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[email protected] Letters in Drug Design & Discovery, 2015, 12, 000-000
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Biological Evaluation of Halogenated Thioureas as Cholinesterases Inhibitors Against Alzheimer’s Disease & Molecular Modeling Studies Jamshed Iqbala,*, Sumera Zaiba, Aamer Saeedb and Muhammad Muddassarc a
Centre for Advanced Drug Research, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan
b
Department of Chemistry, Quaid-I-Azam University, Islamabad, Pakistan
c
Department of Biosciences, COMSATS Institute of Information Technology, Islamabad, Pakistan Abstract: Acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition is thought to be an encouraging approach towards the therapy of Alzheimer’s disease (AD). The current paper targets to give a concise information of mono and dihalo- substituted thioureas similarity with anti-AD potential. The present results represent evaluation of cholinesterase inhibitory potential for halogenated thioureas derivatives. Compound 1t was constituted to be highly potent inhibitor with Ki value 0.12 ± 0.05 M against AChE, while 1b was most the active inhibitor for BChE with Ki value of 0.03 ± 0.001 M. Molecular docking simulations were performed using the homology models of both cholinesterases in order to explore the plausible binding modes of synthesized compounds.
Keywords: Acetylcholinesterase, Alzheimer’s disease, Butyrylcholinesterase, Docking simulations, Halogenated thioureas, Homology models. INTRODUCTION Neurological disorders like Alzheimer’s disease and dementia are in correspondence with clear indication of early cerebral loss that may subsequently lead to acute dementia. Although Alzheimer’s disease suggests a complicated pathology that is still under acute consideration, meaningful verification points in order to achieve the exhaustion of presynaptic cholinergic markers in many phases of the disease [1-5]. Acetylcholine (ACh) is the neurochemical tangled in cholinergic neurotransmission, liberated by presynaptic cholinergic terminus and prompting nicotinic and muscarinic receptors to transform postsynaptic cell activities. Generally, acetylcholinesterase (AChE, EC 3.1.1.7) degrades ACh, thereby terminating its signaling action [6, 7]. Most of the cholinesterase in the human brain is AChE. Nevertheless, at present it is noted that butyrylcholinesterase (BChE, EC 3.1.1.8) has additional widespread dispersal as it was a long ago idea. BChE activity has been positioned in all the areas of normal brain in which regions that acquire cholinergic perceptions [8]. AChE is particular for hydrolysis of ACh, at the same time BChE has the opportunity to metabolize different molecules comprising diverse neuroactive peptides [9]. Alzheimer’s disease (AD) is specified by a failure of cholinergic neurons and their cortical extensions from the nucleus basalis and related areas of basal forebrain. Cholinergic synaptic action seems to be outstandingly responsive to beta-
*Address correspondence to this author at the Centre for Advanced Drug Research, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan; Tel: +92 992 383591 96; Fax: +92 992 383441. E-mail:
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amyloid (A) peptide toxicity, and decline of synaptic cyst on axon terminals can anticipate cholinergic neuronal loss [10]. Enhancement of neurochemical level inside the synaptic cleft due to the reversible inhibition of acetylcholinesterase, definitely influences AD patients and related conditions such as Lewy Body dementia, subcortical vascular dementia and Parkinson's disease [11]. ChEIs have seen to abolish the consequences originated by addictive drugs and can also be given as co-drugs for therapy of addicts [12]. Furthermore, ChEIs development is important in the analysis of the mechanism of cholinergic transmission disorders [13, 14]. Across the few decades, several anti-AChE factors like tacrine [15], donepezil [16], rivastigmine [17] and a short time ago the galantamine [18] have been investigated in the market (Fig. 1). Although, these nitrogen containing anti-AChE drugs have decreased CNS permeability and various adverse effects. Therefore, search for new drugs is mandatory for the therapy of AD with enhanced CNS perforation and fewer adverse consequences. Thiourea are organosulphur compounds [19, 20]. 1-(2aminoethoxy)-3-ar(o)yl(thio)urea’s also have inhibitory activity against acetylcholinesterase [21]. Thiourea’s and its derivatives have other properties like anticancer [22], antimicrobial especially anti-malarial [23], antibacterial [24] and anti-tuberculosis [25]. 7-Methoxytacrine-Adamantylamine thioureas have been proved as inhibitors of human acetyl and butyrylcholinesterase [26]. 1,3-Dialkyl or diarylthioureas revealed noteworthy antifungal activity resistant to plant pathogens Pyriculariaoryzae and Drechsleraoryzae [27]. 1Benzoyl-3-(4,6-disubstituted-pyrimidinyl)thioureas have exhibited potent herbicidal activity [28].
©2015 Bentham Science Publishers
2 Letters in Drug Design & Discovery, 2015, Vol. 12, No. 6
CH3 H3C
N
Iqbal et al.
NH2
CH3 O
H3C
N
CH3 N
O
Tacrine
Neostigmine
O O N
O
N
N
O O
N
O
O
Ensaculine
Ravistigmine
H2N
NH2
N O H3CO
N N
H3CO Donepezil
Propidium
Fig. (1). Chemical structures of some routinely FDA approved cholinesterase inhibitors for the treatment of Alzheimer’s disease.
Isosteric replacement of hydrogen by fluorine to make fluorinated compound has become important approach in drug development and usually blend in the biological recognition site [29]. The lipophilicity of organic unit may enhance due to addition of fluorine and hence the rate at which cell penetrate and drug transport to an active site elevates and more polarizability because of the C–F bond may enhances advance opportunities for receptor binding. Also compounds having fluorine are highly unaffected by metabolic degeneration because of the elevated level of bond energies and heats of formation of bonds between H–O and C– O relative to those of the F–O bond [29-31]. Compounds having fluorinated substitution are highly significant in applications like medicine, agrochemicals and organic electronics. Substitution of fluorine in a drug fragments may impact not only pharmacokinetic properties such as tissue distribution, absorption, rate of secretion and biotransformation at a certain position but also its toxicology, pharmacodynamics and promotes the capability of medicine [32]. Bearing the aforesaid synthetic and biological importance of fluorinated compounds and thioureas in mind, we investigated SAR of fluorinated thioureas and their inhibitory activity against acetyl and butyrylcholinesterases. This can help to behave well towards neurological disorders. MATERIAL AND METHOD A general procedure for the synthesis of 1(fluorobenzoyl)-3-(fluorophenyl)thioureas derivatives (1a1t) has been reported in our recent published paper [33].
DETERMINATION OF AChE AND BChE INHIBITORY ACTIVITIES For the determination of cholinesterase inhibition, electric eel and horse serum were used as source of AChE and BChE, respectively. AChE and BChE inhibition was measured in vitro by the Ellman’s spectrophotometric method with slight modification [34]. Reaction started by mixing 20 μL assay buffer, 10 μL of test compound and 10 μL of enzymes (0.5 and 3.4 U/mg of AChE or BChE, respectively). Then reaction mixture was incubated for 10 min at 25 °C. At the end of the pre-incubation period, 10 L of 1 mM acetylthiocholine iodide or butyrylthiocholine chloride were added to the respective AChE or BChE enzyme solution and 50 L of 0.5 μM, 5,5’-Dithiobis-2-Nitrobenzoic Acid (DTNB) was added as coloring reagent. The mixtures were incubated for 15 min at 25 °C. The formation of enzymatic product was determined by the variation in absorbance measured at 405 nm with microplate reader (Bio-Tek ELx800TM, Instruments Inc., Winooski, VT, USA). In this bioactivity assay, the standard drugs, neostigmine and donepezil were used. The buffer for enzyme dilution comprised of 50 mM Tris-HCl containing 0.1% (w/v) BSA (pH 8). To remove the effect the DMSO on enzymes an blank assay was performed without any enzyme and accounted as non-enzymatic reaction. The analysis of each concentration was done in triplicate and the Ki values were calculated with the linear regression parameters. The computer program used for this purpose is GraphPad Prism 5.0 (San Diego, CA, USA).
Halogenated Thioureas as Cholinesterases Inhibitors
Letters in Drug Design & Discovery, 2015, Vol. 12, No. 6
HO
O
i) SOCl2
O
R'
ii) N
3
S-K+ / dry acetone
S
C
Dry acetone
N
R'
H2N R
HN R
HN S O R'
(1a-t)
a: R'=2-Cl R=2-F, c: R'=2-F R=2-F, e: R'=4-F R=2-F, g: R'=3,4,5-tri-OCH3 R=3-F, i: R'=3-F R=3-F, k: R'=2-Cl R=4-F, m:R'=2-F R=4-F, o: R'=4-F R=4-F, q: R'=3,4,5-tri-OCH3 R=3-Cl, 4-F, s: R'=4-F R=3-Cl, 4-F,
b: R'=3,4,5-triOCH3 R=2-F, d: R'=3-F R=2-F, f: R'=2-Cl R=3-F, h: R'=2-F R=3-F, j: R'=4-F R=3-F, l: R'=3,4,5-tri-OCH3 R=4-F, n: R'=3-F R=4-F, p: R'=2-Cl R=3-Cl, 4-F, r: R'=2-F R=3-Cl, 4-F, t: R'=t-butyl R=3-Cl, 4-F,
Scheme (1). Synthesis of some new 1-(fluorobenzoyl)-3-(fluorophenyl)thioureas.
MOLECULAR MODELING Molecular Structures Preparation The molecular construction and modeling of the synthesized compounds were done using the “Build” tool available in Maestro 2011 suit (Schrödinger Inc.). Then these structures were converted from 2D to 3D geometry using the LigPrep [35] module implemented in molecular modeling Schrodinger package. This program generates energy minimized molecular structures with their tautomers, ionization states, ring conformations, and stereoisomers to produce broad chemical and structural diversity from a single input structure. Therefore we have used all its utilities to prepare our data set for molecular docking simulations. Protein Structure Preparation and Docking Simulations The homology models of acetylcholinesterase (AChE) and butylcholinesterase (BChE) [36], were used for molecular docking simulations of synthesized compounds. Hydrogen atoms were added to model structures and OPLS_2005 force field-based charges were assigned to them. Brief energy minimizations were also performed to relieve steric clashes among the atoms using the protein preparation wizard option in Maestro. These restrained partial minimizations terminated when the root-mean-square deviations (rmsd) reached at a maximum value of 0.3 Å from the native structures of each model. For molecular docking simulations, Glide [37] module implemented in Schrodinger suit 2011 (Schrodinger, Inc.) was used. The amino acid residues located within 15Å from the catalytic triad were defined as the possible binding site for the docking simulations. In the grid, enclosing box (X, Y and Z range, 27.766, 27.766, 27.766) and a scaling factor of 1.00 were set to van der Waals
(VDW) radii of receptor atoms with the partial atomic charge
