HHS Public Access Author manuscript Author Manuscript

Bioorg Med Chem. Author manuscript; available in PMC 2017 November 01. Published in final edited form as: Bioorg Med Chem. 2016 November 1; 24(21): 5633–5638. doi:10.1016/j.bmc.2016.09.027.

Identification of 5,6-dihydroimidazo[2,1-b]thiazoles as a New Class of Antimicrobial Agents Yangmei Lia,*, Nina Biondaa, Renee Fleemanb, Hongjie Wanga, Akihiko Ozawaa, Richard A. Houghtena, and Lindsey Shawb aTorrey

Pines Institute for Molecular Studies, 11350 SW Village Parkway, Port St. Lucie, FL 34987, United State

Author Manuscript

bDepartment

of Cell Biology, Microbiology and Molecular Biology, University of South Florida, 4202 East Fowler Avenue, ISA2015, Tampa, FL 33620, United States

Abstract

Author Manuscript

In an effort to develop novel antimicrobial agents against drug-resistant bacterial infections, 5,6dihydroimidazo[2,1-b]thiazole compounds were synthesized and tested for their antimicrobial activity. Eight compounds comprised by two sub-scaffolds were identified as hits against methicillin-resistant Staphylococcus aureus (MRSA). These hits were modified at 6-position by replacing (S)-6 to (R)-6 configuration and the (R)-isomers increased their antimicrobial activities by two-fold. The most active compound showed a MIC90 value of 3.7 µg/mL against MRSA in a standard microdilution bacterial growth inhibitory assay. This compound protected wax moth worms against MRSA at a dose of 5× MIC using a worm infectious model. This compound also exhibited inhibition of DNA gyrase activity in a DNA gyrase supercoil assay, suggesting the 5,6dihydroimidazo[2,1-b]thiazoles may target DNA gyrase for the antimicrobial action.

Graphical Abstract

Author Manuscript

Keywords heterocyclic; antimicrobial; MRSA; DNA gyrase; imidazothiazole

*

Corresponding author. Tel.: +1-772-345-4725; fax: +1-772-345-3649; [email protected]. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Li et al.

Page 2

Author Manuscript

1. Introduction

Author Manuscript

Over the last decade, methicillin-resistant Staphylococcus aureus (MRSA) strains have emerged as the major concern of multidrug-resistant pathogens in nosocomial and community settings.1 To treat MRSA infections, therapeutic alternatives such as vancomycin, linezolid, tigecycline, and daptomycin have been introduced into clinical practice.2–5 Vancomycin was the ‘antibiotic of last resort’ to treat many serious bacterial infections; but vancomycin-resistant bacteria have already emerged and spread,6 resulting in vancomycin-resistant MRSA (as VRSA) and vancomycin-resistant enterococci (VRE). Linezolid and tigecycline are only approved for certain indications and resistance has already been reported.7 Daptomycin is used in the treatment of systemic and life-threatening MRSA infections, but it binds avidly to pulmonary surfactant; therefore, it cannot be used in the treatment of MRSA pneumonia,8 a common MRSA-caused infection. Even worse, MRSA has already developed resistance to daptomycin during therapy with this agent.9 Thus, even though therapies currently are still available to treat MRSA infections, the need for new antibiotics to fight this ever-expanding resistance is pressing.

Author Manuscript

Imidazole and thiazole are important pharmacophores exhibiting many biological properties, including antimicrobial activity, antitumor activity, et cetera.10–11 Imidazothiazoles fusing imidazole with thiazole may integrate the biological properties of both. In addition, 5,6dihydroimidazo[2,1-b]thiazole may be more attractive because these compounds also incorporate chiral center(s). Numerous examples show the biological activities and specificities of chemical compounds change with a single alternation of a chiral residue either from S- to R- or R- to S-;12 therefore, chiral compounds generally are more selective and specific towards a biological target. To discover the therapeutic potentials of these compounds, we synthesized a imidazothiazole library and tested their antimicrobial activity using bacterial growth inhibitory assays. Herein, we report the discovery of two subscaffolds with imidazothiazole compounds exhibiting potent antimicrobial activities against Gram-positive bacteria.

2. Results and discussion 2.1. Chemistry

Author Manuscript

The imidazothiazole compounds were synthesized using a method combining solid-phase synthesis with microwave radiation (Scheme 1). A reaction intermediate, 4-substitituedimidazolidine-2-thione (5), was synthesized by solid-phase synthetic method as we described.13 Briefly, a Boc-protected amino acid was coupled to the MBHA resin using standard Bocchemistry to generate a resin bound amino acid (2). After exhaustive reduction of the resin bound amino acid with BH3•THF, a resin bound diamine (3) was formed. The resin bound diamine was reacted with 1, 1’-thiocarbonyldiimidazole to form the 4substititued-imidazolidine-2-thione (4) on resin. The 4-substititued-imidazolidine-2-thione (5) was released from the resin after treatment with anhydrous HF.

Bioorg Med Chem. Author manuscript; available in PMC 2017 November 01.

Li et al.

Page 3

Author Manuscript

The 4-substititued-imidazolidine-2-thione (5) was then reacted with an α-bromoketone in ethanol at 85 °C for 10 min under microwave radiation to form the final product 2,3,6substituted-5,6-dihydroimidazo[2,1-a]thiazole (6). The imidazothiazole compounds were analyzed by LC-MS and the majority of the compounds had a yield of 90% and a purity of 85% or higher; therefore, the crude compounds were tested for the antimicrobial activity. 2.2. Antimicrobial activity

Author Manuscript

An initial screening was carried out toward MRSA ATCC 33591 in a bacterial growth inhibitory assay. All compounds were tested in duplicate at a concentration of 64 µg/mL. A set of 8 compounds, LS-1 to LS-8 (Figure 1), was identified exhibiting >80% growth inhibition of S. aureus compared to a control culture. These compounds comprise either phenyl group at both R2 and R3 or a naphthyl group at R2 and hydrogen at R3, indicating the two sub-scaffolds of 5,6-dihydroimidazo[2,1-b]thiazole may have inhibitory activity towards MRSA. The library was also screened towards E. Coli, but no compound was found exhibiting significant growth inhibition at the concentration of 64 µg/mL. The screening results suggested the imidazothiazole scaffold may selectively inhibited Gram positive bacteria over Gram negative bacteria.

Author Manuscript

These compounds were purified by HPLC and their antimicrobial activity was tested against MRSA strains using a standard microdilution broth assay. Two S. aureus strains, MRSA ATCC 33591 and multidrug-resistant BAA-44, were tested. BAA-44 strain is a multidrugresistant strain resistant to almost all classes of antibiotics including β-lactams, macrolides, tetracyclines, and fluoroquinolones. Minimum inhibitory concentration (MIC90) value of each compound towards each strain was determined; most of the compounds exhibited an MIC90 value in a range of 8–64 µg/mL, or 20–200 µM (Table 1), indicating these compounds are moderately potent against the two resistant S. aureus strains. These S enantiomers generally showed ~2-fold higher inhibitory activity towards the BAA-44 strain than the ATCC33591 strain.

Author Manuscript

To identify compounds having higher antimicrobial activity, the R enantiomer analogs, DR-1 to DR-8, were synthesized by the same synthetic method but using D-amino acid instead of L-amino acid. Other than the configuration at the 6-position, the S and the R enantiomers are identical in structure. These R enantiomers were tested for their antimicrobial activity in the bacterial growth inhibitory assays using the standard microdilution broth method. Three S. aureus strains, MRSA ATCC 33591, multidrug-resistant BAA-44, and a vancomycinintermediate S. aureus strain VISA ATCC 700699 were tested. The MIC90 values of the R enantiomers against the BAA-44 strain were generally comparable to those against the ATCC 33591 strain, yet the MIC90 against the VISA strain was about 2-fold higher (Table 1). Vancomycin-resistant S. aureus strains have significantly thickened cell walls;14 the thickened cell walls may hinder the compounds from diffusion to the cell, therefore may decrease their antimicrobial activity towards VISA. The MIC90 value of the R enantiomer was about half the value of its corresponding S enantiomer, indicating of this imidazothiazole scaffold R configuration at 6-position had increased antimicrobial activities towards MRSA. Bioorg Med Chem. Author manuscript; available in PMC 2017 November 01.

Li et al.

Page 4

2.3. Hemolytic activity and cytotoxicity

Author Manuscript

To evaluate the therapeutic potential of these antimicrobial compounds, their cytotoxicity toward human red blood cells and HEK cells was determined. All compounds including both S and R enantiomers were tested for their hemolytic activity, and all of them exhibited < 2% hemolysis at 128 µg/mL, indicating the imidazothiazole compounds were non-hemolytic. Cytotoxicity of the R enantiomers was also assessed by a standard MTT cell viability assay in HEK cells; inhibition of cell viability by each compound was analyzed and the IC50 value was determined (Table 1). A therapeutic index (TI, a ratio of IC50 to MIC90) was then calculated for each compound (Table 1) by comparing the IC50 value to the MIC90 value. Compounds DR-1, DR-3, and DR-5 showed TI values around 5 towards the BAA-44 strain, while DR-1 displayed the highest average TI value towards all three multidrug-resistant MRSA strains, suggesting DR-1 is the best anti-MRSA hit compound of the current study.

Author Manuscript

2.4. Inhibitory activity against DNA gyrase The mechanism of antimicrobial action of the imidazothiazoles was then investigated. As these imidazothiazole-scaffold compounds have some structural similarity to the benzimidazole urea compounds recently reported as DNA gyrase inhibitors,15–16 we considered that our compounds may also target DNA gyrase. DNA gyrase/topoisomerase is a crucial enzyme that catalyzes the ATP-dependent negative supercoiling of double-stranded closed-circular DNA.17 DNA gyrase is essential in all bacteria but absent from higher eukaryotes and has been clinically validated as the target for developing antibiotics such as the fluoroquinolones.18

Author Manuscript

The best hit DR-1 was then investigated for the inhibitory effect on S. aureus DNA gyrase through DNA gyrase supercoiling assays. To eliminate the possibility of direct interaction between relaxed DNA and DR-1, gels in the absence and presence of gyrase were run sideby-side (Figure 2). Samples including ciprofloxacin at 32 µg/ml as a positive control, relaxed pHOT-1 alone, and assay buffer as a negative control are shown in the last 3 lanes. The electrophoresis data showed a dose-dependent inhibitory effect of DR-1 on gyrase supercoiling activity among the concentration range of 0.23 to 7.5 µg/mL, suggesting DNA gyrase may be a target for the antimicrobial activity of DR-1. 2.5. Antimicrobial efficacy in wax moth worms

Author Manuscript

The in vivo antimicrobial efficacy of DR-1 was also tested against a hospital-isolated MRSA strain using a wax moth worm (Galleria mellonella) model of infection.19 Wax moth worm model has been generally accepted as a useful preliminary model for assessing the in vivo efficacy of candidate anti-staphylococcal agents before proceeding to mammalian studies.20 Four groups of the worms were used in the test. One group of worms was treated with DMSO only as a non-MRSA-infected negative-control group (Group 1). Three groups of worms were inoculated with MRSA during the 3-day-efficacy test (Group 2–4). After 1 day cultivation, the 3 groups of worms were inoculated with lethal dose 5 µL of MRSA (~ 2 × 109 CFU mL−1) into the last left proleg using a 50 µL Hamilton syringe. At 2 h post inoculation, the two infected groups of 10 worms were treated with DR-1 in DMSO at doses of 5× MIC (Group 2) and 10× MIC (Group 3), respectively. The last group of the infected 10

Bioorg Med Chem. Author manuscript; available in PMC 2017 November 01.

Li et al.

Page 5

Author Manuscript

worms was treated with DMSO only (Group 4). Compared with the control groups, compound DR-1 at 5× and 10× MIC significantly increased survival of worms infected with MRSA (Figure 3). This was determined to be statistically significant (5× MIC: p128 µg/mL for ATCC33591, and >128 µg/mL

d

51(16 ± 1.1)

69(24 ± 1.3)

10(8.3 ± 0.8)

21(9.4 ± 0.2)

111(42 ± 2.9)

38(15.7 ± 0.6)

20(9 ± 0.3)

12(8 ± 0.1)

ATCC33591

MIC90, µM (µg/mL)a

338(128 ± 12)

9.5(2.4 ± 0.4)

31(7.9 ± 0.5)

11(3.7 ± 0.2)

BAA-44

The minimum concentration that required for inhibiting 50% of human HEK cells proliferation in a MTT assay.

c Therapeutic Index: IC50/MIC90 (BAA-44).

b

MIC90 values are the mean of three independent experiments, each experiment conducted in duplicate.

a

R2

R1

Author Manuscript

Antimicrobial activity of the imidazothiazole compounds

Author Manuscript

Table 1 Li et al. Page 14

Bioorg Med Chem. Author manuscript; available in PMC 2017 November 01.