by Michelle Poore and John J. Beck

Introduction

(Z)-Ligustilide, (1 in Figure 1), is a natural product isolated from the commonly used medicinal herb Ligusticum porteri, more commonly known as Oshá. Ligustilide has been shown to exhibit antimicrobial and antiviral properties. (1) This finding has lead to the synthesis of a simpler version of ligustilide: 3-benzylidenephthalide (2 in Figure 1).

Oshá is marketed as a treatment for headaches, anemia, strokes, irregular menstruation, and colds. Previous studies have determined one of the reactive centers to be at the end of the conjugated lactone system. Ligustilide and its conjugate lactone moiety is purported to be the reason for the medicinal qualities of the Ligusticum species.The conjugated lactone system allows for nucleophilic attack to ligustilide, which can be easily used by the body. A number of bioassays have supported this theory with activity against the bacteria Bacillus subtilis, Staphylococcus aureus, and Klebsellia pneumoniae. Confirmed activity has also been proven against the fungi Candida albicans and Sacharromyces cervevisiae.

Several compounds were
synthesized and then subjected
to structure-activity relationship studies in order to develop a new line of bioactive analogs. All products and their synthetic intermediates will be tested for antimicrobial properties. Bioactivity of the analogs will be influenced by electron donating groups (EDG) or electron withdrawing groups (EWG) located on the ipso carbon of the benzlidene ring. Electron withdrawing groups should activate the reactive center by pulling electron density away from the carbon, allowing it to be more reactive. This is considered to be a favorable interaction (1 in Figure 4).

.

An electron donating group will contribute electron density to the reactive site. This should decrease the bioactivity of the compound, creating an unfavorable reaction (2 in Figure 4).

Results and Discussion

Recrystalizing with ethyl acetate and cold hexanes afforded purified intermediates in the form of a white or yellow solid. The products were identified using GC-MS. The alcohol intermediates (3-6) had a fragmentation pattern of 134 and 105, similar to that of phthalide except with a different retention time. An experiment with D2O confirmed the spectral breakdown. The synthesis of 3 resulted in a white solid with a 49% yield. The synthesis of 4 resulted in a white solid with a 29% yield. 5 resulted in a yellow solid with a 23% yield and 6 resulted in a white solid with a 7% yield.

The synthesis of 7 resulted in a purified yield of 10%. The synthesis of 8 resulted in a purified yield of 75%. Recrystalizing with a minimal amount of ethyl acetate and cold hexanes purified compounds 7 and 8, the results of which were white crystals with a slight orange tint. Difficulties were encountered with recrystalization; pet ether, ethanol, methanol, and isopropanol afforded no crystals. Compounds 7 and 8 were identified using GC-MS. The final derivatives (7-8) had an M+ peak of their molecular weight.

Experimental
General Experimental Procedure:
Reactions were completed in oven-dried glassware and flushed with argon gas. THF was distilled over Na°/benzophenone. Starting materials were purchased from Aldrich Chemical Company and used without further purification. A 2:1 Hexane/EtOAc eluent was used for the TLCs. SiO2 TLC plates were used.

Instruments Used:
60mhz NMR
GC-MS

GC-MS Methodology:
The injector temperature was set at 280°C. The initial oven temperature was 150°C, initial time 1 minute, which was increased to 280°C at a rate of 18°C min-1. Final time was 5 minutes.

Alcohol Intermediate Synthesis (3-6)
LDA was generated at -78 °C in a 25 ml rbf containing a magnetic stir bar. Diisopropylamine ( 1.54 ml, 1.1 eq) was added to n-BuLi (7.69 ml, 1.1 eq) in THF (1.914 ml, 0.44 M) and stirred well. Phthalide (1.34 g, 1.0 eq) was dissolved in THF (25 ml, 0.4 M), and slowly added to the LDA. The solution was allowed to stir until a yellow precipitate formed. The substituted benzaldehyde (1.914 ml, 1.1 eq) was added at the rate of approximately one drop/minute to the phthalide slurry. After the slurry was allowed to dissolve, the reaction was quenched with ice chunks (~2 g). The mixture was acidified to pH = 1 with 1.0 M HCl. The product was extracted into ethyl acetate, and the organic layer was washed with sodium bicarbonate and then brine, dried over MgSO4, filtered, and concentrated in vacuo, to afford a white solid.

Dehydration Synthesis (7-8)
The Alcohol Intermediate (0.308 g, 1.0 eq) was dissolved in THF (2 ml, 0.15M) at 0 °C, and pyridine (0.32 ml, 4.0 eq). Methanesulfonyl Chloride (0.16 ml, 2.0 eq) was slowly added. The mixture was allowed to stir at 0 °C for 5 minutes. The mixture was removed from the bath and allowed to warm to room temperature. Pyridine (3 ml, 0.1M) was then added and the mixture was heated at a gentle reflux for 2 hours. The mixture was then cooled to room temperature, acidified to pH = 1 using 2.0M HCl, extracted into ethyl acetate, washed with sodium bicarbonate and then brine, dried over MgSO4, filtered, and concentrated in vacuo to produce the final derivative.

End Note
¹ J.J. Beck. Investigation of the Bioactive Constituents of Several Herbal Medicines, Doctoral Dissertation, Colorado State University, (1996).

return to table of contents

This page updated February 27, 2004