54

CHAPTER –IV

Synthesis And Biolgical Evaluation of

Schiff Bases Bearing Pyrimidine Moiety.

55

INTRODUCTION

Pyrimidines:

Pyrimidine is a heterocyclic aromatic organic compound similar to

benzene and pyridine, containing two nitrogen atoms at positions 1

and 3 of the six-member ring. It is isomeric with two other forms

of diazine. Pyrimidine is a colourless compound, melting point

(22.5o C), boiling point (124oC). Pyrimidines are aromatic, basic and

water soluble compounds and pyrimidine is a much weaker base

than pyridine.

Three pyrimidine derivatives that are integral part of DNA

and RNA well as to structure of medicinally active agents.

Thymine may also referred as 5-methyl uracil. The substituted

pyrimidines are complex molecules because of the nature of the

substituents. Uracil and thymine may be considered to contain the

neutral urea unit or the acidic imide moiety.

The metabolism of these unique pyrimidines is important from the

standpoint of both biochemical utilization of these compounds and

drug metabolism of pyrimidine derivatives. Uracil is converted into

a useful compound uridylic acid needed for synthesis of RNA. In a

56

similar manner cytosine is conjugated with PRPP to yield cytidine-

5- monophosphate or cytidylic acid. Thymine is metabolized by

conjugation via salvage pathway with PRPP to the thymine ribosyl

–5-phosphate. This form of thymidylic acid can be utilized in

specific RNA molecules.

Two special pyrimidines are barbituric acid and substituted

barbiturates. The substituted barbiturates represent a special

class of compounds. Which have been used for their sedative

hypnotic action.

Barbituric acid is a fairly strong acid with a Pka of 4.12, but

upon substitution at the 5 positions, the Pka rises dramatically,

the 5,5- disubstituted barbiturates react with sodium hydroxide to

form a salt that is quite water-soluble.

Properties of Pyrimidines

Aromaticity1:

A new σ-п separatability criterion is used to divide the total

energy of planner ring system into two parts. A σ part and п part.

The behavior of these parts under distortions towards resonance

structure is investigated. The Callen showed that the σ energy

tends towards bond equalization. The total structure depends on

the relative dominance of “σ vs п” electron tends. The properties of

57

benzene referred to as aromaticity as due to a forced п-electrons

delocalization.

Oxidation: All the three peracids2 (U, T, C) giving N- oxides but

care must be taken with pyrimidines3 due to the relative instability

of the products under the acidic conditions. Pyrazines form N, N1 –

dionides most easily, but pyridazine4 requires forcing conditions5.

Alkylation:

The pyrimidines react with alkyl halides to give mono

quaternary salts, through somewhat less readily than comparable

to pyridines.

Pyrimidines of Pharmacological interest:

Certain pyrimidine derivatives are also known to display

antimalarial6, antifilarial7, and anti-leishmanial8 activities. The

biodynamic property of this ring system prompted us to design

pyrimidine derivatives stimulating pharmacophores and

substituents responsible for diverse pharmacological activities.

Nandeeshaiah et al.,9 reported the synthesis of (1) which

showed the blood platelet disaggregating property.

58

SeNN

N

NHR

R=H

(1)

Hansa et al.,10 have reported the synthesis of (2) which

showed potential anti-microbial activity.

(2)

Upadhyay et al.,11 have reported the synthesis of (3)

pyrimidine and azo pyrimidines as biodynamic agents.

(3)

Heidal et al.,12 prepared 5-Fluorouracil (4) and

demonstrated its significant tumour inhibiting activity.

(4)

Parmar et al.,13 have reported the synthesis of thiazolidinones

(5) from hydrazino pyrimidine as potential anti-microbial agent.

(5)

Sondhi et al.,14 have reported anti-inflammatory and

analgesic activity of synthesized pyrimidine derivatives (6,7).

59

( 6 ) ( 7 )

RIVEW OF LITERATURE

Rajesh et al.,15 synthesized some 2- amino- 4, 6-

diarylsubstituted pyrimidines (8) and then screened them for

antibacterial and herbicidal activity.

( 8 )

Laura et al., 16 synthesized some new amino derivatives of 1,

2, 3- triazole [4, 5- d] pyrimidines (9) and then screened them for

their affinity towards A1 and A2A adenosine receptors.

( 9 )

Erric et al., 17 synthesized analogues of 4- benzylamino-2-

7H- pyrrolo [2, 3- d] pyrimidines (10) and then screened them for

their anxiolytic activity.

(10)

Silvana et al., 18 synthesized novel purine and pyrimidines

nucleoside analogues containing 2, 3- epoxypropyl, 3-amino- 2-

hydroxyl or 2, 3- epoxypropyl ether moieties (11) and then

screened them for their anti-tumour and antiviral activity.

60

(11)

Munchhof et al., 19 synthesized thieno pyrimidines and

thieno pyridines (12) and then screened them for anticancer

activity.

(12)

Nakashima et al., 20 synthesized fused pyrimidine

derivatives (13), and screened them for blood oxygen partial

pressure amelioration.

(13)

John et al., 21 synthesized thieno [3, 2- d] pyrimidines and

furo [3, 2- d] pyrimidines (14) and then screened them for

purinergic receptor antagonists.

(14)

61

SCHEME-II

CHO

FH3C

O

O

O

CH3

Piperidine

CH3COOH

F

COOCH3

H2N

S

HN

CH3

2

H2SO4

F

HN

N

O

OCH3

CH3

CH3

SH3C

F

N

N

O

OCH3

CH3

CH3

SH3C

mCPBA

MDC

F

N

N

O

OCH3

CH3

CH3

SH3C

O O

Methanol

CH3NH2

F

N

N

O

OCH3

CH3

CH3

NH

H3C

F

N

N

O

OCH3

CH3

CH3

NH3C

NaH \ DMF

CH3SO2Cl

SH3C

O

O

DIBAL-H

Toluene

F

N

NCH3

CH3

NH3C

SH3C

O

O

OH

F

N

N

CHO

CH3

CH3

NH3C

SH3C

O

O

DDQ

Toluene

Toluene

MnO2

F

N

NCH3

CH3

NH3C

SH3C

O

O

N

R-NH2

CH3

HMPA

R

(SA-1)

(SA-2)(SA-3)(SA-4)

(SA-5) (SA-6)(SA-7I)

(SA-8a-j)

CH3COOH

p- FluroBenzaldehyde

Methyl isobutyrylacetate

O

62

EXPERIMENTAL

Procedure for the preparation of Compound SA-1:

P-fluorobenzaldahyde (5.94g, 0.1 mol), methylisobutyryl acetate

(6.33g, 0.1 mol), piperidine (0.42g, 0.01mol) and acetic acid (0.2ml)

was placed in a round bottom flask fitted with a condenser and the

mixture was heated at 85-90o C for 3hrs. Cooled the reaction

mixture and added chloroform (100ml). The separated chloroform

layer was washed with 0.1N HCl (50ml), 5 % aq. Sodium

bicarbonate (50ml) and finally with water (100ml). The solvent was

evaporated completely. The crude mass (12g) was purified by

Column chromatography. (Yield-70%, b.p- 320oC).

Procedure for the preparation of Compound SA-2:

Compound SA-1 (10g, 1mol), S-methyl urea hydrogen sulphate

(7.93g, 0.8mol) and hexamethyl phosphoramide (15ml) was placed

in a round bottom flask and the mixture were heated at 120o-

125oC for 24hrs. Cooled the reaction mixture to 80o C and added

toluene (50ml), 5% aq.Sodium bicarbonate (50ml). The mixture

was stirred for 30 min at 80-85o C. Cooled the reaction mixture to

25 o C and separated the toluene layer. It was washed with water

(50ml). The solvent was evaporated under reduced pressure, to get

the crude mass. Fresh toluene (50ml) was added to the crude mass

in a round bottom flask to which added a solution of 2,3-Dichloro-

63

5,6-dicyno-1,4-benzoquinone(DDQ) (4.7g, 0.5mol) in toluene(50ml)

slowly for 15min at 25-30o C. The reaction mixture was stirred for

2hrs. Filtered the 2,3-Dichloro-5,6-dicyno-1,4-hydroquinone

(DDHQ) and washed the DDHQ with toluene (10ml) and filtrate

was washed with 5 % aq. NaHCO3 (4×50ml) and water (100ml). The

solvent was evaporated to get the solid. The solid was recrystallised

from ethanol. (m.p.- 85oC, yield - 76%.)

Procedure for the preparation of Compound SA-3:

In to a clean dry round bottomed flask introduced Compound

SA-2 (10g,0.1mol) in Dichloromethane(100ml) to which was added

a solution of m-choloroperbenzoic acid (13.5g, 0.25mol) in

dichloromethane (50ml) for 30min at 0-5oC. Slowly warmed the

mixture to 25-30oC and stirred for 2hrs at same temperature (m-

chlorobenzoic acid precipitated out). Filtered the m-chlorobenzoic

acid and the filtrate was washed with 5 % aq. NaHCO3 (2×50ml)

and with water (100ml). The solvent was evaporated to get the

crude mass from which pure solid was obtained by the addition of

hexane (50ml). The separated solid was recrystallised with ethanol.

(m.p.- 120 oC, yield-68%.).

64

Procedure for preparation of Compound SA-4:

The compound SA-3 (10g) in menthol (50ml) was placed in a

round bottom flask and cooled the solution to 0-5oC. Added a

solution of 25 % methylamine in methanol (100ml) at 0-5o C for

1hr. and it was refluxed for 2hrs. The solvent was evaporated to get

the solid and the solid was recrystallised with ethanol. (m.p- 95oC,

yield- 80 %.).

Procedure for the preparation of Compound SA-5:

The compound SA-4 (10g, 0.1mol) in DMF (50ml) was placed

in a round bottom flask and cooled the solution to 0-5oC. Added

60 % Sodium hydride (1.5g, 0.12mol) under nitrogen. Stirred the

reaction mixture for 30 min at 0-5o C, added a solution of methane

sulfonyl chloride (3.76g, 0.1mol) in DMF (25ml) for 10min at 0-5

oC. Stirred the mixture for 1hr. Then the reaction mixture was

added to ice cold water with stirring. Filtered the separated crude

solid and washed with 5 % aq. NaHCO3 (50ml) and water (50ml).

The product was recrystallised from ethanol. (m.p.- 109oC, yield -

50%.).

Procedure for the preparation of Compound SA-6:

The compound SA-5 (10g, 0.1mol) was dissolved in toluene (150ml)

in a clean dry round bottom flask. Cooled the reaction mixture to -

65

10 oC under nitrogen atmosphere. Slowly added a solution of 25 %

DIBAL-H in toluene (41.5ml, 0.25mol) for 30min at -10 oC. Added

water (100ml) and stirred for 30min and filtered the reaction

mixture, and washed with hot toluene (50ml). The water was

separated from filtrate. The toluene was evaporated completely to

get solid. The solid was recrystallised with ethanol. (mp.- 79 oC ,

yield-72%.).

Procedure for the preparation of Compound SA-7:

The compound SA-6 (10g) in toluene (100ml) and active Mno2 (20g)

were placed in a round bottom flask and stirred for 15 hrs at 25-30

oC. Filtered the Mno2 and washed with hot toluene (50ml). Then

the solvent was evaporated completely to get the solid. The solid

was recrystallised with ethanol. (m.p.- 62 oC, yield- 65 %.).

Procedure for the preparation of Compound SA-8a-j:

The compound SA-7 (0.1mol) and substituted amines (0.1mol) in

glacial acetic acid (25ml) were placed in a round bottom flask fitted

with a condenser and refluxed for 30 hrs. The mixture was added

to ice cold water with stirring, filtered the separated solid and

washed with water until the filtrate is neutral. The product was

recrystallised from ethanol and (Yields were in the range 60-70 %).

66

BIOLOGICAL ACTIVITY

All the synthesized compounds were evaluated for their

antibacterial, anti inflammatory activity by following the standard

procedure.

ANTIBACTERIAL ACTIVITY:

All the compounds synthesized in the present investigation

were screened for their anti-bacterial activity by subjecting the

compounds to standard procedures. Antibacterial activities were

tested on nutrient agar medium against streptococci and

pseudomonas aureus which are representative types of gram

positive and gram negative organisms respectively. The

antibacterial activity of the compounds was assessed by disc-

diffusion method22.

67

TABLE-6: Antibacterial activity of compounds SA-8a-j.

Sl. No

Name of the compounds

Mean zone of inhibition (in mm)

Streptococci Pseudomonas aureus

50g 100g 50g 100g

01 Streptomycin 21 25 20 23

02 SA-8a 14 19 14 19

03 SA-8b 11 20 15 20

04 SA-8c 13 19 15 19

05 SA-8d 15 18 19 21

06 SA-8e 16 17 16 21

07 SA-8f 15 20 14 16

08 SA–8g 16 21 15 17

09 SA–8h 15 21 11 16

10 SA–8i 15 20 12 17

11 SA–8j 16 21 13 18

Average triplicate ± Standard deviation

Note:- “ – “denote no activity, 06 – 07mm poor activity, 08 –

10mm moderate activity, 11-12mm good activity.

68

ANTI INFLAMMATORY ACTIVITY:

Determination of acute toxicity (LD50):

The acute toxicity of synthesized compounds was

determined by using albino mice of either sex (20-30 gm) those

maintained under standard husbandry conditions. The animals

were fasted overnight prior to the experiment and fixed dose

(OECD guideline No. 420) method of CPCSEA was adopted for

toxicity studies. 1/5th of the lethal dose was taken as effective

dose ED50 (Therapeutic dose).

Anti inflammatory activity:

Inflammation is a normal protective response to tissue injury

caused by physical trauma, noxious chemicals or microbiologic

agents. Inflammation is body‟s response for tissue repair 23.

Inflammation is triggered by the release of chemical mediators

from the injured tissues and migrating cells. The specific chemical

mediators vary with the type of inflammatory process and include

amines such as histamine, serotonin, and lipids such as

prostaglandins and small peptides such as kinins 24. The acute

inflammatory response has 3 main functions.

The affected area is occupied by a transient material

called the acute inflammatory exudates. The exudates

69

carry proteins, fluid and cells from local blood vessels

into the damaged area to mediate local defenses.

If an infective causative agent (e.g. bacteria) is present in

the damaged area, it can be destroyed and eliminated by

components of the exudates.

The damaged tissue can be broken down and partially

liquefied, and the debris removed from the site of

damage.

Mechanism of inflammation:

In and around the inflamed tissue, there is an accumulation of

oedema fluid in the interstitial compartment which comes from

blood plasma by its escape through the endothelial wall of

peripheral vascular bed. In initial stage the escape of fluid i.e. due

to vasodilatation and consequent elevation in hydrostatic pressure,

the characteristic inflammatory oedema, and exudates appear by

increased vascular permeability of micro circulation. Inflammatory

diseases including different types of rheumatic diseases are a

major cause of morbidity of the working force throughout the

world. Many drugs produced a dramatic symptomatic improvement

in rheumatic processes, but all of them shared the common

undesirable effect i.e., gastrointestinal irritation 25.

70

Procedure 26, 27:

Carrageenan induced rat paw oedema model:

Albino rats of either sex weighing 150-200 gm were selected.

They were maintained on standard pellet diet and free access to

water.

The animals were divided into 12 groups each having six

animals. The various groups were treated as follows:

Group 1 - Normal Control (treated with 0.2mlof 5% gum acacia

p.o.)

Group 2 - Diclofenac (50 mg / kg, p.o.)

Group 3 - Compound SA-8a (50 mg/ kg, p.o.)

Group 4 - Compound SA-8b (50 mg/ kg, p.o.)

Group 5 - Compound SA-8c (50 mg/ kg, p.o.)

Group 6 - Compound SA-8d (50 mg/ kg, p.o.)

Group 7 - Compound SA-8e (50 mg/ kg, p.o.)

Group 8 - Compound SA-8f (50 mg/ kg, p.o.)

Group 9 - Compound SA-8g (50 mg/ kg, p.o.)

Group 10 - Compound SA-8h (50 mg/ kg, p.o.)

Group 11 - Compound SA-8i (50 mg/ kg, p.o.)

Group 12 - Compound SA-8j (50 mg/ kg, p.o.)

The normal control, diclofenac and test compounds were

administered to the rats 30 minutes before the injection of 0.1ml of

1% Carrageenan suspension in normal saline. Carrageenan

suspension was injected into the sub-planar region of the left hind

71

paw, and the right hind paw served as reference. Immediately there

after the oedema volume of the injected paws were measured

plethysmographically by mercury displacement method.

For comparison purpose, the volume of oedema at various

prefixed time intervals was measured. The difference between paw

volumes of the treated animals was measured and the mean

oedema volume was calculated.

Percentage reduction in oedema volume was calculated by using

the formula,

Vo - Vt Percentage reduction = x 100 Vo

Where, Vo = Volume of the paw of control at time„t‟.

Vt = Volume of the paw of drug treated at time„t‟.

From the data obtained, the mean oedema volume with

standard error (SEM), standard deviation (S.D.), percentage

reduction in oedema was calculated. The anti-inflammatory activity

of synthesized compound is given in the Table No-7.

72

TABLE-7:Anti-inflammatory activity of compounds SA-8a-j.

Sl.

No.

Name

Compounds

Dose

mg/kg

Mean difference in paw

volume S.E. after

3hr(ml)

Percentage of inhibition

01 Control -- 0.80 0.024* --

02 Standard

(Diclofenac Na) 50 0.16 0.01* 80.00

03 SA-8a 50 0.48 0.038* 40.00

04 SA-8b 50 0.31 0.027* 61.25

05 SA-8c 50 0.34 0.020* 58.00

06 SA-8d 50 0.50 0.033* 37.50

07 SA-8e 50 0.45 0.020* 43.75

08 SA-8f 50 0.54 0.032* 33.00

09 SA-8g 50 0.42 0.019* 47.50

10 SA-8h 50 0.29 0.026* 63.75

11 SA-8i 50 0.55 0.025* 31.25

12 SA-8j 50 0.49 0.021* 38.75

73

Result and Discussions:

All the synthesized pyrimidine derivatives were evaluated for

their antibacterial activity against the organisms Streptococci,

Pseudomonas aureus by disc diffusion method. Streptomycin

was used as a standard drug for compression. Antibacterial

evolution results indicate that the compounds are moderately

active against both the organism at the concentration 50µg/ml and

100µg/ml. Though the schiff‟s bases are known to show broad

spectrum of biological activity, perhaps the steric hindrance due to

the different crowed groups may hinder the activity of the

compounds irrespective of nature of substituents.

Among the series of the compounds SA-8a-SA-8j screened

for anti-inflammatory activity the compounds SA-8b, SA-8c and

SA-8h showed significant activity when compared with rest of the

compounds of the series. However these active compounds proved

to posse‟s moderate activity when compared with standard

Diclofenac sodium.

74

REFERENCES

1. Jug, Karl, Koester and Andreas M. J Am Chem Soc

1990; 112: 67727.

2. Koelsch CF and Gumprecht WH. J Org Chem 1958;

23: 1603.

3. Yamahaka H, Ogawa S and Sakamoto T. Heterocycles

1981; 16: 573.

4. Sazuki I, Nakadate M and Sueyoshi S. Tetrahedron

Lett 1968; 9(15): 1855-1858.

5. Salim AWS and Shakhir M. Chem Abstr 1994; 120:

280314.

6. Falco EA. Brit J Pharmacology 1961; 6: 185.

7. Howells RE, Tinslu J, Devancy E and Smith G. Chem

Abstr 1982; 96: 384k.

8. Ram VJ. J Prakt Chem 1989; 331: 893.

9. Nandeeshwariah SK and Sarvottam Ambekar. Ind J

Chem 1998; 37B: 995.

10. Manish Shah, Khyati Parikh and Hansa Parekh. Ind J

Chem 1998; 37B: 73.

11. Upadhyay DN and Vishnu J Ram. Ind J Chem 1999;

38B: 173.

12. Filler R. J Fluorine Chem 1986; 33: 362.

75

13. Pramar JM, Modha JJ and Parikh AR. Ind J Chem

1999; 38B: 440.

14. Sondhi SM and Verma RP. Ind Drugs 1999; 36(1): 50.

15. Rajesh Vyas, Udaibhan Gahlot S and Verma BL. Ind J

Heterocyclic Chem 2003; 13: 15.

16. Laura Betti, Guiliana Biagi, Gino Giannaccini, Irene

Giorgi, Oreste Livi, Antonio Lucacchini, Clementina

Maner and Valerio Scarto. Eur J Med Chem 1999; 34:

86.

17. Erric Meade A, Marcos Sznaidman, Gerald Pollard T,

Lilia Beauchamp M, James Howard L and Geraid

Pollard T. Eur J Med Chem 1998; 33: 363.

18. Silvana RaicMalic, Mira Grdisa, MIaden Mintas and

Kresimir Pavelic. Eur J Med Chem 1999; 34: 405.

19. Munchhof, Michel John, Sobolovjaynes and Susabeth.

US Appl 1997; 651191.

20. Nakashima, Yoshiharu, Fujita, Takashi, Hizuka,

Michiyo, Ikawa, Hiroshi and Toru. Eur Pat Appl 1991;

EP 899, 263.3.

21. John, Lerpiniere, Joanne Dawson Claine, Elizabeth

Gaur, Suneel, Pratt and Robert Mark. PCT Int Appl

2002; W 00255, 524, 18.