TY - JOUR
AU1 - Zhu, Jiuxiang
AU2 - Song, Xueqin
AU3 - Lin, Ho-Pi
AU4 - Young, Donn C.
AU5 - Yan, Shunqi
AU6 - Marquez, Victor E.
AU7 - Chen, Ching-Shih
AB - Abstract Background: The cyclooxygenase-2 (COX-2) inhibitor celecoxib is thought to act as a chemopreventive agent by sensitizing cancer cells to apoptotic signals. Other COX-2 inhibitors, such as rofecoxib, are two orders of magnitude less potent than celecoxib at inducing apoptosis. The molecular structures of celecoxib and rofecoxib were used as starting points to examine the structural features that contribute to this discrepancy. Methods: We used a systematic chemical approach to modify the structures of celecoxib and rofecoxib to produce a series of compounds that were tested for their effects on the viability of human prostate cancer PC-3 cells and their ability to induce apoptosis in these cells. Cell viability was measured by the trypan blue dye exclusion assay, and apoptosis was measured by an enzyme-linked immunosorbent assay that quantifies DNA cleavage and by western blot detection of poly(ADP-ribose) polymerase (PARP) cleavage. Western blotting was used to monitor the effects of the compounds on phosphorylation of the serine/threonine kinase Akt and extracellular signal-regulated kinase 2 (ERK2), two components of celecoxib-induced apoptosis signaling. Monte Carlo simulations were used to molecularly model the surface electrostatic potential and electron density of selected compounds. All statistical tests were two-sided. Results: The structural requirements for the induction of apoptosis in PC-3 cells were different from those for COX-2 inhibition. Structure–function analysis indicated that the induction of apoptosis by compounds derived from COX-2 inhibitors required a bulky terminal phenyl ring, a heterocyclic system with negative electrostatic potential, and a benzenesulfonamide or benzenecarboxamide moiety. These derivatives mediated apoptosis by facilitating the dephosphorylation of Akt and ERK2, irrespective of their COX-2 inhibitory activities. Conclusion: A new class of compounds that induce apoptosis by targeting Akt and ERK2 signaling pathways in human prostate cancer cells can be synthesized by modifying existing COX-2 inhibitors. Results of recent epidemiologic and animal model studies have suggested that nonsteroidal anti-inflammatory drugs (NSAIDs) act as chemopreventive agents, especially for colon cancer (1–9). Several lines of evidence have attributed the antitumor activities of NSAIDs to their ability to sensitize cancer cells to apoptosis by blocking cyclooxygenase-2 (COX-2) enzyme activity (10–13). It is well documented that COX-2 is constitutively overexpressed in many types of human cancers and that decreased prostaglandin E2 production as a result of COX-2 inhibition is associated with the modulation of various pro- and anti-apoptotic factors, such as Bcl-2 (14), prostate apoptosis-response gene (Par-4) (15), and caspase-3 (16). In addition, knockout of the COX-2 gene suppresses tumorigenesis in mice that have a genetic predisposition to form polyps (17). Recently, the U.S. Food and Drug Administration approved the use of the COX-2 inhibitor celecoxib for the adjuvant treatment of familial adenomatous polyposis, an inherited syndrome that predisposes individuals to colon cancer. In addition, celecoxib has also been tested in numerous clinical trials (18) for its chemopreventive effect on a variety of epithelial malignancies including colon, esophagus, skin, and bladder cancers. However, an expanding body of evidence suggests that COX-2 inhibition may not play a role in NSAID-mediated apoptotic cell death (19). For example, sulindac sulfide and sulindac sulfone, which are metabolites of the NSAID sulindac, have been reported to mediate apoptosis in cancer cells via the inhibition of cyclic GMP phosphodiesterase (20–23). which is a COX-2-independent mechanism (24). In addition, we have used a tetracycline-inducible antisense COX-2 expression plasmid to demonstrate that the sensitivity of prostate cancer cells to COX-2 inhibitor-induced apoptosis is independent of the expression status of COX-2 (25). The finding that these two pharmacologic effects of NSAIDs—COX-2 inhibition and apoptosis induction—are separable has considerable therapeutic implications and provides molecular underpinnings for the design of a new class of anticancer compounds whose mode of action is different from that of conventional chemotherapeutic agents. Celecoxib induces apoptosis in prostate cancer cells by interfering with multiple signaling targets, including the serine/threonine kinase Akt, extracellular signal-regulated kinase 2 (ERK2), and endoplasmic reticulum Ca2+-ATPases (26,27). Disruption of these signaling pathways results in the loss of regulation of cellular functions that govern cell growth and survival, leading to rapid apoptotic death. This rapid induction of apoptosis, however, is unique to celecoxib because other COX-2 inhibitors that have the same COX-2 inhibitory potencies as celecoxib, including rofecoxib (Vioxx®), NS398, and DuP697, display apoptosis-inducing activities that are nearly two orders of magnitude lower than that displayed by celecoxib (26,27). Here we use celecoxib and rofecoxib as molecular starting points from which to understand the structural basis underlying this discrepancy and to optimize the apoptosis-inducing potency of celecoxib in prostate cancer cells. Materials And Methods Materials Celecoxib and rofecoxib were extracted with ethyl acetate followed by recrystallization in a mixture consisting of ethyl acetate and hexane from Celebrex® and Vioxx®, respectively, which were obtained from Amerisource Health (Malvern, PA). NS398 was purchased from Calbiochem (San Diego, CA). Rabbit polyclonal antibodies against Akt, phospho-473Ser Akt, p44/42 ERKs, and phospho-p44/42 ERKs were purchased from Cell Signaling Technologies (Beverly, MA). A rabbit polyclonal anti-poly(ADP-ribose) polymerase (PARP) antibody was obtained from Pharmingen (San Diego, CA). Other chemical and biochemical reagents used were obtained from Sigma-Aldrich (St. Louis, MO), unless otherwise mentioned. Synthesis of Compounds In this article, we discuss 50 compounds. The full chemical name of each of these compounds is provided in Table 1. We used published procedures to synthesize compounds 1, 1-NH2, 2–29, and 40–46.(28–31). Proton nuclear magnetic resonance (1H NMR) spectroscopy, high-resolution mass spectrometry (HRMS), and elemental analysis were used to validate the identity of each of the synthetic compounds. The procedures we used to synthesize compounds 30–39 and 47–50 are presented below. Our results and the properties of these chemicals are summarized in Table 2. 4-[5-(4-Chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl] benzenecarboxyamide (compound 30) was synthesized in two steps. In the first step, 4,4,4-trifluoro-1-(4-chlorophenyl)butane-1,3-dione was prepared as follows. To a solution of ethyl trifluoroacetate (1.08 g, 7.61 mmol) in 5 mL of methyl tert-butyl ether [MTBE] was added 25% sodium methoxide in methanol (1.8 mL) over a period of 2 minutes. A solution of 4′-chloroacetophenone (1 g, 6.46 mmol) in 2 mL of MTBE was then added to that mixture in a dropwise fashion over a period of 5 minutes. The resulting solution was stirred for 16 hours, 3 N HCl (3.4 mL) was added, and the aqueous and organic layers of the resulting mixture were allowed to separate. The organic layer was collected, washed with a saturated NaCl solution, dried over magnesium sulfate, and concentrated under vacuum in a rotary evaporator. The resulting yellow-orange solid was recrystallized from hexane, yielding 4,4,4-trifluoro-1-(4-chlorophenyl)butane-1,3-dione (1.18 g, 86% yield). In the second step, (4-carbamoylphenyl)hydrazine hydrochloride (228 mg, 1.21 mmol) was added to a stirred solution of the aforementioned dione (300 mg, 1.21 mmol) in 20 mL of ethanol. The resulting mixture was stirred under reflux for 24 hours, cooled to room temperature, and then concentrated to dryness under vacuum in a rotary evaporator. The resulting residue was dissolved in ethyl acetate, washed with a saturated NaCl solution, dried over magnesium sulfate, and concentrated under vacuum in a rotary evaporator. The resulting light brown solid was recrystallized from ethyl acetate and hexane to produce compound 30 (350 mg, 80% yield). 4-[5-(2,4-Dichlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenecarboxyamide (compound 31) was synthesized from 2′,4′-dichloroacetophenone using the two-step procedure described for the synthesis of compound 30, in which 2′,4′-dichloroacetophenone was used in place of 4′-chloroacetophenone (52% overall yield). 4-[5-(2,5-Dichlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenecarboxyamide (compound 32) was synthesized from 2′,5′-dichloroacetophenone using the two-step procedure described for the synthesis of compound 30, in which 2′,5′-dichloroacetophenone was used in place of 4′-chloroacetophenone (60% overall yield). 4-[5-(3,4-Dichlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenecarboxyamide (compound 33) was synthesized from 3′,4′-dichloroacetophenone using the two-step procedure described for the synthesis of compound 30, in which 3′,4′-dichloroacetophenone was used in place of 4′-chloroacetophenone (55% overall yield). 4-[5-(4-Methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl] benzenecarboxyamide (compound 34) was synthesized from 4′-methylacetophenone using the two-step procedure described for the synthesis of compound 30, in which 4′-methylacetophenone was used in place of 4′-chloroacetophenone (65% overall yield). 4-[5-(4-Trifluoromethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenecarboxyamide (compound 35) was synthesized from 4′-trifluoromethylacetophenone using the two-step procedure described for the synthesis of compound 30, in which 4′-trifluoromethylacetophenone was used in place of 4′-chloroacetophenone (53% overall yield). 4-[5-(4-Ethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl] benzenecarboxyamide (compound 36) was synthesized from 4′-ethylacetophenone using the two-step procedure described for the synthesis of compound 30, in which 4′-ethylacetophenone was used in place of 4′-chloroacetophenone (44% overall yield). 4-[5-(2,4-Dimethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenecarboxyamide (compound 37) was synthesized from 2′,4′-dimethylacetophenone using the two-step procedure described for the synthesis of compound 30, in which 2′,4′-dimethylacetophenone was used in place of 4′-chloroacetophenone (62% overall yield). 4-[5-(2,5-Dimethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenecarboxyamide (compound 38) was synthesized from 2′,5′-dimethylacetophenone using the two-step procedure described for the synthesis of compound 30, in which 2′,5′-dimethylacetophenone was used in place of 4′-chloroacetophenone (58% overall yield). 4-[5-(3,4-Dimethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenecarboxyamide (compound 39) was synthesized from 3′,4′-dimethylacetophenone using the two-step procedure described for the synthesis of compound 30, in which 3′,4′-dimethylacetophenone was used in place of 4′-chloroacetophenone (56% overall yield). 3-(4-Methylsulfonylphenyl)-4-phenyl-2(5H)-furanone (compound 47) was synthesized in two steps. In the first step, a mixture of 4′-(methylsulfonyl)acetophenone (5.5 g, 27.8 mmol), morpholine (2.5 mL), and sulfur (0.89 g, 27.8 mmol) was refluxed for 10 hours and then poured into ice where it formed a precipitate. The precipitate was collected by filtration and washed with cold ethyl acetate. The precipitate was added to 10% sodium hydroxide (55 mL), and the mixture was then heated to 84 °C for 12 hours, forming an alkaline solution that was acidified to pH 3 with 12 N HCl. The solid that resulted from acidification was collected by filtration, dried, and recrystallized from a solution containing equal volumes of hexane and ethyl acetate to give 4-methylsulfonylphenylacetic acid (a white solid; 4.2 g, 52% overall yield). In the second step, 2-bromoacetophenone (1.02 g, 5.12 mmol) dissolved in acetonitrile (28 mL) was added to triethylamine (1.74 mL), followed by the addition of 4-methylsulfonylphenylacetic acid (1 g, 4.67 mmol). The mixture was stirred at room temperature for 1.5 hours and then 1,8-diazabicyclo[5,4,0]undec-7-ene (1.67 mL) was added. The mixture was stirred for another hour, after which 1 N HCl (35 mL) was added. The end product was extracted from the mixture with ethyl acetate, dried over sodium sulfate, and recrystallized from ethyl acetate–hexane (vol/vol) to give compound 47 (880 mg, 60% overall yield). 3-(4-Sulfamoylphenyl)-4-phenyl-2(5H)-furanone (compound 48) was synthesized from 4-sulfamoylphenylacetic acid and 2-bromoacetophenone in a manner similar to that described for compound 47, with a 40% yield. 3-(4-Sulfamoylphenyl)-4-(2,4-dichlorophenyl)-2(5H)-furanone (compound 49) was synthesized from 4-sulfamoylphenylacetic acid and 2-bromo-1-(2′4-dichlorophenyl)acetophenone in a manner similar to that described for compound 47, with a 32% yield. 3-(4-sulfamoylphenyl)-4-(3,4-dichlorophenyl)-2(5H)-furanone (compound 50) was synthesized from 4-sulfamoylphenylacetic acid and 2-bromo-1-(3′4-dichlorophenyl)acetophenone in a manner similar to that described for compound 47, with a 30% yield. Cell Culture The human prostate cancer cell lines LNCaP and PC-3 were purchased from the American Type Culture Collection (ATCC; Manassas, VA). Bcl-2-overexpressing PC-3 cells were prepared as previously described (26). Cells were cultured in RPMI-1640 medium (Gibco, Grand Island, NY) supplemented with 10% fetal bovine serum (FBS; Gibco) at 37 °C in a humidified incubator containing 5% CO2. Cells were replenished daily with fresh medium and were harvested by trypsinization and split at a 1 : 4 ratio with fresh medium every 3 days. Cell Viability Analysis Prostate cancer cells were grown in 10% FBS-supplemented RPMI-1640 medium for 48 hours to approximately 60% confluency. The cells were then washed in serum-free RPMI-1640 and incubated in serum-free RPMI-1640 medium that contained various concentrations of celecoxib, rofecoxib, or test agent, each dissolved in 0.1% dimethyl sulfoxide (DMSO). Control cell cultures were washed in serum-free RPMI-1640 and then incubated in serum-free RPMI-1640 that contained the same concentration of DMSO as the celecoxib-treated cells. Floating cells were recovered from culture medium by centrifugation at 3200g for 5 minutes, and adherent cells were harvested by trypsinization. Both the floating and adherent cells were observed for morphologic changes with a light microscope at ×200 magnification. We combined the adherent and floating cells and measured their viability by using a trypan blue dye exclusion assay. Assessment of Apoptosis Enzyme-linked immunosorbent assay (ELISA) to detect DNA fragmentation. We used the Cell Death Detection ELISA kit (Roche Diagnostics, Mannheim, Germany), according to the manufacturer’s instructions, to measure the induction of apoptosis in human prostate cancer cells treated with various compounds. This assay quantifies cytoplasmic histone-associated DNA fragments (both mono- and oligonucleosomes) that result from the induction of apoptosis. In brief, 2.5 × 106 PC-3 cells were plated in T-75 flasks and incubated for 24 hours. The cells were washed twice with 5 mL of serum-free RPMI-1640 medium and then incubated with serum-free medium containing the test compounds as described above. We then collected and pooled the floating and adherent cells, as described above, and counted them. Cell lysates equivalent to 104 cells were used for the ELISA analysis. Histone-associated DNA fragments were quantitated spectrophotometrically using antibodies against DNA and histones in a colorimetric assay. Western blot analysis of PARP cleavage. PC-3 cells treated with DMSO or the various compounds as described above were collected, washed with ice-cold phosphate-buffered saline (PBS), and resuspended in 50 μL of lysis buffer (20 mM Tris–HCl [pH 8], 137 mM NaCl, 1 mM CaCl2, 10% glycerol, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 100 μM 4-(2-aminoethyl)benzenesulfonyl fluoride, 10 μg/mL leupeptin, and 10 μg/mL aprotinin) for 10 minutes. Soluble cell lysates were collected after centrifugation at 1500g for 5 minutes. Protein concentrations of the lysates were determined by using a Bradford protein assay kit (Bio-Rad, Hercules, CA); equivalent amounts of protein from each lysate were resolved in 10% SDS–polyacrylamide gels and then transferred to nitrocellulose membranes. Western blotting with an anti-PARP antibody was carried out as described below, and apoptosis was detected by monitoring proteolysis of the 116-kd native PARP enzyme to the apoptosis-specific 85-kd fragment. Western Blot Analysis of Apoptosis Signaling Components Treated cells collected as described above, washed with PBS, resuspended in SDS gel-loading buffer (100 mM Tris–HCl [pH 6.8], 4% [wt/vol] SDS, 0.2% [wt/vol] bromophenol blue, 20% [vol/vol] glycerol, and 200 mM dithiothreitol), sonicated with an ultrasonic sonicator for 5 seconds, and boiled for 5 minutes. After brief centrifugation, equivalent amounts of soluble protein, as determined by the Bradford method, were resolved in 10% SDS–polyacrylamide minigels and transferred to nitrocellulose membranes with the use of a semidry transfer cell (Bio-Rad). The membranes were washed twice with TBS (0.3% [wt/vol] Tris, 0.8% [wt/vol] NaCl, and 0.02% [wt/vol] KCl) containing 0.05% Tween 20 (TBST) and then incubated with TBS containing 5% nonfat dry milk for 60 minutes to block nonspecific antibody binding. Each membrane was then incubated at 4 °C for 12 hours with a primary antibody specific for Akt, phospho-Akt, ERKs, phospho-ERKs, or PARP, which was diluted 1 : 1000 in TBS containing 1% nonfat dry milk. The membranes were washed twice with TBST and then incubated at room temperature for 1 hour with a horseradish peroxidase-conjugated goat anti-rabbit immunoglobulin G (IgG) diluted 1 : 5000 in TBS containing 1% nonfat dry milk. The membranes were washed twice with TBST, and bound antibody was visualized by enhanced chemiluminescence using ECLTM western blotting detection reagents (Amersham Pharmacia Biotech, Little Chalfont, U.K.). Unphosphorylated Akt and ERK2, as immunostained by anti-Akt and anti-ERK2 antibodies, were used as internal standards for the comparison of phospho-Akt and phospho-ERK2 levels among samples of different exposure intervals. Molecular Modeling Experiments Molecular structures of compounds 47–50, as well as celecoxib and rofecoxib, were initially subjected to 1000 steps of Monte Carlo simulation using the Merck Molecular Force Field program available as part of Macromodel 7.0 (Schrodinger, Portland, OR; http://www.schrodinger.com). The minimum conformation reached by the Monte Carlo simulations was then fully optimized at a density functional theory level of B3LYP/6–31G* with Gaussian 98A7 (Gaussian, Inc., Pittsburgh, PA) (32). All the fully optimized structures were confirmed by normal mode analysis; no negative frequencies were found. Computations for electrostatic potential and electron density were then carried out for each of the fully optimized structures with a grid of 216 000 points using Gaussian 98A7. The electrostatic potential maps for each compound were generated by gOpenMol (http://staff.csc.fi/~laaksone/gopenmol/gopenmol.html) (33,34) and are presented with the electrostatic potential mapped onto the electron density. The electron density isosurface value was 0.0004 with a range of –0.03 to 0.03 for the electrostatic potential. Statistical Analysis Each experiment was performed in triplicate and was repeated at least two times on different occasions. Analyses included nonparametric and parametric techniques to include analysis of variance (ANOVA) for linear models of dose response, ANOVA with Scheffé post hoc comparisons, Kruskal–Wallis nonparametric ANOVA, and Spearman rank correlation. A two-sided alpha of 0.05 was considered statistically significant. We used two descriptive terms—the T50% and the apoptosis index—to express the apoptosis-inducing activity of individual compounds in PC-3 cells. T50% denotes the time required for eliciting apoptotic death in 50% of the cells when they are exposed to a specific concentration of the test compound. The apoptosis index is a semiquantitative indication of the apoptosis-inducing activity of a compound and was defined according to the T50% for cells exposed to test compound at 50 μM. Compounds were classified into the following four categories of apoptosis index (in descending order of apoptosis-inducing activity): +++, T50% is less than or equal to 2 hours; ++, T50% is greater than 2 hours but less than or equal to 4 hours; +, T50% is greater than 4 hours but less than or equal to 24 hours; and −, induced no appreciable apoptosis at 24 hours. Cut points to define the categories of apoptosis indices were determined by first performing ANOVA for all T50% values for the compounds that elicited an apoptotic effect. Although there were statistically significant differences in apoptosis index among the different compounds (P<.001), no differences were seen among LNCaP, PC-3, and Bcl-2-overexpressing PC-3 cells (P = .49). To simplify the identification of the apoptosis index categories, we separated groups of compounds into those that had an apoptosis index of 2 hours or less and those that had an apoptosis index of greater than 2 hours, using the rounded group midpoint value of 2 hours that was determined from a conservative Scheffé post hoc comparison. Because compounds were evaluated in all three cell lines only at a dose of 50 μM, no dose–response relationship was analyzed. The purpose of using these two descriptive terms was twofold. First, these two parameters, along with the IC50 (concentration for 50% inhibition) for COX-2 inhibition, allowed us to confirm our previous finding that COX-2 inhibition and apoptosis induction were independent. Second, the use of these terms allowed us to identify compounds that had high apoptosis-inducing potencies. Results Chemistry and Overall Strategy The objective of this structure–function analysis of celecoxib and rofecoxib was twofold: to elucidate the structural basis underlying the discrepancy in apoptosis-inducing potency between the COX-2 inhibitors celecoxib and rofecoxib and to optimize the activity of celecoxib with regard to apoptosis induction. To attain these goals, we systematically altered the structure of celecoxib using the strategies to modify the moieties depicted in Fig. 1, A. In strategy A, we either modified the terminal aromatic ring of celecoxib with various substituents to produce compounds 2–23, or replaced the terminal aromatic ring with different ring systems to produce compounds 24–29 (see Fig. 3 for structures). In strategy B, we substituted the carboxamide group for the sulfonamide group of various apoptosis-active celecoxib derivatives to produce compounds 30–39 (see Fig. 5 for structures). In strategy C, we modified the heterocyclic system of celecoxib to produce compounds 40–46 (Fig. 1, B). Molecular structures of celecoxib and rofecoxib were also subjected to computer modeling to examine the effect of surface potential on apoptosis; compounds 47–50 were prepared, on the basis of computer modeling of those compounds (see Fig. 1, B, and 6, C, for structures). All compounds were evaluated for their ability to induce apoptotic cell death in human prostate cancer PC-3 cells, which are androgen-independent and deficient in p53 expression. Compounds that induced apoptosis in PC-3 cells were further evaluated for their ability to induce apoptosis in two additional cell lines—human prostate cancer LNCaP cells, which are androgen-dependent and express wild-type levels of p53, and Bcl-2-overexpressing PC-3 cells (26). For each compound tested, the T50% value and apoptosis index obtained in each of the three cells lines were similar, indicating that the induction of apoptosis was not dependent on androgen sensitivity, p53 functional status, or the level of Bcl-2 expression. We therefore present cell viability and apoptosis data only for studies performed using PC-3 cells. Role of the Sulfamoyl Moiety of Celecoxib in the Induction of Apoptosis We tested COX-2 inhibitors that have similar IC50 values for COX-2 inhibition for their apoptosis-inducing activities in PC-3 cells and found that these COX-2 inhibitors exhibited widely discrepant apoptosis-inducing activities (Fig. 2, A). On the basis of their respective apoptosis indices (AIs) in PC-3 cells, we classified these COX-2 inhibitors into two groups—those that induced apoptosis (e.g., celecoxib) and those that did not (e.g., rofecoxib, NS398, DuP697, and the terphenyl derivative of DuP697, compound 1) (Fig. 2). Structurally, these COX-2 inhibitors could be classified into two groups according to the type of sulfonyl (-SO2-) functionality they contained, i.e., sulfonamide (-SO2NH2) or methylsulfone (-SO2CH3). As shown in Fig. 2, A, celecoxib contains a sulfonamide group, whereas rofecoxib, NS398, DuP697, and compound 1 all contain a methylsulfone group. This structural difference suggests that the sulfamoyl moiety of celecoxib may play a role in its apoptosis-inducing activity. This possibility was strengthened by our demonstration that the methylsulfone-containing counterpart of celecoxib was a less potent inducer of apoptosis than celecoxib (Fig. 2, B, left panel). Moreover, modification of compound 1 to compound 1-NH2(28) resulted in an increase in apoptosis-inducing activity by an order of magnitude (Fig. 2, B, right panel). The conversion of rofecoxib and DuP697 to their sulfonamide counterparts, however, did not substantially affect the apoptosis-inducing activities of those compounds (data not shown), indicating that structural elements other than the sulfonamide group contributed to the activation of the apoptosis machinery by celecoxib and compound 1-NH2. Effect of the Terminal Phenyl Ring on the Apoptosis-Inducing Activity of Celecoxib We synthesized a series of celecoxib derivatives (i.e., compounds 2–29) to assess the role of the terminal phenyl ring on the apoptosis-inducing activity of celecoxib (Fig. 3). We found that the structural requirements for the induction of apoptosis by these compounds included a certain degree of bulkiness and hydrophobicity in the 5-aryl ring and were distinct from those required for COX-2 inhibition (29). For example, when we reduced the size of the 5-aryl ring (i.e., from a CH3 group in celecoxib to an H group in compound 2 or an F group in compound 3) or increased its polarity (e.g., from a CH3 group to an OH group in compound 11, an NH2 in compound 12, or an NO2 group in compound 13), the apoptosis indices of the resulting compounds decreased precipitously. Among the 28 derivatives we examined, compounds 8, 10, 14, and 19–21 had an apoptosis index of +++ in PC-3 cells; compounds 6, 7, 9, 15, and 18 had an apoptosis index of ++; and the remaining compounds displayed no appreciable apoptosis induction at 24 hours (i.e., an apoptosis index of −). Celecoxib induces apoptosis by a mechanism that involves the concomitant dephosphorylation of Akt and ERK2 (25–27). We therefore examined whether the compounds with the most potent apoptotic indices induced cell death by a mechanism similar to that of celecoxib. Fig. 4, A, depicts the time- and/or dose-dependent effects of one of the active compounds generated by strategy A, compound 10, on cell viability. ELISA analysis of the lysates from drug-treated cells revealed the time-dependent formation of oligonucleosomes as a result of DNA degradation (Fig. 4, B, left panel). In addition, immunoblot analysis of PARP indicated that exposure of PC-3 cells to compound 10 led to the rapid cleavage of the 116-kd native enzyme to form the apoptosis-specific 85-kd fragment (Fig. 4, B, right panel). It is also noteworthy that the mechanism by which compound 10 caused apoptosis, i.e., dephosphorylation of Akt and ERK2 (Fig. 4, C), was the same as that by which celecoxib causes apoptosis. Similar results were obtained with other active compounds in this group. The dose–response relationship for compound 10, with respect to cell viability, was statistically significant (ANOVA, P<.001 for time, dose, and cell viability). Fig. 4, B, shows that PC-3 cells treated with compound 10 displayed statistically significantly higher levels of ELISA-detectable nucleosome formation, an indicator of apoptotic cell death, over time than did PC-3 cells treated with DMSO (ANOVA; P<.001; mean readings [n = 3] at OD450 at 10, 20, and 30 minutes were 2.6 [95% CI = 2.5 to 2.7], 4.1 [95% CI = 4.0 to 4.2], and 4.1 [95% CI = 4.0 to 4.2], respectively, for drug-treated cells and 0.21 [95% CI = 0.18 to 0.24], 0.22 [95% CI = 0.20 to 0.24], and 0.16 [95% CI = 0.15 to 0.17], respectively, for DMSO-treated cells. Because many of the compounds generated by strategy A are potent COX-2 inhibitors (29), we evaluated the relationship between COX-2 inhibition and induction of apoptosis by performing a nonparametric Spearman rank correlation analysis of the IC50 values of these compounds and the time they took to achieve a 50% reduction in cell viability at a concentration of 50 μM. We found no correlation (P = .44). A similar nonparametric Kruskal–Wallis test showed no differences in the IC50 values among compounds in the four apoptosis index groups (P = .67). Although some compounds displayed high potency in inducing apoptosis, they lacked substantial COX-2 inhibitory activity and vice versa. This lack of correlation between apoptosis induction and COX-2 inhibition was consistent with our preliminary data (25) and confirmed that these two pharmacologic activities could be separated. Apoptosis-Inducing Activities of Celecoxib Analogues That Contain a Carboxamide Moiety in Place of the Sulfamoyl Moiety Although both the sulfonamide and methylsulfone pharmacophores showed comparable potency in COX-2 inhibition (29), the apoptosis-inducing activity of celecoxib was abrogated when the sulfamoyl moiety was replaced by a methylsulfonyl group. The result suggested that the sulfonamide pharmacophore conferred optimal potency with regard to apoptosis induction. We further investigated whether this functional group could be replaced by a carboxamide moiety without abrogating apoptosis-inducing activities of the resulting compounds. Accordingly, we determined the apoptosis indices for compounds 30–39, which possess a carboxamide group in place of the sulfonamide group present in compound 6, compounds 8–10, celecoxib, compound 15, and compounds 18–21. As shown in Fig 5, A, replacement of the sulfonamide group in compounds 8, 9, 10, and 19 with a carboxamide group to produce compounds 31, 32, 33, and 37, respectively, had no substantial effect on the potency of the resulting compounds in apoptosis induction. However, for the rest of the compounds examined (i.e., compounds 30, 34, 35, 36, 38, and 39), the replacement of the sulfonamide group with a carboxamide group resulted in a substantial reduction in their apoptosis-inducing activities. This observation suggested that these two pharmacophores (i.e., carboxamide and sulfonamide) may cause some compounds that contain them to interact differently with the target protein(s) that affect apoptosis. With compound 37 as a representative of this class of derivatives, Fig. 5, B, shows evidence of drug-induced apoptotic death, which included time-dependent effects on nucleosomal formation (treated versus control, ANOVA, P<.001) and PARP cleavage. Moreover, the structural modification of compound 19 to compound 37 did not alter the mechanism by which this carboxamide-containing compound mediated apoptosis, i.e., by facilitating Akt and ERK2 dephosphorylation (Fig. 5, C). Similar results were obtained with the other active compounds in this group. Contributions of the Heterocyclic System to the Apoptosis-Inducing Activity of Celecoxib The sulfonamide-containing counterparts of rofecoxib and DuP697 did not show substantial apoptosis-inducing activities in PC-3 cells (data not shown). This finding indicated that structural components other than the sulfonamide group may play a role in interacting with the target protein(s) responsible for apoptosis. To shed light on this issue, we further examined the effect of a number of benzenesulfonamides with different heterocyclic rings (i.e., compounds 40–46) on the viability of PC-3 cells. Despite the presence of the sulfonamide group, none of these compounds displayed appreciable apoptosis induction at 24 hours (data not shown). Both replacement of the pyrazole ring with other heterocyclic systems and removal of the trifluoromethyl moiety from the pyrazole ring of celecoxib eliminated the apoptosis-inducing activity of the resulting compounds. This finding indicates the effect of the heterocyclic system on the apoptosis-inducing activity. Molecular Modeling The above data prompted us to examine how the heterocyclic ring might contribute to the interaction of celecoxib with the signaling target(s) responsible for apoptosis. We therefore conducted a molecular modeling analysis of the two prototypic drugs celecoxib and rofecoxib to examine the electrostatic potentials that surround the heterocyclic systems in these compounds (Fig. 6, A and B). The electron density of individual areas is colored blue to indicate negative electrostatic potentials and red to indicate positive electrostatic potentials. Changes in electrostatic potential from negative to positive are seen in transition from blue to red. As shown in Fig. 6, A and B, the pyrazole and lactone rings had opposite electron density profiles. This finding suggests that the heterocyclic ring in rofecoxib is more electropositive than the heterocyclic ring in celecoxib. On the basis of this computer modeling data, we attempted to alter the surface potential of rofecoxib to mimic that of celecoxib by repositioning the lactone carbonyl group in the opposite orientation (Fig. 6, C). The total electrostatic potential map of the resulting isomer, compound 47, was similar to that of celecoxib (Fig. 6, A versus C). However, because compound 47, which contained a methylsulfone group, showed poor activity in eliciting apoptosis in PC-3 cells (apoptosis index, −) (data not shown), we also modeled the surface electrostatic potentials of the corresponding sulfonamide-containing compound, compound 48, and its dichloro- analogues, compounds 49 and 50. Compound 49, which had a substantially higher apoptosis index (T50% at 50 μM = 15 hours; apoptosis index, +) than rofecoxib, compound 48, or compound 50 (each of which showed no appreciable apoptosis induction at 24 hours; apoptosis index, – [data not shown]), had an electrostatic potential map that strongly resembled that of its pyrazole counterpart, compound 8 (apoptosis index, +++) (Fig. 3). In addition, the similarity in the electrostatic potential profile of compound 49 and compound 8 (Fig. 6, D and E) was akin to the similarity in the profiles of celecoxib and compound 47, but improved on the 5-aryl moiety. These data demonstrate that a clear understanding of the stereo-electronic characteristics of the entire conjugated system may provide a novel way of associating structural changes with the apoptosis-inducing activities of these compounds. Discussion Our previous demonstration that the effects of COX-2 inhibitors on apoptosis were distinct from their effects on COX-2 inhibition (25) suggested that effects on signaling targets other than COX-2, i.e., those involved in apoptosis, could be pharmacologically exploited to generate novel apoptosis-inducing agents. This study was thus aimed at identifying the key structural elements of the COX-2 inhibitor celecoxib that contribute to its apoptosis-inducing activity in human prostate cancer cells. On the basis of our structure–function data, we propose a working model that outlines the structural features essential for the apoptotic effects of celecoxib (Fig. 7). We found that the structural requirements for apoptosis induction are different from those for COX-2 inhibition. In particular, the induction of apoptosis in PC-3 cells by compounds derived from COX-2 inhibitors had stringent requirements with regard to the heterocyclic system and the amide moiety (in the form of sulfonamide or carboxamide groups). These structural features highlight the potential importance of surface electrostatic potential and hydrogen bonding for interactions between these compounds and the protein target(s), and they may account for the discrepancy in the apoptosis-inducing activities of celecoxib and the other COX-2 inhibitors we examined. We also found that apolar substituents at the terminal phenyl function had a profound effect in that enlargement of the hydrophobic aryl group of celecoxib enhanced the apoptosis-inducing activity of the resulting compound. This flexibility in functionality is in sharp contrast to the strict requirements necessary to maintain good COX-2 inhibitory activity (29). This model was validated by our structure–function data for converting rofecoxib to compound 49. Computer modeling analysis of celecoxib versus rofecoxib suggested a link between the surface electrostatic potential surrounding the heterocyclic system and apoptosis-inducing potency. A comparison of the surface electrostatic potentials of celecoxib (apoptosis index, ++) and rofecoxib (apoptosis index, −) revealed important differences in electron density. We therefore modified the structure of rofecoxib to approximate the surface electrostatic potential of celecoxib, which resulted in the design of structural variants compounds 47–50. Among the variants, compound 49 showed a substantial increase in apoptosis-inducing activity. Our statistical analysis of data from three different prostate cancer cell lines suggests that the induction of apoptosis by the active compounds was not dependent on androgen sensitivity, p53 functional status, or the level of Bcl-2 expression. The effectiveness of these agents against androgen-independent prostate cancer (i.e., PC-3) cells is especially noteworthy. Metastatic prostate cancers are lethal because they are heterogeneously composed of both androgen-dependent and androgen-independent malignant cells (35,36). Because androgen-independent prostate cancer cells are resistant to the induction of apoptosis by androgen ablative therapy, an important strategy in developing effective chemotherapy for metastatic prostate cancer is to specifically eliminate androgen-independent cells by targeted apoptosis (35,36). The apoptotic action of these celecoxib derivatives against androgen-independent prostate cancer cells underscores their unique signaling mechanism in disrupting multiple signaling pathways (i.e., Akt and ERK2) that are essential to cancer cell survival (26,27). In addition to its effects on apoptosis induction, celecoxib has effects on angiogenesis (37–40). It has been reported that celecoxib suppresses corneal blood vessel formation in a rat model via a COX-2-dependent mechanism (38). However, our preliminary data indicate that celecoxib derivatives that can induce apoptosis in prostate cancer cells but that lack COX-2 inhibitory activity can also inhibit angiogenesis in the yolk sac of chicken embryos, with potencies comparable with or higher than that of celecoxib (Kulp S, Lin H, Zhu J, Ward P, Chen K, and Chen C: unpublished results). This finding suggests that the anti-angiogenic activity of celecoxib may, in part, be attributable to a COX-2-independent pathway. In view of the crucial role of Akt and ERK2 signaling in embryonal angiogenesis (41), it is possible that the anti-angiogenic effects of celecoxib and its derivatives are mediated through a mechanism similar to the one that induces apoptosis, i.e., Akt and ERK2 dephosphorylation. It is important to note that, when given at therapeutic doses (oral administration of 400–800 mg per day), celecoxib reaches peak plasma concentrations of 3–8 μM (42), severalfold lower than the concentrations required to induce apoptosis in prostate cancer cells in serum-free medium (i.e., 25 μM or higher). It is conceivable that the observed in vivo antitumor activity of celecoxib may arise from the concerted action of multiple mechanisms that include both the induction of apoptosis and the inhibition of angiogenesis. Consequently, our current research focuses on discerning the relative contributions of these mechanisms to the in vivo effects of celecoxib and its derivatives on tumor growth. In addition, investigations of the pharmacokinetic, pharmacodynamic, and toxicity profiles of these apoptosis-inducing agents are under way. Supported by Public Health Service grants CA92307 and CA94829 from the National Cancer Institute (NCI), National Institutes of Health (NIH), Department of Health and Human Services (DHHS); by Army grant DAMD17-02-1-0117 (to C.-S. Chen); and by grant P30 CA16058 from the NCI, NIH, DHHS (to the Comprehensive Cancer Center, The Ohio State University). Table 1. List of compounds used in this study Designation  Full chemical name  compound 1  1,2-difluoro-4-(4-fluorophenyl)-5-[4-(methylsulfonyl)phenyl]benzene  compound 1-NH2  4-[4,5-difluoro-2-(4-fluorophenyl)phenyl]benzenesulfonamide  compound 2  4-[5-phenyl-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 3  4-[5-(4-fluorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 4  4-[5-(2,4-difluorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 5  4-[5-(2,5-difluorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 6  4-[5-(4-chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 7  4-[5-(2-chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 8  4-[5-(2,4-dichlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 9  4-[5-(2,5-dichlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 10  4-[5-(3,4-dichlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 11  4-[5-(4-hydroxyphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 12  4-[5-(4-aminophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 13  4-[5-(4-nitrophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 14  4-[5-(4-azidophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 15  4-[5-(4-trifluoromethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 16  4-[5-(4-cyanophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 17  4-[5-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 18  4-[5-(4-ethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 19  4-[5-(2,4-dimethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 20  4-[5-(2,5-dimethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 21  4-[5-(3,4-dimethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 22  4-[5-(2,4-dimethoxyphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 23  4-[5-(2,5-dimethoxyphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 24  4-[5-(2-pyridinyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 25  4-[5-(1-cyclohexen-1-yl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 26  4-[5-(2-furanyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 27  4-[5-(2-thienyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 28  4-[5-(5-chloro-2-thienyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 29  4-[5-(2,5-dichloro-3-thienyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 30  4-[5-(4-chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenecarboxyamide  compound 31  4-[5-(2,4-dichlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenecarboxyamide  compound 32  4-[5-(2,5-dichlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenecarboxyamide  compound 33  4-[5-(3,4-dichlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenecarboxyamide  compound 34  4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenecarboxyamide  compound 35  4-[5-(4-trifluoromethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenecarboxyamide  compound 36  4-[5-(4-ethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenecarboxyamide  compound 37  4-[5-(2,4-dimethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenecarboxyamide  compound 38  4-[5-(2,5-dimethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenecarboxyamide  compound 39  4-[5-(3,4-dimethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenecarboxyamide  compound 40  4-[5-(4-methylphenyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 41  4-[5-methyl-3-(4-methylphenyl)-4-isoxazolyl]benzenesulfonamide  compound 42  2-(4-methylphenyl)-5-(4-sulfamoylphenyl)thiophene  compound 43  4-[2,3-dihydro-3-(4-methylphenyl)-2-oxo-4-oxazolyl]benzenesulfonamide  compound 44  4-[2,3-dihydro-5-methyl-3-(4-methylphenyl)-2-oxo-4-oxazolyl]benzenesulfonamide  compound 45  4-[2,3-dihydro-3-(3,4-dichlorophenyl)-2-oxo-4-oxazolyl]benzenesulfonamide  compound 46  4-[2,3-dihydro-5-methyl-3-(3,4-dichlorophenyl)-2-oxo-4-oxazolyl]benzenesulfonamide  compound 47  3-(4-methylsulfonylphenyl)-4-phenyl-2(5H)-furanone  compound 48  3-(4-sulfamoylphenyl)-4-phenyl-2(5H)-furanone  compound 49  3-(4-sulfamoylphenyl)-4-(2,4-dichlorophenyl)-2(5H)-furanone  compound 50  3-(4-sulfamoylphenyl)-4-(3,4-dichlorophenyl)-2(5H)-furanone  Designation  Full chemical name  compound 1  1,2-difluoro-4-(4-fluorophenyl)-5-[4-(methylsulfonyl)phenyl]benzene  compound 1-NH2  4-[4,5-difluoro-2-(4-fluorophenyl)phenyl]benzenesulfonamide  compound 2  4-[5-phenyl-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 3  4-[5-(4-fluorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 4  4-[5-(2,4-difluorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 5  4-[5-(2,5-difluorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 6  4-[5-(4-chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 7  4-[5-(2-chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 8  4-[5-(2,4-dichlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 9  4-[5-(2,5-dichlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 10  4-[5-(3,4-dichlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 11  4-[5-(4-hydroxyphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 12  4-[5-(4-aminophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 13  4-[5-(4-nitrophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 14  4-[5-(4-azidophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 15  4-[5-(4-trifluoromethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 16  4-[5-(4-cyanophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 17  4-[5-(4-methoxyphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 18  4-[5-(4-ethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 19  4-[5-(2,4-dimethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 20  4-[5-(2,5-dimethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 21  4-[5-(3,4-dimethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 22  4-[5-(2,4-dimethoxyphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 23  4-[5-(2,5-dimethoxyphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 24  4-[5-(2-pyridinyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 25  4-[5-(1-cyclohexen-1-yl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 26  4-[5-(2-furanyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 27  4-[5-(2-thienyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 28  4-[5-(5-chloro-2-thienyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 29  4-[5-(2,5-dichloro-3-thienyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 30  4-[5-(4-chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenecarboxyamide  compound 31  4-[5-(2,4-dichlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenecarboxyamide  compound 32  4-[5-(2,5-dichlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenecarboxyamide  compound 33  4-[5-(3,4-dichlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenecarboxyamide  compound 34  4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenecarboxyamide  compound 35  4-[5-(4-trifluoromethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenecarboxyamide  compound 36  4-[5-(4-ethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenecarboxyamide  compound 37  4-[5-(2,4-dimethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenecarboxyamide  compound 38  4-[5-(2,5-dimethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenecarboxyamide  compound 39  4-[5-(3,4-dimethylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenecarboxyamide  compound 40  4-[5-(4-methylphenyl)-1H-pyrazol-1-yl]benzenesulfonamide  compound 41  4-[5-methyl-3-(4-methylphenyl)-4-isoxazolyl]benzenesulfonamide  compound 42  2-(4-methylphenyl)-5-(4-sulfamoylphenyl)thiophene  compound 43  4-[2,3-dihydro-3-(4-methylphenyl)-2-oxo-4-oxazolyl]benzenesulfonamide  compound 44  4-[2,3-dihydro-5-methyl-3-(4-methylphenyl)-2-oxo-4-oxazolyl]benzenesulfonamide  compound 45  4-[2,3-dihydro-3-(3,4-dichlorophenyl)-2-oxo-4-oxazolyl]benzenesulfonamide  compound 46  4-[2,3-dihydro-5-methyl-3-(3,4-dichlorophenyl)-2-oxo-4-oxazolyl]benzenesulfonamide  compound 47  3-(4-methylsulfonylphenyl)-4-phenyl-2(5H)-furanone  compound 48  3-(4-sulfamoylphenyl)-4-phenyl-2(5H)-furanone  compound 49  3-(4-sulfamoylphenyl)-4-(2,4-dichlorophenyl)-2(5H)-furanone  compound 50  3-(4-sulfamoylphenyl)-4-(3,4-dichlorophenyl)-2(5H)-furanone  View Large Table 2. 1H-NMR, high resolution mass spectrometry and elemental analysis data for compounds 30–39 and 47–50 Designation  Chemical shift, ppm*  Splitting pattern†  Coupling constant (J), Hz  No. of protons  Actual mass  Theoretical mass  Molecular formula  Actual composition, %  Theoretical composition, %  *Compounds 33, 35, and 48–50 used DMSO-d6 as a solvent in a 250MHz 1H-NMR; all the other compounds used CDCl3 as a solvent in a 250MHz 1H-NMR. – = not applicable.  †The abbreviations s, bs, d, dd, t, q, m, and J used in describing 1H-NMR parameters denote singlet, broad singlet, doublet, doublet doublet, triplet, quartet, multiplet, and coupling constant, respectively.  Compound 30  6.77  s  –  1  365.0522  365.0535  C17H11CIF3N3O  C = 55.72  C = 55.88    7.16  d  8.4  2        H = 3.00  H = 3.04    7.34  d  8.4  2        N = 11.45  N = 11.51    7.40  d  8.4  2              7.83  d  8.4  2            Compound 31  6.79  s  –  1  399.0138  399.0145  C17H10Cl2F3N3O  C = 51.23  C = 51.13    7.22  d  8.2  1        H = 2.76  H = 2.53    7.27  d  8.2  1        N = 10.32  N = 10.53    7.35  d  8.6  2              7.45  d  2.0  1              7.78  d  8.6  2            Compound 32  6.79  s  –  1  399.0150  399.0145  C17H10Cl2F3N3O  C = 49.99  C = 51.13    7.37–7.40  m  –  5        H = 2.85  H = 2.53    7.80  d  8.5  2        N = 10.16  N = 10.53  Compound 33  7.37  s  –  1  399.0162  399.0145  C17H10Cl2F3N3O  C = 51.12  C = 51.13    7.55  d  8.5  2        H = 2.76  H = 2.53    7.66  d  8.5  2        N = 10.43  N = 10.53    7.71  d  2.0  1              7.95  d  8.5  2            Compound 34  2.37  s  –  3  345.1057  345.1081  C18H14F3N3O  C = 62.59  C = 62.48    6.74  s  –  1        H = 4.31  H = 4.37    7.10  d  8.2  2        N = 12.14  N = 12.24    7.15  d  8.2  2              7.41  dd  1.8, 6.7  2              7.80  dd  1.8, 6.7  2            Compound 35  7.45  s  –  1  399.0791  399.0798  C18H11F6N3O  C = 53.28  C = 54.12    7.54  d  8.0  2        H = 2.98  H = 2.78    7.62  d  8.0  2        N = 10.45  N = 10.52    7.79  d  8.4  2              8.03  d  8.4  2            Compound 36  1.24  t  7.6  3  359.1247  359.1238  C19H16F3N3O  C = 63.51  C = 63.49    2.67  q  7.6  2        H = 4.67  H = 4.49    6.74  s  –  1        N = 11.69  N = 11.70    7.13  dd  2.2, 6.2  4              7.42  d  8.5  2              7.81  d  8.5  2            Compound 37  1.94  s  –  3  359.1240  359.1238  C19H16F3N3O  C = 63.58  C = 63.49    2.35  s  –  3        H = 4.88  H = 4.49    6.65  s  –  1        N = 11.47  N = 11.70    7.03  bs  –  1              7.08  d  8.2  2              7.35  d  8.6  2              7.73  d  8.6  2            Compound 38  1.90  s  –  3  359.1268  359.1238  C19H16F3N3O  C = 63.59  C = 63.49    2.32  s  –  3        H = 4.74  H = 4.49    6.65  s  –  1        N = 11.57  N = 11.70    7.08  m  –  3              7.36  d  8.5  2              7.73  d  8.5  2            Compound 39  1.91  s  –  3  359.1257  359.1238  C19H16F3N3O  C = 63.57  C = 63.49    2.34  s  –  3        H = 4.63  H = 4.49    6.67  s  –  1        N = 11.67  N = 11.70    7.08  m  –  3              7.36  d  8.5  2              7.73  d  8.5  2            Compound 47  3.08  s  –  3  314.0632  314.0605  C17H14O4S  C = 64.86  C = 64.96    5.24  s  –  2        H = 4.61  H = 4.61    7.18–7.30  m  –  5        N = 0  N = 0    7.66  dd  1.9, 6.7  2              7.96  dd  1.9, 6.7  2            Compound 48  5.43  s  –  2  315.0573  315.0557  C16H13NO4S  C = 60.45  C = 60.94    7.36–7.45  m  –  5        H = 4.29  H = 4.16    7.76  d  8.5  2        N = 4.81  N = 4.44    7.96  d  8.5  2            Compound 49  5.42  s  –  2  382.9764  382.9778  C16H11Cl2NO4S  C = 49.22  C = 50.01    7.26  dd  2.5, 8.5  1        H = 2.83  H = 2.89    7.42  bs  –  1        N = 3.27  N = 3.65    7.55  d  8.5  1              7.71  d  8.5  2              7.86  d  8.5  2            Compound 50  5.42  s  –  2  382.9785  382.9778  C16H11Cl2NO4S  C = 50.31  C = 50.01    7.26  dd  2.5, 8.5  1        H = 2.60  H = 2.89    7.42  bs  –  1        N = 3.40  N = 3.65    7.55  d  8.5  1              7.71  d  8.5  2              7.86  d  8.5  2            Designation  Chemical shift, ppm*  Splitting pattern†  Coupling constant (J), Hz  No. of protons  Actual mass  Theoretical mass  Molecular formula  Actual composition, %  Theoretical composition, %  *Compounds 33, 35, and 48–50 used DMSO-d6 as a solvent in a 250MHz 1H-NMR; all the other compounds used CDCl3 as a solvent in a 250MHz 1H-NMR. – = not applicable.  †The abbreviations s, bs, d, dd, t, q, m, and J used in describing 1H-NMR parameters denote singlet, broad singlet, doublet, doublet doublet, triplet, quartet, multiplet, and coupling constant, respectively.  Compound 30  6.77  s  –  1  365.0522  365.0535  C17H11CIF3N3O  C = 55.72  C = 55.88    7.16  d  8.4  2        H = 3.00  H = 3.04    7.34  d  8.4  2        N = 11.45  N = 11.51    7.40  d  8.4  2              7.83  d  8.4  2            Compound 31  6.79  s  –  1  399.0138  399.0145  C17H10Cl2F3N3O  C = 51.23  C = 51.13    7.22  d  8.2  1        H = 2.76  H = 2.53    7.27  d  8.2  1        N = 10.32  N = 10.53    7.35  d  8.6  2              7.45  d  2.0  1              7.78  d  8.6  2            Compound 32  6.79  s  –  1  399.0150  399.0145  C17H10Cl2F3N3O  C = 49.99  C = 51.13    7.37–7.40  m  –  5        H = 2.85  H = 2.53    7.80  d  8.5  2        N = 10.16  N = 10.53  Compound 33  7.37  s  –  1  399.0162  399.0145  C17H10Cl2F3N3O  C = 51.12  C = 51.13    7.55  d  8.5  2        H = 2.76  H = 2.53    7.66  d  8.5  2        N = 10.43  N = 10.53    7.71  d  2.0  1              7.95  d  8.5  2            Compound 34  2.37  s  –  3  345.1057  345.1081  C18H14F3N3O  C = 62.59  C = 62.48    6.74  s  –  1        H = 4.31  H = 4.37    7.10  d  8.2  2        N = 12.14  N = 12.24    7.15  d  8.2  2              7.41  dd  1.8, 6.7  2              7.80  dd  1.8, 6.7  2            Compound 35  7.45  s  –  1  399.0791  399.0798  C18H11F6N3O  C = 53.28  C = 54.12    7.54  d  8.0  2        H = 2.98  H = 2.78    7.62  d  8.0  2        N = 10.45  N = 10.52    7.79  d  8.4  2              8.03  d  8.4  2            Compound 36  1.24  t  7.6  3  359.1247  359.1238  C19H16F3N3O  C = 63.51  C = 63.49    2.67  q  7.6  2        H = 4.67  H = 4.49    6.74  s  –  1        N = 11.69  N = 11.70    7.13  dd  2.2, 6.2  4              7.42  d  8.5  2              7.81  d  8.5  2            Compound 37  1.94  s  –  3  359.1240  359.1238  C19H16F3N3O  C = 63.58  C = 63.49    2.35  s  –  3        H = 4.88  H = 4.49    6.65  s  –  1        N = 11.47  N = 11.70    7.03  bs  –  1              7.08  d  8.2  2              7.35  d  8.6  2              7.73  d  8.6  2            Compound 38  1.90  s  –  3  359.1268  359.1238  C19H16F3N3O  C = 63.59  C = 63.49    2.32  s  –  3        H = 4.74  H = 4.49    6.65  s  –  1        N = 11.57  N = 11.70    7.08  m  –  3              7.36  d  8.5  2              7.73  d  8.5  2            Compound 39  1.91  s  –  3  359.1257  359.1238  C19H16F3N3O  C = 63.57  C = 63.49    2.34  s  –  3        H = 4.63  H = 4.49    6.67  s  –  1        N = 11.67  N = 11.70    7.08  m  –  3              7.36  d  8.5  2              7.73  d  8.5  2            Compound 47  3.08  s  –  3  314.0632  314.0605  C17H14O4S  C = 64.86  C = 64.96    5.24  s  –  2        H = 4.61  H = 4.61    7.18–7.30  m  –  5        N = 0  N = 0    7.66  dd  1.9, 6.7  2              7.96  dd  1.9, 6.7  2            Compound 48  5.43  s  –  2  315.0573  315.0557  C16H13NO4S  C = 60.45  C = 60.94    7.36–7.45  m  –  5        H = 4.29  H = 4.16    7.76  d  8.5  2        N = 4.81  N = 4.44    7.96  d  8.5  2            Compound 49  5.42  s  –  2  382.9764  382.9778  C16H11Cl2NO4S  C = 49.22  C = 50.01    7.26  dd  2.5, 8.5  1        H = 2.83  H = 2.89    7.42  bs  –  1        N = 3.27  N = 3.65    7.55  d  8.5  1              7.71  d  8.5  2              7.86  d  8.5  2            Compound 50  5.42  s  –  2  382.9785  382.9778  C16H11Cl2NO4S  C = 50.31  C = 50.01    7.26  dd  2.5, 8.5  1        H = 2.60  H = 2.89    7.42  bs  –  1        N = 3.40  N = 3.65    7.55  d  8.5  1              7.71  d  8.5  2              7.86  d  8.5  2            View Large Fig. 1. View largeDownload slide Structure of celecoxib and some derivatives. A) Overall strategy for the structural modification of celecoxib. A, B, and C denote the three modification strategies that target the terminal phenyl ring, the sulfonamide group, and the heterocyclic system, respectively. The numbers 1, 3, and 5 indicate the position of substituents on the pyrazole ring. B) Chemical structures of compounds 40–46 and 48–50. Fig. 1. View largeDownload slide Structure of celecoxib and some derivatives. A) Overall strategy for the structural modification of celecoxib. A, B, and C denote the three modification strategies that target the terminal phenyl ring, the sulfonamide group, and the heterocyclic system, respectively. The numbers 1, 3, and 5 indicate the position of substituents on the pyrazole ring. B) Chemical structures of compounds 40–46 and 48–50. Fig. 2. View largeDownload slide Effect of sulfonamide versus methylsulfone on the apoptosis-inducing activity of various COX-2 inhibitors in PC-3 cells. A) Structures, IC50 values for COX-2 inhibition, and apoptosis indices of various COX-2 inhibitors. PC-3 cells were exposed to the test agent (50 μM) in serum-free RPMI-1640 medium to assess the effects of the agent on apoptosis. IC50 represents the concentration required to inhibit 50% of the COX-2 enzyme activity. The apoptosis index was determined as described in the “Materials and Methods” section. B) Comparison of the effects of sulfonamide versus methylsulfone pharmacophores on cell viability. Cell viability was analyzed by the trypan blue dye exclusion assay. Left panel, celecoxib (R = NH2) and its methylsulfone counterpart (R = CH3); right panel, compound 1 (R = CH3) and compound 1-NH2 (R = NH2). Data are presented as the mean values of three experiments; bars represent 95% confidence intervals. Fig. 2. View largeDownload slide Effect of sulfonamide versus methylsulfone on the apoptosis-inducing activity of various COX-2 inhibitors in PC-3 cells. A) Structures, IC50 values for COX-2 inhibition, and apoptosis indices of various COX-2 inhibitors. PC-3 cells were exposed to the test agent (50 μM) in serum-free RPMI-1640 medium to assess the effects of the agent on apoptosis. IC50 represents the concentration required to inhibit 50% of the COX-2 enzyme activity. The apoptosis index was determined as described in the “Materials and Methods” section. B) Comparison of the effects of sulfonamide versus methylsulfone pharmacophores on cell viability. Cell viability was analyzed by the trypan blue dye exclusion assay. Left panel, celecoxib (R = NH2) and its methylsulfone counterpart (R = CH3); right panel, compound 1 (R = CH3) and compound 1-NH2 (R = NH2). Data are presented as the mean values of three experiments; bars represent 95% confidence intervals. Fig. 3. View largeDownload slide Apoptosis induction and COX-2 inhibition data for celecoxib and compounds 2–29. The general structure of these molecules is shown at the top. Ar represents different aromatic ring structures contained on the compounds. The apoptosis index (AI) was determined as described in the “Materials and Methods” section. IC50 represents the concentration required to inhibit 50% of the COX-2 enzyme activity; IC50 values were taken from (29). N.D. = not determined. Fig. 3. View largeDownload slide Apoptosis induction and COX-2 inhibition data for celecoxib and compounds 2–29. The general structure of these molecules is shown at the top. Ar represents different aromatic ring structures contained on the compounds. The apoptosis index (AI) was determined as described in the “Materials and Methods” section. IC50 represents the concentration required to inhibit 50% of the COX-2 enzyme activity; IC50 values were taken from (29). N.D. = not determined. Fig. 4. View largeDownload slide Induction of apoptosis in PC-3 cells by compound 10. A) Time- and dose-dependent effects of compound 10 on the cell viability of PC-3 cells in serum-free RPMI-1640 medium. Values obtained from three replicates were plotted for each time point at the indicated concentration of compound 10. Control PC-3 cells were treated with a dimethyl sulfoxide (DMSO) vehicle. B) Evidence of apoptotic death in compound 10-treated PC-3 cells. Left panel, time course of the formation of cytoplasmic nucleosomal DNA in PC-3 cells treated with DMSO vehicle or compound 10 (50 μM). The formation of cytoplasmic nucleosomal DNA was quantitatively measured by a cell death detection enzyme-linked immunosorbent assay with lysates equivalent to 104 cells for each assay. Data are presented as means; error bars represent 95% confidence intervals (n = 3). Right panel, induction of poly(ADP-ribose) polymerase (PARP) cleavage by compound 10 in PC-3 cells. PC-3 cells were treated with 50 μM compound 10 for the indicated times. PARP proteolysis to the apoptosis-specific 85-kd fragment was monitored by western blotting. O.D. = optical density. C) Time-dependent effects of 50 μM compound 10 on Akt and ERK2 phosphorylation. PC-3 cells were treated with 50 μM compound 10 for the indicated times and lysed, and proteins in the resulting supernatants were resolved on sodium dodecyl sulfate–polyacrylamide gels and subjected to western blot analysis. The phosphorylation status of Akt and ERK2 was determined by immunoblotting with the respective phospho-specific antibodies. Unphosphorylated Akt and ERK2, as immunostained by anti-Akt and anti-ERK2 antibodies, were used as internal standards for the comparison of phospho-Akt and phospho-ERK2 levels among samples of different exposure intervals. The blots are representative of three independent experiments. p-Akt = phosphorylated Akt; p-ERK2 = phosphorylated ERK2. Fig. 4. View largeDownload slide Induction of apoptosis in PC-3 cells by compound 10. A) Time- and dose-dependent effects of compound 10 on the cell viability of PC-3 cells in serum-free RPMI-1640 medium. Values obtained from three replicates were plotted for each time point at the indicated concentration of compound 10. Control PC-3 cells were treated with a dimethyl sulfoxide (DMSO) vehicle. B) Evidence of apoptotic death in compound 10-treated PC-3 cells. Left panel, time course of the formation of cytoplasmic nucleosomal DNA in PC-3 cells treated with DMSO vehicle or compound 10 (50 μM). The formation of cytoplasmic nucleosomal DNA was quantitatively measured by a cell death detection enzyme-linked immunosorbent assay with lysates equivalent to 104 cells for each assay. Data are presented as means; error bars represent 95% confidence intervals (n = 3). Right panel, induction of poly(ADP-ribose) polymerase (PARP) cleavage by compound 10 in PC-3 cells. PC-3 cells were treated with 50 μM compound 10 for the indicated times. PARP proteolysis to the apoptosis-specific 85-kd fragment was monitored by western blotting. O.D. = optical density. C) Time-dependent effects of 50 μM compound 10 on Akt and ERK2 phosphorylation. PC-3 cells were treated with 50 μM compound 10 for the indicated times and lysed, and proteins in the resulting supernatants were resolved on sodium dodecyl sulfate–polyacrylamide gels and subjected to western blot analysis. The phosphorylation status of Akt and ERK2 was determined by immunoblotting with the respective phospho-specific antibodies. Unphosphorylated Akt and ERK2, as immunostained by anti-Akt and anti-ERK2 antibodies, were used as internal standards for the comparison of phospho-Akt and phospho-ERK2 levels among samples of different exposure intervals. The blots are representative of three independent experiments. p-Akt = phosphorylated Akt; p-ERK2 = phosphorylated ERK2. Fig. 5. View largeDownload slide Effect of carboxamide derivatives on apoptosis in PC-3 cells. A) Activities of compounds 30–39 in inducing apoptosis in PC-3 cells. The general structure of these molecules is shown at the top. PC-3 cells were exposed to the test agent (50 μM) in serum-free RPMI-1640 medium. Ar represents different aromatic ring structures contained on the compounds. The apoptosis index was determined as described in the “Materials and Methods” section. The cell viability was analyzed by trypan blue dye exclusion assay. B) Evidence for apoptotic death in compound 37-treated PC-3 cells. Left panel, time course of the formation of cytoplasmic nucleosomal DNA in PC-3 cells treated with dimethyl sulfoxide (DMSO) vehicles or compound 37 (50 μM) [ANOVA, P<.001; treated versus DMSO at 10, 20, and 30 minutes]. Data are presented as the mean values of three experiments; bars represent 95% confidence intervals. Right panel, induction of poly(ADP-ribose) polymerase cleavage by compound 37 in PC-3 cells. C) Time-dependent effect of 50 μM compound 37 on Akt and ERK2 phosphorylation. The blots are representative of three independent experiments. All of the above experiments were carried out in a manner similar to that described for Fig. 4. Fig. 5. View largeDownload slide Effect of carboxamide derivatives on apoptosis in PC-3 cells. A) Activities of compounds 30–39 in inducing apoptosis in PC-3 cells. The general structure of these molecules is shown at the top. PC-3 cells were exposed to the test agent (50 μM) in serum-free RPMI-1640 medium. Ar represents different aromatic ring structures contained on the compounds. The apoptosis index was determined as described in the “Materials and Methods” section. The cell viability was analyzed by trypan blue dye exclusion assay. B) Evidence for apoptotic death in compound 37-treated PC-3 cells. Left panel, time course of the formation of cytoplasmic nucleosomal DNA in PC-3 cells treated with dimethyl sulfoxide (DMSO) vehicles or compound 37 (50 μM) [ANOVA, P<.001; treated versus DMSO at 10, 20, and 30 minutes]. Data are presented as the mean values of three experiments; bars represent 95% confidence intervals. Right panel, induction of poly(ADP-ribose) polymerase cleavage by compound 37 in PC-3 cells. C) Time-dependent effect of 50 μM compound 37 on Akt and ERK2 phosphorylation. The blots are representative of three independent experiments. All of the above experiments were carried out in a manner similar to that described for Fig. 4. Fig. 6. View largeDownload slide Molecular modeling of celecoxib (A), rofecoxib (B), compound 47 (C), compound 49 (D), and compound 8 (E). The electrostatic potential of individual areas is coded in color: blue and red denote negative and positive electrostatic potentials, respectively. The pyrazole and lactone rings of celecoxib (panel A) and rofecoxib (panel B) were colored blue and red, respectively, in the chemical structures and indicated by arrows in the electrostatic potential images. Other colors, such as green and yellow, reflect transitions between negative and positive electrostatic potentials (and vice versa). Fig. 6. View largeDownload slide Molecular modeling of celecoxib (A), rofecoxib (B), compound 47 (C), compound 49 (D), and compound 8 (E). The electrostatic potential of individual areas is coded in color: blue and red denote negative and positive electrostatic potentials, respectively. The pyrazole and lactone rings of celecoxib (panel A) and rofecoxib (panel B) were colored blue and red, respectively, in the chemical structures and indicated by arrows in the electrostatic potential images. Other colors, such as green and yellow, reflect transitions between negative and positive electrostatic potentials (and vice versa). Fig. 7. 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TI - Using Cyclooxygenase-2 Inhibitors as Molecular Platforms to Develop a New Class of Apoptosis-Inducing Agents
JF - JNCI: Journal of the National Cancer Institute
DO - 10.1093/jnci/94.23.1745
DA - 2002-12-04
UR - https://www.deepdyve.com/lp/oxford-university-press/using-cyclooxygenase-2-inhibitors-as-molecular-platforms-to-develop-a-giE3bEkdGj
SP - 1745
EP - 1757
VL - 94
IS - 23
DP - DeepDyve
ER -