Biological evaluation and molecular docking studies ...

International Immunopharmacology 43 (2017) 129–139

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Biological evaluation and molecular docking studies of nitro benzamide derivatives with respect to in vitro anti-inflammatory activity Tugba B. Tumer a,⁎, Ferah Comert Onder b, Hande Ipek a, Tugba Gungor b, Seda Savranoglu a, Tugba Taskin Tok c, Ayhan Celik d,1, Mehmet Ay b,⁎ a

Department of Molecular Biology & Genetics, Faculty of Art and Science, Çanakkale Onsekiz Mart University, Terzioğlu, 17020 Çanakkale, Turkey Natural Products and Drug Research Laboratory, Department of Chemistry, Faculty of Art and Science, Çanakkale Onsekiz Mart University, Terzioğlu, 17020 Çanakkale, Turkey Department of Chemistry, Faculty of Art and Science, Gaziantep University, Şehitkamil, 27310 Gaziantep, Turkey d Department of Chemistry, Faculty of Science, Gebze Technical University (GTU), Gebze, 41400 Kocaeli, Turkey b c

a r t i c l e

i n f o

Article history: Received 15 June 2016 Received in revised form 30 November 2016 Accepted 6 December 2016 Available online xxxx Keywords: Nitro substituted benzamides Anti-inflammatory Nitric oxide iNOS Molecular docking

a b s t r a c t A series of nitro substituted benzamide derivatives were synthesized and evaluated for their potential anti-inflammatory activities in vitro. Firstly, all compounds (1–6) were screened for their inhibitory capacity on LPS induced nitric oxide (NO) production in RAW264.7 macrophages. Compounds 5 and 6 demonstrated significantly high inhibition capacities in a dose-dependent manner with IC50 values of 3.7 and 5.3 μM, respectively. These two compounds were also accompanied by no cytotoxicity at the studied concentrations (max 50 μM) in macrophages. Molecular docking analysis on iNOS revealed that compounds 5 and 6 bind to the enzyme more efficiently compared to other compounds due to having optimum number of nitro groups, orientations and polarizabilities. In addition, 5 and 6 demonstrated distinct regulatory mechanisms for the expression of the iNOS enzyme at the mRNA and protein levels. Specifically, both suppressed expressions of COX-2, IL-1β and TNF-α significantly, at 10 and 20 μM. However, only compound 6 significantly and considerably decreased LPS-induced secretion of IL-1β and TNF-α. These results suggest that compound 6 may be a multi-potent promising lead compound for further optimization in structure and as well as for in vivo validation studies. © 2016 Published by Elsevier B.V.

1. Introduction Inflammation is a general physiological process of the body for the modulation of immune response against a diverse range of triggering factors including infectious agents, allergens, free radicals, defective dietary habits rich in refined foods and sedentary lifestyle. A micro environment activated persistently and dominated with low levels of proinflammatory factors for a long time may predispose to chronic inflammation [1]. It is now well established that a state of low-grade chronic inflammation is an underlying cause of various metabolic disorders like obesity, insulin resistance, metabolic syndrome, type II diabetes and even cancer at the long term period [2–5]. Excess production of nitric oxide (NO) by inducible nitric oxide synthase (iNOS) and prostaglandins by cyclooxygenase 2 (COX-2) as well as increased release of proinflammatory cytokines (e.g. TNF-α, IL-1β and IL-6) under inflammatory conditions, all contribute to intracellular cascades of molecular ⁎ Corresponding authors. E-mail addresses: [email protected], [email protected] (T.B. Tumer), [email protected] (M. Ay). 1 Left from GTU in September 2016.

http://dx.doi.org/10.1016/j.intimp.2016.12.009 1567-5769/© 2016 Published by Elsevier B.V.

degeneration [6,7]. Therefore invention of new anti-inflammatory agents with multifactorial mode of action could be a good strategy in fighting chronic inflammation and associated metabolic diseases. A considerable number of drugs and other physiologically active agents are in nitroaromatic structure, such as nilutamide (non-steroidal antiandrogen), nitrendipine (antihypertensive), nitrazepam (anticonvulsant, hypnotic) and nitromide (antibacterial) [8–11]. In addition, some nitro benzamides, such as 5-aziridinyl-2,4-dinitrobenzamide (CB1954), its analog (nitrogen mustards SN23862), and another compound ledakrin (1-nitro-9[(dimethylamino) propylamino]acridine), have special attention as anti-tumor agents due to their remarkable effects obtained from in vitro/in vivo studies and also from clinical trials during treatment of some cancers [12–14]. Although medicinal chemists generally remove nitro containing compounds from their screening collections since they are classified as “structural alert”, they cannot be totally disregarding because of their high efficacy. For example Nimesulide, the sole nitro group containing non-steroidal anti-inflammatory drug (NSAIDs), has been effectively utilized for N30 years due to its multifactorial therapeutic actions and very good gastrointestinal tolerability. Herein inspired by nitro aromatic compounds, a series of benzamide derivatives (1–6) were synthesized and evaluated for their

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potential anti-inflammatory activities. Additionally, interaction mechanisms among these compounds and iNOS at the molecular level were identified by molecular docking studies. We started to investigate the compounds for their activities with respect to inhibiting NO release in lipopolysaccharide (LPS) induced RAW 264.7 macrophages. The excess production of NO by iNOS under inflammatory conditions is critical for the development of intracellular degenerative processes. It has been known for a long time that higher concentration of NO synthesized by iNOS reacts with oxygen and generates various reactive nitrogen oxide intermediates (RNOIs) [15]. These reactive compounds can attack to other biomolecules, especially proteins result in conformational changes and lead to either inhibition or inactivation through different mechanisms particularly, S-nitrosylation (reviewed in [16]). These events trigger deleterious pathological cascades of inflammatory processes. Recently, Yang et al., showed that in the setting of obesity, which is characterized by chronic metabolic inflammation (termed as metaflammation), increased iNOS activity causes S-nitrosylation of some key proteins such as UPR regulator and IREα, and thus contributes to impaired ER function and prolonged ER stress [17]. It was also reported that reversal of iNOS mediated Snitrosylation can promote ER homeostasis and result in substantial metabolic benefits. Therefore, design of new drugs acting on excessive NO production through iNOS is a promising therapeutic intervention against metabolic diseases originating from persistent inflammatory conditions. In the present study; among six synthesized nitro-substituted benzamide derivatives, two molecules were selected as our lead compounds (5 and 6) due to having considerably low half maximal inhibitory concentration (IC50; 3.7 vs 5.3 μM, respectively) for NO inhibition and compatible molecular docking studies for iNOS. Although non-steroidal anti-inflammatory drugs (NSAIDs) have less serious side effects compared to steroidal drugs, the latter have been recognized as the most effective treatment in the inflammatory diseases for N50 years [18]. The potency of steroidal is derived from their nonspecific inhibition capacity for the production of many proinflammatory cytokines [19]. For this reason, the elucidation of new non-steroidal molecules having multi-factorial inhibitory effects on major proinflammatory factors is still desirable. Considering this fact, in the current work, the above-mentioned two compounds (5 and 6) were additionally investigated for their multi-inhibitory effects on the LPS-induced release of IL-1β and TNF-α, expression level of iNOS mRNA and protein as well as expression level of COX-2, IL-1β and TNF-α mRNAs. The cytotoxic effects of these lead compounds were also evaluated in RAW 264.7 macrophages. 2. Experimental section 2.1. General All reagents were obtained from various sources at highest purity possible. Reagents for synthesis from Merck, Fluka and Sigma–Aldrich were used as supplied without prior purification. Melting points were determined with X-4 Melting Point Apparatus and are uncorrected. Synthesis reactions were monitored by TLC on 0.25 mm silica gel plates (60 GF254) and visualized with ultraviolet light. Infrared spectra were measured using ATR techniques on a Perkin Elmer Spectrum 100 FTIR spectrophotometer. The 1H NMR and 13C NMR spectra were recorded at Varian Mercury 500 MHz High Performance Digital FT-NMR spectrometer and Jeol NMR-400 MHz using TMS as an internal standard.

at reflux temperature for 10 h [20]. Similarly, compounds 2 and 4 were obtained at reflux conditions for 1 h at pyridine/benzene mixture [21]. Moreover, compounds 3 and 6 were prepared from 4-nitrobenzoyl chloride and 3-nitroaniline (for compound 3) or 3,5-dinitroaniline (for compound 6) at the conditions that used triethylamine as base in acetone at room temperature for 30 h [22]. In addition, compound 5 was obtained using triethylamine in DMF at 125 °C for 40 h [23]. The general amidation reaction was applied to 4-nitro-N-(2-nitrophenyl) benzamide and 4-nitro-N-(4-nitrophenyl)benzamide (2 and 4) with moderate yields for the first time. While compound 3 was synthesized according to the known procedure, this method was applied for the first time for synthesis of compound 6. DMF was used for the synthesis of 4-nitro-N-(2,4-dinitrophenyl)benzamide (5) as different from the literature method. 2.2.1. Compound 1 White solid, 64%, m.p. 214–215 °C (lit. m.p. 213–214 °C) [24], 1H NMR (400 MHz, DMSO) δ 7.10 (1H, t, J = 6.85 Hz), 7.34 (2H, t, J = 7.96 Hz), 7.74 (2H, d, J = 8.65 Hz), 8.14 (2H, d, J = 8.95 Hz), 8.33 (2H, d, J = 8.93 Hz), 10.53 (1H, s); 13C NMR (100 MHz, DMSO) δ 120.99, 124.09, 124.71, 129.26, 129.74, 139.21, 141.15, 149.66, 164.42. 2.2.2. Compound 2 Yellow solid, 63.5%, m.p. 222 °C (lit. m.p. 223 °C) [25], 1H NMR (500 MHz, DMSO) δ 7.48 (1H, t, J = 8.08 Hz), 7.75 (2H, m, J = 8.12 Hz), 8.05 (1H, d, J = 6.79 Hz), 8.19 (2H, d, J = 9.71 Hz), 8.41 (2H, d, J = 6.78 Hz), 11.04 (1H, s); 13C NMR (125 MHz, DMSO) δ 124.65, 125.62, 126.94, 129.91, 131.24, 134.51, 139.74, 144.04, 150.26, 164.38. 2.2.3. Compound 3 Yellow solid, 68%, m.p. 227–228 °C (lit. m.p. 227–228 °C) [26]; 1H NMR (500 MHz, DMSO) δ 7.67 (1H, t, J = 8.2 Hz), 7.98 (1H, d, J = 5.92 Hz), 8.21 (3H, m, J = 8.52 Hz), 8.37 (2H, d, J = 8.64 Hz), 8.77 (1H, s), 10.97 (1H, s); 13C NMR (125 MHz, DMSO) δ 115.12, 119.22, 124.26, 126.74, 129.94, 130.85, 140.26, 148.14, 149.77, 164.85. 2.2.4. Compound 4 Yellow solid, 68.8%, m.p. 270 °C (lit. m.p. 274 °C) [26], 1H NMR (500 MHz, DMSO) δ 8.06 (2H, d, J = 7.49 Hz), 8.23 (2H, d, J = 7.08 Hz), 8.27 (2H, d, J = 7.49 Hz), 8.38 (2H, d, J = 7.09 Hz); 13C NMR (125 MHz, DMSO) δ 120.43, 123.72, 125.44, 130.08, 140.71, 143.32, 149.98, 165.92. 2.2.5. Compound 5 Yellow solid, 35%, m.p. 194–195 °C (lit. m.p.199–200 °C) [23], 1H NMR (500 MHz, DMSO) δ 8.06 (1H, d, J = 8.99 Hz), 8.22 (2H, d, J = 8.85 Hz), 8.43 (2H, d, J = 8.84 Hz), 8.61 (1H, dd, J = 7.97 Hz), 8.77 (1H, d, J = 7.97 Hz), 11.48 (1H, s); 13C NMR (125 MHz, DMSO) δ 121.64, 124.41, 126.73, 129.06, 129.96, 136.91, 138.76, 142.07, 143.86, 150.31, 164.59. 2.2.6. Compound 6 Yellow powder solid, 55%, m.p. N250 °C; 1H NMR (500 MHz, DMSO) δ 8.24 (2H, d, J = 8.71 Hz), 8.40 (2H, d, J = 9.69 Hz), 8.57 (1H, s), 9.08 (2H, s), 11.32 (1H, s); 13C NMR (125 MHz, DMSO) δ 113.61, 120.09, 124.22, 129.87, 139.45, 141.29, 148.60, 150.11, 165.13. All spectroscopic data of compounds were consistent with literature values (seen in supp. part).

2.2. Preparation of compounds (1–6)

2.3. Effects of synthesized compounds on inflammatory mediators

Nitro substituted aromatic amides (1–6) were prepared by substitution reaction of 4-nitrobenzoyl chloride with nitro containing aniline derivatives (2-nitroaniline, 3-nitroaniline, 4-nitroaniline, 2,4-dinitroaniline, 3,5-dinitroaniline) in the different reaction conditions. Compound 1 was synthesized in pyridine that used as base and solvent

2.3.1. Cell culture and sample treatment All reagents used in cell culture experiments were supplied from Sigma–Aldrich Co. (St. Louis, MO) unless otherwise noted. Murine RAW macrophage cell line (RAW 264.7, ATCC®TIB-71™) was routinely maintained in Dulbecco's modified Eagle's medium (DMEM) (Caisson,


North Logan, UT) supplemented with 100 U/mL penicillin, 100 μg/mL streptomycin, and 10% fetal bovine serum (FBS). Cells were incubated at 37 °C in humidified air containing 5% CO2 and sub-cultured by cell scraping. For treatments, cells were plated at a density of 4 × 105 cells/mL in 24-well plates in phenol red free DMEM supplemented with 10% FBS and then incubated with or without LPS (1 μg/mL) and synthesized compounds (1–6) or with positive control (1400 W, N-[[3(aminomethyl)phenyl]methyl]-ethanimidamide, dihydrochloride) as triplicate for 24 h. Compounds were dissolved in 10% dimethyl sulfoxide (DMSO) and final concentration of DMSO was 0.1% in each well. All biological experiments were repeated at least three times, and the data were represented as mean values (means ± SD). 2.3.2. Nitric oxide production assay At the end of treatment period, as described above, media was collected from each well and centrifuged at 14,000g at 4 °C for 10 min. Supernatant was assayed immediately or stored at − 80 °C. Nitric oxide production was determined in duplicate by using Griess reagent [27]. Briefly, 100 μL cell supernatant was transferred onto a 96-well plate and incubated with 100 μL of Griess reagent containing 1% sulphanilamide and 0.1% naphthylethylenediamine in 5% phosphoric acid solution for 10 min under light protected conditions. The nitrite standard reference curve was prepared by a serial dilution (0 to 100 μM) from 0.1 M NaNO2. Absorbance was read at 540 nm by using a microplate reader (Tecan Infinite® 200 PRO, Switzerland). 2.3.3. IL-1β and TNF-α secretion For IL-1β and TNF-α secretion, the supernatant was assayed separately in duplicate with Mouse IL-1β and TNF-α Platinum ELISA kit (eBioscience, Affymetrix) according to the procedure described by manufacturer. To determine the concentration of IL-1β and TNF-α, a reference curve was prepared by using standards provided with the kit. Absorbance was read at 450 nm. Concentrations of NO, IL-1β and TNF-α were normalized to the cellular protein content as quantified by the Pierce BCA Protein Assay Kit (Thermo Scientific, Rockford, IL, USA). 2.3.4. Cell viability assay The effect of treatments on cell viability was examined by using MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyltetrazolium bromide] (TCI, Portland, OR) [28]. MTT working solution (5 mg/mL) was dissolved in PBS (1 ×, Cayman Chemical, Ann Arbor, MI), filtered through a 0.22 μm sterile syringe filter (Corning, NY, USA) and added to treated cells during the last 2 h of treatment. Media were carefully aspirated cells were dissolved in DMSO and the absorbance was read at 570 nm. 2.3.5. Gene expression analyses by quantitative PCR Total RNA was isolated from RAW 264.7 macrophage cells for quantitative determination of iNOS (NOS2), TNF-α, IL-1β and COX-2 gene expressions by using PureLink® RNA mini kit plus on-column DNase treatment (Applied Biosystems, Foster City, CA) according to manufacturer's specifications. The protocol has been tailored for the isolation and purification of RNA from RAW264.7 cell line using TRIzol. Briefly, 180 μL chloroform was added to TRIzol harvested samples (900 μL). Samples were vigorously mixed for 15 s, incubated at room temperature for 3 min, and centrifuged at 12,000g for 15 min at 4 °C. After that, the upper phase (colorless, 400 μL) was collected and continued with the instructions written in kit's protocol. Samples were allowed to dry and re-suspended in RNAase/DNAase free-water. The integrity of the RNA was evaluated by running RNA on a 1% agarose gel. RNA was then treated with Deoxyribonuclease I Amplification grade (Life Technologies), following the manufacturer's guidelines. RNA quality was checked on the Tecan's Nano Quant Plate (Tecan Infinite® 200 PRO, Switzerland). A ratio of OD 260/280 ≥ 2.0 and OD 260/230 ≥ 1.8 was considered to be good quality. First-strand cDNA was synthetized from 3000 ng total RNA using the high capacity cDNA reverse transcription kit (with oligo-d(T)s as primers) plus RNase inhibitor (Applied

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Biosystems, Foster City, CA). The thermal cycle program was set as follows: 25 °C for 10 min; 37 °C for 120 min; 37 °C; 85 °C for 5 s, and final hold at 4 °C. Synthesized cDNAs were diluted 20-fold and the diluted sample (5 μL) was used for quantitative PCR with specific TaqMan® Gene Expression Assay for each gene (Life Technologies, Thermo Fisher Scientific). Quantitative PCR amplifications were performed on Step One Plus® Real-Time PCR Systems (Applied Biosystems, Thermo Fisher Scientific). β-Actin was used to normalize target gene expression. The effects of compounds on gene expression levels of inflammatory markers were evaluated by the comparative ΔΔCt method. 2.3.6. Western blot analysis RAW 264.7 macrophages were plated at a density of 1.5 × 106 cells/mL in 6-well plates in phenol red free DMEM supplemented with 10% FBS and then incubated without LPS (vehicle) and with 1 μg/mL LPS including compounds 5 and 6 as triplicate for 6 h. After incubation period, cells were washed with ice-cold phosphate buffered saline (PBS, Caisson Lab) and collected and lysed by using radio-immuno assay buffer (RIPA) containing (150 mM sodium chloride, 1.0% NP-40 or Triton X-100 0.5%, sodium deoxycholate, 0.1% Sodium dodecyl sulphate (SDS), 50 mM Tris, pH 8.0), 1× protease (Amresco) and phosphatase inhibitor (Cell Signaling) cocktails. The cell lysate centrifuged at 14.000 g at 4 °C for 15 min. The protein content of supernatant was quantified with the BCA protein assay kit (Pierce). Supernatants (60 μg) were separated on 4% stacking gel and 8.5% SDS-PAGE in a discontinuous buffer system as described by [29] and subjected to Western blotting. Nitrocellulose membrane was incubated with iNOS and β-actin primary antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) overnight while shaking at 4 °C. After washing with TBST buffer (500 mM NaCl, 20 mM Tris pH 7.4, 0.05% Tween 20). The membrane incubated with HRP-conjugated secondary antibody for 1 h at room temperature and visualized with western blotting luminol reagents (Santa Cruz Biotechnology, Santa Cruz, CA) on C-DiGit Chemiluminescence Western Blot Scanner (Licor, NE, USA). The abundance of protein was determined by the densitometric analysis of bands by using Image Studio Lite Software 5.0. 2.4. Molecular docking studies Molecular docking was performed with Discovery Studio [30] (DS) 2017 to provide an insight into the benzamide derivatives (1–6). Firstly, ligand(s) and enzyme were prepared using Gaussian 09 (G09) [31] and DS 2017 software for molecular docking study. The nitro functional group containing aromatic amide compounds (Fig. 1, Scheme 1) were constructed and geometrically optimized using DFT/B3LYP/6-31G* basis set as implemented in G09. Their conformational analyses were performed using DS 2017. The protein crystal structures of iNOS (PDB codes: 3NW2) was selected for this study. Hydrogen and missing heavy atoms were added to the protein structure, and atom types and partial charges were assigned. Hem group and the tetrahydrobiopterin cofactor were kept. The protein binding sites were defined using those occupied by the co-crystallized ligand MPW1 for iNOS. Additionally, 1400 W was used as reference ligand to examine its interactions with iNOS deeply. Their positions of iNOS were subsequently optimized using CHARMm force field and the adopted-basis Newton-Raphson (ABNR) method [32] available in the DS 2017 protocol until the root mean square deviation (RMSD) gradient was b 0.05 kcal/mol Å2. The binding site was defined from literatures about iNOS [33] as given detailed information about the binding site of iNOS on Figs. S1 and S2 in supplementary part. During docking analysis, the ligands were flexible and iNOS enzyme was held rigid. Dock Ligands (CDOCKER) was performed using the default settings. The docking parameters were as follows: Top Hits: 50; Random Conformations: 10; Random Conformations Dynamics Step: 1000; Grid Extension: 8.0; Random Dynamics Time Step: 0.002. After this step, all docked poses were scored by applying Analyze Ligand Poses sub-protocol in Discovery Studio 2017. Finally, binding energies

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Fig. 1. Synthesized nitro-substituted aromatic amide compounds (1–6).

were also calculated by applying Calculate Binding Energy sub-protocol in DS 2017 using in situ ligand minimization step in the ABNR method. The binding between ligands and iNOS were evaluated with Binding Energy, CDOCKER energy, CDOCKER interaction energy values.

2.5. Statistical analysis Statistical analyses were performed using the SPSS Windows Version release 16.0. Equality of variances was analyzed by Levene's test. Group means were compared by a Student's t-test for the independent samples. P-values b 0.05 were considered indicative of significance. All experiments and analyses were repeated at least three times.

3. Results and discussion In this study, we synthesized aromatic amide compounds containing nitro functional group(s) (1–6, Fig. 1) by using different reaction conditions. Structures of the compounds were characterized by melting point, FT-IR, 1H NMR and 13C NMR spectral analysis. A convenient method for synthesizing nitro-substituted aromatic amide compounds was given in Scheme 1. FT-IR data of the compounds generally show a band at ~3410 and/or 3324 cm− 1 for N\\H stretching of amide and a band at ~1670 cm−1 corresponds to C _O stretching of amide linkage. Two intensive bands were observed at ~1530 and 1350 cm−1 correspond to asymmetrical and symmetrical stretching of –NO2 groups, respectively. In 1H NMR spectrum, all singlet protons for -NH groups were observed between ~ 11.48 and 7.10 ppm. Characteristic aromatic ring protons were observed between 9.08 and 7.10 ppm. The spectral values of the compounds 1–6 are given in the Experimental section and supplementary data (Figs. S4–S15). Compounds 1–6 have poor solubility in water. Their solubility is below 5 mg/mL at 25 °C.

3.1. Identification of lead compounds We have identified two lead compounds among six nitro substituted benzamide derivatives as promising anti-inflammatory agents according to results obtained from NO inhibition assay in LPS induced RAW 264.7 macrophages and molecular docking studies performed on iNOS enzyme. For each assay, a control (vehicle alone), and an induction control (1 μg/mL LPS) were used to set a baseline data. Previous works demonstrated that LPS, a component of the gram-negative bacterial cell walls, can trigger the inflammatory process by binding to the CD14 toll-like receptor-4 (TLR-4) complex at the surface of innate immune cells and subcutaneous infusion of LPS to animals resulted in the same metabolic abnormalities induced by the high-fat diet [34]. For this reason, in recent works LPS have been successfully used and generally preferred due to its dual role in inducing both acute and low-level chronic inflammatory responses [34,35].

3.2. Screening of compound by NO inhibition As shown in Fig. 2 all compounds (1–6) were first screened for their effect on LPS induced NO production in RAW 264.7 macrophage cells at 20 μM. 1400 W was used as a positive control. Tested compounds exhibited no cytotoxic effect (N 85 cell viability) on cells at 20 μM, except compound 1. The relative cell viability for compound 1 was 52% (data not shown). Among synthesized six compounds, 5 and 6 demonstrated considerably high effects -N50% inhibition- against NO production at 20 μM with inhibition rates of 62% and 54%, respectively (p b 0.05 for both). When cells were simultaneously treated with different concentrations of these two molecules (5–50 μM) and LPS (1 μg/mL), as seen in Fig. 3, NO production was significantly inhibited in a dose-dependent manner with N70% and 90% inhibitions at 50 μM for compounds 5 and 6, respectively. At all tested doses including the highest 50 μM, the

Scheme 1. Synthesis of 4-Nitro-N-(4-phenyl) benzamide (1) and its derivatives.


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Fig. 2. Screening of all synthesized compounds (1–6) at 20 μM for inhibiting LPS-induced NO production in RAW 264.7 macrophage cells. Ctrl: Control (vehicle alone). 1400 W: positive control at 5 μM. Values show relative change of NO (%) in production of NO compared to vehicle with LPS. Each bar represents the mean ± SEM (n = 3). *** = p b 0.001, ** = p b 0.01, * = p b 0.05.

cellular viability were N90% for both 5 and 6 eliminating the doubt that the observed considerable decrease in LPS induced NO production could be related to cytotoxic effects (Fig. 3). The IC50 values of these two molecules were investigated for NO production at nine different doses ranging between 1 and 20 μM, resulted in 3.7 μM for 5 and 5.7 μM for 6. In recent years, it has been shown that increased production of NO by iNOS is responsible for some acute pathological conditions such as stroke and septic shock, as well as for a diverse array of metabolic disorders originating from chronic low grade inflammation [17,36]. Therefore, the inhibition capacities of 5 and 6 on inducible NO production at quite low micro molar ranges (3.7 and 5.7 μM) compared to reported IC50 values of 1) L-NIL (L-N6(1-iminoethyl)-lysine [37]: 27.1 μM, a selective inhibitor of iNOS, and 2) Indomethacin [37] (65.3 μM, a prescribed NSAID, make these two molecules promising as lead anti-inflammatory compounds. 3.3. Molecular docking studies of lead compounds on iNOS Molecular docking calculations were implemented with one crystallographic complex obtained from the Protein Data Bank (PDB) [36] which is the structure of human inducible nitric oxide synthase (iNOS) complexed with 2-[2-(4-methoxypyridin-2-yl)ethyl]-3H–

imidazo[4,5-b]pyridine (MPW1). The docking results on iNOS exhibited that obtained conformation of MPW2 was superimposed with MPW1 that were nearly identical for the crystallographic crystal complex. A potent, selective inhibitor of iNOS, N-(3(Aminomethyl)benzyl)acetamidine (1400 W) was used to describe an insight into the iNOS-1400 W complex in molecular docking studies. As mentioned in upper part, two molecules (5 and 6) have remarkably low half maximal inhibitory concentration for LPS induced NO production. Based on this finding, docking studies were performed to determine why compounds 5 and 6 demonstrated relatively efficient inhibition capacity compared to others. All six models were flexibly docked with iNOS by using CDOCKER. Of the ten poses produced, the best pose of each ligand was selected based on CDOCKER top score and the target structure was selected for further analysis. The ligand poses were analyzed and a heat map was produced to count H bonds made by the poses to the enzyme molecule using Analyze Ligand Poses sub-protocol in DS 2017 (data were shown in supplementary part, Tables S1–S2). For comparison of orientations of compounds 1–6, we superimposed each compound's best pose, which was obtained by locating the lowest binding energy, the largest minus CDOCKER energy and the lowest minus CDOCKER interaction energy, (see Table S2 in supp. part). Additionally, the synthesized compounds (1–6) including

Fig. 3. Screening of compounds 5 and 6 at different doses 5–50 μM for inhibiting LPS-induced NO production in RAW 264.7 macrophage cells. Ctrl: Control (vehicle alone). Each bar represents relative change of NO (%) as compared to vehicle with LPS. Each dot on the line shows relative survival rate of cells in case of different treatments as compared to vehicle with LPS. Values represent the mean ± SEM (n = 3). *** = p b 0.001, ** = p b 0.01, * = p b 0.05.

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ortho, meta, para and two nitrophenyl combination were evaluated based on N-(phenyl)-4-nitrobenzamide (1) with binding site of the enzyme, as given Figs. S1 and S2 in supplementary part. Hydrogen bonding and lipophilic interactions have great importance in any biological system. Numerical values demonstrating these interactions among benzamide derivatives 5–6 and iNOS were presented in Table S6 (for H-bonds and lipophilic interactions) and Table S4 (Non-bonding interactions data of the 1–4 ligands). Specifically for compound 5, as can be seen in Fig. 4A, while the Arg260 residue formed two hydrogen bonds with the 4-nitrophenyl moiety, the ortho-nitro group made single hydrogen bond (2.471 Å) with Gln486 residue of the enzyme. Single hydrogen bond (2.0296 Å) is also observed with Asn115 residue. Among compound 5 and iNOS five lipophilic interactions can be described: one of them is between 2.4-dinitrophenyl ring and NH1 moiety of Arg382 residue, the other four interactions are observed between Hem group of the enzyme and N7 atom of the compound 5 (Fig. 4A and Table S4). The orientation of compound 6 is quite different from the orientation of compound 5, therefore hydrogen bonds and lipophilic interactions are different. The hydrogen bonds for compound 6 were observed with Arg260, Asn348 and Glu488 residues (1.981 Å, 2.193 Å, 2.222 Å, respectively). In case of lipophilic interactions, compound 6 formed eight different interactions between Hem group and Phe280, Tyr367 and Arg382 residues of the enzyme as shown in Fig. 4B. Based on docking results and relative orientation of ligands for crystallographic complex (seen in Fig. 5A, B), compounds 5 and 6 seem to be good inhibitors of iNOS as compared to other compounds (1–4). When two lead molecules are superimposed with the reference ligand (1400 W), the results showed that they are quite similar (Fig. 5C). The phenyl moieties are almost parallel to Hem group, so that a series of π-π and π-cation interactions can be observed as shown in Fig. 4 and Table S6. Fig. 6A-C also represent that reference ligand (1400 W), compounds 5 and 6 are all at the same conformation on the protein. However, other compounds (1–4) are in perpendicular conformation to Hem group at the binding site of iNOS, thus they cannot make lipophilic interactions with this prosthetic group on the protein. These results suggest that our lead compounds might be potential iNOS inhibitors as determined experimentally by their IC50 values which resulted in 3.7 μM for 5 and 5.7 μM for 6. Considering all compounds, the results demonstrated that for the efficient inhibition of

NO production, the optimum number of nitro group, determined as three, in the binding site of the enzyme has great importance rather than the position of these groups. For this reason, compounds 5 and 6 showed remarkable results. Furthermore, orientations and polarizabilities of lead compounds have very dominant properties for binding willingness on the enzyme. Gray labelled two rows on Table 1 indicate these statuses with numerical values. The compound 5 have higher numerical polarizability than compound 6 (351.811 vs 319.119, respectively). It means that compound 5 exhibits suitable polarizability and orientation for active site of the enzyme as compared to compound 6. This difference might explain slightly lower IC50 value obtained for compound 5 according to compound 6. 3.4. Effects of lead compounds on iNOS mRNA/protein expression The molecular docking studies gave us detailed information about the inhibitory potential of two lead compounds on iNOS enzyme. However, to elucidate the action mechanism of compounds 5 and 6 on the inhibition of NO production in a detailed way, we carried out further studies. To understand whether these two lead molecules inhibit NO production at the level of transcription or protein expression, we determined the relative level of iNOS gene by qPCR and iNOS protein by Western blot in LPS activated macrophages. In both qPCR and Western blot analyses β-actin was used as housekeeping gene/protein to normalize the expression data. Firstly, as shown in Fig. 7 (A-C), the treatment of the macrophages with LPS (1 μg/mL) markedly increased the expression of both iNOS protein and mRNA levels compared to control group (−LPS). In the qPCR analyses, co-treatment with 5 did not result in a decrease in the mRNA expression level at both 10 and 20 μM. Although a significant decrease in protein expression was observed by co-treatment of compound 5 at a ratio of 19% for 10 μM and 24% for 20 μM as seen on Fig. 7A and B, these decreases may not be biologically relevant due to relatively low ratios. Taken together; the present data showed that compound 5 inhibits NO production without effecting mRNA or protein level of iNOS. The very low IC50 value of 5 for NO production may be explained by direct inhibitory action of this compound on catalytic activity of iNOS enzyme. Some of the iNOS inhibitors such as aminoguanidine and S,S′-1.4-phenylene-bis(1.2-ethanediyl)bisisothiourea (PBIT) do not influence the synthesis of iNOS [38,39]. Importantly, the administration of PBIT, a selective iNOS inhibitor that

A

B

Fig. 4. The orientations of compounds A) 5 and B) 6 including hydrogen bonds (left side) and lipophilic interactions (right side) with Hem and residues of iNOS enzyme. 5 and 6 are shown as orange and pink color, respectively. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)


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A

B

C

Fig. 5. A) The relative orientation of ligands MPW1 (blue color), MPW2 (gray color) and 1400 W (green color) in relation to the Hem and H4B groups. B) Docking poses of 1, 2, 3, 4 (stick) and 5, 6 (ball and stick, pink and orange) in iNOS. C) The compounds 5, 6 and ligand 1400 W in iNOS. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

decreased NO generation without affecting iNOS expression significantly suppressed carcinogen-induced tumor development in several cancer models [39,40]. Co-treatment of cells with compound 6 reduced LPS induced iNOS mRNA expression in a dose dependent manner such that while 10 μM decreased the expression 28%, 20 μM resulted in 51% suppression (p b 0.05 for both, Fig. 7C). Furthermore, co-treatment of cells with 6 at both 10 and 20 μM markedly suppressed the LPS induced protein level at nearly same levels 41% vs 45%, respectively (Fig. 7A and B).

These results were found to be consistent with the NO generation data and suggest that in contrast to compound 5, the mechanism for the NO inhibitory activity of compound 6 might be correlated to the suppression of both iNOS gene and protein expression. 3.5. Effects of lead compounds on TNF-α and IL-1β release We investigated the multi-potent inhibitory activity of lead compounds 5 and 6 on secretion of some major proinflammatory factors

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A)

B)

C)

Fig. 6. A, B) Binding site of iNOS with the surface occupied by ligand MPW1and 1400 W is shown in light pink with compounds 5 and 6 inside it. C) The compounds 5 and 6 in the iNOS cavity, showing the aliphatic moiety coming out. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

under inflammatory response. Both IL-1β and TNF-α secretion have been identified decades ago as hallmarks of proinflammatory response in obesity-associated chronic metabolic diseases like insulin resistance and type II diabetes [41,42]. In the current work, treatment of macrophage cells with compound 5 did not result in any change in the amount of LPS induced IL-1β secretion at both 10 and 20 μM doses (Fig. 8A). However, treatment with compound 6 at 10 μM and 20 μM resulted in 57% and 65% inhibition, respectively. Similar trends were also observed for LPS-induced TNF-α secretion. Compound 5 did not present any effect on the secretion of LPS-induced TNF-α at both 10 and 20 μM doses. However, treatment with compound 6 exhibited 46% and 55% inhibition at 10 and 20 μM (Fig. 8B).

44]. In the present study, to assess the effects of compounds 5 and 6 on COX-2, IL-1β and TNF-α expression by LPS-inflamed macrophages, we measured their gene expression level by qPCR. As seen on Fig. 8, in LPS induced macrophages the expression of these three markers were markedly increased compared to control cells. However, co-treatment of cells both by compounds 5 and 6 with LPS resulted in a significant downregulation in the mRNA expression of all genes. The magnitude of suppression for compound 5 was changing between 37 and 50% at 10 and 20 μM in three different genes (Fig. 9). In case of compound 6, the more effective downregulation (around 60%, at 10 μM) has been observed, particularly in the expression of IL-1β. This value increased up to 88% at 20 μM in a dose dependent manner (Fig. 9).

3.6. Effects of lead compounds on IL1β, TNF-α and COX-2 mRNA expression

4. Conclusion

Unregulated overexpression of IL1β, TNF-α and COX-2 along with various other proinflammatory factors have been connected to metastatic cells and contribute to development and progression of cancer [43,

In the current study, six (1–6) nitro substituted benzamide derivatives have been synthesized and addressed for their potential anti-inflammatory activities for the first time. All compounds were screened

Table 1 Orientations and polarizabilities of the compounds (1–6). Index

Ligand names

Pose numbers

Exact polarizabilities

96 7 22 56 42 62

1 2 3 4 5 6

5 1 1 5 1 1

280.866 301.144 206.112 300.322 351.811 319.119

−3.880 4.718 −60.517 5.319 3.731 0.123

158.001 194.257 309.624 181.637 205.421 205.102

4.059 −7.216 0.000 8.906 −6.062 −5.009

−2.235 7.771 0.000 −9.506 8.830 15.199

61.726 63.345 57.842 68.935 68.801 75.274


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Fig. 7. The effects of 5 and 6 on the LPS induced iNOS protein and gene expression level. (A) Western blot analysis for iNOS and β-actin (endogenous control) protein expression (B) Bands were quantified by densitometry and expressed by relative iNOS/β-actin ratio compared to vehicle with LPS. (C) iNOS mRNA expression was determined by qPCR using probe. Values show relative gene expression compared to vehicle with LPS evaluated by comparative ΔΔCt analysis. Each bar represents the mean ± SEM (n = 3). *** = p b 0.001, ** = p b 0.01, * = p b 0.05.

Fig. 8. Relative change of A) IL-1β (%) and B) TNF-α secretion as compared to vehicle with LPS. Each bar represents the mean ± SEM (n = 3). *** = p b 0.001, ** = p b 0.01.

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and spectral analyses of the compounds. Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/ 10.1016/j.intimp.2016.12.009. References

Fig. 9. mRNA expressions for TNF-α, COX-2 and IL-1β were determined by qPCR using probes. Values show relative gene expression compared to vehicle with LPS evaluated by comparative ΔΔCt analysis. Each bar represents the mean ± SEM (n = 3). *** = p b 0.001, ** = p b 0.01, * = p b 0.05.

for their inhibitory capacity on LPS induced NO production in RAW264.7 macrophages at 20 μM. In this screening, only compounds 5 and 6 demonstrated N50% inhibition. A detailed examination of these two lead compounds for NO inhibition resulted in a very clear dose-response relations and quite low IC50 values. Both compounds did not demonstrate cytotoxic effects at tested concentrations (max. 50 μM) in macrophage cells. Molecular docking studies performed on iNOS enzyme with all synthesized compounds provided support for the observed superiority of compounds 5 and 6 for NO inhibition over other compounds. Accordingly, results showed that rather than the position of nitro groups, their optimum number interacting with amino acid residues in the enzyme has the importance. In compounds 5 and 6, all three nitro groups are making hydrogen bonding with Arg, Asn and Gln residues and various lipophilic interactions with Hem group of the enzyme. However, besides the number of nitro groups, the orientations and polarizabilities of both 5 and 6 are also important properties making these compounds dominant over the others for binding willingness on the enzyme. Although, molecular docking studies and the results obtained from Western blot and qPCR analyses have identified the mechanism of NO inhibitory activity of these two lead compounds to some extent, further studies are warranted to elucidate their mechanism of action as therapeutic agents. Collectively, our preliminary results provide support for the hypothesis that while compound 5 has predominantly inhibitory activity on NO production, 6 possesses multi-potent anti-inflammatory activities manifested as decreased iNOS, COX-2, IL-1β and TNF-α expression and NO production. These results should be supported by in vivo validation studies in chronic and acute inflammation models for further therapeutic applications. In addition, as mentioned before the studies relating the nitro benzamide derivatives with anti-inflammatory activity are extremely scarce. In this regard, the present work also introduce a new starting point for the design and synthesis of more potent nitro substituted benzamide derivatives as potential anti-inflammatory agents. Further research studies along with this line and also with in vivo validation studies are on the way with the new projects of our research group. Acknowledgement This work was partially supported by the Scientific and Technological Research Council of Turkey in the scope of two different projects (TUBITAK, Project numbers: 110T754 and 113Z706). Studies related with bioactivity part were partially supported by Canakkale Onsekiz Mart University, Scientific Research Project (FYL-2015-434). Appendix A. Supplementary data Supplementary data associated with this article can be found in the attached files. These data are related with molecular modelling studies

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