Peroxisome proliferator-activated receptor-γ mediates the anti-inflammatory effect of 3-hydroxy-4-pyridinecarboxylic acid derivatives: Synthesis and biological evaluation

Article abstract of DOI:10.1016/j.ejmech.2013.01.024

Seven 3-hydroxy-4-pyridinecarboxylic acid derivatives (HPs), aza-analogues of salicylic acid and structurally close to other potent inflammatory pyridine compounds such as aminopyridinylmethanols and aminopyridinamines, were synthesized, and their anti-inflammatory activity was evaluated. The synthesis was performed by adopting a general procedure involving an intramolecular Diels-Alder cycloaddition of oxazoles with acrylic acid to form various substituted pyridinic acids. The newly synthesized HPs did not exhibit cytotoxic activity on human monocytes-derived macrophages at concentrations up to 10 ² μM. Anti-inflammatory activity of the compounds was screened in vitro by evaluating the capability to inhibit cytokines release from lipopolysaccharide (LPS) stimulated human macrophages. 3-Hydroxy-1-methyl-4- pyridinecarboxylic acid (24) was found to be the most active HP. At 10 μM concentration, HP 24 reduced LPS-induced and nuclear factor-κB activation and cyclooxygenase-2 expression, while increased intracellular reactive oxygen species generation and peroxisome proliferator-activated receptor (PPAR-γ) mRNA transcript level. Indeed, pre-treatment of LPS-exposed human macrophages with PPAR-γ specific antagonist completely prevented HP 24-induced TNF-α and IL8 down regulation, demonstrating that the PPARγ pathway is mandatory for the HP 24 anti-inflammatory effect. Finally, daily treatment with HP 24 ameliorated the outcome of DSS-induced colitis in mice, significantly reducing colonic MPO activity and IL-1β tissue levels.

Full text of DOI:10.1016/j.ejmech.2013.01.024

European Journal of Medicinal Chemistry 62 (2013) 486e497
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Peroxisome proliferator-activated receptor-g mediates the anti-
inflammatory effect of 3-hydroxy-4-pyridinecarboxylic acid
derivatives: Synthesis and biological evaluation
Paola Brun a, Annalisa Dean b, Valerio Di Marco b, Pathak Surajit c, Ignazio Castagliuolo a,
,
Davide Carta d, Maria Grazia Ferlin d
*
a Department of Molecular Medicine, University of Padova, Padova, Italy
b Department of Chemical Sciences, University of Padova, Padova, Italy
c Department of Surgical, Oncological and Gastroenterological Sciences, University of Padova, Padova, Italy
d Department of Pharmacological of Pharmaceutical Sciences, University of Padova, Marzolo 5, 35131 Padova, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Seven 3-hydroxy-4-pyridinecarboxylic acid derivatives (HPs), aza-analogues of salicylic acid and struc-
turally close to other potent inflammatory pyridine compounds such as aminopyridinylmethanols and
aminopyridinamines, were synthesized, and their anti-inflammatory activity was evaluated. The syn-
thesis was performed by adopting a general procedure involving an intramolecular DielseAlder cyclo-
addition of oxazoles with acrylic acid to form various substituted pyridinic acids. The newly synthesized
HPs did not exhibit cytotoxic activity on human monocytes-derived macrophages at concentrations up to
Received 28 September 2012
Received in revised form
11 December 2012
Accepted 19 January 2013
Available online 26 January 2013
102
to inhibit cytokines release from lipopolysaccharide (LPS) stimulated human macrophages. 3-Hydroxy-1-
methyl-4-pyridinecarboxylic acid (24) was found to be the most active HP. At 10 M concentration, HP 24
reduced LPS-induced and nuclear factor- B activation and cyclooxygenase-2 expression, while increased
intracellular reactive oxygen species generation and peroxisome proliferator-activated receptor (PPAR-
mRNA transcript level. Indeed, pre-treatment of LPS-exposed human macrophages with PPAR- specific
antagonist completely prevented HP 24-induced TNF- and IL8 down regulation, demonstrating that the
PPAR pathway is mandatory for the HP 24 anti-inflammatory effect. Finally, daily treatment with HP 24
ameliorated the outcome of DSS-induced colitis in mice, significantly reducing colonic MPO activity and
IL-1 tissue levels.
mM. Anti-inflammatory activity of the compounds was screened in vitro by evaluating the capability
Keywords:
aza-Salicylates
Hydroxypyridinecarboxylic acids
m
k
PPAR-
g
g
)
Anti-inflammatory agents
Macrophage
Inflammatory bowel disease
g
a
g
b
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
management of rubor, calor, dolor, and tumour, the classical hall-
marks elicited by inflammatory mediators encountered in epi-
thelial, stromal, endothelial, and haematopoietic cells [1,2]. At low
doses (5 mg/kg), the acetylated form of salicylic acid, acetylsalicylic
acid (ASP, universally known as aspirin) prevents phosphorylation
Non-steroidal anti-inflammatory drugs (NSAIDs) are the most
common agents used for the treatment of inflammatory diseases.
Synthetic anti-inflammatory compounds are effective in
and subsequent degradation of the inhibitor I
blocks nuclear factor- B (NF- B)-dependent gene activation such as
cyclooxygenase (COX)-2, cytokines and chemokines [3]. The inhi-
bition of NF- B activity and the modulation of inflammatory cyto-
kines putatively represent the main mechanisms of the anti-
inflammatory effect of 5-aminosalicylic acid (5-ASA) and sulfasa-
lazine commonly used for the treatment of chronic inflammatory
bowel diseases (IBDs), ulcerative colitis (UC) and Crohn’s disease
(CD) [4,5]. In vitro and in vivo studies however identified perox-
kB-a (IkB-a) and
k
k
Abbreviations: HP, hydroxypyridinecarboxylic acid; LPS, lipopolysaccharide;
k
PPAR-
g, peroxisome proliferator-activated receptor; DSS, dextran sulphate sodium;
NSAIDs, non-steroidal anti-inflammatory drugs; NF-
k
B, nuclear factor kappa-light-
chain-enhancer of activated B cells; TNF, tumour necrosis factor; IL8, interleukin-8;
MPO, myeloperoxidase; ASP, acetylsalicylic acid; 5-ASA, 5-aminosalicylic acid; IBD,
inflammatory bowel disease; TNBS, trinitrobenzene sulfonate; UC, ulcerative colitis;
CD, Crohn’s disease; COX, cyclooxygenase; rosig, rosiglitazone; SA, salicylic acid;
CMT, mouse; C57BL/ICRFat, rectum, carcinoma; ROS, reactive oxygen species; FACS,
fluorescence-activated cell sorting; RT-PCR, reverse transcription polymerase chain
reaction; PPREs, peroxisome proliferator response elements.
isome proliferator-activated receptor (PPAR-g) as a new target in
5-ASA anti-inflammatory activity. PPARs are members of the
nuclear receptor’s superfamily and functions as ligand-activated
* Corresponding author. Tel.: þ39 0498271603; fax: þ39 0498275366.
E-mail address: mariagrazia.ferlin@unipd.it (M.G. Ferlin).
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.01.024

P. Brun et al. / European Journal of Medicinal Chemistry 62 (2013) 486e497
487
transcriptional regulator of genes involved in several physiological
processes [6]. Thus, PPAR- maintains intestinal mucosal integrity
and prevents inflammatory damage during experimental colitis
[7,8]. Indeed, PPAR- heterozygous mice develop a more severe
16e20, 23, and 24, and the mechanisms of action, were evaluated in
primary human macrophages and mouse intestinal cell lines. As
well, in the attempt to find out a new therapeutic approach for the
treatment of inflammatory bowel disease (IBD), the anti-
inflammatory activity of HP 24 was investigated in an exper-
imental animal model of colitis. The formulae of the investigated
HPs are reported in Table 1.
g
g
þ/ꢀ
inflammation upon administration of trinitrobenzene sulfonate
(TNBS), whereas UC patients exhibit reduced expression of PPAR-
in colonic epithelial cells [9,10]. Worthy to note is the discovery of
PPAR- expression in the macrophage lineage and its up-regulation
during the monocytesemacrophages activation. Thus, PPAR-
g
g
g
2. Chemistry
levels are low in bone-marrow derived macrophages, and increase
in peritoneal macrophages activated by thioglycolate injection [11].
Synthetic route leading to 3-hydroxy-4-pyridinecarboxylic acids
(HPs) are depicted in Schemes 1A and B. For the synthesis of HPs, we
adopted a standard procedure used for obtaining some analogues
pyridine derivatives [21,22]. Such procedure exploits the oxazole
property to give intramolecular DielseAlder cycloaddition with
alkenes to form various substituted pyridines [23,24]. Firstly
(Scheme 1A), we synthesized the appropriate aminoacid derivatives
6e9 starting from the commercial aminoacids 1 and 2 that were
transformed into the corresponding ethoxycarbonyl derivatives by
heating with HCl 37% in absolute ethanol. The esters 3, 4 and the
commercial ethyl ester 5 (diethylaminomalonate) were treated
with triethyl orthoformate [25] at refluxing to give the formylamino
compounds 6e8 in high yields. Also, the diester 5 was transformed
into the corresponding acetyl amino-derivative 9 by reacting with
triethylorthoacetate.
Though the physiological role of PPAR-g in macrophages remains
poorly defined, evidences suggest that it may be a negative mod-
ulator of inflammation affecting macrophage gene expression [12].
In agreement with Murcko’s analysis of Comprehensive Me-
dicinal Chemistry (CMC) [13], pyridine ring is the most common
heteroaromatic ring overall, accounting for almost 25% of all het-
eroaromatics. Pyridine derivatives are of high interest in several
areas of medicinal chemistry. Specifically, some pyridine com-
pounds such as aminopyridinylmethanols and aminopyridin-
amines [14] have been considered as analgesic, as well as anti-
inflammatory agents and for treating Alzheimer’s disease [15].
The pyridine ring characterizes niflumic acid and flunixin [16], two
standard NSAIDs belonging to the class of fenamates (Fig. 1). These
drugs are derivatives of N-aryl substituted anthranilic acid (2-
amino-3-pyridinecarboxylic acid), and they are commonly used
as analgesic, anti-inflammatory and anti-pyretic agents, like salic-
ylates. Flunixin meglumine [17] is a substituted derivative of nic-
otinic acid (3-pyridinecarboxylic acid) and is unique structurally
when compared to other NSAIDs (Fig. 1).
Table 1
Formula and pKa values (if available) of the tested HPs.
HP
Reference
pKCOOH
pKNH
pKOH
Following the analogue-based drug discovery approach, we
became interested in evaluating the anti-inflammatory activity of
some 3-hydroxy-4-pyridinecarboxylic acid derivatives (HPs). These
molecules have the ortho hydroxy-carboxylic acid functions, like
salicylic acid derivatives, but on the pyridine ring. Moreover, they
are structurally related to the above mentioned anti-inflammatory
pyridine compounds. The simplest HP, 3-hydroxy-4-pyr-
idinecarboxylic acid (Fig. 1), was reported as non toxic anti-
inflammatory drug [18] and proposed as anti-phlogistic and anti-
erythematosus with a LD50 > 1000 mg/kg by Tendeloo in 1973
[19]. Some other HPs have been considered as novel chelating
agents for Fe(III) and Al(III), useful in treatment of various disorders,
including Alzheimer’s disease [20]. In this work we report the
synthesis of novel HPs 16e19. The anti-inflammatory activity of HPs
COOH
HO
20
0.47
5.64
11.18
N
16
COOH
HO
n.a.
n.a.
n.a.
N
17
COOH
HO
HO
20
20
26
0.48
4.93
10.64
N
18
COOH
HO
0.59
0.64
5.32
4.71
11.09
10.30
COOH
HO
COOH
CH3
N
H
N
H
N
19
CF3
CF3
COOH
HO
N
N
N
20
Flunixin
Niflumic acid
COOH
COO
N
HO
HO
COOH
COOH
20
27
0.40
0.28
7.70
6.63
N
OH
Cl-
OH
23
zwitterion 23
N
Salicylic acid
3-Hydroxy-4-pyridinecarboxylic acid
Fig. 1. Structures of some known anti-inflammatory drugs and of 3-hydroxy-4-
pyridinecarboxylic acid.

A) Synthesis of intermediate aminoacid derivatives 6-9
H
C
R
1-2
H
H
O
a
b
HOOC
C2H5OOC
C
R
NH2
C2H5OOC
C
R
NH2
NH C
H
3-5
6-8
H
C
R
c
C2H5OOC
5
NHCOCH3
R = CH3 (1, 3, 6); C2H5 (2, 4, 7);
COOC2H5 (5, 8, 9)
9
Reagents and conditions:
a) C2H5OH, HCl 37%, reflux, 5 h, 90-95%;
b) CH(OC2H5)3, reflux, 1.5 h, 90%;
c) CH3COC(OC2H5)3, reflux, 1.5 h, 80-90%.
B) Synthesis of the hydroxypyridinecarboxylic acid derivatives 16-24
a
R6
O
6-9
OCH2CH3
N
c
R2
10-13
COOH
R6
O
c
H3CH2CO
R2
b
OCH2CH3
12, 13
N
O
N
R6
R2
14, 15
COOH
COOH
COOH
OH
OH
OH
e
d
R6
N
R2
R6
N
R1
R2
I-
R6
N
R1
R2
Cl-
23, 24
21, 22
16-20
R2
R1
R6
Reagents and conditions:
10, 16
11, 17
12
-
-
-
-
-
-
-
CH3
H
H
a) P2O5, MgO; anhydrous CHCl3, rt, 20 h, 40-60%;
b) LiAlH4, diethyl ether, rt, 3-4 h, 50-70%;
c) CH=CHCOOH, rt, 15-30 min, 45-65%;
d) CH3I, KOH, aceton, 0°C, 4 h, 50-80%;
e) HCl 2N, athyl acetate.
C2H5
COOC2H5
COOC2H5
CH2OH
CH2OH
H
H
13,
CH3
H
14, 18
15, 19
20
CH3
H
21, 23
22, 24
CH3
CH3
CH3
H
H
H
Schemes 1. A) Synthesis of intermediate aminoacid derivatives 6e9. B) Synthesis of the hydroxypyridinecarboxylic acid derivatives 16e24.

P. Brun et al. / European Journal of Medicinal Chemistry 62 (2013) 486e497
489
In Scheme 1B, aminoacid derivatives 6e9 were submitted to the
24 significantly inhibited LPS-induced TNF
a
production, whereas
cyclization reaction in the presence of P2O5 and MgO in anhydrous
chloroform to obtain the oxazole derivatives 10e13. It is worth-
while to point out that the reaction didn’t work in the absence of
MgO acting as a Lewis acid. Oxazoles 12 and 13 were reduced by
LiAlH4 in anhydrous diethyl ether to give the corresponding oxazole
methyl alcohols 14 and 15. Then, the intramolecular DielseAlder
cycloaddition of oxazoles 10, 11 and 14, 15 with acrylic acid
quickly proceeded at room temperature to yield unstable bicycle
adducts which spontaneously turned into the desired pyridine
derivatives. The variously substituted pyridines 16e19 were
obtained with good yields (45e65%). Hydroxypyridinecarboxylic
acid derivative 16 [22] was methylated at the pyridinic N by means
of CH3I to the corresponding iodide 21, and then the latter was
transformed into chloride 23 by treatment with concentrated HCl.
In Scheme 1B, the previously synthesized compounds 20 [26], 22
and 24 [27] are also included.
HPs 19, 23, and 24 prevented LPS-induced IL-1b and IL8 release
(p < 0.05). The amplitude of anti-inflammatory activity of HPs was
comparable to the effects registered for SA and ASP. HPs 16, 17, 18
and 20 failed to significantly affect LPS-induced cytokines release.
The absence of activity for the latter compounds confirms that the
phenyl/pyridinyl replacement can significantly modify the bio-
logical properties of these compounds.
These results allow to draw some conclusions regarding the
relations between the HPs structure and their anti-inflammatory
activity. The substitutions on the pyridine ring at the positions 2
and/or 6 didn’t promote the anti-inflammatory activity, whereas
the 1-methyl substitution, leading to the HPs 23 and 24, turned out
to exert significant anti-inflammatory effects. The effect was par-
ticularly relevant for HP 24. A possible explanation of the different
activity of the examined HPs lies in their acidebase properties. The
pKa values for the HPs 16, 18, 19, 20, 23, 24 have been exper-
imentally obtained in previous works and are summarized in
Table 1 (references are given in the Table). The pKa values for HP 17
are expected to be very similar to those of HP 16. These values
demonstrate that only HPs 23 and 24 may exist in the form of
neutral zwitterions at physiological pH values and also inside
macrophages (it has been reported that the activated macrophages
pH is slightly acid [28]). This form represents the major or a sig-
nificant fraction of all possible protonated forms at these condi-
tions. For the other HPs the predominant form at neutral pH is
negatively monocharged, because the neutral one can exist only at
significantly lower pH values (in all cases below 6 or even below 5).
Therefore, it can be suggested that the structural requirement for
the anti-inflammatory activity of HPs is their neutrality at physio-
logical pH. This seems to be a peculiar property of HPs, as for
example salicylic acid is active in its charged form.
3. Results and discussion
3.1. Assessment of HPs cytotoxicity on human macrophages
Cytotoxicity was evaluated by MTTassayon human macrophages
treated for 72 h with HPs (concentration range 100
reported in Fig. 2, compounds 16,18, 20 and 24 exhibited significant
cytotoxic effects at 105 or 104
M, whereas no effects on cell viability
were observed at concentrations below 100 M for all tested HPs.
mMe0.1 mM). As
m
m
3.2. Evaluation of HPs anti-inflammatory activity
The anti-inflammatory activity of HPs was evaluated in vitro
using human monocyte-derived macrophages, a key cellular pop-
ulation involved in the inflammatory process. Macrophages were
As seen, HPs 23 and 24 appear to be the most active anti-
inflammatory compounds. Therefore, doseeresponse experiments
pre-treated for 30 min with 10
with 100 ng/mL LPS. Conditioned medium was collected and TNF
IL-1 , and IL-8 released were quantified by ELISA.
As reported in Fig. 3, LPS increased production and secretion of
pro-inflammatory cytokines in human macrophages. Thus, TNF
IL-1 , and IL-8 protein levels increased in LPS-stimulated cells as
mM HPs and then challenged for 24 h
(concentration range 100e0.001
mM) were performed on these
a
,
compounds in order to identify the lowest effective dose which
inhibits LPS-induced cytokine release. As reported in Table 2, only
b
HP 24 prevented LPS-induced TNF
tions up to 10 M. Therefore, additional experiments were per-
formed using HP 24 at a concentration of 10 M.
The anti-inflammatory activity of HP 24 was assessed also at
gene transcriptional level by evaluating the TNF- and IL8 mRNA
transcripts by quantitative RT-PCR. As reported in Fig. 4, HP 24
treatment significantly inhibited LPS-induced TNF- and IL8 mRNA
a and IL8 release at concentra-
a
,
m
b
m
compared to non-stimulated cells or cells treated with test com-
pounds alone (p < 0.02). Pre-treatment of human macrophages
a
with 10
mM SA and ASP significantly prevented the pro-
inflammatory activation triggered by LPS (p < 0.05). HPs 23 and
a
transcripts accumulation in human macrophages (p < 0.02 vs LPS-
treated cells).
3.3. HP 24 inhibits LPS-induced NF-
kB activation
During inflammation the NF- B transcriptional pathway repre-
k
sents the turning point in regulating the responses elicited by both
cytokines and LPS. Indeed, sulfasalazine inhibits LPS-induced NF-
k
B-dependent activation at concentrations as high as 1 mmol/L
[29]. To test whether HP 24 anti-inflammatory effects engage the
NF- B signalling pathway, CMT-93 cells were pre-treated with
10 M HP 24 and then stimulated with LPS as described above. As
reported in Fig. 5 panel A, LPS activated the NF- B pathway as
indicated by increased p65 phosphorylation and reduced IkB
protein levels. HP 24 prevented LPS-induced NF- B p65 phos-
phorylation and IkB degradation. Data were confirmed by luci-
ferase assay in CMT-93 cells transfected with a luciferase reporter
construct carrying an NF- B-responsive element. As reported in
k
m
k
a
k
a
k
Fig. 5 panel B, LPS increased the luciferase activity in CMT-93 cell
line as compared to untreated cells (p < 0.02). Pre-treatment of
mouse epithelial cells with 10 mM HP 24 significantly reduced the
Fig. 2. Cytotoxic effects of HPs. Human monocyte-derived macrophages were incu-
bated with the HPs 16e20, 23 and 24 for 72 h. Cellular viability was determined by
MTT test. Results are expressed as percentage of non treated cells (100% viability).

490
P. Brun et al. / European Journal of Medicinal Chemistry 62 (2013) 486e497
Fig. 3. Anti-inflammatory effects of HPs 16e20, 23 and 24. Human macrophages were pre-treated with 10
medium was collected and TNF (panel A), IL-1
(panel B), and IL-8 (panel C) levels were determined by using ELISA test kits. *Denotes p < 0.02 vs no stimulated cells; ꢁdenotes
p < 0.05 vs cells treated with LPS.
mM of each HP and challenged for 24 h with LPS (100 ng/mL). Conditioned
a
b
LPS-induced luciferase activity (p < 0.05). Suppression of the NF-
k
B
PPAR-
involved in inhibition of pro-inflammatory transcription pathways
such as AP-1, NFAT, C/EBP, Smad3 or NF- B [30,31]. To assess the role
of PPAR- in the anti-inflammatory effect of HP 24, it was first
evaluated whether the selected HP induces PPAR- gene expression
in human macrophages, and secondly whether this compound en-
hances PPAR- transcriptional activity in CMT-93 cells. As reported
in Fig. 6 panel A, HP 24 increased PPAR- mRNA transcript levels in
human macrophages as compared to untreated cells. A similar effect
was displayed by 30 mM 5-ASA (reported to recruit the peroxisome-
proliferator response elements for the anti-inflammatory activity
[9]) whereas rosiglitazone induced a huge increase in mRNA
g interacted with or competed for the recruitment of cofactors
signalling pathway leads to several intracellular effects such as
reduced expression of cytokines, COX-2, and adhesion molecules.
As reported in Fig. 5 panel C, the LPS-induced COX-2 over-
expression was dampened by the pre-treatment of mouse colonic
k
g
g
epithelial CMT-93 cells with 10
3.4. HP 24 anti-inflammatory effect requires PPAR-
Recently, PPAR- has been identified as a target of 5-amino-
mM HP 24.
g
g
g
activation
g
salicylic acid (5-ASA), suggesting an additional mechanism of anti-
inflammatory action in the colon. Upon troglitazone stimulation
expression ofPPAR-
PD68235, a specific PPAR-
anti-inflammatory effect of HP 24 (Fig. 6 panel B). The PD68235
withdrew the HP 24-induced TNF- mRNA transcript levels down
regulation following exposure to LPS. However, PPAR- antagonist
did not hamper the production and release of IL8 (Fig. 6 panel C).
Finally, HP 24 directly stimulates PPAR- transcriptional activity in
CMT-93 cells transfected with the luciferase reporter construct car-
rying PPAR- -responsive elements (PPAREs). As shown in Fig. 6
g. Pre-exposure of human macrophages to 20
mM
g
antagonist, completely prevented the
Table 2
Inflammatory cytokines levels in presence of HPs 23 and 24 measured in con-
ditioned medium of human macrophages using ELISA test kits. *Denotes p < 0.02 vs
untreated cells; ꢁdenotes p < 0.05 vs cells treated with LPS.
a
g
TNFa
(ng/mL)
IL8 (ng/mL)
Untreated
LPS
181 ꢂ 41
1.70 ꢂ 0.23
7.86 ꢂ 0.17*
4.43 ꢂ 0.32ꢁ
4.03 ꢂ 0.18ꢁ
5.40 ꢂ 0.07ꢁ
6.40 ꢂ 0.13
7.34 ꢂ 0.01
7.34 ꢂ 0.09
7.32 ꢂ 0.27
7.04 ꢂ 0.15
7.56 ꢂ 0.12
7.23 ꢂ 0.07
7.98 ꢂ 0.31
7.45 ꢂ 0.18
g
1682 ꢂ 190*
1069 ꢂ 161ꢁ
901 ꢂ 79ꢁ
100
m
M þ LPS
24
23
24
23
24
23
24
23
24
23
24
23
g
1084 ꢂ 143ꢁ
1404 ꢂ 150
1738 ꢂ 295
1394 ꢂ 117
1651 ꢂ 201
1368 ꢂ 191
1654 ꢂ 179
1418 ꢂ 341
1689 ꢂ 224
1626 ꢂ 227
panel D, LPS-induced inflammatory responses dampened the ac-
tivity of PPREs (p < 0.02 vs untreated cells) following 6 h of incu-
bation. HP 24 slightly induced PPAR-g promoter activity in CMT-93
cells as compared to not treated cells (no statistical difference)
whereas upon stimulation with LPS HP 24 completely prevented
10
m
M þ LPS
M þ LPS
M þ LPS
5
1
m
m
10ꢀ2
m
M þ LPS
M þ LPS
LPS-mediated inhibitory effect on PPAR-
cells). As positive control, murine colonic epithelial cells incubated
with 10 M rosiglitazone greatly recovered the PPAR activity
decreased by LPS stimulation (p < 0.001 vs cells treated with LPS).
g (p < 0.02 vs LPS-treated
10ꢀ3
m
m
g

P. Brun et al. / European Journal of Medicinal Chemistry 62 (2013) 486e497
491
Fig. 4. Effects of HP 24 on cytokine specific mRNA transcript levels. Human macrophages were pre-treated with 10
mM HP 24 and stimulated with LPS for 24 h. RNA was extracted
and specific mRNA transcript levels were evaluated by quantitative real-time PCR. *Denotes p < 0.02 vs untreated cells; xdenotes p < 0.02 vs cells treated with LPS.
3.5. HP 24 induces intracellular ROS generation
increased mucosal IL-1b and MPO levels (Fig. 8). Daily treatment
with HP 24 ameliorated the outcome of DSS colitis reducing by
about 56% of colonic MPO activity, similarly to the equal dose of 5-
aminosalicylic acid (5-ASA). Moreover, HP 24 reduced tissue levels
Intracellular reactive oxygen species (ROS) act as transduction
molecules to elicit several signalling pathways, including induction
of PPAR-
g
activity [32]. To assess the capability of HP 24 to generate
M HP 24,
of IL-1b whereas 5-ASA did not.
intracellular ROS, CMT-93 cells were treated with 10
m
challenged with LPS and loaded with the fluorescent probe DCFDA.
ROS generation was finally evaluated by FACS analysis.
4. Conclusion
As reported in Fig. 7 panel A, ROS increased in CMT-93 cells
treated with HP 24 as compared to cells treated with DCFDA alone.
Moreover the LPS challenge further increased the intracellular ROS
levels, as reported elsewhere [33]. To assess the role of ROS in HP
24-induced anti-inflammatory activity, CMT-93 were cultured with
The 3-hydroxy-4-pyridinecarboxylic acid derivatives (HPs)
shown in Table 1 were synthesized and studied as aza-analogues of
salicylic acid, the universally known anti-inflammatory drug
belonging to the NSAIDs class. HPs did not exhibit any cytotoxic
effect at concentrations up to 100
assay on human primary macrophages. On screening their anti-
inflammatory activity at a 10 M concentration, only HP 24 was
able to prevent LPS-induced TNF and IL8 up-regulation on human
mM when subjected to the MTT
the antioxidant agent
As reported in Fig. 7 panels B and C, the
dampened the anti-inflammatory effects of HP 24, similarly to the
PPAR- antagonist PD68235.
a
-tocopherol and treated with HP 24 and LPS.
a
-tocopherol treatment
m
a
g
macrophages as assessed by ELISA and quantification of specific
mRNA transcripts. In the attempt to find out new potent thera-
peutic approach for the treatment of inflammatory bowel disease
(IBD), HP 24 was investigated on mouse colonic epithelial cell line
and in animal model of colitis. HP 24 prevented activation of the
3.6. HP 24 reduces severity of DSS-induced colitis in mice
The anti-inflammatory effect of HP 24 was assessed on exper-
imentally induced colitis. Mice receiving 3.5% w/vol DSS exhibited
within 5 days a wasting syndrome, associated with diarrhoea and
NF-kB pathway and reduced COX-2 protein levels triggered by LPS,
comparable to NSAIDs. Furthermore, the anti-inflammatory activity
Fig. 5. Effects of HP 24 on NF-
Phosphorylation levels of p65, expression of IKB
transfected with NF- -responsive element and incubated as described. Results are reported as fold increase over untreated cells (panel B). *Denotes p < 0.02 vs untreated cells;
ꢁdenotes p < 0.05 vs cells treated with LPS.
kB signalling pathway and COX-2 expression. Colonic epithelial cells CMT-93 were pre-treated with 10
mM HP 24 and stimulated with LPS for 6 h.
a
(panel A) and of COX-2 (panel C), were assessed by Western blot analysis. Luciferase activity was measured in CMT-93 cells
k

492
P. Brun et al. / European Journal of Medicinal Chemistry 62 (2013) 486e497
Fig. 6. PPAR
g
activation is involved in HP 24 anti-inflammatory effect. Human macrophages were pre-treated with 10
(panel A) and TNF (panel B) mRNA transcript levels were evaluate by quantitative real-time PCR after respectively 6 and 24 h of incubation with
LPS 100 ng/mL. At 24 h IL8 protein levels were measured in the conditioned medium of human macrophages by ELISA test kits (panel C). Luciferase activity was assessed in CMT-93
mM HP 24, 30 mM 5-ASA, 10 mM rosiglitazone (rosig), or 20 mM
PPAR
g
antagonist PD68235. PPAR
g
a
cells transfected with PPAR-g
-(PPRE-Luc)-responsive element and stimulated with LPS for 6 h (panel D). *Denotes p < 0.02 vs untreated cells; ꢁdenotes p < 0.05 vs cells treated with
LPS 100 ng/mL; xdenotes p < 0.02 vs cells treated with LPS 100 ng/mL;^denotes p < 0.001 vs cells treated with LPS 100 ng/mL.
Fig. 7. Generation of intracellular reactive oxygen species by HP 24. CMT-93 cells were loaded with the redox sensitive dye 20,70-dichlorofluorescein diacetate for 30 min and
incubated with 10
m
M HP 24 and LPS 100 ng/mL. Fluorescent signal relative to intracellular ROS generation was evaluated by FACS analysis (panel A). Cells were incubated for 30 min
M antioxidant agent -tocopherol (antiox), and 20 M PPAR antagonist PD68235. CMT-93 cells were then challenged with 100 ng/mL LPS for 24 h. IL-1
a
(panel C) protein levels were measured in the conditioned medium by ELISA test kits. *Denotes p < 0.02 vs untreated cells; ꢁdenotes p < 0.02 vs cells treated
with 10 M HP 24, 100
m
m
a
m
g
b
(panel B) and TNF-
with HP 24 þ LPS.

P. Brun et al. / European Journal of Medicinal Chemistry 62 (2013) 486e497
493
Fig. 8. Effects of HP 24 on the severity of experimentally induced colitis. Mice were intragastrically treated for 7 days with HP 24 or 5-ASA (400 mg/kg body weight/day). At the 2nd
day, drinking water was supplemented with 3.5% (w/vol) dextran sodium sulphate (DSS). At the 7th day animals were scarified and severity of colitis was assessed by measurement
of MPO activity (panel A) and IL-1
b
protein levels (panel B) in homogenates of colonic samples. *Denotes p < 0.05 vs control animals; ꢁdenotes p < 0.05 vs animals administered
with DSS; xdenotes p < 0.02 vs animals administered with DSS.
of HP 24 requires indirect activation of PPAR-g. Thus, since anti-
5.2. Chemistry
oxidant agent prevented the HP 24-mediated anti-inflammatory
effects we are led to think that ROS generation mainly induced
PPAR-g activity. Indeed, preventive intragastric administration of
5.2.1. Synthesis of aminoacid ethyl esters hydrochlorides 3e5
As a typical procedure, a solution of commercial amino butanoic
acid (3.47 g, 33.67 mmol) in 150 mL absolute ethanol and 3 mL HCl
37% was refluxed for 5 h. The solvent was then evaporated and the
oily residue was dried under vacuum.
HP 24 reduced colonic myeloperoxidase activity in DSS-induced
colitis in the same way to 5-ASA, that is widely used to treat pa-
tients with IBD. Therefore, HP 24 shares many features with the
most common salicylic acid derivative, demonstrating a potent
anti-inflammatory effect both at circulating and local levels. Indeed,
following in vivo HP 24 administration, circulating macrophages
5.2.1.1. Ethyl 2-aminobutanoate (4). Yield 95%, transparent oil, solid
at RT; 1H NMR (400 MHz, D2O):
(q, J ¼ 7.25 Hz, 2H, eOCH2CH3), 1.70 (d, J ¼ 7.23 Hz, 3H, eCHCH3),
1.41 (t, J ¼ 7.25 Hz, 3H, eOCH2CH3).
d
4.47 (q, J ¼ 7.23 Hz, 1H, CH), 4.28
over-expressing PPAR-
g
could be recruited at the mucosal colonic
lesions and promote anti-inflammatory effects. Concluding, com-
pound 24 with its high anti-inflammatory effect mainly on
inflamed bowel mucosa merits further investigations as a potential
anti-inflammatory drug.
5.2.1.2. Diethyl 2-aminomalonate (5). Yield 93%, transparent oil,
solid at RT; 1H NMR (400 MHz, D2O):
d
4.39 (q, J ¼ 7.15 Hz, 2H, e
OCH2CH3), 4.15 (t, J ¼ 6.24 Hz, 1H, CH), 2.08 (m, J ¼ 7.63 Hz and
J ¼ 6.24 Hz, 2H, eCHCH2CH3), 1.38 (t, J ¼ 7.15 Hz, 3H, eOCH2CH3),
1.10 (t, J ¼ 7.63 Hz, 3H, eCHCH2CH3).
5. Experimental protocols
5.1. Materials and methods
5.2.2. Synthesis of ethyl formylamino derivatives 6e8
As
a
typical procedure,
4
g
4
(26.04 mmol) of ethyl 2-
and 13 mL (78.12 mmol,
Melting points were determined on a Gallenkamp MFB 595
010M/B capillary melting point apparatus, and are uncorrected.
Infrared spectra were recorded on a PerkineElmer 1760 FTIR
spectrometer with potassium bromide pressed disks; all values are
expressed in cmꢀ1. UVeVis spectra were recorded on a Perkine
Elmer Lambda UV/VIS spectrometer. 1H NMR spectra were deter-
mined on Bruker 300 and 400 MHz spectrometers, with the sol-
aminobutanoate hydrochloride
13.09 g) of triethyl orthoformate were refluxed for 1.5 h. The pro-
duced ethanol and excess orthoformate were removed by atmo-
spheric distillation to give a light orange oil. 7 g (26.04 mmol) of
diethylaminomalonate hydrochloride (5) and 30 mL (78.12 mmol,
11.58 g) of triethyl orthoformate were refluxed for 3 h.
vents indicated; chemical shifts are reported in
d (ppm) downfield
5.2.2.1. Ethyl 2-formylamino butanoate (7). Yield 85%, light orange
from tetramethylsilane as internal reference. Coupling constants
are given in Hertz. In the case of multiplets, chemical shifts were
measured starting from the approximate centre. Integrals were
satisfactorily in line with those expected on the basis of compound
structure. Elemental analyses were performed in the Micro-
analytical Laboratory, Department of Pharmaceutical Sciences,
University of Padova, on a PerkineElmer C, H, N elemental analyzer
model 240B, and analyses indicated by the symbols of the elements
were within ꢂ0.4% of the theoretical values. Mass spectra were
obtained on a Mat 112 Varian Mat Bremen (70 eV) mass spec-
trometer and Applied Biosystems Mariner System 5220 LC/MS
(nozzle potential 250.00). Column flash chromatography was per-
formed on Merck silica gels (250e400 mesh ASTM); chemical re-
actions were monitored by analytical thin-layer chromatography
(TLC) on Merck silica gel 60 F-254 glass plates. Solutions were
concentrated on a rotary evaporator under reduced pressure.
Starting materials were purchased from Aldrich Chimica and Acros,
and solvents from Carlo Erba, Fluka and Lab-Scan.
oil; 1H NMR (400 MHz, CDCl3):
d 8.18 (s, 1H, CHO), 7.20 (s, 1H, NH),
4.60 (q, J ¼ 7.20 Hz, 1H, eCHCH3), 4.21 (q, J ¼ 7.25 Hz, 2H, e
OCH2CH3), 1.43 (d, J ¼ 7.20 Hz, 3H, eCHCH3), 1.28 (t, J ¼ 7.25 Hz,
3H, eOCH2CH3).
5.2.2.2. Diethyl 2-formylaminomalonate (8). Yield 80%, orange oil;
1H NMR (400 MHz, CDCl3):
d 8.13 (s, 1H, CHO), 6.52 (bs, 1H, NH),
3.50 (q, J ¼ 7.06 Hz, 2H, eOCH2CH3), 1.70 (m, J ¼ 7.44 Hz and
J ¼ 7.06 Hz, 2H, eCHCH2CH3), 1.21 (t, J ¼ 7.06 Hz, 1H, eCHCH2CH3),
1.13 (t, J ¼ 7.06 Hz, 3H, eOCH2CH3), 0.83 (t, J ¼ 7.44 Hz, 3H, e
CHCH2CH3).
5.2.3. Synthesis of diethyl 2-acetamidomalonate (9)
In a 250 mL round bottom flask, to 4 g (18.90 mmol) of
diethylaminomalonate hydrochloride
5 were added 9.43 mL
(0.885 g mLꢀ1) of triethylorthoacetate, and the reaction mixture
was refluxed for 2 h (oil bath). In the meantime, the colourless

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P. Brun et al. / European Journal of Medicinal Chemistry 62 (2013) 486e497
solution became yellow-brown. At the end, the solution was
concentrated in a rotary evaporator giving a light yellow oily
product.
CDCl3): d 152.65 (2-C), 136.95 (5-C), 124.98 (4-C), 68.56 (e
OCH2CH3), 52.57 (eCH2OH), 14.79 (eOCH2CH3).
Yield 87%, yellow oil; 1H NMR (400 MHz, CDCl3): 6.47 (bs, 1H,
NH), 5.79 (s, 1H, CH), 4.15 (q, J ¼ 7.11 Hz, 4H, eOCH2CH3), 1.91 (s, 3H,
eCOCH3), 1.15 (t, J ¼ 7.11 Hz, 6H, eOCH2CH3).
5.2.5.2. (5-Ethoxy-2-methyloxazol-4-yl)methanol (15). Yield 65%;
Rf ¼ 0.85 (CHCl3/MeOH, 95:5); 1H NMR (400 MHz, CDCl3):
d 5.26
(bs, 1H, eOH), 4.59 (s, 2H, eCH2OH), 4.06 (q, J ¼ 7.11 Hz, 2H, e
OCH2CH3), 2.19 (s, 3H, eCH3),1.15 (t, J ¼ 7.11 Hz, 3H, eOCH2CH3); 13
C
5.2.4. Synthesis of oxazole derivatives 10e13
NMR (101 MHz, CDCl3): d 166.06 (2-C), 137.54 (5-C), 127.21 (4-C),
As a typical procedure, in a three necked oven dried 500 mL
round bottom flask, a suspension of P2O5 (20 g), celite (6 g) and
MgO (6 g) in 150 mL CHCl3 was vigorously stirred at room tem-
perature under inert atmosphere (N2), until it became homoge-
neous. A solution of ethyl N-formyl-2-aminobutanoate 6 (4.33 g,
27.19 mmol) dissolved in 50 mL CHCl3 was slowly added drop wise
and the stirring was continued for 30 min. The mixture was heated
to reflux for 20 h. A saturated solution (300 mL) of NaHCO3 was
slowly added to the cooled reaction mixture and then filtered to
remove the solids. The filtrate was extracted with chloroform, the
organic phase was washed with water, and dried with anhydrous
Na2SO4. On removing the solvent in a rotary evaporator, a dark
brown oil was obtained.
67.65 (eOCH2CH3), 52.05 (eCH2OH), 14.90 (eOCH2CH3).
5.2.6. Synthesis of 3-hydroxy-4-pyridinecarboxylic acid derivatives
16e20
As a typical procedure, in a 100 mL round bottom flask, 3.33 g
(23.6 mmol) of oxazole 11 were reacted with 3 g (2.86 mL,
41.23 mmol) of acrylic acid at room temperature. After 15 min the
formation of a precipitate was observed which resulted complete
after some hours at 4 ꢁC. The thin white crystalline product was col-
lected, re-crystallized from water and dried under vacuum at 40 ꢁC.
5.2.6.1. 2-Ethyl-3-hydroxy-4-pyridinecarboxylic acid (17). Yield 40%,
white solid; m.p. decomposition; 1H NMR (400 MHz, D2O/NaOD):
d
7.46 (d, J ¼ 4.96 Hz, 1H, 6-H), 6.88 (d, J ¼ 4.96 Hz, 1H, 5-H), 2.67 (q,
5.2.4.1. 5-Ethoxy-4-ethyloxazole (11). Yield 50%; Rf ¼ 0.80 (CHCl3/
J ¼ 7.63 Hz, 2H, eCH2CH3), 1.08 (t, J ¼ 7.63 Hz, 3H, eCH2CH3); 13C
MeOH, 9:1); 1H NMR (400 MHz, CDCl3):
d
7.34 (s, 1H, CH), 4.12 (q,
NMR (101 MHz, D2O/NaOD): d
178.84 (eCOOꢀ), 157.74 (3-C), 156.37
J ¼ 7.06 Hz, 2H, eOCH2CH3), 2.40 (q, J ¼ 7.63 Hz, 2H, eCH2CH3), 1.13
(2-C), 135.89 (6-C), 131.41 (4-C), 120.24 (5-C), 29.91 (eCH2CH3),
25.46 (eCH2CH3); Anal. Calcd. for C8H9NO3 (167.16): C 57.48, H
5.42, N 8.39, found: C 57.30, H 5.39, N 8.29; HRMS (ESI, 140 eV): m/z
[M þ Hþ] calcd for C8H10NO3: 168.0661, found: 168.0571.
(t, J ¼ 7.06 Hz, 3H, eOCH2CH3), 1.48 (t, J ¼ 7.63 Hz, 3H, eCH2CH3);
13C NMR (101 MHz, CDCl3):
d 151.43 (2-C), 138.12 (5-C), 125.44 (4-
C), 68.40 (eOCH2CH3), 17.73 (eCH2CH3), 14.88 (eOCH2CH3), 13.05
(eCH2CH3).
5.2.6.2. 3-Hydroxy-2-(hydroxymethyl)-4-pyridinecarboxylic
acid
5.2.4.2. Ethyl 5-ethoxyoxazole-4-carboxylate (12). Yield 60%;
(18). Yield 40%, white solid; m.p. decomposition; 1H NMR
Rf ¼ 0.68 (CHCl3/MeOH, 9:1); 1H NMR (400 MHz, CDCl3):
d
7.30 (s,
(400 MHz, D2O/NaOD):
d
7.20 (d, J ¼ 5.77 Hz, 1H, 6-H), 6.40 (d,
1H, CH), 4.53 (q, J ¼ 7.06 Hz, 2H, eCOOCH2CH3), 4.30 (q, J ¼ 7.25 Hz,
J ¼ 5.77 Hz, 1H, 5-H), 4.25 (s, 2H, eCH2OH); 13C NMR (101 MHz,
2H, eOCH2CH3), 1.40 (t, J ¼ 7.06 Hz, 3H, eCOOCH2CH3), 1.33 (t,
D2O/NaOD): d
178.16 (eCOOꢀ), 158.31 (3-C), 149.66 (2-C), 137.52 (6-
J ¼ 7.25 Hz, 3H, eCH2CH3); 13C NMR (101 MHz, CDCl3):
d
168.19 (e
C), 132.06 (4-C), 121.49 (5-C), 62.29 (eCH2OH); Anal. Calcd. for
C7H7NO4 (169.13): C 49.71, H 4.17, N 8.28, found: C 49.67, H 4.23, N
8.17; HRMS (ESI, 140 eV): m/z [M þ Hþ] calcd for C7H8NO4:
170.0453, found: 170.0424.
COOe), 153.24 (2-C), 136.92 (5-C), 127.32 (4-C), 67.19 (eOCH2CH3),
60.94 (eCOOeCH2CH3), 14.85 (eOCH2CH3), 14.10 (eCOOeCH2CH3).
5.2.4.3. Ethyl
5-ethoxy-2-methyloxazole-4-carboxylate
(13).
Yield 52%; Rf ¼ 0.57 (CHCl3/MeOH, 9:1);
d
4.20 (q, J ¼ 7.11 Hz, 2H, e
5.2.6.3. 3-Hydroxy-2-(hydroxymethyl)-6-methyl-4-pyridinecarboxylic
COOeCH2CH3), 4.13 (q, J ¼ 7.05 Hz, 2H, eOCH2CH3), 2.36 (s, 3H, e
CH3), 1.25 (t, J ¼ 7.11 Hz, 3H, eCOOeCH2CH3), 1.16 (t, J ¼ 7.05 Hz, 3H,
acid (19). Yield 30%, white solid; m.p. decomposition; 1H NMR
(400 MHz, D2O/NaOD):
d
6.65 (s,1H, 5-H), 4.43 (s, 2H, eCH2OH), 2.06
eOCH2CH3); 13C NMR (101 MHz, CDCl3):
d
167.94 (eCOOe), 165.06
(s, 3H, eCH3); 13C NMR (101 MHz, D2O/NaOD):
d
177.94 (eCOOꢀ),
(2-C), 138.06 (5-C), 126.38 (4-C), 69.04 (eOCH2CH3), 61.06 (eCOOe
CH2CH3), 25.50 (eCH3), 14.79 (eOCH2CH3), 14.23 (eCOOeCH2CH3).
155.90 (3-C), 148.55 (6-C), 141.07 (2-C), 138.79 (4-C), 121.35 (5-C),
62.53 (eCH2OH), 21.79 (eCH3); Anal. Calcd. for C8H9NO4 (183.16): C
52.46, H 4.95, N 7.65, found: C 52.19, H 4.84, N 7.62; HRMS (ESI,
140 eV): m/z [M þ Hþ] calcd for C8H10NO4: 184.0610, found:
184.0608.
5.2.5. Synthesis of oxazole derivatives 14 and 15
As a typical procedure, in a two necked oven dried 250 mL round
bottom flask, 3.00
g
(16.20 mmol) of 4-carboxyethyl-5-
ethoxyoxazole 12 dissolved in 50 mL of anhydrous diethyl ether
were added to a suspension of 0.80 g (21.08 mmol) of LiAlH4 in
150 mL of anhydrous diethyl ether. The resulting solution was kept
under stirring at room temperature for about 20 h, maintaining the
anhydrous environment. At the end of the reaction, 200 mL of
a saturated solution of NH4Cl were cautiously added and the mix-
ture was then filtered. The filtrate was extracted with dichloro-
methane (3 ꢃ 50 mL), the organic extracts were combined and
washed with distilled H2O and dried with anhydrous Na2SO4. The
extract was concentrated in rotary evaporator and was brought to
dryness under vacuum obtaining a yellow oily residue.
5.2.7. Synthesis of 1-methyl-3-hydroxy-4-pyridinecarboxylic acid
derivatives 23 and 24
According to a typical procedure, in a 100 mL round bottom
flask, 2 g (13.06 mmol) of the acid 16 were added to 30 mL of
acetone. The mixture was alkalized with a 20% NaOH solution to
completely dissolve the acid. 0.82 mL (13.1 mmol, 1.87 g) of com-
mercial CH3I were then added and the reaction mixture was stirred
at room temperature for about 4 h. The solvent was moved off in
a rotary evaporator and the residue was taken up in a small volume
of distilled water (30 mL), acidified with 2 M HCl and stirred with
slight heating for about 30 min. After concentration of the solution
to about a half volume in a rotary evaporator, the formed solid
product was collected by filtration, and a white residue was
obtained. To remove the NaCl present as an impurity, the residue
was treated with a small volume of absolute ethanol (15 mL), the
suspension was filtered, and to the filtrate was added an excess of
5.2.5.1. (5-Ethoxyoxazol-4-yl)methanol (14). Yield 40%; Rf ¼ 0.75
(CHCl3/MeOH, 95:5); 1H NMR (400 MHz, CDCl3):
d 7.40 (s, 1H, CH),
5.39 (bs, 1H, eOH), 4.65 (s, 2H, eCH2OH), 4.12 (q, J ¼ 7.08 Hz, 2H, e
OCH2CH3), 1.34 (t, J ¼ 7.08 Hz, 3H, eOCH2CH3); 13C NMR (101 MHz,

P. Brun et al. / European Journal of Medicinal Chemistry 62 (2013) 486e497
495
diethyl ether, obtaining the precipitation of the desired compound.
This was collected and dried under vacuum.
solution, PBS) was added to each well. Plates were incubated at
37 ꢁC for 4 h. Tetrazolium crystals were dissolved by adding 100
m
L
of DSS 10% w/vol in HCl 2 M. Optical densities at 620 nm were
measured and recorded using SunRise plate reader and Magellan
software (Tecan Group Ltd, Mannedorf, Switzerland).
5.2.7.1. 3-Hydroxy-1,2-dimethyl-4-pyridinecarboxylic acid chloride
(23). Yield 90%, white solid; m.p. decomposition; 1H NMR
(400 MHz, D2O/NaOD):
d
7.50 (d, J ¼ 6.10 Hz, 1H, 6-H), 6.98 (d,
J ¼ 6.10 Hz, 1H, 5-H), 3.70 (s, 3H, eNCH3), 2.90 (s, 3H, eCH3); 13C
5.3.3. Anti-inflammatory activity
NMR (101 MHz, D2O/NaOD):
d
175.30 (eCOOꢀ), 161.98 (3-C), 148.39
Monocytes-derived human macrophages were grown in 96-
(2-C), 140.38 (6-C), 127.90 (4-C), 121.49 (5-C), 46.59 (eNCH3), 30.27
(eCH3); Anal. Calcd. for C8H10NO3þ (168.17): C 57.14, H 5.99, N 8.33,
found: C 56.94, H 5.87, N 8.28; HRMS (ESI, 140 eV): m/z [M þ Hþ]
calcd for C8H10NO3: 168.0661, found: 168.0591.
well culture plates and pre-treated with 10 mM HP, salicylic acid
(SA), and acetylsalicylic acid (ASP) for 30 min at 37 ꢁC. Cells were
then challenged with 100 ng/mL lipopolysaccharide (LPS, from
Salmonella enteritidis serotype Olll:B4; Sigma, Milan, Italy) and
incubated for 24 h. Conditioned medium was collected and TNF
a,
5.2.7.2. 3-Hydroxy-1-methyl-4-pyridinecarboxylic acid chloride (24).
IL-1 , and IL-8 were quantified using commercially available ELISA
b
Yield 88%, white solid; m.p. decomposition; 1H NMR (400 MHz,
kits (eBioscience; Prodotti Gianni, Milan, Italy). Optical densities
were measured using an ELISA plate reader (SunRise, Tecan) at
450 nm. Cytokine levels were expressed as nanogram or picogram
per millilitre. Sensitivities of the assays ranged from 3 to 12 pg/mL.
D2O/NaOD):
5-H), 7.92 (s, 1H, 2-H), 4.07 (s, 3H, eCH3); 13C NMR (101 MHz, D2O/
NaOD):
175.28 (eCOOꢀ), 161.49 (3-C), 153.19 (2-C), 141.06 (6-C),
d
9.10 (d, J ¼ 4.98 Hz, 1H, 6-H), 8.38 (d, J ¼ 4.98 Hz, 1H,
d
127.89 (4-C), 121.80 (5-C), 45.62 (eNCH3); Anal. Calcd. for
þ
C7H8NO3 (154.14): C 54.54, H 5.23, N 9.09, found: C 54.36, H 5.14,
5.3.4. RNA extraction and quantitative real-time polymerase chain
reaction
The SV total RNA isolation system (Promega, Milan, Italy) was
used to extract total RNA according to the manufacturer’s protocol.
Samples were homogenized in lysis buffer. Contaminating DNA was
þ
N 9.10; HRMS (ESI, 140 eV): m/z [M þ Hþ] calcd for C7H8NO3
:
154.0504, found: 154.0494.
5.3. Biology
removed by DNase I treatment (Promega), and total RNA (3 mg) was
5.3.1. Cell culture
reverse transcribed to cDNA using random primers and Moloney
murine leukaemia virus reverse transcriptase (Applied Biosystems,
Milan, Italy). Real-time quantitative polymerase chain reaction
(qPCR) was performed on an ABI PRISM 7700 Sequence Detection
System (Perkin Elmer, Norwalk, CT) for 40 cycles at 60 ꢁC of
annealing temperature. Reactions were performed using TaqMan
Universal PCR Master Mix and Universal Probe Library (UPL, Roche,
Milan, Italy) carrying a fluorescent dye, according to the manufac-
turer’s instructions. For each gene, standard curves were obtained
by amplification of the corresponding cDNAs sub-cloned into the
pGEM-T vector (Promega). Expression of the target genes was
normalized to the endogenous control gene, glyceraldehyde-3-
phosphate dehydrogenase (GAPDH). The oligonucleotide se-
quences and probes are listed in Table 3. As positive control cells
Monocytes were isolated from freshly collected buffy-coat
preparations of whole human blood. Blood was diluted 1:1 (vol:-
vol) in endotoxin-free RPMI 1640 medium (BioWhittaker, Wal-
kersville, MD) and overlaid onto FicolleHypaque gradient
(Pharmacia, Uppsala, Sweden). After centrifugation (20 min at 1200
revolutions per minute, rpm), peripheral blood mononuclear cells
(PBMC) were collected, washed and cultured (3 ꢃ 106 cells/mL) in
RPMI 1640 medium supplemented with 10% vol/vol heat inacti-
vated foetal calf serum (FCS), 100 U/mL penicillin and 100 mg/mL
streptomycin (all provided by Invitrogen, Milan, Italy). Two hours
later, non adhering cells were removed by extensive washing with
endotoxin-free RPMI 1640 medium, and adhering monocytes were
differentiated in mature macrophages in presence of 2 ng/mL re-
combinant human Macrophage Colony Stimulating Factor (rh M-
CSF; ImmunoTools GmbH, Friesoythe, Germany) for 10 days. Cul-
ture medium was renewed every three days. At 10th day of culture,
purity of macrophages was greater than 95% as determined by
nonspecific esterase, CD14 staining and overall morphology. Via-
bility was greater than 99% by trypan blue exclusion.
were treated for 6 h with 10 mM rosiglitazone (Tocris, Leeds, UK).
5.3.5. Western blot analysis
Monocytes-derived human macrophages were grown in 6-well
culture plates for 10 days. Then the cells were washed and incu-
bated in fresh medium in presence or absence of HP 24 for 30 min
and then stimulated with LPS (100 ng/mL) for 6 h at 37 ꢁC. At the
end of incubation, macrophages were washed twice with ice-cold
PBS and treated for 45 min at 4 ꢁC with non-denaturing RIPA
lysis buffer (1% vol/vol Triton X-100, 0.5% w/vol deoxycholic acid,
The mouse colonic epithelial cell line CMT-93 (European Collec-
tion of Cell Cultures, Porton Down, UK) was maintained in Dulbecco’s
modified Eagle medium supplemented with 10% vol/vol heat-
inactivated FCS, 100 U/mL penicillin and 100 mg/mL streptomycin.
10 mM EDTA, 1
mM leupeptin, 150 nM aprotinin, 500 mM 4-(2-
5.3.2. Cytotoxicity assay
HPs were dissolved in DMSO at 10 M stock solution following by
dilution in 1 M TriseHCl (pH 8.00) prepared using endotoxin-free
aminoethyl)benzenesulfonylfluoride in PBS). Particulate material
was removed by centrifugation (15,000 g for 10 min, at 4 ꢁC). Su-
pernatants were collected, and protein concentrations were
water. Each HP working solution (1 mM, pH 7.40) was tested for
the endotoxin contamination using the BioWhittaker QCL-1000
chromogenic limulus amoebocyte lysate test kit according to the
manufacturer’s instructions (BioWhittaker) and as reported else-
where [34]. Endotoxin concentrations were below the minimum
value detectable by the test (3 pg/mL) in all samples.
Table 3
The list of genes evaluated by quantitative RT-PCR.
Gene (accession number) Primer sequences
hGADPH (NM_002046)
UPL probe Ta (ꢁC)
fw: 50-cgggaagcccatcacca-30
60
60
60
60
59
rv: 50-ccggcctcaccccatt-30
fw: 50-gacaagcctgtagcccatgt-30
rv: 50-tctcagctccacgccatt-30
Macrophages cultured in 96-wells tissue culture plates were
hTNF
hIL8 (NM_000584)
hPPAR (NM_138712)
a (NM_000594)
79
treated with each HP (concentration range 100
mMe0.1 mM) at
37 ꢁC for 72 h. To exclude cytotoxic effects, macrophage viability
was assessed by the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-
diphenyl tetrazolium bromide; Sigma, Milan, Italy) test [35].
fw: 50-gagcactccataaggcacaaa-30 72
rv: 50-atggttccttccggtggt-30
fw: 50-ttgctgtcattattctcagtgga-30
rv: 50-gaggactcagggtggttcag-30
g
1
Briefly, 10
mL of MTT solution (5 mg/mL in phosphate buffer

496
P. Brun et al. / European Journal of Medicinal Chemistry 62 (2013) 486e497
determined using the Bicinchoninic acid (BCA) Protein Assay Kit
(Thermo Scientific, Rockford, USA). Equal amounts of proteins
allocated to receive intragastrically 400 mg/kg body weight/day HP
24, 5-ASA, or vehicle alone (100 L) [9]. Eight to ten animals per
m
(30
6.8, 10% vol/vol glycerol, 2% w/vol sodium dodecyl sulphate, 5% vol/
vol -mercaptoethanol, and 0.1% w/vol bromophenol blue) and
m
g) were added to the sample loading buffer (62.5 mM Tris, pH
group were treated. After 2 days, their drinking water was sup-
plemented with 3.5% (w/vol) dextran sodium sulphate (DSS) (TdB
Consultancy, Uppsala, Sweden) to induce colitis. Animal’s body
weight was measured daily, and 5 days after colitis induction ani-
mals were killed. The proximal colon was removed, flatly opened
and washed in PBS. Full-thickness colonic samples were processed
b
incubated for 5 min at 100 ꢁC. Proteins were then fractionated
through a 10% w/vol SDS-polyacrylamide gel and immobilized on
a PVDF membrane (BioRad, Milan, Italy). Nonspecific binding sites
were blocked by incubating PVDF membranes for 1 h at 22 ꢁC in 5%
w/vol non-fat dry milk in PBS containing 0.05% vol/vol Tween 20
(PBS-T). The PVDF membranes were then incubated overnight at
to determine myeloperoxidase (MPO) activity and IL-1
b protein
levels as previously described [30].
4 ꢁC with anti-phospho NF-
Euroclone, Milan, Italy), I
k
B p65 (mouse, 1:1000, Cell Signalling;
5.4. Statistical analysis of biological data
k
Bh (rabbit, 1:1000, Cell Signalling), or
cyclooxigenase (COX)-2 (goat, 1:1000, Santa Cruz Biotechnology
Inc., Heidelberg, Germany) antibodies. After extensive washings in
PBS-T, the bound antibody was detected by incubating the PVDF
membrane with horseradish peroxidase-conjugated goat anti-
rabbit (Sigma). Finally, the enzymatic reaction was developed us-
ing ECL detecting reagents (Millipore; Milan, Italy) and exposed to
Data are expressed as mean values ꢂ standard error (SEM).
Statistical analyses were performed using the unpaired Student’s t-
test or the one-way ANOVA test followed by a Bonferroni’s multi-
comparison test, using GraphPad Prism 3.03 (San Diego, California,
USA). Statistical significance was considered when P < 0.05.
Hyper film MP (GE Healthcare). Anti-
used as a loading control.
b
-actin antibody (Sigma) was
Acknowledgements
This work was supported by grants from University of Padova.
S.P. was recipient of a fellowship from “Associazione Farini per la
Ricerca Grastroenterologica”.
5.3.6. Transfection and luciferase assay
Nuclear factor (NF)- B and PPAR-
ferase reporter plasmids have been previously described [30].
k
g-(PPRE-Luc)-responsive luci-
Cytomegalovirus b-galactosidase (CMV-b-Gal) reporter plasmid was
purchased from Promega. CMT-93 cells (1 ꢃ 105/well) were seeded
in 12-well culture plates and transfected 24 h later with the speci-
fied plasmid using Lipofectamine 2000 (Promega). Twenty four
hours after transfection, cells were incubated with HP or 30 mM 5-
ASA for 30 min and stimulated with 100 ng/mL LPS. After additional
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