Evaluation of bisindole as potent β-glucuronidase inhibitors: Synthesis and in silico based studies

Bioorganic & Medicinal Chemistry Letters 24 (2014) 1825–1829

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Evaluation of bisindole as potent b-glucuronidase inhibitors: Synthesis and in silico based studies Khalid Mohammed Khan a,⇑, Fazal Rahim b, Abdul Wadood c, Muhammad Taha d,e, Momin Khan f, Shagufta Naureen a, Nida Ambreen a, Shafqat Hussain a, Shahnaz Perveen g, Mohammad Iqbal Choudhary a a

H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan Department of Chemistry, Hazara University, Mansehra, Pakistan Computational Medicinal Chemistry Laboratory, Department of Biochemistry, Abdul Wali Khan University Mardan, Mardan 23200, Pakistan d Atta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor, Malaysia e Faculty of Applied Science UiTM, 40450 Shah Alam, Selangor, Malaysia f Department of Chemistry, Abdul Wali Khan University, Mardan, Mardan 23200, Pakistan g PCSIR Laboratories Complex, Karachi, Shahrah-e-Dr. Salimuzzaman Siddiqui, Karachi 75280, Pakistan b c

a r t i c l e

i n f o

Article history: Received 21 November 2013 Revised 28 January 2014 Accepted 6 February 2014 Available online 14 February 2014 Keywords: Bisindole b-Glucuronidase inhibition Molecular docking SAR

a b s t r a c t Bisindole analogs 1–17 were synthesized and evaluated for their in vitro b-glucuronidase inhibitory potential. Out of seventeen compounds, the analog 1 (IC50 = 1.62 ± 0.04 lM), 6 (IC50 = 1.86 ± 0.05 lM), 10 (IC50 = 2.80 ± 0.29 lM), 9 (IC50 = 3.10 ± 0.28 lM), 14 (IC50 = 4.30 ± 0.08 lM), 2 (IC50 = 18.40 ± 0.09 lM), 19 (IC50 = 19.90 ± 1.05 lM), 4 (IC50 = 20.90 ± 0.62 lM), 7 (IC50 = 21.50 ± 0.77 lM), and 3 (IC50 = 22.30 ± 0.02 lM) showed superior b-glucuronidase inhibitory activity than the standard (D-saccharic acid 1,4-lactone, IC50 = 48.40 ± 1.25 lM). In addition, molecular docking studies were performed to investigate the binding interactions of bisindole derivatives with the enzyme. This study has identified a new class of potent b-glucouronidase inhibitors. Ó 2014 Elsevier Ltd. All rights reserved.

The indole analogs are frequently encountered and are considered to be an important scaffold in medicinal chemistry.1 Variety of compounds having indole moiety, exhibited antitumor activity.2 Bis(indolyl)methanes is the important class of heterocyclic compounds with a variety of pharmacological activities, some of them play a key role in the treatment of chronic fatigue, fibromyalgia and irritable bowel syndrome.3,4 Some of these are responsible for beneficial estrogen metabolism, and induce apoptosis in cancer cells of human.5 The bis(indolyl)alkanes from marine and terrestrial sources are found to posses diverse activities, like coronary dilatory properties, antibacterial activity and genotoxicity.6 Some bisindole alkaloids, such as macrocarpamine, macralstonine acetate and villastonine possess significant antiprotozoal activity in vitro against Plasmodium falciparum and Entamoeba histolytica.7,8 Some bis(indolyl)methane derivatives are also used as dietary supplements for humans.9 They exhibit inhibitory activity against bladder cancer,10 and renal cell carcinoma growth,11 and inhibit lung12 and colon cancers.13,14 Bis(indolyl)methanes have inhibitory activity on phenobarbital-induced hepatic CYP mRNA expression15 and also act as cytodifferentiating agents.16 Some bis ⇑ Corresponding author. Tel.: +92 2134824910; fax: +92 2134819018. E-mail addresses: [email protected], [email protected] (K.M. Khan). http://dx.doi.org/10.1016/j.bmcl.2014.02.015 0960-894X/Ó 2014 Elsevier Ltd. All rights reserved.

(indolyl)methanes derivatives have DDPH radical scavenging,17 antimetastatic18,19 and analgesic activities.20 In present study the synthesis of bis(indolyl)methane analogs 1–17 and their in vitro b-glucuronidase inhibition activity has been achieved. b-Glucuronidase is an enzyme which catalyze the glucuronosyl-O-bonds cleavage.21 This is also up regulated in different pathological conditions, such as infection of urinary tract22–25 renal disease26 rejection of transplantation27 epilepsy28 larynx and breast.29 Besides, this enzyme is also involved in inflammatory joint diseases, like rheumatoid arthritis30,31 Some hepatic diseases and AIDS is also reported due to over-expression of the enzyme. Literature report showed that the bacterial b-glucuronidase inhibitor lead to a decrease in carcinogen induced colonic tumors.32 The current study is focused on the discovery of b-glucuronidase inhibitors of pharmacological importance. Our current research group is continuously working on the chemistry and biological of new heterocyclic compounds,33 sulfur containing compounds34 and hydrazones.35 Recently we have published benzothiazole as potent inhibitor of b-glucuronidase. Due to close resemblance of indole moiety with benzothiazole we envisaged that bis(indolyl)methanes may have b-glucuronidase inhibitory and observed results proved our hypothesis.

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K. M. Khan et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1825–1829

R1

R1

O N H

+

R2

H

NaBrO3 /NaHSO3 H 2O

R2

HN

R1 NH

Scheme 1. Synthesis of bis(indolyl)methane derivatives 1–17.

Bis(indolyl)methane analogs 1–17 were synthesized by treating commercially available substituted indole with different aldehydes in water. In a typical reaction, to a stirring mixture of substituted indole (3 mmol) and different substituted aromatic aldehydes (3 mmol) in water, the 10 mol % of sodium bromate and sodium hydrogen sulfite was added for 2 h.36 The reaction completion was monitored by TLC. The bis(indolyl)methane derivatives 1–17 are obtained in good yields after completion of the reaction. By washing through hot n-hexane or in some cases through column chromatography (silica gel) using 3:7 acetone and n-hexane as eluent afforded pure products. The structures of compounds 1–17 were deduced by using various spectroscopic techniques, including 1 H NMR and EI mass spectroscopy (Scheme 1). b-Glucuronidase belong to the glycosidase family of enzymes which catalyzes this breakdown of complex molecules of carbohydrates. Enzyme inhibition is one of the most significant tools in pharmaceutical research as well as in the field of drugs discovery. During this study, we have synthesized seventeen (17) derivatives and evaluated their b-glucuronidase enzyme inhibition activity. Out of seventeen (17), ten (10) compounds showed a potent inhibition superior to standard inhibitor of b-glucuronidase. Compounds 1–17 exhibited a varying degree of b-glucuronidase inhibitory activity with IC50 values between 1.62 ± 0.04 and 22.30 ± 0.02 lM when compared with standard D-saccharic acid 1,4-lactone (IC50 = 48.40 ± 1.25). Compound 1 (IC50 = 1.62 ± 0.040 lM), 6 (IC50 = 1.86 ± 0.05 lM), 10 (IC50 = 2.80 ± 0.29 lM), 9 (IC50 = 3.10 ± 0.28 lM), 14 (IC50 = 4.30 ± 0.08 lM), 2 (IC50 = 18.40 ± 0.09 lM), 19 (IC50 = 19.90 ± 1.05 lM), 4 (IC50 = 20.90 ± 0.62 lM), 7 (IC50 = 21.50 ± 0.77 lM), and 3 (IC50 = 22.30 ± 0.02 lM) showed excellent b-glucuronidase inhibitory activity higher than the standard (D-saccharic acid 1,4-lactone, IC50 = 48.40 ± 1.25 lM).37 Moreover the remaining compounds showed less than 50% inhibition and thus were not further evaluated for IC50 values. We checked

our bioassay result either its due to aggregate formation or by compounds themselves.38 In order to study the binding affinity of bisindole derivatives within the b-D-glucuronidase binding pocket and to understand their structure–activity relationship, in silico study was performed. In this study, 3D structure of human b-D-glucuronidase (having 80% sequence similarity with bovine b-D-glucuronidase) was used, as 3D structure of bovine b-D-glucuronidase has not been reported yet. The sequence similarity of bovine b-D-glucuronidase was searched by online server Blastp (http://blast.ncbi.nlm.nih.gov/), the obtained results showed 80% sequence similarity with human b-D-glucuronidase and thus 3D structure of human b-D-glucuronidase can be used for molecular docking. From the Protein Data Bank the X-ray crystallographic structure of human b-D-glucuronidase was retrieved39 (PDB Code 1BHG)40 for the docking simulation.41 Docking simulation indicated that the top ranked conformation of the most active compound 1 (Fig. 1A) from three hydrogen bonds between the phenolic group of the compound and the active site residues (His 385, Asp 207, and Tyr 508) of the enzymes. Additionally the indole moiety of the compound forms arene-arene interactions with the phenyl ring of the active site residue (Tyr 504). Glu 540, Glu 541, His 509, Trp 587, and Lys 606 are the other remains that alleviate the requisite of the compound 1 in the active site of b-D-glucuronidase. The strong hydrogen bonding network formed by the phenolic groups of the compound 1 and the active site residues might be the reason for the highest activity of compound 1 in the series (Table 1). For example, if we compare the observed activities (Table 1) and predicted interactions of compounds 1, 2, 4, 6, 7, and 9, the compounds having phenolic moiety showed more activity (compounds 1, 6 and 9), as compared to those lacking this group (compounds 2, 3, 4, and 7). This difference in activities might be due to additional hydrogen bonding between phenolic compounds with active site residues of the enzyme as observed in docking simulation (Fig. 1A and B). When the activities (Table 1) and predicted binding modes (Fig. 2A and B) of compounds 10, 14 and 17 was compared, it was observed that the methoxy group might play an important role in their activities and binding interactions. From the docked conformations of compounds 10 and 14 with phenolic groups, it was observed that these compounds form strong hydrogen

Figure 1. Predicted binding interactions of bisindoles (1) and (17) in the active site of human b-D-glucuronidase.

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K. M. Khan et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1825–1829 Table 1 In vitro b-glucuronidase of bis(indolyl)methanes 1–17 Compound no.

R1

1

5-Br

Table 1 (continued)

R2

IC50 ± SEMa (lM)

HO

Compound no.

R1

15

5-CN

R2

1.62 ± 0.04

Cl

OH 2

5-Br

3

5-Br

F

IC50 ± SEMa (lM)

Cl

NAb

16

5-Br

NO2

NAb

17

5-CN

NO2

19.90 ± 1.05

18.40 ± 0.09

22.30 ± 0.02 D-Saccharic

Cl

acid 1,4-lactone

—

—

48.40 ± 1.25

a

4

5-Br

5

5-Br

6

Me

20.90 ± 0.62

OMe

5-Br

NAb

1.86 ± 0.05

OH

7

5-Br

Cl

8

21.50 ± 0.77

Cl

NAb

5-Br

NO2 9

OH

5-Br

3.10 ± 0.28

Cl 10

OH

5-CN

2.80 ± 0.29

OMe

11

NAb

5-Br

S

12

Me

5-Br

OH

13

NAb

NAb

5-CN

S

14

OMe

Me

5-CN

OH

OMe

SEM is the standard error of the mean. NA not active, D-saccharic acid 1,4-lactone standard inhibitor for b-glucouoronidase activity. b

4.30 ± 0.08

bonding with active site residues Asp 484, and two hydrophobic interactions with residues Asp 207 and Tyr 508, whereas the compound 17 lacking methoxy group was unable to make hydrogen bond with the active site residues, and developed only the hydrophobic interaction with active site residues Tyr 508 as shown in Figure 2B. This lack of hydrogen bonding might be one of the reasons of low activity of compound 17, as compared to compounds 10 and 14 (Fig. 2A and B). The top ranked docked conformations of inactive compound 15 is shown in Figure 3. From the analysis of the predicted binding interactions of inactive compounds it was observed that these compounds do not form hydrogen bonds with the important active site residues of the enzyme, as observed in our previous study.41 This lack of hydrogen bonding might be one of the reasons for their low activity. X-ray crystallographic structure of human b-D-glucuronidase39 was recovered from the protein data bank. In docking procedure, the way for receptor and ligands structures, was used, as explain in our earlier publication.41 Structure of protein was checked for missing atoms, contacts and bonds. The B-chain of protein and hetero-atoms, together with co-factors was distant from the original file of protein data bank. Hydrogen atoms then added to the protein structure by means of the biopolymer module in SYBYL7.3 software package.42 All the docking simulation studies were performed on IntelÒ XeonÒ Quad™ Core processor 3.0 GHz Linux workstation running under open SUSE11.3, equipped with GOLD3.0 as the docking software. Molecular docking studies were carried out via GOLD software version 3.0 from the Cambridge Crystallographic Data Centre.43 Gold Score was selected as a fitness score and the standard default settings were utilized in all docking calculations. The configuration file was defined by the following process: docking site contains all the atoms within 10 Å of a specified centroid (x, y, z coordinates: 80.43, 84.41, 90.48). For each of the 100 independent genetic algorithm runs, a default maximum of 100,000 genetic operations was performed by using the default operator weights and a population size of 100 chromosomes. Operator weights for cross over, mutation and migration were set to 100 and 0, respectively. To allow poor non-bonded contacts at the start of each GA run, the maximum distance between hydrogen donors and fitting points were set to 5.0 Å, and non-bonded VdW energies were selected at cut-off value of 10 Å. All single bonds were treated as rotatable. Results differing by less than 1.5 Å in ligand-all atom RMSDs were

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Figure 2. Predicted binding interactions of bisindoles 10 and 17 in the active site of human b-D-glucuronidase.

Figure 3. Predicted binding interaction of bisindole derivatives having low % of inhibition (Compound 15) in the active site of human b-D-glucuronidase.

clustered together. After docking, thirty poses were saved for each ligand. On the basis of score, top scored docked poses were visually inspected for each ligand. The molecular interactions were visualized using LIGPLOT, implemented in Molecular Operating Environment (MOE).44 A reliable model of the complexed structure of b-D-glucuronidase was prepared using the method mentioned in our previous publication.41 The modeled structure was then used for the prediction of favorable binding interaction of our new synthesized compounds to b-D-glucuronidase by docking simulation (Table 2). Three-dimensional (3D) structures of the ligands were modeled by using standard bond lengths and angles from the SYBYL7.3 fragment library. Partial charges were assigned according to the Gasteiger–Hückel method.45 Geometry optimizations were carried out using the Tripos force field with a distance-dependent dielectric and the Powell conjugate gradient algorithm. Bisindole analogs were synthesized and their inhibitory potential was evaluated against b-D-glucuronidase enzyme. Out of seventeen (17) derivatives, ten (10) compounds showed b-D-glucuronidase inhibitory potential. Compounds 1, 6, 10, 9, 14, 2, 19, 4, 7 and 3 exhibited an excellent inhibition with IC50 values

Table 2 Synthesized compounds with b-D-glucuronidase inhibitory potential and docking simulation

a b

Compounds

Goldscore

IC50 ± SEMa (lM)

p-Nitrophenyl-b-D-glucuronidase 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17

58.37 64.48 59.68 60.78 56.33 51.28 58.36 57.78 53.71 63.25 63.36 50.07 55.23 53.17 68.46 56.86 54.66 62.61

1.62 ± 0.04 18.40 ± 0.09 22.30 ± 0.02 20.90 ± 0.62 NAb NAb 21.50 ± 0.77 NAb 3.10 ± 0.28 2.80 ± 0.29 NAb NAb NAb 4.30 ± 0.08 NAb NAb 19.90 ± 1.05 48.40 ± 1.25

SEM is the standard error of the mean. NA Not active.

K. M. Khan et al. / Bioorg. Med. Chem. Lett. 24 (2014) 1825–1829

between 1.62 ± 0.04 and 22.30 ± 0.02 lM. Consequently in silico studies was performed to recognize the binding mode of these compounds. The planned scaffold of b-D-glucuronidase inhibitors offers the possibility of expedient additional modifications that could give rise to lead structures with enhanced inhibitory activity and selectivity towards the enzyme. Acknowledgments The authors are thankful to Organization for Prohibition of Chemical Weapons (OPCW), Netherlands (Project No. L/ICA/ICB/ 173681/12), for financial support. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2014.02. 015. References and notes 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19.

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