Synthesis of oxadiazole-coupled-thiadiazole derivatives as a potent β-glucuronidase inhibitors and their molecular docking study

Bioorganic & Medicinal Chemistry 27 (2019) 3145–3155

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Synthesis of oxadiazole-coupled-thiadiazole derivatives as a potent β-glucuronidase inhibitors and their molecular docking study

T

Muhammad Tahaa, , Syahrul Imranb, Munther Alomaric, Fazal Rahimd, Abdul Wadoode, Ashik Mosaddika, Nizam Uddinf, Mohammed Gollapallig, Mohammed A. Alqahtanig, Yasser A. Bamaroufg ⁎

a

Department of Clinical Pharmacy, Institute for Research and Medical Consultations (IRMC), Imam Abdulrahman Bin Faisal University, P.O. Box 31441, Dammam, Saudi Arabia b Atta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor D. E., Malaysia c Department of Stem Cell Research, Institute for Research and Medical Consultations, Imam Abdulrahman Bin Faisal University, P. O. Box. 1982, Dammam 31441, Saudi Arabia d Department of Chemistry, Hazara University, Mansehra 21120, Pakistan e Department of Biochemistry, Abdul Wali Khan University Mardan, Mardan 23200, Pakistan f Department of Chemistry, University of Karachi, Karachi 75270, Pakistan g Department of Computer Information Systems, College of Computer Science & Information Technology (CCSIT) Imam Abdulrahman Bin Faisal University P.O. Box 1982, Dammam 31441, Saudi Arabia

ARTICLE INFO

ABSTRACT

Keywords: Oxadiazole-thiadiazole β-Glucuronidase inhibition Molecular docking study SAR

A new series of oxadiazole with thiadiazole moiety (6–27) were synthesized, characterized by different spectroscopic techniques and evaluated for β-glucuronidase inhibitory potential. Sixteen analogs such as 6, 7, 8, 9, 10, 12, 13, 14, 17, 18, 20, 23, 24, 25, 26 and 27 showed IC50 values in the range of 0.96 ± 0.01 to 46.46 ± 1.10 μM, and hence were found to have excellent inhibitory potential in comparison to standard Dsaccharic acid 1,4-lactone (IC50 = 48.4 ± 1.25 μM). Two analogs such as 16 and 19 showed moderate inhibitory potential while analogs 11, 15, 21 and 22 were found inactive. Our study identifies new series of potent β-glucuronidase inhibitors for further investigation. Structure activity relationships were established for all compounds which showed that the activity is varied due to different substituents on benzene ring. The interaction of the compounds with enzyme active site were confirmed with the help of docking studies, which reveals that the electron withdrawing group and hydroxy group make the molecules more favorable for enzyme inhibition.

1. Introduction β-Glucuronidase is an exoglycosidase enzyme which catalyzes the cleavage of glucuronosyl-O-bonds.1 The enzyme is present in many organs and body fluids such as kidney, spleen, bile, serum, urine, respectively.2 Enhanced activity of this enzyme has been reported in a variety of pathological conditions, including urinary tract infection,3–6 renal diseases,7 transplantation rejection,8 epilepsy,9 neoplasm of bladder10 larynx and breast.11 Furthermore, β-glucuronidase is reported to be released into the synovial fluid in inflammatory joint diseases, such as rheumatoid arthritis.12,13 The over-expression of the enzyme is also reported in some hepatic diseases and AIDS.14 β-Glucuronidase is also found to be involved in the etiology of colon cancer and higher

⁎

intestinal level of the enzyme associated with higher incidence of colon carcinoma.15,16 In addition, this enzyme is attributed to the toxicity study of anti-inflammatory drug in mice17 and pathological condition of neonatal hepatic disorders.18 The 1,3,4-Oxadiazoles are a class of heterocyclic compounds with broad-spectrum of biological activities. Compounds bearing 1,3,4-oxadiazole nucleus are known to exhibit anticancer activity.19 1,3,4-Oxadiazoles have been reported to possess insecticidal,20 herbicidal,21 antibacterial,22 antifungal,23 analgesic,24 anti-inflammatory,25 antimalarial,26 antiviral,27 anti-HBV,28 antianxiety,29 anticancer,30 antiHIV,31 antitubercular32 and anticonvulsant activities.33 Similarly, the 1,3,4-thiadiazole framework, a well-known heterocyclic scaffold has drawn special interest because of its inherent and diverse biological

Corresponding author. E-mail address: [email protected] (M. Taha).

https://doi.org/10.1016/j.bmc.2019.05.049 Received 9 January 2019; Received in revised form 16 May 2019; Accepted 31 May 2019 Available online 04 June 2019 0968-0896/ © 2019 Elsevier Ltd. All rights reserved.

Bioorganic & Medicinal Chemistry 27 (2019) 3145–3155

M. Taha, et al. O O O

O H N S R1 N O H

OH

O

O

N N H

N N

N N

among the series having chloro groups at 2,4,6 positions on the phenyl ring. The greater potential shown by this analog might be due to electron withdrawing chloro group. If we compare analog 26 with other chloro analogs like 17, 18 and 19 having one chloro group on phenyl ring, the analog 26 is superior. The greater potential shown by analog 26 might be due to greater number of chloro group on phenyl ring which makes molecule more polar as compare to mono-substituted chloro-analogs (Fig 2). The compound 6, a 3,4-dihydroxy analog was found to be the second most active among the series with IC50 value 1.40 ± 0.05 μM. The greater potential shown by this compound might be due to intramolecular hydrogen bonding which allows proton to have good interaction with enzyme. If we compare analog 6 with analog 7 having IC50 value 6.20 ± 0.10 μM, although both have two hydroxy groups at the phenyl ring, but position of the hydroxy groups is different. In analog 6 the two hydroxy groups are present at 3 and 4 positions while in analog 7 the two hydroxy groups are present at 2 and 5 positions (Fig. 3). The difference in the activity of these two analogs is due to the difference position of the hydoxyl on the phenyl ring. In case of compound 7 intramolecular hydrogen bonding is not possible as compare to compound 6. If we compare analog 20 having IC50 value12.30 ± 0.40 μM with analog 27 having IC50 value 28.10 ± 0.58 μM, although both analogs have one hydroxy and one methoxy group at the phenyl ring but position of the hydroxy and methoxy groups are different. In analog 20 the hydroxy group is present at 2 position and methoxy group is present at 5 position while in analog 27 the hydroxy group is present at 3 position and methoxy group is present at 4 position (Fig. 4). The difference in the activity of these two analogs may be due to the difference in positions of the hydroxy; in case of analog 20 methoxy and hydroxyl groups are very far which allows enzyme to have better hydrogen bonding, with comparison to analog 27 in which methoxy group next to hydroxyl have steric effect and reduced the activity. Similar pattern was also observed for other analogs having different substitution on phenyl ring. It was concluded from this study that position, nature and number of substituents play a critical role in this inhibition. To understand the binding interaction of the most active analogs molecular docking study was performed. This study reveals that the compounds having hydroxy group showed good activity due to having ability to form hydrogen bonding on the other hand molecules having electron withdrawing group like Cl, F and NO2 showed good activity due to polarization of molecules lastly the group which are not involved in hydrogen bonding or polarization of molecules showed moderated to weak activity like Me, pyridine ring.

R2

R5

H N

O

N

N N

R3

S N N

H N R4

R1, R2, R3 = Aryls and R4 and R5 = Me, Cl, Br, .... etc

Fig. 1. Previously reported Oxadiazole and thiadiazole as β-glucuronidase inhibitors.

response.34 Thiadiazoles are bio-isosteres of pyrimidines, oxadiazoles, oxazoles, benzene and its derivatives have been reported to display diverse range of biological and pharmacological properties such as antimicrobial,35 anticonvulsant,36 analgesic and anti-inflammatory,37 antiviral,38 anticancer,39 antitubercular activities as well as alzheimer’s disease.40,41 Our research group is continuously in struggle to explore the biological potential of heterocyclic molecules.42–48 We have previously reported various heterocyclic classes as a potent β-glucuronidase inhibitors such as bis-indolylmethanes, benzothiazole, and benzimidazole derivatives.49 In this study we have planned to synthesize hybrid scaffold of 1,3,4-oxadiazole and 1,3,4- thiadiazole analogs in order to explore its β-glucuronidase potential. We have reported oxadiazole49–51 and thiadiazole as52 as β-glucuronidase but in this time we are reporting hybrid of both which is more promising than previous one (Fig. 1). 2. Results and discussions 2.1. Chemistry The synthetic route of compounds 6–27 is shown in Scheme 1. Compound 1 was synthesized by treating methyl 4-hydroxy-2-methoxybenzoate with hydrazine hydrate. Compound 1 was then reacted with methyl 4-formylbenzoate in methanol with catalytic amount of acetic acid to afford hydrazone 2, which was then subjected through an oxidative cyclization using phenyl iododiacetate (PhI(OAc)2) in dichloromethane to form oxadiazole 3. The ester functional group of compounds 3 was further converted into hydrazide by treating with hydrazine hydrate to afford compound 4, which was then refluxed with Lawesson's reagent in toluene yield corresponding thio-analogue 5. Thiohydrazide 5 was then treated with various benzaldehydes in the presence of POCl3 to form the targeted oxadiazole and thiadiazole derivatives 6–27 (Table 1). The structures of the oxadiazole and thiadiazole derivatives 6–27 were elucidated using spectroscopic techniques such as NMR, MS and were further confirmed using CHN analysis.

2.3. Docking study Molecular docking studies were performed to explore the binding mode of the inhibitors. The interaction details of all the active compounds are given in Table 2. Compounds 26, 6, 24 and 18 were classified as the most active compounds based on their IC50 values. The docking results predicted that all the inhibitors were bound deeply in the active site of the target enzyme. From the docking conformation of the most potent compounds, compound 26 (IC50 = 0.96 ± 0.01) showed docking score of −11.6176 and numerous binding interactions with the active residues of the binding pocket as shown in the Fig. 5. This compound formed three polar, one arene-cation and one arenearene interaction with the Asn 484, Tyr 508, Ser 597 and Arg 600 active residues of the enzyme. Asn 484 formed hydrogen bonds with the –N atoms of thiadiazole moiety of the compound. Tyr 508 is making π-π interaction with the benzene moiety and Ser 597 formed hydrogen bond with the –OH group of the moiety of the same compound. Arg 600 was observed making an arene cation linkage with the oxadiazole moiety. The strong bonding network may be one of the reasons of the high potency of this compound. The availability of the electron rich species Cl and OH might be one of the reasons that make the compound potent and active.

2.2. Biological activity We have synthesized twenty two analogs of oxadiazole and thiadiazole, which have varied degree of β-glucuronidase inhibition ranging in between 0.96 ± 0.01 to 58.16 ± 1.40 μM when compared with the standard inhibitor D-saccharic acid 1,4 lactone having IC50 value 48.4 ± 1.25 μM. Out of these twenty two analogs, sixteen analogs 6, 7, 8, 9, 10, 12, 13, 14, 17, 18, 20, 23, 24, 25, 26 and 27 showed outstanding β-glucuronidase inhibitory potential with IC50 values ranging 1.40–28.10 μM respectively which is many folds better than the standard D-saccharic acid 1,4 lactone. Two analogs such as 16 and 19 showed moderate β-glucuronidase inhibitory potential with IC50 values58.16 ± 1.40 and 56.10 ± 1.45 μM respectively. Four analogs 11, 15, 21 and 22 were found inactive (Table 1). Analog 26 (IC50 = 0.96 ± 0.01 μM) showed potent inhibition 3146

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Scheme 1. Synthesis of Oxadiazole and Thiadiazole Derivatives.

Compound 6 is the second most active inhibitor (IC50 = 1.40 ± 0.05 µM) of the enzyme and it also displayed good docking score (−10.3982). This compound was observed that it formed two polar and two hydrophobic interactions with the active residues of the enzyme. Asn 486 and Thr 523 are involved in hydrogen bond interactions with the –OH groups of the pyrocatechol moiety of the compound. Tyr 508 and His 509 formed π-π linkage with the oxadiazole and thiadiazole moieties respectively of the compound as shown in the Fig. 6. The strong hydrogen bonding network formed by the –OH groups of the compound 6 and the availability of proper delocalized system of electrons in the compound may lead to the good activity of the compound 6 in the series (Table 1). Compound 24 is the third most active ligand (IC50 = 2.60 ± 0.05 µM) among the series of the compound. The binding mode of this compound showed that it formed good interactions with a docking score of −9.6464. Asn 484 formed hydrogen bond with the thiadiazole moiety and Thr 523 showed H bond acceptor interaction with OH group of the 3-methoxyphenol moiety of the compound. His 509 made an arene-arene interaction with the oxadiazole moiety as shown in the Fig. 7. The binding mode of compound 18 (IC50 = 5.14 ± 0.15), which is the fourth one potent compound in the series predicted that it formed arene cation and hydrophobic interaction with the Arg 600 and Asn 484 active residues of the binding pocket as shown in Fig. 8. The docking score of this compound is −9.2517. The potency of the compound 24 and 18 might be due to the existence of the electron withdrawing groups (F, Cl) at one end and electron donating groups (OH, OCH3) on the other end which enhances the polarizability of the molecule as a whole. Furthermore, the physicochemical properties of all the compounds were calculated by using MOE-2016 (www.chemcomp.com). The

predicted drug like properties of the compounds showed that all the compounds have acceptable drug like properties (Table 3). 3. Materials and methods Melting points were determined using Sinosource SGW X-4 melting point apparatus with microscope (Guangzhou, China). IR spectra obtained using PerkinElmer Spectrum 100 FTIR Spectrometer (Waltham, MA, USA) equipped with a diamond crystal Attenuated Total Reflectance (ATR) accessory by PIKE Technologies (Madison, WI, USA). NMR spectroscopy was obtained using Bruker Ultra Shield FT NMR 500 MHz and Avance III 600 Ascend spectrometer (Wissembourg, France). EI-MS spectroscopic analysis had been obtained using Finnigan-MAT-311-A instrument (Bremen, Germany). CHN analysis were carried out by Carlo Erba Strumentazion-Mod-1106, Italy. Thin layer chromatography (TLC) was performed using precoated silica gel plates (Merck, Kieselgel 60F-254, 0.20 mm). 3.1. Synthetic procedure for 4-hydroxy-2-methoxybenzohydrazide (1) Methyl 4-hydroxy-2-methoxybenzoate (9.10 g, 50 mmol) and 20 mL of hydrazine hydrate were mixed in methanol (50 mL). The mixture was refluxed for 6 h. Methanol was then evaporated, and the product was rinsed with plenty of water to remove excess hydrazine hydrate. The product formed was left to dry at room temperature and yielded 8.19 g (90%).50

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Table 1 Synthesis of Oxadiazole and Thiadiazole Derivatives and their β-Glucuronidase Inhibitory Potential. IC50 ± SEMa (μM)

No.

6

1.40 ± 0.05

17

34.16 ± 0.78

7

6.20 ± 0.10

18

5.14 ± 0.15

8

8.20 ± 0.20

19

56.10 ± 1.45

9

27.60 ± 0.30

20

12.30 ± 0.40

10

7.60 ± 0.25

21

N. A.

11

N.A.

22

N. A.

12

46.46 ± 1.10

23

26.14 ± 0.65

13

17.20 ± 0.55

24

2.60 ± 0.05

14

28.60 ± 0.58

25

15.30 ± 0.35

15

N. A.

26

0.96 ± 0.01

16

58.16 ± 1.40

27

28.10 ± 0.58

No.

D-saccharic

a

R

acid 1,4 lactone

R

IC50 ± SEMa (μM)

48.4 ± 1.25 μM

The Standard mean error.

crystallized from ethanol and gives solid product, (12.59 g, 91%).50

3.2. Synthetic procedure for methyl (E)-4-((2-(4-hydroxy-2methoxybenzoyl)hydrazono) methyl)benzoate (2)

3.3. Synthetic procedure for methyl 4-(5-(4-hydroxy-2-methoxyphenyl)1,3,4-oxadiazol-2-yl)benzoate(3)

A mixture of compound 1 (7.28 g, 40 mmol), methyl 4-formylbenzoate (6.56 g, 40 mmol) and catalytic amount of acetic acid in methanol (50 mL) was refluxed for 3 h. The solvent was evaporated and the residue (2) was washed with diethyl ether, filtered, dried, and then

A mixture of compound 2 (11.48 g, 35 mmol) and equivalent amount of PhI (OAc)2 was stirred in dichloromethane (100 mL) at room 3148

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J = 8.5 Hz), 7.90 (d, 1H, J = 9.0 Hz), 7.20 (d, 1H, J = 1.5 Hz), 6.93 (dd, 1H, J = 2 Hz), 6.80 (d, 1H, J = 2.0 Hz), 6.75 (s, 1H), 6.72 (dd, 1H, J = 2.0 Hz, 8.0 Hz), 3.81 (s, 3-H). 13C NMR (125 MHz, DMSO-d6): δ165.4, 165.4, 163.9, 160.3, 159.2, 158.6, 148.6, 145.8, 131.5, 130.2, 127.5, 127.5, 126.9, 126.9, 125.1, 124.9, 121.5, 121.0, 116.2, 111.1, 111.1, 100.9, 56.3; HR-MS for C23H16N4O5S + H, calculated 461.0920 and found 461.0946; Anal. calcd. for C23H16N4O5S: C, 59.99; H, 3.50; N, 12.17; Found: C, 59.98; H, 3.49; N, 12.16. 3.6.2. 2-(5-(4-(5-(4-hydroxy-2-methoxyphenyl)-1,3,4-oxadiazol-2-yl) phenyl)-1,3,4-thia-diazol-2-yl) benzene-1,4-diol (7) Yield: 89%; M.p.: 288 °C; 1H NMR (500 MHz, DMSO-d6): δ 9.99 (br s, 1-OH), 9.64 (br s, 1-OH), 9.23 (br s, 1-OH), 8.24 (d, 2H, J = 8.0 Hz), 8.12 (d, 2H, J = 8.0 Hz), 7.90 (dd, 1H, J = 8.5 Hz), 6.91 (d, 1H, J = 3.0 Hz), 6.80 (d, 1H, J = 2.0 Hz), 6.72–6.80 (m, 3H), 3.83 (s, 3-H). 13 C NMR (125 MHz, DMSO-d6): δ164.5, 163.9, 160.3, 159.7, 158.6, 158.3, 153.4, 149.7, 131.5, 130.2, 127.5, 127.5, 126.9, 126.9, 125.1, 120.9, 118.8, 118.3, 114.8, 111.1, 111.1, 100.9, 56.3; HR-MS for C23H16N4O5S + H, calculated 461.0920 and found 461.0919; Anal. calcd. for C23H16N4O5S: C, 59.99; H, 3.50; N, 12.17; Found: C, 59.97; H, 3.48; N, 12.15.

Fig. 2. The most active analog 26.

temperature overnight. The solvent was evaporated and the residue (3) was washed with diethyl ether, filtered, dried, and then crystallized from ethanol to gives white solid product, (9.75 g, 85%).50 3.4. Synthetic procedure for 4-(5-(4-hydroxy-2-methoxyphenyl)-1,3,4oxadiazol-2-yl) benzohydrazide (4)

3.6.3. 4-(5-(4-(5-(4-hydroxyphenyl)-1,3,4-thiadiazol-2-yl) phenyl)-1,3,4oxadiazol-2-yl)-3- methoxyphenol (8) Yield: 83%; M.p 278 °C; R1H NMR (500 MHz, DMSO-d6): δ 9.88 (br s, 1-OH), 9.69 (br s, 1-OH), 8.17 (d, 2H, J = 8.0 Hz), 8.09 (d, 2H, J = 8.5 Hz), 7.54 (d, 2H, J = 8.5 Hz), 6.78 (d, 1H, J = 2.0 Hz), 6.74 (d, 1H, J = 8.5 Hz), 6.80 (d, 2H, J = 8.0 Hz), 6.75 (dd, 1H, J = 2.5 Hz, 9.0 Hz), 3.84 (s, 3H), 13C NMR (125 MHz, DMSO-d6): δ166.6, 165.6, 163.9, 160.3, 159.7, 158.6, 158.2, 131.5, 130.2, 129.2, 1129.2, 127.5, 127.5, 126.9, 126.9, 125.1, 120.6, 116.1, 116.1, 111.1, 111.1, 100.9, 56.3; HR-MS for C23H16N4O4S + H, calculated 445.0971 and found 445.0962; Anal. calcd. for C23H16N4O4S: C, 62.15; H, 3.63; N, 12.61; Found: C, 62.14; H, 3.62; N, 12.60.

Compound 3 (8.15 g, 25 mmol) and 15 mL of hydrazine hydrate were mixed in methanol (50 mL). The mixture was refluxed for 6 h. Methanol was then evaporated, and the product formed was being rinsed with plenty of water to remove excess hydrazine hydrate. The product formed (4) was left to dry at room temperature and yielded 7.17 g (88%).50 3.5. Synthetic procedure for 4-(5-(4-hydroxy-2-methoxyphenyl)-1,3,4oxadiazol-2-yl) benzothiohydrazide (5) Compound 4 (5.21 g, 16 mmol) and equivalent amount of Lawesson's reagent were mixed in toluene (100 mL). The mixture was refluxed for 5 h. The product formed (5) was filtered and left to dry at room temperature and yielded 4.27 g (83%).

3.6.4. 4-(5-(4-(5-(3-hydroxyphenyl)-1,3,4-thiadiazol-2-yl) phenyl)-1,3,4oxadiazol-2-yl)-3- methoxyphenol (9) Yield: 87%; M.p.: 276 °C; 1H NMR (500 MHz, DMSO-d6): δ 9.88 (br s, 1-OH), 9.27 (br s, 1-OH), 8.23 (d, 2H, J = 8.5 Hz), 8.15 (d, 2H, J = 8.5 Hz), 7.96 (d, 1H, J = 8.5 Hz), 7.24 (t, 1H, J = 7.5 Hz), 7.20 (s, 1H), 7.11 (d, 1H, J = 7.0 Hz), 6.85 (d, 1H, J = 7.0 Hz), 6.80 (s, 1H), 6.70 (dd, 1H, J = 2.0 Hz, 8.0 Hz), 3.84 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ166.4, 165.6, 163.9, 160.3, 159.7, 158.6, 158.0, 131.5, 130.7, 130.6, 130.2, 127.5, 127.5, 126.9, 126.9, 125.1, 119.3, 118.6, 113.2, 111.1, 111.1, 100.9, 56.3; HR-MS for C23H16N4O4S + H, calculated 445.0971 and found 445.0964; Anal. calcd. for C23H16N4O4S: C, 62.15; H, 3.63; N, 12.61; Found: C, 62.13; H, 3.61; N, 12.59.

3.6. General synthetic procedure for oxadiazole and thiadiazole derivatives (6–27) Equimolar quantities (1 mmol) of compound 5 and substituted benzaldehydes (1 mmol) in methanol (25 mL) were refluxed for 3 h, in the presence of POCl3. The resulting solid was filtered and recrystallized from methanol in good yields. 3.6.1. 4-(5-(4-(5-(4-hydroxy-2-methoxyphenyl)-1,3,4-oxadiazol-2-yl) phenyl)-1,3,4-thia-diazol-2-yl) benzene-1,2-diol (6) Yield: 91%; M.p.: 296 °C; 1H NMR (500 MHz, DMSO-d6): δ 9.93 (br s, 1-OH), 9.58 (br s, 2-OH), 8.22 (d, 2H, J = 8.5 Hz), 8.10 (d, 2H,

3.6.5. 4-(5-(4-(5-(2-hydroxyphenyl)-1,3,4-thiadiazol-2-yl) phenyl)-1,3,4oxadiazol-2-yl)-3- methoxyphenol (10) Yield: 84%; M.p.: 272 °C; 1H NMR (500 MHz, DMSO-d6): δ 9.91 (br

Fig. 3. Structure of analog 6 and 7. 3149

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Fig. 4. Structure of analog 20 and 27.

s, 1-OH), 9.64 (br s, 1-OH), 8.17 (d, 2H, J = 8.0 Hz), 8.12 (d, 2H, J = 8.0 Hz), 7.55 (d, 1H, J = 7.5 Hz), 7.30 (m, 3H) 6.93 (t, 2H, J = 8.5 Hz), 6.75 (dd, 1H, J = 2.0 Hz, 8.0 Hz), 3.84 (s, 3-H). 13C NMR (125 MHz, DMSO-d6): δ164.5, 163.9, 161.0, 160.3, 159.7, 158.6, 154.7, 131.5, 130.2, 128.8, 127.7, 127.5, 127.5, 126.9, 126.9, 125.1, 119.7, 116.8, 116.5, 111.1, 111.1, 100.9, 56.3; HR-MS for C23H16N4O4S + H, calculated 445.0971 and found 445.0954; Anal. calcd. for C23H16N4O4S: C, 62.15; H, 3.63; N, 12.61; Found: C, 62.12; H, 3.60; N, 12.58.

s, 1-OH), 8.20 (d, 2H, J = 8.0 Hz), 8.10 (d, 2H, J = 8.0 Hz), 7.91 (d, 1H, J = 8.0 Hz), 7.83 (d, 1H, J = 7.5 Hz), 7.32–7.24 (m, 3H), 6.80 (s, 1H), 6.72 (dd, 1H, J = 2.0 Hz, 8.0 Hz), 3.80 (s, 3H), 2.40 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ164.5, 163.9, 160.3, 159.7, 159.0, 158.6, 136.4, 132.2, 131.9, 131.5, 131.4, 130.2, 129.4, 127.5, 127.5, 127.2, 126.9, 126.9, 125.1, 111.1, 111.1, 100.9, 56.3, 21.2; HR-MS for C24H18N4O3S +H, calculated 443.1178 and found 443.1165; Anal. calcd. for C24H18N4O3S: C, 65.14; H, 4.10; N, 12.66; Found: C, 65.13; H, 4.09; N, 12.65.

3.6.6. 3-methoxy-4-(5-(4-(5-(3-nitrophenyl)-1,3,4-thiadiazol-2-yl) phenyl)-1,3,4-oxadiazol-2-yl) phenol (11) Yield: 80%; M.p.: 302 °C; 1H NMR (500 MHz, DMSO-d6): δ 9.96 (br s, 1-OH), 8.25 (d, 1H, J = 8.0 Hz), 8.18–8.14 (m, 5H), 7.90 (d, 1H, J = 8.5 Hz), 7.74 (t, 1H, J = 8.0 Hz), 6.80 (d, 1H, J = 2.0 Hz), 6.77 (d, 1H, J = 2.0 Hz), 6.70 (dd, 1H, J = 2.0 Hz, 8.0 Hz), 3.84 (s, 3H), 13C NMR (125 MHz, DMSO-d6): δ165.6, 164.4, 163.9, 160.3, 159.7, 158.6, 148.1, 133.6, 131.5, 130.2, 129.5, 127.9, 127.5, 127.5, 126.9, 126.9, 125.1, 124.7, 122.9, 111.1, 111.1, 100.9, 56.9; HR-MS for C23H15N5O5S + H, calculated 474.0872 and found 474.0860; Anal. calcd. for C23H15N5O5S: C, 58.35; H, 3.19; N, 14.79; Found: C, 58.34; H, 3.18; N, 14.78.

3.6.10. 3-methoxy-4-(5-(4-(5-m-tolyl-1,3,4-thiadiazol-2-yl) phenyl)-1,3, 4-oxadiazol-2-yl) phenol (15) Yield: 84%; M.p.: 260 °C; 1H NMR (500 MHz, DMSO-d6): δ 9.92 (br s, 1-OH), 8.23 (d, 2H, J = 8.0 Hz), 8.10 (d, 2H, J = 8.5 Hz), 7.90 (d, 1H, J = 8.0 Hz), 7.56 (s, 1H), 7.54 (d, 1H, J = 7.0 Hz), 7.37 (t, 1H, J = 7.0 Hz), 7.24 (d, 1H, J = 7.5 Hz), 6.80 (d, 1H, J = 2.0 Hz), 6.73 (dd, 1H, J = 2.0 Hz, J = 8.0 Hz), 3.83 (s, 3H), 2.38 (s, 3H); 13C NMR (125 MHz, DMSO-d6): δ165.6, 165.6, 164.4, 163.9, 160.3, 159.7, 158.6, 142.8, 132.8, 132.4, 131.5, 131.5, 131.2, 130.2, 127.5, 127.5, 126.9, 126.9, 124.3, 111.1, 111.1, 100.9, 56.3, 22.0; HR-MS for C24H18N4O3S +H, calculated 443.1178 and found 443.1163; Anal. calcd. for C24H18N4O3S: C, 65.14; H, 4.10; N, 12.66; Found: C, 65.12; H, 4.08; N, 12.64.

3.6.7. 3-methoxy-4-(5-(4-(5-(4-nitrophenyl)-1,3,4-thiadiazol-2-yl) phenyl)-1,3,4-oxadiazol-2-yl) phenol (12) Yield: 71%; M.p.: 309 °C; 1H NMR (500 MHz, DMSO-d6): δ 9.93 (br s, 1-OH), 8.29 (dd, 2H, J = 7.8, 0.9 Hz), 8.08 (dd, 2H, J = 7.8, 0.9 Hz), 8.05 (m, 4H), 7.67 (dd, 1H, J = 7.2, 0.9 Hz), 6.59 (dd, 1H, J = 7.2, 1.2 Hz), 6.63 (s, 1H), 3.84 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ164.5, 163.9, 160.3, 159.7, 158.6, 156.5, 147.9, 131.5, 130.7, 130.2, 130.2, 129.5, 127.6, 127.5, 127.5, 127.5, 126.9, 126.9, 124.3, 111.1, 111.1, 100.9, 56.3; HR-MS for C23H15N5O5S + H, calculated 474.0872 and found 474.0858; Anal. calcd. for C23H15N5O5S: C, 58.35; H, 3.19; N, 14.79; Found: C, 58.33; H, 3.17; N, 14.77.

3.6.11. 3-methoxy-4-(5-(4-(5-p-tolyl-1,3,4-thiadiazol-2-yl) phenyl)-1,3,4oxadiazol-2-yl) phenol (16) Yield: 82%; M.p.: 270 °C; 1H NMR (500 MHz, DMSO-d6): δ 9.87 (br s, 1-OH), 8.23 (d, 2H, J = 8.5 Hz), 8.16 (d, 2H, J = 8.0 Hz), 7.92 (d, 1H, J = 8.0 Hz), 7.62 (d, 2H, J = 7.5 Hz), 7.32 (d, 2H, J = 8.0 Hz), 6.81 (d, 1H, J = 2.0 Hz), 6.74 (dd, 1H, J = 2.5 Hz, J = 8.5 Hz), 3.85 (s, 3H), 2.38 (s, 3H); 13C NMR (125 MHz, DMSO-d6): δ165.5, 163.8, 161.0, 160.4, 159.8, 158.7, 138.6, 131.6, 131.1, 130.8, 130.3, 130.3, 128.8, 128.8, 127.5, 127.5, 126.8, 126.8, 125.1, 111.1, 111.1, 100.9, 56.3, 21.4; HR-MS for C24H18N4O3S + H, calculated 443.1178 and found 443.1167; Anal. calcd. for C24H18N4O3S: C, 65.14; H, 4.10; N, 12.66; Found: C, 65.11; H, 4.07; N, 12.63.

3.6.8. 3-methoxy-4-(5-(4-(5-(2-nitrophenyl)-1,3,4-thiadiazol-2-yl) phenyl)-1,3,4-oxadiazol-2-yl) phenol (13) Yield: 69%; M.p.: 282 °C; 1H NMR (500 MHz, DMSO-d6): δ 9.91 (br s, 1-OH), 8.14–8.09 (m, 6H), 7.90 (d, 1H, J = 8.5 Hz), 7.80 (s, 1H), 7.71 (s, 1H), 6.77 (d, 1H, J = 2.0 Hz), 6.73 (dd, 1H, J = 2.0 Hz, 8.0 Hz), 3.84 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ164.5, 163.9, 160.3, 159.7, 158.6, 156.5, 147.9, 131.5, 130.7, 130.2, 130.2, 129.5, 127.6, 127.6, 127.5, 127.5, 126.9, 126.9, 124.3, 111.1, 111.1, 100.9, 56.3; HR-MS for C23H15N5O5S+H, calculated 474.0872 and found 474.0864; Anal. calcd. for C23H15N5O5S: C, 58.35; H, 3.19; N, 14.79; Found: C, 58.32; H, 3.16; N, 14.76.

3.6.12. 4-(5-(4-(5-(4-chlorophenyl)-1,3,4-thiadiazol-2-yl) phenyl)-1,3,4oxadiazol-2-yl)-3- methoxyphenol (17) Yield: 88%; M.p.: 274 °C; 1H NMR (500 MHz, DMSO-d6): δ 9.90 (br s, 1-OH), 8.25 (d, 2H, J = 8.0 Hz), 8.17 (d, 2H, J = 8.0 Hz), 7.92 (d, 1H, J = 8.5 Hz), 7.75 (d, 2H, J = 8.5 Hz), 7.52 (d, 2H, J = 8.5 Hz), 6.80 (d, 1H, J = 2.0 Hz), 6.73 (dd, 1H, J = 2.5 Hz, J = 8.0 Hz), 3.80 (s, 3H); 13C NMR (125 MHz, DMSO-d6): δ166.9, 165.6, 163.9, 160.3, 159.7, 158.6, 136.4, 131.5, 130.2, 129.6, 129.4, 129.4, 128.2, 128.2, 127.9, 127.9, 127.2, 127.2, 125.3, 111.1, 111.1, 100.9, 56.3; HR-MS for C23H15ClN4O3S+H, calculated 463.0632 and found 463.0622; Anal. calcd. for C23H15ClN4O3S: C, 59.68; H, 3.27; N, 12.10; Found: C, 59.67; H, 3.26; N, 12.09.

3.6.9. 3-methoxy-4-(5-(4-(5-o-tolyl-1,3,4-thiadiazol-2-yl) phenyl)-1,3,4oxadiazol-2-yl) phenol (14) Yield: 81%; M.p.: 265 °C; 1H NMR (500 MHz, DMSO-d6): δ 9.96 (br 3150

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Table 2 Docking scores and report of the predicted interactions of docked conformations. Compounds

Docking scores (S)

Interaction Report Ligand

Receptor

Interaction

Distance

E (kcal/mol)

6

−10.3982

O 30 O 32 O 26 6-ring

O ASN 486 O THR 523 CE1 HIS 509 CB GLU 451

H-donor H-donor pi-pi pi-pi

2.78 3.19 4.81 3.91

−2.8 −0.5 −0.4 −0.2

7

−9.1275

O 18 O 31 6-ring 6-ring 6-ring 5-ring 6-ring

OG1 THR 523 O GLU 451 CB ALA 453 CE1 HIS 455 CB ASN 486 CB ASN 486 CG GLN 520

H-donor H-donor pi-H pi-H pi-H pi-H pi-H

3.10 3.15 4.45 4.20 4.29 4.49 4.45

−1.3 −0.7 −0.2 −0.3 −0.7 −0.1 −0.3

8

−8.9745

C 29 N 21 C 17 6-ring 6-ring

O LEU 412 CE1 HIS 455 5-ring HIS 509 CG GLN 520 CB GLN 524

H-donor H-acceptor H-pi pi-H pi-H

3.58 3.21 4.23 4.59 4.87

−0.1 −0.6 −0.2 −0.1 −0.1

9

−8.0189

C 38 C 32 C 13 6-ring

O TYR 205 O GLN 520 5-ring HIS 509 CB GLU 451

H-donor H-donor H-pi pi-H

3.27 3.50 4.72 3.88

−0.4 −0.1 −0.3 −0.1

10

−9.0028

N 22 N 21 C 32 6-ring 6-ring

NE2 HIS 455 NE2 HIS 455 6-ring TYR 508 CA PHE 206 CB ALA 413

H-acceptor H-acceptor H-pi pi-H pi-H

3.47 3.45 4.65 4.42 3.61

−0.5 −1.9 −0.3 −1.0 −0.2

11

NA

12

−7.4201

6-ring 6-ring

CA PHE 206 CB ALA 413

pi-H pi-H

3.97 3.52

−0.9 −0.3

13

−8.1200

C 31 C 31 C 25 6-ring 6-ring 5-ring 6-ring 6-ring 5-ring

O ASN 484 OD1 ASN 486 6-ring TYR 508 CB GLU 451 CB TYR 508 CB TYR 508 CB HIS 509 CB GLN 520 5-ring HIS 509

H-donor H-donor H-pi pi-H pi-H pi-H pi-H pi-H pi-pi

3.74 3.53 4.53 3.76 4.74 4.50 4.67 4.64 3.48

−0.1 −0.1 −0.7 −0.1 −0.1 −0.2 −0.3 −0.1 −0.0

14

−7.9956

C 10 C 17 C 32 6-ring 6-ring 6-ring

CE1 HIS 455 5-ring HIS 455 6-ring TYR 508 CB ALA 413 CB ASN 484 ND2 ASN 484

H-acceptor H-pi H-pi pi-H pi-H pi-H

2.89 4.92 3.85 4.35 4.23 4.64

−1.2 −0.1 −0.1 −0.1 −0.2 −0.1

15

NA

16

−7.0140

N 20 6-ring 5-ring

CE1 HIS 455 CB GLU 451 CB ASN 486

H-acceptor pi-H pi-H

2.74 4.43 3.72

−1.1 −0.4 −2.1

17

−7.6534

C 14 O 18 C 32 CL 3 6-ring 6-ring 6-ring

O ASN 484 OG1 THR 523 OD1 ASN 486 CA ALA 413 ND2 ASN 484 CA GLN 520 CG GLN 520

H-donor H-donor H-donor H-acceptor pi-H pi-H pi-H

3.57 3.32 3.26 3.55 3.54 4.62 3.47

−0.1 −0.4 −0.1 −0.1 −0.1 −0.1 −0.1

18

−9.7345

C 14 S 23 C 32 C 21

O ASN 484 O ASN 484 O GLU 451 5-ring HIS 509

H-donor H-donor H-donor H-pi

3.28 3.82 3.58 4.33

−0.2 −0.1 −0.1 −0.1

19

−7.4312

C 28 C 32 C 17 C 25 6-ring 5-ring

O TYR 205 OD1 ASN 486 5-ring HIS 509 6-ring TYR 508 CB GLU 451 5-ring HIS 509

H-donor H-donor H-pi H-pi pi-H pi-pi

3.57 2.81 4.49 4.59 3.78 3.75

−0.1 −0.4 −0.2 −0.4 −0.7 −0.0

(continued on next page)

3151

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Table 2 (continued) Compounds

Docking scores (S)

Interaction Report Ligand

Receptor

Interaction

Distance

E (kcal/mol)

6-ring 5-ring 6-ring 5-ring 6-ring

CB TYR 508 CB TYR 508 CB HIS 509 5-ring HIS 509 6-ring TYR 508

pi-H pi-H pi-H pi-H pi-pi

4.66 4.07 4.54 3.74 3.87

−0.4 −0.3 −0.4 −0.0 −0.0

20

−8.8790

21 22

NA NA

23

−8.1101

C 31 C 31 C 25 6-ring 6-ring 6-ring 5-ring

O ASN 484 OD1 ASN 486 6-ring TYR 508 CB GLU 451 CB HIS 509 CB GLN 520 5-ring HIS 509

H-donor H-donor H-pi pi-H pi-H pi-H pi-pi

3.71 3.58 4.62 3.83 4.93 4.68 3.49

−0.2 −0.1 −0.6 −0.1 −0.1 −0.1 −0.0

24

−9.6464

N 20 O 30 6-ring

O ASN 484 OG1 THR 523 CB HIS 509

H-donor H-donor pi-pi

3.80 3.63 3.63

−0.2 −0.1 −0.0

25

−8.5492

O 18 N 20 6-ring 5-ring

OG1 THR 523 CD2 TYR 508 CB GLU 451 5-ring HIS 509

H-donor H-acceptor pi-H pi-pi

3.12 3.35 4.60 3.69

−0.1 −0.1 −0.3 −0.0

26

−11.6176

C 17 N 20 N 22 N 10 6-ring

OG1 SER 597 CB ASN 484 CB ASN 484 ND1 ARG 600 CE1 TYR 508

H-donor H-donor H-acceptor pi-H pi-pi

3.84 3.91 3.34 4.09 3.64

−0.1 −0.1 −0.8 −0.1 −0.0

27

−7.8903

C2 O 18 C 26 O 30 C 32 6-ring 6-ring 5-ring

O ALA 413 O ALA 413 OD1 ASN 486 OD1 ASN 486 O GLU 451 CB ALA 453 CE1 HIS 455 5-ring HIS 509

H-donor H-donor H-donor H-donor H-donor pi-H pi-H pi-pi

3.39 2.87 3.29 3.31 3.29 4.35 4.05 3.91

−0.2 −3.1 −0.1 −0.5 −0.1 −0.1 −0.6 −0.0

Fig. 5. Docking conformation of compound 26 in the active site of β-glucuronidase.

Fig. 6. Docking conformation of compound 6in the active site of β-glucuronidase.

3.6.13. 4-(5-(4-(5-(2-chlorophenyl)-1,3,4-thiadiazol-2-yl) phenyl)-1,3,4oxadiazol-2-yl)-3- methoxyphenol (18) Yield: 87%; M.p.: 256 °C; 1H NMR (500 MHz, DMSO-d6): δ 9.95 (br s, 1-OH), 8.20 (d, 2H, J = 8.0 Hz), 8.16 (d, 2H, J = 8.0 Hz), 8.03 (d, 1H, J = 7.0 Hz), 7.92 (d, 1H, J = 9.0 Hz), 7.51 (d, 1H, J = 7.0 Hz), 7.42 (d, 2H, J = 7.0 Hz), 6.80 (d, 1H, J = 2.0 Hz), 6.72 (dd, 1H, J = 2.0 Hz, J = 8.0 Hz), 3.82 (s, 3H); 13C NMR (125 MHz, DMSO-d6): δ164.8, 163.7, 160.4, 160.1, 159.8, 158.5, 132.7, 132.1, 131.9, 131.2, 130.9, 130.2, 130.2, 128.0, 127.4, 127.4, 126.8, 126.8, 125.0, 111.2, 111.2,

100.7, 56.1; HR-MS for C23H15ClN4O3S + H, calculated 463.0632 and found 463.0619; Anal. calcd. for C23H15ClN4O3S: C, 59.68; H, 3.27; Cl, 7.66; N, 12.10; Found: C, 59.66; H, 3.25; Cl, 7.64; N, 12.08. 3.6.14. 4-(5-(4-(5-(3-chlorophenyl)-1,3,4-thiadiazol-2-yl) phenyl)-1,3,4oxadiazol-2-yl)-3- methoxyphenol (19) Yield: 89%; M.p.: 267 °C; 1H NMR (500 MHz, DMSO-d6): δ9.92 (br s, 1-OH,) 8.20 (d, 2H, J = 8.0 Hz), 8.13 (d, 2H, J = 8.0 Hz),7.99 (d, 1H, 2.0 Hz), 7.95 (dd, 1H, J = 8.0, 2.0, Hz,), 7.65 (dd, 1H, J = 8.0, 2.0 Hz,), 3152

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127.2, 126.3, 126.3, 124.1, 123.6, 111.4, 111.4, 101.1, 56.8; HR-MS for C23H15ClN4O3S + H, calculated 463.0632 and found 463.0618; Anal. calcd. for C23H15ClN4O3S: C, 59.68; H, 3.27; Cl, 7.66; N, 12.10; Found: C, 59.65; H, 3.24; Cl, 7.63; N, 12.07. 3.6.15. 2-(5-(4-(5-(4-hydroxy-2-methoxyphenyl)-1,3,4-oxadiazol-2-yl) phenyl)-1,3,4- thiadiazol-2-yl)-4-methoxyphenol (20) Yield: 79%; M.p.: 284 °C; 1H NMR (500 MHz, DMSO-d6): δ 9.91 (br s, 1-OH), 9.26 (br s, 1-OH), 8.20 (d, 2H, J = 8.0 Hz), 8.14 (d, 2H, J = 8.0 Hz), 7.90 (d, 1H, J = 8.5 Hz), 7.10 (d, 1H, J = 2.0 Hz), 6.92 (d, 1H, J = 8.0 Hz), 6.83 (d, 1H, J = 9.0 Hz), 6.78 (d, 1H, J = 2.0 Hz), 6.70 (dd, 1H, J = 2.0 Hz, 8.0 Hz), 3.79 (s, 3H), 3.85 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ165.5, 164.9, 162.3, 160.7, 159.6, 159.3, 155.9, 150.1, 131.4, 130.6, 127.4, 127.4, 126.8, 126.8, 125.3, 119.2, 118.4, 117.9, 113.7, 111.1, 111.1, 100.3, 55.3, 53.8; HR-MS for C24H18N4O5S +H, calculated 475.1076 and found 475.1059; Anal. calcd. for C24H18N4O5S: C, 60.75; H, 3.82; N, 11.81; Found: C, 60.74; H, 3.81; N, 11.80.

Fig. 7. Docking conformation of compound 24 in the active site of β-glucuronidase.

3.6.16. 3-methoxy-4-(5-(4-(5-(pyridin-3-yl)-1,3,4-thiadiazol-2-yl) phenyl)-1,3,4-oxadiazol-2-yl) phenol (21) Yield: 84%; M.p.: 250 °C; 1H NMR (500 MHz, DMSO-d6): δ9.92 (br s, 1-OH), 9.26 (s, 1H), 8.73 (dd, 1H, J = 7.3, 0.9 Hz), 8.45 (dd,1H, J = 7.8, 0.8 Hz), 8.02 (m, 4H), 7.61 (d, 1H, 7.2 Hz), 7.59 (dd, 1H, J = 7.8, 7.3 Hz), 6.64 (d, 1H, J = 0.9 Hz), 6.57 (dd, 1H, J = 7.2, 1.2 Hz), 3.82 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ165.6, 163.9, 160.3, 159.7, 158.6, 152.5, 149.1, 148.2, 138.0, 134.7, 131.5, 130.2, 127.2, 127.2, 126.7, 126.7, 125.0, 120.4, 110.9, 110.9, 100.7, 56.2; HR-MS for C22H15N5O3S + H, calculated 430.0974 and found 430.0961; Anal. calcd. for C22H15N5O3S: C, 61.53; H, 3.52; N, 16.31; Found: C, 61.52; H, 3.51; N, 16.30. 3.6.17. 3-methoxy-4-(5-(4-(5-(pyridin-4-yl)-1,3,4-thiadiazol-2-yl) phenyl)-1,3,4-oxadiazol-2-yl) phenol (22) Yield: 87%; M.p.: 256 °C; 1H NMR (500 MHz, DMSO-d6): δ 9.84 (br s, 1-OH), 8.29 (d, 2H, J = 8.0 Hz), 8.18 (d, 2H, J = 7.5 Hz), 7.97 (d, 2H, J = 8.5 Hz), 7.90 (d, 1H, J = 8.0 Hz), 7.70 (d, 2H, J = 8.0 Hz), 6.81 (d, 1H, J = 2.0 Hz), 6.77 (dd, 1H, J = 2.0 Hz, 8.0 Hz), 3.85 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ166.3, 163.7, 162.6, 161.3, 160.7, 159.6, 152.4, 138.3, 132.5, 131.2, 128.5, 128.5, 127.9, 127.9, 127.9, 125.2, 125.2, 122.9, 111.2, 111.2, 100.8, 56.7; HR-MS for C22H15N5O3S+H, calculated 430.0974 and found 430.0959; Anal. calcd. for C22H15N5O3S: C, 61.53; H, 3.52; N, 16.31; Found: C, 61.51; H, 3.50; N, 16.29.

Fig. 8. Docking conformation of compound 18 in the active site of β-glucuronidase. Table 3 Predicted chemical properties of compounds. S. No

Molecular Weight (g/mol)

donor

Acceptor

LogP

LogS

6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27

460.47 460.47 444.47 444.47 444.47 473.47 473.47 473.47 442.50 442.50 442.50 462.92 462.92 462.92 474.50 429.46 429.46 429.46 446.46 446.46 531.81 474.50

3 3 2 2 2 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 1 2

8 8 7 7 7 6 6 6 6 6 6 6 6 6 8 7 7 7 6 6 6 8

3.99 4.05 4.48 4.51 4.54 5.04 5.01 5.17 5.48 5.48 5.48 5.65 5.73 5.69 4.45 4.28 4.28 4.34 5.16 5.05 7.12 4.41

−6.63 −6.65 −6.90 −6.92 −6.95 −7.69 −7.67 −7.78 −7.56 −7.56 −7.56 −7.87 −7.93 −7.89 −7.02 −6.88 −6.88 −6.94 −7.40 −7.32 −9.29 −7.00

3.6.18. 3-methoxy-4-(5-(4-(5-(pyridin-2-yl)-1,3,4-thiadiazol-2-yl) phenyl)-1,3,4-oxadiazol-2-yl) phenol (23) Yield: 78%; M.p.: 243 °C; 1H NMR (500 MHz, DMSO-d6): δ9.90 (br s, 1-OH), 8.26 (d, 2H, J = 8.0 Hz), 8.15 (d, 2H, J = 8.0 Hz), 8.01 (d, 1H, J = 8.0 Hz), 7.95 (d, 1H, J = 7.5 Hz), 7.91 (t, 1H, J = 7.5 Hz), 7.45 (t, 1H, J = 6.0 Hz), 6.80–6.74 (m, 2H), 6.76 (dd, 1H, J = 2.0 Hz, 8.0 Hz), 3.80 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ168.7, 165.5, 164.9, 161.3, 159.8, 158.7, 150.4, 146.4, 137.0, 132.5, 130.2, 128.5, 127.9, 127.9, 126.1, 126.1, 124.4, 121.6, 111.4, 111.4, 100.3, 56.8; HR-MS for C22H15N5O3S + H, calculated 430.0974 and found 430.0928; Anal. calcd. for C22H15N5O3S: C, 61.53; H, 3.52; N, 16.31; Found: C, 61.50; H, 3.49; N, 16.28. 3.6.19. 4-(5-(4-(5-(2-fluorophenyl)-1,3,4-thiadiazol-2-yl) phenyl)-1,3,4oxadiazol- 2-yl)-3-methoxyphenol (24) Yield: 79%; M.p.: 286 °C; 1H NMR (500 MHz, DMSO-d6): δ 9.95 (br s, 1-OH), 8.24 (d, 2H, J = 8.0 Hz), 8.14 (d, 2H, J = 8.0 Hz), 7.92 (d, 1H, J = 8.0 Hz), 7.65–7.60 (m, 3H), 7.31 (s, 1H), 6.80 (d, 1H, J = 2.0 Hz), 6.76 (dd, 1H, J = 2.0 Hz, 8.0 Hz), 3.84 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ164.5, 163.4 (J = 263.0 MHz), 161.3, 160.3, 159.7, 158.6, 132.1, 131.1, 130.4, 129.5, 128.2, 127.4, 127.4, 126.7, 126.7, 125.1,

7.49–7.40 (m, 2H), 6.60 (s, 1H), 6.55 (dd, 1H, J = 7.0, 2.0 Hz,), 3.84 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ165.4, 165.4, 164.3, 162.9, 160.1, 159.7, 158.2, 135.3, 132.4, 131.6, 131.1, 130.4, 129.1, 127.2, 3153

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123.6, 119.3, 115.6, 111.0, 111.0, 101.2, 55.3; HR-MS for C23H15FN4O3S+H, calculated 447.0927 and found 447.0911; Anal. calcd. for C23H15FN4O3S: C, 61.88; H, 3.39; F, 4.26; N, 12.55; found: C, 61.87; H, 3.38; N, 12.54.

For each ligand ten conformations were generated. The top-ranked conformation of each compound was used for further analysis.

3.6.20. 4-(5-(4-(5-(4-fluorophenyl)-1,3,4-thiadiazol-2-yl) phenyl)-1,3,4oxadiazol-2-yl) −3-methoxyphenol (25) Yield: 86%; M.p.: 298 °C; 1H NMR (500 MHz, DMSO-d6): δ 9.95 (br s, 1-OH), 8.28 (d, 2H, J = 8.0 Hz), 8.16 (d, 2H, J = 8.0 Hz), 7.78–7.72 (m, 2H), 7.64 (d, 1H, J = 7.2 Hz), 7.37–7.32 (m, 2H), 6.63 (d, 1H, J = 2.0 Hz), 6.57 (dd, 1H, J = 7.0, 2.0 Hz,), 3.83 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ165.2, 165.2, 162.9 (J = 263.0 MHz), 161.3, 158.7, 157.6, 132.5, 131.2, 130.1, 129.4, 129.4, 127.1, 127.1, 126.9, 126.9, 125.2, 116.8, 116.8, 116.2, 111.8, 111.8, 100.2, 56.9; HR-MS for C23H15FN4O3S+H, calculated 447.0927 and found 447.0916; Anal. calcd. for C23H15FN4O3S: C, 61.88; H, 3.39; F, 4.26; N, 12.55; found: C, 61.86; H, 3.37; F, 4.24; N, 12.53.

β-glucuronidase activity was determined in accordance to method used55 by measuring absorbance at 405 nm of p-nitrophenol formed substrate by spectrophotometric method. 250 µL was the volume of total reaction. Reaction mixture containing 5 µL of test compound solution, 185 µL of 0.1 M acetate buffer and 10 µL of enzyme solution were incubated for 30 min at 37 °C. At 405 nm the plates were recorded on multiplate reader (SpectaMax plus 384) after the addition of 50 µL of 0.4 mM p-nitrophenyl-β-D-glucuronide. Experiments were performed for triplicate.56 To avoid precipitation, compound concentrations were decreased, and the volume of reaction was increased (200 µL). Precipitation probability was less thus addition of detergents was not needed.

3.6.21. 3-methoxy-4-(5-(4-(5-(2,4,6-trichlorophenyl)-1,3,4-thiadiazol-2yl) phenyl) −1,3,4-oxadiazol-2-yl) phenol (26) Yield: 88%; M.p.: 311 °C; 1H NMR (500 MHz, DMSO-d6): δ 9.90 (br s, 1-OH), 8.20 (d, 2H, J = 8.0 Hz), 8.15 (d, 2H, J = 8.0 Hz), 7.63 (d,1H, J = 7.0 Hz), 7.59 (s, 2H), 6.60 (s, 1H), 6.59 (dd,1H, J = 7.0, 2.0 Hz), 3.84 (s, 3H). 13C NMR (125 MHz, DMSO-d6): δ164.5, 163.7, 161.3, 159.3, 158.9, 157.4, 135.0, 133.7, 133.7, 132.5, 131.2, 130.4, 128.3, 128.3, 127.4, 127.4, 126.3, 126.3, 125.2, 111.1, 111.1, 100.9, 56.3; HR-MS for C23H13Cl3N4O3S + H, calculated 530.9852 and found 530.0984; Anal. calcd. for C23H13Cl3N4O3S: C, 51.95; H, 2.46; N, 10.54; Found: C, 51.94; H, 2.45; N, 10.53.

4. Conclusion

3.6.22. 5-(5-(4-(5-(4-hydroxy-2-methoxyphenyl)-1,3,4-oxadiazol-2-yl) phenyl) −1,3,4-thiadiazol-2-yl)-2-methoxyphenol (27) Yield: 90%; M.p.: 295 °C; 1H NMR (500 MHz, DMSO-d6): δ 9.94 (br s, 1-OH), 9.49 (br s, 1-OH), 8.18 (d, 2H, J = 8.0 Hz), 8.12 (d, 2H, J = 8.0 Hz), 7.67 (d, 1H, J = 7.0 Hz), 7.36 (dd, 1H, J = 7.5, 2.0 Hz), 7.14 (s, 1H), 6.87 (d, 1H, J = 7.8 Hz), 6.64 (s, 1H), 6.59 (dd, 1H, J = 7.0, 2.0 Hz), 3.88 (s, 3H), 3.85 (s, 3H). 13C NMR (125 MHz, DMSOd6): δ166.3, 163.6, 160.2, 157.7, 154.6, 148.1, 147.5, 132.5, 131.2, 127.3, 127.3, 126.7, 126.7, 124.1, 120.3, 118.9, 117.1, 112.1, 112.1, 110.0, 100.8, 54.7, 43.2; HR-MS for C24H18N4O5S + H, calculated 475.1076 and found 475.1062; Anal. calcd. for C24H18N4O5S: C, 60.75; H, 3.82; N, 11.81; Found: C, 60.74; H, 3.81; N, 11.80.

Acknowledgements

3.7. Molecular docking

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3.8. β-Glucuronidase assay

In conclusion, we have synthesized a series of 1,3,4-Oxadiazoles with 1,3,4-thiadiazole moiety (6–27) and evaluated against β-glucuronidase inhibitory potential. Several analogs such as 6, 7, 8, 9, 10, 12, 13, 14, 17, 18, 20, 23, 24, 25, 26 and 27 showed outstanding inhibitory potential which is many folds better than the standard D-saccharic acid 1,4 lactone. This series can serve as lead for the development of novel class of β-glucuronidase inhibitors. Further optimization based on SAR is required to genuinely lead to a range of compounds to explore their β-glucuronidase inhibiting potentials.

We would like to thank IRMC for the lab facility for this work and we acknowledge for the funding from DSR of Imama Abdulrahman bin Faisal Univervisty (project no. 2018-066 and 2018-090). Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.bmc.2019.05.049. References

The molecular docking program is widely used to predict the binding interaction of the compounds in the binding pocket of the enzyme. The three-dimensional (3D) crystal structure of the β-glucuronidase was retrieved from the protein databank (PDB ID: 1BHG).52 The B-chain of protein and hetero-atoms including cofactors were removed from the original protein data bank file. Then energy minimization was carried out after 3D protonation using default parameters [the Amber 99 forcefield and gradient: 0.05] of the MOE (Molecular Operating Environment) software (www.chemcomp.com). The structures of the synthesized compounds (6–27) were built in MOE and then energy minimization was carried out. Prior to docking of the synthesized compounds, p-nitrophenyl β-D-glucuronide, which is a known substrate molecule, was docked first into the active site of the enzyme by utilizing docking program MOE-Dock. Like our previous results,53,54 the docked conformation of substrate bound structure of human β-D-glucuronidase displayed that the glycoside bond of p-nitrophenyl b-D-glucuronide was appropriately oriented towards the catalytic residues such as Glu540, Glu451, and Tyr504. The active site of the target enzyme was selected by using the site-finder module. The synthesized compounds were docked into the active site of the target enzyme in MOE by the default parameters i.e., Placement: Triangle Matcher, Rescoring: London dG. 3154

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