COX-2 (IC50 = 1 nM); COX-1 (IC50 >500 μM)

CAY10404 (compound 7) does not inhibit COX-1 (IC50>500 µM)[1]. With a mean 50% inhibitory concentration (IC50) of 60-100 µM, CAY10404 (10-100 µM; for 3 days) suppresses the development of NSCLC cell lines in a concentration-dependent manner [3]. For three days, CAY10404 (20–100 µM) causes NSCLC cells to undergo apoptosis [3]. The concentration-dependent reduction in the levels of pAkt, pGSK-3β, and anti-apoptotic proteins (Bcl-2 and Bcl-XL) is induced by CAY10404 (80 µM) over three days [3]. The ability of H460 cells to form colonies in anchorage-independent growth is inhibited in a concentration-dependent manner by CAY10404 (20, 50, 80, and 100 µM; for 14 days) [3].

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Treatment with CAY10404 in the range of 10-100 microM caused dose-dependent growth inhibition, with an average 50% inhibitory concentration (IC(50)) of 60-100 micromol/L, depending on the cell line. Western blot analysis of CAY10404-treated cells showed cleavage of poly (ADP-ribose) polymerase (PARP) and procaspase-3, signifying caspase activity and apoptotic cell death. CAY10404 treatment inhibited the phosphorylation of Akt, glycogen synthase kinase-3beta and extracellular signal-regulated kinases 1/2 in H460 and H358 cells. Conclusions: These results suggest that CAY10404 is a potent inducer of apoptosis in NSCLC cells, and that it may act by suppressing multiple protein kinase B/Akt and mitogen-activated protein kinase pathways [3].

In HTV mice, the intraperitoneal injection of 50 mg/kg/day of CAY10404 ameliorates lung inflammation and ventilator-induced lung damage [2].

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Inhibition of COX-2 Attenuates Ventilator-Induced Lung Injury [2]
Mice were treated with the COX-2–specific inhibitor CAY10404 (50 mg/kg/day, intraperitoneal) × 4 days before the onset of mechanical ventilation. Treatment with the COX-2–specific inhibitor CAY10404 (50 mg/kg/day for 4 days) attenuated cyclooxygenase activity, significantly decreasing BAL PGE2 and 6-keto PGF1α (Figure 3). Likewise, systemic COX-2 inhibition decreased plasma PGE2 by 66% compared with untreated HTV mice (P < 0.05). The pharmacologic inhibition of COX-2 with CAY10404 significantly decreased alveolar–capillary leakage caused by HTV mechanical ventilation (Figure 1, dark bars; P < 0.05). Treatment with CAY10404 exerted no significant effect on tissue EBD or BAL protein in control or LTV mice. Likewise, inhibiting COX-2 decreased lung inflammation in HTV mice (Figures 2A–2D, dark bars; P < 0.05), decreasing BAL leukocytes, tissue PMNs, tissue MPO, and BAL IL-6 compared with untreated HTV mice. Treatment with CAY10404 exerted no significant effect on BAL cell count, PMN score, or IL-6 in control or LTV mice, although a nonsignificant trend toward decreased lung MPO was evident in LTV mice receiving COX-2 inhibition. COX-2 inhibition exerted divergent effects on leukocyte adhesion molecules, markedly decreasing ICAM-1 expression in both ventilation groups, but increasing VCAM-1 in both ventilation groups (Figure 2E, bottom). Of note, the treatment of control mice with CAY10404 increased basal VCAM-1 expression.

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Mice were mechanically ventilated at low and high tidal volumes, in the presence or absence of pharmacologic cyclooxygenase-2-specific inhibition with 3-(4-methylsulphonylphenyl)-4-phenyl-5-trifluoromethylisoxazole (CAY10404). Lung injury was assessed using markers of alveolar-capillary leakage and lung inflammation. Cyclooxygenase-2 expression and activity were measured by Western blotting, real-time PCR, and lung/plasma prostanoid analysis, and tissue sections were analyzed for cyclooxygenase-2 staining by immunohistochemistry. High tidal volume ventilation induced lung injury, significantly increasing both lung leakage and lung inflammation relative to control and low tidal volume ventilation. High tidal volume mechanical ventilation significantly induced cyclooxygenase-2 expression and activity, both in the lungs and systemically, compared with control mice and low tidal volume mice. The immunohistochemical analysis of lung sections localized cyclooxygenase-2 expression to monocytes and macrophages in the alveoli. The pharmacologic inhibition of cyclooxygenase-2 with CAY10404 significantly decreased cyclooxygenase activity and attenuated lung injury in mice ventilated at high tidal volume, attenuating barrier disruption, tissue inflammation, and inflammatory cell signaling. This study demonstrates the induction of cyclooxygenase-2 by mechanical ventilation, and suggests that the therapeutic inhibition of cyclooxygenase-2 may attenuate ventilator-induced acute lung injury [2].

Cyclooxygenase Inhibition Studies. [1]
All compounds described herein were tested for their ability to inhibit COX-1 and COX-2 using a COX-(ovine) inhibitor screening kit. Briefly, cyclooxygenase catalyzes the first step in the biosynthesis of arachidonic acid (AA) to PGH2. PGF2α, produced from PGH2 by reduction with stannous chloride, is measured by enzyme immunoassay. This assay is based on the competition between PGs and a PG−acetylcholinesterase conjugate (PG tracer) for a limited amount of PG antiserum. The amount of PG tracer that is able to bind to the PG antiserum is inversely proportional to the concentration of PGs in the wells because the concentration of the PG tracer is held constant while the concentration of PGs varies. This antibody−PG complex binds to a mouse antirabbit monoclonal antibody that has been previously attached to the well. The plate is washed to remove any unbound reagents, and then Ellman's reagent, which contains the substrate to acetylcholinesterase, is added to the well. The product of this enzymatic reaction produces a distinct yellow color that absorbs at 405 nm. The intensity of this color, determined spectrophotometrically, is proportional to the amount of PG tracer bound to the well, which is inversely proportional to the amount of PGs present in the well during the incubation:  absorbance ∝ [bound PG tracer] ∝ 1/PGs. Percent inhibition was calculated by comparison of the compound treated to various control incubations. The concentration of the test compound causing 50% inhibition (IC50, μM) was calculated from the concentration−inhibition response curve (duplicate determinations).

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COX-2 Inhibition [2]
CAY10404 (3-(4-methylsulphonylphenyl)-4-phenyl-5-trifluoromethylisoxazole) at 50 mg/kg/day was administered by intraperitoneal injection daily for 3 days plus 1 hour before the initiation of ventilation. This dose was chosen to give an optimal balance of efficacy and toxicity, based on our preliminary dose range studies and previous published studies.

Cell Viability Assay [3]
Cell Types: Non-small cell lung cancer (NSCLC) cells (H1703, H358, H460)
Tested Concentrations: 10-100 µM
Incubation Duration: 3 days
Experimental Results: Inhibited the growth of NSCLC cell lines in a certain concentration-dependent manner.

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Apoptosis analysis[3]
Cell Types: H460 Cell
Tested Concentrations: 20, 50, 100 µM
Incubation Duration: 3 days
Experimental Results: Induction of apoptosis.

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Western Blot Analysis [3]
Cell Types: NSCLC cells (H358, H460)
Tested Concentrations: 80 µM
Incubation Duration: 3 days
Experimental Results: Concentration-dependent decrease in the levels of induced anti-apoptotic proteins (Bcl-2 and Bcl-XL) and pAkt and pGSK-3β without changing the levels of pro-apoptotic protein (Bax) and total Akt and GSK-3β protein levels.

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Cell proliferation assays [3]
To measure the effects of CAY10404 on proliferation of NSCLC cells, 3 × 103 cells/well (H-1703, H-358, H-460) were plated in 96-well plates and allowed to adhere overnight at 37°C. The following day, cells were transferred to fresh medium containing 10% serum and a range of concentrations of CAY10404 in DMSO (final concentration, 0.1%). Control cells were treated with 0.1% DMSO. At the end of the incubation period, cell proliferation was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Six replicate wells were used for each analysis. The percentage inhibition of growth was calculated from the equation: percentage inhibition of growth = (1 − At/Ac) × 100, where At and Ac represent the absorbance values for treated and control cultures, respectively. The drug concentration causing 50% inhibition of cell growth (IC50) was determined by interpolation from dose–response curves. At least three independent experiments were performed.

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Anchorage-independent growth assay [3]
The NSCLC cells were mixed with low-temperature melting agarose (0.5%) and placed over solidified agarose (1%) in six-well plates at 2000 cells/well. Both the lower and upper agarose layers contained either 0.1% DMSO (as control) or CAY10404 at different concentrations. The cell-containing upper agarose layer was allowed to solidify at 4°C and the plates were then incubated in a humidified 95% air/5% CO2 atmosphere at 37°C for 14 days. RPMI 1640 plus 10% FBS (with or without CAY10404; 0.5 mL) was placed on top of the agarose after 3 days and replaced every 3 days thereafter. At the end of the experiments, colonies >125 µm in diameter were counted under an inverted microscope (×40).

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Apoptosis assays [3]
The NSCLC cells were exposed to various concentrations of CAY10404 or to 0.1% DMSO and then allowed to grow in medium containing 10% serum for 3 days. Apotosis was measured using the APO-BrdU staining kit, a modified terminal deoxynucleotidyl transferase dUTP nick end labelling (TUNEL) assay, as described.22 Briefly, floating and adherent cells were collected and fixed with 1% paraformaldehyde followed by 70% ethanol. DNA breaks were detected by terminal deoxyuridine transferase-induced incorporation of BrdU triphosphate (Br-dUTP) into the 3’-OH ends of DNA strands. Cells were analysed on a FACScan flow cytometer equipped with a 488-nm argon laser and CellQuest software. A dual display of DNA content (linear red fluorescence) and Br-dUTP incorporation (FITC-PRB-1) was used to determine the percentage of apoptotic cells in the population. Apoptotic cells were identified as the proportion of FITC-positive cells in the total of 10 000 gated cells.

Animal/Disease Models: Adult male C57Bl/6J mice, body weight 24-30 g[2]
Doses: 50 mg/kg
Route of Administration: IP; daily; continued for 4 days
Experimental Results: Cyclooxygenase activity diminished, BAL PGE2 and 6 -ketone PGF1α was Dramatically diminished. Reduces lung inflammation (climax volume; 20 ml/kg; 4 hrs (hrs (hours)) duration) and ventilator-induced lung injury in HTV mice.

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In Vivo Model of Acute Lung Injury/Ventilator-Induced Lung Injury [2]
Adult male C57Bl/6J mice weighing 24–30 g were anesthetized with ketamine/xylazine, intratracheally intubated, and ventilated with room air at either low tidal volume (LTV, 7 ml/kg) or high tidal volume (HTV, 20 ml/kg) for 4 hours at a respiratory rate of 160 breaths/minute, with 3 cm H2O positive end-expiratory pressure. Control animals were anesthetized and allowed to breathe spontaneously. External dead space was applied to the HTV mice. All ventilated animals received an intravenous bolus of 0.5 ml sterile Ringer’s lactate at the onset of mechanical ventilation to prevent hypotension.

[1]. Design and synthesis of 4,5-diphenyl-4-isoxazolines: novel inhibitors of cyclooxygenase-2 with analgesic and antiinflammatory activity. J Med Chem. 2001 Aug 30;44(18):2921-7.

[2]. The role of cyclooxygenase-2 in mechanical ventilation-induced lung injury. Am J Respir Cell Mol Biol. 2012 Sep;47(3):387-94.

[3]. Effects of CAY10404 on the PKB/Akt and MAPK pathway and apoptosis in non-small cell lung cancer cells. Respirology. 2009 Aug;14(6):850-8.

Isoxazole, 3-[4-(methylsulfonyl)phenyl]-4-phenyl-5-(trifluoromethyl)- is a sulfonic acid derivative.

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4,5-Diphenyl-4-isoxazolines (13a-k) possessing a variety of substituents (H, F, MeS, MeSO2) at the para position of one of the phenyl rings were synthesized for evaluation as analgesic and selective cyclooxygenase-2 (COX-2) inhibitory antiinflammatory (AI) agents. Although the 4,5-phenyl-4-isoxazolines (13a-d,f), which do not have a C-3 Me substituent, exhibited potent analgesic and AI activities, those compounds evaluated (13a, 13b, 13h, and 13k) were not selective inhibitors of COX-2. In contrast, 2,3-dimethyl-5-(4-methylsulfonylphenyl)-4-phenyl-4-isoxazoline (13j) exhibited excellent analgesic and AI activities, and it was a potent and selective COX-2 inhibitor (COX-1, IC(50) = 258 microM; COX-2, IC(50) = 0.004 microM). A related compound 13k having a F substituent at the para position of the 4-phenyl ring was also a selective (SI = 3162) but less potent (IC(50) = 0.0316 microM) inhibitor of COX-2 than 13j. A molecular modeling (docking study) for 13j showed that the S atom of the MeSO2 substituent is positioned about 6.46 A inside the entrance to the COX-2 secondary pocket (Val(523)) and that a C-3 Me (13j, 13k) central isoxazoline ring substituent is crucial to selective inhibition of COX-2 for this class of compounds. [1]

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Background and objective: Lung cancer is the most common cause of cancer death in men and women worldwide. The mechanism of cell death induced by CAY10404, a highly selective cyclooxygenase-2 inhibitor, was evaluated in three non-small cell lung cancer (NSCLC) cell lines (H460, H358, H1703).
Methods: To measure the effects of CAY10404 on proliferation of NSCLC cells, 3 x 10(3) cells/well were plated in 96-well plates and allowed to adhere overnight at 37 degrees C. After treatment with CAY10404 for 3 days, cell proliferation was measured by the 3- (4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In the H460 NSCLC cells, evidence of apoptosis was sought using the terminal deoxynucleotidyl transferase deoxyuridine triphosphate (dUTP) nick end labelling (TUNEL) assay and western blot analysis. [3]

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The reason for the sensitivity of the H460 and H358 cells to CAY10404 is not fully understood. However, it may be attributable to the induction of multiple pro-apoptotic effects. Previous studies demonstrated that SCH66336, a farnesyl transferase inhibitor, and NS-398, a specific COX-2 inhibitor, decreased the levels of Bcl-2/Bcl-XL in SqCC/Y1 head and neck squamous carcinoma cells and murine B lymphoma A20 cells, respectively. However, deguelin did not change Bcl-2 protein levels in H322 NSCLC cells.32 In this study, CAY10404 treatment decreased the levels of the anti-apoptotic proteins, Bcl-2 and Bcl-XL, without affecting the level of the pro-apoptotic protein Bax, resulting in a decrease in the ratio of anti-apoptotic to pro-apoptotic proteins. This difference may be attributable to differences in cell type or COX-2 inhibitor. Nevertheless, the present results may explain the pro-apoptotic effect of CAY10404.
As part of the ongoing effort to identify the mechanism of the effects of CAY10404 on NSCLC cells, the involvement of the PKB/Akt and MAPK pathways was studied. MAPK and Akt enzymes play important roles in regulating cell apoptosis and proliferation. It has been demonstrated that PKB/Akt and MAPK have potent inhibitory effects on apoptosis.33 Phosphorylation of Akt has recently been implicated in COX-2 mediated lung cancer and survival of hepatocellular carcinoma cells. In the present study, the consequences of decreased Akt activity in H460 and H358 cells treated with CAY10404 for 3 days were lower levels of phosphorylated Akt substrates, including pGSK-3β, and these changes may also contribute to increased apoptosis. In addition, this study has demonstrated that CAY10404 decreased the levels of pAkt and pGSK-3β in H460 and H358 cells in a concentration-dependent manner, whereas total Akt and GSK-3β protein levels were not changed. In molecular work to identify the role of the MAPK pathway, expression of phospho-Erk1/2 diminished slightly after CAY10404 treatment for 3 days, whereas total Erk1/2 protein levels were not changed. These results suggested that CAY10404 preferentially affects the PKB/Akt signalling pathway, which is important in regulating cell apoptosis and proliferation. This is the first report that CAY10404 induces apoptosis in NSCLC cells by inhibiting the signal transduction mechanism involved in cellular proliferation and survival. Other reports indicated that insulin-like growth factor (IGF) binding protein-3 suppressed the IGF-I-induced activation of PI3K/Akt/PKB and MAPK pathways in NSCLC cells, and overexpression of the dnp85α regulatory subunit of PI3K-induced apoptosis, whereas LY294002 or overexpression of phosphatase and tensin homolog-induced proliferative arrest in H1299 NSCLC cells.37 In premalignant HBE cells, deguelin inhibited PI3K activity and reduced pAkt levels and activity but had minimal effects on the MAPK pathway.38 Therefore, we evaluated whether CAY10404 inhibited signal transduction pathways that suppressed PI3K and MAPK in NSCLC cells.
Many experimental and clinical studies have provided evidence that specific COX-2 inhibitors may be useful in the prevention and treatment of a variety of malignancies. However, recent studies have shown that long-term use of high concentrations of COX-2 inhibitors significantly increased the risk of cardiotoxicity. One approach to overcome this limitation is to use lower doses of COX-2 inhibitors in combination with other established drugs. The synergistic effect of two drugs used simultaneously may enable the use of the specific COX-2 inhibitor, CAY10404, at lower and safer concentrations, and pave the way for more effective treatment in human NSCLC. Future studies should investigate the simultaneous use of COX-2 inhibitors and other established drugs to improve cancer control.[2]

C17H12NO3F3S

367.342

367.049

C, 55.58; H, 3.29; F, 15.52; N, 3.81; O, 13.07; S, 8.73

340267-36-9

10429020

White to off-white solid powder

1.4±0.1 g/cm3

498.6±45.0 °C at 760 mmHg

196.47 °C(Predicted)

255.3±28.7 °C

0.0±1.2 mmHg at 25°C

1.538

2.92

0

7

3

25

547

0

CS(=O)(=O)C1=CC=C(C=C1)C2=NOC(=C2C3=CC=CC=C3)C(F)(F)F

KKBWWVXRKULXHF-UHFFFAOYSA-N

InChI=1S/C17H12F3NO3S/c1-25(22,23)13-9-7-12(8-10-13)15-14(11-5-3-2-4-6-11)16(24-21-15)17(18,19)20/h2-10H,1H3

3-[4-(Methylsulfonyl)phenyl]-4-phenyl-5-(trifluoromethyl)-isoxazole

CAY 10404; 340267-36-9; CAY10,404; CAY 10,404; 3-(4-methylsulphonylphenyl)-4-phenyl-5-trifluoromethylisoxazole; 3-(4-methylsulfonylphenyl)-4-phenyl-5-trifluoromethylisoxazole; 3-(4-methylsulfonylphenyl)-4-phenyl-5-(trifluoromethyl)-1,2-oxazole; CHEMBL97943; 3-[4-(Methylsulfonyl)phenyl]-4-phenyl-5-(trifluoromethyl)-isoxazole; CAY-10404; CAY10404

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~100 mg/mL (~272.23 mM)

Solubility in Formulation 1: 2.5 mg/mL (6.81 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: 2.5 mg/mL (6.81 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.81 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)