rational design and microwave assisted synthesis of some novel phenyl thiazolyl clubbed s triazine derivatives as antimalarial antifolate

Accepted Manuscript Rational Design and Microwave Assisted Synthesis of Some Novel Phenyl Thiazolyl Clubbed s-Triazine Derivatives as Antimalarial Antifolate Jun Moni Kalita, Surajit Kumar Ghosh, Supriya Sahu, Mayurakhi Dutta PII: S2314-7245(16)30046-2 DOI: 10.1016/j.fjps.2016.09.004 Reference: FJPS 23 To appear in: Future Journal of Pharmaceutical sciences Received Date: 12 May 2016 Revised Date: 15 August 2016 Accepted Date: 26 September 2016 Please cite this article as: Kalita JM, Ghosh SK, Sahu S, Dutta M, Rational Design and Microwave Assisted Synthesis of Some Novel Phenyl Thiazolyl Clubbed s-Triazine Derivatives as Antimalarial Antifolate, Future Journal of Pharmaceutical sciences (2016), doi: 10.1016/j.fjps.2016.09.004 This is a PDF file of an unedited manuscript that has been accepted for publication As a service to our customers we are providing this early version of the manuscript The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain ACCEPTED MANUSCRIPT Rational Design and Microwave Assisted Synthesis of Some Novel Phenyl Thiazolyl Clubbed s-Triazine Derivatives as Antimalarial Antifolate a AC C EP TE D M AN U SC RI PT Jun Moni Kalitaa*, Surajit Kumar Ghosha, Supriya Sahua, Mayurakhi Duttab Department of Pharmaceutical Sciences, Dibrugarh University Dibrugarh, Assam,India b Department of Pharmaceutical Sciences, Assam University Silchar, Assam,India *Corresponding Author: Jun Moni Kalita, Email: pjmk84@gmail.com Phone: +91 9508980893 ACCEPTED MANUSCRIPT Rational Design and Microwave Assisted Synthesis of Some Novel Phenyl Thiazolyl Clubbed s-Triazine Derivatives as Antimalarial Antifolate RI PT Abstract: Rational approach to drug design is the process to find new potent molecules on the basis of a known target and available ligands for the target Compared to the traditional system of drug design and discovery, that involves blind testing of different chemicals in vitro and in vivo in cultured cells and animals, rational approach is totally based on the knowledge of the target and the pathway of action Recent developments in the field of rational approach to drug design can be credited to the development in the areas of computer science, molecular SC biology, biophysics, biotechnology and statistics Designing of new molecules based on the knowledge of receptor and the available ligands is well-known as Structure Based Drug Design (SBDD) The branch of rational approach that uses computer as a tool to design and screen design molecules is called as Computer M AN U Aided Drug Design (CADD) In this work computer was used to design and screen the designed molecules virtually Among the 60 designed molecules 10 were selected on the basis of their binding affinity to the receptor molecule Synthesis of the selected molecules was done and In-vitro antimalarial activity was evaluated AC C EP TE D Keywords: Antifolate, Antimalarial, Docking, Phenylthiazole ACCEPTED MANUSCRIPT Introduction Malaria is a mosquito born disease caused by a single celled organism known as protozoa Among the five types of malaria, the disease caused by Plasmodium falciparum is the most common and virulent Although malaria is less common in the developed countries, yet it is a life threatening infectious disease in the developing Asian and African countries While there are numbers of antimalarial drugs available, today an emergency occurred in the area of antimalarial drug search because of resistance occurred by the parasites RI PT against the available drugs [1, 2] Resistance to antimalarial drugs has been reported for only two species of parasite among the five viz P falciparum and p vivax Among the two species P falciparum acquired resistance to almost all the antimalarial drugs available, however the extent of resistance varies from drug to drug The geographical distribution of resistant parasite depends upon the population movement from a resistant place to a nonresistant SC one At present chloroquine resistant P.falciparum strain hass been reported everywhere throughout the world [3-6] Molecules containing thiazole nucleus as a part are reported to have a diverse activities such as anticonvulsant [10], analgesic and anti- inflammatory[11, 12], antitubercular[13] and M AN U antimicrobial [7-9], antican-cer[14-16] It is been also reported that thiazole containing molecules are easily metabolised inside the body without the production of any toxic biproducts[17] Molecules containing a thiazole ring attached with a substituted triazine nucleus were reported to have antimalarial activity as it can block DHFR (Dihydro Folate Reductase), which is a key enzyme responsible for metabolic activity in malarial parasites [18] Rational drug design is also sometimes referred as drug design or rational design In the era of modern TE D drug design and discovery, computer aided drug design played a major rule In contrast to the traditional method of drug discovery, which relies on the trial and error testing rational drug design begins with a hypothesis that modulation in a specific target can give a desired pharmacological activity With the advancement in the technology, it is now possible to simulate in vitro as well as in vivo condition within a computer using any sophisticated software Accordingly molecules can be virtually screened for their activities as well as probable resources [19-22] EP toxicities prior to a real laboratory work This type of virtual screening enables the proper use of time and AC C In the last two decades microwave assisted synthesis become very popular in pharmaceutical and academic areas because of its technology enabling a fast and steady chemical synthesis Further advancement has been achieved in case of Enhanced Microwave Synthesis (EMS), where the reaction vessel is simultaneously cooled during the reaction time Short reaction time and a wide range of reaction scope have enabled microwave assisted synthesis very popular among the researchers and industrial persons [23, 24] ACCEPTED MANUSCRIPT Materials and Methods 2.1 Insilico studies: After a thorough literature review, 60 molecules were designed and these designed molecules were tested for their probable molecular property and expected toxicity Properties of the molecules were calculated by feeding the structures in an online java based program molinspiration property calculator RI PT (http://www.molinspiration.com/cgi-bin/properties) Molecular properties such as miLogP, Total Polar Surface Area (TPSA), No of Atoms, Molecular Weight (MW), No of Hydrogen Bond Donor (HBD), No of Hydrogen Bond Acceptor (HBA), No of Rotatable Bonds, Molecular Volume were calculated Cut off for these properties were kept according to the lipinski’s rule of five and number of violations was calculated Molecules with any Table Estimated property of the designed molecules miLogP TPSA (c Å) No of atoms 66B 3.08 104.883 26.0 67B 3.08 104.883 26.0 68B 2.791 121.873 24.0 69B 3.043 116.322 29.0 72B 2.628 115.641 21.0 78B 4.425 88.094 79B 3.023 81B 3.875 82B 4.471 84B 3.839 MW (Dalton) No of O, N No of OH, NH No of rot bonds Volume (c Å) Violations M AN U Molecule No SC violation were discarded from the study Table 4 319.725 388.888 4 319.725 363.834 292.197 432.941 361.728 319.781 249.462 27.0 403.899 333.982 113.672 25.0 376.877 313.143 90.887 27.0 402.915 337.4 82.098 28.0 416.942 354.343 102.326 30.0 446.968 379.403 AC C EP TE D 388.888 Cut off values for the properties: miLogP: 5, TPSA: 400 c Å, MW: 500 Dalton, No of O, N: 10, No of OH, NH: 5, Volume: 800 c Å After the end of the first property calculation, the qualified designed molecules were further passed through another virtual filter Here different probable toxicities like Mutagenicity, Carcinogenicity, Tumorogenicity and Teratogenicity of the molecules were calculated using another online java based program called Osiris Property Explorer (http://www.organic-chemistry.org/prog/peo/) (Table 2) Molecules reported with good score by both the filter were retained and were considered for docking studies ACCEPTED MANUSCRIPT Table Estimated toxicological properties of the designed molecules Molecule Mutagenic Irritant Reproductive effective 66B 67B 68B 69B 78B 79B 81B 82B 84B 2.1.1 Preparation of protein: moderately toxic highly toxic M AN U Not toxic SC RI PT 72B The crystal structure of wild type Pf-DHFR-TS complex was obtained from protein data bank using Accelrys’ Discovery studio version 2.5 (PDB entry code: 1J3I) Water molecules, co-crystallized ligand (WR99210) were removed and cofactors NADPH and dUMP were allowed to retain Protein was cleaned to TE D remove any extra conformation and binding site was analysed Finally, protein was prepared according to the requirements of the docking protocol 2.1.2 Preparation of ligand: Structures of the designed ligands were prepared by Marvin sketch tool as supported by Sanjeevani EP online program Then the 3D structures of the ligands were imported to Discovery Studio workplace and energy minimization was done by applying CharmM forcefield Further possible ligand conformations were generated by considering an in-silico PH of 7-7.4 Ligand with lowest energy was selected and docked at the active site of AC C the enzyme protein 2.1.3 Molecular Docking: To validate the docking protocol, all atoms RMSD (Root Mean Square Deviation) of the docked ligand with respect to the co-crystallized ligand was calculated The RMSD value for LigandFit protocol was found to be 0.2426 Ao which is less than that of Ao Figure RI PT ACCEPTED MANUSCRIPT Figure All atoms RMSD (co-crystallized ligand in dark blue colour and docked ligand shown in elemental SC colour) Then the Prepared Ligands were docked at the active site of the prepared protein using LigandFit protocol in Discovery Studio 2.5 package At last binding energy of ligand protein complex was calculated with M AN U in situ ligand minimization and non-bond list radius of 14.0 Å by using calculate binding energy protocol Binding pose and ligand orientation at the active site was studied and the molecules were ranked according to their estimated binding energy 2.1 Synthesis TE D Reactions were carried out using dry, freshly distilled solvents under anhydrous conditions, unless otherwise noted Melting points were determined by open capillary tubes using Buchi M-560 melting point apparatus and were uncorrected FTIR spectra of the powdered final compounds were recorded using ATR with a Bruker FTIR spectrophotometer 1H NMR spectra were recorded on a Bruker Advance II spectrophotometer using TMS as an internal reference (Chemical shift represented in δ ppm) Mass spectra were recorded on MS EP ZQMAA255 System Purity of the compounds was checked on TLC plates using silica gel G as stationary phase and was visualized using iodine vapors AC C 2.1.1 Synthesis of substituted phenyl thiazole Synthetic scheme for the preparation of 4-(4-chlorophenyl) thiazol-2-amine is depicted in Scheme chloro acetophenone was reacted with thiourea in the presence of strong oxidising agents like sulfuryl chloride And then the reaction mixture was allowed to reflux for hrs [25] Colour: Light yellow, Nature: Amorphous powder, Yield: 84%, m.p 166o C; FTIR (cm-1): 3433.60 and 3244.40 (NH2), 3114.41 (Ar-H), 1H NMR (400 MHz, DMS0): δ 4.0 (NH2) 6.6 (CH thiazole) 7.32, 7.48 (CH aromatic) 13C NMR (DMSO) δ 100.0, 127.0, 128.8, 129.0, 148.2, 168.4 MS (EI) m/z 209 (M +1) ACCEPTED MANUSCRIPT H2N H3C S O N S sulfuryl chloride + H2N NH2 105o C hr Cl Thiourea RI PT Cl 4-chloro acetophenone Phenyl thiazole Scheme Synthesis of p-chloro phenyl thiazole amine SC 2.1.2 Nucleophillic substitution at triazine ring: Nucleophillic substitution substitution of different selected amines was carried out in three steps as shown in Scheme The first chlorine of cyanuric chloride was substituted at a temperatue of 0-5º C taking M AN U ether as solvent The second chlorine was substituted at a temperature of 40º C with the help of microwave synthesizer where acetone as solvent, whereas the third chlorine atom of cyanuric chloride was substituted by at 110 º C with microwave irradiation taking dioxane as solvent [25-27] Structure and physicochemical properties of synthesized molecules are shown in Table 2.2 Chemistry TE D N2-(4-(4-chlorophenyl) thiazol-2-yl)-6-(piperazin-1-yl)-1, 3, 5-triazine-2, 4-diamine (66B) FTIR (cm-1) 3368.34, 3359.67 (N-H primary, Str.); 1640.02, 851.83(N-H primary, Bend.); 2949.05 (C-H Str.); 1279.57, 1185.64 (CN Aro.) 1H NMR (CDCl3): δ,ppm: 2.76(t 4H, piperazine ring); 3.67 (m, 4H, piperazine ring); 7.12 (s, 1H, thiazole ring); 7.50-7.70 (m, 8H, CH2, phenyl ring) 13 CNMR (CDCl3): δ,ppm: EP 34.21, 45.15, 46.02-46.31, 101.62 , 127.38, 128.56, 134.67, 142.30, 150.73 MS (EI) m/z 387.12 (M +1) N2-(4-(4-chlorophenyl) thiazol-2-yl)-6-(4-methylpiperazin-1-yl)-1,3,5-triazine-2,4-diamine (67B) AC C FTIR (cm-1) 3369.11, 3359.52 (N-H primary, Str.); 1640.22, 850.83(N-H primary, Bend.); 2950.15, 2889.00 (C-H Str.); 1279.12, 1184.64 (CN Aro.) 1H NMR (CDCl3): δ,ppm: 2.39 (s, 3H, CH3); 2.66 (t, 4H, piperazine ring); 3.2(m, 4H, pierazine ring); 7.04 (s, 1H, thiazole ring); 7.52-7.71 (m, 8H, phenyl ring) 13 CNMR (CDCl3): δ,ppm: 44.20, 46.10, 54.32, 125.10, 127.38, 130.61, 134.56, 142.30, 148.73, 167.23 MS (EI) m/z 401.322 (M +1) 2-(4-amino-6-(4-(4-chlorophenyl)thiazol-2-ylamino)-1,3,5-triazin-2-ylamino)ethanol (68B) FTIR (cm-1) 3442.69 (O-H, Str.); 3369.01, 3339.52 (N-H primary, Str.) 1600.92, 842.97 (N-H primary, Bend.); 2953.74 (C-H Str.); 1353.17, 1304.3891 (C-N Ar, Str.) 1H NMR (MeOD): δ,ppm: 3.48(t, 4H, amino ethanol); 7.32 (s, 1H, thiazole ring); 7.59 (d, 2H, phenyl); 7.69 (d, 2H, phenyl) 13 CNMR (MeOD): δ,ppm: 46.34, 57.80, 102.71 , 127.74, 128.60, 134.62, 148.11, 160.56 MS (EI) m/z 362.274 (M +1) ACCEPTED MANUSCRIPT 2-(4-(4-amino-6-(4-(4-chlorophenyl)thiazol-2-ylamino)-1,3,5-triazin-2-yl)piperazin-1-yl)ethanol (69B) FTIR (cm-1) 3424.91, 3353.66, 3289.60 (N-H primary, Str.); 1562.70, 844.93 (N-H primary, Bend.); 1512.49 (N-H secondary, Bend.); 2857.27(C-H Str.); 1198.18, 1331.91 (CN Aro., Str.) 1H NMR (MeOD): δ,ppm: 2.61 (t, 4H, piperazine); 2.71(t, 2H,ethanol); 3.58(m, 6H, piperazine and ethanol); 7.24 (s, 1H, thaizole); 7.48-7.62 (m, 4H, phenyl) 13 CNMR (MeOD): δ,ppm: 42.03, 50.01, 53.36, 62.20, 100.93, 129.25, 129.07, N2-(4-(4-chlorophenyl)thiazol-2-yl)-1,3,5-triazine-2,4,6-triamine (72B) RI PT 160.70, 165.70 MS (EI) m/z 431.01 (M +1) FTIR (cm-1) 3364.43, 3306.57 (N-H primary, Str.); 1530 (N-H secondary, Bend.); 2857.51(C-H Str.); 1352.94, 1254.06 (C-N Aro., Str.), 1H NMR (MeOD): δ,ppm: 4.26 (s, 4H, NH); 7.59 (d, 2H, phenyl); 7.69 (d, 13 2H, phenyl) C NMR (MeOD): δ,ppm: 102.36 , 127.66 , 129.56, 135.23 151.10, 165.92 MS (EI) m/z +1 SC 318.015 (M ) N2-(4-(4-chlorophenyl) thiazol-2-yl)-N4-methyl-6-morpholino-1,3,5-triazine-2,4-diamine (78B) M AN U FTIR (cm-1) 3324.24(N-H secondary, Str.); 1532.17(N-H secondary, Bend.); 2942.45, 2851.74(C-H Str.); 1301.66, 1272.60 (CN Aro., Str.), H NMR (MeOD): δ,ppm: 2.62 (s, 3H, methyl); 3.52-3.64 (sextet, 8H, morpholine); 7.22 (s, 1H, thiazole); 7.59 (d, 2H, phenyl); 7.69 (d, 2H, phenyl) 13 CNMR (MeOD): δ,ppm: 27.56, 43.07, 64.84,102.54, 127.71, 128.58, 166.56, 167.613 MS (EI) m/z 402.362 (M +1) N2-(2-aminoethyl)-N4-(4-(4-chlorophenyl)thiazol-2-yl)-N6-methyl-1,3,5-triazine-2,4,6-triamine (79B) FTIR (cm-1) 3367.34, 3378.29 (N-H, Str), 1563.39, 770.62 (N-H primary, Bend.); 3294.28 (N-H TE D secondary, Str.); 1514.41(N-H secondary, Bend.); 2928.51, 2863.89 (C-H Str.); 1332.94, 1253.72 (CN Aro., Str.), 1H NMR (MeOD):δ,ppm: 2.62 (s, 3H, methyl); 2.82 (t, 2H, ethylamine); 3.27 (t, 2H, ethylamine); 7.22 (s, 1H, thiazole); 7.59 (d, 2H, phenyl); 7.69 (d, 2H, phenyl) 13CNMR (MeOD): δ,ppm: 28.31, 42.52, 57.93, 61.32, 102.69, 126.00, 127.73, 128.61, 165.35 MS (EI) m/z 374.812 (M +1) EP N2-(4-(4-chlorophenyl)thiazol-2-yl)-N4,N4-dimethyl-6-(piperazin-1-yl)-1,3,5-triazine-2,4-diamine (81B) FTIR (cm-1) 3366.34 (N-H secondary, Str.); 1640.02 (N-H Bend.); 2949.05 (C-H Str.); 1279.57, AC C 1185.64 (CN Aro) 1H NMR (MeOD): δ,ppm: 2.82 (t, 4H, piperazine); 3.22 (t, 4H, piperazine); 7.24 (s, 1H, thiazole); 7.59 (d, 2H, phenyl); 7.69 (d, 2H, phenyl) 13 CNMR (MeOD): δ,ppm: 35.95, 45.35, 52.74, 60.38, 102.55, 127.98, 128.64, 150.44 MS (EI) m/z 415.181 (M +1) N2-(4-(4-chlorophenyl)thiazol-2-yl)-N4-methyl-6-(4-methylpiperazin-1-yl)-1,3,5-triazine-2,4-diamine (82B) FTIR (cm-1) 3289.82(N-H secondary, Str.); 1514.93(N-H secondary, Bend.); 2921.71, 2853.30 (C-H Str.); 1353.86, 1290.74(CN Aro.,tr.), 1H NMR (MeOD):δ,ppm: 2.41 (s, 3H, CH3, piperazine); 2.45 (t, 4H, piperazine); 2.61(s, 3H, methyl); 3.56 (t, 4H, piperazine); 7.24 (s, 1H, thiazole); 7.59 (d, 2H, phenyl); 7.69 (d, 2H, phenyl) 13 CNMR (MeOD): δ,ppm: 35.97,41.67, 52.39, 60.41, 102.59, 127.91, 128.65, 150.41, 165.29 MS (EI) m/z 414.827 (M +1) 2-(4-(4-(4-(4-chlorophenyl)thiazol-2-ylamino)-6-(methylamino)-1,3,5-triazin-2-yl)piperazin-1-yl)ethanol (84B) ACCEPTED MANUSCRIPT FTIR (cm-1) 3326.52(N-H secondary, Str.); 1534.93(N-H secondary, Bend.); 2821.75, 2842.38 (C-H Str.); 1341.72, 1291.95(CN Aro.,Str.), 1H NMR (MeOD): δ,ppm: 2.60 (s, 3H, methyl); 2.77 (t, 2H, ethyl); 3.58(m, 6H, piperazine and ethanol); 7.24 (s, 1H, thiazole); 7.59 (d, 2H, phenyl); 7.69 (d, 2H, phenyl) 13CNMR (MeOD): δ,ppm: 27.22, 45.72, 57.31, 62.91, 102.57 , 127.81, 128.74, 150.14, 165.35 MS (EI) m/z 445.019 (M +1 ) Table Structures and physic-chemical properties of the synthesized molecules Colour Rf value TLC* % Yield Melting point! Light yellow 0.72 65 131 o C brown 0.79 NH2 N HN 66B N N N S N Cl NH NH2 N HN N N S 67B N Cl N NH2 N HN N N N S 68B NH Cl HO NH2 N HN N N S N Cl 82 144 o C Brownish yellow 0.53 76 119 o C yellow 0.62 67 118 o C Brownish yellow 0.68 56 143 o C brown 0.74 89 122 o C Light yellow 0.76 62 110 o C brown 0.68 72 147 o C yellow 0.69 79 127 o C N TE D 69B N M AN U N RI PT Structure SC Molecule OH NH2 N HN N N 72B N S EP Cl NH2 NH N HN N N N S 78B O AC C Cl N NH N HN N N 79B N S NH Cl NH2 HN N HN 81B N N N S N Cl NH NH N HN N N 82B S Cl N N N ACCEPTED MANUSCRIPT NH N HN S 84B Yellow sticky N N N N Cl 0.58 85 o C 63 N OH TLC solvent system: Hexane:Ethyl Acetate, 1:1 Cl N Cl R1 N Cl R2 R1 N 0-5 º C Eether N Cl N Cl R1 acetone 40º C, 20 min, µw psi N N R2 N Cl H 2N SC N RI PT * 110º C, min, µw R1 N 1,4-Dioxane psi R2 NH2 Piperazine 67 B NH2 Methyl piperazine 68B NH2 Ethanolamine 69B NH2 Piperazine ethanol 72B NH2 NH2 78B NH2CH3 Morpholine 79B NH2CH3 Ethylene diamine 81B NH2CH3 82B NH2CH3 84B NH2CH3 Cl Phenyl thiazole R1 N N R2 N HN TE D M AN U 66B S N Piperazine Methyl piperazine EP Piperazine ethanol Final Compound Cl Melting point reported according to Pharmacopoeia: Temperature at which the sample melted completely AC C ! S Scheme Synthesis of test compounds by neucleophillic substitution 2.4 In-vitro antimalarial activity: All the in-vitro antimalarial testing was done in the Regional Malarial Research Centre (RMRC), Lahoal, Dibrugarh Supported by Indian Council of Medical Research (ICMR) In-vitro antimalarial assay was carried out according to microassay of Reickmann and co-workers in 96 well-microtitre plates, with minor modifications A stock solution of 5mg/ml of each test compound was prepared in DMSO and subsequent dilutions were prepared with culture medium The test compounds in 20 µl volume at µg/ml and 50µg/ml ACCEPTED MANUSCRIPT concentrations in triplicate wells were incubated with parasitized cell preparation at 37 0C in a CO2 incubator set at 37 0C and 5% CO2 level After 40 hr of incubation, the blood smears were prepared from each well and stained with Giemsa stain The level of paracitemia in tems of % dead rings and trophozoites was determined by counting a total of 100 asexual parasites (both live and death) microscopically using chloroquine as standard drug All the synthesized molecules were evaluated for their In-vitro antimalarial efficacy against CQ-sensitive (3D7) strain of P.falciparum [28] and selected molecules were further studied against chloroquine resistant RI PT strain (RKL2) Table Table Results of in vitro antimalarial activity % paracitemia (RKL2) IC50 (RKL2) 50 µg/ml µg/ml 50 µg/ml 66B 16.5 77.5 37 26.97 67B 23 35.5 * * * 68B 28.5 40.5 19 53.79 69B 0 * * * 72B 3.1 * * * 78B 19 * * * 79B 19 47 11 32 32.5 81B 15.5 37 16.5 43.8 82B * * * 84B 10 * * * TE D M AN U µg/ml Results and Discussion: EP % paracitemia (3D7) SC Molecule The present study results in a systematic and rational plan of work that was carried out in order to AC C overcome the different problems of the classical approach of drug discovery Among the 60 different molecules designed, 10 were selected based on their affinity towards the target protein Though there are some molecules among the selected, which were found to bind at the active site with different binding modes as that of 66B and 79B, they were selected for the study as because they were found to bind with other important amino acids of the same active site Among the ten synthesised molecules, three molecules exhibited good result with wild type Plasmodium falciparum starin (3D7) Molecules found to show good inhibiting activity on the growth of 3D7 strain were further tested with RKL2 strain which is a chloroquine mutant strain IC50 value of molecule 66B suggests that can be further studied and modified to have new active antimalarial antifolate SC RI PT ACCEPTED MANUSCRIPT Figure Structural similarities between active site and the designed ligand mesh structure (a) Blue mesh indicates the 3D view of the active site (b) Mesh structure of ligand 66B (-)Binding energy (Kcal/mol) H-Bond Non bonded interactions 66B 238.804 Asp 54, 1.74 Ao Ile 164, 2.431Ao π - (+) Phe 58, 6.81 Ao , π - (+)Phe 58 6.02 Ao 67B 172.864 Ser 111, 2.31 Ao π - π Phe 116, 4.22 Ao 68B 134.850 Lys 49, 1.93 Ao Ser 111, 1.76 Ao ********* 69B 210.751 Ser 111, 2.10 Ao Asp54, 1.73 Ao Cys 15, 2.28 Ao π - π Phe 116, 4.86 Ao 72B 129.757 Ser 111, 1.75 Ao ********* 78B 130.225 Ser 111, 1.82 Ao Asp 54, 1.94 ********* 79B 246.493 Asp 54, 1.74 Ao Asp 54, 2.36 Ao Thr 185, 2.00 Ao Cys 15, 2.42 Ao π - (+) Phe 58, 5.49 Ao 81B 224.888 Asp 54, 1.95 Ao Asp 54, 2.29 Ao Ser 111, 1.81 Ao Ile 164, 2.17Ao π - (+) Phe 58, 5.40 Ao AC C EP TE D Sl No M AN U Table Bonded and non-bonded interactions of the docked molecules ACCEPTED MANUSCRIPT 158.739 Asp54, 1.81 Ao Ser 111, 2.10 Ao π - (+) Phe58, 5.12 Ao 84B 174.373 Asp54, 1.79 Ao Ser 111, 2.47 Ao Ile 14, 2.09 Ao Ile 164, 2.10 Ao π - (+) Phe 58, 5.54 Ao WR 99210 152.023 Asp54, 1.85 Ao Asp54, 2.16 Ao Ile 14, 2.09 Ao Ile 164, 2.10 Ao π - (+) Phe 58, 5.04 Ao π - (σ) Phe 58, 2.80 Ao RI PT 82B SC Asp-Aspartic acid, Ile-Isoleucine, Ser-Serine, Lys-Lysine, Cys-Cysteine, Thr-Threonine, Phe-Phenylalanine After the comparison of surface mesh property of the designed molecules and three dimensional mesh structure of the active site it can be concluded that, there is a large extent of similarity between them This M AN U simmilarity can be a major reason for active binding of the designed ligands with the receptor protein(Figure 2) It was also noticed that the binding pattern of the designed molecules at the receptor active site is appreciably similar with that of WR99210 Binding energy of the designed molecule suggests that they are more tightly bound with the active site amino acids as compared to WR99210 The molecules were found to interact with key AC C EP TE D amino acids viz Asp 54, Phe 58, Ile 164, Ser 108, Ser 111, Thr 185 (Table 5), Figure Figure Binding pattern of molecules 66B and 79B at the active site ACCEPTED MANUSCRIPT Acknowledgement: Financial support for this work from the DBT, Govt of India is gratefully acknowledged Authors also acknowledge the support obtained from SAIF 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