INTRODUCTION:

Compounds having Imidazole moiety are known from the beginning of twentieth century and the continued interest in the molecule shows the great potential of it in the field of research in medicinal chemistry.

Industrial production of imidazole began in the 1950s; a wide range of derivatives is now available. The recent interest in this old molecule is because of newer application and putting them through newer assays for the activities. It is evident from the recent Patent applications and publications related to imidazoles about its vitality.

Imidazole nucleus forms the main structure of some well-known components of human organisms, i.e. the amino acid histidine, Vit-B₁₂, a component of DNA base structure and purines, histamine and biotin. It is also present in the structure of many natural or synthetic drug molecules, i.e. cimetidine, azomycin, clotrimazole and metronidazole. The synthesis, reactions and biological properties of substituted imidazole constitute a significant part of modern heterocyclic chemistry. Imidazole derivatives are found to be pharmacologically active and are widely published for their analgesic, anti-inflammatory and antiarthritic, anticancer, antihelminthic, antibacterial, antifungal, Histamine H₃ receptor antagonist, and nitric oxide synthase inhibitor activity.

Literature findings, especially on its analgesic, anti-inflammatory activities^1-12 and the continued interest in the imidazole molecule after its discovery more than century ago have prompted the synthesis of similar compounds and screen them for their biological activity. An imidazole derivative, UR-8880 is found to be the new COX-2 inhibitor. It has high (COX-2/COX-1) selectivity in human cell lines and is nine times more potent than Celecoxib and four times more effective than Rofecoxib¹³.

2-substituted-4,5-diphenyl imidazole derivatives were synthesized by reacting benzil and aldehyde derivatives with ammonium acetate in glacial acetic acid. The 2-substituted 4,5-diphenyl imidazole compounds obtained were stirred with NaH in THF and after the addition of the benzyl chloride refluxed for 3-45 h to get 1-benzyl-2-substituted-4, 5-diphenyl-1H-imidazole derivatives.

Alternatively N-alkylation of heterocyclic compounds bearing an acidic hydrogen atom attached to nitrogen is accomplished under phase transfer catalysis (PTC) conditions in microwave oven, in which halides react directly with heterocyclic compounds without converting them to salts in preliminary steps¹⁴. Since many heterocycles exhibit ambident behavior as nucleophiles, alkylation can occur at carbon as well as nitrogen. For selective N-alkylation PTC procedures are found to be most useful in terms of mildness of conditions, yield, and convenience. The reactions were carried out by simply mixing an azaheterocycle compound with 50% excess of an alkyl halide and a catalytic amount of tetrabutyl ammonium bromide (TBAB). The mixtures were either absorbed on the mixture of potassium carbonate and potassium bromide or potassium carbonate, and then irradiated in an open vessel in domestic microwave oven for 1-10 minutes. The work-up procedure is reduced to treatment with an appropriate solvent.

The best results were achieved using K₂CO₃ or Cs₂CO₃ and a few drops of N,N-dimethylformamide or N-methyl-2-pyrrolidinone. These reactions present noteworthy advantages over those carried out employing conventional heating.^15-17

Tetra substituted imidazoles can also be synthesized by N-alkylation of the synthesized tri-substituted imidazoles⁶.

Among the many methods used for screening of antiinflammatory drugs, one of the most commonly employed techniques is based upon the ability of such agents to inhibit the edema produced in the hind paw of the rat after injection of a phlogistic agents (irritants) have been used, such as brewer’s yeast, formaldehyde, dextran, egg albumin, kaolin, aerosil, sulfated polysaccharides like carageenan or naphthoylheparamine¹⁸. Rat paw edema method is one of the widely used, simple model to evaluate the anti-inflammatory activity.

MATERIALS AND METHODS:

MATERIALS:

Chemicals and Apparatus

The AR grade chemicals were used for the synthesis of organic compounds. Various solvents were used for TLC, extraction and purification.

Borosil glass vials: Irradiation was done in tall borosil glass vials since the dielectric heating by microwave irradiation is dependent on the shape and size of the reaction vessel.

Chromatography column: Glass columns were used for purification of the crude compounds by flash chromatography or column chromatography.

Instruments:

Microwave oven: Most of the reactions were carried out in BPL-SANYO Microwave oven under high settings with intermittent cooling during microwave irradiation depending upon the reaction conditions.

Thin Layer Chromatography: TLC was used for monitoring the progress of the reaction and to examine the purity of the compounds. TLC was performed on silica gel plates (Merck). After the development of chromatogram, the spots were detected by placing the plates in UV chamber.

Liquid Chromatography-Mass Spectrometry: Mass of the synthesized compounds and their purity was determined by Agilent 1100 series LC-MSD trap.

Nuclear Magnetic Resonance Spectroscopy: Proton NMR spectra of the synthesized compounds were taken using Bruker ACF- 300MHz spectrometer with tetra methyl silane (TMS) as internal standard. The spectra were recorded in CD₃OD, CD₃COCD₃ and CDCl₃.

Infrared spectroscopy: The infrared spectra of the compounds were taken using SHIMADZU FTIR 8400 spectrometer using potassium bromide pellet technique.

Melting Point Determination: The melting points of the compounds were determined by BÜCHI-535 Melting Point Apparatus.

METHODS:

Two step synthesis of Tetra-substituted Imidazoles: In this method 2,4,5-trisubstituted imidazole is synthesized in a single step, followed by N-alkylation, which forms tetra-substituted imidazole.

Step 1: Synthesis of 2-substituted-4,5-diphenyl imidazole derivatives

This synthesis is a single step three-component reaction wherein the cyclization occurs by microwave irradiation to form imidazole ring.

0.210g (1 millimole) of benzil, 0.152g (2 millimoles) of ammonium acetate and aldehydes (1 millimole) were taken in 1 ml dry DMF in a long necked glass vial. The reaction mixture was irradiated in domestic microwave oven for 3 minutes following the cycle of 30 seconds heating and 10 seconds cooling. The progress of the reaction was monitored by TLC, using 10% methanol in chloroform as the mobile phase. The reaction mixture was allowed to come back to room temperature and 2g of crushed ice was added. This mixture was then stirred continuously as the solid precipitates out. After allowing to stand for 1 hour, the solids were filtered and the precipitate was washed 2-3 times with cold water. After the precipitate dries completely, the product was recrystallized with 20% ethyl acetate in hexane.

Step 1

Step 2: N-Alkylation of The Trisubstituted imidazoles synthesized in step 1 to form 1,2,4,5- Tetrasubstituted Imidazoles

These recrystallized compounds were taken in 1 millimolar quantity and triturated with 0.12ml (1 millimole) of benzyl chloride, 20 millimolar potassium hydroxide and 20 millimolar potassium carbonate. This mixture was heated in domestic microwave oven for two and a half minutes following the cycle of 30 seconds heating, 10 seconds cooling followed by 10 seconds heating and 10 seconds cooling. The reaction progress was monitored using 15% ethyl acetate in hexane as the mobile phase. The reaction mixture was extracted with chloroform 3-4 times and the organic layer was evaporated to dryness using rotary evaporator. The residue obtained was recrystallized using aqueous ethanol.

Screening For Antiinflammatory Activity: Formalin Induced Rat Paw Edema Model:

Experiments were carried out on female Wistar rats of albino strain. They were obtained from National Institute of Mental Health and Neurosciences, Bangalore. The animals were housed in polypropylene cages of dimension 16”x11”x6” for rats. Paddy husk was provided as bedding material, which was changed every day. The cages were maintained clean. The rats were kept 3 to 5 per cage. They were fed with standard pellet diet (Amrut Laboratory Feed, Pranav Agro Industries Ltd, Sangli) and water ad libitum. They were kept in a well-aerated room and a 12-hour light and dark cycle was maintained. The room temperature was maintained at 22±2°C.

Drug treatment:

The drugs were prepared as a suspension using 0.3% sodium carboxy methylcellulose (Na CMC). Standard drug, Rofecoxib 2mg/kg (human single dose 25mg) was used as positive control. Since this dose did not give good results, a higher dose of Rofecoxib 25mg/kg was used for better comparison with the synthesized compounds.

Drugs and vehicle were administered orally one hour before the induction of inflammation.

Procedure:

Rats were given the drug suspension orally and after one hour, the rats were challenged by a subcutaneous injection of 0.1 ml of 1% v/v formalin solution into the plantar region of left hind paw. The paw was marked with ink at the level of lateral malleolus and immersed in mercury up to this mark. The paw volume was measured plethysmographically immediately after injection and every hour for four hours. Change in the mercury level was measured by traveling microscope as change in height (in cm). This value obtained was converted into change in volume (in ml) by interpolating them on plethysmograph calibration curve.

Statistical evaluation:

The increase of paw volume (in ml) after every hour was calculated and compared with the paw volume measured immediately after injection of the irritant. Treated groups show less edema when compared to vehicle control group. The difference in mean values between treated and control groups were compared by Two-Way ANOVA and unpaired student t-test. p value < 0.05 was considered significant.

RESULTS AND DISCUSSION:

Five 2-substituted 4,5-diphenyl compounds were synthesized using five different aldehydes following above scheme in parallel method and the yield was found to be 80-90%. The detailed description of aldehydes used (name, amount taken) and the synthesized compounds is tabulated in Table 1.

Table 1: 2-Substituted-4,5-Diphenyl Imidazole derivatives

S. No.	Aldehydes	Amount Taken (g or ml)	Compounds	Mass	NMR	HPLC Purity (%)	M. Pt. (°C)	Solubility
	4-Methoxy benzaldehyde	0.136		326	HNMR (CD₃OD) = 3.89 (s, 3H); 7.05(d,2H); 7.9(d,2H); 7.3, 7.4, 7.5 (m, 10H)	100	235	Insoluble in water, chloroform; soluble in acetone, methanol.
2	4-Flouro benzaldehyde	0.124		314	HNMR (CDCl₃) = 7.12, 7.27, 7.29, 7.38, 7.87, 7.92 (multiplets,14 H)	99	257	Insoluble in water, ethanol: Soluble in methanol
3	4-hydroxy benzaldehyde	0.122		312	HNMR (CD₃OD= 1.98(s,1H); 6.9(d,2H); 7.3, 7.4, 7.5, 7.8(14H)	96.5	264	Insoluble in water: Soluble in methanol
4	4-hydroxy-3- Methoxy benzaldehyde	0.152		342	HNMR (CD₃OD)= 3.9(s,3H); 6.9(d,2H); 7.3, 7.4, 7.5, 7.6(13H)	100	80.4-90.5	Insoluble in water: Soluble in methanol
5	Benzo(b) thiophen- 3-aldehyde	0.162		352	HNMR (CD3OD)= 7.3, 7.4, 7.5, 7.6(15H)	99.4	285	Insoluble in water, ethanol; soluble in methanol

Table-2: N-Substituted Imidazoles Synthesized By Novel N-Alkylation Method in Domestic Microwave Oven

S. No.	IMIDs used	Amount Taken (g)	Compound /Lab Code	Mass	NMR	HPLC Purity (%)	M.Pt. (°C)	Solubility
1		0.326	IMID-4	416	HNMR (CDCl3) = 3.83 (s,3H); 5.09 (s,2H); 7.05 (d,2H); 6.8 (d,2H); 6.9 (d,2H); 7.1, 7.2, 7.3, 7.5 (m, 15H)	100	155	Insoluble in water, ethanol; soluble in chloroform, methanol
2		0.342	IMID-5	432	HNMR (CDCl3) = 3.74 (s,3H); 5.1 (s,2H); 6.9, 7.1, 7.2, 7.3, 7.5 (m,18H)	88.8	201.5-202.7	Insoluble in water, ethanol; soluble in chloroform, methanol
3		0.314	IMID-8	404	HNMR (CDCl3) = 5.08 (s,2H); 7.05 (d,2H); 6.8 (d,2H); 7.1, 7.2, 7.3, 7.5, 7.6 (m,19H)	54	189	Insoluble in water, ethanol; soluble in chloroform, methanol
4		0.312	IMID-12	402	HNMR (CDCl3) = 5.1 (s,2H); 7.05 (d,2H); 6.8 (d,2H); 6.9 (d,2H); 7.3, 7.5 (m,19H)	87	245-249	Insoluble in water, ethanol; soluble in methanol, acetone, chloroform
5		0.352	F1	442	HNMR (CDCl3) = 5.1 (s,2H); 6.8 (d,2H); 7.2, 7.3, 7.4, 7.6 (m,20H)	100	290-298	Insoluble in water, ethanol; soluble in methanol, acetone

Table 3: Anti-Inflammatory Activity of Synthesized Compounds By Rat Paw Edema Method.

Treatment Group		Increase in Paw volume (ml) (Mean ± SEM)				% Inhibition of edema
Treatment Group		1st Hr	2nd Hr	3rdHr	4th Hr	1st Hr	2nd Hr	3rdHr	4th Hr
Vehicle Control		0.354 ± 0.025	0.439 ± 0.027	0.462 ± 0.037	0.568 ± 0.033	--	--	--	--
Rofecoxib (25 mg/kg)		0.098 ± 0.025^a	0.043 ± 0.023^a	0.026 ± 0.014^a	0.005 ± 0.003**	72.31638	90.20501	94.37229	99.11972
	IMID-4	0.059 ± 0.029^a	0.064 ± 0.035^a	0.025 ± 0.012^a	0.001 ± 0.0001^a	83.33333	85.42141	94.58874	99.82394
IMID-5		0.086 ± 0.013^a	0.032 ± 0.013^a	0.035 ± 0.016^a	0.064 ± 0.023^a	75.70621	92.71071	92.42424	88.73239
	IMID-8	0.018 ± 0.006^a	0.014 ± 0.007^a	0.005 ± 0.001^a	0.007 ± 0.004^a	94.91525	96.81093	98.91775	98.76761
	IMID-12	0.127 ± 0.021^a	0.080 ± 0.040^a	0.109 ± 0.049^a	0.039 ± 0.029^a	64.12429	81.77677	76.40693	93.1338
F-1		0.218 ± 0.033^b	0.179 ± 0.046^a	0.082 ± 0.038^a	0.064 ± 0.033^a	38.41808	59.22551	82.25108	88.73239

CONCLUSION:

The synthesis of imidazole by the conventional route takes longer time and tedious work-up. Here we have standardized methods for fast synthesis of Imidazole derivatives under Microwave reaction conditions. The reaction conditions are standardized for the parallel synthesis for higher throughput.

These compounds are produced in excellent yields and purities under extremely short reaction times.

All the five compounds showed good activity when given orally. The study was done by formalin induced rat paw edema method.

No conclusions could be drawn for probable mechanism of action and structure activity relationship. Further studies are necessary to establish ED₅₀and toxicity.

REFERENCES:

1. Fitzi K., Imidazole derivatives in the treatment of pain. United States Patent, 1976, 3,940,486.

2. Fitzi K., 2-Substituted -4(5)-(aryl)-5(4)-(2,3 or 4-pyridyl)-imidazoles. 1975, 3,929,807.

3. Lombardino G J., Antiinflammatory imidazoles. United States Patent, 1972, 3,707,475.

4. Adams LJ., Boehm CJ, Gallaghar FT., Substituted imidazole compounds. United States Patent, 2000, 6,046,208.

5. Fatimi J., Lagorce JF, Duroux JL., 1,4,5-Trialkyl imidazole system anti-inflammatory properties of new substituted derivatives. Chem Pharm Bull (Tokyo), 1994, 42:698-701.

6. Uçucu U, Karaburun GN, Isikdag I, Synthesis and analgesic activity of some 1-Benzyl-2-substituted-4,5-diphenyl-1-H-imidazole derivatives. IL Farmaco, 2000, 56:285-290.

7. Sallmann A. Trisubstituted imidazole derivatives, Pharmaceutical preparations containing them, and their use. United States Patent, 1984. 4,447,431.

8. Sharpe TR, Cherkofsky SC, Hewes WE, Preparation and anti arthritic and analgesic activity of 4,5-Diaryl-2-(substituted thio)-1H-imidazoles and their sulfoxide and sulfones. J Med Chem., 1985, 28:1188-94.

9. Khanna IK, Yu Y, Huff RM, Weier RM, Selective cyclooxygenase – 2 inhibitors: heteroaryl modified 1,2-Diaryl imidazoles are potent, orally active antiinflammatory agents. J Med Chem., 2000. 43:3168-85.

10. Bender PE, Hill DT, Offen PH., 5,6-Diaryl-2,3-dihydroimidazole[2,1-b]thiazoles: a new class of immunoregulatory anti-inflammatory agents. J Med Chem., 1985. 28:1169-77.

11. Shorbagi I, Sakai S, Gendy MA, Omar NM, Farag HH. Imidazo[2,1-b] Benzothiazoles, Chem. Pharm. Bull. 1989, 38: 4760-4768.

12. Tsukamoto G, Yoshino K, Kohno T., Synthesis and anti-inflammatory activity of some 2- (substituted-pyridinyl)benzimidazoles. J Med Chem. 1980. 23:734-8.

13. Almansa C, Alfon J, Cavalcanti LF, Discovery of UR-8880: A new COX-2 Selective inhibitor, Albany Molecular Research, 2001, 6:5-7.

14. Bogdal D, Pielichowski J, Jaskot K, Remarkable Fast N-Alkylation of Azaheterocycles under Microwave Irradiation in Dry Media. Heterocycles, 1997. 715-722, 45.

15. María Sol Shmidt, Ana María Reverdito, Lautaro Kremenchuzky, Isabel Amalia Perillo* and María Mercedes Blanco. Simple and Efficient Microwave Assisted N-Alkylation of Isatin. Molecules 2008, 13, 831-840.

16. Z.-G. Le, Z.-C. Chen, Y. Hu, Q.-G. Zheng, Organic Reactions in Ionic liquids: N-Alkylation of Phthalimide and Several Nitrogen Heterocycles. Synthesis., 2004, 208-212.

17. K. Brokaitė, V. Mickevičius and R. Vaickelionienė N-alkylation products of substituted imidazoles and dihydropyrimidinediones with dibromoalkanes Chemistry of Heterocyclic Compounds, 2008, 44: 25-28.

18. Vogel HG, Drug Discovery and Evaluation; Pharmacological Assays, 2002, pp. 759-762.

INTRODUCTION:

Screening For Antiinflammatory Activity: Formalin Induced Rat Paw Edema Model:

Aldehydes

Mass

NMR

Solubility

NMR

14. Bogdal D, Pielichowski J, Jaskot K, Remarkable Fast N-Alkylation of Azaheterocycles under Microwave Irradiation in Dry Media. Heterocycles, 1997. 715-722, 45.

16. Z.-G. Le, Z.-C. Chen, Y. Hu, Q.-G. Zheng, Organic Reactions in Ionic liquids: N-Alkylation of Phthalimide and Several Nitrogen Heterocycles. Synthesis., 2004, 208-212.

17. K. Brokaitė, V. Mickevičius and R. Vaickelionienė N-alkylation products of substituted imidazoles and dihydropyrimidinediones with dibromoalkanes Chemistry of Heterocyclic Compounds, 2008, 44: 25-28.