Insilico design and docking of Novel chemical compounds

INSILICO DESIGN, SYNTHESIS, CHARACTERIZATION AND BIOLOGICAL EVALUATION OF SOME NOVEL 1-HYDROXYNAPHTHALEN-2-YL DERIVATIVES

Presented By\S.SHAH FAISAL 12T01R0050T.NAGAIAH 12Y01R0051 P.SIREESHA 12Y01R0073S.AYESHA SULTANA 12Y01R0077 S.ANWAR HUSSAIN 13Y05R0011

INSILICO DESIGN, SYNTHESIS, CHARACTERIZATION AND BIOLOGICAL EVALUATION OF SOME NOVEL 1-HYDROXYNAPHTHALEN-2-YL DERIVATIVES

UNDER THE SUPERVISION OFMr. T. RAJ KUMAR, M.Pharm Associate Professor,Dept. of Pharmaceutical Chemistry, CES College of Pharmacy, Kurnool.

CONTENTSIntroductionReview of literatureObjectives of studyPlan of the workExperimental workBiological ActivityAnti-Microbial ActivityAnti-Inflammatory ActivityResults& DiscussionSummary and Conclusion Future scopeReferences

Chalconeis anaromaticketoneand anenonethat forms the central core for a variety of important biological compounds, which are known collectively as chalcones orchalconoids. Benzylideneacetophenone is the parent member of the chalcone series. The alternative name given to chalcone are phenyl styryl ketone, benzalacetophenone, -phenylacrylophenone,-oxo-, -diphenyl--propylene and -phenyl--benzoylethylene.Chalcones and their derivatives demonstrate wide range of biological activities such as anti-diabetic, anti-neoplastic, anti-hypertensive, anti-retroviral, anti-inflammatory, anti-parasitic, anti-histaminic, anti-malarial, anti-oxidant, anti-fungal, anti-obesity, anti-platelet, anti-tubercular, immunosuppressant, anti-arrhythmic, hypnotic, anti-gout, anxiolytic, anti-spasmodic, anti-nociceptive, hypolipidemic, anti-filarial, anti-angiogenic, anti-protozoal, anti-bacterial, anti-steroidal, cardioprotective, etc .[1]

INTRODUCTION

SYNTHESISChalcones can be prepared by an aldol condensation between benzaldehyde and acetophenone in the presence of sodium hydroxide as a catalyst.

Mechanism

Structure of Chalcone

REVIEW OF LITREATUREBhagat et al. (2006), Used Commercially available LiOHH2O as a highly efficient dual catalyst for Claisen-Schmidt condensation of various aryl methyl ketones with aryl/hetero aryl aldehydes by providing an easy synthesis of 1,3-diaryl-2-propenones under mild conditions.Biswajit Chandra Das et al synthesized substituted chalcone derivatives and evaluated Anthelmintic and Anti-microbial activities.Rajendra Prasad Y et al (2006) synthesized Five novel chalcones by condensing 2-hydroxy-1-acetonaphthone with aldehyde derivatives in dilute ethanolic potassium hydroxide solution at room temperature.M.R.JayapalandN.Y.Sreedhar (1997)proposed:- Novelmethodforthesynthesisof2,6dihydroxysubstitutedchalconesviaaldolcondensation.inthepresenceofSOCl2/EtOHasacatalyst.Yogesh S et al.(1978) synthesized twelve new chalcones by Claisen-Schmidt condensation and studied for their insect antifeedant activity against the mealy bug of cotton (Phenacoccus solanopsis). All the chalcones have exhibited significant activity.

Venkatesan J et al (2007) Synthesised and evaluated biological activities of 4, 6-diaryl substituted-4,5-dihydro-2-amino pyrimidinesVibhute Y et al (2011) reported an efficient and operationally simple reaction is shown between substituted 2-acetyl-1-naphthol/2-acetyl-1-naphthol and different substitutedbenzaldehydes in presence of base afford chalcones in quantitative yield using grindstone technique.Alka NC et al (2011) made a wide search program towards new and efficient antimicrobial agents; substituted chalcones have been synthesized by condensingbenzaldehyde derivatives with acetophenone derivatives in dilute ethanolic sodium hydroxide solution at room temperature.Rahman MA (2011) presented the review highlights of the recently synthesized chalcones and their derivatives possessing important pharmacological activities.Chetana B. Patil (2009) highlighted antioxidant potential of chalcone, mechanism ofantioxidant activity of chalcones and structure activity relationship of chalcone derivatives for antioxidant ability and different methods to evaluate antioxidant activity of chalcone, anti-inflammatory, cytotoxic and anti-hyperglycemic activity of chalcones is also discussed in this review article.Rajitha G (2015) synthesized a series of N-(naphthalene-2-yl)-3-(substituted phenyl) prop-2-enamides were synthesized by reaction of acetyl amino naphthalene with substituted aromatic aldehydes in presence of absolute ethanol and 30% NaOH.

Bhandarkar SE et al (2014) 2-acetyl-1-naphthol 2 is prepared by Modified Nenchis method which on treatment with furfuraldehyde and KOH gives 1-(1-hydroxy naphthalen-2-yl)-3-(furan-2-yl) prop-2-ene-1-ones 3 in excellent yield.Siva sanker reddy L et al (2015) In pursuit of compounds which show antimicrobial activity, synthesis of novel morpholine linked substituted chalcones done by condensing different aldehydes with diazotized 4-amino acetophenone coupled to morpholine and evaluated them for in-vitro anticancer, anti-inflammatory and antibacterial activities.Siva sanker reddy L et al (2016) In search of molecules that possess biological activity, synthesis of novel pyrrolidines chalcones (AP-JP) obtained by condensing different aldehydes with diazotized 4-amino acetophenone coupled to pyrrolidine and evaluate them for in vitro anticancer, anti-inflammatory and antibacterial activities was undertaken.Siva sanker reddy L et al (2016) , synthesized pyrazolines and chalcones were screened for anticancer activity against human breast cancer cell lines-MCF-7 and MDAMB-468 in the range of 100 nm to 100 m. Inhibition of bovine albumin denaturation and heat-induced hemolysis in vitro methods were followed toscreen for anti-inflammatory activity.

OBJECTIVES OF STUDY

In an effort to continually develop potent anti-inflammatory agents, novel series of napthalene chalcones were synthesized and their inhibitory effects on the bovine albumin and Heat induced hemolysis were evaluated in-vitro.It was thought worthwhile to design fewSynthesis of 1-(1-hydroxynaphthalen-2-yl) ethanone from -napthol. To synthesize 1-hydroxynapthalen-2-yl derivatives by reacting 1-(1-hydroxynapthalen-2-yl) ethanone with substituted benzaldehydes.The chalconeswere prepared by Claisen-Schmidt condensation (Scheme I)

PLAN OF WORKThe work plan is divided into 4 parts.1. To synthesize 1-(1-hydroxynaphthalen-2-yl) ethanone from -napthol. 2. To synthesize 1-hydroxynaphthalen-2-yl derivatives by reacting with 1-(1-hydroxynaphthalen-2-yl) ethanone with substituted benzaldehydes3. To characterize derivatives using NMR, Mass and IR spectrometry.4. To evaluate the Anti-inflammatory and Anti-bacterial activity.

EXPERIMENTAL WORK

INTERNET BASED IN-SILICO DESIGN TOOLS AND MATERIALS USED

In our present study we used the biological databases like Protein Data Bank (PDB) and soft wares like ACD Chem Sketch. ACD/Chem Sketch is the powerful all-purpose chemical drawing and graphics package from ACD/Labs developed to help Chemists quickly and easily draw molecules, reactions and schematic diagrams, calculate chemical properties and design professional reports and presentations. ACD Chem Sketch can convert SMILES notations to Structure and also can convert ACD Chem Sketch Structure to SMILES. [19]The Protein Data Bank (PDB) is the single worldwide archive of structural data of biological macromolecules established in Brookhaven National Laboratories (BNL) in 1971.

pkCSMpredicting small-molecule pharmacokinetic properties using graph-based signaturesThe pkCSM signatures were successfully used across five main different pharmacokinetic properties classes to develop predictive regression and classification models. DOCKING BY HEX8.0

Hex is an interactive molecular graphics program for calculating and displaying feasible docking modes of pairs of protein and DNA molecules. Hex can also calculate protein-ligand docking, assuming the ligand is rigid, and it can superpose pairs of molecules using only knowledge of their 3D shapes. Hex has been available for about 12 years now, but as far as I know, it is still the only docking and superpostion program to use spherical polar Fourier (SPF) correlations to accelerate the calculations, and its still one of the few docking programs which has built-in graphics to view the results. Also, as far as I know, it is the first protein docking program to be able to use modern graphics processor units (GPUs) to accelerate the calculations

Procedure for Docking Studies using HEX:

The parameters used in HEX for the docking process were;Correlation type Shape onlyFFT Mode 3D fast liteGrid Dimension 0.6Receptor range 180 Ligand Range 180Twist range 360 Distance Range 40.

TargetsTable 1: Structure of targets

1IS21W073COX4COX6C0X

Ligands

The structures were drawn in ACD Labs and then converted to PDB format using Open Babel GUI 2.2.1 and further used for docking studies.

Table 2: CHEMICALS AND SOLVENTS

S.NONAME OF CHEMICALSNAME OF SUPPLIER12-Chloro BenzaldehydesS.D.Fine Chemicals(India)24-Nitro BenzaldehydesS.D.Fine Chemicals(India)32,3-DiMethoxy BenzaldehydesS.D.Fine Chemicals(India)43,4-DiMethoxy BenzaldehydesS.D.Fine Chemicals(India)53,4,5-TriMethoxy BenzaldehydesS.D.Fine Chemicals(India)6VanillinS.D.Fine Chemicals(India)7DMSOS.D.Fine Chemicals(India)8EthanolS.D.Fine Chemicals(India)9Potassium hydroxideS.D.Fine Chemicals(India)10-naptholS.D.Fine Chemicals(India)11Acetic acidS.D.Fine Chemicals(India)12Zinc chlorideS.D.Fine Chemicals(India)

Table 3: INSTRUMENTS USEDS.NOEQUIPMENTSMAKER1NMR SpectrophotometerBRUKER 400MHZ2Mass Spectrophotometer3UV SpectrophotometerUV Pharma Spec. 1700 (SHIMADZU)4Hot air OvenVision Lab Equipments5Magnetic stirrerVision Lab Equipments6Vacuum pumpVision Lab Equipments7Precision weighing BalanceContech (0.1 mg precison)8Precision Melting point ApparatusVision Lab Equipments9IR SpectrophotometerBRUKER10UV ChamberVision Lab Equipments

EXPERIMENTAL SECTION Step I: Synthesis of 1-(1-hydroxynaphthalen-2-yl)ethanone from -napthol

In hot glacial acetic acid (80ml), fused ZnCl2(50 gm) was added and refluxed till dissolved, then powdered 1-naphthol (30gm )was added and the mixture was refluxed for about 8 hours then cooled & poured in acidulated water. The solid obtained was filtered, washed, dried and recrystallized from rectified spirit to obtain compound 2.

GAA

- NAPTHOL1-(1-hydroxynaphthalen-2-yl)ethanone

Step II: Synthesis of ChalconesTo a 20 ml solution of 20% Potassium hydroxide in distilled water, acetophenone (0.001 mol) was added and dissolved. To this benzaldehyde (0.001 mol) was added in small amounts with continuous stirring with the help of a mechanical stirrer or about 2 h. The mixture was left in refrigerator for 24 h. Then the yellowish product thus formed is filtered, dried and recrystallized from ethanol. The synthesized compounds were monitored by TLC. Physical data of the compound is given in table 4

+1-(1-hydroxynaphthalen-2-yl) ethanone Substituted aromatic aldehyde

KoH

List of Synthesized moleculesCodeStructureIUPAC NameA(2E)-1-(1-hydroxynaphthalen-2-yl)-3-(4-nitrophenyl)prop-2-en-1-one

B(2E)-3-(4-chlorophenyl)-1-(1-hydroxynaphthalen-2-yl)prop-2-en-1-one

C(2E)-3-(4-hydroxy-3-methoxyphenyl)-1-(1-hydroxynaphthalen-2-yl)prop-2-en-1-one

D(2E)-1-(1-hydroxynaphthalen-2-yl)-3-(3,4,5-trimethoxyphenyl)prop-2-en-1-one

E(2E)-3-(3,4-dimethoxyphenyl)-1-(1-hydroxynaphthalen-2-yl)prop-2-en-1-one

Table no :-4

Table 5:List of Synthesized moleculesS.NoSMILE NotationA[O-][N+](=O)c1ccc(cc1)/C=C/C(=O)c3ccc2ccccc2c3OBClc3ccc(/C=C/C(=O)c2ccc1ccccc1c2O)cc3COc1ccc(cc1OC)/C=C/C(=O)c3ccc2ccccc2c3ODCOc1c(cc(cc1OC)/C=C/C(=O)c3ccc2ccccc2c3O)OCECOc1cc(ccc1OC)/C=C/C(=O)c3ccc2ccccc2c3O

Table 6: Type of Chalcones SubstitutionsProductR1R2A4-NO2B4-ClC4-OCH33-OHD3-OCH34-OCH3, , 5-OCH3E3-OCH34-OCH3

ANTIBACTERIAL STUDIESAll the extracts were evaluated for antimicrobial activity using 2-fold serial broth dilution method in duplicates. This method depends upon the inhibition of growth of a microbial culture in a uniform solution of antibiotic in a liquid medium that is favourable to its rapid growth in the absence of the antibiotic. Minimum Inhibitory Concentration (MIC) of the all extracts was determined. The MIC is the lowest concentration of tested compounds that completely inhibited the growth of the test organisms after 24 and 48 h of incubation at 37 C and 27 C for bacteria and fungi, respectively.

Table 7: Solubility of drugs in DMSO and water for antimicrobial studiesS.NoCompoundSoluble1.Compound A-E100% Sterile DMSO, Distilled Water,Ethanol.

Instruments: Incubator Hot air oven PH Meter (324) Refrigerator Laminar flow Autoclave Ultra low temperature cabinet

Chemicals Malt extract powder for bacteriology Glucose crystalline Pure Peptone type-1 used as a culture media ingredients Agar powder for bacteriology Hydrochloric acid (0.1N), Sodium Hydroxide (0.1N)Dimethyl sulfoxide (DMSO) All the appropriately weighed quantities of ingredients were dissolved in the specified quantity of distilled water. The pH was adjusted to 7.2 7.4 and was sterilized in autoclave at 15 lbs pressure and 121 C for 20 minutes.

Material and methods:Trimethoprim was used as standard drug for the comparison of antibacterial activity. Stock solutions of each 5 ml were prepared in sterile DMSO (Dimethyl sulfoxide) at a concentration of 4mg/ml.

The following ingredients were used for preparation of media. Table 8 :Nutrient Broth media.

INGRIDIENTS(Single Strength) (Double Strength)Beef extract1.0 g 2.0 gYeast extract 2.0 g 4.0 gPeptone5.0 g 10.0 gSodium Chloride 5.0 g 10.0 gDistilled water 1.0 L 1.0 LpH7.2 7.4 7.2-7.4

All the appropriately weighed quantities of ingredients were dissolved in the specified quantity of distilled water. The pH was adjusted to 7.2 7.4 and was sterilized in autoclave at 15 lbs pressure and 121 C for 20 minutes.

:

Table 9: Details of bacterial strains used for determination of antibacterial activityNameEscherichia coli NCIM 2810Bacillus substillsMTCC441Growth MediumNutrient broth mediumNutrient broth mediumGrowth ConditionAerobic30 DaysIncubation Temperature37oC37oC

Incubation Time24 Hours24 Hours

Subculture30 Days30 Days

Preparation of Inoculum The suspension of the organisms obtained after revival and it was used for streaking over nutrient agar plates and subjected to incubation at 30 C for 7 days. From the cultures obtained from the streaking in Petri dishes culturing and sub culturing was performed until pure isolated colonies were obtained. From these isolated colonies fresh sterile nutrient broth media were re inoculated and all the test tubes were incubated at 30 C for 7 days. These nutrient broth cultures served as inocula for the determination of antimicrobial activity of the compounds. Cup-plate/Cylinder- plate method Stock solutions of all compounds Compound A-E were prepared in the concentrations of 5mg/ml with sterile DMSO and measured quantities of stock solutions were diluted to prepare conc. of 1000, 500, 250, 125, 62.5 and 31.25 g / ml of the extract.Forty eight hours cultures of required strains were maintained. Half ml (for 100 ml of media) of 48 hrs culture of was inoculated into the nutrient agar media at 40-50 0C.

Inoculated media (30 ml) was poured in to the Petri plates up to 4-5 mm thickness and allowed to solidify. Cylinders/cups were prepared by using sterile cork borer of 5 mm internal diameter. About 0.1ml of the different concentrations of the extract was added to the cups. The well on Petri plates were loaded with solvent (100% sterile DMSO, 15% sterile DMSO and sterile water).The samples were allowed to diffuse uniformly, by keeping it in refrigerator for 1 hour. The Petri plates were incubated for 48 hours at 30oC and the zone of inhibition was measured.

INVITRO ANTI INLAMMATORY ACTIVITYAssessment of invitro anti-inflammatory activityInhibition of albumin denaturation The anti-inflammatory activity was studied by using inhibition of albumin denaturation technique which was studied according to Mizushima et al and Sakat et al followed with minor modifications. The reaction mixture was consists of test samples of 100-500 g/ml and 1% aqueous solution of bovine albumin fraction, pH of the reaction mixture was adjusted using small amount of 1N HCl. The samples were incubated at 37 C for 20 min and then heated to 51 C for 20 min, after cooling the samples the turbidity was measured at 660nm.( UVVisible Spectrophotometer Model 371, Elico India Ltd) The experiment was performed in triplicate. The Percentage inhibition of protein denaturation was calculated as follows: Percentage inhibition = (Abs Control Abs Sample) X 100/ Abs control

Membrane stabilizationPreparation of Red Blood cells (RBCs) suspension The Blood was collected from healthy human volunteer who has not taken any NSAIDs (Non-Steroidal Anti-Inflammatory Drugs) for 2 weeks prior to the experiment and transferred to the centrifuge tubes. The tubes were centrifuged at 3000 rpm for 10min and were washed three times with equal volume of normal saline. The volume of blood was measured and re constituted as 10% v/v suspension with normal saline.

Heat induced haemolysis The reaction mixture (2ml) consisted of 1 ml test sample of different concentrations (100 - 500 g/ml) and 1 ml of 10% RBCs suspension, instead of test sample only saline was added to the control test tube. Ibuprofen was used as a standard drug. All the centrifuge tubes containing reaction mixture were incubated in water bath at 56 C for 30min. At the end of the incubation the tubes were cooled under running tap water. The reaction mixture was centrifuged at 2500 rpm for 5 min and the absorbance of the supernatants was taken at 560 nm. The experiment was performed in triplicates for all the test samples. The Percentage inhibition of Haemolysis was calculated as follows: Percentage inhibition = (Abs control Abs sample) X 100/ Abs control

RESULTS & DISCUSSION Table 9: Synthesis and CharacterizationCompoundMol. FormulaMol. StructureMol. weightYield ( % )M.PTLCAC19H15NO4321.328060-80c0.7BC19H15ClO2310.777290c0.64CC21H20O4336.3866110c0.74DC22H22O5366.4060119c0.72EC21H20O4336.3864110c0.73

PkCSM Molecule Depiction- Compound A

Table 10: Molecule properties of Compound ADescriptorValueMolecular Weight321.332LogP3.5537Rotatable Bonds4Acceptors4Donors1Surface Area138.267

Table :11 Pharmacokinetic properties of Compound APropertyModel NamePredicted ValueUnitAbsorptionWater solubility-5.233Numeric (log mol/L)AbsorptionCaco2 permeability1.107Numeric (log Papp in 106cm/s)AbsorptionIntestinal absorption (human)95.849Numeric (% Absorbed)AbsorptionSkin Permeability-2.813Numeric (log Kp)AbsorptionP-glycoprotein substrateYesCategorical (Yes/No)AbsorptionP-glycoprotein I inhibitorYesCategorical (Yes/No)AbsorptionP-glycoprotein II inhibitorNoCategorical (Yes/No)

Table 12:DistributionPropertyModel NamePredicted ValueUnitDistributionVDss (human)-0.308Numeric (log L/kg)DistributionFraction unbound (human)0.015Numeric (Fu)

DistributionBBB permeability-0.15Numeric (log BB)DistributionCNS permeability-1.917Numeric (log PS)

Table 13:ExcretionPropertyModel NamePredicted ValueUnit

ExcretionTotal Clearance0.131Numeric (log ml/min/kg)ExcretionRenal OCT2 substrateNoCategorical (Yes/No)

Table 14: MetabolismPropertyModel NamePredicted ValueUnitMetabolismCYP2D6 substrateNoCategorical (Yes/No)MetabolismCYP3A4 substrateYesCategorical (Yes/No)MetabolismCYP1A2 inhibitiorYesCategorical (Yes/No)MetabolismCYP2C19 inhibitiorYesCategorical (Yes/No)MetabolismCYP2C9 inhibitiorNoCategorical (Yes/No)MetabolismCYP2D6 inhibitiorNoCategorical (Yes/No)MetabolismCYP3A4 inhibitiorNoCategorical (Yes/No)

Table 15: ToxicityPropertyModel NamePredicted ValueUnit ToxicityAMES toxicityYesCategorical (Yes/No) ToxicityMax. tolerated dose (human)1.205Numeric (log mg/kg/day) ToxicityhERG I inhibitorNoCategorical (Yes/No) ToxicityhERG II inhibitorYesCategorical (Yes/No) ToxicityOral Rat Acute Toxicity (LD50)2.343Numeric (mol/kg) ToxicityOral Rat Chronic Toxicity (LOAEL)2.567Numeric (log mg/kg_bw/day) ToxicityHepatotoxicityNoCategorical (Yes/No) ToxicitySkin SensitisationNoCategorical (Yes/No) ToxicityT.Pyriformistoxicity1.027Numeric (log ug/L) ToxicityMinnow toxicity-0.376Numeric (log mM)

Compound B

Compound C

Compound D

Compound E

COMPOUNDS1IS21W073COX4COX6COXC-1 (C19H15ClO2 )-366.4-320.55-313.04-87.95-148.99C-2 (C19H15NO4)-343.41-328.1-324.77-98.09-128.87C-3 (C21H20O4)-340.44-362.55-329.09-56.14-147.58C-4 (C21H20O4)-327.9-313.38-325.02-90.59-151.85C-5 (C22H22O5)-374.18-320.76-337.09-45.04-165.93

Table 16: HEX DOCKING SCORES OF COMPOUNDS E-Minimum Scores

COMPOUNDS1IS21W073COX4COX6COXC-1 (C19H15ClO2 )564.79585.33485.10315.50147.99C-2 (C19H15NO4)546.19446.54504.41263.87228.04C-3 (C21H20O4)628.34530.60471.59243.56250.08C-4 (C21H20O4)588.19484.21516.65412.50320.16C-5 (C22H22O5)562.48659.37556.27242.45256.07

Table 17: E-Maximum Scores

Fig. 1:E-Minimum Value of C5 at 1IS2

Fig. 2: E-Minimum Value of C3 at 1W07

Fig.4: E-Minimum Value of C2 at 4COXFig.3:E-Minimum Value of C5 at 3COX

Fig.5: E-Minimum Value of C5 at 6COX

Table 18: FT-IR spectral data of ChalconesCOMPOUNDWAVE NUMBER(cm-1)FUNCTIONAL GROUPC 11605170628511197134413601290 3422C=C (Stetching)C-O Group stretchingCH-Aromatic stretchingCO-CH2 stretching(m) NO symmetric stretch nitro compoundsOH stretch, HbondedC 21600165828361182756.68C=C (Stetching)C-O Group stretchingCH-Aromatic stretchingCO-CH2 stretchingAromatic bending ClC 312281636344715813447OH BendingC-O stretchingCH-Aromatic stretchingCH-CH strechingOH stretch, HbondedC 43252 168415871504, 14593442OH stretchingC=O stretchingCH-CH strechingring C=COH stretch, HbondedC 51580.861656.633372.0110623372C=C (Stetching)C-O Group stretchingCH-Aromatic stretchingCO-CH2 stretching OH stretch, Hbonded1-(1-hydroxynaphthalen-2-yl)ethanone1580.861456-3565C=C (Stetching)C-O Group stretchingOH stretch1017CO-CH2 stretching

Table 19: NMR Spectroscopy of Synthesized Chalcones.COMPOUND1HNMR(CDC13,ppm)Compound A7.602-7.952(1H;d;CH-Ar) ,6.920-6.922 (1H;d;CO-H=), 6.532-8.219(8H;m,Ar-H), 8.026 ( OH)Compound B3.69 (H;s;OCH3 ,6.912-6.916 (1H;d;CO-H=), 8.00-9.45 (1H;d;CH-Ar)Compound C7.604-9.553(1H;d;CH-Ar) ,7.219-7.432 (1H;d;CO-H=) ,7.145-8.577(8H;m,Ar-H) , 3.69(9H;s;OCH3)Compound D7.96-7.98(1H;d;CH-Ar) ,7.96-8.62 (8H;m,Ar-H) 8.62 ( OH) ,3.664(9H;s;OCH3)Compound E7.604-7.659(1H;d;CH-Ar) ,7.219-7.432 (1H;d;CO-H=)7.145-8.577(8H;m,Ar-H) ,8.855 ( OH)3.73(9H;s;OCH3)

COMPOUNDMOL. weightMASS FRAGMENTSCompound A321.32144, 156, 149Compound B310.77144, 166, 112Compound C366.38144, 56, 178Compound D366.40184, 168, 198,128,158Compound E336.38128, 156, 198,182

Table 20: MASS Spectroscopy of Synthesized Chalcones

Fig.6: FTIR Spectra of 1-(1-hydroxynaphthalen-2-yl)ethanone

Fig.7: FTIR Spectra of Compound A

Fig.8: FTIR Spectra of Compound B

Fig.9: FTIR Spectra of Compound C

Fig.10: FTIR Spectra of Compound D

Fig.11: FTIR Spectra of Compound E

Fig.12: Mass Spectra of Compound A

Fragmentation Pattern of Compound A

Fig.13: Mass Spectra of Compound B

Fragmentation Pattern of Compound B

Fig.14: Mass Spectra of C

Fragmentation Pattern of Compound C

Fig.15: Mass Spectra of D

Fragmentation Pattern of Compound D

Fig.16: Mass Spectra of Compound E

Fragmentation Pattern of Compound E

Fig.17: NMR Spectra of A

Fig.18: NMR Spectra of B

Fig.19: NMR Spectra of C

Fig.20: NMR Spectra of D

Fig.21: NMR Spectra of E

ANTI-MICROBIAL STUDIES1000(g/ml)500(g/ml)250(g/ml)125 (g/ml)62.5(g/ml)31.25(g/ml)A16129742B15118330C13109430D14118642E1189420

Table 21: Zone of inhibition (ZOI) of compounds against Escherichia coli

Comparison of Zone of inhibition (Diameter in mm) of all synthesized compounds against Escherichia coli using conc. 1000, 500, 250, 125 and 62.5g / ml.

Conc. of Trimethoprim(g/ml) --1000 mlAvg. zone of inhibition(Diameter in mm)-28mmFig.22: Zone of inhibition (ZOI) of Trimethoprim against Escherichia coli


Table 22:Zone of inhibition (ZOI) of compounds against Bacillus substillsFig.23:Zone of inhibition (ZOI) of Trimethoprim against Bacillus substillsComparison of Zone of inhibition (Diameter in mm) of all synthesized compounds against Bacillus substills using conc. 1000, 500, 250, 125 and 62.5g / ml.

Conc. of Trimethoprim(g/ml) --1000 mlAvg. zone of inhibition(Diameter in mm)-30mm


Table 23:Zone of inhibition (ZOI) of compounds against Aspergillus niger

Fig.24:Zone of inhibition (ZOI) of Ciprofloxacin against Aspergillus niger

Comparison of Zone of inhibition (Diameter in mm) of all synthesized compounds against Aspergillus nigerusing conc. 1000, 500, 250, 125 and 62.5g / ml.

Conc. of Ciprofloxacin(g/ml) --1000 mlAvg. zone of inhibition(Diameter in mm)-26mm


Table 24: Zone of inhibition (ZOI) of compounds against Candida albicans Fig.25: Zone of inhibition (ZOI) of Ciprofloxacin against Candida albicans Comparison of Zone of inhibition (Diameter in mm) of all synthesized compounds against Candida albicans using conc. 1000, 500, 250, 125 and 62.5g / ml.

Conc. of Ciprofloxacin(g/ml) --1000 mlAvg. zone of inhibition(Diameter in mm)-26mm

Concentrationg/mlSample-ASample-BSample-CSample-DSample-ESTD10060.5259.6847.74960.6783.7032.7220069.5169.6572.0469.5187.91585.9430097.8893.9563.9091.00568.480.6140098.5898.6491.8498.4483.9874.9950099.1599.0629.63298.7860.975.27

table 25: Percentage Inhibition of Albumin Denaturation StudiesConcentration g/mlSample-ASample-BSample-CSample-DSample-ESTD10075.11880.7268.5879.3688.19077.2420082.75859.24665.3574.9479.8786.32230073.0890.8256.0273.16666.03679.9540075.1189.2059.5073.25161.96278.51450072.5773.0874.4369.2657.7175.88

ANTI INFLAMMATORY STUDIES

Table 26: Percentage Inhibition of Heat induced hemolysis

SUMMARY & CONCLUSIONThe novel chalcones have been synthesized by ClaisenSchmidt condensation reaction. The synthesis of the chalcone is a single step method. The synthesized chalcone derivatives were undergone physicochemical characterization and the obtained results are given inTable 6. The yields of the synthesized compounds were found to be significant.The structure of the synthesized compounds was confirmed by IR, Mass and 1HNMR.

ConclusionThe synthesized compounds were characterized by TLC, melting point, IR spectroscopy, 1HNMR Spectroscopy and mass spectroscopy. The results obtained from this study confirmed that the product has formed. Henceforth viewing these characteristic properties more compounds can be synthesized and subjected to pharmacological evaluation. These Chalcone derivatives have biological activities like anti-bacterial and anti-inflammatory may be a pave for synthesis and characterization of some new chalcone derivatives.

All the synthesized compounds were tested forin vitroantibacterial activity by agar dilution method. The MIC of the compounds against 16 gram-positive bacterial strains is presented in[Table 21-26]. All the Compounds showed good activity against all bacterial strains than trimethoprim and ciprofloxacin taken as reference standards. The compounds showed moderate to efficient activity against all bacterial strains taken for the screening.

The in vitro anti-inflammatory activity was performed by inhibition of bovine albumin denaturation method and heat induced hemolytic method.

The inhibitory activity of the compounds was compared with the control and the significance factor p was less than 0.001 for all the compounds. The inhibitory activity of the compounds was compared with the control and the significance factor p was less than 0.001 for all the compounds. The result of the anti-inflammatory activity was given in table 25&26.

In an Albumin Denaturation Studies, Sample-A Shown 82.758% inhibition at 200 g/ml, Sample-B 90.82 % at 300 g/ml, Sample-C 74.43 % at 500 g/ml, Sample-D 79.36 % at 100 g/ml & Sample-E 88.190 % at 100 g/ml.In Heat induced hemolysis, Sample-A Shown 99.15 % inhibition at 500 g/ml, Sample-B 99.06 % at 500 g/ml, Sample-C 91.84 % at 300 g/ml, Sample-D 98.78 % at 500 g/ml & Sample-E 85.94 % at 200 g/ml.

FUTURE SCOPEThe existing designed analogs (Compound A-E) can be further modified so as to include substituted pyrazolines, pyrimidines, Oxazoles, hydrazones, benzodiazepines etc which can produce further substituted derivatives. Such analogs can be synthesized, and evaluated for their Anthelmentic and anti-tubercular activities and other activities.It would be interesting to deduce the structure of potent compounds for the activity claimed, they may be our lead molecules and there is a possibility to extend the lead molecule towards drug design using various drug design softwares like maestro, glide, scigress, autodock etc.

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