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Savaia, Bharat S., 2011, “Synthesis and Characterization of New Heterocyclic

Compounds & Their Application”, thesis PhD, Saurashtra University

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Synthesis and Characterization of New

heterocyclic compounds & their

applications

A THESIS SUBMITTED TO

THE SAURASHTRA UNIVERSITY

IN THE FACULTY OF SCIENCE

FOR THE DEGREE OF

Doctor of Philosophy

IN

CHEMISTRY

BY

Bharat S. Savalia

UNDER THE GUIDANCE OF

PROF. ANAMIK SHAH

DEPARTMENT OF CHEMISTRY

(DST-FIST FUNDED AND UGC-SAP

SPONSORED)

SAURASHTRA UNIVERSITY

RAJKOT – 360 005

GUJARAT (INDIA)

UNDER THE CO-GUIDANCE OF

PROF. ERIK VAN DER EYCKEN

LABORATORY FOR ORGANIC &

MICROWAVE-ASSISTED CHEMISTRY

(LOMAC).

DEPARTMENT OF CHEMISTRY,

UNIVERSITY OF LEUVEN

(K.U.LEUVEN), BELGIUM

JANUARY-2011

Statement under O. Ph. D. 7 of Saurashtra University The work included in the thesis is done by me under the supervision of

Prof. Anamik K. Shah and Co-supervision of Prof. Erik Van der Eycken

and the contribution made thereof is my own work. The work was carried

out at Department of Chemistry, Saurashtra University, Rajkot under

UGC meritorious Fellowship and at Laboratory for Organic &

Microwave-Assisted Chemistry (LOMAC), Department of Chemistry,

University of Leuven (K.U.Leuven), Belgium under Erasmus Mundus

Scholarship.

Date:

Place: Bharat S. Savalia

The Blessed Lord said: Fearlessness, purification of one's existence, cultivation of

spiritual knowledge, charity, self-control, performance of sacrifice, study of the

Vedas, austerity and simplicity; non-violence, truthfulness, freedom from anger;

renunciation, tranquillity, aversion to faultfinding, compassion and freedom from

covetousness; gentleness, modesty and steady determination; vigour, forgiveness,

fortitude, cleanliness, freedom from envy and the passion for honour--these

transcendental qualities, O son of Bharata, belong to godly men endowed with

divine nature.

Bhagvad Gita, Chapter-16, Verses-1-3.

To my Family,

With Love

Acknowledgment This thesis arose in part out of years of research that has been done since I came to Prof.

Anamik Shah’s group. By that time, I have worked with a great number of people whose

contribution in assorted ways to the research and the making of the thesis deserved special

mention. It is a pleasure to convey my gratitude to them all in my humble acknowledgment.

In the first place I would like to record my gratitude to Prof. ANAMIK SHAH for his

supervision, advice, and guidance from the very early stage of this research as well as giving me

extraordinary experiences through out the work. Above all and the most needed, he provided

me unflinching encouragement and support in various ways. His truly scientific intuition has

made him as a constant oasis of ideas and passions in science, which has exceptionally, inspire

and enrich my growth as a student, a researcher and a scientist want to be. I am indebted to

him for more than he knows.

I gratefully acknowledge My Co-Supervisor Prof. ERIK VAN DER EYCKEN Laboratory

for Organic & Microwave-Assisted Chemistry (LOMAC), Department of Chemistry,

University of Leuven (K.U.Leuven), Belgium, for his advice, supervision, and crucial

contribution, which made him a backbone of this research and so to this thesis. His

involvement with his originality has triggered and nourished my intellectual maturity that I

will benefit from, for a long time to come. Erik, I am grateful in every possible way and hope

to keep up our collaboration in the future.

I am very thankful to the faculties of Department of Chemistry namely, Professor and Head

Dr. P.H. Parasaniya, Prof. V. H. Shah, Dr. H. S. Joshi, Dr. Shipra Baluja, Dr. M. K. Shah,

Dr. Yogesh Naliapara, Dr.U.C.Bhoya and Dr. R. C. Khunt for their encouragement and ever

green support.

The Department of Chemistry and LOMAC has provided the support and equipment I have

needed to produce and complete my thesis and the UGC and Erasmus Mundus has funded my

studies. Many Thanks.

Many thanks go in particular to Dr.Denis Ermolet’v. I am much indebted to Denis for his

valuable advice in scientific discussion and synthetic part of this thesis.

To the role model for hard workers in the lab, Dr. Vaibhav Mehta, I would like to thank him

for many reasons. I would also acknowledge Sachin, Rupesh, Ripula, Dipak, Punit, Bari, Jigo,

Nigam, Nick, Prof. BK, and Claudia (Lab Mummy) for their advice and their willingness to

share their bright thoughts with me, which was very fruitful for shaping up my ideas and

research. Thanks to each and every members of LOMAC.

Collective and individual acknowledgments are also owed to my colleagues at Saurashtra

University whose presence somehow perpetually refreshed, helpful, and memorable. Many

thanks go in particular to Vaibhav, Bunty, Ravi, Sailesh, Shrey, Dhiru, Abhay, Bapu, Nilay,

Manisha, Harshad, Ronny, Mrunal, Rakshit, Ashish, Priti, Fatema, Ketaki, Dilip, Pratik,

Vishwa and Paresh. I was extraordinarily fortunate in having Rahul, Matre, Jigo, Bharat,

Suresh, Pankaj, Atul, Raju, Naval, Naimish, Dodia, Ghetiya Satish and Mahesh as my

friends. Thank s to all dear.

To Prashant Shah, thank for giving me the opportunity to work on your O2h in Ahmedabad.

To Cnp, Tusharbhai, Jigo, Pradip, Hemal, Nileshwari, Shabu, PP, Hiral, Tejas, Pareshbhai

and Vikas thank for your technical assistances and fun during the work we had in O2h. It is

also a pleasure to mention.

I am tempted to individually thank all of my friends which, from my childhood until graduate

school, have joined me in the discovery of what is life about and how to make the best of it.

However, because the list might be too long and by fear of leaving someone out, I will simply

say thank you very much to you all.

It is a pleasure to express my gratitude wholeheartedly to Anamik Shah’s family for their kind

hospitality during my stay in Rajkot for Ph.D. Specially thanks to Ranjanmam and Aditya.

Where would I be without my family? My parents deserve special mention for their

inseparable support and prayers. My Father Savdasbhai, in the first place is the person who

put the fundament of my learning character, showing me the joy of intellectual pursuit ever

since I was a child. My Mother Hansaben, is the one who sincerely raised me with her caring

and gently love. My special gratitude is due to my dadi, my brother Kalpesh, Bhabhi

Jayshreeben, my sisters Manisha, Jiju Rajnibhai, Sweet Miral, Mahek, Nancy, & Man, my

uncle Manshukhbhai and his family for their loving support.

Words fail me to express my appreciation to my wife Trupti whose dedication, love and

persistent confidence in me, has taken the load off my shoulder. I owe her for being unselfishly

let her intelligence, passions, and ambitions collide with mine. Therefore, I would also thank

Tupi’s family for letting me take her hand in marriage, and accepting me as a member of the

family, warmly. Furthermore, to Sorathiya family with their thoughtful support, thank you.

I would like to thank everybody who was important to the successful realization of thesis, as

well as expressing my apology to those whose name I could not mention individually.

Finally, I bow my head before almighty GOD, who has inspired ME in each and every stage

of my life.

BHARAT SAVALIA

CONTENTS

CHAPTER – 1

Microwave Assisted Organic Synthesis

1. Microwave Assisted Organic Synthesis 1

1.1 Introduction 1

1.2 Microwave Frequency 2

1.3 Principle 2

1.4 Heating Mechanism 3

1.4.1 Dipolar Polarisation 3

1.4.2. Conduction Mechanism 4

1.5 Effects of solvents 5

1.6 Conventional vs Microwave Heating 6

1.7 Application of microwave in organic synthesis 7

1.8 Studies on 2-Aminoimidazoles 8

1.9 2-Aminoimidazole Alkaloids 11

1.10 Methods for the preparation of 2-aminoimidazoles 15

1.11 The 2-Aminoimidazole as a Pharmacophore 21

1.11.1 Antibiotics Activity 21

1.11.2 Sodium Hydrogen Exchanger-1 (NHE-1) Antagonists. 22

1.11.3 Anti HIV Activity 22

1.11.4 α-adrenoreceptor agonists. 24

1.11.5 Anti Cancer Activity 24

1.11.6 BACE-1 Inhibitors 25

1.11.7 Anti Biofilm Activity 26

1.12 Introduction to Biofilm 29

1.12.1 Properties of biofilms 30

1.12.2 Biofilm Growth Cycle 31

1.12.3 Biofilm Drug Resistance 31

1.13 Recent Publications on 2-aminoimidazoles 33

1.14 Conclusion 33

1.15 References 34

CHAPTER – 2

Microwave Assisted Synthesis & Structure Activity Relationship of 2-Hydroxy-2-

phenyl-2,3-dihydro-imidazo[1,2-a]pyrimidinium Salts and 2N-Substituted 4(5)-

Phenyl-2-Amino-1H-imidazoles as Inhibitors of the Biofilm Formation by

Salmonella Typhimurium and Pseudomonas aeruginosa

2.1 Introduction 40

2.2 Reaction scheme 41

2.3 Proposed Mechanism 42

2.4 Results and discussion 43

2.5 Biofilm activity 50

2.5.1 2N-substituted 2-aminopyrimidines 48

2.5.2 2-hydroxy-2,3-dihydro-imidazopyrimidinium salts 48

2.5.2.1 n-Alkyl or iso-alkyl substituents at 1-position 48

2.5.2.2 Further Modification in the most active core structure 51

2.5.2.3 Cyclo-alkyl substituents at 1-position 53

2.5.3 2N-substituted 2-aminoimidazoles 54

2.6 Conclusion 56

2.7 Experimental 57

2.8 Biological Assays 58

2.9 Experimental Protocols 60

2.10 Spectral Characterization 62

2.11 Representative NMR spectra 87

2.12 References 90

CHAPTER – 3

Structure Activity Relationship of N1-Substituted 2-Aminoimidazoles as

Inhibitors of Biofilm Formation by Salmonella Typhimurium and Pseudomonas

aeruginosa

3.1 Introduction 93

3.2 Reaction scheme 94

3.3 Proposed Mechanism 95


3.5 Biofilm activity 98

3.5.1 N1-alkyl 2-aminoimidazoles 99

3.5.2 Further Modification in the most active core structure 102

3.5.3 Cyclo-alkyl 2-aminoimidazoles 104

3.6 Conclusion 106

3.7 Experimental 107

3.8 Experimental protocols 108



3.11 References 124

CHAPTER – 4

Copper Catalyzed Microwave Assisted One Pot Synthesis of 2-Aminoimidazole-

Triazole Framework via Dimorth Rearrangement and Click Reaction.

4.1 Introduction 127

4.2 Optimization Study 128


4.4 Possible Mechanism 133

4.5 Conclusion 134


4.7 Experimental Protocols 135



4.10 References 151

CHAPTER – 5

Synthesis, Characterization, Crystal and molecular structure analysis of DP-7: A

new Multi Drug Resistance Reverter Lead Molecule

5.1 Introduction 154

5.2 Reaction Scheme 155

5.3 Method of crystallization 155

5.4 Crystal structure determination 155


5.6 Conclusion 160




5.10 References 165

SUMMARY

CONFERENCES/SEMINARS/WORKSHOPS ATTENDED

LIST OF PUBLICATIONS

Abbreviations

Ac Acetyl

2-AI 2-aminoimidazole

aq Aqueous

Boc tert-butyloxycarbonyl

n-BuLi n-butyllithium

t-BuOK potassium tert-butoxide

Cbz Benzyloxycarbonyl

CI chemical ionization

m-CPBA m-chloroperoxybenzoic acid

DAST diethylaminosulfur trifluoride

DBBA 5,5-dibromobarbituric acid

DCM Dichloromethane

DIBAL diisobutylaluminium hydride

DIEA Diisopropylethylamine

DIPA Diisopropanolamine

DMAP 4-dimethylaminopyridine

DMF Dimethylformamide

DMSO dimethyl sulfoxide

EDCI 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide

ESI electrospray ionization

equiv Equivalent

h Hour

HMBC heteronuclear multiple bond correlation

HRMS high resolution mass spectrometry

LAH lithium aluminium hydride

MAOS microwave-assisted organic synthesis

MeCN Acetonitrile

min Minute

MS mass spectrometry

Ms Mesyl

MW Microwave

NBS N-bromosuccinimide

NCS N-chlorosuccinimide

NMM N-methylmorpholine

NOESY nuclear Overhauser effect spectroscopy

PCC pyridinium chlorochromate

Pd Palladium

Pd2(dba)3 tris(dibenzylideneacetone)dipalladium

PDC pyridinium dichromate

PPA polyphosphoric acid

rt room temperature

SPOS solid-phase organic synthesis

TBDMS tert-butyldimethylsilyl

TBDPS tert-butyldiphenylsilyl

TEA Triethylamine

TFA trifluoroacetic acid

THF Tetrahydrofuran

TLC thin layer chromatography

TMS Tetramethylsilane

Ts Tosyl

W Watt

Microwave Assisted Organic

Synthesis

1. Microwave Assisted Organic Synthesis:

1.1 Introduction

Synthesis of new chemical entities is major bottleneck in drug discovery.

Conventional methods for various chemical synthesis is very well documented and

practiced.1 The methods for synthesis (Heating process) of organic compounds has

continuously modified from the decade. In 1855, Robert Bunsen invented the burner

which acts as energy source for heating a reaction vessel, this was latter superseded

by isomental, oil bath or hot plate, but the drawback of heating, though method

remain the same. Microwave Assisted Organic Synthesis (MAOS), which has

developed in recent years, has been considered superior to traditional heating.

Microwave assisted organic synthesis2 (MAOS) has emerged as a new “lead” in

organic synthesis. The technique offers simple, clean, fast, efficient, and economic for

the synthesis of a large number of organic molecules. In the recent year microwave

assisted organic reaction has emerged as new tool in organic synthesis. Important

advantage of this technology include highly accelerated rate of the reaction,

Reduction in reaction time with an improvement in the yield and quality of the

product. Now day’s technique is considered as an important approach toward green

chemistry, because this technique is more environmentally friendly. This technology

is still under-used in the laboratory and has the potential to have a large impact on the

fields of screening, combinatorial chemistry, medicinal chemistry and drug

development. Conventional method of organic synthesis usually need longer heating

time, tedious apparatus setup, which result in higher cost of process and the excessive

use of solvents/ reagents lead to environmental pollution. This growth of green

chemistry holds significant potential for a reduction of the by product, a reduction in

waste production and a lowering of the energy costs. Due to its ability to couple

directly with the reaction molecule and by passing thermal conductivity leading to a

rapid rise in the temperature, microwave irradiation has been used to improve many

organic syntheses.

1.2 Microwave frequency

Microwave heating refers the use of electromagnetic waves ranges from 0.01m to 1m

wave length of certain frequency to generate heat in the material. These microwaves

lie in the region of the electromagnetic spectrum between millimeter wave and radio

wave i.e. between I.R and radio wave. They are defined as those waves with

wavelengths between 0.01metre to 1meter, corresponding to frequency of 30GHz to

0.3GHz.

1.3 Principle

The basic principle behind the heating in microwave oven is due to the interaction of

charged particle of the reaction material with electro magnetic wavelength of

particular frequency. The phenomena of producing heat by electromagnetic

irradiation are ether by collision or by conduction, some time by both.

All the wave energy changes its polarity from positive to negative with each cycle of

the wave. This cause rapid orientation and reorientation of molecule, which cause

heating by collision. If the charge particles of material are free to travel through the

material (e.g. Electron in a sample of carbon), a current will induce which will travel

in phase with the field. If charge particle are bound within regions of the material, the

electric field component will cause them to move until opposing force balancing the

electric force.3-9

1.4 Heating Mechanism

In microwave oven, material may be heated with use of high frequency

electromagnetic waves. The heating arises from the interaction of electric field

component of the wave with charge particle in the material. Two basic principal

mechanisms involve in the heating of material

1.4.1 Dipolar Polarisation

Dipolar polarisation is a process by which heat is generated in polar molecules. On

exposure to an oscillating electromagnetic field of appropriate frequency, polar

molecules try to follow the field and align themselves in phase with the field.

However, owing to inter-molecular forces, polar molecules experience inertia and are

unable to follow the field. This results in the random motion of particles, and this

random interaction generates heat. Dipolar polarisation can generate heat by either

one or both the following mechanisms:

1. Interaction between polar solvent molecules such as water, methanol and ethanol

2. Interaction between polar solute molecules such as ammonia and formic acid

The key requirement for dipolar polarisation is that the frequency range of the

oscillating field should be appropriate to enable adequate inter -particle interaction. If

the frequency range is very high, inter-molecular forces will stop the motion of a

polar molecule before it tries to follow the field, resulting in inadequate inter-particle

interaction. On the other hand, if the frequency range is low, the polar molecule gets

sufficient time to align itself in phase with the field. Hence, no random interaction

takes place between the adjoining particles. Microwave radiation has the appropriate

frequency (0.3-30 GHz) to oscillate polar particles and enable enough inter-particle

interaction. This makes it an ideal choice for heating polar solutions.

In addition, the energy in a microwave photon (0.037 kcal/mol) is very low, relative

to the typical energy required to break a molecular bond (80-120 kcal/mol).

Therefore, microwave excitation of molecules does not affect the structure of an

organic molecule, and the interaction is purely kinetic.

1.4.1. a Interfacial Polarization

Interfacial polarization is an effect, which is very difficult to treat in a simple manner,

and easily viewed as combination of the conduction and dipolar polarization effects.

This mechanism is important for system where a dielectric material is not

homogenous, but consists of conducting inclusion of one dielectric in other.

1.4.2. Conduction mechanism

The conduction mechanism generates heat through resistance to an electric current.

The oscillating electromagnetic field generates an oscillation of electrons or ions in a

conductor, resulting in an electric current. This current faces internal resistance, which

heats the conductor.

The main limitation of this method is that it is not applicable for materials that have

high conductivity, since such materials reflect most of the energy that falls on them.

1.5. Effects of solvents

Every solvent and reagent will absorb microwave energy differently. They each have

a different degree of polarity within the molecule, and therefore, will be affected

either more or less by the changing microwave field. A solvent that is more polar, for

example, will have a stronger dipole to cause more rotational movement in an effort

to align with the changing field. A compound that is less polar, however, will not be

as disturbed by the changes of the field and, therefore, will not absorb as much

microwave energy. Unfortunately, the polarity of the solvent is not the only factor in

determining the true absorbance of microwave energy, but it does provide a good

frame of reference. Most organic solvents can be broken into three different

categories: low, medium, or high absorber, as shown in Figure 6. The low absorbers

are generally hydrocarbons while the high absorbers are more polar compounds, such

as most alcohols.

Absorbance level Solvents

High

DMSO, EtOH, MeOH, Propanols,

Nitobenzen, Formic Acid, Ethylene

Glycol

Medium

Water, DMF, NMP, Butanol,

Acetonitrile, HMPA, Methy Ethyl

Ketone, Acetone, Nitromethane,

Dichlorobenzene, 1,2-Dichloroethane,

Acetic Acid, trifluoroacetic Acid,

Low

Chloroform, DCM, Carbon tetrachloride,

1,4-Dioxane, Ethy Acetate, Pyridine,

Triethyamine, Toluene, Benzene,

Chlorobenzene, Pentane, Nexane and

other hydrocarbons

1.6 Conventional vs Microwave Heating

Microwave heating is different from conventional heating in many respects. The

mechanism behind microwave Synthesis is quite different from conventional

synthesis. Points enlisted in Table 1, differ the microwave heating from conventional

heating.10-26

No CONVENTIONAL MICROWAVE

1

Reaction mixture heating proceeds

from a surface usually inside surface of

reaction vessels

Reaction mixture heating proceeds

directly inside mixture

2

The vessel should be in physical

contact with surface source that is at a

higher temperature source (e.g. mental,

oil bath, steam bath etc.)

No need of physical contact of

reaction with the higher temperature

source. While vessel is kept in

microwave cavities.

3 By thermal or electric source heating

take place.

By electromagnetic wave heating take

place.

4 Heating mechanism involve-

conduction

Heating mechanism involve-

dielectric polarization and conduction

5

Transfer of energy occur from the wall,

surface of vessel, to the mixture and

eventually to reacting species

The core mixture is heated directly

while surface (vessel wall) is source

of loss of heat

6

In conventional heating, the highest

temperature (for a open vessels) that

can be achieved is limited by boiling

point of particular mixture.

In microwave, the temperature of

mixture can be raised more than its

boiling point i.e. superheating take

place

7

In the conventional heating all the

compound in mixture are heated

equally

In microwave, specific component

can be heated specifically.

8 Heating rate is less Heating rate is several fold high

1.7 Application of microwave in organic synthesis

Following reactions have been performed through microwave heating.

Sr. Reaction Ref. Sr Reaction Ref.

1 Acetylation reaction 27 18 Diel’s-Alder reaction 44

2 Addition reaction 28 19 Dimerization reaction 45

3 Alkylation reaction 29 20 Elimination reaction 28

4 Alkynes metathesis 30 21 Estrification reaction 46

5 Allylation reaction 31 22 Enantioselective reaction 47

6 Amination reaction 32 23 Halogenation reaction 48

7 Aromatic nucleophillic

substitution reaction 33 24 Hydrolysis reaction 59

8 Arylation reaction 34 25 Mannich reaction 50

9 Carbonylation reaction 35 26 Oxidation reaction 51

10 Combinatorial reaction 36 27 Phosphorylation synthesis 52

11 Condensation reaction 37 28 Polymerization reaction 53

12 Coupling reaction 38 29 Rearrangement reaction 54

13 Cyanation reaction 39 30 Reduction reaction 55

14 Cyclization reaction 40 31 Ring closing synthesis 56

15 Cyclo-addition reaction 41 32 Solvent free reaction 57

16 Deacetylation reaction 42 33 Transestrification reaction 46

17 Dehalogenation reaction 43 34 Transformation reaction 58

Figure 1.2: General View of Monomode CEM Discover (left) and Multimode

MILESTONE Start (right) Systems

1.8 Studies on 2-Aminoimidazoles

Much effort has been dedicated to the study of molecular architecture and its

relationship to biological activity. From decades of high throughput screening of both

natural and synthetic small molecules we have clued in on structural features that

impart a high probability of biological efficacy.59 Recently natural products have

enjoyed a renaissance in lead generation for discovery based research. This realization

has further been folded into the concept of Biology-oriented synthesis (BIOS) which

relies on the core structures of natural products as valuable pre-validated scaffolds.60

These so-called “privileged” pharmacophores are often the basis of synthetic

collections aimed at increasing the success rate of small molecule screening ventures.

In particular, marine natural products derived from sponges have provided valuable

leads for therapeutic small molecules.61-62 Surprisingly the large majority of these

compounds have been isolated from organisms of the class Dermospongiae. In the

mid 1980’s chemists noted that the other major sponge class, Calcarea, had rarely

been subject to chemical investigations. A flurry of efforts through the mid-1990’s

helped to establish biogenetic relationships among these sponges. Isolated to explore

these interconnections and not necessarily for specific biological responses the

activities of these natural products have remained largely uncovered. Since these

initial investigations, an emerging structural class has recurrently been identified

through bioassay guided isolation which contains the 2-aminoimidazole core. From

the viewpoint of small molecule discovery this review will highlight alkaloids isolated

from Leucetta sp. This small skeletal family has been shown to interrogate an

incredibly diverse range of biological processes and thus represents an important

discovery scaffold for both medicinal and discovery based research.

In 1958, a nitrogen rich antibiotic, consequently named azomycin, was isolated from a

presumptive Streptomyces sp.63 This new antibiotic displayed good antimicrobial

activity against B. subtilis (6 μg/mL) and E. coli (25 μg/mL) and was relatively well

tolerated in mice (LD50 = 80 mg/kg). Eventually the structure of azomycin was

revealed to be 2-nitroimidazole (2)64, which along with its synthetic derivative

metronidiazole (3) have become clinically relevant antiprotozoal therapeutics (Fig.

1.8.1).65 With the parallel observation that several microbes were able to oxidize

aromatic amines, Lancini’s group showed some Streptomyces species are able to

oxidize 2-aminoimidazole itself, along with a series of 4-alkyl-2-aminoimidazoles, to

their corresponding 2-nitroimidazoles.66 This suggested that the 2-aminoimidazole

might be the direct biosynthetic precursor to azomycin and in 1970 Okami’s group

isolated the parent heterocycle (1) from Streptomyces eurocidicus.67 This study also

showed that supplementing the growth medium with L-Arg substantially increased the

production of 1, suggesting that arginine catabolism is critical to its biosynthesis. It is

unknown whether a similar pathway exists in marine organisms that produce more

complex 2-aminoimidazoles, however 1 has been discovered in sponges of the family

Halicondriidae.68

L-ArgN

NH

NH2

NNH

NO2N

N

Me

NO2

OH

1 2 3

2-Aminoimidazole Azomycin Metronidazole

Fig.1.8.1 Biosynthetic transformations of 2-aminoimidazole.

Studies on azomycin’s (2) and metronidazole’s (3) mechanism of action have

revealed that the nitro group is reduced in vivo to the nitroso radical which is

responsible for its selective anaerobic antiprotozoal activity.69 Subsequent studies

have shown that hypoxic conditions in tumors are also capable of reducing the 2-

nitorimidazole, prompting it to serve as an important prodrug for 2-aminoimidazoles

in hypoxic environments.70-72

The ambivalent reactivity of the key structural motif 2-amino-1H-imidazole (4) is

responsible for the molecular diversity observed in this group of alkaloids. The

electrophilic or nucleophilic reactivity at the same position C-4(5) is dependent on the

tautomeric isomer involved (Scheme 1.8.1).73

nucleophilic position

HN N

NH2

N N

NH

5

H H1

23

4 5

N N

NH2

5

electrophilic position

4

Scheme-1.8.1 The Tautomerism and Ambivalent Reactivity of 2-

Aminoimidazole

It is understood that the 2-aminoimidazole occupies an important region of chemical

space as its hydrogen bond donor-acceptor pattern is capable of recreating that of the

guanidine.74 More importantly, it occupies a unique pKa range between the more

acidic 2-aminopyridinium ion75 and less acidic guanidinium ion76 (Fig.1.8.2).

Furthermore, the pKa’s of 2-aminoimidazolium ions show a predictable trend with

substitution, allowing it to be finely tuned for medicinal applications as illustrated in

the examples below.77

N NH2 N

HN

NH2

2-Aminopyridine 2-aminobenzimidazole

pKa = 6.71 7.18

NHN

NH2

NHN

NH2

NHN

NH2

Me MeMe

pKa = 8.46 8.65 9.21

HN NH2 HN

NH2

NH2

pKa = 12.4 13.4

amidine guanidine

2-aminoimidazole

Fig.1.8.2 pKa’s of common guanidine mimetics

This dramatic difference in the pKa of 2-aminoimidazoles (~4 pKa units) with that of

guanidine has led to some debate concerning the ability of the 2-aminoimidazole to

directly mimic a guanidine. These arguments, based on a lowered pKa and electronic

dissimilarity are, however, precisely what makes the 2-aminoimidazole a unique

guanidine mimic for medicinal chemistry.-77-78 Most importantly, their pKa range

(pKa ~7-9) allows a significant fraction of compound to be uncharged at

physiological pH thus increasing the likelihood of cellular penetration. It also permits

this unique heterocycle to cross the blood brain barrier and may decrease the

susceptibility of these compounds to Pgp mediated efflux vide infra.79

1.9 2-Aminoimidazole Alkaloids

The 2-aminoimidazole framework is emerging as an important pharmacophore,80 and

is widely found in numerous biologically active marine alkaloids. Over the last 25

years, several hundreds of diverse 1-unsubstituted and 1-substituted 2-

aminoimidazoles have been characterized. 1-Unsubstituted 2-aminoimidazole

alkaloids (Fig. 1.9.1) have been isolated essentially from various species of marine

sponges (Phylum Porifera) of Caribbean and South Atlantic regions.81 Oroidin (1) and

the dimeric sceptrin (3) are the most abundant members of the pyrrole-imidazole

family.

N

HN

NH

N

H2N

H2N

HN

HN

O

O

HN

NH

Br

Br

NH

N

NH

HNO

HO

H

Me

O

Br

HN

N

H2N

NNH

N

HN Br

Br

H2N

NH

NHN

H2N

O

NH

N

NH2H2N

Cl

OH

NH

Br

Br

Oroidin (1)

Ageladine A (4)

Girolline (2)

Dragmacidin E (6)Palau'amine (5)

Sceptrin (3)

N

N

N

HO

H

NH2

H

Cl

HNN

HO

H2N

HNH2N

Fig.1.9.1 Some representative 1-unsubstituted 2-aminoimidazoles from marine

sponges.

In turn, most of the 1-substituted 2-aminoimidazole alkaloids (Figure 1.9.2) have been

isolated from the Calcarea group of the genera Leucetta and Clathrina from the Red

Sea and the Indo-Pacific regions.82

N

NH2N

MeOMe

OH

Naamine A (7)

Kealiinine A (11)

N

NH2N

OH

Isonaamine A (8)

OH

N

NH2N

Me OO

OO

Leucettamine A (10)

N

NH2N

Me

OH

OMe

OMe

N

NH2N

Me

OMe

OMeO

HO

Calcaridine A (9)

N

NH2N

Me OO

Preclathridine A (12)

Fig.1.9.2 Some representative 1-substituted 2-aminoimidazoles from

marine sponges.

Both classes of 1-substituted and 1-unsubstituted 2-aminoimidazoles possess

interesting biological properties. Due to their high cytotoxicity many polysubstituted

2-aminoimidazole alkaloids are involved in chemical defense of marine sponges

against predators, in the prevention of the settlement of fouling and pathogenic

microorganisms or in the suppression of epibiotic bacteria and spatial competition.

Recently group of Erik83 described a novel, short and efficient synthesis of diverse 2-

aminoimidazoles from readily available polysubstituted secondary propargylamines

and thioureas (Scheme-1.9.1). Both the guanylation and the cyclization steps can be

carried out either in a stepwise manner with a carbodiimide activator and an AgI

catalyst or in a one-pot process with a recoverable AgI salt as a promoter and catalyst.

This protocol was successfully applied to the total synthesis of all trisubstituted 2-

aminoimidazole naamine alkaloids. Furthermore, we demonstrated the potential of

polysubstituted 2-aminoimidazoles as inhibitors of bacterial biofilm formation.

R1

R2

R3

R4

CHOR5

NH

allylMe

CuBr (10 mol %)

100 oC, MW

Toluene, 25 min

R1

R2

R3

R4

R5

N(Me)Allyl

[Pd(PPh3)4] (5 mol%)

DMBA (3.5 equiv)

DCM, Reflux, 25 min

R1

R2

R3

R4

R5

NH(Me)AgNO3 (1.4 equiv)

Et3N (2 equiv)

MeCN, RT, 5 min

6aN

NN

BocMe

BocR5

R1

R2

R3

R4

N

NH2N

Me

R5

R1

R2

R3

R4

1) TFA/DCM RT, 3h

2) H2-Pd/C MeOH, RT

Scheme 1.9.1 Short total synthesis of alkaloid of the naamine family

Pierre Potier proposed biogenetic scheme, various hypothetical constituents can be

predicted. It is clear that the above chemical pathway suggests that these compounds

may be more widespread than presently known. The isolation and characterisation of

new pyrrole-imidazole metabolites will certainly allow us to explore additional

transformations which could support this hypothesis. As chemical diversity,

enzymatic catalysis variation and mutagenesis are closely related, the ‘‘prebiotic

chemistry’’ presented here can also provide strong evidence for a multifunctional

enzyme-mediated biosynthesis. Such multiprotein systems would be particularly

needed by the living fixed sponges for their adaptation to environmental influences,

and for self-defence.

Fig.1.9.3 Arrangement of all known oroidin alkaloids in structural groups.

1.10 Methods for the preparation of 2-aminoimidazoles

Several methods for the formation of 2-aminoimidazoles are known with widely

differing degrees of synthetic utility, but no current method as of yet has a broad

range of applicability in complex molecule synthesis. Harsh reaction conditions

and/or synthetically difficult and labile precursors are often required for such task.

The methods to prepare 2-aminoimidizoles may be categorized into four main classes:

1) Condensation reactions,

2) Ambivalent addition of 2-aminoimidazole,

3) Direct formation of C-N bond.

4) Heterocyclic exchange reactions.

Functionalized 2-aminoimidazoles can show a wide range of stability issues, the free

base of many substrates is susceptible to nucleophilic or base mediated degredation.

This instability has thus far precluded a unified strategy to access this heterocycle, but

the variety of methods can often lead to success in a complex molecule setting.

1.10.1 Condensations

By far the most common way to prepare the 2-aminoimidazoles originates with the

condensation of α-amino or α -haloketone with cyanamide or a guanidine derivative,

respectively. This strategy was first disclosed by Norris and McKee who explored the

condensation of α -aminoacetophenones with N-cyanoguanidines (scheme-1.10.1).84

O

NH2.HCl

Cl

NH

N

NH

R1

R2

NC

NH

NNH

NHN R1

R2

Cl150-200 oC

Scheme-1.10.1 Norris and McKee‘s condensation.

Subsequently Lawson was able to show that aminoacetaldehyde diethylacetal can

undergo acid catalyzed addition to cyanamide giving the masked guanidine

acetaldehyde (Scheme-1.10.2).85

EtONH2

OEt

NH2CN

AcOHEtO

HN

OEt

NH2

NH

NH

N

NH2.HClHCl

H2O

Scheme-1.10.2 Lawson’s synthesis.

Kruetzberger found that condensation of 1,2-hydrazinedicarboxamidine with benzoin

derivatives in alkali media will yield symmetrically substituted azoimidazoles after

spontaneous oxidation of the bis-imidazolhydrazine (Scheme-1.10.3).86 Reductive

cleavage of the azo group by hydrogenolysis gives two mole equivalents of the 2-

aminoimidazole. This methodology is useful but limited to the preparation of 4,5-

diaryl substituted 2-aminoimidazoles.

H2NNH

HN NHNH2

HN

Ar

O

Ar

OH

NaOH

NNH

ArAr

N

N N

HNAr

Ar

H2

Pd/CHN

N

ArAr

NH2

Scheme-1.10.3 Kruetzberger’s condensation.

In 1966, Lancini and Lazzari successfully expanded Lawson’s strategy to include α -

aminoketones, including the use of N-alkylamines (Scheme-1.10.4).87-88 They were

the first to observe the strict pH dependence of this condensation. At very low pH the

cyanamide is quickly converted to urea. At high pH, dimerization of the aminoketone

to the piperazine competes with cyanamide condensation. Having established the

optimum pH of the reaction to be ~4.5, these conditions are now routinely adopted. In

1925 Pyman and Burtles had reported the reaction of �α-bromoacetone and guanidine

to give a mixture of uncharacterizable compounds, leading them to abandon this

approach and develop their diazonium coupling described below.89 Seventy years

later, Weber discovered that N-acetylguanidine smoothly undergoes displacement and

condensation with α -haloketones and aldehydes.90

OHN

NH2CN

pH = 4.5N

N

NH2

Ph

Scheme-1.10.4 Lancini and Lazzari’s expanded strategy

R1

O

R2

XH2N NAc

NH2

HNN

R1

R2

NHAc

NH2NH2

H2SO4

OR HNN

R1

R2

NH2

Scheme-1.10.5 Weber’s condensation with N-acetylguanidine.

In the late 1970’s Nishimura reported the condensation of substituted α-diketones

with guanidine to yield a variety of useful products (Scheme-1.10.6).91 This report

illuminates the variety of chemistry that is possible around this heterocyclic core.

Treatment of a diketone with guanidine in dioxane and methanol yields the substituted

bicyclic bis-guanidine . Alternatively, aprotic reaction conditions can selectively

deliver the 2-aminoimidizo-4-ol. This intermediate can undergo a base catalyzed

pinacol-type rearrangement to form the 2-aminoimidizo-4-one. This same

intermediate can give rise to the 4,5-diol upon exposure to aqueous hydrochloric acid.

A similar hydration reaction with anhydrous HCl affords the di-methoxylated

derivative. Hydrogenolysis of 2-aminoimidizo-4-ol cleanly gives the 4,5-dialkyl-2-

aminoimidazole. These laboratory transformations foreshadow potential biosynthetic

relationships between the 2-aminoimidazole natural products isolated from marine

sponges.

OR1

R2 O

H2N

HN

NH2

N

NR1OH

R2

NH2

dioxane

MeOH

HN N

HN N

NH2

NH2

R1R2

NH

NO

NH2

R2

R1

NH

NNH2

R1

HO

HO

R2NH

NNH2

R1

MeO

MeO

R2 NH

NNH2

R1

R2

NaOHMeOH conc.HCl

MeOHHCl

H2/Pd-CMeOH

Scheme-1.10.6 Nishimura’s diketone condensation

1.10.2 Ambi-valent Addition of 2-Aminoimidazole

Depending on reaction conditions, the parent heterocycle, 2-aminoimidazole, can be

influenced to react either at N-2 or C-4. In Potier’s synthesis of girolline, 2-

aminoimidizole was added directly to the α-chloroaldehyde. Under the influence of

sodium carbonate, the imidazole adds preferentially through the 4-position (Scheme-

1.10.7 ).92 As revealed in Horne’s synthesis of stevensine, methanesulfonic acid was

used to add the 2-aminoimidazole across an olefin.93-94 Horne has also shown that

Friedel-Crafts type alkylation of the activated ethers can proceed with attack by C-4

in the presence of methanesulfonic acid to give stevensine (Scheme-1.10.7).

Alternatively, triflouroacetic acid persuades N-2 addition to give the N-alkylated 2-

aminoimidazole.

HN

NH2N

OCl

N

O

O

HN

NH2N

NH2

HO

Cl

Na2CO3

HCl

HCl, H2OGirroline

Scheme-1.10.7 Potier’s Ambi-valent additions of 2-aminoimidazole

HN

NH2N

NH

Br

Br

NH

MeOBr

O

NH

Br

BrNH

O

NHN

H2N

MsOH90 oCTFA rt.

NH

Br

BrNH

HN Br

O

NHN

Stevensine

Scheme-1.10.8 Horne’s Ambi-valent additions of 2-aminoimidazole.

1.10.3 Formation of C-N bond.

It is now clear that, condensation reactions are applicable to the preparation of mono-

and di-substituted 2-aminoimidazoles. To generate more highly substituted 2-amino

imidazole cores, the direct introduction of N-2 on an appropriately substituted

imidazole is frequently applied. The first method for forming 2-aminoimidazoles, in

this manifold, was reported in 1925 by Burtles and Pyman who showed that azo-

bromoaniline and dimethylimidazole underwent aromatic substitution to form the

hydrazone (Scheme-1.10.9).The aryl hydrazone was then readily reduced by Zn dust

and aqueous acetic acid or stannous chloride and hydrochloric acid.

Br

N2

HNN

NaHCO3

H2O

HNN

N N

Br

SnCl2

100 oCHN

N

NH2.HCl

Scheme-1.10.9 Pyman’s synthesis.

Hassner was the first to report the reaction of imidazole lithium with a vinyl azide,

hydrolysis of the intermediate triazine gives the aminoimidazole.95 Since that report,

Potier has popularized the use of lithiated imidazoles.96 Potier reported that lithiation

with LDA followed by azide transfer from TsN3 efficiently generated the 2-

azidoimidazoles (Scheme-1.10.10). Hydrogenolysis of the azide proceeds smoothly to

give the 2-aminoimidazole, exemplified in his synthesis of keramidine.97

N

NTr

HN

O

HN

Br

LDA, TsN3

-70 oC

N

NTr

HN

O

HN

Br

N3

N

NTr

HN

O

HN

Br

H2N10 % Pd/C

H2

Scheme-1.10.10 Potier’s method via lithiation.

Lithiated imidazoles can also be trapped by disulfides to give the 2-thioimidazoles.

Oxidation to either the sulfone or sulfoxide creates a leaving group capable of

displacement with azide (Scheme-1.10.11).98 This strategy has been utilized by

Weinreb in the synthesis of ageladine A.99-100

NN

Me SO2Me

EtOOC

COOEt

NaN3, DMF

87 oC, 13 h NN

Me N3

EtOOC

COOEt

Scheme-1.10.11 Anderson’s method of synthesis.

To avoid lithiation, Ohta has shown that 2-bromoimidazoles can undergo palladium

catalyzed coupling with trimethylsilylazide (Scheme-1.10.12). Reduction again

provides the 2-aminoimidazole. This strategy has been implemented by Ohta’s group

to access several Leucetta alkaloids discussed below. Also, noteworthy, Ohta has

shown that during reduction of the azide it can be directly protected as its

benzylidenimine.101-102

N

N

Br

TMSN3

PdCl2(PPH3)2

80 oC N

N

N3

H2, Pd-C

N

N

NH2

Scheme-1.10.12 Ohta’s palladium catalyzed synthesis.

1.10.4 Heterocyclic Exchange Reactions

The transformation of 3-amino-1,2,4-oxadiazoles to 2-aminoimidazoles can be carried

out by reaction with fluorinated ß-dicarbonyls to give the ß-hemiaminal (Scheme-

1.10.13).103 This can undergo subsequent conversion to the 2-amidoimidazole through

a Boulton-Katritzky Rearrangement. This intermediate can then be reduced to give

Intermediate. The benzamide can also be removed via acid catalyzed hydrolysis to

give the trifluoromethyl amino-imidazole. An interesting reaction, however the

synthesis of the intermediate ß-enaminocarbonyl is low yielding and initial results

suggest limited substituent scope, as the initial condensation requires the presence of

the trifluoromethyl ketone.

N

ON

Ph

NH2

CF3O

RO

Montmorillonite K-10, PhMe, 100 oC;

N

ON

Ph

HN

F3C OH

R

O

N

ON

Ph

HN

F3C OH

R

OH

N

NH

CF3

O

RNH

Ph

O

HClEtOHreflux

N

NH

CF3

O

RH2N

N

NH

CF3

OH

RNH

Ph

O

NaBH4THFreflux

Scheme-1.10.13 Boulton-Katritzky rearrangment of aminooxadiazoles.

Another route to 4(5)-acyl-2-amino-1H-imidazoles is based on the recyclization of 5-

acyl-2-amino-1,3-oxazoles (Scheme-1.10.14).104 For example, 5-acetyl-2-amino-

oxazole gave upon heating in water solution with ammonia or aliphatic amines 2-

amino-1H-imidazoles in 32% (R = H) and 43-62% (R = alkyl or cycloalkyl) yields.

NH3 or Amine

H2O, heating NH

NNHR

Me

O

O

NNH2Me

O

Scheme-1.10.14 Mularski’s recyclization method.

Methodology to prepare 2-aminoimidzoles has greatly advanced due to the rising

demand in natural product synthesis and medicinal chemistry. However, the

development of methods to succinctly generate a highly functionalized 2-

aminoimidazole core from stable precursors is warranted.

1.11 The 2-Aminoimidazole as a Pharmacophore

The 2-aminoimidazole has been utilized as a building block has led to the

development of several medicinally relevant small molecules.

1.11.1 Antibiotics Activity

Researchers at Zeneca delivered a series of broad spectrum antibiotics a-c, inspired by

the well studied cephalosporins.106-106 They were further able to show that substitution

on N1 of the aminoimidazole led to inactive compounds, again suggesting that this

motif may make an acid hydrogen bond contact (Fig. 1.11.1). Interestingly they were

able to correlate the pKa of the 2-aminoimidazole with activity. Against Gram

positive organisms they found that a slight elevation in the pKa led to a dramatic

decrease in activity. For Gram-negative bacteria, there was little difference,

suggesting pKa may alter the membrane permeability.

N

S

N

S

N

S

HOOC

O

H

NH

NHN

N

S

N

S

N

S

HOOC

O

H

NH

NHN

N

S

N

S

N

S

HOOC

O

H

NH

NHN

pKa = 7.1

S.aureus = 8

E Coli = 0.03

pKa = 7.4 pKa = 8.05

S.aureus = 16 S.aureus = 256

E Coli = 0.015 E Coli = 0.03

a b c

Fig-1.11.1 2-aminoimidazole modified cephalosporins.

1.11.2 Sodium Hydrogen Exchanger-1 (NHE-1) Antagonists.

Scientists at Bristol-Meyers Squibb have noted the ability of the 2-aminoimidazole to

function as a bioisostere of acylguanidines.106 Looking to produce novel inhibitors of

the sodium hydrogen exchanger-1 (NHE-1) they aimed at replacing the acylguanidine

in their lead compounds (Fig. 1.11.2). They surveyed a number of heterocyclic

analogues that would conserve the pKa of the acylguanidine as a cationic species,

necessary for antagonism of the Na+ transporter.

Cl

HNN

NH2

Cl

HNN

NH2

Cl

HNN

NH2

Cl

HNN

NH2Cl Cla b c d

0.75 0.039 0.18 0.008

Fig.- 1.11.2 NHE-1 antagonists. (NHE-1 Inhibition IC50 (µm))

1.11.3 Anti HIV Activity

Much effort has been dedicated to the development of HIV protease (HIV PR)

inhibitors for the treatment of AIDS. The protease is responsible for the processing of

nascent polypeptides to mature proteins comprising the viral particle. Without the

formation of the mature particle, viral replication of HIV has been shown to be

inhibited. HIV PR has therefore been an active biochemical target for the

development of small molecule therapeutics.

Wilkerson and co-workers at DuPont-Merck had developed a cyclic urea-based HIV

PR inhibitor scaffold which possessed an acceptable pharmacokinetic profile, but

lacking the potency necessary to advance the candidate.107 Biochemical evaluation of

this series of compounds against the HIV PR dimer revealed that a and b were

extremely potent inhibitors Ki = 0.023 and 0.012 nM respectively. The compounds

were also able to inhibit viral replication in vitro as measured by the ability of the

compounds to inhibit RNA synthesis by 90% (a) IC90 = 13.2 nM; (b) IC90 = 26.2 nM).

Interestingly, QSAR on this series of cyclic ureas suggested the optimum lipophilicity

for protease inhibition (ClogP = 4.4) is different from the inhibition of viral

replication (ClogP = 6.36) as revealed by the reversal of Ki and IC90 selectivities for

(a) and (b). This makes it difficult to optimize both parameters within the same

molecule, but suggests that while (a, b) display the same hydrogen bonding pattern at

the terminal urea, the lipophilicity disparity between the 2-aminoimidazole and the

benzimidazole has a significant effect on their pharmacokinetic profile. With these

results, (a) and (b) were advanced for pharmacokinetic studies in female Beagles. As

seen in the pharmacokinetic profiles (Fig. 1.11.3) the decreased lipohilicity of (b) had

a significant impact on the clearance (CL) and half-life (t1/2) of the compound,

advancing its candidacy based on this improved PK profile.

N

O

N

NH2

PhHO OH

Ph

O

HN

N

O

N

NH2

PhHO OH

Ph

O

HN

N

NH

N

NH

a b

Fig.- 1.11.3 Unsymmetrical HIV PR inhibitors.

Parameter a b

Ki (nM) 0.023 0.012

IC90 (nM) 13.2 26.2

Dose (mg/kg) 5.0 5.0

CL, L/h/kg 0.8 0.5

Vss (L/Kg) 1.0 4.0

T1/2 (h,iv) 0.9 5.6

Table-1.13.1: Pharmacokinetic profiles of HIV PR inhibitors

1.11.4 α-adrenoreceptor agonists.

Agents that can stimulate α-andrenoceptors modulate a variety of physiological

processes including the reduction of blood pressure, sedation and the inhibition of

intestinal fluid secretion.108 Scientists at Allergan Pharmaceuticals targeted the α –

andrenoceptor to reduce intraocular pressure(IOP), a common condition in patients

suffering from glaucoma.109 The challenge in designing such agents stems from the

ability of α –andrenoceptor subtypes to modulate different biological processes, most

concerning are those in the central nervous system (CNS). One approach to

minimizing CNS effect is topical application, minimizing access of the compound to

the CNS. From the known α-andrenoceptor agonist clonidine,110 modifications to

minimize CNS access generated the lead compounds p-aminoclonidine and AGN-

191103 (21) (Fig. 1.13.4). The The alternative approach is to discover compounds

displaying enough selectivity between the α-andrenoceptor subtypes such that they

exhibit an enhanced therapeutic index. This led to replacing the imidazolidin-imine

with a 2-aminoimidazole. This compound (a) was selected for further evaluation.

Binding assays demonstrated (a) bound to the α2A-receptor with a Ki = 1.7 nM and

was 1200-fold selective for the α2A –receptor over the α1-receptor, 50-fold selective

for the α2A –receptor over the α2B –receptor, and 10- �fold selective for the α2A –

receptor over the α2C –receptor. Compound (a) was found to be effective in vivo with

an EC50 = 8.7 nM and importantly remained effective when administered

intravenously, demonstrating its capability of crossing the blood-brain barrier.

NH

HN N

Cl

Cl

NH

HN N

NH

HN N

Cl

Cl

NH2

Clonidine p-aminoclonidine AGN-191103

N

N

N

HN

HN

O

O

Compound (a)

Fig.- 1.11.4 α-adrenoreceptor agonists.

1.11.5 Anti Cancer Activity

Cancer has been the leading cause of death worldwide. Chemotherapy remains one of

the major treatment options available for cancer, andmost of the currently used

anticancer drugs are administered to patients via a parenteral infusion or bolus

injection. Clinical complications with the parenteral administrations have been

documented. Extra care is needed because of inappropriate patient compliances, and

extra cost associated with hospitalization is necessary. Efforts in searching for orally

active anticancer agents have been extensive, including the anticancer drug category

of tubulin binding agents from which only injectable drugs are available such as

taxanes and vinca alkaloids. Recently Chiung-Tong Chen111 reported the synthesis

and biological functions of 2-amino-1-arylidenaminoimidazoles as orally active

anticancer agents. Selective compounds had shown potent effects in the interference

on the colchicines binding to tubulins, inhibition of tumor cell proliferation, and

induction of human cancer cell apoptosis.

N

N

H2N

R3

R4

N

R1

R2

IC50 = 0.05- >10 (µM)

1.11.6 BACE-1 Inhibitors

Alzheimer’s disease (AD) is the largest unmet medical need in neurobiology and

accounts for the majority of dementia diagnosed in the elderly. AD is characterized by

a progressively slow decline in cognitive function that leaves the end-stage patient

dependent on custodial care with death occurring on the average of 9 years after

diagnosis. The lack of an effective treatment for AD has stirred an intense search for

novel therapies based on the amyloid hypothesis. This hypothesis states that a gradual

and chronic imbalance between the production and clearance of Aß peptides results in

their accumulation in the brain. These secreted peptides polymerize into neurotoxic

oligomers that disrupt neuronal function and lead to cell death and memory loss that is

phenotypical of AD. ß-secretase (BACE-1) is an as partyl protease representing the

ratelimiting step in the generation of these Aß peptide fragments. Given the central

role of Aß in AD pathology, inhibition of BACE-1 has become an attractive target for

the treatment of Alzheimer’s disease. Vacca112 and Hills113 developed a series of

aromatic heterocycles as BACE-1 inhibitors. The potency of the weak initial lead

structure was dramatically enhanced using traditional medicinal chemistry. These

inhibitors were shown to bind in a unique fashion that allowed for potent binding in a

non-traditional fashion.

N

NH

R2R1

H2NN

N

R2

R1

H2N

MeN

NH2N

N

R2

R1

Bace-1 IC50 = 4-6 (µM) Bace-1 IC50 = 0.47-2 (µM) Bace-1 IC50 = 0.47-2 (µM)

Fig. 1.11.5 BACE-1 inhibitors

1.11.7 Anti Biofilm Activity

Bacterial biofilms are defined as a surface attached community of ignalling sms

that are protected by an extracellular matrix of biomolecules. Within a biofilm state,

bacteria are more resistant to antibiotics and are inherently insensitive to antiseptics

and basic host immune responses. The NIH has estimated that 65-80% of all

microbial infections are biofilm-based. Biofilm infections of indwelling medical

devices are also of major concern, as once the device is colonized, infection is

virtually impossible to eradicate. Bacterial biofilms114-116 underlie the persistent

colonization of hospital facilities, which perpetuates nosocomial infections. Hospital-

acquired infections place a $10 billion burden on the U.S. healthcare system annually.

Given the prominence of biofilms in infectious diseases, there has been an increasing

effort toward the development of small molecules that will modulate bacterial biofilm

development and maintenance. This, coupled with the spread of multi-drug antibiotic

resistance across many of these bacteria, has put a tremendous burden on the scientific

and medical community to alleviate biofilm-related problems. In this review, we will

highlight the development of small molecules that inhibit and/or disperse bacterial

biofilms through non-microbicidal mechanisms. The review will be segmented into

providing a general overview of how bacteria develop into biofilm communities, why

they are important, and the difficulties associated with their control. This will be

followed by a discussion of the numerous approaches that have been applied to the

discovery of lead small molecules that mediate biofilm development. These

approaches are grouped into: 1) discovery/synthesis of molecules based upon

naturally occurring bacterial ignalling molecules, 2) chemical library screening, and

3) discovery/synthesis of natural product and natural product analogues.

Very recently the research group of Melander has been studying widely the ability of

simple analogues of the marine natural products bromoageliferin and oroidin (Fig.

1.11.6) to control biofilm development. Bromoageliferin is a sponge-derived alkaloid

that was reported to inhibit the formation of bacterial biofilms from marine sources,

presumably as a defense mechanism against biofouling.117 They reasoned that core

structures derived from this complex molecule could be used as structural inspiration

for the development of potent and synthetically accessible anti-biofilm agents. One of

these scaffolds developed was based upon an aryl framework.118 From a medicinal

chemistry stand point, this scaffold was deemed a promising platform to develop

further functionalized 2-aminoimidazole (2-AI) derivatives as diversity could rapidly

be introduced through manipulation of the benzene ring. They described the synthesis

and anti-biofilm activity of these aryl 2-AI derivatives against three commonlystudied

Gram-negative bacteria.

N

NH

H2N

HN

NHN

H2N

O

NH

NH

OHN

Br

Br

Br

Bromoageliferin

N

NH

NH

H2N

OHN

Br

Br

Oroidin

N

NH

H2N

R

Aryl 2-AI scaffold

Fig. 1.11.6 2-Aminoimidazole-based anti-biofilm molecules.

The research group of melander has recently developed several novel libraries of 2-

aminoimidazole small molecules inspired by the marine alkaloids bromoageliferin

and oroidin. These natural products were reviously reported to inhibit biofilm

formation of the marine arotobacterium Rhodospirillum salexigens.119 TAGE and

CAGE were the first documented 2-aminoimidazoles with anti-biofilm activity against

Pseudomonas aeruginosa biofilms.

Analogues of the structurally simpler alkaloid oroidin were also pursued to develop

novel classes of small molecules with anti-biofilm activity. Recently, an oroidin

inspired library was constructed utilizing click chemistry to generate a diverse library

of 2-aminoimidazole/triazole conjugates (2-AIT).These compounds displayed the

widest spectrum of anti-biofilm activity observed within the 2-aminoimidazole class

(Fig. 1.11.7).120-121

N

NH

H2N

HN

NHN

H2N

O

NH

NH

OHN

Br

Br

Br

Bromoageliferin

N

NH

NHH2N

O HN

Br

Br

Oroidin

N

NH

H2NHN O

NH

HN

OHN

Br

Br

Br Br

N

NH

H2N

NH2

NH2

TAGE

N

NH

H2N

NH2

NH2

CAGE

N

NH

H2N N NN R

2-AIT Library

Fig. 1.11.7 Representative 2-aminoimidazoles derived from the marine natural

products bromoageliferin and oroidin.

The synthetic approach to this 2-AIT derivatization is outlined in (Scheme 1.11.1). In

their previous synthesis of 2-AIT conjugates, they employed the alkyne-derived 2-AI

as a precursor for the CuI-mediated[3+2] alkyne/azide cycloaddition (click reaction).

Although this reaction worked well, purification of the resulting product was

cumbersome, due to the need for copious amounts of ammonia saturated methanol for

column chromatography. Therefore, they decided to revise the route by employing a

Boc-protected 2-AI alkyne that would allow more traditional means of purification

(i.e. methanol/dichloromethane columns). The Boc-protected scaffold was

synthesized from oct-7-ynoic acid by treatment with oxayl chloride followed by

diazomethane and quenching of the resulting α-diazo ketone with HBr to generate the

intermediate a-bromo ketone. Cyclization with Boc-guanidine then delivered the

target 2-AI alkyne..

a) i : (COCl)2,CH2Cl2, DMF (cat.), ii : CH2N2,Et2O/CH2Cl2, iii : HBr (82%) ; b) Boc-guanidine, DMF, RT(59%) ; c) azide, CuSO4, Na ascorbate, EtOH, CH2Cl2,H2O; d) TFA, CH2Cl2 (58-94 % over two steps)

OH

O

N

NH2N

Boc

N3

HN

R

N

NH

H2N N NN H

NRc,da,b

Scheme-1.11.1 Melander’s 2-AI-T conjugate synthesis.

It was explained by earlier work that once target 2-AI alkyne had been synthesized,

assembled a diverse array of azido amides to employ in the click reaction to create

pilot collection of 2-AIT conjugates. Briefly, 2-bromo-ethylamine was treated with

sodium azide to deliver 2-azido-ethylamine, which following acylation (via the

respective acid chloride) generated the azido amides for elaboration into the 2-AIT

collection. Each azido amide was then subjected to the click reaction with target 2-AI

alkyne. Boc-deprotection (TFA/CH2Cl2) followed by counterion exchange

(trifluoroacetate for chloride) delivered the target 2-AIT compounds for antibiofilm

screening.

1.12 Introduction to Biofilm

Biofilms play a significant role in infection disease and account for up

to 80% of microbial infections in the body.

Bacterial biofilms are defined as a surface-attached community of bacteria

that are surrounded by a protective extracellular matrix.

On a global scale, biofilm related costs incur 6-10 billions of Euros to the

agricultural, engineering, and medical sectors of economy.

Despite the focus of modern microbiology research on pure culture,

planktonic bacteria, it is now recognized that most bacteria found in

natural, clinical, and industrial settings persist in biofilms.

Within a biofilm, bacteria are upward of 1000-times more resistant to

conventional antibiotic treatment

Biofilms grow virtually everywhere, in almost any environment where

there is a combination of moisture, nutrients, and a surface.

1.12.1 Properties of biofilms

A biofilm community can be formed by a single bacterial species, but in nature

biofilms almost always consist of rich mixtures of many species of bacteria, as well as

fungi, algae, yeasts, protozoa, other microorganisms, debris and corrosion products.

Biofilms are held together by sugary molecular strands, collectively termed

"extracellular polymeric substances" or "EPS." The cells produce EPS and are held

together by these strands, allowing them to develop complex, three-dimensional,

resilient, attached communities. Biofilms cost the U.S. literally billions of dollars

every year in energy losses, equipment damage, product contamination and medical

infections. But biofilms also offer huge potential for bioremediating hazardous waste

sites, biofiltering municipal and industrial water and wastewater, and forming

biobarriers to protect soil and groundwater from contamination

1.12.2 Biofilm Growth Cycle

1) Planktonic bacteria reversibly attach to a surface suitable for growth and

Bacteria begin secretion of the extra cellular polymeric substances (EPS) and

attachment becomes irreversible.

2) The maturing biofilm begins to take on a 3-dimensional shape. Biofilm

communities can develop within hours.

3) The biofilm fully matures, and complex architecture is observed. Bacteria

disperse from the biofilm to reinitiate biofilm colonization of a distal surface.

1.12.3 Biofilm Drug Resistance

The figure below shows a model of biofilm resistance to killing based on persister

survival.

Initial treatment with antibiotic kills normal cells (coloured green) in both planktonic

and biofilm populations.

The immune system kills planktonic persisters (coloured green), but the biofilm

persister cells (coloured pink) are protected from the host defences by the exopolymer

matrix.

After the antibiotic concentration is reduced, persisters resuscitate and repopulate the

biofilm and the infection relapses.

Biofilm infections are highly recalcitrant to antibiotic treatment. However, planktonic

cells that are derived from these biofilms are, in most cases, fully susceptible to

antibiotics. Importantly, biofilms do not actually grow in the presence of elevated

concentrations of antibiotics, therefore biofilms do not have increased resistance

compared with planktonic cells.122 But if biofilms are not resistant to antibiotics, how

do biofilm bacteria avoid being killed by antibiotic treatments? The resistance of

biofilms to drug therapy has been one of the more elusive problems in microbiology,

but the analysis of a simple dose-response experiment provided an unexpected insight

into the puzzle.122-124

Most of the cells in a biofilm are highly susceptible to bactericidal agents such as

fluoroquinolone antibiotics or metal oxyanions, which can kill both rapidly dividing

and slow- or non-growing cells.124-126 This is important, as cells in the biofilm are

slow-growing, and many are probably in the stationary phase of growth. The

experiment also revealed a small sub-population of cells that remain alive irrespective

of the concentration of the antibiotic (persisters). The number of surviving persisters

was greater in the non-growing stationary phase. In vitro, a stationary culture seems to

be more tolerant than a biofilm to antibiotics. However, this situation is probably

reversed in vivo. Antibiotic treatment will kill most biofilm and planktonic cells,

leaving persisters alive. At this point, the similarity with an in vitro experiment

probably ends. The immune system can mop up and kill remaining planktonic

persisters, just as it eliminates nongrowing cells of a bacterial population that is

treated with a bacteriostatic antibiotic. However, the biofilm exopolymer matrix

protects against immune cells,127-129 and persisters that are contained in the biofilm

can survive both the onslaught of antibiotic treatment and the immune system. When

the concentration of antibiotic reduces, persisters can repopulate the biofilm, which

will shed off new planktonic cells, producing the relapsing biofilm infection. The

problem of biofilm resistance72 to killing by most therapeutics probably defaults to

persisters. Interestingly, yeast biofilms also form tolerant persisters.

1.13 Recent publications on 2-aminoimidazoles.

The core structure 2-aminoimidazole has assumed importance in various synthesis

related to heterocyclic in drug synthesis as appeared in Sci-finder search results on the

basis of number of publications in medicinal and synthetic chemistry journals.

Fig.1.10 Sci-finder search results by heating term 2-aminoimidazole.

1.14 Conclusion

Bacterial biofilms pose a serious threat to the healthcare system of our society as they

are responsible for the morbidity and mortality of a myriad of diseases. The

development of antibiotic resistance among many bacterial strains has put a

tremendous pressure on the medical community to find alternative approaches for the

treatment of biofilm-mediated diseases. In this present work of thesis a microwave

assisted synthesis of small molecules which shows ability to controlling bacterial

antibiofilm has been reported.

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[122] Lewis, K. Riddle. (2001), Antimicrob.Agents Chemother. 45, 999–1007.

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[130] For biofilm resistance mechanism see, http://www.ncbi.nlm.nih.gov/entrez/query.fcg

Microwave Assisted Synthesis & Structure

Activity Relationship of 2-Hydroxy-2-phenyl-

2,3-dihydro-imidazo[1,2-a]pyrimidinium Salts

and 2N-Substituted 4(5)-Phenyl-2-Amino-1H-

imidazoles as Inhibitors of the Biofilm

Formation by Salmonella Typhimurium and

Pseudomonas aeruginosa

Chapter-2 Microwave Assisted Synthesis & Structure Activity Relationship of 2-

Hydroxy-2-phenyl-2,3-dihydro-imidazo[1,2-a]pyrimidinium Salts and 2N-Substituted 4(5)-Phenyl-2-Amino-1H-imidazoles as Inhibitors

of the Biofilm Formation by Salmonella Typhimurium and Pseudomonas aeruginosa

N

N

N R1

OHBr

R2

N

NH

HNR1

R2

In the current chapter a library of 1-substituted 2-hydroxy-2-phenyl-2,3-dihydro-

imidazo[1,2-a]pyrimidinium salts and 2N-substituted 4(5)-phenyl-2-amino-1H-

imidazoles was synthesized via microwave assisted protocol and tested for the

antagonistic effect against biofilm formation by Salmonella Typhimurium and

Pseudomonas aeruginosa.

Publications:

[1] One-pot microwave-assisted protocol for the synthesis of substituted 2-amino-

1H-imidazoles. Mol Divers. (2010) DOI 10.1007/s11030-010-9270-5

[2] Structure Activity Relationship of 2-Hydroxy-2-phenyl-2,3-dihydro-

imidazo[1,2-a]pyrimidinium Salts and 2N-Substituted 4(5)-Phenyl-2-Amino-

1H-imidazoles as Inhibitors of the Biofilm Formation by Salmonella

Typhimurium and Pseudomonas aeruginosa Bioorganic & Medicinal

Chemistry. Submited (2010)

2.1 Introduction

As described in chapter-1 the class of 2-aminoimidazoles has recently been given

particular interest due to various biological properties of these compounds. 2-

Aminoimidazole alkaloids and their metabolites, isolated from the marine sponges

Hymeniacidon sp., have been described as potent antagonists of serotonergic1 and

histaminergic2 receptors. Naamine and isonaamine alkaloids from the marine sponges

Leucetta sp. exhibit antiviral and anticancer activity.3,4 Because of these interesting

biological activities, numerous synthetic routes to 1-substituted and 1-unsubstituted 2-

aminoimidazoles have been reported. Modern synthetic methods for accessing 1-

unsubstituted 2-amino-1H-imidazoles can be roughly classified as heterocyclization

of substituted or protected guanidines with 1,2-dielectrophiles,5a-c heteroaromatic

nucleophilic substitution5c,6 and recyclization of 2-aminooxazoles.7 Although different

substituted guanidines are readily available and can be prepared in situ (e.g. from

cyanamines8), the high basicity of guanidines together with non-regioselectivity often

leads to multiple products.9 Protection by acetyl5a and Boc-groups5c requires, in turn,

acidic deprotection conditions. Another procedure is the cyclocondensation of

aldehydes and guanidine nitrate using sodium cyanide and supported aluminum oxide,

which provides symmetric 2-aminoimidazoles.10

Recently, we described two new approaches to the synthesis of substituted 2-amino-

1H-imidazoles. The first approach is based on the cleavage of imidazo[1,2-

a]pyrimidines11 with hydrazine12 In the second approach we reported microwave

assisted one pot synthesis.(scheme-3)13

2.2 Reaction scheme:

2.2.1 2-amino pyramidine: (Scheme-1)

N

N

Cl N

N

NH

RR NH2

TEA, EtOH

MW120 oC, 25 min

+

2.2.2 Synthesis of substituted 2-amino 1 H-imidazole. (Scheme-2)

N

NH

HNR2

100 °C, 10 min

64% N2H4

N

N

N R1

R2

OHBr

N

N

NH

R1

H

Br O

R2

+

MeCN

80 °C, 30 min

MW MeCN

MW

R1

2.2.3 One-Pot approach: (Scheme-3)

N

NH

HNR22. N2H4, MW 90 °C

N

N

NH

R1

H

Br O

R2

+MeCN, 30 min

MeCN, 10 min

R1

1.MW, 80 °C

2.3 Proposed Mechanism:

Regarding the mechanism of the transformation of 2-hydroxy-2,3-dihydro-1H-

imidazo[1,2-a]pyrimidinium salts into 2-amino-1H-imidazoles, we presume that the

reaction proceeds via an unusual Dimroth-type rearrangement14-16 (Scheme-4).

Scheme-4: Proposed Mechanism for the Dimroth-type Rearrangement of 2-

Hydroxy-2,3-dihydro-1H-imidazo[1,2-a]pyrimidinium Salts III-12

N

N

N R1

R2 R3

OHBr

N

NH

R1HNR2

R3

N

N

N R1

R2 R3

OH

H2NHN NNH

-

HN

HNNH

R1

R2 R3

O

N

NH

R1HNR2

R3

OH

N2H4

- H2O

slow

fast

N

N

N R1

R2 R3

OHH2NHN

N N

NH2

R2 R3

OHR1

1 A B

C D E 2

In the first step, the 2-hydroxy-2,3-dihydroimidazo[1,2-a]pyrimidinium salt (1)

undergoes cleavage of the pyrimidine ring, resulting in the generation of pyrazole and

2-amino-5-hydroxyimidazolidine (C), which is in equilibrium with the open form (D).

This can cyclize again leading to the isomeric 2-amino-5(4)-hydroxyimidazolidines

(E). Both isomers (C) and (E) were detected by mass-spectrometry. Final

dehydratation upon microwave irradiation, results in the rearranged 2-amino-1H-

imidazoles 2. This dehydratation step, as judged by mass-spectrometry, was found to

be the slowest step of the transformation. While some 2-amino-5(4)-

hydroxyimidazolidines (E) lost water spontaneously at room temperature, other

required higher temperature upon microwave irradiation. Therefore all reactions were

run at 100 °C, preventing the sequence from stopping after the first step.

2.4 Results and discussion:

Initially careful investigation of the formation and dehydratation of salt (3) resulting

in the formation of salt (4) under conventional heating conditions as well as upon

microwave irradiation has been studied. As a proof of concept, the condensation of 2-

methylaminopyrimidine (1, R1 = Me) and α-phenacylbromide (2, R2 = Ph) was

studied (Table 1). A sealed vial containing a solution of the starting compounds in

acetonitrile was conventionally heated with an oil bath (Table 1, No-1-6) or irradiated

with microwaves (Table 1, No-7-12) at different temperatures for 30−60 min. The

formation of the 2-hydroxy salt (3) was faster under microwave irradiation and this

compound was obtained in 88% yield within 30 min. Further increase of the

temperature up to 120 °C using conventional heating or microwave irradiation led to a

mixture of salts (3) and (4) (Table 1). Interestingly, using microwave irradiation at

120 °C for 60 min, we were able to drive the reaction completely to the formation of

the imidazo[1,2-a]pyrimidinium salt (4) (Table 1, No-12).

N

N

N R1

R2

OHBrN

N

NH

R1

H

Br O

R2

+

1 2 3

+ N

N

N R1

R2

Br

4

Conditions

(R1 = Me, R2 = Ph)

No. Conditions Time (min) T (°C) 3 (yield %)b 4 (yield %)b

1

Conventional

30 80 64 0

2 60 80 77 0

3 30 100 81 Traces

4 60 100 85 Traces

5 30 120 68 17

6 60 120 53 28

7

Microwave

30 80 88 0

8 60 80 85 0

9 30 100 48 33

10 60 100 45 35

11 30 120 12 79

12 60 120 traces 84

aAll reactions were carried out on a 1 mmol scale of 2-methylaminopyrimidine (1, R1 = Me) with

1.35 equiv of α-phenacylbromide (2, R2 = Ph) in MeCN (5 Ml); bisolated yield after recrystallization

from MeCN.

Table-1: Investigation of the condensation under conventional heating and

microwave irradiationa

As the transformation of salts (3) into salts (4) is relatively rapid at the temperatures

above 100 °C, the synthesis of salts 3 was conducted at 80 °C. Treatment of

substituted 2-aminopyrimidines (1) with 15 mol % excess of α-bromoketones (2) in

MeCN under microwave irradiation for 30 min generated the intermediate salts (3)

which, in most cases, precipitated from the reaction mixture upon cooling (Table-2).

Reaction progress was monitored by mass-spectrometry. In all cases examined, the

reaction appeared to be complete after 30 min. The cleavage step was performed

under microwave irradiation at 90 °C, using 7 equivalents of hydrazine hydrate. Based

on the data given in (Table-2), the reaction appears to be compatible with both aryl

and alkyl substitutions. All the reactions were clean, smooth, and provided the

products in good and high yields. The compounds were purified by column

chromatography using 5−10% MeOH in CH2Cl2 as the eluent. All the final 2-amino-

1H-imidazoles were characterized by 1H and 13C NMR spectroscopy. Their

composition was also confirmed by HRMS.

N

NH

HNR1

R2

No. Compound

Code R1 R2 Yield

1 BS-136* CH3 2,4-(di-OCH3) 48

2 BS-137* (benzo[d][1,3]dioxol-6- naphthalen-1-yl 72

yl)methyl

3 BS-140* C3H7 3,4-(di-Cl) 70

4 BS-141* cyclohexane 4-SO2Me 43

5 BS-143* C8H17 4-Cl 43

6 BS-146* C5H11 4-Cl 57

7 BS-147* C6H13 4-F 61

8 BS-148* 4-methoxyphenethyl 3-Br 44

9 BS-154* cyclooctane 4-SO2Me 53

10 BS-139* 2-methoxyethyl 4-CN 55

11 BS-258 C8H17 4-OCH3 78

12 BS-259 C8H17 4-F 69

13 BS-260 C8H17 3,4-(di-Cl) 72

14 BS-261 C8H17 3-Br 79

15 BS-262 C8H17 naphthalen-1-yl 68

16 BS-263 C8H17 4-NO2 66

17 BS-264 C8H17 4-(4-NO2Ph) 75

18 BS-265 C8H17 SO2Me 54

19 BS-292 i-Bu 4-NO2 70

20 BS-293 i-Bu H 71

21 BS-294 i-Bu 4-Br 69

22 BS-295 i-Bu 4-Cl 73

*Compounds have been synthesized via One-pot protocol (Scheme-3).

All compounds are either oily or amorphous solid.

Table-2: Microwave assisted synthesis of substituted 2-amino-1H-imidazoles.

2.5 Biofilm activity:

Bacteria are able to switch between a planktonic life style and a biofilm mode of

growth, in which they live as structured communities of bacterial cells enclosed in a

self-produced polymeric matrix and adherent to an inert or living surface.17 As

bacteria in biofilms are more resistant to challenges from predators, antibiotics,

disinfectants and host immune systems,18-21 serious problems and high costs have

been associated with biofilms, both in medicine and industrial settings. According to

the National Institutes of Health, more than 80% of microbial infections are related to

biofilms.22 Especially the use of indwelling medical devices comprises a high risk for

the development of biofilm related infections.19 This high prevalence of biofilm

related infections is particularly problematic given the fact that bacteria in biofilms

can be up to 1000-fold more resistant to antibiotics.23 In industry, biofilms have been

implicated e.g. in the contamination of installations in food industry, mild steel

corrosion, decreased passage through pipelines by colonization of the interior of the

pipes, and enhanced resistance of vessels by initiation of “biofouling” on the vessel

hulls. The yearly economic loss caused by ‘biofouling’ in the marine industry is

estimated at $ 6.5 billion.24

One way to deal with this problem is the development of small molecules that are able

to prevent or destroy biofilm formation25. Only a few molecular scaffolds have been

identified to date, among which the best-studied examples are (1) the halogenated

furanones, which were originally isolated from the seaweed Delisea pulchra,26-27 (2)

analogues of the homoserine lactone signalling molecules28 and (3) analogues of the

sponge-derived marine natural alkaloids oroidin and bromoageliferin.29-36 A

particularly valuable approach is the development of small molecules that specifically

target the biofilm formation in a non-toxic manner, as it is expected that resistance

against these compounds will emerge much slower than against classical

microbiocidal compounds. As a consequence, non-toxic biofilm inhibitors have the

potential to be used in a preventive treatment of a wide diversity of industrial and

medical surfaces. Furthermore, the potential to co-dose biofilm inhibitors and

classical antibiotics for the treatment of biofilm infections is also an attractive

option.27

Salmonella enterica serovar Typhimurium and Pseudomonas aeruginosa are two well

studied organisms in terms of biofilm formation. Salmonella enterica is worldwide

one of the most important causes of foodborne infections. Salmonella is able to form

biofilms on a variety of both biotic surfaces (such as gallstones,37 plant surfaces,38-39

and epithelial cell layers40) and abiotic surfaces (such as concrete, plastics, glass,

polystyrene, … )41-42. These biofilms are an important survival strategy in all stages

of infection, from transmission to chronic infection. Severe non-typhoid Salmonella

infections are commonly treated with fluoroquinolones and third-generation

cephalosporins. Unfortunately, there are alarming reports concerning the development

of resistance against these antibiotics43, underlining the urgent need of alternative

anti-Salmonella strategies. Given the importance of biofilms in the spread of

Salmonella, the development of Salmonella biofilm inhibitors seems a promising

approach. P. aeruginosa is an opportunistic pathogen implicated in a myriad of

infections. Patients with compromised host defenses, such as burn patients, HIV

patients and cystic fibrosis patients (80% colonization rate of P. aeruginosa44) are

particularly susceptible to P. aeruginosa infections.45 P. aeruginosa biofilms have

been related to recurrent ear infections, chronical bacterial prostatitis and lung

infections in CF patients, the latter being extremely harmful as P. aeruginosa

colonization and chronic lung infection is the major causative agent of morbidity and

mortality in CF patients.46 Moreover, P. aeruginosa can colonize as biofilms a

variety of medical devices such as intravascular catheters and urinary catheters.

Obviously there is an urgent need of agents that can prevent or eradicate P.

aeruginosa biofilms on infected tissues and on medical devices.

2.5.1 2N-substituted 2-aminopyrimidines

Some members of the 2-aminopyrimidine class of compounds have previously been

shown to possess antibacterial and antifungal activity.47-48 These molecules are the

precursors of the N1-substituted diazo[1,2-a]pyrimidinium salts in our synthesis

pathway (scheme-2). The availability of a broad array of 2-aminopyrimidines,

substituted with n-alkyl, cyclo-alkyl and aromatic groups at the N2-position prompted

us to investigate the potential of this class of compounds as biofilm inhibitors. The

influence of compounds on the biofilm formation of S. Typhimurium was tested at

25°C. Remarkably, none of the compounds does have an effect on the biofilm

formation at 400 µM, which was the highest concentration tested.

2.5.2 2-hydroxy-2,3-dihydro-imidazopyrimidinium salts

A broad array of 1-substituted 2-hydroxy-2-phenyl-2,3-dihydro-imidazopyrimidinium

salts were synthesized via previously established chemistry from our laboratory and

their ability to prevent the biofilm formation of S. Typhimurium and P. aeruginosa

was tested. These molecules differ in the substitution pattern of the 2-phenyl ring and

in the nature of the substituent at the 1-position i.e. n-alkyl, iso-alkyl, cyclo-alkyl.

2.5.2.1 n-Alkyl or iso-alkyl substituents at 1-position

In first instance, a series of 2-hydroxy-2-phenyl-2,3-dihydro-imidazopyrimidinium

salts with a broad variety of n-alkyl or iso-alkyl substituents at the 1-position, with

lengths ranging from 1 carbon atom to 14 carbon atoms was synthesized. The 2-

phenyl group of these salts is either unsubstituted, para-substituted with a chlorine

atom, a nitro group, a fluorine atom, a bromine atom or a methoxy group, or 3,4-

disubstituted with chlorine atoms. Each compound was assayed for the ability to

inhibit S. Typhimurium ATCC14028 biofilm formation at 25 °C. As depicted in

figure 1A, a clear correlation was found between the length of the alkyl substituent

and the biofilm inhibitory activity. In general the activity of the molecules with a

short alkyl chain was found to be very low (IC50 > 400 µM). Within a series of

molecules with the same substitution of the 2-phenyl ring, in general a gradual

increase in biofilm inhibitory activity was observed by raising the length of the alkyl

chain from 1 to 8 carbon atoms. The compounds with an octyl substituent show a

maximal activity with IC50 values in the range of 6-11 µM. By further raising the

alkyl chain length from 8 to 14 carbon atoms, a gradual decrease in biofilm inhibitory

activity was observed. For the molecules with a heptyl, octyl, nonyl and decyl side

chain (which are the most active molecules), the nature of the substituent of the 2-

phenyl group does not have a substantial effect on the biofilm inhibitory activity.

Next the influence of a subset of the 2-hydroxy-2-phenyl-2,3-dihydro-

imidazopyrimidinium salts for their ability to prevent the biofilm formation of P.

aeruginosa was studied. As represented in table-4 and figure-1B, a similar structure

activity relationship was found as in the case of Salmonella biofilm inhibition.

Compounds with short alkyl substituents (C1-6) in general only have a slight effect on

the biofilm formation (IC50’s >80 µM), while compounds with medium length side

chains (C7-C10) have IC50 values between 20-40 µM and compounds with long side

chains (C11-C14) have IC50 values higher than 800 µM. However, it should be

mentioned that some of the compounds with long side chain do reduce the biofilm

formation to a certain extent, but their dose response curves reach a steady state level

of 25 to 45 % biofilm inhibition starting from concentrations between 25 and 50 µM.

N

N

N R1

OHBr

R2

No. Code R2 R1 S. Typhimurium IC50

P. aeruginosa IC50

1 BS-289 H i-Bu 131.2 289.4 2 BS-083 H n-Pen 278.2 565.4 3 BS-058 H n-Hex 102.0 405.8 4 BS-049 H n-Hep 23.4 25.5 5 BS-052 H n-Oct 9.8 14.4 6 BS-061 H n-Non 8.3 26.8 7 BS-055 H n-Dec 15.0 29.9 8 BS-064 H n-Und 24.6 >800 9 BS-067 H n-Dod 27.5 >800 10 BS-070 H n-Trd 81.6 >800 11 BS-073 H n-Tet 179.6 >800 12 BS-291 4-Cl i-Bu 31.4 83.4 13 BS-099 4-Cl n-Pen 32.6 163.9 14 BS-092 4-Cl n-Hex 13.7 79.8 15 BS-089 4-Cl n-Hep 6.7 36.9 16 BS-090 4-Cl n-Oct 7.0 15.7 17 BS-093 4-Cl n-Non 18.3 20.2 18 BS-091 4-Cl n-Dec 51.3 83.3 19 BS-094 4-Cl n-Und 61.9 >800 20 BS-095 4-Cl n-Dod 122.1 >800 21 BS-096 4-Cl n-Trd 316.1 >800 22 BS-097 4-Cl n-Tet >800 >800 23 BS-288 4-NO2 i-Bu 322.5 191.7 24 BS-290 4-Br i-Bu 5.2 6.3 IC50: Concentration of inhibitor needed to inhibit biofilm formation by 50%.

Table-4: Influence of 1-alkylated 2-hydroxy-2,3-dihydro-imidazo[1,2-

a]pyrimidinium salts on the biofilm formation of S. Typhimurium ATCC14028 and P.

aeruginosa PA14 at 25°C.

N

N

N R1

OHBr

N

N

N R1

OHBr

Cl

Figure-1A: Effect of the length of the n-alkyl chain at the 1-position of 2-hydroxy-2-

phenyl-2,3-dihydro-imidazo[1,2-a]pyrimidinium salts, substituted with Cl and H at

the para-position of the 2-phenyl ring, on the IC50 (µM) for inhibition of biofilm

formation of S. Typhimurium ATCC14028.

2.5.2.2 Further Modification in the most active core structure

Since the compounds with a n-octyl chain substituted at the 1-position have the

highest activity against Salmonella biofilms and also have a high activity against

Pseudomonas biofilms, we decided to synthesize the additional 1-octyl-2-hydroxy-2-

phenyl-2,3-dihydro-imidazopyrimidinium salts with more variation in the substitution

pattern of the 2-phenyl ring. As depicted in Table 5 these compounds generally inhibit

biofilm formation of S. Typhimurium at low concentrations. However, no improved

activity was observed in comparison with the previously described compounds (Table

2). The effect of the n-octyl substituted compounds on the biofilm formation of P.

aeruginosa strongly depends on the substitution pattern of the 2-phenyl ring, as some

of the compounds have low IC50 values (10-40 µM), while the other compounds only

have a moderate activity (IC50 = 100-400 µM).

N

N

N C8H17

OHBr

R

SR CODE R S. Typhimurium

IC50

P. aeruginosa

IC50

1 BS-180 4-NO2 6.6 12.1

2 BS-176 4-F 7.6 nd

3 BS-175 4-OMe 10.5 nd

4 BS-177 3,4-diCl 7.1 nd

5 BS-187 4-SMe 13.2 36.5

6 BS-179 Naphtyl 14.7 15.1

7 BS-185 4-(4-NO2Ph) 31 215.2

8 BS-186 4-SO2Me 60.3 99.9

9 BS-188 4-([1,1’-biphenylyl]-4-yl) 52.8 336.1

10 BS-178 3-Br 7.1 29

Table-5: Influence of N1-octyl 2-hydroxy-2, 3-dihydro-imidazo[1,2-a]pyrimidinium

salts on the biofilm formation of S. Typhimurium ATCC14028 and P. aeruginosa

PA14 at 25°C.

2.5.2.3 Cyclo-alkyl substituents at 1-position

A series of 2-hydroxy-2-phenyl-2,3-dihydro-imidazopyrimidinium salts with a broad

variety of cyclo-alkyl substituents at the 1-position was synthesized with lengths

ranging from 3 to 12 carbon atoms (Table 6). The 2-phenyl group of these salts was

either unsubstantiated or para-substituted with a chlorine atom. Similarly to the

structure-activity relationship delineated for the n-alkyl substituted salts, a gradual

increase in the inhibitory activity against Salmonella biofilms was observed by rising

the length of the cyclo-alkyl chain from 3 to 8 carbon atoms. However, in contrast to

the salts with n-dodecyl chain at the 1-position, the salts with a cyclo-dodecyl chain

do have a very strong effect against Salmonella biofilms (IC50 values < 6.25 µM). By

analogy with (1) the effect on Salmonella biofilms and (2) the activity of the n-alkyl

substituted salts, we found that all the cyclo-alkyl substituted salts with a short side

chain do only have a slight effect on the biofilm formation of P. aeruginosa (IC50’s

>150 µM), while the compounds with a medium length side chain have a stronger

biofilm inhibitory activity. The salts with a cyclo-dodecyl chain do drastically reduce

the Pseudomonas biofilm formation (IC50’s ~7 µM), in sharp contrast to the

compounds with n-dodecyl side chain.

N

N

N R1

OHBr

R2

SR CODE R2 R1 S. Typhimurium

IC50

P. aeruginosa

IC50

1 BS-079 H c-Bu 493.0 >400

2 BS-076 H c-Hex 139.0 22.4

3 BS-046 H c-Hep 84.2 191.3

4 BS-043 H c-Oct 58.4 48.6

5 BS-100 4-Cl c-Bu 120.8 371.5

6 BS- 4-Cl c-Pen 41.8 41.9

7 BS-098 4-Cl c- Hex 33.0 16.4

8 BS-088 4-Cl c- Hep 27.9 76.2

9 BS-087 4-Cl c-Oct 12.6 22.7

10 BS-086 4-Cl c-Doc 5.6 6.8

Table-6: Influence of 1-cyclo-alkyl-2-hydroxy-2,3-dihydro-imidazo[1,2-a]

pyrimidinium salts on the biofilm formation of S. Typhimurium ATCC14028 and P.

aeruginosa PA14 at 25°C.

2.5.3 2N-substituted 2-aminoimidazoles

In the present it was interesting to investigate whether introduction of n-alkyl, iso-

alkyl, cyclo-alkyl or aromatic group at the 2N-position of the 4(5)-phenyl-2-amino-

1H-imidazoles could also enhance their biofilm inhibitory activity. Therefore a broad

range of 2N-substituted 4(5)-phenyl-2-amino-1H-imidazoles were synthesized.

An array of 4(5)-phenyl-2-aminoimidazoles 2N-substituted with either a short n- or

iso-alkyl chain (C1-C5) or n-octyl chain was synthesized. As depicted in table7, a

broad diversity of (substituted) 4(5)-phenyl groups were included. The compounds

with a iso-butyl substitution at the 2N-position were found to be in general more

active than their 2N-unsubstituted counterparts, with respect to both the Salmonella

and Pseudomonas biofilm formation.

N

NH

HNR1

R2

SR R1 R2 S. Typhimurium IC50

P. aeruginosa IC50

1 CH3 2,4-(di-OCH3) Nd Nd

2 (benzo[d][1,3]dioxol-6-yl)methyl

naphthalen-1-yl 261.0 10.5

3 C3H7 3,4-(di-Cl) 10.9 27.7 4 Cyclohexane 4-SO2Me Nd Nd 5 C8H17 4-Cl >400 Nd 6 C5H11 4-Cl >400 Nd 7 C6H13 4-F >400 12.5 8 C8H17 4-OCH3 >400 >800 9 C8H17 4-F >400 Nd 10 C8H17 3,4-(di-Cl) 118.3 >800 11 C8H17 3-Br 44.6 >800 12 C8H17 naphthalen-1-yl 238.4 >800 13 C8H17 4-NO2 10.9 25.0 14 C8H17 4-(4-NO2Ph) Nd Nd 15 C8H17 SO2Me 41.8 Nd 16 i-Bu 4-NO2 3.8 22.9 17 i-Bu H 4.9 1.2 18 i-Bu 4-Br 2.9 1.2 19 i-Bu 4-Cl 2.0 0.9

Table-7: Influence of 2N-alkylated 2-aminoimidazoles on the biofilm formation and

the planktonic growth of S. Typhimurium ATCC14028 and P. aeruginosa PA14 at

25°C.

2.6 Conclusion

In the present study, the potential of these 1-substituted 2-hydroxy-2,3-dihydro-

imidazopyrimidinium salts and 2N-subsituted 2-amino-1H-imidazoles as inhibitors of

the biofilm formation by S. Typhimurium and P. aeruginosa was thoroughly

investigated. We found that 2-hydroxy-2,3-dihydro-imidazopyrimidinium salts with

intermediate length n-alkyl chains (C7-C10) substituted at the 1-position in general

prevent the biofilm formation of both species at low micromolar concentrations (IC50

= 5-50 µM). For these molecules, the nature of the substituent of the 2-phenyl group

does not have a substantial effect on the biofilm inhibitory activity. Salts with a

shorter (C1-C5) or longer (C11-C14) n-alkyl chain at the 1-position were found to be

much less potent. Consistent with this, we observed that salts with an intermediate

length cyclo-alkyl chain are much more active against biofilm formation of both

species as compared to the salts with a short cyclo-alkyl chain. However, remarkably

salts with a long cyclo-dodecyl side chain were found to have even better activities

than salts with an intermediate length cyclo-alkyl side chain.

In the framework of the 2-aminoimidazoles, be the introduction of a butyl, pentyl or

hexyl side chain at the 2N-position of the 2-aminoimdazoles results in an enhanced

biofilm inhibitory activity against both species, while introduction of a shorter n-alkyl

chain reduces the biofilm inhibitory activity. The effect of introduction of longer n-

alkyl chains however seems to be strongly dependent on the substitution pattern of the

5-phenyl ring and the bacterial species studied.

In conclusion, the 2N-substituted 2-aminoimidazoles and 2-hydroxy-2-phenyl-2,3-

dihydro-imidazopyrimidinium salts of the present study are valuable candidates in the

development of therapeutics and sanitizers for the combat of biofilm formation by S.

Typhimurium, P. aeruginosa and possibly other pathogenic bacteria.

2.7 Experimental

2.7.1 General Methods

All chemical reagents were used without further purification. Solvents for column

chromatography and TLC were laboratory grade and distilled before use. For thin-

layer chromatography (TLC), analytical TLC plates (Alugram SIL G/UV254 (E. M.

Merk) were used. Column chromatography was performed with flash silica gel (100-

200 mesh) or neutral alumina oxide (50-200 micron). 1H and 13C NMR spectra were

recorded on a Bruker Avance 300 (300 MHz) or a Bruker AMX-400 (400 MHz)

spectrometers. NMR samples were run in the indicated solvents and were referenced

internally. Chemical shift values were quoted in ppm and coupling constants were

quoted in Hz. Chemical shift multiplicities were reported as s = singlet, d = doublet, t

= triplet, q = quartet, m = multiplet and br = broad. Low-resolution mass spectra were

recorded on a HEWLETT-PACKARD instrument (CI or EI) and LCQ Advantage

instrument (ESI). High-resolution mass spectra (EI) were recorded on a KRATOS

MS50TC instrument. Melting points were determined using Reichert-Jung Thermovar

apparatus and were uncorrected.

2.7.2 Microwave Irradiation Experiments

Microwave irradiation experiments were carried out in a dedicated CEM-Discover

mono-mode microwave apparatus or Milestone MicroSYNTH multi-mode microwave

reactor (Laboratory Microwave Systems). Microwave apparatuses were used in the

standard configuration as delivered, operating at a frequency 2.45 GHz with

continuous irradiation power from 0 to 400 W. The reactions were carried out in 10,

20, 30 and 50 mL glass tubes. The temperature was measured with an IR sensor on

the outer surface of the process vial or fibre optic sensor inside the process vial. After

the irradiation period, the reaction vessel was cooled rapidly (2-5 min) to ambient

temperature by air jet cooling.

2.8 Biological assays

2.8.1 Static peg assay for prevention of Salmonella Typhimurium and

Pseudomonas aeruginosa biofilm formation

The device used for biofilm formation is a platform carrying 96 polystyrene pegs

(Nunc no. 445497) that fits as a microtiter plate lid with a peg hanging into each

microtiter plate well (Nunc no. 269789). Two-fold serial dilutions of the compounds

in 100 µl liquid broth (Tryptic Soy Broth diluted 1/20 (TSB 1/20)) per well were

prepared in the microtiter plate (2 or 3 repeats per compound). Subsequently, an

overnight culture of S. Typhimurium ATCC14028 (grown in Luria-Bertani medium)

or P. aeruginosa (grown in TSB) was diluted 1:100 into the respective liquid broth

and 100 µl (~106 cells) was added to each well of the microtiter plate, resulting in a

total amount of 200 µl medium per well. The pegged lid was placed on the microtiter

plate and the plate was incubated for 24 h at 25°C without shaking. During this

incubation period biofilms were formed on the surface of the pegs. After 24 h, the

optical density at 600 nm (OD600) was measured for the planktonic cells in the

microtiter plate using a VERSAmax microtiter plate reader (Molecular Devices). This

gives a first indication of the effect of the compounds on the planktonic growth. For

quantification of biofilm formation, the pegs were washed once in 200 µl phosphate

buffered saline (PBS). The remaining attached bacteria were stained for 30 min with

200 μl 0.1% (w/v) crystal violet in an isopropanol/methanol/PBS solution (v/v

1:1:18). Excess stain was rinsed off by placing the pegs in a 96-well plate filled with

200 μl distilled water per well. After the pegs were air dried (30 min), the dye bound

to the adherent cells was extracted with 30% glacial acetic acid (200 µl). The OD570 of

each well was measured using a VERSAmax microtiter plate reader (Molecular

Devices). The IC50 value for each compound was determined from the concentration

gradient by using the GraphPad software of Prism.

2.8.2 Bioscreen assay for measuring Salmonella Typhimurium and P.

aeruginosa growth inhibition

The Bioscreen device (Oy Growth Curves Ab Ltd) was used for measuring the

influence of the chemical compounds on the planktonic growth of Salmonella

Typhimurium and P. aeruginosa. The Bioscreen is a computer controlled

incubator/reader/shaker that uses 10x10 well microtiter plates and measures light

absorbance of each well at a specified wave length in function of time. An overnight

culture of S. Typhimurium ATCC14028 (grown up in LB medium) or P. aeruginosa

(grown up in TSB) was diluted 1:200 in liquid broth (TSB 1/20). 300 µl of the diluted

overnight culture was added to each well of the 10x10 well microtiter plate.

Subsequently, serial dilutions of the chemical compounds were prepared in DMSO or

EtOH. 3 µl of each diluted stock solution was added to the wells (containing the 300

µl bacterial culture) in 3-fold. As a control 3 µl of the appropriate solvent was also

added to the plate in 3- or 4-fold. The microtiter plate was incubated in the Bioscreen

device at 25°C for at least 24 h, with continuous medium shaking. The absorbance of

each well was measured at 600 nm each 15 min. Excel was used to generate the

growth curves for the treated wells and the untreated control wells.

The effect of each compound concentration on the planktonic growth was classified

into one of the following categories:

1) The planktonic growth is not or only slightly affected, indicated by the symbol

‘-‘.

2) The planktonic growth is retarded, indicated by the symbol ‘+’.

3) The planktonic growth is completely or almost completely inhibited, indicated

by the symbol ‘o’.

The following criterium was used to decide between the first and the second category:

If the absorbance (measured at 600 nm) of the bacterial culture treated with the

compound is at least 0.5 (for Salmonella) or 0.8 (for Pseudomonas) units lower

than the absorbance of the untreated culture during 4 consecutive hours, then the

effect on the planktonic growth is classified in category 2.

2.9 Experimental protocol:

2.9.1 General Procedure for the Preparation of Substituted 2-

Aminopyrimidines

In a 50 mL microwave vial were successively dissolved in EtOH (20 mL) 2-

chloropyrimidine (3.43 g, 30 mmol), amine (39 mmol, 1.3 equiv) and triethylamine

(6.2 mL, 45 mmol, 1.5 equiv). The reaction tube was sealed, and irradiated in the

cavity of a Milestone MicroSYNTH microwave reactor at a ceiling temperature of

120 °C at 100 W maximum power for 25 min. After the reaction mixture was cooled

with an air flow for 15 min, it was diluted with water (100 mL), extracted with DCM

(2×150 mL) and dried over Na2SO4. The solvent was evaporated in vacuo, and the

crude mixture was purified by silica gel flash chromatography using 0-5%

MeOH−DCM as the eluent.

13 new substituted 2-aminopyrimidine derivatives were synthesized in similar

manner. (Table-1)

Table-1 Compound synthesized

N

N

NH

R

ENTRY CODE R Yield

1 BS-021 cyclododecane 66

2 BS -024 cyclooctane 60

3 BS -025 cycloheptane 64

4 BS -030 cyclobutane 61

5 BS-029 C6H13 88

6 BS -026 C7H15 87

7 BS -027 C8H17 86

8 BS -033 C9H19 86

9 BS -028 C10H21 87

10 BS -034 C11H23 88

11 BS -035 C12H25 89

12 BS -036 C13H27 88

13 BS -037 C14H19 88

2.9.2 General Procedure for the Preparation of Salts.

To a solution of 2-substituted aminopyrimidine (6 mmol) and α-bromoacetophenone

(7.2 mmol, 1.2 equiv) in acetonitrile (12 mL) was added 4-dimethylaminopyridine (6

mg, 0.05 mmol). After being stirred at 85 °C for 5 h, the reaction mixture was diluted

with acetone (20 mL) and the precipitate was filtered and washed with acetone (2×20

mL), ether (2×20 mL) and dried over P2O5 to give salt as a white solid.

10 new compounds were synthesized in similar manner.

2.9.3 General Procedure for the Preparation of Salts. (Microwave)

In a 50 mL microwave vial were successively dissolved 2-substituted

aminopyrimidine (6 mmol) and α-bromoacetophenone (7.2 mmol, 1.2 equiv) in

acetonitrile (12 mL) was added 4-dimethylaminopyridine (6 mg, 0.05 mmol). The

reaction tube was sealed, and irradiated in the cavity of a Milestone MicroSYNTH

microwave reactor at a ceiling temperature of 80 °C at 100 W maximum power for 30

min. After the reaction mixture was cooled with an air flow for 15 min, the reaction

mixture was diluted with acetone (20 mL) and the precipitate was filtered and washed

with acetone (2×20 mL), ether (2×20 mL) and dried over P2O5 to give salt as a white

solid. 34 new compounds were synthesized in similar manner.

2.9.4 General Procedure for the Preparation of 2-Amino-1H-imidazoles.

To a suspension of salt (2 mmol) in MeCN (5 mL) hydrazine hydrate (0.7 mL, 14

mmol of a 64% solution, 7 equiv) was added, and the mixture was irradiated in the

sealed Milestone MicroSYNTH microwave reactor for 10 min at a ceiling

temperature of 100 °C at 150 W maximum power. After cooling down hydrazine

hydrate was evaporated with toluene (3×20 mL). The resulting residue was purified

by column chromatography (silica gel; MeOH−DCM 1:4 v/v with 5% of 6M NH3 in

MeOH) to afford 2-amino-1H-imidazole as an amorphous material.


2.9.5 General procedure for the one-pot two-step microwave-assisted synthesis

of 2-amino-1H-imidazoles.

In a microwave vial (10 mL) were successively dissolved in dry MeCN (3 mL) the

corresponding 2-aminopyrimidine (4 mmol) and α-bromoketone (4.6 mmol). The

reaction tube was sealed and irradiated in a microwave reactor at a ceiling

temperature of 80 °C and a maximum power of 50 W for 30 min. After the reaction

mixture was cooled with an air flow for 15 min, hydrazine hydrate (0.9 mL, 28 mmol

of a 64% solution, 7 equiv) was added, and the mixture was irradiated at a ceiling

temperature of 90 °C and a maximum power of 50 W for another 10 min. After the

reaction mixture was cooled, hydrazine hydrate was removed by distillation with

toluene (3×20 mL). The resulting residue was purified by column chromatography

(silica gel; MeOH-DCM 1:9 v/v with 0.5 % of 6N ammonia in MeOH) to afford 2-

amino-1H-imidazole as amorphous solid.


2.10 Spectral Characterization

N-Cyclooctylpyrimidin-2-amine (BS-024)

N

N

NH

Yield: 60%. 1H NMR (300 MHz, CDCl3): δ = 8.25 (d, J = 4.77 Hz, 2H), 6.46 (t, J =

4.8 Hz, 1H), 5.07 (br s, 1H), 4.04 (m, 1H), 1.93 (m, 2H), 1.63 (m, 12H). 13C NMR

(75.5 MHz, CDCl3): δ =161.6, 158.0 (×2), 110.0, 50.6, 31.7 (×2), 27.5 (×2), 25.3,

23.5 (×2). HRMS (EI): C12H19N3, calcd 205.2994, found: 205.2982.

N –Cycloheptylpyrimidin-2-amine (BS-025)

N

N

NH


4.77 Hz, 1H), 5.09 (br s, 1H), 4.01 (m, 1H), 2.02 (m, 2H), 1.55 (m, 10H). 13C NMR

(75.5 MHz, CDCl3): δ =161.6, 158.0 (×2), 110.0, 51.6, 35.0 (×2), 28.2 (×2), 23.9

(×2). HRMS (EI): C11H17N3, calcd 191.2728, found: 191.2742.

N –Heptylpyrimidin-2-amine (BS-026)

N

N

NH

C7H15

Yield: 87%. 1H NMR (300 MHz, CDCl3): δ = 8.27 (d, J = 4.8 Hz, 2H), 6.50 (t, J = 4.8

Hz, 1H), 5.07 (br s, 1H), 3.40 (m, 2H), 1.61 (m, 2H), 1.31 (m, 8H), 0.85 (m 3H). 13C

NMR (75.5 MHz, CDCl3): δ =162.5, 157.9 (×2), 110.1, 41.5, 31.8, 29.6, 29.0, 26.9,

22.6, 14.0. HRMS (EI): C11H19N3, calcd 193.2887, found: 193.2894.

N –Octylpyrimidin-2-amine (BS-027)

N

N

NH

C8H17


4.77 Hz, 1H), 5.11 (br s, 1H), 3.40 (m, 2H), 1.60 (m, 2H), 1.27 (m, 10H), 0.87 (m

3H). 13C NMR (75.5 MHz, CDCl3): δ =162.4, 158.0 (×2), 110.1, 41.5, 31.8, 29.6,

29.3, 29.2, 26.9, 22.6, 14.1. HRMS (EI): C12H21N3, calcd 207.3152, found: 207.3131.

N –Decylpyrimidin-2-amine (BS-028)

N

N

NH

C10H21


Hz, 1H), 5.07 (br s, 1H), 3.38 (m, 2H), 1.60 (m, 2H), 1.26 (m, 14H), 0.87 (t, J = 6.93,

3H). 13C NMR (75.5 MHz, CDCl3): δ =162.4, 158.0 (×2), 110.1, 41.5, 31.8, 29.6 (×2),

29.5, 29.3 (×2), 26.9, 22.6, 14.1. HRMS (EI): C14H25N3, calcd 235.3684, found:

235.3670.

N –Cyclobutylpyrimidin-2-amine (BS-030)

N

N

NH

Yield: 61%. 1H NMR (300 MHz, CDCl3): δ = 8.27 (m, 2H), 6.51 (t, J = 4.77 Hz, 1H),

5.29 (br s, 1H), 4.46 (m, 1H), 2.42 (m, 2H), 1.89 (m, 2H), 1.76 (m, 2H). 13C NMR

(75.5 MHz, CDCl3): δ =161.6, 158.0 (×2), 110.4, 46.3, 31.5 (×2), 15.0. HRMS (EI):

C8H11N3, calcd 149.1930, found: 149.1917

N –Nonylpyrimidin-2-amine (BS-033)

N

N

NH

C9H19


4.77 Hz, 1H), 5.07 (br s, 1H), 3.40 (m, 2H), 1.61 (m, 2H), 1.26 (m, 12H), 0.87 (t, J =

6.9, 3H). 13C NMR (75.5 MHz, CDCl3): δ =162.5, 158.0 (×2), 110.1, 41.5, 31.8, 29.6,

29.5,29.4, 29.2, 27.0, 22.6, 14.1. HRMS (EI): C13H23N3, calcd 221.3418, found:

221.3400.

N –Undecylpyrimidin-2-amine (BS-034)

N

N

NH

C11H23



6.87, 3H). 13C NMR (75.5 MHz, CDCl3): δ = 162.5, 158.0 (×2), 110.2, 41.5, 31.9,

29.6 (×4), 29.3 (×2), 26.9, 22.6, 14.1. HRMS (EI): C15H27N3, calcd 249.3950, found:

249.3938.

N –Dodecylpyrimidin-2-amine (BS-035)

N

N

NH

C12H25



6.84, 3H). 13C NMR (75.5 MHz, CDCl3): δ = 162.4, 158.0 (×2), 110.2, 41.5, 31.9,

29.6 (×5), 29.3 (×2), 26.9, 22.6, 14.1. HRMS (EI): C16H29N3, calcd 263.4216, found:

263.4202.

N –Tridecylpyrimidin-2-amine (BS-036)

N

N

NH

C13H27



6.9, 3H). 13C NMR (75.5 MHz, CDCl3): δ = 162.4, 158.0 (×2), 110.1, 41.5, 31.9, 29.6

(×6), 29.3 (×2), 27.0, 22.7, 14.1. HRMS (EI): C17H31N3, calcd 277.4481, found:

277.4491.

N –Tetradecylpyrimidin-2-amine (BS-037)

N

N

NH

C14H29



6.9, 3H). 13C NMR (75.5 MHz, CDCl3): δ = 162.4, 158.0 (×2), 110.2, 41.5, 31.9, 29.6

(×7), 29.3 (×2), 26.9, 22.7, 14.1. HRMS (EI): C18H33N3, calcd 291.4747, found:

291.4723.

1-Heptyl-2-hydroxy-2-phenyl-2,3-dihydro-1H-imidazo[1,2-a]pyrimidin-4-ium

bromide amine (BS-049)

N

N

N C7H15

OHBr

Yield: 75 %. 1H NMR (300 MHz, CDCl3): δ = 9.03 (m, 1H), 8.85 (m, 1H), 7.80 (s,

1H), 7.77 (m, 2H), 7.49 (m, 3H), 7.31 (m, 1H), 4.82 (s, 2H). 3.17 (m, 2H), 1.36 (m,

2H), 1.09 (m, 8H), 0.80 (t, J = 6.84 Hz, 3H).

2-Hydroxy-1-octyl-2-phenyl-2,3-dihydro-1H-imidazo[1,2-a]pyrimidin-4-ium

bromide (BS-052)

N

N

N C8H17

OHBr


1H), 7.75 (m, 2H), 7.47 (m, 3H), 7.31 (t, J = 5.49 Hz, 1H), 4.83 (s, 2H). 3.17 (m, 2H),

1.36 (m, 2H), 1.09 (m, 10H), 0.83 (t, J = 6.72 Hz, 3H).

1-Decyl-2-hydroxy-2-phenyl-2,3-dihydro-1H-imidazo[1,2-a]pyrimidin-4-ium

bromide (BS-055)

N

N

N C10H21

OHBr

Yield: 78 %. 1H NMR (300 MHz, CDCl3): δ = 9.03 (d, J = 4.62 Hz, 1H), 8.87 (d, J =

6.18 Hz, 1H), 7.81 (s, 1H), 7.74 (m, 2H), 7.47 (m, 3H), 7.31 (m, 1H), 4.83 (s, 2H).

3.17 (m, 2H), 1.09 (m, 16H), 0.85 (t, J = 6.51 Hz, 3H). 13C NMR (75.5 MHz, CDCl3):

δ =167.5, 154.5, 148.2, 138.2, 129.2, 128.3 (×2), 126.9 (×2), 111.1, 90.6, 62.6, 40.7,

31.2, 28.7, 28.6, 28.5, 28.2, 27.3, 25.8, 22.0, 13.9.

1-Hexyl-2-hydroxy-2-phenyl-2,3-dihydro-1H-imidazo[1,2-a]pyrimidin-4-ium

bromide (BS-058)

N

N

N C6H13

OHBr


1H), 7.74 (m, 2H), 7.47 (m, 3H), 7.31 (m 1H), 4.83 (s, 2H). 3.17 (m, 2H), 1.34 (m,

2H), 1.09 (m, 6H), 0.78 (t, J = 6.48 Hz, 3H).

2-Hydroxy-1-nonyl-2-phenyl-2,3-dihydro-1H-imidazo[1,2-a]pyrimidin-4-ium

bromide (BS-061)

N

N

N C9H19

OHBr


1H), 7.74 (m, 2H), 7.47 (m, 3H), 7.31 (t, J = 6.21 Hz, 1H), 4.83 (s, 2H). 3.17 (m, 2H),

1.09 (m, 14H), 0.84 (t, J = 6.63 Hz, 3H). 13C NMR (75.5 MHz, CDCl3): δ =167.5,

154.5, 148.2, 138.2, 129.2, 128.3 (×2), 126.9 (×2), 111.1, 90.6, 62.6, 40.7, 31.1, 28.5,

28.4, 28.2, 27.3, 25.8, 22.0, 13.9.

2-Hydroxy-2-phenyl-1-undecyl-2,3-dihydro-1H-imidazo[1,2-a]pyrimidin-4-ium

bromide (BS-064)

N

N

N C11H23

OHBr

Yield: 80 %. 1H NMR (300 MHz, CDCl3): δ = 9.03 (d of d, J = 1.89, 4.68 Hz, 1H),

8.87 (d, J = 6.06 Hz, 1H), 7.80 (s, 1H), 7.75 (m, 2H), 7.47 (m, 3H), 7.31 (m 1H), 4.83

(s, 2H). 3.17 (m, 2H), 1.09 (m, 18H), 0.85 (t, J = 6.42 Hz, 3H).

1-Dodecyl-2-hydroxy-2-phenyl-2,3-dihydro-1H-imidazo[1,2-a]pyrimidin-4-ium

bromide (BS-067)

N

N

N C12H25

OHBr


1H), 7.74 (m, 2H), 7.46 (m, 3H), 7.31 (m, 1H), 4.84 (s, 2H). 3.17 (m, 2H), 1.22 (m,

20H), 0.85 (t, J = 6.24 Hz, 3H). 13C NMR (75.5 MHz, CDCl3): δ =167.5, 154.5,

148.2, 138.2, 129.2, 128.3 (×2), 126.9 (×2), 111.1, 90.6, 62.6, 40.7, 31.2, 28.9 (×2),

28.8, 28.6 (×2), 28.2, 27.3, 25.8, 22.0, 13.9.

2-Hydroxy-2-phenyl-1-tridecyl-2,3-dihydro-1H-imidazo[1,2-a]pyrimidin-4-ium

bromide (BS-070)

N

N

N C13H27

OHBr


1H), 7.76 (m, 2H), 7.46 (m, 3H), 7.31 (m 1H), 4.83 (s, 2H). 3.17 (m, 2H), 1.22 (m,

22H), 0.85 (m, 3H).

2-Hydroxy-2-phenyl-1-tetradecyl-2,3-dihydro-1H-imidazo[1,2-a]pyrimidin-4-ium

bromide (BS-073)

N

N

N C14H29

OHBr


6.27 Hz, 1H), 7.81 (s, 1H), 7.77 (m, 2H), 7.46 (m, 3H), 7.32 (m 1H), 4.84 (s, 2H).

3.17 (m, 2H), 1.23 (m, 24H), 0.85 (m, 3H).

1-Cyclobutyl-2-hydroxy-2-phenyl-2,3-dihydro-1H-imidazo[1,2-a]pyrimidin-4-

ium bromide (BS-079)

N

N

NOH

Br


1H), 7.65(m, 2H), 7.46(m, 3H), 7.34 (m 1H), 4.76 (s, 2H). 3.90 (m, 1H), 2.76 (m,

2H), 1.88 (m, 2H), 1.69 (m, 2H).

2-Hydroxy-1-pentyl-2-phenyl-2,3-dihydro-1H-imidazo[1,2-a]pyrimidin-4-ium

bromide (BS-083)

N

N

N C5H11

OHBr


1H), 7.77 (m, 2H), 7.47 (m, 3H), 7.31 (m 1H), 4.83 (s, 2H). 3.17 (m, 2H), 1.36, (m,

2H), 1.09 (m, 4H), 0.73 (t, J = 6.72 Hz, 3H).

2-(4-Chlorophenyl)-1-cycloheptyl-2-hydroxy-2,3-dihydro-1H-imidazo[1,2-

a]pyrimidin-4-ium bromide (BS-088)

N

N

N

OHBr

Cl


8.85 (m, 1H), 7.92 (s, 1H), 7.79 (d, J = 8.64 Hz, 2H), 7.56 (d, J = 8.58 Hz, 2H), 7.28

(m, 1H), 4.74 (m, 2H). 3.10 (m, 1H), 2.19 (m, 2H), 1.40 (m, 10H).

2-(4-Chlorophenyl)-1-heptyl-2-hydroxy-2,3-dihydro-1H-imidazo[1,2-


N

N

N C7H15

OHBr

Cl


5.85 Hz, 1H), 7.90 (s, 1H), 7.78 (d, J = 8.61 Hz, 2H), 7.55 (d, J = 8.58 Hz, 2H), 7.32

(m, 1H), 4.82 (s, 2H). 3.17 (m, 2H), 1.09 (m, 10H), 0.81 (t, J = 7.17 Hz, 3H).

2-(4-Chlorophenyl)-2-hydroxy-1-octyl-2,3-dihydro-1H-imidazo[1,2-a]pyrimidin-

4-ium bromide (BS-090)

N

N

N C8H17

OHBr

Cl



(m, 1H), 4.82 (m, 2H). 3.17 (m, 2H), 1.09 (m, 12H), 0.83 (t, J = 6.75 Hz, 3H).

2-(4-Chlorophenyl)-1-decyl-2-hydroxy-2,3-dihydro-1H-imidazo[1,2-a]pyrimidin-


N

N

N C10H21

OHBr

Cl


6.0 Hz, 1H), 7.89 (s, 1H), 7.81 (d, J = 8.37 Hz, 2H), 7.55 (d, J = 8.4 Hz, 2H), 7.32 (m,

1H), 4.82 (m, 2H). 3.17 (m, 2H), 1.09 (m, 16H), 0.85 (t, J = 6.51 Hz, 3H). 13C NMR

(75.5 MHz, CDCl3): δ = 167.7, 154.6, 148.2, 137.3, 134.1, 129.0 (×2), 126.3 (×2),

111.1, 90.2, 62.5, 40.7, 31.2, 28.8, 28.6 (×2), 28.2, 27.2, 25.8, 22.0, 13.9.

2-(4-Chlorophenyl)-1-hexyl-2-hydroxy-2,3-dihydro-1H-imidazo[1,2-a]pyrimidin-


N

N

N C6H13

OHBr

Cl


6.3 Hz, 1H), 7.90 (s, 1H), 7.78 (d, J = 8.55 Hz, 2H), 7.56 (m, 2H), 7.33 (t, J = 5.0 Hz,

1H), 4.82 (m, 2H). 3.17 (m, 2H), 1.05 (m, 8H), 0.79 (t, J = 6.30 Hz, 3H).

2-(4-Chlorophenyl)-2-hydroxy-1-nonyl-2,3-dihydro-1H-imidazo[1,2-a]pyrimidin-


N

N

N C9H19

OHBr

Cl


8.86 (m, 1H), 7.89 (s, 1H), 7.78 (d, J = 8.64 Hz, 2H), 7.55 (d, J = 8.58 Hz, 2H), 7.32

(m, 1H), 4.82 (m, 2H). 3.17 (m, 2H), 1.09 (m, 14H), 0.84 (t, J = 6.63 Hz, 3H).

2-(4-Chlorophenyl)-2-hydroxy-1-undecyl-2,3-dihydro-1H-imidazo[1,2-


N

N

N C11H23

OHBr

Cl



(m, 1H), 4.82 (m, 2H). 3.17 (m, 2H), 1.22 (m, 18H), 0.85 (t, J = 6. 3 Hz, 3H). 13C

NMR (75.5 MHz, CDCl3): δ = 167.5, 154.6, 148.2, 137.3, 134.1, 129.0 (×2), 128.3

(×2), 111.1, 90.2, 62.5, 40.7, 31.2, 28.9, 28.8, 28.6 (×2), 28.2, 27.2, 25.8, 22.0, 13.9.

2-(4-Chlorophenyl)-1-dodecyl-2-hydroxy-2,3-dihydro-1H-imidazo[1,2-


N

N

N C12H25

OHBr

Cl


8.87 (d, J = 6.06 Hz, 1H), 7.90 (s, 1H), 7.78 (d, J = 8.64 Hz, 2H), 7.55 (d, J = 8.61

Hz, 2H), 7.32 (m, 1H), 4.82 (m, 2H). 3.17 (m, 2H), 1.22 (m, 20H), 0.85 (t, J = 6. 33

Hz, 3H).

2-(4-Chlorophenyl)-2-hydroxy-1-tridecyl-2,3-dihydro-1H-imidazo[1,2-


N

N

N C13H27

OHBr

Cl


8.87 (d of d, J = 1.77, 6.27 Hz, 1H), 7.90 (s, 1H), 7.78 (d, J = 8.61 Hz, 2H), 7.55 (d, J

= 8.61 Hz, 2H), 7.32 (m, 1H), 4.82 (m, 2H). 3.17 (m, 2H), 1.23 (m, 22H), 0.85 (t, J =

6. 33 Hz, 3H). 13C NMR (75.5 MHz, CDCl3): δ = 167.5, 154.6, 148.2, 137.2, 134.1,

129.0 (×2), 128.3 (×2), 111.2, 90.2, 62.5, 40.7, 31.2, 28.9 (×3), 28.8, 28.6 (×2), 28.2,

27.2, 25.8, 22.0, 13.9.

2-(4-Chlorophenyl)-2-hydroxy-1-tetradecyl-2,3-dihydro-1H-imidazo[1,2-


N

N

N C14H29

OHBr

Cl

Yield: 90 %. 1H NMR (300 MHz, CDCl3): δ = 9.03 (d, J = 4.56 Hz, 1H), 8.87 (d J =


(t, J = 4.95 Hz, 1H), 4.82 (m, 2H). 3.17 (m, 2H), 1.23 (m, 24H), 0.85 (t, J = 6. 09 Hz,

3H).

2-(4-Chlorophenyl)-1-cyclohexyl-2-hydroxy-2,3-dihydro-1H-imidazo[1,2-


N

N

NOH

Br

Cl



= 8.58 Hz, 2H), 7.30 (m, 1H), 4.72 (m, 2H). 3.02 (m, 1H), 2.13 (m, 2H), 0.75 (m, 8H).

2-(4-Chlorophenyl)-2-hydroxy-1-pentyl-2,3-dihydro-1H-imidazo[1,2-


N

N

N C5H11

OHBr

Cl



= 8.61 Hz, 2H), 7.32 (m, 1H), 4.81 (m, 2H). 3.17 (m, 2H), 1.10 (m, 6H), 0.75 (t, J = 6.

81 Hz, 3H).

2-(4-Chlorophenyl)-1-cyclobutyl-2-hydroxy-2,3-dihydro-1H-imidazo[1,2-

a]pyrimidin-4-ium (BS-100)

N

N

NOH

Br

Cl



(t, J = 5.22 Hz, 1H), 4.64 (m, 2H), 3.89 (m, 1H), 2.73 (m, 2H), 1.91 (m, 2H), 1.66 (m,

2H).

2-Hydroxy-2-(4-methoxyphenyl)-1-octyl-2,3-dihydro-1H-imidazo[1,2-


N

N

N C8H17

OHBr

OMe

Yield: 77 %. 1H NMR (300 MHz, CDCl3): δ = 9.02 (m, 1H), 8.84 (d, J = 6.3 Hz, 1H),

7.71 (s, 1H), 7.65 (d, J = 8.79 Hz, 2H), 7.30 (m, 2H), 7.01 (d, J = 8.79 Hz, 1H), 4.79

(s, 2H). 3.79 (s, 3H), 3.17 (m, 2H), 1.10 (m, 12H), 0.83 (t, J = 6.66 Hz, 3H). 13C NMR

(75.5 MHz, CDCl3): δ = 167.4, 159.7, 154.4, 148.2, 129.8, 128.4 (×2), 113.5 (×2),

111.0, 90.5, 62.5, 55.2, 40.5, 31.0, 28.2 (×2), 27.3, 25.8, 22.0, 13.8.

2-(4-Fluorophenyl)-2-hydroxy-1-octyl-2,3-dihydro-1H-imidazo[1,2-a]pyrimidin-


N

N

N C8H17

OHBr

F


8.84 (d of d, J = 1.65, 6.3 Hz, 1H), 7.86 (s, 1H), 7.82 (m, 2H), 7.33 (m, 3H), 4.82 (s,

2H). 3.17 (m, 2H), 1.10 (m, 12H), 0.83 (t, J = 6.72 Hz, 3H). 13C NMR (75.5 MHz,

CDCl3): δ = 167.4, 164.1, 160.8, 154.5, 134.5 (×2), 129.5 (d), 115.3 (d), 111.2, 90.2,

62.5, 40.6, 31.0, 28.2 (×2), 27.3, 25.8, 21.9, 13.8.

2-(3,4-Dichlorophenyl)-2-hydroxy-1-octyl-2,3-dihydro-1H-imidazo[1,2-


N

N

N C8H17

OHBr

Cl

Cl


8.88 (d of d, J = 1.74, 6.27 Hz, 1H), 8.07 (s, 1H), 8.02 (s, 1H), 7.78 (s, 2H), 7.33 (m,

1H), 4.83 (m, 2H). 3.23 (m, 2H), 1.11 (m, 12H), 0.83 (t, J = 6.75 Hz, 3H). 13C NMR

(75.5 MHz, CDCl3): δ = 167.4, 154.6, 148.0, 139.4, 132.1, 131.2, 130.4, 129.4, 127.3,

111.3, 89.6, 62.3, 40.6, 31.0, 28.2 (×2), 27.2, 25.7, 21.9, 13.8.

2-(3-Bromophenyl)-2-hydroxy-1-octyl-2,3-dihydro-1H-imidazo[1,2-a]pyrimidin-


N

N

N C8H17

OHBr

Br


8.87 (d of d, J = 1.62, 6.27 Hz, 1H), 8.01 (s, 1H), 7.93 (s, 1H), 7.77 (d, J = 7.86 Hz,

1H), 7.66 (d, J = 8.4 Hz, 1H), 7.46 (t, J = 7.92 Hz, 1H) 7.33 (m, 1H), 4.82 (m, 2H).

3.22 (m, 2H), 1.36 (m, 2H), 1.11 (m, 10H), 0.83 (t, J = 6.72 Hz, 3H). 13C NMR (75.5

MHz, CDCl3): δ = 167.4, 154.6, 148.1, 140.9, 132.1, 130.4, 129.9, 126.0, 121.7,

111.2, 89.8, 62.5, 40.6, 31.0, 28.2 (×2), 27.2, 25.7, 21.9, 13.8.

2-Hydroxy-2-(naphthalen-1-yl)-1-octyl-2,3-dihydro-1H-imidazo[1,2-a]pyrimidin-


N

N

N C8H17

OHBr


5.25 Hz, 1H), 8.38 (s, 1H), 8.02 (m, 4H), 7.86 (d, J = 7. 61 Hz, 1H), 7.60 (m, 2H),

7.38 (t, J = 5.64 Hz, 1H), 4.99 (s, 2H). 3.26 (m, 2H), 1.39 (m, 2H), 1.00 (m, 10H),

0.76 (t, J = 6.6 Hz, 3H). 13C NMR (75.5 MHz, CDCl3): δ = 167.4, 154.7, 148.3,

135.4, 132.9, 132.1, 128.4, 128.1, 127.5, 127.0, 126.6 (×2), 124.2, 111.2, 90.8, 62.5,

40.7, 30.9, 28.2 (×2), 27.3, 25.8, 21.9, 13.8.

2-Hydroxy-2-(4-nitrophenyl)-1-octyl-2,3-dihydro-1H-imidazo[1,2-a]pyrimidin-4-


N

N

N C8H17

OHBr

NO2

Yield: 75 %. 1H NMR (300 MHz, CDCl3): δ = 9.06 (m, 1H), 8.89 (m, 1H), 8.37 (d, J

= 8.7 Hz, 2H), 8.12 (s, 1H), 8.05 (d, J = 8.76 Hz, 2H), 7.36 (m, 1H), 4.86 (m, 2H).

3.22 (m, 2H), 1.39 (m, 2H), 1.09 (m 10H), 0.81 (t, J = 6.66 Hz, 3H). 13C NMR (75.5

MHz, CDCl3): δ = 167.5, 154.7, 148.2, 147.9, 145.2, 128.7 (×2), 123.0 (×2), 111.4,

90.0, 62.3, 40.8, 30.9, 28.2 (×2), 27.2, 25.7, 21.9, 13.8.

2-Hydroxy-2-(4’-nitrobiphenyl-4-yl)-1-octyl-2,3-dihydro-1H-imidazo[1,2-


N

N

N C8H17

OHBr

NO2


6.2 Hz, 1H), 8.32 (d, J = 8.7 Hz, 2H), 8.03 (d, J = 8.76 Hz, 2H), 7.94 (m, 5H), 7.34 (t,

J = 4.98 Hz, 1H), 4.87 (s, 2H). 3.24 (m, 2H), 1.43 (m, 2H), 1.10 (m 10H), 0.76 (t, J =

6.45 Hz, 3H). 13C NMR (75.5 MHz, CDCl3): δ = 167.5, 154.6, 148.2, 146.9, 145.5,

138.9, 138.4, 127.9 (×3), 127.2 (×2), 124.1 (×3), 111.2, 90.5, 62.6, 40.8, 31.0, 28.2

(×2), 27.3, 25.8, 21.9, 13.8.

2-Hydroxy-2-(4-(methylsulfonyl)phenyl)-1-octyl-2,3-dihydro-1H-imidazo[1,2-


N

N

N C8H17

OHBr

SO2Me

Yield: 48 %. 1H NMR (300 MHz, CDCl3): δ = 9.06 (d of d J = 1.65, 4.47 Hz, 1H),

8.94 (d of d, J = 1.56, 6.21 Hz, 1H), 8.08 (s, 1H), 8.05 (s, 4H), 7.36 (m, 1H), 4.87 (m,

2H). 3.27 (s, 3H), 3.24 (m, 2H), 1.40 (m, 2H), 1.11 (m, 10H), 0.82 (t, J = 6.66 Hz,

3H). 13C NMR (75.5 MHz, CDCl3): δ = 167.4, 154.7, 148.2, 143.7 (×2), 141.6, 128.1

(×2), 127.0, 111.4, 90.3, 62.5, 43.3, 40.9, 31.0, 28.2 (×2), 27.4, 25.9, 21.9, 13.8.

2-Hydroxy-2-(4-(methylthio)phenyl)-1-octyl-2,3-dihydro-1H-imidazo[1,2-


N

N

N C8H17

OHBr

SMe


1H), 7.66 (d, J = 8.52 Hz, 2H), 7.33 (m, 3H), 4.79 (m, 2H). 3.33 (s, 3H), 3.22 (m,

2H), 1.39 (m, 2H), 1.10 (m, 10H), 0.83 (t, J = 6.66 Hz, 3H). 13C NMR (75.5 MHz,

CDCl3): δ = 167.4, 154.7, 148.2, 143.7 (×2), 141.6, 128.1 (×2), 127.0, 111.4, 90.3,

62.5, 43.3, 40.9, 31.0, 28.2 (×2), 27.4, 25.9, 21.9, 13.8.

5-(2,4-Dimethoxyphenyl)-N-methy-1H-imidazol-2-amine (BS-136)

N

NH

HNO

O

Yield : 48 %. 1H NMR (300 MHz, DMSO-d6): δ = 10.63 (br, 1H), 7.43 (d, J = 3.0

Hz, 1H), 7.07 (s, 1H), 6.90 (d, J = 8.8 Hz, 1H), 6.63 (d of d, J = 3.1, 3.1 Hz, 1H), 5.73

(q, J = 5.1 Hz, 1H), 3.80 (s, 3H). 3.71 (s, 3H), 2.77 (d, J= 5.1 Hz, 3H). 13C NMR

(75.5 MHz, DMSO-d6): δ =153.1, 151.0, 149.5, 129.3, 123.4, 114.8, 111.8, 111.2,

110.1, 55.5, 55.2, 29.8. HRMS (EI) C12H15N3O2, calcd 233.1164, found : 233.1173.

N-((Benzol[d][1,3]dioxol-6-yl)-5-(naphthalene-3-yl)-1H-imidazol-2-amine (BS-

137)

N

NH

HN

O

O

Yield : 72 %. 1H NMR (300 MHz, DMSO-d6) δ = 10.61 (br, 1H), 8.06 (s, 1H), 7.83-

7.79 (m, 4H), 7.44-7.37 (m, 2H), 7.19 (s, 1H), 6.99 (s, 1H), 6.87-6.86 (m, 2H), 6.32 (t,

J = 6.4 Hz, 1H), 5.96 (s, 2H), 4.34 (d, J = 6.4 Hz, 2H). 13C NMR (75.5 MHz,

DMSO-d6) δ =151.2, 147.0, 145.8, 134.6, 133.5, 131.3, 127.5 (x 2), 127.4, 127.3,

126.0,125.6, 124.6, 123.2, 120.4, 108.0 (x 2),107.8, 100.6 (x 2), 46.1. HRMS EI

C21H17N3O2, calcd 343.1321 , found : 343.1340.

5-(3,4-Dichlorophenyl)-N-propyl-1H-imidazol-2-amine(BS-140)

N

NH

HNC3H7

Cl

Cl

Yield : 70 %. 1H NMR (300 MHz, DMSO-d6) δ = 10.55 (br, 1H), 7.82 (s, 1H), 7.58

(s, 1H), 7.50 (d, J = 8.2 Hz, 1H), 7.22 (s, 1H), 5.83 (t, J = 6.0 Hz, 1H), 3.10 (q, J = 8.2

Hz, 2H), 1.54 (m, 2H), 0.90 (t, J = 7.35 Hz, 3H). 13C NMR (75.5 MHz, DMSO-d6) δ

=151.6, 130.9 (x 2), 130.3 (x 2), 126.3, 124.6, 123.4, 104.2, 44.7, 22.6, 11.4. HRMS

EI C12H13Cl2N3, calcd 269.0487, found : 269.0480.

N-Cyclohexyl-5-(4-(methylsulfonyl)phenyl)-1H-imidazol-2-amine (BS-141)

N

NH

HN

SO

O

Yield : 43 %. 1H NMR (300 MHz, DMSO-d6) δ = 10.54 (br, 1H), 7.83-7.76 (m, 4H),

7.31 (s, 1H), 5.75 (d, J = 8.4 Hz, 1H), 3.16 (s, 3H), 2.49 (s, 1H), 1.90 (s, 2H), 1.70 (m,

2H), 1.20 (m, 6H). 13C NMR (75.5 MHz, DMSO-d6) δ =151.1, 140.1, 136.1, 127.2(x

4), 123.4 (x 2), 104.2, 51.1, 43.7, 33.0(x 2), 25.4, 24.6(x 2). HRMS EI C16H21N3O2S,

calcd 319.1354. , found : 319.1337.

5-(4-Chlorophenyl)-N-octyl-1H-imidazol-2-amine (BS-143)

N

NH

HNC8H17

Cl

Yield : 43%. 1H NMR (300 MHz, DMSO-d6) δ = 10.43 (br, 1H), 7.63 (d, J = 8.3 Hz,

2H), 7.31 (d, J = 8.4 Hz, 2H), 7.09 (s, 1H), 5.71 (t, J = 4.9 Hz, 1H), 3.12 (m, 2H),

1.48-1.51 (m, 2H), 1.25 (m, 10H), 0.85 (t, J = 4.2 Hz, 3H). 13C NMR (75.5 MHz,

DMSO-d6) δ =151.5, 128.8 (x 2), 128.1 (x 3), 124.9 (x 2), 100.7, 42.9, 31.2, 29.4,

28.8, 28.7, 26.4, 22.0, 13.9. HRMS EI C17H24ClN3, calcd 305.1659, found : 305.1646.

5-(4-Chlorophenyl)-N-pentyl-1H-imidazol-2-amine (BS-146)

N

NH

HNC5H11

Cl

Yield: 57 %. 1H NMR (300 MHz, DMSO-d6) δ = 10.44 (br, 1H), 7.65 (d, J = 7.3 Hz,

2H), 7.31 (d, J = 8.3 Hz, 2H), 7.09 (s, 1H), 5.71 (t, J = 5.1 Hz, 1H), 3.12 (m, 2H),

1.51 (t, J = 1.7 Hz, 2H), 1.30 (m, 4H), 0.88 (t, J = 6.7 Hz, 3H).13C NMR (75.5 MHz,

DMSO-d6) δ =151.5, 128.9 (x 2), 128.1 (x 3), 124.9 (x 2), 104.1, 42.8, 29.1, 28.6,

21.9, 13.9.HRMS EI C14H18ClN3, calcd 263.1189. , found: 263.1180.

5-(4-Fluorophenyl)-N-hexyl-1H-imidazol-2-amine (BS-147)

N

NH

HNC6H13

F

Yield: 61 %. 1H NMR (300 MHz, DMSO-d6) δ = 10.38 (br, 1H), 7.62 (m, 2H), 7.09

(t, J = 8.8 Hz, 2H), 6.99 (s, 1H), 5.66 (m, 1H), 3.13-3.10 (m, 2H), 1.51 (m, 2H), 1.28

(m, 6H), 0.87 (t, J = 6.4 Hz, 3H).13C NMR (75.5 MHz, DMSO-d6) δ =161.6, 158.5,

151.4, 125.0, 124.9, 115.0, 114.7, 104.1 (x 2), 42.9, 31.0, 29.4, 26.1, 22.1, 13.8.,

HRMS EI C15H20FN3, calcd 261.1641. , found: 261.1631.

N-(4-Methoxyphenethyl)-5-(3-bromophenyl)-1H-imidazol-2-amine(BS-148)

N

NH

HNBr

O

Yield : 44 %. 1H NMR (300 MHz, DMSO-d6) δ = 10.56 (br, 1H), 7.82 (s, 1H), 7.65

(d, J = 6.5 Hz, 2H), 7.24-7.16 (m, 5H), 6.88 (d, J = 8.4 Hz, 2H), 5.77 (m, 1H), 3.72 (s,

3H), 3.35 (s, 2H), 2.77 (t, J = 7.6 Hz, 2H). 13C NMR (75.5 MHz, DMSO-d6) δ

=157.5, 151.3, 131.6 (x 2), 130.3, 129.6 (x 3), 127.3, 125.7, 122.1, 121.9, 113.6 (x 3),

54.9, 44.8, 34.6.,HRMS EI C18H18BrN3O, calcd 371.0633. , found : 371.0658.

N-Cyclooctyl-5-(4-(methylsulfonyl)phenyl)-1H-imidazol-2-amine (BS-154)

N

NH

HN

SO2Me

Yield : 53 %. 1H NMR (300 MHz, DMSO-d6) δ = 10.49 (br, 1H), 7.86 (d, J = 8.5 Hz,

2H), 7.79 (d, J = 8.5 Hz, 2H), 7.31 (d, J = 1.5 Hz, 1H), 5.74 (d, J = 8.4 Hz, 1H), 3.64

(s, 1H), 3.16 (s, 3H), 1.80 (m, 2H), 1.53 (m, 12H). 13C NMR (75.5 MHz, CDCl3) δ =

151.5, 138.8, 136.2, 127.7 (x 3), 124.0 (x 2), 105.1, 53.7, 44.6, 32.3 (x 2), 27.2 (x 2),

25.2, 23.4 ( x 2). HRMS EI C18H25N3O2S, calcd 344.1667. , found : 344.1644.

4-(2-(2-Methoxyethylamino)-1H-imidazole-5-yl)benzonitrile(BS-139)

N

NHN

H

ON

Yield 85 %. 1H NMR (300 MHz, DMSO d6) δ = 10.6 (br, 1H), 7.7 (m 4H), 7.3 (s 1H),

5.8 (s 1H), 3.5 (t 2H), 3.4 (m 2H), 3.2 (s 3H). 13C NMR (300 MHz, DMSO d6) δ

=151.3, 140.1, 134.2, 132.2(x 2), 123.8, 119.5, 110.8, 106.4, 104.1, 70.9, 57.9, 54.8,

43.3. HRMS EI C13H14N4O, calcd 242.1168 , found : 242.1167.

5-(4-Methoxyphenyl)-N-octyl-1H-imidazol-2-amine (BS-258 )

N

NH

NH

C8H17

O

Yield : 78 %. 1H NMR (300 MHz, DMSO-d6): δ = 10.91 (br s, 1H), 7.51 (d, J = 8.57

Hz, 2H), 6.95 (s, 1H), 6.87 (d, J = 8.75 Hz, 2H), 6.05 (m, 1H), 3.74 (s, 3H), 3.16 (m,


CDCl3): δ =149.2, 131.1, 129.5, 129.3 (×2), 128.8 (×2), 128.2, 123.4, 43.1, 31.6, 29.5,

29.0, 28.9, 26.4, 22.2, 14.0. HRMS (EI): C18H27N3O, calcd 301.2154, found:

301.2142.

5-(4-Fluorophenyl)-N-octyl-1H-imidazol-2-amine (BS-259)

N

NH

NH

C8H17

F

Yield : 69% . 1H NMR (300 MHz, DMSO-d6): δ = 10.44 (br s, 1H), 7.61 (t, J = 5.85

Hz, 2H), 7.08 (t, J = 8.78 Hz, 2H), 6.99 (s, 1H), 5.67 (t, J = 6.03 Hz, 1H), 3.10 (m,

2H), 1.51 (m, 2H), 1.25 (m, 10H), 0.83 (m, 3H). 13C NMR (75.5 MHz, CDCl3): δ

=163.8, 160.5, 148.7, 130.2, 128.4, 127.0, 122.9, 115.5, 104.7, 43.0, 31.6, 29.5, 29.0,

28.9, 26.4, 22.5, 14.0. HRMS (EI): C17H24FN3, calcd 289.1954, found:. 289.1964.

5-(3,4-Dichlorophenyl)-N-octyl-1H-imidazol-2-amine (BS-260)

N

NH

NH

C8H17

Cl

Cl

Yield : 72 %. 1H NMR (300 MHz, DMSO-d6): δ = 10.52 (br s, 1H), 7.82 (s, 1H),

7.58 (m, 1H), 7.50 (d, J = 8.13 Hz, 1H), 7.22 (s, 1H), 5.78 (m, 1H), 3.11 (m, 2H), 1.51

(m, 2H), 1.28 (m, 10H), 0.85 (t, J = 6.94 Hz, 3H). 13C NMR (75.5 MHz, CDCl3): δ

=149.6, 132.8, 131.1, 131.0, 130.6, 129.5, 127.1, 127.0, 124.2, 43.3, 31.6, 29.5, 29.0,

28.9, 26.4, 22.5, 14.0. HRMS (EI): C17H23Cl2N3, calcd 339.1269, found:. 339.1279.

5-(3-Bromophenyl)-N-octyl-1H-imidazol-2-amine (BS-261)

N

NH

NH

C8H17Br


7.59 (s, 1H), 7.22 (m, 1H), 7.14 (s, 1H), 5.73 (t, J = 5.66 Hz, 1H), 3.11 (m, 2H), 1.51


=149.2, 133.0, 130.8, 130.1 (×2), 128.0, 126.5, 123.8, 122.6, 43.2, 31.6, 29.5, 29.0,

28.9, 26.4, 22.5, 14.0. HRMS (EI): C17H24BrN3, calcd 349.1154, found:. 249.1138.

5-(Naphthalen-1-yl)-N-octyl-1H-imidazol-2-amine (BS-262)

N

NH

NH

C8H17


7.80 (m, 3H), 7.39 (m, 3H), 7.17 (s, 1H), 5.73 (m, 1H), 3.16 (m, 2H), 1.54 (m, 2H),

1.26 (m, 10H), 0.83 (t, J = 6.86 Hz, 3H). 13C NMR (75.5 MHz, CDCl3): δ =149.1,

133.4, 132.4, 129.5, 128.3, 128.2, 127.9, 127.7, 126.6, 126.4 (×2), 126.0, 123.0, 43.3,

31.6, 29.5, 29.0, 28.9, 26.4, 22.5, 14.0. HRMS (EI): C21H27N3, calcd 321.4592,

found:. 321.4578.

5-(4-Nitrophenyl)-N-octyl-1H-imidazol-2-amine (BS-263)

N

NH

NH

C8H17

NO2


Hz, 2H), 7.84 (d, J = 8.92 Hz, 2H), 7.43 (s.1H), 5.91 (t, J = 5.54 Hz, 1H), 3.16 (m,


CDCl3): δ =163.8, 160.5, 148.7, 130.2, 128.4, 127.0, 122.9, 115.5, 104.7, 43.0, 31.6,

29.5, 29.0, 28.9, 26.4, 22.5, 14.0. HRMS (EI): C17H24N4O2, calcd 316.1899,

found:.316.1909.

5-(4’-Nitrobiphenyl-4-yl)-N-octyl-1H-imidazol-2-amine (BS-264)

N

NH

NH

C8H17

NO2


Hz, 2H), 7.96 (d, J = 8.71 Hz, 2H), 7.75 (s.4H), 7.18 (s. 1H), 5.76 (t, J = 5.88 Hz,

1H), 3.16 (m, 2H), 1.53 (m, 2H), 1.26 (m, 10H), 0.86 (t, J = 6.75 Hz, 3H). 13C NMR

(75.5 MHz, CDCl3): δ =163.8, 160.5, 148.7, 130.2, 128.4, 127.0, 122.9, 115.5, 104.7,

43.0, 31.6, 29.5, 29.0, 28.9, 26.4, 22.5, 14.0. HRMS (EI): C23H28N4O2, calcd

392.2212, found:.392.2201.

5-(4-(Methylsulfonyl)phenyl)-N-octyl-1H-imidazol-2-amine (BS-265)

N

NH

NH

C8H17

S

O

O


Hz, 2H), 7.76 (d, J = 8.35 Hz, 2H), 7.32 (s.1H), 5.83 (t, J = 5.97 Hz, 1H), 3.16 (m,

5H), 1.52 (m, 2H), 1.25 (m, 10H), 0.85 (t, J = 6. 56 Hz, 3H). 13C NMR (75.5 MHz,

CDCl3): δ =150.7, 138.1, 136.6, 127.9 (×2), 127.6, 127.4 (×2), 126.0, 44.5, 43.6, 31.6,

29.6, 29.0, 28.9, 26.5, 22.5, 14.0. HRMS (EI): C18H27N3O2S, calcd 349.4909,

found:.249.4918.

N-Isobutyl-5-(4-nitrophenyl)-1H-imidazol-2-amine (BS-292)

N

NH

NH NO2


Hz, 2H), 7.84 (d, J = 8.50 Hz, 2H), 7.42 (s, 1H), 5.99 (t, J = 5.93 Hz, 1H), 2.99 (t, J =

6.44 Hz, 2H), 1.83 (m, 1H), 0.89 (d, J = 6.70 Hz, 6H). 13C NMR (75.5 MHz, DMSO-

d6): δ =152.2 (×2), 143.8, 141.6, 123.9 (×2), 123.3, 104.1 (×2), 50.4, 27.9, 20.1 (×2).

HRMS (EI): C13H16N4O2, calcd 260.1273, found: 260.1287

N-Isobutyl-5-phenyl-1H-imidazol-2-amine (BS-293)

N

NH

NH


Hz, 2H), 7.28 (t, J = 7.47 Hz, 2H), 7.09 (t, J = 7.25 Hz,1H), 7.04 (s,1H), 5.92 (t, J =

5.71 Hz, 1H), 2.98 (t, J = 6.37 Hz, 2H), 1.82 (m, 1H), 0.89 (d, J = 6.59 Hz, 6H). 13C

NMR (75.5 MHz, DMSO-d6): δ =151.2, 134.0, 133.1, 128.2 (×2), 125.1, 123.4(×2),

104.1, 50.5, 27.9, 20.1 (×2). HRMS (EI): C13H17N3, calcd 215.1422, found:215.1411.

5-(4-Bromophenyl)-N-isobutyl-1H-imidazol-2-amine (BS-294)

N

NH

NH Br




d6): δ =151.6, 134.1, 133.0 (×3), 125.3 (×2), 117.2, 104.1, 50.6, 27.9, 20.1 (×2).

HRMS (EI): C13H16BrN3, calcd 293.0528, found:293.0529.

5-(4-Chlorophenyl)-N-isobutyl-1H-imidazol-2-amine (BS-295)

N

NH

NH Cl




d6): δ =151.5, 133.6, 133.2, 128.9, 128.0 (×2), 124.9 (×2), 104.1, 50.5, 27.9, 20.1

(×2). HRMS (EI): C13H16ClN3, calcd 249.1033, found:249.1047.

2.11 Representative NMR spectra

2.11.1 1H and 13C NMR spectra of bs-026

1.85

47

0.94

85

0.88

93

1.97

02

2.60

79

8.30

61

3.00

00

Inte

gral

8.27

478.

2590

7.26

29

6.51

666.

5003

6.48

46

5.07

93

3.42

603.

4028

3.38

343.

3790

3.35

95

1.63

161.

6102

1.58

701.

3698

1.35

221.

3403

1.30

581.

2876

0.90

350.

8959

0.88

150.

8583

0.00

03

(ppm)

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5

N

N

NH

C7H15

162.

5113

157.

9984

110.

1675

77.5

117

77.0

862

76.6

644

41.5

103

31.8

118

29.6

081

29.0

662

26.9

680

22.6

114

14.0

875

(ppm)

0102030405060708090100110120130140150160170

N

N

NH

C7H15


0.97

79

0.94

65

0.95

15

1.99

10

1.92

42

0.96

36

1.90

70

1.76

11

16.2

14

3.00

00

Inte

gral

9.04

769.

0376

8.88

828.

8687

7.89

777.

8105

7.78

167.

5826

7.55

447.

3454

7.32

847.

3084

4.82

03

3.33

093.

2179

3.19

593.

1689

3.15

20

2.50

24

1.20

251.

0976

0.87

480.

8535

0.83

09

(ppm)

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5

N

N

N C10H21

OHBr

Cl

167.

5253

154.

6121

148.

2155

137.

3205

134.

1059

129.

0584

128.

3529

111.

2795

90.2

424

62.5

177

40.7

023

31.2

220

28.6

219

28.2

364

27.2

800

25.8

145

22.0

544

13.9

231

(ppm)

0102030405060708090100110120130140150160170

N

N

N C10H21

OHBr

Cl


0.74

29

1.71

33

2.00

12

0.79

88

1.34

79

2.06

69

1.98

83

10.1

74

3.00

00

10.4

329

7.65

497.

6273

7.31

227.

2845

7.09

69

5.71

73

3.12

883.

1068

2.50

24

2.08

63

1.51

131.

4893

1.25

84

0.85

790.

8353

-0.0

001

(ppm)

0.01.02.03.04.05.06.07.08.09.010.0

N

NH

HNC8H17

Cl

151.

5720

128.

8948

128.

1056

124.

9601

100.

7882

42.9

279

31.2

511

29.4

365

28.8

183

28.7

164

26.4

691

22.0

835

13.9

304

(ppm)

0102030405060708090100110120130140150

N

NH

HNC8H17

Cl

2.12 References

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3590.

[2] Kobayashi J, Ohizumi Y, Nakamura H, Hirata Y, (1986) Tetrahedron 42, 1176-1177.

[3] (a) Bedoya Zurita M, Ahond A, Poupat C, Potier P (1989) Tetrahedron. 45, 6713-6720. (b)

Gross H, Kehraus S, Koenig GM, Woerheide G, Wright AD. (2002) J. Nat. Prod. 65, 1190-

1193 (c) Hassan W, Edrada R, Ebel R, Wray V, Berg A, Van Soest R, Wiryowidagdo S,

Proksch P. (2004) J. Nat. Prod. 67, 817-822.

[4] (a) Copp BR, Fairchild CR, Cornell L, Casazza AM, Robinson S, Ireland CM. (1998) J. Med.

Chem. 41, 3909-3911. (b) Colson G, Raboult L, Lavelle F, Zeaial A. (1992) Biochem.

Pharmacol. 43, 1717-1723. (c) Pitts WJ, Wityak J, Smallheer JM, Tobin AE, Jetter JW,

Buynitsky JS, Harlow PP, Solomon KA, Corjay MH, Mousa SA, Wexler R, Jadhav PK.

(2000) J. Med. Chem. 43, 27-40. (d) Batt DG, Petraitis JJ, Houghton GC, Modi DP, Cain GA,

Corjay MH, Mousa SA, Bouchard PJ, Forsythe MS, Harlow PP, Barbera FA, Spitz SM,

Wexler RR, Jadhav. (2000) J. Med. Chem. 43, 41-58.

[5] (a) Little TL, Webber SE. (1994) J. Org. Chem. 59, 7299-7305. (b) Abou Jneid R, Ghoulami

S, Martin MT, Dau ETH, Travert N, Al-Mourabit A. (2004) Org. Lett. 6, 3933-3936. (c) Gore

VK, Ma VV, Norman MH, Ognyanov VI, Xi N. (2006) US Patent, US 2006/0084640

[6] Stamford AW, Craig CD, Huang Y. (2001) Int. Pat. WO 01/44201

[7] (a) Lamattina JL, Mularski CJ (1984)Tetrahedron Lett. 25, 2957-2960 (b) Lamattina JL.

(1985) Eur. Pat. Appl. EP 138464 A2.

[8] Katritzky AR, Rogovoy B (2005) Arkivoc. Part Iv, 49-87 and references herein.

[9] Beyer H, Pyl T, Lahmer H. (1961) Chem. Ber. 94, 3217.

[10] Thangavel N, Murgesh K. (2005) Asian J. Chem. 17, 2769.

[11] Ermolat’ev DS, Giménez V N, Babaev EV, Van der Eycken E. (2006) J. Comb. Chem. 8, 659-

663.

[12] Ermolat’ev DS, Svidritsky EP, Babaev EV, Van der Eycken E. (2009) Tetrahedron Lett. 50,

5218-5220.

[13] Ermolat’ev D. S., Savaliya B, Shah A, Van der Eycken E, (2010) Molecular Diversity.

accepted.

[14] Ashry EI, Kilany Y, Rashed N, Assafi H. (1999) Adv. Heterocycl. Chem. 75, 79;

[15] Brown D. J. “Mechanisms of Molecular Migrations”, Vol. 1, Thyagarajan, B. S. (1968) (Ed.),

Wiley-Interscience, New York, pp. 209.

[16] Ermolat’ev, D. S. and Van der Eycken, E. (2008) J. Org. Chem. 73, 6691.

[17] Costerton, J. W, Stewart P. S, Greenberg,E. P. (1999) Science 284, 1318-22.

[18] Stewart P. S, Costerton J. W. (2001) Lancet. 358, 135-8.

[19] Donlan R. M, Costerton J. W. (2002) Clin Microbiol Rev. 15, 167-93.

[20] Mah T. F, Pitts B, Walker G. C, Stewart P. S, O'Toole G. A. (2003) Nature 426, 306-10.

[21] Matz C, McDougald D, Moreno A. M, Yung P. Y, Yildiz F. H, Kjelleberg S. B. (2005) Proc

Nat. Acad. Sci. USA. 102, 16819-24.

[22] Davies D. U. (2003) Nat Rev Drug Discov. 2, 114-22.

[23] Olson M. E, Ceri H, Morck D. W, Read R. R. (2002) J. Vet. Res. 66, 86-92.

[24] Blanke S. R, Yang R. (2002) Enviromental Institute of Houston

[25] Musk D. J, Hergenrother P. (2006) J. Curr. Med. Chem. 13, 2163-77.

[26] De Nys R, Kumar N, Kjelleberg S, Steinberg P. D. (2006) Prog Mol Subcell Biol. 42.

[27] Janssens J. C, Steenackers H, Robijns S, Gellens E, Levin J, Zhao H, Hermans K, De Coster

D, Verhoeven T. L, Marchal K, Vanderleyden J, De Vos D. E, De Keersmaecker S. C. (2008)

Appl Environ Microbio. 74, 6639-48.

[28] Geske G. D, Siegel A. P, Blackwell H. E. (2005) J. Am. Chem. Soc. 127, 12762-3.

[29] Huigens R. W, Richards J. J, Parise G, Ballard T. E, Zeng W, Deora R, Melander C. (2007) J.

Am. Chem. Soc. 129, 6966-7.

[30] Richards J. J, Ballard T. E, Melander C. (2008) Org. Biomol. Chem. 6, 1356-63.

[31] Richards J. J, Huigens Iii R. W, Ballard T. E, Basso A, Cavanagh J, Melander C. (2008)

Chem. Commun. (Camb). 1698-700.

[32] Huigens R. W, Ma L, Gambino C, Moeller P. D, Basso A, Cavanagh J, Wozniak D. J,

Melander C. (2008) Mol. Biosyst. 4, 614-21.

[33] Richards J. J, Ballard T. E, Huigens R. W, Melander C. (2008) Chem. bio. chem. 9, 1267-79.

[34] Rogers S. A, Melander C. (2008) Angew. Chem. Int. Ed. 47, 5229-31.

[35] Ballard T. E, Richards J, Wolfe A. L, Melander C. (2008) Chemistry. 14, 10745-61.

[36] Sullivan J. D, Giles R. L. (2009) Current Bioactive Compounds. 5, 39-78.

[37] Prouty A. M, Schwesinger W. H, Gunn J. S. (2002) Infect Immun. 70, 2640-9.

[38] Barak J. D, Charkowski A. O. (2002) Appl. Environ Microbiol. 68, 4758-63.

[39] Brandl M. T, Mandrell R. E. (2002) Appl. Environ Microbiol. 68, 3614-21.

[40] Boddicker J. D, Ledeboer N. A, Jagnow J, Jones B. D, Clegg S. (2002) Mol Microbiol. 45,

1255-65.

[41] Romling U, Sierralta W. D, Eriksson K, Normark S. (1998) Mol Microbiol. 28, 249-64.

[42] Latasa C, Roux A, Toledo-Arana A, Ghigo J. M, Gamazo, C, Penades J. R, Lasa I. (2005)

Mol. Microbiol. 58, 1322-39.

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[44] Govan J. R, Deretic V. (1996) Microbiol Rev. 60, 539-74.

[45] Wagner V. E, Iglewski B. H. P. (2008) Clin. Rev. Allergy Immunol. 35, 124-34.

[46] Gomez M. I, Prince A. (2007) Curr. Opin. Pharmacol. 7, 244-51.

[47] Ghannoum M, Abu Elteen K, el-Rayyes N. R. (1989) Microbios. 60, 23-33.

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(1995) Eur. J. Med. Chem. 30, 123-132.

Structure Activity Relationship of N1-

Substituted 2-Aminoimidazoles as Inhibitors of

Biofilm Formation by Salmonella Typhimurium

and Pseudomonas aeruginosa

Chapter-3

Structure Activity Relationship of N1-Substituted 2-Aminoimidazoles as Inhibitors of Biofilm Formation by Salmonella Typhimurium and

Pseudomonas aeruginosa

N

NH2N

R1R2

A library of N1-substituted 4(5)-phenyl-2-aminoimidazoles was synthesized and

tested for the antagonistic effect against biofilm formation by Salmonella

Typhimurium and Pseudomonas aeruginosa. The substitution pattern of the 4(5)-

phenyl group and the nature of the N1-substituent were found to have a major effect

on the biofilm inhibitory activity. The most active compounds of this series were

shown to inhibit the biofilm formation at low micromolar concentrations.

Publication:

[1] Structure Activity Relationship of 4(5)-Phenyl-2-amino-1H-imidazoles, N1-

Substituted 2-Aminoimidazoles and Imidazo[1,2-a]pyrimidinium Salts as

Inhibitors of Biofilm Formation by Salmonella Typhimurium and

Pseudomonas aeruginosa. J. Med.Chem. (2010) DOI: 10.1021/jm1011148

3.1 Introduction

In previous chapter-2 the dimorth rearrangement was performed to generate

substituted 2-aminoimidazoles. The 2-aminoimidazole (2-AI) ring structure is of

particular interest especially within the realms of medicinal chemistry. For example,

several classes of marine natural products possessing this structure were recently

discovered and identified.l Many of these compounds display a broad range of

biological properties including antibacterial, anti-inflammatory, anticancer, and

antiviral activity. Synthetic 2-AI derivatives, including 2-amino-histamine have been

shown to have HI- and H2-receptor agonist and antagonist activity.2 Other unrelated

2-AI derivatives are selective 5-HT3 receptor antagonists, which may be

potentially useful in the treatment of chemotherapy induced emesis3. Novel

cephalosporins with incorporated 2-AI rings display both Gram-positive and Gram-

negative antibacterial activity as well as good ß-lactamase stability4. In addition, 2-AI

can serve as an important starting material in the preparation of 2-nitroimidazole

(azomycin) a naturally occurring antibiotic5. Other synthetic analogs of 2-

nitroimidazole have been found to be cytotoxic toward hypoxic tumor cells and act as

hypoxic cell radiosensitizers.6

The methods reported to prepare imidazoles are numerous, although only a limited

number describe the direct synthesis of the corresponding 2-amino derivatives. The

preparation of 2-AI was accomplished by the intramolecular cyclization of N-(2,2-

diethoxyethyl)guanidine upon acid hydrolysis.7,8 The reaction of α-amino aldehydes

or ketones with cyanamide appears to be the most commonly used direct approach.8,9

However, this reaction is pH sensitive and can lead to different products. In one

case the formation of lH-imidazo[l,2-a]imidazoles was observed.8 The dimerization

and cyclization of a-amino aldehydes or ketones to symmetrical pyrazines is also a

common problem associated with this method.9a,10 A classical, although indirect

approach to 2-AI's relies upon the formation of 2-arylazo derivatives via diazonium

coupling, followed by reduction.2a,7,11 The synthesis of 2-amino-4,5-diarylimidazoles

was accomplished by the reduction of symmetrical 2,2'-azoimidazoles.13 A more

recent procedure was reported where 2-AI's were formed by the reaction of α -

diketones with guanidine, followed by catalytic hydrogenation.14 Other indirect

approaches to 2-AI's involve ipso type substitutions of appropriate C-2 substituted

imidazoles, where the addition of a suitable nitrogen nucleophile is followed by the

elimination of an activated leaving group such as a halogen or alkyl sulfoxide or

sulfone. This method has been applied with limited success to electron-poor

imidazoles or when fluorine is the leaving group.4,15 An interesting synthetic route to

functionalized 2-AI's, which were difficult to obtain by other methods, has been

achieved using a base catalyzed rearrangement of 3-amino-1,2,4-oxadiazoles.16

Another type of heterocyclic rearrangement involves the reaction of 2-aminooxazoles

with ammonia or formamide.17 Most recently, the synthesis of 2-amino-4(5)-(α-

hydroxyalkyl)imidazoles has been demonstrated where 2-AI was reacted with

aldehydes under neutral conditions.18

The microwave-assisted procedure for the synthesis of 1,4,5-substituted 2-

aminoimidazoles (Scheme-1) is two-step protocol based on the cyclocondensation of

2-aminopyrimidines and bromocarbonyl compounds at 130-150 °C, followed by the

cleavage of the intermediary imidazo[1,2-a]pyrimidin-1-ium salts with an excess of

hydrazine.

3.2 Reaction Scheme

Scheme-1

aq. HClO4

N

N

N R1

R2

OHBrN

N

NH

N

NH2N

R2 N

N

N R1

R2

PPA, 150 °C

ClO464% N2H4

R1

H

Br O

R2

+

100 °C, 10 min

MeCN

MW

MeCN

80 °C, 30 min

MW

R1

3.3 Proposed Mechanism

A possible mechanism for the pyrimidine ring cleavage upon reaction of salts (1) with

amines, which involves the formation of azabutadienes (Scheme 2), Initially, the

imidazo[1,2-a]pyrimidinium salt (1) undergoes a nucleophilic attack at its C-5

position by a first molecule of amine. After rearrangement of the intermediate (A) and

isomerisation of the resulting (B), (E, E)-azabutadiene (2) is formed. Initiated by

protonation, the azabutadiene intermediate (C) in turn undergoes 1,4-addition with a

second molecule of amine, finally resulting in the formation of 2-aminoimidazole (3)

together with diazapentadienium salt (4) (Scheme-2).

Consistent with this mechanism, the corresponding imidazo[1,2-a]pyrimidinium salts

(1) can directly be cleaved to 2-aminoimidazoles (3) upon stirring with a large excess

of secondary amine (6 or more equiv) at room temperature, while the isolated

azabutadienes (2) appeared to be stable to 20 and more equivalents of piperidine in

refluxing acetonitrile for 1 h. In contrary, azabutadienes (2) were quantitatively

cleaved to 2-aminoimidazoles (3) in the presence of 1.5 equiv triethylamine

hydrochloride salt (Scheme-3.5).

Scheme-2

N

N

N R1

R2

N

N

N R1

R2

R'2NR'2NH

N

N

N R1

R2

R'2N

N

NN

R1

R2

R'2N

N

NHN

R1

R2

R'2N

HN

NHN

R1

R2

R'2N

R'2NH R'2NH N

N

R1

H2NR2

+ NR'2

NR'2

H-

BA

C D

12

3 4

3.4 Result and Discussion

It was found that the cyclocondensation of 2-alkylaminopyrimidines with α-

bromoacetophenones at 80 °C first led to stable 2-hydroxy-2,3-dihydroimidazo[1,2-

a]pyrimidinium bromides. The bicyclic nature of the structures clearly followed from 1H NMR data, indicating the doublet of doublets of the methylene fragments, as well

as from 13C NMR spectra.

The 2-hydroxy-2,3-dihydroimidazo[1,2-a]pyrimidinium salts were obtained in good

yields, and only for sterically hindered 2-alkylaminopyrimidines the yields slightly

decreased. Dehydration of the 2-hydroxy-2,3-dihydroimidazo[1,2-a]pyrimidinium

salts was successfully achieved by short (15 min) heating in polyphosphoric acid

(PPA) at 150 °C. The corresponding imidazo[1,2-a]pyrimidinium salts were isolated

and characterized as perchlorates, and in most cases the yields were almost

quantitative. The structures of aromatic salts were completely consistent with their 1H,

and 13C NMR data.

The cleavage step was performed under microwave irradiation at 100 °C, using 7

equivalents of hydrazine hydrate. Resulting 2-amino-1H-imidazoles were obtained in

high yields. Using this protocol we have synthesized library of substituted 2-

aminoimidazole (Table-1)

N

NH2N

R1R2

No. Compound

Code R1 R2 Yield % m.p. oC

1 BS-042 cyclododecane H 53 228-230

2 BS-045 cyclooctane H 53 152-154

3 BS-048 cycloheptane H 54 180-182

4 BS-078 cyclohexane H 64 153-155

5 BS-081 cyclobutane H 47 77-79

6 BS-085 C5H11 H 53 123-125

7 BS-060 C6H13 H 53 -

8 BS-051 C7H15 H 61 -

9 BS-054 C8H17 H 55 -

10 BS-063 C9H19 H 58 -

11 BS-057 C10H21 H 53 -

12 BS-066 C11H23 H 52 -

13 BS-069 C12H25 H 52 -

14 BS-072 C13H27 H 58 44-46

15 BS-075 C14H19 H 64 44-46

16 BS-116 cyclododecane Cl 42 221-223

17 BS-117 cyclooctane Cl 55 207-209

18 BS-118 cycloheptane Cl 51 180-182

19 BS-128 cyclohexane Cl 37 179-181

20 BS-130 cyclobutane Cl 40 156-158

21 BS-129 C5H11 Cl 38 96-98

22 BS-122 C6H13 Cl 53 76-78

23 BS-119 C7H15 Cl 54 80-82

24 BS-120 C8H17 Cl 57 76-78

25 BS-123 C9H19 Cl 50 76-78

26 BS-121 C10H21 Cl 52 89-91

27 BS-124 C11H23 Cl 57 78-80

28 BS-125 C12H25 Cl 57 79-81

29 BS-126 C13H27 Cl 56 83-85

30 BS-127 C14H19 Cl 51 82-83

Yields are isolated yields over 2 steps.

Table-1: compound library of substituted 2-aminoimidazole

N

NH2N

C8H17

R1

No. Compound

Code R1 Yield %

1 BS-199 4-OCH3 56

2 BS-200 4-F 71

3 BS-201 3,4-di Cl 64

4 BS-202 3-Br 68

5 BS-203 Naphthalen-1-yl 59

6 BS-204 4-NO2 66

7 BS-205 4-(4-NO2-Ph) 52

8 BS-206 4-SO2Me 61

9 BS-207 4-SMe 40

10 BS-208 4-(4-biphenyl)_ 31

Yields are isolated yields over 2 steps.

Table -2: 2-aminoimidazole with octyl substitution at N-1 position

3.5 Biofilm Activity

The preliminary results from our lab the basic compound (A) with a non-substituted

phenyl group at the C(5) position of the imidazole ring shows a moderate biofilm

inhibitory activity (IC50 = 130 µM). Interestingly, substitution of the para- position of

the phenyl ring with a chlorine (B) atom enhances the biofilm inhibitory activity

about ten times (IC50 = 16 µM).

N

NH

H2NCl

N

NH

H2N

A B

In an attempt to improve their biofilm inhibitory activity, we substituted the N1-

position of some of the para-substituted 4(5)-phenyl-2-aminoimidazoles with n-alkyl

and cyclo-alkyl groups by using our previously established chemistry.

3.5.1 N1-alkyl 2-aminoimidazoles

In first instance a broad variety of alkyl groups, with lengths ranging from one carbon

atom to 14 carbon atoms, were introduced at the N1-position of the 4(5)-

monosubstituted 2-aminoimidazoles (A) and (B) resulting in compounds A-1 to A-

14, and B-1 to B-13 respectively (Table-3). Each compound was assayed for the

ability to inhibit S. Typhimurium ATCC14028 biofilm formation at 25 °C. As

depicted in figure 1, a clear correlation was found between the length of the alkyl

substituent and the biofilm inhibitory activity. In general, compounds with a short

alkyl chain substitution (C1, C2, C3) on the N1-position have a lower activity as

compared to their respective unsubstituted counterparts. This effect is most

pronounced for the methyl substituent, which causes a reduction in the activity of at

least 4 times. To a lesser extent, ethyl and isopropyl substituents also cause a

reduction in activity. Substitution with a butyl group does not have an unequivocal

effect. In the case of compound A-4, the activity is enhanced three-fold, while in the

other cases the activity is reduced. Remarkably, all the compounds substituted with

an alkyl group with an intermediate length between 5 and 10 carbon atoms are very

active biofilm inhibitors, with IC50 values in general below 12 µM. The effect of

substitution with a longer alkyl chain drastically depends on the nature of the

substituent on the C5-position of the ring (phenyl or p-chlorophenyl). Indeed,

replacement of the N1-hydrogen of compound (A) (phenyl on C5 of the ring) by a

long alkyl chain (C11-C14) raises the activity drastically, from an IC50 of 130 µM for

compound (A) to values below 8 µM for the substituted compounds (A-11 to A-14).

In the case of compound (B) (p-chlorophenyl on C5 of the ring), substitution with an

undecyl (B-10) and dodecyl (B-11) group results in very active compounds IC50

values below 5 µM), while introduction of a tridecyl group (B-12) does not have a

clear effect on the biofilm inhibitory activity and introduction of a tetradecyl (B-13)

group drastically reduces the activity (IC50 = 453 µM). Subsequently, we investigated

the influence of some of the most active representatives of this class of compounds on

the planktonic growth curve of S. Typhimurium at 25°C. Most compounds only show

a very small concentration range with a specific effect on biofilm formation and no

effect on the planktonic growth (Table-3). Therefore we cannot exclude the possibility

that the biofilm inhibitory effect of these compounds (at the higher concentrations) is

partially due to a reduction of the planktonic growth of the bacteria in the growth

medium used in the set-up to monitor biofilm formation. Interestingly, compound A-

14 with a long alkyl chain, does not follow this general trend as no effect on the

growth was observed at 80 µM while the biofilm formation was inhibited at 9 µM.

As depicted in table-3 and figure 1, a similar structure activity relationship was found

for the inhibition of P. aeruginosa biofilm formation at 25°C. Again, introduction of

a short alkyl chain at the N1-position generally results in a decreased activity (except

for compound (A-2), while all the compounds with an intermediate alkyl chain length

show very strong activities. The effect of substitution with a long alkyl chain is in this

case also dependent on the nature of the C5-subsitutent. In the case of compound (A),

substitution with a long alkyl chain results in very active compounds (A-10 to A-14),

The activity of compound (B) is also decreased after introduction of a long N1-alkyl

chain, (compounds B-9 to B-13). Interestingly, growth curve analysis revealed that

these compounds are much less toxic to P. aeruginosa than to S. Typhimurium. Most

compounds show a broad concentration range with only biofilm inhibition and no

effect on the planktonic growth (table -3).

N

NH2N

R1R2

No. Compound

Code R1 R2

S. Typhimurium

IC50

P. aeruginosa

IC50

1 A-1 Me H 431.5 95.1

2 A-2 Et H 206.0 20.6

3 A-3 i-pr H 175.5 4.5

4 A-4 n-Bu H 51.9 2.1

5 A-5 n-Pen H 48.3 2.0

6 A-6 n-Hex H 39.6 19.1

7 A-7 n-Hep H 12.2 6.8

8 A-8 n-Oct H 11.8 18.5

9 A-9 n-Non H 5.9 8.3

10 A-10 n-Dec H 6.1 15.0

11 A-11 n-Und H 7.1 16.2

12 A-12 n-Dod H 7.1 12.8

13 A-13 n-Trd H 5.9 27.2

14 A-14 n-Tet H 8.6 9.7

15 B-1 Me Cl 108.1 111.8

16 B-2 Et Cl 53.1 11.3

17 B-3 n-Bu Cl 57.8 16.9

18 B-4 n-Pen Cl 8.0 6.0

19 B-5 n-Hex Cl 4.9 5.3

20 B-6 n-Hep Cl 9.3 2.6

21 B-7 n-Oct Cl 5.8 3.9

22 B-8 n-Non Cl 3.9 8.0

23 B-9 n-Dec Cl 4.0 22.3

24 B-10 n-Und Cl 4.0 42.9

25 B-11 n-Dod Cl 6.1 49.7

26 B-12 n-Trd Cl 19.5 33.8

27 B-13 n-Tet Cl 453.3 19.0

IC50: concentration of inhibitor needed to inhibit biofilm formation by 50%.

Explanation Note: Number of compound code is also corresponding to the number of

carbon chain at R1 position. (ie. A-1 is Methyl substituted )

Table-3: Influence of N1-alkylated 2-aminoimidazoles on the biofilm formation and

the planktonic growth of S. Typhimurium ATCC14028 and P. aeruginosa PA14 at

25°C.

N

NH2NCl

N

NH2N

R R

Figure-1 Effect of introduction of alkyl chains with different lengths (1-14 C-

atoms; at the N1-postion of compound A (Blue) and B (Red) on the IC50 (µM) for

inhibition of the biofilm formation of S. Typhimurium ATCC14028 at 25°C in TSB

1/20.

3.5.2 Further Modification in the most active core structure

The introduction of a n-octyl side chain at the N1-position of compounds (A) and (B)

does result in very active inhibitors of both the Salmonella and Pseudomonas biofilm

formation, we also decided to introduce an n-octyl chain at the N1-position of some

other 4(5)-phenyl-2-amino-1H-imidazoles and evaluate the biofilm inhibitory activity

of the substituted compounds (table 4). All the compounds (C8-1 to C8-10) are

active inhibitors of both the S. Typhimurium and P. aeruginosa biofilm formation at

25°C, except for compounds C8-7 and C8-10. These compounds have very bulky

C5-substituents, which could diminish the solubility of the compounds in the growth

medium, reduce the uptake into the cell or cause steric hindrance in the putative

receptor binding pocket. Growth curve analysis confirmed the previously described

finding that the N1-alkylated 2-aminoimidazoles in general show a broad

concentration range in which they specifically inhibit the biofilm formation of P.

aeruginosa, without reducing the planktonic growth of the bacteria, while this

concentration range is smaller in the case of S. Typhimurium. Compounds C8-7 and

C8-10 are exceptions to this rule as we found them to slow down the planktonic

growth of P. aeruginosa at the IC50 for biofilm inhibition.

N

NH2N

C8H17

R1

Sr. Compound

Code R1

S.

Typhimurium

IC50

P. aeruginosa

IC50

1 C8-1 4-OCH3 8.5 1.2

2 C8-2 4-F 10.7 4.8

3 C8-3 3,4-di Cl 3.7 -

4 C8-4 3-Br 11.1 40.6

5 C8-5 Naphthalen-1-yl 6.5 17.6

6 C8-6 4-NO2 35.4 -

7 C8-7 4-(4-NO2-Ph) 116.6 >800

8 C8-8 4-SO2Me 29.1 4.9

9 C8-9 4-SMe 6.6 2.7

10 C8-10 4-(4-biphenyl) >800 >800


Table-4: Influence of N1-octyl-2-aminoimidazoles on the biofilm formation and the

planktonic growth of S. Typhimurium ATCC14028 and P. aeruginosa PA14 at 25°C.

3.5.3 Cyclo-alkyl 2-aminoimidazoles

To determine whether the introduction of an intermediate or long cyclo-alkyl chain

could also enhance the activity of the compounds, a broad variety of cyclo-alkyl

groups, with lengths ranging from 4 to 12 carbon atoms, were introduced at the N1-

position of the 4(5)-monosubstituted 2-aminoimidazoles (A) and (B) (table 5). The

compounds were tested against biofilm formation of S. Typhimurium and P.

aeruginosa at 25°C. As depicted introduction of an intermediate cyclo-alkyl chain

does improve the biofilm inhibitory activity of compound (A) against S. Typhimurium

and even more drastically against P. aeruginosa. In the case of Salmonella,

introduction of a cyclo-heptyl chain yields the most active compound (compound A-

Cy-5) with an IC50 of 27 µM, as compared to 130 µM for compound (A). In the case

of Pseudomonas, cyclo-hexyl and cyclo-octyl are the best substituents as introduction

of these groups improves the activity drastically from an IC50 of 73 µM. All

compounds derived from (B) by introduction of an intermediate length side chain are

very strong inhibitors of the Salmonella and Pseudomonas biofilm formation (table

5), although their activity is not markedly better than the activity of the unsubstituted

compounds. Introduction of a dodecyl side chain abolishes the activity of all three

compounds against P. aeruginosa biofilm formation, while the effect of a cyclo-

dodecyl substituent on the Salmonella biofilm inhibition depends on the nature of the

C5-group. Indeed, compound A-Cy-7, derived from (A) by introduction of a cyclo-

dodecyl group, is a very strong biofilm inhibitor, while compound B-Cy-6, derived

from (B), is a moderate biofilm inhibitor. As in the case of the n-alkyl substituents,

growth curve analysis revealed that most compounds with cyclo-alkyl substituents

only possess a very small concentration range with a selective effect on the

Salmonella biofilm formation and no effect on the planktonic growth. An exception

is compound A-Cy-7, with a cyclo-dodecyl side chain, which does not affect the

planktonic growth of Salmonella at 80 µM (the highest concentration tested), while

the IC50 for biofilm inhibition is 15 µM. Interestingly, the concentration range

between biofilm inhibition and growth inhibition is much broader in the case of P.

aeruginosa (table-5).

N

NH2N

R1R2

No. Compound

Code R1 R2

S. Typhimurium

IC50

P. aeruginosa

IC50

1 A-Cy-1 c-Pr H 89.6 35.0

2 A-Cy-2 c-Bu H 116.3 19.9

3 A-Cy-3 c-Pen H 70.7 12.6

4 A-Cy-4 c-Hex H 85.1 4.6

5 A-Cy-5 c-Hep H 26.8 14.2

6 A-Cy-6 c-Oct H 40.4 4.5

7 A-Cy-7 c-Dod H 14.5 >800

8 B-Cy-1 c-Pr Cl 54.8 13.0

9 B-Cy-2 c-Bu Cl 11.5 6.0

10 B-Cy-3 c-Hex Cl 33.2 11.2

11 B-Cy-4 c-Hep Cl 15.6 4.0

12 B-Cy-5 c-Oct Cl 12.8 48.6

13 B-Cy-6 c-Dod Cl 120.2 >800


Explanation Note: Number of compound code is also corresponding to the ring size of

cyclic substitution at R1 position. (ie. A-Cy-1 is Cyclopropyl substituted )

Table-5: Influence of N1-cyclo-alkyl-2-aminoimidazoles on the biofilm formation

and the planktonic growth of S. Typhimurium and P. aeruginosa PA14 at 25°C.

3.6. Conclusion

In the present study, we investigated the effect of the different substitution on 2-

aminoimidazoles on the biofilm formation of S. Typhimurium and P. aeruginosa. We

found that our simplest 2-aminoimidazole structure, 4(5)-phenyl-2-amino-1H-

imidazole (A), showed a moderate biofilm inhibitory activity both against S.

Typhimurium and P. aeruginosa. Substitution of the para-position of the 4(5)-phenyl

ring with a chlorine atom (B) enhanced the biofilm inhibitory activity about ten times.

We were able to delineate a relationship between the length of the N1-alkyl or N1-

cyclo-alkyl chain of the N1-substituted 2-aminoimidazoles and their biofilm

inhibitory activity. In general, introduction of a short alkyl chain at the N1-position

resulted in a decreased activity against S. Typhimurium and P. aeruginosa biofilm

formation, while all compounds with an intermediate alkyl or cyclo-alkyl chain length

showed very strong activities in both test systems. The introduction of a long alkyl or

cylco-alkyl chain could either drastically enhance the activity or totally abolish the

activity, depending on the nature of the C5-subsitutent and the bacterial species tested.

Growth curve analysis revealed that the N1-(cyclo-)alkylated 2-aminoimidazoles

show a broad concentration range in which they specifically inhibit the biofilm

formation of P. aeruginosa, without retarding the planktonic growth of the bacteria.

However, these compounds were much more toxic to S. Typhimurium. Therefore, we

cannot exclude the possibility that a part of the inhibitory effect against Salmonella

biofilms (at the higher concentrations) is due to a reduction of the planktonic growth

of the bacteria in the growth medium surrounding the surface on which the biofilms

are formed.

In conclusion, the 2-aminoimidazoles and the imidazopyrimidinium salts of the

present study are valuable candidates in the development of therapeutics and

sanitizers for the combat of biofilm formation by S. Typhimurium, P. aeruginosa and

possibly other pathogenic bacteria.

3.7 Experimental


















mono-mode microwave apparatus or Milestone MicroSYNTH multi-mode microwave

reactor (Laboratory Microwave Systems). Microwave apparatuses were used in the







3.8. Experimental protocols

3.8.1 General Procedure for the Preparation of hydroxy Salts. (Microwave)

According to process described in chapeter-2 40 new hydrocy salts were

synthesized

3.8.2 General Procedure for the Preparation of HClO4 Salts.

A mixture of 84% polyphosphoric acid (3 g) and hydroxy salts (3 mmol) was heated

in 50 ml beaker upon intensive stirring at 150 °C for 15 min. After cooling to the

room temperature the resulting viscous mass was dissolved in 30 ml of water and 1 ml

(10 mmol) of 70% HClO4 was added dropwise upon mild stirring. The white

precipitate was washed with distilled water (3×10 mL), ether (2×10 mL) until a

neutral reaction of the pH paper and then dried over P2O5 to give salt as fine white

crystals.

In similar manner 40 new compounds were synthesized.

3.8.3 General Procedure for the Microwave-Assisted Synthesis of 2-

aminoimidazoles.

To a suspension of HClO4 salt (2 mmol) in MeCN (5 mL) hydrazine hydrate (0.7 mL,

14 mmol of a 64% solution, 7 equiv) was added, and the mixture was irradiated in the

sealed Milestone MicroSYNTH microwave reactor for 10 min at a ceiling

temperature of 100 °C at 150 W maximum power. After cooling down the reaction

was worked up by diluting with DCM (300 mL), washing with sat. NH4Cl solution

(150 mL), brine (150 mL) and water (2×150 mL) and drying over anhydrous Na2SO4.

After filtration and concentration the resulting residue was purified by column

chromatography (silica gel; DCM−MeOH, 9:1 v/v with 3% TEA) to afford the

product as a colourless solid.

40 new compounds were synthesized In similar manner.


1-Cyclododecyl-5-phenyl-1H-imidazol-2-amine (BS-042)

N

NH2N

Yield: 42 %. Mp: 228-230 °C. 1H NMR (300 MHz, CDCl3): δ = 7.38 (m, 5H), 6.59 (s,

1H), 4.16 (m, 1H), 3.98 (br, 2H), 1.88 (m, 5H), 1.22(m, 14H), 0.90 (m, 3H). 13C NMR

(75.5 MHz, CDCl3): δ =148.4, 131.4, 131.2, 129.8 (× 2), 128.3 (×2), 127.5, 122.1,

50.0, 29.1(×2), 24.0 (×2), 23.9 (×2), 22.6 (×2), 21.9 (×2), 21.5. HRMS (EI):C21H31N3,

calcd 325.2518, found: 325.2511.

1-Cyclooctyl-5-phenyl-1H-imidazol-2-amine (BS-045)

N

NH2N

Yield: 53 %. Mp: 152-154 °C. 1H NMR (300 MHz, CDCl3): δ = 7.43-7.28 (m, 5H),

6.60 (s, 1H), 4.24 (m, 1H), 3.98 (br, 2H), 2.11 (m, 2H), 1.87 (m, 4H), 1.50 (m, 7H),

1.25 (m, 1H). 13C NMR (75.5 MHz, CDCl3): δ =148.1, 131.2, 129.5, 129.0 (×2),

128.4 (×2), 127.3, 122.7, 54.5, 33.1(×2), 26.2, 25.8 (×2), 25.3 (×2). HRMS (EI):

C17H23N3, calcd 269.1892, found: 269.1893.

1-Cycloheptyl-5-phenyl-1H-imidazol-2-amine (BS-048)

N

NH2N


6.59 (s, 1H), 4.09 (m, 3H), 2.13 (m, 2H), 1.95 (m, 2H), 1.73 (m, 2H), 1.54 (m, 6H). 13C NMR (75.5 MHz, CDCl3): δ =148.2, 131.1, 129.6, 128.9 (×2), 128.5 (×2), 127.3,

122.6, 54.6, 33.3(×2), 27.3 (×2), 25.7 (×2). HRMS (EI): C16H21N3, calcd 255.1735,

found: 255.1759.

1-Heptyl-5-phenyl-1H-imidazol-2-amine (BS-051)

N

NH2N

C7H15

Yield: 61 %. 1H NMR (300 MHz, CDCl3): δ = 7.43-7.31 (m, 5H), 6.67 (s, 1H), 3.95

(br, 2H), 3.75(t, J = 7.5 Hz, 2H), 1.59 (m, 2H), 1.17 (m, 8H), 0.84 (t, J = 6.5 Hz, 3H). 13C NMR (75.5 MHz, CDCl3): δ =149.0, 131.0, 129.4, 128.6 (×2), 128.2 (×2), 127.2,

122.6, 43.1, 31.5, 29.5, 28.6, 26.4, 22.4, 14.0. HRMS (EI): C16H23N3, calcd 257.1892,

found: 257.1908.

1-Octyl-5-phenyl-1H-imidazol-2-amine (BS-054)

N

NH2N

C8H17


(br, 2H), 3.75(t, J = 7.3 Hz, 2H), 1.59 (m, 2H), 1.17 (m, 10H), 0.85 (t, J = 6.6 Hz,

3H). 13C NMR (75.5 MHz, CDCl3): δ =149.1, 131.0, 129.4, 128.6 (×2), 128.2 (×2),

127.2, 122.6, 43.1, 31.6, 29.5, 29.0, 28.9, 26.4, 22.2, 14.0. HRMS (EI): C17H25N3,

calcd 271.2048, found: 271.2035.

1-Decyl-5-phenyl-1H-imidazol-2-amine (BS-057)

N

NH2N

C10H21


(br, 2H), 3.75(t, J = 7.3 Hz, 2H), 1.58 (m, 2H), 1.21 (m, 14H), 0.87 (t, J = 6.5 Hz,

3H). 13C NMR (75.5 MHz, CDCl3): δ =148.9, 131.0, 129.4, 128.6 (×2), 128.3 (×2),

127.2, 122.6, 43.1, 31.8, 29.5, 29.4, 28.3, 29.2, 28.9, 26.4, 22.2, 14.1. HRMS (EI):

C19H29N3, calcd 299.2361, found: 299.2365.

1-Hexyl-5-phenyl-1H-imidazol-2-amine (BS-060)

N

NH2N

C6H13


(br, 2H), 3.75(t, J = 7.4 Hz, 2H), 1.59 (m, 2H), 1.18 (m, 6H), 0.82 (t, J = 6.4 Hz, 3H). 13C NMR (75.5 MHz, CDCl3): δ =149.0, 131.0, 129.4, 128.6 (×2), 128.2 (×2), 127.2,

122.8, 43.1, 31.1, 29.5, 26.1, 22.4, 13.8. HRMS (EI): C15H21N3, calcd 243.1735,

found: 243.1730.

1-Nonyl-5-phenyl-1H-imidazol-2-amine (BS-063)

N

NH2N

C9H19

Yield: 58 %. 1H NMR (300 MHz, CDCl3): δ = 7.31 (m, 5H), 6.67 (s, 1H), 3.95 (br,

2H), 3.75(t, J = 7.5 Hz, 2H), 1.58 (m, 2H), 1.17 (m, 12H), 0.86 (t, J = 6.6 Hz, 3H). 13C NMR (75.5 MHz, CDCl3): δ =152.6, 134.6, 132.9, 132.1 (× 2), 131.7 (× 2), 130.7,

126.4, 46.6, 35.3, 33.0, 32.8, 32.6, 32.5, 30.0, 26.1, 17.6. HRMS (EI): C18H27N3, calcd

285.2205, found: 285.2222.

5-Phenyl-1-undecyl-1H-imidazol-2-amine (BS-066)

N

NH2N

C11H23


(br, 2H), 3.75 (t, J = 7.0 Hz, 2H), 1.59 (m, 2H), 1.17 (m, 16H), 0.87 (t, J = 6.4 Hz,

3H). 13C NMR (75.5 MHz, CDCl3): δ =149.1, 131.0, 129.4, 128.6 (×2), 128.2 (×2),

127.1, 122.9, 43.1, 31.8, 29.5 (× 2), 29.4, 29.3, 29.2, 28.9, 26.4, 22.6, 14.1. HRMS

(EI): C20H31N3, calcd 313.2518, found: 313.2512.

1-Dodecyl-5-phenyl-1H-imidazol-2-amine (BS-069)

N

NH2N

C12H25


(br, 2H), 3.75 (t, J = 7.2 Hz, 2H), 1.58 (m, 2H), 1.17 (m, 18H), 0.88 (t, J = 6.4 Hz,

3H). 13C NMR (75.5 MHz, CDCl3): δ =149.0, 131.1, 129.4, 128.6 (×2), 128.2 (×2),

127.1, 123.0, 43.1, 31.9, 29.5 (× 3), 29.4, 29.3(× 2), 28.9, 26.4, 22.6, 14.1. HRMS

(EI): C21H33N3, calcd 327.2674, found: 327.2663.

5-Phenyl-1-tridecyl-1H-imidazol-2-amine (BS-072)

N

NH2N

C13H27

Yield: 58 %. Mp: 44-46 °C. 1H NMR (300 MHz, CDCl3): δ = 7.40-7.31 (m, 5H), 6.67

(s, 1H), 3.99 (br, 2H), 3.75 (t, J = 7.3 Hz, 2H), 1.58 (m, 2H), 1.17 (m, 20H), 0.88 (t, J

= 6.4 Hz, 3H). 13C NMR (75.5 MHz, CDCl3): δ =148.9, 131.0, 129.4, 128.6 (×2),

128.2 (×2), 127.1, 122.7, 43.1, 31.9, 29.5 (× 7), 28.9, 26.4, 22.6, 14.1. HRMS (EI):

C22H35N3, calcd 341.2831, found: 341.2827.

5-Phenyl-1-tetradecyl-1H-imidazol-2-amine (BS-075)

N

NH2N

C14H29

Yield: 64 %. Mp: 44-46 °C. 1H NMR (300 MHz, CDCl3): δ = 7.41-7.30 (m, 5H), 6.67

(s, 1H), 4.00 (br, 2H), 3.75 (t, J = 7.4 Hz, 2H), 1.58 (m, 2H), 1.17 (m, 22H), 0.88 (t, J


128.3 (×2), 127.2, 122.8, 43.1, 31.9, 29.6 (× 8), 28.9, 26.4, 22.6, 14.1. HRMS (EI):

C23H37N3, calcd 355.2987, found: 355.3016.

1-Cyclohexyl-5-phenyl-1H-imidazol-2-amine (BS-078)

N

NH2N


6.60 (s, 1H), 3.89 (m, 3H), 1.88 (m, 6H), 1.64 (m, 1H), 1.18 (m, 3H). 13C NMR (75.5

MHz, CDCl3): δ =148.4, 131.2, 130.0, 129.0 (×2), 128.5 (×2), 127.3, 122.6, 55.1,

31.2(×2), 25.9 (×2), 25.1. HRMS (EI): C15H19N3, calcd 241.1579, found: 241.1589.

1-Cyclobutyl-5-phenyl-1H-imidazol-2-amine (BS-081)

N

NH2N

Yield: 47%. Mp: 77-79 °C. 1H NMR (300 MHz, CDCl3): δ = 7.41-7.28 (m, 5H), 6.58

(s, 1H), 4.61 (m, 1H), 4.07 (br, 2H), 2.51 (m, 2H), 2.27 (m, 2H), 1.75 (m, 2H). 13C

NMR (75.5 MHz, CDCl3): δ =149.1, 131.3, 129.7, 128.6 (×2), 128.3 (×2), 127.1,

122.9, 49.2, 29.5 (×2), 15.0. HRMS (EI): C13H15N3, calcd 213.1266, found: 213.1268.

1-Pentyl-5-phenyl-1H-imidazol-2-amine (BS-085)

N

NH2N

C5H11


6.67 (s, 1H), 4.00 (br, 2H), 3.75 (t, J = 7.5 Hz, 2H), 1.59 (m, 2H), 1.19 (m, 4H), 0.81

(t, J = 6.7 Hz, 3H). 13C NMR (75.5 MHz, CDCl3): δ =149.0, 131.0, 129.4, 128.6 (×2),

128.2 (×2), 127.2, 122.8, 43.1, 29.2, 28.6, 22.0, 13.8. HRMS (EI): C14H19N3, calcd

229.1579, found: 229.1594.

5-(4-Chlorophenyl)-1-cyclododecyl-1H-imidazol-2-amine (BS-116)

N

NH2NCl

Yield: 42 %. Mp: 221-223 °C 1H NMR (300 MHz, CDCl3): δ = 7.38 (m, 5H), 6.59 (s,

1H), 4.16 (m, 1H), 3.98 (br, 2H), 1.88 (m, 5H), 1.22(m, 14H), 0.90 (m, 3H). 13C NMR

(75.5 MHz, CDCl3): δ =148.4, 131.4, 131.2, 129.8 (×2), 128.3 (×2), 127.5, 122.1,

50.0, 29.1(×2), 24.0 (×2), 23.9 (×2), 22.6 (×2), 21.9 (×2), 21.5. HRMS (EI):C21H31N3,

calcd 325.2518, found: 325.2118.

5-(4-Chlorophenyl)-1-cyclooctyl-1H-imidazol-2-amine (BS-117)

N

NH2NCl

Yield: 55 %. Mp: 207-209 °C. 1H NMR (300 MHz, CDCl3): δ = 7.37 (d, J = 8.4 Hz,

2H), 7.20 (d, J = 8.4 Hz, 2H), 6.59 (s, 1H), 4.18 (m, 1H), 4.02 (br, 2H), 2.12 (m, 2H),

1.85 (m, 4H), 1.50 (m, 7H), 1.25 (m, 1H). 13C NMR (75.5 MHz, CDCl3): δ =148.6,

131.1, 130.0 (× 2), 129.7, 128.7 (×2), 128.3, 123.4, 56.7, 33.3(×2), 27.2 (×3), 25.7

(×2). HRMS (EI): C17H22ClN3, calcd 303.1502, found: 303.1519.

5-(4-Chlorophenyl)-1-cycloheptyl-1H-imidazol-2-amine (BS-118)

N

NH2NCl


2H), 7.18 (d, J = 8.4 Hz, 2H), 6.59 (s, 1H), 4.00 (m, 3H), 2.12 (m, 2H), 1.92 (m, 2H),

1.75 (m, 2H), 1.55 (m, 6H). 13C NMR (75.5 MHz, CDCl3): δ =148.6, 131.1, 130.0

(×2), 129.7, 128.7 (×2), 128.3, 123.4, 56.7, 33.3(×2), 27.2 (×2), 25.7 (×2). HRMS

(EI): C16H20ClN3, calcd 289.1346, found: 289.1364.

5-(4-Chlorophenyl)-1-heptyl-1H-imidazol-2-amine (BS-119)

N

NH2N

C7H15Cl

Yield: 54 %. Mp: 80-82 °C. 1H NMR (300 MHz, CDCl3): δ = 7.35 (d, J = 8.4 Hz, 2H),

7.23 (d, J = 8.4 Hz, 2H), 6.66 (s, 1H), 4.02 (br, 2H), 3.72(t, J = 7.5 Hz, 2H), 1.57 (m,

2H), 1.18 (m, 8H), 0.84 (t, J = 6.5 Hz, 3H). 13C NMR (75.5 MHz, CDCl3): δ =149.4,

133.0, 129.6, 129.3 (×2), 128.8 (×2), 128.1, 123.5, 43.1, 31.5, 29.5, 28.6, 26.4, 22.5,

14.2. HRMS (EI): C16H22ClN3, calcd 291.1502, found: 291.1507.

5-(4-Chlorophenyl)-1-octyl-1H-imidazol-2-amine (BS-120)

N

NH2N

C8H17Cl


7.23 (d, J = 9.3 Hz, 2H), 6.67 (s, 1H), 3.95 (br, 2H), 3.73(t, J = 7.2 Hz, 2H), 1.57 (m,


131.1, 129.5, 129.3 (×2), 128.8 (×2), 128.2, 123.4, 43.1, 31.6, 29.5, 29.0, 28.9, 26.4,

22.2, 14.0. HRMS (EI): C17H24ClN3, calcd 305.1659, found: 305.1649.

5-(4-Chlorophenyl)-1-decyl-1H-imidazol-2-amine (BS-121)

N

NH2N

C10H21Cl


7.33 (d, J = 9.0 Hz, 2H), 6.67 (s, 1H), 3.97 (br, 2H), 3.72 (t, J = 7.4 Hz, 2H), 1.57 (m,


133.0, 129.5, 129.3 (×2), 128.8 (×2), 128.2, 123.5, 43.1, 31.8, 29.5, 29.4 (×2), 29.2,

28.9, 26.4, 22.6, 14.1. HRMS (EI): C19H28ClN3, calcd 333.1972, found: 333.1999.

5-(4-Chlorophenyl)-1-hexyl-1H-imidazol-2-amine (BS-122)

N

NH2N

C6H13Cl




133.0, 129.5, 129.3 (×2), 128.8 (×2), 128.2, 123.5, 43.1, 31.1, 29.5, 26.1, 22.4, 13.9.

HRMS (EI): C15H20N3, calcd 277.1346, found: 277.1353.

5-(4-Chlorophenyl)-1-nonyl-1H-imidazol-2-amine (BS-123)

N

NH2N

C9H19Cl




133.1, 129.5, 129.3 (× 2), 128.8 (× 2), 128.2, 123.6, 43.1, 31.7, 29.6, 29.3, 29.1, 28.9,

26.4, 22.6, 14.0. HRMS (EI): C18H26ClN3, calcd 319.1815, found: 319.1808.

5-(4-Chlorophenyl)-1-undecyl-1H-imidazol-2-amine (BS-124)

N

NH2N

C11H23Cl




133.1, 129.5, 129.3 (×2), 128.8 (×2), 128.2, 123.6, 43.1, 31.8, 29.6, 29.5, 29.4 (×2),

29.3, 28.9, 26.4, 22.6, 14.1. HRMS (EI): C20H30ClN3, calcd 347.2128, found:

347.2136.

5-(4-Chlorophenyl)-1-dodecyl-1H-imidazol-2-amine (BS-125)

N

NH2N

C12H25Cl


7.23 (d, J = 8.9 Hz, 2H), 6.67 (s, 1H), 3.99 (br, 2H), 3.72 (m, 2H), 1.58 (m, 2H), 1.17

(m, 18H), 0.88 (m, 3H). 13C NMR (75.5 MHz, CDCl3): δ =149.1, 133.1, 129.5, 129.3

(×2), 128.8 (×2), 128.2, 123.6, 43.1, 31.9, 29.6 (× 3), 29.4 (× 2), 29.3, 28.9, 26.4,

22.6, 14.1. HRMS (EI): C21H32ClN3, calcd 361.2285, found: 361.2281.

5-(4-Chlorophenyl)-1-tridecyl-1H-imidazol-2-amine (BS-126)

N

NH2N

C13H27Cl




133.1, 129.5, 129.3 (×2), 128.8 (×2), 128.2, 123.6, 43.1, 31.9, 29.6 (×4), 29.5, 29.4,

29.3, 28.9, 26.4, 22.6, 14.1. HRMS (EI): C22H34ClN3, calcd 375.2441, found:

375.2446.

5-(4-Chlorophenyl)-1-tetradecyl-1H-imidazol-2-amine (BS-127)

N

NH2N

C14H29Cl



2H), 1.17 (m, 22H), 0.87 (m, 3H). 13C NMR (75.5 MHz, CDCl3): δ =149.1, 133.1,

129.5, 129.3 (×2), 128.8 (×2), 128.2, 123.6, 43.1, 31.9, 29.6 (×5), 29.5, 29.4 (×2),

29.0, 26.4, 22.7, 14.1. HRMS (EI): C23H36ClN3, calcd 389.2598, found: 389.2602.

5-(4-Chlorophenyl)-1-cyclohexyl-1H-imidazol-2-amine (BS-128)

N

NH2NCl


2H), 7.18 (d, J = 8.4 Hz, 2H), 6.60 (s, 1H), 4.00 (br, 2H), 3.81 (m, 1H), 1.87 (m, 6H),

1.66 (m, 1H), 1.23 (m, 3H). 13C NMR (75.5 MHz, CDCl3): δ =148.8, 133.2, 130.2

(×2), 129.8, 128.9, 128.7 (×2), 123.7, 55.2, 31.2(×2), 25.9 (×2), 25.1. HRMS (EI):

C15H18ClN3, calcd 275.1189, found: 275.1187.

5-(4-Chlorophenyl)-1-pentyl-1H-imidazol-2-amine (BS-129)

N

NH2N

C5H11Cl




133.0, 129.5, 129.3 (×2), 128.8 (×2), 128.2, 123.4, 43.2, 29.3, 28.6, 22.1, 13.8. HRMS

(EI): C14H18ClN3, calcd 263.1189, found: 263.1185.

5-(4-Chlorophenyl)-1-cyclobutyl-1H-imidazol-2-amine (BS-130)

N

NH2NCl

Yield: 40%. Mp: 156-159 °C. 1H NMR (300 MHz, CDCl3): δ = 7.33 (d, J = 8.4 Hz,

2H), 7.19 (d, J = 8.5 Hz, 2H), 6.57 (s, 1H), 4.55 (m, 1H), 4.00 (br, 2H), 2.47 (m, 2H),

2.27 (m, 2H), 1.76 (m, 2H). 13C NMR (75.5 MHz, CDCl3): δ =149.4, 133.0, 129.8,

129.7 (×2), 128.6 (×2), 128.4, 123.5, 49.2, 29.5 (×2), 15.0. HRMS (EI): C13H14ClN3,

calcd 247.0876, found: 247.0874.

5-(4-Methoxyphenyl)-1-octyl-1H-imidazol-2-amine (BS-199)

N

NH2N

C8H17O

Yield: 56 %. 1H NMR (300 MHz, CDCl3): δ = 7.25 (m, 2H), 6.95 (d, J = 8.7 Hz, 2H),

6.60 (s, 1H), 3.97 (br s, 2H), 3.84 (s, 3H), 3.70 (t, J = 7.8 Hz, 2H), 1.56 (m, 2H), 1.17

(m, 10H), 0.86 (t, J = 7.05 Hz, 3H). 13C NMR (75.5 MHz, CDCl3): δ =149.2, 131.1,

129.5, 129.3 (×2), 128.8 (×2), 128.2, 123.4, 43.1, 31.6, 29.5, 29.0, 28.9, 26.4, 22.2,

14.0. HRMS (EI): C18H27N3O, calcd 301.4265, found: 301.4271.

5-(4-Fluorophenyl)-1-octyl-1H-imidazol-2-amine (BS-200)

N

NH2N

C8H17F


1H), 3.96 (br s, 2H), 3.70 (t, J = 6.8 Hz, 2H), 1.56 (m, 2H), 1.17 (m, 10H), 0.86 (t, J =

7.05 Hz, 3H). 13C NMR (75.5 MHz, CDCl3): δ =163.8, 160.5, 148.7, 130.2, 128.4,

127.0, 122.9, 115.5, 104.7, 43.0, 31.6, 29.5, 29.0, 28.9, 26.4, 22.5, 14.0. HRMS (EI):

C17H24FN3, calcd 289.3910, found: 289.3901.

5-(3,4-Dichlorophenyl)-1-octyl-1H-imidazol-2-amine (BS-201)

N

NH2N

C8H17Cl

Cl


7.98 Hz, 1H), 7.16 (d of d, J = 2.0, 6.2 Hz, 1H), 6.71 (s, 1H), 4.05 (br s, 2H), 3.73 (t, J

= 7.68 Hz, 2H), 1.59 (m, 2H), 1.19 (m, 10H), 0.86 (t, J = 7.05 Hz, 3H). 13C NMR

(75.5 MHz, CDCl3): δ =149.6, 132.8, 131.1, 131.0, 130.6, 129.5, 127.1, 127.0, 124.2,

43.3, 31.6, 29.5, 29.0, 28.9, 26.4, 22.5, 14.0. HRMS (EI): C17H23Cl2N3, calcd

340.2906, found: 240.2918.

5-(3-Bromophenyl)-1-octyl-1H-imidazol-2-amine (BS-202)

N

NH2N

C8H17

Br


1H), 4.02 (br s, 2H), 3.74 (t, J = 7.71 Hz, 2H), 1.59 (m, 2H), 1.19 (m, 10H), 0.86 (t, J


128.0, 126.5, 123.8, 122.6, 43.2, 31.6, 29.5, 29.0, 28.9, 26.4, 22.5, 14.0. HRMS (EI):

C17H24BrN3, calcd 350.2966, found:250.2978.

5-(Naphthalen-1-yl)-1-octyl-1H-imidazol-2-amine (BS-202)

N

NH2N

C8H17


1H), 4.04 (br s, 2H), 3.83 (t, J = 7.62 Hz, 2H), 1.62 (m, 2H), 1.15 (m, 10H), 0.82 (t, J

= 6.99 Hz, 3H). 13C NMR (75.5 MHz, CDCl3): δ =149.1, 133.4, 132.4, 129.5, 128.3,

128.2, 127.9, 127.7, 126.6, 126.4 (×2), 126.0, 123.0, 43.3, 31.6, 29.5, 29.0, 28.9, 26.4,

22.5, 14.0. HRMS (EI): C21H27N3, calcd 321.4592, found: 321.4576.

5-(4-Nitrophenyl)-1-octyl-1H-imidazol-2-amine (BS-204)

N

NH2N

C8H17NO2


Hz, 2H), 6.89 (s, 1H), 4.13 (br s, 2H), 3.82 (t, J = 7.8 Hz, 2H), 1.63 (m, 2H), 1.21 (m,

10H), 0.85 (t, J = 6.96 Hz, 3H). 13C NMR (75.5 MHz, CDCl3): δ =163.8, 160.5,

148.7, 130.2, 128.4, 127.0, 122.9, 115.5, 104.7, 43.0, 31.6, 29.5, 29.0, 28.9, 26.4,

22.5, 14.0. HRMS (EI): C17H24N4O2, calcd 316.3981, found: 316.3993.

5-(4'-Nitrobiphenyl-4-yl)-1-octyl-1H-imidazol-2-amine (BS-205)

N

NH2N

C8H17NO2

Yield: 52 %. 1H NMR (300 MHz, CDCl3): δ = 8.33 (m, 1H), 7.78-7.35 (m, 6H), 7.13

(m 1H), 6.77 (s, 1H), 4.07 (br s, 2H), 3.79 (m, 2H), 1.63 (m, 2H), 1.19 (m, 10H), 0.84

(m, 3H). 13C NMR (75.5 MHz, CDCl3): δ =163.8, 160.5, 148.7, 130.2, 128.4, 127.0,

122.9, 115.5, 104.7, 43.0, 31.6, 29.5, 29.0, 28.9, 26.4, 22.5, 14.0. HRMS (EI):

C23H28N4O2, calcd 392.4940, found:392.4928.

5-(4-(Methylsulfonyl)phenyl)-1-octyl-1H-imidazol-2-amine (BS-206)

N

NH2N

C8H17SO2Me


7.98 Hz, 2H), 6.83 (s, 1H), 4.09 (br s, 2H), 3.82 (t, J = 7.68 Hz, 2H), 3.09 (s 3H), 1.62


=150.7, 138.1, 136.6, 127.9 (×2), 127.6, 127.4 (×2), 126.0, 44.5, 43.6, 31.6, 29.6,

29.0, 28.9, 26.5, 22.5, 14.0. HRMS (EI): C18H27N3O2S, calcd 349.4909, found:

349.4920.

5-(4-(Methylthio)phenyl)-1-octyl-1H-imidazol-2-amine (BS-207)

N

NH2N

C8H17SMe


(br s, 2H), 3.73 (t, J = 7.98 Hz, 2H), 2.51 (s, 3H), 1.58 (m, 2H), 1.18 (m, 10H), 0.86

(t, J = 6.99 Hz, 3H). 13C NMR (75.5 MHz, CDCl3): δ =163.8, 160.5, 148.7, 130.2,

128.4, 127.0, 122.9, 115.5, 104.7, 43.0, 31.6, 29.5, 29.0, 28.9, 26.4, 22.5, 14.0. HRMS

(EI): C18H27N3S, calcd 317.4921, found: 317.4935.

5-(Triphenyl-4-yl)-1-octyl-1H-imidazol-2-amine (BS-208)

N

NH2N

C8H17

Yield: 31 %. 1H NMR (300 MHz, CDCl3): δ = 7.70-7.64 (m, 7H), 7.46-7.40 (m 5H),

6.74 (s, 1H), 4.03 (br s, 2H), 3.80 (m, 2H), 1.63 (m, 2H), 1.20 (m, 10H), 0.86 (m, 3H). 13C NMR (75.5 MHz, CDCl3): δ =148.9, 140.5, 140.2, 139.2 (×2), 130.0, 129.2, 128.8

(×2), 128.5, 127.5 (×2), 127.4, 127.3, 127.2 (×4), 127.0, 126.9, 123.1, 43.2, 31.7,

29.6, 29.0, 28.9, 26.5, 22.5, 14.0. HRMS (EI): C29H33N3, calcd 423.5924, found:

423.5941.



N

NH2N

C9H19

4.90

05

0.93

60

1.45

13

2.10

67

1.96

69

12.3

26

3.00

00

Inte

gral

7.42

637.

3999

7.37

807.

3252

7.31

717.

3102

7.26

25

6.67

62

3.99

05

3.78

213.

7570

3.73

12

1.61

101.

5890

1.56

58

0.89

170.

8691

0.84

65

-0.0

002

(ppm)

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.0

152.

6248

134.

6350

132.

9477

132.

1549

131.

8022

130.

7185

126.

4311

81.0

402

80.6

220

80.2

038

46.6

571

35.3

221

33.0

857

32.8

639

32.6

638

32.5

038

30.0

128

26.1

509

17.6

160

(ppm)

0102030405060708090100110120130140150

N

NH2N

C9H19


N

NH2N

C12H25Cl

2.02

07

2.64

88

0.91

25

1.46

87

1.99

26

1.95

95

18.1

69

3.00

00

Inte

gral

7.38

537.

3570

7.26

297.

2334

6.67

22

3.99

093.

7536

3.72

853.

7028

1.57

50

1.24

921.

1802

0.90

210.

8808

0.85

76

0.00

02

(ppm)

0.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.0

N

NH2N

C12H25Cl

149.

1738

133.

1114

129.

5949

129.

3876

128.

8857

128.

2966

123.

6310

77.4

765

77.0

510

76.6

292

43.1

952

31.9

039

29.6

019

29.4

965

29.4

019

29.3

292

28.9

983

26.4

927

22.6

890

14.1

359

0.00

08

(ppm)

0102030405060708090100110120130140150160

3.11 References

[1] (a) Alvi K. A, Peters B. M, Hunter L. M, Crews P. (1993) Tetrahedron. 49,329. (b) Chan G.

W, Mong S, Hemling M. E, Freyer A. J, Offen P. H, DeBrosse C. W, Sarau H. M, Westley J.

W. (1993) J. Nat. Prod. 56, 116. (c) Tsuda M, Shigemori H, Ishibashi M, Kobayashi J. (1992)

Tetrahedron Lett. 33, 2597. (d) Ishibashi M, Tsuda M, Ohizumi Y, Sasaki T, Kobayashi

(1991) J. Ezperientia, 47, 299. (e) Kobayashi J, Tsuda M, Ohizumi Y. (1991) Experientia 47,

301. (f) Keifer P. A, Schwartz R. E, Koker M. E. S, Hughes R. J, Rittschof D, Rinehart K. L.

(1991) J. Org. Chem. 56, 2965. (g) Morales J. J, Rodriguez A. D. (1991) J. Nat. Prod. 54,

629. (h) Alvi K. A, Crews P, Loughhead D. G. (1991) J. Nat. Prod. 54, 1509. (i) Wright A. E,

Chiles S, Cross S. (1991) J. Nat. Prod. 54, 1684. (j) Kobayashi J, Tsuda M, Murayama T,

Nakamura H, Ohizumi Y, Ishibashi M, Iwamura M, Ohta T, Nozoe S. (1990) Tetrahedron

46, 5579. (k) Bedoya-Zurita M, Ahond A Poupat C, Potier P. (1989) Tetrahedron 45, 6713.

(l) Walker R. P, Faulkner D. J, Van Engen D, Clardy J. (1981) J. Am. Chem. SOC. 103,

6772.

[2] (a) Nagai W, Kirk K. L, Cohen L. A. (1973) J. Org. Chem. 38, 971. (b) Dismukes K, Rogers

M, Daily J. W. (1976) J. Neurochem. 26, 785. (c) Lipinski C. A, LaMattina J. L, Hohnke L.

A. (1986) J. Med. Chem. 28, 1628. (d) Impicciatore M, Morini G, Chiavarini M, Plazzi P. V,

Bordi F, Vitali F. (1986) Agents Actions 18, 134. (e) Haaksma J, DonnB-Op den Kelder M,

Timmerman H, Weinstein H, (1991) In New Perspectives in Histamine Research;

BirkhEiuser Verlag: Basel, 315-324.

[3] See Ann. Drug Data Rep. 1993,15, 513.

[4] Jung F, Boucherot D, Delvare C, Olivier A, Davies G. M, Betts M. J, Brown R, Stevenson R,

Joseph M, Kingston F, Pittam, (1993) J. Antibiot. 46, 992.

[5] (a) Beaman A. G, Tautz W, Gabriel T, Duschinsky R. (1966) J. Am. Chem. SOC. 87, 389.

(b) Lancini C, Lazzari E, Arioli V, Bellani P. (1969) J. Med. Chem. 12, 775.

[6] For example see: Jenkins C, Naylor A, O'Neill, Threadgill D, Cole S, Stratford J, Adams

G. E, Fielden M, Suto J, Stier A. J. Med. Chem. lSW, 33, 2603. Adams E, Stratford J.

(1986) Biochem. Pharm. 35, 71.

[7] Storey B. T, Sullivan W. W, Moyer C. L. (1986) J. Org. Chem. 29, 3118.

[8] Lawson, A. (1956) J. Chem. SOC. 307.

[9] (a) Lancini G. C, Lazzari E. (1966) J. Heterocycl. Chem. 3, 1 (b) Cavalleri B, Ballotta R,

Lancini G. C. (1979) J. Heterocycl. Chem. 979.

[10] See: Porter A. E. A. (1984) In Comprehensive Heterocyclic Chemistr Pyrazines and Their

Benzo Derivatives; Boulton A. J, McKillop A Eds.; Pergamon Press: New York. 3, 184-185.

[11] Fargher R. G, Pyman F. L. (1919) J. Chem. SOC. 217.

[12] Kirk K. L. (1978) J. Org. Chem. 43, 4381.

[13] Kreutzberger A. (1962) J. Org. Chem. 27, 886.

[14] Nishimura T, Kitajima K. (1979) J. Org. Chem. 44, 818.

[15] Takahashi K, Kirk K. L, Cohen L. A. J. Org. Chem. 1984, 49, 1951. Jarosinski M. A,

Anderson K. (1991) J. Org. Chem. 56, 4058.

[16] (a) Ruccia M, Vivona N, Cusmano G. (1974) Tetrahedron 30, 3859. (b) Braun M, Buchi G,

Bushey F. (1978) J. Am. Chem. SOC. 100, 4208. (c) Sehgal K, Agrawal C. (1982) J. Pharm.

Sci. 71, 1203. (d) Berry A, Gilbertsen B, Cook D. (1986) J. Med. Chem. 29, 2034.

[17] LaMattina L, Mularski C. J. (1984) Tetrahedron Lett. 25, 2957.

[18] Xu Y, Yakushijin K, Horne A. (1993) Tetrahedron Lett. 34, 6981

Copper Catalyzed Microwave Assisted One Pot

Synthesis of 2-Aminoimidazole-Triazole

Framework via Dimorth Rearrangement and

Click Reaction

Chapter-4

Copper Catalyzed Microwave Assisted One Pot

Synthesis of 2-Aminoimidazole-Triazole Framework

via Dimorth Rearrangement and Click Reaction.

ABSTRACT

N

N

N

R1OH

N3

R2

nNH

N

NHR1

R2

nN

NN R3

One-pot Microwave-Assisted Synthesis

Br

MW, 100 oC, 2-5 min

R3

22 examples,upto 90% yieldn = 1,2

Cu(OAc)2 (5 mol %)N2H4 (2 equiv)

(1.2 equiv)

A microwave assisted one-pot Cu(II)-catalyzed protocol was developed for the

construction of 2-AI-T framework. This process combining two consecutive steps of

dimorth rearrangement and click reaction represents a useful protocol for the smooth

synthesis of novel 2-aminoimidazole-triazole derivatives.

Publication

[1] Copper Catalyzed Microwave Assisted One Pot Synthesis of 2-

Aminoimidazole-Triazole Framework Org.lett (manuscript in preparation)

4.1 Introduction

There are only a few approaches that describe the direct synthesis of 2-

aminoimidazoles and their biological activity1-5. The earliest method involves

condensation of α-aminocarbonyl compounds with cyanamide or their synthetic

equivalents6,7. This method is most commonly used for the direct construction of the

2-aminoimidazole ring. Other general applicable strategies are cyclocondensation of

α-bromoketone with N-acetylguanidine in acetonitrile8, iminophosphorane-mediated

cyclization of α –azido esters9, ammonolysis of 2-amino-1,3-oxazol-3-iumsalts10,

sequential functionalization of 1,2-diprotected imidazole ring with different

electrophiles11. Most of them involve long experimental procedures, the use of

unstable precursors and tiresome workup process. Accordingly, the development of

straightforward and general procedures for the synthesis of diversely substituted 2-

aminoimidazoles from readily available precursors is highly warranted. Herein, we

report a rapid and highly efficient Cu-mediated synthesis of 2-aminoimidazole-

triazole framework via click reaction and dimorth rearrangement.

“Click chemistry” has emerged as a fast and efficient approach to synthesis of novel

compounds with desired function making use of selected “near perfect” reactions.12

(very fast, selective, high-yield, and wide scope).

The Dimroth rearrangement13 is an isomerization of heterocycles that consists in a

translocation of endo- or exocyclic heteroatoms through a ring-opening-ring-closure

sequence. It can be catalyzed by acids, bases, heat or light.

Recently group of Erik14 reported a new one pot microwave assisted synthesis of

substituted 2-amino-1H- imidazoles from readily available 2-aminopyrimidine (1) and

α-bromoketone (2). The first step was performed by heating N-(3-

azidopropyl)pyrimidin-2-amine (1) and 2-bromo-1-phenylethanone (2) at 75 oC for 3

h resulting in the formation of hydroxyl salt (3) which undergoes dimorth type

rearrangement by 7 equiv of NH2NH2.H2O resulting in the formation of N-(2-azido)-

1H-imidazol-2-amine. Final step of click reaction was performed under microwave-

irradiation using phenylacetylene (1.5.equiv), Cu(Oac)2 (10 mol %)as catalyst at 100 oC 10 min with a maximum power of 40W to obtain 2-AI-T scaffold (5). (Scheme 1)

N

N

N

R1OH

N3

R2

n

NHN

NHR1

R2n

N

NN R3

N

N

NH

N3

nBr

R2

O

R1

MeCN, 75 oC,3 Hrs

Br

MW. 100 oC

10 min, 80 %

+

N

HN

NH

N3

R1

R2MW. 100 oC

MeCN, 10 minN2H4 (7 equiv)

R3

Cu(OAc)2

n

One Pot MW ?

1 2

34

5

Scheme-1 Sequential Synthesis of 2-AI-T Framework

In continuation of earlier work on two step synthesis of 2-AI-T scaffold with excellent

yield. now an ameliorated and convenient one-pot protocol for the synthesis of 2-AI-T

scaffold under microwave conditions has been reported.

4.2 Optimization Study

Looking to the current exploration in the synthesis and microwave-assisted decoration

of the 2-aminoimidazole14 and click reaction15 and preliminary success of the

sequence, it was very interesting to investigate whether one-pot protocol for the

construction of 2-AI-T scaffold should be possible. The test reaction was performed

with hydroxy salt {3} and phenyl acetylene as a model system. Different Cu catalyst,

equiv of NH2NH2.H2O, temperature and reaction time were screened to derive an

optimized protocol (Table 1).

First, the application of Cu(OAc)2 as catalyst for click reaction and different

equivalents of aqueous NH2NH2.H2O for simultaneous dimorth rearrangement (Table-

1, No-1-3) was evaluated. However in case of 1 equiv. of hydrazine used, yield was

only 51 %. (Table-1, No-1). Next study of different reaction time (Table-1, No-3-7)

performed. It was observed that reaction could complete even in very short time

(Table-1, No- 6). Next reaction temperature (Table-1, No-8-9) was investigated.

Decrease of the reaction temperature resulted in lover yield (Table-1, No-9). Then the

examination the catalyst effect on a reaction (Table-1, No-7-13) was done. However

replacement of Cu(Oac)2 with CuSO4 resulted in lower yield (Table-1, No-11). While

in case of Cu Powder (200 mesh) used only traces amount of desire product observed

(Table-1, No-13). Moreover it was observed that the catalyst was mandatory for

successfully performing the reaction, as no product was formed in the absence of the

catalyst (Table-1, No-12). Finally optimum reaction conditions were achieved when a

mixture of hydroxy salt, 1.2 equiv of phenyl acetylene, 5 mol % of Cu(Oac)2 in 1 ml

of Ethenol : water (4: 1) was irradiated for 2 min at a ceiling temperature of 100 oC

applying a maximum power of 36 W, affording the desired compound in 90 % yield

(Table 1 No-8). The protocol was checked with conventional condition providing

lower yield of 24 % of after 24 hrs.

N

N

N

PhOH

Br Ph (1.2 equiv.)

N3

NHN

NH

N NN

PhPh

EtOH : Water (4:1, 1ml)

Cu salt (mol %)

N2H4 (equiv.)

One-Pot microwave-assisted

35

No. time

(min) temp (oC) Cu salt (mol %) yield (%)b

1c 20 100 °C Cu(Oac)2, 10 51

2d 20 100 °C Cu(Oac)2, 10 84

3 20 100 °C Cu(Oac)2, 10 86

4 10 100 °C Cu(Oac)2, 10 80

5 5 100 °C Cu(Oac)2, 10 90

6 2 100 °C Cu(Oac)2, 10 90

7 1 100 °C Cu(Oac)2, 10 53

8 2 100 °C Cu(Oac)2, 5 90

9 2 90 °C Cu(Oac)2, 5 73

10 2 100 °C Cu(Oac)2, 2 57

11 2 100 °C CuSO4.5H2O, 5 67

12 2 100 °C - -

13 2 100 °C Cu Powder (200 mesh) Traces

14e -f 25 °C Cu(Oac)2, 5 24

a All reactions were run on a 0.7 mmol scale of 3, applying phenyl acetylene (1.2

equiv) in ethanol: water (4:1, 1 mL) under microwave irradiation. The mixture was

irradiated in a sealed tube (CEM Discover@) at a ceiling temperature of 100 oC and 35

W maximum power for the stipulated time. b isolated yields. c 1.0 equiv of

NH2NH2.H2O used. d 5.0 equiv of NH2NH2.H2O used. e reaction carried out at room

temperature. f time 24 h.

Table-1: Copper Catalyzed One-Pot protocol: Catalyst, equiv of hydrazine, reaction

time and Temperature Effects on the model reaction to obtain 2-AI-T scaffolda.

4.3 Results and Discussion

Encouraged by these findings, the scope of this protocol was further explored. An

array of hydroxy salts 3 (a-j) (Table-2) were reacted with various acetylene using our

optimized conditions. In most cases, the reactions proceeded well affording the one

pot product in good to excellent yield (Table-2)

N

N

N

R1OH

N3

R2

n

R3

NHN

NHR1

R2n

N

NN R3

One-pot Microwave-assisted

N

N

NH

N3

n

Br

R2

O

R1

MeCN, 75 oC,3 Hrs

Br Cu(OAc)2 (5 mol. %)

MW. 100 oC

2-5 min, 85-90 %

+N2H4

1

2

3 {a-j}4 {a-v}

No. Product, Yieldb (%) No. Product, Yieldb (%)

1 NH

N

NH

N

NN

Br

OMe

4-a (66)

12 NH

N

NH

N NN

Cl

Cl 4-l (90)

2 NH

N

NH

N

NN

4-b (84)

12 NH

N

NH

N

NN

Br 4-m (95)

3 NH

N

NH

N

NN

Br

Me

4-c (73)

14 NH

N

NH

N NN

Br 4-n (74)

4 NH

N

NH

N

NN Bu

4-d (64) 15

NHN

NH

N NN

O

N

O 4-o (29)

5 NH

N

NH

N

NN Hep

Br 4-e (73)

16 NH

N

NH

N

NN

O

N

O 4-p (29)

6 NH

N

NH

N NN

Br Pen 4-f (80)

17 NH

N

NH

N

NN

F 4-q (68)

7 NH

N

NH

N

NN

ClCl

Hep

4-g (80)

18 NH

N

NH

N

NN

F 4-r (80)

8 NH

N

NH

N NN

Cl

Cl

HN

4-h (81)

19 NH

N

NH

N

NN

Cl 4-s (16)

9 NHN

NH

N NN

Br

H2N

4-I (75)

20 NH

N

NH

N NN

Cl

Cl 4-t (85)

10 NH

N

NH

N

NN

ClCl 4-j (85)

21 NH

N

NH

N NN

Cl

Cl

S

4-u (91)

11 NH

N

NH

N

NN

ClCl 4-k (80)

22 NH

N

NH

N

NN

Br 4-v (80) a All reactions were run on a 0.7 mmol scale of 3 {a-j} ethanol: water (4:1) (1 ml) with

phenyl acetylene (1.2 equiv), Cu(OAc)2 (5 mol % ) and hydrazine hydride (2 equiv).

The mixture was irradiated in a sealed tube (CEM Discover@) at a ceiling temperature

of 100 oC and 35 maximum power for the stipulated time. b isolated yields. Yields are

shown in ().

Table-2: Microwave assisted one pot decoration of 2-AI-T Frameworka.

A diverse set of 2-AI-T scaffold were synthesized with these optimized conditions.

Ten different hydroxy salt 3 {a-j} (n = 1 or 2) were reacted with different (hetero)

aromatic or alkyl acetylenes (Table-2) to test the generality of this new microwave-

assisted one pot method (Table 2). All reactions were completed within 5 min using

microwave irradiation at ceiling temperature of 100 oC and 35W power. The yields

varied from good to excellent, although in some cases lower yields were observed

(Table 2, No-15,16 and 19). However in case of morpholine substitution (table-2, No-

15-16) probably due to decomposition of starting hydroxy salt 3 {h} lower yields

were observed. Interestingly when 4,5-disubstituted hydroxy salt react with acetylene

only 16% of desired product 4{s} was observed due to steric hindrance of 4-Me-

phenyl substitution on dimorth rearrangement. Motivated by the importance of the

final target to develop new general method toward widely functionalized 2-AI-T

framework, we next examined the extension of this procedure to other acetylene. The

reaction proceeded effectively with aliphatic and aromatic terminal acetylenes to give

the corresponding 2-AI-T (Table 2). Thus, a variety of substituted 2-AI-T containing

aromatic, aliphatic, cyclic and heterocyclic substitution at the C-4 position of the

triazole ring were obtained.

4.4 Possible Mechanism

Regarding the mechanism of transformation of 2-hydroxy 2,3-dihydro-1H-

imidazo[1,2-a]pyrimidin-4-ium salts 3 into 2-amino-1H-imidazole-triazole 4, we

presume that the reaction proceeds via an unusual Dimroth type rearrangement

(Scheme-3).

N

N

N

R1

OH

N3

R2

n HN

N

NHR1

R2

nBr

NH2NH2

N

N

N

R1

OH

N3

R2

n

NH2NH2Br

N

NH2

N

R1

OH

N

R2

n

NN

R3

Cu(OAc)2

CuNP

HN

NH2

N

R1

O

N

R2

n

NN

R3

N

NN

R3

R3 "Click" Reaction

Generation of"nano-Cu"

H2ORing Opening

Dimroth Rearrangement

MW

NNH ·HBr

25 oC

3

a b

c

4

Scheme-3: Proposed mechanism for One-pot reaction:

In the first step the 2-hydroxy-2,3-dihydroimidazo[1,2-a]pyrimidinium salt (3)

undergoes cleavage of the pyrimidine ring (a), resulting in the generation of pyrazole

and 2-amino-5-hydroxyimidazolidine (b), meanwhile copper nanoparticle16 generated

by reduction of Cu(OAc)2 using 1 equiv of NH2NH2.H2O takes part in click reaction.

This intermediate (b), which is in equilibrium with the open form c can cyclize again

leading by dehydration upon microwave irradiation, results in the rearranged 2-

amino-1H-imidazoles triazole (4).

4.5 Conclusion

In conclusion microwave-assisted Cu-catalyzed one pot protocol for the synthesis of

2-aminoimidazole-triazole framework via two consecutive steps of dimorth

rearrangement and click reaction was demonstrated. Library of 22 compounds have

been synthesized and were subjected to test for antibiofilm activity. However the

antibiofilm activities of these classes of compounds are under pipeline.

4.6 Experimental


















mono-mode microwave apparatus or Microwave apparatuses were used in the







4.7 Experimental protocols

4.7.1 General Procedure for the Preparation 3-Azido-propylamine

To a solution of 3-chloropropan-1-amine hydrochloride (3.2 g, 14.6 mmol) in water

(10 mL) was slowly added NaN3 (3.2 g, 49.2 mmol) as a solution in water (15 mL).

The resulting solution was allowed to stir at reflux overnight. After cooling to room

temperature, about 2/3 of the water was removed by evaporation and the remaining

residue diluted with ether (50 mL). This biphasic mixture was cooled to 0 °C and

KOH pellets (4.0 g) were slowly added. The phases separated and the aqueous layer

extracted with ether (2 x 30 mL). All organics combined, dried over Na2SO4, and

concentrated to afford 3-Azido-propylamine (1.17 g, 80%) as yellow oil

4.7.2 General Procedure for the Preparation of N-(3-azidopropyl)pyrimidin-2-

amine:

In a 50 mL microwave vial were successively dissolved in EtOH (20 mL) 2-

chloropyrimidine (3.43 g, 30 mmol), 3-Azido-propylamine (48 mmol, 1.6 equiv) and

triethylamine (6.2 mL, 45 mmol, 1.5 equiv). The reaction tube was sealed, and

irradiated in the cavity of a Milestone MicroSYNTH microwave reactor at a ceiling

temperature of 120 °C at 100 W maximum power for 60 min. After the reaction

mixture was cooled with an air flow for 15 min, it was diluted with water (100 mL),

extracted with DCM (2×150 mL) and dried over Na2SO4. The solvent was evaporated

in vaccuo, and the crude mixture was purified by silica gel flash chromatography

using 0-5% MeOH−DCM as the eluent.

4.7.3 General Procedure for the Preparation of Salts.

To a solution of N-(3-azidopropyl)pyrimidin-2-amine (6 mmol) and α-

bromoacetophenone (7.2 mmol, 1.2 equiv) in acetonitrile (12 mL) was added 4-

dimethylaminopyridine (6 mg, 0.05 mmol). After being stirred at 85 °C for 5 h, the

reaction mixture was diluted with acetone (20 mL) and the precipitate was filtered and

washed with acetone (2×20 mL), ether (2×20 mL) and dried over P2O5 to give salt as

a white solid.

4.7.4 General Procedure for the preparation of 2-AI-T.

Cool the suspension of hydrazine hydride (2 equiv), in ethanol (0.8 ml.). To the

suspension were added Cu(OAc)2 (5 mol %) in water (0.2 ml). and stirred it for 2 min

at 0 oC. To this were added acetylene (1.5 eq) and hydroxyl salt (1 eq.) The reaction

mixture was irradiated at 100 0 oC at the maximum power 35 W for 2-5 min. After

completion of the reaction, the solvent was removed under reduce pressure. The crude

product was purifier by column chromatography over silica gel using DCM /

methanol / 7N methanolic NH3 (96:3:1) as the eluent.


2-Azidoethanamine (BS-167)

H2NN3

Yield: 41 %. 1H NMR (300 MHz, CDCl3): δ = 3.37 (t, J = 5.61 Hz, 2H), 2.88 (t, J =

5.50 Hz, 2H), 1.53 (Br s, 2H).

3-Azidopropan-1-amine (BS-220)

N3H2N

Yield: 45 %. 1H NMR (300 MHz, CDCl3): δ = 3.38 (t, J = 6.93 Hz, 2H), 2.81 (t, J =

6.84 Hz, 2H), 1.73 (m, 2H), 1.36 (Br s, 2H).

N-(2-Azidoethyl)pyrimidin-2-amine (BS-168)

N

N

NH

N3

Yield: 67 %. 1H NMR (300 MHz, CDCl3): δ = 8.30 (d, J = 4.74 Hz, 2H), 6.58 (t, J =

4.74 Hz, 1H), 5.44 (Br s, 1H), 3.64 (m, 2H), 3.54 (t, J = 5.58 Hz, 2H), 13C NMR

(75.5 MHz, CDCl3): δ =162.0, 158.1, 158.0, 111.1, 50.8, 40.7. HRMS (EI) C6H6N6,

calcd 164.0810, found :.

N-(3-Azidopropyl)pyrimidin-2-amine (BS-221)

N

N

NH

N3

Yield: 69 %. 1H NMR (300 MHz, CDCl3): δ = 8.28 (d, J = 4.80 Hz, 2H), 6.54 (t, J =

4.80 Hz, 1H), 5.30 (Br s, 1H), 3.52 (m, 2H), 3.42 (t, J = 6.57 Hz, 2H), 1.90 (m, 2H).

1-(3-Azidopropyl)-2-hydroxy-2-phenyl-2,3-dihydro-1H-imidazo[1,2-a]pyrimidin-


N

N

NOH

Br

N3

Yield: 72 %. 1H NMR (300 MHz, DMSO-d6) : δ = 9.05 (m, 2H), 7.93 (s, 1H), 7.80

(m, 2H), 7.48 (m, 3H), 7.37 (t, J = 4.86 Hz, 1H), 4.95 (m, 2H), 3.41 (m, 4H), 1.64 (m,

2H). 13C NMR (75.5 MHz, DMSO-d6): δ = 167.4, 154.6, 148.2, 138.1, 129.3, 128.4

(×2), 126.9 (×2), 111.4, 90.6, 62.7, 47.9, 38.2, 27.0.

1-(2-Azidoethyl)-2-hydroxy-2-phenyl-2,3-dihydro-1H-imidazo[1,2-a]pyrimidin-4-


N

N

NOH

Br

N3


(m, 2H), 7.49 (m, 3H), 7.41 (t, J = 4.92 Hz, 1H), 4.95 (m, 2H), 3.38 (m, 4H). 13C

NMR (75.5 MHz, DMSO-d6): δ = 167.5, 154.7, 148.4, 137.8, 129.4, 128.4 (×2), 126.9

(×2), 111.8, 90.6, 62.8, 48.1 (×2).

1-(2-Azidoethyl)-2-hydroxy-2-(naphthalen-1-yl)-2,3-dihydro-1H-imidazo[1,2-


N

N

NOH

Br

N3


(s, 1H), 8.02 (m, 3H), 7.88 (m, 1H), 7.59 (m, 2H), 7.44 (m, 1H), 5.05 (s, 2H), 3.42 (m,

4H). 13C NMR (75.5 MHz, DMSO-d6): δ = 167.5, 154.9, 148.5, 135.0, 132.9, 132.1,

128.4, 128.2, 127.5, 127.1, 126.7 (×2), 124.1, 111.9, 90.8, 62.6, 48.2 (×2).

1-(3-Azidopropyl)-2-(4-bromophenyl)-2-hydroxy-2,3-dihydro-1H-imidazo[1,2-


N

N

NOH

Br

N3

Br

Yield: 76 %. 1H NMR (300 MHz, DMSO-d6) : δ = 9.06 (d of d, J = 1.62, 4.60 Hz,

1H), 8.98 (d of d, J = 1.62, 6.24 Hz,1H) 8.00 (s, 1H), 7.72 (m, 4H), 7.37 (m, 1H),

4.85 (m, 2H), 3.37 (m, 4H), 1.66 (m, 2H). 13C NMR (75.5 MHz, DMSO-d6): δ =

167.5, 154.7, 148.2, 137.6, 131.4(×2), 129.3 (×2), 122.9, 111.5, 90.3, 62.5, 47.9, 38.2,

27.0.

1-(2-Azidoethyl)-2-(3,4-dichlorophenyl)-2-hydroxy-2,3-dihydro-1H-imidazo[1,2-


N

N

NOH

Br

N3

Cl

Cl


(s, 1H), 7.79 (m, 2H), 7.43 (m, 1H), 4.93 (m, 2H), 3.42 (m, 4H). 13C NMR (75.5

MHz, DMSO-d6): δ = 167.5, 154.9, 148.3, 139.0, 132.2, 131.3, 130.5, 129.5, 127.4,

112.1, 89.8, 62.5, 48.0 (×2).

1-(3-Azidopropyl)-2-(3,4-dichlorophenyl)-2-hydroxy-2,3-dihydro-1H-

imidazo[1,2-a]pyrimidin-4-ium bromide (BS-274)

N

N

NOH

Br

N3

Cl

Cl

Yield: 81 %. 1H NMR (300 MHz, DMSO-d6) : δ = 9.07 (m, 1H), 8.97 (m,1H) 8.12 (s,

1H), 8.08 (Br s, 1H), 7.97 (m, 2H), 7.38 (t, J = 4.92 Hz, 1H), 4.86 (m, 2H), 3.37 (m,

4H), 1.68 (m, 2H). 13C NMR (75.5 MHz, DMSO-d6): δ = 167.5, 154.7, 148.1, 139.2,

132.2, 131.3, 130.6, 129.3, 127.4, 111.6, 89.8, 62.4, 47.8, 38.3, 26.9.

1-(2-Azidoethyl)-2-(4-chlorophenyl)-2-hydroxy-3-p-tolyl-2,3-dihydro-1H-

imidazo[1,2-a]pyrimidin-4-ium bromide (BS-299)

N

N

NOH

Br

N3

Cl

Yield: 42 %. 1H NMR (300 MHz, DMSO-d6) : δ = 9.19 (d, J = 3.06 Hz, 1H), 8.55 (d,

J = 5.43 Hz, 1H), 7.71 (d, J = 8.52 Hz, 2H), 7.70 (s, 1H), 7.55 (d, J = 8.49 Hz, 2H),

7.38 (m, 1H), 7.42 (d, J = 7.80 Hz, 2H), 7.08 (d, J = 7.80 Hz, 2H), 6.10 (s, 1H), 3.36

(m, 4H), 2.33 (s, 3H) 13C NMR (75.5 MHz, DMSO-d6): δ = 168.2, 156.1, 146.9,

139.5, 135.9, 134.4, 129.4 (×6), 128.4 (×2), 124.4, 112.8, 93.5, 74.9, 47.9, 41.0, 20.7.

1-(2-Azidoethyl)-2-(4-fluorophenyl)-2-hydroxy-2,3-dihydro-1H-imidazo[1,2-


N

N

NOH

Br

N3

F

Yield: 79 %. 1H NMR (300 MHz, DMSO-d6) : δ = 9.09 (d, J = 4.53 Hz, 1H), 9.01 (d,

J = 6.00 Hz, 1H), 8.04 (s, 1H), 7.85 (m, 2H), 7.37 (m, 3H), 4.92 (m, 2H), 3.38 (m,

4H). 13C NMR (75.5 MHz, DMSO-d6): δ = 167.5, 164.1, 160.9, 154.7, 148.4, 134.0

(d), 129.4 (d), 115.4, 115.1, 111.9, 90.3, 62.7, 48.1 (×2).

5-(4-Bromophenyl)-N-(2-(4-(4-methoxyphenyl)-1H-1,2,3-triazol-1-yl)ethyl)-1H-

imidazol-2-amine (BS-219)

NH

N

NH

N

NN

Br

O

Yield: 66 %. 1H NMR (300 MHz, DMSO-d6): δ = 10.62 (br s, 1H), 8.45 (s, 1H), 7.43

(d, J = 8.8 Hz, 2H), 7.57 (d, J = 7.9 Hz, 2H), 7.43 (d, J = 8.8 Hz, 2H), 7.10 (s, 1H),

6.98 (d, J = 8.8 Hz, 2H), 6.02 (m, 1H), 4.60 (t, J = 5.90 Hz, 2H), 3.78 (s, 3H), 3.68

(m, 2H). 13C NMR (75.5 MHz, DMSO-d6): δ =158.8, 150.0, 146.0, 131.0, 126.4

(×4), 125.5 (×2), 123.4, 120.7, 117.5, 114.2 (×4), 55.0, 49.2. 42.8. HRMS (EI)

C20H19BrN6O, calcd 438.0804, found :438.0811.

5-Phenyl-N-(3-(4-phenyl-1H-1,2,3-triazol-1-yl)propyl)-1H-imidazol-2-amine (BS-

238)

NH

N

NH

N NN


(d, J = 7.2 Hz, 2H), 7.59 (d, J = 7.2 Hz, 2H), 7.44 (t, J = 7.7 Hz, 2H), 7.27 (m, 3H),

7.08 (m, 2H), 6.09 (br s, 1H), 4.50 (t, J = 7.1 Hz, 2H), 3.21 (m, 2H), 2.21 (m, 2H). 13C

NMR (75.5 MHz, DMSO-d6): δ = 150.3, 146.2, 133.0, 132.3, 130.8, 128.8 (×2),

128.3, 127.7, 125.6, 125.0 (×2), 123.6 (×2), 121.4, 110.4, 104.1, 47.3, 28.8 (×2).

HRMS (EI) C20H20N6, calcd 344.1749, found : 344.1734.

5-Phenyl-N-(2-(4-phenyl-1H-1,2,3-triazol-1-yl)ethyl)-1H-imidazol-2-amine (BS-

228)

NH

N

NH

N

NN


(d, J = 7.3 Hz, 2H), 7.61 (m, 2H), 7.43 (t, J = 7.6 Hz, 2H), 7.28 (m, 3H), 7.06 (m,

2H), 6.01 (br s, 1H), 4.63 (t, J = 6.1 Hz, 2H), 3.70 (m, 2H). 13C NMR (75.5 MHz,

DMSO-d6): δ = 150.3, 146.1, 130.4, 128.8 (×4), 128.2, 127.7, 125.1, 125.0 (×4),

123.4, 121.7, 104.1, 49.3. 42.9. HRMS (EI) C19H18N6, calcd 330.1593, found :

330.1582.

5-(4-Bromophenyl)-N-(2-(4-p-tolyl-1H-1,2,3-triazol-1-yl)ethyl)-1H-imidazol-2-

amine (BS-231)

NH

N

NH

N

NN

Br


(d, J = 8.07 Hz, 2H), 7.57 (d, J = 8.31 Hz, 2H), 7.43 (d, J = 8.56 Hz, 2H), 7.22 (d, J =

8.07 Hz, 2H), 7.13 (s, 1H), 6.03 (br s, 1H), 4.61 (t, J = 5.80 Hz, 2H), 3.68 (m, 2H),

2.32 (s, 3H). 13C NMR (75.5 MHz, DMSO-d6): δ = 150.4, 146.1, 136.9 (×2), 130.9

(×2), 129.3 (×4), 128.0, 125.3, 124.9 (×3), 121.2, 117.4, 49.2, 42,8, 20.7. HRMS (EI)

C20H19BrN6, calcd 422.0855, found : 422.0863.

N-(2-(4-(4-Butylphenyl)-1H-1,2,3-triazol-1-yl)ethyl)-5-(naphthalen-1-yl)-1H-


NH

N

NH

N

NN C4H9


(s, 1H), 7.73 (m, 6H), 7.37 (m, 2H), 7.23 (m, 3H), 6.01 (m, 1H), 4.68 (m, 2H), 3.74

(m, 2H), 2.58 (t, J = 7.4 Hz, 2H), 1.56 (m, 2H), 1.32 (m, 2H), 0.89 (t, J = 7.4 Hz, 3H). 13C NMR (75.5 MHz, DMSO-d6): δ = 150.7, 146.2, 141.8, 133.5, 131.3, 128.7 (×3),

128.3, 127.5, 127.3 (×3), 126.0, 125.0 (×3), 124.6, 123.2, 121.3, 120.2, 49.3, 42.9,

34.5, 32.9, 21.7, 13.7. HRMS (EI) C27H28N6, calcd 436.2375, found : 436. 2349.

5-(4-Bromophenyl)-N-(2-(4-heptyl-1H-1,2,3-triazol-1-yl)ethyl)-1H-imidazol-2-

amine (BS-277)

NH

N

NH

N

NN C7H15

Br


(d, J = 8.2 Hz, 2H), 7.42 (d, J = 7.9 Hz, 2H), 7.13 (s, 1H), 5.93 (br s, 1H), 4.51 (t, J =

5.90 Hz, 2H), 3.60 (m, 2H), 2.57 (d, J = 7.7 Hz, 2H), 1.57 (m, 2H), 1.25 (m, 8H), 0.85

(t, J = 6.9 Hz, 3H). 13C NMR (75.5 MHz, DMSO-d6): δ = 150.5, 146.7 (×2), 131.0

(×3 ), 125.3, 121.9 (×2), 117.4, 104.1, 48.9, 42.9, 31.1, 28.9, 28.5, 28.4, 25.0, 22.0,

13.9. HRMS (EI) C20H27BrN6, calcd 430.1481, found : 430.1488.

5-(4-Bromophenyl)-N-(3-(4-(4-pentylphenyl)-1H-1,2,3-triazol-1-yl)propyl)-1H-


NH

N

NH

N NN

BrC5H11


(d, J = 8.06 Hz, 2H), 7.53 (d, J = 8.06 Hz, 2H), 7.38 (d, J = 8.27 Hz, 2H), 7.23 (d, J =

8.06 Hz, 2H), 7.11 (s, 1H), 5.96 (m, 1H), 4.47 (t, J = 6.57 Hz, 2H), 3.19 (m, 2H), 2.59

(t, J = 7.63 Hz, 2H), 2.14 (m, 2H), 1.59 (m, 2H), 1.30 (m, 4H), 0.86 (t, J = 6.78 Hz,

3H). 13C NMR (75.5 MHz, DMSO-d6): δ = 146.3, 142.9, 131.0 (×2), 128.7 (×4),

128.3, 125.4, 124.0 (×4), 121.0, 117.3 (×2), 47.3, 39.9, 34.8, 30.8, 30.5, 30.6, 21.9,

13.8. HRMS (EI) C25H29BrN6. calcd.492.1637, found: 492. 1621.

5-(3,4-Dichlorophenyl)-N-(2-(4-(4-heptylphenyl)-1H-1,2,3-triazol-1-yl)ethyl)-1H-

imidazol-2-amine ( BS-279)

NH

N

NH

N

NN

Cl

Cl

C7H15


(s, 1H), 7.71 (d, J = 8.3 Hz, 2H), 7.62 (m, 1H), 7.48 (d, J = 8.3 Hz, 1H), 7.22 (d, J =

8.3 Hz, 3H), 6.06 (m, 1H), 4.61 (t, J = 5.77 Hz, 2H), 3.69 (m, 2H), 2.58 (t, J =

7.58Hz, 2H), 1.57 (m, 2H), 1.28 (m, 8H), 0.86 (m, 3H). 13C NMR (75.5 MHz,

DMSO-d6): δ = 146.1, 141.8, 130.9 (×2), 130.3, 128.6 (×3), 128.2 (×2), 126.5,124.9

(×3), 124.8, 124.7, 121.2, 49.2, 42.8, 34.8, 31.1, 30.7, 28.5, 28.4, 22.0, 13.8. HRMS

(EI) C26H30Cl2N6, calcd 496.1909, found : 496.1919.

5-(3,4-Dichlorophenyl)-N-(3-(4-((methylamino)methyl)-1H-1,2,3-triazol-1-

yl)propyl)-1H-imidazol-2-amine (BS-283)

NH

N

NH

N NN

HN

Cl

Cl


(s, 1H), 7.58 (d, J = 8.7 Hz, 1H), 7.48 (d, J = 8.2 Hz, 1H), 7.23 (s, 1H), 6.01 (m, 1H),

4.41 (t, J = 6.95 Hz, 2H), 3.67 (m, 2H), 3.14 (m, 2H), 2.26 (s, 3H), 2.07 (t, J = 6.5 Hz,

2H), 1.86 (s,1H) 13C NMR (75.5 MHz, DMSO-d6): δ = 151.4, 145.9, 135.9, 130.9

(×2), 130.3, 126.4 (×2), 124.6, 123.4, 122.6, 47.0 (×2), 41.0, 35.4, 30.3. HRMS (EI)

C16H19Cl2N7, calcd 389.1079, found: 389.1064.

N-(3-(4-(2-Aminopropan-2-yl)-1H-1,2,3-triazol-1-yl)propyl)-5-(4-bromophenyl)-

1H-imidazol-2-amine (BS-287)

NH

N

NH

N NN

BrH2N


(d, J = 8.3 Hz, 2H), 7.42 (d, J = 8.3 Hz, 2H), 7.11 (s, 1H), 5.94 (t, J = 5.6 Hz, 1H),

4.38 (t, J = 7.0 Hz, 2H), 4.10 (m, 2H), 3.16 (m, 2H), 2.07 (m, 2H), 1.37 (s, 6H). 13C

NMR (75.5 MHz, DMSO-d6): δ = 156.0, 151.3, 134.0, 131.0 (×3), 125.3 (×2), 120.1

(×2), 117.3, 54.8, 48.5, 47.1, 30.8 (×2), 30.1. HRMS (EI) C17H22BrN7, calcd

403.1120, found: 403.1131.

5-(3,4-Dichlorophenyl)-N-(2-(4-cyclohexyl-1H-1,2,3-triazol-1-yl)ethyl)-1H-


NH

N

NH

N

NN

Cl

Cl


(s, 1H), 7.60 (m, 1H), 7.48 (d, J = 8.3 Hz, 1H), 7.24 (s, 1H), 6.00 (m, 1H), 4.50 (t, J =

5.76 Hz, 2H), 3.61 (m, 2H), 2.61 (m, 1H), 1.91 (m, 2H), 1.69 (m, 3H), 1.32 (m,5H). 13C NMR (75.5 MHz, DMSO-d6): δ = 151.9, 150.6, 131.0 (×2), 130.3, 126.6 (×2),

124.7, 123.4, 120.7, 104.1, 54.8, 49.0, 42.8, 34.5, 32.4 (×2), 25.5 (×2).HRMS (EI)

C19H22Cl2N6, calcd 404.1283., found: 404.1278.

5-(3,4-Dichlorophenyl)-N-(2-(4-propyl-1H-1,2,3-triazol-1-yl)ethyl)-1H-imidazol-

2-amine (BS-302)

NH

N

NH

N

NN C3H7

Cl

Cl

Yield: 90 %. 1H NMR (300 MHz, DMSO-d6): δ = 10.80 (br s, 1H), 7.84 (d, J = 1.7

Hz, 1H), 7.82 (s, 1H), 7.59 (d of d, J = 8.4, 1.7 Hz, 1H), 7.53 (d, J = 8.4 Hz, 1H), 7.26

(s, 1H), 6.14 (br s, 1H), 4.51 (t, J = 6.04 Hz, 2H), 3.62(m, 2H), 2.56 (t, J = 7.49 Hz,

2H), 1.57 (m, 2H), 0.89 (t, J = 7.25 Hz, 3H). 13C NMR (75.5 MHz, DMSO-d6): δ =

150.4, 146.5, 135.0, 131.0 (×2), 130.4 126.8 (×2), 124.8, 123.5, 122.0, 48.8, 42.8,

27.0, 22.0, 13.6. HRMS (EI) C16H18Cl2N6, calcd 364.0970., found: 364.0980.

5-(3,4-Dichlorophenyl)-N-(3-(4-propyl-1H-1,2,3-triazol-1-yl)propyl)-1H-


NH

N

NH

N NN

C3H7

Cl

Cl


(d, J = 1.7 Hz 1H), 7.58 (d of d, J = 8.3, 1.7 Hz, 1H), 7.52 (s, 1H), 7.49 (s, 1H), 6.13

(br s, 1H), 4.38 (t, J = 6.98 Hz, 2H), 3.15 (m, 2H), 2.57 (t, J = 7.51 Hz, 2H), 2.06 (m,

2H), 1.58 (m, 2H), 0.89 (t, J = 7.51 Hz, 3H). 13C NMR (75.5 MHz, DMSO-d6): δ =

150.0, 146.6, 135.1, 131.0 (×2), 130.3 126.8 (×2), 124.8, 123.5, 121.7, 54.8, 46.9,

30.0, 27.0, 22.2, 13.5. HRMS (EI) C17H20Cl2N6, calcd 378.1127., found : 378.1114.

5-(4-Bromophenyl)-N-(2-(4-propyl-1H-1,2,3-triazol-1-yl)ethyl)-1H-imidazol-2-

amine (BS-304)

NH

N

NH

N

NN C3H7

Br

Yield: 95 %. 1H NMR (300 MHz, DMSO-d6): δ = 10.63 (br s, 1H), 7.87(s, 1H), 7.62

(m, 2H), 7.48 (d , J = 8.6 Hz, 2H), 7.17 (s, 1H), 5.98 (t, J = 5.3 Hz, 1H), 4.59 (t, J =

6.04 Hz, 2H), 3.66 (m, 2H), 2.61 (t, J = 7.44 Hz, 2H), 1.62 (m, 2H), 0.94 (t, J = 7.21

Hz, 3H). 13C NMR (75.5 MHz, DMSO-d6): δ = 150.5, 146.5, 134.6, 131.0 (×3),

125.4 (×2), 122.0 (×2), 117.4, 48.9, 42.9, 27.0, 22.2, 13.6. HRMS (EI) C16H19BrN6,

calcd 374.0855., found: 374.0844.

5-(4-Bromophenyl)-N-(3-(4-propyl-1H-1,2,3-triazol-1-yl)propyl)-1H-imidazol-2-

amine (BS-305)

NH

N

NH

N NN

C3H7

Br


(d , J = 8.1 Hz, 2H), 7.42 (d , J = 8.4 Hz, 2H), 7.11 (s, 1H), 5.93 (t, J = 5.25 Hz, 1H),

4.38 (t, J = 6.83 Hz, 2H), 3.14 (m, 2H), 2.48 (t, J = 7.62 Hz, 2H), 2.06 (m, 2H), 1.58

(m, 2H), 0.90 (t, J = 7.35 Hz, 3H). 13C NMR (75.5 MHz, DMSO-d6): δ = 151.1,

146.6, 134.6, 131.0 (×3), 125.4 (×2), 121.7 (×2), 117.4, 47.9, 30.0, 27.0 (×2), 22.2,

13.5. HRMS (EI) C17H21BrN6, calcd 388.1011., found: 388.1023.

(2-(3-(4-Propyl-1H-1,2,3-triazol-1-yl)propylamino)-1H-imidazol-5-

yl)(morpholino)methanone (BS-306)

NH

N

NH

O

N

N NN

C3H7

O


(s, 1H), 6.05 (m, 1H), 4.35 (t, J = 6.8 Hz, 2H), 3.75 (m, 4H), 3.57 (t, J = 4.31 Hz, 4H),

3.10 (m, 2H), 2.57 (t, J = 7.42 Hz, 2H), 2.05 (m, 2H), 1.60 (m, 2H), 0.90 (t, J = 7.42

Hz, 3H). 13C NMR (75.5 MHz, DMSO-d6): δ = 161.2, 150.1, 146.6, 122.8, 121.7

(×2), 66.3 (×2), 54.8 (×2), 46.9, 44.4, 29.9, 27.0, 22.2, 13.5. HRMS (EI) C16H25N7O2,

calcd 347.2070., found: 347.2085.

(2-(2-(4-Propyl-1H-1,2,3-triazol-1-yl)ethylamino)-1H-imidazol-5-

yl)(morpholino)methanone (BS-307)

NH

N

NH

N

NN

O

N

C3H7

O


(s, 1H), 5.98 (m, 1H), 4.45 (m, 2H), 3.74 (m, 3H), 3.58 (m, 6H), 3.16 (m, 1H), 2.56

(m, 2H), 1.57 (m, 2H), 0.89 (t, J = 7.25 Hz, 3H). 13C NMR (75.5 MHz, DMSO-d6): δ

= 161.4, 149.7, 146.5 (×2), 121.9 (×2), 66.3 (×2), 48.7 (×2), 44.4, 44.7, 27.0, 22.2,

13.6. HRMS (EI) C15H23N7O2, calcd 333.1913., found: 333.1920.

N-(2-(4-(Cyclopentylmethyl)-1H-1,2,3-triazol-1-yl)ethyl)-5-(4-fluorophenyl)-1H-


NH

N

NH

N

NN

F

Yield: 68 %. 1H NMR (300 MHz, DMSO-d6): δ = 10.63 (br s, 1H), 7.82 (s, 1H),

7.63 (q, J = 2.9, 5.7 Hz, 2H), 7.11 (t , J = 8.7 Hz, 2H), 7.03 (s, 1H), 5.96 (t, J = 5.4

Hz, 1H), 4.51 (t, J = 5.99 Hz, 2H), 3.61 (m, 2H), 2.59 (d, J = 7.08 Hz, 2H), 2.07 (m,

1H), 1.66 (m, 6H), 1.70 (m, 2H). 13C NMR (75.5 MHz, DMSO-d6): δ = 161.8, 158.6,

150.3, 146.2, 146.1, 130.9, 125.2, 125.1, 122.2, 115.1, 114.8, 48.9, 42.9, 31.8 (×3),

31.0, 24.5 (×2). HRMS (EI) C19H23FN6, calcd 354.1968., found: 354.1974.

N-(2-(4-Cyclopropyl-1H-1,2,3-triazol-1-yl)ethyl)-5-(4-fluorophenyl)-1H-imidazol-

2-amine (BS-309)

NH

N

NH

N

NN

F

Yield: 80 %. 1H NMR (300 MHz, DMSO-d6): δ = 10.55 (br s, 1H), 7.79 (s, 1H),

7.64 (m, 2H), 7.10 (t , J = 8.7 Hz, 2H), 7.02 (s, 1H), 5.90 (m, 1H), 4.99 (t, J = 5.85

Hz, 2H), 3.61 (m, 2H), 1.91 (m, 1H), 0.85 (m, 2H), 0.69 (m, 2H). 13C NMR (75.5

MHz, DMSO-d6): δ = 161.8, 158.6, 150.4, 148.6 (×2), 131.1, 125.1, 125.0, 120.9,

115.1, 114.8, 48.9, 42.9, 7.6(×2), 6.5. HRMS (EI) C16H17FN6, calcd 312.1499., found:

312.1487.

5-(4-Chlorophenyl)-N-(2-(4-cyclopentyl-1H-1,2,3-triazol-1-yl)ethyl)-4-p-tolyl-1H-

imidazol-2-amine(BS-310-B)

NH

N

NH

N

NN

Cl


(m, 2H), 7.29 (m, 4H), 7.14 (m, 2H), 5.87 (m, 1H), 4.52 (t, J = 5.87 Hz, 2H), 3.64 (m,

2H), 3.06 (m, 1H), 2.29 (s, 3H), 1.95 (m, 2H), 1.60 (m, 6H). 13C NMR (75.5 MHz,

DMSO-d6): δ = 150.9, 150.1, 135.7, 130.0, 128.9 (×3), 128.1 (×3), 128.0 (×2), 127.2

(×3), 121.1, 104.1, 49.0, 42.7, 36.1, 32.7 (×2), 24.6 (×2), 20.7. HRMS (EI)

C25H27ClN6. calcd.446.1986, found: 446.1990.

N-(3-(4-(4-Tert-butylphenyl)-1H-1,2,3-triazol-1-yl)propyl)-5-(3,4-

dichlorophenyl)-1H-imidazol-2-amine (BS-340):

NH

N

NH

N NN

Cl

Cl


(s, 1H), 7.73 (d, J = 7.9 Hz, 2H), 7.57 (m, 1H), 7.43 (m, 3H), 7.25 (s, 1H) 6.02 (m,

1H), 4.48 (t, J = 6.73 Hz, 2H), 3.20 (m, 2H), 2.14 (m, 2H), 1.30 (s, 9H). 13C NMR

(75.5 MHz, DMSO-d6): δ = 150.2, 146.2, 131.0, 130.3, 128.0, 126.6, 125.5 (×3),

124.8 (×3), 124.7, 123.4, 121.0 (×2), 104.1, 47.3, 39.6, 34.2, 31.0 (×3), 29.9.HRMS

(EI) C24H26Cl2N6, calcd 468.1596, found: 468.1602.

5-(3,4-Dichlorophenyl)-N-(3-(4-(thiophen-3-yl)-1H-1,2,3-triazol-1-yl)propyl)-1H-

imidazol-2-amine (BS-341):

NH

N

NH

N NN

Cl

Cl

S


(m, 2H), 7.63 (m, 1H), 7.58 (m, 1H), 7.51 (m, 2H), 7.26 (s, 1H) 6.02 (m, 1H), 4.47 (t,

J = 6.8 Hz, 2H), 3.19 (m, 2H), 2.14 (m, 2H). 13C NMR (75.5 MHz, DMSO-d6): δ =

151.3, 142.8, 132.1 (×2), 131.0, 130.0, 127.0, 126.5, 125.7, 124.7, 123.4, 121.1, 120.6

(×2), 104.1, 47.3, 39.5, 29.9. HRMS (EI) C18H16Cl2N6S, calcd 418.0534, found:

418.0522.

5-(4-Bromophenyl)-N-(2-(4-cyclopropyl-1H-1,2,3-triazol-1-yl)ethyl)-1H-imidazol-

2-amine(BS-342):

NH

N

NH

N

NN

Br


(d, J = 7.3 Hz, 2H), 7.43 (d, J = 7.3 Hz, 2H), 7.14 (s, 1H), 5.93 (m, 1H), 4.49 (t, J =

5.48 Hz, 2H), 3.59 (m, 2H), 3.16 (d of d, J = 5.2, 1.4 Hz, 1 H), 0.87 (m, 2H), 0.69 (m,

2H). 13C NMR (75.5 MHz, DMSO-d6): δ = 150.4, 148.7 (×3), 131.0, 130.9, 125.3,

120.9 (×3), 117.4, 48.9, 42.8, 7.5 (×2), 6.5. HRMS (EI) C16H17BrN6, calcd 372. 0698,

found: 372.0689.



N

N

NOH

Br

N3

Br

167.

5180

154.

7139

148.

2373

137.

6005

131.

4039

129.

3129

122.

9491

111.

5523

90.3

697

62.5

832

47.9

135

40.0

332

39.7

568

39.4

768

39.2

005

38.9

205

27.0

036

(ppm)

0102030405060708090100110120130140150160170

N

N

NOH

Br

N3

Br

1.99

07

1.08

90

3.91

24

0.97

92

2.00

00

8.69

96

1.94

39

Inte

gral

9.06

869.

0591

9.05

358.

9901

8.98

448.

9694

8.96

37

8.00

157.

7800

7.75

057.

7216

7.69

217.

3908

7.37

457.

3701

7.35

44

4.95

934.

9109

4.85

694.

8086

3.46

173.

4378

3.37

883.

3437

3.32

173.

2991

1.68

351.

6609

1.63

77

(ppm)

0.01.02.03.04.05.06.07.08.09.0


NH

N

NH

N

NN C3H7

Br0.

7987

0.99

51

1.87

38

1.97

16

0.92

64

0.94

33

1.94

49

1.96

90

1.95

86

1.95

63

3.00

00

Inte

gral

10.6

535

7.87

677.

6231

7.51

397.

4857

7.17

31

6.00

695.

9887

5.97

05

4.58

904.

5689

4.54

88

3.69

653.

6764

3.65

693.

6368

2.64

142.

6169

2.59

18

1.67

731.

6528

1.62

831.

6032

0.97

240.

9479

0.92

35

(ppm)

0.01.02.03.04.05.06.07.08.09.010.011.0

NH

N

NH

N

NN C3H7

Br

150.

5865

146.

5318

134.

0549

131.

0403

125.

4219

122.

0327

117.

4725

48.9

026

42.9

570

40.3

132

39.7

568

39.4

805

39.2

005

38.6

441

27.0

764

22.2

144

13.6

286

(ppm)

0102030405060708090100110120130140150160

4.10 References

[1] For a general review of the synthesis and activity of 2-aminoimidazole alkaloids, see: (a) S.

M. Weinreb, (2007) Nat. Prod. Rep. 24, 931–948. (b) H. Hoffmann, T. Lindel, (2003)

Synthesis. 1753–1783. (c) J. D. Sullivan, R. L. Giles, R. E. Looper. (2009) Curr. Bioactive

Cmpds. 5, 39-78.

[2] For the anti-biofilm activity of 2-aminoimidazoles, see: (a) R. W. Huigens III, J. J. Richards,

G. Parise, T. E. Ballard, W. Zeng, R. Deora, C. Melander, (2007) J. Am. Chem. Soc. 129,

6966–6967. (b) S. A. Rogers, C.Melander, (2008) Angew. Chem. 120, 5307–5309., (2008)

Angew. Chem. Int. Ed. 47, 5229–5231. (c) T. E. Ballard, J. J. Richards, A. L. Wolf, C.

Melander, (2008) Chem. Eur. J. 14, 10745–10761.

[3] (a) M. S. Malamas, J. Erdei, I. Gunawan, J. Turner, Y. Hu, E. Wagner, K. Fan, R.Chopra, A.

Olland, J. Bard, S. Jacobsen, R. L. Magolda, M. Pangalos, A. J. Robi- chaud. (2010) J. Med.

Chem. 53, 1146–1158. (b) M. S. Malamas, J. rdei, I. Gunawan, K. Barnes, M. Johnson, Y.

Hui, J. Turner, Y.Hu, E. Wagner, K. Fan, A. Olland, J. Bard,A. J. Robichaud. (2009) J. Med.

Chem. 52, 6314–6323.

[4] (a) R. S. Coleman, E. L. Campbell, D. J. Carper. (2009) Org. Lett. 11, 2133–2136. (b) M.

Nodwell, A. Pereira, J. L. Riffell, C. Zimmermann, B. O. Patrick, M. Roberge, R. J. Andersen.

(2009) J. Org. Chem. 74, 995–1006.

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Heterocycl. Chem. 3, 152–154.

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Catimel, E. C. Nice, K. G. Watson. (2007) Bioorg. Med. Chem. Lett. 17,3741–3744.

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Heterocycles. 53, 1939–1955.

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(b) H. C. Kolb, K. B. Sharpless. (2003) Drug Discovery Today. 8, 1128 1137. (c) C. W.

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Chem. 75, 79. (c) D. J. Brown, Mechanisms of Molecular Migrations. Thyagarajan, B. S.,

(1968) Wiley-Interscience: New York, Vol. 1, 209.

[14] For synthesis and anti biofilm activity see (a) D. S. Ermolat’ev, J. B. Bariwal, H. P. L.

Steenackers, S. C. J. De Keersmaecker, E. V. Van der Eycken, (2010) Angew. Chem. Int. Ed.

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Crystal and Molecular Structure Analysis of

DP-7: A New Multi Drug Resistance Reverter

Lead Molecule

Chapter-5 “Crystal and Molecular Structure Analysis of DP-7: A New

Multi Drug Resistance Reverter Lead Molecule”

NH

OO

O

The synthesis of this 3,5-Dibenzoyl-4-(3-methoxyphenyl)-1,4-dihydro-2,6-dimethyl

pyridine DP7, bearing dibenzoyl groups at C(3) and C(5), respectively, has been

achieved by applying the modified Hantzsch-type condensation of 1,3-Diketone (2

equiv) and aromatic aldehydes (1 equiv) using ammonium carbonate as nitrogen

source. The product obtained was characterized by spectroscopic techniques & in

order to correlate structure activity relationship, the X-ray crystallographic study of

DP-7 has been carried out.

Publication:

[1] Synthesis, Characterization, Crystal and molecular structure analysis of DP-7:

A new Multi Drug Resistance Reverter Lead Molecule Cryst. Res. Technol. (2010)

Manuscript in preparation.

5.1 Introduction

The membrane efflux protein P-glycoprotein, a member of the ATP binding cassette

(ABC) family, recognizes and transports structurally, chemically, and

armacologically diverse hydrophobic compounds, giving rise to a phenomenon known

as multidrug resistance (MDR). Development of MDR is one of the main reasons of

failure in cancer chemotherapy, as tumour cells, by increasing drug efflux, acquire

cross-resistance to many structurally and functionally unrelated anticancer agents,

which therefore never achieve effective intracellular concentrations1. In the past few

years, extensive studies have been performed with the aim of developing effective

chemo sensitizers to overcome MDR of human cancer cells. Potent P-glycoprotein

inhibitors have been tested in clinical trials so far, including Ca2+ channel blockers

such as verapamil and dihydropyridines2,3. However, clinical application of these

agents has not been extensively pursued to date, owing to their unwanted and

sometimes life threatening cardiovascular side effects such as atria-ventricular block

and hypotension. As a consequence, in the last few years, much attention has been

focused on congeners of these first generation-MDR inhibitors, in order to develop

compounds characterized by appropriate potency and selectivity but also by reduced

cardiovascular toxicity.

Very recently, 3,5-Dibenzoyl-4-(3-phenoxyphenyl)-1,4-dihydro-2,6-dimethylpyridine

(DP7) was proposed as a new MDR reverter. It was found that DP7, at a

concentration two order of magnitude higher than its IC50 as a P-glycoprotein

inhibitor, was devoid of cardiovascular effects in in vitro rat preparations4,5. These

data were not considered sufficient evidence of DP7 safety before this drug could be

subjected to clinical investigation. Several 1,4 dihydropyridine calcium antagonists

are substrate of CYP enzymes. In particular, CYP3A4 metabolizes dihydropyridines

into the corresponding pyridines6. The dihydropyridine can also inhibit the activity of

CYP3A4. For example, Katoh7 reported that nicardipine, benidipine, maidipine and

barnidipine strongly inhibited human CYP3A4 activity, while nivaldipine, nifedipine,

nitrendepine and amlodipine exhibited a weak inhibition.

The DP-7 was very well explored for variety of biological activity8. It was very

important to study the X-ray Crystal structure of this molecule which was not studied

so far and therefore development of single crystal was taken up in the current work.

Thus the synthesis was optimized and studied.

5.2 Reaction Scheme

5.2.1 Scheme-1 1-phenylbutane-1,3-dione

O

O

O

Ph

O O

CH3ONa

0 oC

Ph

5.2.2 Scheme-2 DP-7

NH

Ph

O

Ph

O

OPh

(NH4)2CO3

MeOH

18 Hrs refluxPh

O

O

O

OPh

O

Ph

O

5.3 Method of Crystallization

0.1gm of DP-7 was taken in 25 ml of methanol into 50ml single neck RBF and

Charcoal (2 g) was added. The solution was heated up to reflux temperature on a

water bath for 45 min. The solution was filtered, while hot through Whatmann 42

filter paper. The resulting solution was kept in a stopper conical flask slightly opened.

After 10 days, crystals grew in cubic shape due to thin layer evaporation. They were

filtered and washed with chilled methanol.

5.4 Crystal structure determination: A single crystal of suitable size was chosen for X-ray diffraction studies. The data

were collected at room temperature on a DIPLabo Image Plate system with graphite

monochromated radiation MoKα. Each exposure of the image plate was set to a period

of 400 s. Thirty-six frames of data were collected in the oscillation mode with an

oscillation range of 5˚ and processed using Denzo9. The reflections were merged with

Scalepack. All the frames could be indexed using a primitive trilinic lattice. The

structure was solved by direct methods using SHELXS-9710. Least-squares refinement

using SHELXL-9710with isotropic displacement parameters for all the non-hydrogen

atoms converged the residual to 0.1999. Subsequent refinements were carried out with

anisotropic thermal parameters for the non-hydrogen atoms. After eight cycles of

refinement the residuals converged to 0.0578. The hydrogen atoms were fixed at

chemically acceptable positions, and were allowed to ride on their parent atoms.

5.5 Results and discussion: The details of crystal data and refinement are given in Table 1. The bond lengths and

bond angles of all the non-hydrogen atoms (Table 2 and 3.) and are in good agreement

with the standard values10. Figure 1 represents the ORTEP12 diagram of the molecule

with thermal ellipsoids drawn at 50% probability.

In the title compound C33H27NO3, the 1,4-dihydropyridine ring is in boat

conformation, with atoms N1 and C4 deviating from the plane13 (Cremer and Pople,

1975) defined by the atoms N1/C2/C3/C4/C5/C6 by 0.1793(2)Å and 0.271(2)Å,

respectively. The 1,4-dihydropyridine ring is puckered. The puckering parameters are

Q = 0.406(2)Å, θ =106.0(3)˚ and Φ =2.4(3)˚. The dihedral angle between the least

squares planes of the ring 1 and 2 is 87.70(1)˚, indicating that the ring 2 is nearly

orthogonal to the dihydropyridine ring. The dihedral angles between the plane (1,3),

(1,4) and (3,4) are 60.74(1)˚, 53.54˚ and 79.49˚, respectively. The structural

conformation of carbomyl groups at C3 and C5 position is described by the torsion

angle values of 50.09˚ and 161.10˚ and have -syn-periplanar and -anti-periplanr

conformations. The structure exhibits coplanarity of the carbomyl group (C=O) with

the conjugated double bond of the 1,4-dihydropyridine ring. The structure exhibit

both inter and intramolecular hydrogen bonds of the type N–H. . .O and C–H. . .O, the

hydrogen bonds bind the molecules into infinite one- dimensional chain. The

observed hydrogen bonds are listed in Table 4. The packing of the molecules down b

axis is shown in the Figure 2.

Empirical formula C33H27NO3

Formula weight 485.56 Temperature 293 k Wavelength 0.71073 Å

Crystal system Triclinic Space group Pī

Cell dimensions a = 7.4310(6Å), b =12.2580(16)Å, c =

14.4180(17)Å, α = 92.066(3)˚, β=95.319(7)˚, γ= 96.235(7)˚

Volume 1298.5(3)Å3 Z 2

Density(calculated) 1.242Mg/m3 Absorption coefficient 0.079 mm-1

F000 512 Crystal size 0.30 x 0.27 x 0.25 mm

Theta range for data collection 3.07˚ to 25.02˚ Index ranges -8<=h<=8, -14<=k<=14, -17<=l<=17

Reflections collected 6548 Independent reflections 4085 Absorption correction None Refinement method Full-matrix least-squares on F2

Data / restraints / parameters 4085 / 0 / 337 Goodness-of-fit on F2 1.035

Final R indices [I > 2σ(I)] R1 = 0.0578, wR2 = 0.1493 R indices (all data) R1 = 0.0728, wR2 = 0.1624

Extinction coefficient 0.045(8) Largest diff. peak and hole 0.446 and -0.315e.Å-3

Deposition number CCDC Table-1: Experimental crystallographic data

Atoms Length(Å) Atoms Length (Å) N1-C2 1.374(2) C17-C18 1.369(5) N1-C6 1.392(3) C18-C19 1.354(6) C2-C3 1.377(3) C19-C20 1.331(7) C2-C8 1.495(3) C20-C21 1.334(6) C3-C30 1.456(3) C22-O23 1.217(3) C3-C4 1.509(3) C22-C24 1.502(3) C4-C5 1.524(3) C24-C25 1.385(3) C4-C9 1.535(3) C24-C29 1.390(3) C5-C6 1.343(3) C25-C26 1.383(4) C5-C22 1.480(3) C26-C27 1.365(5) C6-C7 1.497(3) C27-C28 1.370(6) C9-C10 1.377(3) C28-C29 1.376(5) C9-C14 1.390(3) C30-O31 1.241(2) C10-C11 1.388(3) C30-C32 1.493(3) C11-C12 1.358(4) C32-C33 1.388(3)

C12-C13 1.372(4) C32-C37 1.390(3) C13-C14 1.383(4) C33-C34 1.382(3) C13-O15 1.392(3) C34-C35 1.372(4) O15-C16 1.387(3) C35-C36 1.379(4) C16-C17 1.353(4) C36-C37 1.378(3) C16-C21 1.362(5)

Table-2: Bond lengths (Å)

Atoms Angle(˚) Atoms Angle(˚) C2-N1-C6 122.62(2) C21-C16-O15 117.5(3) N1-C2-C3 118.10(2) C16-C17-C18 118.2(3) N1-C2-C8 113.33(2) C19-C18-C17 119.5(4) C3-C2-C8 128.53(2) C20-C19-C18 121.0(4) C2-C3-C30 126.66(2) C19-C20-C21 120.7(4) C2-C3-C4 118.06(2) C20-C21-C16 119.1(3) C30-C3-C4 115.09(2) O23-C22-C5 123.7(2) C3-C4-C5 108.93(1) O23-C22-C24 119.5(2) C3-C4-C9 114.15(2) C5-C22-C24 116.65(2) C5-C4-C9 109.54(2) C25-C24-C29 118.8(3) C6-C5-C22 122.16(2) C25-C24-C22 121.4(2) C6-C5-C4 118.62(2) C29-C24-C22 119.7(2) C22-C5-C4 119.04(2) C26-C25-C24 120.6(3) C5-C6-N1 117.99(2) C27-C26-C25 119.9(3) C5-C6-C7 127.0(2) C26-C27-C28 120.0(3) N1-C6-C7 114.96(2) C27-C28-C29 120.9(3)

C10-C9-C14 117.8(2) C28-C29-C24 119.8(3) C10-C9-C4 123.18(2) O31-C30-C3 118.58(2) C14-C9-C4 118.97(2) O31-C30-C32 117.36(2) C9-C10-C11 120.7(2) C3-C30-C32 123.99(2) C12-C11-C10 121.3(3) C33-C32-C37 119.10(2) C11-C12-C13 118.6(2) C33-C32-C30 118.92(2) C12-C13-C14 121.0(2) C37-C32-C30 121.67(2) C12-C13-O15 116.8(2) C34-C33-C32 120.4(2) C14-C13-O15 122.0(3) C35-C34-C33 120.1(2) C13-C14-C9 120.5(2) C34-C35-C36 119.9(2)

C16-O15-C13 119.0(2) C37-C36-C35 120.6(2) C17-C16-C21 121.5(3) C36-C37-C32 119.9(2) C17-C16-O15 120.8(3)

Table-3: Bond angles (˚)

Atoms Length (Å) Angle(˚) Symmetry codes

N1–H1. . .O31 2.999(2) 156 1 + x, y, z Table-4: Hydrogen bonding geometry

Fig.-1: ORTEP of the molecule with thermal ellipsoids drawn at 50% probability. Fig.-2: Packing of the molecules when viewed down the a axis. The dashed line

represents the hydrogen bond.

Fig.-3: Packing of the molecules when viewed down the b axis. The dashed line

represents the hydrogen bond.

Fig.-4: Packing of the molecules when viewed down the c axis. The dashed line represents the hydrogen bond.

5.6 Conclusion:

3,5-Dibenzoyl-4-(3-methoxyphenyl)-1,4-dihydro-2,6-dimethyl pyridine (DP-7) has

been shown to be a powerful P-gp inhibitor, almost devoid of cardiovascular effects,

but capable of inhibiting liver CYP3A, thus it can be conclude that DP-7 is a good

lead compound for the synthesis of new dihydropyridines. As DP-7 shown promising

MDR activity and looking to the scientific publications of DP-7, studied of X-ray

crystal structure of DP-7 performed.

5.7 Experimental:

5.7.1 Preparation of 1-phenylbutane-1,3-dione

Cool peaces of freshly prepared sodium methoxide (6 gm) at 0 oC using ice cold

water. To that added ethyl acetate (100 ml) slowly and stirred at 0 oC for 10 min. to

that solution added acetophenone (30 ml) drop wise over 45 min and stirred for

another 12 hrs. After completion of reaction filtered the reaction mass, washed with

diethyl ether and dried in rotavap. After that dissolved the crude product in to water

and filter it to removed unwanted crude material. To that filtrate added gl. acetic acid

to set Ph 3. Solid formed was filtered and dried to give titled compound as white

powder (mp. 61 oC).

5.7.2 Synthesis of 3,5-Dibenzoyl-4-(3-methoxyphenyl)-1,4-dihydro-2,6-dimethyl

pyridine(DP-7)

To a solution of 1-phenylbutane-1,3-dione (20mmol) in dry methanol (25ml), 3-

phenoxybenzaldehyde (10mmol) and ammonium carbonate (1.5g) was added and the

reaction mixture was refluxed for 18hrs with vigorous stirring. After completion of

the reaction, reaction mixture was cooled to RT and was poured on to ice-water

mixture (100ml) and extracted with dichloromethane (2 x 100ml). The organic

extracts were combined and dried over sodium sulfate and the solvent was removed at

reduced pressure. The residue was subjected to the column chromatography (silica

gel; Hexane-Ethyl acetate, 5:5 v/v) to give titled compound as light yellow crystals

(M.P. 190-192°C).

5.8 Characterization

3,5-Dibenzoyl-4-(3-methoxyphenyl)-1,4-dihydro-2,6-dimethyl pyridine(DP-7)

NH

Ph

O

Ph

O

OPh

1H NMR (400 MHz CDCl3): δ = 7.57-7.53 (m 4H), 7.46-7.42 (m 2H), 7.36-7.31 (m

4H), 7.28-7.24 (m 2H), 7.10-7.02 (m 2H), 6.86-6.84 (m 2H), 6.72-6.70 (d of d 2H, J =

2,2), 6.67 (t 1H, J =2), 5.68 (s 1H), 5.15 (s 1H), 1.92 (s 6H). 13C NMR (400 MHz

CDCl3): δ = 197.8 (×2), 157.2 (×2), 147.8 (×2), 140.3 (×2), 131.5, 129.8 (×3), 129.2

(×2), 128.4 (×4), 128.2 (×4), 122.8, 122.3, 118.5, 117.9 (×2), 117.1, 112.6 (×2), 43.7,

19.0 (×2). MS (m/z): 485 (M+, 62%), 316 (100%). IR (KBr, cm-1): 3396(N–H

stretching), 3030(C-H stretching, Asymmetric), 2930(C–H stretching, Symmetric),

1703(C=O stretching, ester), 1670(C=O stretching, ketone), 1589(N–H deformation).


5.9.1 1H NMR spectra of bs-DP-7

5.9.2 13C spectra of bs-DP-7

5.10 References [1] Fusi F., Saponara S., Valoti M., Dragoni S., D'Elia P., Sgaragli T., Alderighi D., Kawase M.,

Shah A., Motohashi N., Sgaragli G. (2006)Curr. Drug Targets. 8, 949–959.

[2] Bates, S.E., Regis, J.I., Robey, R.W., Zhan, Z., Scala, S., Meadows, B.J. (1994)

Chemoresistance in the clinic: overview. Bull. Cancer 81 (suppl 2), 55–61.

[3] Bellamy, W.T. (1996) Annu. Rev. Pharmacol. Toxicol. 36, 161–183

[4] Saponara S., Kawase M., Shah A., Motohashi N., Molnar J., Ugocsai K., Sgaragli G., Fusi F.

(2004) Br. J. Pharm. 141, 415-422.

[5] Saponara S., Ferrara A., Gorelli B., Shah A., Kawase M., Motohashi N., Molnar J., Sgaragli

G., Fusi F. (2007) Eur. J. Pharmacol. 563, 160–163.

[6] Guengerich, F.B., Brian, W.R., Iwasaki, M., Sari, M.A., Baarnhielm, C., Berntsson, P. (1991)

J. Med. Chem. 34, 1838–1844.

[7] Katoh, M., Nakajima, M., Shimada, N., Yamazaki, H., Yokoi, T. (2000) Eur. J. Clin.

Pharmacol. 55, 843–852

[8] For synthesis and biological activity see (a) Paolo D'Elia , Francesco De Matteis , Stefania

Dragoni , Anamik Shah ,Giampietro Sgaragli, Massimo Valoti. (2009) European Journal of

Pharmacology. 614, 7–13 (b) Masami Kawase, Anamik Shah, Harsukh Gaveriya, Noboru

Motohashi, Hiroshi Sakagami, Andreas Vargae, Joseph Molnar. (2002) Bioorganic &

Medicinal Chemistry. 10, 1051–1055. (c) Prashant S. Kharkar, Bhavik Desai, Harsukh

Gaveria, Bharat Varu, Rajesh Loriya, Yogesh Naliapara, Anamik Shah, Vithal M. Kulkarni.

(2002) J. Med. Chem. 45, 4858-4867

[9] Otwinowski, Z. Minor, W. In: Methods in Enzymology, 276, Carter, C. W. Jr., Sweet, R. M.,

(1997) New York: Academic Press, 307-326.

[10] Sheldrick, G.M. (2008) Acta Cryst., A64, 112-122.

[11] Allen, F.H., Kennard, O., Watson, D.G., Brummer, L., Orpen, A.G., Taylor, R. (1987) J.

Chem. Soc. Perkin Trans, II:S1.

[12] Spek, A.L. (2003) J. Appl. Cryst. 36, 7-13.

[13] Cremer, D., and Pople, J.A. (1975) J. Am. Chem. Soc. 97, 1354-1358.

SUMMARY

The work represented in the thesis entitled “Synthesis and Characterization of New

heterocyclic compounds & their application” is divided into five chapters which can

be summarized as under.

Chapter-1 covers basic introduction to Microwave assisted organic synthesis

including comparison of conventional vs microwave assisted reaction and

introduction of 2-aminoimidazole and their presence as naturally occurring marine

alkaloids described. In this part various synthetic approaches toward 2-

aminoimidazole and short review on 2-aminoimidazole as pharmacophores also

presented. Finally basic concept of anti biofilm activity described.

Chapter-2 deals with microwave assisted one pot synthesis and Structure Activity

Relationship of 2-Hydroxy-2-phenyl-2,3-dihydro-imidazo[1,2-a] pyrimidinium Salts

and 2N-Substituted 4(5)-Phenyl-2-Amino-1H-imidazoles as Inhibitors of the Biofilm

Formation by Salmonella Typhimurium and Pseudomonas aeruginosa.

Chapter-3 entitled “Structure Activity Relationship of N1-Substituted 2-

Aminoimidazoles as Inhibitors of Biofilm Formation by Salmonella Typhimurium

and Pseudomonas aeruginosa.” In this chapter a library of N1-substituted 4(5)-phenyl-

2-aminoimidazoles was synthesized and tested for the antagonistic effect against

biofilm formation by Salmonella Typhimurium and Pseudomonas aeruginosa. The

substitution pattern of the 4(5)-phenyl group and the nature of the N1-substituent were

found to have a major effect on the biofilm inhibitory activity. The most active

compounds of this series were shown to inhibit the biofilm formation at low

micromolar concentrations.

Chapter-4 deals with microwave-assisted Cu-catalyzed one pot protocol for the

synthesis of 2-aminoimidazole-triazole framework via two consecutive steps of

dimorth rearrangement and click reaction was demonstrated. Library of 22

compounds have been synthesized and were subjected to test for antibiofilm activity.

However, the antibiofilm activities of these classes of compounds are under pipeline.

After careful study of Structure Activity Relationship, a fresh libraries of N1-

substituted 2-aminoimidazoles and N1-unsubstituted 2-aminoimidazoles have been

synthesized and tested for anti biofilm activity. In Chapter-4 microwave assisted one

pot protocol for synthesis of 2-aminoimidazole-triazoel framework has been described

and all newly synthesized compounds were tested for antibiofilm activity. However

activities of these calls of compounds are under pipeline.

Chapter-5 entitled “Crystal and molecular structure analysis of DP-7: A new Multi

Drug Resistance Reverter Lead Molecule” deal with crystal and molecular structure

analysis. 3,5-Dibenzoyl-4-(3-methoxyphenyl)-1,4-dihydro-2,6-dimethyl pyridine

(DP-7) has been shown to be a powerful P-gp inhibitor, almost devoid of

cardiovascular effects, but capable of inhibiting liver CYP3A, thus it can be conclude

that DP-7 is a good lead compound for the synthesis of new dihydropyridines. As DP-

7 demonstrated promising MDR activity, several scientific publications on this

molecule appeared. However study of X-ray crystallography was done first time and

presented in the current work.

All newly synthesized compounds have been characterized by 1H NMR, 13C NMR

and HRMS. Some representative copy of 1H and 13C NMR spectra also presented.

CONFERENCES/SEMINARS/WORKSHOPS ATTENDED

“International Conference On The Interface of Chemistry-Biology In

Biomedical Research” jointly organized by ISCBC and Birla Institute of

Technology & Science, Pilani. February, 22-24, 2008

“National Workshop On Management And Use Of Chemistry Database

And Patent Literature” organized by GUJCOST & Dept. of Chemistry of

Saurashtra University, Rajkot, (Gujarat), February, 27-29, 2008.

“A National Workshop On Updates In Process And Medicinal Chemistry”

jointly organized by National Facility for Drug Discovery through New

Chemicals Entities Development & Instrumentation support to Small

Manufacturing Pharma Enterprises and DST FIST, UGC-SAP & DST-DPRP

Funded Department of Chemistry, Saurashtra University, Rajkot March, 3-4,

2009.

“National Conference On Selected Topics In Spectroscopy And

Stereochemistry” organized by the Department of Chemistry, Saurashtra

University, Rajkot, March, 18-20, 2009.

“12th Belgian Organic Synthesis Symposium” (BOSS XII) Namur, Belgium

July 11-16, 2010.

Paper/Poster presented at the International Conference:

“Microwave-Assisted One Pot Synthesis Of Anti-Biofilm Active 2-

Aminoimidazole -Triazole Conjugates Using Copper Nanoparticles”

Bharat S. Savalia, Denis S. Ermolat’ev, Anamik K. Shah and Erik V. Van der

Eycken.“12th Belgian Organic Synthesis Symposium” (BOSS XII) Namur,

Belgium (2010)

“A General Approach To 2-Aminoimidazole Marine Sponge Alkaloids

From Leucetta sp.”

Denis S. Ermolat’ev, Bharat S. Savalia, Jitender B. Bariwal and Erik V. Van

der Eycken,“12th Belgian Organic Synthesis Symposium” (BOSS XII)

Namur, Belgium (2010)

“Crystal Structure Of DP-7: A New MDR Reverting Agent Against

Tumor Cells”

Manisha Parmar, Bharat Savaliya, Harsukh Gaveriya, Lakshminarayana B.N.

J. Shashidhara Prasad, M. A. Sridhar and Anamik Shah. ISCBC.

Lucknow,India, 2010.

“Rapid Synthesis Of Fused Bicyclic Thiazolo-Pyrimidine And Purimido-

Thiazine Derivatives Devoid Of Use Of Catalyst By Microwave Assisted

Method.”

Bharat S.Savaliya, Vijay Virsodia, Punit Rasadia, Nikhil Vekaria, Rupesh

Khunt And Anamik Shah.ISCBC., BITS-Pilani, India.2008

PUBLICATIONS [1] One-Pot Microwave-Assisted Protocol For The Synthesis Of Substituted 2-

Amino-1H-Imidazoles. Denis S. Ermolat’ev, Bharat Savaliya, Anamik Shah

and Erik V. Van der Eycken. Molecular Diversity. (2010) DOI

10.1007/s11030-010-9270-5

[2] Structure Activity Relationship of 2-Hydroxy-2-phenyl-2,3-dihydro-

imidazo[1,2-a]pyrimidinium Salts and 2N-Substituted 4(5)-Phenyl-2-Amino-

1H-imidazoles as Inhibitors of the Biofilm Formation by Salmonella

Typhimurium and Pseudomonas aeruginosa. P.L. Steenackers, Denis S.

Ermolat’ev, Bharat Savaliya, Ami De Weerdt, David De Coster, Anamik

Shah, Erik V. Van der Eycken, Dirk E. De Vos, Jozef Vanderleyden and

Sigrid C.J. De Keersmaecker. Bio. & Med. Chem. Submitted (2010)

[3] Structure Activity Relationship of 4(5)-Phenyl-2-amino-1H-imidazoles, N1-

Substituted 2-Aminoimidazoles and Imidazo[1,2-a]pyrimidinium Salts as

Inhibitors of Biofilm Formation by Salmonella Typhimurium and

Pseudomonas aeruginosa. Hans P.L. Steenackers, Denis S. Ermolat’ev,

Bharat Savaliya, Ami De Weerdt, David De Coster, Anamik Shah, Erik V.

Van der Eycken, Dirk E. De Vos, Jozef Vanderleyden and Sigrid C.J. De

Keersmaecker. J. Med. Chem. (2010) DOI: 10.1021/jm1011148

[4] Copper Catalyzed Microwave Assisted One Pot Synthesis of 2-

Aminoimidazole-Triazole Framework. Bharat Savaliya, Denis S. Ermolat’ev,

Anamik Shah and Erik V. Van der Eycken. (2010) Manuscript in preparation.

Org. lett.

[5] Crystal And Molecular Structure Analysis Of DP-7: A New Multi Drug

Resistance Reverter Lead Molecule. Bharat Savaliya, Manisha Parmar,

Vaibhav Ramani, Harsukh Gevariya, B.N. Lakshminarayana, J. Shashidhara

Prashad, M. A. Sridhar and Anamik Shah. (2010) Manuscript in preparation.

[6] Catalyst-Free, Rapid Synthesis of Fused Bicyclic Thiazolo-Pyrimidine and

Pyrimido-Thiazine Derivatives by a Microwave-Assisted Method.

Vijay R. Virsodia, Nikhil R. Vekariya, Atul T. Manvar,Rupesh C. Khunt,

Bhavin R. Marvania, Bharat S.Savalia and Anamik K. Shah. Phosphorus,

Sulfur, and Silicon, 184:34–44 (2009)

Mol DiversDOI 10.1007/s11030-010-9270-5

FULL-LENGTH PAPER

One-pot microwave-assisted protocol for the synthesisof substituted 2-amino-1H -imidazoles

D. S. Ermolat’ev · B. Savaliya · A. Shah ·E. Van der Eycken

Received: 19 May 2010 / Accepted: 7 August 2010© Springer Science+Business Media B.V. 2010

Abstract An efficient microwave-assisted one-pot two-stepprotocol was developed for the construction of disubstituted2-amino-1H -imidazoles. This process involves the sequen-tial formation of 2,3-dihydro-2-hydroxyimidazo[1,2-a]pyri-midinium salts from readily available 2-aminopyrimidinesand α-bromoketones, followed by cleavage of the pyrimi-dine ring with hydrazine.

Keywords Heterocycles · 2-Aminoimidazoles ·Microwave-assisted synthesis · Ring opening ·Rearrangement

Introduction

The class of 2-aminoimidazoles has recently attracted partic-ular interest due to various biological properties of these com-pounds. 2-Aminoimidazole alkaloids and their metabolites,isolated from the marine sponges Hymeniacidon sp., havebeen described as potent antagonists of serotonergic [1] andhistaminergic receptors [2]. Naamine and isonaamine alka-loids from the marine sponges Leucetta sp. exhibit antiviraland anticancer activity [3–10]. Because of these interesting

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11030-010-9270-5) contains supplementarymaterial, which is available to authorized users.

D. S. Ermolat’ev · B. Savaliya · E. Van der Eycken (B)Laboratory for Organic and Microwave-Assisted Chemistry(LOMAC), Department of Chemistry, Katholieke UniversiteitLeuven, Celestijnenlaan 200F, 3001 Leuven, Belgiume-mail: [email protected]

B. Savaliya · A. ShahDepartment of Chemistry, Saurashtra University,Rajkot 360 005, Gujarat, India

N

N

N

R2

OH

R1Br

3

NH

R1HNN R2

12

R1 = H

A BN

N

N

R2

Scheme 1 Synthesis of substituted 2-amino-1H -imidazoles

biological activities, numerous synthetic routes to 1-substi-tuted and 1-unsubstituted 2-aminoimidazoles have beenreported. Modern synthetic methods for accessing 1-unsub-stituted 2-amino-1H -imidazoles can be roughly classifiedas heterocyclization of substituted or protected guanidineswith 1,2-dielectrophiles [11,12], heteroaromatic nucleophilicsubstitution, and recyclization of 2-aminooxazoles [13].Although different substituted guanidines are readily avail-able and can be prepared in situ (e.g., from cyanamides [14]),the high basicity of guanidines together with non-regioselec-tivity often leads to multiple products. Protection by acetyl[11] and Boc-groups [12] requires, in turn, acidic deprotec-tion conditions.

Recently, we have described two new approaches to thesynthesis of substituted 2-amino-1H -imidazoles 1. The firstapproach is based on the cleavage of imidazo[1,2-a]pyrimi-dines [15] 2 with hydrazine (Scheme 1, route A) [16]. In thesecond approach, the hydrazinolysis of 2-hydroxy-2,3-dihydroimidazo[1,2-a]pyrimidinium salts 3 followedby Dimroth-type rearrangement afforded trisubstituted2-amino-1H -imidazoles 1 (Scheme 1, route B) [17].

2-Hydroxy-2,3-dihydroimidazo[1,2-a]pyrimidinium salts3 can be easily prepared from 2-aminopyrimidines 4 withα-bromoketones 5 under microwave irradiation (Scheme 2).We found that the outcome of this reaction strongly dependson the temperature [18]. The microwave-assisted reaction

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Mol Divers

Scheme 2 Competitiveformation of 1-unsubstitutedand 1-substituted2-amino-1H -imidazoles uponmicrowave irradiation

N

N

N

R2

NH

R1HNN R2

OH

R1Br

13N

N

NHR1

O

R2

Br+

4

5N

N

N

R2

R1

6

NH2N

N

R2

7R1

MW

N2H4

>100 °C

<100 °C

MW

Br

N2H4

MW

Table 1 Investigation of thecondensation underconventional heating andmicrowave irradiation

All reactions were carried out ona 1 mmol scale of2-methylaminopyrimidine(1, R1 = Me) with 1.35 equiv. ofα-phenacylbromide (2, R2 = Ph)in MeCN (5 mL)a Isolated yield afterrecrystallization from MeCN

N

N

N

Ph

OH

MeBr

3

N

N

N

Ph

Me

6

+N

N

NHMe+ Br

Ph

O

4 (R1 = Me) 5 (R2 = Ph)

ConditionsBr

Entry Conditions Time (min) T ( ◦C) 3 (Yield %)a 6 (Yield %)a

1 � 30 80 64 0

2 60 80 77 0

3 30 100 81 Traces

4 60 100 85 Traces

5 30 120 68 17

6 60 120 53 28

7 MW 30 80 88 0

8 60 80 85 0

9 30 100 48 33

10 60 100 45 35

11 30 120 12 79

12 60 120 Traces 84

between 2-aminopyrimidines 4 and α-bromoketones 5 per-formed at 80–100 ◦C gave 2-hydroxy-2,3-dihydroimi-dazo [1,2-a]pyrimidinium salts 3, while irradiation of thereaction mixture at temperatures above 100 ◦C providedaromatic 1-substituted imidazo[1,2-a ]pyrimidinium salts6 (Scheme 2). The hydrazinolysis of the salts 3 and 6 leads tothe corresponding 1-unsubstituted and 1-substituted2-amino-1H -imidazoles 1 and 7. Thus, by performing thereaction at 130−150 ◦C, we were able to prepare a library of1,4,5-trisubstituted 2-aminoimidazoles 7 via a one-pot two-step protocol [18].

In continuation of our ongoing studies, herein we reporta simple one-pot procedure for the synthesis of 1-unsubsti-tuted 2-amino-1H -imidazoles 1 from 2-aminopyrimidines 4and α-bromoketones 5 under microwave irradiation.

Results and discussion

We carefully investigated the formation and dehydratation ofsalt 3 resulting in the formation of salt 6 under conventionalheating conditions as well as upon microwave irradiation.As a proof of concept, the condensation of 2-methylamino-pyrimidine (4, R1 = Me) and α-phenacylbromide (5, R2 = Ph)was studied (Table 1). A sealed vial containing a solution ofthe starting compounds in acetonitrile was conventionallyheated with an oil bath (Table 1, entries 1–6) or irradiatedwith microwaves (Table 1, entries 7–12) at different tem-peratures for 30–60 min. The formation of the 2-hydroxysalt 3 was faster under microwave irradiation, and this com-pound was obtained in 88% yield within 30 min. Furtherincrease of the temperature up to 120 ◦C using conventional

123

Mol Divers

Table 2 Microwave-assistedsynthesis of substituted2-amino-1H -imidazoles 1

NH

R1HNN R2N

N

NHR1

O

R2

Br+

1. MW 80 °C MeCN 30 min

2. N2H4, MW 90 °C MeCN 10 minN

N

N

R2

OH

R1

13

4

5

Br

entry 2-aminopyrymidine 4 α–bromoketone 5 product 1 yield

(%)a

1 N

N

NH2

Br

O

Ph

N

NH

H2N

Ph

1{1}

75

2 N

N

NH2

Br

O

F

N

NH

H2N

F

1{2}

79

3 N

N

NH2

Br

O

Cl

N

NH

H2N

Cl

1{3}

88

4 N

N

NH

Me

Br

O

OMe

OMe

N

NH

HN

OMe

1{4}

MeO

Me

48

5 N

N

NH

Piperonyl

Br

O

N

NH

HN

1{5}

Piperonyl

72

6 N

N

NH

Pr

Br

O

Cl

ClN

NH

HN

Cl

1{6}

ClPr

70

heating or microwave irradiation led to a mixture of salts 3and 6 (Table 1). Interestingly, using microwave irradiation at120 ◦C for 60 min, we were able to drive the reaction com-pletely to the formation of the imidazo[1,2-a]pyrimidiniumsalt 6 (Table 1, entry 12).

As the transformation of salts 3 into salts 6 is relativelyrapid at the temperatures above 100 ◦C, the synthesis ofsalts 3 was conducted at 80 ◦C. Treatment of substituted

2-aminopyrimidines 4 with 15 mol% excess of α-bromok-etones 5 in MeCN under microwave irradiation for 30 mingenerated the intermediate salts 3 which, in most cases, pre-cipitated from the reaction mixture upon cooling (Table 2).Reaction progress was monitored by mass-spectrometry.In all the cases examined, the reaction appeared to be com-plete after 30 min. The cleavage step was performed undermicrowave irradiation at 90 ◦C, using seven equivalents of

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Table 2 continued

9 N

N

NH

PentylBr

O

Cl

N

NH

HN

1{9}

Cl

Pentyl 61

10 N

N

NH

HexylBr

O

F

N

NH

HN

1{10}

Hexyl

F

69

11 N

N

NH

PMB

Br

O

Br

N

NH

HN

1{11}

PMB Br 44

12 N

N

NH

Cyclooctyl Br

O

SO2Me

N

NH

HN

1{12}

SO2Me

Cyclooctyl 66

13 N

N

NH

Bu

Br

O

N

NH

HN

1{13}

Bu 83

14 N

N

NH

PMBBr

O

NO2

N

NH

HN

1{14}

NO2

PMB 89

7 N

N

NH

Br

O

SO2Me

N

NH

HN

1{7}

SO2Me

73

8 N

N

NH

OctylBr

O

Cl

N

NH

HN

1{8}

Cl

Octyl 79

hydrazine hydrate. Based on the data given in Table 2, thereaction appears to be compatible with both aryl and alkylsubstitutions. All the reactions were clean, smooth, and pro-vided the products 1{1–18} in good and high yields. Thecompounds were purified by column chromatography using5–10% MeOH in CH2Cl2 as the eluent. All the final 2-amino-1H -imidazoles 1{1–18} were characterized by 1H and 13CNMR spectroscopy. Their compositions were also confirmedby HRMS.

In conclusion, we have developed a simple and practicalmicrowave-assisted procedure for the preparation of substi-tuted 2-amino-1H -imidazoles. We have investigated a one-pot synthesis and cleavage of 2-hydroxy-2,3-dihydroimidazo[1,2-a]pyrimidinyl-4-ium bromides by hydrazine hydrate andfound microwave irradiation to be very effective in this regard.A small library of 2-amino-1H -imidazoles was synthesizedfor screening for potential antiviral and antibacterialactivities.

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Table 2 continued

All the reactions were carriedout on a 4 mmol scale of2-alkylaminopyrimidine 4 with1.15 equiv of α-bromoketone 5in MeCN (3 mL)a Isolated yield after columnchromatography

15 N

N

NH

BuBr

O

1{15}

N

NH

HN

Bu 88

16 N

N

NH

Br

O

SMe

N

NH

HN

SMe

1{16}

53

17 N

N

NH

OMeBr

O

NO2

1{17}

N

NH

HN

OMe

NO2

61

18 N

N

NH

Br

O

OMe1{18}

N

NH

HN

OMe

60

Experimental section

Microwave irradiation experiments

A monomode CEM-Discover microwave reactor (CEM Cor-poration, P.O. Box 200, Matthews, NC 28106) was usedin the standard configuration as delivered, including propri-etary software. All the experiments were carried out in sealedmicrowave process vials (10 mL) at the maximum power andtemperature, as indicated in the tables. After completion ofthe reaction, the vial was cooled to 50 ◦C via air-jet coolingbefore it was opened.

General procedure for the one-pot two-step microwave-assisted synthesis of 2-amino-1H -imidazoles 1{1–18}

In a microwave, vial (10 mL) was successively dissolvedin dry MeCN (3 mL), the corresponding 2-aminopyrimidine(4 mmol) and α-bromoketone (4.6 mmol). The reaction tubewas sealed and irradiated in a microwave reactor at a ceilingtemperature of 80 ◦C and a maximum power of 50 W for30 min. After the reaction mixture was cooled with an airflow for 15 min, hydrazine hydrate (0.9 mL, 28 mmol of a64% solution, 7 equiv.) was added, and the mixture was irra-diated at a ceiling temperature of 90 ◦C and a maximumpower of 50 W for another 10 min. After the reaction mixturewas cooled, hydrazine hydrate was removed by distillationwith toluene (3×20 mL). The resulting residue was purifiedby column chromatography (silica gel; MeOH-DCM 1:9 v/vwith 0.5% of 6 N ammonia in MeOH) to afford 1 as amor-phous solid.

Acknowledgments The support for this project was provided by theResearch Fund of the University of Leuven and by the F.W.O. (Fundfor Scientific Research—Flanders (Belgium)). E.D.S. is grateful to theUniversity of Leuven for obtaining a postdoctoral fellowship, and S.B.to Erasmus Mundus External Cooperation Window Lot 15 India(EMECW15) for obtaining a scholarship. The authors thank Ir.B. Demarsin for HRMS measurements.

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