Abstract
Analogs of furanonaphthoquinone (FNQ) from Tecoma ipe Mart had MICs ranging from 1.56 to 25 μg/ml against gram-positive bacteria. FNQ showed significantly lower MICs against methicillin-resistant Staphylococcus aureus than against methicillin-sensitive S. aureus. FNQ inhibited Helicobacter pylori with an MIC of 0.1 μg/ml. Fungi, including pathogenic species, were sensitive to FNQ with MICs similar to those of amphotericin B.
Kigelinone, 5- or 8-hydroxy-2-(1-hydroxyethyl)naphtho [2,3-b]furan-4,9-dione, a phytochemical analog of naphtho [2,3-b]furan-4,9-dione (furanonaphthoquinone [FNQ]) com- pounds, was isolated from the inner bark of the South American trumpet tree, Tecoma ipe Mart (syn. Tabebuia impetiginosa, Tabebuia cassinoides, and Tecoma avellanedae), or Kigelia pinnata, which is known to have antitumor activity (2, 6, 7, 16). Because little is known about the bioactivity of FNQ analogs, we isolated FNQ and synthesized the isomeric derivatives shown in Fig. 1. They were selectively toxic to human cancer compared with the corresponding normal cells (5, 12). One of the FNQ analogs, 2-methylnaphtho[2,3-b]furan-4,9-dione (FNQ3), was toxic to mitochondria of HeLa cells at 3 to 5 μg per ml, whereas normal cells were unaffected at that concentration but were damaged at a concentration of 20 μg per ml (12). In our continuing search for bioactive FNQ derivatives, we found some with potent inhibitory activity against bacteria and fungi. These activities were not affected by the chemical structures of hydroxy at position 5 (or 8) of naphtho[2,3-b] furan-4,9-dione (Fig. 1). A synthesized analog, FNQ3, has been found in the bark of Tecoma ipe Mart (4). However, 2-methyl-5(or 8)-hydroxynaphtho[2,3-b]furan-4,9-dione (FNQ13) is a newly synthesized substance which has not been reported previously (9).
FIG. 1.
Chemical structure of FNQ3 (a) and FNQ13 (b).
FNQ3 was synthesized by Lee’s method (4). FNQ13 was synthesized by mixing 1 g of 3-hydroxyphthalic anhydride, 0.7 g of 2-acetyl-5-methylfuran, and 2.5 g of aluminum chloride with 5 ml of nitrobenzene and heating to 100°C for 18 h. The synthesized FNQ3 and FNQ13 were recrystallized from methanol as yellow needles. FNQ analogs were dissolved in dimethyl sulfoxide at a concentration of 1 to 5 mg/ml and then diluted in phosphate-buffered saline for assay of antimicrobial activity.
Microorganisms used in this study are described in Table 1 (bacteria) and Table 2 (fungi).
TABLE 1.
Antibacterial activity of FNQ13
| Strain | FNQ13 MIC (μg/ml) |
|---|---|
| Gram positive | |
| Staphylococcus aureus | |
| FAD 209P | 3.13 |
| 1840 | 12.5 |
| MRSA N133 | 1.56 |
| MRSA OF4 | 1.56 |
| MRSA N295 | 6.25 |
| Staphylococcus epidermidis IFO3762 | 6.25 |
| Streptococcus pneumoniae | |
| Type 1 | 6.25 |
| 88031 | 3.13 |
| SP8 | 25 |
| Streptococcus pyogenes E-14 | 25 |
| Streptococcus mutans RIMD 3125001 | 6.25 |
| Streptococcus salivarius GIFU 8326 | 25 |
| Enterococcus faecium IFO3128 | 25 |
| Enterococcus faecalis IFO12580 | 25 |
| Bacillus subtilis IFO3134 | 1.56 |
| Clostridium perfringens NCTC4696 | 25a |
| Gram negative | |
| Escherichia coli K-12 | |
| ATCC 14621 | >100a |
| NIHJJC-2 | >100 |
| Citrobacter freundii TN474 | >100 |
| Enterobacter cloacae GN5788 | >100 |
| Serratia marcescens | |
| IFO12648 | >100 |
| B315 | >100 |
| Klebsiella pneumoniae IFO3321 | >100 |
| Klebsiella oxytoca TN1711 | >100 |
| Proteus vulgaris IFO3988 | >100 |
| Morganella morganii IFC3168 | >100 |
| Acinetobacter calcoaceticus IFO13006 | >100 |
| Pseudomonas aeruginosa IFO3445 | >100 |
| Neisseria gonorrhoeae NCTC8375 | >100a |
| Haemophilus influenzae NN400 | 0.78 |
| Moraxella catarrhalis BN-2 | 0.78 |
| Campylobacter jejuni NCTC11351 | 1.25a |
| Helicobacter felis CN-1 | 0.05a |
| Helicobacter pylori | |
| NCTC11637 | 0.1a |
| HCM1 | 0.1a |
| HCM3 | 0.1a |
| HCM5 | 0.1a |
| TN2 | 0.1 |
| TN58 | 0.1 |
FNQ3 gave results similar to those of FNQ13.
TABLE 2.
Antifungal activity of FNQ13 versus those of fluconazole, amphotericin B, and flucytosine
| Strain | MIC (μg/ml) ofa:
|
|||
|---|---|---|---|---|
| FNQ13 | FLCZ | AMPH-B | 5FC | |
| Candida albicans | ||||
| TA | 4 | 0.03 | 1 | 0.25 |
| TIMM1756 | 2 | 0.06 | 1 | 0.25 |
| TIMM1850 | 2 | 0.06 | 1 | >64 |
| TIMM0239 | 2 | 0.13 | 1 | 0.25 |
| IFO0583 | 4 | 0.03 | 1 | 0.25 |
| CA382 | 2 | 8 | 1 | 0.5 |
| R38509-2 | 2 | 8 | 1 | 0.06 |
| MCV7 | 2 | 32 | 1 | 1 |
| CA383 | 2 | >64 | 1 | 0.13 |
| S78941 | 4 | >64 | 0.5 | 0.13 |
| CA385 | 8 | >64 | 1 | 0.13 |
| T77042 | 2 | >64 | 1 | 0.13 |
| DUMC136 | 4 | >64 | 1 | >64 |
| Candida tropicalis | ||||
| IFO587 | >8 | 0.25 | 2 | 0.13 |
| IFO10241 | 8 | 0.25 | 1 | 0.13 |
| Candida glabrata IFO0622 | 4 | 16 | 1 | <0.03 |
| Candida krusei | ||||
| IFO0584 | >8 | 64 | 2 | 8 |
| IFO1162 | 8 | 64 | 2 | 8 |
| Candida utilis IFO0619 | 2 | 2 | 1 | 1 |
| Cryptococcus neoformans | ||||
| TIMM1740 | 0.5 | 4 | 1 | 64 |
| TIMM1850 | 1 | 8 | 2 | 4 |
| Saccharomyces cerevisiae IFO0209 | 2 | 8 | 1 | 0.06 |
| Aspergillus fumigatus | ||||
| 437 | 1 | >64 | 1 | 2 |
| TIMM1728 | 4 | >64 | 1 | 2 |
| IFO6344 | 4 | >64 | 1 | 2 |
| Aspergillus niger IFO4414 | 4 | >64 | 1 | 2 |
| Trichophyton mentagrophytes ATCC 9533 | 6 | NDb | 1 | ND |
| Trichophyton rubrum DSM4163 | 6 | ND | 1 | ND |
FLCZ, fluconazole; AMPH-B, amphotericin B; 5FC, flucytosine.
ND, not done.
Mueller-Hinton agar (Difco) containing 5% Filder’s peptic digest of blood or 5% defibrinated horse blood was used primarily for culture of gram-positive and gram-negative bacteria, and, except for Clostridium perfringens, bacteria were cultivated aerobically at 37°C. For Campylobacter jejuni, Helicobacter felis, and Helicobacter pylori, brucella agar medium (Becton Dickinson Microbiology Systems) with 5% horse serum was used with microaerobic incubation at 37°C for 3 days as described previously (11). For fungi, RPMI 1640 medium containing 0.15 M morpholinepropanesulfonic acid (MOPS; pH 7.0) and 1% agar was used. Fungi were cultivated aerobically for 1 to 5 days at 35°C.
MICs were determined by the agar dilution method as described previously (11). The initial inoculum was 1 × 104 to 5 × 104 organisms per ml for bacteria and about 1,000 CFU per ml for fungi.
Table 1 shows the MICs of FNQ determined for 39 strains of gram-positive or gram-negative bacteria by the agar dilution method. FNQ inhibited 16 strains of gram-positive bacteria belonging to Staphylococcus, Streptococcus, Enterococcus, Bacillus, and Clostridium species with MICs ranging from 1.56 to 25 μg/ml. Because methicillin-resistant Staphylococcus aureus (MRSA) strains seemed to be more sensitive to FNQ than methicillin-susceptible S. aureus (MSSA) strains (Table 1), we compared the antibacterial activity of FNQ for 11 strains each of MSSA and MRSA from clinical specimens. MICs of FNQ3 for MRSA (mean ± standard deviation, 5.97 ± 3.55 μg/ml) were significantly lower than those for MSSA (11.93 ± 5.20 μg/ml) (P < 0.01). To examine whether a suboptimal amount of FNQ augments the susceptibility of MRSA to various antibiotics, MICs were determined using 96-well air-dried microplates (HP-Plates; Eiken Chemical Co., Ltd., Tokyo, Japan) containing various amounts of antibiotics with or without 0.5 μg of FNQ3 per ml as previously described (8). As shown in Table 3, the MICs of antibiotics such as ampicillin, cefaclor, levofloxacin, minocycline, and vancomycin for MRSA decreased in the presence of FNQ, while the MICs of these antibiotics for MSSA were affected little by addition of FNQ. To define the effect of vancomycin-FNQ interaction on activity against MRSA, checkerboard tests were carried out. The fractional bactericidal concentration index was 0.5 to 0.6, suggestive of an additive effect of vancomycin and FNQ on activity against MRSA.
TABLE 3.
Effect of FNQ3 on MICs of various antibiotics for MRSA and MSSA
| Antibiotic | MIC (μg/ml) for:
|
|||
|---|---|---|---|---|
| MRSA
|
MSSA
|
|||
| − FNQ3 | + FNQ3 | − FNQ3 | + FNQ3 | |
| Ampicillin | 32 | 4 | 32 | 16–32 |
| Cefaclor | 64 | 16 | 1 | 1 |
| Cefotiam | 32 | 16–32 | 0.25–0.5 | 0.25 |
| Clarithromycin | 16 | 16 | 0.5 | 0.5–1.0 |
| Roxithromycin | 16 | 16 | 1 | 1 |
| Levofloxacin | 8 | 4 | 0.25 | 0.25 |
| Minocycline | 8 | 2–4 | 0.25 | 0.25 |
| Vancomycin | 2.5 | 0.6–1.25 | 0.6 | 0.6 |
In contrast to the relatively low MICs of FNQ for gram-positive bacteria, gram-negative bacteria belonging to the genera Escherichia, Citrobacter, Enterobacter, Serratia, Klebsiella, Proteus, Morganella, Acinetobacter, Pseudomonas, and Neisseria were not inhibited by FNQ at concentrations higher than 100 μg/ml. However, strains belonging to the genera Haemophilus, Moraxella, Campylobacter, and Helicobacter had MICs between 0.05 and 1.25 μg/ml. Since the MIC of FNQ for H. pylori was markedly low, we determined MICs for five strains from different human specimens. FNQ inhibited the growth of those strains of H. pylori with an MIC of 0.1 μg/ml (Table 1). H. pylori lives in the mucus layer overlying the human gastric epithelium. The pH of gastric juice and sites within the mucosa may be an important factor that potentially affects drug activity. A wide range of antimicrobial agents are active against H. pylori when tested in vitro at neutral pH. However, the MICs of some antibiotics are known to decrease by 1/10 or 1/100 in acidic culture medium (pH 5.5) (3). We found no effect of culture medium pH values between 5.5 and 7.2 on the FNQ MIC for H. pylori. Since addition of FNQ to several antibiotics decreased their MICs against MRSA, similar experiments were carried out with H. pylori. MICs of ampicillin, cefaclor, and levofloxacin were reduced one-fourth to one-half by the addition of FNQ (0.05 μg/ml) (data not shown). H. pylori has been implicated as being responsible for gastritis, duodenal ulcers, and possibly neoplasia (1, 10, 13–15). Thus, FNQ may be useful as a chemotherapeutic agent against H. pylori infection.
Table 2 shows the MICs of FNQ against 28 strains of various species of fungi, including Candida, Cryptococcus, Aspergillus, and Saccharomyces species, together with MICs of fluconazole, amphotericin B, and flucytosine. FNQ inhibited the growth of those strains of fungi with almost the same MICs as fluconazole, amphotericin B, and flucytosine. In addition, the growth of Tricophyton strains which commonly cause superficial or subcutaneous mycoses was also inhibited by FNQ, with an MIC of 6 μg.
In conclusion, FNQ may be useful as another chemotherapeutic agent against MRSA, H. pylori, and pathogenic fungi.
Acknowledgments
We gratefully acknowledge the Pharmaceutical Research Division of Takeda Chemical Industries Ltd. (Osaka, Japan) for providing data concerning antibacterial and antifungal activities of FNQ.
This work was partly supported by Grants for Collaborative Research from Kanazawa Medical University (P95-20 and C96-6).
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