New Synthetic Approaches to Multifunctional Phenazinium Salt Derivatives

Two different approaches are offered for the synthesis under mild conditions of disubstituted phenazinium and benzo[a]phenazinium salts. Direct nucleophilic substitutions by primary and secondary amines in quaternary phenazinium salts containing an additional positive charge in the aliphatic part of the molecule were carried out. The substitution proceeds successively in positions 2 and 7, which allows selective introduction of different substituents into the heterocycle. Direct nucleophilic substitution in quaternary 2-N-alkyl-acetamidophenazinium and 5-alkoxy-benzo[a]phenazinium salts with different amines can also serve as a convenient method for the introduction of two different substituents.


Introduction
Phenazine and phenazine-like molecules have a wide spectrum of biological activity [1,2]. Several naturally occurring phenazine-containing antibiotics are known to react effectively with biopolymers in vivo [3][4][5]. Some phenazine derivatives are capable of generating hydroxyl radicals under mild conditions, which enables them to be used as reagents for site-directed DNA scission [6]. Introduction of fragments containing substituted phenazines into biopolymers endows the latter with new, unique properties that allow one to use such conjugates for specific modification of biopolymers. Thus, the addition of phenazine at 3'-and/or 5'-terminal phosphates of oligonucleotides bearing alkylating groups significantly enhances the stability of oligonucleotide duplexes [7]. These considerations stimulate the search for new methods of polyfunctionalization of the phenazine ring, especially under conditions where biopolymers retain their biological activity.

Results and Discussion
Despite the availability of a number of methods for the synthesis of various phenazine derivatives [8], direct nucleophilic substitution of the hydrogen atom, which is characteristic of azine quaternary salts [9] and usually proceeds under mild conditions, is the most interesting. The reactivity of phenazine derivatives in this reaction can be estimated by comparing the charge electron density (kinetic control) and nucleophilic localization energies (thermodynamic control) [10]. According to the literature data on the distribution of the electron density on an N-methylphenazinium salt, substitution of the hydrogen atom should occur at positions 2 or 4 [11]. However, the substitution at position 4 is thermodynamically unfavorable and hence only the hydrogen atom in position 2 should be substituted, which fully agrees with the experimental results [7,8]. To estimate the relative reactivity of substituted quaternary phenazinium salts, we calculated the charge distribution for positions 2 and 7 in compounds 1-3,7 and 8 and for positions 5 and 8 in benzo[a]phenazinium derivatives 4-6 ( Figure 1).
These PM3 quantum-mechanical calculations were performed for free cations in vacuo because, under real conditions of nucleophilic substitution of hydrogen, a nucleophile attacks either a solvated cation or an ionic pair separated by solvent molecules. The contribution of solvation to the charge distribution may be taken, in a first approximation, as the same for all quaternary phenazinium salts.
The calculations show that the introduction of the electron-donor amino group at position 2 of a quaternary salt (compound 2) significantly decreases the positive charge in the symmetrical position 7, thus precluding introduction of a second substituent. This suggests that the phenazine ring can be activated for nucleophilic substitution of hydrogen in 2-amino derivatives by acylating the amino group (compound 3). Accordingly, in benzo[a]phenazinium salts the 8 position can be activated by alkylating the oxo group at position 5 (compound 6). This approach, based on a decrease in the donor effects of a substituent, has now been realized by us experimentally (Schemes 1 and 2). The interaction of 9-methylphenazinium methylsulfate (1) with methylamine and the methyl ester of β-alanine easily gives 2-methylamino-and 2-[2-(methoxycarbonyl)ethyl]amino-9-methylphenazinium perchlorates (9 and 10) in high yields (Scheme 1). The electronic spectra of compounds 9 and 10 are similar to those characteristic of 2-aminophenazinium salts (cf. Table 1 in the Experimental), and the 1 H-NMR spectra display signals in the 6.5-6.7 ppm range with a pronounced coupling constant of J=2 Hz that is characteristic of aromatic protons in the meta-position.
As expected, acetylation of compounds 9 and 10 occurs with difficulty, and an acid catalyst is necessary to carry out the reaction. The best results were obtained with boron trifluoride etherate. The products, 9-methyl-2-N-methylacetamido-and 2-N-[2-(methoxycarbonyl)-ethyl)acetamido-9-methylphenazinium perchlorates (11 and 12) have spectral characteristics corresponding to the structures suggested. For example, in their 1 H-NMR spectra, one can observe the downfield shift of the signals for the protons at position 1 by ~1.5 ppm as compared to compounds 9 and 10, which suggests an increase in the acceptor character of the phenazine cycle. However, insufficient stability precluded the isolation of the analytical samples of these compounds, and they were used in subsequent syntheses without additional purification.
The reaction of acetamides 11 and 12 with methylamine, morpholine, and methyl ester of β-alanine were investigated. The reactivity of acetamides in these reactions was approximately one order of magnitude lower than that of the starting quaternary salt 1 (by TLC). Nevertheless, acetamide 11 is completely consumed in the reaction with methylamine even after 6 h at room temperature, and compounds 14 and 13, the 9-methyl-2,7-bis-methylamino-and 9-methyl-2-N-methylacetamido-7methylaminophenazinium perchlorates, were isolated from the reaction mixture. Their spectral and analytical data correspond well to those expected. The formation of compound 14 in the course of the reaction attests to the fact that 13, the product of direct nucleophilic substitution, undergoes partial deacylation under the conditions chosen, probably, in a reaction with excess methylamine. This fact allows one to avoid an additional deacetylation stage by merely increasing the reaction time, though this leads to a certain decrease in the yield of the target product, which was exemplified with the synthesis of 9-methyl-2-methylamino-7-morpholinophenazinium perchlorate 16 (procedures A and B). 7-[2-(Methoxycarbonyl)ethyl]amino-9-methyl-2-methylaminophenazinium perchlorate (15) was obtained by reaction of methyl ester of β-alanine with acetamide 11 or of methylamine with acetamide 12 in 51 and 46 % yields, respectively. As was demonstrated by the chromatographic analysis of the reaction mixture, the relatively low yield of diamine 15 is probably due to aminolysis of the ester group and side reactions, which occur upon prolonged exposure of quaternary phenazinium salts to an excess of amines. The direction of these reactions were studied in more detail using the interaction of compound 11 with morpholine as an example. Along with the target compound 16, isolated in 36 % yield (procedure B), a series of substituted phenazines was obtained, which were identified by mass spectrometry and 1 H-NMR spectroscopy ( Table 2 in the Experimental). As can be seen from the table, the main side processes, which occur in the nucleophilic substitution of hydrogen in quaternary phenazinium salts, are dequaternization of the phenazinium salt and substitution of the Nmethylacetamido group. When substituents resistant to acid hydrolysis are introduced, the reduction in the reaction time is justified as well as the involvement of an additional stage of acid hydrolysis (procedure A for compound 16).
The general synthesis of disubstituted benzo[a]phenazine derivatives is outlined in Scheme 2. The interaction of compound 4 with sodium hydroxide gives 5-oxo-11-(2-hydroxyethyl)benzo[a]phenazine (5), admixed with benzo[a]phenazine (17). Further alkylation of compound 5 by methyl bromoacetate in the presence of silver perchlorate gives product 18. Interaction of 18 with 1,3-diaminopropane at room temperature gives 8-(3-aminopropyl)amino-5-[(methoxycarbonyl)-methyl]hydroxy-11-(2hydroxyethyl)benzo[a]phenazinium perchlorate (19) in high yield. Compounds 5 and 18 are stable and can be isolated, but in our case we have used them in subsequent reactions without isolation and purification. It should be noted that the substitution in quaternary phenazinium salts proceeds via an intermediate that is to be oxidized with oxygen. Thus, the preparative synthesis requires the use of low reactant concentrations and intense flow of air. Otherwise, a considerable amount of side products is formed. It should also be noted that according to the calculations, a similar or even more pronounced effect of activation can be attained by introducing an additional positive charge into the substituent at the quaternary nitrogen atom (cf. Figure 1).
To check this assumption experimentally, we synthesized quaternary phenazinium salt 7, bearing an additional positive charge in the aliphatic fragment of the molecule, and studied its behavior in reactions with aliphatic amines. Salt 7 was synthesized from 9-acetyl-10-(2-bromoethyl)-9,10dihydrophenazine (20) [12] (Scheme 3). Treatment of compound 20 with an excess of 1,4diazabicyclo[2.2.2]octane gives compound 21 in quantitative yield (TLC). The latter was treated in situ with conc. HNO 3 to give dinitrate 7, solvated with two НNО 3 molecules. Prolonged storage of this solvate in vacuum over alkali reduces the content of НNО 3 to 1.2-1.5 acid molecules per molecule of compound 7. In the solid state this compound is stable but its aqueous and alcoholic solutions rapidly darken, especially when exposed to light. The reactions of bisquaternary salt 7 with nucleophiles were studied using 1,3-diaminopropane, morpholine, and dimethylamine as examples. Reaction mixtures were analyzed by HPLC. Figure 2 shows kinetics of the reaction of compound 7 with a tenfold excess of 1,3-diaminopropane. It can be seen that the content of a monosubstituted product (compound 22) in the reaction mixture (~95%) is a maximum after 30 min. The reaction rate at position 7 of product 22 is much lower, so that the percentage of the disubstituted product 24 does not exceed 4-5% in that time. The reaction can be stopped in this stage to isolate the monosubstituted product and introduce another nucleophile, which allows one to obtain a product containing different substituents in position 2 and 7. The possibility of synthesizing asymmetrically substituted phenazine derivatives was exemplified in successive introduction of the morpholine and dimethylamine residues (compound 25, Here we also demonstrated the possibility of these compounds to react with different biopolymers. A dinucleotide (thymidylyl(3'→5')thymidine phosphate tetrabutylammonium salt; pTpT) and tripeptide (histidyl-β-alanyl-histamine; His-β-Ala-HA) were used as models of oligonucleotides and peptides respectively (Scheme 4). At the first stage of synthesis a dinucleotide derivative with a primary amino group was prepared. For this purpose a 1,4-diaminobutane residue was attached to the terminal phosphate group of pTpT which had been preliminary activated by triphenylphosphine and 4,4'-dipyridyl disulfide in the presence of 1-methylimidazole [13]. The yield of this reaction was almost quantitative. Treatment of 26, the amino derivative of pTpT, with an excess of the phenazinium salt 7 gives compound 27 with a high yield (>95% HPLC). The electronic spectra of 27 have absorption maxima corresponding to the dinucleotide part at 267 nm and to the phenazinium part at 236, 293, 397 and 524 nm. His-β-Ala-HA was synthesized by condensation of histamine with the activated pentafluorophenyl esters of the corresponding BOC-protected amino acids using standard methods [14].    (11).  (14) and 9-methyl-2-N-methylacetamido-7methylaminophenazinium perchlorate (13). Gaseous methylamine was passed through a suspension of compound 11 (0.2 g, 0.6 mmol) in dry methanol (10 mL) for 6 h. The precipitate was filtered off and washed with hot EtOH. The mother liquor was concentrated, dissolved in the minimal amount of water, and applied onto a 2x30 cm column containig a polymeric dextran gel (Molselect G-10  Method B: Gaseous methylamine was passed through a suspension of compound 12 (0.1 g, 0.2 mmol) in anhydrous methanol (10 mL) for 6 h. The reaction mixture was allowed to stand for 72 h. and then processed as in procedure A. The yield of compound 12 was 0.045 g (46 %).

9-Methyl-2-methylamino-7-morpholinophenazinium реrchlorate (16).
Method A: Morpholine (0.1 mL, 1.2 mmol) was added to a suspension of compound 11 (0.1 g, 0.3 mmol) in dry methanol (10 mL) and the reaction mixture was stirred for 3 h. The precipitate was filtered off and washed with hot methanol. The filtrate was concentrated, and the residue was dissolved in methanol. A mixture of compound 16 and 9-methyl-2-/V-methylacetamido-7-morpholinophenazinium perchlorate was precipitated with ether. The sediment was dissolved with heating in 0. Method B: Morpholine (0.1 mL, 1.2 mmol) was added to a suspension of compound 11 (0.1 g, 0.3 mmol) in dry methanol (10 mL) and the reaction mixture was stirred for 72 h. The precipitate was filtered off and washed with hot methanol. The filtrate was concentrated, and the residue was dissolved in methanol. Compound 16 was precipitated with ether. After crystallization from 0.1 M HClO 4 , the yield of compound 16 was 0,04 g (36 %). The mother liquor after precipitation with ether was concentrated, the residue was dissolved in a minimal amount of chloroform and chromatographed on a 1х20 cm column with Silasorb-600 (15-20 µm, detection at 350 nm) using gradient elution СНСl 3 → СНCl 3 -МеОН, 5:1. The main fractions were concentrated, and phenazine, 2-morpholinophenazine, 2-/V-methylacetamido-7-morpholino-phenazine, 2-/V-methylacetamidophenazine, and 9-methylphenazin-2-one were obtained in yields of 6-12 mg. The main characteristics of the compounds obtained are given in Table 2.