Synthesis and biological evaluation of licofelone derivatives as anticancer and anti-inflammatory agents

Article abstract of DOI:10.2174/157018011797655223

Two C5-substituted licofelone derivatives were developed and investigated for cytotoxicity against mammary (MCF-7 and MDA-MB 231) as well as colon carcinoma (HT-29) cancer cells. Both compounds were at least 2-fold more active than 5-fluorouracil (5-FU) and licofelone against mammary carcinoma cells. At HT-29 cells, they were less active, but nevertheless distinctly as active as 5-FU and still 2-fold more active than licofelone. However, variation of the C5- carboxylic group results in an occasionally remarkable decrease of anti-inflammatory potency in in vitro and in vivo.

Full text of DOI:10.2174/157018011797655223

Letters in Drug Design & Discovery, 2011, 8, 911-917
911
Synthesis and Biological Evaluation of Licofelone Derivatives as
Anticancer and Anti-inflammatory Agents
Wukun Liu1,2, Jinpei Zhou*,1, Huibin Zhang1, Hai Qian1, Jiahan Yin1, Kerstin Bensdorf2 and
Ronald Gust*,2,3
1Department of Medicinal Chemistry, China Pharmaceutical University, Tongjia Xiang 24, 210009 Nanjing, PR China
2Institute of Pharmacy, Freie Universität Berlin, Königin-Luise-Str, 2+4, 14195 Berlin, Germany
3Institut für Pharmazie, Universität Innsbruck, Innrain 52, A-6020 Innsbruck, Austria
Received June 07, 2011: Revised August 18, 2011: Accepted August 19, 2011
Abstract: Two C5-substituted licofelone derivatives were developed and investigated for cytotoxicity against mammary
(MCF-7 and MDA-MB 231) as well as colon carcinoma (HT-29) cancer cells. Both compounds were at least 2-fold more
active than 5-fluorouracil (5-FU) and licofelone against mammary carcinoma cells. At HT-29 cells, they were less active,
but nevertheless distinctly as active as 5-FU and still 2-fold more active than licofelone. However, variation of the C5-
carboxylic group results in an occasionally remarkable decrease of anti-inflammatory potency in in vitro and in vivo.
Keywords: Anti-inflammatory activity, COX inhibition, Cytotoxicity, Licofelone, Synthesis.
INTRODUCTION
COX-2, implying attractive and thus far unique molecular
pharmacological dynamics as an inhibitor of COX-1, 5-
LOX, and mPGES-1 [10, 11]. In addition, it enhanced
apoptosis in prostate cancer cells as well as HCA-7 colon
cancer cells through the mitochondrial pathway [12, 13].
Non-steroidal anti-inflammatory drugs (NSAIDs) are
drugs with anti-inflammatory, anti-pyretic and analgesic
effects. In recent years numerous experimental,
epidemiologic, and clinical studies suggest that NSAIDs are
promising anticancer agents. NSAIDs restore normal
apoptosis in human adenomatous colorectal polyps and in
various cancer cell lines that have lost adenomatous
polyposis coli gene function. NSAIDs also inhibit
angiogenesis in cell culture and rodent models of
angiogenesis. Many epidemiologic studies have found that
long-term use of NSAIDs is associated with a lower risk of
cancers [1-5].
Moreover, we recently reported a novel series of C5-
substituted licofelone derivatives. The acetic acid of
licofelone was replaced by an acetyl residue which was
further substituted with lipophilic groups (alkyl or chlorine),
formate, acetate, propionate, benzoates, and methylben-
zoates. Dependent on the C5-substituent, the compounds
showed high selectivity for MCF-7 cells. Especially acetyl
benzoate derivatives were inactive at the MDA-MB 231 cell
line and as active as 5-FU at MCF-7 cells. C5-acetyl, -acetyl
formate, -acetyl acetate and –acetylpropionate derivatives
showed growth inhibition at both cell lines, comparable with
cisplatin [15].
Recently, several companies were running drug-
discovery programs to develop dual cyclooxygenase
/lipoxygenase (COX/LOX) inhibitors as NSAIDs with an
improved gastrointestinal safety. To date, various structural
families of dual COX/LOX inhibitors have been designed
and several compounds are currently undergoing clinical
development as anti-inflammatory drugs [6-15]. One of these
compounds, [2,2-dimethyl-6-(4-chlorophenyl)-7-phenyl-2,3-
dihydro-1H-pyrrolizin-5-yl]acetic acid) (licofelone, Scheme
1) showed in clinical trials anti-inflammatory and analgesic
activity in osteoarthritis comparable to conventional NSAIDs
Based on the encouraging antitumor activity of licofelone
and its C5-substituted derivatives, we continue our efforts to
vary the substituents at C5 of licofelone. In this paper, the
acetic acid was replaced by an acetyl residue which was
further substituted with methoxy group and 1,2,4-triazole.
The novel compounds were tested for cytotoxicity against
MCF-7, MDA-MB 231 breast cancer and HT-29 colon
cancer cells as well as for anti-inflammatory potency in in
vitro and in vivo.
with
a safer gastrointestinal profile. It is a potent,
competitive inhibitor of 5-LOX, COX-1 and COX-2 [6-14].
Researches also addressed that licofelone appears to
suppress inflammatory prostaglandin E2 (PGE2) formation
preferentially by inhibiting microsomal prostaglandin E2
synthase-1 (mPGES-1) at concentrations that do not affect
MATERIALS AND METHODS
Materials
All reagents were purchased from Shanghai Chemical
Reagent Company. Licofelone and 5-benzyl-3,3-dimethyl-
3,4-dihydro-2H-pyrrole (2) were synthesized according to
previous method [8, 9, 11, 16-18]. Column chromatography
(CC): silica gel 60 (200–300 mesh). Thin-layer chromato-
graphy (TLC): silica gel 60 F254 plates (250 mm; Qingdao
Ocean Chemical Company, China). Melting point: capillary
*Address correspondence to these authors at the Department of Medicinal
Chemistry, China Pharmaceutical University, Tongjia Xiang 24, 210009
Nanjing, PR China; Tel: +86 25 83471532; Fax: +86 25 83271480;
E-mail: jpzhou668@163.com
Ronald Gust, Institut für Pharmazie, Universität Innsbruck, Innrain 52, A-
6020 Innsbruck, Austria; Tel: +43 512 507 5245 Fax: +43 512 507 2940;
Email: ronald.gust@uibk.ac.at
1570-1808/11 $58.00+.00
© 2011 Bentham Science Publishers

912 Letters in Drug Design & Discovery, 2011, Vol. 8, No. 10
Liu et al.
1
tube; uncorrected. IR spectra: Shimadzu FTIR-8400S
spectrophotometer. 1H-NMR spectra: Bruker ACF-300 Q
apparatus at 300 MHz (internal standard, TMS). Mass
spectrometry (MS): Hewlett–Packard 1100 LC/MSD
spectrometer; in m/z. Elemental analyses: CHN-O-Rapid
instrument.
1725, 1711, 1450, 832, 699; H-NMR (CDCl3, 300 MHz): ꢀ
1.30 (s, 6H, 2-CH3), 2.85 (s, 2H, -CH2-), 3.58 (s, 2H, -CH2-),
3.75 (s, 2H, -CH2-), 7.03-7.26 (m, 9H, Ar-H); Anal. calcd.
for C23H22ClNO2: C, 72.72; H, 5.84; N, 3.69%; Found C,
72.59; H, 5.98; N, 3.82%.
2-Chloro-1-(2-(4-chlorophenyl)-6,6-dimethyl-1-phenyl-6,7-
dihydro-5H-pyrrolizin-3-yl)ethanone (5)
6-(4-chlorophenyl)-2,2-dimethyl-7-phenyl-2,3-dihydro-1H-
pyrrolizine (3)
The reactions were performed in a 100 mL three neck
round bottom flask fitted with a gas inlet port. The flask was
charged with 50 mL of absolute diethyl ether, 0.4 g (1.27
mmol) of 3, 0.25 mL of corresponding chloroacetyl chloride
and 3.72 mmol of Lewis acid (AlCl3 or BF3-Et2O). The
resulting solution was magnetically stirred at room
The 5-benzyl-3,3-dimethyl-3,4-dihydro-2H-pyrrole (2)
(10 g) was dissolved in absolute ethanol (130 mL). The
solution was cooled to 20 °C and 2-bromo-1-(4-
chlorophenyl)ethanone (13 g) was added. After stirring for
30 min, NaHCO3 (6 g) was added and the reaction was
continued at 20-25 °C with protection from light. After about
24 h, the yellowish suspension was then filtered under
suction and the solid was washed with a small amount of
cold ethanol. The dried product was suspended in water at
40-45 °C. After stirring for 30-35 min, the product was then
filtered under suction and dried in vacuo at 35-40 °C.
temperature and purged by nitrogen gas for
3 h.
Subsequently, the reaction mixture was hydrolyzed with 60
mL of water and the product was extracted with diethyl
ether. The organic layer was dried over Na2SO4 and the
solvent was distilled off to give the crude product, which
was then purified by flash column chromatography with
mixture eluent of petroleum ether and ethyl acetate (40:1) to
give a white solid.
Yield 25%; MS (ESI, m/z): [M+H]+ 322.2; IR (KBr, cm-
1
1): 2956, 2917, 2869, 1699, 1600, 832, 765, 699; H-NMR
(CDCl3, 300MHz): ꢀ 1.26 (s, 6H, 2-CH3), 2.79 (s, 2H, -CH2-
), 3.73 (s, 2H, -CH2-), 6.67 (s, 1H, =CH-); 7.03-7.26 (m, 9H,
Ar-H); Anal. calcd. for C21H20ClN: C, 78.37; H, 6.26; N,
4.35%; Found C, 78.56; H, 6.15; N, 4.31%.
Yield (40.1% (AlCl3) or 59.3 % (BF3-Et2O)); mp 157-
159 °C; MS (ESI, m/z): [M+H]+ 398.2, [M+Na]+ 420.2; IR
(KBr, cm-1): 2919, 2839, 1645, 1595, 1450, 1012, 748, 704;
1H-NMR (CDCl3, 300 MHz): ꢀ 1.31 (s, 6H, 2-CH3), 2.84 (s,
2H, -CH2-), 3.85 (s, 2H, -CH2-), 4.24 (s, 2H, -CH2-), 6.95-
7.38 (m, 9H, Ar-H). Anal. calcd. for C23H21Cl2NO: C, 69.35;
H, 5.31; N, 3.52%; Found C, 69.07; H, 5.41; N, 3.71%.
[2,2-dimethyl-6-(4-chlorophenyl)-7-phenyl-2,3-dihydro-1H-
pyrrolizin-5-yl]acetic acid (licofelone)
3 (4 g, 12.7mmol) was dissolved by stirring in dry THF.
The assembly was blanketed with N2 atmosphere. The
yellow colored solution was cooled to 10-15 °C and oxalyl
chloride (2.8 ml, 19.6 mmol) was slowly added below 15 °C
over a period of 30 min. The green colored solution was
further stirred at 18-25 °C for 20-30 min after complete
addition. The internal temperature did not exceed 20 °C, the
reaction mixture was then quenched carefully with ice (50
g). The reaction mixture was then stirred at 20-30 °C for 5-
10 min and was further diluted with diethylene glycol (32
ml) and hydrazine (6 ml, 120 mmol). The solvent THF was
distilled off during the process until the gradual rise in
internal temperature reaches 75-80 °C. The suspension was
then cooled to 50-55 °C and KOH (9.04 g, 161 mmol) was
added portion wise over a period of 30 min. The now
yellowish liquid was then heated to 95-110 °C gradually.
The temperature was then raised slowly to 140-145 °C and
during the process aqueous distillate was collected. The
batch was held at 130-145 °C for 2-3 h. The reaction
temperature was then cooled to 35-40 °C and water and
diethyl ether were added. The mixture was stirred vigorously
for 15-20 min and until the stirring switched off the layers
were allowed to settle. The aqueous phase was separated
cooled below 5 °C and acidified to pH 1 using a solution of
HCl maintaining temperature below 10 °C. The separated
solid was taken up in diethyl ether and the ethereal extract
was washed thoroughly with water. The ethereal extract was
charcoalized before being evaporated under vacuo below 20
°C. The solid was slurried in heptane and filtered under
suction and washed with heptane. The product was dried in
vacuum at 45-55 °C.
1-(6-(4-chlorophenyl)-2,2-dimethyl-7-phenyl-2,3-dihydro-
1H-pyrrolizine-5-yl)-2-(1,2,4-triazole-1-yl)-ethanone (6)
The flask was charged with 10 mL of absolute acetone,
0.14 g (2 mmol) of 1H-1,2,4-triazole and 0.21 g (2 mmol) of
sodium carbonate, and 0.4 g (1.00 mmol) of 5. The resulting
solution was stirred at room temperature for over 24 h. Then
the solvent was distilled off to give the crude product, which
was then purified by flash column chromatography with
mixture eluent of petroleum ether and ethyl acetate (1:1) and
followed by recrystallization from acetone to give a white
solid.
Yield: 75.0%; mp: 121-122 °C; MS (ESI, m/z): [M+H]+
431, [M+Na]+ 453; IR (KBr, cm-1): 3113, 2969, 2854, 1640,
1
1490, 972, 883, 669; H-NMR (CDCl3, 300MHz): ꢀ 1.30 (s,
6H, 2-CH3), 2.85 (s, 2H, -CH2-), 4.19 (s, 2H, -CH2-), 4.69 (s,
2H, -CH2-), 6.97-7.42 (m, 9H, Ar-H), 7.92 (s, 1H, =CH-),
8.02 (s, 1H, =CH-); Anal. Calcd. for C25H23ClN4O: C, 69.68;
H, 5.38; N, 13.00%; Found C, 69.72; H, 5.31; N, 12.87%.
1-(6-(4-chlorophenyl)-2,2-dimethyl-7-phenyl-2,3-dihydro-
1H-pyrrolizine-5-yl)-2-methoxy-ethanone (7)
The flask was charged with 10 mL of absolute methanol,
0.1 g (2 mmol) of sodium methoxide and 0.4 g (1.00 mmol)
of 5. The resulting solution was stirred at room temperature
for over 24 h. Then the solvent was distilled off to give the
crude product, which was then purified by flash column
chromatography with mixture eluent of petroleum ether and
ethyl acetate (20:1) to give a white solid.
Yield: 72.5%; mp 128-130 °C; MS (ESI, m/z): [M+H]+
Yield 55.6%; mp 157 °C; MS (ESI, m/z): [M+H]+ 380,
394.2; IR (KBr, cm-1): 3437, 2959, 1650, 1515, 967, 923,
[M+Na]+ 402; IR (KBr, cm-1): 3402, 2958, 2917, 2900, 2849,

Synthesis and Biological Evaluation of Licofelone Derivatives
Letters in Drug Design & Discovery, 2011, Vol. 8, No. 10 913
773, 699, 644. 1H-NMR (CDCl3, 300MHz): ꢀ 1.31 (s, 6H, 2-
CH3), 2.82 (s, 2H, -CH2-), 3.23 (s, 3H, -CH3), 3.76 (s, 2H, -
CH2-), 4.23 (s, 2H, -CH2-), 6.95-7.37 (m, 9H, Ar-H); Anal.
Calcd. for C24H24ClNO2: C, 73.18; H, 6.14; N, 3.56%; Found
C, 73.07; H, 5.98; N, 3.84%.
Dulbecco’s modified Eagle’s medium (DMEM) with 4.5 g/L
glucose (PAA Laboratories, Austria), supplemented with 5%
fetal bovine serum (FBS; Biochrom, Germany) in a
humidified atmosphere (5% CO2) at 37 °C.
Cytotoxicity
PHARMACOLOGYꢀ
Cell Culture
The experiments were performed according to
established procedures with some modifications [15]. In 96
well plates 100 ꢁL of a cell suspension in culture medium at
7500 cells/mL (MCF-7 and MDA-MB 231) or 3000
cells/mL (HT-29) was plated into each well and was
incubated for three days under culture conditions. After the
The human MCF-7, MDA-MB 231 breast cancer cell
lines and HT-29 colon cancer cell line were obtained from
the American Type Culture Collection. All cell lines were
maintained as a monolayer culture in L-glutamine containing
(a)
(b)
Cl
CN
Cl
N
N
1
3
2
(c)
(d)
Cl
Cl
3
N
N
COOH
COOH
O
Licofelone
4
(f)
(e)
Cl
Cl
N
N
3
CH2Cl
N
O
O
N
N
(g)
5
6
Cl
N
O
OCH3
7
Scheme 1. Synthetic routes of licofelone, 6 and 7.a
a
Reagents and conditions: (a) benzylmagnesium chloride (Grignard species provided in situ from benzylchloride and Mg 1:1), initially
absolute Et2O, 2 h, reflux, then toluene, 3 h, reflux, 70%; (b) 2-bromo-1-(4-chlorophenyl) ethanone, absolute ethanol, NaHCO3, 36 h, rt,
.
25%; (c) oxalyl chloride, THF, 10-15 °C, then add H2O; 25-30 °C, 20 mins; (d) N2H4 H2O, KOH, ethylene diglycol, 85 °C, 5 h, then to 140-
145 °C, 2 h, 55%.; (e) ClCH2COCl, Lewis acid, absolute Et2O, rt, 3 h, (40% (AlCl3) and 59% (BF3-Et2O)). (f) 1-H-1,2,4-triazole, Na2CO3,
absolute acetone, rt, over overnight, 75%. (g) NaOCH3, absolute CH3OH, rt, overnight, 72.5%.

914 Letters in Drug Design & Discovery, 2011, Vol. 8, No. 10
Liu et al.
addition of various concentrations of the test compounds,
cells were incubated for up to 144 hours. Then the medium
was removed, the cells were fixed with glutardialdehyde
solution (1%) and stored under phosphate buffered saline
(PBS) at 4 °C. Cell biomass was determined by a crystal
violet staining, followed by extraction of the bound dye with
ethanol and a photometric measurement at 590 nm. Mean
values were calculated and the effects of the compounds
were expressed as % Treated/Controlcorr values according to
the following equations:
0.05. The analysis of data was performed by software SPSS
13.0.
RESULTS AND DISCUSSION
Chemistry
6,7-Diaryl-2,3-dihydro-1H-pyrrolizine (3) and licofelone
were synthesized according to previously published methods
[8, 9, 11, 16-18] and outlined in Scheme 1.
We condensed 1 with a commercially available benzyl-
Grignard, and accomplished ring closure to rather unstable
5-benzyl-3,3-dimethyl-3,4-dihydro-2H-pyrrole (2). 6,7-
Diaryl-2,3-dihydro-1H-pyrrolizine (3) was cyclized with the
T ꢁ C0
C ꢁ C0
T
[%] =
ꢀ100
Ccorr
(C0 control cells at the time of compound addition; C
control cells at the time of test end; T probes/samples at the
time of test end)
2-bromo-1-(4-chlorophenyl)ethanone and
2 in ethanol/
aqueous NaHCO3 solution at room temperature (rt) for 36 h
with moderate yields. Friedel-Craft acylation of 3 with
oxalyl chloride and subsequent Wolff–Kishner reduction
with hydrazine hydrate yielded licofelone (Scheme 1).
The IC50 value was determined as the concentration
causing 50% inhibition of cell proliferation and calculated as
mean of at least three independent experiments (OriginPro
8).
Analogously, 5 was obtained by reaction of 3 with
chloroacetyl chloride by means of BF3-Et2O or AlCl3 as
catalysts (Scheme 1). The reaction yield depended on the
catalyst, while in the case of 5 both catalysts work as well
(40% (AlCl3) and 59% (BF3-Et2O)). Compound 6 was
obtained by reaction of 5 with 1H-1,2,4-triazole and sodium
carbonate, while compound 7 was obtained by reaction of 5
with sodium methoxide in methanol solution.
Inhibition of COX Enzymes
The inhibition of isolated ovine COX-1 and human
recombinant COX-2 was determined with 10 μM of the
respective compounds by ELISA (“COX inhibitor screening
assay”, Cayman Chemicals). Experiments were performed
according to the manufacturer’s instructions. Absorption was
measured at 415 nm (Victor2, Perkin Elmer). Results were
calculated as the means of duplicate determinations.
1
Under these synthetic conditions IR, ESI-MS, H NMR
of 3, licofelone, 5-7 were recorded. The IR spectra of
compounds 5-7 are very similar. In accordance with the
structure, the IR spectra showed ꢀ(CO) bands at about 1650
1
cm-1. The H NMR of 6 showed two triazole protons at ꢁ
Xylene-Induced Ear Edema
7.92 ppm and 8.02 ppm compared with 5.While the 1H NMR
of 7 showed three methyl protons with single peaks at ꢁ 3.23
ppm compared with 5. Both compounds were further
supported by positive mode ESI mass spectra, which
documented a base peak corresponding to the [M+H]+
fragment for 6 and 7.
Animals
The experiments with animals were approved by
Research Ethic Committee of Jiang-Shu province, China.
Kunming male mice of approximately 20 g were obtained
from experimental animal center of China Pharmaceutical
University, and fed with rat food and water ad libitum. All
animals fasted for 12 h before the experiments. The
temperature (25 °C) and humidity (60%) in the animal room
were well controlled.
BIOLOGICAL ACTIVITY
In vitro cytotoxicity assays were performed to obtain an
insight into the antitumor activity of licofelone and its
derivatives. In addition to licofelone and its derivatives, the
established antitumor drug 5-FU was screened against
hormone dependent MCF-7, hormone independent MDA-
MB 231 breast cancer and HT-29 colon cancer cell lines.
MCF-7 cells have a basal level of COX-1 and a barely
detectable and transient COX-2- inducible expression,
whereas MDA-MB 231 cells show a low expression of
COX-1 but a constitutive level of COX-2 [19]. HT-29
human colon cancer cells constitutively express COX-2 [20].
Therefore, their growth is sensitive to NSAID treatment [1-
3].
Method
Mice were allotted to groups of 6 animals each. Thirty
minutes after i.p. injection of 6, 7, licofelone, ibuprofen or
celecoxibe, 0.015 ml of xylene was applied to the anterior
and posterior surfaces of the right ear. The left ear was
considered as control. Two hours after xylene application,
mice were killed and both ears were removed. Circular
sections were taken, using a cork borer with a diameter of 7
mm, weighed and measured. The degree of ear swelling was
calculated based on the weight and thickness of left ear
without xylene.
The experiments were performed according to
established procedures [15]. A number of known cells were
exposed to increasing concentrations of compounds on a 96-
well tissue culture plate and incubated for a given period of
time. Due to the poor solubility of the substances, DMSO
stock solutions had to be used. Because of the cytotoxicity of
Statistical Analysis
The measurement data were expressed as the mean ± SD.
Data were subjected to one-way analysis of variance
(ANOVA), followed by multiple comparison with least
significant differences (LSD) test or Dunnett’s test as
appropriate. Statistical significance was considered with P <

Synthesis and Biological Evaluation of Licofelone Derivatives
Letters in Drug Design & Discovery, 2011, Vol. 8, No. 10 915
Table 1. Antiproliferative Effects Against MCF-7, MDA-MB 231 and HT-29 Cells
Cytotoxicity IC50, [μM]a
MCF-7
Compound
MDA-MB 231
HT-29
6
7
4.8 ± 0.1
5.3 ± 0.1
36.7 ± 3.2
9.6 ± 0.3
2.2 ± 0.2
2.0 ± 0.2
5.5 ± 0.6
4.7 ± 0.4
6.0 ± 0.2
11.8 ± 0.3
22.6 ± 1.2
7.3 ± 1.0
Licofelone
5-FU
aThe IC50 values represent the concentration which results in a 50% decrease in cell growth after 72 h incubation.
DMSO in higher concentrations, final DMSO concentrations
were limited to 0.1% in all samples. IC50 values for these
compounds were calculated (OriginPro 8) and are presented
in Table 1. Doses dependent antiproliferative effects of
compounds in three cell lines are showed in Fig. (1).
231 (IC50 = 36.7 ꢀM) and HT-29 cells (IC50 = 22.6 ꢀM), it
was only marginally active indicating at least 4-fold
selectivity for MCF-7 cells.
Both novel compounds 6 and 7 showed promising
antiproliferative effects at MCF-7 cells (IC50 = 2.2 μM and
2.0 μM, respectively) and MDA-MB 231 cells (IC50 = 4.8
μM and 5.3 μM, respectively), approximately twice as active
as 5-FU (IC50 = 4.7 μM, MCF-7; IC50 = 9.6 μM, MDA-MB
231). At HT-29 cells, compounds 6 and 7 were less active
(IC50 = 6.0 μM and 11.8 μM, respectively) but nevertheless
distinctly as active as 5-FU (IC50 = 7.3 μM)
160
Licofelone MCF-7
Licofelone MDA-MB 231
Licofelone HT-29
6 MDA-MB 231
140
120
100
80
In comparison with the results of licofelone, exchange of
the C5-acetic acid moiety enormously increased the growth
inhibitory effects. Both novel compounds were at least 2-
fold as active as licofelone in three cell lines. However, the
high selectivity for MCF-7 cells was lost. Moreover, the
cytotoxicity of both compounds was comparable to C5-
acetyl, -acetyl formate, -acetyl acetate and –acetylpropionate
derivatives at MCF-7 and MDA-MB 231 cells [15].
60
40
20
0
-20
-40
The time-activity curves of 6 presented in Fig. (2) show a
marginal recuperation of the tumor cells after a prolonged
exposition. Because exponential cell growth is guaranteed
for at least 140 h of incubation, the rise of the growth curve
can be explained by the development of drug resistance. The
onset of antiproliferative effects was observed early. 6
showed its maximum activity after 48 h, therefore we
evaluated the IC50 value of the compounds after an
incubation time of 72 h.
0
10
20
30
40
50
Concentration (μM)
120
6 HT-29
7 MCF-7
7 MDA-MB 231
7 HT-29
100
80
60
40
20
0
In order to estimate a possible participation of the COX
inhibition on the cytotoxic properties of licofelone and its
derivatives, their influences against the isolated isoenzymes
were determined (Fig. 2). During the recent years, COXs,
especially COX-2 became very interesting targets in cancer
chemotherapy. In addition to the well-established
pathophysiological role that COX-2 plays in inflammation,
recent evidence implies that this isoform may also be
involved in multiple biological events throughout the
tumorigentic process. Further, COX-2 is chronically
overexpressed in many premalignant, malignant, and
metastatic human cancers, and levels of overexpression have
been shown to significantly correlate to invasiveness,
prognosis, and survival in some cancers. Pharmacological
studies consistently demonstrate that COX-2 inhibitors show
dose dependent inhibition tumor growth and metastasis in
various relevant animal models of cancer. Importantly,
several investigators have also shown that COX-2 inhibitors
may act additively or synergistically with currently used
cytotoxics and molecularly targeted agents [1-4, 19-22].
-20
0
10
20
30
40
50
Concentration (μM)
Fig. (1). Dose dependent antiproliferative effects of the novel
compounds 6 and 7 as well as licofelone at MCF-7, MDA-MB 231
and HT-29 cells. In some cases the error bars are hidden behind the
symbols.
Licofelone showed at the MCF-7 cell line an IC50 = 5.5
ꢀM very similar to 5-FU (IC50 = 4.7 ꢀM). Against MDA-MB

916 Letters in Drug Design & Discovery, 2011, Vol. 8, No. 10
Liu et al.
120
70
60
50
40
30
20
10
0
100
80
60
40
20
0
COX-1
COX-2
Licofelone
6
7
0
20
40
60
Incubation time (h)
0.63 μM 1.25 μM
80
100
120
140
Fig. (3). Inhibition of COX-1 (ovine) and COX-2 (human
recombinant) activity after treatment with compounds in the
concentration of 10 μM (negative control (DMSO) was set as 0%).
0.31μM
2.5 μM
5 μM
Fig. (2). Time dependent antiproliferative effects of compound 6 at
MCF-7 cells.
licofelone influenced the ear swelling in a dose-dependent
manner, with an about 50% inhibition at a dose of 100 mg/kg
as well as NSAIDs ibuprofen and celecoxib. Similar to the
results of COX inhibition, 6 and 7 lowered the in vivo
activity with anti-inflammatory effects of about 7-33%.
A drug concentration of 10 ꢀM was used for the
experiments since licofelone inhibited the COX at this
concentration by about 50% (COX-1 (60.6%) and COX-2
(45.8%)). Therefore, we used this concentration and
performed the ELISA without calculation of the IC50 values.
Under the same test conditions, compounds 6 and 7 lowered
the activity of COX-1 (14% and 24%, respectively) and
COX-2 (8% and 12%, respectively) (Fig. 3). In each case,
the COX-1 isoenzyme was more affected by the inhibitor
CONCLUSION
Two licofelone C5-substituted derivatives were
developed and investigated for their biological activity. Both
compounds exhibited high antiproliferative potencies and
reduced anti-inflammatory effects. The anti-inflammatory
properties and the COX-1/2 inhibition did not correlate with
the growth inhibition of cancer cells. However, these results
clearly documented that modification at position 5 of the 2,3-
dihydro-1H-pyrrolizine core allows an optimization of
licofelone for an effective tumor therapy. Additional
investigations to get an insight into the mechanism of action
(such as LOX inhibition [22] or other molecular pathways
[15]) as well as into structure–activity relationships are in
progress and will be part of a forthcoming paper.
than COX-2 indicating
a comparable selectivity as
demonstrated for licofelone. This finding is in accordance
with previous investigations on the derivatization of
licofelone [11, 15, 18]. Variation of the C5-carboxylic group
results in an occasionally remarkable decrease of COX-
activity. However, a correlation of COX inhibition and
cytotoxicity against cancer cells was not visible.
Nevertheless, the anti-inflammatory effects of the
licofelone derivatives were studied in in vivo using the
xylene-induced ear swelling in mice. As shown in Table 2,
Table 2. Effect of the Compounds on Xylene-Induced Ear Swelling in Mice (n = 6,
± S)
x
Dose
[mg/kg]
Swollen extent; weight
[mg]
Inhibition (%)
Swollen extent; thickness
[mm]
Inhibition
(%)
Control
5.7 ± 1.4
3.8 ± 2.2*
0.138 ± 0.017
0.070 ± 0.032**
0.066 ± 0.013**
0.030 ± 0.029**
0.092 ± 0.019**
0.096 ± 0.034*
0.063 ± 0.006**
0.072 ± 0.013*
Licofelone
25
33.3%
43.9%
77.2%
7.0%
49.2%
52.2%
74.6%
33.3%
30.4%
54.3%
47.9%
100
200
100
100
100
100
3.2 ± 0.8**
1.3 ± 1.9**
5.3 ± 0.4
6
7
4.2 ± 0.5*
26.3%
51.2%
46.3%
Ibuprofen
Celecoxib
2.78 ± 0.37**
3.06 ± 0.49**
*P < 0.05, **P < 0.01. Data were subjected to one-way ANOVA.

Synthesis and Biological Evaluation of Licofelone Derivatives
Letters in Drug Design & Discovery, 2011, Vol. 8, No. 10 917
[11]
Liedtke, A.J.; Keck, P.R.; Lehmann, F.; Koeberle, A.; Werz, O.;
Laufer, S.A. Arylpyrrolizines as inhibitors of microsomal
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Narayanan, N.K.; Nargi, D.; Attur, M.; Abramson, S.B.;
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Tavolari, S.; Bonafe, M.; Marini, M.; Ferreri, C.; Bartolini, G.;
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ACKNOWLEDGEMENTS
The authors are grateful for the financial supports of
National Natural Science Foundation of China (No.
30973638). The China scholarship council and the Deutsche
Forschungsgemeinschaft (FOR 630) are gratefully
acknowledged.
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