Quaternary Ammonium Compounds (QACs) and Ionic Liquids (ILs) as Biocides: From Simple Antiseptics to Tunable Antimicrobials

1For many years, quaternary ammonium compounds (QACs) have been included in most antiseptics and disinfectants and used in various areas, from household and agriculture to medicine and industry.

2The COVID-19 pandemic that broke out in 2020 led to a significant increase in the widespread use of sanitizers, including QACs. Recent studies have shown that more than 90% of the dust samples analyzed during the pandemic contained QACs, and their average concentration doubled compared to the pre-COVID period. It is to be expected that with the further progression of the pandemic, this number will increase, although the virucidal effect of QACs on SARS-CoV-2 requires further research.

3The constant presence of subinhibitory concentrations of QACs on various working surfaces, together with the frequent use of QACs, increases the risk of the development of a resistant bacterial environment, which will lead to a plummet of the effectiveness of popular antiseptics and disinfectants. The solution to this problem can be found in the synthesis of new QACs, which exhibit superior antibacterial, antifungal, and antiviral properties.

4The structure of QACs consists of a positively charged nitrogen atom with four or three substituents and one double bond. The core QAC structure can contain one (mono-QAC), two (bis-QAC), or more (multi-QAC, poly-QAC) charged nitrogen atoms, including those in heterocyclic compounds (piperidine, pyridine, imidazole, etc.). One or more of the substituents are usually long aliphatic chains containing at least ten carbon atoms. In the case of bis-QACs, multi-QACs, and poly-QACs, the structure that connects the charged nitrogen atoms (the head or nucleus fragment) is called a spacer or linker, and the alkyl chains extending from the heads (if they are present in the molecule) are called tails (Figure 1). QACs are generally water-soluble and stable. The counterion in these compounds usually does not affect the biological activity but often impacts the solubility of the biocide. The majority of the registered QACs contain chloride or bromide as anions. Due to their amphiphilic nature, QACs are able to form micelles. The critical concentration of micelle formation (CCM) is one of the important characteristics of these substances.

5The first studies of QACs as antibacterial agents were carried out at the beginning of the 20th century. Hexamethylenetetramine derivatives exhibited an in vitro bactericidal effect. With the discovery of benzalkonium chloride (BAC) in 1935, QACs found application in medical practice. Subsequently, the study of this class of compounds has led to the discovery of many valuable properties of QACs, due to which they are now used as surfactants, personal hygiene products, cosmetics, softeners, dyes, biological dyes, and, of course, antiseptics and disinfectants with a wide spectrum of action.

6Therefore, QACs belong to the group of biocides–chemical compounds designed to neutralize, suppress, or prevent the action of harmful organisms by chemical or biological means. As an example, in 2019, QACs accounted for ca. 11% of the whole biocide market in the United States, which equals ca. $192 million (Figure 2).

7The U.S. biocide market has grown by ca. 12% since 2016. The global trade of biocides, including QACs, is expected to grow by 3.9% annually and to reach $10.5 billion in 2027, thus evidencing the relevance and popularity of the topic. In other countries, similar trends can be expected due to the unquestionable significance of QACs.

8Biocides are used in a wide variety of fields. Approximately 50% of biocide applications in the global market are in the water purification and paint industry (Figure 3). However, they also play an important role in the medical field.

9This review focuses on the main QACs exhibiting the characteristics of biocides, the latest discoveries and issues of this field, and is separated into two parts. The first part presents the main commercial QACs currently used as active substances in antiseptics and disinfectants. The second part describes the scientific research of this class of compounds. Due to the ever-increasing demand for new bactericides and fungicides, the search for compounds active against newly arisen resistant strains of pathogenic bacteria and fungi is one of the most important areas of modern pharmaceutics. Of special concern is the emergence of multidrug-resistant strains (so-called “superbugs”). Therefore, we also discuss the possibilities of applying ionic liquids (ILs) as antimicrobial compounds. ILs, some of which can be classified as QACs, comprise a class of substances with vast molecular diversity. These compounds have been shown to possess a wide range of biological activities, including impressive antimicrobial properties. A summary of the bactericidal and fungicidal activities of common ILs, bis-charged ILs, and poly-ILs is provided in the corresponding subsections.

10A significant step in the development of biologically active QACs was the discovery of benzalkonium chloride 1 (BAC) by Domagk in 1935. BAC is a mixture of mono-QACs with benzyl, methyl, and alkyl substituents with different chain lengths from C8 to C18 (Figure 4). This drug is the first active QAC compound approved by the US Environmental Protection Agency in 1947, and it has been widely used to date. More details about the most important discoveries of that time in the QAC field can be found in the review by Rahn and Van Eseltine.

11The biological activity of benzalkonium salts depends on the length of the alkyl side chains. It is known that the C12-C14 compounds exhibit stronger bactericidal effects. Due to its broad antibacterial activity and low toxicity, a mixture of benzalkonium derivatives is used in washing disinfectants for hands and face, mouthwashes, creams, and other cleansing and disinfecting products. BAC exhibits bactericidal activity against Staphylococcus, Streptococcus, Gram-negative bacteria (E. coli, Pseudomonas aeruginosa, Proteus, Klebsiella, etc.), anaerobic bacteria, fungi, and molds. It is also efficient against bacterial strains resistant to antibiotics and chemotherapeutic drugs; it inhibits Staphylococcus plasma coagulase and hyaluronidase. BAC prevents secondary wound infection with hospital strains. In addition, a 0.2% aqueous solution of BAC was shown to inactivate the SARS-CoV-2 virus within 15 s.

12Further study of this class of compounds led to the discovery of several currently widely known QACs with similar structures: alkyltrimethylammonium bromides. The most famous of them are cetyltrimethylammonium bromide (CTAB) 2 and dialkyldimethylammonium chloride, the main representative of the latter being dimethyldidecylammonium chloride (DDAC) 3. The addition of the second long aliphatic chain increased the biological activity of the substance against S. aureus up to 8 times but, at the same time, increased its toxicity against red blood cells.

13Miramistin 4 is a nonheterocyclic alkyl QAC and one of the most popular antibacterial agents in antiseptics used in Russia. Miramistin demonstrates a moderate antiseptic effect against pathogenic fungi and viruses. Its aqueous solutions are used in the treatment of pyo-inflammatory diseases in surgery, obstetrics, gynecology, dermatology, urology, dentistry, and ophthalmology. Miramistin-containing drugs have a pronounced bactericidal effect on Gram-positive (Staphylococcus spp., Streptococcus spp., Streptococcus pneumoniae, etc.), Gram-negative bacteria (Pseudomonas aeruginosa, Escherichia coli, Klebsiella spp., etc.), aerobic, and anaerobic bacteria, both in the form of monocultures and microbial associations, including hospital strains polyresistant to antibiotics. Moreover, miramistin demonstrates antiviral activities (hepatitis, HIV), prevents wound and burn contamination, and facilitates the recovery of damaged tissues.

14Along with the majority of nonheterocyclic QACs on the antiseptic and disinfectant market, there are also examples of heterocyclic QACs, especially pyridine-based QACs (Figure 5).

15The simplest of them is mono-QAC cetylpyridinium chloride 5 (CPC). First described shortly after BAC in 1939, CPC has been extensively used in many mouthwashes and products for oral care. In addition, CPC works as a preservative agent due to its outstanding inhibition properties of bacterial growth.

16The second antiseptic of the subgroup is octenidine dihydrochloride 6 (OCT). Its dimeric structure is more complex than that of the other typical substances of this class. Here, two pyridinic nitrogen atoms linked via an alkyl bridge have alkylamine substituents in the para-position. OCT exists in pyridinic and imino forms. Due to its molecular structure, it demonstrates a broad spectrum of antibacterial activity, affecting S. aureus, S. epidermidis, P. mirabilis, K. pneumoniae, E. coli, P. aeruginosa, etc.. Two cation-active centers divided by the long aliphatic carbon chain facilitate molecule binding to negatively charged surfaces of microbial cells. Strong interactions between octenidine and lipids (in particular, cardiolipins) in the bacterial cell membrane have been detected. OCT has an intense residual effect on the skin, which is observed even 24 h after the last application. Due to its antimicrobial properties and skin compatibility, OCT can be used for various local applications where fast action and long-term effects are required, e.g., for disinfecting the skin of patients or treating acute and chronic wounds spontaneously colonized or locally infected by pathogenic bacteria. OCT can also be used for treating surgical equipment, injection sites of central catheters, infected root canals of teeth, candidiasis, acne, and nail infections.

17A number of other biocides that play an important role in the modern market of antiseptics and disinfectants should also be mentioned. The antiseptics chlorhexidine bigluconate 7 (CHG), alexidine 9, and polyhexamethylene biguanide 8 (PHMB) (Figure 6) are guanidine derivatives from the cationic biocide family, as well as the abovementioned QACs.

18CHG is a symmetrical bis-biguanide connected by an alkyl chain; it carries two positive charges at physiological pH. Developed in the early 1950s during the screening for antimalarial drugs, CHG has since recommended itself as a broad-spectrum antibacterial drug. CHG is one of the first antiseptics used on the skin and for decontamination of wounds. It is typically applied in the form of bigluconate, gluconate, dichloride, and acetate salts. Antiseptic drugs, which contain chlorhexidine bigluconate as an active substance, have a fairly wide spectrum of action. They are active against Gram-positive bacteria but not Gram-negative bacteria and mycobacteria or fungi. CHG is widely used in surgery and hand washing in the treatment of wound sepsis. It is also used in various oral hygiene products, as an anti-plaque agent, and in periodontal treatments. Similar activities were exhibited by aleksidine (Figure 6).

19PHMB is an alkyl biguanide polymer that can be used in a soluble form as chloride. It is an effective alternative to traditional antiseptics due to its low toxicity and superior antibacterial and antifungal activity. It is used for treating swimming pools and fabrics, in cleaning products, and as a disinfectant for contact lenses and mouthwashes.

20The simplicity of synthesis, vast structural diversity, and high biological activity drive numerous scientific studies on QACs. Over the past 85 years, after the emergence of the class of cationic biocides, the number of publications on the topic has been arising significantly (Figure 7). According to SciFinder, more than 700 articles on QAC properties were published in 2020.

21The scientific society proposes various synthetic procedures and applications for QACs, analyzes their structural fragments, and establishes the relations between the efficiency and molecular structure. The last approach, known since the 19th century, is widely used in quantitative studies on various activities of chemical substances (QSAR, quantitative structure–activity relationship).

22Judging from the basic structure (Figure 1), one can change several parts in a given QAC to determine their impact on its activity:

23Head. The number of charged nitrogen atoms (mono-, bis-, multi-QAC), as well as the head structure (non-heterocyclic, heterocyclic, aromatic), can be changed.

24Spacer. The structure (aliphatic, aromatic, saturated, unsaturated, mixed, etc.) can be changed.

25Tail. The structure (saturated, unsaturated, branched, unbranched) and the length of the aliphatic chain can be changed.

26Substituents. A desired group can be introduced into any of the abovementioned fragments of the QAC molecule.

27Hereafter, we will focus on representative examples of synthetic biocidal QACs obtained by various scientific groups in recent years. The effect of the structural fragments of the biocides on their biological activity will also be considered. The material is presented sequentially, depending on the QAC charge (mono-QAC, bis-QAC, poly-QAC). Additional information on studies on antimicrobial activity, surfactant properties, usage, and synthesis can be found in recent reviews on the topic.

28Thorsteinsson and colleagues developed “softer” analogues of the existing QAC biocides. While “hard drugs” (CPC, BAC) are specified as drugs that are not subject to in vivo changes, “soft drugs” are metabolized to nontoxic compounds (Figure 8).

29Due to the introduction of amide and ether groups, the synthesized QAC molecules 10-13 are deactivated and decomposed into amides, fatty acids, and alcohols. Compounds without alkyl chains or with short chains (C2, C3) were found to be inactive. Substances with C12–C18 alkyl tails exhibited antibacterial activity comparable to a known analog (BAC 1) against E. coli, S. aureus, and P. aeruginosa. Additionally, some compounds from series 11 showed activity against herpes simplex virus (HSV-1).

30Miklas and colleagues carried out the synthesis and studied the biological properties of QACs based on camphorsulfonic acid (CSA) 14-16 (Figure 9).

31Upon changing the QAC core from ammonium to a less saturated heterocyclic structure (imidazole), the antimicrobial activity of the compounds gradually decreased. Salts with alkyl tails exhibited better activity than their ester and amide counterparts. The optimal chain length was found to be C12-C14.

32In a recent work, Ali and colleagues developed new pyridine-based QACs from Schiff bases of nicotine hydrazines (Figure 10).

33These substances had good water solubility, most likely due to the presence of hydrazide groups. Despite the shorter alkyl chains (compared to typical QACs), a series of substances 17 showed high activity against colonies and biofilms of E. coli and S. aureus. According to this study, the presence of donor groups in the phenyl ring of the R substituent increased the bactericidal activity.

34In the works of Liu and colleagues, the effect of combining two biocidal fragments (N-chloramines and alkyl QACs) in one molecule 18-19 on bactericidal properties was studied (Figure 11).

35Chloramines act on bacterial cells through the oxidative transfer of chlorine to biological receptors which leads to cell lysis. The attachment of the QAC molecule with a positive charge allowed anchoring of the N-chloramine moiety on the surface of the bacterial cell, thus enhancing the effect. The introduction of a long alkyl chain into the compound leads to the rupture of the bacterial membrane, penetration of the biocide into the cell, and a subsequent enhancement of the bactericidal effect. At the same time, Li and colleagues combined a pyridinic QAC with N-chloramine 20 (Figure 11). The antibacterial activity of this compound was similar to that presented by Liu.

36In the works of Wang and Hou, a similar approach to changing the structure of QAC by adding biologically active fragments to the molecule was used (Figure 12).

37Initially, guided by the hypothesis that hydroxy groups should stimulate membrane penetration and cell destruction, a series of hydroxy-QACs 22 with different alkyl chain lengths was synthesized. All the resulting compounds exhibited lower antibacterial activity than CHG; they also demonstrated antifungal activity with an optimal tail length of C12. It should be noted that the toxicity of the compounds correlated with their activity. Then, a fragment of oxadiazole derivatives 23-24, benzothiazole (X=S) 21, and benzoxazole (X=O) 21 was introduced into the QAC molecule, which led to an increase in bactericidal and fungicidal activity and a decrease in toxicity in epithelial cells and erythrocytes.

38Bogdanov and colleagues explored the microbiological effect of isatin-based QACs (Figure 13).

39As seen from the figure, the structures of these ammonium 25 and pyridine 26-27 salts contain no long alkyl chains. Therefore, the cytotoxicity of these compounds is significantly lower than that of typical QACs. However, the antibacterial activity is markedly reduced in the absence of quaternary nitrogen tails. Thus, none of the compounds from this series showed a biocidal effect against the Gram-negative bacteria E. coli and P. aeruginosa. On the other hand, these salts inhibited the growth of Gram-positive bacteria (S. aureus and B. cereus) and fungi (C. albicans) at concentrations comparable to modern antibiotics (chloramphenicol and norfloxacin). Overall, QACs with pyridinium nuclei and donor substituents in the aromatic part of isatin 27 turned out to be more active than the others.

40Rusew and colleagues presented a work, in which long lipophilic tails in QACs were replaced by more compact aryl-containing substituents (Figure 14).

41The results of a broad antibacterial screening appeared to be nontypical for cationic biocides. Compounds with biphenyl and 1,3-dimethoxyphenyl 29 substituents selectively inhibited the growth of E. coli (Gram-negative) and S. aureus (Gram-positive) but no other Gram-positive and Gram-negative bacteria. In a quantitative sense, the inhibiting zones of these substances were similar to kanamycin.

42Kuca and Soukup studied the biological activity of picolinic QAC with methyl substituents 30 (Figure 15).

43It was found that the position of the substituent did not significantly affect the biocidal effect of methylpicolinates, possibly due to the small size of the methyl substituent. Overall, picolinates showed a comparable or even superior bacteriostatic effect compared to BAC on a wide range of pathogens. The optimal tail length was C14-C16, and higher activity was observed in Gram-positive bacteria than in Gram-negative bacteria, as with most QACs.

44Shtyrlin and his colleagues created a pyridoxine-based QAC library, including bis-derivatives, which will be discussed in the corresponding part of the review (Figure 16).

45Pyridoxin functional derivatives 31-36 exhibited a broad spectrum of antibacterial and antifungal activity; at that time, they were more active against Gram-positive bacteria than Gram-negative bacteria. It should be mentioned that a combination of the antifungal drug terbinafine with pyridoxin-based QAC 36 was efficient against mixed colonies of pathogenic bacteria and fungi. This example proved the advantage of combining two different biocide fragments in one molecule.

46A significant contribution to the development of QACs as a class of cationic biocides was made by the groups of Wuest and Minbiole (Figure 17).

47It was found that close structural analogs of BAC 37 containing amide and ester groups exhibited comparable activity and lower toxicity than BAC. QAC derivatives of natural compounds (quinine 38 and nicotine 39) demonstrated a wide spectrum of antibacterial action, thus justifying the search for other platforms of natural origin to expand the library of active QAC compounds.

48An overview of the antibacterial activity of mono-QACs, analyzed in the review, is shown in Table 1.

49ILs are organic salts that generally exist in liquid form at a wide range of temperatures. The most common ILs are composed of a bulky organic cation and a more compact anion (Figure 18). Due to its broad applications in chemistry, this class of compounds has been studied thoroughly, and the chemical and physicochemical properties, as well as biodegradation potential, of various ILs have been determined.

50Initially, ILs were considered green solvents that could replace traditional toxic organic solvents in various chemical processes. However, when evidence of the high biological activity of various classes of ILs has emerged, these substances have quickly become candidates for new drugs and drug-like molecules. In particular, the antimicrobial activity of ILs has attracted much attention, and their possible medical and environmental applications have been proposed.

51A subclass of ILs with quaternary ammonium cations (which includes several of the above-discussed QACs) has promptly been established as a promising alternative to traditional antimicrobial substances. ILs with other cations have also demonstrated prominent bactericidal and fungicidal activities. Some of these ILs (e.g., N-hexadecylpyridinium chloride, or cetylpyridinium chloride, CPC, which is also classified as a QAC) have been extensively used as antiseptics for a long time. The first successful results of studies on the antimicrobial activities of various ILs have led to the rapid development of API-ILs (active pharmaceutical ingredient–ionic liquid), that is, known commercial drugs in an ionic liquid form.

52An overview of the antimicrobial activities of various members of common IL classes is provided in Table 2 and Table S1. In most cases, there is a direct relation between the length of the alkyl side chain in the cation and the IL antimicrobial activity. ILs with relatively short side chains (ethyl, butyl, hexyl) usually demonstrate weak activity (see Table S1), whereas those with long side chains (dodecyl, tetradecyl, hexadecyl) can be strong inhibitors of some bacterial and fungal species, including biofilm-forming and drug-resistant species (see, e.g., entries for [CnMim][A], n = 12–16, and [CnPy], n = 12–16, in Table 2). For instance, 1-dodecyl-3-methylimidazolium bromide ([C12Mim][Br]), N-dodecyl-N-methylpyrrolidinium bromide ([C12C1Pyr][Br]), and N-dodecyl-N-methylpiperidinium bromide ([C12C1Pip][Br]) demonstrated both high antimicrobial and low hemolytic activity, thus allowing their successful application in medicinal practice. Cholinium-based ILs with long alkyl chains, in particular, N-(2-hydroxyethyl)-N,N-dimethyl-N-tetradecylammonium bromide, N-(2-hydroxyethyl)-N,N-dimethyl-N-hexadecylammonium bromide, and N-(2-hydroxyethyl)-N,N-dimethyl-N-octadecylammonium bromide, efficiently inhibited the growth of various bacterial strains, including antibiotic-resistant strains (see entries for [HOC2C1,1,nN][Br], n = 14–18, in Table 2). Surface-active cholinium ILs with the dodecylbenzenesulfonate anion demonstrated significant activity against Gram-negative and Gram-positive bacteria, fungi, and single-cell algae; these ILs were proposed to be used as coatings for the prevention of biofilm formation on stone surfaces.

53It should be noted that the anion can also have a significant impact on the antimicrobial activity. Thus, the antibacterial activity of 1-butyl-3-methylimidazolium ILs with different anions against pathogenic and semipathogenic Gram-negative and Gram-positive bacteria varied significantly depending on the anionic nature. In particular, 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide ([C4Mim][NTf2]) demonstrated the highest activity against E. coli (see entries for [C4Mim][A] in Table 2 and Table S1); however, its anti-adhesive activity was significantly lower than that of several other ILs tested. A different picture was observed in the case of 1-hexyl-3-methylimidazolium IL, among which 1-hexyl-3-methylimidazolium nitrate ([C6Mim][NO3]) demonstrated the highest activity against E. coli and several other microorganisms tested (see entries for [C6Mim][A] in Table S1). Interestingly, it was demonstrated that for ILs with tris(pentafluoroethyl)trifluorophosphate anions, the antimicrobial activity decreased upon increasing the alkyl side chain length.

54Of special interest are ILs containing antimicrobial moieties in their anions or cations. The API-IL concept allows simultaneously solving two common issues of traditional drugs: low solubility in aqueous media and tendency to form polymorphs. Examples of bactericidal API-ILs are given in Figure 19, Table 3, and Table S2. Thus, API-ILs bearing ampicillin as their anion in combination with cetylpyridinium or 1-hexadecyl-2,3-dimethylimidazolium as their cation demonstrated improved activity against several Gram-negative and Gram-positive bacterial strains, including ampicillin-resistant E. coli strains, compared to the ampicillin sodium salt (see the corresponding entries in Table 3).

55Bis-QAC (or so-called “twin surfactants”) is a subclass of synthetic amphiphiles that contain two cationic nitrogen atoms, a spacer linking them, and two lipophilic alkyl substituents. These are common characteristics of typical bis-QAC, the exact structure of which can vary greatly. The intense development of bis-QACs began later than that of mono-QACs in the 1980s with the discovery of octenidine (see the Commercial QACs section). Nonetheless, there are many publications on the synthesis and biocide properties of bis-QACs.

56A significant number of alkyl bis-QACs were synthesized to test the effect of the total charge of the molecule on the activity (Figure 20).

57Bis-QACs with ester spacer 46 showed better activity than their mono analogues, both against Gram-positive and Gram-negative bacteria and fungi. It is worth noting that the activity against E. coli was nonlinear and plummeted upon increasing the alkyl chain length from C12 to C14. This relationship, which is known for the biocidal action of amphiphils on Gram-negative bacteria, is called the “cut-off” effect. It was described by Devinsky and colleagues as a consequence of membrane penetration. The addition of a second charged nitrogen atom increased the activity 3-fold in S. aureus and 4-fold in E. coli in the work of Hodye (substance 47). The activity also correlated with the distance between the heads, with the optimal spacer length being C6. Wuest and Minbiole and colleagues studied the biocidal action of QACs based on polyamines 43-44. Tetramethylethylenediamine derivatives (TMEDAs) 42 turned out to be an extremely promising class of biocides because of their simple synthesis, cheap starting materials, and high activity. In all the above-mentioned studies, the biological effect on pathogenic bacteria increased 3–4 times, especially for Gram-negative strains, compared to mono-QACs.

58Changing the spacer in the bis-QAC structure is one of the key factors in the design of target molecules. Thus, the aforementioned alkyl bis-QACs can contain aromatic spacers (Figure 21).

59A study by LaDow and colleagues showed that bis-QACs 48-52 inhibited the growth of Gram-positive bacteria at approximately the same concentration as their mono analogs. However, bis-QACs had a much stronger effect on Gram-negative bacteria, which was confirmed by other studies. In continuation of their work on the study of pyridoxine QAC derivatives, Shtyrlun and colleagues noted a clear dependence of the activity of compounds 54 on their lipophilicity. Thus, the values of the lipophilicity coefficient for the most active compounds (C10, C12) were in the range of 1 to 3; at values higher than 6 or lower than 0, the activity decreased sharply. Forman and colleagues studied QAC derivatives of malachite green 53, comparing its mono- and bis-QACs. Analogs with two long alkyl chains were generally comparable to mono-QACs but were more efficient against resistant bacteria.

60Similar to mono-QACs, the head of bis-QACs can have a saturated heterocyclic structure (Figure 22).

61Kourai and colleagues, in their study of bis-QAC derivatives of piperazine 57, found that compounds with different spacer structures but the same lipophilicity exhibited different activities. This fact suggested that the dependence of the biocidal action on lipophilicity was valid only for the series of QACs differing in the length of the tail. Kontos and colleagues tested the dependence of the activity of 58-59 on the rigidity of the structure. The initial assumption that a more flexible structure would provide easier passage through the bacterial membrane and accelerate cell lysis turned out to be erroneous. Thus, derivatives of the more rigid amine structure 59 of diazobicyclooctane (DABCO) were most active in the series. A series of heterocyclic QACs based on cardanol 60 was developed by Ma and colleagues. Along with moderate antibacterial activity, the compounds appeared to be good surfactants.

62There are several examples of mixed bis-QACs carrying two different heterocycles or heterocyclic and alkyl parts (Figure 23).

63In the continuation of the work on preparation of the above-mentioned QAC derivatives of quinine and nicotine, the usual “activation” of the second nitrogen charged center did not lead to a significant increase in the activity of 61-62. Presumably, the total charge of the molecule does not affect the activity as strongly as the addition of the second alkyl chain. In the work of Schallenhammer and colleagues, hybrid bis-QACs 63-64 combining CPC 5 and BAC 1 showed higher activity against Gram-negative bacteria than each of the commercial “source drugs” applied separately. At the same time, hybrid monoderivatives did not show such a result. Piperazine bis-QAC derivatives 65 and their “soft” analogs 66 showed similar relationships with the previous bis-QACs.

64Additionally, there is a range of interesting works concerning QACs with polynuclear heterocycles with several heteroatoms (Figure 24).

65Thomas and colleagues synthesized QACs based on bis-thiazole 67, bis-imidazole 68 and bis-triazole 69. While thiazole derivatives with an alkyl spacer and without lipophilic tails 67 did not show high activity, bis-QACs with nitrogen heterocycles 68-69 demonstrated MIC values lower than that of CHG.

66In contrast, in the work of Shirai and colleagues, thiazole bis-QACs with alkyl tails 71 (Figure 25) exhibited a wide spectrum of antibacterial and antifungal effects. This is additional evidence that the tails in the QAC structure are strong inducer of the biological effect against pathogens. Shrestha and colleagues studied the antibacterial and antifungal activity of bis-triazole QAC based on benzoquinone 72 (Figure 25).

67Inspired by the success of octenidine on the market of cationic biocides, scientists have begun to actively develop a class of bispyridinium salts with various types of spacers (Figure 26).

68In the work of Minbiole and colleagues, bispiridinium QAC derivatives of paraquats 73-75 and bis-QACs without a spacer between pyridinium heads were studied. The activity of meta-75 and parameta-analogs 74 was more pronounced. Cyclovoltamperometric analysis showed the predisposition of paraquats 73 to reversible oxidation-reduction processes and the formation of “superoxide”. This presumably increases the toxicity, while metaquats 75 and parametaquats 74 are not subject to this possibility and thus can be less toxic. In addition, given the high activity of parameta-derivatives 74, this indicates the incoherence between the increase in the biocidal action of QACs and their redox capacity. A study on the dependence of the activity on the rigidity of the structure for bispyridinium-QACs with alkyl spacers with different saturations 76-78 showed ambiguous results. While this dependence was not observed for QACs with alkyl chains as tails, and the MIC values remained approximately at the same level, in the case of bis-QACs with amide bridges in the tails, a sharp decrease in the activity was observed upon increasing the structural rigidity. The authors showed that in such rigid structures, the bis-QAC activity decreased as the charged heads moved away from each other.

69In the last few years, new biocidal pyridine-based bis-QACs containing an aromatic fragment in a spacer have been synthesized (Figure 27). Thus, bis-QACs with 1,4-dioxophenyl as spacer 79 were significantly more active than commercial QACs (BAC 1, CHG 7). Vereshchagin’s group studied the dependence of the activity of biocides on the size of the aromatic spacer of salts, as well as the location of the spacer relative to the charged pyridinium nitrogen 79-83. It was discovered that the QAC activity increased upon increasing the length of the aromatic spacer. The activity increased in the following order: mono- 79 < bi- 80 < terphenyl 82. It can be assumed that in such structures, the activity increases with an increase in the distance between the nitrogen atoms. It is worth noting that the optimal length of the alkyl tails also varied in this series: C12 for phenyl 79, C10 for biphenyl 80, and C8 for terphenyl 82. The influence of the position of substitution in pyridine turned out to be ambiguous. In the case of biphenyl 80, the meta-salts turned out to be slightly more active than the para-derivatives, while the opposite was observed for the more mobile biphenyl ether 81. The ortho-salts showed strikingly lower activity. However, this was not the case for QACs of 2,7-dihydroxynaphthalene derivatives 83, and the biocidal effect of the orthosalts was extremely high. From the viewpoint of their activity, the leading compounds from the series of bis-QACs with aromatic spacers were superior to the widely used QACs, such as CHG 7, CPC 5, BAC 1, and miramistin 4, and were comparable to OCT 6 (Figure 27).

70There is a broad variety of structures of bispyridinium salts containing mixed spacers (Figure 28).

71Kourai and colleagues initiated studies on bis-pyridine salts 84, 86-88. Later, Obando and colleagues proposed the synthesis of biologically active bis-QACs containing mixed alkyl-aromatic spacers 89. In their recent investigation, Hao and colleagues performed a comprehensive physical-chemical and biological analysis of bis-QACs with amide bridges 85.

72Pentaerythritol-based bis-QACs 90-91 (Figure 29) were developed by Yamamoto and colleagues. These substances revealed a broad scope of antibacterial and antifungal activities. At that time, the substances with condensed hydroxy groups 90 had higher activity than those with free hydroxy groups 91. The biocompatibility of the series leaders was similar to or higher than that of the common antiseptics (BAC, CPC, OCT, PHMB). Furthermore, Vereshchagin presented a synthetic route and microbiological study of pentaerythritol bis-QACs as OCT analogues 92. The salts were active towards MRSA and E. coli (Figure 29).

73An overview of the antibacterial activity of bis-QACs, analyzed in the review, is shown in Table 4.

74A number of dicationic ILs have been tested for their antimicrobial activity (see Figure 30, Table 5, and Table S3 for several examples). The high bactericidal activity of some of these ILs (in particular, nitro-substituted imidazolium salts) suggests their possible medical applications (see Table 5).

75Multi-QACs are salts with three or more charged nitrogen atoms in one molecule. This biocide group is rather underexplored compared to mono- and bis-QACs, probably because of the more complicated synthesis and the lack of low-cost platforms for multicharged QAC structures.

76Wuest and Minbiole developed a simple synthetic route for obtaining tris- and tetra-QACs on the basis of polyamine platforms 93-97 (Figure 31). The activity of multi-QACs was significantly higher than that of mono-QACs but was comparable to that of bis-QACs.

77Several multi-QACs with aromatic fragments in the structure were also obtained (Figure 32). Forman and colleagues demonstrated that tris-derivatives of crystal violet with one alkyl tail 98 had lower activity than mono-QACs. However, analogs containing ethyl groups at the charged nitrogen instead of methyl groups were more active. Gallagher and colleagues found that tris-QACs with two alkyl tails 99 were more effective against Gram-negative bacteria than tris-QACs with one alkyl tail. Tris-pyridinium salts 100 and tetrapyridinium salts 101 also comprised an efficient group of biocides with a broad spectrum of action and surpassed the activity of the well-known pyridinium antiseptic CPC 5 several times.

78An overview of the antibacterial activity of multiple QACs, analyzed in the review, is shown in Table 6.

79Polymer structures with quaternary nitrogen occupy a large niche in the field of cationic biocides. QACs exhibiting antimicrobial activity can be incorporated into polymer structures in several ways:

80Ring-opening polymerization. Chain-growth polymerization, in which one end of the polymer chain carries an active site for adding cyclic monomers. The terminal groups of the resulting polymer depend on the initiator used and the termination reaction.

81Controlled radical polymerization. Continuous polymerization includes several stages: Initiation, growth, and chain termination.

82Click reaction. Polymerization that utilizes methods of click chemistry.

83Similar to other types of QACs, the structure of poly-QACs can vary depending on the monomer composition (homogeneous poly-QACs (Figure 33) in the case of the same monomers, or copolymers (Figure 34) in the case of different monomers) and the polymerization type.

84Lu and colleagues studied the biological properties of poly-QACs with benzyl substituents and ether groups in side chain 102. The activity of the polyderivatives was significantly higher than that of the corresponding monomers; it increased upon increasing the length of the alkyl substituent. Guo and colleagues compared polymers with quaternary nitrogen in the side 103 and main 104 chains. The presence of charged nitrogen atoms in the main polymer chain enhanced the antibacterial effect on Gram-positive and Gram-negative bacteria by several times. The carbohydrate-based poly-QACs obtained by Badawy’s 108 and Shaban’s 107 groups also exhibited biocidal activity. Polymer salts consisting of monomers with DABCO-containing heterocyclic QACs 106 were obtained by Mathias’ group. Researchers observed an increase in bactericidal activity with the growth of alkyl chains. It should be noted that the monomer did not exhibit antibacterial activity. Polymerization may be the key to achieving the required biocidal effect for inactive QAC molecules. Timofeeva and colleagues developed an approach to the synthesis of quaternary poly(diallyldialkylammonium) salts with various substituents 105. The researchers noted that the antibacterial effect, but not the antifungal effect, became more pronounced upon increasing the mass of the polymer.

85Kallitsis and colleagues studied single- 109-110 and two-charged 111 copolymeric QACs in their work. The peculiarity of this study was in the fact that the polymer chain in one of the target compounds 110 was an anion, while the cation was a conventional mono-QAC alkyl cation of CTAB type 2, whereas compound 111 was poly-QAC bearing both cations and anions. This composition had a positive impact on the biocidal effect against a wide range of bacteria. The optimal structure was established as 75% ionic and 25% covalent bonds of the polymer with QAC. Jie and colleagues combined the QAC and N-chloramine 113 molecules in one polymer. A similar successful approach was pursued by Liu and colleagues. Bai and colleagues synthesized a polymer combining amino and QAC groups 112, which showed excellent bacteriostatic potential.

86The diversity of homogeneous and copolymeric QACs is very high and is beyond the scope of this review; only exemplary biologically active representatives of this class are presented here. More detailed information on poly-QACs can be found in other reviews.

87An overview of the antibacterial activity of poly-QACs, analyzed in the review, is shown in Table 7.

88According to the strict definition, poly-ILs are ionic polymers with complete ionicity. However, ionic polymers with lower levels of ionicity are often considered poly-ILs in publications. In recent years, poly-ILs have been extensively studied as advantageous materials for antibacterial coatings and surfaces. Exemplary poly-ILs with tested antibacterial activity are listed in Table 8 and Figure 35. Note that the table includes substances 103 and 104, which are also considered poly-(QACs).

89Antibacterial coatings on the basis of 3-(2-(methacryloyloxy)ethyl)-1-alkylimidazolium ILs showed high bactericidal activity against E. coli (see entries 114-116 in Table 8). In the case of 1-alkyl-3-vinylimidazolium-based poly-ILs, the alkyl side chain length and charge density were directly related to the antimicrobial activity against E. coli and S. aureus (see entries 117-119, 121, and 123-126 in Table 8). In contrast, the bactericidal activity of the corresponding poly-IL membranes increased upon increasing the charge density but decreased upon increasing the alkyl chain length. A similar picture was observed for pyrrolidinium-based ILs and membranes. The homopolymeric ILs were active against S. aureus and E. coli, and their antimicrobial activity increased upon increasing the alkyl side chain length in the monomer (see entries 123-126 and 127-131 in Table 8). The opposite was observed for the corresponding poly-IL-based membranes, which also demonstrated good hemocompatibility and low cytotoxicity. Of note, nanoparticles on the basis of 1-alkyl-3-vinylimidazolium poly-ILs showed significantly higher antimicrobial activity than the original poly-ILs (see entries 119-122 in Table 8).

90(2-Ethylhexyl)ethylenediaminium bis(trifluoromethanesulfonyl)imide-loaded ionogel surface coatings efficiently inhibited the growth of various microorganisms, including those from the ESKAPE list, and prevented the formation of biofilms. Microneedle patches on the basis of salicylic acid-containing API-poly-IL were successfully tested in the treatment of Propionobacterium acnes skin infections. Ionic graft copolymers on the basis of [2-(methacryloyloxy)ethyl]trimethylammonium chloride were studied as possible delivery systems for ionic drugs (p-aminosalicylate and clavunate). IL-grafted wound dressings on the basis of 1-vinyl-3-methylimidazolium bromide demonstrated good antimicrobial activity and low cytotoxicity.

91QACs also find application in the composition of bioactive materials and antibacterial coatings. This topic is more relevant than ever due to the growing part of the paint and coatings industry in the biocide market. Thus, research on the application of QACs at surfaces continues to expand.

92Antimicrobial films based on surface-modified microfibrillated cellulose grafted with mono-QACs showed high antibacterial activity against S. aureus and E. coli even at low concentrations. Silica nanoparticles functionalized with quaternary ammonium silane inhibited the growth of Gram-negative bacteria due to the synergistic effect of hydrophobicity and antibacterial activity. QACs with N-halamine coated onto cotton fibers were active against S. aureus. Similarly, the combination of these biocides was highly effective in macroporous cross-linked antimicrobial polymeric resin. An antibacterial coating of immobilized QACs tethered on hyperbranched polyuria demonstrated high contact-killing efficacies toward adhering staphylococci. Antimicrobial acrylic coatings with a QAC-containing perfluoroalkyl monomer were synthesized by using a self-stratification strategy via one-step UV curing. Polyvinylidene fluoride membranes modified by QACs possess antibiofouling effects. Bacterial cellulose incorporated with QACs showed strong and long-term antimicrobial activity against S. aureus and S. epidermidis. QAC-based silver nanocomposites demonstrated synergistic antibiofilm properties along with a low hemolysis rate. More examples of QACs immobilized on material surfaces with antibacterial activities can be found elsewhere.

93Usage in bactericidal surface coatings seems one of the most promising applications of antibacterial ILs in medicine and other areas. Thus, the number of publications on the topic has been increasing steadily in recent years. As already mentioned above, ILs are proposed to be used as components of ionogels, films, and membranes that demonstrate considerable antimicrobial and antifouling activities (see, e.g.,). Cellulose nanofibers grafted with ammonium ILs and silver ions demonstrated significant antimicrobial activity against S. aureus MRSA and E. coli. Zinc ion-coordinated poly-IL membranes with bactericidal properties were efficiently used for wound healing. A conductive hydrogel wound dressing composed of a poly-IL (1-vinyl-3-(aminopropyl)imidazolium tetrafluoroborate) and konjac glucomannan demonstrated long-lasting bactericidal activity against S. aureus and E. coli. Similarly, promising results were obtained with a poly-IL (1-vinyl-3-butylimidazolium bromide)/poly(vinyl alcohol) wound dressing, a reusable 1-vinyl-3-butylimidazolium bromide-grafted cotton gauze wound dressing, and molecular brushes with 3-(12-mercaptododecyl)-1-methylimidazolium bromide. Composite membranes composed of bacterial cellulose and cholinium poly-ILs with amino acid anions were active against Gram-negative and Gram-positive bacteria and fungi. Poly(vinylidene fluoride) (PVDF) materials grafted with ILs (1-vinyl-3-butylimidazolium chloride, 1-vinyl-3-ethylimidazolium tetrafluoroborate) showed activity against both common bacteria and “superbugs”. Calcium phosphate–IL (1-alkyl-3-methylimidazolium chloride) materials with bactericidal properties were proposed to be used for implants. Halloysite nanotubes functionalized with various ILs demonstrated antimicrobial activity.Coatings based on dicationic imidazolium ILs efficiently inhibited bacterial growth on titanium surfaces. TiO2 nanomaterials coated with poly-IL brushes on the basis of imidazolium ILs demonstrated antibacterial and antifouling properties. Cholinium salicylate-containing gelatin films with bactericidal activity were proposed to be used in food packaging. In addition, 1-butyl-3-methylimidazolium bis(trifluoromethanesulfonyl)imide ([C4Mim][NTf2]) was tested as a bactericidal additive in orthodontic adhesive and was shown to reduce biofilm formation.

94Despite the vast diversity of the available QAC structures, there are certain structural criteria designating the biocidal activity of the compounds.

95Usually, the optimal alkyl tail length is within C10-C14; it can vary depending on the number of charges: C12 and longer for mono-QACs and C10-C12 for bis-QACs. Nevertheless, in some series of compounds, those with tails of C10 and shorter demonstrated the highest activity. This observation suggests that the optimal chain length is specific for each set of structures and is related to the other fragments of the molecule.

96In general, QACs with two or more charges (bis-QACs, multi-QACs, poly-QACs) have superior biocidal effects compared to mono-QACs. Moreover, many mono-QACs show little or no activity against Gram-negative bacteria. However, the addition of the second charged nitrogen without an alkyl chain does not always increase the activity, whereas the addition of the second and third alkyl chains increases the toxicity. The introduction of ether or amide bridges into QACs decreases both the toxicity and activity of the corresponding substances.

97The combination of two bactericidal fragments with different mechanisms of action in one QAC has been proven to be a successful approach. These biocides have antibacterial and antifungal effects on a wide range of pathogens.

98The assessment of the direct relation between the presence of aromatic and heterocyclic fragments/substituents in QAC molecules and their activity is complicated because this factor is highly specific for some structures. Relatively speaking, pyridine QACs, especially bis-pyridine salts with broad antibacterial/antifungal activity, are the most advanced and promising among all heterocyclic QACs. Aromatic structures are often used in QACs due to their strong reactivity. They can be spacers, substituents, tails, head parts, etc.

99In 2016, in his report on antibacterial resistance, O’Neill predicted that by 2050, 10 million people would die because of resistant bacteria annually. Moreover, SARS-CoV-2 aggravated the issue. During the current pandemic, antibacterial drugs are being used rather indiscriminately. It should be expected that the threat from resistant bacteria will increase significantly in the next few years. To avert this danger, the next generation of antibacterial drugs, including QACs, should be developed in the near future.

100In this review, we analyze some of the structure–activity dependences and provide a general overview of the current situation in the research on antimicrobial QACs. In addition, a brief overview of the antimicrobial activities of various subclasses of ionic liquids, which are often considered advantageous antimicrobial agents, is also provided. We hope that it will serve as a highlight for future studies on these classes of biocides.