Phd in MOLECULAR DESIGN AND CHARACTERIZATION FOR THE
PROMOTION OF HEALTH AND WELL-BEING: FROM DRUG TO FOOD

PhD Course in Molecular design and characterization for the promotion of health and well-being: from drug to food

XXXVII Cycle

PhD student Fabrizio Masciulli

Tutor Professor Luisa Mannina

1st year report

Untargeted and targeted analyses on different food matrices

During this first year my research activity was focused on two main projects: “FITO-BIO” financed by CIVIS Open Lab Sapienza and “Ri-cicloHorto” funded and supported by the Lazio region.

The aim of the “FITO-BIO” project is to evaluate the difference in chemical and metabolomic profile between ecotypes of the same medicinal plant species. This is because plants have variability in secondary metabolites due to multiple exogenous factors including cultivation method and climatic conditions. I focused my work on two ecotypes of Taraxacum officinale, organically grown and wild species. Both harvested in autumn and provided by farm “Fibreno Officinali”. Since both the aerial part and the root constitute the dandelion drug, I treated the sample separating the two parts and freeze drying them, separately. The lyophilized samples were extracted for untargeted nuclear magnetic resonance (NMR) analysis by Bligh-Dyer protocol. The obtained hydroalcoholic extracts were analyzed using Jeol spectrometer operating at a frequency of 600 MHz obtaining one-dimensional and two-dimensional spectra. The spectra assignments led to the identification of metabolites belonging to different classes: sugars, organic acids, amino acids, and other compounds such as choline and trigonelline. The quantified metabolites were expressed as mg/100 g of dried sample. This analysis showed the difference, in terms of quantity, of the metabolomic profile between ecotypes of the same wild and biologically cultivated species. In particular, sugars and other compounds levels were abundant in the organically grown taraxacum. Organic acids content did not have a constant trend among two ecotypes in both parts of plant. Arginine was not detected in the aerial part of dandelion (the highest amount was measured in the sample of organically grown roots). The root part of the organically grown ecotype showed the highest quantity of amino acids, except leucine, isoleucine, and valine, which were found to be more abundant in spontaneous one. The opposite trend was observed in the leaves, the spontaneous ecotype presented a higher amino acid content, except for Tyrosine. Instead, extracts for high-performance liquid chromatographic (HPLC-DAD) analysis were obtained by a green extraction protocol using a mixture of ethanol and water at percentage v/v 70/30. This targeted analysis allowed the separation of polyphenols, and the obtained chromatograms showed the presence of Phenolic compounds such as Catechin, Chlorogenic acid, Caffeic acid, Chicoric acid, Naringin and Rutin in the aerial part and roots of both ecotypes. The root of spontaneous ecotype was the only one that exhibited the polyphenols: Protocatechuic acid and Hydroxybenzoic acid. The Syringic acid was identified in the root extracts of organically grown ecotypes and leaves of spontaneous one. This multi-methodological approach allows for diversification between spontaneous and organically grown dandelion and makes it possible to delineate the differences among ecotypes both to support agronomic research to develop the best production technique for health and nutraceutical use.

The aim of the “Ri-cicloHorto” project is to ensure the valorisation of food-waste generated along agricultural supply chain. This target supports the circular economy and the use of non-edible portion of food, performing chemical characterization of the chosen matrices in order to identify and isolate molecules with health benefits. I focused my work on cauliflower by-product (leaves, stems and flower-waste) supplied by F.lli Calevi Alberto e Stefano (Viterbo, Lazio, Italy). The Brassica olearacea belonging to Cruciferae family is rich in bio-active compounds useful to formulate dietary supplement for human health. The HPLC-DAD analysis were performed to identify a class of bio-active secondary metabolites, the glucosinolates (organic compounds derived from glucose and an amino acid that contain sulphur and

nitrogen). The obtained chromatograms showed the presence of Glucoiberin, Sinigrin and Glucobrassicin in all three non-edible part.

Posters

• “COMPARISON BETWEEN SPONTANEOUS AND ORGANICALLY GROWN ECOTYPES OF Tarassacum officinale APPLYING NMR APPROACH” (Workshop NMR and food “Applicazione della risonanza magnetica nucleare nella scienza degli alimenti”, June 2022)

• “NMR CHEMICAL CHARACTERIZATION OF Acheta domesticus FLOUR” (Italian-French International Conference On Magnetic Resonance, September 2022)
 

PhD student Donatella Ambroselli

Tutor Professor Luisa Mannina

I° year report

During my first year of my PhD, I was mainly involved in two research fields: the first one is within FITO-BIO project supported by Civis regarding phytochemical characterization of Melissa officinalis ecotypes; the second one as part of Lazio Region project on the production of biostimulants from artichoke and cauliflower waste.

The aim of Melissa officinalis study was to define a metabolomic profile for three ecotype of lemon balm, the organically grown ecotype, the spontaneous ecotype grown in the lowland, and the spontaneous ecotype grown in the mountains. Ecotypes were differentiated by applying magnetic nuclear resonance spectroscopy in order to obtain a complete phytochemical characterization of plant matrix. The one-dimensional proton NMR spectral assignments of Bligh-Dyer hydroalcoholic extracts were performed using 2D experiments and literature data from other plant matrices analyzed under the same experimental conditions. Nine amino acids, five sugars, five organic acids, choline, chlorogenic and caffeic acids, and uracil were identified in all of Bligh-Dyer's hydroalcoholic extracts from upstream and downstream cultivations of lemon balm. Some metabolites, such as threonine and aspartate, chlorogenic acid and uracil, were not identified in the organic cultivation. The signals obtained from the identifications are quantified. Glutamine was the most abundant amino acid derivative detected in the 1H NMR spectra of lemon balm extracts, reaching the highest amount in the downstream cultivation sample. Many amino acids such as proline, valine, isoleucine, leucine and alanine were significantly present in the organic cultivation compared to the spontaneous (upstream and downstream cultivation). The upstream cultivation presented high amounts of sugars, except for galactose, which reached higher amounts in the organic cultivation. Regarding organic acids, tartaric was found to be the most abundant in the three different ecotypes, particularly in the sample of upstream cultivation. Citric, malic and succinic reached higher amounts in organically grown than in conventionally grown (upstream and/or downstream). Among other metabolites, choline had a similar trend for all three cultivation types. Regarding polyphenols: chlorogenic acid, which was found to be abundant from the conventional cultivation of lemon balm (upstream and downstream), seems not to have been detected in the samples of organic cultivation; on the contrary, caffeic acid reached more abundant amounts for organic lemon balm.

In the second project the goal was to characterize the chemical composition of by-products/waste from artichoke and cauliflower production to identify potentially useful components for the production of biostimulants and to evaluation of the chemical-nutritional-biological profile of formulations prepared as possible biostimulants. Consulting literature data, a preparation protocol was developed for obtaining the protein hydrolysate of cauliflower (from stems, flowers and leaves) and artichoke (from stems and leaves) separately. A protease from Aspergillus oryzae was used to constitute the protein hydrolase. Mixes were made for the formulation of two biostimulants, one obtained from cauliflower waste matrices, other one obtained from artichoke waste matrices. In the respective biostimulants, the composition percentages of each matrix were based on the percentages of waste obtained from the selected agro-food supply chains.

This composition ratio was maintained with each formulation to ensure reproducibility in crop trials. The resulting biostimulants could be easily reconstituted in the appropriate amounts of water and applied in open-field treatment (barley and wheat), germination treatment, and greenhouse treatment.

Papers

• Modulatory Properties of Food and Nutraceutical Components Targeting NLRP3 Inflammasome Activation. Nutrients 2022, 14, 490. https://doi.org/10.3390/nu14030490

Poster

• “NMR METABOLOMIC PROFILING OF FRESH GOJI BERRIES FROM TWO VARIETIS GROWN IN LAZIO” Applicazioni della risonanza magnetica nella scienza degli alimenti – 23-24 June 2022, Rome

• “CAULIFLOWER BY-PRODUCTS VALORIZATION BY APPLYING NMR BASED METABOLOMICS” Italian-French International Conference on Magnetic Resonance – 27-30 September 2022, Milan
 

PhD student: Francesca Arrighi

Tutor Professor: Daniela Secci

1° year report Title

Design and synthesis of novel hMAOs inhibitors based on methyl 2-(benzylthio) benzoate scaffold.

Design and synthesis of novel nicotinamide derivatives active against melanoma and non melanoma skin cancer My first year PhD project focused on the design and synthesis of novel hMAOs inhibitors based on the methyl 2-(benzylthio)benzoate scaffold. Monoamine oxidases are mitochondrial bound flavoenzymes which catalyse the oxidative degradation of amines. Two different isoforms called A and B, respectively, have been observed in human beings. The two isoforms possess a dimeric organization structure and are quite similar sharing ~70% sequence identity. However, there are also some differences in tissue/organs distribution and substrate specificity. They play an important role in many neurodegenerative conditions such as Parkinson’s disease. Parkinson’s disease (PD) is a neurodegenerative, hereditary/sporadic condition distinguished by progressive and debilitating nervous system dysfunction leading to altered movement and cognitive impairment. Nowadays, many available therapies are in use like: L-DOPA, domapine receptor agonist and also, hMOAs inhibitors. In particular, for what concerns the role of hMAOs inhibitors, they are responsible of the block of dopamine breakdown in order to preserve the supply of central dopamine, and in addition, cover a neuroprotective effect by the reduction of hydrogen peroxide levels. In the last years our research group actively worked on the development of hMAO-B inhibitors as co-adjuvants in the treatment of neurodegenerative diseases, in particular Parkinson’s disease. Therefore, we developed a series of 20 compounds based on the benzo[b]thiophen-3-ol scaffold that exhibited a preference for the hMAO-B with IC50 in the sub-micromolar range. With the aim to expand our knowledge about hMAOs inhibitors, we designed a novel series of compounds based on the “opened form” of the benzo[b]thiophen-3-ol one, also in order to evaluate if a more flexible structure could lead to a more potent and selective inhibition on hMAOB. Our work has been focused on the assessment of different sulfur oxidation states and also on the study of different substituents located at the ortho, meta and para position of the aromatic ring, thus designing three different libraries. For the synthesis of Libraries I we started from 2-mercaptobenzoate and the proper parasubstituted α-bromo acetophenone that reacted in DMF, at ice-water temperature by employing potassium carbonate as base. The compounds belonging to the Library I were treated in the next oxidative reaction with meta-chloroperbenzoic acid (mCPBA) in order to obtain the two oxidation products, the sulfoxide (Libraries IA) and the sulfone (Libraries IB), in the same reaction by controlling the amount of oxidant added during the synthesis. By this synthetic procedure we arrived to a series of 21 compounds, divided in three different sub-libraries, which contains respectively the sulfur, the sulfoxide and the sulfone group. All the novel synthesised compounds will be characterized by means

NMR, IR spectroscopy and evaluated for their hMAO-A and hMAO-B inhibitory activity by employing previously reported methods. The compounds exhibiting the best hMAO-B inhibition will be further evaluated for their ability to act as antioxidant, radical scavenger, and metal-chelating properties. Another part of my first year PhD project has been based on the design and synthesis of novel nicotinamide derivatives active against melanoma and non-melanoma skin cancer. Nicotinamide is a main player in human’s energy metabolism as a precursor of NAD. For this reason, it is also involved in many skin pathological conditions like: acne, rosacea, atopic dermatitis. In the last years, nicotinamide has been studied as an anticancer agent for its ability to repair UV-induced DNA damage. In particular, it has been seen that UV-induced overexpression of EGFR and also bring to higher concentration of COX-2 leading to DNA damage, cell cycle arrest, apoptosis suppression and uncontrolled cell proliferation. Therefore, we focused our effort on the design of four classes of novel derivatives, starting from nicotinamide. For the first three Libraries we developed a multistep synthetic procedure, while the last Library, based on the Pyridine-acylsulfonamide scaffold, will be obtained through a single step coupling reaction. Based on these studies, for the next steps, we will work on the optimization of synthetic procedure and the evaluation of structural modification. In addition, Nicotinamide represents an interesting opportunity thanks to its safety profile and crucial role in metabolism and human health, therefore these novel compounds could open a new pathway in the study of innovative target therapy in this wide field.

Papers and posters:

“Design, synthesis and human monoamine oxidase inhibitory activity of 2-aroylbenzofuran-3-ol and 2- aroylbenzofuran derivatives: a new route towards hMAOs inhibition”, MEDCHEM2022, Barcelona.

PhD student: Shilashi Badasa Oljira

Tutor: Prof.ssa Alessia Ciogli

Annual progress report: 1st year

Development of Amino-organocatalysts for asymmetric synthesis and their use in heterogeneous phase & Stability studies of enantiomers by dynamic and off-column HPLC.

The work done in this PhD project is classified in to two sections. In the first part, C3-symmetric aminoorganocatalyst has been synthesized as indicated below (Scheme:1; A to L). In this work, aminoorganocatalyst containing three equal catalytic units in a C3-symmetrical fashion has been developed. This novel chiral aminoorganocatalyst was synthesized from commercially available (1R,2R)-(+)-1,2-diamino-1,2-diphenylethane and sources of C3-symmetric group. Di-tert-butyl decarbonate has been used as protecting group to achieve targeted molecule. The first step of this synthesis has been started from monoprotection of the starting material (A). Deprotection and reduction reaction with borane dimethyl sulfide (B) afforded the targeted C3-symmetric aminoorganocatalyst (L) with purity (over 92%) and yield (91%). Over a decade, several excellent enantioselective syntheses of important Warfarin using asymmetric reactions mediated by chiral amino organocatalysts have been took place. To evaluate the catalytic activities of the synthesized molecule: N-(3,5-bis-[(2-amino-1,2-diphenyl-ethylamino)-methyl]-benzyl)-1,2-diphenyl-ethane-1,2-diamine (Fig-1, L), asymmetric Michael reactions between 4-hydroxycoumarin and α, β-unsaturated ketones has been proposed in the next activities. Inconclusion, procedures and proposed reaction protocols allowed the synthesis of C3-symmetric amino-organocatalyst called N-(3,5-bis-[(2-amino-1,2-diphenyl-ethylamino)-methyl]-benzyl)-1,2-diphenyl-ethane-1,2-diamine based (1R,2R)-(+)-1,2-diamino-1,2-diphenylethane with high purity. In second section of this work, the interconversion of atropisomers involve rotation about N−N hydrazide bonds has been studied by dynamic and off-column HPLC. Chiral HPLC stationary phase has been used for resolution of enantiomers. Dynamic-HPLC studies has been performed on both molecules (a) & (b) (Figure:1). Furthermore, the interconversion of enantiomers has been studied by off-column for compound (a)(Figure:1). Among the studied compounds, (a) and, (b) were completely resolved in two peaks at 25 °C (UV 254nm; CD 230nm) and 0 °C (UV 254nm, CD 244nm)

respectively, and enantiomeric ratio has been obtained from the relative area. The obtained CD chromatogram have opposite directions, confirming the enantiomeric relationship of the two atropisomers. For dynamic HPLC studies, variable-temperature HPLC separation on CPSP chiral stationary phase was performed. In case of compound (a), (R, R)-Whelk-O1chiral stationary phase was used with hexane/2-propanol (98:2) and monitored by UV-CD detection while for compound (b), chromatographic profiles were obtained on two different CSP (Chiralpak IB and Lux 5μm cellulose-phenomenex) with hexane/2-propanol (98:2). In both molecules (a) and (b), as the temperature increased, decrease in retention time and formation of plateau were observed. For compound (b), the two peaks approached to coalesce at 25 °C but didn’t form a single peak. Energy barriers of interconversion of N-N chiral hydrazides have been evaluated through line shape simulation of the chromatograms recorded at different temperature.

For compound (a), almost optically pure enantiomer (97%) was collected at room temperature and incubated, and the racemization reaction was monitored by CSP-HPLC-(R, R) Whelk-O1(250x 4.6 mm). Racemization has been studied in both 2-propanol and decalin in a range of temperatures between 50 °C and 80 °C. Enantiomerization process of compound (a) was almost similar in both 2-propanol and decalin. But the results indicated below are concern the racemization of (a) in 2-propanol at 50 °C,57°C, 62 °C ,71 °C & 80°C monitored over time by CSP-HPLC. The kinetic rate constants of the racemization were derived based on reaction time and logarithm of the enantiomeric excess. Regarding compound (a), the obtained rotational barrier for the off-column enantiomerization at 50 °C (ΔG‡= 24.49 kcal mol–1);57 °C (ΔG‡= 24.68 kcal mol–1);62 °C (ΔG‡= 24.73 kcal mol–1);71 °C (ΔG‡= 25.09 kcal mol–1);80 °C (ΔG‡= 25.40 kcal mol–1) while the interconversion barrier and apparent rate constant for on-column experiment at temperatures between 25 °C and 70 °C are at 25 °C(ΔGap1-2=22.47 kcal mol–1, ΔGap2-1=22.53 kcal mol–1; 60 °C (ΔGap1-2=24.94 kcal mol–1, ΔGap2-1=25.49 kcal mol–1);65 °C(ΔGap1-2 =25.08 kcal mol–1,ΔGap2-1=25.43 kcal mol–1),70 °C(ΔGap1-2=25.09kcal mol–1, ΔGap2-1=25.12 kcal mol–1) ,and exact agreement between results obtained by calculation and line shape simulations. For compound (b), the formation of plateaus caused by interconversion at temperatures between 0 °C and 25 °C are at 0 °C (ΔGap2-1 =20.18 kcal mol–1,ΔGap2-1 =21.09 kcal mol–1);5 °C(ΔGap2-1 =20.28 kcal mol–1 ,ΔGap2-1=20.44 kcal mol–1 );10 °C( ΔGap2-1=20.30 kcal mol–1, ΔGap2-1=20.44 kcal mol–1); 25 °C (ΔGap2-1=20.60 kcal mol–1, ΔGap2-1= 20.61 kcal mol–1 ). From the above results, we have seen that the free energy(ΔG‡) of N−N hydrazide molecule substituted by naphthyl at both side (a) is higher than that of the benzoyl substituted (b).

Finally, the kinetic constant and rotational barrier of these two molecules were properly investigated by dynamic HPLC and off-column analysis. Based on the obtained results the free energy (ΔG‡) of N−N hydrazide substituted by naphthyl at both side (a) is higher than that of the benzoyl substituted one (b).
 

PhD student Stefano Dessalvi

Tutor Professor Giancarlo Fabrizi

2022 year report

Valorization of Cashew Nut Shell Liquid (CNSL)

Industrial ecology is one of the most important aspects of Green Chemistry and consists in the development of processes able to advantageously use the waste materials produced.

The fruit industry appears to be one of the most promising areas for the development of this concept. In fact, although the fruit industry is collecting large quantities of raw materials, it uses only a minimal percentage. For example, while the production of cashews exceeds 5 million tons (2018 data), their full use is only partial: cashew is used in the production of liqueurs and edible material.

It is known that from the shells of cashews, through an extraction with hexane, it is possible to obtain a very dense dark brown liquid (CNSL) characterized by an interesting composition under a chemical point of view, as it consists of a series of aromatic compounds, which indicates that the CNSL can be considered an

important renewable source: to demonstrate this, there are numerous articles that have described its application in different fields [1]. An important aspect to consider is that connected to the possibility of chemically transforming the precious compounds in the CNSL: this renewable source consists of a series of phenols substituted with alkenyl or alkyl chains of 15 carbon atoms, potentially transformable into monomers for the synthesis of green polymeric materials, and in surfactants in the industrial, cosmetic and pharmaceutical fields. Specifically, CNSL mainly contains 4 classes of phenols (figure 1) which differ in the number and position of the C-C double bonds on the alkenyl chains, namely: 1) anacardic acid; 2) cardolo; 3) cardanol; 4) 2-methyl cardol. As part of the topic described, it was decided, in agreement with the industrial partner P&G, to study the reduction reaction of the phenolic system to cyclohexanolic, in order to obtain a substrate to be subsequently modified to modulate its surfactant properties.In particular, in order to optimize the conditions of the hydrogenation reaction, it was decided to use commercial cardanol as a model system, consisting of a mixture of 3-substituted phenols with alkyl and alkenyl chains with 15 carbon atoms (1). The hydrogenation reactions were carried out in an autoclave at a pressure ranging from 2 to 10 bar and at a temperature between 25 and 100 ° C (figure 2). The results obtained showed that the optimization of the reaction conditions allowed to obtain the reduction products, that is the 3-alkylcyclohexanone (2) and the cis and trans stereoisomers of the 3-alkylcyclohexanol (3) with a satisfactory selectivity and yield proper chemistry. The reduction products were also purified by chromatography and spectroscopically characterized by NMR and GC-MS.

Bibliografia

1.Andrianarivo Irène Rahobinirina et all., Tetrahedron Letters 2017;58,2284-2289;

2.Gopalakrishna S, Nevaditha NT, Mythili CVJ. Chem Pharm Res.2011;3:490-497;

3.Menon RR, Pillai CKS, Nando GB. Polymer.1998;39:4033-4036;

4.Mythili V, Malarretna A, Gopalakrishnan S. Sci Bull Master. 2004;27:235-241
 

PhD student Maria Gioia Fabiano

Tutor Professor Maria Carafa

1st year report

Nanodispersed essential oils as biodegradable and biocompatible treatments to improve the sanitization and fermentation of the organic fraction of municipal solid waste

During last decades, the production of large amounts of municipal solid waste (MSW) due to an extensive urbanization has become a very serious problem that large cities have to solve because the incorrect wastes treatment could produce an increase of the disease and environmental problems. Organic waste produces bad smells due to the anaerobic process produced by microbial populations, because of the low oxygen concentration within the waste. In fact, the organic wastes are characterized by the presence of numerous pathogenic microorganisms that spread in the surrounding environment. The microbial population can, also, cooperates in the formation of the biofilm and it can reduce the activity of various antibacterial drugs, including antibiotics. It’s very important to make them harmless by specific biological treatments, such as composting and biostabilization, because they can be dangerous for human health, food safety and environmental quality. The composting process converts organic waste into a high organic carbon substances with biological activity and that improves the physical, chemical, and biological properties of the soil and increases the crops productivity. However, it’s important to focus the attention on the quality of the compost because a poorly stabilized and sanitized compost can lead to serious risks to public health, food safety and environmental quality. Biostabilization, on the other hand, makes the organic fraction of urban waste stable and not biologically active before disposal of the waste in landfills. Our goal is to treat wastes with “green” formulations with antimicrobial properties. Essential oils (EOs) have

significant antimicrobial properties and low potential to develop a microbial resistance due to their multicomponent nature and their multi-target activity against multi-drug resistant (MDR) bacteria and they smell good. The idea is the nebulize EOs overthe organic fraction of urban solid waste (MSW).but they show some problems: for examples they contain very volatile, unstable and reactive molecules and they also have low penetration properties and this could make them ineffective. These problems can be overcome by formulating a Nanoemulsions (NE). In general, the emulsions are colloidal dispersions composed by oil, water, and surfactants and they can be oil in water (O/W) or water in oil (W/O). In the emulsion family we can find microemulsions and nanoemulsions. The most important difference is that the first can be formed spontaneously without using external energy, while nanoemulsions can be formed only using a high energy method (like sonication). We have selected Thymus Essential Oil (TEO) for its antimicrobial, antiseptic, antiviral, nematicidal and antioxidative properties. Two ternary diagrams have been constructed: the first one with TEO, Tween 20 and water, and the second with TEO Tween 80 and water. The phase diagram is an important tool to understand the behavior of the three components of the nanoemulsion and after the preparation it is possible to choose the perfect combination between oil, water, surfactant for a specific application. In fact, we selected the formulation containing Tween 80 because allowed us to choose a formulation at the maximum oil concentration: 25% of TEO, 25% of Tween 80 and 50% of water. After, I have finalized the preparation using a high energy apparatus (Ultra-Turrax) to obtain nanoemulsion with suitable droplet size. We have chosen Ultra-Turrax in sigh of an industrial scaling-up. I have characterized the sample in terms of dimensions by using Dynamic Light Scattering (DLS), I monitored the stability for three months at 25°C, I tested the sample nebulizing resistance, and I characterized oil droplets features (microviscosity, polarity and fluidity) by using lipophilic fluorescent probes. The next step will be to try to replace Tween 80 with a biodegradable surfactant, TERGITOL 15-S-9. I prepared the ternary diagram, but the results were not completely acceptable. Tween 80 has been added to the formulation and I chose a new nanoemulsion to be tested: 25% of TEO, 25% surfactant mixture (Tergitol:Tween 80 – 7:3) and 50% of water. Finally, I finalized the preparation by using the high-energy method and then I started the physico-chemical characterization. Future perspectives are:

• To test the stability of formulation at the composting chamber conditions

• To perform release experiment

• To test other oils with antimicrobial

• To mix the Thyme essential oil with another oil with antibacterial activity, to check if it is possible to prepare a nanoemulsion with a lower percentage of

• To introduce in the formulation a natural and completely biodegradable surfactant

Posters:

1. “Nanoemulsions of Satureja Montana Essential Oil: Antimicrobial and Antibiofilm Activity against Avian Escherichia coli Strains” at First Symposium for Young Chemists: Innovation and Sustainability Rome, 20-23 June 2022

2. “Surfactant-based nanoencapsulation of cannabidiol oil: preparation, characterization and preliminary evaluation of antibacterial activity” at XXI Scuola Dottorale in Tecnologia Farmaceutica Rome, 5-7 September 2022
 

PhD student: Michela Guida

Tutor Professor: Giovanna Poce

First year report

Development of potential transmission-blocking agents against malaria

Malaria is an infectious disease that is still one of the leading causes of death in the world. It is spread through the bites of infected Anopheles mosquitos, that introduces the Plasmodium parasites from the

mosquito's saliva into a person's blood through the bite. Five species of Plasmodium can infect humans; most deaths are caused by P. falciparum. The strategies currently used against malaria are the use of anti-malarial drugs and vector control strategies (insecticide, bed nets and indoor spraying); in 2021 has been approved the first malaria vaccine, but it shows a low efficacy and is only available for children. The majority of approved malaria treatments target the asexual replication cycle, which is responsible for the clinical symptoms. The insurgence of drug-resistant parasites, however, has become a serious health problem and the need for new antimalarial candidates is growing. Ideally, these new candidates should target different stages of the parasite life cycle. Transmission-blocking compounds are promising candidates that target the sexual stage of the parasite’s life cycle; by doing so, they interrupt the parasite life cycle, and they could also limit the spread of drug-resistant parasites. In a recent screening of the Medicines for Malaria Venture Pandemic Response Box an antitubercular compound derived from the BM212, a MmpL3 inhibitor, resulted active as a transmission-blocking compound. The finding that some antitubercular agent could act on the Plasmodium parasites led to a collaboration with our group to investigate the possibility of modification around the BM212 pyrrole core. My objective for my first year was to explore the core by synthesising and evaluating new compounds, modifying the substituents on the position N1, C3 and C5 of the pyrrole core, and by doing so trying to answer to some key question about how the substitution on these position could influence on cytotoxicity, lipophilicity and activity properties of the molecule. The synthetic pathway of the pyrroles involves a Stetter reaction between an aldehyde and the methyl vinyl ketone, that in turn is cyclized to pyrroles by a Paal-Knorr reaction. At the end, a Mannich reaction on the pyrrole gives the final products. The biological evaluation of our compounds is led by the group of Prof. Lyn-Marie Birkholtz of the University of Pretoria, South Africa. Some of the molecules that I have synthesised in this year resulted active from the preliminary data that we have obtained. In the near future we will synthesise other analogues by modification on different position to improve the antiplasmodial profile and to achieve a more accurate SAR. We will also investigate the possibility to substitute the pyrrole core with an imidazole core.

Papers and posters Poster:

Guida, M., Hanieh, P. N., Consalvi, S., Forte, J., Cabiddu, G., De Logu, A., Rinaldi, F., Biava, M., Carafa, M., Marianecci, C., Poce, G.. Nano-Based Drug Delivery Systems of Potent MmpL3 Inhibitors for Tuberculosis Treatment. Novel frontiers in nanocarriers preparation and characterization Workshop, Roma.
 

PhD student: Luca Paoletti

Tutor: Prof. Pietro Matricardi

I st year report

Development of polysaccharide-based nano and macrosystems for drug delivery and tissue engineering purposes.

During the first year of my PhD, I had the opportunity to deepen the study of polysaccharides as extremely versatile materials that are widely used in pharmaceutical technology, both as carrier for the delivery of bioactive molecules and as scaffold and support for cell proliferation in tissue engineering. In particular, I have been able to carry out two projects that fit perfectly into these two themes. In the first project, in collaboration with the University Miguel Hernandez of Alicante, hyaluronic acid was used to develop nanosystems able to deliver anti-inflammatory drugs to the posterior segment of the eye as a treatment for some retinopathies. In fact, the anatomy and physiology of the eye have always been a limit to the local delivery of therapeutics since the drug permeation, and therefore the possibility to reach the intraocular tissues, is strongly hindered. In this light, the use of a hyaluronic acid-based nanocarrier, biocompatible, biodegradable, and non-toxic, appear as an attractive strategy to deliver substances by intravitreal injection. Hyaluronic acid (HA) is an anionic polysaccharide which can be easily derivatize thanks to the

presence of a carboxyl group for each repetitive unit. Its functionalization with a small hydrophobic molecule, cholesterol (CH), allowed to obtain an amphiphilic derivative (HA-CH) able to spontaneously self-assemble in aqueous solution. In fact, by autoclave treatment of a homogeneous dispersion of this derivative, a rearrangement of the chains results in the formation of nanoparticles in the range of 200-300 nm which are called nanogels (NHs). These nanosystems, previously developed by our research group, have been evaluated as possible carriers for the delivery of three drugs. At first, our colleagues in Alicante suggested the encapsulation of a salt of acetylsalicylic acid with lysine, which is extremely soluble in water. The drug was then solubilized in a suspension of NHs and loaded by magnetic stirring for one hour. However, after purification of the formulation, no encapsulation of the molecule was obtained. Considering this, the loading of salicylic acid, a hydrolysis product of acetylsalicylic acid characterized by a lower solubility in water, was tested. The molecule was loaded in a two-step procedure, in which a dispersion of HA-CH was placed under magnetic stirring for one hour in contact with a film of the drug and then autoclaved. However, also in this case poor results were obtained since only 10% of the starting salicylic acid was encapsulated into NHs. In the light of the obtained results, a hydrophobic drug was selected as a promising candidate thanks to its affinity to the internal hydrophobic domains of NHs. The chosen molecule is the salsalate (SS), a prodrug of the salicylic acid, very interesting since the enzymatic or chemical hydrolysis of one mole of it leads to two moles of salicylate. In this case it was possible to obtain a loading of 80%, corresponding to a concentration of 267 μg of SS per mL of formulation. Focusing on the latter formulation, drug release experiments were performed mimicking the physiological conditions (37° C, pH 7.4). The obtained profile showed a fast release in the first two hours but comparing the hydrolysis profile of free SS with that of the SS within the formulation, it was possible to notice that NHs seem to protect the drug and delay its hydrolysis over time. Further release experiments under different pH and ionic force conditions are planned, along with mucoadhesion tests and in vivo experiments in Alicante. The second project was dedicated to the modulation of the mechanical properties of a semiinterpenetrate system (semi-IPN) as a potential scaffold for tissue engineering applications. The system, previously developed by our research group, consists of two polysaccharides: dextran, which was previously methacrylate (DexMa), and gellan sodium (GeNa). When a homogeneous mixture of the two polymers is exposed to UV light, in the presence of a photoinitiator, a chemical crosslinking between the methacrylic groups of the dextran chains occurs, leading to the formation of a semi-IPN, in which the chains of the non-crosslinked GeNa are interpenetrated in the crosslinked DexMa matrix. Although these materials are very soluble in water, an unconventional solvent, glycerol, was chosen for its properties and advantages in terms of safety, availability, and affordability. In addition, glycerol can significantly reduce the irradiation time needed to obtain gels, acting as a chain transfer agent. However, if such solvent is present at a concentration higher than 10%, it may affect cell growth and viability. The purpose of my work was trying to modify the mechanical properties of the semi-IPN by introducing different crosslinkers, characterized by a different number of methacrylic groups and, therefore, able to modulate the crosslinking density. These molecules were tested at three different molar ratios compared to the moles of methacrylic groups in the DexMa (0.1:1, 0.25:1 and 0.5:1), exposing the mixtures to UV light for 1 to 5 minutes. Initially, the effect of crosslinkers was observed only in the presence of DexMa: the polysaccharide was solubilized in glycerol at 80°C, crosslinkers and photoinitiator were added to the solution and the final gels were obtained by photocrosslinking at 365 nm. The characterization of such systems was performed using a texture analyser by measuring the force in compression. Two parameters have been extrapolated and used as a yardstick for the gels with and without crosslinkers: the Young’s modulus, which gives information on the rigidity and elasticity of the system, and the maximum stress applied before the breakdown of the gel, which gives indications on the resistance of the semi-IPN to stresses. The use of crosslinkers in a 0.1:1 molar ratio has no influence on the properties of the system, unlike the systems prepared with a ratio of 0.25:1 and 0.5:1, which are much more rigid than semi-IPN without crosslinkers. In addition, the presence of crosslinkers in the ratio of 0.25:1 led to the formation of a much more resistant gel. Finally, the two crosslinkers in the two major ratios were also tested in the presence of GeNa. The simi-IPNs were prepared by photocuring a

mixture of the two polysaccharides in a ratio 1:1. The systems obtained with a 0.5:1 ratio showed an increase in Young’s modulus after 5 minutes of crosslinking, however the characterization of semi-IPN with a 0.25:1 ratio is still ongoing. A rheological characterization of all systems will be carried out in the coming months, together with the development of a glycerol removal and exchange procedure with an aqueous solvent, in order to make the systems biocompatible.
 

PhD student Luca Pisano

Tutor Professor Bruno Botta

1° year report

Design and synthesis of quinoline-based caging compounds with selective thiol-reactive linker

A caged molecule is an inert but photosensitive molecule that is transformed by photolysis into a biologically active molecule at high speed (typically 1 msec) (Figure 1). The process is referred to as photorelease. The spatial resolution of photorelease is limited by the properties of light; submicrometer resolution is potentially achievable. Therefore, focal photorelease of caged molecules enables one to control biological processes with high spatio-temporal precision (1). The aim of the project is to develop a synthetic strategy for the synthesis of a quinoline-based caged system, functionalized with a thiol-selective linker capable to bind the cysteine residue of the inner cavity of ferritin, the delivery agent that we have chosen for our delivery purposes. The system will be characterized by a quinoline core functionalized in position 2 with a methylcarbonate, a methylcarbamate or an ether bond depending on the bound biological effector, and in position 8 with a piperazine linker characterized by a pentafluorobenzensulfonamide group capable to selectively react with the thiol functions of the cysteine in the inner cavity of ferritin (Figure 2).

The synthetic strategy currently ongoing is reported in Figure 3 and at the moment we have managed to obtain compound 16.

The first step is based on the Skraup synthesis (2) to construct the quinoline core endowed with a methyl group in position 2 starting from 2-bromoaniline and crotonaldehyde. The reaction was neutralized after 6h, the crude was extracted three times using ethylacetate and then purified using a flash silica column with an eluent mixture of hexane and ethylacetate in a ratio 8:2, leading to a 55% yield of compound 3. The second step was performed using selenium dioxide to oxidize the methyl in position 2 of compound 3 (3). The reaction was almost quantitative, and the crude was filtered through a silica-packed buchner filter to remove the excess of selenium dioxide, obtaining compound 4 with an yield of 80%. The third step consisted in a reduction of the aldehyde group made by sodium borohydride (4) and the crude material was extracted three times using ethylacetate in order to obtain compound 5 with an yield of 84%. Without any further purification compound 5 was employed in the fourth step, which consists in the protection of the alcoholic group using 3,4-Dihydropyran (DHP) in dichloromethane (DCM), catalyzed by the presence of pyridinium p-toluenesulfonate (5). Compound 6 has been obtained after extraction with DCM and then a flash silica column with an eluent mixture of hexane and ethylacetate in a ration 9:1 leading to an yield of 73%. Compound 15 was obtained through a Buchwald-Hartwig reaction catalyzed by PEPPSI I-Pent, a pre-formed catalyst that showed impressive yields in literature and allowed us to increase the yield of our reaction from 18% (using the common palladium-based catalyst) to 70% (6). The crude reaction was then extracted three times using DCM and purified on flash silica column with an eluent mixture of ethylacetate and diethylether in a ration 9:1 with 1% of triethylamine. Compound 15 was then deprotected using potassium hydroxide in methanol at 65 °C and obtained with low yields (under 20%) and many side products have been observed. New optimization must be conducted in order to increase the yields of the deprotection reaction and to have enough compound 16 to carry on the synthetic strategy until the obtaining of the final quinoline-based caged system.
 

PhD student Enrico Romano

Tutor Professor Luisa Mannina

First year report

Metabolomic characterisation of Burdock by NMR spectroscopy and analysis of chlorophylls, carotenoids and anthocyanins in artichoke and cauliflower waste

I mainly followed two projects: RI-CICLO HORTO, a project about valorization of agri-food waste for biostimulants for agriculture and supplements for human health production and the project FITO-BIO

that proposes a multimethodological study that involves the production and the chemical characterization of medicinal plant.

FITO-BIO

The FITO-BIO project, funded by alliance CIVIS, was born from the collaboration between Sapienza University of Rome and three companies of medicinal plants field (Sarandrea, Fibreno Officinalis and Herba Sapiens) and aims to respond to some very specific requests concerning this sector, namely: (i) development of sustainable extraction procedures and (ii) in-depth knowledge of the composition of phytotherapeutic obtained extracts.

I worked with one plant, namely burdock

My work has been to characterize the metabolimonic profile of Burdock roots, and to assess the different amounts of metabolites in the spontaneous and organic ecotypes, by the application of untargeted NMR. For NMR analysis, the Bligh-Dyer extraction protocol was applied using MeOH/CHCl3/H2O in a 2/2/1.8 v/v/v ratio to obtain two immiscible phases. Finally, the hydroalcoholic fraction was dried with a stream of N2 at room temperature and stored at -20 ◦C. The hydroalcoholic fraction was solubilised in 1mL of 400 mM phosphate buffer in deuterated water (D2O) with TSP as internal standard at a concentration of 2 mM and EDTA. It was then transferred into 1.5mL eppendorf tubes and centrifuged at 25°C for 5 minutes at 7000 rpm to allow any precipitated material to settle. 0.7mL of the supernatant was then withdrawn and placed into 5 mm NMR tubes. 1H NMR spectra were recorded at 25 °C on a JEOL spectrometer operating at a proton frequency of 600 MHz. To perform the signal assignment in the acquired NMR spectra, a series of two-dimensional spectra were performed: 1H-1H Total Correlated Spectroscopy (TOCSY), 1H-13C Heteronuclear Single Quantum Coherence (HSQC) and 1H-13C Heteronuclear Multiple Bond Correlation (HMBC).

Organically cultivated burdock showed a higher amount of identified amino acids, except for glutamine, which showed slightly higher concentrations in wild-grown than in organically grown

Also, regarding organic acids, we find slightly higher concentrations in organically cultivated burdock, except for fumaric acid, which was more abundant in wild cultivation

A different trend is noted for sugars, as they were found to be higher in the wild-grown than in the organic burdock.

As future goals, we have the analysis of the third burdock ecotype, the conventional upstream wild burdock, and an HPLC analysis of polyphenols. I have already proceeded with the extraction of the polyphenols: 100 mg sample1 ml EtOH/H2O 70:30 v/vSonication 30 min, 25°CCentrifugation (7830 rpm, 10 min, 25°C). I used methanol and a 3% aqueous solution of acetic acid as mobile phases. The next step will be to use the standards to carry out peak assignment and quantification of the polyphenols.

RI-CICLO HORTO

As part of the 'RI-CYCLOHORTO' project, the aim was to determine the content of chlorophylls, carotenoids and total anthocyanins in artichoke and cauliflower waste.

The wastes that were considered were artichoke stems and leaves and cauliflower stems, leaves and flowers.

For the extraction of the chlorophyll and carotenoid pigments, this analytical protocol was followed: following trituration with liquid nitrogen, a quantity of between 30-35 mg was weighed for each sample, which was kept at -80°C until use; each sample was then homogenised in a mortar with 3 ml of a CHCl3/CH3OH solution (2: 1 v/v) twice in the presence of about 20 mg of MAGNESIUM OXIDE as a protective agent of the chlorophyll against pheophytinisation. The extract obtained was filtered and then a volume of double-distilled water equal to 20% of the volume of organic extract was added. This was centrifuged for 20 minutes at 20°C (4000 rpm) and then the hydroalcoholic phase obtained was separated from the organic phase (chloroform) containing chlorophyll and carotenoids. The absorption spectrum of the organic phase (1 ml) was acquired with the spectrophotometer in the wavelength range 350- 800 nm at 20°C. The Wellburn equations were used for pigment quantification.

The protocol followed for the extraction of total anthocyanins is that of Abdel-Aal and Huel (1999). For each gram of ground sample (about 65-70 mg tot), 8 mL of acidified ethanol (CH3CH2OH/HCl 1 N, 85:15 v/v) was added. The pH of the resulting mixture was adjusted to 1 with HCl 4 N. To aid the extraction of anthocyanins, the samples were shaken for 30 minutes in the dark. They were then centrifuged at 4000 rpm for 30 minutes at 5°C. The supernatant was taken and made up to a volume of 15 mL with acidified ethanol. The absorbance was measured at 535 nm.

Based on the preliminary results obtained so far, it could be verified that both artichoke and cauliflower leaves contain a higher amount of carotenoids and chlorophylls than stems, in accordance with the fact that these plant parts are subjected to a higher degree of sun exposure. Comparing the concentrations of chlorophyll a and chlorophyll b in the leaves, chlorophyll a is almost twice as high as chlorophyll b, which is typical for plants growing in a sunny open field. In contrast, chlorophyll b is found in greater quantities in plants growing in shady conditions. Regarding anthocyanins, the highest concentration was found in artichoke leaves in which, on a macroscopic level, violet streaks can be identified. In cauliflower flowers, on the other hand, the amount of anthocyanins detected is relatively low.

These first data obtained are a good indicator that the analytical protocol developed is very efficient for the analysis of pigments in artichoke and cauliflower waste. With a view to the future, the interest in recovering these compounds stems from their health value (antioxidant properties). In fact, these pigments will play a very important role in the second part of this project, i.e. the production of supplements for human use, given their innumerable beneficial health properties, always bearing in mind, however, that these supplements may be a valuable aid, but they cannot replace the extraordinary complexity of micronutrients contained in these foods.

Workshops and conferences:

• RI-CICLO HORTO workshop

Oral presentation “CLOROFILLE, CAROTENOIDI E ANTOCIANINE NEGLI SCARTI DI CAVOLFIORE E CARCIOFO”.

• FITO-BIO workshop

Oral presentation “Caratterizzazione metabolomica della Bardana (Arctium lappa L.) mediante spettroscopia NMR”.

• VII Workshop - Applicazioni della Risonanza Magnetica nella Scienza degli Alimenti

Poster “CARATTERIZZAZIONE METABOLOMICA DELLA GENTIANA LUTEA L. MEDIANTE SPETTROSCOPIA NMR”

Research period in industry:

• Two months (July and August) at the SARANDREA company, Via D'Alatri, 03010 Collepardo (Fr).
 

PhD student Lucretia Rotari

Tutor Professor Barbara Chiavarino

First year report

Structural characterization of flavonoids elucidated by IRMPD spectroscopy, CID-MS and computational methods

During first year of the PhD course, I studied the bare protonated or deprotonated forms of selected flavonoids, delivered into the gas phase by electrospray ionization (ESI), by mass spectrometry-based methods to obtain different information. I focused my attention on three natural phytophenols with beneficial health effects: Naringenin (Nar), its structural isomer Naringenin Chalcone (ChNar), and Genistein (Geni). In both studies, a detailed structural characterization and conformational analysis of the ionic forms of the analytes, isolated in the gas phase, has been performed exploiting a combined approach based on ESI-(high resolution) mass spectrometry, collision-induced dissociation (CID) measurements, IR multiple photon dissociation (IRMPD) action spectroscopy, theoretical DFT calculations and ion mobility-mass spectrometry (IMS). Literature reports many efforts aimed at differentiating and characterizing individual flavonoids by tandem MS. 1 In particular, CID-MS studies have successfully characterized several isomeric flavonoids belonging to distinct subgroups, through their different fragmentation patterns. 2 However, the discrimination of Nar and ChNar, in complex mixtures by mass spectrometry appears to be a difficult task, without preliminary separation. The main purpose of my first study was to find a way to discriminate the two isomers Nar and ChNar (Figure 1) by the combined mass spectrometry-based methods approach.

The study was carried out on protonated species to avoid possible naringenin/naringenin chalcone isomerization during the ionization processes in the ESI source or in solution. While IMS and variable collision-energy CID experiments hardly differentiate the two isomers, IRMPD spectroscopy turned out as an efficient method in distinguishing naringenin from its chalcone. In particular, the spectral range between 1400 and 1700 cm−1 appeared highly specific in discriminating between the two isomers in protonated form. We also performed, by IRMPD spectroscopy, methanolic extracts of commercial tomatoes peel and grapefruits albedo, chosen for their higher concentration in flavonoids. Selected vibrational signatures in the IRMPD spectra have allowed us to identify the nature of the metabolite present in methanolic natural extracts. Experiments suggest a prevalence of naringenin chalcone in the tomatoes peel, while grapefruit albedo contains mainly naringenin and a small amount of chalcone. Furthermore, comparisons between experimental IRMPD and calculated IR spectra have clarified the geometries adopted by the two protonated isomers allowing a conformational analysis of the sampled species. The second study was focused on Genistein (Figure 2) an isoflavone with estrogenic properties and proven antioxidant effects. Particularly, the radical scavenging activity of Geni is carried out by the way of mechanisms based on sequential proton-loss electron transfer. For this reason, we decided to elucidate the structural feature of the deprotonated form of Geni. Our experimental evidence reveals that genistein is deprotonated at the most acidic site (7-OH) and benefits from a strong intramolecular H-bond interaction between 5-OH and the adjacent carbonyl oxygen in the most stable arrangement. Moreover, comparisons between experimental and calculated IR spectra suggests the contribute of an admixture of two close-lying rotamers deprotonated at the 7-OH (Aring) position in the sample ion populationDay.

Papers:

[1] R. Paciotti, B. Chiavarino, C. Coletti, D. Scuderi, N. Re, D. Corinti, L. Rotari, S. Fornarini, M. E. Crestoni, IRMPD Spectroscopy of Bare Monodeprotonated Genistein, an Antioxidant Flavonoid, ACS Omega 2022 7 (23), 19535-19544. DOI: 10.1021/acsomega.2c01236

[2] D. Corinti, L. Rotari, M. E. Crestoni, S. Fornarini, J. Oomens, G. Berden, A. Tintaru, B. Chiavarino, Protonated forms of Naringenin and Naringenin Chalcone: proteiform bioactive species elucidated by IRMPD spectroscopy, IMS, CID-MS, and computational approaches. (Submitted)

Oral Presentation:

[1] 10th MS J-Day. IRMPD action spectroscopy of bare deprotonated genistein, a natural antioxidant isoflavone. University of Teramo. May 27, 2022.

Schools:

[1] EU FT-ICR MS 9 th Short Course. Complex mixture analysis with FT-ICR MS. April 19-22, 2022. University of Eastern Finland.
 

PhD student Karim Ullah

Tutor Professor Giancarlo Fabrizi

1° year report

Cu/Pd-Catalyzed synthesis of 1,2,3-triazole-fused Heterocycles

Fused heterocycles are structurally significant motifs with many applications in material and medicinal chemistry [1]. In particular, nitrogen-fused polycyclic molecules are extensively present in pharmaceuticals and natural products [2]. The quinoline moiety is a preferred scaffold among nitrogen heterocyclic compounds that appear in several natural products and is a widely employed heterocyclic ring for the synthesis of bioactive molecules [3]. Another significant family of five-member nitrogen heterocycles is the 1,2,3-triazole [4], which has drawn much interest from synthetic and medicinal chemists for its high stability, ready availability, and wide range of uses in organic, pharmaceutical chemistry, and functional materials [5]. As compounds bearing fused 1,2,3-triazoles have gained importance in the pharmaceutical industry, new motifs and novel strategies are highly desired to synthesize this class of heterocycles. Here, we have synthesized quinoline and dihydroquinoline fused-1,2,3-triazole through an efficient and convenient approach. The synthetic strategy we sought to employ for the synthesis of the fused-1,2,3-triazoles involved three crucial steps: the addition of terminal alkyne, CuAAC, and Pd-catalysed annulation. For the first time, we present quinoline and dihydroquinoline fused-1,2,3-triazole in good to high yields by palladium-catalysed C-H functionalization at the C-5 position of the triazole moiety.
 

XXXVI Cycle

PhD student Cristine D’Agostino

Tutor Professor Franco Mazzei

2° year report

Development of Modifed Biosensors for Clinical, Ecotoxilogical and Food Analysis

The studies carried out during this second year of the PhD course aimed to investigate and develop two electrochemical biosensors for the rapid and accurate detection of faecal calprotectin and polyphenols in coffee samples. Calprotectin (CLP) is a calcium-zinc binding protein secreted by neutrophils and closely related to the inflammatory response. Many studies have reported that calprotectin is associate with the severity of inflammatory bowel disease (IBD) and can be used as a specific biomarker to predict diagnosis, prognosis and relapse of these diseases. The conventional method to detect faecal calprotectin is by the

Enzyme Linked Immunosorbent Assay (ELISA); although this method possesses high accuracy and robustness, the application suffers the shortcomings of unsatisfactory sensitivity, tedious test turnaround times, expensive labels. The biosensor developed therefore aims to determine calprotectin with a miniaturized system, with a rapid and sensitive response and lower analysis costs. The immunosensor configuration is based on the Self Assembled Monolayer (SAM) formation of 11-mercaptoundecanoic acid (11-MUA) on the working electrode of a gold Screen Printed Electrode (Au-SPE). The COOH groups exposed by thiol functionalisation are activated by EDC/NHS coupling to covalently immobilise the anti-CLP solution. An incubation time is required for maximising the interaction with the antigen CLP and improving the sensitivity of the sensor. The measurements are performed in Differential Pulse Voltammetry (DPV) with a portable potentiostat connected to a smartphone using as probe the redox couple ferro/ferricyanide [Fe(CN)6]-3/-4 as a function of the concentration of standard samples. Thirty real samples from Paediatric Gastroenterology and Hepatology Department of Policlinico Umberto I Hospital in Rome were analysed with both the ELISA reference method and the biosensor obtaining a good correspondence between the two methods and according to diagnosis of IBD. The second work is focused on the detection of the total polyphenol content in coffee, which is rich in chlorogenic acid, ferulic acid, caffeic acid, n-cumaric acid, representing one of the principal source of polyphenols in the diet. Depending on the coffee variety Arabica (Coffea Arabica L) and Robusta (Coffea Canephora), the polyphenol content is different; when no roasting processes are carried out the coffee is called “green” and specifically the Robusta quality shows the maximum polyphenol content around 7–14.4% of the dry weight, which is higher than that for Arabica variety.Another important factor affecting the polyphenol content in coffee is the roasting degree of the beans, both the temperature and time. In fact, the green coffee presents the highest content of polyphenols, while high temperature treatments induce the polyphenol degradation. Therefore, I studied an easy-to-use bio-electrochemical platform for the detection and quantification of polyphenols in coffee samples. Specifically, the influence on the total polyphenol content of both the roasting degree and the variety of the coffee analysed have been evaluated.

1. Rosaceleste Zumpano, Francesca Polli, Cristine D'Agostino, Riccarda Antiochia, Gabriele Favero, Franco Mazzei, Nanostructure-Based Electrochemical Immunosensors as Diagnostic Tools, Electrochem (2021), 2(1), 10-28, https://doi.org/10.3390/electrochem2010002

2. Chiara Salvitti, Anna Troiani, Franco Mazzei, Cristine D'Agostino, Rosaceleste Zumpano, Chiara Baldacchini, Anna Rita Bizzarri, Alessandra Tata, Federico Pepi, The Use of A commercial esi z-spray source for ambient ion soft landing and microdroplet reactivity experiments, International Journal of Mass Spectrometry 2021, 468, 116658, https://doi.org/10.1016/j.ijms.2021.116658

3. Rosaceleste Zumpano, Manuela Manghisi, Francesca Polli, Cristine D’Agostino, Federica Ietto, Gabriele Favero, Franco Mazzei, Label-free magnetic nanoparticles-based electrochemical immunosensor for atrazine detection, Analytical Bioanalytical Chemistry 2021, https://link.springer.com/article/10.1007/s00216-021-03838-y

4. Francesca Polli, Cristine D’Agostino, Rosaceleste Zumpano, Viviana De Martinob, Gabriele Faveroc, Luciano Colangelo, Salvatore Minisola, Franco Mazzei, Au@ Mnps-Based Electrochemical Immunosensor for Vitamin D3 Serum Samples Analysis, Talanta 2022, https://dx.doi.org/10.2139/ssrn.4055772.

PhD student Federico Marrone

Tutor Professor Giancarlo Fabrizi

2nd year report

Palladium-Catalyzed Synthesis of Nitrogen Containing Heterocycles from 2-iodoaryl-iminoenones

Enaminones are well known as platform building blocks in organic synthesis, because of their multifunctional character they have been used in a variety of intra- and intermolecular reactions. A number of transition-metal-promoted and-catalyzed processes have also been described for the construction of heterocyclic compounds, especially N-heterocycles. Therefore, because of our continuing interest in N-heterocycles synthesis, during my second year as PhD student, I focalized my studies on the reactivity of enaminone derivatives. In particular N-(2- iodoaryl)iminoenone derivative turned out to be a suitable and attractive precursor of both 3H-indole and quinoline cores, very important structural units found in nature and many biologically active molecules. At first a retrosynthetic analysis was carried out that suggested the employment of aryl-chlorodiazirines as carbene precursor to obtain iminoenone derivatives from enaminones. Due to the incompatibility of diazirines towards many organic solvents, the best reaction conditions were readily found using MeCN as solvent, K2CO3 as base, and carrying out the reaction at 80°C. Aryl-chlorodiazirines were easily prepared via the single step Graham oxidation of amidine precursors, that are widely commercially available, obtained with a simple treatment of amidine derivative with bleach in hexane. Thus a series of variously substituted N-(2-iodoaryl)iminoenone derivatives were synthesized achieving overall good yields with both EW and ED substituents. Then a screening, to obtain a regiodivergent cyclization of the iminoenone derivative, concerning the choice of solvent, base, catalyst and temperature was made; the results showed that Pd(PPh3)4 is the best catalyst and the base is mandatory, furthermore tuning the reaction conditions was possible to obtain selectively the 3H-indole core and the quinoline core. The quinoline core was obtained using Cs2CO3 as base, DMF as solvent at 120°C, unfortunately with the 47% of yield was isolated a quinoline derivative that doesn’t preserve the benzoyl moiety while the benzoylated quinoline derivative was isolated just with 27% of yield. Unlike the quinoline core, the 3H indole core was obtained with excellent yield using HCOOK as base and MeCN as solvent at 120°C. Then the scope of reaction was analysed and a collection of variously substituted 3H-indoles derivatives was obtained mostly in satisfactory yields (60-90%, 8 examples) showing that the reaction conditions are compatible both with EW and ER groups. In conclusion, starting from the same material, it was possible to prepare different exo/endo regioisomers just by appropriately tuning the reaction conditions. New efficient procedures for the synthesis of 2-Iodoaryl-iminoenone and 3H-indole derivatives were designed. Furthermore the research group is investigating the possibility to extend the synthetic method to obtain a series of quinoline derivatives maintaining the benzoyl moiety attached and to obtain a series of 3H-indoles derivatized with various nucleophiles.
 

PhD student Jessica Sebastiani

Tutor Professor Giuseppe La Regina

II year report

New pyrrole derivates as Anti-Lymphoma Agents and Ferroptosis Inducers in GBM and OC Cancers

During the second year of PhD, my research focused on the design and synthesis of new anticancer compound targeting tubulin polymerization. Microtubules play an essential role in various cell functions such as motility, cell division, cell shape maintenance and intracellular transport. Interference with the dynamic equilibrium of microtubule assembly has proven successful in killing cancer cells becoming an important target for the development of new anticancer agents.

In previous work we obtained a potent inhibitors of tubulin polymerization by replacing different group on the phenyl in position 1 of the pyrrole. Data showed that the presence of an ammino group can significantly improve the cellular activity. Considering that the replace of a carbon with a nitrogen atom can affect potency, selectivity, and physicochemical properties, including improved water solubility, we decided to the synthesize the pyrrole derivate RS6077. The new Compound inhibited the growth of multiple cancer cell

lines, with IC50 values in the nM range, without affecting the growth of non-transformed cells. Also the time-lapse video recording revealed a remarkable selectivity in cell death induction by RS6077, in particular, in RPE-1 non-transformed cells mitotic arrest induced was not necessarily followed by cell death; contrarly in HeLa transformed cell lines, cell death was effectively induced during mitotic arrest in cells that fail to complete mitosis. The anti-cancer activuty was also confirmed inTMD8 xenograft model. Thus, all these data allowed us to consider this as a good lead compound to study new anti-lymphoma drugs.

The second project aimed to find a new antimitotic compounds with a different ability to induce cell death. So far inducing apoptosis was considered the main way to cause cancer cell death in conventional treatments. However, to overcome the possible drug insensitivity for drug resistance, increasing studies have reported that inducing ferroptosis can significantly improve the efficacy of killing cancer cells, indicating that ferroptosis could be another important target in treatment of cancer. Thus, in this work we decide to explore the role in ferroptosis in two cancer type: Glioblastoma multiforme and ovarian cancer. Starting from a potent pyrrole agent, we replaced the 1-(methylphenyl) group with a pyridine or pyrimidine ring , in different positions, and kept fixed the hydrogen, phenyl or furan-2-yl moiety at position 4 of the pyrrole. The most active derivate against the tubulin polymerization showed also to strongly inhibit the U-87 MG, OVCAR-3 and MCF-7 cancer cells. Analysis of the levels of LPO, MDA, lactic acid, T-GSH and ATP suggested that the in vivo inhibition of cancer cell proliferation by the new derivate went through stimulation of oxidative stress injury and Fe2+ accumulation. Furthermore, qPCR analysis of the mRNA expression in U-87 MG and SKOV-3 tumor tissues from treated mice showed the presence of different genes correlated with ferroptosis in both groups. The biological profile of new pyrrole derivate, together with its stability in the presence of human liver microsome enzymes, highlight it as a robust lead compound for further optimization to provide new anticancer drugs based on alternative mechanisms of action.

Research period abroad

(01/08/2022-in progress) Carlo Ballatore’s research group, Skaggs School of Pharmacy and Pharmaceutical Sciences (PSB 3268), University of California, San Diego.

Papers

1. Sebastiani, J.; Puxeddu, M.; Nalli, M.; Bai, R.; Altieri, L.; Rovella, P.; Gaudio, E.; Trisciuoglio, D.; Spriano, F.; Lavia, P.; Fionda, C.; Hamel, E.; Bertoni, F.; Silvestri, R.; La Regina, G. RS6077 induces mitotic arrest and selectively activates cell death in human cancer cell lines and in lymphoma tumor in vivo. J. Med. Chem. 2022, submitted.

2. Michela Puxeddu, M.; Wu, J.; Bai, R.; D’Ambrosio, M.; Nalli, M.; Sebastiani J.; La regina G. et al. Induction of Ferroptosis in Glioblastoma and Ovarian Cancers by a New Pyrrole Tubulin Assembly Inhibitor. J. Med. Chem. 2022, accepted.

3. Puxeddu, M.; Shen, H.; Bai, R.; Coluccia, A.; Bufano, M.; Nalli, M.; Sebastiani, J. et al. Discovery of Pyrrole Derivatives for the Treatment of Glioblastoma and Chronic Myeloid Leukemia. Eur. J. Med. Chem. 2021, 221, e113532.

4. Dycke, J. V.; Puxeddu, M.; La Regina, G.; Mastrangelo, E.; Tarantino, D.; Rymenants, J.; Sebastiani J. et al. Discovery of a Novel Class of Norovirus Inhibitors with High Barrier of Resistance. Pharmaceuticals 2021, 14, 1006.

Posters

ESMEC 2022. Poster communication: RS6077 Induces Mitotic Arrest and Selectively Activates Cell Death in Human Cancer Cell Lines and in Lymphoma Tumor In Vivo.

AMYC 2021. Poster communication: New pyrrole derivatives for the treatment of glioblastoma and chronic myeloid leukemia.

MYCS 2021. Poster communication: Novel class of norovirus inhibitors.
 

PhD student: Andrea Sorato

Tutor: Prof.ssa Alessia Ciogli

2nd year report

Application and investigation of catalytic cycle by ESI-MS of new chiral C3-aminocatalyst and study of stereochemistry stability of atropoisomers by HPLC.

In the second year of the PhD Course, after synthesizing a multifunctional catalyst, with C3-symmetry, we focused on the synthesis of warfarin analogues and on the investigation of its catalytic cycle by ESI-MS experiments. The synthesis of warfarin analogues from coumarin derivatives and different unsaturated alpha-beta ketones provided products with good yields and %ee. In parallel, we evaluated catalytic cycle analyzing by ESI-MS two aliquots taken from the crude of warfarin reaction: the first one at t0, where the presence of the enamine intermediate can be noted (in addition to the presence of the starting reagents); the second aliquot after 4 hours was taken, highlighted the presence of intermediates with the product. The most important observation concerned with the detection of the active intermediate made on one molecule of catalyst and two molecules of ketone attesting the dual catalytic sites works at the same time. In addition, the catalyst was anchored on silica particles, to evaluate the catalyst in heterogeneous phase reactions, and the possibility of reuse. In parallel, the stereochemical stability of unconventional atropoisomers was evaluated (1). A study has been started on atropoisomers generated by the rotation of the N-N bond. A preliminary study was carried out on compound 1,2-di-boc-1,2-dibenzyl hydrazine (Figure 2) a symmetrical molecule and lacking chiral centers that generates two atropoisomers due only to the rotation of the N-N bond. A kinetic study was then carried out to monitor the racemization of these atropoisomers using an off-column HPLC approach. Results showed energy values of 23 kcal/mol (at 25 °C). The replacement of a benzyl group with two other substituents generates a family of derivatives, such as in two cases (MC54 and MC38) one more stereogenic element (a center) (Figure 3). For the compound MC54, a study of kinetics was made, with an off-column HPLC approach, which has shown energy values of 28,5 kcal/mol (at 100 °C) for the interconversion of diastereomeric pair.

Research period abroad:

I am carrying out my research period abroad, in Netherlands (Amsterdam) at UvA (University of Amsterdam) in Faculty of Science, working in NRG (Noël Research Group); period May 2022- November 2022.

During my period I’m working on ‘’Metal-free C(sp3)–C(sp3) cross-coupling via a photocatalytic deoxygenative alkylation of N-sulfonyl hydrazones’’. In particular after having established this one-pot C(sp3)−H alkylation procedure (Figure 4), we questioned whether the instalment of an ester moiety would be feasible through use of an ethyl glyoxylate-derived sulfonyl hydrazone (Figure 5). For the cleavage step, a solvent switch to ethanol proved to be essential to ensure reproducible results. Capitalizing on our two-step process, we discovered that various aliphatic ethers and amines could be readily coupled, delivering the corresponding products in good yields. Notably, our method provides also immediate access to various β-amino acids, common moieties in many marketed therapeutics.

Papers:

1) Portolani, C., Centonze, G., Luciani, S., Pellegrini, A., Righi, P., Mazzanti, A., Ciogli, A., Sorato, A., Bencivenni, G. (2022). Synthesis of Atropisomeric Hydrazides by One‐Pot Sequential Enantio‐and Diastereoselective Catalysis. Angewandte Chemie. https://doi.org/10.1002/ange.202209895

2) Salvitti, C., de Petris, G., Troiani, A., Managò, M., Villani, C., Ciogli, A. Sorato , A., Ricci, A, Pepi, F. (2022). Accelerated d-Fructose Acid-Catalyzed Reactions in Thin Films Formed by Charged Microdroplets Deposition. Journal of the American Society for Mass Spectrometry, 33(3), 565-572. 10.1021/jasms.1c00363
 

XXXV Cycle

PhD student Federico Appetecchia

Tutor Professor Giovanna Poce

Third year report

NEW PYRROLE AND PYRAZOLE BASED COMPOUNDS ACTIVE AS ANTIMALARIAL DRUGS

In the past three years we addressed the urgent requirement for novel anti-inflammatory drugs and antimalarial interventions: The beneficial effects of carbon monoxide (CO) gained much interest in research and offer new potential treatments of vascular- and inflammatory-related diseases. However, the medical application of this gas has been hampered by the complexity of the administration route. This problem has been overcome with the discovery of CO-releasing molecules (CORMs), which are an effective tool to deliver CO safely and precisely to the target locations. Particularly, metal-based CORMs are emerging for their striking anti-inflammatory properties that are amplified by the transition metal and are being progressively improved in view of novel future applications. We developed novel dual-active metal-based CORMs containing a pyrrole or a pyrazole core with the potential to be used as therapeutic agents in tendon-derived diseases. Specifically, we designed and synthesized dicobalt(0)hexacarbonyl (DCH)-CORMs containing structural fragments of COX-2 selective inhibitors and tested them for the CO release kinetic (myoglobin release assay) and antiinflammatory/cytoprotective effects on hydrogen peroxide-stimulated human primary-derived tenocytes by taking in account the PGE2 secretion as a readout. The 1,5-diaryl-pyrrolic compounds have shown the most beneficial effects on tenocytes and have been identified as promising therapeutic agents for the treatment of chronic inflammation from tendinopathies. Malaria drug research and development efforts have recently resurged in the last decade following deceleration rate of mortality and malaria cases in endemic regions. Inefficiency of malaria interventions are largely driven by the spreading resistance of the Plasmodium falciparum parasite to the current drug regimens and from the malaria vector – mosquito Anopheles – to insecticides. In response to the new eradication agenda, the development of drugs that act by breaking the malaria transmission cycle (transmissionblocking drugs) has been recognized as an important and additional target for intervention. These drugs take advantage of the susceptibility of Plasmodium population bottlenecks before transmission (gametocytes) and in the mosquito vector (gametes, zygotes, ookinetes, oocysts, sporozoites). In this context, we sought to address the urgent requirement for novel antimalarial interventions by developing and aiding future discovery of transmission-blocking drugs. In collaboration with Birkholtz’s research group at University of Pretoria (Department of Biochemistry Genetics and Microbiology) we started a research project to find new transmission-blocking compounds. Then, we designed and synthesized analogues of the pyrazole MMV1580843 (Figure 2), recently discovered in a high-throughput screening as a potent and selective gametocytocidal compound. SAR studies of these compounds hold promise for improved chemical modifications to progress to a hit-to-lead campaign. Particularly, we found that the pyrazole core allows a variety of substitutions that maintain potent activity towards late-stage gametocytes along with favourable physicochemical and safety profiles. Current studies are focusing on evaluating a new set of designed compounds carrying diverse substitutions and also exploring the importance of the pyrazole core by analysing pyrroles-based compounds. Target identification and comprehensive mechanistic studies will be a priority to better understand the SAR needed for the development of a lead compound. In parallel to this, following a collaboration with Catteruccia’s research group at Harvard T.H. Chan School of Public Health (Department of Immunology and Infectious Diseases), we performed phenotypic screening of compounds belonging to an in-house library and we discovered new pyrazole- and pyrrole-based compounds endowed

with selective activity towards ring and trophozoite stages in the P. falciparum asexual cycle and potential activity against sporogonic stages in mosquitoes. The transmission-blocking potential was assessed by performing topical exposure assays on females Anopheles Gambiae mosquitoes of selected hits and found compound CL191 and CL89 (Figure 3) to significantly decrease the parasite development in the mosquito midgut. Target deconvolution studies are underway.

Research period abroad

Dates: 02/21 – 02/22 Research group: Flaminia Catteruccia’s lab, Harvard TH Chan School of Public Health, Department of Immunology and Infectious Diseases

Papers

1. Consalvi, S., Tammaro, C., Appetecchia, F., Biava, M., & Poce, G. (2022). Malaria transmission blocking compounds: a patent review. Expert Opinion on Therapeutic Patents, 32(6), 649-666.

2. Appetecchia, F., Consalvi, S., Berrino, E., Gallorini, M., Granese, A., Campestre, C., ... & Poce, G. (2021). A novel class of dual-acting DCH-CORMs counteracts oxidative stress-induced inflammation in human primary tenocytes. Antioxidants, 10(11), 1828.

3. Appetecchia, F., Consalvi, S., Scarpecci, C., Biava, M., & Poce, G. (2020). SAR analysis of small molecules interfering with energy-metabolism in Mycobacterium tuberculosis. Pharmaceuticals, 13(9), 227.

4. Appetecchia, F.; Biava, M.; Poce, G. Natural Flavonoid and Chalcone Scaffolds as Leads for Synthetic Antitubercular Agents; Medicinal Chemistry: Lessons from Nature; submitted in Medicinal Chemistry Lessons from Nature; Vol. 1.

Posters and Presentations

- Poster: Appetecchia, F.; Paton, D.; Probst, A; De Vries, L; Tammaro, C; Consalvi, S; Wirth, F. D.; Catteruccia, F; Poce, G. Novel pyrazole- and pyrrole- based compounds active against multiple stages of P.falciparum. Poster presentation presented at 3rd MMCS: Shaping Medicinal Chemistry for the New Decade; July 2022; Rome, Italy.

- Presentation: Appetecchia, F. Development of targeted transmission-blocking agents against malaria. Presented at Terzo workshop sulla Ricerca; June 2022; Dip. Chimica e Tecnologie del Farmaco - La Sapienza Università di Roma; Rome, Italy
 

PhD student: Silvia Cammarone

Tutor: Professor Bruno Botta

3 rd year report

A Route to Lipid ALC-0315: a Key Component of a COVID-19 mRNA 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). 1, 2 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. 3 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 (scheme 1) 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. Based on this evidence, the aim of this research has been to adopt a safe, faster, and cheaper route for the synthesis of lipid ALC-0315, employing much cleaner reactions with the possibility to facilitate purification and, above all, avoid early-stages purification. 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. Key aspects of the work include the use of (i) a protected form of a poorly organic-soluble aminoalcohol, (ii) a TEMPO/bleach oxidation to produce crucial aldehydes, and (iii) a more organic-soluble triacyloxyborohydride reagent.

Period abroad:

Visiting PhD Student at The University of British Columbia, (Vancouver, Canada) under the supervision of Prof. Dr. Marco A. Ciufolini, 08/2021 - 06/2022 Scientific

Publications:

P1. Calcaterra, A.; Polli, F.; Lamelza, L.; Del Plato, C.; Cammarone, S.; Ghirga, F.; Botta, B.; Mazzei, F.; Quaglio, D. Resorc[4]arene-modified gold-decorated magnetic nanoparticles for immunosensors development. ACS Bioconjugate, 2022, submitted.

P2. Polli, F.; Cianfoni, G.; Elnahas, R.; Mangiardi, L.; Scaramuzzo, F. A.; Cammarone, S.; Quaglio, D.; Calcaterra, A.; Mazzei, F.; Zanoni, R.; Botta, B.; Ghirga, F. Resorc[4]arene modifiers for supramolecular site-directed immobilization of antibodies on multi-walled carbon nanotubes. ACS Bioconjugate, 2022, submitted.

P3. Picarazzi, F.; Zuanon, M.; Pasqualetto, G.; Cammarone, S.; Romeo, I.; Young, M.; Brancale, A.; Bassetto, M.; Mori, M. Identification of small molecular chaperones binding P23H mutant opsin through an in-silico structure-based approach. Journal of Chemical Information and Modeling, 2022, under review.

P4. Spano, M.; Di Matteo, G.; Ingallina, C.; Sobolev, A.; Giusti, A. M.; Vinci, G.; Cammarone, S.; Tortora, C.; Lamelza, L.; Prencipe, S.; Gobbi, L.; Botta, B.; Marini, F.; Campiglia, E.; Mannina, L. The effect of agronomical practices on the chemical profile of industrial hemp (Cannabis sativa L.) inflorescences: the case study of Ferimon cultivar. Food Chemistry, 2022, submitted.

P5. 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. (Very Important Paper, VIP).

P6. Casciaro, B.; Ghirga, F.; Cappiello, F.; Vergine, V.; Loffredo, M.R.; Cammarone, S.; Puglisi, E.; Tortora, C.; Quaglio, D.; Mori, M.; Botta, B.; Mangoni, M.L. The Triprenylated Anthranoid Ferruginin A, a Promising Scaffold for the Development of Novel Antibiotics against Gram-Positive Bacteria. Antibiotics, 2022, 11, 84.

P7. Platella, C.; Ghirga, F.; Zizza, P.; Pompili, L.; Marzano, S.; Pagano, B.; Quaglio, D.; Vergine, V.; Cammarone, S.; Botta, B.; Biroccio, A.; Mori, M.; Montesarchio, D. Identification of Effective Anticancer G-Quadruplex-Targeting Chemotypes through the Exploration of a High Diversity Library of Natural Compounds. Pharmaceutics, 2021, 13, 1611.

P8. Spano, M.; Di Matteo, G.; Ingallina, C.; Botta, B.; Quaglio, D.; Ghirga, F.; Balducci, S.; Cammarone, S.; Campiglia, E.; Giusti, A.M.; Vinci, G.; Rapa, M.; Ciano, S.; Mannina, L.; Sobolev, A.P. A Multimethodological Characterization of Cannabis sativa L. Inflorescences from Seven Dioecious Cultivars Grown in Italy: The Effect of Different Harvesting Stages. Molecules, 2021, 26, 2912.

P9. Ghirga, F.; Quaglio, D.; Mori, M.; Cammarone, S.; Iazzetti, A.; Goggiamani, A.; Ingallina, C.; Botta, B.; Calcaterra, A. A unique high-diversity natural product collection as a reservoir of new therapeutic leads, Organic Chemistry Frontiers, 2021; 8, 996-1025.

P10. Quaglio, D; Mangoni, M. L.; Stefanelli, R.; Corradi, S.; Casciaro, B.; Vergine, V.; Lucantoni, F.; Cavinato, L.; Cammarone, S.; Loffredo, M. R.; Cappiello, F.; Calcaterra, A.; Erazo, S.; Ghirga, F.; Mori, M.; Imperi, F.; Ascenzioni, F.; Botta, B. Ent-Beyerane diterpenes as a key platform for the development of ArnT-mediated colistin resistance inhibitors. The Journal of Organic Chemistry, 2020, 85, 10891-10901.

P11. Cappiello, F.; Loffredo, M.R.; Del Plato, C.; Cammarone, S.; Casciaro, B.; Quaglio, D.; Mangoni, M.L.; Botta, B.; Ghirga, F. The Revaluation of Plant-Derived Terpenes to Fight AntibioticResistant Infections. Antibiotics, 2020, 9, 325.

List of Oral Presentations:

1. Cammarone, S.; Cianfoni, G.; Ghirga, F.; Quaglio, D.; Calcaterra, A.; Mazzei, F.; Zanoni, R.; Botta, B. Resorc[4]arene-functionalized MWCNTS for the development of new highly sensitive immunosensors. MedChem 2022 (XI Meeting of the Paul Ehrlich Euro-PhD Network), Barcelona, Spain, July 14th -16th , 2022.

2. Cammarone, S.; Mangoni, M. L.; Quaglio, D.; Ghirga, F.; Ascenzioni. F.; Botta, B. Development of ArnT-mediated colistin resistance diterpene-based inhibitors. List of Poster Presentations. XLV "A. Corbella" International Summer School on Organic Synthesis, Gargnano, Italy, June 14th -18th , 2021.

3. Cammarone, S.; Quaglio, D.; Zhdanovskaya, N.; Screpanti, I.; Passarella, D.; Botta, B.; Palermo, R.; Mori, M.; Ghirga, F. Chalcones and Chalcone-mimetic Derivatives as Notch blocking agents in T-cell acute lymphoblastic leukemia. Poster with Short Oral communication. COST Action 17104 (STRATAGEM) WG2 Meeting and International Online Symposium on “Synthesis and nanodelivery strategies for new therapeutic tools against Multidrug Resistant Tumours. December, 15th , 2019.

List of Poster Presentations:

1. Cianfoni, G.; Polli, F.; Elnahas, R.; Ghirga, F.; Scaramuzzo, F. A.; Cammarone, S.; Quaglio, D.; Calcaterra, A.; Mazzei, F.; Zanoni, R.; Botta, B. Resorc[4]arene-functionalized MWCNTs for the development of new highly sensitive electrochemical immunosensors. 1 st International Supramolecular Chemistry Summer School. Santa Margerita di Pula, Italy, May 29th -June 2nd , 2022.

2. Cammarone, S.; Quaglio, D.; Zhdanovskaya, N.; Screpanti, I.; Passarella, D.; Botta, B.; Palermo, R.; Mori, M.; Ghirga, F. Chalcones and Chalcone-mimetic Derivatives as Notch blocking agents in T-cell acute lymphoblastic leukemia. COST Action 17104 (STRATAGEM) WG2 Meeting and International Online Symposium on “Synthesis and nanodelivery strategies for new therapeutic tools against Multidrug Resistant Tumours. December 15th , 2019.
 

PhD student Giacomo Di Matteo

Tutor Professor Luisa Mannina

Third year report

Metabolomics in the study of foods and officinal plants

In my third year of PhD I continue with the metabolomic studies of food matrices and officinal plants through the use of Nuclear Magnetic Resonance (NMR), for the determination of the complete metabolomic profiles, and of High Performance Liquid Chromatography (HPLC), for the targeted analysis of some compound classes. In particular, I have focused on the conclusion of two topics: the study of pumpkin preservation through the use of different commercial biofilms and the chemical characterization of the metabolites distribution in the Gentiana Lutea L. plant from the Majella National Park (of the Abruzzo region). Pumpkin was studied in a huge project called ORTOPACKHEALTH, which required to determine the best commercial biofilm for storing fresh pumpkins. The studied commercial biofilms were: Naturflex, Ecopounch and PLA. In addition, a conventional polyethylene film was used as a control. I have realized the metabolomic characterization, through NMR methodologies, of the pumpkin samples at time zero and after 10 days of storage in the different biofilms. The results have showed a metabolites alteration of the pumpkin based on the biofilm used for its conservation. From the biology group of the project, we knew that the better biofilm for the pumpkins storage is the PLA biofilm, since it led to the lower microbiological count in the stored pumpkin pieces. Hence, seeing the chemical results, obtained with the NMR analysis, of the pumpkins stored with the PLA biofilm and comparing these results with the two controls, the fresh pumpkins at time zero and the pumpkins conserved with the polyethylene film, we have that: a few number of metabolites had a significant increase in their concentration in the pumpkins stored with PLA biofilm respect with the two controls, another few number of metabolites had a significant concentration decrease, a high number of metabolites had a significant and small concentration change, while the majority of the metabolites didn’t present a significant change in their concentration. Based on these chemical results and the biological results, we have concluded that the PLA biofilm was the better commercial biofilm for the pumpkin storage. Regarding the second topic, I have realized a targeted analysis by the HPLC-DAD instrument of the peculiar class of compounds of the Gentiana Lutea plant parts (roots, leaves and flowers), namely iridoids (loganic acid), xanthones (mangiferin, isogentisine) and secoiridoids (sweroside, swertiamarin, amarogentina, gentiopicroside ). Methanolic extracts were obtained by sonication of the Gentiana Lutea parts.. The liquor, obtained by macerating of the Gentiana Lutea root in 11% v/v alcohol for 40 days, was also analysed. Moreover, the NMR analysis of the Bligh-Dyer extracts of Gentiana Lutea parts by the NMR methodology was also realized. In this way, we obtained the metabolites distribution in the plant parts from roots to leaves and flowers, finding a possible utilization for the leaves. The leaves are rich of iridoids, secoiridoids and xanthones, hence it could be used for the extraction of these metabolites.

Research period abroad

I have spent six months in the University of Valencia for the realization of part of the project about Gentiana Lutea L. I have stayed in Valencia from 01/09/2021 to 28/02/2022 in the research group of Food Science and Toxicology of professor Jordi Mañes. In this period I have realized an in vitro digestion of the Gentiana Lutea flowers to find a possible food utilization. On the obtained gastric and duodenal extracts I have applied the same HPLC-PDA and NMR protocols, developed for the Gentiana Lutea parts in Rome, to calculate the metabolites bioaccessibility. The metabolites bioavailability was also calculated realizing an intestinal cells mono-layer to analyze the metabolites passage. Then the toxicity of the duodenal extract was studied through the MTT assay finding a no-toxicity of the extract for all the tested extract dilutions. Moreover the mycotoxins protective activity was also tested through qPCR analysis monitoring genes that are marker of the intestinal barrier integrity, of the apoptosis and of the response to oxidative stress. We have concluded that the Gentiana flower extract was a good ingredient for the formulation of a functional food with anti-mycotoxin properties.

Posters

• G. Di Matteo, M. Spano, L. Mannina. “ NMR characterization of roots, leaves and flowers of Gentiana Lutea L. from Majella National Park”. Joint Italian-French meeting on Magnetic Resonance, Milan, Italy, 27-30 September 2022

Oral communications

• “Italian local products characterization by NMR methodologies”. MRFOOD2022 – 15th International Conference on the Applications of Magnetic Resonance in Food Science, Aarhus University, Aarhus, Denmark, 7-10 June 2022

• “A multi-methodological approach for the hemp seed oils characterization”. SISSG – Oli e grassi alimentari: innovazione e sostenibilità nella produzione e nel controllo, Perugia, 15-17 June 2022

• “NMR methodologies for the valorisation of Italian local products”. SYNC2022 – First Symposium for Young Chemists: Innovation and Sustainability. Sapienza University of Rome, Rome, Italy, 20-23 June 2022.

• “Profilo NMR metabolomico di campioni di zucca protetti da biofilm”. VII Workshop – Applicazioni della Risonanza Magnetica nella scienza degli alimenti. Sapienza University of Rome, Rome, Italy, 23-24 June 2022.

• “Gentiana Lutea L. : analisi della distribuzione dei metaboliti dalla radice a foglie e fiori”. Caratterizzazione multi-metodologica di piante officinali. Sapienza University of Rome, Rome, Italy, 9 September 2022.

Papers:

• Di Matteo, G. et al. Commercial Bio-Packaging to Preserve the Quality and Extend the Shelf-Life of Vegetables : The Case-Study of Pumpkin Samples Studied by a Multimethodological Approach. 1–24 (2021).

• Farinon, B. et al. Effect of malting on nutritional and antioxidant properties of the seeds of two industrial hemp (Cannabis sativa L.) cultivars. Food Chem. 370 (2022).

• Ullah, H. et al. Hydroethanolic Extract of Prunus domestica L .: Metabolite Profiling and In Vitro Modulation of Molecular Mechanisms Associated to Cardiometabolic Diseases. Nutrients 14, (2022).

• Spano, M. et al. Modulatory Properties of Food and Nutraceutical Components Targeting NLRP3 Inflammasome Activation. Nutrient 14, 1–44 (2022).
 

PhD student: Diego Alejandro Dri

Tutor: Prof. Carlotta Marianecci

Third year report

Regulatory Approach to Innovation in Clinical Trials: Nanomedicines and Machine Learning

Scientific and technical innovation is currently impacting many areas of Clinical Trials (CTs), such as drug discovery, development and delivery, protocol design, study conduction, regulatory assessment and authorisation process.

Data of CTs authorised in 2018 by AIFA, the Competent Authority (CA) in Italy, were retrieved, consolidated and analysed during the first year of the PhD course, those of CTs authorised in 2019 during the second year, and during the third year, data for the full period 2018-2020 were consolidated and analysed[i]. The quality issues detected during the assessment of IMPs that may be classified as nanomedicines are shared, discussing for the first time the results of the assessment reports elaborated by the quality assessors at the Clinical Trials Office.

In addition, we confirmed that the same legal requirements for safety, efficacy, and quality apply to COVID-19 IMPs, including vaccines, such as for any other medicinal product assessed during the evaluation of the benefit–risk balance of a CT application. Quality issues were, in general, noticed across almost all sections of the IMPD, denoting that quality standards and the level of compliance in the documentation submitted by all sponsors should definitely be increased.

We also highlighted that there is limited availability of guidelines supporting submission and assessment of CTs applications, and that no structured data is available in the CTA form to collect detailed information on nanotechnology-based IMPs/machine learning (ML). Submission cover letters for CTs and Substantial Amendments were therefore updated. Guidelines are fragmented and not aligned across different Regulatory Authorities. During the third year we confirmed that additional efforts are needed to define Critical Quality Attributes and requirements for the characterization of nanomedicines and nanocarriers, and that those should be coded in a dedicated regulatory framework for CTs.

Regarding ML and Artificial Intelligence (AI) in a CT setting, during the first and second year we focused on missing information related to the use of ML or AI in a CT, highlighting the need for specific guidelines to support the introduction and the assessment of innovative ML/AI elements in a CT, elaborating the first guide in EU for the submission of a request for authorisation of a CT involving the use of AI or ML systems, published by the Clinical Trials Office on the institutional website of AIFA[ii]. We therefore reviewed current information available on the regulatory approach to CTs and ML and provided inputs and indications for regulators to support the evolution in this field within a strong regulatory framework, focusing on patient safety, health protection and fostering immediate access to effective treatments. During the third year we contributed to support with the elaboration of a multistakeholder expert opinion on priorities for methodology, regulatory affairs, ethics and training providing a national perspective on the implementation of Decentralised Clinical Trials in Italy. We also actively participated to the elaboration of EU guidelines as member of the drafting group of the "Recommendation Paper on the Implementation and Conduct of Decentralized Elements in Clinical Trials with Investigational Medicinal Products in the European Union", as part of the EU decentralised clinical trials project (EU DCT project). Capitalizing on the assessment expertise, it was also proposed a new supporting method to assess CTs impacted by ML methods.

Publications

1 Dri DA, Marianecci C, Carafa M, Gaucci E, Gramaglia D. Surfactants, Nanomedicines and Nanocarriers: A Critical Evaluation on Clinical Trials. Pharmaceutics. 2021 Mar 13;13(3):381. doi: 10.3390/pharmaceutics13030381. PMID: 33805639; PMCID: PMC7999832.

1 Dri DA, Gaucci E, Torrieri I, Carafa M, Marianecci C, Gramaglia D. Critical Analysis and Quality Assessment of Nanomedicines and Nanocarriers in Clinical Trials: Three Years of Activity at the Clinical Trials Office. Pharmaceutics. 2022;14(7):1438. doi:10.3390/pharmaceutics14071438.

1 Dri DA, Praticò G, Gaucci E, Marianecci C, Gramaglia D. Quality Assessment of Investigational Medicinal Products in COVID-19 Clinical Trials: One Year of Activity at the Clinical Trials Office. Pharmaceuticals (Basel). 2021;14(12):1321. doi:10.3390/ph14121321.

1 Ufficio Sperimentazione Clinica. Aggiornamento modulistica autorizzazione sperimentazioni cliniche ed emendamenti sostanziali. AIFA, May 3rd 2021. Available online: https://www.aifa.gov.it/modulistica-sperimentazione-clinica.

• Modelli di lettere di trasmissione per la domanda di autorizzazione alla sperimentazione clinica di fase I

• Modelli di lettere di trasmissione per la domanda di autorizzazione di emendamento sostanziale alla sperimentazione clinica di fase I

• Modelli di lettere di trasmissione per la domanda di autorizzazione alla sperimentazione clinica di fase II, III, IV

• Modelli di lettere di trasmissione per la domanda di autorizzazione di emendamento sostanziale alla sperimentazione clinica di fase II, III, IV

1 Dri DA, Rinaldi F, Carafa M, Marianecci C. Nanomedicines and Nanocarriers in Clinical Trials: Surfing through Regulatory Requirements and Physicochemical Critical Quality Attributes. Drug Delivery and Translational Research. 2022, under review.

1 Gramaglia D, Dri DA, Massella M, Verrelli NM, Praticò G, Petraglia S, Foggi P, Di Marzo M, Agricola E. Guide to the submission of a request for authorisation of a Clinical Trial involving the use of Artificial Intelligence (AI) or Machine Learning (ML) systems. AIFA, May 24th 2021. Available online: https://www.aifa.gov.it/documents/20142/871583/Guide_CT_AI_ML_v_1.0_date_24.05.2021_EN.pdf.

1 Dri DA, Massella M, Gramaglia D, Marianecci C, Petraglia S. Clinical Trials and Machine Learning: regulatory approach review. Reviews on Recent Clinical Trial. 2021, 16, 341-350. doi: 10.2174/1574887116666210715114203.

1 Trogu P, Cagnazzo C, Collamati S, Corrao G, Daniele D, Dri DA, Galliccia F, Primiero P, Serafini E. Implementing Decentralized Clinical Trials in Italy: why and how? Multistakeholder expert opinion on priorities for methodology, regulatory affairs, ethics and training. Book Chapter: Management of digital (and other) data. Tendenze nuove Rivista semestrale online. Issn: 2239-2378. Special Issue 2/2022.

1 Dri DA, Massella M, Gramaglia D. Regulatory Considerations on the use of Machine Learning based tools in Clinical Trials. Health and Technology. 2022, under review.

PhD student Andrea Serraiocco

Tutor Professor Giancarlo Fabrizi

III year report

Studies towards the total synthesis of Amidochelocardin and synthesis of substitued 2,3-dihydropyrazin[1,2-a]indol-4(1H)-ones and of indolo[2,1- a]isoquinolines

During the first part of the third year of Ph.D, I worked with Prof. Kalesse’s group, at Leibniz University in Hannover, to set a total synthesis of the Amidochelocardin, an uncommon and interesting natural tetracycline, which mechanism of action seems to overcome issues and problems connected to bacterial resistance. We envisaged the possibility to obtain the tetracyclic core through a coupling between two fragments: the eastern fragment, characterized by a substituted decalin core, and a western fragment, which has a lactone structure. The key step of the synthesis of the eastern fragment consists of a Diels-Alder reaction between the commercially available Benzoquinone and the TBS protected crotonaldehyde. After the decalin core is established, bearing two OH substituents in position 1 and 4, the other important step is the Tsuji-Trost reaction to insert the NH2 group, protected as Cbz derivative. Eventually the eastern fragment is obtained after a 1,3-cycloaddition using acetaldoxime and NBS, to insert a dihydroisoxazole moiety, and subsequent deprotection and oxidation steps. Regarding the western fragment, the synthesis starts from the 3-methyl salicylic acid, which is methylated on the OH group and consequently transformed into the corresponding diethylamide which undergoes a reaction with sBuLi and formaldehyde and a subsequent intramolecular cyclization to the lactone compound. With these in hands, we manage to perform the fragment coupling obtaining the tetracyclic core in high yields. Further studies are ongoing in the lab to get the natural product. During the second part of the year I investigated the possibility to synthesized the 2,3- dihydropyrazin[1,2-a]indol-4(1H)-ones, starting from (1H-indol-2-yl)methyl acetate.

Molecules bearing this core shows anticancer, anticonvulsant, anxiolytic, depressant of the SNC properties. Indeed, from our previous studies, we think that this class of indole can generate in situ and highly reactive aza-o-QDM intermediate that can be trapped by a nucleophile, in this case an activated amminoacid. The nucleophile attack is followed by an intramolecular cyclization to the desired compounds. After screening of reaction conditions, we manage to synthesized a series of 2,3- dihydropyrazin[1,2-a]indol-4(1H)-ones with different amminoacids and indole, using MeCN as solvent, K2CO3 as base and 5 equiv. of amminoacid activated as ester derivative (10 compounds, 44-76% yield). Eventually, we are currently studying the reactivity of 2-(2-bromophenyl)-1-(propa1,2-dien-1-yl)-1H-indoles, towards the synthesis of indolo[2,1-a]isoquinolines, which have a great importance not only under the pharmaceutical point of view but also under the material science’s one. We hypothesized that the indolo-isoquinoline nucleus could be obtained after an intramolecular palladium-catalyzed cyclization in presence of a selected nucleophile such as H- , generated in situ starting from HCOOK, piperazines, phenols, sulfinates and carbon nucleophiles. Screening reactions are still under investigation with the aim to find the best conditions for the synthesis of these cyclic compounds: preliminary studies allow us to obtain the desired isoquinoline core starting from 2-(2-bromophenyl)-1-(propa-1,2-dien-1-yl)-1H-indole and HCOOK, morpholine, sodium benzensulfinate and ethyl 3-methyl-acetoacetate. With these promising results in hand we aim to go further in the synthesis in order to obtain a series of substituted indolo[2,1-a]isoquinolines.