Sci Pharm www.scipharm.at Research article Open Access Synthesis and Antimicrobial Activity of

3,4-Epoxy-2-dodecanone, a major component in the preorbital gland of the African grey duiker (Sylvicapra grimmia), showed antimicrobial activity in preliminary tests. The C11 to C17 homologues of this compound were prepared and their activity against several pathogenic dermal bacteria and fungi was tested. 3,4-Epoxy-2-dodecanone and 3,4-epoxy-2-tridecanone inhibited the growth of Trichophyton mentagrophytes at 25 μg/mL. Moderate inhibition of the growth of the bacteria Propionibacterium acnes and the lipophilic yeast, Pityrosporum ovale, was seen for several of the homologues.


Introduction
Compounds with antimicrobial activity have been documented from the skin and skin glands of a number of mammalian species.(E)-3-Tridecen-2-one (1c, n=7) the major compound from interdigital glands of the black-tailed deer (Odocoileus hemionus columbianus) has significant antimicrobial activity [1][2][3].Nonanal, octanal and heptanal were found in the hair of Mexican free-tailed bats (Tadarida brasiliensis mexicana) at concentrations known to inhibit the growth of pathogenic skin fungi [4].Hair from the reticulated giraffe (Giraffa camelopardalis reticulata) also has nonanal, octanal and heptanal at high concentrations and p-cresol, indole, tetradecanoic acid and hexadecanoic acid, compounds that inhibit the growth of pathogenic skin fungi and bacteria [5].Several antimicrobial long-chain alcohols were found in the interdigital glands of the American pronghorn (Antilocapra americana) [6].In search of new antimicrobials from animal skin glands, 3,4-epoxy-2-dodecanone (2b, n=6), a major component in the African grey duiker's (Sylvicapra grimmia) preorbital skin gland [7] was prepared and assayed.It showed antifungal activity in preliminary tests, so the C 11 to C 17 , 3,4-epoxy-2-alkanones (2a-2g, Scheme 1) were prepared and tested for antimicrobial activity.The activity of these compounds was compared to antimicrobial activity of the (E)-3-alken-2-ones (1a-1g) from which they were prepared [3].

Antimicrobial Activity
The minimum inhibitory concentrations (MIC) for Trichophyton mentagrophytes, Propionibacterium acnes and Pityrosporum ovale with the 3,4-epoxy-2-alkanones are listed in Table 1 . 3,, the compound from the grey duiker, and 3,4-epoxy-2-tridecanone (2c) inhibited the growth of T. mentagrophytes at the same concentration, 25 μg/mL.Activity against this fungus decreased at longer or shorter chain lengths.For the bacterium, P. acnes, the C 11 to C 15 homologues (2a-2e) were active at 100 μg/mL, but the C 16 and C 17 homologues (2f and 2g) were inactive.With the lipophilic yeast, P. ovale, the shorter chain-length compounds [C 11 to C 16 (2a-2f)] were not active at 800 μg/mL, but the C 17 compound (2g) was active at 200 μg/mL.No activity was observed with the bacterium Staphylococcus aureus and the yeast Candida albicans.For all microorganisms tested, a control using the dissolution medium, dimethylformamide (DMF), had no activity.
Table 2 has the comparable inhibitory activity of the (E)-3-alken-2-ones (1a-1g) [3], the compounds that were used to prepare the 3,4-epoxy-2-alkanones in this report.The inhibition of T. mentagrophytes for both sets of compounds is similar, with activity for the C 11 to C 14 homologues, which decreases starting with the C 15 compound.This suggests that these compounds have a similar mode of action with T. mentagrophytes.The inhibition of growth for P. acnes is quite different for these two series of compounds.The (E)-3-alken-2-ones (1a-1g) have activity increase with increasing carbon chain length.The shorter chain-length 3,4-epoxy-2-alkanones are not as active as the corresponding (E)-3-alken-2-ones and the longer homologues do not inhibit P. acnes.This suggests that these compounds have a different mode of action.The 3,4-epoxy-2-alkanones were almost inactive with the yeast P. ovale.Only the C 17 compound showed slight activity.The activity of the (E)-3-alken-2-ones against P. ovale is moderate for the C 11 to C 14 homologues and ceases at longer chain lengths (C 16 to C 17 ).This also suggests that these compounds have different modes of action.Both types of compound were not active against S. aureus.The epoxide and double bond at the 3,4-position of the 3,4-epoxy-2-tridecanone (2c) and (E)-3-tridecen-2-one (1c) are important to the activity of these two compounds, since 2-tridecanone was inactive on bioassay with these microorganisms [2].Thus, a possible mode of action for these two compounds may be an irreversible inhibition of an enzyme due to a nucleophilic attack at the 4-position.With (E)-3-tridecen-2-one this would be a 1,4-addition (Michael addition) and with 3,4-epoxy-2-tridecanone an epoxide opening.Other homologous 2-alkanones have not been tested, so a comparison with other compounds in this report was not possible.

Antimicrobial Assay
The 3,4-epoxy-2-alkanones were screened against some mammalian skin pathogens: the bacteria Staphylococcus aureus, ATCC 12598, Propionibacterium acnes ATCC 11827; and the fungi Candida albicans ATCC 24433, Pityrosporum ovale ATCC 14521 and Trichophyton mentagrophytes ATCC 18748.The minimum inhibitory concentration (MIC in μg/mL) of these 3,4-epoxy-2-alkanones was done using a two-fold serial broth dilution.Two replicates were done with each microorganism and the MIC was determined as the lowest concentration for each compound at which no growth was observed.The highest concentration used in these tests was 800 µg/mL.
Each test compound (80 mg) was dissolved in 1.0 mL of dimethylformamide (DMF) and 30 μL of this stock solution was dissolved in 3 mL of the applicable medium.Two-fold serial dilution of the resulting 800 µg/mL solution, gave solutions of 400, 200, 100, 50, 25, 12.5, 6.25, and 3.12 μg/mL.In addition, for each microorganism, a control of 30 μL DMF in 3 mL of medium was tested.Finally, to each of the diluted test solutions was added a 30 μL sample of microorganism culture.The cultures of S. aureus, P acnes and C. albicans were examined for turbidity (OD at 660 nm).P. ovale was examined visually for growth at two days and T. mentagrophytes at five days.
T. mentagrophytes lyophile was reconstituted in sterile water and grown on media containing 1 % peptone and 4 % glucose.A seven-day-old plate of well sporulating pure culture was washed and resuspended in 1 L of media.A 30 μL sample of this culture was dispensed into each 3 mL sample of the diluted test solutions (1 % peptone and 4 % glucose).The 13 × 100 mm glass culture tubes with Morton cap closures were incubated at 30°C.

Conclusions
Preorbital glands are common among ungulates, which usually mark twigs and grass with their contents as a chemical cue to other members of the species [9,10].Because of scent marking in cases like this, almost all animal skin glands are usually referred to as "scent glands."While the semiochemical function of these animal skin glands has been the focus of research in this area, a further function of these glands may be to produce antimicrobial compounds against dermal pathogens.Antimicrobial compounds found in these glands may be biosynthesized by the animal itself, or by microbes that live in these glands.
Prospecting for antimicrobial compounds from animal skin glands followed by synthesis of appropriate analogues is an area of drug discovery that has been largely unexplored and has the potential of producing new antimicrobial agents against pathogenic dermal organisms.