Phthalic Hydrazide Synthesis Essay

Abstract

Inhibitors of soluble epoxide hydrolase (sEH) represent one of the novel pharmaceutical approaches for treating hypertension, vascular inflammation, pain and other cardiovascular related diseases. Most of the potent sEH inhibitors reported in literature often suffer from poor solubility and bioavailability. Toward improving pharmacokinetic profile beside favorable potency, two series of 4-benzamidobenzoic acid hydrazide derivatives with hydrazide group as a novel secondary pharmacophore against sEH enzyme were developed. The designed compounds were synthesized in acceptable yield and their in vitro assay was determined. Most of the synthesized compounds have appropriate physical properties and exhibited considerable in-vitro sEH inhibitory activity in comparison with 12-(3-Adamantan-1-yl-ureido)- dodecanoicacid (AUDA), a potent urea-based sEH inhibitor. 4-(2-(4-(4-chlorobenzamido) benzoyl)hydrazinyl)-4-oxobutanoic acid 6c was found to be the most potent inhibitor with inhibitory activity of 72% targeting sEH enzyme.

Key Words: Synthesis, Docking, Benzamidobenzoic acid hydrazide, Soluble epoxide hydrolase, Physical properties

Introduction

Human soluble epoxide hydrolase enzyme converts epoxyeicosatrienoic acids (EETs) to their corresponding hydrated products by catalyzing the addition of water to the epoxide moiety (1, 2). EETs have a wide range of physiological effects. e. g., increase sodium renal excretion, relax vascular conduit and dilate renal afferent arterioles and coronary resistance vessels (3, 4). In addition, EETs modulate leukocyte adhesion, platelet aggregation, vascular smooth muscle cell migration and thrombolysis in preclinical animal models (5). Therefore, inhibition of sEH might be a promising new treatment in hypertension, vascular inflammation, pain and other cardiovascular related diseases (3-6).

The previous studies revealed the hydrolase catalytic pocket of sEH consists of two tyrosine and an aspartate residues which act essential role in epoxide ring opening (6). It has been recognized that amide or urea groups fit well in the hydrolase catalytic pocket to interact with mentioned residues. Specifically, the carbonyl oxygen of the amide or urea is engaged in a hydrogen bond interaction with tyrosine and the N–H moiety acts as a hydrogen bond donor to aspartate. Therefore, various urea and amide analogues have been developed as reversible sEH inhibitors (2-3, 7). Urea, carbamate, and amide compounds substituted with hydrophobic groups are potent and stable sEH inhibitors. However, poor physical properties of these compounds, such as low solubility and high melting points, lead to limited in-vivo availability (8). Solubility and bioavailability improved with the addition of a polar functional group on specific positions of one of the urea or amide moiety (9-11). Therefore, 12-(3-adamantan-1-ylureido) dodecanoic acid (AUDA) and 1-adamantan-1-yl-3-{5-[2-(ethoxyethoxy) ethoxy]pentyl} urea (AEPU) were developed (Figure 1) (12).

Figure 1

Chemical structures of known sEH inhibitors

According to the pharmacophore model suggested for sEH inhibitors (1, 13, 14), we designed, synthesized and biologically evaluated two series of 4-benzamidobenzoic acid hydrazide derivatives as novel soluble epoxide hydrolase inhibitors (Figure 2). The amide group in the represented structures is considered as the primary pharmacophore (P1) and the hydrazide group is the secondary pharmacophore (P2). Phenyl ring joins P1 and P2 together as a lipophilic spacer. Oxobutanoic acid and carbonylbenzoic acid moieties play the role of terminal pharmacophore (L2/P3). Phenyl ring with various hydrophobic or hydrophilic substitutes in the R position were added to the main structure.

Figure 2

General scaffold of the synthesized analogues

Experimental

Chemistry

All laboratory grade reagents were obtained commercially from Aldrich or Merck Company. The reactions were monitored by thin layer chromatography (TLC) performed on commercially available Merck precoated plates (silica gel 60 F254, 0.25 mm). The structures of the synthesized compounds were confirmed by IR, LC/MS and 1HNMR. Perkin Elmer 843 IR and Agilent 6410 (QQQ) LC/MS were used to obtain IR and Mass spectra respectively.1HNMR spectra were recorded on a Bruker advance II (500 MHz) spectrophotometer using [D6] DMSO as a solvent. Water solubility was determined experimentally in 1.0 mL of sodium phosphate buffer (0.1 M, pH 7.4) at 25 ± 1 °C (9, 15). The logP (octanol/water partition coefficient (P)) values were calculated by Crippen’s method using CS ChemBioDraw Ultra version 12.0 software and melting points were taken on a Electrothermal 9100 apparatus and are uncorrected. The designed compounds were synthesized as shown in Figure 3.

Figure 3

Schematic representation of synthesis of the designed compounds. Reagents and conditions: (a) 4-aminobenzoic acid, anhydrous Na2CO3, THF, rt, 6-12 h, 60-85%; (b) H2SO4, EtOH, reflux, 24 h, 58-65%; (c) NH2NH2.H2O, EtOH, rt, 12 h, 70-80%; (d) phthalic anhydrides,...

General procedure for the preparation of 4-(4-substitutedbenzamido)benzoic acid (2a-2e)

A solution of 4-aminobenzoic acid (1.68 mmol) and para substituted benzoylchlorides (1.68 mmol) in dry THF, in presence of anhydrous Na2CO3 (1.68 mmol) was stirred at room temperature for 6-12 h. The solvent was evaporated and the precipitate was washed with water and recrystallized from ethanol 96% to give final products.

4-benzamidobenzoic acid (2a)

Yield: 85%; white crystalline powder; mp: 150-151 °C, IR (KBr): υ (cm-1) 2887-3045 (OH), 3340 (NH), 1675, 1650 (C=O); LC-MS (ESI) m/z = 242 (M+1, 25%), 264 (M+23, 100%).

4-(4-fluorobenzamido)benzoic acid (2b)

Yield: 80%; white crystalline powder; mp: 155-156 °C, IR (KBr): υ (cm-1) 2857-3125 (OH), 3379 (NH), 1696, 1685 (C=O); LC-MS (ESI) m/z = 260 (M+1, 30%), 282 (M+23, 100%).

4-(4-chlorobenzamido)benzoic acid (2c)

Yield: 75%; white crystalline powder; mp: 138-140 °C, IR (KBr): υ (cm-1) 2730-3045 (OH), 3320 (NH), 1695, 1670 (C=O); LC-MS (ESI) m/z = 276 (M+1, 60%), 298 (M+23, 100%).

4-(4-methylbenzamido)benzoic acid (2d)

Yield: 80%; white crystalline powder; mp: 145-147 °C, IR (KBr): υ (cm-1) 2630-3145 (OH), 3340 (NH), 1670, 1690 (C=O); LC-MS (ESI) m/z = 256 (M+1, 30%), 278 (M+23, 100%).

4-(4-methoxybenzamido)benzoic acid (2e)

Yield: 60%; cream crystalline powder; mp: 150-152 °C, IR (KBr): υ (cm-1) 2807-3015 (OH), 3320 (NH), 1695, 1660 (C=O); LC-MS (ESI) m/z = 272 (M+1, 40%), 294 (M+23, 100%).

General procedure for the preparation of Ethyl 4-(4-substitutedbenzamido)benzoate (3a-3e)

8.06 mmol of 2 was dissolved in ethanol (15 mL) and concentrated sulfuric acid (0.5 mL) was added. The solution was refluxed for 24 h. Then, ethanol was evaporated and the remnant was alkalized after being cooled in the ice bath with NaOH 20% and extracted with diethyl ether. The diethyl ether phase was washed first with aqueous NaOH 20% and water and dried with anhydrous sodium sulfate and evaporated.

Ethyl 4-benzamidobenzoate (3a)

Yield: 60%; white crystalline powder; mp: 95-97, IR (KBr): υ (cm-1) 3340 (NH), 1720, 1670 (C=O); LC-MS (ESI) m/z = 270 (M+1, 100%), 292 (M+23, 30%).

Ethyl 4-(4-fluorobenzamido)benzoate (3b)

Yield: 65%; white crystalline powder; mp: 90-92 °C, IR (KBr): υ (cm-1) 3330 (NH), 1715, 1670 (C=O); LC-MS (ESI) m/z = 288 (M+1, 100%), 310 (M+23, 55%).

Ethyl 4-(4-chlorobenzamido)benzoate (3c)

Yield: 63%; white crystalline powder; mp: 96-97 °C, IR (KBr): υ (cm-1) 3315 (NH), 1690, 1730 (C=O); LC-MS (ESI) m/z = 304 (M+1, 100%).

Ethyl 4-(4-methylbenzamido) benzoate (3d)

Yield: 65%; white crystalline powder; mp: 99-100 °C, IR (KBr): υ (cm-1) 3340 (NH), 1730, 1680 (C=O); LC-MS (ESI) m/z = 284 (M+1, 50%), 306 (M+23, 100%). Ethyl 4-(4-methoxybenzamido)benzoate (3e)

Yield: 58%; cream crystalline powder; mp: 105-106 °C, IR (KBr): υ (cm-1) 3330 (NH), 1715, 1690 (C=O); LC-MS (ESI) m/z = 300 (M+1, 100%).

General procedure for the preparation of 4-substituted-N-(4-hydrazinecarbonyl) phenyl)benzamide (4a-4e)

7.24 mmol of 3 and 10 mL hydrazine hydrate (200 mmol) were added to 10 mL ethanol. The mixture was stirred for 12 h at room temperature. Afterward, the solvent was evaporated and white precipitates washed with diethyl ether, and recrystallized from a mixture of ethanol and a few drops of water.

N-(4-(hydrazinecarbonyl)phenyl)benzamide (4a)

Yield: 80%; white crystalline powder; mp: 120-122 °C, IR (KBr): υ (cm-1) 3397, 3307 (NH), 1647 (C=O); LC-MS (ESI) m/z = 256 (M+1, 100%).

4-Fluoro-N-(4-hydrazinecarbonyl)phenyl)benzamide (4b)

Yield: 70%; white crystalline powder; mp: 125-126 °C, IR (KBr): υ (cm-1) 3336, 3317 (NH), 1659 (C=O); LC-MS (ESI) m/z = 274 (M+1, 100%).

4-Chloro-N-(4-hydrazinecarbonyl)phenyl)benzamide (4c)

Yield: 75%; white crystalline powder; mp: 127-129 °C, IR (KBr): υ (cm-1) 3340, 3327 (NH), 1670 (C=O); LC-MS (ESI) m/z = 290 (M+1, 100%).

4-Methyl-N-(4-hydrazinecarbonyl)phenyl) benzamide (4d)

Yield: 80%; white crystalline powder; mp: 120-121 °C, IR (KBr): υ (cm-1) 3336, 3307 (NH), 1659 (C=O); LC-MS (ESI) m/z = 270 (M+1, 100%).

4-Methoxy-N-(4-hydrazinecarbonyl) phenyl)benzamide (4e)

Yield: 70%; white crystalline powder; mp: 130-131 °C, IR (KBr): υ (cm-1) 3320, 3307 (NH), 1680 (C=O); LC-MS (ESI) m/z = 286 (M+1, 100%).

General procedure for the preparation of 2-(2-(4-(4-substitutedbenzamido)benzoyl)hydrazinyl)carbonylbenzoic acid (5a-5e )

A solution of 4 (1.68 mmol) and phtalic anhydrides (1.68 mmol) in dry toluene was stirred at room temperature for overnight. The solvent was evaporated and the precipitate was washed with water and recrystallized from ethanol 96%.

2-(2-(4-benzamido)benzoyl) hydrazinylcarbonylbenzoic acid (5a)

Yield: 70%; white crystalline powder; mp: 170 °C, IR (KBr): υ (cm-1) 2665-3315 (OH), 3268, 3231 (NH), 1694, 1666 (C=O); LC-MS (ESI) m/z = 404 (M+1, 100%); 1HNMR (DMSO/500 MHz): 7.57 (6H, m, H3, H4, H5-benzamido, H3, H4, H5-benzoicacid), 7.70 (1H, d, H6-benzoic acid, J = 8.0 Hz), 7.90 (2H, d, H3, H5-benzoyl, J = 8.0 Hz), 7.95 (2H, d, H2, H6-benzoyl, J = 8.0 Hz), 8.06 (2H, dd, H2, H6-benzamido, J = 8.0, 3.5 Hz), 10.35 (3H, s, NH), 12.00 (1H, br s, COOH).

2-(2-(4-(4-fluorobenzamido)benzoyl)hydrazinyl)carbonylbenzoic acid (5b)

Yield: 65%; white crystalline powder; mp: 172 °C, IR (KBr): υ (cm-1) 2657-3330 (OH), 3235, 3224 (NH), 1710, 1667 (C=O); LC-MS (ESI) m/z = 422 (M+1, 20%), 444 (M+23, 100%); 1HNMR (DMSO/500 MHz): 7.34 (2H, t, H3, H5-benzamido, J = 8.7 Hz), 7.58 (3H, m, H3, H4, H5-benzoicacid), 7.74 (1H, d, H6-benzoic acid, J = 8.5 Hz), 7.90 (2H, d, H3, H5-benzoyl, J = 8.5 Hz), 7.95 (2H, d, H2, H6-benzoyl, J = 8.5 Hz), 8.06 (2H, dd, H2, H6-benzamido, J = 8.7, 2.5 Hz), 10.35 (1H, s, NH), 10.45 (2H, br s, NH), 12.01 (1H, br s, COOH).

2-(2-(4-(4-chlorobenzamido)benzoyl)hydrazinyl)carbonylbenzoic acid (5c)

Yield: 70%; white crystalline powder; mp: 173 °C, IR (KBr): υ (cm-1) 2673-3270 (OH), 3250, 2865 (NH), 1677, 1657 (C=O); LC-MS (ESI) m/z = 438 (M+1, 100%); 1HNMR (DMSO/500 MHz): 7.14 (2H, d, H3, H5-benzamido, J = 8.7 Hz), 7.55 (3H, m, H3, H4, H5-benzoicacid), 7.74 (1H, d, H6-benzoic acid, J = 8.7 Hz), 7.80 (2H, d, H3, H5-benzoyl, J = 8.5 Hz), 7.90 (2H, d, H2, H6-benzoyl, J = 8.5 Hz), 8.00 (2H, d, H2, H6-benzamido, J = 8.7 Hz), 10.05 (1H, s, NH), 10.35 (2H, br s, NH), 12.20 (1H, br s, COOH).

2-(2-(4-(4-methylbenzamido)benzoyl)hydrazinyl)carbonylbenzoic acid (5d)

Yield: 65%; white crystalline powder; mp: 174 °C, IR (KBr): υ (cm-1) 2859-3400 (OH), 3308, 3227 (NH), 1700, 1680 (C=O); LC-MS (ESI) m/z = 418 (M+1, 100%), 440 (M+23, 60%); 1HNMR (DMSO/500 MHz): 2.39 (3H, s, CH3), 7.04 (2H, d, H3, H5-benzamido, J = 8.1 Hz), 7.25 (3H, m, H3, H4, H5-benzoicacid), 7.44 (1H, d, H6-benzoic acid, J = 8.7 Hz), 7.70 (2H, d, H3, H5-benzoyl, J = 8.0 Hz), 7.80 (2H, d, H2, H6-benzoyl, J = 8.0 Hz), 7.90 (2H, d, H2, H6-benzamido, J = 8.1 Hz), 10.25 (3H, s, NH), 12.20 (1H, br s, COOH).

2-(2-(4-(4-methoxybenzamido)benzoyl)hydrazinyl)carbonylbenzoic acid (5e)

Yield: 68%; white crystalline powder; mp: 170 °C, IR (KBr): υ (cm-1) 2940-3492 (OH), 3318, 3228 (NH), 1712, 1643 (C=O); LC-MS (ESI) m/z = 434 (M+1, 100%), 456 (M+23, 80%); 1HNMR (DMSO/500 MHz): 3.84 (3H, s, OCH3), 6.95 (2H, d, H3, H5-benzamido, J = Rezaee zavareh E et al. / IJPR (2014), 13 (supplement): 51-59 56

8.1 Hz), 7.05 (3H, m, H3, H4, H5-benzoicacid), 7.20 (1H, d, H6-benzoic acid, J = 8.7 Hz), 7.30 (2H, d, H3, H5-benzoyl, J = 8.5 Hz), 7.60 (2H, d, H2, H6-benzoyl, J = 8.5 Hz), 7.80 (2H, d, H2, H6-benzamido, J = 8.1 Hz), 10.00 (3H, s, NH), 12.00 (1H, br s, COOH).

General procedure for the preparation of 4-(2-(4-(4-substitutedbenzamido)benzoyl)hydrazinyl)-4-oxobutanoic acid (6a-6e)

A solution of 4 (1.68 mmol) and succinic anhydride (1.68 mmol) in dry toluene was stirred at room temperature for 18 h. The solvent was evaporated and the precipitate was washed with water and recrystallized from methanol.

4-(2-(4-benzamidobenzoyl) hydrazinyl)-4-oxobutanoic acid (6a)

Yield: 80%; white crystalline powder; mp: 172 °C, IR (KBr): υ (cm-1) 2890-3402 (OH), 3235, 3021 (NH), 1684, 1667 (C=O); LC-MS (ESI) m/z = 356 (M+1, 100%), 378 (M+23, 50%); 1HNMR (DMSO/500 MHz): 2.40 (4H, s, CH2CH2), 7.57 (3H, m, H3, H4, H5-benzamido), 7.95 (4H, m, H2, H3, H5, H6-benzoyl), 8.27 (2H, dd, H2, H6-benzamido, J = 8.7, 3.5 Hz), 10.18 (3H, s, NH), 12.10 (1H, br s, COOH).

4-(2-(4-(4-fluorobenzamido)benzoyl)hydrazinyl)-4-oxobutanoic acid (6b)

Yield: 72%; white crystalline powder; mp: 170 °C, IR (KBr): υ (cm-1) 2936-3373 (OH), 3306, 3262 (NH), 1685, 1649 (C=O); LC-MS (ESI) m/z = 374 (M+1, 100%), 396 (M+23, 90%); 1HNMR (DMSO/500 MHz): 2.48 (4H, s, CH2CH2), 7.37 (2H, t, H3, H5-benzamido, J = 8.7 Hz), 7.90 (4H, m, H2, H3, H5, H6-benzoyl), 8.07 (2H, dd, H2, H6-benzamido, J = 8.7, 2.5 Hz), 10.18 (1H, s, NH), 10.44 (1H, s, NH), 10.57 (1H, s, NH), 12.10 (1H, br s, COOH).

4-(2-(4-(4-chlorobenzamido)benzoyl)hydrazinyl)-4-oxobutanoic acid (6c)

Yield: 75%; white crystalline powder; mp: 168 °C, IR (KBr): υ (cm-1) 2965-3492 (OH), 3297, 3043 (NH), 1686, 1644 (C=O); LC-MS (ESI) m/z = 390 (M+1, 100%); 1HNMR (DMSO/500 MHz): 2.38 (4H, s, CH2CH2), 7.27 (2H, d, H3, H5-benzamido, J = 8.7 Hz), 7.80 (4H, m, H2, H3, H5, H6-benzoyl), 8.00 (2H, d, H2, H6-benzamido, J = 8.7 Hz), 10.28 (1H, s, NH), 10.40 (1H, s, NH), 10.57 (1H, s, NH), 12.10 (1H, br s, COOH).

4-(2-(4-(4-methylbenzamido)benzoyl)hydrazinyl)-4-oxobutanoic acid (6d)

Yield: 80%; white crystalline powder; mp: 160 °C, IR (KBr): υ (cm-1) 2942-3501 (OH), 3312, 3273 (NH), 1702, 1661 (C=O); LC-MS (ESI) m/z = 370 (M+1, 100%); 1HNMR (DMSO/500 MHz): 2.39 (3H, s, CH3), 2.48 (4H, s, CH2CH2), 7.07 (2H, d, H3, H5-benzamido, J = 8.7 Hz), 7.50 (4H, m, H2, H3, H5, H6-benzoyl), 7.95 (2H, d, H2, H6-benzamido, J = 8.7 Hz), 10.20 (3H, s, NH), 12.00 (1H, br s, COOH).

4-(2-(4-(4-methoxybenzamido)benzoyl)hydrazinyl)-4-oxobutanoic acid (6e)

Yield: 72%; white crystalline powder; mp: 165 °C, IR (KBr): υ (cm-1) 2981-3513 (OH), 3323, 3284 (NH), 1709, 1667 (C=O); LC-MS (ESI) m/z = 386 (M+1, 20%), 408 (M+23, 100%); 1HNMR (DMSO/500 MHz): 2.38 (4H, s, CH2CH2), 3.84 (3H, s, OCH3), 7.00 (2H, d, H3, H5-benzamido, J = 8.3 Hz), 7.30 (4H, m, H2, H3, H5, H6-benzoyl), 7.75 (2H, d, H2, H6-benzamido, J = 8.3 Hz), 10.70 (3H, s, NH), 12.00 (1H, br s, COOH).

Docking studies

The high resolution crystal structure of sEH (PDB code: 3ANS) complexed with 4-cyano-N-[(1S, 2R)-2-phenylcyclopropyl]benzamide was retrieved from RCSB Protein Data Bank. The structures of compounds were investigated using the Lamarckian genetic algorithm search method implemented in AutoDock 4.0 software. The receptors were kept rigid, and ligands were allowed to be flexible. Polar hydrogens and Kollman united atom partial charges were added to the individual protein atoms. Each structure was energy minimized under MM+ method in HyperChem8 software and converted to pdbqt format file using AutoDockTools 4.0 version1.5.4. A docking grid box was built with 40, 40 and 40 points in 25.8460, 24.0730 and 114.8150 directions in the catalytic site of protein and the number of generations and maximum number of energy evaluations was set to 100 and 2,700,000, respectively. Docking results were clustered with a root mean square deviation (RMSD) of 0.5 Å and evaluated by Pymol software.

In-vitro biological activity

Biological evaluation was performed by Cayman fluorescence-based human soluble epoxide hydrolase assay kit (item number 10011671). The enzyme and inhibitors were incubated for 15 min in 25 mM Bis-Tris/HCl buffer (200 μL; pH 7.0) at 30 °C. 3-phenyl-cyano (6-methoxy-2-naphthalenyl) methyl ester-2-oxiraneacetic acid (PHOME) was used as the substrate for assay. The reference inhibitor for assay is AUDA, one of the most effective inhibitors of sEH with 50% inhibitory activity in 1 nM concentration. Test samples were dissolved in DMSO and tested in 1 nM concentration in duplicate to the determination of the inhibitory activity.

Result and Discussion

The designed compounds were synthesized in good yield according to Figure 3. Compounds 2a-2e was prepared from the reaction of 4-aminobenzoic acid with appropriate benzoyl chlorides (16). Hydrazides 4a-4e was synthesized through esterification of the corresponding acids followed by treatment with hydrazine hydrate (17, 18). The final products 5a-5e and 6a-6e were obtained from the reaction of proper hydrazides with phthalic and succinic anhydride respectively. Molecular structures of the synthesized compounds were confirmed by IR, Mass, and 1HNMR spectroscopy. The unusual result was observed in 1HNMR data of the butanoic acid analogues (6a-6e). Two vicinal methylene groups of the butanoyl moiety were singlet. It seems that they have an exactly equal chemical shift accidentally so they couldn’t split each other. Docking study on designed sEH inhibitors confirms that the analogues fit in the hydrolase catalytic pocket of X-ray crystal structure of sEH. As shown in Figure 4, it is obvious that 4-(2-(4-(4-chlorobenzamido) benzoyl)hydrazinyl)-4-oxobutanoic acid (6c) in the active site pocket have a suitable distance from the three amino acids of Tyr383, Tyr466 and Asp335 for effective hydrogen bonding and additional hydrogen bonds could be formed between this compound and Phe497, Lue408 and Lue428 of the catalytic pocket.

Figure 4

The minimized docking model of compound 6c in human sEH. The windows show the location of the amide (P1) and hydrazide (P2) groups in active site

According to Table 1, most of the synthesized compounds show a considerable inhibitory activity in 1 nM concentration in comparison with inhibitory activity of AUDA, the potent urea-based inhibitor with 50 % inhibitory ratio in the equal concentration. The most potent compounds in both series are those with chloro substituent on the 4-position of the phenyl ring (5c and 6c) with inhibitory activity of 47% and 72% respectively. Generally the butanoic acid analogues (6a-6e) were found to be more potent than the corresponding benzoic acid derivatives (5a-5e) and also have a better water solubility property. Since all of the synthesized compounds have carboxylic moiety, the solubility of these compounds could be improved more by synthesis of the corresponding carboxylate salts. It seems the butanoic acid derivatives could be appropriate candidates for more investigation about new therapeutic agents due to acceptable solubility with proper inhibitory activity. In conclusion, new 4-benzamidobenzoic acid hydrazide derivatives against soluble epoxide hydrolase enzyme were investigated. According to the inhibitory activities and solubility properties the designed structures might be valuable lead scaffold to development of the potent inhibitors with improved pharmacokinetic properties.

Table 1

Inhibitory activityof thesynthesized derivatives andthe related physical properties

Acknowledgment

This work was supported by the Iran National Science Foundation (INSF) and the research council of the School of Pharmacy, Shahid Beheshti University of Medical Sciences.

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14. Anandan SK, Webb HK, Chen D, Wang YX, Aavula BR, Cases S, Cheng Y, Do ZN, Mehra U, Tran V, Vincelette J, Waszczuk J, White K, Wong KR, Zhang LN, Jones PD, Hammock BD, Patel DV, Whitcomb R, MacIntyre D E, Sabry J, Gless R. 1-(1-Acetyl-piperidin-4-yl)-3-adamantan-1-yl-urea (AR9281) as a potent, selective, and orally available soluble epoxide hydrolase inhibitor with efficacy in rodent models of hypertension and dysglycemia. Bioorg. Med. Chem. Lett. 2011;21:983–988.[PMC free article][PubMed]

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Contents

Chapter 1 Organic synthesis
1.1 Preparation of m-Dinitrobenzene
1.2 Preparation of m-Nitroaniline
1.3 Preparation of Hippuric Acid
1.4 Preparation of Azlactone
1.5 Preparation of phthalimide
1.6 Preparation of 2, 4-Dihydroxyacetophenone
1.7 Preparation of Anthracene-Maleic anhydride adduct
1.8 Microwave Assisted Synthesis of Anthracene Maleic Anhydride Adduct
1.9 Microwave Assisted Synthesis of Aspirin
1.9.1 P-Bromoacetanilide
1.9.2 Synthesis of P-Bromoaniline
1.9.3 Preparation of 2, 4, 6 Tribromoaniline
1.9.4 Preparation of 1, 3, 5 Tribromobenzene
1.9.5 Preparation of Aspirin
1.9.6 Preparation of Tetrahydrocarbazole
1.9.7 7-Hydroxy4-Methyl Coumarin (Umbelliferon)
1.9.8 Organic Synthesis-1: 2–Phenyl Indole
1.9.9 Organic Synthesis-2: 7-Hydroxy-3-Methyl Flavone
1.10 Organic Synthesis-3: 2, 5 Di hydroxy Acetophenone
1.10.1 Organic Synthesis-4: 4-Chloro Toluene
1.10.2 Organic Synthesis-5: Benzpinacol [Photo reduction]
1.10.3 Organic Synthesis-6: 7-Hydroxy Coumarin
1.10.4 Organic Synthesis-7: Photodimerization of Maleic Anhydride
1.10.5 Organic Synthesis-8: Benzophenone
1.10.6 Organic Synthesis-9: Benzanilide
1.10.7 Organic Synthesis-10: Vanillyl Alcohol
1.10.8 Organic Synthesis-11 Ortho and Para Nitro Phenols
1.10.9 Organic Synthesis-12: Acridone

Chapter 2 Isolation of Natural product
2.0 Isolation of Piperine from Black-pepper
2.1 Isolation of Caffeine from Tea Leaves
2.2 Isolation of Cineole from Eucalyptus Leaves

Chapter 3 Drug synthesis
3.0 synthesis of Paracetamol
3.1 synthesis of Phenytoin
3.2 synthesis of Benzocaine
3.4 synthesis of Synthesis of chlorbutol
3.5 synthesis of Sulphanilamide
3.6 synthesis of flourescein

Chapter 4 Organic mixture analysis
4.0 Analysis of Organic Binary and Ternary Mixture Analysis
4.1 Separation reagent
4.2 Solubility procedure
4.3 Separation procedure
4.4 Solid-liquid or liquid-liquid mixture
4.5 Binary mixture
4.6 Ternary mixture
4.7 Qualitative Analysis of Organic Compounds
4.8 Functional Group Analysis: Analysis of Carboxylic Acids
4.9 Derivative: Amide & Anilide
4.10 Analysis of Phenols
4.10.1 Derivatives
4.10.2 Analysis of Amines
4.10.3 Derivatives
4.10.4 Analysis of Carbonyl compounds
4.10.5 Iodoform test for methyl ketone
4.10.6 Derivatives for carbonyl compound
4.10.7 Analysis of Amides
4.10.8 Derivatives
4.10.9 Anilides
4.10.10 Derivative
4.11 Analysis of Esters
4.11.1 Derivatives
4.11.2 Analysis of Ethers
4.11.3 Derivatives
4.11.4 Analysis of Hydrocarbon
4.11.5 Derivatives
4.11.6 Analysis of Miscellaneous Group
4.11.7 Carbohydrates
4.11.8 Derivatives: Carbohydrates
4.11.9 Analysis of Urea
4.11.10 Derivatives: Urea
4.12 Analysis of Thiourea
4.12.1 Derivatives: Thiourea

Chapter 5 Spectral Analysis and chromatographic techniques
5.0 Spectral Analysis
5.1 order of interpretation of the spectral data
5.2 Analysis of Spectral Data
5.3 Introduction of chromatography
5.3.1 Classification of Chromatographic Techniques
5.3.2 Thin Layer Chromatography–I
5.3.3 Thin Layer Chromatography–II
5.3.4 Thin Layer Chromatography-3
5.3.5 Coloum Chromatography -1
5.3.5.1 Principle
5.3.5.2 Materials Needed
5.3.5.3 Procedure: Dry Packing Method
5.3.5.4 Wet Packing Method
5.3.6 Column Chromatography-II
5.3.6.1 Principle
5.3.6.2 Materials Needed
5.3.6.3 Procedure: Dry Packing Method
5.3.6.4 Wet Packing Method

Bibliography

Chapter 1 Organic synthesis

1.1 Preparation of m-Dinitrobenzene

Aim: To synthesize a pure sample of m-dinitrobenzene by nitration of nitrobenzene.

Principle: Nitration of aromatic compounds containing electron releasing groups (OH, NH2 etc) is carried out under milder conditions because the aromatic nucleus is activated.

In case the aromatic compound containing an electron withdrawing group (NO2, SO3H, CHO, CO2H) the nitration requires drastic conditions (i.e.,) use of fuming HNO3 and concentrated H2SO4 as nitrating agents.

Type of reaction: Electrophilic Substitution Reaction on Aromatic ring (Nitration).

Apparatus: Round bottom flask (RBF), Air condenser, beaker, suction pump.

Chemicals required: 5ml nitrobenzene, 7ml fuming nitric acid, 10ml concentrated H2SO4.

Procedure: 1. Prepare nitrating mixture by placing 7ml of fuming nitric acid in a clean dry Round bottom flask (RBF). To this carefully add, with shaking 10ml of concentrated H2SO4 and a fragment of porcelain. 2. To the nitrating mixture add 5ml nitrobenzene in very small lots with constant shaking. After Adding whole of nitrating mixture shake the Round bottom flask (RBF) for 5 minutes. 3. Fix the air condenser and heat the flask on boiling water bath for 1 hour. Shake the flask vigorously from time to time throughout this period of heating. 4. After heating allow the Round bottom flask (RBF) to cool to room temperature. Finally pour this mixture carefully with vigorous stirring into a beaker containing crushed ice. The heavy oily dinitrobenzene will rapidly solidify.

Re-crystallization: Rectified spirit or alcohol.

Yield: 5.6g.

Melting Point: 89-900C.

Reference: 1.Practical Organic Chemistry by Mann and Saunders. 2. Comprehensive organic chemistry by V.K Ahluwalia and Renu Aggarwal.

1.2 Preparation of m-Nitroaniline

Aim: To synthesize a pure sample of m-nitroaniline by selective reduction of m-dinitrobenzene.

Principle: m-nitroaniline unlike o and p-nitroaniline cannot be prepared by direct nitration of Aniline. It has therefore to be prepared by reducing only one of the nitro groups in m-Dinitrobenzene. This selective reduction can be achieved by boiling an aqueous suspension of m-Dinitrobenzene with sodium disulphide. Sodium disulphide is prepared in situ by the addition of Sulphur to sodium sulphide solution. In these circumstances the dinitrobenzene is readily reduced to m-nitroaniline, the sodium disulphide being oxidized to sodium thiosulphate.

Types of reaction: partial reduction or selective reduction.

Apparatus: 500ml beaker sand bath burette, glass funnel.

Chemicals required: 2.5g m-dinitrobenzene, 1g-sulphur, 4 g-sodium sulphide, 200ml-water.

Procedure: 1. Preparation of sodium disulphide: Add 1 g of finely powdered sulphur to a solution of 4 g of crystalline sodium sulphide in 60ml water. Boil the mixture gently for a few a minutes until a clear solution of sodium disulphide is obtained. 2. Heat 2.5g of pure m-dinitrobenzene in 150ml of water in a 500ml beaker on sand bath until the water boils gently. 3. Transfer the sodium disulphide solution into a burette and clamp the burette in position such that the end of the burette is immediately above the beaker. 4. Allow the sodium disulfide solution to fall drop by drop into boiling water at such a rate that the total addition takes 10-15minutes throughout this period keep the molten dinitrobenzene continuously dispersed as fine drops and not allowed to settle to the bottom. 5. When the addition of sodium disulphide is complete, boil the solution gently for a 20 minutes and quickly filer the solution using a hot water funnel. 6. A small quantity of elementary sulphur remains in the filter paper. The pale brown filtrate rapidly deposits yellow crystals of m-nitroaniline.

Re-crystallization: Hot water.

Melting point: 1140C

Precautions:

1. The crystalline sodium sulphide (Na2S.9H2O) is very deliquescent, and only a sample which has been kept in a well Stoppard bottle and therefore reasonably dry should be used. 2. Avoid using animal charcoal during re-crystallization as it is liable to absorb and appreciable quantity of the m-nitroaniline.

Chemical reaction and its Mechanism:

illustration not visible in this excerpt

Reference: 1. Practical organic chemistry by man and saunters. 2. Jerry March.

1.3 Preparation of Hippuric Acid:

Aim: To synthesize a pure sample of hippuric Acid from glycine and benzoyl chloride.

Principle: Hippuric Acid can be synthesized by N-benzoylation of glycine. The benzoylation of hydroxyl and amino compounds in the presence of excess of cold aqueous base is called Schotte Baumann reaction. In Schotte-Baumann reaction N- Benzoylation or O- Benzoylation is carried out in the presence of cold aqueous NaOH. The excess of sodium hydroxide reacts with excess of un-reacted benzoyl chloride to give NaCl and sodium benzoate, which remains in solution.

Type of reaction: N-benzoylation.

Apparatus: Iodination flask, beaker, litmus paper, watch glass.

Chemicals required: glycin-2.5g, 25ml-10%NaOH, 4.5ml-Benzoyl chloride, CCl4-10ml.

Procedure: 1. in a clean dry iodination flask dissolve 2.5g glycine in 25ml of 10% NaOH 2. To this add 4.5ml benzoyl chloride in two lots. 3. After each addition, the flask is Stoppard and shaken vigorously until all the chloride has reacted. 4. Transfer the solution to a beaker and rinse the conical flask with a little water. 5. Place some crushed ice in the solution and concentrated HCl slowly with stirring until the mixture is acid to litmus paper. 6. Filter at pump. The crystalline benzoyl glycine obtained is contaminated with benzoic acid. 7. Place the solid in a beaker with 10ml CCl4, cover the beaker with a watch glass and boil gently for 10minutes on electric hot water bath. A pinch of animal charcoal may be added at this point. This extracts any benzoic acid which may be present.

Yield: 4.5g

Melting point: 1870 C

Chemical reaction and its Mechanism:

illustration not visible in this excerpt

Reference: 1. Textbook of practical organic chemistry by Vogel. 2. Comprehensive organic chemistry by V. K. Ahluwalia and Renu Agrawal.

1.4 Preparation of Azlactone:

Aim: To synthesize a pure sample of Azlactone from hippuric acid.

Principle:

Cyclization of benzoyl glycine (N-acyl-α-amino acid) with Ac2O yields an oxazolone derivative called Azlactone. The methylene group in this compound is reactive and condensation with benzaldehyde readily yields Azlactone.

Type of reaction: Condensation reaction (Erlenmeyer-Azlactone synthesis).

Apparatus: conical flask, beaker, glass rod.

Chemicals required: Hippuric acid 3g, 1.8ml benzaldehyde, 4.8ml acetic anhydride, anhydrous Sodium acetate 2g, ethanol 6.7ml.

Procedure:

1. Place a mixture of 1.8ml benzaldehyde, hippuric acid 3g and 4.8ml acetic anhydride into a conical flask. 2. Fuse 2g of sodium acetate and it to the mixture in the conical flask. 3. Heat the flask on burner with constant stirring 4. As soon as the mixture has liquefied completely, transfer the flask to a water bath and heat it for one hour. 5. Cool to room temperature and pour in a beaker containing cold water. Azlactone separates out. 6. Add alcohol and allow it to stand for 10 minutes. Filter at pump.

Re-crystallization: the product obtained is almost pure. If required re-crystallization can be carried out using benzene as solvent

Yield: 2.7g

Melting point: the yield of the compound is almost pure with a melting point of 165-1660C. Re-crystallization with benzene raises the melting point to 167-1680C.

Chemical reaction and its Mechanism:

illustration not visible in this excerpt

Reference: 1.Textbook of practical organic chemistry by Vogel. 2. Comprehensive organic chemistry by V.K Ahluwalia and Renu Aggarwal

1.5 Preparation of phthalimide:

Aim: To synthesize a pure sample of phthalimide from phthalic anhydride and urea.

Principle: It is an example of nucleophilic substitution reaction. Amino group of urea acts as Nucleophiles. Two moles of phthalic anhydride react with one mole of urea to give two moles of Phthalimide with the expulsion of CO2 and H2O.

Type of reaction: Nucleophilic substitution reaction.

Apparatus: Round bottom flask (RBF), suction pump.

Chemicals required: phthalic anhydride-3g, urea-0.6g.

Procedure: 1. intimately mix 3g of phthalic anhydride and 0.6g of urea and place it in a Round bottom flask (RBF). 2. Heat the flask on a low flame at 130-1350C. When the contents have melted effervescence commences and gradually increases in vigour. 3. after 3-5 minutes the mixture suddenly froths to about 3 time its original volume (temperature also rises to150-1600C) and becomes almost a solid. 4. Remove the flame from beneath and allow the RBF to cool to room temperature. 5. Add 5ml water to disintegrate the solid in the flask 6. Filter at pump, wash water and dry.

Re-crystallization: Secondary alcohol.

Yield: 3g

Melting point: 2330C.

Chemical reaction and its Mechanism:

illustration not visible in this excerpt

References: 1.Textbook of practical organic chemistry by Vogel.

1.6 Preparation of 2, 4-Dihydroxyacetophenone

Aim: To synthesize a pure sample of 2, 4-dihydroxyacetophenone from resorcinol.

Principle: 2, 4-dihydroxyacetophenone can be prepared from resorcinol by Nencki reaction. Nencki reaction involves the ring acylation of phenols in the presence of anhydrous ZnCl2 and acetic acid.

Type of reaction: Nencki reaction

Apparatus: beaker, sand bath.

Chemicals required: 2 g-resorcinol, 3g-Anhydrous ZnCl2, 2.9ml-glacial acetic acid, and 9 ml-1:1 dilute HCl.

Procedure: 1.Powdered anhydrous ZnCl2 (or freshly fused and powdered ZnCl2) is dissolved in glacial acetic acid by heating in beaker on sand bath. 2. 2g of powered resorcinol is added with stirring to the beaker containing acetic acid at 1400c the solution is heated until it just beings to boil & kept for 5 mines at 150 0c. 3. 9ml of 1:1 dilute HCl is added to the mixture and the solution is cooled to 5 0C. 4. The separated product is filtered, washed with 1:3 dilute HCl.

Re-crystallization: hot water containing few drops of dilutes HCl.

Yield: 2.5gms

Melting point: 142-144 0C

Chemical reaction and its Mechanism:

illustration not visible in this excerpt

Reference: 1.Comprehensive Organic Chemistry by V.K. Ahluwalia and Renu Aggarwal

1.7 Preparation of Anthracene-Maleic anhydride adduct

Aim: To synthesize a pure sample of anthracene-maleic adduct from anthracene and maleic anhydride.

Principle: Anthracene acts as a conjugated diene system. The double bond in maleic Anhydride is activated by the presence of two carbonyl groups. Therefore maleic Anhydride behaves as a dienophile. The thermal (4+2) cycloaddition reaction between diene (anthracene) and dienophile (maleic anhydride) is called Diels alder reaction. The product of Diels alder reaction is called an Adduct.

Type of reaction: Diels- Alder reaction. (Cycloaddition reaction)

Apparatus: Round bottom flask (RBF), condenser.

Chemical’s required: 1g-anthracene, 0.5gm-maleic anhydride, and dry xylene-13 ml.

Procedure: 1. Take 1g of anthracene, 0.5g-maleic anhydride, dry xylene 13 ml in a clean dry Round bottom flask (RBF). 2. Add a porcelain piece and boil under reflux for 20minutes. 3. During the early stages of heating, keep the mixture gently shaken until a clear solution is obtained, otherwise a portion of the reagents may adhere to the base of the flask and darken because of local overheating. 4. After boiling for 20 minutes, immediately filter using hot water funnel. Cool the solution, when the addition product will rapidly crystallize. 5. Filter at the pump and dry.

Note: 1.The crude is almost pure therefore re-crystallization is not required. Otherwise re-crystallization can be done with 50ml xylene. 2. Preheat the funnel if you are re-crystallizing the crude compound because the crystals tend to solidify very quickly.

Yield: 2.7 g

Melting point: 256-2580c

Chemical reaction and its Mechanism:

illustration not visible in this excerpt

Reference: 1. Comprehensive Organic Chemistry by V.K. Ahluwalia and Renu Aggarwal 2. Practical Organic Chemistry by Mann and Saunders

1.8 Microwave Assisted Synthesis of Anthracene Maleic Anhydride Adduct

Chemicals Required: Anthracene, maleic anhydride, digyme, methanol.

Equipment And Glassware: Microwave oven, 250 ml breaker.

Procedure: 1.Thoroughly grinds a mixture of 1.8g of anthracene and 0.98 g of maleic anhydride in a mortar and transfers it to a 250 ml beaker. 2. Add 5ml diglyme and shake the mixture gently. 3. Cover the beaker with a watch glass and place in a microwave oven. The irradiation is to be carried out for 90 seconds at a medium power level. 4. After the beaker is removed from the oven, allow it to cool to room temperature, a adduct will crystallize out and can be filtered. 5. Wash the product with methanol and dry. 6. The expected yield is 80%.

Reference: Collection of interesting general Chemistry experimental By Anil J. Elias.

1.9 Microwave Assisted Synthesis of Aspirin

Chemical Required: Salicylic acid, acetic anhydride, 85% phosphoric acid, ferric chloride

Equipment and Glassware: microwave oven, 100 ml beaker.

Procedure: 1. Take in a 100ml beaker 1.38g of salicylic acid, 3.06 g of acetic anhydride, and one drop of phosphoric acid and mix well. 2. Cover the beaker with a watch glass and place it in microwave oven at power level of 30% (probably level 3) for 5 minutes. 3. Take the beaker out of the microwave oven, allow it to cool to room temperature and place in an ice bath for crystallization. 4. Test the compound for salicylic acid by ferric chloride test. 5. Report the yield and melting point.

Reference: 1.Collection of interesting general chemistry experiments by Anil J Elias.

1.9.1 P-Bromoacetanilide

Aim: To study selective bromination reaction of acetanilide.

Principle: This reaction is an example for Electrophilic substitution. Acetanilide under goes bromination with bromine acetic acid to give p-Bromoacetanilide, in acetanilide NHCOCH3 group is moderately activating and o, p-directing group. Due to steric reason, acetanilide is brominated preferably at para position forming p-Bromoacetanilide.

Chemicals required: Acetanilide- 2.5 g, Glacial acetic acid 10ml

Apparatus required: Conical flask and beaker.

Procedure: Dissolve 2.5 g of finely powdered acetanilide in 10 ml glacial acetic acid taken in a conical flask. To this add bromine in acetic acid slowly with shaking till the reaction mixture turn reddish orange in colour. Allow the reaction mixture to stand at room temperature for 15-20 minutes. Then pour this mixture into about 100ml of cold water. The separated pale yellow para bromo acetanilide is filtered at the pump and washed with cold water. Allow it to dry completely and re-crystallize from methanol.

Melting point: 1630 c

Chemical reaction and its Mechanism: Preparation of P-Bromo Acetanilide

illustration not visible in this excerpt

Reference: 1.Elementary practical organic chemistry by Arthur Vogel pp-267

1.9.2 Synthesis of P-Bromoaniline

Aim: To study the hydrolysis of p-Bromo acetanilide.

Principle: The NH2 group in aniline is strongly activating and o, p-directing group. Therefore the reaction cannot be stopped at mono bromination stage to prepare p-Bromo aniline. The amino group of aniline is first protected and then brominated to give p-Bromoacetanilide which on hydrolysis gives p-Bromo aniline.

Chemicals required: P-Bromo acetanilide 2g , 70% H2SO4 15ml , 25%NaOH

Procedure: Transfer 2g of P-bromo acetanilide into Round Bottom flask and to this add 15 ml of 70% H2SO4 and boil the mixture gently under reflux for 20 minutes. Then pour the clear solution into about 50 ml of cold water. Neutralize the acidic solution with 25% NaOH until precipitation of P-bromo-aniline is complete. Cool the mixture in ice water and filter it. Wash well with water, drain thoroughly and re-crystallize from ethanol.

Melting point: 660C

Chemical reaction and its Mechanism:

illustration not visible in this excerpt

Reference: 1.Elementary practical Organic Chemistry by Arthur Vogel p: 267

1.9.3 Preparation of 2, 4, 6 Tribromoaniline

Aim: To study the bromination of aniline.

Principle: The amino group of aniline activates benzene towards Electrophilic substitution reaction. The substitutions take place in ortho and one para position to gives 2’4’6 Tribromoaniline.

Chemicals required: Aniline- 5ml , Glacial acetic acid 19ml, Bromine in acetic acid

Apparatus required: Conical flask, beaker and glass rod.

Procedure: Take 5 ml of aniline and 19 ml of glacial acetic acid in a flask. Place the flask in ice bath and add carefully bromine in acetic acid till deep red colour persists. Allow the solution to stand at room temperature for 5-10 minutes. Transfer the mixture into a breaker containing ice cold water. A while precipitate of 2, 4, 6 tribromo aniline precipitates out. Filter the product, wash it with water and re-crystallize from ethanol.

Melting point: 1200C

Chemical reaction and its Mechanism:

illustration not visible in this excerpt

Reference: 1. Compressive Organic Chemistry by V.K. Ahluwalia and Renu Aggarwal P: 108

1.9.4 Preparation of 1, 3, 5 Tribromobenzene:

Aim: To study de-amination of 2, 4, 6-Tribromo aniline.

Principle: The amino group in aromatic primary amine can be replaced by hydrogen (de-amination) by boiling the diazonium salt prepared from amine with ethyl alcohol in presence of dry benzene to give 1, 3, 5-Tribromobenzene.

Chemicals required: 2, 4, 6-Tribromo aniline 2g, Ethanol 20ml , Dry benzene 5ml, H2SO4 1ml, Sodium nitrite 1.5 g

Apparatus required: Round Bottom flask and water condenser

Procedure: Dissolve 2g of 2 ,4, 6-tribromo aniline in a hot mixture of 20ml of ethanol and 5ml of dry benzene taken in 150 ml of concentrated H2SO4 slowly to the hot solution with shaking. Attach a water condenser to the flask and heat on water bath until the clear solution boil. Now remove the condenser and add 1.5 gm of dry powdered sodium nitrate. Return the flask to the condenser but not to the water bath. Shake the flask vigorously. The heat of the reaction causes the solution to continue heating for same time. Reflux the reaction mixture for 45 minutes with occasional shaking. Allow the reaction to cool, thoroughly in ice. Filter the product at pump, wash with small quantity of methanol or ethanol and then water (2-3 times) and re-crystallize from methanol.

Melting point: 880 C

Chemical reaction and its Mechanism:

illustration not visible in this excerpt

Reference: 1. Comprehensive Organic Chemistry by V.K. Ahluwalia and Renu Agrawal P: 5

1.9.5 Preparation of Aspirin:

Aim: To study O-acetylation of salicylic acid.

Principle: Salicylic acid undergoes acetylation with acetic anhydride selectively on the Phenolic group in the presence of Concentrated H2SO4 as catalyst.

Chemicals required: Salicylic acid 2g, acetic anhydride 6ml, Concentrated H2SO4 2-3 drops

Procedure: Weight 2 gm of salicylic acid and transfer into a clean conical flask. Add 6 ml of acetic anhydride, stir with a glass rod and add 2-3 drops of concentrated H2SO4 stir the mixture and heat on the water bath for 15 minutes. Filter the product Precipitated at a suction pump. Wash with cold water and dry it. Re-crystallize from acetic acid and water.

Melting point: 1360C

Chemical reaction and its Mechanism:

illustration not visible in this excerpt

Reference: 1. Comprehensive Organic Chemistry by V.K. Ahluwalia and Renu Agrawal pp: 3

1.9.6 Preparation of Tetrahydrocarbazole

Aim: To study the Fischer Indole synthesis.

Principle: This is an example of Fischer Indole synthesis which involves an acid catalyzed rearrangement of a phenyl hydrozone of an aldehyde or Ketone with the elimination of a molecule of ammonia forming Tetrahydrocarbazole.

Chemicals required: Cyclohexanone 2.5ml , Phenyl hydrazine 2ml , Glacial acetic acid 12ml

Procedure: Transfer 2.5 ml of cyclohexanone and 12 ml of glacial acetic acid in a Round Bottom flask and add 2 ml of phenyl hydrazine to it. Fix a reflux condenser and boil the mixture gently for 30 minutes. Filter the separated Tetrahydrocarbazole and re-crystallize from alcohol.

Melting point: 1170C

Chemical reaction and its Mechanism:

illustration not visible in this excerpt

Reference: 1.Elementary practical Organic Chemistry by Arthur Vogel pp: 363

1.9.7 7-Hydroxy 4-Methyl Coumarin (Umbelliferon)

Aim: To study Pechmann condensation.

Principle: Pechmann reaction involves the interaction of phenol with β- ketonic ester in the presence of condensing agent like H2SO4 or AlCl3 or POCl3 or PPA. The mechanism involves the transfer of proton from the acid catalyst to the ketonic group of β-carbonyl ester. This results in the reduction of electron density of the carbonyl carbon atom which then attacks the ortho position of phenol like any Electrophilic reagent. In the next step Cyclization is completed by elimination of water and ethanol.

Chemicals required: Resorcinol 3.7g, Ethyl aceto acetate 4.5ml.

Apparatus required: Beaker and conical flask.

Procedure: Transfer measured amount of H2SO4 into 250 ml beaker and cool in ice till the temperature reaches 50C. Measure ethylacetoacetate into a clean conical flask and add resorcinol in small quantities while stirring. After the complete addition the mixture should be a clear solution. Then add this mixture in small quantities to H2SO4 taken in the beaker by maintaining the temperature of the mixture between 5-100C Stir the reaction mixture well and cool for 20 minutes. After the addition is completed, transfer the reaction mixture onto the crushed ice taken in beaker with stirring, whereby a pale yellow solid of 7-hydroxy-4-methyl Coumarin separate out. Filter the product at suction and re-crystallize from ethanol.

Melting point: 1850C

Chemical reaction and its Mechanism:

illustration not visible in this excerpt

Reference: 1.Comprehensive Organic Chemistry by V.K. Ahluwalia and Renu Agrawal, pp-177.

1.9.8 Organic Synthesis-1: 2–Phenyl Indole

Aim: To synthesize 2-phenyl indole

Chemical Name: 2-phenyl indole.

Structure:

illustration not visible in this excerpt

Principle: Synthesize of 2-phenyl indole involves two steps.

Step1: Formation of Schiff’s base by reaction between acetophenone and phenyl hydrazine

Step2: Cyclization of phenyl hydrazine derivative in presence of polyphosphoric acid to form 2-phenyl indole.

Type of reaction: Fischer indole synthesis

Step 1- Preparation of acetophenone phenyl hydrazone

illustration not visible in this excerpt

Chemicals: 2.5 ml Acetophenone, 3ml phenyl hydrazine, 5ml rectified spirit.

Apparatus required: Round Bottom flask, water condenser.

Procedure: Warm a mixture of 2.5 ml acetophenone and 3ml of phenyl hydrazine, 4ml of glacial acetic acid on a water bath for 1 hour dissolve hot mixture in 5ml of spirit and shake. Stir to introduce crystallization. Cool mixture in ice, filter wash with 1.2ml of rectified spirit. Dry in vacuums desiccators over anhydrous CaCl3 for at least half an hour.

The yield of phenylhydrazone is 1.5gms.

M.P.: 105-106 degree C.

Step2: Preparation of 2-phenyl indole

Chemicals: 1.5 gm of phenyl hydrazone, 8ml of phosphoric acid, 3gm of phosphorous pent oxide.

Procedure: In a conical flask add 1.5gn of acetophenone phenylhydrazone to per heated, (80degree) 10 gm of polyphosphoric acid (8ml phosphoric acid, 3gm of phosphorous pent oxide is added and heated if necessary to get a clear solution of polyphosphoric acid). Stir mixture at 100 degree C for 1hour on water bath. Cool solution, add ice water and filter off grey precipitate. Wash residue with cold water and dry.

Re-crystallization: solvent-ethanol.

Yield: 1gram

M.P.: 185-186degree C.

Chemical reaction and its Mechanism:

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Reference: 1. Elementary practical organic chemistry part- I Arthur, Vogel, pp-361.

1.9.9 Organic Synthesis - 2: 7-Hydroxy-3-Methyl Flavone

Aim: To synthesize Flavone by Baker Venkatraman method.

Chemical name: 7- Hydroxy 3- methyl Flavone.

Structure:

illustration not visible in this excerpt

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