SYNTHESIS AND EVALUATION OF

2(4CHLOROPHENYL)-1 -H INDOLE AS ANTICANCER

AGENT

A REPORT ON THE PRACTICE SCHOOL SUBMITTED FOR

THE PARTIAL FULFILLMENT

OF THE SEVENTH SEMESTER EXAMINATION IN

BACHELOR OF PHARMACY
TO
THE SAVITRIBAI PHULE PUNE UNIVERSITY, PUNE
by

PANKAJ RAVSAHEB PAGAR

under the guidance of

S. N. WAGH

SNJB’s SHRIMANSURESHDADA
JAIN COLLEGE OFPHARMACY
NEMINAGAR, CHANDWAD, NASHIK
2022-2023

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 SNJB's

SHRIMAN SURESHDADA JAIN COLLEGE OF PHARMACY

JAIN GURUKUL, NEMINAGAR, AT/P. CHANDWAD

Tal. Chandwad, Dist. Nashik-423 101

Date: / /2022

CERTIFICATE

This is to certify that Mr.PANKAJ RAVSAHEB PAGARstudying in VII Semester carried

the work described in this report and has completed the assignment in BP706 PS Practice
School as stipulated in the syllabus of B.Pharm course by SavitribaiPhule Pune University
and is now ready for examination.

( )

Name and Signature of Guide

Designation

Date:

Place: Chandwad

(C. D. Upasani)

Principal

SSDJ College of Pharmacy, Chandwad

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 Acknowledgements

Every achievement is the result of several efforts, but these efforts are hampered
by the presence of hands that smooth the path. We offer our heartfelt thanks for
the helping hands that have been provided to us during our work.

We would want to convey our gratitude and appreciation to our guide PROF. S.
N. WAGH SIR for his guidance and scholarly supervision during the course of
Practice School . We appreciate her advice, patience, and unwavering support
and express our heartfelt gratitude to him for illuminating our way.

It is our pleasure to express our gratitude to Dr. C. D. Upasani Professor and

Principal of SNJB’s SSDJ College of Pharmacy, for providing us with the
necessary facilities, encouragement, and guidance.

Collective and individual acknowledgements are also owed to my friends. We

express our wholehearted gratitude and special thanks to our friends Miss.
Shital Ostawal ,Miss.Rutuja Pawar, Miss.Yogita Tope for providing us with
his valuable time.

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Introduction:-

Estrogen Receptor Alpha in Human Breast Cancer:

Breast cancer is most common cancer in women in industrialized countries and is the second
biggest killer after lung cancer. About 1 woman in ten will develop breast cancer at some
stage in her life (1 in 8 in the USA) . The demonstration over a hundred years ago, of the
inhibitory effect of ovariectomy on patients with metastatic disease provided the first
evidence for the importance of estrogens in regulating breast cancer growth. Direct evidence
to show that estrogen controls breast cancer growth was forthcoming in the 1930s and in the
1950s a receptor was identified1 .

STRUCTURE OF ER-Alpha :-

ERα was the first ER to be discovered and cloned. The gene ESR1 that encodes ERα is
located on chromosome . As shown in Figure no -1 , the ERα protein consists of 595 amino
acids with a molecular weight of approximately 66.2kD.The ERα protein contains six
domains (A-F), three of which are functionally significant. The three functional domains are
the N-terminal A/B domain (NTD), the C domain (which includes the DNA-binding domain,
DBD), and the E domain (the ligand-binding domain, LBD). NTD has a low degree of
conservation and contains AF-1, which has the function of transcriptional activation and is
also the main reason for ERα’s endocrine-sensitivity.AF-1 is critical to the transactivation
function and shows the highest variability among ERs. DBD in the C domain is highly
conserved and exerts its function by binding to the estrogen-responsive element (ERE), which
subsequently regulates the expression of target genes. The D domain shows 30% homology
among ERs and links the C and E domains. LBD (also called AF-2) or the E domain, showing
55% homology with other ERs, is mainly involved in protein and estradiol (E2) binding.LBD
combines with estrogen to form a homodimer that regulates gene suppression and activation
and contributes to transcriptional activation.Studies have also shown that LBD is responsible
for nuclear localization. The F domain, which is not conserved and shows only 18%

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homology, is regarded as an extension of the E domain. Although the structure of ERα has
been studied extensively, the function of the F domain has not been clarified. Understanding
the structure of ERα is essential for the treatment of ERα-over-expressing cancers.2

Part:1
2

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Synthesis

Aim: To prepare 2-(4-chlorophenyl)1-H indole from phenyl hydrazine.

Principle:

Ayrl hydrazones are formed from a condensation reaction of an arylhydrazine and an

aldehyde or ketone. Here phenylhydrazine condenses with 4-Chloroacetophenone to produce
4-Chloroacetophenone phenylhydrazone. Finally by Fischer indole synthesis the
arylhydrazone (Chloroacetophenone phenylhydrazone) converts into the 2(4-
Chlorophenylindole) in the presence of an acid catalyst.

Reaction:-

Mechanism:-

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Requirements:-

Chemicals:

i)4-Chloro Acetophenone ii) Phenylhydrazine iii)Glacial acetic acid iv)

Dilute hydrochloric acid v)Ethanol vi) Polyphosphoric acid vii)Calcium chloride

Procedure:-

Prepare acetophenone phenylhydrazone by boiling a mixture of 6.4ml(0.05 mol) of 4-

chloroacetophenone and 4.9 ml (0.05 mol) of phenylhydrazine with ethanol 15ml and a few
drops of glacial acetic acid. Filter the cold reaction mixture, wash the solid with dilute
hydrochloric acid followed by about 5 ml of cold rectified spirit. Recrystallise a small portion
from ethanol and thus obtain a sample of pure acetophenone phenylhydrazone . Place 2 g of
the crude phenylhydrazone in a 250 ml beaker containing 6.4 g of polyphosphoric acid. Heat
on a boiling water bath, stir with a thermometer and maintain at 100-120 °C for 10 min .Add
30 ml of cold water and stir well to complete solution of the polyphosphoric acid. Filter at the
pump and wash well with water. Boil the crude solid under the reflux along with 21 ml of
rectified spirit, add a little decolourising charcoal and filter through a preheated Buchner

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funnel. Wash the residue with 10 ml of hot rectified spirit. Cool the combined filtrates to
room temperature, filter off the 2-phenylindole and wash it three times with 3ml portions of
cold alcohol. Dry in a vacuum desiccator over anhydrous calcium chloride. The yield of pure
2-(4-Chlorophenyl)1-Hindole, m.p. 110°C, is 1.4 g (70%).

Calculation:.

Molecular formula of phenylhydrazine = C6H8N2

Molecular formula of 2-(4Chlorophenyl)1-Hindole = C14H11NCl

Molecular weight of phenylhydrazine = 108 g/mole

Molecular weight of 2-phenylindole = 228 g/mole

Theoretical yield:

108 g phenylhydrazine forms 228 g 2-(4-Chlorophenyl)1-Hindole

Therefore, 18 g phenylhydrazine will form …….? (X) g 2-phenylindole

= 11.18 g

Theoretical yield = 11.18 g

Practical yield =1.4 g

% Yield = (Practical Yield)/(Theoretical Yield) × 100

%Yield=(1.4 g) /(11.18 g) ×100

= 12 %

Conclusion:-

2-(4 Chlorophenyl)1 H indole was synthesized and the percentage yield was found to be…
12..%

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Part 2:- Evaluation

1)Thin layer Chromatography

Introduction :- Kirchner in 1950 was the first who used adsorption chromatography on
impregnated glass-plate coated with silicic acid or alumina. It may be emphasized, however,
that Egon Stahl’s fundamental work stands as a landmark in the world-wide acceptance of
this new technique in the laboratory. Later on, Stahl in 1958 introduced a standard equipment
for preparing uniform thin-layers of known thickness, which eventually led to the ultimate
acceptance of this new technique as an additional modern tool for analytical chemistry.

Principle of TLC:-

It is based on the principle of adsorption chromatography or partition chromatography or

combination of both, depending on adsorbent, its treatment and nature of solvents employed .

i) The components with more affinity towards stationary phase travels slower.
ii) Components with less affinity towards stationary phase. travels faster

Experimental Techniques

The various techniques with regard to thin layer chromatography (TLC) are as stated below,
namely :
Preparation of TlC Plate : -

The paramount importance with regard to the preparation of thin layer is that it must be
uniform and consistent throughout. Various means have been put forward to apply thin layers
of powdered or their suspensions or their slurries to the carrier plates with a view to achieve
an uniform layer throughout the length of the plates.These are namely :

(a) Pouring of Layers : In order to obtain layers of equal thickness, a measured amount of
the suspension or slurry is placed on a given-size plate that is rested on an absolutely
labelled surface.
(b) Dipping : In which two plates at a time back-to-back are dipped together in a slurry
of the adsorbent in either chloroform or chloroformmethanol. However, this particular
methods is not much in use now-a-days.

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 (c) Spraying : Reitsema** was first to develop this method by making use of a small
paint-sprayer for the distribution of the suspension or slurry onto the surface of the
glass-plate.
(d) Spreading : In this particular case, the suspension or slurry is put in an ‘applicator’,
which is subsequently moved either over the stationary glass-plate or vice-versa i.e., it
is held stationary while the glass plate is pulled or pushed through. This technique
termed as ‘spreading’ usually yields uniform thin layers on the glass plates.
(d) TLC-Plates ready-for Use’ (or Pre-coated Plates) :-
E.Merck AG (Darmstadt, West Germany) was pioneer in introducing two types of
ready-for-use TLC plates either on glass or polysheets (ethyleneterephthalate),
namely :
(i) Ready-for-use TLC Plates with Cellulose-F, and
(ii) Ready-for-use TLC Plates with Silica Gel F-254.

Choice Of Adsorbent substance:-

The choice of proper adsorbent in TLC plays a vital role in the separation of components
either belonging to natural origin or to purely synthetic origin. It is chiefly based on certain
crucial informations like :

(i) Solubility of the substance e.g., hydrophilic and lipophilic,

(ii) Nature of the compound i.e., whether it is acidic/basic/neutral/amphoteric

(iii) Reactivity of compound with either the solvent or the adsorbent, and

(iv) Chemical reactivity of compounds with the binders.

In actual practice, the adsorbents are of two types :

1)Inorganic adsorbents
These are namely :
(i) Aluminium oxide- (Al2O3) : The alkali (Na2CO3 ; NaHCO3) present in alumina very
often gives rise to secondary reactions that may be eliminated by washing with dilute mineral
acid or with water, followed finally by methanol and ultimately by heating at 200 °C.

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 (a) acidic (pH ~− 4.0) ; (b) basic (pH ~− 9.0) ; and (c) neutral (pH ~− 7.5).
(ii) Aluminium Silicate : It permits the adsorption of sterols and sterol glycosides from
oils without the use of solvent.

iii) Calcium Silicate : It is employed frequently for the separation of carbohydrates and the
corresponding phenylosazones.

iv) Calcium Sulphate : It is found to be suitable for the separation of steroids and lipids.
(v) Dicalcium Phosphate : It is used for the purification of carotene-the natural red pigment.

(Vii)Magnesium Silicate (Magnesol : MgO 2.5 SiO2.H2O) : It is usually employed for the
separation of sugar acetates ; whereas, magnesium trisilicate is used for the separation of
steroids, acetylate gycosides, esters, glycerides, lactones etc.
Viii) Silica Gel : (pH 6.0) : It is used extensively for the separation of sterols, fatty acids,
glycerides, azoated carbohydrates, sugar acetates, amino acids
.
2)Organic Adsorbents:-

The organic adsorbents are known for their relatively milder action for the separation of good
number of components, namely :

(i) Cellulose and Acetylated Cellulose : These adsorbents are commercially available in
various forms e.g., particle size, degree of acetylation, with or without binders like
starch or Plaster of Paris.
iii) Dextran Gels : Proteins and nucleotides can be separated by using cross-linked
dextran gels available in various types and particle sizes. The molecular weight of
dextran-gels vary considerably depending upon the extent of cross-linked nature.

The choice of solvent or a mixture of solvents used in TLC is solely guided by two
important factors :
(a) the nature of the constituent to be separated i.e., whether it is polar or non-polar ; and
(b) the nature of the process involved i.e., whether it is a case of ‘adsorption’ or
‘partition chromatography’. It has been observed that the rate of migration of a
substance on a given adsorbent depends upon the solvent used ; therefore, the latter

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 may be arranged in order of the elutive power, usually termed as the elutropic series
as shown in the following TableNNote : (i) These series are not always valid in
precisely the same order for all substances,
(ii) These series may be regarded as good guides for selecting a specific solvent only,
and

(iii) These series are good for hydrophyllic adsorbents and not for hydrophobic ones
e.g., charcoals.

From actual experimental results it has been established beyond any reasonable doubt
that the mixtures of two or three solvents of different polarity mostly offer distinct and much
improved separation as compared to chemically homogeneous solvents. Table 2 records the
elutropic series of one and two component solvents.

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Activation of TLC Plate ;-
It is achieved conveniently first by air-drying the TLC plates for a duration of 30 minutes and
then in a hot-air oven maintained at 110 °C for another 30 minutes and subsequently cooling
them in a dessicator. This drying process helps a great extent in rendering the adsorbent layer
active.

Spoting of component:

The following points may be strictly adhered to while spotting the component or mixture of
components on a TLC plate, namely :

(i) The sample is normally applied as a solution in a ‘non-polar solvent’ as far as

possible, since the use of a polar solvent may cause:
(a) spreading out of the starting spot, and
(b) (b) affect directly the Rf value of components,

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(ii) The solvent employed for dissolving the sample must be easily volatile-in-nature so that it
should be removed from the TLC plate before development commences,

(iii) The ‘area of application’ should be smallest as far as possible so as to achieve a sharper
resolution,
(iv) To maintain the size of the spot ‘small’ repeated applications is made by allowing the
solvent to evaporate after each application.
DEVELOPMENT OF THIN LAYERS:-

The spotted TLC plates, after evaporation of the sample solvent, is placed in a closed
chamber saturated with vapours of the developing solvent(s). One end of the plate is then
wetted with the developer by means of either ‘ascending-technique or the ‘descending-
technique

DETECTION OF COMPONENTS
: (i) Coloured Substances : e.g., Xanthophylls, Chlorophylls, Carotenes, etc., may be located
visually.
(ii) Colourless Substances : e.g., alkaloids, steroids, amino acids and the like may be
detected under short-wave UV-light or a long-wave UV-light. These substances may
also be detected as brown/dark brown spots when exposed to I2-vapours in a closed
dessicator.

(iii) Specific Detecting Reagents : A few specific detecting reagents are normally used for

Aniline-phthalate reagent : for carbohydrates ;

Ninhydrin reagent : for amino-acids, and

Dragendorff’s reagent : for alkaloids

(iv) Chromic acid/conc. H2SO4 : These corrosive reagents usually char the organic
material on TLC plates and may be seen as dark brown spots..

Evaluation of Chromatogram:-

Qualitative Evaluation

Rf= Distance travelled by Solute

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 Distance travelled by solvent

1) Starting material Phenylhydrazine, RF value:-

2) Product RF Value:-

Conclusion :- The TLC of 2 (4 Chloro phenyl) 1-H Indole was performed

i) The Rf value Of Starting material A(Phenylhydrazine) =

ii) The Rf value of Final Product B'(2(4-Chlorophenyl) 1-H Indole) =

Part 3 :- Evaluation by Fourier Transform Infrared Spectrophotometer(FTIR)

History:-

i) Chemical IR spectroscopy was emerged as a science in 1800 by Sir William Herschel.

ii) Firstly most IR instrumentation was based on prism or grating monochromators.

iii) Michelson invented interferometer in 1880s .

iv)In 1949 Peter Fellgett obtained the first IR spectrum by using FTIR spectrometer .

v) In 1960s commercial FTIR spectrometers appeared.

vi) In 1966 Cooley- Tukey developed an algorithm, which quickly does a Fourier transform.

Introduction

Infrared Region:-.

Infrared radiation lies between the visible and microwave portions of the electromagnetic
spectrum. Infrared waves have wavelengths longer than visible and shorter than microwaves, and
have frequencies which are lower than visible and higher than microwaves. The Infrared region is
divided into: near, mid and far- Infrared Near-infrared refers to the part of the infrared spectrum
that is closest to visible light and far- infrared refers to the part that is closer to the microwave region
Mid-infrared is the region between these two.

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FT-IR :-

FT-IR stands for Fourier Transform Infrared, the preferred method of infrared spectroscopy. In
infrared spectroscopy, IR radiation is passed through a sample. Some of the infrared radiation is
absorbed by the sample and some of it is passed through (transmitted). The resulting spectrum
represents the molecular absorption and transmission, creating a molecular fingerprint of the
sample. Like a fingerprint no two unique molecular structures produce the same infrared spectrum.
This makes infrared spectroscopy useful for several types of analysis.It can identify unknow
materials.

Principle:-

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 Radiation emitted from the source is split into two light beam with a beamsplitter in the
interferometer. The fixed and moving mirror reflect each of the beam to the beamsplitter, where the
two beams recombine into one and falls on the detector. The two beams combine constructively or
destructively, varying as the optical path difference , when the moving mirror is moved. When the
combined beam is transmitted through the sample, it is detected as an interferogram and contains
all infrared information on the sample. The infrared spectrum is obtained from the interferogram by
the mathematical process of Fourier transformation. Instrumentation Of FT-IR :-

The FT-IR spectrophotometer consist following component-

A) Sources of Radiation:-

1)Nernst Glower -

i) It contains a hollow rod composed of rare earth oxides such as is it zirconium, Yttriaum and
Thoriaum.

ii) The glower is heated to a temperature within the range of 1000-1800° C.

iii) It gives a maximum radiation of 7100 cm-1.

2.Globar source -
i)It is a rod prepared from centred Silicon carbide
ii) It is heated up to a temperature between 1300-1700°C

iii)It emits maximum radiation at 5200cm-1

B)Interferometer:-

The Michelson interferometer is a common configuration for optical interferometry and was
invented by the 19/20th-century American physicist Albert Abraham Michelson. Using a beam
splitter, a light source is split into two arms. Each of those light beams is reflected back toward the
beamsplitter which then combines their amplitudes using the superposition principle. The resulting
interference pattern that is not directed back toward the source is typically directed to some type of
photoelectric detector

C)Detectors:-

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 1) Thermocouples:-
Thermocouples comprise of a pair of junction of two different metals . IR radiation falling on
one junction raises its temperature whereas the other junction is maintained at a constant
temperature .The temperature difference between the two junctions results in a potential
difference between the two which is in proportion to the temperature difference between
them.Several thermovouples connected in seriesare called a thermopile.
2) Pyroelectric detectors - These are made from a single crystalline wafer of a pyroelectric
material such as Deutrated Triglycerine Sulfate (DTGS) and Lithium tantalate . (LiTaO3).

Attachment used in Recording FTIR:-

ATR (attenuated total reflection)

An attenuated total reflection accessory operates by measuring the changes that occur in a totally
internally reflected infrared beam when the beam comes into contact with a sample .

Operating Process:-

i.Ensure clean and dry atmosphere near FT-IR Spectrophotometer.

ii. Always turn on the RESUME switch before turning on the power.

iii. Run Spectra Manager Software package (Two beeps give confirmation).

iv. Connect "ATR unit" to instrument safely.

v. Allow the instrument in stand by condition for 30 minutes.

vi. Set appropriate parameters from Spectra Manager and measure background.

vii. Keep well dried sample about 1-2 mg on ATR crystal (ZnSe).

viii. Tighten the screw of ATR unit clockwise till it just touches the sample and Close

cabinet of instrument,

ix. Set the appropriate parameter in Spectra Manager and click the Measure Sample.

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Result of peak picking

No. Position Intensity No. Position Intensity

1 3320.823 99.5457 2 3107.72 92.2952
3 2787.6 98.2136 4 1648.84 76.957
5 1605.45 83.6113 6 1561.09 73.6984
7 1498.42 74.8977 8 1435.74 72.3002
9 1367.28 83.7952 10 1328.71 83.2664
11 1221.68 68.4895 12 1173.47 89.2666
13 1105.98 94.8687 14 965.198 94.3505
15 834.062 76.4095 16 800.314 81.8672
17 712.569 81.0159 18 678.82 71.5229

Sr
1 3320.82

2 3107.72

3 2787.6

4 1648.84

5
6
7

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