Synthesis, Molecular Docking and β-Glucuronidase Inhibitory Potential of Indole Base Oxadiazole Derivatives

β-glucuronidase is a lysosomal glycosidase enzyme which catalyzes the extracellular matrix of cancer and normal cells and the glycosaminoglycans of the cell membrane, which is important for cancer cell proliferation, invasion, and metastasis. Liver cancer, colon carcinoma, and neoplasm bladder are triggered by the increase of the level of β-glucuronidase activity. The most valuable structures are indole and oxadiazole which has gain immense attention because of its pharmacological behavior and display many biological properties. Twenty-two (1–22) analogs of indole based oxadiazole were synthesized and screened for their inhibitory potential against β-glucuronidase. Majority of the compounds showed potent inhibitory potential with IC50 values ranging between 0.9 ± 0.01 to 46.4 ± 0.9 µM, under positive control of standard drug d-saccharic acid 1,4 lactone (IC50 = 48.1 ± 1.2 µM). Structural activity relationship (SAR) has been established for all synthesized compounds. To shed light on molecular interactions between the synthesized compounds and β-glucuronidase, 1, 4, and 6 compounds were docked into the active binding site of β-glucuronidase. The obtained results showed that this binding is thermodynamically favorable and β-glucuronidase inhibition of the selected compounds increases with the number of hydrogen bonding established in selected compound-β-glucuronidase complexes.


Introduction
β-glucuronidase is an enzyme that catalyzes the extracellular matrix of cancer and normal cells and also catalyzes glycosaminoglycans of the cell membrane. This catalysis is important for cancer cell proliferation, invasion, and metastasis [1,2]. Liver cancer, colon carcinoma and neoplasm bladder are caused by the increase level of β-glucuronidase activity [3][4][5][6]. β-glucuronidase has been used as an important tool in the detection of real time monitoring, clinical therapies, diagnosis in early stage, and used in tumor marker [7,8]. High level of β-glucuronidase is associated with various diseases including epilepsy [9] and other physiological disorder. β-glucuronidase increase level cause liver damage which is a major issue worldwide [10]. The deficiency of GUS in the human body causes mucopolysaccharidosis type VII (MPSVII, known as Sly syndrome) due to the accumulation of glycosoaminoglycans containing glucuronic acid residues, leading to lysosomal storage in the brain [11,12]. It is very important to inhibit this enzyme to prevent such physiological disorder. The heterocyclic compounds cover one of the important fields in medicinal chemistry. They occupy an important position in the structures of a variety of natural products. One of the most valuable structures is indole which has gain immense attention because of its pharmacological behavior and has been considered a vital scaffold [13,14]. A number of indole-based compounds have been reported for their potential anticancer activities [15]. Hydrazono-indole analogs have been reported to induce apoptosis [16], and to interact with tubulin [17][18][19].

Biological Activity
In this study, we have carried out the synthesis of indole based oxadiazole analogs (1- 22) and their evaluation against β-glucuronidase. Majority of the compounds showed potent inhibitory potential with IC 50 values ranging from 0.9 ± 0.01 to 46.4 ± 0.9 µM when compared with the positive control D-saccharic acid 1,4 lactonec (IC 50 = 48.1 ± 1.2 µM).
Structural activity relationship has been established for all compounds. Compound 6 having at the ortho-meta position the dihydroxyl groups on the phenyl ring was found the most active analog among the series. The greater inhibitory potential of this compound may be due to the position as well as the vicinity of the dihydroxy groups.
If we compare analog 6 with other dihydroxy analogs i.e., analog 5 (IC 50 = 11.4 ± 0.30 µM), analog 7 (IC 50 = 1.2 ± 0.01 µM) and analog 8 (IC 50 = 7.2 ± 0.10 µM), it was found that the Compound 6 is much more potent. This higher activity of analog 6 is seems due to the hydroxyl groups position on the phenyl ring which is ortho-meta. Additionally, the activity may also be enhanced when both hydroxyl groups are at the vicinal position. If we compare the dihydroxy analogs with mono-hydroxy analogs i.e., analog 3 (IC 50 = 6.2 ± 0.2 µM), analog 4 (IC 50 = 17.90 ± 0.4 µM), and analog 9 (IC 50 = 11.0 ± 0.4 µM), the dihydroxy analogs were found to be most superior. The greater potential indicates that position and number of the hydroxyl groups on phenyl ring play a vital role in the inhibitory potential.
Comparing the nitro substituted analogs 10 (IC 50 = 40.0 ± 0.7 µM), 11 (NAb) and 13 (IC 50 = 37.3 ± 0.7 µM), the ortho and para substituted analogs 10 and 12 were found active while analog 11 was found inactive. This indicates that the position of the nitro group at ortho and para positions enhances the activity rather than meta position.
If we compared the o-fluoro substituted analog 18 (IC 50 = 5.0 ± 0.1 µM) with m-fluoro analog 19 (IC 50 = 10.5 ± 0.2 µM) and p-fluoro substituted analog 20 (IC 50 = 17.0 ± 0.4 µM), analog 18 was found more superior and the reason for this greater potential is that ortho substituted analog is more favorable for inhibition of the enzyme. Similarly, the ortho substituted chloro analog 21 with IC 50 = 6.0 ± 0.2 µM showed more potency when compared with meta and para substituted analogs 15 (IC 50 = 34.6 ± 0.7 µM) and 22 (IC 50 = 22.2 ± 0.5 µM), respectively. This greater inhibition of Compound 21 is attributed to the position of chloro group at ortho position. Compounds containing pyridine in their structures-i.e., Compounds 15 and 16-were found to be the least active compounds in the series. This less potency may be due to the non-availability of the nitrogen electron lone pair in the pyridine moiety. From the whole study it has been concluded that position, nature and number of the substituents on the phenyl ring play an important role in the inhibitory potential of the synthesized analogs.

Molecular Docking Study
The concentration inhibition IC 50 values of indole based oxadiazole synthesized derivatives as β-glucoronidase inhibitors are presented in Table 1. From Table 1, it is evident that the inhibitory activity of the synthesized derivatives depends mainly on structural factors such as the type, number and position of the functional group on the phenyl ring of the synthesized derivatives. According to inhibitory IC 50 values (Table 1) the synthesized derivatives may be classified into three groups: Highly active group with low IC 50 values (e.g., 6 and 7), moderate active group (e.g., 4 and 5) and a low active group (e.g., 1 and 2). For a better understanding of the experimental results and to emphasize the effects of type, number, and relative position of substituted groups on β-glucoronidase inhibition by the tilted compounds, molecular docking study has been performed to shed light on the established binding modes of eight selected compounds (1 and 3-9) to the closest residues in the active site of β-glucoronidase enzyme. Table 2 summarized (i) the calculated binding energies of the stable complex's ligand-β-glucoronidase, (ii) number of established intermolecular hydrogen bonding between the synthesized compounds (1 and 3-9) and amino acid residues into the active site of β-glucoronidase, and (iii) number of closest residues surrounded the docked compounds (1 and 3-9) within the active binding site of β-glucoronidase. ± 0.2 µM showed more potency when compared with meta and para substituted analogs 15 (IC50 = 34.6 ± 0.7 µM) and 22 (IC50 = 22.2 ± 0.5 µM), respectively. This greater inhibition of Compound 21 is attributed to the position of chloro group at ortho position. Compounds containing pyridine in their structures-i.e., Compounds 15 and 16-were found to be the least active compounds in the series. This less potency may be due to the non-availability of the nitrogen electron lone pair in the pyridine moiety. From the whole study it has been concluded that position, nature and number of the substituents on the phenyl ring play an important role in the inhibitory potential of the synthesized analogs. ± 0.2 µM showed more potency when compared with meta and para substituted analogs 15 (IC50 = 34.6 ± 0.7 µM) and 22 (IC50 = 22.2 ± 0.5 µM), respectively. This greater inhibition of Compound 21 is attributed to the position of chloro group at ortho position. Compounds containing pyridine in their structures-i.e., Compounds 15 and 16-were found to be the least active compounds in the series. This less potency may be due to the non-availability of the nitrogen electron lone pair in the pyridine moiety. From the whole study it has been concluded that position, nature and number of the substituents on the phenyl ring play an important role in the inhibitory potential of the synthesized analogs. ± 0.2 µM showed more potency when compared with meta and para substituted analogs 15 (IC50 = 34.6 ± 0.7 µM) and 22 (IC50 = 22.2 ± 0.5 µM), respectively. This greater inhibition of Compound 21 is attributed to the position of chloro group at ortho position. Compounds containing pyridine in their structures-i.e., Compounds 15 and 16-were found to be the least active compounds in the series. This less potency may be due to the non-availability of the nitrogen electron lone pair in the pyridine moiety. From the whole study it has been concluded that position, nature and number of the substituents on the phenyl ring play an important role in the inhibitory potential of the synthesized analogs. 26.2 ± 0. 8 13 ± 0.2 µM showed more potency when compared with meta and para substituted analogs 15 (IC50 = 34.6 ± 0.7 µM) and 22 (IC50 = 22.2 ± 0.5 µM), respectively. This greater inhibition of Compound 21 is attributed to the position of chloro group at ortho position. Compounds containing pyridine in their structures-i.e., Compounds 15 and 16-were found to be the least active compounds in the series. This less potency may be due to the non-availability of the nitrogen electron lone pair in the pyridine moiety. From the whole study it has been concluded that position, nature and number of the substituents on the phenyl ring play an important role in the inhibitory potential of the synthesized analogs. favorable for inhibition of the enzyme. Similarly, the ortho substituted chloro analog 21 with IC50 = 6.0 ± 0.2 µM showed more potency when compared with meta and para substituted analogs 15 (IC50 = 34.6 ± 0.7 µM) and 22 (IC50 = 22.2 ± 0.5 µM), respectively. This greater inhibition of Compound 21 is attributed to the position of chloro group at ortho position. Compounds containing pyridine in their structures-i.e., Compounds 15 and 16-were found to be the least active compounds in the series. This less potency may be due to the non-availability of the nitrogen electron lone pair in the pyridine moiety. From the whole study it has been concluded that position, nature and number of the substituents on the phenyl ring play an important role in the inhibitory potential of the synthesized analogs. 6.2 ± 0. 2 14 favorable for inhibition of the enzyme. Similarly, the ortho substituted chloro analog 21 with IC50 = 6.0 ± 0.2 µM showed more potency when compared with meta and para substituted analogs 15 (IC50 = 34.6 ± 0.7 µM) and 22 (IC50 = 22.2 ± 0.5 µM), respectively. This greater inhibition of Compound 21 is attributed to the position of chloro group at ortho position. Compounds containing pyridine in their structures-i.e., Compounds 15 and 16-were found to be the least active compounds in the series. This less potency may be due to the non-availability of the nitrogen electron lone pair in the pyridine moiety. From the whole study it has been concluded that position, nature and number of the substituents on the phenyl ring play an important role in the inhibitory potential of the synthesized analogs. favorable for inhibition of the enzyme. Similarly, the ortho substituted chloro analog 21 with IC50 = 6.0 ± 0.2 µM showed more potency when compared with meta and para substituted analogs 15 (IC50 = 34.6 ± 0.7 µM) and 22 (IC50 = 22.2 ± 0.5 µM), respectively. This greater inhibition of Compound 21 is attributed to the position of chloro group at ortho position. Compounds containing pyridine in their structures-i.e., Compounds 15 and 16-were found to be the least active compounds in the series. This less potency may be due to the non-availability of the nitrogen electron lone pair in the pyridine moiety. From the whole study it has been concluded that position, nature and number of the substituents on the phenyl ring play an important role in the inhibitory potential of the synthesized analogs. favorable for inhibition of the enzyme. Similarly, the ortho substituted chloro analog 21 with IC50 = 6.0 ± 0.2 µM showed more potency when compared with meta and para substituted analogs 15 (IC50 = 34.6 ± 0.7 µM) and 22 (IC50 = 22.2 ± 0.5 µM), respectively. This greater inhibition of Compound 21 is attributed to the position of chloro group at ortho position. Compounds containing pyridine in their structures-i.e., Compounds 15 and 16-were found to be the least active compounds in the series. This less potency may be due to the non-availability of the nitrogen electron lone pair in the pyridine moiety. From the whole study it has been concluded that position, nature and number of the substituents on the phenyl ring play an important role in the inhibitory potential of the synthesized analogs. favorable for inhibition of the enzyme. Similarly, the ortho substituted chloro analog 21 with IC50 = 6.0 ± 0.2 µM showed more potency when compared with meta and para substituted analogs 15 (IC50 = 34.6 ± 0.7 µM) and 22 (IC50 = 22.2 ± 0.5 µM), respectively. This greater inhibition of Compound 21 is attributed to the position of chloro group at ortho position. Compounds containing pyridine in their structures-i.e., Compounds 15 and 16-were found to be the least active compounds in the series. This less potency may be due to the non-availability of the nitrogen electron lone pair in the pyridine moiety. From the whole study it has been concluded that position, nature and number of the substituents on the phenyl ring play an important role in the inhibitory potential of the synthesized analogs. 11.4 ± 0. 30 16 favorable for inhibition of the enzyme. Similarly, the ortho substituted chloro analog 21 with IC50 = 6.0 ± 0.2 µM showed more potency when compared with meta and para substituted analogs 15 (IC50 = 34.6 ± 0.7 µM) and 22 (IC50 = 22.2 ± 0.5 µM), respectively. This greater inhibition of Compound 21 is attributed to the position of chloro group at ortho position. Compounds containing pyridine in their structures-i.e., Compounds 15 and 16-were found to be the least active compounds in the series. This less potency may be due to the non-availability of the nitrogen electron lone pair in the pyridine moiety. From the whole study it has been concluded that position, nature and number of the substituents on the phenyl ring play an important role in the inhibitory potential of the synthesized analogs.

Molecular Docking Study
The concentration inhibition IC50 values of indole based oxadiazole synthesized derivatives as β-glucoronidase inhibitors are presented in Table 1. From Table 1, it is evident that the inhibitory activity of the synthesized derivatives depends mainly on structural factors such as the type, number and position of the functional group on the phenyl ring of the synthesized derivatives. According to inhibitory IC50 values (Table 1) the synthesized derivatives may be classified into three groups: Highly active group with low IC50 values (e.g., 6 and 7), moderate active group (e.g., 4 and 5) and a low active group (e.g., 1 and 2). For a better understanding of the experimental results and to emphasize the effects of type, number, and relative position of substituted groups on β-glucoronidase inhibition by the tilted compounds, molecular docking study has been performed to shed light on the established binding modes of eight selected compounds (1 and 3-9) to the closest residues in the active site of β-glucoronidase enzyme. Table 2 summarized (i) the calculated binding energies of the stable complex's ligand-β-glucoronidase, (ii) number of established intermolecular hydrogen bonding between the synthesized compounds (1 and 3-9) and amino acid residues into the active site of β-glucoronidase, and (iii) number of closest residues surrounded the docked compounds (1 and 3-9) within the active binding site of β-glucoronidase.

Molecular Docking Study
The concentration inhibition IC50 values of indole based oxadiazole synthesized derivatives as β-glucoronidase inhibitors are presented in Table 1. From Table 1, it is evident that the inhibitory activity of the synthesized derivatives depends mainly on structural factors such as the type, number and position of the functional group on the phenyl ring of the synthesized derivatives. According to inhibitory IC50 values (Table 1) the synthesized derivatives may be classified into three groups: Highly active group with low IC50 values (e.g., 6 and 7), moderate active group (e.g., 4 and 5) and a low active group (e.g., 1 and 2). For a better understanding of the experimental results and to emphasize the effects of type, number, and relative position of substituted groups on β-glucoronidase inhibition by the tilted compounds, molecular docking study has been performed to shed light on the established binding modes of eight selected compounds (1 and 3-9) to the closest residues in the active site of β-glucoronidase enzyme. Table 2 summarized (i) the calculated binding energies of the stable complex's ligand-β-glucoronidase, (ii) number of established intermolecular hydrogen bonding between the synthesized compounds (1 and 3-9) and amino acid residues into the active site of β-glucoronidase, and (iii) number of closest residues surrounded the docked compounds (1 and 3-9) within the active binding site of β-glucoronidase.

Molecular Docking Study
The concentration inhibition IC50 values of indole based oxadiazole synthesized derivatives as β-glucoronidase inhibitors are presented in Table 1. From Table 1, it is evident that the inhibitory activity of the synthesized derivatives depends mainly on structural factors such as the type, number and position of the functional group on the phenyl ring of the synthesized derivatives. According to inhibitory IC50 values (Table 1) the synthesized derivatives may be classified into three groups: Highly active group with low IC50 values (e.g., 6 and 7), moderate active group (e.g., 4 and 5) and a low active group (e.g ., 1 and 2). For a better understanding of the experimental results and to emphasize the effects of type, number, and relative position of substituted groups on β-glucoronidase inhibition by the tilted compounds, molecular docking study has been performed to shed light on the established binding modes of eight selected compounds (1 and 3-9) to the closest residues in the active site of β-glucoronidase enzyme. Table 2 summarized (i) the calculated binding energies of the stable complex's ligand-β-glucoronidase, (ii) number of established intermolecular hydrogen bonding between the synthesized compounds (1 and 3-9) and amino acid residues into the active site of β-glucoronidase, and (iii) number of closest residues surrounded the docked compounds (1 and 3-9) within the active binding site of β-glucoronidase.

Molecular Docking Study
The concentration inhibition IC50 values of indole based oxadiazole synthesized derivatives as β-glucoronidase inhibitors are presented in Table 1. From Table 1, it is evident that the inhibitory activity of the synthesized derivatives depends mainly on structural factors such as the type, number and position of the functional group on the phenyl ring of the synthesized derivatives. According to inhibitory IC50 values (Table 1) the synthesized derivatives may be classified into three groups: Highly active group with low IC50 values (e.g., 6 and 7), moderate active group (e.g., 4 and 5) and a low active group (e.g ., 1 and 2). For a better understanding of the experimental results and to emphasize the effects of type, number, and relative position of substituted groups on β-glucoronidase inhibition by the tilted compounds, molecular docking study has been performed to shed light on the established binding modes of eight selected compounds (1 and 3-9) to the closest residues in the active site of β-glucoronidase enzyme. Table 2 summarized (i) the calculated binding energies of the stable complex's ligand-β-glucoronidase, (ii) number of established intermolecular hydrogen bonding between the synthesized compounds (1 and 3-9) and amino acid residues into the active site of β-glucoronidase, and (iii) number of closest residues surrounded the docked compounds (1 and 3-9) within the active binding site of β-glucoronidase.

Molecular Docking Study
The concentration inhibition IC50 values of indole based oxadiazole synthesized derivatives as β-glucoronidase inhibitors are presented in Table 1. From Table 1, it is evident that the inhibitory activity of the synthesized derivatives depends mainly on structural factors such as the type, number and position of the functional group on the phenyl ring of the synthesized derivatives. According to inhibitory IC50 values (Table 1) the synthesized derivatives may be classified into three groups: Highly active group with low IC50 values (e.g., 6 and 7), moderate active group (e.g., 4 and 5) and a low active group (e.g ., 1 and 2). For a better understanding of the experimental results and to emphasize the effects of type, number, and relative position of substituted groups on β-glucoronidase inhibition by the tilted compounds, molecular docking study has been performed to shed light on the established binding modes of eight selected compounds (1 and 3-9) to the closest residues in the active site of β-glucoronidase enzyme. Table 2 summarized (i) the calculated binding energies of the stable complex's ligand-β-glucoronidase, (ii) number of established intermolecular hydrogen bonding between the synthesized compounds (1 and 3-9) and amino acid residues into the active site of β-glucoronidase, and (iii) number of closest residues surrounded the docked compounds (1 and 3-9) within the active binding site of β-glucoronidase.

Molecular Docking Study
The concentration inhibition IC50 values of indole based oxadiazole synthesized derivatives as β-glucoronidase inhibitors are presented in Table 1. From Table 1, it is evident that the inhibitory activity of the synthesized derivatives depends mainly on structural factors such as the type, number and position of the functional group on the phenyl ring of the synthesized derivatives. According to inhibitory IC50 values (Table 1) the synthesized derivatives may be classified into three groups: Highly active group with low IC50 values (e.g., 6 and 7), moderate active group (e.g., 4 and 5) and a low active group (e.g ., 1 and 2). For a better understanding of the experimental results and to emphasize the effects of type, number, and relative position of substituted groups on β-glucoronidase inhibition by the tilted compounds, molecular docking study has been performed to shed light on the established binding modes of eight selected compounds (1 and 3-9) to the closest residues in the active site of β-glucoronidase enzyme. Table 2 summarized (i) the calculated binding energies of the stable complex's ligand-β-glucoronidase, (ii) number of established intermolecular hydrogen bonding between the synthesized compounds (1 and 3-9) and amino acid residues into the active site of β-glucoronidase, and (iii) number of closest residues surrounded the docked compounds (1 and 3-9) within the active binding site of β-glucoronidase.

Molecular Docking Study
The concentration inhibition IC50 values of indole based oxadiazole synthesized derivatives as β-glucoronidase inhibitors are presented in Table 1. From Table 1, it is evident that the inhibitory activity of the synthesized derivatives depends mainly on structural factors such as the type, number and position of the functional group on the phenyl ring of the synthesized derivatives. According to inhibitory IC50 values (Table 1) the synthesized derivatives may be classified into three groups: Highly active group with low IC50 values (e.g., 6 and 7), moderate active group (e.g., 4 and 5) and a low active group (e.g ., 1 and 2). For a better understanding of the experimental results and to emphasize the effects of type, number, and relative position of substituted groups on β-glucoronidase inhibition by the tilted compounds, molecular docking study has been performed to shed light on the established binding modes of eight selected compounds (1 and 3-9) to the closest residues in the active site of β-glucoronidase enzyme. Table 2 summarized (i) the calculated binding energies of the stable complex's ligand-β-glucoronidase, (ii) number of established intermolecular hydrogen bonding between the synthesized compounds (1 and 3-9) and amino acid residues into the active site of β-glucoronidase, and (iii) number of closest residues surrounded the docked compounds (1 and 3-9) within the active binding site of β-glucoronidase.

Molecular Docking Study
The concentration inhibition IC50 values of indole based oxadiazole synthesized derivatives as β-glucoronidase inhibitors are presented in Table 1. From Table 1, it is evident that the inhibitory activity of the synthesized derivatives depends mainly on structural factors such as the type, number and position of the functional group on the phenyl ring of the synthesized derivatives. According to inhibitory IC50 values (Table 1) the synthesized derivatives may be classified into three groups: Highly active group with low IC50 values (e.g., 6 and 7), moderate active group (e.g., 4 and 5) and a low active group (e.g ., 1 and 2). For a better understanding of the experimental results and to emphasize the effects of type, number, and relative position of substituted groups on β-glucoronidase inhibition by the tilted compounds, molecular docking study has been performed to shed light on the established binding modes of eight selected compounds (1 and 3-9) to the closest residues in the active site of β-glucoronidase enzyme. Table 2 summarized (i) the calculated binding energies of the stable complex's ligand-β-glucoronidase, (ii) number of established intermolecular hydrogen bonding between the synthesized compounds (1 and 3-9) and amino acid residues into the active site of β-glucoronidase, and (iii) number of closest residues surrounded the docked compounds (1 and 3-9) within the active binding site of β-glucoronidase.

Molecular Docking Study
The concentration inhibition IC50 values of indole based oxadiazole synthesized derivatives as β-glucoronidase inhibitors are presented in Table 1. From Table 1, it is evident that the inhibitory activity of the synthesized derivatives depends mainly on structural factors such as the type, number and position of the functional group on the phenyl ring of the synthesized derivatives. According to inhibitory IC50 values (Table 1) the synthesized derivatives may be classified into three groups: Highly active group with low IC50 values (e.g., 6 and 7), moderate active group (e.g., 4 and 5) and a low active group (e.g ., 1 and 2). For a better understanding of the experimental results and to emphasize the effects of type, number, and relative position of substituted groups on β-glucoronidase inhibition by the tilted compounds, molecular docking study has been performed to shed light on the established binding modes of eight selected compounds (1 and 3-9) to the closest residues in the active site of β-glucoronidase enzyme. Table 2 summarized (i) the calculated binding energies of the stable complex's ligand-β-glucoronidase, (ii) number of established intermolecular hydrogen bonding between the synthesized compounds (1 and 3-9) and amino acid residues into the active site of β-glucoronidase, and (iii) number of closest residues surrounded the docked compounds (1 and 3-9) within the active binding site of β-glucoronidase.  (1, 4, and 6)

Molecular Docking Study
The concentration inhibition IC50 values of indole based oxadiazole synthesized derivatives as β-glucoronidase inhibitors are presented in Table 1. From Table 1, it is evident that the inhibitory activity of the synthesized derivatives depends mainly on structural factors such as the type, number and position of the functional group on the phenyl ring of the synthesized derivatives. According to inhibitory IC50 values (Table 1) the synthesized derivatives may be classified into three groups: Highly active group with low IC50 values (e.g., 6 and 7), moderate active group (e.g., 4 and 5) and a low active group (e.g ., 1 and 2). For a better understanding of the experimental results and to emphasize the effects of type, number, and relative position of substituted groups on β-glucoronidase inhibition by the tilted compounds, molecular docking study has been performed to shed light on the established binding modes of eight selected compounds (1 and 3-9) to the closest residues in the active site of β-glucoronidase enzyme. Table 2 summarized (i) the calculated binding energies of the stable complex's ligand-β-glucoronidase, (ii) number of established intermolecular hydrogen bonding between the synthesized compounds (1 and 3-9) and amino acid residues into the active site of β-glucoronidase, and (iii) number of closest residues surrounded the docked compounds (1 and 3-9) within the active binding site of β-glucoronidase.  (1, 4, and 6)

Molecular Docking Study
The concentration inhibition IC50 values of indole based oxadiazole synthesized derivatives as β-glucoronidase inhibitors are presented in Table 1. From Table 1, it is evident that the inhibitory activity of the synthesized derivatives depends mainly on structural factors such as the type, number and position of the functional group on the phenyl ring of the synthesized derivatives. According to inhibitory IC50 values (Table 1) the synthesized derivatives may be classified into three groups: Highly active group with low IC50 values (e.g., 6 and 7), moderate active group (e.g., 4 and 5) and a low active group (e.g ., 1 and 2). For a better understanding of the experimental results and to emphasize the effects of type, number, and relative position of substituted groups on β-glucoronidase inhibition by the tilted compounds, molecular docking study has been performed to shed light on the established binding modes of eight selected compounds (1 and 3-9) to the closest residues in the active site of β-glucoronidase enzyme. Table 2 summarized (i) the calculated binding energies of the stable complex's ligand-β-glucoronidase, (ii) number of established intermolecular hydrogen bonding between the synthesized compounds (1 and 3-9) and amino acid residues into the active site of β-glucoronidase, and (iii) number of closest residues surrounded the docked compounds (1 and 3-9) within the active binding site of β-glucoronidase.

Molecular Docking Study
The concentration inhibition IC50 values of indole based oxadiazole synthesized derivatives as β-glucoronidase inhibitors are presented in Table 1. From Table 1, it is evident that the inhibitory activity of the synthesized derivatives depends mainly on structural factors such as the type, number and position of the functional group on the phenyl ring of the synthesized derivatives. According to inhibitory IC50 values (Table 1) the synthesized derivatives may be classified into three groups: Highly active group with low IC50 values (e.g., 6 and 7), moderate active group (e.g., 4 and 5) and a low active group (e.g ., 1 and 2). For a better understanding of the experimental results and to emphasize the effects of type, number, and relative position of substituted groups on β-glucoronidase inhibition by the tilted compounds, molecular docking study has been performed to shed light on the established binding modes of eight selected compounds (1 and 3-9) to the closest residues in the active site of β-glucoronidase enzyme. Table 2 summarized (i) the calculated binding energies of the stable complex's ligand-β-glucoronidase, (ii) number of established intermolecular hydrogen bonding between the synthesized compounds (1 and 3-9) and amino acid residues into the active site of β-glucoronidase, and (iii) number of closest residues surrounded the docked compounds (1 and 3-9) within the active binding site of β-glucoronidase.  Binding energies of the docked selected compounds (Table 2) within the active site β-glucoronidase show negative binding energies, which indicates the ability of the docked compound to inhibit β-glucoronidase, and that the inhibition is spontaneous. The docking results in Table 2 and Figure 1 showed that the activity of synthesized compounds may return to the number of hydrogen bonding established between functional groups of the docked ligands and amnio acid residues of the β-glucoronidase into its binding site. However, the binding energy of the formed complex between the docked compounds and β-glucoronidase, and the number of closest residues to the docked ligands has no significant contribution on the inhibitory activity of the titled compounds (Table 2 and Figure 1). The lower activity of 1 compared with 3-6 is mainly returned to the nature of the substituted groups in the aromatic ring (Scheme 1). The Compound 1 is substitute with a methyl group, while 3-6 are substituted with hydroxyl groups. Docking results show that the methyl group in 1 form alkyl and Pi-Alkyl weak interactions with ALA A 218, TYR A 242, and TYR A 243 residues into the binding site of β-glucoronidase, while in 3-6 the substituted hydroxyl groups forms strong hydrogen bonding interactions with amino acid residues into the binding site of β-glucoronidase (Figure 1).
The aromatic ring in 5-8 is substituted with two hydroxyl groups, while in 3, 4, and 9 is substituted only with one hydroxyl group. It is obvious from docking results that the increase of hydroxyl groups increases the number of hydrogen bonds that can be established with amino acid residues into the binding site of β-glucoronidase (Figure 1). For instance, in the case of the active Compound 6, four hydrogen bonding are established between residues of β-glucoronidase and Compound 6, while in 4 only two hydrogen bonds are formed (Figure 1). In 6, the strongest hydrogen bond is formed between GLU173 amino acid and the hydrogen atom of NH of indole ring with a distance of 1.89 Å. The second strongest hydrogen bond is relatively weaker than the first one and is established between the hydrogen atom of hydroxyl of catechol oxygen in the meta position of the catechol group and GLU245 with a distance of 1.90 Å. The third strongest hydrogen bond is relatively weaker than the two first ones, and is established between the hydrogen atom of hydroxyl of catechol oxygen in ortho position of catechol group and GLU245 with a distance of 2.02 Å. The fourth hydrogen bond is relatively weak than the previous ones, and it is established between TYR243 amino acid and the hydrogen atom of hydroxyl of catechol oxygen in the ortho position of the catechol group with a distance of 2.90 Å. In case of Compound 9 with only one hydroxyl groups, three hydrogen bonding are established in the complex formed between 9 and β-glucoronidase, while for the synthesized Compound 1 only a weaker hydrogen bond formed, which is established between GLU173 amino acid and the hydrogen atom of NH of indole with a distance of 2.30 Å. As mentioned above the position of the substituted groups on the aromatic ring may have a strong influence on β-glucoronidase inhibition. Compound 7 is substituted with two hydroxyl groups on meta and para position, while 8 the two hydroxyl groups are substituted in ortho and para position. The higher activity of 7 compared with 8 may refer to the number of hydrogen bonds that are formed in 7 compared with 8. Indeed, the two hydroxyl groups in 7 forms four hydrogen bonds with amino acids of β-glucoronidase, while in 8 the two hydroxyl groups formed only three hydrogen bonding with amino acid residues of β-glucoronidase. Molecules 2019, 24, 963 6 of 13 (1) (4) (9) (6) (7) (8) Figure 1. 2D closest interactions between active site residues of β-glucoronidase and synthesized compounds 1, 4, 9, 6, 7, and 8.

Molecular Docking Details
To determine the binding modes between the active site residues of β-glucuronidase and docked selected synthesized indole based oxadiazole derivatives (1 and 3-9), molecular docking has been performed by using the Lamarckian genetic algorithm as applied in the Autodock package [37]. The 3D structure of β-glucuronidase and the original docked ligand N-alkyl cyclophellitol aziridine were downloaded from the RCSB data bank website (PDB code 5G0Q) [38]. After removing the water molecules, Kollman charge and polar hydrogen atoms have been added to the extracted receptor structure by manipulating the AutoDock Tools 4.2 (The Scripps Research Institute, La Jolla, CA, USA). The re-docking of the original ligand N-alkyl cyclophellitol aziridine into the active site of β-glucuronidase is well reproduced with an RMSD value lower than 2 Å. 1 and 3-9 structural geometries were optimized using Merck molecular force field 94 as implemented in Hyperchem Software (HyperChem Release 7.52, Hypercube Inc., Gainesville, FL, USA, 2002), and saved as protein data bank (pdf) files. For each docked ligand, non-polar hydrogen's were merged and rotatable bonds were defined. Docking run was performed by the Lamarckian genetic algorithm, with 500 as a total number of run for originated ligand and 100 run for 1, 4, and 6 derivatives. A total population of 150 individuals with 27,000 generations and 250,000 energy evaluations were utilized in each particular run. Operator weights for crossover, mutation, and elitism were set to 0.8, 0.02, and 1, respectively. The binding site was defined using a grid of 40 × 40 × 40 points each with a grid spacing of 0.375 Å. Intel (R) core (M) 15-3770 CPU @ 3.40 GHz workstation is used to carry out docking calculation.

Conclusions
β-glucuronidase is a member of the enzyme lysosomal glycosidase. The function of this enzyme is to catalyze the extracellular matrix of cancer and normal cells and also catalyze glycosaminoglycans of the cell membrane. This catalysis is important for cancer cell proliferation, invasion and metastasis. Liver cancer, colon carcinoma, and neoplasm bladder are caused by the increase level of β-glucuronidase activity. The most valuable structures are indole and oxadiazole which has gain immense attention because of its pharmacological behavior and display many biological properties. The synthesized analogs (1-22), indole based oxadiazole analogs were evaluated for their inhibitory potential against the β-glucuronidase enzyme. Majority of the compounds showed potent inhibitory potential with IC 50 values ranging from 0.9 ± 0.01 to 46.4 ± 0.9 µM when compared with the standard drug D-saccharic acid 1,4 lactonec (IC 50 = 48.1 ± 1.2 µM). Structural activity relationship (SAR) has been established for all synthesized compounds. Molecular docking of 1, 4, and 6 into the active site of β-glucuronidase showed that the β-glucuronidase inhibition by the tilted compounds is thermodynamically favorable and increases with the number of hydrogens bonding intermolecular interactions.