ABSTRACT
The synthesis of ten new alkynylated derivatives of angular phenoxazine and alkynylated
naphthoquinone was thoroughly investigated. The first intermediate, 6-chloro-5Hbenzo[
a]phenoxazin-5-one was obtained by the condensation of 2-aminophenol with 2,3-
dichloro-1,4- naphthoquinone in the presence of anhydrous sodium tricarbonate (IV) .
Thereafter, the intermediate and 2,3-dichloro-1,4-naphthoquinone were each suggested to
Sonogashira cross–coupling reaction under copper-, amine-, and solvent free conditions at 80
oC with five different terminal alkynes using PdCL2(PPh3)2 and tetrabutylammonium
trihydrate (TBAF.3H2O) as the catalyst and ligand respectively to afford the alkynylated
angular phenoxazines and alkynylated naphthoquinone derivatives in good to excellent yield .
Structures of synthesized compounds were confirmed with Uv-visible, Fourier Transform –
Infrared (FT-IR), 1H-NMR and 13C-NMR spectroscopy. The synthesized compounds were
screened against five (5) micro-organisms viz: Staphylococcus aureus, Pseudomonas
aeruginosa, Klebsiella pneumonia, Escherichia coli 1 and Escherichia coli 12 using agar
well diffusion technique. The results showed significant improvement in antimicrobial
activities compared with gentamycin and ampicillin (standard drugs).
TABLE OF CONTENTS
Title Page – – – – – – – – – i
Approval Page – – – – – – – – – ii
Certification – – – – – – – – – iii
Dedication – – – – – – – – – iv
Acknowledgement – – – – – – – – – v
Abstract – – – – – – – – – vii
Table of Content – – – – – – – – – viii
List of Abbreviations – – – – – – – – – xii
List of Tables – – – – – – – – – xiv
List of Figures – – – – – – – – – xv
CHAPTER ONE – – – – – – – – – 1
1.0 Introduction – – – – – – – – – 1
1.1 Background of the study – – – – – – – 2
1.2 Statement of the problem – – – – – – – 6
1.3 Objective of the study – – – – – – – – 6
1.4 Justification of study – – – – – – – – 8
CHAPTER TWO – – – – – – – – – 9
2.0 Literature Review – – – – – – – – 9
2.1 Angular phenoxazines – – – – – – – 9
2.1.1 Benzo[a]phenoxazine – – – – – – – 9
2.1.2 Benzo[c]phenoxazine – – – – – – – 16
2.1.3 Dibenzophenoxazine ring system – – – – –
– 17
10
2.1.3.1 Dibenzo[a,h]phenoxazine – – – – – –
– 17
2.1.3.2 Dibenzo[a,i]phenoxazine – – – – – – – 18
2.1.3.3 Dibenzo[a,j]phenoxazine – – – – – – – 19
2.1.4 Biological Activity of Angular Phenoxazine – – – – 22
2.2 Sonogashira Cross-Coupling reaction – – – – – 23
2.2.1 Mechanism – – – – – – – – – 24
2.2.2 Advantages of Sonogashira cross coupling reaction- – – – 25
2.2.3 Limitations of the Sonogashira Cross-Coupling – – – 25
2.2.4 Efforts to Modify Reaction Conditions – – – – – 26
2.2.5 Applications of Sonogashira Cross-Coupling reaction- – – – 28
2.3 Antimicrobial activities of Naphthoquinones – – – – 33
CHAPTER THREE – – – – – – – – – 37
3.0 Experimental Section – – – – – – – – 37
3.1 General – – – – – – – – – – 37
3.2 6-Chloro-5H-benzo[a]phenoxazin-5-one – – – – – 38
3.3 General procedure for preparation of the derivatives using palladium-
Catalyzed Sonogashira Cross-Coupling reactions under Copper- ,amine-, and
solvent-free conditions – – – – – – – – 38
3.3.1 6-(Pheylethynyl)-5H-benzo[a]phenoxazin-5-one, 35a – – – 39
3.3.2 6-(3-Hydroxy-3-methylbut-1-yn-1-yl)-5H-phenoxazin-5-one, 35b – 40
3.3.3 6-(Hex-1-yn-1-yl)-5H-benzo[a]phenoxazin-5-one, 35c – – – 41
3.3.4 6-(3-hydroxypro-1-yn-1-yl)-5H-phenoxazin-5-one, 35d – – – 42
3.3.5 6-(Oct-1-yn-1-yl)-5H-benzo[a]phenoxazin-5-one, 35e – – – 43
3.3.6 2-Chloro-3-(phenylethynyl)-1,4-naphthoquinone, 33a – – – 44
11
3.3.7 2-Chloro(3-hydroxy-3-methylbut-1-yn-1-yl)-1,4-naphthoquinone, 3b – 45
3.3.8 2-Chloro-3-(hex-1-yn-1-yl)-1,4-naphthoquinone, 33c – – – 46
3.3.9 2-Chloro-3-(3-hydroxyprop-1-yn-1-yl)-1,4-naphthoquinone, 33d – – 47
3.3.10 2-Chloro-3-(Oct-1-yn-1-yl)-1,4-naphthoquinone, 33e – – – 48
3.4 Antimicrobial Activity – – – – – – – 49
3.4.1 Sensitivity Testing of synthesized Compounds – – – – 48
3.4.2 Minimum Inhibitory Concentration of the synthesized compounds – – 49
CHAPTER FOUR – – – – – – – – – 50
4.0 Results and Discussions – – – – – – – 50
4.1 6-Chloro-5H-benzo[a]phenoxazin-5-one – – – – – 50
4.1.1 6-(phenylethynyl)-5H-benzo[a]phenoxazin-5-one, 35a – – – 51
4.1.2 6-(3-Hydroxy-3-methyl but-1-yn-1-yl)-5H-beno[a]phenoxazin-5-one, 35b – 52
4.1.3 6-(Hex-1-yn-1-yl)-5H-benzo[a]phenoxazin-5-one, 35c – – – 52
4.1.4 6-(3-Hydroxyprop-1-yn-1-yl)-5H-benzo[a]phenoxazin-5-one, 35d – – 53
4.1.5 6-(Oct-1-yn-1-yl)-5H-benzo[a]phenoxazin-5-one, 35e – – – 54
4.1.6 2-Chloro-3-(phenylethynyl)-1,4-naphthoquinone, 33a – – – 55
4.1.7 2-Chloro-3-(3-hydroxy-3-methyl-but-1-yn-1-yl)-1,4-naphthoquinone, 33b – 55
4.1.8 2-Chloro-3-(hex-1-yn-1-yl)-1,4-naphthoquinone, 33c – – – 56
4.1.9 2-Chloro-3-(3-hydroxyprop-1-yn-1-yl)-1,4-naphthoquinone, 33d – – 57
12
4.1.10 2-Chloro-3-(oct-1-yn-1-yl)-1,4-naphthoquinone, 33e – – – 57
4.2 Evaluation of the synthesized alkynylated angular phenoxazines and
alkynylated naphthoquinones for antimicrobial activity – – – 59
4.2.1 Results of Sensitivity Testing of the Synthesized 6-alkynylated
-5H-benzo[a]phenoxazin-5-one – – – – – – 60
4.2.2 Results of sensitivity testing of the synthesized.2-chloro-3-alkynylated
-1,4-napthoquinone – – – – – – – – 60
4.2.3 Results of Inhibition zone diameter (IZD) – – – – – 61
4.2.4 Results of minimum inhibition concentration (MIC) – – – – 63
4.3 Conclusion – – – – – – – – – 64
REFERENCES – – – – – – – – – 65
13
CHAPTER ONE
1.0 INTRODUCTION
The chemistry of phenoxazine and its derivatives have been of considerable interest over the
years because of their important and impressive number of applications1 particularly as dyes
and drugs2,3. Phenoxazines are a pharmaceutically important class of tricyclic nitrogenoxygen
heterocycles4. They show tremendous pharmacological activities as anti-epileptic5,
antitumour6,7, anticancer8, antituberculosis9, antibacterial10,11, anthelminthic12, spasmolytic,
central nervous system (C.N.S) depressants,13,14 herbicides tranquilizers, sedatives15 and
parasiticidal agents16. Other applications of phenoxazine derivatives include their use as
antioxidants17, biological stains18,19, acid-base indicators20, and bromometric and
stannometric redox indicators21-25. Phenoxazine itself has been used as a stabilizer for the
polymerization of vinylpyridines26, polyethylene and polystyrene27. Some of its derivatives
were also reported as having radioprotective and antioxidative actions28.
Naphthoquinones are secondary metabolites largely found in plants, micro-organisms,
and some animals29. These compounds have been widely used as colourants for comestics30,
fabrics31, foods and for pharmacological activities such as antitumor, anti-inflammatory,
antibacterial, antiviral, antiproliferative, antiparasitic, cytotoxic activities and others32-34.
They can be prepared synthetically and are widely produced by the chemical industry as
organic dyes35. The scientific community has explored the biological and toxicological
activities of napthoquinones in attempts to discover and develop new drugs.
20
1.1 BACKGROUND OF THE STUDY
Since the discovery of the parent ring phenoxazine 1, which was synthesized first by
Bernthsen36 in 1887, many structural modifications have been carried out to enhance its
biological activities, minimize undesirable effects37 and open new areas of applications.
O
N
H
1
Such molecular modifications had yielded derivatives such as compounds 2, 3, 4, 5, 6 and 7.
O
N
H
O
N
O
N
O
N
O
N
O
N
2
3 4
5
6
7
H H
H
H H
Compounds 2, 3, 4, and 5 are described as “angular” phenoxazines because of the non-linear
arrangement of the ring systems38. These possess fused rings at positions a39, c40, h and j
bonds of the phenoxazine.
21
There are also systems in which naphthalene is attached to two different positions in
the parent compound. Such structures include dibenzo[a,h]phenoxazine 841,
dibenzo[a,i]phenoxazine 9 and dibenzo[a, j]phenoxazine 10.
O
N
8
H
O
N
H
9
O
N
H
10
There are variations of “angular” phenoxazine in which one of the ring carbon atoms has
been replaced with oxygen, known as benzopyrano[3,4-b]benzoxazine 11, and structures in
which the attached benzene ring possesses a substituent. Example of the later is compound
12.
H
O
N
O
H
O
N
O
O
12
11
Many derivatives of non-linear phenoxazine formed by fusion of benzene ring in the [a]
position have been reported. These compounds such as 2 have been used as dye stuff and
suitable indicators42.
A number of intermediates including naphthoquinones 13 derivatives have been used
for the synthesis of non- linear phenoxazines. Naphthoquinone 13 and its derivatives have
been the subject of much research due to their pharmacological activities. Quinone and
naphthoquinone fragments are often encountered in natural biologically active compounds.
Natural naphthoquinone derivatives
22
O
O
13
found in plants, such as 2-hydroxy-1,4-naphthoquinone, have antibacterial effect on several
species of aerobic and anaerobic organism43-44. Some 1,4-naphthoquinone derivatives possess
biological activities45-46. 2-Hydroxy-1,4-naphthoquinone 83 (Lawsone) is a naphthoquinone
dye isolated from leaves of Lawsonia inermis, the Henna plant used for preparing decorative
hair and skin dyes. It also demonstrates antimicrobial and antioxidant effects43. Baker and coworkers
in 1990 isolated naphthoquinone from culture extracts of Fusarium oxysporum and
Fusarium solani47. Brandelli and co-workers in 2004 also reported that the presence of an
imino group instead of a keto group in the position 1 or 4 in 1,4-naphthoquinone results in the
loss of antimicrobial activity48. This may indicate that both free groups are required for full
activity49. The incidence of bacterial infections is an important and challenging problem due
to the emerging new infectious diseases and increasing multi-drug resistance of microbial
pathogens50. For critically ill people with a compromised immune system including AIDS
patients, burn victims, individuals undergoing chemotherapy as well as organ transplant
recipients taking immunosuppressive drugs, fungal infections are a serious concern51.
Modern organic synthesis has been greatly improved by the use of reactions catalyzed
by transition metal complexes especially palladium, and this has led to the development of
new methods of constructing carbon-carbon bonds and carbon-heteroatom bonds52-55. The
transition metal-catalyzed C-C bond forming reactions have gained increasing importance
over the last decade. The development and finetuning of reaction parameters for known and
newly discovered metal–catalyzed transformations have had an important impact on
23
successes in the synthesis of natural and non-natural biologically active compounds and as
theoretically interesting molecules of high complexity56a-c. In addition, process development
for valuable intermediates in the pharmaceutical and agrochemical industry as well as
research towards new materials have benefited a great deal.
The increasing popularity of processes harnessing coupled catalysis is highlighted by
the number of recent reviews in this area, especially the well-documented work on Pdcatalyzed
C-C bond formation57-60. One of the most general and widely used palladium–
catalyzed cross–coupling reactions is the alkynylation of the aryl halides using terminal
alkynes, generally known as the Sonogashira cross–coupling reaction61a-c. Other palladium
catalyzed coupling reactions that have changed the face of organic synthesis include Heck-
Mizoroki coupling reaction, Buchwald-Hartwig coupling reaction, Suzuki- Miyaura reaction
and Negishi reaction.
There are two different approaches to the application of transition metal- catalyzed
reactions to the chemistry of heterocyclic compounds62a-c. One of them, involves the building
of the heterocyclic backbone whereas in the other aspect, the heterocyclic fragment is used as
one of the reaction components. These examples are given in (i) and (ii) below, respectively.
X
Hg
N
+ I
N
20 oC
[]Pd], IX
X =s; 2-py, 3-py, 4-py
i)
ii)
N
I
Cl
1) ArIB(OH)2, 2) ArIIB(OH)2
1% Pd(PPh3)4, K2CO3 100 oC, 4h N
Ar
Ar
I
II
24
1.2 STATEMENT OF THE PROBLEM
Although many naphthoquinones and phenoxazines have been synthesized, only
limited number of their derivatives have been prepared and their biological activities studied.
Intensive research has been in progress in the search for more derivatives of those with highly
improved pharmacological and biological activities.
So far, literature has yielded no results on work been done using Sonogashira cross–
coupling reaction under copper-, amine-, and solvent-free conditions in the synthesis of
alkynylated angular phenoxazines and alkynylated naphthoquinones. Consequently, the
antimicrobial properties of these alkynylated angular phenoxazines and alkynylated
naphthoquinones have not been reported.
1.3 OBJECTIVE OF THE STUDY
The specific objectives of the study were to:
1. Synthesize 6-chloro-5H-benzo[a]phenoxazin-5-one to function as an intermediate in
the synthesis of.
NH2
OH
O
O
Cl
Cl
+ NaOAc, Benzene
Reflux, 6hrs,
80oC, 92% O
N
O
Cl
2-amino phenol
2,3-dichloro-
1,4-napthoquinone 6-chlorobenzo[a]
phenoxazin-5-one
2. Couple 6-chloro-5H-benzo[a]phenoxazin-5-one with various terminal alkynes (i – v)
to give 6-substituted alkynylated-5H-benzo[a]phenoxazin-5-one via Sonogashira
cross-coupling reactions under copper-, amine-, and solvent-free conditions.
25
O
N
O
Cl
+
R
3mol% PdCl2 (PPh3)2
TBAF, 80oC, Nitrogen
O
N
O
R
CH3
OH
2-methyl-3-butyn-2-ol
(i) phenylacetylene
(ii)
OH
(iii)
(iv) C4H10
(v) C6H13
Propargyl alcohol
Hexyne
Octyne
1-
6-chloro-5H-benzo[a]phenoxazin-5-one
6-alkynylated-5H-benzo[a]phenoxazin-5-one
where R is:
3. Couple 2,3-dichloro-1,4-naphthoquinone with the above listed terminal alkynes to
give 2-chloro-3-substituted alkynylated-1,4-naphthoquinones via Sonogashira crosscoupling
reaction under copper-, amine-, and solvent-free conditions.
O
O
Cl
Cl
+ R
3mol% PdCl2 (PPh3)2
TBAF, 80oC, Nitrogen
O
O R
Cl
2,3-dichloro-1,4-naphthoquinone 2-chloro-3-substituted
alkynylated-1,4-naphthoquinone.
26
4. Characterize the new compounds using Uv, IR, NMR (1H &13C) spectroscopic
techniques.
5. Carry out antimicrobial screening of the synthesized compounds.
1.4 JUSTIFICATION OF THE STUDY
Although several phenoxazines compounds have been reported, methods are often not
available for the preparation of the wide derivatives. The wide range of applications of
phenoxazine and naphthoquinone derivatives, especially biological applications, and the need
to synthesize new derivatives which have better and desirable properties motivated this work.
27
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