Ion-molecular reactions of nucleogenic phenyl cations with the nucleophilic centers of 1,4-diazines have been investigated for the first time. Previously unknown tritium labeled N-phenyl quaternary derivatives of pyrazine and quinoxaline, which are potential radioactive biomarkers, have been obtained by nuclear-chemical method.
Diazines attribute to the six-ring heterocycles with two heteroatoms both of which are nitrogen atoms. Cyclic pyrazine system forms common structural fragment of 1, 4-diazines. Benzopyrazine or quinoxaline has pyrazine cycle annulated with the benzene ring. 1,4-Diazines and condensed systems with pyrazine ring are known as an important type of heterocyclic derivatives with high biologic activity [1-4]. Synthetic pyrazines exhibit a wide range of physiological activities including antibacterial, antimycobacterial, antiprotozoal and antitumor [5-9]. These promissing results stimulate chemists and biologists for further research in the field of pyrazine derivatives over the last few years [10-22].
Modern progress in biology, biochemistry and molecular genetics along with the experimental medicine is largely determined by the wide application of labeled, especially tritium labeled compounds, for the sensitive direct studies of the biochemical reaction mechanisms and metabolic pathways of the essential pharmaceuticals [23-36].
Owing to this the development of easy and unusual methods for synthesis of new 1,4-diazine derivatives along with tritium labeling becomes an urgent task. Previously we have applied the elaborated nuclear-chemical method based on the consequences of tritium b-decay for the one-step preparation of unknown and hardly available derivatives of monoazines, effective tritium labeled biological markers [37-41].
Benzene double labeled with tritium was obtained from 1, 4-dibromobenzene (3.4 mg, 0.014 mmol), and n-Bu3N (5.0 ml, 0.020 mmol) in hexane, and tritium gas (3.3 Ci, 0.054 mmol) by dehalogenation on a 5% Pd/BaSO4 catalyst for 1 h at room temperature [
Crystals of the stabilizing salt KBF4 (~200 mg) were placed in 0.5 ml glass ampoules, then 0.055 mmol of substrates: pyrazine (4.4 mg,) or quinoxaline (7.2 mg) dissolved in ether were placed on the salt crystals. The ether was distilled off in vacuum, and after cooling the ampoules with liquid nitrogen, a C6H4T2 hexane solution (1 ml) was added. The C6H4T2 concentration was selected so that the ratio of labeled benzene to substrate was not less than ~1:1000. The ampoules were sealed, and stored for 1 - 2 months at −15˚C for accumulation of the nuclear-chemical synthesis products. The ampoules were opened, the contents were transferred to special vials, and benzene (0.5 ml) was added, followed by the carrier acetone solution (0.5 ml), (1 mg/ml). Due to the absence of isotopic carriers the unlabeled N-phenylpyridinium and quinolinium salts [37,42] were used. Unreacted C6H4T2 was removed by distillation in vacuum. Acetone (0.5 ml) was added to the dry residue and samples (about 5 ml) were taken for isolation of labeled compounds by TLC.
Radiochromatography of the obtained tritiated compounds was carried out on glass plates with Analtech TLC Uniplates C18 Reverse Phase silica gel (Fluorescent Indicator) in MeCN. Bands of the chromatographic adsorption layer measuring 0.5 cm in length were removed into dioxane scintillator and their b-radioactivity measured with the aid of a Rack Beta (Finland) liquid scintillation counter. Typical radiochromatograms are shown on Figures 1 and 2.
First peaks on the radiochromatograms correspond to the quaternary salts. Relative yields of ion-molecular reactions products were determined as a ratio of the radioactivity of an individual compound towards the sum of all tritium labeled products.
Generation of nucleogenic phenyl cations (cations formed by radioactive decay) occurs by spontaneous β-decay of tritium in the labeled benzene. Nuclear-chemical method provides unique opportunity for unusual formation of carbocations along with the several essential advantages: 1) obtained phenyl cations are free, that means without counterion; 2) investigated ion-molecular reactions take place on the surface of stabilizing salt without a solvent; 3) all reactions are carried out in the very mild conditions since decay processes are independent from temperature, pressure and so on.
The presence of at least two atoms of tritium in the benzene molecule leads to the preparation of cations labeled with tritium (Scheme 1).
The proposed scheme (Scheme 2) for the ion-molecular interactions of nucleogenic phenyl cations with quinoxaline (for example) may be represented in the following manner.
Upon the interactions of free phenyl cations with the investigated diazines, quaternary salts are formed by AdE (electrophilic addition) as well as substitution products formed by SE (electrophilic substitution) are produced.
The radiochemical yields of the quaternary salts for the investigated 1,4-diazines are shown in
Nitrogen atom in azines possesses a pair of electrons, which is not involved in the formation of σ or π bonding orbitals. Those electrons may form a bond between the
Scheme 1. Generation of nucleogenic phenyl cations.
Scheme 2. Pathways of ion-molecular interactions of nucleogenic phenyl cations with quinoxaline.
nitrogen atom and a carbon atom, which causes the nitrogen atom to become quaternary [
Pyrazine and quinoxaline are weakly basic (pKa ~ 0.6) [20,21,44], therefore the diquaternarization reaction of such derivatives for a long time wasn’t available in practice. It was suspected that these failures arise from the expected reduction in nucleophilicity of the second nitrogen attendant upon quaternization of the first. However, the use of more potent trialkyloxonium fluoroborates as alkylating agents led to the formation of several pyrazinium diquats [
It is well-known that most N-phenyl quaternary salts of azines are not prepared by direct quaternization but rather the nitrogen substituent is introducing before the ring closure [
Further research will be undertaken for the detail investigations of ion-molecular interactions of nucleogenic phenyl cations with the nucleophilic centers of 1,4-diazines.
Elaborated nuclear-chemical method of synthesis enables a new way of the direct nitrogen atom phenylation by the nucleogenic phenyl cations in 1,4-diazines, furnishing previously unknown N-phenyl quaternary derivatives.
The authors gratefully acknowledge the financial support of the Russian Foundation for Basic Research (Project No. 10-03-00685a).