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ACS Omega Feb 2020Triethylphosphinegold(I) complexes [Au(HL1)P(CHCH)]PF (), [Au(HL2)P(CHCH)]PF (), and [Au(HL3)P(CHCH)]PF () were obtained with...
Triethylphosphinegold(I) complexes [Au(HL1)P(CHCH)]PF (), [Au(HL2)P(CHCH)]PF (), and [Au(HL3)P(CHCH)]PF () were obtained with ()-2-(1-(2-methyl-5-nitro-1H-imidazol-1-yl)propan-2-ylidene)hydrazinecarbothioamide (), ()--methyl-2-(1-(2-methyl-5-nitro-1H-imidazol-1-yl)propan-2-ylidene)hydrazinecarbothioamide (), and ()-2-(1-(2-methyl-5-nitro-1H-imidazol-1-yl)propan-2-ylidene)--phenylhydrazinecarbothioamide (). All compounds were assayed for their cytotoxic activities against HCT-116 colorectal carcinoma cells under normoxia and hypoxia conditions and against nonmalignant HEK-293 human embryonic kidney cells under normoxia conditions. The thiosemicarbazone ligands - were inactive against HCT-116 cells under hypoxia but while was inactive, and proved to be cytotoxic to both cell lineages under normoxia conditions. Complexes (-) and the triethylphosphinegod(I) precursor proved to be active against both cell lineages in normoxia as well as in hypoxia. While and revealed to be active against HEK-293 and HCT-116 cells, being approximately as active against HCT-116 cells in normoxia as under hypoxia, complex () proved to be more active against HCT-116 cells under hypoxia than under normoxia conditions, and more active against HCT-116 cells than against the nonmalignant HEK-293 cells, with the selectivity index, calculated as SI = IC/IC, equal to 3.7, similar to the value obtained for the control drug tirapazamine (tirapazamine (TPZ), SI = 4). Although the compounds showed distinct cytotoxic activities, the electrochemical behaviors of - were very similar, as were the behaviors of complexes (-). Complex () deserves special interest since it was significantly more active under hypoxia than under normoxia conditions. Hence, in this case, selective reduction of the nitro group in a low oxygen pressure environment, resulting in toxic reactive oxygen species (ROS) and damage to DNA or other biomolecules, might operate, while for the remaining compounds, other modes of action probably occur.
PubMed: 32095716
DOI: 10.1021/acsomega.9b03778 -
Pharmaceuticals (Basel, Switzerland) Jan 2019: Benzotriazine-1,4-dioxides (BTDOs) such as tirapazamine (TPZ) and its derivatives act as radiosensitizers of hypoxic tissues. The benzotriazine-1-monoxide (BTMO)...
: Benzotriazine-1,4-dioxides (BTDOs) such as tirapazamine (TPZ) and its derivatives act as radiosensitizers of hypoxic tissues. The benzotriazine-1-monoxide (BTMO) metabolite (SR 4317, TPZMO) of TPZ also has radiosensitizing properties, and via unknown mechanisms, is a potent enhancer of the radiosensitizing effects of TPZ. Unlike their 2-nitroimidazole radiosensitizer counterparts, radiolabeled benzotriazine oxides have not been used as radiopharmaceuticals for diagnostic imaging or molecular radiotherapy (MRT) of hypoxia. The radioiodination chemistry for preparing model radioiodinated BTDOs and BTMOs is now reported. : Radioiodinated 3-(2-iodoethoxyethyl)-amino-1,2,4-benzotriazine-1,4-dioxide (I-EOE-TPZ), a novel bioisosteric analogue of TPZ, and 3-(2-iodoethoxyethyl)-amino-1,2,4-benzotriazine-1-oxide (I-EOE-TPZMO), its monoxide analogue, are candidates for in vivo and in vitro investigations of biochemical mechanisms in pathologies that develop hypoxic microenvironments. In theory, both radiotracers can be prepared from the same precursors. : Radioiodination procedures were based on classical nucleophilic [I]iodide substitution on Tos-EOE-TPZ (P1) and by [I]iodide exchange on I-EOE-TPZ (P2). Reaction parameters, including temperature, reaction time, solvent and the influence of pivalic acid on products' formation and the corresponding radiochemical yields (RCY) were investigated. : The [I]iodide labeling reactions invariably led to the synthesis of both products, but with careful manipulation of conditions the preferred product could be recovered as the major product. Radioiodide exchange on P2 in ACN at 80 ± 5 °C for 30 min afforded the highest RCY, 89%, of [I]I-EOE-TPZ, which upon solid phase purification on an alumina cartridge gave 60% yield of the product with over 97% of radiochemical purity. Similarly, radioiodide exchange on P2 in ACN at 50 ± 5 °C for 30 min with pivalic acid afforded the highest yield, 92%, of [I]I-EOE-TPZMO exclusively with no trace of [I]I-EOE-TPZ. In both cases, extended reaction times and/or elevated temperatures resulted in the formation of at least two additional radioactive reaction products. : Radioiodination of P1 and P2 with [I]iodide leads to the facile formation of [I]I-EOE-TPZMO. At 80 °C and short reaction times, the facile reduction of the N-4-oxide moiety was minimized to afford acceptable radiochemical yields of [I]I-EOE-TPZ from either precursor. Regeneration of [I]I-EOE-TPZ from [I]I-EOE-TPZMO is impractical after reaction work-up.
PubMed: 30609671
DOI: 10.3390/ph12010003 -
Tumour Biology : the Journal of the... Aug 2016In this work, the in vitro tests of biological activity of benzimidazoles were conducted. This group of benzimidazole derivatives was evaluated as potential bioreductive...
In this work, the in vitro tests of biological activity of benzimidazoles were conducted. This group of benzimidazole derivatives was evaluated as potential bioreductive agents and their characteristic pro-apoptosis activity and cell cycle interruption on the human lung adenocarcinoma A549 cells were discussed. Their toxicity on the healthy human erythrocytes and their influence on the healthy human erythrocytes acetylcholinesterase enzyme (AChE) were established. Their apoptosis activity on A549 cells line was determined by Annexin V-APC test, and it was visualized by Hoechst test. In the next stage, their influence on the cell cycle interruption was determined by using the ribonuclease reagent. The AChE inhibition test was defined by the Ellman method, and the red blood cell lysis was defined by erythrotoxicity test. The results proved the pro-apoptosis properties of all tested compounds in normoxia and hypoxia. The DNA content assay showed that the benzimidazoles possess the ability to interrupt S phase of tumor cell cycle. The best activity in this action was presented by compound 1, especially in hypoxia, and it proves that the N-oxide analogs are predispositioned to the hypoxic target. In this study, the benzimidazoles were found as potentially biocompatible and their inhibition of acetylcholinesterase was lower than tirapazamine and much lower than tacrine which constitutes their desired effect of potential biological activity.
Topics: Antineoplastic Agents; Apoptosis; Benzimidazoles; Cell Cycle; Cell Line, Tumor; Drug Screening Assays, Antitumor; Erythrocytes; Flow Cytometry; Humans; Inhibitory Concentration 50; Microscopy, Confocal; Molecular Structure; Structure-Activity Relationship
PubMed: 26932526
DOI: 10.1007/s13277-016-4828-1 -
Molecules (Basel, Switzerland) Jul 2019Extracellular acidification is an important feature of tumor microenvironments but has yet to be successfully exploited in cancer therapy. The reversal of the pH...
Extracellular acidification is an important feature of tumor microenvironments but has yet to be successfully exploited in cancer therapy. The reversal of the pH gradient across the plasma membrane in cells that regulate intracellular pH (pHi) has potential to drive the selective uptake of weak acids at low extracellular pH (pHe). Here, we investigate the dual targeting of low pHe and hypoxia, another key feature of tumor microenvironments. We prepared eight bioreductive prodrugs based on the benzotriazine di-oxide (BTO) nucleus by appending alkanoic or aminoalkanoic acid sidechains. The BTO acids showed modest selectivity for both low pHe (pH 6.5 versus 7.4, ratios 2 to 5-fold) and anoxia (ratios 2 to 8-fold) in SiHa and FaDu cell cultures. Related neutral BTOs were not selective for acidosis, but had greater cytotoxic potency and hypoxic selectivity than the BTO acids. Investigation of the uptake and metabolism of representative BTO acids confirmed enhanced uptake at low pHe, but lower intracellular concentrations than expected for passive diffusion. Further, the modulation of intracellular reductase activity and competition by the cell-excluded electron acceptor WST-1 suggests that the majority of metabolic reductions of BTO acids occur at the cell surface, compromising the engagement of the resulting free radicals with intracellular targets. Thus, the present study provides support for designing bioreductive prodrugs that exploit pH-dependent partitioning, suggesting, however, that that the approach should be applied to prodrugs with obligate intracellular activation.
Topics: Cell Hypoxia; Cell Line, Tumor; Chemical Phenomena; Dose-Response Relationship, Drug; Drug Design; Humans; Hydrogen-Ion Concentration; Models, Biological; Molecular Structure; Neoplasms; Oxidation-Reduction; Oxides; Prodrugs; Triazines
PubMed: 31295864
DOI: 10.3390/molecules24142524 -
ACS Omega Jul 2020The process selection and subsequent development of a reliable, scalable synthesis of the anticancer prodrug tirapazamine (SR259075) is described in this paper. Reaction...
The process selection and subsequent development of a reliable, scalable synthesis of the anticancer prodrug tirapazamine (SR259075) is described in this paper. Reaction of benzofuroxan with cyanamide in acetonitrile in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene at 20-25 °C afforded, after an acidic workup, the targeted molecule in good yield at a kilogram scale. Notable critical parameters and safety enhancements are defined and successfully implemented to produce three consecutive validation batches in a reproducible manner.
PubMed: 32685820
DOI: 10.1021/acsomega.0c01250 -
International Journal of Nanomedicine 2016Reductive drug-functionalized gold nanoparticles (AuNPs) have been proposed to enhance the damage of X-rays to cells through improving hydroxyl radical production by...
Reductive drug-functionalized gold nanoparticles (AuNPs) have been proposed to enhance the damage of X-rays to cells through improving hydroxyl radical production by secondary electrons. In this work, polyethylene glycol-capped AuNPs were conjugated with tirapazamine (TPZ) moiety, and then thioctyl TPZ (TPZs)-modified AuNPs (TPZs-AuNPs) were synthesized. The TPZs-AuNPs were characterized by transmission electron microscopy, ultraviolet-visible spectra, dynamic light scattering, and inductively coupled plasma mass spectrometry to have a size of 16.6±2.1 nm in diameter and a TPZs/AuNPs ratio of ~700:1. In contrast with PEGylated AuNPs, the as-synthesized TPZs-AuNPs exhibited 20% increment in hydroxyl radical production in water at 2.0 Gy, and 19% increase in sensitizer enhancement ratio at 10% survival fraction for human hepatoma HepG2 cells under X-ray irradiation. The production of reactive oxygen species in HepG2 cells exposed to X-rays in vitro demonstrated a synergistic radiosensitizing effect of AuNPs and TPZ moiety. Thus, the reductive drug-conjugated TPZs-AuNPs as a kind of AuNP radiosensitizer with low gold loading provide a new strategy for enhancing the efficacy of radiation therapy.
Topics: Gold; Hep G2 Cells; Humans; Hydroxyl Radical; Metal Nanoparticles; Microscopy, Electron, Transmission; Polyethylene Glycols; Radiation-Sensitizing Agents; Tirapazamine; Triazines; Tumor Microenvironment; X-Rays
PubMed: 27555772
DOI: 10.2147/IJN.S105348 -
The Journal of Clinical Investigation Jun 2021Hypoxia, a hallmark feature of the tumor microenvironment, causes resistance to conventional chemotherapy, but was recently reported to synergize with poly(ADP-ribose)...
Hypoxia, a hallmark feature of the tumor microenvironment, causes resistance to conventional chemotherapy, but was recently reported to synergize with poly(ADP-ribose) polymerase inhibitors (PARPis) in homologous recombination-proficient (HR-proficient) cells through suppression of HR. While this synergistic killing occurs under severe hypoxia (<0.5% oxygen), our study shows that moderate hypoxia (2% oxygen) instead promotes PARPi resistance in both HR-proficient and -deficient cancer cells. Mechanistically, we identify reduced ROS-induced DNA damage as the cause for the observed resistance. To determine the contribution of hypoxia to PARPi resistance in tumors, we used the hypoxic cytotoxin tirapazamine to selectively kill hypoxic tumor cells. We found that the selective elimination of hypoxic tumor cells led to a substantial antitumor response when used with PARPi compared with that in tumors treated with PARPi alone, without enhancing normal tissue toxicity. Since human breast cancers with BRAC1/2 mutations have an increased hypoxia signature and hypoxia reduces the efficacy of PARPi, then eliminating hypoxic tumor cells should enhance the efficacy of PARPi therapy.
Topics: Animals; Cell Hypoxia; Cell Line, Tumor; DNA Damage; Female; Homologous Recombination; Humans; Mice; Mice, Nude; Neoplasms, Experimental; Poly(ADP-ribose) Polymerase Inhibitors; Reactive Oxygen Species; Xenograft Model Antitumor Assays
PubMed: 34060485
DOI: 10.1172/JCI146256 -
Angewandte Chemie (International Ed. in... Sep 2020Tirapazamine (TPZ) has been tested in clinical trials on radio-chemotherapy due to its potential highly selective toxicity towards hypoxic tumor cells. It was suggested...
Tirapazamine (TPZ) has been tested in clinical trials on radio-chemotherapy due to its potential highly selective toxicity towards hypoxic tumor cells. It was suggested that either the hydroxyl radical or benzotriazinyl radical may form as bioactive radical after the initial reduction of TPZ in solution. In the present work, we studied low-energy electron attachment to TPZ in the gas phase and investigated the decomposition of the formed TPZ anion by mass spectrometry. We observed the formation of the (TPZ-OH) anion accompanied by the dissociation of the hydroxyl radical as by far the most abundant reaction pathway upon attachment of a low-energy electron. Quantum chemical calculations suggest that NH pyramidalization is the key reaction coordinate for the reaction dynamics upon electron attachment. We propose an OH roaming mechanism for other reaction channels observed, in competition with the OH dissociation.
PubMed: 32543771
DOI: 10.1002/anie.202006675 -
Molecules (Basel, Switzerland) Oct 2020Hypoxia is an adverse prognostic feature of solid cancers that may be overcome with hypoxia-activated prodrugs (HAPs). Tirapazamine (TPZ) is a HAP which has undergone...
Hypoxia is an adverse prognostic feature of solid cancers that may be overcome with hypoxia-activated prodrugs (HAPs). Tirapazamine (TPZ) is a HAP which has undergone extensive clinical evaluation in this context and stimulated development of optimized analogues. However the subcellular localization of the oxidoreductases responsible for mediating TPZ-dependent DNA damage remains unclear. Some studies conclude only nuclear-localized oxidoreductases can give rise to radical-mediated DNA damage and thus cytotoxicity, whereas others identify a broader role for endoplasmic reticulum and cytosolic oxidoreductases, indicating the subcellular location of TPZ radical formation is not a critical requirement for DNA damage. To explore this question in intact cells we engineered MDA-231 breast cancer cells to express the TPZ reductase human NADPH: cytochrome P450 oxidoreductase (POR) harboring various subcellular localization sequences to guide this flavoenzyme to the nucleus, endoplasmic reticulum, cytosol or inner surface of the plasma membrane. We show that all POR variants are functional, with differences in rates of metabolism reflecting enzyme expression levels rather than intracellular TPZ concentration gradients. Under anoxic conditions, POR expression in all subcellular compartments increased the sensitivity of the cells to TPZ, but with a fall in cytotoxicity per unit of metabolism (termed 'metabolic efficiency') when POR is expressed further from the nucleus. However, under aerobic conditions a much larger increase in cytotoxicity was observed when POR was directed to the nucleus, indicating very high metabolic efficiency. Consequently, nuclear metabolism results in collapse of hypoxic selectivity of TPZ, which was further magnified to the point of reversing O dependence (oxic > hypoxic sensitivity) by employing a DNA-affinic TPZ analogue. This aerobic hypersensitivity phenotype was partially rescued by cellular copper depletion, suggesting the possible involvement of Fenton-like chemistry in generating short-range effects mediated by the hydroxyl radical. In addition, the data suggest that under aerobic conditions reoxidation strictly limits the TPZ radical diffusion range resulting in site-specific cytotoxicity. Collectively these novel findings challenge the purported role of intra-nuclear reductases in orchestrating the hypoxia selectivity of TPZ.
Topics: Antineoplastic Agents; Cell Engineering; Cell Hypoxia; Cell Line, Tumor; Cell Membrane; Cell Nucleus; Cell Survival; Copper; DNA Damage; Humans; Hypoxia; Models, Biological; NADPH-Ferrihemoprotein Reductase; Oxygen; Prodrugs; Tirapazamine
PubMed: 33105798
DOI: 10.3390/molecules25214888 -
Hypoxia (Auckland, N.Z.) 2017There is an urgent need to develop effective therapies and treatment strategies to treat hypoxic tumors, which have a very poor prognosis and do not respond well to...
PURPOSE
There is an urgent need to develop effective therapies and treatment strategies to treat hypoxic tumors, which have a very poor prognosis and do not respond well to existing therapies.
METHODS
A novel hypoxia-targeting agent, KEMTUB012-NI2, was synthesized by conjugating a 2-nitroimidazole hypoxia-targeting moiety to a synthetic tubulysin, a very potent antimitotic. Its hypoxic selectivity and mode of action were studied in breast cancer cell lines.
RESULTS
KEMTUB012-NI2 exhibited a similar selectivity for hypoxic cells to that of tirapazamine, a well-established hypoxia-targeting agent, but was >1,000 times more potent in cell cytotoxicity assays. The hypoxia-targeting mechanism for both KEMTUB012-NI2 and tirapazamine was selective and mediated by one-electron reductases. However, while cytochrome p450 reductase (POR) downregulation could inhibit tirapazamine cytotoxicity, it actually sensitized hypoxic cells to KEMTUB012-NI2.
CONCLUSION
KEMTUB012-NI2 is a potent new agent that can selectively target hypoxic cancer cells. The hypoxia selectivity of KEMTUB012-NI2 and tirapazamine appears to be differentially activated by reductases. Since reductases are heterogeneously expressed in tumors, the different activation mechanisms will allow these agents to complement each other. Combining POR downregulation with KEMTUB012-NI2 treatment could be a new treatment strategy that maximizes efficacy toward hypoxic tumor cells while limiting systemic toxicity.
PubMed: 28580362
DOI: 10.2147/HP.S132832