DNA-PK (IC50 = 2 nM); p110α (IC50 = 5.8 nM); p110γ (IC50 = 76 nM); p110δ (IC50 = 510 nM); p110β (IC50 = 1.3 μM); hsVPS34 (IC50 = 2.6 μM); PI3KC2β (IC50 = 1 μM); PI3KC2α (IC50 = 10 μM); mTORC1 (IC50 = 1 μM); mTORC2 (IC50 = 10 μM); ATM (IC50 = 2.3 μM); ATR (IC50 = 21 μM); PI4KIIIβ (IC50 = 50 μM)

PIK-75 还抑制 p110δ、PI3KC2β、mTORC1、ATM、hsVPS34、PI3KC2α、mTORC2、ATR 和 PI4KIIIβ，IC50 为 510 nM、~1 μM、~1 μM、2.3 μM、2.6 μM、~10 μM、~10 μM、分别为 21 μM、~50 μM[1]。 PIK-75 单独抑制 Thr 308 的磷酸化，L6 肌管中的 IC50 为 1.2 M，3T3-L1 脂肪细胞中的 IC50 为 1.3 M[1]。 PIK-75（1–1000 nM；5 分钟）的 IC50 值为 78 nM，以剂量依赖性方式抑制 CHO-IR 细胞中胰岛素诱导的 PKB Ser473 和 Thr308 磷酸化。通过诱导胰腺癌细胞凋亡，PIK-75（0.1-1000 nM；48 小时）可阻止其生长和存活[3]。此外，当 PIK-75 (0.1–1000 nM) 存在时，胰腺癌 MIA PaCa-2 和 AsPC-1 细胞形成的集落较少[3]。

PIK-75 (2 mg/kg) 增强吉西他滨 (20 mg/kg) 的体内抗癌活性。单独使用吉西他滨 (20 mg/kg) 或 PIK-75 (2 mg/kg) 均能显着减缓肿瘤生长。 PIK-75 和吉西他滨的组合显然具有积极作用，因为它显着减缓体内肿瘤的生长，同时对小鼠的体重没有负面影响[3]。

---

PIK-75增强吉西他滨的体内抗癌活性[3]
体内小鼠异种移植物模型进一步证明了PIK-75/吉西他滨组合的效果。携带MIA PaCa-2肿瘤的小鼠服用吉西他滨（20mg/kg）、PIK-75（2mg/kg）或两种药物的组合。由于PIK-75是一种可逆抑制剂，因此每周给药5次PIK-75以确保保持足够的抑制作用。吉西他滨每周给药两次。如图7A所示，吉西他滨或PIK-75对肿瘤生长的抑制程度相似。PIK-75/吉西他滨的有益作用是显而易见的，因为这种组合显著降低了体内肿瘤的生长，而不影响小鼠的体重（图7B）。

将 PI3K 抑制剂 PIK-75 以 10 mM 的浓度溶解在二甲亚砜中，并保存在 -20°C 下直至使用。 PI3K 酶活性在 50 μL 20 mM HEPES（pH 7.5）和含有 180 μM 磷脂酰肌醇的 5 mM MgCl2 中测定，反应通过添加 100 μM ATP（含有 2.5 μCi 的 [γ-32P]ATP）开始。室温孵育 30 分钟后，添加 50 μL 1 M HCl 终止酶反应。然后用 100 μL 氯仿/甲醇 [1:1 (v/v)] 和 250 μL 2 M KCl 提取磷脂，然后进行液体闪烁计数。将抑制剂稀释在 20% (v/v) 二甲基亚砜中，生成浓度与抑制酶活性的曲线，然后使用 Windows 版 Prism 5.00 版进行分析，计算 IC50。对于动力学分析，使用测量 ATP 消耗的发光测定法。 PI3K 酶活性在 50 μL 20 mM HEPES（pH 7.5）和 5 mM MgCl2 以及不同浓度的 PI 和 ATP 中测定。室温孵育 60 分钟后，加入 50 μL Kinase-Glo 终止反应，然后再孵育 15 分钟。然后使用 Fluostar 读板器读取发光。使用 Prism 分析结果。

在使用或不使用抑制剂的 TGFβ 刺激 48 小时后，使用 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑 (MTT) 测定评估线粒体活性。将收获的洗涤细胞重悬于DMEM-10% FCS中并等分（500 μL）到24孔簇板中，然后一式两份连续稀释（1:2）。稀释细胞后，立即向每个孔中添加 100 μL 适当浓度的 MTT（溶解在 PBS 中，并在使用前通过 0.2 μm 过滤器过滤，以除去任何蓝色甲臜产物），然后在 37 °C 下孵育 3.5 小时。通过向每个孔中添加 500 μL 10% 十二烷基硫酸钠 (SDS) 的 0.01 M HCl 溶液，在 37 °C 下将所得蓝色甲臜产物溶解过夜（16 小时）。将每个重复孔中的样品 (150 μL) 转移至 96 孔微孔板，并通过自动分光光度法对照试剂空白（无细胞）测定光密度。在 570 nm 的测试波长和 690 nm 的参考波长下测量吸光度。对于每个原代细胞培养物，对每次处理的三到六个孔的结果进行平均，并且数据表示为 570 到 690 nm 的吸光度。细胞：A2780、A2780/cp70、2780AD、HCT116、HT29、WIL、CALU-3、MCF7、PC3 和 HS852 细胞。

Dissolved in DMSO and then diluted in PBS.; ≤1 μM; i.p.
RAMTLn3 cells are injected into the right fourth mammary fat pad from the head of female severe-combined immunodeficient/NCr mice.

---

Tumor xenograft study [3]
MIA PaCa-2 cells (∼1.7×106 cells/mouse) mixed with Matrigel were injected subcutaneously into the flank of male athymic nude (Foxn1nu) mice aged 6-weeks. Gemcitabine (50 mg/ml) was dissolved in PBS and PIK-75 (20 mg/ml) was dissolved in DMSO. Injection solution was made as 10% of Cremophor® EL and 3% of poly(ethylene glycol) 400 in sterile water. Before administration of compounds, gemcitabine was further diluted in PBS and DMSO or PIK-75 was further diluted in the injection solution and sterilized by 0.2 μm filter unit. These diluents were mixed with 1:1 ratio and administered into peritoneal cavity of the mouse. Gemcitabine (20 mg/kg) or gemcitabine (20 mg/kg)/PIK-75 (2 mg/kg) combination was administered twice per week and vehicle control and PIK-75 (2 mg/kg) were administered 5 times per week. The body weights and tumor sizes were measured 3 times per week. Tumor volumes were calculated as width (mm) × length (mm) × height (mm)/2.

---

[1]. A pharmacological map of the PI3-K family defines a role for p110alpha in insulin signaling. Cell. 2006 May 19;125(4):733-47.

[2]. Evidence for functional redundancy of class IA PI3K isoforms in insulin signalling. Biochem J. 2007 Jun 15;404(3):449-58.

[3]. Inhibition of NRF2 by PIK-75 augments sensitivity of pancreatic cancer cells to gemcitabine. Int J Oncol. 2014 Mar;44(3):959-69.

N-[(6-bromo-3-imidazo[1,2-a]pyridinyl)methylideneamino]-N,2-dimethyl-5-nitrobenzenesulfonamide is a sulfonamide.
PIK-75 is a preferential p110 alpha/gamma PI3K inhibitor.

---

Phosphoinositide 3-kinases (PI3-Ks) are an important emerging class of drug targets, but the unique roles of PI3-K isoforms remain poorly defined. We describe here an approach to pharmacologically interrogate the PI3-K family. A chemically diverse panel of PI3-K inhibitors was synthesized, and their target selectivity was biochemically enumerated, revealing cryptic homologies across targets and chemotypes. Crystal structures of three inhibitors bound to p110gamma identify a conformationally mobile region that is uniquely exploited by selective compounds. This chemical array was then used to define the PI3-K isoforms required for insulin signaling. We find that p110alpha is the primary insulin-responsive PI3-K in cultured cells, whereas p110beta is dispensable but sets a phenotypic threshold for p110alpha activity. Compounds targeting p110alpha block the acute effects of insulin treatment in vivo, whereas a p110beta inhibitor has no effect. These results illustrate systematic target validation using a matrix of inhibitors that span a protein family.[1]

---

Recent genetic knock-in and pharmacological approaches have suggested that, of class IA PI3Ks (phosphatidylinositol 3-kinases), it is the p110alpha isoform (PIK3CA) that plays the predominant role in insulin signalling. We have used isoform-selective inhibitors of class IA PI3K to dissect further the roles of individual p110 isoforms in insulin signalling. These include a p110alpha-specific inhibitor (PIK-75), a p110alpha-selective inhibitor (PI-103), a p110beta-specific inhibitor (TGX-221) and a p110delta-specific inhibitor (IC87114). Although we find that p110alpha is necessary for insulin-stimulated phosphorylation of PKB (protein kinase B) in several cell lines, we find that this is not the case in HepG2 hepatoma cells. Inhibition of p110beta or p110delta alone was also not sufficient to block insulin signalling to PKB in these cells, but, when added in combination with p110alpha inhibitors, they are able to significantly attenuate insulin signalling. Surprisingly, in J774.2 macrophage cells, insulin signalling to PKB was inhibited to a similar extent by inhibitors of p110alpha, p110beta or p110delta. These results provide evidence that p110beta and p110delta can play a role in insulin signalling and also provide the first evidence that there can be functional redundancy between p110 isoforms. Further, our results indicate that the degree of functional redundancy is linked to the relative levels of expression of each isoform in the target cells.[2]

---

We describe the potential benefit of PIK-75 in combination of gemcitabine to treat pancreatic cancer in a preclinical mouse model. The effect of PIK-75 on the level and activity of NRF2 was characterized using various assays including reporter gene, quantitative PCR, DNA-binding and western blot analyses. Additionally, the combinatorial effect of PIK-75 and gemcitabine was evaluated in human pancreatic cancer cell lines and a xenograft model. PIK-75 reduced NRF2 protein levels and activity to regulate its target gene expression through proteasome-mediated degradation of NRF2 in human pancreatic cancer cell lines. PIK-75 also reduced the gemcitabine-induced NRF2 levels and the expression of its downstream target MRP5. Co-treatment of PIK-75 augmented the antitumor effect of gemcitabine both in vitro and in vivo. Our present study provides a strong mechanistic rationale to evaluate NRF2 targeting agents in combination with gemcitabine to treat pancreatic cancers.[3]

C16H14BRN5O4S

452.28

450.994

C, 42.49; H, 3.12; Br, 17.67; N, 15.48; O, 14.15; S, 7.09

372196-67-3

PIK-75 hydrochloride;372196-77-5

10275789

Light brown to brown solid powder

1.7±0.1 g/cm3

1.701

3.84

121.24

0

7

4

27

679

0

BrC1=CN2C(/C=N/N(C)S(C3C=C([N+]([O-])=O)C=CC=3C)(=O)=O)=CN=C2C=C1

QTHCAAFKVUWAFI-DJKKODMXSA-N

InChI=1S/C16H14BrN5O4S/c1-11-3-5-13(22(23)24)7-15(11)27(25,26)20(2)19-9-14-8-18-16-6-4-12(17)10-21(14)16/h3-10H,1-2H3/b19-9+

N-[(E)-(6-bromoimidazo[1,2-a]pyridin-3-yl)methylideneamino]-N,2-dimethyl-5-nitrobenzenesulfonamide

PIK 75; PIK75; PIK-75; 372196-67-3; 945619-31-8; UNII-9058I8S63D; (E)-N'-((6-bromoimidazo[1,2-a]pyridin-3-yl)methylene)-N,2-dimethyl-5-nitrobenzenesulfonohydrazide; 9058I8S63D; PIK-75

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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