Synthesis and antifouling Evalution of nature-inspired compounds

Francisca Isabel Alves da Silva Carvalhal

SYNTHESIS AND ANTIFOULING EVALUATION OF NATURE-INSPIRED COMPOUNDS

2nd _{Cycle Studies -Master Degree in Pharmaceutical Chemistry}

Faculdade de Farmácia, Universidade do Porto

Advisor: Professora Doutora Marta Correia da Silva Co-advisor: Professora Doutora Emília Sousa

ACCORDING TO THE LEGISLATION, THE REPRODUCTION OF ANY PART OF THIS DISSERTATION IS NOT AUTHORIZED.

DE ACOROD COM A LESGISLAÇÃO EM VIGOR, NÃO É PERMITIDA A REPRODUÇÃO DE QUALQUER PARTE DESTA DISSERTAÇÃO

________________________________________________ (Advisor: Professora Doutora Marta Correia da Silva)

________________________________________________ (Co-advisor: Professora Doutora Emília Sousa)

________________________________________________ (Master Course Director: Professora Doutora Madalena Pinto)

This work was developed in Laboratório de Química Orgânica e Farmacêutica, Departamento de Ciências Químicas, Faculdade de Farmácia da Universidade do Porto. This research was developed under the project PTDC/AAG-TEC/0739/2014 supported through national funds provided by Fundação da Ciência e Tecnologia (FCT/MCTES, PIDDAC) and European Regional Development Fund (ERDF) through the COMPETE – Programa Operacional Factores de Competitividade (POFC) programme (POCI-01-0145-FEDER-016793) and– Reforçar a Investigação, o Desenvolvimento Tecnológico e a Inovação (RIDTI, Project 9471) and INNOVMAR - Innovation and Sustainability in the Management and Exploitation of Marine Resources, reference NORTE-01-0145-FEDER-000035, Research Line NOVELMAR.

COMMUNICATIONS

The results presented in this dissertation are part of the following scientific communications:

Oral Communications in Conferences

Carvalhal, F. Marine Sulfated Steroids. Summer curse – Pharmaceutical Drugs: How to obtain, how do they work and why (a perspective in pharmaceutical medicinal chemistry) Porto, Portugal, July 19-21, 2016.

Poster Communications in Conferences

Internationals:

Carvalhal, F.; Sousa, E.; Neves, A. R.; Correia-da-Silva, M.; Pinto, M. Synthesis of 3,4-dihydroxyxanthone derivatives: hit optimization. 2ª Edição da Escola de Inverno de Farmácia, Porto, Portugal, January 19-27, 2017, P.21.

Nationals:

Carvalhal, F.; Sousa, E.; Neves, A. R.; Correia-da-Silva, M.; Pinto, M. Synthesis and structural elucidation of 3,4-dihydroxyxanthone derivatives. 10th _{Meeting of Young}

Researchers of University of Porto (IJUP17), Porto, Portugal, February 8-10, 2017.

Abstract in Conference Proceeding

Internationals:

Carvalhal, F.; Sousa, E.; Neves, A. R.; Correia-da-Silva, M.; Pinto, M. Synthesis of 3,4-dihydroxyxanthone derivatives: hit optimization. 2ª Edição da Escola de Inverno de Farmácia, P.21 pp.72-73.

Nationals:

Carvalhal, F.; Sousa, E.; Neves, A. R.; Correia-da-Silva, M.; Pinto, M. Synthesis and structural elucidation of 3,4-dihydroxyxanthone derivatives. 10th _{Meeting of Young}

Paper in preparation:

Carvalhal, F., Correia-da-Silva, M., Sousa, E., Pinto, M. Biological activities of marine sulfated steroids, to be submitted

INDEX

FIGURES INDEX ... xiii

TABLES INDEX ... xv

SCHEMES INDEX... xvii

ACKNOWLEDGEMENTS ... xix ABSTRACT ... xxi RESUMO ... xxiii ABBREVIATIONS ... xxv OUTLINE OF DISSERTATION...xxvii CHAPTER 1 - INTRODUCTION ... 1 1.1. Marine Biofouling ... 3

1.2. Biofouling outcomes for maritime industry ... 3

1.3. Toxicity of antifoulants in use ... 4

1.4. Nature as an alternative ... 4

1.5. Nature-inspired synthetic compounds ... 6

1.5.1. Structure-diverse sulfated polyphenols ... 6

1.5.2. Oxygenated xanthones ... 7

1.6. Strategy and aims ... 8

CHAPTER 2 - RESULTS AND DISCUSSION ... 11

2.1. SYNTHESIS ... 13

2.1.1. Total synthesis of 3,4-dihydroxyxanthone (5) ... 13

2.1.1.1. Synthesis of benzophenone intermediate 3 ... 14

2.1.1.2. Cyclization of benzophenone intermediate 3 ... 15

2.1.1.3 Demethylation of 3,4-dimethoxyxanthone (4) ... 17

2.1.2. Synthesis of 3,4-di(carboxymethoxy)xanthone (7) ... 18

2.1.2.1. Nucleophilic substitution of 3,4-dihydroxyxanthone (5) with methyl bromoacetate ... 18

2.1.2.2. Alkaline hydrolysis of 3,4-di(2-methoxy-2-oxoethoxy) xanthone (6) ... 19

2.1.3. Synthesis of 3(2,3,4,6-tetra-sulfate-O-β-D-glucopyranosyl)xanthone (10)... 20

2.1.3.1. Glycosylation of 3,4-dihydroxyxanthone (5) ... 20

2.1.3.2. Desacetylation of 3,4-di(2,3,4,6-tetra-O-acetyl-O-β-D-glucopyranosyl)xanthone (8) ... 21

2.1.4. Sulfation of Stigmasterol (11) ... 23

2.2 STRUCTURE ELUCIDATION ... 24

2.2.1. 3,4-Dihydroxyxanthone (5) ... 24

2.2.2. 3, 4-Dihydroxyxanthone derivatives: 6 and 7 ... 25

2.3.3. 3, 4-Dihydroxyxanthone derivatives: 8, 9, and 10 ... 29

2.2.4. Stigmasterol derivative 12 ... 36

2.3. SEAWATER SOLUBILITY ... 38

2.4. ANTIFOULING ACTIVITY ... 40

CHAPTER 3 - MATERIAL AND METHODS ... 43

3.1. GENERAL MATERIALS AND METHODS ... 45

3.2. SYNTHESIS ... 46 3.2.1. Synthesis of 3, 4-dihydroxyxanthone (5) ... 46 3.2.1.1. Synthesis of 2-hydroxy-2’,3,4-trimethoxybenzophenone (3) ... 46 3.2.1.2. Synthesis of 3,4-diemthyl-9H-xanthen-9-one (4) ... 46 3.2.1.3. Synthesis of 3, 4-dihydroxy-9H-xanth-9-one (5) ... 47 3.2.2. Synthesis of 3,4-di(2-methoxy-2-oxoethoxy)-9H-xanth-9-one (6) ... 47 3.2.3. Synthesis of 3,4-di(carboxymethoxy)xanthone (7) ... 48

3.2.4. Synthesis of 3,4-di(2,3,4,6-tetra-O-acetyl-O- β-D-glucopyranosyl)-9H-xanth-9-one (8) ... 48

3.2.5. Synthesis of 3,4-di(O-β-D-glucopyranosyl)-9H-xanth-9-one (9) ... 49

3.2.6. Synthesis of 3,4-di(2,3,4,6-tetra-sulfate-O-β-D-glucopyranosyl)-9H-xanth-9-one (10) ... 50

3.2.6. Synthesis of Stigmasta-5,22-dien-3-β-sulfate (12) ... 51

3.3. SEAWATER SOLUBILITY ... 51

3.3.1. 3,4-Di(carboxymethoxy)xanthone (7) ... 51

3.3.2. Xanthone 3-O-β-D-glucopyranosil persulfate (10) ... 52

3.4. ANTIFOULING ACTIVITY ... 52

CHAPTER 4 - CONCLUSIONS ... 53

CHAPTER 5 - REFERENCES ... 57

CHAPTER 6 - APPENDICES ... 63

APPENDIX I – Paper in preparation ... 65

APPENDIX II – 1_{H and} 13_{C spectra of compound 5 (DMSO-d} 6). ... 91

APPENDIX III – 1_{H and} 13_{C spectra of compound 6 (CDCl} 3). ... 93

APPENDIX IV – 1_{H and} 13_{C spectra of compound 7 (DMSO-d} 6). ... 95

APPENDIX V – 1_{H and} 13_{C spectra of compound 8 (CDCl} 3). ... 97

APPENDIX VI – 1_{H and} 13_{C spectra of compound 9 (DMSO-d}

6). ... 99

APPENDIX VII – 1_{H and} 13_{C spectra of compound 10 (DMSO-d}

FIGURES INDEX

Figure 1 – Marine sulfated steroids with AF effects. ... 5 Figure 2 – Penicillixanthone. ... 6 Figure 3 – Three hit sulfated polyphenols. Gallic acid persulfate, AGS; rutin 2’’, 2’’’,3’,3’’,3’’’,4’,4’’,4’’’,7-nonasulfate, RS; 3,6-bis(β-D-glucopyranosyl)xanthone persulfate, XGS. ... 7 Figure 4 – 3,4-Dihydroxyxanthone, DOHX. ... 7 Figure 5 – Chromatogram obtained from 3,4-dihydroxyxanthone (5) at 236 nm, in a concentration of 50 M. Mobile phase water/ acetonitrile/ trifluoroacetic acid (80:20:0.1). ... 18 Figure 6 – Chromatogram obtained from 3,4-di(carboxymethoxy)xanthone (7) at 239 nm,

in a concentration of 0.05 mg/ml. Mobile phase water/ acetonitrile/ trifluoroacetic acid (80:20:0.1). ... 20 Figure 7 – Chromatogram obtained from 3-O-β-D-glucopyranosil persulfate (10) at 250 nm, in a concentration of 1 mg/ml. Mobile phase tetrabutylammonium bromide / acetonitrile (38:62). ... 23 Figure 8 – 3, 4-Dihydroxyxanthone (5). ... 24 Figure 9 – 1_{H and} 13_{C NMR data of 3, 4-dihydroxyxanthone (5). ... 25}

Figure 10 – 3,4-Dihydroxyxanthone derivatives: 3,4-di(2-methoxy-2-oxoethoxy)xanthone (6) and 3,4-di(carboxymethoxy)xanthone (7). ... 25 Figure 11 – 1_{H and} 13_{C NMR data of 3,4-di(2-methoxy-2-oxoethoxy)xanthone (6). ... 26}

Figure 12 – Main connectivities found in HMBC for compound 6. ... 28 Figure 14 – Connectivities found in HMBC for compound 7 that evidence the position of the substituents in xanthone. ... 29 Figure 15 – 3,4-Dihydroxyxanthone derivatives:

3,4-di(2,3,4,6-tetra-O-acetyl-O-β-D-glucopyranosyl)xanthone (8), 3,4-di(O-β-D-glucopyranosyl)xanthone (9), and 3(2,3,4,6-tetra-sulfate-O-β-D-glucopyranosyl)xanthone (10)... 30 Figure 16 – 1_{H and} 13_{C NMR data of 3,4-di(2,3,4,6-tetra-O-acetyl-O-β-D-glucopyranosyl)}

xanthone (8). ... 31 Figure 17 – Main connectivities found in HMBC for compound 8. ... 33 Figure 18 – 1_{H and} 13_{C NMR data of 3,4-di(O-β-D-glucopyranosyl)xanthone (9). ... 34}

Figure 19 – Connectivities found in HMBC for compound 9 that evidence the position of the substituents in xanthone. ... 35 Figure 20 – 1_{H and} 13_{C NMR data of 3(2,3,4,6-tetra-sulfate-O-β-D-glucopyranosyl)}

Figure 21 – Connectivities found in HMBC for compound 10 that evidence the position of the sulfated substituent in xanthone. ... 36 Figure 22 – Stigmasta-5,22-dien-3- β –ol (11) and the derivative stigmasta-5,22-dien-3- β

–sulfate (12). ... 37 Figure 23 – Compounds 6-10 and 13-18 inspired in 3,4-dihydroxyxanthone (5) and 3,6-bis(β-D-glucopyranosyl)xanthone persulfate (19): 3,4-di(2-methoxy-2-oxoethoxy)xanthone (6), 3,4-di(carboxymethoxy)xanthone (7), 3,4-di(2,3,4,6-tetra-O-acetyl-O-β-D-glucopyranosyl)xanthone (8), 3,4-di(O-β-D-glucopyranosyl)xanthone (9), 3-(2,3,4,6-tetra-sulfate-O-β-D-glucopyranosyl)xanthone (10), 3,4-disulfatexanthone (13), 3,4-di(carboxymethoxy)xanthone (14), 6-methoxy-2-carboxyxanthone (15), 8-methoxy-2-carboxyxanthone (16), 6,8-dimehtyl-2-carboxyxanthone (17), 5,7-dimehtyl-2-6,8-dimehtyl-2-carboxyxanthone (18). ... 40 Figure 24 – Anti-settlement activity of compounds 5-10 and 13-19 towards plantigrades of the mussel M. galloprovincialis at a concentration of 50 M. ... 41

TABLES INDEX

Table 1 – IR data of 3,4-dihydroxyxanthone (5). ... 24 Table 2 – IR data of 3,4-dihydroxyxanthone derivatives: 6 and 7. ... 25 Table 3 – HSQC data of 3,4-di(2-methoxy-2-oxoethoxy)xanthone (6). ... 27 Table 4 – Main differences of proton and carbon chemical shifts of compounds 5, 6, and

7. ... 29 Table 5 – IR data of 3,4-dihydroxyxanthone derivatives: 8, 9, and 10. ... 30 Table 6 – HSQC data of 3,4-di(2,3,4,6-tetra-O-acetyl-O-β-D-glucopyranosyl) xanthone (8). ... 32 Table 7 – Main differences of aromatic proton and carbon chemical shifts of compounds 5, 8, 9, and 10. ... 36 Table 8 – IR data of stigmasterol (11) and stigmasterol sulfate (12). ... 37 Table 9 – Solubility values of dihydroxyxanthone (5) and two derivatives,

SCHEMES INDEX

Scheme 1 – Synthetic analogues planned in this dissertation. ... 8 Scheme 2 – Reactional route used to obtain 3,4-dihydroxyxanthone (5) ... 13 Scheme 3 – Synthesis of 2-hydroxy-2’,3,4-trimethoxybenzophenone (3) from trimethoxybenzene (1) and 2-methoxybenzoyl chloride (2). r.t. - room temperature. 14 Scheme 4 – Mechanism of 2-hydroxy-2’,3,4-trimethoxybenzophenone (3) from trimethoxybenzene (1) and 2-methoxybenzoyl chloride (2)... 15 Scheme 5 – Synthesis of 3,4-dimethyl-9H-xanthen-9-one (4). ... 16 Scheme 6 – Synthesis of 3,4-dimethyl-9H-xanthen-9-one (4) by base-catalysed

intramolecular nucleophilic aromatic substitution. ... 16 Scheme 7 – Synthesis of 3,4-dihydroxyxanthone (5). ... 17 Scheme 8 – Mechanism of the synthesis of 3, 4-dihydroxyxanthone (5), by demethylation of 3,4-dimethyl-9H-xanthen-9-one (4). ... 17 Scheme 9 – Synthesis of 3,4-di(2-methoxy-2-oxoethoxy)xanthone (6). r.t. - room temperature. ... 19 Scheme 10 – Synthesis of 3,4-di(carboxymethoxy) xanthone (7). r.t. - room temperature.

... 19 Scheme 11 – Synthesis of 3,4-di(2,3,4,6-tetra-O-acetyl-O-β-D-glucopyranosyl) xanthone (8). TBAB = tetrabutylammonium bromide; r.t. - room temperature. ... 21 Scheme 12 – Synthesis of 3,4-di(O-β-D-glucopyranosyl)xanthone (9). r.t. = room

temperature. ... 21 Scheme 13 – Synthesis of xanthone 3(2,3,4,6-tetra-sulfate-O-β-D-glucopyranosyl)

xanthone (10). SO3.TEA – triethylamine-sulfur trioxide adduct; DMA =

dimethylacetamide. ... 22 Scheme 14 – Synthesis of stigmasta-5,22-dien-3-β–sulfate (12). SO3.TEA –

ACKNOWLEDGEMENTS

Este trabalho não ficaria completo sem um especial agradecimento às pessoas que me acompanharam e que contribuíram de forma indispensável ao longo deste mestrado.

Em primeiro lugar quero agradecer à minha orientadora, Professora Doutora Marta Correia da Silva que, pela forma de encarar as oportunidades e os desafios profissionais, me cativaram e impulsionaram a querer mais. Obrigada por ter estado sempre presente para esclarecer todas as dúvidas e pela exigência ao longo do ano. Graças a si, sinto que evoluí muito a nível químico e laboratorial.

À Professora Doutora Emília de Sousa, minha coorientadora, agradeço toda a disponibilidade ao longo do ano, exigência e incentivos.

Agradeço à Professora Doutora Madalena Pinto pela transmissão de conhecimento e por todas as perguntas difíceis, que me motivaram a querer saber mais.

À Doutora Joana Reis de Almeida pela oportunidade que nos deu de realizarmos os ensaios de anti-incrustação no Centro Interdisciplinar de Investigação Marinha e Ambiental (CIIMAR) e por toda a amabilidade e simpatia com que nos recebeu.

À Mestre Ana Rita Neves obrigada por toda a ajuda no laboratório e pela boa disposição de todos os dias e à Cátia Vilas Boas obrigada pela assistência no HPLC.

À Dra Sara Cravo agradeço pela ajuda técnica ao longo deste ano e pela disponibilidade no esclarecimento de qualquer dúvida.

À Gisela Adriano e Liliana Mesquita, obrigada pela vossa simpatia e assistência sempre que necessária.

Aos meu colegas do Mestrado em Química Farmacêutica, aos estudantes de Projeto I do Mestrado Integrado em Ciências Farmacêuticas e aos bolseiros do Laboratório de Química Orgânica e Farmacêutica (LQOF), quero agradecer pelas brincadeiras e pelas amizades que tornaram este ano memorável.

Ao Diogo, obrigada por toda a dedicação. O teu apoio e amizade têm sido imprescindíveis para mim.

Aos meus pais e irmão um especial obrigado por me apoiarem incondicionalmente e por me fazerem ver sempre o lado positivo. Obrigada por me incentivarem todos os dias a fazer mais e melhor e por estarem presentes em todos os momentos. O vosso suporte e carinho é o que torna tudo possível.

ABSTRACT

Marine biofouling, resulting from colonization and subsequent conglomeration of micro and macroorganisms, can be very detrimental when formed in man-made structures that are located in marine waters. To prevent biofouling, a diversity of processes has been used through the years, nonetheless all with negative impact to the environment. In order to develop an antifouling strategy to prevent the development of marine biofouling without harming the surrounding environment, synthesis of nature inspired-derivatives is a strategy that has been explored in LQOF. In this direction, a series of xanthones and sulfated polyphenols were synthesized and screened for antifouling activity and some promising compounds were found. The aim of this work was to synthesize derivatives of the previous hit compounds in order to obtain some insights on structure-antifouling activity relationship. Nine compounds were synthesized, six of which were obtained and characterized for the first time (6-10 and 12), by IR, NMR, and HRMS techniques. Following the synthesis of the hit compound 3,4-dihydroxyxanthone (5) via benzophenone route, synthesis of the carboxylated derivative 7 was achieved in two steps, first by nucleophilic substitution using methyl bromoacetate, followed by alkaline hydrolysis. Sulfated xanthone glycoside 10 was obtained in three steps, through Michael reaction, following Zemplén deacetylation, and sulfation with sulfur trioxide adducts. A sulfated derivative of stigmasterol was also obtained using sulfur trioxide adducts.

Seawater solubility of carboxylated and sulfated derivatives (7 and 10) was tested and compared with the parent compound 5. Both derivatives 7 and 10 presented higher solubility in seawater than the parent compound 5.

Antifouling activity was evaluated for eleven xanthone derivatives (6-10 and 13-18) at 50 μM using an in vivo anti-settlement test with Mytilus galloprovincialis plantigrades larvae. The compounds with the most promising antifouling activity (6, 7, 14, 17, and 18) were selected for future investigations concerning antifouling effectiveness versus toxicity, and general ecotoxicity.

RESUMO

A anti-incrustação marinha, resultado da colonização e subsequente conglomeração de micro e macroorganismos, pode ser prejudicial quando formada em estruturas artificiais que estão localizadas no mar. Para prevenir a bioincrustação, uma diversidade de processos tem sido usado ao longo dos anos, não obstante todos com dano para a biota marinha. Com o intuito de diversificar estratégias de anti-incrustação para prevenir o desenvolvimento de bioincrustação sem prejudicar o ambiente circundante, a síntese de agentes anti-incrustantes inspirados na natureza é uma estratégia que tem sido explorada no LQOF. Nessa direção, uma série de xantonas e polifenóis sulfatados foram sintetizados e avaliados para atividade anti-incrustante e foram encontrados alguns compostos promissores.

O objetivo desde trabalho foi sintetizar derivados dos compostos hit, com o objetivo de obter alguma conclusão sobre relação de estrutura-atividade anti-incrustante. Nove compostos foram sintetizados, seis dos quais foram obtidos e caracterizados pela primeira vez (6-10 e 12), através de técnicas de IV, RMN e EM. Seguindo a síntese do composto hit 3,4-dihidroxixantona (5) via benzofenona, a síntese do derivado carboxilado 7 foi obtida em dois passos, primeiro por substituição nucleofílica usando bromoacetato de metilo, seguido de hidrólise alcalina. A xantona glicosilada sulfatada 10 foi obtida em três passos, através da reação de Michael, seguida pela desacetilação de Zemplén e sulfatação com aducto de trióxido de enxofre e trietilamina. Um derivado sulfatado do estigmasterol foi também obtido usando aducto de trióxido de enxofre e trietilamina.

A solubilidade em água do mar das xantonas carboxilada e sulfatada (7 e 10) foi testada e comparada com o composto de partida (5). Ambos os derivados 7 e 10 apresentaram maior solubilidade em água do mar que o composto de partida 5.

A atividade anti-incrustante foi avaliada para onze compostos (6-10 e 13-18) a 50 μM usando um teste de anti-incrustação in vivo com larvas Mytilus galloprovincialis plantigrades. Os compostos com atividade anti-incrustante mais promissora (6, 7, 14, 17 e 18) foram selecionados para mais investigações relativas à eficácia de anti-incrustação vs. toxicidade, e ecotoxicidade geral.

ABBREVIATIONS

AF - Antifouling activity AlCl3 - Aluminium chloride

Cs2CO3 - Cesium carbonate

d - duplet

dd - double duplet

ddd - double double duplet DMA - Dimethylacetamide DMF - Dimethylformamide

EC50 - Half maximal effective concentration

EM - Espetroscopia de massa HCl - Hydrochloric acid

HMBC - Heteronuclear multiple bond correlation


HPLC-DAD - High-performance liquid chromatography with Diode-array detection HRMS - High resolution mass spectrometry

HSQC - Heteronuclear single quantum correlation IC50 - Half maximal inhibitory concentration

IR - Infrared IV - Infravermelho

LC50 - Median lethal dose

LQOF - Laboratório de Química Orgânica e Farmacêutica m – multiplet

MeOH - Methanol

NaOH - Sodium hydroxide

NMR - Nuclear magnetic resonance RMN - Ressonância magnética nuclear s - singlet

SO3.TEA - Triethylamine-sulfur trioxide adduct

TBT - Tributyltin TEA - Triethylamine

TLC - Thin-layer chromatography UV-Vis - Ultraviolet-visible spectroscopy

OUTLINE OF DISSERTATION

The present dissertation is structured in 6 chapters:

CHAPTER 1 - INTRODUCTION

The first chapter includes a brief introduction of biofouling and its outcomes and presents some strategies used in LQOF to combat biofouling. The strategy and the aims of this work are presented at the end of this chapter.

CHAPTER 2 – RESULTS AND DISCUSSION

In this chapter, results obtained from the synthesis, structure elucidation, seawater solubility studies and antifouling activity evaluation will be presented and discussed.

CHAPTER 3 - MATERIAL AND METHODS

In this chapter, the experimental procedures for the synthesis, structure characterization, solubility studies and antifouling activity evaluation of the synthesized compounds are explained.

CHAPTER 4 – CONCLUSIONS

This chapter includes the main conclusions of the developed work.

CHAPTER 5 – REFERENCES

In this chapter, the references cited throughout the thesis are presented. The main bibliographic research motors were PubMed, Scopus and Google Scholar.

CHAPTER 6 – APPENDICIES

The last chapter contains a review paper that is in preparation concerning Marine Sulfated Steroids as also RMN spectra of the synthesized compounds.

CHAPTER 1 – INTRODUCTION AND AIMS

1.1. Marine Biofouling

Marine biofouling is the temporary or permanent adhesion of organisms on water submerged natural or man-made surfaces. Natural surfaces consist in non-living natural substrates (underwater rocks, reefs, hard ground) and active substrates (macroalgae and animals). Artificial surfaces include metal, plastic, concrete and wood.1 _Generally,

biofouling involves a sequence starting by the formation of a conditioning film composed by organic material (proteins and carbohydrates),2 _{followed by bacteria, cianobacteria,}

unicellular algae and protozoa colonization (microfouling) forming a biofilm.2-4 _{This biofilm}

layer facilitates the adhesion and colonization of macro-organisms, including macroalgal spores and invertebrate larvae (macrofouling), by providing biochemical stimulus for settlement and increasing their adherence to the substrate.5-6 _{It is important to be noted}

that biofouling is not necessarily a successional process, as the properties of the formed biofilms attract specific macrofouling species and repel others that may be attracted by other biofilms or may settle on a surface completely free of biofilm.6-8

1.2. Biofouling outcomes for maritime industry

Submerged or partially submerged artificial surfaces, such as ships, pipelines, oil platforms, bridge pillars and fishing devices are seriously affected by biological settlement.6, 9 _{Maritime industries spend billions annually to control biofouling, which}

constitute an economic burden for shipping industries.10-12 _{The adverse effects consist in}

high frictional resistance, due to generated roughness, which leads to speed loss.10 _The

overload of ship’s weight, clogging in cooling systems, reduction of propeller propulsion are some other problems associated with biofouling in ships that accelerate the higher fuel consumption and increase emissions of harmful gases.13-14 _{On the other hand, money and}

time is also spent in cleaning and maintenance.15 _{Biofouling in ships may also lead to the}

introduction of indigenous species where they were not naturally present, reducing the marine biodiversity and/ or spreading diseases.16 _{Thus biofouling control is essential for}

1.3. Toxicity of antifoulants in use

A variety of toxic materials, like copper, lead, arsenic and mercury, were used in coatings to control fouling in hull vessels.13 _{Copper was an effective and widely used}

biocide, until the discover of tributyltin (TBT), introduced as a biocide in marine antifouling (AF) paints in the early 1960s. TBT self-polishing copolymer coatings had an effective broad spectrum AF biocide and a long lifetime (5 years).17 _{Although, problems concerning}

non-target species and human health risks were rapidly noticed,18 _{which led the}

International Maritime Organization (IMO) to prohibit their application to ships, effective from 2008.19 _{This way, copper has once again become the predominant AF biocide, but}

its use is under several restrictions,20 _{as high concentrations of sediment copper have}

been detected, increasing the risk of bioavailable copper in the water, by resuspension of the sediment, and consequently the risk to aquatic life.21 _{Since copper is active against a}

small range of fouling organisms compared to TBT, most copper AF paints are fortified with ‘booster’ biocides to target micro- and macroalgae hull colonization.20 _{Among the}

most used boosters are Irgarol 1051 and diuron, herbicides that have negative effects on the growth rate of photosynthetic organisms,22-23 _{dichloro-octyl.isothiazolin (DCOIT, Sea}

nine 211),24 _{zinc and copper pyrithionine (Zinc and Copper Omadine}_{), zineb, among}

others.25 _{Additionally, new biocides have become of interest such as triphenylborane}

pyridine (TPBP, Japan),26 _{Econea (US),}27 _{Capsain (China) and medetomidine}

(Sweeden),28 _{considered as ‘emerging’ biocides, but few scientific data have been}

published about them.21, 29 _{Increase awareness of the impacts resulting from the use of AF}

paints, have increased the efforts towards developing environmental-friendly alternatives, which include foul-release coatings with silicone elastomers, waxes or silicone oils incorporated, or replacing environmentally persistent toxins with naturally derived, degradable repellent compounds used by marine organisms against epibionts.12, 30-32

1.4. Nature as an alternative

In marine environment is possible to observe species that are always heavily fouled by epibionts, while other species are free of epiphytic growth.33 _{It is believed that}

echinoderms have evolved an effective mechanism of protection from fouling, as few reports of fouled echinoderms are described.34 _{Natural alternatives including primary or}

secondary metabolites from a multitude of species have been found to inhibit the settlement of different biofouling species.6

Substances extracted from various marine organisms (including seaweeds, sessile marine invertebrates, and marine bacteria, fungi and cyanobacteria) have been investigated, particularly polyketide-related compounds.8, 35 _{In this work, the biological}

activities reported for marine sulfated steroids were extensively reviewed and are presented in Appendix I. A few sulfated steroids are known for their use in chemical protection against predators, and pathogenic or fouling microorganisms,36 _{like the}

carboxylated sterols sulfate (Figure 1), isolated from the sponge Toxadocia zumi, that showed antimicrobial activity against Bacillus subtilis and Staphylococcus aureus and AF activity against a possible fouling organisms, polychaete Salmacina tribanchiata.37 _Three

polyhydroxylated sterols sulfate (Figure 1), isolated from the starfish Luidia clathrata, showed significant activity inhibiting the attachment of barnacle larvae (Balanus amphitrite), while other compounds, also isolated from the same starfish, like the asterosaponins thornasteroside A, ophydianoside F, regularoside B and marthasteroside B (Figure 1) showed complete inhibition of larvae attachment at the same concentration.33

Recently, a xanthone, penicillixanthone (Figure 2), isolated from a marine-derived fungus, Aspergillus terreus SCSGAF0162, exhibited potent AF activity against larvae of the barnacle B. amphitrite, with EC50 values of 17.10.8 g/ mL.38 Nonetheless, little

information on structure–antifouling activity relationships and ultimately on the mechanism of action of polyketide-related compounds and xanthones in particular is available.

Figure 2 – Penicillixanthone.

1.5. Nature-inspired synthetic compounds

A variety of seaweeds and marine invertebrates have been explored as potential sources of AF agents, however a serious problem was encountered in the development of AF paints, that is the need for large amounts of supply.30, 39 _{To overcome this problem,}

nature-inspired synthesis have been explored.40

Based on the above and as a part of LQOF efforts to discover innovative antifoulants, two libraries of in-house synthetic compounds were investigated for their AF potential:

-Structure-diverse sulfated polyphenols - Oxygenated xanthones

1.5.1. Structure-diverse sulfated polyphenols

Three of thirteen sulfated polyphenols, investigated for their AF effects, showed highly significant differences (p ≤ 0.001, Dunnet’s test) in the percentage of settled larvae against the control for the two concentration tested in the first screening (500 and 200 M).40 _{The assessment of effectiveness versus toxicity of these promise compounds}

showed EC50 values ranging from 8.4 to 36.84 µg/mL, LC50 levels higher than 500 µM,

and LC50/EC50 ratios between 21.56 and 26.61 M. None of the selected compounds

showed significant ecotoxicity to A. salina after 24 h of exposure. From these three compounds (Figure 3), gallic acid persulfate (AGS) EC50 value of 17.65 µM, LC50 levels

higher than 500 µM, and LC50/EC50 of 26.61 µM) was selected for further studies, namely

Figure 3 – Three hit sulfated polyphenols. Gallic acid persulfate, AGS; rutin 2’’, 2’’’,3’,3’’,3’’’,4’,4’’,4’’’,7-nonasulfate, RS; 3,6-bis(β-D-glucopyranosyl)xanthone persulfate, XGS.

1.5.2. Oxygenated xanthones

Six of twenty-two oxygenated xanthones showed highly significant differences (p ≤ 0.001, Dunnet’s test) in the percentage of settled larvae against the control for the two concentration tested in the first screening (500 and 200 M). The assessment of effectiveness versus toxicity of these six promise compounds showed EC50 values ranging

from 1.25 to 12.40 µg/mL, LC50 levels higher than 500 µM, and therapeutic ratio

(LC50/EC50) between 17.42 and 141.64 (unpublished results). However, four of the six

compounds showed significant ecotoxicity to A. salina after 24 h of exposure. From the other two compounds, 3,4-dihydroxyxanthone (DOHX, Figure 4) (EC50 value of 11.53 µM,

LC50 levels higher than 500 µM, and LC50/EC50 of 43.37) was selected for further studies,

namely immobilization in coatings and biodegradation studies, due to its feasible synthesis.

1.6. Strategy and aims

In this work, the synthesis of derivatives of the previous hit compounds XGS and DOHX was planned based on previous structure-activity relationship evidences.

Considering the promising AF activity of xanthone XGS, with sulfated glycosides in positions 3 and 6 (Scheme 1, a),40 _{and xanthone DOHX with two hydroxyl groups in}

positions 3 and 4, the synthesis of a xanthone with two sulfated glycosides at positions 3 and 4 was planned (Scheme 1, a). Additionally, inspired by some examples of marine carboxylated and sulfated steroids with AF activity, the synthesis of carboxylated xanthone derivatives was planned (Scheme 1, b) as also the synthesis of a sulfated derivative of stigmasterol, a commercially available sterol, isolated from the marine microalgae Navicula incerta.41

Scheme 1 – Synthetic analogues planned in this dissertation.

Therefore, the principal aims of this work were:

i. synthesize 3,4-dihydroxyxanthone in order to obtain enough amounts for incorporation in coatings and as building block for molecular modifications;

ii. synthesize derivatives of 3,4-dihydroxyxanthone (DOHX) namely carboxylated and glycosylated derivatives,

iii. synthesize a sulfated derivative of a marine sterol; iv. determine solubility of derivatives in seawater;

v. evaluate AF activity of the synthesized derivatives, 3,4-di(carboxymethoxy) xanthone (7) and 3(2,3,4,6-tetra-sulfate-O-β-

D-glucopyranosyl)xanthone (10), and

CHAPTER 2 - RESULTS AND DISCUSSION

2.1. SYNTHESIS

2.1.1. Total synthesis of 3,4-dihydroxyxanthone (5)

Xanthones can be synthesized by three main methods: the Grover, Shah and Shah (GSS) reaction, the benzophenone route and the synthesis via diphenyl ether intermediates.42-43

The GSS reaction requires two aromatic blocks, a salicylic acid derivative and an appropriate phenol that are heated with zinc chloride in phosphoryl chloride as solvent allowing to provide xanthones in only one step. This method is the oldest and well known route, however is associated with numerous limitations.44

The benzophenone route is carried out in two steps: benzophenone synthesis by Friedel-Crafts acylation of a convenient substituted benzoyl chloride with a phenolic derivative, and posterior cyclization. High yields are normally obtained with this route and so, is one of the most used multi-step approaches.45

The diaryl ether method consists in the ether linkage between two aromatic rings, followed by the cyclization to give the xanthone. The yields obtained with this methodology are usually lower.46

The benzophenone route was selected to synthessize 3,4-dihydroxyxanthone (5), in three steps (Scheme 2), accordingly to previous experience in our group.43

2.1.1.1. Synthesis of benzophenone intermediate 3

Benzophenone intermediates are suitable as precursors for cyclization of xanthones. The synthesis of benzophenone intermediates is achieved at room temperature by Friedel-Crafts acylation of methoxybenzene derivatives with the properly substituted benzoyl chloride in the presence of a Lewis acid, namely aluminium chloride (AlCl3). To obtain the benzophenone intermediate 3 (Scheme 3), AlCl3 anhydrous was

added to a dry diethyl ether solution of trimethoxybenzene (1) and 2-methoxybenzoyl chloride (2). This reaction must be carried out under anhydrous condition since the interaction with humidity of the atmosphere can change the AlCl3 to non-reactive form

Al(OH)3 and HCl gas. 47

Scheme 3 – Synthesis of hydroxy-2’,3,4-trimethoxybenzophenone (3) from trimethoxybenzene (1) and 2-methoxybenzoyl chloride (2). r.t. - room temperature.

After completing the reaction, the suspension was acidified with HCl 10% to convert the phenolate group of the benzophenone derivative to the non-ionized form. Then, the non-ionized form of the benzophenone derivative was extracted with dichloromethane. A brown oil which contains the final product was obtained and used without further purification processes to the next reaction step.

Synthesis of 2-hydroxy-2’,3,4-trimethoxybenzophenone (3) occurs by electrophilic aromatic substitution. Friedel-Crafts acylation is catalysed by the Lewis acid, AlCl3, which

activates the acylating reagent, 2-methoxybenzoyl (2, Scheme 4), to the acylium cation, a strong electrophile. The weak nucleophile, trimethoxybenzene (1, Scheme 4), attacks the carbonyl carbon of the acylium ion, forming a stabilized resonance intermediate. A proton is lost to the medium rich in chloride ions, which re-establishes the aromaticity of the ring. Three products are formed (3a, 3 and 3b), a methylated in both positions (3a) and two of them obtained by demethylation of the methoxyl group at positions 2 and 2’ (3 and 3b). Demethylation of 3a occurs by nucleophilic substitution at ortho of carbonyl. 3a product acts as a nucleophile and attacks the hydrogen of the hydrochloric acid in the medium,

forming a positively charged intermediate. Then the chloride ion attacks the methyl group, forming the product 3 or 3b and chloromethane. 45, 48 _{Theoretically, due to steric hindrance}

the main product corresponds to 2-hydroxy-2’,3,4-trimethoxybenzophenone (3).

Scheme 4 – Mechanism of hydroxy-2’,3,4-trimethoxybenzophenone (3) from trimethoxybenzene (1) and 2-methoxybenzoyl chloride (2).

2.1.1.2. Cyclization of benzophenone intermediate 3

The benzophenone intermediate 3 contained in the crude product formed in the previous reaction was cyclized to a xanthone intermediate through an intramolecular nucleophilic aromatic substitution45 _(S

NAr, Scheme 5). The cyclization of the

benzophenone intermediate was performed under conventional heating and the reaction was kept in reflux for 25h. The reaction of cyclization undergone in basic solution, contributing to the deprotonation of acidic phenolic hydroxyl group and the subsequent

nucleophilic substitution. A yellow solution was formed containing the xanthone intermediate 4. After completing the reaction, a solid was trapped by a vacuum filtration. The intermediate 4 contained in the formed solid was washed with water. A white solid was obtained and used in the next reaction step.

Scheme 5 – Synthesis of 3,4-dimethyl-9H-xanthen-9-one (4).

In order to obtain a xanthone from a benzophenone, positions 2 and 2’ need to be connected by an ether bridge. Thus, the presence of at least one hydroxyl group in one of these positions is essential. On the other hand, the other position will define the type of cyclization strategy. Scheme 6 shows the presence of a hydroxyl group in position 2 and a methoxy in 2’, which can act as a good leaving group. Therefore, an intramolecular nucleophilic aromatic substitution base-catalysed occurs in two steps. The first step corresponds to the attack of the nucleophile (-O-_{) on the carbon of the aromatic ring}

connected to the leaving group (-OCH3), followed by the expelling of the leaving group. 45

Scheme 6 – Synthesis of 3,4-dimethyl-9H-xanthen-9-one (4) by base-catalysed intramolecular nucleophilic aromatic substitution.

2.1.1.3 Demethylation of 3,4-dimethoxyxanthone (4)

The demethylation of xanthone intermediate 4 was performed under reflux by nucleophilic substitution, in the presence of AlCl3 as Lewis acid (Scheme 7). To obtain the

3,4-dihydroxyxanthone (5), crude material 4 and AlCl3 anhydrous was added to the

previously dried toluene. After completing the reaction, the AlCl3 was eliminated by

vacuum filtration and the filtrate was acidified with HCl 10% to protonate the complex formed with the AlCl3, to give the final product 3, 4-dihydroxyxanthone (5), with a yield of

59%.

Scheme 7 – Synthesis of 3,4-dihydroxyxanthone (5).

Demethylation of 3,4-dimethyl-9H-xanthen-9-one (4) was carried out using AlCl3,

Lewis acid, capable of accepting electron density of methoxy group (electron density donor). In Scheme 8 the demethylation mechanism is proposed. The electron pairs of the oxygen atoms of the methoxy groups attack the aluminium central metal (Al) and one of the chlorine groups leaves the AlCl3 as Cl-. Besides good leaving group, chloride ion is

also a nucleophile that attacks the methyl group, forming chloromethane and xanthone-AlCl2 complex. The addition of HCl, in the treatment of reaction, lead to protonation of

oxygen and expulsion of AlCl2-.

Scheme 8 – Mechanism of the synthesis of 3, 4-dihydroxyxanthone (5), by demethylation of 3,4-dimethyl-9H-xanthen-9-one (4).

The purity of 5 was evaluated by HPLC-DAD in 98% (Figure 5).

Figure 5 – Chromatogram obtained from 3,4-dihydroxyxanthone (5) at 236 nm, in a concentration of 50 M. Mobile phase water/ acetonitrile/ trifluoroacetic acid (80:20:0.1).

To obtain 2.0 g of xanthone 5, it was necessary to start from 11.2 g of 2-methoxybenzoyl chloride (2), the limiting reagent, so the overall yield of these three steps synthesis was 14%.

2.1.2. Synthesis of 3,4-di(carboxymethoxy)xanthone (7)

For the synthesis of 3,4-di(carboxymethoxy)xanthone (7), the synthesis of 3,4-di(2-methoxy-2-oxoethoxy) xanthone (6) was necessary to be accomplished first by nucleophilic substitution of 3,4-dihydroxyxanthone (5) with methyl bromoacetate 49-50 _as

follows.

2.1.2.1. Nucleophilic substitution of 3,4-dihydroxyxanthone (5) with

methyl bromoacetate

The synthesis of 3,4-di(2-methoxy-2-oxoethoxy)xanthone (6, Scheme 9) was accomplished by a bimolecular nucleophilic substitution (SN2). The reaction occurred

under room temperature using acetone as solvent.49-50 _{3,4-Dihydroxyxanthone (5) was}

deprotonated with cesium carbonate (Cs2CO3), forming a good nucleophile. Methyl

bromoacetate was subsequently added and attacked by the phenoxide ion, forming 3,4-di(2-methoxy-2-oxoethoxy)xanthone (6). The 3,4-3,4-di(2-methoxy-2-oxoethoxy)xanthone (6) was obtained for the first time in 39% yield, after filtration to eliminate Cs2CO3 followed by

addition of ice to the concentrated filtrate, which allowed the insolubilization of the pure product.

Scheme 9 – Synthesis of 3,4-di(2-methoxy-2-oxoethoxy)xanthone (6). r.t. - room temperature.

2.1.2.2. Alkaline hydrolysis of 3,4-di(2-methoxy-2-oxoethoxy)

xanthone (6)

Carboxylic acids could be obtained from their derivatives by nucleophilic acyl substitution, that occurs through the tetrahedral intermediate which collapses with the formation of the products. The reaction condition depends on the reactivity of the derivative, related to the stability of the starting material and the leaving group. For an ester hydrolysis to happen is required the presence of a catalyst, therefore NaOH was added (Scheme 10) to the previously synthesized 3,4-di(2-methoxy-2-oxoethoxy) xanthone (6). The hydroxyl ion acts as a nucleophile and the final step, the deprotonation of the carboxylic acid, is fundamental to move the reaction equilibrium to the formation of the carboxylate ion.51 _{After 3 hours of reaction at room temperature, addition of HCl 5M to}

the aqueous layer, protonated the carboxylate ion into carboxylic acid, obtaining the 3,4-di(2-methoxy-2-oxoethoxy)xanthone (6), for the first time, in a 74% yield.

Xanthone 7 purity was evaluated by HPLC-DAD in 98% (Figure 6).

Figure 6 – Chromatogram obtained from 3,4-di(carboxymethoxy)xanthone (7) at 239 nm, in a concentration of 0.05 mg/ml. Mobile phase water/ acetonitrile/ trifluoroacetic acid (80:20:0.1).

2.1.3.

Synthesis

of

3(2,3,4,6-tetra-sulfate-O-

β-D

-glucopyranosyl)xanthone (10)

For the synthesis of 3(2,3,4,6-tetra-sulfate-O-β-

D-glucopyranosyl)xanthone (10),

2.1.3.1. Glycosylation of 3,4-dihydroxyxanthone (5)

Glycosylation may occur by three methods: Michael, Fischer, and Koenigs-Knorr reactions.

The Michael reaction consists in the addition of stabilized anions to ,  -unsaturated carbonyl compounds. Normally associated to good yields.52

The Fischer reaction is normally used for the preparation of simple O-glycosides, through the linkage of a free sugar to an alcohol under acidic conditions. The main advantage is that the use of protecting groups is not required. However, this reaction is not stereoselective, providing a mixture of anomers.53

Koenigs-Knorr reaction is one of the most useful reactions in obtaining O-glycosides. Normally, silver salts and drying agents are required.53-54

3,4-di(2,3,4,6-tetra-O-acetyl-O-β-D-glucopyranosyl) xanthone (8) was synthesized for the first time by the Michael glycosidation,53-54 _{from the reaction of}

3,4-dihydoxyxanthone (5) with acetobromoglucose in the presence of Cs2CO3 and

tetrabutylammonium bromide. After 5 hours, a vacuum filtration was carried out to eliminate Cs2CO3 followed by flash chromatographies to isolate xanthonoside 8 in a 36%

yield.

Scheme 11 – Synthesis of 3,4-di(2,3,4,6-tetra-O-acetyl-O-β-D-glucopyranosyl) xanthone (8). TBAB = tetrabutylammonium bromide; r.t. - room temperature.

2.1.3.2. Desacetylation of 3,4-di(2,3,4,6-tetra-O-acetyl-O-

β-

-glucopyranosyl)xanthone (8)

3,4-Di(O-β-D-glucopyranosyl)xanthone (9) was obtained for the first time using a Zemplén deacetylation with sodium methoxide MeOH at room temperature (Scheme 12).55 _{Zemplén deacetylation was selected because of the use of catalytic amounts of}

base (sodium methoxide), short reaction times, and excellent yields.56-57

After 1 hour at room temperature the reaction was complete, and neutralization using an ion exchange resin was carried out to afford compound 9 in 83% yield.

2.1.3.3. Sulfation of 3,4-di(O-

β-

-glucopyranosyl)xanthone (9)

3(2,3,4,6-Tetra-sulfate-O-β-D-glucopyranosyl)xanthone (10) was obtained for the first time when applying triethylamine-sulfur trioxide adduct (SO3.TEA) in

dimethylacetamide (DMA) conditions for 17 hours at 70ºC (Scheme 13).58-59

Sulfur trioxide adducts are successfully applied in the polysulfation and persulfation of polyhydroxyl molecules and are mild reagents when comparing with sulfuric acid-derived reagents.59 _{The use of SO}

3.TEA was selected due to large

experience in our group.60

At the end of the reaction, triethylamine (TEA) was added to convert the sulfated xanthone into salts of TEA, which were able to be insolubilized under oil form with ether. A flash chromatography was performed in order to obtain the major product, 3(2,3,4,6-tetra-sulfate-O-β-D-glucopyranosyl) xanthone (10). Sulfated xanthone 10 was converted in sodium salts by adding an aqueous solution of 30% sodium acetate and further purified from inorganic salts by dialysis. 3(2,3,4,6-Tetra-sulfate-O-β-D-glucopyranosyl) xanthone (10) was obtained in 25% yield.

Scheme 13 – Synthesis of xanthone 3(2,3,4,6-tetra-sulfate-O-β-D-glucopyranosyl) xanthone (10). SO3.TEA –

Purity of 10 was evaluated by HPLC-DAD in 96% (Figure 7)

Figure 7 – Chromatogram obtained from 3-O-β-D-glucopyranosil persulfate (10) at 250 nm, in a concentration of 1 mg/ml. Mobile phase tetrabutylammonium bromide / acetonitrile (38:62).

2.1.4. Sulfation of Stigmasterol (11)

Stigmasta-5,22-dien-3-β–sulfate (12) was obtained for the first time when applying SO3.TEA to a solution of stigmasta-5,22-dien-3-β–ol (11) and dimethylformamide (DMF)

for 7 hours at 70ºC (Scheme 14).61-63

Filtration to eliminate DMF was performed, followed by a flash chromatography in order to isolate stigmasta-5,22-dien-3-β–sulfate (12). Conversion to sodium salts was performed by a cation ion exchange resin (Na+ _{form), and the factions were evaporated}

until dryness. Stigmasta-5,22-dien-3-β–sulfate (12) was obtained in 2% yield.

Scheme 14 – Synthesis of stigmasta-5,22-dien-3-β–sulfate (12). SO3.TEA – triethylamine-sulfur trioxide

adduct; DMF – dimethylformamide.

Although we still do not have NMR elucidation, compound 12 presented a different chromatographic behaviour (lower Rf) from the parent compound 11, and IR bands of 12 presented bands from sulfation bonds, in opposition to compound 11.

2.2 STRUCTURE ELUCIDATION

The structure characterization of the new derivatives of 3, 4-dihydroxyxanthone ( 6-10) was established by melting points determination, IR, NMR and HRMS techniques. In this section, IR, 1_{H and} 13_{C nuclear magnetic resonance (NMR) data for 3,}

4-dihydroxyxanthone (5) and their derivatives, will be presented.

2.2.1. 3,4-Dihydroxyxanthone (5)

Structure elucidation of 3,4-dihydroxyxanthone (5, Figure 8) was established by comparison the IR and NMR data with the data already reported in the literature for xanthone 5.43, 64

Figure 8 – 3, 4-Dihydroxyxanthone (5).

IR spectrum of derivative 5 showed two strong bands at 3530 and 3396 cm-1

typical of O-H stretching vibration which suggest the presence of hydroxyl groups (Table 1).

Table 1 – IR data of 3,4-dihydroxyxanthone (5).

Group  (cm-1₎ 5 O-H phenol 3530, 3396 C-H aliphatic 2926 C-C aromatic 1594, 1527, 1459

1_{H and} 13_{C NMR spectra are presented in APPENDIX II and protons and carbons}

were assigned to their respective chemical shifts accordingly to the literature (Figure 9).43, 64

Figure 9 – 1_{H and} 13_{C NMR data of 3, 4-dihydroxyxanthone (5).}

2.2.2. 3, 4-Dihydroxyxanthone derivatives: 6 and 7

The structure of di(2-methoxy-2-oxoethoxy)xanthone (6, Figure 10) and 3,4-di(carboxymethoxy)xanthone (7, Figure 10) was established by IR, NMR, and HRMS.

Figure 10 – Dihydroxyxanthone derivatives: di(2-methoxy-2-oxoethoxy)xanthone (6) and 3,4-di(carboxymethoxy)xanthone (7).

IR spectrum of compound 6 showed a band at 1767 cm-1 _{characteristic of a C=O}

ester stretch and three bands at 1279, 1204, 1183 cm-1 _{correspondent to ether group}

(Table 2), which is in accordance with the molecular modification performed. IR spectrum of compound 7 showed a band of C=O carboxylic acid stretch at 1744 cm-1 _{and a band at}

3414 cm-1 _{of O-H stretch, suggesting the presence of a carboxylic acid group (Table 2).}

Table 2 – IR data of 3,4-dihydroxyxanthone derivatives: 6 and 7.

Group

 (cm-1₎  (cm-1)

6 7

O-H carboxylic acid - 3414

C-H aliphatic 2958, 2927 2958, 2921

C=O ester 1767 -

C=O carboxylic acid - 1744

Group

 (cm-1₎  (cm-1)

6 7

C-C aromatic 1606, 1471, 1447 1606, 1467, 1453

C-O ether 1279, 1204, 1183 1262, 1104

1_{H and} 13_{C spectra of compounds 6 and 7 are presented in APPENDIX III and IV,}

respectively.

Analysis of 1_H, 13_{C, HSQC, and HMBC spectra analysis allowed the assignment of}

proton and carbon chemical shifts to 3,4-di(2-methoxy-2-oxoethoxy)xanthone (6, Figure 11). The assignments of carbon atoms directly bonded to proton atoms were achieved from HSQC experiments (Table 3) and the chemical shifts of the carbon atoms not directly bonded to proton atoms were deduced from HMBC correlations (Figure 12).

Figure 11 – 1_{H and} 13_{C NMR data of 3,4-di(2-methoxy-2-oxoethoxy)xanthone (6).}

1_{H and} 13_{C NMR spectra of compound 6 presented ten more proton signals and six}

more carbon signals than 1_{H and} 13_{C NMR spectra of compound 5, which indicated the}

presence of two carbomethoxymethyl groups, namely signals corresponding to methyl groups (δH 3.82 and 3.85; δC 52.5 and 52.2) and to methylene bridge (δH 4.87 and 4.91; δC

In the HSQC spectra, the following correlations were observed:

Table 3 – HSQC data of 3,4-di(2-methoxy-2-oxoethoxy)xanthone (6).

HSQC (ppm) 1_H _(ppm) 13_C 8.32 (dd, J= 8.0 and 1.7 Hz) H-8 126.7 C-8 8.08 (d, J= 9.0 Hz) H-1 122.7 C-1 7.73 (ddd, J= 8.6, 7.1 and 1.5 Hz) H-6 134.8 C-6 7,56 (d, J= 8.1 Hz) H5 118.2 C-5 7.40 (ddd, J= 7.5, 7.5 and 0.9 Hz) H-7 124.2 C-7 6.89 (d, J= 9.0 Hz) H-2 109.7 C-2 4.91 (s) 4-OCH2 70.0 4-OCH2 4.87 (s) 3-OCH2 65.9 3-OCH2 3.85 (s) (4)-COCH3 52.2 (4)-COCH3 3.82 (s) (3)-COCH3 52.5 (3)-COCH3

Carbon signals of C-9, C-10a, C-4a, C-8a and C-9a were assigned to _{C 176.3,} 156.1, 150.4, 121.5 and 117.7 ppm by deduction of HMBC correlations (Figure 12). The C-9a and C-4a signals of compound 7, assigned to C 117.7 and 150.4 ppm, were

deshielded when compared to the same carbons signals in the parent compound 5 (C

114.8 and 146.6 ppm) (Table 4).

The position of the carbomethoxymethyl groups on the xanthone was further confirmed by the correlations observed in HMBC spectrum between the signal of methylene protons at δ_H 4.87 and 4.91 ppm and the signals of C-3 and C-4 (C 154.9 and

Figure 12 – Main connectivities found in HMBC for compound 6.

Aromatic protons H-2 and H-1 appeared at H 6.89 and 8.08 ppm, respectively,

whereas the corresponding signals of the same protons in the parent compound 5 appeared at H 6.96 and 7.58 ppm (Table 4).

Carbon C-2 was assigned to C 113.3 ppm, in compound 5, whereas this carbon

was at C 109.8 ppm, in compound 6. In contrast, C-1 in the hydroxylated xanthone 5 was

assigned to C 116.7 ppm, whereas in derivative 6 this carbon was at C 122.7 ppm

(Table 4).

Molecular formula of xanthone 6 was also confirmed by HRMS as C₁₉H₁₆O₈.

Proton and carbon assignments of derivative 7 are presented in Figure 13.

Figure 13 – 1_{H and} 13_{C NMR data of 3,4-di(carboxymethoxy)xanthone (7).}

When comparing with compound 6, the compound 7 spectra did not show the singlet signals around _{H 3.82-3.85 ppm neither the signal corresponding to –CH3 carbons} of acetate groups at C 52.2-52.5 ppm.

The position of the substituents on ring A of xanthone was once more confirmed by the correlations observed in HMBC spectrum between the signal of methylene protons at _H 4.97 and 4.84 ppm and the signals of C-3 and C-4 (C 155.2 and 134.5 ppm),

respectively (Figure 14).

Figure 14 – Connectivities found in HMBC for compound 7 that evidence the position of the substituents in xanthone.

Molecular formula of xanthone 7 (C₁₇H₁₂O₈) was confirmed by HRMS.

Table 4 – Main differences of proton and carbon chemical shifts of compounds 5, 6, and 7.

Position 1_{H (ppm)} 13_{C (ppm)} 5 6 7 5 6 7 1 7.58 8.08 7.90 116.7 122.7 121.2 2 6.69 6.89 7.18 113.3 109.8 110.5 3 - - - 151.7 154.9 155.2 4 - - - 132.7 135.3 134.5 4a - - - 146.5 150.4 149.7 9a - - - 114.8 117.7 116.2

2.3.3. 3, 4-Dihydroxyxanthone derivatives: 8, 9, and 10

Structure elucidation of compounds 3,4-di(2,3,4,6-tetra-O-acetyl-O- β-D-glucopyranosyl) xanthone (8), 3,4-di(O-β-D-glucopyranosyl)xanthone (9) and 3(2,3,4,6-tetra-sulfate-O-β-D-glucopyranosyl)xanthone (10) were established for the first time (Figure 15).

Figure 15 – 3,4-Dihydroxyxanthone derivatives: 3,4-di(2,3,4,6-tetra-O-acetyl-O-β-D-glucopyranosyl)xanthone (8), 3,4-di(O-β-D-glucopyranosyl)xanthone (9), and 3(2,3,4,6-tetra-sulfate-O-β-D-glucopyranosyl)xanthone (10).

IR spectrum of derivative 8 showed a strong band at 1756 cm-1 _{typical of C=O}

ester stretching vibration which suggest the presence of acetyl groups (Table 5). On the other hand, IR spectrum of derivative 9 did not showed the band correspondent to the ester, but a band of O-H stretch at 3408 cm-1 _{(Table 5), which suggest that acetyl groups}

were removed. IR spectrum of derivative 10 showed bands from the sulfate groups at 1262 cm-1 _{(S=O), 1029 cm}-1 _{(C-O-S), and 807 cm}-1 _{(S-O), suggesting that sulfation was}

successful (Table 5).

Table 5 – IR data of 3,4-dihydroxyxanthone derivatives: 8, 9, and 10.

Group

 (cm-1₎  (cm-1)  (cm-1)

8 9 10

O-H phenol - - 3451

O-H carboxylic acid - 3408 -

C-H aliphatic 2920 2918 2917 C=O ester 1756 - - C=O ketone 1663 1645 1625 C-C aromatic 1605, 1467, 1447 1606, 1469, 1448 - S=O - - 1262 C-O-S - - 1029 S-O - - 807

1_{H and} 13_{C spectra of compounds 8, 9, and 10 are presented in APPENDIX V, VI,}

and VII, respectively.

1_H, 13_{C, HSQC, and HMBC spectra analysis allowed the assignment of proton and}

carbons chemical shifts to 3,4-di(2,3,4,6-tetra-O-acetyl-O-β-D-glucopyranosyl)xanthone (8, Figure 16). The assignments of carbon atoms directly bonded to proton atoms were achieved from HSQC experiments (Table 6) and the chemical shifts of the carbon atoms not directly bonded to proton atoms were deduced from HMBC correlations (Figure 17).

Figure 16 – 1_{H and} 13_{C NMR data of 3,4-di(2,3,4,6-tetra-O-acetyl-O-β-D-glucopyranosyl) xanthone (8).}

1_{H spectrum of compound 8 showed fourteen more signals characteristic of}

aliphatic protons (the sugar moiety) between _{H 3.82-5.51 ppm. Additionally, two singlets} integrated for three protons each and a multiplet integrated for eighteen protons between H2.12-1.92 ppm were observed and assigned to -CH3 protons of eight acetyl groups. The

coupling constants values of protons H-1’ and H-1’’ (H 5.41-5.21 and 5.51 ppm,

respectively) was of 7.7 Hz, characteristic of  linkage.

13_{C NMR spectrum showed twelve signals between} 

C 61.7-100.3 ppm

170.5-169.3 ppm, and seven signals between C 20.8-20.5 ppm were also observed, which were

assigned to eight C=O of the acetyl groups and to eight –CH3 groups, respectively.

In the HSQC spectra, the following correlations were observed:

Table 6 – HSQC data of 3,4-di(2,3,4,6-tetra-O-acetyl-O-β-D-glucopyranosyl) xanthone (8).

HSQC (ppm) 1_H _(ppm) 13_C 8.33 (dd, J= 8.1 and 1.6 Hz) H-8 126.7 C-8 8.10 (d, J= 9.0 Hz) H-1 122.9 C-1 7.76 (ddd, J= 8.5, 7.0 and 1.6 Hz) H-6 135.1 C-6 7,62 (d, J= 7.9 Hz) H5 118.2 C-5 7.42 (ddd, J= 7.5, 7.5 and 1.0 Hz) H-7 124.5 C-7 7,13 (d, J= 9.1 Hz) H-2 113.3 C-2 5.51 (d, J= 7.3 Hz) H-1’’ 100.3 C-1’’ 5.41-5.21 (7H, m) H-1’ H-2’-H-4’ H-2’’-H-4’’ 99.2, 72.3, 71.2, 68.4, 68.0 C-1’ C-2’-C-4’ C-2’’-C-4’’ 4.31 (dd, J= 5.3 and 12.4 Hz) H-6 acetoglucose 61.70 C-6 acetoglucose 4.25 (dd, J= 5.2 and 12.3 Hz) H-6 acetoglucose 62.1 C-6 acetoglucose 4.18-4.07 (2H, m) H-6 acetoglucose 62.1, 61.70 C-6 acetoglucose 3.91-3.82 (2H, m) H-5’; H-5’’ 72.5, 72.2 C-5’; C-5’’ 2.12 (3H, s) COCH3 20.8 OCOCH3 2.07-2.05 (18H, s) COCH3 20.7 OCOCH3 1.92 (3H, s) COCH3 20.5 OCOCH3

HMBC correlations allowed to deduce the carbon signals of 9, 10a, 4a, C-8a and C-9a that were assigned to _{C 176.2, 156.0, 150.4, 121.4 and 118.8 ppm (Figure} 17). The C-9a and C-4a signals of compound 8, assigned to C 118.8 and 150.4 ppm,

were deshielded when compared to the same carbon signals in compound 5 (C 114.8

The position of the O-acetyl--D-glucopyranoside groups on derivative 8 was further confirmed by the correlations observed in HMBC spectrum between the signal of H-1’’ at _H 5.51 ppm and the signal of C-4 (C 134.0 ppm) (Figure 17).

Figure 17 – Main connectivities found in HMBC for compound 8.

Characteristic signals of the aromatic protons H-2 and H-1 appeared at H 7.13

and 8.10 ppm, respectively, whereas the corresponding signals of the same protons appeared at H 6.89 and 7.58 ppm in the parent compound 5 (Table 7).

In 13_{C NMR spectrum of derivative 8, C-1 was assigned to} 

C 122.9 ppm, whereas

in the parent compound 5 this carbon was at C 116.7 ppm (Table 7).

Structure of xanthone 8 was confirmed by HRMS as C₄₁H₄₄O₂₂.

In Figure 18 is presented the proton and carbon chemical shifts, that were attributed to the protons and carbons of compound 9.

Figure 18 – 1_{H and} 13_{C NMR data of 3,4-di(O-β-D-glucopyranosyl)xanthone (9).}

1_{H and} 13_{C NMR spectra of xanthone derivative 9 did not show the signals of -CH} 3

(H 2.12-1.92; C 20.8-20.5) neither of C=O of acetyl groups (C 170.5-169.3). Although,

eight signals between H 4.39-5.39 ppm are present in 1H spectrum, which is in

accordance with the presence of hydroxyl protons.

In 13_{C NMR spectrum of compound 9, carbons C-2’, C-2’’, C-3’, C-3’’, C-5’ and C-5’}

were assigned to signals between _C 77.3-74.3 ppm, whereas these carbons were at C

68.0-73.0 ppm in compound 8.

The position of the sugar moiety on derivative 9 was further confirmed by the correlations observed in HMBC spectrum between the signal of H-1’ and H-1’’ at _H 5.08 and 5.06 ppm and the signals of C-3 and C-4 (_{C 155.8 and 133.9 ppm), respectively} (Figure 19).

Figure 19 – Connectivities found in HMBC for compound 9 that evidence the position of the substituents in xanthone.

Molecular formula of xanthone 9 was confirmed by HRMS as C₂₅H₂₈O_14.

The assignment of proton and carbons of 3(2,3,4,6-tetra-sulfate-O- β-D-glucopyranosyl)xanthone (10) are shown in Figure 20.

Figure 20 – 1_{H and} 13_{C NMR data of 3(2,3,4,6-tetra-sulfate-O-β-D-glucopyranosyl) xanthone (10).}

When comparing with the starting material, compound 9, signals of hydroxyl protons were not observed in the 1_{H NMR spectrum of compound 10. Furthermore, only}

seven signals between _H 5.29-3.88 ppm were present in compound 10, while fourteen signals were presented in 9. A signal at _H 9.40 ppm integrated for one proton was also observed in the 1_{H NMR spectrum of compound 9, characteristic of phenolic proton.}

In 13_{C NMR spectrum of compound 10, carbons C-4’ and C-6’ were assigned}

between C 71.3-76.9 and 67.3 ppm, whereas in compound 9 these carbons were at C