university of medicine and pharmacy "grigore t. popa

UNIVERSITY OF MEDICINE AND PHARMACY "Grigore T.

Popa"

FACULTY OF PHARMACY

RESEARCHES ON DEVELOPMENT OF NEW

COMPOUNDS WITH HETEROCYCLIC STRUCTURE AND

WITH BIOLOGICAL POTENTIAL

Summary of PhD thesis

SCIENTIFIC COORDINATOR

PROF. DR. Lenuţa PROFIRE

PHD student

Maria WOLSZLEGER (DRĂGAN)

Invest in people!

Project co-financed by the European Social Fund Operational Programme Human Resources Development 2007 -

2013

Priority Axis 1 "Education and training in support of growth and development of knowledge based society"

Area of Intervention 1.5 "Doctoral and post-Doctoral research support"

Title: Strategic partnership to improve the quality of medical research in universities through doctoral and

postdoctoral scholarships - DocMed.Net_2.0

Contract No .: HRD / 159 / 1.5 / S / 136893

Beneficiary: University of Medicine and Pharmacy "Iuliu Hatieganu" Cluj-Napoca

IASI, 2015

Membership of the doctoral committee:

PRESIDENT: Decan Prof. univ. dr. Monica Hăncianu

University Of Medicine And Pharmacy "Grigore T. Popa" Iasi

SCIENTIFIC COORDINATOR: Prof. univ. dr. LenuţaProfire


OFFICIAL REVIEWERS:

Prof. univ. dr. Ileana Chiriţă

University Of Medicine And Pharmacy „Carol Davila”, Bucharest

C.S.I. Dr. Cornelia Vasile

Institute of Macromolecular Chemistry „Petru Poni” Iasi

Associate prof. dr. Cătălina Daniela Stan


PhD thesis contains 149 pages, 40 tables and 77 figures in

the sentence.

The numbering of figures, tables and contents of the

summary are kept in the same form as in the sentence.

Scientific results obtained during doctoral studies are due

and stock status within the project "Strategic partnership to improve the

quality of medical research in universities through doctoral and postdoctoral

scholarships - DocMed.Net_2.0" HRD/159/1.5/S/136893, status that we

had in the period april 2014 - june 2015.

1

TABLE OF CONTENTS

Abbreviations v

ACKNOWLEDGEMENT viii

KNOWLEDGE 1

Chapter 1 1

FERULIC ACID 1

1.1. Overview 1

1.2. Ferulic acid from vegetal sources 2

1.3. The daily intake of ferulic acid 3

1.4. Pharmacokinetic properties 3

1.4.1. The absorption of ferulic acid 3

1.4.2. Ferulic acid metabolism 4

1.4.3. The distribution of ferulic acid 4

1.4.4. Disposal and ferulic acid excretion 4

1.5. Pharmacodynamic properties 5

1.5.1. Ferulic acid and free radicals 5

1.5.2. Ferulic acid and antioxidant enzymes 6

1.5.3. New ferulic acid formulations 6

1.6. Ferulic acid and potential therapeutic applications 7

1.6.1. Alzheimer's Disease 7

1.6.2. Neoplastic diseases 8

1.6.3. Cardiovascular 10

1.6.4. Diabetes mellitus 11

1.6.5. Photoprotection 12

Chapter 2 13

OXIDATIVE STRESS AND NEURODEGENERATIVE

DISEASES

13

2.1. The role of oxidative stress in the aging process 13

2.2. Oxidative stress and neurodegenerative diseases 14

2.3. Oxidative stress and Parkinson's disease 14

2.4. Oxidative stress and Huntington disease 15

2.5. Oxidative stress and amyotrophic lateral sclerosis 16

2.6. Oxidative stress and Alzheimer's disease 17

Chapter 3 19

HYDRAZONE DERIVATIVES WITH

THERAPEUTICAL POTENTIAL

19

3.1. Overview 19

2

3.2. Methods for the synthesis of hydrazone derivatives 20

3.3. Biological effects of the hydrazone compounds 22

3.3.1. Anticonvulsant effect 22

3.3.2. Antidepressive effect 23

3.3.3. Analgesic, anti-inflammatory and antiplatelet 23

3.3.4. Antimalarial effect 25

3.3.5. Antimicrobial effect 26

3.3.6. Effect antimycobacterial 27

3.3.7. Antitumor effect 28

3.3.8. Vasodilator effect 28

3.3.9. Antiviral effect 29

Chapter 4 30

THIAZOLIDINE-4-ONE DERIVATIVES AND THIR

THERAPEUTIC ROLE

30

4.1. Overview 30

4.2. Methods for the synthesis of compounds with

thiazolidin-4-one structure

31

4.3. Biological effects of the compounds with thiazolidin-4-

one structure

32

4.3.1. Antioxidant effect 33

4.3.2. Antimicrobial and antifungal effect 33

4.3.3. Inflammatory and analgesic effect 35

4.3.4. Anticonvulsant effect 36

PERSONAL CONTRIBUTIONS 38

Chapter 5 38

THE MOTIVATION REASONS AND THE

OBJECTIVES FOR PERSONAL RESEARCH

38

Chapter 6 42

THE SYNTHESIS AND CHARACTERIZATION OF

NEW FERULIC ACID DERIVATIVES

42

6.1. Material and methods 42

6.1.1. The synthesis of ferulic acid derivatives with

hydrazone structure

42

6.1.1.1. Method of the synthesis for chloride 3- (4-

hydroxy-3-methoxy-phenyl) acrylic acid

42

6.1.1.2. Method of the synthesis for 3- (4-hydroxy-3-

methoxy-phenyl) acryloyl hydrazine

42

3

6.1.1.3. Method of the synthesis for hydrazones

of 3-(4-hydroxy-3-methoxyphenyl)acrylic acid

43

6.1.2. The synthesis of ferulic acid derivatives with

thiazolidin-4-one structure

43

6.1.2.1. General procedure for the synthesis of

derivatives of 3- (4-hydroxy-3-methoxy-

phenyl) acrylic acid with the structure of the

thiazolidin-4-one

43

6.1.3. Physico-chemical characterization of ferulic acid

derivatives

44

6.2. Results and discussion 44

6.2.1. The synthesis and characterization of ferulic acid

derivatives

45

6.2.1.1. The synthesis and characterization of the

chloride of 3-(4-hydroxy-3-methoxy-phenyl)

acrylic acid

45

6.2.1.2. The synthesis and characterization of 3-(4-

hydroxy-3-methoxy-phenyl) acryloyl

hydrazine

46

6.2.1.3. The synthesis and characterization of

derivatives of N-[3-(4-hydroxy-3-methoxy

phenyl)acryloyl]-N-(R-benzylidene)

hydrazine

47

6.2.2. The synthesis and characterization of derivatives of

2-(R-phenyl)-3- [3-(4-hydroxy-3-methoxy-phenyl)

acrylamido]-thiazol-4-one

49

6.3. Conclusion 50

Chapter 7 52

THE CHEMICAL STRUCTURE CONFIRMATION OF

THE FERULIC ACID DERIVATIVES

52

7.1. Material and methods 52

7.1.1. Infrared Spectrum (IR) 52

7.1.2. Nuclear Magnetic Resonance Spectrum (NMR) 53

7.2. Results and discussion 53

7.2.1. Infrared Spectrum (IR) 53

7.2.1.1. The IR spectrum of chloride of 3-

(4-hydroxy-3-methoxy-phenyl)acrylic acid

53

4

7.2.1.2. The IR spectrum of the of 3-(4-hydroxy-

3-methoxy-phenyl) acryloyl hydrazine

54

7.2.1.3. The IR spectrum of N- [3-(4-hydroxy-

3-methoxyphenyl)acryloyl]-N-(R

benzylidene)hydrazine

55

7.2.1.4. The IR spectrum of 2- (R-phenyl)-3-[3-

(4-hydroxy-3-methoxyphenyl)

acrylamido] -thiazol-4-one

60

7.2.2. Nuclear Magnetic Resonance Spectrum (NMR)

7.2.2.1. 1H-RMN spectrum for N-[3-(4-hydroxy-

3-methoxyphenyl)acryloyl]-N-(R-

benzyliden)hydrazine

7.2.3. 1H-RMN spectrum of 2-(R-phenyl)-3-[3-(4-

hydroxy-3-methoxyphenyl)acrylamido]-

thiazolyn-4-one

64

64

67

Chapter 8 71

THE BIOLOGICAL EVALUATION OF

FERULIC ACID DERIVATIVES

71

8.1. The antioxidant potential evaluation 71

8.1.1. Material and methods 71

8.1.1.1. Determination of the antiradical against

DPPH radical

71


cation radical ABTS·+

72

8.1.1.3. Determination of total antioxidant capacity 73

8.1.1.4. Determination of reducing power 73

8.1.2. Results and discussion 74


DPPH radical

74


cation radical ABTS·+

79

8.1.2.3. Determination of total antioxidant capacity 82

8.1.2.4. Determination of reducing power 86

8.1.3. Conclusion 90

8.2. Evaluation of anti-inflammatory potential in vitro 92


8.2.1.1. Inhibition distortion of serum albumin 92

8.2.1.2. Stabilizing test of erythrocyte membrane 93

5


8.2.2.1. Inhibition distortion of serum albumin 93

8.2.2.2. Stabilizing test of erythrocyte membrane 96


8.3. Toxicology screening. Determination of acute toxicity in

vivo

99




8.4. Assessment of in vivo anti-inflammatory potential 102


8.4.1.1. Model of acute inflammation induced by

carrageenan in rats

102

8.4.1.2. Chronic inflammation model induced in

rats - granuloma test

105


8.4.2.1. Anti-inflammatory effect on acute

inflammation model

106

8.4.2.2. Anti-inflammatory effect on chronic

inflammation model

108

8.4.2.3. Conclusion 114

8.5.The evaluation of biochemical and hematological

parameters on the model of chronic inflammation

115


8.5.1.1. The assessment of biochemical parameters 115

8.5.1.2. The evaluation of hematological

parameters

120


8.5.2.1. The assessment of biochemical parameters 121

8.5.2.2. The evaluation of hematological

parameters

127

8.5.3. Conclusions 129

Chapter 9 130

GENERAL CONCLUSIONS 130

BIBLIOGRAPHY 134

ANNEX - LIST OF WORKS 148

6

ABBREVIATIONS

1H-NMR

ABTS

AD

AIDS

ALS

ALT

AST

CBC- CCl4

CNS

COX

DCM

DM

DMFA

DMSO

DNA

DPPH

EC50

FA

IL-6

IL-1β

IR

LD50

LDH

m.p.

MeOH

MIC

NMR

NSAIDs

ppm

ROS

RT

SOD

TNF-α

TLC

Proton-Nuclear magnetic resonance

2,2-azino-bis(3-ethylbenzothiazolyn-6-sulfonic) acid

Alzheimer Disease

Acquired immune deficiency syndrome

Amyotrophic lateral sclerosis

Alanine aminotransferase

Aspartatamino-transferase

Complete blood count

Carbon tetrachloride

Central nervous siystem

Ciclooxigenase

Dichloromethan

Diabet mellitus

Dimethylformamide

Dimethyl sulfoxide

Deoxyribonucleic acid

2,2-diphenyl-1-picrylhydrazil

Efficient Concentration 50

Ferulic acid

Interleukin 6

Interleukin 1β

Infrared

Lethal dose 50

Lactate dehydrogenase

Melting point

Methanol

Minimal inhibitory concentration

Nuclear magnetic resonance

Nonsteroidal anti-inflammatory drugs

Parts per million

Reactive oxygen species

Reverstranscriptase

Superoxide dismutase

Tumor necrosis factor α

Thin layer chromatography

7

ACKNOWLEDGEMENT

Completing the PhD thesis represents the ending moment of an

important step in my professional training and the result of a sustained effort

over four years of research. This moment is due also to those who helped

me, supported me, forming together a professionally and collegially team.

I owe special gratitude to Mrs. Univ. Prof. Dr. Lenuţa Profire, as

scientific coordinator for her effort and patience showed in the permanent

and competent guidance during the development and the realization of this

thesis.

Sincere thanks to Mrs. Univ. Prof. Dr. Monica Hăncianu - Dean of

the Faculty of Pharmacy, for her support over the years.

I also want to thank to the distinguished official reviewers Mrs.

Univ. Prof. Dr. Ileana Chiriţă - University of Medicine and Pharmacy "Carol

Davila"-Bucharest, Mrs. Univ. Prof. Dr. Cornelia Vasile - Institute of

Macromolecular Chemistry "Petru Poni"- Iasi and Mrs. Univ. Lect. Dr.

Catalina Daniela Stan - University of Medicine and Pharmacy "Grigore T.

Popa" Iasi.

I wish to thank also Mrs. Univ. Prof. Dr. Rodica Cuciureanu, from

the discipline of Environmental Chemistry and Food, Mrs. Univ. Prof. Dr.

Anca Miron, from the discipline of Pharmacognosy and Mr. Lect. Univ. Dr.

Dan Lupaşcu, from the discipline of Pharmaceutical Chemistry as members

of the committee of guidance for the suggestions and moral support offered,

which contributed to the completion of this scientific endeavor.

Sincere thanks to Mrs. Univ. Prof. Dr. Elena Catalina Lupuşoru,

from the discipline of Pharmacology, Faculty of Medicine, for her support

in achieving the toxicological and pharmacological studies.

Special thanks, in particular, to the management team of the

project "Strategic partnership to improve the quality of medical research in

universities through doctoral and postdoctoral scholarships -

DocMed.Net_2.0" HRD/159/1.5/S/136893, for scientific support and given

material.

Warm thanks goes to Drug Industry and Pharmaceutical

Biotechnology department and to Pharmaceutical Chemistry departemenfor

for their help and understanding showed during these four years.

I express my gratitude to my dear husband and son and to my

family for their understanding and moral support shown all these years,

when I had to work exclusively for my professional research.

8

CHAPTER 5

THE MOTIVATION REASONS AND THE OBJECTIVES

FOR PERSONAL RESEARCH

Ferulic acid (4-hydroxy-3-methoxy-cinnamic acid) is a

phenolic compound widely distributed in the plant world; among the

richest sources are: wheat bran, whole grains, citrus, coffee,

eggplant, bamboo shoots, sugar beet, kale, spinach and broccoli. It is

primarily known as a potent antioxidant, is able to protect DNA and

the biological lipids by oxidative stress.

If we refer only to neurodegenerative diseases such as

Alzheimer's, Parkinson's, Huntington's and amyotrophic lateral

sclerosis (ALS), which in Europe affects more than ten million

people, a number that will probably double in the coming decades,

the potential use of ferulic acid to treat these conditions is

particularly important.

An important feature of the Alzherimer disease (AD) is the

inflammation mediated by the activation of microglial and

astroglial (astrocytes) cells and is an important source of oxidative

stress by inducing the formation of superoxide anion. Inflammation

is located around the amyloid plaques and it is characterized by the

release of proinflammatory substances from the activated microglial

cells. The most important molecules released in the inflammatory

process are the reactive oxygen species, prostaglandins, interleukin

1β (IL-1β), interleukin 6 (IL-6) and tumor necrosis factor α (TNF) -

α.

The oxidative stress is also considered to be a major trigger

for autoimmune diseases, cancer, aging, inflammation, and in recent

years more and more research suggests that it plays an important

role in the pathology of neurodegenerative diseases.

It is not known with certainty whether oxidative stress is

the trigger for neurodegeneration or occurs as a side effect of other

illnesses, but there is insufficient evidence supporting its

involvement in the evolution of cellular damage.

Although ROS can cause cell death by three main

mechanisms - lipid peroxidation, protein oxidation and oxidation of

DNA, cells have developed their own defense mechanisms against

oxidative stress and for cell repairement. The endogenous

9

antioxidants are the first line of cell defense including antioxidant

enzymessuch ad superoxide dismutase type, catalase, glutathione

peroxidase, and small molecules as vitamins E and C. Data from the

literature argue that the effectiveness of natural antioxidants

decreases with age while oxidative stress intensifies thus playing a

major role in the neurodegenerative process.

Compared to other tissues, the CNS is extremely vulnerable

to free radicals of oxygen because is a major oxyen consumer, has a

high fat content and in the same time has a relative defficiency in

antioxidant system.

On the other hand, the hydrazone and thiazolidine-4-one

structures are some of the most exploited structures in organic

synthesis. Data from the literature shows that the derivatives with

hydrazone or thiazolidine-4-one structure have significant biological

effects such as antioxidant effect, anti-inflammatory, analgesic,

antimicrobial, antifungal, anti-mycobacterial and anticonvulsant

effect, anti-viral and anti-HIV, anti-tumor and also hypoglycemic

effect.

The main goal of this research was the synthesis of new

ferulic acid derivatives with hydrazone structure (4a-k) and

thiazolidin-4-one structure (5a-l), the resulting derivatives being

obtained by structural modulation of the ferulic acid at the free

carboxyl group. The synthesized compounds have the theoretical

premises for use as potential therapeutic agents in the treatment of

diseases in which inflammation and oxidative stress plays a major

role, among them occupy an important place the neurodegenerative

disorders.

Fig. 5.1. Ferulic acid derivatives with hydrazone structure (4a-k) and

thiazolidin-4-one structure (5a-l)

The achievement for this personal research had the following

objectives:

10

Synthesis and characterization of ferulic acid derivatives

with hydrazone and thiazolidin-4-one structure, where we follow:

the optimization synthesis of intermediate and final

derivatives in order to obtain compounds in high

yield and purity;

the purification of the synthesized compounds by

various methods: precipitation or recrystallization to /

from suitable solvents, separation on silica gel;

physico-chemical characterization of intermediates

and final compounds (molecular formula, melting

point, yield, solubility in different solvents);

confirmation of the chemical structure of the

synthesized compounds by spectroscopic methods

(IR, NMR).

The evaluation of biological potential of ferulic acid

derivatives, where it was followed:

evaluation of the antioxidant potential derivatives

synthesized by determining the antiradical against

DPPH and ABTS, the total antioxidant capacity and

the reducing power;

assessing the toxicity of the compounds synthesized

by determining the lethal dose 50 (LD50);

in vitro evaluation of potential anti-inflammatory,

which aimed the determination of the inhibition of

serum albumin denaturation and erythrocyte

membrane stability compounds synthesized;

the potential anti-inflammatory in vivo evaluation for

the synthesized compounds on a model of acute

inflammation induced by carrageenan and a model of

chronic inflammation - granuloma test;

evaluation of biochemical and hematological

parameters, the model of chronic inflammation

induced in rats - granuloma test.

The scientific results obtained during doctoral studies are due to the

status of scholarship fallower within the project "Strategic partnership to

improve the quality of medical research in universities through doctoral and

postdoctoral scholarships - DocMed.Net_2.0" HRD/159/1.5/S/136893 status

that we had in the period april 2014 - june 2015.

11

CHAPTER 6

THE SYNTHESIS AND CHARACTERIZATION OF NEW


The synthesis of ferulic acid (3-(4-hydroxy-3-methoxy-phenyl)

acrylic acid) with the structure of the hydrazone and thiazolidine-4-

one was carried out in several steps (Fig. 6.1 and

6.2).

R = 4-F(a), 4-Br(b), 4-Cl(c), 2-NO2(d), 3-NO2(e), 4-NO2(f), 2-OH(g), 3-OH(h),

4-OH (i), 2-OCH3(j), H(k)

Fig. 6.1. The general scheme for the preparation of ferulic acid derivatives

with hydrazone structure

R = H(a), 4-Cl(b), 4-F(c), 4-Br(d), 4-NO2(e), 2-NO2(f), 2-OCH3(g), 2-OH(h),

2,6-diCl(i), 4-N(CH3)2(j), 2,3-diOH(k), 4-OH-3-OCH3(l)

Fig. 6.2. The general scheme for the preparation of ferulic acid

derivatives with thiazolidin 4-ones structure.

The synthesis of intermediates and final compounds was

monitored by thin layer chromatography (TLC), the layer of silica

gel on the aluminum support, using as eluent the following solvent

systems: ethyl acetate: methanol: acetone: water and

dichloromethane: methanol, with varying degrees, depending on the

polarity of the compounds. The spots observation on the

chromatograms was done under UV light at 254 nm wavelength.

12

Physical-chemical characterization of intermediates and

final compounds had included the definition of the molecular

formula, relative mass, the melting temperature, solubility in water

and in different organic solvents, and the reaction yield (Table 6.3

and 6.4). Tabel 6.3. The physico-chemical properties of the compounds 4a-k

Comp.

R

Moleculare

formula

Mr

(g/mol)

m.p.

(0C)

ɳ

(%) Solubility

4a -F(4) C17H15FN2O3 314,31 189-194 66,35

readily soluble in

DMFA and DMSO, sparingly

soluble in absolute

ethanol, methanol, chloroform,

acetone, dioxane,

and insoluble in distilled water and

diethyl ether

4b -Cl(4) C17H15ClN2O3 330,08 203-205 69,18

4c -Br(4) C17H15BrN2O3 375,22 210-213 68,81

4d -NO2(2) C17H15N3O5 341,32 196-198 52,04

4e -NO2(3) C17H15N3O5 341,32 185 61,38

4f -NO2(4) C17H15N3O5 341,32 223 60,17

4g -OH(2) C17H16N2O4 312,32 186 55,89

4h -OH(3) C17H16N2O4 312,32 236 25,85

4i -OH(4) C17H16N2O4 312,32 179 84,56

4j -OCH3(2) C18H18N2O4 326,35 200 48,83

4k -H C17H16N2O3 296,32 110 80,53

Tabel 6.4. The physico-chemical properties of the compounds 2- (R-phenyl)

-3- [3- (4-hydroxy-3-methoxyphenyl) acrylamido] -thiazol-4-one (5a-l) Co

mp.

R Moleculare

formula

Mr

(g/mo)

m.p.

(0C)

ɳ

(%) Solubil.

5a -H C19H18N2O4S 370.24 102 15.96

readily

soluble in DMFA,

DMSO and

acetone,

sparingly soluble in

absolute

ethanol, methanol

5b -Cl(4) C19H17ClN2O4S 404.06 190 31.63

5c -F(4) C19H17FN2O4S 388.09 106-110 19.95

5d -Br(4) C19H17BrN2O4S 449.32 173-176 41.24

5e -NO2(4) C19H17N3O6S 415.08 98-102 9.31

5f -NO2(2) C19H17N3O6S 415.08 140-142 36.31

5g -OCH3(2) C20H20N2O5S 400.45 118-122 29.01

5h -OH(2) C19H18N2O5S 386.42 211-215 50.97

5i -Cl(2,6) C19H16Cl2N2O4S 439.31 229-230 82.82

5j N(CH3)2(4) C21H23N3O4S 413.14 160 30.94

5k -OH(2,3) C19H18N2O6S 402.42 212 11.13

5l OH(4),OCH3(3 C20H20N2O6S 416.45 140 15.49

13

CHAPTER 7

THE CHEMICAL STRUCTURE CONFIRMATION OF THE


The structure of the synthesized compounds, intermediates and

final with hydrazone and thiazolidine-4-one structure, was

confirmed by spectral methods: IR spectroscopy (IR) and nuclear

magnetic resonance spectroscopy of proton (1H-NMR).

Infrared Spectroscopy (IR)

The acidic chloride of ferulic acid was confirmed by identifying

the IR characteristics bands found in the base structure (ferulic acid).

The chemical group (CO-Cl) of the acidic chloride appears in the IR

spectrum as an absorption band of high intensity, narrow at the

wavelength of 1724 cm-1. Aromatic ring has been highlighted by the

absorption bands at wavelengths 2949 cm-1 and 854 cm-1,

characteristic for the stretching vibration or deformation of the bond

=CH- and at the wavelength bands 1599 cm-1 and 1508 cm-1

attributable to the stretching vibration of the bond -C=C-.

The formation of ferulic acid hydrazide was confirmed by the

presence of NH-NH2, which is supported by the appearance of

absorption bands in the IR spectrum with an high intensity,

characteristic for -NH2 (3304 cm-1, 3252 cm-1) and the -NH- group

(3182 cm-1, 3149 cm-1). The amide group (-CO-NH) has been

identified at the absorption bands at 1634 cm-1, corresponding to the

C=O, and at 1537 cm-1, characteristic for -NH- bound deformation

vibration.

In the IR spectrum of hydrazone compounds it was observed the

disappearance of the characteristic absorption bands of -NH2 group,

showing only the vibration characteristics for -NH- group, identified

in the region of 3065 cm-1-3263 cm-1 and a CN bond appears at 1227

cm-1 to 1286 cm-1. Another difference from the hydrazide is the

appearance of the absorption band characteristic to the azomethine

bond -C=N-, identified, depending on the compound, in the region

of 1508 cm-1-1607 cm-1. The absorption band specific for the keto

group (CO) of the amide linkage (-CO-NH-) was detected in the

region of 1564 cm-1-1651 cm-1. Halogens were revealed by the

absorption bands at 1095 cm-1 (F), 814 cm-1 (Cl), and 858 cm-1 (Br).

14

Nitro groups were revealed by the symmetric valence vibrations in

the range of 1330 cm-1 - 1353 cm-1, and asymmetrical in the region

of 1380 cm-1 - 1569 cm-1. As the characteristic absorption band for

the phenolic hydroxyl group overlaps with the specific vibration for

-NH- bond , from the spectrum analysis it was only obseved the -CO

bond characteristic band at wavelength 1195 cm-1 (2-hydroxy), 1197

cm-1 (3-hydroxy) or 1164 cm-1 (4-hydroxy). The methoxy group is

identified by the appearance of specific vibration for the-CO bond at

1155 cm-1 (2-methoxy).

Condensation of ferulic acid hydrazide with thioglycolic acid and

various aromatic aldehydes leaded to the obtaining of thiazolidin-4-

one derivatives (5a-l). This cyclization was confirmed by the

appearance of the IR absorption bands for C-S bond (650 cm-1 -704

cm-1) and for the ketone group (1620 cm-1- 1715 cm-1) of the series

of thiazolidine-4- one.

Fig. 7.7. IR spectrum for N-[3-(4-hydroxi-3-metoxiphenyl)acryloyl]-N-

(benzyliden) hydrazine(4k).

Fig. 7.11. IR spectrum for 2-(2,3-dihydroxi-phenyl)-3-[3-(4-hydroxi-3-

metoxiphenyl)acrylamido]-tiazolidin-4-one (5i).

15

7.2.2. Nuclear Magnetic Resonance Spectrum (NMR)

7.2.2.1. 1H-NMR spectrum of the derivatives of N- [3-(4-hydroxy-3-

methoxyphenyl)acryloyl] -N-(R-benzylidene) hydrazine 1H-NMR spectra analysis recorded for ferulic acid hydrazones (4-k)

provided the following information (Table 7.3):

azomethine group(-CH = N) proton appears as a singlet in the

range of 8.57 to 9.01 ppm, with the integrale value of one;

the signals for the aromatic protons appeared in the range of

6.55 to 8.57 ppm as a doublet (d), triplet (t), doublet of

doublet (dd), triplet of doublets (TD) or multiplet, depending

on the structure of hydrazine;

the signal corresponding to the three protons from OCH3

(methoxy) group, characteristic of the structure of ferulic acid

was found in the range 3.74 to 3.85 pm, as a singlet with

integral three.

Table 7.3. 1H-NMR spectral characteristics of the compounds N- [3-(4-hydroxy-3-

methoxyphenyl)acryloyl]-N-(R-benzylidene) hydrazine (4a-k)

Nr. R 1H-NMR (400 MHz, DMSO-d6, δ ppm)

4a 4-F 3.83 (s, 3H, OCH3), 6.79-6.89 (m, 2H, Ar-H), 6.99-7.03 (m, 1H, Ar-H), 7.11 (s, 1H, Ar-H), 7.29-7.37 (m, 3H, Ar-H), 7.81 (dt, 2H,

Ar-H), 8.83 (s, 1H, CH=N)

4b 4-Cl 3.82 (s, 3H, OCH3), 6.80-6.85 (m, 2H, Ar-H), 6.95-7.04 (m, 1H,

Ar-H), 7.19 (s, 1H, Ar-H), 7.37 (dd, 1H, Ar-H), 7.52 (dd, 2H, Ar-H), 7.77 (dd, 2H, A-H), 8.84 (s, 1H, CH=N)

4c 4-Br 3.85 (s, 3H, OCH3), 6.77-6.85 (m, 2H, Ar-H), 6.93-6.99 (m, 1H, Ar-H), 7.13 (s, 1H, Ar-H), 7.30-7.37 (m, 1H, Ar-H), 7.58-7.63 (m,

2H, Ar-H), 7.72-7.75 (m, 2H, A-H), 8.81 (s, 1H, CH=N)

4d 2-NO2 3.83 (s, 3H, OCH3), 6.69-6.75 (m, 1H, Ar-H), 6.88-6.93 (m, 1H,

Ar-H), 6.99 (dd, 1H, Ar-H), 7.16 (s, 1H, Ar-H), 7.35-7.43 (m, 1H,

Ar-H), 7.59-7.64 (m, 1H, Ar-H), 7.91-7.98 (m, 2H, Ar-H), 8.09 (dd, 1H, Ar-H), 8.78 (s, 1H, CH=N)

4e 3-NO2 3.81 (s, 3H, OCH3), 6.75-6.84 (m, 1H, Ar-H), 6.89-6.99 (m, 2H, Ar-H), 7.11 (s, 1H, Ar-H), 7.32-7.36 (m, 1H, Ar-H), 7.78-7.85 (m,

1H, Ar-H), 8.15-8.22 (m, 2H, Ar-H), 8.52-8.57 (m, 1H, Ar-H),

8.80 (s, 1H, CH=N)

16

Nr. R 1H-NMR (400 MHz, DMSO-d6, δ ppm)

4f 4-NO2 3.82 (s, 3H, OCH3), 6.79-6.85 (m, 1H, Ar-H), 6.89-7.00 (m, 2H, Ar-H), 7.12 (s, 1H, Ar-H), 7.30-7.39 (m, 1H, Ar-H), 8.09-8.14 (m,

2H, Ar-H), 8.33-8.39 (m, 2H, Ar-H), 8.70 (s, 1H, CH=N)

4g 2-OH 3.81 (s, 3H, OCH3), 6.55-6.59 (m, 1H, Ar-H), 6.80-6.87 (m, 1H,

Ar-H), 7.00-7.08 (m, 1H, Ar-H), 7.20-7.29 (m, 2H, Ar-H), 7.33-

7.35 (m, 1H, Ar-H), 7.60-7.65 (m, 1H, Ar-H), 7.72-7.79 (m, 1H, Ar-H), 7.94 (s, 1H, Ar-H), 9.01 (s, 1H, CH=N)

4h 3-OH 3.83 (s, 3H, OCH3), 6.79-6.89 (m, 2H, Ar-H), 6.99-7.05 (m, 2H, Ar-H), 7.16 (s, 1H, Ar-H), 7.25-7.30 (m, 1H, Ar-H), 7.37-7.46 (m,

3H, Ar-H), 8.59 (s, 1H, CH=N)

4i 4-OH 3.79 (s, 3H, OCH3), 6.79-6.83 (m, 1H, Ar-H), 6.86-6.90 (m, 3H,

Ar-H), 6.99 (dd, 1H, Ar-H), 7.12 (s, 1H, Ar-H), 7.34-7.42 (m, 1H,

Ar-H),7.78 (dd, 2H, Ar-H), 8.57 (s, 1H, CH=N)

4j 2-OCH3 3.83 (s, 6H, OCH3), 6.79-6.84 (m, 1H, Ar-H), 6.89-6.99 (m, 2H, Ar-H),7.08-7.15 (m, 2H, Ar-H), 7.37-7.42 (m, 1H, Ar-H), 7.58-

7.64 (m, 2H, Ar-H), 7.72-7.79 (m, 1H, Ar-H), 9.01 (s, 1H, CH=N)

4k -H 3.74 (s, 3H, OCH3), 6.79-6.82 (m, 1H, Ar-H), 6.98-7.12 (m, 2H,

Ar-H), 7.39-7.43 (m, 1H, Ar-H), 7.48-7.52 (m, 1H, Ar-H), 7.54-

7.60 (m, 3H, Ar-H), 7.65-7.68 (m, 2H, Ar-H), 8.71 (s, 1H, CH=N)

7.2.2.2. 1H-NMR spectrum of the derivatives of 2- (R-phenyl) -3- [3-

(4-hydroxy-3-methoxyphenyl) acrylamido] -thiazol-4-one

The structure confirmation for the derivatives 2- (R-phenyl)

-3- [3- (4-hydroxy-3-methoxyphenyl) acrylamido] -thiazol-4-one

(5a-l) is supported by the presence in the 1H-NMR spectrum of the

protons signals for the thiazolidin-4-one scaffold, the aromatic ring

and the ferulic acid (Table 7.4).

The proton signals characteristics to the thiazolidin-4-one were

found in the following frequencies:

proton of the group CH-N (methin) has been identified in

the 5.74 to 5.95 ppm as a singlet or doublet signal, with a

coupling constant 1.05-2.5 Hz or as a multiplet, in both

cases wit the integral one;

the protons from CH2S group were identified in the ranfe

from 3.62 to 4.03 ppm as doublet, doublet of doublet,

triplet of doublet or multiplet, with integral two.

The signals for the protons included in the aromatic ring

derived from the aromatic aldehydes were superimposed with

the signals of aromatic protons from the ferulic acid structure

and have been identified in the range from 6.64 to 8.33 ppm.

17

Proton chemical shifts were influenced by graft substitutes on

the aromatic ring.

The signal corresponding to the three protons from the OCH3

(methoxy) group, characteristic to the structure of ferulic acid

was found in the range 3.69 to 4.04 ppm as a singlet with three

as a integrale.

Table. 7.4. 1H-NMR spectral characteristics of 2- (R-phenyl) -3- [3- (4-hydroxy-3-

methoxyphenyl) acrylamido] -thiazol-4-one (5a-l)

Nr.

R 1H-NMR (400 MHz, DMSO-d6, δppm)

5a -H 3.83 (s, 3H, OCH3), 3.85-3.95 (m, 2H, CH2-S), 5.92 (s, 1H, CH-N),

6.79 (dt, 1H, Ar-H), 6.89-6.93 (m, 1H, Ar-H), 6.99-7.04 (m, 1H, Ar-H), 7.16 (s, 1H, Ar-H), 7.36-7.49(m, 5H, Ar-H), 7.82-7.91 (m,

1H, NH-N)

5b 4-Cl 3.76-3.80 (m, 2H, CH2-S), 3.83 (s, 3H, OCH3), 5.92 (s, 1H, CH-N),

7.17 (t, 1H, Ar-H), 7.16 (p, 1H, Ar-H), 7.34-7.39 (m, 3H, Ar-H), 7.41 (p, 1H, Ar-H), 7.51-7.55 (m, 2H, Ar-H), 7.62 (t, 1H, Ar-H),

7.82-7.91 (m, 1H, NH-N)

5c 4-F 3.62-3.75 (m, 2H, CH2-S), 4.04 (s, 3H, OCH3), 5.74-5.80 (m, 1H,

CH-N), 7.13 (t, 1H, Ar-H), 7.16 (p, 1H, Ar-H), 7.23 (t, 1H, Ar-H),

7.31 (p, 1H, Ar-H), 7.37 (t, 1H, Ar-H), 7.42 (t, 1H, Ar-H), 7.44-7.50 (m, 2H, Ar-H), 7.58 (dt, 1H, Ar-H), 7.88-7.95 (m, 1H, NH-N)

5d 4-Br 3.80 (s, 3H, OCH3), 3.85-3.95 (m, 2H, CH2-S), 5.92 (s, 1H, CH-N), 6.79 (t, 1H, Ar-H), 6.89 (p, 1H, Ar-H), 6.97 (t, 1H, Ar-H), 7.12 (dt,

2H, Ar-H), 7.16-7.20 (m, 1H, Ar-H), 7.37 (dd, 1H, Ar-H), 7.85 (dt,

2H, Ar-H), 8.15-8.25 (m, 1H, NH-N)

5e 4-NO2 3.69 (s, 3H, OCH3), 3.85-3.92 (m, 2H, CH2-S), 5.81-5.97 (m, 1H,

CH-N), 6.69 (s, 1H, Ar-H), 7.12-7.25 (m, 1H, Ar-H), 7.58-7.72 (m, 2H, Ar-H), 7.87(dd, 1H, Ar-H), 8.14-8.18 (m, 2H, Ar-H), 8.22 (s,

1H, Ar-H), 8.33 (s, 1H, Ar-H)

5f 2-NO2 3.83 (s, 3H, OCH3), 3.84-3.91 (m, 2H, CH2-S), 5.92 (s, 1H, CH-N),

6.79 (t, 1H, Ar-H), 6.85-6.89 (m, 1H, Ar-H), 6.99-7.04 (m, 1H, Ar-H), 7.16 (s, 1H, Ar-H), 7.37 (dd, 1H, Ar-H), 7.45-7.49 (m, 1H, Ar-

H), 7.52-7.58 (m, 1H, Ar-H), 7.72-7.78 (m, 1H, Ar-H), 7.96-8.01

(m, 1H, Ar-H), 8.09-8.15 (m, 1H, NH-N)

5g 2-OCH3 3.83 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 3.95-4.03 (m, 2H, CH2-S),

5.95 (s, 1H, CH-N), 6.77-6.84 (m, 2H, Ar-H), 6.89-6.92 (m, 3H, Ar-H), 6.98-7.06 (m, 1H, Ar-H), 7.12 (s, 1H, Ar-H), 7.16 (dd, 1H,

Ar-H), 7.37 (dd, 1H, Ar-H), 8.02-8.09 (m, 1H, NH-N)

18

Nr. R 1H-NMR (400 MHz, DMSO-d6, δppm)

5h 2-OH 3.83 (s, 3H, OCH3), 3.82-3.89 (m, 2H, CH2-S), 5.92 (s, 1H, CH-N),

6.69-6.80 (m, 5H, Ar-H), 6.89-7.00 (m, 2H, Ar-H), 7.16 (s, 1H, Ar-H), 7.35-7.39 (m, 1H, Ar-H), 8.02 (s, 1H, NH-N)

5i 2,6-diCl 3.80 (s, 3H, OCH3), 3.75-3.84 (m, 2H, CH2-S), 5.87 (s, 1H, CH-N), 6.69-6.83 (m, 4H, Ar-H), 6.89-6.95 (m, 1H, Ar-H), 6.99-7.03 (m,

1H, Ar-H), 7.15 (s, 1H, Ar-H), 7.32-7.37 (m, 1H, Ar-H), 7.86-7.91

(m, 1H, NH-N)

5j 4-

N(CH3)2

3.06 (s, 6H, N-(CH3)2, 3.80 (s, 3H, OCH3), 3.84-3.93 (m, 2H, CH2-

S), 5.86-5.92 (m, 1H, CH-N), 6.64-6.79 (m, 3H, Ar-H), 6.80-6.88

(m, 1H, Ar-H), 6.93-7.05 (m, 3H, Ar-H), 7.16(s, 1H, Ar-H), 7.34-

7.40 (m, 1H, Ar-H), 8.33 (s, 1H, Ar-H)

5k 2,3-diOH 3.83 (s, 3H, OCH3), 3.84-3.90 (m, 2H, CH2-S), 5.90 (s, 1H, CH-N),

6.59-6.72 (m, 3H, Ar-H), 6.79-6.84 (m, 1H, Ar-H), 6.89-6.93 (m, 1H, Ar-H), 6.99-7.05 (m, 1H, Ar-H), 7.16(s, 1H, Ar-H), 7.37-7.40

(m, 1H, Ar-H), 8.15 (s, 1H, NH-N)

5l 3-OCH3-

4-OH

3.83 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 3.88-3.94 (m, 2H, CH2-S),

5.95 (s, 1H, CH-N), 6.72-6.79 (m, 2H, Ar-H), 6.89-6.95 (m, 1H,

Ar-H), 6.99-7.07 (m, 1H, Ar-H), 7.16 (s, 1H, Ar-H), 7.34-7.37(m, 2H, Ar-H), 7.45-7.48 (m, 1H, Ar-H), 8.02-8.09 (m, 1H, NH-N)

19

CHAPTER 8

THE BIOLOGICAL EVALUATION OF


8.1. The antioxidant potential evaluation

The antioxidant activity was evaluated by two antiradical

methods against DPPH radicals (2,2-diphenyl-1-picrilhidrazil) and

ABTS•+ (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid)),

and the results were expressed by calculating the percentage

inhibition (I%), by the graphical representation of the percentage of

inhibition and the calculation of EC50. The other two tests include

the determination of total antioxidant capacity and reducing power,

the results obtained were expressed in terms of graphic

representation of the absorbance and the calculation of EC50.

8.1.2. Results and discussion

8.1.2.1. Determination of the antiradical effect against radical

DPPH

In the series of derivatives of N-[3-(4-hydroxy-3-

methoxyphenyl)acryloyl]-N-(R-benzylidene)hydrazine (4a-k), it was

observed that most of the compounds studied are more active than

ferulic acid (FA) obtained at all dilutions of 2 mg/mL stock solution

(Fig. .8.2).

Fig. 8.2a. Inhibation capacity (I%) of DPPH free radicals by the ferulic

acid hydrazones (4a-k), at different concentrations after 30minutes.

20

From these, the most active compounds were found to be

the hydrazones resulting from the condensation reaction of ferulic

acid hydrazide with benzaldehyde (4k), 4-nitrobenzaldehyde (4f), 2-

hydroxybenzaldehyde (4g) and 4-hydroxybenzaldehyde (4i). The

percentage inhibition recorded for the compounds at the

concentration of 133µg/mL was I% = 98.44 ± 0.06% after 30

minutes and I% = 98.55 ± 0.09% after 60 minutes for the compound

4k, I% = 98.31 ± 0.15% after 30 minutes and I% = 99.07 ± 0.17%

after 60 minutes for 4f, I% = 98.30 ± 0.01% after 30 minutes and I%

= 98.42 ± 0.03% after 60 minutes for 4g and I% = 98.04 ± 0.06%

after 30 minutes and I% = 98.39 ± 0.07% after 60 minutes for 4i

compound.

For the series of 2- (R-phenyl) -3- [3- (4-hydroxy-3-

methoxy-phenyl) acrylamido] -thiazol-4-one (5a-l) derivatives (Fig.

8.3a) it has been shown that the insertion of thiazolidin-4-one core

on the ferulic acid molecule by the amide bond formation has as

result the enhancing of its antioxidant action. Similar with the ferulic

acid hydrazones, these compounds showed that the antiradical effect

towards DPPH free radicals is directly proportional with the

concentration of the compound and it increases in time.

At all dilutions of the 2 mg/mL stock solution, most of the

tested compounds had an activity similar or bigger than ferulic acid.

For example, at a concentration of 133 µg/mL (in the sample), the

reprezentative compound of the series 5a, resulting from the

condensation reaction of ferulic acid hydrazide, thioglycolic acid

and benzaldehyde the percent of DPPH free radicals inhibition was

I% = 95.76 ± 0.03% after 30 minutes and I% = 97.00 ± 0.03% after

60 minutes. In similar circumstances, the antiradical effect against

the DPPH radical for the ferulic acid was expressed as inhibition

percentage with the value I%=94.56 ± 0.12 after 30 minutes,

respectively I% = 95.72 ± 0,1 after 60 minutes. Regarding the

influence of the substitutition on the aromatic moiety from the

thiazolidin-4-one structure, it was found to have the most favorable

influence the following substituents: 2,3-dihydroxy (5k), 4-hydroxy-

3-methoxy (5l), 4-nitro (5f) and 4-fluoro (5c); the antioxidant effect

of these compounds are slightly more intense than the unsubstituted

derivative 5a. For these compounds the inhibition percentages were

I% = 98.46 ± 0.06% after 30 minutes and I% = 98.66 ± 0.02% after

60 minutes (5k), I%=97.85 ± 0 07% after 30 minutes and I%=98.22

21

± 0.019 after 60 minutes (5l), I%=97.68 ± 0.03% after 30 minutes

and I%=98.28 ± 0.01% after 60 minutes for (5f) compound and

I%=96.60 ± 0.06% after 30 minutes and I%=97.54 ± 0.07% after 60

minutes (5c).

It should be noted that for 5k and 5l derivatives the

antiradical action is maintained at the concentration of 0.5 mg/mL

and they were the only ones in the series which presented at this

concentration the antiradical effect. The data presented show the

positive influence of hydroxy and methoxy substituents on the

antiradical action of ferulic acid derivatives with thiazolidin-4-one

structure.

Fig. 8.3a. The inhibition capacity(I%) of DPPH free radicals for the ferulic acid

derivatives with thiazolidin-4-one structure (5a-l), at different concentr. after 30 min.

8.1.2.2. The antiradical effect determination against

ABTS·+radical cation.

For both series of compounds it was found that the

structural modulation of the ferulic acid at the free carboxyl group

conducted to the maintaining and even the enhancing of its

antiradical action. The compounds analyzed are more active as the

percent inhibition is bigger and the EC50 values are smaller.

In the series of N-[3-(4-hydroxy-3-methoxyphenyl)

acryloyl]-N-(R-benzylidene) hydrazine (4a-k) derivatives, it was

observed that for all the compounds tested the antiradical activity is

directly proportional with the concentration (Fig. 8.5).

At the concentration of 133 mg/mL, the compounds

resulting from the condensation reaction with 4-nitrobenzaldehyde

(4f, I% = 93.33 ± 0.56%) and 2-hydroxybenzaldehyde (4g, I% =

96.89 ± 0.06 %) showed an increased activity than the ones of

ferulic acid (FA I%= 89.31 ± 0.09%). Proved to be more active are

22

the compounds resulted from the condensation reaction with

benzaldehyde (4k, I% = 99.44 ± 0.03%) and 4-hydroxy-

benzaldehyde (4i, I% = 99.59 ± 0.02%).

Fig. 8.5. The antiradical activity towards the ABTS·+ radical ( Inhibition

percentage %) of the N- [3- (4-hydroxy-3-methoxyphenyl) acryloyl] -N- (R-

benzylidene) hydrazine compounds(4a-k), at different concentrations.

In the series of 2- (R-phenyl) -3- [3- (4-hydroxy-3-methoxy-

phenyl) acrylamido] -thiazol-4-one derivatives (5a-l) (fig. 8.6) at the

concentration of 2 mg/mL (different dilutions), the most active

derivative proved to be the one resulting from the condensation of

ferulic acid hydrazide, thioglycolic acid and 2,3-

dihydroxybenzaldehyde (5k).

Fig. 8.6. The antiradical activity towards the ABTS·+ radical ( Inhibition percentage %) of the 2-(R-phenyl)-3-[3-(4-hydroxi-3-metoxyphenil)acrylamido]-thiazolidin-4-

one (5a-l), at different concentrations.

At the concentration of 133 µg/mL, the percent of inhibition

recorded for the compound 5k was I = 97.67% ± 0.15%, while under

similar experimental ferulic acid (FA) showed a percent of

23

inhibition I % = 88.61 ± 0.09%. Higher antiradical activity than the

one of ferulic acid had the derivative substituted on the aromatic ring

with 4-hydroxy-3-methoxy, where I% = 93.89 ± 0.20%.

The compounds obtained by the reaction of condensation with

benzaldehyde (5a, I% = 91.85 ± 0.25%), 2,6-dichlorobenzaldehyde

(5i, I% = 91.31 ± 0.19%), 4-fluorobenzaldehyde (5c, I% = 90.69 ±

0.24), 4-nitrobenzaldehyde (4f, I= 90.64 ± 0.29% ) experienced a

comparable antiradical activity with the one of ferulic acid (I% =

88.61 ± 0 , 09%). (Fig. 8.6).

8.1.2.3. The determination of total antioxidant capacity

Total antioxidant capacity is inversely proportional with the

value of EC50 and directly proportional with the absorbance. It is

estimated that a compound with an increased total antioxidant

capacity has a small EC50 value and a high absorbance value.

Regarding the results obtained at different concentrations is was

observed that the absorbance of the tested compounds increases with

the concentration increase.

For the series of derivatives of N- [3- (4-hydroxy-3-

methoxyphenyl) acryloyl] -N- (R-benzylidene) hydrazine (4a-k) (Fig.

8.7) it is observed that, except for the compounds 4c and 4j

containing 4-Br or 2-OCH3 as substituent on the aromatic ring, all

the other compounds were more active than ferulic acid.

Fig. 8.7. The total antioxidant capacity of the compounds N- [3- (4-

hydroxy-3-methoxyphenyl) acryloyl] -N- (R-benzylidene) hydrazine (4a-k),

at different concentrations.

The biggest absorbance increase, concentration dependent

(from 9.09 mg/mL to 54.54 mg/mL) was recorded for the following

24

compounds 4a (R = 4-F, from 0.2133 ± 0.0013 to 2.1519 ± 0.0032),

4b (R = 4-Cl, from 0.1658 ± 0.0027 to 1.7425 ± 0.013, 4d (R = 2-

NO2, from 0.3069 to 1.9635 ± 0.00015 ± 0.0113), 4e (R = 3-NO2,

from 0.252 ± 0.0017 to 1.9635 ± 0.0113), 4f (R = 4-NO2, from

0.2985 ± 0.0013 to 1.4527 ± 0.0018), 4g (R = 2-OH, from 0.4768 ±

0.0009 to 1.8399 ± 0.00025), 4h (R = 3-OH, from 0.2299 to 1.4577

± 0.00029 ± 0.0005), 4i (R = 4-OH, from 0.2683 ± 0.0004 to 1.6515

± 0.0027) 4k (R = H, from 0.1866 ± 0.0006 to 1.9519 ± 0.0032). For

these compounds the values of absorbance were increased 4-10

times at the concentration of 54.54 mg/mL compared to the values

obtained at the concentration of 9.09 mg/mL.

In the series of derivatives of 2-(R-phenyl)-3-[3-(4-

hydroxy-3-methoxy phenyl)acrylamido]-thiazol-4-one (5a-l) (Fig.

8.8), the largest increase in the values of absorbance, concentration-

dependent (from 9.09 mg/mL to 54.54 mg/mL) was recorded for 5c

(R = 4-F, from 0.1047 ± 0.0004 to 0.9487 ± 0.0006 ), 5h (R = 2-OH,

from 0.1052 ± 0.0008 to 0.9181 ± 0.0011), 5g (R = 2-OCH3, from

0.1520 ± 0.0009 to 1.1615 ± 0.0008), 5i (R = H, from 0.1757 ±

0.0006 to 1.2742 ± 0.0012), 5k (R = 2,3-diOH, from 0.1772 ±

0.0006 to 1.1367 ± 0.0011), 5b (R = 4-Cl, from 0.1493 ± 0.0004 to

0.9019 ± 0.0013), 5e (R = 2-NO2, from 0.1793 ± 0.0006 to 0.9917 ±

0.0017) and 5l (R = 4-OH-3-OCH3, from 0.2116 ± 0.0008 to 1.0699

± 0.0003). The values of absorbance were increased 4.3 to 9.4 times

at the concentration of 54.54 mg/mL compared to the values

obtained at the concentration of 9.09 mg/ mL.

Fig. 8.8. The total antioxidant capacity of the derivatives of 2- (R-phenyl) -3- [3- (4-

hydroxy-3-methoxyphenyl) acrylamido] -thiazol-4-one (5a-l) at various

concentrations.

25

8.1.2.4. Determination of reducing power

Reducing power is inversely proportional with the value of

EC50 and directly proportional with the absorbance. It is estimated

that a compound has an intense reducing power if the EC50 value is

lower and the absorbance values are higher.

The analysis of the results, expressed as effective

concentration 50 values (EC50) showed that the ferulic acid

structural modulation by the condensation reaction of the

corresponding hydrazide with various aromatic aldehydes resulted in

the majority of cases of enhancing the reducing power of ferulic acid

(Table 8.7). The less active derivative was resulted from the

condensation reaction with 4-bromobenzaldehyde (4c), thereby it is

highlighted the negative influence of the substitution on the aromatic

ring with bromine in the para position.

The most active compounds were found to be: 4h (R = 3-

OH, EC50 = 0.1284 ± 0.01 mg/mL) and 4f (R = 4-NO2, EC50 =

0.1431 ± 0.01 mg/mL ), the compounds being 1,5 times (4h) and 1,3

times more active than ferulic acid (EC50 = 0.1898 ± 0.02 mg/mL).

The following compounds showed considerable activity: 4a (R = 4-

F, EC50 = 0.1537 ± 0.04 mg/mL) and 4d (R = 2-NO2, EC50 = 0.1539

± 0.03 mg/mL) 4i (R = 4-OH, EC50 = 0.1545 ± 0.04 mg/mL) and 4b

(R = 4-Cl, EC50 = 0.1581 ± 0.03 mg/mL), which is about 1,2 times

more active than ferulic acid.

Table 8.7. The EC50 values (mg/mL) of N- [3- (4-hydroxy-3-

methoxyphenyl) acryloyl] -N- (R-benzylidene) hydrazine derivatives (4a-k) Comp. R EC50 (mg/mL)* Comp. R EC50 (mg/mL)

Ferulic acid (FA) 0,1898 ± 0,02 4f -NO2(4) 0,1431 ± 0,01

4a -F(4) 0,1537 ± 0,04 4g -OH(2) 0,1834 ± 0,04

4b -Cl(4) 0,1581 ± 0,03 4h -OH(3) 0,1284 ± 0,01

4c -Br(4) 0,9310 ± 0,07 4i -OH(4) 0,1545 ± 0,04

4d -NO2(2) 0,1539 ± 0,03 4j -OCH3(2) 0,1631 ± 0,09

4e -NO2(3) 0,1685 ± 0,02 4k -H 0,1572 ± 0,02

Ascorbic acid 0,0517 ± 0,015

* EC50 values represent the average of three determinations ± standard deviation.

The analysis of the results expressed as effective

concentration values 50 (EC50) (Table 8.8) revealed that the most

active compound it proved to be the one resulting from the

condensation reaction of ferulic acid hydrazide, thioglycolic acid

26

and 2,3-dihydroxybenzaldehyde; 5k compound (R = 2,3-OH, EC50 =

0.0899 ± 0.001 mg/mL) proved to be about 4.2 times more active

than ferulic acid (EC50 = 0.3812 ± 0.09 mg/mL).

A significant activity have also shown 5j (R = 4-N(CH3)2,

EC50 = 0.2571 ± 0.04 mg/mL), 5f (R = 4-NO2, EC50 = 0.2937 ± 0.02

mg/mL) and 5e (R = 2-NO2, EC50 = 0.3357 ± 0.09 mg/mL), the

compounds being about 1.5 times (5j), 1.3 times (5f) and 1.2 times

(5e) more active than ferulic acid.

Less active were the following compounds 5l (R = 4-OH-3-

OCH3, EC50 = 0.37106 ± 0.65 mg/ml) and 5a (R = H, EC50 = 0.3716

± 0.3 mg/mL), their activity is comparable to that of ferulic acid

(EC50 = 0.3812 ± 0.09 mg/mL).

Table 8.8. The EC50 values (mg/mL) of 2- (R-phenyl) -3- [3- (4-hydroxy-

3-methoxyphenyl) acrylamido] -thiazol-4-one derivatives (5a-l) Comp. R EC50 (mg/mL)* Comp. R EC50 (mg/mL)

Ferulic acid (FA) 0,3812 ± 0,09 5g -OCH3(2) 1,6725 ± 0,03

5a -H 0,3716 ± 0,03 5h -OH (2) 1,2106 ± 0,05

5b -Cl(4) 1,7269 ± 0,15 5i -Cl (2,6) 0,6923 ± 0,03

5c - F(4) 0,4228 ± 0,09 5j - N(CH3)2(4) 0,2571 ± 0,04

5d - Br(4) 1,823 ± 0,11 5k -OH(2,3) 0,0899 ± 0,001

5e - NO2(2) 0,3357 ± 0,09 5l -OH(4),OCH3(3) 0,37106 ± 0,05

5f - NO2(4) 0,2937 ± 0,02 Ascorbic acid 0,0516 ± 0,01

* EC50 values represent the average of three determinations ± standard deviation.

8.2. Evaluation of anti-inflammatory potential in vitro

8.2.2.1. Inhibition of serum albumin denaturation

The inhibition percent of serum albumin distortion for the

ferulic acid derivatives with hydrazone structure (4a-k) and

thiazolidin-4-one (5a-l) at different concentrations (100 µg/mL, 200

µg/mL, 500 µg/mL) obtained from the stock solution with a

concentration of 10 mg/ml are represented in fig. 8.11 and 8.12. It is

considered that the derivative with an intense capacity to inhibit the

serum albumin distortion has lower values of the absorbance, which

means a bigger inhibition percent.

From the results analysis it is observed that for all the studied

derivatives the denaturation inhibition of bovine serum albumin

increases with the concentration, the highest percentage of inhibition

was achieved at a concentration of 500 µg/mL.

27

For the series of of N- [3- (4-hydroxy-3-methoxyphenyl)

acryloyl] -N- (R-benzylidene) hydrazine derivatives (4a-k) has been

observed that the majority of the compounds had at the

concentration of 500 µg/mL, an comparable effect to that of

diclofenac, used as a positive control for inhibition of the percentage

of distortion of bovine serum albumin with the value 98.61 ±

0.001% (Fig. 8.11).

Regarding the influence of aromatic ring substitution on the

effect of inhibition for protein denaturation was observed that the

most favorable influence is exerted by fluorine substitution on the

aromatic ring in the para position with nitro and hydroxy in the meta

and para. For these compounds, the percentages of inhibition were

98.23 ± 0.002% (4a, R = -F(4)), 98.76 ± 0.0045% (4e, R = NO2 (3)),

98.38 ± 0.0012% (4f, R = NO2(4)), 98.38 ± 0.0017% (4h, R = -OH

(3)) and 98.23 ± 0.0023% (4i, R = -OH(4).

Fig. 8.11. The inhibiting activity of the bovine serum albumin distortion for N- [3- (4-hydroxy-3-methoxyphenyl) acryloyl] -N- (R-benzylidene) hydrazine derivatives

(4a-k), at different concentrations.

In the series of derivatives of 2- (R-phenyl) -3- [3- (4-

hydroxy-3-methoxyphenyl) acrylamido] -thiazol-4-one (5a-l), it was

observed that the biggest ability to inhibit the protein denaturation

was presented for 5f and 5h compounds, obtained from the

condensation of ferulic acid hydrazide, thioglycolic acid and 2-

nitrobenzaldehyde or 2-hydroxybenzaldehyde, highlighting the

importance of substitution of the aromatic ring from thiazolidin-4-

one structure in ortho position (Fig. 8.12).

At a concentration of 500 µg/mL g/mL the inhibition

percent for this compounds was 98.38 ± 0.012% (5f) or 99 ± 0.017%

(5h), as compared with the recorded value of 97.88 ± 0.001 for the

diclofenac. A comparable effect with the one of sodium diclofenac it

28

was presented for the compounds resulting from cyclization with 4-

nitrobenzaldehyde (5e, 96.32 ± 0.0098%) and 2-methoxy

benzaldehyde (5g, 96.76 ± 0.023%).

Fig. 8.12 The inhibiting activity of bovine serum albumin distortion for

N- [3- (4-hydroxy-3-methoxyphenyl) acryloyl] -N- (R-benzylidene) hydrazine

derivatives (4a-k), at different concentrations.

8.2.2.2. Erythrocyte membrane stability test

The results analysis shows that all the studied derivatives

the erythrocyte membrane stability increases with the concentration,

the biggest stability has been achieved at a concentration of 500

µg/mL.

The ability to stabilize the red blood cell membrane,

expressed as a percentage, for the ferulic acid derivatives with

hydrazone structure (4a-k) and thiazolidin-4-one (5a-l), tested at

different concentrations (100 µg/mL, 200 µg/mL, 500 µg/mL)

obtained from a stock solution with the concentration of 10 mg/ml

are plotted in Fig. 8.13 and 8.14.

In the series of N-[3-(4-hydroxy-3-methoxyphenyl)

acryloyl] -N- (R-benzylidene) hydrazine derivatives (4a-k), the most

favorable influence on the stability of red cell membrane was

observed for the 4k derivative, obtained from the condensation

reaction of ferulic acid hydrazide with benzaldehyde. For this

compound the membrane stability of the erythrocyte, expressed as

percentage it was comparable to that of diclofenac at 200 µg / mL

and 500 µg/mL (99.1308 ± 0.0794 vs 99.39873 ± 0.0950

respectively, 99.4063 ± 0.0125 vs 99.5623 ± 0.0451) and even

slightly increased at the concentration of 100 µg/mL (96.4874 ±

0.09609 vs 95.9794 ± 0.0289.

For the other compounds, even though the stability of the

erythrocyte membrane was reduced compared with diclofenac, it

29

remains in appreciable amounts, higher than 90% at all

concentrations tested and far superior compared with ferulic acid

(FA), in particular at the concentration of 200 µg/mL and 500

µg/mL.

Fig. 8.13. The percentage of red blood cell membrane stability for the derivatives of

N- [3- (4-hydroxy-3-methoxyphenyl) acryloyl] -N- (R-benzylidene) hydrazine (4a-k) at different concentrations.

In the series of derivatives of 2- (R-phenyl) -3- [3- (4-

hydroxy-3-methoxyphenyl) acrylamido] -thiazol-4-one (5a-l) (Fig.

8.14) has been observed that the majority of the compounds studied

had an erythrocyte membrane stability comparable to or greater than

that of diclofenac and superior to the one of ferulic acid (FA) at all

three concentrations tested 100 µg / mL, 200 µg / mL and 500 µg /

mL.

Fig. 8.14 The percentage of red blood cell membrane stability of the derivatives of

2- (R-phenyl) -3- [3- (4-hydroxy-3-methoxyphenyl) acrylamido] -thiazol-4-one (5a-l)

at various concentrations.

At a concentration of 100 µg/mL and 200 µg/mL, with the

exception of the compounds 5l (R = 4-OH, 3-OCH3), 5i (R = 2,6-

diCl), 5h (R = 2-OH), 5e (R = 4-NO2), all the other compounds

exhibited a greater stability than diclofenac (95.9794 ± 0.02886 to

30

100 µg/mL respectively 99.3987 ± 0.0950 to 200 µg/mL ) and than

ferulic acid (93.1372 ± 0.0116 at 100 µg/mL, respectively 93.4981 ±

0.0091 to 200 µg/mL).

At a concentration of 500 µg/mL all of the studied

compounds, derivatives of thiazolidine-4-one showed a higher red

blood cell membrane stability than the one of diclofenac (99.5623 ±

0.0451) and than the one ferulic acid (95.2087 ± 0.0578).

8.3. Toxicology screening. Determination of acute toxicity in vivo

The LD50 determination shows the toxicological profile of

the administered substance, being inversely proportional to its

toxicity. If the LD50 value is small than the tested compound is toxic.

The LD50 values for the 2- (R-phenyl) -3- [3- (4-hydroxy-3-

methoxyphenyl)acrylamido]-thiazol-4-one compounds are presented

in Table 8.3.

Table 8.3. The lethal dose 50 (LD50 in mg / kg) calculated for the compounds 2- (R-phenyl) -3- [3- (4-hydroxy-3-methoxyphenyl) acrylamido] -thiazol-4-one (5a-l)

compared with ferulic acid

Comp. R LD50

(mg/kg corp) Comp. R

LD50

(mg/kg corp)

5a -H 987,5 5g -OCH3(2) 1350

5b -Cl(4) 1590 5h -OH(2) 1250

5c -F(4) 4812,5 5i -Cl(2,6) 1550

5d -Br(4) 1490 5j -N(CH3)2(4) 1750

5f -NO2(2) 5000 5l -OH(4),OCH3(3) 925

Ferulic acid 2875

After the results analysis presented in Table 8.2. we can say

that all the studied derivatives can be classified as moderately toxic,

with LD50 values in the range of 500-5000 mg/kg.

8.4. Assessment of in vivo anti-inflammatory potential

Based on the results obtained from the evaluation of anti-

inflammatory effect using in vitro methods and based on the assess

to determine the toxicity degree, in the further study, the 2- (R-

phenyl)-3-[3-(4-hydroxy-3-methoxyphenyl) acrylamido] thiazolidine

-4-one derivatives (5a-l), obtained by modulating the structure of the

ferulic acid were included in the pharmacological screening sought

to determine the anti-inflammatory effect in vivo:

31

on the model of carrageenan-induced acute inflammation in

rats;

the model of chronic inflammation induced in rats -

granuloma test.

8.4.2.1. Anti-inflammatory effect on acute inflammation model

In this study the 2- (R-phenyl) -3- [3- (4-hydroxy-3-

methoxyphenyl) acrylamido] -thiazol-4-one derivatives (5a-l) have

been tested at a dose of 1/10 of LD50, the results are analyzed

compared to diclofenac sodium and indomethacin, used as reference

substances.

In Fig. 8.17 are graphical represented the results obtained:

the volume of acute inflammatory edema induced in the rat paw at

different time intervals (2 hours, 4 hours, 6 hours, 24 hours), for the

groups treated with the studied compounds (5a-l) and for the groups

treated with ferulic acid, diclofenac, indomethacin and control

(treated with Tween 80). It is estimated that as the acute

inflammatory edema volume is smaller than the studied compound

has an intense anti-inflammatory effect.

Fig. 8.17. Changes in inflammatory edema of the paw volume of rats in the groups treated with 5a-l derivatives, ferulic acid, diclofenac sodium, indomethacin and the

control group at 2 hours, 4 hours, 6 hours and 24 hours.

From the analysis of the results it is found that for all the

studied compounds the maximum effect anti-inflammatory

translated by the reducing of the volume of rat paw edema was

recorded 24 hours after administration, similar to diclofenac and

indomethacin, two well-known anti-inflammatory drugs. In this case

we can say that the synthesized derivatives can be included in terms

of pharmacokinetic in the long-acting compounds category.

32

The best results were obtained for 5h (R = 2-OH) and 5g

(2-OCH3) derivatives, when the effect is more intense than that of

diclofenac and indomethacin. An anti-inflammatory effect

comparable to the reference substances were recorded for the

compounds 5j (4-N(CH3)2), and 5l (4-OH-3-OCH3).

These statements are supported by the inhibition percentage

values of the acute inflammatory edema, recorded for the studied

compounds (5a-l), calculated against the value of the lot control,

results which are presented in Table 8.6.

Table 8.6. Anti-inflammatory effect (% inhibition of inflammatory swelling) of the tested compounds (5a-l, L1-10) compared with ferulic acid (L11), diclofenac sodium

(L12) and indomethacin (L13) at different time intervals

Lot/comp. % inhibition of inflammatory swelling

2h 4h 6h 24h

L1/5a 18,98 ± 2,6 17,85 ± 5,14 20,03 ± 2,45 87,17 ± 6,33

L2/5b 34,17 ± 4,87 27,38 ± 3,06 22,35 ± 2,26 73,07 ± 5,86

L3/5c 31,64 ± 6,49 30,35 ± 4,28 32,94 ± 3,01 73,07 ± 4,87

L4/5d 48,10 ± 5,04 34,52 ± 7,95 57,64 ± 4,07 47,43 ± 6,21

L5/5f 56,96 ± 5,35 38,09 ± 9,82 22,35 ± 4,81 76,92 ± 2,56

L6/5g 34,68 ± 2,94 31,42 ± 3,21 32,23 ± 2,77 95,38 ± 8,70

L7/5h 55,69 ± 2,21 32,14 ± 1,61 47,05 ± 2,16 97,43 ± 7,09

L8/5i 56,32 ± 2,31 50,01 ± 8,44 50,58 ± 5,61 78,84 ± 4,33

L9/5j 53,16 ± 4,09 46,42 ± 3,10 57,64 ± 3,79 94,87 ± 6,61

L10/5l 27,84 ± 8,54 2,38 ± 1,98 23,52 ± 5,50 91,02 ± 9,12

L11/ Ferulic acid 51,89 ± 2,20 47,61 ± 2,41 76,51 ± 9,27 92,30 ± 8,25

L12/ Sodic diclofenac 54,43 ± 3,48 53,57 ± 2,43 43,52 ± 2,25 94,87 ±11,61

L13/Indomethacin 73,41 ± 2,70 64,28 ± 1,81 75,29 ± 2,15 96,15 ±11,10

8.4.2.2. Anti-inflammatory effect on chronic inflammation model

Regarding the effect of the tested compounds on the

formation of granulation tissue correlated with dry pellets weight

was observed that all the compounds studied have reduced the

granulation tissue formation compared to the control group (treated

with Tween 80); for some of them the effect is comparable to that of

sodium diclofenac and indomethacin (table 8.9, 8.10).

The most important effect of inhibiting formation of

granulation tissue and therefore the most important anti-

inflammatory effect was recorded for compound 5f (R = 2-NO2).

This compound inhibited the formation of granulation tissue at a rate

of 79.85%, the effect is comparable to that of diclofenac (89.49%)

33

and of indomethacin (81.25%). A considerable effect presented the

compounds: 5c (R = 4-F), 5l (R = 4-OH, 3-OCH3) and 5d (R = 4-

Br). These compounds inhibited cell proliferation and the formation

of granulation tissue, in a proportion of 71.05% (5c), 73.35% (5l)

and 74.49% (5d). In similar experimental conditions ferulic acid

presented an appreciable anti-inflammatory effect, inhibiting the

formation of granulation tissue at the rate of 77.94%, the effect is

comparable to that of indomethacin.

Table 8.10. The effect of 2- (R-phenyl) -3- [3- (4-hydroxy-3-methoxyphenyl)

acrylamido] -thiazol-4-one compounds (5a-l) on the proliferativ process (granulation

tissue formation) in chronic inflammatory edema induced in rats

8.5. The evaluation of biochemical and hematological

parameters on the model of chronic inflammation

The compounds included in the study of anti-inflammatory

effect on the model of chronic inflammation were the compounds 2-

(R-phenyl) -3- [3- (4-hydroxy-3-methoxyphenyl) acrylamido] -

thiazol-4-one (5a-l) and they were further studied to determine the

effect on the following parameters:

hematological - blood count;

biochemical - alanine aminotransferase (ALT),

aspartatamino-transferase (AST), total bilirubin, direct

Comp. R

Administrated

dose (mg/kg

body/day)

Average weight of

dry pellets (mg)

%

Inhibition

5a -H 98,75 0,617 21,31

5b -Cl(4) 159,00 0,768 2,05

5c -F(4) 481,25 0,227 71,05

5d -Br(4) 149,00 0,200 74,49

5f -NO2(2) 500,00 0,158 79,85

5g -OCH3(2) 135,00 0,528 32,66

5h -OH(2) 125,00 0,463 40,95

5i -Cl(2,6) 155,00 0,563 28,19

5j -N(CH3)2(4) 175,00 0,698 10,97

5l -OH(4)OCH3(3) 92,50 0,209 73,35

Ferulic acid 287,50 0,173 77,94

Diclofenac sodic 5,00 0,153 89,49

Indometacin 1,50 0,147 81,25

Tween 80 0,5 mL/100 g 0,784 -

34

bilirubin, lactatedhydrogenase (LDH), urea, creatinine, uric

acid, total cholesterol, LDL cholesterol, HDL cholesterol,

triglycerides.

Biochemical parameters for the evaluation of liver function

The results obtained in this study are presented in Table

8.19, 8.20, 8.21 and 8.22.

From the results analysis it can be seen that liver enzymes

(AST, ALT, LDH) have been elevated in the group with chronic

inflammation induced by granuloma test (lot 14) compared to the

healthy group (lot 15), which shows the negative influence of the

inflammatory phenomenon on liver function. The values recorded

for lot 14 were 269.5 IU/L (AST), 51 IU/L (ALT) and 1364 IU/L

(LDH) compared to 100 IU/L (AST), 46.15 IU/L (ALT) and 400.6

IU/L (LDH) recorded for the healthy control group.

The reduced liver damage was recorded for the group 2

treated with compound 5b (2- (4-chlorophenyl) -3- [3- (4-hydroxy-

3-methoxy-phenyl) acrylamido] -thiazol-4-one). In this case the

values of liver enzymes were similar to those recorded for reference

NSAIDs used in the study (diclofenac and indomethacin) and

comparable to the values for the healthy control group.

Regarding the concentration of the enzyme ALT,

considered the most relevant indicator of normal functioning of liver

function (hepatic cytolysis indicator) it was found that the less toxic

compounds in the liver were 5b (R = 4-Cl, lot 2) and 5l ( R = 4-OH,

3-OCH3, lot 12), which highlights the favorable influence of

substitution on the aromatic ring at position 4 of the thiazolidin-4-

one cycle with chlorine and with hydroxy and methoxy in 4 and 3

position. For these compounds, the amount of the enzyme ALT were

50.5 IU/L (5b) and 40 IU/L (5l), values comparable to those seen for

diclofenac (49 IU/L), indomethacin (51 IU/L) and healthy control

group (46.15 IU/L).

Except compound 5b (lot 2, LDH = 617 IU/L) for all the

other studied derivatives the LDL enzyme value was higher

compared with healthy control group (lot 15, LDL = 400 IU/L) and

groups treated with diclofenac sodium (LDL = 1116 IU/L) and

indomethacin (LDL = 908.5 IU/L).

35

Table 8.19. The parameters values of liver function in rats from the groups

1 (5a), 2 (5b), 3 (5c) and 4 (5d) Biochemical

parameter

Lot/Compound

Lot 1/5a Lot 2/5b Lot 3/5c Lot 4/5d

AST (UI/L) 244 ± 0,28 124,5 ± 1,90 272,5 ± 0,63 212,5 ±1,48

ALT (UI/L) 58 ± 0,56 50,5 ± 0,63 96,5 ± 1,06 70,5 ±0,35

LDH (UI/L) 2734,5 ± 0,34 617 ± 3,52 3778 ± 0,25 1826,5 ± 2,92

Bilirubin tot. (mg/dL) 0,11 ± 0,014 0,135 ± 0,01 0,115 ± 0,01 0,105 ± 0,01

Bilirubin dir. (mg/dL) 0,045 ± 0,007 0,025 ± 0,02 0,035 ±0,01 0,045 ± 0,01

Tabel 8.20. The parameters values of liver function in rats from the groups

5 (5f), 6 (5g), 7 (5h) şi 8 (5i)

Biochemical

parameter

Lot/Compound

Lot 5/5f Lot 6/5g Lot 7/6h Lot 8/5i

AST (UI/L) 545 ± 7.70 185,5 ± 5,72 211 ± 0,28 207,5 ± 1,62

ALT (UI/L) 130,5 ± 5,86 52,5 ± 1,20 56 ± 0,84 67,5 ± 0,07

LDH (UI/L) 1181 ± 7,72 1866,5 ± 9,76 2450,5 ± 0,71 2046,5 ± 0,71

Bilirubina tot. (mg/dL) 0,13 ± 0,09 0,105 ± 0,01 0,085 ± 0,01 0,095 ± 0,02

Bilirubina dir. (mg/dL) 0,045 ± 0,01 0,03 ± 0,01 0,006 ± 0,03 0,003 ± 0,01


9 (5j), 10 (5l), 11 (FA –ferulic acid) şi 12 (Diclofenac sodic).

Biochemical

parameter

Lot/Compound

Lot 9/5j Lot 10/5l Lot 11/AF Lot 12

/Diclofenac

AST (UI/L) 256,5 ± 0,21 177,5 ± 0,07 198,5 ± 2,47 133,5 ± 2,61

ALT (UI/L) 69,5 ± 0,07 40 ± 0,28 65,5 ± 1,48 49 ± 0,56

LDH (UI/L) 2555 ± 4,52 1824,5 ± 0,16 2020 ± 5,68 1116 ± 6,15

Bilirubina tot. (mg/dL) 0,075 ± 0,03 0,095 ± 0,01 0,11 ± 0,01 0,115 ± 0,02

Bilirubina dir. (mg/dL) 0,055 ± 0,02 0,035 ± 0,01 0,055 ± 0,02 0,035 ± 0,01


13(Indometacin), 14 (C1inflam,Tween 80) şi 15 (C2healthy,Tween 80)

Biochemical

parameter

Lot/Compound

Lot 13/

Indomethacin

Lot 14inflam

/tween 80

Lot 15healthy/

tween 80

AST (UI/L) 124,5 ± 0,35 269,5 ± 1,62 100 ± 1,08

ALT (UI/L) 59,5 ± 1,90 51 ± 1,41 46,15 ± 1,33

LDH (UI/L) 908,5 ± 4,16 1364 ± 4,78 400 ± 0,83

Bilirubina tot. (mg/dL) 0,175 ± 0,05 0,115 ± 0,04 0,085 ± 0,04

Bilirubina dir. (mg/dL) 0,045 ± 0,01 0,042 ± 0,01 0,03 ± 0,02

36

CAPITOLUL 9

GENERAL CONCLUSIONS

The PhD thesis researches brings original contributions in

the field of aryl-acrylic acid derivatives. The representatives of this

class of compounds, including ferulic acid which has an important

place, are known primarily for the important antioxidant effects.

With the discovery of the involvement of oxidative stress in

the pathology of many diseases including autoimmune diseases,

neoplastic diseases, inflammation, neurodegenerative diseases, etc.,

an interest in the development of new compounds with antioxidant

potential has increased considerably.

1. Structural modulation which targeted the free carboxyl

group from the structure of 3-(4-hydroxy-3-methoxy-phenyl) acrylic

acid (ferulic acid) leaded to 2 intermediates and two new final series

of compounds, ferulic acid derivatives with hydrazone structure (11

final compounds) and ferulic acid derivatives with thiazolidin-4-one

structure (12 final compounds).

2. The intermediates and the final compounds were physico-

chemical characterized determining the molecular formula, relative

mass, the melting point, solubility in water and various organic

solvents and the reaction yield.

3. The structure of ferulic acid derivatives, intermediates and

final, was confirmed by spectral methods - infrared spectroscopy

(IR) and nuclear magnetic resonance spectroscopy of proton (1H-

NMR), identifying all the structural elements characteristic of the

compounds.

4. Antioxidant potential of the two series of compounds

(hydrazone and thiazolidin-4-one) was assessed by four in vitro

methods that determine the antiradical effect against DPPH radicals

and ABTS• +, the total antioxidant capacity and reducing power.

The compounds with a hydrazone or thiazolidin-4-one

structure showed a more intense antioxidant effect than the base

37

structure (ferulic acid); for some derivatives the activity is

comparable to that of the positive control, ascorbic acid.

In the series of N- [3-(4-hydroxy-3-methoxyphenyl)

acryloyl]-N-(R-benzylidene) hydrazine derivatives (4a-k), the most

intense antiradical effect was observed for the compound resulting

from the condensation reaction with benzaldehyde (4k). A

pronounced antiradical effect was observed for 4g (R = 2-OH), 4i (R

= 4-OH) and 4f (R = 4-NO2) derivatives, highlighting the favorable

impact of substitution on the aromatic ring of the hydrazones

structure with -OH (in the ortho and para), and -NO2 (para).

In the case of derivatives of 2- (R-phenyl) -3- [3- (4-

hydroxy-3-methoxyphenyl) acrylamido] -thiazol-4-one (5a-l), the

most active compound proved to be the one obtained from

condensation reaction of ferulic acid hydrazide, thioglycolic acid,

and 2,3-dihydroxybenzaldehyde (5k) or 4-hydroxy-3-methoxy-

benzaldehyde (5l). These results support the favorable effect exerted

by radicals R = 2,3-diOH and 4-OH, 3-OCH3 on the antioxidant

effect of ferulic acid.

It was also found that thiazolidine-4-one derivatives are

more active than hydrazone derivatives, which supports the

favorable influence of the thiazolidin-4-one cycle on enhancing the

antioxidant effects of ferulic acid.

5. The results of the acute toxicity determination by

establishing the lethal dose 50 (LD50) support the thiazolidin-4-one

derivatives classification in the category of the compounds with

moderate toxicity. The toxicity is influenced by the nature of the

substituent on the aromatic ring of the thiazolidin-4-one structure,

the most favorable effect was exerted by the fluorine substitution in

the para position (compound 5c) and the nitro substitution in the

ortho position (compound 5f).

6. The anti-inflammatory potential of ferulic acid derivatives

was evaluated by in vitro and in vivo methods.

The anti-inflammatory effect in vitro examine the influence

of ferulic acid derivatives with hydrazone (4a-k) and thiazolidin-4-

one (5a-l) structure on the inhibition of distortion of bovine serum

albumin and the stability of red cell membrane. Similar to the results

obtained in the study assessing the antioxidant effect, it was noticed

38

that thiazolidine-4-one derivatives (5a-l) had a more favorable effect

than hydrazone derivatives (4a-k), on both methods: the inhibition

of protein distortion and erythrocyte membrane stability.

Given the findings in vitro, the ferulic acid derivatives with

thiazolidin-4-one structure (5-l) were evaluated in vivo using two

models of rat induced inflammation: carrageenan-induced acute

inflammation and chronic inflammation induced by granuloma test.

In acute inflammation model, all of the compounds studied

showed a maximum anti-inflammatory effect at 24 hours after

administration, suggesting that regarding the pharmacokinetics they

may be included in the category of long-acting compounds. The

most active compound of the series proved to be the compound 2-

(2-hydroxyphenyl)-3-[3- (4-hydroxy-3-methoxyphenyl) acrylamido]

-thiazol-4-one (5h), formed using the reaction of condensation of

ferulic acid hydrazide, thioglycolic acid and 2-

hydroxybenzaldehyde. At 24 hours after administration the effect of

the compound was slightly more intense than that of sodium

diclofenac and indomethacin, used as reference substances.

In the case of the chronic inflammation it has been

observed that the compounds studied (5a-l), similar with the

reference substances (diclofenac sodium, indomethacin), had a more

intense effect of inhibiting the proliferative component, namely the

formation of granulation tissue than on transudative component of

chronic inflammation. The most important effect of inhibition of the

proliferative process was registered for the compound 5f (2-(2-

nitrophenyl)-3-[3-(4-hydroxy-3-methoxyphenyl)acrylamido]-thiazol

-4-one), the effect being comparable to that of diclofenac and

indomethacin .

7. The study of anti-inflammatory effect of the thiazolidine-4-

one derivatives (5a-l) on the model of chronic inflammation has

been complemented with biochemical and hematological study that

looked after the effect of compounds on liver and kidney function

and after hemoleucogram (CBC- complete blood count).

Liver function was assessed by determining the activity of

liver enzymes spectophotometric: alaninamino-transferase (ALT),

aspartate aminotransferase (AST) and lactate dehydrogenase (LDH)

and by the determination of the total bilirubin concentration and

direct bilirubin. The least toxic compound was shown to be the

39

compound 5b (2-(4-chlorophenyl)-3-[3-(4-hydroxy-3-methoxy

phenyl) acrylamido]-thiazol-lidin-4-one), for which the value of

enzymes ALT, AST and LDH was comparable with the healthy

control group and with the values for diclofenac sodium and

indomethacin. Also the studied compounds presented values of total

and direct bilibubinei comparable to the values for healthy control

group, diclofenac sodium and indomethacin which suggests a similar

toxicological profile with these classics anti-inflammatory.

Renal function, assessed by biochemical parameters -

creatinine, urea, uric acid, was not seriously affected by the

administration of the studied compounds, those parameter values, in

most of the cases, are comparable to the values for the healthy

control group and diclofenac sodium and indomethacin group.

The evaluation of lipid profile it was done using the

following parameters: total cholesterol, LDL-cholesterol, HDL-

cholesterol showed for the studied compounds a lipid profile similar

with the one of sodium diclofenac and indomethacin.

The blood counts values - leukocytes, erythrocytes,

hemoglobin, hematocrit, mean corpuscular volume, mean red blood

cell hemoglobin, mean hemoglobin concentration were within

normal limits, with no significant differences compared to the

healthy control group. In terms of the number of platelets

(thrombocytes) was an increase from the values recorded for the

control group, which was expected because the experiment was

conducted on chronic model of the inflammation, which, as is

known, is correlated with an increase in the number of platelets.

8. Considering the originality of the structures for the

synthesized compounds, the physico-chemical, spectral and

biological characterization we can say that it was made important

contributions in the field of heterocyclic compounds with potential

application in the treatment of inflammatory diseases and other

diseases where inflammation and oxidative stress plays an important

role (neurodegenerative diseases, neoplastic disorders, etc.).

40

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45

LIST OF OF SCIENTIFIC PAPERS

Published papers as first author (from the PhD thesis):

1. Wolszleger (Drăgan) M, Stan CD, Pânzariu A, Jităreanu A,

Profire L. New thiazolidin-4-ones of ferulic acid with

antioxidant potential. Farmacia 2015, 63 (1): 150-154 (ISI,

FI=1,251).

2. Wolszleger (Drăgan) M, Stan CD, Apotrosoaiei M, Vasincu I,

Pânzariu A, Profire L. New hidrazones of ferulic acid:

synthesis, characterization and biological activity. Rev. Med.

Chir. Soc. Med. Nat., Iaşi 2014, 118; 4:1150-1156. (BDI, B+).

Other works:

Papers published in ISI:

1. Apotrosoaei M, Vasincu I, Drăgan M, Buron F, Routier S,

Profire L, Design, synthesis and the biological evaluation of

1,3-thiazolidine-4-ones based on 4-amino-2,3-dimethyl-1-

phenyl-3-pyrazolin-5-one scaffold. Molecules 2014; Doi:

10.3390/molecules190913824: 13824-13847. (ISI, FI=2,095)

2. Stan CD, Tătărîngă G, Gafiţanu C, Drăgan M, Braha S, Popescu

MC, Lisă G, Ştefanache A. Preparation and characterization

of niosomes containing metronidazole. Farmacia 2013, 61;

6:1178-1185. (ISI, FI=1,251).

3. Stan CD, Coromelci-Pastravanu C, Creţescu I, Drăgan M.

Treatment of pesticides in wastewater by heterogeneous and

homogeneous photocatalysis. International Journal of

Photoenergy 2012, Doi:10.1155/2012/194823. (ISI, FI=2,663).

4. Stan CD, Ştefanache A, Tuchiluş C, Diaconu DE, Drăgan M,

Profire L. Influence of extraction solvent on the erythromycin

ethylsuccinate separation from oral suspendable powder.

Farmacia 2011, 59; 3:396-401. (ISI, FI=0,669).

5. Zavastin D, Creţescu I, Bezdadea M, Bourceanu M, Drăgan M,

Lisă G, Mangalagiu I, Vasic V, Savic J. Preparation,

characterization and applicability of cellulose acetate –

polyurethane blend membrane in separation techniques.

Colloids and Surface A: Physicochemical and Engineering

Aspects 2010, 370; 1-30:120-128. (ISI, FI=2,236).

6. Bezdadea M, Bourceanu M, Wolszleger M. The permeation of

protein solution at ultrafiltration through indigenous

46

polyurethane membranes. Roumanian Biotechnological Letters

2006, 11; 5:2905-2931. (ISI, FI=0,291).

Papers published in journals listed BDI / B +

1. Stan CD, Drăgan M, Diaconu DE, Ştefanache A. Posibilităţi de

acilare a acidului 7-aminocefalosporanic în scopul obţinerii unor

cefalosporine. Rev. Med. Chir. Soc. Med. Nat., Iaşi 2014, 118;

1:244-249.

2. Stan CD, Ştefanache A, Drăgan M, Corciovă AM. Development

and validation of a spectrophotometric method for quantitative

determination of new preservatives from pharmaceutical forms.

Rev. Med. Chir. Soc. Med. Nat., Iaşi 2013, 117; 4:1014-1020.

3. Stan CD, Ştefanache A, Drăgan M, Poiată A, Diaconu DE,

Profire L. Cercetări privind îmbunătăţirea condiţiilor de acilare

în cazul obţinerii penicilinelor antistafilococice. Rev. Med. Chir.

Soc. Med. Nat., Iaşi 2011, 115; 3:972-977.

Papers published in conference volumes

1. Drăgan M, Stan CD, Pânzariu A-T, Dragostin OM, Vasincu I-

M, Apotrosoaiei M, Profire L. Noi hidrazone ale acidului

ferulic. Congresul Naţional de Farmacie din România cu

participare internaţională, ed. a XV-a, 24 – 27 septembrie 2014.

2. Stan CD, Tătărîngă G, Drăgan M, Ştefanache A. Descoperirea

vaccinurilor – piatra de temelie a medicinii modern. Al IV-lea

Colocviu International de Istoria Farmaciei şi a XXIII-a

Reuniune Naţională Anuală – 10 Ani de la înfiinţarea secţiei

Brăila a SRIF (2004-2014), Brăila, 5-7 iunie 2014.

3. Stan CD, Drăgan M, Ştefanache A. Descoperirea rapamicinei-

un imunosupresiv de succes. Pagini din istoria farmaciei, Ed.

“Gr. T. Popa” U.M.F. Iaşi, p. 218-220, 2013.

4. Stan CD, Drăgan M, Ştefanache A. Metode clasice şi moderne

de obţinere a vitaminei C. Timp şi semnificaţie în istoria

medicine, Ed. “Gr.T.Popa”, UMF, p. 231-234, 2012.

5. Stan CD, Drăgan M, Poiată A, Ştefanache A. Testarea activităţii

antimicrobiene a unor derivaţi ai acidului mandelic. Actualităţi

şi perspective în cercetarea farmaceutică, Craiova, p. 83-84,

2012.

6. Drăgan M, Poulios I, Stan CD. Degradarea pesticidelor organo-

persistente prin fotocataliză eterogenă (condiţii optime).

47

Conferinţa Naţională de Fitoterapie, ed. aV-a, Iaşi, p.108-111,

2011.

7. Dragostin OM, Vasile C, Lupaşcu F, Drăgan M, Profire L.

Sinteza şi caracterizarea unor noi hidrazone cu structură

sulfonamidică. Conferinţa Naţională de Fitoterapie, ed. aV-a,

Iaşi, p.105-107, 2011.

8. Stan CD, Ştefanache A, Drăgan M, Dumitrache M, Profire L.

Patentarea primului microorganism transgenic. A XV-a

Reuniune Naţională de Istoria Farmaciei, Galaţi, 2009.

9. Stan CD, Ştefanache A, Poiată A, Dumitrache M, Wolszleger

M, Nastase V, Profire L. The testing of preservative action for

some mandelic acid esters in cosmetics. 14th Panhellenic

Pharmaceutical Congress, Atena, Grecia, 2009.

10. Wolszleger M, Bezdadea M, Creţescu I, Doniga E. Controlul

medicamentelor prin cromatografie în strat subţire, folosind noi

faze staţionare cu structură polimeră. The first conference of

phd students in Medicine and Pharmacy, Târgu-Mureş, Rev. De

Medicină şi Farmacie 2008, 54; 3: 538-540.

university of medicine and pharmacy "grigore t. popa

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