Open Access Repository of Indian Theses

Neigapula, Sankara Rao

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i 
 
The thesis entitled 
 
“Cinnamide/Naphthalimide/Chalcone/Pyrazoline Hybrides as Potential Anticancer 
Agents and Quinoline Derivatives as Antimicrobial Agents” has been divided into four 
chapters. 
Chapter-I: General introduction of Cancer, Tubulin and Tuberculosis  
Cancer is a class of diseases or disorders characterized by uncontrolled/ abnormal 
division of cells and the ability of these to spread, either by direct growth into adjacent tissue, 
or by implantation into distant sites by metastasis, in which cancer cells are transported 
through the bloodstream or lymphatic system. Small molecules that disrupt 
microtubule/tubulin dynamics are used widely in cancer treatment. Hence discovery and 
development of new small molecules useful in cancer chemotherapy has identified tubulin as 
possible molecular target medicinal chemists for past few years. Microtubules are one of the 
key structural components of the cytoskeleton in eukaryotic cells comprising of α and β-
tubulin heterodimers. They play a crucial role in various cellular processes and have emerged 
as an attractive and viable target in the development of anticancer drugs mainly due to their 
indispensability in mitotic cell division.  
Tuberculosis (TB) is an ancient, contagious disease caused by the pathogen 
Mycobacterium tuberculosis (M. tb) and is responsible for more human deaths than any other 
single infectious disease. According to World Health Organization (WHO), TB has become a 
more prevalent chronic disease causing 2-3 million deaths annually and infecting one third of 
the world population. Its deadly synergy with HIV has further aggravated the mortality and 
spread of this disease. In recent years, chemotherapy of TB has evolved in due course of 
time. Though there are many drugs available for the treatment of TB the emergence of 
resistance such as multi drug resistance (MDR) and extensive drug resistance (XDR) pose 
threat to the human life. Advances in research such as genetic engineering of M. tb have 
offered many validated targets to develop chemical libraries that may eventually lead to new 
drugs to combat TB. 
Statement of Problem 
Chapter-II: Arylcinnamido-Propionone Hybrids as Tubulin Polymerization Inhibitors 
and Apoptotic inducers. 
Cinnamides are another class of anticancer agents, its natural analogues are known for 
the treatment of cancer for over centuries. Phenylcinnamides are shown to bind to tubulin, 
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thereby causing an inhibition of its polymerization and alteration in the tubulin-microtubule 
equilibrium. They are known to possess an α,β-unsaturated carbonyl moiety, which can be 
considered as a Michael acceptor, employed as a powerful tool in the design of antimitotic 
agents. Some of the potent hybrid/conjugate molecules that have been recently developed as 
new anticancer agents are obtained by the combination of different pharmacophores.
 
Structural features of designed molecules, including the trimethoxyphenyl moiety (found in 
colchicine and 8H), suggested that these molecules exerted cytotoxic action through 
microtubule binding and mitotic arrest. Based on these observations we describe 
modifications on 8H scaffold which contain trimethoxyphenyl moiety without disturbance 
and further congeners were generated by conjugates with substituted arylpropynones to 
cinnamide scaffolds. In this context, we have designed, synthesized some newer cinnamido-
propionone conjugates and evaluated them for their cytotoxic potential apart from their effect 
on the inhibition of tubulin polymerization. 
 
Scheme: Synthesis of arylcinnamido-propionone hybrids 
 
Reagents and Conditions: (a) (i) (COCl)2, dry DCM, 0 °C to rt, 3 h; (ii) Trimethoxy aniline, 
Et3N, dry THF, 3 h; 69−71%; (b) Zn, HCO2NH4, MeOH, rt, 6 h; 60−70%; c) 
                                                                                                                     Synopsis                                                                                                                                      
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ethynylmagnesium bromide, THF, 0°C−rt, 8−9 h; (d) 2-iodoxybenzoic acid, DMSO, rt, 5 h, 
73−88%; (e) EtOH, 3-4 h; 76−88%. 
Results and discussions 
A series of arylcinnamido-propionone conjugates (7-9a-h and 10a-f) has been 
designed, synthesized and evaluated for their anticancer potential against the four human 
cancer cell lines. Among them, conjugates 9d and 9g have showed significant cytotoxic 
activity against prostate cancer cells (DU-145) displaying IC50 of 7.48 and 8.91 µM 
respectively. Studies to understand the mechanism of action of 9d and 9g indicates that 
inhibit the tubulin polymerization thereby arresting the cell cycle in G2/M phase. 
Furthermore, studies like mitochondrial membrane potential and Annexin V-FITC assay 
suggested that these conjugates 9d and 9g induced cell death by apoptosis. The molecular 
docking studies suggested that the binding of these conjugates at the colchicine site of the 
tubulin protein. 
Conclusions 
Cinnamido-propionone conjugates (7-9a-h and 10a-f) were synthesized and evaluated 
for their cytotoxic activity. Conjugates 9d and 9g showed significant cytotoxic activity 
against human prostate cancer cell line, (DU-145). The flow cytometric analysis revealed that 
these conjugates cause cell cycle arrest at G2/M phase. Furthermore, they effectively 
inhibited microtubule assembly. Moreover, the triggering of the apoptotic cell death after 
mitotic arrest was investigated by mitochondrial membrane potential and Annexin V FITC 
assays suggest that these conjugates induced apoptosis. The molecular modeling study carried 
out on the colchicine binding site of tubulin demonstrated that these molecules are involved 
in a series of interactions with the protein thereby binding well with the tubulin. Therefore, 
the work reported herein could be considered of significant importance to provide valuable 
insights in the development newer leads for the treatment of cancer. 
Statement of Problem 
Chapter IIIA: Naphthalimide-Amidobenzothiazole and Naphthalimide-Cinnamide 
Hybrids as Potential Anticancer Agents. 
Naphthalimides represent an important chemical class of DNA-intercalating 
topoisomerase II inhibitors. These agents consist of flat chromophores, generally π-deficient 
aromatic or hetero aromatic system, which binds to DNA by insertion between the base pairs 
of the double helix. They are also characterized by the presence of basic side chains attached 
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to the chromophore. Charge transfer and stacking interactions are the main driving forces for 
binding. Intercalation causes the base pairs to separate vertically, thereby distorting the sugar 
phosphate backbone and changing the degree of rotation between successive base pairs, and 
finally disrupts the cell division. The most potent drugs in this series were amonafide and 
mitonafide both tested in humans as anticancer agents. Amonafide, a 5-amino substituted 
naphthalimide is a novel inhibitor of Topo II that intercalates into DNA and induces apoptotic 
signalling by blocking Topo II binding to DNA. To improve therapeutic properties of 
naphthalimides extensive efforts have been made including the modification of side chain, 
aromatic ring system, and the substituents on the ring. The promising biological activity 
exhibited by these naphthalimides prompted us to develop some newer hybrid molecules by 
linking the naphthalimide pharmacophore with aminobenzothiazole and cinnamide scaffold 
with a view to enhance their anticancer activity. Thus a library of twenty three new class of 
naphthalimide-amidobenzothiazole, naphthalimide-cinnamide hybrids were designed, 
synthesized and evaluated for their anticancer potential.  
Scheme: Synthesis of naphthalimide-amidobenzothiazole and naphthalimide-cinnamide 
hybrids 
 
Reagents and Conditions: (a) EtOH, Reflux, 60°C, 3h; (b) 2-bromoethyl acetate, K2CO3, 
DMF, 24 h;  (c) LiOH, THF, H2O, 12 h; (d) Substituted benzothiazoles, EDCI, HOBt, 
CH2Cl2, 24 h; (e) Substituted cinnamic acids, EDCI, HOBt, CH2Cl2, 24 h. 
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Results and discussions 
These conjugates (4a-g, 5a-b and 6a-n) were synthesized and evaluated for their 
antiproliferative activity against NCI-60 Cell line panel. Among them 5a and 5b showed 
remarkable growth inhibition on all the tested cell lines with GI50 values in the range of 
activity ranging 1.38-43.1 μM and 1.19-14.1 μM respectively. Initial DNA interaction studies 
of these compounds (by CD, UV/vis, and the fluorescence spectroscopy) expectedly 
suggested the π-π stacking interactions facilitate DNA intercalation activity. Furthermore, 
representative compounds were proved to possess strong inhibition against topoisomerase II. 
Therefore DNA and topoisomerase are the main targets of the agents. 
Conclusions 
A series of new naphthalimides were designed and synthesized and their cytotoxic 
activities were evaluated in vitro. Majority of these naphthalimides potentially inhibited the 
growth of HT-29, A549 cancer cell lines selectively. Interestingly hybrid compounds 5a, 5b 
that have an amide bond at 6-position displayed promising cytotoxic activity than reference 
drug amonafide. We were also investigated antitumor mechanism of action of 5a and 5b. The 
most active compounds potentially inhibit topo II. Moreover DNA interaction of the 
representative compounds was studied by CD spectra, UV-Visible Spectra and fluorescence 
spectra. Studies   demonstrating that these compounds intercalate with DNA. 
Statement of Problem 
Chapter IIIB: Chalcone/Pyrazoline based 2-(4-aminophenyl) benzothiazole Hybrids as 
Potential Anticancer Agents. 
Chalcones (3-diaryl-2-propen-1-ones) are biosynthetic products of the shikimate 
pathway, belonging to flavanoid family is precursors of open chain flavonoids and 
isoflavonoids, which are abundant in edible plants. Chalcones are also key precursors in the 
synthesis of many biologically important heterocycles such as benzothiazepine, pyrazolines, 
1,4-diketones, and flavones. Chemically, they consist of open chain flavanoids in which the 
two aromatic rings are joined by a three carbon α, β- unsaturated carbonyl system. Chalcones 
and pyrazolines have attracted increasing attention due to numerous pharmacological 
applications. They have displayed a broad spectrum of pharmacological activities, among 
which antimalarial, anticancer, antiprotozoal (antileishmanial and antitrypanosomal), anti-
inflammatory, antibacterial, antifilarial,
 
antifungal, antimicrobial, anticonvulsant and 
antioxidant activities have been reported. Similarly 2‐(4‐aminophenyl)benzothiazoles 
                                                                                                                     Synopsis                                                                                                                                      
vi 
 
constitute a new and simple class of antitumor molecules possessing diverse biological 
properties. These biologically active conjugates further encouraged to synthesize some newer 
conjugates that are likely to enhance the anticancer activity. In this context we designed and 
synthesized some new chalcone/pyrazoline-2-(4-aminophenyl) benzothiazole conjugates 15-
16(a-f) as potential anticancer agents.  
Scheme: Synthesis of chalcone/pyrazoline based 2-(4-aminophenyl) benzothiazole 
hybrids 
 
Reagents and conditions: (a) aq. KOH, ethanol, 6h; (b) NH2NH2.H2O, Acetic acid, reflux, 
14h; (c) 2-bromoethyl acetate, K2CO3, DMF, 12h; (d) LiOH. H2O, THF, MeOH, H2O, 14h; 
(e) Pyridine, reflux, 3 h; (f) Lawesson’s reagent, toluene, reflux, 8 h; (g) K3Fe(CN)6, 
aq.NaOH, EtOH, 90 °C, 2-3 h; (h) SnCl2.2H2O, EtOH, 3 h; (i) EDCI/HOBT, DCM, 14-16h. 
Conclusions 
In conclusion, a series consisting of 10 analogs have been synthesized by conjugating 
with biologically active heterocyclic scaffold 2-phenylbenzothiazole with chalcone and 
pyrazoline. The synthesized hybrids were decorated with diverse array of substituents on 2-
phenyl benzothiazoles to deduce the structure activity relationship (SAR). These conjugates 
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vii 
 
were tested for their anticancer potential against a panel of five different human cancer cell 
lines. Compounds 16a was found to be the most effective hybrid from the series displaying 
IC50 value of 0.25 ± 0.01 μM against the lung cancer cell line A549. Further studies to probe 
the mode of action of this conjugate are under progress. Moreover another two promising 
compounds 15a and 15b have shown potent cytotoxicity in B16 cells at 1.29 µM and 1.12 
µM. 
Statement of Problem 
Chapter IV: New Quinoline Linked Chalcone and Pyrazoline Conjugates: Molecular 
Properties Prediction, Antimicrobial and Antitubercular Activities. 
The quinoline nucleus is an important heterocyclic structure found in several natural 
and synthetic products with a wide range of pharmacological activities such as anti-
tuberculosis, antimalarial,
 
anti-inflammatory, anticancer, antibiotic, anti-hypertensive, and 
anti-HIV functions. Fluoroquinolones, such as gatifloxcin and moxifloxacin, target DNA 
topoisomerase IV and DNA gyrase and can be used as anti-TB agents, however, they often 
suffer from resistance. Quinoline-based anti-TB compound (TMC207), bearing a bulky biaryl 
side chain at position C3 is a highly potent anti-TB agent and is currently in phase II clinical 
trials. On the other hand, chalcones, which are important class of natural compounds, have 
been reported to possess diverse and interesting biological properties such as antioxidant, 
antifungal, antitumor, analgesic, anti-tuberculosis and anti-HIV. In addition, there have been 
numerous reports on the synthesis of pyrazoline derivatives owing to their wide ranging 
biological activities and their associated therapeutic potential as anti-HIV, anti-inflammatory, 
anticancer and anti-microbial agents. Therefore, considering the antimicrobial potential of 
quinoline, chalcone and pyrazoline derivatives we have developed a new structural motif 
containing all these structures to find potent antimicrobial agents. In the present investigation, 
two new series comprising of forty three conjugates (5a-j, 6a-i, 7a-d, 8b-f, 9a-f, 10a-d and 
11a-d) have been synthesized by conjugating quinoline scaffold with chalcone as well as 
pyrazoline moieties. 
 
 
 
 
 
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viii 
 
Scheme: Synthesis of quinoline linked chalcone and pyrazoline conjugates 
 
 
Reagents and conditions: (a) CH3CN, dil. HCl, 3 h, 79-81%; (b) EtOH, KOH, rt, 3 h, 80-
93%; (c) N2H4.H2O, EtOH, 8 h , reflux, 71-83%; (d) N2H4.H2O, AcOH, 8 h, reflux, 77-83%. 
Results and discussions 
All the synthesized conjugates were tested for their antibacterial, antifungal and 
antitubercular activities. It is important to note that some of these compounds such as 5e, 5f, 
5i, 5j, 7a, 8b, 8c, 8e, 8f, 11b and 11c showed potent antibacterial effects against both Gram 
positive and Gram negative pathogenic strains to variable extents. The conjugates 5j and 8b 
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demonstrated excellent antibacterial activity with the MIC values ranging from 4-8 µg/mL 
against the Gram positive bacterial strains. The active compounds 5j and 8b showed very 
good inhibitory zone at 20 µg/mL concentration when compared to standard reference drugs. 
Moreover MIC values of antifungal activity showed similar trend as antibacterial activity. 
Compound 5j was found to be the most promising candidate among the tested conjugates 
demonstrating MIC 4 µg/mL against both H37RV as well as Rif
R
 strains. 
Conclusions 
In conclusion two new series comprising of forty two compounds have been 
conveniently synthesized by linking the quinoline scaffold with the chalcone, pyrazoline 
moieties and evaluated them for prediction of their molecular properties through online 
software in order to find suitable molecules for the antitubercular, antimicrobial and 
antifungal activities with anticipation of generating new structural leads serving as potent 
anti-infective agents. Some of these compounds exhibited appreciable antibacterial activity 
against the tested bacterial strains. Compounds 5j and 8b were found to be the promising 
candidates exhibiting MIC 4 µg/mL against B. subtilis MTCC 121 and M. luteus MTCC 
2470. In addition, several compounds showed good inhibition of certain fungal organisms. 
Further, some of the compounds displayed good to appreciable anti-tubercular activity 
against both H37RV as well as Rif
R
 strains. Moreover several potent compounds were 
evaluated for their cytotoxic effect in MRC-5 Lung fibroblast cells and results of this study 
demonstrated that active compounds showed relatively low level of cytotoxicity. Selectivity 
index value for compound 5j was ≥5 which indicated that promising antitubercular 
compound. 
The binding modes and effect of the C4 quinoline substitution on the activity have been 
studied using molecular modeling. Methyl group was found to be more suitable than the 
aromatic phenyl group which was supported by the antimicrobial activity data. Therefore, 
compounds such as 5j and 8b could be considered as potential leads to generate chemical 
libraries that aid the process of development of newer antimicrobial agents to combat 
antibiotic resistance. 
 


English

Cinnamide/Naphthalimide/Chalcone/Pyrazoline Hybrids as

Potential Anticancer Agents and Quinoline Derivatives as

Antimicrobial Agents

Rao Y., Mahesh Kumar

Abstract

Characterization of Aspergillus carbonarius mutant in relation to carotenoid production and its application

Fungi are a unique group of organisms, different from all others in their behavior and cellular organization. They have an enormous range of activities – as pathogens of crop plants or humans, as decomposer organisms, as experimental “model organisms” for investigating genetics and cell biology and as producers of many important metabolites. Thousands of secondary metabolites have been described from fungi. For example, the penicillins (from Penicillium chrysogenum), the cephalosporins (from Cephalosporium or Acremonium species), and griseofulvin (from P. griseofulvum) are antibiotics produced commercially from fungi. Some secondary metabolites are plant hormones, like gibberellins produced by the parasitic fungus Gibberella fujikuroi. Secondary metabolites are marketed as pharmaceuticals, including ciclosporin A which suppresses organ rejection in transplant surgery. Further examples include the mycotoxins, such as the aflatoxins produced by Aspergillus flavus and A. parasiticus and the ergot alkaloids produced by Claviceps purpurea and related species. 

Many fungi have the potential to synthesize pigments as secondary metabolites. Most fungal pigments are melanins and are associated with sporulation structures.  These compounds help to protect cells from damage caused by reactive oxygen species such as hydrogen peroxide and superoxide. They are generally dark brown pigments formed by oxidative polymerization of phenolic compounds and are deposited in the cell wall. Species of Aspergillus glaucus, reported to produce pigments like erytroglaucin (red), auroglaucin (orange) and flavoglaucin (yellow). Species of Aspergillus tawarii and Aspergillus sydowii are well known for the production of Xanthomegnin (yellow) and viomellein (Yellow) pigments. Some filamentous fungi such as Phycomyces blakesleeanus, Mucor circinelloides, and Blakeslea trispora possess the ability to synthesise carotenoid pigments. Many microorganisms were exploited for the production of carotenoids such as Blakeslea trispora for β-carotene, Rhodosporidium for γ-carotene, Mycobacterium lacticola & Phaffia rhodozyma for astaxanthin, Brevibacterium for canthaxanthin and Monascus purpureus and Streptomyces coelicolor for polyketides,. Canthaxanthin has been used as food and feed additive for eggyolk, fish and crustacean farms, in cosmetics and as an orally administered pigmenting agent for human skin in pharmaceutical applications. It plays important roles in animal displays of maturity and in the protection of tissues against oxidizing free radicals.

In this laboratory a mutant Aspergillus carbonarius isolated for polygalacturonase over production in shake flasks secreted copious quantities of the enzyme when the growth medium acidified to low pH. During growth under these conditions, the mutant fungus showed features different from that of the wild type. Importantly, it synthesized a yellow pigment, partially saturated canthaxanthin (PSC) which found pharmaceutical applications. This thesis describes studies on the mutant A. carbonarius.

The characterization of the mutant fungus and the possible mechanism involved in its ability to grow in shake flasks at extreme acidic conditions are described. Though the mutant was morphologically distinct from the wild type due to yellow pigmented mycelia, RAPD analysis confirmed its identity as the variant of the wild type A. carbonarius. The pigment (partially saturated canthaxanthin) production and deposition in the mycelia membrane caused a permeability barrier to H+ ions for intracellular pH homeostasis. Thus the mutant was able to grow in extremely acidic conditions and secrete the enzyme under such condition. Probable synthetic pathway of the pigment suggested that the carotenoid originated from the mevalonate pathway. Some other intermediates, not phytoene, were apparently involved in the pigment synthesis. Occurrence of other cyclases, instead of lycopene cyclase, involved in the cyclization reactions to cause pigment synthesis. Results also suggested the possible occurrence of mutation in the isoprenoid pathway for higher production of carotenoid as well as ergosterol. Increased ergosterol concentration with decreased PSC concentration during growth in presence of diphenylamine and vice versa, when grown in the medium containing nicotine, revealed a flow of carbon either for sterol biosynthesis or for carotenoids. 

	For studies on the absorption and bioavailability of A. carbonarius carotenoid, PSC was fed to Wistar rats. These results showing increased ascorbic acid and decreased lipid peroxides in liver suggested the importance of PSC as a dietary compound. The reduction in total cholesterol in the rats fed with PSC was apparently due to decreased serum LDL cholesterol. Dietary PSC in rats showed elevated levels of catalase and glutathione transferase apparently activating endogenous antioxidant defense mechanism. Dietary intake of PSC even at 0.25%, did not result in any treatment related adverse effects in Wistar rats. Thus its possible use in food and therapeutical applications appear feasibility. Upon oral administration, the occurrence of maximum concentration of PSC in the plasma of rats within short period of 3 – 6 h, suggested its consumption to exert health benefits. 

Based on the results above, potential food applications of the pigment, as nutraceutical, were explored.  Preparation of spray dried product with antioxidant property and shelf life are explained. The spray drying of PSC resulted in a free flowing, dry yellow to light brown coloured powder and showed greater storage stability and improved antioxidant property.




Stereoselective Synthesis of (+)-Brefeldin A and (-)-Fumagillol using α-Chloro Sulfide as an Intermediate for C-C bond formation

Thuniki, Naveen Reddy

Introduction 
 
 1
 
The goal of a drug delivery system is to achieve and sustain therapeutic blood 
levels of a drug. Various routes of administration are used in which, per-oral 
route is most preferred, being cost-effective and patient friendly. However per 
oral delivery presents many hurdles starting from drug dissolution in 
gastrointestinal (GIT) fluid to first pass metabolism and also due to various 
physicochemical and bio-pharmaceutical problems. With the result, oral 
therapy has limitations of large variability in the rate and extent of absorption 
of a therapeutic agent into the systemic circulation. 
Maximizing bioavailability of an orally administered drug is therapeutically 
important because the extent of bioavailability directly influences plasma 
concentrations, as well as therapeutic and toxic outcomes. Poorly bioavailable 
drugs are inefficient because a major portion of a dose never reaches the 
plasma or exerts its pharmacological effect. The inter-subject variability in 
bioavailability also remains correlated with the extent of bioavailability. 
Therefore low bioavailability leads to high variability and poor control of 
plasma concentration and effects. Inter-subject variability is particularly of 
concern for a drug with a narrow therapeutic margin or a steep dose vs. effect 
profile [1].  
Considerable evidence is accumulating to suggest that many clinically 
important anticancer drugs are not optimally utilized when taken orally. 
Majority of anticancer drugs show poor and variable bioavailability after their 
oral administration: many even with higher bioavailability show unpredictable 
variability [2, 3]. If systemic availability of a drug averages 20%, for example, 
then 80% of a dose is simply wasted. For drugs that are expensive to produce 
and beyond the reach of millions, wasting 80% of the precious substance is not 
comprehensible. There is, thus, a need to search for alternative drug forms 
having better action profile in terms of oral availability and efficacy.  
Since the beginning of its clinical development 40 years ago, etoposide has 
remained an important and widely used agent in clinical oncology. Its integral 
role in the treatment of germ cell tumors and small-cell lung cancer seems 
unlikely to diminish in the future, and its use in non-Hodgkin's lymphoma and 
Introduction 
 
 2
 
in various high dose regimens will probably continue to increase. Etoposide 
can be given using i.v. and oral formulations. The i.v. dose ranges from 100 to 
120 mg/m2, whereas the oral dose ranges from 200 to 240 mg/m2, reflecting the 
fact that etoposide has an oral bioavailability of ׽50%.  After oral 
administration, the absorption of etoposide is erratic and incomplete: both the 
rate and extent of absorption vary considerably among individuals, and even 
within the same individual, during chronic or multiple dosing, there is wide (25 
- 80%) intra-patient and inter-patient variability in systemic exposure and 
clearance [2, 4-12].      
Improving oral absorption and bioavailability of etoposide has therefore 
remained an important issue within the pharmaceutical industries. Investigation 
for newer active etoposide combination regimens continues for achieving 
improvement in its clinical activity with respect to its optimal dose and 
schedule.  
In recent times a new dimension in chemotherapeutics is being witnessed with 
regard to the development of drug enhancing technologies which will have 
revolutionary shift in the way medicines are administered. The global focus 
therefore is on newer approaches to reduce the drug dosage (and consequently 
cost), making treatment more affordable to a larger section of the society, along 
with lesser toxicity concerns.  
In this context herb-herb and herb-drug interactions have gained a major focus, 
with increasing trend towards the use and understanding of natural products as 
an alternative to the conventional treatment or as an additional medicine to 
boost the conventional therapy.  A vast literature is now available with respect 
to important pharmacokinetic interactions of wide variety of natural products 
(or their analogues) primarily derived from reputed medicinal herbs which 
have shown that such interactions could produce potential effect in vivo,  
among which drug bioavailability is one important pharmacological 
consequence [13,14].  
Introduction 
 
 3
 
In the recent past considerable research has been going on at CSIR- Indian 
Institute of Integrative Medicine (I.I.I.M.), Jammu in order to identify and 
develop bioenhancers which could effectively enhance the bioavailability of 
clinically important drugs which show poor bioavailability after oral 
administration. In this regard multiple bioenhancer moieties derived from 
natural (herbal) sources as well as their analogues have been investigated.  
During the last decade concerted research efforts have resulted in identifying 
an active alkaloid molecule piperine (1-piperoyl piperidine) from piper species, 
and also some other natural substances which have shown to increase the 
bioavailability of a large number of drugs and pharmaceutical agents, and other 
pharmacologically active substances (phenytoin, propranolol, theophylline, 
nevirapine, beta-lactam antibiotics, curcumin, coenzyme Q10, and 
epigallocatechin) in animals and human volunteers. A vast amount of 
information on the role of piperine as bioenhancer has been documented in 
many published reports [15-22]. The mode of bioenhancer action of piperine 
has been understood in its ability to influence the intestinal permeability, and a 
potential CYP 3A4/ P-gp inhibitory role [23-25]. 
In a significant development, CSIR-I.I.I.M., Jammu in public - private 
partnership with Cadila Pharmaceutical Ltd, Ahmedabad, has released a new 
drug formulation against tuberculosis called ‘risorine’, (containing a reduced 
dose of rifampicin along with isoniazid and piperine), which has been found to 
be bioequivalent to standard rifampicin regimen. Launched in November 2009, 
this indigenously developed drug formulation has been approved for marketing 
by Drug Controller General of India after successful completion of all the 
phased clinical trials.  
Recently a novel series of substituted piperine analogues have been synthesized 
at CSIR- IIIM, Jammu [26], and these analogues (including piperine) have 
been evaluated for identifying potential leads as enhancers of drug 
bioavailability with special emphasis on anti-cancer agents. During the 
preliminary investigations one such analogue namely, 4-ethyl 5-(3, 4-
methylenedioxyphenyl)-2E, 4E-pentadienoic acid piperidide, (PA-1) (Fig. 1) 
Introduction 
 
 4
 
was found to be a promising entity as bioavailability enhancer for the drug 
etoposide.   
 
Fig. 1: 4-Ethyl 5-(3, 4 methylenedioxyphenyl)-2E, 
4E - pentadienoic acid piperidine 
 
 
The present study deals with a detailed investigation on the pharmacokinetic 
interaction of PA-1 with etoposide in mice, which has resulted in a significant 
enhancement of bioavailability of orally, administered etoposide. A 
mechanistic evaluation has also been undertaken to examine the mode of action 
of PA-1 by studying some key biochemical events such as absorption/ efflux, 
and metabolism of this drug, as influenced by P-glycoprotein (P-gp), and 
selected cytochrome P450 (CYP) enzymes, which are now recognized as major 
regulators of oral drug bioavailability. 
 


Bio-inspired Strategy to Assemble Graphene Oxide Based

Materials with Inorganic Nanostructures for Photocatalytic

Applications

Vudhgiri, Srikanth

 
 
 
 
 
Synopsis
  Synopsis 
i 
 
Introduction 
Glycolipids were discovered, isolated from brain tissue and named by Ernst Klenk in 
1942. Glycolipids are glycosyl derivatives of lipids. They are collectively a part of larger 
family of substances known as glycoconjugates. Glycolipids are a structurally ubiquitous 
molecules found in all living organisms from prokaryotic to eukaryotic cells, from bacteria to 
humans. Glycolipids are essential constituents of cellular membranes and cell signalling 
molecules. This class of compounds has been attracting an increasing amount of attention up 
to today, on account of playing essential roles in diverse biological functions and bio 
surfactant applications due to their biodegradability. Inspite of a wide range of functions, it is 
surprising that these compounds are not exploited commercially. Possible reasons for this 
could be the lack of availability of natural resources rich in these glycolipids or commercially 
viable process for their isolation/purification as these lipids are found to be complex mixtures 
in their respective natural sources. Furthermore, to understand the complete physico-chemical 
and biological properties of glycolipids, pure glycolipids were required. Thus, in order to 
produce novel glycolipids with enhanced biological applications present study will be mainly 
focused on the entitling “Design, synthesis and biological evaluation of novel glycolipid 
mimics”.  
 
Chapter 1: Introduction to Glycolipids 
 
This Chapter gives introduction about classification of glycolipids, nomenclature, 
structure and different applications. Salient features of various methods available in literature 
for the synthesis of glycoglycerolipids, galactoceramides and phenolic, steryl and 
heterocyclic-based glycosides using chemical and enzymatic process are briefly reviewed. 
Various glycosylation methods used for the synthesis of different type of glycolipids are also 
briefly reviewed. The chapter also gives information on biological evaluation of various type 
of glycolipids isolated and/or synthesized either by partial or total synthesis.  
 
Chapter 2: Synthesis of Dihydrosterculic acid-based Monoglucosyl Diacylglycerol and 
its Analogues and their Biological Evaluation 
 
Among the available broad spectrum of lipids, glycoglycerolipids have gained significant 
attention in view of their diverse properties. They are major constituents present in 
chloroplast membranes of photosynthetic cells and in bacterial cell walls. Glycolipids are 
well-studied molecules exhibiting different biological activities. However, all these activities 
  Synopsis 
ii 
 
are strictly dependent on their fatty acyl chain length and olefinic nature. Simultaneously, 
earlier reports suggest that the site of attachment of the sugar to the glycerol moiety to derive 
an anomeric configuration of the sugar seemed to play negligible roles on improving the 
antitumor potential. 
 
 
Figure 1. Structures of GL1 and Target Molecule 
 
 
  Synopsis 
iii 
 
Recently, Sauvageau and coworkers reported the isolation and characterization of 
dihydrosterculic acid-based glycolipids from Lactobacillus plantarum (GL1) with α-
configuration (Figure 1a). Considering the above facts, in the present study, we herein report 
the synthesis of dihydrosterculic acid-based glycolipids with β-configuration (Figure 1b) 
using trichloroacetimidate method (Scheme 1 to Scheme 3) with good yields for the first 
time. The β-configuration of the GL1 molecule was unambiguously assigned by NMR studies 
using 2D-ROESY (NOE) and J-coupling analysis. Dihydrosterculic acid was synthesized 
using Furukawa’s reagent and the selective esterification of dihydrosterculic acid at C-3 
position of glycerol was achieved with EDC-HCl at 0 °C. All the products were characterized 
by FT-IR, NMR and MS analysis. 
 
 
 
 
 
 
In vitro cytotoxicity of the GL1 molecule and its fatty acid analogues were evaluated 
against DU145, A549, SKOV3 and MCF7 cell lines. Among all the synthesized molecules, 
the GL1 molecule and compound 7d showed moderate activity, while the compound 7b 
showed promising activity against all the tested cell lines with IC50 values of 20.1, 18.2, 19.1 
and 17.6 µM, respectively. In addition, all tested compounds showed poor cytotoxicity 
against normal HUVEC cells. The MCF7 cells when treated with compound 7b showed 
lower bromodeoxyuridine incorporation levels as compared to untreated cells, suggesting that 
the compound 7b was highly effective and inhibited the cell proliferation. In addition, the 
  Synopsis 
iv 
 
compounds showed significant increase in caspases 3 and 9 levels by inducing apoptosis in 
MCF 7 cells. 
 
Chapter 3: Synthesis and Cytotoxic Evaluation of Novel Glycosylated N-Fattyacyl-L-
Tyrosine Derivatives 
 
In general, glycosides are biologically active compounds and the glycosidic residue 
dictates their biological activity. In some cases, the glycosidic residue or overall molecular 
structure improves the pharmacokinetic parameters. Especially, the combination of 
carbohydrate moiety to aglycone plays a functional role in the bioactivity. On the other hand, 
many reports are available on biologically active lipoamino acids and fatty amino acid 
conjugates. These endogenous substances exhibit multiple biological activities such as 
analgesic, anti inflammatory and inhibition of cell proliferation. Based on the above facts, it 
was necessary to place the amino acid acylated with fatty acids and glycosylated with 
carbohydrates into a separate class of lipid derivatives which are worthy to investigate.  
 
 
 
Hence, in the present study, a series of different fatty acids-based (short, medium and long 
unsaturated chains) glycosylated N-fattyacyl-L-tyrosines and N-lipoyl-L-tyrosine methyl 
esters were synthesized and evaluated for their cytotoxic and antimicrobial activities. The 
aglycone moiety was synthesized from L-tyrosine methyl ester. This methyl-L-tyrosinate (1) 
was further derivatized to afford N-fattyacyl-L-tyrosine methyl esters (2-6) with different 
fatty acids using EDC-HCl and HOBt coupling reagents (Scheme 1). The glycosylation of 
aglycone moiety with different carbohydrates was performed using Lewis acid, BF3.Et2O 
(Scheme 2 and 3). The glucose and galactose N-fattyacyl-L-tyrosines were formed in β–
configuration and the mannose and rhamnose N-fattyacyl-L-tyrosines were formed in α–
  Synopsis 
v 
 
configuration due to NGP participation of C2-acetate group of the sugar during the 
glycosylation. 
 
 
 
 
  Synopsis 
vi 
 
All the synthesized compounds were tested against a panel of four cancer cell lines, 
namely, A549, PC3, MDA-MB-231 and HepG2. Among all the tested compounds, 
glycosylated N-fattyacyl-L-tyrosines showed moderate activity against all the cell lines 
irrespective of carbohydrate and lipid moiety and the IC50 values were in the range of 15.6-
45.6 µM. However, the oleic acid analogues (5a, 5d) exhibited minimum IC50 values of 15.6 
and 17.6 µM, respectively, against MDA-MB-231 cell line. In case of N-lipoyl-L-tyrosine 
methyl esters, 6b-6d showed promising activity against all the tested cell lines and the IC50 
values ranged between 9.4-13.8 µM. The compound 6d exhibited significant cytotoxicity 
with IC50 values 10.5, 9.4, 10.9 and 12.1 µM against A549, PC3, MDA-MB-231 and HepG2 
cell lines respectively. It was observed that among all the synthesized compounds, N-
fattyacyl-L-tyrosine methyl esters (2-6) showed a poor antimicrobial activity against both 
bacterial and fungal strains. However, N-fattyacyl-L-tyrosines (7-11) exhibited promising 
antimicrobial activity against both bacterial and fungal strains. 
 
Chapter 4: Design, Synthesis and Cytotoxic Evaluation of Threonine-based Galacto- 
ceramide with Aromatic Groups and Various Fattyacyl Chains 
 
Galactoceramide and its derivatives have been identified as potential anti-tumour 
compounds. From the last two decades, α-GalCer and its synthetic analogues were gained 
much attention in the scientific community due to their high potential antitumor activity in 
the view of versatile utility as invariant natural killer T (iNKT) cell activators and adjuvants 
in many diseases like malaria, HIV, tuberculosis, tumour immunotherapy. Moreover, the 
Phase I clinical trials proved that α-GalCer was ineffective in the treatment of solid tumors 
due to less selectivity toward either Th1 or Th2 cytokines (hormonal messengers) responses. 
Therefore, new α-GalCer analogues will require increasing toward either Th1 or Th2 
cytokines responses. In addition, the SAR study reported in literature on α-GalCer showed 
that modification on the sphingosine OH groups leads to only change in the binding effect but 
not destroy the total activity of α-GalCer. Hence, the modification of fatty acyl group on 
amide moiety or phytosphingosine group allowed for more selective Th2 response.  
 
Based on the above findings, in the present study, threonine-based β-galactoceramide and 
its derivatives were prepared by modifying the fatty acyl group on amide moiety with 
different unusual, branched, saturated and unsaturated fattyacyl moieties and aromatic acids 
employing trichloroacetimidate methodology. 
  Synopsis 
vii 
 
 
 
 
The threonine-based ceramide part was synthesized (Scheme 1) in multi-step process 
using different reagents. This ceramide part was glycosylated with galactose using 
trichloroacetimidate (Scheme 2) as donor. Further, all the synthesized compounds were 
evaluated for in vitro cytotoxicity against three cancer cell lines and one normal cell line. All 
the compounds exhibited moderate to good activity against all the tested cancer cell lines   
  Synopsis 
viii 
 
with IC50 values in the range 14.08 to 64.05 µM. In aromatic derivatives, compound 8i 
exhibited promising activity against MCF7, A549 and HeLa cancer cell lines with IC50 values 
14.08, 14.78 and 16.70 µM, respectively. In fatty acid derivatives, the compound 8m and 
compound 8n exhibited a promising activity against HeLa and MCF7 cancer cell lines with 
IC50 values 16.34 and 18.05 µM, respectively. Based on structure activity relationship, 
aromatic acid derivatives exhibited better activity compared to fatty acid derivatives. As well, 
influence of some of the key factors such as spacer chain length between aromatic residue 
and amide functional group, methoxy substituents on aryl group, terminal unsaturation of 
fatty acid and branching chain effect on the cytotoxicity was described in this study. 
 
Chapter 5: Synthesis and Biological Evaluation of Phenolic, Thio and Steryl Glycosides  
 
Section A: Synthesis and Biological Evaluation of Marumoside A and its Lipid 
Derivatives  
 
Moringa oleifera (horseradish tree) is a fast growing, multipurpose tree native to tropical 
and subtropical areas, for human, animal feeding and medicinal uses. The green pods, flowers 
and leaves are used as vegetables in many countries. In folklore medicine, the various parts of 
the plant have long been recognized for ailments like pain and inflammation. From the 
different parts of Moringa oleifera plant, several types of bioactive compounds have been 
isolated. Recently, Marumoside A and other compounds were isolated from the leaves of 
Moringa oleifera of Thai origin. In Marumoside A, 4-hydroxyphenylethanamide was 
glycosylated at anomeric hydroxyl group of L-rhamnose in α-configuration. Further,             
4-hydroxyphenylethanamide is structurally related to bioactive compounds like homovanillic 
acid amides (Capsaicin) and hydroxyphenylacetamides. These compounds have profound anti 
inflammatory, antinociceptive, analgesic and antiirritant activities.  
 
 
  Synopsis 
ix 
 
 
Considering the above facts, in the present study, Marumoside A was synthesized for the 
first time using trichloroacetimidate donor as a key step. The aglycone 4-hydroxy 
phenylacetamide was prepared from 4-hydroxyphenyl acetic acid using oxallyl chloride and 
aqueous ammonia. The lipid derivatives of Marumoside A were synthesized (Scheme 1 and 
2) using different fatty amines. The antiinflammatory activity of the Marumoside A and its 
lipid derivatives was evaluated for the inhibition of TNF-α and IL-1β secretion levels. 
Among all the synthesized molecules, the oleyl amine lipid (unsaturated) derivative showed 
significant inhibition of TNF-α and IL-1β secretion with IC50 value of 16.7 µM and 23.4 µM, 
respectively when compared to Marumoside A and its saturated lipid derivatives.  
 
Section B: Synthesis and Biological Evaluation of 5-Fattyacylamido-1, 3, 4-Thiadiazole-
2-Thioglycosides  
 
Recently thioglycosides have gained much attention because of their broad spectrum of 
properties such as antitumour, antidiabetic, biological inhibitors, inducers, in vitro inhibitory 
effects on the replication of a number of DNA viruses. Many of the thioglycoside derivatives 
are having common glycosylthio moiety attached to carbon linked to nitrogen either in an 
open chain or cyclic form (heterocyclic ring). These glycosylthio moiety attached to the 
  Synopsis 
x 
 
heterocyclic ring is known as glycosylthio heterocycles. In these glycosylthio heterocycles, 
glycone and aglycone (different heterocyclic rings) were modified for extensive biological 
activities.1, 3, 4-Thiadiazoles and its derivatives having a wide range of biological 
applications in different fields such as agriculture, petroleum and medicine. Many drugs are 
available in the market with thiadiazole nucleus such as sulfamethazole, methazolamide, 
acetazolamide etc. 
 
 
 
 
Based on the above facts, in the present study, the synthesis of 1, 3, 4-thiadiazole-based 
thioglycosides were accomplished in good yields. In Scheme 1, 5-Amino-1, 3, 4-thiadiazole-
2-thiol was synthesized and this was followed by synthesis of a series of 5- fattyacylamido-1, 
3, 4-thiadiazole-2-thiols with fatty acid chlorides. In Scheme 2, the glycosylation of 5- 
fattyacylamido-1, 3, 4-thiadiazole-2-thiols were achieved with trichloroacetimidate 
methodology. All the compounds were in β–configuration. This was supported by 1H-NMR 
analysis, the signals of H-1ʹ proton was observed as a doublet at δ = 4.97 ppm, J = 10.07 Hz. 
Anti microbial and anti cancer activities of 1, 3, 4-thiadiazole-based thioglycosides were 
  Synopsis 
xi 
 
evaluated against three Gram-positive and three Gram-negative bacterial strains and four 
cancer and one normal cell line. Among the entire tested compounds lauric acid and myristic 
acid derivatives showed good antimicrobial activity against Gram-negative bacterial strain 
Klebsiella pneumonia with MIC values 12.5, 25 µg/mL and moderate activity exhibited 
against Gram-positive bacterial strain Bacillus subtilis with MIC values 25 and 50 µg/mL, 
respectively. In case of cytotoxicity, the free hydroxyl lauric (9e) and oleic acid derivative 
(9i) exhibited promising activity against cervical cancer cell line with IC50 values 8.7, 8.8 
µM, respectively. Further, the free hydroxyl compounds 9a, 9c-9j did not show any toxicity 
towards normal CHO-K1 cell line and acylated compounds 8a-8j exhibited toxicity against 
CHO-K1 cell line.    
 
Section C: Synthesis and Cytotoxic Evaluation of Cholesteryl 6-O-acyl-β-D-
Galactopyranosides 
 
In recent years, the isolation and/or synthesis of steryl glycosides have been motivated 
because of their remarkable biological functions, including regulation of host defences 
against pathogens, lipid metabolism and imunomodulatory properties. These steryl glycosides 
are widely found in various plants especially vascular plants, fungi, some animals and a few 
bacteria. Two major glycolipids were isolated from Borrelia burgdorferi, in 2001 they were 
initially identified as galactosyl diacyl glycerols. Later, these glycolipids were corrected as 
cholesteryl 6-O-acyl-β-D-galactopyranoside (BbGL1) and 1, 2-di-O-acyl-3-O-α-D-
galactopyranosyl-sn-glycerol (BbGL2) with major fatty acid composition oleate and 
palmitate. Among these compounds, BbGL1 is a steryl glycoside, it is antigenic lipid 
component of Borrelia burgdorferi, and the research is in progress on these BbGL1 to 
develop potential vaccines against Lyme disease. Moreover, recent evidence reveals that 
cholesterol may directly mask glycosphingolipids and it protects the carbohydrate from toxin-
derived proteins. 
 
Based on the above findings, in the present study, Cholesteryl 6-O-acyl-β-D-
galactopyranosides were synthesized using trichloroacetimidate methodology (Scheme 1) 
with unusual, short, medium, long and unsaturated fatty acids. The anomeric acetyl group of 
galactose penta acetate was chemo selectively removed by hydrazine acetate and this was 
followed by preparation of trichloroacetimidate using trichloroacetonitrile in the presence of 
DBU. Glycosylation of cholesterol was achieved using trichloroacetimidate donor and 
  Synopsis 
xii 
 
TMSOTf as promoter. Acetate groups were selectively deprotected with Zemplen deacylation 
and subsequently, Cholesteryl 6-β-D-galactopyranoside was selectively esterified at C-6 
position with EDC. HCl. All the compounds were evaluated for cytotoxicity against four 
cancer and one normal cell lines, namely human ovarian cancer (SKOV3), cervical cancer 
(HeLa), breast cancer (MDAMB-231), human prostate cancer (DU145) cell lines and normal 
Chinese hamster ovary cancer (CHO-K1) cell line. Among all the tested compounds long 
chain saturated palmitic and stearic acid derivatives (6g, 6h) exhibited significant cytotoxicity 
against cervical cancer cell line with IC50 values 17.3 and 20.3 µM respectively. All the tested 
compounds did not show any toxicity towards normal CHO-K1 cells. 
 
 
 
 
 
 


DESIGN, SYNTHESIS AND BIOLOGICAL EVALUATION OF NOVEL GLYCOLIPID MIMICS

LINGAYYA, RAJAKA

Synopsis 
 
i 
 
Synopsis 
The work carried out in the research tenure has been accumulated in the form of thesis 
entitled as “UTILIZING QUINAZOLINONES FOR THE Ru-, Rh-, Pd-CATALYZED 
C–C, C–N, C–O BOND FORMATION REACTIONS: SYNTHESIS AND 
ANTICANCER ACTIVITIES OF N-HETEROCYCLES”. The main aim of this work 
was the functionalization of quinazolinones by fine-tuning transition metal-catalysis to obtain 
heterocycles of biological interest. Further, synthesis of 2,3-dihydroquinazolin-4(1H)-ones, 
(E)-3-aryl-1-(5-(2-arylimidazo[1,2-a]pyridin-3-yl)thiophen-2-yl)prop-2-en-1-ones and 
evaluation of their anticancer activity. The thesis has been divided into four chapters. 
CHAPTER-1: Describes the general introduction of transition metal-catalyzed cross-
coupling/annulation, carbenoid insertion, alkenylation and heteroarylation reactions. 
CHAPTER-2: Describes the ruthenium as a single catalyst for two steps: one-pot 
ruthenium(II)-catalyzed aerobic oxidative dehydrogenation of dihydroquinazolinones and 
cross-coupling/annulation to give N-fused polycyclic heteroarenes. 
CHAPTER-3: This chapter consists of two sections. 
SECTION-A: This section deals with the rhodium(II)-catalyzed carbenoid insertion of N-
tosylhydrazones into amide N−H bonds: an efficient approach to N
3
-benzyl/alkyl-2-
arylquinazolinones. 
SECTION -B: This section describes the palladium(II)-catalyzed direct O-alkenylation of 2-
arylquinazolinones with N-tosylhydrazones: an efficient route to O-alkenylquinazolines. 
CHAPTER-4: This chapter consists of two sections. 
SECTION-A: This section deals with convenient and scalable synthesis of 2,3- 
dihydroquinazolin-4(1H)-one derivatives and their anticancer activities. 
SECTION-B: This section illustrates palladium(0)-catalyzed direct C–H heteroarylation of 
2-arylimidazo [1,2-a]pyridines with (E)-1-(5-bromothiophen-2-yl)-3-arylprop-2-en-1-ones 
and their anticancer activity. 
CHAPTER-1 
General Introduction of Transition Metal-Catalyzed Cross-Coupling/Annulation, 
Carbenoid Insertion, Alkenylation and Heteroarylation Reactions. 
Introduction: Quinazolinone as a building block occurs widely in natural products that 
exhibit a broad range of useful biological and pharmacological activities. It is assigned as a 
privileged skeleton in drug discovery.
[1]
 The construction of new carbon–carbon and carbon–
heteroatom bonds is the most vital tool in organic chemistry, critical for the synthesis of 
organic compounds that exhibit significant biological, pharmaceutical and material 
Synopsis 
 
ii 
 
properties.
[2]
 In this chapter, we discussed different types of transition metal-catalyzed cross- 
coupling/annulation, carbenoid insertion, alkenylation and heteroarylation reactions with 
examples and recent applications of these reactions. 
CHAPTER-2 
Ruthenium as a Single Catalyst for Two Steps: One-Pot Ruthenium(II)-Catalyzed 
Aerobic Oxidative Dehydrogenation of Dihydroquinazolinones and Cross-
Coupling/Annulation to give N-Fused Polycyclic Heteroarenes. [Asian J. Org. Chem. 
2015, 4, 462–469] 
Introduction: Transition metal-mediated C−H bond functionalizations for C−C and 
C−heteroatom bond formation has evolved as a powerful tool for constructing complex 
structures.
[3]
 Due to highly-attractive green and sustainable advantages, molecular oxygen 
finds increasing use in both the commodities chemical industry and the academic labs.  
Statement of problem: Ruthenium(II)-catalyzed alkynes annulations with directing group 
containing arenes reported thus far required the use of additional oxidants, such as copper(II) 
or silver(I) salts, thereby leading to the formation of undesired stoichiometric metal-
containing by-products. 
Methodology used: 
 
Results and Discussion: One-pot oxidative dehydrogenation of dihydroquinazolinones 
followed by cross-coupling/annulation with alkynes to access N-fused polycyclic 
heteroarenes was performed in the presence of [RuCl2(p-cymene)]2 (5 mol%), O2 (balloon), 
Na2CO3 (2 equiv) in DCE (2 mL) at 80 °C for 12 h. First, we investigated the 
dihydroquinazolinones scope (Scheme 1). Products (1-9) were obtained in good to excellent 
yields with excellent functional-group tolerance. Electron-rich substrates gave high yields. 
The structure of the product 6 was confirmed by single crystal X-ray diffraction. Moreover, 
6-chloro-2-phenyl-2,3-dihydroquinazolin-4(1H)-one derivatives bearing electron-neutral, -
donating and -withdrawing groups were tested (10-18). We further tested heterocyclic 
dihydroquinazolinones, corresponding products (19-24) were obtained in good to excellent 
yields (Scheme 2). Scope of alkynes was then investigated using A1 as the coupling partner 
(Scheme 3). Aryl alkynes (B2) as well as more challenging alkyl alkynes (B3-B5) are also 
tolerated (25-28). Regioselectivity of the C−H activation was evaluated (Scheme 4), with 
Synopsis 
 
iii 
 
A25 produced exclusively one product 29 in 85% yield. When unsymmetrical alkyne was 
employed, two regioisomeric products 30a and 30b were obtained in 82% and 10% yields  
 
Scheme 1 
 
Scheme 2 
 
Scheme 3 
 
Scheme 4 
Synopsis 
 
iv 
 
respectively. For alkynes and dihydroquinazolinones, intermolecular competition 
experiments were conducted. Results revealed that the aromatic alkyne and 
dihydroquinazolinone with electron-donating group showed higher reactivity. Mechanistic 
investigations revealed that CONH group serves as directing group and is essential for the 
annulation process. Further in two steps one-pot process, oxidative dehydrogenation occurs 
first, followed by cross-coupling/annulation. Based on the experimental results, a plausible 
mechanism has been illustrated. 
In summary, ruthenium(II)-catalyzed, aerobic oxidative dehydrogenation, cross-
coupling/annulation via C−H/N−H bond activation of dihydroquinazolinones with alkynes in 
one-pot to access N-fused polycyclic compounds was described. 
CHAPTER-3 (Section-A) 
Rhodium(II)-Catalyzed Carbenoid Insertion of N-Tosylhydrazones into Amide N–H 
Bonds: An Efficient Approach to N
3
-Benzyl/Alkyl-2-arylquinazolinones. [Adv. Synth. 
Catal. 2016, 358, 81–89] 
Introduction: The transition metal-catalyzed insertion of metal-carbenoids into C–H or X–H 
(X = heteroatom) bonds represents an excellent and powerful method for the formation of C–
C or C–X bonds.
[4]
  
Statement of problem: Carbenoid insertion into N–H bonds is a direct and potentially 
powerful method of forming C–N bonds, which is an underexploited process with great 
potential for further development. 
Methodology used: 
 
Results and Discussion: Upon screening a variety of reaction conditions, we identified a 
standard condition as follows: A1 (0.25 mmol), B1 (0.3 mmol), K2CO3 (0.5 mmol), 
[Rh2(OAc)4] (1 mol%), in 1,4-dioxane at 90 °C for 4 h. We subsequently investigated the 
preparative scope of dihydroquinazolinones (A1-A16) with benzaldehyde derived N-
tosylhydrazone (B1) to afford the corresponding N
3
-benzyl-2-arylquinazolin-4(3H)-ones (1-
16) with good to excellent yields (Scheme 1). The structure of the product 4 was confirmed 
by single crystal X-ray diffraction. Next, the scope of N-tosylhydrazones (B2-B12) was 
investigated using 2-phenyl-2,3-dihydroquinazolin-4(1H)-one (A1) as the insertion partner 
Synopsis 
 
v 
 
(Scheme 2). The desired products (17-27) were isolated in good yields. We also conduct this 
reaction in one-pot starting from benzaldehyde; product 1 could be isolated in 80% yield. 
 
Scheme 1 
 
Scheme 2 
Further, 2-isopropyl-2,3-dihydroquinazolin-4(1H)-one (A17), 3-phenyl-3,4-dihydro-2H-
benzo[e][1,2,4]thiadiazine 1,1-dioxide (A18) and disubstituted N-tosylhydrazone (B13) were 
chosen as the substrates (Scheme 3). All reactions proceeded smoothly to form desired 
products (28-30) in 78-81% yield.  
 
Scheme 3 
To elucidate the reaction mechanism, we conducted control experiments. Results 
demonstrate that at first the amine N–H group be would involved in oxidative 
Synopsis 
 
vi 
 
dehydrogenation, and then insertion of rhodium-carbenoid into amide N–H bond would take 
place. A plausible reaction mechanism was illustrated. 
In summary, we have demonstrated a general strategy for direct synthesis of N
3
-
benzyl/alkyl-2-arylquinazolinones via rhodium(II)-carbenoid involved insertion of N-
tosylhydrazones into the amide N–H bonds of quinazolinones.  
CHAPTER-3 (Section-B) 
Palladium(II)-Catalyzed Direct O-Alkenylation of 2-Arylquinazolinones with N-
Tosylhydrazones: An Efficient Route to O-Alkenylquinazolines. [Chem. Commun. 2017, 
53, 1672–1675] 
Introduction: The quinazoline moiety, an abundant core of nitrogen-containing heterocycles, 
is exemplified as a privileged structure that exhibits diverse biological activities. N-
tosylhydrazones were utilized as versatile coupling partners for cross-coupling reactions.
[5]
  
Statement of problem: Direct synthesis of O-alkenylatedquinazolines starting from 2-aryl-
quinazolinones catalyzed by palladium has until now been unreported with N-
tosylhydrazones. 
Methodology used: 
 
Results and Discussion: After extensive optimization studies, we identified a standard 
condition as follows: A1 (0.5 mmol), B1 (0.5 mmol), Pd(OAc)2 (2.5 mol%), PPh3 (5 mol%),  
N
NH
O
Ph R
NNHTs Pd(OAc)2 (2.5 mol%)
PPh3 (5 mol%)
Cs2CO3 (1 equiv)
t-AmOH, 80 C, 12 h
N
N
O
Ph
(A1) (B1-B17) (1-17)
B1 R = C6H5 1 R = C6H5 (90%)
B2 R = 4-MeC6H4 2 R = 4-MeC6H4 (91%)
B3 R = 4-OMeC6H4 3 R = 4-OMeC6H4 (86%)
B4 R = o-FC6H4 4 R = o-FC6H4 (82%)
B5 R = m-MeC6H4 5 R = m-MeC6H4 (85%)
B6 R = 4-FC6H4 6 R = 4-FC6H4 (92%)
B7 R = 4-ClC6H4 7 R = 4-ClC6H4 (89%)
B8 R = 4-BrC6H4 8 R = 4-BrC6H4 (87%)
B9 R = 4-PhC6H4 9 R = 4-PhC6H4 (73%)
B10 R = 2-Naphthyl 10 R = 2-Naphthyl (69%)
B11 R = 2,4-diClC6H3 11 R = 2,4-diClC6H3 (82%)
B12 R = 3,5-diFC6H3 12 R = 3,5-diFC6H3 (89%)
B13 R = 4-CF3C6H4 13 R = 4-CF3C6H4 (78%)
B14 R = 4-NO2C6H4 14 R = 4-NO2C6H4 (68%)
B15 R = 2-Phenylethane 15 R = 2-Phenylethane (71%)
B16 R = Propane 16 R = Propane (68%)
B17 R = Cyclohexane 17 R = Cyclohexane (66%)
R
 
Scheme 1 
Synopsis 
 
vii 
 
 
Scheme 2 
Cs2CO3 (1 equiv.), in t-AmOH (3 mL), at 80 °C, open to air for 12 h. We then investigated 
the scope of N-tosylhydrazones (B1-B17). Aryl as well as aliphatic N-tosylhydrazones 
reacted smoothly, to give desired products (1-17) in 66-92% yields (Scheme 1). Next, the 
scope of 2-aryl-quinazolinones was examined using B1 as the olefin source (Scheme 2). The 
reaction is significantly affected by the position of substituent on the aromatic ring. It is 
noteworthy that chloro and bromo substituents are tolerated, which is advantageous for 
further transformations. Moreover, hetero aryl bearing quinazolinones also worked well in 
the reaction (26-27). 2-Phenylthieno[3,2-d]pyrimidin-4(3H)-one also successfully employed 
as substrate (29). Gram-scale synthesis of 1 was performed in excellent yield. Preliminary 
mechanistic experiments were conducted, a plausible mechanism was illustrated. 
In conclusion, we have developed an efficient Pd(II)-catalyzed direct O-alkenylation 
reaction of 2-aryl-quinazolinones with simple ketone-derived N-tosylhydrazones as the 
alkene source to give O-alkenylated quinazolines. 
CHAPTER-4 (Section-A) 
Convenient and Scalable Synthesis of 2,3-Dihydroquinazolin-4(1H)-one Derivatives and 
Their Anticancer Activities [Synth. Commun. 2015, 45, 1893–1901] 
Introduction: Cancer is a systemic disease characterized by uncontrolled growth of 
abnormal cells and is a leading cause of fatality worldwide.
[6]
 2,3-Dihydroquinazolin-4(1H)-
one derivatives are an important class of fused N-containing heterocycles with various 
biological, medicinal, and pharmacological activities. 
Statement of problem: Synthesis of 2,3-dihydroquinazolin-4(1H)-one derivatives requires 
harsh reaction conditions, excess catalyst loading, and prolonged time. 
 
Synopsis 
 
viii 
 
Methodology used: 
 
Results and Discussion: We carried out the cyclocondensation reaction between 2-
aminobenzanilide (A1) and benzaldehyde (B1) under various conditions. It was found that 
0.5 mol% of InBr3 efficiently catalyzed the reaction. We extended the scope of this method 
by synthesizing an array of 2,3-dihydroquinazolin-4(1H)-one derivatives (1-25) bearing 
either electron-donating or -withdrawing groups on the aromatic ring (Scheme 1). Further, 
aliphatic aldehydes were also tested in the reaction (26 and 27). Finally, the reaction is 
accessible on a high scale (10 mmol of 2-aminobenzanilide and benzaldehyde), requiring 
only 60 min to achieve excellent yield of 92%. A plausible mechanism was illustrated to 
account for the InBr3-catalyzed reaction. Synthesized compounds were evaluated for their 
X
N
H
O
NH2
R2 H
O InBr3 (0.5 mol%)
CH3CN, rt, 10-60 min
R1 X
N
H
N
O
R2
R1
(A1-A2) (B1-B15) (1-27)
A1 X = H; R1 = Ph B1 R2 = C6H5 1 X = H; R
1 = Ph; R2 = C6H5 (94%)
A1 X = H; R1 = Ph B2 R2 = 4-MeC6H4 2 X = H; R
1 = Ph; R2 = 4-MeC6H4 (96%)
A1 X = H; R1 = Ph B3 R2 = 4-IsopropylC6H4 3 X = H; R
1 = Ph; R2 = 4-IsopropylC6H4 (97%)
A1 X = H; R1 = Ph B4 R2 = 4-MeOC6H4 4 X = H; R
1 = Ph; R2 = 4-MeOC6H4 (98%)
A1 X = H; R1 = Ph B5 R2 = 3,4-diMeOC6H3 5 X = H; R
1 = Ph; R2 = 3,4-diMeOC6H3 (95%)
A1 X = H; R1 = Ph B6 R2 = 3,4,5-triMeOC6H2 6 X = H; R
1 = Ph; R2 = 3,4,5-triMeOC6H2 (96%)
A1 X = H; R1 = Ph B7 R2 = 4-FC6H4 7 X = H; R
1 = Ph; R2 = 4-FC6H4 (96%)
A1 X = H; R1 = Ph B8 R2 = 2,4-diClC6H3 8 X = H; R
1 = Ph; R2 = 2,4-diClC6H3 (94%)
A1 X = H; R1 = Ph B9 R2 = 4-BrC6H4 9 X = H; R
1 = Ph; R2 = 4-BrC6H4 (97%)
A1 X = H; R1 = Ph B10 R2 = 4-PhC6H4 10 X = H; R
1 = Ph; R2 = 4-PhC6H4 (96%)
A1 X = H; R1 = Ph B11 R2 = 4-CNC6H4 11 X = H; R
1 = Ph; R2 = 4-CNC6H4 (93%)
A1 X = H; R1 = Ph B12 R2 = 1-Naphthyl 12 X = H; R1 = Ph; R2 = 1-Naphthyl (94%)
A2 X = Cl; R1 = H B1 R2 = C6H5 13 X = Cl; R
1 = H; R2 = C6H5 (97%)
A2 X = Cl; R1 = H B2 R2 = 4-MeC6H4 14 X = Cl; R
1 = H; R2 = 4-MeC6H4 (95%)
A2 X = Cl; R1 = H B3 R2 = 4-IsopropylC6H4 15 X = Cl; R
1 = H; R2 = 4-IsopropylC6H4 (96%)
A2 X = Cl; R1 = H B4 R2 = 4-MeOC6H4 16 X = Cl; R
1 = H; R2 = 4-MeOC6H4 (98%)
A2 X = Cl; R1 = H B5 R2 = 3,4-diMeOC6H3 17 X = Cl; R
1 = H; R2 = 3,4-diMeOC6H3 (96%)
A2 X = Cl; R1 = H B6 R2 = 3,4,5-triMeOC6H2 18 X = Cl; R
1 = H; R2 = 3,4,5-triMeOC6H2 (95%)
A2 X = Cl; R1 = H B7 R2 = 4-FC6H4 19 X = Cl; R
1 = H; R2 = 4-FC6H4 (94%)
A2 X = Cl; R1 = H B8 R2 = 2,4-diClC6H3 20 X = Cl; R
1 = H; R2 = 2,4-diClC6H3 (93%)
A2 X = Cl; R1 = H B9 R2 = 4-BrC6H4 21 X = Cl; R
1 = H; R2 = 4-BrC6H4 (96%)
A2 X = Cl; R1 = H B10 R2 = 4-PhC6H4 22 X = Cl; R
1 = H; R2 = 4-PhC6H4 (95%)
A2 X = Cl; R1 = H B11 R2 = 4-CNC6H4 23 X = Cl; R
1 =H; R2 = 4-CNC6H4 (93%)
A2 X = Cl; R1 = H B12 R2 = 1-Naphthyl 24 X = Cl; R1 =H; R2 = 1-Naphthyl (95%)
A2 X = Cl; R1 = H B13 R2 = 4-NO2C6H4 25 X = Cl; R
1 = H; R2 = 4-NO2C6H4 (94%)
A1 X = H; R1 = Ph B14 R2 = Butyl 26 X = H; R1 = Ph; R2 = Butyl (75%)
A1 X = H; R1 = Ph B15 R2 = Pentyl 27 X = H; R1 = Ph; R2 = Pentyl (79%)  
Scheme 1 
in vitro cytotoxicity against four different human cancer cell lines, i.e., lung cancer (A549), 
breast cancer (MCF7), cervical cancer (HeLa), and prostate cancer (DU145). Doxorubicin is 
used as a reference drug in this study. Compounds 24, 15, 21, and 14 exhibited promising 


Utilizing quinazolinones for the Ru-, Rh-, Pd-catalyzed C-C, C-N-C-O bond formation reactions: Synthesis and anticancer activities of N-heterocycles

SAJJA, YASODAKRISHNA

i 
 
Abstract 
The thesis entitled “A new templates of 6,7,8,9-Tetrahydro-5-
benzocycloheptenones and their biological evaluation, Total synthesis 
of Dendrodolide-I and Regioselectively derived Pyranocoumarins” has 
been divided into five chapters.  
CHAPTER I: This chapter further divided into section A and B.  
        Section A: A brief review on 6,7,8,9-Tetrahydro Benzocyclоheptenоnes 
 This chapter deals with the literature survey of  6,7,8,9-Tetrahydro-
5-Benzocycloheptenones (Benzosuberone) wherein The synthesis of 
benzosuberone and its fused heterocyclic derivatives is a topic of growing 
interest especially in medicinal chemistry. Efficient introduction of this 
moiety into bioactive molecules, especially in the positions responsible for 
their physiological profile, becomes a very important direction in 
pharmaceutical studies that stimulates work directed to elaboration of 
synthetic methodology for various compounds containing benzosuberone 
based fused heterocycles. The existing methods for direct synthesis of fused 
heterocycles do not allow always for the introduction of benzosuberone 
based fused heterocyclic moiety in the required position of the molecule. As 
a result, a more flexible synthetic approach is required based on the 
application of simple and readily available benzosuberone derivatives is a 
good supplement for direct synthetic methods and is now a days gaining 
importance. Hence, we turned our interest towards the development of an 
environmentally benign protocol for the synthesis of diverse fused 
ii 
 
benzosuberone derivatives that might have an extensive scope from both 
industrial and biological significance. 
Section B: A Convenient Synthesis and screening of benzosuberone-
linked triazoles against Mycobacterium tuberculosis. 
 This chapter deal with introduction to triazoles and Literature review 
оn antitubercular activity оf triazole cоntaining cоmpоunds. The present 
work part discuss about the A series of benzosuberone bearing 1,2,3-
triazoles were rationally designed and alkyl/aryl groups appended on 1,2,3-
triazole derivatives were synthesized using click chemistry and evaluated for 
their in vitro antimycobacterial activity against Mycobacterium tuberculosis 
H37Rv (ATCC27294). 
 
 
 
 
iii 
 
 
CHAPTER II: This chapter further divided into section A and B.  
Section A: Three-component, one-pot synthesis of 
benzo[6,7]cyclohepta[1,2-b]pyridine derivatives under catalyst-free 
conditions and evaluation of their anti-inflammatory and Anti-
proliferative activity. 
 This section deals with a very brief introduction to pyridine, 
Benzocyclоhepta[1,2-b]pyridines and Previous work fоr synthesis оf 
benzocyclоhepta[1,2-b]pyridines by MCRs. The present work part, we 
discuss about the synthesis of benzo[6,7]cyclohepta[1,2-b]pyridine 
derivatives using β-chloroacroleins, 1,3-dicarbonyls and  ammonium 
acetate under catalyst free conditions by using ethanol as reaction media 
and evaluation of their anti-inflammatory and Anti-proliferative activity. 
 
Section B: Design, synthesis and molecular docking studies of  
benzo[6,7]cyclohepta[1,2-b]pyridine -1,3,4- oxadiazole hybrids as 
antituberculosis Agents. 
 This chapter deal with introduction to oxadiazoles and Literature 
review оn antitubercular activity of 1,3,4-Oxadiazоles clubbed with pyridine 
containing Compounds. The present work part discuss about the Design, 
iv 
 
synthesis and molecular docking studies of  benzo[6,7]cyclohepta[1,2-
b]pyridine -1,3,4-oxadiazoles and evaluated for their in vitro 
antimycobacterial activity against Mycobacterium tuberculosis H37Rv 
(ATCC27294). 
 
CHAPTER III: Design, synthesis and Molecular docking studies of 
benzo[6,7]cyclohepta[1,2-b]pyridine -1,2,3- triazole hybrids as 
antituberculosis Agents. 
 This chapter deals with the introduction to Mycоbacterium 
tuberculоsis and literature survey on 1,2,3-Triazole derivatives fоr 
treatment оf tuberculоsis. The present work part discuss about the Design, 
synthesis and molecular docking studies of  benzo[6,7]cyclohepta[1,2-
v 
 
b]pyridine -1,2,3-Triazole and evaluated for their in vitro antimycobacterial 
activity against Mycobacterium tuberculosis H37Rv (ATCC27294). 
 
CHAPTER IV: Tоtal synthesis оf Dendrоdolide I 
 This chapter deals with the introduction to macrolides, literature 
survey on natural prоducts cоntaining 12-member ring lactones and Earlier 
synthetic apprоache for Dendrоdolide I. The present work part describes the 
total synthesis of Dendrоdolide I starting from commercially available 
propylene oxide and 3-buten-1-ol. The key steps involved in the synthetic 
sequence of Dendrоdolide I are the Jacobsen hydrolytic kinetic resolution 
(HKR), Brown allylation, Yamaguchi esterification and ring-closing 
metathesis.  
vi 
 
 
CHAPTER V: Lewis Acid - Catalyzed Cyclization of 4-
Hydroxycoumarine with Propargylic alcohols: Regioselective 
synthesis of Pyranocoumarins. 
 This section deals with a very brief introduction to pyranocoumarins 
and previous synthetic approaches for pyranocoumarins. The present work 
part, details the BF3.Et2O catalyzed synthesis of novel pyranocoumarins. 
7 
 
 


A NEW TEMPLATES OF 6,7,8,9-TETRAHYDRO-5-

BENZOCYCLOHEPTENONES AND THEIR BIOLOGICAL EVALUATION, TOTAL SYNTHESIS OF DENDRODOLIDE-I AND REGIOSELECTIVELY DERIVED PYRANOCOUMARINS

VANAPARTHI, SATHEESHVARMA

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Abstract 
Abstract 
 
i 
 
ABSTRACT 
The thesis entitled “Synthesis and Biological Evaluation of Benzo[4,5]thiazolo[1,2-
a]pyrimidine and 1, 2, 3-Triazole heterocycles as Anticancer agents” has been divided 
into four chapters. 
CHAPTER I:   General Introduction:  Micro review on Benzothiazolopyrimidine 
 and 1,2,3- Triazole  
CHAPTER II:     
Section-A :    Synthesis of novel benzo[4,5]thiazolo[1,2-a]pyrimidine-3-
 carboxylate derivatives and biological evaluation as potential 
 anticancer agents                                                   
Section-B:  Synthesis and anti cancer activity of benzo[4,5]thiazolo[3,2-    
 a]pyrimidine-3-carbonyl)piperazine-1-carbodithioate derivatives 
CHAPTER III:  
Section-A:  Synthesis of novel 1,2,3-triazole substituted chalcone acetamide 
 derivatives as potential antitumor agents 
Section-B:     Synthesis and evaluation of anti proliferative activity of novel 
 1,2,3-triazole substituted pyrazole derivatives 
CHAPTER IV: Design and Synthesis of 1,2,3-triazole substituted  benzo[d]  
   imidazole and 1,2,3-triazole substituted quinazolin-4(3H)-one  
   derivatives as  potential anti proliferative agents 
 
 
 
Abstract 
 
ii 
 
Chapter I:   
General Introduction 
Benzo[4,5]thiazolo[1,2-a]pyrimidines: 
Pyrimidines have a long distinguished history extending from the days of their 
discovery as important constituents of nucleic acids to their current use in the 
chemotherapy of AIDS. The pyrimidine ring is found in vitamins like thiamine, 
riboflavin and folic acid. During the last two decades, several pyrimidine derivatives have 
been developed as chemotherapeutic agents and have found wide clinical applications. 
Benzothiazoles represent a class of heterocyclic compounds of great importance in 
biological chemistry. They exist in many condensed fused systems that were found to 
possess a wide range of activity. Various literature reports display numerous fused 
pyrimidine ring systems and their chemotherapeutic activities as anticancer, antibacterial  
and antiviral  agents. Since the two heterocyclic moieties, benzothiazoles and 
pyrimidines, constitute two active pharmacophores that are highly active against 
antitumour and antimicrobial, combining the two is expected to have a  synergistic effect 
on their biological properties. Also substituted thiazolopyrimidine ring systems were 
reported to possess antitumour activity. The reported significance of such synthons 
generated the interest to exploit this valuable structure in the designing and the synthesis 
of new benzo [4,5]thiazolo[1,2-a]pyrimidine-3-carboxylate derivatives. 
1,2,3-Triazoles: 
The chemistry of heterocyclic compounds continues to be an exploring field in the 
organic and pharmaceutical chemistry. 1, 2, 3-Triazoles constitute an important class of 
nitrogen heterocycles in the field of organic and medicinal chemistry. Medicinally, they 
have been shown to possess a wide range of diverse interesting pharmacological 
properties such as antituberculosis,
 
anti-HIV, antimalarial, antiepileptic, antiallergic, 
antileishmanial, antifungal, anticancer and antibacterial
 
activities. In addition, these 
molecules have been utilized as proton transport facilitators,
  
glycoside cluster arrays,
 
spacers or linkers to dendrimers, DNA cleaving agents, structural components in hyper 
Abstract 
 
iii 
 
branched polymers
 
 and most importantly in liquid crystals.  Synthesis of 1,2,3-triazoles 
is mainly the copper(I) catalyzed 1,2,3-triazole forming reaction between azides and 
terminal alkynes is called   ‘click chemistry’. So a programme on structural modification 
and development a novel building blocks of 1, 2, 3-triazole undertaken in the present 
work. Since all the products were evaluated for their biological activity, in this context 
report studies on the synthesis and chemistry of 1, 2, 3-triazole to study their structure 
activity relationship. Synthesis of novel series of 1,2,3-triazole based building blocks, 
their biological evaluation of their preliminary cytotoxicity against the human cancer cell 
lines in vitro.  During antitumor screening of the structural analogues of the title 
compounds, we synthesized most of the compounds shown antitumor activity against 
human cancer cell lines. 
Chapter II 
Section-A: Synthesis of novel benzo[4,5]thiazolo[1,2-a]pyrimidine-3-carboxylate                                       
derivatives and biological evaluation as potential anticancer agents 
In this section we developed a novel benzo[4,5]thiazolo[1,2-a]pyrimidine-3-
carboxylate derivatives which have anti tumour activity. Analysis of structure-activity 
relationships identified the benzothiazole and pyrimidine nuclei as being essential for 
potent activity, and substitution at the carboxylic acid 3’-position of the 
benzothiazolopyrimidine ring when condensed with methyl piperazine and piperidine 
moieties increases the cytotoxicity activity against human breast cancer cell lines MDA-
MB-231 and MCF-7 more efficiently, when compare to the secondary amines which 
shows moderate activity with the above cell line in vitro. When chain length was 
increased with the condensation of β-alanine at the third position of 
benzothiazopyrimidine ring, decreases the cytotoxicity compare to piperidine and 
methylpiperazine moieties Significantly, (2-methyl-4-phenyl-4H-benzo[4,5]thiazolo [3,2-
a]pyrimidin-3-yl)(piperidine-1-yl) methanone and (2-methyl-4-phenyl-4H-
benzo[4,5]thiazolo[3,2-a]pyrimidin-3-yl)(4-methylpiperazin-1-yl)methanone out per 
formed their best cytotoxicity activity against breast cancer cell lines. 
Abstract 
 
iv 
 
In another reaction, ethyl-3-(2-methyl-4-phenyl-4H-benzo[4,5]thiazolo[3,2-
a]pyrimidine-3-carboxamido)propanoate was obtained in good yield by the condensation 
of pyrimidine-3- carboxylic acid with β-alanine ethyl ester in the presence of HOBt, 
EDC.HCl and triethylamine and the compound  was confirmed based on appearance of 
bands at 1642, 1623 cm
-1
 for C=O and ester carbonyl stretching in IR spectrum. The 
1
H 
NMR spectra showed the characteristic ester signals at δ1.22 as triplet representing three 
hydrogens and δ 3.42-3.52 as a multiplet representing for two protons of ester -CH2. 
Upon hydrolysing the compound was converted to 3-(2-methyl-4-phenyl-4H-
benzo[4,5]thiazolo[3,2-a]pyrimidine-3-carboxamido) propanoic acid using KOH, ethanol 
at reflux temperature. Treatment of compound acid with piperidine in the presence of 
HOBt, EDC.HCl and triethylamine at room temperature gave the targeted methyl-N-(3-
oxo-3-(piperidin-1-yl)propyl)-4-phenyl-4H-benzo[4,5]thiazolo[3,2-a]pyrimidine-3-
carboxamide in good yield.  
 
Section-B: Synthesis and anti cancer activity of benzo[4,5]thiazolo[3,2-       
a]pyrimidine-3-carbonyl)piperazine-1-carbodithioate derivatives 
In this topic, we developed a novel benzo[4,5]thiazolo[1,2-a]pyrimidine-3-carbonyl 
piperazine carbodithioate derivatives. The key intermediate used for the synthesis of final 
compounds of both the series was 2-methyl-4-phenyl-4H-benzo[4,5]thiazolo[3,2-
a]pyrimidine-3-carboxylic acid, which in turn was prepared by one-pot condensation of 
2-aminobenzothiazole with substituted benzaldehyde  and ethyl acetoacetate in the 
presence of ethylene glycol by catalysing with TBAHS to give compound which was 
Abstract 
 
v 
 
hydrolyzed using KOH, ethanol refluxed to get the targeted compound, 2-methyl-4-
phenyl-4H-benzo[4,5]thiazolo[3,2-a]pyrimidine-3-carboxylic acid in quantitative yield. 
Treatment of pyrimidine-3-carboxylic acid  with 1-BOC piperazine in the presence of 
HOBt, EDC.HCl and triethylamine at room temperature gave the tert-butyl 4-(2-methyl-
4-phenyl-4H-benzo[4,5]thiazolo[3,2-a]pyrimidine-3-carbonyl)piperazine-1-carboxylate 
which was treated with trifluoro acetic acid in dichloromethane will give the compound 
(2-methyl-4-phenyl-4H-benzo[4,5]thiazolo[3,2-a]pyrimidin-3-yl)(piperazin-1-
yl)methanone in good yield.  In another reaction, the compound (2-methyl-4-phenyl-4H-
benzo[4,5]thiazolo[3,2-a]pyrimidin-3-yl)(piperazin-1-yl)methanone treated with  2-
bromo-N-phenylacetamides in the presence of carbondisulfide by using base potassium 
phosphate in acetone will yield the targeted benzo[4,5]thiazolo[3,2-a]pyrimidine-3-
carbonyl)piperazine-1-carbodithioate derivatives in good to excellent yields.  
 
Chapter-III 
Section-A:  Synthesis of novel 1, 2, 3-triazole substituted chalcone acetamide 
derivatives as potential anti tumor agents 
Synthesis of novel 1, 2, 3-triazole substituted chalcone acetamide derivatives is 
accomplished starting from 1-substituted 1,2,3-triazol-4-yl-methanol. The 1-substituted -
1, 2, 3-triazol-4-yl-methanol was oxidized to corresponding aldehyde by using Jones 
reagent followed by the reaction with 4-aminoacetophenone in ethanol by using sodium 
Abstract 
 
vi 
 
hydroxide as base at room temperature furnished amino chalcone derivative. The (E)-1-
(4-aminophenyl)-3-(1-substituted-1H-1, 2, 3-triazol-4-yl) prop-2-en-1-one was reacted 
with bromoacetyl bromide in dichloromethane in presence of potassium carbonate at 0
o
C 
to RT will get the corresponding 1, 2, 3-triazole substituted chalcone bromo acetamide 
derivatives which was treated with piperazine like compounds in the presence of 
acetonitrile by using triethylamine as base will get the targeted 1,2,3-triazole substituted 
chalcone acetamide derivatives. 
 
Section-B: Synthesis and evaluation of anti proliferative activity of novel 1, 2, 3-
triazole substituted pyrazole derivatives 
 In our present work, we developed a series of 1 ,2, 3-triazole substituted pyrazole 
derivatives as potent anti cancer agents. The key intermediate used for the synthesis of 
final compounds was The 1,4- substituted triazole  aldehydes  which in turn prepared by 
oxidation of triazole alcohol with jones reagent. The compound  which on further reacted 
with substituted acetophenones in the presence of 10% sodium hydroxide in ethanol will 
give corresponding triazole chalcones which on treated with hydrazine hydrate in acetic 
acid at  80 
o
C will give the desired compounds obtained in good yields. 
Abstract 
 
vii 
 
 
CHAPTER IV: Design and Synthesis of 1, 2, 3-triazole substituted 
benzo[d]imidazole and 1,2,3-triazole substituted quinazolin-4(3H)-one derivatives as 
potential anti proliferative agents 
 The synthesis of triazole substituted benzimidazole derivatives can be obtained by 
the reaction sequence,  the propargyl alcohol on reaction with various alkyl azides under 
Sharpless conditions through click chemistry concept gave exclusively 1,4-disubstituted 
1,2,3-triazole alcohols and then oxidized to aldehydes followed by reaction with 
phenylene diamines resulted 1, 2, 3-triazole substituted benzo[d]imidazole. 
In another scheme, the propargyl alcohol on reaction with ethyl 2-azidoacetate 
under Sharpless conditions through click chemistry concept gave exclusively 1,4-
disubstituted 1,2,3-triazole   which on oxidized to give ester of triazole aldehyde. The 
aldehyde was further reacted with phenylene diamine resulted 1, 2, 3-triazole substituted 
benzo[d]imidazole  ester  which on further treated with hydrazine hydrate gave the 
hydrazide  compound. The compound hydrazide  was further reacted with various 
aldehydes obtained  (E)-N'-(substituted benzylidene)-2-(4-(4-oxo-3,4-dihydroquinazolin-
2-yl)-1H-1,2,3-triazol-1-yl)acetohydrazides. 
Abstract 
 
viii 
 
 
Quinazolinones: 
The synthesis of triazole substituted quinazolin-4(3H)-one derivatives can be obtained by 
the reaction sequence,  the propargyl alcohol on reaction with various alkyl azides under 
Sharpless conditions through click chemistry concept gave exclusively 1,4-disubstituted 
1,2,3-triazole alcohols. The compounds were oxidized to aldehydes  followed by reaction 
with anthranilamide resulted quinazolinone derivatives.  
In another scheme, the propargyl alcohol on reaction with ethyl 2-azidoacetate 
under Sharpless conditions through click chemistry concept gave exclusively 1,4-
disubstituted 1,2,3-triazole   which on oxidized to give aldehyde. The aldehyde was 
further reacted with anthranilamide resulted quinazoline triazole ester  which on further 
treated with hydrazine hydrate gave the hydrazide compound. The compound hydrazide  
was further reacted with various aldehydes obtained  (E)-N'-(substituted benzylidene)-2-
(4-(4-oxo-3,4-dihydroquinazolin-2-yl)-1H-1,2,3-triazol-1-yl)acetohydrazides. The 
structures of all the synthesized compounds were confirmed by spectral data. 
Abstract 
 
ix 
 
 


SYNTHESIS AND BIOLOGICAL EVALUATION OF BENZO[4,5] THIAZOLO[1,2-a]PYRIMIDINE AND 1, 2, 3-TRIAZOLE HETEROCYCLES AS ANTICANCER AGENTS

VALLETI, REDDI RANI

 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Synopsis 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Synopsis 
 
 i 
SYNOPSIS 
The thesis entitled “DEVELOPMENT OF ENYNE-ASSISTED ANNULATIONS 
TO SUBSTITUTED THIOPHENES, BENZOTHIOPHENES, INDOLES AND 
CARBAZOLES” has been alienated into three chapters.  
 
CHAPTER-I  
SECTION A :  This section describes “The introduction on enyne-
assisted annulation reactions”. 
SECTION B :  This section describes “A thioannulation approach to       
 substituted thiophenes from MBH acetates of acetylenic                                                    
 aldehydes”.          
  
CHAPTER-II  :  Chapter II describes “A novel [4+2]-benzannulation of 
3-alkenyl thiophenes/pyrroles with propargylic alcohols 
to access substituted benzothiophenes/indoles. 
 
CHAPTER-III :  Chapter III describes “One-pot sequential 
propargylation, cycloisomerization strategy to diversely 
substituted carbazoles and aryl or heteroaryl 
annulated[a]carbazoles. 
 
 
 
 
 
 
Synopsis 
 
 ii 
CHAPTER-I 
SECTION A: “Introduction to Enyne assisted annulation reactions” 
  The annulation reaction is a powerful atom economic method to access 
multisubstituted cyclic structures including both carbocycles and heterocycles from 
acyclic or cyclic precursors. Annulation reactions offer several advantages over 
classical linear substitution strategies, mainly in the preparation of highly substituted 
target molecules. These annulation routes frequently avoid the regiochemical 
ambiguities associated with aromatic substitution reactions and provide access to 
substitution patterns that cannot be obtained via the conventional routes. In the 
literature, there are different types of annulation reactions such as Diels-Alder 
reactions, acid-catalyzed polyolefin cyclizations, photo-chemical, radical and thermal 
cyclization reactions, which involves the unsaturated precursors like aldehydes, 
ketones, alkenes, alkynes, α, β-unsaturated compounds and enynes. Among these, 
enynes are suitable precursors for the construction of highly substituted target 
molecules and also offers simplifying strategies to complex molecules. 
 Organic molecules with combination of alkene (ene) and alkyne (yne) 
functionalities are known as enynes. These are important and versatile synthetic 
intermediates and discovered to be one of the most efficient starting materials to 
variety of cyclic structures, in addition to offering differentiated π-systems for 
selective functionalization. From the past few decades enynes are playing a significant 
role in annulation reaction, as it offers a facile access to wide range of useful 
heterocyclic and carbocyclic compounds. 
 Based on the position of ene and yne functions, enynes are usually addressed 
as 1,n enynes such as short tethered, 1,3 and 1,4-enynes and long-tethered, 1,5-, 1,6-, 
and 1,7-enynes. The behaviour of 1,n-enynes influenced by other functional groups 
such as alcohols, aldehydes, ethers, alkenes or alkynes thus enhancing the molecular 
complexity of the synthesized products. 
Generally,1,n-enynes participated in intramolecular and intermolecular 
annulation reactions. If the enyne participated in annulation with the functional group 
present in the same substrate is called intramolecular enyne annulation.
 
If the enyne 
reacted with other reactants having functional groups such as alkenes, alkynes, 
Synopsis 
 
 iii 
amines, alcohols and carbonyl compounds to give cyclized products is known as 
intermolecular enyne annulations. 1,n Enyne annulation reactions, enyne-assisted 
construction of few heterocycles and enyne cyclisation as the key reaction in their 
total synthesis are also discussed in this section. 
SECTION B: “A thioannulation approach to substituted thiophenes from MBH 
acetates of acetylenic aldehydes” 
 Thiophenes are significant scaffolds in several biologically active natural 
products as well as pharmaceuticals. Due to their structural rigidity and distinct 
electronic properties thiophene derivatives also serve in organic material science. 
Their potential in chemistry and biology has consistently inspired the pursuit for new 
methods for thiophene synthesis. The majority of the methods in the literature 
involves the substitution of thiophene ring via metalation or halogenations and suffer 
from harsh reaction conditions,
 
while thiophene syntheses from the corresponding 
acyclic precursors are limited, which prompted us to develop a novel thioannulation 
approach. In this chapter, we reported a novel thioannulation of Morita-Baylis-
Hillman acetates of acetylenic aldehydes with KSAc under mild and metal-free 
reaction conditions. This approach offers a significant advantage over the known 
thioannulation methods. 
 During our recent studies,
 
we envisioned that an intermolecular allylic 
substitution of MBH-acetate of acetylenic aldehyde with a suitable thio-reagent and 
subsequent deacetylative intramolecular 5-exo-dig-thiocycloisomerization in one-pot 
(two C-S bond formations) would be a convenient process for the synthesis of 
substituted thiophenes (Figure 1.1). 
 
 
 
 
Figure 1.1: [4+1]-Thioannulation of MBH acetates of acetylenic aldehydes 
Synopsis 
 
 iv 
 To find the suitable reaction condition for the proposed thioannulation, MBH-
acetate 1, derived from the reaction of 3-phenylpropiolaldehyde with methyl acrylate, 
was taken as a model substrate. From the optimization studies, it was found that the 
reaction of 1 with KSAc in the presence of K2CO3 produced the thiophene 2 in 94% 
yield. 
 Under the optimized conditions, the scope of this annulation reaction using a 
variety of MBH-acetates of acetylenic aldehydes was investigated and results are 
summarised in Table 1.1. The results revealed that the approach serves as a general 
and efficient method for the synthesis of diversely substituted thiophenes in good 
yields.  
Table 1.1: Synthesis of 2,4-disubstituted thiophenes
a
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Synopsis 
 
 v 
Interestingly, thioannulation of substrate 25 which containing two MBH 
acetate groups tethered to phenyl ring, with KSAc in the presence of K2CO3 in MeOH 
resulted in a smooth reaction to deliver bis-thiophene 26 in 68% yield (Scheme 1.1). 
Scheme 1.1: Synthesis of bis-thiophene derivative 
 
 
 
 
We next evaluated the reactivity of diversely substituted MBH-acetates 
derived from different activated olefins and the results are summarized in Table 1.2.  
Table 1.2: Thioannulation of MBH-acetates of acetylenic aldehydes with KSAc 
 
 
 
 
 
 
 
 
  
 Inspired by these results, we expected that the present reaction could find 
potential applications in organic synthesis because the obtained thiophenes have two 
reactive sites: i) C-5 position and ii) C-4 ester group, as a handle for additional 
diversification. Indeed, this assumption was proved by further investigation using 2 as 
a substrate (Scheme 1.2). 
 
Synopsis 
 
 vi 
Scheme 1.2: C5-Functionalization reactions of 2 
 
 
 
 
 
 
Reagents and conditions: (a) Diphenyl methanol, BF3.Et2O (5 mol%), CH2Cl2, rt, 1 
h, 95%; (b) 4-nitro benzaldehyde, BF3.Et2O (20 mol%), CH2Cl2, reflux, 24 h, 79%;  
(c) 4-iodo toluene, Pd(OAc)2 (10 mol%), DMA, 80 
o
C, 12 h, 65%; (d) NBS, CH3CN, 
0 
o
C-rt, 24 h, 81%. 
 Additionally, thiophene 2 was conveniently utilized in the synthesis of 4H-
thieno [3, 2-c] chromene (43) and thieno [3, 2-c] dihydroquinoline (44) as shown in 
Scheme 1.3. Firstly, the ester group of 2 was reduced using DIBAL-H to alcohol 37, 
which upon treatment with PBr3 in CH2Cl2 provided the corresponding bromide 38 
(Scheme 1.3). The bromide 38 was used in O-alkylation of 2-bromophenol (39) to get 
41 and subsequent cyclization through an intramolecular Pd-catalyzed C-H activation 
offered the desired 4H-thieno [3, 2-c] chromene (43) in 78% yield. Similarly, N-
alkylation of 2-iodo N-tosylaniline (40) with 38 to 42 followed by cyclization gave 
thieno [3, 2-c] dihydroquinoline (44) in good yield (85%). 
 
 
 
Scheme 1.3 
Scheme 1.3: Synthesis of 4H-thieno[3,2-c]chromene (43) and thieno[3,2-
c]dihydroquinoline (44) 
 
Synopsis 
 
 vii 
 
 
 
 
 
 
 
 
 
 In conclusion, we have developed a new, versatile and metal-free method for 
the synthesis of substituted thiophenes from MBH-acetates of acetylenic aldehydes. A 
simple base (K2CO3) promoted the [4+1]-thioannulation of KSAc with MBH-acetates 
through a tandem allylic substitution followed by deacetylative thiocyclo 
isomerization (two C-S bond formations). 
CHAPTER-II: A novel [4+2]-benzannulation of 3-alkenyl thiophenes/pyrroles 
with propargylic alcohols to access substituted benzothiophenes/indoles 
 Indoles and benzothiophenes are an important class of structural frameworks 
among the benzo-fused heteroaromatic compounds due to their presence in various 
bioactive natural products and diverse range of pharmaceutically related small 
molecules. Both have found applications in agrochemicals, optoelectronic materials 
and pigments. Given this diverse utility, it is highly desirable to develop novel method 
for the synthesis of these heterocycles. Most of the reported protocols are through the 
construction of five-membered heterocyclic ring on a pre-functionalized benzene ring. 
Apart from this, construction of benzene ring on a suitably substituted pyrrole or 
thiophene are less in number. Herein, we have developed a novel (4+2) 
benzannulation approach to substituted indoles and benzo thiophenes with 1-
aryl/heteroaryl propargylic alcohols in one pot involving sequential propargylation 
followed by DBU mediated cycloisomerization.    
Synopsis 
 
 viii 
 Our interest on the development of novel [4+2]-benzannulations and the 
utilization of 1-aryl propargylic alcohols as handy synthons towards the synthesis of 
polycyclic aromatic and heteroaromatic compounds inspired us to explore the present 
strategy. Accordingly, we envisaged that 2-propargylated 3-alkenyl pyrroles/2-
propargylated 3-alkenyl thiophenes would undergo cycloisomerization to give the 
corresponding Indoles/Benzo thiophenes (Fig 2.1). 
 
 
 
 
Figure 2.1: [4+2]- Benzannulation reaction of 3-alkenyl pyrrole/3-alkenyl thiophene 
 To test the hypothesis, alkenyl pyrrole 2 and 1,3-diphenylprop-2-yn-1-ol 1 
were chosen as model substrates. From the optimization studies, we were happy to 
found that the formation of [4+2]-benzannulation product, indole 3 in 90% yield using 
5 mol% of BF3
.
Et2O in acetonitrile and DBU reaction conditions.  
 Encouraged by the above result, we examined the substrate scope of this 
reaction using 3-alkenyl pyrrole 2 with variety of 1-aryl and hereroaryl propargylic 
alcohols to obtain indoles having diverse substituents and results are summarised in 
Table 2.1. Interestingly the reaction of 3-alkenyl pyrrole 2 with alcohol 10 underwent 
the desilylative six-membered lactonization during the [4+2]-benzannulation to give 
dihydropyrano[3,4-f]indol-5(1H)-one 11 in 79% yield (entry 5, Table 2.1). 
Table 2.1: Scope of propargylic alcohols in [4+2]-benzannulation with 2 
 
 
 
 
 
Synopsis 
 
 ix 
 
 
 
 
 
 
 
 
 
 
 
  
  
 
 
 
  
 
  
 Next the reactivity of diversely substituted 3-alkenylpyrroles having different 
electron-withdrawing groups with 1,3-diphenylprop-2-yn-1-ol 1 under optimized 
conditions was evaluated and the results are summarized in Scheme 2.1.  
 
Synopsis 
 
 x 
Scheme 2.1: Benzannulation of 3-alkenylpyrroes with 1 
 
 
 
 
 
 Further, we extended the present [4+2]-benzannulation reaction for the 
synthesis of substituted benzothiophenes from the suitably substituted 3-alkenyl 
thiophene 24 with various 1-aryl/heteroaryl propargylic alcohols under the optimized 
reaction conditions to obtain a wide variety of substituted benzothiophenes, as shown 
in Table 2.2. 
Table 2.2: Scope of propargylic alcohols in [4+2]-benzannulation with 24 
 
  
 
 
 
 
 
 
 
 
 
 
Synopsis 
 
 xi 
 
 
 
 
 
 
 
 
 
 We also examined the scope of this [4+2]-benzannulation with a diverse array 
of 2-alkenyl thiophenes as the diene units and 1,3-diphenylprop-2-yn-1-ol 1 as the 
two-carbon partner under optimized conditions (Scheme 2.2). 
Scheme 2.2: [4+2]-Benzannulation of 3-alkenyl thiophenes with 1 
 
 
 
 
 
 Encouraged by the results, we tried to apply the benzannulation of 2-
alkenylpyrrole 2 with a diynyl alcohol, 5-phenyl-1-(1-tosyl-1H-indol-3-yl)penta-2,4-
diyn-1-ol (40) towards the double cyclization product successfully and observed the 
formation of diaza[5]helicene 43 (82%) via the mono-cyclized indole 41 (81%, 
Scheme 3.3). Similarly, the reaction of 2-alkenylbenzothiophene 24 with 2,4-diyn-1-
ol 40 underwent the sequential propargylation followed by double cycloisomerization 
(dibenzannulation) to yield 44 (83%) via benzothiophene 42 (79%. Scheme 2.3).  
Synopsis 
 
 xii 
Scheme 2.3: Synthesis of aza[5]helicenes through dibenzannulation 
 
 
 
 
 
 
 
  
 In summary, we have developed an efficient method for the synthesis of 
multisubstituted indoles/benzo thiophenes via the [4+2] benzannulation of 3-alkenyl 
pyrrole/3-alkenyl thiophene with 1-aryl propargylic alcohols. This method has several 
advantages such as mild and metal-free reaction conditions, wide substrate scope from 
readily accessible starting materials. In addition, an efficient dibenzannulation of 2,4-
diyn-1-ol was also recognized, thus providing a facile access to aza[5]helicenes.   
CHAPTER III: One-pot sequential Propargylation/Cycloisomerization strategy 
to diversely substituted carbazoles and aryl or heteroaryl annulated[a]carbazoles 
 The carbazole scaffold represents an important structural constituent in 
numerous natural products and synthetic compounds showing a wide variety of 
biological and pharmaceutical activities. In addition, they also useful as 
photorefractive materials as well as organic dyes for solar cells. Thus, the synthesis of 
carbazole derivatives has attracted much attention and various strategies have been 
developed.
 
Among these, benzannulation of indoles has received substantial attention 
to construct the substituted carbazoles due to the easy accessibility of indole 
substances. In particular, the alkyne/allene-assisted formation of benzene ring on 
indole moiety is attractive. Herein, we report a novel [4+2]-benzannulation of 2- 
alkenyl/aryl indoles with 1-aryl propargylic alcohols. This method offers a substantial 
Synopsis 
 
 xiii 
advantage by providing an access to diverse carbazoles as well as aryl or heteroaryl-
annulated[a]carbazoles from readily accessible substrates. 
 
 
 
 
 Figure 3.1: Intermolecular annulation of alkenyl indoles with propargylic alcohols 
 Based on our studies on enyne-based annulation reactions and use of 1-aryl 
propargylic alcohols as useful synthon, we envisioned that 3-propargylated 2-alkenyl 
indoles would undergo cycloisomerization to give the corresponding carbazoles 
(Figure 3.1). For testing this hypothesis, the 2-alkenyl indole 1 and 1, 3-
diphenylprop-2-yn-1-ol 11 were chosen as starting materials. The propargylation of 
alkenyl indole 1 was verified in the presence of few acid-catalysts such as BF3
.
Et2O (5 
mol%), FeCl3, pTSA, I2 and AlCl3. Next, the cycloisomerization was tested using 
different bases and found that 5 mol% BF3
.
Et2O followed by DBU in CH3CN was the 
suitable condition for one-pot synthesis of carbazole 2. Encouraged by this result, a 
range of diversely substituted propargylic alcohols was investigated with 1 as a 
typical reaction partner. All propargylic alcohols (bearing aryl, heteroaryl, alkyl and 
cyclopropyl groups on the alkyne), and having aryl, substituted aryl, heteroaryl groups 
at the C1-position) gave the corresponding carbazoles in good yields (Scheme 3.1). 
 
 
 
 
 
 
 
Synopsis 
 
 xiv 
Scheme 3.1: Scope of propargylic alcohols in [4+2]-benzannulation with 1 
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 
 The scope of this [4+2]-benzannulation was also examined with a diverse 
array of 2-alkenyl indoles as the diene units and 1,3-diphenylprop-2-yn-1-ol 11 as the 
two-carbon partner under optimized conditions as shown in Table 3.1. 
 
 
 
Synopsis 
 
 xv 
Table 3. 1: [4+2]-Benzannulation of 2-alkenyl indoles with 11 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
   
  
 The above-mentioned successful results encouraged us to extend the present 
method for the synthesis of aryl and hetero aryl-annulated[a]carabazoles as presented 
Synopsis 
 
 xvi 
in Table 3.2, core of several bioactive natural products, were also synthesized with 
the present protocol from the respective 2-aryl/heteroaryl indoles.  
Table 3.2: [4+2]-Benzannulation of 2-aryl/heteroaryl indoles with 2 
 
  
 
 
 
 
 
 
 
  
 
 
 
 
 
 
 
 Interestingly, we have developed a three-component coupling reaction with 
the help of alkenyl indole, propargylic aldehyde and N-Methyl indole under 
BF3
.
Et2O/DBU in CH3CN conditions to provide 5-indoloyl carbazoles (Scheme 3.2) 
in good yields. 
Synopsis 
 
 xvii 
Scheme 3.2: Three-component approach to 5-indoloyl carbazoles 
 
 
 
 
 
 
 
  
 The application of the present protocol was explored for the construction of 
carbazole-based azahelicene, valuable motifs in various applications. The use of 1,5-
diphenylpenta-2,4-diyn-1-ol (53) in reaction with 1 facilitated the double cyclization 
to give the aza [5]helicene 61 via the monocyclized intermediate 60 (Scheme 3.3). 
Scheme 3.3: Synthesis of aza [5] helicene 55 
 
 
  
 
 
 
 
 In summary, an effective method for the synthesis of multisubstituted 
carbazoles via the [4+2] benzannulation of 2-alkenyl/aryl/heteroaryl indoles with 1-
aryl propargylic alcohols has been developed. This method offers several advantages 
such as mild and metal-free reaction conditions, wide substrate scope from easily 
accessible starting materials. A library of structurally varied carbazoles, in particular 
aryl or heteroaryl-annulated[a]carabazoles should be easily accessible. An interesting 
three-component reaction to indole-substituted carbazoles was established. The 
method was also gave a new entry to aza[5]helicene.  


DEVELOPMENT OF ENYNE-ASSISTED ANNULATIONS TO SUBSTITUTED THIOPHENES, BENZOTHIOPHENES,INDOLES AND CARBAZOLES

SHAIK, SIDDIQ PASHA

Synopsis 
i 
The thesis entitled “Synthesis of Imidazo[2,1-b]thiazole and Benzo[d]imidazo[2,1-b]thiazole 
Conjugates as  Potential Microtubule Targeting Agents” has been divided into four chapters. 
 
Chapter-I: General introduction of cancer and tubulin: 
Cancer is a class of diseases or disorders characterized by uncontrolled/ abnormal division of 
cells and the ability of these to spread, either by direct growth into adjacent tissue, or by implantation into 
distant sites by metastasis, in which cancer cells are transported through the bloodstream or lymphatic 
system. Small molecules that disrupt microtubule/tubulin dynamics are used widely in cancer treatment. 
Hence discovery and development of novel small molecules useful in cancer chemotherapy has identified 
tubulin as a possible molecular target. Microtubules are one of the key structural components of the 
cytoskeleton in eukaryotic cells comprising of α and β-tubulin heterodimers. They play a crucial role in 
various cellular processes and have emerged as an attractive and viable target in the development of 
anticancer drugs mainly due to their indispensability in mitotic cell division. Generally, drugs that target 
microtubules bind to one of the three main sites of tubulin which includes the taxane-site for the 
microtubule stabilizing agents, the vinca domain and the colchicine domain for the destabilizing agents. 
The agents that interfer with the dynamic stability of the microtubules, act as spindle poisons arresting the 
dividing cells in G2/M phase of the cell cycle, causing mitotic catastrophy and finally leading to apoptotic 
cell death. 
 
Statement of Problem: 
Chapter-IIA: Design and Synthesis of Imidazo[2,1-b]thiazole linked Triazole Conjugates: 
Microtubule-Destabilizing Agents. 
In recent years there is considerable interest in the development of new hybrid molecules that 
have been recently developed as anticancer agents were obtained by the combination of different 
pharmacophores. The promising biological activity exhibited by these hybrids prompted us to develop 
some newer hybrid molecules by linking the imidazo[2,1-b]thiazole with triazole scaffold with a view to 
enhance their anticancer activity. Thus a library of twenty four imidazo[2,1-b]thiazole linked triazole 
conjugates (4a-x)  were designed, synthesized and evaluated for their cytotoxic potential. 
Scheme: 
 
Results and discussions: A series of new imidazo[2,1-b]thiazole linked triazole conjugates (4a-x) were 
synthesized and evaluated for their cytotoxic  potency against HeLa, DU-145, A549, MCF-7 and HepG2 
cell lines. Among them, conjugates 4g and 4h exhibited most cytotoxic potency effect against A549 cells 
with IC50 values of 0.92 and 0.78 µM respectively. FACS analysis revealed that these conjugates induced 
cell cycle arrest in G2/M phase in A549 lung cancer cells. The tubulin polymerization assay and 
immunofluorescence analysis showed that these conjugates effectively inhibit microtubule assembly in 
cell-free and cell-based (A549) experiments respectively. Moreover, the apoptosis inducing properties 
were evaluated by Hoechst staining, mitochondrial membrane potential and Annexin V-FITC assay. 
Further, western blot analysis was performed for proapoptotic protein Bax and antiapoptotic protein Bcl-
2, and the results demonstrated that there was up regulation of Bax and down regulation of Bcl-2 
suggesting that these compounds induce apoptosis in human lung cancer cells, A549.   
Conclusion: In conclusion, a series of imidazo[2,1-b]thiazole linked triazole conjugates were synthesized 
and evaluated for their antiproliferative activity, wherein conjugates 4g and 4h showed significant 
cytotoxic activity. The FACS results revealed that these conjugates cause cell cycle arrest at G2/M phase, 
oreever they effectively inhibit microtubule assembly and disrupt the microtubule organization in the lung 
cancer cells. The molecular docking studies revealed that these conjugates bind at the colchicine site of 
Synopsis 
ii 
the tubulin. Further, Hoechst staining, mitochondrial membrane potential and Annexin V FITC assay 
suggest that they induce cell death by apoptosis. Bax and Bcl-2 results also suggest that these conjugates 
induce apoptosis in A549 cells. The SAR provided a useful insight that could be utilized in developing 
improved newer leads for the treatment of lung cancer based on such scaffolds.  
 
Statement of Problem:  
Chapter-IIB: Design and Synthesis of 1,2,3-Triazolo Linked Benzo[d]imidazo[2,1-b]thiazole 
Conjugates as Tubulin Polymerization Inhibitors 
Nitrogen-bridge head fused heterocycles containing an imidazole and benzothiazole ring are a 
common structural moiety in many pharmacologically important molecules that display a wide range of 
activities for diverse targets. Among them, benzo[d]imidazo[2,1-b]thiazole class of compounds like 2-
arylbenzo[d]imidazo[2,1-b]thiazole derivative YM-201627 was found to be orally active for the treatment 
of solid tumors. Another derivative AC220 was found to be highly active against FMS-like tyrosine 
kinase-3 (FLT3) and is in phase III clinical trials. Whereas, 1,2,3-triazoles have attracted the medicinal 
chemists, due to their wide range of biological activities and high metabolic stability as well as  hydrogen-
bonding capability with bimolecular targets. Herein we report the synthesis of 1,2,3-triazolo linked 
benzo[d]imidazo[2,1-b]thiazole conjugates (5a-v) and were evaluated for their cytotoxic potential with a 
view to produce promising anticancer agents. 
Scheme: 
 
Results and discussions: These newly designed 1,2,3-triazolo linked benzo[d]imidazo[2,1-b]thiazoles 
conjugates (5a-v) were synthesized and evaluated for their cytotoxic potency against the DU-145, HeLa, 
MCF-7, HepG2 and A549 cancer cells. Among them, conjugates like 5f and 5k exhibited significant 
antiproliferative effect against MCF-7 cells with IC50 values of 0.60 and 0.78 µM respectively. FACS 
analysis of the cell cycle demonstrated an increase in the percentage of cells in the G2/M phase which was 
further authenticated by elevation of cyclin B1 protein levels. Immunocytochemistry revealed loss of 
intact microtubule structure in cells treated with 5f and 5k, and western blot analysis revealed that these 
conjugates accumulated more tubulin in the soluble fraction. Moreover, the conjugates caused apoptosis 
of the cells that was confirmed by mitochondrial membrane potential and Annexin V-FITC assay. 
Molecular docking studies indicated that these conjugates occupy the colchicine binding site of the 
tubulin protein. 
 
Conclusion: In the present investigation, a new class of 1,2,3-triazolo linked benzo[d]imidazo[2,1-
b]thiazoles conjugates were synthesized and evaluated for their cytotoxic activity. Among them, 
conjugates 5f and 5k showed significant cytotoxic activity against the MCF cell line. The FACS analysis 
revealed that these conjugates cause cell cycle arrest at G2/M phase. The presence of increased levels of 
the cyclin B1 protein and tubulin in the soluble fraction of cells corroborate well with the tubulin 
polymerization inhibition. Furthermore, they effectively inhibit microtubule assembly and disrupt the 
microtubule organization in the breast cancer cells. The molecular docking studies revealed that these 
conjugates bind at the colchicine site of the tubulin. The induction of apoptosis is associated with 
mitochondrial membrane potential and Annexin V FITC assay. Thus these conjugates could be 
considered as potential scaffolds for the development of new leads as chemotherapeutics for breast 
cancer. 
 
 
 
Synopsis 
iii 
Statement of Problem:  
Chapter–IIIA: Design and synthesis of C3-Oxindole Linked Benzo[d]imidazo[2,1-b]thiazole 
Conjugates as Cytotoxic and Apoptotic Inducing Agents 
Pharmacophore hybridization is an effective tool to design highly active new chemical entities by 
covalently combining two or more active pharmacophores into a single molecule. These hybrids may act 
on multiple targets and offer the possibility of overcoming drug resistance, and in addition, hybrids may 
also often show synergetic action. In view of the above considerations and inspired by the promising 
anticancer activity of benzo[d]imidazo[2,1-b]thiazole and oxindole, we synthesized a series of C3 
oxindole linked benzo[d]imidazo[2,1-b]thiazole conjugates (5a-ad) by combining benzo[d]imidazo[2,1-
b]thiazole with oxindoles via knoevenagel condensation with a view to produce promising anticancer 
agents. 
Scheme: 
 
Results and discussions: A series of C3 oxindole linked benzo[d]imidazo[2,1-b]thiazole conjugates (5a-
ad) were designed, synthesized and evaluated for their cytotoxic potency against some human cancer cell 
lines like A549 (lung), DU-145 (prostate), MCF-7 (breast) and HT-29 (colon). Preliminary results 
revealed that some of these conjugate like 5b exhibited significant antiproliferative effect against human 
DU-145 (prostate) cancer cells with IC50 values of 0.889 µM. FACS analysis of the cell cycle 
demonstrated an increase in the percentage of cells in the G2/M phase. Moreover, the apoptosis inducing 
effect of the compound was studied using Hoechst staining, mitochondrial membrane potential, Annexin 
V-FITC assay. Based on these studies, 5b  have been identified as promising new molecule that have the 
potential to be developed as leads. 
 
Conclusion: In summary, a new series of C3 oxindole linked benzo[d]imidazo[2,1-b]thiazole conjugates 
(5a-ad) were synthesized and screened for cytotoxic potency against four human cancer cell lines. 
Conjugate 5b showed significant potency against DU-145 cells. FACS analysis indicates that this 
conjugate induce cell cycle arrest at G2/M phase.  
 
Statement of Problem:  
Chapter IIIB: Design and Synthesis of 3-Arylaminopropenone Linked 2-Arylbenzo[d]imidazo[2,1-
b]thiazole Conjugates as Cytotoxic and Apoptotic Inducing Agents 
Benzo[d]imidazo[2,1-b]thiazole scaffold containing compounds like YM-201627 and AC220 
were found as potent anticancer agents. Whereas, propenones are considered as suitable linkers for the 
hybridization of promising compounds. Based on this background it was considered of interest to 
synthesize 3-arylaminopropenone linked 2-arylbenzo[d]imidazo[2,1-b]thiazole conjugates (4a-ae) with a 
view to explore their cytotoxic potential.  
Scheme: 
 
Synopsis 
iv 
Results and discussions: A series of 3-arylaminopropenone linked 2-arylbenzo[d]imidazo[2,1-b]thiazole 
conjugates were synthesized and screened for their cytotoxic activity against four human cancer cell lines. 
Some conjugates like 4e, 4h, 4m and 4u exhibited good antiproliferative activity and the insight to their 
structure activity relationship was developed. Among them, conjugate 4e showed significant potential 
particularly against HeLa cell line with IC50 value 1.6 µM. FACS analysis indicates that conjugate 4e 
induce cell cycle arrest at G1 phase. In addition, the potential mechanism of cell growth inhibition and 
apoptotic induction by this conjugate was investigated in HeLa cancer cells using cell-based assays, 
including wound healing assay and Hoechst staining. Moreover, conjugate 4e led to the collapse of 
mitochondrial membrane potential (DΨm) and increased levels of reactive oxygen species (ROS) were 
noted. 
 
Conclusion: In summary, a new series of 3-arylaminopropenone linked 2-arylbenzo[d]imidazo[2,1-
b]thiazole conjugates were screened for their cytotoxic potency against four human cancer cell lines. 
Interestingly, conjugate 4e showed significant potency against HeLa cells. FACS analysis indicates that it 
induces cell cycle arrest at G1 phase and also induces apoptosis in HeLa cells by using wound healing 
assay, Actin Filaments and Hoechst staining. It also causies collapse of DΨm and elevation of ROS 
production. Overall, these results demonstrate that conjugate 4e has the potential to be developed as a lead 
and its further structural modification may produce promising anticancer agents for HeLa cancer cells. 
 
Statement of Problem:  
Chapter IVA: Design and Synthesis of Chalcone linked Benzo[d]imidazo[2,1-b]thiazole Conjugates 
as Potential Tubulin Polymerization inhibitors 
Pharmacophore conjugation is an effective tool to design highly active new chemical compounds 
by keeping drug resistance and synergetic action. In this point of view and inspired by the promising 
anticancer activity of benzo[d]imidazo[2,1-b]thiazoles and chalcones, we synthesized a series of chalcone 
linked benzo[d]imidazo[2,1-b]thiazole conjugates (4a-x) with a view to produce promising cytotoxic 
agents. 
 
Results and discussions: Chalcone linked benzo[d]imidazo[2,1-b]thiazole conjugates (4a-x) were 
designed, synthesized and evaluated for their cytoxic potency against some human cancer cell lines like 
cervical (HeLa), breast (MCF-7), liver (HepG2) and lung adenocarcinoma (A549). Preliminary results 
revealed that some of these conjugates like 4b and 4c exhibited significant antiproliferative effect against 
HeLa cells with IC50 values of 1.6 and 2.1 µM respectively. FACS analysis revealed that they arrest the 
cell cycle at the G2/M phase and further authenticated by elevation of cyclin B1 protein levels. 
Immunocytochemistry revealed loss of intact microtubule structure in cells treated with 4b and 4c and 
western blot analysis revealed that these conjugates accumulate more tubulin in the soluble fraction. 
Molecular docking studies indicated that these conjugates occupy the colchicine binding site of the 
tubulin protein. 
Conclusion: In summary, a series of chalcone linked benzo[d]imidazo[2,1-b]thiazole conjugates (4a-x) 
were screened for cytotoxic potency against four human cancer cell lines. Among them, conjugate 4b and 
4c showed significant potency against HeLa cells. FACS analysis indicates that these conjugates induce 
Synopsis 
v 
cell cycle arrest at G2/M phase and they inhibit microtubule assembly and disrupt the microtubule 
organization in the HeLa cancer cells. 
 
Statement of Problem:  
Chapter IVB: Regioselective Oxidative Cross-Coupling of Benzo[d]imidazo[2,1-b]thiazoles with 
Styrenes: A New Route to C3-Dicarbonylation of Benzo[d]imidazo[2,1-b]thiazoles 
The oxidative cross-coupling of two different C-H bonds for the construction of specific C-C bonds in the 
development of effective synthetic strategies to functionalize biologically important pharmacophores is an 
ongoing interest in organic and medicinal chemistry. 1,2-Dicarbonyl compounds are valuable starting 
materials and are important synthetic intermediates in the preparation of many fine chemicals that could 
be readily converted to many other functional groups and  have become attractive targets. Therefore, 
development of metal-free, peroxide-free, oxidant-free and environmentally benign methods with 
terminal alkenes via C(sp
2
)-H bond cleavage is still challenging and highly desirable for 1,2-dicarbonyl 
functionalization via C-H bond cleavage to obtained biologically active heterocyclics.  
Scheme: 
 
Results and discussions: A novel I2 promoted, highly efficient metal-free and peroxide-free greener 
domino protocol for C3-dicarbonylation of benzo[d]imidazo[2,1-b]thiazoles (IBT) with styrenes has been 
developed via oxidative cleavage of C(sp
2
)-H bond, followed by C3-nucleophilic attack of IBT and 
oxidation under mild conditions. Interestingly, under these conditions 2-(benzo[d]imidazo[2,1-b]thiazol-
2-yl)aniline gave the benzo[4',5']thiazolo[2',3':2,3]imidazo[4,5-c]quinoline derivative via oxidative 
cleavage of C(sp
2
)-H bond, followed by pictet spengler cyclization and aromatization. By using this 
simple method a library of C3-dicarbonylated benzo[d]imidazo[2,1-b]thiazole derivatives have been 
synthesized in moderate to good yields and screened for their antoproliferative activity. Among them 3l 
showed significant cytotoxicity with an IC50 of 5.26 µM against MCF-7 cell line. 
Conclusion: In conclusion, we have successfully established a highly efficient molecular I2 mediated, 
metal-free, peroxide-free and greener domino approach for site selective oxidative C3-dicarbonylation of 
IBTs with styrenes via oxidative cleavage of C(sp
2
)-H bond, followed by electron rich C3 attack of IBT 
and oxidation. This method proceeds under mild conditions with high regioselectivity and broad substrate 
scope. Furthermore, using this simple method a library of C3-dicarbonylated benzo[d]imidazo[2,1-
b]thiazole derivatives have been synthesized in moderate to good yields and screened them for 
antiprolifirative activity. Among them 3l showed significant cytotoxicity with an IC50 of 5.26 µM against 
MCF-7 cell line. 


Synthesis of Imidazo[2,1-b]thiazole and Benzo[d]imidazo[2,1-

b]thiazole Conjugates as Potential Microtubule Targeting Agents

Mondal, Tanmoy

Summary of work done 
 
Role of Drosophila Argonaute-1 in apoptosis 
 
 
Introduction 
 
Apoptosis is collection of multiple perturbations of the cellular structure and architecture that do not 
promote only programmed cell death but incubate the unwanted, old or abnormal cells to be removed 
via phagocytes and reduce innumerous immune responses. It also plays an initial role in tissue 
homeostasis and proper development of an organism. Organ structure, shape and size determination 
too depends on programmed cell death.  Programmed cell death allows the apoptotic cells to be 
dismantled in a programmed manner that minimizes damage and disruption of adjacent cells. These 
events are orchestrated intensely by the activation of series of cysteine proteases family members 
referred as caspases. A number of diseases including cancer, immune deficiency disorders and 
neurodegenerative disease develop as a result of deregulated cell death. 
Argonaute (AGO) family proteins are highly conserved across both animal and plant kingdom. In 
mammals, both AGO and PIWI includes four members in their subfamily. Structurally AGO proteins 
contain four domains- an amino terminal domain which is relatively less conserved in nature and 
highly conserved mid domain, PAZ domain & PIWI domain. AGO proteins are the direct binding 
partners of existing small RNA. As member of mammalian and Drosophila AGO family, both AGO1 
and AGO2 are well studied, because of its role in microRNA (miRNA) biogenesis and as a major 
representative of miRNA effector complex (RISC complex). miRNAs are generated after processing 
of long double stranded precursors RNAs and control gene expression by post-transcriptional 
regulation. By interacting with AGO proteins miRNA guide the RISC (RNA-induced silencing 
complex) to its mRNA targets to induce translational repression or degradation. As a post-
transcriptional regulator for a large number of vital genes, miRNAs play crucial role in controlling a 
wide range of cellular processes including cell proliferation, migration, cell death and differentiation. 
Therefore mis expression of miRNAs leads to the development of various diseases including cancer 
and neurodegenerative disease. miRNAs can act as a tumor inducer or an inhibitor of tumor by 
altering the expression of tumor suppressor genes or oncogenes. Altered expression of miRNA 
biogenesis associated proteins such as DROSHA and DICER is frequently observed during 
tumorigenesis and cancer progression. As AGO is the prime factor for functional regulation of 
miRNA, it is speculated that its misexpression affect vital cellular processes such as cell growth, cell 
division, cellular differentiation and programmed cell death.  
 
 Statement of problem 
 
When we had planned this study, the role of Ago-1 on RNAi was well known but no literature was 
available explaining Ago-1 as a regulator of apoptosis. This lack of knowledge provokes us to 
investigate the role of Ago-1 on the very crucial process of any organism’s life, apoptosis. Moreover, 
at that time no suitable in vivo screening platform was there to identify micro RNA inhibitor small 
molecules, synthesized by our chemists in our institute. That scenario encourages us to develop an 
efficient in vivo screening platform to test micro RNA inhibitor small molecules using Drosophila 
model system which allows us to extend our study into genetic level, instead restricting to 
conventional screening work. 
The first aim of this study was to find, Ago-1 is related to any kind of apoptosis or not. After 
getting the answer of this question our next query was how Ago-1 regulates apoptosis and which 
pathways are involved there? Then, our next aim was to identify micro RNAs (miRs) which are 
involved in this process. After acquiring answers of above mentioned query; our final goal was to 
develop an efficient in vivo screening platform to test micro RNA inhibitor small molecules using 
Drosophila model system and to identify some small molecules to target miRs which has a potential 
to inhibit Ago-1 induced apoptosis.  
 
 
Objectives of the study 
 
1.    Identify the role of Ago-1 as a regulator of apoptosis and dissecting the pathway related to Ago-1 
induced apoptosis. 
 
2.  To identify the micro RNAs (miRs) involved in Ago-1 induced apoptosis and their functional roles. 
 
3.   Identifying small molecules that modulate target miRs having potential to inhibit Ago-1 induced 
apoptosis. 
 
 
Methodologies Used and Sample Results 
 
In order to understand the role of Ago-1 in apoptosis, we developed Drosophila lines by using well 
established and powerful biochemical tool of UAS/GAL4 system to ectopically overexpress Ago-1 
gene in different organs of Drosophila using specific GAL4 drivers. GAL4 and the UAS are 
important for any ectopic expression in Drosophila as their ectopic expression or overexpression does 
not interfere with other processes in the organs. In this method, Drosophila modular protein GAL4 is 
fused under the control of different endogenous driver promoters. GAL4 is therefore only expressed in 
the cells in which driver promoters are active. GAL4 protein binds to the UAS sequences to activate 
fused target gene expression. In this study, we used GAL4/ UAS Ago-1 transgene that is expressed 
ectopically in a tissue specific manner. UAS Ago-1 flies were crossed with GMR-GAL4 driver, which 
expressed GAL4 under the control of GMR promoter, thereby ectopically expressing Ago-1 in the 
developing eye. Interestingly, ectopic overexpression of Ago-1 lead to phenotypic changes in the adult 
eyes that resembled apoptotic cell death compared to the control flies that contained endogenous copy 
of Ago-1.  To understand whether these deformities in the adult eyes were due to any developmental 
apoptotic cell death we performed Acridine Orange (AO) test that gives a measurement of apoptosis 
in eye imaginal discs of third instar larvae arising from stocks that contained overexpressed Ago-1, 
mutated Ago-1 and normal. A large number of positively stained apoptotic cells were observed in the 
discs arising from larvae in which Ago-1 is overexpressed (UAS Ago-1/GMR GAL4). Suppression of 
this phenotype by dominant negative mutation of fly JNK, basket clearly indicates the involvement of 
JNK pathway with this process. Followed by real time PCR, Western blotting and immunostaining 
further confirms the involvement of pro-apoptotic genes, apoptotic inhibitor proteins and caspases. 
Chromatin immunoprecipitation (ChIP) assay and nuclear extract immunoprecipitation (IP) revealed 
the interaction between AGO1 and RNA Pol II at the promoter region of tak-1 (TGF-β activated 
kinase-1) gene which is an upstream player of JNK signaling. 
 
 
 Figure 1: Result figures for objective-1- Ago-1 induces caspase dependent apoptosis in developing 
fly organ through phosphorylation of fly JNK, basket (bsk) by interacting with RNA Pol II at the 
promoter region of TGF-β activated kinase 1 (tak1). 
 
Micro RNAs (miRs) are endogenously produced tiny (~21nt) non-coding RNAs that are involved in 
the expression of genes in many developmental processes. A large number of miRNAs have been 
identified in all organisms including fly and human that control around 30–40% of total genes. 
Various reports on functional analysis of micro RNA showed their significant role in modulation and 
fine-tuning of different biological activity including apoptosis. To understand the change of miR 
expression in response to altered Ago-1 expression micro RNA microarray profiling was carried out. 
Involvement of dme-miR-14 and bantam was confirmed from array data, realtime PCR and genetic 
crosses. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Figure 2: Result figures for objective-2- Ago-1 down regulates apoptotic inhibitor micro RNA, miR-
14 and bantam to induce apoptosis. 
 
For above mentioned reasons, micro RNAs are considered as attractive drug targets against different 
diseases including cancer. Small molecule mediated targeting of miRNAs, particularly targeted 
inhibition of oncogenic micro RNAs (oncomiRs) serve a novel approach to discover better 
therapeutics against cancer. 
 In our present study, miR microarray analysis was carried out to know the changes of miR level in 
altered Ago-1 expression. From that result, miR-14 and bantam has been identified as modulator miR 
which has a link with Ago-1 related apoptosis. Next, we have synthesized and characterized a small 
molecule library to inhibit the function of these oncomiRs for better therapeutics. Triazole derivative 
small molecule, DCPTN-PT (1-(4-(3, 4-Dichlorophenyl)-1, 2, 3, 4-tetrahydronaphthalen-1-yl)-4-
pentyl-1H-1, 2, 3-triazole) and Triazole linked benzoxazole derivatives has been identified as an 
inhibitor of bantam and miR-14. These small molecule inhibitors induce apoptosis by targeting 
microRNAs (bantam, miR-14, miR-2 and miR-13) that suppress apoptotic process in fly. Fly genome 
sequencing showed that around 77% of human genes that are associated with various diseases are 
conserved in Drosophila. Ectopic expression of human Parkinson's disease (PD) causing mutant 
alpha-synuclein gene (SNCA) in Drosophila melanogaster results in loss of dopaminergic neurons. 
Fly orthologs of human cancer causing genes can induce cancer-like phenotypes in Drosophila, 
including high rate of cell proliferation, defective apoptosis and deregulation in the process of 
differentiation. So, it is very obvious that small molecule inhibitors which can inhibit fly oncomiRs 
will have the potential to inhibit its human counterpart also.    
To screen these small molecules as inhibitors of miRs “miR Sensor” approach has developed. Control 
sensor fly always express GFP under tubulin (tub) control and miR sensor express GFP depending on 
miR activity, as miR sensor lines carry miR binding site at its 3’ UTR (Un Translated Region). When 
one molecule inhibits the activity of any miR; GFP expression of that particular sensor will increase at 
that time as a result of the release of miR induced repression of GFP trans-gene by that particular miR. 
In this study, initially we’ve screened miR inhibitor small molecules using this miR Sensor approach, 
followed by detailed study has been carried out by over expressing those miRs using UAS/GAL4 
system. As miR-14 has a role in the regulation of mitochondrial Reactive Oxygen Species (mtROS) 
level, so changes in mtROS level was measured using MitoSOX staining. The expression of pro-
apoptotic genes and activation of caspases has been determined by semi-quantitative & quantitative 
PCR and western blot analysis.  
In conclusion, an efficient in vivo screening method has been developed that not only identifies miR 
inhibitor small molecules, but also classifies their elaborate biological functions in cellular and 
developmental pathways. And these newly identified small molecules can function as unique 
modulator for understanding the role of miRs in apoptosis which probably opens a new way for 
alternate use of small molecules to regulate oncomiRs and brings a new light of hope in the efficient 
development of a modern therapeutic strategy to treat cancer. 
 
 
Figure 3: Result figures for objective-3- Small molecule DCPTN-PT, “13j” and “13h” inhibits 
apoptotic inhibitor miR, bantam and miR-14 to induce apoptosis.  “13j” and “13h” also induces 
reaper (rpr) induced apoptosis by increasing mitochondrial reactive oxygen species accumulation. 
 
 


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