Synthesis, Characterization and Evaluation of some newer Pyrimidine derivatives as Anti-inflammatory Agents

 

Kalpana Divekar*, Rekha. S, Murugan. Vedigounder, Shivaprakash. H

College of Pharmaceutical Sciences, Dayananda Sagar University, Shavige Malleshwara Hills,

Kumarswamy Layout, Bangalore.

 

ABSTRACT:

Pyrimidine is an important baseone of the base formed by hydrolysis of nucleosides. It is an interesting molecule in the medicinal chemistry because  of its diversified biological activities. Alloxan which is an oxidation product of Uric acid is also a pyrimidine derivative  of interest to a medicinal chemist. Several pyrimidines are  reported as antimicrobial, analegesic, anti-inflammatory, antibacterial, and antiparasitic agents. Pyrimidine scaffold is considered as an interesting one due to its various pharmacological properties. In this scheme, an attempt is made to carry out synthesis of some new pyrimidine derivatives. The Starting material Chalcone is synthesized by condensation of various aromatic aldehyde and aromatic ketone. Chalcone is then treated with thiourea and KOH in presence of ethanol to yield pyrimidine derivatives. Then those pyrimidine derivatives were subjected to alkylation and acetylation. The synthesized compounds were characterized and confirmed by IR and ¹HNMR spectroscopy and then evaluated for their anti-inflammatory activity. The anti-inflammatory activity of newly synthesized pyrimidine derivatives were carried out by the carrageenan induced rat hind paw edema method by taking Diclofenac sodium as standard.

 

 

 

INTRODUCTION:

Anti-inflammatory agents are compounds that reduce the pain and swelling associated with inflammation¹. Inflammation is a complex phenomenon involving interrelationship of humoral and cellular reactions through a number of inflammatory mediators; it is a response of the body to injuries such as a blow or a burn. The swelling of the affected region of the body occurs because fluid is directed to that region. The inflammatory response can slow down the healing process in conditions like rheumatoid arthritis. The anti-inflammatory agents can reduce the discomfort experienced by of arthritic, patients and also other conditions like asthma. Cortico steroids and nonsteroidal anti-inflammatory agents are two types of anti-inflammatory agents.

 

When the COX enzyme is inhibited by NSAIDs, there i production of prostaglandins and thromboxanes is inhibited. COX-1 is a constitutive enzyme and COX-2 is induced in inflammatory cells. The anti-inflammatory action of NSAIDs is due to inhibition of COX-2 and gastric irritation is due to inhibition of COX-1which is an unwanted effect. NSAIDs can also act as antipyretics because of inhibition in prostaglandin production in hypothalamus². Various pyrimidines have been reported for their various biological activites.^3,4,5, Anti-inflammatory^6,7, Analgesic⁸ and antibacterial^9,10 agents. Pyrimidines have gained lot of interest due to their of diverse chemical and biological activities.

 

MATERIALS AND METHODS:

Materials:

In the present work the chemicals which were used were of AR grade and LR grade, purchased from Loba Chemie, Qualigens, sigma, Ranchem, and Merck. Melting points determination was by open capillary tubes and Thiel`s melting point apparatus. Thin layer chromatography was carried out using silica gel G as stationary phase and different proportions of mobile phase. The spots resolved were visualized by using UV and iodine chamber. The compounds are synthesized by both Microwave irradiation and by classical heating. The synthesized compounds were characterized by IR spectra and ¹H-NMR spectra. The IR spectra were recorded on a Fourier Transform IR spectrometer (model Shimadzu 8400s) in the range of 400-4000 using KBr pellets and the valuesof Vmax are reported in cm^{-1 1}H-NMR spectra were recorded in  Brookfield 200 MHz-NMR spectrometer (Astrazeneca India Ltd) using CDCl₃ and chemical shifts (δ) are reported in parts per million downfield from internal reference Tetramethylsilane (TMS).

 

 

Methods:

1. Synthesis:

Step-I Preparation of Chalcones¹¹:

5.5moles of sodium hydroxide was dissolved in 200ml of water and 100g (122.5ml) of rectified spirit was cold. 52g (0.43mol) of aromatic ketone which was distilled freshly was added with stirring, and then 46g (44ml, 0.43mol) aromatic aldehyde was added drop wise with stirring. The mixture was stirred vigorously until the mixture is so thick that stirring is no longer effective (2-3 hrs), by maintaining the  temperature of the mixture at about 25°C, the reaction mixture was removed and kept in the refrigerator overnight. The product was filtered with suction on a Buchner funnel, washed with cold water until the washings are neutral to litmus, then with 20ml of ice-cold rectified spirit. The recrystallization of crude chalcone was using rectified spirit.

IR  KBr (cm^-1): 3047(Ar, C-H), 1656(C=O), 1593(C=C), 1317(CH=CH), 758 (C-Cl).¹H NMR (δppm): 7.26(2H,S, CH=CH), 7.4-7.9(m, 9H,aromatic)

 

 

Step-II Preparation of pyrimidine derivatives¹²:

chalcone (0.01mol), thiourea (0.01mol) and potassium hydroxide (1gm) were mixed  in ethanol (30ml) and refluxed for 4hrs. This was slowly poured into 400ml cold dil.HCl solution with continues stirring. After 1hr, This was kept in a refrigerator for 1 day. The precipitate solid was rinsed with water, filtered, dried and using pet ether the product was recrystallized.

 

IR  KBr (cm^-1): 2920 (SH), 3180 (Aromatic, C-H), 1676 (C=N), 1554 (C=C, aromatic), 759 (C-Cl).

 

¹H NMR (δ ppm ): 3.2 (S, 1H, SH), 5.2(1H, S, CH=C-), 7.2-8.1(m, 9H, aromatic)

 

Step- III   Preparation of Alkylated Pyrimidine derivatives¹³:

 

A solution of Substituted Pyrimidine derivative (3.7g, 0.02mole) in dry benzene (60ml) was cooled to 0-5°C. Chloroacetyl chloride (5ml. 0.04mole) dissolved in dry benzene (20ml) was slowly added to the solution with vigorous stirring. The reaction mixture was refluxed for 3hrs.Benzene was removed and  the residue was washed with 5% NaHCO₃, and later with water. The crude product was dried and crystallized from ethanol.

 

IR  KBr (cm^-1): 1683 (C=N), 1730 (C=O ), 1579 (C=C, aromatic), 761 (C-Cl).¹H NMR (δ ppm ) 5.75(S, 1H, CH=C-), 3.8(2H, S, CH₂-Cl), 7.3-8.06 (m, 9H, aromatic)

 

Step IV:  Preparation of Acetylated Pyrimidine derivatives⁶:

Pyrimidine derivatives (0.005 mol) and acetic anhydride (10ml) and pyridine (5ml) was refluxed for 4 hours. The residue obtained after removing the excess acetic anhydride poured into ice-cold water, solid was filtered and using a mixture of DMF and ethanol the product was recrystallised.

IR  KBr (cm^-1): 3061 (C-H), 1689 (C=O), 1585 (C=N), 1491 (C=C).

 

Table No.1 Physical Properties of the Alkylated Pyrimidine compounds.

 

 

Table No.2 Physical Properties of the Acetylated Pyrimidine compounds.

 

2. Pharmacological Studies:

(A) Acute toxicity studies (lD₅₀):

The acute toxicity study of pyrimidine derivatives was carried out using Albino swiss mice (23-25gm). The animals were fasted for 24 hrs prior to the experiment and up and down procedure (OECD guideline no. 425) method of CPCSEA was adopted for acute toxicity studies.

 

Procedure:

The procedure was divided into two phases, Phase I(observation made on day one), and Phase II (observed the animals since next 14 days). Two groups of healthy female rats (each group of 3 rats) were used for the experiment. First group animals were divided and fasted for 18 hours deprived from food, water withdrawn before 4 hours of the dosing, body weights were noted before and after dosing pyrimidine derivative (1000mg/kg) orally. Individually animals were observed for 4 hours to see any clinical symptoms, any change in behavior or mortality. 6 hours post dosing again body weights is recorded. From the next day onwards, each day for 1 hour the behavioral change, clinical symptoms or mortality was observed in the same animals for next 14 days and animal body weights were recorded on 8^th and 14^th day. The same procedure was repeated with another group of animal to minimize the errors.

 

(B) Anti Inflammatory studies¹⁴:

The anti-inflammatory activity of the newly synthesized pyrimidine derivatives were carried out using Carrageenan induced rat hind paw edema method.

 

Principle:

Inflammation is a tissue-reaction to infection, irritation or foreign substance. It is a part of the host defense mechanism. The inflammatory reaction is readily produced in rats in the form of paw edema with the help of irritants. Substances such as carrageenan, formalin, bradykinin, histamine, mustard when injected to the dorsum of the foot of rats they produce acute paw edema within a few min of injection. Carrageenan is a sulphated polysaccharide obtained from sea weed (Rodophyceae) and by causing the release of histamine, 5-HT, Bradykinin and prostaglandin it produces inflammation and edema.

 

Procedure:

Rats were assigned into ten groups of 6 animals each. They were marked with picric acid for individual animal identification. The animals were deprived of food overnight (allowed free access to water ad libitium) and the synthetic compounds were administered once before 30 mins the injection of carrageenan. Group I was the solvent control  which received normal saline in a volume not exceeding 0.5ml/100gm orally. Group II was a Positive control which received Diclofenac Sodium (50mg/kg) in a volume not exceeding 0.5ml/100gm orally. Group III to group X Received synthesized Pyrimidine Derivatives at a dose of 100mg/kg suspended in 0.5%w/v CMC in a volume not exceeding 0.5ml/100gms orally. After 30 min. of test compound administration, 0.1ml of 1%w/v of carrageenan in normal saline was injected into the sub plantar region of the left hind paw of the rat. Immediately after the carrageenan injection, the volume of its displacement was measured using plethysmometer. The readings were recorded at 0, 60, 120 and 180 mins. The % inhibition of edema was calculated at the end of 180 mins by using the formula

 

% inhibition = 100 x (1-Vt /Vc)

 

Vt/Vc =edema volume in the rat treated with test drug and control respectively.

 

Table No.3: Effect of Pyrimidine derivatives on carrageenan induced hind paw edema in rats.

Compound	Dose (mg/kg)	Increased paw volume(mm)			% decrease in paw volume after 180 min.
		After 60 min.	After 120  min.	After 180 min.
Control	Saline Soln	44±0.01	51±0.003	59±0.03
Diclofenac Sod	50	11±0.03***	14±0.009***	16±0.03***	72.88
1	100	30±0.02*	42±0.09*	56±0.04*	05.84
2	100	32±0.04**	44±0.04**	55±0.01**	06.70
3	100	15±0.04***	20±0.03***	25±0.01***	57.62
4	100	16±0.02***	20±0.003***	23±0.01***	61.01
5	100	14±0.02***	22±0.03***	24±0.01***	59.32
6	100	18±0.02***	26±0.03***	30±0.03***	49.15
7	100	17±0.02***	24±0.03***	29±0.03***	50.84
8	100	34±0.02**	48±0.03**	55±0.03**	6.70

Values are expressed as mean ± SEM, [number of animal (n)=6].Values were analyzed one way ANOVA followed by Tukey-Kramer’s test. Where, * represents mild significant at P<0.05,

** represents moderate significant at P<0.01,

*** represents highly significant at P<0.001,

ns - represents non significant at P>0.05 Vs control.

 

RESULTS AND DISCUSSION:

The designed compounds were synthesized according to the procedures as given in the methodology. The reactions were monitored by TLC. The physical constants like melting point and solubility were determined for all the intermediate and final products. The compounds were further characterized by IR and ¹HNMR. All the titled compounds were evaluated for their anti-inflammatory activity. The test compounds were screened for anti-inflammatory activity by carrageenan induced rat hind paw edema method using Diclofenac sodium as standard. The animal study of anti-inflammatory activity screening suggested that at the end of 180 min among alkylated pyrimidine compounds 4-methoxyphenyl and 2,4-dichlorophenyl derivatives showed high significance(P<0.001), 4-chlorophenyl derivative showed moderate significance(P<0.01), and diphenyl derivative showed mild significance(P<0.05) compared to the standard Diclofenac sodium. And among acetylated compounds bis-4-chlorophenyl, 4-bromophenyl and bis-2,4-dichlorophenyl derivatives showed high significance (P<0.001), and 4-chlorophenyl derivative showed moderate significance (P<0.01) compared to the standard Diclofenac sodium at the end of 180 min.

 

CONCLUSION:

The aim of this work was to synthesize, purify, characterize and evaluate anti-inflammatory activities of newer alkylated and acetylated pyrimidine derivatives. A series of titled compounds, i.e. [1-8] have been synthesized using appropriate synthetic procedure, as per the methodology. The yields of the synthesized compounds were found to be in the range from 81%-93%. Structures of synthesized compounds were characterized and confirmed with the help of analytical data such as IR and ¹HNMR. Anti-inflammatory activity studies were carried out using carrageenan induced rat hind paw edema method. Among the synthesized compounds screened for anti-inflammatory activity, the synthesized alkylated compounds 4-methoxyphenyl and 2, 4-dichlorophenyl derivatives showed highly significant (P<0.001) anti-inflammatory activity in comparable with the standard Diclofenac sodium at the end of 180min. And among acetylated compounds bis-4-chlorophenyl, 4-bromophenyl and bis-2,4-dichlorophenyl derivatives showed highly significant (P<0.001) anti-inflammatory activity in comparable with the standard Diclofenac sodium at the end of 180min.Hence, newly synthesized pyrimidine derivatives do possess considerable anti-inflammatory activity and further lead optimization should be carried out for the better expected anti-inflammatory activity.

 

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Received on 14.01.2020           Modified on 16.04.2020

Accepted on 02.06.2020         © RJPT All right reserved