Synthesis of Chromen-4-One-Oxadiazole Substituted Analogs as Potent β-Glucuronidase Inhibitors

Chromen-4-one substituted oxadiazole analogs 1–19 have been synthesized, characterized and evaluated for β-glucuronidase inhibition. All analogs exhibited a variable degree of β-glucuronidase inhibitory activity with IC50 values ranging in between 0.8 ± 0.1–42.3 ± 0.8 μM when compared with the standard d-saccharic acid 1,4 lactone (IC50 = 48.1 ± 1.2 μM). Structure activity relationship has been established for all compounds. Molecular docking studies were performed to predict the binding interaction of the compounds with the active site of enzyme.


Introduction
β-Glucuronidase (E.C.3.2.1.31) is one of the most extensively studied enzymes in the metabolic hydrolysis of conjugated compounds, and eliminates large number of toxic compounds from body as glucuronides [1]. It acts as a glycoside hydrolase enzyme which induces to breaking of glucuronosyl-O-bonds [2]. In human body β-glucuronidase is present in many body fluids and organs [3]. Increased activity of β-glucuronidase has resulted in various health issues that include renal disorders [4], urinary tract infections [5], epilepsy [6], renal transplant rejection [7], neoplasm of bladder [8] and breast cancers [9]. Bacterial β-glucuronidase and β-glucosidase in the human colon are involved in the metabolism and activation of xenobiotics derived from dietary compounds [10]. The enzymes are identified to be mediators of colon cancer (CRC). Many carcinogenic dietary compounds are metabolized in the liver and then conjugated to glucuronic acid before being excreted with bile into the small intestine. In the colon, bacterial β-glucuronidase hydrolyzes the conjugates, there by releasing the parent compounds or their activated hepatic metabolites [11]. Epidemiological studies have shown that populations at high risk for colorectal cancer have high levels of fecal βglucuronidase activity and CRC patients have significantly higher levels of the enzyme than healthy controls [12].
Our research group has been working on synthesis of variety of heterocycles and explored a huge biological potential of these compounds [26][27][28][29][30][31][32]. Present work belongs to a simple and reliable synthetic protocol that has been developed for the synthesis of a series of chromone substituted oxadiazole pharmacophores in another effort towards developing more novel agents against βglucuronidase enzyme. This is beyond any doubt that combination of two or more biologically active compounds grants newer molecules with complementary pharmacophoric potentials or with different mechanisms of actions. The unity of pharmacophoric approach, as a result, could possibly address the active site of different targets more effectively and could offer the possibility to overcome the issue of drug resistance and unwanted effects [33][34][35][36]. The outcomes of this merger have already been the subject of attention in finding new antimicrobials [37][38][39][40][41].

Chemistry
The synthesis of the novel series of chromene-based oxadiazole β-glucuronidase inhibitors 1-19 commenced with the reaction of 2-hydroxyacetophenone and p-formylbenzoic acid in ethanolic potassium hydroxide at room temperature (Scheme 1).  [31].  The adduct I underwent oxidative cyclization to form 4-substituted chromenone analog II. This acid analog II was esterified to give III and reacted with hydrazine hydrate to form the corresponding hydrazide IV which upon reaction with the corresponding aldehydes under the described conditions formed the desired oxadiazoles 1-19 Figure 1. All synthesized compounds were characterized using various spectroscopic methods. The adduct I underwent oxidative cyclization to form 4-substituted chromenone analog II. This acid analog II was esterified to give III and reacted with hydrazine hydrate to form the corresponding hydrazide IV which upon reaction with the corresponding aldehydes under the described conditions formed the desired oxadiazoles 1-19 Figure 1. All synthesized compounds were characterized using various spectroscopic methods.

β-Glucuronidase Activity
4-Substituted chromenone-based oxadiazole derivatives 1-19 were synthesized and evaluated for β-glucuronidase inhibitory potential. All the synthesized analogs showed potent inhibitory potential, ranging between 0.8 ± 0.1 µM to 42.3 ± 0.8 µM when compared with the standard d-saccharic acid 1,4 lactone (IC 50 value = 48.1 ± 1.2 µM) ( Table 1). The structure activity relationship has been established on the basis of substitution pattern on the phenyl ring. The adduct I underwent oxidative cyclization to form 4-substituted chromenone analog II. This acid analog II was esterified to give III and reacted with hydrazine hydrate to form the corresponding hydrazide IV which upon reaction with the corresponding aldehydes under the described conditions formed the desired oxadiazoles 1-19 Figure 1. All synthesized compounds were characterized using various spectroscopic methods.

β-Glucuronidase Activity
4-Substituted chromenone-based oxadiazole derivatives 1-19 were synthesized and evaluated for β-glucuronidase inhibitory potential. All the synthesized analogs showed potent inhibitory potential, ranging between 0.8 ± 0.1 μM to 42.3 ± 0.8 μM when compared with the standard Dsaccharic acid 1,4 lactone (IC50 value = 48.1 ± 1.2 μM) ( Table 1). The structure activity relationship has been established on the basis of substitution pattern on the phenyl ring. The adduct I underwent oxidative cyclization to form 4-substituted chromenone analog II. This acid analog II was esterified to give III and reacted with hydrazine hydrate to form the corresponding hydrazide IV which upon reaction with the corresponding aldehydes under the described conditions formed the desired oxadiazoles 1-19 Figure 1. All synthesized compounds were characterized using various spectroscopic methods.

β-Glucuronidase Activity
4-Substituted chromenone-based oxadiazole derivatives 1-19 were synthesized and evaluated for β-glucuronidase inhibitory potential. All the synthesized analogs showed potent inhibitory potential, ranging between 0.8 ± 0.1 μM to 42.3 ± 0.8 μM when compared with the standard Dsaccharic acid 1,4 lactone (IC50 value = 48.1 ± 1.2 μM) ( Table 1). The structure activity relationship has been established on the basis of substitution pattern on the phenyl ring. The adduct I underwent oxidative cyclization to form 4-substituted chromenone analog II. This acid analog II was esterified to give III and reacted with hydrazine hydrate to form the corresponding hydrazide IV which upon reaction with the corresponding aldehydes under the described conditions formed the desired oxadiazoles 1-19 Figure 1. All synthesized compounds were characterized using various spectroscopic methods.

β-Glucuronidase Activity
4-Substituted chromenone-based oxadiazole derivatives 1-19 were synthesized and evaluated for β-glucuronidase inhibitory potential. All the synthesized analogs showed potent inhibitory potential, ranging between 0.8 ± 0.1 μM to 42.3 ± 0.8 μM when compared with the standard Dsaccharic acid 1,4 lactone (IC50 value = 48.1 ± 1.2 μM) ( Table 1). The structure activity relationship has been established on the basis of substitution pattern on the phenyl ring. The adduct I underwent oxidative cyclization to form 4-substituted chromenone analog II. This acid analog II was esterified to give III and reacted with hydrazine hydrate to form the corresponding hydrazide IV which upon reaction with the corresponding aldehydes under the described conditions formed the desired oxadiazoles 1-19 Figure 1. All synthesized compounds were characterized using various spectroscopic methods.

β-Glucuronidase Activity
4-Substituted chromenone-based oxadiazole derivatives 1-19 were synthesized and evaluated for β-glucuronidase inhibitory potential. All the synthesized analogs showed potent inhibitory potential, ranging between 0.8 ± 0.1 μM to 42.3 ± 0.8 μM when compared with the standard Dsaccharic acid 1,4 lactone (IC50 value = 48.1 ± 1.2 μM) ( Table 1). The structure activity relationship has been established on the basis of substitution pattern on the phenyl ring. The adduct I underwent oxidative cyclization to form 4-substituted chromenone analog II. This acid analog II was esterified to give III and reacted with hydrazine hydrate to form the corresponding hydrazide IV which upon reaction with the corresponding aldehydes under the described conditions formed the desired oxadiazoles 1-19 Figure 1. All synthesized compounds were characterized using various spectroscopic methods.

β-Glucuronidase Activity
4-Substituted chromenone-based oxadiazole derivatives 1-19 were synthesized and evaluated for β-glucuronidase inhibitory potential. All the synthesized analogs showed potent inhibitory potential, ranging between 0.8 ± 0.1 μM to 42.3 ± 0.8 μM when compared with the standard Dsaccharic acid 1,4 lactone (IC50 value = 48.1 ± 1.2 μM) ( Table 1). The structure activity relationship has been established on the basis of substitution pattern on the phenyl ring. The adduct I underwent oxidative cyclization to form 4-substituted chromenone analog II. This acid analog II was esterified to give III and reacted with hydrazine hydrate to form the corresponding hydrazide IV which upon reaction with the corresponding aldehydes under the described conditions formed the desired oxadiazoles 1-19 Figure 1. All synthesized compounds were characterized using various spectroscopic methods.

β-Glucuronidase Activity
4-Substituted chromenone-based oxadiazole derivatives 1-19 were synthesized and evaluated for β-glucuronidase inhibitory potential. All the synthesized analogs showed potent inhibitory potential, ranging between 0.8 ± 0.1 μM to 42.3 ± 0.8 μM when compared with the standard Dsaccharic acid 1,4 lactone (IC50 value = 48.1 ± 1.2 μM) ( Table 1). The structure activity relationship has been established on the basis of substitution pattern on the phenyl ring. The adduct I underwent oxidative cyclization to form 4-substituted chromenone analog II. This acid analog II was esterified to give III and reacted with hydrazine hydrate to form the corresponding hydrazide IV which upon reaction with the corresponding aldehydes under the described conditions formed the desired oxadiazoles 1-19 Figure 1. All synthesized compounds were characterized using various spectroscopic methods.

β-Glucuronidase Activity
4-Substituted chromenone-based oxadiazole derivatives 1-19 were synthesized and evaluated for β-glucuronidase inhibitory potential. All the synthesized analogs showed potent inhibitory potential, ranging between 0.8 ± 0.1 μM to 42.3 ± 0.8 μM when compared with the standard Dsaccharic acid 1,4 lactone (IC50 value = 48.1 ± 1.2 μM) ( Table 1). The structure activity relationship has been established on the basis of substitution pattern on the phenyl ring. The adduct I underwent oxidative cyclization to form 4-substituted chromenone analog II. This acid analog II was esterified to give III and reacted with hydrazine hydrate to form the corresponding hydrazide IV which upon reaction with the corresponding aldehydes under the described conditions formed the desired oxadiazoles 1-19 Figure 1. All synthesized compounds were characterized using various spectroscopic methods.

β-Glucuronidase Activity
4-Substituted chromenone-based oxadiazole derivatives 1-19 were synthesized and evaluated for β-glucuronidase inhibitory potential. All the synthesized analogs showed potent inhibitory potential, ranging between 0.8 ± 0.1 μM to 42.3 ± 0.8 μM when compared with the standard Dsaccharic acid 1,4 lactone (IC50 value = 48.1 ± 1.2 μM) ( Table 1). The structure activity relationship has been established on the basis of substitution pattern on the phenyl ring. The adduct I underwent oxidative cyclization to form 4-substituted chromenone analog II. This acid analog II was esterified to give III and reacted with hydrazine hydrate to form the corresponding hydrazide IV which upon reaction with the corresponding aldehydes under the described conditions formed the desired oxadiazoles 1-19 Figure 1. All synthesized compounds were characterized using various spectroscopic methods.

β-Glucuronidase Activity
4-Substituted chromenone-based oxadiazole derivatives 1-19 were synthesized and evaluated for β-glucuronidase inhibitory potential. All the synthesized analogs showed potent inhibitory potential, ranging between 0.8 ± 0.1 μM to 42.3 ± 0.8 μM when compared with the standard Dsaccharic acid 1,4 lactone (IC50 value = 48.1 ± 1.2 μM) ( Table 1). The structure activity relationship has been established on the basis of substitution pattern on the phenyl ring. The adduct I underwent oxidative cyclization to form 4-substituted chromenone analog II. This acid analog II was esterified to give III and reacted with hydrazine hydrate to form the corresponding hydrazide IV which upon reaction with the corresponding aldehydes under the described conditions formed the desired oxadiazoles 1-19 Figure 1. All synthesized compounds were characterized using various spectroscopic methods.

β-Glucuronidase Activity
4-Substituted chromenone-based oxadiazole derivatives 1-19 were synthesized and evaluated for β-glucuronidase inhibitory potential. All the synthesized analogs showed potent inhibitory potential, ranging between 0.8 ± 0.1 μM to 42.3 ± 0.8 μM when compared with the standard Dsaccharic acid 1,4 lactone (IC50 value = 48.1 ± 1.2 μM) ( Table 1). The structure activity relationship has been established on the basis of substitution pattern on the phenyl ring. The adduct I underwent oxidative cyclization to form 4-substituted chromenone analog II. This acid analog II was esterified to give III and reacted with hydrazine hydrate to form the corresponding hydrazide IV which upon reaction with the corresponding aldehydes under the described conditions formed the desired oxadiazoles 1-19 Figure 1. All synthesized compounds were characterized using various spectroscopic methods.

β-Glucuronidase Activity
4-Substituted chromenone-based oxadiazole derivatives 1-19 were synthesized and evaluated for β-glucuronidase inhibitory potential. All the synthesized analogs showed potent inhibitory potential, ranging between 0.8 ± 0.1 μM to 42.3 ± 0.8 μM when compared with the standard Dsaccharic acid 1,4 lactone (IC50 value = 48.1 ± 1.2 μM) ( Table 1). The structure activity relationship has been established on the basis of substitution pattern on the phenyl ring. The adduct I underwent oxidative cyclization to form 4-substituted chromenone analog II. This acid analog II was esterified to give III and reacted with hydrazine hydrate to form the corresponding hydrazide IV which upon reaction with the corresponding aldehydes under the described conditions formed the desired oxadiazoles 1-19 Figure 1. All synthesized compounds were characterized using various spectroscopic methods.

β-Glucuronidase Activity
4-Substituted chromenone-based oxadiazole derivatives 1-19 were synthesized and evaluated for β-glucuronidase inhibitory potential. All the synthesized analogs showed potent inhibitory potential, ranging between 0.8 ± 0.1 μM to 42.3 ± 0.8 μM when compared with the standard Dsaccharic acid 1,4 lactone (IC50 value = 48.1 ± 1.2 μM) ( Table 1). The structure activity relationship has been established on the basis of substitution pattern on the phenyl ring. The adduct I underwent oxidative cyclization to form 4-substituted chromenone analog II. This acid analog II was esterified to give III and reacted with hydrazine hydrate to form the corresponding hydrazide IV which upon reaction with the corresponding aldehydes under the described conditions formed the desired oxadiazoles 1-19 Figure 1. All synthesized compounds were characterized using various spectroscopic methods.

β-Glucuronidase Activity
4-Substituted chromenone-based oxadiazole derivatives 1-19 were synthesized and evaluated for β-glucuronidase inhibitory potential. All the synthesized analogs showed potent inhibitory potential, ranging between 0.8 ± 0.1 μM to 42.3 ± 0.8 μM when compared with the standard Dsaccharic acid 1,4 lactone (IC50 value = 48.1 ± 1.2 μM) ( Table 1). The structure activity relationship has been established on the basis of substitution pattern on the phenyl ring. The adduct I underwent oxidative cyclization to form 4-substituted chromenone analog II. This acid analog II was esterified to give III and reacted with hydrazine hydrate to form the corresponding hydrazide IV which upon reaction with the corresponding aldehydes under the described conditions formed the desired oxadiazoles 1-19 Figure 1. All synthesized compounds were characterized using various spectroscopic methods.

β-Glucuronidase Activity
4-Substituted chromenone-based oxadiazole derivatives 1-19 were synthesized and evaluated for β-glucuronidase inhibitory potential. All the synthesized analogs showed potent inhibitory potential, ranging between 0.8 ± 0.1 μM to 42.3 ± 0.8 μM when compared with the standard Dsaccharic acid 1,4 lactone (IC50 value = 48.1 ± 1.2 μM) ( Table 1). The structure activity relationship has been established on the basis of substitution pattern on the phenyl ring. The adduct I underwent oxidative cyclization to form 4-substituted chromenone analog II. This acid analog II was esterified to give III and reacted with hydrazine hydrate to form the corresponding hydrazide IV which upon reaction with the corresponding aldehydes under the described conditions formed the desired oxadiazoles 1-19 Figure 1. All synthesized compounds were characterized using various spectroscopic methods.

β-Glucuronidase Activity
4-Substituted chromenone-based oxadiazole derivatives 1-19 were synthesized and evaluated for β-glucuronidase inhibitory potential. All the synthesized analogs showed potent inhibitory potential, ranging between 0.8 ± 0.1 μM to 42.3 ± 0.8 μM when compared with the standard Dsaccharic acid 1,4 lactone (IC50 value = 48.1 ± 1.2 μM) ( Table 1). The structure activity relationship has been established on the basis of substitution pattern on the phenyl ring. The adduct I underwent oxidative cyclization to form 4-substituted chromenone analog II. This acid analog II was esterified to give III and reacted with hydrazine hydrate to form the corresponding hydrazide IV which upon reaction with the corresponding aldehydes under the described conditions formed the desired oxadiazoles 1-19 Figure 1. All synthesized compounds were characterized using various spectroscopic methods.

β-Glucuronidase Activity
4-Substituted chromenone-based oxadiazole derivatives 1-19 were synthesized and evaluated for β-glucuronidase inhibitory potential. All the synthesized analogs showed potent inhibitory potential, ranging between 0.8 ± 0.1 μM to 42.3 ± 0.8 μM when compared with the standard Dsaccharic acid 1,4 lactone (IC50 value = 48.1 ± 1.2 μM) ( Table 1). The structure activity relationship has been established on the basis of substitution pattern on the phenyl ring. The compound 6 (IC50 = 0.8 ± 0.1 μM), an ortho-fluoro analog was found to be the most potent among the series. The greater potential shown by this analog might be due to the electron withdrawing fluoro group. If we compare it with other fluoro analogs like compound 4 (IC50 = 1.1 ± 0.05 μM) a para-fluoro analog and compound 5 (IC50 = 3.8 ± 0.2 μM), a meta-fluoro analog, the analog 6 was found superior than the analogs 4 and 5. The little bit difference in their potential is appears to 8 The compound 6 (IC50 = 0.8 ± 0.1 μM), an ortho-fluoro analog was found to be the most potent among the series. The greater potential shown by this analog might be due to the electron withdrawing fluoro group. If we compare it with other fluoro analogs like compound 4 (IC50 = 1.1 ± 0.05 μM) a para-fluoro analog and compound 5 (IC50 = 3.8 ± 0.2 μM), a meta-fluoro analog, the analog 6 was found superior than the analogs 4 and 5. The little bit difference in their potential is appears to The compound 6 (IC50 = 0.8 ± 0.1 μM), an ortho-fluoro analog was found to be the most potent among the series. The greater potential shown by this analog might be due to the electron withdrawing fluoro group. If we compare it with other fluoro analogs like compound 4 (IC50 = 1.1 ± 0.05 μM) a para-fluoro analog and compound 5 (IC50 = 3.8 ± 0.2 μM), a meta-fluoro analog, the analog 6 was found superior than the analogs 4 and 5. The little bit difference in their potential is appears to The compound 6 (IC 50 = 0.8 ± 0.1 µM), an ortho-fluoro analog was found to be the most potent among the series. The greater potential shown by this analog might be due to the electron withdrawing fluoro group. If we compare it with other fluoro analogs like compound 4 (IC 50 = 1.1 ± 0.05 µM) a para-fluoro analog and compound 5 (IC 50 = 3.8 ± 0.2 µM), a meta-fluoro analog, the analog 6 was found superior than the analogs 4 and 5. The little bit difference in their potential is appears to be due to the difference in the position of fluoro groups on phenyl ring Figure 2. A closer look upon the structures of compounds and their corresponding IC 50 values revealed that the presence of different substitutions i.e., fluoro. chloro, methyl, nitro, methoxy and pyridinyl on the aromatic side chain is of great importance. It was also observed that either an EWG or EDG on the phenyl part showed great potential but the slight difference in potential was mainly affected by the position of the substituent.
Further to above, it is evident from the results that the presence of halogens or alkyl groups on benzene ring influenced the inhibitory potentials significantly. Similarly, nitro substitutions reduced the potential of inhibition. Apart from direct interactions of these attachments, it can be easily argued that these substitutions significantly affected the electronic distributions and intensities at the active sites. In many cases, oxadiazole part also played a significant role in forming hydrogen bond interactions with the active site of enzyme. Most important of these positions are Asp 207, Glu 451 and Glu 540. To better understand the binding interactions of the most active analogs, molecular docking analysis were performed.

Molecular Docking Studies
Experimental results were further validated by carrying out molecular docking studies and more information was obtained concerning binding modes of synthesized compounds within the active site of β-Glucuronidase. The X-ray structure of β-glucuronidase was retrieved from the Protein Data Bank with PDB ID: 1BHG. All synthesized compounds were docked into the active site of protein and conformations selected for each docked compound were of least energy and were pictured to further understand the interactions between ligands and receptor.
Binding mode of compound 6, the most active member (IC 50 = 0.80 ± 0.10 µM) among the series was analyzed. Three conventional hydrogen bond interactions were observed between the fluorine atom at ortho position and side chain amino acids including Tyr 508 and Lys 606 with a internuclear distance of 2.63 Å, 2.66 Å and 1.83 Å respectively. Meanwhile, C-H and N-H groups of Asn 484 were involved in forming carbon hydrogen bond with ether oxygen of chromene-4-one ring (2.88 Å) and π-donor hydrogen bond with the central arene ring of compound 6 (3.14 Å). Significant residues like Asp 207, Glu 451 and Glu 540 were not involved in hydrogen bonding due to absence of hydrogen bond donor groups on compound 6 instead, these residues were involved in π-anion interactions as shown in Figure 3a. Furthermore, the ligand /receptor complex was further stabilized by π-π T-shaped and π-π Stacked interactions between indole ring of Trp 587 and phenol ring of Tyr 508 respectively.
Compound 4, the second most active member with IC 50 = 1.10 ± 0.05 µM was evaluated and a conventional hydrogen bond between fluorine atom at para position and N-H group of Tyr 487 with a internuclear distance of 2.22 Å and carbon hydrogen bond between nitrogen atom of pyrazole ring of compound 4 and C-H (at imidazole ring) of His 509 (2.58 Å) were detected. Meanwhile, the catalytic residue Glu 451, as in case of compound 6 was also involved in forming two π-Anion interactions with the chromene-4-one ring of compound 4. Side chain residues involved in π-π T-Shaped interactions with compound 4 were Tyr 504 and Tyr 508 as shown in Figure 3b.
Analysis of interactions between third most active compound 3 (IC 50 = 2.10 ± 0.10 µM) and active site of β-Glucuronidase revealed that it was not involved in conventional hydrogen bonding. It was found that the ether oxygen atom of chromene-4-one ring and central arene ring of compound 3 were involved in forming carbon hydrogen bond (2.77 Å) and π-Donor hydrogen bond (3.26 Å) with the C-H and N-H groups of Asn 484 respectively. The chlorine atom presents at ortho position formed so-called halogen bond of distance 2.94 Å and bond angle~153 • with the carboxyl oxygen of Glu 451. Other interactions shown by compound 3 were almost the same as those shown by compound 6 (Figure 3c).
Experimental results were further validated by carrying out molecular docking studies and more information was obtained concerning binding modes of synthesized compounds within the active site of β-Glucuronidase. The X-ray structure of β-glucuronidase was retrieved from the Protein Data Bank with PDB ID: 1BHG. All synthesized compounds were docked into the active site of protein and conformations selected for each docked compound were of least energy and were pictured to further understand the interactions between ligands and receptor.
Binding mode of compound 6, the most active member (IC50 = 0.80 ± 0.10 μM) among the series was analyzed. Three conventional hydrogen bond interactions were observed between the fluorine atom at ortho position and side chain amino acids including Tyr 508 and Lys 606 with a internuclear distance of 2.63 Å, 2.66 Å and 1.83 Å respectively. Meanwhile, C-H and N-H groups of Asn 484 were involved in forming carbon hydrogen bond with ether oxygen of chromene-4-one ring (2.88 Å) and π-donor hydrogen bond with the central arene ring of compound 6 (3.14 Å). Significant residues like Asp 207, Glu 451 and Glu 540 were not involved in hydrogen bonding due to absence of hydrogen bond donor groups on compound 6 instead, these residues were involved in π-anion interactions as shown in Figure 3a. Furthermore, the ligand /receptor complex was further stabilized by π-π Tshaped and π-π Stacked interactions between indole ring of Trp 587 and phenol ring of Tyr 508 respectively.
Compound 4, the second most active member with IC50 = 1.10 ± 0.05 μM was evaluated and a conventional hydrogen bond between fluorine atom at para position and N-H group of Tyr 487 with a internuclear distance of 2.22 Å and carbon hydrogen bond between nitrogen atom of pyrazole ring of compound 4 and C-H (at imidazole ring) of His 509 (2.58 Å) were detected. Meanwhile, the catalytic residue Glu 451, as in case of compound 6 was also involved in forming two π-Anion interactions with the chromene-4-one ring of compound 4. Side chain residues involved in π-π T-Shaped interactions with compound 4 were Tyr 504 and Tyr 508 as shown in Figure 3b.
Analysis of interactions between third most active compound 3 (IC50 = 2.10 ± 0.10 μM) and active site of β-Glucuronidase revealed that it was not involved in conventional hydrogen bonding. It was found that the ether oxygen atom of chromene-4-one ring and central arene ring of compound 3 were involved in forming carbon hydrogen bond (2.77 Å) and π-Donor hydrogen bond (3.26 Å) with the C-H and N-H groups of Asn 484 respectively. The chlorine atom presents at ortho position formed socalled halogen bond of distance 2.94 Å and bond angle ~153° with the carboxyl oxygen of Glu 451. Other interactions shown by compound 3 were almost the same as those shown by compound 6 (Figure 3c).

Conclusions
Chromen-4-one based oxadiazole derivatives 1-19 have been synthesized and evaluated for β-glucuronidase inhibitory potential. All analogs displayed potent β-glucuronidase inhibitory potential ranging between 0.8 ± 0.1 to 42.3 ± 0.8 µM as a compare to standard d-saccharic acid 1,4 lactone (IC 50 = 48.1 ± 1.2µM). The fluoro derivatives were found to be the more potent among the series. A molecular docking study indicated that the top ranked conformations of almost all compounds were well accommodated inside the active site of β-glucuronidase enzyme and they were involved in various types of interactions with the active site residues of the β-glucuronidase enzyme.

General Procedure for Synthesis of Flavone-Based Oxadiazoles
Compound IV (0.5 mmol) and a substituted arylcarboxylic acid (0.5 mmol) were mixed in a 50 mL round bottom flask then POCl 3 (5 mL) was added dropwise [33]. The mixture was refluxed for 4-5 h while the reaction progress was monitored using TLC until completion, when the reaction mixture cooled to room temperature and poured onto crushed ice. NaHCO 3 solution was added and the resulting solid mass that precipitated out was filtered, dried, and recrystallized from methanol in good to excellent yields.