ISSN: 0975-766X CODEN: IJPTFI Available Online … synthesizer helps in synthesizing an array of ligands using the template ... ture and then activated by the heating at ... Dulbecco’s

Subin Mary Zachariah* et al. International Journal Of Pharmacy & Technology

IJPT| March-2016 | Vol. 8 | IssueNo.1 | 11221-11235 Page 11221

ISSN: 0975-766X

CODEN: IJPTFI

Available Online through Research Article

www.ijptonline.com IN SILICO DESIGN, SYNTHESIS, CHARACTERIZATION AND IN VITRO EVALUATION

OF SOME NOVEL ANTI-LUNG CANCER MOLECULES *Subin Mary Zachariah, Mridula Ramkumar, Namy George, Mohammad Salam Ashif, Alexeyena Varghese

Department of Pharmaceutical Chemistry & Analysis,Amrita School of Pharmacy,

Kochi Amrita Vishwa Vidyapeetham Amrita University, India.

Email: [email protected],[email protected]

Received on 04-03-2016 Accepted on 27-03-2016

Abstract

Azetidinones are four membered heterocycles with nitrogen as the heteroatom. In silico molecular modelling predicts

anticancer property of azetidinone derivatives. The azetidinone derivatives can be synthesized via simple and efficient 2-

step reaction. This involves production of Schiff’s base which then reacts with ketene to form azetidinone. However the

reaction is highly stereo selective and the reaction carried out in this study yielded acetamide derivative. The acetamide

derivative showed good activity against lung cancer cell lines. The current research work deals with an In silico ap-

proach in designing azetidinone derivatives, problems faced during the synthesis of azetidinones and the possible

scheme via which the product deferred.

Key Words: 2-azetidinone, Staudinger reaction, Anti-cancer, Acetamide.

Introduction

Azetidinones

β-Lactam antibiotics, are the most commonly prescribed antibiotics in medicine. The parent ring system present

in these antibiotics is a four membered azetidine ring. It consists of a carbonyl group on the second position

and nitrogen on the first position. Modifications in the azetidine ring in many studies have shown lower toxici-

ty and enhanced activity. These are currently used for chemotherapy of bacterial infections. The selective inhibition

during cell wall synthesis of bacteria is responsible for its lethal and efficacious antibacterial action[1]. In general it

has also been proved that the pharmacological activity is mainly due to the presence of the heterocyclic ring

system that has given impetus to investigate 2-azetidinone derivatives for its activity against lung cancer.



Structural features of 2-Azetidinones

Physical Properties of Azetidinones

Azetidin-2-ones are hydrolytically sensitive colorless solid with a melting point of 73 - 74°C. The X-ray crystallographic

studies revealed that the ring is essentially planar with N2 atom slightly out of the mean plane of its substituent’s except

where the steric factors enforce greater deviations from planarity. In normal amides C=O show a distance of 1.32Å, but

azetidinones shows a distance of 1.38 Å. The increased distance is the reason for angle strain and to inhibition of normal

amide resonance by interaction with the N-aryl substituents[2].

Spectroscopy

In IR spectra, stretching absorption in the region of 1870-1640 cm-1

is shown by carbonyl group present in the azetidi-

none ring. IR spectra with increasing intensity and no interfering bands was obtained. Thus it is the easiest band to be

recognized in the spectra. [3]

NMR Studies

Protons present on C3 and C4 under goes a chemical shift. This explains the effect of substituent’s on these carbon.

Substituents like chlorides, azido which has electronegative properties will reduce the electron density around the pro-

tons, and therefore deshielding of protons take place. As the electronegative property of substituent’s increases, more

strong deshielding takes place and chemical shift of protons take place [4].

Reactivity of Azetidinones

Addition of amide group to azetidinones causes angle strain. This amide linked β-lactam is more susceptible to nucleo-

philic attack at carbonyl group. The bacterial cell wall synthesis is inhibited by the strained bicyclic β-lactam ring which

acylates transpeptidase enzyme. Unstrained β-lactams has IR spectrum absorption of 1600cm-1

, whereas the strained β-

lactams have IR absorption of 1735-1765cm-1

. The strained rings, therefore increase the electrophilicity of carbonyl

groups attached.

Lung Cancer

Lung cancer is a malignant tumor of lung that is characterized by uncontrolled cell growth and differentiation in tissues

of the lung. It is more commonly known as pulmonary carcinoma also. The two major classification of lung cancer are

small cell lung carcinoma and non-small cell lung carcinoma, abbreviated as SCLC and NSCLC respectively. The



common symptoms of lung cancer are coughing usually accompanied with blood, weight loss , shortness of breath and

chest pains. Novel compounds that rationally act on abnormal molecular cells causing cancer can be designed because of

the recent advances in tumor genesis and metastasis[5] . The chemotherapeutic molecules containing heterocyclic moie-

ties have played an important role in the growth of cancer research. Such compounds are capable of reversing the

process of carcinogenesis and also inhibit the growth of tumor cells.

Drug designing

Designing of new chemical scaffolds with a heterocyclic ring , for the prevention of cancer cell growth have been

a present trend .Various bioinformatic tools have been intensively used in the area of drug designing . In the

present era, drug designing is done by computational techniques. The binding affinity of a ligand or drug to the

target can be studied by using various docking softwares. Ligands can be selected from different databases. Li-

gands can either be a metabolite, phytochemical , enzyme or can be developed from a template. Ligands having

maximum complimentarily and minimum energy should be selected. While selecting proteins, those with maximum sta-

bility should be considered. This is because biological systems are considered as smart systems and they have the ten-

dency to self-assemble. Structural analysis can be done for the selected proteins, which will give us information re-

garding the functionality of the proteins. Cavities are observed in the targets due to mutational pressure. Cavity with

the largest size is selected as it indicates stabilized mutation. Combinatorial library is synthesized using library

synthesizer. The synthesizer helps in synthesizing an array of ligands using the template provided. Screening

should be done by using filters. Finally a lead compound will be obtained [6]

Advantages of Drug designing

Drug designing prevent decoys. The quality of the compound synthesized can be maintained. Information regarding

proteins can be easily obtained, which is otherwise very difficult[7]. Helps us to know how error passes in meta-

bolism [8]. Various synthetic approaches for the synthesis of the ligands will also be mentioned.

Materials and Methods

Experimental

Computational Platform:

All the computational work was carried out on the server Dell (inspiron 15) in windows 8.



Softwares used: ChemSketch, Corina, Mol inspiration, Arguslab, PDB, Protparam Expasy.

Methodology of molecular docking

Ligand designing and optimization

Ligands were designed by giving different substitutions at positions of aromatic ring on an azetidinone derivative. Mo-

lecular structure was drawn by using Chem Sketch and 3D structure was generated by using online software CORINA.

The ligands were optimized by calculating the lipophilicity, molecular weight, size and shape of the ligand using Lipins-

ki rule of five[9].

Lipinski rule of five was analysed by using Molinspiration online software. Open Molinspiration home page. Click cal-

culation of molecular properties and drug likeness. Draw the required structure in JME window. Click and calculate

properties. Save the properties.

The crystallographic structures of the target were obtained from Protein Data Bank(PDB). The selected target for anti-

cancer activity was 1TUB.

The identified proteins were put for primary and secondary analysis. Primary structural analysis was done using PROT-

PARAM. The FASTA sequence is pasted in PROTPARAM and then the various descriptors will be mentioned.

Tubulin receptor

Tubulin is one of the prominent targets for antitumor agents. The α- and β-tubulins, two subunits of this protein, under-

go polymerization reaction to give microtubules. Tubulin plays a role in control of cell division processes.

The α, β- dimer structure of tubulin is deformed as a result of various drugs binding at the colchicine binding site of tu-

bulin. Apoptotic cell death is brought by preventing the assembly of tubulin into microtubules.[10].

Pocket identification

Pocket can be identified by using CastP tool. This gives all the information about the pockets in the protein. As the size

of pocket increases the chance for the entry of ligand also increases.

Method of Synthesis

First step is the synthesis of substituted Schiffs base. Schiffs base is synthesized by conventional method. This step in-

volves nucleophilic addition reaction between p-anisidine and substituted aromatic aldehyde which result in the forma-

tion of substituted schiffs base.



The second step is carried out using microwave assistance. The second step leads to the formation 2- azetidinone deriva-

tives by cycloaddition reaction.

One of the smooth and well-documented route for the synthesis of substituted ß – lactam derivatives is the ketene-imine

cycloaddition, as reported by Staudinger and hence also known as Staudinger’s reaction. This involves the addition of

ketene, which is formed in situ, to the imine[11].

Scheme of synthesis [12]

Mechanism

p-anisidine

substituted schiff's base

2-azetidinone derivative

NH2OCH3

Ar-CHO/ CH3OH

OCH3 N CH

Ar

ClCH2COCl/(C

2H

5)3N

OCH3 N

O

ClAr



Thin Layer Chromatography

TLC plates prepared using pour plate method. Silica gel G (Particle size 40 microns)was used to prepare glass plate of

size 3 × 7 cm. A thickness of 0.25 mm was achieved by the uniform spreading. The plates were dried at room tempera-

ture and then activated by the heating at 1210C in hot air oven for one hr and stored over a dessicator until used. Rf value

was generated from the TLC.

Spectral Analysis [13]

IR spectroscopy

The range of electromagnetic radiation between 0.8 and 500 µm was referred as infrared radiation, which was

represented with percent transmittance as the ordinate and the wave number (cm-1

) as the abscissa. It was an important

record which gives sufficient information about the structure of a compound [14].

NMR spectroscopy

NMR spectrum gives almost detailed information about the molecular structure. 1H and

13C NMR helps to determine

the number of proton and carbon atoms present in the molecule. NMR spectral study was done with Bruker Fourier-

NMR spectrometer. TMS was used as an internal standard and MeOD was used as a solvent.

Mass spectroscopy

It was the major tool used to determine the molecular mass of the compound and its elimination composition. The ioni-

zation method used is electron spray ionization. Mass spectra of the samples were recorded on a Waters e 2695-Waters

3100 instrument, which had ESI-PMT arrangement as the mode for ionization and type of detection respectively.

In vitro studies

MTT Assay

MTT is a colorimetric assay. 3-(4, 5dimethythiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) is reduced by the

enzyme mitochondrial succinate dehydrogenase. This reduction is measured by the MTT assay. Since this reaction can

only take place in metabolically active cells, it is a measure of viability of cells [15,16].

In vitro cytotoxic effect of synthesized derivatives on cultured L929 cell lines.

Dulbecco’s modified eagles media is used to maintain the L929 (Fibroblast cells) cell lines, at a temperature of 37°C in a

CO2 incubator. After trypsinizing the cells for 2 minutes, they are passed to T flasks. All the reactions are carried in



complete aseptic conditions. Extracts are added to cultured cells from a stock of 1mg/ml, at a final concentration of 10,

50 and 100µg/ml. It is then incubated for a period of 24 hours. The percentage difference in viability of cells was deter-

mined by after the incubation period by using standard MTT assay.

Determination of anti-proliferative effect of synthesized derivatives on A549 cell lines.

Dulbecco’s modified eagles media is used to maintain A549 lung carcinoma cell lines at a temperature of 37°C in CO2

incubator. The cells are trypsinized for 2 minutes and then passed to T flasks, all carried in complete aseptic conditions.

From a stock of 1mg/ml the extracts are added to the cultured cells at a concentration of 6.25, 12.5, 25, 50 and

100µg/ml. It is then incubated for 24 hours. After the incubation period standard MTT assay is used to determine the

percentage difference in viability of cells.

Results and Discussion

The current research work deals with the in silico design and synthesis of 2-Azetidinone derivatives by simple efficient

process. The results of the molecular docking are given in table 1. The compounds showed good docking score against

tubulin receptor when compared to the standard drug. Drug-likeness assessment carried out is also reported in table 2.

The value of all the derivatives relied within the optimal range. All the compounds had molecular weight less than 500

Daltons and the hydrogen bond donors and acceptors for both derivatives are below the values of 5 and 10 respectively.

The values of partition coefficient and the number of rotatable bonds were under the limit of 5 and 10 respectively.

Moreover none of the analogues exhibited Total polar surface area greater than 140Å .All these data indicated that it has

no more violations and both the selected derivatives could be orally active drugs. The selected azetidinone derivatives

were then synthesized. The appearance, melting points and Rf values of the synthesized compounds were noted and as

displayed in table 3. However on spectral analysis, the product was found to be 2-chloro-N-(4-

methoxyphenyl)acetamide. The results of IR, NMR and mass spectra are displayed in table 4. Since only one product

was formed in both the cases, only one set of values have been displayed in the table. The proposed mechanism for dev-

iation of the product from azetidinone derivative to acetamides is shown in figure 1. Formation of this product is attri-

buted to hydrolysis reaction instead of cyclo addition reaction.

It was noted that the formation of 4-membered β-lactam ring required stringent conditions such as nitrogen atmosphere

and moisture-less environment as the reaction is highly stereoselective. Interestingly, the synthesized compounds pos-



sessed significant level of anticancer activity against lung cancer cell lines. From the studied compounds it was noticed

that CS1 showed comparatively better activity.

This revealed that some impurity existed in either of the compounds or some difference in the procedure resulted in mi-

nor difference in the activities. The results of the invitro anti-cancer activity are depicted in figure 2. It is abundantly

clear from this study as well as the other research work carried out so far, that acetamide derivatives make good anti-

cancer agents.

Conclusion

Azetidin-2-one is one of the medicinally important scaffold with interesting biological activities. The current research

work was based on the anticancer activity of 2-azetidinone derivatives. The derivatives were synthesized by 2 step reac-

tion involving nucleophilic addition reaction and cycloaddition reaction. The first step was carried out by conventional

method and the second step by microwave assisted synthesis.

The yield of the synthesized compounds was found to be significant. However on spectral analysis the product was

found to be 2-chloro-N-(4-methoxyphenyl)acetamide. Formation of this product was attributed to pericyclic reaction

instead of cycloaddition reaction. The formation of 4-membered β-lactam ring requires stringent conditions such as ni-

trogen atmosphere and moisture-less environment as the reaction is highly stereoselective.

Hence inspite of azetidinone derivatives showing good in silico activity and the simple 2-step reaction, the product

formed was 2-chloro-N-(4-methoxyphenyl)acetamide. Any synthetic activities involving azetidinone ring should be pro-

ceeded with provision of proper environment to facilitate the 4-membered ring formation. Since the formed compound

showed good activity against lung cancer cell lines, the acetamide derivatives can be designed and optimized to serve as

good anti-cancer agents and further research can be taken up in this area.

Tables and Figures

Table 1: Selected target and its PDB ID.

SL.NO TARGETS PDB ID

1 Tubulin 1TUB



Table 2: Compounds undergone docking.

SL.

NO

COMPOUND

CODE STRUCTURE

CHEMICAL

NAME

1 CS1

N

O

ClOCH3

Br

4-(3-bromophenyl)-

3-chloro-1-(4-

methoxyphenyl)

azetidin-2-one

2

CS2 N

O

ClOCH3

OH

3-chloro-4-

(2-hydroxyphenyl)-

1-(4-

methoxyphenyl)

azetidin-2-one

Table 3: The docking scores of the selected compounds obtained from the preliminary docking program.

Table-4: Drug-likeness assessments of the selected 2-azetidinone derivatives.

Sl.

No.

Compd

Code Molecular Formula

Mol.Wt

(g/mol)

No. of

HBA

No. of

HBD CLogP

No. of

rot. B TPSA (Å

2)

1 CS1 C16H13BrClNO2 366.642 3 0 4.239 3 49.771

2 CS2 C16H14ClNO3 303.745 4 1 3.394 3 29.543

SL.

NO.

COMPOUND

CODE

DOCKING SCORE (kcal/mol)

ARGUSLAB AUTODOCK

1. CS1 -10.0565 -6.83

2. CS2 -9.1273 -5.91

3. Colchicine -8.6115 -5.41



Table-5: Physical data of compounds.

Sl.no

Compound

Code

Colour and

Appearance

M.P

Percentage

Yield

Solvent system Rf value

1 CS1

Brownish crys-

tal

79-80°C 25.41%

Benzene:

Ethylacetate(9:1)

0.68

2 CS2

Black-grey

Crystals

64-65°C 36%

Benzene:

Ethylacetate(9:1)

0.65

Table 6: Spectral Datas of the Synthesized Derivatives.

IR (cm-1

)

1H NMR (MeOD,

400MHz)

13C NMR (MeOD,

400MHz)

Mass spectra

3061cm1(ar C-H str)

1882cm-1

(C=O str)

1246cm-1

(C-N )

1656cm-1

(C=C)

2922cm-1

(C-H str)

690cm-1

(C-Br)

790.81cm-1

(C-Cl)

δ 7.479 (d, 2H, J = 8.8

Hz), 6.907(d, J = 8.8 Hz,

2H), 4.053 (s, 1H), 3.675

(s, 2H), 2.052 (s, 3H)

δ

167.23,158.38,132.1

6,123.26,115.07,55.9

0,43.94.

Base peak- 123.10

Mol wt. 199

Figure 1: CS1 binding to tubulin receptor.



Figure 2: CS2 binding to tubulin receptor.

Figure 3: Proposed mechanism for deviation.

O

CH3

N

Ar

Cl

O

Cl

O

CH3

N+

O Cl

Ar

HO

H

O

CH3

N

O Cl

Ar

OH

O

CH3

NHCl

O

2-chloro-N-(4-methoxyphenyl)acetamide

Figure 4: Cytotoxic activity of compounds on A549 cell lines and L929 cell lines respectively.



Figure 5: Cytotoxic activity of CS1 and CS2 with control against lung cancer cell lines.

CS1

6.25µg/ml 12.5µg/ml

25 µg/ml 50µg/ml

100µg/ml



CS2

6.25µg/ml 12.5µg/ml

25µg/ml 50µg/ml

100µg/ml control

Figure 6: cytotoxic activity of CS1 and CS2 with control against normal cell lines.

Pictorial representation

CS1

10µg/ml 50µg/ml



100 µg/ml

CS2

10 µg/ml 50µg/ml

100µg/ml control

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Corresponding Author:

Dr Subin Mary Zachariah,

Email: [email protected]

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ISSN: 0975-766X CODEN: IJPTFI Available Online … synthesizer helps in synthesizing an array of ligands using the template ... ture and then activated by the heating at ... Dulbecco’s