ESR studies of structure and kinetics of radicals from hydroxyurea: An antitumor drug directed against ribonucleotide reductase

doi:10.1016/0891-5849(89)90050-6

Free Radical Biology and Medicine

Volume 6, Issue 3, 1989, Pages 241-244

https://doi.org/10.1016/0891-5849(89)90050-6 Get rights and content

Abstract

Hydroxyurea (HU) is a clinically applied antineoplastic drug, which quenches tyrosine radicals in the active stie of ribonucleotide reductase (RR) and inhibits DNA synthesis in proliferating cells. Under oxidizing conditions (Cu²⁺ or H₂O₂) long-lived radicals from HU have been found by ESR. The structure of HU radicals was established to be;

. The kinetics of formation and decay of HU radicals after reaction of HU with H₂O₂ is complex; it exhibits a lag-phase, a maximum, and a decay, all depending on the concentration of HU. Biological consequences of HU radicals for the inhibition of RR as wll as their role in cytotoxic events during chemotherapy of cancer are discussed.

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Altered forms of mammalian nucleotide diphosphate reductase from mutant cells lines
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Hydroxyurea
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Meso-α-β-diphenylsuccinate and hydroxyurea as inhibitor of deoxycytidilate synthesis in extracts of Ehrlich ascites and L-cells
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Characterization of the free radical of mammalian ribonucleotide reductase
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Protection of L5178Y cells by vitamin E against acute hydroxyurea toxicity does not change the efficiency of ribonucleotide reductase-mediated hydroxyurea-induced cytotoxic events
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Hydroxyurea has the capacity to induce to human erythrocytes which can be modified by radical scavengers
Biochem. Biophys. Res. Commun.
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The ribonucleotide reductases—a unique group of metalloenzymes essential for cell proliferation
Struct, Bonding
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Cited by (38)

Oxidation mechanism of hydroxamic acids forming HNO and NO. implications for biological activity
2015, Advances in Inorganic Chemistry
Citation Excerpt :
Oxidation of HXs forming HNO and NO involves the loss of 2 and 3 electrons, respectively. One-electron oxidation of RC(O)NR′OH (37–43) and hydroxyurea (26–28) to their respective transient nitroxide radicals using various oxidizing systems has been previously shown by electron paramagnetic resonance (EPR) spectroscopy. Pulse radiolysis has been used only to oxidize desferrioxamine (Scheme 1) by OH, CO3−, and NO2 radicals, but the mechanism of the decomposition of the respective transient nitroxide radical has not been studied (44,45).
Hydroxamic acids (RC(O)NHOH, HXs) display interesting chemical and biological properties. The pharmacological effects of HXs have been partially attributed to their ability to serve as HNO- or NO-donors under oxidative stress. We have demonstrated that one-electron oxidation of HXs by radiolytically borne radicals yields the respective transient nitroxide radicals, which decompose bimolecularly forming HNO. HXs oxidation by the metmyoglobin and H₂O₂ reactions system involves relatively milder oxidants such as ferryl myoglobin, which oxidize not only HXs but also the nitroxide radical, HNO and NO. The distinction between the effects of NO and HNO in vivo is masked by the reversible redox exchange between these two congeners and due to the Janus-faced behavior of NO and HNO. Acetohydroxamic acid potentiates H₂O₂-induced killing of Escherichia coli, whereas suberoylanilide hydroxamic acid (SAHA) protects mammalian cells subjected to oxidative stress. These results are similar to those observed for NO implying that in these systems HXs serve as NO-donors. In contrast, the effect of acetohydroxamic acid on Bacillus subtilis subjected to oxidative stress is similar to that of HNO. SAHA, but not valproic acid lacking the hydroxamate moiety, enhances the radiosensitization of hypoxic tumor cells in vitro. Given that NO, but not HNO, is a hypoxic cell radiosensitizer, the enhancement of tumor radioresponse by SAHA might involve its ability to serve as a NO-donor. It is concluded that HXs form HNO under oxidative stress, but can be considered as NO-donors if HNO oxidation competes with N₂O formation and with its reaction with potential biological targets.
Investigation of the scavenging mechanism of tyrosyl radical by hydroxybenzohydroxamic acid derivatives: A DFT study
2013, Computational and Theoretical Chemistry
Citation Excerpt :
Experimental studies have revealed that HU and hydroxybenzohydroxamic acid destroy the tyrosyl radical. The structure of the HU radical was established by ESR to be H2NCON(H)O [12], so it is called a “free radical quencher”. TX and DX have radical scavenging activity and can inhibit the tyrosyl radical [13].
The free radical scavenging activity of a series of hydroxybenzohydroxamic acid derivatives have been studied in gas phase, water and benzene environments, using the density functional theory. Different mechanisms of reactions have been considered: single electron transfer (SET), hydrogen atom transfer (HAT). Rate constants were determined to know if the radical scavenging activity of these compounds proceeds via an H-atom or an electron-transfer mechanism. Calculations showed that the presence of the adjacent hydroxyl groups on phenyl ring increases the radical stability through intramolecular hydrogen bonds. The calculated bond dissociation enthalpy (BDE) values for hydroxyl groups on phenyl ring of studied compounds revealed the important role of resonance in stability of radical obtained from the hydrogen atom abstraction by free radical. Results show that the radical scavenging capacity of trimidox is more than the other molecules. The results demonstrate that the H-atom transfer mechanism for scavenging of free radical is more preferable than the single-electron transfer in considered environments.
The mechanism underlying nitroxyl and nitric oxide formation from hydroxamic acids
2012, Biochimica et Biophysica Acta - General Subjects
The pharmacological effects of hydroxamic acids (RC(O)NHOH, HX) are partially attributed to their ability to serve as HNO and/or NO donors under oxidative stress. Given the development and use of HXs as therapeutic agents, elucidation of the oxidation mechanism is needed for more educated selection of HX-based drugs.
Acetohydroxamic and glycine-hydroxamic acids were oxidized at pH 7.0 by a continuous flux of radiolytically generated ^·OH or by metmyoglobin and H₂O₂ reactions system. Gas chromatography and spectroscopic methods were used to monitor the accumulation of N₂O, N₂, nitrite and hydroxylamine.
Oxidation of HXs by ^·OH under anoxia yields N₂O, but not nitrite, N₂ or hydroxylamine. Upon the addition of H₂O₂ to solutions containing HX and metmyoglobin, which is instantaneously and continuously converted into compound II, nitrite and, to a lesser extent, N₂O are accumulated under both anoxia and normoxia.
Oxidation of HXs under anoxia by a continuous flux of ^·OH, which solely oxidizes the hydroxamate moiety to RC(O)NHO^·, forms HNO. This observation implies that bimolecular decomposition of RC(O)NHO^· competes efficiently with unimolecular decomposition processes such as internal disproportionation, hydrolysis or homolysis. Oxidation by metmyoglobin/H₂O₂ involves relatively mild oxidants (compounds I and II). Compound I reacts with HX forming RC(O)NHO^· and compound II, which oxidizes HX, RC(O)NHO^·, HNO and NO. The latter reaction is the main source of nitrite.
HXs under oxidative stress release HNO, but can be considered as NO-donors provided that HNO oxidation is more efficient than its reaction with other biological targets.
Catalytic oxidation of hydroxyurea to bound NO+/ NO<inf>2</inf>- Mediated by pentacyano(L)ferrates. Characterization of the nitroxide radical, bound C-nitrosoformamide and NO• as reaction intermediates
2011, Inorganica Chimica Acta
Hydroxyurea (HU, NH₂CONHOH), reacts with several oxidizing agents (O₂, H₂O₂, ${IO}_{4}^{-}$ , Fe(III) complexes) in aqueous solutions containing either of the pentacyano(aqua)ferrate(II,III) ions, [Fe^II,III(CN)₅H₂O]^3,2−. In the initial 1-electron step, HU reacts with [Fe^III(CN)₅H₂O]²⁻ through an outer-sphere process, leading to [Fe^II(CN)₅H₂O]³⁻ and the EPR active nitroxide radical, NH₂C(O)NHO. This is further oxidized to C-nitrosoformamide, NH₂C(O)NO, in a similar 1-electron step. The latter compound binds to [Fe^II(CN)₅H₂O]³⁻ giving the [Fe^II(CN)₅(NOC(O)NH₂)]³⁻ ion (I³⁻). I³⁻ has been isolated as a solid, Na₃[Fe^II(CN)₅(NOC(O)NH₂)]·2H₂O, (I), and was identified by chemical analysis, IR and UV–Vis spectroscopies (λ_max = 465 nm, ε = 4.0 ± 0.3 × 10³ M⁻¹ cm⁻¹). I and I³⁻ are very stable species in anaerobic media, as solids and in aqueous solutions with a pH close to 7. I³⁻ decomposes in alkaline media through hydrolysis, giving CO₂, NH₃ and, presumably, the nitroxyl complex [Fe^II(CN)₅NO]⁴⁻. The latter ion appears as a source of N₂O, detected by mass spectrometry, and affords, in oxygenated media, two subsequent 1-electron processes, without the rupture of the NO bond, giving the radical intermediate [Fe^II(CN)₅NO]³⁻ (which was identified by UV–Vis and EPR), and finally the nitroprusside ion, [Fe(CN)₅NO]²⁻. At pHs > 10, the latter ion may convert to [Fe^II(CN)₅NO₂]⁴⁻, and leads to [Fe^II(CN)₅H₂O]³⁻ through the release of nitrite. Thus, the aqua-ion might afford a new 4-electron oxidation process if HU and the external oxidant were in excess.
Oxidation of hydroxyurea with oxovanadium(V) ions in acidic aqueous solution
2006, Journal of Inorganic Biochemistry
Hydroxyurea (HU) effectively reduces vanadium(V) into vanadium(IV) species (hereafter V^V and V^IV species, respectively) in acidic aqueous solution via the formation of a transient complex followed by an electron transfer process that includes the formation and subsequent fading out of a free radical, U (U ≡ H₂N–C(=O)N(H)O). The electron paramagnetic resonance (EPR) spectra of U in H₂O/D₂O solutions suggest that the unpaired electron is located predominantly on the hydroxamate hydroxyl-oxygen atom. Visible and V^IV–EPR spectroscopic data reveal HU as a two-electron donor, whereas formation of U, which reduces a second V^V, indicates that electron transfer occurs in two successive one-electron steps. At the molarity ratio [V^V]/[HU] = 2, the studied reaction can be formulated as: 2 V^V + HU → 2 V^IV + 0.98 CO₂ + 0.44 N₂O + 1.1 NH₃ + 0.1 NH₂OH. Lack of evidence for the formation of NO is suggested to be a consequence of the slow oxidation of HNO due to the too low reduction potential of the V^V/V^IV couple under the experimental conditions used.
The nuclear magnetic resonance (⁵¹V-NMR) spectral data indicate protonation of $(H_{2} O)_{4} V^{V} O_{2}^{+}$ , and the protonation equilibrium constant was determined to be K = 0.7 M⁻¹. Spectrophotometric titration data for the V^V–HU system reveal formation of (H₂O)₂V^VO(OH)U⁺ and (H₂O)₃V^VOU²⁺. Their stability constants were calculated as K₁₁₀ = 5 M⁻¹ and K₁₁₁ = 22 M⁻², where the subscript digits refer to $(H_{2} O)_{4} V^{V} O_{2}^{+}$ , HU and H⁺, respectively.
Nitric oxide generation from hydroxyurea: Significance and implications for leukemogenesis in the management of myeloproliferative disorders
2006, Blood
The use of myelosuppressive agents to reduce the risk of thrombosis in patients with polycythemia vera (PV) and essential thrombocythemia (ET) has been associated with an increased risk of transformation to acute myeloid leukemia (AML). Whereas chlorambucil, busulfan, and radiophosphorus (³²P) have been demonstrated to increase the risk of transformation, the leukemogenic potential of hydroxyurea (HU) continues to be a matter of debate. Clinical studies have suggested that HU may cause a small increase in the risk of AML, but it has proven difficult to establish whether AML is actually caused by HU or arises during the natural progression of PV and ET. Reports that HU undergoes metabolic activation to species that induce mutation appear to support the notion that it is leukemogenic. Here, we suggest that the ability of HU to induce mutation in cell culture studies results from the generation of nitrogen dioxide via the autoxidation of nitric oxide, a product of HU metabolism. However, we argue that autoxidation would not occur in vivo, leading to the conclusion that generation of the mutagen nitrogen dioxide is peculiar to cell culture systems and has little relevance to the use of HU in the management of PV and ET.

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Free Radical Biology and Medicine

Abstract

References (19)

Altered forms of mammalian nucleotide diphosphate reductase from mutant cells lines

Pharmacol. Ther.

Hydroxyurea

Mutation Res.

Meso-α-β-diphenylsuccinate and hydroxyurea as inhibitor of deoxycytidilate synthesis in extracts of Ehrlich ascites and L-cells

J. Biol. Chem.

Electron spin resonance of the iron-containing protein B2 from ribonucleotide reductase

J. Biol. Chem.

Nature of the free radical in ribonucleotide reductase from Escherichia coli

J. Biol. Chem.

Characterization of the free radical of mammalian ribonucleotide reductase

J. Biol. Chem.

Protection of L5178Y cells by vitamin E against acute hydroxyurea toxicity does not change the efficiency of ribonucleotide reductase-mediated hydroxyurea-induced cytotoxic events

Cancer Letters

Hydroxyurea has the capacity to induce to human erythrocytes which can be modified by radical scavengers

Biochem. Biophys. Res. Commun.

The ribonucleotide reductases—a unique group of metalloenzymes essential for cell proliferation

Struct, Bonding

Cited by (38)

Oxidation mechanism of hydroxamic acids forming HNO and NO. implications for biological activity

Investigation of the scavenging mechanism of tyrosyl radical by hydroxybenzohydroxamic acid derivatives: A DFT study

The mechanism underlying nitroxyl and nitric oxide formation from hydroxamic acids

Catalytic oxidation of hydroxyurea to bound NO<sup>+</sup>/ NO<inf>2</inf><sup>-</sup> Mediated by pentacyano(L)ferrates. Characterization of the nitroxide radical, bound C-nitrosoformamide and NO<sup>•</sup> as reaction intermediates

Oxidation of hydroxyurea with oxovanadium(V) ions in acidic aqueous solution

Nitric oxide generation from hydroxyurea: Significance and implications for leukemogenesis in the management of myeloproliferative disorders

Original contributionESR studies of structure and kinetics of radicals from hydroxyurea: An antitumor drug directed against ribonucleotide reductase

Abstract

Pharmacol. Ther.

Mutation Res.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Cancer Letters

Biochem. Biophys. Res. Commun.

The ribonucleotide reductases—a unique group of metalloenzymes essential for cell proliferation

Struct, Bonding

Original contribution
ESR studies of structure and kinetics of radicals from hydroxyurea: An antitumor drug directed against ribonucleotide reductase