1 Introduction
Calixarenes are a very important class of compounds in supramolecular chemistry 〚1–5〛. As shown in Fig. 1, calixarenes are cavity-shaped cyclic molecules made up of phenol units linked via the ortho positions by methylene bridges. Calixarenes are synthetic macrocycles produced by condensation of p-substituted phenols with formaldehyde. They have an original molecular architecture and are considered to be important starting materials in the design of novel host-molecules for molecular recognition.
Representation of the calixarenes and designation of the faces.
In the past ten years, parent calixarenes have been widely used as molecular platforms for the synthesis of selective molecular receptors for cations, anions and neutral molecules, in order to develop applications 〚6〛 in the fields of separation 〚7〛 purification 〚8〛, recovery of metals 〚9,10〛, pollution control 〚11〛 membranes for electronic devices 〚12,13〛, and phase transfer agents 〚14〛.
Although some calixarenes have been quoted for their antitumor and antiviral properties, very few publications appeared in the literature on their biological activities. Only recently, in August 2000, a communication 〚15〛 on the cellular toxicity of calixarenes stated that calixarene sulfonates, the calix〚6〛 and 〚8〛 derivatives display the same level of toxicity as glucose, while slight toxicity associated with the calix〚4〛arene sulfonate does exist.
In the present study 57 kinds of calixarenes were screened to find antimicrobially active compounds toward a variety of fungal and bacterial microorganisms. These novel compounds are not only important as new antimicrobial substances but are also attractive from a materials-based perspective in that calixarenes have metal binding properties.
2 Experimental
Materials
The calixarenes synthesized for these experiments are listed in Table 1, along with the different side chain moieties and/or substitution groups they contain.
Synthesized calixarenes having different side chain moieties and/or substitution groups (Ph: phenyl; Ad: adamantyl; Py: pyridyl; bPy: bipyridyl).
| Calixarenes | x | R1 | R2 | Calixarenes | x | R1 | R2 | |||||||
| 1 | 1 | C(CH3)3 | H | 30 | 1 | N=N–Ph | H | |||||||
| 2 | 1 | C(CH3)3 | H | 31 | 1 | NO2 | H | |||||||
| Homooxa | ether bridge | |||||||||||||
| 3 | 2 | C(CH3)3 | H | 32 | 1 | CH2–CH=CH2 | H | |||||||
| 4 | 3 | C(CH3)3 | H | 33 | 1 | C-ethyl bridges | ||||||||
| Resorcin. | ||||||||||||||
| 5 | 4 | C(CH3)3 | H | 34 | 1 | C-nonyl bridges | ||||||||
| Resorcin. | ||||||||||||||
| 6 | 5 | C(CH3)3 | H | 35 | 1 | C-benzylbridges | ||||||||
| Resorcin. | ||||||||||||||
| 7 | 6 | C(CH3)3 | H | 36 | 1 | CH(CH3)2 | ||||||||
| 8 | 7 | C(CH3)3 | H | 37 | 3 | N=N–Ph | H | |||||||
| 9 | 1 | H | H | 38 | 1 | CH(CH3)2 | CH3 | |||||||
| 10 | 2 | H | H | 39 | 3 | CH(CH3)2 | CH3 | |||||||
| 11 | 3 | H | H | 40 | 1 | CH(CH3)2 | CH2–CH3 | |||||||
| 12 | 5 | H | H | 41 | 1 | CH(CH3)2 | CH2–CH=CH2 | |||||||
| 13 | 1 | CH(CH3)2 | H | 42 | 3 | CH(CH3)2 | ||||||||
| 14 | 1 | CH(CH3)2 | H | 43 | 1 | CH(CH3)2 | ||||||||
| Homooxa | ether bridge | |||||||||||||
| 15 | 3 | CH(CH3)2 | H | 44 | 3 | CH(CH3)2 | ||||||||
| 16 | 5 | CH(CH3)2 | H | 45 | 5 | CH(CH3)2 | ||||||||
| 17 | 4 | CH2–CH3 | H | 46 | 1 | CH(CH3)2 | ||||||||
| 18 | 3 | CH2–CH2CH3 | H | 47 | 5 | CH(CH3)2 | ||||||||
| 19 | 5 | CH2(–CH2)6–CH3 | H | 48 | 3 | CH(CH3)2 | ||||||||
| 20 | 2 | C(CH3)2–CH2– | H | 49 | 1 | CH(CH3)2 | ||||||||
| C(CH3)3 | ||||||||||||||
| 21 | 3 | C(CH3)2–CH2– | H | 50 | 1 | CH(CH3)2 | ||||||||
| C(CH3)3 | ||||||||||||||
| 22 | 5 | C(CH3)2–CH2– | H | 51 | 1 | C(CH3)3 | ||||||||
| C(CH3)3 | ||||||||||||||
| 23 | 5 | CH2–(CH2)12–CH3 | H | 52 | 1 | CH(CH3)2 | 3 CH3, 1H | |||||||
| 24 | 3 | CH2–(CH2)16–CH3 | H | 53 | 1 | N=N-Ph-NO2 | H | |||||||
| 25 | 1 | SO3H | H | 54 | 1 | C(CH3)3 | 2 Py + 2 bPy | |||||||
| 26 | 3 | SO3H | H | 55 | 1 | C(CH3)3 | 2 Py + 2bPy,Cu(I) | |||||||
| 27 | 5 | SO3H | H | 56 | 1 | 2 NO2,2 C(CH3)3 | H | |||||||
| 28 | 3 | N=N–Ph–SO3H | H | 57 | 1 | 3 C(CH3)3,1 H | ||||||||
| 29 | 1 | N=N–Ph–SO3H | H |
All even numbered parent calixarene samples 1, 4, 6, 8, 13, 15, 16 have been synthesized according to the Gutsche procedures 〚16–18〛. Odd numbered parent calixarene samples 3, 5, 7 were produced according to modified synthesis procedures 〚19,20〛. Dihomooxacalixarenes 2, 14 were synthesized in basic conditions by optimised synthetic procedures 〚21〛, while C-alkylcalix〚4〛resorcinarene 33, 34, 35 were obtained by acid-catalysed procedures 〚1〛. The upper rim tert-butyl groups were removed 〚22〛 by retro Friedel–Crafts reactions to form H-calixarenes 9–12.
Additionally, the alkylated upper rim calixarene samples 17–23, 29, 32 were obtained by Friedel–Crafts alkylation of H-calixarenes with alkyl chlorides in the presence of AlCl3 in CHCl3 solution 〚3〛. Sulfonato-calixarene samples 25–27 were produced by ipso-sulfonation 〚23〛, and phenylazocalixarene samples 28–30, 37, 53 were formed via the diazo coupling scheme 〚24,25〛. Functional groups were introduced into the lower rim of isopropylcalixarenes by means of Williamson-type OH-modifications. Literature procedures were used to introduce on the p-isopropylcalix〚4〛arene lower rim, ether, ester, acyl, ketone and amine groups for samples 36, 38, 40, 41, 43, 46, 49, 50. The exhaustive functionalisation of p-isopropylcalix〚6〛 and 〚8〛arene samples 39, 42, 44, 45, 47, 48, utilised literature procedures that were subsequently modified and optimised for these specific reactions 〚14〛.
3 Measurement
3.1 Antimicrobial activity towards bacteria
The antimicrobial activity of calixarenes was assessed against a variety of microorganisms, including several kinds of bacteria 〚26〛 and fungi, listed in Table 2. Phytoanthorogenic microorganisms were mainly used to evaluate antimicrobial activity of these compounds. Factors for these choices included ease of experiment, non-toxic quality and prevalent existence in nature.
Microorganisms examined in the present study.
| Microorganisms | Strain number of MAFF |
| Bacteria from Plant pathogens | |
| Agrobacterium tumefaciens | 302307 |
| Clavibacter michiganensis subsp. michganensis | 301368 |
| Pseudomonas cichorii | 118079 |
| Fungi from Plant pathogens | |
| Colletotrichum dematium | 840066 |
| Fusarium solani f. sp .mori | 840046 |
| Rosellinia nocatrix | 840051 |
3.2 Antimicrobial activity against bacteria
Initial antimicrobial studies screened 57 types of calixarenes against the specific microbe, Corynebacterium, chosen for its high sensitivity to a wide variety of antimicrobial substances, such as Ag ions, and phytoalexin. After screening, the antimicrobial activity of the calixarenes was examined against many strains of bacteria and fungi.
From a Corynebacterium cell culture with a cell density of 100 cells/ml, 2 ml were taken and mixed with 25 ml agarose containing King B growth medium (Wako Pure Chemical Industries, Ltd.) at 55 °C, poured into a glass Petri dish and allowed to solidify at 25 °C. The calixarenes (approximately 0.5 mg) in powder form were placed onto the surface of the solid gel and incubated at 25 °C for two days. The antimicrobial activity was evaluated by measuring the radius of growth inhibition around the sample.
3.3 Antimicrobial activity against fungi
The antimicrobial activity of various calixarenes against several fungi, including Rosellinia sclerotiorum (R-8), Colletotrichum dematium (C.d.8901) and Fusarium solani f. sp. mori (F.s.26) was evaluated according to the following procedures. Solidified potato sucrose agar medium (A medium) was prepared by pouring liquid potato sucrose agar into a glass Petri dish at 25 °C. One of the fungi selected was inoculated at 25 °C on the surface of the fresh A medium. All fungal culture strains were thus inoculated on the A medium and allowed to grow at room temperature until a mycelia growth radius of approximately 3 cm had been reached (usually four days after inoculation). Another batch of solidified potato sucrose agar (B medium) was prepared according to the same methods as those described above. From the initial mycelia growths, a small portion from the mycelial periphery (size: 0.5 mm × 0.5 mm × 0.5 mm) was removed with a scalpel and transferred to a different place on the surface of the solidified B medium. Approximately 0.5 mg of calixarenes in powder form was then placed on these mycelia to assess antifungal activity. Growth rate (GR) and inhibition rate (IR) were both monitored for three days as a measure of the antifungal activity according to the equation below. The results presented here are the average of four mean values.
Growth Rate (GR)
B: diameter size of fungal colony with exposure to calixarene material.
Inhibition Rate (IR)
B: diameter size of fungal colony with exposure to calixarene material.
3.4 Antimicrobial activity
A preliminary evaluation of the antimicrobial activity of calixarenes against Corynebacterium was performed and gauged according to the following three stage criteria:
++ high antimicrobial activity; size of inhibition zone is approximately 3.5 mm;
+ relatively high antimicrobial activity; size of the inhibition zone is approximately 2.5 mm;
± weak antimicrobial activity; size of inhibition zone is less than 1.5 mm;
– no antimicrobial activity.
4 Results and discussion
For the preliminary screening studies of the 57 calixarenes, the antimicrobial activities of all samples were determined against Corynebacterium. Corynebacterium is useful because of its high sensitivity toward a wide variety of microorganisms of which the results are listed in Table 3.
Preliminary screening data for antimicrobial activity of calixarenes against Corynebacterium.
| Sample No. | 25 | 26 | 27 | 28 | 30 | 34 | 50 |
| Activity | + | + | + | + | ± | ± | ± |
From Table 3, it was found that the calixarenes exhibited a range of antimicrobial activities from complete growth inhibition to little or no inhibition. Sample numbers 25–28 all exhibit relatively high antimicrobial behaviour, and contain a common SO3H group in the basic form, which might play a role in this behaviour. The other calixarenes do not show any antimicrobial activity against Corynebacterium. It is of interest to note that calixarenes are generally able to selectively combine with metals; this feature could possibly lead to additional significant increases in the antimicrobial activity already displayed.
The extent of antimicrobial activity as a function of material concentration was also examined using the various samples in aqueous solution (Table 4). The 0.1% calixarene solution concentration corresponds to 1000 ppm.
Antimicrobial activity of selected calixarenes against Corynebacterium as a function of material concentration.
| Concentration (%) | 25 | 26 | 27 | 28 |
| 1 | + | + | + | ++ |
| 0.1 | – | – | – | + |
| 0.01 | – | – | – | – |
| 0.001 | – | – | – | – |
As it is clear from Table 4, calixarene sample numbers 25–27 have slight antimicrobial activity against Corynebacterium when the sample concentration is above 1%, while calixarene sample number 28 has a much stronger antimicrobial activity beyond concentrations of 0.1%. Therefore, calixarene 28 has the strongest antimicrobial potential toward Corynebacterium among the 57 calixarenes studied. From an application-based perspective, the desired concentration for an effective pesticide is considered to be less than 100 ppm. The experimental data indicate that these calixarenes do not display antimicrobial activity below 0.1%, 1000 ppm. Therefore, it seems that these compounds cannot be used as effective pesticides at the moment; however, we should bear in mind that most of the calixarenes listed in Table 1 are safe for the human body.
Growth rate and inhibition rate of fungi from plant
The comparison of the growth and inhibition rates is an important measure to distinguish the antimicrobial activity of the samples against specific fungi and bacteria. Figs. 2 and 3 demonstrate the growth and inhibition rates of various calixarenes against differing fungal strains, respectively.
Growth rates of various calixarenes against differing fungal strains.
Inhibition rates of various calixarenes against differing fungal strains.
Calixarene sample numbers 25–27 and 50 displayed a range of antimicrobial activity against Fusarium solani f. sp. mori (F.s.-26), having an inhibition rate of approximately 60–70%, while calixarene sample numbers 25–28 showed excellent and selective antimicrobial activity against fungal strains Rosellinia necatrix (R-8), and Colletotrichum dematium (C.d.8901).
5 Conclusion
Approximately 57 calixarenes were screened to determine compounds having antimicrobial activity against fungal and bacterial microorganisms. Sample numbers 25–28 all showed slight antimicrobial behaviour, and contain a common SO3H group in the basic form, which might play a role in this aspect. The compounds in Table 5 were found to be the prominent calixarenes exhibiting antimicrobial activity.
Calixarenes exhibiting prominent character of antimicrobial activity.
| Sample No. | Name | Character | n |
| 25 | p-sulfonatocalix〚4〛arene | OH, SO3H | 4 |
| 26 | p-sulfonatocalix〚6〛arene | OH, SO3H | 6 |
| 27 | p-sulfonatocalix〚8〛arene | OH, SO3H | 8 |
| 28 | p-phenylazocalix〚6〛arene | OH, N=N–Ph–SO3H | 6 |
| 30 | p-phenylazocalix〚4〛arene | OH, N=N–Ph | 4 |
| 34 | C-nonylresorcin〚4〛arene | OH, OH | 4 |
| 50 | Aminocarbonyl p-iPr-calix 〚4〛arene | CH2CON(C2H5)2 | 4 |
Here we report the slight antimicrobial behaviour of calixarene sample numbers 25–28, which all contain a common SO3H group in the basic form. Additionally interesting is the fact that the antimicrobially active samples 28 and 30 contain a common –N=N–phenyl group.
This essential preliminary information should be further investigated as to why the above calixarenes are antimicrobially active towards microorganisms, as well as to elucidate the activity mechanisms leading to these observations.
These experimental results indicate that several kinds of calixarenes possess antimicrobial activity towards Fusarium solani f. sp. mori (F.s-26). Because F.s.-26 is a strong pathogen and produces harmful plant injuries, these calixarenes are very important substances to consider when developing new functional pesticides. At present, there are no pesticides against F.s-26, indicating that calixarenes comprise an exciting family of compounds that could be a leading materials substance for new pesticide developments.
Normally pesticides that are effective for plants contain a large quantity of harmful halogens such as sulphur and chloride. The advantage presented by these compounds for pesticide production is that calixarenes do not contain halogens in such high amounts. Therefore, it is hypothesized that calixarenes operate under a different antimicrobial mechanism as compared with that of commercialised pesticides. Since calixarenes combine specifically with metal ions, we can produce new antimicrobially active materials based from calixarenes combined with metals, such as Ag. Ag-combined calixarene could offer a safe alternative pesticide, which would be non-toxic to the human body, and a more durable antimicrobial material, which could be used widely in several applications. With this information in place, it will be possible to develop new techniques and materials utilizing antimicrobial calixarenes in the industrial and medical fields.
Acknowledgements
This work was supported by the COE (Centre of Excellence), Special Coordination Funds for promoting Science and Technology, of the Science and Technology Agency, Japan.