Accepted Manuscript Synthesis of Novel Derivatives of Oxindole, their Urease Inhibition and Molecular Docking Studies Muhammad Taha, Nor Hadiani Ismail, Ajmal Khan, Syed Adnan Ali Shah, Ammarah Anwar, Sobia Ahsan Halim, M. Qaiser Fatmi, Syahrul Imran, Fazal Rahim, Khalid Mohammed Khan PII: DOI: Reference:

S0960-894X(15)00543-0 http://dx.doi.org/10.1016/j.bmcl.2015.05.069 BMCL 22759

To appear in:

Bioorganic & Medicinal Chemistry Letters

Received Date: Revised Date: Accepted Date:

3 February 2015 21 May 2015 23 May 2015

Please cite this article as: Taha, M., Ismail, N.H., Khan, A., Shah, S.A.A., Anwar, A., Halim, S.A., Qaiser Fatmi, M., Imran, S., Rahim, F., Khan, K.M., Synthesis of Novel Derivatives of Oxindole, their Urease Inhibition and Molecular Docking Studies, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/ j.bmcl.2015.05.069

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Synthesis of Novel Derivatives of Oxindole, their Urease Inhibition and Molecular Docking Studies Muhammad Tahaa,b, Nor Hadiani Ismaila,b, Ajmal Khanc, Syed Adnan Ali Shaha,d, Ammarah Anware, Sobia Ahsan Halimf, M. Qaiser Fatmie, Syahrul Imrana,b, Fazal Rahimg and Khalid Mohammed Khani a

Atta-ur-Rahman Institute for Natural Product Discovery (AuRIns), Universiti Teknologi MARA Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor D. E. Malaysia. b

Faculty of Applied Science UiTM, 40450 Shah Alam, Selangor, Malaysia Department of Chemistry, COMSATS Institute of Information Technology, Abbottabad-22060, Pakistan d Faculty of Pharmacy, Universiti Tecknologi MARA Puncak Alam, 42300 Bandar Puncak Alam, Selangor D. E. Malaysia. c

e

Department of Biosciences, COMSATS Institute of Information Technology, Park Road, Chak Shahzad, Islamabad, Pakistan. f

National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore 53700, Pakistan. g

Department of Chemistry, Hazara University, Mansehra-21120, Pakistan

i

H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan Abstract: We synthesized a series of novel 5-24 derivatives of oxindole. The synthesis started from 5-chlorooxindole, which was condensed with methyl 4-carboxybezoate and result in the formation of benzolyester derivatives of oxindole which was then treated with hydrazine hydrate. The oxindole benzoylhydrazide was treated with aryl acetophenones and aldehydes to get target compounds 5-24. The synthesized compounds were evaluated for urease inhibition; the compound 5 (IC50 = 13.00 ± 0.35 µM) and 11 (IC50 = 19.20 ± 0.50 µM) showed potent activity as compared to the standard drug thiourea (IC50 = 21.00 ± 0.01 µ M). Other compounds showed moderate to weak activity. All synthetic compounds were characterized by different spectroscopic techniques including 1H-NMR,

13

C-NMR, IR and EI MS. The molecular

interactions of the active compounds within the binding site of Urease enzyme were studied through molecular docking simulations.

Keywords: Oxindole, urease activity, docking studies, novel derivatives 

Correspondence and reprints E-mail: [email protected] [email protected], Tel: 0060193098141

1

Oxindoles have been recognized as probes for investigation of biological processes and, therefore, lead to many drug discovery systems [1,2]. Many examples of oxindole such as 3hydroxy oxindole have great importance because of their occurrence in natural products, and their structures are also valuable in medicinal chemistry. Many 3-hydroxyoxindoles possess useful biological and pharmacological activities [3]. For example, the compounds TMC-95A-D consist of a multifunctional 3-hydroxyoxindole structure, and they inhibit the proteolytic activity of proteasome. This is a protease complex which is targeted in the design of new drugs for many diseases such as cancer and autoimmune diseases [4]. A large number of synthetic compounds such as growth hormone secretagogues [5], analgesic [6], anti-inflammatory compounds [7], SNC active agents like serotonergics [8], the anti-Parkinson’s drug ropirinole [9] etc are known to contain oxindoles moiety and possess useful pharmaceutical properties. Among these compounds most of them contain a variety of substituents at the C-3 position of oxindole, and many of them are 3-spirooxindoles, p-glycoprotein-mediated multiple drug resistance inhibitors [10], antibacterial, antiprotozoal [11], anti-inflammatory agents [12], serotonergics [13], antitumor agents and as inhibitors of the human NKI receptor [14] and antiglycation [15]. Benzohydrazones have many applications in medicinal and analytical chemistry [16-18]. Benzohydrazones were recently reported to possess diuretic [19], antioxidant [20-23], antiglycation [24-26] α-glucosidase [27] and antileishmanial [28] activities. Urease (E.C 3.5.1.5) is an important protein causing virulence as well as determinant in pathogenesis of many diseases in human. It is involved in the production of infectious stones in addition to pathogenesis including urolithiasis, pyelonephritis, and hepatic encephalopathy. The urease enzyme aids Helicobacter pylori (HP) to endure at low pH of the stomach during colonization, thus plays a vital role in the pathogenesis of the gastric as well as peptic ulcers which may causes cancer [29,30]. Additionally, urease causes kidney stones formations [31]. In agriculture, during urea fertilization, high urease activity results in significant environmental pollution as well as economic losses by discharging of abnormally huge amounts of ammonia in atmosphere. This also leads to plant damage by depriving them from essential nutrients,

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secondary ammonia toxicity and increase in pH of the soil [32]. Urease inhibition, therefore, has been identified as first line of treatment of diseases caused by ureolytic bacteria [33]. In recent past many papers are reported for urease inhibition, having Schiff base [34,35] and their metal complexes with tin (IV), Cu, Ni, Zn and Co [36]. We design our project to synthesize novel Schiff bases to evaluate their urease inhibition as well as to study the molecular basis of inhibition. The development of novel drugs is certainly one of the most demanding task of today’s science that is provoked by the collective endeavor of the pharmaceutical industry, biotechnological companies, regulatory authorities, academic researchers, and other private and public sectors. The augmentation of new drugs is a very intricate and challenging interdisciplinary practice [37]. In modern drug designing, molecular docking is successfully used for insightful drug-receptor interaction. It provides valuable information about drug receptor interactions, and is repeatedly used to foretell the binding orientation of ligands to their protein targets in order to evaluate the binding affinity, stability and activity of the drug candidate [38]. Novel derivatives of oxindole (5-24) were synthesized from 6-chlorooxindole (1) and 4formylbenzoic acid (2). They were condensed to form (E)-4-((5-chloro-2-oxoindolin-3ylidene)methyl)benzoic acid (3). The crude Product was purified by column chromatography before further reaction and obtained pure E isomer. (E)-4-((5-chloro-2-oxoindolin-3ylidene)methyl)benzoic

acid

(3)

was

converted

to

(E)-4-((6-chloro-2-oxoindolin-3-

ylidene)methyl)benzohydrazide by reported procedure [39] (4). Scheme-1:

The (E)-4-((6-chloro-2-oxoindolin-3-ylidene)methyl)benzohydrazide was treated with different acetophenones and aryl aldehydes in the presence of acetic acid to give desired products (5-24) Scheme-2. 3

Insert Table-1 Here Based on a wide range biological activity of oxindole and benzohydrazones a series of new derivatives were synthesized. Oxindole derivatives 5-24 were evaluated against urease enzyme, according to the protocol published in the literature [40, 41]. A varying degree of urease inhibitory activity with IC50 values between 13.00- 148.50 µM was observed and compared with the standards, thiourea (IC50 = 21 ± 0.11 µM) and acetohydroxamic acid (IC50 = 42.00 ± 1.26 µM), respectively, (Table 1). The compound 5 and 11 exhibited similar inhibitory activity when compared to both standards. The compound 6 displayed better activity comparable to the second standard while compounds 7, 8 and 24 showed close activity to the second standards. The compounds 10, 14, 18, 20 and 21 posses good activity while compounds 9, 12, 13, 15-17, 19, 22 and 23 showed moderate activity. All 20 synthesized oxindole derivatives were drawn in Chem-Draw Ultra 12.0 followed by their geometry optimization using force field methods. The optimized structures were saved in .mol2 format, and a database was generated in .mdb format. Afterwards ligands were allowed to adjust their fragments, tautomers, protonation state, polar hydrogens and coordinates. Partial charges were added to each ligand in the database, followed by total energy minimization of ligands so that they can attain a stable conformation. Protein 3D structure of urease from Helicobacter Pylori (PDB accession code 1E9Y) was obtained from RCSB PDB (an information portal to biological macromolecular structures). The water molecules were removed from the file, and the protein was

4

protonated in 3D to add polar hydrogen’s. Binding pocket was identified using site finder, and the respective residues were selected. Docking parameters were set to default values and scoring algorithm, The London dG was applied. The docking runs were retained to 30 conformations per ligand. The docked protein structures were saved in .moe format, and ligand's conformations were investigated one by one. Complexes with best conformations were selected on the basis of highest score, lowest binding energy and minimum RMSD values table 2. Insert Table-2 Here Many proteins perform their biological function more efficiently by binding respective protein or ligand at their specific binding site. Identification of interacting residues with ligands is a necessary step towards rational drug designing, understanding of molecular pathway and mechanistic action of protein. In this context, the protein was docked in succession with all 20 synthesized ligands, and 20 stable protein-ligand complexes were predicted based on their lowest binding energy, highest scoring and lowest RMSD values. The complexes were visually inspected as well. A recent study pointed out that the source of most of the connections between protein-ligand is hydrogen bonds, ionic and van der Waals interactions [42], and therefore, they were specifically focused for this particular study. Molecular docking was carried out on MOE between rigid receptor protein and the flexible ligands. Table 2 shows the details of the docking results including RMSD and binding energy values of protein-ligand complexes. The ligands 1, 2 and 7 bind strongly to urease as inferred by their minimum binding energy values i.e. -13.8, -12.9 and -12.3 kcal/mol, respectively. This is in good agreement with the experimentally observed IC50 values for these compounds. Figure 1(a) shows the best conformation of all three ligands forming a cluster into the binding pocket of urease. Insert Figure-1 Here Hydrogen bonds act as an important factor for contributing in protein-ligand stability. They typically posses a distance of less than 3.5 Å between the H-donor and the Hacceptor heavy atoms which are located within an angle range of 0-90°. In case of urease

5

docked complexes with all 20 ligands, the best conformational clusters exhibited an average of 4 hydrogen bonds (excluding multiple H-bonds) with optimum bond distances range from 1.93 to 3.33Å. This observation is also in line with the Lipinski's rule of 5 (RO5) [43, 44] defined for hydrogen bond for oral bio-availability. Figure 1(b, c and d) displays hydrogen bond interactions between protein and ligands. Bond distances in Angstroms were calculated using VMD and LigPlot+, and are shown in Table 3 along with residue name and IDs. Insert Table-3 Here The amino acid side chains have varying tendencies to interact with each other and with ligands. These interactions contribute to the overall stability of the complex, and are necessary for appropriate function of the protein. Likewise hydrogen bonding, the van der Waals interactions also play a vital role in the protein stability. The amino acid residues that are involved in van der Waals interactions are all hydrophobic in nature as expected. The current study reveals that Ala365, Met366, Gly279, Gly280 and Ala169 participate in van der Waals interactions with the ligands; the average distances are 4.25 Å, 5.52 Å, 5.44 Å , 5.33 Å and 4.29 Å, respectively. The binding models of the ligands with urease indicate Ni2+, Ni2+, KCX219, Asp362, His248, Asp223, Arg338, Glu254, Glu222, His322, His274, Met366, Cys321, Thr254, Ala169, His221, Gly279, Gly280 and Ala365 as important residues involved in the ligand-protein interactions, and therefore, designate their significance in ligand binding as well as in probable catalytic actions (Figure 1a). These residues have also been reported previously as catalytic residues in the active site [45, 46], and therefore, they may have the essential role in lead optimization and thus aid in enhanced ligand affinity with the protein

6

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22) Aziz, A.N.; Taha, M.; Ismail, N.H. Anouar, E.H.; Yousuf, S.; Jamil, W.; Awang, K.; Ahmat, N.; Khan, K.M.; Kashif, S. M. Molecules 2014, 19, 8414. 23) Khan, K.M.; Muhammad Taha, F. Naz, S. Ali, S. Perveen, M. Iqbal Choudhary. Med. Chem. 2012, 8, 705. 24) Taha, M.; Ismail, N.H.; Jamil, W.; Rashwan, H.; Kashif, S.M.; Sain, A.A.; Adenan, M.I.; Anouar, E.H.; Ali, M. Rahim, F. Khan, K.M. Eur J Med Chem. 2014, 84, 731. 25) Taha, M.; Naz, H.; Rasheed, S.; Ismail, N.H.;Rahman, A. A.; Yousuf, S.; Choudhary M.I. Molecules 2014, 19, 1286. 26) 1. Khan, K.M.; Taha, M.; Rahim, F.; Fakhri, M.I.; Jamil, W.; Khan, M.; Rasheed, S.; Karim, A. Perveen, S.; Choudhary. M.I. J. Pak. Chem Soc. 2013, 35, 929. 27) Taha, M.; Ismail, N.H.; Lalani, S.; Fatmi, M.Q.; Siddiqui, S. Khan, K.M.; Imran, S. Choudhary, M.I. Eur J Med Chem. 2015, 92, 387. 28) a) Taha, M.; Baharudin, M.S.; Ismail, N.H.; Khan, K.M.; Jaafar, F.M.; Samreen; Siddiqui, S.; Choudhary, M.I. Bioorg. Med Chem. Lett. 2013, 23, 3463; b) Taha M, Ismail NH, Ali M, Khan KM, Jamil W, Kashif S.M.; Asraf, M. Med. Chem. Res. 2014, 23, 5282. 29) Weatherburn, M. W. Anal Chem. 1967, 39, 971. 30) Martelli, A.; Buli, P.; Cortecchia, V. Eur Urol. 1981, 7, 291. 31) Mobley, H. L. T.; Hausinger, R. P. J. Microbial Rev. 1989, 53, 85. 32) Arfan, M.; Ali, M.; Ahmad, H.; Anis, I.; Khan, A.; Choudhary, M. I.; Shah, M. R. J. Enz. Inhib. Med. Chem. 2010, 25, 296. 33) Aslam, M.A.S.; Mahmood, S.; Shahid, M.; Saeed, A.; Iqbal, J. J. Eur. Med. Chem. 2011, 46, 5473.

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45) Azizian, H.; Bahrami, H.; Pasalar, P.; Amanlou, M. J. Mol. Graph. Model. 2010, 28, 626. 46) Mishra, H.; Parrill, A.L.; Williamson, J. S. Anti-microbial agents and Chemother, 2002, 46, 2613.

Scheme, Table, Figures Captions Scheme-1: Synthesis of Oxoindole Chalcone Derivatives Scheme-2: Synthesis of oxindolebenzoylhydrazones derivatives. Table-1. In vitro Urease Inhibitory Activity of Compounds 5-24. Table 2: Docking results of Urease (PDB accession code 1E9Y). Table 3: The formation of hydrogen bonds between protein and ligand along with their distance in Angstrom. Figure 1: (a) Cluster formation of the lowest energy conformation for compounds 5, 6 and 11 indicated by green, magenta and blue CPK models located in the binding pocket of urease enzyme. The binding site residues have been labeled. The lower panels (b), (c), and (d) displays H-bond interactions between protein residues and ligands 5, 6 and 11, respectively.

11

Scheme-1:

The (E)-4-((6-chloro-2-oxoindolin-3-ylidene)methyl)benzohydrazide was treated with different acetophenones and aryl aldehydes in the presence of acetic acid to give desired products (5-24) Scheme-2.

12

Table-1:

Entry

R1

IC50 Entry (M ± SEMa)

R2

R1 1"

6"

5

H

13.00 ± 0.35

15

R1

IC50 (M ± SEMa)

H

119.80 ±1.90

H

143.60 ± 2.05

H

110.50 ±2.17

CH3

71.80 ±1.87

H

145.1±2.7

H

82.00 ± 1.840

H

92.40 ±1.160

H

148.50 ±1.350

OH

5"

3" 4"

6

CH3

35.40 ± 0.55

1"

6"

7

H

16

OH

5"

42.90 ± 1.60

3"

17

5"

8

H

Cl

1"

OH 3"

HO

1"

6"

53.20 ± 1.44

18

5"

OH 3"

4"

4"

1"

6"

9

2" 3"

OH 6"

1"

6"

125.50 ±3.57

H

19

5"

OH 3"

OCH3 1"

6"

10

H

2"

70.80 ± 1.20

5"

20

OH OH 1"

6"

11

19.20 ± 0.50

H HO

6"

12

6" 2"

H

4"

1"

OH

5"

2" 3"

NO2

OH

109.40 ± 2.30

5" 4"

21

1"

OH

22

6"

13

H

1"

5" 4"

14

H

2"

102.30 ± 3.40

23

H

84.80 ± 1.04

24

H

NAb

OH

Standard Drug Thiourea

21 ± 0.11 µM

Standard Drug acetohydroxamic acid SEMa is the standard error of the mean, NAb Not active, Thiourea,c standard inhibitor for urease activity.

42.00 ± 1.26µM

49.10 ±1.330

Table-2: S. No.

Ligand names

1

5

RMSD value in Angstrom 1.93

2

6

2.1

-12.9

3

7

2.61

-10.61

4

8

2.41

-10.59

5

9

1.6

-10.7

6

10

1.2

-10.04

7

11

1.5

-12.3

8 9 10 11 12 13 14 15 16 17 18 19 20

12

1.6 1.45 1.92 2.07 1.8 1.74 2.30 1.07 0.97 2.35 1.14 1.4 4.0

-11.7 -11.9 -10.42 -10.75 -11.3 -10.9 -10.01 -11.5 -9.31 -10.43 -9.11 -9.2 -10.9

13 14 15 16 17 18 19 20 21 22 23 24

Binding Energy in Kcal/mol -13.8

Table-3: S. No.

Ligand No.

1

5

2.5

2

6

2.6

3

7

2.03

3.04

4

8

2.03

3.04

5

9

6

10

7

11

8

12

1.94

3.23

9 10 11 12 13 14 15 16 17 18 19 20

13 14 15 16 17 18 19 20 21 22 23 24

1.98 2.04 1.98 1.99 1.98 1.95

3.06 3.06 3.06 3.08 3.11 3.06

1.99 1.98

3.05 3.07 2.71

Ni 1

Ni 2

His 248

Gly 279

Interacting residues (Distance in Angstrom) His Asp Ala Kcx His Glu Asp Met 322 36 365 219 138 254 223 366 2 2.3 2.2 2.9 2.5 2.1 2.4 2.0 2.7

Ala 169

Glu 222

2.3

2.1

2.9

2.1

3.1/4.5

4.8

3.28 2.34 3.15

3.10

Arg 338

3.42

2.04

3.06

3.02 2.94

3.12

2.19

2.40

2.70

2.06

3.07 3.01 2.97 3.01 3.02 3.06

2.87

2.13

3.33

2.38

2.42

3.04

1.96 2.13

3.13

2.65

Figure-1:

Muhammad Tahaa,b, Nor Hadiani Ismaila,b, Ajmal Khanc, Syed Adnan Ali Shaha,d, Ammarah Anware, Sobia Ahsan Halimf, M. Qaiser Fatmie, Syahrul Imrana,b, Fazal Rahimg and Khalid Mohammed Khani a

Atta-ur-Rahman Institute for Natural Product Discovery (AuRIns), Universiti Teknologi MARA Puncak Alam Campus, 42300 Bandar Puncak Alam, Selangor D. E. Malaysia. b

Faculty of Applied Science UiTM, 40450 Shah Alam, Selangor, Malaysia Department of Chemistry, COMSATS Institute of Information Technology, Abbottabad-22060, Pakistan d Faculty of Pharmacy, Universiti Tecknologi MARA Puncak Alam, 42300 Bandar Puncak Alam, Selangor D. E. Malaysia. c

e

Department of Biosciences, COMSATS Institute of Information Technology, Park Road, Chak Shahzad, Islamabad, Pakistan. f

National Centre of Excellence in Molecular Biology, University of the Punjab, Lahore 53700, Pakistan. g

Department of Chemistry, Hazara University, Mansehra-21120, Pakistan

i

H.E.J. Research Institute of Chemistry, International Center for Chemical and Biological Sciences, University of Karachi, Karachi 75270, Pakistan

*Corresponding author : [email protected], Tel : +923359528343 ____________________________________________________________________



Correspondence and reprints E-mail: [email protected] [email protected], Tel: 0060193098141

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