Biological Activities of 2-Mercaptobenzothiazole Derivatives: A Review

2-Mercaptobenzothiazoles are an important class of bioactive and industrially important organic compounds. These compounds are reported for their antimicrobial and antifungal activities, and are subsequently highlighted as a potent mechanism-based inhibitor of several enzymes like acyl coenzyme A cholesterol acyltransferase, monoamine oxidase, heat shock protein 90, cathepsin D, and c-Jun N-terminal kinases. These derivatives are also known to possess antitubercular, anti-inflammatory, antitumor, amoebic, antiparkinsonian, anthelmintic, antihypertensive, antihyperlipidemic, antiulcer, chemoprotective, and selective CCR3 receptor antagonist activity. This present review article focuses on the pharmacological profile of 2-mercaptobenzothiazoles with their potential activities.


Introduction
The main objective of organic and medicinal chemistry is the design, synthesis, and production of molecules having value as human therapeutic agents. During the past decade, heterocyclic structures received special attention as they belong to a class of compounds with proven utility in medicinal chemistry. There are numerous biologically active bicyclic molecules containing two hetero atoms. 2-Mercaptobenzothiazole (MBT) (1, Figure 1) is an important scaffold known to be associated with several biological activities, and its derivatives are manufactured worldwide for a wide variety of applications. S-acethydrazide hydrazone [1] and S-acyl [2] derivatives of MBT were reported to possess antifungal and antibacterial activities, and were also found to be useful in the leather industry [3]. 2-(Thiocyanomethylthio)benzothiazole [4] is a potential contact fungicide for several economically important crops such as barley, cotton, corn, and wheat. 2,2′-Dithiobisbenzothiazole is used as a fungicide, insecticide, sensitizer, and antiscorching agent in the vulcanization of rubber [5]. Structure of 2-mercaptobenzothiazole A number of methods for the synthesis of 2-mercaptobenzothiazoles (MBTs) have been reported. Among these, classical approaches involve the reaction of thiocarbanilide with sulfur or the interaction of o-aminothiophenol with carbon disulfide under high pressure [6][7][8][9][10][11][12][13]. Several groups reported synthesis of MBTs by the nucleophilic aromatic substitution reaction of a potassium/sodium o-ethyl dithiocarbonate with o-haloanilines followed by a subsequent cyclization [14][15][16][17][18][19]. Recently, two efficient approaches from 2haloaniline precursors were applied for the synthesis of MBTs. The first approach [20] involves a copper-catalyzed condensation reaction of the 2-iodoaniline with thiols in the presence of potassium carbonate. The second [21] involves the reaction of the o-haloanilines with carbon disulfide in the presence of 1,8-diazabicyclo [5.4.0]undec-7-ene (DBU).

Biologically active 2-mercaptobenzothiazoles
Antimicrobial activity 2-Mercaptobenzothiazoles exert adverse effects on viruses and also act on yeasts and fungi. The antiviral screening results of MBT showed significant activity against two out of three viruses tested [48]. The anti-Candida activity of MBT was studied [49] against 15 Candida strains and the results showed 50% growth inhibition at concentrations varying Sci Pharm. 2012; 80: 789-823 between one and 78 mg L −1 . The antifungal effects of MBT were also tested against Aspergillus niger with a suspension of spore-free mycelium homogenate as inoculum, and a 33 mg L −1 MBT concentration was the lower limit for 100% growth inhibition after five days of cultivation. Similar results, although obtained under other conditions, are described [50] for the fungus Trichophyton rubrum. It was observed that for complete growth inhibition of Microsporum gypseum and Epidermophyton floccosum, MBT concentration had to exceed 50 mg L −1 . The results of a study [51] suggested that the thiol group of MBT is essential for its toxicity, since benzothiazole (BT) was not an active fungicide. However, in another experiment [52] the presence of zinc destroyed the fungicidal activity of MBT, and this contradicts what was suggested above.
The antifungal activity of S-thiocyanomethyl derivatives 2 ( Figure 2) of MBT, which in turn were prepared by reacting a metal salt of MBT or substituted MBTs with chloromethyl thiocyanate in an alcohol solution, is described [53].
In a separate communication [68], an author disclosed the anti-candida activity of 3-(2-alkylsulfanyl-6-benzothiazolylaminomethyl)-2-benzothiazolethiones, however compounds were not stable enough when stored for a longer period of time. Replacement of one sulfur atom in the heterocyclic system by an oxygen atom solved the stability of products, and various benzoxazolethiones 6 ( Figure 3) were prepared [69,70] by the reaction of 2-alkylsulfanyl-6-aminobenzothiazoles with 3-hydroxymethyl-2-benzoxazolethione in ethanol. A derivative having a benzyl group at second position of the MBT ring showed maximum inhibition of the oxygen evolution rate in spinach chloroplasts. Photosynthetic-inhibiting activity of a novel series of 2-(6-acetamidobenzothiazolethio)acetic acid esters 7 ( Figure 3) was also reported [71]. Compounds were synthesized by acetylation of 2-(alkoxycarbonylmethylthio)-6-aminobenzothiazoles with acetic anhydride. Compounds having a hexyl acetate group on the second position of the MBT ring exhibited maximum inhibition (IC 50 Fig. 3.

Analogues of MBT that have shown inhibition of oxygen evolution in spinach chloroplasts
MBT also exerts adverse effects on bacteria and for this reason, the compound was under investigation as a potential nitrification inhibitor in soils [72]. Another example of the antibacterial activity of MBT is given [50] and it was considered to have a strong inhibitory effect on Mycobacterium tuberculosis [73][74][75]. Extensive studies [76] clearly indicated that MBT inhibited the growth of bacteria and yeast, but its effects were bacteriostatic rather than bactericidal, and were different for different species. MBT was investigated [77] as an inhibitor of dopamine β-hydroxylase, which converts dopamine into the neurotransmitter noradrenaline. In some reports, the effect of MBT on specific bacterial enzymes is also studied [78,79].
MBT derivatives have been pointed to as promising antibacterial agents. For example, the benzothiazol moiety linked by sulfur to the 3-position of carbapenems 8 ( Figure 4) displayed significant potency against methicillin-resistant Staphylococcus aureus (MRSA) [80], leading to the hypothesis that the 2-mercaptobenzothiazole moiety is a "binding element". It was observed that the introduction of a 1-β-methyl group at position one enhanced the human renal dehydropeptidase-1 (DHP-1) stability of compounds at least six-fold more compared to the 1-β-hydrogen analogues. Further, the introduction of a 1-β-methyl group appeared to enhance potency against MRSA.

Fig. 4.
Analogues of MBT linked to carbapenem and chroman-4-one with promising antibacterial activity A series of compounds 9 ( Figure 4) containing a MBT nucleus linked to the chroman-4-one moiety have been prepared and evaluated for their antimicrobial activities [81]. Derivatives bearing 6-Cl or 6,7-dimethyl substituents on the chroman-4-one moiety exhibited significant activity against the Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis, and against Mycobacterium tuberculosis. The same compounds showed potent activity against Candida albicans and Saccharomyces cerevisiae. MBT derivatives 10 ( Figure 5) possessing both 1,3,4-thiadiazole and 3-chloro-2-azetidinone moieties have also been reported to possess antibacterial activities [82,83]. Derivatives having phenyl or 4-chlorophenyl substituents at fourth position of the azetidinone nucleus were found to be the most potent compounds against both Gram-positive and Gram-negative bacterial strains.
Novel 4-substituted phenyl-3-chloro-1-[(benzothiazolylthio)acetamidyl]-2-azetidinones 11 ( Figure 5) have been synthesized by both conventional and microwave methods [84]. The synthesized compounds were screened for their antibacterial and antifungal activities by the paper disc diffusion method. Compounds having 4-OH-C 6 H 4 and 2-Cl-C 6 H 4 groups at fourth position of the azetidinone nucleus were most active (inhibition zone 16-22 mm) against the tested Gram-positive bacteria Staphylococcus aureus and fungus Aspergillus niger, whereas compounds possessing 3-OH-C 6 H 4 and 3-Cl-C 6 H 4 groups exhibited significant activity (inhibition zone 16-22 mm) against Candida albicans. In a separate communication, the synthesis and antimicrobial activities of 2-benzylsulfanyl derivatives 13 ( Figure 6) of MBT are also described. Compounds were synthesized [86] in 94-98% yields by the reaction of MBT and benzyl bromide in refluxing acetone, in the presence of K 2 CO 3 . Synthesized compounds were found to be either weakly active or inactive against Escherichia coli. All of the tested compounds showed no activity against Bacillus subtilis, Micrococcus luteus, and Pseudomonas aeruginosa. Tested compounds were also found to be either weakly active or inactive against Candida albicans and Aspergillus niger.

Structure of various congeners of MBT that have shown antibacterial property
A series of compounds 16 ( Figure 7) containing a MBT nucleus linked to the 1,3-thiazolidine-4-one moiety through a acetamido group have been synthesized by both microwave and conventional methods using ZnCl 2 as a catalyst. The synthesized compounds were screened for their in vitro antimicrobial activities [88]. Screening results revealed significant inhibitory activity (inhibition zone 20-25 mm) of derivatives having a 2-OCH 3 -C 6 H 4 group at second position of the thiazolidine ring against Escherichia coli, Candida albicans, and Candida parapsilosis, whereas derivatives bearing 4-NO 2 -C 6 H 4 , 2-OH-C 6 H 4 , 4-OH-C 6 H 4 , 4-OCH 3 -C 6 H 4 , 2-Cl-C 6 H 4, and 4-Cl-C 6 H 4 groups were found to have moderate activity (inhibition zone 15-20 mm) against Bacillus subtilis, Staphylococcus aureus, and Escherichia coli. The rest of the tested compounds were found to be either weakly active or inactive. On the other hand, derivatives bearing 4-NO 2 -C 6 H 4 , 4-OH-C 6 H 4 , 4-OCH 3 -C 6 H 4, and 4-Cl-C 6 H 4 groups also exhibited moderate inhibitory activity (inhibition zone 15-20 mm) against Candida albicans, Candida krusei, and Candida parapsilosis.

Anti-inflammatory activity
MBT is the important pharmacodynamic heterocyclic nucleus, which when incorporated in different heterocyclic templates has been reported to possess potent anti-inflammatory activity. For example, MBT derivatives possessing 4-oxothiazolidines 17 and their 5-arylidenes 18 ( Figure 8), showed moderate to weak anti-inflammatory activity in the carrageenan-induced paw edema in rats [89] at an oral dose of 50 mg kg −1 body weight. In a similar work, novel 1,3-thiazolidin-4-ones 19 and their 5-arylidenes 20 ( Figure 8) have been synthesized and evaluated for their antimicrobial activity by the paper disc diffusion method. Compounds were also evaluated for their anti-inflammatory activity by the carrageenan-induced paw edema in rats [90]. Authors disclosed significant antibacterial and antifungal activities for a few compounds, but no appreciable increase in the antiinflammatory activity was observed.
In attempt to improve the potency and selectivity towards the cyclooxygenase-2 (COX-2) enzyme, 2-{[2-alkoxy-6-pentadecylphenyl)methyl]thio]-1H-benzothiazoles 21 ( Figure 9) has been synthesized [91] by reaction of 2-alkoxy-6-pentadecylbenzyl alcohol with thionyl chloride followed by the condensation with MBT in the presence of tetrabutyl ammonium bromide. The synthesized compounds were screened for their human COX-2 inhibitory activity. The compound bearing a OCH 3 group at the second position of the phenyl ring was found to be 470-fold more selective towards COX-2 compared to COX-1.
Recently, novel bis-heterocycles 22 (Figure 9) encompassing 2-mercaptobenzothiazole and 1,2,3-triazoles were synthesized [92] by the cycloaddition reaction of 2-(prop-2ynylthio)benzo[d]thiazole with various azides. The synthesized compounds were evaluated for their anti-inflammatory activity by the carrageenan-induced hind paw edema in rats. The structure-activity relationship revealed that the aromatic ring attached to the triazollyl moiety is essential for potential activity when compared to aliphatic/alicyclic rings. Electron withdrawing groups, when substituted on an aromatic ring mostly at the para position, exhibited potent anti-inflammatory activity compared to the standard drug ibuprofen, without causing any ulceration. The COX-2 inhibitory potential of compounds having a 4-fluorophenyl group at first position of the triazole ring and ulcerogenic studies further conclude that these kinds of molecules can be considered as potent anti-inflammatory agents.
17  Figure 10) were synthesized and evaluated both in vivo and in vitro for their 5-lipoxygenase inhibitory activity [94]. Compounds were prepared by the sequential treatment of 3-(2-benzothiazolylthio)carboxylic acids with oxalyl chloride and the appropriate amine nucleophiles. It was observed that the hydroxyl group of the hydroxyurea must be unsubstituted in order to be active. N'-methyl substituents imparted superior activity in vivo, however, this correlation was not as pronounced in vitro. A three carbon chain was found to be optimum for activity. In general, the compounds were surprisingly sensitive to position and nature of the substituent R 1 on the benzo portion of the benzothiazole nucleus. The 5-chloro substituent present in the benzo portion markedly enhanced the activity both in vitro and in vivo, however, the 7-chloro analogue showed diminished activity in vitro and was inactive in vivo. Replacing the 5-chloro substituent with a 5-trifluoromethyl caused a > 40-fold loss of activity in vitro and virtually a total loss of activity in vivo. Other substitutions such as 6-ethoxy and 6-isopropyl also resulted in inferior activity relative to the unsubstituted analogues. In search of compounds that can selectively inhibit neutrophil activation by inflammatory mediators, compound 25 ( Figure 11) was identified [95] by means of high throughput, which inhibited the respiratory burst (RB) of human neutrophils in response to soluble inflammatory mediators, such as TNF and formylated methionyl-leucyl-phenylalanine (fMLF), but not in response to phobol myristate acetate (PMA), and did not suppress the antibacterial activity of neutrophils.   Figure 14) were found to be effective anthelmintics [100] when tested in mice infested with Hymenolepsis nana or Nematospiroides dubius, in rats infested with Fasciola hepatica, and in fowl infested with Ascaridia galli. The same compounds were also tested for their antimicrobial activities against different strains of Gram-positive and Gram-negative bacteria and against ten strains of fungi. Some of the tested compounds exhibited significant anthelmintic and antibacterial activities.
A series of novel, substituted phenoxyacetyl as well as propionyl-2-mercaptobenzothiazoles 30 ( Figure 14) were synthesized [101] by reaction of aryloxyacetyl chloride with an ice cold alkaline solution of MBT in acetone. Synthesized compounds were tested for their anthelmintic, analgesic, and antimicrobial activities. Compounds having 2,4,6trichloro or 2,4,6-tribromo groups in the phenyl ring and propionyl group as a linker between aryloxy and the MBT ring cleared 40-70% of worms in hamsters infested with Ancyclostoma ceylanicum and Nipostrongylus brasiliensis. The same compounds also showed significant analgesic activity using the tail flick method in rats. Some of the compounds, particularly di and trihalo derivatives, showed marked inhibitory activity (MIC 1-2.5 µg mL −1 ) against Bacillus anthracis and Candida albicans.

Miscellaneous
With the aim of developing novel compounds with improved potential for treating hypertension, propanolamine analogues of MBT 33 ( Figure 15) were investigated [104]. Compounds were synthesized by the reaction of aminoalchohol with MBT, or substituted MBT in organic solvent in the presence of base below 30 °C, under nitrogen. Most of the compounds exhibited significant prolonged antihypertensive effect without noticeable β-adrenergic receptor blocking activity.
In the year 1973, carbonic anhydrase inhibitory activity of a number of aryl-substituted benzothiazole-2-sulfonamides 34 (Figure 16) was described [105]. All of the tested compounds showed potent carbonic anhydrase inhibitory activity and one of these, the 6-ethoxybenzothiazole-2-sulfonamide, produced a clinically useful diuresis. In another report, synthesis of analogues of benzothiazole-2-sulfonamides 35 (Figure 16) as carbonic anhydrase inhibitors was described [106]. Compounds were tested topically for their ability to reduce intraocular eye pressure in glaucoma. Among these, 6-chlorobenzothiazole-2sulfonamide was found to be highly effective.  Analogues of MBT 36 ( Figure 17) containing a substituted hydroxypropyl carbamate group at the second position have been synthesized [107] and evaluated for the inhibition of transient lower esophageal sphincter relaxations, and for the treatment of gastroesophageal reflux disease, and found to have high affinity and potency for the GABA B receptors as revealed by low IC 50 and EC 50 in the 3 H GABA radioligand binding and ileum assay, respectively.
In another study, MBT analogues 37 and 38 ( Figure 18) have been synthesized and evaluated [108] for their ability to displace radiolabeled rosiglitazone (BRL 49653) from a peroxisome proliferator-activated receptor glutathione-S-transferase (PPARγ-GST) fusion protein. Compounds were identified as PPARγ activators and found to be useful agents for the treatment of obesity and related disorders associated with undesirable adipocyte maturation.  Figure 18) were synthesized and evaluated for their human PPARγ transactivation activity [109]. Overall, the potencies of some newly designed agonists were slightly higher than those of typical fibrates, such as clofibrate. While unsubstituted derivatives proved inactive, the overall effect of the introduction of substituents in the 5 and 6 positions improved PPARα agonistic activity. Among the synthesized compounds, the 5-bromo derivative (R 1 = Br, R 2 = H, R 3 and R 4 = CH 3 ) with an EC 50 value of 2.5 μmol was found to be 10-20 times more effective than other compounds of the same series.
In search of an orally active non-peptide antagonist selective for the CCR3 receptors, the 2-(benzothiazolylthio)acetamide derivative 40 (R = R 1 = R 2 =H, Figure 19) was identified as the leading compound which on further derivatization led to the identification of potent and selective antagonists [110,111]. The 7-acetamidobenzothiazole derivative 40 (R = -NHCOCH 3 , R 1 = R 2 = Cl) with an IC 50 of 1.5 nM and a 3600-fold selectivity over that of the CCR1 receptor was found to be the best compound of this series.   Figure 20) containing a MBT nucleus linked at the second position to an arylpiperazine by different alkyl chains, were prepared [112] by reacting the appropriate 1-ω-chloroalkyl-4-arylpiperazine with MBT or 6-chloro-2-mercaptobenzothiazole in acetone at reflux, in the presence of potassium carbonate and potassium iodide. They were tested in radioligand binding experiments to evaluate their affinity for 5-HT 1A and 5-HT 2A receptors. Compounds possessing a 2-nitrophenylpiperazine group showed lower affinity for the 5-HT 1A receptor with respect to the 2-methoxyphenylpiperazine analogues. In addition, compounds characterized by a propyl side chain between the terminal fragment and the arylpiperazine portion exhibited higher affinity toward 5-HT 1A with respect to the analogues containing an ethyl chain as linker. In particular, among propyl derivatives, compounds possessing 2-methoxyphenylpiperazine showed an affinity for the 5-HT 1A receptor (R) in the subnanomolar range (K i 0.29 nM), coupled to a high selectivity over α 1 -adrenergic receptors (K i α 1 -adrenergic R/K i 5-HT 1A R=114). Recently, another group of researchers investigated the binding affinities of these compounds 41 to 5-HT 1A and α 1 -adrenergic receptors in terms of structural requirements [113]. The binding affinity was found to be the function of the cumulative effect of different structural features which were identified in terms of individual descriptors. The synthesis was carried out by reaction of a carboxylic acid or its reactive derivative with an aniline derivative to give an amide derivative, which on further treatment with MBT gave anilides 42. Authors disclosed acyl coenzyme A cholesterol acyltransferase (ACAT) inhibition activity in rabbits, which is therefore useful as an anti-hyperlipidemic agent [114,115]. Apart from acyl coenzyme A cholesterol acyltransferase (ACAT) inhibition activity, serum cholesterol and triglyceride lowering activities are also reported [116] to be associated with MBT derivatives 43 ( Figure 21). A shift in the ratio of α-lipoproteins and β-lipoproteins in the direction of increasing α-lipoproteins was also observed. The chemoprotection of thymocyte apoptosis induced by dexamethasone and γ-irradiation is described [121] for the pifithrin-α analogue 2-(1,3-benzothiazol-2-ylsulfanyl)-1-(4-methylphenyl)ethanone (48 Figure 25), which in turn was prepared by reaction of 2-bromo-4'methylacetophenone with MBT, resulting exclusively in the S-alkylation product. Compound 48 showed cytoprotective activity, with an average of 16% cells remaining when challenged with dexamethasone.  The discovery of benzothiazolothiopurines 49 ( Figure 26) as potent heat shock protein 90 (Hsp90) inhibitors is an important development. Authors described the structure-activity relationship [122]. The benzothiazole moiety was found to be exceptionally sensitive to substitutions on the aromatic ring with a 7′-substituent essential for activity. Some of these compounds exhibited low nanomolar inhibition activity in a Her-2 degradation assay Cathepsin D, a lysosomal aspartyl protease, has been implicated in the pathology of Alzheimer's disease as well as breast and ovarian cancer. Impressed by these facts, MBT analogues 50 ( Figure 27) were synthesized and screened as a cathepsin D inhibitor [123]. It was observed that the heteroatom linker between the two rings can be either sulfur or oxygen, while substitution of the middle ring resulted in a slight increase in activity when a lipophilic substituent (chlorine, methyl, trifluoromethyl) is added ortho to the heteroatom linker. The overall potency of these analogues seems on track with the lipophilicity of the side-chain.   Figure 29) through systematic structural modifications of clofibric acid and evaluated them for human PPARα transactivation activity, with the aim of obtaining new hypolipidemic compounds [125]. Overall, the potencies of some newly designed agonists were slightly higher than those of typical fibrates, such as clofibrate. While unsubstituted derivatives proved inactive, the overall effect of the introduction of substituents in the five and six positions improved PPARα agonistic activity. Among the series, the 5-bromine derivative (R 1 = Br, R 2 = H, R 3 and R 4 = CH 3 ) with an EC 50 value of 2.5 μM was found to be 10-20 times more effective than other compounds of the same series.
In continuation of the above work, N-(phenylsulfonyl)amides 53 ( Figure 29) containing the MBT scaffold were synthesized [126] by structural modification of clofibric acid. Synthesis involves direct condensation of carboxylic acid with benzensulfonamide in the presence of 1-ethyl-3-[3-dimethylaminopropyl]carbodiimide hydrochloride and 4-dimethylaminopyridine. Phenylsulfonamides were evaluated in vitro against the agonistic effect of GW7647; they showed an inhibitory effect on PPARα activation, with the best compounds revealing a dose-dependent antagonistic profile. Among compounds bearing an alkyl chain in α-position to the carboxylic group, the antagonistic activity seems to improve shifting from n-propylic derivatives (IC 50 31.0 µM and IC 50 22.2 µM) to the n-butylic derivative (IC 50 19.9 µM). Further elongation of the substituent gave rise to the agonistic activity, whereas the introduction of a branched chain (i-pro) slightly decreased the antagonistic activity (IC 50 27.9 µM). Finally, when the alkyl chain was replaced with α-phenyl group, this resulted in increased antagonistic activity (IC 50 6.5 µM). The different substitution pattern of the benzothiazole system did not significantly affect the activity. The synthesis and c-Jun N-terminal kinase (JNK) inhibition activity of a novel series of 2-thioether-benzothiazoles 54 and 55 ( Figure 30) is described in literature [127]. Compounds were synthesized by nucleophilic substitution of 2-bromo-5-nitrothiazole with the corresponding thiol of benzothiazole in the presence of sodium ethoxide in methanol at room temperature. The compound bearing a 2-nitrothiazole moiety at the second position of the MBT nucleus showed a promising result with an IC 50 of 1.8 and 0.16 µmol in the Lantha screen kinase and pepJIPI DELFIA displacement assays, respectively. The activity was similar with or without methoxy, however when a chloro or ethoxy was present at the five or six position, compounds were inactive, which was likely due to steric hindrance.  2-Mercaptobenzothiazoles have been widely explored for industrial applications since their discovery. However, the biological activity of this class of compounds deserves further investigation. This becomes clear when microbial infections are considered. Although the research on this subject is incipient, the number of reports disclosing the effects of MBTs on pathogens of clinical interest has recently been increasing. 2-Mercaptobenzothiazole compounds have been shown to be promising, which calls for the design of more efficient antimicrobial, anthelmintic, anti-inflammatory, and anti-allergic agents. Future studies will undoubtedly uncover unexpected properties and applications. Advances in this field will require analyses of the structure-activity relationships of MBTs, as well as the mechanisms of action of these compounds.