Synthesis of quinazolinone derivatives containing an acyl hydrazone skeleton as potent anti-urease agents enzyme kinetic studies and anti-oxidant properties

This paper covers the synthesis, in vitro urease inhibition, enzyme kinetic parameters, and anti-oxidant studies of a novel series of quinazolinone derivatives containing an acyl hydrazone skeleton. Compounds 3a, 3b, 5a, and 5b, having IC50 values ranging from 1.86 ± 0.07 to 6.38 ± 0.11 µg mL−1, show greater inhibitory activity than the standard inhibitor, thiourea. Among the products, (2-[2-(3-methoxybenzyl)-4-oxoquinazolin-3(4H)-yl]acetohydrazide) proves to be the most potent, exhibiting enzyme inhibition activity with an IC50 value of 1.86 ± 0.07 µg mL−1. Kinetic studies involving the Lineweaver–Burk plots reveal that the inhibition mechanism of the most active compounds (3a, 3b, 5a, and 5b) on urease activity are found to be in competitive mode. Also, the anti-oxidant activity and radical-scavenging properties of the synthesized compounds are evaluated using cupric reducing anti-oxidant activity, ferric reducing anti-oxidant capacity, 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid), and 2,2-diphenyl-1-picrylhydrazyl assays. Compounds 3a, b and 5a, b have good anti-oxidant properties and radical-scavenging activity at various final concentrations.


Introduction
The urease enzyme, EC 3.5.1.5, which belongs to the super family of amidohydrolases and phosphotriesterases, catalyzes the hydrolysis of urea into ammonia and a carbamate in micro-organisms, animals and various plant species. 1,2 This reaction promoted by the urease enzyme, results in a sudden increase in the overall pH, which causes negative effects on the health of animals and humans. The enzyme activity has been shown to have a link with several clinical conditions such as pyelonephritis, kidney stones, and gastrointestinal ulcers. Urease inhibitors are impressive against a number of serious infections caused by the secretion of urease by Helicobacter pylori. These bacteria may be responsible for numerous gastroduodenal disorders, together with gastric cancer, duodenal ulcers, peptic ulcers, urolithiasis, and even urinary catheter encrustation. 3,4 Difficulties associated with these kinds of infectious illnesses and other diverse sickness conditions are the synthesis and development of novel pharmacologically active compounds with minimum side effects. Urease inhibitors are known to be potent anti-ulcer drugs. Such potent urease inhibitors are generally divided into two classes as follows: (1) substrate structural analogs like hydroxamic acid and (2) those which affect the mechanism of action, like phosphoramidate, lansoprazole, omeprazole, thiol compounds, quinines, and the Schiff base derivatives. 4 Quinazolinone derivatives containing an acyl hydrazone skeleton are a class of fused heterocycles that is of considerable interest because of their diverse pharmacological properties and activities. Such activity includes anti-fungal, anti-hypertensive, anti-microbial, anti-viral, anti-tumor, anti-convulsant, anti-inflammatory, anti-cancer, and so on. [5][6][7][8][9][10] Enzyme inhibition studies, which are important in pharmaceutical research, have led to the discovery of a wide variety of drugs that are useful in treating a number of diseases. According to recent studies, several quinazolinone derivatives with diverse side groups have anti-urease activity. [11][12][13][14][15][16] In our recent studies, we investigated quinazolinone derivatives, that were very active inhibitors of jack bean urease. However, there is still a considerable need for alternative or novel treatments of H. pylori associated with diseases. [13][14][15] This work describes the synthesis some new quinazolinone derivatives containing an acyl hydrazone skeleton with different side groups (methyl and methoxy) to determine the inhibition properties of these compounds, the enzyme kinetic parameters, and to investigate their antioxidant activities.

Chemistry
In this study, a novel series of quinazolinone derivatives containing acyl hydrazone skeletons was synthesized according to the pathways outlined in Scheme 1. A total of 12 compounds, 10 of which are novel, were synthesized and the structures were confirmed by 1 H NMR, 13 C NMR, and elemental analysis. A literature method was used for the synthesis; 16 however, ethylimido(3-methoxy)benzyl acetate hydrochloride and ethylimido(3-methyl)benzyl acetate hydrochloride were used as the starting compounds instead of ethylimidobenzyl acetate hydrochloride. Ethylimido(3-methoxy)benzyl acetate hydrochloride and ethylimido(3-methyl)benzyl acetate hydrochloride, as starting compounds, were prepared according to the literature. 16,17 In the second step of our experimental studies, compounds 1a and 1b, which are known in the literature, albeit prepared via a different method, 17 were obtained from the reaction of 2-aminobenzamide and ethylimido(3methoxy/methyl)benzyl acetate hydrochloride in absolute methanol at room temperature. In the next step, the corresponding quinazolinone ester derivatives 2a and 2b were obtained through removal of the acidic NH proton of compounds 1a and 1b with K 2 CO 3 followed by the nucleophilic attack on ethyl 2-bromoacetate.
Next, the acetohydrazide compounds 3a and 3b were obtained as a result of nucleophilic attack of hydrazine hydrate on the ester carbonyl carbon of the 2a and 2b. In the final step, the Schiff base quinazolinone compounds 4a, 4b, 5a, 5b, 6a, and 6b were obtained as a result of the reactions of compounds 3a and 3b with the corresponding aldehydes in the presence of a catalytic amount of acetic acid. The hydrazones exist as E/Z geometric isomers about the C=N imine bond and as cis/trans amide conformers. The literature studies show that in the NMR spectra of such compounds, the E isomers are found as pairs of cis/trans conformers, and in these sets, the cis conformer is more dominant than the trans conformer in polar solvents such as DMSO. 18 Also, the location of the amide conformers (syn and anti-periplanar) influences neighboring signals. For this reason, we see some signals in the NMR spectra as binary signals, for methylene and methyl groups. The signals of the all synthesized compounds observed in the 1 H NMR and 13 C NMR spectra occurred at the expected chemical shift values and the obtained values confirmed the structure of our products.

Cupric reducing anti-oxidant activity and ferric reducing anti-oxidant capacity anti-oxidant activity results
The anti-oxidant capacities of the newly synthesized compounds were determined using ferric reducing anti-oxidant capacity (FRAP) and cupric reducing anti-oxidant activity (CUPRAC) assays. The CUPRAC and FRAP of mainly electron transfer-based methods were applied for the synthesized and natural compounds. The CUPRAC method is based on the absorbance measurement of the CUPRAC chromophore, Cu(I)-neocuproine (Nc) chelate, formed as a result of the redox reaction of anti-oxidants with the CUPRAC reagent, a bis(neocuproine)copper(II) cation [Cu(II)-Nc], where the absorbance is recorded at the maximum light absorption wavelength of 450 nm. The orangeyellow color is due to the Cu(I)-Nc charge-transfer complex formed. Increased absorbance of the reaction mixture indicates a higher reducing ability. 19 The anti-oxidant effects were classified into two groups; compounds 3a and 3b, being the most effective, were in the first group and compounds 5a and 5b, being moderately active, were in the second ( Table 1). The remaining compounds showed weak anti-oxidant activity in the CUPRAC assay. In the FRAP assays, compounds 3a and 3b showed the best iron(III) reduction activity. The results of the other compounds were within the range of 0.048-0.757 mM FeSO 4 .7H 2 O/g compound (Table 1).

DPPH and ABTS radical-scavenging assays
The DPPH (2,2-diphenyl-1-picrylhydrazyl) method is based on the fact that the free radical is purple in color and that the purple color of DPPH decays in the presence of an anti-oxidant. The color changes from purple to yellow after reduction, which can be quantified by a decrease in the absorbance at a wavelength of 517 nm. The results are expressed as % radical-scavenging activity at three different final concentrations of 100, 25, and 6.25 µg mL −1 ( Table 2). Compounds 3a and 3b showed greater scavenging activity than the other compounds at the 100, 25, and 6.25 µg mL −1 final concentrations. According to the anti-oxidant test results, compounds having a high CUPRAC value, such as 3a and 3b, exhibited good DPPH radical-scavenging activity. In addition to this observation, it was found that the other compounds had weak scavenging activity at the 100, 25, and 6.25 µg mL −1 final concentrations.
The pre-formed radical monocation of 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS • +) is generated by the oxidation of ABTS with potassium persulfate and is reduced in the presence of hydrogen-donating antioxidants. The influence of both the concentration of the anti-oxidant and the duration of the reaction on the inhibition of the radical cation absorption is taken into account when determining the anti-oxidant activity. 20 Compounds 3a and 3b showed higher scavenging activity than the other products at the 100, 25, and 6.25 µg mL −1 final concentrations, similar to the DPPH method (Table 2). In this study, compounds 3a and 3b in particularly showed radical-scavenging activity almost the same as butylated hydroxytoluene (BHT), which we use as a standard in the ABTS assay (Table 2). Scheme 1. Synthetic pathway toward the target compounds.

Urease inhibition assay and kinetic parameters
The newly synthesized compounds were examined in terms of their urease inhibition potential. The percentage relative activities versus inhibitor concentrations were plotted separately for each organic molecule. The IC 50 values were determined for the urease inhibitory activity of the synthesized compounds and for that of thiourea (Table 3). Compound 3b had the highest inhibitory effect among those tested. Lower IC 50 values indicate higher inhibition. Along with this result, the inhibitory activities of compounds 3a, 3b, 5a, and 5b were significantly lower than that of thiourea. To determine the type of enzyme inhibition of the compounds and thiourea, their urease activity was analyzed by the Lineweaver-Burk plots using data derived from the enzyme assay containing different concentrations of urea in the absence or presence of each inhibitor ( Figure  1(a)-(e) and Table 4). In the presence of compounds 3a, 3b, 5a, and 5b and thiourea, the K m value increased and the V max value (24.814 mM min −1 ) remained the same. From this point of view, the analysis of the Lineweaver-Burk plots indicates that the type of inhibition of these compounds and the standard was via the competitive mode (Figure 1(a)-(e)). The inhibitory activity of the compounds and the standard against the enzyme can be listed as follows, from the most active to the least active molecule: 3b > 3a > 5b > 5a > thiourea (Tables 3 and 4 and Figure  1(a)-(e)). Researchers have reported that 2,3-disubstituted quinazolin-4(3H)-one derivatives showed good urease inhibitory properties, with IC 50 values in the range of 2.90 ± 0.11 to 8.77 ± 0.15 µg mL −1 . 16 In this study, the IC 50 values of our newly synthesized quinazolinone derivatives were found to be in the range of 1.86 ± 0.07 to 23.89 ± 0.10. The IC 50 values reported in many urease enzyme inhibition studies in the literature are close and similar to the results we have obtained. [12][13][14][15][16][20][21][22] Conclusion A novel series of 2-(3-methyl/methoxybenzyl)quinazolin-4(3H)-one derivative containing ester, hydrazide, and acyl hydrazones functional groups on the second nitrogen atom has been synthesized and then their anti-urease, enzyme kinetic studies, and anti-oxidant activities have been screened. The anti-oxidant activities and free radical-scavenging properties of the synthesized compounds were clarified using various assays. Products 3a, 3b, 5a, and 5b showed excellent anti-oxidant activities in DPPH • scavenging and cupric reducing/anti-oxidant capacity tests. The other compounds exhibited moderate anti-oxidant activities. Several of the prepared compounds (3a, 3b, 5a, and 5b) showed excellent anti-urease activity with IC 50 values ranging between 1.86 ± 0.07 and 6.38 ± 0.11 μg mL −1 compared with the standard inhibitor, thiourea (IC 50 = 14.86 ± 0.23 μg mL −1 ). 2-[2-(3-Methoxybenzyl)-4oxoquinazolin-3(4H)-yl]acetohydrazide (3b) has the best inhibitory effect against jack bean urease with an IC 50 value of 1.86 ± 0.07 μg mL −1 . Also, compounds 3a, 3b, 5a, and 5b exhibited excellent competitive inhibitory activity against the urease, in the presence of urea as the substrate. The results show that acyl hydrazone-derived quinazolinones have a positive effect on urease inhibitory activities as well as potent anti-oxidant activities.

Experimental
All the chemicals were supplied from Merck (Darmstadt, Germany), Sigma-Aldrich (St. Louis, MO, USA), and Fluka (Morris Plains, NJ 07950-2546 USA). The progress of all reactions was determined by TLC (aluminum sheets, silica gel 60F 2.54-0.2 mm thickness). Melting points were determined using a Stuart SMP30 melting point apparatus and are uncorrected. 1 H NMR and 13 C NMR (attached proton test (APT)) spectra were recorded on a Varian-Mercury 400 MHz spectrometer in dimethyl sulfoxide-d 6 (DMSO-d 6 ) using tetramethylsilane as an internal standard chemical shifts (δ values) are given in ppm. The elemental compositions were determined on a Carlo Erba 1106 CHN analyzer. The composition values were in agreement (±0.4%) with calculated values . Compounds 1a, 1b, 2a, 2b, 3a, and 3b were prepared according to the literature. 21         General procedure compounds 4a, b, 5a, b and 6a, b