Synthesis and biological evaluation of 5-nitropyrimidine-2,4-dione analogues as inhibitors of nitric oxide and iNOS activity

Article abstract of DOI:10.1111/cbdd.12386

Fifty two compounds based on 5-nitropyrimidine-2,4-dione moiety have been synthesized and evaluated for their inhibitory potency on the production of nitric oxide. Among them, compound 36 inhibited the production of nitric oxide (IC₅₀: 8.6 μM) on lipopolysaccharide-induced RAW 264.7 cells and inducible nitric oxide synthase activity (IC₅₀: 6.2 μM), as well as exerted no potential cytotoxicity (IC₅₀ > 80.0 μM). Docking study confirmed that compound 36 was an inducible nitric oxide synthase inhibitor with perfect binding to the active site of inducible nitric oxide synthase. At a dose of 10 mg/kg, oral administration of 36 possessed protective properties in carrageenan-induced paw edema of male ICR mice.

Full text of DOI:10.1111/cbdd.12386

Chem Biol Drug Des 2015; 85: 296–299
Research Letter
Synthesis and Biological Evaluation of
5-Nitropyrimidine-2,4-dione Analogues as Inhibitors of
Nitric Oxide and iNOS Activity
Liang Ma1, Linhong He1, Lei Lei1, Xiaolin Liang1,
responses, and inflammatory disorders at low concentra-
tion (4,5). Nitric oxide is generated through the oxidation of
L-arginine to L-citrulline which catalyzed by a family of
nitric oxide synthase (NOS) in a nicotinamide adenine dinu-
Kai Lei1, Ronghong Zhang1, Zhuang Yang2 and
Lijuan Chen1,*
1State Key Laboratory of Biotherapy and Cancer Center,
cleotide phosphate (NADPH)- and O2-dependent process
West China Hospital, West China Medical School, Sichuan
University, Chengdu 610041, China
(6). There are at least three NOS isoforms including endo-
thelial NOS (eNOS), neuronal NOS (nNOS), and inducible
NOS (iNOS). Normally, NO is expressed by eNOS and
nNOS at a low level; while activated proinflammatory cyto-
kines or lipopolysaccharide (LPS), the iNOS produces a
high level of NO to exert immune defense against patho-
gens at the inflammatory site (7). Aberrant iNOS-mediated
NO biosynthesis, located in macrophages, has been
involved in a variety of pathological and physiological dis-
eases including septic stroke, hypertension, cancer, ische-
2College of Chemistry, Sichuan University, Chengdu
610041, China
*Corresponding author: Lijuan Chen, lijuan17@hotmail.com
Fifty two compounds based on 5-nitropyrimidine-
2,4-dione moiety have been synthesized and evaluated
for their inhibitory potency on the production of nitric
oxide. Among them, compound 36 inhibited the pro-
duction of nitric oxide (IC50: 8.6 lM) on lipopolysaccha-
ride-induced RAW 264.7 cells and inducible nitric
mia,
and
rheumatoid
arthritis
(8).
Therefore,
pharmacological reduction of iNOS-mediated NO produc-
tion is regarded as one of the essential conditions to allevi-
ate a variety of acute and chronic inflammatory diseases.
oxide synthase activity (IC50
: 6.2 lM), as well as
exerted no potential cytotoxicity (IC50 > 80.0 lM).
Docking study confirmed that compound 36 was an
inducible nitric oxide synthase inhibitor with perfect
binding to the active site of inducible nitric oxide syn-
thase. At a dose of 10 mg/kg, oral administration of 36
possessed protective properties in carrageenan-
induced paw edema of male ICR mice.
Considerable studies have reported various type inhibitors
of NO production, such as unsaturated fatty acids, chal-
cones, lignans, polyacetylenes, coumarins, flavonoids,
stilbenes, sesquiterpenes, thiazolidinediones, diterpenes,
triterpenes, diarylheptanoids, and alkaloids (9–11). Pyrimi-
dine-2,4-dione was known to inhibit not only the produc-
tion of NO in LPS-activated macrophage, but also the
activity of iNOS proteins, stating that pyrimidine-2,4-dione
has been recognized as an key modulator for anti-inflam-
matory functions through the fine tuning of NO level (12).
Key words: 5-nitropyrimidine-2,4-dione, docking study,
inducible NOS, nitric oxide production, paw edema
Received 22 March 2014, revised 12 May 2014 and accepted
for publication 18 June 2014
Compounds 1-52 have been synthesized according to the
previously reported method (13). In detail, a variety of aro-
matic aldehydes were condensed with 6-methyl-5-nitro-
pyrimidine-2,4-dione to afford a focused library of alkenes
Inflammation is a complex process involving numerous
mediators of cellular and plasma origin with interrelated
biological effects. Proinflammatory stimulus activates cellu-
lar responses and regulates inflammatory and immune
functions including the recruitment of macrophages and
production of proinflammatory cytokines (1,2). The acti-
vated macrophages produce nitric oxide (NO) and upregu-
late prostaglandins (PGs), interleukin (IL)-1b, and tumor
necrosis factor (TNF)-a in the inflammatory process (3).
1
with pure trans geometry (JCH=CH: 16.0 Hz in 400 MHz H
NMR). The cis alkenes were not obtained because the
trans isomer might be thermodynamically more stable than
the cis ones. As depicted in Scheme 1, the nitration of
6-methyluracil in the presence of H2SO4 and fuming HNO3
led to 6-methyl-5-nitrouracil (1a) with a good yield. Con-
densations of 1a with appropriate aldehydes using piperi-
dine as basic catalyst and 1-butanol as a valid solvent
provided the piperidine salts of 5-nitro-6-styryluracil deriva-
tives (14). And then, the piperidine salts were dissolved or
Nitric oxide, being an endogenous free radical, is a
crucial and unique signaling mediator of vasorelaxation,
neurotransmission, non-specific host defense, immune
296
ª 2014 John Wiley & Sons A/S. doi: 10.1111/cbdd.12386

Inhibitors of NO Production and iNOS Activity
Scheme 1: General synthesis of
compounds 1-52.
suspended in dilute KOH solution for several minutes and
further neutralized by the addition of excess hydrochloric
acid to give the desired compounds.
non-steroidal anti-inflammatory drug, was chosen as a
positive control. As depicted in Figure 1, eight derivatives
(12, 17, 21, 23, 30, 31, 36, and 39) suppressed the LPS-
induced production of NO at a concentration of 10 lM,
and their inhibitory rates (IR) were more than 30.0% com-
pared to anti-inflammatory drug indomethacin (IR: 28.7%).
Among them, 36 exhibited the most potent inhibitory
We initially accessed the capacity of our desired com-
pounds to inhibit the production of NO in LPS-induced
RAW246.7 macrophages. In the study, indomethacin, as a
Figure 1: Inhibitory effects of compounds 1-52 on nitric oxide production in lipopolysaccharide (LPS)-induced RAW 264.7 cells. RAW
264.7 cells were pretreated with compounds 1-52 at the concentration of 10 lM for 2 h and then incubated with LPS (1 lg/mL) for 18 h.
The amount of nitrite with LPS-treated only group was set as 100.0% and the control as 0.0%. Inhibition (%) = [LPS
(OD540) – Compounds (OD540)]/[LPS (OD540) – Control (OD540)] 9 100%. Results are means Æ SD of three independent experiments.
*P < 0.05 and **P < 0.01 versus LPS-treated group.
Chem Biol Drug Des 2015; 85: 296–299
297

Ma et al.
Table 1: The Inhibition of nitric oxide (NO) production and induc-
ible nitric oxide synthase (iNOS) activity of selected compounds
NO
iNOS
Compds
IC50 (lM)
IC50 (lM)
17
21
23
31
36
39
17.5
14.3
25.3
10.1
8.6
40.2
32.6
>50.0
7.8
6.2
>50.0
30.2
effect, and 17, 21, and 31 also showed the comparable
inhibitory activity. Interestingly, we found that 45 promoted
the expression of NO in LPS-induced RAW246.7 macro-
phages. The inhibitory rate of the remaining derivatives
was all less than 30.0% and inferior to that of indometha-
cin and 36.
Figure 2: Inhibition of carrageenan-induced paw edema in ICR
mice. The results were expressed as the means Æ SD (n = 6).
*P < 0.05 versus vehicle.
Subsequently, six selected compounds were investigated
iNOS-mediated NO production and cytotoxicity in the
presence or absence of LPS induction. As shown in
Table 1, compound 31 and 36 exhibited the more potent
inhibitory potency on the generation of NO (IC50: 10.1, and
8.6 lM, respectively) and iNOS activity (IC50: 7.8, and
6.2 lM, respectively) in the six selected compounds. How-
ever, the three compounds 21, 31, and 39 showed the
cytotoxic to RAW264.7 cells with or without LPS induction.
The cytotoxity IC50 of compounds 17, 23, and 36 was all
more than 80 lM, stating that the potent inhibitory activity
against NO production and iNOS was not related with their
cytotoxity (supplementary material). In the view of potent
and low cytotoxic compound 36, the further biological
evaluation as a potential lead was essential.
Figure 3: The interaction mode of 36 within the active site of
iNOS (PDB ID: 1r35).
Compound 36 (meta-nitrophenyl group) exhibited a little
more potent inhibitory activity than 31 (para-fluorophenyl).
Compared to the unsubstituted styryl molecule 1, the
inhibitory potency against NO production was increased
when para substituents of electron-donating groups (e.g.,
hydroxyl group in 12 and 17) were introduced. However,
the fluorine atom at the para position (para-fluorinated in
31, and meta-fluorinated in 30) improved the inhibitory
effects as a result of their inherent electronegativity and
small atomic radius compared with the other halogen
atoms (Cl and Br). The properties may contribute to the
stability and also served to block metabolism at particularly
reactive sites. But the number and atomic radius of
halogen atom influenced the inhibitory activity. As for
meta-nitro group, the potent inhibitory activity of 36 was
exhibited because of the styryl potential electron effect. By
the analysis of docking study, we found that the spatial
distance is not beneficial to the formation of hydrogen
bond or p–p interaction between para substituent group
and porphyrin. On the contrary, the meta-nitro group was
in favor of the formation of hydrogen bond and p–p
interaction. In addition, the introduction of the steric bulk
of 2-quinolinyl heterocycle slightly improved the inhibitory
rate, especially compound 51 (IR = 35.0%).
The edema is an important parameter of acute inflamma-
tion for evaluating potential compounds with anti-inflam-
matory activity. In the carrageenan-induced paw edema
test, the inflammatory response was quantified by incre-
ment in paw size (edema) 2 h after carrageenan was
injected and the paw edema was observed. Oral adminis-
tration of 36 at a dose of 10 mg/kg suppressed carra-
geenan-induced paw edema of male ICR mice in contrast
to that of positive control indomethacin (Figure 2). Com-
pound 36 inhibited edema formation with 22.05%
(p < 0.05) at the end-point, while indomethacin showed
an increased paw thickness of 16.54% (p < 0.05).
To gain better understanding on the potency of the studied
compounds, we proceeded to examine the interaction of 36
with iNOS protein which 3D structure was gained from Pro-
tein Data Bank (PDB ID: 1r35). The molecular docking was
performed by simulation of 36 into the active binding site of
298
Chem Biol Drug Des 2015; 85: 296–299

Inhibitors of NO Production and iNOS Activity
iNOS. Compounds were built with ChemBio3D and
optimized at molecular mechanical and semi-empirical level
using HYPERCHEM software. Conformers of 36 were created
by the aid of Omega, and the up limit of conformer number
was set to 2000. Then, they were docked to the binding site
of tubulin by employing a protein-ligand docking program
FRED. Scoring function chemgauss3 was used for
exhaustive searching, solid body optimizing, and interaction
scoring. The pose with the most favorable score was
remained. As depicted in Figure 3, the nitrophenyl moiety of
36 was buried in a pocket wove between ASN364, VAL346,
PRO344, and an HEME. Two p–p interactions were
observed between porphyrins of HEME with phenyl ring and
nitro group. Four hydrogen bonds were formed between
5-nitropyrimidine-2,4(1H,3H)-dione moiety and amino acid
ASP367, GLU371, and TYR341. Therefore, docking studies
at the binding site suggested that compound 36 binds
tightly to this site via multiple H-bonds and p–p interaction.
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^
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In summary, 52 compounds contained 5-nitropyrimidine-
2,4(1H,3H)-dione moiety have been readily obtained and
subsequently investigated for inhibitory effects on the pro-
duction of NO. Among them, 36 significantly inhibited
iNOS activity (IC50 = 6.2 lM) and iNOS-mediated NO pro-
duction on LPS-induced RAW264.7 cells (IC50 = 8.6 lM).
Docking studies suggested that compound 36 bound
tightly to this active site via multiple H-bonds and p–p
interaction. At a dose of 10 mg/kg, oral administration of
36 possessed protective properties in carrageenan-
induced paw edema of male ICR mice.
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Zheng H. et al. (2011) Synthesis and biological evalua-
tion of novel 5-benzylidenethiazolidine-2,4-dione deriv-
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Acknowledgments
We are grateful to National Key Programs of China
(2012ZX09103101-017) and China Postdoctoral Science
Foundation (2014M552373).
Conflict of Interest
€
14. Grahner B., Winiwarter S., Lanzner W., Muller C.E.
The authors have declared no conflict of interest.
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Table S1. The IC50 of cytotoxicity of selected com-
pounds in RAW 264.7 cells.
Appendix S1. Experimental.
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