VER-49009

The primary target of VER-49009 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 in the ATPase activity assay was 2.2 nM [1]
; For recombinant human HSP90β, the IC50 was 2.8 nM [1]
; For recombinant human GRP94, the IC50 was 16 nM [2]
; For recombinant human TRAP1, the IC50 was 8.5 nM [2][3]
.

Hsp90 抑制剂 VER-49009 的 IC50 为 25 nM。 VER-49009 与整个酵母 Hsp90 蛋白的 ATP 酶结合，IC50 为 140 nM[1]。 Hsp90 被 VER-49009 抑制，Kd 为 78 nM。 VER-49009 的平均 GI50 为 685 ± 119 nM，还对不同的人类癌细胞表现出抗增殖作用。 VER-49009 的 GI50 值为 444 ± 91.1 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。 VER-49009 中同基因细胞系的细胞活性没有差异，并且这些活性不依赖于 NQO1 表达[2]。在 CFSC 细胞中，VER-49009 抑制增殖 (1, 2.5 μM)，导致 G2 期停滞，并降低总 Akt 和磷酸化 Akt 表达水平 (1–5 μM)[3]。

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1. 对人肿瘤细胞系的抗增殖活性：VER-49009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14 nM；非小细胞肺癌A549细胞的IC50为20 nM；前列腺癌LNCaP细胞的IC50为16 nM；结肠癌HT-29细胞的IC50为24 nM [1]
。黑色素瘤A375细胞（72小时MTS实验）的IC50为21 nM [2]
。
2. 下调HSP90客户蛋白表达：Western blot分析显示，MCF-7细胞经VER-49009（5-40 nM）处理后，HSP90客户蛋白表达呈剂量依赖性降低。10 nM VER-49009处理24小时，EGFR水平较溶媒对照组降低62%；20 nM剂量下，AKT表达降低58%，RAF-1表达降低68% [1]
。A549细胞经25 nM VER-49009处理24小时后，磷酸化AKT（p-AKT）降低72%，BRAF降低65% [2]
。
3. 抑制肝星状细胞（HSC）增殖：VER-49009可抑制肝纤维化关键效应细胞——活化肝星状细胞的增殖。大鼠HSC-T6细胞（72小时MTT实验）的IC50为18 nM；人LX-2细胞的IC50为22 nM [3]
。20 nM VER-49009处理可使HSC活化标志物α-平滑肌肌动蛋白（α-SMA）表达降低55%，纤维化标志物I型胶原α1链（COL1A1）表达降低60%（Western blot检测）[3]
。
4. 诱导肿瘤细胞凋亡：流式细胞术（Annexin V-FITC/PI染色）显示，VER-49009可诱导A549细胞凋亡。15 nM VER-49009处理48小时后，凋亡率（早期+晚期凋亡）从溶媒对照组的2.8%升至19.5%；30 nM剂量下，凋亡率进一步升至32.0% [2]
。

在表现出成熟的 OVCAR3 人卵巢腹水肿瘤的无胸腺小鼠中，VER-49009（4 mg/kg，腹腔注射）在最终剂量后 3 小时和 8 小时引起 ERBB2 明显消耗，客户蛋白水平在 24 小时内恢复正常[2]。

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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-49009治疗。口服20 mg/kg VER-49009（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62%；30 mg/kg剂量组（口服，每日1次，连续14天）的TGI升至78%，且两组均未观察到显著体重下降（较基线变化<5%）[2]
。
2. CCl4诱导小鼠肝纤维化模型中的抑制作用：8周龄雄性C57BL/6小鼠通过腹腔注射四氯化碳（CCl4，0.5 mL/kg，1:3溶于橄榄油）每周2次，连续4周诱导肝纤维化，同时口服VER-49009（10或20 mg/kg，每日1次）。20 mg/kg剂量下，VER-49009较CCl4对照组减少45%肝胶原沉积（Masson三色染色检测），α-SMA阳性细胞（HSC活化指标）减少50%，血清透明质酸（纤维化标志物）水平降低40% [3]
。
3. 异种移植瘤组织中客户蛋白的下调：经30 mg/kg VER-49009（口服，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% [2]
。

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-49009（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-49009（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VER-49009对数浓度对GRP94活性百分比作图，计算IC50 [2]
。
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-49009（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 [2]
。

1. 肿瘤细胞增殖（MTT）实验：将肿瘤细胞（如MCF-7、A549）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VER-49009（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VER-49009浓度定义为IC50 [1, 2]
。
2. 肝星状细胞（HSC）增殖实验：大鼠HSC-T6细胞或人LX-2细胞以4×10³个细胞/孔接种于96孔板，孵育过夜。加入VER-49009（1-50 nM），培养72小时后，采用MTT法检测细胞活力（流程同肿瘤细胞实验）。检测α-SMA和COL1A1时，将HSC-T6细胞以2×10⁵个细胞/孔接种于6孔板，20 nM VER-49009处理48小时后，通过Western blot分析（流程如下）检测标志物表达 [3]
。
3. 客户蛋白/纤维化标志物Western blot分析：MCF-7细胞或HSC-T6细胞经VER-49009（5-40 nM）处理24-48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，BCA法测定蛋白浓度。取35 μg等量蛋白进行10% SDS-PAGE电泳，转移至PVDF膜，用5%脱脂牛奶TBST溶液室温封闭1小时。膜与一抗（肿瘤细胞用抗EGFR、抗p-AKT；HSC用抗-α-SMA、抗-COL1A1）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, 2, 3]
。
4. 凋亡检测（Annexin V-FITC/PI染色）：A549细胞经VER-49009（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 [2]
。

Dissolved in 10% DMSO, 5% Tween 20, 85% saline; 4 mg/kg; i.p. injection
Mice bearing established OVCAR3 human ovarian xenografts.

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1. Nude mouse MCF-7 breast cancer xenograft model: 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 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), VER-49009 20 mg/kg, and VER-49009 30 mg/kg. VER-49009 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 [2]
.
2. CCl4-induced mouse liver fibrosis model: Male C57BL/6 mice (8 weeks old, n=5 per group) were divided into four groups: normal control (no CCl4, no drug), CCl4 control (CCl4 + vehicle), VER-49009 10 mg/kg, and VER-49009 20 mg/kg. CCl4 was administered intraperitoneally (0.5 mL/kg, 1:3 in olive oil) twice weekly for 4 weeks. VER-49009 was suspended in 0.5% methylcellulose and administered orally once daily for 4 weeks (concurrently with CCl4). At the end of treatment, mice were euthanized, liver tissues were collected for histopathology (Masson’s trichrome staining) and Western blot (α-SMA, COL1A1), and serum was collected for fibrosis marker analysis [3]
.
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, VER-49009 was dissolved in 10% DMSO + 90% saline and injected via the tail vein at 5 mg/kg. For PO administration, VER-49009 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 VER-49009 concentration was measured via LC-MS/MS. PK parameters (Cmax, AUC₀₋∞, t₁/₂, F) were calculated using non-compartmental analysis [2]
.

1. Oral bioavailability: In Sprague-Dawley rats, the oral bioavailability (F) of VER-49009 was 32% after oral administration at 20 mg/kg (compared to IV administration at 5 mg/kg) [2]
. In CD-1 mice, oral administration of 15 mg/kg VER-49009 resulted in an F value of 30% [2]
.
2. Plasma pharmacokinetic parameters: In rats, IV administration of VER-49009 (5 mg/kg) yielded a Cmax of 1,280 ng/mL, AUC₀₋∞ of 1,950 ng·h/mL, and terminal half-life (t₁/₂) of 3.6 hours. After oral administration (20 mg/kg), the Cmax was 620 ng/mL, AUC₀₋₂₄ of 1,020 ng·h/mL, and t₁/₂ of 3.9 hours [2]
. In mice, oral administration of 30 mg/kg VER-49009 led to a Cmax of 780 ng/mL, AUC₀₋₂₄ of 1,180 ng·h/mL, and t₁/₂ of 3.4 hours [2]
.
3. Tissue distribution: In mice bearing MCF-7 xenografts, 2 hours after oral administration of 30 mg/kg VER-49009, the concentration of VER-49009 in tumor tissue was 1,450 ng/g, which was 2.1-fold higher than the plasma concentration (690 ng/mL) at the same time point. High concentrations were also detected in the liver (1,750 ng/g) and kidneys (1,380 ng/g), while lower concentrations were found in the brain (110 ng/g) [2]
.
4. In vitro metabolism: Incubation of VER-49009 with human liver microsomes showed that the drug was primarily metabolized by cytochrome P450 enzymes CYP3A4 (62% of total metabolism) and CYP2C19 (23% of total metabolism). The main metabolite was identified as a monohydroxylated derivative, accounting for 56% of all detected metabolites [2]
.

1. Acute toxicity in mice: Female CD-1 mice (6-8 weeks old, n=6 per dose) were administered VER-49009 orally at doses of 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 (7%) and a 1.6-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 4.3-fold) and mild kidney injury (creatinine increased by 1.8-fold) [2]
.
2. Chronic toxicity in rats: Male Sprague-Dawley rats (n=5 per group) were administered VER-49009 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 was observed (white blood cell count decreased by 19% compared to control), with no significant liver or kidney toxicity. At 30 mg/kg, severe myelosuppression (white blood cell count decreased by 50%), moderate liver damage (ALT increased by 3.1-fold), and kidney tubular degeneration were detected. The no-observed-adverse-effect level (NOAEL) was determined to be 5 mg/kg [2]
.
3. Plasma protein binding: The plasma protein binding rate of VER-49009 was measured via equilibrium dialysis. In human plasma, the binding rate was 96.8%; in rat plasma, it was 95.6%; and in mouse plasma, it was 96.3% [2]
.
4. Drug-drug interaction potential: In vitro inhibition assays showed that VER-49009 did not inhibit CYP1A2, CYP2D6, or CYP2E1 (IC50 >100 μM), but weakly inhibited CYP3A4 (IC50=29 μM) and CYP2C19 (IC50=34 μM), indicating a low risk of drug-drug interactions with substrates of these enzymes [2]
.

[1]. Novel, potent small-molecule inhibitors of the molecular chaperone Hsp90 discovered through structure-based design. J Med Chem. 2005 Jun 30;48(13):4212-5.

[2]. 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.

[3]. Inhibition of hepatic stellate cell proliferation by heat shock protein 90 inhibitors in vitro. Mol Cell Biochem. 2009 Oct;330(1-2):181-5.

5-(5-chloro-2,4-dihydroxyphenyl)-N-ethyl-4-(4-methoxyphenyl)pyrazole-3-carboxamide is an aromatic amide obtained by formal condensation of the carboxy group of 5-(5-chloro-2,4-dihydroxyphenyl)-4-(4-methoxyphenyl)pyrazole-3-carboxylic acid with the amino group of ethylamine. It has a role as a Hsp90 inhibitor. It is a member of pyrazoles, a member of resorcinols, a member of monochlorobenzenes, a monomethoxybenzene and an aromatic amide.

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1. Chemical class and design background: VER-49009 is a synthetic resorcinylic pyrazole amide analog, developed through structure-based drug design targeting the ATP-binding pocket of HSP90. Its structure incorporates a resorcinol moiety (critical for hydrogen bonding with HSP90’s N-terminal domain) and a pyrazole amide scaffold (enhancing potency and aqueous solubility), representing an improvement over earlier HSP90 inhibitors (e.g., geldanamycin) in oral bioavailability and reduced hepatotoxicity [1, 2]
.
2. Mechanism of action: VER-49009 exerts its biological effects by binding to the N-terminal ATP-binding pocket of HSP90, inhibiting HSP90 ATPase activity. This leads to the destabilization and proteasomal degradation of HSP90 client proteins (e.g., EGFR, AKT in tumors; α-SMA, COL1A1 in activated HSCs), thereby suppressing tumor cell proliferation/apoptosis and HSC activation (anti-fibrotic effect) [1, 2, 3]
.
3. Therapeutic potential in liver fibrosis: VER-49009 is the first HSP90 inhibitor shown to inhibit hepatic stellate cell proliferation and liver fibrosis in a preclinical CCl4-induced model. Its ability to target activated HSCs (key mediators of fibrosis) suggests potential for treating chronic liver diseases associated with fibrosis [3]
.
4. Broad-spectrum antitumor activity: VER-49009 exhibited inhibitory activity against tumor cell lines with diverse genetic aberrations, including those harboring EGFR mutations (H1975 lung cancer cells, IC50=23 nM), HER2 amplification (SK-BR-3 breast cancer cells, IC50=15 nM), and KRAS mutations (HCT116 colon cancer cells, IC50=24 nM), supporting its potential as a broad-spectrum antitumor agent [1, 2]
.

C19H18CLN3O4

387.82

387.098

558640-51-0

940289-57-6

4369536

White to off-white solid powder

1.4±0.1 g/cm3

620.2±55.0 °C at 760 mmHg

328.9±31.5 °C

0.0±1.9 mmHg at 25°C

1.642

0.96

108

4

5

5

27

503

0

HUNAOTXNHVALTN-UHFFFAOYSA-N

InChI=1S/C19H18ClN3O4/c1-3-21-19(26)18-16(10-4-6-11(27-2)7-5-10)17(22-23-18)12-8-13(20)15(25)9-14(12)24/h4-9,24-25H,3H2,1-2H3,(H,21,26)(H,22,23)

3-(5-chloro-2,4-dihydroxyphenyl)-N-ethyl-4-(4-methoxyphenyl)-1H-pyrazole-5-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: ≥ 2.75 mg/mL (7.09 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 27.5 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: ≥ 2.75 mg/mL (7.09 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 27.5 mg/mL 澄清 DMSO 储备液加入900 μL 玉米油中，混合均匀。

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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。