Open Access Repository of Indian Theses

MIRAJKAR, SUBHASH

SYNOPSIS
      
The title of the thesis is “Application of Prins cyclisation to Stereoselective Total Synthesis of natural products: Aculeatin A and B, Xestodecalactone C and epi-Sporostatin, (10S, 12S)-10-Hydroxy-12-methyl-1-oxacyclododecane-2, 5-Dione.” The quantum of thesis work carried out is divided into three chapters.

CHAPTER I: This chapter is divided into two sections A and B.
Section A: This section describes introduction to Prins cyclisation, previous approaches to the key intermediate 1, 3 diols.
Section B: This section elaborates the introduction and Stereoselective total synthesis of Aculeatin A and B via Prins cyclisation.
	
CHAPTER II: This chapter is further divided into two sections A and B.	
SECTION A: This section describes introduction, known synthetic approaches of Xestodecalactone C and Sporostatin.
SECTION B:	This section describes Stereoselective total synthesis of Xestodecalactone C and epi-Sporostatin via the Prins cyclisation.

CHAPTER III: The chapter is also depicted in two sections A and B.
SECTION A: This section describes introduction, known synthetic approaches of 12-membered lactone.	
SECTION B: This section describes the Stereoselective total synthesis of (10S, 12S)-10-hydroxy-12-methyl-1-oxacyclododecane-2, 5-Dione via the Prins cyclisation.

CHAPTER I:
Section A: This section describes introduction to Prins cyclisation, previous approaches to the key intermediate 1, 3 diols.
Section B: This section elaborates the introduction and Stereoselective total synthesis of Aculeatin A and B via Prins cyclisation.
                                  The Aculeatins A-D (Figure 1, 1-4) represent a novel type of natural compounds displaying the unusual, previously unreported 1, 7-dioxadispiro [5.1.5.2] pentadecane system.  The Aculeatins A (1) and B (2) are two epimeric spiroacetals isolated from the terrestrial plant species Amomum aculeatim Roxb. (fam. Zingiberaceae).  A more complex variant, Aculeatin C (3), was also isolated from the same plant. Later, the same authors reported the isolation of fourth member of this compound family, named Aculeatin D and assigned structure and relative configuration 4. Aculeatin A (1) was found to be amongst the most cytotoxic of the compounds tested and was further evaluated in an in vivo hollow fiber assay. It was found to be active against MCF-7 (human breast cancer) cells implanted intraperitoneally at doses of 6.25, 12.5, 25, and 50 mg/kg. However, when Aculeatin A (1) was tested using P388 lymphocytic leukemia and human A2780 ovarian carcinoma in vivo models, it was deemed to be inactive at the doses used.
As Aculeatins have a novel framework and significant antiprotozoal, antibacterial and antitumoral activities, inspired by the cytotoxic effects and having fascinated spiroacetal frame work in the structure we investigated the synthesis of Aculeatin A and B via Prins cyclisation. Our group has made a significant effort to explore the utility of Prins cyclisation in the synthesis of various polyketide intermediates and applied it in the synthesis of some natural products. As a part of this ongoing programme, we have accomplished a stereoselective total synthesis of the Aculeatin A and B via Prins cyclisation.











Figure1
RETROSYNTHESIS:













Scheme1
The retrosynthetic analysis for the synthesis of Aculeatin A and B is outlined in scheme I.
                                Before the synthesis, a careful examination was made on retrosynthetic analysis (Scheme1). We have simplified the molecule into aliphatic fragment 5 and aromatic fragment 6. The aliphatic part is envisaged to be constructed from 7 which in turn could be drawn from pyranyl methanol 8. Pyranyl methanol 8 could be easily constructed via Prins cyclisation between homoallylicalcohol (HAA) 9 and tetradecanal following our recently developed methodology.
Synthesis of aliphatic fragment:








Scheme II
             The synthesis of aliphatic fragment is initiated from epichlorohydrin as described in Scheme II. Epichlorohydrin 10 on treatment with benzyl alcohol in tetrahydrofuran (THF) yielded benzyl glycidyl ether 11 using a well documented procedure. The Jacobsen HKR of compound 11 using (R, R)-(salen) cobalt (II) precatalyst, aceticacid (AcOH) and H2O (0.51 equiv) for 22 hours resulted in (S)-benzyl glycidyl ether 12 in 46% yield. Reaction of 12 with vinyl magnesium bromide in THF, in presence of CuCN resulted the coupled product 13 which on debenzylation gave homoallylic alcohol 9 in 75% yield. (Scheme II)  
                                                






Scheme III
               
                      Subjection of homoallylic alcohol 9 to crucial Prins cyclisation with tetradecanal  using trifluoroacetic acid (TFA) in CH​​2​Cl2 ​resulted in trisubstituted pyran 15 in 52% yield after hydrolysis of corresponding trifluoroacetate 14 using Potassium carbonate (K2CO3) in methanol.. The stereochemistry was assumed to be in accordance as it was well examined and established previously. However, it was proved after elaborating the compound 15 to the target molecule which in all respects was identical with the reported one. Tosylation with 1.1 equiv of tosyl chloride in the presence of TEA in DCM produced the corresponding primary tosylate 16 in 95% yield followed by secondary alcohol protection with TBSCl, DMAP and imidazole provided the corresponding TBS ether 17 in 92% yield. Exposure of compound 17 to sodium iodide (NaI) in refluxing acetone gave the corresponding iodo compound 18 in 95% yield. (Scheme III).   

 Scheme IV
                    Iodo compound 18 on exposure to unactivated Zn in refluxing ethanol furnished key open chain intermediate 19 with 96% yield. Alcohol 19 when subjected to standard Mitsunobu inversion conditions (DEAD, PPh3, p-nitro benzoic acid in THF then K2CO3, MeOH) yielded 20 in 86% yield followed by secondary alcohol protection with TBSCl, DMAP and imidazole provided the corresponding di TBS ether 21 in 99%yield. Ozonolytic cleavage of the olefinic bond of 21 followed by further oxidation of the resulting crude aldehyde with NaClO2 and NaH2PO4 furnished acid 22 in 90%yield, which was converted to the amide 5 by treatment with morpholine and diisopropyl carbodiimide (DIC). (SchemeIV).  

Synthesis of Aromatic fragment:

Scheme V
          Aromatic fragment was synthesized from 4-Hydroxy benzaldehyde 23 was protected as benzyl ether 24 using BnBr and K2CO3 in 95% yield.  Aldehyde was subsequently treated with CBr4 and TPP to afford the dibromo olefin 25 in 87% yield.  The dibromo olefin was converted into alkyne 6 under Grignard reaction condition by treating with EtMgBr at 0 oC in 92% yield. (Scheme V).  
Synthesis of Aculeatin A and B:

                          Scheme VI
It was thought that the synthesis had reached to the stage for homologation on the other terminal. Hence, treatment of 5 with lithiated 4-benzyloxyphenyl acetylene 6 furnished alkynone 26 in 85% yield, further Catalytic hydrogenolysis with 10% Pd-C/H2 in methanol gave 27 in 88% yield. The later on desilylated with TBAF which without purification treated with Phenyliodonium (III) bis (trifluoroacetate) in Acetone –Water (9:1) yielded a mixture of Aculeatins A and B in a ratio of 2.5:1, which were smoothly separated by column chromatography. Synthetic compound 1 and 2 has showed spectral and analytical data (1H NMR, 13C NMR, IR, Rf and D) identical to the previously synthesized sample.
                      
CHAPTER II: In this chapter the work is presented into two sections A and B.
	
SECTION A: This section describes introduction, known synthetic approaches of Xestodecalactone C and Sporostatin 
SECTION B: This section describes Stereoselective synthesis of Xestodecalactone C and epi-sporostatin via the Prins cyclisation.
                                Marine microorganisms have evolved as a source for novel bioactive compounds, more than 800 microorganisms have so far been isolated from marine sediments and organisms.  Xestadecalactones A, B, C (Figure 2)were isolated from the fungus penicillium cf. montanense which in turn was isolated from Xestospongia exigua.These new compounds were structurally related to a number of compounds isolated from terrestrial fungi. One of those, Sporostatin (1) and Curvularins (5).
                 Sporostatin (1) is an inhibitor isolated from a fungus of Sporormiella sp. M5032 as an inhibitor of cyclic adenosine 3’5’-monophosphate Phosphodietererase. All these compounds constitute a 10-membered macrolide derivative with a 1, 3-dihydroxybenzene ring. Xetodecalactones A-C (2, 3, 4) have been found to exhibit antibacterial and antifungal activities. Inhibitory activity of Sporostatin (1) against cAMP-PDE from bovine heart expressed in terms of 50% inhibition (IC50) was 41µg/ml, and it was noncompetitive against cAMP. It was found to be a specific inhibitor of epidermal growth factor (EGF) receptor tyrosine kinase in vitro. In view of their biological activity, we were interested in the total synthesis of Xestadecalactone C and epi-Sporostatin via Prins cyclisation.
           










Figure 2
 Sporostatin (1), Xestadecalactone A (2), Xestadecalactone B, C (3, 4), Curvularins (5).
                  In our retrosynthetic analysis (SchemeVII) we envisaged that the target molecules 1, 4 could be achieved from a common intermediate 5 through AlI3 reaction. Compound 5 was viewed to be obtained from Mitsunobu product 6 using intramolecular Friedel-Crafts reaction. It was proposed to obtain the 1, 3 diol 7 from 2, 4, 6 substituted pyran 8 which in turn  would be obtained through Prins cyclisation of homoallylic alcohol 9 and Acetaldehyde.
          
SchemeVII
                                 The stereoselective total synthesis of Xestadecalactone (4) and epi-Sporostatin (1) started with chiral homoallyl alcohol (9).Cu mediated regioselective opening of S-(-)-benzyl glycidyl ether with vinylmagnesiumbromide followed by treatment with Li or Na in liquid ammonia debenzylation produced homoallylic alcohol 9.


Scheme VIII
               Prins cyclisation of homoallylic alcohol 9 with Acetaldehyde in the presence of                                                                                 TFA followed by hydrolysis of the resulting trifluoroacetate gave trisubstituted pyran 8 in 52% yield. Stereochemistry was assumed to be in anticipated line as it was well examined and established previously. However, it was proved after elaborating the compound 8 to the target molecule which in all respects was identical with the reported one. Tosylation with 1.1 equiv of tosyl chloride in the presence of TEA in DCM produced the corresponding primary tosylate 12 in 96% yield. TBS protection of the secondary alcohol in 12 with TBSCl, DMAP and imidazole provided the corresponding TBS ether 13 in 99% yield. Treatment of tosylate 13 with NaI in refluxing acetone gave the corresponding iodo compound 14 in 94% yield. (Scheme VIII).  

                          
Scheme IX
                           Iodo compound 14 on exposure to unactivated Zn in refluxing ethanol furnished key open chain intermediate 7 with the required anti-1, 3- diol system in 96% yield. Alcohol 7 when subjected to Mitsunobu reaction conditions DEAD, PPh3, 3,5,dimethoxyphenyl acetic acid in THF yielded 6 in 86% yield. Ozonolytic cleavage of the olefinic bond of 6 followed by further oxidation to acid with NaClO2 and NaH2PO4 gave the corresponding acid 16 in 90% yield. (Scheme IX).           



Scheme X
                  The macrolide 5 was obtained by intramolecular Friedel- Crafts reaction of the carboxylic acid 16 with a mixture of trifluoroacetic acid and trifluoroacetic acid anhydride in 41% yield (rt, 30min). Deprotection of the methoxy groups of 5 using freshly prepared AlI3 furnished the target molecules Xestodecalactone C (AlI3, 100C, 45min, 96%), epi-Sporostatin (AlI3, rt, 12h, 94%).
               
CHAPTER III: This chapter is also further divided into two sections A and B.
SECTION A: This section describes introduction, known synthetic approaches of 12-membered lactone.	
SECTION B: This section describes the Stereoselective total synthesis of (10S, 12S)-10-Hydroxy-12-methyl-1-oxacyclododecane-2, 5-Dione via the Prins cyclisation.
                                     The (10S, 12S)-10-hydroxy-12-methyl-1-oxacyclododecan-2, 5-dione, whose absolute stereochemistry of the two hydroxyl functionalities at C10 and C12 was originally assigned as depicted in 1 (Figure 3), was isolated from the endophytic fungal strain Cladosporium tenuissimum LR463 of Maytenus hookeri. It is found to induce rapid systematic defense responses in plants i.e. the production of peroxidase, phenylalanine ammonia lyase, lignin and salicylic acid. 

As a part of our interest on the synthesis of biologically active molecules, here in we report the synthesis of (10S, 12S)-10-hydroxy-12-methyl-1-oxacyclododecan-2, 5-dione (1).


Figure 3
               In our retrosynthetic analysis (Scheme XI), we envisaged that the target molecule could be achieved from 2 through Ring Closing Metathesis (RCM). Compound 2 could be obtained from 3 and 4 by esterification reaction. It is proposed to obtain the 1, 3-diol 3 from 2, 4, 6-substituted pyran 5 which in turn could be obtained via Prins cyclisation of homoallylic alcohol 9 and acetaldehyde. Compound 4 could be obtained from the Pent-4-en-1-ol 6.

Scheme XI
                              Accordingly, the total synthesis of 1 began with chiral homoallyl alcohol 9. Cupper (Cu) mediated regioselective opening of S-(-)-benzyl glycidyl ether (7) with vinylmagnesium bromide followed by debenzylation with sodium (Na) in liquid ammonia gave homoallyl alcohol 9. Prins cyclisation of compound 9 with acetaldehyde in the presence of TFA followed by hydrolysis of the resulting trifluoroacetate gave trisubstituted pyran 5 in 52% yield. The stereochemistry of compound 5 was assumed to be in anticipated line as it was well examined and established previously. Furthermore, it was proved after elaborating the compound 5 to the target molecule which was identical in all respects with the reported one. Tosylation of compound 5 with 1.1 equiv of tosyl chloride in the presence of TEA in DCM afforded the corresponding primary tosylate 10 in 96% yield. TBS protection of the secondary alcohol 10 with TBSCl, DMAP and imidazole gave the corresponding TBS ether 11. Treatment of tosylate 11 with NaI in refluxing acetone gave the corresponding iodo compound 12.


Scheme XII
                   Exposure of compound 12 to unactivated zinc in refluxing ethanol gave the key intermediate 13 (anti-1, 3-diol) in 96% yield. Esterification of free OH group of 13 with 4-benzyloxy-hept-6-enoic acid 4 in the presence of DCC and a catalytic amount of DMAP gave the diene 9 in 85% yield and set a stage for macrocyclization by ring closing metathesis.

Scheme XIII
                  The compound 4 was synthesized starting from pent-4-en-1-ol 4. Accordingly, compound 6 was protected as its tetrahydropyranyl ether 16, which upon epoxidation with m-CPBA resulted in the terminal epoxide 17 in 85% yield. Regioselective opening of the epoxide 17 with vinylmagnesium bromide gave the secondary alcohol 18 in 92% yield. Protection of the free hydroxy group as its benzyl ether 19 in 94% yield using BnBr in the presence of Sodiumhydride (NaH) and a catalytic amount of TBAI followed by deprotection of the THP ether with PTSA in MeOH gave the primary alcohol 20. Oxidation of the primary alcohol with DMP in DCM followed by further oxidation of the resulting crude aldehyde with NaClO2 and NaH2PO4 furnished benzyloxy acid 4 (90%), which was the key component used in the bis-olefin formation.

Scheme XIV

              The compound 2 possessed all the structural requirements as well as the sense of chirality to be converted into target macrolide via the Ring Closing Metathesis (RCM) reaction. The RCM reaction provides a remarkable scope for the synthesis of medium sized rings and macrocyclic products and is being used extensively. The presence of a suitable functionality and its distance from the olefins play key roles in the success of the metathesis reaction. It has been reported that the RCM reaction proceeds satisfactorily with substrates possessing a 6-heptenoate moiety, which is present in the dienic derivative 2. 
Scheme XV
                  Thus treatment of the diene 2 with 5 mol% Grubb’s II catalyst under high dilution conditions (0.001 M in DCM) gave compound 13 exclusively in 65% yield. Ttreatment of the cyclic ester 13 with Pd/C in MeOH afforded 14 (85%) wherein the two reactions, benzyl deprotection along with the reduction of the double bond were accomplished by a single reaction. 


Scheme XVI
          The resulting secondary hydroxyl was oxidized using Dess-Martin periodinane in CH2Cl2 to gave the ketone 15 (92%). Subsequent deprotection of TBS ether by TBAF in THF afforded macrolactone 1 (83%) as a white solid, m. p: 70-72 oC; [α]D25 +68.5 (c 1.0, MeOH) {lit.2 []25D +68.1 (c 0.25, MeOH )} which was identical in all respects to the reported value.
















English

MASS SPECTROMETRY OF SOME MODEL AROMATIC AND ANTIAROMATIC SYSTEMS

MIRAJKAR, SUBHASH P.

KUNIYIL, SIVADSAN

VII 
 
SYNOPSIS 
Cancer is a disease, causes due to failure of control of growth and 
cell proliferation mechanisms. There are genetic control systems, 
regulate the balance between cell birth and death in response to growth 
signals, growth-inhibiting signals, and death signals in normal 
conditions. So, the losses of cellular regulation and corresponding 
genetic damage give rise to cancer. The ultimate goal of cancer therapy is 
to destroy cancer cells selectively without affecting the normal tissues of 
patients. But, most of the currently available anticancer agents (as well 
as radiation therapy) do not possess higher selectivity towards cancer 
cells and destroy rapidly dividing normal cells including vital tissues 
such as lympho-hematopoetic tissues, gonad, hair follicles and the lining 
epithelium of gastrointestinal tract and mouth. So, adverse side effect of 
anticancer agents limits the doses applied to cancer patients causing 
incomplete killing of cancer cells. As a result, prolong treatment is 
needed and that makes cancer cells ‘un-sensitized’ towards drugs. 
Recurrence of cancer is also an unavoidable outcome of this incomplete 
killing. 
A lot of developmental researches across the globe are going on to 
overcome these limitations with alternative approaches towards cancer 
therapy. Targeted cancer therapy using lipid-based delivery systems and 
subsequent gene delivery is an important strategy for selective cancer 
VIII 
 
eradication. It is also possible to reduce the drug dose using suitable 
adjuvant to overcome the adverse effect of drugs on normal cells and 
make cells sensitized towards drug molecules. 
In targeted cancer therapy, receptors are targeted using suitable 
delivery systems. Certain receptors are over-expressed in various cancers 
but not in normal cells. So, by targeting those receptors, cancer cell 
specific delivery of anticancer agents is possible. On the other hand, 
some receptors behave differently in cancer and normal cells. Those 
receptors can also be targeted to kill cancer cells selectively leaving the 
normal tissues unaffected. The development of a receptor-targeted 
delivery of anticancer agent is described in the second chapter of the 
present thesis. 
Chapter 1A: 
In This chapter, the development of synthetic bivalent inhibitors of 
N-end rule ubiquitin pathway and their in-vitro and in-vivo biological 
effect of inhibition is described. N-end rule pathway is a subset of 
ubiquitin-dependent proteolysis pathway. The N-end rule is a relation 
between the metabolic stability and fate of a protein and the identity of 
its N-terminal residue, which is called N-degron. So, the in-vivo half life 
of a protein depends upon its N-terminal residue. Proteins contain some 
specific N-terminal amino acid residues like Arg, His, Phe, Lys, Leu, Ile, 
called destabilizing residue are degraded rapidly. There are also some 
IX 
 
stabilizing N-terminal residues (e.g. Met, Val) are not degraded in this 
pathway and are stable. The proteins bearing basic N-terminal residues 
(e.g. Arg, Lys, His) are type 1 substrates and the proteins bearing bulky 
hydrophobic N-terminal residues (e.g. Phe, Leu, Ile) are type 2 substrates 
of the N-end rule pathway. There is one more type of substrate, available 
in mammals, called type 3. The proteins bearing internal amino acids 
like Ala, Ser, Thr at a specific position, also recognized by N-end rule 
pathway as a type 3 substrates. The protein degradation pathway 
consists of two successive steps: (a) covalent attachment of multiple 
ubiquitin molecules to the target protein (ubiquitination) through 
recognition by a class of proteins E3, called N-recognin and (b) 
degradation of the ubiquitin-tagged protein by the 26S proteasome. N-
recognins are the E3 ubiquitin ligases involved in N-end rule pathway 
that recognize and bind to the target proteins depending on the presence 
of N-terminal destabilizing residues of proteins. Then they attach poly-
ubiquitin chains to target proteins. So, N-recognins are worthwhile to be 
targeted for selective inhibition of the N-end rule pathway of protein 
degradation. 
Recent biochemical and proteomic studies identified many 
components of the mammalian N-end rule pathway. The studies in 
various species revealed its essential role in various vital physiological 
processes, ranging from cardiovascular development and mitosis/meiosis 
to the pathogenesis of human genetic diseases. Studies indicated that 
X 
 
the increasing rates of ubiquitin conjugation via the activation of N-end 
rule pathway activation enhanced the rapid proteins degradation in 
skeletal muscles. The rapid loss of muscles accompanies many diseases 
like cancer, sepsis, diabetes etc. N-end rule pathway plays an important 
role in cardiogenesis. Recent studies showed its involvement in the 
regulation of apoptosis. So, the N-end rule inhibitor is gaining 
importance in recent days. 
The structural characterization of N-recognins reveals the presence 
of at least two spatially proximal binding sites: type 1 site binds to basic 
N-terminal residues (e.g. Arg, Lys, His) and the type 2 site binds to bulky 
hydrophobic N-terminal residues (e.g. Phe, Leu, Trp, Ile). RF-C11 was 
synthesized by our group as one of the model compounds, L1L2-Cn, 
which are composed of three replaceable components: ligand (L1L2), 
linker (Cn), and core (lysine). In RF-C11, two C11 hydrocarbon chains, 
attached with core lysine, were conjugated to the type-1 substrate Arg (R) 
and the type-2 substrate Phe (F). If the off-target interaction of the linker 
and ligand with themselves and other cellular macromolecules is ignored, 
then the N-end rule inhibitory effect of RF-C11 hetero-bivalent inhibitor 
can be mainly featured to the linker length.  
To this end a series of molecules (RF-Cn) have been synthesized 
with varying chain length. Arg and Phe were conjugated to hydrocarbon 
linkers (n= 1, 2, 4, 7, 8, and 14), which in turn were linked to the core 
XI 
 
Lys moiety by its ε- and α- amine group respectively. The carboxylic acid 
group of Lys is kept esterified to prevent further reactions. We got a 
series of compounds namely, RF-C2, RF-C3, RF-C5, RF-C8, RF-C9, and 
RF-C15 respectively. Two homo-bivalent inhibitors, RR-C5 (where, n=4, 
& bearing Arg at its both termini) and FF-C5 (where, n=4, & bearing Phe 
at its both termini), were also synthesized to observe the effects of homo-
bivalent interactions compared to hetero-bivalent interaction. A 
structural control, GV-C5, bearing two stabilizing amino acid residues 
Gly and Val at its termini, had been synthesized to look at the non-
selective interaction with N-recognins. The structures of the synthesized 
hetero-bivalent inhibitor molecules are represented in scheme 1. 
 
 
 
 
 
 
 
Scheme 1: Structures of synthesized bivalent inhibitors. 
O
O
HN
H
N
N
H
O
N
HO
O
NH3
N
H
NH2
NH2
O
NH3
O
O
HN
H
N
N
H
O
2
N
HO
2
O
NH3O
NH3
O
O
HN
H
N
N
H
O
2
N
HO
2
O
NH3
N
H
NH2
NH2
O
NH3
NH
NH2H2N
Cl
Cl
Cl
Cl
Cl
O
O
HN
H
N
N
H
O
2
N
HO
2
O
O
Cl
Cl
Cl
RF-Cn (where n = 1, 2, 4, 7, 8 & 14) GV-C5
NH3
NH3
Cl
Cl
RR-C5 FF-C5
Cl
n
n
XII 
 
All the synthesized bivalent inhibitors were examined for their 
efficiency in inhibiting the N-end rule pathway mediated protein 
degradation in vitro and in vivo. The structure-activity relationship study 
was done with synthesized RF-Cn molecules in a time- and dose-
dependent manner. Among the inhibitors the maximum protein 
degradation inhibiting activity was observed with RF-C5 and RF-C9 both 
in type 1 and type 2 models for longer time. All the hetero-bivalent 
inhibitors were more effective in inhibiting the degradation of both type 1 
and type 2 N-end rule substrates than that of di-peptides bearing N-
terminal destabilizing residues, used as control molecule. As RF-C5 was 
one of the most efficient inhibitor, corresponding homo-bivalent control 
molecules RR-C5 and FF-C5 were synthesized.  RR-C5 showed very 
limited inhibition of degradation of type 1 substrates only, though at 
higher concentrations in comparison to RF-C5. Similarly, FF-C5, at 
higher concentrations, inhibited the degradation of type 2 substrates 
only. Structural control molecule GV-C5 showed no inhibition of 
degradation of both type 1 & type 2 substrates.   
Chapter 1B: 
 This chapter describes the development of hetero-bivalent N-
end rule pathway inhibitor as an adjuvant for drug molecules in various 
cancer cells. As discussed earlier, N-end rule pathway has a great 
importance in animal physiology. It had been shown that N-end rule 
XIII 
 
regulates apoptosis, a process of programmed cell death in multicellular 
organism. Multiple apoptosis and cell cycle-related factors are 
ubiquitinated by N-end rule pathway. Caspases cleave various target 
proteins to give protein fragments of destabilizing N-degron residues, 
which are recognized and degraded via N-end rule pathway.  
On the other hand, the destructive potential of caspases is 
restrained by Inhibitors of Apoptosis Proteins (IAPs). IAPs exhibit self-Ub 
ligase activity which is important for its inhibitor of apoptosis function as 
it complexes with equimolar amount of caspases for the ubiquitination 
and degradation. During apoptosis IAPs are cut by activated caspases 
and the fragmented parts of IAPs are degraded through N-end rule 
pathway. Incidentally, XIAP, the mammalian counterpart of IAP, is 
fragmented into a Type 3 N-end rule substrate by caspases. However, our 
hypothesis was to decipher this pathway’s role in functional regulation of 
XIAP in cancer. 
 In cancer, XIAP has another critical role to play. PTEN, which 
regulates Akt/PKB kinase pathway, is ubiquitinated and degraded by 
XIAP. This Akt/PKB kinase pathway regulates phosphorylation of Mdm2 
and hence, regulates the recognition and ubiquitination of nuclear wild 
type p53 present in 50% of all cancer cells.     
Additionally, N-end rule pathway also maintains the chromosomal 
stability by degrading a cohesin complex sub-unit (SCC1/RAD21, SCC1 
counterpart in mammals) cleaved by separase at the metaphase to 
XIV 
 
anaphase transition. This also clues to the fact that antagonizing N-end 
rule pathway might lead to chromosomal instability in normal 
functioning cells. This chromosomal instability makes cells ‘sensitized’ 
towards drug molecules. 
The present study was designed to see the sensitizing effect of our 
novel, synthetic hetero-bivalent N-end rule pathway inhibitor RF-C11, 
previously designed and characterized in our lab. RF-C11 was co-treated 
with existing anticancer drugs on cells. We observed that the co-
treatment significantly enhanced drug sensitivity in cancer cells through 
simultaneous reduction of XIAP and in vivo stabilization of RAD21. But, 
there was no such effect on normal cells. Wild type p53 up-regulation 
was observed due to the down-regulation of XIAP via N-end rule pathway. 
Co-treatment group induced apoptosis in cancer cells and also cell cycle 
arrest at G2/M phase. So, altogether we got significant cytotoxicity effect 
of co-treatment in cancer cells but not in normal cells. These data have 
given new insights into how N-end rule pathway may be manipulated for 
the development of anticancer therapeutics.  
Chapter 2: 
This chapter of present thesis consists of the targeted liposomal 
delivery of micro-RNA precursor in plasmid form into cancer cells. Micro-
RNAs (miRNAs) are 19-24 nucleotide long non-coding RNAs, regulate 
gene expression by binding with mRNAs imperfectly or perfectly. miRNAs 
are encoded by the specific genes and sometimes derived from the 
XV 
 
introns of pre-mRNAs. If miRNAs have nearly perfect complementarities 
to mRNA target sequences positioned in either coding or 3’ untranslated 
(3’UTR) regions (in plants), then the perfect base pairing triggers mRNA 
degradation through a mechanism similar to that of RNA interference 
(RNAi). But, in animals, with very few exceptions, miRNAs do not posses 
perfect base pairing like plants. In animals, they regulate gene 
expression by imperfect base pairing to the 3’ UTR of target mRNAs and 
inhibit protein synthesis or causing mRNA degradation. miRNAs have 
important role in physiological processes, as they regulate gene 
expression.  
Here, we delivered artificial miRNA (amiR) against one of the most 
abundant and ubiquitously express cellular chaperon protein Hsp90 
(amiR-Hsp90) in the form of plasmid. Hsp90 as chaperon maintains the 
structural integrity of many of its client proteins, which are involve in 
cellular growth, differentiation and apoptotic pathways. So, by targeting 
one protein expression it could be possible to target many other proteins 
involving in various cellular processes. Here, we used amiR-Hsp90 
plasmid in-stead of small molecules which have some limitations like 
non-specific toxicity, bio-availibility, and solubility.  
 Cationic lipid-based delivery system, liposome, is a good choice to 
overcome the limitations created by small molecules. Dexamethasone 
associated lipoplex (liposome plasmid complex, DX) was used here as 
XVI 
 
delivery system, which acts via glucocorticoid receptor (GR). GR is also a 
client protein of Hsp90 and it maintains the structural integrity of 
ligand-binding domain (LBD) of GR. The LBD of GR allows only two 
molecules of synthetic GR-ligand, dexamethasone (Dex), as it is tightly 
regulated by Hsp90 in normal cells. Our group recently discovered that 
DX could gain GR-mediated nuclear access only in cancer cells and 
eventually expresses the delivered gene, though the size of DX is many 
folds bigger than that of LBD of GR. Similar GR-mediated nuclear 
delivery of DX lipoplex could not be observed in normal cells unless 
Hsp90 was inhibited. This proved that functional activity of Hsp90 must 
be somewhat compromised in cancer cells. GR helps nuclear delivery DX 
only in cancer cells using the compromised functional activity of Hsp90, 
though the cellular uptake of DX was same for both cancer and normal 
cells. This encouraged us to use this delivery system (DX) to deliver 
amiR-Hsp90 plasmid in cancer cells. The DX-mediated delivery to cancer 
cells & selective processing of plasmid in those cells is expected to be 
more effective in comparison to that in normal cells.  
Artificial miRNAs (amiRs), in plasmid form, utilize the cellular 
machinery for expression, processing and targeting of mRNAs to repress 
their protein level expression. Artificial miRNA in plasmid form (pri-
miRNA) has to be processed in nucleus by Drosha following the 
processing in cytoplasm by Dicer to form mature miRNA. Therefore, this 
targeted delivery system (DX) has been used to deliver genetic cargo like 
XVII 
 
amiR-Hsp90 into nucleus of cancer cells to get the repressor effect of 
miRNA against Hsp90 protein. 
Unlike amiRs in plasmid form, mature miRNAs need not to be 
processed from nucleus. Cytoplasmic delivery of mature miRNAs is 
enough to get their effect. As far as cytoplasmic delivery is concerned, 
our delivery system DX cannot differentiate between cancer cells and 
normal cells. But DX can differentiate between cancer cells and normal 
cells as far as GR-mediated nuclear delivery of genetic cargo is 
concerned. On the basis of this important observation, we planned to use 
amiR-Hsp90 in plasmid form to target specific deliver and process it to 
down-regulate Hsp90 in cancer specific manner. 
  The plasmid bearing the artificial miRNA gene (amiR-Hsp90) was 
then complexed with GR-targeted cationic liposomal delivery system DX 
(DX-amiR-hsp90). The anticancer efficacy of lipid/DNA complex was first 
tested in cells in vitro. The down-regulation of Hsp90 at the mRNA level 
was observed by RT-PCR analysis. The comparison of the levels of Hsp90 
and its cancer implicated client proteins was accomplished by western 
blot analysis. We observed down-regulation of Akt1 and corresponding 
phospho-Akts, anti-apoptotic protein bcl-2 and VEGF-R2 & up-
regulation of apoptotic protein p53 and Caspases. Overall western blot 
data indicated the induction of apoptosis and repression of cells 
proliferation through the down-regulation of cancer related client 
XVIII 
 
proteins of Hsp90. The in-vivo studies in two different tumor models 
(mouse melanoma & human lung adenocarcinoma) showed significant 
tumor regression. This, to our knowledge, is the first evidence of GR-
assisted targeted gene therapy against Hsp90 for eliciting selective and 
highly efficient anticancer response. 
         


EXPANSION COEFFICIENTS IN POLYELECTROLYTES

SAYYED , B.A.

SYNTHESIS AND CHARACTERISATION OF CATALYTIC ALLY IMPORTANT IRON OXIDE SYSTEMS

KULKARNI, SURESH

CHAPTER 5  SUMMARY AND CONLUSIONS 
188 
 
CHAPTER 5 
 
SUMMARY AND CONCLUSIONS 
 
Around 300 microorganisms were isolated from enriched petroleum-contaminated soil and 
sludge samples. These isolates were further screened for biosurfactant producers by employing 
three screening methods and eleven promising strains were short-listed. Initially drop collapse 
method was employed for screening in which 11 isolates exhibited positive result. These eleven 
isolates were promising as they reduced the surface tension of culture medium to values below 
42 mN m
-1
. The eight isolates among these 11 isolates produced the biosurfactants which were 
more effective than SDS as they were capable of reducing the surface tension to below that of 
1% SDS solution. Two strains, BS-161R and BS-223, were found to be potent as they were able 
to reduce the surface tension of culture medium to the value below of 27 mN m
-1
. Only two 
isolates, BS-161R and BS-243, showed β-hemolysis on blood agar among the 11 isolates. 
 
All of the tested strains exhibited good emulsification activity against N-hexadecane, hexane, 
xylene, mineral oil, olive oil and castor oil suggesting that the biosurfactants produced can act as 
emulsifiers and this property would be useful in various applications like biodegradation of 
hydrocarbons or other water-immiscible substrates as well as in enhanced oil recovery. But few 
of them, even though possessing good surface tension lowering property, were unable to form 
good stable emulsions. From the analysis of screening methods it can be concluded that the 
direct screening methods like drop collapse assay and surface tension measurement were more 
reliable than indirect methods like hemolytic activity and emulsification capability because of the 
probability of getting more false positives and/or negatives in indirect methods due to their poor 
correlation with surface tension reduction property. The direct surface tension measurement is 
final confirmation method to assess surface tension lowering property, however, the 
measurement of surface tension was inconvenient to use for screening of a large number of 
isolates as it was time consuming and required large volume of sample for analysis. Thus 
sequential screening was carried out to short list the isolates and finally based on surface tension 
measurement, 11 promising isolates were fished out. Furthermore, low values of CMC were 
recorded for the crude biosurfactant produced by these strains ranged between 25 and 190 mg L
-
CHAPTER 5  SUMMARY AND CONLUSIONS 
189 
 
1
. From an environmental perspective, possessing low values of CMC and high capacity to 
reduce surface tension suggest their possible use to promote remediation of contaminated 
subsurface environments by increasing the hydrocarbon mobility. Besides exhibiting surface 
active properties, all of the 11 strains exhibited flocculating activity and among them, three 
isolates BS-2, BS-161R and BS-243 were found to be potent. 
 
The strains were identified up to species level by morphological, physiological, biochemical 
characteristics and 16S rDNA sequence analysis. The isolated microorganisms belonged to 
diverse genera including Bacillus, Brevibacillus, Pseudomonas, Ochrobactrum, Kocuria, 
Nocardia and Microbacterium. Two new microorganisms, namely, Ochrobactrum sp. and 
Microbacterium sp. were identified as new additions to the list of biosurfactant producing 
microorganisms, while Microbacterium sp. exhibiting flocculating activity was identified as new 
addition to the list of bioflocculant-producers. 
 
The crude biosurfactant produced from P. aeruginosa BS-161R had an excellent heat tolerance 
which could withstand at higher temperatures and had broad pH stability. These findings suggest 
that the robust characteristics of the crude biosurfactant which are beneficial for applications 
carried out under extreme conditions of temperature and pH, such as in oil recovery and in the 
bioremediation of a polluted environment. The strain, BS-161R, was capable of producing 
rhamnolipids in presence of both water-soluble and water-insoluble carbon sources. However, 
glycerol was found to be little more effective at promoting the slight increase in the production 
of rhamnolipids. The bio-glycerol which is a by-product of biodiesel production acted as a cheap 
raw material for rhamnolipid production. The optimum concentration of carbon source was 
found to be 25 g L
-1
, while sodium nitrate 4 g L
-1
 was found to be suitable nitrogen source. The 
maximum production of rhamnolipids was observed at C:N ratio of 15 and C-to-Fe ratio of 
28350. The optimum pH and temperature for the highest yield of rhamnolipids ranged between 
pH 7.5 and 8, while temperatures of 34 and 36°C. The agitation speed also influenced the 
production of rhamnolipids as the yield of rhamnolipids was gradually increased with speed and 
reached maximum at 200 rpm. After optimization of nutritional and environmental conditions 
the production of rhamnolipids was increased from 0.369 to 3.312 g L
-1
 which is approximately 
9.0 fold. The optimum medium for Pseudomonas aeruginosa BS-161R consisted of the 
CHAPTER 5  SUMMARY AND CONLUSIONS 
190 
 
following components (g L
-1
): MgSO4, 0.2; CaCl2, 0.02; KH2PO4, 1.0; K2HPO4, 1.0; NH4NO3, 
1.0; FeCl3 .001, NaNO3, 4.0 and bio-glycerol, 25 while the optimum environmental conditions 
were follows as: pH 7.5, temperature, 35°C and agitation speed, 150 rpm. 
 
Thin-layer chromatography of the crude biosurfactant produced from P. aeruginosa BS-161R 
revealed the presence of three major spots. These compounds were purified successfully from 
crude biosurfactant using silica gel column chromatography. The chemical structure of purified 
compounds was deduced successfully as phenazine-1-carboxylic acid (PCA), rhamnolipid-1 
(RL-1) and rhamnolipid-2 (RL-2) based on the 
1
H and 
13
C NMR, FT-IR and mass spectral data 
analyses. 
 
Crude extracts of biosurfactant producing isolates, except BS-323, BS-206 and BS-207, 
exhibited broad-spectrum antimicrobial activity. Among all the isolates, crude extract of BS-
161R showed good activity against all indicator strains. Purified rhamnolipids and PCA from 
isolate BS-161R exhibited a broad spectrum antimicrobial activity against a wide range of 
pathogens, including both Gram-positive and -negative bacteria and Candida albicans. 
Rhamnolipid-1, which is a precursor of rhamnolipid-2, showed more antimicrobial activity than 
rhamnolipid-2. The least MIC value was recorded against Escherichia coli for rhamnolipid-1 at 
16 µg mL
-1
, while rhamnolipid-2 exhibited highest activity against Escherichia coli (64 µg mL
-1
) 
and PCA exhibited highest activity against Klebsiella planticola at 16 µg mL
-1
. The anti-
proliferative activity of purified rhamnolipids and PCA was evaluated against a panel of human 
cancer cell lines in culture, since no published toxicity data on purified rhamnolipids was 
available. The rhamnolipid-2 exhibited more cytotoxic activity as compared to rhamnolipid-1 
and exhibited least IC50 against MDA-MB-231 cell line at a concentration of 54 µM. The PCA 
exhibited the most efficient cytotoxic potency against A549 (IC50, 7 µM), followed by MCF-7 
(IC50, 9.2 µM), HepG2 (IC50, 11.1 µM) and HeLa (IC50, 30 µM), among the cell lines tested. 
 
Silver nanoparticles were successfully synthesized using an eco-friendly approach by using a 
reverse microemulsion technique in which purified rhamnolipid mixture (RL-1 and RL-2) acted 
as stabilizing agent where sodium borohydride acted as a reducing agent. The synthesized 
nanoparticles were characterized by UV-visible spectroscopy, transmission electron microscopy, 
CHAPTER 5  SUMMARY AND CONLUSIONS 
191 
 
and energy dispersive X-ray spectroscopy (EDS). These formed nanoparticles had a sharp 
adsorption peak at 410 nm, which is a characteristic surface plasmon resonance of the silver 
nanoparticles. The nanoparticles were mono-dispersed, with an average particle size of 15.1 nm 
(σ = ±5.82 nm) and spherical in shape. The EDS analysis revealed the presence of elemental 
silver signal in the synthesized nanoparticles. The FTIR and EDS results indicated that the 
possible capping ligands were rhamnolipids. The formed silver nanoparticles exhibited good 
antibiotic activity against both Gram-positive and Gram-negative pathogens and Candida 
albicans, suggesting their broad-spectrum antimicrobial activity. 
 
The culture supernatant of Pseudomonas aeruginosa isolate BS-161R was used for green 
synthesis of silver nanoparticles with silver nitrate at room temperature. It was proposed that the 
reduction of the silver ions may plausibly be due to the protein component contributed by the 
enzyme nitrate reductase, since nitrate reduction is the phenotypic biochemical property of this 
culture. The nanoparticles formed were stabilized probably by the capping of rhamnolipid 
present in the culture supernatant as revealed by FT-IR spectral analysis. The UV-visible 
spectrum showed a surface plasmon resonance peak at around 430 nm, which is characteristic of 
silver nanoparticles. The silver nanoparticles formed were spherical in shape with an average 
particle size of 13 nm, crystalline in nature, and the particle surface was anionic; these properties 
were confirmed by TEM, XRD and zeta potential analysis. The silver nanoparticles had a broad 
spectrum antimicrobial activity against various Gram-positive and Gram-negative bacteria and 
different Candida species.  


ANALYTICAL METHODS IN ORGANIC CHEMISTRY

TAMBE, SANJEEV

Abstract 
vi 
ABSTRACT 
Present developments in the field of materials chemistry show that, though 
inorganic materials are still the choices for many devices, interest in organic 
materials is growing day-by-day in view of the latter’s adaptability to various kinds 
of applications. The field of nonlinear optics has attracted lot of interest in the past 
three decades not only because of the possible numerous applications in 
telecommunications, optical data storage etc., but also because of the fundamental 
research connected to issues like charge transfer, conjugation, polarization and 
crystallization into noncentrosymmetric lattices. In principle, nonlinear optics (NLO) 
deals with the interaction of applied electromagnetic fields in various materials to 
generate new electromagnetic fields altered in frequency, phase or other physical 
properties.  A variety of systems including inorganic materials, organometallic 
materials, organic molecules and polymers have been studied for NLO activity. 
Owing to the combination of chemical tunability and choice of synthetic strategies, 
organic molecules in particular have received much attention.  
The purpose of the present research is the design of novel nonlinear optical 
(NLO) materials, capable of exhibiting enhanced Two-Photon Absorption (TPA) 
behavior. These materials have potential applications in biological imaging, 
microfabrication, sensing, photodynamic cancer therapy, optical limiting and 
ultrafast switching. Currently, only a limited number of 2PA fluorophores, 
specifically tailored for direct labeling of biomolecules for Two Photon induced 
fluorescence imaging studies, hence organic fluorescent probes with high Two 
Photon Absorption are highly desirable.  
The objective of this work is to understand how the chemical structures of 
molecules relate to their electronic and optical properties, and use this 
understanding to design and synthesize novel organic arene compounds for two 
photon absorption which can be useful for bio-imaging applications. The 
requirements of molecules for these applications is high Two Photon Absorption 
cross sections, High fluorescence quantum yields, large transition dipolemoments 
from ground to first excited state and from first excited to the higher excited state 
with low energy gaps. 
Abstract 
vii 
This thesis entitled “Design and synthesis of organic arene compounds for 
Two Photon Absorption” is divided into four chapters.  
CHAPTER 1 
  Chapter 1 provides an introduction of nonlinear optics, their origin, applications 
and various types of nonlinear optical effects. A special emphasis is given to the 
third order nonlinear optical property Two Photon Absorption (TPA) and their 
applications are discussed. Theoretical methods to determine the Two Photon 
Absorption cross-section (δ) and experimental techniques to estimate the 
magnitude of TPA activity are presented. The structure property relationships that 
can enhance the Two Photon absorption were described. 
 CHAPTER 2 
In view of the importance of heterocycle-based chromophores and the 
interest in the development of TPA materials, in this chapter 2, five novel dipolar 
(D-π-A) molecules (TA1-TA5) based on the 1, 3, 5-triazine core as acceptor, 
including substituted anthracene as donor with a ethylenic bridge(C═C) as a 
spacer are synthesized. Linear and nonlinear optical property evaluations are 
presented. Experimental one photon absorption results have been compared with 
theoretical observations and fluorescence, electrochemical studies are carried out. 
Theoretical studies on TPA cross-sections were carried out based on the SACCI 
method. These molecules are with low TPA cross-sections and with negligible 
quantum yields due to the excitation in the molecule transfers to ICT state and 
subsequently to TICT state, which might be finally relaxing to the triplet state via 
cross-transfer process, this result in the weak PL emission.  As high quantum 
yields are the prerequisite for bio-imaging applications these compounds may be 
useful as optical limiters and aimed for the synthesis of compounds with high 
quantum yields in the next chapters. 
CHAPTER 3 
Chapter 3 deals with the synthesis of bis (carbazole fluorene ethynyl) arene 
compounds C1─C5. Their electrochemical and photophysical properties are 
evaluated and compared with the theoretically calculated data. All compounds are 
Abstract 
viii 
having high fluorescence quantum yields. All the compounds are quite thermally 
stable (355-490 °C) in the incident laser environment. Two Photon Absorption 
cross-section values are calculated theoretically and they showed large TPA cross-
section values in the range of 728-5882 GM in the order of C3 > C1 > C5 > C4 > 
C2. Compound C3 with fluorene as a central bridge having high TPA value and 
compound C2 with anthracene as a bridge having low TPA value of 728 GM it may 
be due to lower transition dipole moments from the first excited sate to the lowest 
allowed TPA state. As these compounds are with high fluorescence quantum 
yields and large theoretically predicted TPA cross-sections these compounds can 
be used as promising candidates for TPA with an application in bio-imaging. 
CHAPTER 4 
Chapter 4 explains about synthesis, photo physical and electrochemical properties 
of fluorenyl ethynyl pyrene derivatives (P1-P5) with different donor or acceptor 
substituents as a new series of pyrene based TPA chromophores. Their linear and 
nonlinear optical properties were investigated theoretically. The photophysical and 
electrochemical and theoretical data revealed that the energy gap decreases with 
increasing number of branches from P1─P3, i.e from mono to di to tetra substituted 
compounds. P3─P5 the difference in energy gap is similar. All the molecules 
P1─P5 showed high quantum yields of fluorescence. One photon and two photon 
absorption properties of these five newly synthesized compounds are theoretically 
analyzed by using SACCI method. The maximal one-photon absorption intensities 
for these compounds appear in the first excited state. It reveals that these are 
HOMO-1 to LUMO transitions. With the increase in the number of branches, the 
transition energies show red shift with increase in the intensities. From P1 (180 
GM) to P2 (585 GM), the δ  increased by 3-times and is increased by 4-times in P3 
(705 GM) and 7 and 8-times in P4 (1113 GM) and P5 (1408 GM). It indicates that 
the increase in size of the molecule with increased conjugation enhances TPA 
activity. Strong acceptor group like –NO2 can enhance the transition dipole 
moment and decrease the transition energy which in turn enhances the magnitude 
of TPA cross-section. Compounds with acceptor substitution have large TPA 
cross-section than compounds with donor group substitution. This shed light into 
the significance of the pyrene derivatives as promising fluorescent probes with high 
TPA cross-sections which can be useful for bio-imaging application due to high 
fluorescence quantum yields. 


A STOCHASTIC APPROACH TO THE ANALYSIS OF CHEMICALLY REACTING SYSTEMS

PATIL, RAJKUMAR

  
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
Synopsis 
 
 
i 
 
The thesis entitled “Enantioselective Synthesis of (–)-8,4ʹ-Oxyneolignans & Synthesis 
and Bioevaluation of Nitrogen Containing Heterocyclic Compounds” has been divided 
into two parts, Part A and Part B, and each part is further divided into chapters. Each 
chapter except chapter-I (Introduction), is further sub-divided into the following sections: 
Introduction, Present work, Results and discussion, Experimental section, Conclusions, 
References and Spectroscopic data. 
PART A: Deals with the Enantioselective Synthesis of (–)-8,4ʹ-Oxyneolignans, and is 
divided into Chapter I and Chapter II.  
 
Chapter I: Introduction 
This chapter deals with the introduction of important plant metabolites viz., 
lignans, neolignans and 8,4ʹ-oxyneolignans and summarizes some of the reported 
approaches towards the (–)-8,4ʹ-oxyneolignans. This chapter also deals with the 
introduction of the glycolate Evans aldol reaction and our current approach of its 
application in the enantioselective synthesis of (–)-8,4ʹ-oxyneolignans.  
OH
O
OMe
OH
O
OMe
OMe
OH
O
OMe
OMe
OH
O
OMe
OMe
OMe
() - Virolin (1) () - Surinamensin (2) () - Raphidecursonol B (3) () - Polysphorin (4)
anti-schistosomial
anti-oxidant
anti-fungal
anti-schistosomial
anti-leukimic
anti-fungal
anti-malar ialanti-malar ial
OMe
OMe
OMe
OMe
OMe OMe
OMe
OMe
Figure 1: Srtuctures of ()-virolin (1), ()-surinamensin (2), ()-raphidecursinol B (3) and ()-polysphorin (4).
OMe
 
Chapter-II: Enantioselective Synthesis of (–)-8, 4ʹ-Oxyneolignans 
 This chapter details our efforts in the enantioselective synthesis                             
of four biologically active 8,4ʹ-oxyneolignans., (–)-Virolin, (–)-Surinamensin,                             
(–)-Raphidecursinol B and (–)-Polysphorin. 
(–)-Virolin, (–)-surinamensin, (–)-raphidecursinol B and (–)-polysphorin are 
optically active 8,4ʹ-oxyneolignans, found in various medicinally important natural 
Synopsis 
 
 
ii 
 
products. These compounds, isolated from Myristicaceae and other primitive plant 
families in neotropical regions, belong to one of the major class of neolignans containing 
a chiral aryloxy C(8)-O-C(4ʹ) ether linkage. Virolin (1) and surinamensin (2), isolated 
from the leaves of Virola surinamensis (Myristicaceae), show activity against 
leishmaniasis, fungal and other vector-brone diseases. Raphidecursinol B (3) isolated 
from the seeds (nut meg) of Myristica Fragrans Houtt (Myristicaceae) exhibit activity 
against plasmodium falciparum. Polysphorin (4) isolated from piper polysphorum C in 
China and also from Rhaphidopora decursiva in Vietnam, display anti-malarial activity. 
The four biologically active 8,4ʹ-Oxyneolignans - (–)-virolin (1), (–)-surinamensin (2),   
(–)-raphidecursinol B (3) and (–)-polysphorin (4) - were synthesized using glycolate 
Evans syn-aldol reaction as key step. 
 Scheme 1: Retro-synthetic strategy and the building blocks
OH
O
R2
R1
OMe
OMe
R3
OMe
O
R2
R1
OMe
OMe
R3
OMe
O
AUX
O
O
R2
R1
OMe
A
B
AUX =
N O
O
Ph
Ph
OH
O
R2
R1
OMe
OMe
R3
OMe
OH
+
I II III IV
V
OH
O
OH
R2
R1
OMe
VI
 
Our strategy is depicted retrosynthetically in Scheme 1. It was envisaged that the 
reductive deoxygenation of the primary hydroxyl group of the diol (II) resulting from the 
auxiliary assisted aldol reaction of suitable substrates would afford the 1-aryl-2-
aryloxypropanol (I) moiety in the required stereochemistry. The auxiliary linked chiral 
precursor (III) could easily be generated from aryloxyacetic acid (V), which in turn could 
be readily prepared from commercially available starting materials (VI).  
The aryloxyacetic acid derivative (7), A-ring precursor for virolin and 
surinamensin was assembled starting from vanillin (5). The aryloxyacetic acid derivative 
(10), A-ring building block of raphidecursinol B and polysphorin was generated starting 
from 2,6-dimethoxyphenol (8) (Scheme 2).  
Synopsis 
 
 
iii 
 
OMe
OH
OHC
OMe
O
i) (CH3CN)2PdCl2
MeOH, rt, 24 h
97% (2 steps)
ii) NaOH, MeOH/CH2Cl2
r t, 3 h, 97%
OMe
O
i) BrCH2CO2Et, K2CO3
acetone, reflux, 4 h, 98%
ii) Ph3PCHCH3, K2CO3
1,4-dioxane, reflux, 4 h
5 6 7
CO2Et CO2H
Scheme 2: Preparation of aryloxy acetic acid derivatives 7 & 10
OMe
OH
OMe
OMe
OH
OMe
i) BrCH2CO2Et, K2CO3
acetone, reflux, 5 h, 99%
ii) NaOH, MeOH/CH2Cl2
rt, 3 h, 97%
OMe
O
OMe
CO2H
i) allyl bromide, K2CO3
acetone, reflux, 6 h, 99%
ii) Bi(OTf)3, CH3CN
reflux, 4 h, 97%
8 9 10
 
With both A- and B-ring counterparts at our disposal, we proceeded to the crucial 
auxiliary assisted aldol reaction. Reaction of lithium salt of the auxiliary (11) with 
aryloxyacetic acid chlorides 12 and 13 (prepared from 7 & 10) afforded the 
corresponding N-acyl-imidazolidinone derivatives 14 and 15 (Scheme 3). 
O
N
O
O
OH
OMeR1
R2
R3
OMe
OMe
OPh
Ph
O
NH
OPh
Ph
O
N
OPh
Ph
O
O
R2
MeO
i) n-BuLi, THF, 0 °C
ii) 12 or 13, THF. 0 °C - rt
O
O
R
2
MeO
R1
Cl
OMe
R3
OMeO
i) n-Bu2BOTf, DIPEA
CH2Cl2, 0 °C - rt
ii) 16 or 17, -78 °C - rt
OH
O
OMe
R
3
OMe
OMe
R1
R
2
LAH, Et2O, rt, 2 h
OH
OH
O
OMe
R3
OMe
OMe
R1
R2
i) TsCl, cat. DMAP
CH2Cl2, 0 °C - rt,3 h
ii) LAH, Et2O
rt, 2 h
R
1
14 R
1
= H, R
2
= (E)-propenyl; 92%
15 R
1
= OMe, R
2
= allyl; 92%
18 R
1
= H, R
2
= (E)-propenyl, R
3
= H; 86%
19 R1 = H, R2 = (E)-propenyl, R3 = OMe; 87%
20 R
1
= OMe, R
2
= allyl, R
3
= OMe; 87%
1 R
1
= H, R
2
= (E)-propenyl, R
3
= H; 98%
2 R1 = H, R2= (E)-propenyl, R3 = OMe; 98%
3 R
1
= OMe, R
2
= allyl, R
3
= OMe; 98%
12 R
1
= H, R
2
= (E)-propenyl
13 R
1
= OMe, R
2
= allyl
11
16 R
3
= H
17 R
3
= OMe
21 R1 = H, R2 = (E )-propenyl, R3 = H; 97%
22 R
1
= H, R
2
= (E)-propenyl, R
3
= OMe; 98%
23 R1 = OMe, R2 = allyl, R3 = OMe; 96%
Scheme 3: Synthesis of ()-Virolin (1), ()-Surinamensin (2), ()-Raphidecursinol B (3)
 
The aldol reaction of the boron enolates, generated from imidazolinones 14 and 
15 by treating with di-n-butyl boron triflate in presence of Hünig’s base (i-Pr2NEt) with 
aldehyde afforded only a single diastereomer (>99% from 
1
H-NMR of the crude 
Synopsis 
 
 
iv 
 
product). The reductive removal of the auxiliary from the adducts (18, 19 & 20) gave the 
corresponding diols (21, 22 & 23). Deoxygenation of primary hydroxyl group from the 
diols by the tosylation followed by the reductive removal of the tosylate moiety afforded 
(–)-virolin (1), (–)-surinamensin (2) and (–)-raphidecursinol B (3) (Scheme 3). 
OH
O
OMe
OMe
OMe
OMe
OMe
OH
O
OMe
OMe
OMe
OMe
OMe
PdCl2, MeOH
rt, 6 h, 97%
() - Raphidecursinol B (3) () - Polysphorin (4)
Scheme 4: Preparation of ()-polysphorin (4)  
Finally, the synthesis of (–)-polysphorin (4) was achieved by the isomerization of 
the double bond in (–)-raphidecursinol B (3) (Scheme 4). The spectral characteristics of 
the natural products prepared compare favorably with the literature reported spectral data. 
Structural integrity of 1, 2, 3 and 4 was further confirmed by the specific rotation 
analysis, which matched well with literature reported values. Employing this protocol, the 
overall yields achieved for the final compounds (–)-virolin (1), (–)-surinamensin (2), (–)-
raphidecursinol B (3) and (–)-polysphorin (4) are 70%, 75%, 67% and 65% respectively 
starting from 5 and 8. 
Thus our study resulted in efficient and concise asymmetric total synthesis of 
biologically important plant metabolites (–)-virolin, (–)-surinamensin, (–)-raphidecursinol 
B and (–)-polysphorin has been described using two common intermediates, beginning 
from readily available starting materials. Since these compounds exhibit useful biological 
properties, present strategy facilitates the synthesis of non-natural congeners of the 
natural compounds with better biological profiles. 
 
Part-B: Deals with the synthesis of various heterocyclic compounds and their 
biological evaluation, and is divided into Chapter-III and Chapter-IV. 
 
 
Synopsis 
 
 
v 
 
Chapter-III: Synthesis and Selective Cytotoxic Activity of Novel Hybrid Chalcones 
against Prostate Cancer (PC-3) Cells 
This chapter deals with the introduction to natural and synthetic bioactive 
chalcones and their reported structural modifications for the development of new drug 
candidates. It also describes our design strategy, synthetic execution and biological 
evaluation of new hybrid chalcones.  
Design and synthesis of new types of pharmacologically interesting hybrid 
chalcone analogs for drug discovery have gained much attention during recent years. 
Several structural modifications to the chalcone template, particularly replacement of 
either A or B-phenyl ring or both with heterocyclic groups have been shown to enhance 
their biological profiles. Even though dihydrofuro- and pyrano-chalcones are also 
available in nature along with their prenyl- counterparts with good biological properties, 
less attention was drawn towards these classes of compounds. In nature, the dihydrofuro 
and -pyrano fused chalcones are believed to be formed by oxidative cyclization of the 
prenyl analogs. The nature prefers six membered pyrano fused structures to five 
membered furo- fused ones as exemplified by the relatively high abundance of natural 
pyrano- fused chalcones. The less abundant furo fused chalcone derivatives have been 
reported to possess antioxidant and antimicrobial properties in addition to being chemo- 
preventive and cytotoxic.
 
Against this backdrop, we designed and synthesized a new class 
of chalcones with dihydrobenzofuran moiety as A-ring and either substituted phenyl or 
pyrazole moiety as B-ring and evaluated their cytotoxic activity (fig 1). 
O OBn
O
R2
O OBn
O
N
N
Ph
R2 2a-f
R1
BnO O
O
2g-l
N
N
Ph
R2
R1
1g-l
BnO O
O
R2
1a-f
Figure 1: Chemical sructures of new hybrid chalcones 1a-l and 2a-l
R1 R1
 
The new class of hybrid chalcones was prepared through the ethanones 8 and 10. 
Synthesis of the ethanones 8 and 10 was started from resacetophenone 4
 
which could be 
Synopsis 
 
 
vi 
 
accessed easily from resorcinol (3) (Scheme 1). Mono-allylation of 4 with 3-chloro-2-
methylpropene gave the allyl-aryl ether 5 which on Claisen rearrangement afforded 6 in 
good yield. Acid catalyzed cyclization of the rearranged product 6 followed by the 
benzylation of the resulting benzofuran 7 afforded the ethanone 8 in very good yield. 
Compound 10, the regioisomer of 8, was prepared by benzylation of the 4-hydroxy group 
of 6 followed by acid catalyzed cyclization.  
HO OH
O
O OH
O
HO OH
3-chloro-2-
methylprop-1-ene
K2CO3, NaI, acetone
reflux, 6 h, 65%
ZnCl2, AcOH
140 °C, 88%
3 4 5
Scheme 1: Synthesis of ethanones 8 and 10
HO OH
O
N, N-diethylaniline
220 °C, 4 h, 85%
6
O OH
O
O OBn
O
p-TSA, CHCl3
rt, 6 h, 90%
C6H5CH2Br
K2CO3
acetone, reflux
6 h, 94%
7 8
HO OH
O
OH
O
BnO
OBnO
O
6
7
1
23
4
5
C6H5CH2Br,K2CO3
acetone, reflux
6 h, 90%
p-TSA, toluene
reflux, 3 h, 96%
6 9 10
6
7
1
2 3
4
5
 
The target 1,3-disubstituted-2-propen-1-ones (1a-l and 2a-l) were prepared by the 
Claisen-Schmidt condensation
 
of ethanone 8 or 10 withs various benzaldehydes (11a-f) 
and pyrazolaldehydes
 
(12a-f) in ethanolic NaOH (Scheme 2). The cytotoxic potential of 
all newly synthesized hybrid chalcones was evaluated in vitro against a panel of four 
tumor cell lines prostate cancer (PC-3), colon cancer (HT-29), lung cancer (NCI-H460) 
and mouse macrophages (B-16) using MTT assay
 
based on mitochondrial reduction of 
yellow MTT tetrazolium dye to a highly colored blue formazan product.
 
Doxorubicin was 
used as the reference drug.  
The new class of compounds showed significant to moderate cytotoxic activity on 
PC-3 cell lines (IC50 = 5.9-50 μM). Among the two regioisomeric core structures 8 and 
10, the derivatives of 10 showed more significant cytotoxic activity. It is also worthy to 
note that, the derivatives with pyrazole moiety as B-ring exhibited better activity over the 
Synopsis 
 
 
vii 
 
phenyl counterparts. Derivatives 2i, 2j and 2l exhibited significantly higher cytotoxicity 
with IC50 values 8.4, 7.9 and 5.9 μM respectively and most notably showed very low or 
no activity on the other three cell lines.  
O OBn
O
BnO
O
O
O OBn
O
BnO
O
O
11, 20% NaOH, EtOH
rt, 12 h, then 1M HCl
11, 20% NaOH, EtOH
rt, 12 h, then 1M HCl
R2R1
R2R
1
11a R1 = H, R2 = H
11b R1 = H, R2 = F
11c R1 = H, R2 = Cl
11d R1 = H, R2 = Br
11e R1 = H, R2 = OMe
11f R1 = Cl, R2 = Cl
1a-f
2a-f
R1 R2
OHC
11
8
10
N
N
Ph
OHC
R2
O OBn
O
BnO
O
O
O OBn
O
BnO
O
O
12, 20% NaOH, EtOH
rt, 12 h, then 1M HCl
N
N
N
N
Ph
Ph
R2
R1
R2
R1
12, 20% NaOH, EtOH
rt, 12 h, then 1M HCl
1g-l
2g-l
R1
12a R1 = H, R2 = H
12b R1 = H, R2 = Me
12c R1 = H, R2 = Cl
12d R1 = H, R2 = OMe
12e R1 = H, R2 = NO2
12f R1 = NO2, R
2 = H
12
8
10
Scheme 2: Synthesis of hybrid chalcones  
In order to evaluate the effect of phenolic benzyl moiety, which initially was 
introduced as a protecting group to enable the synthesis, we prepared the O-debenzyl 
analogs of a few selected compounds with better activity and tested for their cytotoxic 
activity against PC-3 cell lines. The debenzylated compounds found to be less potent 
compared to the corresponding benzylated counterparts, presumably due to their 
decreased hydrophobicity. 
In conclusion, the present chapter described the synthesis and cytotoxic activity of 
a new class of hybrid-chalcones against four cell lines (PC-3, HT-29, B-16 and NCI-
H460). Most of the synthesized compounds are found to be selective towards PC-3 cell 
line. As the selectivity of the drug is an important parameter in cancer chemotherapy, the 
Synopsis 
 
 
viii 
 
newly synthesized compounds particularly 2i, 2j and 2l (IC50 = 8.4, 7.9 and 5.9 μM) 
could be potential anticancer drug candidates after further structure optimization. 
Chapter-IV: Synthesis and Biological Evaluation of New Rhodanine-3-acetamide 
Derivatives 
 This chapter deals with the introduction to biological importance of pyrazole 
amines with trifluoromethyl substitution, rhodanine-3-aceticetamide derivatives and 
describes the design, synthesis and biological (Cytotoxic & Larvicidal) evaluation of new 
pyrazolyl rhodanine acetamide derivatives. 
Synthesis and bioevaluation of rhodanine and rhodanine-3-acetic acid derivatives 
were studied extensively by many research groups. Very few reports on the synthesis and 
biological utility of rhodanine-3-acetamide derivatives are documented in the literature, 
in spite of their attractive biological profiles. Combination of two active pharmacophores 
within a single molecule is one of the strategies more often employed in the search for the 
lead molecule. The resulted new molecule would enhance the activity by an additive or 
synergistic effect. Inspired by this and the biological profiles of both 4-amino-3-trifluoro-
pyrazoles and rhodanine-3-acetic acid derivatives reported in the literature, we reasoned 
that incorporation of the structural characteristics of both these moieties to result a new 
rhodanine-3-acetamide derivatives with newer and/or better biological profiles. In the 
present study the design and synthesis of new rhodanine-3-acetamide derivatives and 
their biological evaluation have been carried out. 
O
CF3CO2Et, NaOMe
Et2O, 0 °C-rt
O
CF3
O
aq.NaNO2, AcOH
-5 °C-rt
O
CF3
O
N
HO
1 2 3
Scheme 1: Synthesis of 4-amino-1,5-diphenyl-3-trifluoromethyl-pyrazole (5).
N N
CF3
NO
Ph
PhNHNH2
dil. HCl, 0 °C-rt
N N
CF3
NH2
Ph
5% Pd-C/H2
EtOH, 0 °C-rt
4 5
 
Synopsis 
 
 
ix 
 
Synthesis of 1,5-diphenyl-3-(trifluoromethyl)-1H-pyrazol-4-amine (5) was started 
from 1,3-diketone (2) (Scheme 1), which in turn  was prepared from acetophenone (1) 
and ethyl trifluoromethyl acetate in presence of sodium methoxide in dry ether. 
Treatment of 1,3-diketone (2) with aqueous NaNO2 in acetic acid gave the diketo oxime 
(3) in good yield. Reaction of diketo oxime (3) with phenylhydrazine in dilute HCl 
afforded the nitroso-pyrazole (4) as a green solid. Reduction of the nitroso group by 
hydrogenation in presence of 10% Pd-C gave the aminopyrazole (5).  
Scheme 2: Synthesis of pyrazolyl rhodanine acetamide derivatives (9a-m)
N N
CF3
NH2
Ph
N
S
O
S
Cl
O Et3N, Et2O
0 °C - rt, 4h
N
N
Ph
CF3
N
H
Ph
N
S
O
S
O
+
5 6 7
piperidine, EtOH, rt
N
N
Ph
CF3
N
H
Ph
N
S
O
S
O
R-CHO (8a-m)
9a-m
R R = aryl,
hetero aryl
 
Treatment of the 4-aminopyrazole (5) with rhodanine-3-acetylchloride (6) gave 
the corresponding amide (7) in good yield. The acid chloride (6) was prepared from the 
corresponding rhodanine-3-acetic acid which in turn was prepared from glycine, carbon 
disulphide and sodium salt of chloroacetic acid using a reported procedure. Treatment of 
rhodanine-3-acetamide (7) with different aldehydes (8a-m) in presence of catalytic 
amount of piperidine in ethanol at room temperature gave the methylidine derivatives 
(9a-m) in good yields (Scheme 2).  
The cytotoxic potential of all the newly synthesized rhodanine-3-acetamide 
conjugates were evaluated in vitro against prostate (PC-3 & DU-145) and breast (MCF-7 
& MDA-MB-231) cancer cell lines using MTT assay
 
based on mitochondrial reduction of 
yellow MTT tetrazolium dye to a highly colored blue formazan product.
 
Doxorubicin was 
used as the reference drug. Most compounds, the heterocyclic conjugates in particular, 
displayed cytotoxicity against prostate cancer cell lines. The parent pyrazole amine 7 
exhibited appreciable activity against breast cancer cell lines. Compound 9e is potent 
against prostate cancer cell lines. The pyrazole conjugate 9k showed substantial 
Synopsis 
 
 
x 
 
cytotoxicity towards all four cell lines. The derivatives 9e and 9k are the most promising 
with IC50 values 12.9 & 12.5 μM against PC-3 cell line.  
The larval toxicity study of all newly synthesized compounds was performed 
using standard protocol as described by World Health Organization (WHO, 1963), on 4
th
 
instar mosquito, C. quinquefasciatus larvae. The LC50 values of newly synthesized 
compounds (5, 7, 9a-m) were determined. Among these, only compound 9i showed 
toxicity against C. quinquefasciatus (LC50 = 8.42 ppm, 24 h). All other derivatives 
including 5 and 7 showed very low / no toxicity after 24 h and 48 h.  
In summary a new class of rhodanine-3-acetamide conjugates were synthesized 
and evaluated for their larvicidal and cytotoxic properties. Compound 9i showed toxicity 
against 4
th
 instar mosquito, C. quinquefasciatus larvae. The cytotoxic activity of these 
new derivatives were tested against prostate (PC-3 & DU-145) and breast (MCF-7 & 
MDA-MB-231) cancer cell lines. The derivatives 9e and 9k are the most promising 
compounds with IC50 values 12.9 & 12.5 μM against PC-3 cell lines.   
 


STUDIES ON CELLULASES

SRIVASTAVA, ANJALI

A STUDY ON THE APPLICATION OF THE LIQUID MIX TECHNIQUE AS A PREPARATIVE METHOD FOR SOME USEFUL ELECTROCERAMICS

OTIV, S. R.

Abstract

Some Aspects of Optical Devices and Protocols in Scientific Parallel Computing

Chapter 1:  This chapter highlights the need of scientific parallel computing in order to solve
complex scientific problems like Genome sequencing, design of atom bomb, re-entry
trajectories of space vehicles etc. that demand large scale computations. Many scientific
programs are also listed that were initiated to develop such large infrastructure. This led to
the evolution of various architectures for communication technologies as a result today the
computation speeds of scientific parallel computers are observed to have surpassed the peta
FLOPS mark. The chapter makes a significant mark on the Indian contributions to the
development of communication technologies in scientific parallel computing especially the
Floswitch at the Flosolver lab of National Aerospace Laboratories, Bangalore. The chapter
concludes with the mention on emerging challenges, new paradigms and possibilities in the
development of the Floswitch.
Chapter 2: The chapter spans the development of different versions of Parallel computers
at Flosolver and the evolution of Floswitch with its significance to various
computationally challenging problems like TSP (Transonic Small Perturbation) equations
which is non-linear partial differential equations, the solution to Navier-stokes equation
etc. whose importance in CFD (Computational Fluid Dynamics) is difficult to overstate.
The salient features of the Floswitch are also listed and the chapter concludes by stating
the scope of the thesis and the problem definition.
Chapter 3: The chapter focuses on the scalability issues of Flosolver series of parallel
computers and discusses the role of optical communication in its realisation. Existing
state of technological development in the Flosolver programme is also briefly described.
The chapter concludes by stating the scope of the work for the present thesis that is
outlined and explained in the subsequent chapters.
Chapter 4: The chapter sketches the next stage of logical development which forms the
core of the present investigation. The traditional approach of using off the shelf
processor for Floswitch characterised in the earlier series of Floswitch is discarded and
the fresh approach is suggested which turns out to be efficient by an order of magnitude.
A brief discussion is presented for such a qualitative change.
Chapter 5: The chapter presents the detailed design methodology for the new improved
architecture of the Floswitch and its stepwise implementation techniques. It describes
the data flow mechanism in the new architecture and presents the analysis supported by
the waveforms. It concludes by stating that in order to put the achievement in a broader
perspective, there is a need for analysis of communication times of various standard
communication devices for large scale computations.
Chapter 6: The chapter presents an analysis of a legacy code VARSHA that
involves large scale computations, on a parallel computing environment using
communication devices like Ethernet, InfiniBand and the Floswitch. The graph
obtained suggests that the performance of the new improved architecture of
Floswitch competes with that of the InfiniBand which is a significant achievement.
Chapter72: The chapter details the results obtained and the conclusions drawn based on the
comparative analysis of the performance statistics of the Floswitch along with the other
standard available communication devices, Ethernet and InfiniBand. The research work is
the significant contribution in the improvement in existing technology that forms the
fundamental block of future Large Scale Computing systems




STUDIES IN SYNTHETIC DYES:NEW CHROMOPHORIC SYSTEMS

SHUKLA, P.G

Annexure I 
 
Abstract/Synopsis 
 Reptiles are ectothermic amniotes, providing the key link between 
anamniotic ectotherms, fishes and amphibians, and amniotic endotherms, birds and 
mammals. A greater understanding of reptilian immunity will provide important insights 
into the evolutionary history of vertebrate immunity. Hence, this study was undertaken to 
examine the effect of melatonin and testosterone hormones and photoperiodic modulation 
on immune responses of splenic macrophages. Also, seasonal as well as circadian 
variation in immune responses of splenic macrophages in the fresh-water snake was 
studied. Following immune parameters were taken into consideration: 
 Spleen mass and cellularity 
 Splenic macrophage Phagocytosis 
 Quantitative NBT reduction   
 Nitrite release  
 Splenic lymphocyte proliferation 
 
 To study the effect of melatonin hormone, melatonin injections in saline vehicle 
(dose: 5 µg /g body weight and 10 µg /g body weight) were given during evening hours, 
and alteration in lymphoid organ mass, splenic macrophage phagocytosis, Nitric oxide 
(NO) production (Nitrite assay), superoxide production (NBT reduction assay) and 
lymphoproliferation (MTT assay) were studied on the day following 10
th
 and 20
th
 
injection. No consistent and significant change was observed in phagocytic response of 
splenic macrophages harvested from snakes treated with either dose of melatonin for 
different duration. Nitrite release and superoxide production by splenic macrophages 
were significantly (p<0.05) higher in snakes receiving melatonin injections. Exogenous 
melatonin enhanced the mitogen-induced splenic lymphocyte proliferation only at the 
dose 10 µg/ g body weight, but not at that of 5 µg one.  In vitro melatonin also invariably 
has enhanced the mitogen-induced splenic lymphocyte proliferation, and vigorous 
response has been observed in splenocytes harvested from snakes receiving 10 µg /g 
body weight melatonin injection for 20 days, when splenocytes were induced by 
mitogens: 10 µg mL
-1
 of Con A and PHA and by 20 µg mL
-1
 of LPS. Taken together, the 
results of the present study demonstrate that in vivo melatonin treatment enhances innate 
immunity, in general except phagocytosis, in the fresh-water snake, N. piscator.  
 
To study seasonal variation in immune functions, the requisite number of animals 
were sacrificed under mild anaesthesia during mid of each month.  In all assay, sample 
size was 5 to 6. Analysis of variance reveals that there was no significant variation in 
splenic mass of the fresh-water snake during different months of study period, though 
spleen size has a trend to be high in autumn and winter months and low in spring and 
summer. Spleen cellularity was recorded high in winter months and again in September; 
while it remained low during rest of the year. No definite pattern was observed in 
phagocytosis by splenic macrophages. The percent phagocytosis varied between 42 to 60 
%, being highest in month of February. Analysis of variance revealed that free radical 
production and lymphoproliferation varied significantly during different months. Super 
oxide production, as judged by NBT reduction, was found to be high during spring 
season. Nitrite release by splenic macrophages was high in months of January, May and 
September - October. The splenic lymphocyte proliferation, both basal as well as Con A 
induced, was highest in month of May, followed by a little low in January and October: it 
remained low during rest of the year. 
 
 In the study of circadian variation, Cosinor analysis of the data revealed that no 
significant circadian rhythm was validated in fluctuation of splenosomatic index and 
spleen cellularity in all the three seasons. The percentage phagocytosis had significant 
rhythm of 24 h in summer and spring seasons, but not in winter; and no significant 
circadian rhythm was validated in phagocytic index. NBT reduction and nitrite release by  
splenic macrophages of fresh-water snake also varied significantly in circadian manner 
during winter season. But, significant circadian rhythm was absent in NBT reduction and 
nitrite release during summer season. A significant circadian rhythm was observed in 
splenocyte proliferation (Basal) and splenocyte proliferation (Con A10 stimulated) in all 
three seasons: summer, winter and spring. A significant phase shift in splenocyte 
proliferation (Con A10 stimulated) was obtained in all three seasons with a trend of 
delayed phase shift from winter to spring and spring to summer.  
  Most formal studies on seasonality have focused on day lengths i.e., photoperiod, 
as the environmental cue used by animals to coordinate intrinsic rhythms with extrinsic 
seasonal environmental changes. To study the effect of photoperiodic manipulation on 
immune parameters, snakes were maintained in 24h dark (DD), 24h light (LL) and at 
normal day length (10L:14D). Spleen mass was increased, but insignificantly, in the 
animals subjected to DD. No effect was observed in the animals kept under continuous 
light, i.e. LL, when compared with control ones. On other hand, spleen cellularity was 
also increased, but insignificantly, in the animals of both groups kept in DD as well as 
LL. No significant (p<0.05) change was observed in percentage phagocytosis and 
phagocytic index in animals kept under DD or LL, as compared with animals kept in 
10L: 14D. Super oxide production, as measured by NBT reduction assay, was reduced 
significantly (p<0.05) in the animals kept either in 24D or 24L. Production of nitric oxide 
by snakes’ splenic macrophages, as measured by nitrite assay, was significantly (p<0.05) 
decreased in animals kept under LL; while no change in nitrite release was observed in 
animals kept in DD, when compared with animals kept in 10L:4D. There was significant 
(p<0.05) enhancement of mitogen induced splenocytes proliferation in cultures obtained 
from animals kept under DD. To study whether melatonin could induce the splenocytes 
proliferation, splenocytes cultures obtained from all group animals were treated with in 
vitro melatonin (500 pg ml
−1
, final concentration). In vitro melatonin significantly 
(p<0.05) enhanced the splenocyte proliferation of the cultures obtained from animals kept 
in LL, as compared with cultures from 10L:14D animals, but not in those of DD animals.  
    
To study the effect of in vitro testosterone, splenic macrophages monolayer 
prepared on slides was exposed to different concentrations male sex hormone, (0.1, 1, 10, 
50 and 100 ng ml
–1
). After 4 hour of incubation, the monolayers were washed three times 
to remove testosterone, and phagocytosis was allowed to proceed. Effect of different 
concentrations of in vitro testosterone was also studied on NBT reduction, nitrite release 
and splenic lymphocyte proliferation (basal as well as mitogen induced). It was observed 
that percentage phagocytosis decreased significantly in correlation with the in vitro 
testosterone concentration. Maximum reduction of percentage phagocytosis was obtained 
at 100 ng ml
–1
 concentration of testosterone. Effect of in vitro testosterone on NBT 
reduction was found to be differential: as NBT reduction was decreased at 10 ng ml
–1
 
concentrations, but no decrease at 100ng ml
–1
, and again decreased at 1000 ng ml
–1
. The 
decrease was maximum at 1000 ng ml
−1
 testosterone concentration. Nitrite release was 
also significantly (p<0.05) reduced by in vitro testosterone treatment in a concentration 
dependent manner. When the macrophages were pre-incubated with testosterone receptor 
antagonist, Cyproterone Acetate (CPA), the decrease in Nitrite release was alleviated, as 
Nitrite release was comparable to that of control splenocytes incubated in medium alone. 
There was no change in basal proliferation of splenic lymphocytes in relation to in vitro 
testosterone treatment; while mitogen induced proliferative response reduced 
significantly (p<0.05) by in vitro testosterone. Present finding reveals testosterone as 
inhibitory to splenocytes immune responses in the fresh-water snake, Natrix piscator.     
 
Based on the results in the present work, it may be suggested that immune function in 
reptiles varies in different seasons. Pineal gland, by diurnal rhythm of synthesis and 
release of its principal hormone, melatonin, transmits signals to the immune system, and 
thus, regulates the immune responses. Melatonin injection enhanced the innate immune 
functions, and thus may be considered as an immunoregulatory factor in the development 
and maturation of immune system and in the progression of the immune response in our 
animal model, Natrix piscator. Our study also suggests there is circadian variation in 
innate immune parameters, some of which also show a clear phase shift according to the 
season. Further, in vitro testosterone reduces immune response in a manner dependent to 
testosterone concentration used. Differential effect of photoperiodic regimens, in immune 
parameters studied, is also established in our study. It is concluded that relationship exists 
between external environmental factors, melatonin, sex steroids and immune system 
which might have led to the adaptations to best fit the animal in the challenging 
conditions present. 
 
 
 
 


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MASS SPECTROMETRY OF SOME MODEL AROMATIC AND ANTIAROMATIC SYSTEMS

MASS SPECTROMETRY OF SOME MODEL AROMATIC AND ANTIAROMATIC SYSTEMS

EXPANSION COEFFICIENTS IN POLYELECTROLYTES

SYNTHESIS AND CHARACTERISATION OF CATALYTIC ALLY IMPORTANT IRON OXIDE SYSTEMS

ANALYTICAL METHODS IN ORGANIC CHEMISTRY

A STOCHASTIC APPROACH TO THE ANALYSIS OF CHEMICALLY REACTING SYSTEMS

STUDIES ON CELLULASES

A STUDY ON THE APPLICATION OF THE LIQUID MIX TECHNIQUE AS A PREPARATIVE METHOD FOR SOME USEFUL ELECTROCERAMICS

STUDIES IN SYNTHETIC DYES:NEW CHROMOPHORIC SYSTEMS

STUDIES IN ENCAPSULATION:ENCAPSULATION OF CARBOFURAN USING STRACH MATRIX

506

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