untitled ISSN 2 Synthes docking Mohamed Subraman Saiyad Mu 1 Post Graduate 2 Centre of Adva 3 Department of 4 Post Graduate 5 Post Graduate * Corresponding Tel.: +91.97.904 ARTICLE IN DOI: 10.5155/e Received: 31 Oc Accepted: 19 No Published onlin Printed: 31 Dec KEYWORDS Molecular dock Anticancer activ Drug binding po Antimicrobial a Hydrophobic in Fluorescence re 1. Introduct Malignan mutations of cell death. T (IARC) has e each year as affected som The treat cancer patien the organiza procedures methodology be more defe solid onlooke doxorubicin, constraining ences for the 2153‐2249 (Prin sis, charac g studies o d Ahadu Sha ni Karthikey usthafa 5 and and Research Depa anced Study in Crys f Medical Physics, A and Research Depa and Research Depa g author at: Post Gr 405029. Fax: +91.44 FORMATION eurjchem.7.4.454- ctober 2016 ovember 2016 ne: 31 December 20 cember 2016   S king vity ocket activity nteraction esonance energy tr tion ncy is charact f abnormal cell The Internation estimated that s a result of tu mewhere around tment options i nt with advanc ation of cytotox of cell divisio y is that quickly enseless to the er cells. Norma , paclitaxel and g symptoms bec e exceedingly m E nt) / ISSN 2153‐ h Euro cterizatio of novel th reef 1,*, Moh yan 3, Singara d Jamal Moh artment of Chemist tallography and Bi Anna University, Ch artment of Chemist artment of Zoology raduate and Resear 4.28352883. E‐mai -462.1505 016 ransfer terized by un ls and also whi nal Agency for 7.6 million ind umor and 4 mi d 30 and 69 yea include the use ced stages, whic xic operators th on. The basis y partitioning m cytotoxic impa ally utilized trea d cis‐platin, ca cause of their u multiplying cell uropean Journal Europ 2257 (Online)  http://dx.doi.org/1 pean Jo Journal we n, antican hiophene hamed Must avelu Ganes hamed 5 try, The New Colleg iophysics, University ennai‐600025, Tam try, Jamal Mohame y, The New College, rch Department of l address: jasshaali ABSTRACT 2‐((4‐Methylp (piperazin‐1‐y thiophene‐2‐c good antibact binding chara confirmed by thiophene‐2‐c has been inv molecular dis derivatives) ac environment o technique and bonding, hydr compound. Cite this: Eur. ncontrolled re ich leads to ult Research on C ividuals overal illion individua ars [1‐3]. of chemothera ch normally inv hat follow up o for this trea malignancy cell acts of the drugs atments, for exa ause serious d unfavorable con ls in specific ti l of Chemistry 7 pean Journal of C 2016 Atlanta Pub 10.5155/eurjchem ournal ebpage: www ncer activ e‐2‐carbox thafa 1, Deva san 3, Syed A ge, Chennai‐600014 ty of Madras, Chenn mil Nadu, India ed College, Tiruchir Chennai‐600014, T f Chemistry, The New i@gmail.com (M.A. piperazin‐1‐yl)(th yl(thiophen‐2‐yl) arboxaldehyde d terial as well as acteristics of the y optical spectr arboxaldehyde estigated by st tance (r) betwe ccording to Fors of HSA has also d the molecular rophobic intera J. Chem. 2016, 7 sidual timate Cancer ll died als are py for volves on the tment ls will s than ample, dosage nsequ‐ ssues, incl The alte (EG for invo app pro gen kina par Thi cru are sma sign (4) (2016) 454‐ Chemistry blishing House LL m.7.4.454-462.15 of Che w.eurjchem.co vity, optica xaldehyde adasan Velm Ali Padusha 4 4, Tamil Nadu, Indi nai‐600025, Tamil N rappalli‐620020, Ta Tamil Nadu, India w College, Chennai Shareef). hiophen‐2‐yl)m )methyl)hydraz derivatives have s antifungal acti ese novel comp roscopic, antica derivatives to c tudying its que een donor (HSA ster’s theory of n been studied by r docking techn action between 7(4), 454‐462 luding the bone erefore, it is an ernatives to the Protein kinas GFR) are the m cancer therapy olved in cell sig proximately 2% otein kinases, w nome sequenci ases catalyze ex rticle to tyrosin s procedure a cial cell forms seen in a vari all molecule inh naling [12]. ‐462 LC ‐ All rights rese 505 emistry om al spectro e derivati murugan 2, 4, ia Nadu, India amil Nadu, India i‐600014, Tamil Na ethyl)hydrazine inecarboxamide e been synthesiz ivity and also l pounds and pha ancer and doc carrier protein, nching mechan A) and acceptor non‐radiative ene y using synchron nique has been the human se e marrow, hear n urgent need existing chemo e and epiderm most well‐know y. Protein kinas gnal transductio % of eukaryoti which are iden ng [10] and b xchange the γ‐p ne, serine or th assumes a key [11]. Improper ous types of ca hibitors are des erved ‐ Printed in y oscopic an ives adu, India. ecarboxamide e (L2) from ed. These new c ess toxic in nat armacokinetic m king studies. T Human Serum nism, binding k r (thiophene‐2‐ ergy transfer (FR nous fluorescen used to explor erum albumin w rt and Gastro‐I to develop safe otherapy option mal growth fa wn and validate e is the largest on [4‐9]. They a c genes and m ntified based o biochemical st phosphate bunc hreonine depos part in direct r activation of p ancers and in s signed to target n the USA nd (L1) and (2‐ the family of compounds have ture. Exemplary mechanism were The binding of Albumin (HSA) kinetics and the carboxaldehyde RET). The micro ce spectroscopy e the hydrogen with L1 and L2 Intestinal tract. fe and effective ns. actor receptor ed drug targets enzyme family are encoded by more than 500 on the human tudies. Protein ch from an ATP its in proteins. ting numerous protein kinases such scenarios, t/inhibit kinase ‐ f e y e f ) e e o y n 2 . e r s y y 0 n n P . s s , e Shareef et al. / European Journal of Chemistry 7 (4) (2016) 454‐462 455 Figure 1. Synthesis of compound 2‐[(4‐methylpiperazin‐1‐yl)(thiophen‐2‐yl)ethyl]hydrazinecarboxamide (L1) and 2‐[piperazin‐1‐yl(thiophen‐2‐ yl)methyl]hydrazinecarboxamide (L2). The epidermal development element receptor is derived from three auxiliary spaces: an extracellular ligand‐tying area, a transmembrane area, and an intracellular space. At the point when an agonist ties to its ligand binding site, dimerization of the epidermal growth factor receptor is activated. This actuates the inborn kinase area, prompting auto phosphory‐ lation on particular tyrosine deposits in the C‐terminal [13]. Subsequently, signal transduction falls are started, which advance DNA union and cell expansion. Thus, the epidermal growth factor receptor is a factor responsible for cell relocation, bond, multiplication and resistant reactions in a few cell phenotypes in the human skin [14]. Mostly, the chemotherapeutic agents are administrated intravenously to deliver the drugs to the target organ selecti‐ vely. Serum albumins are the significant local bearer found in the blood and are included in the basic carrying of exogenous and endogenous materials (unsaturated fats, supplements, steroids, and an assortment of restorative drugs). The active‐ tion of drug mainly depends on solubility, bio‐distribution and their interaction which is highly affected by their binding nature and interaction with protein. For instance, solid binding can diminish the grouping of free drugs in plasma while feeble binding might prompt poor conveyance and short lifetime [15‐ 17]. This unmistakably demonstrates the binding of drug with protein might modify the pharmacokinetics and cytotoxic impacts. Consequently the study of the interaction of drug with protein is crucial to outline a new drug and enhance the therapeutic efficacy. Computational biology and bioinformatics play a major role in designing the drug molecules and also in speeding up the drug discovery process. Molecular docking of drug molecule with the receptor (target organ) gives impor‐ tant information about the drug receptor interactions. Herein, we aim to determine the receptor interactions and binding orientation of ligand compounds by molecular docking with two proteins EGFR KINASE TKIs‐L858 (PDB ID: 2ITZ) and Human Serum Albumin (HSA; PDB ID: 4L8U). Also, the human serum albumin with newly synthesized novel ligands binding kinetics was studied by fluorescence spectroscopy techniques. 2. Experimental 2.1. Reactants Analytical grade solvents and reactants were used. N‐ Methyl piperazine, piperazine and thiophene‐2‐carbox‐ aldehyde were purchased from Merck Products and used as such. 2.2. Instrumentations Elemental analyses and characterization studies were carried out at Sophisticated Analytical Instrument Facility (SAIF), Indian Institute of Technology, Madras, Tamil Nadu and India. Melting points of synthesized compounds were measured by electric melting point apparatus SMP1. 1H NMR spectra of the samples were recorded on 300 MHz using DMSO‐d6 with TMS as the internal standard. The homogeneity of the compounds was monitored by Thin Layer Chromato‐ graphy (TLC) Silica‐Gel coated on glass plate and visualized by iodine vapor. The absorption in the UV‐Vis region was recorded by Perkin Elmer Lambda 35 Spectrophotometer using DMF/DMSO as solvents. IR spectra were recorded using KBr pellets with a Nicolet model Impact 470 FTIR spectro‐ photometer in the range of 4000‐400 cm‐1 at the Regional Sophisticated Instrumentation Centre, Indian Institute of Technology, Madras, Tamil Nadu, India, using tetracyano‐ ethylene (TCNE) as the internal standard. Anti‐cancer and cytotoxic studies were carried out at Royal Bio Research Centre, Velachery, Chennai, Tamil Nadu and India. 2.3. Synthesis Thiophene‐2‐carboxaldehyde, N‐methylpiperazine and semicarbazide hydrochloride were taken in 1:1:1 mol ratio and allowed to react as shown in Figure 1. Semicarbazide (11.2 g, 0.1 mol) was taken in a round bottom flask and 10 mL of water was added. To this solution 10.0 mL (0.1 mol) N‐methyl piperazine was added and stirred well for 15 min by keeping the reaction mixture on a magnetic stirrer. Thiophene‐2‐ carboxaldehyde (9.3 mL, 0.1 mol) was added to the above mixture and stirring was continued under ice cold condition. The colorless solid formed was filtered, washed and recrystallized using ethanol. The same procedure was followed for the synthesis of the rest of the compounds as shown in Figure 1 [18‐22]. 2‐((4‐Methylpiperazin‐1‐yl)(thiophen‐2‐yl)methyl)hydrazi‐ necarboxamide (L1): Yield: 53%. M.p.: 150‐152 °C. FT‐IR (KBr, ν, cm‐1): 3301 (N1H2), 1548 (NH, amide II band), 1622 (C=O, amide I band). 1H NMR (400 MHz, CDCl3, δ, ppm): 7.88 (s, 2H, 456 Shareef et al. / European Journal of Chemistry 7 (4) (2016) 454‐462 NH2), 7.45‐7.44 (m, 3H,thiophene), 7.13 (d, 1H, NH(C=O)), 4.15 (s, 1H, CH(NH)), 3.90 (s, 3H, CH3 (piperazine)), 2.51‐2.50 (m, 8H, piperazine), 1.92 (s, 1H, NH(CH)). 13C NMR (100 MHz, CDCl3, δ, ppm): 172.45, 150.02, 132.26, 130.13, 121.89, 114.97, 56.15. HRMS (ESI, m/z) calcd. for C11H19N5OS [M‐H]:269.13; Found: 269.2312. Anal. calcd. for C11H19N5OS: C, 49.05; H, 7.11; N, 26.00. Found: C, 49.08; H, 7.08; N, 26.01%. 2‐(Piperazin‐1‐yl(thiophen‐2‐yl)methyl) hydrazinecarbox‐ amide (L2): Yield: 52%. M.p.: 149‐153 °C. FT‐IR (KBr, ν, cm‐1): 3245 ν(N1H2), 3153 ν(N2H), 1599 δ(N3H), 1719 (C=O). 1H NMR (400 MHz, CDCl3, δ, ppm): 9.95 (s, 2H, NH2), 7.85 (s, 1H, NH), 7.13 (m, 3H, Thiophene), 4.18 (s, 1H, CH), 2.56 (m, 8H, piperazine), 2.40 (d, 1H, NH (CH)), 1.92 (s, 1H, NH of piperazine). 13C NMR (100 MHz, CDCl3, δ, ppm): 170.26, 150.02, 139.75, 130.18, 129.79, 121.89, 114, 99, 40.50, 40.00. HRMS (ESI, m/z) calcd. for C10H17N5OS [M‐H]: 255.12; Found: 255.3000. Anal. calcd. for C10H17N5OS: C, 47.04; H, 6.71; N, 27.43. Found: C, 47.08; H, 6.08; N, 26.22%. 2.4. Antimicrobial studies 2.4.1. Antibacterial activity To study the antibacterial activity of newly synthesized compounds, nutrient agar was used as a medium. The agar medium was prepared by dissolving 5 g of yeast extract, 10 g meat extract, 5 g of peptone, 5g of sodium chloride and 20 g of agar in 100 mL of distilled water in a clean conical flask and the pH was maintained at 7. The solution was boiled to dissolve the medium completely and sterilized by autoclaving at 7 kg pressure (121 °C) for 15 minutes. After sterilization 20 mL media was poured in to the sterilized petri plates. These petri plates were kept at room temperature for some time. After a few minutes, the medium got solidified in the plate. Then, it was incubated for 12 h. After the incubation, it was inoculated with microorganisms, using simile swabs. All these manipulations were carried out with atmospheric air under aseptic condition. 2.4.2. Antifungal activity The potato dextrose agar (PDA) is used as a medium to determine antifungal activity of newly synthesized ligands. The PDA was prepared by dissolving 20 g of potato extract, 20 g of agar and 20 g of dextrose in one liter of distilled water in a clean conical flask. The solution was boiled to dissolve the media completely and sterilize by autoclaving with 7 kg pressure (121 °C) for 30 minutes. After sterilization, 20 mL media was poured into the sterilized petri plates. These petri plates were kept at room temperature for some time. After a few minutes, the medium gets solidified in the plate.0.5 mL of DMSO was used as solvent and 10μg of Amphotericin B as control. In a typical procedure, a well‐made agar medium was inoculated with microorganism and it was filled with 50μL of test solution using a micro pipette. Later, the plates were incubated at 35 °C for 72 h. During this period, the test solution diffuses and affects the growth of the inoculated. 2.5. Human serum albumin (HSA) Human serum albumin (HSA) was purchased from Hi Media laboratory Pvt. Limited, Mumbai, India and it was used without any further purification. Millipore water was used for preparing solution throughout the experiments. HSA solution was prepared in phosphate buffer solution of pH = 7.4. HSA solution was kept in the dark at 4 °C. L1 and L2 are newly synthesized compounds, and the stock solutions of these derivatives were also prepared using the same buffer. 2.6. Fluorescence spectroscopy studies The steady states fluorescence emission measurements were obtained using a commercially available spectrofluoro‐ meter (Fluoromax‐2, ISA; Jobin‐Yuvon‐Spex, Edison, NJ) and spectral band passes were kept at 5 nm in both excitation and emission monochromators. The emission spectrum was recorded in the wavelength region 300‐540 nm at 280 nm excitation. Synchronous fluorescence spectra were recorded by simultaneously scanning the excitation (λex) and emission (λem) monochromators with two different constant wavelength intervals (Δλ) such as 15 and 60 nm between the excitation and emission monochromators. This was carried out with the help of Fluoromax‐2 equipped with an excitation source (150W ozone free Xenon arc lamp) coupled to the monochro‐ matic delivering light to the sample spot at desired wave‐ length. The fluorescence emission from the sample was collected by an emission monochromatic to photomultiplier tube (R928; Hamamatsu, Shizuoka‐Ken, Japan). In addition, using the absorption spectrum of L1 and L2 fluorescence spectrum of HSA (concentration ratio of drug and protein is 1:1 at pH = 7.4), the fluorescence resonance energy transfer and energy transfer efficacy of HSA with various concentration of drug were also evaluated. 2.7. Molecular docking studies Molecular docking is a powerful tool in understanding different protein functions. The X‐ray crystal structures of the Protein EGFR KINASE TKIs ‐ L858 (2ITZ) and human serum albumin protein (Monomer with 585 amino acids) (PDBID: 4L8U) obtained from protein data bank database. Energies of protein structures were minimized using protein preparation wizard panel to include hydrogens [23‐25]. The charge state of protein residues is important for result generation by Glide. Optimized potential of liquid simulations (OPLS) force field was used for minimization process. Protein preparation facility consists of two steps, preparation and refinement. Energy minimization reorients side‐chain hydroxyl groups and alleviates potential steric clashes. These structures were energy minimized using two algorithms with steepest descent and conjugate gradient. Compounds were docked in two drug binding sites using the induced fit docking (IFD) protocol. Here, both the ligand and protein are flexible to dock, and hence many conformations are generated for an individual ligand. Based on the docking score, glide energy and hydrogen bond interaction, the best conformation is sorted and results were analyzed. 3. Results and discussion 3.1. Synthesis Physicochemical characterization of the synthesized compounds were done by the analytical methods such as melting point, TLC, elemental analysis, and spectral methods such as UV‐Visible, IR, 1H NMR, 13C NMR and Mass, Figure 2. 3.2. Antimicrobial studies Agar well dispersion strategy was applied to determine the antibacterial and antifungal activities of these novel compounds. All blended mixtures were screened for their in‐ vitro antifungal movement against Candida albicans, Penicillium notatum and Aspergillus flavus and in‐vitro antibacterial action against Gram‐negative Escherichia coli, Gram‐positive Staphylococcus aureus, Vibrio para haemolyticus, Bacillus subtilis, Klebsiella and Pseudomonas aeruginosa. Amphotericin (20 µg/plate) and streptomycin (20 µg/circle) were utilized as standard references for antibacterial and antifungal activity, respectively. Table 1. Antim Compound TNS Streptomycin Amphotericin Solvent Table 2. Antim Compound TNS Streptomycin Amphotericin Solvent After hat conformed t tions were p inhibition ar showed sign antifungal a activity com amide moie exhibited co microbial studies of Zone of inhibit Bacteria S. aureus B. sub 16 10 20 11 ‐ ‐ ‐ ‐ microbial studies of Zone of inhibit Bacteria S. aureus B. sub 15 10 20 11 ‐ ‐ ‐ ‐ tching, the dista to the depressi precisely measu re presented in ificant antibact activity. L1 and mparable with ety showed m mparable activ Shareef et al f compound L1. tion (mm) btilis V. parahae 10 13 ‐ ‐ f compound L2. tion (mm) btilis V. parahae 8 13 ‐ ‐ ance across of t ons and plate ured in mm. Th n Table 1 and terial activity bu d L2 exhibited standard dru moderate anti vity against all l. / European Jou emolyticus emolyticus Figure 2. Mas he zone of hind of standard m he observed zon 2. Both compo ut weak to mod d good antibac g while L2 be ifungal activit tested fungal s urnal of Chemistry E. coli Klebsiella 7 7 7 6 ‐ ‐ ‐ ‐ E. coli Klebsiella 7 5 7 6 ‐ ‐ ‐ ‐ ss spectrum of com drance edica‐ nes of ounds derate cterial earing ty. L1 strains and and inh 3.3. mec spe ry 7 (4) (2016) 45 a P. aeruginosa 9 7 ‐ ‐ a P. aeruginosa 5 7 ‐ ‐ mpound L1 and L2. d showed good d Penicillium n ibition against a . Fluorescence The binding chanism were ectroscopy. 54‐462 Fungi C. albicans 9 ‐ 6 ‐ Fungi C. albicans 4 ‐ 6 ‐ zone of inhibi notatum while all the selected spectroscopy s affinities of d carried out by A. niger A. Flavus 8 8 ‐ ‐ 7 8 ‐ ‐ A. niger A. Flavus 6 6 ‐ ‐ 7 8 ‐ ‐ ition against Ca e L2 showed w fungal strains. studies rug and prote y using fluoresc 457 s P. notatum 11 ‐ 9 ‐ s P. notatum 8 ‐ 9 ‐ (a) (b) andida albicans weak zone of ein interaction cence emission s f n n 458 Figure 3. Fluor Volmer plot [L1 The HSA tyrosine, an molecules w fluorescence of different c HSA resulte emission at and the shap this decrease fluorophore fluorescence both the dru as quencher decrease in that, upon bi the HSA com To estim Stren‐Volme F0 / F = 1 + K rescence emission 1 (b), L2 (e)]. The pl A emission sp nd phenylalan when bound to e intensity due t concentrations ed in a decrea 347 and 349 n pe of the peaks e in fluorescenc with in protei e quenching of H ugs. Here, both t r and bind in t the fluorescenc inding of comp mplex was forme mate the natur er equation is us KSV [D] = 1+Kq [ Sh n spectra of HSA‐L1 lot of log (F0‐F)/F v pectrum come ine amino ac o HSA would c to the tryptoph of compound L ase in the fluo nm, respectively remained almo ce intensity due in local enviro HSA depend on the drugs (com the vicinity of ce emission. Th pound L1 and L2 ed. re of the quen sed. D]; Kq hareef et al. / Eur (a (b ( 1 (a), HSA‐L2 (d) ( versus Log [Q] [L1( s from trypto cid residues. change the int han residue. Titr L1 and L2 (0‐2 µ orescence max y (Figure 3a an ost unchanged. H e to the interact nment, and als the concentrat mpound L1 and L Trp214 leading hese results in 2 drugs into the nching phenom = Ksv/τ0 ropean Journal of a) b) c) pH = 7.4). HSA (0 (c), L2(f)]. ophan, Small trinsic ration µM) to ximum nd 3d) Hence tion of so the tion of L2) act g to a dicate e HSA, menon (1) whe afte que the the be 1 fluo and and grea mac that L2 i que 3.4. des mat f Chemistry 7 (4) .1×10‐5 M) in the p ere F0 and F a er the additio enching constan concentration molecule with 1×10‐8 s. The Stern‐Vo orescence by co d 3e. Stern‐Volm d 1.578×1013 L ater than the l cromolecular co t probable quen interaction wa enching [26]. . Binding const The binding tinations can thematical state ) (2016) 454‐462 presence of L1, L2 are the fluores on of the que nt; Kq is the qu of the quenche hout quencher a olmer plots o ompound L1 an mer quenching L/mol. The res limiting diffusi omplexes (2.0 × nching mechan as initiated and tant and the nu constant Ka a be ascertained ement [27]. 2 (0 to 2×10‐5 M), re scence intensit encher, KSV is uenching rate c er; τ0 is the aver and its value is of the quenc nd L2 are show constants Kq w sults showed K on rate consta ×1010 L/mol), w nism of HSA‐Com d this may be umber of bindi and the quant d utilizing the (d) (e) (f) espectively. Stern‐ ies before and the dynamic constant; [D] is rage life time of s considered to ching of HSA wn in Figure 3b were 1.291×1013 Kq to be much ant of both the which indicates mpound L1 and e due to static ing sites ity of binding accompanying ‐ d c s f o A b 3 h e s d c g g Figure 4. Synch µM) L1 and L2, r Log [(F0/F)/ A plot o (Figure 3c an From the p 9.13×105 L/m two compou that there is could be bou 3.5. Synchro Synchron concentratio pound L2 (F conformation spectra give in the region the maximu conformation emission wa the synchron information respectively complex. Th tyrosine res residue, whi dibly to the n and L2. Incon which indica after the ad maximum em 3.6. Energy t Forster r apparatus to proteins (do hronous fluorescen respectively. F] = Log Ka+ n L of log [(F0‐F)/F nd 3f) of HSA co plot we can o mol and 6.25×1 unds n = 1.3 and one class of bin und and transpo onous fluoresce nous fluoresce ons of compoun Figure 4b and 4 nal changes of H the informatio n of the chromo um emission nal changes of H avelengths were nous fluoresce about tyros [28] for both he quenching sidue is better ch shows that natural fluoresc nsiderable blue ates maximum ddition of bot mission of trypt transfer from H resonance ener o decide the s onor and an acc Shareef et al nce and its spectra Log [D] F] vs log [D] g ompound with obtain the bin 105 L/mol. and d 1.1 respective nding site for dr orted by HSA to ence spectrosco nce spectra of nd L1 (Figure 4 4d) were meas HSA. The synch n about the mo spheres of mole wavelengths w HSA. The excita e located at Δλ nce spectra giv sine and try the compound of the fluore r when comp tyrosine residu cence of HSA fo e shift in the flu m of tyrosine r th compound tophan remaine HSA to compou rgy transfer (F separation betw ceptor) further l. / European Jou ( ( a, at ∆λ = 15 nm [L gives a straigh compound L1 a ding constant d binding site fo ely. The results rug in HSA and o the body. opy f HSA with dif 4a and 4c) and sured to explor hronous fluores olecular environ ecules, and chan which indicate ation wavelengt = 15 and 60 nm ve the characte yptophan res L1 and L2 with escence intensi ared to trypto ue contributes or both compou uorescence em residue was no L1 and L2. Bu ed unchanged. und L1and L2 FRET) is an eff ween two poin rmore FRET dis urnal of Chemistry (a) c) 1 (a), L2(c)] and ∆λ (2) ht line and L2. Ka = or the show drugs fferent d com‐ re the scence nment nge in e the th and m and eristic idues, h HSA ity of ophan incre‐ und L1 mission oticed ut the fective nts in stance betw con pro ene the fluo of t abs the effic E=1 whe pre don ene effic don whe 50% acce whe ave the inte and is g J λ ry 7 (4) (2016) 45 λ = 60 nm [TNS (b) ween the point nformational ch otein associatio ergy transfer th following con orescence quan the fluorescenc orption spectru donor and a ciency E is defin 1‐(F/F0) ere F is the f sence of accept nor. The fluore ergy transfer to ciency is relat nor distance and ere R0 is the cri % and r is the eptor. 8.79 10 ere k2 is the spa erage refractive donor in the egral of the flu d the absorption iven by followin F λ ε 54‐462 ), TPS (d)]) of HSA ts have been bro hanges upon l ons. According heory, the ener nditions: (1) T ntum yield, (2) ce emission spe um of the accep acceptor is wi ned by the follo fluorescence in tor and F0 is the escence intensi o compound L1 ted to distance d critical energy itical distance w e binding dista atial orientation e index of wate absence of th uorescence emi n spectrum of t ng equation λ λ dλ A (0.1×10‐5 M) in th oadly used to co ligand binding to the Forster rgy transfer sho he donor shou The overlap sh ectrum of the d ptor (3) The dis thin 8 nm. En owing equation ntensity of the e fluorescence i ty of HSA is r 1 and L2. The e e between the y transfer dista when the transf ance between t n factor of the er, φD is the qu he acceptor. J ission spectrum the acceptor in 459 (b) (d) he presence of (0‐2 ontemplate the g and protein‐ r non‐radiative ould happen at uld have more hould be >30% donor with the stance between nergy transfer . (3) e donor in the intensity of the reduced by the energy transfer e acceptor and ance (R0). (4) fer efficiency is the donor and (5) dipole, N is the uantum yield of is the overlap m of the donor units M‐1cm3. J (6) 2 e ‐ e t e % e n r e e e r d s d e f p r J 460 where FD (λ wavelength λ unity and ε acceptor at overlap to t absorption s (Figure 5b). of the accept calculate the 10‐19 M‐1cm3 nm, 0.247 n φD=0.118, r= lower than th the donor a Figure 5. The ove Figure 6. The fluo λ) is the fluor λ to λ + λ, wit εA (λ) is the m wavelength ( the fluorescenc pectrum of bot From the overl tor and fluoresc e J value of bot , 1.2148 × 10‐20 nm, E = 0.109 =0.577 nm, 0.3 he maximum st and acceptor Sh erlap of the fluores orescence contour escence intens th the total inte molar extinctio (λ). Figure 5 ce emission spe th compounds L lapping of the a cence spectrum th compound L 0 M‐1cm3 and th , 0.146, k2 = 2 331 nm. Both tandard values distance r < hareef et al. / Eur scence emission sp maps of (a) HSA, ( sity of the don ensity normaliz on coefficient o shows conside ectrum of HSA L1 (Figure 5a), a absorption spe m of the donor w 1 and L2 as 2.4 he value of R0 = 2/3, N = 1.336 R0 and r value for R0 (< 10 nm 8 nm. So, the ropean Journal of (a) pectrum of HSA and (b) L1‐HSA and (c) nor at zed to of the erable A with and L2 ctrum we can 148 × 0.406 6 and es are m) and ere is pos and 3.7. stud it c qua the HSA sho fluo (L1 com f Chemistry 7 (4) d the absorption co L2‐HSA. Concentra ssibility of ener d L2 [29,30]. . Three dimens The three dim dy the conform can give add u alities by chang same time. Th A, HSA‐compou own in Figure 6 orescent intens and L2) in all mplex formation ) (2016) 454‐462 oefficient spectrum ation of HSA is 1 µM rgy transfer bet sional fluoresc mensional spec mational change p to data with ging the excita he excitation, e und L1 and HS 6a‐c, respectivel ity of peak dec systems and t n between drug 2 m of L1 (a) and L2 (b (a) (b) (c) M and L1, L2 are 2 µ tween HSA and ence spectrosc ctra is a power es of protein an h respect to th ation emission emission floresc SA‐compound L ly. As shown in creased with ad the possible re g and protein. (b) b). µM. d compound L1 copy studies rful method to nd additionally he fluorescence wavelength at cent spectra of L2 complex are n the figure, the ddition of drug eason is due to 1 o y e t f e e g o Table 3. Glide e Compound Glide energy do L1 L2 Glide energy do L1 L2 Figure 7. Cryst amino camptot The resu compound L that in free Trp and Tyr between HSA pocket. The d peak togeth reveals that induced som confirmation 3.8. Molecul Molecula were carried programme to understan the middle o energy docking sco Dock score (K ocking score of hum ‐6.496 ‐6.106 ocking score of hum ‐5.002 ‐3.023 tal structure of (a hecin. Figur ult indicates th L1, HSA‐compou HSA, in which may be reduce A and the drug decrease of fluo her with the t the interactio me changes in n of protein [31 lar docking stu ar docking stud d out using M so as to suppor nd in subtle ele of ligand and pr Shareef et al ore of human lung Kcal/mol) man lung cancer pr man serum albumi ) EGFR‐Kinase dom e 8. LIGPLOT for E hat fluorescent und L2 complex the polarity ar ed. This means g is located with orescence emis synchronous on of drug (L1 n the micro‐en ‐33]. udy ies of synthesiz Maestro Versio rt bioorganic m ements, the dif rotein at the ac l. / European Jou cancer protein (PD Glide energy rotein (PDB ID: 2IT ‐43.713 ‐42.542 in (PDB ID: 4L8U) ‐44.670 ‐43.124 (a) main l858r mutati EGFR kinase domai t intensities of xes were lower round both res that the bindin hin this hydrop ssion intensity o fluorescence 1 and L2) with nvironment an zed ligand comp on 9.3.5. as do movement result fferent interact ctive site of res urnal of Chemistry DB ID: 2ITZ), huma y (Kcal/mol) TZ) with L1 and L2 with L1 and L2 ) ion in complex wi in (a) L1 (b) L2 and f HSA‐ r than idues, ng site phobic of this result h HSA d the plexes ocking ts and tion in sidues. The ID: (PD ‘Site acti liga com affin rece hyd In t with and wer sco hum rev anti ry 7 (4) (2016) 45 an serum albumin Hydroge ARG 841 ARG 841 LEU 115 LEU 115 ith iressa (PDB ID d human serum alb e X‐ray crystal s 2ITZ) (Figure DBID: 4L8U) we e Finder’ tool ive site. Docki and and the bes mplexes was s nity scoring fu eptor ligand drogen bonding the overall mo h human lung d human serum re shown toget re is very minim man serum albu ealed that the icancer activity 54‐462 (PDB ID: 4L8U) w en bond (D‐H…A) 1 & ASP 855 1 & ASP 855 5 & ARG 114 (A) 5 & ARG 117(A) : 2ITZ) (b) Human bumin (c) L1 (d) L2 structure of hum e 7a) and hum ere used for do of the program ing procedures t conformation selected based nction ∆G was complexes. Th g strength of L1 lecular interac cancer protein m albumin (Figu ther in Figure 8 mal as the bind umin and huma ligands have p y. with compound L1 a ) Dis <1. <1. <3. <3. n serum albumin complex. man lung cance man serum alb ocking study (F m was used to s were perfor n of each of the l on energetic used to asses he docking sc and L2 are sho ction of the liga n (Figure 8a (L1 ure 8c (L1) and 8. It is found ou ding interaction an lung cancer potential bindin 461 and L2. stance (Å) .7 .7 .0 .0 (b) complexed with 9 er protein (PDB bumin protein Figure 7b). The o search for its rmed for each ligand receptor grounds. The s and rank the cores and the own in Table 3. ands L1 and L2 1) and 8b (L2)) 8d (L2)) which ut that docking n forces of both protein results ng capacity and 9 B n e s h r e e e . 2 ) h g h s d 462 Shareef et al. / European Journal of Chemistry 7 (4) (2016) 454‐462 4. Conclusions In the present study, the amide is an important functional group and due to its electron properties, it is able to interact and bind with a number of receptors. Therefore, the reason for the wide spread occurrence of amides in modern pharma‐ ceuticals and biologically active compounds is obvious. The properties of the amide moiety can be easily modified by various substitutions. Thus the presence of an amide‐like moiety is characteristic for various anticancer, antibacterial and antifungal agents. We have performed antimicrobial activity and the results showed very potent activity against many Gram positive and negative microbes and the binding interactions of ligands and amino acids receptors were experimentally confirmed with docking scores. Presently we have made an attempt to determine the molecular interactions of ligands and protein receptors using molecular docking studies and optical spectroscopic fluorescence studies. Acknowledgements The authors would like to express their gratitude to the Head, Department of Chemistry, Principal and Management committee of The New College, for providing necessary facilities. We are thankful to Sophisticated Analytical Instru‐ ment Facility (SAIF), Indian Institute of Technology ‐ Chennai for compound characterization studies, Department of Biotechnology supported Bioinformatics Infrastructure Facility, University of Madras for molecular docking studies and Royal Bio Research Centre, Velachery, Chennai for Technical Support, Fluorescence spectroscopy study was supported. We also thank the Board of Research in Nuclear Sciences, Department of Atomic Energy, Government of India, for permitting us to carry out Fluorescence Spectroscopy study. References [1]. Bray, F.; Ren, J. S.; Masuyer, E.; Ferlay, J. Cancer. 2013, 132(5), 1133‐ 1145. [2]. Ferlay, J.; Soerjomataram, I.; Ervik, M.; Dikshit, R.; Eser, S.; Mathers, C.; Rebelo, M.; Parkin, D.M.; Forman, D.; Bray, F. 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