Antonella Peduto

Evaluation of alternative strategies to optimize ketorolac transdermal delivery

In the present study, 2 alternative strategies to optimize ketorolac transdermal delivery, namely, prodrugs (polyoxyethylene glycol ester derivatives, I–IV) and nanostructured lipid carriers (NLC) were investigated. The synthesized prodrugs were chemically stable and easily degraded to the parent drug in human plasma. Ketorolac-loaded NLC with high drug content could be successfully prepared. The obtained products formulated into gels showed a different trend of drug permeation through human stratum corneum and epidermis. Particularly, skin permeation of ester prodrugs was significantly enhanced, apart from ester IV, compared with ketorolac, while the results of drug release from NLC outlined that these carriers were ineffective in increasing ketorolac percutaneous absorption owing to a higher degree of mutual interaction between the drug and carrier lipid matrix. Polyoxyethylene glycol esterification confirmed to be a suitable approach to enhance ketorolac transdermal delivery, while NLC seemed more appropriate for sustained release owing to the possible formation of a drug reservoir into the skin.

Potent inhibition of human 5-lipoxygenase and microsomal prostaglandin E2 synthase-1 by the anti-carcinogenic and anti-inflammatory agent embelin

Embelin (2,5-dihydroxy-3-undecyl-1,4-benzoquinone) possesses anti-inflammatory and anti-carcinogenic properties in vivo, and these features have been related to interference with multiple targets including XIAPs, NFκB, STAT-3, Akt and mTOR. However, interference with these proteins requires relatively high concentrations of embelin (IC₅₀>4 μM) and cannot fully explain its bioactivity observed in several functional studies. Here we reveal human 5-lipoxygenase (5-LO) and microsomal prostaglandin E₂ synthase (mPGES)-1 as direct molecular targets of embelin. Thus, embelin potently suppressed the biosynthesis of eicosanoids by selective inhibition of 5-LO and mPGES-1 with IC₅₀=0.06 and 0.2 μM, respectively. In intact human polymorphonuclear leukocytes and monocytes, embelin consistently blocked the biosynthesis of various 5-LO products regardless of the stimulus (fMLP or A23187) with IC₅₀=0.8-2 μM. Neither the related human 12- and 15-LO nor the cyclooxygenases-1 and -2 or cytosolic phospholipase A₂ were significantly affected by 10 μM embelin. Inhibition of 5-LO and mPGES-1 by embelin was (I) essentially reversible after wash-out, (II) not impaired at higher substrate concentrations, (III) unaffected by inclusion of Triton X-100, and (IV) did not correlate to its proposed antioxidant properties. Docking simulations suggest concrete binding poses in the active sites of both 5-LO and mPGES-1. Because 5-LO- and mPGES-1-derived eicosanoids play roles in inflammation and cancer, the interference of embelin with these enzymes may contribute to its biological effects and suggests embelin as novel chemotype for development of dual 5-LO/mPGES-1 inhibitors.

Design, synthesis, biophysical and biological studies of trisubstituted naphthalimides as G-quadruplex ligands

A series of trisubstituted naphthalimides have been synthesized and evaluated as telomeric G-quadruplex ligands by biophysical methods. Affinity for telomeric G-quadruplex AGGG(TTAGGG)(3) binding was first screened by fluorescence titrations. Subsequently, the interaction of the telomeric G-quadruplex with compounds showing the best affinity has been studied by isothermal titration calorimetry and UV-melting experiments. The two best compounds of the series tightly bind the telomeric quadruplex with a 2:1 drug/DNA stoichiometry. These derivatives have been further evaluated for their ability to inhibit telomerase by a TRAP assay and their pharmacological properties by treating melanoma (M14) and human lung cancer (A549) cell lines with increasing drug concentrations. A dose-dependent inhibition of cell proliferation was observed for all cellular lines during short-term treatment.

Structure–activity relationship study of arbidol derivatives as inhibitors of chikungunya virus replication

Chikungunya virus (CHIKV), a mosquito-borne arthrogenic Alphavirus, causes an acute febrile illness in humans, that is, accompanied by severe joint pains. In many cases, the infection leads to persistent arthralgia, which may last for weeks to several years. The re-emergence of this infection in the early 2000s was exemplified by numerous outbreaks in the eastern hemisphere. Since then, the virus is rapidly spreading. Currently, no drugs have been approved or are in development for the treatment of CHIKV, which makes this viral infection particularly interesting for academic medicinal chemistry efforts. Several molecules have already been identified that inhibit CHIKV replication in phenotypic virus-cell-based assays. One of these is arbidol, a molecule that already has been licensed for the treatment of influenza A and B virus infections. For structural optimization, a dedicated libraries of 43 indole-based derivatives were evaluated leading to more potent analogues (IIIe and IIIf) with anti-chikungunya virus (CHIKV) activities higher than those of the other derivatives, including the lead compound, and with a selective index of inhibition 13.2 and 14.6, respectively, higher than that of ARB (4.6).

Structure-based design, synthesis and preliminary anti-inflammatory activity of bolinaquinone analogues

As a part of our drug discovery efforts we developed a series of simplified derivatives of bolinaquinone (BLQ), a hydroxyquinone marine metabolite, showing potent anti-inflammatory activity. Thirteen new hydroxyquinone derivatives closely related to BLQ were synthesized and tested on mouse macrophage-like RAW 264.7 cell line in order to investigate their ability to modulate the production of Prostaglandin E2 (PGE2). This optimization process led to the identification of three strictly correlated compounds with comparable and higher inhibitory potency than BLQ on PGE2 production. To evaluate the affinity of BLQ and its analogues for hsPLA2, surface plasmon resonance (SPR) experiments were performed.

Synthesis and Cytotoxic Activity of New β-Carboline Derivatives

On the basis of harmine and 1-methoxy-canthin-6-one chemical structures, a series of novel 1,4-disubstituted and 1,4,9-trisubstituted β-carbolines and tetracyclic derivatives were designed and synthesized. Cytotoxic activities of these compounds in vitro were investigated in a human tumor cell line panel. Almost all compounds demonstrated interesting cytotoxic activities in particular against prostate cancer cells PC-3 with IC50 in the low micromolar range. Compound X was found to be the most potent one with IC50 value of 8.0 µM; this suggests further studies with models of prostate cancer.

Novel series of benzoquinones with high potency against 5-lipoxygenase in human polymorphonuclear leukocytes

5-Lipoxygenase (5-LO) is a potential target for pharmacological intervention with various inflammatory and allergic diseases. Starting from the natural dual 5-LO/microsomal prostaglandin E2 synthase (mPGES)-1 inhibitor embelin (2,5-dihydroxy-3-undecyl-1,4-benzoquinone, 2) that suppresses 5-LO activity in human primary leukocytes with IC50 = 0.8-2 μM, we synthesized 48 systematically modified derivatives of 2. We modified the 1,4-quinone to 1,2-quinone, mono- or bimethylated the hydroxyl groups, and varied the C11-n-alkyl residue (C4- to C16-n-alkyl or prenyl) of 2. Biological evaluation yields potent analogues being superior over 2 and obvious structure-activity relationships (SAR) for inhibition of 5-LO. Interestingly, conversion to 1,2-benzoquinone and bimethylation of the hydroxyl moieties strongly improves 5-LO inhibition in polymorphonuclear leukocytes versus 2 up to 60-fold, exemplified by the C12-n-alkyl derivative 22c (4,5-dimethoxy-3-dodecyl-1,2-benzoquinone) with IC50 = 29 nM. Regarding inhibition of mPGES-1, none of the novel benzoquinones could outperform the parental compound 2 (IC50 = 0.21 μM), and only modest suppressive effects on 12- and 15-LOs were evident. Together, our detailed SAR study reveals 22c as highly potent 5-LO-selective lead compound in intact cells that warrants further preclinical evaluation as anti-inflammatory agent.

Design of inhibitors of influenza virus membrane fusion: synthesis, structure-activity relationship and in vitro antiviral activity of a novel indole series.

The fusion of virus and endosome membranes is an essential early stage in influenza virus infection. The low pH-induced conformational change which promotes the fusogenic activity of the haemagglutinin (HA) is thus an attractive target as an antiviral strategy. The anti-influenza drug Arbidol is representative of a class of antivirals which inhibits HA-mediated membrane fusion by increasing the acid stability of the HA. In this study two series of indole derivatives structurally related to Arbidol were designed and synthesized to further probe the foundation of its antiviral activity and develop the basis for a structure-activity relationship (SAR). Ethyl 5-(hydroxymethyl)-1-methyl-2-(phenysulphanylmethyl)-1H-indole-3-carboxylate (15) was identified as one of the most potent inhibitors and more potent than Arbidol against certain subtypes of influenza A viruses. In particular, 15 exhibited a much greater affinity and preference for binding group 2 than group 1 HAs, and exerted a greater stabilising effect, in contrast to Arbidol. The results provide the basis for more detailed SAR studies of Arbidol binding to HA; however, the greater affinity for binding HA was not reflected in a comparable increase in antiviral activity of 15, apparently reflecting the complex nature of the antiviral activity of Arbidol and its derivatives.

Cyclohexa-2,5-diene-1,4-dione-based antiproliferative agents: design, synthesis, and cytotoxic evaluation

BackgroundTumors are diseases characterized by uncontrolled cell growth and, in spite of the progress of medicine over the years, continue to represent a major threat to the health, requiring new therapies. Several synthetic compounds, such as those derived from natural sources, have been identified as anticancer drugs; among these compounds quinone represent the second largest class of anticancer agents in use. Several studies have shown that these act on tumor cells through several mechanisms. An important objective of this work is to develop quinoidscompounds showing antitumor activity, but with fewer side effects. The parachinone cannabinol HU-331, is a small molecule that with its core 4-hydroxy-1,4-benzoquinone, exhibits a potent and selective cytotoxic activity on different tumor cell lines. A series of derivatives 3-hydroxy-1,4-benzochinoni were thus developed through HU-331 chemical modifications. The purpose of the work is to test the ability of the compounds to induce proliferative inhibition and study the mechanisms of cell death.MethodsThe antitumor activities were evaluated in vitro by examining their cytotoxic effects against different human cancer cell lines. All cell lines tested were plated in 96-multiwell and treated with HU-100-V at different concentrations and cell viability was evaluated byMTT assay. Subsequently via flow cytometry (FACS) it was possible to assess apoptosis by the system of double labeling with PI and Annexin-V, and the effect of the compounds on ROS formation by measuring the dichlorofluorescein fluorescence.ResultsThe substitution by n-hexyl chain considerably enhanced the bioactivity of the compounds. In details, 2-hexyl-5-hydroxycyclohexa-2,5-diene-1,4-dione (V), 2,5-Dimethoxy-3-hexyl-2,5-cyclohexadiene-1,4-dione (XII) and 2-hydroxy-5-methoxy-3-hexyl-cyclohexa-2,5-diene-1,4-dione (XIII) showed most prominent cytotoxicity against almost human tumour cell lines. Compound V was further subjected to downstream apoptotic analysis, demostrating a time-dependent pro-apoptotic activity on human melanoma M14 cell line mediated by caspases activation and poly-(ADP-ribose)-polymerase (PARP) protein cleavage.ConclusionsThese findings indicate that 2-hexyl-5-idrossicicloesa-2,5-diene-1,4-dione can be a promising compound for the design of a new class of antineoplastic derivatives.Carmen Petronzi, Michela Festa, Antonella Peduto and Maria Castellano: equally contributed equally to this work.

Elucidation of the molecular mechanism and the efficacy in vivo of a novel 1,4-benzoquinone that inhibits 5-lipoxygenase

1,4‐Benzoquinones are well‐known inhibitors of 5‐lipoxygenase (5‐LOX, the key enzyme in leukotriene biosynthesis), but the molecular mechanisms of 5‐LOX inhibition are not completely understood. Here we investigated the molecular mode of action and the pharmacological profile of the novel 1,4‐benzoquinone derivative 3‐((decahydronaphthalen‐6‐yl)methyl)‐2,5‐dihydroxycyclohexa‐2,5‐diene‐1,4‐dione (RF‐Id) in vitro and its effectiveness in vivo.