
Vitamin D is a secosteroid hormone synthesized in human skin from 7-dehydrocholesterol in the presence of ultraviolet light. It is also commonly obtained from dietary sources, such as fortified dairy products. Vitamin D is primarily metabolized in the liver and subsequently in the kidney, yielding calcitriol, the biologically active metabolite of vitamin D.
The first chapter of this thesis focuses on the design and synthesis of new vitamin D analogues aimed at enhancing their therapeutic efficacy while reducing side effects, particularly hypercalcemia. This condition represents a major challenge in the clinical use of compounds like calcitriol and its derivatives, as it can lead to severe complications, such as soft tissue calcification and renal dysfunction, thus limiting the prolonged administration of these substances at high doses.
Calcitriol plays a crucial role in regulating calcium and phosphate homeostasis. Additionally, it has antiproliferative and differentiation-promoting properties, making it a promising candidate for the treatment of various diseases, including cancer. However, its tendency to elevate blood calcium levels restricts its clinical use at high doses. As a result, the development of analogues that retain the therapeutic benefits of calcitriol without inducing hypercalcemia has been investigated.
The design approach for these analogues involves structural modifications to the calcitriol molecule to optimize its biological stability, affinity for the vitamin D receptor (VDR), and safety profile for prolonged use. The most significant modification focuses on the molecule's side chain, which is crucial in determining its pharmacological properties, including VDR affinity and the ability to modulate gene transcription.
A key advancement in this research has been the development of Gemini-type analogues, which feature two side chains attached to C-20, allowing additional control over their biological activity. These modifications enable more precise regulation of interactions with the VDR, enhancing antiproliferative effects while minimizing hypercalcemic effects. The introduction of dual chains has proven particularly effective in increasing the selectivity of these analogues toward specific pathways, reducing interaction with non-target tissues and thereby minimizing side effects.
The synthetic approach for calcitriol and its analogues involves a Wittig-Horner coupling reaction between a phosphine oxide anion (the precursor for ring A) and a Grundmann ketone (carrying the CD bicyclic system and side chain).
Among the most notable compounds synthesized is UVB1, a Gemini-type calcitriol analogue, which has shown significant antitumor activity alongside low toxicity in various studies. The synthesis of UVB1 was optimized to improve yield and scalability, facilitating broader biological testing and potential clinical development. The new synthetic strategy involves the initial construction of UVB1's modified side chain, followed by the synthesis of the second natural calcitriol side chain.
In the 1990s, LEO Pharmaceutical Products introduced a new class of calcitriol-derived compounds known as 20-epi vitamin D analogues. These analogues, featuring altered stereoisomerism at the C-20 position, exhibited new biological profiles. C-20 epimerization in calcitriol significantly reorients the six-carbon side chain without altering its affinity for the VDR, but increases its antiproliferative activity by 200 to 7,000 times compared to calcitriol, while maintaining relatively constant calcemic activity. Based on this, UG-650, a non-Gemini analogue of UVB1, was synthesized with epimerization at C-20. This modification was designed to explore how the inversion of configuration at this position affects the compound's biological properties. The focus was on UVB1's modified side chain, its interaction with the VDR, and its ability to regulate gene transcription in both tumor and non-tumor cells.
Another relevant approach involved the synthesis of UVB1 analogues incorporating a cyclopropyl ring in the modified side chain, such as UG-480 and UG-481. A key aspect of this synthesis was the Simmons-Smith cyclopropanation, which involves the formation of a carbenoid intermediate to facilitate olefin addition. The introduction of these cyclopropyl rings aimed to investigate how changes in the structural rigidity of the molecule affect its biological activity. The rigidity provided by the cyclopropyl ring could stabilize the interaction between the analogue and the VDR.
Additionally, the introduction of heteroatoms into the vitamin D structure is another key strategy for modulating its biological activity. Calcitriol analogues containing nitrogen or sulfur atoms in their side chain were synthesized as an innovative approach to alter their electronic properties, potentially influencing VDR binding affinity and compound stability in the body. For example, the UG-635 derivative incorporates a nitrogen atom at the C-25 position, which could prolong its activity by reducing the compounds metabolic degradation. Furthermore, the design and synthesis of Gemini-type analogues containing a nitrogen atom at C-25, such as UG-760 and UG-764, were explored. The key step common to the synthesis of these derivatives is the Pd(0)-catalyzed allylic alkylation, also known as the Tsuji-Trost reaction. This transformation generated by-products through side reactions, such as syn-ß-hydride elimination and O-alkylation, presenting challenges in terms of reproducibility and process optimization.
Moreover, the synthesis of derivatives containing an amine group (1.16, 1.18, 1.21) or amide group (1.17, 1.19, 1.22) at C-25 position was abandoned due to degradation during purification following the reduction of the nitro group. Additionally, the synthesis of the Gemini analogue 1.20, containing a nitro group at C-25 and epimerization at C-20, was also discontinued due to the low reproducibility of the Tsuji-Trost reaction. This reaction exhibited extremely low yields (5%), and in many cases, Pd black precipitation occurred, blocking the reaction. Notably, the stereochemical configuration at C-20 increased steric hindrance, making it difficult to form the -allyl complex necessary for the reaction. As a result, the synthesis of the intermediate 1.130, crucial for compound 1.20, was unattainable, leading to the discontinuation of its synthesis.
The analogue containing a sulfur atom was designed with a similar goal. By incorporating a sulfur atom into the side chain, it is expected that these analogues will exhibit higher binding affinity for the VDR due to sulfurs electronic properties. This could result in stronger interactions with the receptors active site, thereby enhancing the biological activity of these compounds. The introduction of sulfur at the C-22 position was achieved via an efficient method developed by the Chugai group, through a stereospecific [3,3]-sigmatropic rearrangement on the less hindered face. One challenge in this synthetic route was the elongation of the second side chain. Although alternative methods for C-21 functionalization were explored, attempts to elongate the natural chain were unsuccessful. Various strategies, such as mesylation and Wittig and Corey-Fuchs reactions, did not produce satisfactory results. However, elongation via reaction with the anion of triethyl((2-methylbut-3-yn-2-yloxy)silane was successful, leading to the discontinuation of the initially proposed 1.23 analogue synthesis and the proposal of the UVB1 16-ene analogue with sulfur at C-22.
Chapter II. Evaluation of Vitamin D Analogues.
This chapter presents a detailed evaluation of several calcitriol analogues synthesized in Chapter I. It describes studies conducted in collaboration with various research groups investigating the biological and structural activity of the synthesized compounds. The results obtained, from molecular docking assays to cell proliferation studies and crystal structure analyses, are discussed regarding their relevance for the development of potential therapies in diverse medical areas.
The focus of the chapter is on the biological and chemical evaluation of synthesized vitamin D analogues, specifically UG-480, UG-481, UG-635, UG-650, UG-760, UG-764, and UVB1. Several studies, including molecular docking, molecular dynamics simulations, in vitro studies, and structural analyses, were employed to investigate the interaction of these analogues with the VDR and their biological activity in different cell models.
In the in silico studies, computational techniques were applied to compare the binding of calcitriol with the analogues UG-480 and UG-481 to the VDR, assessing their pharmacodynamic behavior and ability to induce allosteric modulation related to bioactivity. The pharmacodynamic interaction pattern between calcitriol and its analogues has been extensively studied, identifying key pharmacophoric contacts essential in most calcitriol analogues with bioactivity. Notably, the interaction between the 3ß-OH group of calcitriol and residues Ser237 and Arg274, as well as the interaction between the 1-OH and Ser278 and Tyr143, were emphasized. A third interaction with His305 and His397 was also identified, mediated by a hydroxyl group in the side chain of calcitriol. The UG-480 analogue demonstrated more stable interactions with key receptor residues, such as His305 and His397, suggesting it may possess greater bioactivity compared to UG-481.
Cellular assays were conducted in vitro using two key cell lines: SH-SY5Y (human neuroblastoma) and HepG2 (human hepatocellular carcinoma). Both UG-635 and UG-650 exhibited cytotoxicity profiles comparable to calcitriol, significantly reducing cell viability at concentrations above 10 M in HepG2 and 12.5 M in SH-SY5Y. In addition to cytotoxic effects, the neuroprotective effects of these analogues were evaluated under oxidative stress conditions induced by compounds such as FeNTA and tBHP. The results indicated that UG-635 and UG-650 significantly improved cell viability at non-toxic concentrations, suggesting their potential as neuroprotective agents in neurodegenerative diseases associated with oxidative stress, such as Alzheimer s and Parkinsons disease.
Further assays were conducted to evaluate the antiproliferative effects of the analogues in breast cancer cells (MCF-7) and osteoblastic cells (MC3T3-E1). The results from the analysis of vitamin D analogues in MCF-7 and MC3T3-E1 cells revealed significant antiproliferative activity in both cell lines. In MCF-7 cells, the analogues UG-480 and UG-635 exhibited activity comparable to calcitriol, while other analogues, such as UG-481 and UG-764, showed lower efficacy. Similarly, in MC3T3-E1 cells, UG-635 also showed calcitriol-like activity, whereas UG-764 was less active. Notably, the compounds UG-650 and UG-760 demonstrated antiproliferative activity comparable to or even greater than that of calcitriol in both cell lines, suggesting they could be promising therapeutic candidates for breast cancer and bone conditions such as osteoporosis.
Additionally, structural studies were performed using X-ray crystallography to analyze the binding structures of UG-480, UG-481, and UG-650 to the VDR. Understanding how calcitriol analogues bind to the VDR is essential for elucidating the specific interactions that determine their biological efficacy and safety profiles. This knowledge is crucial for designing analogues with enhanced therapeutic effects and reduced side effects. X-ray crystallography was employed to investigate the binding mode of the analogues to the VDR, allowing for the determination of the three-dimensional structure of the protein-ligand complexes. The results showed that UG-480 effectively stabilizes the active conformation of the VDR, inducing more pronounced conformational changes in the H6-H7 region compared to other analogues. In the case of UG-650, a stable binding conformation was observed, indicating a strong interaction with the receptor. These structural studies are critical for understanding how the analogues dock to the receptor at the atomic level and how these structural changes may influence their biological activity.
Finally, the antiproliferative effects of the combination of UVB1 with Paclitaxel (PTX) were evaluated in murine (4T1) and human (MDA-MB-231) breast cancer cells. Although chemotherapy is a primary therapeutic option for aggressive breast cancer, its effectiveness is limited by resistance and adverse effects. Given the expression of the VDR in triple-negative breast cancer (TNBC), its role in the antitumor action of PTX in TNBC cells was investigated. Previous studies had demonstrated that UVB1 reduces cell viability in TNBC-derived xenografts. This study focused on evaluating the antitumor effects of UVB1, either alone or in combination with low concentrations of PTX. The results showed a synergistic effect between both compounds, suggesting that combining UVB1 with other chemotherapeutic agents could enhance treatment efficacy in breast cancer while reducing the dose of each drug, thereby minimizing side effects. Furthermore, molecular docking studies indicated that PTX binds near the AF-2 region of the VDR and that both compounds can bind simultaneously. Dynamic simulations confirmed the stability of this interaction.
It is important to highlight that, in the evaluation studies of the synthesized derivatives, the effects of the new analogues were compared to calcitriol, considered the reference standard in vitamin D studies. As mentioned, calcitriol is an active form of vitamin D that has shown beneficial effects in the treatment of various diseases. However, one of the main objectives of this study was to demonstrate that the new analogues could have similar or superior effects compared to calcitriol, but with an improved safety profile.
Chapter III. Development of Novel Inverse Agonists for CB2 Receptor.
Plants of the Cannabis genus have been used for thousands of years for their medicinal and psychoactive effects. In recent decades, the total number of natural compounds identified or isolated from Cannabis sativa L. has continued to increase, reaching a total of 545 compounds to date, of which 104 are classified as phytocannabinoids. The identification of 9-THC as the main psychoactive compound in Cannabis sativa L. led to the characterization of a lipid signaling network known as the endocannabinoid system (eCB). This system, which includes CB1 and CB2 receptors, regulates various physiological processes such as metabolism, appetite regulation, inflammation, pain, and cancer.
Studying the CB2 receptor requires developing ligands with high selectivity for CB1 since CB2 expression is much lower in most tissues, especially in the central nervous system. In recent years, the CB2 receptor has gained particular relevance due to its predominant distribution in immune system cells and its involvement in inflammatory processes and neurodegenerative diseases. Unlike the CB1 receptor, which is more related to the psychoactive effects of cannabinoids, CB2 represents an attractive therapeutic target for developing treatments that modulate inflammatory responses and chronic pain, thereby avoiding adverse effects associated with CB1 activation.
For the design of new inverse agonists for CB2, the construction of a high-affinity structural anchor subunit was facilitated through an efficient modular synthetic strategy based on Friedel-Crafts alkylation of resorcinols. Additionally, recent advances in X-ray crystallography of cannabinoid receptors bound to ligands have marked a milestone in the research of these receptors, providing unprecedented insight into CB2 activation. This has enabled the rational design of ligands for agonists, antagonists, or inverse agonists, considering both selectivity and mode of action.
A comparative study between the active and inactive states of CB2 revealed that the inactive state presents a larger lipophilic space adjacent to the gem-dimethyl group at C-2. This subpocket is of particular interest as it contains the "switch" residue Trp258, a highly conserved structural motif in G protein-coupled receptors. Interaction with this switch in CB2 is sufficient to trigger activation and signaling at the cellular level.
This chapter specifically details the rational design and synthetic approach of a series of inverse agonists for the CB2 receptor, based on the compound HU-308, a cannabinoid known for its selectivity towards this receptor. The design approach included the introduction of alkyl and phenyl substituents in the gem-dimethylheptyl side chain, allowing modifications of the pharmacological properties of the compounds and enhancing their selectivity and affinity for the CB2 receptor.
In this context, the Carreira Group has recently developed a ligand platform using a chimeric combination of the ligands HU-308 and AM841, aiming to generate a selective covalent binding chemical probe for CB2. The developed synthetic route comprises an optimal number of steps, starting from the commercially available substrate (3,5-dimethoxyphenyl)acetonitrile.
Once the synthesis was completed, various studies were conducted on the obtained compounds, yielding significant findings that expanded our understanding of the eCB system. These investigations ranged from in silico design of probes to in vitro and in vivo pharmacological assays. The final synthesis of the enantiomers (S)-3.40 and (R)-3.40, derived from the combination of structural elements from HU-308 and AM841 (3.6), and the introduction of a phenyl group at C-2, enabled comprehensive pharmacological studies. The results revealed that both compounds act as highly selective inverse agonists of CB2, demonstrating notable affinity for the receptor and the ability to specifically modulate associated cellular signaling pathways.
Finally, the proposal to use fluorescent probes as an ultimate application emphasizes the importance of improving existing probes in terms of modular design, selectivity, affinity, and specificity for CB2, as well as optimal properties and applicability under various experimental conditions. These advances not only contribute to the understanding of CB2 but also provide valuable tools for future experimental studies. The results of this study suggest that these inverse agonists could have therapeutic applications in a wide range of diseases, including inflammatory disorders, neuropathic pain, and cancer, highlighting the importance of further exploring the therapeutic potential of the eCB system.
