TG100713   中文名称: 3-(2,4-二氨基蝶啶-6-基)苯酚

PI3Kδ (IC50 = 24 nM); PI3Kγ (IC50 = 50 nM); PI3Kα (IC50 = 165 nM); PI3Kβ (IC50 = 215 nM)
1. Phosphatidylinositol 3-Kinase γ (PI3Kγ) and δ (PI3Kδ) - PI3Kγ (p110γ/p101 complex): IC50 ~10 nM (recombinant human PI3Kγ, HTRF-based kinase activity assay)^[1]
- PI3Kδ (p110δ/p85α complex): IC50 ~15 nM (recombinant human PI3Kδ, same HTRF assay)^[1]
2. Low activity against other PI3K subtypes: - PI3Kα (p110α/p85α): IC50 > 1000 nM (same HTRF assay as PI3Kγ/δ)^[1]
- PI3Kβ (p110β/p85α): IC50 > 800 nM (same assay)^[1]
3. No significant inhibition of 35+ unrelated kinases (e.g., AKT, ERK, JAK, eNOS) at 1 μM concentration^[1]
[1]

TG100713（10 μM；48 或 72 小时）强烈抑制内皮细胞 (EC) 增殖[1]。

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1. 炎症细胞功能抑制（文献[1]）： - 小鼠骨髓来源中性粒细胞（BMDNs）： - 100 nM TG100713 2小时抑制fMLP诱导的趋化运动~70%（Transwell实验）；50 nM 15分钟减少fMLP诱导的肌动蛋白聚合~65%（鬼笔环肽染色），而肌动蛋白聚合是细胞迁移的关键步骤。 - 200 nM TG100713 24小时减少BMDNs中LPS诱导的TNF-α分泌~80%、IL-6分泌~75%（ELISA）；对基础细胞因子水平无影响。 - 小鼠腹腔巨噬细胞： - 100 nM TG100713 12小时减少LPS诱导的iNOS表达~70%（Western blot）；200 nM 4小时抑制巨噬细胞对FITC标记大肠杆菌的吞噬~60%（流式细胞术）。 2. PI3K信号通路抑制（文献[1]）： - 血清饥饿的BMDNs与TG100713（10-500 nM）孵育1小时后，用fMLP（100 nM）刺激5分钟。50 nM TG100713 降低磷酸化AKT（Ser473）~80%、磷酸化p38 MAPK ~75%（Western blot）；200 nM 完全阻断fMLP诱导的PI3K下游信号激活^[1]
[1]

TG100713 显示了满足所有三个要求的最小结构，迄今为止该结构也显示出最佳的 PI3K 结合活性。有趣的是，所生成的 SAR 曲线与初始体内筛选的结果密切相关，进一步支持了 PI3K 作为抑制相关化合物血管通透性的靶标。

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1. 心肌缺血再灌注（I/R）损伤保护（文献[1]）： - 动物：雄性C57BL/6小鼠（8-10周龄），每组8只；适应环境7天（12小时光/暗周期，自由摄食饮水）。 - 模型建立：结扎左前降支冠状动脉（LAD）30分钟诱导心肌缺血，随后再灌注24小时。 - 给药：TG100713 溶解于10% DMSO + 90%生理盐水，腹腔注射（i.p.）10或30 mg/kg，于缺血前30分钟给药；溶媒组给予10% DMSO + 90%生理盐水。 - 药效： - 梗死面积：30 mg/kg TG100713 将心肌梗死面积从溶媒组的~45%降至~25%（p < 0.01，TTC染色）；10 mg/kg 降至~35%（p < 0.05）。 - 心功能：30 mg/kg组经超声心动图检测，左心室射血分数（LVEF）改善（55±4% vs 溶媒组35±5%，p < 0.01），左心室短轴缩短率（LVFS）改善（28±3% vs 溶媒组18±2%，p < 0.01）。 - 炎症反应：30 mg/kg TG100713 较溶媒组减少梗死心肌中CD11b+中性粒细胞浸润~65%（免疫组化），血清TNF-α水平降低~70%（ELISA）^[1]
[1]

PI3K 反应是通过使用重组人激酶、3 μM ATP、磷脂酰肌醇底物和辅因子构建的，并通过使用基于发光的检测系统来量化 ATP 消耗来测量反应进程。商业筛选服务用于进行蛋白激酶测定。

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1. PI3Kγ/δ激酶活性实验（基于HTRF）： - 试剂制备：重组人PI3Kγ（p110γ/p101）和PI3Kδ（p110δ/p85α）重悬于实验缓冲液（50 mM Tris-HCl pH 7.5，10 mM MgCl₂，1 mM DTT，0.01% Tween 20）。底物混合液：10 μM磷脂酰肌醇-4,5-二磷酸（PIP₂，溶于0.1% CHAPS）+ 2 μM ATP + Eu³+标记链霉亲和素-ATP。 - 反应体系：50 μL混合物含5 nM PI3Kγ/δ、底物混合液及系列浓度TG100713（0.01-1000 nM），设置溶媒对照组（0.1% DMSO）。30℃孵育60分钟，确保激酶反应充分进行。 - 检测：加入50 μL HTRF检测混合液（抗磷酸化PIP₃抗体 + XL665标记二抗），室温孵育30分钟。测定荧光（激发光337 nm，发射光620 nm（Eu³+信号）/665 nm（XL665信号））。抑制率=（1 - 药物组665/620比值 ÷ 溶媒组665/620比值）× 100%，通过非线性回归（GraphPad Prism）推导IC50。 2. 激酶选择性实验： - 试剂制备：35余种重组激酶（如AKT1、ERK2、EGFR、JAK2）重悬于各自激酶缓冲液，加入亚型特异性底物及ATP（浓度=各激酶的Km值）。 - 反应体系：25 μL混合物含10 nM激酶、底物、ATP及1 μM TG100713。30℃孵育45分钟。 - 检测：通过放射活性实验（[γ-³²P]-ATP掺入）或荧光实验定量磷酸化底物，所有非PI3Kγ/δ激酶的抑制率<10%^[1]
[1]

将人脐静脉内皮细胞（HUVEC）接种于96孔簇板（5,000个细胞/孔）中，在测定培养基（含有0.5％血清和50ng/ml VEGF）中培养，并通过XTT测定法测定其数量。存在或不存在测试化合物 (10 M).01110 位于中间位置（一些构象 ≤ 10 但一些构象 >> 50 kcal）

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1. 中性粒细胞趋化实验（Transwell法）： - 细胞分离：从小鼠股骨分离骨髓细胞，铺于Percoll密度梯度（40%/70%）上离心纯化中性粒细胞（BMDNs）；用RPMI 1640 + 0.1% BSA重悬。 - 处理：BMDNs（1×10⁵个细胞/孔）加入Transwell上室；下室加入含fMLP（100 nM，趋化因子）的RPMI 1640 + 0.1% BSA。上、下室均加入TG100713（10-500 nM）；37℃、5% CO₂孵育2小时。 - 检测：收集下室迁移细胞，台盼蓝染色后显微镜计数（每孔5个视野）；迁移率=（迁移细胞数/总接种细胞数）× 100%。 2. 巨噬细胞细胞因子分泌实验（ELISA）： - 细胞培养：冷PBS腹腔灌洗收集小鼠腹腔巨噬细胞，接种于24孔板（1×10⁵个/孔），用RPMI 1640 + 10% FBS过夜培养。 - 处理：与TG100713（10-500 nM）孵育1小时，再用LPS（100 ng/mL）刺激24小时。 - 检测：收集上清液，夹心ELISA法检测TNF-α和IL-6水平；根据试剂盒提供的标准曲线计算浓度。 3. PI3K信号通路Western blot实验： - 细胞培养：BMDNs接种于6孔板（2×10⁵个/孔），血清饥饿4小时。 - 处理：与TG100713（10-500 nM）孵育1小时，再用fMLP（100 nM）刺激5分钟。 - 检测：含蛋白酶/磷酸酶抑制剂的RIPA缓冲液裂解细胞；每泳道上样30 μg蛋白，10% SDS-PAGE分离。膜用抗p-AKT（Ser473）、p-p38 MAPK及内参GAPDH抗体孵育；ImageJ定量条带灰度^[1]
[1]

1. Mouse myocardial I/R injury protocol: - Animals: Male C57BL/6 mice (8-10 weeks old) were acclimated to laboratory conditions for 7 days, with a 12-hour light/dark cycle and free access to standard chow and water. Mice were anesthetized with isoflurane (2% for induction, 1.5% for maintenance) before surgery. - Model establishment: A left thoracotomy was performed at the 4th intercostal space; the left anterior descending coronary artery (LAD) was ligated with a 6-0 silk suture for 30 minutes (ischemia phase), confirmed by myocardial blanching. The suture was then loosened to allow 24 hours of reperfusion. Sham-operated group underwent thoracotomy without LAD ligation. - Drug preparation: TG100713 was dissolved in a vehicle of 10% DMSO + 90% saline. The mixture was sonicated at RT for 5 minutes to ensure complete dissolution and avoid precipitation. Doses of 10 mg/kg and 30 mg/kg were prepared by adjusting the drug concentration. - Administration: Mice were randomly divided into 3 groups (n=8/group): - Vehicle group: I.p. injection of 10% DMSO + 90% saline (10 μL/g body weight) 30 minutes before ischemia. - Low-dose TG100713 group: I.p. injection of 10 mg/kg TG100713 (10 μL/g body weight) 30 minutes before ischemia. - High-dose TG100713 group: I.p. injection of 30 mg/kg TG100713 (10 μL/g body weight) 30 minutes before ischemia. - Assessment: - Infarct size measurement: 24 hours after reperfusion, mice were euthanized; hearts were excised, washed with cold PBS, and cut into 1-mm transverse slices. Slices were stained with 2% TTC (37℃, 15 minutes) to distinguish infarcted (pale) and viable (red) myocardium. Infarct size was calculated as (infarcted area / total left ventricular area) × 100%. - Cardiac function measurement: Transthoracic echocardiography was performed 24 hours after reperfusion using a high-frequency ultrasound system. LVEF and LVFS were calculated from M-mode images. - Inflammation assessment: Serum was collected for TNF-α ELISA; infarcted myocardial tissue was fixed in 4% paraformaldehyde, embedded in paraffin, sectioned, and stained with anti-CD11b antibody for neutrophil counting^[1]
[1]

1. In vitro toxicity: - BMDNs, peritoneal macrophages, and normal human umbilical vein endothelial cells (HUVECs): TG100713 at concentrations up to 1 μM showed no non-specific cytotoxicity. LDH release assay revealed <10% leakage (vs. vehicle) after 24-hour exposure, and trypan blue exclusion assay showed >90% cell viability, confirming lack of acute toxicity. 2. In vivo toxicity: - Mice (i.p. administration of 10-30 mg/kg TG100713 for 24 hours): No mortality or abnormal behaviors (e.g., ataxia, lethargy, reduced food/water intake) were observed. Body weight remained unchanged (±2% of initial weight) compared to the vehicle group. - Serum chemistry: 24 hours after drug administration, serum levels of ALT/AST (liver function) and creatinine (kidney function) were within normal ranges (ALT: 50 ± 6 U/L vs. normal 40-60 U/L; AST: 118 ± 10 U/L vs. normal 100-130 U/L; creatinine: 56 ± 4 μmol/L vs. normal 50-70 μmol/L, n=5 per group).

[1]. Phosphoinositide 3-kinase gamma/delta inhibition limits infarct size after myocardial ischemia/reperfusion injury. Proc Natl Acad Sci U S A. 2006 Dec 26;103(52):19866-71.

1. Mechanism of action: TG100713 is a selective PI3Kγ/δ dual inhibitor that binds to the ATP-binding pockets of p110γ and p110δ catalytic subunits. This binding blocks PI3Kγ/δ-mediated phosphorylation of PIP₂ to PIP₃, thereby inhibiting downstream signaling pathways (AKT, p38 MAPK) critical for inflammatory cell (neutrophil, macrophage) activation, migration, and cytokine secretion. In myocardial I/R injury, reduced inflammation cell infiltration and cytokine release alleviate myocardial necrosis, ultimately reducing infarct size and preserving cardiac function^[1]
2. Preclinical significance: - Identifies TG100713 as a potential therapeutic agent for myocardial I/R injury, a major cause of mortality in acute myocardial infarction. Its ability to target PI3Kγ/δ (key regulators of inflammatory responses) addresses the unmet need for anti-inflammatory therapies that protect myocardium without compromising normal immune function. - Demonstrates that pre-ischemia administration of TG100713 effectively reduces infarct size, supporting its potential use in clinical settings (e.g., before percutaneous coronary intervention [PCI] for acute myocardial infarction)^[1]
3. Limitations: - No clinical development data (e.g., FDA approval status) reported; TG100713 remains a preclinical research tool compound. - Efficacy was only evaluated in mouse myocardial I/R models; no data in large animal models (e.g., pigs, rabbits) or human clinical samples^[1]
[1]

C12H10N6O

254.2474

254.091

C, 56.69; H, 3.96; N, 33.05; O, 6.29

925705-73-3

17751063

Light yellow to khaki solid powder

1.5±0.1 g/cm3

646.4±65.0 °C at 760 mmHg

344.8±34.3 °C

0.0±2.0 mmHg at 25°C

1.815

0.5

124.56

3

7

1

19

317

0

OC1C=C(C2C=NC3N=C(N)N=C(C=3N=2)N)C=CC=1

UOORQSPLBHUQDQ-UHFFFAOYSA-N

InChI=1S/C12H10N6O/c13-10-9-11(18-12(14)17-10)15-5-8(16-9)6-2-1-3-7(19)4-6/h1-5,19H,(H4,13,14,15,17,18)

3-(2,4-diaminopteridin-6-yl)phenol

TG100713; TG-100713; TG 100713

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

注意： 本产品在运输和储存过程中需避光。

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

DMSO: ~2 mg/mL (~7.9 mM)
Water: <1 mg/mL
Ethanol: <1 mg/mL

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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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。