synthetic studies on nitrogenated fused heterocycles and

Synthetic Studies on Nitrogenated Fused Heterocycles and

their Applications

THESIS

Submitted in partial fulfilment

of the requirements for the degree of

DOCTOR OF PHILOSOPHY

by

UDAY KUMAR TOGITI

ID. No. 2013PHXF0405H

Under the supervision of

Prof. Anupam Bhattacharya

BIRLA INSTITUTE OF TECHNOLOGY AND SCIENCE, PILANI

2019

ii

BIRLA INSTITUTE OF TECHNOLOGY AND SCIENCE, PILANI

CERTIFICATE

This is to certify that thesis entitled “Synthetic Studies on Nitrogenated Fused Heterocycles

and Their Applications” submitted by UDAY KUMAR TOGITI (ID No. 2013PHXF0405H)

for award of Ph. D. of this institute embodies original research work done by under my supervision.

Signature of the supervisor :

Name in capital letters : Dr. ANUPAM BHATTACHARYA

Designation : Associate Professor

Date :

iii

Abstract

The main aim of this research work was development of newer methods for the synthesis of diverse

fused nitrogenated heterocyclic compounds and to look at their applications.

Chapter 1 [Importance of fused heterocyclic systems]: This chapter looks at the importance of

fused chromones, coumarins and quinoxalines based organic molecules. In addition, main

objectives of this research work along with required materials and methods are also stated in this

chapter.

Chapter 2 [Iron (III) catalyzed synthesis of fused chromeno-quinoline scaffolds]: This chapter

describes the synthesis of 6-substituted chromeno[3,2-c]quinolin-7-one by using FeCl3 caytalyzed

reaction between aromatic aldehydes and 2-(2-aminophenyl)-4H-chromen-4-one.

Chapter 3 [Synthesis and anticancer activity evaluation of fused chromeno-thieno/furo-pyridines]:

A linear route for the preparation of chromeno-thio-pyridine system as potential analogs for

anticancer compound lamellarin D is reported in this chapter via systematic application of Suzuki

coupling and modified Pictet-Spengler reaction. The chapter also describes the anticancer activity

screening of the target molecules2 against three different cell lines.

Chapter 4a [Synthesis of diverse 2-acylpyrroles from chalcones using polyphosphoric acid–

mediated regiospecific acyl migration]: This chapter describes the acyl rearrangement on pyrrole

ring.

Chapter 4b [Application of polyphophoric acid mediated rearrangement for the synthesis of

pyrrolo[1,2-a]quinoxalines]: Synthesis of pyrrolo[1,2-a]quinoxalines from chalcones by using

polyphosphoric acid facilitated acyl rearrangement on pyrrole ring is the main focus of this chapter.

Chapter 5 [Expanding the scope of fused pyrrolo-quinoline system for selective sensing of iron]:

This chapter discusses development of an iron sensor, 2-(3H-pyrrolo[2,3-c]quinolin-4-yl)aniline

(APQ). The fluorophore facilitates micromolar detection of Fe3+/Fe2+ in the presence of various

iv

cations, including well-known interfering cations Co2+and Cu2+ by the process of fluorescence

quenching.

[Summary, conclusions and future perspectives]: Main conclusions of the research work done

are discussed along with the future perspectives.

v

Declaration

The research work embodied in this thesis entitled “Synthetic Studies on Nitrogenated

Fused Heterocycles and Study of Their Applications” has been carried under the supervision

of Prof. Anupam Bhattacharya, Department of Chemistry, Bits-Pilani Hyderabad campus, India.

This work is original and has not been submitted in part or full for any degree to this or any other

universities.

Date (UDAY KUMAR TOGITI)

vi

Acknowledgments

Completion of this doctoral dissertation was possible with the support of several people. Firstly I

would like to express my sincere gratitude to my advisor Prof. Anupam Bhattacharya for his

unconditional support, scholarly input continuous and tremendous support throughout my Ph. D.

program. His valuable guidance, constant encouragement and motivation helped me in research

and writing the thesis.

I would also like to thank Dr. P. Srihari (Senior Principal Scientist, IICT-Hyderabad) and Prof.

Nidhi Jain (IIT-Delhi) for evaluating my thesis and giving their valuable comments and inputs

I would like to thank my Doctoral Advisory Committee (DAC) members Prof. Manab

Chakravarty (HOD) and Prof. K.V.G. Chandra Sekhar for their valuable comments,

suggestions.

I am grateful to Prof. Amit Nag and Ms. Swetha Pawar, for their help in fluorescence studies,

Dr. Balaram Ghosh and Ms. Yamini Bodbe for helping me in anticancer activity studies, Dr.

Durba Roy for computational studies and Prof. Krishnan Rangan for single crystal X-Ray

analysis of my compounds.

My sincere thanks to Prof. Bijendranath Jain, Former Vice Chancellor; Prof. Souvik

Bhattacharya, Vice Chancellor, BITS-Pilani; Prof. G. Sundar, Director BITS-Pilani Hyderabad

Campus; Prof. V. S. Rao, former Director BITS-Pilani, Hyderbad Campus.

I would like to thank Doctoral Research Committee (DRC) and other faculty members for their

encouragement and advice during different phases of my research work.

Special thanks to both past (Prof. Vidya Rajesh) and present (Prof. Venkata Vamsi Krishna

Venuganti) Associate Dean of AGSRD (Academic-Graduate Studies and Research Division) for

their help with various official requirements.

I thank my research group members Dr. Mahesh Akula, Dr. Yadagiri Thigulla and all the

research scholars in the department of chemistry for making my stay enjoyable and full of learning.

Immense help which I have received from Mr. Ashok Krishna, Ms. Shantha Kumari, Mr.

Sudheer Reddy, Mr. Gangadhar, Mr. Uppalaiah, Mr. Kumar, Mr. Mallesh and Mr.

Narasimha is highly appreciated.

Finally I would like to acknowledge the financial support that I have received from Council of

Scientific & Industrial Research (CSIR) project junior research fellow, and BITS-Pilani,

Hyderabad Campus for providing Ph.D. Fellowship.

vii

Table of contents

CONTENTS Page No.

Certificate ii

Acknowledgements iii

Abstract iv

Table of contents vi

List of Tables ix

List of Figures x

Abbreviations xii

Chapter 1

1 Introduction 1

1.1 Fused chromone system 1

1.2 Fused coumarin system 4

1.3 Fused pyrrolo-quinoxaline system 7

1.4 Objectives 9

1.5 Materials and methods 10

Chapter 2

2 Introduction 12

2.1 Results and discussions 16

2.2 Conclusions 21

2.3 Experimental 22

2.4 General procedure for the synthesis of fused chromeno[3,2-c]quinolin-7-one 26

2.5 Extra data 42

viii

Chapter 3

3 Introduction 41

3.1 Results and discussion 56

3.2 MTT Assay 53

3.3 Conclusions 56

3.4 Procedure for the synthesis of chromeno[3,4-b]thieno/furo[3,2-d]pyridine-6-one 59

Chapter 4A

4.1 Introduction 79

4.1. 1 Results and discussion 83

4.1.2 Conclusions 91

4.1.3 General procedure for the synthesis of 2,4-disubstitued pyrroles 103

Chapter 4B

4.2 Introduction 116

4.2.1 Results and discussion 120

4.2.2 Conclusions 123

4.2.3 General procedure for the synthesis of fused pyrrolo[1,2-a]quinolxilines 124

Chapter 5

5 Introduction 139

5.1 Results and discussion 140

5.2 Conclusions 157

5.3 Experimental 158

ix

Conclusions 166

Summary 168

Bibilography 174

`

x

List of Tables

S. No. Page No.

1 Table 2.1 Screening of catalysts for the synthesis of chromeno[3,2-

c]quinolin-7-one’s

18

2 Table 2.2: Substrate screening 20

3 Table 3.1: Optimization of reaction conditions 50

4 Table 3.2: Synthesis of diverse 4-substituted-6H-chromeno[3,4-

b]thieno/furo[3,2-d]pyridin-6-one

52

5 Table 4.1.1: Screening of reaction conditions for acyl migration 86

6 Table 4.1.2: Synthesis of diverse 2-acyl pyrroles 90

7 Table 4.2.1: Optimizatin of reaction condition 123

8 Table 4.2.2 Synthesis of various pyrrole[1,2-a]quinoxalines 124

xi

List of figures

S. No. Page No.

1 Figure 1.1. Evaluation of chromone to fused chromone systems 1

2 Figure 1.2. Diverse fused chromones isolated from natural

sources as well as synthesized

3

3 Figure 1.3. Evolution of coumarin to fused coumarin systems 4

4 Figure 1.4. Diverse fused coumarins isolated from natural

sources as well as synthesized

6

5 Figure 1.5. Evolution of pyrrolo quinoxaline systems from

quinoxaline and pyrrole

7

6 Figure 1.6. Diverse pyrrolo quinoxalines with different

constructs

9

7 Figure 2.1 Chemical structures of brosimone I and

cycloartocarpin

13

8 Figure 2.2 Structure of proposed aldimine intermediate 17

9 Figure 3.1: Structure of lamellarin D and the target molecule 42

10 Figure 3.2: The ORTEP single crystal diagram of compound 43,

the thermal ellipsoids are drawn at 50% probability.

49

11 Figure 3.3: Anticancer activity of novel compounds by MTT

assay.

54

12 Figure 3.4: Structure of BG-45, used in MTT assay as positive

control.

54

13 Figure 3.5, 3.6 & 3.7 The IC50 value determination of the more

active compounds on DU-145, B16F10, MCF-7 & HEK cell line

55, 56 &

57

14 Figure 4.1.1 Examples of biologically active 2-acylpyrrole

compounds

80

15 Figure 4.1.2 Comparision of 1H-NMR of compounds 21 and 44 87

16 Figure 4.1.3 Comparision of 13C-NMR of compounds 21 and 44 88

17 Figure 4.1.4 The ORTEP diagram of compound 44 88

xii

18 Figure 4.1.5. Mass analysis for the understanding the

mechanism of the reaction

93

19 Figure 4.2.1. Pyrrolo[1,2-a]quinoxaline compounds as 5HT3

receptor glucogen receptor antagonist

117

20 Figure 5.1. Diverse molecules for selective sensing of Iron 141

21 Figure 5.2. Quinoline based molecules as metal sensors 142

22 Figure 5.3. UV & Fluorescence spectrum of APQ and HPQ 144

23 Figure 5.4. Fluorescence behavior of APQ and HPQ on

incubation with Fe3+

146

24 Figure 5.5. Fluorescence behavior of APQ on incubation with

various metals

146

25 Figure 5.6. Competitive selectivity 147

26 Figure 5.7. Job’s plot to find out stoichiometry between metal

and ligand

148

27 Figure 5.8. Limit of detection 149

28 Figure 5.9. MALDI 150

29 Figure 5.10. Structure of ligands 21, 22, 23 & 24 150

30 Figure 5.11 UV-Study for the compounds 21, 22, 23 & 24 151

31 Figure 5.12 Fluorescence quenching behavior of 21 on


152



153



154



155

35 Figure 5.16 Truth table incorporating logic functions 156

36 Figure 5.17 Bright field and fluorescence confocal images of

span-80 niosomes loaded with APQ with and without Fe3+

158

xiii

List of Abbreviations

13C-NMR Carbon Nuclear Magnetic Resonance

1H-NMR Proton Nuclear Magnetic Resonance

5-HT3R 5-Hydroxytryptamine Receptor 3

Bn Benzyl

CCDC Cambridge Crystallographic Data Centre

CDCl3 Chloroform-d

CSA Camphor sulphonic acid

CuTC Copper (I)thiophene-2-carboxylate

DCB 1,2-Dichlorobenzene

DCE 1,2 Dichloroethane

DCM Dichloromethane

dd Doublet of doublet

DDQ 2,3-Dichlro-5,6-dicyano-1,4-benzoquinone

DFT Density Functional Theory

DNA Deoxyribonucleic acid

EDTA Ethylenediaminetetraacetic acid

ESI Electrospray Ionization

ESIPT Excited-State Intramolecular Proton Transfer

GPER G-Protein coupled Estrogen Receptor

HOMO Highest Occupied Molecular Orbital

HRMS High Resolution Mass Spectroscopy

Hz Hertz

IBX 2-Iodoxybenzoic acid

IC50 Half Maximal Inhibitory Concentration

IR Infrared Spectroscopy

J Coupling Constant

KHMDS Potassium bis(trimethylsilyl)amide

xiv

LCMS Liquid Chromatography-Mass Spectrometry

LDA Lithium diisopropylamide

LRMS Low Resolution Mass Spectrometry

LUMO Lowest Unoccupied Molecular Orbital

m Multiplet

m.p. Melting Point

MALDI Matrix-Assisted Laser Desorption Ionization

MAOs Monoamine oxidase

MDR Multi Drug Resistant

mg Milligram

MHz Mega Hertz

ml Millilitre

MS Mass Spectrum

MTT Assay [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide ]

nm Nanometre

nM Nanomolar

NMR Nuclear Magnetic Resonance

ºC Degree symbol

ORTEP Oakridge Thermal Ellipsoid Plot

PPA Polyphosphoric acid

PTSA Para-toluene sulfonic acid

q Quartet

t Triplet

TBAF Tetrabutylammoniumfluoride

TBQ Tetrachloro-o-benzoquinoline

TEA Triethylamine

TFA Trifluoroaceticacid

TLC Thin Layer Chromatography

xv

TMS Tetramethylsilane

TNF Tumor Necrosis Factor

TosMIC Toluenesulfonylmethylisocyanide

UV Ultraviolet

WSC N-(3-dimethylaminopropyl)-N’-ethylcarbodiimide

g Microgram

M Micromolar

ФF Quantum yield

Chapter 1

Importance of Fused Heterocyclic Systems

1

Introduction

Synthesis of heterocyclic compounds in a stand-alone or fused format remains an important

challenge in the field of organic chemistry. These systems remain some of the most sought after

scaffolds owing to their diverse material and biological properties. In a fused environment with

other aromatic or heteroaromatic units, these molecules acquire additional attributes owing to the

changing electronic environment. Indole, quinoline and isoquinoline remain the most frequently

encountered examples in the area of fused heterocycles. Currently, a lot of research work is

reported in the literature on the synthesis of various fused heterocyclic compounds and exploration

of their biological and physical properties.

Work discussed in this thesis mainly focusses on the synthesis of fused nitrogenated

heterocycles by the application of various synthetic techniques. An additional aspect is also

exploration of anticancer activity and metal sensing property of the synthesized molecules. Three

main types of fused heterocycles were studied during the course of this research work: (a)

chromone-quinoline systems; (b) coumarin-pyridine systems; (c) pyrrole-quinoxaline systems.

Main aim of this chapter is to look at various biological and photophysical properties of similar

fused molecules, to highlight their significance and also to rationalize the reason behind taking up

this research work.

1.1. Fused chromone systems

Figure 1.1 Evolution of chromone to fused chromone systems.

Possible sites to generate fused

chromone scaffold

chromone ring

2

Chromones are a major class of oxygenated heterocyclic compounds, which have been

extensively studied for their various biological and photophysical properties (Figure 1.1).[1-3]

Biologically active fused chromone systems are also well known in literature. Natural products

artonin B (1), cyclochampedol (2) and brosimone I (3) bearing annulated pyran ring display

cytotoxicity and anti-inflammatory activity.[4, 5] Gonzalez et al. reported the isolation of chromone

ptaeroglycol (4) from Cneorum tricoccum and C. pulverulentum . The compound showed

inhibitory activity (ID50 5M) on HeLa cells.[6] Chromone (5) isolated from Comantheria rotula

inhibited cancer tumor cell growth in the 60-cell line panel of National Cancer Institute. The GI50

values ranged from 1.6-18.2 M.[10] Folmer et al. in a study on Comanthus parvicirrus reported

isolation of fused chromones 6a and 6b. Both the compounds completely inhibited TNF-α-induced

NF-КB activation and NF-КB-DNA binding at MIC values 303 and 284 M, respectively.[11] Che

and co-workers reported isolation of thiopyranochromenone 7 from fungus Preussia africana

possessing the 3,4-dihydrothiopyrano[2,3-c]chromen5(2H)-one skeleton. The compound showed

significant cytotoxicity (IC50 8.34 M) against A549 (lung carcinoma epithelial cells) cell lines.[12]

Sayed et al. reported isolation of furochromones khellin (8a) and visnagin (8b) from Cyperus

rotundus L. The compounds were active against L5178y mouse lymphoma cells.[10]

Coumaronochromone (9) isolated by Wu and co-workers from Euchresta formosana displayed

cytotoxic behaviour against 59T(human hepatoma cell) and SCM-1(stomach adenocarcinoma cell)

cell lines.[11] Synthetic molecules have also been reported which exhibit diverse biological

activities. El-Helw et al. reported anticancer activity of a chromeno[2,3-c]pyrazole molecule (10)

against A594(lung) and HCT-116 (colon) cancer cell lines with IC50 values (4.65 and 4.90 M,

respectively) very close to that of doxorubicin (3.90 and 4.20 M, respectively) (Figure 1.2).[12]

3

Compared to regular chromones, their fused congeners are less frequently explored for

photophysical activity. In a recent report Yushchenko and co-workers have reported synthesis of

several fused chromones. Among the synthesized compounds fluorophore 2-(6-

dimethylaminobenzofuryl)-3-hydroxybenzochromone (11) displayed superior ratiometric

response to the polarity of the dye environment.[13]

Figure 1.2 Diverse fused chromones isolated from natural sources as well as through organic

synthesis.

4

1.2. Fused coumarin systems

Figure 1.3 Evolution of coumarin to fused coumarin systems.

Coumarin derivatives are well known for their biological and photophysical attributes (Figure

1.3).[14, 16] Currently, fused coumarin systems are also getting a lot of attention from the scientific

community across the world. Some of the most important biologically active compounds

possessing these structures include naturally occurring gilvocarcin V (12) and arnottin I (13),

known for their antibacterial and antitumor activities.[17, 18] Various fused coumarin compounds

have been prepared to explore their pharmacological properties. Wei et al. reported the synthesis

and antifungal activity of diverse oxazole fused coumarin derivatives.[19] Compound 14a and 14b

bearing ethyl and propyl substituent gave the highest activity against Chenopodium album

compared to commercially available herbicide acetochlor. Moro and co-workers developed potent

protein kinase CK2 inhibitors bearing fused coumarin skeleton by linking ellagic acid and 3,8‐

dibromo‐7‐hydroxy‐4‐methylchromen‐2‐one.[20] Among the compounds screened, bromo-3,8-

dihydroxy-benzo[c]chromen-6-one (15) displayed the best activity with IC50 value of 0.015 M.

Methyl-2,2-dimethyl-8-oxo-3,8-dihydro-2H-furo[2,3-h]chromene-6-carboxylate (16) was

reported as a potent soybean lipoxygenase inhibitor by Symeonidis et al.[21] Shafiee and co-

workers have reported the synthesis and AChE/BuChE inhibitory activity screening of 5-oxo-4,5-

dihydropyrano[3,2-c]chromenes bearing N-benzylpyridinium scaffold.[22] Most potent anti-AChE

activity (IC50 = 0.038 M) and highest AChE/BuChE selectivity (SI> 48) was shown by 1-(4-

Possible sites to generate fused

coumarin scaffold

coumarin ring

5

fluorobenzyl)pyridinium derivative (17). The same group also reported the synthesis and

AChE/BuChE inhibitory activity screening of chromeno[4,3-b]chromene compounds.[23] The

compound bearing 3-hydroxyphenyl substituent (18) gave the highest IC50 value against both

AChE (3.28 M) and BuChE (2.19 M).

Fused coumarins have recently found their way onto photonic-oriented applications.[24]

Fluorescent nature of pyrrolocoumarin 19 was exploited by Sames and co-workers to develop a

probe for monoamine oxidases (MAOs).[25] Conversion of aminoethyl-coumarin 20 to 19 oxidized

by MAOs was used as the reaction to develop the sensing protocol. A benzo[h]coumarin based

molecule (21) was used by Minyaeva et al. for dual sensing of metal ions.[26] The fluorophore

selectively detected Cu(II) and Co(II) in DMSO, whereas Mg(II) and Ba(II) were detected in

acetonitrile as well as toluene. Bizzari and co-workers developed a toolbox of fused coumarin

derivatives as polarity sensitive dyes for in vivo imaging applications.[27] Among the compounds

screened, dye 22 displayed an emission shift (bathochromic/red) of 10 nm (476-486 nm), decrease

in the quantum yield (F= 0.92-0.04) and an absorption shift of 10 nm (404-414 nm) depending

on the polarity of the solvents. Selective cyanide sensing was reported by Kim and co-workers

using 3-cyano-benzo[f]coumarin (23).[28] The probe emitted blue florescence at 450 nm with

Stokes shift of 70 nm. The 1, 4-addition of cyanide ions to probe resulted in fluorescence

quenching (Figure 1.4).

6

Figure 1.4 Diverse fused coumarins isolated from natural sources and via organic synthesis

7

1.3. Fused pyrrolo-quinoxaline systems

Figure 1.5 Evolution of pyrrolo-quinoxaline systems from quinoxaline and pyrrole.

Pyrrole and to a lesser extent quinoxaline ring are well-known for their myriad properties

(Figure 1.5). Diverse structures which emerge from their systematic combination are also equally

bestowed and are highly preferred for their applications. While several arrangements are possible,

most common structures found in literature are pyrrolo[1,2-a]quinoxaline and pyrrolo[2,3-

b]quinoxaline. Aiello and co-workers in a study to identify novel breast cancer inhibitors specific

for GPER (G-protein-coupled estrogen receptor) expressing cells identified a pyrrolo[1,2-

a]qunoxaline molecule 24 as the most promising candidate.[29] Guillon et al. reported synthesis

and biological evaluation of 4-substituted pyrrolo[1,2-a]quinoxaline compounds as

antileishmanial agents.[30] Compounds 25a-b bearing alkenyl side chain at the 4th position gave the

best results against parasites Leishmania amazonensis and Leishmania infantum. Same group also

explored synthesis and antimalarial activity of ferrocenic pyrrolo[1,2-a]quinoxaline derivatives.[31]

Best results were obtained with compounds 26a-d against FcB1, K1 and F32 strains of

Plasmodium falciparum. Desplat et al. investigated the potential of pyrrolo[1,2-a]quinoxaline as

inhibitors of Akt kinase in human leukemic cell lines K562, U937 and MCF7.[32] Best results were

Possible structures generated

by combining pyrazine half of

quinoxaline and pyrrole ring

[1,2-a]

[2,3-b]

Most frequently seen types of

pyrrolo-quinoxaline: pyrrolo[1,2-a]/

[2,3-b]quinoxaline

[3,4-b]

[1, 2, 3-de]

8

obtained for compound 27 which inhibited K562 cell line (IC50 = 4.5 M), and 28 which inhibited

U937 (IC50 = 5 M) and MCF7 (IC50 = 8 M). Halophenyl pyrrolo[2,3-b]quinoxaline derivatives

were explored by Manta et al. for evaluation of their antiviral activity.[33] Compound 29a inhibited

vaccina virus at an EC50 value of 2 M, whereas compound 29b inhibited Sindbis virus at an EC50

value of 4 M.

Patil et al. reported synthesis of pyrrolo[1,2-a]quinoxalines based bipolar host materials for

efficient red phosphorescent organic light emitting diodes (OLEDs).[34] Maximum external

quantum efficiency of 15.1% was obtained for compound 30 when it was incorporated in a red

phosphorescent device. Campiani and co-workers reported the use of pyrrolo[1,2-a]quinoxalines

as fluorescent probes for Aβ1-42 amyloid fibrils.[35] Among the compounds screened 31a-c bearing

substituenst on C-7 position of pyrrolo-quinoxaline scaffold did not alter the Aβ fibrils stability

and also gave the best result as staining agents. Ostrowska et al. used pyrrolo[2,3-b]quinoxaline

with 2-(2-aminoethyl)pyridine chain for selective sensing of Zn2+ ion.[36] The compound (32)

exhibited a dual fluorescence and aggregation induced emission enhancement (AIEE) in

crystalline state (Figure 1.6).

9

Figure 1.6 Diverse pyrrolo-quinoxalines with different constructs.

1.4. Objectives

The main objective of this thesis is to develop newer methods for the synthesis of diverse fused

nitrogenated heterocyclic compounds and their applications.

Chapter II: Development of a simple route for the synthesis of fused chromeno-quinoline scaffold

using iron catalyzed carbon-carbon bond formation as a key step.

Chapter III: Linear synthesis of fused 2H-chromen-2-one-thieno/furo-pyridine system as a mimic

for anticancer compound lamellarin D starting from easily available 4-hydroxy coumarin and

biological activity evaluation of the synthesized compounds.

10

Chapter IV: An alternative route to 2,4-disubstituted pyrrolo-quinoxalines from chalcones via

polyphosphoric acid mediated acyl rearrangement on pyrrole ring.

Chapter V: Fluorescence assisted selective iron sensing using pyrrolo-quinoline system.

1.5. Material and Methods

All starting materials were purchased from Aldrich, Alfa Aesar, Acros, Spectrochem, SRL,

AVRA and Sd Fine (India) and used directly without further purification. Solvents were dried

using standard methods and distilled before use. Visualization on TLC was achieved by use of UV

light (254 nm) or iodine. Melting points were recorded on a Stuart SMP 30 and Krüss melting

point apparatus. 1H NMR (300 MHz and 400 MHz) and 13C (75 MHz and 101 MHz) spectra were

recorded in CDCl3 and DMSO solution with TMS as internal standard. IR spectra were recorded

as KBr plates on Jasco FT/IR-4200 spectrometer. Mass spectra were recorded on Shimadzu

LCMS-2020. High resolution mass spectra were recorded on Bruker microTOF and Agilent

1100/LC MSD Trap SL version QII instrument. Column chromatography was performed on silica

gel (100–200 mesh, SRL. India).

Cell line culture and drug treatment (done in collaboration with Dr. Balaram Ghosh,

Department of Pharmacy, BITS-Pilani Hyderabad campus)

The prostate cancer (DU-145), murine skin melanoma (B16F10) and human breast cancer

(MCF-7) cell lines were used to determine anticancer activity of the novel compounds by MTT

assay. Dulbecco’s modified Eagle’s media (DMEM), supplemented with 1% antibiotic solution

and 10% fetal bovine serum (FBS) was used to culture the cells. MTT [3-(4,5-dimethylthiazol-2-

yl)-2,5-diphenyltetrazolium bromide], a yellow dye was used for the assay. All chemicals were

purchased from Himedia (Mumbai, India).

11

Chapter-2

Iron(III) Catalyzed Synthesis of Fused Chromeno-quinoline

Scaffolds

12

Introduction

Chromones and their C-2 phenyl substituted analogues (flavones) are important organic

molecules bestowed with diverse medicinally beneficial attributes. Some of the important

pharmacological properties displayed by these compounds include antimicrobial, antitumor and

anti-inflammatory activity.[1-3] Currently, a lot of attention is also garnered by chromone-fused

heterocycles, available from natural as well as synthetic sources[4-7] Well known examples of this

class include brosimone I 1 and cycloartocarpin 2, known for their tyrosinase inhibitory activity

(Figure-2.1).[8-9]

Figure 2.1 Chemical structures of brosimone I and cycloartocarpin.

In continuation with our efforts on synthetic and biological studies of fused-heterocycles,

synthesis of chromeno fused quinoline was conceived. Literature search revealed lack of any

synthetic approach to these molecules. To the best of our knowledge, there are no reports on these

molecules in the literature. Based on our prior experience with oxazolo[4,5-

c]quinoline/imidazo[4,5-c]quinoline synthesis,[10-11] and their apparent similarity with chromones,

it was felt that the most straightforward route to the target molecules would involve cyclization at

the unreactive C-3 position of chromone. Thus a Pictet-Spengler inspired approach was envisaged

for the synthesis of diverse chromeno[3,2-c]quinolin-7-one’s (Scheme-2.1).

13

Scheme 2.1 Similarities between our previous results and our current attempt.

A general method for the functionalization of chromone at C-3 position usually involves

lithiation followed by reaction with an electrophile or Heck coupling between 3-halochromenes

and appropriate alkenes.[12-16] While both the above mentioned techniques work efficiently and

provide ample flexibility for obtaining structurally diverse 3-substituted chromones, prior

activation of the C-3 site can sometimes impact the overall yield of the reaction. Reaction at C-3

position by direct C-H functionalization using Pd(II) based complexes have also been reported by

several research groups. Most of these approaches rely on nucleophilic attack of carbon-3 on Pd(II)

catalyst as the first step to form C-3 palladated chromone, followed by subsequent step involving

an appropriate coupling partner.[17] Kim et al., in their research efforts have reported Pd(II)

catalyzed intermolecular alkenylation at the C-3 position of chromone (Scheme 2.2).[18]

Scheme 2.2 Palladium catalysed oxidative C3 alkenylation of chromones.

Zhang and coworkers reported Pd(OAc)2 catalyzed coupling of chromone with

polyfluoroarenes (Scheme 2.3).[19] Reaction was facilitated by the addition of excess amount of

iPr2S, which helped in improving the overall yield of the reaction. The developed protocol was

also extended to other heteroatom-substituted enones.

14

Scheme 2.3 Palladium catalysed dehydrogenative cross coupling reaction between enones and

polyfluoroarenes.

Hong and co-workers in a paper have reported the synthesis of benzofuran-fused chromones

via C-3 selective functionalization of flavones and subsequent C-O cyclization (Scheme 2.4). The

reaction was catalyzed by Zn(OTf)2 in the presence of Cu(OAc)2.[20]

Scheme 2.4 Copper catalysed intramolecular C-O cyclisation of flavones.

The same group also reported formation of flavone-fused benzopyran moiety in the presence of

catalytic system comprising of Pd(OAc)2, Cu(OAc)2, Cs2CO3 and Al2O3 (Scheme 2.5).[21] Initially,

the C-3 alkenylated chromone was generated, which subsequently underwent intramolecular

cyclization to the target molecule.

Scheme 2.5 Palladium-copper catalysed consecutive alkenylation and cyclisation of chromones.

15

Zheng et al. carried out C-3 sulfenylation of chromene using DMSO in the presence of

NH4I (Scheme 2.6).[22]

Scheme 2.6 Ammonium iodide assisted C-3 sulfenylation of chromene.

Patel and co-workers reported Fe-(III) catalyzed C-3 functionalization of flavone using tert-

butyl peroxybenzoate (TBPB)/potassium persulphate (K2S2O8) as oxidant combinations (Scheme

2.7).[23]

Scheme 2.7 Fe(III) catalyzed C-3 functionalization of flavone.

For our project, none of the existing methods looked attractive as we wanted to avoid final step

involving both pre-functionalization as well as C-H activation mediated direct C-C bond coupling.

Given the inclusion of easy to oxidize aldehydes in the final step, we were particularly hesitant to

use palladium-based catalysts as all the reported reactions also included stoichiometric amounts of

additives as oxidants. As articulated previously, we decided to explore a Pictet-Spengler inspired

approach for the synthesis of the target molecules. Accordingly, a reterosynthetic strategy was

proposed as depicted in Scheme 2.8. Target molecule chromeno[3,2-c]quinolin-7-one (i) was

supposed to be assembled from flavone upon C-3 functionalization of the chromone ring.

16

Intermediate ii was to be sourced from easily available 2-(2-nitrophenyl)-4H-chromen-4-one (iii)

via reduction of its nitro functionality (Scheme 2.8).

Scheme 2.8 Retrosynthetic analysis of 6-substituted chromeno[3,2-c]quinolin-7-one.

Results and Discussion

Our efforts started from the synthesis of 2-(2-nitrophenyl)-4H-chromen-4-one (28a) which was

prepared using well-established literature protocols.[24] Subsequently, reduction of the nitro group

with Fe/NH4Cl lead to the formation of 2-(2-aminophenyl)-4H-chromen-4-one (29a, Scheme 2.9).

Scheme 2.9 Synthetic route to 2-(2-aminophenyl)-4H-chromen-4-one

With compound 29a in hand, efforts were focused on screening appropriate conditions to

generate the target molecules. Herein, the reaction between 29a and benzaldehyde (31) was used

17

as the model reaction (Table 2.1). Initially, reactions were attempted with diverse Lewis and

Bronsted acids (entry 1-6). While some of the reactions did not proceed beyond the formation of

aldimines, (Figure 2.2, 30) however with Yb(OTf)3 and FeCl3, target molecules were obtained in

20% and 32% yields, respectively. (entry 4 and 6).

Figure 2.2 Structure of proposed aldimine intermediate.

Subsequent reactions (entry 7-10) were attempted with FeCl3 to screen for the appropriate

solvent. The reaction was successful only with nitrobenzene as a solvent, which gave compound

34 in 56% yield. Further adjustment of the amount of catalyst did not improve the overall yield

(entry 11-12). Reactions when carried out with FeCl3 (20 mol%) in the presence of various

oxidizing agents such as TBPB, TBHP as well as in the combination of TBPB-Oxone or TBPB-

K2S2O8 did not result in the formation of the final product (entry 13). Using Fe(acac)3, as a catalyst

(entry 14), found suitable by Patel and co-workers in their synthetic studies on C-3

functionalization of flavones[23], did not yield the final product. When the reaction was attempted

under air with FeCl3 (20 mol%) as catalyst (entry 15), fused chromeno-quinoline molecule was

obtained in 44% yield. Thus, after thorough screening best results were obtained with 20 mol%

FeCl3 as catalyst and nitrobenzene as a solvent at 180 ºC under nitrogen atmosphere (entry 10).

18

Table 2.1: Screening of catalysts for the synthesis of chromeno[3,2-c]quinolin-7-one’s.

S.No

.

Catalyst

(20 mol%) Solvent

Temp.

(ºC)

Time

(in hr)

Yield

(%)

1 PTSA 1,4-Dioxane 90 12 -

2 CF3SO3H 1,4-Dioxane 90 12 -

3 CH3CO2 H - 110 12 -

4 Yb(OTf)3 1,4-Dioxane 90 12 20

5 Cu(OTf)2 1,4-Dioxane 90 12 -

6 FeCl3 1,4-Dioxane 90 12 32

7 FeCl3 DMF 140 12 -

8 FeCl3 DMSO 140 12 -

9 FeCl3 CH3CN 90 12 -

10 FeCl3 Nitrobenzene 180 6 56

11 FeCl3a Nitrobenzene 180 6 52

12 FeCl3b Nitrobenzene 180 6 56

13 FeCl3c Nitrobenzene 180 12 -

14 Fe(acac)3 Nitrobenzene 180 12 -

15 FeCl3 Nitrobenzene 180 6 44d

With optimized conditions in hand, we set out to demonstrate the substrate scope of

the newly developed methodology. As shown in Table 2.2, wide array of aromatic aldehydes were

examined in the synthesis of chromeno[3,2-c]quinolin-7-one 32-46. In general, aldehydes

possessing electron donating groups (such as -OMe, -OH) resulted in moderate yields of the

corresponding fused-chromeno products. Surprisingly, aldehyde bearing p-methyl group as the

substituent gave comparatively better yield than the aforementioned aldehydes. Screening

Important points about screening All the reactions were carried out under nitrogen atmosphere unless mentioned. a Catalyst used 10 mol% b Catalyst used 30 mol% c Three equivalents of oxidant such as TBPB/TBHP/oxone/K

2S

2O

8 were used along with the catalyst

whenever two oxidants were used the ratio 1:1(1.5 equivalents each) d Reaction was carried out under air atmosphere

19

substrates with electron-withdrawing substitutions (e.g. F, Cl, Br) yielded the cyclized products in

moderate to good yields. The yields were found to be affected by the electronegativity of the

halogens. Best yield was obtained using p-fluorobenzaldehyde, whereas, the yield was

compromised in the case of p-bromobenzaldehyde. Extending the scope of halogenated

benzaldehydes to their di-chlorinated congeners yielded the target compounds in modest yield.

Interestingly, more sterically hindered 2,6-dichlorobenzaldehyde gave better yield than its 2,4-

disubstituted isomer. Subsequent reactions with p-phenylbenzaldehyde, thiophene-2-

carboxaldehyde and pyridine-2-carboxaldehyde gave the final compounds in yields ranging from

25 to 47%. Scope of the reaction was also evaluated using 2-(2-aminophenyl)-6-methyl-4H-

chromen-4-one (29b) and 2-(2-aminophenyl)-6-chloro-4H-chromen-4-one (29c) as substrates.

Reaction carried out between 29b and benzaldehyde (31), gave the corresponding chromeno[3,2-

c]quinolin-7-one analogue in 45% yield, whereas with p-bromobenzaldehyde the yield of the final

product was 40%. With 2-(2-aminophenyl)-6-chloro-4H-chromen-4-one (29c) and benzaldehyde

(31) as substrate the final product was obtained in 52% yield. Our attempts with aliphatic

aldehydes (acetaldehyde, propionaldehyde, isovaleraldehyde and phenylacetaldehyde) were not

fruitful and no products were obtained.

20

Table 2.2 Substrate screening

A plausible mechanism was proposed to rationalize the product formation. The

reaction appears to proceed through cationic intermediate, similar to our previous observations

(Scheme-2.10).[4] The electrophilic attack of chromone ring via position 3 followed by oxidative

aromatization are envisioned as key steps in the synthesis of the target molecules. An alternative

21

mechanism, based on thermal 6π electrocyclization of imine intermediate followed by oxidative

aromatization can also be considered. A similar mechanism was suggested by Khan et al. in order

to rationalize the synthesis of coumarin fused quinolines/dihydroquinolines.[25]

Scheme 2.10 Plausible mechanism for the formation of the target molecule.

Relatively modest to low yields and the requirement of comparatively high reaction temperature,

prompted us to study the putative aldiimine intermediate by DFT calculations. For this purpose

the structure of the intermediate was optimized with the Gaussian 09 [DFT/cam-b3lyp/6-

311++g(d,p)]. The optimized geometry was employed to calculate the Mulliken charge

distribution, the Highest occupied molecular orbital (HOMO) and Lowest unoccupied molecular

orbital (LUMO) energy values [See extra data on page No. 37-39]. The charge distribution

showed comparable charges on the two reaction centers, which in our opinion rationalize the

modest to low yields seen in the reaction.

Conclusions

In conclusion, we have developed a direct iron-catalyzed route involving functionalization of

C-3 position of flavones for the synthesis of 6-substituted chromeno[3,2-c]quinolin-7-one. The

22

developed method helps in coupling two Csp2

centers bearing similar charges and works with easily

available aromatic aldehydes having electron rich and electron deficient substituents. Given the

biological importance of flavones and quinolines, we feel that the devised method will find a lot

of applications in the domain of medicinal chemistry. Additionally, since many chromone/flavone

based natural products are C-3 substituted, the possibility of synthesizing these molecules without

the aid of any additional activation or pre-functionalization step will prove to be an attractive

feature.

Experimental

All the starting materials and reagents required were purchased from commercial sources and

were used without further purification. Solvents were dried and distilled using standard protocols,

prior to use. 1H NMR (400 MHz) and 13C (101 MHz) spectra were recorded in CDCl3 and DMSO

using (CH3)4Si as internal standard. IR spectra were recorded as KBr plates on Jasco FT/IR-4200

instrument. Melting points were recorded on a Biotech India melting point apparatus and are

uncorrected. Mass spectra were recorded on Agilent 6545 Q-TOF LC/MS.

Synthesis of 2-(2-nitrophenyl)-4H-chromen-4-one (28a)

Step 1: To a solution of o- hydroxy acetophenone (0.88 ml) and o-nitro benzaldehyde (1.11 gram,

7.35 mmol) in ethanol (10 ml) was added 10% KOH (0.5 ml) and the reaction mixture was stirred

at room temperature for 30 minutes. Subsequently it was poured into water (10 ml) and extracted

with ethyl acetate (10 ml x 2). The organic layers were then combined, dried with anhydrous

Na2SO4 and concentrated under the reduced pressure. The resulting product was used as a substrate

for the next step without any purification.

23

Step 2: Chalcone was dissolved in DMSO (8ml) and 20mol% of I2 was added to it, the reaction

mixture was allowed to stir for 12 hr at 120 ºC. On completion of the reaction as confirmed by

TLC, mixture was poured into water (10 ml) and extracted with ethyl acetate(10 ml x 2). The

organic layers were then combined, dried with anhydrous Na2SO4 and concentrated under the

reduced pressure. The resulting residue was purified by column chromatography to give the

corresponding product.

Similar procedure had been followed for compounds 6-methyl-2-(2-nitrophenyl)-4H-chromen-4-

one (28b) and 6-chloro-2-(2-nitrophenyl)-4H-chromen-4-one (28c).

2-(2-Nitrophenyl)-4H-chromen-4-one (28a):

Yield: 70%; Brown solid; Rf: 0.5[3:2 hexanes: ethyl acetate];

m.p. 168-172 ºC; νmax (KBr)/cm-1: 3039, 1655, 1433, 1190,

895; 1H NMR (400 MHz, DMSO – d6) δ 6.81 (d, J = 2.8 Hz,

1H), 7.55 (ddd, J = 8.0, 3.4, 1.4 Hz, 2H), 7.82 - 7.88 (m, 1H),

7.90 (dd, J = 7.9, 1.8 Hz, 1H), 7.95 (td, J = 7.4, 1.3 Hz, 1H),

7.99 (dd, J = 7.6, 1.7 Hz, 1H), 8.10 (dd, J = 8.1, 1.7 Hz, 1H),

8.22 (dd, J = 8.0, 1.2 Hz, 1H); 13C NMR (101 MHz, DMSO)

δ 111.4, 118.7, 123.6, 125.5, 126.4, 126.9, 132.1, 133.1,

134.6, 135.3, 148.0, 156.2, 162.3, 177.2; ESI-MS (m/z):

268.06 [M+H]+

6-Methyl-2-(2-nitrophenyl)-4H-chromen-4-one (28b):

24

Yield: 30%; White solid; Rf: 0.5[7:3 hexanes: ethyl acetate];

m.p. 128-134 ºC; νmax (KBr)/cm-1: 3134, 1645, 1524, 1364,

791; 1H NMR (400 MHz, DMSO – d6) δ 2.44 (s, 3H), 6.76 (s,

1H), 7.44 (d, J = 8.5 Hz, 1H), 7.64 (dd, J = 8.9, 2.2 Hz, 1H),

7.85 - 7.90 (m, 2H), 7.92 (dd, J = 7.5, 1.4 Hz, 1H), 7.96 (dd,

J = 4.9, 1.5 Hz, 1H), 8.20 (dd, J = 8.0, 1.2 Hz, 1H); 13C NMR

(101 MHz, DMSO – d6) δ 20.9, 111.2, 118.4, 123.3, 124.7,

125.4, 127.0, 132.0, 133.0, 134.5, 136.1, 136.2, 148.0, 154.5,

162.1, 177.2; ESI-MS (m/z): 282.07 [M+H]+

6-Chloro-2-(2-nitrophenyl)-4H-chromen-4-one (28c):

Yield: 66%; Light yellow solid; Rf: 0.5[2:3 hexanes: ethyl

acetate]; m.p. 209-211 ºC; νmax (KBr)/cm-1: 3139, 1659, 1524,

1436, 754; 1H NMR (400 MHz, CDCl3) δ 6.59 (s, 1H), 7.36

(d, J = 8.9 Hz, 1H), 7.66 (dd, J = 25.5, 7.9 Hz, 2H), 7.77 (dd,

J = 13.1, 7.5 Hz, 2H), 8.11 (d, J = 7.8 Hz, 1H), 8.21 (s, 1H);

13C NMR (101 MHz, CDCl3) δ 111.3, 119.8, 124.8, 125.1,

127.6, 131.2, 131.6, 132.1, 133.6, 134.4, 148.0, 154.7, 162.6,

176.8; ESI-MS (m/z): 302.02[M+H]+

Synthesis of 2-(2-aminophenyl)-4H-chromen-4-one (29a)

To a solution of compound 28a in 20 ml 1, 4 - dioxane-H2O (4: 1), were added Fe powder (0.54

gm, 9.92 mmol) and ammonium chloride (0.54 gm, 9.92 mmol). The reaction mixture was then

allowed to stir at 90 ºC for 3 hr. On completion of the reaction as confirmed by TLC, the reaction

mixture was poured into distilled water and ethyl acetate and filtered through celite pad.

25

Subsequently the organic layer was separated, dried and concentrated under the reduced pressure.

The residue was then washed with ethyl acetate: hexane (1:9; 20ml x 2) and dried.

Similar procedure was followed for the synthesis of compounds 29b and 29c.

2-(2-Aminophenyl)-4H-chromen-4-one (29a):

Yield; 89%; White solid; Rf: 0.4[1:1 hexanes: ethyl acetate];

m.p. 148-150 ºC; νmax (KBr)/cm-1: 3050, 1643, 1537, 1246,

820; 1H NMR (400 MHz, CDCl3) δ 4.82 (s, 2H), 6.63 (s, 1H),

6.81 (d, J = 8.8 Hz, 2H), 7.27 (t, J = 7.1 Hz, 1H), 7.46 (d, J =

7.0 Hz, 2H), 7.54 (d, J = 8.2 Hz, 1H), 7.71 (t, J = 6.9 Hz, 1H),

8.20 (d, J = 7.0 Hz, 1H); 13C NMR (101 MHz, CDCl3) δ

109.9, 116.2, 117.0, 117.8, 117.9, 123.6, 125.2, 125.3, 129.3,

132.1, 133.7, 145.8, 156.1, 165.0, 177.9; HRMS-ESI(m/z):

calcd for C15H12NO2 [M+H]+ 238.0863 found 238.0837.

2-(2-Aminophenyl)-6-methyl-4H-chromen-4-one (29b)

Yield: 87%; Yellow solid; Rf: 0.5[3:2 hexanes : ethyl acetate];

m.p. 137-142 ºC; νmax (KBr)/cm-1: 3210, 1617, 1561, 1484,

1248, 818; 1H NMR (400 MHz, DMSO – d6) δ 2.44 (s, 3H),

5.69 (s, 2H), 6.51 - 6.54 (m, 1H), 6.64 - 6.69 (m, 1H), 6.83

(dd, J = 8.3, 1.0 Hz, 1H), 7.19 - 7.25 (m, 1H), 7.42 (dd, J =

7.9, 1.6 Hz, 1H), 7.60 - 7.62 (m, 2H), 7.81 - 7.86 (m, 1H); 13C

NMR (101 MHz, DMSO – d6) δ 20.9, 109.8, 115.8, 116.6,

26

117.1, 118.9, 123.5, 124.4, 129.9, 132.3, 135.2, 135.3, 147.6,

154.7, 165.2, 177.39; ESI-MS (m/z): 252.10 [M+H]+

2-(2-Aminophenyl)-6-chloro-4H-chromen-4-one (29c):

Yield: 66%; Light yellow solid; Rf: 0.5[3:2 hexanes : ethyl


1451, 1243; 1H NMR (400 MHz, CDCl3) δ 8.20 (d, J = 2.6

Hz, 1H), 7.63 (dd, J = 8.9, 2.6 Hz, 1H), 7.49 – 7.45 (m, 2H),

7.30 (ddd, J = 8.2, 7.3, 1.6 Hz, 1H), 6.88 – 6.83 (m, 1H), 6.79

(dd, J = 8.2, 0.9 Hz, 1H), 6.67 (s, 1H); 13C NMR (101 MHz,

CDCl3) δ 110.4, 116.6, 117.2, 118.7, 119.6, 124.8, 125.3,

129.6, 131.3, 132.5, 133.9, 145.2, 154.6, 165.1, 176.9, ESI-

MS (m/z): 272.04[M+H]+.

General synthetic procedure for 6-substituted-7H-chromeno[3,2-c]quinolin-7-one’s (32-46):

To a solution of 2-(2-aminophenyl)-4H-chromen-4-one 5 (63 mmol) in nitrobenzene (3 ml) was

added the appropriate aldehyde (63 mmol) and FeCl3 (20 mol%). The reaction mixture was

allowed to stir at 180 ºC for 6 hours under N2 atmosphere. After completion of the reaction as

confirmed by TLC, the reaction mixture was poured into water (10 ml). The precipitate thus

obtained was filtered and dried. It was subsequently purified by washing with 10% ethyl

acetate/hexane (10ml x 2), following which the final compound was obtained.

6-Phenyl-7H-chromeno[3,2-c]quinolin-7-one (32):

27

Yield: 56%; Light yellow solid; Rf: 0.4 [9:1 hexanes : ethyl


1228, 758; 1H NMR (400 MHz, DMSO-d6): δ 7.41 - 7.50 (m,

3H), 7.52 - 7.59 (m, 3H), 7.87 (ddd, J = 8.2, 6.9, 1.1 Hz, 1H),

7.92 - 7.99 (m, 2H), 8.04 (ddd, J = 8.4, 6.9, 1.4 Hz, 1H), 8.12

(dd, J = 10.9, 7.9 Hz, 2H), 8.71 (dd, J = 8.3, 0.8 Hz, 1H); 13C

NMR (101 MHz, DMSO-d6): δ 111.9, 118.2, 118.7, 123.1,

124.2, 125.9, 126.5, 127.7, 128.1, 128.4, 129.3, 129.6, 133.7,

135.8, 141.9, 148.4, 154.9, 159.9, 160.4, 175.2; HRMS-

ESI(m/z): calcd for C15H11N2O3 [M+H]+ 293.0921 found

293.092.

6-(p-Tolyl)-7H-chromeno[3,2-c]quinolin-7-one (33):



1220, 758; 1H NMR (400 MHz, CDCl3) δ 2.46 (s, 3H), 7.32

(d, J = 7.8 Hz, 2H), 7.43 (t, J = 7.9 Hz, 1H), 7.49 (d, J = 8.1

Hz, 2H), 7.70 (dd, J = 15.0, 7.8 Hz, 2H), 7.78 (t, J = 8.6 Hz,

1H), 7.90 (t, J = 7.7 Hz, 1H), 8.23 (dd, J = 28.2, 8.2 Hz,

2H),8.60 (d, J = 8.3 Hz, 1H); 13C NMR (101 MHz, CDCl3): δ

21.5, 111.8, 117.7, 118.2, 122.5, 124.3, 125.3, 127.0, 127.2,

128.4, 128.6, 129.7, 132.9, 134.7, 138.3, 138.6, 148.7, 154.8,

160.5, 160.6, 175.4; HRMS-ESI(m/z): calcd for C23H15NO2

[M+H]+ 338.1176 found 338.1142.

28

6-(4-Methoxyphenyl)-7H-chromeno[3,2-c]quinolin-7-one (34):



1245, 759; 1H NMR (400 MHz, CDCl3): δ 3.90 (s, 3H), 7.04

(d, J = 8.7 Hz, 2H), 7.44 (t, J = 7.9 Hz, 1H), 7.58 (d, J = 8.7

Hz, 2H), 7.69 (dd, J = 12.0, 6.5 Hz, 2H), 7.76 - 7.81 (m, 1H),

7.87 – 7.92 (m, 1H), 8.18 (d, J = 8.4 Hz, 1H), 8.28 (dd, J =

7.9, 1.4 Hz, 1H), 8.59 (d, J = 8.9 Hz,1H); 13C NMR (101

MHz, CDCl3): δ 55.3, 111.7, 113.4, 117.7, 118.1, 122.5,

124.3, 125.2, 127.0, 127.1, 129.7, 130.2, 132.8, 133.9, 134.7,

148.8, 154.8, 159.9, 160.6, 175.5; HRMS-ESI(m/z): calcd for

C23H15NO3 [M+H]+ 354.1125 found 354.1106.

6--(2-Hydroxyphenyl)-7H-chromeno[3,2-c]quinolin-7-one (35):



1464, 753; 1H NMR (400 MHz, DMSO-d6): δ 6.85 (d, J = 8.0

Hz, 1H), 6.92 (t, J = 7.3 Hz, 1H), 7.27 (t, J = 8.5 Hz, 1H),

7.33 (d, J = 7.4 Hz, 1H), 7.54 (ddd, J = 8.0, 5.3, 2.9 Hz, 1H),

7.85 (t, J = 7.5 Hz, 1H), 7.93 (d, J = 2.5 Hz, 2H), 8.02 (t, J =

8.1 Hz, 1H), 8.11 (t, J = 8.0 Hz, 2H), 8.68 (d, J = 8.0 Hz, 1H),

9.33 (s, 1H); 13C NMR (101 MHz, DMSO-d6): δ 113.2, 115.3,

118.3, 118.7, 118.9, 123.0, 123.9, 125.9, 126.4, 128.0, 129.5,

29

129.6, 129.7, 130.1, 133.4, 135.7, 148.8, 154.9, 155.3, 157.9,

159.3, 174.8; HRMS-ESI(m/z): calcd for C22H13NO3 [M+H]+

340.0968 found 340.0942.

6-(4-Fluorophenyl)-7H-chromeno[3,2-c]quinolin-7-one (36):



1225, 758; 1H NMR (400 MHz, CDCl3): δ 7.17 - 7.23 (m,

2H), 7.46 (ddd, J = 8.1, 7.2, 1.0 Hz, 1H), 7.56 - 7.62 (m, 2H),

7.71 (dd, J = 8.4, 0.8 Hz, 1H), 7.75 (ddd, J = 8.2, 7.0, 1.1 Hz,

1H), 7.82 (ddd, J = 8.6, 7.1, 1.7 Hz, 1H), 7.94 (ddd, J = 8.4,

7.0, 1.5 Hz, 1H), 8.21 (d, J = 8.4 Hz, 1H), 8.27 (dd, J = 8.0,

1.6 Hz, 1H), 8.64 (dd, J = 8.1, 1.1 Hz, 1H); 13C NMR (101

MHz, CDCl3): δ 111.7, 114.9 (2JC-F = 22.2 Hz), 117.7, 118.3,

122.5, 124.1, 125.4, 127.0, 127.5, 129.6, 130.5 (3JC-F = 8 Hz),

133.1, 134.9, 148.5, 154.8, 159.3, 160.7, 163.1 (1JC-F = 248

Hz), 175.4; HRMS-ESI(m/z): calcd for C22H12FNO2 [M+H]+

342.0925 found 342.0899.

6-(4-Chlorophenyl)-7H-chromeno[3,2-c]quinolin-7-one (37):



1226, 764; 1H NMR (400 MHz, CDCl3): δ 8.63 (d, J = 7.4 Hz,

1H), 8.28 – 8.18 (m, 2H), 7.93 (ddd, J = 8.4, 7.0, 1.4 Hz, 1H),

30

7.81 (ddd, J = 8.7, 7.1, 1.7 Hz, 1H), 7.78 – 7.67 (m, 2H), 7.54

(d, J = 8.5 Hz, 2H), 7.46 (dd, J = 11.3, 7.8 Hz, 3H); 13C NMR

(101 MHz, CDCl3): δ 111.6, 117.8, 118.3, 122.5, 123.9,

124.1, 125.5, 126.9, 127.6, 128.1, 129.7, 130.0, 133.1, 133.5,

134.9, 154.8, 159.1; HRMS-ESI(m/z): calcd for

C22H12ClNO2 [M+H]+ 358.0629 found 358.0608.

6-(4-Bromophenyl)-7H-chromeno[3,2-c]quinolin-7-one (38):

Yield: 52%; White solid; Rf: 0.5 [9:1 hexanes : ethyl acetate];

m.p. 269- 271 ºC; νmax (KBr)/cm-1: 1672, 1462, 1227, 1424,

763; 1H NMR (400 MHz, CDCl3): δ 7.48 (dt, J = 8.1, 1.9 Hz,

3H), 7.62 - 7.65 (m, 2H), 7.71 (d, J = 7.6 Hz, 1H), 7.75 (ddd,

J = 8.2, 7.0, 1.1 Hz, 1H), 7.81 (ddd, J = 8.6, 7.1, 1.7 Hz, 1H),

7.93 (ddd, J = 8.4, 7.0, 1.4 Hz, 1H), 8.20 (d, J = 8.4 Hz, 1H),

8.27 (dd, J = 8.0, 1.6 Hz, 1H), 8.63 (d, J = 8.3 Hz, 1H); 13C

NMR (101 MHz, CDCl3): δ 111.6, 117.8, 118.4, 122.5, 123.0,

124.0, 125.5, 126.9, 127.6, 129.6, 130.3, 131.0, 133.2, 135.0,

140.2, 148.5, 154.8, 159.1, 160.6, 175.3; HRMS-ESI(m/z):

calcd for C22H12BrNO2 [M+H]+ 402.0124 found 402.0097

(404.0079).

6-(2, 4-Dichlorophenyl)-7H-chromeno[3,2-c]quinolin-7-one (39):

31


m.p. 248-251 ºC; νmax (KBr)/cm-1: 1662, 1619, 1464, 1424,

896; 1H NMR (400 MHz, CDCl3): δ 7.33 - 7.43 (m, 2H), 7.48

(d, J = 8.0 Hz, 2H), 7.68 (d, J = 8.4 Hz, 1H), 7.75 - 7.81 (m,

2H), 7.91 - 7.97 (m, 1H), 8.26 (d, J = 8.1 Hz, 2H), 8.64 (d, J

= 8.3 Hz, 1H); 13C NMR (101 MHz, CDCl3): δ 112.5, 117.9,

118.7, 122.6, 123.6, 125.4, 126.9, 127.9, 128.0, 129.6, 129.9,

132.9, 133.5, 135.1, 139.4, 149.2, 155.1, 155.2, 160.1, 174.9;

HRMS-ESI(m/z): calcd for C22H11Cl2NO2 [M+H]+ 392.024

found 392.0198.

6-(2, 6-Dichlorophenyl)-7H-chromeno[3,2-c]quinolin-7-one (40):

Yield: 48%; White solid; Rf: 0.5 [4:1 hexanes : ethyl acetate]; m.p.

230-232 ºC; νmax (KBr)/cm-1: 1660, 1611, 1465, 1221, 756; 1H NMR

(400 MHz, CDCl3): δ 7.44 (ddd, J = 16.7, 13.8, 8.1 Hz, 3H), 7.53 (d,

J = 1.8 Hz, 1H), 7.72 (d, J = 8.4 Hz, 1H), 7.77 - 7.86 (m, 2H), 7.93

- 8.02 (m, 1H), 8.21 - 8.32 (m, 2H), 8.67 (d, J = 8.3 Hz, 1H); 13C

NMR (101 MHz, CDCl3): δ 112.6, 117.9, 118.8, 122.7, 123.6, 125.7,

126.9, 127.3, 128.3, 129.0, 129.9, 133.4, 135.3, 155.0, 156.6, 174.9;

HRMS-ESI(m/z): calcd for C22H11Cl2NO2 [M+H]+ 392.024 found

392.0229.

6-([1,1’-Biphenyl]-4-yl)-7H-chromeno[3,2-c]quinolin-7-one (41):

32

Yield: 35%; White solid; Rf: 0.4 [7:3 hexanes : ethyl acetate]; m.p.

246-248 ºC; νmax (KBr)/cm-1: 3110, 1662, 1619, 1489, 1222, 764;

1H NMR (400 MHz, CDCl3): δ 7.37 (t, J = 7.4 Hz, 1H), 7.43 - 7.49

(m, 3H), 7.72 (ddd, J = 11.8, 8.4, 4.7 Hz, 8H), 7.81 (ddd, J = 8.6,

7.2, 1.6 Hz, 1H), 7.93 (ddd, J = 8.4, 7.1, 1.3 Hz, 1H), 8.21 - 8.32

(m, 2H), 8.63 (d, J = 9.1 Hz, 1H); 13C NMR (101MHz, CDCl3): δ

111.8, 117.7, 118.3, 122.5, 124.1, 125.4, 126.7, 127.0, 127.3, 127.5,

128.7, 129.1, 129.5, 133.1, 134.9, 140.1, 141.1, 141.4, 148.5, 154.8,

160.1, 160.7, 175.4; HRMS- ESI(m/z): calcd for C28H17NO2

[M+H]+ 400.1332 found 400.1312.

6-(Thiophen-2-yl)-7H-chromeno[3,2-c]quinolin-7-one (42):


m.p. 204-206 ºC; νmax (KBr)/cm-1: 3132, 1661, 1403, 1222,

768; 1H NMR (400 MHz, CDCl3): δ 7.17 (dd, J = 5.1, 3.7 Hz,

1H), 7.41 - 7.46 (m, 1H), 7.52 (dd, J = 5.1, 1.1 Hz, 1H), 7.60

- 7.67 (m, 2H), 7.68 (dd, J = 3.7, 1.1 Hz, 1H), 7.77 (ddd, J =

8.6, 7.2, 1.7 Hz, 1H), 7.85 (ddd, J = 8.4, 7.0, 1.4 Hz, 1H),

8.11 (d, J = 8.4 Hz, 1H), 8.29 (dd, J = 7.9, 1.6 Hz, 1H), 8.51

(dd, J = 8.3, 0.9 Hz, 1H); 13C NMR (101 MHz, CDCl3): δ

111.6, 117.5, 118.0, 122.5, 124.2, 125.3, 127.0, 127.1, 127.3,

128.1, 129.5, 129.9, 132.9, 134.7, 142.9, 148.5, 152.9, 154.5,

160.6, 175.4; HRMS-ESI(m/z): calcd for C20H11NO2S

[M+H]+ 330.0583 found 330.0575.

33

6-(Pyridin-2-yl)-7H-chromeno[3,2-c]quinolin-7-one (43):


m.p. 266-268 ºC; νmax (KBr)/cm-1: 1659, 1606, 1565, 1223,

897; 1H NMR (400 MHz, CDCl3) δ 7.43 (dd, J = 12.0, 5.2 Hz,

2H), 7.69 (d, J = 8.0 Hz, 2H), 7.72 - 7.81 (m, 2H), 7.88 - 7.97

(m, 2H), 8.19 - 8.28 (m, 2H), 8.64 (d, J = 8.1 Hz, 1H), 8.74

(d, J = 4.6 Hz, 1H); 13C NMR (101 MHz, CDCl3): δ 112.1,

117.8, 118.7, 122.5, 122.7, 123.1, 124.1, 125.3, 126.9, 127.7,

129.9, 132.9, 134.9, 136.5, 148.7, 148.9, 155.0, 158.7, 159.2,

160.3, 175.2; HRMS- ESI(m/z): calcd for C21H12N2O2

[M+H]+ 325.0972 found 325.0951.

9-Methyl-6-phenyl-7H-chromeno[3,2-c]quinolin-7-one (44):



1436, 774; 1H NMR (400 MHz, CDCl3) δ 2.46 (s, 3H), 7.50

(d, J = 1.8 Hz, 1H), 7.51 (s, 1H), 7.57 (d, J = 1.7 Hz, 1H),

7.60 (d, J = 1.3 Hz, 2H), 7.54 (d, 2H), 7.73 (dd, J = 11.2, 4.1

Hz, 1H), 7.92 (ddd, J = 8.4, 7.0, 1.4 Hz, 1H), 8.05 (s, 1H),

8.22 (d, J = 8.4 Hz, 1H), 8.62 (d, J = 8.3 Hz, 1H); 13C NMR

(101 MHz, CDCl3): δ 20.9, 96.1, 111.7, 117.5, 122.5, 126.5,

127.9, 128.4, 133.0, 135.4, 136.0, 153.0, 160.5; HRMS-

34

ESI(m/z): calcd for C23H15NO2 [M+H]+ 338.1176 found

338.1157.

6-(4-Bromophenyl)-9-methyl-7H-chromeno[3,2-c]quinolin-7-one (45):


m.p. 266-268 ºC; νmax (KBr)/cm-1: 1660, 1617, 1482, 1432, 810;

1H NMR (400 MHz, TFA-d1): δ 3.15 (s, 3H), 8.14 (d, J = 8.5

Hz, 2H), 8.41 (d, J = 8.5 Hz, 2H), 8.46 (d, J = 8.6 Hz, 1H),

8.53 (d, J = 8.7 Hz, 1H), 8.70 (s, 1H), 8.78 (t, J = 7.6 Hz, 1H),

8.88 - 8.96 (m, 2H), 9.59 (d, J = 8.3 Hz, 1H); 13C NMR (101

MHz, TFA-d1): δ 19.78, 111.8, 118.5, 120.0, 120.9, 122.3,

124.7, 126.5, 128.8, 130.0, 132.1, 133.1, 138.7, 140.2,

140.41, 154.1, 160.4, 165.8, 176.9; HRMS-ESI(m/z): calcd

for C23H14BrNO2 [M+H]+ 416.0281 found 416.0255

(418.0236)

9-Chloro-6-phenyl-7H-chromeno[3,2-c]quinolin-7-one (46):

Yield: 52%; Light yellow solid; Rf: 0.5 [9:1 hexanes: ethyl


1436, 774; 1H NMR (400 MHz, CDCl3): δ 7.51 (dd, J = 5.1,

1.7 Hz, 3H), 7.57 (dd, J = 6.5, 3.2 Hz, 2H), 7.67 (d, J =

8.9 Hz, 1H), 7.78 - 7.82 (m, 2H), 7.94 (ddd, J = 8.4, 7.0, 1.4

Hz, 1H), 8.23 (dd, J = 5.4, 2.9 Hz, 2H), 8.61 (d, J = 8.3 Hz,

1H); 13C NMR (101 MHz, CDCl3): δ 111.5, 118.1, 119.4,

122.4, 125.2, 126.5, 127.5, 127.9, 128.4, 128.6, 129.9, 131.3,

35

133.2, 134.9, 141.1, 148.8, 153.2, 160.3, 160.5, 174.2;

HRMS-ESI(m/z): calcd for C22H12NO2 [M+H]+ 358.0629

found 358.0625.

1H NMR (400 MHz, DMSO-d6): 6-Phenyl-7H-chromeno[3,2-c]quinolin-7-one (32)

13C NMR (101 MHz, DMSO-d6): 6-Phenyl-7H-chromeno[3,2-c]quinolin-7-one (32)

36

Extra data

Mass spectrum showing the presence of imine intermediate (unstable compound, could not

be separated by column chromatography even after repeated attempts)

37

Table for Mulliken atomic charges (in au)

1 C 0.003506 21 H 0.176804

2 C -0.689940 22 C -0.114557

3 C 1.045852 23 C -0.113138

4 C -0.796968 24 H 0.174626

5 C -0.286063 25 H 0.174396

6 H 0.183501 26 H 0.183023

7 C -0.166520 27 N 0.307558

8 C 0.435252 28 C 0.870816

9 H 0.224525 29 C -0.560915

10 C -0.244714 30 C -0.362009

11 C -0.291829 31 C -0.473307

12 H 0.208185 32 H 0.165891

13 H 0.175674 33 C -0.127608

14 H 0.182486 34 H 0.177033

15 O 0.044609 35 C -0.364736

16 O -0.292598 36 H 0.177414

17 C 0.730476 37 H 0.200393

18 C -0.117968 38 H 0.171168

19 C -0.300996 39 C -0.400197

20 C -0.458168 40 H 0.149043

38

Mulliken charge distribution for the intermediate

39

HOMO of the intermediate

LUMO of the intermediate

40

Chapter-3

Synthesis and Anticancer Activity Evaluation of Fused

Chromeno-thieno/furo-pyridines

41

Introduction

Ubiquitous 2H-chromen-2-one, naturally sourced as well as of synthetic origin, represents an

important pharmacophore found in various medicinally important compounds.[1-5] This scaffold is

also present in a fused format in diverse biologically important natural products ranging from

millettonine and lamellarines A/C/D/H/L, to ningalin B.[6-13] Among the diverse lamellarins known

in the literature, lamellarin D [Figure-3.1(a)] has received a lot of attention due to its potent

anticancer activity against multidrug-resistant (MDR)tumor cell lines and prostate cancer cells.[14-

17] Based on the above examples, we wanted to carry out synthesis of a fused 2H-chromen-2-one-

thieno/furo-pyridine system [Figure-3.1(b)] as potential mimics of lamellarin D. The proposed

system was designed by adjustments to the core structure of lamellarin D. We envisaged the

replacement of 1-phenylpyrrolo[2,1-a]isoquinoline with 7-substituted-thieno/furo[2,3-c]pyridine

system. It was felt that removal of the isoquinoline ring can be compensated by introducing

comparatively bulky aromatic rings at the 7th position of the target molecule.

Figure 3.1 Structure of lamellarin D and the target molecule

It is worth mentioning that several literature reports are available describing structural changes

in lamellarin D and studying its effect on anticancer activity. Opatz et al., has explored efficacy of

a D-ring contracted analogue of lamellarin D towards a wild type and a multidrug resistant cancer

cell line(Scheme 3.1).[18]

42

Scheme 3.1 Synthesis of Lamallarin D-analogue

It was found to inhibit growth of tumour cells at submicromolar concentrations and displayed a

lower relative resistance in the MDR cell line compared to well-known anticancer drug

camptothecin. Pal and co-workers have reported one-pot synthesis of quinoxaline fused

pyrano[3,4-b]indole, which is the central core of lamellarin D (Scheme 3.2).[19]

Scheme-3.2 AlCl3 mediated synthesis of pyrano [3,4-b]indole fused quinoxalines

43

Several derivatives formed showed promising growth inhibition of lung and cervical cancer cells

and good in silico inhibition of human topoisomerase I. Diazaindeno[2,1-b]phenanthrene

analogues of lamellarin D were synthesized and studied by Dallavalle et al. (Scheme 3.3).[20] These

molecules displayed cytotoxicity in sub-micromolar level against human lung cancer H460 cell

line and also poisoning activity on topoisomerase I.

Scheme 3.3 Synthesis of diazaindeno[2,1-b]phenanthrene analogues of lamellarin D

Thasana and co-workers have explored synthesis and anticancer activity evaluation of

azalamellarins (Scheme 3.4).[21]

44

Scheme 3.4 Synthesis of Cu mediated azalamellarins

Several compounds exhibited good cytotoxity against cancer cell lines HuCCA-1, A-549, HepG2

and MOLT-3.

Literature search did not reveal any report of the proposed target molecules, however few

articles on systems bearing fused 2H-chromen-2-one-pyridine system are known. Peng and co-

workers reported methanesulfonic acid promoted one-pot three-component reaction leading to

functionalized pyrido[2,3-c] coumarin derivatives (Scheme 3.5).[22]

Scheme 3.5 Synthesis of various pyrido[2, 3-c]coumarin derivatives

45

The reaction involved application of inverse electron demand Diels-Alder reaction and the final

product were obtained in modest to good yields. Recently, Jamal and co-workers reported two step

synthesis of chromeno[4′,3′-4,5]pyrido[1,2-a]pyrazines/diazepines.[23] (Scheme 3.6)

Scheme 3.6 Synthetic route to chromeno[41,31:4,5]pyrido[1, 2-a]pyrazine-1,3-carboxylates

The molecules were synthesized by the reaction of substituted dimethyl 2-(3-acetyl-2-oxo-

2H-chromen-4-yl)fumarates with 1,n-diamines. Importantly the reactions were carried out at room

temperature and in the absence of any metal catalyst. While reported methods looked useful,

incorporating thiophene/furan ring in the target molecule was not possible using either of them.

Thus a different approach was envisaged for the proposed systems (Scheme-3.7). It was felt that

easily available 4-hydroxy coumarin 34 can be used as the starting compound, followed by its

nitration and chlorination to 4-chloro-3-nitro-2H-chromen-2-one 36. Introduction of chlorine

would allow application of Suzuki coupling to introduce thieno/furo ring at the fourth position of

compound 36. Subsequent steps would involve reduction followed by modified Pictet-Spengler

reaction with appropriate aldehyde to generate fused chromeno-thieno/furo-pyridines.

46

Scheme-3.7 Retrosynthetic approach for synthesis of the target molecules

Results and discussion

Our synthetic efforts started with nitration of 4-hydroxy coumarin (34) to yield 4-hydroxy-3-

nitro-2H-chromen-2-one (35),[24] which was treated with POCl3 to yield the chlorinated compound

(36) (Scheme-3.8).[25]

Scheme-3.8: Synthesis of 4-chloro-3-nitro-2H-chromen-2-one

Suzuki coupling was then used to combine compound 36 with 37/38, which yielded the coupled

product (39/40). In a subsequent step, the existing nitro group was reduced to the corresponding

amine 41/42 (Scheme-3.9).

47

Scheme-3.9 Suzuki coupling and its nitro reduction

Compound 41 was thoroughly characterized and a screening program was initiated to find out

the most appropriate reaction condition for their conversion to the final molecule. For this purpose

reaction between 41 and benzaldehyde was taken as the model reaction (Table-1). Preliminary

reactions were carried out with FeCl3 (20 mol%) as catalyst and ethanol, THF, DMF and 1,4-

dioxane as solvents (Table 1, entry 1-4). Desired product was obtained in all the cases except when

the reaction was carried out in THF, in which case it was difficult to isolate as the conversion was

incomplete and most of the starting material remained unreacted. Best yield (89%) was obtained

when the reaction was carried out at 90 °C using 1, 4-dioxane as solvent. The product was

thoroughly characterized by 1H and 13C NMR, prior to further optimization of the reaction

conditions. . In 1H-NMR spectrum of the final compound, disappearance of proton peak at δ7.02

indicated the formation of C-C bond at C-11 position of thiophene. Additionally, the presence of

a phenyl group as a substituent was indicated by the presence of five extra protons in the 1H-NMR

spectrum of the compound. 13C-NMR data was also used to further substantiate this conclusion as

shift of C-11 peak was noticed from δ128.1 to 140.6 and an additional peak appeared at δ155.2

indicative of C(14)=N bond formation in the target molecule. Four extra peaks (δ126.1, 128.8,

128.9 and 133.8) were also seen in the 13C-NMR, which are indicative of the presence of phenyl

ring in the molecule. X-ray crystallographic analysis was also carried out to ascertain the final

structure (Figure-3.2). The compound crystallized in triclinic crystal system with P-1 space group

(CCDC number 1863299). Two molecules and a single solvent [2(C20H11O2NS) + CHCl3]

appeared in an asymmetric unit during crystal structure solving with slightly varying bond lengths

and bond angle parameters. The molecule is composed of a central pyridine ring fused C2 and C3

position with a coumarin ring and fused to a thiophene ring at C4 and C5 position, further a phenyl

48

ring substitution at the C6 position. The dihedral angle representing between the pyridine ring and

coumarin ring N1-C8-C9-O1 and N1’-C8’-C9’-O1’ are 178.05º and 178.16º respectively. The

dihedral angle between the pyridine ring and the fused thiophene ring N1-C12-C11-S1 and N1’-

C12’-C11’-S1’ are 172.75º and 178.10º respectively. The phenyl ring substituted to the pyridine

rings deviated N1-C12-C15-C20 = 36.96º and N1’-C12’-C15’-C20’ = 45.47º from the planarity.

The slight change in the crystal structure geometrical arrangements may be attributable for the

adjustment adopted during crystal packing and the presence of a solvent molecule CHCl3 which

further establish weak non-covalent interaction Cl1---H18 = 2.906 Å, Cl2---H18 = 2.947 Å, Cl3--

-H5’ = 2.941 Å, H21---O1’ = 2.445 Å and H21---O2’ = 2.396 Å which are shorter than their sum

of the corresponding van der Waals radii.

The high resolution mass spectrum (HRMS) data of 40 was found to be 330.0551 (M + H+),

which was used as the final piece of evidence to confirm the molecule as 4-phenyl-6H-

chromeno[3,4-b]thieno[3,2-d]pyridin-6-one.

Figure 3.2 The ORTEP single crystal diagram of compound 43, the thermal ellipsoids are drawn

at 50% probability.

49

Reaction was also carried out with FeCl3 under microwave reaction conditions by using DMF

as solvent, which provided the target molecule in 65% yield (Table 3.1, entry 5). Reaction carried

out in the absence of catalyst (Table 3.1, entry 10), did not yield the target molecule. Switching to

other catalysts (Table 3.1, entry 6-9) such as Yb(OTf)3, CH3COOH, AlCl3 and PTSA resulted in

the synthesis of the target molecule, albeit in low yields in cases except in CH3COOH, whence the

target molecule could not be isolated.

Table 3.1 Optimization of reaction conditions

All the reactions were carried out for 16 hours and under nitrogen atmosphere. a Isolated yields. b Product not isolated.

Entry Catalyst Solvent Temp. (ºC) Yield (%)a

1 FeCl3 Ethanol 90 29

2 FeCl3 THF 60 b

3 FeCl3 DMF 120 57

4 FeCl3 1,4-dioxane 90 89

5 FeCl3 DMFc 200 65

6 Yb(OTf)3 PhNO2 180 25

7 CH3COOH DMF 120 Trace

8 AlCl3 1,4-dioxane 90 16

9 PTSA DMF 120 36

10 No catalyst 1,4-dioxane 90 d

50

c Reaction was carried out under microwave condition for 20 minutes d No reaction

In order to establish the scope and limitations of the optimized conditions, reactions were

carried out between 41 and various benzaldehydes. Initial attempts with aldehydes containing both

electron releasing and donating groups gave the final products (44-49) in 42-84% yields. Higher

yields in case of aldehydes with electron withdrawing groups clearly indicated importance of

increasing electrophilicity of carbonyl carbon in aldehydes. Subsequently, reactions were also

carried out with benzaldehydes containing diverse halogens as substituents (50-52), which resulted

in formation of the target molecule in 73-84% yields. These conversions further affirmed the

importance of carbonyl activation due to withdrawal of electron density from benzene ring. On

extending the optimized conditions to aldehydes bearing heterocycles and other aromatic systems,

the final compounds (53-55) were generated in modest to good yields (47-79%).

The optimized protocol was also extended to 3-amino-4-(furan-3-yl)-2H-chromen-2-one (42),

where reactions gave higher yields compared to thienyl system (41). This can be attributed to

higher reactivity of furan systems in comparison to thiophene systems. Interestingly, with

benzaldehyde, p-trifluoromethylbenzaldehyde and anthracene-9-carbaldehyde, final compounds

56, 61 and 68 were obtained in less yields (78%, 70% and 46%, respectively) compared to their

thiophene congeners (Table 3.2).

Table 3.2 Synthesis of diverse 4-substituted-6H-chromeno[3,4-b]thieno/furo[3,2-d]pyridin-6

51

Final compounds after thorough characterization were screened for their anticancer activity

using MTT assay (Figure 3.3).[12] BG-45, an anticancer molecule (Figure-3.4) was used in the

assay as positive control.

Anticancer activity was carried out in minithroughput way by taking 10 µM and 100 µM

doses in duplicate wells for 48 hours against DU-145 (prostate cancer cells). MTT assay results

revealed two very important points, no dependence on electron donating/withdrawing ability of

the substituents on the activity of the molecules and superiority of fused furo-coumarin framework

52

over corresponding thieno-coumarin molecules. Among the compounds screened, eight most

active compounds (44, 48, 57, 58, 61, 62, 63 and 66) were further explored against DU-145

(prostate cancer cells), B16F10 (murine melanoma cells) and MCF-7 (breast cancer cells) with

longer range of concentration to find out their IC50 value (Figure-3.5 and 3.6). During the

subsequent IC50 assays BG-45 (Figure-3.4) was used again as a positive control. The most

important observation that emerged out of this study was superior efficacy of some of the

developed compounds against MCF-7 in comparison to DU-145 and B16F10 cell lines. Against

DU-145, compounds 58/61/66 were most potent with highest activity of 20.88 M displayed by

4-(4-(trifluoromethyl)phenyl)-6H-chromeno[3,4-b]furo[3,2-d]pyridin-6-one (61). Studies on

B16F10 cell lines on the other hand gave 57/62/63 as the compounds with best activity, here 4-(4-

fluorophenyl)-6H-chromeno[3,4-b]furo[3,2-d]pyridin-6-one (62) showed the highest IC50 value of

12.98 M. Highest activity as shown by compounds 61 and 62 against DU-145 and B16F10 cell

lines also conforms with the general observation of increase in potency of a molecule, due to the

presence of fluorine.13 In the case of MCF-7 cell lines, compounds 58/62/63 were found to be most

potent, with highest activity (6.83 M) displayed by 4-(4-methoxyphenyl)-6H-chromeno[3,4-

b]furo[3,2-d]pyridin-6-one (58). Comparable IC50 value as shown by compounds 62 and 63 against

MCF-7 cell lines also demonstrate lack of any significant effect of electronegativity as well as the

size of the halogen substituent on the anticancer activity. According to us, presence of bromine in

compound 63 will allow the development of other structural analogues via diverse coupling

protocols. Interestingly, against all the three cell lines compounds synthesized were more potent

than BG-45. Besides as already highlighted previously, no clear distinction was seen between

molecules bearing electron donating or withdrawing substituents.

53

MTT assay was also performed to evaluate the toxicity of the selected eight compounds against

normal cells. Human embryonic cell (HEK293) was used for the experiment. A series of higher

doses ranging from 1.9 µM to 1000 µM for 48h treatment were used for the cytotoxicity dose

response experiment using the same experimental protocol discussed above (Figure-3.7). The

cytotoxic IC50 values (193 µM to 327 µM) of the compounds revealed that the title compounds are

safe and less toxic to the normal cell lines in comparison to the cancer cell lines tested.

MTT ASSAY

DU-145 MTT Assay 48 h

Contr

ol43 43 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60 61 62 63 64 65 66 67 68

BG-4

5

0

20

40

60

80

100

120

100 µM

10 µM

Compounds

% C

ell

Via

bilit

y

Figure 3.3 Anticancer activity of novel compounds by MTT assay.

Prostate cancer (DU-145) cells were treated with all compounds at two doses; 100µM and 10

µM in duplicate. Data represents mean ±SD (n=2; triplicate wells in each assay).

Figure 3.4 Structure of BG-45, used in MTT assay as positive control

54

DU-145 IC50 Assay 48 h

3 4 5 60

20

40

60

80

100

120

61 [IC50=20.88M]

62[IC50=31.83M]

63[IC50=34.81M]

66[IC50=25.30M]

44[IC50=53.39M]

48 [IC50=35.59M]

57[IC50=27.92M]

58[IC50=23.79M]

BG-45[[IC50=32.40M]

Log concentration (nM)

% C

ell

Via

bilit

y

Figure 3.5 The IC50 value determination of the more active compounds on DU-145.

B16F10 IC50 Assay 48 h

3 4 5 60

20

40

60

80

100

120 44 [IC50=25.27 M]

48 [IC50=25.01 M]

57 [IC50=15.43 M]

58 [IC50=17.85 M]

61 [IC50=18.48 M]

62 [IC50=12.98 M]

63 [IC50=13.28 M]

66 [IC50=16.64 M]

BG 45 [IC50=34.58 M]


% C

ell

Via

bilit

y

55

MCF-7 IC50 Assay 48 h

3 4 5 60

20

40

60

80

100

120

44 [IC50=26.53 M]

48 [IC50=24.29 M]

57 [IC50=6.83 M]

58 [IC50=20.58 M]

61 [IC50=13.33 M]

62 [IC50=19.34 M]

63 [IC50=8.03 M]

66 [IC50=8.00 M]

BG 45 [IC50=34.33M]


% C

ell

Via

bilit

y

Figure 3.6 The IC50 value determination of the more active compounds on B16F10 and MCF-7

with ten different doses (0.781 µM - 200 µM) for 48 hours treatment. Cell viability was measured

using MTT assay. Data shown as mean ± SD (n =2). Compounds found to show anticancer activity

with IC50 values in the range of 6.5 µM to 53 µM.

HEK IC50 Assay 48 h

0 1 2 3 40

20

40

60

80

100

120 44[IC50=327.5M]

48 [IC50=249.4M]

57[IC50=344.1M]

58[IC50=116.8M]

61 [IC50=255.3M]

62[IC50=193.0M]

63[IC50=.244.2M]

66[IC50=213.5M]

Log concentration (M)

% C

ell

Via

bilit

y

56

Figure 3.7 IC50 results of the selected eight compounds (44, 48, 57, 58, 61, 62, 63 and 66) by

MTT assay. Human embryonic kidney (HEK-293) cell lines were treated with the compounds at

concentration range of 1.9 µM -1000 µM for 48hrs. Data shown as mean ± SD (n =2).

Conclusions

In conclusion, a simple and efficient route has been devised to provide direct access to a

library of fused tetracyclic scaffolds by systematic incorporation of coumarin, thiophene and

pyridine ring. The method established can be easily extended for the synthesis of corresponding

furan analogues. The synthetic strategy developed is amenable to biologically important and

sensitive functional groups. In vitro anticancer activity evaluation against three different cancer

cell lines revealed modest activity by several compounds, with highest IC50 of 6.83 μM displayed

by compound 57 and the compounds are not toxic to the normal cells. The results obtained indicate

ample scope for exploring relatively untapped structures containing fused chromeno-thieno/furo-

pyridines as potential anti-cancer compounds.

57

Experimental

General information

All starting materials were purchased from various chemical manufacturers and were used directly.

Solvents were dried and distilled using standard methods, before use. Column chromatography

was performed on silica gel (100–200 mesh, SRL. India). Visualization on TLC was achieved by

use of UV light (254 nm) or iodine. 1H NMR (300MHz and 400 MHz) and 13C (75 MHz and 100

MHz) spectra were recorded in CDCl3, DMSO-d6 and TFA-d1 solution with TMS as internal

standard. IR spectra were recorded as KBr plates on Jasco FT/IR-4200 instrument. Melting points

were recorded on a Stuart SMP 30 melting point apparatus and are uncorrected. Mass spectra were

recorded on Agilent 6545 Q-TOF LC/MS.

Procedures for preparation of the starting compounds

Synthetic scheme commenced from 4-hydroxycoumarin which was procured from Spectrochem-

India and was used without any further purification.

Synthesis of 4-hydroxy-3-nitro-2H-chromen-2-one (35): Reference [29]

4-Hydroxy coumarin (1 gm, 6.17 mmol) was taken in a round bottomed flask under nitrogen

atmosphere. Conc. HNO3 (4 ml) and glacial acetic acid (15 ml) were added to it, the resulting

mixture was then allowed to stir at 60 °C for 3 hours. Afterwards the mixture was poured in a

beaker containing crushed ice and the precipitate thus obtained was filtered. Pure product was

obtained in 83% (1.06 gm) yield and did not require any purification step. Light yellow solid; Rf:

0.6 (1:4 ethyl acetate: hexanes); m.p. 128-130 ºC.

Synthesis of 4-chloro-3-nitro-2H-chromen-2-one: (36) Reference [29]

POCl3 (0.49 ml, 5.31 mmol) was added dropwise to ice cold N, N-dimethyl formamide (0.41 ml,

5.31 mmol) and the resulting solution was stirred for 15 minutes. To this, a solution of 4-hydroxy-

58

3-nitrocoumarin (0.92 gm, 4.46 mmol) dissolved in DMF (2 ml) was added and the resulting

reaction mixture was stirred for an additional 15 minutes. On completion of the reaction as

indicated by TLC, the mixture was poured into ice water. The precipitate thus obtained was filtered

and dried, to obtain the final compound in 81% (0.87 gm) yield. White solid; Rf : 0.4 (1:4 ethyl

acetate: hexanes); m.p. 158-160 ºC.

Synthesis of 3-nitro-4-(thiophen-3-yl)-2H-chromen-2-one (39)

1, 4-dioxane (22ml) was taken in a round bottom flask and kept under nitrogen atmosphere. 4-

Chloro-3-nitro-2H-chromen-2-one (2.3 gm, 10 mmol), 3-thienyl boronic acid (1.5 gm, 12 mmol)

and K2CO3 (2.83 gm, 20 mmol) were subsequently added to it and the reaction mixture was stirred

for 5 minutes. Afterward, Pd(PPh3)4 (0.2 gm, 0.2 mmol) was added and the reaction mixture was

heated at 90 ºC for 3 hours. On completion of the reaction as indicated by TLC, the reaction mixture

was diluted with water (50 ml) and extracted with EtOAc (3 x 40 ml). The organic layer was

washed with brine solution, dried over anhydrous Na2SO4 and concentrated under reduced

pressure. Crude product obtained was subjected to column chromatography (Silica gel, 10% ethyl

acetate-hexane) to afford the compound as white solid. Yield: 2.1 gm (75%); m.p. 162-168 ºC.

Synthesis of 3-nitro-4-(furan-3-yl)-2H-chromen-2-one (40): Same procedure was followed

and the compound was obtained as white solid in 89% yield; m.p. 124-130 ºC.

Synthesis of 3-amino-4-(thiophen-3-yl)-2H-chromen-2-one/3-amino-4-(furan-3-yl)-2H-

chromen-2-one (41 & 42)

Appropriate nitro compound (7 mmol) was dissolved in 16 ml of 1, 4-dioxane and 4 ml of distilled

water was added to it. To this mixture Fe powder (35 mmol) and ammonium chloride (35 mmol)

were added and the reaction mixture was allowed to stir at 90 ºC for 3 hr. On completion, the

reaction was poured into 30 ml distilled water and ethyl acetate (1:1) and filtered through celite

59

pad. The organic layer from the filterate was subsequently separated, dried over anhydrous Na2SO4

and concentrated under reduced pressure. The crude product obtained was washed with 10 % ethyl

acetate/hexane (20ml x 2) and dried.

The 3-amino-4-(thiophen-3-yl)-2H-chromen-2-one was obtained in 52% yield, while 3-amino-4-

(furan-3-yl)-2H-chromen-2-one (42) was obtained in 60% yield.

General synthetic procedure for 4-substituted-6H-chromeno[3,4-b]thieno/furo[3,2-

d]pyridin-6-one(43-68)

To a mixture of 3-amino-4-(thiophen-3-yl)-2H-chromen-2-one/3-amino-4-(furan-3-yl)-2H-

chromen-2-one (0.5 mmol) and appropriate aldehyde (0.6 mmol) in 2 ml of 1,4–dioxane was added

FeCl3 (20 mol%). The solution was then stirred at 90 °C for 16 hrs under nitrogen atmosphere. On

completion of the reaction as indicated by TLC, the reaction mixture was poured into water (10

ml) and extracted with ethyl acetate (3 x 10 ml). The separated organic layer was subsequently

dried over anhydrous Na2SO4 (1 gm) and concentrated in vacuo. The resulting residue was purified

by column chromatography using hexane/ethyl acetate as the eluent to give the corresponding

product.

4-Hydroxy-3-nitro-2H-chromen-2-one (35):

Yield: 70%; White solid; 1H NMR (400 MHz, DMSO – d6)

δ 7.18 - 7.28 (m, 2H), 7.55 (ddd, J = 8.2, 7.3, 1.7 Hz, 1H),

7.91 (dd, J = 7.8, 1.7 Hz, 1H), 11.64 (s, 1H); 13C NMR (101

MHz, DMSO – d6) δ 116.6, 121.8, 123.7, 126.0, 132.9,

152.9, 157.7, 167.1; ESI-MS(m/z): 208.02[M+H]+

4-Chloro-3-nitro-2H-chromen-2-one (36):

60

Yield: 74%; White solid; Rf: 0.4[1:4 Hexanes: Ethyl acetate];

1H NMR (400 MHz, CDCl3) δ 7.47 (d, J = 8.4 Hz, 1H), 7.53

(t, J = 7.7 Hz, 1H), 7.79 (t, J = 7.4 Hz, 1H), 8.01 (d, J = 7.7

Hz, 1H); 13C NMR (101 MHz, CDCl3) δ 116.0, 117.5, 126.3,

127.2, 141.9, 151.6, 151.9; LRMS-ESI: (m/z):

226.5932[M+H]+

3-Nitro-4-(thiophen-3-yl)-2H-chromen-2-one (39):

Yield:75%; White solid; Rf: 0.4[1:4 Hexanes: Ethyl acetate];

m.p. 164-166 ºC; νmax (KBr/cm-1): 3094, 1689, 1722, 1289,

1460; 1H NMR (400 MHz, DMSO – d6) δ 7.28 (dd, J = 5.0,

1.4 Hz, 1H), 7.46 - 7.49 (m, 2H), 7.63 (d, J = 8.2 Hz, 1H),

7.81 - 7.86 (m, 1H), 7.90 (dd, J = 5.0, 2.9 Hz, 1H), 7.96 (dd,

J = 2.9, 1.4 Hz, 1H); 13C NMR (101 MHz, CDCl3) δ 117.3,

117.5, 125.6, 127.2, 127.9, 127.9, 127.9, 128.8, 134.2, 142.4,

152.5, 153.3; LRMS-ESI(m/z): 274.2718[M+H]+

4-(Furan-3-yl)-3-nitro-2H-chromen-2-one (40):

Yield: 89%; White Solid; Rf: 0.4 [1:4 Hexanes: Ethyl acetate];

m.p. 126-128 ºC; νmax (KBr/cm-1): 3923, 1726, 1603, 1542,

1276; 1H NMR (400 MHz, DMSO – d6) δ 6.77 (dd, J = 1.9,

0.9 Hz, 1H), 7.46 - 7.52 (m, 1H), 7.60 (dd, J = 8.4, 0.9 Hz,

1H), 7.69 (dd, J = 8.1, 1.5 Hz, 1H), 7.83 (ddd, J = 8.6, 7.3, 1.6

Hz, 1H), 8.03 (t, J = 1.7 Hz, 1H), 8.18 (dd, J = 1.5, 4 Hz, 1H);

13C NMR (101 MHz, DMSO – d6) δ 111.2, 113.9, 117.3,

61

117.7, 126.3, 129.1, 135.0, 136.5, 139.6, 144.4, 145.9, 152.7,

153.7; LRMS-ESI(m/z): 258.2062[M+H]+

3-Amino-4-(thiophen-3-yl)-2H-chromen-2-one (41)

Yield: 44%; Rf: 0.5[1:4 Hexanes:Ethyl acetate]; m.p. 134-136

ºC; νmax (KBr/cm-1): 3452, 3344, 1704, 1615, 1173, 1451; m.p.

132-138 0C; 1H NMR (300 MHz, DMSO – d6) δ 5.17 (s, 2H),

7.05 (d, J = 7.2 Hz, 1H), 7.20 (t, J = 5.5 Hz, 2H), 7.27 - 7.45

(m, 2H), 7.72 (s, 1H), 7.83 (dd, J = 4.7, 2.9 Hz, 1H); 13C NMR

(101 MHz, DMSO – d6) δ 115.3, 116.3, 122.2, 123.9, 124.9,

126.5, 126.6, 128.1, 128.8, 130.5, 133.0, 147.9, 158.7;

LRMS-ESI(m/z): 244.2889[M+H]+

3-Amino-4-(furan-3-yl)-2H-chromen-2-one (42)

Yield: 46%; Rf: 0.6 [1:4 Hexanes:Ethyl acertate]; m.p. 116-

118 ºC; νmax (KBr/cm-1): 3435, 1704, 1622, 1167, 1018; 1H

NMR (400 MHz, CDCl3) δ 4.11 (s, 2H), 6.58 (dd, J = 1.7, 0.9

Hz, 1H), 7.16 - 7.20 (m, 1H), 7.28 (dd, J = 8.0, 1.8 Hz, 1H),

7.32 (d, J = 8.9 Hz, 2H), 7.68 (dt, J = 2.3, 1.5 Hz, 2H); 13C

NMR (101 MHz, CDCl3) δ 111.1, 113.4, 116.4, 116.9, 121.4,

124.0, 124.5, 126.8, 129.8, 141.8, 144.4, 148.4, 158.9;

LRMS-ESI(m/z): 228.2233[M+H]+

4-Phenyl-6H-chromeno[3,4-b]thieno[3,2-d]pyridin-6-one (43):

62

Yield: 89%; white solid; Rf: 0.5 [7:3 Hexanes:Ethyl acetate];

m.p. 242-244 °C; νmax (KBr/cm-1): 3034, 1735, 1603, 1458,

1178, 1097; 1H NMR (400 MHz, CDCl3): δ 7.45 – 7.54 (m,

2H), 7.57 – 7.63 (m, 4H), 8.12 (d, J = 5.6 Hz, 1H), 8.16 – 8.21

(m, 2H), 8.44 (d, J = 5.7 Hz, 1H), 8.52 (d, J = 7.5 Hz, 1H);

13C NMR (101 MHz, CDCl3): δ 117.8, 118.1, 124.2, 124.7,

126.0, 126.3, 128.8, 128.9, 130.7, 133.9, 134.8, 138.3, 140.2,

140.6, 151.1, 155.2, 159.7; HRMS-ESI(m/z): calcd for

C20H11NO2S [M + H]+ 330.0583 found 330.0551.

4-(p-Tolyl)-6H-chromeno[3,4-b]thieno[3,2-d]pyridin-6-one (44):

Yield: 42%; light yellow solid; Rf: 0.5 [7:3 Hexanes:Ethyl

acetate]; m.p. 194-196 °C; νmax (KBr/cm-1): 3069, 1736, 1458,

1178, 1094; 1H NMR (400 MHz, TFA-d1): δ 2.86 (s, 3H),

7.95 (d, J = 7.9 Hz, 2H), 8.02 - 8.12 (m, 2H), 8.21- 8.29 (m,

3H), 9.12 (d, J = 8.3 Hz, 1H), 9.26 (d, J = 5.5 Hz, 1H), 9.36

(d, J = 5.5 Hz, 1H); 13C NMR (101 MHz, TFA-d1): δ 20.2,

114.8, 122.9, 125.7, 126.6, 126.7, 127.6, 129.2, 129.4, 131.1,

134.63, 141.9, 144.5, 147.8, 148.4, 151.2, 153.9, 158.4;

HRMS-ESI(m/z): calcd for C21H13NO2S [M + H]+ 344.0740

found 344.0696.

4-(4-Methoxyphenyl)-6H-chromeno[3,4-b]thieno[3,2-d]pyridin-6-one (45):

63

Yield: 67%; light yellow solid; Rf: 0.5 [3:2 Hexanes:Ethyl


1248, 1035; 1H NMR (400 MHz, CDCl3): δ 3.94 (s, 3H), 7.11

(d, J = 8.4 Hz, 2H), 7.45-7.62 (m, 3H), 8.11 (d, J = 5.6 Hz,

1H), 8.19 (d, J = 8.2 Hz, 2H), 8.43 (d, J = 5.7 Hz, 1H), 8.51

(d, J = 8.1 Hz, 1H); 13C NMR (101 MHz, CDCl3): δ 50.7,

109.5, 113.2, 113.4, 119.5, 119.9, 121.2, 125.7, 125.8, 126.1,

129.0, 129.8, 135.0, 135.9, 146.2, 150.1, 155.0, 156.6;

HRMS-ESI(m/z): calcd for C21H13NO3S [M + H]+ 360.0689

found 360.0652.

4-(2-Hydroxyphenyl)-6H-chromeno[3,4-b]thieno[3,2-d]pyridin-6-one (46):

Yield: 49%; light yellow solid; Rf: 0.5 [3:2 Hexanes : Ethyl


1465, 1179, 1103; 1H NMR (400 MHz, CDCl3): δ 7.06 (dd, J

= 16.8, 9.4 Hz, 1H), 7.22 (d, J = 8.2 Hz, 1H), 7.43 – 7.53 (m,

3H), 7.61 (t, J = 7.5 Hz, 1H), 8.20 (s, 1H), 8.33 (d, J = 7.9

Hz, 1H), 8.48 (d, J = 9.0 Hz, 2H); 13C NMR (101 MHz,

CDCl3): δ 117.3, 118.3, 118.7, 118.9, 119.4, 124.2, 125.0,

125.7, 126.0, 127.9, 130.4, 131.0, 132.7, 135.4, 137.3, 141.5,


C20H11NO3S [M + H]+ 346.0532 found 346.0496.

4-(6-Oxo-6H-chromeno[3,4-b]thieno[3,2-d]pyridin-4-yl)benzonitrile (47):

64



1607, 1173, 1095; 1H NMR (400 MHz, TFA-d1): δ 7.99 - 8.10

(m, 2H), 8.19 - 8.25 (m, 1H), 8.47 (dd, J = 26.7, 7.3 Hz, 4H),

9.08 (d, J = 7.7 Hz, 1H), 9.28 (s, 1H), 9.38 (d, J = 4.7 Hz,

1H); 13C NMR (101 MHz, TFA-d1): δ 118.9, 123.6, 126.7,

127.0, 127.7, 130.5, 133.4, 133.9, 135.1, 142.3, 145.3, 149.1,

150.9, 151.3, 157.8; HRMS-ESI(m/z): calcd for C21H10N2O2S

[M + H]+ 355.0536 found 355.0488.

4-(4-(Trifluoromethyl)phenyl)-6H-chromeno[3,4-b]thieno[3,2-d]pyridin-6-one (48):

Yield: 75%; white solid; Rf: 0.5 [7:3 Hexanes : Ethyl

acetate]; m.p. 218-220 °C; νmax(KBr/cm-1): 3088, 2356, 1746,

1690, 1324, 1169, 1117; 1H NMR (400 MHz, TFA-d1): δ 8.61

- 8.72 (m, 2H), 8.83 (t, J = 7.9 Hz, 1H), 8.97 (d, J = 8.1 Hz,

2H), 9.07 (d, J = 8.1 Hz, 2H), 9.71 (d, J = 8.2 Hz, 1H), 9.89

(d, J = 5.5 Hz, 1H), 9.99 (d, J = 5.5 Hz, 1H); 13C NMR (101

MHz, TFA-d1): δ 115.2, 123.8, 124.1, 127.3, 127.5, 127.8,

128.3, 130.8, 132.4, 135.6, 137.6(2JC-F = 34.4 Hz), 142.9,


C21H10F3NO2S [M + H]+ 398.0444 found 398.0432.

4-(4-Fluorophenyl)-6H-chromeno[3,4-b]thieno[3,2-d]pyridin-6-one (49):

65

Yield: 84%; white solid; Rf: 0.4 [7:3 Hexanes : Ethyl


1227, 1172, 1097; 1H NMR (400 MHz, CDCl3): δ 7.29 (s, 2H,

merged with residual CHCl3 peak of CDCl3), 7.46 - 7.52 (m,

2H), 7.59 – 7.63 (m, 1H), 8.13-8.16 (two merged broad

singlets, 3H), 8.44(broad singlet, 1H), 8.52(broad singlet);

13C NMR (101 MHz, CDCl3): δ 111.2, 113.0, 113.4, 119.6,

120.1, 121.4, 121.7, 126.1, 126.2, 129.1, 129.6, 130.0, 136.0,


C20H10FNO2S [M + H]+ 348.0489 found 348.0458.

4-(4-Bromophenyl)-6H-chromeno[3,4-b]thieno[3,2-d]pyridin-6-one (50):

Yield: 79%; light yellow solid; Rf: 0.4[7:3 Hexanes : Ethyl


1460, 1176, 1096; 1H NMR (400 MHz, TFA-d1): δ 8.08-8.13

(m, 2H), 8.23 - 8.33 (m, 5H), 9.15 (bs, 1H), 9.31 (s, 1H), 9.41

(s, 1H); 13C NMR (101 MHz, TFA-d1): δ 114.7, 123.2, 126.6,

126.9, 127.1, 127.7, 129.9, 130.5, 130.9, 133.9, 134.9, 142.1,


C20H10BrNO2S [M + H]+ 407.9688 found 407.9637

(409.9616).

4-(2,4-Dichlorophenyl)-6H-chromeno[3,4-b]thieno[3,2-d]pyridin-6-one (51):

66

Yield: 73%; white solid; Rf: 0.5 [7:3 Hexanes : Ethyl acetate];

m.p. 246-248 °C; νmax (KBr/cm-1): 3059, 1740, 1594, 1457,

1185, 1106, 754; 1H NMR (400 MHz, CDCl3): δ 7.39 – 7.72

(m, 6H), 8.14 (s, 1H), 8.45-8.55 (two broad singlets merging,

2H); 13C NMR (101 MHz, CDCl3): δ 117.7, 118.2, 124.3,

125.0, 126.4, 127.6, 130.0, 131.2, 132.1, 133.9, 135.4, 136.4,

140.0, 151.2; HRMS-ESI (m/z): calcd for C20H9Cl2NO2S [M

+ H]+ 397.9804 found 397.9753.

4-(Thiophen-2-yl)-6H-chromeno[3,4-b]thieno[3,2-d]pyridin-6-one (52):



1164, 1100; 1H NMR (400 MHz, CDCl3): δ 7.26 (t, J = 4.0

Hz, 1H), 7.42 – 7.48 (m, 2H), 7.56 (t, J = 7.6 Hz, 1H), 7.62

(d, J = 4.9 Hz, 1H), 8.07 (d, J = 3.5 Hz, 1H), 8.10 (d, J = 5.3

Hz, 1H), 8.38 (d, J = 5.3 Hz, 1H), 8.43 (d, J = 7.9 Hz, 1H);

13C NMR (101 MHz, CDCl3): δ 117.7, 118.1, 124.2, 124.8,

125.8, 126.0, 128.21, 128.2, 130.1, 130.6, 133.4, 134.2,

137.2, 140.8, 142.6, 148.5, 150.9, 159.1; HRMS-ESI(m/z):

calcd for C18H9NO2S2 [M + H]+ 336.0147 found 336.0103.

4-(Pyridin-2-yl)-6H-chromeno[3,4-b]thieno[3,2-d]pyridin-6-one (53):

67



1465, 1184, 1102; 1H NMR (400 MHz, CDCl3): δ 7.44 – 7.48

(m, 2H), 7.51 (dd, J = 8.2, 1.3 Hz, 1H), 7.57 – 7.62 (m, 1H),

7.96 (td, J = 7.8, 1.7 Hz, 1H), 8.19 (d, J = 5.7 Hz, 1H), 8.38

(d, J = 5.8 Hz, 1H), 8.54 (d, J = 8.1 Hz, 1H), 8.83 (d, J = 5.6

Hz, 1H), 8.97 (d, J = 8.0 Hz, 1H). 13C NMR (101 MHz,

CDCl3): δ 118.1, 122.6, 122.9, 124.6, 124.7, 126.5, 127.4,

130.8, 133.0, 137.0, 137.9, 139.0, 141.4, 147.7, 150.7, 151.1,

154.2, 159.7; HRMS-ESI(m/z): calcd for C19H10N2O2S [M +

H]+ 331.0536 found 331.0502.

4-([1,1'-Biphenyl]-4-yl)-6H-chromeno[3,4-b]thieno[3,2-d]pyridin-6-one (54):


m.p. 238-240 °C; νmax (KBr/cm-1): 3104, 1741, 1605, 1420,

1177, 1097; 1H NMR (400 MHz, TFA-d1): δ 7.74 – 7.86 (m,

3H), 8.00 – 8.15 (m, 4H), 8.21 – 8.29 (m, 1H), 8.36 – 8.41

(m, 2H), 8.44 – 8.51 (m, 2H), 9.11 – 9.17 (m, 1H), 9.26 – 9.32

(m, 1H), 9.36 – 9.42 (m, 1H); 13C NMR (101 MHz, TFA-d1):

δ 112.7, 113.7, 114.8, 123.1, 126.6, 126.7, 126.9, 127.1,

127.6, 128.9, 129.2, 129.3, 129.5, 129.8, 134.7, 138.6, 141.9,

144.6, 148.4, 149.2, 151.2, 153.5, 158.4; HRMS-ESI(m/z):

calcd for C26H15NO2S [M + H]+ 406.0896 found 406.0838.

4-(Anthracen-9-yl)-6H-chromeno[3,4-b]thieno[3,2-d]pyridin-6-one (55):

68



1446, 1240; 1H NMR (400 MHz, TFA-d1): δ 7.29 (d, J = 8.7

Hz, 2H), 7.63 (dt, J = 14.8, 7.2 Hz, 4H), 7.84 - 7.97 (m, 2H),

8.07 (t, J = 7.7 Hz, 1H), 8.32 (d, J = 8.1 Hz, 2H), 8.96 - 9.16

(m, 4H); 13C NMR (101 MHz, TFA-d1): δ 114.5, 118.6,

118.9, 121.8, 123.5, 125.9 126.2, 126.4, 127.3, 129.0, 129.2,

130.2, 130.4, 130.8, 133.6, 134.6, 144.2, 145.5, 148.8, 151.0,

153.1, 157.9; HRMS-ESI(m/z): calcd for C28H15NO2S [M +

H]+ 430.0896 found 430.0845.

4-Phenyl-6H-chromeno[3,4-b]furo[3,2-d]pyridin-6-one (56):


m.p. 220-222 °C; νmax (KBr/cm-1): 3111, 1744, 1602, 1328,

1145; 1H NMR (400 MHz, CDCl3): δ 7.46-7.49(m, 2H), 7.54-

7.61(m, 4H), 7.65 (s, 1H), 8.15 (s, 1H), 8.31 (s, 1H), 8.54 (d,

J = 4.0 Hz, 2H); 13C NMR (101 MHz, DMSO-d6): δ 102.7,

113.2, 113.1, 120.0, 120.4, 121.6, 123.9, 124.5, 124.9, 125.7,

126.1, 127.6, 129.8, 139.8, 144.5, 146.4, 154.3; HRMS-ESI

(m/z) calcd for C20H11NO3 [M + H]+ 314.0812 found

314.0772.

4-(p-Tolyl)-6H-chromeno[3,4-b]furo[3,2-d]pyridin-6-one (57):

69



1458, 1154; 1H NMR (400 MHz, CDCl3): δ 2.46 (s, 3H), 7.36

(d, J = 7.7 Hz, 2H), 7.45 (d, J = 8.4 Hz, 2H), 7.55 (t, J = 8.0

Hz, 1H), 7.61 (s, 1H), 8.11 (s, 1H), 8.26 (d, J = 7.6 Hz, 1H),

8.43 (d, J = 7.7 Hz, 2H); 13C NMR (101 MHz, CDCl3): δ 21.5,

107.4, 117.9, 124.7, 125.1, 126.0, 129.1, 129.4, 129.5, 130.7,

131.8, 132.3, 140.8, 144.5, 149.2, 151.0, 151.8, 159.2;

HRMS-ESI (m/z) calcd for C21H13NO3 [M + H]+ 328.0968

found 328.0937.

4-(4-Methoxyphenyl)-6H-chromeno[3,4-b]furo[3,2-d]pyridin-6-one (58):



1455, 1251; 1H NMR (400 MHz, CDCl3): δ 3.93 (s, 3H), 7.08

(s, 2H), 7.40 – 7.68 (m, 4H), 8.13 - 8.26 (two merged broad

singlets, 2H), 8.54 (s, 2H); 13C NMR (101 MHz, DMSO-d6):

δ 50.8, 102.8, 109.4, 113.1, 113.2, 120.0, 120.3, 120.9, 122.5,

124.7, 125.8, 126.1, 127.4, 139.4, 144.5, 146.2, 146.8, 154.5,

156.7; HRMS-ESI (m/z) calcd for C20H13NO4 [M + H]+

344.0917 found 344.0881.

4-(2-Hydroxyphenyl)-6H-chromeno[3,4-b]furo[3,2-d]pyridin-6-one (59):

70


acetate]; m.p. 286-288 °C; νmax (KBr/cm-1): 3148, 1746,

1606.4, 1465, 1157; 1H NMR (400 MHz, TFA-d1): δ 7.77 (s,

2H), 8.13 (s, 3H), 8.27 (s, 1H), 8.55 (s, 1H), 8.97 (s, 1H), 9.14

(s, 1H), 9.38 (s, 1H); 13C NMR (101 MHz, TFA-d1): δ 109.9,

114.9, 118.1, 121.8, 123.4, 126.1, 127.8, 128.1, 132.8, 134.7,

136.5, 138.1, 141.1, 150.5, 151.3, 157.4, 157.6, 158.4;

HRMS-ESI (m/z) calcd for C20H11NO4 [M + H]+ 330.0761

found 330.0726.

4-(6-Oxo-6H-chromeno[3,4-b]furo[3,2-d]pyridin-4-yl)benzonitrile (60):



1510, 1210; 1H NMR (400 MHz, TFA-d1): δ 8.09 (d, J = 8.4

Hz, 1H), 8.15 (t, J = 7.8 Hz, 1H), 8.31 (t, J = 7.9 Hz, 1H),

8.52 (d, J = 8.5 Hz, 2H), 8.63 (d, J = 2.2 Hz, 1H), 8.75 (d, J =

8.5 Hz, 2H), 8.97 (d, J = 8.1 Hz, 1H), 9.20 (d, J = 2.1 Hz, 1H);

13C NMR (101 MHz, TFA-d1): δ 110.6, 114.4, 116.8, 123.9,

126.2, 127.8, 129.8, 130.2, 131.3, 133.8, 135.5, 138.2, 140.9,

151.1, 151.3, 157.2, 160.3; HRMS-ESI (m/z) calcd for

C21H10N2O3 [M + H]+ 339.0764 found 339.0733.

4-(4-(Trifluoromethyl)phenyl)-6H-chromeno[3,4-b]furo[3,2-d]pyridin-6-one (61):

71


m.p. 224-226 °C; νmax (KBr/cm-1): 3137, 1745, 1610, 1326,

1109; 1H NMR (400 MHz, TFA-d1): δ 8.08 (d, J = 8.4 Hz,

1H), 8.11 - 8.16 (m, 1H), 8.27 - 8.32 (m, 1H), 8.41 (d, J = 8.4

Hz, 2H), 8.60 (d, J = 2.2 Hz, 1H), 8.70 (d, J = 8.3 Hz, 2H),

8.96 (dd, J = 8.2, 1.2 Hz, 1H), 9.19 (d, J = 2.1 Hz, 1H); 13C

NMR (101 MHz, TFA-d1): δ 114.5, 123.2, 123.6, 126.2,

127.0, 127.1, 127.8, 127.9, 129.8, 130.9, 135.3, 137.3(2JC-F =

34.4 Hz), 137.9, 142.0, 151.1, 151.3, 157.5, 160.1; HRMS-

ESI (m/z) calcd for C21H10F3NO3 [M + H]+ 382.0686 found

382.0652.

4-(4-Fluorophenyl)-6H-chromeno[3,4-b]furo[3,2-d]pyridin-6-one (62):

Yield: 88%; white solid; Rf: 0.5 [3:2 Hexanes: Ethyl acetate];

m.p. 196-198 °C; νmax (KBr/cm-1): 3131, 1747, 1604, 1460,

1229, 1155; 1H NMR (400 MHz, CDCl3): δ 7.25 (t, J = 8.5

Hz, 2H), 7.45 (t, J = 8.0 Hz, 2H), 7.57 (t, J = 7.6 Hz, 1H),

7.64 (s, 1H), 8.15 (s, 1H), 8.27 (d, J = 7.7 Hz, 1H), 8.56 (dd,

J = 8.4, 5.5 Hz, 2H); 13C NMR (101 MHz, DMSO) δ 102.8,

110.9, 111.1, 113.1 (2JC-F = 19.19 Hz), 120.1, 120.4, 121.5,

124.9, 126.0 (4JC-F = 3.03 Hz), 126.1, 126.6 (3JC-F = 9.09 Hz),

127.5, 138.5, 144.6, 146.6, 146.8, 154.3, 159.5 (1JC-F = 252.50

Hz).HRMS-ESI (m/z) calcd for C20H10FNO3 [M + H]+

332.0717 found 332.0685.

72

4-(4-Bromophenyl)-6H-chromeno[3,4-b]furo[3,2-d]pyridin-6-one (63):



1458, 1146, 762; 1H NMR (400 MHz, TFA-d1): δ 8.17(d, J =

8 Hz, 1H), 8.23 (t, J = 7.2 Hz, 1H), 8.36 – 8.42 [ two merged

doublets, J = ~8 Hz(each), 3H], 8.56 (d, J = 7.2 Hz, 2H), 8.67

(s, 1H), 9.05 (d, J = 7.5 Hz, 1H), 9.26 (s, 1H); 13C NMR (101

MHz, TFA-d1): δ 114.5, 118.8, 123.3, 123.4, 126.1, 127.7,

129.3, 131.3, 131.7, 133.8, 135.1, 137.5, 142.6, 150.9, 151.2,

157.6, 159.6; HRMS-ESI (m/z) calcd for C20H10BrNO3

[M+H]+ 391.9917 found 391.9874 (393.9856).

4-(2,4-Dichlorophenyl)-6H-chromeno[3,4-b]furo[3,2-d]pyridin-6-one (64):


m.p. 260-262 °C; νmax (KBr/cm-1): 3169, 1749, 1603, 1461,

1143; 1H NMR (400 MHz, CDCl3): δ 7.37 (bs, 1H), 7.47 –

7.54 (m, 2H), 7.60 (s, 1H), 7.62 – 7.70 (singlet and multiplet,

3H), 8.11 (s, 1H), 8.36-8.38 (d, J = 8.3 Hz, 1H); 13C NMR

(101 MHz, DMSO): δ 102.9, 113.3, 120.2, 120.7, 122.8,

124.4, 124.9, 126.6, 128.3, 129.7, 131.7, 138.8, 145.1, 146.5,

147.3, 154.2; HRMS-ESI (m/z) calcd for C20H9Cl2NO3

[M+H]+ 382.0032 found 381.9996.

4-(Thiophen-2-yl)-6H-chromeno[3,4-b]furo[3,2-d]pyridin-6-one (65):

73



1453, 1141, 199; 1H NMR (400 MHz, CDCl3): δ 7.26 (s, 1H),

7.46 (d, J = 8.2 Hz, 2H), 7.53 – 7.59 (m, 1H), 7.63 (s, 2H),

8.15 (s, 1H), 8.26 (d, J = 6.9 Hz, 1H), 8.29 (d, J = 2.8 Hz,

1H); 13C NMR (101 MHz, CDCl3): δ 107.6, 117.8, 117.9,

124.8, 125.0, 125.8, 128.4, 129.1, 129.9, 130.2, 130.7, 132.2,

139.2, 139.9, 149.4, 149.8, 150.9, 158.7; HRMS-ESI(m/z)

calcd for C18H9NO3S [M+H]+ 320.0736 found 320.0343.

4-(Pyridin-2-yl)-6H-chromeno[3,4-b]furo[3,2-d]pyridin-6-one (66):



1523, 1144; 1H NMR (400 MHz, CDCl3): δ 7.46 (s, 3H),

7.59(s, 1H), 7.66 (s, 1H), 7.96 (s, 1H), 8.24 (s, 1H), 8.32 (s,

1H), 8.68 (s, 1H), 8.89 (s, 1H), 13C NMR (101 MHz, CDCl3):

δ 107.1, 117.7, 118.0, 124.4, 124.5, 124.9, 125.4, 127.4,

130.7, 131.2, 131.9, 137.1, 143.1, 149.3, 150.5, 151.2, 151.9,

154.0, 158.9; HRMS-ESI (m/z) calcd for C19H10N2O3 [M+H]+

315.0764 found 315.0725.

4-([1,1'-Biphenyl]-4-yl)-6H-chromeno[3,4-b]furo[3,2-d]pyridin-6-one (67):

74


m.p. 218-224 °C; νmax (KBr/cm-1): 2355, 1737, 1606,

1249, 1101; 1H NMR (400 MHz, TFA-d1): δ 7.49 (dd, J = 8.0,

6.3 Hz, 1H), 7.55 (t, J = 7.5 Hz, 2H), 7.55 - 7.81 (m, 3H),

7.85 (t, J = 7.8 Hz, 1H), 7.99 (t, J = 7.9 Hz, 1H), 8.11 (d, J =

7.3 Hz, 2H), 8.29 (d, J = 1.6 Hz, 1H), 8.42 (d, J = 8.1 Hz, 2H),

8.67 (d, J = 8.1 Hz, 1H), 8.89 (d, J = 2.0 Hz, 1H); 13C NMR

(101 MHz, TFA-d1): δ 109.9, 114.2, 118.3, 122.6, 122.7,

125.6, 126.7, 127.3, 128.4, 128.5, 128.8, 129.0, 130.3, 134.5,

136.7, 138.1, 142.8, 149.2, 150.6, 150.7, 157.4, 158.9;

HRMS-ESI (m/z) calcd for C26H15NO3 [M+H]+ 390.1125

found 390.1093.

4-(Anthracen-9-yl)-6H-chromeno[3,4-b]furo[3,2-d]pyridin-6-one (68):



1460, 1148; 1H NMR (400 MHz, TFA-d1): δ 7.33 (d, J = 8.7

Hz, 2H), 7.62 (dt, J = 15.3, 7.4 Hz, 4H), 7.83 (d, J = 8.3 Hz,

1H), 7.91 (t, J = 7.7 Hz, 1H), 8.05 (t, J = 7.9 Hz, 1H), 8.29 (d,

J = 8.5 Hz, 2H), 8.39 (s, 1H), 8.71 (s, 1H), 8.79 (d, J = 8.1

Hz, 1H), 8.98 (s, 1H); 13C NMR (101 MHz, TFA-d1): δ 114.3,

121.8, 123.8, 125.8 (two peaks: 125.83 and 125.88), 127.4,

129.0, 129.2, 129.9, 130.8, 130.9, 133.7, 134.8, 137.1, 143.3,

75

150.9, 153.0, 157.3, 159.5; HRMS-ESI (m/z) calcd for

C28H15NO3 [M+H]+ 414.1125 found 414.1087.

1H NMR (400 MHz, CDCl3): 4-phenyl-6H-chromeno[3,4-b]thieno[3,2-d]pyridin-6-one (43)

13C NMR (101MHz, CDCl3): 4-phenyl-6H-chromeno[3,4-b]thieno[3,2-d]pyridin-6-one (43)

76

1H NMR (400 MHz, CDCl3): 4-phenyl-6H-chromeno[3,4-b]furo[3,2-d]pyridin-6-one (56)

13C NMR (101 MHz, CDCl3): 4-phenyl-6H-chromeno[3,4-b]furo[3,2-d]pyridin-6-one (56)

77

Anticancer activity determination by MTT assay:

The prostate cancer (DU-145), murine skin melanoma (B16F10) and human breast cancer

(MCF-7) cell lines were used to determine anticancer activity of the novel compounds by MTT

assay. Dulbecco’s modified Eagle’s media (DMEM), supplemented with 1% antibiotic solution

and 10% fetal bovine serum (FBS) was used to culture the cells. MTT [3-(4,5-dimethylthiazol-2-

yl)-2,5-diphenyltetrazolium bromide], a yellow dye was used for the assay. All chemicals were

purchased from Himedia (Mumbai, India).

Briefly, DU-145 cells were seeded in 96 well plate with the cell density of 1× 104 per 100

µL/well and were allowed to attached for overnight. Following day, the medium was removed and

cells were treated with novel compounds at concentration of 100µM and 10 µM in 100 µL

complete medium in triplicate wells. After 48 hours of incubation, medium was removed and 50µL

of 5 mg/mL MTT solution was added in to each well and kept for 4 hours which is sufficient time

to form the formazan crystals. MTT solution was removed after 4 hours and 150 µL of DMSO was

added to dissolve formed formazan crystals after gentle shaking. Absorbance intensity of the

solution in the 96 well plate was measured by using a microplate reader (Spectramax™, microplate

reader, Molecular Devices, US) at 570 nm and 650 nm. The same procedure was followed for all

3 cell lines.

It was observed from the initial two dosage screening of anticancer activity that eight

compounds out of twenty eight novel compounds showed more promising activity and they were

further screened to determine IC50 values. The various concentrations, 200 µM, 100 µM, 50 µM,

25 µM, 12.5 µM, 6.25 µM, 3.125 µM, 1.562 µM, 0.781 µM and 0.390 µM were used. The

experiment was repeated following the same protocol but with different batch of cells and cell

viability was measured by MTT assay as discussed.12

78

Chapter 4 (A)

Synthesis of Diverse 2-Acylpyrroles from Chalcones Using

Polyphosphoric Acid–Mediated Regiospecific Acyl Migration

79

Introduction

2-Acylpyrroles on their own or as part of a larger framework are known for a multitude of

biological/biomedical properties ranging from non-steroidal anti-inflammatory drugs (NSAID),

antibiotics to anticancer compounds.[1-5]. Some important examples of this type of compounds

include tolmetin, ketorolac, pyoluteorin, calcimycin and diverse marinopyrroles (Figure-4.1.1).

Figure 4.1.1 Examples of biologically active 2-acylpyrrole compounds

Acyl migrations within the molecular framework represent an important organic

transformation used in multitude of synthetic strategies. Mamer et al., in a paper reported acid

catalyzed acyl rearrangement on a taxane, 9-dihydro-13-acetylbaccatin-III (Scheme 4.1.1).[6]

Ashfield and co-workers applied the chemoselective O- to N- acyl migration in the Staudinger

ligation for the synthesis of diverse amides (Scheme 4.1.2).[7] In a paper by Arganat and co-

workers, Friedel-Crafts acyl rearrangement which is primarily a C- to C-acyl migration, has

been used for the synthesis of diverse acyl fluoranthene compounds (Scheme 4.1.3).[8] The

reported reactions were carried out in polyphosphoric acid (PPA) at various temperatures and

resulted in the regioselective formation of the target molecules.

80

Scheme 4.1.1 Acid rearrangement on 9-dihydro-13-acetylbaccatin III

Scheme 4.1.2 Acyl migration leading to diverse amides

Scheme 4.1.3 Rearrangement of 3-acetyl fluoranthene to 8-acetyl fluoranthene

81

Several other type of acyl rearrangement reactions are also known, which include base-

induced Baker-Venkataraman and acid catalyzed Fries rearrangements. Yu et al. reported the

synthesis of multi‐functionalized chromeno[2,3‐c]pyrrol‐9(2H)‐ones from 1,3‐diaryl‐1,3‐

diketones and amino acids using 4‐(dimethylamino)pyridine‐catalyzed Baker–Venkataraman

rearrangement as a key step.[9]

Similar acyl migration was used by Fougerousse et al. for the synthesis of

dibenzoylmethanes.[10] Fries rearrangement was used by Jeon and Magnion for the synthesis

of hydroxyl aryl ketones using methanesulfonic acid and methanesulfonic anhydride.[11]

Maeda et al. reported application of photo Fries rearrangement on pyrenyl esters.[12] (Scheme

4.1.4) Reported reaction was amenable to the presence of electron releasing/withdrawing

groups as well as diverse heteroaromatic carboxylates.

Scheme 4.1.4 Photo-Fries rearrangement of phenyl 1-pyrene carboxylate.

Base promoted C→N acyl migration was used by Vicario and co-workers for the

synthesis of non proteinogenic tertiary and quaternary N-alkyl α-amino acids.[13] In a recent

work by Kong et al. indolo[3,2-c]quinolinones were synthesized via a Pd/Cu catalysed 1,2-

acyl migration, starting from indole-2-carboxamides.[14] (Scheme 4.1.5)The methods reported

thus clearly demonstrate the applicability of acyl rearrangement in diverse synthetic routes

82

Scheme 4.1.5 Palladium catalysed 1,2-acyl migration

However, acyl rearrangement has been rarely used as a method for the preparation of

2-acylpyrroles. One of the very first examples of acyl rearrangement on pyrrole systems was

reported by Palmer et al. in their attempt to cyclize various 3-(2-pyrrolyl)propanoic acids.[15]

It was noticed that in the presence of PPA, both the desired compound 4H-

cyclopenta[b]pyrrole-4-ones as well as corresponding pyrrole-6-ones were obtained. Acyl

rearrangement on pyrrole systems was also studied by Carson and Davis.[16] In their work,

rearrangement of N-alkyl-2-acylpyrroles to corresponding N-alkyl-3-acylpyrroles was noticed

in the presence of anhydrous trifluoroacetic acid (TFA). However, 2 or 3-acylpyrroles bearing

hydrogen on nitrogen resulted in an equilibrium mixture of both the acylated pyrroles, with

their ratio dependent on the nature of the reaction medium. Dellemagne et al. in a separate

paper has explored TFA mediated synthesis 1-phenylpyrrole-3-carboxaldehydes from their 2-

isomers using dichloroethane as a solvent.[17] The final products were obtained in good yields

with very high regioselectivity. Acyl rearrangement of 2-acyl pyrroles to their C-3 isomers

was also noticed by Pina et al. in their attempt to carryout acylation of 1-nitrophenyl pyrroles,

using acid anhydrides in the presence of catalytic amount of orthophosphoric acid.[18]

Interestingly, most of the examples show rearrangement of 2-acyl systems to their 3-acyl

congeners and very few papers have highlighted the reverse process. Jefford and coworkers

have reported the synthesis of 2-acylpyrroles by intramolecular delivery of acylium ion from

83

their N-substituted mixed anhydrides to C-2 position of pyrroles.[19] These reactions were

performed using the stoichiometric amount of AlCl3 and dry Et2O as a solvent in modest to

good yields.

Based on the above reports we initiated studies on synthesis of diverse 4-substituted-2-

acylpyrroles (a) using rarely implemented acyl rearrangement. The simplicity of such a

transformation and the possibility of introducing acyl group by avoiding unselective Friedel-

Crafts/Vilsmeier-Haack reaction was an added attraction. A straightforward retrosynthetic strategy

was conceived to start our investigation in this domain. Compound (b) was envisaged as the

substrate for the final step leading to c. Synthesis of compound b was supposed to be carried out

by reaction between easily accessible chalcones (c) via van Leusen’s pyrrole synthesis.[20-23]

Scheme 4.1.6 Retrosynthetic analysis of the target molecules

Results and discussion

Our initial synthetic efforts started from chalcones, which were converted to corresponding di-

substituted pyrroles by reaction with well-known isonitrile source TosMIC in the presence of

potassium tert-butoxide and DMSO as solvent at the room temperature for 0.5 hours (Scheme

4.1.7).

Scheme 4.1.7 Synthesis of 3,3’-disubstituted pyrroles

84

After fully characterizing the products, we initiated a screening program to identify the most

appropriate condition for their conversion to the target molecules (Table-4.1.1). For this purpose

phenyl(4-phenyl-1H-pyrrol-3-yl)methanone (21) was chosen as the model substrate. As 1,2-acyl

shift/migration was the desired goal of this work, choice of reaction conditions was based on Fries

and other 1,2-acyl rearrangements reported in literature.[8, 24-28] Thus, reactions were attempted

with Lewis acids such as AlCl3, ZnCl2 and FeCl3 (Entry 1-3). While no conversion was noticed

in case of AlCl3 and ZnCl2, FeCl3 gave the target molecule in 66% yield. The purified product was

thoroughly characterized by 1H and 13C NMR, prior to further optimization of reaction conditions.

1H NMR of compound 44 revealed downfield shift of NH peak to 12.25 ppm from 11.64 ppm in

21, which indicated the presence of an electron withdrawing group on the adjacent carbon (Figure-

4.1.2). Additionally, disappearance of C-H peak at position 2 and presence of a proton at carbon-

3 indicates rearrangement of acyl group to 2nd position of pyrrole. 13C NMR further substantiated

the proof for acyl rearrangement as carbonyl peak underwent a 6.5 ppm downfield shift to 184.2

ppm (Figure-4.1.3). Single crystal X-ray crystallography further ascertained the structure of

compound 44 (CCDC number 1859629) as shown in Figure-4.1.4. The pyrrole ring and phenyl

ring substituted to the pyrrole ring are almost coplanar with dihedral angle C11-C10-C12-C13 =

0.36º only. The phenyl ring plane which is attached to the carbonyl group is deviating 40.83º from

the pyrrole ring plane. The molecules form a hydrogen bonded dimer with pyrrole N―H as donor

and the carbonyl oxygen atom as acceptor with N―H…O non-covalent hydrogen bond length and

D―H―A bond angle of 2.03 Å and 155.3º respectively.

85

Table 4.1.1 Screening of reaction conditions for acyl migration

All the reactions were carried out for 12 hours. a Isolated yield. N.R. No reaction b Complete conversion was noticed in 30 minutes.

Entry Acid (20 mol%) Solvent Temp.(ºC) Yield (%)a

1 AlCl3 1,2-DCB 140 NR

2 ZnCl2 1,2-DCB 140 NR

3 FeCl3 1,2-DCB 140 66

4 FeCl3 DCM 40 20

5 FeCl3 THF 60 NR

6 FeCl3 1,4-dioxane 110 NR

7 FeCl3 PhNO2 140 28

8 FeCl3 Ph2O 140 48

9 FeCl3 1,2-DCE 85 NR

10 CH3COOH - 120 NR

11 CH3COOH 1,2-DCB 140 NR

12 CH3COOH THF 140 NR

13 H3PO4 - 80 NR

14 H3PO4 1,2-DCB 140 34

15 CF3SO3H THF 60 NR

16 HCl THF 60 NR

17 PPA 1,2-DCB 140 NR

18b PPA - 110 73

19 PPA PEG-400 110 NR

86

Figure 4.1.2 Comparison of 1H NMR of phenyl(4-phenyl-1H-pyrrol-3-yl)methanone (21) and

phenyl(4-phenyl-1H-pyrrol-2-yl)methanone (44)

87

Figure 4.1.3 Comparison of 13C NMR of phenyl(4-phenyl-1H-pyrrol-3-yl)methanone (21) and

phenyl(4-phenyl-1H-pyrrol-2-yl)methanone (44)

Figure 4.1.4 The ORTEP diagram of compound 44. The thermal ellipsoids are drawn with the

50% probability level

88

Our initial success with FeCl3 using 1,2-DCB as solvent prompted us to explore reactions with

different solvents and reaction temperatures (Entry 4-9). While product formation was observed

with DCM, PhNO2 and Ph2O, the yields obtained were lower compared to initial attempt with

FeCl3. Further screenings were carried out with Brönsted acids (Entry 10-19). Here reaction was

successful only with H3PO4 and PPA (neat), with PPA mediated reaction giving highest yield of

73%. Interestingly, when 1,2-DCB or PEG-400 were used as solvents in reactions involving PPA,

no product formation was noticed. Based on the above results, we established the use of PPA (neat)

at 110 °C for 0.5 hours as the best condition for the formation of 2-acyl pyrrole (44) from 3-acyl

pyrrole (21).

With the optimized conditions in hand, assortments of 4-substituted-3-acylpyrroles were used

to explore the substrate scope of the reaction (Table-4.1.2). Reactions were initially attempted by

varying the substituents at 4th position of 3-benzoylpyrrole (21-27). While all the aforementioned

substrates were compatible, comparatively better yield was obtained with p-tolyl substituent.

Substrate 24 bearing p-nitrophenyl substituent provided the desired product in 53% yield. With

mono/di-halogenated halogen phenyl substituents (25-27), final products could be obtained in

modest to good yields. Subsequent studies were conducted on 3-acylpyrroles bearing bi-phenyl,

anthracen-9-yl and methyl as substituents (28-30). In these studies an inverse relationship was

shown between the size of the substituent and the yield of the reaction. N-substituted systems (31-

32) on the other hand gave modest yield, indicating steric hindrance posed by the presence of

substituent on pyrrole nitrogen.

89

Table 4.1.2 Synthesis of diverse 2-acylpyrroles

Further studies were carried out to ascertain the effect of acyl groups on the feasibility/yield

of the reaction (33-43). Substrates with diverse benzoyl substituents indicated possible

involvement of acyl carbocation in the reaction, as relatively high yields were seen with molecules

bearing methyl and methoxy/di-methoxy groups as substituents (56-58). In case of p-fluoro and p-

chloro substituted benzoyl systems comparatively low yields (25% and 52%, respectively) were

recorded (59-60). Applying the established conditions on naphthalen-2-yl(4-phenyl-1H-pyrrol-3-

44 R = Ph; R’ = Ph; R’’=H; 73% 56 R = (p-CH3)Ph; R’ = Ph; R’’=H; 64%

45 R = Ph; R’ = (p-CH3)Ph; R’’=H; 74% 57 R = (p-OCH3)Ph; R’ = Ph; R’’=H; 53%

46 R = Ph; R’ = (p-OCH3)Ph; R’’=H; 37% 58 R = (m, p-OCH3, OCH3)Ph; R’ = Ph;

R’’=H; 63%

47 R = Ph; R’ = (p-NO2)Ph; R’’=H; 53% 59 R = (p-F)Ph; R’ = Ph; R’’=H; 25%

48 R = Ph; R’ = (p-F)Ph; R’’=H; 55% 60 R = (p-Cl)Ph; R’ = Ph; R’’=H; 52%

49 R = Ph; R’ = (o, p-Cl, Cl)Ph; R’’=H; 65% 61 R = naphthalen-2-yl; R’ = Ph; R’’=H;

55%

50 R = Ph; R’ = (o, p-Cl, F)Ph; R’’=H; 60% 62 R = thiophen-2-yl; R’ = Ph; R’’=H; 77%

51 R = Ph; R’ = (p-Ph)Ph; R’’=H; 65% 63 R = thiophen-2-yl; R’ = (p-OCH3)Ph;

R’’=H; 45%

52 R = Ph; R’ = anthracen-9-yl; R’’=H; 22% 64 R = thiophen-2-yl; R’ = (o, p-Cl, Cl)Ph;

R’’=H; 82%

53 R = CH3; R’ = Ph; R’’=H; 73% 65 R = thiophen-3-yl; R’ = Ph; R’’=H; 79%

54 R = Ph; R’ = Ph; R’’=CH3; 35% 66 R = pyridine-2-yl; R’ = Ph; R’’=H; 15%

55 R = Ph; R’ = Ph; R’’=CH2Ph; 40%

90

yl)methanone (38), resulted in synthesis of the corresponding 2-acyl system (61) in 55% yield.

With 2-/3-thieno and 2-pyrido acyl systems (39-43), rearranged products were obtained in 15-82%

yields. Interestingly, with 3-acylthieno systems only 2-acylpyrrole product was obtained and

possible 2-acylthieno rearranged product was not seen. This result clearly demonstrates suitability

of the method and its regiospecificity while generating rearranged 2-acylpyrrole product, even if

corresponding C-2 positions are available in thiophene systems. Low yield in case of compound

66 indicates destabilization of putative acyl carbocation which is likely to form in the reaction.

Similar observation was also noticed in case of compound 59 containing p-fluoro substituent.

Based on the results obtained in the above studies, an experiment was carried out to

understand the mechanism of acyl migration. Previous literature examples from pyrrole and indole

systems have speculated formation of an acylium ion as well as internal transfer of acyl group.[15,

29] Reaction was carried out on phenyl(4-phenyl-1H-pyrrol-3-yl)methanone (21) in the presence

of anisole, with 1:1 ratio of both the substrates. On completion, the reaction mixture was analyzed

by ESI mass spectroscopy, which indicated formation of (2-

methoxyphenyl)(phenyl)methanone/(4-methoxyphenyl)(phenyl) methanone [M+H+=213] along

with the compound 44 [M+H+=248] (Figure 4.1.5). Based on this observation a plausible

mechanism has been proposed for the reaction (Scheme-4.1.7). It is felt that protonation of carbon

bearing the acyl group is the first step of this rearrangement, followed by cleavage of acyl group

from pyrrole ring. Given the natural tendency of pyrrole ring to undergo electrophilic substitution

reaction at the 2nd position, the final step of this mechanism involves the reaction of acylium cation

at the aforementioned position.

91

Scheme 4.1.7 Plausible mechanism for the conversion of 3-acyl pyrrole to 2-acyl pyrrole

Conclusions

In conclusion, we have developed a simple strategy for the synthesis of diverse 2-acylpyrroles.

Target molecules were synthesized in two steps, starting from easily accessible chalcones. Well

established van Leusen’s pyrrole synthesis was used to perform the first step, whereas final step

involved application of seldom used acid catalyzed acyl migration. The polyphosphoric acid

assisted method developed for the final step gave the desired products in modest to good yields

and could be applied to diverse substituted pyrrole systems. The regiospecificity of the method

towards C-2 position of pyrrole is demonstrated even when corresponding positions were available

in activated phenyl and thiophene systems. A preliminary mechanistic study was also performed

which indicated involvement of acylium ion in generating the rearranged product.

92

Figure 4.1.5 Mass analysis for understanding the mechanism of the reaction

Experimental

All the compounds and reagents required were purchased from commercial sources and were used

without further purification. Solvents were dried and distilled using standard procedures, prior to

use. 1H NMR (300/400 MHz) and 13C (75.5/101 MHz) spectra were recorded in CDCl3 and DMSO

using (CH3)4Si as internal standard. IR spectra were recorded as KBr plates on Jasco FT/IR-4200

instrument. Melting points were recorded on a Biotech India melting point apparatus and are

uncorrected. Single crystal X-ray studies were performed using CrysAlis PRO on a single crystal

Rigaku Oxford XtaLab Pro diffractometer.

93

Synthesis of chalcones:

NaOH (10%, 0.1 ml) was added dropwise to a solution of acetophenone/substituted

acetophenone/2 or 3-acetyl thiophene/2-acetyl pyridine (2.5 mmol) and benzaldehyde/substituted

benzaldehyde (2.5 mmol) in 5 ml of ethanol and the resulting mixture was allowed to stir at room

temperature. After completion of the reaction as indicated by TLC, reaction mixture was poured

into water (15 ml) and extracted with ethyl acetate (3 x 15 ml). The organic layers were pooled

together, dried over anhydrous Na2SO4 and concentrated under reduced pressure. The chalcone,

thus obtained was used without further purification.

Synthesis of 3, 4-disubstituted pyrroles (21-30; 33-43):

To a solution of appropriate chalcone (0.5 mmol) in DMSO (10 ml), TosMIC (136 mg, 0.7 mmol)

and t-BuOK (112 mg, 1 mmol) were added, and the reaction mixture was stirred for 0.5 h at 25 ºC.

After completion of the reaction as indicated by TLC, brine solution (10 ml) was added to the

reaction mixture and it was extracted with ethyl acetate (3 x 15 ml), the combined organic layer

was dried over anhydrous Na2SO4. Further, ethyl acetate was removed under vacuum and the crude

product thus obtained was used for the next step. The compounds were sufficiently pure and did

not require any further steps of purification.

Phenyl(4-phenyl-1H-pyrrol-3-yl)methanone (21)

Yield: 75%; 1H NMR (400 MHz, DMSO-d6): δ 7.09 (s, 1H), 7.14

- 7.19 (m, 1H), 7.23 (s, 1H), 7.24 - 7.28 (m, 2H), 7.39 – 7.36 (m,

2H), 7.44 - 7.49 (m, 2H), 7.54 - 7.59 (m, 1H), 7.72 - 7.75 (m, 2H),


125.9, 126.1, 128.1, 128.5, 128.6, 128.8, 129.4, 131.9, 135.7,

140.4, 190.8. ESI-MS (m/z): 248.02[M+H]+.

94

Phenyl(4-(p-tolyl)-1H-pyrrol-3-yl)methanone (22)

Yield: 72%; 1H NMR (400 MHz, DMSO) δ 2.28 (s, 3H), 7.05

(dd, J = 11.5, 5.0 Hz, 3H), 7.20 (d, J = 2.0 Hz, 1H), 7.27 (d, J =

8.1 Hz, 2H), 7.46 (t, J = 7.5 Hz, 2H), 7.56 (t, J = 7.4 Hz, 1H), -

7.71 - 7.78 (m, 2H), 11.61 (s, 1H); 13C NMR (101 MHz, DMSO-

d6): δ 21.1, 119.7, 121.0, 125.9, 128.4, 128.6, 128.7, 128.8,

129.3, 131.9, 132.7, 135.1, 140.5, 190.1; ESI-MS (m/z): 262.14

[M+H]+.

(4-(4-Methoxyphenyl)-1H-pyrrol-3-yl)(phenyl)methanone (23)


(d, J = 8.8 Hz, 2H), 7.01 (d, J = 2.0 Hz, 1H), 7.19 (d, J = 2.0 Hz,

1H), 7.32 (d, J = 8.8 Hz, 2H), 7.46 (t, J = 7.4 Hz, 2H), 7.56 (t, J

= 7.4 Hz, 1H), 7.70 - 7.76 (m, 2H), 11.57 (s, 1H); 13C NMR (101

MHz, DMSO-d6): δ 55.4, 113.6, 119.4, 120.8, 125.6, 128.1,

128.4, 128.6, 129.3, 129.9, 131.8, 140.6, 158.0, 190.8; ESI-MS

(m/z): 278.17 [M+H]+.

(4-(4-Nitrophenyl)-1H-pyrrol-3-yl)(phenyl)methanone (24)

Yield: 71%; 1H NMR (400 MHz, DMSO-d6): δ 7.31 - 7.38 (m,

2H), 7.50 (t, J = 7.5 Hz, 2H), 7.61 (t, J = 7.4 Hz, 1H), 7.64 - 7.70

(m, 2H), 7.75 - 7.82 (m, 2H), 8.14 (d, J = 8.9 Hz, 2H), 11.91 (s,

1H); 13C NMR (101 MHz, DMSO-d6): δ 121.2, 122.2, 123.5,

123.8, 128.7, 129.3, 129.5, 129.7, 132.3, 140.1, 143.1, 145.6,

190.6; ESI-MS (m/z): 293.08 [M+H]+.

95

(4-(4-Fluorophenyl)-1H-pyrrol-3-yl)(phenyl)methanone (25)

Yield: 70%; 1H NMR (400 MHz, DMSO-d6): δ 7.09 (dd, J = 10.0,

7.9 Hz, 3H), 7.23 (d, J = 1.9 Hz, 1H), 7.39 - 7.43 (m, 2H), 7.47 (t,

J = 7.5 Hz, 2H), 7.57 (t, J = 7.4 Hz, 1H), 7.71 - 7.75 (m, 2H),

11.67 (s, 1H); 13C NMR (101 MHz, DMSO-d6): δ 114.9 (2JC-F =

21.2 Hz), 120.2, 120.9, 124.9, 128.6, 128.8, 129.4, 130.6 (3JC-F =

8.0 Hz), 131.9, 132.1 (4JC-F = 3.0 Hz), 140.4, 161.2 (1JC-F = 242.4

Hz), 190.7; ESI-MS (m/z): 266.09 [M+H]+.

(4-(2,4-Dichlorophenyl)-1H-pyrrol-3-yl)(phenyl)methanone (26)

Yield: 68%; 1H NMR (400 MHz, DMSO-d6): δ 7.02 (d, J = 1.7

Hz, 1H), 7.30 (d, J = 1.7 Hz, 1H), 7.36 (s, 2H), 7.46 (t, J = 7.5 Hz,

2H), 7.51 (s, 1H), 7.56 (t, J = 7.4 Hz, 1H), 7.71 (d, J = 7.2 Hz,

2H), 11.75 (s, 1H); 13C NMR (101 MHz, DMSO-d6): δ 121.0,

121.4, 122.4, 127.1, 127.2, 128.6, 128.8, 129.2, 131.8, 131.9,

133.4, 134.2, 134.5, 139.8, 190.1; ESI-MS (m/z): 316.06 [M+H]+.

(4-(2-Chloro-4-fluorophenyl)-1H-pyrrol-3-yl)(phenyl)methanone (27)

Yield: 70%; 1H NMR (400 MHz, DMSO-d6): δ 6.99 (t, J = 2.2

Hz, 1H), 7.16 (td, J = 8.5, 2.7 Hz, 1H), 7.30 (dd, J = 3.0, 2.0 Hz,

1H), 7.32 - 7.39 (m, 2H), 7.45 (t, J = 7.5 Hz, 2H), 7.55 (t, J = 7.4

Hz, 1H), 7.68 - 7.72 (m, 2H), 11.71 (s, 1H); 13C NMR (101 MHz,

DMSO-d6): δ 13C NMR (101 MHz, DMSO-d6): δ 114.1 (2JC-F =

21.2 Hz), 116.5 (2JC’-F = 24.2 Hz), 120.9, 121.6, 122.5, 127.1,

128.6, 129.2, 131.9, 132.0 (4JC-F = 4.04 Hz), 133.4 (3JC-F = 8.0

96

Hz), 133.9, 134.0 (3HC’-F = 11.0 Hz), 161.1 (1JC-F = 246.4 Hz),

190.2; ESI-MS (m/z): 300.05 [M+H]+.

(4-([1,1'-biphenyl]-4-yl)-1H-pyrrol-3-yl)(phenyl)methanone (28)

Yield: 78%; 1H NMR (400 MHz, DMSO-d6): δ 7.17 (d, J = 1.7

Hz, 1H), 7.25 (d, J = 1.7 Hz, 1H), 7.35 (t, J = 7.3 Hz, 1H), 7.46

(d, J = 7.9 Hz, 2H), 7.49 (s, 2H), 7.51 (s, 2H), 7.57 (s, 2H), 7.59

(s, 1H), 7.68 (d, J = 7.4 Hz, 2H), 7.77 (s, 2H), 11.70 (s, 1H);

13C NMR (101 MHz, DMSO-d6): δ 120.2, 121.0, 125.4, 126.4,

126.8, 127.6, 128.6, 128.8, 129.3, 132.0, 134.9, 137.8, 140.4,

140.5, 190.8; ESI-MS (m/z): 324.13 [M+H]+.

(4-(Anthracen-9-yl)-1H-pyrrol-3-yl)(phenyl)methanone (29)

Yield: 75%; m.p. 168-174 ºC; 1H NMR (400 MHz, DMSO-d6): δ

7.08 (d, J = 1.3 Hz, 1H), 7.37 (dd, J = 11.0, 4.0 Hz, 4H), 7.43 –

7.49 (m, 3H), 7.60 (d, J = 1.3 Hz, 1H), 7.65 (d, J = 7.3 Hz, 2H),

7.86 (d, J = 8.7 Hz, 2H), 8.07 (d, J = 8.4 Hz, 2H), 8.53 (s, 1H),


124.1, 125.4, 125.5, 125.8, 127.2, 127.8, 128.4, 128.6, 128.9,

131.3, 131.4, 131.6, 132.1, 140.3, 189.8; ESI-MS (m/z): 348.15

[M+H]+.

1-(4-Phenyl-1H-pyrrol-3-yl)ethanone (30)

97

Yield: 61%; 1H NMR (400 MHz, DMSO-d6) : δ 2.33 (s, 3H), 6.93

(t, J = 2.2 Hz, 1H), 7.15 - 7.23 (m, 1H), 7.25 - 7.31 (m, 2H), 7.41

(dt, J = 8.2, 1.7 Hz, 2H), 7.70 (dd, J = 3.1, 2.1 Hz, 1H), 11.55 (s,

1H); 13C NMR (101 MHz, DMSO-d6): δ 28.7, 120.3, 122.4,

124.8, 126.2, 127.9, 128.0, 129.3, 136.0, 193.0; ESI-MS (m/z):

186.04 [M+H]+.

Synthesis of (1-methyl-4-phenyl-1H-pyrrol-3-yl)(phenyl)methanone (31):

In a 50 ml round bottom flask maintained at 0oC under nitrogen, NaH(30 mg, 1.21 mmol) was

added slowly to a solution of compound 2a (200mg, 0.8 mmol) in anhydrous DMF (4ml). Further,

methyl iodide (0.06 ml, 0.97 mmol) was added to the reaction mixture and it was allowed to stir

for 30 minutes. On completion of the reaction by TLC, the mixture was poured into 25 ml of

distilled water and extracted with ethyl acetate (3 x 25 ml). The organic layers thus obtained were

pooled together, dried with anhydrous Na2SO4 and concentrated under reduced pressure. The

product obtained was purified by column chromatography and it was obtained as colourless oil.


(d, J = 2.2 Hz, 1H), 7.15 - 7.21 (m, 1H), 7.26 (dd, J = 11.1, 4.6

Hz, 3H), 7.35 - 7.40 (m, 2H), 7.47 (t, J = 7.5 Hz, 2H), 7.53 - 7.59

(m, 1H), 7.73 - 7.77 (m, 2H); 13C NMR (101 MHz, DMSO-d6): δ

98

36.5, 120.8, 123.9, 126.2, 126.4, 128.2, 128.6, 128.7, 129.3,

131.9, 131.9, 135.4, 140.4, 190.3; ESI-MS (m/z): 262.14 [M+H]+.

Synthesis of (1-benzyl-4-phenyl-1H-pyrrol-3-yl)(phenyl)methanone (32):

In a 50 ml round bottom flask maintained at 0oC under nitrogen, NaH(30 mg, 1.21 mmol) was

added slowly to a solution of compound 2a (200mg, 0.8 mmol) in anhydrous DMF (4ml). Further,

benzyl bromide (0.11 ml, 0.97 mmol) was added to the reaction mixture and it was allowed to stir

for 30 minutes. On completion of the reaction by TLC, the mixture was poured into 25 ml of

distilled water and extracted with ethyl acetate (3 x 25 ml). The organic layers thus obtained were

pooled together, dried with anhydrous Na2SO4 and concentrated under reduced pressure. The

product obtained was purified by column chromatography and it was obtained as colourless oil.

Yield: 74%; 1H NMR (400 MHz, DMSO-d6) δ 7.77 – 7.72 (m,

2H), 7.58 – 7.54 (m, 1H), 7.46 (dd, J = 13.2, 5.0 Hz, 3H), 7.41 –

7.33 (m, 6H), 7.33 – 7.29 (m, 1H), 7.28 – 7.23 (m, 2H), 7.17 (tt,

J = 2.6, 1.9 Hz, 2H), 5.20 (s, 2H); 13C NMR (101 MHz, DMSO-

d6) δ 190.3, 140.3, 138.2, 135.2, 132.1, 131.2, 129.4, 129.2, 128.7,

128.2, 128.1, 126.8, 126.3, 123, 121, 52.9; ESI-MS (m/z): 338.15

[M+H]+.

99

(4-Phenyl-1H-pyrrol-3-yl)(p-tolyl)methanone (33):

Yield: 70%; 1H NMR (400 MHz, DMSO) δ 2.37 (s, 3H), 7.08 (s,

1H), 7.16 (t, J = 7.3 Hz, 1H), 7.22 (d, J = 10.4 Hz, 2H), 7.27 (dd,

J = 8.0, 2.0 Hz, 3H), 7.35 - 7.39 (m, 2H), 7.67 (d, J = 8.1 Hz,

2H), 11.60 (s, 1H); 13C NMR (101 MHz, DMSO-d6) δ 21.5,

119.9, 121.1, 125.8, 126.0, 128.1, 128.2, 128.7, 129.2, 129.6,

135.8, 137.7, 142.1, 190.5; ESI-MS (m/z): 262.14 [M+H]+.

(4-Methoxyphenyl)(4-phenyl-1H-pyrrol-3-yl)methanone(34)

Yield: 77%; 1H NMR (400 MHz, DMSO-d6) δ 3.82 (s, 3H), 7.00

(d, J = 8.9 Hz, 2H), 7.09 (t, J = 2.2 Hz, 1H), 7.13 - 7.18 (m, 1H),

7.21 - 7.27 (m, 3H), 7.33 - 7.36 (m, 2H), 7.75 - 7.79 (m, 2H),

11.60 (s, 1H); 13C NMR (101 MHz, DMSO-d6) δ 55.9, 113.9,

119.7, 121.2, 125.7, 125.9, 127.3, 128.2, 128.6, 131.8, 132.8,

135.8, 162.6, 189.7; ESI-MS (m/z): 278.0[M+H]+.

(3,4-Dimethoxyphenyl)(4-phenyl-1H-pyrrol-3-yl)methanone(35)


(s, 3H), 7.01 (d, J = 8.4 Hz, 1H), 7.09 (t, J = 2.2 Hz, 1H), 7.15 (t,

J = 7.3 Hz, 1H), 7.24 (t, J = 5.7 Hz, 2H), 7.26 (d, J = 1.3 Hz, 1H),

7.34 (dt, J = 3.6, 1.8 Hz, 3H), 7.43 (dd, J = 8.3, 2.0 Hz, 1H),

11.56 (s, 1H); 13C NMR (101 MHz, DMSO-d6) δ 55.8, 56.1, 111,

112.3, 119.6, 121.2, 124.0, 125.7, 125.9, 127.1, 128.2, 128.6,

100

132.6, 135.8, 148.6, 152.5, 189.7; ESI-MS (m/z):

308.11[M+H]+.

(4-fluorophenyl)(4-phenyl-1H-pyrrol-3-yl)methanone(36)

Yield: 68%; 1H NMR (400 MHz, DMSO-d6) δ 7.10 (d, J = 2.0

Hz, 2H), 7.17 (t, J = 6.7 Hz, 2H), 7.23 (s, 1H), 7.25 (d, J = 3.5

Hz, 3H), 7.27 (d, J = 1.8 Hz, 4H), 7.29 (s, 1H), 7.33 - 7.39 (m,

4H), 7.77 - 7.85 (m, 4H), 11.69 (s, 1H); 13C NMR (101 MHz,

DMSO-d6) δ 13C NMR (101 MHz, DMSO-d6): δ 115.5 (2JC-F =

21.2 Hz), 120.0, 120.9, 125.9, 126.1, 128.1, 128.3, 128.8, 132.2

(3JC-F = 9.09 Hz), 135.6, 136.9 (4JC-F = 3.03 Hz), 164.5 (1JC-F =

250 Hz), 189.5; ESI-MS (m/z): 266.09[M+H]+.

(4-Chlorophenyl)(4-phenyl-1H-pyrrol-3-yl)methanone(37)


(t, J = 6.9 Hz, 1H), 7.23 - 7.31 (m, 3H), 7.37 (d, J = 7.3 Hz, 2H),

7.52 (d, J = 8.1 Hz, 2H), 7.75 (d, J = 8.0 Hz, 2H), 11.72 (s, 1H);

13C NMR (101 MHz, DMSO-d6): δ 120.2, 120.7, 125.9, 126.2,

128.2, 128.7, 128.8, 131.2, 135.6, 136.7, 139.1, 189.5; ESI-MS

(m/z): 282.06[M+H]+.

Naphthalen-2-yl(4-phenyl-1H-pyrrol-3-yl)methanone(38)

101

Yield: 66%; 1H NMR (400 MHz, DMSO-d6) δ 7.15 (dd, J = 11.9,

4.6 Hz, 2H), 7.26 (t, J = 7.6 Hz, 2H), 7.33 (d, J = 1.8 Hz, 1H),

7.42 (d, J = 7.2 Hz, 2H), 7.61 (dt, J = 20.1, 6.8 Hz, 2H), 7.84

(dd, J = 8.5, 1.5 Hz, 1H), 7.99 (d, J = 8.4 Hz, 2H), 8.08 (d, J =

7.9 Hz, 1H), 8.38 (s, 1H), 11.68 (s, 1H); 13C NMR (101 MHz,

DMSO-d6) δ 120.1, 121.2, 125.8, 126, 126.1, 127.1, 128, 128.2,

128.3, 128.7, 128.8, 129.8, 130.6, 132.4, 134.8, 135.8, 137.7,

190.8; ESI-MS (m/z): 298.0[M+H]+.

(4-Phenyl-1H-pyrrol-3-yl)(thiophen-2-yl)methanone(39):

Yield: 80%; 1H NMR (400 MHz, DMSO) δ 7.11 (d, J = 1.7 Hz,

1H), 7.20 (dd, J = 9.9, 5.4 Hz, 2H), 7.28 (t, J = 7.5 Hz, 2H), 7.37

(d, J = 7.2 Hz, 2H), 7.53 (d, J = 1.7 Hz, 1H), 7.73 (d, J = 3.6 Hz,

1H), 7.94 (d, J = 4.9 Hz, 1H), 11.72 (s, 1H); 13C NMR (101 MHz,

DMSO-d6) δ 120, 120.7, 125.5, 126.1, 127.2, 128.3, 128.6,

128.7, 133.5, 133.8, 135.6, 182.4; ESI-MS (m/z):

254.06[M+H]+.

(4-(4-Methoxyphenyl)-1H-pyrrol-3-yl)(thiophen-2-yl)methanone(40)


(d, J = 8.8 Hz, 2H), 7.02 (t, J = 2.2 Hz, 1H), 7.21 (dd, J = 5.0,

3.7 Hz, 1H), 7.26 - 7.34 (m, 2H), 7.50 (dd, J = 3.1, 2.0 Hz, 1H),

7.72 (dd, J = 3.7, 1.1 Hz, 1H), 7.93 (dd, J = 5.0, 1.1 Hz, 1H),

11.61 (s, 1H); 13C NMR (101 MHz, DMSO-d6) δ 55.5, 113.8,

102

119.4, 120.6, 125.3, 126.9, 127.9, 128.7, 129.7, 133.4, 133.7,

146.2, 158, 182.3; ESI-MS (m/z): 284.07[M+H]+.

(4-(2,4-dichlorophenyl)-1H-pyrrol-3-yl)(thiophen-2-yl)methanone(41)

Yield: 71%; 1H NMR (400 MHz, DMSO-d6) δ 7.03 (t, J = 2.2

Hz, 1H), 7.21 (dd, J = 4.9, 3.8 Hz, 1H), 7.40 – 7.33 (m, 2H), 7.55

(d, J = 2.0 Hz, 1H), 7.63 (dd, J = 3.0, 2.0 Hz, 1H), 7.78 (dd, J =

3.7, 1.1 Hz, 1H), 7.92 (dd, J = 5.0, 1.1 Hz, 1H), 11.79 (s, 1H);

13C NMR (101 MHz, DMSO-d6) δ 121, 121.3, 122.1, 125.9,

127.2, 128.7, 128.9, 131.9, 132.9, 133.4, 133.6, 134.1, 145.2,

181.4; ESI-MS (m/z): 321.98[M+H]+.

(4-Phenyl-1H-pyrrol-3-yl)(thiophen-3-yl)methanone (42)

Yield: 53%; 1H NMR (400 MHz, DMSO-d6) δ 7.10 (t, J = 2.2

Hz, 1H), 7.15 - 7.20 (m, 1H), 7.20 - 7.23 (m, 1H), 7.28 (dd, J =

10.4, 4.8 Hz, 2H), 7.37 (dd, J = 8.2, 1.1 Hz, 2H), 7.53 (dd, J =

2.9, 2.1 Hz, 1H), 7.73 (dd, J = 3.7, 1.0 Hz, 1H), 7.94 (dd, J = 5.0,

1.0 Hz, 1H), 11.67 (s, 1H); 13C NMR (101 MHz, DMSO-d6) δ

120, 120.8, 125.6, 126.1, 128.3, 128.6, 128.7, 133.5, 133.8,

135.6, 146, 182.4; ESI-MS (m/z): 254.06[M+H]+.

(4-Phenyl-1H-pyrrol-3-yl)(pyridin-2-yl)methanone (43)

103

Yield: 42%; 1H NMR (400 MHz, DMSO- d6) δ 7.03 (t, J = 2.2

Hz, 1H), 7.18 - 7.23 (m, 1H), 7.30 (t, J = 7.5 Hz, 2H), 7.43 - 7.46

(m, 2H), 7.54 (ddd, J = 7.5, 4.8, 1.3 Hz, 1H), 7.80 (dd, J = 3.2,

2.1 Hz, 1H), 7.86 (dt, J = 7.9, 1.1 Hz, 1H), 7.95 (td, J = 7.7, 1.7

Hz, 1H), 8.66 (ddd, J = 4.8, 1.6, 0.9 Hz, 1H), 11.69 (s, 1H); 13C

NMR (101 MHz, DMSO-d6) δ 119.6, 119.9, 123.5, 126.2, 126.2,

126.6, 128, 129.2, 131.1, 136.1, 137.6, 148.8, 157.2, 187.9; ESI-

MS (m/z): 249.10[M+H]+.

General procedure for the synthesis of 2,4 disubstituted pyrroles (44-66): To a 25 ml round

bottom flask equipped with a stir bar was added appropriate 3,4-disubstituted pyrrole (0.5 mM)

and polyphosphoric acid (2 ml). The reaction mixture was then allowed to stir for 30 minutes at

110 °C under nitrogen atmosphere. It was subsequently cooled to room temperature and stirred

with water (10 mL) for 30 minutes. The aqueous solution thus obtained was extracted with ethyl

acetate (3 x 10 mL) and the ensuing organic layer was successively washed with brine and dried

over anhydrous Na2SO4. Finally, the dried organic layer was concentrated under reduced pressure

and the residue obtained was purified by column chromatography.

Phenyl(4-phenyl-1H-pyrrol-2-yl)methanone (44)

Yield: 73%; white solid; m.p. 150-152 ºC; Rf 0.3 [hexanes: ethyl

acetate = 9:1]; νmax(KBr)/cm-1: 3264, 1609, 1600, 1560, 1059; 1H

NMR (400 MHz, DMSO-d6): δ 7.16 - 7.21 (m, 2H), 7.34 (t, J =

7.7 Hz, 2H), 7.54 - 7.59 (m, 2H), 7.62 – 7.68 (m, 3H), 7.73 (dd,

J = 3.1, 1.6 Hz, 1H), 7.88 - 7.91 (m, 2H), 12.25 (s, 1H); 13C NMR

(101 MHz, DMSO-d6): δ 116.1, 124.0, 125.4, 126.2, 126.4,

104

128.9, 129.1, 131.7, 132.2, 134.8, 138.8, 184.2; ESI-MS (m/z):

248.0 [M+H]+.

Phenyl(4-(p-tolyl)-1H-pyrrol-2-yl)methanone (45).

Yield: 74%; white solid; m.p. 156-158 ºC; Rf 0.4 [hexanes: ethyl

acetate = 9:1]; νmax (KBr)/cm-1: 3262, 1623, 1571, 1146, 807; 1H

NMR (400 MHz, DMSO-d6): δ 2.28 (s, 3H), 7.14 (dd, J = 4.8,

2.2 Hz, 3H), 7.56 (dd, J = 10.0, 7.9 Hz, 4H), 7.64 (t, J = 7.4 Hz,

1H), 7.69 (dd, J = 3.1, 1.5 Hz, 1H), 7.87 - 7.91 (m, 2H), 12.22

(s, 1H); 13C NMR (101 MHz, DMSO-d6): δ 21.1, 115.9, 123.7,

125.3, 126.2, 128.9, 129.1, 129.7, 131.6, 131.9, 132.1, 135.4,

138.9, 184.1; ESI-MS (m/z): 262.0 [M+H]+.

4-(4-Methoxyphenyl)-1H-pyrrol-2-yl)(phenyl)methanone (46)

Yield: 37%; white solid; m.p. 120-122 ºC; Rf : 0.4 [hexanes:

ethyl acetate = 9:1]; νmax(KBr)/cm-1: 3269, 3012, 1614, 1562,

1147; 1H NMR (400 MHz, CDCl3): δ 3.82 (s, 3H), 6.91 (d, J =

8.8 Hz, 2H), 7.07 (s, 1H), 7.36 (s, 1H), 7.45 (d, J = 8.8 Hz, 2H),

7.49 - 7.54 (m, 2H), 7.59 (t, J = 7.4 Hz, 1H), 7.92 - 7.96 (m, 2H),

9.87 (s, 1H); 13C NMR (101 MHz, CDCl3): δ 55.3, 114.2, 116.1,

121.5, 126.5, 127.0, 127.2, 128.4, 128.9, 131.6, 131.9, 138.3,

158.5, 184.8; ESI-MS (m/z): 278.0 [M+H]+.

105

(4-(4-Nitrophenyl)-1H-pyrrol-2-yl)(phenyl)methanone (47)

Yield: 53%; yellow solid; m.p. 208-210 ºC; Rf: 0.5 [hexanes:


850; 1H NMR (400 MHz, DMSO-d6): δ 7.38 (dd, J = 2.4, 1.6 Hz,

1H), 7.58 (dd, J = 7.2, 6.1 Hz, 2H), 7.64 - 7.70 (m, 1H), 7.91 (t,

J = 1.7 Hz, 1H), 7.92 (s, 1H), 7.95 - 7.98 (m, 1H),7.99 (d, J = 2.1

Hz, 1H), 8.00 (dd, J = 3.3, 1.6 Hz, 1H), 8.17 (dd, J = 6.8, 4.7 Hz,

2H), 12.53 (s, 1H); 13C NMR (101 MHz, DMSO-d6): δ 116.7,

124.0, 124.5, 125.8, 126.0, 129.0, 129.2, 132.3, 132.4, 138.4,

142.0, 145.5, 184.4; HRMS-ESI(m/z): calcd for C17H12N2O3

[M+H]+ 293.0921 found 293.092.

(4-(4-Fluorophenyl)-1H-pyrrol-2-yl)(phenyl)methanone (48)

Yield: 55%; white solid; m.p. 110-112 ºC; Rf: 0.3 [hexanes:

ethylacetate = 9:1]; νmax(KBr)/cm-1: 3259, 1625, 1587, 1562,

1205, 1076, 896; 1H NMR (400 MHz, DMSO-d6): δ 7.16 (dd, J

= 11.1, 5.2 Hz, 3H), 7.56 (t, J = 7.8 Hz, 2H), 7.64 (t, J = 6.8

Hz, 1H), 7.70 (dd, J = 8.6, 5.1 Hz, 3H), 7.89 (d, J = 7.9 Hz, 2H),

12.25 (s, 1H); 13C NMR (101 MHz, DMSO) δ 115.9 (2JC-F = 23.2

Hz), 123.9, 125.2, 127.2(3JC-F = 8.0 Hz), 128.9, 129.2, 131.4(4JC-

F = 2.0 Hz), 131.7, 132.2, 138.8, 161.2 (1JC-F, = 243.4 Hz)),

184.2.

106

(4-(2,4-Dichlorophenyl)-1H-pyrrol-2-yl)(phenyl)methanone (49)

Yield: 65%; white solid; m.p. 94-96 ºC; Rf: 0.4 [hexanes: ethyl

acetate = 9:1]; νmax(KBr)/cm-1: 3264, 1736, 1626, 1561, 1294;

1H NMR (400 MHz, DMSO) δ 7.13 - 7.15 (m, 1H), 7.37 (dd, J

= 8.4, 2.2 Hz, 1H), 7.54 (t, J = 7.4 Hz, 2H), 7.58 - 7.61 (m, 1H),

7.62 (d, J = 2.2 Hz, 1H), 7.66 (dd, J = 3.7, 2.1 Hz, 2H), 7.86 -

7.91 (m, 2H), 12.47 (s, 1H); 13C NMR (101 MHz, DMSO-d6): δ

119.18, 121.87, 126.53, 127.94, 128.96, 129.14, 129.90, 131.03,

131.67, 131.87, 132.00, 132.34, 132.52, 138.65, 184.31; ESI-MS

(m/z): 316.0 [M+H]+.

(4-(2-Chloro-4-fluorophenyl)-1H-pyrrol-2-yl)(phenyl)methanone (50)

Yield: 60%; white solid; m.p. 108-110 ºC; Rf :0.3 [hexanes: ethyl


NMR (400 MHz, DMSO-d6): δ 7.10 (dd, J = 2.4, 1.6 Hz, 1H),

7.23 (td, J = 8.5, 2.7 Hz, 1H), 7.47 (dd, J = 8.9, 2.7 Hz, 1H), 7.55

(t, J = 7.4 Hz, 2H), 7.59 - 7.65 (m, 2H), 7.65 - 7.70 (m, 1H), 7.85

- 7.91 (m, 2H), 12.41 (s, 1H); 13C NMR (101 MHz, DMSO) δ

115.1 (2JC-F = 20.2 Hz), 117.5 (2JC’-F = 25.2 Hz), 119.3, 122.1,

126.3, 128.9, 129.1, 130.2 (4JC-F = 3.0 Hz), 130.9, 131.8 (3JC-F =

10.1 Hz), 132.2 (3JC’-F = 8.0 Hz), 132.3, 138.7, 160.8 (1JC-F =

248.46 Hz), 184.3; HRMS-ESI(m/z): calcd for C17H11ClFNO

[M+H]+ 300.0586 found 300.0588.

107

(4-([1,1'-Biphenyl]-4-yl)-1H-pyrrol-2-yl)(phenyl)methanone (51)

Yield: 65%; light yellow solid; m.p. 200-202 ºC; Rf: 0.5 [hexanes:

ethyl acetate = 4:1]; νmax(KBr)/cm-1: 3266, 1611, 1565, 1287, 760;

1H NMR (400 MHz, DMSO-d6): δ 7.23 - 7.26 (m, 1H), 7.35 (t, J

= 7.3 Hz, 1H), 7.46 (t, J = 7.7 Hz, 2H), 7.58 (t, J = 6.7 Hz, 2H),

7.62 - 7.67 (m, 3H), 7.67 – 7.71 (m, 2H), 7.77 (d, J = 8.4 Hz, 2H),

7.81 (dd, J = 3.1, 1.5 Hz, 1H), 7.95 (m, 2H), 12.31 (s, 1H); 13C

NMR (101 MHz, DMSO-d6): δ 116.1, 124.2, 125.7, 125.9, 126.8,

127.3, 127.6, 129.0, 129.2, 129.3, 131.8, 132.2, 134.0, 138.0,

138.8, 140.3, 184.2; HRMS-ESI(m/z): calcd for C23H7NO [M+H]+

324.1383 found 324.1389.

(4-(Anthracen-9-yl)-1H-pyrrol-2-yl)(phenyl)methanone (52)

Yield: 22%; light yellow solid; m.p. 124-126 ºC; Rf: 0.4 [hexanes:

ethylacetate = 4:1]; νmax(KBr)/cm-1: 3278, 1608, 1572, 3038, 1122;

1H NMR (400 MHz, DMSO) δ 6.98 (dd, J = 2.4, 1.5 Hz, 1H), 7.43

- 7.46 (m, 1H), 7.47 (d, J = 1.6 Hz, 1H), 7.48 - 7.54 (m, 5H), 7.59

(ddd, J = 7.4, 3.9, 1.3 Hz, 1H), 7.93 (d, J = 1.5 Hz, 1H), 7.95 (d, J

= 4.5 Hz, 2H), 7.98 (s, 1H), 8.11 (d, J = 8.2 Hz, 2H), 8.60 (s, 1H),


125.7, 126.1, 126.6, 126.9, 127.8, 128.8, 128.9, 129.1, 130.5, 130.9,


C25H17NO [M+H]+ 348.1383 found 348.1383.

108

(4-Methyl-1H-pyrrol-2-yl)(phenyl)methanone (53)

Yield: 73%; white solid; m.p. 146-148 ºC; Rf: 0.3 [hexanes : ethyl

acetate =9:1]; νmax(KBr)/cm-1: 3198, 1630, 1568, 1217, 938; 1H NMR

400 MHz, (CDCl3 + two drops of DMSO-d6): δ 2.48 (s, 3H), 7.18 (d,

J =9.6 Hz, 1H), 7.20 – 7.26 (m, 1H), 7.36 (dd, J = 16.3, 8.6 Hz, 3H),

7.53 (t, J = 8.5 Hz, 2H), 10.71 (s, 1H); 13C NMR (101 MHz, CDCl3 +

two drops of DMSO-d6): δ 25.6, 113.8, 121.9, 125.2, 126.2, 126.5,

128.8, 132.8, 134.5, 188.1; ESI-MS (m/z): 186.0 [M+H]+.

(1-Methyl-4-phenyl-1H-pyrrol-2-yl)(phenyl)methanone (54)

Yield: 35%; yellow liquid; Rf : 0.4 [hexanes: ethyl acetate =9:1];

νmax(KBr)/cm-1:3087, 1623, 1573, 1198, 723; 1H NMR (400 MHz,

DMSO-d6): δ 4.00 (s, 3H), 7.03 (d, J = 1.9 Hz, 1H), 7.15 - 7.21 (m,

1H), 7.33 (t, J = 7.7 Hz, 2H), 7.55 (ddd, J = 8.1, 5.4, 1.1 Hz, 4H), 7.61

– 7.66 (m, 1H), 7.80 (dt, J = 3.8, 1.7 Hz, 3H); 13C NMR (101 MHz,

DMSO-d6): δ 37.5, 118.8, 123.6, 125.2, 126.4, 128.8, 129.2, 129.3,

129.9, 130.9, 132.1, 134.3, 139.7, 185.5; HRMS-ESI(m/z): calcd for

C18H15NO [M+H]+ 262.1226 found 262.1229.

(1-Benzyl-4-phenyl-1H-pyrrol-2-yl)(phenyl)methanone (55)

Yield: 40%; light yellow liquid; Rf : 0.5 [hexanes: ethylacetate =9:1];

νmax(KBr)/cm-1: 3410, 1614, 1559, 1123, 893; 1H NMR (400 MHz,

DMSO-d6): δ 5.69 (s, 2H), 7.10 (d, J = 1.9 Hz, 1H), 7.21 (dd, J = 9.6,

2.7 Hz, 3H), 7.25 (d, J = 7.3 Hz, 1H), 7.30 - 7.37 (m, 4H), 7.52 (dd,

J = 10.3, 4.6 Hz, 2H), 7.58 - 7.64 (m, 3H), 7.74 –7.79 (m, 2H), 8.00

109

(d, J = 1.9 Hz, 1H); 13C NMR (101 MHz, DMSO-d6): δ 52.0, 119.7,

124.2, 125.3, 126.6, 127.2, 127.7, 128.8, 128.9, 129.2, 129.4, 129.5,

130.4, 132.2, 134.1, 139.2, 139.6, 185.5; ESI-MS (m/z): 338.0

[M+H]+.

(4-tolyl)(4-phenyl-1H-pyrrol-2-yl)methanone (56)


ethyl acetate =9:1]; νmax(KBr)/cm-1: 3264, 3028, 1599, 1559, 894;

1H NMR (400 MHz,CDCl3): δ2.45 (s, 3H), 7.15 (dd, J = 2.5, 1.6

Hz,1H), 7.22 (d, J = 7.4 Hz, 1H), 7.32 (d, J = 7.9 Hz, 2H), 7.36

(t, J = 7.7 Hz, 2H), 7.43 (dd, J = 3.0, 1.5 Hz, 1H), 7.53 (dd, J =

8.2, 1.1 Hz, 2H), 7.87 (d, J = 8.1 Hz, 2H), 10.07 (s, 1H); 13C

NMR (101 MHz, CDCl3): δ 21.6, 116.1, 121.9, 125.3, 126.4,

127.3, 128.8, 129.1, 129.2, 131.8, 134.4, 135.5, 142.7, 184.7;

ESI-MS (m/z): 262.0 [M+H]+.

(4-methoxyphenyl)(4-phenyl-1H-pyrrol-2-yl)methanone (57)



938; 1H NMR (400 MHz, CDCl3): δ 3.90 (s, 3H), 6.99 - 7.01 (m,

1H), 7.02 - 7.03 (m, 1H), 7.14 (dd, J = 2.6, 1.6 Hz, 1H), 7.21 -

7.25 (m, 1H), 7.34 - 7.39 (m, 2H), 7.41 (dd, J = 3.0, 1.5 Hz, 1H),

7.53 - 7.55 (m, 2H), 7.97 - 7.98 (m, 1H), 7.99 - 8.01 (m, 1H),

9.88 (s, 1H); 13C NMR (101 MHz, CDCl3): δ 55.5, 113.7, 115.5,

110

121.4, 125.3, 126.4, 127.2, 128.8, 130.8, 131.2, 131.8, 134.4,

162.9, 183.7; ESI-MS (m/z): 278.0 [M+H]+.

(3,4-dimethoxyphenyl)(4-phenyl-1H-pyrrol-2-yl)methanone (58)


ethylacetate = 4:1]; νmax(KBr)/cm-1: 3257, 1598, 1233, 1138,

812; 1H NMR (400 MHz DMSO-d6): δ 3.86 (d, J = 7.7 Hz,

6H), 7.12 (d, J = 8.4 Hz, 1H), 7.18 (t, J = 7.4 Hz, 1H), 7.22 (d,

J = 3.8 Hz, 1H), 7.34 (t, J = 7.7 Hz, 2H), 7.42 (d, J = 1.9 Hz,

1H), 7.60 (dd, J = 8.3, 1.9 Hz, 1H), 7.64 - 7.70 (m, 3H), 12.16

(s, 1H); 13C NMR (101 MHz, DMSO-d6): δ 55.9, 56.1, 111.4,

112.0, 115.4, 123.3, 123.4, 125.3, 125.9, 126.3, 129.1, 131.2,


C19H7NO3 [M+H]+ 308.1281 found 308.1282.

(4-Fluorophenyl)(4-phenyl-1H-pyrrol-2-yl)methanone (59)

Yield: 25%; white solid; m.p. 120-122 ºC; Rf : 0.3 [hexanes: ethyl


NMR (400 MHz, DMSO-d6): δ 7.18-7.21 (m, 2H), 7.36 (dt, J =

15.5, 8.4 Hz, 4H), 7.65 - 7.69 (m, 2H), 7.75 (dd, J = 3.1, 1.5 Hz,

1H), 7.98 (dd, J = 8.8, 5.6 Hz, 2H), 12.27 (s, 1H); 13C NMR (101

MHz, DMSO-d6): δ 13C NMR (101 MHz, DMSO) δ 115.9 (2JC-F

= 21.2 Hz), 116.2, 124.1, 125.4, 126.2, 126.4, 129.1, 131.5, 131.9

(3JC-F = 9.0 Hz), 134.7, 135.3 (4JC-F = 3.0 Hz), 164.7 (1JC-F = 250.5

Hz), 182.8. ESI-MS (m/z): 266.0 [M+H]+.

111

(4-Chlorophenyl)(4-phenyl-1H-pyrrol-2-yl)methanone (60)

Naphthalen-2-yl(4-phenyl-1H-pyrrol-2-yl)methanone (61)

Yield: 55%; light yellow solid; m.p. 182-184 ºC; Rf 0.4 [hexanes:

ethyl acetate =4:1]; νmax(KBr)/cm-1: 3275, 1600, 1561, 1391,

899; 1H NMR (400 MHz, DMSO-d6): δ 7.19 (t, J = 7.4 Hz, 1H),

7.30 – 7.32 (m, 1H), 7.34 (t, J = 7.7 Hz, 2H), 7.61 - 7.71 (m, 4H),

7.77 (dd, J = 3.1, 1.5 Hz, 1H), 7.94 (dd, J = 8.5, 1.7 Hz, 1H),

8.04 (d, J = 7.9 Hz, 1H), 8.08 (d, J = 8.6 Hz, 1H), 8.20 (d, J =

7.8 Hz, 1H), 8.56 (s, 1H), 12.31 (s, 1H); 13C NMR (101 MHz,

DMSO-d6): δ 116.4, 124.0, 125.4, 125.6, 126.3, 126.4, 127.2,

128.1, 128.5, 128.6, 129.1, 129.9, 130.0, 131.9, 132.6, 134.8,

134.9, 136.1, 184.2; HRMS-ESI(m/z): calcd for C21H15NO

[M+H]+ 298.1226 found 298.1230.


Yield: 52%; white solid; m.p. 152-154 ºC ; Rf : 0.4 [hexanes:


757; 1H NMR (400 MHz DMSO-d6): δ 7.19 (ddd, J = 8.4, 4.6,

1.3 Hz, 2H), 7.34 (t, J = 7.7 Hz, 2H), 7.60 - 7.65 (m, 2H), 7.67

(dd, J = 8.2, 1.1 Hz, 2H), 7.76 (dd, J = 3.1, 1.5 Hz, 1H), 7.88 -

7.95 (m, 2H), 12.30 (s, 1H); 13C NMR (101 MHz, DMSO-d6): δ

116.4, 124.4, 125.4, 126.3, 126.4, 129.0, 129.1, 131.0, 131.4,

134.7, 137.0, 137.4, 182.8; ESI-MS (m/z): 282.0 [M+H]+.

112

Yield: 77%; white solid; m.p.122-124 ºC; Rf: 0.3[hexanes:


500; 1H NMR (300 MHz, CDCl3): δ 7.19 – 7.24 (m, 1H),

7.40 (dd, J = 10.6, 4.6 Hz, 4H), 7.57 (d, J = 7.2 Hz, 2H),

7.68 (d, J = 4.1 Hz, 1H), 7.99 (d, J = 2.8 Hz, 1H), 9.65 (s,

1H); 13C NMR (101 MHz, DMSO-d6): δ 114.5, 123.8, 125.4,

126.4, 129.1, 131.1, 132.9, 133.7, 134.8, 143.5, 175.2; ESI-

MS (m/z): 254.0 [M+H]+.

(4-(4-Methoxyphenyl)-1H-pyrrol-2-yl)(thiophen-2-yl)methanone (63)

Yield: 45%; white solid; m.p.188-190 ºC; Rf: 0.4 [hexanes:

ethyl acetate =4:1]; νmax(KBr)/cm-1: 3275, 1592, 1570, 809,

1247; 1H NMR (400 MHz, DMSO-d6): δ 3.77 (s, 3H), 6.91 -

6.95 (m, 2H), 7.29 (dd, J = 4.9, 3.8 Hz, 1H), 7.48 - 7.51 (m,

1H), 7.62 (dd, J = 3.1, 1.5 Hz, 1H), 7.64 (s, 1H), 7.66 (s, 1H),

8.00 (dd, J = 5.0, 1.0 Hz, 1H), 8.13 (dd, J = 3.8, 1.0 Hz, 1H),

12.14 (s, 1H); 13C NMR (101 MHz, DMSO-d6): δ 55.5,

114.1, 114.5, 123.2, 126.3, 126.6, 127.4, 129.0, 130.9, 132.8,


C16H13NO2S [M+H]+ 284.074 found 284.0739.

4-(2,4-Dichlorophenyl)-1H-pyrrol-2-yl)(thiophen-2-yl)methanone (64)

113

Yield: 82%; white solid; m.p. 158-160 ºC; Rf : 0.5 [hexanes: ethyl


NMR (400 MHz, DMSO-d6): δ 7.28 (dd, J = 4.9, 3.8 Hz, 1H),

7.41 (dd, J = 8.4, 2.2 Hz, 1H), 7.51 (dd, J = 2.4, 1.6 Hz, 1H), 7.62

(d, J = 2.2 Hz, 1H), 7.64 (dd, J = 3.2, 1.5 Hz, 1H), 7.71 (d, J =

8.4 Hz, 1H), 8.00 (dd, J = 5.0, 1.0 Hz, 1H), 8.08 (dd, J = 3.8, 1.0

Hz, 1H), 12.44 (s, 1H); 13C NMR (101 MHz, DMSO-d6): δ 117.5,

122.0, 126.3, 127.9, 129.0, 129.9, 130.5, 131.7, 131.9, 132.1,


C15H9Cl2NOS [M+H]+ 321.9855 found 321.9855.



ethyl acetate = 9:1]; νmax(KBr)/cm-1: 3271, 1583, 1416,

1387, 819; 1H NMR (400 MHz, DMSO-d6): δ 7.20 (t, J =

7.4 Hz, 1H), 7.30 (dd, J = 4.9, 3.8 Hz, 1H), 7.36 (t, J = 7.7

Hz, 2H), 7.58 (dd, J = 2.4, 1.6 Hz, 1H), 7.73 (dd, J = 9.4, 2.2

Hz, 3H), 8.15 (dd, J = 3.8, 1.0 Hz, 1H), 8.01 (dd, J = 5.0, 1.0

Hz, 1H), 12.24 (s, 1H); 13C NMR (101 MHz, DMSO-d6): δ

114.5, 123.8, 125.4, 126.3, 126.4, 129.0, 129.1, 131.1,


C15H11NOS [M+H]+ 254.0634 found 254.063.

114

(4-Phenyl-1H-pyrrol-2-yl)(pyridin-2-yl)methanone (66)


ethyl acetate =9:1]; νmax (KBr)/cm-1: 3267, 1636, 1502, 1133, 888;

1H NMR (400 MHz, DMSO-d6): δ 7.29 - 7.33 (m, 2H), 7.37 (dd, J

= 7.7, 5.6 Hz, 3H), 7.57 (ddd, J = 7.5, 4.8, 1.2 Hz, 1H), 7.81 (d, J =

7.8 Hz, 1H), 7.94 (td, J = 7.7, 1.7 Hz, 1H), 8.14 (d, J = 3.6 Hz, 1H),

8.66 (d, J = 4.2 Hz, 1H), 8.68 (d, J = 4.7 Hz, 1H), 12.45 (s, 1H); 13C

NMR (101 MHz, DMSO-d6): δ 122.2, 123.4, 126.7, 127.6, 128.1,

130.3, 130.7, 132.6, 133.0, 135.9, 137.8, 148.8, 155.9, 186.9,

189.0; HRMS-ESI(m/z): calcd for C16H12N2O [M+H]+ 249.1022

found 249.1024.

115

CHAPTER 4B

Application of Polyphophoric Acid MediatedRearrangement

on Pyrrole for the Synthesis of 2,4-Disubstituted Pyrrolo[1,2

a]quinoxalines

116

Introduction

Pyrrolo[1,2-a]quinoxalines are important synthetic target owing to their diverse bioactivities and

photophysical properties.[1-4] These molecules have shown their potential as anti-leishmanial and

antitumor compounds as well as central dopamine anatagonists.[5-7] Additionally, compounds like

1 and 2 are also known which display 5-HT3 receptor and glucagon receptor antagonist activity,

respectively (Figure 4.2.1).[8,9]

Figure 4.2.1: Pyrrolo[1,2-a]quinoxaline compounds as 5-HT3 receptor and glucagon receptor

antagonist.

Based on the work described in the previous chapter and also in continuance with our interest

in the synthesis and applications of fused heterocyclic systems,[10-14] we wanted to explore the

synthesis of 2,4-disubstituted pyrrolo[1,2-a]quinoxalines. While diverse methodologies are known

for the synthesis of the above mentioned compounds, they can be segregated into two broad

categories. Cyclization carried out on 1-arylpyrroles and conversion of propiolates or N-ylides to

pyrrolo[1,2-a]quinoxalines via 1,3-dipolar cycloadditions.[15-17]

Scheme 4.2.1 Synthesis of novel quinoxalinehydrides

117

Several alternative strategies have also been devised relying mainly on certain specific precursors

or catalysts for the synthesis of the aforementioned compounds. Ammermann et al. have

demonstrated the synthesis of these molecules via annulation of 2-alkylquinoxalines. Unique

feature of this reaction was incorporation of pyrrole ring at the last step of synthesis using Ir(acac)3

as the catalyst.[18]

Scheme 4.2.2 Annulation of 2-alkyl quinoxalines

Synthesis of pyrrolo[1,2-a]quinoxalines was shown by Gueiffier and co-workers by

condensation of 2-methylquinoxalines and ethyl bromopyruvate.[19]

Scheme 4.2.3 Synthesis of pyrrolo[1,2-a]quinoxalines by condensation of 2-methylquinoxalines

and ethyl bromopyruvate.

In a separate work, one-pot three-component synthesis using o-phenylenediamines, 2-alkoxy-2,3-

dihydrofurans and ketones was disclosed by Wang et al.[20]

118

Scheme 4.2.4 One pot synthesis of pyrrolo[1,2 a]quinoxalines

The reaction was catalyzed by BF3.Et2O with nitromethane as solvent. Zhang and co-workers

used iron catalyzed intra-molecular cyclization of 1-(N-arylpyrrol-2-yl)ethanone O-acetyl oximes

for the synthesis of both pyrrolo[1,2-a]quinoxalines as well as their indole congeners.[21]

Scheme 4.2.5 Fe Catalyzed intra-molecular C(sp2)-N cyclisation

A palladium catalyzed one pot approach was put forward by Bruno et al., for the synthesis of

trisubstituted pyrrolo[1,2-a]quinoxalines using 3-substituted-2-chloroquinoxalines, propargyl

bromides and diverse secondary amines as precursors.[22]

Scheme 4.2.6 One pot synthesis of 1,2,4-tri(morpholin-)pyrrolo[1,2 a]quinoxalines

119

In a recent report Subba Reddy and co-workers have reported one pot synthesis of pyrrolo[1,2-

a]quinoxalines 1-(2-aminophenyl)pyrrole and stryrene as precursors and using I2/IBX in DMSO

as oxidant.[23]

Scheme 4.2.7 Synthesis of pyrrolo[1,2-a]quinoxalines from styrenes

The literature search reveals that most of the recent efforts rely mainly on precursors which are

either difficult to synthesize or require expensive/difficult to handle transition metal catalysts.

Additionally, in the cases with simple starting compounds, sufficient structural diversity of the

final product is not possible. Our motivation here was to start with a precursor that can be easily

synthesized and subsequently converted to the target compounds. Accordingly a retrosynthetic

approach was envisaged as shown in Scheme 4.2.8. We decided to start our method with chalcones

for their easy accessibility by synthesis as well as possibility of incorporating diverse terminal

groups. For the second step conversion of chalcones to 3, 3’-disubstituted pyrroles with

toluenesulfonylmethylisocyanide (TosMIC) was to be implemented. Given the requirement of a

2-acyl substituted pyrrole for our proposed route, polyphosphoric acid mediated rearrangement of

3, 3’-disubstituted pyrroles was selected. Subsequent steps proposed were reaction between 2-

fluoronitrobenzene and the pyrrole compound, followed by reduction, to yield the target

compounds.

120

Scheme 4.2.8 Retrosynthetic analysis for the synthesis of 2,4-disubstituted pyrrolo[1,2-

a]quinoxalines.


Our efforts towards synthesis of the target molecules started with conversion of chalcones (26-

37) to corresponding 2,3-disubstituted pyrroles by application of well-known Van Leusen

synthesis.[24, 25] The product bearing an acyl linkage was then treated with polyphosphoric acid at

110 °C to effect acyl rearrangement to C-2 position of the pyrrole ring (Scheme 4.2.9).[26]

Scheme 4.2.9 Synthesis of 2,4-disubstituted pyrrole from chalcones.

Subsequent step involved application of SNAr reaction by using rearranged pyrrole and 1-fluoro-

2-nitrobenzene (62) as substrates in the presence of K+-OC(CH3)3 in DMSO (Scheme-4.2.10).

121

Scheme 4.2.10 Functionalization of pyrrole nitrogen by 1-fluoro-2-nitrobenzene

The product isolated with o-nitrophenyl substituent on pyrrole nitrogen was now suitable to try

the final step. For this reaction (1-(2-nitrophenyl)-4-phenyl-1H-pyrrol-2-yl)(phenyl)methanone

(63) was used as a model substrate and a screening program was initiated to identify the most

suitable condition, to sequentially carry out selective reduction of nitro group in the presence of

ketone functionality and imine formation-cyclization reactions. Initial attempts (Table 4.2.1 entry

1-2) carried out with NaBH4 (5 eqv) and NiCl2.6H2O (0.2 eqv) in CH3CN-H2O (1:2) and (9:1) gave

the target molecule in 20% and 40% yields respectively. Increasing the amount of NiCl2.6H2O to

0.5 equivalents in CH3CN-H2O (9:1) (entry 3) did not improve the yield (38%). Following this

reaction with NaBH4/NiCl2.6H2O [5/0.2] in THF and THF-H2O combination were carried out

(entry 4-6) which gave the highest yield of 40% when THF-H2O ratio was 1:1. Reaction was also

attempted with sodium dithionite (entry 7) based on literature precedence which did not give the

target molecule in isolable yields.27 When Fe was used to facilitate the reduction of nitro group in

acetic acid the final product was obtained in 42% yield (entry-8). However reaction using Fe in

the combination of EtOH:CH3COOH:H2O (2:2:1) was not successful (entry-9). Reduction when

pursued with Fe in combination with either CaCl2 or NH4Cl (entry 10-12) failed to yield the final

compounds. Based on the screening reduction with Fe in acetic acid was identified as the optimized

condition for further attempts on required nitro substrates.

122

Table 4.2.1 Optimization of reaction conditions

Entry Reagent [eq] Solvent Temp. (°C) Time (hr) %Yielda

1 NaBH4/NiCl2.6H2O [5/ 0.2] CH3CN-H2O (1:2) RT 0.5 20

2 NaBH4/NiCl2.6H2O [5/0.2] CH3CN-H2O (9:1) RT 0.5 40

3 NaBH4/NiCl2.6H2O [5/0.5] CH3CN-H2O (9:1) RT 0.5 38

4 NaBH4/NiCl2.6H2O [5/0.2] THF RT 0.5 N.I.

5 NaBH4/NiCl2.6H2O [5/0.2] THF-H2O (1:1) RT 0.5 40

6 NaBH4/NiCl2.6H2O [5/0.2] THF-H2O (9:1) RT 0.5 N.I.

7 Na2S2O4 [5] EtOH-H2O (1:1) RT 0.5 N.I.

8 Fe [5] CH3COOH 90 2 42

9 Fe [5] EtOH : CH3COOH :

H2O (2:2:1) 90 2 N.I.

10 Fe/CaCl2[5/0.2] MeOH 70 12 -

11 Fe/NH4Cl [5/5] 1,4-dioxane-H2O (9:1) 90 12 N.I.

12 Fe/NH4Cl [5/5] THF-H2O (9:1) 90 12 N.I.

aIsolated yields; N.I. conversion to final compound was noticed but isolation of the compound was not possible.

The optimized conditions were subsequently used on various N-substituted pyrrole compounds

for the synthesis of the final compounds. Initial attempts were carried out on substrates 64-66 with

unsubstituted benzoyl moiety on 2nd position and various para substituted phenyl rings on 4th

position. Compounds 76-78 were obtained in 41-58% yields, the outcome of the reaction showed

no dependence on the electron withdrawing or electron donating ability of the substituents, as

comparable yields of 58% and 55% were seen for compounds 77 and 78, respectively. Screening

substrates 67-68 with di-substituted phenyl ring on 4th position of pyrrole and unsubstituted

benzoyl moiety on 2nd position gave compound 79 with 66% yield and 80 with 45% yield. The

123

yields obtained in the above reaction do not show any steric requirement on substituent at 4th

position of pyrrole. Reactions on compounds 69-71 containing substituted benzoyl group on 2nd

position and unsubstituted phenyl ring on 4th position were subsequently attempted which yielded

the target molecules in 39-50% yields. Comparatively better yield of 50% in case of substrate 70

with p-methoxy substituent on benzoyl moiety was surprising as presence of electron donating

group on benzoyl group is generally known to reduce the reactivity of carbonyl carbon towards

nucleophilic addition reaction. On using substrates with acetyl and 2-thienocarbonyl group

products 85 and 86 were obtained in 38% and 30% yields, respectively.

Table 4.2.2 Synthesis of various pyrrolo[1,2-a]quinoxalines

75 R = Ph; R’ = Ph [46%] 76 R = p-Me-Ph; R’ = Ph [41%]

77 R = p-OMe-Ph; R’ = Ph [58%] 78 R = p-F-Ph; R’ = Ph [55%]

79 R = o, p-Cl, F-Ph; R’ = Ph [66%] 80 R = o, p-Cl, Cl-Ph; R’ = Ph [45%]

81 R = Ph; R’ = p-Me-Ph [39%] 82 R = Ph; R’ = p-OMe-Ph [50%]

83 R = Ph; R’ = p-F-Ph [42%] 84 R = Ph; R’ = p-Cl-Ph [48%]

85 R = Ph; R’ = CH3 [38%] 86 R = Ph; R’ = 2-thienyl [30%]

While the yields obtained are modest at best, given the fact that two consecutive reactions can

be performed and the final step of nucleophilic addition reaction was can be successfully carried

out on comparatively unreactive ketone, makes this alternative approach useful in our opinion.

Conclusions

In summary, we have put forward an alternate strategy for the synthesis of pyrrolo[1,2-

a]quinoxalines from easily available chalcones. The method developed allows exclusive synthesis

of 2, 4-disubstituted variant of pyrrolo[1,2-a]quinoxalines in four easy steps to execute and in

124

modest yields. Given the importance of these scaffolds due to their attractive biological and

photophysical properties, we feel that the devised method will find a lot of applications in the

domain of medicinal as well as materials chemistry.

General method for the synthesis of compounds (63-74)

In an oven dried round bottomed flask under nitrogen atmosphere compound 4 (0.80 mmol) and

0.1 ml (0.97mmol) of o-fluoro nitrobenzene were dissolved in DMSO (3 ml). Further potassium

tertiary-butoxide (100 mg; 0.97mmol) was added and the resulting reaction mixture was heated at

120 °C for 6 hours. The reaction was then quenched with water (10 ml) and extracted with ethyl

acetate (3 x 10 ml). The resulting organic layer was dried with anhydrous Na2SO4 and concentrated

under reduced pressure. The concentrate obtained was purified using column chromatography with

silica gel as the stationary phase.

General method for the synthesis of fused pyrrolo[1,2-a]quinolxilines(75-86)

Appropriate N-functionalized pyrrole (5) (0.27 mmol) was dissolved in 3 ml acetic acid under

nitrogen atmosphere and Fe (5 eqv) was added to it. The reaction mixture was then allowed to

reflux at 110 ºC for 3 hours. On completion of the reaction as indicated by TLC, the reaction was

quenched with water (10 ml) and extracted with ethyl acetate (3 x 10 ml). The resulting organic

layer was dried with anhydrous Na2SO4 and concentrated under reduced pressure. The resulting

crude compound was purified using column chromatography.

125

1HNMR of 2,4-Diphenylpyrrolo[1,2-a]quinoxaline (75)

13C NMR of 2,4-Diphenylpyrrolo[1,2-a]quinoxaline (75)

126

(1-(2-Nitrophenyl)-4-phenyl-1H-pyrrol-2-yl)(phenyl)methanone (63)

Yield: 86%; Light yellow solid; m.p. 70-73 ºC; Rf :0.5 [1 : 4 Ethyl

acetate : Hexane]; νmax (KBr)/cm-1: 3133, 1632, 1527, 1402 cm-

1; 1H NMR (400 MHz, CDCl3) δ 7.18 (d, J = 1.9 Hz, 1H), 7.26

(dt, J = 14.8, 1.2 Hz, 2H), 7.33 (d, J = 1.9 Hz, 1H), 7.38 (t, J =

7.7 Hz, 2H), 7.47 (t, J = 7.5 Hz, 2H), 7.53 (d, J = 1.1 Hz, 1H),

7.55 (d, J = 1.5 Hz, 2H), 7.57 (d, J = 1.5 Hz, 1H), 7.62 (dd, J =

8.1, 1.4 Hz, 1H), 7.74 (td, J = 7.7, 1.5 Hz, 1H), 7.83 - 7.86 (m,

2H), 8.13 (dd, J = 8.2, 1.5 Hz, 1H); 13C NMR (101 MHz, CDCl3)

δ 119.9, 125.2, 125.4, 126.6, 126.9, 128.3, 128.9, 129.2, 129.4,

129.8, 132.1, 132.6, 133.4, 133.7, 134.7, 138.5, 145.9, 185.3;

ESI-MS(m/z): 369.15[M+H]+

(1-(2-Nitrophenyl)-4-(p-tolyl)-1H-pyrrol-2-yl)(phenyl)methanone (64)

Yield: 76%; Light yellow solid; m.p. 128-130 ºC; Rf : 0.5 [1 : 4

Ethyl acetate : Hexane]; νmax (KBr)/cm-1: 3133, 1619, 1528.3,

1403.0, 1346.3; 1H NMR (400 MHz, CDCl3) δ 2.35 (s, 3H), 7.16

(dd, J = 9.9, 4.9 Hz, 3H), 7.29 (d, J = 1.8 Hz, 1H), 7.44 (dd, J =

16.4, 8.0 Hz, 4H), 7.53 - 7.57 (m, 2H), 7.60 (dd, J = 8.0, 1.3 Hz,

1H), 7.72 (td, J = 7.7, 1.5 Hz, 1H), 7.82 - 7.86 (m, 2H), 8.11 (dd,

J = 8.1, 1.4 Hz, 1H);13C NMR (101 MHz, CDCl3) δ 21.2, 119.9,

125.2, 125.3, 126.6, 126.7, 128.3, 129.1, 129.4, 129.6, 129.8,

130.6, 132.1, 132.5, 133.7, 134.8, 136.6, 138.5, 145.9, 185.3;

ESI-MS(m/z): 383.15[M+H]+

127

(4-(4-methoxyphenyl)-1-(2-nitrophenyl)-1H-pyrrol-2-yl)(phenyl)methanone (65)


Ethyl acetate : Hexane]; νmax (KBr)/cm-1: 3133, 1632, 1528,

1402, 1249; 1H NMR (400 MHz, CDCl3) δ 3.83 (s, 3H), 6.92 (d,

J = 8.8 Hz, 2H), 7.12 (d, J = 1.9 Hz, 1H), 7.26 (d, J = 0.9 Hz,

1H), 7.44 - 7.48 (m, 4H), 7.54 - 7.58 (m, 2H), 7.61 (dd, J = 8.0,

1.4 Hz, 1H), 7.73 (td, J = 7.7, 1.5 Hz, 1H), 7.82 - 7.85 (m, 2H),

8.12 (dd, J = 8.1, 1.5 Hz, 1H); 13C NMR (101 MHz, CDCl3) δ

55.4, 114.3, 119.8, 125.2, 126.1, 126.3, 126.6, 128.2, 129.1,

129.4, 129.8, 132.0, 132.5, 133.6, 134.8, 138.5, 145.9, 158.7,

185.3; ESI-MS(m/z): 399.13[M+H]+.

(4-(4-Fluorophenyl)-1-(2-nitrophenyl)-1H-pyrrol-2-yl)(phenyl)methanone (66)



1403, 1359; 1H NMR (400 MHz, CDCl3) δ 7.07 (t, J = 8.7 Hz,

2H), 7.12 (d, J = 1.9 Hz, 1H), 7.27 (d, J = 1.9 Hz, 1H), 7.48 (dt,

J = 10.5, 6.3 Hz, 4H), 7.53 - 7.57 (m, 2H), 7.59 - 7.64 (m, 1H),

7.74 (td, J = 7.7, 1.5 Hz, 1H), 7.82 - 7.85 (m, 2H), 8.13 (dd, J =

8.2, 1.5 Hz, 1H); 13C NMR (101 MHz, CDCl3) δ 115.8 (2JC-F =

21.2 Hz), 119.7, 125.2, 125.7, 126.6, 127.0 (3JC-F = 8.0 Hz),

128.3, 129.2, 129.4, 129.6 (4JC-F = 3.0 Hz), 129.8, 132.2, 132.7,

133.7, 134.6, 138.4, 145.9, 161.9 (1JC-F = 246.4 Hz), 185.3; ESI-

MS(m/z): 387.12[M+H]+.

(4-(2-Chloro-4-fluorophenyl)-1-(2-nitrophenyl)-1H-pyrrol-2-yl)(phenyl)methanone (67)

128

Yield: 78%; Light yellow solid; m.p. 148-150; Rf : 0.6 [1 : 4


1402, 1264; 1H NMR (400 MHz, CDCl3) δ 7.01 (ddd, J = 8.7,

7.8, 2.7 Hz, 1H), 7.14 (d, J = 1.8 Hz, 1H), 7.21 (dd, J = 8.5, 2.6

Hz, 1H), 7.40 (d, J = 1.8 Hz, 1H), 7.43 - 7.48 (m, 3H), 7.53 - 7.57

(m, 1H), 7.59 (dd, J = 5.5, 1.4 Hz, 1H), 7.63 (dd, J = 8.0, 1.4 Hz,

1H), 7.74 (td, J = 7.7, 1.5 Hz, 1H), 7.82 - 7.85 (m, 2H), 8.14 (dd,

J = 8.1, 1.5 Hz, 1H); 13C NMR (101 MHz, CDCl3) δ 114.3, 114.5,

117.5, 117.7, 122.5, 125.2, 128.3, 128.8 (4JC-F = 3 Hz), 129.3,

129.4, 129.6, 129.8, 131.2 (2JC-F = 8 Hz), 131.7, 132.2, 132.8

(3JC-F = 10 Hz), 133.7, 134.5, 138.3, 145.9, 161.3 (1JC-F = 250

Hz), 185.2; ESI-MS(m/z): 421.10[M+H]+.

(4-(2,4-Dichlorophenyl)-1-(2-nitrophenyl)-1H-pyrrol-2-yl)(phenyl)methanone (68)



1403, 783; 1H NMR (400 MHz, CDCl3) δ 7.17 (d, J = 1.8 Hz,

1H), 7.26 (dd, J = 6.7, 1.6 Hz, 1H), 7.42 - 7.48 (m, 5H), 7.53 -

7.59 (m, 2H), 7.62 (td, J = 7.9, 1.4 Hz, 1H), 7.75 (td, J = 7.7, 1.5

Hz, 1H), 7.82 - 7.86 (m, 2H), 8.14 (dd, J = 8.1, 1.4 Hz, 1H); 13C

NMR (101 MHz, CDCl3) δ 122.3, 125.2, 127.4, 128.3, 129.4,

129.4, 129.7, 129.8, 130.2, 130.9, 131.1, 131.8, 132.2, 132.6,

133.0, 133.7, 134.5, 138.3, 145.9, 185.2; ESI-MS(m/z):

437.04[M+H]+.

129

(1-(2-Nitrophenyl)-4-phenyl-1H-pyrrol-2-yl)(p-tolyl)methanone (69)



1402, 1348; 1H NMR (400 MHz, CDCl3) δ 2.43 (s, 3H), 7.18 (d,

J = 1.9 Hz, 1H), 7.26 (d, J = 8.2 Hz, 3H), 7.32 (d, J = 1.9 Hz,

1H), 7.37 (t, J = 7.7 Hz, 2H), 7.54 (ddd, J = 5.9, 4.3, 1.2 Hz, 3H),

7.57 - 7.62 (m, 1H), 7.72 (dd, J = 7.7, 1.5 Hz, 1H), 7.76 (d, J =

8.2 Hz, 2H), 8.12 (dd, J = 8.1, 1.5 Hz, 1H); 13C NMR (101 MHz,

CDCl3) δ 21.6, 119.5, 125.2, 125.4, 126.4, 126.6, 126.8, 128.9,

128.9, 129.1, 129.6, 129.8, 132.8, 133.5, 133.6, 134.8, 135.7,

142.8, 145.9, 185.1; ESI-MS(m/z): 383.13[M+H]+.

(4-Methoxyphenyl)(1-(2-nitrophenyl)-4-phenyl-1H-pyrrol-2-yl)methanone (70)

Yield: 55%; Yellow solid; m.p. 123-125 ºC; Rf : 0.5 [2 : 3 Ethyl

acetate : Hexane]; νmax (KBr)/cm-1: 3134, 1625, 1529, 1402, 1256;

1H NMR (400 MHz, CDCl3) δ 3.88 (s, 3H), 6.94 - 6.97 (m, 2H),

7.17 (d, J = 1.9 Hz, 1H), 7.23 - 7.28 (m, 1H), 7.31 (d, J = 1.9 Hz,

1H), 7.38 (t, J = 7.7 Hz, 2H), 7.56 – 7.53 (m, 3H), 7.57 - 7.61 (m,

1H), 7.72 (td, J = 7.7, 1.5 Hz, 1H), 7.87 (d, J = 8.9 Hz, 2H), 8.10

(dd, J = 8.1, 1.4 Hz, 1H); 13C NMR (101 MHz, CDCl3) δ 55.5,

113.6, 118.9, 125.2, 125.4, 126.3, 126.8, 128.9, 129.1, 129.8,

131.0, 131.7, 132.9, 133.6, 134.7, 145.9, 163.0, 184.1; ESI-

MS(m/z): 399.13[M+H]+.

(4-Fluorophenyl)(1-(2-nitrophenyl)-4-phenyl-1H-pyrrol-2-yl)methanone (71)

130

Yield: 64%; White solid; m.p. 102 - 104 ºC; Rf: 0.4 [1: 4

Ethylacetate : Hexane]; νmax (KBr)/cm-1: 1630, 1596, 1530,

1403, 1299; 1H NMR (400 MHz, CDCl3) δ 7.15 (dd, J = 10.0,

7.4 Hz, 3H), 7.28 (d, J = 7.5 Hz, 1H), 7.34 (d, J = 1.9 Hz, 1H),

7.39 (t, J = 7.7 Hz, 2H), 7.55 (td, J = 8.0, 1.3 Hz, 3H), 7.67 –

7.59 (m, 1H), 7.75 (td, J = 7.7, 1.5 Hz, 1H), 7.88 (dd, J = 8.9,

5.4 Hz, 2H), 8.13 (dd, J = 8.2, 1.5 Hz, 1H); 13C NMR (101 MHz,

CDCl3) δ 115.3, 115.5, 119.6, 125.2, 125.4, 126.6, 126.9, 128.6,

133.7, 128.9, 129.2, 129.8, 130.8, 131.3, 131.8, 131.9, 133.3,

ESI-MS(m/z): 387.10[M+H]+.

(4-(4-Chlorophenyl)-1-(2-nitrophenyl)-1H-pyrrol-2-yl)(phenyl)methanone (72)

Yield: 75%; Yellow solid; m.p. 95-97 ºC; Rf: 0.4[1:4 Ethyl

acetate : Hexane]; νmax (KBr)/cm-1: 3132, 1632, 1524, 1402,

1159; 1H NMR (400 MHz, CDCl3) δ 7.15 (d, J = 1.9 Hz, 1H),

7.28 (d, J = 7.4 Hz, 1H), 7.34 (d, J = 1.9 Hz, 1H), 7.38 (t, J = 7.7

Hz, 2H), 7.42 - 7.47 (m, 2H), 7.54 (td, J = 7.9, 1.2 Hz, 3H), 7.60

- 7.65 (m, 1H), 7.74 (dd, J = 7.7, 1.5 Hz, 1H), 7.77 - 7.80 (m,

2H), 8.13 (dd, J = 8.2, 1.5 Hz, 1H), 13C NMR (101 MHz, CDCl3)

δ 119.8, 125.2, 125.4, 126.7, 126.9, 127.1, 128.6, 128.9, 129.3,

129.8, 130.8, 132.3, 133.3, 133.7, 134.5, 136.8, 138.5, 145.9,

183.9; ESI-MS(m/z): 403.08[M+H]+.

1-(1-(2-Nitrophenyl)-4-phenyl-1H-pyrrol-2-yl)ethanone (73)

131

Yield: 81 %; Light yellow solid; m.p. 123-125 ºC; Rf : 0.4 [1 : 4


1526, 1247; 1H NMR (400 MHz, CDCl3) δ 2.46 (s, 3H), 7.23 (s,

1H), 7.27 - 7.32 (m, 1H), 7.43 (dd, J = 14.6, 7.7 Hz, 4H), 7.60

(dd, J = 16.9, 7.7 Hz, 3H), 7.72 (t, J = 7.6 Hz, 1H), 8.15 (d, J =

8.1 Hz, 1H); 13C NMR (101 MHz, CDCl3) δ 26.6, 117.4, 125.1,

125.4, 126.2, 126.5, 126.6, 126.9, 128.9, 129.2, 129.6, 132.5,

133.5, 133.6, 134.7, 134.9, 145.9, 187.8; ESI-MS(m/z):

307.10[M+H]+

(1-(2-Nitrophenyl)-4-phenyl-1H-pyrrol-2-yl)(thiophen-2-yl)methanone (74)

Yield: 68%; White solid; m.p. 109-111 ºC; Rf : 0.4 [1 : 4 Ethyl

acetate : Hexane]; νmax (KBr)/cm-1:1605, 1525, 1402, 1349, 823;

1H NMR (400 MHz, CDCl3) δ 7.16 (dd, J = 4.9, 3.8 Hz, 1H),

7.29 – 7.25 (m, 1H), 7.31 (d, J = 1.8 Hz, 1H), 7.39 (t, J = 7.7 Hz,

2H), 7.47 (d, J = 1.8 Hz, 1H), 7.51 (dd, J = 7.8, 1.4 Hz, 1H),

7.60 – 7.56 (m, 3H), 7.64 – 7.61 (m, 1H), 7.71 (td, J = 7.7, 1.5

Hz, 1H), 8.12 (dd, J = 8.1, 1.5 Hz, 1H), 7.87 (dd, J = 3.8, 1.1 Hz,

1H); 13C NMR (101 MHz, CDCl3) δ 118.1, 125.2, 125.5, 126.7,

126.9, 127.8, 128.9, 129.3, 129.9, 132.3, 132.9, 133.0, 133.5,

133.6, 134.5, 143.3, 145.9, 176.2; ESI-MS(m/z): 375.08[M+H]+.

2,4-Diphenylpyrrolo[1,2-a]quinoxaline (75)

132

Yield: 46%; White solid; m.p. 128-130 °C; Rf : 0.5; [1 : 9 ethyl

acetate : hexane]; νmax (KBr)/cm-1: 1602, 1467, 1416, 1351, 1264;

1H NMR (400 MHz, CDCl3) δ 7.22 (s, 1H), 7.29 (t, J = 7.3 Hz,

1H), 7.43 (dt, J = 15.2, 7.7 Hz, 3H), 7.53 (dd, J = 18.4, 6.6 Hz,

4H), 7.68 (d, J = 7.4 Hz, 2H), 7.89 (d, J = 8.1 Hz, 1H), 8.03 (t, J

= 7.3 Hz, 3H), 8.23 (s, 1H); 13C NMR (101 MHz, DMSO) δ 105.7,

114.0, 115.2, 125.4, 126.1, 126.4, 126.9, 127.5, 128.5, 129.0,

129.1, 129.3, 129.6, 130.1, 130.5, 134.2, 136.0, 138.2, 153.4;

HRMS-ESI(m/z): calcd for C23H17N2 [M+H]+ 321.1392 found

321.1393.

4-Phenyl-2-(p-tolyl)pyrrolo[1,2-a]quinoxaline (76)

Yield: 41%; White solid; m.p. 171-173 °C; Rf : 0.5 [1 : 9 ethyl


1H NMR (400 MHz, CDCl3) δ 2.38 (s, 3H), 7.18 - 7.23 (m, 2H),

7.24 (s, 1H), 7.42 - 7.48 (m, 1H), 7.52 (dd, J = 8.1, 1.5 Hz, 1H),

7.54 - 7.60 (m, 5H), 7.90 (dd, J = 8.2, 1.1 Hz, 1H), 7.99 - 8.06 (m,

3H), 8.22 (d, J = 1.5 Hz, 1H); 13C NMR (101 MHz, CDCl3) δ 21.2,

105.9, 111.1, 113.6, 125.3, 126.1, 126.9, 127.6, 128.6, 128.7,

129.6, 129.8, 129.9, 130.4, 131.3, 136.3, 136.9, 138.5, 154.2;

HRMS-ESI(m/z): calcd for C24H19N2 [M+H]+ 335.1543 found

335.1552.

2-(4-Methoxyphenyl)-4-phenylpyrrolo[1,2-a]quinoxaline (77)

133



1H NMR (400 MHz, CDCl3) δ 3.85 (s, 3H), 6.96 (d, J = 8.8 Hz,

2H), 7.17 (d, J = 1.5 Hz, 1H), 7.45 (td, J = 7.7, 1.3 Hz, 1H), 7.49

- 7.53 (m, 1H), 7.53 - 7.58 (m, 3H), 7.61 (d, J = 8.8 Hz, 2H), 7.89

(d, J = 9.2 Hz, 1H), 7.98 - 8.07 (m, 3H), 8.17 (d, J = 1.4 Hz, 1H);

13C NMR (101 MHz, CDCl3) δ 55.4, 105.7, 110.8, 113.6, 114.4,

125.3, 126.1, 126.9, 127.4, 127.6, 128.6, 128.7, 129.8, 129.8,


C24H19N2O [M+H]+ 351.1492 found 351.1502.

2-(4-Fluorophenyl)-4-phenylpyrrolo[1,2-a]quinoxaline (78)



1H NMR (400 MHz, CDCl3) δ 7.12 (t, J = 8.7 Hz, 2H), 7.17 (d, J

= 1.5 Hz, 1H), 7.45 - 7.50 (m, 1H), 7.55 (ddd, J = 11.6, 6.0, 1.7

Hz, 4H), 7.62 - 7.67 (m, 2H), 7.91 (dd, J = 8.2, 1.3 Hz, 1H), 8.02

(dd, J = 7.6, 2.0 Hz, 2H), 8.05 (dd, J = 8.0, 1.5 Hz, 1H), 8.20 (d,

J = 1.5 Hz, 1H); 13C NMR (101 MHz, CDCl3) δ 105.9, 111.1,

113.6, 115.9 (2JC-F = 21 Hz), 125.5, 126.2, 126.8, 127.8 (3JC-F = 8

Hz), 128.6, 128.7, 129.0, 129.4 (4JC-F = 3 Hz), 129.9, 130.4, 136.3,

138.3, 154.2, 162.2 (1JC-F = 247.4 Hz); HRMS-ESI(m/z): calcd for

C23H16FN2 [M+H]+ 339.1292 found 339.1304.

2-(2-Chloro-4-fluorophenyl)-4-phenylpyrrolo[1,2-a]quinoxaline (79)

134



1H NMR (400 MHz, CDCl3): δ 8.29 (d, J = 1.4 Hz, 1H), 8.06 (dd,

J = 7.9, 1.4 Hz, 1H), 8.02 (dd, J = 7.4, 2.1 Hz, 2H), 7.91 (d, J = 8.0

Hz, 1H), 7.51 - 7.60 (m, 5H), 7.51 – 7.46 (m, 1H), 7.24 - 7.27 (m,

1H), 7.18 (d, J = 1.4 Hz, 1H), 7.02 - 7.09 (m, 1H); 13C NMR (101

MHz, CDCl3): δ 108.9, 113.7, 114.1, 114.4 (2JC-F = 21 Hz), 117.6

(2JC’-F = 25.2 Hz), 125.1, 125.6, 125.9, 126.7, 127.7, 128.6, 128.7,

129.7 (4JC-F = 3 Hz), 129.9, 130.4, 132.1(3JC-F = 9 Hz) , 133.1 (3JC’-

F = 11 Hz), 136.4, 138.3, 154.3, 161.5 (1JC-F = 251 Hz); HRMS-

ESI(m/z): calcd for C23H15ClFN2 [M+H]+ 373.0902 found

373.0914.

2-(2,4-Dichlorophenyl)-4-phenylpyrrolo[1,2-a]quinoxaline (80)

Yield: 45%; White solid; m.p. 200-203 °C; Rf : 0.5 [1 : 9 ethyl acetate

: hexane]; νmax (KBr)/cm-1: 3132, 1608, 1461, 1402, 745; 1H NMR

(400 MHz, CDCl3) δ 7.20 (d, J = 1.5 Hz, 1H), 7.30 (dd, J = 8.4, 2.2

Hz, 1H), 7.48 (s, 1H), 7.50 - 7.53 (m, 2H), 7.53 - 7.58 (m, 4H), 7.91

(dd, J = 8.1, 1.3 Hz, 1H), 8.02 (dd, J = 7.4, 2.2 Hz, 2H), 8.06 (dd, J =

8.0, 1.5 Hz, 1H), 8.33 (d, J = 1.5 Hz, 1H); 13C NMR (101 MHz, CDCl3)

δ 108.8, 113.7, 114.2, 125.2, 125.7, 125.7, 126.7, 127.4, 127.7, 128.6,

128.7, 129.9, 130.2, 130.4, 131.8, 132.0, 132.9, 133.3, 136.4, 138.2,

154.3; HRMS-ESI(m/z): calcd for C23H15N2Cl2 [M+H]+ 389.0607

found 389.0616.

135

2-Phenyl-4-(p-tolyl)pyrrolo[1,2-a]quinoxaline (81)




1H), 7.29 (t, J = 7.4 Hz, 1H), 7.37 (d, J = 7.8 Hz, 2H), 7.42 (dd, J

= 10.4, 4.9 Hz, 2H), 7.46 (dd, J = 7.9, 1.4 Hz, 1H), 7.49 - 7.54 (m,

1H), 7.69 (dd, J = 8.3, 1.2 Hz, 2H), 7.89 (dd, J = 8.1, 1.3 Hz, 1H),

7.93 (d, J = 8.1 Hz, 2H), 8.03 (dd, J = 8.0, 1.5 Hz, 1H), 8.23 (d, J

= 1.5 Hz, 1H); 13C NMR (101 MHz, CDCl3) δ 21.5, 106.0, 111.2,

113.6, 125.4, 126.2, 126.8, 127.1, 127.4, 128.6, 128.9, 129.4,

129.8, 130.3, 134.3, 135.6, 136.4, 140.0, 154.2; HRMS-ESI(m/z):

calcd for C24H19N2 [M+H]+ 335.1543 found 335.1551.

4-(4-Methoxyphenyl)-2-phenylpyrrolo[1,2-a]quinoxaline (82)




2H), 7.24 (d, J = 1.5 Hz, 1H), 7.29 (t, J = 7.4 Hz, 1H), 7.47 – 7.40

(m, 3H), 7.52 – 7.48 (m, 1H), 7.71 – 7.67 (m, 2H), 7.88 (dd, J =

8.1, 1.3 Hz, 1H), 8.03 – 7.99 (m, 3H), 8.22 (d, J = 1.5 Hz, 1H);

13C NMR (101 MHz, CDCl3) δ 55.5, 106.0, 111.2, 113.6, 114.1,

125.4, 126.2, 126.8, 127.1, 127.3, 128.9, 129.8, 130.1, 130.9,

134.3, 136.4, 153.7, 161.1; HRMS-ESI(m/z): calcd for C24H19N2O

[M+H]+ 351.1492 found 351.1500.

4-(4-Fluorophenyl)-2-phenylpyrrolo[1,2-a]quinoxaline (83)

136


acetate : hexane]; νmax (KBr)/cm-1: 3133, 1635, 1401, 1325,

1218; 1H NMR (400 MHz, CDCl3): δ 7.19 (d, J = 1.5 Hz, 1H),

7.25 (t, J = 8.7 Hz, 2H), 7.31 (t, J = 7.4 Hz, 1H), 7.39 - 7.49 (m,

3H), 7.50 - 7.57 (m, 1H), 7.69 (dd, J = 8.2, 1.1 Hz, 2H), 7.90 (dd,

J = 8.2, 1.2 Hz, 1H), 8.03 (dd, J = 8.9, 5.5 Hz, 3H), 8.24 (d, J =

1.5 Hz, 1H); 13C NMR (101 MHz, CDCl3): δ 105.8, 111.4, 113.6,

115.7 (2JC-F = 21 Hz), 125.5, 125.9, 126.2, 126.5, 126.8, 127.2,

127.7, 128.5, 128.9, 130.0, 130.3, 130.5 (3JC-F = 8 Hz), 134.1,

134.5 (4JC-F = 3 Hz), 136.2, 153.0, 163.9 (1JC-F = 258 Hz);

HRMS-ESI(m/z): calcd for C23H16FN2 [M+H]+ 339.1292 found

339.1296.

4-(4-Chlorophenyl)-2-phenylpyrrolo[1,2-a]quinoxaline (84)

Yield: 48%; White solid; m.p. 160-162 °C; Rf :0.4 [1 : 9 ethyl


1H NMR (400 MHz, CDCl3) δ 7.18 (d, J = 1.5 Hz, 1H), 7.31 (t,

J = 7.4 Hz, 1H), 7.40 - 7.49 (m, 3H), 7.50 - 7.57 (m, 3H), 7.68

(dd, J = 8.2, 1.1 Hz, 2H), 7.89 (dd, J = 8.2, 1.1 Hz, 1H), 8.00 –

7.95 (m, 2H), 8.02 (dd, J = 8.0, 1.4 Hz, 1H), 8.23 (d, J = 1.4 Hz,

1H); 13C NMR (101 MHz, CDCl3) δ 105.8, 111.5, 113.6, 125.5,

125.8, 126.2, 126.8, 127.3, 127.8, 128.9, 129.0, 129.9, 130.1,

130.4, 134.1, 135.9, 136.2, 136.8, 152.9; HRMS-ESI(m/z):

calcd for C23H16ClN2 [M+H]+ 355.0997 found 355.1005.

137

4-Methyl-2-phenylpyrrolo[1,2-a]quinoxaline (85)

Yield: 38%; White solid; m.p. 184-190 °C; Rf :0.4 [1 : 9 ethyl


1247; 1H NMR (400 MHz, CDCl3) δ 2.80 (s, 3H), 6.99 (s, 1H),

7.33 (t, J = 7.0 Hz, 1H), 7.44 (t, J = 7.3 Hz, 2H), 7.52 (t, J = 7.6

Hz, 1H), 7.65 (dd, J = 17.9, 7.7 Hz, 3H), 7.86 (d, J = 8.1 Hz,

1H), 8.07 (s, 1H), 8.70 (d, J = 8.2 Hz, 1H); 13C NMR (101 MHz,

CDCl3) δ 14.2, 104.2, 111.2, 113.9, 121.7, 125.7, 126.1, 126.9,

127.4, 128.0, 129.0, 129.6, 130.8, 131.1, 133.6, 138.3. HRMS-

ESI(m/z): calcd for C18H15N2 [M+H]+ 259.1230 found

259.1238.

2-Phenyl-4-(thiophen-2-yl)pyrrolo[1,2-a]quinoxaline (86)



818; 1H NMR (400 MHz, CDCl3) δ 7.22 - 7.25 (m, 1H), 7.31

(t, J = 7.4 Hz, 1H), 7.39 - 7.46 (m, 3H), 7.46 - 7.52 (m, 2H),

7.55 (dd, J = 5.1, 1.0 Hz, 1H), 7.68 - 7.75 (m, 2H), 7.85 (d, J =

8.0 Hz, 1H), 7.95 - 8.02 (m, 2H), 8.20 (d, J = 1.4 Hz, 1H); 13C

NMR (101 MHz, CDCl3) δ 105.3, 111.4, 113.5, 124.8, 125.4,

126.3, 126.7,127.2, 127.5, 127.8, 128.2, 128.8, 128.9, 129.9,


C21H15N2S [M+H]+ 327.0950 found 327.0957.

138

CHAPTER 5

Expanding the Scope of Fused Pyrrolo-quinoline System for

Selective Sensing of Iron

139

Introduction

Iron in both its +2 and +3 oxidation state is one of the most important transition metal in living

systems. It serves as a cofactor in diverse biochemical reactions/processes such as oxidoreductase

catalysis, oxygen transport and electron transport. Both excess and deficiency of this metal is

known to induce wide spectrum of diseases ranging from liver cancer, liver cirrhosis, anaemia to

Parkinson’s disease, arthritis, diabetes, malaria and even heart failure.[1-4] Thus, selective sensing

of iron is of considerable importance to human health. Various techniques such as atomic

absorption spectroscopy,[5] spectrophotometry,[6-8] voltammetry[9-11] and chemiluminescence,[12-13]

have been used for selective detection of iron. Some of these techniques, requiring sophisticated

instrumentation along with elaborate pretreatment procedures are inappropriate for in-field or on-

line monitoring. While potentiometric sensors offer advantages owing to their simple, rapid and

non-destructive characteristics, they are plagued by the problem of low response slopes due to a

charge of analyzed ions. All these concerns are effectively addressed by colorimetric and

fluorescence techniques, which allow sub-ppm level detection along with the possibility of

intracellular monitoring. Additionally, fluorescence sensors are also bestowed with features like

fast response time and technical simplicity. These advantages have catapulted the fluorescence

based analytical techniques at the forefront of selective iron sensing. [14-15]

In a recent report by Lan et al., oligothiophenes (1) were used for selective iron sensing in

aqueous condition.[16] Coumarin based fluorophores (2) were used by Hua et al. and Zhao et al. for

selective turn-OFF fluorescent sensing of Fe3+ in DMSO and in aqueous medium, respectively. [17-

18] In a separate work, Pant and coworkers reported coumarin-triazole compounds (3-4) for

selective iron sensing using turn-OFF fluorescence technique.[19] A chemosensor (5) derived from

vitamin B6 cofactor pyridoxyl-5-phosphate was used by Sharma et al., for selective detection of

140

iron in aqueous medium[20]. 5-hydroxybenzo[g]indoles (6) were used by Pramanik and coworkers

for selective turn-OFF iron sensing.[21] Iron sensors using fluorescence signaling process can be

classified into three types: turn-ON,[16, 22-23] turn-OFF,[17-21, 24] and ratiometric,[25-27] with

significant share garnered by fluorescence turn-OFF techniques. Main reason for such behaviour

is generally attributed to Fe3+ assisted photoinduced electron transfer and/or exited state de-

excitation pathways (via electronic energy transfer).[15]

We have an ongoing interest in the area of ion sensing. Towards this effort, we have looked at

selective Zn2+ and F- sensing by using easy to assemble 4-substituted pyrrolo[2,3-c]quinolines as

fluorophores.[28, 29] The work reported in this communication attempts to expand the scope of these

compounds to selective sensing of Fe3+.

Figure 5.1 Diverse molecules for selective sensing of iron


Given the general tendency of fluorescence based iron detection via quenching pathway and the

141

simplicity of fluorophore requirements, we decided to design fluorophores (7, 8) for this study

with ligands 9 and 10 reported by Ghosh et al. and Zhang et al. (Scheme-5.1).[24, 30]

Scheme 5.1 Design of APQ and HPQ based on known ligands 9 and 10.

Additional inputs for fluorophore design also came from several literature reports on quinoline

based molecules [Figure 5.2(11-13)].[31-34] Kuoxi et al. reported with two acetophenone

conjugated quinolone derivatives which will be used as Fe3+ sensor.[32] Yuanquing et al. had

developed a novel quinoline based Schiff base turn on fluorescent probe which selectively binds

with Cu2+ over the other metal ions.[33] Xiaobin et al. developed highly selective turn-off

fluorescent probe for Cu2+.[34]

Figure 5.2 Quinoline based molecules as metal sensors

142

Our familiarity with pyrrolo[2,3-c]quinoline systems prompted us to initiate this study with

ligands APQ and HPQ bearing o-aniline and o-phenol as substituents. It was felt that presence of

amino and hydroxyl functionality along with pyrrole nitrogen will provide suitable binding sites

to the metal. Molecule HPQ has been already reported by us as a selective fluoride sensor.[29]

Synthesis of the ligands was carried out by using modified Pictet-Spengler reaction as a key step

(Scheme 5.2).[35] The synthetic scheme started with reaction between o-nitrostyrene (14) and

TosMIC in presence of potassium tertiary butoxide to generate o-nitrophenylpyrrole (15). The

product obtained was subsequently converted to o-aminophenylpyrrole (16) in the presence of iron

and hydrochloric acid. Treatment with appropriate aldehyde [salicaldehyde (17) or o-

nitrobenzaldehyde (18)] afforded the required pyrrolo[2,3-c]quinoline scaffold. While HPQ was

formed on reaction with salicaldehyde (17), APQ was formed only after the nitro compound (19)

was reduced.

Scheme 5.2 Synthetic scheme for the ligands APQ and HPQ

143

The compounds were thoroughly characterized prior to UV-visible and fluorescence studies. Our

approach was to look at steady state fluorescence behaviour of APQ and HPQ, individually, in

DMF as a solvent and then carryout the same study by incubating the ligands with Fe3+ ion.

Initial fluorescence screening revealed that while APQ exhibits an intensive fluorescence emission

and a large Stokes shift of 115 nm, due to Excited-State Intramolecular Proton Transfer(ESIPT)

reactions (Scheme 5.3 and 5.4), it was subdued for HPQ with a comparatively low (67 nm) Stokes

shift (Figure 5.3).

Figure 5.3 UV and fluorescence spectrum of APQ and HPQ

Both the ligands displayed fluorescence quenching behaviour with Fe3+, though the relative

fluorescence decrease was less for HPQ in comparison with APQ (Figure-5.4). These primary

results encouraged us to use APQ for further studies. Subsequently, fluorescence behaviour of

APQ was examined by incubation with metals such as Ag+, Ca2+, Cd2+, Co2+, Cu2+, Fe2+, Hg2+, K+,

Mg2+, Na+, Ni2+, Pb2+, Sn2+ and Zn2+ (Figure-5.5). Modest increase in fluorescence intensity was

noticed in case of most of the metals except Ni2+, Sn2+ and Cu2+, whereas Fe2+ also displayed

fluorescence quenching.

APQ HPQ

144

Scheme 5.3 ESIPT mechanism as shown by ligand APQ

Scheme 5.4 Inhibition of ESIPT process on binding with Fe3+

ESIPT on Fe3+

Fluorescence Fluorescence

quenching

325 nm 440 nm

S0

S*

S0

’

S’*

145

Figure 5.4 Fluorescence behaviour of APQ and HPQ on incubation with Fe3+

Figure 5.5 Fluorescence behaviour of APQ on incubation with various metals

To establish the selectivity of the molecule APQ towards Fe3+, competitive fluorescence

sensing experiments were also performed in the presence of other metals (Figure-5.6). The

observations clearly show that APQ is highly selective towards the detection of Fe3+ in presence

146

of most of the other metals. It is noteworthy to mention here that APQ shows prominent selectivity

towards Fe3+, even in presence of well-known interfering cations Co2+and Cu2+.[30]

Figure 5.6 Competitive selectivity (λex = 320 nm and fluorescence was recorded at 435 nm) of

APQ towards Fe3+ in DMF, in presence of other metal ions. 1: only APQ. Black bar represents

APQ + metal ions and red bar represents APQ + metal ions + Fe3+ ions

With these initial results we explored binding stoichiometry between metal and ligand. It

showed ratio of metal to ligand as 2:1. Job’s plot of APQ (in DMF) and Fe3+, where concentrations

of APQ is 10-4 and Fe3+ is 10-2 M, λ ex =320nm and fluorescence was recorded at 440nm (Figure-

5.7). Association constant of the complex and detection limit were subsequently determined as 10

x 106M-2 and 0.4 x 10-6M, respectively, using well established literature reports (Figure-5.8).[36,

37] Stoichiometry of the complex was also confirmed by the MALDI analysis, which showed a

prominent peak at m/z 515.325 [(M + Li+; C17H11Cl4Fe2LiN3+), exact mass m/z 515.856), thus

establishing 2:1, metal to ligand ratio (Figure-5.9).

Further efforts were focused on probing the role played by pyrrole ring nitrogen in APQ. The

main approach here was to introduce minor structural changes in the APQ structure and then

147

examine its effect on Fe3+/Fe2+ sensing. Accordingly, compounds 2-(3-methyl-3H-pyrrolo[2,3-

c]quinolin-4-yl)aniline (21), 2-(thieno[2,3-c]quinolin-4-yl)aniline (22), 2-(4-phenylquinolin-2-

yl)aniline (23) and 3-(3H-pyrrolo[2,3-c]quinolin-4-yl)pyridin-2-amine (24) were synthesized

(Figure-5.10)[35, 38, 39] and their UV absorbance studies were carried out in DMF (Figure-5.11).

Each of these molecules while retaining the basic quinoline core had pyrrole ring either modified

or completely replaced. On screening these compounds with Fe3+ in DMF, fluorescence quenching

was observed in all the cases. It was comparable to APQ in ligands 21 and 22 bearing N-methyl

pyrrole and thiophene ring, respectively (Figure-5.12 and 5.13). Whereas, relative fluorescence

intensity as well as fluorescence quenching was very less for ligand 23 (Figure-5.14). Thus, these

results indicated that presence of pyrrole ring is not an absolute requirement for the selective

sensing of Fe3+ ion.

0.0 0.2 0.4 0.6 0.8 1.0

0.00

0.01

0.02

0.03

0.04

0.05

0.06

[C] *

Inte

nsit

y

Mole fraction

Figure 5.7 Job’s plot to find out binding stoichiometry between metal and ligand

148

-4.4 -4.2 -4.0 -3.8 -3.6 -3.4 -3.20.0

0.2

0.4

0.6

0.8

1.0

I max-I

/I m

ax-I

min

log[Fe3+

]

Figure 5.8 Limit of Detection (calculated for linear range of 10-450 M at wavelength 435 nm)

Plots of (Imax-I)/(Imax-Imin) vs log([Fe3+]), the calculated detection limit of sensor APQ is 43

µM.

Calculations:

Intercept = -4.36

Log [Fe3+] = -4.36

[Fe3+] = Antilog - 4.36 = 4.32 x 10-5M = 43 µM

149

Figure 5.9 MALDI (Matrix: DHB): m/z 515.325 [(M + Li+; C17H11Cl4Fe2LiN3+), Exact mass

m/z 515.856

Figure 5.10 Structure of ligands 21, 22, 23 and 24

150

300 400 500

0

1

2

Absorb

ance

Wavelength (nm)

10-4

M 21

10-4

M 22

10-4

M 23

10-5

M 24

Figure 5.11 UV study for the compounds 21, 22, 23 and 24 in DMF solvent.

Compound 21 shows absorbance at 309 nm




151

Scheme 5.5 Synthetic scheme for 2-(3-methyl-3H-pyrrolo[2,3-c]quinolin-4-yl)aniline (21)

350 400 450 500 550 6000

200

400

600

800

Flu

ore

scence I

nte

nsit

y

Wavelength (nm)

10-5

M 21 in DMF

1 x 10-4

M Fe3+

2 x 10-4

M Fe+3

3 x 10-4

M Fe3+

4 x 10-4

M Fe+3

5 x 10-4

M Fe+3

Figure 5.12 Fluorescence quenching behaviour of ligand 21 on incubation with Fe3+

152

Scheme 5.6 Synthetic scheme for 2-(thieno[2,3-c]quinolin-4-yl)aniline (22)

100 200 300 400 500 600 700 800 900 1000

0

100

200

300

400

500

600

Flu

ore

scen

ce I

nte

nsi

ty

Wavelength(nm)

1 x 10-4 M Fe3+ in DMF

2 x 10-4 M Fe+3 in DMF

3 x 10-4 M Fe3+ in DMF



10-5M 22 in DMF


153

Scheme 5.7 Synthetic scheme for 2-(4-phenylquinolin-2-yl)aniline (23)

400 600 800

0

50

100

150

200

Flu

oresc

en

ce I

nte

nsi

ty

Wavelength(nm)

10-5

M 23 in DMF

1 x 10-4

M Fe3+

in DMF

2 x 10-4

M Fe3+

in DMF

3 x 10-4

M Fe3+

in DMF

4 x 10-4

M Fe3+

in DMF


154

In case of ligand 24, minor modification on aniline half of APQ scaffold was carried out by

replacing benzene with pyridine ring while retaining the relative position of amino functionality.[35]

It also exhibited fluorescence quenching behaviour as APQ, on incubation with Fe3+, showing

criticality of the amino functionality (Figure 5.15).

Scheme 5.8 Synthetic scheme for 3-(3H-pyrrolo[2,3-c]quinolin-4-yl)pyridin-2-amine (24)

200 400 600 800 1000

0

200

400

600

800

Flo

ure

scen

ce I

nte

nsi

ty

Wavelength (nm)

10-5

M 24 in DMF

1 x 10-4

M Fe3+

in DMF

2 x 10-4

M Fe3+

in DMF

3 x 10-4

M Fe3+

in DMF

4 x 10-4

M Fe3+

in DMF

5 x 10-4

M Fe3+

in DMF


Subsequently, the reversibility of Fe3+ binding to APQ was tested by titration with EDTA, a

well- known chelator for Fe3+. While gradual enhancement of fluorescence signal on titration of

155

Fe3+-APQ complex with EDTA solution was noticed, it did not recover fully, which is clearly

indicative of the high association constant of Fe3+-APQ complex. The signal was again quenched

on further addition of Fe3+ solution. This experiment helped us to establish a somewhat reversible

binding behaviour of Fe3+ with APQ.

Given the importance of logic gates in molecular keypad devices and molecular switches,

reversibility experiment involving Fe3+ and EDTA were used as input signals for the same.

Emission intensity at 140 a.u. was taken as the threshold value at wavelength 435 nm. A state OUT

= 0 was given above the threshold value of the emission intensity, while a state OUT = 1 was given

below it (Figure-5.16).

Figure 5.16 (a) Fe3+and EDTA as chemical inputs and their effect on the emission spectra of

APQ. (b) Emission spectra of APQ, EDTA and Fe3+titrations. (c) Truth table incorporating logic

functions

156

In order to explore the practical applications of the developed fluorophore, quenching

experiments were also performed by encapsulating APQ inside span80 (sorbitan ester of oleic

acid) niosome. Niosomes are vesicular structures formed by self-assemblies of non-ionic

surfactants. They mimic cell membranes in several aspects and potentially used as transdermal

carriers for hydrophilic or hydrophobic drugs.[40] We have demonstrated confocal imaging

(Figure-5.17) in the span80 vesicular system by using the fluorescence of APQ with and without

Fe3+. Figures 5a, b showed the bright field and the corresponding fluorescence images of vesicles

loaded with APQ, when excited at 405 nm and emission was collected in the wavelength region

of 460 ± 20nm. As and when, APQ bound with Fe3+, the fluorescence intensity was quenched

significantly and very weak fluorescence was observed from the vesicles (Figure-5.17 d & f).

These observations clearly indicated the interaction of APQ with Fe3+ inside the vesicle membrane,

which can be suitably tailored for biological applications as required.

157

Figure 5.17 Bright field (a, c, e) and fluorescence (b, d, f) Confocal images of span-80 niosomes

loaded with APQ, with and without Fe3+

Conclusions

In summary, we have developed a sensor for selective micromolar detection of Fe3+. The probe

works by blocking the ESIPT reaction of the ligand in the presence of Fe3+ and performs optimally

even in the presence of well-known interfering cations Co2+ and Cu2+. Binding stoichiometry was

found to be 2:1 for metal and ligand. The capability of the developed sensor was further

demonstrated by its encapsulation inside vesicle membrane and detection of Fe3+ ions thereby.

158

Experimental

All the starting materials were purchased and used directly. Solvents were dried and distilled

before use. Visualization on TLC was achieved by use of UV light (254 nm) or iodine. 1H NMR

(300MHz and 400 MHz) and 13C (75 MHz and 100MHz) spectra were recorded in CDCl3 and

DMSO solution with TMS as internal standard. The mass spectrum was recorded on Agilent

1100/LC MSD Trap SL version. Column chromatography was performed on silica gel (100–200

mesh, SRL. India). Fluorescence studies were done on Hitachi F-7000 spectrofluorimeter.

Synthesis of 2-(3H-pyrrolo[2,3-c]quinolin-4-yl)aniline (7-APQ): Synthesis of the compound

was carried out as per the procedure described in reference 31.

1H NMR (300 MHz, DMSO-d6): δ 6.13 (s, 2H), 6.77 (t, J = 7.4 Hz, 1H), 6.92 (d, J = 8.1 Hz, 1H),

7.22 (s, 2H), 7.58 (t, J = 6.6 Hz, 4H), 8.06 – 7.97 (m, 1H), 8.36 – 8.25 (m, 1H), 11.69 (s, 1H). 13C

NMR (101 MHz, DMSO-d6): δ 101.6 (s), 116.7 (s), 120.6 (s), 123.1 (s), 148.0 (s), 123.5 (s), 125.9

(s), 126.2 (s), 127.3 (s), 128.6 (s), 129.1 (s), 129.5 (s), 130.2, 141.7 (s), 147.6 (s); HRMS-ESI(m/z):

calcd for C17H14N3 [M+H]+ 260.1182 found 260.1186.

Synthesis of 2-(3H-pyrrolo[2,3-c]quinolin-4-yl)phenol (8-HPQ): Synthesis of the compound

was carried out as per the procedure described in reference 31. Structure confirmed by IR, 1H, 13C

NMR and mass spectrum and was consistent with those described in the literature.[31]

Synthesis of 2-(3-methyl-3H-pyrrolo[2,3-c]quinolin-4-yl)aniline (21): Synthesis of the

compound was carried out as per the procedure described in reference 31.

1H NMR (300 MHz, DMSO-d6): δ 3.37 (s, 3H), 4.82 (s, 2H), 6.72 (t, J = 7.2 Hz, 1H), 6.85 (d, J

= 8.0 Hz, 1H), 7.12 (d, J = 7.4 Hz, 1H), 7.20 (dd, J = 15.0, 5.1 Hz, 2H), 7.57 (dd, J = 6.1, 3.3 Hz,

3H), 8.00 (dd, J = 6.1, 3.4 Hz, 1H), 8.32 (dd, J = 6.2, 3.2 Hz, 1H). 13C NMR (75 MHz, DMSO-

159

d6): δ 35.2 (s), 99.8 (s), 114.9 (s), 115.9 (s), 122.9 (d, J = 12.8 Hz), 125.59 (d, J = 10.0 Hz), 127.9

(s), 128.9 (s), 129.4 (s), 129.7 (s), 130.2 (s), 133.2 (s), 141.8 (s), 146.4 (d, J = 8.0 Hz); HRMS-

ESI(m/z): calcd for C18H16N3 [M+H]+ 274.1339 found 274.1342.

Synthesis of 2-(thieno[2,3-c]quinolin-4-yl)aniline (22): Synthesis of the compound was carried

out as per the procedure described in reference 34.

1H NMR (400 MHz, DMSO-d6): δ 7.74 - 7.83 (m, 2H), 7.86 (ddd, J = 8.1, 7.4, 1.6 Hz, 1H), 7.96

(td, J = 7.5, 1.2 Hz, 1H), 8.01 (dd, J = 7.6, 1.4 Hz, 1H), 8.07 - 8.12 (m, 1H), 8.24 (dd, J = 8.1, 0.9

Hz, 1H), 8.34 (d, J = 5.3 Hz, 1H), 8.44 (d, J = 5.4 Hz, 1H), 8.60 - 8.64 (m, 1H). 13C NMR (101

MHz, DMSO-d6): δ 116.2, 116.8, 121.9, 123.2, 124.2, 127.1, 128.9, 129.3, 129.5, 130.8, 132.7,

134.1, 143.4, 144.1, 147.8, 154.2; HRMS-ESI(m/z): calcd for C17H13N2S [M+H]+ 277.0799 found

277.0795

Synthesis of 2-(4-phenylquinolin-2-yl)aniline (23): Synthesis of the compound was carried out

as per the procedure described in reference 35.

1H NMR (400 MHz, DMSO-d6): δ 6.62 - 6.68 (m, 1H), 6.87 (dd, J = 8.2, 1.1 Hz, 1H), 7.12 - 7.20

(m, 3H), 7.53 - 7.66 (m, 6H), 7.79 (ddd, J = 8.3, 6.9, 1.4 Hz, 1H), 7.84 (ddd, J = 8.3, 4.2, 1.0 Hz,

2H), 7.88 (s, 1H), 8.12 (d, J = 8.4 Hz, 1H). 13C NMR (100 MHz, DMSO-d6): δ 115.7, 116.6,

119.1, 120.0, 124.0, 125.0, 126.4, 128.5, 128.7, 128.9, 129.5, 129.7, 130.2, 137.5, 146.7, 148.1,

148.6, 158.4; HRMS-ESI(m/z): calcd for C21H17N2 [M+H]+ 297.1392 found 297.1380.

Synthesis of 3-(3H-pyrrolo[2,3-c]quinolin-4-yl)pyridin-2-amine (24): Synthesis of the

compound was carried out as per the procedure described in reference 31.

1H NMR (400 MHz, DMSO-d6): δ 6.81 (dd, J = 7.4, 4.9 Hz, 1H), 6.91 (s, 2H), 7.23 - 7.27 (m, 1H),

7.57 - 7.66 (m, 3H), 8.01 (ddd, J = 9.2, 7.3, 2.8 Hz, 2H), 8.13 (dd, J = 4.9, 1.7 Hz, 1H), 8.33 (dd,

160

J = 6.5, 3.0 Hz, 1H), 11.86 (s, 1H). 13C NMR (101 MHz, DMSO-d6): δ 101.8 (s), 113.0 (s), 115.3

(s), 123.2 (s), 123.5 (s), 126.2 (s), 126.4 (s), 127.2 (s), 128.9 (s), 129.1 (s), 129.7 (s), 138.2 (s),

141.6 (s), 145.8 (s), 148.9 (s); HRMS-ESI(m/z): calcd for C16H13N4 [M+H]+ 261.1135 found

261.1135

Encapsulation of APQ in the vesicle and confocal imaging:

Niosomes were prepared using standard thin layer evaporation method.[36] Span80 surfactant and

cholesterol were taken in the ratio 1:1. Afterward, this was dissolved in 2:1 chloroform/methanol

mixture. These solvents were evaporated in a rotary- evaporator at 100 rpm and under a vacuum

of 20 Hg at 30 ºC to form a thin film, which was further hydrated with water. The suspension was

vortexed and sonicated to get the final niosomal suspension. The prepared niosomes were first

loaded with 10µM of APQ. For quenching experiments, APQ loaded niosomes were further treated

with 0.19 and 0.38 mM Fe3+ solutions. The niosomes were incubated with the formulation for 60

minutes and then imaged using a TCS SP8 spectral laser scanning confocal microscope.

161

Spectra of the ligands

1H and 13C NMR of 2-(3H-pyrrolo[2,3-c]quinolin-4-yl)aniline (7-APQ)

162

1H and 13C NMR of 2-(3-methyl-3H-pyrrolo[2,3-c]quinolin-4-yl)aniline (21)

163

1H and 13C NMR of 2-(thieno[2,3-c]quinolin-4-yl)aniline (22)

164

1H and 13C NMR of 2-(4-phenylquinolin-2-yl)aniline (23)

1H and 13C NMR of 3-(3H-pyrrolo[2,3-c]quinolin-4-yl)pyridin-2-amine (24)

165

3-(3H-pyrrolo[2,3-c]quinolin-4-yl)pyridin-2-amine(24)

166

Conclusions

The main aim of this thesis is to develop newer methods for the synthesis of diverse fused

nitrogenated heterocyclic compounds and their applications.First chapter looks at the importance

of fused chromones, coumarins and quinoxalines based organic molecules.

Chapter two describes the synthetic route to fused chromeno[3,2-c]quinolines via FeCl3

catalysis. The method developed does not require any pre-functionalization to execute the pivotal

coupling reaction at the C-3 position of flavones. The final step involves the consecutive

application of imine formation, Csp2-Csp

2 coupling and oxidation reaction, with aromatic aldehydes

and 2-(2-aminophenyl)-4H-chromen-4-one as the reactants. Presence of electron

donating/withdrawing groups was well tolerated in the aldehydes and the method developed could

also be extended to other substituted 2-(2-aminophenyl)-4H-chromen-4-ones.

In chapter three, we wanted to look at the synthesis of fused coumarin systems as potential

analogs for anticancer compound lamellarin D. Accordingly a linear route for the preparation of

chromeno-thio-pyridine was developed by the systematic application of Suzuki coupling and

modified Pictet-Spengler reaction. The crucial final step was catalyzed using easily available FeCl3

and gave the target molecules in modest to good yields. The method developed was also extended

to furan rings and generally showed higher overall yield compared to the corresponding thiophene

congeners. Target molecules when subjected to anticancer activity screening against three different

cell lines [DU-145 (prostate cancer cells), B16F10 (murine melanoma cells) and MCF-7 (breast

cancer cells)], displayed the highest activity with compounds bearing fused chromeno-furo-

pyridine skeleton, with best IC50 value of 6.83 M obtained against MCF-7 cell line.

167

The main motivation behind chapter four was to develop an exclusive synthetic route for 2,4-

disubstituted pyrrolo[1,2-a]quinoxalines starting from chalcones (α,β-unsaturated ketones). This

chapter is divided into two parts; first part (Part-A) looks at the optimization and mechanism of a

crucial acyl rearrangement reaction on pyrrole ring and the second part (Part-B) discusses the

application of the acyl rearrangement reaction for the synthesis of the target molecules. In part-A

synthesis of the target molecule (rearranged pyrrole compound) started from chalcones and was

carried out in two steps. Initial step involved the conversion of chalcones to corresponding 4-

substituted-3-acylpyrroles by reaction with TosMIC. In the subsequent step, target molecules were

obtained in modest to good yields by polyphosphoric acid-mediated acyl rearrangement of 3-acyl

pyrroles to their 2-acyl congeners. The crucial final step was amenable to diverse substitutions on

pyrrole ring. Preliminary experiment for the determination of mechanism indicated the

involvement of acylium ion. In part-B some selected rearranged pyrrole compounds were treated

with 1-fluoro-2-nitrobenzene and the resultant products were subjected to reduction and

cyclization in the presence of iron (Fe) in acetic acid. The final compounds obtained were 2, 4-

disubstituted pyrrolo[1,2-a]quinoxalines.

Chapter five reports the development of an iron sensor APQ based on pyrrolo[2,3-c]quinoline

structure. The fluorophore facilitates micro molar detection of Fe3+/Fe2+ in the presence of various

cations by blocking the ESIPT reaction. Binding stoichiometry was found to be 2:1 for metal and

ligand. The capability of the developed sensor was demonstrated by its encapsulation inside

vesicle membrane and detection of Fe3+ ions thereby.

168

Summary

The main motivation for undertaking this research work was to explore various facets of

heterocyclic compounds. Heterocycles are the most diverse set of chemical entities possessing

equally diverse set of properties. Initial approach was to develop newer routes for the synthesis of

three different classes of fused heterocyclic compounds. Subsequent efforts were focused on

exploring the anticancer activity of one of the synthesized class of molecules. Selective iron

sensing behaviour of a fused system 2-(3H-pyrrolo[2,3-c]quinolin-4-yl)aniline was also explored.

The thesis is divided into six chapters

Chapter 1: Importance of fused chromeno/coumarin/quinoxaline heterocyclic systems.

Chapter 2: Iron(III) catalyzed synthesis of fused chromeno-quinoline scaffolds.

Chapter 3: Synthesis and anticancer activity evaluation of fused chromeno-thieno/furo-pyridines.

Chapter 4 (A): Synthesis of diverse 2-acylpyrroles from chalcones using polyphosphoric acid–

mediated regiospecific acyl migration.

Chapter 4 (B): Application of polyphosphoric acid mediated rearrangement on pyrrole for the

synthesis of 2,4-disubstituted pyrrolo[1,2 a]quinoxalines.

Chapter 5: Expanding the scope of fused pyrrolo-quinoline system for selective sensing of iron.

169

Chapter 1

Importance of fused chromeno/coumarin/quinoxaline based heterocyclic systems

This chapter looks at the importance of fused chromones, coumarins and quinoxalines based

organic molecules. In addition, main objectives of this research work along with required materials

and methods are also mentioned in this chapter.

Chapter 2

Iron(III) catalyzed synthesis of fused chromeno-quinoline scaffolds

Chromones and their C-2 phenyl substituted analogues (flavones) display important

pharmacological properties such as antimicrobial, antitumor and anti-inflammatory activity.

Currently, chromone-fused heterocycles are getting a lot of attention due to their unique structures

as well as diverse biological activities. This chapter will discuss, our efforts towards the synthesis

of 6-substituted chromeno[3,2-c]quinolin-7-one’s via direct functionalization of the C-3 site of

flavones. There was no literature available on these compounds prior to the work described in this

chapter.

Functionalization of C-3 position of chromone/flavone usually involves lithiation followed by

electrophile addition, Heck coupling between 3-halochromenes and appropriate alkenes or direct

C-H functionalization using palladium (II) or zinc based catalysts. Given several disadvantages

like pre-functionalization or the use of stoichiometric amounts of oxidizing agents, which these

methods possess, the development of an alternative route was felt necessary. The method

developed in this project uses a Pictet-Spengler inspired approach and does not require any pre-

functionalization to execute the crucial coupling reaction at the C-3 position of flavones.

170

Our synthetic efforts started with the reduction of nitro functional group on 2-(2-

nitrophenyl)-4H-chromen-4-one. The final step involved the consecutive application of imine

formation, Csp2-Csp

2 coupling and oxidation reaction, with aromatic aldehydes and 2-(2-

aminophenyl)-4H-chromen-4-one as the reactants and FeCl3 as a catalyst. The method developed

does not require any additives and can tolerate the presence of electron donating/withdrawing

groups on the aldehydes. Based on these studies, a plausible mechanism of the reaction was

proposed and reaction outcomes were also supported by density functional theory (DFT)

calculations.

Chapter 3

Synthesis and anticancer activity evaluation of fused chromeno-thieno/furo-pyridines

Lamellarin D containing 2H-chromen-2-one skeleton has received a lot of attention due to its

potent anticancer activity against multidrug-resistant tumor cell lines and prostate cancer cells.

Taking a clue from these activities, we attempted the synthesis of fused 2H-chromen-2-one-

thieno/furo-pyridine system as potential mimics of lamellarin D

A linear route for the preparation of the target molecule was devised starting from

commercially available 4-hydroxy coumarin.

After systematic application of nitration, chlorination, Suzuki coupling (using thiophene-3-

boronic acid) and reduction, the final step was carried out by using FeCl3 as a catalyst. The target

molecules were obtained in modest to good yields. The method developed was also extended to

furan rings and generally showed higher overall yield compared to the corresponding thiophene

congeners. Target molecules when subjected to anticancer activity screening against three different

cell lines, displayed the highest activity with compounds bearing fused chromeno-furo-pyridine

skeleton, with best IC50 value of 6.83 M obtained against MCF-7 cell lines

171

Chapter 4 (A)

Synthesis of diverse 2-acylpyrroles from chalcones using polyphosphoric acid–mediated

regiospecific acyl migration

A metal-free approach for the synthesis of 2-acylpyrroles is described in this chapter. Synthesis

of the target molecule started from chalcones and was carried out in two steps. Initial step involved

the conversion of chalcones to corresponding 4-substituted-3-acylpyrroles by reaction with

TosMIC. In the subsequent step, target molecules were obtained in modest to good yields by

polyphosphoric acid-mediated acyl rearrangement of 3-acyl pyrroles to their 2-acyl congeners.

The crucial final step was amenable to diverse substitutions on pyrrole ring. Preliminary

experiment for the determination of mechanism indicated the involvement of acylium ion.

Chapter 4 (B)

Application of polyphophoric acid mediated rearrangement on pyrrole for the synthesis of

2,4-disubstituted pyrrolo[1,2 a]quinoxalines

Pyrrolo[1,2-a]quinoxalines are important synthetic target which show their potential as

anti-leishmanial and antitumor compounds as well as inhibitors of antimicrobial resistance. While

diverse strategies are known for the synthesis of these molecules, they are mainly divided into

two broad categories. Conversion of propiolates or N-ylides to pyrrolo[1,2-a]quinoxalines via 1,3-

dipolar cycloadditions and cyclization carried out on 1-arylpyrroles. In this work we have carried

out synthesis of 2, 4-disubstituted pyrrolo[1,2-a]quinoxalines from chalcones. Our approach

started with the conversion of chalcones to 3,3’-disubstituted pyrroles followed by their

conversion to the corresponding 2,4-disubstituted congeners, mediated by polyphosphoric acid.

Subsequently, nitrogen on pyrrole ring was functionalized by SNAr reaction with 1-fluoro-2-

172

nitrobenzene. Final step combined reduction of nitro group and intramolecular cyclization to yield

the final compounds. The overall scheme developed presents an alternative route to the synthesis

of pyrrolo[1,2-a]quinoxalines and can tolerate both electron donating as well as withdrawing

groups on chalcones.

Chapter 5

Expanding the scope of fused pyrrolo-quinoline system for selective sensing of iron

Iron is one of the most important transition metal in living systems. Both excess and deficiency

of this metal is known to induce wide spectrum of diseases. This chapter reports development of a

fluorescence based iron sensor, 2-(3H-pyrrolo[2,3-c]quinolin-4-yl)aniline.

Initial approach involved looking at comparative steady state fluorescence behaviour of two

compounds 2-(3H-pyrrolo[2,3-c]quinolin-4-yl)aniline (APQ) and 2-(3H-pyrrolo[2,3-c]quinolin-

4-yl)phenol (HPQ). Better Stokes shift prompted us to carry out further studies with APQ.

Screening various metals revealed quenching behaviour with only Fe2+ and Fe3+ ions.

Further studies revealed micromolar detection of Fe3+/Fe2+ in the presence of various cations,

including well-known interfering cations Co2+and Cu2+. The ligand to metal ratio was found to

be 1:2 with an association constant of 10 x 106M-2. Stoichiometry of complex was also

confirmed by MALDI analysis. Structural analogues of APQ were used to look at the criticality

of amino and pyrrole moiety in selective iron sensing. The utility of the developed sensor was

also demonstrated by its encapsulation inside the vesicle membrane and detection of Fe3+ ions

thereby.

173

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