A Review of the Biological Activity of Amidrazone Derivatives

Amidrazones are widely used in chemical synthesis, industry and agriculture. We compiled some of the most important findings on the biological activities of amidrazones described in the years 2010–2022. The data were obtained using the ScienceDirect, Reaxys and Google Scholar search engines with keywords (amidrazone, carbohydrazonamide, carboximidohydrazide, aminoguanidine) and structure strategies. Compounds with significant biological activities were included in the review. The described structures derived from amidrazones include: amidrazone derivatives; aminoguanidine derivatives; complexes obtained using amidrazones as ligands; and some cyclic compounds obtained from amidrazones and/or containing an amidrazone moiety in their structures. This review includes chapters based on compound activities, including: tuberculostatic, antibacterial, antifungal, antiparasitic, antiviral, anti-inflammatory, cytoprotective, and antitumor compounds, as well as furin and acetylocholinesterase inhibitors. Detailed information on the compounds tested in vivo, along the mechanisms of action and toxicity of the selected amidrazone derivatives, are described. We describe examples of compounds that have a chance of becoming drugs due to promising preclinical or clinical research, as well as old drugs with new therapeutic targets (repositioning) which have the potential to be used in the treatment of other diseases. The described examples prove that amidrazone derivatives are a potential source of new therapeutic substances and deserve further research.


Introduction
Amidrazones (hydrazones of acid amides) are organic compounds represented by the general structure presented in Figure 1a. These compounds are characterized by three nitrogen atoms (N 1 , N 2 and N 3 ), of which only two, N 1 and N 3 , may be substituted with alkyl or aryl groups. Amidrazones can exhibit tautomerism due to the transfer between the nitrogen atoms N 3 and N 2 [1,2]. Amidrazones are monoacid bases which form salts with inorganic acids, among which the most widely known are the hydrochlorides [2].

Introduction
Amidrazones (hydrazones of acid amides) are organic compounds represented by the general structure presented in Figure 1a. These compounds are characterized by three nitrogen atoms (N 1 , N 2 and N 3 ), of which only two, N 1 and N 3 , may be substituted with alkyl or aryl groups. Amidrazones can exhibit tautomerism due to the transfer between the nitrogen atoms N 3 and N 2 [1,2]. Amidrazones are monoacid bases which form salts with inorganic acids, among which the most widely known are the hydrochlorides [2].  Amidrazones constitute a group of interesting compounds used mainly as precursors for the synthesis of five-, six-and seven-membered heterocyclic systems. Simple meth-active against Mycobacterium kansasii than isoniazid (MIC = 4.2 µM) [17].
Derivative 8, containing an aminoguanidine moiety, showed strong tuberculostatic activity against (MIC = 0.78 µM), and low cytotoxicity to, human embryonic kidney cells. The mechanism of 8 was the inhibition of the enoyl acyl carrier protein reductase enzyme (InhA), which was confirmed in vitro and in computational studies [19].

Antibacterial Activity
Several compounds with antibacterial activities are presented in Figure 3. The previously mentioned compounds 6-7 exhibited a significant antibacterial activity against several Gram-positive bacterial strains (Staphylococcus epidermidis, Micrococcus luteus, Bacillus subtilis, Bacillus cereus and Streptococcus mutans), with MIC values of 0.12-1.95 µg/mL. Additionally, derivative 6 showed an activity against Staphylococcus aureus comparable to ciprofloxacin and vancomycin. Interestingly, the replacement of the pyrrolidine ring found in compound 6 with a morpholine moiety present in compound 7 resulted in an approximately twofold decrease in its anti-tuberculosis and antibacterial activities against Gram-positive strains in comparison with the starting compounds of 6-7 [18]. Compound 9, containing an isatin moiety, demonstrated stronger antibacterial activity against S. aureus (MIC = 4 µg/mL) than ciprofloxacin [20].
Derivative 8, containing an aminoguanidine moiety, showed strong tuberculostatic activity against (MIC = 0.78 µM), and low cytotoxicity to, human embryonic kidney cells. The mechanism of 8 was the inhibition of the enoyl acyl carrier protein reductase enzyme (InhA), which was confirmed in vitro and in computational studies [19].

Antibacterial Activity
Several compounds with antibacterial activities are presented in Figure 3. The previously mentioned compounds 6-7 exhibited a significant antibacterial activity against several Gram-positive bacterial strains (Staphylococcus epidermidis, Micrococcus luteus, Bacillus subtilis, Bacillus cereus and Streptococcus mutans), with MIC values of 0.12-1.95 µg/mL. Additionally, derivative 6 showed an activity against Staphylococcus aureus comparable to ciprofloxacin and vancomycin. Interestingly, the replacement of the pyrrolidine ring found in compound 6 with a morpholine moiety present in compound 7 resulted in an approximately twofold decrease in its anti-tuberculosis and antibacterial activities against Gram-positive strains in comparison with the starting compounds of 6-7 [18]. Compound 9, containing an isatin moiety, demonstrated stronger antibacterial activity against S. aureus (MIC = 4 µg/mL) than ciprofloxacin [20].
Aminoguanidine derivative 27 demonstrated a wide range of antimicrobial activities, with an MIC value of 1 µM/mL against eight strains (including S. aureus, S. mutans, E.coli, C. albicans, MRSA and Quinolone-resistant S. aureus). The inhibition of the dihydrofolate reductase (DHFR) protein is a possible mechanism of action of 27 [27].
Thiazole derivatives 30-31 demonstrated strong bactericidal activity against the S. aureus, MRSA and VRSA bacterial strains (in most cases, MIC = MBC = 2 µg/mL) and were active against MRSA in several animal models. Compound 30 demonstrated resistance to the microsomal cytochrome P450 and stability during metabolism. However, it interacted with enzymes connected to bacterial wall synthesis (such as undecaprenyl diphosphate synthase and undecaprenyl diphosphate phosphatase). Due to its similar activity (but in lower doses) to that of vancomycin in mice, compound 30 may be a new leading structure in the treatment of drug-resistant bacterial strains [30].

Antifungal Activity
Among the previously mentioned amidrazone derivatives 10-14, the strongest fungistatic activity against C. albicans was exhibited by compounds 11 (MIC = 4 µg/mL) and 10 (MIC = 8 µg/mL). Additionally, derivative 11 was fungicidal at a concentration of 16 µg/mL against Aspergillus niger and Aspergillus brasiliensis [32]. The presence of a nitro group in the position R 1 of compound 11 seems to increase its antifungal activity. Contrarily, the addition of a four-nitro substituent in the N 1 -phenyl rings of compounds 12 and 14 decreased their antifungal properties but elevated their antibacterial activity.
Compound 33 ( Figure 4) exhibited antifungal activity against Candida albicans (MIC = 16 µg/mL) [26]. Pyrazinylamidrazone 34 exhibited antifungal activity against the clinical strain C. albicans (MIC = 16 µg/mL). The replacement of the phenyl ring of compound 34 with a hydrogen or a methyl group resulted in the total disappearance of the antifungal activity of the obtained derivatives, which underlines the importance of the phenyl substituent in this position [33].  The imidazolylamidrazone derivatives 35-37 demonstrated fungistatic activity against Candida krusei (MIC = 3.1-6.3 µg/mL) and Candida neoformans (MIC = 2−4 µg/mL) [34]. Derivatives 35-37 also displayed a strong inhibitory effect on biofilm development in the case of Candida spp. biofilms on nanohydroxyapatite substrate, and the strongest effect was observed for compound 36 [35]. The mechanism of action of compounds 35-37 seems to be connected with the production of reactive oxygen species [36]. Amidrazonequinolone hybrids 38-39 showed an antifungal activity in vitro against C. albicans comparable to that of fluconazole [37].
Likewise, aminoguanidine derivatives 46-50 were studied as antiparasitic agents. Robenidine (46) is an antibiotic used in veterinary medicine which, in current research, has shown an antigiardial activity against G. lamblia comparable to that of metronidazole. In contrast to the reference drug, compound 46 completely inhibited the adherence of trophozoides and is a candidate for a new generation of antigiardial drugs [42]. The imidazolylamidrazone derivatives 35-37 demonstrated fungistatic activity against Candida krusei (MIC = 3.1-6.3 µg/mL) and Candida neoformans (MIC = 2-4 µg/mL) [34]. Derivatives 35-37 also displayed a strong inhibitory effect on biofilm development in the case of Candida spp. biofilms on nanohydroxyapatite substrate, and the strongest effect was observed for compound 36 [35]. The mechanism of action of compounds 35-37 seems to be connected with the production of reactive oxygen species [36]. Amidrazone-quinolone hybrids 38-39 showed an antifungal activity in vitro against C. albicans comparable to that of fluconazole [37].
Likewise, aminoguanidine derivatives 46-50 were studied as antiparasitic agents. Robenidine (46) is an antibiotic used in veterinary medicine which, in current research, has shown an antigiardial activity against G. lamblia comparable to that of metronidazole. In contrast to the reference drug, compound 46 completely inhibited the adherence of trophozoides and is a candidate for a new generation of antigiardial drugs [42]. Guanabenz (47) is a known antihypertensive drug currently drawing attention for the purpose of other medicinal uses. It has exhibited antiparasitic activity against the replicative stages of Toxoplasma and Plasmodium falciparum [43]. Guanabenz inhibited the Toxoplasma dephosphorylation enzyme eIF2α. This translational control is critical during infections with both the replicative and latent forms of Toxoplasma [43,44]. In mice models, guanabenz extended the survival of mice acutely infected with Toxoplasma within 2-3 days [44] and reduced the number of brain cysts in chronically infected mice [43].
Aminoguanidine derivatives 48-50 showed antileishmanial activity against amastigotes of Leishmania chagasi (IC50 = 0.6−7.27 µM) comparable to pentamidine (IC50 = 4.4 µM). Compounds 48-50 showed a 50-80 times higher toxicity to amastigotes than to murine macrophages. The mechanism of action of the most promising compound, 50, is probably related to its interaction with the active site of the trypanothione reductase enzyme, interfering in the redox system of L. chagasi amastigotes [45].
The 1,2,4-triazole derivative 51, obtained from amidrazone, showed strong anthelmintic activity (2.475 µg/µL) against Rhabditis nematodes. Due to its stronger activity than albendazole and low toxicity to PBMC, compound 51 could be a candidate for the development of new anthelmintic drugs [46].

Antiviral Activity
Amidrazone derivative 52 ( Figure 6) reduced the number of plaques of herpes simplex type-1 (HSV-1) on Vero cells by 67% [47]. Amidrazon 53, with a pyrazoloisoxazole moiety, showed antiviral activity against two HIV strains studied in two leukemia cell lines (EC50 = 0.17−0.46 nM). Compound 53 was two times more effective than the anti-HIV drug efavirenz and about two times less toxic to uninfected cell lines. Compound 53 exhibited strong inhibitory activity towards HIV reverse transcriptase (HIV-RT). Molecular docking confirmed that compound 53 strongly interacts with the HIV-RT active pocket, which enables its classification as a potential non-nucleoside reverse transcriptase inhibitor [48].  Guanabenz (47) is a known antihypertensive drug currently drawing attention for the purpose of other medicinal uses. It has exhibited antiparasitic activity against the replicative stages of Toxoplasma and Plasmodium falciparum [43]. Guanabenz inhibited the Toxoplasma dephosphorylation enzyme eIF2α. This translational control is critical during infections with both the replicative and latent forms of Toxoplasma [43,44]. In mice models, guanabenz extended the survival of mice acutely infected with Toxoplasma within 2-3 days [44] and reduced the number of brain cysts in chronically infected mice [43].
Aminoguanidine derivatives 48-50 showed antileishmanial activity against amastigotes of Leishmania chagasi (IC 50 = 0.6-7.27 µM) comparable to pentamidine (IC 50 = 4.4 µM). Compounds 48-50 showed a 50-80 times higher toxicity to amastigotes than to murine macrophages. The mechanism of action of the most promising compound, 50, is probably related to its interaction with the active site of the trypanothione reductase enzyme, interfering in the redox system of L. chagasi amastigotes [45].
The 1,2,4-triazole derivative 51, obtained from amidrazone, showed strong anthelmintic activity (2.475 µg/µL) against Rhabditis nematodes. Due to its stronger activity than albendazole and low toxicity to PBMC, compound 51 could be a candidate for the development of new anthelmintic drugs [46].

Antiviral Activity
Amidrazone derivative 52 ( Figure 6) reduced the number of plaques of herpes simplex type-1 (HSV-1) on Vero cells by 67% [47]. Amidrazon 53, with a pyrazoloisoxazole moiety, showed antiviral activity against two HIV strains studied in two leukemia cell lines (EC 50 = 0.17-0.46 nM). Compound 53 was two times more effective than the anti-HIV drug efavirenz and about two times less toxic to uninfected cell lines. Compound 53 exhibited strong inhibitory activity towards HIV reverse transcriptase (HIV-RT). Molecular docking confirmed that compound 53 strongly interacts with the HIV-RT active pocket, which enables its classification as a potential non-nucleoside reverse transcriptase inhibitor [48]. Guanabenz (47) is a known antihypertensive drug currently drawing a the purpose of other medicinal uses. It has exhibited antiparasitic activity aga licative stages of Toxoplasma and Plasmodium falciparum [43]. Guanabenz inhib oplasma dephosphorylation enzyme eIF2α. This translational control is critica fections with both the replicative and latent forms of Toxoplasma [43,44]. In m guanabenz extended the survival of mice acutely infected with Toxoplasma days [44] and reduced the number of brain cysts in chronically infected mice Aminoguanidine derivatives 48-50 showed antileishmanial activ amastigotes of Leishmania chagasi (IC50 = 0.6−7.27 µM) comparable to pentam 4.4 µM). Compounds 48-50 showed a 50-80 times higher toxicity to amastig murine macrophages. The mechanism of action of the most promising comp probably related to its interaction with the active site of the trypanothione r zyme, interfering in the redox system of L. chagasi amastigotes [45].

Antiviral Activity
Amidrazone derivative 52 ( Figure 6) reduced the number of plaques of plex type-1 (HSV-1) on Vero cells by 67% [47]. Amidrazon 53, with a pyraz moiety, showed antiviral activity against two HIV strains studied in two le lines (EC50 = 0.17−0.46 nM). Compound 53 was two times more effective than drug efavirenz and about two times less toxic to uninfected cell lines. Comp hibited strong inhibitory activity towards HIV reverse transcriptase (HIV-RT docking confirmed that compound 53 strongly interacts with the HIV-RT ac which enables its classification as a potential non-nucleoside reverse transcri tor [48].

Anti-Inflammatory Activity
Derivatives of N 1 ,N 3 -substituted 2-pyridylamidrazone 54-57 ( Figure 7) were studied in order to assess their anti-inflammatory activity in mitogen-stimulated peripheral blood mononuclear cells (PBMC). Compound 54 decreased the production of TNF-α by 43% and showed no toxicity to PBMC at a concentration of 100 µg/mL [49].

Anti-Inflammatory Activity
Derivatives of N 1 ,N 3 -substituted 2-pyridylamidrazone 54-57 ( Figure 7) were studied in order to assess their anti-inflammatory activity in mitogen-stimulated peripheral blood mononuclear cells (PBMC). Compound 54 decreased the production of TNF-α by 43% and showed no toxicity to PBMC at a concentration of 100 µg/mL [49].
Compound 55, at a concentration of 10 µg/mL, inhibited the production of the proinflammatory cytokine IL-6 by 35% [50]. The median lethal dose of 55 (i.p.) in mice was identified as 417 mg/kg. Compound 55, at a concentration of 21 mg/kg, reduced rat hind paw edema to a greater extent than diclofenac at a dose of 50 mg/kg. Moreover, derivative 55 demonstrated antinociceptive activity in mice comparable to that of morphine but with a longer duration of action. In summary, compound 55 could be a potential non-steroidal anti-inflammatory drug [50]. Compound 56, at a concentration of 10 µg/mL, inhibited the production of TNF-α in PBMC stimulated by lipopolysaccharide (LPS) by 53% [51]. Compound 57, at a concentration of 50 µg/mL, showed no toxicity but strongly inhibited the proliferation of PBMC activated by anti-CD3 antibodies or phytohaemagglutinin by 90-99%, and the observed effects were comparable to or stronger than those of ibuprofen. The mechanism of action of derivative 57 is cell cycle arrest at the G1 phase [52].
Anti-inflammatory activity was also reported for amidrazone-derived pyrrole-2,5dione derivatives 58-59. Compound 58, possessing two phenyl substituents, significantly reduced the production of IL-6 (by 64%) in LPS-stimulated PBMC cultures. Both compounds 58 and 59 inhibited the proliferation of PBMC even at a low dose of 10 µg/mL, and the strongest effect was observed for the latter, possessing two 2-pyridine rings [54].
The previously mentioned N 1 ,N 3 -substituted amidrazones 38-39 showed an anti-inflammatory activity in protein denaturation assays comparable to that of the sodium salt of diclofenac. Both derivatives showed a stronger antioxidant activity than ascorbic acid [37].
Indoleamidrazone derivatives 60-63 produced a stronger reduction in carrageenaninduced rat paw edema in rats than indomethacin. In general, compounds possessing nitro or methoxy substituents at the para position showed stronger anti-inflammatory effects than derivatives possessing the same groups in the meta position [55]. Compound 55, at a concentration of 10 µg/mL, inhibited the production of the proinflammatory cytokine IL-6 by 35% [50]. The median lethal dose of 55 (i.p.) in mice was identified as 417 mg/kg. Compound 55, at a concentration of 21 mg/kg, reduced rat hind paw edema to a greater extent than diclofenac at a dose of 50 mg/kg. Moreover, derivative 55 demonstrated antinociceptive activity in mice comparable to that of morphine but with a longer duration of action. In summary, compound 55 could be a potential non-steroidal anti-inflammatory drug [50].
Compound 56, at a concentration of 10 µg/mL, inhibited the production of TNF-α in PBMC stimulated by lipopolysaccharide (LPS) by 53% [51]. Compound 57, at a concentration of 50 µg/mL, showed no toxicity but strongly inhibited the proliferation of PBMC activated by anti-CD3 antibodies or phytohaemagglutinin by 90-99%, and the observed effects were comparable to or stronger than those of ibuprofen. The mechanism of action of derivative 57 is cell cycle arrest at the G1 phase [52].
Anti-inflammatory activity was also reported for amidrazone-derived pyrrole-2,5dione derivatives 58-59. Compound 58, possessing two phenyl substituents, significantly reduced the production of IL-6 (by 64%) in LPS-stimulated PBMC cultures. Both compounds 58 and 59 inhibited the proliferation of PBMC even at a low dose of 10 µg/mL, and the strongest effect was observed for the latter, possessing two 2-pyridine rings [54].
The previously mentioned N 1 ,N 3 -substituted amidrazones 38-39 showed an antiinflammatory activity in protein denaturation assays comparable to that of the sodium salt of diclofenac. Both derivatives showed a stronger antioxidant activity than ascorbic acid [37].
Indoleamidrazone derivatives 60-63 produced a stronger reduction in carrageenaninduced rat paw edema in rats than indomethacin. In general, compounds possessing nitro or methoxy substituents at the para position showed stronger anti-inflammatory effects than derivatives possessing the same groups in the meta position [55]. Naphthylamidrazone derivative 64 revealed properties preventing the adverse effects of a chronic inflammatory reaction in the articular chondrocytes through a mechanism involving the ASIC1a channels, which are sensitive to the acidification of the environment. Compound 64, in a concentration range of 6.25-50 µM, caused a significant inhibition of the ASIC1a protein expression in the joint chondrocytes comparable to amiloride (a weak non-selective ASIC1 inhibitor). Additionally, compound 64, at a dose of 25 µM, decreased the number of Ca 2+ ions in the acidic environment of isolated rat articular chondrocytes by 69%, which is almost three times higher than the effect of amiloride at a dose of 100 µM. In summary, it can be stated that compound 64 is a potential drug for rheumatoid arthritis [56].
Aminoguanidine (AG) has been shown to possess strong anti-inflammatory and antioxidant activities in multiple ways. It inhibits the formation of highly reactive advanced glycosylation end products in the course of advanced diabetes. AG passed phase III clinical trials in diabetic patients. Although high doses of AG induced side effects, including liver dysfunction, low doses of AG therapy could be promising for the treatment of renal diseases [57].
Aminoguanidine derivatives 23-26 were studied in tests on xylene-induced ear edema in mice. Compound 23 showed an anti-inflammatory activity similar to indomethacin. However, compound 24, with a bromine atom at position 3, was about two times less effective [25]. Derivatives 25 and 26 were about two times stronger as anti-inflammatory agents than indomethacin [26].
Aminoguanidine derivative 65 was studied in an LPS-stimulated neonatal sepsis mice model. The mechanism of compound 65 was connected to a decreased pro-inflammatory cytokine release and COX-2 expression, as well as the suppression of microglia activation. Additionally, septic mice treated with derivative 65 did not exhibit the cognitive impairment and the anxiety behavior caused by LPS [58].

Cytoprotective Activity
Some aminoguanidine derivatives, such as guanabenz (47), sephin1 (66) and raphin1 (50), possess cytoprotective activities (Figure 8). The effects of those compounds are connected with the reduced deposition of proteins of abnormal conformation, which are present in many neurodegenerative diseases, such as Alzheimer's, Parkinson's, amyotrophic lateral sclerosis (ALS) and others. Guanabenz and sephin1 are inhibitors of the stress-induced transcription factor R15A. They prolong eIF2α (translation initiation factor) phosphorylation and, in consequence, cause the transient attenuation of protein synthesis induced by endoplasmic reticulum (ER) stress [59]. Guanabenz is currently in clinical trials as a method for the management of multiple sclerosis [60] and amyotrophic lateral sclerosis [61,62]. Guanabenz has also been shown to reduce neuroinflammation in mice with latent toxoplasmosis and reversed the behavioral changes in the studied rodents [63]. Sephin1 has passed phase I clinical trials and is being developed for treating Charcot-Marie-Tooth disease [64]. Moreover, sephin1 showed protective activity in a mouse model of multiple sclerosis [65]. Naphthylamidrazone derivative 64 revealed properties preventing the adverse effects of a chronic inflammatory reaction in the articular chondrocytes through a mechanism involving the ASIC1a channels, which are sensitive to the acidification of the environment. Compound 64, in a concentration range of 6.25-50 µM, caused a significant inhibition of the ASIC1a protein expression in the joint chondrocytes comparable to amiloride (a weak non-selective ASIC1 inhibitor). Additionally, compound 64, at a dose of 25 µM, decreased the number of Ca 2+ ions in the acidic environment of isolated rat articular chondrocytes by 69%, which is almost three times higher than the effect of amiloride at a dose of 100 µM. In summary, it can be stated that compound 64 is a potential drug for rheumatoid arthritis [56].
Aminoguanidine (AG) has been shown to possess strong anti-inflammatory and antioxidant activities in multiple ways. It inhibits the formation of highly reactive advanced glycosylation end products in the course of advanced diabetes. AG passed phase III clinical trials in diabetic patients. Although high doses of AG induced side effects, including liver dysfunction, low doses of AG therapy could be promising for the treatment of renal diseases [57].
Aminoguanidine derivatives 23-26 were studied in tests on xylene-induced ear edema in mice. Compound 23 showed an anti-inflammatory activity similar to indomethacin. However, compound 24, with a bromine atom at position 3, was about two times less effective [25]. Derivatives 25 and 26 were about two times stronger as anti-inflammatory agents than indomethacin [26].
Aminoguanidine derivative 65 was studied in an LPS-stimulated neonatal sepsis mice model. The mechanism of compound 65 was connected to a decreased pro-inflammatory cytokine release and COX-2 expression, as well as the suppression of microglia activation. Additionally, septic mice treated with derivative 65 did not exhibit the cognitive impairment and the anxiety behavior caused by LPS [58].

Cytoprotective Activity
Some aminoguanidine derivatives, such as guanabenz (47), sephin1 (66) and raphin1 (50), possess cytoprotective activities (Figure 8). The effects of those compounds are connected with the reduced deposition of proteins of abnormal conformation, which are present in many neurodegenerative diseases, such as Alzheimer's, Parkinson's, amyotrophic lateral sclerosis (ALS) and others. Guanabenz and sephin1 are inhibitors of the stress-induced transcription factor R15A. They prolong eIF2α (translation initiation factor) phosphorylation and, in consequence, cause the transient attenuation of protein synthesis induced by endoplasmic reticulum (ER) stress [59]. Guanabenz is currently in clinical trials as a method for the management of multiple sclerosis [60] and amyotrophic lateral sclerosis [61,62]. Guanabenz has also been shown to reduce neuroinflammation in mice with latent toxoplasmosis and reversed the behavioral changes in the studied rodents [63]. Se-phin1 has passed phase I clinical trials and is being developed for treating Charcot-Marie-Tooth disease [64]. Moreover, sephin1 showed protective activity in a mouse model of multiple sclerosis [65]. Raphin1 is an inhibitor of the constitutively expressed transcription factor R15B, which may be useful when combating a wide range diseases, as it could enable the in- Raphin1 is an inhibitor of the constitutively expressed transcription factor R15B, which may be useful when combating a wide range diseases, as it could enable the increase in the control capacity of the protein quality by transiently increasing eIF2α phosphorylation and translation attenuation. It was effective in a mouse model of Huntington's disease [66]. Moreover, the previously mentioned robenidine showed cytoprotective properties [67].  , respectively). This underlines their strong anti-cancer properties [68]. Moreover, amidrazones 69-70 showed antiproliferative activity against several cancer cell lines, including leukemia K562, breast MCF-7 (Table 1), prostate PC-3 and colon HCT (in all cases, IC 50 = 1.9-3.9 µM) [69].  Amidrazones possessing a thiophenyl (71)(72), flavone (73)(74) or coumarin (75) moiety, as well as bisamidrazone derivative 79, showed antiproliferative activity against the MCF-7 and K562 cancerous cell lines (Table 1). Compounds 72, 76 and 79 had low toxicity to human fibroblasts in vitro. Molecular docking revealed a similarity of compounds 72-76 with imatinib (a drug belonging to the group of tyrosine kinase inhibitors) during interactions with bcr-abl tyrosine kinase, which may indicate a similar mechanism of action of those compounds [70][71][72][73][74][75][76]. Alternatively, according to in silico studies, derivative 79 could act as an effective inhibitor of phosphatidylinositol 3-kinase, the hyperactivity of which was observed in cells of the MCF-7 line [77]. Ciprofloxacin derivatives 18-19 showed antiproliferative activity against the HeLa and MCF-7 cancerous cells [23]. Amidrazones 78-79, which possess a chloroquine moiety, showed antiproliferative activity against the cervix HeLa and MCF-7 cancer cells [23].
Indoleamidrazone 80 inhibited the proliferation of MCF-7 cells by 68% at a concentration of 100 µg/mL [55]. As previously mentioned, the similar compounds 60-63, which possess nitro-or methoxy-phenyl substituents instead of the benzyl observed in 80, were inactive, except for derivative 63, which showed a 61.5% growth inhibition of MCF-7 cells [55].
Aminoguanidine derivative 81 demonstrated strong antiproliferative activity against MCF-7 and an inhibitory effect on tubulin polymerization (IC 50 = 8.4 µM). Molecular docking revealed that the probable mechanism of derivative 81 may be connected with colchicine biding [78]. Compound 82 showed a potent inhibition of ribosomal kinase RSK2 and MCF-7 tumor cell growth inhibition [79].
Computational methods were used to identify compounds with anticancer properties. Aminoguanidine derivative 83 was one of the predicted compounds, with a confirmed antiproliferative activity against HL-60 leukemia cells (IC 50 = 11 µM) and low to towards Vero cells (IC 50 > 100 µM) [80].
Cu(II) complex 90 showed antiproliferative activity against the Colo-205 adenocarcinoma cell line and low toxicity to MRC-5 human lung fibroblasts [86]. Another Cu(II) complex, 91, at concentration 100 µg/mL, showed a similar (almost total) antiproliferative activity to cisplatin against colon CX-1 and colon SW-948 cancer and epidermal A431 cell lines but was about 12-fold less toxic than the reference drug [49].
In 2022, two publications describing the antitumor activity of N 1 -benzylidenepyrazine-2-carbohydrazonamide complexes were published. The strongest activity was reported for the cobalt complex against glioma U87 MG cancerous cells (IC 50 = 7.69 µg/mL) [87,88]. However, the structures of those complexes have not been precisely specified.

Furin Inhibition
Furin is a trans-membrane protein which plays an important role in many bacterial and viral diseases, tumorigenesis, neurodegenerative disorders and diabetes [89]. It has recently been shown that furin inhibitors can be used to successfully block the entry of the SARS-COV-2 virus [90]. Aminoguanidine derivatives 92 and 93 ( Figure 10) showed furin inhibitory activity (K i = 0.46 µM and 0.58 µM, respectively). Additionally, derivative 92 also showed inhibitory activity against trypsin, while compound 93 was also a thrombin inhibitor [89].
Cu(II) complex 90 showed antiproliferative activity against the Colo-205 adenocarcinoma cell line and low toxicity to MRC-5 human lung fibroblasts [86]. Another Cu(II) complex, 91, at concentration 100 µg/mL, showed a similar (almost total) antiproliferative activity to cisplatin against colon CX-1 and colon SW-948 cancer and epidermal A431 cel lines but was about 12-fold less toxic than the reference drug [49].
In 2022, two publications describing the antitumor activity of N 1 -benzylidenepyrazine-2-carbohydrazonamide complexes were published. The strongest activity was reported for the cobalt complex against glioma U87 MG cancerous cells (IC50 = 7.69 µg/mL) [87,88]. However, the structures of those complexes have not been precisely specified.

Furin Inhibition
Furin is a trans-membrane protein which plays an important role in many bacteria and viral diseases, tumorigenesis, neurodegenerative disorders and diabetes [89]. It has recently been shown that furin inhibitors can be used to successfully block the entry of the SARS-COV-2 virus [90]. Aminoguanidine derivatives 92 and 93 ( Figure 10) showed furin inhibitory activity (Ki = 0.46 µM and 0.58 µM, respectively). Additionally, derivative 92 also showed inhibitory activity against trypsin, while compound 93 was also a thrombin inhibitor [89].

Acetylocholinesterase Inhibition
Several compounds were identified as potential acetylcholinesterase (AChE) or butyrylocholinesterase (BChE) inhibitors in the search for potential drug candidates for treating Alzheimer's disease ( Figure 11, Table 2). Compound 75 showed high activity

Acetylocholinesterase Inhibition
Several compounds were identified as potential acetylcholinesterase (AChE) or butyrylocholinesterase (BChE) inhibitors in the search for potential drug candidates for treating Alzheimer's disease ( Figure 11, Table 2). Compound 75 showed high activity against, and selectivity to, BChE and was about 3900 times stronger in its activity against this enzyme than tacrine [91]. against, and selectivity to, BChE and was about 3900 times stronger in its activity agains this enzyme than tacrine [91].
Aminoguanidine derivative 94 showed a threefold stronger AChE inhibitory activity than rivastigmine and no selectivity towards BChE. Compound 95 was a selective inhibi tor of BChE, with an approximately 16-fold lower AChE inhibitory activity, while deriv ative 96 was a selective AChE inhibitor. This proves the great potential of aminoguanidine derivatives, which may, in the future, act as inhibitors of various types of cholinesterases [92].

Summary
We compiled the biological activities of amidrazone derivatives described in the  Aminoguanidine derivative 94 showed a threefold stronger AChE inhibitory activity than rivastigmine and no selectivity towards BChE. Compound 95 was a selective inhibitor of BChE, with an approximately 16-fold lower AChE inhibitory activity, while derivative 96 was a selective AChE inhibitor. This proves the great potential of aminoguanidine derivatives, which may, in the future, act as inhibitors of various types of cholinesterases [92].

Summary
We compiled the biological activities of amidrazone derivatives described in the years 2010-2022. Antimicrobial, antitumor, anti-inflammatory and antiparasitic activities constitute the main kinds of exhibited biological activities. The most important compounds studied in vitro are presented in Table 3, together with their activity details. Due to their advanced stages in preclinical studies, they form an important group, from which new therapeutic substances may emerge. Compounds with known mechanisms of action are summarized in Table 4.  Among the antimicrobial agents, delpazolid showed a low toxicity and high efficacy and is undergoing further clinical trials for the treatment of tuberculosis. The 2pyridylamidrazone moiety determines the anti-mycobacterial properties of compounds 2-7.
It is worth noting that the amidrazones with the unsubstituted nitrogen N 3 (2-7, 9-14 and 33-37) showed stronger antimicrobial properties than amidrazones 54-55, which are N 3substituted with aryl rings [49,50]. In general, aminoguanidine derivatives 22-31 revealed a wider range of antimicrobial activities, as well as stronger antibacterial and antifungal properties than amidrazones 9-21. Moreover, derivative 22, which possesses two aminoguanidine groups, showed the strongest antimicrobial effects. Aminoguanidine derivatives 30-31 showed significant antibacterial effects in various animal models and deserve further research.
Eight derivatives (23, 25-26, 55, 60-63) showed significant anti-inflammatory activity in rodents. Moreover, the anti-inflammatory effect of compound 65, used in the research on the treatment of neonatal anti-sepsis in mice, deserves greater attention.
Amidrazones demonstrated a diverse number of antitumor mechanisms, acting as brc-abl kinase inhibitors (72)(73)(74)(75)(76), an inhibitor of phosphatidylinositol 3-kinase (79), an inhibitor of tubulin polymerization (81) and an inhibitor of ribosomal kinase RSK2 (82), which indicates their potential in the search for new anti-cancer drugs. Compound 72 showed the highest selectivity and may be a future drug candidate for leukemia.
Aminoguanidine derivatives exhibited cytoprotective activity and inhibited cholinesterases. Their possession of both these mechanism simultaneously could be useful in the search for a cure for Alzheimer's disease. The phosphorylation of eIF2α translation initiation factor by guanabenz, sephin1 or raphin1 is promising in regard to the prevention and treatment of many neurodegenerative diseases. For example, guanabenz, an old-generation antihypertensive drug, is currently being studied for new potential medical applications, including the treatment of amyotrophic lateral sclerosis, multiple sclerosis and parasitic toxoplasmosis.
Amidrazones showed moderate toxicity in various models (Table 5). However, among the derivatives with the lowest toxicity, as many as five (44-45, 56-57 and 59) contain an acyl group at atom N 1 , which may be valuable for the synthesis of new derivatives with more advantageous properties.  A useful property of amidrazones is their use as ligands for the synthesis of complexes with metals, which provides researchers with the opportunity to obtain new compounds with anti-tumor (e.g., 86) or antibacterial (32) properties.

Conclusions
Amidrazones remain an interesting area for researchers, as evidenced by the latest works from 2022. Many derivatives described in this review show strong biological activities and deserve more detailed research in this field. We hope that this article, which systematizes the knowledge about the biological activities of amidrazones, will increase the scientific interest in these compounds and, in effect, will encourage the development of novel derivatives and their introduction to research in preclinical and clinical studies.

Conflicts of Interest:
The authors declare no conflict of interest.