David Rodrigues Palharesrepositorium.sdum.uminho.pt/bitstream/1822/41134/1/Master... · 2019. 1. 1. · Declaração Nome: David Rodrigues Palhares Endereço electrónico: [email protected]

School of Sciences

October 2015

David Rodrigues Palhares

Synthesis and biological evaluation of mono andbis-naphthalimide derivatives against SH-SY5Y,human brain cancer cells

Master Thesis

Masters in Medicinal Chemistry

Under the supervision of Dr António Gil Fortes and Dr Maria José Alves

Declaração

Nome: David Rodrigues Palhares

Endereço electrónico: [email protected]

Telefone: (+351) 912566090

Título da dissertação: Synthesis and biological evaluation of mono and bis

naphthalimide derivatives against SH-SY5Y, human brain cancer cells

Orientadores:

Professora Doutora Maria José Alves

Professor Doutor António Gil Fortes

Ano de Conclusão: 2015

Designação do Mestrado: Mestrado em Química Medicinal

DE ACORDO COM A LESGISLAÇÃO EM VIGOR, NÃO É PERMITIDA A REPRODUÇÃO DE

QUALQUER PARTE DESTA TESE/TRABALHO

Universidade do Minho, / /

Assinatura:

mailto:[email protected]

iii

Acknowledgements

I’d like to thank my supervisors, Dr Maria José Alves and Dr António Gil Fortes

whose support me, for their transmitted knowledge and for helping me during this

project.

To the Dr Paul Kong Thoo Lin for his orientation along my stay at Robert Gordon

University, for the support he gave me, his knowledge that he transmitted to me and

always present when I lived in Aberdeen. Also for the help to my project is concluded

with success.

To Dr.a Elisa Pinto and Dr.a Vânia Azevedo for their hard work showed on realization

of Nuclear Magnetic Resonance spectrums.

For all my fellow graduate students for all support and help me along this 2 years

and mainly in this last year.

My truly regards to all my friends, my girlfriend and my brother, who always

supported, guided me in any respect during this last year always with kind words.

Lastly, I’d like to thank my family for their continuous support, especially to my

parents and my godfather. They always believe in me, and help me always possible

and make my travel to RGU a dream come true, thing that I will never forget.

v

Abstract

Naphthalimides (1H-benzo[de]isoquinoline-1,3-(2H)-diones) consists of a flat,

generally π-deficient aromatic or heteroaromatic system and show strong

hydrophobicity. These types of compounds with this moiety demonstrate inherent

fluorescence and biological properties such as anticancer, antimicrobial,

antitrypanosomal, analgesic, antioxidative and antiviral properties. The naphthalimide

compounds are also known to be very good DNA intercalators, since the planar

naphthalimido moiety binds by perpendicular insertion between the base pairs of the

double helix of DNA.

Previous work had already shown that mono and bis naphthalimido derivatives

exhibit strong activity against different cancer cell lines. Here in this work will be

demonstrate that the alkyl chain, i.e. the linker between the naphthalimido groups or

the substituent attached at the end of the linker chain, do have an impact on the

biological and DNA binding properties.

Therefore the synthesis of a series of new mono-naphthalimide derivatives were

prepared in moderate to good yields by reaction of aminonaphthalimides containing

different alkyl chain length with carious aromatic aldehydes. The new bis-

naphthalimides were prepared by an N-alkylation reaction of different linkers with the

corresponding O-tosyl alkylnaphthalimides.

The biological activity of the newly synthesized compounds includes their ability to

bind DNA, their toxicity against SH-SY5Y human brain cancer cells in vitro, cell

morphology and cellular uptake were tested.

As expected the bis-naphthalimide derivatives gave better results when compared

to the mono-naphthalimides in all tests. For the mono naphthalimides, longer the

length of the alkyl chain, better are the results; also the nature of the aromatic

aldehyde interfere with the results. For the bis-naphthalimides the type of linker has

influence on biological activity and binding studies. It has been shown when the linker

has more flexibility, the biological activity and binding studies gave better results.

vii

Resumo

As naftalimidas (1H-benzo[de]isoquinoline-1,3-(2H)-dionas) consistem num sistema

planar aromático ou heteroaromático deficiente em electrões π e mostram forte

hidrofobicidade. Estes tipos de compostos apresentam fluorescência e propriedades

biológicas como anticancerígenos, antimicrobianos, antitrypanosomal, analgésica,

antioxidante e antiviral. Os compostos de naftalimida também são conhecidos como

intercalantes do ADN, pois o seu núcleo naftalimido planar liga-se através de inserção

perpendicular entre os pares de bases da dupla hélice do ADN.

Em trabalhos anteriores mostraram que os derivados mono e bis naftalimidas

exibiram forte atividade contra diferentes linhas celulares cancerígenas. Neste

trabalho foi possível demonstrar que a cadeia alquílica, por exemplo a cadeia ligando,

tem ou não impacto nas propriedades quer biológicas quer na ligação do ADN.

A síntese de novos derivados de naftalimida foram preparados com rendimentos de

moderados a bons através da reação de aminonaftalimidas contendo diferentes

tamanhos de cadeia alquílica com aldeídos aromáticos. As novas bis naftalimidas

foram preparados por uma reação de N-alquilação com diferentes ligandos com os

correspondentes O-tosil alquilnaftalimidas.

A atividade biológica dos novos compostos sintetizados incluindo a capacidade de

ligação ao ADN, sua toxicidade in vitro contra células humanas do cancro do cérebro,

SH-SY5Y, a morfologia celular e a absorção celular foram testadas.

Como esperado os derivados de bis-naftalimida apresentaram melhores resultados

quando comparadas com as mono-naftalimidas em todos os testes efetuados. Para as

mono-naftalimidas, os resultados mostraram que o aumento da cadeia alquílica, tal

como a natureza do aldeído aromático influenciam os resultados na atividade biológica

e nos estudos de ligação ao ADN. Para as bis-naftalimidas, o tipo de flexibilidade do

ligando tem influência nos resultados de atividade biológica e nos estudos de ligação

ao ADN..

ix

Table of Contents

Acknowledgements .................................................................................. iii

Abstract ..................................................................................................... v

Resumo .................................................................................................... vii

List of Figures .......................................................................................... xiii

List of Tables ........................................................................................... xvii

List of Schemes ........................................................................................ xix

Abbreviations and symbols ..................................................................... xxi

Chapter 1 - Introduction

1. Introduction .............................................................................................. 3

1.1 Cancer ................................................................................................................. 3

1.2 Target Therapy .................................................................................................... 4

1.3 Neuroblastoma ................................................................................................... 4

1.4 Cell Death ............................................................................................................ 7

1.5 DNA ..................................................................................................................... 9

1.6 DNA Intercalators ............................................................................................. 14

1.7 Naphthalimides ................................................................................................. 16

1.8 Bis-Naphthalimides ........................................................................................... 22

1.9 Aims .................................................................................................................. 25

Chapter 2 - Results and Discussion

2. Results and Discussion ............................................................................... 29

2.1 Introduction ...................................................................................................... 29

2.1.1 Synthesis of naphthalimides 1a – c .............................................................. 29

x

2.1.2 Synthesis of compounds 2a – p ................................................................... 32

2.1.3 Synthesis of compounds 3a – p ................................................................... 40

2.1.4 Synthesis of naphthalimides 4a – c .............................................................. 44

2.1.5 Synthesis of tosyl compounds 5a – c ........................................................... 46

2.1.6 Synthesis of Mesitylamines 6a and 6b ......................................................... 48

2.1.7 Synthesis of bis-naphthalimide derivatives 7a – c ........................................ 51

2.1.8 Synthesis of 8a – c derivatives ..................................................................... 54

2.2 DNA Binding Studies ......................................................................................... 57

2.2.1 Fluorescence Binding .................................................................................. 57

2.2.2 Discussion of the values obtained for mono-naphthalimide derivatives ...... 59

2.2.3 Discussion of the values obtained for bis-naphthalimide derivatives ........... 67

2.3 Biological Activity.............................................................................................. 70

2.3.1 Cell Morphology .......................................................................................... 70

2.3.2 Cytotoxicity ................................................................................................. 72

2.4 Cellular Uptake ................................................................................................. 79

Chapter 3 - Experimental Procedure

3. Experimental Procedure ............................................................................. 83

3.1 General Details ................................................................................................. 83

3.1.1 Chemical Synthesis – Analytical Techniques ................................................ 83

3.1.2 Binding Studies ............................................................................................ 84

3.1.3 Cell Culture and Biological Activity .............................................................. 84

3.2 General synthesis of mono-naphthalimide derivatives .................................... 85

3.3 Reaction of 1,8-naphthalic anhydride with alkyl diamines ............................... 86

3.3.1 General procedure ...................................................................................... 86

3.4 Reaction of amines 1a – c with aldehydes ........................................................ 88


3.5 Reduction of imines 2a – p ................................................................................ 98


3.6 General synthesis of bis-naphthalimide derivatives ....................................... 108

xi

3.7 Reaction of 1,8-naphthalic anhydride with alkyl aminoalcohols .................... 109

3.7.1 General procedure .................................................................................... 109

3.8 Reaction of 4a – c with p-toluenesulfonyl chloride ......................................... 111


3.9 Reaction of alkyl diamines 10 and 11 with 2-mesitylenesulfonyl chloride (linker

synthesis) .............................................................................................................. 113


3.10 Reaction of compounds 5b and 5c with 6a and 6b (N-Alquilation reaction) . 114

3.10.1 General procedure .................................................................................. 114

3.11 Deprotection reaction of 7a – c ..................................................................... 117

3.11.1 General procedure .................................................................................. 117

3.12 Binding Studies ............................................................................................. 120

3.13 Biological Activity .......................................................................................... 122

3.13.1 Cell Maintenance .................................................................................... 122

3.13.2 Cell Counting ........................................................................................... 123

3.14 Cytotoxicity ................................................................................................... 124

Chapter 4 - Conclusion

4. Conclusion.............................................................................................. 129

Chapter 5 - Future Work

5. Future Work ........................................................................................... 133

Chapter 6 - Bibliography

6. Bibliography ........................................................................................... 137

Appendix................................................................................................. 143

xiii

List of Figures

Figure 1: Stages of cellular response to a stress stimuli and harmful6 ........................................ 8

Figure 2: Cellular characteristics of necrosis (at left) and apoptosis (at right)6 ........................... 9

Figure 3: Components of nucleic acids: bases, sugars and phosphates (adapted)23 ................. 10

Figure 4: Formation of DNA chain23 ......................................................................................... 11

Figure 5: The two common Watson-Crick base pairs of DNA23 ................................................. 12

Figure 6: Representation of major and minor groove of DNA structure ................................... 13

Figure 7: Intercalation model of aromatic ring system into DNA. A) monointercalation; B)

bisintercalation28 .................................................................................................................... 14

Figure 8: Structures of atypical and typical intercalators and groove binders .......................... 15

Figure 9: A – Cytotoxicity compounds considered in the design of Naphthalimides; B – First

series of naphthalimides synthetized for possible antitumor activity1...................................... 19

Figure 10: Structure of mono-naphthalimide derivatives......................................................... 19

Figure 11: Structure of Scriptaid.............................................................................................. 21

Figure 12: General structure of 2-metoxybenzyl derivatives .................................................... 21

Figure 13: General structure of bis-naphthalimides ................................................................. 22

Figure 14: Deformation of the DNA helix by a monointercalating agent (at left) and scheme of

intercalation between a bisintercalator and DNA (at right)30 ................................................... 23

Figure 15: Structure of Elinafide and Bisnafide ........................................................................ 23

Figure 16: Mono and Bis-Naphthalimides derivatives synthetized ........................................... 25

Figure 17: Structure of compounds 1a – c ............................................................................... 30

Figure 18: 1H NMR of imine 2a ................................................................................................ 33

Figure 19: DEPT 135 of imine 2a.............................................................................................. 33

Figure 20: Structure of compounds 2d and 2i .......................................................................... 34

Figure 21: Structure of compounds 2a, 2e and 2j .................................................................... 35

Figure 22: Structure of compounds 2b, 2f and 2k .................................................................... 36

Figure 23: Structure of compounds 2b, 2g and 2l .................................................................... 37

Figure 24: Structure of compounds 2h and 2m ....................................................................... 38

Figure 25: Structure of compounds 2n-p ................................................................................. 39

Figure 26: 1H NMR spectrum of amine 3a ............................................................................... 41

Figure 27: DEPT 135 spectrum of amine 3a ............................................................................. 41

Figure 28: Structures of compounds 3a – p ............................................................................. 42

Figure 29: 1H NMR spectrum of Napthalimidopropanol (4b) ................................................... 45

xiv

Figure 30: Structures of compounds 4a – c.............................................................................. 45

Figure 31: 1H NMR spectrum of tosyl compound 5b ................................................................ 47

Figure 32: Structure of compounds 5a – c ............................................................................... 47

Figure 33: Structure of compound 6a ...................................................................................... 49

Figure 34: Structure of compound 6b ...................................................................................... 50

Figure 35: 1H NMR of compound 7c ........................................................................................ 52

Figure 36: Structure of compounds 7a, 7b and 7c ................................................................... 53

Figure 37: 1H NMR spectrum of compound 8b ........................................................................ 54

Figure 38: Structure of compound 8a ...................................................................................... 55

Figure 39: Structure of compounds 8b and 8c ......................................................................... 55

Figure 40: MS spectrum of compound 8a ................................................................................ 56

Figure 41: MS spectrum of compound 8c ................................................................................ 56

Figure 42: Structure of Ethidium Bromide (EtBr) ..................................................................... 57

Figure 43: Mechanism of action of displacement studies ........................................................ 58

Figure 44: The effect of varying 3i and 3j concentration (0-50 µM) on fluorescence intensity of

EtBr bound DNA ...................................................................................................................... 59

Figure 45: The effect of varying 3k and 3l concentration (0-50 µM) on fluorescence intensity of

EtBr bound DNA ...................................................................................................................... 59

Figure 46: The effect of varying 3m and 3p concentration (0-50 µM) on fluorescence intensity

of EtBr bound DNA .................................................................................................................. 60

Figure 47: The effect of concentration of mono-naphthalimide derivatives with 4 carbon atoms

chain on % of fluorescence intensity ....................................................................................... 60

Figure 48: The effect of varying 3d and 3e concentration (0-50 µM) on fluorescence intensity of

EtBr bound DNA ...................................................................................................................... 62

Figure 49: The effect of varying 3f and 3g concentration (0-50 µM) on fluorescence intensity of

EtBr bound DNA ...................................................................................................................... 62

Figure 50: The effect of varying 3o concentration (0-50 µM) on fluorescence intensity of EtBr

bound DNA ............................................................................................................................. 63

Figure 51: The effect of concentration of mono-naphthalimide derivatives with 3 carbon atoms


Figure 52: The effect of varying 3a and 3b concentration (0-50 µM) on fluorescence intensity of

EtBr bound DNA ...................................................................................................................... 64

Figure 53: The effect of varying 3c and 3n concentration (0-50 µM) on fluorescence intensity of

EtBr bound DNA ...................................................................................................................... 65

xv

Figure 54: The effect of concentration of mono-naphthalimide derivatives with 2 carbons atom


Figure 55: The effect of varying 8a and 8b concentration (0-25 µM) on fluorescence intensity of

EtBr ........................................................................................................................................ 67

Figure 56: The effect of varying 8c concentrations (0-25 µM) on fluorescence intensity of EtBr

............................................................................................................................................... 67

Figure 57: The effect of concentration of bis-naphthalimide derivatives on % of fluorescence

intensity .................................................................................................................................. 68

Figure 58: Morphological changes of brain cancer cells treated with mono-naphthalimide 3l at

different concentrations (0 – 150 µM) (green arrows = viable cells, red arrow = dead cells ..... 71

Figure 59: Morphological changes of brain cancer cells treated with bis-naphthalimide 8c at

different concentrations (0 – 40 µM) (green arrows = viable cells, red arrow = dead cells ....... 72

Figure 60: Reduction in MTT tetrazolium dye with bis-naphthalimide derivatives ................... 73

Figure 61: Concentration variation on % of absorbance intensity in brain cancer SH-SY5Y cells,

treated by mono-naphthalimide derivatives (4 carbons atoms chain) ..................................... 74






treated by bis-naphthalimide derivatives ................................................................................ 77

Figure 65: Fluorescence present in brain cancer, SH-SY5Y, cells after treatments with different

concentrations of mono-naphthalimide derivatives ................................................................ 79

Figure 66: Fluorescence present in brain cancer SH-SY5Y cells after treatments with bis-

naphthalimide derivatives ....................................................................................................... 80

Figure 67: Scheme of chamber in haemocytometer .............................................................. 123

Figure 68: Section to count the cells...................................................................................... 123

Figure 69: Scheme of lay out of a 96-well plate for MTT Assay .............................................. 125

xvii

List of Tables

Table 1: The International Neuroblastoma Staging System (INSS)18 ........................................... 6

Table 2: 1H NMR signals of protons in derivatives 1a – c in CDCl3; J in Hz; δH in ppm at 400 MHz

............................................................................................................................................... 31

Table 3: 13C NMR signals of protons in derivatives 1a – c in CDCl3; δC in ppm at 100.6 MHz...... 31

Table 4: 1H NMR signals of 2d and 2i derivatives in CDCl3, J in Hz, δH in ppm at 400 MHz ......... 34

Table 5: 13C NMR signals of 2d and 2i derivatives in CDCl3, δC in ppm at 100.6 MHz ................. 34

Table 6: 1H NMR signals of 2a, 2e and 2j derivatives in CDCl3, J in Hz, δH in ppm at 400 MHz .... 35

Table 7: 13C NMR signals of 2a, 2e and 2j derivatives in CDCl3, δC in ppm at 100.6 MHz ........... 35

Table 8: 1H NMR signals of 2b, 2f and 2k derivatives in CDCl3, J in Hz, δH in ppm at 400 MHz ... 36

Table 9: 13C NMR signals of 2b, 2f and 2k derivatives in CDCl3, δC in ppm at 100.6 MHz ........... 36

Table 10: 1H NMR signals of 2c, 2g and 2l derivatives in CDCl3, J in Hz, δH in ppm at 400 MHz .. 37

Table 11: 13C NMR signals of 2c, 2g and 2l derivatives in CDCl3, δC in ppm at 100.6 MHz.......... 37

Table 12: 1H NMR signals of 2h and 2m derivatives in CDCl3, J in Hz, δH in ppm at 400 MHz ..... 38

Table 13: 13C NMR signals of 2h and 2m derivatives in CDCl3, δC in ppm at 100.6 MHz ............. 38

Table 14: 1H NMR signals of 2n – p derivatives in CDCl3,J in Hz, δH in ppm at 400 MHz ............. 39

Table 15: 13C NMR signals of 2n – p derivatives in CDCl3, δC in ppm at 100.6 MHz .................... 39

Table 16: 1H NMR signals of 3d and 3i derivatives in CDCl3, J in Hz, δH in ppm at 400 MHz ....... 42

Table 17: 13C NMR signals of 3d and 3i derivatives in CDCl3, δC in ppm at 100.6 MHz ............... 42

Table 18: 1H NMR signals of 3a, 3e and 3j derivatives in CDCl3, J in Hz, δH in ppm at 400 MHz .. 42

Table 19: 13C NMR signals of 3a, 3e and 3j derivatives in CDCl3, δC in ppm at 100.6 MHz ......... 43

Table 20: 1H NMR signals of 3b, 3f and 3k derivatives in CDCl3, J in Hz, δH in ppm at 400 MHz .. 43

Table 21: 13C NMR signals of 3b, 3f and 3k derivatives in CDCl3, δC in ppm at 100.6 MHz ......... 43

Table 22: 1H NMR signals of 3c, 3g and 3l derivatives in CDCl3,J in Hz, δH in ppm at 400 MHz ... 43

Table 23: 13C NMR signals of 3c, 3g and 3l derivatives in CDCl3, δC in ppm at 100.6 MHz........... 43

Table 24: 1H NMR signals of 3h and 3m derivatives in CDCl3, J in Hz, δH in ppm at 400 MHz ..... 44

Table 25: 13C NMR signals of 3h and 3m derivatives in CDCl3, δC in ppm at 100.6 MHz ............. 44

Table 26: 1H NMR signals of 3n – p derivatives in CDCl3, J in Hz, δH in ppm at 400 MHz ............. 44

Table 27: 13C NMR signals of 3n – p derivatives in CDCl3, δC in ppm at 100.6 MHz .................... 44

Table 28: 1H NMR signals of 4a – c derivatives in CDCl3, J in Hz, δH in ppm at 400 MHz ............. 46

Table 29: 13C NMR signals of 4a – c derivatives in CDCl3, δC in ppm at 100.6 MHz .................... 46

Table 30: 1H NMR signals of 5a – c derivatives in CDCl3, J in Hz, δH in ppm at 400 MHz ............. 48

Table 31: 13C NMR signals of 5a – c derivatives in CDCl3, δC in ppm at 100.6 MHz .................... 48

xviii

Table 32: 1H NMR signals of 6a derivatives in CDCl3, J in Hz, δH in ppm at 400 MHz .................. 49

Table 33: 13C NMR signals of 6a derivatives in CDCl3, δC in ppm at 100.6 MHz .......................... 50

Table 34: 1H NMR signals of 6b derivatives in CDCl3, J in Hz, δH in ppm at 400 MHz ................. 50

Table 35: 13C NMR signals of 6b derivatives in CDCl3, δC in ppm at 100.6 MHz .......................... 50

Table 36: 1H NMR signals of 7a derivatives in CDCl3, J in Hz, δH in ppm at 400 MHz .................. 53

Table 37: 1H NMR signals of 7b and 7c derivatives in CDCl3, J in Hz, δH in ppm at 400 MHz ...... 53

Table 38: 13C NMR signals of 7a derivatives in CDCl3, J in Hz, δC in ppm at 100.6 MHz .............. 53

Table 39: 13C NMR signals of 7b and 7c derivatives in CDCl3, J in Hz, δC in ppm at 100.6 MHz ... 53

Table 40: 1H NMR signals of 8a derivatives in DMSO, J in Hz, δH in ppm at 400 MHz ................. 55

Table 41: 13C NMR signals of 8a derivatives in DMSO, δC in ppm at 100.6 MHz......................... 55

Table 42: 1H NMR signals of 8b and 8c derivatives in DMSO, δC in ppm at 400 MHz ................. 55

Table 43: 13C NMR signals of 8a and 8c derivatives in DMSO, δC in ppm at 100.6 MHz ............. 55

Table 44: Mono-naphthalimide derivatives with 4 carbon atoms chain C50 values (µM) .......... 61

Table 45: Mono-naphthalimide derivatives with 3 carbon linker chain C50 values (µM) ........... 64

Table 46: Mono-naphthalimides derivatives with 2 carbons of linker chain C50 values (µM) .... 66

Table 47: Comparison of Mono naphthalimide compounds with the same substituent/chain . 66

Table 48: Bis-naphthalimide derivatives C50 values (µM) ......................................................... 68

Table 49: Mono-naphthalimide derivatives (4 carbon atoms chain) IC50 values (µM) after 24

hours incubation ..................................................................................................................... 74





Table 52: Comparison of mono-naphthalimide derivatives same substituent/chain ................ 77

Table 53: Bis-naphthalimide derivatives IC50 values (µM) after 24 hours incubation ................ 78

Table 54: Preparation of Test Solutions for Fluorescence binding Studies ............................. 121

Table 55: Preparation of Test Solutions for Fluorescence binding Studies ............................. 121

xix

List of Schemes

Scheme 1: General scheme of synthesis of mono-naphthalimide derivatives .......................... 17

Scheme 2: General scheme of synthesis Mitonafide and Amonafide; i) H2SO4, HNO3, 5-20ºC; ii)

N,N,-dimethylethylelediamine, EtOH, reflux; iii) 10% Pd/C, H2, EtOH ....................................... 20

Scheme 3: Synthesis of Scriptaid. a) propanoic acid, aminopropanoic ξ-acid, reflux, 4h; b) Et3N,

THF, 0ºC, 15min, followed by NH2OH, MeOH, r.t., 1h .............................................................. 21

Scheme 4: Synthesis of 2-metoxibezyl derivatives; a) EtOH, reflux, 24h; b) 2-MeOC6H4CHO,

toluene, reflux, 3h; c) NaBH4, EtOH, r.t., 3h ............................................................................. 22

Scheme 5: Synthesis of Elinafide. a) dioxane, reflux ................................................................ 24

Scheme 6: Synthesis of Bisnafide. a) THF, B2H6; b) EtOH, reflux, metasulfonic acid .................. 24

Scheme 7: Reaction between 1,8-napthalic anhydride and alkyl diamines. i) EtOH, reflux ....... 29

Scheme 8: Mechanism between 1,8-naphthalic anhydride and alkyl diamine.......................... 30

Scheme 9: Reaction between naphthalimides 1a – c and aldehydes; i) EtOH, reflux ................ 32

Scheme 10: Mechanism between compounds 1a – c and an aldehyde .................................... 32

Scheme 11: Reduction of imines 2a – p; i) THF:MeOH (1:1), NaBH4 ......................................... 40

Scheme 12: Imines reduction mechanism ............................................................................... 40

Scheme 13: Reaction between 1,8-naphthalic anhydride and alkyl aminoalcohols; i) DMF, 85°C

............................................................................................................................................... 44

Scheme 14: Reaction between 4a – c and p-toluenesulfonyl chloride; i) Py, 4°C ...................... 46

Scheme 15: Mechanism between alcohols 4a – c and tosyl chloride ....................................... 47

Scheme 16: Mesitylation of alkyl diamine; i) Mts-Cl, Py, 0°C .................................................... 48

Scheme 17: Reaction between the mesityl linkers 6a and 6b with tosylnaphthalimides; i) DMF

ii) Cs2CO3 ................................................................................................................................. 51

Scheme 18: Mechanism between linkers n and 6b and the tosylnaphthalimides n and 5c ....... 52

Scheme 19: Deprotection of compounds 7a – c; i) DCM, HBr/gCH3CO2H (20 %) ....................... 54

Scheme 20: Reaction of reduction of MTT............................................................................... 73

Scheme 21: General scheme for the synthesis of mono-naphthalimide derivatives; i) EtOH 3-

4h, reflux; ii) EtOH/Aldehyde 4-6 h, reflux; iii) THF/MeOH/NaBH4 overnight, r.t. ..................... 85

Scheme 22: General scheme for the synthesis of bis-naphthalimide derivatives; i) DMF/DBU

4h, 85 °C; ii) Py/Ts-Cl 12hrs, 4°C; iii) Py/Mts-Cl 1h, 0°C (for 6a and 6b), DMF/Cs2CO3, 12h, 60°C;

iv) DCM/HBr in glacial CH3CO2H, 24h, r.t. .............................................................................. 108

xxi

Abbreviations and symbols

13C NMR Carbon-13 Nuclear Magnetic Resonance spectroscopy

1H NMR Proton Nuclear Magnetic Resonance spectroscopy

A Adenine

CNS Central Nervous System

Comp. Compound

C Cytosine

d Doublet

dd Double Doublet

δ Chemical shift in ppm

DEPT 135 Distortionless Enhancement by Polarization Transfer 135

DBU 1,8-Diazabicyclo-[5.4.0]-undec-7-ene

DCM Dichloromethane

DMF N,N-Dimethylformamide

DMSO Dimethyl Sulfoxide

DNA Deoxyribonucleic Acid

dsDNA Double stranded DNA

equiv Molar equivalents

EtOH Ethanol

Et-Br Ethidium Bromide

FBS Foetal Bovine Serum

G Guanine

h Hours

Hz Hertz

IC50 Half maximal inhibitory Concentration

INSS International Neuroblastoma Staging System

J Coupling constant

m Multiplet

M.p. Melting point

MDR Multiple Drug Resistance

MS Mass Spectroscopy

xxii

Mts-Cl Mesitylenesulfonyl Chloride

MeOH Methanol

MTT 3-(4,5-dimethylthuazol-2-yl)-2,5-diphenyltetrazolium bromide

NB Neuroblastoma

NMR Nuclear Magnetic Resonance

PBS Phosphate Buffered Saline

ppm Parts per million

Py Pyridine

r.t. Room temperature

RNA Ribonucleic Acid

s Singlet

SAR Structure-Activity Relationship

SSC Saline-Sodium Citrate

ssDNA Single stranded DNA

THF Tetrahydrofuran

TLC Thin Layer Chromatography

TOPO II Topoisomerase II

Ts-Cl Toluenesulfonyl Chloride

T Thymine

Chapter 1 Introduction


3

1. Introduction

1.1 Cancer

Nowadays, cancer is one of the highest problems on worldwide health, representing

the 2nd major cause of death around the world and in 2030, nearly 12 million deaths

are estimated due to cancer.1,2

This disease is characterized by uncontrolled cellular growth3, because cancer cells

have this unique ability to replicate indefinitely and spread to others tissues due

invasion or metastasis.4,5 Due to these factors, cancer is now an unsolved problem and

represents a real crisis of public health across the world.4

Neoplasm, or more ordinary, tumour is when the cancer cells have a certain degree

of autonomy and a constant way to increase their size independent of environment

and of the nutritional stage of the host.6

Tumours showed higher frequency on older people. They can be classified into two

types: benign: when the cells are very similar and can behave like normal cells.

Besides, their growth is localized and don’t have the ability to metastasize or invade

other tissues, however these cells start to be problematic when almost all the cells

interfere with the normal behaviour or start to secrete excessive quantities of active

biologic substances as hormones; malignant: are characterized by their rapid cellular

growth more than normal and this cells have the ability to invade and/or

metastasize.7,8

The main reasons that turns the cancer a difficult disease to treat are: there are

over 200 different types of cancer; in most cases their aetiology is unknown; which

results in difficulties in creating/designing selective drugs against the transformed

cells; pre-clinics models are generally difficult to extrapolate; and, in the end, these

type of cells develop resistance against multiple anticancer drugs (MDR).3


4

1.2 Target Therapy

Over the years, the researchers have learned more about the genetic alterations in

cells that cause cancer and with that in mind, were able to develop drugs that target

on these changes. This type of treatment is known as targeted therapy.9

The rationally behind this therapy was formulated 15 years ago and comprises in

the design and applications of drugs that act directly and specifically on targets and

become critical to the survival of the tumor without compromise the normal organs

and tissues.10

These types of drugs are able to attack the cancer cells with less damage to normal

cells. This process is used to prevent the growth and propagation of the cancer, in

other words, this mechanism breaks the process of carcinogenesis (process which the

normal cells turn into cancer cells). The drugs act on certain parts of the cell and

interfere with signals which are needed for the development of cancer growth.

Different drugs are used to treat the cancer and may operate in different areas

depending on the phase in which the cancer is located: a) heal, slow down the growth,

and kill the cancer cells to avoid the spread; b) attenuate the symptoms caused by the

cancer.9,11

1.3 Neuroblastoma

Neuroblastoma (NB) is one of the most common solid tumors which occur in

childhood.12,13 It is the 3rd most common in children getting behind only leukemia and

Central Nervous System (CNS) tumors14,15 and is also responsible for about 10% of all

pediatric cancers. This disease is diagnosed around 17/18 months and can occur

throughout the sympathetic nervous system, and more insidious at adrenal medulla.

12,16

The pathologist J.H. Wright, in 1910, introduce for the first time the term

neuroblastoma, describing as an ensemble of tumors at childhood with characteristics

of neuronal origin. It is believed that NBs derive from immature cells originating in the

neural crest, where these cells differentiate inappropriately and the primary tumors

are mainly found in the adrenal medulla or across the paraspinal sympathetic ganglia.

Clinically, it is a heterogeneous disease which makes a great challenge for the


5

investigation. The most common sites of metastization are the bones, bone marrow

and lymph nodules; however a separate metastatic pattern, confined to the liver and

skin, is seen in children. Multiple factors such as the age at which it is diagnosed, the

stage of the disease and the genetic profile of the tumor, together with other

molecular characteristics determine the clinical outcome of the disease.14,17

The most common primary tumors (65%) appears at abdomen, with at least half of

them appear at medulla adrenal. Chest, pelvis and neck are also usual sites where NB

tumor can appear.13,16

The symptoms of NB are generally diffuse and depend on the primary tumor site

and also the presence and location of metastasis. A patient with a localized disease

sometimes showed several abdominal pain, besides in other cases, the patient doesn’t

have localized symptoms and the tumor is found by chance. In the other hand, patients

with metastatic NB are typically quite ill at the time of diagnosis, showing some

unspecific symptoms like fever, pallor or anorexia. For the metastatic specific

symptoms, these are characterized by bone pain and sometimes even marrow failure

related to bone or bone marrow metastases are also common.16

Another way to diagnosis the NB is due the histopathological characterization of

tumor tissue and also evaluate the levels of catecholamines in urine or serum if they

increased.17

In order to implement one system of consensual staging, in 1986, it was

implemented the International Neuroblastoma Staging System (INSS). In this system,

the age at which the tumor was diagnosed, the extension of the disease, and the

resectability of the tumor, classified the tumors into four stages, 1 till 4, where the

stage 4 is the most aggressive. Also have a special stage called 4s. This stage is for

children with less than 1 year old with primary tumors localized and restricted

metastasis in liver, skin and/or bone marrow. (Table 1)


6

Table 1: The International Neuroblastoma Staging System (INSS)18

Stage Description

1

Localized tumor with complete gross excision, with or without microscopic

residual disease; representative ipsilateral lymph nodes negative for tumor

microscopically (nodes attached to and removed with the primary tumor may

be positive).

2A Localized tumor with incomplete gross excision; representative ipsilateral

nonadherent lymph nodes negative for tumor microscopically.

3B

Localized tumor with or without incomplete gross excision, with ipsilateral

nonadherent lymph nodes positive for tumor. Enlarged contralateral lymph

nodes must be negative microscopically.

3

Unresectable unilateral tumor infiltrating across the midline*, with or

without regional lymph node involvement; or localized unilateral tumor with

contralateral regional lymph node involvement; or midline tumor with

bilateral extension by infiltration (unresectable) or by lymph node

involvement

4 Any primary tumor with dissemination to distant lymph nodes, bone, bone

marrow, liver, skin and/or other organs (except as defined by stage 4S)

4s

Localized primary tumor (as defined for stage 1, 2A or 2B), with

dissemination limited to skin, liver, and/or bone marrow† (limited to infants

< 1 year of age)

These outcomes could be very different between the subtypes of NBs varying from

regressive low risk till aggressive high risk. The low risk is characterized by tumor with

small or no risk and have favorable clinic prognostic. (stage 1 2 and 4s). The NBs with

high risk are aggressive and are metastatic tumors with a poor diagnosis with low or no

response to chemotherapy and show outcomes not favorable (stage 3 and 4).18

Following a general tendency in the evolution of medicine, the treatment of

neuroblastoma requires an interdisciplinary work involving surgeons, radiation

therapists, chemotherapists and chemists. Such an approach is best adapted to the

construction of the treatment programme. The therapy for neuroblastoma is adapted

to each patient depending, for example, on the disease stage, and patient age. The


7

available options range from combined modality of surgery, chemotherapy and

radiotherapy.15

Due of heterogeneity of this disease, it is a challenge to find the cure for the

patients with high risk but also to avoid the overtreatment to the patients with

favorable diagnosis. The surgical removal of primary tumor remains the most

important treatment of NB. For localized tumor, surgery is an option, but for the

metastatic tumor the use is debatable. Chemotherapy is, generally used for the

treatment of subtypes of intermediate NB and high risk that require one systemic

approach due a metastasis. The objective of chemotherapy is reducing the size of the

tumor, to make it easy for the surgical removal and eliminate the metastatic diseases.

This is the predominant treatment for the treating of NB.19 There are many alkylating

drugs use as chemotherapeutic agents in the treatment of NB: cyclophosphamide,

(busulphan, melphalan), vinca-alkaloids (vincristine), antracyclines (doxorubicin), and

platinum analogues (cis-platinum, Carboplatinum).17 Currently, therapy options for

neuroblastoma comprise combined modality of surgery, chemotherapy and

radiotherapy. This approach is individually modified depending on disease stage and

patients age at presentation.16,18

In spite of the advances of the actual treatment, the clinical trials on course and

investigations of basic science NB still to be a complex medical challenge with a clinical

curse unpredictable and dismal overall outcome for advance stage disease.

1.4 Cell Death

The cells, due the extracellular stress or because their necessities, they are continuously

adjusting their function and structure to maintain their normal behavior.6

When the cells are subject to pathological stimuli and physiological stress, they

adapt and continue with their function and duty viability or don’t support this type of

damage and take the process called cell death (Figure 1).

The study of cell death comes over than a century. The increase knowledge of

different mechanisms of the apoptotic process clearly showed the complexity and the

difficulty of distinguishing the different forms of cell death, necrosis, apoptosis and/or

oncosis.20,21


8

Figure 1: Stages of cellular response to a stress stimuli and harmful6

Depending on the type of damage that the cell suffers, the cells could die through

two processes: apoptosis – the way to cell death that is introduced by a suicide

program tightly regulated where the cells are destined to die, and there is activation of

enzymes capable to degrading the nuclear DNA of cells and also the cytoplasmic

proteins. This process is characterized by the shrinking, condensation and margination

chromatin and riffling of the plasmatic membrane, called budding. The cell is divided

into apoptotic bodies, becoming these fragments targets of the phagocytes preventing

the leakage of them; necrosis – this process results from degradative action of

enzymes to damage cells. The main characteristics of this process are, firstly, the

cellular swelling, the condensation of chromatin and the inability to maintain the

integrity of the cell membrane and their constituents are leak out inducing

inflammation. However, these two processes can coexist because the apoptosis induce

pathological stimuli which in turn induces the activation process of necrosis (Figure

2).6,20,21


9

Figure 2: Cellular characteristics of necrosis (at left) and apoptosis (at right)6

The process of cell death is one of the most important processes in the evolution of

diseases of any organ or tissue. Besides that, it is also important that during the

development of the organs, homeostasis is maintained. It is believed that the

development of malign tumors results due to deregulated proliferation or the

incapacity the cells suffer apoptotic cell death.22

The disruption and the anomaly regulation of cell death and cell growth is one of

the reasons that cause cancer. Recent studies showed that the induction of apoptosis

by therapeutics agents in cancer cells is critical to the tumor developing.21

1.5 DNA

The nomenclature known nowadays of deoxyribonucleic acid (DNA) has undergone

a major evolution. The DNA is organized into a double-helical structure and consists on

a very long sequence of base pairs. The nucleic acids are a long chain or a polymer of

repeated subunits, called nucleotides. Each nucleotide consists of 3 parts: one sugar of

5 carbons, one phosphate group and one nitrogenous base. (Figure 3)


10

Figure 3: Components of nucleic acids: bases, sugars and phosphates (adapted)23

The formation of DNA chain occurs in 3 steps: 1st each base is connected to the

sugar in C1’, forming the nucleoside; 2nd when the phosphate group is connected to

the C5’ of the same sugar, the nucleotides are formed; 3rd in the end, these

nucleotides polymerize by condensations reactions to form the DNA chain. (Figure 4).23


11

Figure 4: Formation of DNA chain23

DNA is a nucleic acid polymerized which contains genetic information that specifies

the biological development of all cellular life forms. The molecule of DNA is responsible

for the genetic propagation of all traces, so it also call as molecule of heredity. During

the process of reproduction, DNA is replicated and transmitted to the offspring. The

ribonucleic acid (RNA) is accountable to the transcription process (process where the

DNA sequence is copied), and this molecule are used in protein synthesis to code a

sequence of specific proteins (translation).


12

The double stranded DNA (dsDNA) is the structure in which the DNA presents the

double stranded antiparallel and this structure is held together by hydrogen bound

interactions between complementary base pairs: Adenine (A), Thymine (T), Guanine

(G) and Cytosine (C). The C only can interact with G and A with T. Adenine and Guanine

are purines besides Cytosine and Thymine are pyrimidines (Figure 5). In biological

systems, the DNA structure presents as dsDNA but also can show as single stranded

DNA (ssDNA) but this case is not usual.

Figure 5: The two common Watson-Crick base pairs of DNA23

The existence of several exposed reactive spots on the surface of double helix is one

of the characteristics of DNA (Figure 6). For example, the amino group N2 of Guanine

in minor groove of DNA is liable to action of the drugs. The specific binding of the

drugs to DNA mainly involves the recognition of the G at minor groove and also by

hydrogen bonds interactions of the N2 exocyclic amino group. However, this amino

group is normally stericly hindered that causes a decrease of the affinity. Also in minor

groove, the N3 of Guanine is a good spot for the drugs. but the most reactive spot is

N7 of Guanine where a number of metallic ions and alkylating agents can attack.24


13

Figure 6: Representation of major and minor groove of DNA structure

The key of developing of new chemotherapy therapeutics is understand at

molecular level how the genetic information is expressed and how to stimulate or

prevent the genetic expression. Once the human genome was completely sequenced

there has been a great increase on the study of the genetic sequence of organisms. To

prevent the appearance certain diseases a lot of efforts have been made to control

specific gene expression.

Nowadays, the study of biochemical sensor technologies that focus on the direct

detection of nucleic acids (DNA and RNA) has generated a lot of interest and they play

a major role in different scientific areas like forensics, pharmaceutical applications,

medical diagnosis, genetic screening, rational drug design, diagnosis of drug resistance,

food and agricultural analysis, etc. A new exciting field of research has develop to

understand this principles to the rational design, synthesis and applications of new

DNA intercalators.24

Targeting DNA is a highly important approach to the development of novel

therapies. The interaction with DNA may interfere with transciption and hence with

translation. Also, DNA replication is essential for the cell cycle progression. The nucleic

acids interact reversibly with a great amount of chemical species, including water, and

metallic ions and their complexes, organic molecules and proteins. These molecules

may interact with the duplex nucleic acids in 3 primary forms: a) binding along the

outside of the helix through non-specific interactions and primarily electrostatics; b)


14

specific interactions of groove binding with a bound molecule with the edges of base

pairs like in minor and major groove of NA and c) intercalation of aromatic cyclic

systems between base pairs.25

1.6 DNA Intercalators

The search for new anticancer drugs and the development of new molecules able to

bind to DNA and show anticancer activity has received great attention nowadays.26

DNA is one of the most important biological targets to design anticancer drugs.27 Exist

3 primary ways by which some molecules and ions interact with nucleic acids duplex:

a) binding along the exterior of the helix trough interactions which are usually non-

specific and are primarily electrostatic; b) specific groove-binding interactions of the

bound molecule with the edges of base-pairs in either of the (major or minor) grooves

of nucleic acids; and c) intercalation of planar or approximately planar aromatic ring

systems between base-pairs.28

The process of intercalation consists of inserting molecules between the base pairs.

This is a very important process, especially with regards to the function of many

anticancer drugs and also the way intercalators interact with the DNA structures

represent new potential anticancer drugs (Figure 7).29,30

Figure 7: Intercalation model of aromatic ring system into DNA. A) monointercalation; B) bisintercalation

28


15

The intercalation process begins with the transfer of the intercalating molecule

from an aqueous environment to the hydrophobic space between 2 adjacent base

pairs of DNA. The disruption of the organized shell of water molecules around the

ligand also called the hydrophobic effect causes the positive entropy, which leads to a

process that is thermodynamically favoured.30

In structural terms, the intercalating molecules are typical fused bi/tricyclic ring

structures or atypical molecules with nonfused rings systems. (Figure 8).31

Figure 8: Structures of atypical and typical intercalators and groove binders

These molecules are characterized (Figure 8) by a planar flat π-deficient aromatic or

heteroaromatic ring systems, a moiety of approximately the size and shape of a DNA

base pairs.30,32,33

They insert perpendicularly to the axis of the helix into the DNA, between adjacent

base pairs, without forming any covalent bonds, and the formed complex is stabilized

by hydrophobic interactions, van der Walls forces, hydrogen bonds and charge transfer

forces.30,32 These molecules bind reversibly to double helical DNA. This process leads to

changes at sugar phosphate torsional angles in order to accommodate the

intercalating compound. The DNA back bone conformation suffer some changes

inducing the unwinding, lengthening, and stiffening of the double helix interfering with

DNA protein interaction or may affect the replication processes of the cells, leading to

the cellular death and genotoxic effects or even may lead to the retardation, inhibition


16

of transcription and replication. The DNA intercalators may be mutagenic through

interference with the molecular recognition and function of DNA binding proteins,

such as polymerases, transcription factors, DNA repair systems, and, specially,

topoisomerases.28,31,34

The intercalators are oriented parallel to the base pairs, commonly π-stacking in

major groove, although some bindings seem to occur preferentially in the minor

groove of DNA. In dsDNA helix, the nucleic acids are located in almost coplanar

arrangement, which allows planar aromatic molecules to intercalate between two

base pairs. When intercalated, it is possible note π-stack interactions (intercalated

moiety), hydrogen-bonding, van der Waals interactions, hydrophobic interactions and

steric hindrance effects.3,33

Comparing the DNA intercalators with minor groove binding agents, these are less

sequence selective, and also show propensity for G-C regions. The main reason of this

selectivity is due to complementary hydrophobic or electrostastic interactions, which

are due to substituents attached to the chromophore within the major or minor

grooves.30

In general the stronger the interactions of the intercalator with DNA, the higher the

antitumor activity of the drug will be. In order to use any active substance as a drug,

the recognition of all possible interactions with target molecules is essential.34

1.7 Naphthalimides

An active field of research of searching of new chemotherapeutic agents and also

new approaches to treat cancer is stimulated by the discovery of new biological targets

and the possibility of obtain new drugs without undesirable secondary effects.32

Naphthalimide (1H-benzo[de]isoquinoline-1,3-(2H)-diones) is a polycyclic amide

consisting of a flat, and π-deficient aromatic or heteroaromatic system with high

hydrophobicity (Scheme 1).35,36


17

Scheme 1: General scheme of synthesis of mono-naphthalimide derivatives

Naphthalimide derivatives have been investigated through their great potential in

medicinal chemistry, assembly and supramolecular reorganization and in material

science. This type of structure interacts with many active targets in biological system

by non-covalent forces like π-π stacking or hydrogen bonding with different enzymes

and receptors. The majority of these compounds which contain this moiety are

fluorescents and exhibit biologic properties such as antitrypanosomal, antiviral (herpes

and HIV), antimicrobial, antioxidative,36 anesthetics locals, analgesics, antagonists

activity in serotonin 5-HT3 and 5-HT4 receptors as chemosensory etc. Furthermore

these derivatives are also used in some other non-biologic applications like, optic

brighters, non-biologic sensor, fluorescent probes, fluorescent dyes and for the

synthesis of polymers, lucifer dyes, solar energy, etc.26,34,37

The naphthalimide derivatives can act as photo-reagents, which can induce damage

to DNA molecules and with that they are capable to kill the cells when these are

photo-activated. This opens the possibility of this type of compounds act at photo-

therapy.

In terms of synthesis these compounds are easily synthetized with high purity and in

good yields.26

Many derivatives with different substituents have been synthesized in order to

produce new potential anticancer drugs with cytotoxic activity and low toxicity. These

results also catalyzed the use of derivatives for different pharmacological purposes.

Considering the amount of patents and research articles, it is obvious that the

naphthalimides have many biologic and non-biologic applications. The major

application of these derivatives is related to their capacity to act as anticancer agents.1

The naphthalimides represent an important class of drugs characterized by their

high cytotoxic activity against a great variety of murine and tumoral human cells. They

showed their biologic activity through the formation of ternary complexes DNA-


18

Intercalant-topo II or by inhibiting other enzymes and/or transcption factors which act

upon DNA. The strong interactions with the DNA, as was said before, play an important

rule to their pharmacological properties.38

The naphthalimide derivatives are DNA intercalating agents, because they bind to

DNA through insertion between base pairs of double helix. The main forces to bind the

DNA are the interaction by charge transfer and the stacking between base pairs.25

However, the small molecules can bind to DNA in different ways like: through

binding at grooves (most at minor groove) or externally (especially if the molecules

show good capacity for stacking). It is also possible that the binding mode depends on

the DNA sequence. The technics of absorption of UV/visible and fluorescence

spectroscopy are excellent to monitoring the binding to the nucleic acids.26

Nowadays, the antitumor activity is related to the capacity to inhibit the human

DNA topo II. Besides that, the bis-1,8-naphthalimides analogs kill the eukaryotic cells

through the stabilization of the cleaved complex of topo II with the DNA.39

Numerous derivatives of mono and bis naphthalimides have shown to exhibit

potent anticancer properties against a great variety of murine and human tumor cells.

These observations with structural optimization due to the maintenance the

naphthalimide moiety and add/change appropriate functional groups can help to

reduce the systemic toxicity.25,40,41

The first series of naphthalimide derivatives with biological activity appeared in

1973. These compounds resulted from the combination of important structural

characteristics of existing anti-tumoral drugs as acid aristocholic, tilorone,

ciclohexamide and morpholino-β-thalidomide in one molecule (Figure 9).1,42


19

Figure 9: A – Cytotoxicity compounds considered in the design of Naphthalimides; B – First series of naphthalimides synthetized for possible antitumor activity1

The naphthalimide derivatives that had the greatest impact were the amonafide (3-

amino-1,8-naphthlimide), mitonafide (3-nitro-1,8-naphthalimide) and azonafide.

(Figure 10).28,39,43 Amonafide and mitonafide have been tested in clinic trials for the

treatment of solid tumors. Both entered into phase II clinical trials and showed high

antitumor activity with IC50 values 0.47mM and 8.8mM respectively against HeLa cell

lines.26

Figure 10: Structure of mono-naphthalimide derivatives

Both derivatives bind to dsDNA by intercalation. When they bind to DNA there is an

increased of viscosity of sonicated rod-like DNA fragments and consequent increase in

length. The mode of action of these two drugs is inducing a topo II mediated DNA

cleavage at nucleotide nº 1830 on pBR322 DNA.

The other naphthalimide derivatives didn’t show this specific cleavage. The

explanation is the lacking of the basic side chain because this could interacts sterically

with the enzyme active site in the ternary complex.28


20

Some SAR (structure-activity relationship) studies have shown that some

parameters are essential and influence the anticancer properties of naphthalimide

derivatives. The main characteristics are the presence of a basic terminal group in the

side chain and a 2 or 3 methylene groups separating the N-terminal of the side chain

from the naphthalene ring. These characteristics showed to play a key role in

anticancer activity.1

However, they have never been employed in therapeutics because nearly all clinical

trials failed because of several factors: low water solubility, poor therapeutic index,

dose-limiting bone marrow toxicity (mitonafide) or unexpected central neurotoxicity,

hematotoxicity and limited efficiency (amonafide).30,25, 43

With these results and observations, some changes were made to optimize the

activity and reduce the toxicity by keeping the key naphthalimide moiety intact while

adding suitable functional groups.40 They are also used as leads to design bis-

intercalators.30

Amonafide and Mitonafide can be synthetize in the same reaction. 1,8-naphthalic

anhydride react with the HNO3 in presence of H2SO4 to give the nitro derivative. Next,

was added the amine, N,N,-dimethylethylelediamine, to give the Mitonafide. With the

mitonafide is possible to obtain the amonafide by reduction of nitro group to amino

group. (Scheme 2).43

Scheme 2: General scheme of synthesis Mitonafide and Amonafide; i) H2SO4, HNO3, 5-20ºC; ii) N,N,-dimethylethylelediamine, EtOH, reflux; iii) 10% Pd/C, H2, EtOH

Another naphthalimide derivative, Scriptaid, showed biological activity as a potent

histone deactylase (HDAC) inhibitor because of its structural similarity to hidroxamic

acid-containing HDAC inhibitors (Figure 11).1,44 Also the accumulation proteins as


21

Acetylated H3 and H4 histone in certain type of cells, confirm the effect of Scriptaid as

HDAC inhibitor.45

This compound has used for the treatment of several diseases like cancer, infectious

diseases, immune deficiency ischemic injury, etc1

Figure 11: Structure of Scriptaid

The synthesis of Scriptaid starts with the reaction between 1,8 naphthalic anhydride

with aminopropanoic ξ acid in reflux in acid medium. The resulting compounds, was

treated by ethyl chloroformat in triethylamine. In the end the mixture obtain is treated

with NH2OH to give the Scriptaid (Scheme 3).45

Scheme 3: Synthesis of Scriptaid. a) propanoic acid, aminopropanoic ξ-acid, reflux, 4h; b) Et3N, THF, 0ºC,

15min, followed by NH2OH, MeOH, r.t., 1h

Several naphthalimide derivatives were synthetized using a 2-metoxibenzyl group

as a substituent and variating the alkyl chain between 2 and 10 carbons (Figure 12).

Previous work demonstrated structures with polymethylene chains where the length

of the chain can change may have relevance for the anticancer activity.32

Figure 12: General structure of 2-metoxybenzyl derivatives


22

The synthesis of this type of compounds starts with the reaction between 1,8-

naphthalic anhydride with a alkyldiamine with different chain length. After that the 2-

metoxibenzaldehyde was added creating the imine and after the imine is reduce using

sodium borohydride. (Scheme 4)

Scheme 4: Synthesis of 2-metoxibezyl derivatives; a) EtOH, reflux, 24h; b) 2-MeOC6H4CHO, toluene,

reflux, 3h; c) NaBH4, EtOH, r.t., 3h

1.8 Bis-Naphthalimides

One way to enhance the binding constant was to create bifunctional, or even

polyfunctional intercalators based on their corresponding monomers. (Figure 13) The

idea of synthesizing this type of compounds has stimulated by the idea that the

pharmacological activity of intercalating drugs can be significantly increased with

higher DNA binding constants and a slower dissociation rates from DNA expected for

bisintercalators relative to monointercalators. Another reason may be the increase the

global size occupied by the ligand could afford greater opportunities for sequence

selective but this reason is not clear.28,46

Figure 13: General structure of bis-naphthalimides

Bis-naphthalimide derivatives are a type of compounds that comprise two mono

naphthalimides units and they are connected by a polyamine spacer (linker).3,26 They

showed higher affinity and selectivity in binding when compare to their mono

counterparts. The main reason can be attributed to the increased local concentration

of the active moiety (Figure 14).39,47


23

Figure 14: Deformation of the DNA helix by a monointercalating agent (at left) and scheme of intercalation between a bisintercalator and DNA (at right)30

The two bis-naphthalimido derivatives that deserve more attention and were the

first ones to reach the clinical trials against solid tumors were Elinafide and Bisnafide.

(Figure 15) They exhibited very high in vivo and in vitro activity.48,49

Figure 15: Structure of Elinafide and Bisnafide

Elinafide (LU79553) showed high activity against a variety of human xenograft

models such as LX-1 (lung), CX-1 (colon), and LOX (melanoma).26

The synthesis of this compound consists in the reaction between of 1,8 naphthalic

anhydride with the diamine in dioxane (Scheme 5).


24

Scheme 5: Synthesis of Elinafide. a) dioxane, reflux

Some reports showed that the Bisnafide analogues kill the eukaryotic cells by

stabilizing the cleavage complex of topo II with DNA but this is still controversial since

in another studies, Elinafide was found not to be a poison of topo II.38

The synthesis of Bisnafide start with a reaction between ethylenediamine with a

carbonyl group protected. To the intermediate formed was added acid to remove the

protecting group. To the resulting product was added B2H6 to reduce the carbonyl

group. In the end the diamine produced react with the nitro naphthalimide (Scheme

6).50

Scheme 6: Synthesis of Bisnafide. a) THF, B2H6; b) EtOH, reflux, metasulfonic acid

The presence of positive charges on bis-naphthalimide derivatives creates a good

intercalation because the positive charges interact with the negatively charged

backbone of the DNA providing enhanced DNA stability.48

Some SAR studies and analysis showed that the methylene middle chain is important

for the activity. Longer the methylene middle chain of at least 8 methylene groups


25

between the two naphthalimides rings is important for example for the activity against

anti-Plasmidium.49

The major problem of this type of compounds is that they are very insoluble and it

would be very difficult to test them in biological assays. One way to overcome this

problem is to introduce more than two nitrogen atoms in the linker chain to improve

the solubility without compromise the biological activity.51

1.9 Aims

The aim of this work is to synthesize a series of mono and bis-naphthalimides

derivatives with potential anticancer activity. These novel derivatives will be tested in

brain cancer cells, SH-5YSY, to determine their cytotoxicity and DNA binding properties

(Figure 16).

Figure 16: Mono and Bis-Naphthalimides derivatives synthetized

Chapter 2 Results and Discussion


29

2. Results and Discussion

2.1 Introduction

The aim/objective for the synthesis of these naphthalimides was to test them

against cancer cells, study the effect of modifications in the linker chain, either in

length and substituents. The project included binding studies of the molecules to DNA.

Earlier work showed that certain types of linkers, the length or the nature of the

substituent was extremely important for enhanced solubility and effective biological

activity against colon cancer cells.52,53

2.1.1 Synthesis of naphthalimides 1a – c

Scheme 7: Reaction between 1,8-napthalic anhydride and alkyl diamines. i) EtOH, reflux

1,8-Naphthalimides (1a – c) were obtained by a straight forward method in good

yields (58 – 86 %), combining 1,8-naphthalic anhydride with alkyl diamines (Scheme 7).

The nitrogen of alkyl diamines acts as nucleophile attacking the carbonyl of the

naphthalic anhydride, with ring opening to generate an intermediate stabilized by

resonance. Next, the same nitrogen attacks the other carbonyl group to give the desire

derivatives 1a – c by water elimination. (Scheme 8)

Chapter 2 – Results and Discussion

30

Scheme 8: Mechanism between 1,8-naphthalic anhydride and alkyl diamine

To confirm and determine the structures of the products obtained, 1H and 13C NMR

spectroscopy was used (Figure 17).

Figure 17: Structure of compounds 1a – c

1H NMR showed the presence of the -CH2 attached to the naphthalimide ring in all

derivatives. The protons H-1’ appeared between δH = 4.20 – 4.30 ppm as triplets (t)

with a J vicinal between δH = 6.4 – 7.0 Hz for all derivatives. The H-2’ of 1a appeared as

a triplet at δH = 3.08 ppm with a J vicinal = 6.8 Hz. Compounds 1b and 1c showed

internal methylenic protons as multiplets (m) at δH = 1.77 – 1.95 ppm. The methylene

unit near to the nitrogen of the naphthalimide ring appeared at lower field, due to

deprotection effect of nitrogen atom connected to the amide. At aromatic region the

protons H-4 appeared as double doublets (dd) δH = 8.61 – 8.62 ppm with a J meta = 1.2

Hz and J ortho δH = 7.4 – 8.4 Hz. The H-5 appeared as double doublets δH = 7.76 – 7.77


31

ppm with a J ortho = 7.2 – 8.4 Hz. Finally H-6 appeared as double doublets at δH = 8.21

– 8.22 ppm with a J meta= 0.8 – 1.2 Hz and J ortho = 8.0 - 8.4 Hz. (Table 2)

Table 2: 1H NMR signals of protons in derivatives 1a – c in CDCl3; J in Hz; δH in ppm at 400 MHz

Compound

1H ppm

H-4 H-5 H-6 H-1’ H-2’ H-3’ H-4’

1a 8.62 dd 7.76 dd 8.22 dd 4.29 t 3.08 t --- ---

1b 8.61 dd 7.77 dd 8.22 dd 4.30 t 1.88-1.95 2.78 t ---

1c 8.61 dd 7.76 dd 8.21 dd 4.20 t 1.77-1.82 1.55-1.63 2.79 t

13C NMR spectra, including DEPT 135 confirms the methylene units. In the aliphatic

region all the C-1’ appeared at δc = 37.4 – 43.1 ppm and C-2’ at δc = 25.1 – 40.9 ppm.

The C-3’ peaks of 1b and 1c show up between δc = 30.8 – 39.5 ppm and the C-4’ of the

1c derivative appeared at δc = 41.6 ppm. At the aromatic region signals due to C-4

appeared δc = 131.2 – 131.5 ppm, to C-5 at δc = 126.8 – 126.9 ppm, to C-6 at δc = 133.9

ppm, to C-3a at δc = 122.4 – 122.5 ppm, to C-6a at δc = 131.2 – 131.5 ppm, to C-10 at δc

= 128.0 – 128.1 ppm and to C-1 at δc = 164.2 – 164.5 ppm. (Table 3)

Table 3: 13C NMR signals of protons in derivatives 1a – c in CDCl3; δC in ppm at 100.6 MHz

Comp.

13C ppm

C-1/3 C-4 C-5 C-6 C-3a C-6a C-10 C-1’ C-2’ C-3’ C-4’

1a 164.5 131.3 126.9 133.9 122.5 131.2 128.1 43.1 40.9 --- ---

1b 164.2 131.2 126.8 133.9 122.4 131.5 128.0 37.4 31.9 39.5 ---

1c 164.3 131.5 126.8 133.9 122.4 131.2 128.0 39.8 25.1 30.8 41.6


32

2.1.2 Synthesis of compounds 2a – p

Scheme 9: Reaction between naphthalimides 1a – c and aldehydes; i) EtOH, reflux

Compounds 2a – p were obtained by a straight forward method by reaction of 1a –

c with aldehydes. The respective imines were obtained and isolated in good yields 54 –

97 % (Scheme 9). In this reaction, the amine attacks the C=O group forming the

intermediate showed in Scheme 10. A water molecule was eliminated to give the

iminic derivatives 2a – p.

Scheme 10: Mechanism between compounds 1a – c and an aldehyde


33

The structures of the molecules synthesized were confirmed by NMR spectroscopy.

The 1H NMR of compound 2a is shown in Figure 18 and it’s DEPT 135 in Figure 19.

1H NMR

Figure 18: 1H NMR of imine 2a

DEPT 135

Figure 19: DEPT 135 of imine 2a

H-2’ H-1’

OCH3

Naphthalimide ring

H-4/9

H-5/8

H-6/7 + H-1’’

H-3’’/4’’

H-2’’/5’’

CH2

N=CH


34

Figure 20: Structure of compounds 2d and 2i

For compounds 2d and 2i (Figure 20) the 1H NMR, the main peaks are: at aromatic

region appeared the H-2’’/H-5’’ protons at δH = 7.67 – 7.77 ppm as a doublet (d) with J

ortho = 8.4 Hz and the H-3’’/H-4’’ at δH = 8.06 – 8.15 ppm as a doublet with a J ortho =

7.2 – 8.8 Hz. The iminic proton, H-1’’, in all derivatives appeared between δH = 8.35 –

8.39 ppm as a singlet. (Table 4)

Table 4: 1H NMR signals of 2d and 2i derivatives in CDCl3, J in Hz, δH in ppm at 400 MHz

Compound

1H ppm

H-1’ H-2’’/5’’ H-3’’/4’’

2d 8.35 s 7.67 d 8.06 d

2i 8.39 s 7.77 d 8.15 d

13C RMN spectrum showed the iminic carbon, C-1’’, δC = 160.2 – 161.0 ppm and C-

2’’/C-5’’ and C-3’’/C-4’’ appeared at δC = 128.6 – 129.7 ppm and 124.2 – 125.4 ppm,

respectively. (Table 5)

Table 5: 13

C NMR signals of 2d and 2i derivatives in CDCl3, δC in ppm at 100.6 MHz

Compound

13C ppm

C-1’’ C-1a’’ C-2’’/5’’ C-3’’/4’’ C-3a’’

2d 160.2 129.5 129.0 125.4 148.9

2i 161.0 129.2 128.6 124.2 148.7


35

Figure 21: Structure of compounds 2a, 2e and 2j

For compounds 2a, 2e and 2j (Figure 21) the 1H NMR spectra showed at the

aliphatic region one signal at δH = 3.82 – 3.83 ppm as a singlet corresponding to

methoxyl group. The H-2’’/H-5’’ appeared at δH = 7.30 – 7.66 ppm as a doublet (J ortho

= 8.4 – 8.8 Hz) and H-3’’/H-4’’ appeared at δH = 6.80 – 6.90 ppm as another doublet

with same coupling. The iminic proton, H-1’’, of all derivatives appeared between 8.20

– 8.28 ppm as singlets. (Table 6)

Table 6: 1H NMR signals of 2a, 2e and 2j derivatives in CDCl3, J in Hz, δH in ppm at 400 MHz

Compound 1H ppm

H-1’’ H-2’’/5’’ H-3’’/4’’ OCH3 2a 8.28 s 7.64 d 6.89 d 3.83 s 2e 8.22 s 7.30 d 6.80 d 3.82 s 2j 8.20 s 7.66 d 6.90 d 3.83 s

In the 13C NMR spectra, iminic carbons, C-1’’, appeared at δC = 160.5 – 161.9 ppm,

the methoxyls showed δC = at 50.1 – 55.3 ppm and C-2’’/C-5’’ and C-3’’/C-4’’ appeared

at δC = 129.6 – 129.7 ppm and 113.7 – 113.9 ppm, respectively. (Table 7)

Table 7: 13C NMR signals of 2a, 2e and 2j derivatives in CDCl3, δC in ppm at 100.6 MHz

Compound

13C ppm

C-1’’ C-1a’’ C-2’’/5’’ C-3’’/4’’ C-3a’’ OCH3

2a 161.9 131.6 129.7 113.9 160.1 55.3

2e 161.2 132.2 129.6 113.7 159.8 50.1

2j 160.5 132.0 129.6 113.9 161.4 55.3


36

Figure 22: Structure of compounds 2b, 2f and 2k

For compounds 2b, 2f and 2k, (Figure 22) the protons H-3’’/H-5’’ showed up at δH =

7.29 – 7.34 ppm as a doublet with a J ortho = 7.2 – 8.2 Hz, H-4’’ at δH = 7.18 – 7.23 ppm

as a double doublet with a J ortho = 7.2 and 8.8 Hz (2b and 4f) and 7.6 and 8.4 Hz for

2k. The iminic proton, H-1’’ of all derivatives appeared between 8.45 – 8.51 ppm as

singlets. (Table 8)

Table 8: 1H NMR signals of 2b, 2f and 2k derivatives in CDCl3, J in Hz, δH in ppm at 400 MHz

Compound

1H ppm

H-1’’ H-3’’/5’’ H-4’’

2b 8.49 7.28 d 7.18 dd

2f 8.51 7.33 d 7.22 dd

2k 8.45 7.34 d 7.23 dd

In the 13C NMR, the iminic carbon, C-1’’ appeared at δC = 160.5 – 161.9 ppm, C-4’’,

C-2’’/C-6’’ and C-3’’/C-5’’ at δC = 128.4 – 129.1, 159.3 – 160.1 ppm and 128.1 – 128.3

ppm, respectively. (Table 9)

Table 9: 13C NMR signals of 2b, 2f and 2k derivatives in CDCl3, δC in ppm at 100.6 MHz

Compound

13C ppm

C-1’’ C-1a’’ C-2’’/6’’ C-3’’/5’’ C-4’’

2b 161.5 129.0 160.1 128.1 128.4

2f 161.8 134.1 159.2 128.3 128.8

2k 161.2 132.0 159.3 128.3 129.1


37

Figure 23: Structure of compounds 2b, 2g and 2l

1H NMR pattern of the p-substitued phenyl group in compounds 2c, 2g and 2l is

similar to other substituted phenyl compounds (Figure 23). Due to the Fluor

conjugation forwards the ring the chemical shifts are at slightly higher fields. In the

aromatic zone the H-2’’/H-5’’ appeared at δH = 7.56 – 7.71 ppm as a doublet with a J

ortho = 8.8 – 9.2 Hz and the H-3’’/H-4’’ appeared at δH = 6.95 – 7.08 ppm as a triplet

with a J ortho = 8.4 - 8.8 Hz. The iminic proton, H-1’’ of all derivatives appeared

between 8.25 – 8.31 ppm as a singlet. (Table 10)

Table 10: 1H NMR signals of 2c, 2g and 2l derivatives in CDCl3, J in Hz, δH in ppm at 400 MHz

Compound

1H ppm

H-1’’ H-2’’/5’’ H-3’’/4’’

2c 8.31 s 7.69 d 7.06 t

2g 8.25 s 7.56 d 6.95 t

2l 8.25 s 7.71 d 7.08 t

With the carbon (13C), the iminic carbon, C-1’’ appeared at δC =161.0 – 162.0 ppm.

At the aromatic region the C-2’’/C-5’’ and C-3’’/C-4’’ appeared at δC = 129.6 – 130.0

ppm and δC = 115.5 – 117.0 ppm respectively. (Table 11)

Table 11: 13C NMR signals of 2c, 2g and 2l derivatives in CDCl3, δC in ppm at 100.6 MHz

Compound

13C ppm

C-1’’ C-1a’’ C-2’’/5’’ C-3’’/4’’ C-3a’’

2c 162.0 134.8 130.0 117.0 161.2

2g 161.4 136.2 129.8 115.5 159.6

2l 161.0 142.0 129.6 116.9 160.2


38

Figure 24: Structure of compounds 2h and 2m

The substituted phenyl group in compounds 2h and 2m (Figure 24) are also

electron-rich system: H-3’’/H-4’’ appeared at δH = 6.60 – 6.69 ppm as a doublet with J

ortho = 8.8 Hz and the H-2’’/H-5’’ at δH = 7.50 – 7.59 ppm with the same coupling

constant. The two methyl groups showed up as singlets at δH = 2.99 ppm. The iminic

proton, H-1’’, of all derivatives appeared between 8.16 – 8.30 ppm as a singlets. (Table

12)

Table 12: 1H NMR signals of 2h and 2m derivatives in CDCl3, J in Hz, δH in ppm at 400 MHz

Compound

1H ppm

H-1’’ H-2’’/5’’ H-3’’/4’’ CH3

2h 8.30 s 7.50 d 6.60 d 2.99 s

2m 8.16 s 7.59 d 6.69 d 2.99 s

In the 13C NMR spectra, the iminic carbon, C-1’’ appeared at δC = 151.9 – 161.1 ppm,

the two methyl group at δC = 40.2 ppm and the C-2’’/C-5’’ and C-3’’/C-4’’ at δC = 129.4

– 123.2 ppm and 129.4 – 130.0 ppm, respectively. (Table 13)

Table 13: 13C NMR signals of 2h and 2m derivatives in CDCl3, δC in ppm at 100.6 MHz

Compound

13C ppm

C-1’’ C-1a’’ C-2’’/5’’ C-3’’/4’’ C-3a’’ CH3

2h 161.2 134.5 129.4 111.5 151.9 40.2

2m 161.1 132.2 130.0 111.6 151.9 40.2


39

Figure 25: Structure of compounds 2n-p

For the compounds 2n – p (Figure 25) the 1H NMR spectra showed the pyrrol

protons H-3’’ δH = 6.10 – 6.22 ppm as double doublets with J ortho = 2.4 – 2.8 Hz, H-2’’

at δH = 6.16 – 6.24 ppm as dd with J ortho = 2.4 – 2.8 Hz and a J meta = 3.2 – 3.6 Hz and

H-4’’ at δH = 6.91 – 6.93 ppm as a dd with J ortho = 2.8 Hz and J meta =-3.2 – 3.6 Hz.

The methyl group attached to the nitrogen showed up at δH = 3.51 – 3.96 ppm. The

iminic proton, H-1’’, of all derivatives appeared between δH = 8.14 – 8.31 ppm as a

singlets. (Table 14)

Table 14: 1H NMR signals of 2n – p derivatives in CDCl3,J in Hz, δH in ppm at 400 MHz

Compound

1H ppm

H-1’’ H-2’’ H-3’’ H-4’’ CH3

2n 8.16 s 6.24 dd 6.10 dd 6.93 dd 3.51 s

2o 8.31 s 6.24 dd 6.22 dd 6.91 dd 3.96 s

2p 8.14 s 6.16 dd 6.14 dd 6.98 dd 3.92 s

In the 13C NMR spectra, the iminic carbon, C-1’’, appeared at δC = 160.8 – 161.3 ppm

as usual. The methyl group showed up at δC = 34.2 – 34.8 ppm, and C-2’’, C-3’’ and C-

4’’ appeared at δC = 107.5 – 109.3 ppm, 106.0 – 108.3 ppm and 116-9 – 122.9 ppm

respectively. (Table 15)

Table 15: 13

C NMR signals of 2n – p derivatives in CDCl3, δC in ppm at 100.6 MHz

Compound

13C ppm

C-1’’ C-1a’’ C-2’’ C-3’’ C-4’’ CH3

2n 160.8 151.2 109.3 108.3 116.9 34.2

2o 161.3 151.2 107.9 106.9 122.0 34.2

2p 161.1 152.0 107.5 106.0 122.9 34.8


40

2.1.3 Synthesis of compounds 3a – p

Scheme 11: Reduction of imines 2a – p;

Amines 3a – p were obtained by reduction of 2a – p derivatives with NaBH4, in good

yields (49 – 95 %). (Scheme 11) In this reaction the hydride ion reacts at the

electrophilic carbon atom of the imine function. An anion is formed, which is

subsequently protonated in a second step to generate secondary amines 3a – p.

(Scheme 12)

Scheme 12: Imines reduction mechanism


41

In Figures 26 and 27 are shown 1H and DEPT 135 NMR spectra of compound 3a.

Clearly there are five signals at aromatic region due to the phenyl and naphthalic rings.

The iminic signal disappeared, comparing with the reagent imine. The aliphatic region

shows the two methylenic units attached to the nitrogen atoms of the carbon chain as

in its precursor, a new methylenic group formed by reduction and the methoxyl group.

A DEPT 135 spectrum is consistent with three methylenes, a methyl group attached to

oxygen, and five -CH in the aromatic zone.

Figure 26: 1H NMR spectrum of amine 3a

Figure 27: DEPT 135 spectrum of amine 3a

H-1’’

CH3

H-2’

Naphtalimide

H-1’

H-3’’

H-2’’

CH2

CH3


42

Figure 28: Structures of compounds 3a – p

Tables 16 – 27 show the signals of the different derivatives 3a – p.

Table 16: 1H NMR signals of 3d and 3i derivatives in CDCl3, J in Hz, δH in ppm at 400 MHz

Compound

1H ppm

H-1’’ H-2’’ H-3’’

3d 3.90 s 7.49 d 8.12 d

3i 3.89 s 7.50 d 8.15 d

Table 17: 13

C NMR signals of 3d and 3i derivatives in CDCl3, δC in ppm at 100.6 MHz

Compound

13C ppm

C-1’’ C-1a’’ C-2’’ C-3’’ C-3a’’

3d 53.1 132.1 128.4 123.5 148.3

3i 53.1 132.1 128.5 123.8 148.4

Table 18: 1H NMR signals of 3a, 3e and 3j derivatives in CDCl3, J in Hz, δH in ppm at 400 MHz

Compound

1H ppm

H-1’’ H-2’’ H-3’’ H-3b’’

3a 3.77 s 7.19 dd 6.76 dd 3.72 s

3e 3.69 s 7.21 dd 6.78 dd 3.72 s

3j 3.70 s 7.21 dd 6.83 dd 3.76 s


43

Table 19: 13

C NMR signals of 3a, 3e and 3j derivatives in CDCl3, δC in ppm at 100.6 MHz

Compound

13C ppm

C-1’’ C-1a’’ C-2’’ C-3’’ C-3a’’ C-3b’’

3a 52.7 132.9 129.1 113.5 158.2 55.0

3e 53.1 133.0 129.2 113.5 158.3 55.0

3j 53.2 132.4 129.2 113.7 158.5 55.2

Table 20: 1H NMR signals of 3b, 3f and 3k derivatives in CDCl3, J in Hz, δH in ppm at 400 MHz

Compound

1H ppm

H-1’’ H-3’’ H-4’’

3b 4.10 s 7.20 d 7.18 dd

3f 4.08 s 7.25 d 7.08 dd

3k 4.05 s 7.26 d 7.07 dd

Table 21: 13C NMR signals of 3b, 3f and 3k derivatives in CDCl3, δC in ppm at 100.6 MHz

Compound

13C ppm

C-1’’ C-1a’’ C-2’’ C-3’’ C-4’’

3b 47.9 134.2 158.6 128.2 128.7

3f 48.1 133.8 155.9 128.2 128.7

3k 48.2 135.8 155.9 128.3 128.7

Table 22: 1H NMR signals of 3c, 3g and 3l derivatives in CDCl3,J in Hz, δH in ppm at 400 MHz

Compound

1H ppm

H-1’’ H-2’’ H-3’’

3c 3.69 s 7.26 dd 6.98 t

3g 3.74 s 7.26 dd 6.94 t

3l 3.74 s 7.27 dd 6.98 t

Table 23: 13

C NMR signals of 3c, 3g and 3l derivatives in CDCl3, δC in ppm at 100.6 MHz

Compound 13C ppm

C-1’’ C-1a’’ C-2’’ C-3’’ C-3a’’

3c 53.2 135.7 129.3 115.1 161.4

3g 53.0 136.0 129.5 114.9 161.1

3l 53.1 136.1 129.4 115.1 160.6


44

Table 24: 1H NMR signals of 3h and 3m derivatives in CDCl3, J in Hz, δH in ppm at 400 MHz

Compound

1H ppm

H-1’’ H-2’’ H-3’’ H-3b’’

3h 3.69 s 7.18 dd 6.66 dd 2.90 s

3m 3.69 s 7.17 d 6.69 d 2.93 s

Table 25: 13C NMR signals of 3h and 3m derivatives in CDCl3, δC in ppm at 100.6 MHz

Compound

13C ppm

C-1’’ C-1a’’ C-2’’ C-3’’ C-3a’’ C-3b’’

3h 53.0 132.1 112.4 128.2 149.6 40.6

3m 53.4 132.3 112.7 128.9 148.7 40.7

Table 26: 1H NMR signals of 3n – p derivatives in CDCl3, J in Hz, δH in ppm at 400 MHz

Compound

1H ppm

H-1’’ H-2’’ H-3’’ H-4’’ CH3

3n 3.80 s 6.01 dd 5.98 dd 6.49 dd 3.56 s

3o 3.74 s 6.03 dd 5.97 dd 6.50 dd 3.70 s

3p 3.72 s 6.02 dd 5.99 dd 6.56 dd 3.63 s

Table 27: 13C NMR signals of 3n – p derivatives in CDCl3, δC in ppm at 100.6 MHz

Compound

13C ppm

C-1’’ C-1a’’ C-2’’ C-3’’ C-4’’ CH3

3n 45.1 130.9 107.9 106.3 122.1 33.5

3o 45.3 130.2 107.7 106.3 122.2 33.9

3p 45.4 131.3 107.7 106.1 121.9 33.6

2.1.4 Synthesis of naphthalimides 4a – c

Scheme 13: Reaction between 1,8-naphthalic anhydride and alkyl aminoalcohols; i) DMF, 85°C


45

1,8-Naphthalimides 4a – c were obtained by a straight forward methods by reaction

of 1,8-naphthalic anhydride with alkyl aminoalcohols. (Figure 30) Products were

obtained in good yields, between 81 – 93 % (Scheme 13). The amino group attacks

preferentially at the carbonyl group giving the naphthalimides. The mechanism is the

same as the one depicted in Scheme 8 for the reaction of diamines with naphthalic

anhydride.

The 1H NMR spectrum of compound 4b is shown in Figure 29.

Figure 29: 1H NMR spectrum of Napthalimidopropanol (4b)

The methylenic protons appeared at the expected chemical shifts; two signals show

up at comparable chemicals shifts to 1b (H-1’ and H-2’) and H-3’ is 1ppm lower field in

comparison to compound 1b. This is due to the higher deprotection of the oxygen

atom. The hydrogen attached to the oxygen showed up in compounds 4a, and 4b, but

is absent in compound 4c (Table 28).

Figure 30: Structures of compounds 4a – c

OH H-2’

H-1’

H-3’

Naphthalimide Ring


46

Table 28: 1

H NMR signals of 4a – c derivatives in CDCl3, J in Hz, δH in ppm at 400 MHz

Compound

1H ppm

H-4 H-5 H-6 H-1’ H-2’ H-3’ H-4’ OH

4a 8.60 dd 8.22 dd 7.76 dd 4.47 t 3.99 t --- --- 2.52 s

4b 8.60 dd 8.19 dd 7.76 dd 4.37 t 1.99-2.05 3.62 t --- 3.25 s

4c 8.59 dd 8.20 dd 7.75 dd 4.20 t 1.77-1.82 1.55-1.63 3.79 t ---

Table 29 registered the signals of 13C NMR spectra of these compounds. Again the

carbon neighbor to the oxygen atom appears at lower field than the carbons attached

to the nitrogen in compounds 1a – c.

Table 29: 13C NMR signals of 4a – c derivatives in CDCl3, δC in ppm at 100.6 MHz

Comp.

13C ppm

C-1/3 C-4 C-6 C-5 C-3a C-6a C-10 C-1’ C-2’ C-3’ C-4’

4a 165.1 131.5 127.0 134.2 122.4 131.5 128.2 42.8 61.7 --- ---

4b 164.9 131.6 127.0 134.2 122.3 131.6 128.1 36.7 31.0 58.8 ---

4c 164.2 131.2 126.9 133.9 122.6 131.5 128.1 33.9 24.5 29.9 62.5

2.1.5 Synthesis of tosyl compounds 5a – c

Scheme 14: Reaction between 4a – c and p-toluenesulfonyl chloride; i) Py, 4°C

Toluenesulfonylnaphthalimides 5a – c were obtained by straight forward methods

from 4a – c by reaction with Ts-Cl (Figure 32). The compounds formed in good yields

between 63 – 83 %, after recrystallizion from ethanol (Scheme 14). In terms of

mechanism, the alcohol group acted as a nucleophile attacking the sulfur atom with

displacement of the chloride ion and formation of the respective toluenesulfonate

ester. The pyridine is the base, it removes the proton and the pyridinium formed is

stabilized with the chloride ion. (Scheme 15)


47

Scheme 15: Mechanism between alcohols 4a – c and tosyl chloride

Figure 31 shows the 1H NMR spectrum of compound 5b. The major feature is the

methylene group attached to the tosyl group that is shifted from δH = 3.62 ppm in the

alcohol precursor (4b) to δH = 4.25 ppm (5b)

Figure 31: 1H NMR spectrum of tosyl compound 5b

Figure 32: Structure of compounds 5a – c

CH3

H-1’ and

H-3’

H-2’ H from Tosyl

Naphthalimide


48

The mobile proton of the hydroxyl group in compounds 4 disappear in compounds

5. Protons of the p-methylphenyl group appeared in all compounds, and are registed in

Table 30.

Table 30: 1H NMR signals of 5a – c derivatives in CDCl3, J in Hz, δH in ppm at 400 MHz

Compound

1H ppm

H-2’’ H-3’’ CH3

5a 7.66 d 6.96 d 2.16 s

5b 7.78 d 7.31 d 2.45 s

5c 7.78 d 7.35 d 2.49 s

In the 13C NMR spectrum the methyl group appeared at δC = 21.5 – 21.9 ppm and

the CH of the ring appeared at the expected shifts. (Table 31)

Table 31: 13C NMR signals of 5a – c derivatives in CDCl3, δC in ppm at 100.6 MHz

Compound

13C ppm

C-1’’ C-2’’ C-3’’ CH3

5a 144.5 127.7 129.5 21.5

5b 144.7 127.9 129.8 21.7

5c 144.9 128.0 129.8 21.9

2.1.6 Synthesis of Mesitylamines 6a and 6b

Scheme 16: Mesitylation of alkyl diamine; i) Mts-Cl, Py, 0°C


49

Compounds 6a and 6b were obtained by straight forward methods from the

respective amines and Mts-Cl (Figures 33 and 34). The products were obtained in low

to moderate yields, between 20 – 50 % (Scheme 16). The amino group attacks the

sulfur atom with displacement of the chloride ion. The pyridine is the base, it removes

the proton and the pyridinium formed is stabilized with the chloride ion. The

mechanism is the same as the one depicted in Scheme 15.

Figure 33: Structure of compound 6a

1H NMR spectrum of compound 6a confirms the structure. As the structure has two

symmetry plans (Figure 33) the two methyl groups at para position appeared as a

singlet at δH = 2.34 ppm, and the o-methyls, also as a single singlet at δH = 2.67 ppm. In

the first case the peak integrates for six protons, and in the second case for twelve

protons. Protons directly attached to the benzenic ring showed up at δH = 6.99 ppm as

a singlet with integration for four protons. The protons of the carbon chain are shifted

to lower field in comparison with the starting amine. The main difference refers to the

methylenic group directly attached to the sulfonamide function, and of course of the

proton attached to the nitrogen atom of this function. H-5 showed up as a singlet for

four H-3 under the o-CH3 group signals. (Table 32)

Table 32: 1

H NMR signals of 6a derivatives in CDCl3, J in Hz, δH in ppm at 400 MHz

Compound

1H ppm

NH H-2 H-3 H-4 H-3’ o-CH3/5 p-CH3

6a 7.1 s 2.99 s 1.75 m 2.53 s 6.99 s 2.67/2.68 2.34 s

13C NMR spectrum, confirms the presence of the carbons of the mesityl groups. All

the –CH2, C-2-6 appears at δC = 24.0 – 57.9 ppm and the ones near to the nitrogen

were the more deprotected. The o-CH3 and p-CH3 of Mts appears at 23.0 and 20.9

respectively. At aromatic zone the C-3’ appears at 131.9 ppm. (Table 33)


50

Table 33: 13

C NMR signals of 6a derivatives in CDCl3, δC in ppm at 100.6 MHz

Compound

13C ppm

C-2 C-3/5 C-4 C-1’ C-2’ C-3’ C-3a’ p-CH3 o-CH3

6a 43.3 24.0 57.9 149.9 139.0 131.9 130.9 20.9 23.0

Figure 34: Structure of compound 6b

1H NMR spectrum of compound 6b follows the pattern of compound 6a. (Table 34)

Table 34: 1H NMR signals of 6b derivatives in CDCl3, J in Hz, δH in ppm at 400 MHz

Compound

1H ppm

NH H-2 H-3 H-4/5 H-3’ p-CH3 o-CH3

6b 4.61 s 2.87-2.92 m 1.44 t 1.17-1.22 m 6.99 2.33 s 2.67 s

In Table 35 are described the 13C NMR spectrum of compound 6b. The signals showed

up at the expected chemical shifts, in accordance with those of compound 6a.

Table 35: 13C NMR signals of 6b derivatives in CDCl3, δC in ppm at 100.6 MHz

Compound

13C ppm

C-2 C-3 C-4 C-5 C-2’ C-3’ C-3a’ p-CH3 o-CH3

6b 42.5 29.5 26.4 28.8 142.1 131.7 131.9 20.9 23.1


51

2.1.7 Synthesis of bis-naphthalimide derivatives 7a – c

Scheme 17: Reaction between the mesityl linkers 6a and 6b with tosylnaphthalimides; i) DMF ii) Cs2CO3

The compounds 7a – c were obtained by a straight forward method from 5b and 5c

by reaction with 6a and 6b (Figure 36). Products were obtained in low yields, between

23 – 31 %. (Scheme 17)

In this reaction, Cs2CO3 is the base used to remove the proton attached to the

nitrogen atom of the sulphonamide function, creating a negative charge, which

displaces the O-tosyl group to form the alkylating product. The coupling reaction

mechanism is shown in the Scheme 18. The mesitylenesulfonyl protecting group serves

for two purposes: i) by having the Mts group on the amino, it renders the hydrogen on

the sulphoamide group more acidic and hence in the presence of a base such as

ceasium carbonate, makes the N-alkylation easy (ii) If the Mts group is not present, it

could in principle lead to di N-alkylation. Hence the presence of the Mts group only

allows mono N-alkylation. (Scheme 18)


52

Scheme 18: Mechanism between linkers 6a and 6b and the tosylnaphthalimides 5b and 5c

In Figure 35 is shown the 1H NMR of compound 7c. A very simple pattern is

depicted according to the high symmetry of the compound.

Figure 35: 1H NMR of compound 7c

Naphthalimide rings

H-4’’ H-7’/8’/9’ H-1’ H-4’/6’

H-5’’

H-4’’


53

In Tables 36 and 37 are comprised the 1H NMR signals for compound 7a, 7b and 7c.

The tosyl group disappeared as expected.

Figure 36: Structure of compounds 7a, 7b and 7c

Table 36: 1H NMR signals of 7a derivatives in CDCl3, J in Hz, δH in ppm at 400 MHz

Comp.

1H ppm

H-1’ H-2/6’ H-3’/5’/7’ H-8’ H-3’’ p-CH3 o-CH3

7a 4.29 2.01 3.75 2.97 6.69 2.40 3.01

Table 37: 1H NMR signals of 7b and 7c derivatives in CDCl3, J in Hz, δH in ppm at 400 MHz

Comp. 1H ppm

H-1’ H-2’/7’ H-3’/6’ --- H-8’/9’ H-3’’ p-CH3 o-CH3

7b 4.04 1.86-1.90 2.67 --- 2.87-2.92 6.75 2.17 2.54

H-1’ H-2’/3’ H-4’/6’ H-7’ H-8’/9’ H-3’’ p-CH3 o-CH3

7c 4.13 1.62 3.17-3.26 1.50 1.12 6.89 2.21 2.62

The 13C NMR spectra of compounds 7a, 7b and 7c are comprised in Tables 38 and

39.

Table 38: 13C NMR signals of 7a derivatives in CDCl3, J in Hz, δC in ppm at 100.6 MHz

Comp.

13C ppm

C-1’ C-2’ C-3’/7’ C-5’ C-6’ C-8’ C-1’’ C-3’’ p-CH3 o-CH3

7a 37.5 21.2 45.4 44.4 25.0 39.7 142.3 132.1 23.5 20.5

Table 39: 13C NMR signals of 7b and 7c derivatives in CDCl3, J in Hz, δC in ppm at 100.6 MHz

Comp.

13C ppm

C-1’ C-2’ C-3’ C-4’ C-6’ C-7’ C-

8’/9’ C-1’’ C-3’’

p-CH3

o-CH3

7b 38.2 26.2 46.0 28.9 48.1 29.9 28.9 142.1 131.8 20.7 23.0

C-1’ C-

2’/7’ C-3’ C-4’ C-6’ ---

C-8’/9’

C-1’’ C-3’’ p-

CH3 o-

CH3

7c 38.5 26.2 27.0 39.7 48.8 --- 28.9 142.0 131.9 20.6 23.2


54

2.1.8 Synthesis of 8a – c derivatives

Scheme 19: Deprotection of compounds 7a – c; i) DCM, HBr/gCH3CO2H (20 %)

Compounds 8a – c were obtained as hydrobromic salts by deprotection of

compounds 7a, 7b and 7c under 20% HBr/glacial CH3CO2H (Figure 38 and 39). The

respective products formed in high yields between 92 – 100 %. (Scheme 19)

The removal of the mesityl groups and the appearance of the NH were confirmed

by 1H and 13C NMR spectra. (Figure 37)

Figure 37: 1H NMR spectrum of compound 8b

The Tables 40 and 41 show data of the aliphatic H and C atoms in 1H and 13C NMR

for compound 8a.

Absence of CH of mesityl group

NH


55

.

Figure 38: Structure of compound 8a

Table 40: 1H NMR signals of 8a derivatives in DMSO, J in Hz, δH in ppm at 400 MHz

Comp.

1H ppm

H-1’ H-2’ H-3’ H-4’ H-5’ H-6’ H-7’ H-8’

8a 4.14 2.06 3.02 8.62 3.02 2.06 2.50 2.86

Table 41: 13

C NMR signals of 8a derivatives in DMSO, δC in ppm at 100.6 MHz

Comp.

13C ppm

C-1’ C-2’ C-3’ C-5’ C-6’ C-7’ C-8’

8a 37.5 25.0 45.4 44.4 20.8 44.4 39.7

In Tables 42 and 43 are shown the data of aliphatic signals in 1H and 13C NMR for

compounds 8b and 8c.

Figure 39: Structure of compounds 8b and 8c

Table 42: 1H NMR signals of 8b and 8c derivatives in DMSO, δC in ppm at 400 MHz

Comp.

1H ppm

H-1’ H-2’ H-3’ H-4’ H-5’ H-6’ H-7’ H-8’ H-9’

8b 4.11 2.00 2.99 --- 4.94 2.85 t 1.52 1.23 1.23

8c 4.08 1.69 1.69 2.89 4.51 2.83 t 1.55 1.25 1.25

Table 43: 13C NMR signals of 8a and 8c derivatives in DMSO, δC in ppm at 100.6 MHz

Comp.

13C ppm

C-1’ C-2’ C-3’ C-4’ C-6’ C-7’ C-8’ C-9’

8b 37.5 25.0 45.2 --- 47.2 25.9 26.3 28.7

8c 37.5 45.2 45.2 --- 47.2 40.2 25.9 26.3


56

The compounds 8a and 8c are also characterized by Mass Spectroscopy (MS). In

Figure 40 and 41 showed the mass spec of compounds 8a and 8c. The results support

the identification of the compounds by the matching values between exact mass and

molecular ion.

Compound 8a: HRMS [M+H]: 675.3644; calculate for: C40H47N6O4 = 675.3659

Compound 8c: HRMS [M+H]: 647.3585; calculate for: C40H47N4O4 = 647.3597

Figure 40: MS spectrum of compound 8a

Figure 41: MS spectrum of compound 8c


57

2.2 DNA Binding Studies

Fluorescence binding assays to DNA were obtained by competitive EtBr

displacement studies. This is an in vitro technique which provides a simple and reliable

method to study DNA binding mechanisms to drugs. The stronger the drug affinity to

DNA, the stronger is the binding.

Ethidium Bromide (EtBr) is a known DNA intercalator and was used as a positive

control. The mono and bis-naphthalimides synthetized were tested to evaluate if they

compete with EtBr to DNA and if they intercalate better or worse than EtBr.

2.2.1 Fluorescence Binding

The competitive EtBr displacement, also called, fluorescence binding, is the

selective technique used in this work to evaluate mono and bis-naphthalimide

derivatives intercalation to DNA. These days, a lot of compounds are known to act as

anticancer drugs by intercalation to DNA. The process of intercalation affects the

shape of the DNA and also the activity of some enzymes in their normal processes, e.g.

replication and/or repair of DNA. EtBr compound represented in Figure 42 is a strong

DNA intercalator, binding the purinic and pyrimidinic bases of the DNA helix.

Figure 42: Structure of Ethidium Bromide (EtBr)

When the EtBr binds to DNA emission of fluorescence and can be measure. To

evaluate the mono and bis-naphthalimide derivatives affinity to DNA a solution of EtBr

bounded to DNA with SSC buffer was added a solution of the new drugs. If the new

molecules display larger affinity than EtBr towards DNA, EtBr will be displaced,

resulting in decrease in fluorescence (Figure 43).


58

Figure 43: Mechanism of action of displacement studies

This experiment was carried out with all the mono and bis-naphthalimide

derivatives to see if the drugs have affinity to DNA and compare the results obtained.

The mono- naphthalimide derivatives concentrations in the experiment vary from to 0

– 50 µM and the bis-naphthalimide derivatives concentrations vary from 0 to 5 µM.

The concentration of EtBr is 2 µM and DNA (from Calf Thymus) is 10 µM. The results of

the EtBr displacement are shown in the Figures 44 – 46, 48 – 50, 52 and 53. The

spectrum shows the variation of the fluorescence intensity in function of absorbance

read.

The drug concentration causing a significant decrease in fluorescence can be related

to the biological activity of the drug.


59

2.2.2 Discussion of the values obtained for mono-naphthalimide derivatives

In Figures 44 – 46, 48 – 50, 52 and 53 is shown the effect of addition of mono-

naphthalimides to the DNA-EtBr complex.

For the mono-naphthalimides:

Figure 44: The effect of varying 3i and 3j concentration (0-50 µM) on fluorescence intensity of EtBr bound DNA

Figure 45: The effect of varying 3k and 3l concentration (0-50 µM) on fluorescence intensity of EtBr bound DNA


60

Figure 46: The effect of varying 3m and 3p concentration (0-50 µM) on fluorescence intensity of EtBr bound DNA

With the values obtained at excitation/emission maxima of each concentration, it

was possible to represent the C50 that is the minimum concentration needed to

decrease the fluorescence intensity to 50%, in all the derivatives. It was plotted

fluorescence intensity % to obtain a curve against concentration and read C50 values in

each case (Figure 47).

Figure 47: The effect of concentration of mono-naphthalimide derivatives with 4 carbon atoms chain on % of fluorescence intensity

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% o

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Inte

nsi

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Concentration (µM)

The effect of 4 carbons atom chain on % of Fluorescence Intensity

3i

3j

3k

3l

3m

3p


61

The results obtained with compounds having 4 carbon atoms alkyl chain showed to

have affinity to DNA, displacing the EtBr. Compounds 3i and 3m showed more affinity

than the others to DNA. On the other hand, compound 3l showed the lowest affinity to

DNA even didn’t reach till 50% of maximum concentration.

The result of the compounds 3i was 17.5 µM and 3m is 18.2 µM. Compound 3l did

not reached below 50% so it was not represented in Table 44

Table 44: Mono-naphthalimide derivatives with 4 carbon atoms chain C50 values (µM)

Mono-Naphthalimide derivatives C50 (µM)

3i 17.5

3j 32.8

3k 34

3l ---

3m 18.2

3p 31.2

The C50 values showed that the compounds 3i (17.5 µM) and 3m (18.2 µM) was the

ones that had more affinity to bind the DNA. In this case, the difference of the

substituent of in the benzene ring, the position of the substituent in the benzene ring

and the type of substituent attached to the alkyl chain will affect the affinity to the

DNA. The presence of NO2 and NMe2 at the position 4 of the benzene was important

for binding to DNA while Fluor at same position did not result in a similar binding to

DNA. 2,6-Dichloro group were also ineffective. When the substituent attached to the

alkyl chain is not benzene, but an N-Methylpyrrol, the affinity to DNA was significant

but not as good as benzene. (Table 44)


62

Figure 48: The effect of varying 3d and 3e concentration (0-50 µM) on fluorescence intensity of EtBr bound DNA

Figure 49: The effect of varying 3f and 3g concentration (0-50 µM) on fluorescence intensity of EtBr bound DNA


63

Figure 50: The effect of varying 3o concentration (0-50 µM) on fluorescence intensity of EtBr bound DNA

The same type of correlation concentration vs fluorescence was obtained for 3

carbon chain atom derivatives. (Figure 51)

Figure 51: The effect of concentration of mono-naphthalimide derivatives with 3 carbon atoms chain on % of fluorescence intensity

The results for the compounds with 3 carbons chain (3d – g, 3o) are worse than for

the 4 carbon atom chain compounds. The best compounds are 3e, 3g and 3o. On the

other hand, compound 3d and 3f showed the lowest affinity to DNA (Table 45).

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% o

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Concentration (µM)


3d

3e

3f

3g

3o


64

Table 45: Mono-naphthalimide derivatives with 3 carbon linker chain C50 values (µM)


3d ---

3e 38.5

3f ---

3g 38.5

3o 28.5

C50 values of compounds 3i/3d showed a sharp difference: compound 3i display a

17.5 µM binding and 3d (with the same aromatic substituent) is not possible to

calculate the C50 value with 50 µM concentration. It seems that the resulting in

diminishing the binding was related to the alkyl chain. The same happens for the

compounds 3j and 3e. Comparing the results of 3n and 3o an anomalous result was

obtained. Compound 3o is slightly better binder then 3n. With the substituent of

compounds 3g and 3l, a better binding was also obtained for 3g rather than 3l.

Figure 52: The effect of varying 3a and 3b concentration (0-50 µM) on fluorescence intensity of EtBr bound DNA


65

Figure 53: The effect of varying 3c and 3n concentration (0-50 µM) on fluorescence intensity of EtBr bound DNA

The same plot was obtained for compounds with two carbons atom chain after the

measuring emission vs concentration (Figure 54).

Figure 54: The effect of concentration of mono-naphthalimide derivatives with 2 carbons atom chain on % of fluorescence intensity

The best is the compound 3a with 34.5 µM binding affinity. Generally the 2 carbons

atom chain compounds are weaker binders than longer carbon chain. Compound 3b

case the affinity binding to DNA increase in relation to 3f and 3k. Compound 3a is

better them 3e (3 carbon atoms chain) but worse than 3j (4 carbon atoms chain).

(Table 46)

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% o

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Inte

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Concentration (µM)


3a

3b

3c

3n


66

Table 46: Mono-naphthalimides derivatives with 2 carbons of linker chain C50 values (µM)


3a 34.5

3b 37.2

3c 40

3n 46

With the results obtained it was not possible to drive a definitive conclusion base on

the chain length. The types of substituents used seem to be important, but there is a

few results to make an assumption (Table 47)

SAME SUBSTITUENT/CHAIN

Table 47: Comparison of concentrations (µM) Mono naphthalimide compounds with the same substituent/chain

2 carbons 3 carbons 4 carbons

4-NO2 (3d, 3i) --- a) --- b) 17.5

4-OCH3 (3a, 3e, 3j) 34.5 38.5 32.8

2,6-DiCl (3b, 3f, 3k) 37.2 --- b) 34

4-F (3c, 3g ,3l) 40 38.5 --- (b)

4-N-ME2 (3m) --- a) --- c) 18.2

N-MePy (3n, 3o, 3p) 46 28.5 31.2

a) Not synthetized

b) Not determined for 50 µM

c) Not tested


67

2.2.3 Discussion of the values obtained for bis-naphthalimide derivatives

In Figures 55 and 56 is shown on effect of addition of bis-naphthalimides to the

DNA-EtBr complex.

For Bis-Naphthalimides:

Figure 55: The effect of varying 8a and 8b concentration (0-25 µM) on fluorescence intensity of EtBr

Figure 56: The effect of varying 8c concentrations (0-25 µM) on fluorescence intensity of EtBr

These results were translated to a plot of % of fluorescence intensity against

concentration (Figure 57).


68

Figure 57: The effect of concentration of bis-naphthalimide derivatives on % of fluorescence intensity

The results of the bis-naphthalimide derivatives showed that all the compounds

have a big affinity to DNA displacing EtBr. These compounds compete with the EtBr to

intercalation sites.

The maximum emission decreases to 50% in concentrations varying from 0.91µM

for compound 8c to 1.95 µM for compound 8a (Table 48).

Table 48: Bis-naphthalimide derivatives C50 values (µM)

Bis-Naphthalimide derivatives C50 (µM)

8a 1.95

8b 0.96

8c 0.91

Compounds with the octane (8b and 8c) as a linker chain display a better affinity to

DNA than 8a. The linker for the compound 8a is a piperazine. This chain does not have

the same flexibility as an octanyl and this might be the reason for the lower binding.

The difference between of 8b and 8c is the number of carbons of the chain. The 8c has

2 more carbons than the 8b but this difference does not seem to be significant

because the C50 of them are very similar. Increasing the bis-naphthalimides

concentration do not display much different in terms of binding in all the three

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Concentration (µM)

The effect of bis-naphthalimides derivatives on % of Fluorescence Intensity

8a

8b

8c


69

compounds (8a, 8b and 8c). This is probably connected to the reach of saturation with

the first concentration tested.


70

2.3 Biological Activity

In order to treat the brain cancer cells, SH-SY5Y, the mono and bis-naphthalimide

derivatives were solubilized and store mono-naphthalimide derivatives as 10mM stock

solutions in 50% DMSO/water, and the bis-naphthalimide derivatives as 10mM stock

solutions in DMSO.

To evaluate if the cancer cells dying, the cells were examined by two different

methods: a) by the morphological changes in cells, examined by light microscope; b) by

cytotoxicity assays at different concentration levels.

2.3.1 Cell Morphology

The changes in cell morphology were examined in brain cancer cells (SH-SY5Y)

treated, with the mono and bis-naphthalimide derivatives using the light microscope.

The mono-naphthalimide derivatives were treated in concentrations from 0 - 150 µM

and bis-naphthalimide derivatives from 0 - 40 µM for 24 hours.

In Figure 58 taken by the light microscope shows the morphological changes in cells

after treatment with mono-naphthalimide derivatives. Picture A shows the

morphology of untreated cells. They exhibit like spikes. Morphological changes occur

only above 25 µM after 24 hours treatment (D). Dead cells are smaller and rounded. At

50 µM (E) more than a half of the cells are dead suggesting that the IC50 value would

be between 25 µM and 50 µM. At 150 µM all the cells were dead (F).


71

Mono naphthalimides:

Figure 58: Morphological changes of brain cancer cells treated with mono-naphthalimide 3l at different concentrations (0 – 150 µM) (green arrows = viable cells, red arrow = dead cells

For the bis-naphthalimide derivatives the morphological changes are shown in

Figure 59. Picture A shows normal cells.

Successive increases of drug concentration are shown in pictures B-F. At 2 µM (D)

more than 50% of the cells are dead. At maximum concentration, 40 µM, the cells are

all dead (F).


72

For bis-naphthalimides:

Figure 59: Morphological changes of brain cancer cells treated with bis-naphthalimide 8c at different concentrations (0 – 40 µM) (green arrows = viable cells, red arrow = dead cells

2.3.2 Cytotoxicity

Cell viability of neuroblastoma cells, SH-SY5Y, were treated with different

concentrations of mono and bis naphthalimide derivatives for 24 hours and were

assessed by a colorimetric 3-(4,5-dimethylthiazol-1-yl)-2,5-diphenyltetrazolium

bromide (MTT) assay. The mono naphthalimide derivatives were tested at 0 - 150 µM

and the bis naphthalimide derivatives were tested at 0 - 40 µM.

The principle of this assay is the colour change from yellow to purple in living cells

due to mitochondrial reductase, which is responsible to reduce the MTT to formazin.

After 4h incubation time the yellow solution is removed and substituted by DMSO. The

living cells displayed a purple colour and the dead cells were colourless (Scheme 20).

Measuring the differences in purple shades solution by the UV/visible spectroscopy

absorbance it is possible to quantify the amount of living cells.


73

Scheme 20: Reaction of reduction of MTT

Figure 60 shows different purple shades. The purple colour fades away by

increasing drug concentration.

Figure 60: Reduction in MTT tetrazolium dye with bis-naphthalimide derivatives

The results of the MTT assay were represented in the next figures. These figures

represents the percentage of growth inhibition in NB cells (SH-SY5Y) treated with the

mono and bis naphthalimide derivatives after 24h of incubation at 37°C under an

atmosphere with 5% of CO2.

In Figure 61 are shown the results of the mono-naphthalimide derivatives with 4

carbons atom chain. The graph is expressed by % of absorbance intensity vs

concentration to obtain the IC50 values.


74

For mono-naphthalimides:

Figure 61: Concentration variation on % of absorbance intensity in brain cancer SH-SY5Y cells, treated by mono-naphthalimide derivatives (4 carbons atoms chain)

The best results belong to compounds 3j (15.3 µM) and 3p (14.8 µM). Compound 3i

show a very weak activity. It happens that 3i is the best binder to DNA. The opposite

trend happened to compound 3j its affinity to DNA is weak but display good biological

activity against this type of cells. The results of compound 3k are inconclusive, it

precipitated out forming crystals (Table 49)

Compound 2l at 1µM, showed an increase of the absorbance to a value bigger than

100%, a fast replication of cells, as a defensive mechanism.

Table 49: Mono-naphthalimide derivatives (4 carbon atoms chain) IC50 values (µM) after 24 hours incubation

Compound IC50 (µM)

3i No activity

3j 15.3

3k Crystals

3l 26.4

3m 27.5

3p 14.8

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Concentration (µM)

The effect of concentration variation of mono-naphthalimide derivatives with 4 carbon atoms chain against SH-SY5Y cells

3i 595 nm

3j 595 nm

3l 595 nm

3m 595 nm

3p 595 nm


75

For the mono-naphthalimide derivatives with 3 carbon atoms chain, a plot was

made with absorbance intensity measure in % vs drug concentration. IC50 values were

obtained by this graph. (Figure 62)


In all cases the results obtained from 4 carbon atoms chain compounds are better

than those from 3 carbon atoms chain compounds. Compounds 3d with p-NO2 and 3f

with 2,6-dichloro groups showed the same results as their analogous with 4 carbon

atoms chain. The other compounds showed some activity, but lower than their

analogous with 4 carbon atoms chain. The deeper decrease in activity occurred with N-

Methylpyrrol derivative which is 3 times less active than the 4 carbon atoms chain 3n.

(Table 50)


Compound IC50 (µM)

3d No activity

3e 39

3f Crystals

3g 39

3o 47

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Concentration (µM)


3d 595 nm

3e 595 nm

3g 595 nm

3o 595 nm


76

Mono-naphthalimide with 2 carbon atoms chain results were plotted in Figure 63.

In this graph is represented the % of absorbance intensity vs drug concentration.


Compound 3c showed some activity (86 µM). The main difference was the no formation of

crystals of compound with 2,6-dichloro group (3b) (Table 51).


Compound IC50 (µM)

3a No Activity

3b No activity

3c 86

3n No activity

Comparing the results obtained for all the compounds a conclusion can be derived:

keeping the substituent, the activity of the compounds draft from 4 carbons > 3

carbons > 2 carbons (Table 52)

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3a 595 nm

3b 595 nm

3c 595 nm

3n 595 nm


77

Table 52: Comparison of concentrations (µM) mono-naphthalimide derivatives same substituent/chain

2 carbons 3 carbons 4 carbons

4-NO2 (3d, 3i) --- (a) No activity No activity

4-OCH3 (3a, 3e, 3j) No activity 39 15.3

2,6-DiCl (3b, 3f, 3k) No activity Crystals Crystals

4-F (3c, 3g ,3l) 86 39 26.4

4-N-ME2 (3m) --- (a) --- (b) 27.5

N-MePy (3n, 3o, 3p) No activity 47 14.8

a) Not synthetized

b) Not determined for 50 µM

The results of biological activity of bis-naphthalimide derivatives are represented in

Figure 64, % of absorvance intensity against concentration.


Figure 64: Concentration variation on % of absorbance intensity in brain cancer SH-SY5Y cells, treated by bis-naphthalimide derivatives

After 24h of incubation, bis-naphthalimide derivatives exhibited good cytotoxic

activity with IC50 values ranging from 3.4 – 25.7 µM.

0

15

30

45

60

75

90

105

0 4 8 12 16 20 24 28 32 36 40

% o

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ten

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Concentration (µM)

The effect of concentration variation of bis-naphthalimide derivatives against SH-SY5Y cells

8a 595 nm

8b 595 nm

8c 595 nm


78

Table 53: Bis-naphthalimide derivatives IC50 values (µM) after 24 hours incubation

Compound IC50 (µM)

8a 25.7

8b 3.5

8c 3.4

All bis-naphthalimide derivatives exhibited biological activity after 24 hours with

IC50 values ranging from 3.4 – 25.7 µM. Compounds 8b (3.5 µM) and 8c (3.4 µM)

showed higher cytotoxicity than 8a (25.7 µM) in NB cells after 24 hours. The difference

between these compounds is the between the naphthalimide rings. The presence of

octane as a linker showed a IC50 7 times higher comparing with the effect of the

piperazine. Probably the better response by the octane chain compounds is due to a

better flexibility of the structures.

The best compounds 8b and 8c differ in the size of the alkyl chain attached to the

naphthalimides rings (3 carbon atoms chain in compound 8b and 4 carbon atoms chain

in compound 8c). The difference does not affect cytotoxicity of the compounds (Table

53).

The cytotoxicity results coincide with the results of the DNA binding studies.

In a previous study by Oliveira et al54 compounds 8b tested in colon cancer cells

(Caco-2) gave IC50 values of 6.2 µM the same procedure. The IC50 value of 8b against

SH-5Y-SY cells was 3.4 µM, suggesting that cytotoxicity of bis-naphthalimides is cell line

dependent.


79

2.4 Cellular Uptake

The characteristic fluorescence properties of mono and bis-naphthalimide

derivatives allowed the cellular uptake and distribution of mono-naphthalimide

derivatives at 0 – 150 µM and of bis-naphthalimide derivatives at 0 – 40 µM, to be

examined by fluorescence microscopy.

After 24 hours of incubation, fluorescence could be seen in cells treated with mono

and bis naphthalimide derivatives (Figures 65 and 66). This observation is in agreement

with the toxicity showed. All compounds showed fluorescence after 24 hours

incubation.


When increasing concentration, increased fluorescence, suggesting that the drug

enter the cells.

In Figure 65, picture A, 1µM, showed that the drug entered but the fluorescence is

weak. At 25 µM, B, the drug enter the cells, but the cells are not all dead suggesting

that drug enter into the cells but did not efficiently kill the cells. An increase of

concentration to 50 and 150 µM the fluorescence was higher showing a linear

correlation between the cell death and the cellular uptake.

Figure 65: Fluorescence present in brain cancer, SH-SY5Y, cells after treatments with different concentrations of mono-naphthalimide derivatives


80


The results showed that the cellular uptake is related to the cell death. In Figure 66

are shown different concentrations of bis-naphthalimide in its effects in cells. At lower

concentrations (0.5 and 1.0 µM), the cellular uptake is minimum, but when the drug

concentration increase till 40 µM cells die effectively and the fluorescence is extremely

higher.

A previous study by Barron et al.54 on this very same compound 8b with MD-MB-

231 cells the cytotoxicity is related to uptake. But the same authors conclude that with

MCF-10A the cytotoxicity is not related to the uptake. This shows that the uptake in

bis-naphthalimide derivatives case is cell line dependent.

Figure 66: Fluorescence present in brain cancer SH-SY5Y cells after treatments with bis-naphthalimide derivatives

Chapter 3 Experimental Procedure

Chapter 3 – Experimental Procedure

83

3. Experimental Procedure

3.1 General Details

This section applies to all experimental work described in this thesis. Most of the

compounds prepared were identified by at least three of the following: melting point,

mass spectroscopy, infrared, nuclear magnetic resonance (NMR) spectroscopy and

elemental analysis. For known compounds, the spectroscopic data quoted fundaments

identification of the compounds. Biological tests (MTT assays) were made in triplicate.

For the binding studies the compounds were also tested three times at

displacement tested by spectrofluorophotometer.

3.1.1 Chemical Synthesis – Analytical Techniques

Thin Layer Chromatography (TLC) was performed on silica gel 60 F254 (2cm x 5cm)

(Merck, Germany) in a chloroform:methanol (95:5) mobile phase, and spots visualised

by UV-light (254nm).

Nuclear Magnetic Resonance (NMR) spectroscopy was carried out on a Bruker 400

Ultrashield spectrometer operating at 400 MHz for proton (1H) and 100.6 MHz for

carbon-13 (13C) NMR using the solvent peak as intern reference. NMR solvents

chloroform-d (CDCl3) and dimethyl sulfoxide-d6 (DMSO-d6) were used to dissolve

appropriate reaction intermediates and final products as their corresponding

dihydrobromide salts for the bis-naphthalimide derivatives and as trifluoroacetate salts

for the mono-naphthalimide derivatives.

The multiplicities of the signals registed are: singlet (s), doublet (d), double doublet

(dd), triplet (t), multiplet (m). The coupling constant (J) was obtained in Hertz (Hz) and

the chemical shift (δ) in parts per million (ppm).

The Infrared spectrums (IR) were recorded on a spectrophotometer Bomem MB

104. The samples were prepared as Nujol mull and run in sodium chloride cells.

The melting points were determined on a Gallenkamp melting point apparatus, and

are uncorrected.


84

The MS analysis was carried out at the Engineering and Physical Sciences Research

Council’s (EPSRC) National Mass Spectrometry Service Centre at Swansea University,

Swansea, UK.

Purifications by column chromatography were carried out on Kieselgel 0.060-0.200

mm silica.

The solvents were used as purchased.

3.1.2 Binding Studies

Fluorescent-binding studies were carried out in disposable cuvettes using a RF-5301

PC Spectrofluorophotometer (Shimadzu), at room temperature. The program used to

read the emission was panorama Fluorescence 1.1.

3.1.3 Cell Culture and Biological Activity

All cell culture techniques were carried out under aseptic conditions within a

MicroFlow Class II Safety Cabinet (Thermo Electron Corporation, Germany). All cell

culture equipment used was sterile certified and all pipette tips were sterilised before

use in a Boxer Autoclave (Boxer Lab Equipment, UK). The cells were incubated at 37°C,

in a humidified 5% CO2 atmosphere in a HERAcell 150i, CO2 incubator, and when

necessary were counted using an improved Neubauer Haemocytometer (Assitent,

Germany).

The cells were viewed and photographed using a Leica DMIL The cells were viewed

and photographed using a Leica DMIL inverted light microscope (Leica Microsystems,

UK) or a Leica DMIL inverted fluorescence microscope (Leica Microsystems, UK) using a

UV filter, with a Leica DC 200 camera (Leica Microsystems, UK) attached and, viewed

using IrfanView 4.10 software (Leica Microsystems, UK). For the colorimetric 3-(4,5-

Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay analysis, a 96 well

plate reader (BIO-RAD, iMark, microplate reader) was used with Microplate Manager

Software.


85

3.2 General synthesis of mono-naphthalimide derivatives

The methods used for the synthesis of the mono-naphthalimide derivatives, 3a – p

were based on the methods previously used by Vicenzo Tumiati et al.32 (Scheme 21)

Scheme 21: General scheme for the synthesis of mono-naphthalimide derivatives; i) EtOH 3-4h, reflux; ii) EtOH/Aldehyde 4-6 h, reflux; iii) THF/MeOH/NaBH4 overnight, r.t.


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3.3 Reaction of 1,8-naphthalic anhydride with alkyl diamines

3.3.1 General procedure

To a solution of 1,8-naphthalic anhydride (1.6 – 1.8 g; 8.1 – 9.1 mmol) in ethanol

(150 – 200 mL) was added the amine, ethylenediamine, 1,3-diaminopropane or 1,4-

diaminobutane (1.2 – 3.1 g; 16 – 51 mmol; 1.7 – 3.4 mL) and the resulting mixture was

refluxed for 3 to 4 hours. The solution was left at r. t. and/or in an ice bath until a solid

was formed. The solid was filtered, the solvent of the resulting solution was

evaporated under vacuum and the mixture was left in the freezer to crystalize. The

solid formed was filtered, washed with diethyl ether and dried to give the desired

compound 1a – c (1.7 – 2.1 g; 4.9 – 7.8 mmol; 58 – 86 %).

Synthesis of 2-(2-aminoethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (1a)

1,8-Naphthalic anhydride (1.7 g; 8.5 mmol); solvent 150 mL; ethylenediamine (3.1 g;

51 mmol; 3.4 mL); Time: 4 h.

White solid 1a (1.8 g; 4.9 mmol; 58%); M.p. 126-128 °C; υmax (Nujol) 1664, 3350 cm-

1. δH (400 MHz, CDCl3): 3.08 (2H, t, J = 6.8 Hz, H-2’); 4.29 (2H, t, J = 6.4 Hz, H-1’); 7.76

(2H, dd, J = 7.6 and 8.4 Hz, H-5); 8.22 (2H, dd, J = 1.2 and 8.4 Hz, H-6); 8.62 (2H, dd, J =

1.2 and 7.6 Hz, H-4) ppm. δC (100 MHz, CDCl3): 40.9 (C-2’); 43.1 (C-1’); 122.5 (C-3a);

126.9 (C-5); 128.1 (C-10); 131.2 (C-6a); 131.3 (C-4); 133.9 (C-6); 164.5 (C-1/C-3) ppm.


87

Synthesis of 2-(3-aminopropyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (1b)

1,8-Naphthalic anhydride (1.6 g; 8.1 mmol); solvent 200 mL; 1.3-diaminoproprane

(1.2 g; 16 mmol; 1.7 mL); Time: 3 h.

Green solid 1b (1.7 g; 6.7 mmol; 83%); M.p. 126-129 °C. υmax (Nujol) 1650, 3350 cm-

1. δH (400 MHz, CDCl3): 1.88-1.95 (2H, m, H-2’); 2.78 (2H, t, J = 6.8 Hz, H-3’); 4.30 (2H, t,

J = 6.8 Hz, H-1’); 7.77 (2H, dd, J = 7.2 and 8.0 Hz, H-5); 8.22 (2H, dd, J = 0.8 and 8.0 Hz,

H-6); 8.61 (2H, dd, J = 1.2 and 7.6 Hz, (H-4) ppm. δC (100 MHz, CDCl3): 31.9 (C-2’); 37.4

(C-1’); 39.5 (C-3’); 122.4 (C-3a); 126.8 (C-5); 128.0 (C-10); 131.2 (C-4); 131.5 (C-6a);

133.9 (C-6); 164.2 (C-1/C-3) ppm.

Synthesis of 2-(4-aminobutyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (1c)

1,8-Naphthalic anhydride (1.8 g; 9.1 mmol); solvent: 200mL; 1,4-diaminobutane (1.6

g; 18 mmol; 1.8 mL); Time: 3 h.

Green solid 1c (2.1 g; 7.8 mmol; 86%). M.p. 106-109 °C. υmax (Nujol) 1660, 3349 cm-

1. δH (400 MHz, CDCl3): 1.55 - 1.63 (2H, m, H-3’); 1.77 - 1.82 (2H, m, H-2’); 2.79 (2H, t, J

= 7.2 Hz, H-4’); 4.20 (2H, t, J = 7.0 Hz, H-1’); 7.76 (2H, dd, J = 7.2 and 8.4 Hz, H-5); 8.21

(2H, dd, J = 1.2 and 8.4 Hz, H-6); 8.61 (2H, dd, J = 1.2 and 7.6 Hz, H-4) ppm. δC (100

MHz, CDCl3): 25.1 (C-2’); 30.8 (C-3’); 39.8 (C-1’); 41.6 (C-4’); 122.4 (C-3a); 126.8 (C-5);

128.0 (C-10); 131.2 (C-6a); 131.5 (C-4); 133.9 (C-6); 164.3 (C-1/C-3) ppm.


88

3.4 Reaction of amines 1a – c with aldehydes


To a solution of the amine compound 1a – c (0.10 – 0.21 g; 3.7 – 8.6 mmol) in

ethanol (15 mL) was added a solution of the aldehyde (1.0 equiv). The resulting

mixture was refluxed for 4 – 6 hours, then partially concentrated under vacuum and

left in the freezer until a solid was formed. The solid was filtered, washed with ethanol

and dried under vacuum to afford the corresponding imine 2a – p (0.085 – 0.27 g; 0.21

– 0.79 mmol; 54 – 97 %).

Synthesis of 2-(2-((4-methoxybenzylidene)amino)ethyl)-1H-benzo[de]isoquinoline-

1,3(2H)-dione (2a)

Compound 1a (0.21 g; 0.85 mmol); 4-methoxybenzaldehyde (0.11 g; 0.82 mmol; 0.1

mL); time: 4 h.

White solid 2a (0.27 g; 0.75 mmol; 91%). M.p. 199-203 °C. δH (400 MHz, CDCl3): 3.83

(3H, s, H-3b’’); 3.89 - 3.96 (2H, m, H-2’); 4.54 (2H, t, J = 6.8 Hz, H-1’); 6.89 (2H, d, J = 8.4

Hz, H-3’’/H-4’’); 7.64 (2H, d, J = 8.4 Hz, H-2’’/H-5’’); 7.75 (2H, dd, J = 7.2 and 8.0 Hz, H-

5/H-8); 8.21 (2H, dd, J = 1.2 and 8.4 Hz, H-6/H-7); 8.28 (1H, s, H-1’’); 8.61 (2H, dd, J =

1.2 and 7.2 Hz, H-4/H-9) ppm. δC (100 MHz, CDCl3): 40.9 (C-1’); 55.3 (C-3b’’); 58.5 (C-

2’); 113.9 (C-3’’/C-4’’); 122.6 (C-3a/C-9a); 126.9 (C-5/C-8); 128.2 (C-10); 129.7 (C-2’’/C-

5’’); 131.2 (C-4/C-9); 131.3 (C-6a); 131.6 (C-1a’’); 133.9 (C-6/C-7); 160.1 (C-3a’’); 161.9

(C-1’’); 164.1 (C-1/C-3) ppm.


89

Synthesis of 2-(2-((2,6-dichlorobenzylidene)amino)ethyl)-1H-benzo[de]isoquinoline-

1,3(2H)-dione (2b)

Compound 1a (0.17 g; 0.71 mmol); 2,6-dichlorobenzaldehyde (0.63 mmol; 0.11 g);

Time: 24 h.

White solid 2b (0.23 g; 0.58 mmol; 92%). M.p. 201-203 °C; δH (400 MHz, CDCl3): 4.13

(2H, t, J = 6.8 Hz, H-2’); 4.66 (2H, t, J = 6.8 Hz, H-1’); 7.18 (1H, dd, J = 7.6 and 8.4 Hz, H-

4’’); 7.28 (2H, d, J = 7.6 Hz, H-3’’/H-5’’); 7.77 (2H, dd, J = 7.2 and 8.0 Hz, H-5/H-8); 8.23

(2H, dd, J = 1.2 and 8.4 Hz, H-6/H-7); 8.49 (1H, s, H-1’’); 8.63 (2H, dd, J = 1.2 and 7.6 Hz,

H-4/H-9) ppm. δC (100 MHz, CDCl3): 41.1 (C-1’); 58.7 (C-2’); 122.5 (C-3a/C-9a); 126.9 (C-

5/C-8); 128.0 (C-10); 128.1 (C-3’’/C-5’’); 128.4 (C-4’’); 129.0 (C-1a’); 131.3 (C-4/C-9);

131.5 (C-6a); 133.8 (C-6/C-7); 160.1 (C-2’’/C-6’’); 161.5 (C-1’’); 164.2 (C-1/C-3) ppm.

Synthesis of 2-(2-((4-fluorobenzylidene)amino)ethyl)-1H-benzo[de]isoquinoline-

1,3(2H)-dione (2c)

Compound 1a (0.17 g; 0.71 mmol); 4-fluorobenzaldehyde (0.63 mmol; 0.11 g); Time:

8 h.

Yellow solid 2c (0.23 g; 0.66 mmol; 95%) M.p. 158-160 °C; δH (400 MHz, CDCl3): 3.97

(2H, t, J = 7.2 Hz, H-2’); 4.55 (2H, t, J = 7.2 Hz, H-1’); 7.06 (2H, t, J = 8.8 Hz, H-3’’/H-4’’);

7.69 (2H, d, J = 8.8 Hz, H-2’’/H-5’’); 7.77 (2H, dd, J = 7.2 and 8.0 Hz, H-5/H-8); 8.20 (2H,

dd, J = 0.8 and 8.4 Hz, H-6/H-7); 8.31 (1H, s, H-1’’); 8.63 (2H, dd, J = 0.8 and 7.2 Hz, H-

4/H-9) ppm. δC (100 MHz, CDCl3): 41.5 (C-1’); 56.9 (C-2’); 117.0 (C-3’’/C-4’’); 122.6 (C-


90

3a/C-9a); 127.0 (C-5/C-8); 128.1 (C-10); 130.0 (C-2’’/C-5’’); 131.2 (C-4/C-9); 131,5 (C-

6a); 133.9 (C-6/C-7); 134.8 (C-1a’’); 161.2 (C-3a’’); 162.0 (C-1’’); 164.3 (C-1/C-3) ppm.

Synthesis of 2-(2-(((1-methyl-1H-pyrrol-2-yl)methylene)amino)ethyl)-1H-

benzo[de]isoquinoline-1,3(2H)-dione (2n)

Compound 1a (0.21 g; 0.85 mmol); N-methyl-2-pyrrolecarboxaldehyde (0.095 g;

0.87 mmol; 0.81 mL); Time: 2.5 h.

White solid 2n (0.26 g; 0.79 mmol; 93%); M.p. 170-173 °C; δH (400 MHz, CDCl3): 3.12

(2H, t, J = 7.2 Hz, H-2’); 3.51 (3H, s, H-5’’); 4.53 (2H, t, J = 6.8 Hz, H-1’); 6.10 (1H, dd, J =

2.4 and 3.2 Hz, H-2’’); 6.24 (1H, dd, J = 2.4 Hz, H-3’’); 6.93 (1H, dd, J = 2.8 and 3.2 Hz, H-

4’’); 7.76 (2H, dd, J = 7.2 and 8,0 Hz, H-5/H-8); 8.16 (1H, s, H-1’’); 8.21 (2H, dd, J = 1.2

and 8.4 Hz, H-6/H-7); 8.61 (2H, dd, J = 1.2 and 8.2 Hz, H-4/H-9) ppm. δC (100 MHz,

CDCl3): 34.2 (C-5’’); 40.1 (C-1’); 47.1 (C-2’); 108.3 (C-3’’); 109.3 (C-2’’); 116.9 (C-4’’);

122.5 (C-3a/C-9a); 126.9 (C-5/C-8); 128.1 (C-10); 131.1 (C-4/C-9); 131.3 (C-6a); 133.9

(C-6/C-7); 151.2 (C-1a’’); 160.8 (C-1’’); 164.2 (C-1/C-3) ppm.

Synthesis of 2-(3-((4-nitrobenzylidene)amino)propyl)-1H-benzo[de]isoquinoline-

1,3(2H)-dione (2d)

Compound 1b (0.20 g; 0.79 mmol); 4-nitrobenzaldehyde (0.12 g; 0.79 mmol); time:

6 h.


91

White solid 2d (0.17 g; 0.43 mmol; 54%). M.p. 221-224 °C; δH (400 MHz, CDCl3): 2.29

- 2.24 (2H, m, H-2’); 3.82 (2H, t, J = 6.8 Hz, H-3’); 4.38 (2H, t, J = 6.8 Hz, H-1’); 7.67 (2H,

d, J = 8,8 Hz, H-2’’/H-5’’); 7.73 (2H, d, J = 8,0 Hz, H-5/H-8); 8.06 (2H, d, J = 8.8 Hz, H-

3’’/H-4’’); 8.18 (2H, dd, J = 1.2 and 8.4 Hz, H-6/H-7); 8.35 (1H, s, H-1'’); 8.57 (2H, dd, J =

0.8 and 7.2 Hz, H-4/H-9) ppm. δC (100 MHz, CDCl3): 34.0 (C-2’); 42.3 (C-1’); 56.1 (C-3’);

122.6 (C-3a/C-9a); 125,4 (C-3’’/C-4’’); 127.5 (C-5/C-8); 128.1 (C-10); 129.0 (C-2’’/C-5’’);

129.5 (C-1a’’); 131.2 (C-4/C-9); 131.6 (C-6a); 133.9 (C-6/C-7); 148.9 (C-3a’’); 160.2 (C-

1’’); 164.3 (C-1/C-3) ppm.

Synthesis of 2-(3-((4-methoxybenzylidene)amino)propyl)-1H-benzo[de]isoquinoline-

1,3(2H)-dione (2e)

Compound 1b (0.21 g; 0.82 mmol); 4-methoxybenzaldehyde (0.74 mmol; 1.0 mL);

Time: 2 days.

Beige solid 2e (0.27 g; 0.72 mmol; 97%). M.p. 208-210 °C; δH (400 MHz, CDCl3): 2.16-

2.23 (2H, m, H-2’); 3.71-3.75 (2H, t, H-3’); 3.82 (3H, s, H-3b’’); 4.35 (2H, s, J = 8.8 Hz, H-

1’); 6.80 (2H, d, J = 8.8 Hz, H-3’’/H-4’’); 7.30 (2H, d, J = 8.8 Hz, H-2’’/H-5’’); 7.74 (2H, dd,

J = 7.6 and 8.4 Hz, H-5/H-8); 8.19 (2H, dd, J = 1.2 and 8.4 Hz, H-6/H-7); 8.22 (1H, s, H-

1’’); 8.59 (2H, dd, J = 1.2 and 7.2 Hz, H-4/H-9) ppm. δC (100 MHz, CDCl3): 33.4 (C-2’);

41.2 (C-1’); 50.1 (C-3b’’); 55.7 (C-3’); 113.7 (C-3’’/C-4’’); 122.5 (C-3a/C-9a); 126.7 (C-

5/C-8); 128.1 (C-10); 129.6 (C-2’’/C-5’’); 131.0 (C-4/C-9); 131.3 (C-6a); 132.2 (C-1a’’);

133.8 (C-6/C-7); 159.8 (C-3a’’); 161.2 (C-1’’); 164.2 (C-1/C-3) ppm.


92

Synthesis of 2-(3-((2,6-dichlorobenzylidene)amino)propyl)-1H-benzo[de]isoquinoline-

1,3(2H)-dione (2f)

Compound 1b (0.16 g; 0.63 mmol); 2,6-dichlorobenzaldehyde (0.63 mmol; 0.11 g);

Time: 2 days.

White solid 2f (0.16 g; 0.38 mmol; 60%);M.p. 205-207 °C; δH (400 MHz, CDCl3): 2.18-

2.28 (2H, m, H-2’); 3.87 (2H, t, J = 7.2 Hz, H-3’); 4.39 (2H, t, J = 7.2 Hz, H-1’); 7.22 (1H,

dd, J = 7.2 and 8.8 Hz, H-4’’); 7.33 (2H, d, J = 7.2 Hz, H-3’’/H-5’’); 7.76 (2H, dd, J = 7.2

and 8.0 Hz, H-5/H-8); 8.21 (2H, dd, J = 1.2 and 8.4 Hz, H-6/H-7); 8.51 (1H, s, H-1’’); 8.62

(2H, dd, J = 1.2 and 7.6 Hz, H-4/H-9) ppm. (100 MHz, CDCl3): 30.1 (C-2’); 40.2 (C-1’);

48.2 (C-3’); 122.8 (C-3a/C-9a); 126.7 (C-5/C-8); 127.9 (C-10); 128.3 (C-3’’/C-5’’); 128.8

(C-4’’); 131.2 (C-4/C-9); 131.5 (C-6a); 134.1 (C-1a’’); 136.0 (C-6/C-7); 159.2 (C-2’’/C-6’’);

161.8 (C-1’’); 164.3 (C-1/C-3) ppm.

Synthesis of 2-(3-((4-fluorobenzylidene)amino)propyl)-1H-benzo[de]isoquinoline-

1,3(2H)-dione (2g)

Compound 1b (0.15 g; 0.59 mmol); 4-fluorobenzaldehyde (0.086 g; 0.69 mmol; 0.75

mL); Time: 4 h.

Beige solid 2g (0.14 g; 0.39 mmol; 66%); M.p. 187-189 °C; δH (400 MHz, CDCl3): 2.18-

2.24 (2H, m, H-2’); 3.75 (2H, t, J = 6.8 Hz, H-3’); 4.36 (2H, t, J = 6.8 Hz, H-1’); 6.95 (2H, t,

J = 8.8 Hz, H-3’’/H-4’’); 7.56 (2H, d, J = 9.2 Hz, H-2’’/H-5’’); 7.74 (2H, dd, J = 7.2 and 8.0

Hz, H-5/H-8); 8.19 (2H, dd, J = 1.2 and 8.4 Hz, H-6/H-7); 8.25 (1H, s, H-1’’); 8.58 (2H, dd,


93

J = 1.2 and 7.2 Hz, H-4/H-9) ppm. δC (100 MHz, CDCl3): 29.9 (C-2’); 39.2 (C-1’); 48.2 (C-

3’); 115.5 (C-3’’/C-4’’); 122.8 (C-3a/C-9a); 126.7 (C-5/C-8); 128.0 (C-10); 129.8 (C-2’’/C-

5’’); 131.2 (C-4/C-9); 131.5 (C-6a); 133.9 (C-6/C-7); 136.2 (C-1a’’); 159.6 (C-3a’’); 161.4

(C-1’’); 164.4 (C-1/C-3) ppm.

Synthesis of 2-(3-((4-(dimethylamino)benzylidene)amino)propyl)-1H-

benzo[de]isoquinoline-1,3(2H)-dione (2h)

Compound 1b (0.16 g; 0.63 mmol); N-dimethylaminobenzaldehyde (0.67 mmol,

0.10 g); Time: 24 h.

Beige solid 2h (0.16 g; 0.41 mmol; 65%); M.p. 201-203 °C; δH (400 MHz, CDCl3): 2.14-

2.21 (2H, m, H-2’); 2.99 (6H, s, H-3b’’); 3.70 (2H, t, J = 6.8 Hz, H-3’); 4.33 (2H, t, J = 7.2

Hz, H-1’); 6.60 (2H, d, J = 8.8 Hz, H-3’’/H-4’’); 7.50 (2H, d, J = 9.2 Hz, H-2’’/H-5’’); 7.73

(2H, dd, J = 7.2 and 8.4 Hz, H-5/H-8); 8.18 (2H, dd, J = 0.8 and 8.4 Hz, H-6/H-7); 8.30

(1H, s, H-1’’); 8.58 (2H, dd, J = 1.2 and 7.2 Hz, H-4/H-9) ppm. δC (100 MHz, CDCl3): 29.6

(C-2’); 38.9 (C-1’); 40.2 (C-3b’’); 59.4 (C-3’); 111.5 (C-3’’/C-4’’); 122.8 (C-3a/C-9a); 126.9

(C-5/C-8); 128.1 (C-10); 129.4 (C-2’’/C-6’’); 131.1 (C-4/C-9); 133.7 (C-6/C-7); 134.5 (C-

1a’’); 151.9 (C-3a’’); 161.2 (C-1’’); 164.2 (C-1/C-3) ppm.

Synthesis of 2-(3-(((1-methyl-1H-pyrrol-2-yl)methylene)amino)propyl)-1H-

benzo[de]isoquinoline-1,3(2H)-dione (2o)


94

Compound 1b (0.16 g; 0.62 mmol); N-methyl-2-carboxyaldehyde (0.74 mmol; 0.083

mL); Time: 5 h.

Yellow solid 2o (0.16 g; 0.46 mmol; 74%); M.p. 172-175 °C; δH (400 MHz, CDCl3):

2.01-2.11 (2H, m, H-2’); 3.68 (2H, t, J = 7.2 Hz, H-3’); 3.92 (3H, s, H-5’’); 4.31 (2H, t, J =

7.2 Hz, H-1’); 6.22 (1H, dd, J = 2.4 and 3.6 Hz, H-2’’); 6.24 (1H, dd, J = 2.4 Hz, H-3’’); 6.91

(1H, dd, J = 2.8 and 3.6 Hz, H-4’’); 7.78 (2H, dd, J = 7.2 and 7.6 Hz, H-5/H-8); 8.24 (2H,

dd, J = 1.2 and 7.2 Hz, H-6/H-7); 8.31 (1H, s, H-1’’); 8.60 (2H, dd, J = 1.2 and 7.6 Hz, H-

4/H-9) ppm. δC (100 MHz, CDCl3): 28.7 (C-2’); 34.2 (C-5’’); 39.1 (C-1’); 46.8 (C-3’); 106.9

(C-3’’); 107.9 (C-2’’); 122.0 (C-4’’); 122.4 (C-3a/C-9a); 126,9 (C-5/C-8); 127.9 (C-10);

131.1 (C-4/C-9); 131.3 (C-6a); 133.9 (C-6/C-7); 151.2 (C-1a’’); 161.3 (C-1’’); 164.3 (C-

1/C-3) ppm.

Synthesis of 2-(4-((4-nitrobenzylidene)amino)butyl)-1H-benzo[de]isoquinoline-

1,3(2H)-dione (2i)

Compound 1c (0.10 g; 0.37 mmol); 4-nitrobenzaldehyde (0.39 mmol, 0.06 g); Time:

6 h.

Orange oil 2i (0.12 g; 0.29 mmol; 78%); M.p. 212-215 °C. δH (400 MHz, CDCl3):1.84-

1.89 (4H, m, H-2’/H-3’); 4.76 (2H, t, J = 6.8 Hz, H-4’); 4.24 (2H, t, J = 7.2 Hz, H-1’); 7.77

(2H, d, J = 8.4 Hz, H-2’’/H-5’’); 7.89 (2H, dd, J = 7.6 and 8.4 Hz, H-5/H-8); 8.21 (2H, t, J =

7.2 Hz, H-3’’/H-4’’); 8.24 (2H, dd, J = 0.8 and 8.4 Hz, H-6/H-7); 8.39 (1H, s, H-1’’); 8.60

(2H, dd, J = 1.2 and 7.6 Hz, H-4/H-9) ppm. δC (100 MHz, CDCl3): 28.7 (C-2’); 30.1 (C-3’);

41.2 (C-1’); 49.5 (C-4’); 122.7 (C-3a/C-9a); 124.2 (C-3’’/C-4’’); 127.1 (C-5/C-8); 128.0 (C-

10); 128.6 (C-2’’/C-5’’); 129.2 (C-1a’’); 131.3 (C-4/C-9); 131.6 (C-6a); 133.8 (C-6/C-7);

148.7 (C-3a’’); 161.0 (C-1’’); 164.4 (C-1/C-3) ppm.


95

Synthesis of 2-(4-((4-methoxybenzylidene)amino)butyl)-1H-benzo[de]isoquinoline-

1,3(2H)-dione (2j)

Compound 1c (0.15 g; 0.57 mmol); 4-methoxybenzaldehyde (0.72 mmol, 0.73 mL); -

Time: 2 days.

Beige solid 2j (0.20 g; 0.53 mmol; 93%); M.p. 201-203 °C; δH (400 MHz, CDCl3): 1.80 -

1.85 (4H, m, H-2’/H-3’); 3.63-3.67 (2H, m, H-4’); 3.83 (3H, s, H-3b’’); 4.24-4.28 (2H, m,

H-1’); 6.90 (2H, d, J = 8.8 Hz, H-3’’/H-4’’); 7.66 (2H, d, J = 8.8 Hz, H-2’’/H-5’’); 7.76 (2H,

dd, J = 7.6 e 8.4 Hz, H-5/H-8); 8.21 (2H, dd, J = 1.2 and 8.4 Hz, H-6/H-7); 8.20 (1H, s, H-

1’’); 8.61 (2H, dd, J = 1.2 and 7.6 Hz, H-4/H-9) ppm. δC (100 MHz, CDCl3): 25.9 (C-2’/C-

3’); 28.4 (C-2’/C-3’); 40.1 (C-1’); 55.3 (C-3b’’); 61.2 (C-4’); 113.9 (C-3’’/C-4’’); 122.7 (C-

3a/C-9a); 126.9 (C-5/C-8); 128.2 (C-10); 129.6 (C-2’’/C-5’’); 131.2 (C-4/C-9); 131.6 (C-

6a); 132.0 (C-1a’’); 133.9 (C-6/C-7); 160.5 (C-1’’); 161.4 (C-3a’’); 164.2 (C-1/C-3) ppm.

Synthesis of 2-(4-((2,6-dichlorobenzylidene)amino)butyl)-1H-benzo[de]isoquinoline-

1,3(2H)-dione (2k)

Compound 1c (0.17 g; 0.63 mmol); 2,6-dichlorobenzaldehyde (0.43 mmol, 0.075 g);

Time: 2 days.

Beige solid 2k (0.13 g; 0.31 mmol; 72%);M.p. 200-202 °C; δH (400 MHz, CDCl3): 1.89-


(1H, dd, J = 7.2 and 8.8 Hz, H-4’’); 7.34 (2H, d, J = 8.2 Hz, H-3’’/H-5’’); 7.76 (2H, dd, J =


96

6.8 and 7.6 Hz, H-5/H-8); 8.22 (2H, dd, J = 0.8 and 6.8 Hz, H-6/H-7); 8.45 (1H, s, H-1’’);

8.62 (2H, dd, J = 0.8 and 7.6 Hz, H-4/H-9) ppm. δC (100 MHz, CDCl3): 26.2 (C-2’); 28.4

(C-3’); 40.5 (C-1’); 49.3 (C-4’); 122.7 (C-3a/C-9a); 126.9 (C-5/C-8); 128.1 (C-10); 128.3

(C-3’’/C-5’’); 129.1 (C-4’’); 131.2 (C-4/C-9); 131.6 (C-6a); 132.0 (C-1a’’); 133.9 (C-6/C-7);

159.3 (C-2’’/6’’); 161.2 (C-1’’); 164.3 (C-1/C-3) ppm.

Synthesis of 2-(4-((4-fluorobenzylidene)amino)butyl)-1H-benzo[de]isoquinoline-

1,3(2H)-dione (2l)

Compound 1c (0.17 g; 0.64 mmol); 4-fluorobenzaldehyde (0.70 mmol, 0.086 g,

0.075 mL); Time: 3 h.

Beige solid 2l (0.13 g; 0.35 mmol; 55%); M.p. 182-185 °C; δH (400 MHz, CDCl3): 1.77-


(2H, t, J = 8.4 Hz, H-3’’/H-4’’); 7.71 (2H, d, J = 8.8 Hz, H-2’’/H-5’’); 7.76 (2H, dd, J = 7.2

and 8.4 Hz, H-5/H-8); 8.22 (2H, dd, J = 1.2 and 8.0 Hz, H-6/H-7); 8.25 (1H, s, H-1’’); 8.61

(2H, dd, J = 1.2 and 7.2 Hz, H-4/H-9) ppm. δC (100 MHz, CDCl3): 26.1 (C-2’); 28.9 (C-3’);

40.4 (C-1’); 59.2 (C-4’); 116.9 (C-3’’/C-4’’); 122.7 (C-3a/C-9a); 126.8 (C-5/C-8); 128.0 (C-

10); 129.6 (C-2’’/C-5’’); 131.3 (C-4/C-9); 131.6 (C-6a); 133.8 (C-6/C-7); 142.0 (C-1a’’);

160.2 (C-3a’’); 161.1 (C-1’’); 164.4 (C-1/C-3) ppm.

Synthesis of 2-(4-((4-(dimethylamino)benzylidene)amino)butyl)-1H-

benzo[de]isoquinoline-1,3(2H)-dione (2m)


97

Compound 1c (0.10 g; 0.38 mmol); 4-(dimethylamino)benzaldehyde (0.06 g; 0.40

mmol); Time: 5 h;

Beige solid 2m (0.085 g; 0.21 mmol; 55%); M.p. 201-205 °C; δH (400 MHz, CDCl3):

1.81-1.84 (4H, m, H-2’/H-3’); 2.99 (6H, s, H-3b’’); 3.70-3.75 (2H, m, H-4’); 4.24-4.27 (2H,

m, H-1’); 6.69 (2H, d, J = 8.8 Hz, H-3’’/H-4’’); 7.59 (2H, d, J = 8.8 Hz, H-2’’/H-5’’); 7.74

(2H, dd, J = 7.2 and 8.0 Hz, H-5/H-8); 8.16 (1H, s, H-1’’) 8.20 (2H, dd, J = 1.2 and 8.4 Hz,

H-6/H-7); 8.60 (2H, dd, J = 0.8 and 7.2 Hz, H-4/H-9) ppm. δC (100 MHz, CDCl3): 25.9 (C-

2’); 28.6 (C-3’); 40.1 (C-1’); 40.2 (C-3b’’); 61.2 (C-4’); 111.6 (C-3’’/4’’); 122.7 (C-3a/C-9a);

126,9 (C-5/C-8); 128.1 (C-10); 130.0 (C-2’’/C-5’’); 131.2 (C-4/C-9); 131.6 (C-6a); 132.2

(C-1a’’); 133.8 (C-6/C-7); 151.9 (C-3a’’); 161.1 (C-1’’); 164.2 (C-1/C-3) ppm.

Synthesis of 2-(4-(((1-methyl-1H-pyrrol-2-yl)methylene)amino)butyl)-1H-

benzo[de]isoquinoline-1,3(2H)-dione (2p)

Compound 1c (0.15 g; 0.57 mmol); N-methyl-2-carboxyaldehyde (0.079 g; 0.73

mmol; 0.8 mL); Time: 4 h.

Beige solid 2p (0.12 g; 0.32 mmol; 56%); M.p. 177-179 °C; δH (400 MHz, CDCl3): 1.71-

1.78 (2H, m, H-3’); 1.77-1.84 (4H, m, H-2’/H-3’); 3.55 (2H, t, J = 7.2 Hz, H-4’); 3.92 (3H,

s, H-5’’); 4.25 (2H, t, J = 7.2 Hz, H-1’); 6.14 (1H,dd, J = 2.8 Hz, H-3’’); 6.16 (1H, dd, J = 2.8

and 3.6 Hz, H-2’’) (6.98 (1H, t, J = 2.8 and 3.2 Hz, H-4’’); 7.76 (2H, dd, J = 7.2 and 8.0 Hz,

H-5/H-8); 8.14 (1H, s, H-1’’) 8.22 (2H, dd, J = 1.2 and 8.4 Hz, H-6/H-7); 8.61 (2H, dd, J =

1.2 and 7.2 Hz, H-4/H-9) ppm. δC (100 MHz, CDCl3): 26.1 (C-3’); 27.5 (C-2’); 34.8 (C-5’’);

42.1 (C-1’); 51.2 (C-4’); 106.0 (C-3’’); 107.5 (C-2’’); 122.9 (C-4’’); 122.6 (C-3a/9a); 126.9

(C-5/C-8); 128.0 (C-10); 131.1 (C-4/C-9); 131.4 (C-6a); 133.6 (C-6/C-7); 152.0 (C-1a’’);

161.1 (C-1’’); 164.2 (C-1/C-3) ppm.


98

3.5 Reduction of imines 2a – p


To a solution of the imine 2a – p in THF:MeOH (1:1, 10 mL) was added sodium

borohydride (NaBH4) (2 – 4 equiv.) and the resulting mixture was stirred at r.t. for 1 – 4

days. The solvent was evaporated under vacuum and the solid formed was dissolved in

DCM (30 mL). The solution was washed with H2O (2 x 10 mL), the organic phase was

dried over MgSO4, filtered and the solvent removed under vacuum to afford the

desired amines 3a – p as oils or solids (0.057 – 0.26 g, 0.13 – 0.71 mmol, 49 – 95 %).

Synthesis of 2-(2-((4-methoxybenzyl)amino)ethyl)-1H-benzo[de]isoquinoline-1,3(2H)-

dione (3a)

Compound 2a (0.27 g; 0.75 mmol); NaBH4 (0.060 g); Time: 24 h.

Orange oil 3a (0.26 g; 0.71 mmol; 95%). M.p. 192 - 195 °C. υmax (Nujol) 1025, 1666,

2644 cm-1. δH (400 MHz, CDCl3): 2.99 (2H, t, J = 6.4 Hz, H-2’); 3.72 (3H, s, H-3b’’); 3.77

(2H, s, H-1’’); 4.31 (2H, t, J = 6.8 Hz, H-1’); 6.76 (2H, dd, J = 2.0 and 6.4 Hz, H-3’’); 7.19

(2H, dd, J = 2.0 and 6.8 Hz, H-2’’); 7.66 (2H, dd, J = 7.2 and 8.0 Hz, H-5); 8.11 (2H, dd, J =

1.2 and 8.4 Hz, H-6); 8.50 (2H, dd, J = 1.2 and 7.6 Hz, H-4) ppm. δC (100 MHz, CDCl3):

39.7 (C-1’); 46.8 (C-2’); 52.7 (C-1’’); 55.0 (C-3b’’); 113.5 (C-3’’); 122.4 (C-3a); 126.7 (C-5),

127.9 (C-10); 129.1 (C-2’’); 130.9 (C-4); 131.3 (C-6a); 132.9 (C-1a’); 133.7 (C-6); 158.2

(C-3a’’); 164.1 (C-1/C-3) ppm.


99

Synthesis of 2-(2-((2,6-dichlorobenzyl)amino)ethyl)-1H-benzo[de]isoquinoline-

1,3(2H)-dione (3b)

Compound 2b (0.23 g; 0.58 mmol); NaBH4 (0.060 g); Time: 4 days.

White solid 3b (0.17 g; 0.42 mmol; 73%); M.p. 200-202 °C. υmax (Nujol) 692, 1659,

2650 cm-1. δH (400 MHz, CDCl3): 2.99 (2H, t, J = 6.4 Hz, H-2’); 4.10 (2H, s, H-1’’); 4.31

(2H, t, J = 6.4 Hz, H-1’); 7.18 (1H, dd, J = 7.2 and 8.0 Hz, H-4’’); 7.20 (2H, d, J = 8.0 Hz, H-

3’’); 7.68 (2H, dd, J = 7.2 and 8.0 Hz, H-5); 8,13 (2H, dd, J = 0.8 and 7.6 Hz, H-6); 8.50

(2H, dd, J = 1.2 and 7.2 Hz, H-4) ppm. δC (100 MHz, CDCl3): 39.9 (C-1’); 47.8 (C-2’); 47.9

(C-1’’); 122.4 (C-3a); 126.8 (C-5); 127.9 (C-10); 128.2 (C-3’’); 128.7 (C-4’’); 131.1 (C-4);

131.4 (C-6a); 133.7 (C-6); 134.2 (C-1a’’); 158.6 (C-2’’); 164.1 (C-1/C-3) ppm.

Synthesis of 2-(2-((4-fluorobenzyl)amino)ethyl)-1H-benzo[de]isoquinoline-1,3(2H)-

dione (3c)

Compound 2c (0.20 g; 0.50 mmol); NaBH4 (0.044 g); Time: 2 days.

White solid 3c (0.10 g; 0.42 mmol; 84%); M.p. 175-177 °C. υmax (Nujol) 1109, 1661,

2671 cm-1. δH (400 MHz, CDCl3): 2.81 (2H, t, J = 6.8 Hz, H-2’); 3.69 (2H, s, H-1’’); 4.29

(2H, t, J = 7.2 Hz, H-1’); 6.98 (2H, t, J = 8.4 Hz, H-3’’); 7.26 (2H, dd, J = 7.2 and 8.8 Hz, H-

2’’); 7.70 (2H, dd, J = 7.6 and 8.8 Hz, H-5); 8.19 (2H, dd, J = 1.2 and 7.6 Hz, H-6); 8.52

(2H, dd, J = 1.2 and 8.0 Hz, H-4) ppm. δC (100 MHz, CDCl3): 38.7 (C-1’); 46.7 (C-2’); 53.2

(C-1’’); 115.1 (C-3’’); 122.4 (C-3a); 127.9 (C-5); 128.1 (C-10); 129.3 (C-2’’); 131.0 (C-4);

131.3 (C-6a); 133.7 (C-6); 135.7 (C-1a’’); 161.4 (C-3a’’); 164.2 (C-1/C-3) ppm.


100

Synthesis of 2-(2-(((1-methyl-1H-pyrrol-2-yl)methyl)amino)ethyl)-1H-

benzo[de]isoquinoline-1,3(2H)-dione (3n)

Compound 2n (0.23 g; 0.69 mmol); NaBH4 (0.063 g); Time: 4 h.

Yellow oil 3n (0.16 g; 0.47 mmol; 68%); M.p. 170-173 °C. υmax (Nujol) 1092, 1662 cm-

1. δH (400 MHz, CDCl3): 3.05 (2H, t, J = 6.4 Hz, H-2’); 3.56 (3H, s, H-5’’); 3.80 (2H, s, H-

1’’); 4.34 (2H, t, J = 6.4 Hz, H-1’); 5.98 (1H, dd, J = 2.8 e 3.2 Hz, H-3’’); 6.01 (1H, dd, J =

2.0 e 3.2 Hz, H-2’’); 6.49 (1H, dd, J = 2.0 and 3.2 Hz, H-4’’); 7.72 (2H, dd, J = 7.2 and 8.4

Hz, H-5); 8,17 (2H, dd, J = 1.2 and 8.4 Hz, H-6); 8.55 (2H, dd, J = 1.2 and 7.6 Hz, H-4)

ppm. δC (100 MHz, CDCl3): 33.5 (C-5’’); 39.7 (C-1’); 45.1 (C-1’’); 46.9 (C-2’); 106.3 (C-3’’);

107.9 (C-2’’); 122.1 (C-4’’); 122.4 (C-3a); 126.8 (C-5); 127.9 (C-10); 130.9 (C-1a’’); 131.0

(C-4); 131.4 (C-6a); 133.8 (C-6); 164.2 (C-1/C-3) ppm.

Synthesis of 2-(3-((4-nitrobenzyl)amino)propyl)-1H-benzo[de]isoquinoline-1,3(2H)-

dione (3d)

Compound 2d (0.17 g; 0.43 mmol); NaBH4 (0.054 g); Time: 2 days.

Beige solid 3d (0.081 g; 0.21 mmol; 49%). M.p. 226-228 °C. υmax (Nujol) 1329, 1547,

1657, 2671 cm-1. δH (400 MHz, CDCl3): 1.96-2.03 (2H, m, H-2’); 2.71 (2H, t, J = 6.8 Hz, H-

3’); 3.90 (2H, s, H-1’’); 4.31 (2H, t, J = 6.8 Hz, H-1’); 7.49 (2H, d, J = 8.8 Hz, H-2’’); 7.77

(2H, dd, J = 7.2 and 8.4 Hz, H-5); 8.12 (2H, d, J = 8.8 Hz, H-3’’); 8.23 (2H, dd, J = 1.2 and

8.4 Hz, H-6); 8.60 (2H, dd, J = 1.2 and 7.2 Hz, H-4) ppm. δC (100 MHz, CDCl3): 28.2 (C-

2’); 38.1 (C-1’); 46.4 (C-3’); 53.1 (C-1’’); 122.5 (C-3a); 123.5 (C-3’’); 126.9 (C-5); 128.1 (C-


101

10); 128.4 (C-2’’); 131.3 (C-4); 131.6 (C-6a); 132.1 (C-1a’’); 134.0 (C-6); 148.3 (C-3a’’);

164.3 (C-1/C-3) ppm.

Synthesis of 2-(3-((4-methoxybenzyl)amino)propyl)-1H-benzo[de]isoquinoline-

1,3(2H)-dione (3e)

Compound 2e (0.25 g; 0.67 mmol); NaBH4 (0.063 g); Time: 2 days.

White solid 3e (0.19 g; 0.51 mmol; 75%). M.p. 205-208 °C. υmax (Nujol) 1101, 1653,

2599 cm-1. δH (400 MHz, CDCl3): 1.90-1.98 (2H, m, H-2’); 2.67 (2H, t, J = 6.8 Hz, H-3’);

3.69 (2H, s, H-1’’); 3.72 (3H, s, H-3b’’); 4.22 (2H, t, J = 6.8 Hz, H-1’); 6.78 (2H, dd, J = 2.2

and 6.4 Hz, H-3’’); 7.21 (2H, dd, J = 2.0 and 6.4 Hz, H-2’’); 7.67 (2H, dd, J = 7.2 and 8.0

Hz, H-5); 8.12 (2H, dd, J = 0.8 and 8.4 Hz, H-6); 8.50 (2H, dd, J = 1.2 and 7.2 Hz, H-4)

ppm. δC (100 MHz, CDCl3): 28.1 (C-2’); 38.1 (C-1’); 46.2 (C-3’); 53.1 (C-1’’); 55.0 (C-3b’’),

113.5 (C-3’’); 122.3 (C-3a); 126.7 (C-5); 127.8 (C-10); 129.2 (C-2’’); 130.9 (C-4); 131.3 (C-

6a); 133.0 (C-1a’’); 133.7 (C-6); 158.3 (C-3a’’); 163.9 (C-1/C-3) ppm.

Synthesis of 2-(3-((2,6-dichlorobenzyl)amino)propyl)-1H-benzo[de]isoquinoline-

1,3(2H)-dione (3f)

Compound 2f (0.10 g; 0.25 mmol); NaBH4 (0.036 g); Time: 2 days.


102

White solid 3f (0.70 g; 0.17 mmol; 68%); M.p. 204-206°C. υmax (Nujol) 604, 1660,


4.08 (2H, s, H-1’’); 4.25 (2H, t, J = 7.2 Hz, H-1’); 7.08 (2H, dd, J = 7.6 and 8.4 Hz, H-4’’);

7.25 (2H, d, J = 8,0 Hz, H-3’’); 7.72 (2H, dd, J = 7.6 and 8.4 Hz, H-5); 8.17 (2H, dd, J = 0.8

and 8.4 Hz, H-6); 8.55 (2H, dd, J = 1.2 and 7.6 Hz, H-4) ppm. δC (100 MHz, CDCl3): 28.4

(C-2’); 38.3 (C-1’); 46.3 (C-3’); 48.2 (C-1’’); 122.6 (C-3a); 126.8 (C-5); 128.0 (C-10); 128.2

(C-3’’); 128.7 (C-4’’); 131.1 (C-4); 131.5 (C-6a); 133.8 (C-1a’’); 135.8 (C-6); 155.9 (C-2’’);

164.1 (C-1/C-3) ppm.

Synthesis of 2-(3-((4-fluorobenzyl)amino)propyl)-1H-benzo[de]isoquinoline-1,3(2H)-

dione (3g)

Compound 2g (0.14 g; 0.38 mmol); NaBH4 (0.033 g); Time: 24 h.

Beige solid 3g (0.079 g; 0.22 mmol; 58%). M.p. 182-184 °C. υmax (Nujol) 1055, 1661,


3.74 (2H, s, H-1’’); 4.25 (2H, t, J = 7.2 Hz, H-1’); 6.94 (2H, dd, J = 7.6 and 8.8 Hz, H-3’’);

7.26 (2H, t, J = 8.4 Hz, H-2’’); 7.71 (2H, dd, J = 7.2 and 8.4 Hz, H-5); 8.16 (2H, dd, J = 1.2

and 8.4 Hz, H-6); 8.54 (2H, dd, J = 1.2 and 7.6 Hz, H-4) ppm. δC (100 MHz, CDCl3): 28.1

(C-2’); 38.1 (C-1’); 46.2 (C-3’); 53.0 (C-1’’); 114.9 (C-3’’); 122.4 (C-3a); 126,8 (C-5); 128.0

(C-10); 129.5 (C-2’’); 131.1 (C-4); 131.4 (C-6a); 133.8 (C-6); 136.0 (C-1a’’); 161.1 (C-3a’’);

164.1 (C-1/C-3) ppm.


103

Synthesis of 2-(3-((4-(dimethylamino)benzyl)amino)propyl)-1H-

benzo[de]isoquinoline-1,3(2H)-dione (3h)

Compound 2h (0.12 g; 0.31 mmol); NaBH4 (0.029 g); Time: 24 h.

Yellow solid 3h (0.077 g; 0.20 mmol; 65%); M.p. 205-207 °C. υmax (Nujol) 1201, 1225,

1667, 2612 cm-1. δH (400 MHz, CDCl3): 1.93-1.99 (2H, m, H-2’); 2.69 (2H, t, J = 6.8 Hz, H-

3’); 2.90 (6H, s, H-3b’’); 3.69 (2H, s, H-1’’); 4.25 (2H, t, J = 6.8 Hz, H-1’); 6.66 (2H, dd, J =

2.0 and 6.4 Hz, H-3’’); 7.18 (2H, dd, J = 2.0 and 6.8 Hz, H-2’’); 7.71 (2H, dd, J = 7.2 and

8.4 Hz, H-5); 8.16 (2H, dd, J = 0.8 and 8.0 Hz, H-6); 8.50 (2H, dd, J = 1.2 and 7.6 Hz, H-4)

ppm. δC (100 MHz, CDCl3): 28.2 (C-3’); 38.2 (C-1’); 40.6 (C-3b’’); 46.2 (C-2’); 53.0 (C-1’’);

112.4 (C-2’’); 122.5 (C-3a); 126.7 (C-5); 127.9 (C-10); 128.2 (C-3’’); 131.0 (C-4); 131.4 (C-

6a); 132.1 (C-1a’’); 133.7 (C-6); 149.6 (C-3a’’); 164.0 (C-1/C-3) ppm.

Synthesis of 2-(3-(((1-methyl-1H-pyrrol-2-yl)methyl)amino)propyl)-1H-

benzo[de]isoquinoline-1,3(2H)-dione (3o)

Compound 2o (0.13 g; 0.35 mmol); NaBH4 (0.043 g); Time: 24 hours.

Yellow oil 3o (0.067 g; 0.19 mmol; 54%); M.p. 177-179 °C. υmax (Nujol) 1088, 1659,


3.70 (3H, s, H-5’’); 3.74 (2H, s, H-1’’); 4.26 (2H, t, J = 7.2 Hz, H-1’); 5.97 (1H, dd, J = 2.4

and 3.6 Hz, H-3’’); 6.03 (1H, dd, J = 2.0 and 3.4 Hz, H-2’’); 6.50 (1H, dd, J = 2.4 and 4.0

Hz, H-4’’); 7.73 (2H, dd, J = 8.0 and 8.4 Hz, H-5); 8.18 (2H, dd, J = 1.2 and 8.4 Hz, H-6);

8.56 (2H, dd, J = 1.2 and 8.0 Hz, H-4) ppm. δC (100 MHz, CDCl3): 28.2 (C-2’); 33.9 (C-5’’);


104

38.3 (C-1’); 45.3 (C-1’’); 46.5 (C-3’); 106.3 (C-3’’); 107.7 (C-2’’); 122.2 (C-4’’); 122.5 (C-

3a); 126.8 (C-5); 128.0 (C-10); 130.2 (C-1a’’); 131.1 (C-4); 131.4 (C-6a); 133.8 (C-6);

164.1 (C-1/C-3) ppm.

Synthesis of 2-(4-((4-nitrobenzyl)amino)butyl)-1H-benzo[de]isoquinoline-1,3(2H)-

dione (3i)

Compound 2i (0.12 g; 0.29 mmol); NaBH4 (0.025 g); Time: 24 h.

Orange oil 3i (0.084 g; 0.22 mmol; 76%); M.p. 220-222 °C. υmax (Nujol) 1334, 1555,

1658, 2591 cm-1. δH (400 MHz, CDCl3): 1.62-1.69 (2H, m, H-3’); 1.78-1.85 (2H, m, H-2’);

2.71 (2H, t, J = 6.8 Hz, H-4’); 3.89 (2H, s, H-1’’); 4.20 (2H, t, J = 7.2 Hz, H-1’); 7.50 (2H, t,

J = 8.8 Hz, H-2’’); 7.75 (2H, dd, J = 7.6 and 8.2 Hz, H-5); 8.15 (2H, t, J = 8.0 Hz, H-3’’);

8.20 (2H, dd, J = 0.4 and 8.0 Hz, H-6); 8.57 (2H, dd, J = 0.8 and 7.8 Hz, H-4) ppm. δC (100

MHz, CDCl3): 25.7 (C-2’); 27.4 (C-3’); 40.0 (C-1’); 48.9 (C-4’); 53.1 (C-1’’); 122.6 (C-3a);

123.8 (C-3’’); 126.9 (C-5), 128.1 (C-10); 128.5 (C-2’’); 128.7 (C-6a’); 131.2 (C-4); 131.5

(C-6a); 132.1 (C-1a’); 133.9 (C-6); 148.4 (C-3a’’); 164.2 (C-1/C-3) ppm.

Synthesis of 2-(4-((4-methoxybenzyl)amino)butyl)-1H-benzo[de]isoquinoline-1,3(2H)-

dione (3j)

Compound 2j (0.097 g; 0.25 mmol); NaBH4 (0.025 g); Time: 24h.


105

Orange oil 3j (0.084 g; 0.22 mmol; 86%); M.p. 198-200 °C. υmax (Nujol) 1042, 1658,

2591 cm-1. δH (400 MHz, CDCl3): 1.60-1.64 (4H, m, H-3’); 1.75-1.79 (2H, m, H-2’); 2.67

(2H, t, J = 7.2 Hz, H-4’); 3.70 (2H, s, H-1’’); 3.76 (3H, s, H-3b’’); 4.18 (2H, t, J = 7.6 Hz, H-

1’); 6.83 (2H, dd, J = 2.4 and 6.8 Hz, H-3’’); 7.21 (2H, dd, J = 2.4 and 7.2 Hz, H-2’’); 7.72

(2H, dd, J = 7.2 and 8.4 Hz, H-5); 8.18 (2H, dd, J = 1.2 and 8.4 Hz, H-6); 8.56 (2H, dd, J =

1.2 and 7.2 Hz, H-4) ppm. δC (100 MHz, CDCl3): 25.8 (C-2’); 27.4 (C-3’); 40.1 (C-1’); 48.8

(C-4’); 53.2 (C-1’’); 55.2 (C-3b’’); 113.7 (C-3’’); 122.6 (C-3a); 126.8 (C-5); 128.0 (C-10);

129.2 (C-2’’); 131.1 (C-4); 131.5 (C-6a); 132.4 (C-1a’’); 133.8 (C-6); 158.5 (C-3a’’); 164.1

(C-1/C-3) ppm.

Syntehsis of 2-(4-((2,6-dichlorobenzyl)amino)butyl)-1H-benzo[de]isoquinoline-

1,3(2H)-dione (3k)

Compound 2k (0.080 g; 0.19 mmol); NaBH4 (0.022 g); Time: 24 h.

White solid 3k (0.057 g; 0.13 mmol; 68%); M.p. 204-207 °C. υmax (Nujol) 654, 1663,


(2H, t, J = 7.2 Hz, H-4’); 4.05 (2H, s, H-1’’); 4.18 (2H, t, J = 7.2 Hz, H-1’); 7.07 (2H, dd, J =

7.2 and 8.4 Hz, H-4’’); 7.26 (2H, d, J = 8.0 Hz, H-3’’); 7.72 (2H, dd, J = 7.6 and 8.4 Hz, H-

5); 8.18 (2H, dd, J = 1.2 and 8.4 Hz, H-6); 8.56 (2H, dd, J = 1.2 and 7.2 Hz, H-4) ppm. δC

(100 MHz, CDCl3): 25.9 (C-2’); 27.5 (C-3’); 40.1 (C-1’); 48.2 (C-1’’); 48.5 (C-4’); 122.6 (C-

3a); 126.8 (C-5); 128.0 (C-10); 128.3 (C-3’); 128.7 (C-4’’); 131.1 (C-4); 131.5 (C-6a);

133.8 (C-6); 135.8 (C-1a’’); 155.9 (C-2’’); 164.1 (C-1/C-3) ppm.


106

Synthesis of 2-(4-((4-fluorobenzyl)amino)butyl)-1H-benzo[de]isoquinoline-1,3(2H)-

dione (3l)

Compound 2l (0.10 g; 0.27 mmol); NaBH4 (0.035 g); Time: 2 days.

White solid 3l (0.069 g; 0.18 mmol; 67%); M.p. 190-192 °C. υmax (Nujol) 1024, 1671,


(2H, t, J = 7.2 Hz, H-4’); 3.74 (2H, s, H-1’’); 4.19 (2H, t, J = 7.6 Hz, H-1’); 6.98 (2H, t, J =

8.4 Hz, H-3’’); 7.27 (2H, dd, J = 7.6 and 8.8 Hz, H-2’’); 7.74 (2H, dd, J = 7.2 and 8.0 Hz, H-

5); 8.19 (2H, dd, J = 1.2 and 8.4 Hz, H-6); 8.58 (2H, dd, J = 1.2 and 7.2 Hz, H-4) ppm. δC

(100 MHz, CDCl3): 25.8 (C-2’); 27.4 (C-3’); 40.1 (C-1’); 48.8 (C-4’); 53.1 (C-1’’); 115.1 (C-

3’’); 122.6 (C-3a); 126.9 (C-5); 128.1 (C-10); 129.5 (C-2’’); 131.1 (C-4); 131.5 (C-6a);

133.9 (C-6); 136.1 (C-1a’’); 160.6 (C-3a’’); 164.1 (C-1/C-3) ppm.

Synthesis of 2-(4-((4-(dimethylamino)benzyl)amino)butyl)-1H-benzo[de]isoquinoline-

1,3(2H)-dione (3m)

Compound 2m (0.070 g; 0.18 mmol); NaBH4 (0.020 g); Time: 2 days.

Orange oil 3m (0.058 g; 0.14 mmol; 78%); M.p. 200-203 °C. υmax (Nujol) 1126, 1129,

1661, 2551 cm-1. δH (400 MHz, CDCl3): 1.59 - 1.67 (2H, m, H-3’); 1.75-1.82 (2H, m, H-2’);

2.69 (2H, t, J = 7.2 Hz, H-4’); 2.93 (6H, s, H-3b’’); 3.69 (2H, s, H-1’’); 4.19 (2H, t, J = 7.2

Hz, H-1’); 6.69 (2H, d, J = 8.8 Hz, H-3’’); 7.17 (2H, d, J = 8.8 Hz, H-2’’); 7.73 (2H, dd, J =

7.2 and 8.0 Hz, H-5); 8.19 (2H, dd, J = 1.2 and 8.4 Hz, H-6); 8.58 (2H, dd, J = 1.2 and 7.2

Hz, H-4) ppm. δC (100 MHz, CDCl3): 25.9 (C-2’); 27.5 (C-3’); 30.3 (C-1’); 40.7 (C-3b’’);


107

48.8 (C-4’); 53.4 (C-1’’); 112.7 (C-2’’); 122.6 (C-3a); 126.8 (C-5); 128.1 (C-10); 128.9 (C-

3’’); 131.1 (C-4); 131.5 (C-6a); 132.3 (C-1a’’);133.8 (C-6); 148.7 (C-3a’’); 164.1 (C-1/C-3)

ppm.

Synthesis of 2-(4-(((1-methyl-1H-pyrrol-2-yl)methyl)amino)butyl)-1H-

benzo[de]isoquinoline-1,3(2H)-dione (3p)

Compound 2p (0.11 g; 0.30 mmol); NaBH4 (0.053 g); Time: 24 hours.

Yellow oil 3p (0.085 g; 0.24 mmol; 80%); M.p. 175-178 °C. υmax (Nujol) 1105, 1658

cm-1. δH (400 MHz, CDCl3): 1.60 (2H, t, J = 7.6 Hz, H-3’); 1.77-1.82 (2H, m, H-2’); 2.17

(2H, t, J = 7.2 Hz, H-4’); 3.63 (3H, s, H-5’’); 3.72 (2H, s, H-1’’); 4.19 (2H, t, J = 7.6 Hz, H-

1’); 5.99 (1H, dd, J = 2.4 and 3.0 Hz, H-3’’); 6.02 (1H, dd, J = 2.4 and 3.6 Hz, H-2’’); 6.56

(1H, dd, J = 2.4 and 4.0 Hz, H-4’’); 7.73 (2H, dd, J = 7.2 and 8.0 Hz, H-5); 8,19 (2H, dd, J =

0.8 and 8.4 Hz, H-6); 8.56 (2H, dd, J = 1.2 and 7.2 Hz, H-4) ppm. δC (100 MHz, CDCl3):

25.8 (C-3’); 27.4 (C-2’); 33.6 (C-5’’); 40.1 (C-1’); 45.4 (C-1’’); 48.9 (C-4’); 106.1 (C-3’’);

107.7 (C-2’’); 121.9 (C-4’’); 122.5 (C-3a); 126.8 (C-5); 127.9 (C-10); 131.0 (C-4); 131.3 (C-

1a’’); 131.4 (C-6a); 133.7 (C-6); 164.0 (C-1/C-3) ppm.


108

3.6 General synthesis of bis-naphthalimide derivatives

The methods used for the synthesis of the bis-naphthalimides derivatives, 8a – c,

were based on the methods previously used by J. Oliveira et al.52 (Scheme 22)

Scheme 22: General scheme for the synthesis of bis-naphthalimide derivatives; i) DMF/DBU 4h, 85 °C; ii) Py/Ts-Cl 12hrs, 4°C; iii) Py/Mts-Cl 1h, 0°C (for 6a and 6b), DMF/Cs2CO3, 12h, 60°C; iv) DCM/HBr in glacial

CH3CO2H, 24h, r.t.


109

3.7 Reaction of 1,8-naphthalic anhydride with alkyl aminoalcohols


To a solution of 1,8-naphthalic anhydride (1.5 – 12.4 g, 7.6 – 61 mmol) dissolved in

DMF (30 – 140 mL), was added the alcohols, 2-amino-ethanol, 3-amino-1-propanol or

4-amino-1-butanol, (4.7 mL – 7.8 mL, 7.6 – 130 mmol) followed by DBU (1.7 mL – 14

mL, 11 – 91 mmol). The solution was stirred for 4-6 h at 85°C and the reaction was

monitored by TLC (95:5 DCM:MeOH). The reaction mixture was poured into ice water

(200 mL) and stirred with a glass rod. The solution was left to settle for 15 minutes

until a precipitate formed. The precipitate was filtered off, washed thoroughly with

water and dried in a vacuum-oven overnight to give the desired compounds 4a – c as

solids (1.02 – 5.02 g, 7.06 – 52.3 mmol, 81 – 93 %)

Synthesis of 2-(2-hydroxyethyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (4a)

1,8-Naphthalic anhydride (2.5 g; 13 mmol): DMF (50mL); ethanolamine (7.9 g; 130

mmol; 7.8 mL); DBU (3.0 g; 20 mmol; 2.9 mL; Time: 5 hours.

Yellow solid 4a (2.8 g; 11 mmol; 81%); M.p. 164-167 °C. υmax (Nujol) 1650, 3482 cm-

1; δH (400 MHz, CDCl3): 2.52 (1H, s, OH); 3.99 (2H, t, J = 5.4 Hz, H-2’); 4.47 (2H, t, J = 5.4

Hz, H-1’); 7.76 (2H, dd, J = 7.2 and 8.4 Hz, H-5); 8.22 (2H, dd, J = 0.8 and 8.4 Hz, H-6);

8.60 (2H, dd, J = 1.2 and 7.4 Hz, H-4) ppm. δC (100 MHz, CDCl3): 42.8 (C-1’); 61.7 (C-2’);

122.4 (C-3a); 127.0 (C-5); 128.2 (C-10); 131.5 (C-4); 131.5 (C-6a); 134.2 (C-6); 165.1 (C-

1/C-3) ppm.


110

Synthesis of 2-(3-hydroxypropyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (4b)

1,8-Naphthalic anhydride (12.1 g; 61 mmol): DMF (140mL); 3-amino-1-propanol (4.6

g; 61 mmol; 4.7 mL); DBU (14 g; 91 mmol; 14 mL); Time: 4 h.

Yellow solid 4b (13.3 g; 52 mmol; 86%). M.p. 116-118 °C. υmax (Nujol) 1642, 3430

cm-1. δH (400 MHz, CDCl3): 1.99-2.05 (2H, m, H-2’); 3.25 (1H, s, OH); 3.62 (2H, t, J = 5.6

Hz, H-3’); 4.37 (2H, t, J = 6.0 Hz, H-1’); 7.76 (2H, dd, J = 7.4 and 8.0 Hz, H-5); 8.19 (2H,

dd, J = 0.8 and 8.2 Hz, H-6); 8,60 (2H, dd, J = 1.2 and 7,6 Hz, H-4) ppm. δC (100 MHz,

CDCl3): 31.0 (C-2’); 36.7 (C-1’); 58.8 (C-3’); 122.3 (C-3a); 127.0 (C-5); 128.1 (C-10); 131.6

(C-4); 131.6 (C-6a); 134.2 (C-6); 164.9 (C-1/C-3) ppm.

Synthesis of 2-(4-hydroxybutyl)-1H-benzo[de]isoquinoline-1,3(2H)-dione (4c)

1,8-Naphthalic anhydride (1.5 g; 7.6 mmol): DMF (30mL); 4-amino-1-butanol (0.68

g; 7.6 mmol; 6.9 mL); DBU (1.7 g; 11 mmol; 1.7 mL); Time: 4 h.

White solid 4c (1.9 g; 7.1 mmol; 93%); M.p. 109-111 °C. υmax (Nujol) 1655, 3510 cm-

1. δH (400 MHz, CDCl3): 1.55-1.63 (2H, m, H-3’); 1.77-1.83 (2H, m, H-2’); 3.79 (2H, t, J =

6,4 Hz H-4’); 4.20 (2H, t, J = 6.8 Hz, H-1’); 7.75 (2H, dd, J = 7.4 and 8.2 Hz, H-5); 8.20


MHz, CDCl3): 24.5 (C-2’); 29.9 (C-3’); 33.9 (C-1’); 62.5 (C-4’); 122.6 (C-3a); 126.9 (C-5);

128.1 (C-10); 131.2 (C-4); 131.5 (C-6a); 133.9 (C-6); 164.2 (C-1/C-3) ppm.


111

3.8 Reaction of 4a – c with p-toluenesulfonyl chloride


To a solution of the aminoalcohols 4a – c (0.93 – 5.0 g, 3.5 – 20 mmol) dissolved in

anhydrous pyridine (20 – 100mL) and stirred for 15 minutes at 0°C (on ice), was added

p-toluenesulfonyl chloride (Ts-Cl) (0.99 – 5.6 g, 5.2 – 29 mmol) slowly, over 30 minutes.

The reaction was left in the fridge overnight at 4°C and monitored by TLC (DCM:MeOH

95:5). When the reaction was complete, the solution was poured into ice water (100 -

200 mL), stirred with a glass rod and left to settle for 30 minutes to form a precipitate.

The precipitate was filtered off and washed thoroughly with water and dried under

vacuum at 50°C overnight.

The crude product was recrystallised from ethanol to give the desired compound 5a

– c as solids (1.0 – 5.1 g, 2.4 – 13 mmol, 63 – 83%).

Synthesis of 2-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)ethyl 4-

methylbenzenesulfonate (5a)

Compound 4a (1.0 g; 4.3 mmol): Pyridine 50 mL; p-toluenesulfonyl chloride (1.2 g;

6.4 mmol); Time: 18 hours.

White solid 5a (1.4 g; 3.5 mmol; 83%); M.p. 139-143 °C; δH (400 MHz, CDCl3): 2.16

(3H, s, H-4’’); 4.45-4.52 (4H, m, H-1’/H-2’); 6.96 (2H, d, J = 8.0 Hz, H-3’’); 7.66 (2H, d, J =

7.6 Hz, H-2’’); 7.78 (2H, dd, J = 7.2 and 8.0 Hz, H-5); 8.26 (2H, dd, J = 0.8 and 8.4 Hz, H-

6); 8.55 (2H, dd, J = 0.8 and 7.2 Hz, H-4) ppm. δC (100 MHz, CDCl3): 21.5 (C-4’’); 38.7 (C-

1’); 67.1 (C-2’); 122.3 (C-3a); 127.0 (C-5); 127.7 (C-2’’); 128.1 (C-10); 129.5 (C-3’´); 131.5

(C-4); 132.8 (C-6a); 134.3 (C-6); 144.5 (C-1’’); 163.9 (C-1/C-3) ppm.


112

Synthesis of 3-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)propyl 4-

methylbenzenesulfonate (5b)

Compound 4b (5.0 g; 20 mmol); Pyridine 100 mL; p-toluenesulfonyl chloride (5.6 g;

29 mmol); Time: 18 hours.

White solid 5b (5.1 g; 13 mmol; 63%); M.p. 120-125 °C; δH (400 MHz, CDCl3): 2.16

(2H, m, H-2’); 2.45 (3H, s, H-4’’); 4.21-4.28 (4H, m, H-1’/H-3’); 7.31 (2H, d, J = 8.0 Hz, H-

3’’); 7.78 (2H, d, J = 8.0, H-2’’); 7.80 (2H, dd, J = 7.6 and 8.4 Hz, H-5); 8.25 (2H, dd, J =

0.8 and 8.4 Hz, H6); 8.60 (2H, dd, J = 0.8 and 7.2 Hz, H-4) ppm. δC (100 MHz, CDCl3):

21.7 (C-4’’); 27.7 (C-1’); 37.1 (C-2’); 68.5 (C-3’); 122.4 (C-3a); 127.1 (C-5); 127.9 (C-2’’);

128.1 (C-10); 129.8 (C-3’’); 131.5 (C-4); 132.9 (C-6a); 134.1 (C-6); 144.7 (C-4’); 164.1 (C-

1/C-3) ppm.

Synthesis of 4-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-yl)butyl 4-

methylbenzenesulfonate (5c)

Compound 4c (0.931 g; 3.5 mmol): Pyridine 50 mL; p-toluenesulfonyl chloride (0.99

g; 5.19 mmol); Time: 18 hours.

White solid 5c (1.0 g; 2.4 mmol; 89%); M.p. 119-121 °C; δH (400 MHz, CDCl3): 2.11-

2.24 (4H, m, H-2’/H-3’); 2.49 (3H, s, H-4’’); 4.19-4.30 (4H, m, H-1’/H-4’); 7.35 (2H, d, J =

7.6 Hz, H-3’’); 7.78 (2H, d, J = 8.0 Hz, H-2’’); 7.78 (2H, dd, J = 7.6 and 8.0 Hz, H-5); 8.22


MHz, CDCl3): 21.9 (C-4’’); 27.5 (C-1’); 36.5 (C-2’); 37.2 (C-3’); 68.6 (C-4’); 122.3 (C-3a);


113

127.0 (C-5); 128.0 (C-2’’); 128.1 (C-10); 129.8 (C-3’’); 131.4 (C-4); 132.7 (C-6a); 134.1 (C-

6/C-7); 144.9 (C-1’’); 163.9 (C-1/C-3) ppm.

3.9 Reaction of alkyl diamines 10 and 11 with 2-mesitylenesulfonyl chloride

(linker synthesis)


In a round bottomed flask, 1,4-bis(3-aminopropyl)piperazine (1.5 g, 5.5 mmol) and

1,8-diaminooctane (2.26 g – 1.7 mmol) was dissolved in anhydrous pyridine (30 - 100

mL). The solution was stirred at 0°C (on ice) until fully dissolved, then 2-

mesitylenesulfonylchloride (Mts-Cl) (7.49 – 1.7 mmol, 2.01 excess) was added slowly,

over 15 minutes. The reaction was stirred for 1 hour at 0°C (ice bath) and monitored by

TLC (DCM:MeOH 95:5). The reaction mixture was poured into ice water (200 mL)

stirred with a glass rod and left to settle for 15 minutes to form a precipitate. The

precipitate was filtered off, washed thoroughly with water and dried in a vacuum-oven

to dry for 2 hours.

The solid product was recrystallised from ethanol to give the desired compound 6a

and 6b as solids (0.69 – 2.3 g, 1.36 – 3.4 mmol, 20 – 50 %).

Synthesis of N,N'-(piperazine-1,4-diylbis(propane-3,1-diyl))bis(2,4,6-

trimethylbenzenesulfonamide) (6a)

1,4-bis(3-aminopropyl)piperazine (1.5 g, 1.5 mL; 7.5 mmol): Pyridine 20 mL; 2-

mesitylenesulfonylchloride (3.3 g; 15 mmol); Time: 1.5 hours.


114

White solid 6a (0.86 g; 1.5 mmol; 20%); M.p. 163-167 °C; δH (400 MHz, CDCl3): 1.75

(4H, t, J = 9.2 Hz, H-3); 2.34 (6H, s, H-4’); 2.53 (4H, s, H-4); 2.67-2.68 (20H, m, H-5/H-5’);

2.99 (4H, s, H-2); 6.99 (4H, s, H-3’); 7.02 (2H, s, H-1) ppm. δC (100 MHz, CDCl3): 20.9 (C-

4’); 23.0 (C-5’); 24.0 (C-3/C-5); 43.3 (C-2); 57.9 (C-4); 130.8 (C-3a’); 131.9 (C-3’); 141.9

(C-1’) ppm.

Synthesis of N,N'-(octane-1,8-diyl)bis(2,4,6-trimethylbenzenesulfonamide) (16b)

1,8-diaminooctane (2.3 g; 17 mmol): Pyridine 100 mL; 2-mesitylenesulfonylchloride

(6.1 g; 28 mmol); Time: 1 hours.

White solid 6b (2.3 g; 2.3 mmol; 50%); M.p. 121-124 °C; δH (400 MHz, CDCl3):

1.17-1.22 (8H, m, H-4/H-5); 1.44 (4H, t, J = 7.6 Hz, H-3); 2.33 (6H, s, H-4’); 2.67 (12H, s,

H-5’); 2.87-2.92 (4H, m, H-2); 4.6 (2H, s, H-1); 6.99 (4H, s, H-3’) ppm. δC (100 MHz,

CDCl3): 20.9 (C-4’); 23.1 (C-5’); 26.4 (C-5); 28.8 (C-4); 29.5 (C-3); 42.5 (C-2); 131.9 (C-3’);

133.7 (C-3a’); 139.1 (C-2’); 142.1 (C-1’) ppm.

3.10 Reaction of compounds 5b and 5c with 6a and 6b (N-Alquilation reaction)


To a solution of 6a and 6b (0.36 – 1.5 g, 0.71 – 2.9 mmol) was dissolved in DMF (10 -

40 mL), was added 5b and 5c (0.60 – 2.4 g; 1.4 – 5.9 mmol) (2.01 equiv.) followed by

slow addition of Cs2CO3 (1.2 – 4.8 g; 3.6 – 15 mmol) (5.00 equiv.). The reaction mixture

was stirred overnight at 60°C and reaction was monitored by TLC (DCM:MeOH 95:5).

When the reaction was complete, the solution was poured into ice water (200 mL),

stirred with a glass rod and some drops of dil. HCl were added till the pH turn to acid.


115

The solution was left to settle for 15 minutes to form a precipitate. The solid was

filtered off, washed thoroughly with water and dried in a vacuum-oven for 2 hours.

The solid was recrystallised from ethanol to give the desired compound 7a – c as

solids (0.52 – 2.3; 0.49 – 2.2 mmol; 23 – 31 %).

Synthesis of N,N'-(piperazine-1,4-diylbis(propane-3,1-diyl))bis(N-(3-(1,3-dioxo-1H-

benzo[de]isoquinolin-2(3H)-yl)propyl)-2,4,6-trimethylbenzenesulfonamide) (7a)

Compound 5b (0.6 g; 1.5 mmol): DMF 25 mL; 6a (0.41 g; 7.3 mmol); Time: 24 hours.

White solid 7a (0.52 g; 0.49 mmol; 23 %); M.p. 241-243 °C; δH (400 MHz, CDCl3):

2.01 (8H, m, H-2’/H-6’); 2.40 (6H, s, H-4’’); 2.97 (8H, s, H-8’); 3.01 (12H, s, H-5’’); 3.75

(12H, m, H-3’/H-5’/H-7’); 4.29 (2H, t, J = 6.4 Hz, H-1’); 6.69 (4H, s, H-3’’); 7.79 (4H, dd, J

= 7.4 and 8.0 Hz, H-5); 8.23 (4H, dd, J = 0.8 and 8.4 Hz, H-6); 8.60 (4H, dd, J = 1.2 and

8.4 Hz, H-4) ppm. δC (100 MHz, CDCl3): 20.5 (C-4’’); 21.2 (C-2’); 23.5 (C-5’’); 25.0 (C-6’);

37.5 (C-1’); 39.7 (C-8’); 44.4 (C-5’); 45.4 (C-3’/C-7’); 122.6 (C-3a); 127.7 (C-5); 127.9 (C-

10); 130.8 (C-3a’’); 131.2 (C-4); 131.7 (C-6a); 132.1 (C-3’’); 134.9 (C-6); 142.3 (C-1’’);

164.5 (C-1/C-3) ppm.


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Synthesis of N,N'-(octane-1,8-diyl)bis(N-(3-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-

yl)propyl)-2,4,6-trimethylbenzenesulfonamide) (7b)

Compound 5b (2.4 g; 5.9 mmol): DMF 40 mL; 6b (1.5 g; 2.9 mmol); Time: 20 h.

Beige solid 7b (2.3 g; 2.2 mmol; 25%); M.p. 226-228 °C; δH (400 MHz, CDCl3): 1.54

(8H, s, H-8’/H-9’); 1.86-1.90 (8H, m, H-2’/H-7’); 2.17 (6H, s, H-4’’); 2.54 (12H, s, H-5’’);

3.25-3.34 (8H, m, H-3’/H-6’); 4.04 (4H, t, J = 7.2 Hz, H-1’); 6.75 (4H, s, H-3’’); 7.80 (4H,

dd, J = 7.6 and 8.4 Hz, H-5); 8.26 (4H, dd, J = 0.8 and 8.4 Hz, H-6); 8.59 (4H, dd, J = 0.8

and 7.2 Hz, H-4) ppm. δC (100 MHz, CDCl3): 20.7 (C-4’’); 23.0 (C-5’’); 26.2 (C-2’); 28.9 (C-

8’/C-9’); 29.9 (C-7’); 38.2 (C-1’); 46.0 (C-3’); 48.1 (C-6’); 122.3 (C-3a); 127.1 (C-5); 127.9

(C-10); 131.3 (C-6); 131.8 (C-3’’); 132.9 (C-6a); 134.4 (C-4); 139.0 (C-2’’); 142.1 (C-1’’);

163.9 (C-1/C-3) ppm.

Synthesis of N,N'-(octane-1,8-diyl)bis(N-(4-(1,3-dioxo-1H-benzo[de]isoquinolin-2(3H)-

yl)butyl)-2,4,6-trimethylbenzenesulfonamide) (7c)

Compound 5c (0.61 g; 1.4 mmol): DMF 20 mL; 6b (0.36 g; 7.1 mmol); Time: 19 h.

White solid 7c (0.68 g; 0.67 mmol; 31%); M.p. 216-218 °C; δH (400 MHz, CDCl3):1.12

(8H, s, H-8’/H-9’); 1.50 (4H, s, H-7’); 1.62 (4H, s, H-2’/H-3’); 2.21 (6H, s, H-4’’); 2.62

(12H, s, H-5’’); 3.17-3.26 (8H, m, H-4’/H-6’); 4.13 (4H, t, J = 6.8 Hz, H-1’); 6.89 (4H, s, H-


117

3’’); 7.79 (4H, dd, J = 7.6 and 8.0 Hz, H-5/H-8); 8.25 (4H, dd, J = 1.2 and 8.4 Hz, H-6/H-

7); 8.61 (4H, dd, J = 0.8 and 7.2 Hz, H-4/H-9) ppm. δC (100 MHz, CDCl3): 20.6 (C-4’’);

23.2 (C-5’’); 26.2 (C-2’/C-7’); 27.0 (C-3’); 28.9 (C-8’/C-9’); 30.2 (C-7’); 38.5 (C-1’); 46.4

(C-4’); 48.8 (C-6’); 122.4 (C-3a); 127.0 (C-5); 128.1 (C-10); 131.4 (C-4); 131.9 (C-3’’);

132.9 (C-6a); 134.2 (C-6); 139.2 (C-2’’); 142.0 (C-1’’); 164.2 (C-1/C-3) ppm.

3.11 Deprotection reaction of 7a – c


To a solution of 7a – c (0.52 - 1.0 g; 0.50 – 1.02 mmol) dissolved in DCM (10 – 20

mL), was added HBr/gCH3CO2H (1 mL) drop by drop. The solution was stirred overnight

at r.t. and monitored by TLC (DCM:MeOH 95:5), the precipitate formed was filtered

off, washed with DCM and ether (small amount) and dried under vacuum at 50°C

overnight to give the desired compound 8a – c as solids (0.44 – 0.91 g; 0.53 – 1.2

mmol; 92 – 100 %).

Synthesis of 2,2'-(((piperazine-1,4-diylbis(propane-3,1-

diyl))bis(azanediyl))bis(propane-3,1-diyl))bis(1H-benzo[de]isoquinoline-1,3(2H)-

dione) (8a)

Compound 7a (0.52 g; 0.50 mmol): DCM 10 mL; HBr/CH3CO2H (1 mL); Time: 20 h.

Yellow solid 8a (0.44 g; 0.53 mmol; ~100%); M.p. 290-293 °C; δH (400 MHz, DMSO):

2.06 (8H, m, H-2’/H-6’); 2.50 (4H, s, H-7’); 3.02 (8H, m, H-3’/H-5’); 3.86 (8H, s, H-8’);

4.14 (4H, t, J = 6.4 Hz, H-1’); 7.87-8.52 (12H, m, H-4/H-5/H-6) ppm. δC (100 MHz,

DMSO): 20.8 (C-6’); 25.0 (C-2’); 37.5 (C-1’); 39.7 (C-8’); 44.4 (C-5’/7’); 45.4 (C-3’); 122.6


118

(C-3a); 127.7 (C-5); 127.9 (C-10); 131.3 (C-4); 131.8 (C-6a); 134.9 (C-6); 164.2 (C-1/C-3)

ppm.

HRMS [M+H]: 675.3644; calculate for: C40H47N6O4 = 675.3659

Synthesis of 2,2'-((octane-1,8-diylbis(azanediyl))bis(propane-3,1-diyl))bis(1H-

benzo[de]isoquinoline-1,3(2H)-dione) (8b)

Compound 7b (1.00 g; 1.0 mmol): DCM 15 mL; HBr/gCH3CO2H (0.75 mL); Time: 18 h.

Orange solid 8b (0.91 g; 1.2 mmol; 100%); M.p. 260-263 °C; δH (400 MHz, DMSO):

1.23 (8H, s, H-8’/H-9’); 1.52 (4H, s, H-7’); 2.00 (4H, m, H-2’); 2.85 (4H, t, J = 12 Hz, H-6’);

2.99 (4H, t, J = 11.6 Hz, H-3’); 4.11 (4H, t, J = 6.4 Hz, H-1’); 4.94 (2H, s, H-5’); 7.85-8.50

(12H, m, H-4/H-5/H-6) ppm. δC (100 MHz, DMSO): 25.9 (C-2’); 25.9 (C-8’); 26.3 (C-9’);

37.5 (C-1’); 40.2 (C-7’); 45.2 (C-3’); 47.2 (C-6’); 122.6 (C-3a); 127.7 (C-5); 127.9 (C-10);

131.2 (C-4); 131.8 (C-6a); 134.9 (C-6); 164.2 (C-1/C-3) ppm.

Synthesis of 2,2'-((octane-1,8-diylbis(azanediyl))bis(butane-4,1-diyl))bis(1H-

benzo[de]isoquinoline-1,3(2H)-dione) (8c)

Compound 7c (0.68 g; 0.67 mmol): DCM 10 mL; HBr/CH3CO2H (0.80 mL); time: 20 h.


119

Orange solid 8c (0.49 g; 0.62 mmol; 92%); M.p. 256-258 °C; δH (400 MHz, DSMO):

1.25 (8H, s, H-8’/H-9’); 1.55 (4H, s, H-7’); 1.69 (8H, m, H-2’/H-3’); 2.83 (4H, t, J = 14.8

Hz, H-6’); 2.90 (4H, t, J = 17.2 Hz, H-4’); 4.08 (4H, t, J = 6.4 Hz, H-1’); 4.51 (2H, s, H-5’);

7.86-8.50 (12H, m, H-4/H-5/H-6) ppm. δC (100 MHz, DSMO): 23.7 (C-3’); 25.3 (C-2’);

26.3 (C-8’); 28.7 (C-9’); 39.7 (C-1’); 40.8 (C-7’); 47.0 (C-6’); 122.4 (C-3a); 127.7 (C-5);

127.8 (C-10); 131.3 (C-4); 131.8 (C-6a); 134.9 (C-6); 163.9 (C-1/C-3) ppm.

HRMS [M+H]: 647.3585; calculate for: C40H47N4O4 = 647.3597


120

3.12 Binding Studies

The way to test how the mono and bis-naphthalimides interact with the DNA, was

carried out according to the following experiment: a 200 µM de SSC Buffer was

prepared mixing 250 µL of x 20 SSC Buffer in 475 mL of distillated water. The solution

was sonicated during 10 minutes to guarantee a homogeneous solution. After that was

added more distilled water to reach at 500 mL volume. To prepare the DNA solution,

7.5 mg of Calf Thymus DNA was dissolved in 250 mL of 0.01 SSC Buffer. The solution

was left 24h in to fridge and then sonicated to make sure that all DNA was dissolved. A

200 µM solution of Ethidium Bromide (Et-Br) was provided by another student. The

solutions of mono and bis-naphthalimides were prepared through the stock solution

previously made (10 µM). For the tests, the mono (400 µM) and bis-naphthalimides

(100 µM) solutions were diluted in SSC Buffer.

Test solutions were prepared by adding varying volumes of 0.01 SSC Buffer, 200 µL

of Calf Thymus DNA, 20 µL of Et-Br solution together into disposable cuvettes to give

the final concentrations of: for mono: 0, 5, 10, 15, 20, 25, 30, 40, 50 µM and for bis: 0,

0.25, 0.5, 1, 2, 3, 5 µM. The final concentrations of DNA and Et-Br in the test solutions

were 20 µM and 2 µM respectively (Table 54 and 55). These solutions were analyzed

using a luminescence spectrophotometer. The values of C50 were defined as the

variation of the concentration (µM) needed to generate a 50% of decrease in the

fluorescence of DNA bound Et-Br.


121


Table 54: Preparation of Test Solutions for Fluorescence binding Studies

Volume (μL) of 0.01M SSC Buffer

1780 1775 1770 1760 1740 1720 1680

Volume (μL) of 200μM DNA Solution

200 200 200 200 200 200 200

Volume (μL) of 200μM EtBr Solution

20 20 20 20 20 20 20

Volume (μL) of 100μM BisNaphtalimide

0 5 10 20 40 60 100

Total Volume (μL) 2000 2000 2000 2000 2000 2000 2000

Final Bisnaphta derivative Concentration (μM)

0 0,25 0,5 1 2 3 5


Table 55: Preparation of Test Solutions for Fluorescence binding Studies

Volume (μL) of 0.01M SSC Buffer

1780 1775 1730 1705 1680 1655 1630 1580 1530

Volume (μL) of 200μM DNA Solution

200 200 200 200 200 200 200 200 200

Volume (μL) of 200μM EtBr Solution

20 20 20 20 20 20 20 20 20

Volume (μL) of 400μM Mono-Naphta

0 25 50 75 100 125 150 200 250

Total Volume (μL) 2000 2000 2000 2000 2000 2000 2000 2000 2000

Final Mononaphta derivative

Concentration (μM) 0 5 10 15 20 25 30 40 50


122

3.13 Biological Activity

3.13.1 Cell Maintenance

The SH-SY5Y cells were incubated at 37°C with 5% of CO2. This type of cells grew

quickly, so it was necessary split them every 2/3 days to guarantee they did not

become too confluent. Approximately 1 million cells were required to seed a 75 cm2

tissue culture flask. This type of cells, SH-SY5Y, are adherent cells, so they stick to the

wall of the culture flask and to proceed with any experimentation, the cells need to be

carefully removed with a specific process.

Under the class II Safety Cabinet the medium with serum from the culture flask was

poured off into to a beaker and the adherent cells were washed with Phosphate

Buffered Saline (PBS) twice. Between the washes the PBS was poured off into a beaker.

After the PBS was removed, a small amount of Trypsin was added (approximately 2

mL), enough to cover all cells in the culture flask. The flask was returned to the

incubator at 37°C with 5% of CO2. The trypsin detaches the cells from the walls of the

culture flask. To ensure that all the cells are detached from the wall was given a tap on

the side to lift all the remaining cells. The culture flask was taken to the microscope to

see if all the cells were detached from the culture flask.

The trypsin was poured to a universal (falcon tube) and was added 5-10mL of fresh

medium. The cell solution was centrifuged at 1500 rpm for 5 minutes using a Sigma 2-4

centrifuge (Gillingham Dorset UK). Once finished, in the falcon tube was formed a cell

pellet. The supernatant was poured off carefully and the cell pellet was re-suspended

with 3 mL of fresh medium. The cells were counted using an improved Neubauer

Haemocytometer. For the experimental purposes approx. 100000 cells were seeded

per well in a 96-well plate. To ensure that we have cells for further experiments,

approx. 1000000 cells were seeded into a new tissue culture flask.


123

3.13.2 Cell Counting

To count the SH-SY5Y cells a haemocytometer was used. It consists in 2

components: a) a glass base; b) a thick glass cover slide that has a rulered grid

engraved onto it. The thick glass was placed on top of the glass base to create a

chamber (Figure 67).

Figure 67: Scheme of chamber in haemocytometer

In that chamber, is the place where the cell suspension mix is introduced (approx.

20 µL). Once introduced, the cells were examined and counted under the microscope.

The way to know how many cells that we have is count the number of cells that are

present in the four sections 1, 2, 3 and 4 (Figure 68). The number of cells per mL can

then be calculated using the following equation:

[(1+2+3+4)/4] x 104 x DF = cells per mL

Figure 68: Section to count the cells


124

3.14 Cytotoxicity

The way to test the cytotoxicity of the mono and bis-naphthalimides was using a

colorimetric 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay.

The brain cancer cells, SH-SY5Y, was seeded in 96-well plates (10000 cells per well).

In the columns 1 and 12 and in the lines A and H was added only fresh medium (Figure

69). After 24h, 100 µL of the mono and bis-naphthalimide derivatives was added in to

the wells between the columns 4 and 11 and lines B and G with different

concentrations: for mono: 5, 10, 25, 50, 75, 100, 125, 150 µM and for bis: 0.1, 0.5, 1, 2,

5, 10, 20, 40 µM. In the columns 2 and 3 was added 100 µL of DMSO (200

µM/medium) and medium respectably (Figure 69). The DMSO was used to ensure that

it was not affecting the cells, and the medium was used as control.

The mono-naphthalimides was dissolved in 50% DMSO/H20 and the bis-

naphthalimides were dissolved only in DMSO. After more 24h further the addiction of

the drugs, the solutions in the wells were removed and 100 µL of sterile-filtered (0.22

µM filter) MTT solution (1mg MTT/1mL of medium) was added at each well. The 96-

well plates were incubated again for 4h at 37°C and 5% of CO2. After 4h, the solution

was carefully removed and was added 100 µL of DMSO at each well. In this case the

DMSO allows the dissolution of the metabolized MTT product. The 96-well plates

were shaken for 20 minutes at room temperature and the results of the absorbance

were measured 595 nm on a 96 well plate reader.

The values obtained were express as % of absorbance of the treated cells, where

the cells used as a control, 100% don’t show any inhibition of growth.

The IC50 was defined as the concentration that the drug cause 50% growth

inhibition of the cell population compared to that of control cells.


125

Figure 69: Scheme of lay out of a 96-well plate for MTT Assay

Chapter 4 Conclusion

Chapter 4 – Conclusion

129

4. Conclusion

The main objective of this project was to synthetize new mono- and bis-

naphthalimide derivatives and evaluate their cytotoxicity against brain cancer cells. In

mono-naphthalimides compounds the increase of the chain length and/or nature of

the substituent influence their activity, and in bis-naphthalimides the chain

length/nature of the linker change the activity of the compounds.

Chemical synthesis

The mono and bis-naphthalimides were synthetized in moderate to high yields.

Mono-naphthalimides were obtained by reaction of naphthalimide amine derivatives

with different aldehydes. TFA salts were formed to test their biological activity. Bis-

naphthalimide derivatives were obtained by the reaction of different alkyl amine

linkers with O-tosylalkyl naphthalimides to generate the respective bis-naphthalimides.

DNA Binding studies

The studies of EtBr displacement confirm that almost all derivatives bind to DNA by

intercalation.

As expected the bis-naphthalimide derivatives showed better affinity to DNA than

mono-naphthalimides.

Generally for mono-naphthalimide derivatives, the longer the alkyl chain, the better

the binding; the best results were obtained with compound 3i, bearing a 4 carbons

chain length and an aromatic ring substituted at para position with a NO2 group. For

the bis-naphthalimides the best results were obtained for compounds 8b and 8c,

having an 8 carbon chain linker, which suggest that the flexibility of the linker is

important.

Cytotoxicity

Almost all the compounds synthetized have shown to affect the cellular morphology

and also the cellular viability in brain cancer cells treated after 24h test. The mono-

naphthalimide derivatives showed IC50 values between 14.8 and 86 µM and the bis-

naphthalimide derivatives between 3.4 and 25.7 µM. Some of mono-naphthalimide

Chapter 4 – Conclusion

130

compounds synthetized (with the concentration used the IC50 value did not decrease

50%). The results for mono naphthalimides show that the increase of the chain length

has a positive impact in the activity. For the bis-naphthalimides the presence of linear

alkyl chain is better than a linker with a piperazine ring incorporated. The cellular

uptake showed to be related with the cytotoxicity.

Overall, the bis-naphthalimide derivatives have shown more potential for

anticancer activity than mono-naphthalimides; bis-naphthalimide 8b and 8c show the

best activity and display very good binding to DNA, suggesting that this is their mode of

action. Mono-naphthalimides showed some activity but not as good as bis-

naphthalimides. Comparing the results of binding and cytotoxicity assays of mono-

naphthalimides the results do not match: the best compounds for binding are not the

most active, suggesting that the DNA is not the target for this type of cells/compounds

Chapter 5 Future Work

Chapter 5 – Future Work

133

5. Future Work

For future work, would be useful to study the morphological changes in the cells

after MTT assays, analyzing cell over 24h time interval period. This information would

allow to understand whether or not there is a relationship between the cellular uptake

and cytotoxicity. Another suggestion is to increase incubation’s time for 48h or 72h to

observe possible reduction of IC50. Would be useful to change the cell line type and

evaluate the activity of the compounds on those cells.

It would also be interesting the preparation of new mono-naphthalimide bearing

longer chain length, and bis-naphthalimide with different linkers types.

Chapter 6 Bibliography

Chapter 6 – Bibliography

137

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143

Appendix

Appendix

145

Results of biologic tests (against SH-SY5Y Cells) and DNA Binding Studies

Compound Structure Binding studies

SH-SY5Y Cells

3a

34.5 µM No

activity

3b

37.2 µM No

activity

3c

40 µM 86 µM

3n

46 µM No

activity

3d

No binding

No activity

3e

38.5 µM 39 µM

3f

No binding

Crystals

Appendix

146

3g

28.5 µM 39 µM

3h

---

3o

28.5 µM 47 µM

3i

17.5 µM No

activity

3j

32.8 µM 15.3 µM

3k

34 µM Crystals

3l

No binding

26.4 µM

3m

18.2 µM 27.5 µM

Appendix

147

3p

31.2 µM 14.8 µM

8ª

1.95 µM 25.7 µM

8b

0.96 µM 3.5 µM

8c

0.91 µM 3.4 µM

Documents

David Rodrigues Palharesrepositorium.sdum.uminho.pt/bitstream/1822/41134/1/Master... · 2019. 1. 1. · Declaração Nome: David Rodrigues Palhares Endereço electrónico: [email protected]