Synthesis, anti-inflammatory, bactericidal activities and docking studies of novel 1,2,3-triazoles derived from ibuprofen using click chemistry

Background Nonsteroidal anti-inflammatory drugs are of vast therapeutic benefit in the treatment of different types of inflammatory conditions. 1,2,3-Triazoles and their derivatives have a wide range of applications as anti-bacterial, anti-fungal, anti-tubercular, cytostatic, anti-HIV, anti-allergic, anti-neoplastic and anti-inflammatory (AI) agents. Considering the individual biological and medicinal importance of ibuprofen and 1,2,3-triazoles, we wanted to explore novel chemical entities based on ibuprofen and triazole moieties towards their biological significance. Results Click chemistry has utilized as an ideal strategy to prepare novel ibuprofen-based 1,4-disubstituted 1,2,3-triazole containing molecules. These compounds were screened for their in vivo AI activity, among all the synthesized analogues 13o was shown potent effect than the reference AI drug ibuprofen at the same concentration (10 mg/kg body weight). Compounds 13l, 13g, 13c, 13k, 13i, 13n, 13m and 13j were shown significant AI activity. These triazole analogues were also screened for their bactericidal profile. Compounds 13c, 13i, 13l and 13o exhibited considerable bactericidal activity against gram positive and gram negative strains. In addition to this, molecular docking studies were also carried out into cyclooxygenase-2 active site to predict the affinity and orientation of these novel compounds (13a–q). Conclusions In summary, we have designed and synthesized 1,2,3-triazole analogues of ibuprofen in good yields using Click chemistry approach. AI and bactericidal activities of these compounds were evaluated and shown remarkable results. Electronic supplementary material The online version of this article (doi:10.1186/s40064-016-2052-5) contains supplementary material, which is available to authorized users.


Background
The nonsteroidal anti-inflammatory drugs are extensively applied for the treatment of analgesic, antipyretic, rheumatic arthritis and in high doses these are used to treat inflammatory diseases. Prolonged oral administration of these drugs was reported for frequent adverse effects on the gastrointestinal tract (GIT) (Allison et al. 1992;Lazzaroni and Bianchi Porro 2004) and subsequently leads to obstacles such as kidney damage (Ruiz and Lowenthal 1997), gastric ulcer (Alsarra et al. 2010) and hepatotoxicity (Tan et al. 2007). This is most likely due to the presence of free carboxyl group on Nonsteroidal antiinflammatory drugs (NSAIDs) (Mishra et al. 2008). The GIT mucosal injury problems produced by NSAIDs are commonly believed to be caused by two different mechanisms. One is local irritation produced by free carboxylic acid group and inhibition of prostaglandin biosynthesis in the GIT. The second has indirect effect can be attributed to combination of an ion trapping mechanism of NSAIDs from the lumen into the mucosa. Thus, free acidic group plays a key role in keeping the effectiveness and producing the gastric ulceration as well. Hence, there remains a compelling need for effective NSAIDs with an improved safety profile and strategy for suppressing inflammation with least side effects.
It has been reported (Kalgutkar et al. 2000a(Kalgutkar et al. , b, 2002Shanbhag et al. 1992;Tozkoparan et al. 2000) that conversion of the carboxyl group containing NSAIDs to ester, amide functions and some other modifications retains the anti-inflammatory activity of the parent NSAIDs and makes them more selective towards cyclooxygenase-2 (COX-2) enzyme. These modifications were inspired us to concentrate on the carboxyl side chain of ibuprofen to design novel class of molecules. During the synthesis of these analogues, we planned to utilize Click chemistry as key reaction. Click reaction is one of the most popular reactions for the construction of triazoles. Concept of this reaction discovered by the groups of Sharpless (Rostovtsev et al. 2002) and Meldal (Tornøe et al. 2002) independently. It is a copper(I)-catalyzed 1,3-dipolar cycloaddition (CuAAC) reaction which involves alkyne and azide as key partners to deliver 1,2,3-triazoles. Click chemistry has recently emerged to become a powerful tool in drug discovery. The 1,4-disubstituted 1,2,3-triazoles obtained from CuAAC reactions are found to possess wide applications in several research fields including synthetic organic (Liu et al. 2008;Wacharasindhu et al. 2009), biological (Romeo et al. 2015) and medicinal chemistry (Liang et al. 2015). 1,2,3-Triazole is one of the key structural unit found in a wide variety of bioactive molecules tazobactam (Yang et al. 1999), cefatrizine (Dunn et al. 1976), carboxyamidotriazole (Guo et al. 2008).
In this paper, we framed out our studies to synthesize analogues of ibuprofen, a widely used drug among other clinically existing NSAIDs. Free carboxyl group in ibuprofen could be modified to obtain more potent analogues. Derivatives of ibuprofen with modified carboxylic acid functionality were shown in Fig. 1 (1-3) (Bansal et al. 2015;Yadav et al. 2006). In compound 1, carboxyl group modified as an aromatic motif. Whereas in compounds 2-3, acid functionality diminished by making different substituted aromatic amides. Interestingly, we found that some molecules having 1,2,3-triazoles (4-6, Fig. 1) (Rao et al. 2014;Haftchenary et al. 2015;Shafi et al. 2012) also showed good anti-inflammatory properties. Considering the individual biological activities, medicinal importance of ibuprofen and 1,4-disubstituted 1,2,3-triazoles, we designed novel class of small molecules (7, Fig. 2) having these two pharmacophores in single frame work through an aromatic linker. Resorcinol could be a useful moiety to link ibuprofen and 1,2,3-triazoles via C-C and C-O bonds respectively. The more active position (4th) of resorcinol was linked to carboxyl group of ibuprofen via C-C bond to obtain a novel compound which having ibuprofen as a major structural backbone and key resorcinol moiety. We utilized more reactive hydroxyl functionality, which is present at para position of the linker to make the Fig. 1 Examples of COX-2 inhibitors/anti-inflammatory molecules containing ibuprofen and triazole moieties propargyl handle. Using Click chemistry approach, this was further diversified by treating with different azides to give novel analogues that contain ibuprofen, resorcinol and 1,4-disubstituted 1,2,3-triazoles as substructures.
In recent years, the multidrug resistance of microbial pathogens has heightened the urgency to develop new antibacterial agents. Having the advantages of mono therapy of an anti-inflammatory drug with anti-microbial properties, here we studied bactericidal activity along with anti-inflammatory activity of the newly synthesized triazoles.

Chemistry
As shown in Scheme 1, 1-(2,4-dihydroxyphenyl)-2-(4isobutylphenyl)propan-1-one (10) was prepared by heating of resorcinol (9) and ibuprofen (8) in the presence of freshly fused ZnCl 2 . For O-alkylation, compound 10 was refluxed with propargyl bromide and potassium carbonate in dry acetone for 8 h. In this reaction we got para propargylated compound (11) as major product with 85 % yield. The reason for formation of para propargylated product as major may be explained on the basis of mesomeric effect and steric factors; ortho hydroxy group will have less nucleophilicity than para hydroxyl group. Major product (11) was separated and analyzed by 1 H-NMR spectroscopy, which showed characteristic singlet at δ 12.91 due to presence of chelated phenolic hydrogen of ortho hydroxyl group and singlet for two protons at δ 4.66 (O-CH 2 -), triplet for one proton at δ 2.53 (≡CH) indicates the formations of O-propargylation. With this evidence it is confirmed that the resulting major product was 1-(2-hydroxy-4-(prop-2-yn-1-yloxy)phenyl)-2-(4isobutylphenyl)propan-1-one (11).
Then we synthesized various aliphatic and aromatic azides (12a-q, Fig. 3) by utilizing literature protocols (Lee et al. 2012;Kumar et al. 2014). To prepare alkyl azides, corresponding alkyl halide was heated at 80-90 °C with NaN 3 in DMF. Aromatic azides were prepared from different substituted anilines using diazotization followed by treatment with NaN 3 .

In vivo anti-inflammatory activity
The in vivo anti-inflammatory activity of the synthesized novel triazole moiety containing molecules (13a-q) was determined at the dose of 10 mg/kg body weight using carrageenan-induced rat paw edema model (Winter et al. 1962). Anti-inflammatory activity was calculated at hourly intervals up to 6 h after injection and results were summarized in Table 1 as the mean ± SEM paw volume (mL) and the percentage anti-inflammatory activity. The paw volume differences were compared between the treated animals and the control group. Percentage inhibition was calculated as per the formula, of the paw control at time t, V t = volume of the paw of drug treated at time t. The maximum anti-inflammatory activity was obtained after 3 h which is the time required for reaching the maximum activity, soon after gradually decreased for the next 2 h.
Compounds 13a-q, showed mild to excellent antiinflammatory activities (47.00-94.01 % at 3 h, 55.47-96.35 % at 4 h and 56.87-95.62 % at 5 h). It is interesting to note that presence of electron withdrawing group or atom (NO 2 or Cl) at meta or para (C 3 or C 4 ) positions of benzyl or phenyl ring on triazole leads to significant increase in the activity. Among these triazoles (13a-q), compound 13o bearing a 4-nitrobenzyl group on the triazole moiety exhibited most potent activity 94. 01

Bactericidal activity
According to the results obtained, all the bacterial strains noticed high susceptible nature towards the compounds tested. Among the tested triazoles, compounds 13c, 13i, 13l and 13o exhibited high bactericidal activity. The minimum inhibitory concentration (MIC) of 13o against tested bacterial strains is comparable with that from standard antibiotic drug cefixime (Table 2). On the other hand, compounds 13l, 13c and 13i also produced significant MIC and minimum bactericidal concentration (MBC) values against tested human pathogenic organisms. These results indicates that electron withdrawing group or atom (NO 2 or Cl) at meta or para positions of benzyl or phenyl ring attached to triazole may increasing the bactericidal activity than aliphatic and electron donating aryl substituted triazoles. By the present investigation, it has been understood that the synthesized compounds are highly active against gram positive strain compared to gram negative strains. Among the screened bacterial strains methicillin-resistant Staphylococcus aureus (MRSA) was found most susceptible MIC (12.5) and MBC (15.1) values for compound 13o which were almost nearer to the values <13.5 and <13 of positive control.

Molecular modeling approach
The successful docking has been performed for all newly synthesized target compounds (13a-q) using genetic optimization for ligand docking (GOLD) algorithm version 2.0 (Verdonk et al. 2003). The GOLD program uses a genetic algorithm (GA) to explore the full range of the rotational flexibility of selected receptor hydrogens and ligand flexibility. The 3D crystallographic structure of COX-2 (PDB code 4PH9) (Orlando et al. 2015) was used as template selected from RCSB protein data bank (PDB) for anti-inflammatory activity. The interactions between ligand (13a-q) and receptor in the modeled complexes were investigated and observed the fitness function ability of COX-2 by all newly synthesized inhibitors. The observed Chem score and Gold fitness scores have been produced in Tables 3 and 4. Binding energies shown in Table 5 were calculated with ArgusLab (Thompson 2004) docking software. Discovery studio visualizer has been utilized to visualize the binding conformations of these analogues in the active site of 4PH9 protein and good binding orientation poses were shown in Figs. 5, 6 and 7.
In this investigation, we discovered that the synthesized triazoles which showed good Chem score and Gold score functions, also exhibited good binding energy values. Finally, molecular docking studies showed good correlation between the in vivo anti-inflammatory activity of final compounds and their binding interactions with COX-2 as well as their Gold fitness scores. Among all compounds, 13o and 13l report good affinities with COX-2 (4PH9).

Synthesis and characterization data
Melting points were determined in open capillaries. 1 H-Nuclear magnetic resonance (NMR) spectra were recorded on Bruker AV 300 and 400 MHz instruments, in CDCl 3 using TMS as an internal standard. Chemical shifts are given in (δ) ppm and coupling constants (J) are given in Hz. Combinations of the following abbreviations are used to describe NMR spectra: s = singlet; d = doublet; t = triplet; q = quartet; m = multiplet. Thin layer chromatography (TLC) was carried out on aluminium sheets coated with silica gel 60 F 254 (Merck, 1.05554) and the spots were visualized with UV light at 254 nm or alternatively by staining with aqueous basic potassium permanganate. Flash column chromatography was performed using silica gel (Merck, 60A, 100-200 mesh). Chromatographic purity by HPLC was determined by using area normalization method and the condition specified in each case: column, mobile phase, flow rate, detection wavelength, and retention times. Commercially   available reagents were used as supplied and some of them were distilled before use. All reactions were performed in oven dried glassware. All solvents were removed by evaporation under reduced pressure.
The reaction mixture was heated to 140 °C for 30 min and monitored by TLC for complete conversion of starting materials. The reaction mixture was allowed to room temperature and was poured into ice cold water (100 mL), extracted with ethyl acetate (3 × 50 mL). The combined organic layers were washed with 5 % HCl (50 mL), saturated NaHCO 3 (25 mL) and brine (2 × 25 mL). The organic layer was dried over anhydrous sodium sulphate, filtered and concentrated in vacuo. The crude product was purified by column chromatography using 100-200 mesh silica gel, eluted at 10 % ethyl acetate in pet ether to afford 1-(2,4-dihydroxy-phenyl)-2-phenyl-ethanone as light yellow liquid (2.2 g, 75 %).

Biological evaluation Animals
Wistar strain albino rats of male sex weighing 150-250 g were purchased from National Institute of Nutrition, Hyderabad, India and housed under standard environmental conditions (temperature: 24 ± 1 °C, light/dark cycle: 10/14 h). The rats were fed with standard pellet diet (Amrut laboratory animal feed, Maharashtra, India) and water ad libitum. Animals were acclimatized to laboratory conditions at least 1 week before conducting the experiments according to the guide lines of CPCSEA-New Delhi (Registration No.-915/ac/05/CPCSEA).

In vivo anti-inflammatory activity
The synthesized compounds assessed for their antiinflammatory activity using carrageenan-induced rat paw edema method. Male Wistar rats (150-250 g) were fasted with access of water at least 24 h prior to the experiments and were divided randomly into different groups (control, standard and the test groups) of five rats each. The rat paw edema was induced by subcutaneous injection of 0.1 mL of 1 % freshly prepared saline solution of carrageenan into the right hind paw of rats. The standard drug ibuprofen (10 mg/kg body weight) given orally as a positive control. The control group was administered orally with 0.9 % of 0.1 mL of saline solution only. The test groups were administered orally with the synthesized compounds at the equimolar dosage of the standard drug, 1 h before the administration of carrageenan. The paw volumes were measured using plethysmometer at interval of 1 h.

Determination of bactericidal activity
The assay was conducted to assess the bactericidal activities of synthesized compounds through microtiter plates (Lambert et al. 2001). The assay reaction mixture consisted of phosphate-buffered saline (PBS) [50 mM sodium phosphate, 150 mM NaCl (pH 7.0)], the test compound at various concentrations and the bacterial strains were prepared in sterile 96-well microtiter plates (Nunc, Inc). The wells are filled with 100 µL diluted test compounds in PBS and 50 µL of the diluted bacterial strains. The wells were incubated with gentle shaking (140 rpm) at 37 °C for various incubation periods 0 (baseline), 2, 4, 8, 12 and 24 h (time-kill studies). For positive and negative controls, a separate microtiter plate was prepared and screened for each incubation time studied (0, 2, 4, 8, 12 and 24 h). Following incubation, a 20-µL aliquot from each well was spotted at the top of a square plate containing nutrient agar medium. The plate was labelled and tapped gently to facilitate the movement of the liquid.
There were approximately 200 cells in the spotted (20-µL) sample. Plates were placed uncovered in biohood until the sample liquid dried (ca. 10 min) and incubated overnight at 37 °C. The colony forming units (CFU) for each streak were enumerated after 24 h using a colony counter. The number of CFU at each dilution of test compounds was compared with the average of positive control value to determine the percentage of bacteria killed per well. The percentage of the bacteria killed was plotted graphically, and the percentage of the test compound resulting decrease in the number of CFU (MIC/MBC) was determined.

Molecular modelling approach
In the present study, Hyperchem 8.0, Swiss Protein Data Base Viewer (SPDBV) 3.7 (Johansson et al. 2012) version, GOLD Version 2.0, ArgusLabs 4.0.1 and Discovery studio visualiser 4.1 docking programs were evaluated to determine the interactions, affinities, binding energies and selectivity's of compounds (13a-q). Ligands energy minimization was carried out by using Hyperchem. 8.0 version. The protein-ligand interactions between COX-2 (PDB code 4PH9) and target molecules (13a-q) were prepared for docking studies by adding hydrogen atoms, removing water molecules, co-crystallized inhibitors and refined by using the Deep View/SPDBV. Basic amines were protonated and acidic carboxyl groups were de-protonated prior to charge calculation. Then successful docking has been performed using GOLD 2.0. GOLD was used to evaluate Chem score and Gold fitness functions. ArgusLab 4.0.1 docking software used here to visualize the binding conformations and to calculate the binding energies of the analogues (13a-q). Discovery studio visualizer has been utilized to visualize the best binding poses of the final target analogues (13a-q) within the active site of 4PH9 protein.

Conclusions
We have designed and synthesized a number of hybrid molecules containing ibuprofen-resorcinol-triazole moieties in single molecule using Click chemistry. These synthesized analogues (13a-q) were screened for in vivo anti-inflammatory. Compounds 13l, 13g, 13c, 13k, 13i, 13n, 13m and 13j were shown significant activity. Most of the biological experimental values correlated with docking results. These molecular binding interactions of an in silico data demonstrated that 13o has more specificity towards the COX-2 binding site and could be a potent anti-inflammatory compound. These final derivatives were also evaluated for bactericidal activity. Compounds 13c, 13i, 13l and 13o exhibited good bactericidal profile. Finally, among all compounds 13o, 13c, 13i and 13l showed an interesting dual anti-inflammatory and antibacterial activity. These results gave us positive encouragement to develop further novel chemical entities towards challenging biological agents.