Synthesis and Structure Activity Relationship of Some Indole Derivatives as Potential Anti-inflammatory Agents

A series of fused pyrroles were synthesized and tested for their in vivo anti-inflammatory activity. Among 14 examined derivatives, 5 derivatives (1b–e, g and 5b), showed a promising anti-inflammatory activity equivalent to reference anti-inflammatory drugs (indomethacin and ibuprofen). A molecular docking study was conducted to interpret the biological activities of the tested compounds. The docking results were complementary with the phase of the biological survey and confirmed the biological effects.

Due to the importance of this ring system, 39 we prepared some fused o-aminocyano tetrahydrobenzo[b]pyrrole derivatives as an essential propagation step in our search for new pyrrole and pyrrolopyrimidine derivatives, [40][41][42][43] and evaluated them for the anti-inflammatory activities.In addition, a molecular docking study has been done to explain the activity of the biologically active compounds.
Radwan et al.: Synthesis and Structure Activity Relationship ...

1. Chemistry General Information for Chemicals
All melting points were uncorrected and measured using Electro-thermal IA 9100 apparatus (Shimadzu, Japan).IR spectra were recorded as potassium bromide pellets on a Perkin-Elmer 1650 spectrophotometer (USA), Faculty of Science, Cairo University, Cairo, Egypt. 1 H-NMR and 13 C-NMR spectra were performed on Jeol NMR FXQ-300 MHz and Jeol NMR FXQ-500 MHz spectrometers; chemical shifts are expressed as ppm against TMS as the internal reference (Faculty of Science, Cairo University, Cairo, Egypt).Mass spectra were recorded at 70 eV EI Ms-QP 1000 EX (Shimadzu, Japan), Faculty of Science, Cairo University, Cairo, Egypt.Microanalyses were operated using Vario, Elemental apparatus (Shimadzu, Japan), Organic Microanalysis Unit, Faculty of Science, Cairo University, Cairo, Egypt.Column Chromatography was performed on (Merck) Silica gel 60 (particle size 0.06-0.20 mm).Compounds 1a,b, 3a,b and 5a were prepared as reported in the literature. 44All new compounds yielded spectral data consistent with the proposed structures and microanalysis within ±0.4% of the theoretical values.

General methods for the preparation of 2-oxo-substituded malononitriles I and II
A mixture of 2-chloroketone (0.01 mol) and malononitrile (1 g, 0.016 mol) in CHCl 3 (50 mL), was cooled with stirring to 0-5 °C for 30 min.A cold solution of NaOH (2.5 g in 10 mL of water) was added to the mixture dropwise for 30 min.The stirring was continued for 50 min under ice and the reaction mixture then left for 72 h at room temperature.The precipitate formed was filtered off, recrystallized from appropriate solvent.
Figure 1: Pyrroles and Indoles derivatives as NSAIDs 37,38 their mechanism of action at the molecular level such as cyclooxygenase (COX Method B: A solution of I (1 g, 0.016 mol) in isopropanol (30 mL), the appropriate aromatic amine (0.01 mol) was added.The reaction mixture was refluxed for 72 h (TLC monitored), then cooled to room temperature, and the solvent was removed under reduce pressure.The residue was dissolved in methanol (10 mL), poured into crushed ice (25 g).The precipitate formed was filtered off, dried and recrystallized from appropriate solvent to give compound 1

General methods for the preparation of aryl-pyrrolo [2,3d]pyrimidin-4-ones 5 and 6
A suspension of the appropriate compound 1 or 2 (0.01 mol) in formic acid (20 mL, 85%) was refluxed for 3 h, cooled, poured onto ice-water to give a precipitate which was filtered off, dried, and recrystallized from appropriate solvent to afford 5 and 6.

Animals
Ninety adult male Sprague-Dawley rats (5 rats per group for 14 tested compounds, control (injected with 1 mL DMSO only, 2 standard drugs), weighing 120-150 g, were housed in cages in a temperature-controlled (25 ± 1 °C) environment and provided free access to pelleted food and purified drinking water ad libitum.The protocol of the study was approved by the animal ethics committee of the Faculty of Pharmacy, Helwan University on 10-01-2012.The study was conducted in accordance with the EC.DFT -IRective 86/609/EEC for animal experiments.

Assessment of Anti-inflammatory Activity
Rat paw oedema assay was carried out according to Winter et al. 45 Prepared compounds (equimolar to active dose of the reference drug), control and 2 standard drugs were dissolved in 1mL DMSO and administrated subcutaneously.One hour later, paw oedema was induced by sub -plantar injection of 0.1 mL of 1% carrageenan (Sigma-Aldrich, St. Louis, USA) into the right paw.Paw volume was measured using a water plethysmometer (Basile, Comerio, Italy).The difference between the right and left paw volu-me was measured at 1, 2, 3 and 4 h after induction of inflammation.Control group received 1 mL DMSO (as to evaluate the interference of DMSO itself in biological test) subcutaneously and carrageenan in sub-plantar region.Results were expressed as percentage inhibition of inflammation.Ibuprofen (70 mg/kg) and indomethacin (20 mg/kg) were used as the reference drugs.

Statistical Analysis
Results were expressed as the mean ± SEM, and different groups were compared using one way analysis of variance (ANOVA) followed by Tukey-Kramer test for multiple comparisons, using Graph Pad Instant (version 3.05) as the statistical software.
Calculation: equimolar doses of tested compounds were calculated in relation to these of reference drug: swel = mean difference in rat paw volume between right and left paw ± SE. % inhibition = (1 -rt/rc) × 100 [rt = swel of tested group; rc = swel of control group].

2. Molecular Docking Study MOE 2013.08 Docking
The molecular docking studies were done using MOE 2013.08 and Leadit 2.1.2.All compounds were built and saved as MOE.Rigid receptor was used as a docking protocol.Both receptor-solvent were kept as a »receptor«.Triangle matcher was used as a placement method.Two rescoring were computed, rescoring 1 was selected as London dG.Rescoring 2 was selected as affinity.The force field was used as a refinement.

Leadit 2.1.2 Docking
All compounds were built and saved as Mol2.The crystal structure of COX-2 enzyme complexes with indomethacin was downloaded from protein data bank (PDB: 4COX).The protein was loaded into Leadit 2.1.2and the receptor components were chosen by selection of chain A as the main chain when complexes with indomethacin.Binding site was defined by choosing indomethacin as the reference ligand to which all coordinates were computed.Amino acids within radius 6.5 Å were selected in the binding site.All chemical ambiguities of residues were left as default.Ligand binding was driven by enthalpy (classic Triangle matching).For scoring, all default settings were restored.Intra-ligand clashes were computed by using clash factor = 0.6.Maximum number of solutions per iteration = 200.Maximum of solution per fragmentation = 200.The base placement method was used as the docking strategy.
Attempts made to develop such compounds using the regular reactants (α-chlorocyclohexanone and/or 2-chloroindenone, with certain aromatic amines and malononitrile) were unfruitful. 60We also found that α-chlorocyclohexanone under the reaction conditions EtOH/NaOH (polar basic medium) might undergo what is called Favorskii rearrangement, 61 rearrangement of an α-halo ketone upon treatment with a base; the reaction continues through cyclopropanone intermediate formed by nucleophilic attack [EtO -].In addition to the possibility of condensation with amine to give the anil (Fig. 2b). 62,63aking the previous results into consideration, we tried to diminish the potential of such side reactions.Our plan was to prepare the novel intermediate 2-(2-oxocyclohexyl) malononitrile (I) from the reaction of 2-chlorocyclohexanone with malononitrile in a nonpolar solvent, followed by condensation with the appropriate aromatic amines.
By applying this method, we successfully set up the required tetrahydrobenzo[b]pyrroles 1 with a fair yield.Using the same conditions with 2-chloroindenone it produced 2 (Scheme 1).
3-d]pyrimidines were reported to act as potent anticancer agents, in this work, a series of novel 2-substituted-

2. Biological Results and Discussion
Fourteen of the synthesized compounds were evaluated for their anti-inflammatory activity, using a method established by Harrk et al. 31 4-benzodioxine or pyrrole nucleus are described.All the newly synthesized compounds were examined for their in vitro and in vivo anti-inflammatory activity.Several derivatives, including (S Five of the tested compounds induced significant anti-inflammatory activity, compared with that of ibuprofen and indomethacin.Compound 1b exerted significant activities compared to standard drugs at all time intervals post-carrageenan (≅ 78%, ≅ 80%, ≅ 84% and ≅ 85% inhibition at 1 st , 2 nd , 3 rd and 4 th hour interval post-carrageenan).The activity profile was the same as for standard drugs (response increasing with time).Compound 1c exerted noticeable acti-vities compared to standard drugs at the 1 st and 2 nd hour post-carrageenan (≅ 81% and ≅ 82% inhibition at 1 st and 2 nd hour interval post-carrageenan).The activity profile was the same as standard drugs (response increasing with time), yet the activity showed weak, yet significant activities, decreasing at 3 rd and 4 th hour post-carrageenan (≅ 79% and ≅ 75% inhibition at 3 rd and 4 th hour interval post-carrageenan).Compounds 1d and 5b showed a marked anti -inflammatory effect than standard drugs, from the 1 st hour to 4 th hour post-carrageenan.Compound 5b showed a moderate inhibitory action at the 4 th hour interval: 76% inhibition.Yet, compound 1d showed the unusual profile compared to standard drugs: it showed ≅ 79% inhibition at 2 nd hour post-carrageenan and ≅ 70% inhibition at 3 rd hour post-carrageenan and then decreased to 66% inhibition at 4 th hour post-carrageenan.Compound 1g exerted a moderate activity compared to standard drugs at the 3 rd and 4 th hour post-carrageenan (% inhibition ≅48 at 3 rd hour and 61% at 4 th hour post-carrageenan), it showed no activity at 1 st and 2 nd hour intervals post-carrageenan injection.Compounds 1f, 3c, 3d, 3e, 3f and 5d,e were all inactive over all tested periods, showing % inhibition < 12, 21, 25 and 38 at 1 st to 4 th hours, respectively, and were indicated as inactive in Table 1.

3. Molecular Docking Results
8][69][70] 3D-QSAR and docking studies were carried out on 23 pyrrole derivatives, to model their HIV-1 gp41 inhibitory activities.The 2D, 3D-QSAR studies were performed using CODESSA software package and comparative molecular field analysis  (CoMFA It also helps in the interpretation and explanation of the biological results.Molecular docking is one of these approaches and is used to predict the binding mode of organic compounds. 68A molecular docking study had been done using both MOE 2013.08 71 and Leadit 2.1.2software. 72,73Possible binding modes of the active compounds inside the active site of COX-2 were estimated.Indomethacin and ibuprofen were also docked.The presence of a carboxylic group in both indomethacin and ibuprofen was important for the carboxylate anion to form an electrostatic interaction with the cationic guanidine moiety of Arg 120 residue found in the active site of the COX-2 enzyme.The oxygen atom of carbonyl group found in this carboxylic moiety participated with the formation of a hydrogen bond with the hydrogen atom of -OH group of Tyr 355 residue.The binding affinity of indomethacin and ibuprofen was found to be -30.24kcal/mol and -19.09 kcal/mol, respectively (Fig. 3a).
The clash score was computed with Leadit 2.1.2software indicating that both drugs have a low clash score (Table 2).
The presence of the p-chloro group in the derivative 1c with higher lipophilic contribution value (-10.90)improving the binding modes and interactions, compared with other tested compounds.All compounds with 2-amino-(substituted)-1H-indole-3-carbonitrile shared a hydrogen bond formed between their nitrile groups and the -OH group of Tyr 355.Compound 1g showed a mode of binding in which three hydrogen bonds with Tyr 355, Arg 120 and Ser 530 were formed.Finally, compound 5b showed three hydrogen bonds as well with His 90, Tyr 355 and Ser 530 (Fig. 3b).
Compounds 1d and 1g had the highest clash penalty score (11.54)

Structure-activity Relationships (SAR)
To investigate SAR of any NSAIDs, pathophysiology of inflammation and its treatment have to be well understood.Inflammation represents the response to injury. 74Many processes are involved in the promotion of the inflammatory process, one of them is the secretion of arachidonic acid (AA) from damaged cells membranes.AA is metabolized by enzyme COX into prostanoids (as prostaglandins) and lipoxygenase (leukotrienes). 75The major mechanism of action of NSAIDs was found to be the inhibition of PG synthesis, through inhibition of COXs, that is to say preventing the AA from forming PG. [76][77][78] it has a very short half-live in blood, its oxidization to 15-ketoprostaglandins is catalyzed by 15-hydroxyprostaglandin dehydrogenase (15-PGDH In order to design any structure with pyrrole moiety or its fused form indole, vital considerations must be taken to ensure its anti-inflammatory activity. 32,74,79,80irst, the structure should consist of an acidic moiety (carboxylic acid, enols, ester etc.) attached to a planar, aromatic functional group (appears to correlate with the double bond of AA), 31 4-benzodioxine or pyrrole nucleus are described.All the newly synthesized compounds were examined for their in vitro and in vivo anti-inflammatory activity.Several derivatives, including (S and a polar linking group (which attaches the aromatic ring to a lipophilic group in AA). 30 Addition of a second hydrophobic ring, not coplanar with the original aromatic ring, was found to enhance activity, 35 this second heteroaromatic ring or heterocyclic ring was believed to provide the necessary geometry to attach to AA. 81 Taking indomethacin (benzo[b] pyrrole) as an example, it was found that N-benzoyl moiety seems to play an important role for the COX-1 activity of indomethacin. 32][84] Taking all this into consideration, and to analyze our SAR, two structural components were considered: the nature of the heterocycle nucleus and the character of the side chain (N-substitution).First, the influence of the nature of the aromatic heterocyclic system: fused pyrrole 1a, 1c, and 1d showed the highest activity over fused pyrrolopyrimidine 5b.Regarding the side chain type, addition of bulky heterocyclic ring (anti-pyrine) in compound 1g causes the activity to decrease over the substituted phenyl in compounds 1a, 1c, and 1d.Also methoxy group at para-position in compound 1a has conferred significantly higher activity during all time intervals than the methyl goup at ortho-position in compound 1d.Replacing the antipyrine moiety in compound 1g with the halogen group at para-position in compound 1c has conferred significantly higher activity during 1 st and 2 nd hour time intervals, with decreased activity in 3 rd and 4 th hour post-carrageenan.

Conclusion
A new strategy was developed to prepare a series of 2-amino-1-(aryl)-4,5,6,7-tetrahydro-1H-indole-3-carbonitriles 1a-g and 2-amino-1-(aryl)-4,5,6,7-tetrahydro-1H -indole-3-carbonitriles 2a,b as potential anti-inflammatory agents.Based on their structure, we can conclude that the best aromatic nucleus was the pyrrole with a para substituted phenyl and cyclization to prepare pyrrolopyrimidine derivatives, added some anti-inflammatory activity in the heterocyclic system.The molecular docking study provided the interpretation of the biological activities of the active compounds compared to the two reference drugs indomethacin and ibuprofen.

Scheme 1 :Scheme 2 :
Scheme 1: Synthesis of tetrahydroindoles 1a-g and 2a Figure 3a.Binding modes of both A) indomethacin and B) ibuprofen.This was computed with Leadit 2.1.2
et al.: Synthesis and Structure Activity Relationship ...

Table 1 .
In vivo anti-inflammatory activity results for active compounds.
which affected their fitting in the binding site and resulted in the lowest affinity values in both MOE 2013.08 and Leadit 2.1.2docking results.That could expla-

Table 2 .
The clash score for active compounds and standard drugs using Leadit 2.1.2software.