A novel bioresponsive self-immolative spacer based on aza-quinone methide reactivity for the controlled release of thiols, phenols, amines, sulfonamides or amides.

A stimuli-sensitive linker is one of the indispensable components of prodrugs for cancer therapy as it covalently binds the drug and releases it upon external stimulation at the tumour site. Quinone methide elimination has been widely used as the key transformation to release drugs based on their nucleofugacity. The usual approach is to bind the drug to the linker as a carbamate and release it as a free amine after a self-immolative 1,6-elimination. Although this approach is very efficient, it is limited to amines (as carbamates), alcohols or phenols (as carbonates) or other acidic functional groups. We report here a self-immolative spacer capable of directly linking and releasing amines, phenols, thiols, sulfonamides and carboxyamides after a reductive stimulus. The spacer is based on the structure of (5-nitro-2-pyrrolyl)methanol (NPYM-OH), which was used for the direct alkylation of the functional groups mentioned above. The spacer is metabolically stable and has three indispensable sites for bioconjugation: the bioresponsive trigger, the conjugated 1,6 self-immolative system and a third arm suitable for conjugation with a carrier or other modifiers. Release was achieved by selective reduction of the nitro group over Fe/Pd nanoparticles (NPs) in a micellar aqueous environment (H2O/TPGS-750-M), or by NADH mediated nitroreductase activation. A DFT study demonstrates that, during the 1,6 elimination, the transition state formed from 5-aminopyrrole has a lower activation energy compared to other 5-membered heterocycles or p-aminobenzyl derivatives. The NPYM scaffold was validated by late-stage functionalisation of approved drugs such as celecoxib, colchicine, vorinostat or ciprofloxacin. A hypoxia-activated NPYM-based prodrug (HAP) derived from HDAC inhibitor ST7612AA1 was also produced, which was active in cancer cells under hypoxic conditions.

Serendipitous Identification of Azine Anticancer Agents Using a Privileged Scaffold Morphing Strategy.

The use of privileged scaffolds as a starting point for the construction of libraries of bioactive compounds is a widely used strategy in drug discovery and development. Scaffold decoration, morphing and hopping are additional techniques that enable the modification of the chosen privileged framework and better explore the chemical space around it. In this study, two series of highly functionalized pyrimidine and pyridine derivatives were synthesized using a scaffold morphing approach consisting of triazine compounds obtained previously as antiviral agents. Newly synthesized azines were evaluated against lymphoma, hepatocarcinoma, and colon epithelial carcinoma cells, showing in five cases acceptable to good anticancer activity associated with low cytotoxicity on healthy fibroblasts. Finally, ADME in vitro studies were conducted on the best derivatives of the two series showing good passive permeability and resistance to metabolic degradation.

A Facile Access to Green Fluorescent Albumin Derivatives.

A Morita-Baylis-Hillman Adduct (MBHA) derivative bearing a triphenylamine moiety was found to react with human serum albumin (HSA) shifting its emission from the blue to the green-yellow thus leading to green fluorescent albumin (GFA) derivatives and enlarging the platform of probes for aggregation-induced fluorescent-based detection techniques. A possible interaction of MBHA derivative 7 with a lipophilic pocket within the HSA structure was suggested by docking studies. DLS experiments showed that the reaction with HSA induce a conformational change of the protein contributing to the aggregation process of GFA derivatives. The results of investigations on the biological properties suggested that GFA retained the ability of binding drug molecules such as warfarin and diazepam. Finally, cytotoxicity evaluation studies suggested that, although the MBHA derivative 7 at 0.1 µg/mL affected the percentage of cell viability in comparison to the negative control, it cannot be considered cytotoxic, whereas at all the other concentrations≥0.5 µg/mL resulted cytotoxic at different extent.

Early investigation of a novel SI306 theranostic prodrug for glioblastoma treatment.

Glioblastoma multiforme (GBM) is one of the most aggressive malignancies with a high recurrence rate and poor prognosis. Theranostic, combining therapeutic and diagnostic approaches, arises as a successful strategy to improve patient outcomes through personalized medicine. Src is a non-receptor tyrosine kinase (nRTK) whose involvement in GBM has been extensively demonstrated. Our previous research highlighted the effectiveness of the pyrazolo[3,4-d]pyrimidine SI306 and its more soluble prodrug CMP1 as Src inhibitors both in in vitro and in vivo GBM models. In this scenario, we decided to develop a theranostic prodrug of SI306, ProSI-DOTA(68 Ga) 1, which was designed to target GBM cells after hydrolysis and follow-up on the disease's progression and improve the therapy's outcome. First, the corresponding nonradioactive prodrug 2 was tested to evaluate its ADME profile and biological activity. It showed good metabolic stability, no inhibition of CYP3A4, suboptimal aqueous solubility, and slight gastrointestinal and blood-brain barrier passive permeability. Compound 2 exhibited a drastic reduction of cell vitality after 72 h on two different GBM cell lines (GL261 and U87MG). Then, 2 was subjected to complexation with the radionuclide Gallium-68 to give ProSI-DOTA(68 Ga) 1. The cellular uptake of 1 was evaluated on GBM cells, highlighting a slight but significant time-dependent uptake. The data obtained from our preliminary studies reflect the physiochemical properties of 1. The use of an alternative route of administration, such as the intranasal route, could overcome the physiochemical limitations and enhance the pharmacokinetic properties of 1, paving the way for its future development.

Sweet Cherry Extract as Permeation Enhancer of Tyrosine Kinase Inhibitors: A Promising Prospective for Future Oral Anticancer Therapies.

Although patients would rather oral therapies to injections, the gastrointestinal tract's low permeability makes this method limiting for most compounds, including anticancer drugs. Due to their low bioavailability, oral antitumor therapies suffer from significant variability in pharmacokinetics and efficacy. The improvement of their pharmacokinetic profiles can be achieved by a new approach: the use of natural extracts enriched with polyphenolic compounds that act as intestinal permeability enhancers. Here, we propose a safe sweet cherry extract capable of enhancing oral absorption. The extract was characterized by the HPLC-UV/MS method, evaluated for in vitro antioxidant activity, safety on the Caco-2 cell line, and as a potential permeation enhancer. The sweet cherry extract showed a high antioxidant capacity (ABTS and DPPH assays were 211.74 and 48.65 µmol of Trolox equivalent/g dried extract, respectively), high content of polyphenols (8.44 mg of gallic acid per gram of dry extract), and anthocyanins (1.80 mg of cyanidin-3-glucoside equivalent per g of dry extract), reassuring safety profile (cell viability never lower than 98%), and a significant and fully reversible ability to alter the integrity of the Caco-2 monolayer (+81.5% of Lucifer yellow permeability after 2 h). Furthermore, the ability of the sweet cherry extract to improve the permeability (Papp) and modify the efflux ratio (ER) of reference compounds (atenolol, propranolol, and dasatinib) and selected pyrazolo[3,4-d]pyrimidine derivatives was investigated. The obtained results show a significant increase in apparent permeability across the Caco-2 monolayer (tripled and quadrupled in most cases), and an interesting decrease in efflux ratio when compounds were co-incubated with sweet cherry extract.

Development and validation of derivatization-based LC-MS/MS method for quantification of short-chain fatty acids in human, rat, and mouse plasma.

Short-chain fatty acids (SCFAs), the end products of gut microbial fermentation of dietary fibers and non-digestible polysaccharides, act as a link between the microbiome, immune system, and inflammatory processes. The importance of accurately quantifying SCFAs in plasma has recently emerged to understand their biological role. In this work, a sensitive and reproducible LC-MS/MS method is reported for SCFAs quantification in three different matrices such as human, rat and mouse plasma via derivatization, using as derivatizing agent O-benzylhydroxylamine (O-BHA), coupled with liquid-liquid extraction. First, the instrumental parameters of the mass spectrometer and then the chromatographic conditions were optimized using previously SCFAs derivatives synthetized and used as standards. After that, the best conditions for derivatization and extraction from plasma were studied and a series of determinations were performed on human, rat, and mouse plasma aliquots to validate the overall method (derivatization, extraction, and LC-MS/MS determination). The method showed good performance in terms of recovery (> 80%), precision (RSD <14%), accuracy (RE < ± 10%) and sensitivity (LOQ of 0.01 µM for acetic, butyric, propionic and isobutyric acid) in all plasma samples. The method thus developed and validated was applied to the quantification of major SCFAs in adult and aged mice, germ-free mice and in germ-free recipient mice subjected to fecal transplant from adult and aged donors. Results highlighted how plasma concentrations of SCFAs are correlated with age further highlighting the importance of developing a method that is reliable for the quantification of SCFAs to study their biological role.

Targeting GLI1 and GLI2 with small molecule inhibitors to suppress GLI-dependent transcription and tumor growth.

Aberrant activation of Hedgehog (HH) signaling in cancer is the result of genetic alterations of upstream pathway components (canonical) or other oncogenic mechanisms (noncanonical), that ultimately concur to activate the zinc-finger transcription factors GLI1 and GLI2. Therefore, inhibition of GLI activity is a good therapeutic option to suppress both canonical and noncanonical activation of the HH pathway. However, only a few GLI inhibitors are available, and none of them have the profile required for clinical development due to poor metabolic stability and aqueous solubility, and high hydrophobicity. Two promising quinoline inhibitors of GLI were selected by virtual screening and subjected to hit-to-lead optimization, thus leading to the identification of the 4-methoxy-8-hydroxyquinoline derivative JC19. This molecule impaired GLI1 and GLI2 activities in several cellular models interfering with the binding of GLI1 and GLI2 to DNA. JC19 suppressed cancer cell proliferation by enhancing apoptosis, inducing a strong anti-tumor response in several cancer cell lines in vitro. Specificity towards GLI1 and GLI2 was demonstrated by lower activity of JC19 in GLI1- or GLI2-depleted cancer cells. JC19 showed excellent metabolic stability and high passive permeability. Notably, JC19 inhibited GLI1-dependent melanoma xenograft growth in vivo, with no evidence of toxic effects in mice. These results highlight the potential of JC19 as a novel anti-cancer agent targeting GLI1 and GLI2.

Progress towards Adjuvant Development: Focus on Antiviral Therapy.

In recent decades, vaccines have been extraordinary resources to prevent pathogen diffusion and cancer. Even if they can be formed by a single antigen, the addition of one or more adjuvants represents the key to enhance the response of the immune signal to the antigen, thus accelerating and increasing the duration and the potency of the protective effect. Their use is of particular importance for vulnerable populations, such as the elderly or immunocompromised people. Despite their importance, only in the last forty years has the search for novel adjuvants increased, with the discovery of novel classes of immune potentiators and immunomodulators. Due to the complexity of the cascades involved in immune signal activation, their mechanism of action remains poorly understood, even if significant discovery has been recently made thanks to recombinant technology and metabolomics. This review focuses on the classes of adjuvants under research, recent mechanism of action studies, as well as nanodelivery systems and novel classes of adjuvants that can be chemically manipulated to create novel small molecule adjuvants.

Discovery and Optimization of a Novel Macrocyclic Amidinourea Series Active as Acidic Mammalian Chitinase Inhibitors.

Our research group has been involved for a long time in the development of macrocyclic amidinoureas (MCAs) as antifungal agents. The mechanistic investigation drove us to perform an in silico target fishing study, which allowed the identification of chitinases as one of their putative targets, with 1a showing a submicromolar inhibition of Trichoderma viride chitinase. In this work, we investigated the possibility to further inhibit the corresponding human enzymes, acidic mammalian chitinase (AMCase) and chitotriosidase (CHIT1), involved in several chronic inflammatory lung diseases. Thus, we first validated the inhibitory activity of 1a against AMCase and CHIT1 and then designed and synthesized new derivatives aimed at improving the potency and selectivity against AMCase. Among them, compound 3f emerged for its activity profile along with its promising in vitro ADME properties. We also gained a good understanding of the key interactions with the target enzyme through in silico studies.

Biological Evaluation and In Vitro Characterization of ADME Profile of In-House Pyrazolo[3,4-d]pyrimidines as Dual Tyrosine Kinase Inhibitors Active against Glioblastoma Multiforme.

The therapeutic use of tyrosine kinase inhibitors (TKIs) represents one of the successful strategies for the treatment of glioblastoma (GBM). Pyrazolo[3,4-d]pyrimidines have already been reported as promising small molecules active as c-Src/Abl dual inhibitors. Herein, we present a series of pyrazolo[3,4-d]pyrimidine derivatives, selected from our in-house library, to identify a promising candidate active against GBM. The inhibitory activity against c-Src and Abl was investigated, and the antiproliferative profile against four GBM cell lines was studied. For the most active compounds endowed with antiproliferative efficacy in the low-micromolar range, the effects toward nontumoral, healthy cell lines (fibroblasts FIBRO 2-93 and keratinocytes HaCaT) was investigated. Lastly, the in silico and in vitro ADME properties of all compounds were also assessed. Among the tested compounds, the promising inhibitory activity against c-Src and Abl (Ki 3.14 µM and 0.44 µM, respectively), the irreversible, apoptotic-mediated death toward U-87, LN18, LN229, and DBTRG GBM cell lines (IC50 6.8 µM, 10.8 µM, 6.9 µM, and 8.5 µM, respectively), the significant reduction in GBM cell migration, the safe profile toward FIBRO 2-93 and HaCaT healthy cell lines (CC50 91.7 µM and 126.5 µM, respectively), the high metabolic stability, and the excellent passive permeability across gastrointestinal and blood-brain barriers led us to select compound 5 for further in vivo assays.

Tenebrio molitor as a Simple and Cheap Preclinical Pharmacokinetic and Toxicity Model.

The progression of drugs into clinical phases requires proper toxicity assessment in animals and the correct identification of possible metabolites. Accordingly, different animal models are used to preliminarily evaluate toxicity and biotransformations. Rodents are the most common models used to preliminarily evaluate the safety of drugs; however, their use is subject to ethical consideration and elevated costs, and strictly regulated by national legislations. Herein, we developed a novel, cheap and convenient toxicity model using Tenebrio molitor coleoptera (TMC). A panel of 15 drugs-including antivirals and antibacterials-with different therapeutic applications was administered to TMC and the LD50 was determined. The values are comparable with those already determined in mice and rats. In addition, a TMC model was used to determine the presence of the main metabolites and in vivo pharmacokinetics (PK), and results were compared with those available from in vitro assays and the literature. Taken together, our results demonstrate that TMC can be used as a novel and convenient preliminary toxicity model to preliminarily evaluate the safety of experimental compounds and the formation of main metabolites, and to reduce the costs and number of rodents, according to 3R principles.

Privileged Scaffold Decoration for the Identification of the First Trisubstituted Triazine with Anti-SARS-CoV-2 Activity.

Current therapy against severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) are based on the use of Remdesivir 1, Molnupiravir 2, and the recently identified Nirmatrelvir 3. Unfortunately, these three drugs showed some limitations regarding potency and possible drug-drug interactions. A series of derivatives coming from a decoration approach of the privileged scaffold s-triazines were synthesized and evaluated against SAR-CoV-2. One derivative emerged as the hit of the series for its micromolar antiviral activity and low cytotoxicity. Mode of action and pharmacokinetic in vitro preliminary studies further confirm the role as candidates for a future optimization campaign of the most active derivative identified with this work.

Novel pyrazolo[3,4-d]pyrimidines as dual Src/Bcr-Abl kinase inhibitors: Synthesis and biological evaluation for chronic myeloid leukemia treatment.

The Bcr-Abl tyrosine kinase (TK) is the molecular hallmark of chronic myeloid leukemia (CML). Src is another TK kinase whose involvement in CML was widely demonstrated. Small molecules active as dual Src/Bcr-Abl inhibitors emerged as effective targeted therapies for CML and a few compounds are currently in clinical use. In this study, we applied a target-oriented approach to identify a family of pyrazolo[3,4-d]pyrimidines as dual Src/Bcr-Abl inhibitors as anti-leukemia agents. Considering the high homology between Src and Bcr-Abl, in-house Src inhibitors 8a-l and new analogue compounds 9a-n were screened as dual Src/Bcr-Abl inhibitors. The antiproliferative activity on K562 CML cells and the ADME profile were determined for the most promising compounds. Molecular modeling studies elucidated the binding mode of the inhibitors into the Bcr-Abl (wt) catalytic pocket. Compounds 8j and 8k showed nanomolar activities in enzymatic and cellular assays, together with favorable ADME properties, emerging as promising candidates for CML therapy. Finally, derivatives 9j and 9k, emerging as valuable inhibitors of the most aggressive Bcr-Abl mutation, T315I, constitute a good starting point in the search for compounds able to treat drug-resistant forms of CML. Overall, this study allowed us to identify more potent compounds than those previously reported by the group, marking a step forward in searching for new antileukemic agents.

DEAD-Box Helicase DDX3X as a Host Target against Emerging Viruses: New Insights for Medicinal Chemical Approaches.

In recent years, globalization, global warming, and population aging have contributed to the spread of emerging viruses, such as coronaviruses (COVs), West Nile (WNV), Dengue (DENV), and Zika (ZIKV). The number of reported infections is increasing, and considering the high viral mutation rate, it is conceivable that it will increase significantly in the coming years. The risk caused by viruses is now more evident due to the COVID-19 pandemic, which highlighted the need to find new broad-spectrum antiviral agents able to tackle the present pandemic and future epidemics. DDX3X helicase is a host factor required for viral replication. Selective inhibitors have been identified and developed into broad-spectrum antivirals active against emerging pathogens, including SARS-CoV-2 and most importantly against drug-resistant strains. This perspective describes the inhibitors identified in the last years, highlighting their therapeutic potential as innovative broad-spectrum antivirals.

The Pyrazolo[3,4-d]Pyrimidine Derivative Si306 Encapsulated into Anti-GD2-Immunoliposomes as Therapeutic Treatment of Neuroblastoma.

Si306, a pyrazolo[3,4-d]pyrimidine derivative recently identified as promising anticancer agent, has shown favorable in vitro and in vivo activity profile against neuroblastoma (NB) models by acting as a competitive inhibitor of c-Src tyrosine kinase. Nevertheless, Si306 antitumor activity is associated with sub-optimal aqueous solubility, which might hinder its further development. Drug delivery systems were here developed with the aim to overcome this limitation, obtaining suitable formulations for more efficacious in vivo use. Si306 was encapsulated in pegylated stealth liposomes, undecorated or decorated with a monoclonal antibody able to specifically recognize and bind to the disialoganglioside GD2 expressed by NB cells (LP[Si306] and GD2-LP[Si306], respectively). Both liposomes possessed excellent morphological and physio-chemical properties, maintained over a period of two weeks. Compared to LP[Si306], GD2-LP[Si306] showed in vitro specific cellular targeting and increased cytotoxic activity against NB cell lines. After intravenous injection in healthy mice, pharmacokinetic profiles showed increased plasma exposure of Si306 when delivered by both liposomal formulations, compared to that obtained when Si306 was administered as free form. In vivo tumor homing and cytotoxic effectiveness of both liposomal formulations were finally tested in an orthotopic animal model of NB. Si306 tumor uptake resulted significantly higher when encapsulated in GD2-LP, compared to Si306, either free or encapsulated into untargeted LP. This, in turn, led to a significant increase in survival of mice treated with GD2-LP[Si306]. These results demonstrate a promising antitumor efficacy of Si306 encapsulated into GD2-targeted liposomes, supporting further therapeutic developments in pre-clinical trials and in the clinic for NB.

Antibacterial alkylguanidino ureas: Molecular simplification approach, searching for membrane-based MoA.

The ever-faster rise of antimicrobial resistance (AMR) represents a major global Public Health challenge. New chemical entities with innovative Modes of Action (MoAs) are thus desirable. We recently reported the development of a novel class of broad-spectrum bactericidal agents, the AlkylGuanidino Ureas (AGU). Due to their polycationic structure, they likely target bacterial membranes. In order to better understand their MoA, we synthesized a library of AGU derivatives by structural simplification of selected hit compounds and developed specific assays based on membrane models by means of both analytical and computational techniques. Cell-based assays provided experimental evidence that AGUs disrupt bacterial membranes without showing hemolytic behavior. Hence, we herein report a thorough chemical and biological characterization of a new series of AGUs obtained through molecular simplification, allowing the rational design of potent antibacterial compounds active on antibiotic-resistant strains.

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

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 in vitro 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 in vitro release of NO, and the antiproliferative activity in tumor cell lines are presented.

(Thia)calixarenephosphonic Acids as Potent Inhibitors of the Nucleic Acid Chaperone Activity of the HIV-1 Nucleocapsid Protein with a New Binding Mode and Multitarget Antiviral Activity.

The nucleocapsid protein (NC) is a highly conserved protein that plays key roles in HIV-1 replication through its nucleic acid chaperone properties mediated by its two zinc fingers and basic residues. NC is a promising target for antiviral therapy, particularly to control viral strains resistant to currently available drugs. Since calixarenes with antiviral properties have been described, we explored the ability of calixarene hydroxymethylphosphonic or sulfonic acids to inhibit NC chaperone properties and exhibit antiviral activity. By using fluorescence-based assays, we selected four calixarenes inhibiting NC chaperone activity with submicromolar IC50 values. These compounds were further shown by mass spectrometry, isothermal titration calorimetry, and fluorescence anisotropy to bind NC with no zinc ejection and to compete with nucleic acids for the binding to NC. Molecular dynamic simulations further indicated that these compounds interact via their phosphonate or sulfonate groups with the basic surface of NC but not with the hydrophobic plateau at the top of the folded fingers. Cellular studies showed that the most soluble compound CIP201 inhibited the infectivity of wild-type and drug-resistant HIV-1 strains at low micromolar concentrations, primarily targeting the early steps of HIV-1 replication. Moreover, CIP201 was also found to inhibit the flipping and polymerization activity of reverse transcriptase. Calixarenes thus form a class of noncovalent NC inhibitors, endowed with a new binding mode and multitarget antiviral activity.

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

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.

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

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 Ki values against c-Src, interesting antiproliferative activity on SH-SY5Y cells and a suitable ADME profile.