Alkylated Phenol Derivatives
gives the phenol coefficients of a series of simple alkylated phenol homologs against a range of organisms.16
The germicidal potency increases as the number of methylene groups in the substituted alkyl group increases. After the n
) derivative, a further increase in size of the alkyl chain produces an increase in activity against S aureus
, Mycobacterium tuberculosis
, and Candida albicans
, but the effectiveness against S typhi
declines. The three isomeric cresols (methylphenols) and the four isomers of xylenol (dimethylphenols) show increases in activity of up to 5 times that of phenol, but within each isomer series, there is no apparent difference in activity. These data again serve to indicate at the time that the phenol coefficient against S typhi
cannot be used as a meaningful indicator of general antimicrobial activity. These results are in agreement with those of other workers.17
concluded that substitution in the 4-position of the phenolic ring of an alkyl chain of up to six carbons in length increases the antibacterial action of phenolics, presumably by increasing the surface activity and ability to orient at an interface. Activity falls off after this because of decreased water solubility. Because of the polarity enhancement of polar properties, straight-chain 4-position substitution confers greater activity than branched-chain substitution containing the same number of carbon atoms. Studies with the dermatophyte Trichophyton mentagrophytes
suggested that the optimum alkyl chain length may vary with the organism concerned. Etoh et al19
showed that the lowest minimum inhibitory concentration (MIC
) of a range of 4-n
-alkyl substituted phenols was obtained with the nonyl (C9
) phenol. Introduction of bulky substituent groups in the 2-position of a phenol already substituted with an alkyl group in the 4-position dramatically reduced the antimicrobial activity against this fungus.
One simple alkylated phenol still in commercial use is 4-tert-amylphenol (p
-tert-amylphenol; 4-[2-methyl-2-butyl]-phenol). This derivative is commonly used as an active ingredient in various disinfectant formulations, where it is combined with other phenols to give activity against pseudomonads. Paulus6
reported the microbicidal concentration of this compound alone as 400 mg/L for Salmonella choleraesuis
and 500 mg/L for S aureus
Halogenated-Alkyl Phenol Derivatives
Halogenation of phenolic compounds, preferably by chlorination, leads to a potentiation of antibacterial effectiveness. The combination of alkylation and halogenation has produced a number of highly effective microbiocides. A systematic examination of the relationships between the chemical structure and the microbiocidal action of aliphatic and aromatic substitution derivatives of 2- and 4-chlorophenol was carried out
by Klarmann et al.16 Table 20.2
illustrates the situation found in the case of 2-position alkyl derivatives of 4-chlorophenol; Table 20.3
shows the effect of 4-position alkyl derivatives of 2-chlorophenol. The general trends are summarized as follows:
TABLE 20.2 Microbicidal action of 2-position alkyl derivatives of 4-chlorophenol (phenol coefficients, 37°C)
Salmonella ser typhi
Abbreviation: NA, not available.
Halogen substitution intensifies the microbicidal potency of phenol derivatives; the presence of halogen in the 4-position to the hydroxyl group is more effective in this respect than in the 2-position.
Introduction of aliphatic or aromatic groups into the nucleus of halogen phenols increases the bactericidal potency even further (up to certain limits). The increase depends in the case of alkyl substitution on the number of carbon atoms present in the substituting group or groups.
TABLE 20.3 Microbicidal action of 4-position alkyl derivatives of 2-chlorophenol (phenol coefficients, 37°C)
Salmonella ser typhi
Abbreviation: NA, not available.
As a rule, the intensifying effect on the bactericidal potency of a normal aliphatic chain with a given number of carbon atoms is greater than that of a branched-chain or of two alkyl groups with the same total of carbon atoms.
The 2-alkyl derivatives of 4-chlorophenol are more actively germicidal than 4-alkyl derivatives of 2-chlorophenol.
In addition to the homologous series of alkyl derivatives of 2- and 4-chlorophenol, a series of polyalkyl of 4-chlorophenol also was studied (Table 20.4
). As with the
monoalkyl series described previously (see Table 20.2
), the number of methylene groups in the alkyl substitution group determines the degree of selective action against different microorganisms. With monoalkyl substitution, a compound with five carbon atoms (n
-amyl) in the side groups gives maximum activity against S typhi
. For the polyalkyl derivatives, maximum activity is reached when the compounds have a total of four substituting carbon atoms (6-n
-propyl-3-methyl- and 6-isopropyl-3-methyl). Against the other test microorganisms, the greatest antimicrobial activity in these polyalkyl derivatives is seen when a total of seven carbon atoms are in the side groups (2-diethylmethyl-3,5-dimethyl).
TABLE 20.4 Microbicidal action of polyalkyl derivatives of 4-chlorophenol (phenol coefficients, 37°C)
Salmonella ser typhi
Comparing the activity of the 4-chlorophenol derivatives with a total of four carbon atoms in the substituting groups against S typhi
, it can be seen that substitution by one alkyl group (see Table 20.2
-butyl) leads to a more effective compound than substitution by two groups (see Table 20.4
-propyl-3-methyl). This in turn is better than substitution by three groups (see Table 20.4
; 2-ethyl-3,5-dimethyl) even though it contains the same total number of carbon atoms.
The halogenated alkyl phenolics commonly used today are 4-chloro-3,5-dimethylphenol (p
]), 2,4-dichloro-3,5-dimethylphenol (dichloro-m
]), 4-chloro-3-methylphenol (p
]), and 6-isopropyl-3-methyl-4-chlorophenol (chlorothymol) (see Figure 20.1
Because of its strong microbicidal properties and low toxicity, PCMX
is used today as a broad-spectrum antimicrobial in disinfectants, antiseptics, and soaps.20
It also has been used as a fungicide for adhesives, paints, textiles, paper products, and polishes. It is reasonably soluble in water (0.33 g/L at 20°C), but it is more soluble in alkaline solution and organic solvents. The PCMX
is not sporicidal and alone has some activity against mycobacteria. To improve its solubility and achieve its full antimicrobial potential, correct formulation of PCMX
is essential. The impact of formulation on the activity and stability of antiseptics and disinfectants is considered in further detail later in this chapter and in chapter 6
. But an example of a typical formulation containing a chloroxylenol solution containing soap, terpineol, and ethanol is still included in the 2008 British Pharmacopoeia12
for application as an antiseptic.
has similar properties and spectrum of activity as PCMX6
and has been used in pine-type disinfectants and medicated soaps.21
However, because of its lower aqueous solubility (0.2 g/L at 20°C) and its more distinctive phenolic odor, its use tends to be limited.
The aqueous solubility of PCMC
is greater (3.85 g/L at 20°C) than that of other chlorophenols. Although it appears slightly less active against a range of microorganisms than PCMX
it has gained considerable importance as an industrial preservative in protecting various functional fluids, such as thickeners, adhesives, and pigments as well as textiles and leather. This is principally because
it remains active over a wide pH range (4-8), where, compared with other phenolic derivatives, only PCMC
remains sufficiently soluble.
Chlorothymol makes a satisfactory antiseptic when applied in the form of a solution in alcohol and glycerin.22
Monochloro and dichloro derivatives of carvacrol (5-isopropyl-2-methylphenol) were particularly effective against S aureus
comparing favorably with the corresponding thymol derivatives. More recently, Paulus6
reported that 4-chlorothymol is effective against fungi, less active against bacteria, and insufficient in its activity against Pseudomonas
Among the aromatic halogenated substituted phenols, 2-benzyl-4-chlorophenol (o
]; chlorophene) (see Figure 20.1
) is still used today in combination with other phenolics in various institutional and domestic disinfectant formulations. It is almost insoluble in water (149 mg/L at 25°C), but this is improved in alkaline solution, in organic solvents, or with the aid of saturated vegetable oil soap, such as coconut oil or various anionic detergents. In a suitable formulation, chlorophene shows good broad-spectrum activity.6
The formulation of disinfectants with benzylphenols and their halogen substitution products is the subject of a study by Carswell and Doubly.24
A liquid soap properly formulated with chlorophene compared favorably with one containing the same proportion of hexachlorophene its effectiveness.25
However, there are currently significant safety concerns for chlorophene and dichlorophen,26
which eventually may place it under new regulatory classifications.
Phenylphenols, also referred to as aryl phenols
, are compounds composed of a phenolic group connected directly to another aromatic ring. Two examples of these phenols are 2-phenylphenol (o
]) and 4-phenylphenol (p
]). Both compounds have similar broad-spectrum activity; however, PPP
is not commercially used because of its poor solubility properties and cost.6 OPP
, on the other hand, is well known, is widely used, and has become one of the most important phenolic biocides for application in disinfectants and in industrial preservation.
The solubility of OPP
(see Figure 20.1
) in water is low (0.2 g/L at 20°C), but it has good solubility in various solvents. The MIC
ranges from 50 to 500 mg/L for various bacteria, yeasts, and molds,6
making it reasonably broad spectrum. Its activity against Pseudomonas aeruginosa
is, however, less distinctive (MIC
approximately 1500 mg/L).6
The greatest use for OPP
in its phenol form is in the formulation of general surface disinfectants. It is commonly used as the active ingredient in hospital-type disinfectants because of its strong activity against M tuberculosis
. It may be formulated alone or in combination with other alkyl or halogenated phenolic derivatives. pH and the ratio/concentration of soap or detergents to OPP
and the phenolic concentration are all critical factors in the germicidal potency of phenolic disinfectants. The sodium phenate form of OPP
generally is used in preservation applications because of its high water solubility.28
As a preservative, OPP
is used to prevent the microbial degradation of industrial fluids, cosmetics, adhesives, paints, and textiles.
Bisphenols are compounds that are composed of two phenolic groups connected either directly or by various linkages. Large numbers of this type of compound have been synthesized and have been described by Gump.29
The compounds that exhibit the best activity are those that are separated by -CH2
– or -S- or -O- linkages. If a -CO-, -SO-, or -CH(OH)- group separates the phenyl groups, activity is low. Maximum activity is found with the hydroxyl groups at the 2,2-position of the bisphenol. Increased efficacy is also observed as the degree of halogenation of the phenol molecules increases; this is particularly the case against the gram-positive bacteria. When different members of a bisphenol series were tested in soaps against S aureus
, the superiority of the highly chlorinated members was evident.30
suggested that halogenation in the 4-position of each ring was the optimum position for activity against fungi. Increased halogenation, however, gave a decrease in activity against fungi, but this was considered to be due to a decrease in solubility. Unfortunately, increased halogenation is also accompanied by increased toxicity.6
As a result of this, and other side effects, the practical use of many bisphenols has been abandoned.
All bisphenols have limited water solubility, but they are more soluble in organic solvents and dilute alkali solutions. Although certain of the bisphenols demonstrate high bacteriostatic or fungistatic properties, their bactericidal and fungicidal activity is low. Like many other phenolic agents, activity against Pseudomonas
species is also low but may be improved by formulation effects (see chapter 6
Beginning in 1937, halogenated bisphenols were studied intensively in the laboratories of the Givaudan Corporation. From a long series of compounds that were examined, two had important commercial applications. The first of these was 2,2′-dihydroxy-5,5′-dichlorodiphenyl-methane (alternatively 2,2′-methylene-bis[4-chlorophenol]; dichlorophen) (Figure 20.2
). This was first prepared by Weiler et al31
and later by the improved method of Gump and Luthy.32
Although it exhibited reasonable activity against bacteria, its activity against fungi and yeast was relatively high.6
Because of this and its low skin irritancy, it found application as a preservative for toiletries and as a treatment
for athlete’s foot.7
Other applications include use as a fungicide and mildew-proofing agent for textiles, paper, cardboard, and adhesives; as a slimicide in paper manufacture; as preservative for lubricants and coolants; and to prevent microbial growth in water-cooling towers and humidifying plants. It also has been used in veterinary applications for its antiparasitic (anthelmintic and antiprotozoan, as a dewormer) activity, presumably due to its laxative activity.33
FIGURE 20.2 Structures of bisphenols and bis-(hydroxyphenyl) alkanes.
The other compound of commercial importance that was synthesized and described by Gump29
was bis-(3,5,6-trichloro-2-hydroxyphenyl)-methane (alternatively, 2,2′-methylene-bis[3,4,6-trichlorophenol]; or hexachlorophene) (see Figure 20.2
). Although it was essentially insoluble in water, the outstanding usefulness of this substance at that time was that it could be formulated into antiseptic soaps and detergents without a loss of activity. Like other bisphenols, it was also substantive to the skin, providing continuing bacteriostatic activity, especially against gram-positive bacteria.
Although hexachlorophene also has been used as a microbicide in cosmetics and textiles, its application in this area and as the active ingredient in consumer antimicrobial soap and detergent products has now been limited worldwide because of its potential for absorption through the skin and subsequent toxicity and neurotoxicity, particularly in neonates.34
Another review by Evangelista de Duffard and Duffard35
of chlorinated hydrocarbons, including hexachlorophene, showed it to cause motor dysfunction and developmental neurotoxicity.
A number of hydroxy-halogenated derivatives of diphenyl sulfide have been used as biocidal agents in a number of applications. One, 2,2′-dihydroxy-5,5′-dichlorodiphenyl-sulfide (alternatively, bis-[2-hydroxy-5-chlorophenyl]-sulfide; fentichlor) (see Figure 20.2
), has good bacteriostatic activity against gram-positive bacteria and surprisingly good activity against fungi, yeast, and algae. As a result, it has been used as a preservative in lubricants and coolants to overcome the problem of fungal growth.6
Hugo and Russell7
reported that the chief application of fentichlor was originally in the treatment of dermatophytic conditions; however, this molecule is also a photosensitizer, and hence, its use in this application and as a preservative in cosmetics is now limited. The halogenated analogue of fentichlor, 2,2′-thiobis(2,4-dichlorophenol), which also found application in soaps and cosmetics,36
also caused photosensitization and has been discontinued.
Although strictly a phenoxyphenol, 2,4,4′-trichloro-2′-hydroxydiphenyl ether, triclosan, is included in this section because of its structural similarity to the bisphenols (see Figure 20.2
). Triclosan is sparingly soluble in water (10 mg/L at 20°C), but it is readily soluble in a range of solvents and dilute alkali.37
In terms of its antimicrobial activity, triclosan is principally bacteriostatic with some fungistatic activity. Unlike the classic bisphenols, this inhibitory activity is reasonably broad, being equally effective against gram-positive and most gram-negative bacteria, good activity against Candida
species and mycobacteria, and poor activity against filamentous fungi. Its activity is not affected by organic matter but can be inhibited by formulated surfactant micelles structures. However, its activity against Serratia marcescens
for both >100 mg/L), and P aeruginosa
>1000 mg/L) is low.38
A primary target for triclosan in bacteria is by inhibiting fatty acid synthesis via its binding to the enoyl-acyl carrier protein reductase (ENR) enzyme,39
which is encoded by the gene fabI
. This increases the enzyme’s affinity for nicotinamide adenine dinucleotide (NAD+
) resulting in the formation of a stable complex of ENR-NAD+
-triclosan, making it unable to participate in fatty acid synthesis. These fatty acids are necessary for building and reproducing cell membranes in bacteria, and triclosan can specifically inhibit this biosynthesis.40
Triclosan also has a persistent skin activity similar to chlorhexidine (see chapter 22
), which can be affected pH, surfactants, and ionic nature of the formulation.
Triclosan has been incorporated into a large number of hospital and consumer products. This extensive use led to a number of questions related to the generation of triclosan-tolerant bacteria and its potential linkage to antibiotic resistance.42
This was clearly a cause for concern. Currently, there is a plethora of evidence to indicate that products containing triclosan could lead to the generation of antibiotic-resistant bacteria, particularly under laboratory conditions. Bailey et al43
observed differences in triclosan-dependent transcriptomes of Escherichia coli
and Salmonella ser typhimurium
with increased expression of efflux pump component genes. McBain et al44
observed that sublethal concentrations of triclosan can decrease bacterial diversity and a limited decrease in susceptibility to portions of the population in environmental microcosms. For a review of bacterial resistance to triclosan and its implications with respect to antibiotics, the reader should refer to reviews by Carey and McNamara45
The US Food and Drug Administration (FDA
issued a ruling in 2016 relating to triclosan usage in over-the-counter consumer antiseptic wash products and recommended products containing triclosan should no longer be marketed. This ruling was based on a number of factors: first, lack of direct evidence that antibacterial washes were more effective at preventing the spread of germs than normal washing with soap and water; second, long-term exposure to triclosan could pose health risks, such as bacterial resistance; and/or hormonal effects were not satisfied or sufficient for the agency to find it safe.47
However, it should be noted that triclosan’s use in consumer hand sanitizers, wipes, or health care settings was left intact by the FDA
An analogue of triclosan, triclocarban (or 3-[4-chlorophenyl]-1-[3,4-dichlorophenyl]urea), is another compound that shares some features with bisphenols, such as its skin substantiveness. Although a trichlorocarbanilide, triclocarban is similar enough to bisphenols to be considered here. Triclocarban has found application in antimicrobial soaps, particularly bar soaps, lotions, deodorants, toothpaste, and plastics. Research has suggested triclocarban exerts its effect by inhibiting the activity of enoyl-acyl carrier protein (enoyl-ACP) reductase, widely distributed in bacteria, fungi, and plants. The ACP reductase catalyzes the last step in each cycle of fatty acid elongation in the type II fatty acid synthase systems. This is very similar to the inhibition effects of triclosan described earlier. As a result, this agent interrupts cell membrane synthesis and leads to bacterial growth inhibition.48
As with triclosan, triclocarban is fairly broad spectrum with activity against gram-positive bacteria being greater than against gram-negative bacteria, good activity against Candida
species, and poor activity against filamentous fungi. Its activity is not generally affected by the presence of organic matter.
also issued a ruling in 2016 relating to triclocarban usage in consumer antiseptic wash products and recommended products containing triclocarban could no longer be marketed, similar to that described earlier with triclosan. Again, this ruling was based on two main factors: lack of direct evidence of the benefit over normal washing with soap and water and health risks. In fact, due to its use predominantly in bar soaps, a high percentage of the compound was found in wastewater biosolids,49
which could provide a prime breeding ground for cross-resistance and promotion of antibiotic resistance via genetic exchange. Some recent evidence points to an altered antibiotic tolerance in biosolids containing triclocarban.50
Hydroxycarboxylic Acids and Esters
Historically, various aromatic hydroxycarboxylic acids have found application as preservatives in the food, cosmetic, and pharmaceutical industries. Although they can be classified as “acid” preservatives, because of the appended hydroxyl group, they are considered here under the heading of phenolics.
Salicylic acid (ortho-hydroxybenzoic acid) is a weak acid with a dissociation constant of 1.07 × 10-3
. This compound was the subject of several early investigations, but its efficacy appeared to vary depending on the organism concerned. Cains et al57
found that salicylic acid was less effective than phenol against Yersinia pestis
, whereas Woodward et al58
found it to have a phenol coefficient of 18.3. Since then, because of its low pKa
, salicylic acid has found application as a preservative in acidic products. Its optimum pH for antimicrobial activity lies between 4 and 6. Although it is directed primarily against yeast and mold,59
its antibacterial activity is superior to that of benzoic acid. It is only in its undissociated state that it exhibits antimicrobial activity, but this significantly changes
as the pH varies. At pH 2, 90% is undissociated; at pH 4, this value is 8.6%; and at pH 6, only 0.09% is in the active form.27
The use of salicylic acid as a food preservative has now been abandoned principally because of its toxicity. It still finds application in topical skin care products, often in combination with benzoic acid, for the treatment of fungal infections or acne. This is in addition to its additional keratinolytic properties.7
The European Union is assessing the toxicology of salicylic acid.60
TABLE 20.5 Inhibition of bacteria and fungi by esters of p-hydroxybenzoic acid (percentages)
Salmonella ser typhi
(g/100 g at 15°C)
(g/100 g at 25°C)
Log P (octanol:water)
Abbreviation: NA, not available.
The alkyl esters of p
-hydroxybenzoic acid, or parabens as they are more commonly known, hold a position of considerable importance as antimicrobial agents, notably in the field of preservation. The parabens are relatively stable chemicals, being rapidly hydrolyzed only under extreme conditions, for example, at pH 10 or pH 1.59
They are active over a wide pH range (pH 4-8) and have pKa
values between 8 and 8.5. From the antimicrobial results shown in Table 20.5
, it is clear that the activity of the individual esters increases from methyl to butyl,61
whereas their already low water solubility decreases further over the same series. This decrease in water solubility is mirrored by an increase in the lipid solubility.
Most of the data contributing to the understanding of the parabens activity have come from the early work of Sabalitschka and colleagues.62
As early as 1939, the parabens were regarded as among the most useful preservatives available,68
and this view is held today (see chapter 40
The parabens are generally more active against gram-positive bacteria, fungi, and yeast than against gram-negative bacteria, particularly P aeruginosa
. Fukahori et al69
investigated the relationship between paraben ester alkyl chain length and antimicrobial activity against E coli
. Uptake into the bacterial cell and reduction in the minimum bactericidal concentration (MBC
) were proportional to the number of carbon atoms in the alkyl chain. Interestingly, the actual level of paraben necessary to achieve the desired antibacterial effect decreased logarithmically because the alkyl chain length increased.
The parabens display a low order of acute and subacute toxicity.70
concluded that the parabens approximate the requirements of an “ideal” pharmaceutical preservative as formulated by Gershenfeld and Perlstein.68
This was based on irritation, absorption, and excretion studies carried out in both animals and humans. These conclusions agree with those reached later by Matthews et al.71
In addition, Paulus59
indicated that they are broad spectrum, effective at low concentrations (0.05%-0.2%), stable, odorless, colorless, and effective over a wide range of temperature and pH.
Parabens have a long history in preservation from personal care products to food products. Over the last 10 years or so, the general media has created a myth about the safety and health risk parabens may pose, counter to the published research and global regulatory acceptability. Some of this myth came from a misunderstanding by the media
of a 2004 research study,72
which mistakenly linked parabens to breast cancer. Parabens are normally broken down quite quickly in human body, metabolized, and harmlessly excreted, which seem to refute the aforementioned claim. A number of global organizations have stated acceptance of the use of parabens in personal care products. However, due to this toxicity profile, they were considered less favorable and their use in many applications was reduced. Currently, the FDA
has limitations on the level of use of some parabens; however, these limits are based on the lack of human dermal and associated toxicity data required by this agency. Industry has argued that extensive animal studies should suffice and correlate to the human model, but this argument has been unsuccessful to fate.73
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