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Study of Vanadium Complexes as Novel Inhibitors of Bacillus Pasteurii and Canavalia Ensiformis Urease Enzyme.

Byline: Muhammad Arif Lodhi, Uzma Ashiq, Rifat Ara Jamal and Khalid Mohammed Khan


Oxovanadium(IV) complexes with aroylhydrazine ligands have been synthesized. Structural elucidation of synthesized complexes is carried out with the help of various physical, chemical and spectral analysis. Dimeric nature of synthesized complexes further characterized by effective magnetic moments, spectral, conductance, CHN and metal analysis. In the current study we examined 1c to 10c vanadium (IV)-aroylhydrazine complexes for their mechanism of inhibition with the nickel containing active sites of Canavalia ensiformis (Jack bean) and Bacillus pasteurii ureases. Inhibition of both urease enzymes by compounds 1c-10c are depends on concentration. Lineweaver- Burk as well as Dixon plots and their secondary replots indicated that the mechanisms of inhibition exhibited by vanadium complexes 1c-10c are very diverse.

The Ki values ranging from 15.1-79.3 M for Canavalia ensiformis urease while for Bacillus pasteurii urease 25.2123.7 M. The model presented here is evidenced by biochemical enzyme kinetics data. Efforts are made to obtain significant approach into the pharmacophore needs of urease. The high affinity of these vanadium complexes along with their safe profile against plants could make them promising lead candidates and provides fertile ground for future research.

Key words: Vanadium complexes, Bacillus pasteurii, Canavalia ensiformis (Jack bean), Urease inhibition, Enzyme kinetics, Aroyl hydrazine


Enzymes have a vital function in germination process of plants nitrogen metabolism. Various microorganisms use nitrogen for nourishment from urease (urea amidohydrolase, EC: enzyme, which found in all over the plant and animal kingdom [1, 2]. For increasing yields of crops in form of urea as a fertilizer in agriculture is scammed by occurrence of activity of urease in soil. Degradation of urea fertilizer occurs fastly due to excess limits of urease in soil and in consequence loss of ammonia and phytopathic condition causes [3]. Many diseases in animals and humans like peptic ulcer, pyelonephritis and presence of stones in kidney are caused by urease [4]. Unfortunately for bacterial diseases, the medication and cure is usually useless [5], and just small number of controls has proven clinical practice. Thus the new and different cure and control is required.

For pharmaceutical research, the findings of most powerful and non toxic urease inhibitors can play a very significant role in pathological area. Previously, we have published a new classes of natural and synthesized urease inhibitors alongwith its kinetics and structure function relationship [6-9]. In our random screening we found oxovanadium(IV) complexes as potent inhibitor of medicinally significant enzyme urease [10]. The objective of the present study is to find effective inhibitors of urease to discover the feasible binding connections of these compounds in the protein. This study may guide future drug design to get more better and effective. In this article, we have endeavored to present new exciting findings concerned with vanadium(IV)-aroylhydrazine complex receptor binding events.


Materials and Methods

All reagent-grade chemicals were purchased from Aldrich or Sigma and used without further purification. Molar conductance of complexes were made using HANNA (HI-8633) conductivity meter (Romania). Effective magnetic moments of synthesized complexes were determined on a Sherwood MSB Mk1 magnetic susceptibility balance. Shimadzu 1601 UV-Visible spectrometer in a range of 200 to 900 nm was used to record UV- visible spectra of complex using UVPC v3.9 software. Concentration of complexes was 2.510-4 M. FT-IR spectra at 400-4000 cm-1 were recorded on KBr disks, employing a Shimadzu 460 IR spectrophotometer. Finnigan-MAT-311-A apparatus was used to determine EI-MS of ligands. Proton NMR of synthesized hydrazides at 400 and 500 MHz. were obtained from Bruker spectrometers. A Perkin Elmer 2400 series II CHN/S analyzer was employed in order to carry out CHN (elemental) analysis.

Vanadium contents in the complex were analysed by volumetric analysis based on redox reaction involving iodine [11]. Sulfate was analyzed by gravimetric method [12].

Synthesis of the Aroylhydrazine ligands (1-5)

To a solution of corresponding ester (10 mmol), 75 mL of ethanolic solution of hydrazine hydrate (50 mmol) was added and refluxed it for 4- 5 h. After removal of solvent using rotary evaporator, a Synthesis of the oxovanadium(IV)-aroylhydrazine complexes (1c-5c) In methanolic solution (15 mL) of hydrazide (5 mmol), methanolic solution (10 mL) of vanadyl sulphate (1.27 g, 5 mmol) was added and stirred with heating. The contents were refluxed for 2 2.5 h. A solid product was obtained (Scheme-1). The contents were cooled at room temperature. The resulting product i.e. complex was filtered. Washing of solid complex was done with methanol. Analytical and spectroscopic data of compounds 1c-5c are given below.


is(benzoylhydrazino)dioxovanadium(IV) sulfate [V2O2(1)2(H2O)2]SO4 (1c): Green solid. Anal. Calcd for C14H14N4O2SO4V2O2(H2O)2 (FW=536 gmol-): Cal: C, 31.34; H, 3.35; N, 10.44; V, 19.02 %, SO4, 17.91%. Found: C, 31.45; H, 3.62; N, 9.38; V, 19.10 %, SO4, 17.48 %. FTIR (KBr cm-): 1689 (C=O), 3600-2700 br (OH, NH2 stretch., NH), 1625 (NH2 bend.), 1625, 1516 (C=C), 1415 (C-N), 996 (V=O), 1178, 1070, 896, 710, 666, 544. UV Vis. , nm, (, M-1 cm-1): 262(8929), 301(8121), 427(7146), 559(1100), 771(20), 820(4); Molar conductivity (DMSO): 42.50 O-1cm2 mol-1. Effective magnetic moment: 1.32 B.M. Yield: 56 %. Diaquo--bis(2- nitrobenzoylhydrazino)dioxovanadium(IV) sulfate [V2O2(2)2(H2O)2]SO4 (2c): Blue solid. Anal. Calcd for C14H12N6O6 SO4V2O2(H2O)2 (FW = 626 gmol-): Cal: C, 26.83; H, 2.55; N, 13.41; V, 16.29 %. SO4, 15.33 %.

Found: C, 26.54; H, 2.64; N, 13.22; V, 16.39 %, SO4, 15.27 %. FTIR (KBr cm-): 1633 (C=O), 3600-2600 br (OH, NH2 stretch., NH), 1526 (NH2 bend.), 1584, 1452 (C= C), 1348 (C-N), 973 (V=O), 1162, 1085, 757, 668, 550, 462. UV Vis. , nm, (, M-1 cm-1): 263 (8402), 334 (6877), 504 (927), 766 (64), 823 (48); Molar conductivity (DMSO): 63.06 O-1cm2 mol-1. Effective magnetic moment: 1.21 B.M. Yield: 45 %.


is(4- nitrobenzoylhydrazino)dioxovanadium(IV) sulfate [V2O2(3)2(H2O)2]SO4 (3c): Green solid. Anal. Calcd for C14H12N6O6 SO4V2O2(H2O)2 (FW = 626 gmol-): Cal: C, 26.83; H, 2.55; N, 13.41; V, 16.29 %. SO4, 15.33 %. Found: C, 26.94; H, 2.65; N, 13.39; V, 16.35 %, SO4, 15.70 %. FTIR (KBr cm-): 1642 (C=O), 3600-2700 br (OH, NH2 stretch., NH), 1527 (NH2 bend.), 1613, 1490 (C=C), 1350 (C-N), 984 (V=O), 1187, 1115, 1036, 854, 713, 589, 462. UV Vis. , nm, (, M-1 cm-1 ): 268 (15,999), 451 (7225), 550 (2178), 773 (16), 834 (4); Molar conductivity (DMSO): 60.96 O -1cm2 mol -1. Effective magnetic moment: 1.37 B.M. Yield: 68 %.


is(nicotinoylhydrazino)dioxovanadium(IV) sulfate [V2O2(4)2(H2O)2]SO4 (4c): Green solid. Anal. Calcd for C12H12N6O2SO4V2O2(H2O)2 (FW = 538 gmol-): Cal: C, 26.76; H, 2.97; N, 15.61; V, 18.95 %. SO4, 17.84 %. Found: C, 26.54; H, 2.84; N, 15.49; V, 19.01 %, SO4, 17.86 %. FTIR (KBr cm-): 1654 (C=O), 3700-2500 br (OH, NH2 stretch., NH), 1562 (NH2 bend.), 1609, 1479 (C=C), 1365 (C-N), 974 (V=O), 1175, 1124, 1034, 699, 602, 483. UV Vis. , nm, (, M-1 cm-1): 260 (6946), 290 (4885), 430(4325), 559(756), 769(16), 838(8); Molar conductivity (DMSO): 80.91 O-1cm2 mol-1. Effective magnetic moment: 1.39 B.M. Yield: 59 %.


is(isonicotinoylhydrazino)dioxovanadium(IV) sulfate [V2O2(5)2(H2O)2]SO4 (5c): Green solid. Anal. Calcd for C12H12N6O2SO4V2O2(H2O)2 (FW = 538 gmol-): Cal: C, 26.76; H, 2.97; N, 15.61; V, 18.95 %. SO4, 17.84 %. Found: C, 26.88; H, 2.99; N, 15.75; V, 18.45 %, SO4, 17.82 %. FTIR (KBr cm-): 1656 (C=O), 3700-2700 br (OH, NH2 stretch., NH), 1566 (NH2 bend.), 1609, 1477 (C=C), 1366 (C-N), 980 (V=O), 1124, 1039, 696, 651, 602, 473. UV Vis. , nm, (, M-1 cm-1): 263 (5910), 434(4123), 552(829), 770(32), 837(28); Molar conductivity (DMSO): 46.12 O -1cm2 mol -1. Effective magnetic moment: 1.39 B.M. Yield: 62 %.

Urease Assay and Inhibition

5 L of test compounds were incubated for 15 minutes at 30 C in 96-well plates along with 25 L of enzyme (Canavalia ensiformis and Bacillus pasteurii urease) solution and 55 L of buffers containing urea (2-24 mM for Canavalia ensiformis and Bacillus pasteurii urease) at pH 6.8 (3 mM sodium phosphate buffer. Phenol red (7 g/mL ) as an indicator was used. All readings were taken in a 200 L of final volume. The absorbance at 560 nm was measured against the control after 10 minutes, using a microplate reader (Spectra Max, Molecular Devices, USA) spectrophotometer. The data of change in absorbance per minute were manipulated by using SoftMax Pro software (Molecular Device, USA). The percentage inhibitions were calculated from the following formula:


Thiourea was used as positive control.

Determination of Kinetic Parameters

The IC50 of test compounds (i.e., the compound concentration inhibiting the hydrolysis of substrates (Canavalia ensiformis urease and Bacillus pasteurii ureases) were computed using EZ-Fit Enzyme Kinetics Program (Parella Scientific, Amherst, USA) through examining the percent inhibitory effect of samples at various concentrations. The interaction of 1c-10c with Canavalia ensiformis and Bacillus pasteurii Bacillus ureases are described in the Scheme 2-4. Where ES is the Canavalia ensiformis or Bacillus pasteurii urease-urea complex and P is the product. Interactions of inhibitors with the free Canavalia ensiformis or Bacillus pasteurii urease represented by K1 and the Canavalia ensiformis or Bacillus pasteurii urease-urea complexes represented by AYK1 inhibition constant.

To determine the kinetics parameters, Dixon plot was used to determine inhibition constant (Ki) [15] and Lineweaver-Burk plot [16] over the substrate was used at different concentrations 2.0-2.9 mM urea for Canavalia ensiformis and 4.5-6.3 mM for Bacillus pasteurii urease. In Dixon and Lineweaver-Burk plot, the values of KI, Km and Vmax were determined by non linear regression analysis.

Statistical Analysis

GraFit program was used to plot graphs [17]. Using the same program by linear regression, values of standard errors, correlation coefficients, intercepts and slopes were determined. The mean of three experiments shown in plotted graph.

Results and Discussion

Characterization of Oxovanadium(IV) Complexes of Aroylhydrazine

The structure of the complexes was confirmed by C, H, N, V, SO4 analysis, magnetic moment, conductivity measurements, UV-vis and IR spectroscopy. Dimeric nature of the vanadium- aroylhydrazine complexes was supported by magnetic properties [18, 19]. Synthesized complexes showed magnetic moments in the range of 1.21 to 1.39 which are lesser than that displayed by V(IV) complexes. Numerous reports are present for dimeric oxovanadium(IV) complexes which shows antiferromagnetic coupling [20, 21]. These binuclear complexes show similar properties exhibited by complexes reported herein [22-24]. Constant lower magnetic moments of every complex signify that the vanadium cores are antiferromagnetically coupled.

Molar conductivity of the complexes in DMSO were found in the range of 42.50 to 80.91 ohm-1 cm2 mol.-1 which showed 1:1 ionic ratio [25] suggested sulfate as counter anion. Presence of sulfate ion as counter ion was confirmed by treating solution of synthesized complexes with barium chloride. Coordinated sulfate will not react with barium chloride whereas non-coordinated sulfate will give a precipitate.

UV-Visible Spectroscopy

Solutions of oxovanadium(IV) complexes in DMSO display UV-visible transitions as arranged in experimental. For comparative purpose electronic transitions of ligands are also listed. All the ligands have carbonyl group attached with benzene ring with different substituent groups along with NH-NH2 (Scheme-1). For vanadium complexes, the absorption around 430 nm is tentatively assigned to MLCT (vanadium-aroylhydrazine) transitions. p to p transitions which are also observed in un-complexed ligands in the range of 250-300 nm come up from aromatic C=C. These bands are moved to lower wavelength on complexation with the metal center, which point to lowering of the energy of p-orbital of the aroylhydrazine ligand.

Nevertheless, several vanadium complexes display an additional transition at higher wavelength around 290-334 nm which are absent in un- coordinated ligands. These additional peaks are cautiously dispensed to p to p transitions of C=N group [26] which is at hand in the complex and is missing in the ligand.

In general, three absorption bands are found at room temperature. Each complex accounted in this research found to have DMSO coordination at sixth position confirmed by the presence of weak intensity transitions beyond 700 nm [26-33].

Stability studies of DMSO solution of oxovanadium(IV) complexes with respect to time, reveal very exciting behavior. Fig. 1a and 1b show the spectrum of the vanadium complexes with ligand 11 which has pyridyl ring making exhibiting least steric hindrance. A freshly prepared solution of this complex exhibits four peaks in visible and ultraviolet region. One peak at 263 nm in UV region is ligand based p-p transitions, however, peaks in visible region observed at 552 and 434 nm are cautiously correspondingly consigned to band II and III transitions [32, 34-37]. It was observed that absorbance of this solution increases with time and achieve maximum absorbance after 5 days (Fig. 1a, 1b). At this stage this complex exhibited two more peaks above 700 nm which show the coordination of DMSO at sixth position. It has been noted that no peak above 700 nm was observed in the spectrum taken in between 3 h. It showed that DMSO does not attach at sixth position in 3 h.

After five days a decrease in absorbance was observed at all four wavelengths resulting in a spectrum which is similar to the spectrum observed by dissolving vanadyl sulfate in DMSO. This new complex is attribute to the formation of [VO(DMSO)5].2+ All reported complexes show same behavior i.e. all complexes exhibit band I, II and III transitions. All oxovanadium complexes with diverse aroylhydrazine ligands are found to have band II and III transitions at nearly same positions, which indicates that electronic environment of the vanadium centers are similar and does not show any significant effect of substituents on aroylhydrazine ligands on these transitions. These electronic transitions are dependent upon the geometry around metal center and their similar peak positions indicate similar geometry of all complexes.

Infrared Spectroscopy

IR spectral data of aroylhydrazine ligands and their VO(IV) complexes is given in experimental part. Aroylhydrazine ligands display two reasonably strong stretching vibrations around 3010-3331 cm-1 because of two sorts of N-H vibrational modes. The strong bands in this region are due to hydrogen bonded -NH protons [38]. All vanadium complexes showed a broad absorption bands around 3700-2500 cm-1 which showed the existence of non-hydrogen bonded groups.

The IR bands at 1654 12 associated with (C=O) stretching frequency of the uncoordinated ligand is shifted to lesser energy 1640 14 after complexation, indicating the coordination of carbonyl group through its oxygen atom. Coordination of the oxygen atom of C=O to metal center with concomitant deprotonation of amine nitrogen would result in double bond character of C-N bond which is likely to result in higher stretching frequency. This assumption is favored by the raise in C-N frequency of complex which is 1340 9 cm-1 in free ligand and increased to around 1356 9 cm-1 in complex. Every complex exhibit sharp V=O stretching band around 970 10 cm-1 which are similar to (V=O) stretches accounted for other oxovanadium(IV) complexes [39, 40]. These peaks are not present in the uncoordinated ligands.

Enzyme Kinetics Studies

Hydrolysis of urea is catalyzed by a large heteropolymeric urease by using bimetallic nickel centre. Ureolytic bacteria are reported as responsible of several diseases, at the same time effectiveness of soil nitrogen fertilization with urea is brutally decreased by urease. Thus, for the diminution of environmental pollution there is a need of urease inhibitors, for improved efficiency of urea nitrogen uptake by plant and to enhanced therapeutic approach for handling of infections because of ureolytic bacteria. There is a need of knowledge of enzyme mechanism for structure-based seek of urease inhibitors [41]. High quality data sets for native Bacillus pasteurii urease (2.0 A ) (39), as well urease inhibited with phenylphosphorodiamidate (PPD) (2.0 A ) (3), b-mercaptoethanol (b-ME) (1.65 A ) [42, 43], acetohydroxamic acid (AHA) (1 A ), and b- mercaptoethylamine (b-MEA) (1.84 A ) afford extremely valuable information for the drug designing which is associated with geometry and structure.

The valuable information of the active site is exposed by arrangements of both native and inhibited urease. It gives important information regarding protein surroundings and function of the metal ions in catalysis. Native enzyme contains two Ni ions which are bonded by bridging hydroxide and water molecule [44]. Tetrahedral cluster of solvent molecules is completed by fourth water. Enzymatic hydrolysis of PPD produces diamidophosphoric acid (DAP), which bound to the two Ni ions in a unique mode. Mode of binding of DAP, which is tetrahedral state analogue, to the Ni ions of native and inhibited urease show a new mechanism for enzymatic urea hydrolysis. This mechanism is proved by structural and biochemical data [42, 43]. This mechanism reveals that both Ni are involved in activating and binding the substrate. In the process of urea hydrolysis, bridging hydroxide acted as the nucleophile.

Distinct conformations, of the flap lining the active site cavity, were observed in comparison of the native and DAP-inhibited structures. It gives the better knowledge of a role of this motif to understand the properties of active site, which speed up the reaction by stabilizing catalytic transition states [43].

Using high throughput screening method we are able to identify compounds 1c-10c which strongly inhibiting Canavalia ensiformis and Bacillus pasteurii ureases. Vanadium(IV) complexes study revealed that the onset inhibition of ureases by 1c- 10c vanadium(IV) complexes. Inhibition of ureases by compounds 1c-10c is not time dependent and is perfectly fixed to Michaelis-Menten kinetics. These experiments demonstrated that both ureases were strongly inhibited by studied vanadium complexes 1c-10c. Observations demonstrates that studied vanadium complexes 1c-10c had a lower Ki which shows much higher affinity for plant urease for microbial urease. All vanadium(IV) complexes inhibited urease with Ki values ranging from 15.1- 79.3 M for Canavalia ensiformis urease (Table-1) and 25.2123.7 M for Bacillus pasteurii urease (Table-2).

Inhibitor's interaction to the enzyme is because of an E. I complex isomerization. The early interaction of compounds 1c-10c with the both ureases proposes that there are no considerable hurdles come across for the proper alignment of compounds 1c-10c at the active site of the both ureases. Ki values were calculated in three different ways; first, the slopes of each line in the Lineweaver- Burk plot were plotted against different concentrations of inhibitors, secondly the 1/Vmaxapp was calculated by plotting different fixed concentrations of urease versus V in presence of different fixed concentrations of inhibitors in the respective assays of urease. Then Ki was calculated by plotting different concentrations of inhibitor versus 1/Vmaxapp. Ki was the intercept on the x-axis. In third method, Ki was directly measured from Dixon plot as an intercept on x-axis.

Determination of the inhibition type is critical for the identification of mechanism of inhibition and the sites of inhibitor binding. Lineweaver-Burk, Dixon plots and their replots indicated that these 1c-10c compounds exhibit competitive, non-competitive, un-competitive type of inhibition against Canavalia ensiformis and Bacillus pasteurii ureases. Indeed, inhibition by these vanadium(IV) complexes, it is because of interactions with amino acid residues situated within the active site in some cases while some are interacted little bit away from enzyme's active site. In case of competitive type of inhibition all vanadium(IV) complexes contend with urea in the urea-binding site at lower concentrations of the substrate, and stimulates a decline in substrate hydrolysis, whereas in excess of urea, inhibition is trounce. As expected for a competitive inhibitor, the Vmax is not modified, under these different experimental conditions.

In case of non-competitive inhibition Vmax is decreased while Km is not effected, while in un-competitive type of inhibition both Vmax and Km are decreased. It has been established that, whereas Canavalia ensiformis urease has maximum activity in buffers with near neutral pH urease from the alkaliphilic bacterium Bacillus pasteurii has maximum activity in alkaline buffers. Interaction of vanadium(IV) with protein are most valuable due to its application as therapeutic drug. Vanadium(IV)-aroylhydrazine complexes are small molecules, which interacts with protein's binding sites.

Table-1: Ki (dissociation constant or inhibition constant) was determined from nonlinear regression analysis by Dixon plot and secondary Lineweaver- Burk plot at various concentrations of 1c-10c, Km (Michaelis- Menten constant) is equal to the reciprocal of x-axis intersection, Vmax (maximal velocity) is equal to the reciprocal of y-axis intersection of each line for each concentration of 1c-10c in the Lineweaver-Burk plot. The Vmaxapp is equal to the reciprocal of y-axis intersection of each line for each concentration of 1c-10c in Dixon plot (Each point in Lineweaver-Burk and represents the mean of three determinations). Canavalia ensiformis urease).

Enzyme###Compounds###Ki (M) SEM###Km (mM)###Km (mM) app###Vmax (mol / min)-1###Vmaxapp###Type of Inhibition

###1c###27.0 0.1###2.5###2.5###105###86###Non-Competitive

###2c###31..9 0.6###2.5###2.5###105###69###Non-Competitive

###3c###38.5 0.4###2.5###10.7###105###102###Competitive

###4c###79.3 0.1###2.5###2.4###105###81###Non-Competitive

###5c###45.2 0.5###2.5###8.9###105###103###Competitive

Urease (JB)

###6c###45.0 0.3###2.5###8.6###105###103###Competitive

###7c###29.5 1.1###2.5###10.1###105###107###Competitive

###8c###15.1 0.4###2.5###6.4###105###107###Competitive

###9c###30.1 0.3###2.5###8.0###105###107###Competitive

###10c###24.3 0.0###2.5###2.3###105###77###Non-Competitive

Table-2: Ki (dissociation constant or inhibition constant) was determined from nonlinear regression analysis by Dixon plot and secondary Lineweaver- Burk plot at various concentrations of 1c-10c, Km (Michaelis- Menten constant) is equal to the reciprocal of x-axis intersection, Vmax (maximal velocity) is equal to the reciprocal of y-axis intersection of each line for each concentration of 1c-10c in the Lineweaver-Burk plot. The Vmaxapp is equal to the reciprocal of y-axis intersection of each line for each concentration of 1c-10c in Dixon plot (Each point in Lineweaver-Burk and represents the mean of three determinations). Urease (BP) (Bacillus pasteurii ureases).

###Enzyme###Compounds###Ki (M) SEM###Km (mM)###Km (mM) app###Vmax (mol / min)-1###Vmaxapp###Type of Inhibition











In the present study, we present novel inhibitors against urease by high throughput screening. We reveal that the studied vanadium complexes of various aroylhydrazines are capable to attach and inhibit the activity of the both ureases. The comparatively high Ki value of a few of these vanadium complexes specified their medium affinity for urease. This affinity could be enhanced by slight changes in structure of the vanadium(IV)- aroylhydrazine complexes. Enhancement of the attraction of studied vanadium complexes is of great significance because high-affinity vanadium complexes could be used as enzyme inhibitors in the clinic. The recognition of vanadium complexes of urease may unlock new approach for the improvement of therapeutic drugs, using vanadium complexes as scaffolds for the rational design of new chemical inhibitory molecules.


This study was supported by the Higher Education Commission (HEC) Pakistan for financial support (The National Research Grants Program for Universities', grant No.1862/RandD/10).


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