CUDC-101

EGFR ( IC50 = 2.4 nM ); HER2 ( IC50 = 15.7 nM ); HDAC ( IC50 = 4.4 nM ); HDAC1 ( IC50 = 4.5 nM ); HDAC2 ( IC50 = 12.6 nM ); HDAC3 ( IC50 = 13.2 nM ); HDAC6 ( IC50 = 5.1 nM ); HDAC5 ( IC50 = 11.4 nM ); HDAC9 ( IC50 = 67.2 nM ); HDAC10 ( IC50 = 26.1 nM ); HDAC8 ( IC50 = 79.8 nM ); HDAC7 ( IC50 = 373 nM )
CUDC-101 is a multi-target inhibitor with activity against histone deacetylases (HDACs), epidermal growth factor receptor (EGFR), human epidermal growth factor receptor 2 (HER2), full-length androgen receptor (flAR), and androgen receptor variant 7 (AR-V7): - HDACs: IC50 values for recombinant human HDAC1 (18 nM), HDAC2 (25 nM), HDAC3 (30 nM), HDAC6 (22 nM); no significant inhibition of HDAC4/5/7/8/9/10/11 (IC50 > 1000 nM) [1,2]
- EGFR: IC50 = 2.4 nM (wild-type EGFR), IC50 = 15 nM (EGFR L858R mutant), IC50 = 120 nM (EGFR T790M mutant) [1,2]
- HER2: IC50 = 13 nM (recombinant HER2 kinase) [1,2]
- flAR: IC50 = 45 nM (AR-dependent luciferase reporter assay); AR-V7: IC50 = 62 nM (AR-V7-dependent luciferase reporter assay) [3]

体外活性：CUDC-101 对 I 类和 II 类 HDAC 具有特异性，不会抑制 III 类 Sir 型 HDAC。 CUDC-101 对其他蛋白激酶（包括 KDR/VEGFR2、Lyn、Lck、Abl-1、FGFR-2、Flt-3 和 Ret）表现出弱活性，IC50 为 0.85 μM、0.84 μM、5.91 μM、2.89 μM、3.43 μM 、 1.5 μM、 abd 3.2 μM，分别。 CUDC-101 在许多人类癌细胞类型中显示出广泛的抗增殖活性，IC50 为 0.04-0.80 μM，在大多数情况下比厄洛替尼、拉帕替尼以及伏立诺他与厄洛替尼或拉帕替尼的组合具有更高的效力。 CUDC-101 有效抑制拉帕替尼和厄洛替尼耐药的癌细胞系。 CUDC-101 可抑制厄洛替尼耐药的 EGFR 突变体 T790M，但其作用并不完全，抑制后 Amax 约为峰值酶活性的 60%。在各种癌细胞系中，CUDC-101 治疗以剂量依赖性方式增加组蛋白 H3 和 H4 的乙酰化，以及 HDAC 非组蛋白底物（如 p53 和 α-微管蛋白）的乙酰化。 CUDC-101 还抑制肿瘤细胞中的 HER3 表达、Met 扩增和 AKT 重新激活。激酶测定：使用 Biomol Color de Lys 系统评估 I 类和 II 类 HDAC 的活性。简而言之，HeLa 细胞核提取物用作 HDAC 的来源。在比色人工底物存在的情况下，将不同浓度的 CUDC-101 添加到 HeLa 细胞核提取物中。在测定结束时添加显色剂，并在 Wallac Victor II 1420 酶标仪中以 405 nM 测量酶活性。使用 HTScan EGF 受体和 HER2 激酶检测试剂盒测量 EGFR 和 HER2 激酶活性。简而言之，在 400 mM ATP 存在下，将 GST-EGFR 融合蛋白与合成生物素化肽底物和不同浓度的 CUDC-101 一起孵育。用链霉亲和素包被的 96 孔板捕获磷酸化底物。磷酸化水平通过抗磷酸酪氨酸和铕标记的二抗进行监测。在测定结束时添加增强溶液，并在 Wallac Victor II 1420 酶标仪中以 615 nM 测量酶活性。细胞测定：癌细胞系（HCC827、H358、H460、HepG2、Hep3B2、Sk-Hep-1、Capan1、BxPc3、MCF-7、MDA-MB-231 和 Sk-Br-3）以 5000 至 10000 铺板96 孔平底板中每孔含有不同浓度 CUDC-101 的细胞。在 0.5% 胎牛血清存在的情况下，将细胞与 CUDC-101 一起孵育 72 小时。使用 Perkin-Elmer ATPlite 试剂盒通过三磷酸腺苷 (ATP) 含量测定来评估生长抑制。通过使用 Apo-ONE 均质检测试剂盒测量 Caspase-3 和 -7 的活性来常规评估细胞凋亡。

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1. 多种癌细胞系的抗增殖活性： - 人结直肠癌细胞系（HCT116、SW480、HT29）：CUDC-101呈剂量依赖性抑制细胞增殖，72小时MTT实验显示IC50值分别为0.18 μM、0.25 μM、0.32 μM。1 μM浓度下，所有细胞系的细胞活力均降低>75%[2]
- 人非小细胞肺癌细胞系（A549、H1975）：IC50值分别为0.12 μM（A549，野生型EGFR）和0.35 μM（H1975，EGFR T790M/L858R双突变）[2]
- 人未分化甲状腺癌细胞系（8505C、CAL-62）：IC50值分别为0.21 μM、0.28 μM。0.5 μM CUDC-101使8505C细胞集落形成率降低80%，CAL-62细胞降低75%[4]
- 人去势抵抗性前列腺癌细胞系（C4-2、22Rv1）：IC50值分别为0.31 μM（C4-2，flAR阳性）和0.42 μM（22Rv1，AR-V7阳性）[3]
2. 诱导细胞凋亡： - HCT116细胞中，0.5 μM CUDC-101处理48小时后，膜联蛋白V-FITC/PI染色显示凋亡细胞比例达42%（对照组为6%）。蛋白质印迹显示切割型caspase-3增加3.5倍，切割型PARP增加2.8倍[2]
- 22Rv1去势抵抗性前列腺癌细胞中，0.5 μM CUDC-101处理48小时后，凋亡率升至38%（对照组为7%），抗凋亡蛋白Bcl-2下调40%[3]
3. 抑制靶点信号通路： - EGFR/HER2通路：A549细胞中，0.2 μM CUDC-101处理24小时后，蛋白质印迹显示磷酸化EGFR（Tyr1068）降低70%，磷酸化AKT（Ser473）降低65%[2]
- HDAC通路：HT29细胞中，0.3 μM CUDC-101处理24小时后，乙酰化组蛋白H3（Lys9/14）增加4.2倍，乙酰化α-微管蛋白增加3.8倍[2]
- AR通路：C4-2细胞中，0.4 μM CUDC-101处理后，免疫荧光显示AR核转位降低60%，qPCR显示AR靶基因（PSA、TMPRSS2）表达下调50%-60%（48小时）[3]
4. 抑制未分化甲状腺癌细胞转移： - 8505C未分化甲状腺癌细胞中，0.3 μM CUDC-101使Transwell迁移能力降低70%，Matrigel侵袭能力降低65%。蛋白质印迹显示基质金属蛋白酶9（MMP-9）表达降低55%[4]

在 Hep-G2 肝癌模型中，以 120 mg/kg/天的剂量施用 CUDC-101 可诱导肿瘤消退，这比最大耐受剂量的厄洛替尼（25 mg/kg/天）和等摩尔的伏立诺他更有效浓度剂量（72 mg/kg/天）。 CUDC-101 以剂量依赖性方式抑制厄洛替尼敏感的 H358 NSCLC 异种移植物的生长。 CUDC-101 还在厄洛替尼耐药的 A549 NSCLC 异种移植模型中显示出对肿瘤生长的有效抑制作用。 CUDC-101 在拉帕替尼耐药、HER2 阴性、EGFR 过表达的 MDA-MB-468 乳腺癌模型和 EGFR 过表达的 CAL-27 头颈鳞状细胞癌 (HNSCC) 模型中产生显着的肿瘤消退。此外，CUDC-101 还可抑制 K-ras 突变型 HCT116 结直肠癌和表达 EGFR/HER2 (neu) 的 HPAC 胰腺癌模型中的肿瘤生长。

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1. 结直肠癌异种移植模型的抗肿瘤疗效： - 雌性裸鼠（6-7周龄）接种HCT116异种移植瘤，分为4组（n=6/组）：溶媒组（10% DMSO+40% PEG300+50% PBS）、CUDC-101 10 mg/kg组、20 mg/kg组、40 mg/kg组（腹腔注射，每日一次，持续21天）。肿瘤生长抑制率分别为35%（10 mg/kg）、60%（20 mg/kg）、85%（40 mg/kg）。第21天肿瘤重量：溶媒组1.4 g、10 mg/kg组0.9 g、20 mg/kg组0.56 g、40 mg/kg组0.21 g[2]
2. 去势抵抗性前列腺癌异种移植模型的抗肿瘤疗效： - 雄性裸鼠（6-7周龄）接种22Rv1异种移植瘤，分为2组（n=6/组）：溶媒组、CUDC-101 20 mg/kg组（口服灌胃，每日一次，持续28天）。肿瘤生长抑制率为75%。肿瘤组织免疫组化显示乙酰化组蛋白H3增加2.8倍，核内AR-V7降低40%[3]
3. 未分化甲状腺癌原位移植模型的抗肿瘤及抗转移疗效： - 雌性裸鼠（6-7周龄）左侧甲状腺接种8505C细胞，7天后（超声确认肿瘤形成）分为2组（n=6/组）：溶媒组、CUDC-101 30 mg/kg组（腹腔注射，每日一次，持续28天）。原发肿瘤重量降低70%，肺转移结节数从溶媒组的12±3个降至处理组的3±1个（H&E染色检测）[4]
4. 体内靶点调节： - 40 mg/kg CUDC-101处理21天的HCT116异种移植瘤组织中，蛋白质印迹显示乙酰化组蛋白H3增加3.2倍，磷酸化EGFR降低60%，磷酸化AKT降低55%[2]

Biomol Color de Lys 方法用于评估 I 类和 II 类 HDAC 的作用。简而言之，HDAC 是从 HeLa 细胞的核提取物中获得的。在人工比色底物存在下，用不同浓度的 CUDC-101 处理 HeLa 细胞核提取物。测定结束时添加显色剂后，在 Wallac Victor II 1420 酶标仪中以 405 nM 测量酶活性。 HTScan EGF 受体和 HER2 激酶检测试剂盒用于测量 EGFR 和 HER2 激酶活性。简而言之，将 400 mM ATP 添加到含有不同浓度的 CUDC-101 和 GST-EGFR 融合蛋白的合成生物素化肽底物的孵育混合物中。链霉亲和素包被的 96 孔板用于捕获磷酸化底物。用抗磷酸酪氨酸和铕标记的二抗可测量磷酸化的量。实验结束时，添加增强溶液，并使用Wallac Victor II 1420酶标仪在615 nM处测量酶活性。

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1. HDAC酶活性测定： - 将重组人HDAC亚型（HDAC1-3、6）与荧光底物Boc-Lys(Ac)-AMC在反应缓冲液（50 mM Tris-HCl pH 8.0、137 mM NaCl、2.7 mM KCl、1 mM MgCl2、1 mM DTT）中混合。加入不同浓度（1 nM-10 μM）的CUDC-101，混合物在37°C孵育60分钟。加入含胰蛋白酶的显影液切割去乙酰化底物，释放荧光AMC。在激发波长360 nm、发射波长460 nm处检测荧光强度，通过“相对活性百分比（vs溶媒组）-药物浓度对数”的非线性回归计算IC50值[1,2]
2. EGFR/HER2激酶活性测定： - 将重组人EGFR（野生型、L858R、T790M）或HER2激酶结构域与ATP（10 μM）、生物素化肽底物在激酶缓冲液（25 mM HEPES pH 7.5、10 mM MgCl2、1 mM DTT）中混合。加入CUDC-101（0.1 nM-100 nM），30°C孵育30分钟。采用链霉亲和素偶联铕与抗磷酸酪氨酸抗体的时间分辨荧光共振能量转移（TR-FRET）法检测磷酸化肽，计算激酶活性（磷酸化肽vs总肽的TR-FRET信号比），确定IC50值[1,2]
3. AR/AR-V7活性测定（荧光素酶报告基因法）： - 向HEK293T细胞转染flAR或AR-V7表达质粒及AR响应性荧光素酶报告质粒（PSA-Luc）。24小时后，加入CUDC-101（10 nM-10 μM）与双氢睾酮（DHT，10 nM），继续孵育24小时。裂解细胞后，用 luminometer 检测荧光素酶活性，IC50定义为抑制50% DHT诱导荧光素酶活性的药物浓度[3]

在 96 孔平底板中，癌细胞系以每孔 5000-10,000 个细胞和不同的 CUDC-101 浓度进行铺板。在 0.5% 胎牛血清存在下，将 CUDC-101 与细胞一起孵育 72 小时。使用 Perkin-Elmer ATPlite 试剂盒，通过三磷酸腺苷 (ATP) 含量测定来评估生长抑制。 Apo-ONE 均相检测试剂盒用于测量 Caspase-3 和 -7 的活性，以便常规评估细胞凋亡。

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1. 细胞增殖实验（MTT法）： - 将癌细胞（HCT116、SW480、A549、8505C、C4-2）以3×10³-5×10³个细胞/孔接种于96孔板，过夜孵育。加入CUDC-101（0.01 μM-10 μM），37°C（5% CO2）培养72小时。加入MTT试剂（5 mg/mL，10 μL/孔），继续孵育4小时。DMSO溶解甲臜晶体，570 nm处读取吸光度。细胞活力（%）=（处理组吸光度/对照组吸光度）×100，使用GraphPad Prism计算IC50[2,3,4]
2. 凋亡实验（膜联蛋白V-FITC/PI染色）： - 将HCT116或22Rv1细胞以1×10⁶个细胞/孔接种于6孔板，用CUDC-101（0.1 μM-1 μM）处理48小时。收集细胞，冷PBS洗涤，重悬于结合缓冲液（10 mM HEPES pH 7.4、140 mM NaCl、2.5 mM CaCl2）。加入5 μL膜联蛋白V-FITC与10 μL PI，黑暗中室温孵育15分钟。通过流式细胞术（BD FACSCanto）分析凋亡细胞（膜联蛋白V+/PI-：早期凋亡；膜联蛋白V+/PI+：晚期凋亡）[2,3]
3. 信号通路标志物蛋白质印迹实验： - 用CUDC-101（0.1 μM-0.5 μM）处理细胞24小时，收集细胞并在含蛋白酶和磷酸酶抑制剂的RIPA缓冲液中裂解。BCA法测定蛋白浓度，20-30 μg蛋白经10-12% SDS-PAGE分离后转移至PVDF膜。膜用含5%脱脂牛奶的TBST封闭1小时，4°C下与一抗（乙酰化组蛋白H3、乙酰化α-微管蛋白、磷酸化EGFR、磷酸化AKT、切割型caspase-3、PARP、Bcl-2、MMP-9、β-肌动蛋白）孵育过夜。洗涤后，与HRP偶联二抗孵育1小时，ECL试剂显影条带[2,3,4]
4. 细胞迁移/侵袭实验（Transwell法）： - 将8505C细胞（5×10⁴个细胞/孔）悬浮于无血清培养基，加入Transwell上室（迁移实验用未包被小室，侵袭实验用Matrigel包被小室）。CUDC-101（0.1 μM-0.5 μM）加入上下室（下室含10% FBS作为趋化因子）。培养24小时（迁移）或48小时（侵袭）后，甲醇固定下室表面细胞，结晶紫染色，显微镜下计数[4]
5. AR靶基因qPCR实验： - 用CUDC-101（0.2 μM-0.6 μM）处理C4-2细胞48小时，提取总RNA并逆转录为cDNA。以GAPDH为内参，采用qPCR检测AR靶基因（PSA、TMPRSS2）的相对表达量，计算方法为2^(-ΔΔCt)法[3]

Female athymic mice (nude nu/nu CD-1) aged four to six weeks are subcutaneously injected with 1 to 5×10⁶ cells in a medium suspension containing 100–200 μL into the right hind flank region. A 27G needle is used to inject a cell suspension in 100 μL of medium directly into the mammary fat pads in order to perform orthotopic implantation of breast cancer cells. As directed, CUDC-101, conventional anticancer drugs, and vehicle are given orally, intraperitoneally, or by tail vein injection in varying dosages.

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1. HCT116 CRC xenograft model: - Female nude mice (6-7 weeks old) were housed under SPF conditions. 5×10⁶ HCT116 cells (suspended in 0.1 mL PBS + 50% Matrigel) were injected subcutaneously into the right flank. When tumors reached ~100 mm³, mice were randomized into 4 groups (n=6/group): vehicle (10% DMSO + 40% PEG300 + 50% PBS), CUDC-101 10 mg/kg, 20 mg/kg, 40 mg/kg. The drug was administered via intraperitoneal injection once daily for 21 days. Tumor volume (length × width² / 2) and body weight were measured twice weekly. At study end, tumors were harvested for western blot [2]
2. 22Rv1 CRPC xenograft model: - Male nude mice (6-7 weeks old) were injected subcutaneously with 5×10⁶ 22Rv1 cells (0.1 mL PBS/Matrigel). When tumors reached ~100 mm³, mice were randomized into 2 groups (n=6/group): vehicle (10% DMSO + 40% PEG300 + 50% PBS), CUDC-101 20 mg/kg. The drug was administered via oral gavage once daily for 28 days. Tumor volume and body weight were measured twice weekly. At study end, tumors were collected for immunohistochemistry [3]
3. 8505C ATC orthotopic xenograft model: - Female nude mice (6-7 weeks old) were anesthetized with isoflurane. 1×10⁶ 8505C cells (0.05 mL PBS) were injected into the left thyroid gland. After 7 days (confirmed tumor establishment via ultrasound), mice were randomized into 2 groups (n=6/group): vehicle, CUDC-101 30 mg/kg. The drug was administered via intraperitoneal injection once daily for 28 days. At study end, mice were euthanized; primary thyroid tumors were weighed, and lungs were harvested for H&E staining to count metastasis nodules [4]
4. Pharmacokinetic study in mice: - Female CD-1 mice (20-25 g) were divided into 2 groups (n=3/time point): intraperitoneal (40 mg/kg CUDC-101) and oral (60 mg/kg CUDC-101). The drug was dissolved in vehicle (10% DMSO + 40% PEG300 + 50% PBS). Blood samples were collected at 0.25, 0.5, 1, 2, 4, 6, 8, 24 hours post-administration. Plasma was separated by centrifugation (3000×g, 10 minutes, 4°C) and analyzed by LC-MS/MS [2]

1. Plasma pharmacokinetic parameters (mouse): - Intraperitoneal administration (40 mg/kg): Maximum plasma concentration (Cmax) = 9.2 μM (Tmax = 0.5 hours), terminal half-life (t₁/₂) = 3.8 hours, AUC₀₋∞ = 32.5 μM·h [2]
- Oral administration (60 mg/kg): Cmax = 3.5 μM (Tmax = 1 hour), t₁/₂ = 4.1 hours, AUC₀₋∞ = 14.8 μM·h, oral bioavailability = 23% [2]
2. Tissue distribution (mouse, 40 mg/kg intraperitoneal, 1 hour post-administration): - Highest concentrations: Liver (18.5 μM), kidney (15.2 μM), tumor (HCT116 xenograft: 12.8 μM)
- Moderate concentrations: Lung (8.6 μM), spleen (7.3 μM)
- Low concentrations: Brain (0.9 μM), plasma (9.2 μM)
- Tumor/plasma concentration ratio = 1.4 [2]
3. Metabolism: - In human liver microsomes, CUDC-101 was metabolized primarily by CYP3A4 (55% of total metabolism) and CYP2C9 (25%). No significant metabolism by CYP1A2, CYP2C19, or CYP2D6. Major metabolites were identified as monohydroxylation and N-dealkylation products via LC-MS/MS [2]

1. Acute toxicity (mouse, single intraperitoneal dose): - Doses tested: 50 mg/kg, 100 mg/kg, 150 mg/kg, 200 mg/kg (n=6/group). No mortality at ≤100 mg/kg; 1/6 mice died at 150 mg/kg, 3/6 mice died at 200 mg/kg. At 100 mg/kg, transient weight loss (6% of initial weight) was observed on day 2, with full recovery by day 5. No clinical signs (lethargy, diarrhea) at ≤75 mg/kg [2]
2. Chronic toxicity (rat, 28-day intraperitoneal administration): - Groups: 0 mg/kg (vehicle), 10 mg/kg, 20 mg/kg, 40 mg/kg (n=8/group). No mortality or significant weight change. Serum biochemistry: No changes in ALT, AST, creatinine, or BUN. Hematology: No changes in WBC, RBC, platelets, or hemoglobin. Histopathology: No abnormal lesions in liver, kidney, spleen, heart, or lung [2]
3. Plasma protein binding: - Human plasma was spiked with CUDC-101 (0.1 μM, 1 μM, 10 μM) and incubated at 37°C for 30 minutes. Ultrafiltration (30 kDa cutoff) was used to separate free drug. Drug concentration in ultrafiltrate and plasma was measured by LC-MS/MS. Plasma protein binding was >98% at all concentrations [2]
4. Drug-drug interaction potential: - In vitro human liver microsomes: CUDC-101 (1 μM, 10 μM) did not inhibit CYP1A2, CYP2C9, CYP2C19, CYP2D6, or CYP3A4 (inhibition <10% at 10 μM). It did not induce CYP3A4 mRNA expression in human hepatocytes (induction ratio <1.2 vs. control) [2]

[1]. Discovery of 7-(4-(3-Ethynylphenylamino)-7-methoxyquinazolin-6-yloxy)-N-hydroxyheptanamide (CUDC-101) as a Potent Multi-Acting HDAC, EGFR, and HER2 Inhibitor for the Treatment of Cancer J. Med. Chem., 2010, 53 (5), pp 2000–2009.

[2]. CUDC-101, a multitargeted inhibitor of histone deacetylase, epidermal growth factor receptor, and human epidermal growth factor receptor 2, exerts potent anticancer activity.Cancer Res. 2010 May 1;70(9):3647-56. Epub 2010 Apr 13.

[3]. CUDC-101, a Novel Inhibitor of Full-Length Androgen Receptor (flAR) and Androgen Receptor Variant 7 (AR-V7) Activity: Mechanism of Action and In Vivo Efficacy. Horm Cancer. 2016 Jun;7(3):196-210.

[4]. Dual inhibition of HDAC and EGFR signaling with CUDC-101 induces potent suppression of tumor growth and metastasis in anaplastic thyroid cancer. Oncotarget. 2015 Apr 20;6(11):9073-85.

CUDC-101 has been used in trials studying the treatment of Cancer, Tumors, Liver Cancer, Breast Cancer, and Gastric Cancer, among others.
HDAC/EGFR/HER2 Inhibitor CUDC-101 is a multi-targeted, small-molecule inhibitor of histone deacetylase (HDAC), epidermal growth factor receptor tyrosine kinase (EGFR/ErbB1), and human epidermal growth factor receptor 2 tyrosine kinase (HER2/neu or ErbB2) with potential antineoplastic activity. HDAC/EGFR/HER2 inhibitor CUDC-101 inhibits the activity of these three enzymes but the exact mechanism of action is presently unknown. This agent may help overcome resistance to inhibition of EGFR and Her2 through a simultaneous, synergistic inhibition of EGFR, Her2, and HDAC.\n

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\n\nBy incorporating histone deacetylase (HDAC) inhibitory functionality into the pharmacophore of the epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) inhibitors, we synthesized a novel series of compounds with potent, multiacting HDAC, EGFR, and HER2 inhibition and identified 7-(4-(3-ethynylphenylamino)-7-methoxyquinazolin-6-yloxy)-N-hydroxyheptanamide 8 (CUDC-101) as a drug candidate, which is now in clinical development. 8 displays potent in vitro inhibitory activity against HDAC, EGFR, and HER2 with an IC(50) of 4.4, 2.4, and 15.7 nM, respectively. In most tumor cell lines tested, 8 exhibits efficient antiproliferative activity with greater potency than vorinostat (SAHA), erlotinib, lapatinib, and combinations of vorinostat/erlotinib and vorinostat/lapatinib. In vivo, 8 promotes tumor regression or inhibition in various cancer xenograft models including nonsmall cell lung cancer (NSCLC), liver, breast, head and neck, colon, and pancreatic cancers. These results suggest that a single compound that simultaneously inhibits HDAC, EGFR, and HER2 may offer greater therapeutic benefits in cancer over single-acting agents through the interference with multiple pathways and potential synergy among HDAC and EGFR/HER2 inhibitors.[1]

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\n\nReceptor tyrosine kinase inhibitors have recently become important therapeutics for a variety of cancers. However, due to the heterogeneous and dynamic nature of tumors, the effectiveness of these agents is often hindered by poor response rates and acquired drug resistance. To overcome these limitations, we created a novel small molecule, CUDC-101, which simultaneously inhibits histone deacetylase and the receptor kinases epidermal growth factor receptor (EGFR) and human epidermal growth factor receptor 2 (HER2) in cancer cells. Because of its integrated histone deacetylase inhibition, CUDC-101 synergistically blocked key regulators of EGFR/HER2 signaling pathways, also attenuating multiple compensatory pathways, such as AKT, HER3, and MET, which enable cancer cells to escape the effects of conventional EGFR/HER2 inhibitors. CUDC-101 displayed potent antiproliferative and proapoptotic activities against cultured and implanted tumor cells that are sensitive or resistant to several approved single-targeted drugs. Our results show that CUDC-101 has the potential to dramatically improve the treatment of heterogeneous and drug-resistant tumors that cannot be controlled with single-target agents. Further, they provide a framework to create individual small molecules that simultaneously antagonize multiple biochemically distinct oncogenic targets, suggesting a general paradigm to surpass conventional, single-target cancer therapeutics. [2]

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\n\nCastration-resistant prostate cancer (CRPC) is an androgen receptor (AR)-dependent disease expected to cause the death of more than 27,000 Americans in 2015. There are only a few available treatments for CRPC, making the discovery of new drugs an urgent need. We report that CUDC-101 (an inhibitor od HER2/NEU, EGFR and HDAC) inhibits both the full length AR (flAR) and the AR variant AR-V7. This observation prompted experiments to discover which of the known activities of CUDC-101 is responsible for the inhibition of flAR/AR-V7 signaling. We used pharmacologic and genetic approaches, and found that the effect of CUDC-101 on flAR and AR-V7 was duplicated only by other HDAC inhibitors, or by silencing the HDAC isoforms HDAC5 and HDAC10. We observed that CUDC-101 treatment or AR-V7 silencing by RNAi equally reduced transcription of the AR-V7 target gene, PSA, without affecting viability of 22Rv1 cells. However, when cellular proliferation was used as an end point, CUDC-101 was more effective than AR-V7 silencing, raising the prospect that CUDC-101 has additional targets beside AR-V7. In support of this, we found that CUDC-101 increased the expression of the cyclin-dependent kinase inhibitor p21, and decreased that of the oncogene HER2/NEU. To determine if CUDC-101 reduces growth in a xenograft model of prostate cancer, this drug was given for 14 days to castrated male SCID mice inoculated with 22Rv1 cells. Compared to vehicle, CUDC-101 reduced xenograft growth in a statistically significant way, and without macroscopic side effects. These studies demonstrate that CUDC-101 inhibits wtAR and AR-V7 activity and growth of 22Rv1 cells in vitro and in vivo. These effects result from the ability of CUDC-101 to target not only HDAC signaling, which was associated with decreased flAR and AR-V7 activity, but multiple additional oncogenic pathways. These observations raise the possibility that treatment of CRPC may be achieved by using similarly multi-targeted approaches.[3] \n\n

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1. Mechanism of action: CUDC-101 exerts synergistic anticancer effects via simultaneous inhibition of three key pathways: (1) HDAC inhibition (increases histone acetylation, upregulates tumor suppressor genes); (2) EGFR/HER2 inhibition (blocks oncogenic tyrosine kinase signaling); (3) AR/AR-V7 inhibition (suppresses androgen-dependent and -independent prostate cancer growth). This multi-target activity overcomes single-agent resistance in diverse cancers [1,2,3,4]
2. Preclinical advantages over single-target inhibitors: Compared to single HDAC inhibitors (e.g., vorinostat) or EGFR inhibitors (e.g., erlotinib), CUDC-101 shows enhanced antitumor efficacy in EGFR-overexpressing/HDAC-dependent cancers (e.g., CRC, ATC). For example, in HCT116 xenografts, 40 mg/kg CUDC-101 (85% tumor inhibition) outperformed vorinostat (40% inhibition) and erlotinib (35% inhibition) at equivalent doses [2]
3. Potential clinical indications: Based on preclinical data, CUDC-101 is being evaluated for treatment of: (1) Solid tumors with EGFR/HER2 overexpression (CRC, ATC); (2) Castration-resistant prostate cancer (CRPC) with flAR/AR-V7 expression; (3) EGFR-mutant NSCLC (limited activity in T790M mutants, but synergism with EGFR T790M inhibitors is under investigation) [2,3,4]
4. Formulation considerations: CUDC-101 has low aqueous solubility, requiring formulation in DMSO/PEG300/PBS for in vivo studies. Oral bioavailability (23% in mice) is moderate; future formulation optimization (e.g., nanosuspensions) may improve oral absorption [2]

C24H26N4O4

434.49

434.195

C, 66.34; H, 6.03; N, 12.89; O, 14.73

1012054-59-9

24756910

White to yellow solid powder

1.3±0.1 g/cm3

174-177ºC

1.638

2.84

109.09

3

7

12

32

624

0

O(C1=C(C([H])=C2C(C(=NC([H])=N2)N([H])C2=C([H])C([H])=C([H])C(C#C[H])=C2[H])=C1[H])OC([H])([H])[H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C([H])([H])C(N([H])O[H])=O

PLIVFNIUGLLCEK-UHFFFAOYSA-N

InChI=1S/C24H26N4O4/c1-3-17-9-8-10-18(13-17)27-24-19-14-22(21(31-2)15-20(19)25-16-26-24)32-12-7-5-4-6-11-23(29)28-30/h1,8-10,13-16,30H,4-7,11-12H2,2H3,(H,28,29)(H,25,26,27)

7-[4-(3-ethynylanilino)-7-methoxyquinazolin-6-yl]oxy-N-hydroxyheptanamide

CUDC-101; CUDC 101; 7-(4-(3-Ethynylphenylamino)-7-methoxyquinazolin-6-yloxy)-N-hydroxyheptanamide; CUDC101; 7-[[4-(3-Ethynylphenylamino)-7-methoxyquinazolin-6-yl]oxy]-N-hydroxyheptanamide; CHEMBL598797; 1A7Y9MP123; CUDC101

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)

配方 1 中的溶解度: ≥ 2.08 mg/mL (4.79 mM) (饱和度未知) 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), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 20.8 mg/mL澄清DMSO储备液加入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: ≥ 2.08 mg/mL (4.79 mM) (饱和度未知) 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), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 20.8 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: ≥ 2.08 mg/mL (4.79 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 20.8 mg/mL 澄清 DMSO 储备液添加到 900 μL 玉米油中并混合均匀。

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配方 4 中的溶解度: 15% Captisol: 30mg/mL

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配方 5 中的溶解度: 16.67 mg/mL (38.37 mM) in 50% PEG300 50% 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。
*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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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。