Synthesis, in vitro urease inhibitory potential and molecular docking study of Benzimidazole analogues

Accepted Manuscript

Synthesis, in vitro urease inhibitory potential and molecular docking study of

Benzimidazole analogues

Khalid zaman, Fazal Rahim, Muhammad Taha, Hayat Ullah, Abdul Wadood,

Mohsan Nawaz, Fahad Khan, Zainul Wahab, Syed Adnan Ali Shah, Ashfaq Ur

Rehman, Abdel-Nasser Kawde, Mohammed Gollapalli

PII:

S0045-2068(19)30209-3

https://doi.org/10.1016/j.bioorg.2019.103024

103024

DOI:

Article Number:

Reference:

YBIOO 103024

Bioorganic Chemistry

To appear in:

Received Date:

Revised Date:

Accepted Date:

9 February 2019

21 March 2019

30 May 2019

Please cite this article as: K. zaman, F. Rahim, M. Taha, H. Ullah, A. Wadood, M. Nawaz, F. Khan, Z. Wahab, S.

Adnan Ali Shah, A. Ur Rehman, A-N. Kawde, M. Gollapalli, Synthesis, in vitro urease inhibitory potential and

molecular docking study of Benzimidazole analogues, Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/

j.bioorg.2019.103024

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Synthesis, in vitro urease inhibitory potential and molecular docking study of

Benzimidazole analogues

Khalid zaman^a, Fazal Rahim^a*, Muhammad Taha*^b, Hayat Ullah^a, Abdul Wadood^c, Mohsan

Nawaz^a, Fahad Khan^a, Zainul Wahab^d, Syed Adnan Ali Shah^e,f, Ashfaq Ur Rehman^c, Abdel-

Nasser Kawde^g, Mohammed Gollapalli^h

^aDepartment of Chemistry, Hazara University, Mansehra-21300, Khyber Pakhtunkhwa, Pakistan

b

Department of clinical pharmacy, Institute for Research and Medical Consultations (IRMC), Imam Abdulrahman

Bin Faisal University, P.O. Box 1982, Dammam 31441, Saudi Arabia.

^cDepartment of Biochemistry, Abdul Wali Khan University Mardan, Mardan 23200, Pakistan

^dDepartment of Conservation Sciences, Hazara University, Mansehra-21300, Pakistan.

^eAtta-ur-Rahman Institute for Natural Product Discovery, Universiti Teknologi MARA (UiTM), Puncak Alam

Campus, 42300 Bandar Puncak Alam, Selangor D. E. Malaysia.

^fFaculty of Pharmacy, Universiti Teknologi MARA (UiTM), Puncak Alam Campus, 42300 Bandar Puncak Alam,

Selangor Darul Ehsan, Malaysia

^gChemistry Department, King Fahd University of Petroleum and Minerals, Dhahran, 31261, Saudi Arabia.

^hDepartment of Computer Information Systems, College of Computer Science & Information Technology (CCSIT)

Imam Abdulrahman Bin Faisal University P.O. Box 1982, Dammam 31441, Saudi Arabia.

---------------------------------------------------------------------------------------------------------------------

Abstract

Despite of many diverse biological activities exhibited by benzimidazole scaffold, it is rarely

explored for the urease inhibitory potential. For that purpose, benzimidazole analogues 1-19

were synthesized and screened for in vitro urease inhibitory potential. Structures of all synthetic

analogues were deduced by different spectroscopic techniques. All analogues revealed inhibition

potential with IC₅₀values of 0.90 ± 0.01 to 35.20 ± 1.10 μM, when compared with the standard

thiourea (IC₅₀= 21.40 ± 0.21 μM). Limited SAR suggested that the variations in the inhibitory

potentials of the analogues are the result of different substitutions on phenyl ring. In order to

rationalize the binding interactions of most active compounds with the active site of urease

enzyme, molecular docking study was conducted.

Keywords: Synthesis, Benzimidazole, Urease inhibitory potential, Molecular docking, SAR.

---------------------------------------------------------------------------------------------------------------

^Correspondence and reprints

E-mail: fazalstar@gmail.com, Tel.: 0092-335-9528343(FR); Mtaha@iau.edu.sa; Tel:00966502057370 (MT)

1

2

1.

Introduction

Urease is a nickel containing metallo-enzyme that catalyzes the conversion of urea into ammonia

and carbamate in micro-organism and various plant species [1-5]. The hydrolysis of urea by

urease enzyme occurs speedily at 10¹⁴times faster than the hydrolysis reaction by uncatalyzed

way. This reaction caused by urease enzyme results in sudden increase of the overall pH, which

causes negative effects to agriculture production, animal health and human beings. Urease

enzyme has revealed to be significant lethal factor to cause hepatic coma, urinary catheter

encrustation, pyelonephritis and infection stones in human beings and animals [6, 7]. Further,

urease enzyme can harshly decline the efficacy of urea fertilizers to cause the discharge of large

quantities of ammonia and also induce damage of plant by ammonia toxicity and pH rise in soil

[8]. In this context, urease enzyme inhibition study has attracted growing attention to tackle the

negative effects [9-11]. Moreover, urease inhibitory studies are important in elucidating the

catalytic mechanism of urease which is still unclear as has been extensively discussed in a recent

article [12]. However, the efficiency of currently available inhibitors is low and the complete

potential of urease inhibition has not yet been discovered [13]. Consequently, the ability to

control the degree of enzymatic urea hydrolysis by urease enzyme inhibitors is significant goal to

chase [14].

Nitrogen containing heterocycles have attracted considerable attention due to their wide

occurrence and pharmacological importance. Benzimidazole is an aromatic heterocyclic organic

compound having benzene ring fused with an imidazole ring. Imidazole ring is the part of many

natural products including purine, histamine, histidine, and nucleic acid. Due to its polar and

ionisable ability, it proves to be characteristics pharmacokinetic for lead molecules by enhancing

their solubility [15-17]. Benzimidazole is one of the privileged structures in medicinal chemistry

due to its wide range of activities including analgesic, antidiabetic, anti-inflammatory, antiulcer,

antifungal, antimicrobial, anticancer, antiprotozoal and antiviral activities etc [18-21].

Our research group has been working on design and synthesis of heterocyclic compounds in

search of potential lead compounds since many years and had found promising results [22-29].

We have compared our synthesized analogues with already reported benzimidazole analogues by

Emre Mentese et al., for anti-urease activity [30, 31]. We have also reported benzimidazole

derivatives as α-glucosidase inhibitors [32, 33] and 2-(2՜-Pyridyl) benzimidazole derivatives as

potent inhibitors for urease enzyme [34] but there is still need to explore more compounds for

3

this activity in order to identify lead candidates for more advances research in future. Thus, we

decided to screen a library of substituted benzimidazoles for urease inhibitory activity.

Cl

N

Cl

N

CH₃

O

N

HN

N

R

S

Urease inhibitors [30, 31]

O

N

O

N

HN NH

N

H

HN N

S

HN

R

IC = 8.40 to 179.71 M



IC = 35.83 to 297.99 M



50

-glucosidase inhibitors [33]

a

-glucosidase inhibitors [32]

a

N

H

N H

N

S

O

R₁

X

N

O

R₂

HN N

R

R₃

New Synthetic Analogs 1-19

Potent Urease inhibitors

IC = 19.22 to 77.31 M



50

Urease inhibitors [34]

Fig-1: Benzimidazole based already identified inhibitors of α-glucosidase and urease enzymes

and newly synthesized derivatives as urease inhibitors 1-19

2.0.

2.1.

Results and Discussion

Chemistry

We mixed 1H-benzimidazole-2-thiol (I) (1mmol) with methyl 4-(bromomethyl) benzoate (II)

(1mmol) in acetone in the presence of K₂CO₃and reflux for 5 hrs to give methyl 4-(((1H-

benzimidazol-2-yl)thio)methyl)benzoate (III) as intermediate product. The intermediate product

4

(III) was further treated with hydrazine hydrate in ethanol and reflux for 6 hrs to give 4-(((1H-

benzimidazol-2-yl)thio)methyl)benzohydrazide (IV) as second intermediate product. The

intermediate product (IV) was finally treated and refluxed with different substituted

aldehyde/acetophenone to give the desired products (1-19). Completion of reaction was

monitored by TLC. After completion of reaction; the product was filtered, washed with n-hexane

and dried.

O

H

N

H

N

Acetone

O

S

O

SH

Br

N

K₂CO₃, reflux, 5hrs

N

III

II

I

N₂H₄. H₂O

EtOH

O

R

reflux, 6 hrs

R

NH

H₂N

R

N

S

N NH

O

S

O

N

EtOH/H

H

Rfx, 3-4 hrs

IV

1-19

Scheme-1: Synthesis of benzimidazole analogues (1-19)

Table-1: Different analogues of benzimidazole and their urease inhibitory activity

Code

1

Structure

IC₅₀

O₂N

4.60 ± 0.2

O

N

H

S

N

5

Cl

2

3

8.60 ± 0.3

O

N

H

S

N

NO₂

15.20 ± 0.5

O

N

H

S

N

Cl

4

5

3.20 ± 0.01

O

N

H

N

S

N

13.10 ± 0.3

O

N

H

Br

H

N

S

N

OH

6

1.40 ± 0.01

O

N

H

S

N

6

7

18.6 ± 0.50

O

N

H

S

N

NO₂

8

9

15.70 ± 0.50

O

N

H

S

N

Cl

5.10 ± 0.2

Cl

O

N

H

S

N

HO

10

11

1.20 ± 0.01

O

N

H

S

N

35.20 ± 1.10

O

N

H

S

N

7

12

13

31.20 ± 1.10

13.60 ± 0.40

O

N

H

S

N

O

N

H

S

N

O

14

15

1.90 ± 0.01

7.10 ± 0.20

H

O

N

S

HN N

N

OH

HO

OH

O

N

H

S

N

OH

16

17

6.6 ± 0.20

O

N

H

N

S

N

Cl

8.20 ± 0.20

HO

O

N

Cl

N

H

S

N

8

OH

18

0.90 ± 0.01

OH

O

N

H

S

N

NO₂

19

14.80 ± 0.30

O

N

H

S

N

Thiourea

21.40 ± 0.21 µM

2.2.

In vitro urease inhibitory potential

We have synthesized nineteen benzimidazole analogues (1-19) which have varied degree of

urease inhibition ranging in between 0.90 ± 0.01 to 35.20 ± 1.10 μM when compared with the

standard drug thiourea having IC₅₀value 21.40 ± 0.21 μM. All analogues showed excellent

urease inhibitory potentials. The structure activity relationship (SAR) was mainly based upon by

bring about difference of substituents on phenyl ring.

If we compare analogue 1 (IC₅₀= 4.60 ± 0.2 μM) with analogue 3 (IC₅₀= 15.20 ± 0.5 μM) and

analogue 19 (IC₅₀= 14.80 ± 0.30 μM), all three analogues have nitro group on phenyl ring, but

the arrangement of nitro group is different in them which confirm that the difference in position

of substituents greatly affect the inhibitory potentials of the analogues.

If we compare analogue 2 (IC₅₀= 8.60 ± 0.3 µM) having one chloro group at 4-position on

phenyl ring with analogue 4 (IC₅₀= 3.20 ± 0.01 µM) having two chloro groups at 2,4-position on

phenyl ring and analogue 9 (IC₅₀= 5.10 ± 0.2 µM) also having two chloro groups at 3,4-position

on phenyl ring. All compounds have the same chloro groups but the position as well as number

of the chloro group is different at the phenyl ring. Compound 4 was found to be superior who

showed that position as well as number of substituent also play role in this inhibition.

9

By comparing analogue 6 (IC₅₀= 1.40 ± 0.01 µM) having one methoxy and one hydroxy

substituents with analogue 14 (IC₅₀= 1.90 ± 0.01 µM) also having one methoxy and one hydroxy

substituents on phenyl ring. In analogue 6, the methoxy group is present at 4-position and

hydroxy is present at 3-position and in analogue 14, the methoxy group is present at 3-position

and hydroxy at 4-position on phenyl ring. Both analogues have the same methoxy and hydroxy

groups but the position of the hydroxy and methoxy groups are different on phenyl ring.

Compound 6 was found to be superior who showed that position of substituent also play role in

this inhibition.

In this study, we observed that either electron withdrawing group (EWG) or electron donating

group (EDG) on phenyl ring showed potential but the slight difference in potential was mainly

affected by the position of the substituent as well as in some cases the number of substituent also

play a role. To understand the binding interaction of the most active analogs molecular docking

study was performed.

2.3. Molecular Docking

Molecular Operating Environment (MOE) package [35] was used to perform molecular docking

study to predict the binding mode of the synthesized compounds within the active site of urease

enzyme. First, the 3D structures of the synthesized derivatives were generated by using the

builder tool executed in MOE package. Next, all the compounds were subjected for protonation,

and energy minimized using the default parameters of the MOE (gradient: 0.05, Force Field:

MMFF94X) and saved in mdb (Molecular knowledge Base) file format. The 3D structure of the

target protein retrieved from the protein databank (PDB ID 4UBP). The retrieved protein then

opened in MOE package, all the water molecules removed, later on, 3D protonation carried.

After 3D protonation, the protein was energy minimized to get a stable conformation of the

protein using the default parameters of the MOE package. For docking studies, the default

parameters of the MOE package were used, i.e., Placement: Triangle Matcher, rescoring 1:

London dG, Refinement: Force field, Rescoring 2: GBVI/WSA. For each ligand ten

conformations could be fashioned, and the top-ranked conformations based on docking score

were selected for additional analysis.

2.4.

Molecular docking study

Molecular docking study was carried out through MOE package, in order to illustrate the binding

mode of interaction of the entire synthesized compound enlisted in the current study (1-19),

10

again the urease enzyme to validate the experimental results further. The primarily favorable

docking conformations were ascertained within the active site with proper orientation for all the

compounds. Generally, the ligand binding site of the corresponding enzyme comprises from both

hydrophobic and hydrophilic amino acids. A bunch of total five surface residues (A170, 366,

K169, L319, and C322) makes the hydrophobic region, while the hydrophilic region, totally nine

essential residues G166, 223, R339, D224, 494, H315, 323, 324, and 249 respectively as shown

in Fig. 1A. The two embedded Ni ions (Ni198 & 799) conjointly participate a significant role by

linking the critical amino acid and ligands. However, from the molecular docking study, it has

been observed that all the compounds fit well in the active pocket of the urease enzyme. More

ever, the most promising docked conformation of each compound was evaluated further for

binding mode analysis, based on the scores from the GBVI/WSA binding free energy

calculation. Generally, from the post-docking analysis, it was observed that the enlisted

synthesized compound possesses different substituted groups, like some, have electron

withdrawing (EW), and other have donating (ED) groups, and hence, the position and the

changing groups from EW to ED or vice versa ultimately altered the enzyme activity. The high

potency for most active compound 18 (0.90 ± 0.01) was confirmed from both the fit-well

behavior and ligand-protein interaction (LPI) analysis, that this compound found the only one in

the series which adopts most favorable interactions with the essential active site residues, include

R369, 339, L319, T362, D363, additionally the two embedded Ni ions were found in conjugate

with; Ni799-O¹⁹and Ni-798-O³⁰of the candidate compound.

Furthermore, this Ni799 was observed in ionic coordination with the modified residue KCX220.

The high LPI profile observed for 18 (Fig. 1B), further demonstrate that the high potency of the

candidate compound might be due to the attached EDG (di-hydroxyl). The -OH group has a

more activating effect on some positions; ortho & para around the ring than others means that

incoming groups will go into some positions much faster than they will into others, and hence

enhance the enzyme activity. As the variation occurrence from di- to mono or reverse, as well the

position from -ortho to -para or reveres ultimately enhance or reduce the enzyme activity. The

change as mentioned above was observed in the case of second rank compound 10 (1.20 ± 0.01).

The LPI profile for compound 10 indicates that this compound adopts favorable interaction with

the essential residue like; R369, D363, H249, and KCX220 (Fig. 1C).

11

Additionally, the Ni798 has observed ion ionic interaction with the S of the candidate compound.

The low potency in comparison with the highly potent compound 18 might be due to the attached

EDG (mono-OH)-group at benzene ring. Both the compounds possess the same EDG but hold

differences only on their benzene ring attached group (di-OH and mono-OH). More ever, the

same LPI profile was observed for compound 6 (Fig. 1D) and 14 (Fig. 1E), but less than the

other most active compound. The compound 4, hold the EWG, showed less potency, and LPI

profile again Urease enzyme, like an adopted interaction with H222, 249, and D363 (Fig. 1F).

The high potency of this compound might be due to EWG attached on benzene, hence, withdraw

the electron from the benzene and remain the benzene partial +ve, so further this benzene rings

unable to form pi-interaction.

The docking pose of almost all potent compounds computationally inhibited the catalytic

activities of the urease by binding determinedly through strong hydrogen bonding, hydrophobic,

and polar interactions with key residues, additionally, the compounds hold the EDG showed high

potency as compared to the compounds possess the EWG.

12

Fig-1: The ligand-protein interaction (LPI) profile for synthesized compounds against urease

enzyme. (A) The surface representation of the urease enzyme (PDB ID 4UBP). The

hydrophobic and hydrophilic regions were colored to green and blue, additionally, the two

Nickle ions (798 & 799 were shown in light green spheres. (B) The ligand-protein interaction

pose for the most active compound 18, (C) for 10, (D) for 6, (E) 14, and (F) 4.

2.5.

ADMET Analysis

ADMET pharmacokinetics is a very important method in the designing and screening of drug,

which is responsible for failure of drug. The ADMET properties of the drug candidates are

greatly influenced by the optimum value of the molecular weight, LogP, hydrogen bond donor,

hydrogen bond acceptor, intestinal absorption, water solubility, blood brain barrier (BBB)

penetration level. We used Lipinski’s rule of five to determine the drug-likeness of these

compounds. By this rule, most orally administered drugs have molecular weight less than 500, a

distribution ratio less than 5, less than 5 hydrogen bond donors and less than 10 hydrogen bond

acceptors. The ADMET properties of these newly synthesized compounds 1-19 was measured

using online server PKCM (biosig.unimelb.edu.au/pkcsm/prediction) as a drug reference was

reported in Table 2. From the table-2, it is clear that all our synthesized compounds obeyed the

Lipinski’s rule of 5. These results showed that our compounds are drug-like compounds.

Furthermore, it was also observed that these compounds have the capability to cross the BBB,

have good intestinal absorption and solubility. Overall the ADMET analysis and Lipinski’s rule

of 5 showed that these compounds are drug-like compounds (Table-2).

Table-2: Prediction of ADMET properties of compounds 1-19.

Compound MW

LogP

H bond H bond Solubility Absorption BBB

acceptor donor

1

2

3

4

5

6

7

8

431.477 4.5273

420.925 5.2725

431.477 4.5273

6

4

6

4

5

6

5

6

4

5

4

5

2

3

2

3

2

-2.928

-2.499

-2.929

-2.931

-2.988

-2.963

-2.922

-2.965

-2.931

-2.956

-2.896

-2.925

89.971

77.499

89.991

76.71

78.536

87.313

78.372

91.697

77.215

77.762

80.291

78.371

-0.876

-0.865

-0.887

-1.037

-1.057

-0.974

-0.882

-0.9

-1.032

-1.016

-0.733

-0.892

455.37

5.9259

495.402 5.3902

432.505 4.3333

492.604 6.1981

445.504 4.9174

9

455.37

416.506 4.7148

486.6 6.9255

506.631 6.5882

5.9259

10

11

12

13

14

15

16

17

18

19

429.549 4.6851

432.505 4.3333

432.505 4.4204

402.479 4.3247

471.369 5.6315

418.478 4.0303

431.477 4.5273

5

6

5

6

2

3

4

3

4

2

-2.988

-2.958

-2.925

-2.916

-2.917

-2.9

80.577

84.256

83.458

76.431

76.802

78.984

90.023

-0.72

-0.965

-1.29

-0.898

-1.224

-1.178

-0.895

-2.929

3.0.

Conclusion

In conclusion we have synthesized nineteen benzimidazole analogues (1-19) and screened

against urease inhibitory potential. All analogues showed a varied degree of urease inhibition

with IC₅₀values ranging between 0.90 ± 0.01 to 35.20 ± 1.10 μM when compared with the

standard drug thiourea having IC₅₀value 21.40 ± 0.21 μM. SAR studies were carried out to

investigate the role of substitutions and nature of the functional groups attached to the phenyl

ring which exert imperative influence on the urease inhibitory potential. Molecular docking

study was performed to understand the binding interaction of the most active analogues with

enzyme active site.

4.0.

4.1.

Material and Methods

General procedure for the synthesis of benzimidazole analogues (1-19)