Synthesis, Structure Elucidation and Cytotoxicity of (+)-Usnic Acid Derivatives on U87MG Glioblastoma Cells

Huong Giang Thi Nguyen1, Ngoc Vinh Nguyen1, Van Anh Vo3, Wanjoo Chun3, Fadhil S Kamounah2, Ole Vang2 and Poul Erik Hansen2* 1Institute of Drug Quality Control, 200 Co BAC Street, Ho Chi Minh city, Vietnam 2Department of Science and Environment, Roskilde University, DK-4000 Roskilde, Denmark 3Department of Pharmacology, School of Medicine, Kangwon National University, Chuncheon, Kangwon, South Korea


Introduction
Usnic acid (2,6-diacetyl-7,9-dihydroxy-8,9b-dimethyl-1,3(2H,9bH)-dibenzofurandione; C 18 H 16 O 7 ) (Scheme 1) is a lichen metabolite and both the R(+) and S(-) isomers can be found in nature. (+)-Usnic acid and its derivative exhibit a wide range of biological activities [1] such as antibiotic [2], antiviral [3], apoptotic [4,5], analgesic [6], antipyretic [6] and anti-proliferative [7] activities. The toxicity in vitro and in vivo has been evaluated by Guo et al. [8] and hepatotoxicity but no general toxicity was observed. Antitumor activity of usnic acid was displayed for the first time by Kupchan and Kopperman [9] against Lewis lung carcinoma in mice. In addition, cytotoxicity activity of usnic acid and usnic acid-amine derivatives has been determined against a wide variety of murine and human cancer cell lines [4]. Although the cytotoxicity of usnic acid has been extensively reviewed, no data regarding the effects of usnic acid on glioblastoma cells (formally glioblastoma multiform, GBM) exists. GBM represents both the most common and most malignant variant among a number of primary brain tumors. In this study, usnic acid and ten derivatives of (+)-usnic acid were evaluated on a human glioblastoma -astrocytoma cell line (U87MG) by a MTT assay for cell viability. The N-heterocyclic derivative (1,4-diazepine) showed higher cytotoxicity on this cancer cell line than temozolomide (TMZ), which is the drug currently used against this cancer form.
The reaction between usnic acid and primary amines has been studied in a number of papers [10][11][12][13][14][15][16][17][18][19][20][21] leading to enamines and Schiff bases. One inherent problem with biological testing of usnic acid and some of its derivatives is a very low solubility in water [22]. The aim of this study is to synthesize new (+)-usnic acid derivatives with potential biological activities and better solubility. Ten derivatives of (+)-usnic acid were synthesized with ethylene diamine, series of aromatic amines and hydroxylamine.

Procedure (A) for the preparation of compounds (1-2):
A suspension of (+)-usnic acid (1 mmol) in a mixture of tetrahydrofuran and absolute ethyl alcohol (1:5; 12 ml) was treated with ethylene diamine (1.2 mmol, pure liquid), and heated to reflux for 4 hrs. with stirring. After the end of the reaction, the mixture was concentrated under reduced pressure. The obtained residue was purified by preparative chromatography.    Table 2.

Procedure (B) for the preparation of ketamines 3-7 and (11):
A suspension of (+)-usnic acid (1 mmol) in absolute ethyl alcohol (10 mL) was treated with amines (1.2 mmol, neat) and heated to reflux for 4 hr with stirring. After the end of reaction, the mixture was concentrated under reduced pressure. The obtained residue was purified by preparative chromatography.  Cell viability by the MTT assay: U87MG glioblastoma cells were plated into 12-well culture plates and were incubated in 10% serum culture at 37°C under a 5% CO 2 / 95% humidified air incubator for 24 hrs. Cells were then incubated with TMZ, (+)-usnic acid or compounds 1-10 at indicated concentrations in serum free DMEM for an additional 24 hrs. The compounds were dissolved in DMSO and the level of DMSO in treatments did not exceed 0.1%. Cell viability was determined using the MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay, which yields a blue formazan product in living cells, but not in dead cells or their lytic debris. Then, cells were washed with PBS (phosphate buffered saline) and dissolved in DMSO (dimethyl sulfoxide, Sigma Aldrich). Absorbance was determined at 540 nm. The results were expressed as a percentage of MTT assay activity relative to the non-treated control. CC 50 's of the specific compound was calculated, based on three concentrations using Origin software (www. originlab.com). The concentration of treating compounds at which 50% cell alive compared to non-treated samples was calculated from formula in corresponding to correlation curve formed individually, being considered as CC 50 's of each compound. The experiments were repeated three times independently.

Synthesis
The reaction of (+)-usnic acid (2.0 equivalents) and ethylene diamine (1.0 equivalent) in a mixture of absolute ethyl alcohol and tetrahydrofuran (5:1) at reflux for 4 hr produced compound 1 only [28]. In our hands, the reaction of (+)-usnic acid (1.0 equivalent) and ethylene diamine (1.2 equivalents) gave compound 1 and the N-heterocyclic derivative 2 under the same conditions. The latter was predominant.

Preparative chromatography
High-speed fractionation was performed using a column with a high separation capacity so the obtained compounds could be achieved with high purity. The obtained residue was purified on a Shimadzu LC-20A using a silica column at ambient temperature. Separation was achieved using the mobile phases in Table 3 at a flow rate of 12 ml/min and was monitored using a PDA detector at 254 nm. In the purification step the yields were finally obtained in the range from 33% to 72%. reaction with primary amines occur at the carbonyl group C(11)=O leading to an enamine. This is confirmed in this study. Schiff base formation involves the amine attacking at C+=O. C-11 is part of a triketone system and therefore is more reactive than C-13 being part of an aromatic systems. This means that the Schiff base is formed at C-11.
The assignment of the NMR chemical shifts is central to the assignment of structures. Following the assignments of Ref. [19] it can be seen that the chemical shifts of the A-ring (Scheme 1) of mono substituted derivatives are very similar to those of usnic acid itself. The assignments of the other compounds are based on chemical shifts of usnic acid and this reasoning. For the benzene rings of compounds 3-7, C-F coupling constants are of great help in derivatives 3-5 and the resonances of the benzene rings of compounds 6 and 7 can then be assigned based on substituent effects.
The structure and assignment of compound 2 will be discussed separately as the structure is rather complex. Based upon the HSQC spectrum (Table 1) C-4 can be assigned unambiguously and the methylene carbons can be identified. Based on the HMBC spectrum (Table 1) long-range correlations from OH-7 confirm the assignment of C-6, C-7 and C-8. Knowing the assignment of C-6 the cross-peak from CH 3 -14 assigns H-14 as well as C-13. Knowing the position of C-8, cross-peaks from CH 3 -15 assigns H-15. The cross-peak from OH-9 confirms that the resonances in the HMBC spectrum overlap, although they are separate in the 1D spectrum at a lower field. H-4 helps to assign C-2 and C-4a. As H-10 and H-15 are now assigned, the remaining methyl signal must be those of H-12 and H-14. Crosspeaks from these assign C-13 and C-11. Cross-peaks from H-10 assign C-9a, C-4a and C-1. The latter is very important and the assignment is confirmed by cross-peaks to the CH 2 protons. The chemical shifts in DMF-d 7 are very similar to those in CDCl 3 (Table 1) so we may assume that the structure is similar in the two solvents.
Deuterium isotope effects on chemical shifts have also been studied in order to support the structural assignment. The deuteriation is done by dissolving the compound in CH 3 OD and removing the methanol by rotary evaporation. This type of isotope effect is useful in the study of intramolecularly hydrogen bonded compounds [29,30]. The deuterium isotope effect at C-9 (0.6 ppm) is unusually large. Also C-11 shows large isotope effects although somewhat broad. The large effect at C-9 indicates a strong intramolecular hydrogen bond or an equilibrium system [31,32]. The former is in line with the large OH chemical shift of 15.6 ppm. Both the OH-7 and the NH resonance show splittings due to isotope effects. Based on the above information a structure can be suggested as seen in Figure 2. However, upon cooling to -60°C two broad resonances appear in the 1 H spectrum at 12.7 and 9.95 ppm integrating roughly 1:1. These are ascribed to NH resonances. Furthermore, the CH 2 resonances merge to one broad resonance. In the 13 C spectrum many resonances are now broad: C-1, C-4, C-9, C-10, C-11 and CH 2 CH 2 indicating that an exchange situation is at hand, suggesting that two different tautomer's exist as seen in Figure 1.
A comparison between calculated and experimental data for structure A shows a very good correlation (Figure 2). This correlation is not improved by adding data for structure B, so the former must dominate at ambient temperature. Kutney and Sanchez [10] found that using hydroxylamine hydrochloride in 5.0% methanolic potassium hydroxide -water (2:1) with the dibenzyl ether of usnic acid resulted in oxime formation at carbon 13 although a more logical result would have been at C-11 as amines react at this position (see previously). However, HMBC spectra confirmed that for 10 the reaction had occurred at C-13 (Scheme 1).

Biological activity
The MTT assay [29] was applied to evaluate the cytotoxicity of the derivatives on the human glioblastoma-astrocytoma cell line (U87MG) ( Table 2).
The cytotoxicity of (+)-usnic acid and derivatives has been  Table 2: Cytotoxicity of (+)-usnic acid and of ten derivatives 1-10 on the U87MG cell line.
extensively reviewed [2,4,33,34], but there is no published data regarding its activity against U87MG cells, which was most commonly used to study glioblastoma multiform (GBM), the most aggressive brain cancer, according to WHO (World Health Organization). As seen in Table 2 the cytotoxicity of (+)-usnic acid and its ketamine-derivatives reduce the cell viability of U87MG significantly. Temozolomide (TMZ) is a DNA-alkylating agent approved by the U.S. Food and Drug Administration for treatment of the newly diagnosed GBM. However, the response of U87MG cell to TMZ is lower than that of (+)-usnic acid and its derivatives, whereas derivatives 2, 4 , 8, 10 and (+)-usnic acid show significantly cytotoxic effects on U87MG cells after 24 hr of treatment.
The sensitivity of (+)-usnic acid is varying from one cell line to the next. The CC 50 observed in U87MG cells after 24 hrs. (47 µM) is corresponding to previously published IC 50 values in K-562 and DU145 cells (53 and 57 mM, respectively), but after 72 hrs. [4]. The time effects of (+)-usnic acid need to be addressed. In human keratinocytes (HaCaT) IC 50 was estimated to 70 µM for (+)-usnic acid after 24 hrs. [20]. Both the (+) and (-) isomer of usnic acid are found naturally and the (-)-isomer shows generally a lower cytotoxicity compared to (+)-isomers [4,35]. Although a recent study relating to Mycobacterium tuberculosis showed similar activity for the two isomers [35]. A recent paper by Bruno et al. showed less cytotoxicity in rat myoblasts (L6 cells) of Schiff bases of usnic acid relative to the mother compound itself [19]. Our preliminary bioactivity data suggests that (+)-usnic acid and its derivatives could be potential cancer chemical therapy agent although a profound study is needed to examine the specificity of cytotoxicity on cancer cell lines as well as mechanism of activities in vitro and in vivo.

Conclusions
Condensation of (+)-usnic acid with amines and hydroxylamine gave a series of ketamine derivatives and its oxime derivatives. Of particular interest is the formation of a new type of compound 2. In addition, the cytotoxic activity of (+)-usnic acid and ketamine derivatives was evaluated using a MTT assay. The new compounds 2 and 8 were more active compounds in terms of cytotoxicity and they had an improved solubility. Compound 2 showed better activities (CC 50 =33.14 µM<46.99 µM) than (+)-usnic acid in this study. However, further investigations are needed to evaluate the biological activity of these compounds in relation to U87MG cancer cells and to improve the activity of usnic acid derivatives in vivo.  Ethyl acetate -hexane (5:5)