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Parr and Pearson [24] introduced the chemical hardness as a central ...... 1963, 85(22), 3533–3539; (b) Robert G. P.; Ralph, G. P. J. Am. Chem. Soc., 1983 ...

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Letters in Drug Design & Discovery , 2016, 13, 888-896

RESEARCH ARTICLE ISSN: 1570-1808 eISSN: 1875-628X

Imidazole-based Derivatives as Potential Anti-platelet Inhibitors: DFT and Molecular Docking Study

Impact Factor: 0.974

BENTHAM SCIENCE

Mehbub I. K. Momina, Neil A. Koorbanallya and Bahareh Honarparvarb,* a

School of Chemistry, University of KwaZulu-Natal, Private Bag X54001, Durban, 4000, South Africa; bSchool of Pharmacy and Pharmacology, University of KwaZulu-Natal, Private Bag X54001, Durban, 4000, South Africa

Pe N rs ot on fo al rD U is se tri O bu n tio ly n

Letters in Drug Design & Discovery

888

ARTICLE HISTORY Received: April 20, 2015 Revised: February 09, 2016 Accepted: May 13, 2016

DOI: 10.2174/1570180813666160517162 937

Abstract: Selected synthesized biologically active fluorinated diethyl-2-(benzylthio)-2,3-dihydro1H-imidazole-4,5-dicarboxylate derivatives exhibited promising anti-platelet aggregation activity against thrombin. With a view to understanding their structural features, some common electronic structural characteristics, including polarizability (), ELUMO-HOMO, chemical hardness () and softness (S) were calculated using density functional theory (DFT) with B3LYP functional and 6-31+G* basis set. The high polarizabilities (=139.554 A3, 138.459 A3) with low ELUMO-HOMO (3.761 eV, 3.662 eV), low chemical hardness (= 1.880 eV, 1.831 eV) and high chemical values of softness (S= 0.265 eV, 0.273 eV) are observed for the two most active imidazole-based anti-platelet derivatives with reasonably good IC50 values (0.40 mg mL-1, 0.44 mg mL-1). Moreover, molecular docking analysis of these anti-platelet compounds in the active binding site of the thrombin receptor identified the potential interaction between these inhibitors with the active residues of thrombin. Inspection of the docked structures implies that the antiplatelet inhibition of the most active compounds is mainly due to the possibilities of hydrogen bonding interaction and intramolecular close contacts of some active residues of the thrombin receptor (Gly219, Glu192, Gly216, Trp60, Tyr60A, Trp215, Leu99, His57, Cys191, Lys60, Glu217) with anti-platelet leads. These results suggest that the active imidazole derivatives could be promising chemical scaffolds to target the thrombin receptor.

Keywords: Anti-platelet derivatives, Inhibitory concentration (IC50), density functional theory (DFT), natural bond orbital (NBO) analysis, molecular docking. 1. INTRODUCTION

Antiplatelet therapy has dramatically reduced mortality and morbidity in acute myocardial infarction. Understanding the molecular basis of the role of platelets in cardiovascular thrombosis has enabled the development of better pharmaceutical agents with the potential to further reduce mortality [1]. These inhibitors potentially adhere to injured blood vessels and play an important role in normal hemostasis [2]. They are capable of acting as anti-thrombin agents and are responsible for the inhibition of platelet aggregation [3]. In vivo platelet aggregation can cause serious cardiovascular diseases, including atherosclerosis, ischemia, thrombosis, infarction and stroke [4].

Imidazole based drug-like derivatives show biological anti-coagulant, anti-inflammatory, anti-bacterial, anti-fungal, anti-viral, anti-tubercular, anti-diabetic and anti-malarial activities [5-7]. Imidazole is a planar five-membered ring molecule with two nitrogen atoms and two conjugated dou-

*Address correspondence to this author at the School of Pharmacy and Pharmacology, University of KwaZulu-Natal, Durban 4001, South Africa; Tel: + 27 31 2608482; E-mail: [email protected] 17-;/16 $58.00+.00

ble bonds. The imidazole ring is the basis of several important natural products such as purine, histamine, histidine and nucleic acids [8].

Human -thrombin as serine proteases is an enzyme of the blood coagulation cascade and plays an important role in thrombosis and hemostasis [9, 10]. The activity of thrombin will cause the cleavage of fibrinogen to form fibrin and the activation of platelets via the thrombin receptor [11-14]. Moreover, thrombin interacts with different cells and induces platelet aggregation [15]. Human thrombin contains an A chain of 36 AA and a B chain of 259 AA, linked by a disulphide bridge. This was confirmed by X-ray crystallography [10]. It includes the catalytic triad Asp 102, His 57 and Ser 195 and an insertion loop formed by Tyr 60A-Pro 60B-Pro 60C-Trp 60D, preventing access of inhibitors to the active site segment [16]. Due to the limitations and side-effects of existing antiplatelet drugs, there is a need for the search of new antiplatelet aggregation agents. Further research and development in quantum chemical electronic structure properties that could be related to platelet activation are therefore necessary to design the synthesis of novel antiplatelet lead compounds with improved activities. ©2016 Bentham Science Publishers

Imidazole-based Derivatives as the Potential Anti-platelet Inhibitors O

H

Letters in Drug Design & Discovery, 2016, Vol. 13, No. 9 O

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O O

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O

H N

O S

O

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F3C

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Cl NO2

1 O

H

O O

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F

F

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Fig. (1). The structures of the platelet aggregation inhibitors: 1:Diethyl 2-(4-nitrobenzylthio)-1H-imidazole-4,5-dicarboxylate, 2: Diethyl 2(3-chlorobenzylthio)-1H-imidazole-4,5-dicarboxylate, 3:Diethyl 2-(2,4-bis(trifluoromethyl)benzylthio)-1H-imidazole-4,5-dicarboxylate, 4: Diethyl 2-(perfluorobenzylthio)-1H-imidazole-4,5-dicarboxylate, 5: Diethyl-2-(sulfanyl)-1H-imidazole-4,5-dicarboxylate, 6: Diethyl 2-(3,4difluorobenzylthio)-1H-imidazole-4,5-dicarboxylate, 7: Diethyl 2-(4-(trifluoromethoxy)benzylthio)-1H-imidazole-4,5-dicarboxylate, 8: Diethyl 2-(4-(trifluoromethyl)benzylthio)-1H-imidazole-4,5-dicarboxylate, 9: Diethyl 2-(3-fluorobenzylthio)-1H-imidazole-4,5-dicarboxylate, 10: Diethyl 2-(4-fluorobenzylthio)-1H-imidazole-4,5-dicarboxylate.

In the current research, a quantum chemical investigation, in silico ADMET calculations and molecular docking of the imidazole derivatives as platelet aggregation inhibitors (Fig. 1) were performed to get a structural insight of the substituent effects of these compounds on their observed biological activities (IC50). In line with our ongoing interest in platelet aggregation inhibitors, a range of imidazole-based derivatives were synthesized and tested against thrombin for their ability to inhibit platelet aggregation [17]. From this study, it was found that the most active anti-platelet molecules in the thrombininduced assay were the 4''-nitro (1) and the 3''-chloro (2) derivatives [17]. In some cases in vivo/in vitro measurement of IC50 or EC50 were not made due to low bioavailability or other experimental/instrumental challenges. The novelty of

this work lies in proposing quantum chemical calculations of common activity-based descriptors to estimate the activities of those cases with unknown compounds. Herein, our aim is to assess the impact of the inclusion of different substituents on their electronic structure and in silico ADMET properties and investigate the binding affinities of these ten ligands against thrombin in an attempt to gain insight into their preferred mode of interaction. Hence, the main aim of this work is to calculate common activity-related physicochemical descriptors including ELUMO-HOMO band gap, chemical hardness () and softness (S), derived by natural bond orbital (NBO) analysis, and polarizability (). Furthermore, some 2D in silico drug-like descriptors related to their ADMET properties including

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Momin et al.

partition coefficient (ClogP) and molar refractivity (MR) were measured. To further assess their mode of action, molecular docking analysis against the thrombin receptor was also performed and in each case docked binding energies of the top ranked conformer and the active residues that were interacting with each ligand were explored. 2. MATERIALS AND METHODS 2.1. Quantum Chemical Calculations

Molar refractivity (MR) is a measure of the total polarizability () of a mole of a substance and is dependent on the temperature, the index of refraction, and the pressure. MR is defined as:

4 (4) N  3 A Where N A is Avogadro constant (NA 6.022 x 1023) and  is the mean polarizability of a molecule. According to Eq. 4, MR and the molecular polarizability should be strongly correlated, since they only differ by a constant. The molecular polarizability (), is often approximated as the sum of the Van der Waals coefficients for all atoms in the molecule [31]. MR =

Pe N rs ot on fo al rD U is se tri O bu n tio ly n

All the structures of the diethyl-2-(benzylthio)-2,3dihydro-1H-imidazole-4,5-dicarboxylate derivatives were optimized to local minima using density functional theory)DFT)[18] and 6-31+G* basis set with Gaussian 09 software package [19]. The frequency calculation was followed by optimization to determine if there was a true minimum on the potential energy surface. Natural bond orbital (NBO) analysis [20, 21] was performed to calculate the HOMO and LUMO frontier molecular orbitals. Assuming that biological activities of molecules are highly affected by their electronic structure properties [22], several commonly used activityrelated descriptors such as ELUMO-HOMO, chemical hardness (), softness (S), and polarizability () [22a, 22b, 23], which may responsible for the observed biological activity of the synthesized compounds were calculated.

as the octanol-water partition coefficient. This parameter is known as a suitable index to measure molecular hydrophobicity (or lipophilicity) for rational drug design. Hydrophobic compounds have higher logP values than hydrophilic chemicals [28a, 27, 29].

Parr and Pearson [24] introduced the chemical hardness as a central quantity for use in the study of reactivity and stability, through the hard and soft acids and bases principle [25] as:

IP – EA (1) 2 Where IE and EA are the first vertical ionization energy and electron affinity, respectively. The other activity-based parameters, are approximated in terms of the energies of HOMO and LUMO frontier molecular orbitals as [26]:

=

E LUMO – E HOMO 2 1 S= 2

=

(2)

(3)

2.2. 2D ADMET Properties According to Lipinski's rule of five, an orally active drug has (a) less than 5 hydrogen bond donors (OH and NH groups), (b) less than 10 hydrogen bond acceptors, (c) a molecular weight under 500 g mol-1 (d) a partition coefficient logP under 5 [27]. These features have a significant impact on drug absorption, bioavailability, hydrophobic drugreceptor interactions, metabolism of molecules, and toxicity. In this context, the 2D structural drug-like descriptors related to the absorption, distribution, metabolism, and excretion (ADME) properties [28] including partition coefficient (ClogP), molar refractivity (MR) and polarizability () [29] were calculated using ACD lab/ChemSketch software [30] to determine if our considered anti-platelet molecules possess drug-like properties. Lipophilicity is another drug-like property represented as the logarithm of partition coefficient, P (logP), also known

2.3. Molecular Docking

The concept of three-dimensional binding sites that interact with ligands is known as the core of structure-based drug design. Herein, molecular docking methods play a critical role in identifying the structures and binding pattern of the enzyme–inhibitor complex [32]. In this approach, the known or predicted shape of the binding site is used to optimize the ligand to characterize the steric and electrostatic complementarity between the receptor and the ligand [33]. The ligand– receptor interaction is quantum mechanical in nature and based on the understanding of molecular recognition, which is now replaced by the generally accepted “lock-and-key” or “induced fit” theory [34]. However, due to the complexity of biological systems, quantum theory is not affordable directly. Thus, most methods used in docking and other common computational approaches are more empirical in nature [35]. As for molecular docking, the considered anti-platelet ligands were first superimposed with the experimentally reported ligand inside the active site of the x-ray thrombinligand complex (1PPB.pdb) [10] using the Pymol program [36]. The ligand-thrombin complex was then minimized with shifted non-bonded interactions to provide a reasonable resolution of possible overlapping structures. Finally, the minima are ranked according to their approximate docked binding energies. Molecular docking of these aligned anti-platelet structures inside the active binding site of thrombin were then carried out using Autodock4 software [37, 38] to model the potential interaction between these synthesized anti-platelet compounds and the thrombin receptor. The RMSD values between docked conformers and experimental X-ray structure were calculated to validate the docking results. This program implements a mathematical algorithm, which explores the translations, rotations and internal degrees of freedom of the inhibitor inside the active site. The scoring function plays an important role to distinguish between good and bad docked conformations. The Lamarkian genetic algorithm (LGA) including a traditional GA for global search combined with a Solis and Wets local search procedure was used [37, 39].

Imidazole-based Derivatives as the Potential Anti-platelet Inhibitors

Table 1.

Letters in Drug Design & Discovery, 2016, Vol. 13, No. 9

891

The difference between HOMO and LUMO energy eigenvalues (ELUMO-HOMO), chemical hardness (), softness (S), and polarizability ( ) of the selected imidazole-based anti-platelet agents.

 ELUMO-HOMO) /(ev)

 (eV)

S (eV)

 (A3)

1

3.761

1.880

0.265

139.554

2

3.662

1.831

0.273

138.459

3

4.489

2.244

0.222

144.602

4

4.690

2.345

0.213

131.108

5

12.710

6.355

0.078

89.970

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Anti-platelet Compounds

6

14.081

7.041

0.071

129.267

7

6.344

3.172

0.157

138.247

8

4.572

2.286

0.218

136.612

9

12.196

6.098

0.081

155.583

10

4.685

2.342

0.213

127.330

3. RESULTS AND DISCUSSIONS

We first focus on interpreting the structural criteria including quantum chemical and 2D in silico ADMET properties.

This could be related to the observed biological activities of the synthesized antiplatelet agents. Secondly, the outcome of the molecular docking studies of these compounds inside the active binding site of thrombin will be discussed to address the mode of action for these antiplatelet inhibitors which could be described by their measured IC50 data. 3.1. Analysis of Quantum Chemical Properties

The HOMO and LUMO energy eigenvalues (EHOMO, ELUMO), chemical hardness (), softness (S), and polarizability (), and the selected anti-platelet agents are reported in Table 1.

HOMO (Highest Occupied Molecular Orbital) and LUMO (Lowest Unoccupied Molecular Orbital) energy eigenvalues and the difference between them (ELUMO-HOMO) are important quantities for measurement of the molecule’s stability [40]. HOMO energy eigenvalue (EHOMO) is directly related to ionization potential and the molecule’s potency for electrophilic attack. LUMO energy eigenvalue (ELUMO) is closely related to the electron affinity and shows the molecule’s tendency for nucleophilic attack. A large energy gap implies higher stability and lower chemical reactivity [41]. From the calculations made using DFT at the B3LYP/631+G* basis set level (Table 1), the lowest values for ELUMO-HOMO (3.761 eV, 3.662 eV), indicating potency in antiplatelet activity, correspond to the two most biologically active imidazole-based anti-platelet derivatives with low IC50 (0.40 mg mL-1, 0.44 mg mL-1).

Based on the HSAB principle [42], chemical reactivity and stability of a molecule is related to its global hardness () and global softness (S) calculated according to equation 2 and 3. Increase in hardness, increases movement of the system towards a more stable configuration and low reactivity.

Global softness has an inverse relationship with hardness. In other words, soft molecules undergo changes in electron density more easily than hard molecules and are more reactive than hard molecules [43]. In light of this, low chemical hardness ( =1.880 eV, 1.831 eV) and high chemical values of softness (S = 0.265 eV, 0.273 eV) were assigned to the most active cases. Polarizability is the relative tendency of charge distribution, such as the distortion of an electron cloud of an atom or molecule from its normal shape by an external electric field. This may be caused by the presence of a nearby ion or dipole. Interestingly, the two most active compounds were found to be highly polarized ( =139.554 Å3, 138.459 Å3), which implies that molecular polarizability is an effective physicochemical property in the observed activities of this series of anti-platelet derivatives. The obtained electrostatic potential energy maps characterized the charge distribution over the molecule. The electron rich region was mapped with an isodensity surface of 0.002 a.u. This electrostatic potential surface includes the van der Waals volumes of the individual atoms in the molecule [44]. The electrostatic potential map for the two most active anti-platelet derivatives were obtained using the Molekel 5.4 program [45] (Fig. 2). A comparison of electrostatic potential charge range of the two active anti-platelet derivatives, namely 1: Diethyl 2(4-nitrobenzylthio)-1H-imidazole-4,5-dicarboxylate (electrostatic potential charge range: -4.478 up to 3.075) and 2: Diethyl 2-(3-chlorobenzylthio)-1H-imidazole-4,5-dicarboxylate (electrostatic potential charge range: -4.065 up to 2.738) revealed that these two active compounds have a larger range of electrostatic charge density than other less active compounds such as 5: Diethyl-2-(sulfanyl)-1H-imidazole-4,5dicarboxylate (electrostatic potential charge range: -1.708 to 3.952). The frontier molecular orbitals (HOMO and LUMO) for compounds 1 and 10 obtained by the B3LYP/6-311++G** method are shown in Fig. 3.

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Pe N rs ot on fo al rD U is se tri O bu n tio ly n

(a)

(b)

(c)

Fig. (2). The electrostatic potential map for (a) Diethyl-2-(sulfanyl)-1H-imidazole-4,5-dicarboxylate (electrostatic potential charge range: 1.708 to 3.952) (b) Diethyl 2-(4-nitrobenzylthio)-1H-imidazole-4,5-dicarboxylate (electrostatic potential charge range: -4.478 up to 3.075) and (c) Diethyl 2-(3-chlorobenzylthio)-1H-imidazole-4,5-dicarboxylate (electrostatic potential charge range: -4.065 up to 2.738 obtained by B3LYP/6-311++G** method. Molecular visualization was performed using Molekel 5.4 [55]. (red = electron rich and blue = electron deficient) (The electrostatic potential maps of other stilbene derivatives are provided in supplementary materials).

(a)

(c)

(b)

(d)

Fig. (3). The frontier molecular orbitals (HOMO and LUMO) for (a) HOMO for compound 10 (b) LUMO for 10 and (c) HOMO for compound 1 (d) LUMO for compound 1 obtained by B3LYP/6-311++G** method. (The frontier molecular orbitals of other considered stilbene derivatives are provided in supplementary materials).

From Fig. 3, it can be seen that the HOMO orbitals are localized on the atoms of the 4-nitrobenzylthio and 4-fluorobenzylthio substituents. As HOMO frontier orbitals

is the origin of electron flow (electron flow occurs from HOMO to LUMO), it seems that NO2 and F functional groups could play an essential role in the observed biological

Imidazole-based Derivatives as the Potential Anti-platelet Inhibitors

Table 2.

Letters in Drug Design & Discovery, 2016, Vol. 13, No. 9

893

The calculated 2D drug-like descriptors of the selected anti-platelet agents measured by ACD lab/ ChemSketch package [38]. ClogP

Molar Refractivity (MR) (cm3)

Polarizability () (10-24) (cm3)

1

3.41

93.34

37.00

2

0.71

92.14

36.52

3

4.54

97.29

38.57

4

3.47

87.87

34.83

5

0.71

59.25

23.49

6

3.70

87.54

34.70

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Anti-platelet Compounds

7

3.78

94.04

37.28

8

3.77

92.30

36.59

9

3.73

87.42

34.65

10

3.73

87.42

34.65

activities of these compounds. This observation provides a rationale for the medicinal chemists to synthesize NO2 and F containing imidazole-based derivatives as they present a reasonable model for anti-platelet agents.

structural changes. This observation is consistent with what we discussed in Table 1.

3.2. Analysis of 2D ADMET Properties

Human -thrombin has two polypeptide chains of 36 (Achain) and 259 residues (B-chain) covalently linked via a disulfide bridge.

The calculated 2D drug-like descriptors of the selected anti-platelet agents that could be related to their ADMET properties are listed in Table 2.

Molecular weight of the compound is very crucial in drug action. If the molecular weight is increased beyond a limit (above 500 g mol-1), the bulkiness of the compounds also increases the possibility of the drug action. The molecular weight of the selected compounds is in the range of 244 to 470 g mol-1, which follow Lipinski’s rule of 5. Therefore, the bulkiness of the compounds is at an optimum limit for biological action.

A close study of our molecules shows that they fulfill nearly all requirements of an oral drug. Interestingly, all the selected anti-platelet derivatives show ClogP values in the range of 0.71 to 4.54. This observation indicates that all derivatives follow the Lipinski’s rule of five [27], which states that the ClogP value should be under 5. Amongst the considered derivatives, compound 3 with a CF3 group has the highest ClogP value (ClogP = 4.54), indicating more hydrophobic character than the other compounds. In contrast, compound 2 with a Cl substituent has a ClogP value of 0.71, and is the most hydrophilic of these compounds. The polarizability is closely correlated to molar refractivity (MR) and is an important physicochemical quantity that can provide structural information on the bonding and geometrical features of lead compounds. Our studies show that the highest molar refractivity values (MR=92.14 cm3-97.29 cm3) and polarizability ( =36.52 Å3-38.57 Å3) were found for the most active compounds. Hence, it seems that these compounds have more tendency to be polarized and reasonable potency to undergo

3.3. Molecular Docking Analysis

The active residues in the binding site of the crystal structure of the thrombin-ligand complex (1PPB.pdb) [10] is shown in Table 3. The mode of interaction between all docked anti-platelet structures and the thrombin receptor were created with Ligplot software [46] (Fig. 5). This model was considered as a clue in obtaining a clear picture of the relative arrangement of binding interaction sites, the hydrogen bond interactions (by dashed lines) and hydrophobic contacts, which are represented by an arc with spokes radiating toward the ligand atoms they contact and are colored in green. The docked binding energy values and the active residues near the gate of the active site of thrombin characterized by Autodock4 software [37, 38] are given in Table 3. The RMSD values between docked conformers and experimental X-ray structures were also reported in Table 3 to validate the results. Docked results were visually inspected with Ligplot software to ensure the pattern of an acceptable drug/enzyme interaction. According to the available crystal structure of the thrombin-ligand complex (1PPB.pdb) [10], the residues directly interacting with the ligand comprise Gly219, Glu192, Gly216, Trp60, Trp215, Leu99, His57 and Tyr60A. According to Table 3, the most biologically active antiplatelet agent (compound 1, IC50 = 0.40 mg mL-1) showed the most negative docked binding energy (-31.21 kJ mol-1 ) as well as most negative intra-molecular energy (-41.71 kJ mol-1). Furthermore, Fig. 4 and Table 3 revealed that the active residues near the gate of the active site of thrombin in this docked complex (compound 1), namely, Gly219, Glu192,

894 Letters in Drug Design & Discovery, 2016, Vol. 13, No. 9

Table 3.

Momin et al.

Docking parameters for the selected synthesized imidazole-based anti-platelet agents against thrombin receptor obtained by Autodock4 [46, 47]. The active residues near the gate of the active site of the crystal structure of thrombin-ligand complex (1PPB.pdb) [12] are shown in bold.

Anti-platelet Compounds

IntraMolecular Energy (kJ mol-1)

Docked Binding Energy (kJ mol-1)

1

-41.71

2

-37.94

(Å)

Measured IC50 in Thrombin (mg mL-1)

Active Residues Near the Gate of the Active Site of Thrombin

-31.21

0.780

0.40

Gly219,Glu192,Gly216,Trp60,Tyr60A,Trp215,Leu99, His57,Cys191,Lys60,Glu217

-30.45

0.784

0.44

Trp60,Tyr60A,Trp215,His57,Glu192, Glu217,Ser197,Ser214,Asp189,Cys191,Val213

Pe N rs ot on fo al rD U is se tri O bu n tio ly n

RMSD

3

-37.23

-28.95

0.788

2.06

Glu192, Gly219, His57, Gly216, Trp215, Leu99, Ser214, Ile174, Cys191, Ala190

4

-37.11

-28.86

0.790

2.24

Trp215, Glu192, His57, Leu99, Tyr60, Trp60, Asn98, Gly193, Ser195, Cys191, Glu97

5

-36.73

-28.74

0.876

2.73

Trp215, Glu192,Gly219, Glu217, Ser195, Ser214, Gly216, Cys191, Ala190, Gly226, Asp189, Cys220

6

-25.27

-26.02

0.785

2.96

Trp215, Tyr60, Gly216, Trp96, Ile174, Glu217, Arg97, Asn98

7

-39.12

-25.39

0.787

Not detected

Gly216, Leu99, His57, Trp215, Glu217, Trp148, Glu191, Cys191, Gly226, Ser195, Asp189, Val213, Ser214, Ala190, Ile174

8

-36.40

-28.53

0.783

Not detected

Trp215, Leu99, Glu192, His57, Gly216, Gly219, Ser214, Ser195, Val213, Asp189, Gly226, Ala190, Cys191, Asn98, Ile174

9

-40.04

-26.31

0.826

Not detected

Leu99, Trp215, Trp60, Gly216, Tyr60, His57, Cys191, Glu192, Val213, Try228, Asp189, Gly226, Ala190, Phe227, Ser214,

10

-39.37

-28.15

0.775

Not detected

Trp60, Leu99, Trp215, Gly216, Ser195, Ser214, Val213, Cys191, Asp189, Ala190, Gly219, Glu217

Glu97

Trp148

Leu99

C16

C15

C9

Glu192

C8

C7

Tyr60

O6

Glu217

C6

C5

O5 C14

N

Gly216

Trp215

C3

N

Gly219

C

C13

C1

3.24

N1

O4

N

N2

C2

CA C2

OG

C14

O1

Trp60

CA

CB

O

C3

Gly216

ND1 CA

C13

C

S

N

CA

N2

CE1

CG

C

C10

CD2

O2

O

CB

O C

C21

C

C12

NE2 C11

His57

C5 CA

N6

3.15

N O

2.83

O3 C15

C19

C4

Trp215

O

2.87 N3

C16

C2 0

C17

Ala190

C10

Leu99 CA

OD2

C9

Trp60

C8 N4 OG

C18

Gly219

CG CB

2.83

CA

N3 O2

OD1

NZ N

CA

C

Ser195

C7

N

CB O

C6

C

2.68 N5

Cys191

N1

Ser214

C1

3.11

CB

C12

C11

C4

O

O3

Glu192

O1

2.69

N

His57

CE

Gly226 C

Asp189 O

CG

C

O

CD

Tyr60 CA CB

Cys220

Cys191

(a)

N

Lys60

(b)

Fig. (4). Schematic diagrams of thrombin-ligand interactions in (a) Crystal structure of thrombin-ligand complex (1PPB.pdb) [12] and (b) Structure of docked ligand 1-thrombin complex created by Ligplot software [56] (The plots for other considered complexes with their corresponding 3D structures are provided in the Supplementary material.).

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Gly216, Trp60, Tyr60A, Trp215, Leu99 and His57, are more consistent with the crystal structure of the thrombin-ligand complex (1PPB.pdb). This high binding affinity of compound 1 (IC50 = 0.40 mg mL-1), could be partly due to the hydrogen bonding formation between Gly219 as the active residue of thrombin with the carboxyl group of the ligand (Fig. 4). Compound 2 (IC50 = 0.44 mg mL-1) showed a similar docked binding energy (-30.45 kJ mol-1) as well as the most negative intra-molecular energy (-37.94 kJ mol-1). The RMSD values between each docked conformer and experimental X-ray structure were reasonable (between 0.7-0.8 Å). The least negative docked binding energy (-26.02 kJ mol-1 ) and intra-molecular energy (-25.27 kJ mol-1) was obtained for compound 6 with a higher IC50 of 2.96 mg mL-1. The experimental IC50 in thrombin could not be detected in compounds 7-10, however based on the reported data in Table 3 it is expected that among these compounds with unknown biological activities, the CF3 and F containing imidazolebased derivatives for both compounds 7 and 8 (in the para position) could provide better binding affinities of these compounds against the thrombin receptor. However, in addition to the substituent effect, other factors that can cause higher binding affinity are under investigation and the results will be reported elsewhere.

tives, with molecular dynamic simulations of various imidazole-based anti-platelet derivatives will be carried out in an attempt to understand the dynamic behavior of these inhibitors and their free binding energies against the thrombin receptor. The probable mechanism behind the anti-platelet and anti-thrombotic activity is also worth pursuing. Our continuously improved understanding of various physicochemical, biophysical, and pharmacological effects of anti-platelet compounds offers interesting new opportunities in the drug discovery process for these specific leads.

Taken together, a reasonable correlation could be observed between the experimental IC50 in thrombin and the docked binding and intra-molecular energies of the titled anti-platelet inhibitors. Interestingly, the most biologically active imidazole-based anti-platelet derivatives with low IC50 and reasonably good docked binding energy showed high polarizabilities (=139.554 Å3, 138.459 Å3), low LUMOHOMO (3.761 eV, 3.662 eV), low chemical hardness (=1.880 eV, 1.831) and high chemical values of softness (S= 0.265 eV, 0.273 eV).

Supplementary material is available on the publishers Web site along with the published article.

CONFLICT OF INTEREST

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The authors declare that this article content has no conflict of interest. ACKNOWLEDGEMENTS This work was supported by a grant from the National Research Foundation (NRF), South Africa. We are also grateful to the helpful support of CHPC (www.chpc.ac.za) for providing computational resources. SUPPLEMENTARY MATERIAL

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Investigation of the molecular physico-chemical properties of drug-like compounds is a suitable approach to relate the observed quantities and the chemical behavior of molecules to their calculated electronic structure and experimentally measured biological activity (IC50). In silico ADMET calculations confirmed that all the selected candidates meet Lipinski’s rule and can be regarded as drug-like compounds. Molecular docking identified the possible binding models of these imidazole-based anti-platelet derivatives within the active site of thrombin. A combination of quantum chemical descriptors and in silico ADMET could provide structural information related to their activities. Molecular docking revealed the hydrogen bonding and electrostatic interactions of the imidazole anti-platelet derivatives with active residues of the thrombin receptor. The results of the present study demonstrate that imidazole derivatives exert an anti-platelet aggregation effect. It could also be inferred that that these types of compounds could be accommodated at the binding surface of the complex, indicating that these anti-platelet agents could bind to the thrombin receptor freely. This is consistent with the finding that anti-thrombin compounds could inhibit platelet aggregation in vitro and in vivo. The continuation of this work with activity-related descriptors for a variety of imidazole-based anti-platelet deriva-

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