An overview: metal-based inhibitors of urease

Abstract

Urease is a kind of nickel-dependent metalloenzyme, which exists in the biological world widely, and can catalyse the hydrolysis of urea into ammonia and carbon dioxide to provide a nitrogen source for organisms. Urease has important uses in agriculture and medicine because it can catalyse the production of ammonia. Therefore, in this review, metal-based inhibitors of urease will be summarised according to different transition metal ions. Including the urease inhibition, structure–activity relationship, and molecular docking. Importantly, among these reviewed effective urease inhibitors, most of copper metal complexes exhibited stronger urease inhibition with IC₅₀ values ranging from 0.46 μM to 41.1 μM. Significantly, the collected comprehensive information looks forward to providing rational guidance and effective strategies for the development of novel, potent, and safe metal-based urease inhibitors, which are better for practical applications in the future.

Keywords:

• Urease
• metal-based urease inhibitor
• structure–activity relationship
• molecular docking

Subject classification codes:

• Review

Introduction

Urease (EC 3.5.1.5, urea amide hydrolase) is a natural enzyme strictly dependent on the metal ion nickel, widely distributed in fungi, bacteria, algae, plants, and other species ^1,2. Besides, urease can catalyse the hydrolysis of urea to form carbamic acid and ammonia ^3,4. In 1995, the first complete structures of three-dimensional urease have been reported via the crystallographic study of Klebsiella aerogenes urease⁵. Since then, the researchers have elucidated other structures of ureases from Canavalia ensiformis⁶, Pasteurella⁷, and Helicobacter pylori (H. pylori)⁸. Moreover, the structure of urease contains heteromeric molecules with three subunits α, β, and γ⁹, and the active site of urease is located in the α subunit, which has two Ni²⁺ (Ni₁ and Ni₂) centres¹⁰. Additionally, these two nickel ions are essential in the general mechanism of catalysis by urease⁷. One of the nickel ions is responsible for binding and activating urea, while the other binds and activates nucleophilic water molecules⁷. When the water molecules are replaced, the N atom of amino on the urea combines with Ni₂, which promotes the release of NH₃ and carbamate⁷.

It was reported that urease plays a vital role in various fields^11,12. Literature also has demonstrated that the increase in ammonia concentration will cause an increase in pH, which will have implications for agriculture and medicine^13–16. Therefore, in agriculture, highly active urease releases a large amount of ammonia into the atmosphere in the process of fertilising with urea, causing serious problems including environmental and economic¹⁷. Additionally, the deprivation of essential nutrients and elevated soil pH will harm plants¹⁸. Medically, urease is a major virulence factor in various pathogens of humans, such as ureolytic bacteria, represented by H. pylori, which could lead to human and animal health complications, including pyelonephritis, kidney stone formation, hepatic encephalopathy, and eventually hepatic coma^9,19. What’s more, urease is the leading cause of H. pylori lesions for it allows bacteria to survive during colonisation in an environment that is barely acidic in the stomach. The main reason is that the NH₃ produced by H. pylori urease can neutralise the acid of the stomach. And as the concentration of ammonia increases, the pH suddenly increases, causing obvious negative effects^12,20–23. Therefore, it is clear that the activity of urease is an important determinant of virulence in the pathogenesis of many clinical diseases harmful to human and animal health as well as agriculture²⁴.

Schiff base, an active pharmacophore, has been proven to be an effective antibacterial agent by literature^25,26, and exhibited significant urease inhibition activity²⁷. Additionally, transition metals presented important biological activity in many biochemical processes²⁸. Commonly used transition metals are copper, nickel, cobalt, zinc, and so on. At the same time, complexes formed with various metal ions have been shown to present significant inhibitory activity against various enzymes^29,30. Therefore, combing these two effective agents, metal complexes based on Schiff base as potential urease inhibitors had been synthesised and investigated widely.

Urease has important uses in agriculture and medicine because it can catalyse the production of ammonia. Thus, in this review, heavy ion complexes based on Schiff base ligands as urease inhibitors will be summarised. Besides, some metal complexes with other ligands having urease inhibitory activity will be discussed as well.

Metal-based urease inhibitors

Schiff base metal complexes

Schiff bases (–C = N–) is very well-known for being active pharmacophore, which exhibits potent biological potential³¹. Besides, transition metal complexes based on Schiff base ligands have been investigated broadly for their potential biological potency³². Therefore, a series of transition metal complexes based on Schiff base was prepared.

Schiff base copper metal complexes

Copper, an endogenous biocompatible trace element, plays a key part in the varieties of enzymes in biological systems³³. Two azido-bridged Schiff base copper complexes 1 and 2 were synthesised (Figure 1)³⁴. The bioactivities of these two complexes were performed, and it was displayed that complexes 1 (IC₅₀ = 27.82 ± 0.35 μM) and 2 (IC₅₀ = 25.23 ± 0.70 μM) showed good activities against urease in comparison with the reference compound acetohydroxamic acid (AHA, IC₅₀ = 42.12 ± 0.08 μM). While the corresponding Schiff base ligands had inactive on inhibiting urease. The studies of SAR found Cu atoms in these two complexes were five-coordinate in a square pyramidal configuration, which involved the three N atoms of the Schiff base ligand and two N atoms from two bridging azide ligands. Moreover, it was discovered that the azide ligands adopt an end-to-end bridging mode in complex 1 and an end-on bridging mode in complex 2. Further, the SAR study displayed that ligands had an important role in inhibiting urease. Therefore, modifying the raw structures into new ones could improve urease inhibitory activities effectively. All the results indicated that the ligands’ steric structure influences the coordinate mode of the azido co-ligand.

Figure 1. Schiff base copper metal complexes 1–6.

Two novel copper (II) complexes (complex 3 and 4) were synthesised³⁵. The inhibitory activities of these complexes were carried out and results showed complexes 3 and 4 had moderate anti-urease activities. The IC₅₀ values of 3 and 4 were 37.2 ± 0.5 μM and 41.1 ± 0.7 μM, respectively (Figure 1). Compared with these two novel complexes, the positive compound AHA (IC₅₀ = 46.5 ± 0.3 μM) showed weaker inhibitory activities. Moreover, it was found that Schiff base ligands showed inactive against urease. The SAR delivered that the structure of 3 was an azide coordinated monokaryon while the structure of 4 was a terminal thiocyanate coordinated monokaryon. Furthermore, each copper was a four-coordinated square-planar with a phenolic O, an imine N, an amine N with Schiff base ligand, and a terminal coordinated N which enabled complexes more stable and displayed higher urease inhibitory activity. All research results indicated these two complexes had the potential to be urease inhibitors.

Six transition Cu (II) complexes of Schiff base were synthesised and the inhibitory ability against Jack bean urease had been evaluated³⁶. Urease inhibition displayed that Cu (II) complexes 5 and 6 (IC₅₀ values = 0.52 μM and 0.46 μM) had higher urease inhibition than the positive control AHA (IC₅₀ = 42.12 μM) (Figure 1). Meanwhile, it was also found that the urease inhibition of these two selected effective complexes were equivalent to Cu²⁺ (IC₅₀ = 0.37 μM). The docking descriptions of complexes 5 and 6 illustrated that the atom of phenolic oxygen forms one kind of hydrogen bond in compounds, and there were two kinds of hydrogen bonds formed in the binding model of complex 6. Besides, the formation of hydrophobic interactions of complex 5 with Ala197, Leu196, and Phe273 of urease, and complex 6 could form hydrophobic interactions with Ile172, Leu196, Ala197, Ile220, and Ile247 of Jack bean urease.

Two copper (II) complexes with Schiff base 7 and 8 were synthesised³⁷_. Besides, the urease inhibitions of these complexes were carried out as well. The results of bioactivities revealed that complex 7 (IC₅₀ = 0.986 ± 0.012 μM) showed excellent anti-urease activities (Figure 2), while complex 8 displayed inactive on inhibiting urease with an IC₅₀ value above 100 μM. Obviously, the activity of inhibiting the urease enzyme of 7 was stronger than the positive reference AHA (IC₅₀ = 36.13 ± 0.20 μM). It was discovered from studies of structures of synthesised complexes that compound 7 was a mononuclear Cu (II) complex, while complex 8 consists of a dimer with the two phenolate O atoms linking the two compounds adjacently, which was the main reason for 8 to form a six-coordinate in an octahedral geometry. While the Cu in compound 7 forms a square planar geometry with four-coordinate. Importantly, the difference between these two complexes causes the difference in inhibiting ability against urease. The SAR explained the coordination of the Cu atoms may also be one of the influence factors of the Jack bean urease inhibitory activities of these compounds. In conclusion, complex 7, as a novel urease inhibitor, had great potential to be a urease inhibitor to be further investigated.

Figure 2. Schiff base copper metal complexes 7–11.

A copper (II) complex 9 with N-n-butylsalicylaldiminate of Schiff base was synthesised and the structure was defined by X-ray crystallography³⁸. The inhibition against H. pylori urease of 9 was performed and the result displayed that it had greater urease inhibition with an IC₅₀ value of 0.95 ± 0.04 μM (Figure 2). When compared with the standard reference AHA (IC₅₀ = 42.47 ± 0.19 μM) and Cu²⁺ (IC₅₀ = 1.35 ± 0.13 μM), this novel compound had a higher inhibitory activity of inhibiting urease, while the Schiff base ligand had inactive against urease. Furthermore, a molecular docking study of the copper (II) compound revealed the hydroxyl group of Phe218 of the urease protein formed a hydrogen bond with the oxygen of the complex. Additionally, the complex may form hydrophobic bonds by interacting with urease Leu196 and Ile220. The results displayed that hydrogen bonds and hydrophobic bonds may be the main reasons for inhibiting H. pylori urease effectively. Furthermore, the docking calculations also showed that complex 9 had lower binding free energy in comparison with AHA, which could further explain the excellent inhibitory activity of complex 9 in inhibiting H. pylori urease.

Two kinds of copper (II) complexes with Schiff base (10 and 11) were studied through synthesis, crystal structures, and urease inhibitory activity³⁹. Among these two complexes, complex 10 had a peculiar structure of end-to-end thiocyanato bridged polynuclear while complex 11 was mononuclear. The biological studies proved that both complex 10 (IC₅₀ = 31.3 ± 0.2 μM) and complex 11 (IC₅₀ = 20.5 ± 0.3 μM) showed moderate activities against urease in comparison with the positive compound AHA (IC₅₀ = 37.23 ± 0.27 μM) (Figure 2). In this work, it was further demonstrated copper (II) complex with Schiff base as a potent urease inhibitor had a higher inhibitory rate on inhibiting urease while exploring highly effective urease inhibitors is still needed to develop.

Four copper (II) complexes of Schiff base ligands were synthesised and evaluated their urease inhibitions⁴⁰. It was found complexes 12–15 (IC₅₀ = 1.45–3.59 μM) had strong inhibitory activities against urease (Figure 3). In addition, all these complexes were determined by X-ray and the results showed in the metal centre, they presented the square planar coordination geometry. From the molecular docking study and the structure–activity relationship, it was revealed that the complex molecules were well-fitted in the active centre of the urease. Additionally, the experimental operation was described as that 25 μL jack bean urease (12 kU/L) and 25 μL compounds with different concentrations (dissolved in DMSO/H₂O = 1:1 (v/v) were mixed and cultured at 37 °C for 1 h in a 96-well plate. Next, 0.2 ml, 100 mM Hepes buffer (pH = 6.8, containing 500 mM urea, 0. 002% phenol red) was incubated at 37 °C until the PH was changed from 6.8 to 7.7, and the endpoint of the reaction was monitored by a change in phenol red colour. At last, the absorbance value was measured at 570 nm. All results demonstrated these complexes had great potential to be investigated as urease inhibitors deeply.

Figure 3. Schiff base copper metal complexes 12–15.

Metal Cu (II) complexes as potent inhibitors of Jack bean urease had been reported by varieties literature. Therefore, based on a series of studies, three novel dimeric Cu(II) complexes with Schiff base were synthesised⁴¹. The crystal structures of synthesised complexes were determined by X-ray. These crystal structures are solved directly and optimised for F² by the full matrix least squares method. Besides, infra-red and UV-Vis spectroscopic were prepared for synthesised compounds. Moreover, the anti-urease activities of these complexes were carried out in vitro and the results revealed IC₅₀ values ranging from 7.20 μM to 11.00 μM. Among these effective complexes, complex 16 (IC₅₀ = 7.20 μM) exhibited higher anti-urease activity than other complexes (Figure 4). Compared with the positive compound AHA (IC₅₀ = 63.00 μM), all complexes had great potential to be potent urease inhibitors. Controlled experiments displayed that Schiff base ligand had no activity on inhibiting urease (IC₅₀ > 100 μM). Nevertheless, free Cu²⁺ showed strong urease inhibitory activity with an IC₅₀ value of 10.85 μM. Similar to the inhibitory effect of other heavy metal ions^38,42, the urease inhibitory activity of copper (II) ion might be due to its interaction with the active site of urease. Furthermore, the SAR was also investigated and results demonstrated that the difference in electron-withdrawing property of halogen ligands resulting in copper (II) complexes showed a slight difference in inhibiting urease. Importantly, docking studies found complex 16 could form two kinds of hydrogen bonds via interacting with amino acid residues His593 and His492, respectively.

Figure 4. Schiff base copper metal complexes 16–18 and docking study of most active inhibitor 18.

Six novel Cu(II) complexes based on O, N, O-tridentate of Schiff bases complexes were studied and synthesised⁴³. The solid-state structures of these complexes consist of four-coordinate mononuclear copper (II) units with a slightly distorted square planar geometry. In this literature, anti-urease activities of synthesised compounds were tested, and the results displayed that all complexes showed good inhibition with IC₅₀ values ranging from 2.15 ± 0.11 μM to 32.12 ± 0.65 μM, while the urease inhibitory activity of standard compound AHA (IC₅₀ = 60.22 ± 0.85 μM) was lower than all complexes distinctly. Among these complexes, complex 17 had the strongest inhibitory activity with IC₅₀ of 2.15 ± 0.11 μM (Figure 4). Additionally, the investigation of molecular docking was performed as well, it explained the main reason was the complex filled with the active centre of the urease enzyme. The docking studies revealed that these new complexes with O, N, O-tridentate of Schiff bases had potential great potential to be urease inhibitors, but the inhibitory mechanisms of these kinds of complexes are going to study in the future.

Four kinds of Cu (II) complexes were synthesised⁴⁴, and the urease inhibition of these synthesised complexes was carried out in vitro. The results of inhibiting urease assay showed that all compounds had different degrees of inhibiting urease activities, and the IC₅₀ values were between 1.00 μM and 8.01 μM. Out of these effective compounds, complex 18 (IC₅₀ = 1.00 μM) had excellent urease inhibition than the standard compound acetohydroxamic acid (IC₅₀ = 27.7 μM) and other compounds (Figure 4). Importantly, the mechanism of all synthesised complexes was elaborated that complex 18 was a non-competitive inhibitor. The results of the computer molecular docking showed that these four metal complexes could interact with the centre of the urease, which could be a good reason to further explain the active mode between complexes and urease. Additionally, docking also displayed complex 18 could form a more stable inhibitory binding model via salt bridges with amino acid.

Schiff base nickel metal complexes

Nickel (II) ion is not only an important part of urease but also the complexes of Ni have been widely discussed because of the known various biological activities including antibacterial, antifungal and antiepileptic, and so on⁴⁵.

Five complexes based on Schiff base ligands were synthesised⁴⁰. Among these complexes, nickel metal complex 19 was further investigated in this paper. Furthermore, the anti-urease activity of this compound was performed, and the results showed that complex 19 had moderate urease inhibition with an IC₅₀ value above 50 μM (Figure 5). When compared with the positive compound AHA (IC₅₀ = 63.00 μM), the activity of inhibiting the urease enzyme of this complex was higher than it. Besides, the analysis of the crystal structure of 19 demonstrated that this complex consists of a discrete mononuclear Ni(L³)₂ unit and two lattice DMF molecules, which could further interact with DMF molecules via intramolecular hydrogen bonds.

Figure 5. Schiff base nickel metal complexes 19–22.

A novel nickel complex based on Schiff base 20 was prepared and its structure was characterised by X-ray⁴⁶. Besides, the activity against urease of this complex was evaluated and results demonstrated that complex 20 (IC₅₀ = 3.9 ± 0.2 μM) showed stronger urease inhibition than the positive reference AHA (IC₅₀ = 42.3 ± 0.4 μM) (Figure 5), even more than Ni²⁺ ion (IC₅₀ = 4.6 ± 0.3 μM). Furthermore, the results of docking found complex 20 exhibited a lower binding free energy with K_i of −11.72 kcal/mol than AHA (K_i = 10.07 μM), which also explained the excellent inhibitory activity against H. pylori urease of this compound.

A library of novel Ni(II) complexes was investigated and their structures were characterised by X-ray⁴⁷. In addition, their urease inhibitions were evaluated, and results demonstrated that complexes 21 and 22 were observed to be the most potent urease inhibitors, presenting IC₅₀ values of 1.17 ± 0.12 μM and 1.19 ± 0.41 μM, respectively (Figure 5). Compared with the standard reference thiourea (IC₅₀ = 23.3 ± 11.0 μM), these two effective complexes had excellent urease inhibition. Besides, the cytotoxicity and antileishmanial activity of these complexes were tested, and the Ni complex was shown to have important potential to inhibit both cell lines (BHK-21 and H157) in comparison with N, N, N’-trisubstituted. To conclude, complexes exhibited remarkable anti-cancer cell lines abilities and had no toxicity on normal cell lines. In addition, these synthesised complexes had an effective inhibitory effect on leishmaniasis. Therefore, these complexes had great potential to be candidates as urease inhibitors in the future.

Three Ni(II) complexes based on bis-Schiff base ligands were synthesised and urease inhibitions of these complexes were tested in vitro as well⁴⁸. Results of urease inhibition revealed all complexes had different degrees of activity against urease. Out of these complexes, compound 23 showed stronger activity in inhibiting urease, exhibiting an IC₅₀ value of 11.27 ± 2.08 μM (Figure 6). When compared with nickel acetate (IC₅₀ = 23.23 ± 2.25 μM) and positive compounds AHA (IC₅₀ = 30.73 ± 5.18 μM), urease inhibition of this complex was about more than twice. Moreover, the structure–activity relationship discovered Ni (II) ion interacted with Schiff-based ligands had an important role in physiological activity, it was deduced that due to the good conjugation of the indole ring ineffective compound 23, which could further explain the good urease inhibition of complex 23. Furthermore, the two indoles in complex 23 could form π-π stacking interaction with amino acid residues His594 and form σ–π interaction with Glu493.

Figure 6. Schiff base nickel metal complex 23 and the docking study of this compound.

Schiff base zinc metal complexes

Zinc complexes derived from Schiff bases have attracted the attention of more and more researchers in related fields due to their structural diversity and biological characteristics^49–54. Two new zinc(II) complexes of Schiff base (24 and 25) were synthesised, and the crystal structures of compounds 24 and 25 were characterised⁵⁵. Crystal structures displayed that the Zn atom in each complex was four-coordinated by one phenate O of Schiff base ligand, one imine N atom, and two bromine atoms to form tetrahedral coordination, which forms tetrahedral coordination. Besides, the Jack bean inhibitory activities of these two complexes were tested and results exhibited the inhibition rate of compound 24 was much lower than compound 25. The inhibition rates of complexes 24 and 25 were 23.18 ± 2.22% and 35.63 ± 1.45%, respectively (Figure 7). While compared with these two complexes, Schiff bases and ZnBr₂ had weaker inhibition rates. But all of these compounds showed weaker inhibitory activities than the standard reference AHA with an inhibition rate of 88.23 ± 2.08%.

Figure 7. Schiff base zinc metal complexes 24–27.

Two novel isostructural dinuclear zinc(II) complexes (26 and 27) based on Schiff base were synthesised⁵⁶. It was found that the Schiff base was a tridentate chelating and the Zn atom in complexes was five-coordinated in a square pyramidal geometry. Additionally, the urease inhibitions of complexes 26 and 27 were performed and the results revealed that compared with the reference acetohydroxamic acid (IC₅₀ = 45.37 ± 0.31 μM), both compounds showed poor activities against urease with IC₅₀ values of 72.03 ± 0.54 μM and 86.12 ± 0.23 μM, respectively (Figure 7). Moreover, both complexes showed stronger activities than the Schiff base ligands and zinc salts, which were used as the synthetic starting material.

Schiff base cobalt metal complexes

Cobalt is one of the most investigated transition metals for inhibiting urease activity, and cobalt complexes have been used in antibacterial, antiviral, antifungal and tumour cells, some of which have shown strong anti-urease activity^57–59. A series of novel cobalt (III) complexes were synthesised⁶⁰. In this literature, the inhibitory activities of urease of H. pylori were carried out, and it was shown that complex 28 (IC₅₀ = 0.35 ± 1.52 μM) showed effective activity against urease (Figure 8). Compared with the positive compound AHA (IC₅₀ = 37.2 ± 4.0 μM), the urease inhibition of complex 28 was more than 100 times, and cobalt perchlorate had also inactive on Jack bean urease. Furthermore, the results of molecular docking revealed that complex 28 could cooperate well with the active pocket of urease, and other complexes were located at the entrance of the active pocket. Additionally, this compound could form a hydrogen bond by interacting with amino acid residue His322, and several hydrophobic interactions could be found between the binding model and synthesised urease inhibitors. Moreover, the relationship between complex structure and urease inhibitory activity indicated that a complex with a suitable confirmation and flanks is very important for the inhibitory effect of urease.

Figure 8. Schiff base cobalt metal complexes 28–30 and the docking study of complex 30.

The novel complex of [CoL₂]NO₃ 29, which was based on the barbituric acid Schiff base ligand, 5-((benzyl amino)methylene)-1,3 dimethylpyrimidine-2,4,6(1H,3H,5H)-trione was synthesised⁶¹. The anti-urease bioactivity of complex 29 was screened in this paper, and the results presented that the synthesised ligand and corresponding complex were proved to have potential urease inhibitory activities. What’s more, compared with the standard compound AHA (IC₅₀ = 20.3 ± 0.43 μM), the complex had greater inhibition on urease with IC₅₀ of 16.0 ± 0.54 μM (Figure 8), but the ligand showed weaker urease inhibition. Moreover, it could be summarised from the single crystal X-ray structure of this novel complex that the structure contained six-coordinated Co(III) ion with barbiturate Schiff base ligand to be mononegative N₂O^- tridentate chelate, and the date of X-ray had also shown the structure of new [CoL₂]NO₃ complex was imine-enolate structure instead enamine-doine structure. Additionally, the molecular docking proved that the Co (III) complex of the barbituric acid Schiff base ligand could integrate with the active pocket of urease highly so that this complex could exhibit great inhibitory activity. In short, this kind of complex could be investigated further as a novel inhibitor in the future.

To find more inhibitors of urease, three novel Co(II) complexes based on N₂O₄-donor bis-Schiff base were synthesised⁶². The consequences of inhibiting Jack bean urease indicated that all complexes had potential urease inhibitions with various IC₅₀ values. Amongst these complexes, complex 30 (IC₅₀ = 16.43 ± 2.35 μM) showed the greatest activity against urease (Figure 8). Even more, the urease inhibitory activity of complex 30 was superior to the reference AHA (IC₅₀ = 26.99 ± 1.43 μM) and cobalt acetate (IC₅₀ = 21.36 ± 2.25 μM). In addition, the docking results showed complex 30 could form a more stable structure by forming special σ-π and two types of hydrogen bonds so that it could fit well with the active centre of the urease enzyme.

Other metal complexes

In addition to the transition metal complexes based on Schiff base ligands with urease inhibitory activity, some other ligands metal complexes also have urease inhibition. Such as vanadium, silver, manganese, cadmium, iron, and gold metal complexes.

Vanadium metal complexes

Vanadium complexes with a variety of ligands have been presented to have extensive biological effects such as normalising hyperglycaemia levels and acting as a model for haloperoxidases^63–65. A library of oxovanadium complexes was synthesised²². All urease inhibition rates of these complexes were tested at the concentration of 100 μM, and results showed the inhibition rates were ranging from 18.96% to 90.72%. Out of these effective compounds, complex 31 had the most effective activity against urease with urease inhibition of 90.72% and an IC₅₀ value of 17.35 μM (Figure 9). Compared with the positive compound AHA (inhibition rate: 87.30%, IC₅₀: 46.27 μM), complex 31 showed stronger urease inhibitory activity. Besides, the structure–activity relationship demonstrated that complexes with chloro-substituted were more effective than alkoxy-substituted complexes. Moreover, the results of kinetic studies illustrated complex 31 was a mixed-competitive urease inhibitor. Furthermore, a molecular docking study revealed the hydroxyl O atom, the oxo O atom and the hydroxy group of complex 31 could form three kinds of hydrogen bonds with the amino group of Arg338, His221, and the O of Asn168, respectively.

Figure 9. Vanadium metal complexes 31–33.

There were two oxovanadium (V) complexes (32 and 33) were prepared and their urease inhibitions were also performed⁶⁶. Results of pharmacological activity displayed that the IC₅₀ values of these synthesised complexes were 27.0 μM and 133 μM, respectively (Figure 9). Compared with the reference HAE (IC₅₀ = 40.3 μM), complex 32 showed more effective bioactivity than the positive and complex 33. Moreover, the results of docking further revealed compound 32 could fit well in the active centre of urease and contact with the residues, which exist in the active centre. Additionally, the amino moiety of the acetyl hydroxamic acid ligand was next to the two nickel atoms in the active centre of the urease. Importantly, it could be found that there were two hydrogen bonds between the phenate oxygen and the oxo group of the amino complex with amino acid residues Arg338 and His322. The results of docking could further explain why complex 32 had good urease inhibitory activity.

Sliver metal complexes

For thousands of years, silver as one of the pharmacologically active transition metals has been used for many medical conditions including antibacterial activity⁶⁷. Besides, literature discovered there are three highly conserved amino acid residues (αCys322, αHis323, αMet367) bound to two silver (I) ions in a unique way, which could block the movement of the flap of urease, thereby inhibiting enzyme-catalysed activity⁶⁸. Therefore, based on these important findings, Ag (I) complexes play important roles in inhibiting urease.

Two silver (I) (complexes 34 and 35) were synthesised and their urease inhibitory activities were also evaluated⁶⁹. It was found from the results of urease inhibition that these two complexes showed potent 0.66 μM and 1.10 μM, respectively (Figure 10). In comparison to the reference AHA with IC₅₀ of 63.05 μM, these effective complexes had stronger activities of inhibiting urease. It was obvious that complexes 34 and 35 had the potential to be further investigated as effective urease inhibitors.

Figure 10. Sliver metal complexes 34–37.

A library of novel silver (I) complexes was prepared and the urease inhibitory activities were screened as well⁷⁰. Among these synthesised complexes, complex 36 had the lowest IC₅₀ value of 1.73 ± 0.05 μM (Figure 10). What’s more, the urease inhibitory activity of 36 was better than AHA (IC₅₀ = 63.70 ± 0.13 μM), which was used to be the standard urease inhibitor. Obviously, in comparison with AgNO₃ (IC₅₀ = 3.47 ± 0.16 μM), the results demonstrated that complex 36 proved to be a better Jack bean urease inhibitor. It could be summarised that the chelation of N, N-eten ligands with Ag ions could enhance the biological activity of inhibiting urease. Ag (I) complexes as urease inhibitors with mixed ligands were investigated⁷¹. The general formula for these compounds is Ag(PEt₃)X (X = Cl⁻, Br⁻, I⁻, PEt₃). Urease inhibition revealed discovered that those complexes showed excellent inhibitory activities with nanomolar level. Especially, [Ag(PEt₃)I]₄ (37) (IC₅₀: 27 ± 3 nM) was the strongest urease inhibitor. Besides, enzyme kinetic results further showed that IC₅₀ values for the inhibitory activity of these compounds against urease were in the nanomolar range. Moreover, the analysis of crystal structure provided the structural insights into the mechanism of interaction between urease enzymes and Ag (I)-based inhibitors are further provided.

Manganese metal complexes

There were six complexes with different kinds of transition metal atoms were synthesised and the activities against Jack bean urease were tested⁷². Among these complexes, most compounds were inactive in inhibiting urease, while complex 38 with Mn²⁺ (IC₅₀ = 8.30 ± 0.93 μM) showed potent urease inhibition even stronger than the positive drug acetohydroxamic acid with IC₅₀ of 42.12 ± 0.08 μM (Figure 11). However, complex 39, which had the same metal atoms as 38 but different ligands, showed inactive on inhibiting urease. These results showed that the metal ions and ligands played an important role in urease inhibition. Besides, complex 40 with Mn (III) ions was synthesised⁷³. However, the results of urease inhibition displayed that compound 40 (IC₅₀ = 61.05 μM) showed moderate urease inhibition on this enzyme as compared with thiourea, which was tested as the reference standard with an IC₅₀ value of 21.25 μM (Figure 11).

Figure 11. Manganese metal complexes 38–40.

Cadmium metal complexes

Two isostructural cadmium(II) complexes (41 and 42) were synthesised and structures were characterised by X-ray diffraction⁷⁴. Additionally, the urease inhibition of these two complexes was carried out, and the results displayed that 41 and 42 inhibited urease with moderate activities. The IC₅₀ values for complexes 41 and 42 were 56.51 ± 0.62 μM and 52.17 ± 0.37 μM, respectively (Figure 12). Compared to cadmium nitrate (18.23 ± 0.33 μM) and acetohydroxamic acid (42.12 ± 0.08 μM), these two complexes showed weaker activities against the urease enzyme.

Figure 12. Cadmium metal complexes 41–43.

Complexes with cadmium(II) were synthesised to investigate the activities of inhibiting urease⁷⁵. Amongst these complexes, complex 43 exhibited potent with an IC₅₀ value of 9.11 μM, which was better than the reference standard acetohydroxamic acid (IC₅₀ = 42.12 ± 0.08 μM) (Figure 12). Moreover, the ability of 43 against urease was stronger than cadmium acetate (IC₅₀ = 19.31 ± 0.65 μM). All results displayed that cadmium ions play vital roles in urease inhibition.

Iron metal complexes

Four novel iron metal complexes were synthesised and characterised⁷⁶. Urease inhibitory activities of these synthesised compounds were evaluated. Among them, complex 44 with Fe^II atom showed inactive against urease (Figure 13). Besides, a series of transition metal complexes containing Fe (III) (45) were also prepared⁷⁷ (Figure 13). However, in this literature, in comparison with other complexes and the positive compound acetohydroxamic acid (inhibition rate: 90.2% ± 3.7), compound 45 exhibited much lower inhibition against the urease. The urease inhibition for 45 was 39.5% ± 2.1 at the concentration of 100 μM.

Figure 13. Iron metal complexes 44 and 45.

Gold metal complexes

There were five selected Au (III) compounds were reported with potent inhibition against jack bean urease with IC₅₀ values from 9.0 nM to 31 nM⁷⁸. Amongst these effective complexes, complex 46 (IC₅₀: 9.0 ± 0.2 nM) showed strongest urease inhibitory activities than others (Figure 14). The results of the crystal structure of 46 displayed there were two Au (III) ions bound to the highly conserved amino acid residues αCys322, αHis323, and αMet367. The special binding of gold ions to these amino acid residues could hinder the catalytic activity of urease. Additionally, it was also further found by the same team⁷⁹ that four Au (I)-compounds exhibited excellent urease inhibition with IC₅₀ values in the 30 nM −7 μM range. Especially, complex 47 showed excellent urease inhibition with IC₅₀ of 38 ± 1 nM (Figure 14). The crystal structures demonstrated that there were at least two gold ions bound to the binding domain of urease, and the coordination environment of the two essential nickel(II) ions at the active centre are completely unaffected, thereby eliminating enzyme activity.

Figure 14. Gold metal complexes 46 and 47.

Conclusion

Urease is an important enzyme, which is closely related to the formation of peptic ulcers and infectious stones. In addition, it plays an important part in pyelonephritis, the pathogenesis of urolithiasis, hepatic coma and hepatic encephalopathy, ammonia, and ductal scab as well. Nevertheless, the overexpression of urease will cause great harm to agriculture, animal health, and clinically related diseases. Therefore, the development of potent, effective, and low-toxicity urease inhibitors have attracted the attention of corresponding researchers. Since other inhibitors of urease including thiourea, phosphoramide and sulphonamide, flavone, coumarin, chalcone, indole, hydroxamic acid, benzimidazole, thiazole, barbituric acid, and thiobarbituric acid, quinazolinone, oxadiazole, and triazole have been previously reported⁸⁰, metal-based urease inhibitors are mainly reviewed in this article. Although in comparison with other urease inhibitors, metal complexes used as urease inhibitors showed moderate to potent urease inhibitory activity, this series of compounds are well worth further research. Therefore, we systematically outline the potency, cytotoxicity, SAR, molecular docking, and more details and experimental conditions of metal-based urease inhibitors, which is expected to provide more reasonable and feasible guidance for the later design and synthesis of more effective and safe metal-based urease inhibitors. What’s more, within the scope of this review, it could be found that copper (II) complexes are the most widely investigated and exhibited the lowest IC₅₀ values for urease inhibition. However, it could be found that there were many shortcomings in this class of urease inhibitors. For example, the issue of the stability of compounds in aqueous solutions has not been fully considered, such as azides, thiocyanates, nitrates, bromides, alkoxy groups and several other ligands may dissociate and be replaced by water. Additionally, whether there are differences in the results of the interaction of different sources of urease (such as Jack bean urease, H. pylori and other bacteria ureases) with synthesised metal complexes had not be discussed. More importantly, it was found that the inhibitory activity of synthesised complexes on urease was comparable to that of the corresponding metal ions³⁶. Thus, these issues need to be further solved in later research. To sum up, this review summarised the metal-based complexes inhibitors of urease, hoping to provide reasonable and effective guidance for further research of metal complexes.

Disclosure statement

The authors report no conflicts of interest.

Additional information

Funding

This work was supported by the One Thousand Talents Program of Guizhou Province (the fifth group, [2019]4).