Publications by authors named "Claudio Zamperini"

37 Publications

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

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

Synthesis and Antiproliferative Activity of Nitric Oxide-Donor Largazole Prodrugs.

ACS Med Chem Lett 2020 May 7;11(5):846-851. Epub 2020 Feb 7.

Promidis, Via Olgettina 60, 20132 Milano, Italy.

The marine natural product Largazole is the most potent Class I HDAC inhibitor identified to date. Since its discovery, many research groups have been attracted by the structural complexity and the peculiar anticancer activity, due to its capability to discriminate between tumor cells and normal cells. Herein, we discuss the synthesis and the biological profile of hybrid analogues of Largazole, as dual HDAC inhibitor and nitric oxide (NO) donors, potentially useful as anticancer agents. In particular, the metabolic stability of the modified thioester moiety of Largazole, bearing the NO-donor function/s, the release of NO, and the antiproliferative activity in tumor cell lines are presented.
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http://dx.doi.org/10.1021/acsmedchemlett.9b00643DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7236235PMC
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

Synthesis and Antiviral Activity of Novel 1,3,4-Thiadiazole Inhibitors of DDX3X.

Molecules 2019 Nov 4;24(21). Epub 2019 Nov 4.

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

The human ATPase/RNA helicase X-linked DEAD-box polypeptide 3 (DDX3X) emerged as a novel therapeutic target in the fight against both infectious diseases and cancer. Herein, a new family of DDX3X inhibitors was designed, synthesized, and tested for its inhibitory action on the ATPase activity of the enzyme. The potential use of the most promising derivatives it has been investigated by evaluating their anti-HIV-1 effects, revealing inhibitory activities in the low micromolar range. A preliminary ADME analysis demonstrated high metabolic stability and good aqueous solubility. The promising biological profile, together with the suitable in vitro pharmacokinetic properties, make these novel compounds a very good starting point for further development.
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http://dx.doi.org/10.3390/molecules24213988DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6864647PMC
November 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

DDX3X Helicase Inhibitors as a New Strategy To Fight the West Nile Virus Infection.

J Med Chem 2019 03 21;62(5):2333-2347. Epub 2019 Feb 21.

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

Increased frequency of arbovirus outbreaks in the last 10 years represents an important emergence for global health. Climate warming, extensive urbanization of tropical regions, and human migration flows facilitate the expansion of anthropophilic mosquitos and the emerging or re-emerging of new viral infections. Only recently the human adenosinetriphosphatase/RNA helicase X-linked DEAD-box polypeptide 3 (DDX3X) emerged as a novel therapeutic target in the fight against infectious diseases. Herein, starting from our previous studies, a new family of DDX3X inhibitors was designed, synthesized, validated on the target enzyme, and evaluated against the West Nile virus (WNV) infection. Time of addition experiments after virus infection indicated that the compounds exerted their antiviral activities after the entry process, likely at the protein translation step of WNV replication. Finally, the most interesting compounds were then analyzed for their in vitro pharmacokinetic parameters, revealing favorable absorption, distribution, metabolism, and excretion values. The good safety profile together with a good activity against WNV for which no treatments are currently available, make this new class of molecules a good starting point for further in vivo studies.
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http://dx.doi.org/10.1021/acs.jmedchem.8b01403DOI Listing
March 2019

Alkyl-guanidine Compounds as Potent Broad-Spectrum Antibacterial Agents: Chemical Library Extension and Biological Characterization.

J Med Chem 2018 10 11;61(20):9162-9176. Epub 2018 Oct 11.

Department of Biotechnology, Chemistry, and Pharmacy , University of Siena , I-53100 Siena , Italy.

Nowadays, the increasing of multidrug-resistant pathogenic bacteria represents a serious threat to public health, and the lack of new antibiotics is becoming a global emergency. Therefore, research in antibacterial fields is urgently needed to expand the currently available arsenal of drugs. We have recently reported an alkyl-guanidine derivative (2), characterized by a symmetrical dimeric structure, as a good candidate for further developments, with a high antibacterial activity against both Gram-positive and Gram-negative strains. In this study, starting from its chemical scaffold, we synthesized a small library of analogues. Moreover, biological and in vitro pharmacokinetic characterizations were conducted on some selected derivatives, revealing notable properties: broad-spectrum profile, activity against resistant clinical isolates, and appreciable aqueous solubility. Interestingly, 2 seems neither to select for resistant strains nor to macroscopically alter the membranes, but further studies are required to determine the mode of action.
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http://dx.doi.org/10.1021/acs.jmedchem.8b00619DOI Listing
October 2018

Efficient optimization of pyrazolo[3,4-d]pyrimidines derivatives as c-Src kinase inhibitors in neuroblastoma treatment.

Bioorg Med Chem Lett 2018 11 20;28(21):3454-3457. Epub 2018 Sep 20.

Dipartimento 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, PA 19122, United States; Lead Discovery Siena S.r.l., Via Vittorio Alfieri 31, 53019 Castelnuovo Berardenga, Siena, Italy. Electronic address:

The proto-oncogene c-Src is a non-receptor tyrosine kinase which is involved in the regulation of many cellular processes, such as differentiation, adhesion and survival. c-Src hyperactivation has been detected in many tumors, including neuroblastoma (NB), one of the major causes of death from neoplasia in infancy. We already reported a large family of pyrazolo[3,4-d]pyrimidines active as c-Src inhibitors. Interestingly, some of these derivatives resulted also active on SH-SY5Y NB cell line. Herein, starting from our previous Free Energy Perturbation/Monte Carlo calculations, we report an optimization study which led to the identification of a new series of derivatives endowed with nanomolar K values against c-Src, interesting antiproliferative activity on SH-SY5Y cells and a suitable ADME profile.
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http://dx.doi.org/10.1016/j.bmcl.2018.09.024DOI Listing
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

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

One drug for two targets: Biological evaluation of antiretroviral agents endowed with antiproliferative activity.

Bioorg Med Chem Lett 2017 06 1;27(11):2502-2505. Epub 2017 Apr 1.

Departamento de Química Inorgánica y Orgánica, Universidad Jaume I, E-12071 Castellón, Spain.

AIDS-related cancer diseases are malignancies with low incidence on healthy people that affect mostly subjects already immunocompromised. The connection between HIV/AIDS and these cancers has not been established yet, but a weakened immune system is certainly the main cause. We envisaged the possibility to screen a small library of compounds synthesized in our laboratory against opportunistic tumors mainly due to HIV infection like Burkitt's Lymphoma. From cellular assays and gene expression analysis we identified two promising compounds. These derivatives have the dual action required inhibiting HIV replication in human TZM-bl cells infected with HIV-1 NL4.3 and showing cytotoxic activity on human colon HT-29 and breast adenocarcinoma MCF-7 cells. In addition, preclinical in vitro adsorption, distribution, metabolism, and excretion studies highlighted a satisfactory pharmacokinetic profile.
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http://dx.doi.org/10.1016/j.bmcl.2017.03.097DOI Listing
June 2017

Discovery of in vitro antitubercular agents through in silico ligand-based approaches.

Eur J Med Chem 2016 Oct 20;121:169-180. Epub 2016 May 20.

Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, Via Aldo Moro 2, 53019 Siena, Italy; Sbarro Institute for Cancer Research & Molecular Medicine, Center for Biotechnology, College of Science & Technology, Temple University, BioLife Science Building, Suite 333, 1900 N 12th Street, Philadelphia, PA 19122, USA. Electronic address:

The development of new anti-tubercular agents represents a constant challenge mostly due to the insurgency of resistance to the currently available drugs. In this study, a set of 60 molecules were selected by screening the Asinex and the ZINC collections and an in house library by means of in silico ligand-based approaches. Biological assays in Mycobacterium tuberculosis H37Ra ATCC 25177 strain highlighted (±)-1-(4-chlorophenyl)-2-(1H-imidazol-1-yl)ethyl-4-(3,4-dichlorophenyl)piperazine-1-carboxylate (5i) and 3-(4-chlorophenyl)-5-(2,4-dimethylpyrimidin-5-yl)-2-methylpyrazolo[1.5-a]pyrimidin-7(4H)-one (42) as the most potent compounds, having a Minimum Inhibitory Concentration (MIC) of 4 and 2 μg/mL respectively. These molecules represent a good starting point for further optimization of effective anti-TB agents.
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http://dx.doi.org/10.1016/j.ejmech.2016.05.032DOI Listing
October 2016

Suppression of SRC Signaling Is Effective in Reducing Synergy between Glioblastoma and Stromal Cells.

Mol Cancer Ther 2016 07 11;15(7):1535-44. Epub 2016 May 11.

Dipartimento di Scienze Cliniche Applicate e Biotecnologiche, Università dell'Aquila, L'Aquila, Italy.

Glioblastoma cells efficiently interact with and infiltrate the surrounding normal tissue, rendering surgical resection and adjuvant chemo/radiotherapy ineffective. New therapeutic targets, able to interfere with glioblastoma's capacity to synergize with normal brain tissue, are currently under investigation. The compound Si306, a pyrazolo[3,4-d]pyrimidine derivative, selected for its favorable activity against SRC, was tested in vitro and in vivo on glioblastoma cell lines. In vivo, combination treatment with Si306 and radiotherapy was strongly active in reducing U-87 xenograft growth with respect to control and single treatments. The histology revealed a significant difference in the stromal compartment of tumoral tissue derived from control or radiotherapy-treated samples with respect to Si306-treated samples, showing in the latter a reduced presence of collagen and α-SMA-positive cells. This effect was paralleled in vitro by the capacity of Si306 to interfere with myofibroblastic differentiation of normal fibroblasts induced by U-87 cells. In the presence of Si306, TGF-β released by U-87 cells, mainly in hypoxia, was ineffective in upregulating α-SMA and β-PDGFR in fibroblasts. Si306 efficiently reached the brain and significantly prolonged the survival of mice orthotopically injected with U-87 cells. Drugs that target SRC could represent an effective therapeutic strategy in glioblastoma, able to block positive paracrine loop with stromal cells based on the β-PDGFR axis and the formation of a tumor-promoting microenvironment. This approach could be important in combination with conventional treatments in the effort to reduce tumor resistance to therapy. Mol Cancer Ther; 15(7); 1535-44. ©2016 AACR.
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http://dx.doi.org/10.1158/1535-7163.MCT-15-1011DOI Listing
July 2016

Human DDX3 protein is a valuable target to develop broad spectrum antiviral agents.

Proc Natl Acad Sci U S A 2016 May 26;113(19):5388-93. Epub 2016 Apr 26.

Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena, I-53100 Siena, Italy; Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, Philadelphia, PA 19122; Lead Discovery Siena S.r.l., Castelnuovo Berardenga, 53019 Siena, Italy

Targeting a host factor essential for the replication of different viruses but not for the cells offers a higher genetic barrier to the development of resistance, may simplify therapy regimens for coinfections, and facilitates management of emerging viral diseases. DEAD-box polypeptide 3 (DDX3) is a human host factor required for the replication of several DNA and RNA viruses, including some of the most challenging human pathogens currently circulating, such as HIV-1, Hepatitis C virus, Dengue virus, and West Nile virus. Herein, we showed for the first time, to our knowledge, that the inhibition of DDX3 by a small molecule could be successfully exploited for the development of a broad spectrum antiviral agent. In addition to the multiple antiviral activities, hit compound 16d retained full activity against drug-resistant HIV-1 strains in the absence of cellular toxicity. Pharmacokinetics and toxicity studies in rats confirmed a good safety profile and bioavailability of 16d. Thus, DDX3 is here validated as a valuable therapeutic target.
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http://dx.doi.org/10.1073/pnas.1522987113DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4868442PMC
May 2016

In vitro screening of 2-(1H-imidazol-1-yl)-1-phenylethanol derivatives as antiprotozoal agents and docking studies on Trypanosoma cruzi CYP51.

Eur J Med Chem 2016 May 15;113:28-33. Epub 2016 Feb 15.

Department of "Chimica e Tecnologie del Farmaco", Sapienza University of Rome, Piazzale Aldo Moro, 5, 00185 Rome, Italy.

Sterol 14α-demethylase (CYP51) is a key enzyme involved in the survival and virulence of many parasite protozoa, such as Trypanosoma and Leishmania species, thus representing a valuable drug target for the treatment of Kinetoplastid diseases. A set of azole-based compounds selected from an in-house compound library was in vitro screened against different human protozoan parasites. Several compounds showed selective activity against Trypanosoma cruzi, with compound 7 being the most active (IC50 = 40 nM). Given the structural similarity between the compounds here reported and known CYP51 inhibitors, a molecular docking study was performed to assess their binding with protozoal target and to rationalize the biological activity data.
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http://dx.doi.org/10.1016/j.ejmech.2016.02.028DOI Listing
May 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

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

New 1-phenyl-5-(1H-pyrrol-1-yl)-1H-pyrazole-3-carboxamides inhibit hepatitis C virus replication via suppression of cyclooxygenase-2.

Eur J Med Chem 2015 Jan 27;90:497-506. Epub 2014 Nov 27.

Istituto Pasteur - Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma, Piazzale Aldo Moro 5, I-00185 Roma, Italy. Electronic address:

We report here the synthesis and mechanism of inhibition of pyrazolecarboxamide derivatives as a new class of HCV inhibitors. Compounds 6, 7, 8 and 16 inhibited the subgenomic HCV replicon 1b genotype at EC50 values between 5 and 8 μM and displayed an even higher potency against the infectious Jc1 HCV 2a genotype. Compound 6 exhibited an EC50 of 6.7 μM and selectivity index of 23 against HCV 1b, and reduced the RNA copies of the infectious Jc1 chimeric 2a clone by 82% at 7 μM. Evaluation of the mode of anti-HCV activity of 6 revealed that it suppressed HCV-induced COX-2 mRNA and protein expression, displaying an IC50 of 3.2 μM in COX-2 promoter-linked luciferase reporter assay. Conversely, the anti-HCV activity of 6 was abrogated upon over-expression of COX-2. These findings suggest that 6 as a representative of these pyrazolecarboxamides function as anti-HCV agents via targeting COX-2 at both the transcription and translation levels.
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http://dx.doi.org/10.1016/j.ejmech.2014.11.042DOI Listing
January 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

Indolylarylsulfones carrying a heterocyclic tail as very potent and broad spectrum HIV-1 non-nucleoside reverse transcriptase inhibitors.

J Med Chem 2014 Dec 24;57(23):9945-57. Epub 2014 Nov 24.

Istituto Pasteur - Fondazione Cenci Bolognetti, Dipartimento di Chimica e Tecnologie del Farmaco, Sapienza Università di Roma , Piazzale Aldo Moro 5, I-00185 Roma, Italy.

We synthesized new indolylarylsulfone (IAS) derivatives carrying a heterocyclic tail at the indole-2-carboxamide nitrogen as potential anti-HIV/AIDS agents. Several new IASs yielded EC50 values <1.0 nM against HIV-1 WT and mutant strains in MT-4 cells. The (R)-11 enantiomer proved to be exceptionally potent against the whole viral panel; in the reverse transcriptase (RT) screening assay, it was remarkably superior to NVP and EFV and comparable to ETV. The binding poses were consistent with the one previously described for the IAS non-nucleoside reverse transcriptase inhibitors. Docking studies showed that the methyl group of (R)-11 points toward the cleft created by the K103N mutation, different from the corresponding group of (S)-11. By calculating the solvent-accessible surface, we observed that the exposed area of RT in complex with (S)-11 was larger than the area of the (R)-11 complex. Compounds 6 and 16 and enantiomer (R)-11 represent novel robust lead compounds of the IAS class.
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http://dx.doi.org/10.1021/jm5011622DOI Listing
December 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

New Promising Compounds with in Vitro Nanomolar Activity against Trypanosoma cruzi.

ACS Med Chem Lett 2013 Jun 7;4(6):538-41. Epub 2013 May 7.

Dipartimento di Chimica e Tecnologie del Farmaco, "Sapienza" Università di Roma , P.le A. Moro 5, 00185 Rome, Italy.

The antiparasitic activity of azole and new 4-aminopyridine derivatives has been investigated. The imidazoles 1 and 3-5 showed a potent in vitro antichagasic activity with IC50 values in the low nanomolar concentration range. The (S)-1, (S)-3, and (S)-5 enantiomers showed (up to) a thousand-fold higher activity than the reference drug benznidazole and furthermore low cytotoxicity on rat myogenic L6 cells.
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http://dx.doi.org/10.1021/ml400039rDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4027138PMC
June 2013

CYP-dependent metabolism of antitumor pyrazolo[3,4-d]pyrimidine derivatives is characterized by an oxidative dechlorination reaction.

Drug Metab Pharmacokinet 2014 20;29(6):433-40. Epub 2014 May 20.

Dipartimento di Biotecnologie, Chimica e Farmacia, Università di Siena.

The aim of this study is to investigate the metabolic (cytochrome P450-dependent) behaviour of pyrazolo[3,4-d]pyrimidines 1-10 dual Abl/Src kinase inhibitors. All the compounds demonstrate good metabolic stability both in human liver (HLM) and in rat liver (RLM) microsomes. Moreover, all the tested molecules undergo the same metabolic CYP-dependent reactions, namely oxidative dechlorination and N-dealkylation. These metabolic pathways are fully characterized for compound 1. In HLM, the dehalogenated metabolite accounts for about 87% of the full 1 metabolism, while the N-dealkylated metabolite accounts for 12%. Inhibition studies performed using different CYP-inhibitors indicate that the 3A family is the isoenzyme family most involved in pyrazolo[3,4-d]pyrimidine metabolism. This observation is confirmed by studies performed by using CYP3A selective substrates. Furthermore kinetic analysis performed in RLM, HLM and cDNA CYP3A4 shows that the affinity of CYPs towards compound 1 is similar in all the tested preparations (Km = 32.7, 21.8, and 48.7 µM, respectively).
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http://dx.doi.org/10.2133/dmpk.DMPK-13-RG-094DOI Listing
October 2015
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