Anticancer Assignment

8/6/2019 Anticancer Assignment

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SYNTHESIS OF TYRPHOSTINS AND THEIR INVITRO BIOLOGICAL EVALUATIONUSING MTT ASSAY:

Objectives:

1. Synthesis of2-(4-chlorobenzylidine)malononitrile and recrystallization of obtained crudeproduct using ethanol.

2. In-vitro analysis of anti-neoplastic activity demonstrated by the prepared 2-(4-

chlorobenzylidine)malononitrile on human leukemia cell lines(K562) using MTT assay.

Materials and Methods:

The experimental procedure was divided into two phases.The first phase being the synthesis of the

tyrphostin compound followed by biological evaluation in the later.

Synthesis:A 50 cm3 single round bottom flask was obtained and 10 mmol 4-chlorobenzaldehyde was

transferred into it. Now 7ml of ethanol was added to it along with 2 drops of piperidine.To the

above, 10.4 mmol of malonitrile was added. A few anti-bumping granules were added and then it

was connected to a reflux condenser with water circulation and was refluxed for about 30 minutes.It

was then allowed to cool to room temperature until a white solid appeared. The white was then

collected and transferred to a 100ml conical flask and the weight of the crude crystals was

determined. The crude product was then dissolved in a minimal quantity of hot ethanol and

recrystallized. The recrystallization mixture was then let to cool down in an ice bath to allow

crystals to reform and grow. The crystals were then vacuum filtered and dried using a buchner

funnel. The crystals were rinsed with a small quantity of cold ethanol and left to dry. The melting

point was them determined. A proton and carbon NMR and an IR spectrum was then recorded.

Biological EvaluationThe biological evaluation was carried out using an MTT assay using(3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide. For the evaluation, 2 mg of 2-(4-chlorobenzylidine)malononitrile

was accurately weighed and transferred into a 1.5ml micro-centrifuge tube. The sample was

dissolved in an appropriate quantity of DMSO to make a stock. 100L of the cell suspension was

used to determine the cell concentration using a haemocytometer.

A 100L of the cell solution was transferred into each of the 96 wells labelled as Colunm 2-4 assample 'A',column 5-7 as sample 'B' and column 8-10 as 'C'.the plate was then incubated at 37

degrees with Celsius 5% carbon-dioxide and stored until the addition of drug was carried out. Eight

sterile 1.5mL micro-centrifuge tubes were obtained and 1mL medium(RPMI-1640, with 2mM l-

glutamine, supplemented with a 10% v/v foetal calf serum) was transferred into tube 8 and 0.5mL

into the remaining 7 tubes. 2L of the prepared 50mM drug stock was transferred to tube 8.After

mixing,0.5mL was transferred to next tube.This was continued and serial dilutions were carried out

6 times as above. Nothing was added to the last tube except media.This tube served positive control

for cell proliferation. The drug solutions were then pipetted in triplicate into the 96 well MTT assay

plate. About 100L of solution was transferred into each of the 96 wells and incubated for 5 days at

37 degrees with Celsius 5% carbon-dioxide. On the 5th day, to each well was added 50L of MTT

and incubated for 3 more hours. The MTT and media were then aspirated from all the wells andformazan precipitate in 200L DMSO.The optical density of each of the wells was read at 540 and

690 nm using a plate reader.


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Results and Discussion

Calculation of yield and %yield

ClC6H4CHO + CNCH2CN -------------) C10H5ClN2

4-chlorobenzaldehyde

10mmol of 4-chlorobenzaldehyde was transferred.

10Mmol = 10/1000 or 0.01 molar concentration

mass = n x m.wt

where 'n'is the no of moles and 'm.wt'is the molecular weight.Molecular weight of 4-chlorobenzaldehyde = 140.57 g/mol

Thus: 0.01 x 140.57 = 1.4057g

malanonitrile

10mmol of malanonitrile was transferredMolecular weight ofmalanonitrile = 66.06 g/moldensity ofmalanonitrile = 1.1910 g/mL

10Mmol = 10.4/1000 or 0.0104 molar concentration

mass = n x m.wt Thus: 0.0104 x 66.06 = 1.4057g

volume = mass/density

volume of malanonitrile added = 0.687/1.19 = 0.57 ml

% dry weight(calculation of loss on drying) = weight after drying/weight before drying x 100

weight before drying = 1.97 g

weight after drying = 1.65 g

%dryweight = 1.65/1.97 x 100 = ~ 83.7%

% yield = practical yield/theoretical yiels x 100molecular weight = 188.5 or 1.85 molar

% yield = 1.65/1.85 x 100 =89.1 %

Melting point = 163 C


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Determination of IC50For the determination of the IC50, Ascent was used to interpret the plate.First the MTT

was opened from the file list. The plate was then into the honlder on the plate reader.'Start' was then

pressed to begin the read. In the result, under 'precalc2' tab, the difference in 690 reading from 540

was displayed. All the cells were then highlighted and copied starting from the first cell in

Microsoft Excel with an MTT Graph Template into Cell 1,2.Under doses, the appropriate dose was

entered. The 'Autocalc' tab now gives us the value of IC50.probable sources of error that can be avoided:Care should be taken during the analysis of product. The spectroscopic analysis should be carried

out carefully to avoid contamination with anything to avoid any unwanted peaks in the spectrum.

With an experimenter doing an error, there are always possibilities of personal errors which can

occur. It should be made sure all the instruments have been pre-calibrated. Random errors can occur

quite inconsistently and cannot be avoided in any kind of analytical procedure. Thus to produce

concurrent results, a number of experimental data sets have to be obtained to cut off the error

margin.Thus the experiment could be repeated multiple times to get more accuracy.

Questions

(a) Mechanism of formation of TyrphostinsA. Knoevenagel condensation is a reaction involving an aromatic aldehyde. Bezaldehyde anaromatic aldehyde undergoes electrophilic addition with malanonitrile because of the polar carbonyl

oxygen. Electrophilic reagents like malanonitrile in this case attack the oxygen and undergo

addition to form tyrphostin analogous(fig 1).

fig 1.

The following represents a schematic of a reaction where chloro-substituted bezaldehyde reactswith

malanonitrile in presence of piperidine and forms chlorobenzylidine)malononitrile losing one

molecule of water and undergoing electrophilic addition(fig 2).

(fig 2.)


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(b) Spectroscopic data

IR spectrumpeak functional group

3034.47 = Alkenyl C-H strech

2225.60 = Nitrile strech1487.22 = aromoatic double bond

proton NMR7.4(d) , 7.8 (s) = aromatic hydrogens1.5(s) = alkene group7.6(s) = Cdcl3

Carbon NMR80- c double bond c

150- maybe nitrile group

127,128- aromatic carbons

(c)Acquire all ic50's for all tyrphostins synth'ed in the lab .compare structures and discussbiological activity: (table 1)Ic50 is a quantitative measure of the ability of a compound to interfere with biochemical function of

a physiochemical, physiological process. The ic50 values for various substituted tyrphostins are

mentioned in the table above. When pure benzaldehyde was tested, it showed an IC50 of

11.80M.But when a highly electronegative atom-chlorine atom was substituted, the selective

activity was boster to 1.01M.Nitro derivatives such as nitro benzaldehyde only resulted only in a

slight biological activity. Substitution of aromatic groups resulted in a very low biological activity.

Electron with-drawing groups seem to increase biological activity but with the case of fluoro

substituted benzaldehyde, no activity was observed. In a study by Gazit in 1993 it was observed that

the substitution of hydroxy groups by methoxy or groups did not give very good results(e.g., 3-

methoxy-4,5-dihydroxybenzylidenemalononitrileICs0 (pM) = 62 and 3-methyl-4,5-

dihydroxybenzylidenemalononitrile, IC50(pM) = 5.6).In order to improve activity, the methyl group

in a methoxy-methyl-hydro benzeylidine malonitrile by a substituted thio methyl group which

increased activity.


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Table 1

(d)Based on pharmacophore, which tyrphostin would u synth next?Purely based on the results obtained from the experiment, chloro substituted benzaldehyde seems to

be a compound which could be studied more as it has the best biological activity.It could be

assumed that because it shows such high inhibitory activity, it probably has a structural

conformation that is largely more specific to the EGF-R tyrosine kinase receptor.Compounds in

general in medicinal chemistry, which seem be show good activity for the function they aredesigned for usually have a structural resemblance which not only fits into the receptor with

specificity but also have suitable hydrogen bondings and vandervall radai which allows them to

properly elucidate biological activity.

DiscussionThe proliferation of cells is under the influence of certain growth factor interactions with certain

receptors. These are vital cellular factors constitute a delicate balance in cell proliferation and are

under strict regulatory control. Loss of control over these processes is one of the many hallmarks of

cancer development. These growth factors initiate signal transduction cascades by binding to a set

of analogous membrane bound receptor system through the formation of oligomeric or dimeric

structures. These growth factors for typrsine-kinase receptors mediate paracrine and some autocrinegrowth factors of both normal and malignant cells. This growth factor is a tyrosine kinase

membrane protein which is usually over expressed by malignant many cells including transitional


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and renal cell carcinomas are small molecular weight compounds that have benn found to

preferentially act on these EGF-R receptors. Thus these compounds are able to suppress malignancy

mediated by loss of regulatory control associated with EGF-R. Binding of EGF and its homolouge

transform growth factor (TGF)-alpha which activates EGF-R tyrosine kinase which transduces a

mitotic signal by phosphorylation of certain intracellular substrates.Over-expression of this

molecular mechanism has been notably observed with severe malignancies. Knoevenagel

condensation is a reaction involving the condensation of aromatic aldehydes withmalanonitrile.Malanonitrile has an active methylene group with undergoes cyclo addition in

presence of a catalyst of a strong base which incorporates a nitrogen species.Piperidine was used as

the catalyst.It provides a highly active catalytic site necessary for base catalysed Knovengel

condesation. Usually in a typical synthesis, 1mol of an aromatic benzaldehyde sustituted as per the

product required is mixed with 1mol of malanonitrile with 5ml of absolute ethanol in presence of

piperidine is refluxed for a time until forward reaction reaches completion. The product is

crystallized on cooling and is recrystallized .Samples can be analysed

Conclusion:

Though many different tyrphostins have been synthesized, there is still a lot of scope to exploreother different substituted derivatives with relation to biological activity.Some compounds have

shows good inhibitory activity.In this case chloro-tyrphostin showed good activity to inhibiting

EGR-R tyrosine kinase receptor.There is still scope for further exploration for SAR studies of

tyrphostins

References

A.Gazit et.al. (1993). Tyrphostins. 3. Structure-Activity Relationship Studies of a-Substituted

Benzylidenemalononitrile 5-S-Aryltyrphostinst.Journal of Medicinal Chemistry. 36 (3), 3556-3564.

N.S Vardy et al. (1995). Anti proliferative effects of Tyrosine Kinase inhibitors on human bladder

and renal carcinoma cells.Journal of surgical research. 59 (1), 675-680.

J.Mondal et al. (2011). Highly efficient mesoporous base catalyzed Knoevenagel condensation ofdifferent aromatic aldehydes with malononitrile and subsequent noncatalytic DielsAlder

reactions.Journal of Molecular Catalysis A: Chemical. 335 (1), 236-241.

A.Pande et al.(2005). A Novel Eco-Friendly Process for the Synthesis of 2-Chlorobenzylidenemalononitrile and ITS Analogues Using Water As a Solvent. Organic Process

Research & Development. 9 (1), 133-136.

Stamos, J.(2002) M.X. Sliwkowski, and C. Eigenbrot, Structure of the epidermal growth factorreceptor kinase-domain alone and in complex with a 4-anilinoquinazoline inhibitor. J. Biol.

Chem.,.277(48): p.46265-46272


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Anticancer Assignment