Design and synthesis of novel nitrothiazolacetamide conjugated to different thioquinazolinone derivatives as anti-urease agents

The present article describes the design, synthesis, in vitro urease inhibition, and in silico molecular docking studies of a novel series of nitrothiazolacetamide conjugated to different thioquinazolinones. Fourteen nitrothiazolacetamide bearing thioquinazolinones derivatives (8a-n) were synthesized through the reaction of isatoic anhydride with different amine, followed by reaction with carbon disulfide and KOH in ethanol. The intermediates were then converted into final products by treating them with 2-chloro-N-(5-nitrothiazol-2-yl)acetamide in DMF. All derivatives were then characterized through different spectroscopic techniques (1H, 13C-NMR, MS, and FTIR). In vitro screening of these molecules against urease demonstrated the potent urease inhibitory potential of derivatives with IC50 values ranging between 2.22 ± 0.09 and 8.43 ± 0.61 μM when compared with the standard thiourea (IC50 = 22.50 ± 0.44 μM). Compound 8h as the most potent derivative exhibited an uncompetitive inhibition pattern against urease in the kinetic study. The high anti-ureolytic activity of 8h was confirmed against two urease-positive microorganisms. According to molecular docking study, 8h exhibited several hydrophobic interactions with Lys10, Leu11, Met44, Ala47, Ala85, Phe87, and Pro88 residues plus two hydrogen bound interactions with Thr86. According to the in silico assessment, the ADME-Toxicity and drug-likeness profile of synthesized compounds were in the acceptable range.


Results and discussion
Designing consideration. For the past few years, there are limited reports of quinazolines as urease inhibitors. New series of 2,3-disubstituted quinazolin-4(3H)-ones ( Fig. 1A) were synthesized and exhibited potent urease inhibitory activity in the range of 1.55-2.65 μg/mL. The structure-activity relationship (SAR) indicated that halogen atoms on phenyl ring improved urease inhibition 34 . More recently, Mustafa and co-workers identified another set of quinazolinone-coumarin derivatives and the most potent compound (Fig. 1B) exhibited the IC 50 values of 1.26 ± 0.07 μg/mL. In vitro results showed that the heterocyclic group substituted on N-3 position of quinazolinone ring plays an important role in the inhibitory activity 39 . This research group also developed and synthesized another series of quinazolin-4(3H)-ones (Fig. 1C). Most of the compounds showed excellent activity with IC 50 values ranging between 1.88 ± 0.17 and 6.42 ± 0.23 μg/mL, compared to that of thiourea with an IC 50 value of 15.06 μg/mL. Molecular docking interactions of compound C as the most potent derivative of this set showed key interactions with Arg439, Met637, Gln635 residues 35 . Nitazoxanide (Fig. 1D) and nitazoxanide (Fig. 1E) are known as approved antiparasitic medications with aminonitrothiazole structure. These compounds were shown to have antibacterial activities against both metronidazole-resistant strains and sensitive clinical isolates of H. pylori pathogens. It is noteworthy that strains resistant to metronidazole were susceptible to these drugs 40 . Recently, thiazolbenzamide (Fig. 1F) was reported as a potential urease inhibitor. It was inferred that molecules with thio-substituted groups generally improved the urease inhibition of the target enzyme 41 .
Also, it would be interesting to note that the most effective inhibitors contain functional groups with electronegative atoms such as oxygen, nitrogen, or/and sulfur to form complexes with Ni ions of the enzyme as well as His residues in the active site. Stronger interaction of inhibitors with enzyme active site and higher inhibitory efficiency was observed in sulfur-containing inhibitors compared to the rest of heteroatoms 42,43 .
By considering the structure of the previously reported active agents discussed herein.
• Quinazolinone was utilized as an elegant skeleton to design urease inhibitors. Substitution at the R position of quinazolinone was performed to evaluate the type of substitution against urease. www.nature.com/scientificreports/ • To improve urease inhibitory potency, the nitrothiazole pendant with ensured anti-urease properties was incorporated into the quinazolinone ring. Nitrothiazole can improve hydrogen bonding capability within the enzyme cavity. • Thioacetamide is an ideal candidate to link the quinazolinone and nitrothiazol moiety with the substrate-like structure. It was assumed that sulfur atoms provide better and sometimes selective interactions with critical Ni (I) and Ni (II) coordinated with His519, His545, Lys490, His407, His409, Asp633, and Lys490 45 .
In continuation of our previous effort on designing urease inhibitors [46][47][48] , this work was aimed to report the synthesis of nitrothiazolacetamide conjugated to different thioquinazolinones. The urease inhibitory potential of all derivatives, as well as SAR and molecular docking studies, were also performed.
Chemistry. The synthetic pathway to the target compounds (8a-n) is outlined in Fig. 2. Intermediates 3a-n were synthesized by the method reported in our previous study 44 . Briefly, isatoic anhydride (1) was reacted with different amine (2a-n) in ethanol under reflux conditions for 3 h to obtain compound 3a-n. Carbon disulfide and KOH were added to this solution and the reaction was further refluxed for an extra 3 h. The targeted compounds (4a-n) were obtained after cooling and recrystallizing in ethanol. Compound 7 was prepared by a simple reaction of nitrothiazolamine (5) with 2-chloroacetyl chloride (6) in DMF at room temperature. The crude product was purified by recrystallization in ethanol. Compounds 8a-n were synthesized by the nucleophilic addition of thio-derivatives (4a-n) to intermediate 7 in DMF using K 2 CO 3 as a catalyst at 50 °C. The structures of purified products were confirmed by IR, 1 H NMR, 13 C NMR, elemental analysis, and mass spectroscopy.
Evaluation of urease inhibitory activity and structure-activity relationship. In vitro anti-urease activity of synthesized compounds, 8a-n were performed based on the calorimetric method against urease compared with thiourea as the reference inhibitor. The results of the urease inhibitory assay were shown in Table 1 in the terms of IC 50 . In this series, all compounds had significant inhibition against urease with IC 50 values ranging from 2.22 to 8.43 µM compared with thiourea as a positive control with an IC 50 value of 22.50 µM.
Based on the obtained biological results related to 8a-d, compound 8a as the unsubstituted phenyl pendant displayed an IC 50 value of 4.72 µM with around fivefold improvement in the potency compared to thiourea as a standard inhibitor. Any substitution in this group including electron-withdrawing such as chlorine (8b) or bromine (8c) or even electron-donating group (8d, methoxy) improved urease inhibition and there is snot significant differences in these substituted derivatives.
The evaluations on 8e-g as the methyl-substituted group demonstrated that 8 g (R = methylpyridine) with an IC 50 of 2.50 μM was categorized as the top potent urease inhibitor in this group followed by 8f (R = methyl benzyl) and 8e (R = benzyl). It seems that the presence of heteroatom in the aromatic ring could amend the interactions within the binding site of urease.
Assessments of 8a, 8e, and 8 h analogs showed the importance of the length of the alkyl chain between quinazolinone and aryl moiety. Compound 8h bearing ethyl linker demonstrated an IC 50  www.nature.com/scientificreports/ urease, while 8e (IC 50 = 3.83 μM) possessing methyl linker was less potent compared with 8h followed by 8a with IC 50 = 4.72 μM. It seems that the elongation of the alkyl linker between the quinazolinone and aryl pendant improved urease inhibitory activity.
In the case of compounds containing aliphatic chain substitution (8i-m), it can be seen that in most cases such structural modification reduced the inhibitory potency of compounds (IC 50 ranging from 3.02 to 8.43 μM) compared to aromatic substituted derivatives (IC 50 ranging from 2.22 to 3.85 μM). In this group, the most potent urease inhibitor was 8 k (R = n-butyl) with an IC 50 value of 3.02 µM followed by 8i (R = n-propyl; IC 50 = 4.26 μM), 8j (R = iso-propyl; IC 50 = 6.04 μM) and 8l (R = iso-butyl; IC 50 = 8.43 μM). As can be seen, the longer aliphatic chain demonstrated better inhibitory activity compared to shorter or branch one. Interestingly, compound 8n bearing cyclopentyl group as an aliphatic-ring substitute showed better activity (IC 50 = 2.96 μM) compared to the rest of the aliphatic-chain group. As can be seen in this set of compounds, it seems that aliphatic-ring followed by aliphatic linear chains are more potent than aliphatic branched-chain counterparts.
Kinetic study of the most potent compound 8h. The mechanism of urease inhibition was investigated by enzyme kinetics, following the similar procedure of the urease inhibition assay. Lineweaver-Burk graphics were used to estimate the type of inhibition. Graphical analysis of the reciprocal Lineweaver-Burk plot (Fig. 3) related to compound 8h showed that K m and V max decreased with an increase in inhibitor concentration confirming an uncompetitive inhibition pattern against urease. Furthermore, the plot of the K m versus different concentrations of 8h gave an estimate of the inhibition constant, K i of 1.994 µM which is in accordance with the IC 50 value of 8h (Fig. 4).

Molecular docking simulation.
Jack bean urease (JBU) is a T-shaped metalo-hydrolase enzyme that acts by converting urea into ammoniac within its active site. JBU monomer third structure consists of four main domains (Fig. 5). From the N-terminal of the enzyme sequence, starts by first αβ domain located in the hammer handle. The second αβ domain is located in the hammerhead which is connected through a middle β domain to the other head of the hammer which is (αβ) 8 TIM barrel domain holding the active site of the enzyme 12 .
The enzyme kinetic study showed that the compound 8h acts as an uncompetitive inhibitor of the JBU enzyme in this type of inhibition the inhibitor interacted with the enzyme-substrate ([ES]) complex to form a final enzyme-substrate-inhibitor ([ESI]) complex; hence, the molecular docking study was performed on the [ES] complex. To make the [ES] complex the urea docked into the active site of the JBU enzyme (PDB ID: 4H9M).
In order to find the possible allosteric sites, the protein-substrate complex was treated using mastreo sitemap tool to identify the suitable sites for occupancy of hydrophobic, H-bond donor, and H-bond acceptor ligand www.nature.com/scientificreports/ www.nature.com/scientificreports/   www.nature.com/scientificreports/ groups. Eventually, five possible binding sites were detected which can be suitable as a drug-like binding site. As shown in Fig. 6, five binding sites were detected on the surface of the [ES] complex. Site 1 (purple) and site 2 (magenta) located in the first αβ domain which was showed the suitable size and potential interaction sites, calculated to have the best site scores (1.011 and 0.932 respectively). Site 3 (brown) nearby the canonical active site also had a plausible site score of 0.917. Site 4 (orange) and site 5 (cyan) were the smallest sites and had a few potential H-bond interactions, their site scores were calculated to be 0.724 and 0.510, respectively. Compound 8h as the most potent structure in the series, was docked on all of the potential binding sites of the [ES] complex to form the enzyme-substrate-inhibitor [ESI] complex. Considering the glide score (− 6.78 kcal/ mol) and interactions, site 2 appeared to have the maximum affinity in comparison with other identified sites. As it is shown in Fig. 7, compound 8h well occupied the site, and the following interactions were detected: Thr86 residue acting as both H-bond acceptor and H-bond donor with amide group and quinazolinone ring nitrogen. The pi-cation interaction between Lys10 and the quinazolinone aromatic system and another pi-cation interaction between Arg48 and the thiazole ring was observed. A pi-pi stacking interactions were found among His14 and ethylbenzene moiety moreover several hydrophobic interactions were found between compound 8h and Leu11, Met44, Ala47, Ala85, Phe87, and Pro88 residues.
Antimicrobial and anti-ureolytic activity of tested compounds. Compounds 8c, 8g, and 8h were chosen for their antimicrobial activities against microorganisms including standard species of Cryptococcus neoformans (H99), and clinical isolate of Proteus vulgaris. The results showed that at concentrations ranging from 1 to 512 μg/ml, the examined compounds exhibited no antimicrobial activities against the tested pathogens (MIC > 512 (μg/ml).
Next, the anti-ureolytic activity of highly potent urease inhibitors (8c, 8g, and 8h) against the C. neoformans (H99) and P.vulgaris was visually and spectroscopically measured at 560 nm. Table 2 summarizes the findings. Compound 8h, like our enzymatic assay results, displayed the highest anti-ureolytic activities followed by compound 8c. Notably, 8g exhibited selective urease activity against C. neoformans but not against P. vulgaris at the tested range.
According to the findings, none of the selected derivatives had anti-microbial effects on the tested microorganisms; however, the high activity of tested compounds against ureolytic microorganisms strengthens our hypothesis that the designed pharmacophore can be an ideal candidate for targeting ureolytic microorganisms through urease enzyme inhibition. ADME-toxicity profiles and physicochemical properties. The pkCSM server 45 was used to predict the ADME-Toxicity properties of synthesized compounds. As shown in Table 3. All derivatives showed good human intestinal absorption, low clearance values, and limited toxicity. www.nature.com/scientificreports/ According to the physicochemical properties predicted from the SwissADME website 46 , all compounds had appropriate molecular properties with no drug-likeness rules violations (Table 4).

Conclusion
In summary, fourteen new compounds with thioquinazolinone structures were designed and prepared as antiurease agents. Among them, compound 8h exhibited the most potent inhibitory effect against urease with an IC 50 value of 2.22 μM with around a ten-fold increase in the potency compared to the positive control. In addition, compound 8h possessed the uncompetitive type of inhibition in the enzymatic assay indicating that ligand bonded only to the complex formed between the enzyme and the substrate. The molecular docking study revealed that compound 8h could fit well into the binding site of urease by pi-cation, pi-pi, and H-bond interactions. 8h also demonstrated IC 50 values of 129.4 ± 5.3 and 172.4 ± 8.7 µg/ml against C. neoformans and  www.nature.com/scientificreports/ P.vulgaris on the ureolytic assay. Furthermore, in silico evaluations also found acceptable ADME-Toxicity and drug-likeness profiles.

Material and method
Chemistry. Compounds 3a-n were obtained by reaction of isatoic anhydride (compound 1, 1 mmol) with different amines (compound 2, 1.1 mmol) as the raw materials in ethanol under reflux conditions for 3 h. To the above solution carbon disulfide and KOH were added and the reaction was further refluxed for an extra 3 h to afford compounds 4a-n. Next, the intermediate 7 were synthesized by a simple reaction of nitrothiazolamine (5) with 2-chloroacetyl chloride (6) in DMF at room temperature. Finally, compounds 4a-n were reacted with 2-chloro-N-(5-nitrothiazol-2-yl)acetamide in the presence of K 2 CO 3 to provide the crude products 8a-n which was purified by column chromatography to yield the final products.

2-((3-cyclopentyl-4-oxo-3,4-dihydroquinazolin-2-yl)thio)-N-(5-nitrothiazol-2-yl)acetamide (8n
To gain a better understanding of the active site residue conformational change in the [ES] complex, the induced fit docking method was utilized for docking the urea in the active site of the molecule 54 . AHA was considered as the grid center and the maximum number of 20 poses was calculated with receptor and ligand van der Waals radii of 0.7 A and 0.5 A, respectively. Structures with prime energy levels beyond 30 kcal/mol were eliminated based on standard precious glide docking. The Site map tool was used to find the possible allosteric binding sites of the [ES] complex 55 . The site map was tasked to report up to 5 potential binding sites with at least 15 site points per each reported site by more restrictive definition of hydrophobicity. The grid box was generated for each binding site using entries with a box size of 25 A, compound 8h was docked on binding sites using glide with extra precision and flexible ligand sampling, reporting 10 poses per ligand to form the final [ESI] complex 56 . Antimicrobial activity against ureolytic microorganisms. The antimicrobial activity of compounds against the microorganisms including C. neoformans (H99), and clinical isolate of P.vulgaris was assessed using the microbroth dilution method, as recommended by the Clinical and Laboratory Standards Institute (CLSI) (M07-A9 for bacteria; M27-A3 for yeasts). The compounds were diluted, and stock solutions of 20 mg/ml in DMSO were prepared. Mueller-Hinton Broth (HiMedia) and RPMI-1640 (Sigma) were prepared as recommended for antimicrobial susceptibility testing of bacterial and fungal strains, respectively. Two-fold dilutions were made in the range of 1-512 μg/ml for tested compounds. The microbroth dilution test was accomplished using a 96-well microtiter plate, containing growth control (yeast culture in broth media) and sterility control (broth media without fungal culture). The antimicrobial susceptibility test was accomplished by adding a cell suspension adjusted to the 0.5 McFarland standard (1-2 × 10 8 CFU/mL for bacterial strains; 1−5 × 10 6 cells/ml for yeast) to different concentrations of tested compounds. Following incubation, the minimum inhibitory concentration (MIC) was established as the lowest concentration of compound that completely inhibits the growth of the organism in wells as detected visually. All experiments were performed in duplicates.
Anti-ureolytic activity against ureolytic microorganisms. The colorimetric microdilution technique using urea broth media (Merck, supplemented with glucose; pH = 6 for C. neoformans) was used to examine the urolytic activity of C. neoformans (H99), and clinical isolate of P.vulgaris treated with tested substances. Compounds in the concentration range of 1-512 μg/mL were exposed to ureolytic microorganisms, and the color of the medium was evaluated visually and spectroscopically at 560 nm after three days for C. neoformans and 24 h for P. vulgaris. The positive control, which included ureolytic bacteria but no drugs, changed color from yellow to dark pink or magenta. This shifts, allowing the determination of the inhibitory activity of compounds against urease activity of organisms even without a microtiter plate reader 57,58 . In silico pharmacokinetic properties of synthesized compounds. SwissADME and pkCSM servers were used to determine the physicochemical and drug-likeness properties of the derivatives.

Data availability
The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.