Structure-based design, synthesis, molecular docking, and biological activities of 2-(3-benzoylphenyl) propanoic acid derivatives as dual mechanism drugs.

PURPOSE: 2-(3-benzoyl phenyl)propanohydroxamic acid (2) and 2-{3-[(hydroxyimino)(phenyl)methyl]phenyl}propanoic acid (3) were synthesized from non-steroidal anti-inflammatory drug, ketoprofen as dual-mechanism drugs.
MATERIALS AND METHODS: Structures of the synthesized compounds were established by IR, (1)H NMR, and mass spectroscopy. Both compounds were screened for their anti-inflammatory activity in rat paw edema model and in vitro antitumor activity against 60 human tumor cell lines. Flexible ligand docking studies were performed with different matrix metalloproteinases and cyclooxygenases to gain an insight into the structural preferences for their inhibition.
RESULTS: Compound (2) proved out to be more potent than ketoprofen in rat paw edema model. Both compounds showed moderate anticancer activity ranging from 1% to 23% inhibition of growth in 38 cell lines of 8 tumor subpanels at 10 μM concentration in a single dose experiment. Hydroxamic acid analogue was found to be more potent than ketoximic analogue in terms of its antitumor activity.
CONCLUSION: Analysis of docking results together with experimental findings provide a good explanation for the biological activities associated with synthesized compounds which may be fruitful in designing dual-target-directed drugs that may inhibit cyclooxygenases and MMPs for the treatment of cancer.

Accumulating evidence in the literature suggests that a drug with two or more mechanisms of action targeted at multiple etiologies of the same disease may offer more therapeutic benefit in certain disorders than a drug that targets one disease etiology only. Moreover, such multiple mechanism drugs may exhibit a more favorable side effect profile than a polypharmaceutical combination of several drugs that individually target the same disease etiologies than those identified for a single multifunctional drug.[12]

The matrix metalloproteinases (MMPs) are a family of ubiquitous enzymes that are involved in extracellular matrix degradation and remodeling. They are critical for the processes of morphogenesis and wound healing, but are also implicated in many human diseases including arthritis, metastasis, and cancer.[3-5] This family includes matrilysin (MMP-7), fibroblast collagenase (MMP-1), neutrophil collagenase (MMP-8), stromelysin-1 (MMP-3), stromelysin-2 (MMP-10), stromelysin-3 (MMP-11), gelatinase A (MMP-2), gelatinase B (MMP-9), collagenase-3 (MMP-13), and the membrane type MMP. It is currently believed that expression of these MMPs is causally linked to the invasion and progression of numerous human tumors.[6-8] MMPs have been the target of structure-based drug design since the late 1970s.[9] Many biaryl hydroxamic and carboxylic acid derivatives have attracted medicinal chemists due to anticancer properties associated with them. Among the most efficient compounds, SC-276 is effective as a single agent and in combination with paclitaxel for the treatment of breast cancer while other derivatives such as RS-130830,[10] CGS-27023A[11] and AG3340[12] are under clinical trials as potential anticancer agents acting by inhibition of MMPs [Figure 1].

Figure 1

Design of 2-(3-benzoylphenyl)propanoic acid derivatives as dual mechanism drugs based on some potential inhibitors of matrix metalloproteinases and cyclooxygenases

Numerous experimental, epidemiologic, and clinical studies suggest that nonsteroidal anti-inflammatory drugs (NSAIDs), particularly the highly selective cyclooxygenase (COX)-2 inhibitors, have promise as anticancer agents.[13-15] It is evident that COX-2 subcells locate in the endoplasm and karyotheca, the PG produced enter the nucleus regulating the transcription of target gene and play a significant role in the process of tumorigenesis in different tumors. The whole process is mediated by CUGBP-2, a cytidine-uridineguanosine binding protein-2. In cancer cells, the gene expressing CUGBP-2 is closed, resulting in the enhancement of COX-2 activity, producing the PG excessively, and promoting the gene expression associated with angiogenesis. As a result, COX-2 becomes a potential target for the prevention of tumor.[16]

In the course of identifying various chemical substances which may serve as leads for designing novel antitumor agents, 2-(3-benzoylphenyl)propanoic acid (ketoprofen) derivatives are particularly interesting due to their antiproliferative and COX-inhibiting properties.[17] In the present study, ketoprofen molecule was modified to include structural features required for MMP as well as COX inhibition [Figure 1] and evaluated for their anti-inflammatory activity in rat paw edema model and in vitro anticancer property. Flexible ligand docking studies were performed with different MMPs and COXs to enlighten their binding interactions at the target site which may be fruitful in designing dual-target-directed drugs that may inhibit cyclooxygenases and MMPs for the treatment of cancer.

Materials and Methods

Synthetic starting material, reagents and solvents were of analytical reagent grade or of the highest quality commercially available and were purchased from Aldrich Chemical Co., Merck Chemical Co. and were dried when necessary. The progress of the reactions was monitored by thin layer chromatography with F254 silica-gel precoated sheets (Merck); hydroxamic acid was visualized with FeCl3 aqueous solution. Flash chromatography or Preparative medium pressure liquid chromatography (MPLC) were carried out with glass columns containing 40-63 μm silica gel (Machinery-Nagel Silica Gel 60). The MPLCs were performed using a chromatography apparatus consisting of a Buchi 681 pump, Knauer differential refractometer detector and a Philips PM 8220 pen recorder. IR spectra were recorded, on a Jasco IR Report-100 spectrometer and wave numbers are given in cm-1. The mass spectra were recorded on Trace-GC-Trase-DSQ (Thermo Finnigan). 1H NMR spectra, in DMSO-d6 and CDCl3 solution, were recorded on a Bruker DRX-300 instrument at 298 K. Chemical shifts are reported as ppm relative to TMS as internal standard. Melting points (°C) were determined with Gallenkamp hot-stage apparatus in an open glass capillary tube and are uncorrected. Elemental analyses were performed on Elementar Vario EL III instrument.

Synthesis of 2-(3-benzoyl phenyl)propanohydroxamic acid (2)

Sodium methoxide, prepared from 3.79 g (0.16 mol) of sodium metal in 12 ml of methanol, was treated with a solution of hydroxylamine hydrochloride (7.72 g, 0.11 mol) in 5.6 ml of methanol. After 15 min of stirring, the precipitate so obtained was filtered, 14 g (0.05 mol) of the methyl 2-(3-benzoyl phenyl)propanoate in methanol (20 ml) added with stirring to the fresh filtrate, and the reaction mixture set aside at room temperature for 5 days. Then, methanol was evaporated under reduced pressure to furnish oily residue which was dissolved in a minimum amount of water, acidified cautiously with acetic acid and extracted three times with ether (20 ml). Ether was evaporated to afford compound (2) in pure form, yield: 75%, viscose oil at room temperature.

IR (neat, v cm-1 ): 1677, 1660; 1H NMR (CDCl3, δ): 9.95(br s, 1H, NH), 7.77-7.71 (m, 2H, ArH), 7.58--7.28 (m, 7H, ArH), 3.62 (q, 1H, CH, J = 6.70 Hz), 1.85 (s, 1H, OH), 1.42 (d, 3H, CH3 , J = 6.70 Hz); MS: m/z 269 (M+, 25%), 270 (M+1), 237, 236, 209 (100%), 194, 181, 165, 77; Anal. Calcd for C16H15NO3 : C, 71.36; H, 5.61; N, 5.20. Found: C, 71.32; H, 5.59; N, 5.21.

Synthesis of 2-{3-[(hydroxyimino)(phenyl)methyl]phenyl}propanoic acid (3)

To a solution of ketoprofen (5 g, 0.02 mol) and hydroxylamine hydrochloride (2.83 g, 0.04 mol) in ethanol (40 ml), sodium acetate (4.17 g, 0.05 mol) was added with stirring. After 2 h of refluxing, the mixture was filtered and filtrate was evaporated to give pure compound (3), yield: 90%, viscose oil at room temperature.

IR (neat, v cm-1): 3240, 3030, 2950, 2900, 1690; 1H NMR (CDCl3, δ): 10.2 (br s, 1H, NOH), 7.75-7.35 (m, 9H, ArH), 3.48 (q, 1H, CH, J = 5.1 Hz), 1.89 (s, 1H, OH, COOH), 1.50 (d, 3H, CH 3, J = 3.9 Hz); MS: m/z 269 (M + , 4%), 270 (M+1), 236, 224, 209 (100%), 196, 180, 77; Anal. Calcd. for C16H15NO3 : C, 71.36; H, 5.61; N, 5.20. Found: C, 71.37; H, 5.60; N, 5.18.

Anticancer activity

The human tumor cell lines of the cancer-screening panel are grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM l-glutamine. For a typical screening experiment, cells are inoculated into 96 well microtiter plates in 100 μl at plating densities ranging from 5000 to 40 000 cells/well depending on the doubling time of individual cell lines. After cell inoculation, the microtiter plates are incubated at 37°C, 5% CO2, 95% air, and 100% relative humidity for 24 h prior to addition of experimental drugs. After 24 h, two plates of each cell line are fixed in situ with TCA, to represent a measurement of the cell population for each cell line at the time of drug addition (Tz). Experimental drugs are solubilized in dimethyl sulfoxide at 400-fold the desired final maximum test concentration and stored frozen prior to use. At the time of drug addition, an aliquot of frozen concentrate is thawed and diluted to twice the desired final maximum test concentration with complete medium containing 50 μg/ml gentamicin. Additional four, 10-fold or ½ log serial dilutions are made to provide a total of five drug concentrations plus control. Aliquots of 100 μl of these different drug dilutions are added to the appropriate microtiter wells already containing 100 μl of medium, resulting in the required final drug concentrations. Following drug addition, the plates are incubated for an additional 48 h at 37°C, 5% CO2 , 95% air, and 100% relative humidity. For adherent cells, the assay is terminated by the addition of cold TCA. Cells are fixed in situ by the gentle addition of 50 μl of cold 50% (w/v) TCA (final concentration, 10% TCA) and incubated for 60 min at 4°C. The supernatant is discarded, and the plates are washed five times with tap water and air-dried. Sulforhodamine B (SRB) solution (100 μl) at 0.4 % (w/v) in 1% acetic acid is added to each well, and plates are incubated for 10 min at room temperature. After staining, unbound dye is removed by washing five times with 1% acetic acid and the plates are air-dried. Bound stain is subsequently solubilized with 10 mM trizma base, and the absorbance is read on an automated plate reader at a wavelength of 515 nm. For suspension cells, the methodology is the same except that the assay is terminated by fixing settled cells at the bottom of the wells by gently adding 50 μl of 80% TCA (final concentration, 16% TCA). Using the seven absorbance measurements [time zero, (Tz), control growth, (C), and test growth in the presence of drug at the five concentration levels (Ti)], the percentage growth is calculated at each of the drug concentrations levels. Percentage growth inhibition was calculated as:

[(Ti-Tz)/(C-Tz)] × 100 for concentrations for which Ti ≥ Tz (1)

[(Ti-Tz)/Tz] × 100 for concentrations for which Ti < Tz (2)

Anti-inflammatory activity

Animals

Adult albino rats of both sexes weighing 120–170 g were procured from National Center for Medical Research, Libya. The animals were housed and fed in a laboratory kept at constant temperature of 25 °C and Humidity (55 ± 1%) under the standard conditions (12:12h L:D cycle, standard pellet diet, tap water). Animals were randomly assigned to different experimental groups, and each experimental group consisted of seven animals. All the animals were used only once. The procedures were performed after approval from the Ethics committee of the institute and in accordance with the recommendations for the proper care and use of laboratory animals issued by National Institutes of Health, USA.

Carrageenan-induced rat paw edema

The carrageenan-induced rat paw edema model of inflammation was used to evaluate the anti-inflammatory properties of the tested compounds. Rats were randomly assigned to treatment groups and sterile carrageenan (1% carrageenan in normal saline) was injected sub-planter into right hind paw of the rat according to the method of Winter et al.[18] Synthesized compounds or ketoprofen were given orally 1hr before carrageenan injection. Physiological saline was administered by the same route, to the control group. The development of paw edema was measured using plethysmometer (paw volume meter). The volume of edema was measured at 3 h, % of edema and edema inhibition was calculated according to the formulas: Edema (%) = (N’×100)/N. Edema inhibition % = (N-N’×100)/N. N: paw diameters measured at 3 h after injection of carrageenan to the control group – paw diameters at the beginning. N′: paw diameters measured at 3 h after injection of carrageenan to the test groups – paw diameters at the beginning.

Molecular docking

Three-dimensional coordinates of MMPs and COXs structures were obtained from the Protein Data Bank. Discovery Studio Visualizer and AutoDock Tools packages were used to prepare docking files. For each PDB structural data file containing MMP/COX and inhibitor complex, the inhibitor was extracted from protein complex in PDB file. The partial atomic charges were calculated with the aid of Gasteiger method and after merging non-polar hydrogens, rotatable bonds were assigned. All the amide bonds were considered as non-rotatable. For the protein, the hetero-atoms including cofactors, water molecules and the ligand were removed. The zinc ion at the active site was retained in case of MMPs. For compounds (2) and (3), structures were drawn in ChemBioDraw Ultra 12.0 and converted to their three dimensional structures in ChemBio3D Ultra 12.0, energy miminized by PM3 method using MOPAC Ultra 2009 program, (http://OpenMOPAC.net) and saved as in pdb format. The prepared ligands were used as input files for AutoDock 4.2 in the next step. Lamarckian genetic algorithm method was employed for docking simulations.[19] The standard docking procedure was used for a rigid protein and a flexible ligand whose torsion angles were identified (for 10 independent runs per ligand). A grid of 60, 60, and 60 points in x, y, and z directions was built with a grid spacing of 0.375 Å and a distance-dependent function of the dielectric constant were used for the calculation of the energetic map. The default settings were used for all other parameters. At the end of docking, the best poses were analyzed for hydrogen bonding/π-π interactions and root mean square deviation (RMSD) calculations using Discovery Studio Visualizer 2.5 program. From the estimated free energy of ligand binding (ΔG binding, kcal/mol), the inhibition constant (Ki) for each ligand was calculated.

Results and Discussion

Chemistry

2-(3-Benzoylphenyl)propanohydroxamic acid (2) and 2-{3-[(hydroxyimino)(phenyl)methyl]phenyl}propanoic acid (3) were synthesized from the non-steroidal anti-inflammatory drug, ketoprofen as presented in Scheme 1. Ketoprofen was treated with absolute methanol under reflux in the presence of H2SO4 to give methyl 2-(3-benzoylphenyl) propionate ester (1) which on treatment with NH2OH.HCl in the presence of NaOMe in methanol afforded 2-(3-benzoyl phenyl) propanohydroxamic acid (2). The ketoprofen, when treated with NH2OH.HCl and sodium acetate in a mixture of ethanol and few drops of water under reflux, furnished 2-{3-[(hydroxyimino)(phenyl)methyl]phenyl}propanoic acid (3). The structure of the synthesized compounds was confirmed by elemental analysis and spectral data (IR,1 H NMR, and MS).

Scheme 1

Synthesis of 2-(3-benzoylphenyl)propanoic acid derivatives

The in vitro antitumor screening of the prepared compounds (2) and (3) were performed at the National Cancer Institute (NCI) Bethesda, Maryland, USA. These compounds were subjected to disease oriented human cells screening panel assay[20-23] in which about 38 cell lines of nine tumor subpanels were incubated with 10 μM of each compound and growth inhibition percentage was calculated. The results are summarized in Table 1.

Table 1

Results of the in vitro antitumor screening

Both compounds showed anticancer activity ranging from 1% to 23% inhibition of growth in 38 cell lines of 8 tumor subpanels at 10μM concentration in a single dose experiment. Compound (2) exhibited growth inhibition in 28 cell lines of 8 tumor subpanels with a maximum inhibition of 23% whereas compound (3) inhibited the growth of 31 cell lines in 8 tumor subpanels showing maximum inhibition of 15%. Growth of HS 578T, MDA-MB-468, IGROV1, OVCAR-5, CCRF-CEM, TK-10, and UO-31 was inhibited by compound (2) whereas compound (3) has not exerted any effect on the growth of these cell lines. Compound (2) was found to be inactive in A549/ATCC, HOP-92, NCI-H23, HCC-2998, BT-549, NCI/ADR-RES, K-562, ACHN, SK-MEL-2, and SK-MEL-5 cell lines while compound (3) demonstrated a pronounced anticancer activity in these cells.

The maximum growth inhibition of 23% was revealed by compound (2) whereas 15% growth inhibition was found in case of compound (3) against leukemia. Compound (2) showed 23%, 15%, 14%, 13%, 12%, 11%, 9%, and 7% effectiveness against leukemia, renal cancer, CNS cancer, ovarian cancer, non-small cell lung cancer, breast cancer, melanoma, and colon cancer, respectively. The growth inhibitory efficiency of compound 3 was found to be 15%, 14%, 13%, 10%, 8%, 8%, 7%, and 3% in leukemia, CNS cancer, renal cancer, breast cancer, non-small cell lung cancer, melanoma, colon cancer, and ovarian cancer, respectively. Both compounds were found to be equipotent in case of CNS cancer showing 14% and 1% inhibition in SNB-75 and SNB-19 cell lines, respectively.

The anti-inflammatory activity of the synthesized compounds (2) and (3) was evaluated by the carrageenan-induced paw edema method. The compounds were tested at an intraperitoneal dose of 20 mg/kg body weight and were compared with the standard drug, ketoprofen at the same i.p. dose. The hydroxamic acid derivative (compound (2)), inhibited the carrageenan-induced paw edema by 36% whereas ketoprofen exhibited 22% inhibition which justifies that conversion of carboxylic acid moiety of ketoprofen to hydroxamic acid moiety is beneficial in terms of anti-inflammatory activity [Figure 2]. It seems plausible to state that NSAIDs, when converted to their different derivatives, are better in terms of biological activity as supported by a report Marjanovic et al.,[17] where various derivatives of fenoprofen, ketoprofen, ibuprofen, indomethacin, and diclofenac were synthesized and exhibited significantly higher antiproliferative activities than their corresponding parent compound, probably due to a better cell uptake. However, when ketoprofen was converted to ketoxime derivative (compound 3), the activity declined to 13%. This is in agreement with the report of Levin et al.[24] where it is stated that hydroxamic acid analogues are more effective medicinal agents than carboxylic acid analogues.

Figure 2

Carrageenan-induced rat paw edema test (n = 7). Data is presented as %inhibition of inflammation. *P, 0.01 compared with corresponding value for control group evaluated by t-test. ** P, 0.001 compared with corresponding value for control group evaluated by t-test

To optimize the observed anti-inflammatory and anticancer activities, compounds (2) and (3) were docked into crystal structures of eight different MMPs, COX-1, and COX-2 by using AutoDock 4.2 program.[19] For each docking experiment, the lowest energy docked structure was selected from 10 runs. The binding affinity was evaluated in terms of binding free energies (ΔGb, kcal/mol), inhibition constants (Ki), hydrogen bonding, π-interactions, and RMSD values [Table 2].

Table 2

Docking results of 2-(3-benzoylphenyl) propanoic acid derivatives with MMP and COX

Validation of the accuracy and performance of AutoDock

According to the method of validation cited in literature,[25] where if the RMSD of the best docked conformation is ≤ 2.0 Å from the experimental one, the used scoring function is successful. The RMSD values of the native cocrystallized ligands after docking were ≤ 2.0 Å (data not shown), which confirms the reliability of AutoDock for docking our compounds into crystal structures of MMPs and COXs.

Docking of the synthesized compounds into MMPs

Results of the molecular docking study revealed that the compounds (2) and (3) docked into the active sites of MMP-1, -2, -3, -7, -8, -9, -12, and -13 (PDB entry: 3AYK, 1HOV, 2JT5, 1MMQ, 3DPE, 1GKC, 1Y93, and 2D1N, respectively) exhibited their binding interactions in a similar fashion to the native cocrystallized ligands of the respective enzymes [Figure 3]. All types of MMPs are multidomain enzymes, generally constituted at least by a prodomain and a catalytic domain.[26] The catalytic domain (approximately 170 amino acids) shows the same overall folding in all MMPs and contains two zinc(II) and two or three calcium(II) ions. One of the zinc ions has a catalytic function, while the other metal ions play a structural role. A deep cleft around the catalytic zinc, which includes the so-called hydrophobic S1’ pocket, delineates the active site and substrate binding of all MMPs.[2627] So, three are the main determinants of the inhibitor–protein interaction in the MMP active site: the nature of the catalytic zinc-coordinating group, the presence of inhibitor–enzyme hydrogen bonds, and the hydrophobic interactions between the inhibitor and the S1’ pocket residues.[2627]

Figure 3

Compound (2) (red) and (3) (yellow) docked into MMP-3 receptor site (PDB code: 2JT5) occupying the same position as the native cocrystallized ligand, MLC 88 (green) exhibiting RMSD of 1.21 Å and 0.43 Å, respectively.

Analysis of the docking results revealed that both compounds (2) and (3) are at par of fulfilling two of the structural requirements for their binding with MMPs. Although, the compounds were observed to interact within the catalytic domain of each MMP, neither of them exhibited the typical hydroxamate-Zinc interaction in the active site. Weak in silico interaction of the compounds (2) and (3) with the MMPs may be responsible for their moderate/poor anticancer activity and justifies that hydroxamate-Zinc interaction in the catalytic domain is essential for binding to the target site and exhibiting potent anticancer activity. Considering the binding energies, hydrophobic, hydrophilic, and calculated inhibition constants presented in Table 2, it can be concluded that compound (2) having hydroxamate moiety is better suited for interaction with different MMPs than compound (3) having ketoxime moiety [Figure 4].

Figure 4

Docking of compound (2) with MMP-8. The residues of binding pocket are shown as stick while compound (2) is presented as ball and stick style in cyan color. Dashed lines in green and orange indicate H-bonds and π-π stacking interactions, respectively. Bond distances are given in Å

Docking of the synthesized compounds into COXs

The detailed structural information about the COX-1 and COX-2 active site with high-resolution X-ray analysis from cocrystallized inhibitor enzyme complexes was obtained from protein data bank (1EQG and 1CX2 for COX-1, and COX-2, respectively). Docking studies confirm that the compounds 2 and 3 dock in a similar binding modus like ibuprofen and SC-558 [Figure 5] in COX-1 and COX-2 enzymes, respectively. Both compounds (2) and (3) interacted with COX-1 and COX-2 in a better way than their parent drug, ketoprofen in terms of binding energy, hydrophilic as well as hydrophobic interactions [Figure 6]. The docking results can be well correlated with observed anti-inflammatory activity of compound (2), which was more active than ketoprofen in rat paw edema model. However, in spite of exhibiting superior binding profile in docking experiment, compound (3) failed to enhance anti-inflammatory activity of ketoprofen in rat paw edema model.

Figure 5

Compound (2) (red) and (3) (cyan) docked into COX-2 receptor site (PDB code: 1CX2) occupying the same position as the native cocrystallized ligand, SC 558 (blue) exhibiting RMSD of 1.8 Å and 3.1 Å, respectively

Figure 6

Docking of compound (2) with COX-1. The residues of binding pocket are shown as stick in pink while compound (2) is presented as ball and stick in cyan color. Dashed lines in green indicate H-bonds. Bond distances are given in Å

In summary, ketoprofen molecule was modified to include structural features of COXs as well as MMPs inhibition with the objective of developing dual-target-directed drugs that may possess better pharmacological profile with less adverse effects. Hydroxamic acid analogue was more active than ketoximic analogue in anticancer and anti-inflammatory screening. Analysis of the docking results brought in focus the importance of hydroxamate group in holding the molecule at place (binding) at the active site by extra hydrogen bonds and justifies the experimental findings. These findings may be fruitful in designing dual mechanism drugs for the therapy of inflammation and cancer.