Synthesis, characterization and molecular docking of sulphacetamide

This is our 7th SEM Practice School work related to Chemistry.
This ppt contains various docking, characterization of the anti-bacterial drug Sulfacetamide, one of the Sulfonamides.

1. Presented By, ANAS. H* 170090658 AMEENA KADAR K. A* 170090656 [VII th SEMESTER B. PHARM] GRACE COLLEGE OF PHARMACY SYNTHESIS, CHARACTERIZATION AND MOLECULAR DOCKING OF SULPHACETAMIDE Under the Guidance of , Dr. Baskar L M. Pharm, PhD Head of Pharmaceutical Chemistry Grace College of Pharmacy 1

2. ACKNOWLEDGEMENT It is a great pleasure to express our gratitude to our guide Dr. Baskar L M. Pharm, PhD and Head of Pharmaceutical Chemistry and sincere thanks to the whole CHEMISTRY department . We honestly express warm regards to our principal Dr. Y Haribabu M. Pharm, PhD and vice principal Dr. C I Sajeeth M. Pharm, PhD. We also extend our sincere thanks to all the supportive mentors of our college. 2

3. CONTENTS INTRODUCTION AIM & OBJECTIVES PLAN OF WORK EXPERIMENTAL SECTION RESULT ANALYSIS CONCLUSION BIBILOGRAPHY 3

4. INTRODUCTION A D rug , or a pharmaceutical, is a substance used to prevent or cure a disease or ailment or to alleviate its symptoms . Drugs are very essential for the welfare of human beings. To meet the requirements, several multidisciplinary approaches are required for the process of drug discovery and development . Drug discovery is a multifaceted process, which involves the identification of a drug, chemical therapeutically useful in treating and management of a disease condition The process of drug discovery includes the identification of drug candidates, synthesis, characterization, screening, and assays for therapeutic efficacy . It takes about 12 - 15 years from discovery to the approved medicine. . 4

5. DRUG DISCOVERY 5

6. DRUG DESIGN It is the process of finding new medications based on the knowledge of a biological target . In the most basic sense, drug design involves the design of molecules that are complementary in shape and charge to the biomolecular target with which they interact and therefore will bind to it . Various approaches ; Rational drug design Insilico / Computer Aided Drug Design (CADD) De Novo Drug Design 6

7. COMPUTER AIDED DRUG DESIGN (CADD ) CADD also known as In silico screening has become powerful technique because of its utility in various phases of drug discovery and development. It can help in identifying drug targets via bioinformatics tools. They can also be used to analyse the target structures for possible binding active sites, generate candidate molecules, check for their drug likeness, dock these molecule with the target, rank them according to their affinities, further optimize the molecules to improve binding characteristics. 7

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9. LBDD methods focus on known ligands for a target to establish a relationship between their physico-chemical properties and activities referred to as structure-activity relationship ( SAR ), that can be used for optimization of known drugs or guide the design of new drugs with improved activity. SBDD (structure of 3D target protein is known, hence test ligands calculated based on affinity after docking). 9

10. Molecular Docking It denotes ligand binding to its receptor or target protein. It is used to generate multiple ligand conformation and orientation and the most appropriate ones are selected . Docking is a  molecular modeling technique that is used to predict how a protein (enzyme) interacts with small molecules (ligands ). 10

11. Basic steps involved in Molecular Docking: 11

12. Overview of Molecular Docking 12

13. LIGAND Sulfacetamide is a sulfonamide, that is sulfanilamide acylated on the sulfonamide nitrogen .  It inhibits bacterial folic acid synthesis by competing with para amino benzoic acid . With a broad spectrum of action, it is used as an anti-infective topical agent to treat skin infections and as an oral agent for urinary tract infections. 13

14. Target – DIHYDROPTEROATE SYNTHASE PDB ID – 1AJ0 Dihydropteroate synthase (DHPS) is essential for the de novo synthesis of folate in prokaryotes, in lower eukaryotes such as protozoa and yeast, and in plants. DHPS is absent in mammals. This makes it an ideal drug target . 14

15. SOFTWARES USED IN DOCKING USE SOFTWARE FEATURES Drawing tools Chem Draw Helpful for the analysis of NMR and Mass spectroscopic studies. Useful for MOT, VBT and also for Hybridization. Chem Sketch Gives the physical properties of molecules. Covert structure to SMILE form. 2 . Data Banks RCS PDB Information about the 3D shapes of proteins, nucleic acids, and complex assemblies . TTD Information about the known and explored therapeutic protein and nucleic acid targets, the targeted disease, pathway information and the corresponding drugs directed at each of these targets.  15

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19. Pubchem Freely accessible chemical information Drug Bank Informations of  drug interactions, pharmacology, chemical structures, targets, metabolism ChEMBL ChEMBL is a database of bioactive drug-like small molecules, it contains 2-D structures, calculated properties (e.g. logP , Molecular Weight, Lipinski Parameters, etc.) and abstracted bioactivities (e.g. binding constants, pharmacology and ADMET data) 3. Energy Minimization MOE An integrated computer- aided molecular design platform. Mainly used for the macromolecules. 19

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22. Chem 3D Pro To generate  3D  models  To minimize the energy of ligand. 4. Prediction Pass Prediction A prediction of activity spectra for substances. Swiss Target Prediction To predict the most probable protein targets of small molecules . 5. Protein Validation Ramachandhran Plot Server & PROCHECK Main-Chain Conformational Tendencies of Amino Acids. 22

23. 7. Pharmacokinetic Parameters & toxicity Swiss ADME To compute physicochemical descriptors as well as to predict ADME parameters, pharmacokinetic properties, drug like nature and medicinal chemistry friendliness of one or multiple small molecules to support drug discovery. ADMET SAR Free tool for the prediction of chemical ADMET properties based on QSAR. 8. Chemical Tool Box Open Babel Chemical toolbox designed to speak the many languages of chemical data. 6. Chemoinformatics Molinspiration Molecule manipulation and processing, including SMILES and SD file conversion, generation of tautomers, calculation of various molecular properties needed in QSAR. 23

24. AutoDock Vina Significantly improves the average accuracy of the binding mode predictions compared to AutoDock 4.2. PatchDock An object recognition and image segmentation techniques used in computer vision. Online docking program. SwissDock A web server program dedicated to the docking of small molecules on target proteins 9. Docking Tools AutoDock 4.2 Suite of automated docking tools. It is designed to predict how small molecules, such as substrates or drug candidates, bind to a receptor of known 3D structure. 24

25. 10. Visualization Tools Discovery Studio An interactive package with a broad variety of features for modelling and simulation. PyMol An open source, user-sponsored, molecular visualization system. Chimera Program for the interactive visualization and analysis of molecular structures and related data. 25

26. SYNTHESIS OF SULFACETAMIDE The discovery of sulfonamides is a significant milestone event in the human chemotherapeutic history. Sulfonamides are synthetic compounds that have activity against both gram-positive and gram- negative bacteria. Originally they were synthesized in Germany as azodye known as prontosil . P rontosil breaks down in the human body to produce Sulfanilamide which is the active agent that kills streptococcus bacteria . Sulfacetamide found highly successful use in fighting urinary tract infections starting in 1941 . 26

27. RECRYSTALLIZATION Separation of a mixture of solids or purification of solids are most readily achieved by recrystallization. The basic principles employed in recrystallization is the dissolution of the crude material in a suitable solvent and filtration is the necessary step, to remove any suitable impurities in the solution. FILTRATION TECHNIQUES Technique used to separate solids from liquids, is the act of pouring a mixture onto a membrane (filter paper) that allows the passage of liquid (the filtrate) and results in the collection of the solid. Two filtration techniques are generally used in chemical separations in general chemistry lab: “ gravity filtration” and “ vacuum/suction” filtration . 27

28. Suction filtration is a technique which allows for a greater rate of filtration. Whereas in normal filtration gravity provides the force which draws the liquid through the filter paper, in suction filtration a pressure gradient performs this function. It is used in recrystallization experiments. 28

29. DRYING Drying has been defined as the process whereby moisture is vaporized from a material and is swept away from the surface, sometimes under vacuum, but normally by means of carrier gas which passes through or over the material. THIN LAYER CHROMATOGRAPHY TLC is simple, convenient and useful technique because it is quick, cheap and accurate and easy to use. Useful to separate the required product from the mixture. This technique is helpful to monitor organic reactions, similarly in quality control studies on stability testing, shelf life determination or degradation. 29

30. Overview of TLC Method 30

31. MELTING POINT DETERMINATION The temperature at which a solid melts and becomes a liquid is the Melting Point . Hence , different compounds tend to have different melting points. A pure, nonionic, crystalline organic compound usually has a sharp and characteristic melting point. A mixture of very small amounts of miscible impurities will produce a depression of the melting point and an increase in the melting point range . Consequently, the melting point of a compound is a criterion for purity as well as for identification. 31

32. SPECTRAL CHARACTERIZATION TECHNIQUES The most widely used methods of organic compound identification is that to ensure the interactions of compounds with electromagnetic radiations of different wavelength. There are a number of analytical technique which facilitates to identify and characterize the compound, some of them are: Ultra Violet -Visible Spectroscopy Infrared Spectroscopy UV-Visible Spectrophotometer Fourier Transform Infra-Red Spectrophotometer 32

33. AIM AND OBJECTIVES AIM The purpose of the study is to carry out Synthesis, Characterization And Molecular Docking Of Sulphacetamide. OBJECTIVES To design and characterization of crude drug of Sulphacetamide. Calculation of lipinskis rule and drug likeness score using Molinspiration software. Determination of pharmacokinetic properties and toxicity using Swiss ADME and ADMET SAR. Determination of pharmacological effects by Pass Prediction . Molecular docking of lead molecule towards Dihydropteroate synthase using Autodock 4.2, Autodock Vina, PyRx, Patch dock and Swiss dock . To carry out the spectroscopic (FTIR, UV), TLC and Melting Point of Sulphacetamide. 33

34. PLAN OF WORK 34

35. EXPERIMENTAL SECTION Docking of sulphacetamide by different software AutoDock 4.2 It is a suite of automated docking tools . It is designed to predict how small molecules, such as substrates or drug candidates, bind to a receptor of known 3D structure. It actually consists of two main programs:  autodock  performs the docking of the ligand to a set of grids describing the target protein;  autogrid  pre-calculates these grids. In addition to using them for docking, the atomic affinity grids can be visualised . 35

36. Protein Preparation Available in RCS PDB, TTD, download it in the PDB format. Refine it by using MOE. Protein ID – 1AJ0 Open Autodock window File Read Molecule Open Protein Give Charges: > Edit Charges Add kollman charges Saving file: > Save as PDBQT , where Q Charges & T Type of Atom 36

37. Ligand Preparation The structures were built by ChemDraw Ultra 8.0 version, ACD Chem sketch and saved in PDB format. Energy Minimization - Chem 3D Pro Open Autodock window Ligand Open Choose File Ligand Torsion tree Choose Root Detect Root Choose torsions Set number of torsions. Ligand Output Save as PDBQT. Grid Formation Grid Macromolecule Choose Grid Set map types Choose ligand grid box confine the receptor to the grid box file close saving current Grid Output Save as GPF (Grid Parameter File). 37

38. Docking Docking Macromolecule Set rigid filename Receptor Docking Ligand select default parameters Accept Search parameters Genetic algorithm Accept Docking Lamarckian GA (4.2) Save as Ligand.dpf (Docking Parameter File) Run Autogrid Program path name Select autogrid4.exe.file Launch Run Autodock Select autodock4.exe and select ligand.dpf Launch. Visualization of Docking Result Analyse Docking Open Choose ligand.dlg (docking log file) Analyse Macromolecule Analyse Conformations Load Analyse Docking Show interactions Analyse Clusterings Show 38

39. 2. AutoDock Vina It significantly improves the average accuracy of the binding mode predictions compared to AutoDock 4 . Pre-calculating grid maps (Vina does that internally), and some other implementation tricks, such as pre-calculating the interaction between every atom type air at every distance. It also uses the same type of structure format (PDBQT) for maximum compatibility with auxiliary software . 3. SwissDock It is a docking web server, the structure of the target protein, as well as that of the ligand, can be automatically prepared for docking . The interpretation of docking results and their integration into existing research pipelines is greatly facilitated by the seamless visualization of docking predictions in the UCSF Chimera molecular viewer. 39

40. 4. PatchDock It is a geometry-based molecular docking algorithm. It is aimed at finding docking transformations that yield good molecular shape complementarity . SYNTHESIS OF SULFACETAMIDE 40

41. RECRYSTALLIZATION Dried Drug + few ml of Ethanol/Hot water. FILTRATION Done by Suction/ Vacuum filtration. CHARACTERIZATION OF SULPHACETAMIDE Melting Point Determination Thin Layer Chromatography (TLC) Spectral Studies UV – Visible Spectroscopy Fourier Transform Infra- Red Spectroscopy (FTIR) 41

42. THIN LAYER CHROMATOGRAPHY TLC Plate – Coating the plate with Silica gel Mobile Phase – A mixture of 50 volume of 1-Butanol + 25 volume of ethanol + 25 volume of water + 10 volume of strong ammonia solution. Test Solution – Dilute a suitable volume with water to produce a solution containing 4%w/v of Sulphacetamide. Reference Solution – 0.2%w/v solution of Sulphanilamide in Water. Detecting Method – Iodine Chamber. UV- VISIBLE SPECTROSCOPY Calculate the λ max with the help of “ UV Probe ” software. FOURIER TRANSFORM INFRA-RED SPECTROSCOPY “ IR Solution ” software helps to determine the FTIR peak, which determines the Structural Conformations of the particular drug. 42

43. RESULTS AND DISCUSSION CHEM DRAW 43

44. 2. CHEM 3D PRO 44

45. 3. CHEM SKETCH 45

46. Wireframe Sticks Ball & Stick Different 3D visualization by using Chemsketch 3D Viewer 46

47. 4. PASS PREDICTION 47

48. 5. Swiss Target Prediction 48

49. 6. Molinspiration miLogP TPSA MW No.of H-bond acceptors No.of H-bond donors No. of violations No. of rotatable bonds Volume -0.56 89.26 214.25 5 3 2 174.71 Properties GPCR ligand Ion channel modulator Kinase inhibitor Nuclear receptor ligand Protease inhibitor Enzyme inhibitor -0.46   -0.48   -0.70   -1.28   -0.36   -0.12   Bioactivity 49

50. 7. Lipinski’s Rule of Five 50

51. Formula Mol.Wt No. of Heavy atoms No.of arom . heavy atom No.of Rotatble bonds No.of H-bond acceptors No.of H-bond donors Molar Refractivity TPSA   C 8 H 10 N 2 O 3 S 214.24 g/ mol 14 6 3 3 2 51.75 97.64 A 2 Water Solubility GI Absorption BBB Permeant Bioavailability Score Log Kp Very soluble High No 0.55 -8.29 cm/s 8. SWISS ADME Physicochemical Properties 9. ADMET SAR Human Intestinal Absorption BBB Human Oral Bioavailability Carcinogenicity (binary) Eye corrosion + 0.9939 + 0.9764 + 0.8286 - 0.6429 - 0.9706 51

52. Eye Irritation Hepatotoxicity Acute Oral Toxicity Water Solubility Plasma Protein Binding Biodegradation + 0.6471 + 0.8750 2.261 -1.292 0.533 -0. 8250 10. RAMACHANDRAN PLOT SERVER 52

53. No. of residues in favored regions No. of residues in allowed regions No. of residues in outliner region 240 (97.166%) 5 (2.024%) 2 (0.810%) 11. AUTODOCK Result Analysis Software Protein Ligand Docking Score Amino acid residue AutoDock 4.2 1AJ0 Sulphacetamide -5.87 GLY217 , ILE20 . ARG265, ASN 115 , ILE 117, MET139, PHE190, LYS 221 , SER 219 AutoDock Vina 1AJ0 Sulphacetamide -5.7 SER 219 , LYS 221 , ARG 255, GLY 217 , LEU 215, PHE188, ASP 185, ASN 115 , ILE 20 , PHE 190 53

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56. 12. AUTODOCK VINA 56

57. 13. SWISSDOCK S. No. Compound Full fitness (Kcal/mol) Estimated Δ G (Kcal/mol) 1. Sulphacetamide -1089.31 - 6.93 57

58. 14. Patch Dock ACE (Kcal mol-1) Area Amino acid Residue -239.73 330.20 PRO 145, PRO 190, GLN 149, LYS 192, GLY 65, ARG 63, THR 62, PHE 190, GLY 191, GLY 189, ALA 151, ASN 197, MET 148 58

59. CHARACTERIZATION OF SULPHACETAMIDE Characterization testing is used to gain an understanding of the physical and chemical properties of pharmaceutical materials. Physical Properties Chemical Properties PHYSICAL PROPERTIES PHYSICAL PROPERTIES PROPERTY VALUE Physical state Solid Colour White Powder Molecular Formula C 8 H 10 N 2 O 3 S Molecular Weight 214.243 PH 7.4 Melting Point 183 C Rf Value 0.93 59

60. SPECTRAL STUDIES UV-VISIBLE SPECTROSCOPY λmax = 263nm 60

61. FTIR 61

62. CONCLUSION Sulphacetamide drug had synthesized and characterized which has proven that and leads to the path of its attractive feature of the antimicrobial activity. Drug likeness of the compound was confirmed by the software viz., Molinspiration, Lipinski’s rule of five, and pass prediction . Protein validation was done by the Ramachandhran plot. Molecular docking was performed by using AutoDock 4.2, AutoDock Vina, SwissDock, and PatchDock and found that the drug majorly binds to amino acids like Asparagine, Glycine, and Serine which has been matched and supported with the reference literature. Pharmacokinetic parameters were evaluated by Swiss ADME, ADMET SAR and ADRs are studied by Pass Prediction . 62

63. Sulphacetamide was synthesized from Sulphanilamide and the product was obtained with a good yield. Physicochemical properties like Melting Point, Retention factor were performed and reported. The product was confirmed by FTIR (Functional groups); UV- visible spectroscopy was done to get λ max . In near future, we aim to elaborate on the chemistry and pharmacological background of the present moiety that we had taken it. 63

64. BIBILOGRAPHY Hira Saleem , Arooma Maryam, Saleem Ahmed Bokhari ,  Ayesha Ashiq ,   Sadaf Abdul Rauf ,   Rana Rehan Khalid,   Fahim Ashraf Qureshi ,  and Abdul Rauf Siddiqi : Design, synthesis, characterization and computational docking studies of novel sulfonamide derivatives. EXCLI journal, Published online 2018 Feb 1, Pages: 169-179 . https :// www.technologynetworks.com/drug-discovery/articles/target-identification-validation-in-drug-discovery-312290 https ://slidetodoc.com/aim-preparation-and-evaluation-of-sulphacetamide-sodium-eye/ https://www.molinspiration.com/cgi-bin/properties https://www.uniprot.org/uniprot/P0AC13 64

65. 6. Amol B. Deore *, Jayprabha R. Dhumane , Hrushikesh V Wagh , Rushikesh B. Sonawane : The Stages of Drug Discovery and Development Process. Asian Journal of Pharmaceutical Research and Development, pages: 62-66. 7. Neema Bisht and B. K. Singh: ROLE OF COMPUTER AIDED DRUG DESIGN IN DRUG DEVELOPMENT AND DRUG DISCOVERY. IJPSR, 2018; Vol. 9(4): 1405-1415. 8. http ://dx.doi.org/10.22192/ijarbs.2017.04.02.009 9. Supplementary Table S1: A list of 211 pieces of software for CADD. Number of citations was obtained using Google Scholar, last accessed on 14th April 2021 . 10. http :// dx.doi.org/10.22270/ajprd.v7i6.616 http ://dx.doi.org/10.13040/IJPSR.0975-8232.9(4). 1405-15 12. https://www.elsevier.com/locate/bfopcu 65

66. THANK YOU HAVE A GOOD DAY 66