Synthesis of 2-Amino-N'-aroyl(het)arylhydrazides, DNA Photocleavage, Molecular Docking and Cytotoxicity Studies against Melanoma CarB Cell Lines.

Diacylhydrazine bridged anthranilic acids with aryl and heteroaryl domains have been synthesized as the open flexible scaffold of arylamide quinazolinones in order to investigate flexibility versus rigidity towards DNA photocleavage and sensitivity. Most of the compounds have been synthesized via the in situ formation of their anthraniloyl chloride and subsequent reaction with the desired hydrazide and were obtained as precipitates, in moderate yields. All compounds showed high UV-A light absorption and are eligible for DNA photocleavage studies under this "harmless" irradiation. Despite their reduced UV-B light absorption, a first screening indicated the necessity of a halogen at the p-position in relation to the amine group and the lack of an electron-withdrawing group on the aryl group. These characteristics, in general, remained under UV-A light, rendering these compounds as a novel class of UV-A-triggered DNA photocleavers. The best photocleaver, the compound 9, was active at concentrations as low as 2 µΜ. The 5-Nitro-anthranilic derivatives were inactive, giving the opposite results to their related rigid quinazolinones. Molecular docking studies with DNA showed possible interaction sites, whereas cytotoxicity experiments indicated the iodo derivative 17 as a potent cytotoxic agent and the compound 9 as a slight phototoxic compound.

Effect of Arylazo Sulfones on DNA: Binding, Cleavage, Photocleavage, Molecular Docking Studies and Interaction with A375 Melanoma and Non-Cancer Cells.

A set of arylazo sulfones, known to undergo N-S bond cleavage upon light exposure, has been synthesized, and their activity in the dark and upon irradiation towards DNA has been investigated. Their interaction with calf-thymus DNA has been examined, and the significant affinity observed (most probably due to DNA intercalation) was analyzed by means of molecular docking "in silico" calculations that pointed out polar contacts, mainly via the sulfonyl moiety. Incubation with plasmid pBluescript KS II revealed DNA cleavage that has been studied over time and concentration. UV-A irradiation considerably improved DNA damage for most of the compounds, whereas under visible light the effect was slightly lower. Moving to in vitro experiments, irradiation was found to slightly enhance the death of the cells in the majority of the compounds. Naphthylazosulfone 1 showed photo-disruptive effect under UV-A irradiation (IC50 ~13 µΜ) followed by derivatives 14 and 17 (IC50 ~100 µΜ). Those compounds were irradiated in the presence of two non-cancer cell lines and were found equally toxic only upon irradiation and not in the dark. The temporal and spatial control of light, therefore, might provide a chance for these novel scaffolds to be useful for the development of phototoxic pharmaceuticals.

p-Pyridinyl oxime carbamates: synthesis, DNA binding, DNA photocleaving activity and theoretical photodegradation studies.

A number of p-pyridinyl oxime carbamate derivatives were prepared upon the reaction of the corresponding oximes with isocyanates. These novel compounds reacted photochemically in the presence of supercoiled plasmid DNA. Structure-activity relationship (SAR) studies revealed that the substituent on the imine group was not affecting the extend of the DNA damage, whereas the substituent of the carbamate group was critical, with the halogenated derivatives to be able to cause extensive single and double stranded DNA cleavages, acting as "synthetic nucleases", independently of oxygen and pH. Calf thymus-DNA affinity studies showed a good-to-excellent affinity of selected both active and non-active derivatives. Preliminary theoretical studies were performed, in an effort to explain the reasons why some derivatives cause photocleavage and some others not, which were experimentally verified using triplet state activators and quenchers. These theoretical studies seem to allow the prediction of the activity of derivatives able to pass intersystem crossing to their triplet energy state and thus create radicals able to damage DNA. With this study, it is shown that oxime carbamate derivatives have the potential to act as novel effective photobase generating DNA-photocleavers, and are proposed as new leads for "on demand" biotechnological applications in drug discovery and medicine.

The actinobacterium Tsukamurella paurometabola has a functionally divergent arylamine N-acetyltransferase (NAT) homolog.

Actinobacteria in the Tsukamurella genus are aerobic, high-GC, Gram-positive mycolata, considered as opportunistic pathogens and isolated from various environmental sources, including sites contaminated with oil, urban or industrial waste and pesticides. Although studies look into xenobiotic biotransformation by Tsukamurella isolates, the relevant enzymes remain uncharacterized. We investigated the arylamine N-acetyltransferase (NAT) enzyme family, known for its role in the xenobiotic metabolism of prokaryotes and eukaryotes. Xenobiotic sensitivity of Tsukamurella paurometabola type strain DSM 20162T was assessed, followed by cloning, recombinant expression and functional characterization of its single NAT homolog (TSUPD)NAT1. The bacterium appeared quite robust against chloroanilines, but more sensitive to 4-anisidine and 2-aminophenol. However, metabolic activity was not evident towards those compounds, presumably due to mechanisms protecting cells from xenobiotic entry. Of the pharmaceutical arylhydrazines tested, hydralazine was toxic, but the bacterium was less sensitive to isoniazid, a drug targeting mycolic acid biosynthesis in mycobacteria. Although (TSUPD)NAT1 protein has an atypical Cys-His-Glu (instead of the expected Cys-His-Asp) catalytic triad, it is enzymatically active, suggesting that this deviation is likely due to evolutionary adaptation potentially serving a different function. The protein was indeed found to use malonyl-CoA, instead of the archetypal acetyl-CoA, as its preferred donor substrate. Malonyl-CoA is important for microbial biosynthesis of fatty acids (including mycolic acids) and polyketide chains, and the corresponding enzymatic systems have common evolutionary histories, also linked to xenobiotic metabolism. This study adds to accummulating evidence suggesting broad phylogenetic and functional divergence of microbial NAT enzymes that goes beyond xenobiotic metabolism and merits investigation.

Synthesis of inositol phosphate-based competitive antagonists of inositol 1,4,5-trisphosphate receptors.

Inositol 1,4,5-trisphosphate receptors (IP3Rs) are intracellular Ca(2+) channels that are widely expressed in animal cells, where they mediate the release of Ca(2+) from intracellular stores evoked by extracellular stimuli. A diverse array of synthetic agonists of IP3Rs has defined structure-activity relationships, but existing antagonists have severe limitations. We combined analyses of Ca(2+) release with equilibrium competition binding to IP3R to show that (1,3,4,6)IP4 is a full agonist of IP3R1 with lower affinity than (1,4,5)IP3. Systematic manipulation of this meso-compound via a versatile synthetic scheme provided a family of dimeric analogs of 2-O-butyryl-(1,3,4,6)IP4 and (1,3,4,5,6)IP5 that compete with (1,4,5)IP3 for binding to IP3R without evoking Ca(2+) release. These novel analogs are the first inositol phosphate-based competitive antagonists of IP3Rs with affinities comparable to that of the only commonly used competitive antagonist, heparin, the utility of which is limited by off-target effects.

Pyridine and p-Nitrophenyl Oxime Esters with Possible Photochemotherapeutic Activity: Synthesis, DNA Photocleavage and DNA Binding Studies.

Compared to standard treatments for various diseases, photochemotherapy and photo-dynamic therapy are less invasive approaches, in which DNA photocleavers represent promising tools for novel "on demand" chemotherapeutics. A series of p-nitrobenzoyl and p-pyridoyl ester conjugated aldoximes, amidoximes and ethanone oximes were subjected to UV irradiation at 312 nm with supercoiled circular plasmid DNA. The compounds which possessed appropriate properties were additionally subjected to UVA irradiation at 365 nm. The ability of most of the compounds to photocleave DNA was high at 312 nm, whereas higher concentrations were required at 365 nm as a result of their lower UV absorption. The affinity of selected compounds to calf-thymus (CT) DNA was studied by UV spectroscopy, viscosity experiments and competitive studies with ethidium bromide (EB) revealing that all compounds interacted with CT DNA. The fluorescence emission spectra of the pre-treated EB-DNA exhibited a moderate to significant quenching in the presence of the compounds indicating the binding of the compounds to CT DNA via intercalation as concluded also by DNA-viscosity experiments. For the oxime esters the DNA photocleavage and affinity studies aimed to clarify the role of the oxime nature (aldoxime, ketoxime, amidoxime) and the role of the pyridine and p-nitrophenyl moieties both as oxime substituents and ester conjugates.

Enhanced exercise capacity in mice with severe heart failure treated with an allosteric effector of hemoglobin, myo-inositol trispyrophosphate.

A major determinant of maximal exercise capacity is the delivery of oxygen to exercising muscles. myo-Inositol trispyrophosphate (ITPP) is a recently identified membrane-permeant molecule that causes allosteric regulation of Hb oxygen binding affinity. In normal mice, i.p. administration of ITPP (0.5-3 g/kg) caused a dose-related increase in the oxygen tension at which Hb is 50% saturated (p50), with a maximal increase of 31%. In parallel experiments, ITPP caused a dose-related increase in maximal exercise capacity, with a maximal increase of 57 +/- 13% (P = 0.002). In transgenic mice with severe heart failure caused by cardiac-specific overexpression of G alpha q, i.p. ITPP increased exercise capacity, with a maximal increase of 63 +/- 7% (P = 0.005). Oral administration of ITPP in drinking water increased Hb p50 and maximal exercise capacity (+34 +/- 10%; P < 0.002) in normal and failing mice. Consistent with increased tissue oxygen availability, ITPP decreased hypoxia inducible factor-1alpha mRNA expression in myocardium. It had no effect on myocardial contractility in isolated mouse cardiac myocytes and did not affect arterial blood pressure in vivo in mice. Thus, ITPP decreases the oxygen binding affinity of Hb, increases tissue oxygen delivery, and increases maximal exercise capacity in normal mice and mice with severe heart failure. ITPP is thus an attractive candidate for the therapy of patients with reduced exercise capacity caused by heart failure.

Transition metal(II) complexes of halogenated derivatives of (E)-4-(2-(pyridin-2-ylmethylene)hydrazinyl)quinazoline: structure, antioxidant activity, DNA-binding DNA photocleavage, interaction with albumin and in silico studies.

Two novel halogenated (Br- and F-) quinazoline derivatives, namely [(E)-4-(2-((6-bromopyridin-2-yl)methylene)hydrazinyl)quinazoline] (L1) and [(E)-4-(2-((3-fluoropyridin-2-yl)methylene)hydrazinyl) quinazoline] (L2), were synthesized and characterized. Their interaction with a series of metal(II) ions (= Mn(II), Ni(II), Cu(II), Zn(II) and Cd(II)) resulted in the formation of six mononuclear complexes characterized by spectroscopic techniques and single-crystal X-ray crystallography. The complexes bear the formulae [Ni(L1)2](NO3)2 (1), [Zn(L2)2](NO3)(PF6) (2), [Cd(L2)(H2O)(CH3OH)(NO3)](NO3) (3), [Cu(L2)Cl2] (4), [Ni(L2)2](NO3)2 (5) and [Mn(L2)(CH3OH)(Cl)2] (6). The biological activity of the compounds was further evaluated in vitro regarding their interaction with calf-thymus DNA, their cleavage ability towards supercoiled circular pBR322 plasmid DNA in the absence or presence of irradiation at various wavelengths (UVA, UVB and visible light), their affinity to bovine serum albumin and their ability to scavenge 1,1-diphenyl-picrylhydrazyl and 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radicals and to reduce H2O2. In silico molecular docking calculations were employed to study the behavior of the complexes towards calf-thymus DNA and bovine serum albumin.

A taxonomically representative strain collection to explore xenobiotic and secondary metabolism in bacteria.

Bacteria employ secondary metabolism to combat competitors, and xenobiotic metabolism to survive their chemical environment. This project has aimed to introduce a bacterial collection enabling comprehensive comparative investigations of those functions. The collection comprises 120 strains (Proteobacteria, Actinobacteria and Firmicutes), and was compiled on the basis of the broad taxonomic range of isolates and their postulated biosynthetic and/or xenobiotic detoxification capabilities. The utility of the collection was demonstrated in two ways: first, by performing 5144 co-cultures, recording inhibition between isolates and employing bioinformatics to predict biosynthetic gene clusters in sequenced genomes of species; second, by screening for xenobiotic sensitivity of isolates against 2-benzoxazolinone and 2-aminophenol. The co-culture medium of Bacillus siamensis D9 and Lysinibacillus sphaericus DSM 28T was further analysed for possible antimicrobial compounds, using liquid chromatography-mass spectrometry (LC-MS), and guided by computational predictions and the literature. Finally, LC-MS analysis demonstrated N-acetylation of 3,4-dichloroaniline (a toxic pesticide residue of concern) by the actinobacterium Tsukamurella paurometabola DSM 20162T which is highly tolerant of the xenobiotic. Microbial collections enable "pipeline" comparative screening of strains: on the one hand, bacterial co-culture is a promising approach for antibiotic discovery; on the other hand, bioremediation is effective in combating pollution, but requires knowledge of microbial xenobiotic metabolism. The presented outcomes are anticipated to pave the way for studies that may identify bacterial strains and/or metabolites of merit in biotechnological applications.

Structure and biological profile of transition metal complexes with (E)-4-(2-(pyridin-2-ylmethylene)hydrazinyl)quinazoline.

The interaction of the recently reported quinazoline derivative (E)-4-(2-(pyridin-2-ylmethylene)hydrazinyl)quinazoline (L) with a series of metal(II) (= copper(II), nickel(II), cobalt(II) and cadmium(II)) chlorides or nitrates resulted in the formation of mononuclear complexes which were characterized by spectroscopic techniques and single-crystal X-ray crystallography, i.e. [Cu(L)2]Cl2·4H2O (1·4H2O), [Ni(L)2]Cl2·4H2O (2·4H2O), [Ni(L)2](NO3)2·MeOH (3·MeOH), [Co(L)2]Cl2·4H2O (4·4H2O), [Co(L)2](NO3)2·H2O (5·H2O), [Co(L)2](NO3)3·2.5H2O (6·2.5H2O), [Cd(L)(Cl)2]·H2O (7·H2O) and [Cd(L)(CH3OH)(H2O)(NO3)](NO3) (8). The biological profile of the complexes was further assessed in regard to their binding affinity with calf-thymus DNA, their cleavage ability towards pBluescript II KS plasmid DNA in the absence or presence of irradiation of various wavelengths, their interaction with bovine serum albumin and finally, their ability to scavenge 1,1-diphenyl-picrylhydrazyl and 2,2΄-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radicals and to reduce H2O2.

6-Nitro-Quinazolin-4(3H)-one Exhibits Photodynamic Effects and Photodegrades Human Melanoma Cell Lines. A Study on the Photoreactivity of Simple Quinazolin-4(3H)-ones.

Photochemo and photodynamic therapies are minimally invasive approaches for the treatment of cancers and powerful weapons for competing bacterial resistance to antibiotics. Synthetic and naturally occurring quinazolinones are considered privileged anticancer and antibacterial agents, with several of them to have emerged as commercially available drugs. In the present study, applying a single-step green microwave irradiation mediated protocol we have synthesized eleven quinazolinon-4(3H)-ones, from cheap readily available anthranilic acids, in very good yields and purity. These products were irradiated in the presence of pBR322 plasmid DNA under UVB, UVA and visible light. Four of the compounds proved to be very effective DNA photocleavers, at low concentrations, being time and concentration dependent as well as pH independent. Participation of reactive oxygen species was related to the substitution of quinazolinone derivatives. 6-Nitro-quinazolinone in combination with UVA irradiation was found to be in vitro photodestructive for three cell lines; glioblastoma (U87MG and T98G) and mainly melanoma (A-375). Thus, certain appropriately substituted quinazolinones may serve as new lead photosensitizers for the development of promising biotechnological applications and as novel photochemo and photodynamic therapeutics.

Zn(II) complexes of (E)-4-(2-(pyridin-2-ylmethylene)hydrazinyl)quinazoline in combination with non-steroidal anti-inflammatory drug sodium diclofenac: Structure, DNA binding and photo-cleavage studies, antioxidant activity and interaction with albumin.

The interaction of the novel quinazoline (E)-4-(2-(pyridin-2-ylmethylene)hydrazinyl)quinazoline (L) with Zn2+ was performed in the absence or presence of the non-steroidal anti-inflammatory drug sodium diclofenac (Nadicl) and resulted in the formation of complexes [Zn(L)2](NO3)2·MeOH (1·MeOH) and [Zn(L)(dicl-O)2]·MeOH (2·MeOH), respectively. The two complexes were characterized by IR and 1H NMR spectroscopy and by single-crystal X-ray crystallography. In these complexes, L was tridentately coordinated to Zn(II) via the quinazoline, hydrazone and pyridine nitrogen atoms. Further studies concerning the behavior of the compounds towards calf-thymus (CT) DNA and supercoiled circular pBluescript KS II plasmid DNA (pDNA) have been performed. The complexes may bind to CT DNA via intercalation, with complex 1 showing higher binding affinity than 2. The complexes may cleave pDNA in the absence or presence of irradiation with UVA, UVB or visible light and the most active pDNA-cleavager is compound 1. The binding constants of the compounds for bovine serum albumin were calculated and the subdomain of the albumin where the compounds prefer to bind was determined. The free radical scavenging ability of the compounds was evaluated towards 1,1-diphenyl-picrylhydrazyl and 2,2΄-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) radicals with complex 2 being the most active compound. Thus, complex of type 1 maybe a lead compound for the development of novel DNA-binders and DNA-cleavers or photo-cleavers for medical and biotechnological "on demand" applications, whereas the structure of complex type 2 may provide novel antioxidants and radical scavengers.

Structure and biological evaluation of pyridine-2-carboxamidine copper(II) complex resulting from N'-(4-nitrophenylsulfonyloxy)2-pyridine-carboxamidoxime.

The interaction of Cu(NO3)2·3H2O with the sulfonyl o-pyridine carboxamidoxime N'-(4-nitrophenylsulfonyloxy)picolinimidamide (L) resulted in the mononuclear complex [Cu(L1)2](L2)2 (1), where L1 = pyridine-2-carboxamidine ligand and (L2)- = 4-nitrobenzenesulfonate anion derived from the homolytic cleavage of the NO bond of L. The complex was characterized by diverse techniques including single-crystal X-ray crystallography. From the antimicrobial tests performed, complex 1 seems to be active against gram-negative bacterial strains. The complex binds tightly and reversibly to serum albumins and tightly to calf-thymus DNA via an intercalative mode and also via electrostatic interactions (as expected due to its cationic nature). Additionally, it interacts with (pBluescriptSK(+)) plasmid DNA in a concentration-dependent manner. The results from the present in silico molecular modeling simulations provide useful complementary insights for the elucidation of the mechanism of action of the studied complex at a molecular level. Molecular modeling calculations provide a molecular basis for the understanding of both the impairment of DNA by its binding with the studied complex and the ability of this compound to act as an antibacterial agent, most probably by its activity against DNA-gyrase, as well as for transportation through serum albumins and possible interaction with other protein targets involved in various diseases.

Alkyl and aryl sulfonyl p-pyridine ethanone oximes are efficient DNA photo-cleavage agents.

Sulfonyloxyl radicals, readily generated upon UV irradiation of p-pyridine sulfonyl ethanone oxime derivatives, effectively cleave DNA, in a pH independent manner, and under either aerobic or anaerobic conditions. p-Pyridine sulfonyl ethanone oxime derivatives were synthesized from the reaction of p-pyridine ethanone oxime with the corresponding sulfonyl chlorides in good to excellent yields. All compounds, at a concentration of 100µM, were irradiated at 312nm for 15min, after incubation with supercoiled circular pBluescript KS II DNA and resulted in extended single- and double- strand cleavages. The cleavage ability was found to be concentration dependent, with some derivatives exhibiting activity even at nanomolar levels. Besides that, p-pyridine sulfonyl ethanone oxime derivatives showed good affinity to DNA, as it was observed with UV interaction and viscosity experiments with CT DNA and competitive studies with ethidium bromide. The compounds interact to CT DNA probably by non-classical intercalation (i.e. groove-binding) and at a second step they may intercalate within the DNA base pairs. The fluorescence emission spectra of pre-treated EB-DNA exhibited a significant or moderate quenching. Comparing with the known aryl carbonyloxyl radicals the sulfonyloxyl ones are more powerful, with both aryl and alkyl sulfonyl substituted derivatives to exhibit DNA photo-cleaving ability, in significantly lower concentrations. These properties may serve in the discovery of new leads for "on demand" biotechnological and medical applications.

Natural and synthetic coumarin derivatives with anti-inflammatory/ antioxidant activities.

Several natural products with a coumarinic moiety have been reported to have multiple biological activities. It is to be expected that, in a similar way to isomeric flavonoids, coumarins might affect the formation and scavenging of reactive oxygen species (ROS) and influence processes involving free radical-mediated injury. Coumarin can reduce tissue edema and inflammation. Moreover coumarin and its 7-hydroxy-derivative inhibit prostaglandin biosynthesis, which involves fatty acid hydroperoxy intermediates. Natural products like esculetin, fraxetin, daphnetin and other related coumarin derivatives are recognised as inhibitors not only of the lipoxygenase and cycloxygenase enzymic systems, but also of the neutrophil-dependent superoxide anion generation. Due to the unquestionable importance of coumarin derivatives considerable efforts have been made by several investigators, to prepare new compounds bearing single substituents, or more complicated systems, including heterocyclic rings mainly at 3-, 4- and/or 7-positions. In this review we shall deal with naturally occurring or synthetically derived coumarin derivatives, which possess anti-inflammatory as well as antioxidant activities.

Synthesis and evaluation of the antioxidant and antiinflammatory activities of some benzo[l]khellactone derivatives and analogues.

Treatment of 3-hydroxy-beta-lapachone 4 with ylide 5 gave the coumarin derivative 7a, which was transformed to compounds 10-14. Compound 14 was then transformed to benzo[f]seselin 15 as well as to benzo[l]khellactones 16, 18 from which the title compounds 17, 19(I), 19(II), 20, 21(I) and 21(II) were prepared. All the tested compounds were found to interact with DPPH in a concentration and time dependent manner. All the tested compounds highly inhibited the soybean lipoxygenase, whereas compounds 12, 17 and 19(II) highly compete with DMSO for (*)OH. Compounds 7a, 7b, 12 and 17 induced at 48.7-58.9% protection against carrageenin induced rat paw edema.

Total Synthesis of Apoptolidin: Part 1. Retrosynthetic Analysis and Construction of Building Blocks.

No less than 30 stereogenic elements, a highly unsaturated 20-membered macrocyclic system, four carbohydrate units, and unique biological activity, make the natural occurring apoptolidin (1) a challenging synthetic target. The retrosynthetic analysis revealed five key building blocks-three for the construction of the macrolide ring B and two prospective pendant saccharide units-which were synthesized in a highly convergent manner and then connected. Apoptolidin's rather labile nature proved particularly challenging in the final deprotection, purification, and characterization procedures.

Total Synthesis of Apoptolidin: Part 2. Coupling of Key Building Blocks and Completion of the Synthesis.

No less than 30 stereogenic elements, a highly unsaturated 20-membered macrocyclic system, four carbohydrate units, and unique biological activity, make the natural occurring apoptolidin (1) a challenging synthetic target. The retrosynthetic analysis revealed five key building blocks-three for the construction of the macrolide ring B and two prospective pendant saccharide units-which were synthesized in a highly convergent manner and then connected. Apoptolidin's rather labile nature proved particularly challenging in the final deprotection, purification, and characterization procedures.

Synthesis of stable aromatic and heteroaromatic sulfonyl-amidoximes and evaluation of their antioxidant and lipid peroxidation activity.

We describe herein the synthesis of stable aromatic and heteroaromatic sulfonyl-amidoximes, from the reaction of amidoximes with the corresponding sulfonyl chlorides, in low to excellent yields. Evaluation of their antioxidant activity has shown that 17 out of 28 compounds highly compete DMSO for hydroxyl radicals, while five of them inhibit lipid peroxidation. Combining the reducing and anti-lipid peroxidation ability it seems that compounds 13 and 31 could be used as lead molecules.

Stable tumor vessel normalization with pO2 increase and endothelial PTEN activation by inositol trispyrophosphate brings novel tumor treatment.

Tumor hypoxia is a characteristic of cancer cell growth and invasion, promoting angiogenesis, which facilitates metastasis. Oxygen delivery remains impaired because tumor vessels are anarchic and leaky, contributing to tumor cell dissemination. Counteracting hypoxia by normalizing tumor vessels in order to improve drug and radio therapy efficacy and avoid cancer stem-like cell selection is a highly challenging issue. We show here that inositol trispyrophosphate (ITPP) treatment stably increases oxygen tension and blood flow in melanoma and breast cancer syngeneic models. It suppresses hypoxia-inducible factors (HIFs) and proangiogenic/glycolysis genes and proteins cascade. It selectively activates the tumor suppressor phosphatase and tensin homolog (PTEN) in vitro and in vivo at the endothelial cell (EC) level thus inhibiting PI3K and reducing tumor AKT phosphorylation. These mechanisms normalize tumor vessels by EC reorganization, maturation, pericytes attraction, and lowering progenitor cells recruitment in the tumor. It strongly reduces vascular leakage, tumor growth, drug resistance, and metastasis. ITPP treatment avoids cancer stem-like cell selection, multidrug resistance (MDR) activation and efficiently enhances chemotherapeutic drugs activity. These data show that counteracting tumor hypoxia by stably restoring healthy vasculature is achieved by ITPP treatment, which opens new therapeutic options overcoming hypoxia-related limitations of antiangiogenesis-restricted therapies. By achieving long-term vessels normalization, ITPP should provide the adjuvant treatment required in order to overcome the subtle definition of therapeutic windows for in vivo treatments aimed by the current strategies against angiogenesis-dependent tumors.