Inhibition of iNOS and COX-2 in human whole blood ex vivo and monocyte-macrophage J774 cells by a new group of aminothiopyrimidone derivatives

Article abstract of DOI:10.1016/j.bmc.2009.01.029

We tested a series of 11 new aminothiopyrimidones on the activity of inducible nitric oxide synthase (iNOS) and prostaglandin G/H synthase-1 and 2 (COX-1 and COX-2) in the whole human blood and monocyte-macrophage J774 cell line. To induce COX-2 and iNOS,

Full text of DOI:10.1016/j.bmc.2009.01.029

Bioorganic & Medicinal Chemistry 17 (2009) 1991–1996
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Inhibition of iNOS and COX-2 in human whole blood ex vivo and monocyte-
macrophage J774 cells by a new group of aminothiopyrimidone derivatives
a
b
b
Venera Cardile a, , Laura Lombardo , Giuseppe Granata , Antonio Perdicaro ,
*
Michael Balazy c, Andrea Santagati b
a Department of Physiological Sciences, University of Catania, V.le A. Doria 6, 95125 Catania, Italy
b Department of Pharmaceutical Sciences, University of Catania, V.le A. Doria 6, 95125 Catania, Italy
c Department of Pathology, New York Medical College, Valhalla, NY 10595, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
We tested a series of 11 new aminothiopyrimidones on the activity of inducible nitric oxide synthase
(iNOS) and prostaglandin G/H synthase-1 and 2 (COX-1 and COX-2) in the whole human blood and mono-
cyte-macrophage J774 cell line. To induce COX-2 and iNOS, blood samples and J774 cells were stimulated
with bacterial lipopolysaccharide (LPS) in the absence or presence of the test compounds. After incuba-
tion, the plasma and the supernatants of culture media were collected for the measurement of TxB2 and
PGE2 by a specific enzyme-immunoassay and determination of nitrite by a colorimetric assay. Several
phenylthieno derivatives of substituted pyrimidone inhibited formation of both COX-2 and iNOS derived
products with one of the compounds (compound 11, N-[2-[(2,4-dinitrophenyl)thio]-4-oxo-6-phenylthie-
no[2,3-d]pyrimidin-3(4H)-y]methanesulfonamide) showing a complete inhibition of LPS-stimulated for-
mation of NO and PGE2.
Received 17 October 2008
Revised 12 January 2009
Accepted 14 January 2009
Available online 20 January 2009
Keywords:
Nitric oxide
NSAIDs
Prostaglandins
Aminothiopyrimidone
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
2 inhibitors.19–21 None of the traditional NSAIDs displays selective
COX-2 inhibition, which explains some of the side effects of NSA-
Prostaglandin E2 (PGE2) is one of the principal mediators of
inflammation, which is biosynthesised by the metabolism of the
phospholipase A2 (PLA2)-derived arachidonic acid to PGG2/PGH2
via COX enzymes, and then followed by the action of PGE synthase.
Two COX isozymes have been identified. COX-1 is a constitutively
expressed isoform, which biosynthesizes physiologically relevant
PGs involved in gastric and other organ cytoprotection. High levels
of COX-1 are expressed in platelets,1 vascular endothelial cells,2
stomach,3,4 and renal collecting tubules.3,5,6 COX-1 levels remain
fairly constant, but in some instances, 2- to 4-fold COX-1 protein
increase has been observed following hormonal and/or growth fac-
tor stimulation.7,8 COX-2 is an inducible isoform, which is almost
undetectable in most tissues under physiological conditions, but
its expression can be considerably increased (10- to 80-fold) fol-
lowing induction by many pro-inflammatory stimuli, cytokines,
growth factors and mitogens.3,5,6,9–12 Bacterial lipopolysaccharides
(LPS) stimulate COX-2-dependent PG formation in monocytes13
macrophages14–16 and cause inflammation in synoviocytes17 and
follicles.18
IDS related to the removal of gastric cytoprotection.22 This obser-
vation laid foundation for development of more specific COX-2
inhibitors as potentially better approach to inflammation thera-
peutics. However, recent placebo-controlled trials have revealed
that COX-2 inhibitors pose a small risk of myocardial infarction
and stroke, which resulted in the withdrawal from the market of
some members of this class of drugs.4 These inhibitors cause a sup-
pression of COX-2-derived prostacyclin (PGI2), which counteracts
platelet COX-1-derived thromboxane (TxA2), PGH2, and other
endogenous stimuli. The consumption of celecoxib, rofecoxib,
and valdecoxib has raised concern that many patients may have
suffered adverse cardiovascular reactions from these drugs. Thus,
a new generation of safe COX-2 inhibitors would ideally not only
spare COX-1-derived PGs but also PGI2 to afford endothelial
cytoprotection.
Another component of the inflammatory processes is the mas-
sive production of nitric oxide (NO) by inducible NO synthase
(iNOS). Moreover, many inflammatory stimuli, induce expression
of both iNOS and COX-2 proteins, which bind selectively.23,24
These two enzyme systems appear to interact by a mechanism
in which COX-2 activity increases via iNOS-stimulated nitration
of the COX-2 cysteine residues. This provides a rationale for
development of more refined dual COX-2/iNOS inhibitors that
will block symptoms of inflammation related to PGs and NO
formation.23
While it has been long known that non-steroidal anti-inflam-
matory drugs (NSAIDs) inhibit PG formation, clinically useful NSA-
IDS are either fairly selective COX-1 inhibitors or dual COX-1/COX-
* Corresponding author. Tel.: +39 095 7384040; fax: +39 095 7384217.
E-mail address: cardile@unict.it (V. Cardile).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.01.029

1992
V. Cardile et al. / Bioorg. Med. Chem. 17 (2009) 1991–1996
Over the last few years, we have established new methods for
ml (n = 9) from the control levels of 10 0.6 pg/ml (n = 9) (301% in-
crease). We observed that the compounds synthesized by us either
inhibited (Table 2) or stimulated (Table 3) PGE2 formation by COX-
2. Compounds 4, 8, 9, 10, and 11 inhibited LPS-induced COX-2 both
in blood and J774 cells (Table 2) by 70–100%. The IC50 values for
the high yield synthesis of a number of derivatives containing
the pyrimidin-4-one system with various functional groups.25,26
The choice of the heterocyclic system was based on the remarkable
analgesic and anti-inflammatory actions with no ulcerogenic effect
of methyl and phenyl derivatives of the thieno-, indol- and benzo-
pyrimidin-4-one systems.25,26
the derivatives 4,
8
and 11 were
7
0.4, 5.8 0.2 and
3.4 0.2 M, respectively. Compound 11 was the most potent
l
In this study, we tested the effects of novel aminothiopyrimid-
inone derivatives (1–11) on the generation of COX-2 and iNOS
products in LPS-challenged human whole blood and monocyte-
macrophage J774 cell line by measuring PGE2 production by en-
zyme-immunoassay (EIA) system and formation of nitrite by a col-
orimetric assay. In addition, the compounds were tested on COX-1
activity in human whole blood and compared with the activity of
indomethacin and celecoxib as references of COX-1 and COX-2
inhibitors, respectively.
among these inhibitors showing potency comparable to celecoxib
(Table 2). Compounds 4, 8, 9 and 10 were relatively less potent
but showed preferential specificity for blood (70–90% inhibition)
as compared to J774 cell-derived PGE2 (0–50% inhibition).
The effects of the compounds on COX-1 were evaluated in sam-
ples of human clotted blood drawn from the same healthy volun-
teers by measurement of TxB2 generation, which reflects
maximally stimulated cyclooxygenase activity of platelet COX-1
by endogenously formed thrombin in clotting blood. Similar to
celecoxib, compound 11 did not show a significant inhibition of
the COX-1 activity (4 0.5%). In this assay, indomethacin (10
l
M)
2. Results
inhibited COX-1 by 85%. Compounds 3 and 10 (10
lM) inhibited
COX-1 by 3% and 36%, respectively.
2.1. Effects on COX-2
Interestingly, compounds 1, 2, 5, 6, 7 increased the LPS-induced
PGE2 formation. The J744 cells were more sensitive than blood to
these derivatives. Compound 2 was the most potent among these
derivatives and stimulated PGE2 formation 2- to 3.5-fold in blood
and J744 cells, respectively.
The effects of pyrimidinone derivatives (1–11) were tested on
the formation of PGE2 and NO in human whole blood ex vivo and
mouse monocyte-macrophage J774 cells in culture. The COX-2
activity was evaluated by measurement of PGE2 levels in LPS-trea-
ted whole blood.27 The control blood samples contained
0.28 0.02 ng/ml (n = 9) of PGE2, which increased by
2.2. Effects on iNOS
35.8 5.5 ng/ml (n = 9) following the LPS stimulation (10
lg/ml
for 24 h) (12,785% increase). Stimulation of the J774 cells with
LPS (5 lg/ml for 72 h) increased the PGE2 levels to 30.1 3.5 pg/
The nitrite levels produced by J744 cells before and after LPS
stimulation were
4
0.2
l
M
and 21
3 lM (525% increase),
Table 1
Structures of compounds 1–11
O
5
1
NH2
S
9
O
O
N
H
H
*
C
N
H
NH2
NH2
N
N
H5C6
COOH
H
N
H5C6
S
S
S
N
*
S
C
C OOH
CH
3
O
2
H3C
H3C
NH2
H
O
6
O
N
10
H
*C
N
NH2
H
N
NH2
N
S
S
N
COOH
H
H5C6
N
S
*
S
S
N
C
C OOH
CH
O2N
NO2
3
O
3
O
N
NH2
S
S
7
NH2
O
S
H
*C
S
O
N
N
N
C H
3
11
H
H
H
N
H
COOH
N
N
O
S
H
*
H5C6
C
C OOH
S
S
CH
3
O2N
NO2
4
O
NH2
S
O
N
Br
8
H
*C
N
NH2
N
COOH
N
H
S
*
C
C OOH
CH
3
An asterisk indicates the optically active carbon in the thiopropanoic and thiophenylacetic acid derivatives.

V. Cardile et al. / Bioorg. Med. Chem. 17 (2009) 1991–1996
1993
Table 2
inhibitors rather than selective COX-2 inhibitors as a novel class
of anti-inflammatory drugs.
Comparison of the inhibitory activity of aminothiopyrimidone derivatives with
indomethacin and celecoxib in human whole blood and mouse monocyte-macro-
phage J774 cells stimulated with LPS
Interestingly, several of our thiopyrimidone derivatives stimu-
lated COX-2 activity and, to a lesser extent, iNOS activity. This
observation suggests that a combination of structural elements of
the pyrimidone derivatives may lead to a more refined COX-2
inhibitor that at the same time inhibits iNOS.
Inhibitors 10
concentration
l
M
Inhibition of PGE2 generation (%)
Inhibition of NO
generation (%)
in J774 cells
Human whole blood
J774 Cells
3
4
8
9
10
11
None inhibition
70 2*
None inhibition
50 4*
55 2*
38 2*
50 7*
70 5*
60 5*
100 3*
90 6*
96 8*
The compounds tested contained a basic 2-thio-3-amino-pyr-
imidone-4-on structure that was modified at key atoms based on
molecular modeling and our previous study.26 We identified sev-
eral structural elements that contributed to modulation of the
COX-2 and iNOS activities in blood and J744 cells stimulated with
LPS. These modifications included substitutions at the thiol group,
fusion of the pyrimidone ring and a substitution of the amino
group. Both S substituted phenylacetic and propanoic acids deriv-
atives activated COX-2 with exception of derivatives 4 and 8,
which were mild inhibitors. Synthesis of dinitrophenylthio deriva-
tives resulted in strong inhibitory properties of compounds 10 and
11. Another major effect was observed with the dihydrothieno fu-
sion to the pyrimidone ring. The dimethyl-dihydrothieno deriva-
tive 2 showed potent COX-2 stimulatory effect whereas phenyl
substituted dihydrothieno derivatives 9, 10, and 11 displayed
strong inhibitory effects. A striking effect was observed by creation
of the methanesulfonamide derivative of the N-amino group in
compound 10. This modification generated compound 11, which
substantially increased the ability of compound 11 to inhibit
COX-2 resulting in 100% of COX-2 inhibition relatively to com-
pound 10 (Table 2) in blood and J744 cells, respectively. While
celecoxib and valdecoxib are sulfonamines, the effect of the meth-
anesulfonamide group in compound 11 suggests an important con-
tribution of the amino group to the modulatory effect on COX-2 in
the studied compounds.
COX-2 inhibitors are believed to position themselves inside the
critical catalytic pocket of the enzyme whereby blocking the access
of the substrate, arachidonic acid. The spatial position of many
inhibitors relative to the COX-2 tertiary protein structure is not
known, and, therefore, trial and error approaches have revealed
various chemical structures that can produce an inhibitory effect.
It has been suggested that the preference or selectivity toward
the COX-2 enzyme of the methylsulfonamide derivatives does
not depend solely on the presence of these moieties in their molec-
ular structures, but also on the fact that they possess an overall
molecular profile which allows these groups to occupy a favorable
spatial position during the interaction of the inhibitor with cata-
lytic site, which permits interactions with amino acidic residues
specific for the COX-2 active site.34 In J774 cells, in fact, the com-
pounds 3, 8, 9, and 10, well inhibiting NO production, did not re-
duce in the same manner the PGE2 release.
88 6*
23 1*
90 2*
20 5*
75 5*
None inhibition
100 5*
100 4*
100 5*
100 6*
Indomethacin
Celecoxib
100 3*
100 2*
The values are the mean SEM of three experiments performed in triplicate.
*
Denotes significant difference of control vs LPS treatment (P < 0.01).
Table 3
Stimulatory activity of aminothiopyrimidone derivatives in human whole blood and
mouse monocyte-macrophage J774 cells stimulated with LPS
Activators 10
concentration
l
M
Activation of PGE2 generation (%) Activation of NO
generation (%) in J774 cells
Human whole blood J774 Cells
1
2
5
6
7
21 3*
205 22*
90 12*
28 3*
30 5*
346 17*
250 22*
180 14*
52 12*
5
1*
11 2*
52 7*
35 6*
10 3*
6
0.5*
The values are the mean SEM of three experiments performed in triplicate.
*
Denotes significant difference of control vs LPS stimulation (P < 0.01).
respectively. The same compounds 4, 8, 9, 10 and 11 inhibiting
COX-2 reduced NO production with the exception of compound
3, which was a iNOS inhibitor but not active toward COX-2 (Table
2). COX-2 activators (1, 2, 5, 6 and 7) were also moderate activators
of iNOS in J744 cells (Table 3).
3. Discussion
In this study, a number of heterocyclic derivatives containing
aminothiopyrimidone system with various functional substituents
were tested on COX-2 activity. Our efforts identified several com-
pounds that were excellent COX-2 as well as iNOS inhibitors.
One of these compounds (11) inhibited COX-2-generated PGE2,
and iNOS generated nitrite, and showed similar potency to
celecoxib.
There are several lines of rationale for development of dual
COX-2/iNOS inhibitors as a group of more effective anti-inflamma-
tory and analgesic drugs. Increased levels of both prostaglandins
and NO-derived products such as peroxynitrite and nitrogen diox-
ide mediate the inflammatory reactions and pain.28,29 While bind-
ing of NO to COX heme does not appear to alter its activity,30
several mechanisms may be involved in the activation of COX-2
by NO and iNOS.23,31 These involve stimulation of COX-2 by NO
that involves MAP kinase and superoxide.31 In human colon and
breast cancers, increased expression of iNOS and elevated levels
of prostanoids are associated with COX-2 overexpression, in both
cancer cells and stroma,32 suggesting a close association between
these two enzymes.33 More recently, Kim, Huri and Snyder23 have
reported that iNOS specifically binds to COX-2 in a continuous
In conclusion, our work further supports our original finding
that the aminothio derivatives of pyrimidone could be a novel
promising group of anti-inflammatory drugs and emphasises the
significance of the methylsulfonamide substitution of the amino-
pyrimidine ring. We believe that these new derivatives offer a
new direction for development of safer COX-2 inhibitors having
additional anti-inflammatory effects derived from iNOS inhibition.
4. Experimental
4.1. Chemistry
macrophage cell line (RAW264.7) treated with LPS and IFN-c. iNOS
The structures of the compounds used in this study are summa-
rized in Table 1. The syntheses of amino acid derivatives [(3-ami-
no-4-oxo-3,4-dihydroquinazolin-2-yl)thio](phenyl)acetic acid 1,
[(3-amino-5,6-dimethyl-4-oxo-3,4-dihydrothieno[2,3-d]pyrimidin-
2-yl)thio](phenyl)acetic acid 2, [(3-amino-4-oxo-3,4-dihydrothie-
binding causes S-nitrosylation of COX-2 cysteines, which enhances
COX-2 catalytic activity.23 Selective disruption of the iNOS-COX-2
binding prevents NO-mediated activation of COX-2. This synergis-
tic molecular interaction between two inflammatory systems pro-
vides a strong rationale for development of dual COX-2/iNOS

1994
V. Cardile et al. / Bioorg. Med. Chem. 17 (2009) 1991–1996
no[3,2-d]pyrimidin-2-yl)thio](phenyl)acetic acid 3, [(3-amino-6-
bromo-4-oxo-3,4-dihydroquinazolin-2-yl)thio] (phenyl)acetic acid
4, 2-[(3-amino-4-oxo-6-phenyl-3,4-dihydrothieno[2,3-d]pyrimi-
din-2-yl)thio]propanoic acid 5, 2-[(3-amino-4-oxo-4,5-dihydro-
3H-pyrimido[5,4-b]indol-2-yl)thio]propanoic acid 6, 2-[(3-amino-
4-oxo-3,4-dihydro[1]benzothieno[3,2-d]pyrimidin-2-yl)thio]prop-
anoic acid 7, 2-[(3-amino-4-oxo-3,4-dihydroquinazolin-2-yl)thio]-
propanoic acid 8, has been accomplished according to methods
reported by Santagati et al.25 Amino-thio derivatives 3-amino-2-
(cyclohexylthio)-6-phenylthieno[2,3-d]pyrimidin-4(3H)-one 9, 3-
amino-2-[(2,4-dinitrophenyl)thio]-6-phenylthieno[2,3-d]pyrimi-
din-4(3H)-one 10 and N-[2-[(2,4-dinitrophenyl)thio]-4-oxo-6-
at TLC; mp 239–241 °C dec.; IR: 3324, 3271 and 3185 (NH and
NH2), 1700 and 1661 (C@O) cmÀ1 1H NMR: d 1.57 (d, J = 7.6,
CH3), 4.36 (q, J = 7.6, CH), 5.89 (s, 2H, NH2), 7.17–7.95 (m, 4H,
Ar–H), 11.99 (s, 1H, NH).
The synthesis of 2-[(3-amino-4-oxo-3,4-dihydro[1]benzothie-
no[3,2-d]pyrimidin-2-yl)thio]propanoic acid 7 was reported in
Santagati et al.37
2-[(3-Amino-4-oxo-3,4-dihydroquinazolin-2-yl)thio]propanoic
acid 8, as compound 5, yield 10%, as a white powder, pure at TLC,
mp 260–63 °C dec.; IR: 3313 and 3204 (NH2), 1710 and 1694
(C@O).
;
3-Amino-2-(cyclohexylthio)-6-phenylthieno[2,3-d]pyrimidin-4(3H)-
one 9: A mixture of sodium salt of 3-amino-2,3-dihydro-6-phenyl-
2-thioxothieno[2,3-d]pyrimidin-4(1H)-one (200 mg, 0.67 mmol),
cyclohexyl iodide (0.1 ml, 98% d = 1.624, 0.75 mmol) in dimethyl-
formamide (2 ml) was heated at 80 °C under stirring for 5 h.38 After
cooling to room temperature the mixture was poured in to water
(100 ml): a solid separated. The solid was collected, treated with
sodium hydroxide, collected, washed with water, dried first at
room temperature and then under vacuum to give 9 as a green-yel-
low powder, pure at TLC, mp: 208–10 °C dec.; yield 50%; IR: 3301
phenylthieno[2,3-d]pyrimidin-3(4H)-y]methanesulfonamide
11
have been synthesized according to methods reported by Granata
et al.26 The purity and structures of compounds 1–11 was accom-
plished as described.25,26 The compounds 1–8 were tested as race-
mic mixtures. Indomethacin was purchased from Sigma Chemical
(Milan, Italy) whereas celecoxib was isolated by extraction from
Celebrex (Pharmacia or Pfizer) followed by purification on a chro-
matographic column. Stock solutions of the reference standards or
test compounds were prepared in dimethyl sulfoxide, and an
equivalent amount of dimethyl sulfoxide was included in control
samples.
and 3191 (NH2), 2928 [(CH2)6], 1679 (C@O) cmÀ1
;
1H NMR
(dimethylsulfoxide-d6): d 1.22–2.03 (m, 10H, cyclohexyl), 3.40–
3.79 (m, 1H, S–CH), 5.76 (s, 2H, NH2), 7.34–7.84 (m, 6H, Ar–H
and thiophene-H). Anal. Calcd for C18H19N3OS2 (MM 357): C,
60.50; H, 5.30; N, 11.75; S, 17.92. Found: C, 60.15; H, 5.00; N,
12.00; S, 18.75.
4.2. Synthesis and characterization of derivatives
1H NMR-spectra were recorded at 200 MHz on a Varian Gemini
200 spectrometer; chemical shifts (d) are reported in ppm from tet-
ramethylsilane as internal standard; coupling constants (J) are in
Hertz (Hz). IR spectra were recorded on a Perkin Elmer 1600 Series
FT-IR in potassium bromide disks. Microanalyses for C, H, N and S
were obtained from an EA 1108 elemental analyzer Fisons Carlo-
Erba instrument. Analyses indicated by the symbols of the ele-
ments or functions were within 0.4% of the theoretical values.
Melting points are uncorrected and were determined in open cap-
illary tubes on a SMP1 apparatus (Stuart Scientific Staffordshire).
The mp’s of all crude compounds were within À3 °C, if compared
with the pure product: therefore, as synthetic intermediates they
could be used without further purification. The purity of com-
pounds was checked by thin layer chromatography on Merck silica
gel 60 F-254 plates. All commercial chemicals were purchased
from Aldrich, Fluka, Merck, Lancaster and Carlo-Erba and were
used without further purification.
3-Amino-2-[(2,4-dinitrophenyl)thio]-6-phenylthieno[2,3-d]pyrim-
idin-4(3H)-one 10: To a solution of sodium salt of 3-amino-2,3-
dihydro-6-phenyl-2-thioxo-thieno[2,3-d]pyrimidin-4(1H)-one
(200 mg, 0.67 mmol) in water (10 ml), 2,4-dinitroiodobenzene
(200 mg, 98%, 0.67 mmol) in ethanol (10 ml) and powdery copper
(20 mg) were added; the mixture was heated at 80 °C under stir-
ring for 2 h.38 An orange precipitate formed. The mixture was fil-
tered while warm. The precipitate was washed with warm
ethanol, treated with sodium hydroxide, collected, washed with
water and dried first at room temperature and then at 100 °C to
give 10 as an yellowish powder; yield 40%; mp 213–15 °C dec.;
IR: 3305 and 3189 (NH2), 1681 (C@O) cmÀ1. Anal. Calcd for
C18H11N5O5S2Á2H2O (MM 477): C, 45.28; H, 3.14; N, 14.67; S,
13.41. Found: C, 45.31; H, 3.00; N, 14.30; S, 13.80.
N-[2-[(2,4-dinitrophenyl)thio]-4-oxo-6-phenylthieno[2,3-d]pyrim-
idin-3(4H)-yl]methanesulfonamide 11: To a solution of N-(4-oxo-6-
phenyl-2-thioxo-1,4-dihydrothieno[2,3-d]pyrimidin-3(2H)meth-
anesulfonamide (100 mg, 0.28 mmol), and sodium hydroxide
(30 mg, 0.75 mmol) in water (20 ml) 2,4-dinitroiodobenzene
(90 mg, 98%, 0.3 mmol) in ethanol (5 ml) and powdery copper
(20 mg) were added and the mixture was heated at reflux under
stirring;38 after 6 h the mixture was filtered while hot and the fil-
trate was cooled to room temperature and poured in to water; the
solution was acidified with concentrated hydrochloride acid to pH
3–4: the resulting solid was collected, washed with water, dried
first at room temperature and then under vacuum and crystallized
from ethanol/water to give 11 as an orange powder, pure at TLC;
yield 60%; mp < 130 °C dec.; IR: 3210 br (NH), 3100 (C–H benzene),
1705 (C@O), 1345 and 1160 (N–SO2) cmÀ1; 1H NMR (dimethylsulf-
oxide-d6): d 3.39 (s, 3H, CH3), 7.32–8.89 (m, 9H, Ar–H and H-thio-
phene), 11.68 (s, 1H, NH). Anal. Calcd for C19 H13 N5 07 S3 (MM
519.5): C, 43.88; H, 2.50; N, 13.47; S, 18.47. Found: C, 43.77; H,
2.47; N, 13.75; S, 18.58.
The synthesis of [(3-amino-4-oxo-3,4-dihydroquinazolin-2-
yl)thio](phenyl)-acetic acid 1 was reported in Santagati et al.35
The synthesis of [(3-amino-5,6-dimethyl-4-oxo-3,4-dihydrothi-
eno[2,3-d]pyrimidin-2-yl)thio](phenyl)acetic acid 2 was reported
in Santagati et al.36
[(3-Amino-4-oxo-3,4-dihydrothieno[3,2-d]pyrimidin-2-yl)thio]
(phenyl)acetic acid 3 was prepared according to the same proce-
dure adopted for compound 2; yield 60%, as a white powder, pure
at TLC, mp 113–15 °C dec.; IR: 3313 and 3200 (NH2), 1729 and
1659 (C@O) cmÀ1
.
[(3-Amino-6-bromo-4-oxo-3,4-dihydroquinazolin-2-yl)thio]
(phenyl)acetic acid 4, as compound 3; yield 25%, as white powder,
pure at TLC; mp > 125 °C dec.; IR: 3324 and 3210 (NH2), 1721 and
1678 (C@O) cmÀ1
.
2-[(3-Amino-4-oxo-6-phenyl-3,4-dihydrothieno[2,3-d]pyrimi-
din-2-yl)thio]propanoic acid 5 was prepared according to the same
method adopted for compound 7; yield 35%, as a white powder,
pure at TLC; mp > 265 °C dec.; IR: 3301 and 3191 (NH2), 1715
and 1680 (C@O) cmÀ1
;
1H NMR: d 5.35 (s, 1H, CH), 5.92 (s, 2H,
4.3. Determination of COX-1 activity in human whole blood
NH2), 7.32–7.78 (m, 11H, Ar–H and H-thiophene), 13.00 (s, 1H,
COOH).
2-[(3-Amino-4-oxo-4,5-dihydro-3H-pyrimido[5,4-b]indol-2-yl)
thio]propanoic acid 6, as compound 5; yield 65%, as a powder, pure
Aliquots of 1 ml of whole blood drawn from the volunteers, stu-
dents at the University of Catania, prior to aspirin administration,
were immediately transferred into glass tubes and allowed to clot

V. Cardile et al. / Bioorg. Med. Chem. 17 (2009) 1991–1996
1995
at 37 °C for 60 min. The test derivatives were added in 2
ll of
opportunely diluted provides the top standard of 320 pg/50
ll em-
DMSO, to attain the final concentration of 10 M—blanks received
l
ployed for the serial dilutions (8 standard levels). PGE2 was mea-
sured in the range 2.5–320 pg/well (50–6400 pg/ml).
DMSO. Serum was separated by centrifugation (10 min at
2000 rpm) and kept at –70 °C until assayed for TxB2 production
by specific enzyme-immunoassay kit (RPN 220 Amersham Biosci-
ences, USA). TxB2 was determined according to the instructions
provided by the manufacturer of the kit. The results were ex-
pressed as percent of inhibition of TxB2 production relative to con-
trol (blanks) incubation containing DMSO (vehicle). The detection
limit was 29 pg/ml.
4.7. NO determination
The concentration of nitrite and nitrate in the supernatant of
J774 culture media was quantified by colorimetric assay based
on the Griess40 reaction, as described by Ding et al.41 Briefly,
0.1 ml of supernatant from untreated or treated J774 cultures
was mixed with an equal volume of Griess reagent (a 1:1 mixture
of 0.1% naphthylenediamine dihydrochloride and 1% sulfanilamide
in 5% H3PO4) at room temperature for 10 min. The absorbance was
measured at k 550 nm in a microplate spectrophotometer reader
(Titertek Multiskan, DAS, Italy). Sodium nitrite was used as a stan-
4.4. COX-2 induction and human whole blood assay
Aliquots of 1 ml of whole blood samples drawn from young
healthy volunteers, students at the University of Catania (both
male and female), 48 h after oral administration of 300 mg of aspi-
rin, to suppress the contribution platelet, PGHS-1 (COX-1), and
containing 10 U.I. of sodium heparin, were incubated either in
dard. The detection limit was 1
As control, to verify the effect of an inhibitor of NO generation
on levels of nitrite/nitrate, NG-monomethyl- -arginine (NGMMA)
was used, dissolved in the medium and employed at the concentra-
tion of 500 M (124 g/ml).
lM.
L
the absence or in the presence of LPS (10
37 °C, to obtain the maximum production of PGE2.39 The test com-
pounds were added in 2 l of DMSO before stimulation by LPS to
attain the final concentration of 10 M except blanks, which re-
lg/ml) for 24 h at
l
l
l
l
4.8. Statistical analysis
ceived DMSO. After incubation, the plasma was separated by cen-
trifugation (10 min at 2000 rpm) and kept at À70 °C until assayed
for PGE2 by a specific enzyme-immunoassay kit (Prostaglandin E2,
Biotrak, EIA System, Amersham Biosciences, USA).
Each experiment was repeated at least three times in triplicate
and the mean SEM for each value was calculated. Statistical anal-
ysis of the results was performed using Student’s t-test and one-
way ANOVA by the statistical software package SYSTAT, version
9 (Systat Inc., Evanston IL, USA). A difference was considered signif-
icant at P < 0.01.
4.5. Cell cultures
The J774 cells, a mouse monocyte-macrophage cell line, were
obtained from American Type Culture Collection (Rockville, MD,
USA). The cells were cultured in DMEM containing 10% fetal calf
serum, 4.5 g/l glucose, 1 mM sodium pyruvate, 100 U/ml penicillin,
Acknowledgment
This work was supported in part by grants from the project of
Ministero Istruzione Università Ricerca (MIUR).
100 lg/ml streptomycin, and 25 lg/ml fungizone (InVitrogen, UK)
and incubated at 37 °C and 5% CO2. The medium was changed
every three days and subcultures were performed every 8–10 days.
Experimental J774 cells were plated in 24-well cultures plated at a
density of 2.5 Â 105 cells/ml and allowed to adhere at 37 °C in 5%
CO2/95% O2 for 24 h. After this time, the cells were stimulated with
References and notes
1. Funk, C. D.; Funk, L. B.; Kennedy, M. E.; Pong, A. S.; Fitzgerald, G. A. FASEB J.
1991, 5, 2304.
2. Goppelt-Struebe, M. Biophys. Acta 1995, 1256, 257.
3. Smith, W. L.; Meade, E. A.; DeWitt, D. L. Ann. N.Y. Acad. Sci. 1994, 744, 50.
4. Grosser, T.; Fries, S.; Fitzgerald, G. A. J. Clin. Invest. 2006, 116, 4.
5. Sanchez, T.; Moreno, J. J. Prostaglandins Other Lipid Mediators 1999, 57, 119.
6. Seibert, K.; Zhang, Y.; Leahy, K.; Hauser, S.; Masferrer, J.; Perkins, W.; Lee, L.;
Isakson, P. Proc. Natl. Acad. Sci. U.S.A. 1994, 91, 12013.
7. DeWitt, D. L. Biochim. Biophys. Acta 1991, 1083, 121.
8. Oshima, T.; Yoshimoto, T.; Yamamoto, S.; Kumegawa, M.; Yokoyama, C.;
Tanabe, T. J. Biol. Chem. 1991, 266, 13621.
LPS (5
alone) or presence of the test compounds with concentrations of
10 M. The supernatants were collected for the measurement of
lg/ml) for 72 h to induce COX-2 in the absence (DMSO
l
PGE2 by a specific enzyme-immunoassay kit (Prostaglandin E2,
Biotrak, EIA System, Amersham Biosciences, USA), and the deter-
mination of NO by colorimetric assay.
9. Seibert, K.; Masferrer, J. L. Receptor 1994, 4, 17.
10. Crofford, L. J.; Wilder, R. L.; Ristimaki, A. P.; Sano, H.; Remmers, E. F.; Epps, H. R.;
Hla, T. J. Clin. Invest. 1994, 93, 1095.
4.6. PGE2 determination
11. Kujubu, D. A.; Fletcher, B. S.; Varnum, B. C.; Lim, R. W.; Herschman, H. R. J. Biol.
Chem. 1991, 266, 12866.
12. O’Banion, M. K.; Winn, V. D.; Young, D. A. Proc. Natl. Acad. Sci. U.S.A. 1992, 89,
4888.
13. Fu, J. Y.; Masferrer, J. L.; Seibert, K.; Raz, A.; Needleman, P. J. Biol. Chem. 1990,
265, 16737.
14. Masferrer, J. L.; Zweifel, B. S.; Seibert, K.; Needleman, P. J. Clin. Invest. 1990, 86,
1375.
15. Lee, S. H.; Soyoola, E.; Chanmugam, P.; Hart, S.; Sun, W.; Zhong, H.; Liou, S.;
Simmons, D.; Hwang, D. J. Biol. Chem. 1992, 267, 25934.
16. O’Sullivan, M. G.; Huggins, E. M., Jr.; Meade, E. A.; DeWitt, D. L.; McCall, C. E.
Biochem. Biophys. Res. Commun. 1992, 187, 1123.
17. Sano, H.; Hla, T.; Maier, J. A.; Crofford, L. J.; Case, J. P.; Maciag, T.; Wilder, R. L. J.
Clin. Invest. 1992, 89, 97.
The concentration of PGE2 was measured in the human whole
blood and J774 culture media by biotrak enzyme-immunoassay
(EIA) system according to the manufacturer’s instructions (Amer-
sham Biosciences, USA). The assay is based on competition be-
tween unlabeled PGE2 and a fixed quantity of peroxidase-labeled
PGE2 for a limited number of binding sites on a PGE2 specific anti-
body. Briefly, 50
ll of supernatant was dispensed into 96 wells
microplate and added of 50
l
l of diluted antibody and 50 l of di-
l
luted conjugate. The plate was incubated at room temperature for
1 h. Afterwards, all wells were washed four times with wash buffer
and 150 ll of room temperature equilibrate enzyme substrate was
added to each well. The plate was mixed on a microtitre plate sha-
ker for 30 min at room temperature. The reaction was stopped by
the addition of 100 ll of sulfuric acid 1 M to each well. The optical
density of each sample was measured with a microplate spectro-
photometer reader (Titertek Multiskan, DAS, Italy) at k = 450 nm
within 30 min. A calibration curve was generated by using as a
PGE2 standard, part of the commercial kit and ready for use. This
18. Sirois, J.; Richards, J. S. J. Biol. Chem. 1992, 267, 6382.
19. Masferrer, J. L.; Seibert, K.; Zweifel, B.; Needleman, P. Proc. Natl. Acad. Sci. U.S.A.
1992, 89, 3917.
20. Masferrer, J. L.; Reddy, S. T.; Zweifel, B. S.; Seibert, K.; Needleman, P.; Gilbert, R.
S.; Herschman, H. R. J. Pharmacol. Exp. Ther. 1994, 270, 1340.
21. Vane, J. R.; Bakhle, Y. S.; Botting, R. M. Annu. Rev. Pharmacol. Toxicol. 1998, 38,
97.
22. Bateman, D. N. Lancet 1994, 343, 1051.
23. Kim, S. F.; Huri, D. A.; Snyder, S. H. Science 2005, 310, 1966.
24. Appleton, I.; Tomlinson, A.; Willoughby, D. A. Adv. Pharmacol. 1996, 35, 27.

1996
V. Cardile et al. / Bioorg. Med. Chem. 17 (2009) 1991–1996
25. Santagati, A.; Granata, G.; Marrazzo, A.; Santagati, M. Arch. Pharm. (Weinheim)
2003, 336, 429.
26. Granata, G.; Barbagallo, S.; Marrazzo, A.; Santagati, A.; Lombardo, L.; Cardile, V.
J. Heterocyl. Chem. 2006, 43, 1099.
27. Brideau, C.; Kargman, S.; Liu, S.; Dallob, A. L.; Ehrich, E. W.; Rodger, I. W.; Chan,
C. C. Inflamm. Res. 1996, 45, 68.
28. Balazy, M.; Poff, C. D. Curr. Vasc. Pharmacol. 2004, 2, 81.
29. Kermorvant-Duchemin, E.; Sennlaub, F.; Sirinyan, M.; Brault, S.; Andelfinger,
G.; Kooli, A.; Germain, S.; Ong, H.; d’Orleans-Juste, P.; Gobeil, F.; Zhu, T.;
Boisvert, C.; Hardy, P.; Jain, K.; Falck, J. R.; Balazy, M.; Chemtob, S. Nat. Med.
2005, 11, 1339.
30. Tsai, A. L.; Wei, C.; Kulmacz, R. J. Arch. Biochem. Biophys. 1994, 313, 367.
31. Yang, T.; Zhang, A.; Pasumarthy, A.; Zhang, L.; Warnock, Z.; Schnermann, J. B.
Am. J. Physiol. Renal Physiol. 2006, 291, F891.
33. Gasparini, G.; Longo, R.; Sarmiento, R.; Morabito, A. Lancet Oncol. 2003, 4, 605.
34. Balsamo, A.; Mancini, F.; Milanese, C.; Orlandini, E.; Ortore, G.; Pinza, M.;
Rapposelli, S.; Rossello, A. Il Farmaco 2003, 58, 707.
35. Santagati, A.; Modica, M.; Monsù Scolaro, M.; Santagati, M. J. Chem. Res. (S)
1999, 5, 86.
36. Santagati, A.; Modica, M.; Santagati, M. J. Heterocycl. Chem. 2000, 37, 1161.
37. Santagati, A.; Granata, G.; Marrazzo, A.; Santagati, M. Arch. Pharm. Pharm. Med.
Chem. 2003, 336, 429.
38. Santagati, A.; Marrazzo, A.; Granata, G. J. Heterocycl. Chem. 2003, 40, 869.
39. Patrignani, P.; Panara, M. R.; Greco, A.; Fusco, O.; Natoli, C.; Iacobelli, S.;
Cipollone, F.; Ganci, A.; Creminon, C.; Maclouf, J. J. Pharmacol. Exp. Ther. 1994,
271, 1705.
40. Green, L. C.; Wagner, D. A.; Glogowski, J.; Skipper, P. L.; Wishnok, J. S.;
Tannenbaum, S. R. Anal. Biochem. 1982, 126, 131.
32. Bing, R. J.; Miyataka, M.; Rich, K. A.; Hanson, N.; Wang, X.; Slosser, H. D.; Shi, S.
R. Clin. Cancer Res. 2001, 7, 3385.
41. Ding, A. H.; Nathan, C. F.; Stuehr, D. J. J. Immunol. 1988, 141, 2407.