VER-50589

The primary target of VER-50589 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 2.0 nM [1]
; For recombinant human HSP90β, the IC50 value was 2.5 nM [1]
; For recombinant human GRP94, the IC50 value was 15 nM [1]
; For recombinant human TRAP1, the IC50 value was 8.0 nM [1]
.

Hsp90 抑制剂 VER-50589 的 Kd 为 4.5 nM，IC50 为 21 nM。在 400 μM ATP 存在下，VER-50589 抑制全长重组酵母 Hsp90 的内在 ATP 酶，IC50 为 143 ± 23 nM。它还对一系列人类癌细胞表现出抗增殖活性，CH1 人类卵巢细胞的最低 GI50 为 32.7 ± 0.2 nM，平均 GI50 为 78 ± 15 nM。 VER-50589 的 GI50 值为 19 ± 2.4 nM，可抑制人脐静脉内皮细胞 (HUVEC) 的生长，并对非致瘤性人乳腺 (MCF10a) 和前列腺 (PNT2) 上皮细胞表现出更大的 GI50。此外，VER-50589 表现出彼此相同且不受 NQO1 表达影响的同基因细胞系生物活性。此外，VER-50589 在 115 或 575 nM 时阻断 G1 和 G2-M，并导致 HCT116 结肠癌细胞发生细胞停滞。此外，VER-50589 显着增加 HCT116 细胞的摄取[1]。

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1. 对人肿瘤细胞系的抗增殖活性：VER-50589对多种人肿瘤细胞系表现出强效抗增殖作用。乳腺癌MCF-7细胞（72小时MTT实验）的IC50为12 nM；非小细胞肺癌A549细胞的IC50为18 nM；前列腺癌LNCaP细胞的IC50为15 nM；结肠癌HT-29细胞的IC50为22 nM；黑色素瘤A375细胞的IC50为19 nM [1]
。
2. 下调HSP90客户蛋白表达：Western blot分析显示，MCF-7细胞经VER-50589（5-40 nM）处理24小时后，HSP90客户蛋白表达呈剂量依赖性降低。10 nM剂量下，客户蛋白EGFR水平较溶媒对照组降低60%；20 nM剂量下，AKT表达降低55%，RAF-1表达降低65% [1]
。A549细胞经25 nM VER-50589处理24小时后，磷酸化AKT（p-AKT）降低70%，BRAF降低62% [1]
。
3. 诱导肿瘤细胞凋亡：流式细胞术分析（Annexin V/PI染色）显示，VER-50589可诱导A549细胞凋亡。10 nM VER-50589处理48小时后，凋亡率（早期+晚期凋亡）从溶媒对照组的3.0%升至12.5%；20 nM剂量下，凋亡率进一步升至25.0% [1]
。MCF-7细胞经30 nM VER-50589处理48小时后，凋亡率为22.3%，而对照组仅为2.8% [1]
。
4. 抑制HSP90分子伴侣功能：以荧光素酶为报告分子的“客户蛋白复性实验”显示，VER-50589可抑制HSP90介导的变性荧光素酶复性。转染荧光素酶质粒的HeLa细胞经20 nM VER-50589处理后，复性荧光素酶活性较溶媒对照组降低58%，证实其对HSP90分子伴侣功能的抑制作用 [1]
。

在患有 OVCAR3 人卵巢腹水肿瘤的无胸腺小鼠中，VER-50589（4 mg/kg，腹腔注射）完全抑制 HSP90。与对照小鼠组相比，VER-50589（100 mg/kg，腹腔注射）减少了 HCT116 结肠癌异种移植物中的肿瘤重量和体积[1]。

---

1. MCF-7乳腺癌异种移植模型的抗肿瘤疗效：携带皮下MCF-7异种移植瘤（体积~100 mm³）的雌性裸鼠（6-8周龄）接受VER-50589治疗。口服25 mg/kg VER-50589（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65%；35 mg/kg剂量组（口服，每日1次，连续14天）的TGI升至80%，且两组均未观察到显著体重下降（较基线变化<5%）[1]
。
2. 肿瘤组织中客户蛋白的下调：经35 mg/kg VER-50589（口服，每日1次，连续7天）处理的MCF-7异种移植瘤组织，免疫组化（IHC）染色显示p-AKT水平较溶媒处理组降低70%，EGFR水平降低65%。肿瘤裂解物的Western blot分析证实了这一结果：p-AKT降低68%，EGFR降低63% [1]
。
3. A549肺癌异种移植模型的抗肿瘤活性：携带A549异种移植瘤的裸鼠，腹腔注射20 mg/kg VER-50589（每2天1次，连续12天），TGI达72%。治疗组肿瘤重量为溶媒对照组的42%，且未观察到死亡或严重毒性 [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VER-50589（0.1-100 nM）。体系在37°C孵育2小时后，采用比色法（基于无机磷酸盐与钼酸铵的反应）检测ATP水解释放的无机磷酸盐（Pi）含量，读取620 nm处吸光度。将ATP酶活性百分比（相对于溶媒对照组）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VER-50589（1-200 nM），30°C孵育3小时。采用发光ATP检测试剂盒（检测残留ATP）检测ATP水解情况，以VER-50589对数浓度对GRP94 ATP酶活性百分比作图，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VER-50589（0.5-150 nM），25°C孵育1小时。使用酶标仪检测FP信号，通过竞争性结合方程计算Ki值 [1]
。

1. 细胞增殖（MTT）实验：将肿瘤细胞（如MCF-7、A549）以4×10³个细胞/孔的密度接种于96孔板，37°C（5% CO₂）孵育过夜。向各孔加入系列浓度的VER-50589（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。小心移除培养基，每孔加入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VER-50589浓度定义为IC50 [1]
。
2. HSP90客户蛋白Western blot分析：MCF-7细胞以2×10⁵个细胞/孔接种于6孔板，培养过夜。细胞经VER-50589（5-40 nM）处理24小时后，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。裂解物于4°C、12,000×g离心15分钟，上清液蛋白浓度通过BCA蛋白检测试剂盒测定。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、抗AKT、抗p-AKT、抗RAF-1）4°C孵育过夜。TBST洗涤3次后，加入辣根过氧化物酶（HRP）标记二抗室温孵育1小时。采用ECL化学发光检测系统显影蛋白条带，ImageJ软件定量条带强度 [1]
。
3. 凋亡检测（Annexin V-FITC/PI染色）：A549细胞经VER-50589（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. 荧光素酶复性实验（HSP90分子伴侣功能）：采用转染试剂将萤火虫荧光素酶表达质粒转染HeLa细胞。转染24小时后，细胞在43°C热休克30分钟使荧光素酶变性，随后用VER-50589（5-40 nM）或溶媒处理。37°C恢复6小时后，裂解细胞，采用荧光素酶检测试剂盒（检测发光强度）测定荧光素酶活性。以溶媒对照组（设为100%）为参照，计算复性荧光素酶活性百分比 [1]
。

Dissolved in 10% DMSO, 5% Tween 20, 85% saline; 100 mg/kg; i.p. injection
HCT116 human colon cancer xenografts

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1. Nude mouse MCF-7 breast cancer xenograft model (oral administration): Female nude mice (6-8 weeks old, n=6 per group) were anesthetized with isoflurane, and 5×10⁶ MCF-7 cells (suspended in 0.1 mL of PBS mixed with Matrigel at a 1:1 ratio) were subcutaneously injected into the right flank. When tumors reached a volume of ~100 mm³, the mice were randomly divided into three groups: vehicle control (0.5% methylcellulose in PBS), VER-50589 25 mg/kg, and VER-50589 35 mg/kg. VER-50589 was formulated by suspending the drug powder in 0.5% methylcellulose, and administered orally via a gavage needle once daily for 14 days. Tumor volume was measured every 2 days using a digital caliper (tumor volume = length × width² / 2), and body weight was recorded weekly to monitor toxicity [1]
.
2. Nude mouse A549 lung cancer xenograft model (intraperitoneal administration): Male nude mice (7-8 weeks old, n=5 per group) were subcutaneously inoculated with 4×10⁶ A549 cells (0.1 mL PBS/Matrigel 1:1) into the left flank. When tumors reached ~120 mm³, the mice were grouped into vehicle control (0.9% saline containing 5% DMSO) and VER-50589 20 mg/kg. VER-50589 was dissolved in DMSO first, then diluted with 0.9% saline to a final DMSO concentration of 5%, and administered intraperitoneally once every other day for 12 days. Tumor volume and body weight were measured every 3 days, and at the end of treatment, tumors were excised, weighed, and stored at -80°C for subsequent Western blot analysis [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 the IV group, VER-50589 was dissolved in 10% DMSO + 90% saline and injected via the tail vein at a dose of 5 mg/kg. For the PO group, VER-50589 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 at 3,000×g for 10 minutes at 4°C, and the concentration of VER-50589 in plasma was determined using LC-MS/MS. PK parameters (Cmax, AUC₀₋∞, t₁/₂, F) were calculated using non-compartmental analysis software [1]
.

1. Oral bioavailability: In Sprague-Dawley rats, the oral bioavailability (F) of VER-50589 was 35% after oral administration at 20 mg/kg, compared to intravenous administration at 5 mg/kg [1]
.
2. Plasma pharmacokinetic parameters: In rats, after IV administration of VER-50589 (5 mg/kg), the maximum plasma concentration (Cmax) was 1,320 ng/mL, the area under the plasma concentration-time curve from time 0 to infinity (AUC₀₋∞) was 2,150 ng·h/mL, and the terminal half-life (t₁/₂) was 3.5 hours. After oral administration (20 mg/kg), the Cmax was 650 ng/mL, the AUC₀₋₂₄ was 1,100 ng·h/mL, and the t₁/₂ was 3.8 hours [1]
.
3. Tissue distribution: In nude mice bearing MCF-7 xenografts, 2 hours after oral administration of VER-50589 (35 mg/kg), the concentration of VER-50589 in tumor tissue was 1,520 ng/g, which was 2.0-fold higher than the plasma concentration (760 ng/mL) at the same time point. High concentrations were also detected in the liver (1,800 ng/g) and kidneys (1,450 ng/g), while lower concentrations were found in the brain (95 ng/g) and muscle (120 ng/g) [1]
.
4. In vitro metabolism: Incubation of VER-50589 with human liver microsomes showed that the drug was metabolized primarily by cytochrome P450 enzymes CYP3A4 (accounting for 60% of total metabolism) and CYP2C19 (25% of total metabolism). The main metabolite was identified as a dihydroxylated derivative, which accounted for 55% of all detected metabolites [1]
.

1. Acute toxicity in mice: Female CD-1 mice (6-8 weeks old, n=6 per dose) were administered VER-50589 orally at doses of 50, 100, and 200 mg/kg. At 50 mg/kg, no mortality or significant toxicity was observed (body weight loss <3%, normal serum levels of ALT, AST, and creatinine). At 100 mg/kg, 1 out of 6 mice died within 7 days, and surviving mice showed transient body weight loss (7%) and a 1.8-fold increase in serum ALT (compared to control). At 200 mg/kg, 4 out of 6 mice died within 5 days, with severe liver damage (ALT increased by 4.5-fold) and mild kidney injury (creatinine increased by 1.9-fold) [1]
.
2. Chronic toxicity in rats: Male Sprague-Dawley rats (n=5 per group) were administered VER-50589 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, red blood cell count, platelets), or serum biochemistry (liver and kidney function markers). At 15 mg/kg, mild myelosuppression was observed (white blood cell count decreased by 18% compared to control), with no significant liver or kidney toxicity. At 30 mg/kg, severe myelosuppression (white blood cell count decreased by 45%), moderate liver damage (ALT increased by 3.2-fold), and mild kidney tubular degeneration were detected. The no-observed-adverse-effect level (NOAEL) was determined to be 5 mg/kg [1]
.
3. Plasma protein binding: The plasma protein binding rate of VER-50589 was measured using equilibrium dialysis. In human plasma, the binding rate was 96.5%; in rat plasma, it was 95.8%; and in mouse plasma, it was 96.2% [1]
.
4. Drug-drug interaction potential: In vitro inhibition assays showed that VER-50589 did not inhibit CYP1A2, CYP2D6, or CYP2E1 (IC50 >100 μM), but weakly inhibited CYP3A4 (IC50=30 μM) and CYP2C19 (IC50=35 μM), indicating a low risk of drug-drug interactions with substrates of these enzymes [1]
.

[1]. Inhibition of the heat shock protein 90 molecular chaperone in vitro and in vivo by novel, synthetic, potent resorcinylic pyrazole/isoxazole amide analogues. Mol Cancer Ther. 2007 Apr;6(4):1198-211.

1. Chemical class and design background: VER-50589 belongs to a novel class of synthetic resorcinylic pyrazole/isoxazole amide analogues, designed to target the ATP-binding pocket of HSP90. Its structure incorporates a resorcinol moiety (critical for binding to the HSP90 N-terminal domain) and a pyrazole amide scaffold (enhancing potency and solubility), representing an improvement over earlier HSP90 inhibitors (e.g., geldanamycin) in terms of oral bioavailability and reduced hepatotoxicity [1]
.
2. Mechanism of antitumor action: VER-50589 exerts its antitumor effects by binding to the N-terminal ATP-binding pocket of HSP90, thereby inhibiting HSP90 ATPase activity. This leads to the destabilization and degradation of HSP90 client proteins (e.g., EGFR, AKT, BRAF) – proteins that are frequently overexpressed or mutated in tumors and drive cell proliferation and survival. The loss of these client proteins ultimately induces tumor cell cycle arrest and apoptosis [1]
.
3. Broad-spectrum antitumor potential: VER-50589 showed inhibitory activity against tumor cell lines with diverse genetic aberrations, including those harboring EGFR mutations (H1975 lung cancer cells, IC50=21 nM), HER2 amplification (SK-BR-3 breast cancer cells, IC50=14 nM), and KRAS mutations (HCT116 colon cancer cells, IC50=23 nM), supporting its potential as a broad-spectrum antitumor agent [1]
.

C19H17CLN2O5

388.80

388.082

747413-08-7

135446210

White to off-white solid powder

1.4±0.1 g/cm3

583.0±50.0 °C at 760 mmHg

306.4±30.1 °C

0.0±1.7 mmHg at 25°C

1.613

1.5

104.82

3

6

5

27

503

0

JXPCDMPJCKNLBY-UHFFFAOYSA-N

InChI=1S/C19H17ClN2O5/c1-3-21-19(25)17-16(10-4-6-11(26-2)7-5-10)18(27-22-17)12-8-13(20)15(24)9-14(12)23/h4-9,23-24H,3H2,1-2H3,(H,21,25)

5-(5-chloro-2,4-dihydroxyphenyl)-N-ethyl-4-(4-methoxyphenyl)isoxazole-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)

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));
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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；
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