Wurster aza-crown ethers with N - para-phenylene-phenothiazine or -phenoxazine groups

N -Phenylaza-15-crown-5 3 reacts with phenothiazine 1a , 2-chlorophenothiazine 1b , or phenoxazine 1c in the presence of mild oxidizing agents (I 2 , Fe 3+ or Cu 2+ ) affording new Wurster aza-crown-ethers 4a – 4c . Homolytic processes for the formation of compounds 4a-4c were discussed. Redox properties of these compounds were investigated by cyclic voltammetry. In concentrated sulfuric acid as solvent and oxidant, these compounds give stable radical-cations as proved by electron paramagnetic resonance (EPR). Ionophoric properties of the new compounds 4a-4c were evidenced by cyclic voltammetry with lithium and sodium cations. The relative hydrophobic/hydrophilic character of these compounds was determined by reverse-phase thin-layer chromatography (RP-TLC). Redox and ionophoric properties of the new compounds 4a – 4c may lead to analytical and bioanalytical applications

4][25] In the present case, however, the reaction mechanism in methanol at room temperature in the presence of mild oxidizing agents (I 2 , Fe 3+ or Cu 2+ ) is a homolytic aromatic substitution, involving the N-phenylaza-15-crown-5 3 and the nitrogen atom of the free radicals 2a-2c (Scheme 1), affording compounds 4a-4c (Scheme 2).This statement is based on the following experimental observations: i. as seen in Table 1 for the first oxidation step, compounds 1a-1c are oxidized more easily than N-phenylaza-15-crown-5 3; ii.irrespective of the oxidizing agent (I 2 , Fe 3+ or Cu 2+ ), the dimers 5a, 5c and 6a, 6c (evidenced by TLC, along with other side-products), proved the formation of the neutral free radicals 2a-2c; iii.since the literature mentions that dimers 5a and 6a are formed from phenothiazine 1a and the stable free radical DPPH (2,2-diphenyl-1-picrylhydrazyl) or solid PbO 2 , 3,26,27 we investigated the reaction between 1a and 3 in methylene chloride and each of these two oxidizing agents at room temperature for 24 hrs; along with dimers 5a and 6a in significant amounts, TLC showed the presence of 4a in low concentration; iv. on monitoring by cyclic voltammetry the reaction of 1a with 3 in acetonitrile, one reversible major peak was detected (Figure 1), and TLC of the oxidation products evidenced the electrochemical formation of 1a, 3, 4a and dimers 5a and 6a in the absence of chemical oxidants (see Experimental Part); v. no reaction occurs when phenothiazine 1a is replaced by N-methylphenothiazine; this experiment proves that the N-centered stable free radical 2a is essential for the formation of 4a; vi.sodium acetate, which favors heterolytic processes, 24 lowers the yield in products 4a or 4c, as one can see in Table 2 (the ratios between the aromatic heterocycle, 1a or 1c, Nphenylaza-15-crown-5 3 and oxidizing agent are also displayed in Table 2).When iodine was used as an oxidizing agent for the reaction between 1a-1c and 3, another product (7 in 10-15% yield relatively to 3) was identified, in addition to 4a-4c, 5a-5c, and 6a-6c.The iodo-derivative 7 has been described in the literature as the reaction product between 3 and N-iodosuccinimide, involving probably also a homolytic process. 28We have obtained it as the main reaction product from 3 and iodine in methanol (74% yield, see Experimental Part).
An excess of oxidizing agent (iodine) up to 50% increases the yield in compounds 4 but a larger excess lowers it (the first six experiments in Table 2), so that the other experiments were carried out with equimolar amounts of reactants 1a, 1c and 3, and with 1.5 moles of oxidant per mole of each reactant (Table 2).
When iodine was used as oxidant, about half of the unreacted crown ether 3 could be recovered (20%).
In the presence of water (or hydrated oxidizing agents, Table 2) one obtains from 1a or 1c, along with other side-products, phenothiazin-3-one or phenoxazin-3-one (these products may also be observed when the first step of the work-up involves aqueous solutions 18,19 ).
Two less likely alternatives to the homolytic aromatic substitution mechanism presented in Scheme 2 are: (i) a coupling of two radicals formed by oxidation of both reactants 1 and 3 (as seen from Table 1, whereas 1a-1c have oxidation potentials of 0.3-0.4V, the macrocyclic amine 3 has a higher oxidation potential, 0.5 V); (ii) a homolytic ipso-substitution 29 for the case when iodine was the oxidizing agent, involving the formation of the iodo-derivative 7 that would react with radical 2a-2c displacing the iodine; actually, on reacting in methanol for 24 hrs.at room temperature an equimolar mixture of 1a and 7 with an excess of anhydrous FeCl 3 and then extracting the mixture with methylene chloride and analyzing the products by TLC, 4a was detected in 40% yield.

NMR and IR Spectra of the new Wurster aza-crown-ethers 4a-4c
Both by 1 H-NMR and 13 C-NMR spectra (see Experimental Part), the structures of the new compounds 4a-4c were confirmed.The following remarks should be noted: i. the symmetry of the unsubstituted heterocycle 4a and 4c is reflected by the pairwise equality of δ values for H-1-9, H-2-8, H-3-7, H-4-6 and for C-1-9, C-2-8, C-3-7, C-4-6; however, this symmetry is broken by the 2-chloro-substituent in 4b; ii. 1 H-and 13 C-NMR chemical shift δ values for positions 4 and 6 decrease on replacing the sulfur heteroatom by the more electronegative oxygen heteroatom 4a>4c; iii.for compound 7, NMR data are similar to those reported in the literature 28 and confirm the para-position of the iodo substituent in N-phenylaza-15-crown-5, 3.
Infrared absorption spectra of compounds 4a-4c confirm the absence of the NH stretching frequency in these products.

Redox reactions of the new Wurster aza-crown-ethers 4a-4c
Cyclic voltammetry of compounds 4a-4c using tetra-n-butylammonium perchlorate (TBAP) as supporting electrolyte evidenced two redox reactions (peaks I and II) in reversible processes (Figure 2), corresponding to Scheme 3, similarly to other Wurster compounds.The oxidation potentials (Figure 2) increase in the order 4a<4b≈4c, for each of the oxidation steps (Table 3).When LiClO 4 or NaClO 4 are used as a support electrolyte, the voltammograms indicated also a reversible system (Figure 3).With tetramethylammonium perchlorate (TMAP) as reference, Table 4 indicates that the oxidation potentials decrease with these cations (Li + or Na + ).This fact proves that the lone electron pair of the nitrogen atom in the macrocycle becomes involved in the complex formation.3) and peak potentials (Table 4) demonstrated that the resulting supramolecular assembly (4a-M + complex) is stable, and so is also its mono-oxidation product (8a-M + complex); however, the next oxidation step no longer has a lone electron pair at the macrocyclic nitrogen atom, and therefore this is no longer stable, 8,9 but forms the dication 9a releasing the alkaline metal cation (Scheme 4).The complexation of Li + or Na + with 4a (Table 4) is in agreement with data reported in the literature, 30 regarding the fitting between the ionic diameter and the macrocyclic cavity size (1.2-1.5 Å) of the N-phenylaza-15-crown-5 3 and the ionic diameters for Li + (1.36 Å) and Na + (1.94 Å) respectively.In the case of K + with larger ionic diameter (2.66 Å), 30 our investigations revealed no complex formation.

Spectrophotometry of the new Wurster aza-crown-ethers 4a-4c
The electronic absorption spectra in the 230-325 nm range of methanol solutions of 1a-1c, 3 and 4a-4c are displayed in Table 5.The following observations hold: i. all seven compounds present two distinct absorption maxima at 238-259 nm and 302-323 nm.In addition, compounds 4a-4c have a third band at 265nm appearing as a shoulder for 4a and 4b (on the 257 nm or 259 nm absorption band, respectively), but as distinct maximum for 4c; ii.taking into account that compounds 4a-4c possess both molecular moieties of 1a-1c and 3, one expects the presence of electronic transitions characterizing these moieties, and in addition a band that would be similar to that of para-phenylenediamine; iii. in solution and as TLC spots, compounds 1a-1c and the new Wurster aza-crown-ethers 4a-4c are weakly fluorescent at 366 nm; at present, this property was not investigated more closely.
The red solutions of 4a-4c in concentrated sulfuric acid presenting the EPR spectra shown in Figure 4 reform these compounds on diluting with a 20-times larger amount of water.The workup was extraction with dichloromethane, drying of the colorless organic layer, and evaporating the solvent in vacuum.The pink-colored aqueous phase was neutralized with NaHCO 3 , and then the same procedure was followed.Analysis of both residues by TLC revealed only the presence of compounds 4a-4c, proving that no degradation takes place in H 2 SO 4 and that radicals 8Ha-8Hc have no structural modifications.

Hydrophobicity/hydrophilicity balance of the new Wurster aza-crown-ethers 4a-4c
The hydrophobicity/hydrophilicity property of compounds 4 is important for their possible chemical and biomedical applications.The octanol-water partition coefficient (P) and its logarithm (logP) are the usual parameters 38 for estimating quantitatively these characteristics, and they can be measured or computed.In our case, this property for compounds 4a-4c was studied experimentally by reversed phase TLC [39][40][41][42][43] (RP-TLC) and compared with this property for the starting compounds 1a-1c, 3, and the side-reaction compound 7. Thus, R f values were measured using precoated C 18 -chain layers (RP-18F 254S , Merck), as stationary phases and different acetonitrile-water mixtures as mobile phases (Table 7).The molecular hydrophobicity, R M0 , appreciated as a result of experimental data depending on R M0 values and calculated [39][40][41][42][43] with eqs. 1 and 2, is the R M value extrapolated to zero concentration of the organic component in the acetonitrile-water mixture; b is the change in the R M value caused by increasing the concentration (K) of the organic component in the mobile phase.Statistical analysis involved the correlation coefficient (R), the Fisher parameter (F), and the standard deviation (SD) (Table 7).Five determinations on silica gel RP-18F 254S (Merck), with percent of acetonitrile in mixture acetonitrile-water: A = 95%, B = 90%, C = 85%, D = 80% and E = 75%; b R M0 , b, R, F, and SD are defined by the preceding text and by eqs. 1 and 2.
On attempting to calculate logP values using fragmental constants, 44 a satisfactory correlation with experimental data for R M0 was obtained (Figure 5).The experimental results concerning the hydrophobic/hydrophilic character (R M0 values, Table 7) indicate that the starting heterocycles 1a-1c have a higher hydrophobicity than the compounds 3 and 7 containing the N-phenylaza-15 crown-5, and that the new Wurster azacrown-ethers 4a-4c are the most hydrophobic of all.
Instrumentation. 1 H-NMR (300 MHz) and 13 C-NMR spectra (100 MHz) were recorded with a Varian Inova 400 with an ASW-SW headprobe at 30 ºC. using unidimensional techniques (Dept, Apt) and bidimensional sequences (Gcosy, Ghmqc, Ghmbc, Ghsqc, where G means gradient).IR spectra were recorded with an FT-IR Bruker Vertex 70 equipped with ATR diamond cell.ESI-MS spectra were recorded with a QMD 1000 Carlo Erba instrument.Cyclic voltammetric measurements were performed with a conventional three-electrode glass cell by means of a PAR-273-A potentiostat, and all solutions were prepared by using acetonitrile.As reference electrode, a silver wire immersed in a 0.1M AgNO 3 solution was used, linked to the main compartment of the cell by means of a Vycor plug.A platinum disk (surface area, 0.07 cm 2 ) and a platinum wire were used as the working and counter electrode, respectively.The voltammograms were recorded at a concentration of the investigated compounds of 10 -3 M, within the potential range -0.5 to 1.0 V.As supporting electrolyte, tetra-n-butylammonium perchlorate (TBAP), tetramethylammonium perchlorate (TMAP), NaClO 4 or LiClO 4 , were used, at a concentration of 0.1M.EPR spectra were recorded at room temperature on a JEOL FA 100 spectrometer with 100 kHz modulation frequency, 0.998 mW microwave power, 480 s sweep time, 0.2 G modulation amplitude, time constant 0.3 s.Compounds 1a-1c and 4a-4c were oxidized in H 2 SO 4 to give the corresponding radical-dications 8Ha-8Hc and radical-cations 10a-10c, respectively.A. Oxidation with iodine.Equimolar amounts of aromatic heterocyc1es 1a-1c and Nphenylaza-15-crown-5 3 were dissolved in methanol (30 mL for one gram of mixture) at room temperature.The pale yellow solution was treated with a solution of iodine in methanol (molar ratio 1:1.5 for 1:I 2 , i. e. 40 mL methanol for one gram of iodine) and left for 24 hrs with stirring at room temperature.Then distilled water (about ten times larger volume) was added to the green-brown solution, when a fine brown precipitate was formed.A solution of sodium thiosulfate was added for completely removing iodine, followed by adding ascorbic acid till the pH was 3.5.Solid sodium chloride was added to the grey suspension for obtaining an almost saturated solution in order to facilitate the precipitation.After keeping overnight at 5°C, the precipitate was filtered off with suction on a G3 glass filter and washed with distilled water (from the filtrate one may isolate unreacted N-phenylaza-15-crown-5 3 with 20% yield, by extraction with methylene chloride, evaporation of the solvent, and purification by TLC).The precipitate was dissolved in methylene chloride and the extract was washed with 2% aqueous thiosulfate, and then with 2% aqueous ascorbic acid.The yellow-green organic phase was dried over sodium sulfate and then the solvent was removed under reduced pressure.The crude compounds 4a-4c were purified by preparative TLC (PLC plates Silica Gel 60 F 254 , dichloromethane: toluene: methanol, 5:5:0.2v/v, four times).The extraction from silica gel was performed in a Soxhlet with dichloromethane: methanol (9:1 v/v).Yields: 52% for 4a, 53% for 4b and 51% for 4c.B. Oxidation with anhydrous iron(III) chloride.With the same molar ratios as in the previous procedure, the methanol solution was kept for 24 hrs at room temperature under stirring.Then distilled water (a 10-times larger volume) was added, followed by ascorbic acid to pH = 3.5 and by solid sodium chloride to reach an almost saturated solution.After keeping at 5°C overnight, the solution was extracted with methylene chloride, the organic phase was dried over Na  Monitoring the reaction between 1a and 3 by cyclic voltammetry.An equimolar mixture of compounds 1a and 3 in acetonitrile in the presence of TBAP was monitored analytically by cyclic voltammetry following the redox processes (Figure 1).Then, on adding a tenfold volume of distilled water to the solution, the milky liquid phase was extracted L/L with methylene chloride.The lower phase was separated, dried over Na 2 SO 4 , and concentrated under reduced pressure to about 0.1 mL.The TLC analysis confirmed the mechanism described by Scheme 2 by identifying the starting materials 1a, 3, of the reaction product 4a (traces), and of traces of dimers 5a, 6a resulting from the free radical 2a.
On analytical silica gel 60 F 254 (Merck) plates the three mobile phases (A-C) allowed the identification of unreacted starting materials (1a-1c), reaction products 4a-4c, dimeric sideproducts 5a-5c, 6a-6c, and compound 7. Detection of spots employed UV light (254 nm for non-fluorescent background, or 366 nm for fluorescent background) as well as iodine vapor yielding various colors indicated in Table 8.

Scheme 1 .
Scheme 1.A few of the resonance structures of phenothiazines 1a, 1b and phenoxazine 1c and of the corresponding neutral free radicals 2a-2c: a, X=S, R=H; b, X=S, R=Cl; c, X=O, R=H.
Scheme 4. Reversible redox ionophoric process of 4a in the presence of alkali metal ions (M + =Li or Na)

Table 1 .
Peak Cyclic voltammogram recorded for a mixture of 1a and 3 in acetonitrile with TBAP (10 -1 M) as supporting electrolyte in concentrations of 10 -3 M, at a scan rate of 0.5 V/s; Epa=0.27V and Epc=0.16V.

Table 6 .
EPR a N (or a NH ) and g values, as well as electronic absorption data for radical-dications

Table 7 .
R f values, hydrophobic characteristics (R M0 and b) and calculated logP of the new compounds 4, of the starting compounds 1, 3 and of the secondary reaction product 7. RP-TLC results a,b for acetonitrile-water mixtures (A-E) in methanol was treated with a solution of iodine in methanol (40 mL for one gram of I 2 , molar ratio 1:1.5 for 3:I 2 ).The brown solution was stirred at room temperature for 24 hrs.A tenfold larger volume of distilled water was added, when a fine brown precipitate separated.Then solid sodium thiosulfate was added under stirring till the solution became colorless.After bringing the pH to 3.5 with solid ascorbic acid, the suspension was kept overnight at 5°C.Solid sodium chloride was added to saturation, and the solution was extracted with methylene chloride.After drying over Na 2 SO 4 , the solvent was removed under reduced pressure.Purification was carried out by preparative TLC as outlined above.Yield 74%.