INTRODUCTION:

N,N′-Dicyclohexylcarbodiimide (DCC) is a widely used dehydrating and oxidizing agents for the synthesis of proteins, amides and esters¹. This study reports synthetic application of the DCC to form N-aroylurea analogs and their antioxidant activity². The effect of various concentrations of essential alpha-amino acids on the luminol-sodium hypochlorite chemiluminescence (CL) has been studied. It was found that alpha-amino acids have free radical inhibiting effect³. With this background and keeping in view the good antioxidant activity of N-aroyl- N, N′-dicyclohexyl urea analogs,studies were undertaken to synthesize the amino acid conjugates of N-aroyl- N,N′-dicyclohexyl urea⁴. The N-cyclohexyl-N-(cyclohexylcarbamoyl) benzamide (1) was prepared by reacting N, N′-dicyclohexylcarbodiimide with benzoic acid in the reported manner. It was reacted with chloro acetyl chloride in presence of triethylamine leading to formation of N-((2-chloroacetyl)(cyclohexyl)carbamoyl)N-cyclohexyl benzamide(2) which was treated with tyrosine in presence of triethylamine and potassium carbonate to yield 2-((2-3-benzoyl-1,3-dicyclohexylureido)-2-oxoethyl)amino)-3-(4-hydroxyphenyl) propanoic acid(3a). Other compounds of the series (3b-j) were prepared in a similar manner (Figure 1). The in vitro antioxidant activity and anti-inflammatory were evaluated.

EXPERIMENTAL:

All reagents and solvents were procured and used without further purification. Melting points were determined on a Sisco melting point apparatus and are uncorrected. Crude products were purified by column chromatography on silica gel of 60—120 mesh. Molecular weights of the compounds were determined by Rast’s procedure. IR spectra were obtained on a JASCO FTIR-4100 spectrometer using KBr pellet. NMR spectra were recorded on BRUKER AVANCE-II- 400 MHz spectrometer using CDCl₃. The chemical shifts were reported as ppm down field using tetramethylsilane (TMS) as an internal standard. Elemental analysis was carried out with PERKIN ELMER-2400 analyzer. Mass spectra were recorded on a MICROMASS Q-TOF MICRO spectrometer operating at 70 eV.

N-cyclohexyl-N-(cyclohexylcarbamoyl)benzamide(1). N, N′-dicyclohexyl carbodiimide (2.07g, 1mmol), benzoic acid (1.23g, 1mmol) and triethylamine (1.4mL, 1mmol) in dry toluene (50 mL) was heated up to 110ºC overnight to yield N-benzoyl-N, N′-dicyclohexylurea (1). The purity of the compound was checked with TLC. Yield 74%. Off-white color. mp 112-115°C, Mol Wt. Calcd. 328.22; Found 327.94. ¹HNMR(CDCl3): δ 1.12-1.49(m, 20H), 5.4(s, 1H), 7.6-8.1(m,5H);FT-IR (KBr,cm^-1): 3211.46, 3022.74, 2865.12, 1685.12.

N-((2-chloroacetyl)(cyclohexyl)carbamoyl)N-cyclohexyl benzamide (2).To the well stirred solution of 1 (2 mmol) and triethylamine (2.2mmol) in 50 ml benzene,chloro acetyl chloride (2.2 mmol) was added drop by drop for about 30min.The reaction mixture was stirred at room temperature for about 6h. Progress of the reaction was monitored by TLC.

Figure-1

After completion of reaction, the reaction mass was quenched in ice cold water and extracted in diethyl ether. The ether layer was washed twice with 5% NaHCO₃and twice with distilled water. Finallythe ether layer was dried with anhydrous Na₂SO_4. Yield 70%. mp 121-123°C.White color. Mol Wt.Calcd. 404.19; Found 404.29. ¹HNMR(CDCl3): δ 1.11-1.47(m, 20H), 3.41(m, 1H), 4.17(s, 2H), 7.1-8.25(m,5H); FT-IR (KBr, cm^-1): 3034.55, 2845.58, 1666.74.

(2S)-2-((2-3-benzoyl-1,3-dicyclohexylureido)-2-oxoethyl)amino)-3-(4hydroxyphenyl) propanoic acid (3a). Tyrosine (1.2 mmol) in methanol (25 ml) was neutralized with triethylamine (1.2 mmol). To this K₂CO₃ (600 mg) was added. Later the solution of 2 (1 mmol) in methanol (50 ml) was added drop by drop for 30 min. The reaction mixture was refluxed for 8h. The progress of the reaction was monitored by TLC. The reaction mixture was then desolventized and compound was extracted in ethyl acetate. The ethyl acetate layer was washed with water and dried over anhydrous Na₂SO₄. The product was further recrystallised from ethanol. Other compounds of the series 3b-j were obtained in a similar manner. Yield 65 %.mp 124-126°C. Brown color. Mol Wt.Calcd. 549.28; Found 548.85. ¹HNMR (CDCl3): δ 1.1-1.49 (m, 20H), 2.12 (s, 1H), 3.45-3.47(m, 1H), 3.89 (s, 1H), 5.86(s, 1H), 6.8-8.1(m, 9H); FT-IR (KBr, cm^-1):3442.33, 3029.12, 2854.64, 1669.87.

(2S)-2-((2-(3-benzoyl-1,3-dicyclohexylureido)-2-oxoethyl)amino)-3-phenyl propanoic acid (3b) Yield 64 %. mp 133-135°C. White color. Mol Wt.Calcd. 533.29; Found 532.72. ¹HNMR(CDCl3): δ 1.2-1.45 (m, 20H), 2.15 (s,1H), 3.49-3.51(m,1H),3.74 (s,1H), 7.1-8.1(m, 9H); FT-IR(KBr, cm^-1):3445.69, 2941.23, 2837.85,1671.42.

(2S)-2-((2-(3-benzoyl-1,3-dicyclohexylureido)-2-oxoethyl)amino)-3-hydroxy butanoic acid (3c)- Yield 72%.mp 136-138°C. Off white color. Mol Wt.Calcd. 487.27; Found 487.85. Anal. Calcd. (%):C, 64.05; H, 7.65; N,8.62; Found (%):C, 64.11; H, 8.03; N,8.84; ¹HNMR(CDCl3): δ 1.05-1.48 (m, 20H), 2.08 (s,1H), 3.94-3.99(m,1H),4-4.016 (d,1H), 6(s,1H), 7.26-7.76(m, 5H); FT-IR(KBr, cm^-1):3317.45, 2929.21, 2854.33,1627.03; MS(m/z): 487(M⁺).

(2R)-2-((2-(3-benzoyl-1,3-dicyclohexylureido)-2-oxoethyl)amino)-3-hydroxy propanoic acid (3d)- Yield 55%. mp 117-119°C. Pale yellow. Mol Wt.Calcd. 473.25; Found 473.1. FT-IR(KBr, cm^-1): 3511.21, 3211.55, 2855.89, 1642.32.

(2R)-2-((2-(3-benzoyl-1,3-dicyclohexylureido)-2-oxoethyl)amino)-3-methyl butanoic acid (3e)- Yield 49%. mp 130-132°C. Brown. Mol Wt.Calcd. 485.29; Found 484.79.¹HNMR(CDCl3): δ 0.82-0.93(m,6H),1.12-1.44 (m, 20H), 2.34 (s,1H), 7.31-7.98(m, 5H); FT-IR(KBr, cm^-1): 3487.94.3189.76, 3032.65, 2834.95, 1642.78.

Table-1: Antioxidant and Anti-inflammatory of 2-((2-3-benzoyl-1, 3-dicyclohexylureido)-2-oxoethyl) amino) - 3- (4-hydroxyphenyl) propanoic acid (3a) and its analogues 3a-j.

Group	R	IC 50 (μg/ml)	Anti-inflammatory activity
Group	R	IC 50 (μg/ml)	Increase PV	% inhibition
Control	……..	……..	0.6833±0.03073	………
Standard	………	50	0.1833±0.03073^^*	73.17
3a		326	0.3500±0.05627^^*	48.77
3b		10	0.3167±0.04773^^*	53.65
3c		180	0.3667±0.03333^^*	46.33
3d		169	0.4667±0.03333^^*	31.69
3e		381	0.4000±0.04472^^*	41.46
3f		65	0.3333±0.04216^^*	51.22
3g		254	0.2667±0.04216^^*	60.98
3h		90	0.4333±0.04944^^*	36.58
3i		81	0.4167±0.04773^^*	39.01
3j		332	0.4000±0.02582^^*	41.46

RSA: Radical (DPPH) scavenging activity, Data are represented as mean ± S.E.M Statistical analysis was done with one way analysis of variance (ANOVA). ***P < 0.001 as compared to control (n = 6) in each group)

(2R)-2-((2-(3-benzoyl-1,3-dicyclohexylureido)-2-oxoethyl)amino)-3-(methylthio) propanoic acid (3f)- Yield 56%. mp 141-143°C. White. Mol Wt.Calcd. 503.25; Found 502.978. FT-IR(KBr, cm^-1): 3456.48.3211.13, 3035.21, 2865.45, 1654.89.

(2S)-2-((2-(3-benzoyl-1,3-dicyclohexylureido)-2-oxoethyl)amino)-3-(1H-indol-3-yl) propanoic acid (3g)- Yield 53%. mp 127-129°C .Off-white. Mol Wt.Calcd. 572.3; Found 571.65. FT-IR(KBr, cm^-1): 3445.53,3240.11, 3023.87, 2854.43, 1651.77.

5-amino-(2S)-2-((2-(3-benzoyl-1,3-dicyclohexylureido)-2-oxoethyl)amino)-5-oxo pentanoic acid (3h)- Yield 44%. mp 141-143°C .White. Mol Wt.Calcd. 514.28; Found 513.74. FT-IR(KBr, cm^-1): 3432.67,3278.76,3176.54, 3061.37, 2845.33, 1648.87.

(2R)-2-((2-(3-benzoyl-1,3-dicyclohexylureido)-2-oxoethyl)amino)-3-mercapto propanoic acid (3i)- Yield 58%. mp 131-133°C .Brownish. Mol Wt.Calcd. 489.23; Found 488.86. FT-IR(KBr, cm^-1): 3423.12,3189.65, 3033.79, 2843.34, 1666.54

(2S)-2-((2-(3-benzoyl-1,3-dicyclohexylureido)-2-oxoethyl)amino)-3-(1-H-imidazole-5-yl) propanoic acid (3j)- Yield 59%.Brown color. Mol Wt.Calcd. 523.28; Found 522.86. Anal. Calcd. (%): C, 64.23; H, 7.12; N, 13.37; Found (%):C, 64.42; H, 7.48; N,13.24; ¹HNMR(CDCl3): δ1.1286-1.8826 (m,20H), 2.12(s,1H), 2.54-2.56 (m,2H), 3.39-3.44 (t,1H), 3.77-3.81(m,1H), 7.38-7.85(m,5H), 8.13(s,1H), 8.35(s,1H); FT-IR(KBr, cm^-1): 3407.6, 2930.31, 2855, 1627.83; MS (m/z) : 523.2(M⁺).

In-vitro antioxidant study The free radical scavenging activity of synthesized compounds was evaluated by the method first employed by Blois⁵, Using 1,1-diphenyl-2-picrylhydrazyl (DPPH). To 1ml of each compound of different concentrations (1mg/ml, 2mg/ml, 3mg/ml, 4mg/ml and 5mg/ml) 1ml of 0.1 mmol DPPH was added and incubated in the dark room for 35 min⁶. The absorbance was measured at 517nm and percentage quenching of DPPH was calculated. For all the compounds and standard half inhibition concentration (IC₅₀) was calculated and showed in Table-1.

Anti-inflammatory activity of synthesized compounds Oral acute toxicity of the synthesized compounds was made by guidelines of Organization of Economic Co-operation and. Development (OECD -423) for testing of chemicals. The dose was increased up to 3000 mg/kg and lethality was not observed upto 3000mg/kg indicating the safety of these compounds (3a-j). Acute inflammation was produced by sub plantar injection of 0.1 ml of 1% suspension of carrageenin in normal saline in the right hind paw of the rats. Paw volume was measured plethysmometrically⁷ at 0 and 4 h after carrageenin injection. The animals were treated with the synthesized compounds (50mg/kg), Saline (3 ml/kg, orally) treated animals served as control and acetyl salicylic acid (100 mg/kg, orally) was administered as standard drug. The non-steroidal anti-inflammatory drugs (NSAIDs) such as acetyl salicylic acid (aspirin), diclofenac sodium, and ibuprofen exert their anti-inflammatory effects via inhibition of early steps in the biosynthesis pathway of prostaglandins and are widely used for managing inflammation and pain ^8,9. The drugs were administered simultaneously with carrageenin injection. Mean increase in paw volume was measured and reported in Table 1.

RESULTS AND DISCUSSION:

The IC₅₀ value of compound 3b was lowest among the test compounds followed by that of 3f indicating their good radical scavenging potential. The DPPH radical scavenging action of 3b was found to be better than ascorbic acid. All the test compounds reduced the paw volume. The anti-inflammatory activity was found to be significant (p<0.001) for compound 3a-j. Compounds 3b containing phenyl alanine and 3g containing tryptophan were showing maximum percentage reduction in paw edema primarily due to inhibition of prostaglandin E2 and serotonin relating to acute inflammatory mediator as comparing to Aspirin as standard anti-inflammatory drug. In the oral acute toxicity of the synthesized compounds lethality was not observed up to 3000mg/kg suggesting the lack of toxicity of the test compounds. Hence this study has yielded some very safe new compounds with good anti-inflammatory potential. This study needs to be further explored to maximize the therapeutic potential of the compounds.

CONCLUSION:

The present study indicates that the derived compounds are very much safe from their toxicity studies with very good anti-inflammatory activity and the anti-oxidant activity. Data reveals that the newly synthesized compounds can be explored to maximize their therapeutic potential.

ACKNOWLEDGEMENT:

The authors are thankful to Director, sophisticated analytical instrument facility, Punjab University, Chandigarh for their kind help in analysis.

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Received on 09.08.2011 Modified on 22.08.2011

Asian J. Research Chem. 4(9): Sept, 2011; Page 1485-1488