Medicinal Chemistry Research

, Volume 23, Issue 1, pp 503–517

Novel ibuprofen prodrugs with improved pharmacokinetics and non-ulcerogenic potential

Authors

  • Valmik D. Dhakane
    • Medicinal Chemistry Research Laboratory, School of Chemical SciencesSolapur University
  • Hemant V. Chavan
    • Medicinal Chemistry Research Laboratory, School of Chemical SciencesSolapur University
  • Vishnu N. Thakare
    • Department of PharmacologySinhgad Institute of Pharmaceutical Sciences
  • Laxman K. Adsul
    • Medicinal Chemistry Research Laboratory, School of Chemical SciencesSolapur University
  • Sadanand N. Shringare
    • Medicinal Chemistry Research Laboratory, School of Chemical SciencesSolapur University
    • Medicinal Chemistry Research Laboratory, School of Chemical SciencesSolapur University
Original Research

DOI: 10.1007/s00044-013-0639-8

Cite this article as:
Dhakane, V.D., Chavan, H.V., Thakare, V.N. et al. Med Chem Res (2014) 23: 503. doi:10.1007/s00044-013-0639-8
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Abstract

In the present study, we evaluated the anti-inflammatory activity with pharmacokinetic, ulcerogenic properties of various synthesized prodrugs of ibuprofen in experimental animals. Prodrugs 2, 6, 9, 10, 12, and 14 were found to possess significant anti-inflammatory activity with almost non-ulcerogenic potential than standard drug ibuprofen 1a in both normal and inflammation-induced rats. Metabolic stability of prodrugs 2, 6, 9, 10, 12, and 14 were also studied in rat liver microsomes and oral bioavailability was determined by estimating area under curve (AUC) and plasma concentration of these prodrugs at various time intervals. The experimental findings elicited higher AUC and plasma concentration at 1 and 2 h indicating improved oral bioavailability as compared to parent ibuprofen. These prodrugs are found to have least gastric ulceration with retain anti-inflammatory activity observed in experimental animals. Therefore, present experimental findings demonstrated significant improvement of various pharmacokinetic properties with least ulcerogenic potential of ester prodrugs of ibuprofen an anti-inflammatory agent

Keywords

ProdrugIbuprofenAnti-inflammatory activityNon-ulcerogenicPharmacokinetic properties

Abbreviations

NSAID

Nonsteroidal anti-inflammatory drug

Tris–HCl

Tris(hydroxymethyl)aminomethane hydrochloride

NADPH

Nicotinamide adenine dinucleotide phosphate

HPLC

High performance liquid chromatography

GC

Gas chromatography

RLM

Rat liver microsomes

RP

Rat plasma

API

Active pharmaceutical ingredient

UI

Ulcer index

GI

Gastrointestinal

DMAc

Dimethyl acetamide

PTSA

p-Toluene sulfonic acid

PXRD

Powder X-ray diffraction

RT

Room temperature

TMG

1,1,3,3-Tetramethyl guanidine

18 Crown 6

1,4,7,10,13,16-Hexaoxacyclooctadecane

EP

European pharmacopoeia

Ar

Aromatic

BDL

Below detection limit

Introduction

Ibuprofen, a well-known nonsteroidal anti-inflammatory drug (NSAIDs) most widely used and represents a mainstay in the therapy of acute and chronic pain and inflammation viz. osteoarthritis and rheumatoid arthritis (Coruzzi et al., 2007; Kawail et al., 2005; Klasser and Epstein, 2005). The anti-inflammatory activity of ibuprofen is mainly due to its inhibitory effect on prostaglandin synthase (cyclooxygenase) which is involved in the formation of various inflammatory prostaglandins. However, the use of ibuprofen is frequently associated with an adverse effect, in particular its gastritis associated with ulceration, which are related to inhibiting prostaglandin synthesis. These adverse effects are likely due to the presence of carboxylic group and inhibition of protective cyclooxygenase-1 (COX-1) enzyme in stomach (Rainsford, 1988; Bhosale et al., 2006). Inhibition of cyclooxygenase-2 (COX-2) enzyme in desirable with ibuprofen for its anti-inflammatory activity is concern; however, simultaneously the COX-1 inhibition is documented with ibuprofen which is undesired. Selective inhibition of COX-2 with coxibs (roficoxib, valdecoxib) are of great importance in inflammation. However, their serious cardiovascular effects (stroke) observed from clinical studies and post marketing surveillance, forcing the pharma companies to withdraw these coxibs from the market (Bresalier et al., 2005; Nussmeier et al., 2005). Precondition for the prodrugs possesses good pharmacological (anti-inflammatory) effect, differentiated pharmacokinetic property (appropriate aqueous solubility and lipophilicity) and no ulcerogenic action. Various ester prodrugs of NSAIDs were found to possess potent anti-inflammatory activity with lesser ulcerogenic property on gastrointestinal (GI) mucosa Perioli et al., 2004; Halen et al., 2009; Shanbhag et al., 1992; Chen et al., 2009; Borhade et al.,2012.

The majority of NSAID prodrugs have been produced by masking of carboxylic functional group. The esters have dominated prodrug research due to their ideal characteristic of exhibiting reasonable in vitro chemical stability, allowing them to formulate with desired t1/2. Because of its ability to functions as esterase substrates, esters are suitably liable in vivo condition (Bundgaard, 1986, 1989). Therefore, by the use of prodrug approach, one can mask the carboxylic acid functional group of NSAID, which on hydrolysis, an active parent drug to be released in vivo for its anti-inflammatory activity along with reduction in ulcerogenic property (Gairola et al., 2005; Wallace, 1994; Kim et al., 2006).

Toxicological and pharmacological profiles of ibuprofen guiacol ester (Cioli et al., 1980), synthesized ester prodrugs using N-hydroxymethylsuccinimide and N-hydroxymethylisatin as promoeities to reduce their GI toxicity and improve bioavailability (Mahfouz et al., 1999), 2-formylphenyl esters (Abordo et al., 1998) was studied. However, their release characteristic is found to exhibits variation. And most of the prodrugs of NSAIDs reported in the literatures have poor or limited hydro solubility (Nielsen and Bundgaard, 1988; Shaaya et al., 2003).

The various studies on prodrugs suggested that, nitric oxide (NO) release elicits cytoprotective activity and retained anti-inflammatory activity (Huang et al., 2011). However, the aim of synthesis of prodrugs of NSAIDs is to prevent the adverse effect by protecting GI damage and this effect can be masked by producing inert promoeities. Because there may be a chance of other side effect that might be associated with NO on the other organ system apart from GI.

Based on the scientific reports on ibuprofen prodrugs which are of great interest of reducing GI toxicity and improved biopharmaceutical properties further experiments are required to design and identify prodrugs of ibuprofen, which would be suitable for clinical practice in terms of stability, metabolism, toxicology and side effects. Ibuprofen is presently marketed as a racemic mixture of active substance for its anti-inflammatory and analgesic actions. Therefore, racemic mixture of ibuprofen is taken for the preparation of prodrugs.

The present research work is an attempt to develop prodrugs of ibuprofen 1–15, using novel promoeities 2a–o having high in vivo enzymatic conversion rate, anti-inflammatory, physicochemical and non-ulcerogenic properties.

Experimental section

Materials and methods

All reactions were monitored by thin layer chromatography (TLC) with 0.2 mm Merck silica gel F254 plates and GC. NMR spectra were recorded on Bruker DRX FT NMR at 400 MHz spectrometer in CDCl3 using TMS as internal standard (chemical shifts are expressed as δ values relative to TMS as internal standard). IR spectra were recorded on a FTIR Perkin-Elmer model RXI spectrophotometer. Mass spectra were recorded on an Agilent GCMS-5975C VL MSD spectrophotometer. HPLC analysis was performed using a Shimadzu LC 2010 CHT with UV detector. All prodrugs 1–15 were analyzed by HPLC, and their purity was confirmed to be excess of 98 %. GC analysis was performed on Shimadzu GC-2014 instrument, and purity of promoieties 2a–o was confirmed to be in excess of 97 %. In vivo study was performed using experimental protocols reviewed and approved by the Institutional Animal Ethics Committee (SIPS/IAEC/2011-12/17) and conform to the Indian National Science Academy Guidelines for the use and care of experimental animals in research. The animal house Registration No. with Government of India is 962/c/06/CPCSEA.

General procedure for the preparation of prodrugs (1–15)

Ibuprofen (2 g, 9.67 mmol) was dissolved in dimethyl acetamide (25 mL) at room temperature (25 °C). To this solution, potassium carbonate (4 g, 28.9 mmol) under nitrogen atmosphere was added. The reaction mass was stirred for 20 min at room temperature (25 °C). Then, to the above reaction mass, promoieties 2a–2o (8.7 mmol) was added under stirring and maintained the reaction under stirring for 4–5 h at room temperature. The reaction progress was monitored by TLC and GC. After completion, the reaction mass was added under stirring into the mixture of ethyl acetate (60 mL) and water (100 mL). The organic layer was separated and aqueous layer extracted with ethyl acetate twice (60 mL × 2). Combined organic layer washed with 20 % solution of sodium bicarbonate (100 mL × 2) followed by brine wash (10 % NaCl, 100 mL). Organic layer was dried over anhydrous sodium sulfate, distilled off the solvent under vacuum and desired product collected as viscous oil.

Spectral data of synthesized compounds

Ethyl 2-(2-(4-isobutylphenyl)propanoyloxy)-3-oxobutanoate (1)

Chromatographic purity (HPLC) 99.78 %; bp 298 ºC; MS (ESI+) m/z = 334 [M+]; IR (KBr, cm−1): 2957, 2871, 1745, 1633, 1609, 1514, 1369, 1156, 852; 1H NMR (CDCl3, 400 MHz) δ 0.89 (d, J = 6.8 Hz, 6H, CH(CH3)2), 1.23 (t, J = 7.2 Hz, 3H, OCH2CH3); 1.57 (m, 3H, ArCHCH3), 1.84 (m, 1H, CH(CH3)2), 2.08 (s, 3H, COCH3), 2.45 (d, J = 7.2 Hz, 2H, ArCH2CH), 3.90 (q, J = 7.2 Hz, 2H, OCH2CH3), 4.24 (q, J = 7.2 Hz, 1H, ArCHCH3), 5.43 (s, 1H, OCHCO), 7.10 (d, J = 8 Hz, 2H, ArH), 7.25 (d, J = 8 Hz, 2H, ArH).

4-Acetoxybutyl-2-(4-isobutylphenyl)propanoate (2)

Chromatographic purity (HPLC) 99.60 %; bp > 300 ºC; MS (ESI+) m/z = 320 [M+]; IR (KBr, cm−1): 2945, 2870, 1743, 1614, 1515, 1460, 1151, 843; 1H NMR (CDCl3, 400 MHz) δ 0.88 (d, J = 6.8 Hz, 6H, CH(CH3)2), 1.49 (d, J = 7.2 Hz, 3H, ArCHCH3), 1.54–1.68 (m, 4H, CH2CH2CH2O), 1.84 (m, 1H, CH(CH3)2), 2.02 (s, 3H, COCH3), 2.45 (d, J = 7.2 Hz, 2H, ArCH2CH), 3.69 (q, J = 7.2 Hz, 1H, ArCHCH3), 4.01 (t, J = 6 Hz, 2H, CH2COOCH3), 4.08 (t, J = 6 Hz, 2H, CH2OCOCH), 7.09 (d, J = 8 Hz, 2H, ArH), 7.20 (d, J = 8 Hz, 2H, ArH).

1-Methoxy-1-oxopropan-2-yl-2-(4-isobutylphenyl)propanoate (3)

Chromatographic purity (HPLC) 99.69 %; bp 276 ºC; MS (ESI+) m/z = 292 [M+]; IR (KBr, cm−1): 2955, 2870, 1744, 1614, 1514, 1455, 1160, 1101, 848; 1H NMR (CDCl3, 400 MHz) δ 0.90 (d, J = 6.4 Hz, 6H, CH(CH3)2), 1.42 (d, J = 7.2 Hz, 1H, OCHCH3), 1.53 (d, J = 7.2 Hz, 3H, ArCHCH3), 1.84 (m, 1H, CH(CH3)2), 2.45 (d, J = 7.2 Hz, 2H, ArCH2CH), 3.62 (s, 3H, COOCH3), 3.79 (q, J = 7.2 Hz, 1H, ArCHCH3), 5.08 (q, J = 7.2 Hz, 1H, OCHCH3), 7.10 (d, J = 8 Hz, 2H, ArH), 7.22 (d, J = 8 Hz, 2H, ArH).

2-(Cyclohexyloxy)-2-oxoethyl-2-(4-isobutylphenyl)propanoate (4)

Chromatographic purity (HPLC) 99.91 %; bp > 300 ºC; MS (ESI+) m/z = 346 [M+]; IR (KBr, cm−1): 2938, 2865, 1746, 1614, 1514, 1455, 1150, 845; 1H NMR (CDCl3, 400 MHz) δ 0.86 (d, J = 6.4 Hz, 6H, CH(CH3)2), 1.19–1.34 (m, 6H, 3×CH2), 1.60–1.71 (m, 4H, 2×CH2), 1.41 (d, J = 7.2 Hz, 3H, ArCHCH3), 1.80 (m, 1H, CH(CH3)2), 2.42 (d, J = 7.2 Hz, 2H, ArCH2CH), 3.85 (q, J = 7.2 Hz, 1H, ArCHCH3), 4.61 (s, 2H, OCH2CO), 4.69 (m, 1H, COOCH), 7.12 (d, J = 8 Hz, 2H, ArH), 7.20 (d, J = 8 Hz, 2H, ArH).

2-(2-Ethylbutoxy)-2-oxoethyl-2-(4-isobutylphenyl)propanoate (5)

Chromatographic purity (HPLC) 99.78 %; bp 291 ºC; MS (ESI+) m/z = 348 [M+]; IR (KBr, cm−1): 2962, 2875, 1747, 1513, 1463, 1152, 847; 1H NMR (CDCl3, 400 MHz) δ 0.84–0.88 (m, 12H, 4×CH3), 1.23–1.34 (m, 3H, CHCH2CH3/CHCH2CH3); 1.50 (qu, 2H, CHCH2CH3), 1.41 (d, J = 7.2 Hz, 3H, ArCHCH3), 1.81 (m, 1H, CH(CH3)2), 2.42 (d, J = 7.2 Hz, 2H, ArCH2CH), 3.85 (q, J = 7.2 Hz, 1H, ArCHCH3), 4.05 (t, J = 6.4 Hz, 2H, COOCH2CH), 4.64 (s, 2H, OCH2CO), 7.11 (d, J = 8 Hz, 2H, ArH), 7.20 (d, J = 8 Hz, 2H, ArH).

2-(2-Methoxyethoxy)-2-oxoethyl-2-(4-isobutylphenyl)propanoate (6)

Chromatographic purity (HPLC) 99.77 %; bp 288 ºC; MS (ESI+) m/z = 322 [M+]; IR (KBr, cm−1): 2955, 2870, 1746, 1513, 1458, 1157, 847; 1H NMR (CDCl3, 400 MHz) δ 0.88 (d, J = 6.6 Hz, 6H, CH(CH3)2), 1.54 (d, J = 7.2 Hz, 3H, ArCHCH3), 1.84 (m, 1H, CH(CH3)2), 2.45 (d, J = 7.2 Hz, 2H, ArCH2CH), 3.36 (s, 3H, OCH3), 3.56 (m, 2H, CH2OCH3), 3.81 (q, J = 7.2 Hz, 1H, ArCHCH3), 4.28 (m, 2H, COOCH2), 4.59 (s, 2H, OCH2CO), 7.11 (d, J = 8 Hz, 2H, ArH), 7.22 (d, J = 8 Hz, 2H, ArH).

2-Isopropoxy-2-oxoethyl-2-(4-isobutylphenyl)propanoate (7)

Chromatographic purity (HPLC) 98.34 %; bp 266 ºC; MS (ESI+) m/z = 306 [M+]; IR (KBr, cm−1): 2976, 2870, 1741, 1516, 1462, 1373, 1105, 845; 1H NMR (CDCl3, 400 MHz) δ 0.84 (d, J = 6.8 Hz, 6H, CH(CH3)2), 1.14 (d, J = 6.4 Hz, 6H, OCH(CH3)2), 1.41 (d, J = 7.2 Hz, 3H, ArCHCH3), 1.81 (m, 1H, CH2CH(CH3)2), 2.42 (d, J = 7.2 Hz, 2H, ArCH2CH), 3.85 (q, J = 7.2 Hz, 1H, ArCHCH3), 4.59 (s, 2H, OCH2CO), 4.91 (sept, J = 6.4 Hz, 1H, OCH(CH3)2), 7.12 (d, J = 8 Hz, 2H, ArH), 7.23 (d, J = 8 Hz, 2H, ArH).

2-Butoxy-2-oxoethyl-2-(4-isobutylphenyl)propanoate (8)

Chromatographic purity (HPLC) 99.67 %; bp 275 ºC; MS (ESI+) m/z = 320 [M+]; IR (KBr, cm−1): 2958, 2871, 1747, 1514, 1458, 1153, 846; 1H NMR (CDCl3, 400 MHz) δ 0.80–0.86 (m, 11H, 3×CH3, CH2CH3), 1.25 (qu, J = 6.8 Hz, 2H, OCH2CH2), 1.41 (d, J = 7.2 Hz, 3H, ArCHCH3), 1.80 (m, 1H, CH(CH3)2), 2.42 (d, J = 7.2 Hz, 2H, ArCH2CH), 3.85 (q, J = 7.2 Hz, 1H, ArCHCH3), 3.98 (m, 2H, OCH2CH2), 4.65 (s, 2H, OCH2CO), 7.11 (d, J = 8 Hz, 2H, ArH), 7.20 (d, J = 8 Hz, 2H, ArH).

2-(Benzyloxy)-2-oxoethyl-2-(4-isobutylphenyl)propanoate (9)

Chromatographic purity (HPLC) 98.81 %; bp > 300 ºC; MS (ESI+) m/z = 354 [M+]; IR (KBr, cm−1): 2954, 2848, 1747, 1608, 1514, 1455, 1150, 847, 750, 698; 1H NMR (CDCl3, 400 MHz) δ 0.85–0.90 (m, 6H, CH(CH3)2), 1.52 (d, J = 7.2 Hz, 3H, ArCHCH3), 1.84 (m, 1H, CH(CH3)2), 2.43 (d, J = 7.2 Hz, 2H, ArCH2CH), 3.82 (q, J = 7.2 Hz, 1H, ArCHCH3), 4.54 (s, 2H, COOCH2CO), 4.05–4.10 (m, 2H, OCH2Ph), 7.11 (d, J = 8 Hz, 2H, ArH), 7.22 (d, J = 8 Hz, 2H, ArH), 7.28–7.40 (m, 5H, ArH).

Cyclohexyl-2-(2-(4-isobutylphenyl)propanoyloxy)butanoate (10)

Chromatographic purity (HPLC) 98.88 %; bp > 300 ºC; MS (ESI+) m/z = 374 [M+]; IR (KBr, cm−1): 2941, 2869, 1748, 1615, 1512, 1458, 1153, 844; 1H NMR (CDCl3, 400 MHz) δ 0.81–0.94 (m, 9H, 3×CH3), 1.25–1.39 (m, 6H, 3×CH2), 1.53 (m, 3H, ArCHCH3), 1.65–1.74 (m, 4H, 2×CH2), 1.76–1.87 (m, 3H, CH(CH3)2/CHCH2CH3), 2.43 (d, J = 7.2 Hz, 2H, ArCH2CH), 3.79 (q, J = 7.2 Hz, 1H, ArCHCH3), 4.75–4.90 (m, 2H, OCHCO/OCH(CH2)2), 7.09 (d, J = 8 Hz, 2H, ArH), 7.24 (d, J = 8 Hz, 2H, ArH), 7.28–7.40 (m, 5H, ArH).

2-Ethylbutyl-2-(2-(4-isobutylphenyl)propanoyloxy)butanoate (11)

Chromatographic purity (HPLC) 99.78 %; bp > 300 ºC; MS (ESI+) m/z = 376 [M+]; IR (KBr, cm−1): 2966, 2877, 1748, 1514, 1460, 1151, 848; 1H NMR (CDCl3, 400 MHz) δ 0.73–0.85 (m, 15H, 5×CH3), 1.17–1.30 (m, 4H, CH(CH2CH3)2), 1.46 (m, 1H, OCH2CH), 1.66–1.82 (m, 3H, CH(CH3)2/CHCH2CH3), 1.40 (d, J = 7.2 Hz, 3H, ArCHCH3), 2.42 (d, J = 7.2 Hz, 2H, ArCH2CH), 3.79–3.95 (m, 2H, OCH2CH), 3.96–4.06 (m, 1H, ArCHCH3), 4.85 (m, 1H, OCHCO), 7.11 (d, J = 8 Hz, 2H, ArH), 7.20 (d, J = 8 Hz, 2H, ArH).

2-Methoxyethyl-2-(2-(4-isobutylphenyl)propanoyloxy)butanoate (12)

Chromatographic purity (HPLC) 99.81 %; bp > 300 ºC; MS (ESI+) m/z = 350 [M+]; IR (KBr, cm−1): 2955, 2880, 1743, 1514, 1458, 1383, 1199, 1161, 848; 1H NMR (CDCl3, 400 MHz) δ 0.80–0.94 (m, 9H, 3×CH3), 1.54 (m, 3H, ArCHCH3), 1.79–1.88 (m, 3H, CH(CH3)2/CH3CH2CH), 2.43 (d, J = 7.2 Hz, ArCH2CH), 3.36 (s, 3H, OCH3), 3.47–3.58 (m, 2H, CH2OCH3), 3.80 (m, 1H, ArCHCH3), 4.21–4.30 (m, 2H, COOCH2), 4.90–4.95 (m, 1H, OCHCO), 7.08–7.11 (m, 2H, ArH), 7.21–7.26 (m, 2H, ArH).

Isopropyl-2-(2-(4-isobutylphenyl)propanoyloxy)butanoate (13)

Chromatographic purity (HPLC) 99.97 %; bp 296 ºC; MS (ESI+) m/z = 334 [M+]; IR (KBr, cm−1): 2978, 2937, 2870, 1740, 1513, 1465, 1376, 1103, 847; 1H NMR (CDCl3, 400 MHz) δ 0.81–0.94 (m, 9H, 3×CH3), 1.11 (d, J = 6.4 Hz, 3H, CH3CH), 1.20 (d, J = 6.4 Hz, 3H, CH3CH), 1.51 (d, J = 7.2 Hz, 3H, ArCHCH3), 1.55–1.86 (m, 3H, CH(CH3)2/CH3CH2CH), 2.43 (d, J = 7.2 Hz, ArCH2CH), 3.79 (m, 1H, ArCHCH3), 4.81–4.88 (m, 1H, OCH(CH3)2), 4.95–5.07 (m, 1H, OCHCO), 7.07–7.10 (m, 2H, ArH), 7.22–7.26 (m, 2H, ArH).

Butyl-2-(2-(4-isobutylphenyl)propanoyloxy)butanoate (14)

Chromatographic purity (HPLC) 99.80 %; bp > 300 ºC; MS (ESI+) m/z = 348 [M+]; IR (KBr, cm−1): 2960, 2872, 1746, 1514, 1457, 1154, 847; 1H NMR (CDCl3, 400 MHz) δ 0.76 (t, J = 7.2 Hz, 3H, CH3CH2), 0.82–0.89 (m, 9H, 3×CH3), 1.27 (m, 2H, CH3CH2CH2), 1.40 (d, J = 7.2 Hz, 3H, ArCHCH3), 1.50 (m, 2H, OCH2CH2CH2), 1.68–1.82 (m, 3H, CH(CH3)2/CHCH2CH3), 2.42 (d, J = 7.2 Hz, ArCH2CH), 3.81–3.99 (m, 2H, OCH2CH2), 4.01–4.11 (m, 1H, ArCHCH3), 4.83 (m, 1H, OCHCO), 7.11 (d, J = 8 Hz, 2H, ArH), 7.22 (d, J = 8 Hz, 2H, ArH).

Benzyl-2-(2-(4-isobutylphenyl)propanoyloxy)butanoate (15)

Chromatographic purity (HPLC) 99.59 %; bp > 300 ºC; MS (ESI+) m/z = 382 [M+]; IR (KBr, cm−1): 2955, 2870, 1740, 1608, 1514, 1456, 1383, 1197, 1107, 847, 750, 698; 1H NMR (CDCl3, 400 MHz) δ 0.73 (t, J = 7.2 Hz, 3H, CH3CH2), 0.82 (m, 6H, CH(CH3)2), 1.38 (t, J = 7.2 Hz, 3H, ArCHCH3), 1.66–1.81 (m, 3H, CH(CH3)2, CH3CH2 CH), 2.41 (d, J = 7.2 Hz, 2H, ArCH2CH), 3.83 (q, J = 7.2 Hz, 1H, ArCHCH3), 4.90 (m, 1H, OCHCO), 5.09 (s, 2H, OCH2Ph), 7.09 (d, J = 8 Hz, 2H, ArH), 7.18 (d, J = 8 Hz, 2H, ArH), 7.27–7.38 (m, 5H, ArH).

Preparation of promoieties

Ethyl-2-chloro-3-oxobutanoate (2a)

2-Chloro-3-oxobutanoic acid (20 g, 0.15 mol) was treated with ethanol (9.2 g, 0.2 mol) in the presence of toluene (100 g, 1.08 mmol) as a solvent and PTSA (0.5 gm, 0.0026 mmol) as a catalyst. The reaction mass was maintained at reflux for 2 h. Reaction progress was monitored on GC. After completion, reaction mass cooled to 30 °C and washed twice with 200 mL 20 % sodium bicarbonate solution in water followed by brine and water wash. Organic layer dried over anhydrous sodium sulfate and distilled off the solvent under reduced pressure and pure ethyl 2-chloro 3-oxobutanoate was collected as colorless oil.

Chromatographic purity (HPLC) 97.73 %; bp 200 ºC; MS (ESI+) m/z = 164 [M+], 166 [M+2]; IR (KBr, cm−1): 2985, 1732, 1645, 1621, 1257, 1032; 1H NMR (CDCl3, 400 MHz) δ 1.34 (t, J = 7.6 Hz, 3H, CH2CH3), 2.39 (s, 3H, COCH3), 4.30 (q, J = 7.6 Hz, 2H, CH2CH3), 4.79 (s, 1H, CHCl).

4-Bromobutyl acetate (2b)

4-Bromobutyric acid (20 g, 0.12 mol), methanol (9.5 g, 0.29 mol) in the presence of toluene (100 g, 1.08 mmol) and PTSA (0.5 g, 0.0026 mmol) refluxed for 2 h. Work up of reaction carried out as per the procedure given for 2a.

Chromatographic purity (HPLC) 99.08 %; bp 211 ºC; MS (ESI+) m/z = 194 [M+], 196 [M+2]; IR (KBr, cm−1): 2963, 1739, 1438, 1237, 1042; 1H NMR (CDCl3, 400 MHz) δ 1.81 (qu, J = 6.4 Hz, 2H, CH2CH2Br), 1.93 (qu, J = 6.4 Hz, 2H, CH2CH2O), 2.06 (s, 3H, COCH3), 3.44 (t, J = 6.4 Hz, 2H, CH2Br), 4.10 (t, J = 6.4 Hz, 2H, COOCH2).

Methyl-2-bromopropanoate (2c)

2-Bromopropionic acid (20 g, 0.13 mol), methanol (9.5 g, 0.29 mol) in the presence of toluene (100 g, 1.08 mmol) and PTSA (0.5 g, 0.0026 mmol) refluxed for 2 h. Work up of reaction carried out as per the procedure given for 2a.

Chromatographic purity (HPLC) 97.72 %; bp 145 ºC; MS (ESI+) m/z = 166 [M+], 168 [M+2]; IR (KBr, cm−1): 2968, 2878, 1736, 1458, 1270, 1156; 1H NMR (CDCl3, 400 MHz) δ 1.84 (d, J = 7.6 Hz, 3H, CH2CH3), 3.78 (s, 3H, OCH3), 4.40 (q, J = 7.6 Hz, 1H, CHCH3).

Cyclohexyl-2-bromoacetate (2d)

Bromoacetic acid (20 g, 0.14 mmol), cyclohexanol (12.9 g, 0.12 mmol) in the presence of toluene (100 g, 1.08 mmol) and PTSA (0.5 g, 0.0026 mmol) refluxed for 2 h. Work up of reaction carried out as per the procedure given for 2a.

Chromatographic purity (HPLC) 98.80 %; bp 242 ºC; MS (ESI+) m/z = 221 [M+]; IR (KBr, cm−1):2965, 2877, 1736, 1451, 1233, 1163; 1H NMR (CDCl3, 400 MHz) δ 1.42–1.57 (m, 6H, 2×CH2), 1.72–1.88 (m, 4H, CH(CH2)2), 3.81 (s, 2H, COCH2Br), 4.82 (m, 1H, CH(CH2)2).

2-Ethylbutyl-2-bromoacetate (2e)

Bromoacetic acid (20 g, 0.14 mmol), 2-ethyl butanol (12.86 g, 0.12 mmol) in the presence of toluene (100 g, 1.08 mmol) and PTSA (0.5 g, 0.0026 mmol) refluxed for 2 h. Work up of reaction carried out as per the procedure given for 2a.

Chromatographic purity (HPLC) 98.60 %; bp 261 ºC; MS (ESI+) m/z = 223 [M+]; IR (KBr, cm−1): 2969, 2876, 1742, 1458, 1265, 1162; 1H NMR (CDCl3, 400 MHz) δ 0.90 (t, J = 7.4 Hz, 6H, (CH2CH3)2), 1.37 (qu, J = 7.4 Hz, 4H (CH2CH3)2), 1.56 (septet, J = 6 Hz, 1H, CH(CH2)3), 3.84 (s, 2H, COCH2Br), 4.11 (d, J = 6 Hz, 2H, OCH2CH).

2-Methoxyethyl-2-bromoacetate (2f)

Bromoacetic acid (20 g, 0.14 mmol) 2-methoxyethanol (9.16 g, 0.12 mmol) in the presence of toluene (100 g, 1.08 mmol) and PTSA (0.5 g, 0.0026 mmol) refluxed for 2 h. Work up of reaction carried out as per the procedure given for 2a.

Chromatographic purity (HPLC) 98.74 %; bp 213 ºC; MS (ESI+) m/z = 198 [M+2]; IR (KBr, cm−1):2955, 2891, 2823, 1739, 1452, 1286, 1128, 1034; 1H NMR (CDCl3, 400 MHz) δ 3.40 (s, 3H, OCH3), 3.63 (t, J = 4.4 Hz, 2H, OCH2CH2), 3.89 (s, 2H, COCH2Br), 4.33 (t, J = 4.4 Hz, 2H, COOCH2CH2).

Isopropyl-2-bromoacetate (2g)

Bromoacetic acid (20 g, 0.14 mmol), isopropanol (7.56 g, 0.12 mmol) in the presence of toluene (100 g, 1.08 mmol) and PTSA (0.5 g, 0.0026 mmol) refluxed for 2 h. Work up of reaction carried out as per the procedure given for 2a.

Chromatographic purity (HPLC) 95.89 %; bp 274 ºC; MS (ESI+) m/z = 179 [M+]; IR (KBr, cm−1): 2968, 2881, 1741, 1461, 1272, 1155; 1H NMR (CDCl3, 400 MHz) δ 1.28 (d, J = 6.4 Hz, 6H, CH(CH3)2), 3.80 (s, 2H, COCH2Br), 5.06 (septet, J = 6.4 Hz, 1H, CH(CH3)2).

Butyl-2-bromoacetate (2h)

Bromoacetic acid (20 g, 0.14 mmol), n-bromobutanol (9.60 g, 0.12 mmol) in the presence of toluene (100 g, 1.08 mmol) and PTSA (0.5 g, 0.0026 mmol) refluxed for 2 h. Work up of reaction carried out as per the procedure given for 2a.

Chromatographic purity (HPLC) 97.30 %; bp 92 ºC; MS (ESI+) m/z = 195 [M+]; IR (KBr, cm−1): 2937, 2875, 1738, 1458, 1283, 1170; 1H NMR (CDCl3, 400 MHz) δ 0.94 (t, J = 7.6 Hz, 3H, CH2CH3), 1.40 (sextet, J = 7.6 Hz, 2H, CH2CH2CH3), 1.65 (qu, J = 7.6 Hz, 2H, CH2CH2CH3), 3.83 (s, 2H, COCH2Br), 4.18 (t, J = 6.8 Hz, 2H, OCH2CH2).

Benzyl-2-bromoacetate (2i)

Bromoacetic acid (20 g, 0.14 mmol), Benzyl alcohol (13.72 g, 0.12 mmol) in the presence of toluene (100 g, 1.08 mmol) and PTSA (0.5 g, 0.0026 mmol) refluxed for 2 h. Work up of reaction carried out as per the procedure given for 2a.

Chromatographic purity (HPLC) 96.82 %; bp 287 ºC; MS (ESI+) m/z = 228 [M+]; IR (KBr, cm−1): 3033, 2960, 1738, 1455, 1278, 1156, 971; 1H NMR (CDCl3, 400 MHz) δ 3.84 (s, 2H, COCH2Br), 5.18 (s, 2H, OCH2Ph), 7.35 (m, 5H, ArH).

2-Bromo-butyric acid cyclohexyl ester (2j)

Bromobutyric acid (20 g, 0.14 mmol), cyclohexanol (10.70 g, 0.10 mmol) in the presence of toluene (100 g, 1.08 mmol) and PTSA (0.5 g, 0.0026 mmol) refluxed for 2 h. Work up of reaction carried out as per the procedure given for 2a.

Chromatographic purity (HPLC) 99.00 %; bp 280 ºC; MS (ESI+) m/z = 249 [M+]; IR (KBr, cm−1): 2939, 2860, 1732, 1455, 1262, 1161; 1H NMR (CDCl3, 400 MHz) δ 1.04 (t, J = 7.2 Hz, 3H, CH3CH2), 1.27–1.56 (m, 6H, 3×CH2), 1.70-1.88 (m, 4H, 2×CH2), 1.85–2.15 (m, 2H, CH2CHBr), 4.14 (t, J = 7.2 Hz, 1H, COCHBr), 4.83 (m, 1H, COOCH).

2-Bromo-butyric acid 2-ethyl-butyl ester (2k)

2-Bromobutyric acid (20 g, 0.11 mmol), 2-ethyl butanol (10.11 g, 0.09 mmol) in the presence of toluene (100 g, 1.08 mmol) and PTSA (0.5 g, 0.0026 mmol) refluxed for 2 h. Work up of reaction carried out as per the procedure given for 2a.

Chromatographic purity (HPLC) 98.88 %; bp 292 ºC; MS (ESI+) m/z = 251 [M+]; IR (KBr, cm−1): 2965, 2878, 1741, 1460, 1268, 1156; 1H NMR (CDCl3, 400 MHz) δ 0.90 (t, J = 7.6 Hz, 6H, 2×CH3CH2), 1.02 (t, J = 7.6 Hz, 3H, CH3CH2), 1.38 (qu, J = 7.6 Hz, 4H, 2×CH2CH3), 1.56 (sept, J = 7.6 Hz, 1H, CH(CH3)2), 1.97–2.14 (m, 2H, CH2CHBr), 4.06–4.18 (m, 3H, COOCH2, CH2CHBr).

2-Bromo-butyric acid 2-methoxy-ethyl ester (2l)

2-Bromobutyric acid (20 g, 0.11 mmol), 2-methoxy ethanol (7.5 g, 0.09 mol) in the presence of toluene (100 g, 1.08 mmol) and PTSA (0.5 g, 0.0026 mmol) refluxed for 2 h. Work up of reaction carried out as per the procedure given for 2a.

Chromatographic purity (HPLC) 98.46 %; bp 181 ºC; MS (ESI+) m/z = 225 [M+]; IR (KBr, cm−1): 2975, 2881, 1743, 1457, 1262, 1158; 1H NMR (CDCl3, 400 MHz) δ 1.04 (t, J = 7.2 Hz, 3H, CH3CH2), 1.96–2.15 (m, 2H, CH2CHBr), 3.39 (s, 3H, OCH3), 3.61 (t, J = 4.8 Hz, 2H, CH2CH2O), 4.21 (t, J = 7.2 Hz, 1H, CH2CHBr), 4.32 (t, J = 4.8 Hz, 2H, COOCH2CH2).

2-Bromo-butyric acid isopropyl ester (2m)

2-Bromobutyric acid (20 g, 0.11 mmol), Isopropanol (5.94 g, 0.09 mmol) in the presence of toluene (100 g, 1.08 mmol) and PTSA (0.5 g, 0.0026 mmol) refluxed for 2 h. Work up of reaction carried out as per the procedure given for 2a.

Chromatographic purity (HPLC) 98.19 %; bp 213 ºC; MS (ESI+) m/z = 208 [M+]; IR (KBr, cm−1): 2981, 2939, 2880, 1738, 1458, 1272, 1161, 1105; 1H NMR (CDCl3, 400 MHz) δ 1.02 (t, J = 7.6 Hz, 3H, CH3CH2), 1.26 (d, J = 6.4 Hz, 6H, CH(CH3)2), 1.96–2.13 (m, 2H, CH2CHBr), 4.12 (t, J = 7.6 Hz, 1H, CH2CHBr), 5.06 (sept, J = 6.4 Hz, 1H, CH(CH3)2).

2-Bromo-butyric acid butyl ester (2n)

2-Bromobutyric acid (20 g, 0.11 mmol), n-butanol (7.3 g, 0.09 mmol) in the presence of toluene (100 g, 1.08 mmol) and PTSA (0.5 g, 0.0026 mmol) refluxed for 2 h. Work up of reaction carried out as per the procedure given for 2a.

Chromatographic purity (HPLC) 98.80 %; bp 255 ºC; MS (ESI+) m/z = 223 [M+]; IR (KBr, cm−1): 2963, 2877, 1740, 1458, 1271, 1157; 1H NMR (CDCl3, 400 MHz) δ 0.94 (t, J = 7.6 Hz, 3H, CH3CH2CH2), 1.03 (t, J = 7.2 Hz, 3H, CH3CH2CHBr), 1.40 (sextet, J = 7.6 Hz, 2H, CH3CH2CH2), 1.66 (qu, J = 7.6 Hz, 2H, OCH2CH2), 1.95–2.16 (m, 2H, CH2CHBr), 4.13–4.22 (m, 3H, OCH2, CH2CHBr).

2-Bromo-butyric acid benzyl ester (2o)

2-Bromobutyric acid (20 g, 0.11 mmol), benzyl alcohol (10.72 g, 0.09 mmol) in the presence of toluene (100 g, 1.08 mmol) and PTSA (0.5 g, 0.0026 mmol) refluxed for 2 h. Work up of reaction carried out as per the procedure given for 2a.

Chromatographic purity (HPLC) 98.45 %; bp 295 ºC; MS (ESI+) m/z = 256 [M+]; IR (KBr, cm−1): 2973, 1741, 1455, 1266, 1150; 1H NMR (CDCl3, 400 MHz) δ 1.00 (t, J = 7.2 Hz, 3H, CH3CH2), 1.97–2.15 (m, 2H, CH2CHBr), 4.20 (t, J = 7.2 Hz, 1H, CH2CHBr), 5.20 (s, 2H, OCH2Ph), 7.36 (m, 5H, ArH).

Anti-inflammatory activity evaluation in animal models

Oral acute toxicity study

Acute toxicity of various prodrugs was carried out in Swiss albino mice (20–25 g) as per the OECD guidelines (2006). Different doses of various prodrugs were administered to groups (n = 3) up to 2,000 mg/kg orally and animals were observed for a period of 72 h for behavioral changes, toxic reactions, and mortality if any.

Carrageenan-induced hind paw edema in rats

Dose response study

Rats were divided into various groups (n = 6) and allowed to free access to water ad libitum. Different groups of rats administered with ibuprofen (100 mg/kg, b.w.) and various prodrugs (100 mg/kg, v) orally. One group of rats served as a control and administered with gum acacia (1 %, w/v; 10 mL/kg, b.w., p.o.). One hour after the drug administration, to all rats, hind paw edema was induced by the method of winter et al. (1962) by injecting 0.1 mL of 1 % (w/v) solution of carrageenan subcutaneously into the subplanter region of hind paw.

Time course study

The hind paw edema volume was measured by volume displacement method using plethysmometer (Orchid scientific; 112, India) by immersing the paw till the level lateral malleolus at various time intervals (1, 3 and 6 h) after carrageenan injection.

Cotton pellets-induced granuloma formation in rats

Two sterilised cotton pellets (10 mg) were implanted on ventral regions of rats, procedure described by winter and porter (Winter and Porter, 1957) and divided into various groups (6 rats/group). Different groups of rats administered with ibuprofen (100 mg/kg) and various prodrugs (100 mg/kg) orally for a duration of 8 days. Control group received gum acacia (2 %, 10 mL/kg, p.o.). On the day 9th, rats were sacrificed with excess ether anesthesia. The cotton pellets were removed and freed from extraneous tissue and used for granular tissue formation by recording wet (immediately) and dry weight of pellets. The granular tissue formation was studied by drying cotton pellets at 55 °C for 6 h or till the weight of pellets remains constant. The dry weight was calculated after deducting cotton pellet weight and taken as a granular tissue formation.

Measurement of ulcer index

Ulcer index were evaluated in 2 experimental conditions (a) For the normal 24 h fasted rats were taken and were administered with test compounds orally at 400 mg/kg and 24 h after the drug administration, animals were scarified and stomach were isolated and observed for number of ulcers, (b) in cotton pellets-induced granuloma in rats-Immediately after cotton removal in above experiment, stomach was dissected out, incised along the greater curvature and mucosa were rinsed with cold normal saline solution (pH 7.2) to remove blood contaminant if any. The tissue were kept overnight in 10 % formalin solution. Next day, ulcers were examined under the 10× lens using method described in the literature (Ganguly and Bhatnagar, 1973) on the scale [0 = no ulcer; 1 = hemorrhagic patch; 2 = ulcer <2 mm; 3 = ulcer >2 mm or <4 mm; 4 = ulcer >4 mm]. The ulcer index was calculated as mean ± SEM of 6 rats in each group.

In vivo metabolic stability study (plasma drug concentration determination)

Metabolic stability is an important property of drug candidates, as it affects parameters such as plasma clearance, half-life, and bioavailability. A successful prodrug candidate is expected to undergo rapid, complete conversion to parent compound in the plasma or microsomes within 1–3 h for its desired pharmacological action. The metabolic stability study was carried out in the following method. The male Sprague-Dawley rats were used to compare the bioavailability of prodrugs (2, 6, 9, 10, 12, and 14) with that of ibuprofen (1a) following oral administration. Overnight fasted rats were divided into various groups (n = 6), and they were administered prodrugs (2, 6, 9, 10, 12, and 14) at the dose of 100 mg/kg, orally. Blood sample from each rats were withdrawn through retro orbital plexus in eppendorf tube (containing EDTA 5 % as anticoagulant) at various time intervals viz; 0.5, 1, 2, 3, 4, 5, 6, 7, and 8 h after the drug administration and centrifuged at 5000 rpm for 10 min for plasma separation. To 200 μL of plasma, 200 μL of acetonitrile solution was added and vortexed for 2 min for the precipitation of protein which was separated by centrifugation. The clear supernatant obtained and used for determination of concentration by HPLC technique. The area under curve (AUC) is calculated by
$$ {\text{AUC}} = C_{ 0} \,{ + } \,\frac{{C_{ 1} }}{ 2} \times {\text{Time}}_{{ 1 { - 0}}} $$
where, C0 represents the peak area at time t0 and C1 represents the peak area at time t1.

Microsomal stability

Materials

Nicotinamide adenine dinucleotide phosphate (NADPH) (Sigma, lot no. N 6674); acetonitrile (HPLC grade, Merck, India); Tris–HCl (S.D.Fine-Chem, India); rat liver microsomes (isolated in pharmacology laboratory); prodrugs; and standard ibuprofen.

Requirement

Required items were as follows: rat liver microsomes (10 mg/mL protein concentration), NADPH (10 mM solution), Tris–HCl buffer (pH 7.4), Eppendorf tubes, and solutions of various prodrugs of ibuprofen (5 mM).

Experimental conditions

Incubation period, 60 min; incubation conditions, 37 °C with 60 rpm shaking; protein precipitation solvent, acetonitrile.

Procedure
In 50 μL of rat liver microsomes, 100 μL Tris–HCl buffer was added, with 50 μL of NADPH solution(10 mM), and were mixed thoroughly and vortexed subsequently for 10 s. To this mixture, 5 μL prodrug solution (5 mM) was injected and vortexed well. The 75 μL sample was withdrawn immediately and transferred to the centrifuge tube containing 75 μL ice cold acetonitrile, vortexed, and centrifuged at 14,000 rpm for 10 min. Aliquots of the supernatant was separated and used for HPLC analysis. Then the assay mixture was incubated in a water bath at 37 °C for 60 min. At specific time points (30 and 60 min), the75 μL assay mixture was withdrawn and vortexed in equal volume of cold acetonitrile and centrifuged again at 14,000 rpm for 10 min. Aliquots of the supernatant were separated and used for analysis by HPLC. The percentage of prodrug remaining in rat microsomes was calculated by:
$$ {\text{Prodrug remaining }}(\% ) = \frac{{{\text{Peak area at respective time }}({ \hbox{min} }) \times 100}}{{{\text{Peak area at}}\,0\,{ \hbox{min} }}} $$

Aqueous solubility

The solubility of prodrugs 2, 6, 9, 10, 12, and 14 was determined using boric acid buffer (pH 9.0) at 40 °C, 0.2 mM phosphate buffer (pH 7.4), 0.1 mM phosphate buffer (pH 5.0), 0.1 mM citric acid buffer (pH 3.0), and 0.2 M hydrochloric acid (pH 1.0). The various prodrugs of ibuprofen (5 mg) in buffer solution (10 mL) were incubated at 40 °C for 4 h at 400 rpm on a mechanical shaker. The solution was filtered through 0.20 μM membrane filter and the filtrate was analyzed quantitatively by HPLC at a wavelength of 254 nM for its solubility and hydrolysis.

Metabolic stability

For the HPLC analysis system; water alliance 2695-separation module with 2996 PDA detector, C18 Column, ODS 3 v, 250 mm × 4.6 mm, 5 μM, and mobile phase was A-10 mM potassium dihydrogen phosphate, wavelength, 265 nM and for B-acetonitrile at various ratios (90:10, 30:70, 10:90 and 70:30).

Results and discussion

Chemistry

A new series of ibuprofen ester prodrugs 1–15 (Fig. 1) were readily prepared in good yields by the condensation of ibuprofen 1a with appropriate promoieties 2a–o (Fig. 2), in the presence of potassium carbonate in dimethyl acetamide (DMAc) at room temperature under nitrogen atmosphere (Scheme 1). The structures of all prodrugs were established by IR, 1H NMR, mass spectrometry, and their purity in excess of 99 % was confirmed by HPLC analysis.
https://static-content.springer.com/image/art%3A10.1007%2Fs00044-013-0639-8/MediaObjects/44_2013_639_Fig1_HTML.gif
Fig. 1

Chemical structures of ibuprofen (1a) and produrgs 1–15

https://static-content.springer.com/image/art%3A10.1007%2Fs00044-013-0639-8/MediaObjects/44_2013_639_Fig2_HTML.gif
Fig. 2

Chemical structures of ethyl-2-chloro-3-oxobutanoate (2a), 4-bromobutyl acetate (2b), methyl-2-bromopropanoate (2c), cyclohexyl-2-bromoacetate (2d), 2-ethylbutyl-2-bromoacetate (2e), 2-methoxyethyl-2-bromoacetate (2f), isopropyl-2-bromoacetate (2g), butyl-2-bromoacetate (2h), benzyl-2-bromoacetate (2i), 2-bromo-butyric acid cyclohexyl ester (2j), 2-bromo-butyric acid 2-ethyl-butyl ester (2k), 2-bromo-butyric acid 2-methoxy-ethyl ester (2l), 2-bromo-butyric acid isopropyl ester (2m), 2-bromo-butyric acid butyl ester (2n), 2-bromo-butyric acid benzyl ester (2o)

https://static-content.springer.com/image/art%3A10.1007%2Fs00044-013-0639-8/MediaObjects/44_2013_639_Sch1_HTML.gif
Scheme 1

Synthesis of ibuprofen ester prodrugs 1–15. Reagents and conditions: (i) promoieties 2a–o, K2CO3, DMAc, rt

The syntheses of promoieties 2a–c were outlined in Scheme 2. The promoieties 2d–i were obtained by refluxing 2-bromoacetic acid with respective alcohols in the presence of toluene and catalytic amount of PTSA (Scheme 3). Similarly, promoieties 2j–o were obtained by refluxing 2-bromobutanoic acid with respective alcohols in the presence of toluene and catalytic amount of PTSA (Scheme 4). The structures of all promoieties were established by IR, 1H NMR, and mass spectrometry, and their purity in excess of 97 % was confirmed by HPLC analysis.
https://static-content.springer.com/image/art%3A10.1007%2Fs00044-013-0639-8/MediaObjects/44_2013_639_Sch2_HTML.gif
Scheme 2

Reagents and conditions: (a) PTSA, toluene, reflux, 2 h

https://static-content.springer.com/image/art%3A10.1007%2Fs00044-013-0639-8/MediaObjects/44_2013_639_Sch3_HTML.gif
Scheme 3

Reagents and conditions: (a) PTSA, toluene, reflux, 2 h

https://static-content.springer.com/image/art%3A10.1007%2Fs00044-013-0639-8/MediaObjects/44_2013_639_Sch4_HTML.gif
Scheme 4

Reagents and conditions: (a) PTSA, toluene, reflux, 2 h

Pharmacological evaluation

The therapeutic use of NSAIDs is often restricted by the necessity to deliver the drug to specific sites of target organ or tissue. The use of NSAIDs is also limited by their irritant side effects on the gastro-enteric mucous and by their poor water solubility. A series of ester prodrugs 1–15 of ibuprofen 1a was synthesized with the aim of minimizing these side effects and to enhance the oral bioavailability.

Acute toxicity study

There were no significant behavioral changes observed with all the employed doses of prodrugs (1–15). To all the mice, neither toxic reaction nor mortality observed. Therefore, 2,000 mg/kg, body weight (b.w.), per oral (p.o.) dose was considered as maximum tolerated dose. Based on acute toxicity study, we have selected 100 mg/kg for anti-inflammatory activity evaluation of various prodrugs (1–15).

Anti-inflammatory activities evaluation

In a dose response study in carrageenan edema, there was significant (p < 0.01) increase in paw volume of carrageenan control rats when compared with normal rats. All prodrugs significantly inhibited the edema formation (p < 0.01) than the parent drug ibuprofen. However, the extent of edema inhibition in rats treated with 2, 6, 9, 10, 12, and 14 was much greater at 3 and 6 h than other prodrugs when compared to carrageenan control rats (Table 1). Inhibition of paw edema formation at 6 h is greater due to the inhibition of prostaglandins, which is known to be released at 4–6 h after the carrageenan injection (Crunckhon and Meacock, 1971). Prodrugs elicited greater paw edema inhibition (2, 6, 9, 10, 12, and 14) was taken for further evaluation.
Table 1

Effects of various prodrugs 1–15 on carrageenan-induced paw edema in rats

Treatment and dose (100 mg/kg p.o.)

Paw volume (mL) (Mean ± SEM) at various time interval

1 h

3 h

6 h

Normal group

0.40 ± 0.04

0.35 ± 0.04

0.38 ± 0.06

Carrageenan control

0.94 ± 0.05#

1.17 ± 0.10#

1.69 ± 0.08##

Ibuprofen (1a)

0.84 ± 0.05

1.00 ± 0.07

0.73 ± 0.05**

1

1.20 ± 0.04

0.96 ± 0.10

1.26 ± 0.10

2

1.03 ± 0.10

0.98 ± 0.10

0.84 ± 0.10**

3

1.12 ± 0.06

0.97 ± 0.05

1.20 ± 1.10

4

1.29 ± 0.10

1.11 ± 0.10

1.01 ± 0.10*

5

0.88 ± 0.10

0.98 ± 0.10

1.19 ± 0.10

6

1.03 ± 0.10

1.00 ± 0.10

0.89 ± 0.10**

7

1.11 ± 0.04

1.10 ± 0.03

1.18 ± 0.20

8

1.10 ± 0.04

1.10 ± 0.03

1.18 ± 0.20

9

1.26 ± 0.15

0.68 ± 0.03**

0.75 ± 0.10**

10

0.76 ± 0.10

0.95 ± 0.10*

0.82 ± 0.10**

11

0.93 ± 0.10

0.89 ± 0.10*

1.00 ± 0.10*

12

1.04 ± 0.10

0.93 ± 0.1*

0.22 ± 0.10**

13

1.09 ± 0.10

0.85 ± 0.05*

0.98 ± 0.03*

14

0.80 ± 0.10

1.05 ± 0.10

0.81 ± 0.01**

15

1.03 ± 0.05

0.96 ± 0.10

1.00 ± 0.05*

p < 0.05; ## p < 0.01 when compared with normal control group. ** p < 0.01; * p < 0.05 when compared with carrageenan control group

In cotton pellet-induced granuloma in rats, all the selected prodrugs 2, 6, 9, 10, 12, and 14 were significantly inhibited (p < 0.01) both the exuhdatory and granulatory phases of inflammation than the parent drug ibuprofen (1a). However, greater degree inhibition of these phases was found with prodrugs 2, 12 and 14 when compared with control rats. Ibuprofen treatment also showed similar inhibitory effects on both the phases of inflammation (Table 2).
Table 2

Effects of various prodrugs on cotton pellet-induced granuloma in rats

Treatment and dose (100 mg/kg p.o.)

Weight (mg) (Mean ± SEM)

Wet

Dry

Normal control

256 ± 11.2

153 ± 9.9

Ibuprofen (1a)

148 ± 11.5**

76 ± 4.9

2

174 ± 8.5**

104 ± 3.9

6

170 ± 13.8**

90.4 ± 5.9

9

165 ± 13.2**

92.8 ± 4.6

10

194 ± 4.2*

122.4 ± 3.9

12

178 ± 5.08**

77.4 ± 7.2

14

161 ± 3.9**

79 ± 1.9

** p < 0.01; * p < 0.05 when compared with normal control group

p < 0.05; ## p < 0.01 when compared with normal control group ** p < 0.01; * p < 0.05 when compared with vehicle treated control group

Ulcerogenic assay

Ulcer index in rats during cotton pellet-induced granuloma
In this model, lesser degree of ulcers was observed in rats treated with prodrugs 2, 6, 9, 10, 12, and 14 when compared to standard ibuprofen (Table 3). Administration of ibuprofen 1a and prodrugs 2, 6, 9, 10, 12 and 14 (100 mg/kg, b.w., p.o.) daily for 7 days in rats implanted with cotton pellet-induced ulcers. As the ibuprofen was found to produce irritation and ulceration of the GI mucosa, has arisen in clinical trials (Ashraf et al., 1999; Kean and Buchanan, 2005). The GI ulceration in both the experimental conditions may be due to the presence of carboxylic acid functional group present in ibuprofen, inhibition of protective prostaglandins and inhibition of mucus production. Rats treated with standard drug ibuprofen 1a had ulcer index of about 6 mm lesion and found to be much greater than all the synthesized prodrugs (Fig. 3). Rats treated with 2 (0.1 ulcer), 12 (0.1 ulcer) and 14 (0.1 ulcer) found to developed no ulcerogenic lesions as same that of ibuprofen 1a (6 ulcer). These experimental findings revealed that the presence of functional groups like, 4-bromobutyl acetate, 2-methoxy ethyl-2-bromobutanoate, and butyl-2-bromo-butanoate on carboxylic acid to 2, 12, and 14 respectively. However, other prodrugs, viz. 6, 9, and 10 also showed lesser degree of ulcer formations but comparatively greater than 2, 12, and 14. Therefore, 2, 12, and 14 are most potent anti-inflammatory prodrugs with no or less ulcerogenic potential as compared to parent ibuprofen.
Table 3

Ulcer index of various prodrugs in cotton pellets-induced granuloma in rats

Treatment and dose (400 mg/kg p.o.)

Number of ulcer (Mean ± SEM)

Ulcer index (Mean ± SEM)

Normal control

0.00 ± 0.0

0.10 ± 0.01

Ibuprofen (1a)

5.1 ± 0.20##

6.0 ± 0.30##

2

0.2 ± 0.03**

0.1 ± 0.02**

6

0.7 ± 0.01**

1.0 ± 0.01**

9

0.8 ± 0.04**

1.0 ± 0.01**

10

0.9 ± 0.01**

1.1 ± 0.07**

12

0.2 ± 0.01**

0.1 ± 0.02**

14

0.2 ± 0.01**

0.1 ± 0.01**

p < 0.05; ## p < 0.01 when compared with normal control group

** p < 0.01; * p < 0.05 when compared with ibuprofen group

https://static-content.springer.com/image/art%3A10.1007%2Fs00044-013-0639-8/MediaObjects/44_2013_639_Fig3_HTML.gif
Fig. 3

Ulcer index of various prodrugs in cotton pellets-induced granuloma in rats

Ulcer index in normal rats
Experimental findings demonstrated that rats administered with ibuprofen 1a at 400 mg/kg showed high incidence of ulcers when compared with normal group; however, the lesser degree of ulcer formation was observed in test prodrugs 2, 6, 9, 10, 12, and 14 as compared to standard ibuprofen 1a (Table 4). Rats treated with standard drug ibuprofen 1a had ulcer index of about 10.2 and found to be much greater than the entire group of tested prodrugs (Fig. 4). This gives clear indication that ibuprofen is highly ulcerogenic than other ester prodrugs. Furthermore, compounds 2, 12, and 14 were found to produce least ulcer in normal rats at 400 mg/kg, indicating significant anti-inflammatory activity with no ulcer formation.
Table 4

Ulcer index of various prodrugs in normal rats

Treatment and dose (400 mg/kg p.o.)

Number of ulcer (Mean ± SEM)

Ulcer index (Mean ± SEM)

Normal control

0.00 ± 0.0

0.10 ± 0.01

Ibuprofen (1a)

7.2 ± 0.20##

10.3 ± 1.10##

2

0.2 ± 0.03**

0.20 ± 0.02**

6

0.6 ± 0.01**

0.80 ± 0.01**

9

0.5 ± 0.04**

0.80 ± 0.01**

10

0.7 ± 0.01**

1.20 ± 0.09**

12

0.2 ± 0.01**

0.20 ± 0.01**

14

0.02 ± 0.01

0.20 ± 0.01**

p < 0.05; ## p < 0.01 when compared with normal control group; ** p < 0.01, * p < 0.05 when compared with ibuprofen group

https://static-content.springer.com/image/art%3A10.1007%2Fs00044-013-0639-8/MediaObjects/44_2013_639_Fig4_HTML.gif
Fig. 4

Ulcer index of various prodrugs in normal rats

The present experimental findings demonstrated that 4-bromobutyl acetate, 2-methoxy ethyl-2-bromobutanoate, and butyl-2-bromo-butanoate ester prodrugs of ibuprofen protected the gastric mucosal damage induced by ibuprofen. In this respect, our findings shed new chemical requisite to design new agents of ibuprofen with no ulcerogenic effects on GI system.

In vivo metabolic stability study (plasma drug concentration determination)

The hydrolysis studies were also studied in male Wistar rats. All selected ester prodrugs (2, 6, 9, 10, 12, and 14) were quantitatively hydrolyzed to the parent drug ibuprofen by enzymatic conversion in vivo; their respective release characteristics were observed (Fig. 5). The Cmax (maximum concentration) of standard ibuprofen 1a was 91.2 mg/L and (AUC)0–t was 239, whereas Cmax of 2, 6, 9, 10, 12, and 14 were 70.1, 73.2, 78.0, 80.2, 92.3, 115.2 mg/L respectively with AUC0–t of 2, 6, 9, 10, 12, and 14 were 188, 244, 241, 237, 192 and 335 respectively (Table 5).
https://static-content.springer.com/image/art%3A10.1007%2Fs00044-013-0639-8/MediaObjects/44_2013_639_Fig5_HTML.gif
Fig. 5

Plasma concentration of ibuprofen and prodrugs at various time intervals

Table 5

Area under curve and plasma concentration of various test compounds

Compound

Area under curve (AUC)

Plasma concentration (mg/lit)

Ibuprofen (1a)

239

91.2

2

188

70.1

6

244

73.2

9

241

78.0

10

237

80.2

12

192

92.3

14

335

115.2

Bioavailability was measured by calculating the AUC of the drug concentration time profile. The plasma concentration of compound 14 was found to maximum as compared to parent drug ibuprofen indicating improved bioavailability. Prodrug 10 showed slow released rate than other compounds which indicate that slow enzymatic conversion of prodrug moiety into parent compound. Similarly, compound 12 also showed same characteristic release rate in vivo with Cmax of 92.3 with higher bioavailability compared to all other prodrugs except 14 indicate fast release of prodrug into parent ibuprofen for its pharmacological action.

In vivo metabolic stability study (microsomal stability)

From experimental findings it is found that ester prodrugs of ibuprofen were highly metabolized however, standard ibuprofen was stable up to 60 min in liver microsomes (Table 6).
Table 6

Metabolic stability of various prodrugs of ibuprofen

Compound

Metabolic stability in rat liver microsomes at various time intervals (min)

0

30

60

90

Ibuprofen (1a)

100

92.1

79.2

58.3

2

100

8.1

0

0

6

100

9.2

0

0

9

100

1

0

0

10

100

1.2

0

0

12

100

2.1

0

0

14

100

0

0

0

The percentage of prodrugs remaining in rat microsomes was calculated by Prodrug remaining (%) = peak area at respective time (min)/peak area at 0 min × 100

Physicochemical properties

Aqueous solubility
All the drugs administered by oral route are supposed to stable at various pH environments encountered in the GI to convert the intact prodrug to the systemic circulation and reach to site of action. Prodrugs of ibuprofen candidate (2, 6, 9, 10, 12, and 14) were evaluated for solubility and stability in a series of buffer solutions ranging from pH 1 to 9. All the ibuprofen prodrugs were found to be stable and sparingly soluble in buffer solutions (below detection limit). Aqueous solubility of 9, 10, 12, and 14 was improved; however, it is lower than parent ibuprofen (Table 7). Furthermore, the degradation of prodrugs 9, 10, 12, and 14 indicted a substantially higher chemical stability in an aqueous buffer solution.
Table 7

Aqueous solubility, stability of ibuprofen and various prodrugs (2, 6, 9, 10, 12, 14) in buffer solutions at 40 °C for 4 h

Prodrug

Hydrolysis to parent drug (μg/mL) at various pH

Solubility (μg/mL) at various pH

1.0

3.0

5.0

7.4

9.0

1.0

3.0

5.0

7.4

9.0

Ibuprofen (1a)

BDL

BDL

BDL

BDL

BDL

2

0.0735

0.0122

0.1250

0.1256

0.0090

BDL

BDL

BDL

BDL

BDL

6

0.0936

0.0975

0.2965

0.1252

0.2521

BDL

BDL

BDL

BDL

BDL

9

0.2253

0.0255

0.2600

0.2510

0.2987

BDL

BDL

BDL

BDL

BDL

10

0.2361

0.1258

0.0556

0.0255

0.3565

BDL

BDL

BDL

BDL

BDL

12

0.2990

0.2415

0.0115

0.0241

0.3325

BDL

BDL

BDL

BDL

BDL

14

0.0323

0.0153

0.0026

0.2990

0.1988

BDL

BDL

BDL

BDL

BDL

Limit of detection is estimated by HPLC method; ibuprofen (1a) (0.0081 μg/mL), 2 (0.0162 μg/mL), 6 (0.0615 μg/mL), 9 (0.0071 μg/mL), 10 (0.0121 μg/mL), 12 (0.0072 μg/mL), 14 (0.0411 μg/mL)

BDL below detection limit

Partition coefficient (log P)
The partition coefficients (log P) of ibuprofen 1a and prodrugs 2, 6, 9, 10, 12, 14 were determined by HPLC method in octanol-buffer system. Partition coefficient values for all prodrugs were found to be higher than ibuprofen at pH 7.4. Results of log P at pH 3.0 and pH 7.4 are summarized in Table 8. Higher log P values of all prodrugs at pH 7.4, indicated that prodrugs are more lipophilic than the parent drug.
Table 8

Partition coefficients of ibuprofen and prodrugs (2, 6, 9, 10, 12, 14) in buffer solutions at 20 °C and pH 4 and 7.4

Prodrug

Partition coefficients of prodrugs at various pH

3.0

7.4

Ibuprofen (1a)

3.75

2.45

2

3.68

4.11

6

4.12

3.49

9

4.09

4.21

10

4.76

4.57

12

3.91

4.02

14

4.05

4.25

Results reported are the average of three experiments (n = 3)

Conclusions

A series of novel ester prodrugs of ibuprofen have been synthesized and evaluated in vivo for their anti-inflammatory potential using carrageenan-induced rat hind paw edema and cotton pellet-induced granuloma models inflammation along with non-ulcerogenic potential. These prodrugs emerged out as anti-inflammatory prodrugs with lesser degree of ulcerogenic potential than the parent drug ibuprofen. Ulcer index (UI) studies revealed that the ibuprofen prodrugs 2, 12, and 14 were substantially less ulcerogenic in both normal rats (UI = about 0.1) and inflammation-induced rats (cotton pellets-induced granuloma; UI = about 0.2) than the parent ibuprofen 1a (UI = 6 and 10.3 respectively). Metabolic stability study showed that prodrug 2, 12 and 14 were rapidly transformed enzymatically into parent drug ibuprofen in both rat liver microsomes and rat plasma. Based on these experimental observations, prodrug 12 and 14 are potent and safe anti-inflammatory and analgesic agents with least GI irritation and ulceration therefore, can be preferred over parent drug ibuprofen for the treatment of inflammation

Acknowledgments

The authors also gratefully acknowledge the support extended by Mr. Ashok Hiremath, Managing Director, Astec Lifesciences Ltd. Authors are also thankful to Dr. S. R. Naik and Dr. S.B. Bhise, Sinhgad Institute of Pharmaceutical Sciences, Lonavala for providing necessary facility for pharmacological experimentation.

Copyright information

© Springer Science+Business Media New York 2013