1 Advances in Polymer Science and Technology: An International Journal 2011; 1 (1): 1-9

Original Article

Acrylic and methacrylic hompolymers based on pyramido [4, 5-d] pyrimidine

derivatives: synthesis, characterization and in vitro antimicrobial activity

Aravinda Reddy P1, Ramachandra Reddy G

1, Babul Reddy A

2, Subbarami Reddy N

1 *

1Department of Polymer Science & Technology, Sri Krishnadevaraya University, Anantapur 515055, Andra Pradesh, India 2Department of Chemistry, Sri Krishnadevaraya University, Anantapur 515055, Andra Pradesh, India

*E-Mail: nsubbaramireddy@gmail.com 1Synthetic Polymer Laboratory-II, Department of Polymer Science & Technology, Sri Krishnadevaraya University, Anantapur.

2Department of Chemistry, Sri Krishnadevaraya University, Anantapur

Received 25 July 2011; accepted 16 August 2011

Abstract

The monomers of N-(5-oxo-7-phenyl-5,6-dihydro-pyrimido[4,5-d]-pyrimidin-2yl)-acrylate (PPPA) were synthesized from

acryloyl chloride and N-(5-oxo-7-phenyl-5,6-dihydro-pyrimido[4,5-d]-pyrimidin-2yl)-methacrylate (PPPMA) from methacryloyl chloride. The monomers were used to produce poly [N-(5-oxo-7-phenyl-5,6-dihydro-pyrimido[4,5-d]-pyrimidin-

2yl)-acrylate] (poly (PPPA)) and poly [N-(5-oxo-7-phenyl-5,6-dihydro-pyrimido[4,5-d]-pyrimidin-2yl)-methacrylate] (poly

(PPPMA)), were synthesized by free radical solution copolymerization using benzoyl peroxide as initiator in N, N-

dimethylformamide at 70 oC. The monomers and polymer structures were characterized by infrared and proton nuclear

magnetic resonance spectroscopy. Thermo gravimetric analysis (TGA) of polymers carried out in a nitrogen atmosphere

showed moderate thermal stability in the region 233 to 561 ºC. As the PPPA content in the polymer decreases the activation

energy increases. Antimicrobial activity of the polymers was also investigated against various microorganisms like bacteria

(Pseudomonas Aeruginasa, Escherichia coli, Proteus vulgaris, Salmonella enteridis, Klebsiella pneumoniae and

Staphylococcus aureus) and fungi (Aspergillus niger). The antimicrobial activity of the polymers increases as the PPPA

content increases in the copolymer. This shows that pyrimidopyrimidine moiety place very important role as antimicrobial

agent.

© 2011 Universal Research Publications. All rights reserved Key words: N-(5-oxo-7-phenyl-5, 6-dihydro-pyrimido [4, 5-d]-pyrimidin-2yl)-acrylate, polymerization, Thermal degradation,

Thermogravimetric analysis, Antimicrobial activity.

1.0 INTRODUCTION

It has been well documented that copolymerization is one of

the important techniques used in affecting systematic changes

in the properties of the commercially important polymers

[1,2] for example, the copolymers of acrylic/methacrylic

esters have been used for various application. Acrylate

homopolymers along with their copolymers are used in

various fields such as films, fibers, filaments, coating, lithography, lacquers, adhesives, printing inks and binders [3-

5]. The incorporation of two different monomers in the same

polymer chain in varying proportions leads to formation of

new materials. Acrylic polymers are a class of reactive

polymers that finds extensive applications due to the presence

of electron attracting groups in the aromatic ring [6].

Pyrimidines exhibit a range of pharmacological activity such

as antibacterial [7–9], antifungal [10, 11], anticancer [12, 13],

anti-inflammatory [14, 15] and cardioprotective effects [16].

And also Pyrazolo[3,4-d]pyrimidine[17 – 20] derivatives

were found to be selective ligands with antagonist activity for

A1 adenosine receptors (A1AR). They may have therapeutically use as cognitive enhancers, antidementia

drugs (e.g., for Alzheimer’s disease and cerebrovascular

dementia), psycostimulants, antidepressant drugs, and

ameliorants of cerebral function [21] A1AR antagonists have

also demonstrated promising therapeutic potential for renal

Available online at http://www.urpjournals.com

Advances in Polymer Science and Technology: An International Journal

Universal Research Publications. All rights reserved

2 Advances in Polymer Science and Technology: An International Journal 2011; 1 (1): 1-9

and cardiac failure [22, 23]. The copolymers were

characterized by FT-IR and 1H-NMR spectra.

2.0 EXPERIMENTAL

The homopolymers Poly (PPPA) and Poly (PPPMA) of were prepared by solution blending in dimethyl sulfoxide (DMSO)

at different temperatures.

2.1 Materials

2.1.1 Chemicals and solvents

The chemicals and solvents used were p-hydroxy benzoic

acid (S.D.Fine), sodium hydroxide (S.D.Fine), 1,4-dioxane

(S.D.Fine) and N,N-dimethyl formamide (Merck).

2.1.2 Initiator

Benzoyl peroxide (BPO) from S.D.Fine chemical has been

recrystallized from chloroform–methanol (1:1) mixture.

2.1.3 Reagents The reagents used were benzoyl chloride (S.D.Fine),

methacrylic acid (Merck), acrylic acid (Merck) and

hydroquinone (S.D.Fine).

2.2 Preparation of Reagents

2.2.1 Preparation of Acryloyl Chloride (Scheme 1) Acryloyl chloride (iii) is prepared according to the method of

Stampel et al. [24]. A mixture of acrylic acid (i) (23g, 0.32

mol), benzoyl chloride (ii) (134.9g, 0.96mol) and

hydroquinone (0.5 g) were taken in a round bottom flask and

distilled at a fairly rapid rate and the fraction distilling at 90-

100 oC is collected. Then it is redistilled and the fraction is collected at 72-760C. In order to prevent polymerization of

acrylic acid, hydroquinone is used. The yield is 69% with a

boiling point of 72-76 oC.

Scheme 1: Preparation of acryloyl Chloride

2.2.2 Preparation of Methacryloyl Chloride (Scheme 2)

Methacryloyl chloride (v) was prepared from methacrylic

acid (iv) (21.5g, 0.25mol) and benzoyl chloride (ii) (105.4g,

0.75mol) and hydroquinone (0.5gm). They were taken in a

round bottom flask and distilled at a fairly rapid rate at first,

the fraction boiling at 110-130 oC is collected, which was

then redistilled and collected the fraction boiling at 95-97oC.

The yield of compound (v) 65% with a boiling point of 95-97 oC.

Scheme 1: Preparation of methaacryloyl Chloride

2.3 Preparation of Monomers:

N-(5-oxo-7-phenyl-5,6-dihydro-pyrimido[4,5-d]pyrimidin-

2-yl)-acrylate (PPPA) (Scheme 3)

A solution of 7-amino-2-phenyl-3H-pyrimido[4,5-

d]pyrimidin-4-one (vi) (13.14g, 0.095 mol) and NaOH (7.70g, 0.193mol) in 40 ml of water and 15 ml of 1,4-dioxane

was prepared and cooled to 0-5 oC. Then, acryloyl chloride

(iii) (8.9g, 0.098mol) was added drop wise via dropping

funnel with a continuous stirring at temperature of 0-5 oC.

Upon completion, the temperature was kept at room

temperature for 4 hours. After 4 hours, the reaction mixture

was neutralized with a diluted HCl. The solid white

precipitate was formed. The solid white precipitate which is

N-(5-oxo-7-phenyl-5,6-dihydro-pyrimido[4,5-d]pyrimidin-2-

yl)-acrylate (vii) monomer was filtered, washed with hot

water, dried and recrystalized from methanol-water mixture.

The monomer yield was 77%, which has melting point of 232-233

oC.

N

NH2N N

NH

O

H2CCl

O

+

NaOH, H2O and 1,4-Dioxane

R.T

iii) Acryloyl chloride vi) 7-amino-2-phenyl-3H-pyrimido[4,5-d]pyrimidin-4-one

N

NHN N

NH

O

H2C

O

vii) N-(5-oxo-7-phenyl-5,6-dihydro-pyrimido[4,5-d]-pyrimidin-2yl)-acrylate

Scheme 3: Preparation of PPPA

2.3.1jN-(5-oxo-7-phenyl-5,6-dihydro-pyrimido[4,5-

d]pyrimidin-2-yl)-methacrylate (PPPMA) (Scheme 4)

A solution of 7-amino-2-phenyl-3H-pyrimido[4,5-

d]pyrimidin-4-one (vi) (13.14 g, 0.095 mol) and NaOH (7.70

g, 0.193 mol) in 40 ml of water and 15 ml of 1,4-dioxane was

prepared and cooled to 0-5 oC. Then, methacryloyl chloride

(viii) (8.9 g, 0.098 mol) was added drop wise via dropping

funnel with a continuous stirring at temperature of 0-5 oC.

Upon completion, the addition methacryloyl chloride the

temperature was kept at room temperature for 4 hours. After

4 hours, the reaction mixture was neutralized with a diluted

HCl. A solid white precipitate which is N-(5-oxo-7-phenyl-5,6-dihydro-pyrimido[4,5-d]pyrimidin-2-yl)-methacrylate

(ix) monomer was filtered, washed with hot water, dried and

recrystalized from methanol-water mixture. The monomer

yield was 78%, which has melting point of 184-186 oC

N

NH2N N

NH

O

H2CCl

O

+

NaOH, H2O and 1,4-Dioxane

R.T

viii) methacryloyl chloride vi) 7-amino-2-phenyl-3H-pyrimido[4,5-d]pyrimidin-4-one

N

NHN N

NH

O

H2C

O

ix) N-(5-oxo-7-phenyl-5,6-dihydro-pyrimido[4,5-d]-pyrimidin-2yl)-methacrylate

CH3

CH3

Scheme 4: Preparation of PPPMA

2.4 Homopolymers

2.4.1 Homopolymerization of (PPPA) (Scheme 5)

Homopolymerization of PPPA, (vii) was carried out in N, N-

dimethyl formamide (DMF) using benzoly peroxide (BPO) as

an initiator. Predetermined quantities of PPPA, DMF and

3 Advances in Polymer Science and Technology: An International Journal 2011; 1 (1): 1-9

BPO were mixed in a three necked round-bottomed flask

equipped with stirrer. The reaction mixture was heated at 70 oC with constant stirring for 12 hours. By pouring the

reaction mixture into the non-solvent water stops the

polymerization and precipitated polymer is filtered. The solid

homo polymer was dissolved in DMF and precipitated with water. This process was repeated to get pure polymer. Then

the polymer was dried under vacuum. The homo

polymerization reaction of PPPA (x) is shown in Scheme 5.

NN

NH

N

NH

O

vii) N-(5-oxo-7-phenyl-5,6-dihydro-pyrimido[4,5-d]-pyrimidin-2yl)-acrylate

O

Benzoyl peroxide

H3CN O

CH3NN

HN

N

NH

O

O

H

m

ix) Poly ( N-(5-oxo-7-phenyl-5,6-dihydro-pyrimido[4,5-d]-pyrimidin-2yl)-acrylate)

Scheme 5: Homopolymerization of PPPA

2.4.2 Homopolymerization of PPPMA (Scheme 6)

Homopolymerization of PPPMA (ix) was carried out in N,N-

dimethylformamide (DMF) using benzoly peroxide (BPO) as

an initiator. Predetermined quantities of PPPMA, DMF and

BPO were mixed in a round-bottomed flask equipped with

stirrer. The reaction mixture was heated at 70oC with constant stirring for 12 hours. By pouring the reaction mixture into the

non-solvent water stops the polymerization and precipitated

polymer was filtered. The solid homopolymer was dissolved

in DMF and precipitated with water. This process was

repeated to get pure polymer. Then the polymer was dried

under vacuum. The homopolymerization reaction of PPPMA

(xi) is shown in Scheme 6.

NN

NH

N

NH

O

viii) N-(5-oxo-7-phenyl-5,6-dihydro-pyrimido[4,5-d]-pyrimidin-2yl)-acrylate

O

Benzoyl peroxide

H3CN O

CH3NN

HN

N

NH

O

O

CH3

m

x) Poly ( N-(5-oxo-7-phenyl-5,6-dihydro-pyrimido[4,5-d]-pyrimidin-2yl)-methacrylate)

CH3

Scheme 6: Homopolymerization of PPPMA.

3.0 Characterization of Monomers

Characterization Techniques

Infrared Spectra

IR spectra of the monomers and polymers as KBr pellet or in

chloroform solution were recorded on a JASCO FT/IR-5300

infrared double beam spectrometer.

1H NMR Spectra

High-resolution 1H NMR spectra were obtained with a

Bruker 500 MHz FT-NMR spectrometer at room temperature

in CDCl3/DMSO-D6 solution for all the monomers and

copolymers. Tetramethylsilane (TMS) was used as the internal reference.

3.1.dN-(5-oxo-7-phenyl-5,6-dihydro-pyrimido[4,5-

d]pyrimidin-2-yl)-acrylate (PPPA)

The IR, NMR spectra of PPPA are shown in Figures 1 and 2.

3.1.1 IR:

A strong absorption band observed in region 1726 cm-1

corresponding to the carbonyl stretching frequency. The absorption at 1638 cm-1 region corresponding to the vinylic

C=C stretching vibration. A peak observed at 1506 cm-1

region was corresponded to aromatic C=C stretching

vibration. Aromatic Ar-H stretching vibration was observed

at 3040 cm-1. An absorption band – NH stretching vibration

was observed at 3356 cm-1.

3.1.2 NMR:

Signals (two singlets) at 5.79 and 6.37 ppm due to vinyl

(CH2=C) protons. The aromatic protons were observed as

two doublets in the region 7.25 and 7.48 ppm. The singlet at

8.86 ppm related to NH group and 2.17 ppm corresponds to

the α–methylene group.

3.2. N-(5-oxo-7-phenyl-5,6-dihydro-pyrimido[4,5-d]

pyrimidin-2-yl)-methacrylate (PPPMA):

The IR, NMR spectra of CMPA are shown in Figures 3 and

4.

3.2.1 IR:

A strong absorption band was observed in region 1740 cm-1

corresponds to the carbonyl stretching frequency. The

absorption at 1630cm-1 region corresponds to the vinylic C=C

stretching vibration. The absorption 2972 cm-1 indicates CH3

stretching and 1377 cm-1 corresponds to –CH- bending in methyl group. A peak observed at 1508 cm-1 region

corresponds to aromatic C=C stretching vibration. Aromatic

Ar- H stretching vibration was observed at 3040 cm-1. An

absorption band of -NH stretching vibration is observed at

3336 cm-1.

3.2.2 NMR:

Signals (two singlets) at 5.80 and 6.38 ppm were due to

vinylic protons. The aromatic protons were observed as two

doublets in the region 7.26 and 7.68 ppm. The singlet at 8.64

ppm corresponds to NH group and 2.07 ppm corresponds to

the α–methyl group.

3.3 Characterization of Homopolymers

The data of IR spectra of Poly (PPPA), Poly (PPPMA) were

shown in Figures 5 and 6, and the data is presented in the

Table 1. The NMR spectra of Poly (PPPA), Poly (PPPMA),

are shown in Figures 7 and 8. The NMR spectra data of Poly

(PPPA), Poly (PPPMA), are presented in the Table 2. In the

monomers, two peaks were observed 5.5 and 6.5 ppm which

corresponds C=C but when polymerized, these peaks

disappear between 5.5 and 6.5 which indicate the formation

of homopolymer

4 Advances in Polymer Science and Technology: An International Journal 2011; 1 (1): 1-9

Figure. 1: IR spectrum of PPPA

Figure 2:

1H-NMR spectrum of PPPA

Figure. 3: IR spectrum of PPPA

5 Advances in Polymer Science and Technology: An International Journal 2011; 1 (1): 1-9

Figure 4:

1H-NMR spectrum of PPPA

Figure. 5: IR spectrum of poly(PPPA).

Figure. 6: IR spectrum of poly(PPPMA).

6 Advances in Polymer Science and Technology: An International Journal 2011; 1 (1): 1-9

Figure 7: 1HNMR spectrum of poly(PPPA).

Figure 8:

1HNMR spectrum of poly (PPPMA).

Figure 9: Thermogravimetric analysis curve of PPPA.

Figure 10: Thermogravimetric analysis curve of PPPMA.

7 Advances in Polymer Science and Technology: An International Journal 2011; 1 (1): 1-9

Table 1: IR Spectral data of Poly (PPPA), Poly (PPPMA) Homopolymer Wave number (cm-1)

V (C-H) V (C=0) V (Ar-C=C) N-H

Poly (PPPA) 3082&

2667 1740 1608 & 1514 3386

Poly (PPPMA) 3097 &

2722 1739 1601 & 1505 3369

Table 2:

1HNMR Spectral data of Poly (PPPA), Poly (PPPMA)

Homo Polymer Chemical shift (ppm) in CDCl3+ DMSO

Poly (PPPA) 1.17 (-CH), 2.07 – 2.52 (-CH2), 7.15-8.00 (m, Ar-H), 8.34

(s, NH)

Poly (PPPMA) 1.32 (b, 3H, ά-CH3 ) 2.49(-CH2), 7.05-7.8 (m, Ar-H), 8.85

(s, NH)

4.0 Thermo chemical properties of the polymers

The thermal properties of the two polymers, PPPA and

PPPMA were investigated by thermogravimetric analysis

(TGA) and differential scanning calorimetry (DSC). For TGA

the samples were heated (PerkinElmer Thermal Analysis

System 409) from 25 °C to 85 °C at a rate of 10 °C/min and

at a nitrogen gas flow rate of 80 mL/min. Polymers PPPA

and PPPMA lost weight gradually during the early phase of the experiment. A thermal gravimetric analysis curve of

PPPA is shown in Figure 9. The thermal analysis results

indicated that polymers PPPA and PPPMA have a high

decomposition temperature Td and good thermal stability

above 300 °C, which is very attractive for the fabrication of

stable organic electroluminescent devices. For DSC, the

samples were first heated to melting, then cooled to the

glassy state and reheated to determine the glass transition

temperature (Tg). A representative DSC graph is shown for

PPPA in Figure 11. Tg is calculated from the second heating

curve. As it can be seen from the graph, Tg for the polymer is

154 °C.

5.0 Antimicrobial activity

The biological activities of the monomers and their

homopolymers and were tested against different

microorganisms with DMSO as the solvent. The sample

concentrations were 100 μg. All microorganism strains were

obtained from the Culture Collection of Microbiology

Laboratory of S.K University (Anantapur, India). In this

study, Staphylococcus aureus ATCC 29213, Escherichia coli

ATCC 25922, Pseudomonas aeruginasa ATCC 27853,

proteus vulgaris, Salmonella enteridis, and Klebsiella pneumoniae were used as bacteria. Candida albicans CCM

31 was a fungus. YEPD medium cell culture was prepared as

described by Connerton [25]. Ten milliliters of YEPD

medium were inoculated with each cell from plate cultures.

Yeast extract 1% (w/v), bactopeptone 2% (w/v), and glucose

2% (w/v), was obtained from Difco. Microorganisms were

incubated at 35 ◦C for 24 h. About 1.5 ml of these overnight

stationary phase cultures were inoculated onto 250 ml of

YEPD and incubated at 35 ◦C until OD600 reached 0.5. The

antibiotic sensitivity of the polymers was tested with the

antibiotic disk assay as described [26]. Nutrient Agar (NA)

was purchased from Merck. About 1.5 ml of each prepared different cell culture was transferred into 20 ml of NA and

mixed gently. The mixture was inoculated into the plate. The

plates were rotated firmly and allowed to dry at room

temperature for 10 min. prepared antibiotic discs (50 and 100

μg) were placed on the surface of the agar medium [27]. The

plates were kept at 5 ◦C for 30 min and then incubated at 35

◦C for 2 days. If a toxic compound leached out from the disc,

it means that the microbial growth is inhibited around the

sample. The width of this area expressed the antibacterial or

antifungal activity by diffusion. The zones of inhibition of

microorganism growth of the standard samples monomers and homopolymers, were measured with a millimeter ruler at

the end of the incubation period.

Generally, monomers have higher activity than their

polymers. However, poly(PPA) is more active against some

microorganisms than PPA monomer in this study. This can be

observed for some polymer molecules, because the phenyl

and pyridine content of the poly (PPA) appears to be most

important to impart antimicrobial properties, it is possible

that the conformation of the polymers acquired under

experimental conditions may also be a factor for their

antimicrobial activity. The results were standardized against penicilin, g. and

teicoplanin under the same conditions. All the compounds

exhibited moderate activity comparable to that of the standard

drugs. The data reported in Table 6 are the average data of

three experiments. The results show that the investigated

polymers have good biological activity comparable to that of

standard drugs such as penicilin, g. and teicoplanin. The

results suggest that the monomers, polymers and the some

copolymers have good biological activity on the

Staphylococcus aureus microorganisms in comparison with

standard drugs.

6.0 Conclusions

The synthesis of new monomers (PPPA and PPPMA) having

pendant Pyridine and Pyramidine moieties have been

8 Advances in Polymer Science and Technology: An International Journal 2011; 1 (1): 1-9

Table 6. Antimicrobial effects of the compounds (mm of zones)

compounds Pseudomonas

Aeruginasa

Escherichia

coli

Proteus

vulgaris

Salmonella

enteridis

Klebsiella

pneumoniae

Staphylococcus

aureus

Candida

albicans

PPA 15 16 18 16 14 18 17

PPMA 11 12 11 09 12 16 14

PolyPPA 13 13 12 11 - 15 11

PolyPPMA 10 11 - 14 15 16 09

6/94 12 - 14 16 11 - 16

15/85 15 09 16 - 18 12 11

29/71 - 15 12 12 11 16 18

39/61 13 16 15 14 12 17 15

60/40 13 16 13 14 11 16 -

69/31 18 11 10 09 - - -

Penicillin G 16 12 09 16 18 17 35

Teicoplanin 18 18 11 22 25 12 15

DMSO - - - - - -

reported for the first time. The structure of monomer and its

polymer was characterized by spectroscopic methods.

Polymers of PPPA with acryloyl chloride were prepared by

free-radical polymerization in 1,4-dioxane at 70 ◦C. The

activation energy of the decomposition of the investigated

polymers was calculated by the Ozawa method with the TGA

data. The polymers have good biological activity comparable to that of standard drugs such as penicillin, g. and teicoplanin.

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Source of support: Nil; Conflict of interest: None declared