Synthesis and biological evaluation of curcuminoid pyrazoles as new therapeutic agents in inflammatory bowel disease: Effect on matrix metalloproteinases

Article abstract of DOI:10.1016/j.bmc.2008.12.029

Seven N-unsubstituted curcuminoid pyrazoles have been synthesized from the corresponding β-diketones (including curcumin). We evaluated the possibility of curcuminoid pyrazoles regulating the activity of matrix metalloproteinases (MMPs) by human intestinal epithelial cells in vitro. Zymographic analysis revealed that three compounds significantly down-regulated MMP-9 activity on inflammation-induced intestinal epithelial cells, making them original candidates for the treatment of inflammatory bowel disease (IBD).

Full text of DOI:10.1016/j.bmc.2008.12.029

Bioorganic & Medicinal Chemistry 17 (2009) 1290–1296
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of curcuminoid pyrazoles as new
therapeutic agents in inflammatory bowel disease: Effect on matrix
metalloproteinases
a
a
a
b
c
c,
*
R. M. Claramunt a, , L. Bouissane , M. P. Cabildo , M. P. Cornago , J. Elguero , A. Radziwon , C. Medina
*
a Departamento de Química Orgánica y Bio-Orgánica, Facultad de Ciencias, UNED, Senda del Rey 9, E-28040 Madrid, Spain
b Instituto de Química Médica, CSIC, Juan de la Cierva 3, E-28006 Madrid, Spain
c School of Pharmacy and Pharmaceutical Sciences, Trinity College Dublin, 23 Westland Row, Dublin 2, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history:
Seven N-unsubstituted curcuminoid pyrazoles have been synthesized from the corresponding b-dike-
tones (including curcumin). We evaluated the possibility of curcuminoid pyrazoles regulating the activity
of matrix metalloproteinases (MMPs) by human intestinal epithelial cells in vitro. Zymographic analysis
revealed that three compounds significantly down-regulated MMP-9 activity on inflammation-induced
intestinal epithelial cells, making them original candidates for the treatment of inflammatory bowel dis-
ease (IBD).
Received 10 September 2008
Accepted 8 December 2008
Available online 6 January 2009
Dedicated to Professor M del Carmen Pardo
of the ‘Universidad Complutense de Madrid’
on the occasion of her retirement
Ó 2008 Elsevier Ltd. All rights reserved.
Keywords:
Curcumin
Pyrazole
Inflammatory bowel disease
Crohn’s disease
Ulcerative colitis
Matrix metalloproteinases
Gelatinases
Caco-2 cells
1. Introduction
metastasis and inflammatory reactions. Based on substrate speci-
ficity, MMPs have been classically divided into collagenases, gela-
Inflammatory bowel disease (IBD), both ulcerative colitis (UC)
and Crohn’s disease (CD), is a chronic relapsing condition with
inflammation and tissue remodeling of the gastrointestinal tract
whose etiology remains unknown.1,2 However, it is now apparent
that the intestinal epithelium may play an important role in the
immunomodulatory response of the intestinal mucosa. In addition,
existing therapies in IBD, predominantly aminosalicylates, steroids
and immunosuppressants, can relieve the inflammatory symp-
toms; however, IBD patients may suffer numerous relapses and
there is no cure.
tinases (MMP-9 and MMP-2) and stromelysins. Several lines of
evidence support the involvement of these enzymes in accelerated
breakdown of the ECM in IBD and therefore, contribute to the path-
ogenesis of this condition.3–7 In fact, the gelatinase MMP-9 which
is released by intestinal cells in response to pro-inflammatory
cytokines such as TNF-a and IL-1b, becomes active and it is the
most abundantly MMP expressed in the inflammatory tissues of
IBD patients.3 In addition, we and others have found that pharma-
cological inhibition of MMPs is protective in several experimental
models of colitis.8–12
Matrix metalloproteinases (MMPs) comprise a group of zinc
and calcium-dependent endopeptidases that are involved in the
remodeling and degradation of extracellular matrix (ECM) during
physiological and pathological conditions, such as tumor growth,
The aim of this study was to test new synthetic curcumin
pyrazole derivatives 1–7 in intestinal inflammation. Curcumin 8
[E-100: 1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-3,5-
dione] is the main colorant present in the rhizome of Curcuma
longa. The root of this plant has been used in India as preservative,
colorant, flavoring in meals (curry) and as a traditional medicine
(one significant finding is that the four most frequent neopla-
sias—colon, breast, prostate and lung—are less frequent in India,
where this ingredient is widely used). It is known that 8 acts as
* Corresponding authors. Tel.: +34 913987322; fax: +34 913988372 (R.M.
Claramunt), tel.: +35 318962823 (C. Medina).
E-mail addresses: rclaramunt@ccia.uned.es (R.M. Claramunt), cmedinammar@
hotmail.com (C. Medina).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.12.029

R. M. Claramunt et al. / Bioorg. Med. Chem. 17 (2009) 1290–1296
1291
an antioxidant,13–15 anti-inflammatory,16–18 antitumoral (antian-
H
R
R1
R2
giogenic),16,17,19–21 and that it corrects the genetic mutation that
causes cystic fibrosis,22 which explains the growing interest in this
material. Other studies of the properties of curcumin and related b-
diketones (curcuminoids) include their properties as metal com-
plexants,23 their binding to DNA,24 their proton transfer and tau-
tomerism,25 and their immunopharmacology.26 Three recent
reviews have been devoted to curcumin and the curcuminoids.27–
N N
R1
R
H
H
OH
1
2
3
HO
R2
OCH3
CH3
OCH3
OH
OH
OH
a
CH3
CH3
H
29
Concerning SAR, it appears that the presence of the phenolic
C6H5
4
5
6
rings is a prerequisite for activity.14,30,31
H
N N
R1
A way to modify the curcumin b-diketone skeleton is to trans-
form it into an heterocycle, for instance, reacting it with hydrazines
to form pyrazoles. Three strategies are possible (Scheme 1): (i) to
react curcumin itself with different monosubstituted hydrazines
(including hydrazine itself), (ii) to react curcuminoids (including
curcumin) with hydrazine itself (compound 1 are obtained by both
ways), and (iii) mixing both approaches. Pyrazole 1 derived di-
rectly from curcumin has been prepared many times since
1991.32–38 It is a pale yellow solid that melts at 211–214 °C32 or
215 °C.34
C6H5
C6H5
H
OCH3
OCH3
R
CH3
R2
OCH3
H
OH
b
7
OHC
Figure 1. Pyrazoles studied as new therapeutic agents for IBD.
characterized and the NMR assignments completed as shown in
the Experimental Section.
Discussion on the NH-tautomers (a and b) stability and the tau-
tomerization equilibrium constant for each pyrazole 1–7 is not the
object of the present paper and will be described elsewhere.39
The activity of the curcuminoid pyrazoles covers domains such
as anti-inflammatory (5-lipooxygenase and cyclooxygenase inhib-
itors),32,36 antitumoral (anti-angiogenic),33–35 and drugs for the
treatment of the Alzheimer disease (potent c-secretase inhibitors,
potent ligands for fibrillar Ab42 aggregates, tau aggregation inhib-
itors, and depolymerizing agents for tau aggregates).37,38 In the last
and more recent application (type i approach with 11 different R
including H), curcumin-derived pyrazoles were synthesized in or-
der to minimize the metal chelation properties of curcumin. The
reduced rotational freedom and the absence of stereoisomers
2.2. Pharmacology
Knowing that intestinal epithelial cells may play an important
role in the immunomodulatory response in IBD, we decided to
use Caco-2 cells, human epithelial intestinal cells, in our experi-
ments. Since MMP-9 is one of the main proteolytic enzymes in-
was anticipated to enhance the inhibition of c-secretase with les-
volved in human IBD and TNF-
a and IL-1b are pro-inflammatory
ser secondary effects. Accordingly, the replacement of the 1,3-
dicarbonyl moiety by isosteric heterocycles turned these com-
pounds very interesting candidates for AD research. Finally, it must
be considered that pyrazoles, and amongst them Celecoxib, are
inhibitors of MMP-9.39–41
cytokines that are over-expressed in this condition, we tested the
effect of these cytokines on the activity of gelatinases produced
by caco-2 cells. In fact, MMP-9 seems to play a main role in inflam-
mation-associated tissue destruction both in human IBD and
experimental colitis.3,10,12,43,44 A significant correlation between
the MMP-9 activity and the tissue damage has been previously
found.3,12 Indeed, either pharmacological MMP-9 inhibition or
MMP-9 deficiency have been proven useful in different experimen-
tal models of colitis.10,12,43,44 Therefore, inhibition of this enzyme
in IBD seems to be an excellent therapeutic strategy.
We decided to explore way ii preparing the seven pyrazoles re-
ported in Figure 1 with the aim of studying them in IBD.
2. Results and discussion
2.1. Chemistry
In our experiments, we found that in untreated cells, MMP-2
was the predominant gelatinase, whereas MMP-9 was negligible
as measured by zymography. However, cytokine treatment re-
sulted in a significant dose-dependent up-regulation of MMP-9
activity in intestinal epithelial cells. Nevertheless, MMP-2 re-
mained unchanged by the cytokine treatment (Fig. 2A and B). We
corroborated these results using specific antibodies against
MMP-9 (Fig. 3A and B). Again, cytokine treatment induced an up-
regulation of MMP-9 expression as measured by western-blotting.
These results are completely in agreement with previous stud-
ies.44,45 In fact, gene transcription of MMP-9 is inducible and the
promoter region is highly responsive to most growth factors and
cytokines in several cells. In contrast, MMP-2 is expressed consti-
tutively by most cells and appears to be moderated induced or
repressed.46,47
The synthesis of curcumin pyrazoles 1–7 was achieved follow-
ing the procedure described for the preparation of the parent com-
pound 132,34 starting from the corresponding b-diketones 8–14 and
hydrazine, as depicted in Scheme 1. All compounds have been fully
i:
ii:
O
O
O
O
R
R1
HO
OH
OCH3
curcumin (8)
R2
OCH3
OCH3
Since the concentration of 10 ng/ml TNF-
a and IL-1b was
R-NH-NH2
NH2-NH2
found to be really effective inducing the up-regulation of
MMP-9 activity, further experiments were performed with this
concentration of cytokines. We found that co-treatment with
R
H
N
N
N
N
several compounds, such as 1, 2 and 3 at 50 lM, resulted in a
significant down-regulation of MMP-9 activity as measured by
R
HO
R2
OH
OCH3
R1
zymography (Fig. 4A and B).
1, R = H
OCH3
OCH3
To further explore the optimal concentration of the three com-
pounds that were able to down-regulate MMP-9 activity in vitro,
Scheme 1. Pyrazoles related to curcumin.

1292
R. M. Claramunt et al. / Bioorg. Med. Chem. 17 (2009) 1290–1296
TNFα + IL-1β (ng/ml)
1500
*
C
1
10
100
*
1000
500
0
92 kDa
72 kDa
*
C
1
10
100
Figure 2. Activity of gelatinases in Caco-2 cells in absence (c) or presence of TNF-
molecular weights of 72 and 92 kDa corresponding to MMP-2 and MMP-9, respectively. (B) Quantitative zymographic analysis ( p < 0.05 vs control).
a
and IL-1b (1, 10 and 100 ng/ml). (A) Representative zymogram of gelatinases with
*
*
500
TNFα + IL-1β (ng/ml)
*
400
C
1
10
100
*
300
200
100
0
92 kDa
C
1
10
100
Figure 3. MMP-9 expression in Caco-2 cells in absence (c) or presence of TNF-
weight of 92 kDa. (B) Quantitative western-blot analysis ( p < 0.05 vs control).
a and IL-1b (1, 10 and 100 ng/ml). (A) Representative western-blot of MMP-9 with a molecular
*
HT1080 Control TNFα+IL-1β
1
2
4
3
5
6
7
92 kDa
72 kDa
200
control
TNF+Il1B
1
2
4
3
5
6
7
*
*
100
*
0
Figure 4. Activity of gelatinases in Caco-2 cells stimulated by TNF
a
and IL-1b (10 ng/ml) in the presence of the different curcumin pyrazoles at a concentration of 50
lM. (A)
Representative zymogram of gelatinases, MMP-9 (92 kDa) and MMP-2 (72 kDa), in untreated cells (control), stimulated cells with cytokines (TNF
a
+ IL-1b) and stimulated
cells with cytokines and in the presence of the different curcumin pyrazoles (1–7). Conditioned media of HT1080 human fibrosarcoma cells which express high amount of
*
both gelatinases were used as positive control. B: Quantitative zymographic analysis ( p < 0.05 vs TNFa+IL-1b).
additional experiments were carried out. The dose-response curves
for the most active compounds 1–3 are shown in Figure 5A–C.
Further experiments were carried out with regular curcumin as
it is known that this compound down-regulates MMP-9 activity. In
fact, it has been found that curcumin inhibits MMP-9 in human
articular chondrocytes48 and in blood mononuclear cells.49 The
concentration-response curve is shown in Figure 6. As expected,
curcumin was able to significantly down-regulate MMP-9 activity
in caco-2 cells stimulated by pro-inflammatory cytokines to the
same extent than curcumin-derived pyrazoles. Therefore, curcumi-

R. M. Claramunt et al. / Bioorg. Med. Chem. 17 (2009) 1290–1296
1293
1000
750
500
250
0
3. Conclusion
Control
TNF + Il1B
Three pyrazoles have demonstrated activity in the biological
tests used as models for IBD (Scheme 2).
*
1
*
5
The main points of this study are: (i) although pyrazole 1 had
already been synthesized and biologically tested, its possible appli-
cation in the field of IBD is described here for the first time; (ii) two
new pyrazoles 2 and 3 have shown activities similar to 1; (iii) the
fact that a 3(5)-methyl group is better than a 3(5)-phenyl group
(compounds 4–6) was unexpected and offers interesting possibili-
ties of lead optimization.
10
25
50
*
*
IC50:19.4 0.7
Further studies are guaranteed to test the therapeutic effect and
minor secondary effects of these compounds in vivo.
1000
750
500
250
0
Control
TNF + IL1B
4. Experimental
*
1
*
5
4.1. Chemical procedures
10
25
50
Melting points for pyrazoles 1–7 were determined by DSC on a
Seiko DSC 220C connected to a Model SSC5200H Disk Station and
for the other compounds a hot stage microscope was used. Ther-
mograms (sample size 0.003–0.0010 g) were recorded at the scan-
ning rate of 2.0 °C minꢀ1. Thin-layer chromatography (TLC) was
performed with Merck silica gel (60 F254). Compounds were de-
tected with a 254-nm UV lamp. Silica gel (60–320 mesh) was em-
ployed for routine column chromatography separations. Elemental
analyses for carbon, hydrogen, and nitrogen were carried out by
the Microanalytical Service of the ‘Universidad Complutense de
Madrid’ on a Perkin–Elmer 240 analyzer. The NMR spectra were re-
corded on a Bruker DRX 400 (9.4 T, 400.13 MHz for 1H, and
100.62 MHz for 13C) spectrometer with a 5-mm inverse detection
H-X probe equipped with a z-gradient coil at 300 K. Chemical shifts
(d in ppm) are given from internal solvent, for 1H, CDCl3 (7.26),
[D6]DMSO (2.49), for 13C, CDCl3 (77.0), [D6]DMSO (39.5). 2D
(1H–1H) gs-COSY and inverse proton detected heteronuclear shift
correlation spectra, (1H–13C) gs-HMQC, and (1H–13C) gs-HMBC
*
*
IC50:17.6 0.2
1000
750
500
250
0
Control
TNF + IL1B
*
1
5
*
10
25
50
*
*
IC50:8 0.1
Figure 5. MMP-9 activity in Caco-2 cells stimulated by TNF
in the presence of curcumin pyrazoles 1–3 at different concentrations (1, 5, 10, 25,
a
and IL-1b (10 ng/ml)
50 M). A, B and C represents the quantitative analysis of MMP-9 activity in
untreated cells (Control), stimulated cells with cytokines (TNF + IL-1) and stimu-
l
were acquired and processed using standard Bruker NMR software
50
and in non-phase-sensitive mode.
Commercial curcumin or
lated cells with cytokines and in the presence of the curcumin pyrazoles 1 (A), 2 (B)
(1E,6E)-1,7-bis(4-hydroxy-3-methoxyphenyl)hepta-1,6-diene-3,5-
dione (8) was used after purification and its NMR data have already
been published.51
*
and 3 (C) at different concentrations (1, 5, 10, 25 and 50
1b) and IC50, respectively.
l
M) ( p < 0.05 vs TNF
a+IL-
4.1.1. General procedure for the preparation of pyrazole
derivatives (1–7)
1000
Control
Compounds 1–7 were prepared by reacting the corresponding
b-diketones 8–14 (1 mmol) with hydrazine hydrate 98%
(1.5 mmol) in acetic acid (5 mL). After heating at reflux for 2 h
the reaction mixture was poured into water. The precipitate was
filtered off, washed with water and dried. The solid was purified
by column chromatography using ethyl acetate as eluent. The
NMR atom numbering for pyrazoles 1–7 is reported in Scheme 3.
TNF + IL1B
750
1
*
5
500
10
25
50
*
250
0
*
*
4.1.1.1. (E)-3,5-Bis[b-(4-Hydroxy-3-methoxyphenyl)-ethenyl]-1
H-pyrazole (1). Prepared from purified commercially available
curcumin (8). The compound was recrystallized from H2O–EtOH
to give a colorless solid (1 g, 2.74 mmol, 63%). Mp 217.1 °C, lit.
211–214 °C,32 215 °C.34 Anal. Calcd for C21H20N2O4 (364.14): C,
69.22; H, 5.53; N, 7.69. Found: C, 68.79; H, 5.53; N, 7.70. 1H
NMR ([D6]DMSO): d = 3.82 (s, 3H, 30-OCH3), 6.61 (s, 1H, H-4),
6.76 (d, J3 = 7.5 Hz, 1H, H-50), 6.91 (d, Jtrans = 16.7 Hz, 1H, H-80),
6.93 (d, J3 = 7.5 Hz, 1H, H-60), 7.03 (d, Jtrans = 16.7 Hz, 1H, H-70),
7.13 (s, 1H, H-20), 9.17 (s, 1H, 40-OH), 12.80 ppm (s, 1H, H–N).
13C NMR ([D6]DMSO): d = 55.6 (30-OCH3), 99.3 (C-4), 109.5 (C-20),
112.9 and 118.4 (C-80), 115.6 (C-50), 120.1 (C-60), 128.4 (C-10),
IC50: 8.5 0.2
Figure 6. Quantitative analysis of MMP-9 activity in untreated cells (Control),
stimulated cells with cytokines (TNF+IL-1) and stimulated cells with cytokines and
in the presence of curcumin 8 at different concentrations (1, 5, 10, 25 and 50
lM)
*
( p < 0.05 vs TNF
a
+IL-1b) and IC50.
noid pyrazoles derivatives are as effective as regular curcumin
inhibiting MMP-9 activity on inflammation-induced intestinal epi-
thelial cells. Hence, these new compounds raise the possibility of a
new therapeutic approach to human IBD.

1294
R. M. Claramunt et al. / Bioorg. Med. Chem. 17 (2009) 1290–1296
H
H
H
H
H
H
H
N
N
N
N
N
N
CH3
CH3
H
H
H
H
HO
HO
HO
OH
OCH3
CH3
OCH3
OCH3
OCH3
1
3
2
Scheme 2. The three bioactive pyrazoles.
129.8 (C-70), 142.0 (C-5), 146.8 (C-40), 147.9 ppm (C-30), 151.0 ppm
(C-3).
118.4 (36%) (C-80), 115.6 (64%) and 115. 3 (36%) (C-50), 120.2
(64%) and 122.1 (36%) (C-60), 125.1 (64%) and 125.0 (36%) (C-
o), 127.5 (C-p), 128.1 (C-10), 128.7 (C-m), 130.1 (C-70), 133.6
(64%) and 132.0 (36%) (C-ipso), 142.6 (64%) and 140.3 (36%)
(C-5), 147.1 (64%) and 146.6 (36%) (C-40), 147.9 (C-30), 151.0
(64%) and 151.4 ppm (36%) (C-3).
4.1.1.2. (E)-3(5)-[b-(4-hydroxy-3-methoxyphenyl)-ethenyl]-5(3)
-methyl-1H-pyrazole (2). Prepared from b-diketone 9. The com-
pound was recrystallized from CH2Cl2–hexane–EtOH to give a col-
orless solid (251 mg, 1.1 mmol, 85%). Mp 141.6 °C. Anal. Calcd for
C13H14N2O2 (230.11): C, 67.26; H, 6.44; N, 12.11. Found: C, 67.81;
H, 6.13; N, 12.17. 1H NMR ([D6]DMSO): d = 2.19 (s, 3H, 5(3)-CH3),
3.81 (s, 3H, 30-OCH3), 6.20 (s, 1H, H-4), 6.74 (d, J3 = 8.1 Hz, 1H, H-
50), 6.88 (d, Jtrans = 16.7 Hz, 1H, H-80), 6.91 (br, 1H, H-60), 6.95 (d,
Jtrans = 16.7 Hz, 1H, H-70), 7.12 (s, 1H, H-20), 9.15 (s, 1H, 40-OH),
12.40 ppm (s, 1H, H–N). 13C NMR ([D6]DMSO): d = 11.6 (5(3)-
CH3), 55.6 (30-OCH3), 101.3 (C-4), 109.5 (C-20), 115.7 (C-50), 117.4
(C-80), 119.9 (C-60), 128.6 (C-10), 129.0 (C-70), 140.5 (C-CH3), 146.6
(C-40), 147.9 (C-30), 149.6 ppm (C–C80 @C07).
4.1.1.5. (E)-3(5)-[b-(3,4-dimethoxyphenyl)-ethenyl]-5(3)-phe-
nyl-1H-pyrazole (5). Prepared from b-diketone 12. The com-
pound is obtained as a colorless solid after recrystallization from
CH2Cl2–hexane–EtOH (196 mg, 1.27 mmol, 51%). Mp 173.4 °C.
Anal. Calcd for C19H18N2O2 (306.14): C, 74.49; H, 5.92; N, 9.14.
Found: C, 74.21; H, 5.82; N, 9.16. 1H NMR ([D6]DMSO): d = 3.78
(s, 3H, 40-OCH3), 3.83 (s, 3H, 30-OCH3), 6.87 (s, 1H, H-4), 6.96 (d,
J3 = 8.3 Hz, 1H, H-50), 7.03 (d, Jtrans = 16.3 Hz, 1H, H-80), 7.06 (dd,
J3 = 8.3 Hz, J4 = 1.5 Hz, 1H, H-60), 7.14 (d, Jtrans = 16.3 Hz, 1H, H-70),
7.19 (s, 1H, H-20), 7.32 (br, 1H, H-p), 7.43 (br, 2H, H-m), 7.80 (br,
2H, H-o), 13.21 (60%) and 13.00 ppm (40%) (s, 1H, H–N). 13C NMR
([D6]DMSO): d = 55.45 (40-OCH3), 55.51 (30-OCH3), 99.2 (40%) and
99.8 (60%) (C-4), 108.9 (C-20), 111.9 (C-50), 113.6 (C-80), 119.4
(40%) and 119.9 (60%) (C-60), 125.0 (C-o), 127.4 (C-p), 128.6 (C-
m), 128.9 (40%) and 129.7 (60%) (C-70), 129.4 (C-10), 133.7 (C-ipso),
142.4 (60%) and 142.8 (40%) (C-5), 149.0 (C-30), 149.0 (C-40), 150.9
(60%) and 151.3 ppm (40%) (C-3).
4.1.1.3. (E)-3(5)-[b-(4-hydroxy-3-methoxyphenyl)-ethenyl]-4,5
(3)-dimethyl-1H-pyrazole (3).
Prepared from b-diketone 10.
The compound is obtained as a colorless solid after recrystalliza-
tion from CH2Cl2–hexane–EtOH (180 mg, 073 mmol, 61%). Mp
176.1 °C. Anal. Calcd for C14H16N2O2 (244.12): C, 68.46; H, 6.61;
N, 11.35. Found: C, 68.83; H, 6.60; N, 11.47. 1H NMR ([D6]DMSO):
d = 2.03 (s, 3H, 4-CH3), 2.10 (s, 3H, 5(3)-CH3), 3.83 (s, 3H, 30-OCH3),
6.75 (d, J3 = 8.2 Hz, 1H, H-50), 6.86 (d, Jtrans = 16.6 Hz, 1H, H-80), 6.91
(dd, J3 = 8.2 Hz, J4 = 2.0 Hz, 1H, H-60), 6.95 (d, Jtrans = 16.6 Hz, 1H, H-
70), 7.13 (d, J4 = 1.7 Hz, 1H, H-20), 9.08 (s, 1H, 40-OH), 12.29 ppm (s,
1H, H-N). 13C NMR ([D6]DMSO): d = 8.1 (4-CH3), 10.6 (5(3)-CH3),
55.7 (30-OCH3), 109.6 (C-20), 110.4 (C-4), 114.9 (C-80), 115.6 (C-
50), 119.8 (C-60), 127.9 (C-70), 128.8 (C-10), 141.6 (C-3 or C-5),
146.6 (C-40), 147.9 ppm (C-30).
4.1.1.6. (E)-3(5)-[b-(3,4-dimethoxyphenyl)-ethenyl]-4-methyl-
5(3)-phenyl-1H-pyrazole (6). Prepared from b-diketone 13. The
compound is obtained as a colorless solid after recrystallization
from CH2Cl2–hexane–EtOH (170 mg, 0.53 mmol, 58%). Mp
182.0 °C. Anal. Calcd for C20H20N2O2 (320.15): C, 74.98; H, 6.29;
N, 874. Found: C, 74.28; H, 6.14; N, 8.77. 1H NMR ([D6]DMSO):
d = 2.29 (s, 1H, 4-CH3), 3.77 (s, 3H, 40-OCH3), 3.84 (s, 3H, 30-
OCH3), 6.95 (d, J3 = 8.3 Hz, 1H, H-50), 7.06 (d, Jtrans = 16.7 Hz, 1H,
H-80), 7.07 (dd, J3 = 8.3 Hz, J4 = 1.7 Hz, 1H, H-60), 7.14 (d,
Jtrans = 16.7 Hz, 1H, H-70), 7.25 (d, J4 = 1.7 Hz, 1H, H-20), 7.34 (br,
1H, H-p), 7.45 (br, 2H, H-m), 7.65 (br, 2H, H-o), 12.94 ppm (s, 1H,
4.1.1.4. (E)-3(5)-[b-(4-hydroxy-3-methoxyphenyl)-ethenyl]-5(3)
-phenyl-1H-pyrazole (4). Prepared from b-diketone 11. The
compound is obtained as a colorless solid after recrystallization
from CH2Cl2–hexane–EtOH (228 mg, 0.78 mmol, 77%). Mp
142.9 °C. Anal. Calcd for C18H16N2O2 (292.12): C, 73.95; H,
5.52; N, 10.11. Found: C, 73.95; H, 5.54; N, 9.58. 1H NMR
([D6]DMSO): d = 3.84 (s, 3H, 30-OCH3), 6.78 (d, J3 = 8.1 Hz, 1H,
H-50), 6.88 (s, 1H, H-4), 6.95 (d, Jtrans = 16.5 Hz, 1H, H-80), 6.96
(dd, 1H, J3 = 8.1 Hz, J4 = 1.8 Hz, 1H, H-60), 7.10 (d, Jtrans = 16.5 Hz,
1H, H-70), 7.15 (s, 1H, H-20), 7.31 (t, J3 = J3 = 6.7 Hz, 1H, H-p), 7.43
(m, 2H, H-m), 7.80 (m, 2H, H-o), 9.21 (64%) and 9.10 (36%) (s,
1H, 40-OH), 13.18 (64%) and 12.96 ppm (36%) (s, 1H, H–N). 13C
NMR ([D6]DMSO): d = 55.6 (64%) and 55.5 (36%) (30-OCH3), 99.5
(64%) and 100.4 (36%) (C-4), 109.5 (C-20), 112.7 (64%) and
13
H–N). C NMR ([D6]DMSO): d = 9.4 (4-CH3), 55.5 (40-OCH3), 55.6
(30-OCH3), 109.1 (C-20), 110.7 (C-4), 111.9 (C-50), 114.5 (C-80),
119.9 (C-60), 127.1 (C-o), 127.3 (C-p), 1208.4 (C-70), 128.5 (C-m),
130.0 (C-10), 133.2 (C-ipso), 141.7 (C–C8@C07), 147.1 (C–C6H5),
148.8 (C-40), 149.1 ppm (C-30).
4.1.1.7. (E)-3(5)-[b-4-hydroxy-3-methoxyphenyl)-ethenyl]-5(3)-
vinylbenzaldehyde-1H-pyrazole (7). Prepared from b-diketone
14. The resulting product was a yellow solid after recrystallization
from CH2Cl2–hexane–EtOH (198 mg, 057 mmol, 51%). Mp 228.8 °C.
H
H
2 1
1
2
6'
N N
N N
10'
7'
1'
2'
OCH3
5'
4'
5
5
3
R
R
3
4
4
R1
9'
8'
R2
R2
CHO
3'
R1
OCH3
b
a
Scheme 3. NMR atom numbering of pyrazole derivatives.

R. M. Claramunt et al. / Bioorg. Med. Chem. 17 (2009) 1290–1296
1295
MS (EI) m/z: 347 [M+1]. Anal. Calcd for C21H18N2O3 (346.13): C,
72.82; H, 5.24; N, 8.09. Found: C, 66.41; H, 5.82; N, 11.81. 1H
NMR ([D6]DMSO): d = 3.83 (s, 1H, 30-OCH3), 6.72 (s, 1H, H-4),
6.78 (d, J3 = 8.2, 1H, H-50), 6.93 (d, Jtrans = 16.2, 1H, H-80), 6.94 (d,
J3 = 8.2, 1H, H-60), 7.07 (d, Jtrans = 16.2, 1H, H-70), 7.16 (d, J3 = 9.02,
1H, H-90), 7.16 (d, J3 = 9.02, 1H, H-100), 7.17 (s, 1H, H-20), 7.62 (m,
2H, H-o), 7.67 (64%) and 7.70 (36%) (m, 2H, H-m), 7.98 (64%) and
8.13 (36%) (s, 1H, H-CHO), 9.17 (s, 1H, 40-OH), 11.24 (64%) and
4.1.2.4. Methylation of (E)-5-(4-Hydroxy-3-methoxyphenyl)-1-
phenylpent-4-ene-1,3-dione (11). CH3I (190 l, 2.02 mmol)
l
was added to 11 (600 mg, 2.02 mmol) and K2CO3 (280 mg,
2.02 mmol) in dry acetone and the resulting mixture was stirred
at room temperature for 48 h. Then, acetone was evaporated under
reduced pressure and the residue was purified by column chroma-
tography using chloroform–diethyl ether (95:5) as eluent to give
the b-diketones 12 and 13.
13
11.36 ppm (36%) (H–N). C NMR ([D6]DMSO): d = 55.6 (30-OCH3),
100.1 (C-4), 109.5 (C-20), 112.5 (C-80), 115.6 (C-50), 120.1 (C-60),
126.6 (C-o), 127.1 (64%) and 127.4 (36%) (C-m), 128.3 (C-10),
128.3 (C-90), 128.3 (C-100), 129.9 (C-70), 133.5 (C-p), 138.1 (60%)
and 138.4 (36%) (C-ipso), 142.2 (64%) and 145.2 (36%) (CHO),
146.9 (C-40), 147.9 (C-30), 150.3 ppm (C-5 or C-3).
4.1.2.5. (E)-5-(3,4-Dimethoxyphenyl)-1-phenylpent-4-ene-1,3-
dione (12). The compound is obtained as a yellow solid (100 mg,
032 mmol, 16%). Mp 114–115 °C. 1H NMR (CDCl3): d = 3.91 (s, 3H,
OCH3-40), 3.93 (s, 3H, OCH3-30), 6.33 (s, 1H, CH@COH), 6.53 (d,
1H, Jtrans = 15.8 Hz, H-80), 6.88 (d, 1H, J3 = 8.2 Hz, H-50), 7.09 (d,
1H, J4 = 1.8 Hz, H-20), 7.15 (dd, 1H, J3 = 8.2 Hz, J4 = 1.8 Hz,
H-60), 7.46 (m, 2H, H-m), 7.53 (t, 1H, J3 = 7.3 Hz, H-p), 7.64
(d, 1H, Jtrans = 15.8 Hz, H-70), 7.95 (m, 2H, H-o), 16.27 ppm (s, 1H,
OH).
4.1.2. General procedure for the preparation of diketones 9–14
Appropriate b-diketone (75 mmol), boric anhydride (3.5 g,
54 mmol) and tri-n-butyl borate (23 g, 100 mmol) were mixed in
ethyl acetate (50 mL) at 0 °C with stirring. A solution of vanillin
(3.75 g, 25 mmol) and n-butylamine (0.5 mL) in ethyl acetate was
slowly added for 1.5 h. The reaction mixture was stirred at 0 °C
for 90 min and then overnight at room temperature. Hydrochloric
acid (0.4 N) was then added to the solution, at 60 °C, until pH be-
came acid. The reaction mixture was stirred for an additional hour
and then extracted with ethyl acetate. The organic layer was
washed with water (10 mL, 2 times), dried (Na2SO4) and evapo-
rated under vacuum. The product was isolated by column chroma-
tography with dichloromethane–diethyl ether (95:5) as eluent. The
NMR atom numbering for diketones 9–14 is reported in Scheme 4.
4.1.2.6. (E)-5-(3,4-Dimethoxyphenyl)-2-methyl-1-phenylpent-4
-ene-1,3-dione (13). The compound is obtained as a yellow pale
solid (400 mg, 1.23 mmol, 61%). Mp 112–113 °C. 1H NMR (CDCl3):
d = 1.54 (s, 3H, CH3-100), 3.89 (s, 6H, OCH3-30 and OCH3-40), 6.69 (d,
1H, Jtrans = 15.8 Hz, H-80), 6.83 (d, 1H, J3 = 8.3 Hz, H-50), 7.01 (dd, 1H,
J4 = 1.4 Hz, H-20), 7.10 (dd, 1H, J3 = 8.3 Hz, J4 = 1.4 Hz, H-60), 7.45 (m,
2H, H-m), 7.55 (t, 1H, J3 = 7.6 Hz, H-p), 7.60 (d, 1H, Jtrans = 15.8 Hz,
H-70), 8.00 ppm (m, 2H, H-o).
4.1.2.7. 4-((1E,6E)-7-(4-hydroxy-3-methoxyphenyl)-3,5-dioxoh
epta-1,6-dienyl)
benzaldehyde
(14). Feruloylacetone
9
4.1.2.1. (E)-6-(4-Hydroxy-3-methoxyphenyl)hex-5-ene-2,4-dio
ne (feruloylacetone) (9). From 2,4-pentanedione (7.5 g,
75 mmol) to give a yellow solid (2 g, 8.54 mmol, 34%): mp 141–
143 °C (lit. 146–147 °C42). 1H NMR (CDCl3) d: 2.16 (s, 3H, CH3-
110), 3.94 (s, 3H, OCH3), 5.63 (s, 1H, CH@COH), 5.83 (s, 1H, OH-
40), 6.32 (d, 1H, J = 15.8 Hz, H-80), 6.92 (d, 1H, J = 8.3 Hz, H-50),
7.02 (d, 1H, J4 = 1.9 Hz, H-20), 7.09 (dd, 1H, J3 = 8.3 Hz, J4 = 1.9 Hz,
H-60), 7.53 (d, 1H, J = 15.8 Hz, H-70), 15.46 ppm (s, 1H, OH).
(500 mg, 2.14 mmol), boric anhydride (230 mg, 3.20 mmol) and
tri-n-butyl borate (2.5 g, 10.05 mmol) in DMF (10 ml) were heated
at 80 °C, for 1 h.
A solution of terephthaldicarboxaldehyde
(145 mg, 1.07 mmol) and n-butylamine (0.15 ml) in DMF (2 ml)
was slowly added (60 min). After 5 h, the reaction mixture was
cooled down to 60 °C and hydrochloric acid (0.4 N) was added un-
til the reaction mixture reached pH 2–3. The mixture was stirred
for 1 h and extracted with dichloromethane. The organic layer
was washed with water (10 mL, 2 times), dried (Na2SO4) and evap-
orated under vacuum. The solid residue was purified by column
chromatography (eluent ethyl acetate–hexane 1:1giving com-
pound 14 as an orange solid (194 mg, 0.55 mmol, 52%). Mp 209–
211 °C. MS (EI) m/z: 351 [M+1]. 1H NMR ([D6]DMSO): d = 3.84 (s,
3H, 30-OCH3), 6.18 (s, 1H, H-100), 6.82 (d, 1H, Jtrans = 16.0 Hz, H-
800), 6.83 (d, 1H, J3 = 8.1 Hz, H-50), 7.09 (d, 1H, Jtrans = 15.9 Hz, H-
90), 7.18 (dd, 1H, J3 = 8.1 Hz, J4 = 1.7 Hz, H-60), 7.35 (d, 1H,
J4 = 1.3 Hz, H-20), 7.62 (d, 1H, Jtrans = 16.0 Hz, H-70), 7.93 (m, 2H,
H-o), 7.96 (m, 2H, H-m), 9.71 (s, 1H, 40-OH), 10.03 (s, 1H, CHO),
16.14 ppm (s, 1H, OH).
4.1.2.2. (E)-6-(4-Hydroxy-3-methoxyphenyl)-3-methylhex-5-en
e-2,4-dione
(10). From
3-methylpentane-2,4-dione
(9 g,
75 mmol) to give a yellow solid (800 mg, 3.22 mmol, 13%). Mp
1
138–140 °C. H NMR (CDCl3): d = 2.01 (s, 3H, CH3-100), 2.24 (s,
3H, CH3-110), 3.94 (s, 3H, OCH3-30), 5.88 (s, 1H, OH-40), 6.78 (d,
1H, Jtrans = 15.5 Hz, H-80), 6.92 (d, 1H, J3 = 8.2 Hz, H-50), 7.02 (d,
1H, J4 = 1.5 Hz, H-20), 7.13 (dd, 1H, J3 = 8.2 Hz, J4 = 1.5 Hz, H-60),
7.58 (d, 1H, Jtrans = 15.5 Hz, H-70), 16.45 ppm (s, 1H, OH).
4.1.2.3. (E)-5-(4-Hydroxy-3-methoxyphenyl)-1-phenylpent-4-e
ne-1,3-dione (11). From 1-phenylbutane-1,3-dione (12 g,
75 mmol) to give a yellow solid (6 g, 20.27 mmol, 82%). Mp 158–
4.2. Biological methods
1
161 °C. H NMR (CDCl3): d = 3.95 (s, 3H, OCH3-30), 5.86 (s, 1H,
OH-40), 6.33 (s, 1H, CH@COH), 6.52 (d, 1H, Jtrans = 15.8 Hz, H-80),
6.94 (d, 1H, J3 = 8.3 Hz, H-50), 7.07 (d, 1H, J4 = 1.9 Hz, H-20), 7.14
(dd, 1H, J3 = 8.2, J4 = 1.9 Hz, H-60), 7.47 (m, 2H, H-m), 7.53 (m, 1H,
H-p), 7.63 (d, 1H, Jtrans = 15.8 Hz, H-70), 7.95 (m, 2H, H-o),
16.26 ppm (s, 1H, OH).
4.2.1. Cell culture
Caco-2 cells, human intestinal epithelial cell line, were obtained
from European Collection of Cell Cultures (ECACC). Cells were cul-
tured in 75-cm2 flasks in a humidified atmosphere containing 5%
CO2 at 37 °C.
OH
10'
O
9'
O
OH
13'
12'
7'
7'
6'
6'
10'
11'
R1
1'
2'
OCH3
1'
2'
OCH3
5'
4'
5'
4'
9'
R
R
11'
8'
8'
CHO
R1
R2
R2
3'
3'
Scheme 4. NMR atom numbering of diketone derivatives in the enol forms.

1296
R. M. Claramunt et al. / Bioorg. Med. Chem. 17 (2009) 1290–1296
8. Di Sebastiano, P.; di Mola, F. F.; Artese, L.; Rossi, C.; Mascetta, G.; Pernthaler, H.;
For the in vitro experiments, intestinal cells were seeded into
24-well plates in free serum-medium and cultured in the presence
or absence of the combination of human recombinant TNF- (1, 10
and 100 ng/mL) and IL-1b (1, 10 and 10 ng/mL) for 24 h. In addi-
tion, cells were incubated in the presence of different synthetic
curcumin pyrazole derivatives at different concentrations (1, 5,
Innocenti, P. Digestion 2001, 63, 234.
9. Medina, C.; Santana, A.; Llopis, M.; Paz-Cabrera, M. C.; Antolin, M.; Mourelle, M.;
Guarner, F.; Vilaseca, J.; Gonzalez, C.; Salas, A.; Quintero, E.; Malagelada, J.-R.
Inflamm. Bowel Dis. 2005, 11, 99.
10. Medina, C.; Santana, A.; Paz, M. C.; Diaz, F.; Farre, E.; Salas, A.; Radomski, M. W.;
Quintero, E. J. Leukocyte Biol. 2006, 79, 954.
11. Medina, C.; Videla, S.; Radomski, A.; Radomski, M.; Antolin, M.; Guarner, F.;
Vilaseca, J.; Salas, A.; Malagelada, J.-R. Scand. J. Gastroenterol. 2001, 36, 1314.
12. Medina, C.; Videla, S.; Radomski, A.; Radomski, M. W.; Antolin, M.; Guarner, F.;
Vilaseca, J.; Salas, A.; Malagelada, J.-R. Am. J. Physiol. Gastrointest. Liver Physiol.
2003, 284, G116.
a
10, 25, 50 lM) 1 h before adding cytokines. Afterwards, condi-
tioned medium was collected for the study of MMP activity by
zymography assay. Further experiments were performed with cur-
cumin at the same doses.
13. Jovanovic, S. V.; Steenken, S.; Boone, C. W.; Simic, M. G. J. Am. Chem. Soc. 1999,
121, 9677.
14. Masuda, T.; Hidaka, K.; Shinohara, A.; Maekawa, T.; Takeda, Y.; Yamaguchi, H. J.
Agric. Food Chem. 1999, 47, 71.
15. Weber, W. M.; Hunsaker, L. A.; Abcouwer, S. F.; Deck, L. M.; Vander Jagt, D. L.
Bioorg. Med. Chem. 2005, 13, 3811.
16. Brouet, I.; Ohshima, H. Biochem. Biophys. Res. Commun. 1995, 206, 533.
17. Arbiser, J. L.; Klauber, N.; Rohan, R.; van Leeuwen, R.; Huang, M. T.; Fisher, C.;
Flynn, E.; Byers, H. Mol. Med. 1998, 4, 376.
18. Jacob, A.; Wu, R.; Zhou, M.; Wang, P. PPAR Res. 2007, 89369.
19. Sharma, R. A.; Ireson, C. R.; Verschoyle, R. D.; Hill, K. A.; Williams, M. L.;
Leuratti, C.; Manson, M. M.; Marnett, L. J.; Steward, W. P.; Gescher, A. Clin.
Cancer Res. 2001, 7, 1452.
4.2.2. Zymography
Gelatine zymography was used to detect MMP-9 and MMP-2
activity. Zymographicanalysiswascarriedoutonconditionedmedia
as previously described (12). Briefly, zymography was performed
subjecting samples to 10% sodium dodecyl sulfate-polyacrylamide
gel electrophoresis (SDS-PAGE) with copolymerized gelatin (0.2%;
Sigma Chemical Co, St. Louis, Mo) incorporated as a substrate for gel-
atinolytic proteases. After electrophoresis, the gels were washed
with 2% Triton X-100 (3 times, 20 minutes each), and then incubated
in development buffer (50 mml Tris HCl, 200 mmol NaCl, 10 mmol
20. Füllbeck, M.; Huang, X.; Dumdey, R.; Frommel, C.; Dubiel, W.; Preissner, R. BMC
Cancer 2005, 5, 97.
21. Siwak, D. R.; Shishodia, S.; Aggarwal, B. B.; Kurzrock, R. Cancer 2005, 104, 879.
22. Egan, M. E.; Pearson, M.; Weiner, S. A.; Rajendran, V.; Rubin, D.; Glockner-Pagel,
J.; Canny, S.; Du, K.; Lukacs, G. L.; Caplan, M. J. Science 2004, 304, 600.
23. Sundaryono, A.; Nourmamode, A.; Gardrat, C.; Fritsch, A.; Castellan, A. J. Mol.
Struct. 2003, 649, 177.
24. Zsila, F.; Bikadi, Z.; Simonyi, M. Org. Biomol. Chem. 2004, 2, 2902.
25. Kong, L.; Priyadarsini, I.; Zhang, H.-Y. J. Mol. Struct. (TheoChem) 2004, 684, 111.
26. Kim, G.-Y.; Kim, K.-H.; Lee, S.-H.; Yoon, M.-S.; Lee, H.-J.; Moon, D.-O.; Lee, C.-M.;
Ahn, S.-C.; Park, Y. C.; Park, Y.-M. J. Immunol. 2005, 174, 8116.
27. Aggarwal, B. B.; Kumar, A.; Aggarwal, M. S.; Shishodia, S. In Phytochemicals in
Cancer Chemoprevention; Bagchi, D., Preuss, H. G., Eds.; CRC Press: Boca Raton,
2005; p 349.
28. Maheshwari, R. K.; Singh, A. K.; Gaddipati, J.; Srimal, R. C. Life Sci. 2006, 78,
2081.
29. The Molecular Targets and Therapeutic Uses of Curcumin in Health and Disease;
Aggarwal, B. B., Shishodia, S., Surh, Y.-J., Eds.; Springer Publishers: New York,
2007.
CaCl2 and 1 lmol ZnCl2, pH 7.5) at 37C overnight. Internal standard,
conditioned medium of HT-1080 human fibrosarcoma cells (that
contains high amounts of proMMP-2, MMP-2, proMMP-9 and
MMP-9), were run as controls on the gels. After 48 h development,
gels were fixed and stained in 40% methanol, 10% acetic acid and
0,1% (wt/v) Coomassie Blue R-250 (Sigma Chemical Co, St. Louis,
Mo) for 1 h and then de-stained. Relative molecular weights of clear
bands were analyzed in comparison to the standards using a cali-
brated densitometer (GS-800, BioRad) and QuantityOne Quantita-
tion analysis software (BioRad, version 4). Zymographic activity of
each band was measured in terms of Optical Density (OD)/mm2.
4.2.3. Western-blotting
30. Wright, J. S. J. Mol. Struct. (TheoChem) 2002, 591, 207.
31. Jayaprakasha, G. K.; Jaganmohan Rao, L.; Sakariah, K. K. Food Chem. 2006, 98,
720.
Western-blot analysis of MMP-9 was performed in Caco-2 con-
ditioned media, as previously described.41 Samples were dena-
32. Flynn, D. L.; Belliotti, T. R.; Boctor, A. M.; Connor, D. T.; Kostlan, C. R.; Nies, D. E.;
Ortwine, D. F.; Schrier, D. J.; Sircar, J. C. J. Med. Chem. 1991, 34, 518.
33. Shim, J. S.; Kim, D. H.; Jung, H. J.; Kim, J. H.; Lim, D.; Lee, S.-K.; Kim, K.-W.; Ahn,
J. W.; Yoo, J.-S.; Rho, J.-R.; Shin, J.; Kwon, H. J. Bioorg. Med. Chem. 2002, 10, 2439.
34. Ishida, J.; Ohtsu, H.; Tachibana, Y.; Nakanishi, Y.; Bastow, K. F.; Nagai, M.;
Wang, H.-K.; Itokawa, H.; Lee, K.-H. Bioorg. Med. Chem. 2002, 10, 3481.
35. Ohtsu, H.; Xiao, Z.; Ishida, J.; Nagai, M.; Wang, H. K.; Itokawa, H.; Su, C. Y.; Shih,
C.; Chiang, T.; Chang, E.; Lee, Y. F.; Tsai, M. Y.; Chang, C.; Lee, K. H. J. Med. Chem.
2002, 45, 5037.
tured and loaded (50 lg/lane) onto 10% (vol/vol) polyacrylamide
gels. Following electrophoresis, proteins were transferred onto
nitrocellulose membrane (Protran, Schleider&Schuell) and de-
tected using a rabbit monoclonal anti-MMP-9 antibody (Chemicon,
Ca, USA) at 1:1000 concentration and a chemiluminescent sub-
strate (Pierce, Rockford, USA). To account for the inter-blot varia-
tions in MMP immunoreactivity, internal standard (human
recombinant MMP-9) was used. Relative molecular weights of
clear bands were analyzed in comparison to the standard using a
calibrated densitometer (GS-800, BioRad) and QuantityOne Quan-
titation analysis software (BioRad, version 4). Protein expression
of each band was measured in terms of Optical Density (OD)/mm2.
36. Selvam, C.; Jachak, S. M.; Thilagavathi, R.; Chakraborti, A. K. Bioorg. Med. Chem.
Lett. 2005, 15, 1793.
37. Narlawar, R.; Baumann, K.; Schubenel, R. Neurodegener Dis. 2007, 4, 88.
38. Narlawar, R.; Pickhardt, M.; Leuchtenberger, S.; Baumann, K.; Krause, S.; Dyrks,
T.; Weggen, S.; Mandelkow, E.; Schmidt, E. ChemMedChem 2008, 3, 65.
39. Levin, J. I.; Gu, Y.; Nelson, F. C.; Zask, A.; DiJoseph, J. F.; Sharr, M. A.; Sung, A.; Jin,
G.; Cowling, R.; Chanda, P.; Cosmi, S.; Hsiao, C.-L.; Edris, W.; Wilhelm, J.; Killar,
L. M.; Skotnicki, J. S. Bioorg. Med. Chem. Lett. 2001, 11, 239.
40. Shishodia, S.; Koul, D.; Aggarwal, B. B. J. Immunol. 2004, 2011.
41. Donnini, S.; Monti, M.; Castagnini, C.; Solito, R.; Botta, M.; Schenone, S.;
Giachetti, A.; Ziche, M. Int. J. Cancer 2007, 120, 995.
Acknowledgements
42. Cornago, M. P.; Cabildo, M. P.; Claramunt, R. M.; Bouissane, L.; Pinilla, E.; Torres,
M. R.; Elguero, J. New J. Chem. 2009, 33, 125.
43. Castaneda, F. E.; Walia, B.; Vijay-Kumar, M.; Patel, N. R.; Roser, S.; Kolachala, V.
L.; Rojas, M.; Wang, L.; Oprea, G.; Garg, P.; Gewirtz, A. T.; Roman, J.; Merlin, D.;
Sitaraman, S. V. Gastroenterology 2005, 129, 1991.
44. Santana, A.; Medina, C.; Paz-Cabrera, M.; Díaz-Gonzalez, F.; Farre, E.; Salas, A.;
Radomski, M. W.; Quintero, E. World J. Gastroenterol. 2006, 12, 6464.
45. Gan, X.; Wong, B.; Wright, S. D.; Cai, T.-Q. J. Interferon Cytokine Res. 2001, 21, 93.
46. Huhtala, P.; Tuuttila, A.; Chow, L. T.; Lohi, J.; Keski-Oja, J.; Tryggvason, K. J. Biol.
Chem. 1991, 266, 16485.
This work has been financed by the Spanish MEC (CTQ2006-
02586 and CTQ2007-62113) and by Stokes Science Foundation of
Ireland (SFI) program. Thanks are given to Dr. Sergio Erill and Dr.
Sebastian Videla that promoted the collaborative research.
References and notes
1. Fiocchi, C. Am. J. Physiol. Gastrointest. Liver Physiol. 1997, 273, G769.
2. Podolsky, D. K. N. Engl. J. Med. 2002, 34, 417.
47. Huhtala, P.; Eddy, R. L.; Fan, Y. S.; Byers, M. G.; Shows, T. B.; Tryggvason, K.
3. Baugh, M. D.; Perry, M. J.; Hollander, A. P.; Davies, D. R.; Cross, S. S.; Lobo, A. J.;
Taylor, C. J.; Evans, G. S. Gastroenterology 1999, 117, 814.
4. Heuschkel, R. B.; MacDonald, T. T.; Monteleone, G.; Bajaj-Elliott, M.; Smith, J. A.
W.; Pender, S. L. Gut 2000, 47, 57.
5. Kirkegaard, T.; Hansen, A.; Bruun, E.; Brynskov, J. Gut 2004, 53, 701.
6. Medina, C.; Radomski, M. W. J. Pharmacol. Exp. Ther. 2006, 318, 933.
7. Von Lampe, B.; Barthel, B.; Coupland, S. E.; Riecken, E. O.; Rosewicz, S. Gut 2000,
47, 63.
Genomics 1990, 6, 554.
48. Shakibaei, M.; John, T.; Schulze-Tanzil, G.; Lehmann, I.; Mobasheri, A. Biochem.
Pharmacol. 2007, 73, 1434.
49. Saja, K.; Babu, M. S.; Karunagaran, D.; Sudhakaran, P. R. Int. Immunopharmacol.
2007, 7, 1659.
50. Berger, S.; Braun, S. In 200 More NMR Experiments: A Practical Course; J Wiley &
Sons: New York, 2004.
51. Payton, F.; Sandusky, P.; Alworth, W. L. J. Nat. Prod. 2007, 70, 143.