Carbazole Derivatives as Potential Antimicrobial Agents

Microbial infection is a leading cause of death worldwide, resulting in around 1.2 million deaths annually. Due to this, medicinal chemists are continuously searching for new or improved alternatives to combat microbial infections. Among many nitrogen-containing heterocycles, carbazole derivatives have shown significant biological activities, of which its antimicrobial and antifungal activities are the most studied. In this review, miscellaneous carbazole derivatives and their antimicrobial activity are discussed (articles published from 1999 to 2022).


Introduction
Drug resistance due to overpopulation, increased use, and various other reasons is one of the key factors for most deaths caused by microbial diseases [1,2]. The World Health Organization (WHO) recently estimated that global death caused by antimicrobial-resistant pathogens will reach 10 million people per year by 2050 [3]. There are several critical factors that are responsible for drug resistance, such as a high rate of mutations, ineffectiveness towards available clinical agents, lack of awareness about the overuse of antibiotic drugs that are available over the counter, and, more significantly, the non-availability of newer drugs due to the lack of drug discovery and development strategies; these factors are responsible for drug resistance in microbial diseases [4,5]. Currently, clinically effective treatments with molecular pharmacophores such as macrolides, quinolones, tetracycline, glycopeptide, and cephalosporins have encountered tremendous challenges, including increased resistance due to the above-mentioned issues [6][7][8][9][10][11][12][13][14]. Microorganisms in areas of high drug use, such as hospitals, for example, continue to mutate rapidly and can spread among patients, becoming more resistant to current treatments [10]. These resistant microorganisms will continue to pose unforeseen health problems globally. Drug resistance is continually increasing due to many factors and does not appear to be slowing, making this issue of the utmost importance. Therefore, the antimicrobial research community must continually work together to design and develop new and safer small molecule drugs to overcome amassing drug resistance along with toxicity problems. The medicinal chemistry community works systematically to identify novel pharmacophores or new molecular entities (NMEs) to overcome these problems. Then, researchers combine medicinal, computational, and synthetic chemistry knowledge along with drug resistance to successfully develop efficacious new drug candidates with novel pharmacophoric functionalities.
Carbazole is one of the privileged nitrogen-containing heterocyclic pharmacophores in medicinal chemistry due to its attractive drug-like properties and outstanding biological activities. Carbazole (I) was first isolated by Graebe and Glazer in 1872 from coal tar ( Figure 1) [15]. The carbazole analog, murrayanine (1-methoxy-3-formylcarbazole) (II), was Therapeutically important natural carbazoles have inspired s chemists to design and develop novel (semi)synthetic carbazole derivat moiety is present in several important commercially available drug m lipticine (III), alectinib (IV), midostaurin (V), carvedilol (VI), carazol (VIII), and frovatriptan (IX) (Figure 2). The naturally occurring alkal was first discovered in 1959 as an anticancer agent. It was extracted Ochrosia elliptica and is considered to be one of the initial leading the analogs for the treatment of cancer [21]. Alectinib (IV) was approved for the treatment of advanced non-small cell lung cancer (NSCLC) [22 was permitted by the FDA in 2017 for the treatment of newly diagno leukemia (AML) and for advanced systemic mastocytosis (SM) [23,24]  Therapeutically important natural carbazoles have inspired synthetic medicinal chemists to design and develop novel (semi)synthetic carbazole derivatives. The carbazole moiety is present in several important commercially available drug molecules such as ellipticine (III), alectinib (IV), midostaurin (V), carvedilol (VI), carazolol (VII), carprofen (VIII), and frovatriptan (IX) (Figure 2). The naturally occurring alkaloid ellipticine (III) was first discovered in 1959 as an anticancer agent. It was extracted from the leaves of Ochrosia elliptica and is considered to be one of the initial leading therapeutic carbazole analogs for the treatment of cancer [21]. Alectinib (IV) was approved by the FDA in 2015 for the treatment of advanced non-small cell lung cancer (NSCLC) [22]. Midostaurin (V) was permitted by the FDA in 2017 for the treatment of newly diagnosed acute myeloid leukemia (AML) and for advanced systemic mastocytosis (SM) [23,24]. Carvedilol (VI) was approved in 1995 to treat high blood pressure and congestive heart failure (CHF) [25]. Carazolol (VII) is a truncated analog of carvedilol and was approved to treat cardiovascular disorders in non-human mammals [26]. Carprofen (VIII) is a non-steroidal anti-inflammatory drug (NSAID) and has been used to relieve pain and inflammation in animals [27]. The 5-hydroxytryptamine (5-HT) receptor agonist, frovatriptan (IX), has been approved for the acute treatment of moderate to severe migraines in humans [28]. Even though there exist several carbazole containing-drugs that Carvedilol (VI) was approved in 1995 to treat high blood pressure and congestive heart failure (CHF) [25]. Carazolol (VII) is a truncated analog of carvedilol and was approved to treat cardiovascular disorders in non-human mammals [26]. Carprofen (VIII) is a non-steroidal anti-inflammatory drug (NSAID) and has been used to relieve pain and inflammation in animals [27]. The 5-hydroxytryptamine (5-HT) receptor agonist, frovatriptan (IX), has been approved for the acute treatment of moderate to severe migraines in humans [28]. Even though there exist several carbazole containing-drugs that have been approved to treat various diseases, their full biological potential has yet to be exploited, and therefore, breakthroughs remain to be discovered. This has generated renewed interest in discovering and developing new carbazole analogs with improved efficacy and reduced toxicity for various diseases, including microbial and fungal diseases. The present review discusses the synthesis and biological activities of carbazole analogs as antimicrobial agents.

Antibacterial and Antifungal Activities of Carbazole Derivatives
Compounds containing the carbazole moiety are a favorable class of agents against numerous diseases and are conceivably useful in clinical studies. In 1999, Nair et al. separated carbazole alkaloids (1 and 2a-b) ( Figure 3) from Murraya koenigii leaves. Carbazole alkaloid 1 demonstrated high activity towards C. kruseii, C. parapsilasis, E. coli, S. aureus, and S. pyogenes species, while carbazole alkaloid 2a exhibited low activity against these test species. Carbazole alkaloids 2b and 1 gave minimum inhibitory concentration (MIC) against S. aureus, and S. pyogenes at 25 µg/mL [29]. In 2003, Prasad et al. reported the one-pot preparation and evaluation of carbazole derivatives (3a-e) ( Figure 3) as antimicrobial agents. The chloro-substituted derivative 3d exhibited outstanding antimicrobial activity against E. coli, S. aureus, P. aeruginosa, and B. subtilis, whereas carbazole derivatives 3a and 3c displayed slightly lower activity against all the bacterial species tested. Carbazole derivative 3d demonstrated excellent antifungal activity towards all the fungi screened in comparison with the standard drug carbendazim. This high activity might be due to the presence of chloro and N-oxide groups in the compound [30]. One year later, in 2004, Prasad et al. also reported the antimicrobial activities of isoxazolo-and pyrazolinoannulated carbazoles (4a-e and 5a-e) ( Figure 3) against certain pathogenic bacterial and fungal species. The antimicrobial activity of all the carbazole derivatives (4a-e and 5a-e) has been compared with the standard fungicide bavistin and the bactericide streptomycin. Almost all the carbazole derivatives (4a-e and 5a-e) demonstrated exceptional activity against both the organisms of bacteria and fungi [31]. In another report, Prasad et al. also investigated the synthesis and characterization of carbazole derivatives (6a-d) ( Figure 3). All the compounds (6a-d) were screened for their in vitro antimicrobial activities towards A. hydrophila, P. aeruginosa, B. subtilis, A. niger, and Fusarium species through the disc diffusion method. Among the compounds screened, 6d was found to have higher activity compared to the remaining compounds [32]. Extraction, characterization, and antimicrobial activity of carbazole alkaloid (7) (Figure 3) from Murraya koenigii was explored from Rahman et al. The structure of the carbazole alkaloid (7) was confirmed by various spectroscopic techniques. The carbazole alkaloid (7) was then tested for antimicrobial activities by a microdilution technique and exhibited strong antimicrobial activity towards E. coli, P. vulgaris, A. niger, and C. albicans species [33].
Antimicrobial properties of another series of carbazole derivatives (8a-g and 9a-g) ( Figure 3) were described by Surendiran et al. at a concentration of 25 µg/mL using ciprofloxacin and ketoconazole as a standard. Carbazole derivatives (8a), (8d), (9c), and (9d), having methoxy groups and chloro groups in heterocyclic moieties, demonstrated moderate to good antibacterial activities. Carbazole derivatives (8e) and (9e), having acetyloxy and methoxy groups, displayed more pronounced activities than the other tested compounds. Compounds (9c) and (9d) exhibited excellent antifungal activity against F. porum and A. macrospore species, whereas compounds (8b) and (9b) displayed moderate activity [34].    Macrocyclic compounds have also received significant attention due to their biological activities. Rajakumar et al. evaluated the antimicrobial activity of a series of sulfur and oxygen linkages containing carbazole-based macrocyclic diamides (10a-f) ( Figure 3). The MIC of carbazole amides (10a-f) towards bacterial species was determined to have a concentration range between 5 and 80 µg/mL. The macrocyclic diamide 10f displayed significant antibacterial activity towards all the screened bacterial species when compared to the rest of the macrocyclic diamides and standard antibiotic tetracycline at low concentrations ranging from 5 to 10 µg/mL. In contrast, MICs of the macrocyclic diamides (10a-f) towards fungal species were determined to have a concentration range between 10 and 45 µg/mL. Among the macrocyclic diamides (10a-f) screened, macrocyclic diamide 10f considerably inhibited all four of the fungal species with a concentration range between 10 to 20 µg/mL when compared to the rest of the macrocyclic diamides and standard fungicide, carbendazim [35]. Gu et al. reported the preparation and antimicrobial activities of novel 1H-dibenzo[a,c]carbazoles by dehydroabietic acid. All the prepared carbazoles (11a-m) ( Figure 4) were examined for their activity against four bacteria and three fungal species. Among the screened carbazoles, 11d-f and 11m revealed strong (1.9-7.8 µg/mL) antibacterial activities, whereas carbazoles 11e and 11m displayed moderate antifungal activities. Principally, 11d demonstrated stronger (1.9 µg/mL) antibacterial activity towards B. subtilis when compared to the current standard drug amikacin [36]. In modern drug synthesis, an essential constituent of the search for new leads is the preparation of the library of new molecules. Synthesis of new carbazole derivatives and an evaluation of their antimicrobial activity were investigated by Kaplancikli et al. Antimicrobial activities of all the compounds were established using the microbroth dilution method with chloramphenicol and ketoconazole as the standard drug. Almost all compounds (12a-i) ( Figure 4) were effective towards C. albicans. When compared with ketoconazole, 12c and 12d in particular displayed similar activity, and 12a, 12b, 12e, 12f revealed moderate activity towards C. albicans. Comparable results were obtained from C. glabrata. The antibacterial activity revealed that carbazole derivatives (12a-i) exhibited moderate or slight activity with MIC values within the range of 100->400 µg/mL towards all the bacterial species. By comparing their MIC values with the standard drug chloramphenicol, the carbazole derivatives (12a-i) were less active towards E. coli and S. aureus. Conversely, the carbazole derivatives (12a-i) exhibited comparable or better activities towards P. aeruginosa than did chloramphenicol [37]. Bandgar et al. explored the antimicrobial activities of a series of novel carbazole chalcones (13a-o) ( Figure 4). Carbazole chalcones 13a, 13e and 13m revealed good antifungal activity towards all three fungal growths. Compounds 13b, 13g and 13h exhibited good antibacterial activity against P. vulgaris and E. coli selectively, and compounds 13c and 13o were active against S. aureus. Additionally, the antifungal screening data revealed that compounds 13h and 13m exhibited good antifungal activity, while the remaining compounds were inactive towards C. albicans [38]. Synthesis and spectral characterization of sulfonamide and carbamate derivatives of 4-(oxiran-2-ylmethoxy)-9Hcarbazole (14a-f and 15a-d) (Figure 4) were described by Raju et al. All the synthesized compounds (14a-f and 15a-d) were investigated regarding their in vitro antibacterial (S. aureus, B. subtilis, and E. coli) and antifungal (F. oxysporum, C. albicans, and A. niger) activities through the agar well diffusion technique. All the compounds (14a-f and 15a-d) revealed moderate to strong antimicrobial activities at a concentration of 200 µg/mL, and the results were comparable to the standard drugs ciprofloxacin and fluconazole [39].   The pyrimidine moiety is one of the most noticeable structures found in the nucleic acid. In 2012, Bandgar et al. examined the antimicrobial activity of a series of new carbazole substituted aminopyrimidines (16a-p) (Figure 4) using the disk diffusion method. Carbazole derivatives 16c, 16g, 16j, and 16o displayed modest activity towards all the selected bacterial species at a concentration of 1 mg/mL as compared to the standard drug tetracycline. Carbazole derivative 16o exhibited comparable activity to that of the standard and towards B. subtilis, S. aureus and S. flexenari. On the other hand, compounds 16b, 16c, 16m, and 16o exhibited decent activity against certain fungal organisms at a concentration of 1 mg/mL as compared to the standard drug nystatin. Carbazole derivatives 16m and 16o displayed comparable activity to that of the standard and towards C. albicans and A. niger [40].
Several heteroannulated carbazole compounds have drawn great attention due to their natural occurrence and their comprehensive spectrum of antimicrobial activity.  (20a-h, 21a-h, and 22a-h) (Figure 5) of ursolic acid. All the prepared carbazole derivatives were assessed for their antimicrobial activity towards four bacterial and three fungal strains utilizing the serial dilution method [44]. Carbazole derivatives 20a-f, 21a, 21b, 22a, and 22b demonstrated substantial antibacterial activity towards at least one of the tested bacteria with MIC values of 3.9-15.6 µg/mL. Specifically, carbazole derivative 20b demonstrated pronounced antibacterial activity against B. subtilis with an MIC value of 3.9 µg/mL, near that of the positive control. Furthermore, the activities of these and other carbazole derivatives were closely pertinent to their structural properties [45,46]. Ochung et al. isolated three carbazole alkaloids, koenidine (23), koenimbine (24) and mohanimbine (25), from the stem bark of Alysicarpus ovalifolius through different solvents such as n-hexane, CH 2 Cl 2 , and MeOH, respectively [47]. All three of the isolated compounds (23-25) ( Figure 5) were subjected to in vitro antimicrobial activities against yeast-like and filamentous fungi as well as some Gram-positive and Gram-negative bacteria utilizing the disc diffusion method. Dichloromethane extract showed the strongest activity towards C. albicans and S. aureus with zones of inhibition 13.2 ± 0.1 and 15.3 ± 0.1 mm in diameter, respectively, compared to the standard drugs fluconazole and amoxicillin, with zones of inhibition of 17.3 ± 0.2 and 19.5 ± 0.1 mm. Particularly, compound (25) showed the strongest inhibition towards C. albicans and S. aureus with zones of inhibition of 14.5 ± 0.1 and 13.8 ± 0.1 mm, respectively.    In 2016, Ashok et al. reported a microwave-assisted synthesis of 27 different carbazole derivatives (9 chalcones (26a-i), 9 aurones (27a-i), and 9 flavones (28a-i)) ( Figure 6). This library of compounds was subjected to in vitro biological studies. Two Gram-positive bacterial strains, S. aureus and B. subtilis, two Gram-negative strains, K. pneumonia and E. coli, and three fungal strains, F. oxysporum, A. niger, and A. flavus, were assessed. Although all compounds showed moderate activity against all compounds tested, none of them exhib-ited higher activity than the standards used: ciprofloxacin (for bacteria) and amphotericin-B and clotrimazole (for fungi) [48]. Additionally, in 2016, Addla et al. reported two large sets of (N)-alkyl carbazole derivatives (29a-j and 30a-j). As depicted in Figure 6, compounds 29a-j are alpha-chloro ketone derivatives, while adducts 30a-j are aminothiazoles. All compounds were evaluated against four Gram-positive bacteria (S. aureus (ATCC259223 and N315), B. subtilis, and M. luteus), four Gram-negative bacteria (P. aeruginosa, E. typhosa, B. proteus, and E. coli), and five fungal strains (C. utilis, A. favus, B. yeast, C. albicans, and C. mycoderma) using the standard two-fold serial dilution method [49]. After screening, only compound 30f showed superior growth inhibition against MRSA (N315) with an MIC value of 4 µg/mL, whereas the reference drug chloromycin gave a MIC value of 64 µg/mL, and norfloxacin gave 8 µg/mL. Similarly, Narayana et al. reported a neat synthesis and antimicrobial activity of 10 new pyrano [3,2-c]carbazole derivatives (31a-e, and 32a-e) [50]. In addition to the antiproliferative activity of these 10 adducts, the MIC of each product was obtained for three Gram-positive bacteria, B. subtilis, S. aureus, and S. epidermidis, and three Gram-negative bacteria, P. aeruginosa, K. pneumonia, and E. coli, with penicillin and streptomycin used as the standard drugs. Most of the compounds showed poor antibacterial activity, with only a couple showing moderate to good activity against P. aeruginosa and S. aureus. Carbazole 32b exhibited good activity against P. aeruginosa with an MIC value of 9.37 µg/mL, which is superior in comparison to penicillin (MIC = 12.5 µg/ML) [50]. Winther et al. synthesized 15 N-substituted carbazole derivatives (33a-d, 34a-d, and 35a-g), as depicted in Figure 6. As others have done, the new adducts were screened against seven fungal strains, S. cerevisiae, C. albicans, C. krusei, C. glabrata (ATCC 90030), C. glabrata (Cg003), A. flavus, and A. fumigatus [51]. The fungal growth inhibition MIC was reported using amphotericin-B as the standard drug. Of the compounds evaluated, adduct 33d was unquestionably the most potent antifungal carbazole derivative, with MIC values ranging from 1.2 µM (S. cerevisiae) to 12 µM (A. flavus), which was comparable to that detected by amphotericin-B at 1 µM. In 2018, Farghaly et al. prepared several thiazole-containing carbazoles through microwave-assisted synthesis [52]. The synthesis started from 9-ethyl-9H-carbazole-3-carbaldehyde and thiosemicarbazide to produce intermediate 36, which was then reacted with different hydrazonyl chlorides to afford carbazole derivatives 37a-f and 38a-e ( Figure 6). All the compounds were screened against four Gram-positive bacteria, i.e., B. subtilis, S. aureus, S. epidermidis, and S. pyogenes, three Gram-negative bacteria, namely, P. aeruginosa, K. pneumonia, and S. typhimurium, and two fungi, A. niger and G. candidum. The antimicrobial data revealed that compound 38b was the most reactive across the board. For instance, the MIC value (0.49 µg/mL) for compound 38b against A. niger was superior to the standard drug amphotericin B (MIC = 0.98 µg/mL). Figure 6 also includes a set of carbazole derivatives reported by Zhang et al. Compounds 39a-g, 40a-f, and 41-42 were prepared following a 3-step synthetic protocol starting with commercially available carbazoles and azoles [53]. All adducts were screened against five Gram-positive bacteria (S. aureus, methicillin-resistant S. aureus, E. faecalis, S. aureus (ATCC 25923), and S. aureus (ATCC 29213)), six Gram-negative bacteria (K. pneumonia, E. coli, A. baumannii, P. aeruginosa, P. aeruginosa (ATCC 27853), and E. coli (ATCC 25922)), and five fungal strains (C. albicans, C. tropicalis, A. fumigatus, C. albicans (ATCC 90023), and C. parapsilosis (ATCC 22019)). All adducts showed weak to modest activity again all the tested microorganisms, with the exception of compound 39f. This compound was shown to inhibit the growth of In 2020, Karpoormath et al. reported a relatively large library of carbazole hybrids (43a-p, 44a-l, and 45a-f) (Figure 7). The new compounds were evaluated for their antimicrobial activity against four bacteria (S. aureus, B. subtilis, E. coli, and P. aeruginosa) and four fungal (C. albicans, C. neoformans, C. tropicalis, and A. niger) strains [54]. Among the evaluated adducts, 44g was the most active compound with an MIC value of 3.125 µg/mL against C. neoformans and 1.56 µg/mL against S. aureus. The same year, Baycan prepared three carbazole-fluorene polymers (46a-c). In addition to the standard studies for a polymer (optical, electrochemical, and thermal properties), together with the surface morphology, a small antimicrobial study was reported, this time using three bacteria (S. aureus (ATCC 6538), E. coli (ATCC 1301), and E. coli (ATCC 25922)) and one fungal (C. albicans (ATCC 10231)) strain. Gentamicin and cycloheximide were used as the positive controls for bacterial and fungal tests, respectively. Unfortunately, due to poor solubility, weak antimicrobial activity was observed [55]. Liu et al. synthesized a collection containing 39 compounds consisting of N-alkylated carbazoles with substitutions on position 4 of the carbazole (47a-g, 48a-f, 49a-r, 50a-d, and 51a-d) as depicted in Figure 7 [56]. With these adducts, an antibacterial study was performed using three S. aureus strains (ATCC 29213, MRSA N315, and MRSA NCTC10442). Of all compounds tested, guanidine-containing carbazole derivative 49p arose as the lead compound. It exhibited excellent antibacterial activity with MIC values in the range of 0.78-1.56 µg/mL, comparable to that of the standard drug vancomycin.

Conclusions
This review condenses the known reports of various carbazole derivatives and their antimicrobial activities, which are attractive structural motifs in synthetic organic

Conclusions
This review condenses the known reports of various carbazole derivatives and their antimicrobial activities, which are attractive structural motifs in synthetic organic chemistry because of their tunable electronic and steric properties. As summarized above, the presence of carbazole moieties has proven effective in enhancing the antimicrobial activity of numerous compounds. Some carbazole derivatives exhibited strong in vitro inhibitory activity towards bacteria with comparable or even superior activity when compared to the positive control drugs. Therefore, it is believed that the handy literature regarding carbazole derivatives and their associated antimicrobial activities would be greatly useful in the designing of novel, potent, and safe carbazole derivatives against microbial diseases in the near future.