CAY-10404

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

CAY10404（化合物 7）不会抑制 COX-1 (IC50>500 µM)[1]。 CAY10404（10-100 µM；持续 3 天）的平均 50% 抑制浓度 (IC50) 为 60-100 µM，以浓度依赖性方式抑制 NSCLC 细胞系的发育 [3]。三天内，CAY10404 (20–100 µM) 会导致 NSCLC 细胞发生凋亡 [3]。 CAY10404 (80 µM) 在三天内诱导 pAkt、pGSK-3β 和抗凋亡蛋白（Bcl-2 和 Bcl-XL）水平呈浓度依赖性降低 [3]。 CAY10404（20、50、80 和 100 µM；持续 14 天）以浓度依赖性方式抑制 H460 细胞在不依赖贴壁的生长中形成集落的能力 [3]。

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在10-100微M范围内用CAY10404治疗可引起剂量依赖性生长抑制，平均50%抑制浓度（IC（50））为60-100微mol/L，具体取决于细胞系。CAY10404处理的细胞的蛋白质印迹分析显示聚ADP核糖聚合酶（PARP）和前天冬氨酸蛋白酶-3的切割，表明胱天蛋白酶活性和凋亡细胞死亡。CAY10404处理抑制了H460和H358细胞中Akt、糖原合酶激酶-3β和细胞外信号调节激酶1/2的磷酸化。 结论：这些结果表明，CAY10404是NSCLC细胞凋亡的强效诱导剂，可能通过抑制多种蛋白激酶B/Akt和丝裂原活化蛋白激酶途径发挥作用[3]。

在 HTV 小鼠中，腹腔注射 50 mg/kg/天的 CAY10404 可改善肺部炎症和呼吸机引起的肺损伤 [2]。

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抑制COX-2可减轻呼吸机诱导的肺损伤[2]
在机械通气开始前4天，用COX-2特异性抑制剂CAY10404（50mg/kg/天，腹腔注射）治疗小鼠。使用COX-2特异性抑制剂CAY10404（50mg/kg/天，持续4天）治疗可减弱环氧化酶活性，显著降低BAL PGE2和6-酮PGF1α（图3）。同样，与未经治疗的HTV小鼠相比，全身COX-2抑制使血浆PGE2降低了66%（P<0.05）。CAY10404对COX-2的药理学抑制显著降低了HTV机械通气引起的肺泡毛细血管渗漏（图1，深色条；P<0.05）。CAY10404治疗对对照组或LTV小鼠的组织EBD或BAL蛋白没有显著影响。同样，与未经治疗的HTV小鼠相比，抑制COX-2可减少HTV小鼠的肺部炎症（图2A-2D，深色条；P<0.05），降低BAL白细胞、组织PMN、组织MPO和BAL IL-6。用CAY10404治疗对对照组或LTV小鼠的BAL细胞计数、PMN评分或IL-6没有显著影响，尽管在接受COX-2抑制的LTV小鼠中，肺MPO降低的趋势并不显著。COX-2抑制对白细胞粘附分子产生不同的影响，在两个通气组中显著降低ICAM-1的表达，但在两个通风组中增加VCAM-1（图2E，底部）。值得注意的是，用CAY10404治疗对照组小鼠增加了基础VCAM-1表达。

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在存在或不存在3-（4-甲基磺酰基苯基）-4-苯基-5-三氟甲基异恶唑（CAY10404）对环氧合酶-2-特异性药物抑制的情况下，在低潮气量和高潮气量下对小鼠进行机械通气。使用肺泡毛细血管渗漏和肺部炎症标志物评估肺损伤。通过蛋白质印迹、实时PCR和肺/血浆前列腺素分析测量环氧化酶-2的表达和活性，并通过免疫组织化学分析组织切片中环氧化酶-2的染色。高潮气量通气引起肺损伤，与对照和低潮气量通气相比，肺漏和肺部炎症显著增加。与对照组小鼠和低潮气量小鼠相比，高潮气量机械通气显著诱导了肺和全身环氧合酶-2的表达和活性。肺切片的免疫组织化学分析将环氧化酶-2的表达定位在肺泡中的单核细胞和巨噬细胞上。CAY10404对环氧合酶-2的药理学抑制显著降低了高潮气量通气小鼠的环氧合酶活性，减轻了肺损伤，减轻了屏障破坏、组织炎症和炎性细胞信号传导。本研究证明了机械通气诱导环氧化酶-2，并表明环氧化酶-2的治疗性抑制可能会减轻呼吸机诱导的急性肺损伤[2]。

环氧合酶抑制研究。[1]
使用COX-（绵羊）抑制剂筛选试剂盒测试了本文所述的所有化合物抑制COX-1和COX-2的能力。简而言之，环氧化酶催化花生四烯酸（AA）生物合成为PGH2的第一步。PGF2α由PGH2经氯化亚锡还原产生，通过酶免疫测定法进行测量。该检测基于PG和PG-乙酰胆碱酯酶结合物（PG示踪剂）之间对有限量PG抗血清的竞争。能够结合PG抗血清的PG示踪剂的量与孔中PG的浓度成反比，因为PG示踪剂的浓度保持恒定，而PG的浓度变化。这种抗体-PG复合物与之前附着在孔上的小鼠抗abbit单克隆抗体结合。清洗平板以去除任何未结合的试剂，然后将含有乙酰胆碱酯酶底物的Ellman试剂加入孔中。这种酶反应的产物产生明显的黄色，在405nm处吸收。通过分光光度法测定的这种颜色的强度与结合到孔上的PG示踪剂的量成正比，而结合到孔中的PG示踪剂与孵育过程中孔中存在的PG的量成反比：吸光度├[结合的PG示踪剂]├1/PG。通过比较经不同对照培养处理的化合物来计算抑制百分比。根据浓度-抑制反应曲线（重复测定）计算引起50%抑制的试验化合物的浓度（IC50，μM）。

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COX-2抑制[2]
CAY10404（3-（4-甲基磺酰基苯基）-4-苯基-5-三氟甲基异恶唑）50mg/kg/天，在开始通气前每天腹腔注射3天加1小时。根据我们的初步剂量范围研究和之前发表的研究，选择该剂量是为了在疗效和毒性之间达到最佳平衡。

细胞活力测定 [3]
细胞类型： 非小细胞肺癌 (NSCLC) 细胞（H1703、H358、H460）
测试浓度： 10 -100 µM
孵育时间： 3 天
实验结果： 以一定的浓度依赖性方式抑制 NSCLC 细胞系的生长。

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细胞凋亡分析[3]
细胞类型： H460 细胞
测试浓度： 20、50、100 µM
孵育时间：3天
实验结果：诱导细胞凋亡。

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蛋白质印迹分析 [3]
细胞类型： NSCLC 细胞（H358、H460）
测试浓度： 80 µM
孵育时间： 3 天
实验结果：诱导抗凋亡蛋白（Bcl-2 和 Bcl-XL）和 pAkt 水平随浓度依赖性降低pGSK-3β 不改变促凋亡蛋白 (Bax) 的水平以及总 Akt 和 GSK-3β 蛋白水平。

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细胞增殖试验[3]
为了测量CAY10404对NSCLC细胞增殖的影响，将3×103个细胞/孔（H-1703、H-358、H-460）镀在96孔板上，并在37°C下粘附过夜。第二天，将细胞转移到含有10%血清和DMSO中浓度范围为CAY-10404的新鲜培养基中（终浓度为0.1%）。对照细胞用0.1%DMSO处理。在潜伏期结束时，使用3-（4,5-二甲基噻唑-2-基）-2,5-二苯基溴化四唑（MTT）法测量细胞增殖。每次分析使用六个重复孔。生长抑制百分比由以下方程式计算：生长抑制百分比=（1-At/Ac）×100，其中At和Ac分别表示处理和对照培养物的吸光度值。通过剂量-反应曲线插值确定引起50%细胞生长抑制的药物浓度（IC50）。至少进行了三次独立实验。

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锚地独立生长试验[3]
将NSCLC细胞与低温熔融琼脂糖（0.5%）混合，并以2000个细胞/孔的速度放置在六孔板中的凝固琼脂糖（1%）上。琼脂糖下层和上层均含有不同浓度的0.1%DMSO（作为对照）或CAY10404。将含有上层琼脂糖层的细胞在4°C下固化，然后在37°C下在95%空气/5%二氧化碳的加湿气氛中孵育14天。3天后，将RPMI 1640加10%FBS（含或不含CAY10404；0.5 mL）放置在琼脂糖上，此后每3天更换一次。实验结束时，在倒置显微镜（×40）下计数直径>125µm的菌落。

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细胞凋亡检测[3]
将NSCLC细胞暴露于不同浓度的CAY10404或0.1%DMSO中，然后在含有10%血清的培养基中生长3天。如前所述，使用APO BrdU染色试剂盒（一种改良的末端脱氧核苷酸转移酶dUTP缺口末端标记（TUNEL）测定法）测量细胞凋亡。22简而言之，收集漂浮和贴壁细胞，用1%多聚甲醛和70%乙醇固定。通过末端脱氧尿苷转移酶诱导BrdU三磷酸（Br-dUTP）掺入DNA链的3'-OH末端来检测DNA断裂。在配备488nm氩激光和CellQuest软件的FACScan流式细胞仪上分析细胞。DNA含量（线性红色荧光）和Br-dUTP掺入（FITC-PRB-1）的双重显示用于确定群体中凋亡细胞的百分比。凋亡细胞被鉴定为FITC阳性细胞在10000个门控细胞总数中的比例。

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

68.55

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

注意： 请将本产品存放在密封且受保护的环境中（例如氮气保护），避免吸湿/受潮。

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)

配方 1 中的溶解度: 2.5 mg/mL (6.81 mM) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 悬浮液；超声助溶。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: 2.5 mg/mL (6.81 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 悬浊液; 超声助溶。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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配方 3 中的溶解度: ≥ 2.5 mg/mL (6.81 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液加入到 900 μL 玉米油中并混合均匀。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。