3,5-Dinitro-N -(4’-benzo-15-crown-5)-benzamide Derivatives. Synthesis and Properties

Four new derivatives of benzo-15-crown-5 ( 4 – 7 ) were synthesized from 4’-amino-benzo-15-crown-5 ( 3 ) and 2-chloro-3,5-dinitrobenzoic acid ( 1 ) or its chloride ( 2 ): the amide 4 resulted from 2 and 3 , and the arylamine 5 from 1 and 3 . A compound with two crown ether cavities ( 6 ) resulted from 3 and 4 . Nucleophilic substitution with methoxyamine converted 4 into 7 . Structures were confirmed by 1 H-NMR, 13 C-NMR, and IR-ATR spectroscopy. Geometries of the the new compounds are presented. The hydrophobic character of 5 , 6


Introduction
2][3] An interesting molecular design, which was adopted in the present communication, involves a benzo-crown moiety connected to a "core" moiety possessing reactive groups that can be used for anchoring the desired functions to the crown ether scaffold.5][6][7][8][9] Moreover,` cytostatic activities were found for pyridine-dicarboxamides derived from benzo-15-crown-5. 10 Starting from the commercially available 2-chloro-3,5-dinitrobenzoic acid (1) one can easily prepare 2-chloro-3,5-dinitrobenzoyl chloride (2). 11The chlorine atom attached to the carbonyl group in 2 is more reactive (in S N 2 reactions), 12 and then the chlorine atom attached to the dinitrophenyl group can be substituted by an S N Ar process. 12,14In the present paper we report the synthesis and properties of four new derivatives of benzo-15-crown-5 (4 -7) with a dinitrophenyl core moiety to which we attached one or two crown ether groups having the amine scaffold 4'-aminobenzo-15-crown-5 (3), also commercially available.We will show the special ionophoric properties of the methoxyamino derivative 7 that is acidic enough to afford salts with sodium or lithium hydroxides, in which the alkali cation forms a complex with the crown ether.

Synthesis of compounds 4 -7
By means of a low temperature and short reaction time, compound 4 was obtained by combining compounds 2 and 3 and stopping the reaction by acidifying the reaction mixture (Scheme 1).Compounds 5, 6, and 7 were prepared by S N Ar processes which involve the formation of Meisenheimer adducts, exemplified for 5. 11,13,14 For compound 6, a slightly better yield was achieved when the two steps were combined into a single one, starting from 2 and an excess of 3 in the presence of a tertiary amine.
To facilitate NMR assignments, atoms are numbered in Schemes 1-4 consistently rather than systematically.The new compounds 4 -7 were purified by preparative TLC, and their purity was attested by single spots in TLC.For compound 6 that has two crown ether moieties, the 1 H-NMR and 13 C-NMR spectra were repeated at a higher temperature (55 ºC) but no significant differences were observed relatively to room temperature NMR.Fourier transform infrared (FTIR) spectra were performed in the attenuated total reflection (ATR) mode.
Optimized geometries for compounds 4-7 were calculated using the ArgusLab program (Fig. 1) based on molecular mechanics (MM2). 15-17Optimized geometries of compounds 4 -7 (Fig. 1) lead to the following conclusions: (i) energies are influenced by the torsion energies which are lowest for 4 and highest for 6; (ii) compound 4 followed by 7 presents the closest proximity between the dinitrophenyl and crown rings; (iii) the carboxy group of 5 has the closest vicinity to the macrocyclic ring.In solution one expects various mobile conformations for 4 and 7 having two moieties connected by the CO-NH bridge group, for 5 with two moieties connected by the NH bridge, and for 6 with three cyclic blocks connected by both types of bridges.
(2) The molecular hydrophobicity, R M0 , is the R M value extrapolated to zero concentration of organic component in the alcohol-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). 25Five determinations on silica gel RP-18F 254 (Merck), with per cent of ethanol in the mixture ethanol-water: A = 80%, B = 70%, C = 60%, D = 50%; R M0 = molecular hydrophobicity (eq.2).

Electronic absorption spectra of compounds 5-7
In crystalline state, compounds 5-7 are yellow-orange and their solutions are colored in yelloworange (6) or red-orange (5, 7).We selected acetonitrile as a water-miscible solvent for investigating the complexing abilities of the new compounds, and therefore we report in Table 2 the electronic absorption data in this solvent.The bathochromic effect of the 1-amino nitrogen attached to the 2-carbonyl-4,6dinitrophenyl group decreases in the order 7>6>5.

Table 2. UV-Vis values (λ max and ε) in acetonitrile for compounds 5-7
Compound An interesting behavior of 7 was observed in various solvents.The λ max value reported in Table 2 refers to HCl-containing acetonitrile.Similarly, in methylene chloriide or dioxane,compound 7 presents only one absorption band at 340 or 344 nm, respectively.In acetonitrile without HCl, in methanol, or dimethylsulfoxide, partial ionization of 7 leads to the appearance of a shoulder around 430 nm (ε = 1.410L•mol -1 •cm -1 ).
Using the MOPAC-2007 program, 16 the net atomic charge on the aminic nitrogen atom (NAC N ) was calculated, and by assuming an inverse correlation with the experimental longestwavelength absorption band, the calculated λ max values according to eq. ( 3) present a reasonable correlation with the experimental λ max values (Table 3).Because only three points are involved, statistical data are mainly for orientation.
λ max (calc.)= -332.9NACN + 872.1 A more complete explanation for the λ max presented in Table 2 would require an elaborate quantum-chemical calculation of HOMO-LUMO values.
On treatment with increasing amounts of powdered LiOH or NaOH, compound 7 undergoes gradual changes in its electronic absorption spectrum illustrated in Fig. 3, which reveals isosbestic points (388 nm for LiOH in Fig. 3A, and 386 nm for NaOH in Fig. 3B).On applying Job's method, [31][32][33] it was found that the alkali metal cation and the anion of compound 7 form equimolar complexes 8 in which the ratio 7 : M + is 1:1 for M + = Li + , Na + .Visually, on acidifying complexes 8, the red-orange color becomes less intense due to the reformation of compounds 7 (Scheme 5), which were identified by TLC.
Reversible reaction of 7 with M + OH -(where M + = Li + or Na + ) leading to the reversible formation of complex 8.
By using the Benesi-Hildebrand method, 34 stability constants (log K S ) were determined in acetonitrile at room temperature for the two supramolecular complexes 8.Both have similar stability constants, as seen from Table 4, resulting from five determinations at room temperature.The order K S (Na + ) > K S (Li + ) agrees with the closeness between the diameters of the cation and of the macrocyclic cavity, believed to be similar to that of 15-crown-5, namely 1.7 -2.2 Å. 35,36
A similar treatment of 7 with solid potassium hydroxide did not result quantitatively in complexation probably because the cavity of the 15-crown-5 macrocycle is smaller than the diameter of the potassium cation (2.66 Å). 35,36 This cation can be properly accommodated by the larger cavity of the 18-crown-6 ethers. 2,3,35,36n shaking a dichloromethane solution of 7 with aqueous LiOH or NaOH, the organic layer becomes red, and the aqueous solution stays colorless, proving that the alkali cations are extracted into the organic layer.The process becomes reversible on acidification, as indicated by spectrophotometry.A similar complexation takes place between dichloromethane solutions of 7 which become red on treatment with aqueous sodium chloride, nitrite, or nitrate.
On shaking the orange-red solution of 7 in methylene chloride with an aqueous solution of a basic amino acid (lysine, ornithine or arginine), the organic layer loses its color and the aqueous layer becomes red, evidencing the extraction of 7 as an anion into the aqueous layer which now contains also the conjugate acid of lysine, ornithine or arginine.An aqueous solution of bovine serum albumin or chitosane becomes red on treatment with a solution of 7 in methanol, due to a similar protolytic equilibrium.In the absence of water, on treating a dichloromethane solution of 7 with these solid amino acids (lysine, ornithine or arginine), an intense red color appears; if, after filtering this solution, low-boiling petroleum ether is added (fraction with NBP 30-60° C), brick-red crystals are formed, leaving a colorless solution.Details about these processes will be reported separately.

Conclusions
Using as starting materials the carboxylic acid 1, the acid chloride 2, and the amine 3, compounds 4-7 were synthesized by nucleophilic substitutions of chlorine atoms.Experimental determinations of the molecular hydrophobicity R M0 by means of RP-TLC and theoretical calculations of log P for compounds 5-7 showed that the hydrophobicity decreases in the order 7>5>6.Electronic absorption spectra indicated the formation of a supramolecular complex 8 of compound 7 with an equimolar amount of LiOH or NaOH.The stability constants K S of these complexes in acetonitrile at room temperature are similar (log K S Li + = 3.59, log K S Na + = 3.85); these complexes are also formed in a biphasic dichloromethane/water system, and their formation becomes reverted on acidification.The supramolecular complex 8 (M + = Na + ) was also obtained in solid state.With solid LiOH, NaOH, or basic amino acids (either solid or in aqueous solution) dichloromethane solutions of 7 also yield molecular complexes.The fact that 7 has an acidic proton and presents ionophoric and chromogenic properties recommends it as an analytical and bioanalytical reagent.
Syntheses and Spectra.General Procedure Compound 4. The amine 3 and the acid chloride 2 (molar ratio 3:2 = 2.2) were combined, 11 observing strict temperature and duration conditions.One gram of 3 was dissolved in 15 mL of water-acetone mixture 3:2 v/v (15 mL/ g of 3), and the solution was cooled in an ice-salt bath.Under external cooling and rapid stirring a pre-cooled solution of 2 in acetone (5 mL/g of 2) was added rapidly in one portion.After 40-45 seconds, 50 mL of cooled 1M hydrochloric acid were added in one portion for stopping the reaction.Stirring was continued under cooling for complete precipitation.The precipitate was filtered off (glass filter G3) and washed on the filter with 1M hydrochloric acid and then with water.The solid product was dissolved in methylene chloride and the solution was stirred for one hour with a 10% solution of sodium hydrogen carbonate.The organic layer was washed twice with a saturated solution of sodium chloride in 1M hydrochloric acid, then after drying with anhydrous sodium sulfate the solvent was removed under reduced pressure.The crude 4 was purified by preparative TLC (silica gel GF 254, methylene chloride/methanol 9:1 v/v, twice).The yellow zone was then extracted (Soxhlet), with a methylene chloride/methanol mixture 8:2 v/v, and the solvent mixture was removed under vacuum, affording the pure 4 which gives a single spot by TLC (methylene chloride/methanol mixture 9:1 v/v, twice).(4)  .Compound 5.An equimolar mixture of 1 and 3 in acetonitrile (20 mL/1 g mixture) was stirred for 2 hrs at 90° C in the presence of an excess of powdered sodium carbonate.An orange-red precipitate was formed.To this mixture 1M hydrochloric acid was added till a pH = 2 was reached.After cooling the mixture to 5 °C, the precipitate was filtered off on a G3 glass filter and washed thrice on the filter with 1M hydrochloric acid.Then this first batch of crude 5 was dried on CaCl 2 in a desiccator.A second batch was obtained by extracting the filtrate with dichloromethane, separating the organic phase and evaporating the solvent under reduced pressure for another crop of 5.The combined crude 5 was purified by preparative TLC (silica gel GF 254, methylene chloride/methanol 9:1 v/v, twice).The orange-red zone was then extracted (Soxhlet) with a methylene chloride/methanol mixture 8:2 v/v and the solvent mixture was removed under vacuum, affording the pure 5 which gives a single spot by TLC (silica gel GF 254, methylene chloride/methanol mixture 9:1 v/v, twice).

Compound 6. Variant A.
A solution of compounds 3 and 4 (molar ratio 3:4 = 2.5) in a mixture (9:1 v/v) of methylene chloride and methanol (10 mL solvent mixture for one gram of mixture 3+4) was stirred at room temperature for four days in a closed container in the presence of triethylamine (5 mL for one gram of mixture 3 +4).Then 10 mL of methylene chloride were added to the red reaction mixture, and this mixture was extracted twice with 1M hydrochloric acid.The organic phase was dried on anhydrous sodium sulfate and the solvent was removed under reduced pressure.The crude 6 was purified by preparative TLC (silica gel GF 254, toluene/methylene chloride/methanol 4:5:1 v/v, twice).The yellow zone was then extracted (Soxhlet) with a methylene chloride/methanol mixture 8:2 v/v and the solvent mixture was removed under vacuum, affording the pure 6 which gave a single spot by TLC (silica gel GF 254, toluene/dichloromethane/methanol mixture 4:5:1 v/v, three times).Variant B. A mixture of compounds 2 and 3 (molar ratio 3:2=2.5) in methylene chloride (15 mL for one gram of mixture 2+3), in the presence of triethylamine (5 mL for one gram of mixture 2 +3) was stirred for five days at room temperature in a closed container.Then the workup was as in the previous variant.3,5-Dinitro-N,N'-bis-(4'-benzo-15-crown-5)-anthranylamide (6) ).Supramolecular complex 8 (M + = Na + ).Compound 7 was treated with a methanol solution of sodium hydroxide (molar ratio NaOH:7 = 1.1); 3 mL of methanol was used for one gram of mixture NaOH + 7. The clear red-colored solution was diluted with an equal volume of dichloromethane and then petroleum ether was gradually added, scratching the walls of the contained with a glass rod for initiating crystallization.After 20 minutes at 5 ºC, the red-brown precipitate (8) was filtered off from the colorless solution.Compound 8 was dried over anhydrous CaCl 2 in a vacuum desiccator.Yield 95%.By titration with HCl 1N and UV-Vis spectrophotometry it was confirmed that 7 was re-obtained and that the stoichiometry of the complex was 7:Na + = 1:1.

Table 3 .
Net atomic charges on the amino nitrogen (NAC N ), experimental and calculated λ max (with eq. 3, in nm) for compounds 5-7 in acetonitrile
ARKAT USA, Inc.8 Compound 7. A solution of compound 4 (1 g) in 10 mL of warm ethanol was mixed with a solution of an excess (molar ratio 4:H 3 CO-NH 3 + Cl -= 4.5) of O-methylhydroxylamine hydrochloride in a mixture of dioxane-water (3:2 v:v) (5 mL for one gram of mixture) with sufficient sodium carbonate for neutralizing all the hydrochloric acid.The solution was kept at pH around 8 by adding Na 2 CO 3 , and was stirred at 50 °C for six days.To the resulted red solution an equal volume of dichloromethane was added, and the mixture was extracted with a saturated solution of sodium chloride in 1M hydrochloric acid.The orange-colored organic phase was dried on Na 2 SO 4 and the solvent was removed under reduced pressure.The crude 7 was purified by preparative TLC (silica gel GF 254, toluene/dichloromethane/methanol mixture 4:5:1 v/v, twice).The orange-red zone was then extracted (Soxhlet) with a methylene chloride/methanol mixture 8:2 v/v and the solvent mixture was removed under vacuum, affording the pure 5 which gave a single spot by TLC (silica gel GF 254, toluene/dichloromethane/methanol mixture 4:5:1 v/v, twice).