Novel classes of porphyrazine macrocycles: effect of -delocalization, exocyclic coordination and quaternization processes

The pyrazinoporphyrazine macrocycles having formula [Py8TPyzPzH2], (Figure 1B) and its metal complexes [Py8TPyzPzM], with M = bivalent first transition series and non transition series metal ions, were extensively investigated by our group [1]. Structural and electronic features were studied by single crystal X-ray work, spectral (IR, UV-visible, NMR) and electrochemical investigations, and contributed by detailed theoretical DFT and TDDFT studies. The important role of the external pyridine rings has been examined by considering their capability of electronic contact with the central pyrazinoporphyrazine core. Their ability to coordinate metal centers [2] or to undergo quaternization processes at the N atoms [1b,c] has also been studied. The attention for applicative aspects has been particularly devoted to learn about their behavior as photosensitizers in PDT and their potentialities as multimodal anticancer agents [3, 2b-d]. The present thesis work has selected as the main subject the synthesis of two novel types of porphyrazine macrocycles, strictly related to the above considered “pyrazinoporphyrazines”, but showing a narrower (“restricted” macrocycle) or more extended porphyrazine core (“expanded” macrocycle”). The two compounds, in the form of unmetalated species are schematically shown in Figures 1A and 1C respectively. The targets of the present project were to explain: a) how the change in the expansion or contraction of the central planar or quasi-planar -conjugated skeleton, with respect to that of the pyrazinoporphyrazine core, will modify stability, solubility, tendency to aggregation, general physicochemical and redox behaviour of the new species and their applicative potentialities, with attention centered on their photoactivity properties; b) how the presence of the external pyridine rings, directly attached to the porphyrazine core (“restricted” macrocycle; Figure 1A) or more far away than in the already studied “pyrazinoporphyrazine” macrocycle (“expanded” macrocycle; Figure 1C) added of local metal coordination or quaternization processes, can produce consistent effects on the structural and electronic features of the new macrocyclic skeletons; c) how do the data concerning the response of the new species as photosensitizers compare with those already known for the original “pyrazinoporphyrazine” macrocycles. UV-visible spectral data in the low-donor nonaqueous solvents indicate that there is a progressive highly remarkable bathochromic shift of the barycentre of the overall spectrum in the direction pyridylporphyrazines pyrazinoporphyrazines quinoxalinoporphyrazines, in line with the parallel enhanced extension of the -conjugated system of the macrocycles in the order given. The eight external pyridines allow the accomplishment of exocyclic coordination and formation of pentanuclear species carrying externally PdCl2 and PtCl2. Figure 2 shows the pentametallic species for the pyridyl- (A) and quinoxalinoporphyrazines (B). Data at hand indicate that coordination of the PdII or PtII units takes place in all cases at the pyridine N atoms, and sites of the type N2(pyr)MCl2 (M = PdII, PtII; “py-py” coordination) are generated, displaying a square planar geometry and directed nearly perpendicularly to the plane of the central -conjugated macrocyclic system. External ligation modifies the UV-visible spectrum of the initial mononuclear species, the effect evidencing a bathochromic shift of the original main Q band by an average value of 15-20 nm. This remarkable shift is surprising if account is taken that exocyclic coordination of PdII and PtII, particularly for the pyrazino- and even more for the quinoxalinoporphyrazine compounds, takes place at the extreme periphery of the macrocycle and progressively more far away from the central metal. Further extension of the work focused on the synthesis of supercharged macrocycles for the pyridyl- and quinoxalinoporphyrazines. The new octacationic compounds which are moderately water soluble, were prepared from the mononuclear species upon reaction with CH3I, a process which results in the full N-methylation of the pyridine rings (Figure 3). The UV-visible spectra in water solution and in the low-donor solvents pyridine, DMSO and DMF show interesting effects which parallel those determined by the external metalation. The spectra evidence in the process from neutral to octacationic species a bathochromic shift of the Q bands about of the same order observed for the change mononuclear pentanuclear species. This means that the charged macrocycles enhance their electron deficiency; a fact that should be confirmed by the electrochemical behaviour in terms of the expected less negative half-wave potentials with respect to those pertinent to the neutral mononuclear species. The final challenge of some of the pyridyl- and quinoxalinoporphyrazines, especially those carrying centrally ZnII and MgII (closed shell metal ions) is their measured photoactivity for the generation in DMF of singlet oxygen, 1O2, the cytotoxic agent in the photodynamic therapy of cancer (PDT). The quantum yields of 1O2 measured, particularly for the two pyridylporphyazines having centrally ZnII and MgII, qualify these compounds as excellent photosensitizers. A combination of purity of the samples, water solubility, stability under the appropriate irradiation (600-750 nm), absence of aggregation in solution may be profitable for application in the PDT curative modality. The species carrying outside PtCl2 units may open perspectives for applications as bimodal PDT/cis-platin anticancer agents. Concomitant work was conducted on “pyrazinoporphyrazine” macrocycles carrying externally thienyl rings (Figure 4). The work on the thienyl pyrazinoporphyrazines involved modifications of the peripheral part of the macrocycle, focusing on the coordination properties of the S atoms inserted in the external 2-thienyl rings, in an interesting comparison with those seen for the pyridine rings in the pyridinated “pyrazinoporphyrazines” [4]. On the other hand the singlet oxygen and fluorescence response of the mono- and pentametallic complexes open perspectives for their potential use in PDT of cancer and for medical imaging and diagnosis. References: [1] a) Donzello, M. P.; Ou, Z.; Monacelli, F.; Ricciardi, G.; Rizzoli, C.; Ercolani, C.; Kadish, K. M. Inorg. Chem. 2004, 43, 8626; b) Donzello, M. P.; Ou, Z.; Dini, D.; Meneghetti, M.; Ercolani, C.; Kadish, K. M. Inorg. Chem., 2004, 43, 8637; c) Bergami, C.; Donzello, M. P.; Ercolani, C.; Monacelli, F.; Kadish, K. M.; Rizzoli, C. Inorg. Chem., 2005, 44, 9852; d) Bergami, C.; Donzello M. P.; Monacelli, F.; Ercolani, C.; Kadish, K. M. Inorg. Chem., 2005, 44, 9862. [2] a) Donzello, M. P.; Viola, E.; Cai, X.; Mannina, L.; Rizzoli, C.; Ricciardi, G.; Ercolani, C.; Kadish, K. M.; Rosa, A. Inorg. Chem., 2008, 47, 3903; b) Donzello, M. P.; Viola, E.; Cai, X.; Mannina, L.; Ercolani, C.; Kadish, K. M. Inorg. Chem., 2010, 49, 2447; c) Donzello, M. P.; Viola, E.; Mannina, L.; Barteri, M.; Fu, Z.; Ercolani, C. J. Porphyrins Phthalocyanines, 2011, 15, 984; d) Donzello, M. P.; Vittori, D.; Viola, E.; Manet, I.; Mannina , L.; Cellai, L.; Monti, S.; Ercolani, C. Inorg. Chem., 2011, 50, 7391. [3] Donzello, M. P.; Viola, E.; Bergami, C.; Dini, D.; Ercolani, C.; Giustini, M.; Kadish, K. M.; Meneghetti, M.; Monacelli, F.; Rosa, A.; Ricciardi, G. Inorg. Chem., 2008, 47, 8757. [4] a) De Mori, G.; Fu, Z.; Viola, E.; Cai, X.; Ercolani, C.; Donzello, M. P.; Kadish, K. M. Inorg. Chem., 2011, 50, 8225; b) Donzello, M.P.; De Mori, G.; Viola, E.; Ercolani, C.; Bodo, E.; Mannina, L.; Capitani, D.; Rizzoli, C.; Gontrani, L.; Aquilanti, G.; Kadish, K. M.; D’Angelo, P. Inorg. Chem., 2011, 50, 12116

Novel classes of porphyrazine macrocycles: effect of -delocalization, exocyclic coordination and quaternization processes

The pyrazinoporphyrazine macrocycles having formula [Py8TPyzPzH2], (Figure 1B) and its metal complexes [Py8TPyzPzM], with M = bivalent first transition series and non transition series metal ions, were extensively investigated by our group [1]. Structural and electronic features were studied by single crystal X-ray work, spectral (IR, UV-visible, NMR) and electrochemical investigations, and contributed by detailed theoretical DFT and TDDFT studies. The important role of the external pyridine rings has been examined by considering their capability of electronic contact with the central pyrazinoporphyrazine core. Their ability to coordinate metal centers [2] or to undergo quaternization processes at the N atoms [1b,c] has also been studied. The attention for applicative aspects has been particularly devoted to learn about their behavior as photosensitizers in PDT and their potentialities as multimodal anticancer agents [3, 2b-d]. The present thesis work has selected as the main subject the synthesis of two novel types of porphyrazine macrocycles, strictly related to the above considered “pyrazinoporphyrazines”, but showing a narrower (“restricted” macrocycle) or more extended porphyrazine core (“expanded” macrocycle”). The two compounds, in the form of unmetalated species are schematically shown in Figures 1A and 1C respectively. The targets of the present project were to explain: a) how the change in the expansion or contraction of the central planar or quasi-planar -conjugated skeleton, with respect to that of the pyrazinoporphyrazine core, will modify stability, solubility, tendency to aggregation, general physicochemical and redox behaviour of the new species and their applicative potentialities, with attention centered on their photoactivity properties; b) how the presence of the external pyridine rings, directly attached to the porphyrazine core (“restricted” macrocycle; Figure 1A) or more far away than in the already studied “pyrazinoporphyrazine” macrocycle (“expanded” macrocycle; Figure 1C) added of local metal coordination or quaternization processes, can produce consistent effects on the structural and electronic features of the new macrocyclic skeletons; c) how do the data concerning the response of the new species as photosensitizers compare with those already known for the original “pyrazinoporphyrazine” macrocycles. UV-visible spectral data in the low-donor nonaqueous solvents indicate that there is a progressive highly remarkable bathochromic shift of the barycentre of the overall spectrum in the direction pyridylporphyrazines pyrazinoporphyrazines quinoxalinoporphyrazines, in line with the parallel enhanced extension of the -conjugated system of the macrocycles in the order given. The eight external pyridines allow the accomplishment of exocyclic coordination and formation of pentanuclear species carrying externally PdCl2 and PtCl2. Figure 2 shows the pentametallic species for the pyridyl- (A) and quinoxalinoporphyrazines (B). Data at hand indicate that coordination of the PdII or PtII units takes place in all cases at the pyridine N atoms, and sites of the type N2(pyr)MCl2 (M = PdII, PtII; “py-py” coordination) are generated, displaying a square planar geometry and directed nearly perpendicularly to the plane of the central -conjugated macrocyclic system. External ligation modifies the UV-visible spectrum of the initial mononuclear species, the effect evidencing a bathochromic shift of the original main Q band by an average value of 15-20 nm. This remarkable shift is surprising if account is taken that exocyclic coordination of PdII and PtII, particularly for the pyrazino- and even more for the quinoxalinoporphyrazine compounds, takes place at the extreme periphery of the macrocycle and progressively more far away from the central metal. Further extension of the work focused on the synthesis of supercharged macrocycles for the pyridyl- and quinoxalinoporphyrazines. The new octacationic compounds which are moderately water soluble, were prepared from the mononuclear species upon reaction with CH3I, a process which results in the full N-methylation of the pyridine rings (Figure 3). The UV-visible spectra in water solution and in the low-donor solvents pyridine, DMSO and DMF show interesting effects which parallel those determined by the external metalation. The spectra evidence in the process from neutral to octacationic species a bathochromic shift of the Q bands about of the same order observed for the change mononuclear pentanuclear species. This means that the charged macrocycles enhance their electron deficiency; a fact that should be confirmed by the electrochemical behaviour in terms of the expected less negative half-wave potentials with respect to those pertinent to the neutral mononuclear species. The final challenge of some of the pyridyl- and quinoxalinoporphyrazines, especially those carrying centrally ZnII and MgII (closed shell metal ions) is their measured photoactivity for the generation in DMF of singlet oxygen, 1O2, the cytotoxic agent in the photodynamic therapy of cancer (PDT). The quantum yields of 1O2 measured, particularly for the two pyridylporphyazines having centrally ZnII and MgII, qualify these compounds as excellent photosensitizers. A combination of purity of the samples, water solubility, stability under the appropriate irradiation (600-750 nm), absence of aggregation in solution may be profitable for application in the PDT curative modality. The species carrying outside PtCl2 units may open perspectives for applications as bimodal PDT/cis-platin anticancer agents. Concomitant work was conducted on “pyrazinoporphyrazine” macrocycles carrying externally thienyl rings (Figure 4). The work on the thienyl pyrazinoporphyrazines involved modifications of the peripheral part of the macrocycle, focusing on the coordination properties of the S atoms inserted in the external 2-thienyl rings, in an interesting comparison with those seen for the pyridine rings in the pyridinated “pyrazinoporphyrazines” [4]. On the other hand the singlet oxygen and fluorescence response of the mono- and pentametallic complexes open perspectives for their potential use in PDT of cancer and for medical imaging and diagnosis. References: [1] a) Donzello, M. P.; Ou, Z.; Monacelli, F.; Ricciardi, G.; Rizzoli, C.; Ercolani, C.; Kadish, K. M. Inorg. Chem. 2004, 43, 8626; b) Donzello, M. P.; Ou, Z.; Dini, D.; Meneghetti, M.; Ercolani, C.; Kadish, K. M. Inorg. Chem., 2004, 43, 8637; c) Bergami, C.; Donzello, M. P.; Ercolani, C.; Monacelli, F.; Kadish, K. M.; Rizzoli, C. Inorg. Chem., 2005, 44, 9852; d) Bergami, C.; Donzello M. P.; Monacelli, F.; Ercolani, C.; Kadish, K. M. Inorg. Chem., 2005, 44, 9862. [2] a) Donzello, M. P.; Viola, E.; Cai, X.; Mannina, L.; Rizzoli, C.; Ricciardi, G.; Ercolani, C.; Kadish, K. M.; Rosa, A. Inorg. Chem., 2008, 47, 3903; b) Donzello, M. P.; Viola, E.; Cai, X.; Mannina, L.; Ercolani, C.; Kadish, K. M. Inorg. Chem., 2010, 49, 2447; c) Donzello, M. P.; Viola, E.; Mannina, L.; Barteri, M.; Fu, Z.; Ercolani, C. J. Porphyrins Phthalocyanines, 2011, 15, 984; d) Donzello, M. P.; Vittori, D.; Viola, E.; Manet, I.; Mannina , L.; Cellai, L.; Monti, S.; Ercolani, C. Inorg. Chem., 2011, 50, 7391. [3] Donzello, M. P.; Viola, E.; Bergami, C.; Dini, D.; Ercolani, C.; Giustini, M.; Kadish, K. M.; Meneghetti, M.; Monacelli, F.; Rosa, A.; Ricciardi, G. Inorg. Chem., 2008, 47, 8757. [4] a) De Mori, G.; Fu, Z.; Viola, E.; Cai, X.; Ercolani, C.; Donzello, M. P.; Kadish, K. M. Inorg. Chem., 2011, 50, 8225; b) Donzello, M.P.; De Mori, G.; Viola, E.; Ercolani, C.; Bodo, E.; Mannina, L.; Capitani, D.; Rizzoli, C.; Gontrani, L.; Aquilanti, G.; Kadish, K. M.; D’Angelo, P. Inorg. Chem., 2011, 50, 12116

2,6-Diphenyl-imidazopyridine derivatives as novel prototypes of anticancer agents targeting aldehyde dehydrogenase

Aldehyde dehydrogenase (ALDH) superfamily comprises 19 different enzyme types located in specific subcellular districts, including cytosol and mitocondria. Their main function is to oxidize endogenous and exogenous aldehydes produced in human cells. In particular, isoforms 1A1, 1A2 and 1A3 catalyze the transformation of retinal into retinoic acid, which is a potent differentiation tissue factor for cellular development. Overexpression of these three isoforms in cancer stem cells (CSC), underlined in recent studies, is to date extremely important in cancer field, as it offers the chance to use these proteins both as prognostic marker and as novel targets in the fight against cancer. Here we present a novel series of 2,6-diphenyl-imidazol[1,2-a]pyridines, designed as aldehyde dehydrogenase inhibitors by means of a structured-based optimizations of a previously developed lead, GA11. The novel compounds were evaluated in vitro for their activity and selectivity against the three isoforms of the ALDH1A family, and investigated through crystallization and modeling studies for their ability to interact with the catalytic site of the 1A3 isoform. Tested in vitro on different populations of CSCs, obtained from glioma, colorectal and prostate tissue specimens, they exhibited a relevant anti-proliferative efficacy, thus paving the way for treating cancer by means of the still untapped aldehyde dehydrogenases

Synthesis and evaluation of β-hydroxytriazoles and related compounds as antitubercular agents

A new series of β-hydroxytriazoles were synthesized and evaluated as Mycobacterium tuberculosis inhibitors. Our strategy implied the synthesis of alkyne precursors through a Barbier reaction between benzaldehydes and propargyl bromide followed by click chemistry to afford substituted β-hydroxyl benzyltriazoles. These compounds are also key intermediates either for oxidation reactions leading to α,β-diketotriazoles or for elimination reactions affording styryl triazoles. Evaluation of all new compounds for in vitro antitubercular activity against Mycobacterium tuberculosis H37Rv resulted in compounds with MIC up to 7 μM

Discovery and development of novel salicylate synthase (MbtI) furanic inhibitors as antitubercular agents

We report on the virtual screening, synthesis, and biological evaluation of new furan derivatives targeting Mycobacterium tuberculosis salicylate synthase (MbtI). A receptor-based virtual screening procedure was applied to screen the Enamine database, identifying two compounds, I and III, endowed with a good enzyme inhibitory activity. Considering the most active compound I as starting point for the development of novel MbtI inhibitors, we obtained new derivatives based on the furan scaffold. Among the SAR performed on this class, compound 1a emerged as the most potent MbtI inhibitor reported to date (Ki = 5.3 μM). Moreover, compound 1a showed a promising antimycobacterial activity (MIC99 = 156 μM), which is conceivably related to mycobactin biosynthesis inhibition

Fate of mesoangioblasts in a vaginal birth injury model: influence of the route of administration

Currently cell therapy is considered as an experimental strategy to assist the healing process following simulated vaginal birth injury in rats, boosting the functional and morphologic recovery of pelvic floor muscles and nerves. However, the optimal administration route and dose still need to be determined. Mesangioblasts theoretically have the advantage that they can differentiate in skeletal and smooth muscle. We investigated the fate of mesoangioblasts transduced with luciferase and green fluorescent protein reporter genes (rMABseGFP/fLUC) using bioluminescence, immunofluorescence and RT-PCR in rats undergoing simulated birth injury. rMABseGFP/fLUC were injected locally, intravenously and intra-arterially (common iliacs and aorta). Intra-arterial delivery resulted in the highest amount of rMABseGFP/fLUC in the pelvic organs region and in a more homogeneous distribution over all relevant pelvic organs. Sham controls showed that the presence of the injury is important for recruitment of intra-arterially injected rMABseGFP/fLUC. Injection through the aorta or bilaterally in the common iliac arteries resulted in comparable numbers of rMABseGFP/fLUC in the pelvic organs, yet aortic injection was faster and gave less complications

Shifts of Faecal Microbiota during Sporadic Colorectal Carcinogenesis

Gut microbiota has been implicated in the etiopathogenesis of colorectal cancer. The development of colorectal cancer is a multistep process by which healthy epithelium slowly develops into preneoplastic lesions, which in turn progress into malignant carcinomas over time. In particular, sporadic colorectal cancers can arise from adenomas (about 85% of cases) or serrated polyps through the "adenoma-carcinoma" or the "serrated polyp-carcinoma" sequences, respectively. In this study, we performed 16 S rRNA gene sequencing of bacterial DNA extracted from faecal samples to compare the microbiota of healthy subjects and patients with different preneoplastic and neoplastic lesions. We identified putative microbial biomarkers associated with stage-specific progression of colorectal cancer. In particular, bacteria belonging to the Firmicutes and Actinobacteria phyla, as well as members of the Lachnospiraceae family, proved to be specific of the faecal microbiota of patients with preneoplastic lesions, including adenomas and hyperplastic polyps. On the other hand, two families of the Proteobacteria phylum, Alcaligeneaceae and Enterobacteriaceae, with Sutterella and Escherichia/Shigella being the most representative genera, appeared to be associated with malignancy. These findings, once confirmed on larger cohorts of patients, can represent an important step towards the development of more effective diagnostic strategies

Analogous Mechanisms of Resistance to Benzothiazinones and Dinitrobenzamides in Mycobacterium smegmatis

Tuberculosis is still a leading cause of death worldwide. The selection and spread of Mycobacterium tuberculosis multidrug-resistant (MDR-TB) and extensively drug-resistant strains (XDR-TB) is a severe public health problem. Recently, two different classes of chemical series, the benzothiazinones (BTZ) and the dinitrobenzamide (DNB) derivatives have been found to be highly active against M. tuberculosis, including XDR-TB strains. The target of BTZs is DprE1 protein which works in concert with DprE2 to form the heteromeric decaprenylphosphoryl-β-D-ribose 2′-epimerase, involved in Decaprenyl-Phospho-Arabinose (DPA) biosynthesis. Interestingly, it has been shown that the DNBs block the same pathway thus suggesting that both drugs could share the same target. Moreover, in Mycobacterium smegmatis the overexpression of the NfnB nitroreductase led to the inactivation of the BTZs by reduction of a critical nitro-group to an amino-group. In this work several spontaneous M. smegmatis mutants resistant to DNBs were isolated. Sixteen mutants, showing high levels of DNB resistance, exhibited a mutation in the Cys394 of DprE1. Using fluorescence titration and mass spectrometry it has been possible to monitor the binding between DprE1 and DNBs, achieving direct evidence that MSMEG_6382 is the cellular target of DNBs in mycobacteria. Additionally, M. smegmatis mutants having low levels of resistance to DNBs harbor various mutations in MSMEG_6503 gene encoding the transcriptional repressor of the nitroreductase NfnB. By LC/MS2 analysis it has been demonstrated that NfnB is responsible for DNB inactivation. Taken together, our data demonstrate that both DNB and BTZ drugs share common resistance mechanisms in M. smegmatis

Climate and landscape composition explain agronomic practices, pesticide use and grape yield in vineyards across Italy

Context Worldwide, organic farming is being promoted as one of the main alternatives to intensive conventional farming. However, the benefits of organic agriculture are still controversial and need to be tested across wide environmental gradients. Objective Here, we carried out an observational study to test how agronomic practices, pest management, environmental impact and yield of conventional and organic vineyards changed along wide climatic and landscape gradients across Italy. Methods We used a block design with 38 pairs of conventional and organic vineyards across Italy. Results and conclusions Most agronomic practices did not differ between conventional and organic vineyards. By contrast, landscape composition and climate were strong predictors of management in both systems. First, increasing semi-natural areas around the vineyards reduced pesticide pressure and related environmental impacts, but was also associated with lower yield. Second, irrespective of the farming system, a warm and dry climate was associated with reduced fungicide pressure. Conventional farming had a yield gain of 40% in cold and wet climate compared to organic but the yield gap disappeared in the warmest regions. Significance In both farming systems, we observed a large variability in management practices that was mainly explained by climate and landscape composition. This large variability should be considered when evaluating the benefits and drawbacks of different farming systems under contrasting environmental contexts

Prediction of early distant recurrence in upfront resectable pancreatic adenocarcinoma: A multidisciplinary, machine learning-based approach

Despite careful selection, the recurrence rate after upfront surgery for pancreatic adenocarcinoma can be very high. We aimed to construct and validate a model for the prediction of early distant recurrence (<12 months from index surgery) after upfront pancreaticoduodenectomy. After exclusions, 147 patients were retrospectively enrolled. Preoperative clinical and radiological (CT-based) data were systematically evaluated; moreover, 182 radiomics features (RFs) were extracted. Most significant RFs were selected using minimum redundancy, robustness against delineation uncertainty and an original machine learning bootstrap-based method. Patients were split into training (n = 94) and validation cohort (n = 53). Multivariable Cox regression analysis was first applied on the training cohort; the resulting prognostic index was then tested in the validation cohort. Clinical (serum level of CA19.9), radiological (necrosis), and radiomic (SurfAreaToVolumeRatio) features were significantly associated with the early resurge of distant recurrence. The model combining these three variables performed well in the training cohort (p = 0.0015,HR = 3.58,95%CI = 1.98–6.71) and was then confirmed in the validation cohort (p = 0.0178,HR = 5.06,95%CI = 1.75–14.58). The comparison of survival curves between low and high-risk patients showed a p-value <0.0001. Our model may help to better define resectability status, thus providing an actual aid for pancreatic adenocarcinoma patients’ management (upfront surgery vs. neoadjuvant chemotherapy). Independent validations are warranted