Dihydropyrazole Derivatives Containing Benzo Oxygen Heterocycle and Sulfonamide Moieties Selectively and Potently Inhibit COX-2: Design, Synthesis, and Anti-Colon Cancer Activity Evaluation

Cyclooxygenase-2 (COX-2) as a rate-limiting metabolism enzyme of arachidonic acid has been found to be implicated in tumor occurrence, angiogenesis, metastasis as well as apoptosis inhibition, regarded as an attractive therapeutic target for cancer therapy. In our research, a series of dihydropyrazole derivatives containing benzo oxygen heterocycle and sulfonamide moieties were designed as highly potent and selective COX-2 inhibitors by computer-aided drug analysis of known COX-2 inhibitors. A total of 26 compounds were synthesized and evaluated COX-2 inhibition and pharmacological efficiency both in vitro and in vivo with multi-angle of view. Among them, compound 4b exhibited most excellent anti-proliferation activities against SW620 cells with IC50 of 0.86 ± 0.02 µM than Celecoxib (IC50 = 1.29 ± 0.04 µM). The results favored our rational design intention and provides compound 4b as an effective COX-2 inhibitor available for the development of colon tumor therapeutics.


Instruction
Cyclooxygenase (COX), also known as prostaglandin oxidase reductase, is a bifunctional enzyme with cyclooxygenase and catalase activities, and plays a key rate-limiting role in the conversion of arachidonic acid into prostaglandin. According to the current research, there are three isozymes for cox-oxidase, such as COX-1, COX-2, and COX-3 [1,2]. However, three isozyme types not only have great differences in intracellular quantity and distribution area, but also in physiological functions [3]. COX-1 is a structural primary enzyme, which mainly exists in gastrointestinal tract, kidney and other parts. Its function is to promote the synthesis of physiological prostaglandin and regulate the physiological activities of normal tissue cells by protecting the digestive tract mucosa or changing the vascular tension. COX-3 is a spliced variant of COX-1 isozyme, which is only found in the brain of dogs, but not in the human body [4,5]. Cox-2, by contrast, is an induced isozyme, of which the expressions are generally quite low in normal tissue cells [6]. Only when being in cancer cells or cells are stimulated by inflammatory factors, the expression level of COX-2 can be significantly increased, which can be

Chemistry
The routes to synthesizing the novel dihydropyrazole derivatives containing benzo oxygen heterocycle and sulfonamide moieties 4a-4z are outlined in Scheme 1. According to Experimental Section, the target compounds can be obtained through the three-step reaction. All synthesized compounds structures were exhibited in Table 1, which were reported for the first time and characterized by melting test, 1 H NMR, ESI-MS and elemental analysis. The structural characterization parameters fully conform to the designed structure, and the related spectral data have been proposed in the Supplementary Materials. Meanwhile, single crystal X-ray diffraction structure analysis further identified representative compound to determine the specific type of skeleton structure. These crystal data and corresponding Cambridge Crystallographic Data Centre (CCDC) number were presented in Figure 2 and Table 2, giving a perspective view of these compounds together with the atomic labeling system.

Chemistry
The routes to synthesizing the novel dihydropyrazole derivatives containing benzo oxygen heterocycle and sulfonamide moieties 4a-4z are outlined in Scheme 1. According to Experimental Section, the target compounds can be obtained through the three-step reaction. All synthesized compounds structures were exhibited in Table 1, which were reported for the first time and characterized by melting test, 1 H NMR, ESI-MS and elemental analysis. The structural characterization parameters fully conform to the designed structure, and the related spectral data have been proposed in the Supplementary Materials. Meanwhile, single crystal X-ray diffraction structure analysis further identified representative compound to determine the specific type of skeleton structure. These crystal data and corresponding Cambridge Crystallographic Data Centre (CCDC) number were presented in Figure 2 and Table 2, giving a perspective view of these compounds together with the atomic labeling system. Scheme 1. Synthesis routes of 4a-4z. Reagents and conditions: (i) 1.5 equiv dibromomethane or 1,2dibromoethane, 0.5 equiv anhydrous potassium carbonate, DMF, 70 °C, (ii) 0.5 equiv 40% potassium hydroxide solution, ethanol, 1.0 equiv different substituted acetophenone, (iii) 0.5 acetic acid, ethanol, 1.2 equiv 4-sulfamoylphenylhydrazine hydrochloride.

In Vitro Antiproliferative Activities
All synthetic dihydropyrazole derivatives containing benzo oxygen heterocycle and sulfonamide moieties 4a-4z were evaluated for antiproliferation activities against 5 cancer cell lines (SW620, MCF-7, HeLa, A549 and HepG2) and one noncancer cell line, NCM460 by the MTT assay, in comparison with the reference Celecoxib. As shown in Table 4, the antiproliferative activities of the tested compounds were more pronounced against human colorectal carcinoma SW620 cells than the other cell lines. Compound 4b exhibited the best antiproliferative activity among the tested compounds, with IC 50 values of 0.86 ± 0.02 µM, especially against SW620 cells, compared with positive control drug Celecoxib (1.29 ± 0.04 µM). As for SW620, compounds with 1,3-dioxolane 4a-4j behaved more potent antiproliferative activities than compounds with 1,4-dioxane 4k-4z. Besides, the MTT assay was performed on a non-cancer line NCM460 to assess the cytotoxicity of the test compound to normal colon cells. The results showed that compound 4a-4z and Celecoxib had similar low cytotoxic effect on NCM460, so these series of compounds had desirable safety.

Compound 4b Induced SW620 Cell Apoptosis
To confirm that antiproliferation activities of SW620 were associated with apoptosis, SW620 cell apoptosis induced by compound 4b was determined using flow cytometry with Annexin V-FITC/PI Apoptosis Detection Kit. The outcome was shown in Figure 3, which indicated that the percentage of apoptotic cells was significantly increased in a dose-dependent manner. The percentages of cell apoptosis 7.9%, 19.9%, 45.9%, and 65.0% were responding to the concentration of compound 4b 0 µM, 2.0 µM, 4.0 µM, and 8.0 µM, respectively. a Antiproliferation activity and cytotoxicity of the synthetic compounds were measured using the MTT assay. Data displayed are the mean ± SD of three independent experiments (n = 3).

Compound 4b Induced SW620 Cell Apoptosis
To confirm that antiproliferation activities of SW620 were associated with apoptosis, SW620 cell apoptosis induced by compound 4b was determined using flow cytometry with Annexin V-FITC/PI Apoptosis Detection Kit. The outcome was shown in Figure 3, which indicated that the percentage of apoptotic cells was significantly increased in a dose-dependent manner. The percentages of cell apoptosis 7.9%, 19.9%, 45.9%, and 65.0% were responding to the concentration of compound 4b 0 µM, 2.0 µM, 4.0 µM, and 8.0 µM, respectively. The decisive factor of tumor metastasis is the cell adhesion to fibronectin and laminin. Weak cell adhesion is beneficial to tumor metastasis inhibition, so cell adhesion to fibronectin and laminin assay was used to evaluate the effects of different concentrations of compound 4b and Celecoxib on the

Compound 4b Weakened the Adhesion of SW620 Cells
The decisive factor of tumor metastasis is the cell adhesion to fibronectin and laminin. Weak cell adhesion is beneficial to tumor metastasis inhibition, so cell adhesion to fibronectin and laminin assay was used to evaluate the effects of different concentrations of compound 4b and Celecoxib on the adhesion ability of SW620 cells after 24 h treatment. Results as shown in Figure 4, compound 4b exhibited a similar ability to Celecoxib to reduce the adhesion of SW620 cells to fibronectin and laminin.

Xenograft Model In Vivo
In view of potent cox-2 selective inhibitory activity and anti-colon cancer proliferation activity in vitro, compound 4b was further evaluated for anti-colon cancer activity in vivo. SW620 cells (5 × 10 6 ) were subcutaneously injected into the rightwing nude mice to establish a xenograft model. When the tumor volume grows to the macroscopic size of about 100 mm 3 , 15 tumor-bearing mice were randomly divided into vehicle, Celecoxib (20 mg/kg) and compound 4b (20 mg/kg) groups. Intraperitoneal administration was performed every 2 days and tumor volume changes were recorded for 12 consecutive days. As shown in Figure 5B, tumor volume increased rapidly in the vehicle group, whereas tumor growth was significantly inhibited in two treatment groups. Among them, the tumor inhibition effect of compound 4b (20 mg/kg) group was better than that of Celecoxib (20 mg/kg) group. After 12 days of treatment, the tumor volumes of the two treatment groups were 43.71 mm 3 and 51.69 mm 3 , respectively. Finally, the tumors of each group were removed and weighed to calculate the ratio of tumor weight to body weight. The specific results are shown in Figure 5A and 5D. Compared to the vehicle group with an average ratio of tumor weight to body weight of 0.34, the other two treatment groups showed significant reduction, with compound 4b (20 mg/kg) indicating a lighter average ratio of tumor weight to body weight (0.46). At the same time, no significant weight change was observed in the treatment group, suggesting that the compounds in these mice were nontoxic. In contrast, body weight increased slightly in the vehicle group at the later stage of treatment ( Figure 5C). From the above, these results suggested that compound 4b had potent anticolon cancer activity in vivo. This series of representative compounds 4b, as a selective COX-2 inhibitor for the targeted therapy of colon cancer, had prominent research prospect.

Xenograft Model In Vivo
In view of potent cox-2 selective inhibitory activity and anti-colon cancer proliferation activity in vitro, compound 4b was further evaluated for anti-colon cancer activity in vivo. SW620 cells (5 × 10 6 ) were subcutaneously injected into the rightwing nude mice to establish a xenograft model. When the tumor volume grows to the macroscopic size of about 100 mm 3 , 15 tumor-bearing mice were randomly divided into vehicle, Celecoxib (20 mg/kg) and compound 4b (20 mg/kg) groups. Intraperitoneal administration was performed every 2 days and tumor volume changes were recorded for 12 consecutive days. As shown in Figure 5B, tumor volume increased rapidly in the vehicle group, whereas tumor growth was significantly inhibited in two treatment groups. Among them, the tumor inhibition effect of compound 4b (20 mg/kg) group was better than that of Celecoxib (20 mg/kg) group. After 12 days of treatment, the tumor volumes of the two treatment groups were 43.71 mm 3 and 51.69 mm 3 , respectively. Finally, the tumors of each group were removed and weighed to calculate the ratio of tumor weight to body weight. The specific results are shown in Figure 5A,D. Compared to the vehicle group with an average ratio of tumor weight to body weight of 0.34, the other two treatment groups showed significant reduction, with compound 4b (20 mg/kg) indicating a lighter average ratio of tumor weight to body weight (0.46). At the same time, no significant weight change was observed in the treatment group, suggesting that the compounds in these mice were nontoxic. In contrast, body weight increased slightly in the vehicle group at the later stage of treatment ( Figure 5C). From the above, these results suggested that compound 4b had potent anti-colon cancer activity in vivo. This series of representative compounds 4b, as a selective COX-2 inhibitor for the targeted therapy of colon cancer, had prominent research prospect.

Molecular Docking
In order to better study the binding mode and interaction, molecule docking of dihydropyrazole derivatives containing benzo oxygen heterocycle and sulfonamide moieties 4a-4z and known COX-2 inhibitors Celecoxib about the COX-2 (PDB ID: 3LN1) enzymes were performed together. All simulations were performed around the central region of the already known COX-2 (PDB ID: 3LN1) site where Celecoxib bound. The judgment criterion is to compare the predicted combined interaction energy. The estimated value of the interaction energy of each compound were ranged from −45.96 to −33.41 kcal/mol, as displayed in the histogram of Table 5 and Figure 6. Besides, linear fitting was performed by comparing the binding energy and COX-2 inhibition. It behaved approximate linearity (y =46.82 − 0.86x, R2 = 0.72) exhibited in the Figure7. The R value is approximately equal to 0.85 and approaches to 1, which not only indicates that the fitting curve has a good credibility, but also indicates that our design idea is reasonable. Compared Celecoxib (energy value of −42.57 kcal/mol), compound 4b was relatively superior with predicted combined interaction energy value of −45.96 kcal/mol. And their concrete combination of computer simulation was depicted in Figure 7.

Molecular Docking
In order to better study the binding mode and interaction, molecule docking of dihydropyrazole derivatives containing benzo oxygen heterocycle and sulfonamide moieties 4a-4z and known COX-2 inhibitors Celecoxib about the COX-2 (PDB ID: 3LN1) enzymes were performed together. All simulations were performed around the central region of the already known COX-2 (PDB ID: 3LN1) site where Celecoxib bound. The judgment criterion is to compare the predicted combined interaction energy. The estimated value of the interaction energy of each compound were ranged from −45.96 to −33.41 kcal/mol, as displayed in the histogram of Table 5 and Figure 6. Besides, linear fitting was performed by comparing the binding energy and COX-2 inhibition. It behaved approximate linearity (y = 46.82 − 0.86x, R 2 = 0.72) exhibited in the Figure 7. The R value is approximately equal to 0.85 and approaches to 1, which not only indicates that the fitting curve has a good credibility, but also indicates that our design idea is reasonable. Compared Celecoxib (energy value of −42.57 kcal/mol), compound 4b was relatively superior with predicted combined interaction energy value of −45.96 kcal/mol. And their concrete combination of computer simulation was depicted in Figure 7.   As illustrated in Figure 8A,B, Celecoxib could effectively bind at this site (XYZ axis: 30.99, −22.28 and −16.51; radius: 6.96 Å) through five hydrogen bond receptors and eleven Pi bonds, with the stronger interactional amino acids for Arg-106, Ser-339, Arg-499, Gln-178 and Leu-338. Compared with Celecoxib, compound 4b interacts more with proteins in this region and binds better, as shown in the Figure 4C,D. Compound 4b could effectively bind through nine hydrogen bond receptors and fourteen Pi bonds. In addition to the stronger interactional amino acids bond with Celecoxib, there are stronger interactions with His-75, Phe-504, and Val-102, which bound to the sulfa and 1,3-dioxolane domains, respectively. In general, these simulation results were consistent with the actual activity detection, and compound 4b as a COX-2 inhibitor should be a promising effective drug. The difference in simulation between compound 4b and Celecoxib could explain that compound 4b was a better COX-2 inhibitor.   As illustrated in Figure 8A,B, Celecoxib could effectively bind at this site (XYZ axis: 30.99, −22.28 and −16.51; radius: 6.96 Å) through five hydrogen bond receptors and eleven Pi bonds, with the stronger interactional amino acids for Arg-106, Ser-339, Arg-499, Gln-178 and Leu-338. Compared with Celecoxib, compound 4b interacts more with proteins in this region and binds better, as shown in the Figure 4C,D. Compound 4b could effectively bind through nine hydrogen bond receptors and fourteen Pi bonds. In addition to the stronger interactional amino acids bond with Celecoxib, there

Materials and Measurements
All commercial reagents and solvents were analyzed using products produced by USA Energy Chemical company and Aladdin reagent (Shanghai, China). The melting points of all compounds were determined by an x4mp instrument. All 1 H NMR spectra were recorded in DMSO-d 6

General Procedure for the Synthesis of Compound 2a,2b
The protocatechuic aldehyde 1a (1 mmol) was dissolved in anhydrous DMF (5 mL), anhydrous potassium carbonate (0.5 mmol) added. The solution was then heated to 70 • C and then dibromomethane or 1,2-dibromoethane was added dropwise. The reaction was stirred for 4 h and poured into ice water. A white precipitate (compound 2a, 2b) was formed which was filtered and recrystallized from ethanol [33].

General Procedure for the Synthesis of Compounds 3a-3z
Compound 2a,2b (1 mmol) was added to 15 mL absolute ethanol solution with acetophenone derivative (1 mmol) and 40% sodium hydroxide solution (0.5 mmol). The reaction was stirred at room temperature for 10 h, and then filtered to afford compound 3a-3z without further purification [34].

General Procedure for the Synthesis of Compounds 4a-4z
The compounds 3a-3z (1 mmol), 4-sulfamethylhydrazine hydrochloride (1.2 mmol) and glacial acetic acid (0.5 mmol) were refluxed overnight in ethanol (15 mL). The reaction mixture was diluted with 50 mL water and extracted with ethyl acetate. Saturated sodium bicarbonate was used to wash the organic anhydrous layer, which was then filtered to dry by anhydrous sodium sulfate and concentrated in vacuum. The desired products 4a-4z were purified by flash chromatography.

Docking Simulations
The crystal structure of the protein complex was retrieved from the RCSB protein database (PDB code: 3LN1). Then, the three-dimensional structure of the compound was constructed using Chem 3D Ultra 14.0 software (Cambridge Software, Boston, MA, USA), and then energy minimization was performed using MMFF94, iterating 5000 times with a minimum RMS gradient of 0.10. Compound 4b and Celecoxib molecules were docked into a three-dimensional complex structure of COX-2 using the Discovery Studio (Discovery Studio 4.5, Accelrys, Co. Ltd., Beijing, China) software via the graphical user interface DS-CDOCKER protocol. All bound water and ligand were removed from the protein and polar hydrogen was added to the protein. Briefly, we defined the COX-2 complex as a receptor and then replaced the Celecoxib molecule with compound 4b, mimicking the entry of the compound into the site where COX-2 binds to Celecoxib.

Conclusions
Overexpression of COX-2 was clearly associated with the development of various types of cancer, and the concept of COX-2 as a possible target was a promising therapeutic strategy. Therefore, a series of novel dihydropyrazole derivatives containing benzo oxygen heterocycle and sulfonamide moieties 4a-4z were designed to be COX-2 inhibitors. Most of the synthetic compounds exhibited strong selective and inhibitory activity, and compound 4b exhibited the most potent inhibitory activity against COX-2 and antiproliferative activity on SW620 cells with IC 50 values of 0.86 µM. In further research, representative compound 4b induced apoptosis in SW620 cells in a dose-dependent manner and attenuated its adhesion. Finally, the in vivo anti-colon cancer activity of compound 4b was verified in SW620 xenograft mouse models. In conclusion, it was hoped that this study would be beneficial to the research of COX-2 inhibitors in the treatment for colorectal carcinoma.
Supplementary Materials: The following are available online, Figure S1: The design pathway for novel dihydropyrazole derivatives containing benzo oxygen heterocycle and sulfonamide moieties., Table S1: Structures of compounds 4a-4z, etc.
Author Contributions: All authors contributed to conceptualization, methodology, and writing. X.-Q.Y. conducted data processing. X.-Q.Y. performed data analysis and simulation.

Conflicts of Interest:
The authors declare no conflict of interest.