Design and Synthesis of 2-Pyridone Based ... - ACS Publications

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Design and Synthesis of 2‑Pyridone Based Flexible Dimers and Their Conformational Study through X‑ray Diffraction and Density Functional Theory: Perspective of Cyclooxygenase‑2 Inhibition Sunil K. Rai, Shaziya Khanam, Ranjana S. Khanna, and Ashish K. Tewari* Department of Chemistry (Center of Advanced Study), Faculty of Science, Banaras Hindu University, Varanasi 221005, India S Supporting Information *

ABSTRACT: This paper describes the results of X-ray crystallography of 4-methyl-2-oxo-6-phenyl-1,2-dihydropyridine-3-carbonitrile (1) and its propylene bridged dimers 2 and 3. Influence of inter- and intramolecular interactions on the conformation of propylene linker have been studied through single crystal X-ray crystallography and density functional theory studies. Hirshfeld surface analysis has been employed for the study of intermolecular interactions. However, differential scanning calorimetry analysis of compounds 2 and 3, and thermogravimetric analysis of compound 3 has been performed to determine the thermal stability. Along with molecular packing and thermal analysis, molecular docking has also been performed in the catalytic site of cyclooxygenase-2 to identify the potential anti-inflammatory activity of dimer 2 and 3. The above results suggest that the supramolecular aggregate structures which are formed in solution are of lowest energy. However, cyclooxygenase-2 active site prefers the higher energy conformers.

1. INTRODUCTION 2-Pyridones are found in a wide range of compounds including natural products and medicines having a broad spectrum of biological activity, such as vasodilatory, antimalarial, antiasthma, antiepilepsy, antidiabetic, antimicrobial, antioxidant, and antiviral activity, etc.1−4 Recently, their 2-hydroxypyridine dimers have been reported as active anti-inflammatory agents.5,6 These flexible molecules are highly selective for the COX-2 active site, due to their resemblance with the celecoxib. This remarkable property of flexible dimers has intrigued us leading to the design and development of such types of molecules. The rational design and synthesis of their solid-state architectures are of current interest in the field of supramolecular chemistry and crystal engineering.7−11 Therefore, the study of conventional hydrogen bonding and noncovalent interactions (i.e., C−H···N, C−H···O, C−H···π, and π···π) in their crystal structures is important.12−16 Under the influence of inter- and intramolecular interactions, these flexible models adjust their low energy conformation in solid state, and their conformation is significantly controlled by intramolecular interactions rather than intermolecular interactions.17−21 Sometimes solvent−solute interactions show the significant role in driving flexible molecules into compact conformation and crystallization.22−28 These noncovalent interactions with solvent molecules in crystal packing play a pivotal role in the self-assembly of host molecules.29,30 The formation of well ordered supramolecular assemblies of host molecules guided by guest molecules has been utilized in the design of nanotubes, nanofibrils, and nanospheres.29,31,32 © XXXX American Chemical Society

This report deals with the crystal structure of 4-methyl-2oxo-6-phenyl-1,2-dihydropyridine-3-carbonitrile (1) and its propylene bridged O/O (2) and N/N (3) dimers. The dimers 2 and 3 are free from strong hydrogen bonding sites and have enough flexibility to adjust the almost stable conformation during crystallization. According to the Leonard, core moieties in 2 and 3 are close enough to show the intramolecular noncovalent interactions.33,34 The single crystal X-ray analysis and density functional theory of these molecules will offer insight that may help to predict the conformation of flexible molecules in crystal packing under the influence of weak intraand intermolecular interaction. Additionally, we have shown the advantage of flexibility in the catalytic site of cyclooxygenase-2.

2. EXPERIMENTAL SECTION 2.1. General Methods. All reactions were performed in ordinary conditions at ambient temperature, and reagents were used without further purification. 1H and 13C NMR spectra were recorded on JEOL AL300 FT-NMR spectrometer (300 MHz for 1H and 75 MHz for 13 C). TMS was used as internal reference, and chemical shift values were expressed in δ ppm units. Differential scanning calorimetry (DSC) analysis was performed on a Mettler Toledo TC 15 TA under N2 purge from 30 to 250 °C at a heating rate of 10 °C min−1. Fourier transform infrared (FT-IR) spectra of the synthesized compounds in a KBr pellet were recorded using a Varian 3100 FT-IR Excalibur spectrophotometer. Microanalysis was conducted on a PerkinElmer PE 2400 CHN elemental analyzer for dried samples. Received: December 9, 2014 Revised: January 19, 2015

A

DOI: 10.1021/cg501793v Cryst. Growth Des. XXXX, XXX, XXX−XXX

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Crystal Growth & Design

Figure 1. ORTEP diagram of compound 1 (a), 2 (b), and 3⊃benzene, (c) at 50% probability label. 2.2. Synthesis. 2,2′-{Propane-1,3-diylbis(oxy)}bis(4-methyl-6phenylnicotinonitrile) (2). In 100 mL round-bottom flask, compound 1 (2 g, 9.51 mmol) was dissolved in dry DMF (10 mL), then anhydrous K2CO3 (1.31 g, 9.51 mmol) was added and the reaction mixture was stirred for 15 min. 1,3-Dibromopropane (0.48 mL, 4.76 mmol) was added to the mixture and it was left to stir for 24 h. Completion of the reaction was checked with thin layer chromatography (TLC). The desired compound was obtained by column chromatography by using ethyl acetate/hexane (10/90) mixture as eluent. Mp. 174−175 °C, yield 1.56 g (69%); IR (νmax/cm−1) 3067.97, 2958.47, 2923.20, 2220.57, 1595.89, 1581.52, 1563.60, 1461.23, 1441.92, 1405.61, 1362.14; 1H NMR (300 MHz, CDCl3 298 K, TMS) δH 7.98 (d, 4H, J = 3.6 Hz, o-Ar-H), 7.48−7.44 (m, 6H, m- and p-Ar-H), 7.21 (s, 2H, pyridine ring-H), 4.79 (t, 4H, J = 6, −O-CH2-), 2.48−2.39 (m, 6H for -CH3 + 2H for -CH2-); 13C NMR (75 MHz, CDCl3 298 K, TMS) 163.7, 157.2, 154.7, 136.9, 130.0, 128.6, 127.0, 114.7, 113.8, 95.0, 63.2, 28.5, 20.2; MS m/z 461 (M + 1). Elemental analysis for C29H24N4O2. Calcd: C, 75.63; H, 5.25; N, 12.17. Found: C, 75.60; H, 5.25; N, 12.20. 1,1′-(Propane-1,3-diyl)bis(4-methyl-2-oxo-6-phenyl-1,2-dihydropyridine-3-carbonitrile) (3). In 100 mL round-bottom flask, ethanol was taken (5 mL) and cooled at 0−5 °C. Ethyl cyanoacetate (1 mL, 9.37 mmol) and 1,3-diaminopropane (0.40 mL, 4.69 mmol) were added one by one to the round-bottom flask and kept in ice bath for 2 h. Then aqueous K2CO3 (1.30 g, 9.37 mmol) was added to the previous reaction mixture and stirred for 15 min. In the resulting reaction mixture, benzoylacetone (1.50 g, 9.37 mmol) was added and stirring was continued for further 24 h. Completion of the reaction was checked with TLC. Desired compound was obtained by column chromatography by using ethyl acetate/hexane (40/60) mixture as eluent. Mp. 229−231 °C: yield 1.8 g (83%); IR (νmax/cm−1) 3077.72, 2966.50, 2924.61, 2220.20, 1664.07, 1646.76, 1603.72, 1589.78, 1544.34, 1491.93, 1449.70, 1414.19, 1376.15; 1H NMR (300 MHz, CDCl3 298 K, TMS) δH 7.50−7.48 (m, 6H, m- and p-Ar-H), 7.25 (d, 4H, J = 6.3 Hz, o-Ar-H), 6.00 (s, 2H, pyridone ring-H), 3.74 (t, 4H, J = 7.1, −N-CH2-), 2.41 (s, 6H, -CH3), 1.84 (quint., 2H J = 7.1, -CH2-); 13 C NMR (75 MHz, CDCl3 298 K, TMS) 160.3, 157.9, 153.1, 133.6, 130.1, 128.9, 127.8, 155.2, 110.4, 103.0, 43.6, 27.4, 20.9; FAB MS m/z 461 (M + 1). Elemental analysis for C29H24N4O2. Calcd: C, 75.63; H, 5.25; N, 12.17. Found: C, 75.61; H, 5.24; N, 12.18. 2.3. Crystallization Experiments. Compound 1. In a minimum amount of dry DMF, compound 1 was dissolved by heating and was kept at room temperature for slow evaporation of the solvent. After 2 months, transparent platelike crystals were formed. Compound 2. Crystals of compound 2 were obtained easily in polar solvents such as dichloromethane, chloroform, ethyl acetate, methanol, ethanol, and acetonitrile. Its crystallization in aromatic solvents like benzene, toluene, xylene, chlorobenzene, and mesitylene was failed. Crystals obtained from ethyl acetate and methanol showed the same space group and cell dimensions. Compound 3. In contrast to the compound 2, compound 3 crystallized only in benzene and formed solvates. Crystallization was

also tried with toluene, xylene, chlorobenzene, and mesitylene. Crystallization with a minimum amount of toluene was unsuccessful, but some fine shining fibers were present with white powders. While crystals were obtained in excess of toluene in 7−10 days, single crystal X-ray diffraction showed the benzene solvates. Similarly, compound 3 formed benzene solvates when crystallized in xylene, chlorobenzene, and mesitylene having 2−5% benzene as residual impurity. We did not find any crystals with ethyl acetate, chloroform, dichloromethane, acetonitrile, methanol, and ethanol. 2.4. X-ray Crystallography. Single-crystal X-ray data for compounds 1, 2, and 3 was collected with an Oxford Diffraction Xcalibur CCD diffractometer using graphite monochromated Mo Kα radiation (λ = 0.71073 Å). The structures were determined by direct methods using SHELXS-97 and refined on F2 by a full-matrix leastsquares technique.35 Non-hydrogen atoms were refined anisotropically, and hydrogen atoms were geometrically fixed with thermal parameters equivalent to 1.2 times that of the atom to which they are bonded. Molecular diagrams (Figure 1) for all compounds were prepared using ORTEP, and the packing diagrams were generated using Mercurry version 3.1.36 PLATON was used for the analysis of bond lengths, bond angles, and other geometrical parameters. Crystallographic details of compounds 1, 2, and 3 have been summarized in Table 1. 2.5. Hirshfeld Surface Analysis. The Hirshfeld surface emerged from an attempt to define the space occupied by a molecule in a crystal for the purpose of partitioning the crystal electron density into molecular fragments. Graphical tools based on the Hirshfeld surface and the associated two-dimensional (2D) fingerprint plot offered considerable promise for exploring packing modes and intermolecular interactions in molecular crystals.37 Calculations were performed using the Crystal Explorer package. 2.6. Theoretical Study. In order to investigate the conformational stability and host−guest interaction in gaseous state, single point and optimized energies have been calculated using the DFT-D method equipped in Gaussian 09.38

3. RESULTS AND DISCUSSION 3.1. X-ray Crystallographic Evidence. Single crystal Xray structure of compound 1 (Figure 1a) reveals that tautomeric hydrogen is very close to the ring nitrogen rather than oxygen. The literature has sufficient data to prove the existence of 2-pyridone in polar solvents and 2-hydroxypyridine in nonpolar solvents.39−41 Nucleophilic substitution by compound 1 on 1,3-dibromopropane in K2CO3/DMF at room temperature yields O/O linked dimer (i.e., compound 2) as the major product and N/O in trace amount. Nitrogen linked dimer (compound 3) was prepared by condensation of 1,3-diaminopropane with ethyl cyanoacetate followed by cyclization of benzoyl acetone (Scheme 1). Here, we will discuss about the crystal packing of 1 and its packing will be B


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assist the linear chain formation as well as interlayer connectivity (Figure 2d,e). However, interlayer connectivity is further assisted by C−H···π and π···π interactions. The C−H··· O bond distances for trifurcated hydrogen bonding are 2.55 and 2.59 Å, and angles on hydrogen atoms are 139.8° and 128.7°, while the N−H···O bond distance and angle on hydrogen atom is 18.2 Å and 172.7°, respectively (Figure 2f). Crystal packing of compound 2 has been shown in Figure 3a. Packing is stabilized by alternate π···π and C−H···π layers. Moreover, succeeding π···π layers are orthogonal to each other, but C−H···π layers are in a similar fashion. If we assume first π···π layer as A, CH···π layer as B, and orthogonal π···π layer as C, then the arrangement of layers are like ABCB−ABCB... (Figure 3b). In comparison to compound 1, compound 2 exhibits stronger intermolecular π···π (3.70 Å) interaction which is resulted due to coplanarity of the pyridine ring and phenyl ring. Distinctly, coplanarity also strengthens the C− H···π interactions and becomes 3.24 Å rather than 3.43 and 3.74 Å in compound 1. The orthogonal layers A and C are distinguishable due to ag-conformation (anti-gauch conformation) on the propylene linker. In crystal packing, one pyridine moiety of each dimer shows reverse face to face intermolecular π···π stacking, while other moiety shows intermolecular C− H···π interaction (Figure 3c and d). Another stabilizing interaction in crystal packing is C−H···N, which assemble the molecules in centrosymmetric dimers via 18 and 28 membered rings defined by graph-set notation as R22(18) and R22(28), respectively (Figure 3e and f). Crystals packing of compound 3⊃benzene (Figure 4a) shows that benzene is trapped in the cavity, formed by the host molecules. CPK model (Figure 4b) shows the bowl shape geometry, and in between two bowls benzene is occupied. Further, the host−guest interactions reveal that benzene molecules reinforce the packing by raising the number of C− H···π interactions as shown in Figure 4c. The cavity formed inside the crystal lattice, which can be seen after removal of benzene (Figure 4e), reveals that arrangement of host molecules forms two-dimensional zigzag chain, and these chains are stabilized by host−host and host−guest interactions. Further, these two-dimensional layers are forming threedimensional network by interchain C−H···O interactions (Figure 4d)

Table 1. Crystallographic Parameters of Compound 1, 2 and 3⊃benzene crystal data emp formula formula wt CCDC no. crystal system space group T (K) a (Å) b (Å) c (Å) α (deg) β (deg) γ (deg) Z volume (Å3) Dcalc (g cm−3) μ (mm−1) R-factor (%) Rint theta range (deg) indep reflns measured reflections GOF on F2

compound 1

compound 2

compound 3⊃ benzene

C13H10N2O 210.23 1034535 monoclinic P21/n 298 12.88 (17) 7.23 (8) 12.95 (17) 90 119.16 (18) 90 4 1054.87 1.324 0.09 5.21 0.018 29.1−3.3 2382 4386

C29H24N4O2 460.53 1034536 monoclinic P21/c 298 15.26 (12) 11.69 (9) 13.81 (10) 90 102.42 (7) 90 4 2408.37 1.270 0.08 5.29 0.020 29.1−3.0 5460 11038

C29H24N4O2⊃C6H6 538.63 1034534 monoclinic P2/c 298 19.18 (3) 7.94 (11) 20.32 (2) 90 107.14 (13) 90 4 2957.78 1.210 0.08 6.19 0.042 28.9−3.5 3350 6983

1.02

1.02

0.99

compared with 2 and 3. Packing variation between isomeric compounds 2 and 3 will also be compared. In the crystal structure of compound 1, two molecules are arranged in close proximity, so that intermolecular hydrogen bonds of the N−H···O (1.82 Å) type are formed between the two neighboring moieties, which seems to be typical of 2pyridones with D22(8) graph-set notation. Acute angle (dihedral angle) between plane of pyridone ring and phenyl ring is 24.23°, which are sufficient enough to show the intermolecular parallel stacking. The stacking distance for π···π is 3.70 and 3.82 Å, while for C−H···π is 3.53 and 3.74 Å (Figure 2b). Apart from these interactions, trifurcated (four center) hydrogen bonding at oxygen atom is also observed, i.e., two C−H···O and one N−H···O (Figure 2e,f). This trifurcated hydrogen bonding Scheme 1. Synthesis of Compounds 1, 2 and 3

C


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Figure 2. (a) Crystal packing of compound 1 viewed along the c-axes, (b) C−H···π and π···π interactions between two moleules, (c) dimer formation due to intermolecular hydrogen bonding, (d) 2D layer showing intermolecular hydrogen bonding and π···π stacking between two layers. (e) Trifurcated intermolecular hydrogen bonding and (f) trifurcated hydrogen bonding showing C−H···O (2.55 and 2.59) and N−H···O (1.82) interactions and C−H···O (128.7 and 139.8) and N−H···O (172.7) angles. All distances have been shown in angstroms and angles in degrees.

Figure 3. (a) Crystal packing of compound 2 viewed along the a-axes, (b) alternate layers of π···π and C−H···π interactions, (c) highlighted region showing distances of intermolecular C−H···π interactions, (d) highlighted region showing distances of intermolecular π···π interactions, (e) and (f) showing centrosymmetric dimers via 18- and 28-membered rings defined by graph-set notation as R22(18) and R22(28) respectively.

D


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Figure 4. (a) Crystal packing of compound 3⊃benzene viewed along the c-axes, (b) encapsulation of benzene between two molecules, (c) representation of weak interactions showing distances and angles, (d) showing chain formation through intermolecular CH···O interactions which is defined by graph-set notation as C11(9) (e) supramolecular 2D network showing cavities after removal of benzene and pink color highlighted region showing that alternate cavities are antiparallel to each other and light blue color region showing the inter layer interactions.

3.2. Hirshfeld Surface Analysis. Hirshfeld surface analysis provides the adoption of a “whole of structure” view of intermolecular interactions, rather than concentrating exclusively on assumed “important” (i.e., short) interactions.37 It has encouraged a new way of exploring packing modes and intermolecular interactions in molecular crystals. The 2D fingerprint plots obtained from “Hirshfeld surface analysis” provide a summary of the frequency of each combination of de and di across the surface of molecule and so indicate not only which interactions are present, but also the relative area of the surface corresponding to each interaction. The 2D fingerprint plots and decompose fingerprints are shown in Table S1 in Supporting Information, and percentage contributions of N··· H, O···H, H···H, C···C, and C···H interactions in fingerprints are shown in bar graph (Figure 5). Among all noncovalent interactions, H···O (C−H···O and N−H···O) is stronger (≥1.88 Å) in compound 1, as the fingerprint shows a pair of long sharp spikes characteristic of a strong hydrogen bond (i.e., N−H···O). However, the red area in the fingerprint shows the significant contribution made by the π···π stacking interaction that is dominant over all interactions and frequency is high between di + de = 3.6−3.8 Å, whereas absence of wings in the fingerprint indicates the lack of C−H···π interactions. In compound 2, the disappearance of long sharp spikes indicates the absence of strong H···O (i.e., N−H···O) interactions. Yet, weaker H···O (i.e., C−H···O) interactions persist within the range of 3.6−4.0 Å. However, the appearance of long sharp

spikes indicates the strengthening of weak N···H (i.e., C−H··· N) interactions which ranges from 2.0 to 3.2 Å. The decompose fingerprint of C···C interaction shows a small sharp spike, indicates the strengthening of π···π stacking interaction. No doubt, frequency of π···π interaction is less in compound 2 than 1, which is confirmed by the color intensity of the C···C region. Furthermore, appearance of wings in the fingerprint indicates the presence of C−H···π interactions. The fingerprint plot of compound 3⊃benzene is devoid of sharp spikes for N···H and O···H both. This indicates that only weaker interaction for C−H···O and C−H···N are present. Nevertheless, appearance of prominent wings in the fingerprint indicates the abundance of C−H···π interactions. In contrast to compounds 1 and 2, compound 3⊃benzene shows moderate intensity for C···C interaction, which indicates the absence of absolute π···π stacking. To check the extent of crystallinity of compound 3, the X-ray powder diffraction (XRPD) experiment was performed on the bulk material. Comparison of the powder pattern of compound 3 and its solvate (Figure 6) reveals that the intensity of sharp peaks is higher in benzene solvate than that of pure compound. However, the lack of peak broadening in XRPD lines for the bulk material of pure compound imply that the amorphous content is much less than its crystalline content. Further, the crystalline nature of the pure compound was supported by sharp melting endotherm observed in DSC (Figure 7a). E


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Figure 5. Three molecular representations of compound 3⊃benzene (i.e., 3A, 3B, and 3C) have been shown (above) over which Hirshfeld surfaces have been generated and percentage contributions in fingerprint in compounds 1, 2, and 3⊃benzene (3A, 3B, and 3C) are shown in bar graph (below).

Figure 6. XRPD profile of compound 3 (left) and compound 3⊃benzene (right).

3⊃benzene shows an endothermic peak at 148.21 °C indicating the loss of benzene from the lattice. This has been further supported by TGA (Figure 7). 3.4. Computational Studies. To probe the conformational stability of compounds 2 and 3, relaxed potential energy surface scans were performed at the B3LYP/6-31G** level by partial geometry optimizations in redundant internal coordinates subject to change of dihedral angles (Φ which is indicated by bold line in Figure 8). No symmetry was assumed in potential energy scans. Scanning was started from Φ = 0° to 360° by taking 3° intervals (i.e., 120 stapes) and total 121 scan steps. Energy of the conformation obtained in global minima of both molecules and host−guest interaction in compound 3⊃benzene was refined using the DFT-D method (the B-LYP function with the TZVP basis set and Grimme’s dispersion (D3) correction included). This method is known to describe

3.3. DSC and TGA Analysis. Differential scanning calorimetry (DSC) is frequently a preferred thermal analytical technique because of its ability to provide detailed information about both the physical and energetic properties of a substance, especially for purity of the component and phase changes upon heating.42 DSC analysis of compound 2 revealed that it is pure as well as highly crystalline and stable at high temperature (Figure S7 in Supporting Information). Lack of a phase change (except the melting endotherm) and crystal to crystal transformation signal during the heating process indicates that the solid state structure of compound 2 is the most stable conformation at room temperature as well as at elevated temperature. However, compound 3 and its benzene solvate show melting with decomposition. Sharp melting endotherm at 229.31 (compound 3) and 231.07 (compound 3⊃benzene) indicates that compounds are of crystalline nature. Compound F


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Figure 7. DSC and TGA graph of compound 3 (a) and compound 3⊃benzene (b).

different types of noncovalent interactions reasonably well.43,44 The geometry obtained in global minima of potential energy surface scan-2 of compound 2 closely resembles the crystal structure. In this geometry propylene linker showed agconformation (2c2 in Figure 8), although the geometry obtained in the global minima of scan-1 is of lowest energy and the propylene linker adopted the gg-conformation (2d1 in Figure 8). Since the favored conformation of oxygen terminated alkyl chains are prone to adopt the gauche conformation,45 it was expected that compound 2 would adopt the geometry as in 2d1 in the solid state. The deviation from its natural geometry (i.e., gg-conformation) of propylene linker in crystal packing was induced by the intramolecular C−H···N interaction that is formed between nitrogen atom of the nitrile group and mhydrogen of the phenyl ring. The unexpected intramolecular 16-membered ring formation in the crystal lattice was supposed to be due to the molecular strain and was expected to vanish in an optimized structure. On the contrary, calculations revealed that the C(17)H···N(2) distance in the gaseous state decreased to 2.81 Å, while C(27)H···O(1) remained the status quo (Table 2). This indicates that the ag-conformation (it is stable conformer but has little higher energy than gg-conformer in gaseous state) preferred by compound 2 in crystal packing is devoid of any strain. However, potential energy surface scans of compound 3 showed four minima, among them three were of nearly the same energy. Conformer 3a was of highest energy, while 3b and 3b′ were of nearly same energy but acquire a little higher energy than 3c. Among them conformation of 3b and 3b′ were very close to that of crystal structure, while the structure of 3c showed the folded conformation. The interactions which stabilized the gaseous state folded conformation 3c are C(1)−H···O(1), C(12)−H···N(2), and π···π (pyridone centroids), and the propylene linker adopted the gg-conformation, whereas the solid state solvated structure showed centrosymmetry with aa-conformation. The bowl shape geometry of compound 3 was not only stabilized by intramolecular C(1)−H···O(1) interaction but also intermolecular C(15)−H···π (benzene centroid), C(16)−H···π (nitrile centroid), and C(17)−H···π (pyridone centroid), interactions. The host−guest complex optimized in the orientation as shown in Figure S8 showed the C−H···π distances between 3.74 and 3.87 Å, while in the crystal structure these distances were

between 3.04 and 3.73 Å (Figure S8c) (see Table S2 in Supporting Information for single point and optimized energy). 3.5. Cox-2 In Silico Studies of Compounds 2 and 3. Prior to the simulations, all bound ligands, cofactors, and water molecules were removed from the proteins. The macromolecule was checked for polar hydrogen, and torsion bonds of the inhibitors were selected and defined. Gasteiger charges were computed, and the Auto Dock atom types were defined using Auto Dock version 4.2, the graphical user interface of Auto Dock supplied by MGL Tools.46 The Lamarckian genetic algorithm (LGA), which is considered one of the best docking methods available in Auto Dock, was employed.47,48 This algorithm yields superior docking performance compared to simulated annealing or the simple genetic algorithm and the other search algorithms available in Auto Dock version 4.0. Then, the three-dimensional grid boxes were created by the Auto Grid algorithm to evaluate the binding energies on the macromolecule coordinates. The grid maps representing the intact ligand in the actual docking target site were calculated with Auto Grid (part of the Auto Dock package). Eventually, cubic grids encompassed the binding site where the intact ligand was embedded. Finally, Auto Dock was used to calculate the binding free energy of a given inhibitor conformation in the macromolecular structure, while the probable structural inaccuracies were ignored in the calculations. The search was extended over the whole receptor protein used as blind docking. Molecular docking of compounds was conducted in the COX-2 crystal structure of Protein Data Bank (PDB) entry 3LN1 (2.9 Å resolution). Celecoxib as COX-2 selective drugs (native ligand) in the crystal structure was also docked as references. Table 3 shows the docking scores of celecoxib, compounds 2 and 3 within the active sites of COX-2 (Figure 9). From the structure−activity relationship (SAR) perspective, straight comparison of inhibitory potency and selectivity index profiles of both the compounds revealed that compound 3 shows weaker binding affinity with COX-2 in comparison to that of celecoxib, while compound 2 binds strongly, well inside the pocket of COX-2. No doubt conformations of both the molecules inside the COX-2 pocket are of higher energy when compared to the single crystal structure. Docking simulation revealed that compound 2 possessed eight torsional angles, while six torsional angles were possessed by compound 3. G


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Figure 8. Representation of potential energy surface scan through linker dihedrals (Φ) for compound 2 (upper left) and 3 (upper right). Geometries obtained in local and global minima are shown below with their potential energy. Refined energy for 2c2, 3b, and 3b′ at the B3LYP/TZVP level are shown in bracket.

Table 2. Representation of Key Bonds and Angles in Crystal Structure and Optimized Structures of Compound 2 and Compound 3⊃benzene at the B3LYP/TZVP Level of Theory crystal structure s. no. Compound 2 (1) (2) Compound 3 (1) (2) (3)

optimized structure

D−H···A

H···A (Å)

D···A (Å)

D−H···A (deg)

H···A (Å)

D···A (Å)

D−H···A (deg)

C(17)H···N(2) C(27)H···O(1)

3.05 2.71

3.74 2.91

132.16 92.28

2.81 2.73

3.60 2.94

130.08 89.91

C(17)H···π C(16)H···π C(15)H···π

3.19 3.73 3.04

3.68 4.51 3.50

114.48 142.52 110.98

3.89 3.87 3.74

4.32 4.71 4.27

106.35 136.71 112.43

H


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binding energy. Therefore, the design of such types of flexible molecules is of interest for structural and medicinal chemists.

Table 3. Docking Scores of Celecoxib, Compounds 2 and 3 with the COX-2 Proteina s. no.

a

compound

binding energy (kcal/mol)

1.

Celecoxib

−7.80

2.

compound 2

−8.79

3.

compound 3

−7.59

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ASSOCIATED CONTENT

S Supporting Information *

interacting side chain residues

1 H and 13C NMR, cif files of compounds 1, 2 and 3, and computational details. This material is available free of charge via the Internet at http://pubs.acs.org.

Ala142, Cys32, Glu31, Gly121, Leu138, Tyr116, Tyr121 and Val141 Asn19, Cys32, Gly121, His119, Leu138, Pro140, Try116 and Val118 Ala142, Asn19, Cys32, Gly121, Pro139, Pro140, Ser34, and Tyr122

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

PDB entry 3LN1.

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge DST India Grant SR/S1/381 OC-42/2012 for its financial support of this work. The Department of Chemistry, Faculty of Science of Banaras Hindu University is acknowledged for departmental facilities. S.K.R. thanks CSIR, New Delhi, for SRF.

Because of the higher flexibility, compound 2 deformed its geometry to make close interactions with the neighboring side chain residues, while compound 3 showed relatively weaker interactions with side chain residues. The docking pose showed that Cys32 and Gly121 of COX-2 protein are present in the binding site of all three compounds (i.e., celecoxib, compounds 2 and 3). This reveals that compounds 2 and 3 bind selectively to the COX-2 active site.

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REFERENCES

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4. CONCLUSION In brief, it can be concluded that coplanarity between the pyridone ring and phenyl ring strengthens the parallel π···π stacking and hence close packing of the crystal. Consequently, the calculated crystal density of compounds 1, 2, and 3 was 1.32, 1.27, and 1.21 g/cm3, respectively. If we talk about the linker conformation, it not only depends on intramolecular interactions but also depends on various intermolecular interactions, which predominantly minimized the lattice energy. The evidence from DFT calculations revealed that ggconformation was the lowest energy conformation for compounds 2 and 3, while single crystal structure showed agconformation for compound 2 and aa-conformation for compound 3. Further, docking simulations revealed that dimers 2 and 3 experienced the lowest binding energy at the COX-2 active site, but the conformation adopted was of the high energy. Overall we have demonstrated that flexible molecule 2 which has a higher torsional angle than celecoxib shows lower

Figure 9. Docked pose of compound 2 (left) and 3 (right) within COX-2 (PDB entry 3LN1). I


Article

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