Synthesis of 1,2,3-triazole containing compounds as potential antimicrobial agents

General procedure for the click reactions

In a round bottom flask charged with a stir bar, the alkyne (1.0 equiv.) was dissolved in THF:H₂O (0.08 M, 1:1). In a separate flask, a mixture containing sodium ascorbate (0.50 equiv.), copper (II) sulfate pentahydrate (0.20 equiv.), and THF:H₂O (0.08 M, 1:1) was prepared. To this mixture was added the azide (1.0 equiv.) dissolved in THF (approximately 2–3 mL). The azide mixture was then added all at once to the flask containing the alkyne. The resulting mixture was allowed to stir at room temperature overnight. Work up consisted of partitioning the reaction between ethyl acetate and water, washing the organic layer with water, then brine, drying over anhydrous sodium sulfate, and concentration by rotary evaporation. The crude product was typically purified via flash column chromatography.

Bacterial and fungal strains

All synthesized compounds were tested against several Gram-negative and Gram-positive bacterial strains as well as fungal strains to assess their activity, which were obtained from different sources. Klebsiella pneumoniae ATCC 27736, Salmonella enterica ATCC 14028, and Escherichia coli MC1061 were donated by Professor Paul J. Hergenrother (University of Illinois at Urbana-Champaign, Champaign, IL, USA). The bacterial strain Acinetobacter baumannii ATCC 19606 and the filamentous fungal strain Aspergillus terreus ATCC MYA-3633 were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA). Bacillus anthracis 3452 str. Sterne was provided by Professor Philip C. Hannah (University of Michigan, Ann Arbor, MI, USA). Enterobacter cloacae ATCC 13880, Pseudomonas aeruginosa ATCC 14028, Staphylococcus aureus ATCC 25923, and Staphylococcus epidermidis ATCC 12228 were donated from Professor Dev P. Arya (Clemson University, Clemson, SC, USA). M. smegmatis mc²122 was provided by Professor Sabine Ehrt (Weill Cornell Medical College, New York, NY, USA). The yeast fungal strain Candida albicans ATCC 10231 was acquired from Professor Jon Y. Takemoto (Utah State University, Logan, UT, USA). All bacterial and fungal strains were stored at −80 °C.

Determination of minimum inhibitory concentration (MIC) values

A total of 10 mg/mL stock solutions of compounds were prepared in DMSO. Fluconazole (FLC), kanamycin A (KAN), and ampicillin (AMP) were purchased from VWR (Radnor, PA, USA) and were dissolved in DMSO (FLC) or ddH₂O (KAN and AMP) at final concentrations of 10 mg/mL.

Bacterial strains were streaked on Mueller-Hinton (MH) plates and allowed to grow at 37 °C overnight. 5–10 colonies were scraped from the plate and suspended in MH broth with vortexing to reach an attenuance at 600 nm (OD₆₀₀) of ∼0.5. The resulting culture was diluted 1:1000 and added to the prepared serially doubly diluted compound solutions in 96-well plates. For M. smegmatis, a 1:100 dilution of the bacteria was done in MH. All MIC values were determined using 96-well plates with low evaporation seals at least in duplicate. For each row, one well was reserved for sterile control (which consisted of 200 µL of MH medium) and another well was reserved for growth control (which consisted of 100 µL of MH medium seeded with 100 µL of bacterial culture).

The MIC values of compounds against yeast cells were determined in 96-well plates as described in the CLSI document M27-A3 with minor modifications (Clinical Laboratory Standards Institute, 2008b). A single colony of freshly prepared yeast cells was used to inoculate five mL of yeast extract peptone dextrose (YPD) broth prior to incubation overnight with shaking at 200 rpm at 35 °C. From the actively growing yeast culture, 100 mL were then transferred to 900 mL of RPMI 1640 medium and re-adjusted to achieve OD₆₀₀ of 0.12 (∼1 × 10⁶ CFU/mL). The cell suspension was further diluted to achieve 1:100 in RPMI 1640 medium. 100 mL of cells were added to the wells of a 96-well microtiter plates that contained compounds prior to incubation for 48 h at 35 °C.

MIC for filamentous fungi were determined as previously described in CLSI document M38A2 (Clinical and Laboratory Standards Institute, 2008a). Spores were harvested from sporulating cultures growing on potato dextrose agar (PDA) by filtration through sterile glass wool and enumerated by using a hemocytometer to obtain the desired inoculum size. Serial dilutions of compounds were made in sterile 96-well microplates in RPMI 1,640 medium. Spore suspensions were added to the wells to afford a final concentration of 5 × 10⁵ CFU/mL. The plates were incubated at 35 °C for 48 h. The MIC values of compounds against filamentous fungi were based on the complete inhibition of growth when compared to the growth control.

Synthesis of first generation 1,2,3-triazole compounds

Our primary goal was to synthesize a number of simple compounds that contained a 1,2,3-triazole scaffold in order to determine their potential antimicrobial activity.

The key functional characteristic of our first generation of compounds was the 1,2,3-triazole with a one-carbon linker derived from the corresponding benzyl azide (Fig. 3). In addition, the two aromatic rings possessed a variety of functionality including electron-withdrawing groups (i.e., CF₃, F, etc.) as well as electron-donating groups (i.e., OCH₃, t-Bu, etc.). The key synthetic step involves a copper-catalyzed azide-alkyne cycloaddition between the necessary azide and alkyne (Fig. 3).

The synthesis of compounds 1–23 (Fig. 3) began with the formation of the azide intermediates. While many were commercially available, we chose to synthesize all azides via a substitution reaction between the corresponding benzyl bromide and sodium azide in DMF at room temperature for 3 h (Sá, Ramos & Fernandes, 2006). Since all of our synthesized azide intermediates were known compounds, each was verified solely by ¹H NMR and used without further purification.

With the azides in hand, click reactions were performed with a variety of commercially available alkynes to yield compounds 1–23 (Fig. 3). The synthesized 1,2,3-triazole compounds (1–23) were obtained in moderate to good yield and provided sufficient quantities for biological testing (see Materials & Methods section for the general procedure).

Synthesis of second generation 1,2,3-triazole compounds

After the initial set of compounds were obtained, we decided to probe the linker length. With wanting to keep the molecule relatively rigid, we synthesized compounds 24–26 (Fig. 4) from either 1-(2-azidoethyl)naphthalene or 1-(2-azidoethyl)-4-methoxybenzene in order to increase the linker from one carbon to two carbons and followed an analogous synthesis to those previously mentioned.

We also wanted to investigate the use of a benzyl azide derived from a heterocyclic aromatic compound. Based on commercially available benzyl bromides, we chose to synthesize (2-azidomethyl)quinoline, (2-azidomethyl)pyridine, and 3-(2-azidoethyl)-1H-indole. As all azide intermediates were known compounds, each was verified solely by ¹H NMR and used without further purification. With the heterocyclic azides in hand, we performed the key click reactions with the corresponding alkynes to yield compounds 27–32 (Fig. 4).

Synthesis of third generation 1,2,3-triazole compounds

In our final generation of compounds (Fig. 5), we sought to investigate how a carbonyl/hydroxyl functionality would affect the antifungal activity. Our motivation was two-fold. We anticipated that the added functional group would increase the activity; however, if this was not the case, we foresaw further derivatization whereby the carbonyl could be reduced to the hydroxyl.

To begin, 2-bromo-1-(4-fluorophenyl)ethan-1-one was converted to 2-azido-1-(4-fluorophenyl)ethan-1-one using sodium azide as previously described. Following the substitution reaction, the azide was reacted with the alkyne under analogous conditions to provide the carbonyl containing compounds 33–34 (Fig. 5). To further derivatize, compounds 33–34 were reduced using NaBH₄ to provide the alcohols 35–36 (Fig. 5).

Biological evaluation of synthesized 1,2,3-triazole compounds

Once the structure of each compound was confirmed, it was tested against several Gram-negative and Gram-positive bacterial strains as well as fungal strains to assess antimicrobial activity. When assessing the biological activity of our synthesized compounds, we sought to find compounds that possessed single digit μg/mL antimicrobial activity. To our disappointment, our synthesized compounds did not possess antibiotic or antifungal activity (data tables in supplemental information). Interestingly, compound 14(Fig. 3) showed minimal activity (16 µg /mL) toward the bacteria Mycobacterium smegmatis. This data was surprising to us given the similarities between compound 14 and inactive compound 13. Both compounds are derived from the same azide precursor (1-(azidomethyl)naphthalene) but differ in the placement of the fluorine on the aryl group of the alkyne precursor. Compound 14 bears the fluorine in the ortho position, whereas the fluorine substituent is in the para position in 13. We would like to further investigate this result given the structural similarities of these two compounds.