Departamento de Química - Universidade de Aveiro

Departamento de Química Universidade de Aveiro

2018

Renato Ribeiro

Maia

Catálise biomimética de derivados de flavonas

usando uma porfirina sintética.

Biomimetic catalysis of flavones derivatives using a

synthetic porphyrin.

Departamento de Química Universidade de Aveiro

2018

Renato Ribeiro

Maia

Catálise biomimética de derivados de flavonas

usando uma porfirina sintética.

Biomimetic catalysis of flavones derivatives using a

synthetic porphyrin.

Dissertação apresentada à Universidade de Aveiro para cumprimento dos requisitos necessários à obtenção do grau de Mestre em Bioquímica, ramo de

Bioquímica Clínica, realizada sob a orientação científica da Doutora Diana

Cláudia Gouveia Alves Pinto, Professora Auxiliar do Departamento de Química

da Universidade de Aveiro.

Júri

Presidente

Doutor Brian James Goodfellow Professor Auxiliar da Universidade de Aveiro

Doutora Diana Cláudia Gouveia Alves Pinto Professora Auxiliar da Universidade de Aveiro

Doutora Ana Maria Loureiro da Seca Professora Auxiliar da Universidade dos Açores

Agradecimentos

Para começar à Doutora Diana Pinto, a orientadora deste projeto, por toda a

disponibilidade e paciência demonstrada durante este último ano.

Aos alunos de doutoramento do departamento de Química, Carlos Silva e Vasco

Batista, por todo conhecimento transmitido e toda a ajuda dada quando por vezes as

coisas não corriam tão bem como esperado.

Aos meus colegas de laboratório por todo o apoio, que fizeram com que estes últimos

meses fossem muito mais fáceis de superar.

Um agradecimento à Universidade de Aveiro e ao departamento de Química pela

oportunidade e a todos aqueles que podendo não ter interferido diretamente

contribuíram para que este trabalho fosse possível.

E, por último, mas não menos importante, aos meus Pais, por me terem apoiado e

feito de tudo para que eu pudesse concluir esta etapa académica.

Palavras Chave

Resumo

Flavonas, calconas, catálise biomimética, porfirinas, atividade biológica

Uma vez que tanto as flavonas de origem natural como as obtidas sinteticamente,

apresentam uma diversa e extensa lista de atividades biológicas que incluem antioxidante, anti-inflamatória, anticancerígena, antimicrobiana, antiviral,

antidiabética ou modulação do metabolismo de lípidos, a síntese de novas flavonas

contendo grupos pouco usuais pode ser extremamente relevante. Embora, estes

novos derivados possam ser biologicamente ativos, há uma questão que carece de

resposta, serão estes compostos devidamente metabolizados pelos sistemas biológicos? Estudos biomiméticos podem ser uma boa estratégia para encontrar a

resposta e porfirinas sintéticas podem ser usadas na oxidação biomimética.

Simultaneamente estas oxidações biomiméticas podem ser um novo método de

síntese de novas flavonas.

Neste trabalho foram sintetizadas novas flavonas contendo grupos naftilo, em bons

rendimentos (> 60%). Estas foram obtidas através da ciclização oxidativa das respetivas calconas, que por sua vez foram sintetizadas por condensação de Claisen-

Schmidt e obtidas em muito bons rendimentos (> 70%).

A oxidação biomimética demonstrou que apenas os grupos 2-naftilo foram oxidados,

sendo isto indicativo da maior estabilidade do núcleo benzocromenona. As atividades

antioxidante e anti-colinesterase foram avaliadas, apenas para um dos produtos da

oxidação biomimética, infelizmente os resultados não foram significativos. Todos os compostos sintetizados foram caracterizados por estudos de

espectroscopia de ressonância magnética nuclear (RMN), incluindo espectros de 1H

e 13C, e estudos bidimensionais de correlação espectroscópica heteronuclear (HSQC

e HMBC).

Keywords

Abstract

Flavones, chalcones, biomimetic catalysis, porphyrin, biological activity.

Since flavones present a diverse and vast list of biological activities, including antioxidant, anti-inflammatory, anticancer, antimicrobial, antiviral, antidiabetic, anti-

allergic, activities or modulation of enzyme activity or metabolic pathways like the lipid

metabolism, the synthesis of new flavones bearing uncommon patterns may be

extremely useful. Though, these new flavone derivatives may be bioactive, one

question needs to be answered, are they properly metabolized in biological systems?

To answer that, biomimetic studies are a good strategy and synthetic porphyrins can be used in biomimetic oxidations. Simultaneously, these biomimetic oxidations can be

a new way to obtain new flavones.

In this work new flavones bearing naphthyl groups were obtained in good yields (>

60%). They were synthesised by the oxidative cyclization of the appropriated chalcone

derivatives, which were obtained through a Claisen-Schmidt condensation in very

good yields (> 70%). The biomimetic oxidation showed that only the 2-naphthyl moiety was oxidised,

indicated that the benzochromenone scaffold is very stable. In addition, the biomimetic

oxidation led to the synthesis of new flavone derivatives. One of the oxidation products

was evaluated for its antioxidant and anti-cholinesterase activities, unfortunately the

results showed that its activity is not significant.

The characterization of all synthesised compounds was made by nuclear magnetic resonance (NMR) spectroscopic studies, including 1H and 13C, and two-dimensional

heteronuclear correlated spectroscopy (HSQC and HMBC).

Abbreviations and chemical formulas

13C NMR - 1-dimensional carbon Nuclear Magnetic Resonance

1H NMR - 1-dimensional hydrogen Nuclear Magnetic Resonance

2D NMR - 2-dimensional Nuclear Magnetic Resonance

3D - 3-dimensional

4CL - 4-coumarate:coenzyme A ligase

5-LOX - Arachidonate 5-lipoxygenase

ABTS - 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid)

ACh - acetylcholine

AChE - Acetylcholinesterase

AChEI - Acetylcholinesterase inhibitors

ALA - δ-aminolaevulinic acid or 5- aminolaevulinic acid

ALAS - δ-aminolaevulinic acid synthase

ATChI - Acetylthiocholine iodine

Bak - Bcl-2 homologous antagonist/killer

Bax - Bcl-2 associated X protein

C4H - Cinnamic acid 4-hydrolase

CDCl3 - Deuterated Chloroform

CHI - Chalcone isomerase

CHI - Chalcone isomerase

CHS - Chalcone synthase

CoA - Coenzyme A

COX-1 - Cyclooxygenase 1

COX-2 - Cyclooxygenase 2

CPP32 - Caspase 3

CUPRAC - cupric reducing antioxidant power assay

DMSO - Dimethyl sulfoxide

DPPH - 2,2-diphenyl-1-picrylhydrazyl

DTNB - 5,5-dithio-bis-(2-nitrobenzoic acid)

F3’H - Flavonoid-3’-hydroxilase

F3H - Flavanone-3-hydroxilase

Fe-(TDCPPS)Cl - meso-tetra(2,6-dichloro-3-sulfonatophenyl) porphyrinate of iron(III)

chloride

FNR - Flavanone-4-reductase

FNS – Flavone synthase

FRAP - Ferric reducing antioxidant power method

H2O2 - Hydrogen peroxide

HCl - Hydrochloric acid

HDL - High Density Lipoprotein

HepG2 - Human liver hepatocellular carcinoma cell line

HMBC - Heteronuclear Multiple Bond Correlation

HMEC-1 - Human dermal microvascular endothelium cells

HORAC - Hydroxyl radical averting capacity assay

HPLC - High performance liquid chromatography

HSQC – Heteromolecular Single Quantum Coherence

Hz - Hertz

I2 - Iodine

IC50 - Half maximal inhibitory concentration

IL-1β - Interleukin 1 beta

iNOS - Inducible nitric oxide synthase

IκB-α - Nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor, alpha

JNK - c-Jun N-terminal kinase

KF–Al2O3 - Potassium fluoride in alumina

KOH - Potassium hydroxide

LPS - Lipopolysaccharides

LXRα - Liver X receptor alpha

LXRβ - Liver X receptor beta

MCF-7 - Michigan Cancer Foundation-7 cell line

MDCK - Madin-Darby Canine Kidney cell line

MDR - Multiple drug resistance

MHz - Megahertz

Mn(TDCPP)Cl - 5,10,15,20-tetrakis(2,6-dichlorophenyl) porphyrin manganese(III) chloride

MRSA - Methicillin-resistant Staphylococcus aureus

Na2SO4 - Anhydrous sodium sulphate

NF-κB - Nuclear factor kappa B

NH4I - Ammonium iodide

NMR - Nuclear Molecular Resonance

NO - Nitric oxide

P450 - Cytochromes P450

PAL - Phenylalanine ammonia lyase

PCP - Pentachlorophenol

PFP - Pentafluorophenol

PFRAP - potassium ferricyanide reducing power method

PGE2 - Prostaglandin E2

ppm - parts per million

PTC - Phase Transfer Catalysts

SiO2-I2 - Iodine adsorbed in silica

SREBP-1c - Sterol regulatory element-binding protein 1 isoform

TAL - Tyrosine ammonia lyase

THF - Tetrahydrofuran

TLC - Thin layer chromatography

TMS - Tetramethylsilane

TNF-α - Tumour necrosis factor alpha

TRAP - Total peroxyl radical trapping antioxidant parameter assay

Trolox - 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid

X/XO - Xanthine/Xanthine Oxidase

δ - Chemical shift

Index

1. Introduction .................................................................................................................... 1

1.1. Biosynthesis of flavonoids ...................................................................................... 1

1.2. Synthesis of flavones .................................................................................................. 3

1.3. Flavone biological activities ................................................................................... 8

1.3.1. Antioxidant properties of flavones .................................................................. 8

1.3.2. Anti-inflammatory properties of flavones ..................................................... 11

1.3.3. Anti-cancer properties of flavone derivatives ............................................... 15

1.3.4. Antimicrobial and antiviral activity of flavone derivatives ........................... 17

1.3.5. Lipid metabolism modulation ........................................................................ 20

1.3.6. Anti-diabetes activity ..................................................................................... 20

1.4. Porphyrins and P450 complex .............................................................................. 21

1.4.1. Biosynthesis of porphyrins (heme) ................................................................ 22

1.4.2. Cytochrome P450 complex ........................................................................... 22

1.4.3. Synthetic porphyrins ...................................................................................... 25

1.5. Biomimetic oxidation catalysis ............................................................................. 25

1.6. Objectives ............................................................................................................. 29

2. Results and discussion .................................................................................................. 33

2.1. Chalcone derivatives synthesis ............................................................................. 33

2.2. 1H NMR characterization of the synthesized chalcone derivatives ...................... 34

2.2.1. Chalcone 6 (1-(2-hydroxynaphthalen-1-yl)-3-phenylprop-2-en-1-one) ........ 34

2.2.2. Chalcone 9 (1-(2-hydroxyphenyl)-3-(naphthalen-2-yl)prop-2-en-1-one) ..... 35

2.2.3. Chalcone 10 (1-(2-hydroxynaphthalen-1-yl)-3-(naphthalen-2-yl)prop-2-en-1-

one). 35

2.3. Flavone derivatives synthesis ............................................................................... 37

2.4. 1H NMR and 13C NMR characterization of the synthesized flavone derivatives . 39

2.4.1. Flavone 1 (3-phenyl-1H-benzo[f]chromen-1-one) ........................................ 39

2.4.2. Flavone 2 (2-(Naphthalen-2-yl)-4H-chromen-4-one) ................................... 40

2.4.3. Flavone 3 (3-(naphthalen-2-yl)-1H-benzo[f]chromen-1-one) ....................... 41

2.5. Flavone biomimetic oxidation catalysis................................................................ 44

2.5.1. Oxidation product identification and structure .............................................. 45

2.6. Biological activity tests of flavone 11 .................................................................. 49

2.6.1. Antioxidant activity tests ............................................................................... 49

2.6.1.1. DPPH test ............................................................................................... 50

2.6.1.2. ABTS test ............................................................................................... 50

2.6.2. Anti-acetilcholinesterase activity test ............................................................ 50

3. Materials and methods ................................................................................................. 55

3.1. Chalcone derivatives synthesis ............................................................................. 55

3.1.1. Synthesis and purification of chalcone 6 ....................................................... 55

3.1.2. Synthesis and purification of chalcone 9 ....................................................... 56

3.1.3. Synthesis and purification of chalcone 10 ..................................................... 56

3.2. Synthesis and purification of flavones .................................................................. 56

3.2.1. Synthesis and purification of flavone 1 ......................................................... 56



3.3. Biomimetic catalysis tests ..................................................................................... 57

3.3.1. Biomimetic catalysis of flavone 1 ................................................................. 58



3.4. Biological activity tests ......................................................................................... 59

3.4.1. DPPH test ...................................................................................................... 59

3.4.2. ABTS test ...................................................................................................... 60

3.5. Acetylcholinesterase test ....................................................................................... 60

4. Conclusions and future perspectives ............................................................................ 63

5. Bibliography ................................................................................................................. 67

Figure Index

Figure 1. Flavone backbone structure. .................................................................................. 1

Figure 2. Structure dependency of flavone derivatives antioxidant activity. ..................... 11

Figure 3. Favoured positions and substituent groups that increase anti-inflammatory activity

of flavone derivatives. ......................................................................................................... 15

Figure 4. Effects observed on cancer cells treated with flavones. ...................................... 17

Figure 5. Tetrapyrrolic heterocyclic macrocycle structure. Skeleton of porphyrins. ......... 21

Figure 6. Structure of the heme b (left), and structure of 5,10,15,20-tetrakis (2,6-

dichlorophenyl) porphyrin manganese(III) chloride (right). ............................................... 27

Figure 7. Flavone derivatives bearing naphthyl groups to be used on the biomimetic

catalysis studies. .................................................................................................................. 29

Figure 8. Structure for chalcone 6. ..................................................................................... 34

Figure 9. Structure for chalcone 9. ..................................................................................... 35

Figure 10. Proposed structure for chalcone 10. .................................................................. 36

Figure 11. 1H NMR spectrum of chalcone 10. ................................................................... 36

Figure 12. Aromatic region of the spectrum of chalone 10 (7.15-8.15 ppm). .................... 36

Figure 13. Structure of flavone 1. ....................................................................................... 39



Figure 16. 1H NMR spectrum from flavone 3. ................................................................... 42

Figure 17. 13C NMR spectrum from flavone 3. .................................................................. 43

Figure 18. HSQC NMR spectrum from flavone 3. ............................................................. 43

Figure 19. HMBC NMR spectrum from flavone 3 ............................................................ 44

Figure 20. Structure of the flavone 11, 2-(1a,1b,2a,6b-tetrahydronaphtho[1,2-b:3,4-

b']bis(oxirene)-4-yl)-4H-chromen-4-one. ............................................................................ 48

Figure 21. NMR Spectrum from oxidation product from flavone 3 (top) and NMR spectrum

from flavone 11 (bottom). ................................................................................................... 48

Figure 22. Possible structure for the oxidation product of flavone 3 (2-(1a,1b,2a,6b-

tetrahydronaphtho[1,2-b:3,4-b']bis(oxirene)-4-yl)-1H-benzo[f]chromen-1-one). .............. 49

Figure 23. Graphical presentation of the results for the DPPH test.................................... 50

Figure 24. Graphical presentation of the results for the ABTS test.................................... 51

Scheme Index

Scheme 1. Phenylpropanoid pathway of biosynthesis of flavones. ...................................... 2

Scheme 2. Mechanism of Baker-Venkataraman rearrangement to 1,3-diones, followed by

cyclodehydration catalysed by a strong acid. ........................................................................ 5

Scheme 3. Mechanism of synthesis of chalcones by Claisen-Schmidt condensation........... 7

Scheme 4. Mechanism of synthesis of flavones from chalcones using iodine in DMSO. .... 8

Scheme 5. Pathway for the biosynthesis of heme b. ........................................................... 23

Scheme 6. Examples of oxidation reactions catalysed by synthetic porphyrins. ................ 28

Scheme 7. Reagents and conditions used to synthesize the chalcone derivative 6 (a),

chalcone derivative 9 (b) and chalcone derivative 10 (c). ................................................... 33

Scheme 8. Reagents and conditions used to synthesize the flavone derivative 1 (a), flavone

derivative 2 (b) and flavone derivative 3 (c). ...................................................................... 38

Table index

Table 1. Yields for the chalcone derivatives synthesized. .................................................. 34

Table 2. Yields for the flavones derivatives synthesized. ................................................... 37

Table 3. 1H NMR signals for flavone 2 (left) and the flavone 11 (right). .......................... 46

Table 4. 13C NMR signals for flavone 2 (left) and flavone 11 (right). .............................. 47

Chapter I: Introduction


1

1. Introduction

Flavones, from the Greek word flavus (yellow), are a class of flavonoids, naturally

synthesized by plants and fungi but can also be synthetically produced. Flavones have a 15

carbon atoms backbone (Figure 1) composed of a benzene ring (A) bound to a heterocyclic

pyran ring (C) with a phenyl substitution (ring B) on position 2 of ring C.

Figure 1. Flavone backbone structure.

Flavone derivatives may have several substituent groups linked to rings A and B

carbons, and as well as in the 3-position on ring C (Figure 1). Naturally produced flavones

can be found in two forms, as glycosides, linked to one or more sugar units mostly by β-

linkage, but also α or as aglycones without sugars. Besides the sugar units, many other

substituent groups are common in naturally occurring flavones including the hydroxyl group

(-OH), methoxyl group (-OCH3) or prenyl group (CH2CHC(CH3)2). In synthetic flavones,

other substituent groups, such as halogens (bromide, chlorine and fluorine) or nitro groups

(-NO2) may be found.

In plants, flavones and other flavonoids are responsible for a vast array of functions

including flower and fruit colouration and aroma, protection from different types of stress,

protection from UV radiation, detoxification and defence against microbial threats.1 In

addition to their physiological role, flavones also possess several important biological

activities in animals.

1.1. Biosynthesis of flavonoids

Flavonoids biosynthesis starts with the phenylpropanoid pathway (Scheme 1) that

converts the amino acid L-phenylalanine to 4-coumaroyl-CoA. The amino group is removed

from L-phenylalanine by the enzyme phenylalanine ammonia lyase (PAL) originating (E)-


2

cinnamic acid. Following the removal of the amino group, a hydroxyl group is added by the

enzyme cinnamic acid 4-hydroxylase (C4H), an enzyme from the cytochrome P450 complex

Scheme 1. Phenylpropanoid pathway of biosynthesis of flavones.

that exhibits monooxygenase activity.2 The resulting molecule is the (E)-4-coumaric acid

that can, alternatively, be formed from the amino acid L-Tyrosine by the action of the

enzyme tyrosine ammonia lyase (TAL).3 p-Coumaric acid is then converted by the enzyme


3

4-coumarate:coenzyme A ligase (4CL) to the respective CoA-ester, 4-coumaroyl-CoA. 4-

coumaroyl-CoA can then suffer the action of several enzymes such as trihydroxystilbene

synthase, chalcone synthase or 6’-deoxychalcone synthase. The enzyme trihydroxystilbene

synthase synthesizes the conversion of 4-coumaroyl-CoA and 3 malonyl-CoA to 3,4',5-

trihydroxy-stilbene commonly known as resveratrol present in high amounts in red wine and

known to exhibit extraordinary biological activities such as antioxidant activity. The enzyme

chalcone synthase synthesizes the conversion of 4-coumaroyl-CoA and 3 malonyl-CoA to

2',4,4',6'-tetrahydroxychalcone (naringenin chalcone), while the action of 6’-deoxychalcone

synthase originates 2',4,4'-trihydroxychalcone (Isoliquiritigenin).2 Naringenin chalcone can

then be isomerized to the flavanone naringenin by the action of an extremely efficient and

chalcone specific enzyme, chalcone isomerase (CHI). Flavones are then synthesized from

their precursor, the flavanone naringenin, by the enzymes flavone synthase (FNS) that adds

a double bond between the C-2 and C-3 positions and the flavone created from this process

is apigenin, one the most common and studied flavone. Other flavanones such as eriodictyol

can also be substrate for the flavone synthase enzyme, and in this case the flavone obtained

is luteolin.2 Besides being a substrate to the flavone synthase, naringenin is also a substrate

for several other enzymes such as flavanone-4-reductase (FNR), flavanone-3-hydroxilase

(F3H) or flavonoid-3’-hydroxilase (F3’H), forming respectively flavan-4-ol,

dihydrokaempferol and eriodictyol.4 The action of different enzymes that modify the

structure of naringenin by adding/replacing substituent groups, creates several flavone

derivatives such as O-glycosylated flavones, C-glycosylated flavones or methoxyflavones.

Besides the flavones, and depending of the synthetic pathway followed, naringenin is also

the precursor of several other flavonoid families, including flavanones, flavonols,

proanthocyanidins, anthocyanins or phlobaphenes.4

1.2. Synthesis of flavones

Obtaining flavones from synthetic sources can be achieved by using different

synthesis strategies. Some of those strategies allow for the direct synthesis of flavone5 while

in many other, different molecules such as chalcones6 or 1,3 diones7 are synthesized and are

then used for the synthesis of the respective flavones. Depending from the starting reagents,

flavones with different substituent groups can be obtained. Because of this, many flavones

similar to the natural ones can be obtained, but also many other flavones that are not naturally


4

synthesized. Currently some of the most used techniques to synthesize flavones are the

Baker-Venkataraman rearrangement, and the cyclodehydrogenation of chalcones previously

synthesized. But many other techniques exist with special attention to one pot techniques

and solvent free techniques both usually associated with green chemistry methods. These

techniques usually involve the use of either Baker-Venkataraman rearrangement or Claisen-

Schmidt condensation, but with specially designed conditions, and/or catalysts that usually

make the synthesis faster, reduce the necessity of solvents and produce much less unwanted

residues.

The Baker-Venkataraman rearrangement (Scheme 2) was first described by Wilson

Baker in 19338 and K. Venkataraman in 19349 and is often used to synthesize many

molecules including chromones and flavones. In this method, 2-acetoxyacetophenones react

with a base that removes a hydrogen atom from the alpha carbon, forming an enolate. This

enolate attacks the ester carbonyl carbon originating a cyclic alkoxide, that is relatively

unstable, leading to the opening of the ring generating a phenolate that is then protonated

under acidic conditions originating the final product, a 1,3-diketone. To form the desired

chromone or flavone, this 1,3-diketone needs to undergo cyclodehydration usually with the

help of a strong acid, and the final product is then obtained. More recently several different

approaches to this method of synthesis have been studied to achieve a more efficiently

synthesis, solvent free and/or synthesis that does not involve the usage of dangerous

substances These modified synthesis techniques usually only change the reaction conditions

and not the reaction mechanism, affecting the rearrangement step and/or the

cyclodehydration step.

One example is the modified Baker-Venkataraman rearrangement of 2-

aryloxyacetophenones to 1-(2-hydroxyphenyl)-3-phenyl propane-1,3-diones. These

diketones are important intermediaries in the synthesis of numerous compounds including

flavones, coumaran-3-ones, isoxazoles, pyrimidines, and pyrazolines with relevant

biological activities and therefore pharmacological interest. But this reaction was usually

carried out using a pyridine medium and using bases such as sodamide, sodium hydride or

barium hydroxide, substances that are dangerous and require extreme care while being used.

To avoid the use of said chemicals, Sharma and colleagues10 developed a fast (15 minutes

of reaction time) and solvent free reaction method that could be carried at room temperature.

This method involves the use of pulverized potassium hydroxide under grinding conditions


5

in a mortar and pestle where the product, the corresponding 1,3-diketones, could be easily

recovered by acidification of the reaction mixture in water without the use of organic

solvents.

Scheme 2. Mechanism of Baker-Venkataraman rearrangement to 1,3-diones, followed by

cyclodehydration catalysed by a strong acid.

Other modification is the use of Phase Transfer Catalysts (PTC). This technique

allows the reaction of inorganic and organic ions and many other molecules, usually not

soluble in organic solvents, with organic molecules.11 Instead of a homogeneous reaction

medium there is a two-phase system. The use of PTC allows the migration of molecules

from one phase to the other, allowing the reaction to take place. This method allows the use

of reagents otherwise not miscible in the same solvent, to achieve the desired product. This

technique was already applied to the rearrangement phase of the Baker-Venkataraman

method of synthesis of molecules such as 2-styrylchromonesprecursors12 or synthesis of 1-


6

Polyoxyphenyl-3-(2,6-dioxyphenyl)propane-1,3-diones, 1-polyoxyphenyl-3-(2-

benzyloxyphenyl)propane-1,3-diones and 1-polyoxy-3-(2,6-dibenzyloxyphenyl)propane-

1,3-diones, precursors of flavones.13

Not all modifications involve the rearrangement phase of the Baker-Venkataraman

method of synthesis of flavones and similar molecules. Other modifications involve the

cyclodehydration phase. One example is the use of microwave assisted cyclodehydration,

were 1,3-diketones like 1-(2-hydroxyphenyl)-3-phenyl-1,3-propanedione are transformed

into the respective flavone with a yield of up to 98%.14 This synthesis method is not only

fast, compounds are subjected to microwaves for only 5 mins, but the purification phase only

involves the use of a short silica column, being an easy and quick method of

cyclodehydration.

Yet another method involves the use of a silica-supported Preyssler heteropolyacid,

that can be reusable. This method allows the synthesis of flavones with high yields

(87−94%), without the use of solvents and with short reaction times (7−13 min).7

The other extremely popular method of synthesis of flavones involves the use of the

Claisen-Schmidt condensation that was first reported, independently, by two investigators,

Rainer Ludwig Claisen and J. G. Schmidt in 188115,16. This is a reaction were a ketone reacts

with an aldehyde and can occur in both alkaline and acidic conditions (Scheme 3). This

reaction involves two steps, first the formation of an aldol/aldolate and a second step of

dehydration. Once the chalcone is synthesized, to obtain the respective flavone, this chalcone

must be oxidized to lead to the consequent cyclization. This cyclization step can be achieved

by several methods that include I2 in DMSO, SiO2-I2 mediated cyclization or NH4I mediated

cyclization (Scheme 4).17

Exactly like the Baker-Venkataraman rearrangement so does the Claisen-Schmidt

condensation can be modified to achieve higher yields or faster synthesis. Modifications to

this synthesis method include the use of sulfonic acid−functional ionic liquids that act as

both catalyst and solvent for the synthesis of chalcones with yields of 85-94%.18,19 With a

simple decantation, the chalcones can be separated and the sulfonic acid−functional ionic

liquids can be recovered and reused without significant loss in catalytic activity. Other

catalyst that showed interesting results is the sodium nitrate modified fluorapatite.20 This

catalyst is a bifunctional, recyclable and heterogeneous acid-base catalyst, with high

selectivity for the synthesis of chalcones and high yields at room temperature.


7

Other modification involving the Claisen-Schmidt condensation include the use of

microwave irradiations of an aqueous solution with the aldehyde and ketone and sodium

hydroxide. This modification allows for a short reaction time, reduction in the use of volatile

solvents, high yields and many substituent groups such as nitro, amino, chloro or ether

groups, present in the reagents, remain in the final product.21

Scheme 3. Mechanism of synthesis of chalcones by Claisen-Schmidt condensation.

Another technique involves the use of ultrasound irradiation, with the use of catalysts

in one of the cases, catalysts were prepared by grafting amino groups on sodium and cesium

exchanged X zeolite22 while in the other, the catalyst consisted in pulverized KOH and KF–

Al2O3.23 For the sodium and cesium exchanged X zeolite, no solvent was required and

chalcones were selectively synthesized with very high yields (>95%). While using the

pulverized catalysts, there was the need for an alcoholic solvent and the yields were worse,

but still could reach values above 90%.

As it was already referred earlier, the second step of flavone synthesis using Claisen-

Schmidt condensation involves the cyclodehydrogenation of the chalcone and consequent

cyclization (Scheme 4). This step can be achieved by many different techniques involving

different compounds and solvents with differences in the yields obtained. These techniques

are already extensively revised on17.

Since the chalcones and respective flavones synthesized in this project do not have

substituent groups, that usually reduce synthesis yields, the flavone synthesis was


8

accomplished by cyclodehydrogenation of chalcones that were previously synthesized by

non-catalysed Claisen-Schmidt condensation at room temperature in the presence on sodium

hydrate. The cyclization of the synthesized flavones was conducted using I2 in DMSO at

~165ºC.

Scheme 4. Mechanism of synthesis of flavones from chalcones using iodine in DMSO.

1.3. Flavone biological activities

Flavones and its derivatives have shown to possess antioxidant24–27, anti-

inflammatory28–31, anticancer32–34, anti-ageing35, antimicrobial36,37, antidiabetic38,

neuroprotective39, cardioprotective40 and chemotherapeutic41. Some of these biological

activities can have a huge impact in the way we treat civilizational diseases that are

increasing in prevalence such as cancer, obesity or diabetes. The versatility of their structure

and the diversity of biological activities makes this class of compounds one of the most

promising, and most studied currently worldwide.

1.3.1. Antioxidant properties of flavones

Free radicals like superoxide radical (O2-) or hydroxyl radical (●OH) are produced by cells

as either by-products of normal cellular processes (ex: electron transport chain42) or as

important molecular messengers (ex: inflammation43). In normal concentrations free radicals

are beneficial, but when in excessive amounts, a situation designated as oxidative stress, they

alter the normal cellular behaviour and may become cytotoxic, leading to cell death.44

Oxidative stress and therefore free radicals are linked to numerous diseases including


9

cancer45, obesity and associated diseases46 or chronic diseases47. The development/discovery

of new compounds with antioxidant activity (e.g. scavenging of free radical’s) may be

extremely important to reduce mortality associated with diseases linked to oxidative stress.

Several tests can be used to assess the antioxidant capability of compounds/extracts in vitro,

including spectrometric, chromatographic and electrochemical techniques. From the tests

usually conducted, DPPH and/or ABTS tests, two spectrofotometric techniques are among

the most widely used. Both DPPH and ABTS are free radicals of synthetic origin used to

assess the radical scavenging capability of compounds or extracts. Besides the DPPH and

ABTS tests several other can be conducted to fully understand the antioxidant capability of

the compounds being tested. Among these other tests are the ferric reducing antioxidant

power method (FRAP), the hydroxyl radical averting capacity assay (HORAC), the total

peroxyl radical trapping antioxidant parameter assay (TRAP), the lipid peroxidation

inhibition assay, the potassium ferricyanide reducing power method (PFRAP), the cupric

reducing antioxidant power assay (CUPRAC), Fluorimetry method, Cyclic voltammetry,

amperometric method, the biamperometric method, the biosensors method, the gas

chromatography and high performance liquid chromatography (HPLC).48

Flavones are some of the most promising compounds with antioxidant properties and

many natural flavones like eupatorin33, apigenin49 or chrysin34 and synthetic flavones were

already studied in recent years. According to several studies conducted, both natural and

synthetic flavone derivatives can possess antioxidant activity26,50,51. This antioxidant activity

is due to several processes like free radical scavenging (direct antioxidant activity),

inhibition of radical production (indirect antioxidant activity)50 or chelation of transition

metals such as iron, zinc and copper that show pro-oxidant activity in living cells52.

Masek et al53, using both DPPH and ABTS, showed that flavones with no hydroxyl

groups in its structure, are weak antioxidants when compared to flavones with one or more

hydroxyl groups. The same study demonstrated that the antioxidant activity of flavones is

dependent on their structure, and the existence of a 3-OH group was extremely important

and was responsible for higher radical scavenging capabilities. This was proven by the fact

that unsubstituted flavones, 6-hydroxyflavone and 7-hydroxyflavone were less effective in

scavenging radicals when compared with 3-hydroxyflavone. Besides that, the presence of

3’-OH and 4’-OH groups (ortho dihydroxy configuration) in ring B and the 4-carbonyl group

or 3-OH also result in high antioxidant activity.


10

Using the DPPH method Cottele and co-workers50 showed that the presence of a

catechol or a pyrogallol structure (Figure 2) on the B ring is extremely important for the

scavenging efficiency of superoxide radical (O2-). A second group of flavones with 3-OH

substitution when the B ring was substituted (presence of hydroxyl groups), also had

significant free radical scavenging properties. The same investigation group proved that all

the flavones with hydroxyl groups at positions 3' and 4' or 3', 4' and 5', scavenged superoxide

anions. These results suggest that ortho-di-phenolic groups (catechol moiety) or tri-OH

groups (pyrogallol moiety) on B ring are essential in the free radical scavenging process by

flavones. Besides evaluating free radical scavenging by flavones, Cottele and co-workers

also studied the influence of flavones on O2- production by the Xanthine/Xanthine Oxidase

(X/XO) system. Results indicate that flavones with hydroxyl groups at positions 3' and 4' or

3', 4' and 5' could both scavenge free radicals and inhibit O2- production by X/XO system by

a non-competitive process. When both di and tri-OH groups where absent but a 7-OH group

is present on the A ring, flavones also reduced O2- production by (X/XO) system but by a

competitive process. Molecules containing both 7-OH group and 3'-OH and 4'-OH or 7-OH

group and 3'-OH, 4'-OH and 5'-OH groups showed complementary properties revealing

extraordinary inhibition effect on O2- production by the system X/XO.

Another study conducted by Cao and co-workers25 showed that antioxidant activity

of flavones against peroxyl radicals tends to be proportional to the number of hydroxyl

substitution on their structure. When comparing two flavones both with 4 hydroxyl

substitutions, luteolin (5, 7, 3' and 4' hydroxyl groups) and kaempferol (4, 5, 7 and 4'

hydroxyl groups), the results show higher antioxidant activity for luteolin. This indicates that

the catechol like moiety (ortho dihydroxy configuration) in ring B is extremely important,

as observed in other studies24,50, proving that substitutions in different positions contribute

in a different manner to the antioxidant activity of flavones. In addition, when using luteolin

tetramethyl ether that has no free hydroxyl groups, no antioxidant activity against peroxyl

radicals was detected. This indicates the extraordinary role of hydroxyl groups on

antioxidant activity of flavone derivatives. Cao and co-workers25 also investigated the

antioxidant activity of flavones against hydroxyl radicals (●OH), and results show less

antioxidant activity in this case, most likely due to the fact that ●OH is extremely reactive

and has an extremely low half-life and has more probability to react with any nearby

molecule and not specific molecules.


11

Results presented previously showed significant impact of flavone structure in the

antioxidant properties of flavones. In order to further understand how the structure affects

antioxidant potency, Park et al26 used a three-dimensional (3D) quantitative structure activity

relationship study. Their findings demonstrated that a 3-OH group most likely works as a

hydrogen bond donator, which seems to be a prerequisite for good intermolecular

interaction, increasing antioxidant activity. Moreover, substitution on 3'- or 4'-positions with

both hydroxyl and/or methoxy groups of 3-hydroxyflavones resulted in increased

antioxidant activities. In contrast substitution at the 2'-position significantly reduced flavone

antioxidant activity, indicating that this group has less freedom to interact with other

molecules when compared to substitution on both 3' and 4'-position.

It is possible to conclude that substituent groups that are present/absent assume an

extremely important role on the antioxidant activity of flavone derivatives, with emphasis to

hydroxyl groups. The position of given groups is also of extreme importance, being the 5, 7,

2', 3', 4' and 5'-positions the ones that give rise to the most differences in antioxidant potency

between different flavone derivatives (Figure 2). In addition, the presence of a catechol or

a pyrogallol moiety in ring B seems to be of great importance for high antioxidant activity.

Figure 2. Structure dependency of flavone derivatives antioxidant activity.

1.3.2. Anti-inflammatory properties of flavones

Inflammation is a normal biological process mediated by the immune system cells

and can be caused by the presence of allergens, toxic substances, foreign bodies, pathogens

or tissue damage.54,55 The initial inflammatory response (acute inflammation) is short lived


12

and is beneficial since it helps the body to recover from the injury. If the inflammation does

not resolve in a normal fashion, it progresses to a state called chronic inflammation. Chronic

inflammation, a state of low grade and persistent inflammation, as severe consequences and

can cause cell damage. Just like oxidative stress, also inflammation, especially the persistent

form, is linked to several diseases like cancer and chronic diseases. In the future, the use of

compounds with anti-inflammatory activity may significantly reduce the impact of the

inflammation related chronic diseases. In recent years the anti-inflammatory properties of

several flavone derivatives started to be studied and some information is already available

regarding structure dependency on anti-inflammatory activity and. To study the anti-

inflammatory activity of flavone derivatives, several molecules can be used to track

inflammation progression. Most studies focus on the effects of flavone derivatives in the

prevention of LPS triggered inflammation, so molecules linked to the early stages of

inflammation are the ones focused the most. These molecules include cytokines like TNF-α

or IL-1β, enzymes like COX-1, COX-2, 5-LOX or iNOS, PGE2 (an eicosanoid) or the

transcription factor NF-κB responsible for cytokines expression.

Barik and co-workers28 tested the anti-inflammatory properties of one flavone, (5,7-

dihydroxy-2-(3-hydroxy-4,5-dimethoxy-phenyl)-chromen-4-one) extracted from bruguiera

gymnorrhiza. This flavone showed inhibition of COX-2 mediated PGE2 production, reduced

the activity of 5-LOX enzyme and inhibited the production of the proinflammatory cytokine

TNF-α. Their findings indicate that anti-inflammatory activity of this flavone derivative is

mediated through several mechanisms rather than only one. Inhibition of the metabolism of

arachidonic acid by inhibition of both COX-2 and 5-LOX (as seen in other study56) and

downregulation of pro-inflammatory cytokines expression through NF-κB inhibition are two

of the possible mechanisms of anti-inflammatory action of flavone derivatives.

Anti-inflammatory activity of 7,8-dihydroxyflavone was evaluated by testing the

potential inhibition of LPS-induced NO production by suppression of iNOS expression in

RAW264.7 murine macrophage cells.57 Treatment with 7,8-dihydroxyflavone of LPS

stimulated cells resulted in a decrease in NO production in a dose dependent fashion when

compared with LPS alone. The decrease in NO production was due to the inhibition of

expression of LPS-induced iNOS protein (Figure 6). The same group also evaluated the

impact on PGE2 and IL-1β concentration after treatment with 7,8-dihydroxyflavone. The

treatment resulted in the reversion of the increase of PGE2 caused by LPS by suppression of


13

COX-2 expression and a decrease in release of IL-1β both in a dose dependent fashion. Since

NF-κB acts as transcription factor for several mediators including iNOS, COX-2 and IL-1β

after stimulation with LPS, the effect of 7,8-dihydroxyflavone was also evaluated. This

treatment supressed both nuclear translocation of NF-κB and IκB-α degradation, resulting in

inhibition of the NF-κB signalling pathway (Figure 6) and resulted in anti-inflammatory

effect. The concentration used in assays did not affected cell viability so the anti-

inflammatory activity of 7,8-dihydroxyflavone was not a consequence of cytotoxicity caused

by this flavone.

Wang and co-workers58 tested the anti-inflammatory activity of several mono- and

polyhydroxylated flavones in kidney mesangial cells, by measuring the LPS-induced NO

production by iNOS. High inhibition of NO production was detected within the group of

mono- and dihydroxyflavones like 6-hydroxyflavone and 4',6-dihydroxyflavone while tri-

tetra- and pentahydroxyflavones had low inhibitory activity against LPS-induced NO

production. The most polyhydroxylated flavones like 3,3',4',5,7,8-hexahydroxyflavone

(gossypetin) and 3,3',4',5,5',7- hexahydroxyflavone (myricetin) did not showed significant

inhibitory activity. The results also show that the hydroxyl groups position modulates the

flavone derivatives inhibitory activity for mono- and dihydroxyflavones. The decreasing

order of activity found was 6-OH > 7-OH >> 3-OH and 2'-OH. In addition, adding a 4'-OH

group to 6-hydroxyflavone had no impact on its anti-inflammatory activity while changing

the position of substitution from 6-OH to 5-OH clearly decreased anti-inflammatory activity.

The same group also tested 6-methoxyflavone, 6-acetoxyflavone and flavone 6-sulfate

(because of the potential toxic effects of 6-hydroxyflavone against kidney mesangial cells).

All three flavones were surprisingly potent and were good inhibitors against LPS-induced

NO production. The most potent inhibitor was 6-methoxyflavone, had minimal cytotoxic

effects and was more potent that any of the hydroxyflavones tested. Exactly like the

antioxidant activity of flavone derivatives, also anti-inflammatory activity seems to be

influenced by position, substitution group and number of substitutions.

Tsuji and co-workers59 tested the iNOS inhibition in murine macrophage-like

RAW264.7 cells by several flavonoids including many hydroxy- and methoxyflavones.

Their results showed greater inhibition for 5,3-dimethoxyflavone, and 5,7-

dimethoxyflavone. It was also concluded that the presence of 5- OCH3 facilitated the

inhibition of iNOS and the presence of 5-OH substitution did not, just like observed by Wang


14

et al.58. Another group of investigators29 tested the anti-inflammatory activity of four

dimethoxyflavones (7,2'-dimethoxyflavone, 7,3'-dimethoxyflavone, 7,4'- dimethoxyflavone

and 7,8-dimethoxyflavone) against Carrageenan induced hind paw oedema in rats. All the

four dimethoxyflavones showed dose-dependent anti-inflammatory activity with 7,4'-

dimethoxyflavone has the most active. Further tests showed that these dimethoxyflavones

inhibited both COX-1 and COX-2 activities in a dose-dependent manner, and reduced TNF-

α and IL-1β expression. Both the enzyme inhibition and decrease in cytokines expression

have anti-inflammatory effects and explain the anti-inflammatory activity of this

dimethoxyflavones.

Hsu and co-workers60 tested the anti-inflammatory activity of β-naphthoflavone on

HMEC-1 cells. Results show that this flavone induced the reduction of lymphocyte

infiltration and monocytic migration by reduction of adhesion molecules expression. In

addition, there was also a significant reduction of TNF-α-induced ROS production and TNF-

α-induced NF-κB activity. The TNF-α induces oxidative stress, the expression of cell

adhesion molecules and activates NF-κB, important steps in inflammation genesis. All these

responses are supressed in the presence of this compound, indicating that β-naphthoflavone

acts by supressing TNF-α activity.

Flavones naturally synthesized by plants are commonly found in plants as glycosides.

Enzymatic reactions can occur to hydrolyse the flavone glycosides, obtaining the free

flavone aglycone and the free sugar. Since structure is important in the flavone activity,

Hostetler and co-workers61 tested the influence of the presence of sugars on flavone structure

in both bioavailability and anti-inflammatory activity. To achieve it, several glycosides and

respective aglycones were used. While aglycones significantly reduced expression of TNF-

α the respective glycosides showed no significant effect. Besides that, aglycones like

apigenin, luteolin or chrysoeriol significantly reduced LPS-stimulated NF-κB activity but

the same didn’t occurred for the glycosides. Besides that, this team also proved that the

aglycones are absorbed in the gastrointestinal tract in a greater extent when compared with

the respective glycosides. These results indicate that flavones from plants may not be

extensively absorbed and the conversion to the corresponding aglycone not only increases

the bioavailability but also increases the anti-inflammatory activity of flavone derivatives

naturally present in food. Flavone derivatives clearly show anti-inflammatory properties of

great use against some diseases like cancer or many chronic diseases. Just like in the case of


15

antioxidant activity, the structure and type of substitutions present in flavones also modulates

anti-inflammatory activity. In this case, less substitutions (1 or 2) lead to increased activity

and the presence of methoxyl substitutions also leads to increased activity when compared

to hydroxyflavones. The preferential positions for the substitutions are 6- and 7-positions for

hydroxyflavones, and 3-, 4', 5- and 7-positions for methoxyflavones (Figure 3). In addition,

the use of aglycones in diet instead of glycosides leads to an increase in absorption, increased

bioavailability and anti-inflammatory activity of flavone derivatives.

Figure 3. Favoured positions and substituent groups that increase anti-inflammatory activity

of flavone derivatives.

1.3.3. Anti-cancer properties of flavone derivatives

In 2012, estimations indicate that there were 14.1 million new cancer cases and 8.2

million deaths caused by cancer worldwide. In 2030 the numbers are estimated to increase

to 21.7 million new cancer cases and 12 million deaths worldwide.62 Cancer treatments like

radiotherapy and chemotherapy have severe side effects, that include cardiotoxicity63,

neurotoxicity64 or bone loss65. Cancer cells possess unique characteristic like overexpression

of certain molecules66, altered metabolism67 or physiological abnormalities in blood

vessels68. The development of a targeted cancer treatment that takes advantage of such

characteristics, reduces cancer treatment related side effects and increases both specificity

and efficiency69,70. Several studies conducted suggest that flavone derivatives have

antiproliferative and anticancer activity and can inhibit MDR linked transporters making

these compounds serious candidates for future cancer therapy techniques.

Juvale and co-workers71 tested the inhibitory effect of several flavone derivatives on

BCRP overexpressing MDCK BCRP cells. BCRP is a membrane transport protein and

confers multidrug resistance to cancer cells, making cancer cells immune to several


16

pharmacological treatment options. In this study, flavones, 7,8-naphthoflavones and 5,6-

naphthoflavones with different substituent groups were used. Two groups were used in the

case of flavones, those having hydroxy substituents at position 3 and those having methoxyl

substituents in the same position. 3-hydroxyflavones showed lower inhibition activity when

compared to 3-methoxyflavones. In addition, the presence of methoxyl groups on B ring

increased the inhibitory activity of flavones, especially when this substitution was present in

both 3′- and 4′-positions. When comparing 5-OH with 5-OCH3 substitution, the hydroxyl

substitution had increased activity. The most potent flavone was found to be the compound

with both 3-OCH3 and 5-OH substitutions. For 7,8- and 5,6-naphthoflavones the presence

of a 3-OH substituent led to a decrease in activity, while the presence of a 3- OCH3

substitution increased inhibitory activity. Just like in the case of flavones the presence of

both 3′- OCH3 and 4′- OCH3 groups increased activity. Besides that, in general, 5,6-

naphthoflavones showed to be less potent when compared to 7,8-naphthoflavones. In this

case, the anticancer activity is not directly due to the flavone activity, but their presence

increases the targeted treatment efficiency by inhibition of cancer cell multidrug resistance

activity.

Other studies focused on direct effect of flavones on cancer cell lines, showed

naturally occurring flavones like chrysin could induce cytotoxicity and apoptosis against the

MCF-7 breast cancer cell line in a time- and dose dependent manner34. Another study

indicated that luteolin could induce apoptosis in HepG2 cells72. Several tests were conducted

to understand the mechanism behind the apoptosis induction by luteolin. The results show

that luteolin induces translocation of Bak and Bax to the mitochondria and consequent

translocation of cytochrome c to the cytosol, leading to activation of CPP32/caspase-3 (like)

protease. Besides that, luteolin also induced activation of JNK in a time dependent manner.

JNK is linked to several responses including apoptosis. Activation of both CPP32/caspase-

3 and JNK together with Bak and Bax translocation to the mitochondria, were considered

the critical events in the luteolin-induced apoptosis of human hepatoma HepG2 cells.

Another study focused on the antiproliferative activity of several flavone derivatives on

human hepatoma HepG2 cells. Most of the compounds tested showed promising

antiproliferative activity against HepG2 cells73, especially those with a 3′-OCH3 group.

Results also showed that antiproliferative activity depended on both position and number of

methoxyl substituent groups on ring B, with flavones with more methoxyl groups having


17

higher antiproliferative activity. When a nitro groups was present in ring A, the flavone

would show little to no antiproliferative activity, indicating that electron-negativity in the

benzene ring greatly affected the compound activity. In addition, by comparing flavones

with naphthoflavones, results suggest that the presence of the naphthalene group on

naphthoflavones improved antiproliferative activity against HepG2 cells. With these results

in mind the most potent flavone was tested to understand the antiproliferative activity of

flavones on HepG2 cells. Treatment of HepG2 cells with this compound originated DNA

fragmentation in a dose-dependent manner. DNA fragmentation is a biochemical

characteristic of cells in apoptosis and in this case occurred mainly by the caspase-dependent

intrinsic mitochondria pathway.

These results indicate that direct antiproliferative and anti-cancer activity of flavones

most likely occurs due to induction of programmed cell death (apoptosis) by several

mechanisms including the caspase-dependent intrinsic mitochondria pathway (Figure 4).

The flavone activity in cancer cells can also reduce the efficiency of cell membrane

transporters with MDR activity, usually overexpressed in these cells, with the flavones

exhibiting an indirect anti-cancer effect by increasing efficiency of drugs used in cancer

treatment.

Figure 4. Effects observed on cancer cells treated with flavones.

1.3.4. Antimicrobial and antiviral activity of flavone derivatives

Resistance to antibiotics is a major health problem, and is becoming increasingly

more common, leading to increased rates of infection complications that may include


18

death74. Also, some of the existing antibiotics have severe side-effects and/or toxicity. This

leads to an ever-existing necessity to found new compounds with potent antimicrobial

activity and with less side-effects and toxicity. Natural compounds are becoming

increasingly more important in recent years. Some plants are used in traditional medicine

since many centuries ago and only now we start to understand what compounds are

responsible for their medicinal effects75.

Naik and co-workers conducted a study to test the effect of several synthesized 3-

hydroxyflavones on different microorganisms by determination of inhibition area in vitro36.

The microorganisms used to test antibacterial and antifungal activity consisted of 3 gram-

positive and 3 gram-negative bacterial strains and 2 fungal strains. Some of the tested 3-

hydroxyflavones exhibited potent antimicrobial activity, being more active against gram-

negative bacteria. Another investigation group tested the antimicrobial activity, of one

extract from Lagerstroemia indica leaves76, on Candida albicans yeast and four bacteria that

are considered food pathogenic (Staphylococcus aureus, Salmonella enteritidis, Escherichia

coli, and Listeria monocytogenes). This extract showed antimicrobial activity against these

bacteria strains. After this, the compound responsible for this antimicrobial activity was

purified and characterized. The compound isolated proved to be a flavone derivative, 4'-

methoxy apigenin (acacetin)-8-C-β-D-glucopyranose (cytisoside), a glycoside with a single

sugar moiety. The purified compound was then tested to prove its relevancy in the

antimicrobial activity of this extract. The compound showed potent antimicrobial activity

against all five microorganisms tested, being even more potent at same concentration than

the standard antibiotic used as positive control. Shoaib and co-workers37 tested the

antibacterial activity of several synthesized flavones against both gram-positive and gram-

negative bacterial strains. Their results show a dependency of the antibacterial activity on

the presence of substitutions on rings A and B. For example, the presence of a halogen like

bromide at ring A and trifluoromethyl at ring B led to strong antibacterial activity against

both gram-positive and gram-negative bacteria. In contrast, the presence of both methoxyl

and bromide on ring A and trifluoromethyl on ring B clearly reduced the compound activity.

Besides that, flavones containing methyl groups on ring B and even the base flavone with

no substituent groups also showed significant antibacterial activity. This study concluded

that the presence of halogens on ring A and trifluoromethyl group at ring B generated

flavones with potent antibacterial activity. Addition of methyl and dimethylamino groups


19

also generated compounds with significant antibacterial activity while addition of halogens

on ring B generated flavones with low antibacterial activity.

Since flavones can also be synthesized by fungus, Kaur and co-workers77 tested the

antimicrobial activity of the extracellular culture broth of Penicillium sp., HT-28 against

several gram-positive and gram-negative bacterial strains and two yeast strains. This

extracellular culture broth showed antimicrobial activity against three of the gram-positive

bacteria: Staphylococcus aureus, Staphylococcus epidermidis and MRSA, one gram-

negative bacteria: Klebsiella pneumoniae and one yeast Candida albicans. One of the

compounds purified from this culture broth was a flavone derivative 6-[1,2-dimethyl-6-(2-

methyl-allyloxy)-hexyl]-3-(2-methoxy-phenyl)-chromen-4-one. Subsequent tests conducted

indicate that this compound is responsible for the antibacterial activity of the culture broth

used. Besides that, the compound remained stable even at temperatures as high as 100ºC and

showed neither any cytotoxicity or mutagenicity, being suited for further testing.

Infection can occur due to the presence of several organisms and viral particles. Since

flavones showed potential antibiotic usage against bacteria, Hossain and co-workers78

decided to test if flavones possess antiviral activity. To achieve that, the activity of different

hydroxylated flavones was tested on the influenza A virus. They started to test the effects of

the flavones used on normal MDCK cells, and their cell viability was increased in the

presence of some of the flavones like 3,2-dihydroxyflavone and 3,4-dihydroxyflavone. In

addition, these two flavones were the ones showing the most potent antiviral effects in vitro.

Due to the excellent results in vitro, the same group decided to test the effects of these

flavones on mice infected with influenza A virus and comparing results with both control

and mice treated with Tamiflu. The body weight loss in the infected mice treated with the

flavones was significantly reduced when compared with the infected control mice. Besides

that, the survival of the mice was significantly increased when compared with control (0%

on control, 60% to 70% on mice treated with flavones, 100% on mice treated with Tamiflu).

Flavone treatment clearly increased mice survival rates to influenza A virus infection, when

compared with untreated mouse, showing the possibility of viral infection treatment with

these compounds.

Flavone derivatives show potentially useful antimicrobial and antiviral activities.

These compounds tend to show little to no cytotoxic or mutagenic effects and even increase

the normal cell viability. These properties make flavone derivatives possible excellent


20

antibiotics, but further investigation is still a must since most of the information available

was obtained from in vitro studies.

1.3.5. Lipid metabolism modulation

Besides all the biological activities already presented, there are other less studied

activities that may be extremely important and increase the utility of the flavone derivatives.

Two examples of that are the lipid metabolism and anti-diabetic activity.

Several pathologies are related to alteration on the lipid metabolism such as

dyslipidemias, or cardiovascular diseases. LXRα and LXRβ are transcription factors that are

part of the nuclear receptor superfamily of ligand-activated transcription factors79. LXRβ is

present in almost all the tissues while LXRα is mainly present in metabolic active tissues

such as the liver, kidneys or adipose tissue. When active these transcription factors lead to

the expression of genes that result in increase in HDL levels, reduced cholesterol

biosynthesis, and increased cholesterol metabolism and excretion. In addition, LXRs

activation also leads to an increase in triglyceride levels linked to several cardiovascular

diseases. LXRs are naturally activated by oxysterols, oxidized derivatives of cholesterol

usually present when the levels of cholesterol are higher than normal. Due to the structural

similarities between the LXR ligands, and flavones Francisco and co-workers80 tested the

effect of luteolin, a flavone derivative on LXRα and LXRβ activities. The results show that

luteolin reduces the LXRs activity when these transcription factors are activated by synthetic

agonists. The treatment with both agonist and luteolin results in reduced lipogenesis and

reduced liver lipid content by reducing the expression of SREBP-1c. Since LXRs are also

linked to an anti-inflammatory activity that contributes to a decreased risk in several

cardiovascular diseases, the inhibitory effect of luteolin could possibly induce these diseases.

Since luteolin alone also possesses anti-inflammatory activity, the combination of this

activity with the lipid concentration decrease by inhibition of LXRs makes luteolin

potentially useful in diseases associated with high levels of lipids and to a pro-inflammatory

state. These pathologies include hepatic steatosis, cardiovascular diseases, and diabetes.

1.3.6. Anti-diabetes activity

Diabetes Mellitus is a pathology characterized by the presence of hyperglycemia,

(high blood sugar levels) and it is caused by reduced production of insulin and/or insulin


21

resistance81. When insulin resistance is present, the usage of insulin independent methods to

manage blood glucose levels such as restricted diet need to be used to help manage glucose

homeostasis. Bumke-Vogt82 tested the effect of luteolin and apigenin on cells from human

liver carcinomas. The results showed a significant reduction in expression of gluconeogenic

and lipogenic genes. The fact that these two flavones can reduce the expression of

gluconeogenic genes gives these compounds antidiabetic activity by reducing the hepatic

glucose production. This property can be extremely helpful in controlling the glucose

homeostasis in patients with diabetes were insulin resistance is present.

1.4. Porphyrins and P450 complex

Porphyrins are heterocyclic macrocyclic organic molecules, constituted by four

modified pyrrole subunits, forming a tetrapyrrolic macrocycle, with the four pyrrole subunits

linked via methine bridges (=CH‒) at the α carbons (Figure 5).83 At the centre of the

porphyrin there is enough space for a metallic ion to be accommodated, that will be linked

to the four nitrogen atoms in the centre of the porphyrin.

Figure 5. Tetrapyrrolic heterocyclic macrocycle structure. Skeleton of porphyrins.

Different metallic ions can be present in naturally occurring porphyrins like

magnesium in chlorophyll. The heme group is present in several proteins such as

haemoglobin and in several enzymes with oxidase activity such as enzymes from complex

P450 like catalase or peroxidase.84,85 The complex P450 enzymes are extremely important

in the metabolism of xenobiotic compounds or radical scavenging.85 Besides the naturally

occurring porphyrins, these class of compounds can also be synthetically obtained86 and

different metallic ions can be present, such as manganese87,88. The synthetically produced

porphyrins show oxidase activity in the presence of a co-catalyst, such as ammonium acetate,


22

and an oxidizer such as hydrogen peroxide, and are seemingly extremely useful to reproduce

the activity of naturally occurring enzymes containing heme groups.89–91

1.4.1. Biosynthesis of porphyrins (heme)

The synthesis of heme b starts in the mitochondria with only two compounds, glycine

and succinyl-CoA (Scheme 5).92 By the action of the enzyme, δ-aminolaevulinic acid

synthase (ALAS), an enzyme that requires vitamin B6, δ-aminolaevulinic acid (ALA) that

is also known as 5-aminolevulinic acid is formed. After this first reaction, ALA is

transported to the cytosol where ALA dehydratase (porphobilinogen synthase) will lead to

the dimerization of two molecules of ALA, producing the porphobilinogen, a pyrrole ring

compound. Posteriorly, porphobilinogen deaminase, is involved in the head-to-tail

condensation of four porphobilinogen molecules, forming the linear tetrapyrrolic

intermediate hydroxymethylbilane. Hydroxymethylbilane is then converted to

uroporphyrinogen III, a cyclic molecule, by the enzyme uroporphyrinogen III synthase.

Following cyclization, all the acetate substituent groups from uroporphyrinogen III are then

decarboxylated due to the action of the enzyme uroporphyrinogen decarboxylase, with the

resulting compound, coproporphyrinogen III having methyl instead of the acetate

substituents. After its synthesis, coproporphyrinogen III is transported to the mitochondria

were two propionate residues are decarboxylated to vinyl substituents by the action of the

oxygen dependent enzyme coproporphyrinogen III oxidase, yielding protoporphyrinogen

IX. After that the action of the protoporphyrinogen IX oxidase converts protoporphyrinogen

IX to protoporphyrin IX. Finally, the metallic ion Fe2+ is inserted by the enzyme

ferrochetalase and the final molecule of the synthesis chain is a heme b molecule. Heme b is

the most common heme form, but not the only one, with heme a being present in the

cytochromes of the heme a type like cytochromes of the complex IV of the oxidative

phosphorylation pathway93 and heme c from the cytochrome c complex.94 Both heme a and

heme c are modified iron protoporphyrin IX.

1.4.2. Cytochrome P450 complex

The cytochrome P450 complex of enzymes constitutes a superfamily of

hemoproteins present in almost all living beings. These enzymes present monooxygenase

activity and contain an heme b iron porphyrin prosthetic group. These enzymes catalyse a


23

Scheme 5. Pathway for the biosynthesis of heme b.


24

wide range of reactions such as epoxidation of C=C double bonds, hydroxylation of aromatic

carbons, N-oxidation, deamination or dehalogenation. In some cases, these enzymes can also

catalyse other reactions such as cleavage of C-C bonds or Baeyer-Villiger oxidation.95 Not

only these enzymes have a wide range of activities, they are also able to accept a wide variety

of compounds that include important molecules such as prostaglandins, fatty acids or

steroids, being extremely important in both synthesis and metabolism of these molecules.96

Besides synthesizing and metabolizing naturally occurring molecules, the P450 complex is

also able to accept as substrate poly- and heteroaromatic compounds and xenobiotics that

include many drugs, antibiotics, pesticides, carcinogenic compounds or toxins.97,98

The enzymes from the P450 complex are present in almost all living beings99, even

being present in some virus100, presenting remarkable conserved structural similarities

showing how important these enzymes are at a biological level. The fact that these enzymes

can catalyse regio-, chemo- and stereospecific oxidation of many different molecules may

be of great use for the synthesis of many drugs/compounds, since the synthetic oxidation of

several compounds is usually of low yield and/or originates unwanted products increasing

costs and increases the quantity of organic solvents used. Using these enzymes for the

synthesis of said compounds could not only reduce costs leading to potentially more

profitable synthesis pathways, it can also contribute to a greener approach to organic

synthesis, since these reactions would require lower quantities of starting compound to

achieve similar or even better yields. Besides that, less purification steps would be needed,

due to the specificity of these enzymes, reducing solvents needed and the undesired

inevitable waste by-products of the purification steps.

In addition, these enzymes are also involved in the metabolism of several

xenobiotics, reducing their hydrophobicity, making these compounds easier to be excreted.

These xenobiotics include compounds such as drugs, used for treatment of numerous

diseases and usually the metabolization of such compounds reduces the desired activity or

even completely inactivates these drugs, leading to an increase in the dosage needed to

achieve the desired response.101 Drug design with the help of the P450 complex enzymes

could lead to the production of compounds with similar or higher activity, or even lead to

the development of inactive compounds that would become active after the metabolism by

these enzymes.102 These compounds are known as prodrugs, drugs that need to be

metabolized to become pharmacological active. Usually prodrugs are designed to increase


25

bioavailability on the gastrointestinal tract, requiring lower dosage and having reduced

undesired side effects.103

The vast range of activity and potential uses of the enzymes of the P450 complex,

caught the attention of scientists from diverse areas including organic chemistry and

biotechnology, turning these enzymes some of the most studied currently worldwide. Even

though a lot was already discovered there is still huge potential for the uses of these enzymes

and many things are yet unexplored.

1.4.3. Synthetic porphyrins

The activity of the enzymes of the P450 complex is extremely dependent on the

porphyrin group present in the catalytic centre of these enzymes.104 Because of this, efforts

have been made to produce synthetic analogues of these natural molecules to understand not

only how the P450 complex enzymes work but, most importantly, to replicate their activity

without the need for the whole enzyme.105,106 Synthetic porphyrins have extremely useful

applications such as in the field of biomimetic catalysis, but the potential applications of

these porphyrins go way beyond this single application. Synthetic porphyrins have many

interesting properties that can be useful for many other applications, with ruthenium

porphyrins showing interesting results when used in photodynamic treatment of bladder

cancer107, porphyrins derivatives that can be used as imaging agents for the nuclear imaging

of tumors108 or catalysts for reactions such as the synthesis of cyclic carbonates from

epoxides and CO2.109

1.5. Biomimetic oxidation catalysis

The biomimetic oxidation catalysis tries to replicate the oxidation activity of

enzymes such as the P450 complex enzymes. Since in the catalytic centre of these enzymes

lies a porphyrin, synthetic porphyrins are being tested/used to replicate this oxidation

activity. Many porphyrins can be synthesized in laboratory, having similarities in their

structure with natural porphyrins but also extremely relevant differences (Figure 6). But

while most of the naturally occurring porphyrins have a metallic ion such as iron or

magnesium in the middle cavity, there is a much higher degree of freedom for the synthetic

porphyrins. Currently the porphyrins with the highest potential in the field of biomimetic

oxidation catalysis are porphyrins containing a ruthenium or manganese ion in the middle


26

cavity such as 5,10,15,20-tetrakis(2,6-dichlorophenyl) porphyrin manganese(III) chloride

(Mn(TDCPP)Cl) porphyrin, the one used in this project.

Several studies conducted show an enormous potential for the use of porphyrins,

because of their capability to oxidate different molecules, making them useful for a vast

array of functions (Scheme 6). Tests showed that synthetic water-soluble iron porphyrins

such as meso-tetra(2,6-dichloro-3-sulfonatophenyl)porphyrinate of iron(III) chloride, (Fe-

(TDCPPS)Cl) have potential in the detoxification of environmental compartments like

water, soil and sediments, when used simultaneously with both humic acid substances and

an oxidizer such as hydrogen peroxide.110 These porphyrins acted as a catalyst for the

oxidative coupling reaction between the humic acids and polyhalogenated phenols such as

pentachlorophenol (PCP) or pentafluorophenol (PFP) forming a stable complex. This

evidence was later confirmed when a synthetic water-soluble iron porphyrin was used on

two different highly polluted soils from an industrial site.111 These polluted soils were first

treated with an exogenous humic acid followed by the treatment with the porphyrin that

catalysed the coupling between the humic acid and the pollutants present in the soil. Results

show a dramatic reduction in the quantity of free pollutants in the soil of up to 90% indicating

that this method may be of great use in the remediation of soils and restoring soil quality

reducing the impact that human activities have in the environment. This method also adds

organic matter on the form of humic acids that can be extremely important since

microorganisms, that may use this organic matter, also play a pivotal role in the

detoxification of soils.

Other project shows that synthetic porphyrins such as ruthenium porphyrins are able

to catalyse the oxidation of several organic compounds, including styrenes, cycloalkenes,

allyl substituted alkenes, enones, steroids, benzylic hydrocarbons and aromatic

hydrocarbons.112 Yet another project using manganese porphyrins show that these

porphyrins are extremely versatile and can catalyse diverse oxidation reactions on a variety

of heteroatom containing compounds such as sulphides, amines or phosphites,89 but with

incredible affinity to C=C bonds, including aromatic compounds, and capable of catalysing

oxidation reactions such as epoxidation or hydroxylation.

Even though the potential applications are wide and with extremely relevant results,

arguably the most interesting and most promising application is the study of the oxidative

metabolism of both synthetic and natural compounds that show important biological


27

activities. As said before, porphyrins are capable of catalysing oxidation reactions on diverse

substrates including many biological molecules. All the evidences, so far, indicate that the

reactions catalysed by these porphyrins are the same reactions catalysed by the P450

complex of enzymes responsible for the metabolism of diverse molecules.113,114 Among the

molecules metabolised by these enzymes are several compounds with relevant interest like

several medical drugs or even natural compounds that show biological activity. Knowing

exactly how a compound is metabolised by a living organism is pivotal to understand its life

cycle and get a more deeply understanding of its destiny. Besides that, there is the possibility

to reduce the potential side effects of many drugs, since when metabolised some compounds

both of synthetic and natural origin can become toxic, but at the same time some compounds

become even more active, something that can be exploited in drug synthesis.

Figure 6. Structure of the heme b (left), and structure of 5,10,15,20-tetrakis (2,6-

dichlorophenyl) porphyrin manganese(III) chloride (right).

Due to the fact that all the knowledge of the potential biomimetic activity of

porphyrins is still fairly recent, almost no information of its use is available. One exception

is the use of metalloporphyrins on two compounds that showed interesting biological

activity, piperine and piplartine.90 The results of the oxidation of these compounds showed

that not only the porphyrins can mimic the activity of the P450 enzymes, they can also be

used to produce the derivatives of these compounds. These products can be tested to assess

the potential side effects of the compounds originated from the normal metabolism but also

help to understand the mechanism of action of these compounds since sometimes the

products of the metabolism show higher activity when compared with the starting


28

compound. The overall properties of the synthetic porphyrins make this class of compounds

extremely useful to understand how certain compounds are metabolized in living beings and

can be extremely useful in the process of creating and testing new compounds, including

drugs in the form of prodrugs. Unfortunately, there is still a long path to be run since little

to no information about the actual use of porphyrins on metabolism mimetics and drug

development/testing is known.

Scheme 6. Examples of oxidation reactions catalysed by synthetic porphyrins.


29

1.6. Objectives

The main objective of this project was to obtain flavone derivatives bearing naphthyl

groups (Figure 7) to be used on biomimetic catalysis studies. First it will be discussed the

synthesis of the flavone derivatives precursors, the chalcone derivatives, then the synthesis

of the flavone derivatives from the chalcone derivatives, followed by the biomimetics studies

of these flavones. At the end, some biological activity tests will be addressed.

Figure 7. Flavone derivatives bearing naphthyl groups to be used on the biomimetic

catalysis studies.


30

31

Chapter II: Results and discussion


32


33

2. Results and discussion

2.1. Chalcone derivatives synthesis

The chalcones derivatives were synthesized using the Claisen-Schmidt method with

specific reagents and reactions conditions (Scheme 7). This method of synthesis allowed to

obtain the desired chalcones derivatives with very good yields, 70-89% (Table 1).

Scheme 7. Reagents and conditions used to synthesize the chalcone derivative 6 (a),

chalcone derivative 9 (b) and chalcone derivative 10 (c).

When compared with the literature, these values remain decent and close to the

values usually obtained for these techniques but are still far from the best yields possible.

When using the proper catalyst, yields showed to be >90% for the synthesis of certain

chalcones, specially the most simple ones.19 Without the use of these catalysts, it is almost

impossible to achieve such yields with this method, but relatively high and still good to very

good yields can be achieved. For that to be possible, the reaction conditions and reagents

needed to be taken in consideration, such as making sure that the solvent used, the THF, is

completely dry and that the reaction occurs under nitrogen atmosphere so that the NaH does

not get degraded by the water or oxygen present in both the THF and the air. The degradation

of NaH would certainly be something that would reduce the yield of the reaction by lowering

the availability to react with the desired reagents. The reactions optimization was done by

a)

b)

c)


34

using different reagent equivalents reaction time and temperature. For all the chalcones, the

reaction optimal conditions were extremely similar, with both the NaH and the aldehydes

used (compounds 5 and 8) being added in excess ~2.5-3 equivalents and 1.35 equivalents

respectively when compared with the ketones used (compounds 4 and 7) and the temperature

used was the room temperature (~20ºC). The aspect that had greater impact in the reaction

yield was the reaction time with the optimal reaction time being between 5 and 6 hours.

Table 1. Yields for the chalcone derivatives synthesized.

Compound Yield (%)

Chalcone 6 70

Chalcone 9 89

Chalcone 10 72

2.2. 1H NMR characterization of the synthesized chalcone derivatives

2.2.1. Chalcone 6 (1-(2-hydroxynaphthalen-1-yl)-3-phenylprop-2-en-1-one)

1H NMR (300.13 MHz, CDCl3): δ 7.19 (1 H, d, J = 9.0 Hz, H8’), 7.38-7.46 (4 H, m, H4’,

H3’’, H4’’, H5’’), 7.51 (1 H, d, J = 15.7 Hz, H2), 7.54 (1 H, m, H5’), 7.60-7.66 (2 H, m,

H2’’, H6’’), 7.82 (1 H, dd, J = 8.0 Hz, 1.4 Hz, H6’), 7.93 (2H, d, d, J = 9.0 Hz, J = 15.7 Hz,

H7’, H3), 8.06 (1 H, d, J = 8.5 Hz, H3’), 12.58 (1 H, s, 1’-OH).

Figure 8. Structure for chalcone 6.

The 1H NMR shows characteristic signals from which the signal for the 1’-OH

proton, δ at 12.58 ppm, and the signals for the α and β hydrogens (connected to carbons 2

and 3) both doublets that couple with each other and present a very distinctive coupling

constant of 15.7 Hz, can be highlighted. Other signals also easily identified are hydrogens

7’ and 8’ that present a coupling of 9.0 Hz, both the signals are also doublets and present a

constant characteristic of ortho coupling. The other signals are difficult to distinguish due to

overlap but allowed the confirmation of the structure depicted in figure 8.


35

2.2.2. Chalcone 9 (1-(2-hydroxyphenyl)-3-(naphthalen-2-yl)prop-2-en-1-one)

1H NMR (300.13 MHz, CDCl3): δ 6.98 (1 H, ddd, J = 8.2 Hz, 7.2 Hz, 1.2 Hz, H5’), 7.05 (1

H, dd, J = 8.4 Hz, 1.0 Hz, H3’), 7.49-7.58 (3 H, m, H4’, H5’’, H8’’), 7.79 (1 H, d, J = 15.4

Hz, H2), 7.80-7.93 (4 H, m, H3’’, H4’’, H6’’, H7’’), 7.99 (1 H, dd, J = 8.2 Hz, 1.6 Hz, H6’),

8.02 (1 H, s, H1’’), 8.10 (1 H, d, J =15.4 Hz, H3), 12.87 (1 H, s, 2’-OH).

Figure 9. Structure for chalcone 9.

For this compound, the 1H NMR also shows the same characteristic signals present

in the previous chalcone. The signal for the 2’-OH proton in this case slightly more

unprotected being present at δ 12.87 ppm, and the signals for the α and β hydrogens with a

very distinctive coupling constant of 15.4 Hz. All the other signals share no similarities with

the signals from chalcone 6, but some are also easily identified such as hydrogen 1’’, a broad

singlet at δ 8.02 ppm, presenting no coupling with other hydrogens. It is also to note signals

such as the one at δ 6.98 ppm, a doublet of doublet of doublets, presenting two coupling

constants of 8.2 Hz and 7.2 Hz, both characteristic of ortho coupling and an additional

coupling constant of 1.2 Hz, characteristic of meta coupling. This signal at δ 6.98 ppm most

likely couples with the signal at δ 7.99 ppm, a doublet of doublets that also presents a

coupling constant of 8.2 Hz, these two signals were attributed to hydrogens 5’ and 6’

respectively. Once again, almost all of the remaining signals are difficult to distinguish due

to their overlap, but it is possible to confirm that the obtained compound was the chalcone

depicted in figure 9.

2.2.3. Chalcone 10 (1-(2-hydroxynaphthalen-1-yl)-3-(naphthalen-2-yl)prop-2-

en-1-one).

1H NMR (300.13 MHz, CDCl3): δ 7.21 (1 H, d, J = 9.0 Hz, H8’), 7.43 (1 H, ddd, H4’),

7.52-7.60 (3 H, m, H5’, H5’’, H8’’), 7.62 (1 H, d, J = 15.6 Hz, H2 or H3), 7.74 (1 H, dd, J

= 8.6 Hz, 1.6 Hz, H6’), 7.82-7.96 (5 H, m, H7’, H3’’, H4’’, H6’’, H7’’) , 8.07 (1 H, s, H1’’),

8.10 (1 H, d, J =15.6 Hz, H2 or H3), 8.12 (1 H, d, J = 8.4 Hz, H3’), 12.60 (1 H, s, 1’-OH).


36

Figure 10. Proposed structure for chalcone 10.

This last compound is extremely interesting since it combines two naphthyl moieties

and presents similarities at the level of the 1H NMR spectra (Figure 11 and Figure 12), with

Figure 11. 1H NMR spectrum of chalcone 10.

Figure 12. Aromatic region of the spectrum of chalone 10 (7.15-8.15 ppm).

the previous chalcones. In this chalcone the coupling constant between the α and β hydrogens

is 15.6 Hz. The other characteristic signals are the 1’-OH proton that is present at δ 12.60


37

ppm and the singlet for hydrogen 1’’ at δ 8.10 ppm. Due to similarities with the previous

chalcones other signals were easily identified such as hydrogen 8’ at δ 7.21 ppm and

hydrogen 4’ at δ 7.43 ppm. Once again, almost all of the remaining signals are difficult to

distinguish due to their overlap, but it is possible to confirm that the obtained compound was

the chalcone depicted in figure 10.

2.3. Flavone derivatives synthesis

The synthesis of flavones was achieved by cyclodehydrogenation of the previously

synthesized chalcones, (Scheme 8). This process is usually of lower yields when compared

with many other techniques17, but it is extremely easy, relatively fast and doesn’t require the

use of potentially dangerous reagents, allowing to obtain the desired flavones easily and with

lower risk. Still the desired flavones were obtained with good yields (Table 2) without

unwanted side products, allowing for reuse of the unconverted chalcone on further reactions.

Initially the reactions were conducted at lower temperature (130 ºC) and reaction

time (30 minutes), and even though there was conversion from chalcone to flavone, it was

relatively low. Followed this, optimization of the reaction was conducted to maximize the

yields and due to the similarities of the chalcones, the ideal reaction conditions were

extremely similar. The first conclusion was that the amount of iodine used wasn’t the

limiting factor, after that several attempts at different temperatures and reaction times,

showed that longer reaction times (~2 hours) were the ideal and that the higher temperature

was also having an extremely important effect. Even though temperatures closer to the

boiling point of DMSO (189 ºC) showed better results, the difference wasn’t significant and

lower temperatures of ~165ºC were used, since not only this temperature is much easier to

reach but also to maintain. Since the chalcones could be reused due to the absence of by-

products in the effective yield of the reaction, if run twice or more, is considerably higher

with lower waste of the chalcone.

Table 2. Yields for the flavones derivatives synthesized.

Compound Yield (%)

Flavone 1 62

Flavone 2 77

Flavone 3 65


38

When analysing the yields obtained for both chalcones and flavones, it is possible to

see that the compounds, chalcone 9 and flavone 2, are the compounds obtained with the

highest yields. These compounds are also the only ones that do not contain an additional

aromatic ring on ring A, indicating that the presence of such a bulky substituent group may

have a big impact on synthesis. It is also to be noted that from the six compounds

synthesized, these two, chalcone 9 and flavone 2, were also the ones with the least solubility

issues that can also explain the yield differences. These solubility issues posed a great

problem during the biomimetic studiesfor the flavone derivatives 2 and 3. The use of these

compounds for the biomimetic catalysis tests was difficult, especially flavone 3 that was

almost completely insoluble in ethanol and had low solubility in acetonitrile, the solvent

most commonly used on biomimetic oxidation catalysis studies.

Scheme 8. Reagents and conditions used to synthesize the flavone derivative 1 (a), flavone

derivative 2 (b) and flavone derivative 3 (c).

a)

b)

c)


39

2.4. 1H NMR and 13C NMR characterization of the synthesized flavone derivatives

2.4.1. Flavone 1 (3-phenyl-1H-benzo[f]chromen-1-one)

1H NMR (300.13 MHz, CDCl3): δ 7.00 (1 H, s, H3), 7.52-7.59 (3 H, m, H3’, H4’, H5’),

7.60-7.68 (2 H, m, H10, H7), 7.77 (1 H, ddd, J = 8.6 Hz, 7.0 Hz, 1.5 Hz, H6), 7.92 (1 H, dd,

J = 8.0 Hz, 1.3 Hz, H8), 7.95-8.01 (2 H, m, H2’, H6’), 8.13 (1 H, d, J = 9.0 Hz, H9), 10.09

(1 H, J = 8.6 Hz, d, H5).

13C NMR (300.13 MHz, CDCl3): δ 110.49 (C3), 117.32 (C4a), 117.62 (C10), 126.12 (C2’,

C6’), 126.66 (C7), 127.21 (C5), 128.17 (C8), 129.10 (C3’, C5’), 129.27 (C6), 130.52 (C8a

or C4b), 130.64 (C8a or C4b), 131.42 (C4’), 135.52 (C9), 157.44 (C10a), 160.90 (C2),

180.37 (C4).

Figure 13. Structure of flavone 1.

On the 1H NMR spectra of this compound, it is possible to see significant changes

when compared with the spectra from the chalcone 6, the precursor of this compound. The

most important and relevant changes are the disappearance of the vinylic system and the

proton from the hydroxyl group. This compound also shows two signals easily identified,

the hydrogen 3, a singlet at δ 7.00 ppm, that is the most protected hydrogen of the structure

and hydrogen 5 at δ 10.09 ppm, the most unprotected hydrogen, due to the combined effect

of the aromatic ring but also the effect of the nearby carbonyl oxygen. On the 13C NMR

some signals are also easily identified, especially the ones from the unprotected carbons, the

ones directly connected with oxygen atoms, like carbon 4 at δ 180.37 ppm, carbon 2 δ at

160.90 ppm and carbon 10a at δ 157.44 ppm. The other signal also easily identified is the

most protected carbon, the carbon 3 at δ 110.49 ppm. Most of the remaining signals can’t be

so easily assigned without the help of the two-dimensional NMR spectra. But, with the help


40

of these spectra all signals were assigned and allowed for the confirmation of the structure

depicted in figure 13.

2.4.2. Flavone 2 (2-(Naphthalen-2-yl)-4H-chromen-4-one)

1H NMR (300.13 MHz, CDCl3): δ 6.96 (1 H, s, H3), 7.44 (1 H, ddd, J = 8.0 Hz, 7.1 Hz, 1.2

Hz, H6), 7.55-7.66 (3 H, m, H8, H5’, H8’), 7.73 (1 H, ddd, J = 8.6 Hz, 7.1 Hz, 1.7 Hz, H7),

7.87-8.01 (4 H, m, H4’, H3’, H6’, H7’), 8.26 (1 H, dd, J = 8.0 Hz, 1.2 Hz, H5), 8.49 (1 H, s,

H1’).

13C NMR (300.13 MHz, CDCl3): δ 107.92 (C3), 118.15 (C8), 122.54 (C3’), 124.03 (C4a),

125.28 (C6), 125.77 (C5), 126.96 (C5’), 127.10 (C1’), 127.85 (C4’), 128.05 (C8’), 128.93

C2’), 128.97 (C7’), 129.08 (C6’), 132.92 (C8’a), 133.84 (C7), 134.69 (C4’a), 156.37 (C8a),

163.38 (C2), 178.50 (C4).


Once again, on the 1H NMR spectra of this compound, shows significant changes

when compared to the spectra from the chalcone 9, the precursor of this compound. The

most important and relevant changes are also the disappearance of the vinylic system and

the proton from the hydroxyl group. This compound also shows two signals easily identified,

the hydrogen 3, a singlet at δ 6.96 ppm, that is the most protected hydrogen of the structure

and hydrogen 1’ also a singlet at δ 8.49 ppm. On the 13C NMR similar to the previous

compound, the carbons directly connected with oxygen atoms are easily identified, with

carbon 4 at δ 178.50 ppm, carbon 2 at δ 163.38 ppm and carbon 8a at δ 156.37 ppm. The

last easily identified signal is carbon 3 at δ 107.93 ppm. Most of the remaining signals can

only be assigned with the help of the two-dimensional NMR spectra, that allowed for the

assignment of all signals and the confirmation of the structure depicted in figure 14.


41

2.4.3. Flavone 3 (3-(naphthalen-2-yl)-1H-benzo[f]chromen-1-one)

1H NMR (300.13 MHz, CDCl3): δ 7.12 (1 H, s, H3), 7.55-7.67 (3 H, m, H7, H5’, H8’), 7.70

(1 H, d, J = 9.0 Hz, H10), 7.77 (1 H, ddd, J = 8.6 Hz, 7.0 Hz, 1.5 Hz, H6), 7.87-7.94 (2 H,

m, H8, H4’), 7.95-8.01 (3 H, m, H3’, H6’, H7’) , 8.14 (1 H, d, J = 9.0 Hz, H9), 8.51 (1 H, s,

H1’), 10.09 (1 H, d, J = 8.6 Hz, H5).

13C NMR (300.13 MHz, CDCl3): δ 110.73 (C3), 117.30 (C4a), 117.65 (C10), 122.42 (C3’),

126.60 (C1’ or C7), 126.66 (C1’ or C7), 127.09 (C5 or C5’), 127.22 (C5 or C5’), 127.87

(C4’), 127.94 (C8’), 128.19 (C8), 128.53 (C2’), 128.99 (C7’), 129.02 (C6’), 129.27 (C6),

130.52 (C8a or C4b), 130.65 (C8a or C4b), 132.97 (C8’a), 134.59 (C4’a), 135.56 (C9),

157.51 (C10a), 160.85 (C2), 180.31 (C4).


Exactly like the 1H NMR spectra from the previous flavone derivatives, also the one

from this compound, shows significant changes when compared the precursor of this

compound, with the disappearance of the vinylic system and the proton from the hydroxyl

group. This compound also shows four signals easily identified, the hydrogen 3, a singlet at

δ 7.12 ppm, the hydrogen 1’ also a singlet at δ 8.51 ppm and hydrogens 9 and 10 at δ 8.14

and 7.70 ppm respectively, two hydrogens that couple with each other with a coupling

constant of 9.0 Hz. On the 13C NMR similar to the previous compounds, the carbons directly

connected with oxygen atoms are easily identified, with carbon 4 at δ 180.31 ppm, carbon 2

at δ 160.85 ppm and carbon 10a at δ 157.51 ppm. The other signal easily identified is carbon

3 at δ 107.93 ppm. Once again most of the remaining signals can only be assigned with the

help of the two-dimensional NMR spectra, that allowed for the assignment of all signals and

the confirmation of the structure depicted in figure 15.


42

By analysing all of the NMR spectra obtained (Examples from flavone 3 presented

on Figure 16, Figure 17, Figure 18 and Figure 19) it is possible to see incredible similarities

between the signals identified from the 3 flavones, especially the ones from carbons and

hydrogen from the heterocyclic ring C, such as H3, C4 or C2. The similarities of these signals

can be explained by the similar environment on which these atoms are surrounded, even

though the molecules have structures that have significant differences. All the flavones

present signals quite characteristic from this type of compound such as the H3 signal (6.96

– 7.12 ppm), a hydrogen with higher electronic shielding when compared with the aromatic

hydrogens that suffer the strong de-shielding effect caused by the anisotropic effect of the

aromatic ring. Other signals such as the ones from carbons C4, C8a or C10a, C2 and C3 can

also be easily identified because C3 corresponds to the carbon directly connected with the

H3 hydrogen, while the other three are carbons directly connected with an oxygen atom.

Being directly connected with an oxygen atom, an highly electronegative atom, reduces

these carbons electron shielding, significantly increasing their deprotection and carbons C4

(178.5-180.37 ppm), C8a or C10a (156.37-157.51 ppm), C2 (160.85-163.28 ppm) appear

much more to the far left of the spectra that the rest of the carbons being C4 the most

deprotected due to the fact it’s a carbon connected via a double bond with an oxygen atom.

All spectra signals referred, correspond to typically observed signals on these family of

compounds, being extremely helpful on the compound identification and signal assignment.

Figure 16. 1H NMR spectrum from flavone 3.


43

Figure 17. 13C NMR spectrum from flavone 3.

Figure 18. HSQC NMR spectrum from flavone 3.


44

Figure 19. HMBC NMR spectrum from flavone 3

2.5. Flavone biomimetic oxidation catalysis

After obtaining the desired flavones the next step involved the attempt to

biomimetically oxidate these compounds with the help of the Mn(TDCPP)Cl porphyrin. The

literature available shows that little to no information exists regarding flavones specifically,

but similar compounds, i.e. aromatic compounds, showed some interesting results, being

oxidized using this type of technique. With these in mind, all the three flavones were used

in preliminary tests.

Initially, the conditions used were the ones optimized for similar compounds. Being

used acetonitrile alone at 30 ºC. Unfortunately, all of the 3 flavones, especially flavone 3 and

flavone 1 presented low solubility. The next step involved increasing solvent volume while

also decreasing the amount of compound used maintaining all of the other conditions, but

the problem wasn’t solved. In order to solve the solubility problems without risking to

completely interfere with the stability of the porphyrin and therefore its activity, a mixture


45

of acetonitrile and dichloromethane (1 :1) was used, but due to the extremely low boiling

point of dichloromethane allied to the long reaction time (usually 5 to 6 hours) all of the next

attempts were conducted at room temperature. Even though the solubility problems for

flavone 1 were completely solved, even by slightly increasing the temperature (~25 ºC) and

increasing both porphyrin and oxidizer added, this flavone showed no appearance of an

oxidation product. Due to the lack of oxidation products even after several attempts with

different conditions and reaction times, this flavone was eventually discarded from further

tests and only flavone 2 and 3 remained. For both of these two flavones all the changes to

the conditions used resulted in the detection of at least one oxidation product for each of the

flavones, with the help of NMR spectra from the starting flavone and the whole reaction at

the end. The next step involved the attempt of obtaining these oxidation compound on their

pure state to allow for both NMR characterization but also further tests involving biological

activity. Unfortunately, only the oxidation product from flavone 2 was purified, with all the

attempts to purify the oxidation product from flavone 3 revealing to be futile, most likely

due to the low conversion rate from the original flavone to this oxidation product (< 10%).

Even though this compound wasn’t purified some of the detected signals indicated its

potential structure which cannot be proved but can be theorized supported with all the

information available.

2.5.1. Oxidation product identification and structure

To allow the identification of this compound (Figure 20), from now on called flavone

11, the same NMR techniques used for the identification of the base flavones was used to

identify this compound. The list of signals and their respective identification is presented

next (Table 3 and Table 4).

1H NMR (300.13 MHz, CDCl3): δ 3.79 (1 H, m, H8’), 3.84 (1 H, m, H5’) 4.08 (2 H, H6’,

H7’), 6.84 (1 H, s, H3), 7.45 (1 H, ddd, J = 8.0 Hz, 4.9 Hz, 1.3 Hz, H6), 7.60 (2 H, m, H4’,

H8), 7.73 (1 H, ddd, J = 7.2 Hz, 6.1 Hz, 1.7 Hz, H7), 7.92 (1 H, dd, J = 7.9 Hz, 1.9 Hz, H3’),

8.0 (1 H, d, J = 1.8 Hz, H1’), 8.24 (1 H, dd, J = 7.9 Hz, 1.6 Hz, H5).

13C NMR (300.13 MHz, CDCl3): δ 51.37 (C8’), 51.66 (C5’), 54.80 (C6’ or C7’), 55.00

(C6’ or C7’), 108.15 (C3), 118.13 (C8), 123.97 (C4a), 125.50 (C6), 125.81 (C5), 127.15

(C3’), 129.11 (C1’), 132.17 (C4’), 132.85 (C8’a or C2’), 132.95 (C8’a or C2’), 134.05 (C7),

135.23 (C4’a), 156.22 (C8a), 161.99 (C2), 178.33 (C4).


46

It is immediately possible to see both similarities with the base flavone 2, but also

significant changes. The signals from the atoms H3-H8, C4, C8a and C2 can easily be

identified since they suffered little to no alterations, indicating that their electronic

environment suffered almost no changes, being indicative of no alteration near these atoms.

On the other hand, it is possible to see that four carbons have signals of 51-55 ppm indicative

of loss of aromaticity. The chemical shifts from these carbons are also indicative of the

presence of a direct link from these carbons with an electronegative atom such as oxygen

and nitrogen. The lack of extra hydrogen atoms signals, indicates the inexistence of the

addition of -OH or NH2 groups. Since the chemical shifts of these carbons match the ones

expected for epoxides, that explain both the loss of aromaticity and chemical shifts present,

this molecule was identified as a compound with two epoxides connected to carbons C5’

and C6’ and carbons C7’ and C8’. Since there seems to be no other structural alterations on

the molecule, the structure proposed for this flavone was obtained (Figure 20).

Table 3. 1H NMR signals for flavone 2 (left) and the flavone 11 (right).

The literature shows several examples of the possibility to use porphyrins to add

oxygen atoms on the form of epoxides to compounds containing aromatic rings or simple

double bonds115,116. But so far there are only proof it is possible/easier with smaller and

simpler molecules, and this oxidized flavone is considerably larger, possibly opening the

way for the oxidation of many other compounds using this technique.

Atom Flavone 2 (δ, ppm) Flavone 11 (δ, ppm)

H3 6.96 (s) 6.84 (s)

H5 8.26 (dd) 8.24 (dd)

H6 7.44 (ddd) 7.45 (ddd)

H7 7.73 (ddd) 7.73 (ddd)

H8 7.55-7.67 (m) 7.59-7.62 (m)

H1’ 8.49 (s) 8.00 (d)

H3’ 7.87-8.01 (m) 7.92 (dd)

H4’ 7.87-8.01 (m) 7.59-7.62 (m)

H5’ 7.55-7.67 (m) 3.84 (m)

H6’ 7.87-8.01 (m) 4.08 (m)

H7’ 7.87-8.01 (m) 4.08 (m)

H8’ 7.55-7.67 (m) 3.79 (m)


47

Flavone 3 is one example of an even larger molecules that may be oxidized by this

method. Even though no actual purified oxidation product was obtained, and no

characterization was conducted, there is evidence that shows the existence of two epoxides

on this compound (Figure 21), but no extra signals such as from potential -OH groups were

detected leaving margin for the construction of a theoretical structure. Since flavone 1

showed no alterations with this method and flavone 2 and 3 share the naphthyl group

connected to the carbon C2, the only group that suffered alterations in the case of flavone 2,

one potential structure for this flavone derivative was obtained. In this structure all remains

unchanged except for the naphthyl group which, once again, seems to have present two

epoxides one on carbons C5’ and C6’ and the other on carbons C7’ and C8’ (Figure 22).

With the presence of the compound of oxidation of the flavone 2 alone, there is evidence

Table 4. 13C NMR signals for flavone 2 (left) and flavone 11 (right).

Atom Flavone 2 (δ, ppm) Flavone 11 (δ, ppm)

C2 163.4 162.0

C3 107.9 108.2

C4 178.5 178.3

C4a 124.0 124.0

C5 125.8 125.8

C6 125.3 125.5

C7 133.8 134.1

C8 118.2 118.1

C8a 156.4 156.2

C1’ 127.1 129.1

C2’ 128.9 132.9 or 133.0

C3’ 122.5 127.2

C4’ 127.9 132.2

C4’a 134.7 135.2

C5’ 127.0 51.7

C6’ 129.1 54.8

C7’ 129.0 54.8

C8’ 128.0 51.4

C8’a 132.9 132.9 or 133.0


48

that not only the oxidation of small compounds such as naphthalene is possible but, also to

oxidize compounds containing the naphthalene group (naphthyl group). Unfortunately, it

seems that the oxidation, of these compounds is dependent of the presence of this naphthyl

group which may be a clear problem for the implementation of this porphyrin on the

oxidation of more similar molecules. Since a limited number of compounds and conditions,

namely oxidizer, were tested, this is not by far, a big and solid evidence to discard this

porphyrin or similar ones from biomimetic oxidation catalysis. Although it is proof that even

this method has limitations, limitations that need to be studied and maybe, with changes to

procedure, oxidizer or even porphyrin, even these flavones can present more oxidation

products with potential uses due to the high biological value of flavones and its derivative

compounds.

Figure 20. Structure of the flavone 11, 2-(1a,1b,2a,6b-tetrahydronaphtho[1,2-b:3,4-

b']bis(oxirene)-4-yl)-4H-chromen-4-one.

Figure 21. NMR Spectrum from oxidation product from flavone 3 (top) and NMR spectrum

from flavone 11 (bottom).


49

Figure 22. Possible structure for the oxidation product of flavone 3 (2-(1a,1b,2a,6b-

tetrahydronaphtho[1,2-b:3,4-b']bis(oxirene)-4-yl)-1H-benzo[f]chromen-1-one).

2.6. Biological activity tests of flavone 11

After obtaining the oxidized compound, the natural following step is the study of the

biological activities that this compound may present. Since in the case of unsubstituted

flavones almost always if not always there is present little to no biological activity, or when

present it is much lower than the activity of substituted compounds, only flavone 11 was

tested. The biological activity tests were limited to antioxidant activity (DPPH and ABTS

tests) and anticholinesterase activity.

2.6.1. Antioxidant activity tests

Both DPPH and ABTS tests are spectrophotometric based and serve to evaluate the

radical scavenging capability of tested compounds indicated by the drop on absorbance

registered. These tests are widely used due to the fact that they allow for fast and easy

gathering of data. Even though both tests have clear limitations especially since both DPPH

and ABTS are synthetical radicals with structure that is not similar to naturally occurring

radicals, the data obtained is widely accepted and the results serve as an indication of the

radical scavenging capability of the tested compounds. For both tests, Trolox, (6-hydroxy-

2,5,7,8-tetramethylchroman-2-carboxylic acid) a synthetic analogous of vitamin E was used

as the standard. In order to compare the antioxidant activity of flavone 11 and the standard,

the IC50 for both compounds were calculated and used as a way to compare antioxidant

activity. The results for these tests are also shown on the form of graphics (Figure 23 and

Figure 24) to more easily demonstrate the dependence of antioxidant activity on the

concentration of compound.


50

2.6.1.1. DPPH test

In this test, flavone 11 presented an IC50 of 650.20 ± 3.10 μM, while Trolox showed

a IC50 of 26.06 ± 0.33 μM. These results show that flavone 11 has slight antioxidant activity

but only at relatively high concentrations, with an IC50 25 times higher than the one for the

standard compound Trolox.

Figure 23. Graphical presentation of the results for the DPPH test.

2.6.1.2. ABTS test

In the case of the ABTS test, the IC50 for flavone 11 was 385.74 ± 6.59 μM and the

IC50 for Trolox was 11.74 ± 0.13 μM. Once again, the IC50 for the tested compound is much

higher than the one from the standard compound Trolox, being almost 33 times higher,

indicating much lower antioxidant activity of the flavone 11 when compared with Trolox.

Flavone 11 has no hydroxyl groups present, and these groups showed to be extremely

important on the antioxidant activity of flavone derivative compounds. This fact allows to

explain the extremely lower antioxidant activity on both DPPH and ABTS tests when

compared with Trolox, a molecule often used as a basis to indicate the antioxidant activity

of other molecules. This also indicates that epoxides seem to not contribute for the

antioxidant activity of these compounds.

2.6.2. Anti-acetilcholinesterase activity test

In the case of the anti-acetilcholinesterase activity, no actual effects of the presence

of flavone 11 were seen and the enzyme, showed normal activity. Several studies conducted

demonstrated the potential anti-acetilcholinesterase activity of certain flavone derivatives,

but the presence of specific substituent groups such as hydroxyl or methoxy groups seems

to be extremely important.117,118

y = 0,0766x + 0,4139R² = 0,9992

0

25

50

75

100

0 100 200 300 400 500 600 700

Per

cen

tage

of

inh

ibit

ion

μM Flavone 11

y = 1,9445x - 1,3389R² = 0,9967

0

25

50

75

100

0 5 10 15 20 25 30 35 40 45

Per

cen

tage

of

inh

ibit

ion

μM Trolox


51

Figure 24. Graphical presentation of the results for the ABTS test.

Epoxides seem to have a big impact on the biological activity of certain compounds

such as lipids119, but little to no actual information about biological activity studies, including

anti-acetilcholinesterase activity tests, using flavones whose substituent groups comprise of

epoxide groups can be found. This fact may be explained by the fact that epoxide containing

compounds are usually potentially toxic and are rapidly metabolized by specific enzymes,

the epoxide hydrolases120. This process is usually quick and specific since epoxides are

common products of the activity of enzymes of the P450 complex, or normal metabolism

products and epoxides tend to have low half-life times in a living organism. In the place of

the epoxide, two hydroxyl groups are placed, which in the case of flavones, tend to have

much higher effect on biological activity, but the ring will not possess aromaticity, which

can have a tremendous negative effect on activity, especially activities that rely on the

electron delocalization aromatic compounds provide such as antioxidant activity.

y = 0,1276x + 1,5908R² = 0,9906

0

25

50

75

100

0 100 200 300 400 500 600 700

Per

cen

tage

of

inh

ibit

ion

μM Flavone 11

y = 4,3043x - 1,0302R² = 0,9966

0

25

50

75

100

0 2 4 6 8 10 12 14 16 18 20 22

Per

cen

tage

ofi

nh

ibit

ion

μM Trolox


52

53

Chapter III: Materials and methods


54


55

3. Materials and methods

All the reagents used in this project were used without previous purification, and the

solvents used were either of analytical purity or were purified by distillation when/if

necessary. Tetrahydrofuran (THF) was dried at reflux temperature using metallic sodium in

the presence of benzophenone as indicator. Once the solution became deep blue coloured,

the THF was distilled and used immediately whenever possible or stored in airtight

containers with nitrogen atmosphere. The reactions that were conducted were always

controlled by thin layer chromatography (TLC) using laminated plates covered by gel silica

60 F254 (Merck) incorporated indicator and with 0.25 mm of thickness using the best suited

elution solution. After elution, the plates were observed using an ultraviolet light at 254

and/or 366nm.

Purification of the reactions products was conducted using column chromatography

and/or thin layer chromatography. For the column purification gel silica 60 with 0.063-0.2

mm granule size from Merck was used. Thin layer chromatography was conducted using

glass plates (20 x 20 cm) covered in gel silica 60 GF254 from Merck with 0.5 mm in thickness

activated at 120ºC for 12 hours before usage.

The Nuclear Magnetic Resonance (NMR) spectra for 1H, 13C, HSQC and HMBC

were obtained using a Bruker Avance 300 spectrometer (300.13 MHz and 75.47 MHz for

1H NMR and 13C NMR respectively). All the spectra obtained were conducted at room

temperature using deuterated chloroform as solvent with tetramethylsilane (TMS) as internal

standard.

3.1. Chalcone derivatives synthesis

3.1.1. Synthesis and purification of chalcone 6

In a round bottom flask, 100µL of benzaldehyde and 136.0 mg of 2’-hydroxy-1’-

acetonaphtone were dissolved in approximately 20 mL of anhydrous THF under an inert

nitrogen atmosphere. Once the reagents were dissolved, 43.8 mg of sodium hydride were

added, and the mixture was left reacting at room temperature under magnetic stirring.

Several TLC’s of the reaction mixture were conducted to follow the progress of the reaction,

and after approximately 6 hours the reaction was stopped by pouring the reaction mixture in

ice. The solution pH was then adjusted to approximately 1 using an aqueous solution of HCl


56

20%. A liquid-liquid extraction was then performed using dichloromethane as the organic

solvent and anhydrous sodium sulphate (Na2SO4) was used to dry the organic phase. The

organic solvent was then evaporated in a rotary evaporator and the chalcone was then

purified in a first stage by column chromatography with a mixture of ethyl acetate/hexane (1

: 9), obtaining pure chalcone. The contaminated chalcone remaining after the column was

then purified by thin layer chromatography with a mixture of ethyl acetate/hexane (1 : 1),

and the final yield of the chalcone synthesis was 70%.


The process of synthesis and purification of this chalcone was the same process used

for the chalcone 6, but in this case, 100µL of 2’hydroxyacetophenone, 175.4 mg of 2-

naphthaldehyde and 49.6 mg of sodium hydride were used. The reaction was stopped after

5 hours and the final yield of this chalcone was 89%, considerably higher than the previous

chalcone.


The process of synthesis and purification of this chalcone was the same process used

for the previous chalcones, but in this case, 103.5 mg of 2’-hydroxy-1’-acetonaphtone and

113.2 mg of naphthaldehyde and 35.1 mg of sodium hydride were used. The reaction was

stopped after 5 hours and final yield of this chalcone was 72%.

3.2. Synthesis and purification of flavones

3.2.1. Synthesis and purification of flavone 1

In a round bottom flask, 103.7 mg of chalcone 6 were dissolved in the least amount

possible (~4 mL) of dimethyl sulfoxide (DMSO). A catalytic amount of I2 (~0.05

equivalents) was added and the mixture was placed in a sand bath and heated to 160-170ºC

under constant magnetic stirring. Once the mixture reached the desired temperature, the

reaction was followed by TLC, and 2 hours after the reaction was interrupted by pouring the

solution in ice. Exactly like for the chalcone, a liquid-liquid extraction was performed using

dichloromethane as the organic solvent and anhydrous sodium sulphate (Na2SO4) to dry the

organic phase and the organic solvent was evaporated in a rotary evaporator. The resulting


57

solid was dissolves in the smallest amount possible of ethanol under heating, and the flavone

was left crystallizing for 72 hours. After that period of time the resulting crystals were

filtrated, and the remaining flavone dissolved was purified by thin layer chromatography

with a mixture of ethyl acetate/hexane (1 : 1), and the final yield of the flavone was 62%.


The synthesis and purification of flavone 2 was the same as the flavone 1 with a few

changes. In this case, 102.3 mg of chalcone 9 were dissolved in approximately 3 mL of

DMSO with a catalytic amount of I2 (~0.05 equivalents) and the mixture placed in a sand

bath and heated to 160-170ºC under constant magnetic stirring. Once again, the reaction was

followed by TLC and after 1 and a half hours the reaction was stopped by pouring the

mixture in ice. A liquid-liquid extraction was performed, and the solvent evaporated in a

rotary evaporator. The resulting solid was dissolves in the smallest amount possible of

ethanol under heating, and the flavone was left crystalizing for 48-72 hours. After that period

of time the resulting crystals were filtrated, and the remaining flavone dissolved was purified

by thin layer chromatography with a mixture of ethyl acetate/hexane (1 : 1), and the final

yield of the flavone was 72%.


In the case of flavone 3, 130.5 mg of chalcone 10 were dissolved in approximately 5

mL of DMSO, with a catalytic amount of I2 (~0.05 equivalents) and the mixture placed in a

sand bath and heated to 160-170ºC under constant magnetic stirring. Once again, the reaction

was followed by TLC and stopped after 1 and a half hours by pouring the mixture in ice. A

liquid-liquid extraction was performed, and the solvent evaporated in a rotary evaporator.

To the resulting solid, ethanol was added, and the precipitated residue was filtrated under

reduced pressure and posteriorly identified as the desired flavone. The remaining solution

was purified by thin layer chromatography with a mixture of ethyl acetate/hexane (1 : 1),

and the final yield of the flavone was 65%.

3.3. Biomimetic catalysis tests

The biomimetic catalysis was conducted using a solution of the porphyrin

Mn(TDCPP)Cl prepared in acetonitrile specifically for these tests (1.05 *10-3 M), a diluted


58

solution of commercial H2O2 30% as the oxidizer and ammonium acetate as co-catalyst. The

conditions used were slightly adapted from115 with changes to the substrate amount (flavone)

and run times.

3.3.1. Biomimetic catalysis of flavone 1

In a test tube, 25.3 mg of flavone 1 were dissolved in 4 mL of

acetonitrile/dichloromethane (1 : 1) under magnetic stirring. After the flavone was properly

dissolved, 250 µL of the porphyrin solution and approximately 5 mg of the co-catalyst were

added and left for 5 minutes under magnetic stirring. 20 µL of H2O2 30%/acetonitrile (1:5)

were then added every 15 minutes for 6 hours. After the 6 hours, 25 mL of distilled water

were added, and a liquid-liquid extraction was performed using dichloromethane as the

organic solvent. The organic phase was then dried using anhydrous sodium sulphate and the

solvent evaporated in a rotary evaporator. For this flavone no oxidation product was

detected, with the only compound present being the starting flavone.


In the case of flavone 2, due the much higher solubility in the chosen mixture of

solvents 40.8 mg of flavone 2 were dissolved in 4 mL of acetonitrile/dichloromethane (1 :

1) under magnetic stirring. After the flavone was properly dissolved, 490 µL of the porphyrin

solution and approximately 20 mg of the co-catalyst were added and left for a few minutes

under magnetic stirring. 20 µL of H2O2 30%/acetonitrile (2 : 5) were then added 5 minutes

after the addition of the co-catalyst, and every 15 minutes for 5 hours. After the 5 hours, 35

mL of distilled water were added, and a liquid-liquid extraction was performed using

dichloromethane as the organic solvent. The organic phase was then dried using anhydrous

sodium sulphate and the solvent evaporated in a rotary evaporator. Following the

evaporation, the single product detected was purified by both column chromatography and

thin layer chromatography, and a purified compound was obtained.


Due to the low solubility, 10.3 mg of flavone 3 were dissolved in 4 mL of

acetonitrile/dichloromethane (1 : 1) under magnetic stirring. After the flavone was properly

dissolved, 120 µL of the porphyrin solution and approximately 10 mg of the co-catalyst were


59

added and left for a few minutes under magnetic stirring. 20 µL of H2O2 30%/acetonitrile (1

: 9) were then added 5 minutes after the addition of the co-catalyst, and every 15 minutes for

6 hours. After the 6 hours, 20 mL of distilled water were added, and a liquid-liquid extraction

was performed using dichloromethane as the organic solvent. The organic phase was then

dried using anhydrous sodium sulphate and the solvent evaporated in a rotary evaporator.

Following the evaporation several attempts to purify the single product of the oxidation

reaction, that was detected in NMR spectra were conducted but, potentially due to the low

conversion rate, it was not possible to obtain that product for characterization and for further

studies.

3.4. Biological activity tests

3.4.1. DPPH test

For the DPPH test a solution of DPPH (0.08 mg/mL) was prepared, stored in the cold and

used in the next day. In addition, the solutions for the compound to be tested, standard

(Trolox) and negative control were prepared. Due to the low solubility of the compound, this

was prepared in a concentration of 1 mg/mL in methanol/DMSO (2 : 1). The standard was

prepared in a concentration of 0.5 mg/mL in methanol and the negative control consisted in

a mixture of 50 µL DMSO and 950 µL.

A round bottom 96 wells microplate was prepared by adding 200 µL of methanol to

all wells from column 1 (blank), and 100 µL to all wells from column 3 to 8. To the wells of

column 2, 200 µL of either compound, standard or negative control were added in triplicate.

From each well from column 2, 100 µL were taken and added to the well, in the same row,

on column 3 and an up and down movement was performed to homogenize the solution in

the well. This process is then repeated for column 3 to 4, 4 to 5 and so on until reaching the

row 8 were the 100 µL taken from this well were discarded. After this process that creates a

concentration slope with each well in a row having half the concentration of the previous

well, 100 µL of the DPPH solution are then added to all wells from column 2 to 8. The plate

is then left in the dark at room temperature for 30 minutes and is read in a microplate reader

at 515 nm.


60

3.4.2. ABTS test

The ABTS test is similar to the DPPH test, and the solutions used for the compound

to be tested, standard (Trolox) and negative control had the same concentration and were

prepared in the same solvents. The solution of ABTS was obtained by preparing a solution

of 7 mM of ABTS and 2.45 mM of potassium persulphate in methanol. The solution was

then stored in the dark and left overnight for 15 hours to be used in the next day. After the

period necessary for the formation of the ABTs radicals, the absorbance of this solution was

then adjusted to be approximately 0.700 at the final concentration present on the microplate.

Round bottom 96 wells microplates were prepared by adding 200 µL of methanol to all wells

from column 1 (blank), and 100 µL to all wells from column 3 to 8. To the wells of column

2, 200 µL of either compound, standard or negative control were added in triplicate. From

each well from column 2, 100 µL were taken and added to the well, in the same row, on

column 3 and an up and down movement was performed to homogenize the solution in the

well. This process is then repeated for column 3 to 4, 4 to 5 and so on until reaching the row

8 were the 100 µL taken from this well were discarded. Finally, 100 µL of the ABTS solution

with the desired absorbance were then added to all wells from column 2 to 8. The plate is

then left in the dark at room temperature for 5 minutes and is read in a microplate reader at

730 nm.

3.5. Acetylcholinesterase test

For the anti-acetylcholinesterase activity test, acetylcholinesterase (AChE, E.C.

3.1.1.7, from Electrophorus electricus), 5,5-dithiobis-(2-nitrobenzoic acid) (DTNB) and

acetylthiocholine iodine (ATChI) were purchased from Sigma Aldrich. The tested

compound presented solubility issues in the phosphate buffer, so this compound was

prepared in methanol, and the amount of aqueous buffer solution was minimized, without

risking the activity of the enzyme. In every well from the microplate, 50 µL of the tested

compound was added, followed by 100 µL of a 0.025U/mL of AChE and the plate was left

incubating at 37ºC for 5 minutes. After the 5 minutes had passed, 50 μL of a 2.5 mM solution

of ATChI and 50 μL of a 0.5 mM solution of DTNB were added in sequence. The microplate

was then placed in a microplate reader and the absorbance was read at 415 nm.

61

Chapter IV: Conclusion and future perspectives

Chapter IV: Conclusions and future perspectives

63

4. Conclusions and future perspectives

Flavones and their derivatives are recognised bioactive compounds, consequently

efforts on the synthesis of new ones are welcomed. However, understand their metabolism

in living systems is also an important issue. In this context, the use of a synthetic porphyrin

in the biomimetic oxidation of naphthylflavones was studied.

First the synthesis of naphthylchalcones 6, 9 and 10 was studied and after its

optimization, the desired derivatives were obtained in very good yields. The following step,

these chalcones oxidative cyclization using iodine as catalyst, was also studied and

optimized in order to obtain, in good yields, the naphthylflavones 1, 2 and 3.

Afterwards the obtained naphthylflavones were used in a biomimetic study using

hydrogen peroxide and Mn(TDCPP)Cl (Figure 6). Although several conditions were tested,

the naphthylflavone 1 was not oxidised. In the cases of naphthylflavones 2 and 3, a single

product of their oxidation was obtained and characterized. Moreover, in the case of

naphthylflavone 2, the product of oxidation, the 2-(1a,1b,2a,6b-tetrahydronaphtho[1,2-

b:3,4-b']bis(oxirene)-4-yl)-4H-chromen-4-one 11, was obtained in sufficient amount that its

antioxidant and anti-cholinesterase activity was evaluated.

Even though the results are not exactly the best, it is worth emphasizing that this

work not only allowed the synthesis of a few new compounds such as flavones 3 and 11, but

also demonstrates the potential of porphyrins in biomimetic oxidations. Naturally, the scope

of the methodology should be evaluated and also the study should be extended to other

porphyrin derivatives. Who knows! Another porphyrin may be able to oxidise the

naphthylflavone 1.

Other future directions could be the optimization of the biomimetic oxidative

methodology and/or its application with other flavones, aiming to obtain new derivatives

with interesting substituents. In fact, the epoxide rings can be opened in different conditions

and a wide range new substitution patterns can be obtained.

From this can be concluded that, due to the vast array of biological activities flavones

possess, obtaining new flavones or creating new ways to obtain already studied flavones can

be extremely important, and biomimetic oxidation catalysis may perfectly fit this role due to

the potential to obtain flavones hard to obtain from other procedures.

Chapter V: Bibliography

65



66


67

5. Bibliography

1. Samanta, A., Das, G. & Das, S. Roles of flavonoids in Plants. Int. J. Pharm. Sci. Technol.

6, 12–35 (2011).

2. Jiang, N., Doseff, A. I. & Grotewold, E. Flavones: From biosynthesis to health benefits.

Plants 5, 27 (2016).

3. Alvarez-Álvarez, R., Botas, A., Albillos, S., Rumbero, Á., Martin, J. & Liras, P.

Molecular genetics of naringenin biosynthesis, a typical plant secondary metabolite

produced by Streptomyces clavuligerus. Microb. Cell Factories 14, 178 (2015).

4. Falcone Ferreyra, M. L., Rius, S. P. & Casati, P. Flavonoids: biosynthesis, biological

functions, and biotechnological applications. Front. Plant Sci. 3, 222 (2012).

5. Chee, C. F., Buckle, M. J. C. & Rahman, N. A. An efficient one-pot synthesis of

flavones. Tetrahedron Lett. 52, 3120–3123 (2011).

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