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, Kai Lei1, Ronghong Zhang1, Zhuang Yang2 and Lijuan Chen1,* 1
State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041, China 2 College of Chemistry, Sichuan University, Chengdu 610041, China *Corresponding author: Lijuan Chen, [email protected] Fifty two compounds based on 5-nitropyrimidine2,4-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 lM) on lipopolysaccharide-induced RAW 264.7 cells and inducible nitric 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 synthase. At a dose of 10 mg/kg, oral administration of 36 possessed protective properties in carrageenaninduced paw edema of male ICR mice. Key words: 5-nitropyrimidine-2,4-dione, docking inducible NOS, nitric oxide production, paw edema
study,
Received 22 March 2014, revised 12 May 2014 and accepted for publication 18 June 2014
Inflammation is a complex process involving numerous mediators of cellular and plasma origin with interrelated biological effects. Proinflammatory stimulus activates cellular responses and regulates inflammatory and immune functions including the recruitment of macrophages and production of proinflammatory cytokines (1,2). The activated macrophages produce nitric oxide (NO) and upregulate prostaglandins (PGs), interleukin (IL)-1b, and tumor necrosis factor (TNF)-a in the inflammatory process (3). Nitric oxide, being an endogenous free radical, is a crucial and unique signaling mediator of vasorelaxation, neurotransmission, non-specific host defense, immune
296
responses, and inflammatory disorders at low concentration (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 dinucleotide phosphate (NADPH)- and O2-dependent process (6). There are at least three NOS isoforms including endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS). Normally, NO is expressed by eNOS and nNOS at a low level; while activated proinflammatory cytokines or lipopolysaccharide (LPS), the iNOS produces a high level of NO to exert immune defense against pathogens at the inflammatory site (7). Aberrant iNOS-mediated NO biosynthesis, located in macrophages, has been involved in a variety of pathological and physiological diseases including septic stroke, hypertension, cancer, ischemia, and rheumatoid arthritis (8). Therefore, pharmacological reduction of iNOS-mediated NO production is regarded as one of the essential conditions to alleviate a variety of acute and chronic inflammatory diseases. Considerable studies have reported various type inhibitors of NO production, such as unsaturated fatty acids, chalcones, lignans, polyacetylenes, coumarins, flavonoids, stilbenes, sesquiterpenes, thiazolidinediones, diterpenes, triterpenes, diarylheptanoids, and alkaloids (9–11). Pyrimidine-2,4-dione was known to inhibit not only the production 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-inflammatory functions through the fine tuning of NO level (12). Compounds 1-52 have been synthesized according to the previously reported method (13). In detail, a variety of aromatic aldehydes were condensed with 6-methyl-5-nitropyrimidine-2,4-dione to afford a focused library of alkenes with pure trans geometry (JCH=CH: 16.0 Hz in 400 MHz 1H 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. Condensations of 1a with appropriate aldehydes using piperidine as basic catalyst and 1-butanol as a valid solvent provided the piperidine salts of 5-nitro-6-styryluracil derivatives (14). And then, the piperidine salts were dissolved or 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. We initially accessed the capacity of our desired compounds to inhibit the production of NO in LPS-induced RAW246.7 macrophages. In the study, indomethacin, as a
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 LPSinduced production of NO at a concentration of 10 lM, and their inhibitory rates (IR) were more than 30.0% compared to anti-inflammatory drug indomethacin (IR: 28.7%). Among them, 36 exhibited the most potent inhibitory
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 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 inducible nitric oxide synthase (iNOS) activity of selected compounds
Compds
NO IC50 (lM)
iNOS IC50 (lM)
17 21 23 31 36 39
17.5 14.3 25.3 10.1 8.6 30.2
40.2 32.6 >50.0 7.8 6.2 >50.0
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 macrophages. The inhibitory rate of the remaining derivatives was all less than 30.0% and inferior to that of indomethacin and 36. 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. However, 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. 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 298
Figure 2: Inhibition of carrageenan-induced paw edema in ICR mice. The results were expressed as the means SD (n = 6). *P < 0.05 versus vehicle.
Figure 3: The interaction mode of 36 within the active site of iNOS (PDB ID: 1r35).
of 2-quinolinyl heterocycle slightly improved the inhibitory rate, especially compound 51 (IR = 35.0%). The edema is an important parameter of acute inflammation for evaluating potential compounds with anti-inflammatory activity. In the carrageenan-induced paw edema test, the inflammatory response was quantified by increment in paw size (edema) 2 h after carrageenan was injected and the paw edema was observed. Oral administration of 36 at a dose of 10 mg/kg suppressed carrageenan-induced paw edema of male ICR mice in contrast to that of positive control indomethacin (Figure 2). Compound 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 Protein Data Bank (PDB ID: 1r35). The molecular docking was performed by simulation of 36 into the active binding site of 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. In summary, 52 compounds contained 5-nitropyrimidine2,4(1H,3H)-dione moiety have been readily obtained and subsequently investigated for inhibitory effects on the production of NO. Among them, 36 significantly inhibited iNOS activity (IC50 = 6.2 lM) and iNOS-mediated NO production 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 carrageenaninduced paw edema of male ICR mice.
Acknowledgments We are grateful to National Key Programs of China (2012ZX09103101-017) and China Postdoctoral Science Foundation (2014M552373).
Conflict of Interest The authors have declared no conflict of interest.
References 1. Ruslan M. (2010) Inflammation 2010: new adventures of an old flame. Cell;140:771–776. 2. Ruslan M. (2008) Origin and physiological roles of inflammation. Nature;454:428–435. 3. Carl N. (2002) Points of control in inflammation. Nature; 420:846–852. 4. MacMicking J., Xie Q.W., Nathan C. (1997) Nitric oxide and macrophage function. Annu Rev Immunol;15:323– 350.
Chem Biol Drug Des 2015; 85: 296–299
5. Clancy R.M., Amin A.R., Abramson S.B. (1998) The role of nitric oxide in inflammation and immunity. Arthritis Rheum;41:1111–1151. 6. Hobbs A.J., Higgs A., Moncada S. (1999) Inhibition of nitric oxide synthase as a potential therapeutic target. Annu Rev Pharmacol Toxicol;39:191–220. 7. Alderton W.K., Cooper C.E., Knowles R.G. (2001) Nitric oxide synthases: structure, function and inhibition. Biochem J;357:593–615. ^a T.A., Alves C.C., Castro S.B., Oliveira E.E., 8. Corre Franco L.S., Ferreira A.P., de Almeida M.V. (2013) Synthesis of 1,4-anthracene-9,10-dione derivatives and their regulation of nitric oxide, IL-1b and TNF-a in activated RAW264.7 cells. Chem Biol Drug Des;82:463–467. 9. Wang F., Sun J.R., Huang M.Y., Wang H.Y., Sun P.H., Lin J., Chen W.M. (2013) Design, synthesis and antiinflammatory evaluation of novel 5-benzylidene-3,4-dihalofuran-2-one derivatives. Eur J Med Chem;72:35– 45. 10. Ma L., Xie C.F., Ma Y.H., Liu J., Xiang M.L., Ye X., Zheng H. et al. (2011) Synthesis and biological evaluation of novel 5-benzylidenethiazolidine-2,4-dione derivatives for the treatment of inflammatory diseases. J Med Chem;54:2060–2068. 11. Reddy M.V., Hwang T.L., Leu Y.L., Chiou W.F., Wu T.S. (2011) Inhibitory effects of Mannich bases of heterocyclic chalcones on NO production by activated RAW 264.7 macrophages and superoxide anion generation and elastase release by activated human neutrophils. Bioorg Med Chem;19:2751–2756. sek P., Kra l V., Masters B.S., 12. Raman C.S., Li H., Marta Poulos T.L. (1998) Crystal structure of constitutive endothelial nitric oxide synthase: a paradigm for pterin function involving a novel metal center. Cell;95:939– 950. 13. Talukdar A., Breen M., Bacher A., Illarionov B., Fischer M., Georg G., Ye Q.Z., Cushman M. (2009) Discovery and development of a small molecule library with lumazine synthase inhibitory activity. J Org Chem;74: 5123–5134. €ller C.E. 14. Grahner B., Winiwarter S., Lanzner W., Mu (1994) Synthesis and structure- activity relationships of deazaxanthines: analogs of potent A1- and A2-adenosine receptor antagonists. J Med Chem;37:1526– 1534.
Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1. The IC50 of cytotoxicity of selected compounds in RAW 264.7 cells. Appendix S1. Experimental.
299
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, Kai Lei1, Ronghong Zhang1, Zhuang Yang2 and Lijuan Chen1,* 1
State Key Laboratory of Biotherapy and Cancer Center, West China Hospital, West China Medical School, Sichuan University, Chengdu 610041, China 2 College of Chemistry, Sichuan University, Chengdu 610041, China *Corresponding author: Lijuan Chen, [email protected] Fifty two compounds based on 5-nitropyrimidine2,4-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 lM) on lipopolysaccharide-induced RAW 264.7 cells and inducible nitric 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 synthase. At a dose of 10 mg/kg, oral administration of 36 possessed protective properties in carrageenaninduced paw edema of male ICR mice. Key words: 5-nitropyrimidine-2,4-dione, docking inducible NOS, nitric oxide production, paw edema
study,
Received 22 March 2014, revised 12 May 2014 and accepted for publication 18 June 2014
Inflammation is a complex process involving numerous mediators of cellular and plasma origin with interrelated biological effects. Proinflammatory stimulus activates cellular responses and regulates inflammatory and immune functions including the recruitment of macrophages and production of proinflammatory cytokines (1,2). The activated macrophages produce nitric oxide (NO) and upregulate prostaglandins (PGs), interleukin (IL)-1b, and tumor necrosis factor (TNF)-a in the inflammatory process (3). Nitric oxide, being an endogenous free radical, is a crucial and unique signaling mediator of vasorelaxation, neurotransmission, non-specific host defense, immune
296
responses, and inflammatory disorders at low concentration (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 dinucleotide phosphate (NADPH)- and O2-dependent process (6). There are at least three NOS isoforms including endothelial NOS (eNOS), neuronal NOS (nNOS), and inducible NOS (iNOS). Normally, NO is expressed by eNOS and nNOS at a low level; while activated proinflammatory cytokines or lipopolysaccharide (LPS), the iNOS produces a high level of NO to exert immune defense against pathogens at the inflammatory site (7). Aberrant iNOS-mediated NO biosynthesis, located in macrophages, has been involved in a variety of pathological and physiological diseases including septic stroke, hypertension, cancer, ischemia, and rheumatoid arthritis (8). Therefore, pharmacological reduction of iNOS-mediated NO production is regarded as one of the essential conditions to alleviate a variety of acute and chronic inflammatory diseases. Considerable studies have reported various type inhibitors of NO production, such as unsaturated fatty acids, chalcones, lignans, polyacetylenes, coumarins, flavonoids, stilbenes, sesquiterpenes, thiazolidinediones, diterpenes, triterpenes, diarylheptanoids, and alkaloids (9–11). Pyrimidine-2,4-dione was known to inhibit not only the production 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-inflammatory functions through the fine tuning of NO level (12). Compounds 1-52 have been synthesized according to the previously reported method (13). In detail, a variety of aromatic aldehydes were condensed with 6-methyl-5-nitropyrimidine-2,4-dione to afford a focused library of alkenes with pure trans geometry (JCH=CH: 16.0 Hz in 400 MHz 1H 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. Condensations of 1a with appropriate aldehydes using piperidine as basic catalyst and 1-butanol as a valid solvent provided the piperidine salts of 5-nitro-6-styryluracil derivatives (14). And then, the piperidine salts were dissolved or 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. We initially accessed the capacity of our desired compounds to inhibit the production of NO in LPS-induced RAW246.7 macrophages. In the study, indomethacin, as a
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 LPSinduced production of NO at a concentration of 10 lM, and their inhibitory rates (IR) were more than 30.0% compared to anti-inflammatory drug indomethacin (IR: 28.7%). Among them, 36 exhibited the most potent inhibitory
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 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 inducible nitric oxide synthase (iNOS) activity of selected compounds
Compds
NO IC50 (lM)
iNOS IC50 (lM)
17 21 23 31 36 39
17.5 14.3 25.3 10.1 8.6 30.2
40.2 32.6 >50.0 7.8 6.2 >50.0
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 macrophages. The inhibitory rate of the remaining derivatives was all less than 30.0% and inferior to that of indomethacin and 36. 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. However, 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. 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 298
Figure 2: Inhibition of carrageenan-induced paw edema in ICR mice. The results were expressed as the means SD (n = 6). *P < 0.05 versus vehicle.
Figure 3: The interaction mode of 36 within the active site of iNOS (PDB ID: 1r35).
of 2-quinolinyl heterocycle slightly improved the inhibitory rate, especially compound 51 (IR = 35.0%). The edema is an important parameter of acute inflammation for evaluating potential compounds with anti-inflammatory activity. In the carrageenan-induced paw edema test, the inflammatory response was quantified by increment in paw size (edema) 2 h after carrageenan was injected and the paw edema was observed. Oral administration of 36 at a dose of 10 mg/kg suppressed carrageenan-induced paw edema of male ICR mice in contrast to that of positive control indomethacin (Figure 2). Compound 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 Protein Data Bank (PDB ID: 1r35). The molecular docking was performed by simulation of 36 into the active binding site of 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. In summary, 52 compounds contained 5-nitropyrimidine2,4(1H,3H)-dione moiety have been readily obtained and subsequently investigated for inhibitory effects on the production of NO. Among them, 36 significantly inhibited iNOS activity (IC50 = 6.2 lM) and iNOS-mediated NO production 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 carrageenaninduced paw edema of male ICR mice.
Acknowledgments We are grateful to National Key Programs of China (2012ZX09103101-017) and China Postdoctoral Science Foundation (2014M552373).
Conflict of Interest The authors have declared no conflict of interest.
References 1. Ruslan M. (2010) Inflammation 2010: new adventures of an old flame. Cell;140:771–776. 2. Ruslan M. (2008) Origin and physiological roles of inflammation. Nature;454:428–435. 3. Carl N. (2002) Points of control in inflammation. Nature; 420:846–852. 4. MacMicking J., Xie Q.W., Nathan C. (1997) Nitric oxide and macrophage function. Annu Rev Immunol;15:323– 350.
Chem Biol Drug Des 2015; 85: 296–299
5. Clancy R.M., Amin A.R., Abramson S.B. (1998) The role of nitric oxide in inflammation and immunity. Arthritis Rheum;41:1111–1151. 6. Hobbs A.J., Higgs A., Moncada S. (1999) Inhibition of nitric oxide synthase as a potential therapeutic target. Annu Rev Pharmacol Toxicol;39:191–220. 7. Alderton W.K., Cooper C.E., Knowles R.G. (2001) Nitric oxide synthases: structure, function and inhibition. Biochem J;357:593–615. ^a T.A., Alves C.C., Castro S.B., Oliveira E.E., 8. Corre Franco L.S., Ferreira A.P., de Almeida M.V. (2013) Synthesis of 1,4-anthracene-9,10-dione derivatives and their regulation of nitric oxide, IL-1b and TNF-a in activated RAW264.7 cells. Chem Biol Drug Des;82:463–467. 9. Wang F., Sun J.R., Huang M.Y., Wang H.Y., Sun P.H., Lin J., Chen W.M. (2013) Design, synthesis and antiinflammatory evaluation of novel 5-benzylidene-3,4-dihalofuran-2-one derivatives. Eur J Med Chem;72:35– 45. 10. Ma L., Xie C.F., Ma Y.H., Liu J., Xiang M.L., Ye X., Zheng H. et al. (2011) Synthesis and biological evaluation of novel 5-benzylidenethiazolidine-2,4-dione derivatives for the treatment of inflammatory diseases. J Med Chem;54:2060–2068. 11. Reddy M.V., Hwang T.L., Leu Y.L., Chiou W.F., Wu T.S. (2011) Inhibitory effects of Mannich bases of heterocyclic chalcones on NO production by activated RAW 264.7 macrophages and superoxide anion generation and elastase release by activated human neutrophils. Bioorg Med Chem;19:2751–2756. sek P., Kra l V., Masters B.S., 12. Raman C.S., Li H., Marta Poulos T.L. (1998) Crystal structure of constitutive endothelial nitric oxide synthase: a paradigm for pterin function involving a novel metal center. Cell;95:939– 950. 13. Talukdar A., Breen M., Bacher A., Illarionov B., Fischer M., Georg G., Ye Q.Z., Cushman M. (2009) Discovery and development of a small molecule library with lumazine synthase inhibitory activity. J Org Chem;74: 5123–5134. €ller C.E. 14. Grahner B., Winiwarter S., Lanzner W., Mu (1994) Synthesis and structure- activity relationships of deazaxanthines: analogs of potent A1- and A2-adenosine receptor antagonists. J Med Chem;37:1526– 1534.
Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1. The IC50 of cytotoxicity of selected compounds in RAW 264.7 cells. Appendix S1. Experimental.
299
