Updated Information on Antimicrobial Activity of Hydrazide–Hydrazones

Hydrazide–hydrazones possess a wide spectrum of bioactivity, including antibacterial, antitubercular, antifungal, anticancer, anti-inflammatory, anticonvulsant, antidepressant, antiviral, and antiprotozoal properties. This review is focused on the latest scientific reports regarding antibacterial, antimycobacterial, and antifungal activities of hydrazide–hydrazones published between 2017 and 2021. The molecules and their chemical structures presented in this article are the most active derivatives, with discussed activities having a hydrazide–hydrazone moiety as the main scaffold or as a side chain. Presented information constitute a concise summary, which may be used as a practical guide for further design of new molecules with antimicrobial activity.

Among bioactivity profiles of hydrazide-hydrazones, antimicrobial properties are the most common in the scientific literature . This is especially important due to the fact that bacterial and fungal infections became more and more difficult and sometimes impossible to treat as a result of the increase of antibiotic and chemotherapeutic resistant strains [47]. It is worth mentioning that hydrazide-hydrazone moiety is also present in the chemical structure of medicines with antimicrobial activity, such as nitrofurazone, furazolidone, or nitrofurantoin [3].
This review is an update and continuation of the review, which was previously published in 2017 [3], and focuses on the most recently described (2017-2021) potent hydrazidehydrazones with applications as antibacterial, antimycobacterial, and antifungal agents.

Antibacterial Activity
The most frequently encountered in the scientific literature are hydrazide-hydrazones which possess antibacterial activity. Searching for such compounds among this group seems reasonable due to the fact that many derivatives of this class are highly active even against resistant strains; this is especially important nowadays when many bacteria become resistant to commonly used medicines .
Noshiranzadeh et al. conducted the synthesis of a series of new hydrazide-hydrazones of lactic acid and evaluated their antibacterial activity against four bacterial strains (S. aureus, S. pneumoniae, E. coli, and P. aeruginosa) using the broth microdilution method. Compounds 1 and 2 showed higher antibacterial activity (Minimal inhibitory concentration MIC = 64-128 µg/mL) than the other compounds but lower than gentamicin used as a reference substance (Figure 1). The high antibacterial activity of compound 1 is possibly attributed to the presence of an electronegative NO 2 substituent. The authors concluded that the compounds with electron-withdrawing groups like I, Br, or NO 2 generally showed better antibacterial activity in comparison with the compounds containing electron-donating OCH 3 or OH groups [11]. Heidari et al. published the study which aimed to investigate the effects of subminimum inhibitory concentrations of compound 1 against Pseudomonas aeruginosa PAO1 quorum sensing related virulence factors ( Figure 1). Treated PAO1 cultures in the presence of this hydrazide-hydrazone at subinhibitory concentrations showed significant inhibition of virulence factors, including motility, biofilm formation, alginate and pyocyanin production, and susceptibility to H 2 O 2 (p <0.001). The authors suggested that such action may be the mechanism of activity of this compound against Pseudomonas aeruginosa PAO1 [12].
Olayinka et al. synthesized a series of new hydrazide-hydrazones of 2-propylquinoline-4-carboxylic acid and carried out antibacterial activity screening towards six bacterial strains (P. aeruginosa, S. aureus, E. coli, Proteus vulgaris, Bacillus licheniformis, and Micrococcus varians) using the agar diffusion method. Compound 3 was the hydrazide-hydrazone with the lowest MIC value in the range of 0.39 ± 0.02-1.56 ± 0.02 µg/mL across all the microorganisms screened (Table 1, Figure 2). Authors proved that the presence of electrondonating group (EDG) at position 4 and electron-withdrawing group (EWG) at position 2 in the phenyl ring had a crucial effect on the antibacterial activity [13].  Krátký et al. synthesized a series of new hydrazide-hydrazones of 4-trifluoromethylbenzoic acid and evaluated them as possible antibacterial agents. The majority of obtained hydrazide-hydrazones were only slightly active. The highest activity superior to bacitracin (BAC), used as a reference substance, was shown by compound 4 (Table 2, Figure 3). This substance did not show cytotoxicity towards HepG2 cells (hepatocellular carcinoma cells) and BMMΦ (murine bone marrow culture-derived macrophages) (IC 50 >100 µM) [14].  Abdelrahman et al. synthesized novel hydrazide-hydrazones and evaluated in vitro their antibacterial properties against two Gram-positive bacteria: Streptococcus pneumoniae RCMB 010010, S. aureus RCMB 010028 and two Gram-negative bacteria: P. aeruginosa RCMB 010043, E. coli RCMB 010052. Compounds 5 and 6 displayed significant and higher antibacterial activity when compared with ampicillin and ciprofloxacin, respectively ( Figure 4). Compounds 5 and 6 showed two-fold increased inhibition against S. pneumoniae with MIC = 0.49 µg/mL, compared to ampicillin (MIC = 0.98 µg/mL). The authors proved that compounds bearing electron-donating groups showed better activities than the electronwithdrawing ones. Regarding Gram-negative bacteria, remarkable activity was elicited by the derivatives 5 and 6 against E. coli (MIC = 0.49 µg/mL), showing two-fold the potency of the standard ciprofloxacin (MIC = 0.98 µg/mL). The authors tested synthesized compounds for cytotoxic activities against human lung fibroblast normal cells (WI-38). Hydrazidehydrazones 5 and 6 showed no cytotoxic activity at 0-500 µg/mL concentrations [15].   The results of the study by Popiołek and Biernasiuk indicated that synthesized and in vitro examined hydrazide-hydrazones exhibited a wide spectrum of antibacterial activity against tested reference bacteria. Substances 8, 9, and 10 were especially potent (MIC = 0.002-0.98 µg/mL) against Gram-positive bacteria ( Figure 6). Staphylococcus epidermidis ATCC 12228 was the most sensitive to all synthesized compounds, while Micrococcus luteus ATCC 10240 was the least susceptible. Compounds 8 and 9 showed almost two thousand times higher activity (MIC < 1 µg/mL) than nitrofurantoin (MIC = 3.91 µg/mL) against B. subtilis ATCC 6633 and S. epidermidis ATCC 12228, respectively. The MIC value of compound 9 (MIC = 0.002 µg/mL) was sixty-one times lower than the MIC of ciprofloxacin (MIC = 0.122 µg/mL) against S. epidermidis ATCC 12228. Compound 8 had MIC value (MIC = 0.002 µg/mL) against B. subtilis ATCC 6633, which was almost eight thousand times lower than the MIC of cefuroxime (MIC = 15.63 µg/mL) [17].   Table 4) [19].     Similarly, Polović et al., in 2019, synthesized novel series of hydrazide-hydrazones of nicotinic acid and assessed their antibacterial activity. Antibacterial assays performed in this study showed that compound 17 with nitro group displayed good inhibition of bacterial growth, whereas the compounds without an electron-withdrawing group (chloroand nitro-) showed weak antibacterial activity ( Figure 11, Table 5). According to the authors, this may be due to the fact that electron-withdrawing substituents increase the lipophilicity of the compounds, which leads to higher partitioning of such compounds into the lipophilic phase of a microbial membrane [22].
All the newly synthesized hydrazide-hydrazones by Shaaban et al. were evaluated for their in vitro antibacterial activity against S. aureus RCMB 0100183, B. subtilis RCMB 0100162, S. epidermidis RCMB 0100183, P. aeruginosa RCMB 0100243, P. vulgaris RCMB 010085, and E. coli RCMB 010052. Ampicillin and levofloxacin were used as reference standard antibacterial agents. Compound 18 exhibited moderate activity against Grampositive and Gram-negative bacteria (Table 6, Figure 12) [23].  Hydrazide-hydrazones obtained by Haiba et al. exhibited promising antibacterial activity against bacterial strains. Compound 19 showed two-fold higher antibacterial activity against E. coli (MIC = 12.5 µg/mL) and S. aureus (MIC = 6.25 µg/mL) than ampicillin (MIC = 25 and 12.5 µg/mL, respectively) ( Figure 13). Additionally, compound 19 also showed significant antibacterial activity against MDR clinical isolates of K. pneumoniae (MIC = 12.5 µg/mL) and methicillin-resistant S. aureus MRSA1 (MIC = 3.125 µg/mL). The cytotoxicity of hydrazide-hydrazone 19 was tested in a VERO (African green monkey kidney) cell line. The performed test revealed that 50% cytotoxic concentration value CC 50 equals 125, which, according to the authors, corresponds to good safety profile of this substance. The authors also performed a molecular docking study to find potential mechanism of antibacterial action of synthesized compounds. As a result, it occurred that the antibacterial potency of obtained hydrazide-hydrazones may be connected with strong binding interactions in the DNA gyrase active site [24].   The most active compound among indol-2-one derivatives synthesized by Salem et al. was 21. It showed higher antibacterial activity than tetracycline against B. subtilis, S. aureus, and E. coli ( Figure 15, Table 8). In order to determine the possible mechanism of action of synthesized compounds, the authors performed the inhibitory activity assay against DNA gyrase isolated from S. aureus. Hydrazide-hydrazone 21 showed strong inhibition of gyrase (IC 50 = 19.32 ± 0.99 µM) when compared to ciprofloxacin (IC 50 = 26.43 ± 0.64 µM) [26].  The results of antibacterial activity screening of hydrazide-hydrazones synthesized by Tiwari et al. indicated that the compounds were active against Gram-positive bacteria. The highest activity was shown by compound 22 (Figure 16, Table 9) [27].  The hydrazide-hydrazone 23 obtained by Ewies et al. exhibited promising antibacterial activity against S. aureus, S. typhimurium, and P. aeruginosa ( Figure 17, Table 10) [28].  In 2020, Popiołek     Novel derivatives of 1,2,3-thiadiazole synthesized by Paruch et al. displayed interesting antibacterial properties. Hydrazide-hydrazone 28 showed a bactericidal effect among almost all tested strains ( Figure 20). The MIC values of this substance, which inhibited bacterial growth, ranged from 1.95 µg/mL (for Staphylococcus spp.) to 15.62 µg/mL (for E. faecalis ATCC 29212). The activity of this derivative against S. aureus ATCC 25923 and ATCC 43300 was two-fold greater than for nitrofurantoin. Towards S. aureus ATCC 6538 strain was seven times higher than for nitrofurantoin. This compound also possessed good activity against S. epidermidis ATCC 12228 and M. luteus ATCC 10240, two and eight times higher, respectively, in comparison with nitrofurantoin [31].

Antimycobacterial Activity
On the basis of the survey of scientific literature, it can be concluded that hydrazidehydrazones may also be regarded as promising antitubercular agents, which is especially important when tuberculosis is still a serious threat for people [32].
Among hydrazide-hydrazones of 4-trifluoromethylbenzoic acid synthesized by Krátký et al., compound 29 showed high antitubercular activity comparable to isoniazid against the clinical isolate of Mycobacterium kansasii 6509/96 ( Figure 21, Table 12) [14].  In the study performed by Angelova et al., synthesized hydrazide-hydrazones with 2H-chromene and coumarin scaffold were evaluated in vitro against M. tuberculosis H37Rv. Compound 30 proved to be the most active against tested strain (MIC = 0.13 µM) (Figure 22). Isoniazid and ethambutol showed higher values of MIC 1.45 and 7.64 µM, respectively. Hydrazide-hydrazone 30 showed low cytotoxicity against human embryonal kidney cell line HEK-293T (IC 50 = 90.66 µM) and displayed a favorable selectivity index (SI = 697.38). Authors suggested that this class of hydrazide-hydrazones may be regarded as a promising new candidate for further investigations as an antitubercular agent [33]. In 2017, Angelova et al. published a similar study that concerned the in vitro antimycobacterial activity of benzopyran derivatives against the reference strain of M. tuberculosis H37Rv. The most active was compound 31 (MIC = 0.28 µM) with p-methoxyphenyl substituent ( Figure 23). Its activity was higher than that of isoniazid (MIC = 0.79 µM) and ethambutol (MIC = 1.46 µM). The authors observed that the presence of OH, OCH 3 , and N(CH) 3 functional groups at four position in the phenyl ring enhanced the antimycobacterial activity. This compound was also tested for cytotoxicity against the human embryonic kidney cell line HEK-293T. According to the authors, it showed minimal cytotoxicity (IC 50 = 112.9 µM) and a high value of selectivity index (SI = 403). In order to discover the potential mechanism of antimycobacterial activity of synthesized compounds, the authors performed molecular docking studies and investigated binding to the 2-trans-enoyl-ACP reductase (InhA) enzyme involved in M. tuberculosis cell wall biogenesis. On the basis of obtained results, the authors suggested that the activity of synthesized molecules may be connected with interactions with the inhibitor binding cavity of M. tuberculosis enoyl-ACP reductase and/or related to the ability of the tested compounds to penetrate mycobacterial cells [34]. Hydrazide-hydrazones synthesized and tested for potential antimycobacterial activity by Atta et al. displayed high potency against M. tuberculosis H37Rv. Compound 32 showed the highest activity with MIC = 7.30 µM and was equipotent to ethambutol (MIC = 7.64 µM) and seven times more active than pyrazinamide (MIC = 50.77 µM), which were used as reference compounds (Figure 24). Substance 32 was also examined for cytotoxicity in human embryonic kidney (HEK 293) cell line at the concentration of 50 µg/mL with the use of MTT assay. It showed a lower cytotoxic effect (20.08% inhibition) than standard antimycobacterial medicine-isoniazid (35.60% inhibition) [35].     (Figure 28). The activity of this compound was equal to the activity of isoniazid [38]. Hassan et al. synthesized novel pyrazine derivatives with hydrazide-hydrazone moiety, which appeared to be effective inhibitors of the growth of M. tuberculosis H37Rv ATCC 27294. The highest potency was shown by compound 37, which displayed significant anti-TB activity with MIC value of 0.78 µg/mL, which equals two times the activity of ethambutol (MIC = 1.56 µg/mL) and eight times the activity of pyrazinamide (MIC = 6.25 µg/mL) ( Figure 29). In the in vitro cytotoxicity assay against PBMC (peripheral blood mononuclear cells) normal cell line, this hydrazide-hydrazone showed no cytotoxicity (IC 50 = 846.9 µg/mL) and very high selectivity index (SI = 1085.7). To find a possible mechanism of activity of obtained hydrazide-hydrazones, the authors processed a docking study into the active site of the pantothenate synthetase enzyme. Substance 37 revealed to have favorable binding modes and interaction patterns with the active site of the enzyme [39].  Gürsoy et al., in 2020, obtained novel thiazole derivatives with antitubercular activity. Hydrazide-hydrazone 39 was found to be most active against M. tuberculosis H37Rv (MIC = 16.252 µg/mL) and had no cytotoxicity towards CRFK (Crandell Rees feline kidney) cells (CC 50 > 100 µg/mL). However, its activity was lower than for rifampicin, which was used as a reference substance (MIC = 0.125 µg/mL) (Figure 31) [41]. 56 µg/mL, which was higher when compared with ethambutol (MIC = 3.13 µg/mL). The authors discovered that compounds bearing methoxy groups in the phenyl ring attached to the 1,3-oxazole scaffold displayed better activity compared to the other compounds ( Figure 32). Synthesized substances (40,41) were tested in vitro for cytotoxicity in human embryonic kidney (HEK-293T) cells and did not display changes in cytotoxicity as compared with vehicle (DMSO) [42].
Krátký et al. evaluated hydrazide-hydrazones of 4-trifluoromethylbenzoic acid also as possible antifungal agents. The most active against a panel of fungi was, similarly as against bacterial strains, the hydrazide-hydrazone numbered as 4 ( Figure 3, Table 13). The activity of this molecule (MIC = 1.98 µM) was four times higher than for fluconazole (MIC = 7.81 µM) against Trichophyton mentagrophytes 445 [14]. Compounds 5 and 6 synthesized by Abdelrahman et al. displayed antifungal activity ( Figure 4). Their activity against tested fungi was higher or equal to the activity of Amphotericin B (Table 14) [15]. Analysis of the diameter of zone of inhibition growth (mm) of compounds obtained by Manikandan et al. revealed that only hydrazide-hydrazone 42 had shown good antifungal sensitivity against three species of fungi ( Figure 33, Table 15) [16].  Nitrofurazone analogues synthesized by Popiołek and Biernasiuk also showed interesting antifungal activity. Compounds 43, 44, and 45 displayed good fungicidal or fungistatic properties against Candida spp. ATCC (MIC = 31.25-125 µg/mL, MFC = 125-1000 µg/mL) but they were less active than fluconazole, which was used as a reference substance ( Figure 34) [17]. Benzimidazole derivatives synthesized by Yadav et al. were also tested for their antifungal activity. The best results showed compound 11 (Figure 7). Its activity against C. albicans (MIC = 0.016 µM) and A. niger (MIC = 0.032 µM) was higher than that of fluconazole (MIC = 0.40 and 0.82 µM, respectively) [18].
Molecules synthesized by El-Sayed et al. possessed interesting antifungal activity. Especially compounds 12 and 13 showed good effects against Aspergillus niger (diameter of zone of inhibition growth ZOI = 16 and 19 mm, respectively) in comparison with nystatin (ZOI = 20 mm) (Figure 8) [19].
All yeasts belonging to Candida spp. ATCC were sensitive to hydrazide-hydrazones of isonicotinic acid synthesized by Popiołek    Among novel indol-2-one derivatives with hydrazide-hydrazone moiety compound, 21 was found to have the most potent antifungal properties, even though its activity against C. albicans (MIC = 31.25 µg/mL) was two times and for F. oxysporum (MIC = 125 µg/mL) four times lower than for amphotericin B (MIC = 15.62 and 31.25 µg/mL, respectively) ( Figure 15) [26].
New hydrazide-hydrazones of 5-nitrofuran-2-carboxylic acid obtained by Popiołek et al. also indicated significant anticandidal activity. The values of MIC of compound 26, which contained the 2-iodophenyl substituent, were the lowest (from 7.81 to 15.62 µg/mL), indicating its strong or very strong activity against all reference Candida spp. (Figure 18) [29].
Among novel derivatives of 5-pyrrolidin-2-one, synthesized by Dascalu et al., compounds 48 and 49 with chloride and bromine atom at position 4 in the phenyl ring showed a broad spectrum of antifungal activity ( Figure 37, Table 16) [46].

Conclusions
In conclusion, this article gives an overview of the antibacterial, antitubercular, and antifungal properties of hydrazide-hydrazones published since 2017. As presented in this study, the hydrazide-hydrazone moiety may be found and incorporated in various bioactive molecules. Thus, this review appears to be important for further development of hydrazide-hydrazones as potential antimicrobial agents.