The novel ketoprofen amides - Synthesis and biological evaluation as antioxidants, lipoxygenase inhibitors and cytostatic agents

Article abstract of DOI:10.1111/j.1747-0285.2010.00963.x

The novel amides of ketoprofen and its reduced derivatives (5a-f, 4a-n, 6a-g) with aromatic and cycloalkyl amines or hydroxylamines were prepared and screened for their reducing and cytostatic activity as well as for their ability to inhibit soybean lipox

Full text of DOI:10.1111/j.1747-0285.2010.00963.x

ª 2010 John Wiley & Sons A/S
doi: 10.1111/j.1747-0285.2010.00963.x
Chem Biol Drug Des 2010; 75: 641–652
Research Article
The Novel Ketoprofen Amides – Synthesis and
Biological Evaluation as Antioxidants,
Lipoxygenase Inhibitors and Cytostatic Agents
1
2
Ketoprofen, 2-(3-benzoylphenyl)propionic acid, is a non-steroidal
anti-inflammatory drug (NSAID) with pronounced analgesic and anti-
pyretic properties. Numerous derivatives of ketoprofen and other
NSAIDs have been synthesized in order to minimize side-effects,
prolong plasma half-life and increase water solubility or lipophilicity
(1–3). It has been demonstrated that amidation of NSAIDs improves
selectivity toward COX-2 (4), while modification of carboxylic group
to hydroxamic acid leads to inhibition of both cyclooxygenase and
5-lipoxygenase, two enzymes crucial in inflammatory processes
(5,6). On the other hand, NSAIDs are potential anticancer drugs and
effective chemopreventive agents (7–12). A combination of anti-
inflammatory agents as biochemical modulators with several anti-
cancer drugs has been considered useful as an auxiliary means for
combined modality therapy in cancer (13).
´
Zrinka Rajic , Dimitra Hadjipavlou-Litina ,
Eleni Pontiki2, Marijeta Kralj3, Lidija
Suman and Branka Zorc *
3
1,
ˇ
1Department of Medicinal Chemistry, Faculty of Pharmacy and
Biochemistry, University of Zagreb, HR-10000 Zagreb, Croatia
2Department of Pharmaceutical Chemistry, School of Pharmacy,
Aristotles University of Thessaloniki, GR-54124 Thessaloniki, Greece
3Division of Molecular Medicine, Laboratory of Experimental
Therapy, ''Rudjer Boskovic'' Institute, HR-10000 Zagreb, Croatia
*Corresponding author: Branka Zorc, bzbz@pharma.hr
The novel amides of ketoprofen and its reduced
derivatives (5a–f, 4a–n, 6a–g) with aromatic and
cycloalkyl amines or hydroxylamines were pre-
pared and screened for their reducing and cyto-
static activity as well as for their ability to inhibit
soybean lipoxygenase and lipid peroxidation. 1,1-
Diphenyl-picrylhydrazyl test for reducing ability
revealed that ketoprofen amides were more
potent antioxidants than the amides of the
reduced ketoprofen derivatives. The most active
compound was benzhydryl ketoprofen amide 5f.
Lipoxygenase inhibition of the tested compounds
varied from strong to very weak. The most potent
In our previous articles (14–16), potency of amides, NSAID hydroxa-
mic acids and O-alkylated NSAID hydroxamic acids of ketoprofen
and related NSAIDs as cytostatic and antioxidative agents and as
inhibitors of lipoxygenase and linoleic acid lipid peroxidation was
screened. It was shown that ketoprofen amidation significantly
enhanced antiproliferative activity of the parent compound and that
lipophilicity of the compounds had a significant influence on their
antiproliferative activity. Following these findings, a new series of
lipophilic amides of ketoprofen was prepared. In order to evaluate
the importance of hydroxyl group in the amide moiety, a number of
hydroxylamides was designed. In this article, we report their syn-
thesis and full chemical characterization, together with their biologi-
cal screening.
compound was benzhydryl derivative 6f (IC50
=
20.5 lM). Aromatic and cycloalkyl amides 4 and 5
were more potent lipoxygenase inhibitors than
derivatives with carboxylic group. Aromatic
amides of series 4 and 5 showed excellent lipid
peroxidation inhibition (92.2–99.9%). On the other
hand, the most pronounced cytostatic activity
was exerted by O-benzyl derivative 4i, although in
general all tested reduced and non-reduced lipo-
philic derivatives showed similar activity.
Methods
Chemistry
Key words: amide, antioxidant activity, cytostatic activity, ketopro-
fen derivatives, soybean lipoxygenase
Melting points were determined on a Stuart Melting Point Apparatus
SMP3 (Barworld Scientific, Staffordshire, UK) and were uncorrected.
IR spectra were recorded on a FTIR Perkin Elmer Paragon 500 spec-
trometer (Perkin Elmer, Waltham, MA, USA). 1H and 13C NMR spectra
were recorded on a Varian Gemini 300 spectrometer (Varian, Palo-Alto,
Abbreviations: AAPH, 2,2¢-azobis(2-amidinopropane) dihydrochlo-
ride; Bt, N-1-benzotriazolyl, COX, cyclooxygenase; DMEM, Dulbecco's
modified Eagle's medium; DPPH, 1,1-diphenyl-picrylhydrazyl; FBS, fetal
bovine serum; LOX, soybean lipoxygenase; MTT, 3-(4,5-dimethylthiazol-2-
yl)-2,5-diphenyltetrazolium bromide; NDGA, nordihydroguaiaretic acid;
NSAID, non-steroidal anti-inflammatory drug; PG, percentage of growth;
TEA, triethylamine.
1
CA, USA), operating at 300 and 75.5 MHz for the H and 13C nuclei,
respectively. Samples were measured in dimethyl sulphoxide (DMSO)-
d6 solutions at 20 ꢀC in 5-mm NMR tubes. Chemical shifts (d) in ppm
were referred to tetramethylsilane (TMS). Coupling constants (J ) are
given in Hz. Elemental analyses were performed on CHNS LECO
analyzer and mass spectra on HPLC-MS ⁄ MS (HPLC, Agilent Technolo-
gies 1200 Series; MS, Agilent Technologies 6410 Triple Quad, Santa
Received 20 January 2009, revised 26 December 2009 and accepted 6
February 2010
641

´
Rajic et al.
Clara, CA, USA). For thin-layer chromatography, precoated Merck silica
gel 60 F254 (Merck, Darmstadt, Germany) and solvent systems cyclo-
hexane ⁄ ethyl acetate ⁄ methanol (3:1:0.5), dichloromethane ⁄ methanol
(95:5) and ethyl acetate were used. Spots were visualized by short-
wave UV light and iodine vapor. Column chromatography was per-
formed with silica gel 0.063–0.200 mm (Merck), with ethyl acetate,
cyclohexane ⁄ ethyl acetate (1:1, 2:1, 3:1, 4:1), ethyl acetate ⁄ methanol
(8:1), cyclohexane ⁄ ethyl acetate ⁄ methanol (3:1:0.5), dichlorome-
thane ⁄ methanol (93:7, 97:3) or chloroform ⁄ methanol (93:7, 95:5) as el-
uents. Benzotriazole, triphosgene, triethylamine, cyclopentylamine,
cyclohexylamine, cyclohexanemethylamine, benzylamine, 2-phenyleth-
ylamine, benzhydrylamine, 2-hydroxyethylamine, 3-hydroxypropyl-
amine, diethanolamine, ethylenediamine, and 10% Pd ⁄ C were
purchased from Sigma–Aldrich (St. Louis, MO, USA). Ketoprofen was
obtained as a gift sample from PLIVA (Zagreb, Croatia). All solvents
were of analytical grade purity and were dried prior to use.
temperature for 5 h and purification of the product by trituration
with ether was obtained 0.252 g (82%) of 9a; mp 92–93 ꢀC; IR
(KBr, m ⁄ cm)1) 3274, 3061, 3027, 2962, 2936, 2864, 1635, 1599,
1548, 1448, 785, 731, 711, 699; 1H NMR (300 MHz, DMSO) d
7.85 (d, J = 7.15, 1H), 7.30–7.04 (m, 9H), 3.99–3.87 (m, 3H), 3.52
(q, J = 7.01, 1H), 1.83–1.16 (m, 8H), 1.27 (d, J = 7.03, 3H); 13C
NMR (75.5 MHz, DMSO) d 173.02, 143.11, 141.69, 141.42, 129.09,
128.83, 128.68, 128.03, 127.21, 126.39, 125.26, 50.64, 45.22,
41.67, 32.68, 32.63, 23.96, 23.92, 19.01; MS ⁄ MS m ⁄ z 308.3
(M + H)+, 330.3, (M + Na)+; Elemental analysis: calcd for
C21H25NO: C, 82.04; H, 8.20; N, 4.56. Found: C, 82.00; H, 8.36; N,
4.68.
2-(3-Benzylphenyl)-N-cyclohexylpropanamide (4b)
From the reaction of 0.341 g (1 mmol) 3a, 0.126 mL (1.1 mmol)
cyclohexylamine and 0.696 mL (5 mmol) triethylamine at room tem-
perature for 7 h and purification of the product by trituration with
ether was obtained 0.273 g (85%) of 4b; mp 89–93 ꢀC; IR (KBr,
m ⁄ cm)1) 3297, 3062, 3026, 2980, 2936, 2854, 1639, 1585, 1546,
1448, 779, 750, 698; 1H NMR (300 MHz, DMSO) d 7.74 (d,
J = 7.79, 1H), 7.29–7.04 (m, 9H), 3.90 (s, 2H), 3.52 (q, J = 7.04, 1H),
3.49–3.40 (m, 1H), 1.73–1.54 (m, 6H), 1.21–0.93 (m, 4H), 1.27 (d,
J = 7.03, 3H); 13C NMR (75.5 MHz, DMSO) d 172.59, 143.15,
141.69, 141.41, 129.10, 128.82, 128.67, 128.00, 127.19, 126.37,
125.26, 47.84, 45.27, 41.66, 32.81, 32.78, 25.69, 25.04, 24.96,
19.00; MS ⁄ MS m ⁄ z 322.3 (M + H)+, 344.3, (M + Na)+; Elemental
analysis: calcd for C22H27NO: C, 82.20; H, 8.47; N, 4.36. Found: C,
82.09; H, 8.53; N, 4.42.
1-Benzotriazole carboxylic acid chloride (1), benzotriazolide 3b,
2-(3-benzoylphenyl)-N-cyclohexylpropanamide (5b), N-benzyloxy-2-
(3-benzoylphenyl)propanamide (5g), N-hydroxy-2-(3-benzylphenyl)
propanamide (4m) and 10% Pd ⁄ C(en) were prepared following the
published procedures (16–19). 2-(3-Benzylphenyl)propanoic acid (2a)
and 2-(3-(hydroxy(phenyl)methyl)phenyl)propanoic acid (2b) were
prepared by reduction of ketoprofen using H2 ⁄ Pd ⁄ C ⁄ methanol and
H2 ⁄ Pd ⁄ C(en) ⁄ tetrahydrofurane systems, respectively (the modified
published procedures 21,22).
2-(3-Benzylphenyl)propanoic acid benzotriazo-
lide (3a)
To a solution of 2.403 g (10 mmol) 2a and 1.393 mL (10 mmol) tri-
ethylamine in dry toluene (30 mL) was added dropwise (0.25 h) a
solution of 1.696 g (10 mmol) chloride 1 in dry toluene (30 mL). The
reaction mixture was stirred at room temperature for 1 h and
extracted with water (3 · 60 mL). After drying of the organic layer
(anhydrous sodium sulfate), filtration, evaporation of the solvent,
and trituration of the residue with ether, 2.663 g (78%) of 3a was
obtained; mp 59–60 ꢀC; IR (KBr, m ⁄ cm)1) 3027, 2937, 2918, 2852,
1730, 1597, 1485, 1452, 1390, 1372, 1168, 955, 780, 756, 738, 708,
692. Elemental analysis: calcd for C22H19N3O: C, 77.40; H, 5.61; N,
12.31. Found: C, 77.48; H, 5.88; N, 12.64.
2-(3-Benzylphenyl)-N-(cyclohexanemethyl)propan-
amide (4c)
From the reaction of 0.341 g (1 mmol) 3a, 0.143 mL (1.1 mmol) cy-
clohexanemethylamine and 0.696 mL (5 mmol) triethylamine at room
temperature for 8 h and purification of the product by column chro-
matography (eluent cyclohexane ⁄ ethyl acetate 3:1) was obtained
0.275 g (82%) of 4c; mp 50–54 ꢀC; IR (KBr, m ⁄ cm)1) 3295, 3061,
3027, 2968, 2918, 2851, 1647, 1602, 1585, 1558, 1442, 1221, 1155,
1
711, 698; H NMR (300 MHz, DMSO) d 7.84 (t, J = 5.31, 1H), 7.30–
7.03 (m, 9H), 3.90 (s, 2H), 3.56 (q, J = 6.97, 1H), 2.85 (dd, J = 6.11,
J = 6.50, 2H), 1.66–1.51 (m, 6H), 1.40–0.72 (m, 4H), 1.28 (d,
J = 7.02, 3H); 13C NMR (75.5 MHz, DMSO) d 173.55, 143.10,
141.67, 141.36, 129.07, 128.83, 128.66, 128.11, 127.27, 126.39,
125.31, 45.46, 45.24, 41.65, 37.88, 30.81, 30.77, 26.49, 25.85,
19.02; MS ⁄ MS m ⁄ z 336.3 (M + H)+, 358.3, (M + Na)+; Elemental
analysis: calcd for C23H29NO: C, 82.34; H, 8.71; N, 4.18. Found: C,
82.66; H, 8.65; N, 4.37.
Ketoprofen derivatives 4a–i and 5a–f. General
procedure
A solution of the appropriate benzotriazolide 3a,b (1 mmol), the
appropriate amine (1.1 mmol) and 0.696 mL (5 mmol) triethylamine
in toluene (10 mL) was stirred at room temperature for 1–48 h or at
78 ꢀC for 11–16 h. The reaction mixture was extracted with brine
(5 · 10 mL), hydrochloric acid (w = 1%, 2 · 10 mL) and washed
with water till pH 7. After drying (anhydrous sodium sulfate) and
evaporation of the solvent, the crude product was obtained.
N-Benzyl-2-(3-benzylphenyl)propanamide (4d)
From the reaction of 0.341 g (1 mmol) 3a, 0.120 mL (1.1 mmol)
benzylamine and 0.696 mL (5 mmol) triethylamine at room tempera-
ture for 3.5 h, purification of the product by column chromatography
(eluent cyclohexane ⁄ ethyl acetate 2:1) and trituration with ether
2-(3-Benzylphenyl)-N-cyclopentylpropanamide
(4a)
was obtained 0.290 g (88%) of 4d; mp 70–74 ꢀC; IR (KBr, m ⁄ cm)1
)
From the reaction of 0.341 g (1 mmol) 3a, 0.109 mL (1.1 mmol)
cyclopentylamine and 0.696 mL (5 mmol) triethylamine at room
3292, 3062, 3028, 2971, 2930, 1648, 1602, 1546, 1495, 1453, 722,
697; H NMR (300 MHz, DMSO) d 8.44 (t, J = 5.65, 1H), 7.31–7.06
1
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Chem Biol Drug Des 2010; 75: 641–652

Biological Evaluation of Ketoprofen Amides
(m, 14H), 4.24 (d, J = 5.52, 2H), 3.91 (s, 2H), 3.64 (q, J = 6.96, 1H),
1.34 (d, J = 7.01, 3H); 13C NMR (75.5 MHz, DMSO) d 173.69,
142.88, 141.66, 141.48, 140.00, 129.11, 128.86, 128.67, 128.16,
127.23, 126.41, 125.41, 128.76, 127.51, 127.41, 127.14, 45.52,
42.48, 41.64, 19.04; MS ⁄ MS m ⁄ z 330.3 (M + H)+, 352.2, (M + Na)+;
Elemental analysis: calcd for C23H23NO: C, 83.85; H, 7.04; N, 4.25.
Found: C, 83.74; H, 6.99; N, 4.56.
2-(3-Benzylphenyl)-N-ethoxypropanamide (4h)
From the reaction of 0.341 g (1 mmol) 3a, 0.107 g O-ethylhydroxyl-
amine hydrochloride and 0.696 mL (5 mmol) triethylamine at room
temperature for 46 h and purification of the product by column
chromatography (eluent cyclohexane ⁄ ethyl acetate 1:1) was
obtained 0.130 g (46%) of 4h; mp 114–116 ꢀC; IR (KBr, m ⁄ cm)1
)
1
3170, 3023, 2987, 1671, 1650, 1600, 1492, 1047, 721, 707; H NMR
(300 MHz, DMSO) d 11.07 (s, 1H), 7.31)7.07 (m, 9H), 3.91 (s, 2H),
3.71 (q, J = 7.03, 1H), 3.37 (q, J = 7.07, 2H), 1.30 (d, J = 7.04, 3H),
1.07 (t, J = 7.03, 3H); 13C NMR (75.5 MHz, DMSO) d 170.55,
142.11, 141.62, 129.11, 128.86, 128.82, 127.99, 127.55, 126.41,
125.28, 70.85, 42.51, 41.60, 18.65, 13.83; MS ⁄ MS m ⁄ z 284.2
(M + H)+, 306.2, (M + Na)+; Elemental analysis: calcd for
C18H21NO2: C, 76.29; H, 7.47; N, 4.94. Found: C, 76.01; H, 7.31; N,
4.99.
2-(3-Benzylphenyl)-N-phenethylpropanamide (4e)
From the reaction of 0.341 g (1 mmol) 3a, 0.139 mL (1.1 mmol)
phenylethylamine and 0.696 mL (5 mmol) triethylamine at room tem-
perature for 2 h and purification of the product by trituration with
ether ⁄ petrolether was obtained 0.295 g (86%) of 4e; mp 60–64 ꢀC;
IR (KBr, m ⁄ cm)1) 3306, 3064, 3026, 2925, 1642, 1548, 1496, 1454,
1
753, 712, 698; H NMR (300 MHz, DMSO) d 7.97 (t, J = 5.43, 1H),
7.29–7.05 (m, 14H), 3.91 (s, 2H), 3.52 (q, J = 7.00, 1H), 3.29–3.16
(m, 2H), 2.64 (t, J = 7.23, 2H), 1.27 (d, J = 7.04, 3H); 13C NMR
(75.5 MHz, DMSO) d 173.53, 142.90, 141.67, 141.47, 139.91,
129.13, 128.86, 128.71, 128.12, 127.30, 126.40, 125.40, 129.10,
128.67, 126.44, 45.50, 41.66, 40.83, 35.37, 19.02; MS ⁄ MS m ⁄ z
344.3 (M + H)+, 366.3, (M + Na)+; Elemental analysis: calcd for
C24H25NO: C, 83.93; H, 7.34; N, 4.08. Found: C, 84.08; H, 7.39; N,
4.18.
N-Benzyloxy-2-(3-benzylphenyl)propanamide (4i)
From the reaction of 0.341 g (1 mmol) 3a, 0.176 g O-benzylhydro-
xylamine hydrochloride and 0.696 mL (5 mmol) triethylamine at
78 ꢀC for 11 h and purification of the product by column chroma-
tography (eluent cyclohexane ⁄ ethyl acetate ⁄ methanol 3:1:0.5) was
obtained 0.231 g (67%) of 4i; oil; IR (film, m ⁄ cm)1) 3181, 3062,
3028, 2975, 2934, 2876, 1658, 1601, 1495, 1454, 1027, 749, 725,
698; 1H NMR (300 MHz, DMSO) d 11.18 (s, 1H), 7.34)7.07 (m,
14H), 4.70 (s, 2H), 3.92 (s, 2H), 3.38 (q, J = 7.01, 1H), 1.30 (d,
J = 7.03, 3H); 13C NMR (75.5 MHz, DMSO) d 170.71, 141.99,
141.62, 136.34, 129.36, 128.71, 129.12, 128.86, 128.82, 128.04,
127.56, 126.42, 125.36, 77.06, 42.44, 41.60, 18.59; MS ⁄ MS m ⁄ z
346.3 (M + H)+, 368.2, (M + Na)+; Elemental analysis: calcd for
C23H23NO2: C, 79.97; H, 6.71; N, 4.05. Found: C, 80.08; H, 6.99; N,
4.11.
N-Benzhydryl-2-(3-benzylphenyl)propanamide (4f)
From the reaction of 0.341 g (1 mmol) 3a, 0.190 mL (1.1 mmol)
benzhydrylamine and 0.696 mL (5 mmol) triethylamine at 78 ꢀC for
16 h, purification of the product by column chromatography (eluent
cyclohexane ⁄ ethyl acetate 4:1) and trituration with ether was
obtained 0.284 g (70%) of 4f; mp 122–123 ꢀC; IR (KBr, m ⁄ cm)1
)
3297, 3087, 3064, 3025, 2979, 1667, 1641, 1601, 1586, 1529, 1494,
1452, 757, 741, 718, 703; 1H NMR (300 MHz, DMSO) d 8.86 (d,
J = 8.45, 1H), 7.36–7.04 (m, 19H), 6.07 (d, J = 8.45, 1H), 3.90 (s,
2H), 3.80 (q, J = 6.94, 1H), 1.32 (d, J = 7.00, 3H); 13C NMR
(75.5 MHz, DMSO) d 172.97, 142.95, 141.67, 141.40, 142.86,
142.74, 129.09, 128.85, 128.72, 128.23, 127.28, 126.40, 125.38,
128.68, 127.85, 127.55, 127.41, 127.40, 56.28, 45.06, 41.65, 18.86;
MS ⁄ MS m ⁄ z 406.3 (M + H)+, 428.2, (M + Na)+; Elemental analysis:
calcd for C29H27NO: C, 85.89; H, 6.71; N, 3.45. Found: C, 85.69; H,
7.00; N, 3.76.
2-(3-Benzoylphenyl)-N-cyclopentylpropanamide
(5a)
From the reaction of 0.355 g (1 mmol) 3b, 0.109 mL (1.1 mmol)
cyclopentylamine and 0.696 mL (5 mmol) triethylamine at room
temperature for 1.5 h and trituration with ether was obtained
0.257 g (80%) of 5a; mp 94–95 ꢀC; IR (KBr, m ⁄ cm)1) 3355, 3066,
2964, 2870, 1663, 1648, 1596, 1528, 1449, 1312, 1284, 723, 702,
1
642; H NMR (300 MHz, DMSO) d 7.99 (d, J = 7.01, 1H), 7.72–7.45
(m, 9H), 3.99–3.87 (m, 1H), 3.66 (q, J = 6.73, 1H), 1.83–1.17 (m, 8H),
1.32 (d, J = 7.00, 3H); 13C NMR (75.5 MHz, DMSO) d 196.23, 172.71,
143.34, 137.53, 137.28, 133.14, 131.99, 130.04, 129.03, 128.97, 128.88,
128.41, 50.73, 45.08, 32.68, 23.94, 23.90, 18.95; MS⁄MS m⁄z 322.3
(M + H)+, 344.2, (M + Na)+; Elemental analysis: calcd for C21H23NO2:
C, 78.47; H, 7.21; N, 4.36. Found: C, 78.60; H, 7.56; N, 4.68.
2-(3-Benzylphenyl)-N-methoxypropanamide (4g)
From the reaction of 0.341 g (1 mmol) 3a, 0.092 g O-methylhydro-
xylamine hydrochloride and 0.696 mL (5 mmol) triethylamine at
room temperature for 48 h and purification of the product by
column chromatography (eluent dichloromethane ⁄ methanol 97:3)
was obtained 0.158 g (59%) of 4g; mp 61–64 ꢀC; IR (KBr, m ⁄ cm)1
)
3153, 3083, 2971, 938, 2854, 1661, 1640, 493, 1069, 1054, 720,
706; H NMR (300 MHz, DMSO) d 11.19 (s, 1H), 7.30)7.07 (m, 9H),
2-(3-Benzoylphenyl)-N-(cyclohexanemethyl)propa-
namide (5c)
1
3.91 (s, 2H), 3.38)3.32 (m, 4H), 1.30 (d, J = 7.09, 3H); 13C NMR
(75.5 MHz, DMSO) d 170.48, 141.99, 141.67, 141.62, 129.13,
128.87, 128.00, 127.57, 126.42, 125.30, 63.48, 42.52, 41.59, 18.65;
MS ⁄ MS m ⁄ z 270.2 (M + H)+, 292.2, (M + Na)+; Elemental analysis:
calcd for C17H19NO2: C, 75.81; H, 7.11; N, 5.20. Found: C, 75.50; H,
7.49; N, 5.53.
From the reaction of 0.355 g (1 mmol) 3b, 0.143 mL (1.1 mmol)
cyclohexanemethylamine and 0.696 mL (5 mmol) triethylamine at
room temperature for 2 h and trituration with ether was obtained
0.315 g (90%) of 5c; mp 96–98 ꢀC; IR (KBr, m ⁄ cm)1) 3298, 3094,
2919, 2853, 1676, 1660, 1642, 1597, 1563, 1447, 1289, 1271, 1234,
1
729, 707, 640; H NMR (300 MHz, DMSO) d 7.98 (t, J = 7.98, 1H),
Chem Biol Drug Des 2010; 75: 641–652
643

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Rajic et al.
7.74–7.47 (m, 9H), 3.72 (q, J = 6.99, 1H), 2.87 (dd, J = 6.18,
J = 4.38, 2H), 1.67–1.51 (m, 6H), 1.41–0.73 (m, 4H), 1.35 (d,
J = 7.00, 3H); 13C NMR (75.5 MHz, DMSO) d 196.22, 173.24,
143.41, 137.53, 137.30, 133.17, 132.00, 130.04, 129.03, 128.92,
128.44, 45.30, 45.26, 37.89, 30.78, 30.75, 26.46, 25.84, 18.92;
MS ⁄ MS m ⁄ z 350.3 (M + H)+, 372.3, (M + Na)+; Elemental analysis:
calcd for C23H27NO2: C, 79.05; H, 7.79; N, 4.01. Found: C, 79.49; H,
8.01; N, 4.13.
Ketoprofen derivatives 4j–l. General procedure
solution of 0.341 g (1 mmol) 3a, the appropriate amine
(1.1 mmol) and 0.139 mL (1 mmol) triethylamine in toluene ⁄ ethyl
acetate or toluene ⁄ acetonitrile (2:1, 15 mL) was stirred at room
temperature for 0.25 or 5 h. After evaporation of the solvent and
column chromatography, the crude product was obtained.
A
2-(3-Benzylphenyl)-N-(2-hydroxyethyl)propan-
amide (4j)
N-Benzyl-2-(3-benzoylphenyl)propanamide (5d)
From the reaction of 0.355 g (1 mmol) 3b, 0.120 mL (1.1 mmol)
benzylamine and 0.696 mL (5 mmol) triethylamine at room tempera-
ture for 1 h and trituration with ether was obtained 0.285 g (83%)
of 5d; mp 97–99 ꢀC; IR (KBr, m ⁄ cm)1) 3314, 3287, 3066, 3033,
2926, 2870, 1644, 1551, 1449, 1349, 1015, 712, 701; 1H NMR
(300 MHz, DMSO) d 8.56 (t, J = 5.75, 1H), 7.76–7.49 (m, 9H), 7.29–
7.14 (m, 5H), 4.25 (d, J = 5.84, 2H), 3.80 (q, J = 7.03, 3H), 1,40 (d,
J = 7.03, 3H); 13C NMR (75.5 MHz, DMSO) d 196.22, 173.37,
143.15, 137.51, 137.38, 139.86, 133.17, 132.08, 130.07, 129.04,
129.02, 128.58, 128.70, 127.50, 127.21, 45.36, 42.54, 18.91;
MS ⁄ MS m ⁄ z 344.2 (M + H)+, 366.2, (M + Na)+; Elemental analysis:
calcd for C23H21NO2: C, 80.44; H, 6.16; N, 4.08. Found: C, 80.67; H,
6.30; N, 4.29. Synthesis of the compound 5d was reported previ-
ously (23), but no spectroscopic data were given.
From the reaction of 0.341 g (1 mmol) 3a, 0.066 mL (1.1 mmol)
2-hydroxyethylamine and 0.139 mL (1 mmol) triethylamine in
toluene ⁄ ethyl acetate for 0.25 h and purification of the product by
column chromatography (eluent ethyl acetate) was obtained 0.238 g
(84%) of 4j; oil; IR (film, m ⁄ cm)1) 3414, 3312, 3084, 3027, 2972,
2931, 1648, 1601, 1560, 1540, 1494, 1453, 1363, 1072, 723, 700;
1H NMR (300 MHz, DMSO) d 7.92 (t, J = 5.24, 1H), 7.31–7.04 (m,
9H), 4.64 (t, J = 5.36, 1H), 3.91 (s, 2H), 3.57 (q, J = 7.03, 1H), 3.39–
3.33 (m, 2H), 3.17–3.01 (m, 2H), 1.28 (d, J = 7.03, 3H); 13C NMR
(75.5 MHz, DMSO) d 173.81, 143.11, 141.67, 141.47, 129.13,
128.86, 128.71, 128.14, 127.27, 126.41, 125.37, 60.30, 45.40, 41.98,
41.65, 19.21; MS ⁄ MS m ⁄ z 284.2 (M + H)+, 306.2, (M + Na)+; Ele-
mental analysis: calcd for C18H21NO2: C, 76.29; H, 7.47; N, 4.94.
Found: C, 76.62; H, 7.40; N, 4.82.
2-(3-Benzylphenyl)-N-(3-hydroxypropyl)propan-
amide (4k)
2-(3-Benzoylphenyl)-N-phenethylpropanamide (5e)
From the reaction of 0.355 g (1 mmol) 3b, 0.139 mL (1.1 mmol)
phenylethylamine and 0.696 mL (5 mmol) triethylamine at room tem-
perature for 1 h and trituration with ether ⁄ petrolether was obtained
0.329 g (92%) of 5e; mp 66–69 ꢀC; IR (KBr, m ⁄ cm)1) 3255, 3062,
2936, 2876, 1661, 1634, 1599, 1559, 1543, 1448, 1288, 748, 702;
1H NMR (300 MHz, DMSO) d 8.11 (t, J = 5.45, 1H), 7.75–7.48 (m,
9H), 7.24–7.07 (m, 5H), 3.69 (q, J = 6.94, 1H), 3.32–3.18 (m, 2H),
2.66 (t, J = 7.13, 2H), 1.34 (d, J = 7.02, 3H); 13C NMR (75.5 MHz,
DMSO) d 196.22, 173.22, 143.19, 137.54, 137.32, 139.85, 133.16,
132.09, 130.08, 129.04, 128.99, 128.97, 128.52, 129.10, 128.64,
126.44, 45.33, 40.71, 35.40, 18.91; MS ⁄ MS m ⁄ z 358.2 (M + H)+,
380.2, (M + Na)+; Elemental analysis: calcd for C24H23NO2: C, 80.64;
H, 6.49; N, 3.92. Found: C, 80.90; H, 6.70; N, 4.04.
From the reaction of 0.341 g (1 mmol) 3a, 0.084 mL (1.1 mmol)
3-hydroxypropylamine and 0.139 mL (1 mmol) triethylamine in
toluene ⁄ ethyl acetate for 0.25 h and purification of the product by
column chromatography (eluent ethyl acetate) was obtained 0.229 g
(77%) of 4k; oil; IR (film, m ⁄ cm)1) 3301, 3062, 3027, 2970, 2935,
2877, 1652, 1601, 1558, 1548, 1494, 1453, 721, 699; 1H NMR
(300 MHz, DMSO) d 7.89 (t, J = 5.29, 1H), 7.31–7.04 (m, 9H), 4.38
(t, 1H), 3.91 (s, 2H), 3.53 (q, J = 7.00, 1H), 3.34 (q, J = 6.19, 2H),
3.06 (q, J = 6.51, 2H), 1.54–1.45 (m, 2H), 1.28 (d, J = 7.04, 3H); 13C
NMR (75.5 MHz, DMSO) d 173.64, 143.02, 141.66, 141.47, 129.12,
128.86, 128.72, 128.08, 127.28, 126.40, 125.30, 58.80, 45.50, 41.65,
36.20, 32.83, 19.07; MS ⁄ MS m ⁄ z 298.3 (M + H)+, 320.3, (M + Na)+;
Elemental analysis: calcd for C19H23NO2: C, 76.73; H, 7.80; N, 4.71.
Found: C, 76.80; H, 7.66; N, 4.70.
N-Benzhydryl-2-(3-benzoylphenyl)propanamide
(5f)
2-(3-Benzylphenyl)-N,N-bis(2-hydroxyethyl)pro-
panamide (4l)
From the reaction of 0.355 g (1 mmol) 3b, 0.190 mL (1.1 mmol)
benzhydrylamine and 0.696 mL (5 mmol) triethylamine at 78 ꢀC for
11 h and trituration with ether was obtained 0.306 g (73%) of 5f;
mp 115–119 ꢀC; IR (KBr, m ⁄ cm)1) 3316, 3251, 3057, 3030, 2932,
From the reaction of 0.341 g (1 mmol) 3a, 0.116 mL (1.1 mmol)
diethanolamine and 0.139 mL (1 mmol) triethylamine in tolu-
ene ⁄ acetonitrile for 5 h and purification of the product by column
chromatography (eluent ethyl acetate fi ethyl acetate ⁄ methanol
8:1) was obtained 0.249 g (76%) of 4l; oil; IR (film, m ⁄ cm)1) 3370,
3060, 3026, 2931, 1622, 1494, 1473, 1454, 1442, 1061, 745, 699;
1H NMR (300 MHz, DMSO) d 7.31–7.04 (m, 9H), 4.82 (s, 1H), 4.46
(s, 1H), 4.10 (q, J = 6.74, 1H), 3.91 (s, 2H), 3.56–3.10 (m, 8H), 1.24
(d, J = 6.77, 3H); 13C NMR (75.5 MHz, DMSO) d 173.73, 143.10,
141.89, 141.63, 129.09, 128.86, 128.00, 127.32, 126.40, 125.40,
59.73, 59.07, 50.64, 48.93, 41.64, 41.60, 21.26; MS ⁄ MS m ⁄ z 328.3
(M + H)+, 350.3, (M + Na)+; Elemental analysis: calcd for
1
1656, 1649, 1598, 1528, 1494, 1320, 1285, 723, 704, 696; H NMR
(300 MHz, DMSO) d 8.98 (d, J = 8.46, 1H), 7.78–7.48 (m, 9H), 7.37–
7.08 (m, 10H), 6.08 (d, J = 8.40, 1H), 3.96 (q, J = 6.99, 1H), 1.39 (d,
J = 7.01, 3H); 13C NMR (75.5 MHz, DMSO) d 196.23, 172.64,
143.01, 137.51, 137.35, 142.80, 142.77, 133.16, 132.07, 130.05,
129.03, 128.98, 128.53, 128.88, 128.68, 127.86, 127.45, 127.33,
56.36, 44.98, 18.81; MS ⁄ MS m ⁄ z 420.2 (M + H)+, 442.2, (M + Na)+;
Elemental analysis: calcd for C29H25NO2: C, 83.03; H, 6.01; N, 3.34.
Found: C, 83.30; H, 6.09; N, 3.58.
644
Chem Biol Drug Des 2010; 75: 641–652

Biological Evaluation of Ketoprofen Amides
C20H25NO3: C, 73.37; H, 7.70; N, 4.28. Found: C, 73.44; H, 7.88; N,
4.20.
N-Cyclohexanemethyl-2-(3-(hydroxy(phenyl)methyl)
phenyl)propanamide (6c)
From the reaction of 0.175 g (0.5 mmol) 5c for 7 h and purification
by column chromatography (eluent chloroform ⁄ methanol 95:5) was
obtained 0.141 g (80%) of 6c; oil; IR (film, m ⁄ cm)1) 3312, 3063,
2-(3-Benzylphenyl)propanamide (4n)
1
A suspension of 0.359 g (0.5 mmol) 5g and 0.050 g Pd ⁄ C in metha-
nol (10 mL) was hydrogenated at atmospheric pressure and room
temperature for 17 h. After filtration of the catalyst and evaporation
of the solvent, 0.239 g (100%) of 4n was obtained; mp 76–78 ꢀC;
IR (KBr, m ⁄ cm)1) 3393, 3177, 3084, 3026, 2980, 2926, 1653, 1599,
1451, 1406, 1368, 1288, 1123, 786, 710, 694, 646, 628; 1H NMR
(300 MHz, DMSO) d 7.41)7.04 (m, 9H), 6.78 (s, 2H), 3.91 (s, 2H),
3.53 (q, J = 7.01, 1H), 1.28 (d, J = 7.06, 3H); 13C NMR (75.5 MHz,
DMSO) d 175.69, 143.02, 141.68, 141.46, 129.12, 128.87, 128.15,
127.30, 126.41, 125.41, 45.33, 41.65, 18.97; MS ⁄ MS m ⁄ z 240.2
(M + H)+, 262.2, (M + Na)+; Elemental analysis: calcd for C16H17NO:
C, 80.30; H, 7.16; N, 5.85. Found: C, 80.67; H, 7.08; N, 5.60.
2925, 2852, 1649, 1604, 1550, 1493, 1450, 1025, 731, 701; H NMR
(300 MHz, DMSO) d 7.87 (t, J = 5.56, 1H), 7.37–7.14 (m, 9H), 5.86
(dd, J = 2.84, J = 1.05, 1H), 5.65 (d, J = 3.50, 1H), 3.58 (q,
J = 7.01, 1H), 2.85 (t, J = 6.26, 2H), 1.68–1.51 (m, 6H), 1.39–0.73
(m, 4H), 1.28 (d, J = 6.85, 3H); 13C NMR (75.5 MHz, DMSO) d
173.58, 146.15, 145.93, 142.71, 128.47, 128.28, 127.13, 126.69,
126.03, 125.62, 124.90, 74.82, 45.53, 45.28, 37.87, 30.83, 30.81,
26.49, 25.85, 19.13; MS ⁄ MS m ⁄ z 352.3 (M + H)+, 374.3, (M + Na)+;
Elemental analysis: calcd for C23H29NO2: C, 78.59; H, 8.32; N, 3.99.
Found: C, 78.67; H, 8.49; N, 3.70.
N-Benzyl-2-(3-(hydroxy(phenyl)methyl)phenyl)pro-
panamide (6d)
Ketoprofen derivatives 6a–f. General procedure
A suspension of the amide 5a–f (0.5 mmol) and 0.020 g 10%
Pd ⁄ C(en) in methanol (10 mL) was hydrogenated at atmospheric
pressure and room temperature for 5–9 h. After filtration of the
catalyst and evaporation of the solvent under the reduced pressure,
the crude product was obtained.
From the reaction of 0.172 g (0.5 mmol) 5d for 6 h and purification
by column chromatography (eluent chloroform ⁄ methanol 93:7) was
obtained 0.142 g (82%) of 6d; mp 100–103 ꢀC; IR (KBr, m ⁄ cm)1
)
3331, 3285, 1648, 1603, 1539, 1494, 1454, 1238, 1017, 730, 701;
1H NMR (300 MHz, DMSO) d 8.46 (t, J = 5.57, 1H), 7.41–7.15 (m,
14H), 5.88 (d, J = 3.21, 1H), 5.66 (s, 1H), 4.24 (d, J = 5.74, 2H),
3.66 (q, J = 6.97, 1H), 1.34 (d, J = 6.97, 3H); 13C NMR (75.5 MHz,
DMSO) d 173.71, 146.11, 146.02, 142.50, 139.96, 128.69, 127.53,
128.50, 128.38, 127.14, 126.73, 126.09, 125.67, 124.04, 74.83,
45.59, 42.51, 19.11; MS ⁄ MS m ⁄ z 368.2 (M + Na)+; Elemental analy-
sis: calcd for C23H23NO2: C, 79.97; H, 6.71; N, 4.05. Found: C,
80.08; H, 6.99; N, 3.92.
N-Cyclopentyl-2-(3-(hydroxy(phenyl)methyl)phen-
yl)propanamide (6a)
From the reaction of 0.161 g (0.5 mmol) 5a for 5 h and purification
by trituration with ether was obtained 0.126 (78%) of 6a; mp 120–
125 ꢀC; IR (KBr, m ⁄ cm)1) 3274, 3062, 3031, 2964, 2933, 2866, 1645,
1
1603, 1549, 1453, 1231, 1195, 1021, 730, 701; H NMR (300 MHz,
DMSO) d 7.86 (d, J = 6.92, 1H), 7.36–7.13 (m, 9H), 5.86 (d,
J = 3.83, 1H), 5.65 (d, J = 3.04, 1H), 3.98–3.87 (m, 1H), 3.54 (q,
J = 6.94, 1H), 1.81–1.18 (m, 8H), 1.27 (d, J = 6.99, 3H); 13C NMR
(75.5 MHz, DMSO) d 173.00, 146.16, 145.99, 142.76, 128.47,
128.29, 127.11, 126.69, 126.01, 125.56, 124.81, 74.84, 50.63, 45.29,
32.69, 32.65, 23.97, 23.93, 19.10; MS ⁄ MS m ⁄ z 324.3 (M + H)+,
346.2, (M + Na)+; Elemental analysis: calcd for C21H25NO2: C, 77.98;
H, 7.79; N, 4.33. Found: C, 78.08; H, 7.85; N, 4.69.
2-(3-(Hydroxy(phenyl)methyl)phenyl)-N-phenethyl-
propanamide (6e)
From the reaction of 0.179 g (0.5 mmol) 5e for 6 h and purification
by column chromatography (eluent chloroform ⁄ methanol 93:7) was
obtained 0.131 g (73%) of 6e; oil; IR (film, m ⁄ cm)1) 3412, 3312,
3062, 3028, 2973, 2932, 1652, 1603, 1558, 1540, 1522, 1454, 1368,
1239, 1025, 750, 700; 1H NMR (300 MHz, DMSO) d 8.04 (t,
J = 5.47, 1H), 7.42–7.14 (m, 14H), 5.91 (dd, J = 3.25, J = 0.63, 1H),
5.70 (d, J = 3.75, 1H), 3.58 (q, J = 6.93, 1H), 3.26 (q, J = 7.22, 2H),
2.68 (t, J = 7.28, 2H), 1.32 (d, J = 6.89, 3H); 13C NMR (75.5 MHz,
DMSO) d 173.55, 146.16, 146.02, 142.55, 139.90, 129.10, 128.71,
126.45, 128.49, 128.34, 127.14, 126.73, 126.17, 125.63, 124.95,
74.83, 45.58, 40.74, 35.53, 19.12; MS ⁄ MS m ⁄ z 382.2 (M + Na)+;
Elemental analysis: calcd for C24H25NO2: C, 80.19; H, 7.01; N, 3.90.
Found: C, 79.96; H, 7.25; N, 4.02.
N-Cyclohexyl-2-(3-(hydroxy(phenyl)methyl)phenyl)
propanamide (6b)
From the reaction of 0.166 g (0.5 mmol) 5b for 6 h and purification
by column chromatography (eluent dichloromethane ⁄ methanol 93:7)
was obtained 0.127 g (75%) of 6b; mp 94–98 ꢀC; IR (KBr, m ⁄ cm)1
)
3366, 3308, 3058, 2973, 2924, 2855, 1641, 1603, 1539, 1449, 1015,
1
712, 701; H NMR (300 MHz, DMSO) d 7.74 (dd, J = 5.24, J = 2.39,
1H), 7.35–7.11 (m, 9H), 5.84 (d, J = 3.84, 1H), 5.64 (d, J = 3.57, 1H),
3.53 (q, J = 6.98, 1H), 3.48–3.38 (m, 1H), 1.72–1.46 (m, 6H), 1.27–
0.92 (m, 4H), 1.25 (d, J = 6.56, 3H); 13C NMR (75.5 MHz, DMSO) d
172.59, 146.16, 145.98, 142.82, 128.45, 128.28, 127.11, 126.71,
126.02, 125.55, 124.81, 74.84, 47.83, 45.34, 32.83, 32.80, 25.70,
25.05, 24.98, 19.10; MS ⁄ MS m ⁄ z 338.3 (M + H)+, 360.3, (M + Na)+;
Elemental analysis: calcd for C22H27NO2: C, 78.30; H, 8.06; N, 4.15.
Found: C, 78.55; H, 8.44; N, 4.36.
N-Benzhydryl-2-(3-(hydroxy(phenyl)methyl)phenyl)
propanamide (6f)
From the reaction of 0.210 g (0.5 mmol) 5f for 9 h and crystallization
from acetone ⁄ petrolether was obtained 0.190 g (73%) of 6f; mp 51–
54 ꢀC; IR (KBr, m ⁄ cm)1) 3410, 3304, 3062, 3029, 2975, 2930, 1649,
1
1602, 1531, 1494, 1451, 1028, 743, 699; H NMR (300 MHz, DMSO)
d 8.88 (d, J = 8.39, 1H), 7.44–7.10 (m, 19H), 6.06 (d, J = 8.38, 1H),
5.87 (d, J = 3.86, 1H), 5.65 (d, J = 3.19, 1H), 3.82 (q, J = 6.78, 1H),
Chem Biol Drug Des 2010; 75: 641–652
645

´
Rajic et al.
1.32 (d, J = 6.20, 3H); 13C NMR (75.5 MHz, DMSO) d 173.00, 146.13,
145.97, 142.82, 143.01, 142.36, 128.85, 128.70, 127.81, 127.58,
127.55, 127.41, 127.26, 128.49, 128.33, 127.13, 126.73, 126.05,
125.76, 125.04, 74.83, 56.30, 45.14, 18.95; MS ⁄ MS m ⁄ z 422.2
(M + H)+, 444.3, (M + Na)+; Elemental analysis: calcd for C29H27NO2:
C, 82.63; H, 6.46; N, 3.32. Found: C, 82.88; H, 6.69; N, 3.01.
ette containing phosphate buffer pH 7.4 (0.93 mL, 0.05 M) at room
temperature. The oxidation reaction was initiated under air by the
addition of AAPH solution (50 lL, 40 mM). Oxidation was carried
out in the presence of compounds (10 lL, 0.1 mM). In the assay
with no antioxidant, lipid oxidation was measured in the presence
of the same level of DMSO. The rate of oxidation was monitored
at 37 ꢀC by recording the increase of absorption at 234 nm caused
by conjugated diene hydroperoxides. The results were compared to
the standard inhibitor Trolox.
N-Hydroxy-2-(3-(hydroxy(phenyl)methyl)phenyl)
propanamide (6g)
A suspension of 0.180 g (0.5 mmol) 5g and 0.020 g Pd ⁄ C in metha-
nol (10 mL) was hydrogenated at atmospheric pressure and room
temperature for 3 h. After filtration of the catalyst, evaporation of
the solvent, and crystallization from cyclohexane ⁄ ethyl acetate
Calculation of lipophilicity
Lipophilicity was theoretically calculated as Clog P values in n-octa-
nol-buffer by CLOGP Programme of Biobyte Corp.a
0.099 g (90%) of 6g was obtained; mp 148–152 ꢀC; IR (KBr, m ⁄ cm)1
)
3378, 3214, 2979, 2915, 1645, 1520, 1492, 1447, 1022, 945, 724,
701; 1H NMR (300 MHz, DMSO) d 10.62 (s, 1H), 8.77 (s, 1H),
7.38)7.16 (m, 9H), 5.87 (d, J = 3.90, 1H), 5.66 (d, J = 3.60, 1H), 3.41
(q, J = 7.00, 1H), 1.31 (d, J = 7.00, 3H); 13C NMR (75.5 MHz, DMSO)
d 170.65, 146.12, 146.02, 142.09, 128.52, 128.33, 127.16, 126.73,
126.13, 125.64, 125.11, 74.80, 42.62, 18.77; MS ⁄ MS m ⁄ z 294.1
(M + Na)+; Elemental analysis: calcd for C16H17NO3: C, 70.83; H,
6.32; N, 5.16. Found: C, 70.64; H, 6.06; N, 5.39.
Cytostatic activity
The H 460 (lung carcinoma), MCF-7 (breast carcinoma), HCT 116
and SW620 (colon carcinoma) and MOLT-4 (acute lymphoblastic leu-
kemia) cells (obtained from American Type Culture Collection (ATCC,
Rockville, MD, USA). MCF-7, SW620, HCT 116 and H 460 cells were
cultured as monolayers and maintained in Dulbecco's modified
Eagle medium (DMEM, Gibco, Carlsbad, CA, USA), while MOLT-4
cells were cultured in suspension in RPMI medium, both supple-
mented with 10% fetal bovine serum (FBS, Gibco), 2 mM L-gluta-
mine, 100 U ⁄ mL penicillin and 100 lg ⁄ mL streptomycin (Gibco) in a
humidified atmosphere with 5% CO2 at 37 ꢀC. The growth inhibition
activity was assessed as described previously, according to the
slightly modified procedure of the National Cancer Institute, Devel-
opmental Therapeutics Program (14,16). Briefly, the cells were cul-
tured as monolayers and maintained in DMEM supplemented with
10% FBS, 2 mM L-glutamine, 100 U ⁄ mL penicillin and 100 lg ⁄ mL
streptomycin in a humidified atmosphere with 5% CO2 at 37 ꢀC.
The cells were inoculated onto a series of standard 96-well micro-
titer plates on day 0, at 1 · 104 to 3 · 104 cells ⁄ mL, depending on
the doubling times of specific cell line. Test agents were then
added in consecutive 10-fold dilutions (10)8–10)4 M) and incubated
for a further 72 h. Working dilutions were freshly prepared on the
day of testing. The cell growth rate was evaluated by performing
Biological evaluation
1,1-Diphenyl-2-picrylhydrazyl (DPPH), 2,2¢-azobis(2-amidinopropane)
dihydrochloride (AAPH), nordihydroguaiaretic acid (NDGA), sodium
linoleate, soybean lipoxygenase, caffeic acid and Trolox were
purchased from Sigma-Aldrich.
Interaction with DPPH (24)
To a solution of DPPH (0.05 mM) in absolute ethanol an equal volume
of ethanolic solution of the tested compound (final concentrations
0.05 or 0.1 mM) was added. After 20 and 60 min, the absorbance
was recorded at 517 nm and compared with the appropriate standard
NDGA (Table 1). Ethanol was used as a control. Each experiment in
vitro was performed at least in triplicate, and the standard deviation
of absorbance was less than 10% of the mean.
the
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide
(MTT) assay (Sigma, Taufkirchen, Germany) after 72 h of incubation,
which detects mitochondrial dehydrogenase activity in viable cells.
Soybean lipoxygenase inhibition
Dimethyl sulphoxide solution of the tested compound was incubated
with sodium linoleate (0.1 mM) and soybean lipoxygenase solution
(0.2 mL, 1000 U ⁄ mL in saline) at room temperature. The inhibitory
activity of the compounds was measured at the concentration range
10–1000 lM and IC50 values were determined. The conversion of
sodium linoleate to 13-hydroperoxylinoleic acid was recorded at
234 nm and compared with the standard inhibitor caffeic acid
(Table 1), according to the procedure previously reported (23).
Each test was performed in quadruplicate in three individual experi-
ments. The results are expressed as IC50, which is the concentration
necessary for 50% of inhibition. The IC50 values for each compound
are calculated from concentration–response curves using linear
regression analysis by fitting the test concentrations that give per-
centage of growth (PG) values above and below the reference value
(i.e., 50%). If however, for a given cell line all of the tested concentra-
tions produce PGs exceeding the respective reference level of effect
(e.g., PG value of 50), then the highest tested concentration is
assigned as the default value, which is preceded by a '>' sign.
Inhibition of linoleic acid lipid peroxidation (25)
Oxidation of linoleic acid to conjugated diene hydroperoxide in an
aqueous dispersion was monitored at 234 nm. 2,2¢-Azobis(2-amidi-
nopropane) dihydrochloride was used as a free radical initiator.
Linoleic acid dispersion (10 lL, 16 mM) was added to the UV cuv-
Clonogenic assay
Cell survival after treatment with the compound 4i was deter-
mined by clonogenic assay. Briefly, HCT 116 cells were plated at
646
Chem Biol Drug Des 2010; 75: 641–652

Biological Evaluation of Ketoprofen Amides
Table 1: Interaction with DPPH,
in vitro inhibition of soybean lipoxy-
genase and inhibition of lipid
peroxidation by ketoprofen deriva-
tives 4–6
DPPH
DPPH
60 mina
(%)
LOX LP
20 mina
IC 50 inhib.a
(µM) (%)
Clog P
Compound
(%)
CH
CH
3
H
N
4a
28.5
27.6
29.5
33.9
82
63.2
4.54
O
3
H
N
4b
4c
30.9
31.4
19.9
32.9
37.5 80.4
5.10
5.72
4.63
5.01
O
CH
3
H
N
80
87.9
O
CH 3
4d
H
N
26.6
21.7
74.5 97.0
O
CH
CH
3
H
N
4e
4f
43
50
99.9
98.8
O
3
H
N
34.0
28.5
5.98
O
CH
CH
CH
3
H
N
4g
4h
4i
CH
3
O
O
O
22.8
27.6
29.4
33.0
32.2
32.7
26.1
28.6
29.3
22.5
27.7
23.5
n.d.b n.a.c
3.04
3.57
4.98
2.61
2.14
2.83
O
3
H
N
CH
3
385
100
270
280
400
n.a.
98.7
66.7
32.6
22.8
O
3
H
N
O
3
CH
CH
H
N
4j
OH
O
3
H
N
4k
4l
OH
O
3
CH
CH
OH
N
OH
O
3
4n
NH 2
30.0
38.1
36.1
29.4
24.1
33.8
31.6
35.4
325
23.6
2.83
3.51
4.07
4.69
O
CH 3
O
H
N
5a
61.5 55.8
O
O
CH 3
H
N
5b
120
53
69.7
92.2
O
CH 3
O
5c
H
N
O
CH 3
O
5d
H
N
36.8
35.2
31.2
30.8
87.5 99.0
3.61
3.98
O
CH
O
3
H
N
5e
65
92.2
O
Chem Biol Drug Des 2010; 75: 641–652
647

´
Rajic et al.
Table 1: Continued
DPPH
DPPH
60 mina
(%)
LOX LP
20 mina
IC 50 inhib.a
(µM) (%)
Clog P
Compound
(%)
O
CH
3
5f
H
N
38.5
32.5
64.5 98.6
4.95
O
CH
OH
3
H
N
6a
1.9
2.9
2.9
2.7
2.6
3.4
230
425
295
23.6
43.0
28.1
2.78
3.34
3.96
O
CH 3
OH
H
N
6b
O
CH
CH
OH
3
6c
H
N
O
OH
3
6d
H
N
3.9
1.2
2.6
5.4
200
120
31.2
35.1
2.88
3.26
O
CH 3
OH
H
N
6e
O
CH
CH
OH
3
6f
H
N
3.8
2.1
20.5 n.a.
4.22
O
OH
3
H
N
6g
OH
26.6
6.9
43.0
4.8
200
415
44.3
31.1
51.8
1.56
3.79
O
CH
CH
3
2a
OH
OH
O
OH
3
2b
n.a.
8.1
5.5
7.2
200
130
2.03
2.76
O
Ketoprofen
69.3
63
Trolox
Caffeic acid
600
DPPH, 1,1-diphenyl-picrylhydrazyl; LOX, soybean lipoxygenase; LP, lipid peroxidation.
aConcentration: 1 · 10)4 M.
bn.d. – IC50 value could not be determined: percent of inhibition was 12% and 27.5% at concentrations 100 and
500 lM, respectively.
cn.a. – No activity, under the reported experimental conditions.
3000 cell ⁄ 100-mm dish and 18 h after seeding cells were treated
with 4i at the concentration of 10 lM (ꢀIC50) and 50 lM. The
cultures were washed after the desired length of time (6 or
24 h), fed with complete medium, and 1–2 weeks later surviving
colonies were fixed with ice-cold methanol, stained with 0.1%
w ⁄ v crystal violet and counted. Survival was calculated as the
ratio of the number of colonies from treated versus untreated
samples.
Results and Discussion
Chemistry
Ketoprofen was the starting compound in the synthetic pathway
leading to reduced analogs 2a,b and their novel amides
(Scheme 1). Compounds 2a,b were synthesized by the catalytic
hydrogenation of ketoprofen under different reaction conditions. Fur-
ther on, 2a and ketoprofen in the reaction with 1-benzotriazolide
648
Chem Biol Drug Des 2010; 75: 641–652

Biological Evaluation of Ketoprofen Amides
carboxylic acid chloride (1) gave the corresponding NSAID ben-
zotriazolides 3a,b (17), which were transformed to amides 4a–l and
5a–g. In general, the amidation was performed with 1.1 equivalent
of the appropriate amine in the presence of five equivalents of trieth-
ylamine in toluene. Triethylamine formed a water-soluble salt with
benzotriazole, a by-product of the reaction, which was readily
extracted with water. All amides 4 and 5 were prepared at room
temperature except compounds 4f, 4i and 5f: the reactions with
benzhydrylamine and O-benzylhydroxylamine were too slow at room
temperature, probably because of the steric hindrance of the first and
lower nucleophilicity of the second amine. Reactions with hydrophilic
bifunctional amines (2-hydroxyethylamine, 3-hydroxypropylamine,
diethanolamine) were performed in solvent mixtures toluene ⁄ ethyl
acetate or toluene ⁄ acetonitrile with 1.1 equivalent of the amine and
one equivalent of triethylamine. It was established that under given
reaction conditions hydroxy groups of the amines were not reactive.
On the other hand, hydrogenolysis of the N-benzyloxy-2-(3-benzoyl-
phenyl)propanamide (5g) with 10% Pd ⁄ C(en) was too slow
(Scheme 2). When 10% Pd ⁄ C was used, the reaction rate was sat-
isfactory and the product 6g was obtained in 90% yield. The
amount of the catalyst determined the selectivity of the reaction: a
higher amount of the catalyst led first to hydroxamic acid 4m (16),
and finally to amide 4n.
Structures of the compounds 4a–n, 5a–g and 6a–g were
1
deduced from the analysis of their IR, H and 13C NMR spectra and
confirmed by elemental analysis. The chemical shifts were consis-
tent with the proposed structures of the novel compounds.
Biological studies
Antioxidant activity
Derivatives 6a–f were synthesized by the catalytic hydrogenation
of the compounds 5a–f. Selective reduction of keto to hydroxy
group was achieved using heterogeneous Pd-ethylenediamine
[Pd ⁄ C(en)] complex catalyst (25). Completely reduced products 4a–f
were obtained as by-products in small amounts only.
The interaction of the examined compounds with the stable free
radical DPPH was studied. Interaction with DPPH indicates radical
scavenging ability in an iron-free system. Interactions were moni-
tored after 20 and 60 min at two concentrations of DPPH (0.05 and
0.1 mM). Ketoprofen, the prototype compound, as well as the
CH3
CH3
CH3 R1
N
OH
Bt
1
2
R2
H /Pd/C
2
NHR R , TEA
toluene
BtCOCl, TEA
MeOH
toluene
O
O
O
2a
3a
4a-l
OH
O
CH3
O
CH3
OH
Bt
OH
H /Pd/C(en)
2
THF
O
O
2b
3b
Ket
N
CH3
O
CH3
OH
CH3
O
N
N
H
N
H
N
R
NH R, TEA
2
R
H /Pd/C(en)
2
BtCOCl, TEA
toluene
toluene
MeOH
O
O
Bt
5a-f
6a-f
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
R1
R2
O
CH
3
OH
O
O
CH
3
OH
OH
H
H
H
H
H
H
H
H
H
H
H
OH
5a, 6a
5b, 6b
5c, 6c
5d, 6d
5e, 6e
5f, 6f
R
Scheme 1: Synthesis of ketoprofen derivatives 4a–l, 5a–f and 6a–f.
O
CH3
OH
CH3
H
H
N
N
H /Pd/C
2
O
OH
H /Pd/C
2
MeOH
MeOH
O
O
5g
6g
CH3
CH3
H
N
NH2
H /Pd/C
2
H /Pd/C
2
OH
MeOH
MeOH
O
O
Scheme 2: Hydrogenation of
5g.
4m
4n
Chem Biol Drug Des 2010; 75: 641–652
649

´
Rajic et al.
reduced derivatives 2a and 2b presented very low interaction val-
ues. In general, it seemed that all the tested compounds interacted
with DPPH in a concentration-dependent manner. No significant
changes were observed with time. Compounds 5f, 5a, 5d and 5e
were the most active in the 0.1 mM assay. Lipophilicity does not
seem to influence their interaction with DPPH. The non-reduced ke-
toprofen amides were more potent than the reduced ones (e.g.,
5a > 4a, 5b > 4b, 5d > 4d). The lowest antioxidant activity
showed the hydroxy bearing derivatives 6. The results are pre-
sented in Table 1.
In the series 5, aromatic amides were also more active than the
cycloalkyl ones.
Using the C-QSAR program, an analysis was performed to discover
whether any correlation between antioxidant activity and several
physicochemical parameters (lipophilicity, steric and electronic vari-
ables) existed. For the inhibition of lipid peroxidation, the following
equation was derived:
log LP% ¼ 0:153ð0:0550ÞClog P þ 0:217ð0:130ÞIco þ 1:086ð0:225Þ
ð1Þ
Soybean lipoxygenase inhibition
N = 24, r = 0.829, r2 = 0.688, q2 = 0.594, s = 0.14, F2,21 = 26.43,
a = 0.01
Compounds were further evaluated for inhibition of soybean lipox-
ygenase (LOX) by the UV absorbance-based enzyme assay (26).
Lipoxygenases oxidize certain fatty acids at specific positions to
hydroperoxides, precursors of leukotrienes, which contain a conju-
gated triene structure, i.e., soybean lipoxygenase converts linoleic
to 13-hydroperoxylinoleic acid. Leukotrienes play a role as media-
tors of a variety of inflammatory and allergic processes. Lipoxy-
genases are involved in membrane lipid peroxidation by forming
hydroperoxides in the lipid bilayer (27). Inhibitors of LOX have
attracted attention initially as potential agents for the treatment
of inflammatory and allergic diseases, but their therapeutic poten-
tial has now been expanded to certain types of cancer and car-
diovascular diseases (28). Most of the LOX inhibitors are
antioxidants or free radical scavengers, as lipoxygenation occurs
via a carbon-centered radical (29). Perusal of IC50 values shows
that compound 6f is the most active by far, followed by com-
pounds 4b, 4e, 4f, 5c, 5a, 5e ꢀ 5f, 4d, 4c and 4a
(IC50 = 20.5–82 lM). From Table 1, it is obvious that aromatic and
cycloalkyl amides 4 and 5 are more potent lipoxygenase inhibitors
(IC50 = 37.5–120 lM) than derivatives with carboxylic group (IC50
of ketoprofen, 2b, and 2a are 130, 200 and 415 lM, respec-
tively). Lipophilicity is referred as an important physicochemical
parameter for lipoxygenase inhibition (30,31). However, in this
data set lipophilicity does not seem to affect absolutely the LOX
inhibition, as the most potent compound 6f does not possess the
highest Clog P value within the set.
Lipophilicity is the most significant parameter followed by the indi-
cator variable Ico (taking the value 1 for the compounds having the
characteristic carbonyl group of ketoprofen and the value 0 for the
congeners without this group). According to the leave-one-out pro-
cedure of the program compounds 4j and 6g were not included in
the regression analysis. We may speculate that these compounds
operate by a different biochemical mechanism from the rest. Further
investigation is in progress to delineate the physicochemical proper-
ties implicated in the lipid peroxidation inhibition.
Furthermore, the results indicate that the LOX inhibition in vitro is
correlated with the lipid peroxidation inhibition (r2 = 0.672).
Lipoxygenases play a role in membrane lipid peroxidation by
forming hydroperoxides in the lipid bilayer. Under our experimental
conditions, both activities are correlated according to the following
equation:
log 1=IC50LOX ¼ 1:182ð0:348ÞLP% þ 1:825ð0:612Þ
ð2Þ
N = 26, r = 0.820, r2 = 0.672, q2 = 0.627, s = 0.194, F1,24 = 53.15,
a = 0.01
Cytostatic activity
Compounds 4b,c,e,f,i, 5c,e,f and 6c,f–g were screened for
their potential antiproliferative (cytostatic) effects on a panel of
five human cell lines, which were derived from different cancer
types including HCT 116 and SW620 (colon carcinoma), MCF-7
(breast carcinoma), MOLT-4 (acute lymphoblastic leukemia), and H
460 (lung carcinoma). The tested compounds showed very similar
antiproliferative effect on the presented panel cell lines (Table 2),
except 6g, which was the least active, coinciding again with its
significantly lower lipophilicity, as discussed in our previous paper
(14). Even so, differences in Clog P between all other compounds
do not seem to have an impact on the slight differences in their
antiproliferative activity. Besides, the cytostatic activities do not
correlate with either antioxidative, lipoxygenase inhibitory activity
or lipid peroxidation inhibition. However, some compounds, espe-
cially 5f, precipitated from the cell culture medium, thus reduc-
ing their cell growth–inhibitory activity and causing larger
deviations between experiments. Still, in spite of the precipita-
tion, compound 4i showed the highest and the most consistent
activity.
Inhibition of linoleic acid lipid peroxidation
Azo compounds generating free radicals through spontaneous ther-
mal decomposition are useful for free radical production studies in
vitro. The water soluble azo compound AAPH has been extensively
used as a clean and controllable source of thermally produced al-
kylperoxyl free radicals. In our studies, AAPH was used as a free
radical initiator to follow oxidative changes of linoleic acid to con-
jugated diene hydroperoxide. The results indicated that compounds
4e, 5d, 4f, 4i, 5f, 4d, 5e, and 5c are excellent inhibitors of
lipid peroxidation (92.2–99.9%), significantly higher than ketoprofen
(69.3%) and reduced derivatives 2b and 2a (51.8% and 31.1%,
respectively) (Table 1). In general, methylene derivatives 4 (with
the exception of 4k, 4l, 4n) and keto derivatives 5 were more
potent than the hydroxy bearing derivatives 6. Comparison of the
amide substituents revealed that in the series 4 the activity
decreased as follows: aromatic substituent > cycloalkyl > ali-
phatic ꢀ non-substituted amide > O-substituted hydroxamic acids.
650
Chem Biol Drug Des 2010; 75: 641–652

Biological Evaluation of Ketoprofen Amides
Table 2: Inhibitory effects of ketoprofen derivatives 4–6 on the
growth of malignant tumor cell lines
reduced ketoprofen derivatives. The most active compound was the
benzhydryl ketoprofen amide 5f. Lipoxygenase inhibition of the
tested compounds varied from strong to very weak. The most
potent compound was benzhydryl derivative 6f (IC50 = 20.5 lM).
Aromatic and cycloalkyl amides 4 and 5 were more potent lipoxy-
genase inhibitors (IC50 = 37.5–120 lM) than derivatives with carbox-
ylic group. Aromatic amides of series 4 and 5 showed as excellent
lipid peroxidation inhibitors (92.2–99.9%). In general, methylene
derivatives 4 and keto derivatives 5 were more potent than the
hydroxy bearing derivatives 6. Comparison of the amide substitu-
ents revealed that in the series 4 the activity decreased as follows:
aromatic substituent > cycloalkyl > aliphatic = non-substituted ami-
de > O-substituted hydroxamic acids. In the series 5, aromatic
amides were also more active than the cycloalkyl ones. On the
other hand, the most pronounced cytostatic activity was exerted by
O-benzyl derivative 4i, although in general all tested reduced and
non-reduced lipophilic derivatives showed similar activity.
a
Tumor cell growth [IC50 (lM)]
Compound
HCT 116
SW620
MCF-7
MOLT-4
H 460
4bb
4c
12 € 3
11 € 0.3
15 € 0.6
4 € 3
56 € 43
24 € 2
25 € 5
87 € 10
16 € 0.2
53 € 42
30 € 3
>10c
37 € 8
50 € 40
18 € 2
>100
14 € 1
14 € 0.1
20 € 2
14 € 4
15 € 1
13 € 0.4
17 € 5
‡10c
13 € 0.1
23 € 16
13 € 2
>100
19 € 3
17 € 0.2
11 € 8
40 € 15
5 € 3
15 € 0.8
15 € 3
>10c
15 € 0.4
27 € 15
11 € 4
49 € 31
53 € 46
19 € 1
21 € 0.7
52 € 40
7 € 1
20 € 1
22 € 4
‡10c
32 € 8
57 € 37
15 € 0.1
‡100
4e
4fb
4ib
5c
7 € 5
13 € 5
15 € 2
‡10c
13 € 0.8
18 € 15
13 € 2
‡100
5e
5fb
6c
6eb
6fb
6g
a50% inhibitory concentration or compound concentration required to inhibit
tumor cell proliferation by 50%.
bPrecipitates from the media at 1 · 10)4 lM concentration.
cThe IC50 concentrations could not be calculated precisely because of the
precipitation.
Acknowledgments
Support for this study by the Ministry of Science and Technology of
the Republic of Croatia (Projects No. 006-0000000-3216 and 098-
0982464-2514) is gratefully acknowledged. The authors thank Dr. C.
Hansch and Biobyte Corp. Claremont CA California, USA for free
access to the C-QSAR program.
Table 3: Survival of HCT 116 cells after treatment with 4i, mea-
sured by the clonogenic assay (see Methods for details)
Survival of HCT 116 cells (%)
Treatment with 4i
10 lM
50 lM
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Figure S1. Long-term survival of HCT 116 cells following treat-
ment with 4i at 10, or 50 lM.
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