Publications by authors named "Elena Dreassi"

44 Publications

Bithiazole Inhibitors of Phosphatidylinositol 4-Kinase (PI4KIIIβ) as Broad-Spectrum Antivirals Blocking the Replication of SARS-CoV-2, Zika Virus, and Human Rhinoviruses.

ChemMedChem 2021 Aug 11. Epub 2021 Aug 11.

Dipartimento di Scienze degli Alimenti e del Farmaco, Università degli Studi di Parma, Parco Area delle Scienze, 27/A, 43124, Parma, Italy.

Over half a century since the description of the first antiviral drug, "old" re-emerging viruses and "new" emerging viruses still represent a serious threat to global health. Their high mutation rate and rapid selection of resistance toward common antiviral drugs, together with the increasing number of co-infections, make the war against viruses quite challenging. Herein we report a host-targeted approach, based on the inhibition of the lipid kinase PI4KIIIβ, as a promising strategy for inhibiting the replication of multiple viruses hijacking this protein. We show that bithiazole inhibitors of PI4KIIIβ block the replication of human rhinoviruses (hRV), Zika virus (ZIKV) and SARS-CoV-2 at low micromolar and sub-micromolar concentrations. However, while the anti-hRV/ZIKV activity can be directly linked to PI4KIIIβ inhibition, the role of PI4KIIIβ in SARS-CoV-2 entry/replication is debated.
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http://dx.doi.org/10.1002/cmdc.202100483DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8427023PMC
August 2021

Si113-prodrugs selectively activated by plasmin against hepatocellular and ovarian carcinoma.

Eur J Med Chem 2021 Nov 17;223:113653. Epub 2021 Jun 17.

Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, Via Aldo Moro 2, 53100, Siena, Italy; Lead Discovery Siena S.r.l., Via Vittorio Alfieri 31, 53019, Castelnuovo Berardenga, Siena, Italy; Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology Temple University, BioLife Science Building, Suite 333, 1900 North 12th Street, Philadelphia, PA, 19122, United States.

Si113, a pyrazolo[3,4-d]pyrimidine derivative, gained more attention as an anticancer agent due to its potent anticancer activity on both in vitro and in vivo hepatocellular carcinomas (HCC) and ovarian carcinoma models. But the drawback is the low water solubility which prevents its further development. In this context, we successfully overcame this limitation by synthesizing two novel prodrugs introducing the amino acid sequence D-Ala-Leu-Lys (TP). Moreover, TP sequence has a high affinity with plasmin, a protease recognized as overexpressed in many solid cancers, including HCC and ovarian carcinoma. The prodrugs were synthesized and fully characterized in terms of in vitro ADME properties, plasma stability and plasmin-induced release of the parent drug. The inhibitory activity against Sgk1 was evaluated and in vitro growth inhibition was evaluated on ovarian carcinoma and HCC cell lines in the presence and absence of human plasmin. In vivo pharmacokinetic properties and preliminary tissue distribution confirmed a better profile highlighting the importance of the prodrug approach. Finally, the prodrug antitumor efficacy was evaluated in an HCC xenografted murine model, where a significant reduction (around 90%) in tumor growth was observed. Treatment with ProSi113-TP in combination with paclitaxel in a paclitaxel-resistant ovarian carcinoma xenografted murine model, resulted in an impressive reduction of tumor volume greater than 95%. Our results revealed a promising activity of Si113 prodrugs and pave the way for their further development against resistant cancer.
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http://dx.doi.org/10.1016/j.ejmech.2021.113653DOI Listing
November 2021

Determination of copper by AAS in tear fluid of patients with keratoconus.

Anal Biochem 2021 06 23;623:114174. Epub 2021 Mar 23.

Ophthalmology Unit, Department of Medical, Surgical and Neurosciences, Siena University, Viale M.Bracci, 16, 53100, Siena, Italy.

Keratoconus (KC) is the most common degenerative corneal disease and no single biomarker for KC has been discovered. Its causes have not yet been clarified and this work aims to be a contribution to the deepening of the knowledge of this disease and a preliminary data to the evaluation of the possibility of the use of copper (Cu) concentration in the tear fluid as a specific marker. A tear fluid sampling and Cu determination by spectrometric atomic absorption method was optimized to determine Cu levels in the tear fluid of patients with KC compared to that of healthy patients. Results demonstrate that in the KC subjects (n = 6) the concentration of Cu ions was 325.5 ± 110.7 ng/ml, while in the control group was 141.3 ± 71.1 ng/ml. A significant increase in Cu ion levels in the tear fluid was observed in the KC group compared to the control group (p value < 0.001).
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http://dx.doi.org/10.1016/j.ab.2021.114174DOI Listing
June 2021

Unique Domain for a Unique Target: Selective Inhibitors of Host Cell DDX3X to Fight Emerging Viruses.

J Med Chem 2020 09 18;63(17):9876-9887. Epub 2020 Aug 18.

Istituto di Genetica Molecolare IGM-CNR "Luigi Luca Cavalli-Sforza", Via Abbiategrasso 207, I-27100 Pavia, Italy.

Emerging viruses like dengue, West Nile, chikungunya, and Zika can cause widespread viral epidemics. Developing novel drugs or vaccines against specific targets for each virus is a difficult task. As obligate parasites, all viruses exploit common cellular pathways, providing the possibility to develop broad-spectrum antiviral agents targeting host factors. The human DEAD-box RNA helicase DDX3X is an essential cofactor for viral replication but dispensable for cell viability. Herein, we exploited the presence of a unique structural motif of DDX3X not shared by other cellular enzymes to develop a theoretical model to aid in the design of a novel class of highly selective inhibitors acting against such specific targets, thus limiting off-targeting effects. High-throughput virtual screening led us to identify hit compound , endowed with promising antienzymatic activity. To improve its aqueous solubility, and its two enantiomers were synthesized and converted into their corresponding acetate salts (compounds , , and ). mutagenesis and biochemical and cellular assays further confirmed that the developed molecules were selective for DDX3X and were able to suppress replication of West Nile and dengue viruses in infected cells in the micromolar range while showing no toxicity for uninfected cells. These results provide proof of principle for a novel strategy in developing highly selective and broad-spectrum antiviral molecules active against emerging and dangerous viral pathogens. This study paves the way for the development of larger focused libraries targeting such domain to expand SAR studies and fully characterize their mode of interaction.
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http://dx.doi.org/10.1021/acs.jmedchem.0c01039DOI Listing
September 2020

Antihypertensive, cardio- and neuro-protective effects of Tenebrio molitor (Coleoptera: Tenebrionidae) defatted larvae in spontaneously hypertensive rats.

PLoS One 2020 29;15(5):e0233788. Epub 2020 May 29.

Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, Siena, Italy.

In pre-hypertension, moderate control of blood pressure (BP) can be obtained by a nutritional approach. The effects of a diet enriched with defatted larvae of the mealworm Tenebrio molitor (Coleoptera: Tenebrionidae) (TM) endowed with ACE inhibitory activity was studied in both spontaneously hypertensive rats (SHR) and in the age-matched normotensive Wistar Kyoto strain. These were fed for 4 weeks with standard laboratory rodent chow supplemented with or without TM or captopril. In SHR, the TM diet caused a significant reduction in BP, heart rate and coronary perfusion pressure, as well as an increase in red blood cell glutathione/glutathione disulphide ratio. Rat brain slices of SHR were more resistant to oxidative stress and contained lower levels of inflammatory cytokines, while vascular and liver enzyme-activities were not affected. These results suggest that TM can be considered a new functional food that can lower BP in vivo and thus control cardiovascular-associated risk factors such as hypertension.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0233788PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7259609PMC
September 2020

DDX3X inhibitors, an effective way to overcome HIV-1 resistance targeting host proteins.

Eur J Med Chem 2020 Aug 7;200:112319. Epub 2020 May 7.

Istituto di Genetica Molecolare "Luigi Luca Cavalli - Sforza", IGM-CNR, Via Abbiategrasso 207, I-27100, Pavia, Italy. Electronic address:

The huge resources that had gone into Human Immunodeficiency virus (HIV) research led to the development of potent antivirals able to suppress viral load in the majority of treated patients, thus dramatically increasing the life expectancy of people living with HIV. However, life-long treatments could result in the emergence of drug-resistant viruses that can progressively reduce the number of therapeutic options, facilitating the progression of the disease. In this scenario, we previously demonstrated that inhibitors of the human DDX3X helicase can represent an innovative approach for the simultaneous treatment of HIV and other viral infections such as Hepatitis c virus (HCV). We reported herein 6b, a novel DDX3X inhibitor that thanks to its distinct target of action is effective against HIV-1 strains resistant to currently approved drugs. Its improved in vitro ADME properties allowed us to perform preliminary in vivo studies in mice, which highlighted optimal biocompatibility and an improved bioavailability. These results represent a significant advancement in the development of DDX3X inhibitors as a novel class of broad spectrum and safe anti-HIV-1 drugs.
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http://dx.doi.org/10.1016/j.ejmech.2020.112319DOI Listing
August 2020

Exploring the Implication of DDX3X in DENV Infection: Discovery of the First-in-Class DDX3X Fluorescent Inhibitor.

ACS Med Chem Lett 2020 May 9;11(5):956-962. Epub 2020 Apr 9.

Dipartimento Farmaco Chimico Tecnologico, Università degli Studi di Siena, via Aldo Moro 2, 53100 Siena, Italy.

In the absence of effective drugs or vaccines for the treatment of the five Dengue Virus serotypes, the search for novel antiviral drugs is of primary importance for the scientific community. In this context, drug repurposing represents the most used strategy; however, the study of host targets is now attracting attention since it allows identification of broad-spectrum drugs endowed with high genetic barrier. In the last ten years our research group identified several small molecules DDX3X inhibitors and proved their efficacy against different viruses including novel emerging ones. Herein, starting from a screening of our compounds, we designed and synthesized novel derivatives with potent activity and high selectivity. Finally, we synthesized a fluorescent inhibitor that allowed us to study DDX3X cellular localization during DENV infection . Immunofluorescence analysis showed that our inhibitor colocalized with DDX3X, promoting the reduction of infected cells and recovering the number of viable cells.
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http://dx.doi.org/10.1021/acsmedchemlett.9b00681DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7236276PMC
May 2020

AuNP Pyrazolo[3,4-]pyrimidine Nanosystem in Combination with Radiotherapy against Glioblastoma.

ACS Med Chem Lett 2020 May 5;11(5):664-670. Epub 2020 Mar 5.

Dipartimento Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, Via A. Moro 2, 53100 Siena, Italy.

Gold-nanoparticle (AuNP)-conjugated drugs represent a promising and innovative antitumor therapeutic approach. In our study, we describe the design, the synthesis, the preparation, and the characterization of AuNPs conjugated with the pyrazolo[3,4-]pyrimidine derivative SI306, a c-Src inhibitor. AuNPs-SI306 showed a good loading efficacy (65%), optimal stability in polar media and in human plasma, and a suitable morphological profile: a ζ-potential of -43.9 mV, a nanoparticle diameter of 48.6 nm, and a 0.441 PDI value. The antitumoral activity of AuNPs-SI306 was evaluated in the glioblastoma model, by the low-density growth assay, and also in combination with radiotherapy (RT). Results demonstrated that AuNPs had a basal radiosensitization ability and that AuNPs-SI306, when used in combination with RT, were more effective in inhibiting tumor cell growth with respect to AuNPs and free SI306.
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http://dx.doi.org/10.1021/acsmedchemlett.9b00538DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7236040PMC
May 2020

In vitro characterization, ADME analysis, and histological and toxicological evaluation of BM1, a macrocyclic amidinourea active against azole-resistant Candida strains.

Int J Antimicrob Agents 2020 Mar 20;55(3):105865. Epub 2019 Dec 20.

Department of Biotechnology, Chemistry and Pharmacy, University of Siena, I-53100 Siena, Italy; Lead Discovery Siena s.r.l., Via Vittorio Alfieri 31, I-53019 Castelnuovo Berardenga, Italy; Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, BioLife Science Building, Philadelphia, PA 19122, USA.

Background: Candida species are one of the most common causes of nosocomial bloodstream infections among the opportunistic fungi. Extensive use of antifungal agents, most of which were launched on the market more than 20 years ago, led to the selection of drug-resistant or even multidrug-resistant fungi. We recently described a novel class of antifungal macrocyclic compounds with an amidinourea moiety that is highly active against azole-resistant Candida strains.

Objective: A compound from this family, BM1, was investigated in terms of in vitro activity against various Candida species, including C. auris isolates, interaction with the ABC transporter, CDR6, and in vivo distribution and safety.

Methods: In vitro assays (CYP inhibition, microsomal stability, permeability, spot assays) were used to collect chemical and biological data; animal models (rat) paired with LC-MS analysis were utilised to evaluate in vivo toxicology, pharmacokinetics, and distribution.

Results: The current research shows BM1 has a low in vivo toxicity profile, affinity for the renal system in rats, and good absorption, distribution, metabolism, and excretion (ADME). BM1 also has potent activity against azole-resistant fungal strains, including C. auris isolates and CDR6-overexpressing strains.

Conclusions: The results confirmed low minimum inhibitory concentrations (MICs) against several Candida species, including preliminary data vs. C. auris. BM1 has good ADME and biochemical characteristics, is suitable and safe for daily administration and is particularly indicated for renal infections. These data indicate BM1 and its derivatives form a novel, promising antifungal class.
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http://dx.doi.org/10.1016/j.ijantimicag.2019.105865DOI Listing
March 2020

Multitarget CFTR Modulators Endowed with Multiple Beneficial Side Effects for Cystic Fibrosis Patients: Toward a Simplified Therapeutic Approach †.

J Med Chem 2019 12 3;62(23):10833-10847. Epub 2019 Dec 3.

Dipartimento di Scienze degli Alimenti e del Farmaco , Università degli Studi di Parma , Viale delle Scienze, 27/A , 43124 Parma , Italy.

Cystic fibrosis (CF) is a multiorgan disease caused by mutations of the cystic fibrosis transmembrane conductance regulator (CFTR). In addition to respiratory impairment due to mucus accumulation, viruses and bacteria trigger acute pulmonary exacerbations, accelerating disease progression and mortality rate. Treatment complexity increases with patients' age, and simplifying the therapeutic regimen represents one of the key priorities in CF. We have recently reported the discovery of multitarget compounds able to "kill two birds with one stone" by targeting F508del-CFTR and PI4KIIIβ and thus acting simultaneously as CFTR correctors and broad-spectrum enterovirus (EV) inhibitors. Starting from these preliminary results, we report herein a hit-to-lead optimization and multidimensional structure-activity relationship (SAR) study that led to compound . This compound showed good antiviral and F508del-CFTR correction potency, additivity/synergy with lumacaftor, and a promising in vitro absorption, distribution, metabolism, and excretion (ADME) profile. It was well tolerated in vivo with no sign of acute toxicity and histological alterations in key biodistribution organs.
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http://dx.doi.org/10.1021/acs.jmedchem.9b01416DOI Listing
December 2019

A New Strategy for Glioblastoma Treatment: In Vitro and In Vivo Preclinical Characterization of Si306, a Pyrazolo[3,4-]Pyrimidine Dual Src/P-Glycoprotein Inhibitor.

Cancers (Basel) 2019 Jun 19;11(6). Epub 2019 Jun 19.

Department of Pharmacy, Università degli Studi di Genova, 16132 Genova, Italy.

Overexpression of P-glycoprotein (P-gp) and other ATP-binding cassette (ABC) transporters in multidrug resistant (MDR) cancer cells is responsible for the reduction of intracellular drug accumulation, thus decreasing the efficacy of chemotherapeutics. P-gp is also found at endothelial cells' membrane of the blood-brain barrier, where it limits drug delivery to central nervous system (CNS) tumors. We have previously developed a set of pyrazolo[3,4-]pyrimidines and their prodrugs as novel Src tyrosine kinase inhibitors (TKIs), showing a significant activity against CNS tumors in in vivo. Here we investigated the interaction of the most promising pair of drug/prodrug with P-gp at the cellular level. The tested compounds were found to increase the intracellular accumulation of Rho 123, and to enhance the efficacy of paclitaxel in P-gp overexpressing cells. Encouraging pharmacokinetics properties and tolerability in vivo were also observed. Our findings revealed a novel role of pyrazolo[3,4-]pyrimidines which may be useful for developing a new effective therapy in MDR cancer treatment, particularly against glioblastoma.
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http://dx.doi.org/10.3390/cancers11060848DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6628362PMC
June 2019

Assessing the Efficiency of Molecular Markers for the Species Identification of Gregarines Isolated from the Mealworm and Super Worm Midgut.

Microorganisms 2018 Nov 27;6(4). Epub 2018 Nov 27.

Department of Life Sciences, University of Siena, 53100 Siena, Italy.

Protozoa, of the taxon Gregarinasina, are a heterogeneous group of Apicomplexa that includes ~1600 species. They are parasites of a large variety of both marine and terrestrial invertebrates, mainly annelids, arthropods and mollusks. Unlike coccidians and heamosporidians, gregarines have not proven to have a negative effect on human welfare; thus, they have been poorly investigated. This study focuses on the molecular identification and phylogeny of the gregarine species found in the midgut of two insect species that are considered as an alternative source of animal proteins for the human diet: the mealworm , and the super-worm (Coleoptera: Tenebrionidae). Gregarine specimens were isolated from the gut of both larval and adult stages of specimens, as well as from larvae. The morphological analyses were restricted to the identification of the different parasite morpho-types, likely corresponding either to different life-cycle stages or to alternative gregarine species. The samples were also used for the DNA extraction necessary for their genetic characterization. Finally, the efficiency of different molecular markers (i.e., rDNA gene alone or combined with the Internal Transcribed Spacer 1) was assessed when applied either to gregarine species identification and to phylogenetic inference.
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http://dx.doi.org/10.3390/microorganisms6040119DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6313518PMC
November 2018

Plasmin-Binding Tripeptide-Decorated Liposomes Loading Pyrazolo[3,4-]pyrimidines for Targeting Hepatocellular Carcinoma.

ACS Med Chem Lett 2018 Jul 7;9(7):646-651. Epub 2018 May 7.

Dipartimento Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, Via A. Moro 2, I-53100 Siena, Italy.

Hepatocellular carcinoma (HCC) is one of the most fatal cancer types worldwide. HCC cells were proved to overexpress c-Src and Sgk1, a tyrosine and a serine-threonine kinase, respectively, whose role is crucial for the development and progression of the tumor. Pyrazolo[3,4-]pyrimidine derivatives are a class of tyrosine kinase inhibitors that have shown good activity against HepG2. HCC cells were also proved to overexpress plasmin, which is localized on the cell surface bound to its receptors. In this study, a tripeptide with sequence d-Ala-Phe-Lys, which binds a specific reactive site of plasmin, was synthesized and characterized. This tripeptide was used to decorate liposomes encapsulating three selected pyrazolo[3,4-]pyrimidines. Liposomes bearing tripeptide have been characterized, not showing remarkable differences with respect to the corresponding tripeptide-free liposomes. HepG2 cell uptake profiles and cytotoxicities showed that the presence of the tripeptide on the liposomal membrane surface improves the cell-penetrating ability of liposomes and increases the activity of two of the three tested compounds.
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http://dx.doi.org/10.1021/acsmedchemlett.8b00062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6047171PMC
July 2018

Rhodanine derivatives as potent anti-HIV and anti-HSV microbicides.

PLoS One 2018 5;13(6):e0198478. Epub 2018 Jun 5.

Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Siena, Italy.

Although highly active antiretroviral therapies (HAART) remarkably increased life expectancy of HIV positive people, the rate of novel HIV-1 infections worldwide still represent a major concern. In this context, pre-exposure prophylaxis (PrEP) approaches such as vaginal microbicide gels topically releasing antiretroviral drugs, showed to have a striking impact in limiting HIV-1 spread. Nevertheless, the co-presence of other genital infections, particularly those due to HSV-1 or 2, constitute a serious drawback that strongly limits the efficacy of PrEP approaches. For this reason, combinations of different compounds with mixed antiviral and antiretroviral activity are thoroughly investigated Here we report the synthesis and the biological evaluation of a novel series of rhodanine derivatives, which showed to inhibit both HIV-1 and HSV-1/2 replication at nanomolar concentration, and were found to be active also on acyclovir resistant HSV-2 strains. The compounds showed a considerable reduction of activity in presence of serum due to a high binding to serum albumin, as determined through in vitro ADME evaluations. However, the most promising compound of the series maintained a considerable activity in gel formulation, with an EC50 comparable to that obtained for the reference drug tenofovir. Moreover, the series of compounds showed pharmacokinetic properties suitable for topical formulation, thus suggesting that the novel rhodanine derivatives could represent effective agents to be used as dual anti HIV/HSV microbicides in PrEP approaches.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0198478PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5988308PMC
December 2018

Potent and Selective Carboxylic Acid Inhibitors of Tumor-Associated Carbonic Anhydrases IX and XII.

Molecules 2017 Dec 22;23(1). Epub 2017 Dec 22.

Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, via Aldo Moro 2, I-53100 Siena, Italy.

Selective inhibition of tumor-associated carbonic anhydrase (CA; EC 4.2.1.1) isoforms IX and XII is a crucial prerequisite to develop successful anticancer therapeutics. Herein, we confirmed the efficacy of the 3-nitrobenzoic acid substructure in the design of potent and selective carboxylic acid derivatives as CAs inhibitors. Compound emerged as the most potent inhibitor of the tumor-associated hCA IX and XII ( = 16 and 82.1 nM, respectively) with a significant selectivity with respect to the wide spread hCA II. Other 3-nitrobenzoic acid derivatives showed a peculiar CA inhibition profile with a notable potency towards hCA IX.
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http://dx.doi.org/10.3390/molecules23010017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5943959PMC
December 2017

Prodrugs of Pyrazolo[3,4-d]pyrimidines: From Library Synthesis to Evaluation as Potential Anticancer Agents in an Orthotopic Glioblastoma Model.

J Med Chem 2017 07 18;60(14):6305-6320. Epub 2017 Jul 18.

Consiglio Nazionale delle Ricerche, Istituto di Fisiologia Clinica and Istituto Toscano Tumori, Core Research Laboratory , Via Fiorentina 1, 53100 Siena, Italy.

Pyrazolo[3,4-d]pyrimidines are potent protein kinase inhibitors with promising antitumor activity but suboptimal aqueous solubility, consequently worth being further optimized. Herein, we present the one-pot two-step procedure for the synthesis of a set of pyrazolo[3,4-d]pyrimidine prodrugs (1a-8a and 9a-e) with higher aqueous solubility and enhanced pharmacokinetic and therapeutic properties. ADME studies demonstrated for the most promising prodrugs a better aqueous solubility, a favorable hydrolysis in human and murine serum, and an increased ability to cross cell membranes with respect to the parental drugs, explaining their better 24 h in vitro cytotoxicity against human glioblastoma U87 cell line. Finally, the 4-4a couple of drug/prodrug was also evaluated in vivo, revealing a profitable pharmacokinetic profile of the prodrug associated with a good efficacy. The application of the prodrug approach demonstrated to be a successful strategy for improving aqueous solubility of the parental drugs, determining a positive impact also in their biological efficacy.
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http://dx.doi.org/10.1021/acs.jmedchem.7b00637DOI Listing
July 2017

Pyrazolo[3,4-d]pyrimidines-loaded human serum albumin (HSA) nanoparticles: Preparation, characterization and cytotoxicity evaluation against neuroblastoma cell line.

Bioorg Med Chem Lett 2017 07 17;27(14):3196-3200. Epub 2017 May 17.

Dipartimento di Farmacia, Università degli Studi di Genova, Viale Benedetto XV, 3, 16132 Genova, Italy.

Pyrazolo[3,4-d]pyrimidine derivatives 1-5, active as c-Src inhibitors, have been selected to be formulated as drug-loaded human serum albumin (HSA) nanoparticles, with the aim of improving their solubility and pharmacokinetic properties. The present study includes the optimization of a desolvation method-based procedure for preparing HSA nanoparticles. First, characterization by HPLC-MS and Dynamic Light Scattering (DLS) showed a good entrapment efficacy, a controllable particle size (between 100 and 200nm) and an optimal stability over time, confirmed by an in vitro drug release assay. Then, 1-4 and the corresponding NPs were tested for their antiproliferative activity against neuroblastoma SH-SY5Y cell line. Notably, 3-NPs and 4-NPs were identified as the most promising formulation showing a profitable balance of stability, small size and a similar activity compared to the free drugs in cell-based assays. In addition, albumin formulations increase the solubility of pyrazolo[3,4-d]pyrimidine avoiding the use of DMSO as solubilizing agent.
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http://dx.doi.org/10.1016/j.bmcl.2017.05.015DOI Listing
July 2017

Chemical evaluation of the Rhynchophorus ferrugineus larvae fed on different substrates as human food source.

Food Sci Technol Int 2017 Sep 21;23(6):529-539. Epub 2017 Apr 21.

1 CREA-ABP, Consiglio per la Ricerca in agricoltura e l'analisi dell'economia agraria (CREA), Centro di Ricerca per l'Agrobiologia e Pedologia (ABP), Firenze, Italy.

We investigated the chemical composition of the weevil Rhynchophorus ferrugineus larvae, traditionally used as human food in Asia and known worldwide as one of the most significant pest for palm trees. Total fat content and fatty acid composition were analyzed using standard methodologies in (1) weevil larvae reared on apple fruit slices and wild specimens collected from attacked (2) Phoenix canariensis and (3) Syagrus romanzoffiana palm trees. Total fat content was extremely high in all the specimens (ranged between 57.62 and 60.03% based on dry weight). Despite sharing the same prevalent fatty acids (myristic acid, palmitic acid, stearic acid, palmitoleic acid, oleic acid, α-linoleic acid, and α-linolenic acid), fatty acid composition of the wild weevil larvae significantly differed from that of the specimens raised on apple fruit, due to the presence of other minor compounds. In general, a good balance between unsaturated fatty acids (∼53.68% of total fatty acids) and saturated fatty acids (∼43.41% of total fatty acids) and a low cholesterol content (74.61-152.32 mg/kg based on dry matter) were detected in all the specimens. Conversely, the weevil larvae did not represent a good source of α-tocopherol (14.17-26.22 mg/kg based on dry matter). The ability of the protein extracts obtained from the weevil larvae to inhibit in vitro the angiotensin-converting enzyme, the main enzyme involved in blood pressure regulation, was also investigated. To simulate gastrointestinal digestion process, protein extracts were hydrolyzed by the gastrointestinal enzymes. A significantly lower IC (0.588-0.623 mg/ml) was measured in all the protein extracts after enzymatic hydrolysis versus the corresponding crude protein extracts (3.270-3.752 mg/ml). Given that the weevil larvae are able to provide interesting benefits for human health, this study supports their use as human food not just in the native countries where they are traditionally consumed and farmed but also throughout the world.
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http://dx.doi.org/10.1177/1082013217705718DOI Listing
September 2017

Dietary fatty acids influence the growth and fatty acid composition of the yellow mealworm Tenebrio molitor (Coleoptera: Tenebrionidae).

Lipids 2017 03 12;52(3):285-294. Epub 2017 Jan 12.

Consiglio per la ricerca e la sperimentazione in agricoltura, Research Centre for Agrobiology and Pedology, Cascine del Riccio, Via di Lanciola 12/a, 50125, Florence, Italy.

Fat is the second most abundant component of the nutrient composition of the mealworm Tenebrio molitor (Coleoptera: Tenebrionidae) that represents also an interesting source of PUFA, especially n-6 and n-3 fatty acids, involved in prevention of cardiovascular diseases. This study investigated the possibility of modifying the fat content and the FA composition of yellow mealworms through feeding and how this would be influenced by developmental stages, pupal sex, and generation with the future aim of applying this coleopteran as a diet supplement for human health. Growth rate and cumulative mortality percentage on the different feeding substrates were also evaluated to select the optimal conditions for a mass-raising of this insect species. Despite the different fat content in the six different breeding substrates used, T. molitor larvae and pupae contained a constant fat percentage (>34% in larvae and >30% in pupae). A similar total fat content was found comparing larvae and male and female pupae of the second generation to those of the first generation. On the contrary, FA composition differed both in larvae and pupae reared on the different feeding substrates. However, the exemplars reared on the diets based on 100% bread and 100% oat flour showed SFA, PUFA percentages, and an n-6/n-3 ratio more suitable for human consumption; the diet based on beer yeast, wheat flour, and oat flour resulted in a contemporary diet that most satisfied the balance between a fat composition of high quality and favorable growth conditions.
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http://dx.doi.org/10.1007/s11745-016-4220-3DOI Listing
March 2017

Cuticle-degrading proteases and toxins as virulence markers of Beauveria bassiana (Balsamo) Vuillemin.

J Basic Microbiol 2016 Sep 20;56(9):941-8. Epub 2016 May 20.

Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, Siena, Italy.

Beauveria bassiana is one of the most known entomopathogenic fungal species and its entomopathogenic mechanism involves several bioactive metabolites, mainly cuticle-degrading enzymes and toxic molecules, which are predicted to play a key role as virulence factors. In this study six Beauveria bassiana strains (B 13/I03, B 13/I11, B 13/I49, B 13/I57, B 13/I63, and B 13/I64) were assayed against Tenebrio molitor larvae. Enzymatic activity of total proteases and specifically Pr 1 and Pr 2, as well as the production of toxic compounds were investigated in each fungal strain. Toxins were detected both in vitro-in medium filtrates and mycelia-and in vivo-in Tenebrio molitor larvae infected by the fungal strains tested. B 13/I11 and B 13/I63 strains showed the most significant entomopathogenic activity against Tenebrio molitor larvae (cumulative mortality rate 100 and 97%, respectively; average survival time 5.85 and 6.74 days, respectively). A widely variable and fungal strain-dependent enzymatic activity of total proteases, Pr 1 and Pr 2 was found. Beauvericin, beauvericin A and bassianolide resulted the most prevalent toxins detected in the substrates analyzed. It has been found that an increase of beauvericin content in vivo resulted significantly correlated to a decrease of Tenebrio molitor larvae average survival time in entomopathogenic bioassay (inverse correlation). The involvement of beauvericin in B. bassiana entomopathogenic process is confirmed; in vitro analysis of cuticle degrading proteases activity and toxins production in relation to the methods adopted resulted insufficient for a rapid screening to determine the virulence of B. bassiana strains against Tenebrio molitor larvae.
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http://dx.doi.org/10.1002/jobm.201600022DOI Listing
September 2016

Inhibition of para-Hydroxyphenylpyruvate Dioxygenase by Analogues of the Herbicide Nitisinone As a Strategy to Decrease Homogentisic Acid Levels, the Causative Agent of Alkaptonuria.

ChemMedChem 2016 Apr 7;11(7):674-8. Epub 2016 Mar 7.

Department of Biotechnology, Chemistry and Pharmacy, Università degli Studi di Siena, Via Aldo Moro 2, 53100, Siena, Italy.

Alkaptonuria (AKU) is a rare multisystem metabolic disease caused by deficient activity of homogentisate 1,2-dioxygenase (HGD), which leads to the accumulation of homogentisic acid (HGA). Currently, there is no treatment for AKU. The sole drug with some beneficial effects is the herbicide nitisinone (1), an inhibitor of p-hydroxyphenylpyruvate dioxygenase (4-HPPD). 1 has been used as a life-saving drug in infants with type I tyrosinemia despite severe side effects due to the buildup of tyrosine. Four clinical trials of nitisinone to treat AKU have shown that 1 consistently decreases HGA levels, but also caused the accumulation of tyrosine in blood serum. Moreover, the human preclinical toxicological data for 1 are incomplete. In this work, we performed pharmacodynamics and toxicological evaluations of 1, providing the first report of LD50 values in human cells. Intracellular tyrosinemia was also evaluated. Three additional 4-HPPD inhibitors with a more favorable profile than that of 1 in terms of IC50, LD50, and tyrosine accumulation were also identified among commercially available compounds. These may be promising starting points for the development of new therapeutic strategies for the treatment of AKU.
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http://dx.doi.org/10.1002/cmdc.201500578DOI Listing
April 2016

Development and in Vitro Evaluation of a Microbicide Gel Formulation for a Novel Non-Nucleoside Reverse Transcriptase Inhibitor Belonging to the N-Dihydroalkyloxybenzyloxopyrimidines (N-DABOs) Family.

J Med Chem 2016 Mar 8;59(6):2747-59. Epub 2016 Mar 8.

Dipartimento Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena , Via A. De Gasperi 2, I-53100 Siena, Italy.

Preventing HIV transmission by the use of a vaginal microbicide is a topic of considerable interest in the fight against AIDS. Both a potent anti-HIV agent and an efficient formulation are required to develop a successful microbicide. In this regard, molecules able to inhibit the HIV replication before the integration of the viral DNA into the genetic material of the host cells, such as entry inhibitors or reverse transcriptase inhibitors (RTIs), are ideal candidates for prevention purpose. Among RTIs, S- and N-dihydroalkyloxybenzyloxopyrimidines (S-DABOs and N-DABOs) are interesting compounds active at nanomolar concentration against wild type of RT and with a very interesting activity against RT mutations. Herein, novel N-DABOs were synthesized and tested as anti-HIV agents. Furthermore, their mode of binding was studied by molecular modeling. At the same time, a vaginal microbicide gel formulation was developed and tested for one of the most promising candidates.
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http://dx.doi.org/10.1021/acs.jmedchem.5b01979DOI Listing
March 2016

Improvement of pyrazolo[3,4-d]pyrimidines pharmacokinetic properties: nanosystem approaches for drug delivery.

Sci Rep 2016 Feb 22;6:21509. Epub 2016 Feb 22.

Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, via Aldo Moro 2, 53100, Siena, Italy.

Pyrazolo[3,4-d]pyrimidines are a class of compounds with a good activity against several cancer cell lines. Despite the promising anticancer activity, these molecules showed a poor aqueous solubility. This issue could threat the future development of pyrazolo[3,4-d]pyrimidines as clinical drug candidates. With the aim of improving their solubility profile and consequently their pharmacokinetic properties, we have chosen four compounds (1-4) on the base of their anti-neuroblastoma activity and we have developed albumin nanoparticles and liposomes for the selected candidates. Albumin nanoparticles and liposomes were prepared and characterized regarding size and ζ-potential distribution, polidispersity index, entrapment efficiency and activity against SH-SY5Y human neuroblastoma cell line. The most promising nanosystem, namely LP-2, was chosen to perform further studies: confocal microscopy, stability and drug release in physiological conditions, and biodistribution. Altogether, the obtained data strongly indicate that the encapsulation of pyrazolo[3,4-d]pyrimidines in liposomes represent an effective method to overcome the poor water solubility.
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http://dx.doi.org/10.1038/srep21509DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4761914PMC
February 2016

Phase I trial of thymidylate synthase poly-epitope peptide (TSPP) vaccine in advanced cancer patients.

Cancer Immunol Immunother 2015 Sep 2;64(9):1159-73. Epub 2015 Jun 2.

Department of Medical Biotechnologies, Siena University, Siena, Italy.

Thymidylate synthase (TS) poly-epitope peptide (TSPP) is a 27-mer peptide vaccine containing the amino acidic sequences of three epitopes with HLA-A2.1-binding motifs of TS, an enzyme overexpressed in cancer cells, which plays a crucial role in DNA repair and replication. Based on the results of preclinical studies, we designed a phase Ib trial (TSPP/VAC1) to investigate, in a dose escalation setting, the safety and the biological activity of TSPP vaccination alone (arm A) or in combination with GM-CSF and IL-2 (arm B) in cancer patients. Twenty-one pretreated metastatic cancer patients, with a good performance status (ECOG ≤ 1) and no severe organ failure or immunological disease, were enrolled in the study (12 in arm A, nine in arm B) between April 2011 and January 2012, with a median follow-up of 28 months. TSPP resulted safe, and its maximal tolerated dose was not achieved. No grade 4 toxicity was observed. The most common adverse events were grade 2 dermatological reactions to the vaccine injection, cough, rhinitis, fever, poly-arthralgia, gastro-enteric symptoms and, to a lesser extent, moderate hypertension and hypothyroidism. We detected a significant rise in auto-antibodies and TS-epitope-specific CTL precursors. Furthermore, TSPP showed antitumor activity in this group of pretreated patients; indeed, we recorded one partial response and seven disease stabilizations (SD) in arm A, and three SD in arm B. Taken together, our findings provide the framework for the evaluation of the TSPP anti-tumor activity in further disease-oriented clinical trials.
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http://dx.doi.org/10.1007/s00262-015-1711-7DOI Listing
September 2015

Studies on the ATP Binding Site of Fyn Kinase for the Identification of New Inhibitors and Their Evaluation as Potential Agents against Tauopathies and Tumors.

J Med Chem 2015 Jun 20;58(11):4590-609. Epub 2015 May 20.

†Dipartimento Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, Via A. De Gasperi 2, I-53100 Siena, Italy.

Fyn is a member of the Src-family of nonreceptor protein-tyrosine kinases. Its abnormal activity has been shown to be related to various human cancers as well as to severe pathologies, such as Alzheimer's and Parkinson's diseases. Herein, a structure-based drug design protocol was employed aimed at identifying novel Fyn inhibitors. Two hits from commercial sources (1, 2) were found active against Fyn with K(i) of about 2 μM, while derivative 4a, derived from our internal library, showed a K(i) of 0.9 μM. A hit-to-lead optimization effort was then initiated on derivative 4a to improve its potency. Slightly modifications rapidly determine an increase in the binding affinity, with the best inhibitors 4c and 4d having K(i)s of 70 and 95 nM, respectively. Both compounds were found able to inhibit the phosphorylation of the protein Tau in an Alzheimer's model cell line and showed antiproliferative activities against different cancer cell lines.
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http://dx.doi.org/10.1021/acs.jmedchem.5b00140DOI Listing
June 2015

SRC family kinase inhibition through a new pyrazolo[3,4-d]pyrimidine derivative as a feasible approach for glioblastoma treatment.

J Cell Biochem 2015 May;116(5):856-63

Department of Medicine, Surgery and Neuroscience, University of Siena and Istituto Toscano Tumori (ITT), Siena, Italy.

Glioblastoma (GB) is the most common and aggressive primary tumor of the central nervous system. The current standard of care for GB consists of surgical resection, followed by radiotherapy combined with temozolomide chemotherapy. However, despite this intensive treatment, the prognosis remains extremely poor. Therefore, more effective therapies are urgently required. Recent studies indicate that SRC family kinases (SFKs) could represent promising molecular targets for GB therapy. Here, we challenged four GB cell lines with a new selective pyrazolo[3,4-d]pyrimidine derivative SFK inhibitor, called SI221. This compound exerted a significant cytotoxic effect on GB cells, without significantly affecting non-tumor cells (primary human skin fibroblasts), as evaluated by MTS assay. We also observed that SI221 was more effective than the well-known SFK inhibitor PP2 in GB cells. Notably, despite the high intrinsic resistance to apoptosis of GB cells, SI221 was able to induce this cell death process in all the GB cell lines, as observed through cytofluorimetric analysis and caspase-3 assay. SI221 also exerted a long-term inhibition of GB cell growth and was able to reduce GB cell migration, as shown by clonogenic assay and scratch test, respectively. Moreover, through in vitro pharmacokinetic assays, SI221 proved to have a high metabolic stability and a good potential to cross the blood brain barrier, which is an essential requirement for a drug intended to treat brain tumors. Therefore, despite the need of developing strategies to improve SI221 solubility, our results suggest a potential application of this selective SFK inhibitor in GB therapy.
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http://dx.doi.org/10.1002/jcb.25042DOI Listing
May 2015

Combining X-ray crystallography and molecular modeling toward the optimization of pyrazolo[3,4-d]pyrimidines as potent c-Src inhibitors active in vivo against neuroblastoma.

J Med Chem 2015 Jan 3;58(1):347-61. Epub 2014 Dec 3.

Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena Via Aldo Moro 2, 53100 Siena, Italy.

c-Src is a tyrosine kinase belonging to the Src-family kinases. It is overexpressed and/or hyperactivated in a variety of cancer cells, thus its inhibition has been predicted to have therapeutic effects in solid tumors. Recently, the pyrazolo[3,4-d]pyrimidine 3 was reported as a dual c-Src/Abl inhibitor. Herein we describe a multidisciplinary drug discovery approach for the optimization of the lead 3 against c-Src. Starting from the X-ray crystal structure of c-Src in complex with 3, Monte Carlo free energy perturbation calculations were applied to guide the design of c-Src inhibitors with improved activities. As a result, the introduction of a meta hydroxyl group on the C4 anilino ring was computed to be particularly favorable. The potency of the synthesized inhibitors was increased with respect to the starting lead 3. The best identified compounds were also found active in the inhibition of neuroblastoma cell proliferation. Furthermore, compound 29 also showed in vivo activity in xenograft model using SH-SY5Y cells.
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http://dx.doi.org/10.1021/jm5013159DOI Listing
January 2015

Synthesis of linear and cyclic guazatine derivatives endowed with antibacterial activity.

Bioorg Med Chem Lett 2014 Dec 14;24(23):5525-9. Epub 2014 Oct 14.

Department of Biotechnology, Chemistry and Pharmacy, University of Siena, I-53100 Siena, Italy; Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, BioLife Science Building, Suite 333, 1900 N 12th Street, Philadelphia, PA 19122, USA; Lead Discovery Siena s.r.l, Via Vittorio Alfieri 31, I-53019 Castelnuovo Berardenga, Italy. Electronic address:

Antibiotic resistance has reached alarming levels in many clinically-relevant human pathogens, and there is an increasing clinical need for new antibiotics active on drug-resistant Gram-negative pathogens who rapidly evolve towards pandrug resistance phenotypes. Here, we report on two related classes of guanidinic compounds endowed with antibacterial activity. The two best compounds (9a and 13d) exhibited the most potent antibacterial activity with MIC values ranging 0.12-8 μg/ml with most tested pathogens, including both Gram-positive and Gram-negative bacteria. Interestingly, MIC values were not affected (1-8 μg/ml) when measured using recent clinical isolates with various antibiotic resistance determinants. The results reported herein identify guazatine derivatives as an interesting starting point for the optimization of a potentially novel class of antibacterial agents.
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http://dx.doi.org/10.1016/j.bmcl.2014.09.081DOI Listing
December 2014

GC-EI-MS analysis of fatty acid composition in brain and serum of twitcher mouse.

Lipids 2014 Nov 11;49(11):1115-25. Epub 2014 Sep 11.

Department of Biotechnology, Chemistry and Pharmacy, University of Siena, Viale Bracci, 53100, Siena, Italy.

Globoid cell leukodystrophy or Krabbe disease is an inherited autosomal recessive disorder caused by mutations in the galactosylceramidase gene. The objective of the study was to present information about the fatty acid (FA) composition of the brain and serum of twitcher mice, a mouse model of Krabbe disease, compared to wild type, in order to identify biomarker of disease progression. We defined the FA profiles by identifying the main components present in serum and brain using GC-EI-MS analysis. The FA percentage composition was measured and data were analyzed considering the disease and the mouse age as experimental factors. Significant correlations were established, both in brain and in serum, in the fatty acid percentage composition of twitcher compared to wild type mice. The most abundant saturated fatty acid in brain was the palmitic acid (C16:0) with mean values significantly increased in twitcher mouse (p = 0.0142); moreover, three monounsaturated, three polyunsaturated (PUFA) and a plasmalogen were significantly correlated to disease. In the serum highly significant differences were observed between the two groups for three polyunsaturated fatty acids. In fact, the docosahexaenoic acid (C22:6n3c) content was significantly increased (p = 0.0116), while the C20 PUFA (C20:3n6c and C20:5n3c) were significantly decreased in twitcher serum samples. Our study shows a specific FA profile that may help to define a possible pattern that could distinguish between twitcher and wild type; these data are likely to provide insight in the identification of new biomarkers to monitor the disease progression and thereby permit the critical analysis of therapeutic approaches.
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http://dx.doi.org/10.1007/s11745-014-3945-0DOI Listing
November 2014

Pyrazolo[3,4-d]pyrimidine Prodrugs: Strategic Optimization of the Aqueous Solubility of Dual Src/Abl Inhibitors.

ACS Med Chem Lett 2013 Jul 20;4(7):622-6. Epub 2013 May 20.

Dipartimentodi Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena , Via Aldo Moro 2, 53100 Siena, Italy ; Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University , BioLife Science Building, Suite 333, 1900 North 12th Street, Philadelphia, Pennsylvania 19122, United States.

Design and synthesis of prodrugs of promising drug candidates represents a valid strategy to overcome the lack of favorable ADME properties, in particular aqueous solubility and bioavailability. We report herein the successful application of this strategy with two representative pyrazolo[3,4-d]pyrimidine derivatives (1 and 2), which led to the development of the corresponding and highly water-soluble antitumor prodrugs (7 and 8). In vitro studies confirmed a significant improvement of aqueous solubility and, for compound 8, good plasma stability, suggesting superior in vivo bioavailability. As expected, the uncleaved water-soluble prodrugs 7 and 8 showed no activity toward the enzymatic targets (c-Src and c-Abl) but revealed promising antiproliferative activity in myeloid cell lines, as a consequence of the in vitro hydrolysis of the selected solubilizing moiety, followed by the release of the active compounds (1 and 2).
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http://dx.doi.org/10.1021/ml4000782DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4027536PMC
July 2013
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