Preparation of some derivatives of (+)-usnic acid with aromatic amines under microwave irradiation condition

Up to now, derivatives of usnic acid were only prepared by classical methods of refluxing in organic solvents for hours. In this study, we examined the condensation reaction of usnic acid with o-toluidine under microwave irradiation condition which gave good yield in a short time. The obtained optimal condition was applied to prepare some other derivatives of (+)-usnic acid.


INTRODUCTION
Usnic acid (1), 2,6-diacetyl-7,9-dihydroxy-8,12-dimethyl-1,3-(2H,12H)-dibenzofurandione, is a naturally occurring dibenzofurane derivative found in several lichen species. Usnic acid was identified in many genera of lichens including Alectoria, Cladonia, Evernia, Parmotrema, Ramalia, and Usnea, and it exists in one of two enantiomers which differ in the orientation of the methyl group located in position 12. It was first isolated by the German scientist W. Knop in 1844 [1].  Condensation reaction of (+)-usnic acid with some amines. [3,4] Usnic acid possesses a wide range of interesting biological properties. It is a potent antibiotic effective against Gram-positive bacteria, including Mycobacterium tuberculosis, Staphylococcus, Streptococcus and examined these reactions under microwave irradiation condition with the aim obtaining new compounds by using the reaction conditions which were more friendly with the environment.

Microwave irradiation method.
In a 10 mL test tube of a dedicated microwave oven, 1 (x mM), o-toluidine (y mM), and solvent (ethanol, THF or CPME) were added.
The solution attained naturally pH 5, the optimal pH condition for condensation reactions. Then the solution was irradiated at a power of 60 W. The reaction conditions were changed such as: temperature, time, ratio of substrate and reactant and kind of solvents. After the reaction, the percentage (H %) of the performed product (3) was determined by HPLC. After the reaction, the mixture was cooled and the light yellow precipitate was filtered off, washed with hot water, and dried for 24 hrs in a dessicator. The results were presented in Table 1.
The obtained optimal condition was applied to prepare some derivatives ( Table 2) of (+)-usnic acid.

Conventional heating refluxing method.
A solution of 1 (0.1 mM) and o-toluidine (0.1 mM) in CPME (5 mL) was refluxed and stirred at 500 rounds/min. Twelve different experiments were prepared in order to determine the yield of the product, determined by HPLC, owing to time (112 hours).

Effect of the reaction temperature
The yield increased when the temperature of the solution was raised from 80 to 90 o C (entries 1, 2 of Table 1), but from 90 o C to 110 o C, the yield began to decrease (entries 3,4). This result was due to the formed side-product (4). Therefore, the good yield of reaction was achieved at 90 o C. The result showed that the higher the temperature was, the lower reaction selectivity took place.

Effect of the reaction time
In order to investigate the role of the reaction time, the reaction was carried out within 15-20 minutes at pH 5 and other conditions were kept unchanged (entries 2, 5, 6 of Table 1). The good yield of reaction was achieved in 20 minutes (entry 2).

Effect of the quantity of o-toluidine
The reaction of 1 and o-toluidine with the ratio of (1:1) gave highest yield of 93.9 %. When the ratio of o-toluidine was increased, the excess amine would react with both carbonyl groups, C-14 and C-17 of usnic acid, to create the side product 4.

Effect of solvent
Under microwave irradiation condition, CPME, ethanol or TMF gave appropriate results and among them, CPME gave the best. CPME is a green solvent and is no harmful to the human health and the environment, therefore, it was chosen for the following experiments.

Conventional heating refluxing method
As time increased from 1 to 9 hours, the yield also increased from 11.2 % (1 hour) to 58.2 % (9 hours) and reached the highest 62.2 % at 10 hours. More than 10 hours, the yield was not be ameliorated (60.2 %).
The results showed that the reaction yield performed under microwave irradiation condition was higher and quicker than under the conventional method, therefore this condition was applied to prepare some derivatives of (+)-usnic acid.

Preparation of some derivatives of (+)-usnic acid
The obtained optimal condition was applied to prepare some derivatives of (+)-usnic acid. The results were presented in Table 2  Process for preparing some derivatives of (+)-usnic acid

DISCUSSIONS
The condensation reaction of usnic acid and amine at the ratio of (1:1) gave the enaminoadduct performed at the carbonyl group C-14 of usnic acid. If the molar ratio was (1:2), the sideproduct was performed in which the two carbonyl groups at C-14 and C-17 were attacked. These results were compatible with published data in the literature [3,4]. C-15, as a methyl group adjacent to a ketone group with δC 27.95 in usnic acid, was changed to a methyl adjacent to an alkene group with δC 18.0518.46.
Furthermore, the calculation of the active energy of two structures by DFT:B3LYP with the basis 6-31G(d,p) method in methanol, tetrahydrofuran and gas, the energy of the enamine form is lower than that of the imine one. These results showed that obtained adducts were in the enamine form.

CONCLUSION
Condensation reaction of usnic acid with otoluidine under microwave irradiation condition gave better yield in a short time comparing to the conventional heating method. The best condition of condensation reaction between (+)-usnic acid and o-toluidine (1:1) was irradiated at 90 o C within 20 minutes. Cyclopentyl methyl ether is a good solvent for the reaction with the yield of 96.9 %. Some new derivatives of (+)-usnic acid were prepared and this is the first time their NMR spectral data are reported.