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Enzyme Inhibition study of selected Schiff’s Bases

By Muhammad Zubair

M.Phil Scholar

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SupervisorDr. Muhammad SajidAstt.ProfessorDepartment of Biochemistry

Co-SupervisorDr.Muhammad AshrafAssociate Professor Department Of Biochemistry & Biotechnology Bahawalpur

CONTENTS

INTRODUCTION.

EXPERIMENTAL.

RESULTS AND

DISCUSSIONS.

CONCLUSIONS.

ACKNOWLEDGEMENTS.

INTRODUCTION TO

ENZYME

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Enzyme

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Enzymes are protein molecules composed of amino acids ranging in 62 and 2,500 amino acid residues produced by the living cell. Enzymes are macromolecule which catalyse various chemical transformation in biological system.In a biological system enzyme control almost all essential function. In 1850s, Louis Pasteur revealed that in the presence of a catalyst called ‘’ferment’’ sugar could be fermented into alcohol by yeast. These molecule were called enzyme by Fredrick W.Kuhne. A breakthrough in early studies of enzyme were obtained when in 1926s, James Summer succeeded in isolation and crystallization of urease. In 1965s, D.C Phillips determined the lysozyme structure by single X-ray diffraction technique .

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Lock and key theory

Theories of enzyme

Induced fit theory

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Trypsin

Amylases

Cellulase

proteases, amylases

and lipases

Biological and Industrial Importance of Enzyme

Luciferase enzyme Muscle relaxation and

contraction

HIV integrase and reverse transcriptase

Various Enzyme their associated disease and specific inhibitor

Enzyme Associated disease Inhibitor(drug)

Carbonic anhydrase Glaucoma epilepsy Acetazolamide

Beta-glucoronidase Glaucoma d-saccharic acid 1,-4 lactone

Thymidine phosphorylase Cancer, Agiogenesis Endostatin

Alpha-glucosidase Diabetes Acarbose

Aldose reductase Eye and nerve damage in diabetes Atorvastatin and lovastatin

Reverse transcriptase Aids Zidovudine

Tyrosine kinase Carbonic myeloid leukaemia Tyrphostin

Propyl endopeptidase Cognitive disorder Peptidyl alpha –keto benzothiazole

Phosphodiestrase-4 Chronic obstructive pulmonary disease Rolipram

Lipoxygenase Asthama,inflammation Zileuton

Enzyme Associated disease Inhibitor(drug)

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Types of enzyme inhibitors

Competitive inhibitor of enzymes

Non-competitive inhibitor of enzymes

Mix-Type inhibitor of enzymes

Uncompetitive inhibitor of enzymes

SCHIFF’S BASES

INTRODUCTION

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Chemical compounds in which carbon-nitrogen have a double bond -C=N- and nitrogen atom is attached to aryl, alkyl, hetroaryl , hydrogen or lmetalo (usually Si, Al, B, Sn) are Schiff’s bases. These compounds were first synthesized and reported by Hugo Schiff.The Schiff bases general structure are as follow

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CNR2

R1

R3

R1R2 and R3 = H, Alkyl or aryl1

Significance and application of Schiff’s bases

Schiff’s bases showed antipyretic, antiproliferative, antibacterial, antifungal, anti-inflammatory and antiviral activities. 1-amino-3-hydroxyguanidine tosylate-derived Schiff bases compound is most potent against mouse hepatitis virus (MHV).

Structure of antiviral drug

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HO NHN

NH

NH

HO

.CH3C6H4SO3H

Antimalarial, 5-nitroisoquinolines

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Antibacterial

Antifungal, Piperonyl

N

CF3

NO2

Cl

HO

NN

OHCl

N O

O

R

Introduction to HydrazoneHydrazone is a class of organic compounds and have a structure R1R2C=NNH2. Hydrazone are prepared by the action of hydrazine on ketone or aldehyde.

Biological activityHydrazone have a number of biological activity such as analgesic, anti-inflammatory and antiplatelet, antimalarial, antimicrobial, antitubercular, antitumoral, anticonvulsant and antidepressant activity

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CR1

R2

NNH2

Biological active compoundsAnalgesic, 2-(2-formylfuryl) pyridylhydrazone

Antitumoral, Di-phenolic Hydrazone

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N

HO

NH

NO2O2N

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Alpha glucosidaseAlpha glucosidase enzyme (EC 3.2.1.20) is produced by mammalian small intestine. Alpha glucosidase remain bound to intestinal lining of the small intestine rather than secreted into the intestinal tract where it interacts with large carbohydrate taken in through the diet. Alpha-Glucosidase belongs to GH-family .Mechanism of action of alpha glucosidaseOne of the therapeutic approaches for reducing postprandial hyperglycaemia in patients with DM is to prevent absorption of carbohydrates after food uptake. Only monosaccharides, such as glucose and fructose, can be transported out of the intestinal lumen into the blood stream. Complex starches, oligosaccharides and disaccharides must be broken down into individual monosaccharides before being absorbed in the duodenum and upper jejunum. This digestion is facilitated by enteric enzyme, α-glucosidases that are attached to the brush border of the intestinal cells.

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Alpha glucosidase

clinical inhibitor

VogliboseMiglitol

Acarbose

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Introduction to Urease enzymeIn the history of science urease has a prominent place. James Sumner isolated and crystallized urease from Jack Bean plant (Carnavalia ensiformis) in 1926s.

For this achievement, he received noble prize in the field of chemistry in 1946s.Through his studies he confirmed that protein can be crystallized and also confirmed the proteinaceous nature of enzyme. Urease contain nickel in its active site .It belongs to amido-hydrolase family with (EC 3.3.1.5).Urease catalyse the decomposition of urea into ammonia and carbamate.It is a hydrolysis process. At the physiological pH carbamate hydrolyze spontaneously to carbonic acid and yield another molecule of ammonia. Urease is present in plant, fungi and various types of bacteria

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Importance of urease in pathogenesis and agriculture

Peptic ulcer Urolithiasis Pyelonephritis

AmmoniaNitrogen cycle

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Urease clinical inhibitor

Hydroxamic acid

Biscoumarin

Bismuth complexes

Phosphoramide

Imidazole

Aims and Objective

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Due to its biological importance synthesized Schiff base were examined for enzyme inhibition such as α-glucosidase and urease enzyme.The alpha glucosidase has a role in Type 2 diabetes mellitus.Urease cause peptic ulcer,Dyspepsia,diarrhoea and kidney stone in human being and also causes agricultural problem. The present work is mainly concerned with drug development.

Experimental

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SCHEMEThe Salicyloyl hydrazide Schiff bases synthesis were carried in two steps; firstly Salicyloyl hydrazide synthesis was done by treating methyl salicylate with hydrazine monohydrate in ethanol as a solvent with few drops of acetic acid for 4 hrs at room temp.The product in the first step were treated with different aldehydes (1:1) in EtOH at reflux for 2-3 hrs. HCl (1-2 drops) were added as a catalyst. The reaction progress was confirmed by TLC. The crude product were washed by ethyl alcohol and n-hexane to obtained pure Schiff bases.

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HN

OHO

NH2

O

R H

O

NH

OHN

R

H H2OHClr.t

2-3hr

O

O

Me

HO HO

HN

O

NH2

Acetic Acid

r.t4hr

H2N-NH2.H2O Me-OH

SCHEME

Alpha Glucosidase assay

Percentage inhibition = 100 - (P.R – A.R / Abs of Control) × 100

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70μL Buffer10μL test

compound10μL enzyme

Incubate for 10 minute

P.R at 400 nm

10μL substrate

A.R at 400nm

37 ºC

37 ºC

Incubate for 20 minute

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37ºC

Incubate for 10 minute

P.R at 630 nm 15μL substrate

37ºCIncubate for 15 minute

40μL phenol& 60μL alkali

37ºCIncubate for 15 minute

A.R at 630

Percentage inhibition = 100 - (P.R – A.R / Abs of Control) × 100

urease assay65μL Buffer10μL test compound

10μL enzyme

Result and Discussion

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Synthesis of various schiff,s base analogs 41-84

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S. No R S. No R S. No R

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47 53

42 48 54

43 49 55

44 50 56

45 51 57

46 52 58

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S. No R S. No R S. No R

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65 71

60 66 72

61 67 73

62 68 74

63 69 75

64 70 76

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S. No R S. No R

77 83

78 84

79

80

81

82

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S.No Inhibition (%) at

0.5 mM

IC50 ± SEMa (µM) S.No Inhibition (%) at

0.5 mM

IC50 ± SEMa (µM)

41 24.53±0.12 - 54 71.83±0.26 106.19±0.14

42 59.14±0.25 435.23±0.12 55 13.72±0.19 -

43 92.87±0.21 115.27±0.17 56 26.87±0.12 -

44 2.51±0.25 - 57 57.43±0.27 392.75±0.16

45 98.75±0.29 194.25±0.16 58 86.91±0.27 387.39±0.12

46 95.56±0.27 215.76±0.15 59 23.87±0.16 -

47 64.39±0.25 129.83±0.12 60 47.76±0.14 -

48 12.38±0.19 - 61 13.31±0.19 -

49 58.21±0.21 328.81±0.13 62 83.96±0.11 215.98±0.17

50 12.25±0.21 - 63 87.56±0.12 118.51±0.12

51 3.83±0.27 - 64 14.12±0.48 -

52 21.76±0.19 - 65 31.19±0.55 -

53 2.45±0.13 - Acarbose 92.23±0.14 38.25±0.12

Table showing alpha glucosidase inhibitory potential

Table showing alpha glucosidase inhibitory potential

S.No Inhibition (%) at 0.5

mM

IC50 ± SEMa (µM) S.No Inhibition (%) at 0.5

mM

IC50 ± SEMa (µM)

66 4.11±0.16 - 76 9.11±0.16 -

67 55.16±0.11 369.28±0.12 77 9.16±0.11 -

68 52.97±0.12 386.61±0.18 78 5.77±0.12 -

69 97.31±0.17 127.12±0.17 79 Amount less -

70 12.83±0.15 - 80 23.83±0.15 -

71 56.12±0.28 371.95±0.12 81 8.12±0.18 -

72 8.56±0.13 - 82 Amount less -

73 16.54±0.19 - 83 21.56±0.17 -

74 9.14±0.11 - 84 17.75±0.29 -

75 13.56±0.18 - Acarbose 92.23±0.14 38.25±0.12

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SYNTHESIZED SALICYLOYL HYDRAZONE ALPHA

GLUCOSIDASE INHIBITIONCompound 54, with IC50 value (106.19±0.14) μM

43, with IC50 value (115.27±0.17) μM

63, with IC50 value (118.51±0.12) μM

69, with IC50 value (127.12±0.17 ) μM

47, with IC50 value (129.83±0.12 ) μM

Standard Acarbose with IC50 value (38.25±0.12)μM.

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Table showing urease inhibitory potential

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S. No.Inhibition (%)

at 0.5 mMIC50 (μM) S. No.

Inhibition (%)

at 0.5 mMIC50 (μM)

41 86.12±0.25 51.84±0.11 53 59.74±0.33 173.64±0.29

42 Amount less - 54 88.48±0.23 17.95±0.12

43 92.18±0.24 79.34±0.12 55 62.19±0.27 36.36±0.13

44 81.13±0.31 127.63±0.26 56 54.47±0.35 397.43±0.21

45 89.13±0.38 51.75±0.29 57 76.67±0.32 311.92±0.27

46 87.96±0.26 83.46±0.12 58 Amount less -

47 92.14±0.24 21.72±0.19 59 96.19±0.21 9.14±0.12

48 58.75±0.31 154.43±0.27 60 76.37±0.33 161.63±0.27

49 83.81±0.22 27.84±0.18 61 54.86±0.39 402.36±0.25

50 87.45±0.25 47.63±0.11 62 58.35±0.26 219.18±0.12

51 99.21±0.28 19.32±0.14 63 57.29±0.21 174.53±0.17

52 93.28±0.25 29.32±0.13 64 87.14±0.21 48.56±0.17

Table showing urease inhibitory potential

S. No.Inhibition (%) at

0.5 mMIC50 (μM) S. No.

Inhibition (%) at

0.5 mMIC50 (μM)

65 54.23±0.38 415.75±0.24 76 Amount less -

66 73.51±0.35 195.12±0.21 77 Amount less -

67 63.69±0.33 163.75±0.19 78 Amount less -

68 91.37±0.27 28.97±0.13 79 83.93±0.19 197.42±0.15

69 Amount less - 80 75.84±0.25 114.76±0.11

70 89.23±0.29 71.76±0.15 81 Amount less -

71 76.44±0.26 51.32±0.12 82 55.47±0.35 389.65±0.29

72 62.42±0.31 347.64±0.27 83 87.17±0.28 21.26±0.14

73 Amount less - 84 Amount less -

74 82.16±0.26 132.51±0.11 Standard (thiourea) 98.45±0.87 21.25±0.15

75 75.45±0.21 118.45±0.17

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UREASE INHIBITION

Compound 47, with IC50 (21.72±0.19)μM

51, with IC50 (19.32±0.14)μM

54, with IC50 (17.95±0.12)μM

59, with IC50 (9.14±0.12 )μM

83, with IC50 (21.26±0.14 )μM

Standard Thiourea, with IC50 (21.25±0.15)μM

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CONCLUSIONS

1. Present work was carried out for synthesis of salicyloyl hydrazone Schiff’s base analogs (41-84).

2. Compound were tested against α-Glucosidase and urease inhibition.

3. Results shows that all analogs show excellent to moderate potential.

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ACKNOWLEDGEMENTSAlmighty Allah Dr. Muhammad Sajid (Supervisor) Dr. Muhammad Ashraf (Co-Supervisor) Associate Professor Islamia University BahawalpurProf. Dr. Mukhtiar Hassan (Chairman) Department Of Biochemistry.All Teachers My All Lab FellowsAll FriendsFamily Members

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THANKS TO ALL OF

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