MSC DRUG DESIGN AND BIOMEDICAL SCIENCE/ MSc BIOMEDICAL SCIENCE/

MSc PHARMACEUTICAL SCIENCE/ MSc PHARMACEUTICAL SCIENCE

BMS11105 DRUG DESIGN AND CHEMOTHERAPY 2020

LABORATORY PRACTICAL WRITE-UP TEMPLATE

1. Background: [Maximum word count: 400].

Telomers are present at the ends of DNA sequence which are made of simple
guanine repeats that protect the chromosome from any damage. During cell
apoptosis in tumour cells the length of the DNA is shortened by few base pairs.
After a few rounds of cell division, the telomers get shortened and the
chromosomes become susceptible to damage. To maintain the length of telomers
the enzyme telomerase repairs the sites by adding guanine rich repeated
sequences to the shortened telomers. Telomerase enzyme is a ribonucleoprotein
which has two subunits RNA and with the template sequence along with reverse
transcriptase synthesize DNA tandem repeats for telomeric ends. Enzyme
telomerase plays a vital role in cancer therapy. Telomerase are present in high
levels in cancer patients. This act’s as a biomarker in detection of cancer in
humans. The telomerase activity is detected by the hTERCT protein. In cancer
cells the telomerase enzyme promotes cell division by maintaining the length of
telomers which protects the chromosome from damage. The telomerase was
responsible for promoting the growth of cancer cells; hence it was important to
inhibit the telomerase enzyme activity. Many researchers have been done for
inhibition of telomerase activity. The principle was to inhibit the telomerase activity
in cancer cells without causing any damage to the normal cells. The
demonstration to inhibit the telomerase activity in cancer cells was done using
anthraquinone molecules in g-quadruplex. G-quadruplexes are helical structures
formed by nucleic acids that are rich in guanine. Researchers have found that the
binding of anthraquinones to g-quadruples was effective in inhibiting the
telomerase activity completely in the cancer cells. It also showed that
anthraquinones showed effective binding with quadruplex than with 2 strands of
DNA.

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2. References
1) De Cian A, Lacroix L, Douarre C, Temime-Smaali N, Trentesaux C, Riou JF,
Mergny JL. (2008) Targeting telomeres and telomerase. Biochimie., 90(1),
pp.131-155.

2) Fletcher TM, Cathers BE, Ravikumar KS, Mamiya BM, Kerwin M. (2001)
Inhibition of human telomerase by 7-deaza-2-deoxyguanosine nucleoside
triphosphate analogues: potent inhibition by 6-thio-7-deaza-2-
deoxyguanosine-5-triphosphate. Bioorg. Chem. 29, pp. 36-55.

3)Gilson E, Ségal-Bendirdjian E. (2010) The telomere story or the triumph

of an open-minded research. Biochimie.

4) Perry PJ, Gowan SM, Reszka AP, Polucci P, Jenkins TC, Kelland LR,
Neidle S. (1998). 1,4- and 2,6-disubstituted amidoanthracene-9,10-dione
derivatives as inhibitors of human telomerase. J Med Chem., 41(17),
pp.3253-3260.

5) Röth A, Harley CB, Baerlocher GM. (2010) Imetelstat (GRN163L) -

Telomerase-Based Cancer Therapy. Recent Results Cancer Res. 184, pp.
221-234.

6) Sun D, Thompson B, Cathers BE, Salazar M, Kerwin SM, Trent JO, Jenkins
TC,Neidle S, Hurley LH. (1997) Inhibition of human telomerase by a G-
quadruplex-interactive compound. J Med Chem., 40(14), pp.2113-2116.

7) Tauchi T, Shin-ya K, Sashida G, Sumi M, Okabe S, Ohyashiki JH, Ohyashiki

K. (2006) Telomerase inhibition with a novel G-quadruplex-interactive
agent, telomestatin: in vitro and in vivo studies in acute leukemia.
Oncogene., 25(42), pp. 5719-5725.

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3. Experimental: [Maximum word count: 300].
AIM: Thin Layer Chromatography is used to identify the purification of the given compounds. 

Principle: TLC is a type of planar chromatography it is used to identify the components in a compound

mixture like Alkaloids, Phospholipids and Amino acids. The separation depends on relative affinity of
compounds towards stationary and mobile phase. The compounds under the influence of mobile phase travel
over the surface of the stationary phase. During this movement the compounds with higher affinity to
stationary phase travel slowly while other travel faster. 

Equipment’s: 
TLC Plates 
TLC Chamber 
Mobile phase 

Reagents:  
Chloroform: methanol, 19:1, 
Butanol: acetic acid: water, 4:5:1 

Procedure:  
TLC Plates are marked accordingly with MSc-A, MSc-B, MSc-C and a solvent medium. A very small
amount of MSc-A, MSc-B, MSc-C and solvent medium are placed on their respective markers then the
plates are introduced to TLC chamber and left for 10 minutes until the solvent rises to 1/3 of the TLC plate
after 10 minutes the plates are taken out of the TLC Chamber and observed under UV light. The spots are
marked and Rf Value is calculated  

Rf = distance travelled by solute/distance travelled by solvent

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4. Results and discussion:

(i) T.L.C.:
MSc-A
19:1 – 1.7/3.1 = 0.51
4:5:1 - 2.2/2.5 = 0.88

MSc-B
4:5:1 – 2.1/2.3 = 0.91
19:1- 1.9/3.9 = 0.48

MSc-C
4:5:1 - 1.5/2.5 = 0.6
19:1 – 0.6/3.5 = 0.17

Discussion: MSc-C sample have low Rf value compare to other samples from
the above , so MSc-C have polarity than the both MSc A and MSc B sample.

NOTE: t.l.c. plates must be either scanned into your report (preferred) or you can
attach the actual t.l.c.s to the paper copy of your submitted report.

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(ii) Percentage Yield Calculation (assume your recovered amount of purified

MSc A was 0.0406 g, as indicated in the accompanying write-up guidelines pdf.:

Molecular mass of 1-chloroanthraquinone=243g/mol

Molecular mass of MSc-A =311g/mol
Theoretical yield =311/243x0.25=0.319
Percentage yield of MSc-A = Actual yield/theoretical x100
=0.0406/0.319 x 100
=12.7%
Actual % of yield MSc-A = Theoretical yi eld/percentage yield x 100
=0.319/12.7 x 100
=2.5%

[Show all calculations in full] [Marks: /4]

(iii) Comment on the purity and % yield:

Percentage yield = actual yield / theoretical yield x 100

 TLC is utilized to deciding the purity of compound. In this technique a pure solid
show only one spot on a developed TLC plate. The balance of mixtures stretched
out by decreased the added solvents and change in main impetus by replaced of
water with de-ionized water and maintain a strategic distance from the running of
mixes in arrangements.

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5. Summary conclusion. [Marks: /4]

TLC technique is used to deciding the purity of compounds based on the mobility
of components. In this experiment the synthesis and characterization of new
anthraquinone was done. MSc-A and MSc-B are the compounds manufactured in
lab and analysed the purity test. And observed that they showed similar activity as
anticancer drugs after introducing it into the telomerase.
Question section (show all calculations in full)
Q 1. Q 1. MSc-A 
Molecular Formula: C18H17NO4 
Molecular weight: 311g/mol 
MSc-B   
Molecular formula: C22H26N2O6 
Molecular weight: 414g/mol
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Q2. a) Number of moles = mass/elative molecular mass                      

                               =0.25/242.66 
                               =0.001 moles  
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b) Density = mass/volume 
                   1.4 = mass/3 
               Mass= 1.4 x 3 
               Mass = 4.38g
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c) number of moles= mass/relative molecular mass  
                                 =4.38/104 
                            = 0.042 moles
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d) Limiting reagent in this reaction is 1-chloroanhraquinone                
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 e) Fold of excess reagent used= amount used/amount needed 
                                               =0.0402mol/0.0019mol 
                                              =21.2mol
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f) Excess amount of amine is essential to make sure the oxidation of Anthraquinone
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Q3. Given,  
  a) 0.0016g this is converted into mg to give 0.0016/1000=1.6g 
 1mg-1ml 
1.6-? 
1.6 x 1/1=1.6mg 
So, 1.6ml of DMSO is added to 0.0016g of MSc-B     
 
    b) Molarity of the 1mg/1ml of stock solution of MSc-B  
   Number of moles = amount of substance in grams / Relative molecular mass  
 Given amount in grams -0.001  
  Relative molecular mass of MSc-B is 414 
 Number of moles = 0.001/414
   =0.00000241
Molarity = 0.00000241x1000
=0.00241

    c)  Dilution of 1mg/1ml of stock solution of MSc-B to give 1ml of a 400 milli moles solution of MSc-B  
Given  
1mg-1ml  
400 milli g-? 
  We know that 1mg is equal to 1000 milli grams
1000 milli gram -1ml
400 milli grams -?
                                                                                                                    
In order to make it 1ml we need to add 0.6 of DMSO to 0.4 ml
                                      

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Q4.
a) a) DNA G-quadruplexs are DNA 2˚ structures formed in G-rich sequences.
DNA sequences form G-quadruplexes have been found in regions with
biological significances. DNA G-quadruplexes is a new class of novel molecular
targets for anticancer drugs. DNA G-quadruplexes can form in solutions under
physiological conditions and are globularly folded nucleic acid structures.
The Intramolecular structures of G-quadruplexes appear different from one to
another and it may be regulated and targeted by proteins and drugs differently.
b) Telomerase is a therapeutic target for cancers in humans. Potential
inhibitors designed with computer modelling, which exploit the quadruplex DNA
structural features. 3,6,9-trisubstituted acridine inhibitors interact with human
quadruplex structure, as a means of specifically inhibiting the action of human
telomerase in extending the length of single stranded telomeric DNA.
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b) HPLC technique can be used to confirm the purity of the given
compounds than the TLC.
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c) Telomerase is a potentially molecular target in cancer therapies. 2,7
aminoanthraquinone series were designed and synthesized and their effects
evaluated on telomerase activity, hTERT expression, cell proliferation and
cytotoxicity.
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References for Q 4.
1) Danzhou and keika okamoto, structural insights into G-quadruplexex; towards
new anticancer drugs, Future Med Chem.2010 Apr; 2(4) 619-646.

2) Hsu-Shan Haung, synthesis, Human Telomerase Inhibition and Anti-

Proliferative studies of a Series of 2,7-bis-substituted Amido-Anthraquinone
Derivaties, et al.Bioorg Med Chem.2008 Jul 15;16(14): 6976-86.

3) Hurley LH, Wheelhouse RT, Sun D, Kerwin SM, Salazar M, Federoff OY, Han
FX, Han H, Izbicka E, Von Hoff DD. G-quadruplexes as targets for drug design.
Pharmacol.Ther. (2000) 85(3): 141-158.