Research Methodology
RESEARCH MYETHODOLOGY:
3.1 General Remarks:

Most of the reactions were carried out in oven dried glassware with magnetic stirrer
using dry, freshly distilled solvents.

Reactions were monitored on aluminum coated thin-layer chromatography (TLC)
with UV light (256 nm), iodine, vanillin-sulphuric acid as developing agents.

Separation of the compounds were by column chromatography was carried out with
Silica gel (60-120 mesh ASTM, E.Merck).

Melting points (uncorrected) were determined on Buchi 535 melting point apparatus.

Proton magnetic resonance spectra were recorded on Bruker 400 MHz spectrometers
using tetramethylsilane (TMS) as the internal standard.

Chemical shifts have been expressed in δ values downfield from TMS. Multiplicity of
NMR signals is designated as s (singlet), d (doublet), dd (double doublet), t (triplet), q
(quartet), m (multiplet).

Infrared spectra were recorded on Agilent Technologies FT-IR spectrophotometer.

All solvents and reagents were purified and dried according to procedure given in
Vogel‟s Textbook of Practical Organic Chemistry.
3.1.1. Chemicals Used:
Table No. 3.1.1. List of Chemicals
S.No.
Name of Chemical
Name of Company
1
Absolute alcohol
S.D. fine Chemical
2
Potassium hydroxide
Qualigens
3
Iodine crystals
Merck-Schuchard
4
n-Hexane
Sigma Aldrich
5
Ethylacetate
Sigma Aldrich
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6
Hydrochloric acid
Sigma Aldrich
7
Dimethyl sulphoxide (DMSO)
Sigma Aldrich
8
4-Fluoro benzaldehyde
Sigma Aldrich
9
4-Chloro benzaldehyde
Sigma Aldrich
10
4-Bromo benzaldehyde
Sigma Aldrich
11
2-Hydroxy propiophenone
Merck-Schuchard
12
Benzaldehyde
Merck-Schuchard
13
2-Hydroxy benzaldehyde
Sigma Aldrich
14
4-Methyl benzaldehyde
Merck
15
4-Hydroxy benzaldehyde
Sigma Aldrich
16
2-Bromo benzaldehyde
Sigma Aldrich
17
4-Methoxy benzaldehyde
S.D. fine Chemical
18
3-Methyl benzaldehyde
Sigma Aldrich
19
3-Methoxy benzaldehyde
Sigma Aldrich
20
2-Fluoro benzaldehyde
Sigma Aldrich
21
2-Methyl benzaldehyde
Sigma Aldrich
22
2-methoxy benzaldehyde
Sigma Aldrich
23
Muller Hinton agar media
Hi media laboratories
24
4-Hydroxyacetophenones
Sigma Aldrich
25
4- Methyl benzaldehyde
Sigma Aldrich
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26
4-Bromo benzaldehyde
Sigma Aldrich
27
4-Flouro benzaldehyde
Sigma Aldrich
28
4-Chloro benzaldehyde
Sigma Aldrich
29
4-Methoxy benzaldehyde
Sigma Aldrich
30
2-Hydroxy benzaldehyde
Sigma Aldrich
31
4-Hydroxy benzaldehyde
Sigma Aldrich
32
Benzaldehyde
Sigma Aldrich
33
2-Methyl benzaldehyde
Sigma Aldrich
34
2‟-Hydroxy-4‟-methoxy propiophenone
Sigma Aldrich
35
2‟-Hydroxy-4‟-nitroacetophenone
Sigma Aldrich
36
2‟-Hydroxy-5‟-nitroacetophenone
Sigma Aldrich
3.1.2. Instruments and Software Used:
Table No. 3.1.2. Instruments and Software
Data
Instrument/Software
Melting Point
Shital Scientific industries
UV chamber
Serve-well instruments Pvt Ltd
UV- visible spectrometer
Shimadzu UV-Vis Spectrophotometer UV-1650 PC
FT-IR
Agilent FTIR 8310
GC-MS
Shimadzu GCMS QP5050
Structure Builder
ACD Chem.-Sketch 8.0
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3.2. Experimental
3.2.1. Scheme I:
General procedure for the synthesis of novel dihydro methyl chalcones:
To a solution of 0.01 mole of substituted 2‟-hydroxypropiophenones in 10 ml of 40%
KOH and 20 ml of ethyl alcohol, 0.01mole of substituted benzaldehyde was added and
mixture was stirred for 48-72 hours. The coloured solution was poured into crushed ice
and acidified with 1N HCl at 24-260C.The precipitate so obtained was washed with cold
water, filtered, dried and recrystallized with absolute alcohol .Fifteen substituted
synthesized compounds are enlisted in Table No: 4.1.1. The partition coefficient and
other important physical data are enlisted in Table No:4.1.2 and 4.1.3 respectively.
HO
R1
O
H C
R
H
R2
O
R3
H
CH3
Substituted Benzaldehydes
40% Ethanolic KOH
Room temperature 24-260C.
24-72 hrs,
2-Hydroxy propiophenones
OH
R
O
H
HO
H
CH3
R1
R2
R3
Fig No: 3.2.1.a (1, 3- biphenyl-3-hydroxy-2-methyl propenone analogues.)
Table No: 3.2.1.a Different Substitution
R=
OCH3,
R1 =
Cl, CH3, OCH3
R2 =
OH, OCH3, CH3,Cl, F
R3=
OCH3,,Cl, F, CH3
R4=
OH, Cl, F, OCH3, CH3
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In an attempt to modify the classical Claisen-Schmidt synthesis for chalcones which
involves aldol condensation followed by dehydration. A strategy was envisaged after
proper study based on Prentic Hall Frame Work Molecular Model that instead of
acetophenone, if propiophenones are taken the OH (Hydroxy group) generated from –
CHO of aromatic aldehyde would prefer to be trans to the methyl with the results that no
vicinal trans hydrogen atom will be available for the elimination of a water molecule.
There is restriction of free rotation ( freedom) across.
for vicinal hydrogen & OH to take trans position.
O
H
bond to make it difficult
C C
CH3
Specification:
Prop-2-en-1-one analogues are synthesized by base catalysed Claisen-Schmidt
condensation of substituted 2‟- Hydroxy propiophenone and substituted benzaldehyde to
be generally followed by dehydration is one port reaction. But in the present case no
dehydration took place as predicted to yield only aldol condensation product.
This method is unique and its novelty can be explained by following points:
1. Generally dehydration is acid catalyzed process but these are the only reactions which
undergo base catalyzed dehydration by the activation of α methylene, α to carbonyl
group.
2. The series of products should have followed Claisen-Schimdt type of condensation,
but the molecule appeared to be fairly polar and it could have been due to nondehydration of hydrogen and hydroxyl atoms. This phenomenon appears to be novel.
3. To support our speculation, the series of 15 substituted products were subjected to
both acid and base catalysed dehydration and the results showed that the series of
products are of the type where dehydration is not possible inspite of the fact the
hydroxyl being allylic and the only reason one could conclude that the proton on the
next carbon (geminal to OH) has cis configuration
4. This speculation was predicted and confirmed by the study of Prentic Hall Frame
Work Molecular Model where it was clear that there is no free rotation across three
propane moieties and there is restricted rotation across these three C-C bonds and the
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hydroxyl group (-OH) and methyl (CH3) lies trans to each other. With the result the
OH on carbon remains in cis configuration to adjacent H (geminal to CH3), as
expected no dehydration is possible.
5. Prentic Hall Molecular Model:
Fig No: 3.2.1.b General Stereochemistry of 1-(2‟hydroxy)-1,3-diphenyl propan-1-one
6. Prentic Hall Molecular Model:
These type of model are prepared because, such model take care of van der waals
forces to restrict free rotation accros c-c bonds.
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Stereochemistry of the synthesized compounds:
B
Fig No: 3.1.2.c Steriochemistry of 3- methyl dihydrochalcone
In a base catalyzed reaction between two reactive prochiral molecules, the one like B will
attack as carboanion ion source from the side where the smallest atoms or molecules (
Here H) is a substituent to a carbanion prochiral molecule like A, with the result that
product (C) will be formed as an enantiomeric excess (EE) while in the present case ,it
is diastereoenantiomeric (DE) excess , since two chiral centers are simultaneously
formed , absolutely based on cohn‟s principle of asymmetric synthesis. The molecule
like C thus formed support the speculation as no dehydration takes place in the present
study which otherwise typical of Claisen –Schmidt reaction beside only one major
product between (70-85 % ) was formed . The reason for not having a dehydrated
product is supported by the study of molecular model Prentic Hall Molecular Model
bases on Cohn„s principle. The product C has methyl and hydroxyl group at trans position while the vicinal proton is to methyl, thus acid or base catalysed attempted
dehydration failed to take place. This appears to be first of its kind of reaction.
Spesific Rotation: The compounds (KLD-2 and KLD-4) have shown specific rotation
[α] D = +22.40 and +34.30 (c,100 µg/ml, Chloroform),at 200 C hence proven that all the
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compounds synthesized in this series are opticaly active. Each compound has four
enantiomers with two chiral centres. The wavelength of the light used is 589 nanometre
(the sodium D line), the symbol “D” is used at 200C.
3.4.2. Scheme II: General procedure for the synthesis of trans Chalcones:
To a solution of 0.01 mole of 4‟-hydroxy acetophenone in 10ml of 40% KOH and 20ml
of ethyl alcohol, 0.01mole of substituted benzaldehyde was added and mixture was
stirred or refluxed for 24-72 hours. Completion of the reaction was monitored on TLC
(20% Ethyl acetate in toluene). The coloured solution was poured into crushed ice and
acidified with 1N HCl at 24-26oC .The precipitate so obtained was washed with cold
water, filtered, dried and recrystallized with absolute alcohol. Fifteen substituted
synthesized compounds are enlisted in Table No: 4.1.4. The partition coefficient and
other important physical data are enlisted in Table No: 4.1.5 and 4.1.6 respectively.
R1
O
HO
R2
O
CH3
H C
R3
Substituted benzaldeyde
4'-hyrdoxacetophenones
HCl
40% KOH
Ethanol
24-72 hrs Reflux/stirring
OH
O
HO
R1
H
R2
R3
H
Stirring
RT
O
H
Reflux
O
R1
H R1
R2
HO
H
R3
HO
R2
H
R3
Substituted Chalcones
Substituted Chalcones
Fig No: 3.2.2. General Reaction Scheme.-1
R1= OCH3, H , Br,
R2= CH3,Br Cl, F, CN, H
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Specification:
On the basis of different reaction conditions and spectroscopic data, it has been found
that the compounds synthesized were categorized into two groups. The suggested
compounds were trans at room temperature, whereas some compounds were only
synthesized after heating under reflux. Under these conditions the α hydrogen to the
carbonyl group is under magnetic an isotropic environment and is therefore extra
ordinary downfield. The 1HNMR data shows that different configuration of these
compounds and chemical shift of α and β hydrogen atoms are very close to each other
which is un usual, but could be a special case of protons which are chemically different
but magnetically very close.
3.2.3. Scheme III:
3.2.3.1. Synthesis of bioactive chalcones and their dihydro pyrazole analogues
A) Synthesis of Chalcones
To a solution of 0.01 mole of Substituted acetophenone in 10ml of 40% KOH and 20ml
of ethyl alcohol, 0.01mole of substituted benzaldehyde was added and mixture was
stirred or refluxed for 24-72 hours. Completion of the reaction was monitored on TLC
(20% Ethyl acetate in toluene). The coloured solution was poured into crushed ice and
acidified with 1N HCl .The precipitate so obtained was washed with cold water, filtered,
dried and recrystallized with absolute alcohol. Fifteen substituted synthesized compounds
are enlisted in Table No: 4.1.11. The partition coefficient and other important physical
data are enlisted in Table No: 4.1.7 and 4.1.8 respectively.
OH
O
H
CH2
H
R
O
C
OH
R2
O
H
R2
R3
R3 Stirring,
40% KOH
+
R4 24-72 Hrs
H
R
R1
R5
Substituted 2'-Hydroxyacetophenone
Substituted Benzaldehyde
R4
R1
trans chalcones
Fig No: 3.2.3.1 General Reaction Scheme-2
R = NO2, Cl, F
R3 = OCH3, Methylene dioxide
R1 =NO2
R4 = F, Cl, CH3, OH, Br, Methylene dioxide
R2 = OCH3, Br.
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All the test compounds were synthesized based on classical Claisen-Schmidt reaction and
further under gone reactions with hydrazine hydrate to produce various dihydro pyrazole
analogues.
3.2.3.2. General procedure for synthesis of dihydro pyrazole chalcone analogues:
A mixture of chalcones (0.01mol) and hydrazine hydrate (0.02 mol) in glacial acetic acid
(20-25 ml) was refluxed for 4-8 hrs. Completion of reaction was monitored on TLC (20%
Ethyl acetate in toluene). The coloured solution was poured into crushed ice and acidified
with 1N HCl .The precipitate so obtained was washed with cold water, filtered, dried and
recrystallized with absolute alcohol . Fifteen (15) substituted synthesized compounds are
enlisted in Table No: 4.1.11. The partition coefficient y and other important physical data
are enlisted in Table No: 4.1.10 and 4.1.9 respectively.
O
C CH3
O
H
R2
R3
+
H
R
N
N
R4
R2
Reflux 4-8 Hrs
C
H2
NH2.NH2.H2O
Glacial Acetic
R
Acid
R1
R1
R3
R4
Fig No: 3.2.3.2. Dihydro pyrazole Chalcone Analogues
R = NO2, Cl, F ,
R3 = OCH3, Methylene dioxide
R1 = NO2 R2 = OCH3,Br ,
R4 = F, Cl, CH3, OH, Br, Methylene dioxide,
R2, = OCH3, Br,
3.2.4. General Scheme:
The chalcones are generally synthesized by Claisen-Schmidt condensation having
aldehyde with no α hydrogen atom and ketone should be having at least one α hydrogen
atom .The chalcones though strongly hydrogen bonded are still having free rotation to
yield number of conformers with the result that the activity is reduced. To make it rigid
and introducing basic character can help in making salts which are supported to be
soluble in water.
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General Syntheic Scheme :
OH
O
R3
R4
O
R1
H C
CH2
R5
H
R2
40% KOH
Substituted Benzaldehyde
Ethenol
24-72 hrs Refluxe /stirring at RT
Substituted Acetophenone
OH
OH
O
R1
R3
R4
R5
H
H
R2
reflux for
24-72 hrs
OH
Stirring at
RT
OH
H
R3
O
R4
H R3
R1
R4
H
R1
R2
R5
H
Glacial Acetic Acid
NH2NH2 H2O
trans-3',4'-substituted prop-2-en-1-one analogues
Under similar conditions
R1 = OH, NO2, Cl, F
O
OH
N
N
R5
R2
trans-3',4'-substituted prop-2-en-1-one analogues
R2 = NO2, Cl, H. F
C CH3
R3
R4
R1
O
R5
R3 = Br. OCH3, CH3, etc
R4 = Br. , CH3, Cl, H. F etc
R5 = CH3, Cl, H. F NO2, Methylene
dioxede.etc
R2
Dihydro pyrazol .analogues
Fig. No: 3.2.4. General Scheme
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Chalcones are synthesized by Claisen-Schmidt condensation of aldehyde and ketone by
base catalysed, acid catalysed or free radical mechanism followed by dehydration to yield
chalcones.
3.2.5. Mechanism of the base catalysed synthesis:
Step 1: Generation of carbanion
O
O
+ OH
CH2
H
O
CH2
CH2 + H2O
R'
R'
R'
Step-2: Attack of aldehyde on carbanion:
O
_
O
O
H
CH2
+
R'
R
H
O
NaOH
H
R'
R
_
OH
OH
O
H
O
-H2O
R
H
H
RT
R'
R;
trans -Chalcones
Reflux
-H2O
O
H
R
H
R'
trans-chalcones
Fig No: 3.2.5. Reaction Mechanism-1
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3.2.6. Mechanism of acid catalysed synthesis:
Step: 1
OH
O
+
CH2
+
OH
CH3
H
CH
H
H
H
Acetophenone
OH
CH2
H+
+
Step: 2
OH
O
H
H
H+
+
OH
H
Benzaldehyde
Step: 3
H
OH
OH
CH2
O
H
OH
H
+
ketone
Aldehyde
O
H
O
H
+
H
H
OH2
+
- H2O
+
H
+
H
OH
H+
O
H
H
Chalcone
Fig No: 3.2.6. Reaction Mechanism-2
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3.4.7. Mechanism of Reaction of chalcone with Hydrazine hydrate:
-
O
O
H
H
C
H
C
C
H
R'
R'
R
R
:NH2NH2.H2O
NH2
C
C
O
NH2
NH
C
C
O H
CH
H
CH
-
R'
R
R'
R
NH
-H2O
CH
+
H
CH
H
R
NH
N
NH
N
+
CH3CO
C
H2
R'
R
R
+
CH3CO
O
C
N
CH3
N
C
H2
R
R'
Dihydro pyrazole analogues.
Fig No: 3.2.7.
Reaction Mechanism-3
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3.3. EXPERIMENTAL BIOLOGICAL ACTIVITY.
The experimental parts of the biological activities include
3.3.1 Antioxidant screening by DPPH method and ABTS methods.
3.3.2. In vitro anticancer study by MTT assay against different cell lines
3.3.3..Antimicrobial activity against six bacterial and two fungal strains
The experimental parts of these activities have already been discussed in natural section
(Part -1) of this thesis.
3.3.4. Partition coefficient:
Hydrophobicity is an important physicochemical parameter used in QSAR generating
much interest, excitement and controversy over the last 40 years. Hydrophobic
interactions are of crucial importance in many areas of chemistry. These include enzymeligand interactions, the assembly of lipids in bio membranes, aggregation of surfactants,
coagulation and detergency.
In the early 1960‟s, Hansch and Fujita formulated a linear free energy related approach
(Extra thermodynamic approach) known as Hansch analysis where, the biological activity
is considered as a function of different physicochemical properties in a linear additive
manner.
Log 1/C = a Log P + b, whereas, Log 1/C = Log of inverse molar dose that produces the
biological response.
Log p = Log of partition coefficient.
Hydrophobicity is defined as the partitioning of a compound between an aqueous and
non-aqueous phase. It can be readily determined by measuring the partition coefficient
designated as „P‟. Partition coefficient is defined as the ratio of concentration of solute in
n-Octanol to its concentration in water (aqueous phase) (Taylor, 2005).
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P = [conc.] Octanol/ [conc.] aqueous
Methods to determine partition coefficient:
1. Classical shake flask method.
2. Counter current method (AKUFVE device).
3. Partition chromatographic methods ( TLC, HPLC ).
4. CLOGP software.
In the present study, shake flask method is employed using n-octanol and water system.
Shake flask method:
Principle
In order to determine partition coefficient, equilibrium between all interacting
components of the system must be achieved, and the concentration of the substances
dissolved in two phases must be determined. A study of the literature on this subject
indicates that several different techniques can be used to solve this problem namely, the
thorough mixing of the two phases followed by their separation in order to determine the
equilibrium concentration for the substance being examined.
Experimental
Requirements: Conical flasks, Test tubes, separating funnel, n-Octanol AR grade,
Cuvettes and UV Spectrophotometer.
Working Procedure
1. Preparation of Solvents.
n-Octanol: Partition coefficient experiments are performed by using high purity analytical
grade n-octanol. If any inorganic contaminants are present, they can be removed from
commercial n-octanol by washing with acid and base, drying, and distilling. More
sophisticated methods can be used to separate n-octanol from organic contaminants with
similar vapour pressure if they are present.
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Water distilled or double distilled in glass or quartz apparatus should be employed. For
ionisable compounds, buffer solutions in place of water should be used if justified. Water
taken directly from an ion exchanger should not be used.
2. Pre-saturation of Solvents.
Before partition coefficient was determined, the phases of the solvent system were
mutually saturated by shaking at room temperature. Two large stock bottles of high purity
analytical grade n-octanol or water was shaken with sufficient quantity of other for 24
hours on a mechanical shaker and allowed to stand enough to achieve a saturation state
and proper separation of phase.
3. Preparation of Stock solutions and dilutions.
10 mg of each of the test compounds was taken in 10 ml of A.R grade DMSO (stock
solution) and diluted with pre-saturated n-octanol A.R grade further, to get a
concentration of 10µg/ml (0.5 ml of stock solution diluted to 50ml with n-octanol presaturated).
The λ
max
for each of the test compounds in n-octanol was determined and the absorbance
at that wavelength was noted (B -Absorbance before extraction). 20 ml of the drug
E
solution (10µg/ml) in n-octanol and 20 ml of water were added to 250 ml conical flask,
covered with parafilm and shaken in the mechanical shaker for 24 hours to create an
endogenous environment. The phases were then kept without any disturbances for proper
separation. The absorbance of the separated n-octanol phase was then measured at the
λ
max
specific for that compound. (A - -Absorbance after extraction).
E
P = Corg / Caq = BE / BE-AE
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