Letters in Applied Microbiology 2005, 40, 212–217
doi:10.1111/j.1472-765X.2005.01657.x
Acetophenones with selective antimycobacterial activity L. Rajabi, C. Courreges, J. Montoya, R.J. Aguilera and T.P. Primm Department of Biological Sciences and Border Biomedical Research Center, The University of Texas at El Paso, El Paso, TX, USA 2004/0684: received 15 June 2004, revised 19 November 2004 and accepted 22 November 2004
ABSTRACT L . R A J A B I , C . C O U R R E G E S , J . M O N T O Y A , R . J . A G U I L E R A A N D T . P . P R I M M . 2005.
Aims: Mycobacteria are a serious cause of infections in humans, with limited treatment options, as no new antibiotics have been developed against mycobacteria since the 1960s. In this study, the antimycobacterial activity of a small library of acetophenone (AP) compounds was analysed. Methods and Results: Twenty-three AP derivatives were examined for activity against mycobacteria using a microbroth assay. The compounds were bacteriostatic, with the most effective (cyclohexylacetophenone and piperidinoacetophenone) having minimal inhibitory concentrations of 246 lM. Active compounds tended to be more hydrophobic, and may work by alkylation of as yet undetermined intracellular target protein(s). Cytotoxicity against eukaryotic cells was also determined and appears to be unrelated to the bacteriostatic activity. Significance and Impact of the Study: AP may serve as a novel group of useful therapeutics against the mycobacteria. Keywords: cytotoxicity, minimum bacteriocidal concentration, minimum inhibitory concentration, Mycobacterium smegmatis.
INTRODUCTION Mycobacterium tuberculosis is a serious worldwide health threat, killing three million people annually, with eight million active cases per year. The World Health Organization estimates that a staggering one-third of the world’s population is latently infected (Tufariello et al. 2003). Current therapies are long (6–9 months for fully sensitive cases, longer for drug resistant), the drugs have serious side effects (Shakya et al. 2004) and therapy does not kill latent bacilli. There are c. 70 other species in the genus Mycobacterium, many of which are opportunistic pathogens in animals and humans. Other mycobacterial species, especially Mycobacterium avium, are emerging pathogens in the immunocompromised population, most notably AIDS patients (Primm et al. 2004). These nontuberculous mycobacteria (NTM) are ubiquitous in the environment, and naturally resistant to many disinfection procedures. Treatment options are limited, and no new antibiotics have been Correspondence to: Todd P. Primm, Department of Biological Sciences, The University of Texas at El Paso, 500 West University Avenue, El Paso, TX 79968, USA (e-mail:
[email protected]).
developed against any mycobacteria since the late 1960s. There is an urgent need for new biocides and antibiotics to prevent and treat mycobacterial infections. The caseload of the NTM is rising, and each of the c. 20 clinically important species have different drug susceptibility patterns (ATS 1997). Treatment again is difficult and lengthy. In certain cases, antibiotic therapy is so ineffective that surgical resection is used, i.e. with Mycobacterium scrofulaceum lymph node infections in children (Hazra et al. 1999; Danielides et al. 2002). While clarithromycin has good activity against M. avium, patients with resistant infections have a poor prognosis (Heifets 1996). With such a short list of antibiotics against these emerging pathogens, there is a serious need for development of new antimycobacterials. Acetophenone (AP) (phenyl methyl ketone, hypnone) is used in consumer fragrances and as an industrial solvent. Some AP derivatives have antimicrobial activity against Gram-positive bacteria and fungi (Gul et al. 2002) and others are used as herbicides (Teruyuki et al. 1986). Certain AP with a hydroxyl group at C-2 have antimutagenic activity in Salmonella typhimurium (Miyazawa et al. 2000). Many AP are also natural products in plants (Guyot et al. ª 2005 The Society for Applied Microbiology
ACETOPHENONES ARE BACTERIOSTATIC
1998) and fungi (Wilkins and Scholl 1989). When orally administered to rats, paeonol (2-hydroxy-4-methoxyacetophenone) is rapidly excreted in the urine as sulphated derivatives (Yasuda et al. 1999). No apparent toxicity was exhibited with numerous 50 mg kg)1 doses. The US Environmental Protection Agency administered AP at 423 mg kg)1day)1 in the diet to Osborne–Mendel rats for 17 weeks with no effects on growth, haematology or tissue pathology (Hagan et al. 1967). Chalcones (benzylideneacetophenones) are natural products from plants generated by polyketide synthesis, and M. tuberculosis contains such a synthetic gene cluster (Sirakova et al. 2003), perhaps to generate an immunosuppressant (Ko et al. 2003). Certain chalcones have inhibitory activity against M. tuberculosis (Lin et al. 2002). The compound p-nitro-alpha-acetylaminobeta-hydroxypropiophenone is used in an in vitro growth test (termed the NAP test) to discriminate M. tuberculosis complex from other mycobacteria (Morgan et al. 1985; Rastogi et al. 1989), as it only inhibits growth of the complex at certain concentrations. We are not aware of any published study examining phenones as antimycobacterial agents, and we have therefore focused this study on AP as they may be useful as antibiotics or biocides.
MATERIALS AND METHODS Cultures and compounds All compounds were from Sigma-Aldrich (St Louis, MO, USA), except p-nitrophenol from J.T. Baker Chemical Company (Phillipsburg, NJ, USA). Compounds were dissolved at 20 mg ml)1 in molecular biology-grade dimethylsulphoxide (DMSO; Sigma-Aldrich) to make stock solutions, and stored at )20C. Mycobacterium smegmatis strain mc2-155 (William Jacobs, Albert Einstein College of Medicine, NY, USA) was used for screening. Microbroth screening for minimal inhibitory concentration Bacteria taken from exponential phase growth cultures were diluted to an optical density at 650 nm of 0Æ001 in 7H9ADC media (Becton Dickinson, Franklin Lakes, NJ, USA), to give c. 10 000 bacteria in the 150-ll well)1 volume used in screening. Cells were placed in 96-well transparent polystyrene plates (Costar, Corning, NY, USA) and twofold serial dilutions of compounds were made, with the first well at 6400 lg ml)1, thus exposing bacilli to a 2048-fold range of concentrations (across 12 wells). All minimum inhibitory concentration (MIC) numbers came from at least two separate experiments, each with two dilution series. Plates were incubated stationary at 37C until growth was visible (4 days).
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Minimal bactericidal concentration determination The entire contents of the wells without growth were each plated onto a 7H11-OADC agar plate (Becton Dickinson), and incubated as above. A colony count of 10 or less (99Æ9% lethality) was read as the minimal bactericidal concentration (MBC) . Control experiments indicate that when similar amounts of representative compounds [AP or p-nitroacetophenone (4NAP)] are added to media plates that although colony growth can be delayed, no significant changes in colony numbers occur, thus carryover is not an issue. Survival curve Mycobacterium smegmatis from exponential phase were diluted in fresh media to an optical density at 650 nm of 0Æ05 (c. 5 · 105 CFU ml)1). The 10-ml culture was incubated at 37C and 150 rev min)1 in a culture tube (Becton Dickinson) during exposure. Viability was measured before compound addition, and 1, 2, 4, 8 and 16 h after addition of either 4-nitroacetophenone or 4-piperidinoacetophenone at the indicated concentrations. Viability was determined by plating dilutions onto 7H11-OADC plates and counting colonies after 4 days. Cytotoxicity determination The rapid screening method for cell toxicity is based on the loss or degradation of the nuclear green fluorescence protein (GFP) fluorescence of HeLa cells that constitutively express nuclear GFP as a chimeric histone H2B fusion protein (Kanda et al. 1998; Montoya et al. 2004). In brief, 500 cells were plated on single wells on a 24-well plate in 500 ll of DMEM media containing 10% serum (newborn calf). After 24 h of incubation with a suspected toxic agent, cells were visualized by fluorescence microscopy and the percentage of dying was determined by counting normal and abnormal nuclei (degradation or complete loss of GFP signal). Select compounds were also tested on the nonadherent lymphocyte cell line NFS-70 and mature murine B-cell line Bal-17 by the standard tryphan blue-exclusion assay. Statistical analysis When an r-value is given, Pearson’s correlation coefficient was utilized. When an rs value is given, Spearman’s rank correlation was used. All analyses were performed with SAS for Windows version 7. RESULTS AP and related compounds (Fig. 1) exhibited a range of antimycobacterial activity (Table 1), with MIC ranging
ª 2005 The Society for Applied Microbiology, Letters in Applied Microbiology, 40, 212–217, doi:10.1111/j.1472-765X.2005.01657.x
214 L . R A J A B I ET AL.
O
DISCUSSION
R1
R2
Fig. 1 General structure of acetophenone library
from 246 to 13 310 lM, with three AP showing strong inhibition [4-cyclohexylacetophenone (CHAP), 4NAP and 4-piperidinoacetophenone (PAP)]. DMSO itself does not inhibit the growth of M. smegmatis until it reaches 12Æ5% of the media. Two of the most potent compounds, CHAP and PAP, had identical MIC values when tested in the absence of DMSO (data not shown). Thus the solvent did not significantly affect screening values. However, the two compounds were much less soluble without DMSO.
AP are uniformly bacteriostatic in action, as seen by the large (average of eightfold) differences between MIC and MBC values (Table 1). The base compound, AP, is itself only weakly antibacterial (13 mM MIC). All the compounds together have a mean MIC of 5 mM and mean MBC of 25 mM. Addition of hydroxy, amino or ethyl groups at the third or fourth positions either had no effect or only slightly enhanced activity. The effect of addition of a halide group correlated with size, as Br > Cl > F for antimicrobial activity. With methyl, methoxy and nitro groups, the para-substituted (position 4) compound was more active than the meta (position 3). The 3,4-dimethyl compound was more active than either of the singly methyl-substituted compounds, but the 3,4-methoxy was less active.Compounds with larger hydrophobic substituents [4-morpholinoacetophenone (MAP), CHAP, PAP] exhibited the best activity. The two most potent inhibitory compounds against mycobacteria were CHAP and PAP, effective at 246 lM. By comparison, the major clinical drug against M. avium,
Table 1 Antimycobacterial activity of phenone compounds Compound
R1
R2
MIC
MBC
Abbreviation
Acetophenone 4-Methylacetophenone 3-Methylacetophenone 3,4-Dimethylacetophenone 4-Methoxyacetophenone 3-Methoxyacetophenone 3,4-Dimethoxyacetophenone 4-Ethylacetophenone 4-Chloroacetophenone 3-Chloroacetophenone 3,4-Dichloroacetophenone 4-Bromoacetophenone 3-Bromoacetophenone 4-Flouroacetophenone 4-Triflouromethyl-acetophenone 4-Hydroxyacetophenone 4-Hydroxybenzoic acid 4-Hydroxybenzaldehyde 3-Hydroxyacetophenone 4-Morpholinoacetophenone 4-Cyclohexylacetophenone Benzophenone 4-Aminobenzophenone 4-Aminoacetophenone 4-Nitroacetophenone 3-Nitroacetophenone 4-Nitrophenol 4-Piperidinoacetophenone
H H CH3 CH3 H OCH3 OCH3 H H Cl Cl H Br H H H
H CH3 H CH3 OCH3 H OCH3 CH2CH3 Cl H Cl Br H F CF3 OH
OH H H
H C4H8NO C6H11
H H NO2
NH3 NO2 H
H
C5H10N
13 310 2980 11 920 1350 2660 5330 8880 10 800 10 350 2590 2120 1000 1000 5790 8510 11 750 11 590 820 5870 974 246 1100 2030 11 830 605 2420 2880 246
26 620 47 690 23 850 21 590 10 650 10 650 >35 500 21 600 20 700 20 700 16 930 16 080 16 080 46 340 34 020 >47 000 46 340 26 210 11 750 31 170 >31 640 17 560 32 450 23 670 9690 >38 740 23 000 15 740
AP 4MeAP 3MeAP DMAP 4MoAP 3MoAP DMoAP EAP 4CAP 3CAP DCAP 4BAP 3BAP 4FAP TFMAP 4HAP HBA HB 3HAP MAP CHAP Diphenyl ketone Methanone 4AAP 4NAP 3NAP PNP PAP
R1 is at the C3 position (meta to the aceto group) and R2 is at the C4 position (para). Compounds which are not acetophenones have no entry under R1 or R2. MIC and MBC are both in lM. MBC is 3 log death or higher. ª 2005 The Society for Applied Microbiology, Letters in Applied Microbiology, 40, 212–217, doi:10.1111/j.1472-765X.2005.01657.x
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clarithromycin, has an MIC of 11 lM as determined by microbroth (Heifets 1996). In our system, kanamycin was used as a control drug and exhibited an MIC of 32 lM. There is a strong significant correlation between AP solubility (mg l)1) and MIC (lM) (rs ¼ 0Æ82, P ¼ 0Æ0003); thus more hydrophobic compounds are more inhibitory. This is reasonable in light of the high lipid content of the mycobacterial cell wall. Despite the hydrophobic nature, disruption of cell membranes as a mechanism of action is unlikely, as compounds known to have membrane rupturing effects exhibit rapid (
