WO2016187521A1

WO2016187521A1 - 5-substituted 1 h-tetrazole compounds, methods of synthesizing and therapeutic use

Info

Publication number: WO2016187521A1
Application number: PCT/US2016/033493
Authority: WO
Inventors: Adiel COCA; Elizabeth Lewis ROBERTS
Original assignee: South Connecticut State University
Priority date: 2015-05-21
Filing date: 2016-05-20
Publication date: 2016-11-24

Abstract

A pharmaceutical formulation includes an antibiotic 5-substituted 1H-tetrazole compound in an amount sufficient to treat a bacterial infection, the antibiotic 5-substituted 1H-tetrazole compound being selected from the group consisting of: an aryl tetrazole compound, a heteroaryl tetrazole compound, a vinyl tetrazole compound, and a benzylic tetrazole compound. A method of synthesizing an antibiotic 5-substituted 1H-tetrazole compound includes forming a mixture of an azide, an organonitrile, a catalyst, and a solvent and heating the mixture at a temperature of about 100-160°C for about 30 minutes to 4 hours to synthesize the antibiotic 5-substituted 1H-tetrazole compound.

Description

5-SUBSTITUTED 1 H-TETRAZOLE COMPOUNDS, METHODS OF SYNTHESIZING AND THERAPEUTIC USE

Technical Field

[0001] The present invention relates generally to 5-substituted 1 H-tetrazole compounds and, more specifically, to 5-substituted 1 H-tetrazole compounds with antimicrobial properties, methods of synthesizing same, and therapeutic uses of same.

Background

[0002] A major reason for the increase in life expectancy during the latter part of the 20^th century was the discovery of antibiotics. However, overuse and improper use of antibiotics as well as an increase in travel, has led to a steady increase in and the spread of multi-drug resistant bacterial strains. Approximately 17 million deaths primarily in children and the elderly occur worldwide each year due to infectious diseases. For example, there are around 1 .3 million deaths annually worldwide due to tuberculosis. Moreover, substantial research still needs to be done to fight neglected diseases caused by microorganisms.

[0003] Overcoming bacterial resistance to antibiotics has been a major challenge to researchers ever since the discovery of the first antibiotics. In the last few decades there has been a rapid rise in the number of multi-drug resistant bacteria. The European Centre for Disease Prevention and Control has reported that around 25,000 people within the European Union succumb to infection caused by multiple antibiotic resistant bacteria each year. Over the last four decades, only six new classes of antibiotics have been introduced into the market with all them having little to no activity against Gram-negative bacteria. Most antibiotics developed during this time belong to an already established class. In fact, 73% of antibiotics approved between 1981 and 2005 belonged to one of four well established classes of compounds (i.e., cephalosporins, penicillins, quinolones, and macrolides). Thus, there is a critical need to develop novel scaffolds with antibiotic activity. [0004] Given the currently rapid emergence of multi-drug resistant strains of various microorganisms, the frequency of microbial infections among immunocompromised individuals, and the lack of available classes of antibiotics, there is a need to develop effective new antimicrobial drug leads.

Summary

[0005] An embodiment of the present invention provides a pharmaceutical formulation that includes an antibiotic tetrazole 5-substituted 1 /-/-tetrazole compound in an amount sufficient to treat a bacterial infection, the antibiotic tetrazole compound being selected from the group consisting of: an aryl compound, a heteroaryl tetrazole compound, a vinyl tetrazole compound, and a benzylic tetrazole compound.

[0006] An embodiment of the present invention provides a method of treating a bacterial infection in a subject. The method includes administering an antibiotic 5- substituted 1 /-/-tetrazole compound in an amount sufficient to treat a bacterial infection, the antibiotic tetrazole compound being selected from the group consisting of: an aryl compound, a heteroaryl tetrazole compound, a vinyl tetrazole compound, and a benzylic tetrazole compound.

[0007] An embodiment of the present invention provides a method of synthesizing an antibiotic tetrazole compound. The method includes forming a mixture of an azide, an organonitrile, a catalyst, and a solvent and heating the mixture at a temperature of about 100-160 °C for about 30 minutes to 4 hours to synthesize the antibiotic tetrazole compound.

Detailed Description

[0008] Aspects of the invention are directed to pharmaceutical formulations of one or more tetrazole compounds described herein having antibiotic activity.

Tetrazoles are a class of heterocycles and have the ability to hold large amounts of energy. Compared to carboxylic acids, tetrazole compounds are more lipophilic (i.e., increased biological absorption), more metabolically stable, and bind anions better. Tetrazole compounds are more suitable for cell penetrance than carboxylates due to their higher pKa.

[0009] Embodiments of the present invention are directed to a method of synthesizing 5-substituted 1 /-/-tetrazole compounds. Tetrazole compounds may be synthesized through a [2 + 3] cycloaddition of an azide and a nitrile. The

cycloaddition of an organonitrile with sodium azide is shown below in Equation 1.

[0010] In various embodiments, sodium azide (e.g., 1 -3 equivalent), an organonitrile (e.g., 2 mmol, 1 equivalent), a rare-earth or post-transitional metal catalyst (e.g., 0.1 -0.5 equivalent), and a solvent (e.g., 8 ml_) may be combined and heated. In an embodiment, the reaction may occur in a microwave reactor.

Industrial microwave reactors offer variability controls for wattage and safety features for concerns over pressure release. Further, this heating process minimizes side reactions through elicit manipulation of input radiation and reduces the amount of catalyst required to complete reactions. The reaction may be heated at about 100- 160 °C for about 30 minutes to 4 hours. The product may be extracted by addition of a saturated aqueous sodium bicarbonate solution (e.g., 15 ml_). The aqueous layers may be washed twice with ethyl acetate (e.g. , 15 ml_) and subsequently acidified with a concentrated hydrochloric acid solution. An extraction may be performed twice using ethyl acetate (e.g., 15 ml_ x 2). The organic layers may be combined and dried with anhydrous sodium sulfate. The tetrazole compound may then be concentrated by rotary evaporation using reduced pressure.

[0011] It should be recognized that the azide and nitrile used in the synthesis of the 5-substituted 1 /-/-tetrazole compounds may vary based on the intended application. Both organic and inorganic azides can violently decompose with little input of energy making them heat and shock sensitive. These considerations may be minimized through the use of azides wherein the total number of nitrogen atoms does not exceed the number of carbon atoms. The azide may include, without limitation, sodium, tin, silicon, and organoaluminum azides. Exemplary nitriles include, without limitation, para-substituted aryl nitriles, heteroaryl nitriles, vinyl nitriles, benzylic nitriles, and aliphatic nitriles.

[0012] Additionally, a variety of solvents and catalysts may be used in the synthesis of the 5-substituted 1 H-tetrazole compounds useful in pharmaceutical formulations. For example, the solvent may be an aprotic polar solvent, such as dioxane, dichloromethane (DCM), tetrahydrofuran (THF), or dimethylformamide (DMF), or a protic polar solvent, such as an isopropanol/water mixture. The catalyst may include, for example, rare earth metal catalysts and post-transitional metal catalysts. Exemplary catalysts include, without limitation, zinc salts, ytterbium triflate (Yb(OTf)₃), indium chloride (lnCI₃), indium triflate (ln(OTf)₃), cerium chloride (CeCI₃), scandium triflate (Sc(OTf)₃), bismuth chloride (BiCI₃), bismuth triflate (Bi(OTf)₃), and iron trichloride (FeCI₃). The yield percentage for a given synthesis reaction may vary depending on the catalyst used.

[0013] The types of 5-substituted 1 H-tetrazole compounds useful in

pharmaceutical formulations may vary based on the intended application.

Exemplary 5-substituted 1 H-tetrazole compounds include, without limitation, aryl, heteroaryl, vinyl, benzylic, and aliphatic tetrazole compounds. Exemplary tetrazole compounds include, but are not limited to: 5-(4-nitrobenzo)-1 H-tetrazole (1 a), 1 - (4(1 H-tetrazol-5-yl)phenyl)ethan-1 -one (1 b), 5-(4-bromobenzo)-1 H-tetrazole (1 c), 1 ,4-di(1 H-tetrazol-5-yl)benzene (1 d), 3-(1 H-tetrazol-5-yl)pyrrole (1 e), 4-(1 H-tetrazol- 5-yl)isoquinoline (1f), (E)-1 ,2-di(1 H-tetrazol-5-yl)ethene (1 g), and phenyl(1 H-tetrazol- 5-yl)methanol (1 h). The structural formulas for these compounds are shown below.

[0014] Additional exemplary tetrazole compounds include: 5-(4-chlorophenyl)- 1 H-tetrazole (1 i), 5-(4-methoxyphenyl)-1 H-tetrazole (1j), 5-phenyl-1 H-tetrazole (1 k), phenyl(1 H-tetrazol-5-yl)methanone (1 1), 5-(thiophen-3-yl)-1 H-tetrazole (1 m), and 5- (furan-2-yl)-1 H-tetrazole (1 n). The structural formulas for these compounds are shown below.

[0015] In an aspect of the invention, a pharmaceutical formulation includes, in addition to the one or more tetrazole compounds, one or more additional antibiotic compounds. The combination of the one or more tetrazole compounds with one or more additional antibiotic compounds may result in a synergistic effect on the antimicrobial properties of the combination. Exemplary additional antibiotic compounds include, without limitation, amoxicillin trihydrate, sulfamethoxazole, and trimethoprim. In various embodiments, the mass ratio of the tetrazole compound to the antibiotic compound may be about 1 : 1 , 2: 1 , or 1 :2, although other ratios may be used.

[0016] Aspects of the invention are directed to methods of treating a subject for a bacterial infection with pharmaceutical formulations that include one or more antibiotic tetrazole compounds. The antibiotic tetrazole compound may be a 5- substituted 1 H tetrazole compound as described herein. Further, aspects of the invention are directed to methods of treating a subject for a bacterial infection with pharmaceutical formulations that include one or more antibiotic tetrazole compounds in combination with one or more additional antibiotic compounds.

[0017] When treating bacterial infections, the one or more antibiotic 5- substituted 1 H tetrazole compounds are administered in a dose that achieves levels of the one or more antibiotic tetrazole compounds and/or their active metabolites at the sight of the infection to levels sufficient to have an antibiotic effect to treat and prevent bacterial infections. An antibiotic effect means that the growth of the microorganisms is inhibited. In some embodiments, the method includes

administering at least one antibiotic compound, such as one or more of trimethoprim, sulfamethoxazole, and amoxicillin trihydrate, in addition to the one or more antibiotic tetrazole compounds, each at a dose sufficient to result in a synergistic antibiotic effect to treat and prevent bacterial infections. The dose and duration of treatment required to adequately treat a bacterial infection in a given patient may vary widely due to titration required by the effectiveness of treatment and the rate of clearance. In various embodiments, the dosage of the tetrazole compound or the

tetrazole/antibiotic combination may range from 0.1 mg/kg per day to 1 g/kg per day or from 1 -100 mg/kg per day.

[0018] The pharmaceutical formulations may be administered by standard routes, such as by injection, orally, topically, and transdermally. Accordingly, pharmaceutical formulations may be in a form suitable for the desired route of administration. For example, the pharmaceutical formulations may be dissolved in a liquid for injection, formed into pills or filled into capsules for oral administration, or suspended in an ointment for topical or transdermal administration. The

pharmaceutical formulation may include additional additives pharmaceutically acceptable for the desired route of administration.

[0019] In order to facilitate a more complete understanding of the embodiments of the invention, the following non-limiting examples are provided.

Example 1

[0020] Synthesis of 1-(4(1H-tetrazol-5-yl)phenyl)ethan-1-one (1b). 1 -(4(1 H- Tetrazol-5-yl)phenyl)ethan-1 -one (1 b) (290 mg, 2 mmol), NaN₃ (260 mg, 4 mmol), InC (89 mg, 0.4 mmol), and 8 mL of a 3: 1 isopropanol/water mixture were added to a 30-mL Pyrex microwave vessel and capped. The microwave vessel was then placed in a Milestone Start Synth microwave reactor. The reaction was magnetically stirred and heated for 1 hour at 160 °C. The reaction was monitored by TLC using an ether/hexane mixture (typically 50/50) for development. After cooling, the reaction mixture was diluted with saturated aqueous sodium bicarbonate (20 mL) and washed with ethyl acetate (2 x 15 mL). The aqueous sodium bicarbonate layer was cooled to 0 °C and acidified to a pH of 2 or less with concentrated hydrochloric acid, which was added drop-wise. The precipitate formed was extracted with ethyl acetate (3 x 15 mL). The combined organic layers were dried with anhydrous sodium sulfate and decanted into a tared round bottom flask. The organic layer was concentrated under reduced pressure. The tetrazole product was recrystallized from ethyl acetate and hexane. All reagents mentioned above were used unpurified.

[0021] NMR Spectra. NMR spectra were acquired on a spectrometer at 300 MHz for ¹ H and 75 MHz for ¹³C acquisitions. All ¹ H NMR spectra were taken in DMSO-d6 using DMSO as a standard at 2.52 ppm. All ¹³C NMR spectra were taken in DMSO-d6 using DMSO as a standard at 40.45 ppm. An IR spectrum was obtained using an FTIR spectrophotometer. A melting point was also obtained for the solid products. 1 -(4(1 /-/-Tetrazol-5-yl)phenyl)ethan-1 -one (1 b) is a white solid. IR (KBr, thin film) vmax (cm-1 ): 3400 (br), 1710, 1674, 1242; ¹ H NMR (300 MHz, DMSO-d6, δ): 8.19 (m, 4H), 2.67 (s, 3H); ¹³C NMR (75 MHz, DMSO-d6, δ): 197.5, 155.4 (br), 138.5, 130.2, 129.2, 127.3, 26.9; mp 175-176 °C.

[0022] Minimum Inhibitory Concentration (MIC) Determination. The

antibacterial properties of 1 -(4(1 /-/-tetrazol-5-yl)phenyl)ethan-1-one (1 b) were investigated against Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa. Compound 1 b was screened for antimicrobial activity by a modified Kirby-Bauer assay. Compound 1 b was dissolved in DMSO at concentration of 0.1 mg/mL. Next, 10 μΙ_ of the solution was placed on a nutrient agar plate streaked with a lawn of bacteria of S. aureus (ATTC 27661 ), E. coli (ATCC 14948), or P.

aeruginosa (ATCC 27853). Bioactivity was determined after incubating each plate at 37 °C for 24 hours. A lack of bacterial growth where a tetrazole solution was placed was taken as a sign of bioactivity. Because 1 -(4(1 /-/-tetrazol-5-yl)phenyl)ethan-1 -one (1 b) was found to be active, it was dissolved in a 20% DMSO in nutrient broth at an initial concentration of 10 mg/mL. The tetrazole standard solution was serially diluted with nutrient broth in glass tubes to make solutions with concentrations of 1000, 500, 250, 125, 62.5, 31 .3, 15.6, 7.81 , and 3.91 μg/mL. A solution without any tetrazole present was also prepared. Each solution was inoculated with 10 μΙ_ of an overnight culture of one of the previously mentioned strains of bacteria. After 24 h of incubation at 37 °C, the minimum inhibitory concentration (MIC) for the 1 b compound was determined as the concentration of the test substance, which completely inhibited the growth of the microorganism (solution was 100% transparent).

[0023] 1-(4(1 /-/-Tetrazol-5-yl)phenyl)ethan-1 -one (1 b) was significantly active against Gram-negative E. coli. It performed similarly to trimethoprim and amoxicillin trihydrate against E. coli (see data below in Table 1 ).

Example 2

[0024] Synthesis of para-substituted benzotetrazoles. 5-substituted 1 /-/- tetrazoles were prepared in a multimode microwave reactor employing a modified literature procedure with a 3: 1 isopropanol/water mixture as the solvent, as described in Example 1 . Several p-substituted benzotetrazoles were prepared by heating the corresponding benzonitriles with 2 equivalents of sodium azide using 0.2 equivalent of indium chloride. The corresponding tetrazoles for 4-nitrobenzonitrile, 4- chlorobenzonitrile, 1-(4(1 /-/-tetrazol-5-yl)phenyl)ethan-1 -one (1 b), and 4- cyanobenzophenone reacted in high yield after 1 h at 160 °C. Other p-substituted benzonitriles such as 4-bromobenzonitrile, 4-trifluromethylbenzonitrile, and 4- methoxybenzonitrile, were synthesized after 4 h of hearing at 160 °C for full conversion. The corresponding tetrazoles for these three nitriles were also obtained in high yield. The electron-rich 4-methylbenzonitrile was much less reactive, and the corresponding tetrazole was obtained in a very low yield. 1 ,4-Dicyanobenzene was converted in very high yield when reacted with 4 equivalent of sodium azide and using 0.4 equivalent of indium chloride.

[0025] Synthesis of non para-substituted benzotetrazoles. Several other aryl tetrazoles were synthesized from non para-substituted benzonitriles. Highly electron deficient o-nitrobenzonitrile afforded a high yield of the corresponding tetrazole. Most other ortho-substituted nitriles, such as 2-chlorobenzonitrile, 2- trifluoromethylbenzonitrile, 2-methylbenzonitrile, and 4'-methyl-2-cyanobiphenyl, required 4 h of heating at 160 °C for good conversion. The monosubstituted benzonitriles tested gave a moderated yield of the corresponding tetrazoles after 4 h at 160 °C. 1 ,3-Dicyanobenzene was also converted in good yield after only 1 h by using 0.4 equivalents of indium chloride and 4 equivalents of sodium azide.

[0026] Synthesis of heteroaryl, vinyl, benzylic, and aliphatic tetrazole

compounds. Heteroaryl tetrazole compounds (i.e., tetrazole compounds attached to heteroaromatic rings) were synthesized using the method described in Example 1 with various organonitriles. The time that the reactions were heated and the yields of the tetrazole compounds varied. Several compounds were synthesized with a high yield (e.g., 55-99%) after 1 h of heating. Additionally, several vinyl, benzylic, and aliphatic tetrazole compounds were prepared using the same method. Vinyl, benzylic, and aliphatic tetrazoles are generally slower reacting than aryl tetrazoles. Some tetrazole compounds gave relatively high yields, while others were obtained in yields of less than 50%. For example, tetrazole products corresponding to 4- bromothiopene-2-carbonitrile, 3-bromobenzonitrile, 5-bromo-3-cyanopyridine and 4- cyanobenzaldehyde afforded yields of 84%, 82%, 84% and 79%, respectively.

Example 3

[0027] Minimum Inhibitory Concentration (MIC) Determination of Tetrazole Compounds. The antibacterial properties of the aryl, heteroaryl, vinyl, benzylic, and aliphatic tetrazole compounds were investigated against S. aureus, E. coli, and P. aeruginosa. The minimum inhibitory concentration (MIC) of each tetrazole compound was determined against each line of bacteria using the process described in Example 1. Table 1 shows the MIC ^g/mL) values for the 5-substituted 1/-/- tetrazole compounds: 5-(4-nitrobenzo)-1/-/-tetrazole (1a), 1-(4(1/-/-tetrazol-5- yl)phenyl)ethan-1-one (1b), 5-(4-bromobenzo)-1/-/-tetrazole (1c), 1 ,4-di(1 --tetrazol- 5-yl)benzene (1d), 3-(1 /-/-tetrazol-5-yl)pyrrole (1e), 4-(1/-/-tetrazol-5-yl)isoquinoline (1f), (E)-1,2-di(1H-tetrazol-5-yl)ethene (1g), phenyl(1H-tetrazol-5-yl)methanol (1h), 5- (4-chlorophenyl)-1/-/-tetrazole (1i), 5-(4-methoxyphenyl)-1/-/-tetrazole (1j), 5-phenyl- 1/-/-tetrazole (1k), phenyl(1/-/-tetrazol-5-yl)methanone (11), 5-(thiophen-3-yl)-1/-/- tetrazole (1m), and 5-(furan-2-yl)-1/-/-tetrazole (1n).

Table 1: Antibacterial Activities (MIC, g/mL) of Tetrazole Compounds.

[0028] Some of the tetrazole compounds tested showed significant antibiotic activity. The four compounds 1a, 1b, 1c, and 1d performed similarly to trimethoprim against S. aureus and E. coli as well as amoxicillin trihydrate against E. coli. For example, 5-(4-nitrobenzo)-1 /-/-tetrazole (1a) and 5-(4-bromobenzo)-1 /-/-tetrazole (1c) showed inhibition of Gram-positive S. aureus growth at 250 μg/mL. The following compounds displayed inhibition of Gram-negative E. coli growth at 250 μg/mL: 1 ,4- di(1/-/-tetrazol-5-yl)benzene (1d), 1-(1/-/-tetrazol-5-yl)isoquinoline (1f), and (E)-1,2- di(1 H-tetrazol-5-yl)ethene (1 g). Both 1 -(4-(1 H-tetrazol-5-yl)phenyl)ethan-1 -one (1 b) and phenyl(1/-/-tetrazol-5-yl)methanol (1h) inhibited E. coli growth at 125 μg/mL. These compounds compared well with known antibiotics trimethoprim and amoxicillin trihydrate which both had MIC values of 125 μg/mL against E coli and were more effective than sulfamethoxazole (MIC >1000 μg/mL against all three bacteria lines). The compounds shown in Table 1 had relatively weak activity against P. aeruginosa.

[0029] Minimum Inhibitory Concentration (MIC) Determination of Antibiotic Compounds. Table 2 shows the MIC values for conventional antibiotics including amoxicillin trihydrate, sulfamethoxazole, trimethoprim, and the combination of trimethoprim and sulfamethoxazole (1 : 1 by weight) that were determined against each line of bacteria using the process described in Example 1 .

Table 2: Antibacterial Activities (MIC, g/mL) of Antibiotic Compounds.

[0030] Both amoxicillin and trimethoprim had an MIC value of 125 μg/mL against E. coli, whereas sulfamethoxazole showed insignificant activity against all three bacteria. Trimethoprim also gave an MIC value of 250 μg/mL against S.

aureus. As can be seen in a comparison between Tables 1 and 2, some of the tetrazole compounds were considerably active exhibiting a minimum inhibitory concentration between 125-250 μg/mL, which is comparable to the antibiotic activity of trimethoprim against S. aureus and E. coli.

Example 3

[0031] Minimum Inhibitory Concentration (MIC) Determination

Tetrazole/ Antibiotic Combinations. The eight most active tetrazole compounds from Example 2 were tested in 1 : 1 by weight combinations with one of trimethoprim, amoxicillin trihydrate, or sulfamethoxazole. The eight tetrazole compounds include: 5-(4-nitrobenzo)-1 /-/-tetrazole (1 a), 1 -(4(1 /-/-tetrazol-5-yl)phenyl)ethan-1 -one (1 b), 5- (4-bromobenzo)-1 H-tetrazole (1 c), 1 ,4-di(1 H-tetrazol-5-yl)benzene (1 d), 3-(1 H- tetrazol-5-yl)pyrrole (1 e), 4-(1 /-/-tetrazol-5-yl)isoquinoline (1f), (E)-1 ,2-di(1 /-/-tetrazol- 5-yl)ethene (1 g), and phenyl(1 /-/-tetrazol-5-yl)methanol (1 h). Table 3 shows the MIC values for the tetrazole/antibiotic combinations determined against each line of bacteria. Table 3. Antibacterial Activities (MIC, g/mL) of Antibiotics and Tetrazole/Antibiotic

Combinations.

[0032] As can be seen, a number of the tetrazole/trimethoprim combinations showed synergistic effects against E. coli and S. aureus. There was a synergistic effect when compounds 1 a, 1 b, 1 c, and 1 d were independently used in combination with trimethoprim against Gram-negative E. coli with MIC values ranging from 0.24- 1 .95 μg/mL. Of note, these combinations all performed better against E. coli than the sulfamethoxazole/trimethoprim combination (MIC = 15.6 μg/mL). The trimethoprim combinations with compounds 1 b, 1 c, and 1 d also seemed to show a synergistic effect against Gram-positive S. aureus with MIC values as low as 3.91 μg/mL, which was similar to the MIC value obtained for the

sulfamethoxazole/trimethoprim combination. 5-(4-Bromobenzo)-1 /-/-tetrazole (1 c) with trimethoprim was active against S. aureus and E. coli at 15.6 and 0.98 μg/mL. Trimethoprim in combination with 1 ,4-di(1 /-/-tetrazol-5-yl)benzene (1 d) was active against S. aureus showing a MIC value of 31 .3 μg/mL and against E. coli with an MIC value of 0.98 μg/mL.

[0033] With trimethoprim, 2-(1 /-/-tetrazol-5-yl)pyrrole (1 e) was active at

62.50 μg/mL against S. aureus and 0.98 μg/mL against E coli. A combination of 4-

(1 /-/-tetrazol-5-yl)isoquinoline (1f) and trimethoprim showed MIC values of 7.81 μg/mL against S. aureus and E. coli. Trimethoprim with 1 ,2-di-1 /-/-tetrazole ethene (1 g) performed well against E. coli showing a MIC value of 0.98 μg/mL against E. coli and moderately against S. aureus with a MIC value of 125 μg/mL. Phenyl(1 /-/-tetrazol-5-yl)methanol (1 h) was active against E coli at a concentration of 62.5 μg/mL when used with trimethoprim. A combination of phenyl (1 /-/-tetrazol-5- yl)methanol (1 h) and amoxicillin trihydrate showed inhibition of E. coli with a MIC value of 31 .3 μg/mL but was not very active when evaluated in conjunction with sulfamethoxazole. None of the tetrazoles tested, whether or not in combination with trimethoprim, significantly inhibited the growth of Gram-negative P. aeruginosa.

[0034] The 1-(4(1 /-/-tetrazol-5-yl)phenyl)ethan-1 -one (1 b) and trimethoprim combination proved to be the most effective against both S. aureus and E coli.

Using 1 -(4(1 /-/-tetrazol-5-yl)phenyl)ethan-1-one (1 b) with trimethoprim showed an MIC value of 3.91 μg/mL against S. aureus and 0.24 μg/mL against E coli. Note that, when comparing Tables 2 and 3, it can be seen that the combination of 1 - (4(1 /-/-tetrazol-5-yl)phenyl)ethan-1 -one (1 b) with trimethoprim performed better against E. coli than the combination of sulfamethoxazole and trimethoprim. In addition, (4(1 /-/-tetrazol-5-yl)phenyl)ethan-1 -one (1 b) with trimethoprim was comparable to the sulfamethoxazole/trimethoprim combination against S. aureus. Storing compound 1 b with trimethoprim in wet DMSO did not appear to lead to a chemical reaction between these compounds after 24 hours at room temperature based on NMR spectral data. Compound 1 b was also tested in combination with the β-lactam antibiotic amoxicillin trihydrate, and a small synergistic effect with a MIC value of 31 .3 μg/mL was observed against E coli and to a lesser extent against P. aeruginosa. The combination of compound 1 b with sulfamethoxazole showed no synergistic effect at all.

[0035] These increases in activity when using the trimethoprim or amoxicillin trihydrate combination as compared to the isolated compounds may suggest that the tetrazole compounds are working through a different mechanism than trimethoprim and amoxicillin trihydrate but a similar mechanism as sulfamethoxazole, a sulfa drug. 5-(p-Aminophenyl)tetrazole has been previously examined as an analogue of p- aminobenzoic acid but was inactive against S. aureus and E. coli in concentrations up to 6 x 10^"3 mol/L (-1000 μg/mL). Sulfonamide drugs, such as sulfamethoxazole, possess antibacterial activity because they compete with p-aminobenzoic acid for the enzyme dihydropteroate synthase and, therefore, interrupt the biosynthesis of tetrahydrofolate in bacteria. In that regard, 5-substituted aryl 1 /-/-tetrazoles without a p-amino group (such as the p-amino group in 5-(p-aminophenyl)tetrazole) could potentially serve as structural analogs of sulfonamides.

[0036] While specific embodiments have been described in considerable detail to illustrate the present invention, the description is not intended to restrict or in any way limit the scope of the appended claims to such detail. The various features discussed herein may be used alone or in any combination. Additional advantages and modifications will readily appear to those skilled in the art. The invention in its broader aspects is therefore not limited to the specific details, representative apparatus and methods and illustrative examples shown and described.

Accordingly, departures may be made from such details without departing from the scope of the general inventive concept.

Claims

What is claimed is:

1 . A pharmaceutical formulation comprising:

an antibiotic 5-substituted 1 /-/-tetrazole compound in an amount sufficient to treat a bacterial infection, the antibiotic 5-substituted 1 /-/-tetrazole compound being selected from the group consisting of: an aryl tetrazole compound, a heteroaryl tetrazole compound, a vinyl tetrazole compound, and a benzylic tetrazole compound.

2. The pharmaceutical formulation of claim 1 , wherein the antibiotic 5-substituted 1 /-/-tetrazole compound is selected from the group consisting of: 5-(4-nitrobenzo)-

1 /-/-tetrazole, 1 -(4(1 H-tetrazol-5-yl)phenyl)ethan-1 -one, 5-(4-bromobenzo)-1 H- tetrazole, 1 ,4-di(1 /-/-tetrazol-5-yl)benzene, 3-(1 /-/-tetrazol-5-yl)pyrrole, 4-(1 H-tetrazol- 5-yl)isoquinoline, (E)-1 ,2-di(1 /-/-tetrazol-5-yl)ethene, and phenyl(1 /-/-tetrazol-5- yl)methanol.

3. The pharmaceutical formulation of claim 1 , further comprising:

an additional antibiotic compound in an amount sufficient to have a synergistic antibacterial effect with the antibiotic 5-substituted 1 /-/-tetrazole compound.

4. The pharmaceutical formulation of claim 3, wherein the additional antibiotic compound is selected from the group consisting of: trimethoprim, sulfamethoxazole, amoxicillin trihydrate, and combinations thereof.

5. A method of treating a bacterial infection in a subject comprising:

administering an antibiotic tetrazole compound in an amount sufficient to treat a bacterial infection, the antibiotic 5-substituted 1 /-/-tetrazole compound being selected from the group consisting of: an aryl tetrazole compound, a heteroaryl tetrazole compound, a vinyl tetrazole compound, and a benzylic tetrazole compound.

6. A method of synthesizing an antibiotic 5-substituted 1 /-/-tetrazole compound comprising:

forming a mixture of an azide, an organonitrile, a catalyst, and a solvent; and heating the mixture at a temperature of about 100-160 °C for about 30 minutes to 4 hours to synthesize the antibiotic 5-substituted 1 /-/-tetrazole compound.

7. The method of claim 6, wherein the catalyst is a rare-earth metal catalyst or a post-transitional metal catalyst.

8. The method of claim 6, wherein the catalyst is selected from the group consisting of: ytterbium triflate, indium chloride, indium triflate, cerium chloride, scandium triflate, bismuth chloride, bismuth triflate, and iron trichloride.

9. The method of claim 6, wherein an equivalent amount of the catalyst in the mixture is between 0.1 -0.5.

10. The method of claim 6, wherein the solvent is a polar protic solvent.

1 1. The method of claim 10, wherein the solvent is a 3: 1 isopropanol/water mixture.

12. The method of claim 6, wherein heating the mixture using a microwave reactor.