Stable ester and amide conjugates of some NSAIDs as analgesic and antiinflammatory compounds with improved biological activity

Article abstract of DOI:10.3906/kim-1010-732

A set of ester and amide derivatives of some acidic NSAIDs, including ibuprofen, ketoprofen, and mefenamic acid (1-3), were synthesized and evaluated for their in vivo analgesic and antiinflammatory activity using the p-benzoquinone-induced writhing test

Full text of DOI:10.3906/kim-1010-732

Turk J Chem
35 (2011) , 427 – 439.
¨
˙
c
ꢀ TUBITAK
doi:10.3906/kim-1010-732
Stable ester and amide conjugates of some NSAIDs as
analgesic and antiinflammatory compounds with
improved biological activity
1
2
1
1
˘
¨
Mecit Orhan ULUDAG , Burcu C¸ ALIS¸KAN ERGUN , Duygu Aslı ALKAN , Nilu¨fer ERCAN ,
2
2,∗
¨
˘
Gu¨ler Yag˘mur OZKAN , Erden BANOGLU
1Department of Pharmacology, Faculty of Pharmacy, Gazi University,
06330, Ankara-TURKEY
2Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Gazi University,
06330, Ankara-TURKEY
e-mail: banoglu@gazi.edu.tr
Received: 14.10.2010
A set of ester and amide derivatives of some acidic NSAIDs, including ibuprofen, ketoprofen, and
mefenamic acid (1-3), were synthesized and evaluated for their in vivo analgesic and antiinflammatory
activity using the p-benzoquinone-induced writhing test and the carrageenan-induced paw edema model,
respectively. Among the synthesized compounds, ester derivatives of ketoprofen showed especially potent
analgesic and antiinflammatory activity as compared to the parent drug. In vitro chemical stability studies
revealed that ester and amide derivatives were chemically stable in simulated gastric (pH 1.2) and intestinal
fluids (pH 6.8). In 80% human plasma, the ester derivatives were found to be relatively stable against plasma
esterases over periods of 24 h, indicating that the observed activity was not due to the parent NSAIDs. Most
of the compounds were found to be nonulcerogenic under the tested conditions.
Key Words: Ibuprofen, ketoprofen, mefenamic acid, writhing, carrageenan
Introduction
Nonsteroidal antiinflammatory drugs (NSAIDs) are widely used for the treatment of pain and chronic inflamma-
tory ailments such as rheumatoid arthritis.1 The majority of currently known NSAIDs mainly act peripherally
∗Corresponding author
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Stable ester and amide conjugates of some NSAIDs as..., M. O. ULUDAG, et al.
by blocking the production of prostaglandins (PG) through inhibition of cyclooxygenases (COX-1 and COX-
2) to varying extents.2 However, the prolonged utilization of these drugs limits their therapeutic use since
they cause gastrointestinal and renal side effects3 due to inhibition of constitutive COX-1 responsible for the
production of PGs that are important for gastroprotection and vascular homeostasis.3−5 In addition, a direct
contact mechanism also appears to play a major role in the development of gastrointestinal lesions with acidic
NSAIDs.6 As a result, the gastric disturbance caused by NSAIDs is probably a combination of local irritations
produced by the free carboxyl group in NSAIDs and by inhibition of the cytoprotective PGs on gastric mucosa.
Therefore, there is still growing interest in improved NSAIDs that are biologically effective but devoid of the
side effects inherent to traditional NSAIDs.
Kalgutkar et al. demonstrated that ester and amide derivatives of indomethacin and meclofenamic acid
selectively inhibited COX-2 and showed that certain amide derivatives of indomethacin were potent nonulcero-
genic antiinflammatory agents in an in vivo model of acute inflammation.7−9 These common approaches to
overcome the gastrointestinal side effects of known NSAIDs were also used by many other researchers over the
years as has been reviewed recently.10 For example, 2-formylphenyl esters of indomethacin, ketoprofen and
ibuprofen were shown to have more potent antiinflammatory activity than the parent drugs.11 In addition,
morpholinoalkyl esters of naproxen, indomethacin and diclofenac were reported to have better bioavailability
and be less irritating to the gastric mucosa than the parent compounds.12,13 Moreover, the benzyl ester pro-
drug of ibuprofen showed good gastric tolerability without affecting the pharmacological activity.14 The proline
amides of ibuprofen were reported as neuroprotective agents and were shown to possess antiinflammatory ac-
tivity without ulcerogenic potential.15 The L-cysteine ethyl ester of a series of NSAIDs including ibuprofen
and ketoprofen was also found to be a potent antiinflammatory and antioxidant agent while demonstrating
considerably reduced gastrointestinal toxicity.16 Based on the above findings, it is evident that derivatization
of the free carboxylic acid group in NSAIDs represents a suitable strategy for obtaining novel compounds with
an improved therapeutic index and even the conversion of nonselective COX inhibitor NSAIDs into selective
COX-2 inhibitor drugs.
In the present study, we prepared 2 phenolic esters and 2 secondary amide derivatives of ibuprofen,
ketoprofen and mefenamic acid with the aim that they would have increased pharmacological activity, low
ulcerogenicity and good chemical stability.
Experimental
Chemistry
Ketoprofen, mefenamic acid, ibuprofen, EDC, and amine and phenol derivatives were purchased from Aldrich
(Deisenhofen, Germany) and Merck (Darmstadt, Germany). IR (KBr) spectra were recorded on a PerkinElmer
Spectrum 100N FT-NIR spectrometer. 1 H-NMR spectra were recorded in DMSO-d6 or CDCl3 on a Varian
Mercury 400 MHz High Performance Digital FT-NMR spectrometer using tetramethylsilane as the internal
standard at the NMR facility of the Faculty of Pharmacy, Ankara University. All chemical shifts were recorded
as δ (ppm). HRMS spectra were taken on a Waters LCT Premier XE orthogonal acceleration time-of-flight
(oa-TOF) mass spectrometer using ESI (+) or ESI (-) methods (Waters Corporation, Milford, MA, USA).
428

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Stable ester and amide conjugates of some NSAIDs as..., M. O. ULUDAG, et al.
Melting points were determined with an SMP-II Digital Melting Point Apparatus and are uncorrected. Flash
r
chromatography was performed with a CombiflashꢀRf automated flash chromatography system (Teledyne Isco,
Lincoln, NE, USA) using hexane-ethyl acetate or dichloromethane-methanol solvent gradients.
General synthesis of esters and amide derivatives
To a solution of appropriate carboxylic acid derivative (1 mmol) and phenol or amine derivative (1 mmol) in
DCM (10 mL), DMAP (0.2 mmol) and EDC (1.1 mmol) were added and the resulting solution was stirred
overnight at room temperature. The reaction mixture was quenched with 0.5 N HCl and extracted with DCM.
The organic phase was washed with a 1% NaHCO3 solution and brine, dried over Na2 SO4 , and evaporated
r
under vacuum. The residue was purified by flash column chromatography (CombiflashꢀRf) using hexane-
EtOAc or DCM-MeOH as eluents.
4-Isopropylphenyl 2-(4-isobutylphenyl)propanoate (1a)
Elution with hexane-EtOAc (0%-3%) afforded 1a as a colorless oil (yield 94%); IR (KBr, cm−1): 1754 (C=O);
1 H-NMR (CDCl3 , δ): 0.90 (d, J = 6.8 Hz, 6H, CH(CH3)2), 1.21 (d, J = 7.2 Hz, 6H, CH(CH3)2), 1.59 (d, J =
6.8 Hz, 3H, COCHCH3), 1.86 (m, 1H, CH(CH3)2), 2.47 (d, J = 6.8 Hz, 2H, CH2 CH), 2.87 (m, 1H, CH(CH3)2),
3.92 (q, J = 7.0 Hz, 1H, COCHCH3), 6.90 (m, 2H, ArH), 7.13 (d, J = 8.0 Hz, 2H, ArH), 7.17 (m, 2H, ArH),
7.29 (d, J = 8.0 Hz, 2H, ArH); HRMS calcd. for C22 H28 O2 (M+1): 325.2168, found: 325.2178.
4-Tert-butylphenyl 2-(4-isobutylphenyl)propanoate (1b)
Elution with hexane-EtOAc (0%-5%) afforded 1b as a white solid (yield 61%); mp 54 ◦ C; IR (KBr, cm−1):
1752 (C=O); 1 H-NMR (CDCl3 , δ): 0.90 (d, J = 6.8 Hz, 6H, CH(CH3)2), 1.28 (s, 9H, C(CH3)3), 1.59 (d, J =
6.8 Hz, 3H, COCHCH3), 1.86 (m, 1H, CH(CH3)2), 2.46 (d, J = 6.8 Hz, 2H, CH2 CH), 3.93 (q, J =7.2 Hz, 1H,
COCHCH3), 6.91 (m, 2H, ArH), 7.13 (d, J = 8.0 Hz, 2H, ArH), 7.29 (d, J = 8.0 Hz, 2H, ArH), 7.33 (m, 2H,
ArH); HRMS calcd. for C23 H30 O2 (M+1): 339.2324, found: 339.2325.
1-(4-Tert-butylbenzyl)-4-[2-(4-isobutylphenyl)propanoyl]piperazine (1c)
Elution with DCM-MeOH (0%-5%) afforded 1c as a white solid (yield 65%); mp 282 ◦ C; IR (KBr, cm−1):
1635 (C=O); 1 H-NMR (CDCl3 , δ): 0.91 (m, 6H, CH(CH3)2), 1.31 (s, 9H, C(CH3)3), 1.41 (d, J = 6.8 Hz, 3H,
COCHCH3), 1.86 (m, 1H, CH(CH3)2), 2.47 (d, J = 7.6 Hz, 2H, CH2 CH), 2.40-4.76 (m, 10H, piperazine-H,
NCH2), 7.09-7.49 (m, 8H, ArH); HRMS calcd. for C28 H40 N2 O (M+1): 421.3219, found: 421.3210.
Ethyl 1-[2-(4-isobutylphenyl)propanoyl]piperidine-4-carboxylate (1d)
Elution with DCM-MeOH (0%-4%) afforded 1d as a colorless oil (yield 82%); IR (KBr, cm−1): 1728 (C=O),
1641 (C=O); 1 H-NMR (CDCl3 , δ): 0.87 (m, 6H, CH(CH3)2), 1.21 (m, 3H, OCH2 CH3), 1.42 (d, J = 6.4
Hz, 3H, COCHCH3), 1.58-1.88 (m, 5H, piperidine, CH(CH3)2), 2.37 (m, 1H, piperidine), 2.43 (d, J = 6.8 Hz,
2H, CH2 CH), 2.67-3.00 (m, 2H, piperidine), 3.79 (m, 2H, piperidine, COCHCH3), 4.09 (m, 2H, OCH2 CH3),
429

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Stable ester and amide conjugates of some NSAIDs as..., M. O. ULUDAG, et al.
4.29-4.57 (m, 1H, piperidine), 7.10 (m, 4H, ArH); HRMS calcd. for C21 H31 NO3 (M+1): 346.2382, found:
346.2378.
4-Isopropylphenyl 2-(3-benzoylphenyl)propanoate (2a)
Elution with DCM-MeOH (0%-5%) afforded 2a as a colorless oil (yield 32%); IR (KBr, cm−1): 1754 (C=O),
1659 (C=O); 1 H-NMR (CDCl3 , δ): 1.22 (d, J = 6.8 Hz, 6H, CH(CH3)2), 1.65 (d, J = 7.6 Hz, 3H, CHCH3),
2.89 (m, 1H, CH(CH3)2), 4.03 (q, 1H, J = 7.2 Hz, CHCH3), 6.92 (m, 2H, ArH), 7.18 (m, 2H, ArH), 7.46-7.51
(m, 3H, ArH), 7.59 (m, 1H, ArH), 7.64 (m, 1H, ArH), 7.73 (m, 1H, ArH), 7.80-7.85 (m, 3H, ArH); HRMS calcd.
for C25 H24 O3 (M+1): 373.1804, found: 373.1812.
4-Tert-butylphenyl 2-(3-benzoylphenyl)propanoate (2b)
Elution with hexane-EtOAc (0%-20%) afforded 2b as a colorless oil (yield 65%); IR (KBr, cm−1): 1753 (C=O),
1659 (C=O); 1 H-NMR (CDCl3 , δ): 1.29 (s, 9H, C(CH3)3), 1.65 (d, J = 7.2 Hz, 3H, CHCH3), 4.03 (q, 1H,
J = 7.2 Hz, CHCH3), 6.93 (d, J = 8.8 Hz, 2H, ArH), 7.35 (d, J = 8.8 Hz, 2H, ArH), 7.46-7.51 (m, 3H, ArH),
7.60 (m, 1H, ArH), 7.64 (m, 1H, ArH), 7.72 (m, 1H, ArH), 7.80-7.85 (m, 3H, ArH); HRMS calcd. for C26 H26 O3
(M+1): 387.1960, found: 387.1941.
(3-{2-[4-(4-Tert-butylbenzyl)piperazin-1-yl]-1-methyl-2-oxoethyl}phenyl)(phenyl) methanone (2c)
Elution with DCM-MeOH (0%-2%) afforded 2c as a yellow solid (yield 63%); mp 221 ◦ C; IR (KBr, cm−1):
1647 (C=O), 1659 (C=O); 1 H-NMR (CDCl3 , δ): 1.29 (d, 9H, C(CH3)3), 1.50 (m, 3H, CHCH3), 2.68-4.82
(m, 11H, piperazine-H, NCH2 , CHCH3), 7.40-7.84 (m, 13H, ArH); HRMS calcd. for C31 H36 N2 O2 (M+1):
469.2855, found: 469.2840.
Ethyl 1-[2-(3-benzoylphenyl)propanoyl]piperidine-4-carboxylate (2d)
Elution with DCM-MeOH (0%-5%) afforded 2d as a colorless oil (yield 48%); IR (KBr, cm−1): 1726 (C=O),
1659 (C=O), 1641 (C=O); 1 H-NMR (CDCl3 , δ): 1.22 (m, 3H, OCH2 CH3), 1.47 (m, 3H, COCHCH3), 1.57
(m, 1H, piperidine), 1.66 (m, 2H, piperidine), 1.90 (m, 1H, piperidine), 2.44 (m, 1H, piperidine), 2.75-3.10 (m,
2H, piperidine), 3.80 (m, 1H, piperidine), 3.98 (m, 1H, COCHCH3), 4.11 (m, 2H, OCH2 CH3), 4.34-4.53 (m,
1H, piperidine), 7.43-7.79 (m, 9H, ArH); HRMS calcd. for C24 H27 NO4 (M+1): 394.2018, found: 394.2008.
4-Isopropylphenyl 2-[(2,3-dimethylphenyl)amino]benzoate (3a)
Elution with hexane-EtOAc (0%-5%) afforded 3a as a yellow solid (yield 79%); mp 102 ◦ C; IR (KBr, cm−1):
3337 (N-H), 1689 (C=O); 1 H-NMR (CDCl3 , δ): 1.27 (d, J = 6.8 Hz, 6H, CH(CH3)2), 2.15 (s, 3H, CH3), 2.31
(s, 3H, CH3), 2.95 (m, 1H, CH(CH3)2), 6.73 (m, 1H, ArH), 6.79 (d, J = 8.8 Hz, 1H, ArH), 7.02 ( d, J = 6.8
Hz, 1H, ArH), 7.09 (d, J = 8.0 Hz, 1H, ArH), 7.12-7.17 (m, 3H, ArH), 7.28-7.33 (m, 3H, ArH), 8.19 (dd, J =
8.0 Hz, 1.6 Hz, 1H, ArH), 9.19 (s, 1H, NH); HRMS calcd. for C24 H25 NO2 (M+1): 360.1964, found: 360.1957.
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Stable ester and amide conjugates of some NSAIDs as..., M. O. ULUDAG, et al.
4-Tert-butylphenyl 2-[(2,3-dimethylphenyl)amino]benzoate (3b)
Elution with hexane-EtOAc (0%-5%) afforded 3b as a pale yellow solid (yield 87%); mp 122 ◦ C; IR (KBr,
cm−1): 3343 (N-H), 1690 (C=O); 1 H-NMR (CDCl3 , δ): 1.34 (s, 9H, C(CH3)3), 2.15 (s, 3H, CH3), 2.31 (s,
3H, CH3), 6.73 (m, 1H, ArH), 6.79 (d, J = 8.8 Hz, 1H, ArH), 7.02 (d, J = 6.8 Hz, 1H, ArH), 7.09 (d, J = 8.0
Hz, 1H, ArH), 7.12-7.17 (m, 3H, ArH), 7.31 (m, 1H, ArH), 7.45 (m, 2H, ArH), 8.19 (dd, J = 8.0 Hz, 1.6 Hz,
1H, ArH), 9.19 (s, 1H, NH); HRMS calcd. for C25 H27 NO2 (M+1): 374.2120, found: 374.2115.
N-(2-{[4-(4-Tert-butylbenzyl)piperazin-1-yl]carbonyl}phenyl)-2,3-dimethylaniline (3c)
Elution with DCM-MeOH (0%-5%) afforded 3c as a white solid (yield 70%); mp 123 ◦ C; IR (KBr, cm−1):
3381 (N-H), 1623 (C=O); 1 H-NMR (CDCl3 , δ): 1.31 (s, 9H, C(CH3)3), 2.14 (s, 3H, CH3), 2.31 (s, 3H, CH3),
2.45 (s, 4H, piperazine), 3.47 (s, 2H, NCH2), 3.67 (s, 4H, piperazine), 6.76 (m, 1H, ArH), 6.92 (m, 2H, ArH),
7.00 (s, 1H, NH), 7.03-7.18 (m, 4H, ArH), 7.22 (d, J = 8.0 Hz, 2H, ArH), 7.33 (d, J = 8.4 Hz, 2H, ArH); HRMS
calcd. for C30 H37 N3 O (M+1): 456.3015, found: 456.2999.
Ethyl 1-{2-[(2,3-dimethylphenyl)amino]benzoyl}piperidine-4-carboxylate (3d)
Elution with DCM-MeOH (0%-5%) afforded 3d as a white solid (yield 52%); mp 77 ◦ C; IR (KBr, cm−1): 3361
(N-H), 1725 (C=O), 1625 (C=O); 1 H-NMR (CD3 COCD3 , δ): 1.21 (t, J = 7.2 Hz, 3H, CH2 CH3), 1.65 (m,
2H, piperidine), 1.93 (m, 2H, piperidine), 2.11 (s, 3H, CH3), 2.29 (s, 3H, CH3), 2.64 (m, 1H, piperidine), 3.14
(m, 2H, piperidine), 4.09 (q, J = 7.2 Hz, 2H, CH2 CH3), 4.20 (m, 2H, piperidine), 6.79-6.92 (m, 3H, ArH),
7.04-7.06 (m, 2H, NH+ArH), 7.18-7.25 (m, 3H, ArH); HRMS calcd for C23 H28 N2 O3 (M+1): 381.2178, found:
381.2195.
Chemical stability
The hydrolytic stability of the synthesized ester and amide derivatives was studied in near physiological
conditions in hydrochloric acid (simulated gastric fluid, SGF, pH 1.3) and phosphate buffer (simulated intestinal
fluid, SIF, pH 6.8) containing 1% Tween 20 at 37.4 ◦ C. The ionic strength (μ) for each buffer was maintained at
0.5 by adding a calculated amount of potassium chloride. The reactions were initiated by adding 0.1 mL of stock
solution of the compounds (10 mg/mL) to 1.9 mL of SGF or SIF in screw-capped vials that were preequilibrated
at 37.4 ◦ C. Samples were withdrawn at appropriate intervals for 24 h and analyzed by LC/MS-TOF for residual
prodrug and parent drug concentrations.
Enzymatic stability
The hydrolysis rates of the ester derivatives were studied in 80% human plasma diluted with isotonic phosphate
buffer (pH 7.4) at 37.4 ◦ C. The reactions were initiated by adding 20 μL of stock solutions of the ester derivatives
in DMSO (10 mg/mL) to 480 μL of diluted plasma, incubated at 37.4 ◦ C. At appropriate time intervals, 50
μL of the plasma were taken and deproteinized by the addition of 450 μL of acetonitrile. After mixing and
centrifugation for 10 min at 10,000 rpm, the supernatant was analyzed by LC/MS-TOF for intact ester and
parent drug concentrations.
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Stable ester and amide conjugates of some NSAIDs as..., M. O. ULUDAG, et al.
Pharmacological screening
Animals
Male Swiss albino mice (25-30 g) were purchased from the animal breeding laboratories of the Refik Saydam
Central Institute of Health (Ankara, Turkey). They were housed in a room with controlled temperature (22
1
◦ C), humidity (55
10%), and photoperiod (12:12 h) for at least 1 week before being used. They
were maintained on a standard pellet diet and water ad libitum throughout the experiment. A minimum of
6 animals was used in each group. Throughout the experiments, the animals were processed according to
the suggested international ethical guidelines for the care of laboratory animals under the audit of the Gazi
University Commission of Animal Ethics (Permission No: 53-3044).
Drugs
p-Benzoquinone (PBQ), λ-carrageenan (type IV), and other biochemicals used in this study were purchased
from Sigma-Aldrich Chemical Company (St. Louis, MO, USA).
Analgesic activity
Analgesic activity was measured using the PBQ-induced writhing (abdominal constriction) test in mice.17
According to the protocol, 30 min after the subcutaneous administration of a test sample (100 mg/kg body
weight), the mice were intraperitoneally injected with 0.1 mL/10 g body weight of 2.5% (w/v) PBQ solution
in distilled water. Control animals received an appropriate volume of dosing vehicle. The mice were then kept
individually for the observation and the total number of abdominal contractions (writhing movements) was
counted for the following 15 min, starting 5 min after the PBQ injection. The data represent the average of the
total number of writhing movements observed. Analgesic activity was then expressed as the percentage change
from the writhing controls.
Antiinflammatory activity
The carrageenan-induced hind paw edema model was used for determining antiinflammatory activity.18 Each
group contained a minimum of 7 animals, and 30 min after the subcutaneous administration of a test sample
(50 mg/kg body weight) or dosing vehicle, each mouse was injected with a freshly prepared suspension of
carrageenan (0.5 mg/25 μL) in physiological saline into the subplantar tissue of the right hind paw. As a
control, 25 μL of saline was injected into that of the left hind paw. Paw edema was measured 90, 180, 270,
and 360 min after the carrageenan injections. The difference in hind paw thicknesses was measured by caliber
compasses (Ozaki Co., Tokyo, Japan). Mean values of each treated group were compared with the control group
and analyzed by statistical methods.
Acute toxicity
At the end of these experiments, after 48 h of observation of all animals, no morbidity or mortality was recorded.
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Stable ester and amide conjugates of some NSAIDs as..., M. O. ULUDAG, et al.
Gastric ulcerogenic effect
The ulcerogenicity was investigated as described elsewhere.19 The animals were sacrificed with an overdose of
diethyl ether after 270 min of administration of the compounds. After abdominal dissection, the stomachs of the
animals were slightly taken out. The esophagus was then tied in a knot nearest the cardia by a surgical suture.
From the duodenum side, 2.5 mL of 10% formalin solution was injected into the stomach. The distended
stomach was immediately tied to the pyloric sphincter using another surgical suture to avoid leakage of the
formalin solution. Finally, the stomachs were removed from the abdominal cavity and immersed in the same
solution to fix the outer layer of the stomach. Each stomach was then dissected along the greater curvature,
rinsed with tap water to remove the gastric contents (e.g. food fragments, blood clots), and examined under a
dissecting microscope (20 × 6.3) to assess the formation of ulcers. The sum of the length (mm) of all lesions
for each stomach was used as the ulcer index.
Statistical analysis
The data were expressed as means SEM. The significance of differences between the treatment and the control
group of animals was determined by 1-way ANOVA with Bartlett’s test following a post hoc Student-Newman-
Keuls multiple comparisons test for analgesic activity, and 2-way ANOVA following a post hoc Bonferroni test
for antiinflammatory activity. Values of P < 0.05 were considered statistically significant.
1) EDC, DMAP, CH2Cl2
O
R C R'
1a-d, 2a-d, 3a-d
R'
R COOH
R
2) Phenol or amine derivatives
O
a
1
2
O
O
b
N
N
c
H
N
O
N
O
d
3
Scheme. Synthetic pathway for the synthesis of the target ester and amide derivatives.
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Stable ester and amide conjugates of some NSAIDs as..., M. O. ULUDAG, et al.
Results
The desired ester and amide derivatives of ibuprofen (1), ketoprofen (2), and mefenamic acid (3) were prepared
in a one-pot synthetic procedure as shown in the Scheme. Treatment of parent NSAIDs with the appropriate
phenol or amine derivatives in the presence of N-(3-dimethylaminopropyl)-Nꢀ-ethylcarbodiimide hydrochloride
(EDCI) as the carboxyl group activator and dimethylaminopyridine (DMAP) afforded the desired products
(1a-d, 2a-d, and 3a-d) in 50%-70% yields.
In the pharmacological study, we investigated the analgesic and antiinflammatory activities and the
ulcerogenic potential of acute administration of both the parent NSAIDs (1-3) and the corresponding ester
(1a-b, 2a-b, and 3a-b) and amide (1c-d, 2c-d, and 3c-d) derivatives. Animals were administered 100 mg/kg
body weight doses of test compounds, and the percentage of analgesic activity by means of inhibition of writhing
movements in comparison with parent NSAIDs are given in the Figure and the Table. Closer inspection of the
results indicates that all compounds showed significant analgesic activity as compared with the vehicle-injected
mice. The percentage inhibition of PBQ-induced writhing revealed that the ester and amide derivatives of
ibuprofen displayed similar (1b-d) or reduced (1a) analgesic activities compared to the parent ibuprofen. In
the ketoprofen series, ester derivatives 2a and 2b showed more potent and higher analgesic activity than
ketoprofen, while amide derivatives 2c and 2d resulted in lower activity as compared to the parent drug. In
addition, both ester and amide derivatives of mefenamic acid showed analgesic activity comparable to the parent
NSAID.
The parent NSAIDs (1-3) and their corresponding ester and amide derivatives (1a-d, 2a-d, and 3a-d)
were also evaluated for their in vivo systemic antiinflammatory activity using carrageenan-induced paw edema
in mice at 50 mg/kg body weight doses. All compounds and parent NSAIDs showed a peak antiinflammatory
activity at 270 min, and the inhibitory activity on edema formation started to diminish at that point (Table).
The 4-i-propylphenyl ester (1a) showed the same pattern of inhibition, with ibuprofen also having a peak
inhibition at 270 min (28%). The 4-t-butylphenyl ester of ibuprofen (1b) showed extended inhibitory activity
ranging from 35% to 21%. On the other hand, the piperazine amide (1c) of ibuprofen did not show any notable
activity, while the piperidine amide derivative (1d) was effective until 270 min, although with a reduced activity
as compared to the parent drug. For the ketoprofen (2) series, the phenolic ester (2ab) and piperidine (2d)
amide derivatives resulted in potent antiinflammatory activity, which was extended to all time points and was
higher than or comparable to the parent drug. However, the piperazine (2c) amide derivative showed reduced
antiinflammatory activity and the peak inhibition of edema was only evident for 180 min. With regard to
mefenamic acid (3), it was interesting that only the 4-i-propylphenyl ester (3a) and piperidine amide (3d)
derivatives showed inhibition of edema formation; however, this activity was not as pronounced as that of the
parent drug. Moreover, the compounds that were tested for antiinflammatory activity were further monitored
for their gastric toxicity on acute administration to determine the ulcerogenic potential at the time interval in
which the compounds showed the highest antiinflammatory activity (270 min). Results indicated that all parent
NSAIDs showed a measurable ulcerogenic index in at least 1 or 2 animals after the subcutaneous administration
of 50 mg/kg doses. As seen from the Table, none of the ibuprofen or mefenamic acid prodrugs caused any sign
of gastric lesions under the same conditions. However, the ketoprofen ester derivatives (2a-b), which exhibited
the highest analgesic and antiinflammatory activity of the whole series, caused severe bleeding lesions compared
to the parent drug in the tested animals.
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The chemical stability of the synthesized ester and amide derivatives of selected NSAIDs were studied at
37.4 ◦ C in aqueous buffer solutions of pH 1.2 and 6.8, which were considered as nonenzymatic simulated gastric
fluid (SGF) and nonenzymatic simulated intestinal fluid (SIF), respectively. The reactions were monitored by
mass spectrometry coupled with a UPLC system. The results indicated that the synthesized ester and amide
derivatives were sufficiently stable in SGF (pH 1.2) and SIF (pH 6.8) for a period of 1 day (24 h) at 37.4 ◦ C.
30
25
20
15
10
5
30
20
10
0
B)
A)
**
***
***
***
***
***
***
***
***
***
0
30
25
20
15
10
5
C)
**
***
***
***
***
0
Figure. Writhing responses (means
SEM, n = 6-9) for the A) ibuprofen, B) ketoprofen, and C) mefenamic acid
derivatives compared to the control (**P < 0.01, ***P < 0.001).
435

˘
Stable ester and amide conjugates of some NSAIDs as..., M. O. ULUDAG, et al.
Table. Analgesic and antiinflammatory effects of the test compounds on p-benzoquinone (PBQ)-induced abdominal
constriction test and carrageenan (CG)-induced hind paw edema model in mice, respectively, and ratio of ulceration.
Test
Analgesic activity,
SEMa
Swelling in thickness (×10−2 mm)
SEM (inhibition of edema, %)b
270 min 360 min
86.4 7.3
Ratio of
ulcerationc
0:7
compounds
%
90 min
180 min
69.3 8.4
47.1
Control
1
-
30.0
31.4
32.1
3.5
79.3
67.9
7.4
80.9
46.4
68.1
73.1
74.0
88.7
93.6
94.1
75.9
80.4
62.1
64.2
75.0
52.0
64.3
11.4
13.3
12.0
11.1
9.8
4.6
6.8 (32.0)
5.4 (30.9)
5.1 (35.1)∗
57.9
62.1
64.3
85.0
72.1
6.6 (33.1)∗
5.5 (28.1)∗
6.3 (25.6)
6.1 (14.4)
5.5 (2.7)
6.9 (21.6)
6.6
1:7
1a
1b
1c
3.9
47.9
45.0
63.6
48.6
77.1
62.1
0:7
29.3
32.1
27.1
4.4 (2.4)
3.4
0:7
12.5 (8.3)
5.7 (1.7)
88.6
77.9
52.9
47.5
58.6
87.9
0:7
1d
2
4.7 (9.5)
5.0 (11.9)
4.7 (35.0)
5.3 (51.4)
5.8 (20.5)
6.6 (1.4)
7.3 (7.1)
4.1
6.4 (29.9)
7.5 (16.5)
5.9 (1.8)
5.7 (33.3)∗
5.3 (40.1)∗∗
6.4 (26.1)
0:7
4.5
26.4
19.5
14.6
23.9
29.6
27.9
34.3
3.8 (50.5)∗∗∗
6.2 (60.3)∗∗∗
4.2 (68.0)∗∗∗
7.1 (25.8)
3.9 (53.6)∗∗∗
10 (41.2)∗
4.7 (26.8)
37.9
6.6 (56.2)∗∗∗
6.5 (53.7)∗∗∗
7.1 (56.2)∗∗∗
6.2 (13.2)
4:7
2a
2b
2c
5.3
27.5
22.1
40.0
37.9
2:7
1,5
5:7
11.3
8.5
51.4
32.1
40.7
75.0
7.4
0:7
2d
3
52.1
55.0
70.7
75.7
76.4
66.6
5.7 (39.7)∗∗
11 (36.4)∗∗
7.5 (18.2)
72.1
55.7
7.7 (9.0)
12 (29.7)
9.3
0:7
7.7
2:7
3a
3b
3c
13.7
9.4
39.3
40.0
28.6
22.9
50.7
90.0
80.7
75.0
78.6
0:7
3.5
70.7
3.7
3.2 (12.4)
5.1
0:7
11.1
9.7
3.6 (4.8)
5.4 (23.8)
65.7
48.6
1.0 (5.2)
8.3 (29.9)
12 (11.6)
7.4 (5.4)
7.5 (0.9)
0:7
3d
5.3 (23.0)
0:7
Data obtained from animal experiments were expressed as means
ketoprofen, 3: mefenamic acid.
standard error of mean (SEM); 1: ibuprofen, 2:
a Groups composed of 8 mice were employed for the PBQ-induced writhing test and all test drugs were subcutaneously
administered to mice at doses of 100 mg/kg body weight.
b,c Groups composed of 7 mice were employed for the CG-induced paw edema model and all test drugs were subcutaneously
administered to mice at doses of 50 mg/kg body weight.
*Significantly different from the control (*P < 0.05, **P < 0.01, ***P < 0.001).
Moreover, the enzymatic hydrolysis of ester derivatives 1a-b and 2a-b in human plasma also revealed that
they were relatively stable against plasma esterases, since most of the ester derivatives remained intact after
the 24-h incubation period. The highest conversion rate to parent drug was observed with the isopropylphenyl
ester of ketoprofen (2a), whereby 12% and 51% of 2a was converted to ketoprofen after 270 min and after 24
h of incubation in human plasma, respectively. The tertiarybutylphenyl ester derivative (2b) hydrolyzed more
slowly, with a hydrolysis rate of about 6% and 23% at 270 min and 24 h, respectively. For ibuprofen derivatives
1a and 1b, the conversion rate to parent drug was 7%-8% at 270 min, whereas it was 16%-18% after 24 h of
incubation in human plasma.
Discussion and conclusion
It is already known that a large variety of substituents can be tolerated in the ester and amide groups of
NSAID derivatives, indicating that the free carboxylic acid of NSAIDs is not a significant requirement for an-
tiinflammatory activity.10 Therefore, it appears that the carboxyl group of the parent NSAID is a convenient
functionality for derivatization. We have previously reported that piperazine and piperidine amide derivatives
of certain heteroaromatic structures contribute to the analgesic and antiinflammatory activity.20−24 Mean-
436

˘
Stable ester and amide conjugates of some NSAIDs as..., M. O. ULUDAG, et al.
while, others have also reported that various compounds having partial piperidine carboxylic acid structures
bear potent analgesic and antiinflammatory activity. In addition, during the course of our ongoing studies of
antiinflammatory compounds, we also determined that preparation of i-propyl- and t-butyl-phenol esters and
4-t-butyl-benzylpiperazine and ethyl 4-piperidinecarboxylate amides of pyrazole-3-propanoic acids produced
compounds that effectively inhibited the LTB4 biosynthesis in human leukocytes (unpublished results). There-
fore, we have implemented these results on selected contemporary NSAIDs, namely ibuprofen and ketoprofen as
arylpropionic acid derivatives and mefenamic acid as an N-arylanthranilic acid derivative, and prepared targeted
ester and amide derivatives 1a-d, 2a-d, and 3a-d.
The resulting change of paw volume in mice indicated that all active compounds were able to inhibit the
change in paw volume at 180 and 270 min after carrageenan injection, providing an implication toward their
probable mechanism of action. Since prostaglandins are secreted in the third and fourth hours after carrageenan
injection, it was indicated that all synthesized ester and amide derivatives still maintained the inhibition of the
arachidonic acid pathway effectively. Among the chosen arylpropionic acids, derivatization of ketoprofen to its
lipophilic esters (2a-b) produced more potent derivatives in terms of analgesic and antiinflammatory activity,
while the amidification was not as effective as the esterification. However, the ulcerative potential of these
ester derivatives was also comparable to the parent ketoprofen, indicating that the COX-1 inhibition may
prevail with these compounds. In terms of ibuprofen esters, 1a-b showed comparable activity to ibuprofen
without producing gastric lesions in the tested animals. However, for the mefenamic acid derivatives, neither
esterification nor amidification produced an increase in antiinflammatory activity. Analgesic activity results of
the compounds also showed good correlation with their antiinflammatory activities in that compounds 1b-d,
2a-b, 3a-b, and 3d strongly inhibited the peripheral pain response in the mice, as the amount of writhing
was found to be significantly diminished as compared to the control animals treated with vehicle (Figure). In
addition, most of the compounds were found to be active in comparison with parent drugs, indicating that
esterification and amidification with these NSAIDs improved the analgesic activity.
According to the chemical stability studies, the synthesized ester and amide derivatives were sufficiently
stable at pH 1.2 for 24 h, so no hydrolysis was expected in the stomach. In addition, the chemical stability
of the compounds at pH 6.8 for 1 day suggested that they can be absorbed from the intestines almost intact.
The chemical stability of the reported ester and amide derivatives of ibuprofen, ketoprofen, and mefenamic
acid at pH values simulating gastric and intestinal fluids revealed that the designed compounds might have
increased absorption as compared to parent NSAIDs. Moreover, it is well-evidenced that the direct contact
mechanism appears to play a major role in the production of gastrointestinal lesions upon administration
of NSAIDs.6 Therefore, the chemical stability of the reported compounds might indicate that stable ester
and amide derivatives of an NSAID might also have a potentially improved therapeutic index compared to
contemporary parent drugs. Furthermore, the enzymatic stability of the ester derivatives after 270 min and
24 h of incubation in human plasma establishes evidence that they may act in vivo without prior hydrolysis
of the ester bond. It should be added that the antiinflammatory activity of the compounds in the present
study showed a peak activity at 270 min after carrageenan injection. Therefore, the observed stability of the
synthesized ester derivatives may fulfill the essential requirements for the oral delivery system of NSAIDs, in
which the acid stable ester group might also prevent the direct contact effects with the stomach mucosa.
In conclusion, the prepared ester and amide derivatives of ibuprofen, ketoprofen, and mefenamic acid
437

˘
Stable ester and amide conjugates of some NSAIDs as..., M. O. ULUDAG, et al.
were sufficiently chemically stable in SGF and SIF. Additionally, the increased lipophilicity of the resulting
derivatives may attain intact absorption at a higher rate than the parent drugs. Moreover, the enzymatic
stability of the ester derivatives also indicated that the activities observed with these molecules were not due to
the parent NSAIDs. Therefore, we can conclude that the preparation of suitable ester and amide derivatives of
contemporary NSAIDs may represent a potentially useful method for developing compounds with more potent
analgesic and antiinflammatory activity than the parent compounds, in addition to reduced gastrointestinal
side effects and increased chemical and enzymatic stability.
Acknowledgments
This investigation was supported by Scientific Research Grant 02/2010-21, awarded by Gazi University BAP.
˙
The authors greatly acknowledge Prof. Ilhan Gu¨rbu¨z (Gazi University, Faculty of Pharmacy, Department of
Pharmacognosy) for his assistance in performing the gastric ulceration experiments.
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439

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