Synthesis, Characterization, Anti-Oxidant and Anti Inflammatory Activity Evaluation of Chalcones & Pyrazoline Derivatives

Introduction

In Medicinal chemistry, derivatives of 4-Amino Acetophenone found to have diverse therapeutic applications.  Many  4-Amino  Acetophenone  derivatives  have  been  developed  as  chemo therapeutic  agents  and  are  widely  used.  4-Amino  Acetophenone moiety  carrying  compounds exhibit  various  activities  like  anti-bacterial,  anti-fungal,  anticancer,  anti-convulsion,  anti-inflammatory, anti-oxidantetc^1,2,3,4.

Chalcone  is  an  aromatic  ketone  and  an  enone  that  forms  the  central  core  for  a  variety  of important  biological  compounds,  which  are  known  collectively  as  chalcones  or  chalconoids. These  are  coloured  compounds  because  of  the  presence  of  the  chromophore  -CO-CH=CH-. Chalcones  bears  a  very  good  synthon  so  that  variety  of  novel  heterocycles  with  good pharmaceutical profile can be designed. Chalcones can be prepared by Claisen-Schmidt condensation between an aromatic aldehyde and an aromatic ketone in the presence of sodium hydroxide as a catalyst.

Chalcones are popular intermediates for synthesizing various heterocyclic compounds.  The compounds with  the  backbone  of  chalcones  have  been  reported  to  possess  various  biological activities such as anti-microbial, anti-inflammatory of chemical mediators  release,  inhibition of leukotriene B4, inhibition of tyrosinases an inhibition of aldose reductase activities. The presence of  a  reactive  α,β-unsaturated  keto function in chalcones is found to be  responsible  for  their biological activities⁵.

Heterocyclic compounds are well known  for  their wide  range  of biological  applications out of which  pyrazolines  occupy  unique  position  due  to  dominant  applications.  Pyrazolines are well known and important nitrogen-containing five-membered heterocyclic compounds.  Several pyrazoline  derivatives  have  been  found  to  possess  considerable  biological  activities,  which stimulated research activity in this field. Considerable attention has been focused on Pyrazolines and substituted Pyrazolines due to their interesting biological activities.  They  have  found  to possess  anti-fungal,  anti-depressant,  anti-convulsant,  anti-inflammatory,  anti-bacterial,  anti-cancer,  antioxidant,  anti-pyretic,  anti-neoplastic  activities,  anti-viral,  anti-amoebic,  anti-cholinergic,  antidiabetic,  anti-HIV,  antimalarial,  anaxiolytic,  antiparasitic,  anti-allergic,  anti-microbial,  anti-tuberculosis,  tyrosinase  inhibitor,  hypoglycemic,  hypotensive,  immunosuppressive, anti-tumor properties^6,7. Pyrazoline is dihydropyrazole which is a five membered heterocyclic compound containing two nitrogen  atoms  in  adjacent  position  having  only  one  endocyclic double  bond. Among  all  the pyrazoline derivatives  2-pyrazoline  has  gained  the  most  importance because  of  its  diverse biological  activities.  2-Pyrazolines  display  a  broad  spectrum  of  potential  pharmacological activities and are present in a number of pharmacologically active molecules such as phenazone/ amidopyrene/  methampyrone  (analgesic  and antipyretic),  azolid/  tandearil  (anti-inflammatory), indoxacarb  (insecticidal),  anturane  (uricosuric),  etc.  In addition, pyrazolines have played a crucial part in the development of theory in hetero cyclic chemistry and also used extensively in organic synthesis.

Materials and Methods

The  Identification  and  characterization  of  synthesized  compounds were  carried out by  the  following procedure  to  ascertain  that  all  the prepared  compounds were of different chemical nature  than  the  respective parent  compounds. This  involved  the determination of  the melting  point,  solubility  character’s,  and  their  behaviour  in Thin  Layer Chromatography  (TLC) studies as  compared  to  that  of  their  parent  compounds  and Nuclear Magnetic Resonance  (¹H NMR) data, Infra-red spectroscopy. The melting point was determined for the synthesized compounds were taken in open capillary tubes by using Arson digital melting point apparatus which were uncorrected.

The TLC was done on precoated aluminium plates of silica gel 60 F254 obtained from MERCK and visualization of spots was done by using UV TLC visualization chamber.IR Spectra recorded on BRUKER,¹H NMR spectra of the compounds recorded on BRUKER-AMX 400 MHz, UV  Visible  spectrophotometer  of  Lab  India  was  used  for  screening  the Synthesized compounds for antioxidant activity.

Methodology

Step1: Diazotization and coupling of amine Group in 4-Amino Acetophenone with morpholine:

0.01 mol of 4-Amino acetophenone is dissolved in a mixture of 3ml of Conc. HCl and 6ml water.  Then  dissolve  1.35gm  of  sodium  nitrite  in  6ml  of  water  .  Both the above solutions are cooled to 0-5^oC by keeping in an ice bath and also by the addition of few pieces of ice to the 4-Amino acetophenone solution. Now sodium nitrite solution is added drop wise to the mixture  of  4-Amino  acetophenone  in  Conc. HCl  and water with  continuous  stirring  in order  to avoid rise in temperature during the reaction. After the completion of sodium nitrite addition, morpholine is added to the reaction mixture with continuous stirring at the same temperature i.e., 0-5^oC until a yellow coloured precipitate is obtained. This is then filtered and washed with ice cold water and dried.

Step 2: Preparation of Chalcones

0.005 mol of diazotized 4-Amino Acetophenone coupled with morpholine and equimolar amount of aldehydes in a round bottomed flask containing 50ml of ethanol, and then 1ml of 40% KOH is added at the room temperature with continuous stirring on magnetic stirrer for 2hrs. The reaction mixture is monitored by TLC during the reaction. After the completion of the reaction the reaction mixture is poured into crushed ice with a little amount of dilute HCl in order to neutralize potassium hydroxide. Then the solid is filtered and washed with cold water, dried and recrystallized from ethanol.

Step 3: Preparation of Pyrazolines

0.003mol of chalcones and 0.024mol (1.2ml) of  hydrazine hydrate in a round bottomed flask containing 30ml ethanol as a solvent is refluxed  for  2hrs  at  60^oC  by monitoring  with  TLC,  After the completion, the  reaction mixture  is  poured  into crushed ice and the solid is filtered and dried.

Figure 1: Protection of Amine group in 4-Amino Acetophenone  Click here to View figure

 

Figure 2: Synthesis of Chalcones  Click here to View figure

 

Figure 3: Cyclisation of chalcones to Pyrazolines  Click here to View figure

 

Antioxidant Activity

DPPH Method :(1, 1-diphenyl-2-picrylhydrazyl⁸

To 3 ml of various concentrations of test/ standard solution, 1 ml solution of DPPH 0.1 mM(0.39 mg  in 10 ml methanol) was added. Simultaneously blank samples were prepared for each concentration without addition of 0.1mM of DPPH solution and equal amount of methanol was added to each blank sample. 3 ml of methanol and 1 ml of 0.1mM DPPH was added and used as control. Ascorbic acid was used as standard for comparison. After incubation for 20 minutes in dark, absorbance was recorded at 517 nm. % scavenging was calculated using the formula %Scavenged = [(A-A1)/A] x 100

Where A=Absorbance of the control ; A1=Absorbance of the test or standard

Hydrogen Peroxide Method⁹

The  ability  of  the  synthesized  compounds  to  scavenge  H₂O₂  was  determined  by  the following  procedure. A solution of H₂O₂ (40mM) was prepared in phosphate buffer (pH 7.4). The  concentration  of  H₂O₂  was  determined  by  absorption  at  230  nm  using  a  UV  Visible spectrophotometer. Test solutions were added to a H₂O₂solution (0.6 ml 40mM). The absorbance of H₂O₂ at 230 nm was determined after 10 minutes against blank solution containing phosphate buffer and test compound without H₂O₂. Control solution was prepared by taking a solution of H₂O₂ in phosphate buffer (pH 7.4) and its absorbance was measured. The percentage of H₂O₂ scavenging by the test and the standard was calculated using the following formula. %Scavenged = [(A-A1)/A] x 100.

Where A=absorbance of the control, A1= absorbance of the test /standard

Invitro Anti-Inflammatory Activity

Inhibition of Egg Albumin Denaturation Method¹⁰

To  2ml  of  various  concentrations  of  test  or  standard  solutions  2.8ml  of  normal  saline (PH=7.4) and 0.2ml of 1% egg albumin solution was added. Simultaneously blank samples were prepared for each concentration without addition of 1% egg albumin solution and equal volume of normal saline (PH=7.4) was added to each blank sample. To 4.8ml of normal saline (PH=7.4), 0.2ml  of  1%  egg  albumin  solution was  added  and  used  as  control. The test/standard samples were incubated for 15min at 70^oC. Then the tubes were cooled under running tap water and then absorbance was recorded at 660nm. % inhibition of denaturation of egg albumin was calculated using the following formula.

% Inhibition = [(A-A1)/A] x 100

Where A=absorbance of the control. A1= absorbance of the test /standard.

Heat Induced Haemolytic Method¹¹

To  1ml  of  various  concentrations  of  test  or  standard  solutions,  1ml  of  1%  RBC’s suspension  was  added.  Simultaneously blank samples were prepared for each concentration without addition of 1% RBC’s suspension and equal amount of normal saline was added to each blank sample. Equal amount of 1% RBC’s suspension and normal saline was added and used as control. All these samples were taken into centrifuge tubes and incubated in water bath at 56^oC for 30 min. The tubes were cooled under running tap water and then centrifuged at 2500 rpm for 15 min and absorbance of supernatant was taken at 560 nm. % inhibition was calculated using formula

% Inhibition = [(A-A1)/A] x 100

Where A=absorbance of the control.

A1= absorbance of the test /standard.

Calculation of IC₅₀ values¹²

IC₅₀ was calculated using Graphpad prism software.  In order to calculate IC₅₀ initially XY data table was created. Then the logarithm of the concentration of the inhibitor was entered into X and response was entered into Y. From  the  data  table  click Analyze,  choose  nonlinear regression,  then  choose  the  panel  of  equations  “Dose  response  curves-Inhibition”  and  then choose  the equation “log  (Inhibitor) v/s normalized  response-variable  slope”. Then we will get IC₅₀ values for the given data.

Results and Discussions

Table 1:  Physico-chemical characterization data of synthesized compounds

Solvent used: *   = n-Hexane: Ethyl acetate (6:4)      ** = n-Hexane: Ethyl acetate (4:6)

Compound 2A: (2E)-1-{4-[(E)-morpholin-4-yldiazenyl] phenyl}-3-(4-nitrophenyl) prop-2-en-1-one:

Molecular Formula

C₁₉H₁₈N₄O₄, Molecular Weight: 366.37, Physical state:  crystalline powder, Colour: Green, Melting Point: 216-218^oC, Rf Value: 0.47 (n-hexane: Ethyl acetate; 6:4), Solubility: Chloroform, DMSO, Ethyl acetate, Percentage yield   : 87%.

IR (KBr disk)

γ (cm-¹), C-H Stretching (Morpholine) – 3104.94 Cm^-1, C-H Stretching (Aromatic)  – 2980.08 Cm^-1 , C-H Stretching (olefins) – 3076.89 Cm^-1, C=O – 1658.49 Cm^-1 ,C=C Stretching (Olefin)   – 1598.09 Cm^-1 ,  C=C (Aromatic)   – 1517.45 Cm-1, N=O (Nitro)  – 1431.77 Cm^-1, C-H Bending (Aromatic) – 832.65 Cm^-1C-H Stretching (Morpholine) – 3104.94 Cm^-1, C-H Stretching (Aromatic) – 2980.08 Cm^-1  , C-H Stretching (olefins) – 3076.89 Cm^-1 , C=O – 1658.49 Cm^-1 ,C=C Stretching (Olefin) – 1598.09 Cm^-1 , C=C (Aromatic) – 1517.45 Cm-1 , N=O (Nitro)  – 1431.77 Cm^-1 , C-H Bending (Aromatic) – 832.65 Cm^-1 .

¹H NMR (CDCl₃)

δ(ppm)3.893 to  3.884  (8H,m,morpholine protons),7.575  (2H,d,CC&CE Ar-H,J=8.8Hz), 7.693 (1H,d,Ci Olefinic proton=15.6 Hz), 7.801 (2H,d,C2&C6Ar-H,J=9Hz),  7.841  (1H,d,CiiOlefinic  proton=16),  8.071  (2H,d,CB&CF  Ar-H,J=8.4 Hz), 8.29 (2H,d,C3&C5Ar-H,J=8.4 Hz).

Compound 2B: (2E)-1 -{4-[(E)-morpholin-4-yldiazenyl]phenyl}-3-(3-nitrophenyl)prop-2-en-1 -one

Molecular Formula

C₁₉H₁₈N₄O₄, Molecular Weight  :  366.37, Physical state :  crystalline powder ,Colour: Green ,Melting Point: 136-1 39oC R_fValue : 0.7 (n-hexane:Ethylacetate;4:6) ,Solubility : Chloroform, DMSO, Ethyl acetate, Percentage yield   :   85%.

IR (KBr disk)

γ (cm^-1 ),C-H Stretching (olefins) – 3073.45 Cm^-1,C-H Stretching (morpholine)  – 2963.93 Cm^-1,C-H Stretching (Aromatic)   – 2990.21 Cm^-1,C=C (Aromatic) – 1522.56 Cm^-1,C-H Bending (Aromatic) – 805.79 Cm^-1, C=O – 1660.04 Cm^-1,N=O (Nitro)  – 1437.85 Cm^-1 , C=C Stretching (Olefin)- 1607.01 Cm^-1

¹H NMR (CDCl₃)

δ(ppm)3.911 to 3.885 (8H,m,morpholine protons), 7.593  to7.558    (2H,d,CC&CE Ar-H,J=8.4Hz),  7.633  (1H,t,C5Ar-H,J=8 Hz),  7.705  (1H,d,C6Ar-H,J=8.9 Hz),  7.854  (1H,d, CiOlefinicproton,J=15.6 Hz),  7.929  (1H,d,CiiOlefinic  proton,J=14 Hz), 8.084 (2H,d,CB&CF Ar-H,J=8.8Hz), 8.263 (1H,d,C4 Ar-H,J=8.4Hz), 8.518(1H,s,C6 Ar-H).

Compound 2C: (2E)-3-(4-methylphenyl)-1-{4-[(E)-morpholin-4-yldiazenyl]phenyl}prop-2-en-1-one

Molecular Formula

C2₀H₂₁N₃O₂, Molecular Weight   :  335.39, Physical state  :  crystalline powder, Colour:Orange,Melting Point :137-140oC,R_f Value:0.57(n-hexane:Ethylacetate;6:4) , Solubility: Chloroform, DMSO, Ethyl acetate, Percentage yield  :  70%.

IR (KBr disk)

γ (cm^-1),C-H Stretching (olefins) – 3052.25 Cm^-1, C-H Stretching (morpholine)  – 2971.19 Cm^-1, C-H Stretching (Aromatic) – 2903.18 Cm^-1,C-H Stretching (CH3) – 2854.95 Cm^-1,C=O  – 1649.71 Cm-¹,C=C Stretching (Olefin) – 1588.87 Cm^-1,C=C (Aromatic) – 1562.54 Cm^-1,C-H Bending (Aromatic) – 810.55 Cm^-1

¹H NMR  (CDCl₃)

δ(ppm)  2.396 ( 3H,s,-CH3  protons  on  C4 of aldehyde),  3.876  (8H,m,morpholine protons),7.258(2H,d,CC&CEAr-H,J=8.8Hz),7.505(1H,d,Ci Olefinicproton,J=12Hz),7.560  to 7.531 (4H,m,C2,C3,C5,C6Ar-H,J=8.4Hz), 7.818(1H,d,CiiOlefinic proton,J=14.6Hz), 8.054(2H,d, CB&CF Ar-H,J=8.4 Hz).

Compound 2D :(2E)-3-(3, 4, 5-tri methoxy phenyl)-1-{4-[(E)-morpholin-4-yldiazenyl] phenyl}prop-2-en-1-one

Molecular Formula

C₂₂H₂₅N₃O₅,Molecular Weight  :  411.45, Physical state:  crystalline powder , Colour: Green ,Melting Point  : 126-129oC,Rf Value:  0.52 (n-hexane:Ethylacetate; 4:6) Solubility  : Chloroform, DMSO, Ethyl acetate. Percentage yield   :   86 %.

IR (KBr disk)

γ (cm_-1),C-H Stretching (olefins)-2995.03 Cm^-1,C-H Stretching (morpholine)- 2973.24 Cm^-1, C-H Stretching (Aromatic) – 2937.50 Cm^-1,C=O  – 1656.15 Cm^-1, C=C (Aromatic) – 1602.99 Cm^-1, C=C Stretching (Olefin)  – 1502.80 Cm^-1, C-O Stretching  (Ether) – 1326.88 Cm^-1 , C-O-C Stretching  – 1248.83 Cm^-1, C-H Bending (Aromatic) – 836.89 Cm^-1.

¹H NMR (CDCl₃)

δ(ppm) 3.875 (8H,m,morpholine protons), 3.904 (3H,s,-OCH3 on C4), 3.925 (6H,s,-OCH3 on C3&C5), 6.872 (2H,s, C2&C6Ar-H), 7.453 (1H,d,Ci Olefinicproton,J=15.6Hz), 7.559 (2H,d,CC&CE  Ar-H,J=8.4Hz), 7.742(1H,d,CiiOlefinicproton,J=15.6Hz), 8.052 (2H,d,CB&CF Ar-H,J=8.4Hz).

Compound 2E: (2E)-3-(4-fluorophenyl)-1-{4-[(E)-morpholin-4-yldiazenyl]phenyl}prop-2-en-1-one

Molecular Formula

C₁₉H₁₈FN₃O₂, Molecular Weight: 339.36, Physical state:  Crystalline powder, Colour: Cream, Melting Point: 102-104^oC,R_f Value:  0.6 (n-hexane: Ethylacetate; 6:4), Solubility:   Chloroform, DMSO, Ethyl acetate, Percentage yield:  88.2%

IR (KBr disk)

γ (cm^-1),C-H Stretching (olefins) – 3066.52 Cm^-1,C-H Stretching (morpholine)  – 2974.73 Cm^-1, C-H Stretching (Aromatic) – 2866.95  Cm^-1,C=O   – 1656.66 Cm^-1, C=C Stretching (Olefin)  – 1596.46 Cm^-1,C=C (Aromatic) – 1510.09 Cm^-1, C-F Stretching  – 1332.37 Cm^-1  C-H Bending (Aromatic)  – 827.50 Cm^-1

¹H  NMR  (CDCl₃)

δ(ppm)  3.874  (8H,m,morpholine  protons),  7.218 (2H,d,C3&C5Ar-H,J=8.4 Hz), 7.470 (1H,d,Ci Olefinicproton=15.6 Hz), 7.555 (2H,d,C2&C6Ar-H,J=8.4  Hz),7.654 to 7.619(2H,d,C3&C5Ar-H,J=8.4 Hz),7.796 (1H,d,Cii  Olefinicproton=15.6Hz), 8.051 (2H,d,CB&CF Ar-H,J=8.4Hz).

Compound 3A:4-[(E)-{4-[5-(4-nitrophenyl)-4, 5-dihydro-1H-pyrazol- 3yl] phenyl} diazenyl] morpholine

Molecular Formula

C₁₉H₂₀N₆O₃, Molecular Weight  :  380.4 , Physical state  :  Crystalline powder , Colour  : Light yellow , Melting Point: 140-142^oC , R_f Value : 0.35 (n-hexane:Ethylacetate; 6:4) ,Solubility : Chloroform, DMSO, Ethyl acetate, Percentage yield   :  87.6%.

IR (KBr disk)

γ (cm^-1),C-H Stretching (olefins) – 3355.58 Cm^-1,N-H Stretching (pyrazoline)   – 3105.16 Cm^-1, C-H Stretching (morpholine)  – 3072.35 Cm^-1, C-H Stretching (Aromatic) – 2953.16 Cm^-1, N=O (Nitro)  – 1598.61 Cm^-1, C=C (Aromatic) – 1513.25 Cm^-1, C=N Stretching (Pyrazoline)  – 1437.94 Cm^-1, C5-N1 Stretching (Pyrazoline)- 1156.89 Cm^-1, C-H Bending (Aromatic)   – 843.41 Cm^-1.

¹H NMR  (CDCl₃):  δ(ppm)

3.035  to  2.970  (1H,dd,Hb  on C4  of pyrazoline  ring),  3.611  to  3.544  (1H,dd,Ha  on  C4  of  pyrazoline  ring),  3.863  to  3.819 (8H,m,morpholine  protons),  5.069  to  5.018  (1H,m,  Hx  on  C5  of  pyrazoline  ring),  6.080 (1H,d,N1H proton in pyrazoline ring), 7.264 (2H,d,C2&C6Ar-H,J=8.4Hz), 7.657 to 7.579 (4H,m, CB,CF, CC&CE  Ar-H,J=8.4 Hz), 8.225 (2H,d,C3&C5Ar-H,J=8.Hz), Jab=16.4, Jax=5.2, Jbx =10.8.

Compound 3B:4-[(E)-{4-[5-(3-nitrophenyl)-4, 5-dihydro-1H- pyrazol3yl] phenyl}diazenyl]morpholine

Molecular Formula

C₁₉H₂₀N₆O₃,Molecular Weight  :  380.4 ,Physical state  :  Crystalline powder ,Colour  :  Light yellow ,Melting Point:  76-78^oC  ,R_f Value   :  0.58 (n-hexane:Ethylacetate; 4:6) ,Solubility: Chloroform, DMSO, Ethyl acetate, Percentage yield    :  70%

IR (KBr disk)

γ (cm^-1),C-H Stretching (olefins)-3336.76 Cm^-1, C-H Stretching (morpholine)  – 3308.19 Cm^-1, N-H Stretching (pyrazoline)  – 3085.54 Cm^-1, C-H Stretching (Aromatic) – 2857.31 Cm^-1, C=C (Aromatic) – 1438.17 Cm^-1, N=O (Nitro) – 1524.22 Cm^-1, C=N Stretching (Pyrazoline)  – 1348.84 Cm^-1, C5-N1 Stretching (Pyrazoline)- 1154.79 Cm^-1, C-H Bending (Aromatic)    – 835.42 Cm^-1.

¹H NMR  (CDCl₃):  δ(ppm)  3.050  to  2.985  (1H,dd,Ha  on  C4 of pyrazoline  ring),  3.617  to  3.577  (1H,dd,Hb  on  C4  of  pyrazoline  ring),  3.857  to  3.550 (8H,m,morpholine protons), 5.083  to 5.032 (1H,m, Hx on C5 of pyrazoline ring), 7.481  to 7.460 (2H,d,  CC&CE   Ar-H,J=8.4  Hz),  7.557  to  7.518  (2H,d,  C4&C6  Ar-H,J=8Hz),  7.662  to  7.642 (2H,d, CB&CF Ar-H,J=8 Hz), 7.789  (1H,d,N1H proton  in pyrazoline  ring), 8.163  (1H,t, C5 Ar-H,J=8.1Hz), 8.284 (1H,s, C2 Ar-H), Jab=16 , Jax=6 , Jbx =10.8.

Compound 3C:4-[(E)-{4-[5-(4-methylphenyl)-4, 5-dihydro-1H-pyrazol-3-yl] phenyl}diazenyl]morpholine

Molecular Formula:C₂₁H₂₃N₅O,Molecular Weight  :  349, Physical state  :  Crystalline powder , Colour  :  Dark yellow , Melting Point  : 56-58^oC  , R_f Value   :  0.45 (n-hexane:Ethylacetate; 6:4) ,Solubility   :  Chloroform, DMSO, Ethyl acetate. Percentage yield         :  86.5%

IR (KBr disk)

γ (cm^-1),C-H Stretching (olefins)-3355.61 Cm^-1,C-H Stretching (morpholine)  – 3333.83 Cm^-1, N-H Stretching (pyrazoline)  – 3025.31 Cm^-1, C-H Stretching (Aromatic)   – 2918.99 Cm^-1, C-H Stretching  (CH3) – 2854.54 Cm^-1, C=C (Aromatic)  – 1435.06 Cm^-1, C=N Stretching (Pyrazoline)  – 1348.57 Cm^-1.  C5-N1 Stretching (Pyrazoline) – 1154.81 Cm^-1, C-H Bending (Aromatic) – 814.53 Cm^-1

¹H NMR (CDCl₃): δ(ppm)

2.391 (3H,s,CH3 on C4), 3.072  to 3.010 (1H,dd, Ha on C4 of pyrazoline ring), 3.487 to 3.420 (1H,dd,Hb on C4 of pyrazoline ring), 3.815 (1H,m, Hx on C5 of pyrazoline ring), 3.855 (8H,m,morpholine protons), 7.192 (1H,d,N1H proton in pyrazoline ring), 7.530  to 7.448 (4H,d, C2,C3, C5&C6  Ar-H,J=8.4 Hz), 7.665  to 7.595 (4H,d, CB,CF, CC&CE  Ar-H,J=8.4 Hz), Jab=16.4 , Jax=6.4 , Jbx =10.4.

Compound 3E:4-[(E)-{4-[5-(4-fluorophenyl)-4, 5-dihydro-1H-pyrazol-3-yl] phenyl} diazenyl] morpholine

Molecular Formula:C₁₉H₂₀FN₅O, Molecular Weight :  353.39, Physical state  :  Crystalline powder , Colour  : Dark yellow , Melting Point  :  68-70^oC ,R_f Value:  0.35 (n-hexane:Ethyl acetate; 6:4) , Solubility  : Chloroform, DMSO, Ethyl acetate, Percentage yield  :  88.3%.

IR (KBr disk)

γ (cm^-1),C-H Stretching (olefins)  – 3063.27 Cm^-1, C-H Stretching (morpholine)  – 3036.41 Cm^-1, N-H Stretching (pyrazoline)   – 2967.44 Cm^-1,C-H Stretching (Aromatic)  – 2855.66 Cm^-1,  C=C (Aromatic)  – 1438.58 Cm^-1, C=N Stretching (Pyrazoline)  – 1347.59 Cm^-1, C5-N1 Stretching (Pyrazoline)- 1156.12 Cm^-1, C-F Stretching   – 1015.78 Cm^-1, C-H Bending (Aromatic)  – 839.10 Cm^-1.

¹H NMR  (CDCl₃):  δ(ppm)

3.040  to  2.976  (1H,dd,Ha  on  C4 of pyrazoline  ring),  3.510  to  3.442  (1H,dd,Hb  on  C4  of  pyrazoline  ring),  3.868  to  3.800 (8H,m,morpholine protons), 4.942  to 4.483 (1H,m, Hx on C5 of pyrazoline ring), 7.050  to 7.007 (2H,d,  CC&CE    Ar-H,J=8.8  Hz),7.374  (1H,d,N1H  proton  in  pyrazoline  ring),  7.476  to  7.454 (4H,d, C2,C3, C5&C6  Ar-H,J=8.8 Hz), 7.664  to 7.643  (2H,d, CB&CF Ar-H,J=8.4 Hz), Jab=16.4  , Jax=6.8 , Jbx =10.8.

Table 2: The percentage scavenging activity by DPPH Method

Concμg/ml	% scavenging activity(Mean ± SEM)
	Std	2A	2B	2C	2D	2E	3A	3B	3C	3E
20	84.26  ±0.176	49.29±0.159	45.43±0.088	58.43±0.296	63.50±0.288	55.50±0.288	50.30±0.650	44.46±0.290	58.73±0.218	46.40±0.305
40	85.40±0.208	50.26±0.091	49.35±0.224	64.60±0.305	67.90±0.450	58.73±0.371	51.560.808	49.33±0.333	61.33±0.333	49.96±0.983
60	86.36±0.185	53.63±0.120	50.46±0.218	67.50±0.288	70.40±0.305	62.06±0.560	55.800.416	52.60±0.264	65.86±0.466	54.53±0.290
80	89.30±0.152	56.53±0.088	55.43±0.120	71.66±0.240	73.63±0.272	64.60±0.305	58.730.371	55.80±0.416	71.93±0.520	60.70±0.351
100	90.40±0.115	61.33±0.145	60.63±0.272	72.06±0.581	76.60±0.305	65.86±0.466	64.660.240	61.67±0.338	74.96±0.548	65.93±0.405
120	91.33±0.176	66.40±0.115	62.83±0.120	74.60±0.305	78.86±0.466	69.13±0.592	66.830.440	65.30±0.208	81.63±0.317	69.16±0.088
IC50μg/ml	2.80	58.61	64.20	42.81	36.30	50.62	55.42	60.02	32.34	54.90

Mean ± SEM = Mean ± Standard Error Mean, IC₅₀=Half maximal inhibitory concentration

Table 3: The percentage scavenging activity by Hydrogen Peroxide Method

Conc	% scavenging activity(Mean ± SEM)
μg/ml	Std	2A	2B	2C	2D	2E	3A	3B	3C	3E
20	89.7	44.53	33.46	48.7	50.2	52.6	47.9	37.83	55.6	46.4
	±0.351	±0.290	±0.290	±0.351	±0.099	±0.305	±0.493	±0.440	±0.305	±0.305
40	92.6	46.86	35.06	51.36	55.2	57.767	49.6	40.76	61.86	58.733
	±0.305	±0.466	±0.066	±0.317	±0.416	±0.39	±0.305	±0.145	±0.466	±0.371
60	94.46	50.23	37.73	54.86	59.4	59.267	52.6	42.36	63.63	60.53
	±0.240	±0.392	±0.371	±0.466	±0.305	±0.266	±0.305	±0.317	±0.317	±0.290
80	95.06	53.76	42.7	58.7	61.36	61.567	58.23	45.63	67.46	62.23
	±0.066	±0.392	±0.351	±0.351	±0.317	±0.296	±0.120	±0.3179	±0.290	±0.185
100	95.36	55.83	47.6	60.4	64.93	63.267	59.03	47.26	70.63	62.8
	±0.185	±0.440	±0.305	±0.305	±0.520	±0.133	±0.088	±0.176	±0.202	±0.115
120	96.2	57.83	49.53	63.43	69.4	64.4	60.5	48.56	72.6	65.56
	±0.100	±0.440	±0.290	±0.296	±0.305	±0.305	±0.288	±0.296	±0.305	±0.296
IC50	2.2	62.41	116.02	24.23	20.18	32.41	50.11	120.06	16.28	40.92
μg/ml

Mean ± SEM = Mean ± Standard Error Mean, IC₅₀=Half maximal inhibitory concentration

Figure 4: The percentage scavenging activity by DPPH Method  Click here to View figure

 

Figure 5: The percentage scavenging activity by Hydrogen Peroxide Method  Click here to View figure

 

Table 4: The percentage scavenging activity by Egg Albumin Denaturation Method

Conc	% Inhibition(Mean ± SEM)
μg/ml	Std	2A	2B	2C	2D	2E	3A	3B	3C	3E
20	76.83 ±0.441	25.86 ±0.592	32.56 ±0.260	36.40 ±0.305	33.4	30.6	28.4	23.8	40.7	29.93
					±0.305	±0.305	±0.305	±0.416	±0.351	±0.520
40	78.2 ±0.115	30.46 ±0.290	34.26 ±0.176	37.30 ±0.251	35.7	41.63	33.46	35.46	42.2	31.66
					±0.152	±0.318	±0.290	±0.290	±0.200	±0.176
60	79.30 ±0.173	36.63 ±0.318	37.36 ±0.318	43.26 ±0.176	40.2	43.56	41.63	42.5	45.5	33.26
					±0.416	±0.296	±0.318	±0.288	±0.288	±0.176
80	80.06 ±0.066	43.46 ±0.290	43.33	48.43 ±0.233	47.4	45.16	43.7	44.36	47.5	35.33
			±0.166		±0.305	±0.088	±0.351	±0.272	±0.288	±0.176
100	81.40 ±0.115	46.40 ±0.305	44.53 ±0.290	51.46 ±0.290	55.56	46.56	46.4	45.26	52.36	48.46
					±0.296	±0.296	±0.305	±0.176	±0.318	±0.290
120	82.63 ±0.202	51.30 ±0.351	47.40 ±0.305	56.73 ±0.371	61.8	54.3	51.63	46.6	58.53	53.73
					±0.416	±0.351	±0.318	±0.305	±0.290	±0.371
IC50	1.02	116.32	134.04	86.43	84.22	104.08	114.02	128.4	82.06	106.28
μg/ml

Mean ± SEM = Mean ± Standard Error Mean, IC₅₀=Half maximal inhibitory concentration

Figure 6: The percentage scavenging activity by Egg Albumin Denaturation Method  Click here to View figure

 

Table 5: The percentage scavenging activity by Egg Albumin Denaturation Method

Conc	% Inhibition(Mean ± SEM)
μg/ml	Std	2A	2B	2C	2D	2E	3A	3B	3C	3E
20	53.36	29.6	27.56	35.83	33.7	31.2	31.9	30.43	35.5	34.23
	±0.318	±0.305	±0.296	±0.441	±0.351	±0.115	±0.435	±0.296	±0.288	±0.145
40	62.4	31.53	35.76	40.53	35.86	32.63	34.7	32.26	38.63	35.36
	±0.305	±0.290	±0.393	±0.290	±0.466	±0.318	±0.251	±0.218	±0.318	±0.318
60	67.86	35.43	37.7	48.93	37.56	34.36	36.86	34.6	40.46	37.7
	±0.466	±0.296	±0.351	±0.520	±0.296	±0.318	±0.466	±0.305	±0.290	±0.351
80	71.9	42.46	40.46	50.2	42.2	37.86	39.36	37.5	41.53	40.4
	±0.493	±0.290	±0.290	±0.200	±0.611	±0.466	±0.318	±0.288	±0.290	±0.305
100	74.6	44.2	42.26	51.16	49.46	42.6	43.46	43.73	45.6	42.46
	±0.305	±0.200	±0.176	±0.088	±0.290	±0.305	±0.290	±0.371	±0.305	±0.240
120	75.56	45.86	44.4	52.46	54.8	48.33	49.86	48.53	55.16	50.66
	±0.296	±0.466	±0.305	±0.290	±0.416	±0.333	±0.240	±0.290	±0.166	±0.176
IC50	13.02	128.01	174.51	90.2	102.01	126.04	130.45	142.05	100	164.23
μg/ml

Mean ± SEM = Mean ± Standard Error Mean, IC₅₀=Half maximal inhibitory concentration

Figure 7: The percentage scavenging activity by Heat Induced hemolytic Method  Click here to View figure

 

The data was analyzed by one way ANOVA using Graph pad prism software. The  scavenging activity of the compounds ( Table 2&3) and their IC₅₀ values were compared with the standard  and  the  significance  factor  “p”  was  found  to  be  less  than  0.001  for  most  of  the compounds. Among chalcones compound 2D having 3,4,5-  trimethoxy  substitution and among pyrazolines  compound  3C  having  4-Methyl  has  shown  highest  activity  with  least  IC₅₀ values suggesting that electron donating groups aid in scavenging activity. Among chalcones compound 2B and among pyrazolines compound 3B having 3-Nitro  substitutions has  shown  least activity with highest  IC₅₀values  indicating  that electron withdrawing groups at  the meta position  in  the compounds  have  less  scavenging  activity.  All  the  other  compounds  with  4-Nitro,  4-  fluorosubstituents were found  to have intermediate activity.

Similarly  the  anti  inflammatory  activity  of  the  compounds  and  their  IC₅₀ values  were compared with  the  standard  and  the  significance  factor  p≤  0.001  for most  of  the  compounds ( Table 4 & 5). Among  chalcones  compound 2D having 3,4,5-  trimethoxy  substitution  and  among pyrazolines compound 3C having 4-Methyl has shown highest activity with least IC50values  suggesting that electron  donating  groups  in  the  compounds  aid  in  good  anti  inflammatory  activity.  Among chalcones compound 2B and among pyrazolines compound 3B having 3-Nitro substitutions with highest  IC₅₀values  has  shown  least  activity  indicating  that  electron withdrawing  groups  at  the meta position  in  the compounds have  less anti  inflammatory activity. All the other compounds with 4-Nitro, 4- fluoro substituents were found to have intermediate activity.

Conclusion

4- Amino acetophenone was diazotized then followed by coupling with with morpholine as ameans of protection of amine group in 4-Amino acetophenone. The obtained diazotizedproduct containing the acetyl group is then made to react with different aldehydes in the presence of 40% KOH solution as catalyst to get chalcone derivatives. The sechalcone derivatives are then subjected for cyclization by treating with hydrazine hydrate. All the reactions were monitored by TLC to ascertain the completion of the reaction. All the compoundswere found to have good yields. R_f values and melting points of the synthesized compounds were different with each other indicating the difference between the compounds.  The structures were proposed based on¹H NMR and IR spectral data.

Anti  oxidant  activity  was  performed  for  the  synthesized  compounds  by  DPPH  and Hydrogen Peroxide Method,  and anti inflammatory activity was performed by Inhibition Of Egg Albumin Denaturation Method and Heat Induced Haemolytic Method. Most of the compounds showed significant activity with p≤ 0.001 when the data was subjected to one way ANOVA by using Graph pad prism-5 software. Among  chalcones  compound  2D  having  3,4,5-  trimethoxy substitution and among pyrazolines compound 3C having 4-Methyl has  shown highest activity with  least  IC₅₀values which  is considerable with  the standard, suggesting  that electron donating groups  aid  in  scavenging  activity.  Among  chalcones  compound  2B  and  among  pyrazolines compound  3B  having  3-Nitro  substitutions  has  shown  least  activity  with  highest  IC₅₀ values which is considerable with the standard, suggesting that electron withdrawing groups at the meta position in the compounds have less scavenging activity. All the other compounds with 4-Nitro, 4- fluorosubstituent’s were found to have intermediate activity.

Acknowledgement

The authors are thankful to the Principal, Creative Educational Society’s College of pharmacy for providing the necessary facilities to carry out the research.The authors are also thankful to Dr.Murugesan, SAIF, IIT-Chennai for providing¹H NMR data.

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