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Design and synthesis of isatin analogues
By
Student Surname and Name Student number Cellphone number and
Púcuta Mateus António da
Conceição
200968513 0813908792
Department: Chemistry and Biochemistry
Supervisor: Dr. R. Hans
Submitted in partial fulfillment of the requirements for the degree
Bachelor of Science (honors)
in the
FACULTY OF SCIENCE
at the
UNIVERSITY OF NAMIBIA
Subject:
Research Project (CHM3810)
Date of submission:
03rd December 2014
FACULTY OF SCIENCE
DEPARTMENT OF CHEMISTRY AND BIOCHEMISTRY
Declaration Regarding Plagiarism
I (full names & surname): Mateus António da Conceição Púcuta
Student number: 200968513
Declare the following:
1. I understand what plagiarism entails and am aware of the University’s policy in this
regard.
2. I declare that this assignment is my own, original work. Where someone else’s work
was used (whether from a printed source, the Internet or any other source) due
acknowledgement was given and reference was made according to departmental
requirements.
3. I did not copy and paste any information directly from an electronic source (e.g. a
web page, electronic journal article or CD ROM) into this document.
4. I did not make use of another student’s previous work and submitted it as my own.
5. I did not allow and will not allow anyone to copy my work with the intention of
presenting it as his/her own work.
Signature Date
ii
DEDICATION
A dedication to my loving parents Maria and Jacinto Púcuta and my siblings: Fuca, Perpétua,
José, Jacinto, Pascoalina, Teresa, Isabel and João for their inspiring strength, encouragement,
support, guidance and prayers.
A special feeling of gratitude to my fiancée Tecla Tembo for the encouragement and support
throughout the process.
iii
ACKNOWLEDGEMENTS
I am profoundly grateful to my Lord and my God for the unconditional love, grace, guidance
and mercy He has bestowed upon me during the course of this project.
I would like to thank my supervisor Dr. Renate Hazel Hans for her guidance, support, patience
and encouragement throughout the project. A great debt of gratitude is owed to my family for
their loving support and for their belief in me. Also acknowledged is the valuable
contribution of Mr. P. Shanika for helping out in the supply of resources needed during the
project. My thanks to Mr. N. Gariseb, the project coordinator, for his efforts and his availability
for progress of this project.
I would also like to thank the Faculty of Science in the University of Namibia, and in
particularly the Chemistry and Biochemistry Department for allowing me to complete my
undergraduate studies here, I do not take the knowledge for granted. My special thanks to Prof.
Koch, University of Stellenbosch, Faculty of Science, and Chemistry Department for the NMR
analysis on all the synthesized intermediates.
Finally and with deep appreciation, I would like to thank my lab partners: Cesar Lubongo,
Iyaloo Amadhila, Viktor Ambondo and Eradius Mwaetako for their help and guidance during
the lab works.
My sincerest apologies to all persons whose contribution I might have overlooked or
dealt with inadequately.
iv
LIST OF ABBREVIATIONS
AlCl3 Aluminium chloride
CH2Cl2 : H2O Dichloromethane : Water
DCM Dichloromethane
DMF Dimethylformamide
eq equivalence
EtOAc Ethyl acetate
HIV/AIDS Human Immunodeficiency Virus/Acquired Immune Deficiency
Syndrome
K2CO3 Potassium carbonate
MeOH Methanol
mol moles
mol % mole percentage
mmol millimoles
NaOH Sodium Hydroxide
Na2SO4 Sodium Sulphate
NMR Nuclear Magnetic Resonance
Rf Retardation factor
SARS Severe Acute Respiratory Syndrome
TB Tuberculosis
TLC Thin Layer Chromatography
w/v weight per volume
v
LIST OF FIGURES, SCHEMES AND TABLE
Figure 1: Examples of medicines from plants ....................................................................................... 1
Figure 2: Structure of isatin ................................................................................................................... 2
Figure 3: Sources of natural products .................................................................................................... 4
Figure 4: Example of a natural product isolated from microorganism ................................................. 4
Figure 5: Examples of natural products from marine organisms ........................................................... 5
Figure 6: Examples of natural products from animal sources................................................................ 5
Figure 7: Example of a natural product from plants source ................................................................... 6
Figure 8: Isatinyl thiosemicarbazone derivative .................................................................................... 7
Figure 9: Lamivudine and its derivative ................................................................................................ 7
Figure 10: Target molecule .................................................................................................................... 8
Figure 11: 1H NMR spectrum showing all signals of compound 21 in CDCl3 at 500 MHz ................ 19
Figure 12: 1H NMR spectrum showing all signals of compound 21 in CDCl3 at 500 MHz – expansion
of region 7.06 - 7.60........................................................................................................................... 20
Figure 13: 13C NMR spectrum of compound 21 .................................................................................. 21
Scheme 1: Retrosynthesis of target molecule ....................................................................................... 10
Scheme 2: Mechanism for Aldol condensation .................................................................................... 13
Scheme 3: Proposed mechanism for the Cu(I)-catalyzed azide-acetylene cycloaddition ................... 14
Table 1: Table of synthesized intermediates and target molecules ...................................................... 15
vi
ABSTRACT
HIV/AIDS and TB are infectious diseases responsible for a quarter of all deaths worldwide and
Africa has the highest burden of these diseases. The etiological agents of these infectious
diseases develop resistance against most of the clinically used drugs which increases the need
for more potent drugs with potentially new modes of action.
Natural products are the most consistent, valuable source of drug leads because they provide
greater structural diversity than compounds derived through combinatorial synthesis. This
offers an opportunity for finding novel low molecular weight lead structures that are potentially
active against a wide range of assay targets. Isatin, the natural product scaffold chosen for this
study, and its derivatives have been reported to display antiviral activities against the Severe
Acute Respiratory Syndrome (SARS) virus. There also exist reports on the inhibitory activity
of isatin-β-thiosemicarbazones and other isatin derivatives against HIV replication. The
objective of this study is therefore to synthetically modify the isatin scaffold in order to obtain
novel isatin analogues with potential anti-HIV activity.
Synthesis of the designed isatin analogues was done using reported procedures. For the
characterization of the synthesized analogues physical data, such as melting point and
retardation factor, as well as spectral data – Infrared, 1H NMR and 13C NMR - were obtained.
An acetylenic isatin, O-alkylated benzaldehydes and azido chalcones were synthesized and the
yields of 37, 27, 53, 33, 95, 89, 61, 76, 76, and 96 % respectively were obtained. Three triazole
derivatives (target molecules) were synthesized and obtained they yield of 51%, 48% and 38%,
respectively. Spectral and melting point data confirmed the proposed structures for known
intermediates. After structure confirmation these novel analogues will be submitted for testing
of inhibitory activity against HIV protease and reverse transcriptase at the Chemistry and
Biochemistry Department (UNAM)).
vii
TABLE OF CONTENTS
Dedication .................................................................................................................................. ii
Acknowledgements .................................................................................................................. iii
List of abbreviations ................................................................................................................. iv
List of figures, schemes and table .............................................................................................. v
Abstract ..................................................................................................................................... vi
Table of contents ...................................................................................................................... vii
1. Introduction ........................................................................................................................ 1
2. Motivation of study ............................................................................................................ 3
3. Literature review ................................................................................................................. 4
3.1. Natural products .............................................................................................................. 4
4. Objectives of the study ....................................................................................................... 8
5. Methodology ....................................................................................................................... 8
5.1. Design of target molecules .............................................................................................. 8
5.2. Retrosynthesis of target molecules ................................................................................ 10
5.3. Chemical synthesis ........................................................................................................ 11
5.3.1. Synthesis of acetylenic isatin, 21 ............................................................................ 11
5.3.2. Synthesis of O-alkylated aldehydes, 20a-c ............................................................. 11
5.3.3. Syntheis of O-alkylated chalcones, 19a-c .............................................................. 12
5.3.4. Synthesis of azido chalcones, 18a-c ....................................................................... 12
5.3.5. Synthesis of triazoles, 17a-c ................................................................................... 12
5.4. Mechanisms ................................................................................................................... 13
5.4.1. Mechanism for aldol condensation ......................................................................... 13
5.4.2. Mechanism of the click reaction (1,3- dipolar cycloaddition) ................................ 14
6. Results and discussion ...................................................................................................... 15
6.1. Characterization ............................................................................................................ 18
viii
6.1.1. Spectroscopic analysis ............................................................................................ 18
7. Conclusion ........................................................................................................................ 22
8. References ............................................................................................................................ 23
1
1. INTRODUCTION
According to the World Health Organization, approximately 80 % of the population in
developing countries relies almost entirely on plants for medication (Farnsworth, Akerele,
Bingel, Soejarto, & Guo, 1985). Natural products have been recognized as an important sources
of therapeutically effective medicines. They present a consistent, valuable source of drug leads
and provide greater structural diversity than compounds obtained through standard
combinatorial synthesis. Natural product research also offers major opportunities for finding
novel low molecular weight lead structures that are potentially active against a wide range of
assay targets (Dias, Urban, & Roessener, 2012).
Natural products play a key role in pharmaceutical research because many medicines are either
natural products or derivatives thereof. Indeed, it is estimated that about 40% of all medicines
is either natural products or their semi-synthetic derivatives (Jacob, 2009). Clinical,
pharmacological, and chemical studies of these traditional medicines, which were derived
predominantly from plants, were the basis of most early medicines such as aspirin (1),
morphine (2), quinine (3), pilocarpine (4) and digitoxin (5), figure 1 (Buttler, 2004).
Figure 1: Examples of medicines from plants
1 2 3
4 5
2
Despite competition from other drug discovery methods, natural products are still providing
their fair share of new clinical candidates and drugs (Buttler, 2004). Therefore, in addition to
being a proven and important source of drug leads, natural products derived drugs also
contribute significantly to the profitability of many companies. Natural products research
continues to explore a variety of lead structures, which may be used as templates for the
development of new drugs by the pharmaceutical industry (Patwardhan, Vaidya, & Chorghade,
2004). In addition, natural products display structural diversity that can be exploited and will
therefore continue to play an important role in the discovery of new drugs (Shen, Xu, & Cheng,
2003).
Isatin (5, fig. 2), the natural product scaffold selected for this study, is an indole derivative (1H-
indole-2,3-dione) which is a synthetically versatile substrate. It was selected because it can be
used as the starting material for the synthesis of a large variety of heterocyclic compounds,
such as indoles and quinolines, and as raw material for drug synthesis (Abele, E. & Abele, R.,
2003). It was first obtained by Erdmann and Laurent in 1841 as a product from the oxidation
of indigo dye by nitric acid and chromic acids. It is also isolated from many plants namely
Isatis tictoria (from Central and Western Asia, eastern Siberia and some parts of Central
Europe), Calanthe discolor (Korea, Japan and China) and Couroupita guianesis (from Central
and South America).
Figure 2: Structure of isatin
The key focus of this study is therefore to use isatin as a template to design and synthesize
analogues modelled on it.
5
3
2. MOTIVATION OF STUDY
Natural product-derived drugs have fewer side effects and are readily absorbed compared to
synthetic drugs (Esron, 2002). They are used as templates in drug discovery process because
they offer an opportunity for finding novel low molecular weight lead structures that are
potentially active against a wide range of assay targets (Dias, Urban, & Roessener, 2012).
Isatin, the chosen scaffold and its derivatives reportedly display antiviral activity against SARS
viruses. Previous work reported the inhibitory activity of isatin-β-thiosemicarbazones and
isatin derivatives against HIV replication (Banerjee, et al., 2011). The synthetic modification
of isatin and its derivatives may yield new and improved drugs with enhanced biological
properties.
HIV/AIDS and TB are infectious diseases responsible for a quarter of all deaths worldwide.
Africa has the highest burden of such diseases in the world (Kinghorn, et al., 2012). They were
the second main cause of mortality in the past few years, with HIV/AIDS (Acquired
Immunodeficiency Syndrome), for which there is no cure, being a major contributor. The
causative agents of these infectious diseases develop resistance against prescribed drugs,
therefore there is a need for new anti-infective drugs.
Analogues, briefly put, are chemical derivatives of natural products which, due to minor
structural changes, show a weaker or stronger activity than the parent compounds. It is
therefore envisaged that by synthesizing analogues a more efficient drug with a favorable
solubility/pharmacokinetic profile compared to the parent natural product may be generated.
4
3. LITERATURE REVIEW
3.1. NATURAL PRODUCTS
Several drug candidates have been derived from different natural occurring sources, which can
be broadly divided into four categories as shown in figure 3 below.
Figure 3: Sources of natural products
Microorganisms, as a source of potential drug candidates, were not explored until the discovery
of penicillin in 1929. Since then, a large number of terrestrial and marine microorganisms have
been screened in drug discovery efforts. Microorganisms have a wide variety of potentially
active substances and have led to the discovery of anticancer agents like epirubicin (6, figure
4), (Chin, Balunas, Chai, & Kinghorn, 2006).
Figure 4: Example of a natural product isolated from a microorganism
The first active compounds to be isolated from marine species were spongouridine (7, figure
5) and spongothymidine (8, figure 5) from the Carribean sponge, Cryptotheca crypta in the
1950s. These compounds are nucleotides and show great potential as anticancer and antiviral
Natural Products
Microbes Plants Animals Marine organisms
6
5
agents. Their discovery led to an extensive search for novel drug candidates from marine
sources. About 70 % of the earth’s surface is covered by the oceans, providing significant
biodiversity for exploration of drug sources. Many marine organisms have a sedentary lifestyle,
and thereby synthesize many complex and extremely potent chemicals as a means of defense
against predators (Haefner, 2006). These chemicals can serve as possible remedies for various
ailments, especially cancer. One such example is discodermolide (9, figure 5), isolated from
the marine sponge, Discodermia dissoluta, which has a strong antitumor activity (Huang, et
al., 2006).
Figure 5: Examples of natural products from marine organisms
Animals also serve as a source of drugs and drug leads. Epibatidine, an analgesic agent obtained
from the skin of an Ecuadorian poison frog, is ten times more potent than morphine (Koehn &
Carter, 2005). Venoms and toxins from animals have played a significant role in designing a
multitude of cures for several diseases. Teprotide, for example, extracted from a Brazilian
viper, has led to the development of cilazapril (10, figure 6) and captopril (11, figure 6), which
are effective for the treatment of hypertension (Koehn & Carter, 2005).
10 11
Figure 6: Examples of natural products from animal sources
7 8 9
6
The use of plants as medicines has a long history in the treatment of various diseases. The
earliest known records for the use of plants as drugs stem from Mesopotamia in 2600 B.C.
(Koehn & Carter, 2005). Several important drugs such as taxol, camptothecin, morphine and
quinine (3, figure 1) were isolated from plant sources. The first two are widely used as anti-
cancer drugs, while the remaining are analgesic and antimalarial agents, respectively.
Probably the most famous and well known example to date would be the synthesis of the
anti-inflammatory agent, acetylsalicylic acid better known as aspirin (12, figure 7) derived
from the natural product, salicin and isolated from the bark of the willow tree Salix alba.
12
Figure 7: Example of a natural product from plant source
A literature study revealed that research on isatin and its derivatives were primarily focused on
evaluating their antimalarial (Raj, Gut, Rosenthal, & Kumar , 2014), antitubercular (Hans, et
al., 2011), anticancer (Han, et al., 2014), antitumor (Liang, et al., 2014), antiplasmodial (Hans,
Gut, Rosenthal, & Chibale, 2010) activities. Also reported are their antiviral activities,
specifically against pox virus, vaccinia, rhino virus, moleney leukemia virus and SARS viruses
(Banerjee, et al., 2011).
For the potential treatment of HIV-TB co-infections, an isatinyl thiosemicarbazones
derivatives 13 was found to be the most potent in inhibiting the replication of HIV-1 cells
(Banerjee, et al., 2011). Using lamivudine drug (14, figure 9), more potent analogues such as
15 were obtained. The antiviral activity of lamivudine and its prodrugs against HIV-1 was
determined in vitro in T4 lymphocytes (Sriram, Yogeeswari, & Gopal, 2005).
7
13
Figure 8: Isatinyl thiosemicarbazone derivative
14 15
Figure 9: Lamivudine and its derivative
8
4. OBJECTIVES OF THE STUDY
The objectives of this study are to:
Design analogues modelled on isatin
Synthesize isatin analogues
Characterize the synthesized analogues
5. METHODOLOGY
5.1. DESIGN OF TARGET MOLECULES
The target molecule was designed in such a way that the isatin scaffold was linked with a
chalcone through a triazole ring system and the ketonic carbonyl was reacted with a
semicabazide or thiosemicabazide to form a Schiff base (16).
Triazole linker
Chalcone
R= H, ClX= Semicarbazide, ThiosemicarbazideY= H, OCH3
Figure 10: Target molecule
For designing of the target molecules, the following reports were considered:
The isatin moiety is a scaffold which offers different sites for chemical modification.
Reference has been made to the broad spectrum of biological properties displayed by
its derivatives and its synthetic versatility (Raghu, et al., 2013).
16
9
In recent years, Schiff and Mannich bases of isatin were reported to exhibit
chemotherapeutic properties including antiviral, antitubercular, antifungal, and
antibacterial activities. Investigation of the SARs of isatin derivatives revealed that 5-
halogenation, N-alkylation, N-Mannich base, and 3-thiosemicarbazone formation were
effective in triggering a marked rise in activity against various bacteria, fungi, and
viruses (Raghu, et al., 2013). Notably, Schiff bases of isatin have been reported to
possess anti-HIV, anticonvulsant, antibacterial, antiprotozoal, antifungal, anti-viral,
and anthelmintic activities (Chegyuan, et al., 2014).
Over the past few years the 1,2,3-triazole ring system and derivatives which contain
this ring system, have attracted a great deal of interest due to their diverse biological
activities such as antitubercular, anti-HIV, antifungal, antibacterial, and anticancer
activities. ‘Click chemistry’ allow for easy synthesis of this ring system. The favourable
properties of 1,2,3-triazole ring like moderate dipole character, hydrogen bonding
capability, rigidity and stability under in vivo conditions are evidently responsible for
enhanced biological activities. Moreover, the incorporation of 1,2,3-triazoles as a linker
of two pharmacophores to give bifunctional drugs, have become increasingly useful
and important in constructing bioactive molecules (Kewal, Sunir, Luke, Mandeep , &
Vipan, 2012).
Chalcones are of considerable interest in drug discovery because of the diverse
biological activities displayed by their derivatives and the ease and simplicity of their
synthesis. Moreover, this scaffold allows for the systematic variation of substituents
and or substitution patterns on the aromatic rings (Hans, Jiri, Rosenthal, & Chibale,
2010).
10
5.2. RETROSYNTHESIS OF TARGET MOLECULES
5
Semicarbazide
18
Propargy bromide
R= H, Cl
Dibromoethane
Salicylaldehyde, a
4-hydroxybenzaldehyde, b
vanillin, c
16
20Acetophenone
21
17
N-alkylation
Shiff base
formation
Click
reaction
Funtional Group
Interconversion
Aldol Condensation
O-alkylation
19
Scheme 1: Retrosynthesis of target molecule
Synthesis of target molecule 16 was envisaged through the Schiff base formation
reaction of semicarbazides or thiosemicarbazides with the ketonic carbonyl group of
intermediate 17. Intermediate 17 in turn can be obtained through the click reaction of the azido
chalcone 18 and the acetylenic isatin 21. The acetylenic isatin 21 can be obtained through N-
11
alkylation of isatin with propargyl bromide. The azido chalcone 18 can be obtained through
functional group interconversion of the O-alkylated chalcone 19, and the latter can be accessed
through the Aldol condensation reaction of O-alkylated benzaldehyde derivative 20 and
acetophenone. O-alkylation of a benzaldehyde derivative with 1,2-dibromoethane will give 20.
Friedel’s-Craft acylation method was also attempted in order to synthesize the target molecule.
The method consisted of the acylation of acetanilide with acetyl chloride using anhydrous
AlCl3 as the catalyst and DCM as solvent. Unfortunately, no reaction occurred due to the poor
solubility of the aromatic substrate in the solvent. On the other hand, three target molecules
were envisaged using the procedure outlined above (scheme 1) but with different starting
benzaldehyde derivatives such as salicylaldehyde, 4-hydroxybenzaldehyde and vanillin.
5.3. CHEMICAL SYNTHESIS
5.3.1. SYNTHESIS OF ACETYLENIC ISATIN, 21
Sodium hydride, 60 % suspended in mineral oil (16.99 mmol, 1.5 eq) was added to
commercially available isatin (11.32 mmol, 1.0 eq) in 16.64 mL of anhydrous DMF at 0 °C.
The propargyl bromide, 80 % in toluene, (56.61 mmol, 4.0 eq) was added and the resulting
mixture slowly warmed to 25 °C. Stirring was continued for 1 hour at this temperature under
nitrogen atmosphere. The temperature was then increased to 60 °C and the reaction mixture
stirred for 24 hours at this temperature under nitrogen atmosphere. Ice-cold water was added
to the orange coloured reaction mixture and the precipitate that formed was filtered, washed
with water and recrystallized from MeOH to yield the pure product (Hans R. H., Novel
Antimalarial and Antitubercular Agents Based on Natural Products, 2009).
5.3.2. SYNTHESIS OF O-ALKYLATED ALDEHYDES, 20a-c
Anhydrous K2CO3 (8.50 g, 61.50 mmol, 1.5 eq) was added to a benzaldehyde derivative
(5.0 g, 40.94 mmol, 1.0 eq) dissolved in 25 mL anhydrous DMF and 1,2-dibromoethane, (9.2
g, 48.97 mmol, 1.2 eq) was added to the mixture. The resulting mixture was stirred for 16 hours
at 25 °C under nitrogen atmosphere. After reaction completion, as indicated by TLC, ice-
cold water was added to the reaction mixture. The obtained precipitate was filtered, washed
with water and recrystallized from MeOH to yield the pure product (Hans R. H., Novel
Antimalarial and Antitubercular Agents Based on Natural Products, 2009).
12
5.3.3. SYNTHEIS OF O-ALKYLATED CHALCONES, 19a-c
To a solution of the O-alkylated benzaldehyde derivatives 20a-c (10.61 mmol, 1.0 eq) in MeOH
was added 8.5 mL of methanolic NaOH (3% w/v). The resulting mixture was stirred at room
temperature (at 25 °C) for 30 minutes. A methanolic solution of the commercially available,
acetophenone (10.61 mmol, 1.0 eq) was added and the mixture stirred overnight at the
same temperature under ambient atmosphere. The precipitate that formed was filtered and
washed with cold MeOH. Recrystallization from MeOH afforded the pure product (Hans R.
H., Novel Antimalarial and Antitubercular Agents Based on Natural Products, 2009).
5.3.4. SYNTHESIS OF AZIDO CHALCONES, 18a-c
Sodium azide (2.78 mmol, 2.0 eq) was added to a solution of O-alkylated chalcones, 19a-c
(1.39 mmol, 1.0 eq) in 3 mL of anhydrous DMF. The reaction mixture was stirred at 25 ºC for
18 hours under nitrogen atmosphere. The addition of ice-cold water to the product mixture
resulted in the formation of a precipitate which was filtered and washed with copious amounts
of water. Recrystallization from MeOH afforded the pure product (Hans R. H., Novel
Antimalarial and Antitubercular Agents Based on Natural Products, 2009).
5.3.5. SYNTHESIS OF TRIAZOLES, 17a-c
The azides, 18a-c (0.464 mmol, 1.0 eq) and acetylenic isatin 21 (0.510 mmol, 1.1 eq) were
dissolved in 3 mL of CH2Cl2:H2O (1:1). Copper (II) sulphate pentahydrate (0.0232 mmol, 5
mol %) and sodium ascorbate (0.0696 mmol, 15 mol %) was added to the mixture. The
resulting mixture was stirred for 16 hours at 25 °C under ambient atmosphere. Upon
completion, the product mixture was diluted with water and extracted with EtOAc. The
combined organic layer was washed with water and brine, dried over anhydrous Na2SO4 and
concentrated under reduced pressure to yield the product (Hans R. H., Novel Antimalarial and
Antitubercular Agents Based on Natural Products, 2009).
13
5.4. MECHANISMS
5.4.1. MECHANISM FOR ALDOL CONDENSATION
- +
- OR
Na +
Na+
-
- NaOH
Na + -OH
-Na +
Scheme 2: Mechanism for Aldol condensation (Hans R. H., Novel Antimalarial and
Antitubercular Agents Based on Natural Products, 2009)
The mechanism for the Aldol condensation is depicted in scheme 2. It involves the base-
catalyzed enolization of acetophenone followed by nucleophilic attack of the enolate on the
O-alkylated benzaldehyde derivative. The β-hydroxy ketone so formed undergoes base
catalyzed elimination in a E1cB mechanism to yield the α,β-unsaturated chalcone.
14
5.4.2. MECHANISM OF THE CLICK REACTION (1,3- DIPOLAR
CYCLOADDITION)
-H+
[CuLn]+
Ligand (L)reducing agent
CuSO4
+H+
+
+ -
(i)
(ii)
(iii)
(iv)
Scheme 3: Proposed mechanism for the Cu(I)-catalyzed azide-acetylene cycloaddition 1
(1) Patton, G.C. Development and Application of Click Chemistry, 2004,
15
6. RESULTS AND DISCUSSION
Table 1: Table of synthesized intermediates and target molecules
Intermediate/
Target
Molecule
Chemical
Formula
IUPAC
name
Novel or
Known
Melting
Point
(°C)
Rf
value
Yield
(%)
21
C11H7NO2
1-(prop-2-yn-1-
yl) indoline-2,3-
dione
Known
153
158 a
0.79
(EtOAc:
Hex 1:1)
37
20a
C9H9BrO2
2-(2-
bromoethoxy)
benzaldehyde
Known
125
52 b
0.27
(EtOAc:
Hex 3:7)
27
20b
C9H9BrO2
4-(2-
bromoethoxy)
benzaldehyde
Known
119
61 c
0.77
(EtOAc:
Hex)
53
20c
C10H11BrO3
4-(2-
bromoethoxy)-3-
methoxybenzald
ehyde
Known
178
b.p.356 d
0.73
(EtOAc:
Hex 3:1)
33
19a
C17H15BrO2
(E)-3-(2-(2-
bromoethoxy)
phenyl)-1-
phenylprop-2-en-
1-one
Novel
142-144
0.77
(EtOAc:
Hex 1:1)
95
16
Intermediate/
Target Molecule
Chemical
Formula
IUPAC
name
Novel
or
Known
Melting
Point
(°C)
Rf
value
Yield
(%)
19b
C17H15BrO2
(E)-3-(4-(2-
bromoethoxy)
phenyl)-1-
phenylprop-2-en-
1-one
Novel
176
0.81
(EtOAc:
Hex 1:1)
89
19c
C18H17BrO3
(E)-3-(4-(2-
bromoethoxy)-3-
methoxyphenyl)-
1-phenylprop-2-
en-1-one
Novel
168
0.73
(EtOAc:
Hex 3:1)
61
18a
C17H15N3O2
(E)-3-(2-(2-
azidoethoxy)phe
nyl)-1-
phenylprop-2-en-
1-one
Novel
145
0.80
(EtOAc:
Hex 1:1)
76
18b
C17H15N3O2
(E)-3-(4-(2-
azidoethoxy)phe
nyl)-1-
phenylprop-2-en-
1-one
Novel
184
0.83
(EtOAc:
Hex 1:1)
76
18c
C18H17N3O3
(E)-3-(4-(2-
azidoethoxy)-3-
methoxyphenyl)-
1-phenylprop-2-
en-1-one
Novel
169
0.74
(EtOAc:
Hex 1:1)
96
17
Intermediate/
Target Molecule
Chemical
Formula
IUPAC
name
Novel
or
Known
Melting
Point
(°C)
Rf
value
Yield
(%)
17a
C28H22N4O4
(E)-1-((1-(2-(2-
(3-oxo-3-
phenylprop-1-en-
1-
yl)phenoxy)ethyl
)-1H-1, 2, 3-
triazol-5-
yl)methyl)indoli
ne-2,3-dione
Novel
116
0.62
(MeOH :
DCM
0.2 : 9.8)
51
17b
C28H22N4O4
(E)-1-((1-(2-(4-
(3-oxo-3-
phenylprop-1-en-
1-
yl)phenoxy)ethyl
)-1H-1, 2, 3-
triazol-4-
yl)methyl)indoli
ne-2,3-dione
Novel
137
0.53
(MeOH :
DCM
0.2 : 9.8)
48
17c
C29H22N4O4
(E)-1-((1-(2-(2-
methyl-4-(3-oxo-
3-phenylprop-1-
en-1-
yl)phenoxy)ethyl
)-1H-1, 2, 3-
triazol-4-
yl)methyl)indoli
ne-2,3-dione
Novel
136
0.67
(MeOH :
DCM
0.2 : 9.8)
35
(a). Literature melting point (http://www.chemspider.com/Chemical-Structure.1468549.html (accessed 05:43, Oct 23, 2014)).
(b). Literature melting point (Zhao, Wang, Hu, Ma, & Wang, 2005)
(c). Literature melting point (Zhu, et al., 2014)
(d). Literature boiling point (http://www.chemspider.com/Chemical-Structure.12956958.html (accessed 05:56, Oct 23, 2014)).
18
The yields of acetylenic isatin and the O-alkylated benzaldehydes might have been affected by
the nitrogen gas that was not 100 % dry. To obtain dry N2 gas, a drying tube filled with
anhydrous CaCl2 should have been connected to the nitrogen cylinder. On the other hand, for
the acetylenic isatin synthesis and the O-alkylated benzaldehyde synthesis, the reactions were
conducted for 24 hours at 60 °C and for 16 hours at 25 °C respectively and the presence of
moisture in the reaction mixture might have affected the yields.
The acetylenic isatin synthesized is a known compound and the melting point obtained from
the literature is approximately the same as the one measured. It can be concluded that the
proposed intermediate was indeed obtained. For the O-alkylated benzaldehyde derivatives, the
melting points in the literature have a very strong difference in their magnitudes and this may
be due to the impurities present in the synthesized intermediate.
The azido chalcone and the triazoles are all novel compounds and thus comparisons with
literature melting point values could not be done.
6.1. CHARACTERIZATION
6.1.1. SPECTROSCOPIC ANALYSIS
The spectroscopic data obtained for the desired intermediate 21 is consistent with the proposed
structure. Figures 11, 12 and 13 show the 1H and 13C NMR spectra of representative compound
21. The 1H NMR data (figure 12) showed some key signals of the isatin scaffold appearing in
the aromatic region of the spectrum as multiplets resonating at 7.06 - 7.60 and integrating for
4 protons. These signals were assigned to H-4, H-5, H-6 and H-7. A pair of one-proton singlet
resonating at 4.5 was assigned to the methylene protons H-1’ a/b. The acetylenic proton, H-
3’, resonated at 2.3 and showed coupling to the methylene protons H-1’ a/b.
19
Figure 11: 1H NMR spectrum showing all signals of compound 21 in CDCl3 at 600 MHz
H-1’ a/b
H-3’ H-4, 7
H-5, 6
1
2
33a
4
5
6
7
7a
1'
2'
3'
20
Figure 12: 1H NMR spectrum showing all signals of compound 21 in CDCl3 at 600 MHz –
expansion of region 7.06 - 7.60
The 13C NMR spectrum of compound 21 (figure 13) showed 11 non-equivalent signals which
correlates with the number of carbons expected for the proposed structure. Key signals at 160
and 185 were assigned to carbonyl carbons at C-2 and C3 respectively. Another carbon
signals of interest are the acetylenic carbon C-3’ resonating at 76 and the amine carbon C-1’
resonating at 35. The 13C NMR spectrum also confirms the proposed structure of compound
21.
For the remaining intermediates samples were sent for 1H and 13C NMR analysis but
unfortunately the spectra are not available yet.
H-4, 7 H-5, 6
1
2
33a
4
5
6
7
7a
1'
2'
3'
21
Figure 13: 13C NMR Spectrum of compound 21
C-3’ C-2’
C-2 C-3 C-1’
C-5
1
2
33a
4
5
6
7
7a
1'
2'
3'
22
7. CONCLUSION
In this study, the analogues modelled on isatin have successfully been designed,
synthesized and characterized. The Schiff bases were not synthesized due to time
constraint. The objectives stated for this study were partially fulfilled and therefore it can
be said that the research was successful. A recommendation for the improvement of yields
is that all the nitrogen gas should be completely dry and the reaction medium completely
free of moisture.
For the way forward after all structure confirmation of the analogues, this includes the
advance intermediates and target molecules, will be submitted for testing of inhibitory
activity against HIV protease and reverse transcriptase at the Chemistry and Biochemistry
Department (UNAM)).
23
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