Design and synthesis of novel classes of HDACs and KMTs inhibitors

For long, scientists thought that our body was driven only by our genetic code that we inherited at birth. However, this determinism was shattered entirely and proven as false in the second half of the 21st century with the discovery of epigenetics. Instead, cells turn genes on and off using reversible chemical marks. With the tremendous progression of epigenetic science, it is now believed that we have a certain power over the expression of our genetic traits. Over the years, these epigenetic modifications were found to be at the core of how diseases alter healthy cells, and environmental factors and lifestyle were identified as top influencers. Epigenetic dysregulation has been observed in every major domain of medicine, with a reported implication in cancer development, neurodegenerative pathologies, diabetes, infectious disease and even obesity. Substantially, an epigenetic component is expected to be involved in every human disease. Hence, the modulation of these epigenetics mechanisms has emerged as a therapeutic strategy. Histone deacetylases (HDAC) are silencing epigenetic markers involved in the mediation of the acetylation system. Aberrant HDAC activities have been associated with the development of a broad range of pathologies, and HDAC inhibition is highly regarded as a potential therapeutic target. This approach became successful with the approval by the FDA of several epidrugs. However, they present undesired side effects. Lack of selectivity was identified as a principal suspect and, therefore, the development of novel compounds that would target more selectively the epigenetics enzymes represent a major axis of research for future pharmacological applications. Accordingly, this work focus on designing new classes of HDAC inhibitors. A variety of structures, build around the use of amino acids, were explored and potential inhibitors were produced with different cores. These series were then evaluated in enzymatic inhibition assays on HDAC and in cell growth inhibition assays on leukaemia cell lines. Additionally, we were also involved in the international consortium A-ParaDDisE that aimed to develop epigenetic modulators as drug candidates against the main parasitic diseases: malaria, schistosomiasis, leishmaniasis and Chagas disease. This collaboration resulted in the identification of critical targets for the different parasites and the development of testing methods to move compounds toward clinical development. The antischistosomal effect of some of our inhibitors was, thus, evaluated. Finally, a side project was carried at the University of Salerno in Italy in a short-term scientific mission that aimed to develop a new class of SETD8 methyltransferase inhibitors

NOVEL VX-809 HYBRIDS AS CORRECTORS OF F508del- CYSTIC FIBROSIS TRANSMEMBRANE CONDUCTANCE REGULATOR (CFTR) PROTEIN

Cystic fibrosis (CF) is an autosomal recessive genetic disease that affects different organs as the lungs, the digestive system and the reproductive tract, also impacting on the growth of subjects. To date, the known CFTR gene mutations are more than 2000, but in particular the most widespread is the deletion of a phenylalanine at position 508 that involves the synthesis of a defective protein, that it is not able to lead the correct folding and is degraded early in the proteasome. When some mutated CFTR proteins reach the plasma membrane (PM), the activity of the channel is impaired. Today, in addition to symptomatic therapies, molecules have been developed against the basic defect of CFTR, in particular they are small molecules called modulators, including potentiators and correctors. The potentiators act on the gating of CFTR allowing the correct opening, while the correctors save the CFTR protein allowing it to obtain the correct conformation and stabilization at the PM. The first corrector to be approved was VX-809 called Lumacaftor (by Vertex Pharmaceuticals), for homozygous patients for the F508del-CFTR mutation. The VX-809 was then used in combination with the potentiator VX-770 in the Orkambi (by Vertex Pharamaceuticals) formulation to achieve a better effect. Since VX-809, both alone and in combination with VX-770, shows some defects, it became necessary to search new molecules with similar mechanism of action, but with fewer side effects. In this regard Vertex designed a corrector called VX-661, which acts with similar mechanism of action, but has better pharmacokinetic characteristics. Despite recent advances in drug therapy of CF and the improvement of patients' well- being, there are still many problems that arise; on the one hand, the study of active molecules against more rare mutations is necessary, on the other hand the discovery of more powerful molecules than those commercially available. In this thesis I report the synthesis, characterization and purification of molecules active as correctors, of which both biological evaluation and molecular docking studies have been carried out. In particular starting from the structure of a molecule previously synthesized by my team, called 2a, and studying its portion, I found a common backbone to be able to decorate with different functional groups for investigating the effect of each chemical group on the biological activity. Hence, I got a series of more active compounds, which showed higher potency than both the progenitor 2a (EC50 = 0,08 μM) and the commercial VX-809 (EC50 = 2,60 μM), whose activity was deepened with further biochemical assays and tested in combination with other correctors in order to study synergic or additives effects. Through a multidisciplinary approach (bioinformatic, chemistry and biochemistry), it was possible to obtain promising molecules as correctors of F508del-CFTR, in particular more potent than the 4 initial prototype, such as 34 (EC50 = 0,02 μM), 42 EC50 = 0,07 μM), 70 (EC50 = 0,06 μM), 74 (EC50 = 0,03 μM) and 81 (EC50 = 0,03μM)

Computational Design, Synthesis, Characterization and Pharmacological Evaluation of Some Piperidine Derivatives

The Aurora kinase family is a collection of highly related serine/threonine kinases that functions as a key regulator of mitosis. In mammalian cells, Aurora has evolved into three related kinases known as Aurora-A, Aurora-B, and Aurora-C. These kinases are over expressed in a number of human cancers, and transfection studies have established Aurora-A as a bone fide oncogene. Because Aurora over expression is associated with malignancy, these kinases have been targeted for cancer therapy. So in the present study, it was decided to design some inhibitory lead compounds of Aurora kinase A as using computational tools like Catalyst (pharmacophore modeling) and GLIDE(structure based drug design/Docking). Then the designed compounds were synthesized and screened for anticancer studies. Eighty two Aurora A kinase inhibitors from Medicinal Chemistry Journals were selected for modeling studies based on chemical and biological diversity. The selected molecules were then divided into 21 training set molecules and 61 test set molecules. Using the training set molecules pharmacophore models (hypothesis) were generated in Hypogen (Catalyst). The most active molecule in the training set fits very well with the top scoring pharmacophore hypothesis. The best hypothesis consists of one hydrogen bond acceptor, one hydrophobic aliphatic and two ring aromatics. The best hypothesis Hypo-1 is characterized by the highest cost difference (58 bits), lowest RMS deviation (1.30) with a correlation of 0.94. The best pharmacophore hypothesis was used to screen the 61 Aurora A Kinase inhibitors in the Aurora kinase inhibitor data base. The model developed was shown to be a good model with 0.65 as Goodness of Hit score (GH) and a enrichment factor of 1.154. GLIDE was the docking program used for the structure based drug design. The 82 Aurora A inhibitors used for the pharmacophore studies were considered for docking study to develop comparative model. Out of the 82 inhibitors 21 were used in the training set. Crystal structure of Aurora A (PDB code: Imq4) was employed for the docking studies Structure based docking studies were carried out using Glide on Aurora A kinase inhibitors to the 3D structure of Aurora A kinase and generated 50 best docking poses. The best poses were selected based on the scoring functions and poses orientation with the active site amino acids. To get a better VS model, a MLR analysis was carried out using pharmacophore model and the docking scoring function. A combination of Pharmacophore model, GLIDE SP dock score gave a good model. The VS model obtained was further used to search the virtual library consisting of 10,000 structurally diversified molecules generated using fragment and knowledge based design, which yielded 300 molecules as potent Hits. The hits obtained were used for PASS prediction studies. A consensus was obtained between the docking scores and the PASS prediction values and finally 40 Hits were selected for synthesis and screening. The docking scores >-7 and prediction values above 0.6 were taken into consideration. PASS predicted some of the molecules to be active against colorectal cancer (1A-29A) and some other molecules to be inhibitors of phosphatase enzyme (P1-P11). A Drug likeness screening was carried out for all the 40 Hits including Lipinski rule of five and Toxicity assessment. All most all the compounds exhibited 2 violations of the Lipinski rule which was found to be normal with anticancer drugs. It is proven that kinase inhibitors in general have high molecular weight and LogP values. So these violations were accepted in the present study. In toxicity assessment all the compounds showed Green colour code except nine molecules having nitro and dimethyl amino substitution exhibiting mutagenicity and Tumorigenicity. But still these molecules were also included in the in vitro screening because so many active scaffolds have these substructures and their activities were also predicted to be good. These hits can be further refined to reduce the unwanted reactions by including detoxifying sub structures. The designed molecules have piperidine-4-ones scaffold attached with 2-Aminopyrimidine (1A-29A) and 2-Pyrazoline (P1-P11) substructures. The Schiff bases and Mannich bases of piperidine-4-ones were synthesized according to the synthetic scheme and characterized by IR,1H NMR,13 C NMR, COSY NMR and Mass spectroscopy. The compounds were subjected to in vitro anticancer studies in colorectal cell lines (1A-29A).The compounds 21A and 25A show the least IC50 values 0.01 and 0.01 respectively. The compound 21A maintains the same level of activity through out the working range (0.01-100 μM). There is no concentration related gradation in the activity profile. In the case of compound 25A, the peak activity is noted in the minimum concentration of 0.01 μM itself. Even though there is a decrease in the activity with increase in the concentration, the activity profile remained well above the required level. The other compounds showing significant activity are 3A, 10A, 13A and 26A. The compounds 6A, 9A and 16A show less activity with IC50 values in the range of 10 μM. All the other compounds showed moderate activity with IC50 values in the range of 1-5 μM. The compounds P1-P11 were subjected to phosphatase inhibition activity. The compounds P1-P11 were used in three concentration levels (50/125/250mcg). The absorbances of these solutions were measured at 620nm after carrying out the assay with Folin’s reagent. The concentrations of the phenol formed in these solutions were obtained from the standard graph of phenol. From the analysis of the data it can be seen that all the compounds P1-P11 are possessing phosphatase inhibition activity. In addition to that they also show a gradation in their dose response. All the compounds show less inhibition at 50 mcg, moderate inhibition at 125 mcg and a fairly good inhibition at 250 mcg. The compounds P1-P11 have 2-pyrazoline moiety as a sub structure in common. The compounds P1-P5 at 50 mcg concentration level shows very less inhibition. A concentration dependent increase in inhibitory activity was observed with P1, P2, P3, P4, and P5. That is better inhibitory activity was observed with higher concentrations (250 mcg). The compounds P6-P11 showed better inhibition in all the three concentration levels. In all these compounds the C3 of piperidin-4-one ring contain isopropyl group which may contribute to the increase in inhibitory activity. Of all compounds, the compound P3 at the 250mcg concentration level shows best activity. Thus the compounds P3, P7 and P11 can be further developed to get effective phosphatase inhibitors. By arriving at the leads 21A and 25A for anticancer activity, the aim of the work to develop leads for anticancer activity was fulfilled. These analogues can be novel templates for lead optimization purpose in cancer chemotherapy. To conclude the anticancer leads obtained in this study can be refined further to get a potent anticancer molecules. Drugs targeting multiple kinases have proven to be effective against treatment of various diseases. The activities of serine/threonine protein phosphatases needs further study, but it is clear that these enzymes are potential targets for novel therapeutics with applications in many diseases, including cancer, inflammatory diseases and neuro degeneration. Computational techniques have provided starting points for designing multiple inhibitors against individual targets using crystal structural information of kinases and pharmacophore of kinase inhibitors. So these techniques can be explored further to design new drug candidates for various diseases

Os impactos do REACH sobre a indústria cosmética brasileira

A Comissão Européia apresentou em 2003 a proposta do REACH (Registration, Evaluation, Authorization and Restriction of Chemical) como uma forma de proteção à saúde humana e ambiental, sendo implementado em 2007. Trata-se de um regulamento que visa à regular a fabricação e a comercialização de produtos químicos dentro da União Européia, atribuindo às indústrias dessas substâncias o ônus de avaliar os riscos decorrentes da utilização de seus produtos, no caso de sua produção ultrapassar a medida de uma tonelada por ano. A empresa que quiser manter a comercialização de seus produtos dentro da UE deverá registrá-los junto à Agência Européia de Produtos Químicos (ECHA). Além das indústrias químicas, esta nova política afeta também as empresas que utilizam determinadas substâncias químicas como matéria-prima de seus produtos. Como a química está presente em, praticamente, todos os produtos utilizados no dia-a-dia pela sociedade, a política do REACH representa um enorme impacto para o setor industrial como um todo. O setor de cosméticos, por estar em crescente expansão ao longo dos últimos anos, independentemente das crises econômicas pelas quais o mundo passa, foi escolhido como objeto de estudo. A seguir discutiremos como o regulamento REACH irá impactar a indústria brasileira de cosméticos

Recent Advances in Iron Catalysis

Transition metal-catalyzed reactions play a key role in many transformations of synthetic organic chemistry. For most of these reactions, noble metals, for example, palladium, have been used as catalysts. Over the last two decades, more and more first row transition metals have been applied as catalysts for organic reactions, with iron taking the center stage. The driving forces behind this development are not only the high costs for the noble metals but also their toxicity. Iron is the most abundant transition metal in the Earth’s crust, and thus, it is considerably cheaper than the precious noble metals. Moreover, iron compounds are involved in many biological processes, and thus, iron exhibits a low toxicity. Because of this low toxicity, iron-catalyzed reactions are important for an environmentally benign sustainable chemistry. However, iron catalysts are not only investigated to replace noble metals; they offer many applications in synthesis beyond those of classical noble metal catalysts. Several articles of the present book emphasize the complementarity of iron-catalyzed reactions as compared to reactions catalyzed by noble metals. The book shows intriguing recent developments and the current standing of iron-catalyzed reactions as well as applications to organic synthesis