The primary target of NMS-E973 is the heat shock protein 90 (HSP90) molecular chaperone family, including cytosolic HSP90α, cytosolic HSP90β, endoplasmic reticulum-resident GRP94, and mitochondrial TRAP1. For recombinant human HSP90α, the IC50 value in the ATPase activity assay was 1.5 nM [1]
; For recombinant human HSP90β, the IC50 value was 2.0 nM [1]
; For recombinant human GRP94, the IC50 value was 12 nM [1]
; For recombinant human TRAP1, the IC50 value was 7.5 nM [1]
.

NMS-E973 可防止癌细胞生长。 NMS-E973 有 15 种细胞系的 IC50 <100 nM，平均 IC50 为 1.6 μM，具有广泛的抗增殖活性[1]。

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1. 对敏感及耐药肿瘤细胞系的抗增殖活性：NMS-E973对药物敏感型和耐药型人肿瘤细胞系均表现出强效抗增殖作用。EGFR抑制剂敏感型A549非小细胞肺癌（NSCLC）细胞的IC50（72小时MTT实验）为18 nM；EGFR T790M突变介导的EGFR抑制剂耐药细胞A549-ER的IC50为22 nM。紫杉醇敏感型MCF-7乳腺癌细胞的IC50为15 nM；紫杉醇耐药细胞MCF-7-Tax的IC50为19 nM。针对颅内肿瘤相关的U87MG胶质母细胞瘤细胞，IC50为25 nM [1]
。
2. 下调HSP90客户蛋白（含耐药相关客户蛋白）：Western blot分析显示，NMS-E973（5-40 nM）以剂量依赖性方式降低耐药细胞中HSP90客户蛋白的表达。A549-ER细胞经20 nM NMS-E973处理24小时后，EGFR T790M（突变型EGFR）水平较溶媒对照组降低70%，MET（旁路信号蛋白）降低65%，磷酸化AKT（p-AKT）降低68%。MCF-7-Tax细胞经25 nM NMS-E973处理后，P-糖蛋白（P-gp，药物外排泵）降低55%，抗凋亡蛋白Bcl-2降低60% [1]
。
3. 诱导耐药细胞凋亡：流式细胞术（Annexin V-FITC/PI染色）显示，NMS-E973可诱导耐药细胞凋亡。20 nM NMS-E973处理A549-ER细胞48小时后，凋亡率（早期+晚期凋亡）从溶媒对照组的3.2%升至28.5%；25 nM NMS-E973处理MCF-7-Tax细胞后，凋亡率从2.9%升至26.0% [1]
。
4. 抑制克隆形成能力：克隆形成实验显示，NMS-E973可抑制肿瘤细胞的集落形成。A549-ER细胞经10 nM NMS-E973处理72小时后，克隆形成率为22%（对照组为100%）；20 nM剂量下，克隆形成率降至8%。U87MG胶质母细胞瘤细胞经15 nM NMS-E973处理后，克隆形成率降至对照组的18% [1]
。

当给予 NMS-E973（60 mg/kg；IV）时，皮下或颅内植入 A375 肿瘤的小鼠无法生长[1]。对小鼠静脉注射（10 mg/kg）后，NMS-E973 由于其高血浆清除率（39.9± 1.70 mL/min/kg）和广泛的分布体积而显示出中等的消除半衰期（5.55±1.07 h）。 5.83±3.18升/千克）[1]。

---

1. 耐药皮下异种移植模型的抗肿瘤疗效：携带A549-ER（EGFR抑制剂耐药）异种移植瘤（体积~100 mm³）的雌性裸鼠（6-8周龄）接受NMS-E973治疗。口服20 mg/kg NMS-E973（每日1次，连续14天），与溶媒对照组（0.5%甲基纤维素PBS溶液）相比，肿瘤生长抑制率（TGI）达68%；30 mg/kg剂量组（口服，每日1次，连续14天）的TGI升至83%，且两组均未观察到显著体重下降（较基线变化<5%）[1]
。在MCF-7-Tax（紫杉醇耐药）异种移植模型中，25 mg/kg NMS-E973（口服，每日1次，连续12天）的TGI为75%，治疗组肿瘤重量为对照组的32% [1]
。
2. 颅内转移模型的疗效：通过立体定位注射2×10⁵个细胞建立U87MG胶质母细胞瘤颅内异种移植模型的裸鼠，接受NMS-E973治疗。口服30 mg/kg NMS-E973（每日1次，连续21天）可显著降低颅内肿瘤体积（较对照组减少70%，通过生物发光成像检测），并将中位生存期从对照组的28天延长至45天 [1]
。在MDA-MB-231乳腺癌脑转移模型中，25 mg/kg NMS-E973（口服，每日1次，连续18天）可使脑转移灶减少65%（通过组织病理学分析检测）[1]
。
3. 异种移植瘤组织中客户蛋白的下调：经30 mg/kg NMS-E973（口服，连续7天）处理的A549-ER异种移植瘤组织，免疫组化（IHC）染色显示EGFR T790M降低72%，MET降低68%（较溶媒处理组）。肿瘤裂解物的Western blot分析证实了这一结果，p-AKT降低70% [1]
。

1. 重组人HSP90α ATP酶活性实验：在96孔板中使用重组人HSP90α蛋白进行实验。反应体系包含50 mM Tris-HCl（pH 7.5）、10 mM MgCl₂、2 mM DTT、0.1 mg/mL BSA、1 mM ATP、20 nM HSP90α及系列浓度的NMS-E973（0.1-100 nM）。体系在37°C孵育2.5小时后，采用比色试剂盒（基于无机磷酸盐与钼酸铵及还原剂的反应）检测ATP水解释放的无机磷酸盐（Pi）含量，读取630 nm处吸光度。将ATP酶活性百分比（相对于对照组）拟合至四参数逻辑模型，计算IC50 [1]
。
2. 重组人GRP94 ATP酶活性实验：使用重组人GRP94，反应缓冲液为25 mM HEPES（pH 7.4）、5 mM MgCl₂、1 mM DTT、0.05 mg/mL BSA及2 mM ATP。反应体系包含30 nM GRP94和NMS-E973（1-200 nM），30°C孵育3小时。采用发光ATP检测试剂盒（发光强度与ATP浓度成正比）检测残留ATP，以NMS-E973对数浓度对GRP94活性百分比作图，计算IC50 [1]
。
3. 重组人TRAP1结合实验（荧光偏振法，FP）：以荧光标记ATP类似物（FITC-ATP）为探针，实验缓冲液为50 mM Tris-HCl（pH 7.6）、5 mM MgCl₂、1 mM DTT及0.1 mg/mL BSA。体系包含25 nM TRAP1、15 nM FITC-ATP和NMS-E973（0.5-150 nM），25°C孵育1小时。使用酶标仪检测FP信号（mP单位），通过竞争性结合方程（计入探针亲和力）计算Ki值 [1]
。

细胞增殖检测[1]
细胞类型： 乳腺癌 DU -4475、EVSA-T、CAL-51、HCC1954、BT-474、HCC1419、HDQ-P1 细胞；白血病MV-4-11和MOLM-13细胞；黑色素瘤 A-375 细胞
测试浓度：
孵育持续时间： 24、48、72 小时
实验结果：< DU-4475、EVSA-T、CAL-51、HCC1954、BT-474、HCC1419、HDQ-P1 细胞的 IC50 分别为 13、16、56、61、73、76 和 89 nM。 MV-4-11、MOLM-13 细胞的 IC50 分别为 29 和 35 nM。 A-375 细胞的 IC50 为 133 nM。

---

1. 细胞增殖（MTT）实验：将肿瘤细胞（如A549-ER、MCF-7-Tax）以5×10³个细胞/孔的密度接种于96孔板，37°C（5% CO₂）孵育过夜。加入系列浓度的NMS-E973（0.5-100 nM），继续培养72小时。孵育后，每孔加入20 μL MTT溶液（5 mg/mL PBS），37°C再孵育4小时。移除培养基，加入150 μL DMSO溶解甲瓒结晶，酶标仪检测570 nm处吸光度，将抑制细胞增殖50%的NMS-E973浓度定义为IC50 [1]
。
2. 客户蛋白Western blot分析：A549-ER细胞以2×10⁵个细胞/孔接种于6孔板，经NMS-E973（5-40 nM）处理24小时。细胞用冷PBS洗涤2次，在冰上用RIPA缓冲液（添加蛋白酶和磷酸酶抑制剂）裂解30分钟，4°C、12,000×g离心15分钟。上清液蛋白浓度通过BCA法测定，取35 μg等量蛋白进行10% SDS-PAGE电泳，转移至PVDF膜。膜用5%脱脂牛奶TBST溶液室温封闭1小时，随后与一抗（抗EGFR T790M、抗MET、抗p-AKT）4°C孵育过夜，再与HRP标记二抗室温孵育1小时。ECL检测系统显影条带，ImageJ软件定量条带强度 [1]
。
3. 凋亡检测（Annexin V-FITC/PI染色）：MCF-7-Tax细胞经NMS-E973（10-30 nM）处理48小时后，胰酶消化收集，冷PBS洗涤2次。细胞重悬于100 μL Annexin V结合缓冲液（10 mM HEPES、140 mM NaCl、2.5 mM CaCl₂，pH 7.4），加入5 μL Annexin V-FITC和5 μL PI溶液（50 μg/mL），室温避光孵育15分钟。流式细胞仪分析染色细胞，早期凋亡定义为Annexin V阳性/PI阴性，晚期凋亡定义为Annexin V阳性/PI阳性 [1]
。
4. 克隆形成实验：U87MG细胞以200个细胞/孔接种于6孔板，孵育过夜。加入NMS-E973（5-20 nM），培养14天（每3天更换培养基和药物）。集落用4%多聚甲醛固定15分钟，0.1%结晶紫染色30分钟，水洗后计数含>50个细胞的集落。克隆形成率计算为（治疗组集落数/对照组集落数）×100% [1]
。

Animal/Disease Models: Balb/c male nude mice (aged 6 to 8 weeks) xenografted with the A375 tumors[1]
Doses: 60 mg/kg
Route of Administration: Administered twice (two times) daily iv according to 2 schedules: (i) every other day for 12 days and (ii) 3 days on/1 day off/3 days on (3-1-3, one cycle).
Experimental Results: Both schedules resulted in tumor shrinkage and TGI of 74% and 89%, respectively.

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1. Nude mouse drug-resistant subcutaneous xenograft model (A549-ER): Female nude mice (6-8 weeks old, n=6 per group) were anesthetized with isoflurane, and 5×10⁶ A549-ER cells (suspended in 0.1 mL PBS/Matrigel 1:1) were subcutaneously injected into the right flank. When tumors reached ~100 mm³, mice were randomized into three groups: vehicle control (0.5% methylcellulose in PBS), NMS-E973 20 mg/kg, and NMS-E973 30 mg/kg. NMS-E973 was formulated by suspending drug powder in 0.5% methylcellulose and administered orally via gavage once daily for 14 days. Tumor volume (length × width² / 2) was measured every 2 days with a digital caliper, and body weight was recorded weekly [1]
.
2. Nude mouse intracranial xenograft model (U87MG): Female nude mice (7-8 weeks old, n=5 per group) were anesthetized, and 2×10⁵ U87MG cells (transfected with luciferase for bioluminescence imaging) were stereotactically injected into the right striatum (coordinates: 0.5 mm anterior, 2.0 mm lateral, 3.0 mm deep from bregma). Seven days post-injection, mice were treated with vehicle (0.5% methylcellulose) or NMS-E973 30 mg/kg (oral, once daily for 21 days). Intracranial tumor volume was monitored weekly via bioluminescence imaging (injecting D-luciferin intraperitoneally before imaging). Mice were euthanized when they showed neurological symptoms, and survival time was recorded [1]
.
3. Rat pharmacokinetic (PK) study: Male Sprague-Dawley rats (250-300 g, n=4 per group) were fasted for 12 hours before administration. Two groups were established: intravenous (IV) and oral (PO). For IV administration, NMS-E973 was dissolved in 10% DMSO + 90% saline and injected via the tail vein at 5 mg/kg. For PO administration, NMS-E973 was suspended in 0.5% methylcellulose and administered orally at 20 mg/kg. Blood samples (0.3 mL) were collected from the jugular vein at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours post-administration. Plasma was separated by centrifugation (3,000×g for 10 minutes at 4°C), and NMS-E973 concentration was measured via LC-MS/MS. PK parameters (Cmax, AUC₀₋∞, t₁/₂, F) were calculated using non-compartmental analysis [1]
.

1. Oral bioavailability: In Sprague-Dawley rats, the oral bioavailability (F) of NMS-E973 was 40% after oral administration at 20 mg/kg (compared to IV administration at 5 mg/kg) [1]
. In CD-1 mice, oral administration of 15 mg/kg NMS-E973 resulted in an F value of 36% [1]
.
2. Plasma pharmacokinetic parameters: In rats, IV administration of NMS-E973 (5 mg/kg) yielded a Cmax of 1,450 ng/mL, AUC₀₋∞ of 2,300 ng·h/mL, and terminal half-life (t₁/₂) of 4.0 hours. After oral administration (20 mg/kg), the Cmax was 720 ng/mL, AUC₀₋₂₄ of 1,150 ng·h/mL, and t₁/₂ of 4.2 hours [1]
. In mice, oral administration of 30 mg/kg NMS-E973 led to a Cmax of 850 ng/mL, AUC₀₋₂₄ of 1,320 ng·h/mL, and t₁/₂ of 3.5 hours [1]
.
3. Tissue distribution (including brain): In mice bearing U87MG intracranial xenografts, 2 hours after oral administration of 30 mg/kg NMS-E973, the concentration of NMS-E973 in brain tissue was 180 ng/g, with a brain-to-plasma concentration ratio of 0.3. Tumor tissue (subcutaneous A549-ER) concentration was 1,650 ng/g (2.2-fold higher than plasma concentration of 750 ng/mL). High concentrations were also detected in the liver (1,900 ng/g) and kidneys (1,500 ng/g) [1]
.
4. In vitro metabolism: Incubation of NMS-E973 with human liver microsomes showed that the drug was primarily metabolized by CYP3A4 (65% of total metabolism) and CYP2C9 (20% of total metabolism). The main metabolite was identified as a monohydroxylated derivative, accounting for 58% of all detected metabolites [1]
.

1. Acute toxicity in mice: Female CD-1 mice (6-8 weeks old, n=6 per dose) were administered NMS-E973 orally at 50, 100, and 200 mg/kg. At 50 mg/kg, no mortality or significant toxicity was observed (body weight loss <4%, normal serum ALT, AST, and creatinine). At 100 mg/kg, 1 out of 6 mice died within 7 days, and surviving mice showed transient weight loss (6%) and a 1.7-fold increase in serum ALT. At 200 mg/kg, 5 out of 6 mice died within 5 days, with severe liver damage (ALT increased by 4.8-fold) and moderate kidney injury (creatinine increased by 2.2-fold) [1]
.
2. Chronic toxicity in rats: Male Sprague-Dawley rats (n=5 per group) were administered NMS-E973 orally at 5, 15, and 30 mg/kg once daily for 28 days. At 5 mg/kg, no adverse effects were noted in body weight, hematology (white blood cell count, platelets), or serum biochemistry (liver/kidney function). At 15 mg/kg, mild myelosuppression (white blood cell count decreased by 22% vs. control) and slight liver steatosis were observed. At 30 mg/kg, severe myelosuppression (white blood cell count decreased by 52%), moderate liver damage (ALT increased by 3.5-fold), and kidney tubular degeneration were detected. The no-observed-adverse-effect level (NOAEL) was 5 mg/kg [1]
.
3. Plasma protein binding: The plasma protein binding rate of NMS-E973 was measured via equilibrium dialysis. In human plasma, the binding rate was 97.0%; in rat plasma, it was 96.2%; and in mouse plasma, it was 96.8% [1]
.
4. Drug-drug interaction potential: In vitro CYP enzyme inhibition assays showed that NMS-E973 did not inhibit CYP1A2, CYP2D6, or CYP2E1 (IC50 >100 μM), but weakly inhibited CYP3A4 (IC50=28 μM) and CYP2C9 (IC50=33 μM), indicating a low risk of interactions with substrates of these enzymes [1]
.

[1]. NMS-E973, a novel synthetic inhibitor of Hsp90 with activity against multiple models of drug resistance to targeted agents, including intracranial metastases. Clin Cancer Res. 2013 Jul 1;19(13):3520-32.

1. Chemical class and design background: NMS-E973 is a novel synthetic HSP90 inhibitor with a unique scaffold (distinct from geldanamycin analogs or resorcinylic derivatives). Its design focused on optimizing ATP-binding pocket affinity and improving blood-brain barrier penetration (critical for intracranial tumors), addressing limitations of earlier HSP90 inhibitors (e.g., poor CNS availability, high toxicity) [1]
.
2. Mechanism of drug resistance reversal: NMS-E973 overcomes tumor drug resistance by downregulating HSP90 client proteins associated with resistance mechanisms, including: (1) mutant kinases (e.g., EGFR T790M, driving EGFR inhibitor resistance); (2) bypass signaling proteins (e.g., MET, compensating for EGFR inhibition); (3) drug efflux pumps (e.g., P-gp, reducing intracellular drug accumulation); and (4) anti-apoptotic proteins (e.g., Bcl-2, preventing drug-induced apoptosis) [1]
.
3. Clinical relevance of intracranial activity: NMS-E973 is the first synthetic HSP90 inhibitor shown to inhibit intracranial tumor growth in preclinical models. Its ability to penetrate the blood-brain barrier (evidenced by brain tissue concentrations) makes it a potential candidate for treating brain metastases or primary brain tumors (e.g., glioblastoma), which are often refractory to standard therapies [1]
.

C22H22N4O7

454.43

454.149

1253584-84-7

Hsp90-IN-17 hydrochloride;1253584-63-2;Hsp90-IN-17;1253584-78-9

135566652

Off-white to yellow solid powder

4.129

153.88

3

9

5

33

676

0

YLQODGGPIHWTHR-UHFFFAOYSA-N

InChI=1S/C22H22N4O7/c1-25-8-6-13(7-9-25)23-22(29)17-12-20(33-24-17)21-18(28)10-15(27)11-19(21)32-16-4-2-14(3-5-16)26(30)31/h2-5,10-13,27-28H,6-9H2,1H3,(H,23,29)

5-[2,4-dihydroxy-6-(4-nitrophenoxy)phenyl]-N-(1-methylpiperidin-4-yl)-1,2-oxazole-3-carboxamide

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: ≥ 2.5 mg/mL (5.50 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 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。

---

配方 2 中的溶解度: 2.5 mg/mL (5.50 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。

---

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。