Transferring oxygen isotopes to 1,2,4-benzotriazine 1-oxides forming the corresponding 1,4-dioxides by using the HOF·CH3CN complex

Heterocyclic benzotriazine N-oxides are an interesting class of experimental anticancer and antibacterial agents. Analogs with ¹⁸O incorporated into the N-oxide group may offer useful mechanistic tools. We describe the use of H₂¹⁸OF·CH₃CN in a fast, readily executed and high-yielding preparation of 1,2,4-benzotriazine 1,4-dioxides containing an ¹⁸O-label at the 4-oxide position.     

Oxides of 3-methyl-1,2,4-benzotriazine

The previously prepared 3-methyl-1,2,4-benzotriazine oxide¹ is shown to be the 4-oxide 5. Synthesis and structures of other isomeric and related oxides are described. A modification of a previously described synthesis of 1,2,4-benzotriazines produces purer products in higher yields.     

A mass spectrometry study of tirapazamine and its metabolites. insights into the mechanism of metabolic transformations and the characterization of reaction intermediates

Tandem mass spectrometry methods were used to study the sites of protonation and for identification of 3-amino-1,2,4-benzotriazine 1,4-dioxide (1, tirapazamine), and its metabolites (3-amino-1,2,4-benzotriazine 1-oxide (3), 3-amino-1,2,4-benzotriazine 4-oxide (4), 3-amino-1,2,4-benzotriazine (5), and a related isomer 3-amino-1,2,4-benzotriazine 2-oxide (6). Fragmentation pathways of 3 and 5 indicated the 4-N-atom as the most likely site of protonation. Among the N-oxides studied, the 4-oxide (4) showed the highest degree of protonation at the oxygen atom. The differences in collision-induced dissociation of isomeric protonated 1-, 2- and 4-oxides allowed for their identification by LC/MS/MS. Gas phase and liquid phase protonation of tirapazamine occurred exclusively at the oxygen in the 4-position. A loss of OH radical from these ions (2(+)) resulted in ionized 3. Neutralization-reionization mass spectrometry (NR MS) experiments demonstrated the stability of the neutral analogue of protonated tirapazamine in the gas phase in the micro s time-frame. A significant portion of the neutral tirapazamine radicals (2) dissociated by loss of hydroxyl radical during the NR MS event, which indicates that previously proposed mechanisms for redox-activated DNA damage are reasonable. The activation energy for loss of hydroxyl radical from activated tirapazamine (2) was estimated to be approximately 14 kcal mol(-1). Stable neutral analogues of [3 + H](+) and [5 + H](+) ions were also generated in the course of NR MS experiments. Structures of these radicals were assigned to the molecules having an extra hydrogen atom at one of the ring N-atoms. Quantum chemical calculations of protonated 1, 3, 4 and 5 and the corresponding neutrals were performed to assist in the interpretation of experimental results and to help identify their structures.     

3-amino-1,2,4-benzotriazine 4-oxide: characterization of a new metabolite arising from bioreductive processing of the antitumor agent 3-amino-1,2,4-benzotriazine 1,4-dioxide (tirapazamine).

Tirapazamine (1) is a promising antitumor agent that selectively causes DNA damage in hypoxic tumor cells, following one-electron bioreductive activation. Surprisingly, after more than 10 years of study, the products arising from bioreductive metabolism of tirapazamine have not been completely characterized. The two previously characterized metabolites are 3-amino-1,2,4-benzotriazine 1-oxide (3) and 3-amino-1,2,4-benzotriazine (5). In this work, 3-amino-1,2,4-benzotriazine 4-oxide (4) is identified for the first time as a product resulting from one-electron activation of the antitumor agent tirapazamine by the enzymes xanthine/xanthine oxidase and NADPH:cytochrome P450 oxidoreductase. As part of this work, the novel N-oxide (4) was unambiguously synthesized and characterized using NMR spectroscopy, UV-vis spectroscopy, LC/MS, and X-ray crystallography. Under conditions where the parent drug tirapazamine is enzymatically activated, the metabolite 4 is produced but readily undergoes further reduction to the benzotriazine (5). Thus, under circumstances where extensive reductive metabolism occurs, the yield of the 4-oxide (4) decreases. In contrast, the isomeric two-electron reduction product 3-amino-1,2,4-benzotriazine 1-oxide (3) does not readily undergo enzymatic reduction and, therefore, is found as a major bioreductive metabolite under all conditions. Finally, the ability of the 4-oxide metabolite (4) to participate in tirapazamine-mediated DNA damage is considered.     

Reduction of cyclohexanone 2-nitrophenylhydrazone. Formation of cyclohexane-3-spiro-3,4-dihydro-1,2,4-benzotriazine

The structure of compound C₁₂H₁₅N₃, obtained by Perkin and Riley in 1923, through the reduction of cyclohexanone 2-nitrophenylhydrazone, was reexamined. This compound, considered originally as 3,4-cyclotetramethylene-4,5-dihydro-1,2,5-benzotriazepine (I) and later as 2-aminophenylazocyclohexene (II), is now defined through the nmr spectrum and chemical behaviour as cyclohexane-3-spiro-3,4-dihydro-1,2,4-benzotriazine (V). It is formed by spontaneous oxidation of the cyclic form of cyclohexanone 2-aminophenylhydrazone (namely, cyclohexane-3-spiro-1,2,3,4-tetrahydro-1,2,4-benzotriazine) obtained through amino group addition on the hydrazone double bond.     

Metal-Assisted Oxidative Cyclization of Arylamidrazones I. Synthesis of 3-Acetyl-1,4-dihydro-1-phenyl-1,2,4-benzotriazine

Summary. In the presence of RuCl₃, N-phenylamidrazone underwent oxidative cyclization into 1,4-dihydro-1-phenyl-1,2,4-benzotriazine, the structure of which is established by spectral and X-ray diffraction data.     

Metal-Assisted Oxidative Cyclization of Arylamidrazones I. Synthesis of 3-Acetyl-1,4-dihydro-1-phenyl-1,2,4-benzotriazine

In the presence of RuCl₃, N-phenylamidrazone underwent oxidative cyclization into 1,4-dihydro-1-phenyl-1,2,4-benzotriazine, the structure of which is established by spectral and X-ray diffraction data.     

Simple and efficient synthesis of 2,6-dialkyl-3,5-dialkoxycarbonyl-4-(3-aryl-1-phenyl-pyrazol-4-yl)pyridines using TPAP/NMO as a catalyst under mild conditions

Biologically important pyrazolylpyridines were synthesized in excellent yield by the oxidation of pyrazolyl 1,4-dihydropyridines (pyrazolyl 1,4-DHPs) using tetrapropylammonium perruthenate/N-methylmorpholine-N-oxide (TPAP/NMO) under mild conditions at 0 °C.     

Unexpected valence bond isomerization of [1,2,4]triazolo[3,4-c][1,2,4]benzotriazines under flash vacuum pyrolytic (fvp) conditions

Flash vacuum pyrolysis (fvp) of some substituted [1,2,4]triazolo[3,4-c][1,2,4]benzotriazine derivatives (1a-d) has been studied between 450 and 600 °C. The only transformation observed up to 525 °C was the unexpected valence bond isomerization of the angularly fused starting compounds to the isomeric linearly fused [1,2,4]triazolo[4,3-b][1,2,4]benzotriazine derivatives (9a-d), whereas at higher temperatures fragmentation products such as aromatic nitriles were also formed. Kinetic measurements revealed negative entropies of activation in the isomerization process, which suggest a concerted ring closure reaction to an intermediate antiaromatic diazirine. Reversibiblity of the title isomerization reaction was also proved by FVP experiments.     

New and versatile syntheses of 3-alkyl- and 3-aryl-1,2,4-benzotriazine 1,4-dioxides: preparation of the bioreductive cytotoxins SR 4895 and SR 4941

Palladium-mediated coupling of 3-chloro-1,2,4-benzotriazine 1-oxide with a variety of stannanes in the presence of Pd(PPh₃)₄ gives 3-alkyl derivatives in good yields. Suzuki reaction of the 3-chloro compound with phenylboronic acids gives 3-aryl-1,2,4-benzotriazine 1-oxides. Oxidation of 1-oxides with trifluoroperacetic acid gives the 1,4-dioxides. This method provides a better route to the potential anti-cancer agents SR 4895 and SR 4941.     

Biotransformation of Protoberberine Alkaloids by the Endophytic Fungus Coelomycetes AFKR-3 Isolated from Yellow Moonsheed Plant (Archangelisia Flava (L.) Merr.)

The endophytic fungus Coelomycetes AFKR-3 isolated from young stems of yellow moonshed plant (Archangelisia flava (L.) Merr.) has shown the capability to biotransform berberine into its 7-N-oxide derivative. Further investigations showed that the fungus can also biotransform the protoberberine alkaloid, palmatine into a new derivative palmatine 7-N-oxide in liquid medium of glucose-yeast extract-peptone. Berberine 7-N-oxide displayed the same antimicrobial activity against pathogenic bacteria and pathogenic fungi as berberine     

Heterocyclizations of 1-(Benzothiazol-2-yl)-4-phenylthiosemicarbazide

1-(Benzothiazol-2-yl)-4-phenylthiosemicarbazide reacted with methyl iodide in the presence of sodium acetate in boiling ethanol to give 2,2′-dithiobis[N-(5-methylsulfanyl-4-phenyl-4H-1,2,4-triazol-3-yl)-benzenamine]. The reaction of the title compound with dimethyl acetylenedicarboxylate in dioxane led to the formation of methyl 3-(benzothiazol-2-yl)-2-(2-methoxy-2-oxoethyl)-2,3-dihydro-1,3,4-thiadiazole-2-carboxylate.     

3-Azido-1,2,4-triazine N-Oxides. Syntheses and structure elucidation

Several 3-azido-1,2,4-triazine 1-oxides were prepared by treating the appropriate 3-hy-drazino derivatives with nitrous acid. 3-Azido-1,2,4-triazine 2-oxide was prepared by reaching the corresponding 3-bromo derivative with either tetramethylguanidinium azide in chloroform or sodium azide in aqueous acetone. The azido derivatives which could cyclize to form the tetra-zolo isomers were proven to exist enitrely in the open chain form by a ¹³C nmr, ¹H nmr and infrared spectroscopic study.     

Evaluation of different PAMAM dendrimers as molecular vehicle of 1,2,4-triazine N-oxide derivative with potential antitumor activity

The interaction between a 1,2,4-triazine N-oxide derivative, that holds potential antitumor activity under hypoxic conditions, and diverse polyamidoamine (PAMAM) dendrimers were investigated with the purpose of select the most appropriate macromolecule to act as potential molecular carrier of this active compound. The results shows that dendrimers with amine terminal groups (PAMAM-AT G = 3) and dendrimers with carboxylate terminal groups (PAMAM-CT G2.5 and G4.5) produces triazine derivative hydrolysis, even in buffered medium, and are not suitable as carriers. In contrast, dendrimers with neutral end groups (PAMAM-OHT) shows stable association with the active compound, making this dendrimer a possible medium for triazine carriage.     

Directed lithiation on the quinuclidine ring system: the synthesis of 2,3-difunctionalised quinuclidines

Lithiation of 3-methoxymethyl quinuclidine N-oxide occurs regioselectively to generate the 2-lithio 3-methoxymethyl derivative which can be trapped out with non-enolisable electrophiles to give 2,3-disubstituted quinuclidine N-oxide derivatives in good yield.     

Oxidation of polyfunctional sulfides with chlorine dioxide

3-Benzylsulfanyl-4,5-diphenyl-4H-1,2,4-triazole, 5-methylsulfanyl-1-phenyl-1H-tetrazole, 2-methylsulfanyl-1H-benzimidazole, 2-benzylsulfanyl-1H-benzimidazole, and 1-butylsulfanyl-4-nitrobenzene were oxidized to the corresponding sulfoxides with chlorine dioxide using different modes of oxidant supply. The oxidation process was characterized by high chemoselectivity.     

Protonation of 1,2,4-triazine 4-oxides

The basicity of some 1,2,4-triazine 4-oxides was estimated on a quantitative level, and their probable protonation patterns were ascertained. The dissociation constants of mono- and diprotonated 3-R-6-phenyl-1,2,4-triazine 4-oxides in aqueous buffer solutions and aqueous sulfuric acid solutions were determined (6-phenyl-1,2,4-triazine 4-oxide: $pK_{BH^ + } = 1.1$ , $pK_{BH^{2 + } } = - 6.02$ ). According to the results of DFT calculations (B3LYP/6-311**) and spectral data, first protonation of 1,2,4-triazine 4-oxides involves the N¹ nitrogen atom, and the second proton adds to the N-oxide oxygen atom.     

Chemoselective protection of thiols versus alcohols and phenols. The Tosvinyl group

The conjugate addition of aliphatic and aromatic thiols to ethynyl p-tolyl sulphone (tosylacetylene) has been managed to afford Tosvinyl derivatives chemoselectively (in the presence of oxygen nucleophiles) and stereoselectively (isomers Z) in practically quantitative yields. The conditions of choice are: catalytic amounts of Et₃N (only 0.5-1.0 mol%), a reaction temperature around 0°C and, for the less acidic thiols, CF₃CH₂OH or CH₃CN/CF₃CH₂OH as the solvent. Thus, N-Boc-Cys-OMe has been quantitatively protected as its S-Tosvinyl derivative in the presence of N-Boc-Ser-OMe and N-Boc-Tyr-OMe. This novel protecting group is stable to several basic and acidic conditions; its removal is achieved at rt by treatment with an excess of pyrrolidine or at 0°C with alkanethiolate ions.     

Bergman cycloaromatization of imidazole-fused enediynes: the remarkable effect of N-aryl substitution

A series of N-aryl substituted `imidazole-fused' (Z) 3-ene-1,5-diynes was prepared and kinetic parameters for their Bergman cycloaromatization reactivities were determined. N-Arylation enhanced rates relative to N-alkyl derivatives by up to sevenfold (ANOVA p<0.0001). The greatest enhancement was exhibited by the N-phenyl derivative (sevenfold at 145 °C).     

Probing the metal complexing ability of bis-quinolizidine N-oxides: Synthesis, spectroscopy and crystal structures

New ZnX₂ (X = Cl, Br) complexes with sparteine N1-oxide, sparteine epi-N16-oxide, lupanine (2-oxosparteine) N-oxide and α-isosparteine N-oxide were obtained and characterized by spectroscopic and crystallographic methods. Complexation with N1-oxides involves inversion of the configuration at the N16 atom of sparteine, converting its C ring from a boat into a chair form, whereas the structure of sparteine epi-N16-oxide, typified by the trans boat/chair C/D quinolizidine moiety, remains unchanged upon complexation. Coordination of zinc salts in the above compounds is realized solely through the oxygen atom of the N-O group and is accompanied by protonation of the “not-N-oxide” nitrogen atom. At variance lupanine N-oxide forms bis-monodentate complexes with ZnX₂ utilizing both the N-oxide and the lactam carbonyl oxygen atoms to bridge the neighboring ZnX₂ units, while reaction of α-isosparteine N-oxide with ZnX₂ leads to formation of complex salts of the general formula [H(−)α-Sp(N-oxide)][(ZnX₃)(H₂O)].