The Electrochemistry and Fluorescence Studies of 2,3-Diphenyl Indole Derivatives

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THE ELECTROCHEMISTRY AND FLUORESCENCE STUDIES OF 2,3-DIPHENYL INDOLE DERIVATIVES

A thesis in fulfillment of the requirements for the degree of

Master of Philosophy

Nidup Phuntsho

Supervisors

Prof. Naresh Kumar (UNSW) A/Prof. Steve Colbran (UNSW) Prof. David StC Black (UNSW)

School of Chemistry Faculty of Science University of New South Wales Kensington, Australia

August 2014

PLEASE TYPE THE UNIVERSITY OF NEW SOUTH WALES Thesis/Dissertation Sheet

Surname or Fam1ly name· Phuntsho

First name: N1dup Other name/s:

Abbreviation for degree as g1ven in the University calendar: MSc

School Chemistry Faculty; Science

Title· The electrochemistry and fluorescence studies of 2,3- diphenyl indole derivatives

Abstract 350 words maximum: (PLEASE TYPE)

A series of 2,3-diphenyl-4,6-dimethoxyindole derivatives with C-7 substitutions were successfully synthesized. All the novel indolyl derivatives were fully characterized using 1H NMR, 13C NMR, IR spectroscopy and high-resolution mass spectrometry (HRMS) techniques.

Electrochemical oxidation pathways for indolyl derivatives were explored using cyclic voltammetry (CV), spectroelectrochemistry and electronic paramagnetic resonance (EPR) spectroscopy. The electrochemical mechanisms of 2,3-disubstituted-4,6- dimethoxyindole derivatives were proposed based on results obtained from the above-mentioned techniques.

This thesis also includes the synthesis of novel indolyl ligands and explores their use in metal-binding fluorescence studies. Novel indolyl chemosensors based di-2-picolylamine (DPA) and a range of amino acids were synthesized. A group of biologically relevant metal ions was selected to study their bindin!1 modes and how that affected the fluorescence emission intensity of the ligands. DPA-based indole ligand was found to be Cu • ion selective. The binding mode of the metal and the ligand was most likely 1:1. •

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I also declare that the intellectual content of this thesis is the product of my own work, except to the extent of assistance from other in the project's design and conception or in style, presentation and linguistic expression is acknowledged. '

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'I hereby grant the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all proprietary rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstract International (this is applicable to doctoral theses only). I have either used no substantial portions of copyright material in my thesis or I have obtained permission to use copyright material; where permission has not been granted I have applied/will apply for a partial restriction of the digital copy of my thesis or dissertation.' I

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‘I hereby declare that this submission is my own work and to the best of my knowledge it contains no materials previously published or written by another person, or substantial proportions of material which have been accepted for the award of any other degree of diploma at UNSW or any other educational institution, except where due acknowledgement is made in the thesis. Any contribution made to the research by others, with whom I have worked at UNSW or elsewhere, is explicitly acknowledged in the thesis.

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Abstract

4,6-dimethoxyindole derivatives were proposed based on results obtained from the above-mentioned techniques.

This thesis also includes the synthesis and use of novel indolyl ligands in metal-binding fluorescence studies. Novel indolyl chemosensors based on di-2- picolylamine (DPA) and a range of amino acids were synthesized. The chemosensors were investigated for their fluorescence response and binding modes to a group of biologically relevant metal ions. The DPA-based indole ligand was found to be Cu2+ selective, and the binding mode of the metal to the ligand was predicted to be 1:1.

Acknowledgements

I would like to express my gratitude to my supervisors Prof. Naresh Kumar and

A/Prof Steve Colbran for giving me the opportunity to do my Master’s degree under their supervision. I also thank them for all their assistance, support and encouragement throughout the course of the project.

I wish to thank all the staff of the School of Chemistry at UNSW for all the help they have given me. Special thanks goes to Dr. Jim Hook and Dr. Doug Lawes for the

NMR spectroscopy support.

Many thanks to the past and present members of the Kumar and Black group especially Adeline for her time reading and correcting my thesis. Thanks to Thanh,

Kitty, Samuel, Ray, Ren, Murat, Ibrahim, Hakan, Dr George Iskander and Dr Abel

Salek for their support. Also, I would like to give many thanks to the Colbran group for their help and advice.

I would also like to thank Australian Government (AusAID) for funding me to pursue this Master’s degree, and all the Australian people for being very kind and friendly.

Finally, I would like to thank my family for their love and support throughout this journey.

iii

Table of Contents

Originality Statement ...... i

Abstract ...... ii

Acknowledgements ...... iii

Table of Contents ...... iv

Abbreviations………………………………………………………………………..vii

CHAPTER 1: Introduction ...... 1

1.1. General background to indoles ...... 1

1.2. General methods of indole ring synthesis ...... 2

1.3. Indole chemistry ...... 4

1.3.1. General reactions ...... 4

1.3.2 Radical chemistry of indoles ...... 8

1.3.3 Fluorescence chemistry of indole ...... 8

1.4 Primary aims ...... 9

1.4.1 Develop model compounds for the formation of stable indole radicals and

study their electrochemical properties ...... 9

1.4.2. Synthesize indole-based fluorometric sensors for metal-ion binding studies

...... 10

1.5 References ...... 10

CHAPTER 2: Synthesis of novel indole derivatives ...... 14

2.1 Introduction ...... 14

2.2 Indole derivatives synthesized for electrochemical studies ...... 14

2.2.1 Synthesis of 4,6-dimethoxy-2,3-disubstituted-indoles ...... 15

2.2.3 Oxidative dimerization ...... 17

2.2.4 Vilsmeier-Haack formylation ...... 18

2.2.5 Synthesis of 7-trichloroacetylindoles ...... 19

2.2.6 Synthesis of indole-7-carboxylic acid ...... 21

2.2.7 Synthesis of indole carboxamides ...... 21

2.2.8 Synthesis of indole carboxylic esters ...... 23

2.2.9 Friedel-Crafts acylation ...... 24

2.2.10 N-Methylation of indole and preparation of its derivatives ...... 25

2.3 Indole derivatives for fluorescence studies ...... 26

2.3.1 Synthesis of indole-based di-(2-picolyl)amine (DPA) ligand ...... 27

2.3.2 Synthesis of novel 7-amino acid-substituted indole ligands ...... 28

2.4 Conclusion ...... 35

2.5 References ...... 35

CHAPTER 3: Electrochemical studies of highly substituted indole derivatives ..... 38

3.1. Introduction ...... 38

3.2. Electrochemistry of indoles ...... 38

3.3. Cyclic voltammetry ...... 41

3.3.1. Results and discussion ...... 41

3.4. UV-Vis Spectroelectrochemistry ...... 55

3.4.1 Chemical oxidation of 4,6-dimethoxy-2,3-diphenyl indole ...... 55

3.4.2 UV-vis studies of 4,6-Dimethoxy-2,3-diphenylindole derivatives ...... 56

3.5 Conclusion ...... 61

3.6 References ...... 62

CHAPTER 4: Fluorometric Studies of Novel Indole Ligands for Detection of Biologically Important Metal Ions ...... 64

4.1 Introduction ...... 64

4.2 Designing metal-responsive fluorophores for cellular use ...... 65

4.3 Results and discussion ...... 66

4.3.2 DPA ligands ...... 71

4.3.3 Ion selectivity in fluoroionophore DPI-DPA (69) ...... 72

4.3.4 Fluorescence studies of amino acid indole ligands ...... 75

4.4 Conclusion ...... 80

4.5 References ...... 81

CHAPTER 5: Experimental ...... 84

5.1 General synthetic procedures ...... 84

5.2 Physical measurements ...... 85

5.3 Experimental details ...... 88

5.4 References ...... 111

Abbreviations

• λ – Lambda (symbol of wavelength)

• μ – Micro (10-6)

• δ – Delta (symbol for chemical shifts)

• Å – angstrom

• AcOH – acetic acid

• AgCl – silver chloride

• aq – aqueous

• Ar – Aryl

• a.u – arbitrary unit

• [Bu4N][PF6] – tetrabutyl ammonium hexafluorophosphate

• CV – cyclic voltammetry

• CHCl3 - chloroform

• D2O – deuterated water

• DCM – dichloromethane

• DMF – dimethyl formamide

• DMSO – dimethyl sulphoxide

• DPA – di-(2-picolyl)amine

• DPI – diphenylindole

• ΔE – potential difference

• EDG – electron donating group

• EPR – electron paramagnetic resonance

• ESI – electrospray ionization

• EtOH – ethanol

vii

• Fc – ferrocene

• h – hours

• H2SO4 – sulphuric acid

• HCl – hydrochloric acid

• HRMS – high resolution mass spectrometry

• Hz – hertz

• i – current

• IR – infrared spectroscopy

• K – kelvin

• KCl – potassium chloride

• KOH – potassium hydroxide

• Lit. – literature

• M - molarity

• MeCN - acetonitrile

• MeO - methoxy

• MeOH – methanol

• min – minute

• mL – milliliter(s)

• mmol – millimol

• N2 – dinitrogen

• NaBH3(CN) – sodium cyanoborohydride

• NaHCO3 – sodium bicarbonate

• NH3 – ammonia

• NMR – nuclear magnetic resonance

• Ph – phenyl viii

• POCl3 – phosphoryl chloride

• r.t. – room temperature

• SOCl2 – thionyl chloride

• TEA – triethylamine

• THF – tetrahydrofuran

• TLC – thin layer chromatography

• TMSCl – trimethylchlorosilane

• UV – ultraviolet spectroscopy

• V – volts

CHAPTER 1: Introduction

1.1. General Background to Indoles

Indole 1 is an electron-rich nitrogen containing aromatic heterocyclic organic compound that occurs widely in natural products from plants, fungi and marine organisms[1, 2]. The structure of indole consists of a benzene ring and a pyrrole ring fused together in a bicyclic system. Numerous reviews on the incredible structural diversity of indole alkaloids have been published in the literatures[3-5].

4 3 5 2 6 N1 7 H

Well-known indole compounds include tryptophan 2, an amino acid that is essential to human diet[6, 7], serotonin 3, the neurotransmitter that regulates the function of the central nervous system, bowel motion and bladder control[8], and melatonin 4 that is known for its antioxidant activity against free-radicals, thereby reducing oxidative stress in living systems[9, 10].

O O OH NH2 HN

NH HO 2 O

N N H N H H

2 3 4

Due to the potent biological activity of indole compounds, considerable efforts have been devoted to the synthesis of the pharmacologically active indole alkaloids.

Novel indole alkaloids continue to be isolated from natural sources while new

1 synthetic strategies towards new indolic heterocycles with diverse therapeutic activity are continually being developed. Some successful drugs that contain the indole nucleus are the anti-inflammatory drug, indomethacin 5[11], the anti-HIV reverse transcriptase inhibitor, delavirdine 6[12], the antibiotic, carbazomycin 7[13], and the anti-cancer agent, naphthoindole 8[14].

O O S NH HO N O O N N HN N N H O O Cl 6 5

R O OMe O OH MeO Me NH2 Me N OH N H H O OH

7 8

1.2. General methods of indole ring synthesis

Since the first synthesis of indole in 1866 by Baeyer and Knop, many decades of research have led to the development of a variety of synthetic methods in constructing the indole skeleton[5, 15-19]. Although there are now many different ways of making indoles, the most common and important methods currently used for the preparation of indoles are the Fischer and Bischler indole syntheses[20-25].

The Fischer indole synthesis[5, 24, 25], also known as Fischer indolization is one of the oldest methods of making indoles. It has been the subject of much experimental work and is now considered the most versatile and convenient method for the preparation of

2 indoles. In this method, indole 10 is obtained simply by heating phenylhydrazones 9 in presence of an acid catalyst, with the elimination of ammonia as a by-product

(Scheme 1.1)[26]. Despite its early discovery, the detailed mechanism of this reaction was only established recently[27].

R R 1 1 R2 H+ + NH3 N R2 N N H H

9 10 Scheme 1.1: Fischer indole synthesis.

On the other hand, the Bischler rearrangement method involves the cyclization of amino-ketones 13, prepared in turn from the condensation of aniline 11 with phenacyl bromides 12, to give 2-substituted indoles 14.

Ar OMe Ar OMe a O + O MeO NH 2 Br MeO N H

b 13 11 12 OMe

Ar MeO N H 14

Scheme 1.2: Reagents and conditions: a) EtOH, NaHCO3, reflux, 2 h; b) aniline hydrobromide, silicone oil, inert gas, 130 °C, 3 h.

Recently, a modified Bischler technique has been reported that involves the treatment of 3,5-dimethoxyaniline 11 with halogenated ketones 15 in the presence of an inorganic base such as sodium bicarbonate to afford the substituted phenacyl aniline intermediates 16. The aniline-ketones are then N-protected with acetic anhydride to give the corresponding amides 17, which are then cyclized in the presence of trifluoroacetic acid to generate N-acetylindoles 18. The acetyl group is subsequently deprotected with methanolic potassium hydroxide to afford the desired 3-substituted-

4,6-dimethoxyindoles 19 (Scheme 1.3)[28].

X OMe X OMe a O + O

MeO NH MeO N 2 Br H

11 15 16 b

X X X OMe OMe OMe O d c MeO N MeO N MeO N H O O

19 18 17

Scheme 1.3: Reagents and conditions: a) NaHCO3, EtOH, reflux 2 h; b) Ac2O, r.t., overnight; c) TFA, 100 °C, argon, 2 h; d) MeOH, KOH, r.t., 1 h.

1.3. Indole chemistry

1.3.1. General reactions

Due to its high electron density, indole itself is a nucleophilic heterocycle and reacts very readily with electrophiles. The preferred site of attack is at C-3 rather than at C-

2. The reason for this reactivity is that the π-HOMO orbital at C-3 in indole contains higher electron density, due to the lone pair of electrons present on the nitrogen atom.

Moreover, electrophilic attack at the C-2 position would result in a loss of the aromatic stabilization of the fused benzene ring. Scheme 1.4 illustrates the resonance structures 20–21 of indole.

N N N H H H 1 20 21

Scheme 1.4: Resonance contributors of indole.

Considering this resonance pathway, the resulting cation formed by electrophilic attack at C-3 produces a cation 23 that can benefit from stabilization from the ring nitrogen without disrupting the aromaticity of the benzene ring. However, attack of the electrophile at C-2 to generate cation 22 will result in the disruption of the aromaticity of the benzene ring (Scheme 1.5)[29].

H E H E+ E+

N E N N H H H

22 1 23

Scheme 1.5: Electrophilic substitution reactions of indole

However, if the C-3 position is blocked, as in indole 24, electrophilic substitution can take place at C-2 to generate the cation 25 leading to the formation of the 2- substituted indole 26 (Scheme 1.6)[29, 30].

. R R R E+ H -H+

E E N N N H H H 24 25 26

Scheme 1.6: Electrophilic substitution of indole at C-2.

Depending upon the types of N-1, C-2 and/or C-3 substituents, substitution is also possible on the relatively less active benzenoid ring. 3-Substituted 2-alkylindoles 27, for example, are observed to undergo nitration in sulphuric acid at C-5 on the benzene ring 28 (Scheme 1.7)[31].

R R O2N Alkyl Alkyl N N H H 27 28

Scheme 1.7: Reagents and conditions: HNO3/H2SO4.

Similarly, bromination can also take place on the benzene ring if C-2 and C-3 are substituted. For example, treatment of 2,3-dimethylindole 29 with bromine and sulfuric acid produces 5-bromo-2,3-dimethylindole 30 in the presence of silver sulfate

(Scheme 1.8)[32].

Me Me Br Me Me N N H H 29 30

+ Scheme 1.8: Reagents and conditions: Br2, H , Ag2SO4.

The introduction of electron-donating methoxy groups on the benzene ring can direct electrophilic substitution activity to other positions, depending on the substitution pattern of the indole scaffold. 4,6-Dimethoxy substitution directs reactivity to C-7, while 5,7-dimethoxy substitution directs reactivity to C-4. This modification of the indole moiety has led to the synthesis of variety of augmented indoles, such as indoles

31–34 containing additional rings as the result of their ambident nucleophilic properties (Scheme 1.9)[33-35].

OMe OMe MeO R Ar MeO OMe MeO N N N H H O Ar Ar OH

31 32

OMe R NO2 N CO2Me MeO MeO CHAr N OMe OMe MeO2C RN 33 MeO 34 Scheme 1.9: Augmented indolyl derivatives.

Methoxy-activated indoles are not only capable of yielding a variety of new polycyclic structural compounds, but also show interesting electronic properties due to the electron-donating methoxy groups making the indole nucleus more electron rich and stabilizing the indole radical centres. One of the main aims of the project is to study the oxidative electrochemical decomposition pathways of indole.

1.3.2 Radical chemistry of indoles

Free radicals are known to play an important role in the pathogenesis of diseases, such as myocardial conditions, inflammation, cancer-initiation and aging processes[36-38].

The disintegration of cellular and organellar membranes induced by free radicals have been implicated in various pathological processes. Therefore, free radical scavengers can potentially be used as therapeutic agents for the treatment of these diseases. The study of indole radical chemistry has captivated many researchers over the past few decades due to the crucial biochemical role it plays in living systems. As most indole derivatives are easily oxidized, the antioxidant properties of indoles have been of particular focus. Hence, the antioxidant behaviour of many indoles has been studied using chemical and electrochemical oxidation methods. However, despite extensive research conducted on the redox and acid-base properties of indolyl radicals, very little information is known regarding the nature of their oxidation products. Prior experiments on indole oxidation using different electrochemical techniques, have suggested that the thin film formed at the electrode surface consisted of a polymeric material resulting from radical-initiated chain reactions[39, 40].

1.3.3 Fluorescence chemistry of indole

Fluorescence spectroscopy is a central technique used extensively in fields such as biotechnology, flow cytometry, medical diagnostics, DNA sequencing, forensics and genetic analysis. It is a highly sensitive technique that can reveal the localization and properties of intracellular compounds, sometimes at the level of a single molecule. Fluorescence typically occurs in aromatic molecules. Some common examples of fluorophores are fluorescein 35, acridine orange 36, and rhodamine B 37.

A natural indole molecule that is intrinsically fluorescent is tryptophan 2, and the

8 fluorescence of this amino acid residue has been used to study the conformation and dynamics of proteins. However, the interpretation of the fluorescence results of tryptophan can be difficult due to its complex photophysics[41].

Cl–

1.4 Primary aims

1.4.1 To develop model compounds for the formation of stable indole radicals and to study their electrochemical properties

The first aim of my project is to synthesize a series of indole derivatives that are able to generate stable indolyl radicals or radical cations when oxidized at room temperature, in order to help understand the biological oxidation pathways of indoles.

Electrochemical studies coupled with spectral techniques have been previously used to probe the oxidation chemistry and biochemistry of various biologically significant molecules[42, 43]. Hence, the redox mechanisms of these synthesized compounds will be investigated using electrochemical techniques such as cyclic voltammetry and UV-

Vis spectroelectrochemistry. Furthermore, the oxidation products, such as indole radicals or radical cations, will be characterized by electron paramagnetic resonance,

UV-Vis-NIR and FTIR spectroscopic techniques.

1.4.2. To synthesize indole-based fluorometric sensors for metal-ion binding studies

The second aim of my research is to synthesize indole-based analogues for metal-ion binding studies. Different metal binding sites will be incorporated at C7 of the highly fluorescent 2,3-diphenyl-4,6-dimethoxyindole. These newly synthesized indole ligands will be examined in fluorometric studies with various biologically important metal ions. This study will indicate whether the indole-based ligands can be used for detection of metal ions in living cells and tissues.

1.5 References

1. von Nussbaum, F., Stephacidin B—A New Stage of Complexity within Prenylated Indole Alkaloids from Fungi. Angewandte Chemie International Edition, 2003. 42(27): p. 3068-3071.

2. Li, S.-M., Prenylated indole derivatives from fungi: structure diversity, biological activities, biosynthesis and chemoenzymatic synthesis. Natural Product Reports, 2010. 27(1): p. 57-78.

3. Chen, F.-E. and J. Huang, Reserpine: A Challenge for Total Synthesis of Natural Products. Chemical Reviews, 2005. 105(12): p. 4671-4706.

4. Hibino, S. and T. Choshi, Simple indole alkaloids and those with a nonrearranged monoterpenoid unit. Natural Product Reports, 2002. 19(2): p. 148-180.

5. Van Order, R.B. and H.G. Lindwall, Indole. Chemical Reviews, 1942. 30(1): p. 69-96.

6. Le Floc’h, N., W. Otten, and E. Merlot, Tryptophan metabolism, from nutrition to potential therapeutic applications. Amino Acids, 2011. 41(5): p. 1195-1205.

7. Yao, K., et al., Tryptophan metabolism in animals: important roles in nutrition and health. Front Biosci (Schol Ed), 2011. 3: p. 286-97.

8. Watts, S.W., et al., Serotonin and Blood Pressure Regulation. Pharmacological Reviews, 2012. 64(2): p. 359-388.

9. Bespyatykh, A., O. Burlakova, and V. Golichenkov, Melatonin as an antioxidant: The main functions and properties. Biology Bulletin Reviews, 2011. 1(2): p. 143-150. 10. Dzie˛giel, P., et al., Melatonin stimulates the activity of protective antioxidative enzymes in myocardial cells of rats in the course of doxorubicin intoxication. Journal of Pineal Research, 2003. 35(3): p. 183-187.

11. Maity, P., et al., Indomethacin, a Non-steroidal Anti-inflammatory Drug, Develops Gastropathy by Inducing Reactive Oxygen Species-mediated Mitochondrial Pathology and Associated Apoptosis in Gastric Mucosa. Journal of Biological Chemistry, 2009. 284(5): p. 3058-3068.

12. Scott, L.J. and C.M. Perry, Delavirdine: A Review of its Use in HIV Infection. Drugs, 2000. 60(6): p. 1411-1444.

13. Yamasaki, K., et al., New antibiotics, carbazomycins A and B. III. Taxonomy and biosynthesis. J Antibiot (Tokyo), 1983. 36(5): p. 552-8.

14. Shchekotikhin, A.E., et al., Naphthoindole-based analogues of tryptophan and tryptamine: Synthesis and cytotoxic properties. Bioorganic & Medicinal Chemistry, 2007. 15(7): p. 2651-2659.

15. Shiri, M., et al., Bis- and Trisindolylmethanes (BIMs and TIMs). Chemical Reviews, 2009. 110(4): p. 2250-2293.

16. Gribble, G.W., Recent developments in indole ring synthesis-methodology and applications. Journal of the Chemical Society, Perkin Transactions 1, 2000(7): p. 1045-1075.

17. Cacchi, S. and G. Fabrizi, Synthesis and Functionalization of Indoles Through Palladium-catalyzed Reactions. Chemical Reviews, 2005. 105(7): p. 2873- 2920.

18. Humphrey, G.R. and J.T. Kuethe, Practical Methodologies for the Synthesis of Indoles. Chemical Reviews, 2006. 106(7): p. 2875-2911.

19. Gilchrist, T.L., Synthesis of aromatic heterocycles. Journal of the Chemical Society, Perkin Transactions 1, 2001(20): p. 2491-2515.

20. Fischer, E. and F. Jourdan, Ueber die Hydrazine der Brenztraubensäure. Berichte der deutschen chemischen Gesellschaft, 1883. 16(2): p. 2241-2245.

21. Fischer, E. and O. Hess, Synthese von Indolderivaten. Berichte der deutschen chemischen Gesellschaft, 1884. 17(1): p. 559-568.

22. Bischler, A., Ueber die Entstehung einiger substituirter Indole. Berichte der deutschen chemischen Gesellschaft, 1892. 25(2): p. 2860-2879.

23. Bischler, A. and P. Fireman, Zur Kenntniss einiger α-β- Diphenylindole. Berichte der deutschen chemischen Gesellschaft, 1893. 26(2): p. 1336-1349.

24. Robinson, B., The Fischer Indole Synthesis. Chemical Reviews, 1963. 63(4): p. 373-401. 25. Robinson, B., Studies on the Fischer indole synthesis. Chemical Reviews, 1969. 69(2): p. 227-250.

26. Xu, D.-Q., et al., Fischer indole synthesis catalyzed by novel SO3H- functionalized ionic liquids in water. Green Chemistry, 2009. 11(8): p. 1239- 1246.

27. Murakami, Y., Peculiarity of methoxy group-substituted phenylhydrazones in Fischer indole synthesis. Proc Jpn Acad Ser B Phys Biol Sci, 2012. 88(1): p. 1-17.

28. Black, D., et al., Investigation of the Bischler indole synthesis from 3,5- dimethoxyaniline. Australian Journal of Chemistry, 1980. 33(2): p. 343-350.

29. Jackson, A.H. and A.E. Smith, Electrophilic substitution in indoles—II: The formation of 3,3-spirocyclic indole derivatives from tryptamines and their rearrangement to β-carbolines. Tetrahedron, 1968. 24(1): p. 403-413.

30. Black, D.S.C., et al., Substitution, oxidation and addition reactions at C-7 of activated indoles. Tetrahedron, 1994. 50(35): p. 10497-10508.

31. Noland, W.E., L.R. Smith, and D.C. Johnson, Nitration of Indoles. II. The Mononitration of Methylindoles. The Journal of Organic Chemistry, 1963. 28(9): p. 2262-2266.

32. Kost, A.N., et al., Bromination of 2,3-dimethylindole. Chemistry of Heterocyclic Compounds, 1966. 1(4): p. 426-427.

33. Black, D.S., N. Kumar, and P.S.R. Mitchell, Synthesis of Pyrroloquinolines as Indole Analogues of Flavonols. The Journal of Organic Chemistry, 2002. 67(8): p. 2464-2473.

34. Black, D.S., A.J. Ivory, and N. Kumar, Synthesis of indolo[3,2-b]carbazoles from 4,6-dimethoxyindole and aryl aldehydes 1. Tetrahedron, 1995. 51(43): p. 11801-11808.

35. Condie, G.C., et al., Regioselective reactivity of some 5,7-dimethoxyindoles. Tetrahedron, 2005. 61(21): p. 4989-5004.

36. Hammond, B., H.A. Kontos, and M.L. Hess, Oxygen radicals in the adult respiratory distress syndrome, in myocardial ischemia and reperfusion injury, and in cerebral vascular damage. Canadian Journal of Physiology and Pharmacology, 1985. 63(3): p. 173-187.

37. Halliwell, B. and J.M.C. Gutteridge, Free radicals in biology and medicine, second edition: 540 pp. Free Radical Biology and Medicine, 1991. 10(6): p. 449-450.

38. Ralser, M. and H. Lehrach, Building a new bridge between metabolism, free radicals and longevity. Aging (Albany NY), 2009. 1(10): p. 836-8.

39. Holze, R. and C.H. Hamann, Electrosynthetic aspects of anodic reactions of anilines and indoles. Tetrahedron, 1991. 47(4–5): p. 737-746.

40. Tourillon, G. and F. Garnier, New electrochemically generated organic conducting polymers. Journal of Electroanalytical Chemistry and Interfacial Electrochemistry, 1982. 135(1): p. 173-178.

41. Creed, D., The photophysics and photochemistry of the Near-UV absorbing amino acids-1: Tryptophan and its simple derivatives. Photochemistry and Photobiology, 1984. 39(4): p. 537-562.

42. Martin, K.F., C.A. Marsden, and F. Crespi, In vivo electrochemistry with carbon fibre electrodes: Principles and application to neuropharmacology. TrAC Trends in Analytical Chemistry, 1988. 7(9): p. 334-339.

43. Hammes-Schiffer, S. and A.V. Soudackov, Proton-Coupled Electron Transfer in Solution, Proteins, and Electrochemistry. The Journal of Physical Chemistry B, 2008. 112(45): p. 14108-14123.

CHAPTER 2: Synthesis of novel indole derivatives

2.1 Introduction

The research areas covered and described in this thesis are centered around

2,3-disubstituted-4,6-dimethoxyindoles. Their enhanced reactivity towards aromatic electrophilic substitution reactions at C-7 provides the basis of the synthetic strategy for this project. Based on this, two different areas of indole chemistry were explored: i) to investigate the anodic (oxidative) electrochemical behaviour with a view towards elucidating the decomposition pathway(s) of the novel indoles and; ii) to examine the metal-binding properties of novel indole-based ligands.

As previously described, 4,6-dimethoxy-substituted indoles undergo formylation, acylation, bromination, oxidative coupling and acid-catalysed addition at C-7 to produce a range of 7-substituted indoles[1-5]. Furthermore, they can undergo many other types of reactions to form a variety of augmented indoles containing additional rings, as a result of their ambident nucleophilic properties[6-9]. This chapter will focus on the synthetic strategies for the compounds that were prepared for the project. The synthesized compounds were fully characterized and then further examined for electrochemical and fluorescence studies.

2.2 Indole derivatives synthesized for electrochemical studies

The aim of this project was to synthesize 2,3-diphenyl-substituted 4,6- dimethoxyindoles derivatives that can be oxidized to afford “stable radicals”. While numerous works have been accomplished in terms of the synthesis and biological applications of indoles, the electrochemical behaviour of indoles and their derivatives

14 has only been briefly investigated. There are two methods that can be used in stabilising the indole radicals: the first one aims to close off decomposition pathways by using bulky subtituents (kinetic stabilization), while the second aims to increase the electron density of the radical centre, thus lowering its inherent reactivity

(electronic stabilization).

When two phenyl groups are placed at the C-2 and C-3 positions of the indole nucleus, the decomposition pathways from those relatively high electron-density carbons can be cut off. Moreover, not only is the phenyl group a bulky substituent, they are also electron-donating groups that can help to increase the stabilization of the indole radical. Hence, indole compounds bearing phenyl groups at C2 and C3, and the methoxy groups at C-4 and C-6 were synthesized. The methoxy groups at C-4 and C-

6 positions of the indole nucleus can further increase the overall electron density of the indole moiety. These methoxy groups also facilitated the synthesis of the desired indole derivatives by increasing the electron density at C-7.

Well-developed organic syntheses were used to prepare the novel indole derivatives that fit the criteria for forming stabilized radicals.

OMe R' R' = -Ph, -(4-MeO)Ph, -Me R' R = H+ acceptor MeO N H R

2.2.1 Synthesis of 4,6-dimethoxy-2,3-disubstituted-indoles

To begin with, 4,6-dimethoxy-2,3-diphenylindole 39 was synthesized in major quantities (~20 g) by the one-pot reaction of 3,5-dimethoxyaniline 11 with benzoin 38

(Scheme 2.1). After refluxing overnight in glacial acetic acid, the product was obtained as a white solid that did not require further purification[10, 11].

OMe O OMe + OH MeO NH2 MeO N H

11 38 39

Scheme 2.1: Reagents and conditions: aniline, CH3COOH, reflux 12 h.

Similarly, indoles 41 was synthesized from 3,5-dimethoxyaniline 11 and anisoin 38 in

79% yield, while indole 43 was synthesized from 3,4,5-trimethoxyaniline 42 and benzoin 38 in 50% yield (Scheme 2.2).

OMe OMe OMe O OMe + HO OMe MeO NH2 MeO N OMe H

11 40 41

OMe OMe MeO O + MeO OH MeO NH2 MeO N H

42 38 43

Scheme 2.2: Reagents and conditions: 3,5-dimethoxyanilium hydrochloride, silicone oil, 140 °C, 3 h.

The investigation also included the synthesis of 4,6-dimethoxy-2,3-dimethylindole 45, which was synthesized from 3,5-dimethoxyaniline 11 and 3-chlorobutanone 44 in a one-pot procedure developed by Black et al. (Scheme 2.3)[10]

OMe OMe Me Cl + Me Me Me O MeO N MeO NH2 H 11 44 45

Scheme 2.3: Reagents and conditions: NaHCO3, LiBr, abs. EtOH, reflux 3 h.

2.2.3 Oxidative dimerization

4,6-Dimethoxy-2,3-diphenylindole 39 was then used as the primary starting compound to produce a variety of 7-substituted indole derivatives. 7,7′-Bi-indolyl 46 was obtained in high yield (97%) upon the treatment of indole 39 with 1,4- benzoquinone and HCl in tetrahydrofuran (THF) (Scheme 2.4). The use of chloranil or dichlorodicyanoquinone gave lower yields of 70% and 60%, respectively.

OMe

OMe MeO N H H OMe MeO N N H

39 OMe

Scheme 2.4: Reagents and conditions: 1,4-benzoquinone, H+, THF, r.t. 3 h.

1H NMR spectrum analysis of compound 46 showed the disappearance of the proton at C-7, while mass spectrometry revealed the anticipated molecular mass of m/z 656

(M+ 100%)[12].

2.2.4 Vilsmeier-Haack formylation

The standard Vilsmeier reagent consists of a mixture of N,N-dimethylformamide

(DMF) and phosphoryl chloride, which can generate the chloroiminium species at low temperature (0-5°C). This species reacts with indoles via an electrophilic substitution mechanism. The addition of an aqueous base (NaOH) then results in the formation of indole carbaldehydes. The presence of 2,3-disubstituted groups ensures that formylation can only take place at the C-7 position. Black et al. have reported that

4,6-dimethoxy-2,3-diphenylindole 39 can be directly formylated at C-7 to give indole

47 (Scheme 2.5)[13].

OMe OMe

MeO N MeO N H H H O

39 47

Scheme 2.5: Reagents and conditions: POCl3, DMF, 25°C, 2 h.

The crude product was recrystallised from DCM/n-hexane to obtain the final product, whose 1H NMR spectrum showed the disappearance of the H-7 peak at 6.53 ppm and the appearance of the new –CHO peak as a singlet at 10.38 ppm.

Subsequently, 7-formylindole 47 was reduced to the corresponding alcohol 48 by refluxing the aldehyde with excess NaBH4 in absolute ethanol (Scheme 2.6). The solvent was then evaporated, and water and aqueous 2M NaOH were added. The resulting white precipitate was filtered, washed with water, dried and collected.

OMe OMe

MeO N MeO N H H H O OH

47 48

Scheme 2.6: Reagents and conditions: NaBH4, abs. EtOH, reflux 3 h

2.2.5 Synthesis of 7-trichloroacetylindoles

Like acid chlorides, trihalomethyl ketones are strong electrophiles. Their importance in organic synthesis lies in their ability to be readily hydrolysed, and many reactions utilize the trihalomethyl moiety as a leaving group[13, 14]. The trihaloacetylation reactions of indoles are normally carried out under mild conditions. For instance, trifluoroacetic anhydride (TFAA) reacts readily with indole 1 at room temperature to give a mixture of 3-trifluoroacetylindole 49, N-trifluoroacetylindoles 50 and N- trifluoroacetyl-2,2′-bi-indole 51 (Scheme 2.7)[15].

H O N CF3 + N + N N H O O N F C F C H 3 3 1 49 50 51

Scheme 2.7: Reagents and conditions: TFAA, THF, r.t.

Black et al. have observed that the treatment of 2,3-diphenylindole 39 with trifluoroacetic anhydride in THF produced 7-trifluoroacetylindole 52 in good yield

(Scheme 2.8)[13].

OMe OMe

MeO N MeO N H H F C O 3 39 52

Scheme 2.8: Reagents and conditions: TFAA, THF.

However, in the present study, it was found that the corresponding trichloroacetylated indole could not be prepared using trichloroacetic anhydride even under vigorous conditions. Therefore, trichloroacetyl indole 53 was instead prepared via treatment of indole 39 with an excess of the trichloroacetyl chloride (TCAC) in refluxing chloroform (Scheme 2.9). 7-Trichloroacetylindole 53 was successfully afforded as a single product, which was chromatographed through a silica column for purification[16].

OMe OMe

MeO N MeO N H H Cl3C O

39 53

Scheme 2.9: Reagents and conditions: TCAC, CHCl3, reflux overnight.

Trichloroacetylindole 53 was subsequently used as the starting material to prepare analogues of other indole derivatives as described in the following sections.

2.2.6 Synthesis of indole-7-carboxylic acid

Indole carboxylic acid can be prepared in several ways. One approach involves the hydrolysis of the trichloroacetyl group to yield the corresponding carboxylic acid.

Thus, treatment of indole 53 with aqueous alcoholic sodium hydroxide gave the indole-7-carboxylic acid 54 in 87% yield[13].

OMe OMe

MeO N MeO N H H

Cl3C O HO O

53 54

Scheme 2.10: Reagents and conditions: NaOH, EtOH/H2O, reflux, 1 h.

2.2.7 Synthesis of indole carboxamides

The reactions of trichloroacetyl derivatives with ammonia, primary or secondary amines are known to proceed rapidly at room temperature to yield the respective amides (Scheme 2.11)[17].

O O

R' + CHCl3 CCl3

X X

R′ = RNH, NH2, R2N; R = alkyl

Scheme 2.11: Reagents and conditions: amine, CHCl3, r.t.

Similarly, treatment of indole 53 with an excess of amines in acetonitrile gave the respective amides in 55-85% yields[16].

OMe OMe

MeO N MeO N H H Cl C O 3 R O

53 55

55a R = NH2, 85% 55b R = NHCH , 80% 3 55c R = N(CH ) , 59% 3 2 55d R = N(C2H5)2, 55% 55e R = NH(CH2)Bz, 75%

Scheme 2.12: Reagents and conditions: a) 32% (w/w) aqueous ammonia; b) 40% (w/w) aqueous methylamine; c) 60% (w/w) aqueous dimethylamine d) diethylamine; e) benzylamine.MeCN, r.t.

However, when the reaction shown in Scheme 2.12 was repeated with aryl amines such as aniline, only the starting materials were recovered. Changing the solvent to dichloroethane and increasing the temperature of the reaction to reflux failed to give the desired product. This might be due to the weaker nucleophilicity of aryl amines compared to aliphatic amines because of the electron-withdrawing effect of the phenyl group.

OMe OMe

MeO N MeO N H H Cl3C O R' O 53 56

Scheme 2.14: Reagents and conditions: a) aniline; b) N-methylaniline. MeCN, reflux.

The reaction of 7-trichloroacetylindole 53 with acetic anhydride in acetonitrile, however gave 7-acetylindole 57 (Scheme 2.13), which was used as a model compound in the electrochemistry studies (Chapter 3).

2.2.8 Synthesis of indole carboxylic esters

Black et al.[10] reported the synthesis of methyl indole-2-carboxylate 59 by the treatment of indole-2-carboxylic acid 58 with dimethylsulfate (DMS) in acetone.

OMe OMe OH OMe

MeO N O MeO N O H H

58 59

Scheme 2.15: Reagents and conditions: DMS, acetone.

The same procedure using DMS in acetone was applied to indole-7-carboxylic acid

54, giving the methyl indole-7-carboxylate 60 in 80% yield[11].

OMe OMe

MeO N MeO N H H HO O MeO O

54 60

Scheme 2.16: Reagents and conditions: 1) DMS, acetone, reflux, 3 h.

The 1H NMR spectrum of compound 60 displayed the existence of three methoxy peaks at 3.78, 3.99 and 4.00 ppm, while the 13C NMR spectrum also showed the presence of three methoxy carbon signals at 51.75, 55.28 and 57.32 ppm.

Alternatively, indolyl esters could also be synthesized by the alcoholysis of trichloroacetyl indole 53 in the presence of a base such as a tertiary amine, an alkoxide or potassium carbonate[18]. This method was efficient at introducing the methyl and benzyl ester groups, thus generating indole esters 60 and 61 in high purity and in yields of 71% and 64% respectively (Scheme 2.17)[11].

OMe OMe

MeO N MeO N H H

Cl3C O R O

53 60 R = O-Me, 71% 61 R = O-CH2-Ph, 64%

Scheme 2.17: Reagents and conditions: 1) MeOH, TEA; 2) benzyl alcohol, K2CO3.

Overall, both methods of indole esterification were found to be relatively simple and effective.

2.2.9 Friedel-Crafts acylation

The synthesis of indolyldeoxybenzoins 63 was carried out by the addition of anhydrous tin(IV) chloride to a mixture of 4,6-dimethoxy-2,3-diphenylindole 39 and phenylacetyl chloride 62 in anhydrous toluene (Scheme 2.18). The yield of the product was 31%.

OMe Cl OMe O + MeO N H

O MeO N H

63 62 39

Scheme 2.18: Reagents and conditions: SnCl4, toluene, r.t., 24 h.

Analysis of the 1H NMR spectrum of the indolyldeoxybenzoin 63 showed the disappearance of H-7, along with the appearance of additional aromatic protons from

[11] the benzene ring and a singlet peak at 4.45 ppm corresponding to the CH2 protons .

2.2.10 N-Methylation of indole and preparation of its derivatives

N-Methylation of indole 39 was achieved with methyl iodide in DMSO in the presence of KOH, to yield indole 64 in 95% yield (Scheme 2.19)[19].

OMe OMe

N MeO N MeO H Me

39 64

Scheme 2.19: Reagents and conditions: MeI, KOH, DMSO, r.t, 0.5 h

The 1H NMR spectrum of the methylated product 64 showed the disappearance of the

NH proton at 8.10 ppm and the appearance of a new singlet at 3.93 ppm corresponding to the N-methyl group. Similarly, the 13C NMR spectrum showed the appearance of the N-methyl carbon at 32.05 ppm.

Formylation and trichloroacetylation of N-methyl indole 64 were undertaken in a similar manner as that for indole 39 to yield the 7-formyl and 7-trichloroacetyl indole derivatives 65 and 66, respectively. However, due to increased steric hindrance from the methyl group, the reactions were carried out at higher temperatures over a longer duration of time. 7-Trichloroacetylindole 66 was subsequently reacted with methylamine to yield the amide derivative 67 (Scheme 2.20).

OMe a)

MeO N Me OMe H O 65

MeO N Me OMe c) OMe 64 b)

N MeO N MeO Me Me H CHN O Cl3C O 3 67 66

Scheme 2.20: Reagents and conditions: a) POCl3, DMF, 70°C, 3 h; b) Cl3COCl, CHCl3, reflux 24 h; c) 40% (w/w) aqueous methyl amine, MeCN, reflux 12 h.

2.3 Indole derivatives for fluorescence studies

Indole-based acyclic and macrocyclic chelating systems have been extensively studied for decades. These nitrogen heterocycles have also been frequently assembled into polydentate ligands, and their metal complexes have been employed to mimic the structures and functions of metalloenzymes. The following sections describe the synthesis of a series of indole-based fluorescent chemosensors for metal-binding studies.

2.3.1 Synthesis of indole-based di-(2-picolyl)amine (DPA) ligand

Numerous coordination complexes of di-(2-picolyl)amine (DPA) have been reported[20-24]. The different coordination modes available to the ligand result in an unusual structural diversity, and the luminescence properties of its complexes render them suitable as metal-detecting tools in biochemistry.

The indole-based DPA ligand 69 was synthesized for the purpose of metal-binding studies for the first time. The reductive amination of indole-7-carbaldehyde 47 with

DPA 68 using sodium cyanoborohydride in the presence of glacial acetic acid was used to generate the novel ligand 69 in 58% yield (Scheme 2.21). However, the use of sodium triacetoxyborohydride as a reducing agent resulted in mixture of products.

OMe

OMe H N MeO N + H

N N MeO N N H N H O 68 N 47

+ Scheme 2.21: Reagents and conditions: NaBH3(CN), H , MeOH, under N2, 72 h.

The reaction took several days to complete as monitored by TLC. Heating the reaction mixture resulted in the formation of many by-products. Therefore, the reaction was carried out at room temperature under a nitrogen atmosphere.

12.43 8.17 8.17 8.17 8.17 8.16 8.16 8.15 8.15 7.45 7.45 7.43 7.42 7.42 7.35 7.33 7.33 7.29 7.26 7.19 7.18 7.17 7.15 7.15 7.15 6.97 6.96 6.95 6.95 6.94 6.94 6.92 6.92 6.14 3.88 3.81 3.76 3.58

OMe

MeO N H N N N

12 11 10 9 8 7 6 5 4 ppm

1.01 1.89 4.01 2.00 2.09 6.62 2.01 1.00 2.10 3.92 3.04 3.00

1 Figure 2.1: H NMR spectrum of the indole ligand 69 in CDCl3.

Identification of the ligand was performed through 1H and 13C NMR spectroscopy and mass spectrometry data. The 1H NMR spectrum of compound 69 (Figure 2.1) showed the disappearance of the aldehyde proton at 10.38 ppm. Two new peaks corresponding to the three methylene groups appeared as singlets at 3.81 (2-pyridyl

CH2 groups) and 3.88 ppm (indolyl CH2 group), along with the characteristic pyridyl proton peaks at 6.92 and 8.16 ppm. Furthermore, the chemical shift of the indole NH proton shifted from 10.57 to 12.43 ppm.

2.3.2 Synthesis of novel 7-amino acid-substituted indole ligands

A series of 7-amino acid-substituted indole ligands were prepared to study their relative affinities for different metal ions and to understand their metal ion-ligand interactions. These ligands contain standard amino acids attached to the indole motif via a carbonyl linkage. This sub-section will describe the general synthetic procedure and characterizations of the compounds.

2.3.2.1. Synthesis of amino acid alkyl esters

The commercially acquired amino acids were converted to their corresponding methyl esters hydrochlorides in order to prevent the acid group from taking part in the coupling reaction. Methanol/trimethylchlorosilane (TMSCl) has been shown to be a convenient reagent for the preparation of methyl esters of various carboxylic acids[25,

26]. Using this reagent, the esterification of the amino acids was carried out with methanol at room temperature (Scheme 2.22).

R OH R O H2N H2N Me HCl O O

70 71 71a R = CH-(CH2)4NH2 71b R = CH-CH2COOH 71c R = CH-CH2Ph 71d R = CH(CH)2SCH3 71e R = CH2NH-COCH2NH2

Scheme 2.22: Reagents and conditions: TMSCl, MeOH, r.t.

The TMSCl/MeOH system of esterification was not only convenient from an operational point of view, but the yields achieved were also comparable to those obtained with the thionyl chloride/MeOH or conc. HCl or H2SO4/MeOH system. For example, in the thionyl chloride/MeOH method, the temperature has to be strictly maintained below 0°C and HCl gas must be continuously passed through the refluxing mixture[27]. The use of TMSCl is also known to minimize racemization problems that are common in the synthesis of amino acid esters[28].

Table 2.1: Esterification of amino acids with methanol in the presence of TMSCl.*

Reported Substrate Product Time Yield yields (h) (%) (%) O O H N H N Me (71a) [29] 2 OH 2 O 12 87 92 NH2 NH2 O O HO O Me [30] OH Me O (71b) 15 82 86 O NH2 O NH2 NH2 NH2 [31] OH O (71c) 10 85 88 Me

O O O O S S Me OH O (71d) 15 78 - NH2 NH2 O O H H N N Me H N O (71e) 12 80 - H2N OH 2 O O

*Glycine ethyl esters and leucine ethyl ester hydrochlorides were commercially acquired.

1 13 H and C NMR spectra were recorded in DMSO-d6, and the proton NMR analysis showed the disappearance of the carboxylic acid proton peak and the appearance of a singlet corresponding to the methyl protons. 13C NMR spectroscopy further confirmed the formation of the product with the appearance of a new methoxy carbon signals

(see Chapter 5 for details).

2.3.2.2 Synthesis of indole amino esters

The method described for the reaction between 7-trichloroacetylindole 53 with various amines was employed in the amino acid-indole conjugation. The free amino group on the amino acid ester acts as a nucleophile and attacks the electron-deficient carbonyl carbon, resulting in the formation of an amide bond. All of the amino acid

30 esters were coupled with indole 53 in anhydrous acetonitrile in the presence of triethylamine (TEA) as base to neutralize the hydrochloride salt (Scheme 2.23). In general, 1.5 equivalents of the amino acid esters were used. In case of lysine methyl ester, two equivalents of the indole 53 were used because of the presence of two free amino groups on the ester. The reactions were conducted under ambient temperature with a gradual temperature increase as required by monitoring the progress of the reaction on TLC. All reactions were carried out under an inert atmosphere of argon gas.

OMe OMe

MeO N MeO N H H O O Cl3C O Me NH O

R 72 53

Scheme 2.23: Regents and conditions: amino acid ester hydrochloride salt, TEA, MeCN.

Table 2.2: Synthesis of 7-substituted amino acid indole ligands.

Entry Substrate (R) Product Time Temperature Yield (h) (°C) (%) O

H2N C2H5 (72a) 3 22–25 62 O 1 O

C2H5 (72b) 12 85 22 O 2 NH2 O

H2N (72c) 24 85 30 OH 3 NH2 O O Me (72d) 12 85 30 Me O 4 O NH2

NH2 O (72e) 12 85 36 Me 5 O O S Me (72f) 12 85 29 O 6 NH2 O H N Me (72g) 12 85 56 H2N O 7 O

Compound 72a was obtained after a relatively short period of time (3 h), and the product precipitated out as a white solid upon addition of water. Structural determination by 1H and 13C NMR spectroscopy data revealed the formation of indole-glycine ester as the sole product (Figure 2.2). The characteristic amide proton was found at 8.63 ppm as a triplet (J = 4.85 Hz), the methylene group appeared as a

32 singlet at 4.45 ppm, and the ethyl protons appeared at 1.32 ppm (triplet, J = 7.29 Hz) and 4.25 ppm (quartet, J = 7.29 Hz).

11.20 8.67 8.65 8.64 7.38 7.31 7.30 7.30 7.27 7.26 7.23 7.20 6.23 4.31 4.29 4.29 4.27 4.24 4.08 3.77 1.36 1.33 1.31

OMe

MeO N H HN O O C2H5 O

11 10 9 8 7 6 5 4 3 2 1 ppm 0.95 0.97 1.00 3.77 3.09 3.02 2.90 10.19

1 Figure 2.2: H NMR spectrum of indole ligand 72a in CDCl3.

The yields obtained were generally less than 40%. However, in the case of glycine ethyl ester and glycyl-glycine methyl esters, higher yields of 62% and 56% respectively were obtained. This is probably due to the less sterically hindered amino group of glycine in contrast to the NH2 group of other amino acid esters where the adjacent carbon is bonded to sterically bulky substituents. Another reason for the lower yields for the other amino acid esters derivatives could be due to the side reactions that might be occurring as a result of the presence of different functional groups on their side chains.

The work-up procedure for the rest of the compounds 72b-g was performed differently. After the reaction was complete, the solvent (MeCN) was partially

33 evaporated and the organic fraction was extracted with DCM. The crude product obtained by evaporation of the solvent was purified by gravity column chromatography. In contrast to the other amino acids, lysine has two primary amino groups, which led to the formation of an interesting product 72c whereby two indoles were linked to lysine via amide bridges. The 1H and 13C NMR, HRMS and elemental analysis confirmed the structure of this unusual product. In particular, the 1H NMR spectrum of the bi-indolyl ligand 72c showed the presence of two sets of protons for the indole rings clearly indicating the reaction of two equivalents of indole with one equivalent of lysine ester.

11.33 11.19 8.66 8.63 8.12 7.39 7.38 7.27 7.20 6.10 6.07 4.89 4.88 4.86 4.86 4.16 4.13 4.13 4.11 4.08 3.96 3.89 3.79 3.70 3.68 3.55 3.53 3.50 3.48

OMe

MeO N H MeO O HN O

NH HN

MeO OMe

11 10 9 8 7 6 5 4 ppm 1.19 1.24 1.10 1.11 1.00 1.13 1.16 1.81 3.54 3.69 3.85 3.34 3.30 2.58

25.73

1 Figure 2.3: H NMR spectrum of indole ligand 72c in CDCl3.

2.4 Conclusion

Utilizing the C-7 reactivity of 4,6-dimethoxyindoles, a series of novel indole derivatives were successfully prepared. In addition to indoles bearing simple functional groups, such as amide, acid and aldehyde groups at C-7 of the parent 2,3- diphenylindole nucleus, a series of new amino acid-indole based compounds were also synthesized. The structural identification of each compound was performed using

1H NMR, 13C NMR, UV-vis, IR spectroscopy and HRMS data, and are described in detail in Chapter 5 of this thesis.

2.5 References

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26. Brook, M.A. and T.H. Chan, A simple procedure for the esterification of carboxylic acids. Synthesis, 1983. 1983(03): p. 201,203.

27. Hosangadi, B.D. and R.H. Dave, An efficient general method for esterification of aromatic carboxylic acids. Tetrahedron Letters, 1996. 37(35): p. 6375- 6378. 28. Chen, B.-C., et al., A facile method for the transformation of N-(tert- Butoxycarbonyl) α-amino acids to N-unprotected α-amino methyl esters. The Journal of Organic Chemistry, 1999. 64(25): p. 9294-9296.

29. Driffield, M., D.M. Goodall, and D.K. Smith, Syntheses of dendritic branches based on l-lysine: is the stereochemistry preserved throughout the synthesis? Organic & Biomolecular Chemistry, 2003. 1(14): p. 2612-2620.

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31. Carmi, C., et al., 5-Benzylidene-hydantoins as new EGFR inhibitors with antiproliferative activity. Bioorganic & Medicinal Chemistry Letters, 2006. 16(15): p. 4021-4025.

CHAPTER 3: Electrochemical studies of highly substituted indole derivatives

3.1. Introduction

In this chapter, the investigations of the redox chemistry of several highly substituted indole derivatives carried out by electrochemical experiments, including cyclic voltammetry, UV-Vis spectroelectrochemistry and electron paramagnetic resonance

(EPR) spectroscopy will be discussed. Electrochemistry deals with behavior of oxidation and reduction reactions connected by an external electric circuit to understand each process. Redox and electrochemical processes involve electron transfer to/from a molecule. This reaction can occur by the application of a voltage or by the release of chemical energy. Applications of electrochemistry include the determination of electrode oxidation and reduction mechanisms[1, 2]. Knowing that there is a resemblance between electrochemical and biological reactions, it can be considered that the oxidation or reduction reactions taking place at the electrode and in the body may have similar mechanisms.

3.2. Electrochemistry of indoles

Electrochemical studies of the oxidation of indole derivatives have been reported using different electrodes: glassy carbon[3-5], pyrolitic graphite electrode (PGE)[6-8], carbon paste[9, 10], gold [3], platinum [11] and boron doped diamond[12]. Although there are some known pathways for the oxidations of indole and indole derivatives[6, 13-15], more work needs to be done to fully characterize the mechanisms of the electrochemical oxidations of indole derivatives.

Previous electrochemistry studies of indole derivatives include investigations of biologically relevant, C-3-substituted indoles such as indol-3-acetamide (IAM), tryptamine, gramine, indole acetic acid (IAA) and tryptophan[3, 4, 15]. Oliveira-Brett et al. have reported the electrochemical oxidation of indole by differential pulse voltammetry at a glassy carbon electrode (GCE) over a wide pH range between 1.0 and 12.0. Indole was reported to show a single, irreversible oxidation step at all pHs and the maximum peak current was observed in electrolytes with neutral pH. A multielectron process leading to a quinonoid product was also observed and reaction mechanism in Scheme 3.1 was proposed to account for the observations.

-e, -H+ 21 N • N H H

+H2O 1

N + H OH -e, -H -e, -H O +H O 2 73 + +H 2 O

N H O O N 74 H O 75 +/- 2e, 2H+ +/- 2e, 2H+ OH

N HO N H H OH OH 76 77

Scheme 3.1: Proposed mechanism for oxidation of indole at a glassy carbon electrode[15]

The initial oxidation afforded the radical, which was followed by nucleophilic attack of water leading to hydroxylation of the benzene ring at C-7. Further oxidation gave the quinonoid products[15]. Oliveira-Brett et al. also investigated the electrochemical oxidations of indole derivatives with a substituent at C-3 position. The redox behaviour was found to be complex, pH dependent, and irreversible. It was suggested that the substitution at C-3 led to oxidation at C-2 position on the pyrrole ring, which was followed by hydroxylation at C-7 on the benzene moiety (Scheme 3.2). Again multi-electron oxidation resulted in quinonoid products.

R R R + -e, -H -e, -H+ O N N • N H H 79 24 78 O +H2O H2C NH2 R R = H C CH O 2 N 3 N 80 H CH3 + -e, -H

-e, -H O 2 +H H C NH +H + 2 2 2 O O R R O

O O H C OH N 2 81 O N 82 H H O O

+ + OH +/- 2e, 2H +/- 2e, 2H H2C O

OH R R O O O H2C OH HO N 84 N 83 H H OH OH

Scheme 3.2: Proposed oxidation mechanism of C-3 substituted indoles[15]

It is notable that from the study electrochemical behaviour of indole and its derivatives using glassy carbon electrode in aqueous medium resulted in no radical coupling or dimerization of cation radical species[15].

3.3. Cyclic voltammetry

3.3.1. Results and discussion

Initial information about 4,6-dimethoxy-2,3-diphenylindole (DPI, 39) and its derivatives was obtained through cyclic voltammetry studies. The oxidation and reduction peak potential data from cyclic voltammograms (CVs) for the various indole derivatives was obtained at a scan rate of 100 mV/s and at 295 K (Table 3.1).

The reduction potential (E1/2) and peak potential separation (ΔEp) data observed for the redox processes is presented in Table 3.2.

Table 3.1 Potential data (V vs ferrocenium-ferrocene) for peaks from cyclic n voltammograms recorded in MeCN – 0.1 M [Bu 4N][PF6] at room temperature.

Conditions: 1 mm glassy carbon disc working electrode, Ag/AgCl(aq) reference electrode, 100 mV/s scan rate.

Compound Anodic potential peaks (Ea,p/V) Cathodic potential peaks (Ec,p/V) I II III I* II* III* 39 0.74 0.9 1.25 0.63 0.83 1.14

41 0.68 0.78 1.11 0.55 0.71 1.03

43 0.91 1.26 1.74 0.83 1.12 1.63

45 0.6 0.76 1.25 0.5 0.7 1.05

46 0.69 0.9 1.25 0.63 0.83 1.12

47 1.05 1.34 - 0.97 1.23 -

48 0.76 1.15 - 0.68 1 -

54 1.05 1.24 - 0.96 1.15 -

55e 0.95 1.28 - 0.89 1.2 -

61 0.97 1.2 - 0.89 1.1 -

63 1.01 1.3 - 0.94 1.22 -

56 1 1.28 - 0.92 1.2 -

64 0.78 0.9 1.3 0.65 0.84 1.19

65 1 1.31 - 0.92 1.25 -

Table 3.2 Potential data (V vs ferrocenium (Fc+)-ferrocene (Fc)) for processes observed in n cyclic voltammograms recorded in MeCN – 0.1 M [Bu 4N][PF6] at room temperature

(1 mm glassy carbon disc working electrode, Ag/AgCl(aq) reference electrode, 100 + mV/s scan rate). ΔEp (Fc /Fc) = 69 mV.

Compound E1/2(I/I*) ΔEp(I/I*) E1/2(II/II*) ΔEp(II/II*) E1/2(III/III*) ΔEp(III/III*) (V) (mV) (V) (mV) (V) (mV) 39 0.685 115 0.87 70 1.20 110

41 0.615 130 0.745 70 1.07 80

43 0.87 80 1.19 140 1.685 110

45 0.55 100 0.73 60 - 200

46 0.66 60 0.865 70 1.185 130

47 1.01 80 1.285 110 - -

48 0.72 80 1.075 150 - -

54 1.005 90 1.195 90 - -

55e 0.92 60 1.24 80 - -

61 0.93 80 1.15 100 - -

63 0.975 70 1.26 80 - -

56 0.96 80 1.24 80 - -

64 0.715 130 0.87 60 1.245 110

65 0.96 80 1.28 60 - -

3.3.1.1 Cyclic voltammetry studies of 4,6-dimethoxy-2,3- diphenylindole and its dimers

The parent indole 39 was the first compound studied using CV experiments. Indole 39

showed three oxidation processes at +0.74 V (E1/2(I/IV*)), 0.87 V (E1/2(II/II*)), and

1.20 V (E1/2(III/III*)); see Figure 3.1. From the peak-to-peak separations (Table 3.2),

43 it was immediately apparent that the II/II* process was an electrochemically reversible (Nernstian) process whereas the III/III* couple was considerably broader indicating less electrochemical reversibility[16].

OMe

MeO N H

Figure 3.1: Cyclic voltammogram of indole 39. Experimental conditions:

[39] = 2 mM in MeCN (anhydrous); 0.1 M [Bu4N][PF6] electrolyte; 295 K, 0.5 mm diameter glassy carbon minidisk working electrolyte, against Ag/AgCl reference electrode

The peak current for the anodic wave I is almost double that for the II/II* and III/III* couples and the peak is ‘sharp’; Ep(I) – Ep/2(I) = 50 mV (<69 mV for the one-electron

Fc/Fc+ couple for ferrocene standard). In comparison, the peak for the cathodic wave

(IV*) is considerably broader: Ep(IV) – Ep/2(IV) = 75 mV, and appeared at a potential more negative than that expected if it and I* were a couple, which leads to the large

ΔEp(I/IV*) value (115 mV, see Table 3.2). These observations led to a suspicion, which was soon confirmed (see below), that wave I was a multi-electron process and peak IV* was from the re-reduction of a product species that formed rapidly upon the multi-electron oxidation of indole 39 at peak I. Further confirmation that peak I is a multielectron process and that I and IV* are not waves from a couple comes from a

1/2 plot of ip vs v for peak I; Figure 3.2. The linear fit suggests that the peak current is limited by bulk diffusion; i.e. the underlying process is electrochemically fast and

Nernstian. Therefore, the observation of ΔEp(I/IV*) = 115 mV is inconsistent with peaks I and IV* being a couple. The broad peak IV* was later identified as belonging to the anodic half of the first oxidation couple for dimer 46. Furthermore, conclusive spectro-electrochemical and chemical evidence for quantitative dimerization of 39 to afford 46 upon oxidation are presented later in this chapter.

1/2 Figure 3.2: Plot of peak currents (ip) versus square root of scan rates (ν ) for 39.

Also noted in the CV traces in Figure 3.1 are small cathodic peaks at -0.25 V and -0.5

V that may be identified belonging to the oxidized degradation products produced from the III/III* couple. Figure 3.3 presents a CV of 39 that was switched before the third couple. The disappearance of signals at -0.25 V and -0.5 V reveals that the third oxidation was accompanied by some complex chemistry (or decomposition).

Figure 3.3: CV of 39 showing the disappearance of cathodic peaks at -0.25 and -0.5 V when the scan is cut-off before the third oxidation process; scan rate = 1000 mV/s.

From the above observations, a mechanism for the first oxidation process of 39 can be proposed (Scheme 3.3). Upon oxidation, indole 39 loses an electron to form the indole radical cation species 39+. Species 39+ is highly energetic and very unstable species with appreciable radical character at the C-7 position. Once formed, the 39+ radical cations rapidly dimerise and spontaneously deprotonate to form the dimer 46.

The CVs of 46, presented below, show that it will be spontaneously oxidized at the potential for its formation from 39 to afford the cation 46+.

OMe OMe OMe - 2e 2 2 (b) MeO N (a) MeO N H H MeO N H H 37

(c)

OMe OMe OMe

+/- e - 2H+ MeO N MeO N MeO N H H H H H H H H OMe OMe N (e) N (d) N OMe

OMe OMe OMe 46•+ 46

Scheme 3.3: A proposed mechanism for the dimerization of 39 to produce 46 that takes place at oxidation peak in the CV of 39. The steps are:(a) 1e-oxidation of 39 to form a radical cation species; (b) electron delocalization and the resonance structures indicating the positions of highest reactivity; (c) rapid dimerization; (d) formation of the dimer by elimination of two protons; (e) the spontaneous reversible oxidation of dimer.

Conventional cyclic voltammetry interrogates reactions within ~ 0.1 ms time scales.

The rate of dimerization of 39+ must be more rapid than this (otherwise a cathodic peak corresponding to reduction of 39+ would be observed). The first oxidation couple for dimer 46 is 25 mV lower than that for monomer 39 so there should be appreciable oxidation of 46 as it forms from 39. Therefore, the first oxidation process of 39 should be an 1.5-electron process, which is consistent with the peak current observed in the CVs for 39. In short, upon oxidation of 39 at wave I, dimer 46 is rapidly formed by radical coupling and is spontaneously oxidized to the radical cation

46•+. Thus, all other waves in the CV of 39 corresponded to waves in the CV of 46;

47 i.e. the II/II* and III/III* couples and the anodic wave IV* are identical to those in the

CV of the genuine dimer 46 (see next).

Since the experimental evidence for the oxidation of parent indole 39 was highly suggestive of the formation of the 7,7‘-dimer 46, this 7,7‘-bis-indole was synthesized and its electrochemical behaviour was studied to confirm the dimerization of 39•+.

Figure 3.4 shows CVs of dimer 46.

OMe

MeO N H

H N OMe

OMe

Figure 3.4: Cyclic voltammograms of 46. Conditions: same as for 39 in Figure 3.1

The CVs of the dimer show three redox couples. The first and second couples are chemically reversible, Nernstian processes (ΔE(I/I*) = 60 mV, ΔE(II/II*) = 70 mV), but the third process was both electrochemically quasi-reversible (ΔE(III/III*) = 130

c a mV) and had poor chemical reversibility (ip /ip = 0.34 at a scan rate of 100 mV/s).

Traversing the third process led to daughter peaks at -0.45 V and -1.70 V in the return cathodic scan. These peaks disappeared if the scan direction was switched before the

III/III* couple. It was immediately obvious that the II/II* and III/III* couples for dimer 46 were identical in both potential and in behaviour to those observed in the

CVs of monomer 39. The cathodic wave I* for 46 and IV* for 39 are one and the same as they are equally broad and appear at exactly the same potential of 0.63 V.

These observations confirmed that 39 upon its initial oxidation rapidly forms by dimerization to afford 46. Scheme 3.4 summarises the electrochemistry of dimer 46.

+ Couple I/I* 46 46 + e E1/2 = +0.66 V

+ 2+ Couple II/II* 46 46 + e E1/2 = +0.87 V

2+ + Couple III/III* 46 46 + e E1/2 = +1.19 V

46 ? (decomposition)

Scheme 3.4: A summary of the processes observed in the CVs of dimer 46.

3.3.1.2 Increasing the electron density of the indole nucleus by modifying substituents of the 4,6-dimethoxy-2,3-disubstituted indoles

Changing substituents of the indole nucleus will change the electron density of the system and may also change the electrochemistry properties. It is known that electron donating groups (EDG) may increase the stability of the cation/cationic radical oxidation products. Indole 39 is a highly substituted and electron-rich compound in itself. However, derivatives 41, 43 and 45 have EDGs at different positions and so were examined to further see the effect EDGs have on the electrochemical behaviour.

In case of indoles 41 and 45, the pyrollic ring was modified by the addition of methoxy groups to the para-position of 2- and 3-phenyl groups and by changing these phenyl groups to methyl groups, respectively. An extra methoxy donor substituent was added to the indole phenyl ring to afford indole 43. Figure 3.9 shows CVs for indoles 41, 43 and 45.

OMe

4139 OMe OMe MeO N H

OMe Me

4543 Me MeO N H

41 OMe 43 MeO

MeO N H

Figure 3.5: Cyclic voltammograms of indoles with modified substituents.

Conditions: same as 37 (Fig. 3.6); scan rate = 100 mV/s.

Noticeably, there are no remarkable changes in the CVs of the indole derivatives 41 and 45 in comparison those of the parent indole 39. The peak potentials of the anodic waves, as expected, have decreased by 10 mV and 20 mV for 41 and 45, respectively, due to the increase in the electron density of the indole nucleus. The overall of oxidation behaviour for both the compounds is very similar to that of the parent 39.

However, the change in the CV of 43 is quite notable. The first anodic peak potential has increased by 170 mV compared to that for parent indole 39. This anodic shift in potential is in opposition to the methoxy group being an EDG. Also of note is that the

50 first oxidation couple, ‘I/I*’, for indole 43 has become almost a completely reversible,

Nernstian process. This result suggests that the radical cation formed from the first oxidation must be sufficiently stable to prevent its dimerization within the CV timescale. A further study of indole 43 is definitely warranted, as the radical appeared stable on the CV timescale (the chemically reversible CV couple I/I*). However, this result was obtained at the end of this study, and due to time constraints further investigations could not be completed.

3.3.1.3 C-7 Substituted indole derivatives N-Benzyl-4,6-dimethoxy- 2,3-diphenyl-1H-indole-7-carboxamide

Another way of stabilizing the indole radical produced by one-electron oxidation of a species based on 39 is by blocking the C-7 position with a substituent to prevent dimerization. Therefore, indole derivatives with the 7-position substituted with various groups were made and examined by cyclic voltammetry. The results are presented here.

A series of derivatives of 39 with the indole C-7 position substituted by carboxylic acid, aldehyde, ketone, amide, alcohol or ester functionalities was synthesized. The 7- substituents were selected to mimic a peptide bond (the amide group), and also to have some degree of hydrogen bonding with the indole N-H in order to investigate how this might affect the oxidation of indole.

The CVs of the benzylamido-derivative 55e were typical of those seen for all of the

7-substituted indole derivatives. The CVs showed a first oxidation couple at +0.92 V

(ΔEp = 60 mV) followed by a second oxidation couple at +1.24 V (ΔEp = 80 mV) that has half the current of the first oxidation couple irrespective of scan rate. Neither couple was fully chemically reversible in that the cathodic peaks were always smaller than the anodic peaks for each process.

OMe

MeO N H HN O

Figure 3.6: Cyclic voltammograms of 55e. Conditions: same as for 39 (Fig. 3.1)

If the CVs were switched before the second oxidation (Figure 3.8), the first process

became completely chemically reversible (ip,c/ip,a= 1.0) and the daughter peak in the

return negative scan at ~ -0.46 V disappeared. This revealed that the chemical

irreversibility arises from the second oxidation, as the first oxidation is a fully

chemically reversible, Nernstian process.

Figure 3.7: CV of 55e when the scan is cut-off before the second oxidation process showing the first process is fully chemically reversible. Conditions: same as for 39 (Fig. 3.1)

Given the chemical reversibility for the first oxidation, the second process must arise from a second oxidation of the cation and not from oxidation of a product formed by an intermediary reaction step that follows the initial oxidation of the parent indole.

This suggests that the first oxidation is a two-electron process as it has twice the current of the second process. The conclusion of a two-electron primary oxidation process for 55e is further backed up by the spectroelectrochemical results to be presented below (Section 3.4). The characteristic bands for indole radical species were not observed. However, during electrolysis at the first oxidation potential in situ in the EPR spectrometer, an EPR signal for an intermediate radical species was observed (see below). The signal for the radical disappeared immediately when the in situ electrolysis was ceased. This result suggests that a radical species is an intermediate in the two-electron oxidation process, but is very short-lived.

Figure 3.8 shows CVs for the other derivatives of parent indole 39, which all have an additional substituent at the 7-position. The CVs are all similar to those for 55e: namely they show a two-electron oxidation process followed by another process with less current. The potential of the first oxidation increased by about 250 mV (to ~1 V) compared to parent indole 39 for the carbonyl-substituted derivatives 47, 54, 63 and

61. In contrast, the methanol-substituted indole derivative 48, showed only a 20 mV increase in oxidation potential. The higher oxidation potentials for the –C(O)X substituted derivatives is consistent with the electron-withdrawing effect of the carbonyl and carboxyl substituents. The peaks in the CV of 48 are noticeably broader than those CVs of the other of the derivatives. This is possibly because of differences in the extent of hydrogen bonding to the N-H group with the alcohol-substituent compared to with amide, ester, acid, aldehyde or ketone groups.

47 54 C C H O HO O

63 C 61 C O O O

48 C

Figure 3.8: CVs of various 7-substituted indole derivatives.

Conditions: Same as those for the CVs of 39 (Fig. 3.1).

3.3.2.3 Effect of N-methylation: 4,6-Dimethoxy-1-methyl-2,3- diphenyl-1H-indole

The cyclic voltammetry study of the N-methylated indole 64 was performed in order to find out whether N-deprotonation of indole might have affected the electrochemical pathways, leading to different products. Figure 3.8 shows the CVs of N-methyl indole

64 carried out under the same conditions as for the CVs of 39. Not surprisingly, the

CVs of 64 turned out to be very similar as the parent indole 39, confirming that deprotonation at N1 did not occur in the electrochemical oxidation pathways for 39 and 46. Thus, the electrochemistry of 64 can also be summarized by Scheme 3.3, but with all structures modified to bear a N-methyl group.

OMe

MeO N Me

Figure 3.9: Cyclic voltammogram of 64. Conditions: same as for 39 (Fig. 3.1)

3.4. UV-Vis Spectroelectrochemistry

3.4.1 Chemical oxidation of 4,6-dimethoxy-2,3-diphenyl indole

Ceric ammonium nitrate (CAN) was chosen as an oxidizing agent as it is a strong one-electron oxidizing-agent. The parent indole 39 was reacted with one or two molar equivalents of CAN while keeping the other conditions same. This was done to investigate whether the indole oxidation was a one or two electron reaction. The reactions were all performed in an inert atmosphere of nitrogen to avoid oxygen interference and the solvents used were dried to minimize other unwanted side- reactions due to water.

When 39 was reacted with one equivalent of CAN in MeCN, after one hour of stirring at room temperature, all the starting material was converted to form one single product which was characterized to be the 7-7’-dimer 46 of DPI. However, when excess CAN was used, further reactions consumed 46; perhaps oligomers and small polymers resulting from the oxidation of the 7,7'-dimer 46 formed but this was not definitively established.

3.4.2 UV-vis studies of 4,6-Dimethoxy-2,3-diphenylindole derivatives

Figure 3.11 shows the in situ UV-vis spectroelectrochemistry for the oxidation of 39 at an applied electrolysis potential of +0.80 V ( vs Fc/Fc+), which is higher than the primary anodic peak (I) at +0.74 V (vs Fc/Fc+). The UV-vis spectra showed three sharp isosbestic points at 250, 272 and 340 nm indicating a clean transformation of 39 with UV-vis bands at 245 and 321 nm to a product that has UV-Vis bands at 258 and

328 nm. There are no bands below 400 nm. Indole radical cations and neutral indole radicals typically show low-energy bands at ~ 450 and ~ 650 nm [17]. The absence of these characteristic peaks for a radical species indicates that such a species does not build in to any appreciable concentration during the electrolysis.

Figure 3.10: UV-vis spectroelectrochemistry for oxidation of 39. Conditions: 0.2 mM of 39 in acetonitrile with 0.1 M [n-Bu4N][PF6] at 298 K; applied potential = +0.80 V

(vs. Fc+-Fc). The in-set shows the broadening of the spectra suggestive for the slow loss of the primary electrolysis product upon extended electrolysis.

The UV-vis spectroelectrochemistry of the bis(indole)-dimer 46 was also recorded using the same electrolysis conditions as just described for parent indole 39. The spectra are presented in Fig. 3.11. Isosbestic points are observed at 255, 273 and 345 nm with no peaks appearing at energies below 450 nm. The similarity of the spectra for dimer 46 and the primary oxidation of 39 confirms that indole 39 dimerized to 46 upon oxidation.

Figure 3.10: UV-vis spectroelectrochemistry for oxidation of 46. Conditions:

Conc. 0.2 mM of 46 in acetonitrile with 0.1 M [n-Bu4N][PF6] at 298 K under nitrogen; applied potential = +0.80 V (vs. Fc+-Fc).

Figure 3.12 presents the UV-vis spectroelectrochemistry for oxidation of indole 55e, which is substituted at C-7 by a benzylamido group. A clean transformation of indole

55e with UV-vis bands at 248, 316 and 333 nm to a single, as yet unidentified, product with bands at ~ 230, 262 and 332 nm was observed. Extended electrolysis lead to broadening of the peaks for the oxidation product was consistent with it undergoing decomposition. Despite the C-7 substitution of indole 55e, peaks consistent with an indolyl radical were not observed.

Figure 3.12: UV-Vis spectroelectrochemistry for oxidation of 55e.

Conditions: 0.2 mM of 55e in acetonitrile with 0.1 M [n-Bu4N][PF6] at 298 K under dinitrogen; applied potential = +0.80 V (vs. Fc+-Fc).

Figure 3.13 shows an EPR spectrum acquired during in situ oxidation of 7- benzylamido-derivative 55e within an EPR spectroelectrochemical cell sited in the microwave cavity of an EPR spectrometer. The spectrum shows a featureless isotropic signal for an organic radical centred at g = 2.0052. Despite adjusting the spectrometer acquisition parameters to optimize the spectrum, hyperfine couplings could not be observed. The radical was very short lived. It was only observed during electrolysis, and when the electrolysis was ceased and an EPR spectrum immediately taken, the EPR signal did not appear. The observation of the rapid loss of the radical is consistent with it rapidly decomposing, perhaps according to the proton-dependent disproportionation: 2 55e+ 55e + (55e – H+)+ + H+. A rapid proton-dependent disproportion such as this would account for the observation of a two-electron primary oxidation process in the electrochemistry (see above).

Figure 3.13: X-band EPR spectrum of the radical formed during oxidation of 7- benzylamide DPI. Instrument settings: frequency 9.767 GHz, power 0.630 mW, modulation amplitude 5.00 G, time constant 20.480 ms, and temperature 295 K.

Additionally, the UV-vis spectroelectrochemistry of another C-7-substituted indole, namely 63 with a benzoyl substituent, was recorded. The spectra are shown in Fig.

3.14. The primary oxidation product has bands at 266 and 329 nm. The isosbestic points at 256 and 288 nm suggest the oxidation is a clean 1: 1 conversion. The spectra obtained are noticeably similar to those obtained during oxidation of indole 55e and differ from those obtained upon oxidation of the parent indole 39. This is consistent with the substitution at the indole C-7 position preventing dimerization of the intermediate radical cation. The identities of the ultimate two-electron oxidation products of 55e and 63 remain unknown.

Figure 3.14: UV-Vis spectroelectrochemistry for oxidation of 63.

Conditions: Conc. 0.2 mM of 62 in acetonitrile with 0.1 M [n-Bu4N][PF6] at 298 K, under Nitrogen flow; applied potential = at +0.80 V (vs. Fc+-Fc).

3.5 Conclusion

The redox chemistry of a series of highly substituted indoles has been studied by a combination of cyclic voltammetry and by in situ UV-vis and EPR spectroelectrochemistry experiments, coupled with some studies of chemical oxidation. The peak potential data for the primary oxidation processes of the indoles has been obtained and compared. The parent indole 39 rapidly and irreversibly dimerises upon its one-electron oxidation. The 7,7’-dimer 46 is the product. In contrast, indoles already substituted at the 7-position show a primary two-electron oxidation couple in their CVs. The EPR spectroelectrochemistry for oxidation 55e suggests short-lived radicals as intermediates in these oxidation processes. However, overall, no stable indolyl radical products were observed. 61

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15. Enache, T.A. and A.M. Oliveira-Brett, Pathways of Electrochemical Oxidation of Indolic Compounds. Electroanalysis, 2011. 23(6): p. 1337-1344.

16. Kissinger, P.T. and W.R. Heineman, Cyclic voltammetry. Journal of Chemical Education, 1983. 60(9): p. 702.

17. Crespo, A., A.G. Turjanski, and D.o.A. Estrin, Electronic spectra of indolyl radicals: a time-dependent DFT study. Chemical Physics Letters, 2002. 365(1–2): p. 15-21.

CHAPTER 4: Fluorometric Studies of Novel Indole Ligands for Detection of Biologically Important Metal Ions

4.1 Introduction

A biosensor is an analytical device that is capable of detecting a specific target using a biological recognition element (biochemical receptor) that is directly attached onto a physical or physicochemical transducer. Examples of biochemical receptors are enzymes, antibodies, membrane receptors, nucleic acids, cell receptors, microorganisms, organelles and DNA fragments, all of which are used to recognize and interact specifically with desired target. The response resulting from the interaction of the receptor with a biological element, which the transducer senses, allows translation of the biological interaction into a signal that can be more easily measured and quantified. Electrochemistry, fluorescence, interferometry, electromagnetic resonance and reflectometry are techniques that are most frequently used to measure transduction[1-4].

In comparison to many other conventional analytical measurement schemes, biosensors offer many advantages in terms of simplicity, lower limits of detection, higher sensitivity, measurement of non-polar molecules (that do not respond to most measurement devices), and allowing rapid continuous control of the system. During the past two decades there has been a remarkable growth in the use of fluorescence in the biochemical sciences. Just a few years ago, fluorescence spectroscopy and time- resolved fluorescence were considered as primary research tools in biochemistry and biophysics. Fluorescence is now more widely used in environmental monitoring, clinical chemistry, DNA sequencing, biological imaging and genetic analysis, to name just a few areas of application[5-8]. Additionally, fluorescence has been used for cell

64 identification and sorting in flow-cytometry, and in cellular imaging to reveal the localization and movement of intracellular substances by means of fluorescence microscopy. Due to the high sensitivity of fluorescence detection, and the expense and difficulties of handling radioactive substances, there is a continuing development of medical tests based on the phenomenon of fluorescence[9-14].

4.2 Designing metal-responsive fluorophores for cellular use

Effective fluorescent probes for imaging metal ions in living cellular systems must meet several strict requirements[15-17]. A sensor should essentially be selective for a specific metal ion over other biologically abundant cations, including those that exist at much higher cellular concentrations (Na+, K+, Mg2+, Ca2+, Fe2+, Cu2+ and Zn2+ ions). The principles of coordination chemistry, including preferred donor numbers and ligand field geometries, are critical for designing and obtaining metal-selective responses. A turn-on emission increase or a shift in excitation/emission profiles is preferred over a turn-off emission-quenching response to maximize spatial resolution in light microscopy. Furthermore, high optical brightness values is preferred in order to lower the amount of dye needed for cellular applications, which minimizes the potential for altering endogenous cellular distributions of metal ions. Dyes that have visible-light excitation and emission are desirable in order to minimize sample damage and reduce auto-fluorescence. Finally, probes must also be compatible with biological systems: they must be water soluble for examination of extracellular, intracellular or subcellular regions, and they must be nontoxic. Addressing the challenge of meeting both chemical and biological constraints is critical for developing useful tools for cellular applications.

4.3 Results and discussion

In this chapter, results from preliminary experiments to interrogate the interaction of some biologically important metal ions with the novel indole ligands are described and discussed. Tryptophan 2 is a fluorescent amino acid, and it had been noted for some time that 4,6-dimethoxy-2,3-diphenylindole (DPI, 39) was highly fluorescent.

Therefore, it was anticipateded that a series of fluorescent chemosensors could be made based on the parent 2,3-diphenylindole core as the fluorophore (the signal transducing centre) with selected amino acid ligands as the metal ion-binding group (a biomimetic ionophore). Therefore, the fluorescence properties of the parent indole 39, its dipicolinylamine derivative 69 and some new amino acid-substituted derivatives

(72a-g) were studied in the absence and presence of some biologically relevant metal ions. The syntheses and characterizations of the compounds are described in

Chapter 2.

In the fluorescence experiments described below, a fixed concentration for the fluorophore of 74 μM was used, as it is the minimum concentration at which all fluorophores gave reasonable and reproducible fluorescence emission spectra. The fixed concentration of fluorophore also facilitates comparisons in fluorescent intensity between compounds. The combination of THF/water (3:7 v/v) at room temperature

(298 K) was used as the solvent system. This is due to the fact that the compounds were not soluble in water alone and 30% THF was added as co-solvent to ensure complete dissolution. Metal nitrate salts were used as the metal ion sources, because the nitrate counter ion dissociates from the metal ions in aqueous solution and should not influence the fluorescence properties.

4.3.1 Relative fluorescent intensities

Figure 4.1 presents fluorescence spectra which show how fluorescent parent diphenylindole (DPI, 39) and its various derivatives are in relation to the standard dye fluorescein. These fluorescence emission spectra were carried out without metal ions in order to compare the intrinsic fluorescence emission intensity of each compound relative to fluorescein. The excitation wavelengths were employed to give the maximum fluorescence intensity (Table 4.1). While fluorescein showed a strong fluorescence emission band at 524 nm, under the identical standard conditions employed, the emission peaks for all of the DPI derivatives were observed at 400-425 nm, which distinguished the DPI fluorophores from fluoroscein. The numerical data obtained from the spectra in Figure 4.1 are listed in Table 4.1.

Figure 4.1: Fluorescence spectra of parent indole 39 and the new ligands based on it along with that of fluorescein. All dyes were 74 μM, and the solvent was 3:7 v/v THF/H O at 298 K. 2

As had been noted visually, the parent indole was highly fluorescent exhibiting a fluorescence emission band at 423 nm with about 60% intensity relative to the standard dye fluorescein under identical conditions. Noticeably, the derivatives of the parent indole 39 are less fluorescent: the dipicrylamine derivative 69 showed only 61% of the fluorescence intensity of 39 whereas the maximum fluorescence intensity of the amino-acid-substituted derivatives 72a-g was 9–19% of that of 39.

Table 4.1: Excitation wavelength, emission wavelength and relative fluorescent intensity data for fluorescein, parent indole 39 and each ligand 72a–g.

Compound Excitation Emission Relative wavelength (nm) wavelength (nm) fluorescent intensity (%) Fluorescein 450 524 167

DPI (39) 330 423 100

DPA-DPI (69) 325 423 61

Gly-DPI(72a) 327 405; 552 19; 9

Leu-DPI(72b) 320 421 18

Lys-DPI(72c) 325 408; 541 10; 5

Asp-DPI(72d) 322 403; 560 11; 6

Phe-Ala-DPI (72e) 330 406; 557 14; 11

Met-DPI(72f) 327 415; 554 16; 5

Gly-Gly-DPI (72g) 340 406; 549 14; 5

For the amino acid-substituted indole derivatives 72a-g, the low emission intensity observed could be, in part, related to dimer formation and emission from an excited state – ground state dimeric complex; i.e. due to excimer formation and emission.

Figure 4.2 presents expansions of the emission spectra of the amino acid-indole conjugates (shown in Figure 4.1). The emission spectrum of Leu-DPI 72b is similar to

68 that of parent DPI 39 and that of the DPI-DPA derivative 69 in that it showed a single fluorescence band at ca. 423 nm, albeit with a considerably weaker fluorescence intensity. All other amino acid-DPI conjugates showed a primary fluorescence emission band that was hypsochromically shifted by ~20 nm to ca. 405 nm and a broad secondary fluorescence emission band at ca. 550 nm.

Figure 4.2: Fluorescence spectra of the indole-amino acid conjugates. Conditions are exactly those listed in the caption to Figure 4.1.

The hypsochromic (blue) shift of the primary fluorescence emission band and the appearance of the broad secondary fluorescence band at lower energy provides two strong and independent pieces of evidence for excimer formation. A hypsochromic shift and weak emission is characteristic for formation of an “H-aggregate” (H is short for hypsochromic) of the dye molecules. This occurs when the dyes form face-to-face

π-stacked dimers and is common for porphyrin or merocyanine dyes[18-20]. In

69 comparison, J-type dye aggregates form when the dye molecules align head-to-tail and they typically show intense fluorescence bands with a bathochromic (red) shift.

Figure 4.3 shows a depiction of the energy states upon formation of H- and J-type dye aggregates. According to exciton theory, the dye molecule is regarded as a point of dipole and the excitonic state of the dye aggregate splits into two levels through the interaction of transition dipoles. A transition to the upper state in parallel aggregates having parallel transition moments (H-aggregates) and to a lower state in a head–to– tail arrangement with perpendicular transition moments (J-aggregates) leads to hypsochromic (blue) and bathochromic (red) shifts, respectively.

Figure 4.3:Diagramatic representation of the relationship between chromophore arrangement and spectral shift based on the molecular exciton theory.According to exciton theory, the slip-angle (α) is <54.7° for J-aggregates and >>54.7° for H-aggregates.

The broad lower energy band arises from emission from the lowest energy S1 excited state of the excimer. Although strictly optically forbidden for H-aggregates, incorporation of vibrational coupling leads to allowed transitions and, hence, a broad weak fluorescence band is typically observed at much lower energy. This is the

70 observed secondary fluorescence emission band at ca. 550 nm of the diphenylindole- amino acid conjugates 72a and 72c–72g. That excimer formation was observed for the DPI-amino acid aggregates 72a and 72c–72g, but not for the parent DPI 39 or the

DPI-dpa derivative 69 or the leucine-DPI conjugate 72b, implies that the amide linkage and the amino acid/peptide group control aggregation. Presumably, excimer emission does not occur for the Leu-DPI conjugate 72b because the bulky isobutyl

(CH2CH(CH3)2) sidechain of leucine prevents face-to-face aggregate formation.

4.3.2 DPA ligands

Many coordination complexes of di-2-picolylamine (DPA) and its substituted derivatives have been prepared and structurally characterized[21-26]. The DPA group is known to possess a remarkable binding selectivity for some transition metal ions, especially Zn2+, which makes it an ideal ionophore for incorporation into chemo- and biosensors for biological applications. Some examples of the DPA-based fluorophores are given below (Figure 4.4)[21-26].

Anthracene-substituted fluorescent sensor 85 was designed to function as an off-on- off switch for protons, and as an off-on fluorescent switch for post-transition metal ions[21]. The turn-on fluorescent sensor 86 was designed as a metal ion-responsive fluorophore for cellular use[26, 27]. Comprising a dichlorofluorescein reporter functionalized with two DPA receptors, this probe was reported to exhibit a three-fold fluorescence increase with near-unity quantum efficiency (Φ = 0.87) upon Zn2+

[26, 27] binding with nanomolar affinity (Kd = 0.7 nM) . The stilbene-based DPA fluoroionophore 87 was designed to detect copper ion in both reduced (Cu+) and oxidized environment (Cu2+). The chemosensor is a D-π-A (Donor-π-Acceptor) system and displayed an “on-off” mode of fluorescence sensing of copper ions[22].

N N N N N N N N N

HO O O

Cl Cl COOH 85

86 N NC N

Figure 4.4: Examples of DPA-based fluoroionophores

4.3.3 Ion selectivity in fluoroionophore DPI-DPA (69)

Selectivity is the most important characteristic of fluoroionophores as it determines the utility of the fluoroionophores in real sample measurement. The ability of DPI-

DPA 69 (74 μM) to selectively detect the metal ions such as Na+, K+, Cr3+, Fe2+, Co2+,

Cu2+, Ni2+, Zn2+, Cd2+ and Pb2+ by simple fluorescence measurements was carried out in 3:7 v/v THF/H2O. Excess metal ion (5.0 equivalents) compared to compound 69 was used in this survey. The fluorescence spectrum of 69 was measured in the absence and in the presence of metal ions, see Figure 4.5.

OMe OMe

MeO N M2+ MeO N H H

N N N M2+ N

N N

Scheme 4.1: Schematic representation of a metal ion binding with fluoroionophore 69 {DPI (bright green) is the fluorophore and DPA (blue) is the ionophore}

In the fluorescence spectra of 69 alone, maximum excitation and emission wavelengths were recorded at 325 and 423 nm, respectively. An excess of the metal ions, other than the Cu2+ ion, did not perturb the fluorescence of the DPI-DPA fluoroionophore 69 (Figure 4.5). Zn2+ ion is among those that did not influence the fluorescence of 69.

Figure 4.5: Overlaid fluorescence emission spectra of DPA-DPI 69 (74

μM) at 298 K in 3:7 v/v THF/H2O without (blue trace) and with 370 μM (5.0 equivalents) of the metal ions Na+, K+, Cr3+, Fe2+, Co2+, Ni2+, Zn2+, Cd2+ and Pb2+ (green traces).

Interestingly the change in the fluorescence of 69 was observed as the concentration of the copper ions is increased (Figure 4.6). After addition of 0.2 equivalents (10.0

μM) of Cu2+ ion, the fluorescence emission intensity of 69 was quenched by ~15%.

The drop in the fluorescence emission intensity for 69 decreased exponentially with increasing Cu2+ concentration (see the inset to Fig. 4.6). It is likely that Cu2+ binds 69 in a 1:1 ratio, but a full study to determine the binding ratio and affinity could not be done due to time constraints.

Figure 4.6: Fluorescence emission spectra of DPI-DPA 69 (Conc. = 74

μM, 298 K, THF/H2O-3:7, v/v) in the presence of increasing concentrations of Cu2+. The concentration of Cu2+ for curves top to

bottom are 0.0, 5.0, 10.0, 15.0, 20.0, 25.0, ….., 75.0 μM. Inset: Plot of relative fluorescence intensity of 69 with increasing Cu2+ concentration

at λem =425 nm

4.3.4 Fluorescence studies of amino acid indole ligands

Interactions of amino acids with metal ions are a fundamental process in biological systems. Peptides have attracted much attention because they can specifically bind to many different metal ions[28-30]. For example, electrochemical and spectroscopic investigations on the interactions between short peptides and metal ions have been reported for the detection of ultra trace Cu2+ ion[28-30]. These investigations showed that peptidyl motifs like Gly-His and Gly-Gly-His strongly bind to Cu2+ ion.

However, the interactions between the metal ions and the peptides are not always clear. Moreover, prediction of the interactions of peptides with metal ions, including their metal ion affinities and selectivity, is difficult. Thus, an experimental/empirical approach is often used to find fluorophore-amino acid ionophore conjugates that selectively detect a metal ion.

In this section, results are described from fluorometric experiments that were carried out with the DPI-amino acid ligands 72a–g to establish how selected, biologically important, metal ions affected their fluorescence emission spectra.

4.3.4.1 Amino acid-indole ligands with no fluorescent selectivity of the metal ions

Figure 4.7 presents typical fluorescence emission spectra of the amino-acid-indole conjugates 72c–e, which were found to have no selectivity towards metal ions. The experiments were conducted under the same conditions as those used in the study of the DPI-DPA fluoroionophore 69. The fluorescence spectra of the 72c–e were run without and with three equivalents of the metal ions Na+, K+, Cr3+, Fe2+, Co2+, Cu2+,

Ni2+, Zn2+, Cd2+ and Pb2+. Unfortunately, no change in fluorescence bands or their intensity was observed when the metal ions were added. This indicated that these

DPI-amino acid conjugates were unperturbed by metal ions and, presumably, did not selectively bind to any of the metal ions surveyed either alone as the fluoroionophore monomer or as an aggregate.

(a) (b)

(e)

Figure 4.7: Fluorescence emission spectra of the diphenylindole-amino acid conjugates: (a) Glycyl-DPI (72a), (b) Phenylalanine-DPI (72e), (c) Aspartic acid-

DPI (72d), (d) Lysine-DPI (72c), (e) Methionine-DPI (72f) measured alone and in + + 3+ 2+ 2+ 2+ 2+ 2+ 2+ 2+ the presence of Na , K , Cr , Fe , Co , Cu , Ni , Zn , Cd and Pb (3.0 equiv.). Other experimental conditions are the same as those given in the caption for Fig. 4.5. 76

4.3.4.2 Amino acid-diphenylindole ligands that exhibit metal ion- selectivity

Figure 4.7 shows the combined fluorescence spectra of the leucine-diphenylindole fluorophore 72b obtained with and without the metals ions Na+, K+, Cr3+, Co2+, Cu2+,

Ni2+, Zn2+, Cd2+ and Pb2+ (under the same experimental conditions as used to obtain spectra of 69 and 72a). Fluorophore 72b was excited at 320 nm. The fluorescence of

72b is unaffected by these metal ions.

Figure 4.8: Fluorescence emission spectra of Leu-DPI 72b with Na+, K+, Cr3+, Co2+, Cu2+, Ni2+, Zn2+, Cd2+ and Pb2+ions. Experimental conditions: same as for 69.

Excitation wavelength: λex = 320 nm.

Figure 4.9 shows the fluorescence spectra of Leu-DPI 72b with increasing concentrations of Fe2+ ion. The result obtained was similar to that of fluoroionophore

69 with Cu2+ ions. As the concentration of Fe2+ ions was increased, the fluorescence

77 emission intensity of 72b decreased exponentially (Figure 4.9 inset). Again, time constraints prevented a detailed study of Fe2+ ion with 72b being undertaken.

Figure 4.9: Fluorescence emission spectra of Leu-DPI 72b (Conc. = 74 μM, 298 K,

THF/H2O-3:7, v/v, slit width = 5 nm) in the presence of increasing concentration of Fe2+. The concentration of Fe2+ for curves top to bottom are 0.0, 5.0, 10.0, 15.0, 20.0, 25.0, ….., 75.0 μM. Inset: relative fluorescence intensity of 72b with increasing 2+ Fe concentration at λem = 421 nm

Another amino-acid derivative of diphenylindole that showed Fe2+ selectivity is peptide Gly-Gly-DPI conjugate 72g. Gly-Gly-DPI exhibited primary and secondary emission peaks at 406 and 549 nm, respectively, which are characteristic for H- aggregate formation, as discussed above. The fluorescence emission from the H- aggregate of 72g was unaffected by excess (3 equiv.) of the metal ions Na+, K+, Cr3+,

Co2+, Cu2+, Ni2+, Zn2+, Cd2+ and Pb2+ (Figure: 4.10). However, increasing the Fe2+ ion led to a decrease in the fluorescence emission intensity (Figure 4.11). The behavior

78 was very similar to that found for DPI-DPA 69 and Leu-DPA 72g, except that in this case it is the emission from the H-aggregate (of 72b) that is quenched. Figure 4.11- inset shows that the quenching of the fluorescence emission from the Gly-Gly-DPI H- aggregate scales exponentially with the concentration of Fe2+ ion.

Figure 4.10: Fluorescence emission spectra of Gly-Gly-DPI 72g with Na+, K+, Cr3+, Co2+, Cu2+, Ni2+, Zn2+,Cd2+andPb2+ ions. Experimental conditions: same as

for 69. Excitation wavelength: λex = 340 nm.

Figure 4.11: Fluorescence emission spectra of Gly-Gly-DPI. 72g (Conc. = 74

μM, 298 K, THF/H2O-3:7, v/v, slit width = 5 nm) in the presence of increasing concentration of Fe2+. The concentration of Fe2+ for curves top to bottom are 0.0, 5.0, 10.0, 15.0, 20.0, 25.0, ….., 45.0 μM. Inset: relative fluorescence intensity of 2+ 72g with increasing Fe concentration at λem =406 nm

The in-sets in Figures 4.6, 4.9 and 4.11 have in common that the fluorescence intensity of the ligands decreases exponentially until they reach a minimum at one equivalent of metal ion added [Cu2+ ion for DPA-DPI ligand 69, Fe2+ ions for Leu-

DPI ligand 72b, and Gly-Gly-DPI ligand 72g], which likely indicates a 1:1 binding ratio. However, due to time constraint, further experiments to confirm binding stoichiometry and to ascertain binding affinities of the metal ion quencher to these fluorophores could not be performed.

4.4 Conclusion

The Cu2+-sensitive fluoroionophore 69 was synthesized by modifying the parent

80 diphenylindole (DPI) with a di-2-picolylamine (DPA) ligand group. Significant fluorescence emission quenching was observed with Cu2+ions indicating a high sensitivity and specificity of the DPI-DPA chemosensor for Cu2+ions. The presence of metal ions such as Na+, K+, Cr3+, Fe2+, Co2+, Cu2+, Ni2+, Zn2+, Cd2+ and Pb2+ ions had little influence on the intensity of the fluorescence emission of 69.

The fluorescence spectra of the amino acid-diphenylindole conjugates provided evidence for face-to-face H-aggregation of the indole fluorophores mediated by the amide linkage and amino acid(s). The fluorescence emission experiments revealed that the fluorescence emission bands of Leu-DPI 72b and Gly-Gly-DPI 72g were selectively quenched by Fe2+ ion. The fluorescence emission intensity of both 72b and

72g decreased exponentially with increasing Fe2+ concentration up to 1 equivalent of

Fe2+ added. Addition of Na+, K+, Cr3+, Co2+, Cu2+, Ni2+, Zn2+, Cd2+ and Pb2+ ions did not affect on the fluorescence emission bands of 72b and 72g, even when these ions were added in excess.

4.5 References

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2. Borisov, S.M. and O.S. Wolfbeis, Optical Biosensors. Chemical Reviews, 2008. 108(2): p. 423-461.

3. Wang, J., Electrochemical Glucose Biosensors. Chemical Reviews, 2007. 108(2): p. 814-825.

4. Kim, D.-K., et al., Label-Free DNA Biosensor Based on Localized Surface Plasmon Resonance Coupled with Interferometry. Analytical Chemistry, 2007. 79(5): p. 1855-1864.

5. Berezin, M.Y. and S. Achilefu, Fluorescence Lifetime Measurements and Biological Imaging. Chemical Reviews, 2010. 110(5): p. 2641-2684.

6. Rogers, K.R., et al., Detection of Low Dose Radiation Induced DNA Damage Using Temperature Differential Fluorescence Assay. Analytical Chemistry, 1999. 71(19): p. 4423-4426.

7. Xu, X., et al., Fluorescence Recovery Assay for the Detection of Protein−DNA Binding. Analytical Chemistry, 2008. 80(14): p. 5616-5621.

8. Leu, W., Ph.D. Thesis, UNSW. 2008.

9. Strianese, M., et al., Fluorescence-based biosensors. Methods Mol Biol, 2012. 875: p. 193-216.

10. Qi, X., et al., New BODIPY derivatives as OFF-ON fluorescent chemosensor and fluorescent chemodosimeter for Cu2+: cooperative selectivity enhancement toward Cu2+. J Org Chem, 2006. 71(7): p. 2881-4.

11. Nolan, E.M. and S.J. Lippard, A "turn-on" fluorescent sensor for the selective detection of mercuric ion in aqueous media. J Am Chem Soc, 2003. 125(47): p. 14270-1.

12. Yoon, S., et al., Screening mercury levels in fish with a selective fluorescent chemosensor. J Am Chem Soc, 2005. 127(46): p. 16030-1.

13. Hirano, T., et al., Improvement and biological applications of fluorescent probes for zinc, ZnAFs. J Am Chem Soc, 2002. 124(23): p. 6555-62.

14. Wu, Y., et al., Boron dipyrromethene fluorophore based fluorescence sensor for the selective imaging of Zn(II) in living cells. Org Biomol Chem, 2005. 3(8): p. 1387-92.

15. Domaille, D.W., E.L. Que, and C.J. Chang, Synthetic fluorescent sensors for studying the cell biology of metals. Nat Chem Biol, 2008. 4(3): p. 168-175.

16. Que, E.L., D.W. Domaille, and C.J. Chang, Metals in Neurobiology: Probing Their Chemistry and Biology with Molecular Imaging. Chemical Reviews, 2008. 108(5): p. 1517-1549.

17. Zhang, J., et al., Creating new fluorescent probes for cell biology. Nat Rev Mol Cell Biol, 2002. 3(12): p. 906-918.

18. Nandy, R., et al., Intramolecular π-Stacking Interaction in a Rigid Molecular Hinge Substituted with 1-(Pyrenylethynyl) Units‡. The Journal of Organic Chemistry, 2007. 72(3): p. 938-944.

19. Kaiser, T.E., V. Stepanenko, and F. Wu rthner, Fluorescent J-Aggregates of Core-Substituted Perylene Bisimides: Studies on Structure−Property Relationship, Nucleation−Elongation Mechanism, and Sergeants-and-Soldiers Principle. Journal of the American Chemical Society, 2009. 131(19): p. 6719- 6732.

20. Consiglio, G., et al., Supramolecular Aggregation/Deaggregation in Amphiphilic Dipolar Schiff-Base Zinc(II) Complexes. Inorganic Chemistry, 2010. 49(11): p. 5134-5142.

21. de Silva, S.A., et al., A fluorescent photoinduced electron transfer sensor for cations with an off-on-off proton switch. Tetrahedron Letters, 1997. 38(13): p. 2237-2240.

22. Zhu, M.-Q., et al., A stilbene-based fluoroionophore for copper ion sensing in both reduced and oxidized environments. Talanta, 2010. 81(1–2): p. 678-683.

24. Benoist, E., et al., A Click procedure with heterogeneous copper to tether technetium-99m chelating agents and rhenium complexes. Evaluation of the chelating properties and biodistribution of the new radiolabelled glucose conjugates. Carbohydrate Research, 2011. 346(1): p. 26-34.

25. Shimazaki, Y., et al., Metal complexes involving indole rings: Structures and effects of metal–indole interactions. Coordination Chemistry Reviews, 2009. 253(3–4): p. 479-492.

26. Walkup, G.K., et al., A New Cell-Permeable Fluorescent Probe for Zn2+. Journal of the American Chemical Society, 2000. 122(23): p. 5644-5645.

27. Burdette, S.C., et al., Fluorescent sensors for Zn(II) based on a fluorescein platform: synthesis, properties and intracellular distribution. J. Am. Chem. Soc., 2001. 123: p. 7831-7841.

28. Yang, W.R., Jaramillo D, Gooding J J, Hibbert D B, Zhang R, Willett G D, Fisher K J, Chem. Commun.,, 2001: p. 1982-1983.

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CHAPTER 5: Experimental

5.1 General synthetic procedures

All commercially available reagents were purchased from Fluka, Aldrich, Acros

Organics, Alfa Aesar and Lancaster and were used without further purification. All reactions requiring anhydrous conditions were performed under an argon atmosphere.

Molecular sieve 4A (pore size of 4Å) were used for the in situ drying of solvents wherever water was formed as a by-product of a reaction.

Commonly used solvents such as methanol (MeOH), ethanol (EtOH), ethyl acetate, ether, acetone, chloroform, toluene, dichloromethane (DCM), n-hexane (light petroleum) were obtained from commercial sources. Anhydrous solvents such as acetonitrile (MeCN), MeOH, CHCl3, and tetrahydrofuran (THF) were obtained using a PureSolv MD Solvent Purification System.

Pressure column chromatography refers to the use of compressed air at the top of the column to force the solvent down through the column; suction column chromatography refers to the use of suction at the base of the column, via a water aspirator, to draw the solvent down through the column; gravity column chromatography relies on the earth’s gravitational force to draw the solvent down through the column. Pressure column chromatography was carried out using Merck

230-400 mesh ASTM silica gel. Suction column chromatography was carried out using Merck 60H silica gel. Gravity column chromatography was carried out using

Merck 70-230 mesh ASTM silica gel. Preparative thin layer chromatography was carried out on 3 × 200 × 200 mm glass plates coated with Merck 60GF254silica gel.

Reactions were monitored using thin layer chromatography, performed on Merck DC aluminium foil coated with silica gel GF254. Compounds were detected by short and

84 long wavelength ultraviolet light, charring with vanillin or permanganate solutions and with iodine vapour.

Reactions were monitored using thin-layer chromatography performed on Merck DC aluminium foil coated with silica gel GF254. Compounds were detected by short and long wavelength ultraviolet light and with iodine vapour.

5.2 Physical measurements

1H and 13C NMR spectroscopy

1H and 13C NMR spectra were obtained in the designated solvents on a Bruker

Avance DPX300 (300 MHz) or a Bruker DMX600 (600 MHz) spectrometer at the designated frequency and were internally referenced to the solvent peaks. 1H NMR spectral data are reported as follows: chemical shift measured in parts per million

(ppm) downfield from TMS (δ); multiplicity observed; coupling constant (J) in Hertz

(Hz); proton count; assignment. Multiplicities are reported as singlet (s), broad singlet

(br s), doublet (d), triplet (t), quartet (q), quintet (p), multiplet (m), doublet of doublets

(dd), doublet of doublet of doublets (ddd), broad (br), buried, and combinations of these. 13C NMR chemical shifts are reported in ppm downfield from TMS (δ), and identifiable carbons are given. Acid-free deuterated solvents (CDCl3, d-DMSO, D2O) were obtained from a commercial source.

IR spectroscopy

Infrared spectra were recorded with a Thermo Nicolet 370 FTIR spectrometer using

KBr disks. Absorptions are described as: s, strong; m, medium; w, weak; brs, broad strong; brm, broad medium.

UV-visible spectroscopy

Ultraviolet and visible spectra were recorded using a Varian Cary 100 Scan

Spectrometer in the designated solvents and data reported as wavelength (λ) in nm and adsorption coefficient (ε) in cm-1M-1.

High resolution mass spectrometry

High resolution mass spectra were recorded on either a Bruker FT-ICR MS (EI) or a

Micromass ZQ2000 (ESI) mass spectrometer at the School of Chemistry, UNSW. The high resolution mass values are reported to 4 decimal places.

Cyclic voltammetry

Electrochemical measurements were recorded using a Pine Instrument Co. AFCBP1

Bipotentiostat interfaced to and controlled by a PentiumTM computer. Data were transferred to a Macintosh computer and processed using IgorProTM 6.22A software.

A standard three-electrode configuration was used for cyclic voltammetric experiments. The reference electrode comprised a commercial Ag/AgCl electrode

(Cyprus Systems, Inc. EE008) filled with AgCl saturated 3M KCl (aq) solution, the working electrode consisted of a glassy carbon (0.5 mm diameter) commercial mini- electrode (Cyprus Systems, Inc. EE041) and a Pt wire was used as the auxiliary electrode. For a smooth and uniform surface area, the working electrode was pre- treated by grinding with SiC emery paper (600 mesh), then successively polishing with 6 and 1 μm diamond slurries, and finally with 0.2 μm alumina slurry. The electrodes were rinsed with deionized water, acetone and then the solvent to be used and thoroughly dried.

High quality grade anhydrous acetonitrile sealed under dinitrogen was used as a solvent for electrochemical measurement. The supporting electrolyte was tetra-n-

86 butylammonium hexafluorophosphate (0.1 M). An electrochemical scan of the solvent-electrolyte (blank) system was always recorded prior to the addition of the compound to ensure high reproducibility and avoid spurious signals. All potentials are quoted relative to the ferrocenium/ferrocene (Fc/Fc+) couple, which was measured in situ as an internal reference.

UV-Vis spectroelectrochemistry

The in situ ultraviolet-visible (UV-Vis) spectroelectrochemical study on the indole derivatives was performed. This required the use of a modified quartz cuvette of 1 mm width and 1.7 mL sample size. Pt gauze was used as the working electrode, the reference electrode was Ag/AgCl and the auxiliary electrode was a single Pt wire. A three electrode system was used and a Cary 500 spectrometer measured the change in absorbance as a voltage was applied using a Model Princeton Applied Research 173

Potentiostat/Galvanostat at a value selected from the CV of the compound of interest.

Anhydrous acetonitrile stored in SuresealTM bottles under nitrogen from Aldrich

Chemical Co was used as a solvent for electrochemical measurement. The supporting electrode was 0.1 M tetra-n-butylammonium hexafluorophosphate. The working electrode and cell were cleansed by washing with dilute nitric acid solution, rinsing with water, then with acetone, and oven-dried. A scan of the baseline was run before each experiment.

Electronic paramagnetic resonance (EPR)spectroscopy

EPR spectra for paramagnetic species were recorded on a Bruker EMX10 EPR spectrometer interfaced to a PentiumTM computer and controlled by Bruker WinEPR

Acquisition (version 2.2) software. The spectra in this thesis were reproduced using

IgorProTM 6.22A software.

Fluorescence spectroscopy

Fluorometric measurements were carried out on a Perkin Elmer Luminescence

Spectrometer LS 50B (slit width, 5mm; 450W Xe lamp). A sealed quartz cuvette(1 x

1 x 5 cm) was used.

5.3 Experimental details

4,6-Dimethoxy-2,3-diphenylindole (39)

Finely ground 3,5-dimethoxyaniline (8.0g, 52 mmol) and OMe benzoin (11.0 g, 52 mmol) were combined and heated for

MeO N 2 h at 140 °C. The resulting brown oil was then dissolved H in glacial acetic acid (100 mL). Unsubstituted aniline (3 mL) was added and the resulting mixture was refluxed for 4 h. Upon cooling, a white precipitate appeared in the dark brown solution. The precipitate was filtered, washed with water followed by methanol to yield the title compound as a fine white powder (12 g, 70%). M.p. 242–

[1] 1 243 °C (from methanol), Lit. 240–241 °C (from CHCl3/n-hexane 60–80°). H NMR

(300 MHz, CDCl3): δ 3.68 (s, 3H, OMe), 3.86 (s, 3H, OMe), 6.22 (d, J = 1.92 Hz,

1H, H5), 6.53 (d, J = 1.92 Hz, 1H, H7), 7.19–7.35 (m, 10H, Ph), 8.10 (br s, 1H, NH).

4,6-Dimethoxy-2,3-bis(4-methoxyphenyl)-1H-indole (41)

A stream of hydrochloric acid gas was passed OMe through a suspension of 3,5-dimethoxyaniline 11 OMe (9.85 g, 64.3 mmol) in anhydrous diethyl ether (80 OMe mL) for 0.5 h. The resulting slurry was filtered and MeO N H washed with ether (100 mL) to give 3,5-dimethoxyanilinium hydrochloride as a

88 yellow solid (11.50 g, 94%). The 3,5-dimethoxyanilinium hydrochloride salt was then added to a mixture of 3,5-dimethoxyaniline 11 (2.14 g, 14.0 mmol) and anisoin 40

(1.90 g, 7.01 mmol) in silicone oil (10 mL) and the mixture was heated at 130–140 °C for 4 h. After cooling, the mixture was washed with light petroleum (2 × 20 mL), extracted with DCM (3 × 15 mL) and concentrated under vacuum to yield the title

1 compound as a yellow solid (4.30 g, 79 %). H NMR (300 MHz, CDCl3): δ 3.70 (s,

3H, OMe), 3.79 (s, 3H, OMe), 3.86 (s, 3H, OMe), 3.95 (s, 3H, OMe), 6.21 (d, J =1.94

Hz, 1H, H5), 6.50 (d, J = 1.94 Hz, 1H, H7), 7.28 (m, 8H, Ph), 8.03 (br s, 1H, NH). IR

(KBr): νmax 3322 s, 3026 m, 3004 m, 2946 w, 2837 w, 1731 m, 1685 m, 1634 m, 1584 s, 1541 s, 1459 w, 1388 m, 1360 m, 1343 m, 1284 w, 1202 s, 1151 m, 1129 m, 1069 w, 1048 w, 997 w, 986 w, 942 w, 904 m, 804 s, 776 m, 763 w, 704 m cm–1.

4,5,6-Trimethoxy-2,3-diphenyl-1H-indole (43)

A mixture of 3,4,5-trimethoxyaniline 42 (1 g, 5.46 mmol)

OMe and benzoin 38 (1.4 g, 6.6 mmol) was heated with stirring MeO at 140 °C for 2 h. On cooling, aniline (0.8 mL, 8.78 MeO N H mmol) and acetic acid (10 mL) were added and the mixture was heated under reflux for 5 h. Methanol (20 mL) was then added to the cooled mixture and the resulting precipitate was filtered, washed with methanol and n-hexane to yield the title

[2] 1 compound 43 as a grey solid (0.98 g, 50%) . H NMR (300 MHz, CDCl3): δ 3.44 (s,

3H, OMe), 3.88 (s, 3H, OMe), 3.93 (s, 3H, OMe), 6.72 (s, 1H, H7), 7.28 (m, 10H,

Ph), 8.11 (br s, 1H, NH). IR (KBr): νmax 3363 br m, 3005 w, 2936 w, 2834 w, 1624 s,

1601 s, 1496 m, 1466 s, 1426 s, 1286 s, 1265 m, 1232 m, 1141 m, 1106 s, 1045 w,

768 w, 694 w cm–1.

4,6-Dimethoxy-2,3-dimethyl-1H-indole (45)

A mixture of 3,5-dimethoxyaniline (5.00 g, 33.0 mmol), 3- OMe Me chlorobutanone (3.50 g, 33.0 mmol), sodium hydrogen MeO N Me carbonate (5.50 g, 65.0 mmol) and lithium bromide (1.75 g, H

44.0 mmol) was suspended in absolute ethanol (50 mL) and the mixture was heated under reflux for 3 h. The mixture was cooled to room temperature and the resulting solid was filtered, washed with water to yield the title compound 4 as a white solid

(3.81 g, 56 %). M.p. 115–116 °C (from ethanol), Lit.[3] 124–125 °C; 1H NMR (300

MHz, CDCl3): δ 2.25 (s, 3H, Me), 2.33 (s, 3H, Me), 3.80 (s, 3H, OMe), 3.85 (s, 3H,

OMe), 6.15 (bs, 1H, H5), 6.34 (bs, 1H, H7), 7.51 (br s, 1H, NH). IR (KBr): νmax 3400 s, 3382 s, 2958 w, 2921 w, 1844 w, 1630 s, 1596 s, 1568 s, 1513 m, 1461 m, 1338 m,

1306 m, 1248 m, 1214 m, 1199 m, 1147 s, 1125 s, 811 m, 795 m cm–1.

4,4',6,6'-Tetramethoxy-2,2',3,3'-tetraphenyl-1H,1'H-7,7'-biindole (46)

2,3-Diphenylindole 39 (0.5 g, 1.52 mmol) was dissolved in tetrahydrofuran (40 mL) containing OMe concentrated HCl (4 mL) and 1,4-benzoquinone MeO N H H (0.16 g). After 3 h of vigorous stirring, water was N OMe added to the solution and the product was OMe extracted with dichloromethane (3 x 30 mL). The combined dichloromethane extract was dried over anhydrous sodium sulfate and evaporated to dryness. The crude product was recrystallized from dichloromethane/n- hexane (1:1) as a white solid compound (0.5 g, 100%). M.p. 291–294 °C. 1H NMR

(300 MHz, CDCl3): δ 3.80 (s, 6H, OMe), 3.84 (s, 6H, OMe), 6.49 (s, 2H, H5), 7.28

(m, 20H, Ph), 8.00 (br s, 2H, NH). IR (KBr): νmax 3461 w, 3445 w, 3046 w, 3018 w,

2948 w, 2831 w, 2999 w, 1598 s, 1500 m, 1462 m, 1424 m, 1329 s, 1256 m, 1201 m,

1168 m, 1138 m, 993 m, 760 w, 697 m cm–1.

4,6-Dimethoxy-2,3-diphenyl-1H-indole-7-carbaldehyde (47)

Indole 37 (1.99 g, 6.05 mmol) was added to an ice-cooled mixture of phosphorus oxychloride (0.7 mL, 7.65 mmol) OMe and DMF (20 mL). The reaction mixture was allowed to MeO N H warm to room temperature and stirred for further 2.5 h. H O The reaction was quenched with water (100 mL) and made strongly basic with aqueous sodium hydroxide solution (1 M, 10 mL). The resulting precipitate was filtered off and recrystallized from DCM/light petroleum to give the title compound

47 as a yellow solid (1.98 g, 92%). M.p. 180–182 °C. Lit.[5] 183-184 °C; 1H NMR

(300 MHz, CDCl3): δ 3.79 (s, 3H, OMe), 3.97 (s, 3H, OMe), 6.13 (s, 1H, H5), 7.22–

7.35 (s, 10H, Ph), 10.38 (s, 1H, CHO), 10.57 (br s, 1H, NH). IR (KBr): νmax 3299 w,

1628 s, 1597 s, 1548 s, 1450 m, 1399 m, 1372 w, 1367 m, 1250 s, 1222 s, 1128 m,

991 m, 764 w,750 m, 696 s cm–1.

(4,6-Dimethoxy-2,3-diphenyl-1H-indol-7-yl)methanol (48)

To a suspension of 7-formyl indole 47 (0.30 g, 0.8 mmol) in absolute ethanol (20 mL) was added sodium OMe borohydride (0.20 g, 2.6 mmol) and the mixture was MeO N H refluxed for 3 h. After cooling to room temperature, the OH solvent was evaporated, and aqueous NaOH (0.1 M, 30 mL) was added to the residue.

The resulting white precipitate was filtered off, washed with water and dried under 91 reduced pressure to give the alcohol 48 as a white solid (0.27 g, 94%). 1H NMR (300

MHz, CDCl3): δ 3.12 (br s, 1H, OH), 3.89 (s, 3H, OMe), 3.99 (s, 3H, OMe), 4.76 (s,

13 2H, CH2), 6.37 (s, 1H, H5), 7.20–7.41 (m, 10H, 2Ph), 11.36 (br s, 1H, NH); C NMR

(75 MHz, CDCl3): δ 54.17 (CH2-OH), 55.43 (OMe), 56.67 (OMe), 88.36 (C-5),

103.11 (C-7), 127. 05 (Ph-C), 127.55 (Ph-C), 127.87 (Ph-C), 128.21 (Ph-C), 128.30

(Ph-C), 128.97 (Ph-C), 131.76 (C), 134.54 (C-2), 136.05 (C-3), 137.40 (C), 145.81

(C-4), 151.74 (C-6).IR (KBr): νmax3357 s br, 3274 s, 1598 s, 1514 s, 1537 m, 1410 w,

1400 m, 1302 w, 1330 m, 1215 s, 1207 s, 1115 w, 940 m, 650 w, 752 m, 693 s cm–1.

+ HRMS (+ESI): Found m/z 359.1450, [M] ; C23H21NO3 required 359.1521.

2,2,2-Trichloro-1-(4,6-dimethoxy-2,3-diphenyl-1H-indol-7-yl)ethanone (53)

Trichloroacetyl chloride (2 mL, 17.9 mmol) was added dropwise to a solution of 4,6-dimethoxy-2,3- OMe diphenylindole (1.00 g, 3 mmol) in chloroform (20 mL). MeO N H The mixture was heated at reflux under a nitrogen Cl3C O atmosphere overnight. Upon cooling, the mixture was quenched with 50 mL of water.

The organic layer was extracted with DCM (2 × 20 mL) and the solvent was evaporated under reduced pressure to yield a dark orange solid. The crude product was column filtered to obtain the title compound as a bright yellow solid (0.9 g,

[6] 1 75%) . H NMR (300 MHz, CDCl3): δ 3.82 (s, 3H, OMe), 3.99 (s, 3H, OMe), 6.19

13 (s, 1H, H5), 7.28 (m, 10H, Ph), 10.42 (br, s, 1H, NH); C NMR (75 MHz, CDCl3):

55.50 (OMe), 55.62 (OMe), 87.77 (C-5), 98.36 (CCl3), 98.67 (C-7), 126.38 (Ph-C),

127.42 (Ph-C), 127.54 (Ph-C), 127.91 (Ph-C), 128.54 (Ph-C), 131.31 (Ph-C), 132.08

(C-2), 133.00 (C-3), 135.33 (C), 139.03 (C), 160.31 (C-6), 181.15 (C=O).

4,6-Dimethoxy-2,3-diphenyl-1H-indole-7-carboxylic acid (54)

A mixture of 7-trichloroacetylindole 53 (100 mg, 0.21 mmol) and NaOH (50 mg, 1.25 mmol) in ethanol/water OMe

(3:1; 25 mL) was heated under reflux for 1 h. After MeO N cooling, the mixture was acidified with concentrated HCl H HO O to afford the title compound 52 as a white solid (64 mg, 82%)[4]. M.p. 190–192 °C; 1H

NMR (300 MHz, CDCl3): δ 3.80 (s, 3H, OMe), 4.14 (s, 3H, OMe), 6.25 (s, 1H, H5),

7.28 (m, 10 H, Ph), 10.64 (s, 1H, -COOH), 10.89 (br s, 1H, NH); 13C NMR (75 MHz,

CDCl3): 56.02 (OMe), 56.74 (OMe), 87.24 (C-5), 95.17 (C-7), 127.27 (Ph-C), 127.85

(Ph-C), 128.22 (Ph-C), 131.82 (Ph-C), 134.21 (C-2), 136.00 (C-3), 139.51 (C), 152.10

(C-4), 161.92 (C-6), 171.06 (C=O). IR (KBr): νmax 3387 m, 3325 m, 3240 m, 1701 s,

1596 s, 1388 w, 1347 m, 1246 m, 1220 m, 1176 m, 1142 w, 985 w, 751 m, 700 w cm–

4,6-Dimethoxy-2,3-diphenyl-1H-indole-7-carboxamide (55a)

To a solution of 7-trichloroacetylindole 53 (30 mg,

0.063 mmol) in MeCN (5 mL), aqueous concentrated OMe ammonia solution (32% w/w, 0.5 mL, 12 mmol) was MeO N added drop wise. The mixture was stirred at room H H2N O temperature for 30 min. After completion of the reaction as monitored on TLC, water was added and the mixture was stirred for another 30 min. The resulting white precipitate was filtered off, washed with water and dried to afford the title compound

1 55a (20 mg, 85%). M.p. 199–202 °C; H NMR (300 MHz, CDCl3): δ 3.79 (s, 3H,

OMe), 4.07 (s, 3H, OMe), 6.24 (s, 1H, H5), 6.48 (br s, 1H, NH2), 7.20–7.35 (m, 10H,

13 Ph), 8.11 (br s, 1H, NH2), 10.88 (s, 1H, NH); C NMR (75 MHz, CDCl3): 56.37

(OMe), 56.80 (OMe), 87.03 (C-5), 96.54 (C-7), 127.34 (Ph-C), 127.66 (Ph-C), 128.09 93

(Ph-C), 128.95 (Ph-C), 131.21 (Ph-C), 132.91 (C), 134.88 (C-2), 136.00 (C-3), 136.79

(C), 150.34 (C-4), 156.03 (C-6), 168.99 (C=O). IR (KBr): νmax 3456 s, 3409 s, 2876 m, 2804 w, 1735 s, 1704 s, 1601 m, 1535 w, 1500 m, 1422 w, 1288 s, 1230 m, 1128 m, 1073 w, 996 m, 902 w, 762 w cm–1.

4,6-Dimethoxy-N-methyl-2,3-diphenyl-1H-indole-7-carboxamide (55b)

To a solution of 7-trichloroacetyl indole 53 (100 mg,

0.21 mmol) in MeCN (10 mL), aqueous methylamine OMe (40% w/w, 1 mL, 17 mmol) was added slowly. The

MeO N mixture was stirred for 2 h and a white precipitate was H

H3CHN O obtained. Water (20 mL) was added to the mixture and the precipitate was filtered off to give the title compound as a white solid (65 mg,

1 80%). M.p. 186-187 °C; H NMR (300 MHz, CDCl3): δ 3.04 (d, J = 4.34 Hz, 3H,

NHCH3), 3.76 (s, 3H, OMe), 4.04 (s, 3H, OMe), 6.22 (s, 1H, H5), 7.28 (m, 10H, Ph),

13 8.05 (d, J = 4.48 Hz, 1H, NHCH3), 11.36 (s, 1H, NH); C NMR (75 MHz, CDCl3): δ

26.13 (CH3), 55.30 (OMe), 56.87 (OMe), 87.61 (C-5), 97.51 (C-7), 113.61 (C),

113.84 (C), 125.99 (Ph-C), 127.01 (Ph-C), 127.39 (Ph-C), 127.94 (Ph-C), 128.32 (Ph-

C), 131.51 (C-2), 136.34 (C-3), 158.29 (C), 168.38 (C=O). IR (KBr): νmax 3428 s,

3335 s, 3018 w, 2942 w, 1638 s, 1594 s, 1541 m, 1507 m, 1466 w, 1357 w, 1259 s,

1230 s, 1218 w, 1147 m, 1110 s, 985 m, 796 w, 771 m, 698 m cm–1.

4,6-Dimethoxy-N,N-dimethyl-2,3-diphenyl-1H-indole-7-carboxamide (55c)

To a solution of 7-trichloroacetyl indole 53 (100 mg,

0.21 mmol) in MeCN (10 mL), aqueous OMe dimethylamine (60% w/w, 1 mL, 12 mmol) was added MeO N H slowly. The mixture was stirred for 5 h with gentle (H3C)2N O heating (45 °C). After completion of the reaction, water was added and the resulting solid was filtered off to obtain the title compound as a yellowish-white solid (50 mg,

1 59%). M.p. 192-193°C; H NMR (300 MHz, CDCl3): δ 3.01 (s, 6H, (-NCH3)2), 3.76

(s, 3H, OMe), 3.84 (s, 3H, OMe), 6.50 (s, 1H, H5), 7.15–7.37 (m, 10H, 2Ph), 10.56

13 (br s, 1H, NH); C NMR (75 MHz, CDCl3): δ 34.59 (CH3N), 55.76 (OMe), 56.20

(OMe), 84.38 (C-5), 96.31 (C-7), 126.23 (Ph-C), 128.35 (Ph-C), 129.44 (Ph-C),

132.32 (Ph-C), 133.45 (C-2), 134.81 (C-3), 134.62 (C), 136.00 (C), 156.49 (C-4),

157.94 (C-6), 169.53 (C=O). IR (KBr): νmax 3411 s, 3329 s, 3125 w, 2962 w, 1648 s,

1562 s, 1539s, 1507 m, 1426 w, 1357 w, 1259 s, 1251 s, 1208 w, 1137 m, 1110 s, 985 m, 747 w, 702 m, 653s cm–1.

N,N-Diethyl-4,6-dimethoxy-2,3-diphenyl-1H-indole-7-carboxamide (55d)

Diethylamine (1 mL, mmol) was added dropwise to a solution of 7-trichloroacetyl indole 53 (100 mg, 0.21 OMe mmol) in MeCN (10 mL). The mixture was refluxed

MeO N for 4 h. After the completion of the reaction, distilled H

(C2H5)2N O water (30 mL) was added and the resulting white solid was filtered off to obtain the title compound 55d (47 mg, 55%). M.p. 185-187 °C; 1H

NMR (300 MHz, CDCl3): δ 1.32 (t, 6H, CH2CH3) 3.26 (s, 3H, OMe), 3.57 (s, 3H,

OMe), 4.01 (s, 4H, CH2CH3), 6.59 (s, 1H, H5), 7.19–7.37 (m, 10H, 2Ph), 11.51 (s,

13 1H, NH); C NMR (75 MHz, CDCl3): δ 12.06 (CH3), 42.21 (CH2), 55.34 (OMe),

57.88 (OMe), 88.22 (C-5), 97.86 (C-7), 127.72 (Ph-C), 128.02 (Ph-C), 128.56 (Ph-C),

128.80 (Ph-C), 129.23 (Ph-C), 134.86 (C-2), 136.03 (C-3), 136.89 (C), 168.90 (C=O).

IR (KBr): νmax 3462 s, 3306 s, 3279 m, 2937 m, 1745 s, 1609 s, 1558 m, 1584 w,

1516 m, 1350 s, 1275 m, 1210 w, 1118 m, 1017 w, 974 m, 852 s, 744 m, 705 w,

640 m cm–1.

N-Benzyl-4,6-dimethoxy-2,3-diphenyl-1H-indole-7-carboxamide (55e)

Benzylamine (0.5 mL, 4.6 mmol) was added to a solution of 7-trichloroacetylindole 53 (200 mg, 0.4 OMe mmol) in MeCN (15 mL). The mixture was stirred at

MeO N room temperature for 1 h and the resulting yellow H HN O solid was filtered to obtain the title compound 60 (140 mg, 75%). M.p. 201-203 °C; 1H NMR (300 MHz,

CDCl3): δ 3.77 (s, 3H, OMe), 4.00 (s, 3H, OMe), 4.73 (d, J = 5.68, 2H, PhCH2NH),

6.23 (s, 1H, H5), 7.28 (m, 15H, Ph), 8.46 (t, J = 5.64, 1H, C(O)NH), 11.34 (s, 1H,

13 NH); C NMR (75 MHz, CDCl3): 43.17 (CH2), 55.34 (OMe), 56.98 (OMe), 87.74

(C-5), 95.79 (C-7), 126.00 (Ph-C), 127.05 (Ph-C), 127.18 (Ph-C), 127.28 (Ph-C),

127.40 (Ph-C), 128.02 (Ph-C), 128.33 (Ph-C), 128.70 (Ph-C), 131.14 (Ph-C), 132.97

(C-3), 134.88 (C-2), 138.61 (C), 139.14 (C), 156.63 (C-4), 157. 60 (C-6), 167.68

(C=O). IR (KBr): νmax 3407 s, 3370 s, 3046 w, 3005 w, 2932 w, 2837 w, 1630 s, 1537 s, 1508 s, 1460 m, 1356 m, 1262 s, 1232 s, 1189 s, 1114 s, 990 s, 794 s, 955 s, 699 m,

637 m, 605 m, 574 m, 485 m cm–1.

1-(4,6-Dimethoxy-2,3-diphenyl-1H-indol-7-yl)ethanone (56)

A solution of indole 53 (0.2 g, 0.6 mmol) in MeCN OMe (15 mL) was added to an ice-cold solution of acetic anhydride (1 mL) in MeCN (10 mL). The mixture MeO N H Me O was stirred for 1 h. The precipitate formed was filtered and washed with water followed by aqueous NaHCO3 solution to obtain the title compound as a yellow solid (0.17 g, 76%). M.p. 167-169 °C; 1H NMR (300

MHz, CDCl3): δ 2.72 (s, 3H, CH3C=O), 3.80 (s, 3H, OMe), 4.02 (s, 3H, OMe), 6.20

(s, 1H, H5), 7.25–7.36 (m, 10H, 2Ph), 11.15 (br s, 1H, NH). 13C NMR (75 MHz,

CDCl3): δ 31.54 (CH3), 56.23 (OMe), 56.79 (OMe), 87.06 (C-5), 95.98 (C-7), 127.07

(Ph-C), 127.68 (Ph-C), 127.95 (Ph-C), 128.64 (Ph-C), 132.08 (C), 134.83 (C-2),

146.07 (C-3), 138.00 (C), 152.32 (C-4), 156.22 (C-6), 186.98 (C=O). IR (KBr):

νmax3401 s, 3318 s, 2950 m, 2835 w, 1735 s, 1634 s, 1688 w, 1541 m, 1445 w, 1366 s, 1218 m, 1175 m, 1117 w, 1083 s, 1033 w, 982 m, 800 m, 736 w, 648 m cm-1.

Methyl 4,6-dimethoxy-2,3-diphenyl-1H-indole-7-carboxylate (60)

Method A: OMe A solution of dimethyl sulfate (0.02 g, 0.15 mmol) in acetone (1 mL) was added dropwise with stirring to a MeO N H MeO O solution of the indole-7-carboxylic acid 55 (56 mg, 0.15 mmol) in acetone (10 mL) containing anhydrous K2CO3 (20.7 mg, 0.15 mmol). The mixture was refluxed for 3 h and the mixture was allowed to cool to room temperature. Water (40 mL) was added to the mixture and the resulting yellow solid was filtered, washed with water and dried to yield the title compound (45 mg, 80%).

1 M.p. 212-215 °C; H NMR (300 MHz, CDCl3): δ 3.78 (s, 3H, OMe), 3.99 (s, 3H,

OMe), 4.00 (s, 3H, OMe), 6.24 (s, 1H, H5), 7.28 (m, 10H, Ph), 10.37 (br s, 1H, NH);

13 C NMR (75 MHz, CDCl3): 51.75 (OMe), 55.28 (OMe), 57.32 (OMe), 88.92 (C-7),

126.14 (Ph-C), 127.17 (Ph-C), 127.43 (Ph-C), 127.91 (Ph-C), 128.47 (C), 132.54 (C),

132.84 (C-2), 135.70 (C-3), 160.06 (C), 168.02 (C=O). IR (KBr): νmax 3403 s, 3053 w, 2996 w, 2936 w, 1658 s, 1600 s, 1467 m, 1431 m, 1373 m, 1265 s, 1264 s, 1238 s,

1188 m, 1146 m, 1102 m, 986 m, 798 m, 748 m, 700 w cm–1.

Method B:

7-Trichloroacetylindole 53 (0.66 g, 1.40 mmol) was partially dissolved in anhydrous methanol (40 mL) containing triethylamine (1 mL). The mixture was heated under reflux for 2 h and allowed to cool to room temperature. The resulting yellow precipitate was filtered off, washed with methanol and dried to afford the title compound (0.39 g, 71%).

Benzyl 4,6-dimethoxy-2,3-diphenyl-1H-indole-7-carboxylate (61)

A mixture of 7-trichloroacetylindole 53 (0.66 g, 1.40

OMe mmol), benzyl alcohol (1 mL) and anhydrous K2CO3

(3.0 g) was refluxed for 2 h. The mixture was then MeO N H extracted with diethyl ether (3 x 15 mL) and the organic O O layer was washed with water and dried over anhydrous sodium sulfate and concentrated to a small volume.

Light petroleum was added to the organic layer until a white solid formed. The precipitate was filtered and dried to afford the title compound (0.41 g, 64%). M.p.

1 203-205 °C; H NMR (300 MHz, CDCl3): δ 3.77 (s, 3H, OMe), 4.00 (s, 3H, OMe),

13 5.45 (s, 2H, CH2), 6.24 (s, 1H, H5), 7.28 (m, 10H, Ph), 10.15 (br s, 1H, NH); C

NMR (75 MHz, CDCl3): 53.26 (OMe), 54.15 (OMe), 65.22 (CH2), 87.12 (C-5), 92.35

(C-7), 127.03 (Ph-C), 128.50 (Ph-C), 128.65 (Ph-C), 128.97 (Ph-C), 129.04 (Ph-C),

131.41 (Ph-C), 132. 07 (C-2), 134.75 (Ph-C), 134.99 (C-3), 139.03 (C), 168. 66

(C=O). IR (KBr): νmax 3465 s, 3321 m, 2901 w, 2853 w, 2745 w, 1728 s, 1653 s, 1435 m, 1403 s, 1321 m, 1203 m, 1146 s, 1102 s, 1022 s, 984 m, 946 m, 829 m, 705 m, 698

–1 + m, 642w, 613m cm . HRMS (+ESI): Found m/z 463.1739, [M] ; C30H25NO4 required

463.1784.

1-(4,6-Dimethoxy-2,3-diphenyl-1H-indol-7-yl)-2-phenylethanone (63)

A solution of tin(IV) chloride (1.71 g, 6.6 mmol) in anhydrous toluene (10 mL) was added dropwise to a OMe solution of indole 39 (1.18 g 3.6 mmol) and MeO N H phenylacetyl chloride (0.91 g, 4.9 mmol) in anhydrous O toluene (25 mL). The mixture was stirred at room temperature for 30 h and quenched with ice water (50 mL) followed by 10% aqueous sodium hydroxide. The resulting mixture was extracted with dichloromethane (3 × 20 mL) and the combined organic extract was washed with water (3 × 15 mL), dried over anhydrous sodium sulfate and concentrated under reduced pressure. The crude product was purified by column chromatography (dichloromethane/light petroleum, 3:7) to yield the title compound

62 as a yellow powder (0.5 g, 31%). M.p. 143–144 °C; Lit.[3]; 145-146 °C; 1H NMR

(300 MHz, CDCl3): δ 3.80 (s, 3H, OMe), 4.02 (s, 3H, OMe), 4.45 (s, 2H, CH2), 6.21

13 (s, 1H, H5), 7.28 (m, 15H, Ph), 11.92 (s, 1H, NH); C NMR (75 MHz, CDCl3):

δ51.27 (CH2), 54.20 (OMe), 55.37 (OMe), 87.13 (C-5), 104.12 (C-7), 113.14 (C),

125.98 (Ph-C), 126.31 (Ph-C), 126.96 (Ph-C), 127.30 (Ph-C), 127.81 (Ph-C), 128.22

(Ph-C), 128.45 (Ph-C), 131.62 (Ph-C), 132.57 (C-2), 136.06 (C-3), 136.34 (C), 159.34 99

(C-4), 160.86 (C), 168.08 (C=O). IR (KBr): νmax 3380 s, 3295 s, 1625 s, 1590 m,

1543 m, 1463 w, 1376 m, 1224 s, 1200 s, 1163 m, 1074 w, 992 m, 765 m cm-1.

4,6-Dimethoxy-1-methyl-2,3-diphenyl-1H-indole (64)

Finely ground KOH (1.0 g, 17 mmol) was added to dimethyl sulfoxide (25 mL) and the resulting mixture OMe was stirred at room temperature for 10 min, followed by the addition of diphenyl indole 39 (1.0 g, 3 mmol). The MeO N CH3 mixture was stirred at room temperature for further 30 min. Methyl iodide (1 mL, 16 mmol) was added and the mixture was stirred for another hour. The reaction was quenched with ice water, and the resulting white precipitate was filtered to yield the title compound 63 as fine white powder (1.1 g,

1 98%). M.p. 152-153 °C; H NMR (300 MHz, CDCl3): δ 3.78 (s, 3H, OMe), 3.81 (s,

3H, OMe), 3. 93 (s, 3H, N-CH3), 6.44 (d, J = 1.93, 1H, H5), 6.79 (d, J = 1.93, 1H,

13 H7), 7.23–7.35 (m, 10H, 2Ph); C NMR (75 MHz, CDCl3): 32.05 (N-CH3), 55.69

(OMe), 56.23 (OMe), 86.10 (C-5), 91.27 (C-7), 127.30 (Ph-C), 127.93 (Ph-C), 128.06

(Ph-C), 129.73 (Ph-C), 131.81 (Ph-C), 135.50 (C), 135.94 (C-3), 137.39 (C-2), 139.00

(C).

4,6-Dimethoxy-1-methyl-2,3-diphenyl-1H-indole-7-carbaldehyde (65)

Indole 63 (2.00 g, 5.83 mmol) was added to a mixture of phosphorus oxychloride (0.8 mL, 8.74 mmol) in DMF OMe (20 mL). The resulting mixture was heated at 50 °C for

4 h before being quenched with water (100 mL) and MeO N CH3 H O made strongly basic with aqueous sodium hydroxide solution (1 M). The resulting precipitate was collected and recrystallized from

100

DCM/light petroleum to yield the title compound 66 as a yellow solid (1.23 g, 57%).

1 M.p. 180-183 °C; H NMR (300 MHz, CDCl3): δ 3.80 (s, 3H, OMe), 3.83 (s, 3H,

13 OMe), 3.87 (s, 3H, N-CH3), 6.96 (s, 1H, H5), 7.23–7.40 (m, 10H, 2Ph). C NMR (75

MHz, CDCl3). IR (KBr): νmax 3444 w, 2935 w, 2843 w, 1658 s, 1582 s, 1568 s, 1452 m, 1398 m, 1360 w, 1253 s, 1211 s, 1134 s, 1084 s, 1047 s, 758 m, 703 m cm–1.

2,2,2-Trichloro-1-(4,6-dimethoxy-1-methyl-2,3-diphenyl-1H-indol-7-yl)ethanone

(66)

Trichloroacetyl chloride (1 mL, 8.95 mmol) was added dropwise to a solution of indole 63 (1.00g, 2.9 mmol) OMe in chloroform (20 mL). The reaction mixture was MeO N heated at reflux under a nitrogen atmosphere Me Cl3C O overnight. Upon cooling, the mixture was quenched with 50 mL of water and the organic layer was extracted with dichloromethane (2 ×

20 mL). The combined organic layer was evaporated under reduced pressure and the crude mixture was column chromatographed to obtain the title compound as a bright

1 yellow solid (0.52 g, 35%). M.p. 159-161 °C; H NMR (300 MHz, CDCl3): δ 3.81 (s,

3H, OMe), 3.84 (s, 3H, OMe), 3.90 (s, 3H, NCH3), 6.87 (s, 1H, H5), 7.19–7.37 (m, 10

13 H, 2Ph); C NMR (75 MHz, CDCl3): 42.5 (CH3), 57.6 (OMe), 55.8 (OMe), 87.7 (C-

7), 95.0 (CCl3), 108.9 (C), 120.7 (C), 128.0 (Ph-C), 130.2 (Ph-C), 133.6 (Ph-C), 134.6

(C-2), 135.9 (C-3), 138.5 (C), 151.3 (C), 176.5 (C=O). HRMS (+ESI) Found m/z

+ 487.0486, [M] ; C25H20Cl3NO3 required 487.0509

101

4,6-Dimethoxy-N,1-dimethyl-2,3-diphenyl-1H-indole-7-carboxamide (67)

To a solution of N-metyl 7-trichloroacetyl indole 65

(100 mg, 0.20 mmol) in MeCN (10 mL), aqueous OMe methylamine (40% w/w, 1 mL, 17 mmol) was added MeO N slowly. The mixture was refluxed for 24 h followed by CH3 H3CHN O addition of water (20 mL). The crude mixture was extracted with DCM (3 x 10 mL) and the combined extract was concentrated and chromatographed to yield the title compound as a white solid (45 mg, 56%). M.p.

1 184-185 °C; H NMR (300 MHz, CDCl3): δ 3.07 (d, J = 4.95, 3H, CH3NH), 3.57 (s,

3H, OMe), 3.71 (s, 3H, OMe), 3.91 (s, 3H, NCH3), 6.08 (d, J = 4.95, 1H, NH), 6.29

(s, 1H, H5), 7.16–7.27 (m, 10H, Ph). HRMS (+ESI): Found m/z 400.1754, [M]+;

C25H24N2O3 required 400.1787

1-(4,6-Dimethoxy-2,3-diphenyl-1H-indol-7-yl)-N,N-bis(pyridin-2- ylmethyl)methanamine (69)

An anhydrous solution of sodium cyanoborohydride OMe (0.1 g, 1.59 mmol) in methanol (5 mL) was added slowly to a mixture of di-2-picolylamine (DPA) MeO N H (0.38 g, 1.9 mmol), 7-formyl-2,3-diphenylindole 47 N N (0.68 g, 1.9 mmol) and glacial acetic acid (0.22 mL, N 3.7 mmol) in anhydrous methanol (20 mL) under a nitrogen atmosphere for 5 days. The reaction was monitored by thin layer chromatography. Upon completion of the reaction, distilled water (50 mL) was added and the mixture was extracted with DCM (2 × 25 mL). The organic layer was dried over anhydrous Na2SO4 and the solvent was evaporated under reduced pressure. The remaining crude mixture was column chromatographed (MeOH:n-hexane) and

102 recrystallized to yield the title compound 68 as a yellow solid (0.6 g, 58%). M.p. 204-

1 207 °C; H NMR (300 MHz, CDCl3): δ3.58 (s, 3H, OMe), 3.76 (s, 3H, OMe), 3.81 (s,

4H, 2 x CH2), 3.88 (s, 2H, CH2), 6.14 (s, 1H, H-5), 6.92–6.96 (m, 2H, Py-H), 7.15–

7.45 (m, 14H, 2Ph + Py-H), 8.15–8.17 (m, 2H, Py-H), 12.43 (br s, 1H, NH); 13C

NMR (75 MHz, CDCl3): δ 48.84 (CH2), 55.46 (OMe), 57.64 (OMe), 59.62 (2CH2),

89.56 (C5), 98.21 (C-7), 113.70 (PyC), 122.05 (Py-C), 125.60 (Ph-C), 126.55 (Ph-C),

127.26 (C), 128.21 (C-2), 128.47 (C-3), 131.66 (C), 133.31 (Py-C), 136.58 (Py-C),

137.90 (C-4), 154.33 (C-6), 154.56 (Py-C).IR (KBr): νmax 3160 m, 3126 m, 3065 m,

3031 m, 3002 m, 2932 w, 2837 w, 2803 m, 2356 w, 1509 s, 1461 m, 1432 m, 1351 s,

1324 s, 1215 m, 1201 s, 1164 s, 1140 s, 1107 s, 1094 s, 1000 m, 789 m, 762 w, 695 w

–1 + cm . HRMS (+ESI): Found m/z 540.2517, [M] ; C35H32N4O2 required 540.2525.

General procedure for the synthesis for amino acid methyl ester hydrochlorides

Chlorotrimethylsilane (25 μL, 0.2 mmol) was added slowly to a solution of the amino acid (0.1 mmol) in methanol (10 mL), and the resulting mixture was stirred at room temperature for 1 h. After the reaction was complete, the solvent was removed on a rotary evaporator to yield the desired amino acid methyl ester hydrochloride salt[7].

(S)-Methyl 2,6-diaminohexanoate (71a) [Lysine methyl ester]

1H NMR (300 MHz, DMSO): δ1.42–1.49 (m, 2 H, O H N Me 2 O CH2), 1.59 (p, J = 7.52 Hz, 2 H, CH2), 1.84 (q, J = NH2

6.75 Hz, 2 H, CH2), 2.74 (br s, 2H, CH2), 3.43 (br s, 1 H, CH), 3.75 (s, 3 H, OMe),

13 3.96 (t, J = 6.43 Hz, 1 H, CH), 8.26 (br s, 2 H, NH2), 8.78 (br s, 2 H, NH2); C NMR

(75 MHz, CDCl3): δ 23.2 (CH2), 27.8 (CH2), 32.5 (CH2), 40.8 (CH2), 53.2 (CH), 55.9

(OMe), 164.7 (C=O).

103

(S)-Dimethyl 2-aminosuccinate (71b) [Aspartic acid methyl ester]

1 H NMR (300 MHz, CDCl3): δ2.68 (br s, 2H, CH2), 3.09

(s, 3H, OMe), 3.18 (s, 3H, OMe), 3.83 (p, J = 6.35 Hz, O O Me 13 Me O 1H, CH), 7.71 (br s, 2H, NH2); C NMR (75 MHz, O NH2

CDCl3): δ 33.6 (CH2), 47.2 (CH), 50.6 (OMe), 150.9 (C=O), 157.3 (C=O).

(R)-Methyl 2-amino-3-phenylpropanoate (71c) [Phenylalanine methyl ester]

1 H NMR (300 MHz, CDCl3): δ 3.53 (q, J = 7.54 Hz, 1H, NH2 O CH2), 3.69 (q, J = 6.89 Hz, 1H, CH2), 3.72 (s, 3H, OMe), Me O 4.34 (t, J = 5.92 Hz, 1H, CH), 7.19–7.32 (m, 5H, Ph, 8.34

13 (br s, 2H, NH2); C NMR (75 MHz, CDCl3): δ35.30 (C), 53.21 (Me), 55.29 (C),

127.31 (Ph-C), 129.20 (Ph-C), 130.84 (Ph-C), 141.57 (Ph-C), 167.92 (C=O).

(S)-Methyl 2-amino-4-(methylthio)butanoate (71d) [Methionine methyl ester]

1 H NMR (300 MHz, D2O): δ1.46 (s, 3H, CH3), 1.60 (q, J = O S Me O 6.35 Hz, 2H, CH2), 2.04 (br s, 2H, CH2), 3.19 (s, 3H, OMe), NH2 13 3.66 (br s, 1H, CH), 7.72 (br s, 2H, NH2); C NMR (75 MHz, CDCl3): δ 17.9

(CH3S), 32.6 (CH2), 33.7 (CH2), 52.5 (CH), 53.7 (OMe), 152.0 (C=O).

Methyl 2-(2-aminoacetamido)acetate (71e) [Glycyl-

O glycine methyl ester] H N Me H N O 1H NMR (300 MHz, CDCl ): 1.73 (br s, 2H, NH ), 3.62 2 3 δ 2 O

104

13 (s, 3H, OMe), 4.02 (s, 2H, CH2), 4.27 (s, 2H, CH2), 9.07 (br, s, 1H, NH); C NMR

(75 MHz, CDCl3): δ37.39 (CH2), 41.97 (CH2), 54.28 (OMe), 163.25 (C=O), 168.03

(C=O).

Ethyl 2-(4,6-dimethoxy-2,3-diphenyl-1H-indole-7-carboxamido)acetate (72a) [glycine ethyl ester-Indole Ligand]

Glycine ethyl ester hydrochloride salt (100 mg, 0.72 mmol) was added to a solution of 7-trichloroacetyl indole OMe

53 (400 mg, 0.84 mmol) in MeCN (20 mL) containing MeO N triethylamine (2 mL). The reaction mixture was stirred at H HN O room temperature for 3 h and the progress of the reaction O C2H5 O was monitored by thin layer chromatography. After completion of the reaction, water (50 mL) was added and the resulting precipitate was filtered to obtain the title compound 71a as white solid (350 mg, 76%). M.p. 235-237

1 °C; H NMR (300 MHz, CDCl3): δ 1.32 (t, J = 7.29 Hz, 3H, CH2CH3), 3.76 (s, 3H,

OMe), 4.07 (s, 3H, OMe), 4.25 (q, J = 7.29 Hz, 2H, CH2CH3), 4.45 (s, 2H, CH2), 6.23

(s, 1H, H5), 7.25–7.39 (m, 10H, Ph), 8.63 (t, J =4.85 Hz, 1H, NHC=O), 11.18 (br s,

13 1H, NH); C NMR (75 MHz, CDCl3): δ 14.25 (CH3), 41.71 (CH2), 55.32 (OMe),

57.00 (OMe), 61.44 (CH2), 87.70 (C-5), 97.05 (C-7), 126.00 (Ph-C), 127.04 (Ph-C),

127.39 (Ph-C), 131.50 (C-2), 132.61 (C), 132.29 (C-3), 135.94 (C), 138.53 (C),

157.02 (C-4), 157.84 (C-6), 167.64 (C=O), 170.48 (C=O). IR (KBr): νmax 3380 s,

3031 w, 2987 w, 1732 s, 1625 s, 1596 m, 1526 s, 1501 m, 1429 m, 1345 s, 1283 m,

1224 s, 1154 m, 1065 w, 1022 w, 993 m, 805 m, 775 w, 759 w, 702 w, 643 w cm–1.

+ HRMS (+ESI): Found m/z458.1826, [M] ; C27H26N2O5 required 458.1842.

105

Ethyl 2-(4,6-dimethoxy-2,3-diphenyl-1H-indole-7- carboxamido)-4-methylpentanoate (72b) [Leucine OMe ethyl ester-Indole Ligand] L-Leucine ethyl ester hydrochloride salt (130 mg, 0.72 MeO N H mmol) was added to a solution of 7-trichloroacetyl HN O O indole 53 (400 mg, 0.84 mmol) in MeCN (20 mL) C2H5 O containing triethylamine (2 mL). The reaction mixture was stirred at room temperature for 3 h and the progress of the reaction was monitored by thin layer chromatography. After completion of the reaction, water (50 mL) was added and the resulting precipitate was filtered to obtain the title compound

1 72b as white solid (95 mg, 22%). M.p. 254-256 °C; H NMR (300 MHz, CDCl3): δ

0.91 (m, 1H, CH), 1.04 (t, J = 5.37 Hz, 6H, CH3), 1.35 (t, J = 4.69 Hz, 3H, CH3), 1.83

(m, 1H, CH), 3.80 (s, 3H, OMe), 4.11 (s, 3H, OMe), 4.29 (q, J = 7.03 Hz, 2H, CH2),

4.86 (q, J = 6.49 Hz, 1H, CH), 6.27 (s, 1H, H5), 7.21–7.40 (m, 10H, 2Ph), 8.56 (d, J =

13 3.74 Hz, 1H, NH), 11.24 (br s, 1H, NH); C NMR (75 MHz, CDCl3): δ 14.25 (CH3),

22.33 (CH3), 22.92 (CH3), 24.53 (CH), 40.04 (CH2), 55.33 (OMe), 57.19 (OMe),

61.19 (CH2), 87.65 (C-5), 96.23 (C-7), 125.97 (Ph-C), 127.36 (Ph-C), 128.09 (Ph-C),

128.30 (Ph-C), 131.51 (C), 134.32 (C-2), 136.03 (C-3), 152.32 (C-4), 158.65 (C-6),

167.32 (C=O), 170.43 (C=O). IR (KBr): νmax 3297 s, 3250 m, 3073 w, 2864 w, 2844 w, 1827 s, 1752 s, 1707m, 1659m, 1529s, 1492w, 1452 w, 1379 s, 1250 m, 1170 s,

1039 m, 972 s, 945 m, 840 s, 729 m, 702 w, 633 w cm–1. HRMS (+ESI): Found m/z

+ 514.2432, [M] ; C31H34N2O5 required 514.2468.

106

Methyl 2,6-bis(4,6-dimethoxy-2,3-diphenyl-1H-indole-7-carboxamido)hexanoate (72c) [Lysine methyl ester-Indole Ligand]

L-lysine methyl ester OMe hydrochloride salt (100 mg,

MeO N 0.51 mmol) was added to a H MeO O HN O solution of 7-trichloroacetyl NH HN indole 53 (400 mg, 0.84 O mmol) in acetonitrile (20 mL) MeO OMe containing triethylamine (1 mL). The mixture was heated at reflux for 24 h, and after completion of the reaction, the crude mixture was diluted with water (50 mL), extracted with dichloromethane and purified using gravity column chromatography to yield the title compound as a light-pink solid (50 mg, 30%). M.p. 267-269 °C; 1H

NMR (300 MHz, CDCl3): δ 0.86 (m, 2H, CH2), 1.74 (m, 2H, CH2), 1.89 (m, 2H,

CH2), 3.68 (s, 3H, OMe), 3.70 (s, 3H, OMe), 3.79 (s, 3H, OMe), 3.89 (s, 3H, OMe),

3.96 (s, 3H, OMe), 4.13 (q, J = 6.80 Hz, 2H, CH2), 6.07 (s, 1H, H5), 6.10 (s, 1H, H5),

7.19–7.38 (m, 20H, 4Ph), 8.12 (t, J = 5.39 Hz, 1 H, NH), 8.64 (d, J = 7.44 Hz, 1 H,

13 NH), 11.19 (br s, 1H, NH), 11.32 (s, 1H, NH); C NMR (75 MHz, CDCl3): δ 22.13

(CH2), 29.75 (CH2), 30.17 (CH2), 39.07 (CH2), 52.10 (OMe), 52.38 (OMe), 55.19

(OMe), 55.22 (OMe), 56.98 (OMe), 87.28 (C-7), 94.57 (C), 127.38 (Ph-C), 127.91

(Ph-C), 127.98 (Ph-C), 128.33 (Ph-C), 131.50 (Ph-C), 131.83 (C), 133.03 (Ph-C),

134.25 (C-2), 135.10 (C-3), 151.47 (C-4), 152.66 (C-6), 167.59 (C=O), 172.90

(C=O). IR (KBr): νmax 3383 s, 2939 m, 2844 w, 1740 m, 1631 s, 1595 s, 1533 s, 1461 m, 1357 m, 1284 m, 1233 s, 1188 s, 1113 m, 991 m, 797 m, 694 m cm–1.HRMS

+ (+ESI): Found m/z 870.3613, [M] ; C53H50N4O8 required 870.3629.

107

(R)-Dimethyl 2-(4,6-dimethoxy-2,3-diphenyl-1H-indole-7-carboxamido)succinate (72d) [Aspartic acid methyl ester-Indole Ligand]

The indole ligand 72d was prepared according to OMe the method of preparation of compound 72b using indole 53 (400 mg, 0.84 mmol) and MeO N H aspartic acid methyl ester hydrochloride salt (160 O HN O Me O O Me mg, 1.00 mmol). The title compound was O obtained as an orange solid (130 mg, 30%). M.p. 275-277 °C; 1H NMR (300 MHz,

CDCl3): δ 2.99–3.19 (m, 2H, CH2), 3.72 (s, 3H, OMe), 3.76 (s, 3H, OMe), 3.80 (s,

3H, OMe), 4.08 (s, 3H, OMe), 6.23 (s, 1H, H5), 7.20–7.36 (m, 10H, 2Ph), 9.13 (d, J =

13 6.92, 1H, NH), 11.15 (br s, 1H, NH); C NMR (75 MHz, CDCl3): δ 37.52 (CH2),

50.97 (OMe), 51.28 (OMe), 52.37 (CH), 56.71 (OMe), 56.91 (OMe), 87.09 (C-5),

95.10 (C-7), 127.06 (Ph-C), 127.72 (Ph-C), 129.20 (Ph-C), 131.83 (Ph-C), 122.94

(C), 134.75 (Ph-C), 134.95 (C-2), 136.06 (C-3), 136.73 (C), 150.33 (C-4), 158.32 (C-

6), 167.53 (C=O), 168.59 (C=O), 173.20 (C=O). IR (KBr): νmax 3404 m, 3373 m,

3046 w, 2945 w, 2844 w, 1740 s, 1728 s, 1630 s, 1628 s, 1596 m, 1518 m, 1503 m,

1436 m, 1367 m, 1283 s, 1256 m, 1232 m, 1180 s, 1168 s, 1129 s, 1150 s, 1006 s, 990 s, 795 m, 776 m, 753 m, 702 m, 645 m cm–1. HRMS (+ESI): Found m/z 516.1873,

+ [M] ; C29H28N2O7 required 516.1897.

(R)-Methyl 2-(4,6-dimethoxy-2,3-diphenyl-1H- indole-7-carboxamido)-3-phenylpropanoate (72e) OMe [Phenylalanine methyl ester-Indole Ligand]

MeO N H HN O OMe

O 108

The indole ligand 72e was prepared according to the method of preparation of compound 72b using indole 53 (400 mg, 0.84 mmol) and phenylalanine methyl ester hydrochloride salt (220 mg, 1.00 mmol). The title compound was obtained as a light

1 yellow solid (160 mg, 36%). M.p. 215-217 °C; H NMR (300 MHz, CDCl3): δ 3.26

(dd, J = 2.53, 2H, CH2), 3.75 (s, 3H, OMe), 3.77 (s, 3H, OMe), 3.87 (s, 3H, OMe),

5.10 (q, J = 5.65, 1H, CH), 6.19 (s, 1H, H5), 7.19–7.36 (m, 10H, 2Ph), 8.55 (d, J =

13 7.48 Hz, 1H, NH), 11.18 (br s, 1H, NH); C NMR (75 MHz, CDCl3): δ 37.99 (CH2),

52.29 (OMe), 53.47 (OMe), 55.32 (OMe), 86.89 (C-7), 125.99 (Ph-C), 127.05 (Ph-C),

127.38 (Ph-C), 128.04 (Ph-C), 128.32 (Ph-C), 128.53 (Ph-C), 128.53 (C), 129.43 (C),

131.49 (C-2), 135.93 (C-3), 136.41 (C), 157.05 (C=O), 162.32 (C=O). IR (KBr): νmax

3386 s, 3372 m, 3053 w, 2945 w, 2847 w, 2363 w, 2334 w, 1731 m, 1634 s, 1628 s,

1599 m, 1529 m, 1434 w, 1251 s, 1233 s, 1188 m, 1162 m, 1118 m, 1116 s, 988 w,

–1 + 701 w cm . HRMS (+ESI): Found m/z 534.2137, [M] ; C33H30N2O5 required

534.2155.

Methyl 2-(4,6-dimethoxy-2,3-diphenyl-1H-indole-7-carboxamido)-4- (methylthio)butanoate (72f) [Methionine methyl ester-Indole Ligand]

The indole ligand 72f was prepared according to

OMe the method of preparation of compound 72b using indole 53 (400 mg, 0.84 mmol) and MeO N H methionine methyl ester hydrochloride salt (200 HN O Me OMe mg, 1.00 mmol). The title compound was S O obtained as an orange solid (125 mg, 29%). M.p. 277-279 °C; 1H NMR (300 MHz,

CDCl3): δ 2.07 (s, 3H, MeS), 2.16 (m, 2H, CH2), 2.70 (m, 2H, CH2), 3.65 (s, 3H,

OMe), 3.78 (s, 3H, OMe), 3.80 (s, 3H, OMe), 4.56 (t, J =5.65, 1H, CH), 6.52 (s, 1H,

109

H5), 7.18–7.36 (m, 10H, 2Ph), 8.97 (br s, 1H, NH(C=O)), 11.20 (br s, 1H, NH); 13C

NMR (75 MHz, CDCl3): δ 21.26 (CS), 31.21 (CS), 31.07 (CH2), 55.97 (OMe), 57.80

(OMe), 59.27 (OMe), 87.13 (C-5), 97.29 (C), 127.89 (Ph-C), 128.23 (C-2), 131.33

(C-3), 134.42 (Ph-C), 135.69 (C), 136.88 (C), 141.16 (C), 143.28 (C), 146.54 (C),

150.31 (C-4), 158.68 (C-6), 167.01 (C=O), 177.41 (C=O). IR (KBr): νmax 3323 m,

3279 m, 3046 w, 2947 w, 2742 w, 1649 s, 1600 s, 1529 s, 1510 s, 1496 m, 1421 m,

1403 m, 1300 m, 1297 m, 1253 s, 1201 m, 1158m, 1109 s, 797 m, 706 m, 653 m, 602

–1 + m, 594 m cm . HRMS (+ESI): Found m/z 518.1820, [M] ; C29H30N2O5S required

518.1875.

Methyl 2-(2-(4,6-dimethoxy-2,3-diphenyl-1H-indole-7- carboxamido)acetamido)acetate (72g) [Glycylglycine methyl ester-Indole Ligand]

Glycine-glycine methyl ester (100 mg, 0.68 mmol) was added to the solution 7- OMe trichloroacetyl indole 53 (400 mg, 0.84 MeO N H mmol) in acetonitrile (20 mL) containing O HN O H N triethylamine (1 mL). The mixture was MeO O heated under reflux overnight under a nitrogen atmosphere. The crude mixture was diluted with water and extracted with DCM. The crude mixture was column chromatographed to yielded the titled compound as a white solid (240 mg, 49.9 %).

1 M.p. 284-285 °C; H NMR (300 MHz, CDCl3): δ 3.75 (s, 3H, OMe), 3.78 (s, 3H,

OMe), 4.09 (s, 3H, OMe), 4.10 (d, J = 4.27 Hz, 2H, CH2), 4.23 (d, J = 3.95 Hz, 2H,

CH2), 6.25 (s, 1H, H5), 6.88 (t, J = 6.29 Hz, 1H, NH), 6.87–7.37 (m, 10H, 2Ph), 8.65

13 (t, J = 5.10 Hz, 1H, NH), 11.15 (br s, 1H, NH); C NMR (75 MHz, CDCl3): δ 41.20

110

(CH2), 43.75 (CH2), 52.41 (OMe), 55.35 (OMe), 56.92 (OMe), 87.56 (C-5), 95.23

(C), 127.41 (Ph-C), 127.98 (Ph-C), 128.39 (Ph-C), 131.47 (C-2), 132.43 (C-3), 134.73

(ArC), 134.97 (C), 136.00 (C), 150.12 (C-4), 158.43 (C-6), 167.34 (C=O), 169.53

(C=O), 171.87 (C=O). IR (KBr): νmax 3357 s, 3256 w, 2907 w, 2707 s, 1895 s, 1800 m, 1549 s, 1431 m, 1404 m, 1305 s, 1283 m, 1224 s, 1154 m, 1071 w, 1027 w, 944 m,

903 m, 857 s, 805 m, 759 w, 721 w, 702 w, 648 w cm–1. HRMS (+ESI): Found m/z

+ 501.1865, [M] ; C28H27N3O6 required 501.1900.

5.4 References

1. Black, D., N. Kumar, and L. Wong, Synthesis of 4,6-Dimethoxyindoles. Australian Journal of Chemistry, 1986. 39(1): p. 15-20.

2. Khandimir, H., PhD. Thesis, School of Chmistry, UNSW. 2011.

3. Leu, W., Ph.D. Thesis, UNSW. 2008.

4. Black, D., M. Bowyer, M. Catalano, A. Ivory, P. Keller, N. Kumar, S. Nugent, Substitution, oxidation and addition reactions at C-7 of activated indoles. Tetrahedron, 1994. 50(35): p. 10497-10508.

5. Ivory, A.J., Ph.D Thesis, UNSW. 1992.

6. Bambang, D., Ph.D Thesis, UNSW. 1998.

7. Li, J. and Y. Sha, A Convenient Synthesis of Amino Acid Methyl Esters. Molecules, 2008. 13(5): p. 1111-1119.

111