Please download to get full document.

View again

of 11
All materials on our website are shared by users. If you have any questions about copyright issues, please report us to resolve them. We are always happy to assist you.
Similar Documents
Information Report

Instruction manuals


Views: 0 | Pages: 11

Extension: PDF | Download: 0

  Original article Synthesis, cytotoxicity and molecular modelling studies of newphenylcinnamide derivatives as potent inhibitors of cholinesterases Aamer Saeed a , Parvez Ali Mahesar a , Sumera Zaib b , Muhammad Siraj Khan b ,Abdul Matin c , Mohammad Shahid d , Jamshed Iqbal b , e , * a Department of Chemistry, Quaid-I-Azam University, Islamabad 45320, Pakistan b Centre for Advanced Drug Research, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan c Institute of Biomedical and Genetic Engineering, PO Box 2891, Sector G-9/1, Islamabad, Pakistan d Department of Bioinformatics, Fraunhofer Institute SCAI, Sankt Augustin, Germany e Department of Pharmaceutical Sciences, COMSATS Institute of Information Technology, Abbottabad 22060, Pakistan a r t i c l e i n f o  Article history: Received 12 October 2013Received in revised form15 February 2014Accepted 6 March 2014Available online 7 March 2014 Keywords: Phenylcinnamide derivativesAlzheimer ’ s diseaseCholinesterases inhibitorsCytotoxicityMolecular docking a b s t r a c t The present study reports the synthesis of cinnamide derivatives and their biological activity as in-hibitors of both cholinesterases and anticancer agents. Controlled inhibition of brain acetylcholinesterase(AChE) and butyrylcholinesterase (BChE) may slow neurodegeneration in Alzheimer ’ s diseases (AD). Theanticholinesterase activity of phenylcinnamide derivatives was determined against  Electric Eel  acetyl-cholinesterase (EeAChE) and horse serum butyrylcholinesterase (hBChE) and some of the compoundsappeared as moderately potent inhibitors of EeAChE and hBChE. The compound 3-(2-(Benzyloxy)phenyl)-N-(3,4,5-trimethoxyphenyl)acrylamide ( 3i ) showed maximum activity against EeAChE with anIC 50  0.29    0.21  m M whereas 3-(2-chloro-6-nitrophenyl)-N-(3,4,5-trimethoxyphenyl)acrylamide ( 3k  )was proved to be the most potent inhibitor of hBChE having IC 50 1.18  1.31  m M. To better understand theenzyme e inhibitor interaction of the most active compounds toward cholinesterases, molecularmodelling studies were carried out on high-resolution crystallographic structures. The anticancer effectsof synthesized compounds were also evaluated against cancer cell line (lung carcinoma). The compoundsmay be useful leads for the design of a new class of anticancer drugs for the treatment of cancer andcholinesterase inhibitors for Alzheimer ’ s disease (AD).   2014 Elsevier Masson SAS. All rights reserved. 1. Introduction Acetylcholine (ACh) is a cholinergic neurotransmitter, releasedpresynaptically by cholinergic terminals, and it interacts witheither nicotinic or muscarinic receptors thereby affecting functionof postsynaptic cells. Signalling action of ACh is terminated by theaction of acetylcholinesterase (AChE) and butyrylcholinesterase(BChE) [1,2]. Both enzymes are widely distributed throughout thebody; however, AChE remains the major cholinesterase within thehuman brain. All the brain parts that are innervated by cholinergictransmission in a normal brain hold AChE and BChE activity [3].Alzheimer ’ s diseaseisalonglastingneurodegenerativedisorderthat brings about irreversible memory loss in elderly individuals.Furthermore, this disease has not only been reported of havingde fi ned accumulation of amyloid- b  peptide plaques at extracellularlevel but also characterized with the intracellular neuro fi brillarytangles (NFTs) in the brains of suffering patients. With the pro-gression of disease, the prominent indications are the continuousmemory loss, confusion, petulance, anger and the lack of vigour inbody to function evenly which eventually become the ultimatecause of death [4 e 6]. It is estimated that 24.3 million people weresuspected to have Alzheimer ’ s disease (AD) in 2005; also the pro- jected number of individuals with AD at world level in 2020 and in2040 would be 42.3 and 81.1 million, respectively [7]. In this situ-ation,globalescalatingoccurrenceofADhascompelledresearchersto undertake studies on neurodegenerative disorders (NDDS) [8].Cholinesterases belong to a familyof serinehydrolases that splitacetylcholine into choline and acetic acid, an unavoidable step inretaining the function of cholinergic neuron [9]. AChE has  fi veimportant regions within active site and these are important tounderstand substrate and inhibitor binding pattern. These regionsare, 1) catalytic triad residues, 2) acyl pocket, 3) oxyanion hole, 4)anionic site(AS), 5) and a peripheral anionic site(PAS) [10 e 15]. The *  Corresponding author. Centre for Advanced Drug Research, COMSATS Instituteof Information Technology, Abbottabad 22060, Pakistan. E-mail addresses:  drjamshed@ciit.net.pk, jamshediqb@gmail.com (J. Iqbal). Contents lists available at ScienceDirect European Journal of Medicinal Chemistry journal homepage: http://www.elsevier.com/locate/ejmech http://dx.doi.org/10.1016/j.ejmech.2014.03.0150223-5234/   2014 Elsevier Masson SAS. All rights reserved. European Journal of Medicinal Chemistry 78 (2014) 43 e 53  AChE binds to PAS resulting in the formation of amyloid- b  peptideplaques and acetylcholinesterase inhibitors (AChEIs) bind to thePAS to prevent amyloid- b  peptide plaques formation [16,17].However, such treatment (using AChEIs) only relieves the symp-toms for short term and do not eradicate the disease eternally [18].The major role of acetylcholinesterase (AChE) is to catalyze thehydrolysis of acetylcholine (ACh) in cholinergic synapses andreplacement of AChE function in Alzheimer ’ s brains [19]. Some of the important cholinesterase inhibitors which are employed in thetreatment of AD are rivastigmine, galantamine, tacrine, ensaculinand donepezil. Among these remedies the rivastigmine is broaderin its action as it non-speci fi cally inhibits AChE as well as BChE,whereas galantamine and donepezil speci fi cally inhibit AChE thusincreasing ACh level at neuronal level and relieving AD symptomsbut they all cannot stop progress of dementia [20]. Various com-pounds like carbamates (e.g., neostigmine and physostigmine etc),organophosphates, coumarine and cinnamide derivatives are re-portedin literature asinhibitors ofcholinesterases[18,21,22].Apartfrom anticholinesterase and cytotoxic activity cinnamide de-rivatives (N-(carboxyaryl)-phenylcinnamide) have shown theiractivity against leukotriene B 4  (LTB 4 ), which is a metabolic productof arachidonic acid and a potent in fl ammatory mediator [23,24].LTB 4  has important role in a range of diseases, like psoriasis, adultrespiratory distress syndrome, in fl ammatory bowel disease andrheumatoid arthritis, it makes cinnamide derivatives as potenttherapeutic agents against in fl ammatory disorders [25 e 29].The aim of the present study was to synthesize and investigatephenylcinnamide derivatives as anticholinesterase as well as anti-cancer agents. 2. Results and discussion  2.1. Chemistry We envisioned that Doebner e Knoevenagel condensation wasthe best availableroute tosynthesize cinnamic acidsderivatives  (1)(a e  y)  [30,31]. It was subsequent one pot activation and couplingwhich led to formation of corresponding amides with 2,4,6-trichloro-1,3,5-triazine [32] that ultimately afforded the desiredproducts phenylcinnamide  (3a e  y)  in a good to excellent yield.Gratifyingly, we came to know that cyanuric chloride (TCT) wascost-effective and stable reagent for amide formation, moreimportantly it was easily handled and transformation took place atambient temperature. All of the synthesized compounds werecharacterized with  1 H and  13 C NMR along with IR (Scheme 1).  2.2. In vitro inhibition studies of EeAChE and huBChE In vitro  inhibitory studies of synthesized phenylcinnamide de-rivativeswerecarriedoutonEeAChEandhBChE.Inhibitionpotencyof compounds expressed as IC 50  values is shown in Table 1. Amongthe phenylcinnamide derivatives,  3i  showed maximum inhibitoryactivity against EeAChE because of its electron donating benzyloxy( e OCH 2 Ph) group at  ortho  position, although the same groupattached to  meta  and  para  position showed little activity. However,when e OCH 2 CHCH 2  group was attached to the same  ortho  positionthenadecreaseintheactivityofthecompound( 3m )wasobserved, 3a : R = 3-OCH 2 Ph 3n : R = 3,4-dihydroxy 3b : R = 3-OCH 3 , 4-OOCCH 3 3o : R = 3-NO 2 3c : R = 4-NHOOCCH 3 3p : R = 4-Cl 3d : R = 3-OCH 3 , 4-OCH 2 Ph 3q : R = 3-OCH 3 3e : R = 3-OCH 2 Ph, 4-OCH 3 3r : R = 2-OH, 3-OCH 3 3f : R = 4-OCH 2 CHCH 2 3s : R = 3-OH, 4-OCH 3 3g : R = 3-OOCCH 3 3t : R = 2,5-dihydroxy 3h : R = 2-OH, 4-OCH 2 Ph 3u : R = 2,3-dihydroxy 3i : R = 2-OCH 2 Ph 3v : R = 2,4-dihydroxy 3j : R = 4-OOCCH 3 3w : R = 2-F,6-Cl 3k : R = 2-Cl, 6-NO 2 3x : R =4-OCH 2 Ph 3l : R = 2-OH, 6-NO 2 3y : R = 3-NO 2 ,4-OCH 3 3m : R = 2-OCH 2 CHCH 2 Scheme 1.  Synthesis of substituted phenylcinnamide derivatives.  Table 1 Inhibitory potential of phenylcinnamide derivatives against EeAChE and hBChE.Compounds EeAChE hBChEIC 50   a SEM ( m M)/(%Inhibition) 3a  6.52  0.39 3.93  1.44 3b  4.98  0.27 (31.1) 3c  12.54  0.65 3.85  0.91 3d  12.61  1.48 (32.8) 3e  6.02  2.95 (31.1) 3f   (33.6) (32.6) 3g   (29.2) 1.25  0.08 3h  1.59  1.45 (27.2) 3i  0.29  0.21 (32.1) 3j  (36.5) (37.4) 3k   (34.2) 1.18  1.31 3l  12.77  1.06 (35.4) 3m  1.41  0.77 (24.8) 3n  (36.4) (34.6) 3o  3.24  2.16 (26.2) 3p  (31.2) (25.7) 3q  12.91  0.98 (41.8) 3r   6.22  0.33 5.19  0.78 3s  4.22  0.03 7.65  0.51 3t  (30.3) (36.2) 3u  (31.2) (41.2) 3v   (36.6) (18.21) 3w   (34.1) 1.32  0.15 3x   3.72  0.13 (33.5) 3y   6.69  4.0 (28.1) Neostigmine  22.0  3.01 49.1  6.0 Donepezil  0.03  0.003 6.37  0.32 a SEM shows standard error of mean of three experiments.  A. Saeed et al. / European Journal of Medicinal Chemistry 78 (2014) 43 e 53 44  same was the case when  e OCH 2 CHCH 2  group was attached to thesame  para  position ( 3f  ). In addition, the value of IC 50  furtherdecreasedwhena e NO 2 groupwasattachedat the meta positiontothe benzene ring ( 3o ). Introduction of two hydroxy substituents tobenzene ring further decreased inhibition against the EeAChE for 3n, 3t, 3u  and  3v  . As for as the action of the phenylcinnamide de-rivatives against the hBChE was concerned,  3k   having  e Cl and  e NO 2  groups at  ortho  positions exhibited maximum inhibitory po-tency against hBChE. The attachment of the  e OOCCH 3  groupinstead of   e Cl and  e NO 2  groups ( 3g  ) to the  meta  position slightlyreducedtheinhibitorypotency.Electronwithdrawinggroupslike e Cland e Fsimultaneouslyat ortho positions,asincaseofcompound 3w   also resulted in potent inhibitor of hBChE but relatively lesspotent than  3k   and  3g  .  2.3. Cytotoxicity of 3 (a e  y) against lung carcinoma (H157) cells In the present study we investigated the toxic effects of thesecompounds (in dose dependent manner) against lung carcinoma(H157) cells through SRB assay in order to assess their behaviours.The vincristine a standard anticancer drug was used as referenceforcomparisontothetestcompounds.Inthepresentstudy,at1 m Mend concentration in the experiment, 3w and 3y demonstratedalmost 66% cytotoxicity, exhibiting highest antiproliferative activ-ities. Compounds 3e and 3t were the next most potent compoundsshowing about 61% and 60% cytotoxicity respectively at the sameconcentration (1  m M). The Fig. 1 reveals the cytotoxic effects of phenylcinnamide derivatives against lungs cancer cells. Anti-proliferativeactivityofthepositivecontrolvincristinetowardslungcarcinoma (H157) cell line was about 46% at  fi nal concentration of 100  m M, whereas the result revealed that these compounds weremany folds potent in comparison topositive control. Therefore, it isshown that phenylcinnamide derivatives exhibited more cytotoxiceffects than standard anticancer drug (vincristine) even at lowesttested concentration.  2.4. Docking and pose ranking  To investigate possible ligand e AChE interactions, dockingstudies were performed to generate binding modes. For acetyl-cholinesterase, the compounds were docked to TcAChE ( Torpedocalifornica  PDB Code: 3I6Z) and for butyrylcholinesterase a high-resolution crystal structure of 2.00   A huBChE (human PDB Code:1P0I) was selected for docking studies. The results of re-dockingexperiments showed that the structures were well prepared andthedockingprogramreproducedthenativelyboundconformationsof the co-crystallized bound ligands. An RMSD of 1.80   A was ob-tained for the re-docked ligand (galantamine) of TcAChE thatindicated a good prediction by the docking program. However, there-docking experiment of using galantamine as a reference ligandin huBChE did not produce satisfactory results in terms of RMSD,however, the orientation of the predicted conformation of the re-docked structure was  fl ipped at 180  that resulted in larger RMSDvalues of 8   A and higher.Similarly,the docking results for these compounds  3(a e  y) wereobtainedforbothenzymes,whichshowedthatthereweredifferentconformations(poses)predictedforeachcompound.Table2showsthe docking scores obtained with LeadIT and the rank number of best pose obtained with NNScore algorithm for the compoundsscreened against both TcAChE and huBChE enzymes. Dockingscores of the compounds range from  20 to  39 for both enzymes,however, fi lteringtheposesforthecompoundsbyNNScoreshowedthere could be other conformations that might interact favourablywith the enzymes. The docking scores for the  fi rst conformationand the selected conformation ranks are given in Table 2.  2.5. Binding mode analysis A common binding mode was observed for the all these com-pounds docked into both structures of TcAChE and huBChE en-zymes. The compounds interact in a similar way like the reference Fig.1.  Antiproliferative activity of the synthesized phenylcinnamide derivatives towards lung carcinoma (H157) cell line at various concentrations. Vincristine was used as standarddrug (100  m M).  A. Saeed et al. / European Journal of Medicinal Chemistry 78 (2014) 43 e 53  45  ligand galantamine as shown in Figs. 2 and 3. In both enzymes, the predicted docked conformations of the compounds adapt similarorientations and form exactly the same interactions with the activesiteresidues. As showninTable 2, the poses with preferred bindingmodes that are selected by the external scoring functions occupytheactivesiteof theenzymeand interact withthesameresiduesinboth enzymes. Therefore, the preferred binding modes selected byexternal scoring function were in accordance to the interactionsmade by the reference ligand galantamine in the active site of TcAChE as shown in Figs. 2 and 3. As it appears in Fig. 2, the tri- methoxybenzoic moiety of the compounds is involved withhydrogen bond interaction with Ser200, that is anchored betweenTrp233 and Trp84 and the benzene ring is making aromaticstacking interactions with Phe290. Tyr121 is involved in bothhydrogen bonding interactions as well as in aromatic interactionswith the phenyl moiety. The tails of the compounds are positionedin the opening of the enzyme pocket and adapt  fl exibleconformations and is involved in a variety of interactions in whichthe interaction with Trp279 is common. The hydrogen bond net-works formed by most of the compounds include hydrogenbonding with Tyr70, Tyr121 and Ser200 in TcAChE.  2.6. Molecular dynamics simulations In total, 50 individual molecular dynamics simulations wereconducted for all the selected poses of the compounds against bothenzyme structures of TcAChE and huBChE. Each enzyme e ligandcomplex was parameterized for the high-ranking pose  fi ltered andselected by the external scoring function NNScore. The pre-equilibration steps of density and  fi nal equilibration results(Figs. 4 and 5) showed that the complexes were equilibrated well, and the protein e ligand complexes can be further simulated toobtainenoughsamplesforcomputingfreeenergyofbindingvaluesaswellasdeterminethestabilityofthebindingmodes.Fig.6showsthe RMSD deviations of ligand atoms in the production MD runs of 5ns simulation time. The ligand atoms of all compounds do notshow deviations during the simulation time and suggests that thepredicted docked conformations remain stable during the simula-tion and the selected binding poses are favoured inside the activesite pockets of the enzymes.  2.7. Calculations of binding free energy The binding free energies ( D G) were estimated by using Me-chanics/Poisson e Boltzmann Surface Area (MM/PBSA) method forall compounds against both TcAChE and huBChE enzymes, thevalues are given in Table 3. The estimated  D G values showed thatthe activities of compounds predicted against TcAChE are higher ascomparedtohuBChEenzymeformostofthecompounds.However,some of the compounds have similar binding energies estimatedfor both enzymes. The observed correlation of estimated  D G andexperimental  D G is positive (i.e. 0.41) for TcAChE but no correlationis observed between the experimental and estimated  D G for huB-ChE (i.e. correlation of 0.02). The reason for observing no correla-tion in the case of huBChE enzyme may be the structuraldifferences between the horse BChE and human BChE enzymes.These results show that MM/PBSA based method to systematicallyevaluate free energy of binding for a series of compounds canpredict satisfactorily the relative binding free energies when themodelling and experimental evaluation is performed on the samestructures. Fig. 2.  Preferred binding modes for the compounds dockedintothe activesite pocketof TcAChE. (Left): Predicted conformations of compounds  3e ,  3f  ,  3g  ,  3h ,  3i  and  3j  shown in CPKmodel. The reference ligand Galantamine (G6X) is shown in green. (Right): Predicted conformations of compounds  3p ,  3q ,  3r  ,  3s ,  3t  inside the active site of TcAChE. Compounds areshown in CPK model and the reference ligand Galantamine is shown in green colour. (For interpretation of the references to colour in this  fi gure legend, the reader is referred to theweb version of this article.)  Table 2 Dockingscoresand bestposeranksforthecompoundsscreenedagainstTcAChEandhuBChE.Compound Docking scores (kcal/mol) Pose ranks in top 10TcAChE huBChE TcAChE huBChE 3a   27.25   35.69 10 2 3b   34.52   25.07 6 7 3c   31.38   39.34 9 8 3d   28.43   25.75 4 8 3e   28.66   31.62 9 3 3f    26.38   22.25 7 7 3g    34.07   28.15 10 6 3h   29.81   26.19 3 1 3i   30.16   25.57 1 4 3j   31.09   27.63 10 8 3k    36.33   29.52 7 6 3l   27.13   21.52 7 2 3m   26.18   28.91 6 3 3n   35.64   30.99 6 4 3o   33.44   39.59 3 1 3p   25.11   20.57 6 2 3q   26.89   25.72 2 10 3r    29.13   27.03 4 4 3s   27.60   26.89 8 1 3t   28.70   28.24 5 5 3u   30.83   28.80 10 10 3v    29.41   26.19 10 10 3w    25.83   24.31 5 3 3x    27.78   28.05 7 9 3y    33.32   32.67 5 4  A. Saeed et al. / European Journal of Medicinal Chemistry 78 (2014) 43 e 53 46  Fig. 3.  Interaction diagrams of reference ligand (Galantamine, left) and compound  3f   (right). Hydrogen bond interactions are shown with red dotted lines and hydrophobicinteracting residues are shown in the regions surrounding by green lines. (For interpretation of the references to colour in this  fi gure legend, the reader is referred to the webversion of this article.) Fig. 4.  Density plots showing density equilibrations for all individual protein e ligand complexes. Density plots for all compounds during pre-equilibration step for TcAChE com-plexes (Left). Density plots for all compounds during pre-equilibration step for huBChE complexes (Right). Fig. 5.  Equilibration plots for all enzyme e ligand complexes of TcAChE and huBChE. RMSD deviations in protein backbone atoms show that the complexes start to equilibrate after2ns of MD simulation.  A. Saeed et al. / European Journal of Medicinal Chemistry 78 (2014) 43 e 53  47
We Need Your Support
Thank you for visiting our website and your interest in our free products and services. We are nonprofit website to share and download documents. To the running of this website, we need your help to support us.

Thanks to everyone for your continued support.

No, Thanks

We need your sign to support Project to invent "SMART AND CONTROLLABLE REFLECTIVE BALLOONS" to cover the Sun and Save Our Earth.

More details...

Sign Now!

We are very appreciated for your Prompt Action!