A novel 2,3-diphenyl-4H-pyrido[1,2-a]pyrimidin-4-one derivative inhibits endothelial cell dysfunction and smooth muscle cell proliferation/activation

Article abstract of DOI:10.1016/j.ejmech.2013.11.021

Hyper-proliferation and migration of vascular smooth muscle cells and endothelial cell dysfunction are central events in the development of neo-intimal lesions. Pursuing our interest in the synthesis of bioisosters of flavonoids, we studied in depth a novel synthetic 2,3-diphenyl-4H-pyrido[1,2-a] pyrimidin-4-one derivative, examining its effects in vitro on induced-cell proliferation and activation in human aortic smooth muscle cells (HAoSMCs) and in human umbilical vein endothelial cells (HUVECs). Compared with two well known flavonoids, apigenin and quercetin, the novel compound, 2-(3,4-dimethoxyphenyl) -3-phenyl-4H-pyrido[1,2-a]pyrimidin-4-one, 3, was not toxic for HUVECs, even at high concentrations and for long incubation times, while the two flavonoids were not tolerated, even at concentrations as low as 10 μmol/L. Compound 3 inhibited selectively, and in a concentration-dependent manner, the proliferation of HAoSMCs but not that of HUVECs. In HUVECs, it inhibited the cytokine-induced vascular cell adhesion molecule-1 expression, but not the cyclooxygenase-2 (COX-2) expression. Instead, in HAoSMC, it inhibited the induction of COX-2 expression and the relative release of prostaglandin E ₂. In addition, it inhibited the transcription of the matrix metalloproteinase-9 and its activity. Thanks to its multiple and tissue-specific function, 2-(3,4-dimethoxyphenyl)-3-phenyl-4H-pyrido[1,2-a]pyrimidin-4-one might replace or assist the action of current drugs eluted by coronary stents, in order to promote a functional repair of damaged wall.

Full text of DOI:10.1016/j.ejmech.2013.11.021

European Journal of Medicinal Chemistry 72 (2014) 102e109
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
A novel 2,3-diphenyl-4H-pyrido[1,2-a]pyrimidin-4-one derivative
inhibits endothelial cell dysfunction and smooth muscle cell
proliferation/activation
Serena Del Turco a, Stefania Sartini b, Cassandra Sentieri a, Chiara Saponaro a,
,
Teresa Navarra a, Bianca Dario b, Federico Da Settimo b, Concettina La Motta b
**
,
*
,
Giuseppina Basta a
a CNR, Institute of Clinical Physiology, Via G. Moruzzi, 1, 56124 Pisa, Italy
b Department of Pharmacy, University of Pisa, Via Bonanno, 6, 56126 Pisa, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Hyper-proliferation and migration of vascular smooth muscle cells and endothelial cell dysfunction are
central events in the development of neo-intimal lesions.
Pursuing our interest in the synthesis of bioisosters of flavonoids, we studied in depth a novel syn-
thetic 2,3-diphenyl-4H-pyrido[1,2-a]pyrimidin-4-one derivative, examining its effects in vitro on
induced-cell proliferation and activation in human aortic smooth muscle cells (HAoSMCs) and in human
umbilical vein endothelial cells (HUVECs).
Received 18 September 2013
Received in revised form
14 November 2013
Accepted 21 November 2013
Available online 1 December 2013
Compared with two well known flavonoids, apigenin and quercetin, the novel compound, 2-(3,4-
dimethoxyphenyl)-3-phenyl-4H-pyrido[1,2-a]pyrimidin-4-one, 3, was not toxic for HUVECs, even at
high concentrations and for long incubation times, while the two flavonoids were not tolerated, even at
Keywords:
2,3-Diphenyl-4H-pyrido[1,2-a]pyrimidin-4-
ones
concentrations as low as 10 mmol/L. Compound 3 inhibited selectively, and in a concentration-dependent
2-(3,4-Dimethoxyphenyl)-3-phenyl-4H-
pyrido[1,2-a]pyrimidin-4-one
Human aortic smooth muscle cells
Human umbilical vein endothelial cells
Cell proliferation
manner, the proliferation of HAoSMCs but not that of HUVECs. In HUVECs, it inhibited the cytokine-induced
vascular cell adhesion molecule-1 expression, but not the cyclooxygenase-2 (COX-2) expression. Instead, in
HAoSMC, it inhibited the induction of COX-2 expression and the relative release of prostaglandin E2. In
addition, it inhibited the transcription of the matrix metalloproteinase-9 and its activity.
Thanks to its multiple and tissue-specific function, 2-(3,4-dimethoxyphenyl)-3-phenyl-4H-pyrido[1,2-
a]pyrimidin-4-one might replace or assist the action of current drugs eluted by coronary stents, in order
to promote a functional repair of damaged wall.
Cell dysfunction
Ó 2013 Elsevier Masson SAS. All rights reserved.
1. Introduction
inflammatory cytokines, enhanced expression of adhesion proteins
and cyclooxygenase-2 (COX-2) [3,4]. Injured and inflamed vessel
Percutaneous coronary intervention remains an effective ther-
apy for the revascularization of occluded arteries [1,2]. However, at
the site of endovascular intervention, we observe endothelial
denudation and dysfunction, characterized by an increase in
wall attracts platelets and leukocytes that release growth factors
and cytokines, promoting also activation of vascular smooth muscle
cells, which migrate to de-endothelialized vessel surface and pro-
liferate. All this leads to neo-intimal tissue formation which, in turn,
may lead to vessel restenosis [4e6]. Therefore, injured vessel wall
should be profitably treated with agents possessing vascular pro-
tective properties, improving vascular healing, protecting the
endothelial layer, and down-regulating smooth muscle cell prolif-
eration and migration [7].
Abbreviation: HAoSMCs, human aortic smooth muscle cells; HUVECs, human
umbilical vein endothelial cells; COX-2, cyclooxygenase-2; TNF-
a, tumor necrosis
factor- ; PDGF-BB, platelet derived growth factor-BB; ECGF, endothelial cell growth
a
The development of neointima can be critically induced by cy-
tokines and growth factors such as tumor necrosis factor (TNF-a)
and platelet derived growth factor-BB (PDGF-BB), as well estab-
lished in arterial injury models [8,9]. Therefore, the inhibition of
factor; VCAM-1, vascular cell adhesion molecule-1; MMP-9, matrix metal-
loproteinase-9; PGE2, prostaglandin E2; PGI2, prostacyclin; PMA, phorbol 12-
myristate 13-acetate.
* Corresponding author. Tel.: þ39 050 2219593; fax: þ39 050 2219605.
** Corresponding author. Tel.: þ39 050 3152216; fax: þ39 050 3152166.
E-mail addresses: concettina.lamotta@farm.unipi.it (C. La Motta), lapina@ifc.cnr.it
(G. Basta).
TNF-a-induced endothelial dysfunction and PDGF-BB-stimulated
vascular smooth cell proliferation and phenotypic modulation,
0223-5234/$ e see front matter Ó 2013 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2013.11.021

S. Del Turco et al. / European Journal of Medicinal Chemistry 72 (2014) 102e109
103
may represent an important point of therapeutic intervention in
restenosis after angioplasty.
reported elsewhere [20]. The target compound, 2-(3,4-
dimethoxyphenyl)-3-phenyl-4H-pyrido[1,2-a]pyrimidin-4-one 3,
in particular, was obtained from the key intermediate 2-(3,4-
dimethoxyphenyl)-4H-pyrido[1,2-a]pyrimidin-4-one [19]. A sus-
pension of the key intermediate (1.00 mmol) and N-bromosucci-
nimide (1.00 mmol) in chloroform was refluxed under stirring until
the disappearance of the starting material (TLC analysis). The sol-
vent was then removed in vacuo and the crude product, 3-bromo-
2-(3,4-dimethoxyphenyl)-4H-pyrido[1,2-a]pyrimidin-4-one, was
purified by crystallization from EtOH. The pure bromo-derivative
(1.00 mmol) was then added to a suspension of bis(triphenyl
phosphine)palladium(II) dichloride (0.20 mmol) in EtOH/toluene,
followed by phenylboronic acid (1.50 mmol) and an aqueous so-
lution of Na2CO3 (2 M, 3.0 mL). The resulting reaction mixture was
refluxed under stirring until the disappearance of the starting
material (TLC analysis). After being cooled to room temperature,
the mixture was evaporated to dryness under reduced pressure and
the residue was purified by crystallization from MeOH, to afford the
target compound, 3, as a white solid. Yield: 90%. P. f.: 182e184 ꢁC. 1H
However, the currently available anti-proliferative drugs eluted
from coronary stents, while inhibiting neo-intimal hyperplasia,
impair the antithrombotic functions of endothelial cells, hindering
the re-endothelialization and thereby increasing the risk of stent
thrombosis [2,10,11]. Accordingly, novel and more effective thera-
peutic agents are highly welcome.
Flavonoids, a family of natural polyphenolic compounds, show
many biological and pharmacological effects, resulting from their
antioxidant, anti-inflammatory, and anti-angiogenetic activities
[12]. Although they are characterized by low bioavailability
[13,14], a huge amount of experimental data, acquired through
both in vitro assays and in animal models, clearly demonstrate
their ability to modulate key cellular and molecular mechanisms,
related to cardiovascular diseases and some types of cancer [8,15e
18]. Accordingly, they represent an intriguing source of inspiration
for medicinal chemists, who are involved in a continuous pro-
duction of structurally related synthetic analogs, developed with
the aim of achieving clinically effective compounds endowed with
suitable pharmacokinetic properties.
Pursuing our interest in the synthesis of bioisosters of flavo-
noids, we moved from our previously developed anti-oxidant de-
rivatives, characterized by a 2-phenyl-4H-pyrido[1,2-a]pyrimidine
core [19] and, through the insertion of an additional phenyl ring in
the position 3 of the heterocyclic scaffold, we obtained a novel class
of 2,3-diphenyl-4H-pyrido[1,2-a]pyrimidin-4-ones (DPPPs, Chart
1). The synthesized compounds, designed as anti-oxidant prod-
ucts provided with anti-inflammatory activity, turned out to be
effective and viable agents exploitable in the management of
vascular dysfunctions [20].
Here we describe the functional evaluation of the main repre-
sentative of this novel class of compounds, 2-(3,4-dimethoxy
phenyl)-3-phenyl-4H-pyrido[1,2-a]pyrimidin-4-one, 3, whose ef-
ficacy as a novel drug candidate for the treatment of vessel walls
subjected to endovascular intervention has been studied in depth,
through in vitro assays on both endothelial cells and smooth
muscle cells.
NMR,
d
, ppm, Hz: 8.96 (d, 1H, J ¼ 7.08), 7.95 (t, 1H, J ¼ 8.79), 7.73 (d,
1H, J ¼ 8.79), 7.34e7.10 (m, 7H), 6.88e6.75 (m, 2H), 3.72 (s, 3H),
3.36 (s, 3H).
2.3. Cell cultures and treatments
Human umbilical vein endothelial cells (HUVECs) were isolated,
characterized and maintained as described [21]. Cells were ob-
tained from discarded umbilical vein and treated anonymously
conforming to the principles outlined in the Declaration of Helsinki.
Cells were used up to the fifth passage from primary culture. Hu-
man aortic smooth muscle cells (HAoSMCs) were obtained from the
American Type Culture Collection (CRL 1999; ATCC, USA), cultured
with HAM’S F-12K medium supplemented with 10% fetal bovine
serum, as described previously [22] and used between passages
20e30.
If not otherwise indicated, the experiments were performed
when the cells reached the confluence, and cells were pre-treated
for 1 h with 1, 10 and 25
mM of compound, or vehicle alone,
before each challenge. The stimulation occurred with phorbol 12-
myristate 13-acetate (PMA) (10 nM), PDGF-BB (20 ng/mL), endo-
2. Experimental protocol
thelial cell growth factor (ECGF) (20 ng/mL) or TNF-a (10 ng/mL).
2.1. Materials
2.4. Cell toxicity assay
All reagents were purchased from SigmaeAldrich (St. Louis, MO)
except where specified. Cell Proliferation Reagent WST-1 (4-[3-(4-
iodophenyl)-2-(4-nitrophenyl)-2H-5-tetrazolio]-1,3-benzene
disulfonate), and Cell Proliferation ELISA, BrdU (colorimetric) were
obtained from Roche Diagnostics (Mannheim, Germany). The goat
anti-COX-2 polyclonal antibody was purchased from Tema Ricerca
Srl (Bologna, Italy). PGI2 (as 6-keto-PGF1a) and prostaglandin E2
(PGE2) (enzyme immunoassay, EIA) kits were purchased from
Cayman Chemical Company (Ann Arbor, MI, USA).
Stock solutions of apigenin, quercetin and test compounds were
dissolved in sterile dimethyl sulfoxide (DMSO) and stored at ꢀ80 ꢁC
at the maximum solubility of 50 mM. Since the final concentration
of DMSO in the culture medium never exceeded 0.1% (vol/vol),
DMSO (0.1%) alone served as the control. At this concentration,
DMSO alone, used as the control, did not show any effect on cell
viability, cell proliferation, or related molecular mechanisms (data
not shown).
Cellular toxicity by quercetin, apigenin and the novel synthe-
sized compounds, 2,3-diphenyl-4H-pyrido[1,2-a]pyrimidin-4-ones,
was checked for concentrations up to 50 mM through phase contrast
microscopy of cell morphology and WST-1 assay.
The quantitative cell viability was assessed with the compound
WST-1 (Roche Diagnostics) following the manufacturer’s protocol.
This assay reflects the activity of mitochondrial dehydrogenase
present in living cells. During the assay, the yellow tetrazolium salt
WST-1 is reduced to a highly colored formazan dye by mitochon-
drial enzymes in cells. Because these mitochondrial enzymes are
inactivated shortly after cell death, the orange colored formazan
dye only appears in viable cells. Cell grown on 96-well plates were
treated with quercetin, apigenin or 2,3-diphenyl-4H-pyrido[1,2-a]
pyrimidin-4-ones for 24 h or 72 h, and subsequently treated with
premix solution (10 mL) for further 2 h. Afterward, the absorbance at
450 nm was read using a microplate reader.
2.2. Chemical synthesis of test compounds
2.5. Cell proliferation assay
2,3-Diphenyl-4H-pyrido[1,2-a]pyrimidin-4-ones (Chart 1), were
synthesized by S.S., B.D., and C.L.M. (Department of Pharmacy) as
A colorimetric immunoassay (Roche Diagnostic) based on the
measurement of BrdU incorporation during DNA synthesis was

104
S. Del Turco et al. / European Journal of Medicinal Chemistry 72 (2014) 102e109
Table 1
RT-PCR primer pairs.
used. Prior to stimulation, cells, at a confluence of about 70%, were
synchronized by overnight serum starvation. Then, cells pre-
treated with 3 were stimulated with PDGF-BB or ECGF for 48 h.
Subsequently cells were labeled with BrdU, fixed, and stained ac-
cording to the manufacturer’s instructions. Finally, the absorbance
at 450 nm was read using a microplate reader.
Genes
Primers (50-30)
Length Annealing Cycles
(bp)
(ꢁC)
(n)
VCAM-1 F: 50-ATGACATGCTTGAGCCAGG-30
R: 50-GTGTCTCCTTCTTTGACACT-30
COX-2
260
55ꢁ (60 s) 30
52ꢁ (30 s) 28
64ꢁ (30 s) 35
55ꢁ (60 s) 30
F: 50-TGGGAAGCCTTCTCTAACCTCTCCT-30 388
R:50-CTTTGACTGTGGGAGGATACATCTC-30
MMP-9 F: 50-GGCGCTCATGTACCCTATGT-30
R: 50-TCAAAGACCGAGTCCAGCTT-30
GAPDH F: 50-GGTCTCCTCTGACTTCAACAGCG-30
R: 50-GGTACTTTATTGATGGTACATGAC-30
468
354
2.6. Detection of cell surface VCAM-1 expression
HUVECs, grown in 96-well plates, were pre-treated with the
novel synthesized compounds, 2,3-diphenyl-4H-pyrido[1,2-a]pyr-
imidin-4-ones, and stimulated with TNF-a overnight. The VCAM-1
expression was measured by a cell surface enzyme immunoassay,
as previously described by us [23].
2.9. Measurement of PGI2 and PGE2 production
Cells grown in 96-well plates were exposed to 3 and stimulated
with PMA or TNF- . The conditioned medium was then collected,
2.7. COX-2 protein expression assay
a
centrifuged and concentrations of PGI2 (as 6-keto-PGF1a, the stable
metabolite of PGI2) and PGE2 were determined by competitive
enzyme immunoassays (Cayman Chemical Company), according
to manufacturer’s instructions. Values obtained were expressed in
pg/mL.
Cells were grown in 96-well plates and exposed to 3 before
PMA, PDGF-BB or TNF-
a overnight stimulation. A modified EIA
procedure was used to measure the cellular expression of COX-2.
Briefly, cell monolayer was washed once with cold phosphate-
buffered saline before being fixed and permeabilized on ice with
cold acetone for 2 min. EIA were then carried out by incubating the
monolayer first with saturating concentrations (1 mg/mL) of goat
2.10. Gelatin zymography
anti-COX-2 antibody (Tema Ricerca Srl), then with HRP-conjugated
rabbit anti-goat IgG antibody. The plates were washed three times
between each incubation step and at each time the monolayer
integrity was monitored by microscopy. The peroxidase substrate,
3,30,5,50-tetramethylbenzidine was then added to the micro-well.
After 30 min, the reaction was stopped by adding 0.18 M sulfuric
HAoSMCs grown in 21 cm2 dishes were treated in serum-free
medium with PMA in presence/absence of 3 for 24 h. The condi-
tioned media were collected and analyzed for MMP-9 activity by
gelatin zymography. The protein content of the samples was
measured by the colorimetric method using serum albumin as the
acid (50
mL) and COX-2 cellular content was finally quantified
standard. Proteins (1 mg) were loaded on SDS-PAGE gel (11%) con-
spectrophotometrically, reading the absorbance at 450 nm.
taining gelatine (0.1%) for electrophoresis at constant voltage
(125 V) for approximately 1.5 h in cold room (4 ꢁC). Gels were
washed twice with 2.5% Triton X-100 (2.5%) and incubated (24 h at
37 ꢁC) in zymographic buffer [TRISeHCl (0.5 M) pH 7.5, CaCl2
(50 mM), NaCl (2 M), Brij35 (0.2%)]. Subsequently, gels were stained
with Coomassie Brilliant Blue R-250 (0.5%), de-stained with
methanol (5%) in acetic acid (10%), and visualized. Clear areas on
the Coomassie-stained gel, which indicated the presence of pro-
teolytic activity, were quantified by densitometric scanning.
Densitometry of destained areas was quantified with the use of
Scion imageÔ.
2.8. Total RNA extraction and reverse-transcription PCR (RT-PCR)
Cells were cultured in 21 cm2 dishes, exposed to 3 and stimu-
lated with PMA, PDGF-BB or TNF-a overnight (for COX-2 and matrix
metalloproteinase-9 [MMP-9] mRNAs) or for 4 h (for VCAM-1
mRNA). Total RNA was isolated using QIAzol lysis reagent (QIA-
GEN, Hilden, Germany) and quantified spectophotometrically at
260 nm. The ratio of O.D. values at 260 nm and 280 nm provided an
estimate of RNA purity. For each sample, the RNA integrity was
evaluated by electrophoresis in a 1.5% agarose gel.
2.11. Statistical analysis
Next, RNA (1
mg) was reverse-transcribed to cDNA by iScript
cDNA Synthesis Kit (Biorad, Hercules, CA, USA) in 20
m
L of total
The results were expressed as mean ꢂ SD or mean ꢂ SEM.
Multiple comparisons were performed by one-way ANOVA fol-
lowed by Bonferroni’s post-hoc tests. Values of P < 0.05 were
considered statistically significant. Data were analyzed with the use
of statistical software SPSS 13.0 (SPSS Inc, Chicago, IL, USA).
reaction volume, according to the manufacturer’s instructions in a
GeneAmp PCR System 9700 thermal cycler (Perkin Elmer, Lockport
Place, Lorton).
The gene expression of VCAM-1, MMP-9 and COX-2 was per-
formed using specific primers and conditions (Table 1) as previ-
ously described [21,24,25]. Human glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) gene was amplified as an internal control
(Table 1) in the same reaction of the target genes. All PCR reactions
3. Results
were performed in a 50
(Qiagen, Germany) (2.5 U), deoxyribonucleotide triphosphate
(dNTP) (0.2 mM), MgCl2 (1.5 mM) and forward and reverse primers
m
L total volume, containing Taq polymerase
3.1. Effects of 2,3-diphenyl-4H-pyrido[1,2-a]pyrimidin-4-ones,
apigenin and quercetin on endothelial cell survival
(1
m
M each). The PCR products were separated by electrophoresis
We started our functional study evaluating the toxicity of the
novel synthesized 2,3-diphenyl-4H-pyrido[1,2-a]pyrimidin-4-
ones on HUVECs, exploiting the WST-1 assay. None of the test
compounds displayed any evident toxicity (data not shown). As
on an agarose gel (2%) in Tris/acetate/EDTA buffer and visualized at
260 nm after staining with ethidium bromide. After gel image
acquisition by a digital camera (Kodak DC290 Digital camera Sys-
temÔ; Eastman Kodak, Rochester, NY, USA), band intensity of PCR
DNA was quantified using densitometric analysis by Scion imageÔ.
The relative mRNA expressions of VCAM-1, MMP-9 and COX-2 were
normalized to GAPDH expression.
an example, data obtained with 3 (10, 25 and 50 mM) in HUVECs,
with respect to apigenin and quercetin, are reported in Fig. 1A.
HUVECs treated with 3, for 24 h and 72 h, showed no significant
difference in the viability when compared to the untreated cells,

S. Del Turco et al. / European Journal of Medicinal Chemistry 72 (2014) 102e109
105
Table 2
whereas apigenin and quercetin resulted in a strong decrease in
cell viability, as evaluated by the WST-1 assay. The effects on
HUVECs morphology after 72 h of treatment were assessed with
phase-contrast microscopy. As shown by photomicrographs
(Fig. 1B) 3 did not cause cell detachment while apigenin and
quercetin markedly reduced the number of adherent cells
(Fig. 1B). Although 3 proved to have no toxic effects on HUVECs up
Effects of 2,3-diphenyl-4H-pyrido[1,2-a]pyrimidin-4-one derivatives, 1e8, on
VCAM-1 expression.
R4
O
N
R3
R2
N
to 50 mM, in the following studies we chose to exploit up to 25 mM
R1
of compound, being clinically more compatible. Since flavonoids
were toxic at these concentrations and with less exposure time,
we cannot compare their efficacy with that of 3. On the other
hand, at lower concentrations they had no effect (data not
shown).
N
R1
R2
R3
R4
% of inhibitiona
1
2
3
4
5
6
7
8
H
H
H
OCH3
H
H
H
OCH3
H
H
H
H
H
OCH3
H
H
H
H
H
OCH3
OCH3
OCH3
OCH3
OCH3
8.5 ꢂ 3.2
8.9 ꢂ 3.3
37 ꢂ 8.5
12 ꢂ 4.1
12 ꢂ 3.6
9.6 ꢂ 3.9
10.3 ꢂ 4.2
15 ꢂ 3.3
OCH3
OCH3
H
3.2. 2,3-Diphenyl-4H-pyrido[1,2-a]pyrimidin-4-ones
transcriptionally modulates endothelial VCAM-1 expression
H
OCH3
OCH3
OCH3
We tested the effect of the novel 2,3-diphenyl-4H-pyrido[1,2-a]
pyrimidin-4-one derivatives on TNF-a induced VCAM expression,
used here as a molecular marker of inflammation and endothelial
cell dysfunction. As shown in Table 2, all the test compounds
OCH3
a
Values ꢂ SEM, determined in HUVECs pre-treated with the test compound
(25
m
M) for 1 h, stimulated with TNF- (10 ng/mL) overnight and assayed for cell-
a
surface VCAM-1 expression.
A
24 h
72 h
290
*
*
*
240
#
#
*
*
#
190
140
90
#
#
#
#
40
10
25
50
0
10
25
3
50
µM
10
25
50
Apigenin
Quercetin
B
Compound 3
Apigenin
Quercetin
0
10
25
50
µM
Fig. 1. Effects of 3, apigenin and quercetin on endothelial cell survival A) HUVECs were pretreated with 3, apigenin and quercetin for 24 h and 72 h, and a WST-1 assay was
performed. Each bar represents the mean of three independent experiments, each performed in n ¼ 6 replicates. ANOVA with Bonferroni’s post hoc comparison, *P < 0.01 and
#P < 0.001 vs. control. B) Representative photomicrographs show the effect of the three compounds on HUVEC monolayer after 72 h of treatment.

106
S. Del Turco et al. / European Journal of Medicinal Chemistry 72 (2014) 102e109
proved to modulate endothelial VCAM-1 expression, showing
percentage inhibition values ranging from 8.5 to 37. Among all the
test compounds, 3 exhibited the highest efficacy, demonstrating to
concentrations of 3 (10e25 mM) did not show any effects on cell
proliferation (Fig. 3B).
inhibit TNF-
a induced VCAM-1 expression in a concentration-
3.4. COX-2 expression: compound 3 has differential actions on the
two cell types
dependent manner (Fig. 2A). To determine whether the inhibitory
effect of 3 on VCAM-1 was due to changes in mRNA levels, a RT-PCR
analysis of VCAM-1 gene expression was performed on HUVECs
treated with TNF-
transcripts were not found expressed in untreated cells while the
addition of TNF- up-regulated VCAM-1 mRNA (Fig. 2B).
Treatment with 3 (10e25 M) resulted in a significant reduction
of VCAM-1 m-RNA expression (Fig. 2B). Conversely, this treatment
did not effect the mRNA levels of the constitutively expressed gene
GAPDH (Fig. 2B). The effects of 3 on VCAM-1 mRNA levels reflect
the results obtained with measurements of cell surface expression
of VCAM-1.
Since the activation of COX-2 is critically involved in cell pro-
liferation, growth and inflammatory responses [27e29], we
explored the effect of our compound on COX-2 protein expression
elicited by various stimuli. In HUVECs, COX-2 protein expression
a in the presence/absence of 3. VCAM-1 mRNA
a
m
was up-regulated by PMA or TNF-a but 3 did not alter the COX-2
expression induced from both compounds (Fig. 4A). Results of
COX-2 mRNA, analyzed by RT-PCR, confirmed those of the protein
assay (Fig. 4B). According to the irrelevant effect of 3 on COX-2
expression in HUVECs, our compound did not affect the COX-2
activity stimulated by TNF-
PGE2 release (Fig. 4C).
a, as assessed by measuring the PGI2/
3.3. Compound 3 inhibits the smooth muscle cell proliferation but
not that of endothelial cells
Dissimilar results were obtained on HAoSMCs, pre-treated with
3. COX-2 protein expression in HAoSMCs was highly induced by
PDGF-BB (Fig. 5A). Derivative 3 inhibited the PDGF-BB-induced
COX-2 expression in a concentration-dependent manner (Fig. 5A).
The inhibitory effect occurred at transcriptional levels, as shown by
the COX-2 mRNA evaluation (Fig. 5B).
The inhibitory effect of 3 on COX-2 protein and gene expression
stimulated by PMA was also investigated, and results obtained
showed the same inhibitory pattern of 3 on COX-2 expression
induced by PDGF-BB (Fig. 5A and B). In accordance with reduced
COX-2 protein expression, 3 suppressed PMA-stimulated COX-2
activity, as assessed by measuring the production of PGE2 in culture
media (Fig. 5C).
The development of restenosis is largely due to vascular
smooth muscle cell hyper-proliferation [26]. To determine
whether 3 has a growth inhibition effect on vascular cells, the
BrdU incorporation assay was performed. The test compound
inhibited HAoSMCs proliferation, stimulated by the potent growth
factor PDGF-BB, in a concentration-dependent manner (Fig. 3A).
On the contrary, the proliferation of HUVECs slightly increased
after treatment with 1 mM 3, alone or in the presence of ECGF,
when compared to the untreated cells. Treatment with higher
A
- TNF-α
600
+ TNF-α
A
1400
- PDGF-BB
500
*
1200
+ PDGF-BB
*
400
**
1000
§
*
300
800
200
100
°
600
#
400
0
compound 3 (µM)
0
1
10
25
200
0
compound 3 (µM)
0
1
10
25
B
150
100
50
*
#
B
2500
2000
1500
1000
500
- ECGF
+ ECGF
*
0
VCAM-1
(260bp)
GAPDH
*
(354 bp)
compound 3 (µM)
Control
0
1
10
25
M
0
TNF-α (10 ng/mL)
compound 3 (µM)
0
1
10
25
Fig. 2. Effects of 3 on VCAM-1 expression A) HUVECs were pre-treated with 3 for 1 h,
and stimulated with TNF- (10 ng/mL) overnight. Each bar represents the mean of
three independent experiments, each performed in n ¼ 8 replicates. ANOVA with
Bonferroni’s post hoc comparison, *P < 0.001 vs. TNF- alone. B) VCAM-1 mRNA,
analyzed by RT-PCR, in HUVECs pre-treated with 3 for 1 h and stimulated with TNF-
Fig. 3. Effects of 3 on cell proliferation evaluated with the BrdU cell incorporation
assay A) Preconfluent HAoSMC pre-treated with 3 for 1 h were grown in the presence/
absence of PDGF-BB (20 ng/mL) for 48 h. Each bar represents the mean of three
separate experiments each performed in n ¼ 8 replicates. ANOVA with Bonferroni’s
post hoc comparison, *P < 0.01, **P < 0.001 and xP < 0.0001 vs. PDGF-BB alone,
ꢁP < 0.01 and #P < 0.001 vs. control. B) Preconfluent HUVECs pre-treated with 3 for
1 h, were grown in the presence/absence of ECGF (20 ng/mL) for 48 h. Each bar rep-
resents the mean of three separate experiments each performed in n ¼ 8 replicates.
ANOVA with Bonferroni’s post hoc comparison, *P < 0.01 vs. ECGF or control.
a
a
a
for 4 h. PCR DNA bands shown are representatives of three determinations. Bar graphs
of all values were expressed as the mean ꢂ SEM of three determinations, ANOVA with
Bonferroni’s post hoc comparison, *P
M ¼ markers.
< < 0.01 vs. TNF-a alone.
0.05 and #P

S. Del Turco et al. / European Journal of Medicinal Chemistry 72 (2014) 102e109
107
A
A
450
400
350
300
250
200
150
1150
1100
*
1050
*
°
1000
#
§
950
900
850
800
750
0
1
10
25
compound 3 (µM)
Control
0
1
10
25
700
TNF-
α
(10 ng/mL)
PMA (10 nM)
compound 3 (µM)
0
1
10
25
Control
0
1
10
25
PDGF (20 ng/mL)
PMA (10 nM)
B
B
150
100
50
150
100
50
*
*
°
§
°
0
0
COX-2
(388 bp)
COX-2
(388 bp)
compound 3 (µM)
Control
0
1
10 25
(10 ng/mL)
Control 0
1
10
25
M
TNF-
α
PMA (10 nM)
Control
0
1
10
25
M
Control
0
1
10
25
compound 3 (µM)
PDGF (20 ng/mL)
PMA (10 nM)
C
105
C
105
104
103
PGI
2
104
PGE
2
*
#
§
103
102
102
Control
0
1
10
25 Control
0
1
10
25 compound 3 (µM)
TNF-
α
(10 ng/mL)
TNF-α
(10 ng/mL)
10
compound 3 (µM)
Control
0
1
10
25
Fig. 4. Effects of 3 on COX-2 expression and prostanoid production in HUVECs A)
HUVECs were pre-treated with 3 for 1 h, and stimulated overnight with TNF- or PMA.
PMA (10 nM)
a
The COX-2 protein expression was measured as described in the Methods. Each bar
represents the mean of three independent experiments, each performed in n ¼ 6
replicates. B) COX-2 m-RNA, analyzed by RT-PCR, in HUVECs pre-treated with 3 for 1 h,
Fig. 5. Effects of 3 on COX-2 expression and PGE2 production in HAoSMCs A) HAoSMCs
were pre-treated with 3 for 1 h, and stimulated overnight with PDGF-BB or PMA. The
COX-2 protein expression was measured as described in the Methods. Each bar rep-
resents the mean of three independent experiments, each performed in n ¼ 6 repli-
cates. ANOVA with Bonferroni’s post hoc comparison, *P < 0.01, and xP < 0.001 vs.
PDGF-BB alone; ꢁP < 0.01 and #P < 0.001 vs. PMA alone. B) COX-2 m-RNA, analyzed by
RT-PCR, in HAoSMCs pre-treated with 3 for 1 h and stimulated overnight with PDGF-
BB or PMA. ANOVA with Bonferroni’s post hoc comparison, *P < 0.05, ꢁP < 0.01 and
xP < 0.001 vs. PDGF-BB alone; *P < 0.05, ꢁP < 0.01 vs. PMA alone. M ¼ markers. C) PGE2
production in HAoSMCs was measured in cell culture medium after overnight stim-
ulation with PMA. Data are means ꢂ SEM of three independent experiments per-
and stimulated overnight with TNF-
and PGE2 production in HUVECs were measured in cell culture medium after overnight
stimulation with TNF-
. Data are means ꢂ SEM of three independent experiments
performed in duplicate.
a
or PMA. M ¼ markers C). PGI2 (as 6-keto-PGF1
a)
a
3.5. Compound 3 inhibits MMP-9 gene transcription and its
enzymatic activity in smooth muscle cells
formed in duplicate. ANOVA with Bonferroni’s post hoc comparison, *P
#P < 0.001 and xP < 0.0001 vs. PMA alone.
< 0.01,
MMPs may be a potential therapeutic target for the treatment of
restenosis or atherosclerosis, as they play a key role in smooth
muscle cell migration and neointima formation after vascular
injury.
4. Discussion
We evaluated the effect of 3 on gene transcription of MMP-9, the
main protease involved in the above-mentioned process. HAoSMCs
were pre-treated with increasing concentrations of test compound
before PMA stimulation, and then mRNA expression of the target
proteins was evaluated. As shown in Fig. 6A, the up-regulation of
In this study, we describe the functional evaluation of a novel
heterocyclic compound, 2-(3,4-dimethoxyphenyl)-3-phenyl-4H-
pyrido[1,2-a]pyrimidin-4-one, 3, which emerged as the most
effective one among a number of parent analogs developed as drug
candidates for the treatment of vascular dysfunction [20]. Since the
test compound was structurally inspired by the flavonoid scaffold,
we preliminarily evaluated its effects on endothelial cell vitality
with respect to two main flavonoids, apigenin and quercetin, whose
anti-proliferative and anti-inflammatory properties have been
associated with the prevention and therapy of cardiovascular dis-
eases and cancer [30,31].
MMP-9 m-RNA induced by PMA was inhibited by 3 at 10 and 25 mM.
To determine whether 3 affected the levels of proteolytic ac-
tivity of MMP-9, using gelatin zymography, we examined the
conditioned medium from HAoSMCs exposed to PMA with or
without 3 (10 and 25 mM) for 24 h.
Zymography analysis of HAoSMCs treated with PMA alone
revealed the presence of MMP-9 (82 kDa) compared to cells treated
with DMSO alone (Fig. 6B). The supernatants harvested from
Surprisingly, in our experimental conditions, the two known
flavonoids turned out to be toxic to endothelial cells after 72 h of
HAoSMCs pre-treated with 3 (25
mM) showed reduced induction of
incubation, even at concentration of 10
mM, while 3 was well-
MMP-9 (Fig. 6B).
tolerated even at 50 M and for longer time. Therefore, by
m

108
S. Del Turco et al. / European Journal of Medicinal Chemistry 72 (2014) 102e109
formation, even at low concentrations, without altering endothelial
A
cell vitality, ensuring both tissue homeostasis and the healing of the
injured vessel wall.
150
100
50
Smooth muscle cell hyperplasia during atherosclerosis and
restenosis is associated with the induction of MMP expression that
remodel the surrounding extracellular matrix [32,33]. Over-
expression of MMP-9 in smooth muscle cells in injured vascular
wall induces considerable changes such as an increase in smooth
muscle cell migration, expansive remodeling, and a decrease in
extracellular matrix [32]. Therefore, the ability of our compound to
also inhibit the MMP-9 up-regulation and activity can result in a
lesser SMC migration and proliferation.
*
#
0
MMP-9
(468bp)
This study also showed that 3 reduced the cytokine-induced
expression of COX-2, followed by reduction of PGE2 production in
HAoSMCs. On the contrary, in endothelial cells, 3 affected neither
COX-2 protein and gene expression nor the production of prosta-
noids, particularly the prostacyclin PGI2 that acts synergistically
with nitric oxide to maintain normal vascular functions, i.e., athero-
protection, inhibition of platelet activation, and vascular smooth
muscle contraction [34].
Since the expression of COX-2 is associated with mitogen-
activated protein kinase and mediates smooth muscle cell prolif-
eration [35,36], we suppose that the inhibitory effect of 3 on
HAoSMCs cell proliferation may be due to the inhibition of COX-2
gene expression. On the other hand, the null effects of 3 on both
COX-2 expression and proliferation in HUVECs, confirm the COX-2-
dependent anti-proliferative response of 3 in HAoSMCs. Nonethe-
less, we cannot exclude other cell-signaling pathways by which 3
exerts its inhibitory effects on cell growth.
GAPDH
(364 bp)
M
compound 3 (µM)
0
1
10
25
Control
PMA (10 nM)
B
Pro-MMP-9 (92 kDa)
Act-MMP-9 (82 kDa)
compound 3 (µM)
Control
0
10
25
PMA (10 nM)
Fig. 6. Effects of 3 on MMP-9 gene expression and activity in HAoSMCs A) MMP-9 m-
RNA, analyzed by RT-PCR, in HAoSMCs pre-treated with 3 for 1 h and stimulated
overnight with PMA. The bands of PCR DNA shown are representatives of three de-
terminations. Bar graphs of all values were expressed as the mean ꢂ SEM from three
determinations. ANOVA with Bonferroni’s post hoc comparison, *P
< 0.05 and
#P < 0.01 vs. PMA alone. M ¼ markers B) MMP-9 activity was determined by gelatin
zymography assay. The bands of enzymatic activity shown are representative of three
determinations.
5. Concluding remarks
In conclusion, we have shown that the novel heterocyclic
compound, 2-(3,4-dimethoxyphenyl)-3-phenyl-4H-pyrido[1,2-a]
pyrimidin-4-one, 3, regulates events involved in the remodeling of
the vessel wall. Accordingly, it might provide a new way to
modulate the vascular remodeling under pathologic conditions.
Thanks to the multiple and tissue-specific function of 3, i.e., se-
lective down-regulation of HAoSMC proliferation, inhibition of the
endothelial cell dysfunction and maintenance of the endothelial
cell athero-protective action, we can assume that it could become a
novel therapeutic candidate for restenosis.
suitably modifying the heterocyclic core of flavonoids we suc-
ceeded in obtaining an effective compounds, devoid of any toxic
effects and endowed with improved vasoprotective and anti-
apoptotic properties. Most cell culture-based studies investigating
the inhibitory effects of polyphenols on cell proliferation generally
exploit supra-physiological concentrations of specific compounds,
thus treating cells with very high apoptotic concentrations (until to
100
mM) [30,31]. On the contrary, our compound inhibited the
proliferation of HAoSMCs, responsible for the abnormal neointimal
This compound might replace or assist the action of current
drugs eluted by coronary stents, in order to promote physiological
repair of the injured wall, restoring its compatibility with the
circulating blood, and thus limiting the incidence of thrombosis
and restenosis after revascularization.
A better understanding of mechanisms underlying the anti-
proliferative and athero-protective effects of 3 in vascular wall
cells may provide strategies for improving the outcome of reste-
nosis treatment. Animal model studies are needed to more defin-
itively test the biological mechanisms and the actual therapeutic
effectiveness of our compound.
OH
O
O
OH
O
O
OH
OH
OH
HO
HO
OH
Apigenin
Quercetin
Conflicts of interest
R4
O
N
The authors declare no conflict of interest.
Acknowledgment
N
R3
R2
R1
DPPPs
R1, R2, R3, R4 = H, OCH3
This work was supported by the Fondazione Pisa, within the
framework of the MICRO-Vast project (www.microvast.it) (Grant
153/09).
The authors thank Alison Frank, for her assistance in editing the
English language.
Chart 1. Quercetin, apigenin and 2,3-diphenyl-4H-pyrido[1,2-a]pyrimidin-4-ones,
DPPPs.

S. Del Turco et al. / European Journal of Medicinal Chemistry 72 (2014) 102e109
109
selective aldose reductase inhibitors exhibiting antioxidant activity, J. Med.
Chem. 50 (2007) 4917e4927.
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