Synthesis of Analogs of Amathamide A Their Preliminary Antimicrobial Activity

Syntheses of three non-brominated analogs of amathamide A (1), a natural alkaloid isolated from the Tasmanian marine bryozoan Amathia wilsoni, are described. Antimicrobial activity against Bacillus subtilis, Staphylococcus aureus, Escherichia coli, Pseudomona aeruginosa, and Candida albicans was tested. Test results for amathamide A (1) showed a weak activity against C. albicans and E. coli. The three non-natural analogs 2-4 proved to be inactive compounds.


Introduction
Amathamide A (1) is a brominated alkaloid isolated from the Tasmanian bryozoan Amathia wilsoni Kirkpatrick [1,2]. Biological activity of this type of enamide alkaloids has not been well studied, and only nematocidal, antifungal, and antibacterial activitity has been described for amathamides A (1), B, G, H, and I, isolated from A. wilsoni and A. convolute species [3]. Our previous work relating to the synthesis of two natural amathamides (A and B) [4] prompted us to investigate the synthesis and biological evaluation of analogs of this type of alkaloid. Therefore, we selected amathamide A (1) as a lead compound and we carried out the synthesis of three non-brominated analogs 2-4, maintaining the N-methyl or substituting it by a single hydrogen or t-butylcarbamate ( Figure 1). Antimicrobial activity was then determined against two Gram-positive bacteria (B. subtilis and S. aureus), two Gram-negative bacteria (E. coli and P. aeruginosa) and a yeast (C. albicans).  H H

Results and Discussion
Amathamide A (1) was synthesized as previously reported [4]. For the three non-natural analogs, the synthetic route was modified as shown in Scheme 1. m-Anisaldehyde was condensed with nitromethane in 81 % yield in the presence of ammonium acetate and then thiophenol was incorporated via Michael addition with a catalytic amount of N-isopropylcyclohexylamine to obtain the product 6 in 90 % yield. Nitro group reduction was achieved in 45% yield in the conventional manner [5] using Zn in AcOH/HCl. Acylation of the amine with (S)-N-t-butoxycarbonylproline in presence of DCC/HOBt gave 8 in 20 % yield. Removal of the Boc (t-butoxycarbonyl) protective group from compound 8 affords 9 in very good yield and acylation of the latter with formaldehyde and reduction with NaBH 3 CN afford 10 (80 % yield). Elimination of thiophenol of 10 via oxidation with NaIO 4 to the sulfoxide and refluxing in toluene/K 2 CO 3 , gave the non-brominated analogue 2. On the other hand, elimination of thiophenol from 8 led to analogue 3, and removal of Boc afforded 4. In this manner, we obtained the three non-natural analogs of amathamide A (1).
The preliminary antimicrobial activity of amathamide A (1) and the three non-natural analogs 2-4 was determined against the Gram-positive bacteria S. aureus and B. subtuilis, the Gram-negative bacteria E. coli and P. aeruginosa and the yeast C. albicans by using the agar dilution-streak assay (Mitscher method) [6]. Only compound 1 was active in the bioassay at the final concentration of 200 µg/mL. Minimal inhibitory concentrations (MIC) for 1 were determined [7,8]. The MICs of 1 for C.

Conclusions
The non-brominated analogues of amathamides obtained following a simpler synthetic route proved to be inactive compounds. Thus, we conclude that the presence of bromine on the aryl ring contributes to the antimicrobial activity of amathamide A (1). According to Narkowicz et al. [3], the E orientation of the double bond of amathamide A (1) conferred the highest biological activity compared with the less constrained saturated version, as in amathamide I, or the Z orientation of the double bond as in amathamide B. This information leaves only the possibility of altering the acyl group and/or substitution on the amide nitrogen in an effort to obtain analogues with enhanced antimicrobial activity for further biological testing. The effect of other aryl substituents could also be explored.

Acknowledgments
We gratefully acknowledge support for this project by CONACYT grant E130,1517 (2001).

Bioassay Evaluation Procedures.
Antimicrobial activity against Gram-positive bacteria Staphylococcus aureus ATCC 6538 and Bacillus subtilis ATCC 6633, Gram-negative bacteria Escherichia coli ATCC 8739 and Pseudomona aeruginosa ATCC 9027, and yeast Candida albicans ATCC 10231 was determined by the agar Mitscher method as previously described [5]. Test samples were dissolved (2 mg/mL) in 10 mL nutrient agar medium No. 1 (Merck) for bacterial cultures and Sabouraud agar (Merck) supplemented with glucose (4 %) for yeast, and added aseptically to each Petri dish and swirled carefully until the agar began to set. After 24 h. of incubation (sterility test) the bacteria or yeast in a concentration of approximately 0.5 McFarland were streaked in radial patterns on the agar plates containing the samples, incubated at 37 o C for 24 h, except for C. albicans which was incubated at 25 o C. Complete inhibition of bacterial growth was expected for a sample to be declared active. Inhibition was present at 200 µg/mL only for amathamide A (1). Minimal inhibitory concentrations (MICs) values were determined by the dilution method previously described [6,7]. Two-fold serial dilutions were tested in nutrient broth (Merck) for B. subtilis, S. aureus, E. coli and P. aeruginosa, and Sabouraud-glucose (2 %) nutrient broth (Merck) for C. albicans. An initial concentration of 1 mg/mL of sample was prepared by dissolving in dimethyl sulphoxide. Serial dilutions (100-0.1 µg/mL) of sample were prepared, and the liquid medium was inoculated by cultures in stationary phase at concentration of 10 5 CFU/mL. After overnight incubation, the MIC was determined as the lowest concentration of compound preventing any visible growth. Streptomycin sulfate (Sigma) (1 µg/mL) and nystatin (Sigma) (3 µg/mL) were used as positive controls.