Bioorganic & Medicinal Chemistry Letters 27 (2017) 4409–4414
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, anti-inflammatory activity, and molecular docking studies of perimidine derivatives containing triazole Hong-Jian Zhang a, Xiu-Zhi Wang b, Qi Cao c, Guo-Hua Gong b,⇑, Zhe-Shan Quan a,⇑ a Key Laboratory of Natural Resources and Functional Molecules of the Changbai Mountain, Affiliated Ministry of Education, College of Pharmacy, Yanbian University, No. 977, Park Road, Yanji, Jilin 133002, China b Affiliated Hospital of Inner Mongolia University for Nationalities, Tongliao 028000, Inner Mongolia, China c Department of Pathology, 306 Hospital of PLA, Beijing 100101, China
a r t i c l e
i n f o
Article history: Received 22 February 2017 Revised 6 June 2017 Accepted 8 August 2017 Available online 9 August 2017 Keywords: Synthesis Perimidine Anti-inflammatory activity Molecular docking study
a b s t r a c t We report here the design, synthesis, and anti-inflammatory activities of a series of perimidine derivatives containing triazole (5a–s). The chemical structures of the synthesized compounds have been assigned on the basis of IR, 1H NMR, 13C NMR, and HRMS spectral analyses. The anti-inflammatory properties of the synthesized perimidine derivatives were evaluated in a lipopolysaccharide (LPS)-stimulated inflammation model. Among the tested compounds, compound 7-(3-methylbenzyl)-7H-[1,2,4]triazolo [4,3-a]perimidine (hereafter referred to as 5h) and compound 7-(2-fluorobenzyl)-7H-[1,2,4]triazolo [4,3-a]perimidine (hereafter referred to as 5n) caused a reduction in the levels of the pro-inflammatory cytokines—tumor necrosis factor (TNF)-a and interleukin (IL)-6—in RAW264.7 cells. The anti-inflammatory potential of compounds 5h and 5n was also evaluated in vivo in a xylene-induced ear inflammation model. Compound 5n showed the most potent anti-inflammatory activity with an inhibition of 49.26% at a dose of 50 mg/kg. This activity is more potent than that of the reference drug ibuprofen (28.13%), and slightly less than that of indometacin (49.36%). To further elucidate the mechanisms underlying these inhibitory effects, LPS-induced nuclear factor-jB (NF-jB) activation and mitogen-activated protein kinase (MAPK) phosphorylation were studied. The results of western blotting showed that the extract obtained from compound 5n inhibited NF-jB (p65) activation and MAPK (extracellular signal-regulated kinase (ERK) and p38) phosphorylation in a dose-dependent manner. Moreover, the results of a docking study of compound 5n into the COX-2 binding site revealed that its mechanism was possibly similar to that of naproxen, a COX-2 inhibitor. The effect of compound 5n on COX-2 antibody was showed it could significantly inhibit COX-2 activity. Ó 2017 Elsevier Ltd. All rights reserved.
Introduction Inflammation is a common condition in which body tissues are affected by heat, redness, swelling, and pain.1 Inflammation is generally caused by hay fever, periodontitis, atherosclerosis, rheumatoid arthritis, and even cancer.2 Commonly, non-steroidal antiinflammatory drugs (NSAIDs) are used as analgesics and antipyretics, as well as anti-inflammatory agents at higher doses. Nevertheless, the long-term use of NSAIDs is known to cause gastrointestinal complications, such as bleeding, ulcer, perforation, obstruction, and so on.3 Therefore, it is necessary to study and develop safe anti-inflammatory drugs.
⇑ Corresponding authors. E-mail addresses: [email protected] (G.-H. Gong), [email protected] (Z.-S. Quan). http://dx.doi.org/10.1016/j.bmcl.2017.08.014 0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
As previously reported, various structures of compounds containing triazole have shown anti-inflammatory properties, such as 2-ethylthio-6-formyl-7-(4-methoxyphenylamino)-5-oxo-1phenyl-1,5-dihydro-1,2,4-triazolo[1,5-a]pyridine-8-carbonitrile (I), 6-(m-tolyloxy)-[1,2,4]triazolo[3,4-a]phthalazine-3-carboxamide (II), (1H-perimidin-2-ylthio)(phenyl)methanol (III), and (Z)-2(1-(6-methoxynaphthalene-2-yl)ethyl)-5-((5-methylthiophene-2yl)methylene)thiazolo[3,2-b]-1,2,4-triazole-6(5H)-one (IV).4,5,2,6 Bassyouni et al. reported that 1H-perimidine derivatives had anti-inflammatory activities (V). In search for new potentially active substances, we committed to obtaining target compounds containing 1H-perimidine and triazole molecules. Accordingly, the target compounds 5a–s were designed (Fig. 1).7 The synthesis of the target compounds is presented in Scheme 1. Compound 1 was prepared by reacting 1,8-diaminonaphthalene with carbamide via a melting reaction. Compound 1 reacted under POCl3 conditions and yielded compound 2. Compound 2 was then
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Fig. 1. Structures of some compounds in containing triazole and design of target compounds.
Scheme 1. Synthesis of the target compounds.
reacted with hydrazine hydrate in ethanol to produce compound 3. Compound 3 reacted with formic acid under reflux conditions to yield compound 4. Compound 4 was then reacted with alkyl halide or substituted with benzyl chloride in the presence of sodium hydride in N,N-dimethylformamide (DMF) to produce compounds 5a–s. The structures of the synthesized compounds were confirmed via IR, 1H NMR, 13C NMR, and high-resolution mass spectroscopy (HRMS) analyses. The in vitro anti-inflammatory activities of the synthesized compounds were evaluated using a LPS-stimulated inflammation model. The results of the perimidine derivatives containing triazole were presented in Fig. 2. Compound 5a, 5d, 5e, 5f, 5g, 5i, 5l, 5m, and 5s showed cell toxicity, and the other seven compounds reduced cell viability significantly, except for 5a and 5l (p < 0.001) at a dose of 50 lg/mL. Obviously, the length of the carbon chain was closely related with the compounds’ toxicity, especially when the length of the carbon chain was greater than five. Furthermore, when RAW264.7 cells were treated with LPS and synthesized compounds at the same time, the viability of cells treated with compounds 5b, 5e, and 5l decreased to a greater extent than when treated with other compounds. TNF-a, IL-6, and other pro-inflammatory cytokines play an important role in biological injury and inflammation.8 TNF-a has a wide range of biological activities, and participates in inflammation, immune response, anti-tumor activities, and engages in the pathological process of endotoxin shock. It can cause fever, shock, among other abnormalities, as well as increase the levels of IL-6 and other cytokines, thus aggravating the inflammatory response if an excessive amount is released or produced.9 Generally, IL-6 is produced after TNF-a stimulation, and is
closely related to the pathological and physiological processes of various diseases. It is also an important, sensitive indicator which reflects the severity of the inflammation and tissue damage.10,11 TNF-a and IL-6 are involved in the body’s inflammatory response as important inflammatory mediators.12 Under the physiological conditions, TNF-a and IL-6 levels remain low; however, in a pathological state, a large number of TNF-a and IL-6 molecules are secreted. The preliminary screening showed compounds 5c, 5h, 5j, 5k, 5n, 5o, 5p, 5q, and 5r displayed lower toxicity by MTT assay. Then, we measured their concentration of TNF-a and IL-6 at dose of 50 lg/mL, respectively. We found only two compounds 5h and 5n could decrease the concentration of two inflammatory factors. Thus, compounds 5h and 5n were chose for the following test. The effects of compounds 5h and 5n on pro-inflammatory cytokine production are shown in Fig. 3. With regard to TNF-a and IL-6, levels in the control group were regard as 100%. The fold of test group was the ratio of the content of test group with that of the control group. There were statistically significant differences between the control group and the LPS group. This suggests that LPS induced an increase in TNF-a and IL-6 secretion. Fortunately, compounds 5h and 5n could inhibit this increase in TNF-a and IL-6 in a dose-dependent manner. Compounds 5h or 5n at doses of 25 lg/mL, and 50 lg/mL significantly reduced the levels of TNF-a. Among them, TNF-a levels reduced most noticeably at a treatment dose of 50 lg/mL, with the mean fold of TNF-a at 146.24% and 124.87%. The positive drug group had no significant difference compared with that of LPS group. Compounds 5h and 5n reduced IL-6 levels in the same conditions, as was expected. The effects of compounds 5n were obviously stronger than those of compound 5h. IL-6-fold were reduced by
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Fig. 2. The effect of compound 5a–s and LPS for cell viability. Black column: only compound, Gray column: compound + LPS; Data are means ± SD; *: 0.01 < p < 0.05, p < 0.001 compared with Control group; #: 0.01 < p < 0.05, ##: 0.001 < p < 0.01, ###: p < 0.001 compared with LPS group.
***
:
Fig. 3. The effect of compound 5h and 5n on pro-inflammatory cytokine production in RAW 264.7 cells. The TNF-a and IL-6 production in the supernatants was determined by ELISA. Values were the mean ± S.D. (fold) of triplicate experiments. ###: p < 0.001 compared with Control group; *: 0.01 < p < 0.05, **: 0.001 < p < 0.01, ***: p < 0.001 compared with LPS group.
compound 5n to 71.80%, 68.23%, and 47.21% at doses of 12.5 lg/ mL, 25 lg/mL, and 50 lg/mL, respectively. The xylene-induced ear-edema test was used to evaluate acute anti-inflammatory activity.13,14 The anti-inflammatory activities of compounds 5h and 5n were also evaluated in vivo and the results are shown in Table 1. In this test, compounds were screened in a xylene-induced ear-edema test in mice, with ibuprofen and indomethacin used as the reference drugs. Anti-inflammatory activity was assessed based on the ability of the test compounds to prevent edema. The data revealed that compounds 5h and 5n showed significant anti-inflammatory effects at 25 mg/kg and 50 mg/kg administered i.p. When compared with the control group, these two compounds showed anti-inflammatory activities at a dose of 12.5 mg/kg, with lower inhibition rate, but this was not statistically significant. Compound 5n exhibited the strongest inhibition of inflammation at 49.26%, which was higher than ibuprofen
Table 1 Anti-inflammatory activity of compounds 5h and 5n administrated i.p. Compound
Dose (mg/kg)
Edema mean ± S.D. (mg)
Inhibition rate (%)
Control Ibuprofen Indometacin 5h
– 50 50 12.5 25 50 12.5 25 50
10.13 ± 0.62 7.28 ± 0.65* 5.13 ± 1.20*** 8.54 ± 0.44 7.11 ± 0.63* 5.78 ± 0.84*** 8.39 ± 0.57 6.59 ± 0.73** 5.14 ± 0.72***
– 28.13 49.36 15.70 29.81 42.94 17.18 34.95 49.26
5n
*
: 0.01 < p < 0.05, **: 0.001 < p < *0.01, **: p < 0.001 compared with Control group.
(28.13%) and lower than indomethacin (49.36%) at a dose of 50 mg/kg.
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The release of pro-inflammatory cytokines (e.g.: TNF-a and IL6) and mediators (e.g.: iNOS and COX-2) is related to the MAPK pathway in LPS-stimulated macrophages.15–17 MAPKs include ERK, c-Jun N-terminal kinase (JNK), and p38, and their signaling pathways play important roles in several biological processes. Accordingly, the effects of compound 5h on the activation of ERK and p38 were investigated. The results were presented in Fig. 4. The phosphorylation levels of ERK and p38 significantly decreased, and the effects of compound 5n were dose-dependent. However, the levels of ERK and p38 were unaffected after stimulation. These results suggested that compound 5n could inhibit
pro-inflammatory mediator and cytokine expression via MAPK pathways. NF-jB is an important transcription factor, which can regulate the expression of pro-inflammatory mediators in activated macrophages.18 The NF-jB protein comprises p65 and p50 subunits. Under normal conditions, a functional NF-jB dimer is exists as a complex with the inhibitor protein IjB in the cytosol.19 The activation of NF-jB in response to LPS stimulation leads to the degradation of IjB, followed by the release and nuclear translocation of NF-jB. After nuclear translocation, p65 and p50 regulate the production of TNF-a, IL-6, and mediators.20–23 Therefore, the inhi-
Fig. 4. The effect of compound 5n on the protein level of MAPKs in RAW 264.7 cells. The protein levels of MAPKs were determined using western blot assay. Data were means ± SD; ###: p < 0.001 compared with Control group; **:0.001 < p < 0.01, ***: p < 0.001 compared with LPS group.
Fig. 5. The effect of compound 5n on the protein level of NF-jB in RAW 264.7 cells. The protein levels of NF-jB were determined using western blot assay. Data were means ± SD; ##: 0.001 < p < 0.01 compared with Control group; *: 0.05 < p < 0.01, **: 0.001 < p < 0.01, compared with LPS group.
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bition of NF-jB activation has been regarded as a therapeutic approach for autoimmune and inflammatory conditions.24,25 As shown in Fig. 5, the effects of the extract obtained from compound 5n on NF-jB activation were investigated in LPS-stimulated RAW264.7 cells. The phosphorylation levels of p65 reduced significantly, and IjB levels increased dramatically in LPS-stimulated RAW264.7 cells in a dose-dependent manner. These results indicate that compound 5h inhibits the activation of NF-jB by preventing IjB degradation in LPS-stimulated RAW264.7 cells. In recent years, a large number of scholars engaged in COX-2 receptor-related research.26–30 Naproxen is a powerful, non-selective non-steroidal anti-inflammatory drug that is extensively used as a prescription and over-the-counter medication. It interacts with COX as reported by Duggan et al.31 The atomic coordinates and structure factors (codes 3NT1) have been deposited in the Protein Data Bank. Naproxen is a relatively simple molecule with a naphthyl scaffold, similar to compound 5n. A docking study of compound 5n into the COX-2 binding site was therefore performed. As shown in Fig. 6, there are some cavities around the naproxen molecule in the COX-2 binding pocket. Compound 5n could shrink the cavity space, thereby enhancing the interaction force. Moreover, the triazole ring can interact with the upper right hydrogen bond donor, in a manner equivalent to that of the carbonyl group of naproxen acting as hydrogen bond acceptor. The detailed interaction of compound 5n and naproxen with COX-2 is shown in Table 2. The results revealed that compound 5n shows
a stronger interaction with COX-2. This may result in a better inhibitory effect on COX-2. In order to verify our speculation, the effect of compound 5n on COX-2 was measured. The result was present in Fig. 6. The expression of COX-2 has significant difference between control group and LPS group. The value of COX-2/GAPDH was significant decrease treatment with compound 5n. Especially, the fold of COX-2/GAPDH was reduced to 45.19% at dose of 50 lg/mL. It suggested that compound 5n was liked to display anti-inflammatory activity related with the site of COX-2 as a kind of COX-2 inhibitors. In conclusion, a series of perimidine derivatives containing triazole (5a–s) were synthesized and their anti-inflammatory activities were evaluated using the MTT assay in a LPS-stimulated inflammation model. Compounds 5h and 5n reduced the levels of pro-inflammatory cytokines TNF-a and IL-6 in RAW264.7 cells. Compound 5n showed the most potent anti-inflammatory activity with an inhibition rate of 49.26% at a dose of 50 mg/kg, which is more potent than that of the reference drug ibuprofen (28.13%) and slightly less than that of indometacin (49.36%) in a xyleneinduced ear inflammation model. The results of western blotting showed compound 5n inhibited NF-jB (p65) activation and MAPK (ERK and p38) phosphorylation in a dose-dependent manner. Moreover, the results of the docking study of compound 5n into the COX-2 binding site and COX-2 inhibitory test revealed that its mechanism was possibly similar to that of naproxen, a COX-2 inhibitor.
Fig. 6. The docking result of compound 5n (left) and reported naproxen pose (right) into the COX-2 binding site.
Table 2 The interaction of compound 5n and naproxen with COX-2.
Compound 5h
Distance (Angstrom)
Types
Amino acid residues
Types
Distance (Angstrom)
2.81 3.52, 3.93, 4.01, 4.54, 5.00 5.06 5.11 5.95 – –
Carbon Hydrogen bond Pi-Alkyl Amide-Pi Stacked Pi-Alkyl Pi-Alkyl Pi-Pi T-shaped Pi-Alkyl Pi-Alkyl Pi-Sulfur – –
SER353 VAL523 GLY526 ALA527 LEU352 TYR355 VAL349 VAL116 MET522 LEU359 GLU524
– Pi-Alkyl Amide-Pi Stacked Pi-Alkyl Pi-Alkyl Pi-Alkyl Pi-Alkyl Alkyl – – Negative-Negative
– 5.12 4.24, 4.91, 3.35 4.64, 5.46 4.99 462, 4.94 5.24 – 4.46 5.28
4.41, 4.69, 4.33, 4.72,
4.67 5.40 4.46, 5.32 4.89, 5.24
Naproxen
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Acknowledgment This work was supported by the National Natural Science Foundation of China (No. 81360468 and 81660837). A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.bmcl.2017.08. 014. References 1. Jubert Alicia, Massa Ne’stor E, Te’vez Leonor Lo’pez, Okulik Nora Beatriz. Vibrational and theoretical studies of the non-steroidal anti-inflamatory drugs niflumic [2-3((3-trifluoromethyl)phenylamino)-3-pyridinecarboxylic acid]; diclofenac [[2-(2,6-dichlorophenyl)amino]-benzeneacetic acid] and indometacin acids [1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indole-3acetic acid]. Vib Spectrosc. 2005;37:161–178. 2. Paprocka R, Wiese M, Eljaszewicz A, et al. Synthesis and anti-inflammatory activity of new 1,2,4-triazole derivatives. Bioorg Med Chem Lett. 2015;25:2664–2667. 3. García Rodríguez LA, Cattaruzzi C, Troncon MG, Agostinis L. Risk of hospitalization for upper gastrointestinal tract bleeding associated with ketorolac, other nonsteroidal anti-inflammatory drugs, calcium antagonists, and other antihypertensive drugs. Arch Intern Med. 1998;158:33–39. 4. Mekheimer RA, Sayed AA, Ahmed EA, Sadek KU. Synthesis and characterization of new 1,2,4-triazolo[1,5-a]pyridines that extend the life span of caenorhabiditis elegans via their anti-inflammatory/antioxidant effects. Arch Pharm (Weinheim). 2015;348:650–665. 5. Liu DC, Gong GH, Wei CX, Jin XJ, Quan ZS. Synthesis and anti-inflammatory activity evaluation of a novel series of 6-phenoxy-[1,2,4]triazolo[3,4-a] phthalazine-3-carboxamide derivatives. Bioorg Med Chem Lett. 2016;26:1576–1579. 6. Sarigol D, Uzgoren-Baran A, Tel BC, et al. Novel thiazolo[3,2-b]-1,2,4-triazoles derived from naproxen with analgesic/anti-inflammatory properties: Synthesis, biological evaluation and molecular modeling studies. Bioorg Med Chem. 2015;23:2518–2528. 7. Bassyouni FA, Abu-Bakr SM, Hegab KH, et al. Synthesis of new transition metal complexes of 1H-perimidine derivatives having antimicrobial and antiinflammatory activities. Res Chem Intermed. 2012;38:1527–1550. 8. Wilson MR, Choudhury S, Takata M. Pulmonary inflammation induced by highstretch ventilation is mediated by tumor necrosis factor signaling in mice. Am J Physiol Lung Cell Mol Physiol. 2005;288:L599–L607. 9. Campbell IK, Roberts LJ, Wicks IP. Molecular targets in immune-mediated diseases: the case of tumour necrosis factor and rheumatoid arthritis. Immunol Cell Biol. 2003;81:354–366. 10. Spielmann S, Kerner T, Ahlers O, Keh D, Gerlach M, Gerlach H. Early detection of increased tumour necrosis factor alpha (TNFalpha) and soluble TNF receptor protein plasma levels after trauma reveals associations with the clinical course. Acta Anaesthesiol Scand. 2001;45:364–370. 11. Pape HC, van Griensven M, Rice J, et al. Major secondary surgery in blunt trauma patients and perioperative cytokine liberation: determination of the clinical relevance of biochemical markers. J Trauma. 2001;50:989–1000. 12. Murano M, Maemura K, Hirata I, et al. Therapeutic effect of intracolonically administered nuclear factor kappa B (p65) antisense oligonucleotide on mouse dextran sulphate sodium (DSS)-induced colitis. Clin Exp Immunol. 2000;120:51–58.
13. Cheng J, Ma T, Liu W, et al. In in vivo evaluation of the anti-inflammatory and analgesic activities of compound Muniziqi granule in experimental animal models. BMC Complement Altern Med. 2016;16:20. 14. Eidi A, Oryan S, Zaringhalam J, Rad M. Antinociceptive and anti-inflammatory effects of the aerial parts of Artemisia dracunculus in mice. Pharm Biol. 2016;54:549–554. 15. Coskun M, Olsen J, Seidelin JB, Nielsen OH. MAP kinases in inflammatory bowel disease. Clin Chim Acta. 2011;412:513–520. 16. Kim KN, Ko YJ, Yang HM, et al. Anti-inflammatory effect of essential oil and its constituents from fingered citron (Citrus medica L. var. sarcodactylis) through blocking JNK, ERK and NF-kappa B signaling pathways in LPS activated RAW 264.7 cells. Food Chem Toxicol. 2013;57:126–131. 17. Yang CP, Huang GJ, Huang HC, et al. A new butanolide compound from the aerial part of Lindera akoensis with anti-inflammatory activity. Molecules. 2012;17:6585–6592. 18. Hanada T, Yoshimura A. Regulation of cytokine signaling and inflammation. Cytokine Growth Factor Rev.. 2002;13:413–421. 19. Schmitz ML, Bacher S, Kracht M. I kappaB-independent control of NF-kappa B activity by modulatory phosphorylations. Trends Biochem Sci. 2001;26:186–190. 20. Liu F, Morris S, Epps J, Carroll R. Demonstration of an activation regulated NFkappaB/I-kappaBalpha complex inhuman platelets. Thromb Res. 2002;106:199–203. 21. Lappas M, Permezel M, Georgiou HM, Rice GE. Nuclear factor kappa B regulation of proinflammatory cytokines in human gestational tissues in vitro. Biol Reprod. 2002;67:668–673. 22. Verma IM, Stevenson JK, Schwarz EM, Van Antwerp D, Miyamoto S. Rel/NFkappa B/I kappa B family: intimate tales of association and dissociation. Genes Dev. 1995;9:2723–2735. 23. Song SM, Ham YM, Ko YJ, et al. Anti-inflammatory activities of the products of supercritical fluid extraction from Litsea japonica fruit in RAW 2647 cells. J Funct Foods. 2016;22:44–51. 24. Bufalo MC, Ferreira I, Costa G, et al. Propolis and its constituent caffeic acid suppress LPS-stimulated pro-inflammatory response by blocking NF-kappaB and MAPK activation in macrophages. J Ethnopharmacol. 2013;149:84–92. 25. Lee Y, Shin DH, Kim JH, et al. Caffeic acid phenethyl ester-mediated Nrf2 activation and IkappaB kinase inhibition are involved in NF-kappaB inhibitory effect: structural analysis for NFkappaB inhibition. Eur J Pharmacol. 2010;643:21–28. 26. Abdel-Aziz AA, Abou-Zeid LA, ElTahir KE, Ayyad RR, El-Sayed MA, El-Azab AS. Synthesis, anti-inflammatory, analgesic, COX-1/2 inhibitory activities and molecular docking studies of substituted 2-mercapto-4(3H)-quinazolinones. Eur J Med Chem. 2016;121:410–421. 27. Chen L, Teng H, Fang T, Xiao J. Agrimonolide from Agrimonia pilosa suppresses inflammatory responses through down-regulation of COX-2/iNOS and inactivation of NF-jB in lipopolysaccharide-stimulated macrophages. Phytomedicine. 2016;23:846–855. 28. Cha SM, Cha JD, Jang EJ, Kim GU, Lee KY. Sophoraflavanone G prevents Streptococcus mutans surface antigen I/II-induced production of NO and PGE2 by inhibiting MAPK-mediated pathways in RAW 264.7 macrophages. Arch Oral Biol. 2016;68:97–104. 29. Park JM, Han YM, Lee JS, et al. Nrf2-mediated mucoprotective and antiinflammatory actions of Artemisia extracts led to attenuate stress related mucosal damages. J Clin Biochem Nutr. 2015;56:132–142. 30. Niu X, Mu Q, Li W, Yao H, Li H, Huang H. Esculentic acid, a novel and selective COX-2 inhibitor with anti-inflammatory effect in vivo and in vitro. Eur J Pharmacol. 2014;740:532–538. 31. Duggan KC, Walters MJ, Musee J, et al. Molecular basis for cyclooxygenase inhibition by the non-steroidal anti-inflammatory drug naproxen. J Biol Chem. 2010;285:34950–34959.
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevier.com/locate/bmcl
Design, synthesis, anti-inflammatory activity, and molecular docking studies of perimidine derivatives containing triazole Hong-Jian Zhang a, Xiu-Zhi Wang b, Qi Cao c, Guo-Hua Gong b,⇑, Zhe-Shan Quan a,⇑ a Key Laboratory of Natural Resources and Functional Molecules of the Changbai Mountain, Affiliated Ministry of Education, College of Pharmacy, Yanbian University, No. 977, Park Road, Yanji, Jilin 133002, China b Affiliated Hospital of Inner Mongolia University for Nationalities, Tongliao 028000, Inner Mongolia, China c Department of Pathology, 306 Hospital of PLA, Beijing 100101, China
a r t i c l e
i n f o
Article history: Received 22 February 2017 Revised 6 June 2017 Accepted 8 August 2017 Available online 9 August 2017 Keywords: Synthesis Perimidine Anti-inflammatory activity Molecular docking study
a b s t r a c t We report here the design, synthesis, and anti-inflammatory activities of a series of perimidine derivatives containing triazole (5a–s). The chemical structures of the synthesized compounds have been assigned on the basis of IR, 1H NMR, 13C NMR, and HRMS spectral analyses. The anti-inflammatory properties of the synthesized perimidine derivatives were evaluated in a lipopolysaccharide (LPS)-stimulated inflammation model. Among the tested compounds, compound 7-(3-methylbenzyl)-7H-[1,2,4]triazolo [4,3-a]perimidine (hereafter referred to as 5h) and compound 7-(2-fluorobenzyl)-7H-[1,2,4]triazolo [4,3-a]perimidine (hereafter referred to as 5n) caused a reduction in the levels of the pro-inflammatory cytokines—tumor necrosis factor (TNF)-a and interleukin (IL)-6—in RAW264.7 cells. The anti-inflammatory potential of compounds 5h and 5n was also evaluated in vivo in a xylene-induced ear inflammation model. Compound 5n showed the most potent anti-inflammatory activity with an inhibition of 49.26% at a dose of 50 mg/kg. This activity is more potent than that of the reference drug ibuprofen (28.13%), and slightly less than that of indometacin (49.36%). To further elucidate the mechanisms underlying these inhibitory effects, LPS-induced nuclear factor-jB (NF-jB) activation and mitogen-activated protein kinase (MAPK) phosphorylation were studied. The results of western blotting showed that the extract obtained from compound 5n inhibited NF-jB (p65) activation and MAPK (extracellular signal-regulated kinase (ERK) and p38) phosphorylation in a dose-dependent manner. Moreover, the results of a docking study of compound 5n into the COX-2 binding site revealed that its mechanism was possibly similar to that of naproxen, a COX-2 inhibitor. The effect of compound 5n on COX-2 antibody was showed it could significantly inhibit COX-2 activity. Ó 2017 Elsevier Ltd. All rights reserved.
Introduction Inflammation is a common condition in which body tissues are affected by heat, redness, swelling, and pain.1 Inflammation is generally caused by hay fever, periodontitis, atherosclerosis, rheumatoid arthritis, and even cancer.2 Commonly, non-steroidal antiinflammatory drugs (NSAIDs) are used as analgesics and antipyretics, as well as anti-inflammatory agents at higher doses. Nevertheless, the long-term use of NSAIDs is known to cause gastrointestinal complications, such as bleeding, ulcer, perforation, obstruction, and so on.3 Therefore, it is necessary to study and develop safe anti-inflammatory drugs.
⇑ Corresponding authors. E-mail addresses: [email protected] (G.-H. Gong), [email protected] (Z.-S. Quan). http://dx.doi.org/10.1016/j.bmcl.2017.08.014 0960-894X/Ó 2017 Elsevier Ltd. All rights reserved.
As previously reported, various structures of compounds containing triazole have shown anti-inflammatory properties, such as 2-ethylthio-6-formyl-7-(4-methoxyphenylamino)-5-oxo-1phenyl-1,5-dihydro-1,2,4-triazolo[1,5-a]pyridine-8-carbonitrile (I), 6-(m-tolyloxy)-[1,2,4]triazolo[3,4-a]phthalazine-3-carboxamide (II), (1H-perimidin-2-ylthio)(phenyl)methanol (III), and (Z)-2(1-(6-methoxynaphthalene-2-yl)ethyl)-5-((5-methylthiophene-2yl)methylene)thiazolo[3,2-b]-1,2,4-triazole-6(5H)-one (IV).4,5,2,6 Bassyouni et al. reported that 1H-perimidine derivatives had anti-inflammatory activities (V). In search for new potentially active substances, we committed to obtaining target compounds containing 1H-perimidine and triazole molecules. Accordingly, the target compounds 5a–s were designed (Fig. 1).7 The synthesis of the target compounds is presented in Scheme 1. Compound 1 was prepared by reacting 1,8-diaminonaphthalene with carbamide via a melting reaction. Compound 1 reacted under POCl3 conditions and yielded compound 2. Compound 2 was then
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Fig. 1. Structures of some compounds in containing triazole and design of target compounds.
Scheme 1. Synthesis of the target compounds.
reacted with hydrazine hydrate in ethanol to produce compound 3. Compound 3 reacted with formic acid under reflux conditions to yield compound 4. Compound 4 was then reacted with alkyl halide or substituted with benzyl chloride in the presence of sodium hydride in N,N-dimethylformamide (DMF) to produce compounds 5a–s. The structures of the synthesized compounds were confirmed via IR, 1H NMR, 13C NMR, and high-resolution mass spectroscopy (HRMS) analyses. The in vitro anti-inflammatory activities of the synthesized compounds were evaluated using a LPS-stimulated inflammation model. The results of the perimidine derivatives containing triazole were presented in Fig. 2. Compound 5a, 5d, 5e, 5f, 5g, 5i, 5l, 5m, and 5s showed cell toxicity, and the other seven compounds reduced cell viability significantly, except for 5a and 5l (p < 0.001) at a dose of 50 lg/mL. Obviously, the length of the carbon chain was closely related with the compounds’ toxicity, especially when the length of the carbon chain was greater than five. Furthermore, when RAW264.7 cells were treated with LPS and synthesized compounds at the same time, the viability of cells treated with compounds 5b, 5e, and 5l decreased to a greater extent than when treated with other compounds. TNF-a, IL-6, and other pro-inflammatory cytokines play an important role in biological injury and inflammation.8 TNF-a has a wide range of biological activities, and participates in inflammation, immune response, anti-tumor activities, and engages in the pathological process of endotoxin shock. It can cause fever, shock, among other abnormalities, as well as increase the levels of IL-6 and other cytokines, thus aggravating the inflammatory response if an excessive amount is released or produced.9 Generally, IL-6 is produced after TNF-a stimulation, and is
closely related to the pathological and physiological processes of various diseases. It is also an important, sensitive indicator which reflects the severity of the inflammation and tissue damage.10,11 TNF-a and IL-6 are involved in the body’s inflammatory response as important inflammatory mediators.12 Under the physiological conditions, TNF-a and IL-6 levels remain low; however, in a pathological state, a large number of TNF-a and IL-6 molecules are secreted. The preliminary screening showed compounds 5c, 5h, 5j, 5k, 5n, 5o, 5p, 5q, and 5r displayed lower toxicity by MTT assay. Then, we measured their concentration of TNF-a and IL-6 at dose of 50 lg/mL, respectively. We found only two compounds 5h and 5n could decrease the concentration of two inflammatory factors. Thus, compounds 5h and 5n were chose for the following test. The effects of compounds 5h and 5n on pro-inflammatory cytokine production are shown in Fig. 3. With regard to TNF-a and IL-6, levels in the control group were regard as 100%. The fold of test group was the ratio of the content of test group with that of the control group. There were statistically significant differences between the control group and the LPS group. This suggests that LPS induced an increase in TNF-a and IL-6 secretion. Fortunately, compounds 5h and 5n could inhibit this increase in TNF-a and IL-6 in a dose-dependent manner. Compounds 5h or 5n at doses of 25 lg/mL, and 50 lg/mL significantly reduced the levels of TNF-a. Among them, TNF-a levels reduced most noticeably at a treatment dose of 50 lg/mL, with the mean fold of TNF-a at 146.24% and 124.87%. The positive drug group had no significant difference compared with that of LPS group. Compounds 5h and 5n reduced IL-6 levels in the same conditions, as was expected. The effects of compounds 5n were obviously stronger than those of compound 5h. IL-6-fold were reduced by
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Fig. 2. The effect of compound 5a–s and LPS for cell viability. Black column: only compound, Gray column: compound + LPS; Data are means ± SD; *: 0.01 < p < 0.05, p < 0.001 compared with Control group; #: 0.01 < p < 0.05, ##: 0.001 < p < 0.01, ###: p < 0.001 compared with LPS group.
***
:
Fig. 3. The effect of compound 5h and 5n on pro-inflammatory cytokine production in RAW 264.7 cells. The TNF-a and IL-6 production in the supernatants was determined by ELISA. Values were the mean ± S.D. (fold) of triplicate experiments. ###: p < 0.001 compared with Control group; *: 0.01 < p < 0.05, **: 0.001 < p < 0.01, ***: p < 0.001 compared with LPS group.
compound 5n to 71.80%, 68.23%, and 47.21% at doses of 12.5 lg/ mL, 25 lg/mL, and 50 lg/mL, respectively. The xylene-induced ear-edema test was used to evaluate acute anti-inflammatory activity.13,14 The anti-inflammatory activities of compounds 5h and 5n were also evaluated in vivo and the results are shown in Table 1. In this test, compounds were screened in a xylene-induced ear-edema test in mice, with ibuprofen and indomethacin used as the reference drugs. Anti-inflammatory activity was assessed based on the ability of the test compounds to prevent edema. The data revealed that compounds 5h and 5n showed significant anti-inflammatory effects at 25 mg/kg and 50 mg/kg administered i.p. When compared with the control group, these two compounds showed anti-inflammatory activities at a dose of 12.5 mg/kg, with lower inhibition rate, but this was not statistically significant. Compound 5n exhibited the strongest inhibition of inflammation at 49.26%, which was higher than ibuprofen
Table 1 Anti-inflammatory activity of compounds 5h and 5n administrated i.p. Compound
Dose (mg/kg)
Edema mean ± S.D. (mg)
Inhibition rate (%)
Control Ibuprofen Indometacin 5h
– 50 50 12.5 25 50 12.5 25 50
10.13 ± 0.62 7.28 ± 0.65* 5.13 ± 1.20*** 8.54 ± 0.44 7.11 ± 0.63* 5.78 ± 0.84*** 8.39 ± 0.57 6.59 ± 0.73** 5.14 ± 0.72***
– 28.13 49.36 15.70 29.81 42.94 17.18 34.95 49.26
5n
*
: 0.01 < p < 0.05, **: 0.001 < p < *0.01, **: p < 0.001 compared with Control group.
(28.13%) and lower than indomethacin (49.36%) at a dose of 50 mg/kg.
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The release of pro-inflammatory cytokines (e.g.: TNF-a and IL6) and mediators (e.g.: iNOS and COX-2) is related to the MAPK pathway in LPS-stimulated macrophages.15–17 MAPKs include ERK, c-Jun N-terminal kinase (JNK), and p38, and their signaling pathways play important roles in several biological processes. Accordingly, the effects of compound 5h on the activation of ERK and p38 were investigated. The results were presented in Fig. 4. The phosphorylation levels of ERK and p38 significantly decreased, and the effects of compound 5n were dose-dependent. However, the levels of ERK and p38 were unaffected after stimulation. These results suggested that compound 5n could inhibit
pro-inflammatory mediator and cytokine expression via MAPK pathways. NF-jB is an important transcription factor, which can regulate the expression of pro-inflammatory mediators in activated macrophages.18 The NF-jB protein comprises p65 and p50 subunits. Under normal conditions, a functional NF-jB dimer is exists as a complex with the inhibitor protein IjB in the cytosol.19 The activation of NF-jB in response to LPS stimulation leads to the degradation of IjB, followed by the release and nuclear translocation of NF-jB. After nuclear translocation, p65 and p50 regulate the production of TNF-a, IL-6, and mediators.20–23 Therefore, the inhi-
Fig. 4. The effect of compound 5n on the protein level of MAPKs in RAW 264.7 cells. The protein levels of MAPKs were determined using western blot assay. Data were means ± SD; ###: p < 0.001 compared with Control group; **:0.001 < p < 0.01, ***: p < 0.001 compared with LPS group.
Fig. 5. The effect of compound 5n on the protein level of NF-jB in RAW 264.7 cells. The protein levels of NF-jB were determined using western blot assay. Data were means ± SD; ##: 0.001 < p < 0.01 compared with Control group; *: 0.05 < p < 0.01, **: 0.001 < p < 0.01, compared with LPS group.
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bition of NF-jB activation has been regarded as a therapeutic approach for autoimmune and inflammatory conditions.24,25 As shown in Fig. 5, the effects of the extract obtained from compound 5n on NF-jB activation were investigated in LPS-stimulated RAW264.7 cells. The phosphorylation levels of p65 reduced significantly, and IjB levels increased dramatically in LPS-stimulated RAW264.7 cells in a dose-dependent manner. These results indicate that compound 5h inhibits the activation of NF-jB by preventing IjB degradation in LPS-stimulated RAW264.7 cells. In recent years, a large number of scholars engaged in COX-2 receptor-related research.26–30 Naproxen is a powerful, non-selective non-steroidal anti-inflammatory drug that is extensively used as a prescription and over-the-counter medication. It interacts with COX as reported by Duggan et al.31 The atomic coordinates and structure factors (codes 3NT1) have been deposited in the Protein Data Bank. Naproxen is a relatively simple molecule with a naphthyl scaffold, similar to compound 5n. A docking study of compound 5n into the COX-2 binding site was therefore performed. As shown in Fig. 6, there are some cavities around the naproxen molecule in the COX-2 binding pocket. Compound 5n could shrink the cavity space, thereby enhancing the interaction force. Moreover, the triazole ring can interact with the upper right hydrogen bond donor, in a manner equivalent to that of the carbonyl group of naproxen acting as hydrogen bond acceptor. The detailed interaction of compound 5n and naproxen with COX-2 is shown in Table 2. The results revealed that compound 5n shows
a stronger interaction with COX-2. This may result in a better inhibitory effect on COX-2. In order to verify our speculation, the effect of compound 5n on COX-2 was measured. The result was present in Fig. 6. The expression of COX-2 has significant difference between control group and LPS group. The value of COX-2/GAPDH was significant decrease treatment with compound 5n. Especially, the fold of COX-2/GAPDH was reduced to 45.19% at dose of 50 lg/mL. It suggested that compound 5n was liked to display anti-inflammatory activity related with the site of COX-2 as a kind of COX-2 inhibitors. In conclusion, a series of perimidine derivatives containing triazole (5a–s) were synthesized and their anti-inflammatory activities were evaluated using the MTT assay in a LPS-stimulated inflammation model. Compounds 5h and 5n reduced the levels of pro-inflammatory cytokines TNF-a and IL-6 in RAW264.7 cells. Compound 5n showed the most potent anti-inflammatory activity with an inhibition rate of 49.26% at a dose of 50 mg/kg, which is more potent than that of the reference drug ibuprofen (28.13%) and slightly less than that of indometacin (49.36%) in a xyleneinduced ear inflammation model. The results of western blotting showed compound 5n inhibited NF-jB (p65) activation and MAPK (ERK and p38) phosphorylation in a dose-dependent manner. Moreover, the results of the docking study of compound 5n into the COX-2 binding site and COX-2 inhibitory test revealed that its mechanism was possibly similar to that of naproxen, a COX-2 inhibitor.
Fig. 6. The docking result of compound 5n (left) and reported naproxen pose (right) into the COX-2 binding site.
Table 2 The interaction of compound 5n and naproxen with COX-2.
Compound 5h
Distance (Angstrom)
Types
Amino acid residues
Types
Distance (Angstrom)
2.81 3.52, 3.93, 4.01, 4.54, 5.00 5.06 5.11 5.95 – –
Carbon Hydrogen bond Pi-Alkyl Amide-Pi Stacked Pi-Alkyl Pi-Alkyl Pi-Pi T-shaped Pi-Alkyl Pi-Alkyl Pi-Sulfur – –
SER353 VAL523 GLY526 ALA527 LEU352 TYR355 VAL349 VAL116 MET522 LEU359 GLU524
– Pi-Alkyl Amide-Pi Stacked Pi-Alkyl Pi-Alkyl Pi-Alkyl Pi-Alkyl Alkyl – – Negative-Negative
– 5.12 4.24, 4.91, 3.35 4.64, 5.46 4.99 462, 4.94 5.24 – 4.46 5.28
4.41, 4.69, 4.33, 4.72,
4.67 5.40 4.46, 5.32 4.89, 5.24
Naproxen
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