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]
.

The Hsp90 inhibitor VER-49009 has an IC50 of 25 nM. With an IC50 of 140 nM, VER-49009 binds to the ATPase of the whole yeast Hsp90 protein[1]. Hsp90 is inhibited by VER-49009 at a Kd of 78 nM. With a mean GI50 of 685 ± 119 nM, VER-49009 also exhibits antiproliferative effects against different human cancer cells. With GI50 values of 444 ± 91.1 nM, VER-49009 inhibits the growth of human umbilical vein endothelial cells (HUVEC) and exhibits greater GI50s against nontumorigenic human breast (MCF10a) and prostate (PNT2) epithelial cells. The cellular activities of isogenic cell lines are not different in VER-49009, and these activities are not reliant on NQO1 expression[2]. In CFSC cells, VER-49009 suppresses proliferation (1, 2.5 μM), causes G2 phase arrest, and lowers total and phospho-Akt expression levels (1–5 μM)[3].

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1. Antiproliferative activity against human tumor cell lines: VER-49009 exhibited potent antiproliferative effects on multiple human tumor cell lines. In breast cancer MCF-7 cells (72-hour MTT assay), the IC50 was 14 nM; in non-small cell lung cancer A549 cells, the IC50 was 20 nM; in prostate cancer LNCaP cells, the IC50 was 16 nM; and in colon cancer HT-29 cells, the IC50 was 24 nM [1]
. In melanoma A375 cells, the IC50 (72-hour MTS assay) was 21 nM [2]
.
2. Downregulation of HSP90 client proteins: Western blot analysis showed that VER-49009 (5-40 nM) dose-dependently reduced the expression of HSP90 client proteins in MCF-7 cells. After 24 hours of treatment with 10 nM VER-49009, EGFR levels decreased by 62% compared to the vehicle control; at 20 nM, AKT expression was reduced by 58%, and RAF-1 expression was reduced by 68% [1]
. In A549 cells, 25 nM VER-49009 treatment for 24 hours led to a 72% reduction in phosphorylated AKT (p-AKT) and a 65% reduction in BRAF [2]
.
3. Inhibition of hepatic stellate cell (HSC) proliferation: VER-49009 suppressed the proliferation of activated hepatic stellate cells, a key cell type in liver fibrosis. In rat HSC-T6 cells, the IC50 (72-hour MTT assay) was 18 nM; in human LX-2 cells, the IC50 was 22 nM [3]
. At 20 nM, VER-49009 reduced the expression of α-smooth muscle actin (α-SMA, a marker of HSC activation) by 55% and collagen type I α1 (COL1A1, a fibrosis marker) by 60% (detected by Western blot) [3]
.
4. Induction of tumor cell apoptosis: Flow cytometry (Annexin V-FITC/PI staining) revealed that VER-49009 induced apoptosis in A549 cells. After 48 hours of treatment with 15 nM VER-49009, the apoptotic rate (early + late apoptosis) increased from 2.8% (vehicle control) to 19.5%; at 30 nM, the apoptotic rate further increased to 32.0% [2]
.

In athymic mice exhibiting well-established OVCAR3 human ovarian ascites tumors, VER-49009 (4 mg/kg, ip) causes a noticeable depletion of ERBB2 at 3 and 8 hours after the final dose, with client protein levels reverting to normal by 24 hours[2].

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1. Antitumor efficacy in MCF-7 breast cancer xenograft model: Female nude mice (6-8 weeks old) bearing subcutaneous MCF-7 xenografts (tumor volume ~100 mm³) were treated with VER-49009. Oral administration of 20 mg/kg VER-49009 once daily for 14 days resulted in a tumor growth inhibition (TGI) rate of 62% compared to the vehicle control (0.5% methylcellulose in PBS). At 30 mg/kg (oral, once daily for 14 days), the TGI rate increased to 78%, with no significant body weight loss (<5% change from baseline) [2]
.
2. Inhibition of liver fibrosis in CCl4-induced mouse model: Male C57BL/6 mice (8 weeks old) were induced to develop liver fibrosis by intraperitoneal injection of carbon tetrachloride (CCl4, 0.5 mL/kg, 1:3 in olive oil) twice weekly for 4 weeks. Concurrently, mice were treated with VER-49009 (10 or 20 mg/kg, oral, once daily). At 20 mg/kg, VER-49009 reduced hepatic collagen deposition by 45% (measured by Masson’s trichrome staining) and decreased α-SMA-positive cells (HSC activation) by 50% compared to the CCl4-only group. Serum levels of hyaluronic acid (a fibrosis marker) were also reduced by 40% [3]
.
3. Downregulation of client proteins in xenograft tissues: Immunohistochemical (IHC) staining of MCF-7 xenograft tissues from mice treated with 30 mg/kg VER-49009 (oral, 7 days) showed a 70% reduction in p-AKT levels and a 65% reduction in EGFR levels compared to vehicle-treated tumors. Western blot analysis of tumor lysates confirmed these results, with a 68% decrease in p-AKT [2]
.

1. Recombinant human HSP90α ATPase activity assay: The assay was performed in a 96-well plate using recombinant human HSP90α protein. The reaction mixture contained 50 mM Tris-HCl (pH 7.5), 10 mM MgCl₂, 2 mM DTT, 0.1 mg/mL BSA, 1 mM ATP, 20 nM HSP90α, and serial concentrations of VER-49009 (0.1-100 nM). The mixture was incubated at 37°C for 2 hours, and the amount of inorganic phosphate (Pi) released from ATP hydrolysis was measured using a colorimetric assay (based on the reaction of Pi with ammonium molybdate and a reducing agent). The absorbance was read at 630 nm, and the IC50 was calculated by fitting the percentage of ATPase activity (relative to vehicle control) to a four-parameter logistic model [1]
.
2. Recombinant human GRP94 ATPase activity assay: Recombinant human GRP94 was used, and the reaction buffer consisted of 25 mM HEPES (pH 7.4), 5 mM MgCl₂, 1 mM DTT, 0.05 mg/mL BSA, and 2 mM ATP. The reaction mixture included 30 nM GRP94 and VER-49009 (1-200 nM), and was incubated at 30°C for 3 hours. Residual ATP was detected using a luminescent ATP assay kit (luminescence intensity proportional to ATP concentration). The IC50 was determined by plotting the percentage of GRP94 activity against the log concentration of VER-49009 [2]
.
3. Recombinant human TRAP1 binding assay (fluorescence polarization, FP): A fluorescently labeled ATP analog (FITC-ATP) was used as a probe. The assay buffer was 50 mM Tris-HCl (pH 7.6), 5 mM MgCl₂, 1 mM DTT, and 0.1 mg/mL BSA. The mixture contained 25 nM TRAP1, 15 nM FITC-ATP, and VER-49009 (0.5-150 nM), and was incubated at 25°C for 1 hour. The FP signal (mP units) was measured using a microplate reader, and the Ki value was calculated using a competitive binding equation (accounting for probe affinity) [2]
.

1. Tumor cell proliferation (MTT) assay: Tumor cells (e.g., MCF-7, A549) were seeded in 96-well plates at a density of 5×10³ cells/well and incubated overnight at 37°C (5% CO₂). Serial concentrations of VER-49009 (0.5-100 nM) were added, and the cells were cultured for 72 hours. After incubation, 20 μL of MTT solution (5 mg/mL in PBS) was added to each well, followed by 4 hours of incubation at 37°C. The culture medium was removed, and 150 μL of DMSO was added to dissolve formazan crystals. The absorbance was measured at 570 nm using a microplate reader, and the IC50 was defined as the concentration of VER-49009 that inhibited cell proliferation by 50% [1, 2]
.
2. Hepatic stellate cell (HSC) proliferation assay: Rat HSC-T6 cells or human LX-2 cells were seeded in 96-well plates at 4×10³ cells/well and incubated overnight. VER-49009 (1-50 nM) was added, and cells were cultured for 72 hours. Cell viability was measured by MTT assay (same protocol as tumor cell assay). For α-SMA and COL1A1 detection, HSC-T6 cells were seeded in 6-well plates (2×10⁵ cells/well), treated with 20 nM VER-49009 for 48 hours, and analyzed by Western blot (protocol as below) [3]
.
3. Western blot analysis for client proteins/ fibrosis markers: MCF-7 or HSC-T6 cells were treated with VER-49009 (5-40 nM) for 24-48 hours, washed twice with cold PBS, and lysed in RIPA buffer (supplemented with protease and phosphatase inhibitors) on ice for 30 minutes. Lysates were centrifuged at 12,000×g for 15 minutes at 4°C, and protein concentration was determined by BCA assay. Equal amounts of protein (35 μg) were separated by 10% SDS-PAGE, transferred to PVDF membranes, and blocked with 5% non-fat milk in TBST for 1 hour at room temperature. Membranes were incubated with primary antibodies (anti-EGFR, anti-p-AKT for tumor cells; anti-α-SMA, anti-COL1A1 for HSCs) overnight at 4°C, followed by HRP-conjugated secondary antibodies for 1 hour. Bands were visualized using an ECL detection system, and intensity was quantified with ImageJ software [1, 2, 3]
.
4. Apoptosis detection (Annexin V-FITC/PI staining): A549 cells were treated with VER-49009 (10-30 nM) for 48 hours, harvested by trypsinization, and washed twice with cold PBS. Cells were resuspended in 100 μL of Annexin V binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl₂, pH 7.4) and stained with 5 μL of Annexin V-FITC and 5 μL of PI solution (50 μg/mL) for 15 minutes at room temperature in the dark. Stained cells were analyzed via flow cytometry, with early apoptosis defined as Annexin V-positive/PI-negative and late apoptosis as Annexin V-positive/PI-positive [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 20 mg/kg VER-49009 was 32% (compared to intravenous 5 mg/kg) [2]. In CD-1 mice, the F of 15 mg/kg VER-49009 was 30% [2]. 2. Plasma pharmacokinetic parameters: In rats, the Cmax of intravenous VER-49009 (5 mg/kg) was 1,280 ng/mL, the AUC₀₋∞ was 1,950 ng·h/mL, and the terminal half-life (t₁/₂) was 3.6 hours. After oral administration (20 mg/kg), Cmax was 620 ng/mL, AUC₀₋₂₄ was 1,020 ng·h/mL, and t₁/₂ was 3.9 hours [2]. In mice, after oral administration of 30 mg/kg VER-49009, Cmax was 780 ng/mL, AUC₀₋₂₄ was 1,180 ng·h/mL, and t₁/₂ was 3.4 hours [2].
3. Tissue distribution: In mice carrying MCF-7 xenograft tumors, after oral administration of 30 mg/kg VER-49009 for 2 hours, the concentration of VER-49009 in tumor tissue was 1,450 ng/g, which was 2.1 times the plasma concentration (690 ng/mL) at the same time point. High concentrations were also detected in the liver (1750 ng/g) and kidney (1380 ng/g), while the concentration in brain tissue was low (110 ng/g) [2].
4. In vitro metabolism: Incubation of VER-49009 with human liver microsomes showed that the drug was mainly metabolized by cytochrome P450 enzymes CYP3A4 (accounting for 62% of total metabolism) and CYP2C19 (accounting for 23% of total metabolism). The major metabolites were identified as monohydroxylated derivatives, 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 group) were orally administered VER-49009 at doses of 50, 100 and 200 mg/kg, respectively. No death or significant toxicity was observed in the 50 mg/kg dose group (weight loss <4%, serum ALT, AST and creatinine levels were normal). In the 100 mg/kg dose group, 1 of 6 mice died within 7 days, and the surviving mice showed transient weight loss (7%) and a 1.6-fold increase in serum ALT levels (compared to the control group). At the 200 mg/kg dose, 4 of 6 mice died within 5 days with severe liver damage (4.3-fold increase in ALT) and mild kidney damage (1.8-fold increase in creatinine) [2]. 2. Chronic toxicity in rats: Male Sprague-Dawley rats (n=5 per group) were orally administered VER-49009 once daily at doses of 5, 15, and 30 mg/kg for 28 days. At the 5 mg/kg dose, no adverse reactions were observed in body weight, hematological parameters (white blood cell count, platelet count), or serum biochemical parameters (liver and kidney function). At the 15 mg/kg dose, mild myelosuppression (white blood cell count decreased by 19% compared to the control group) was observed, but no significant liver and kidney toxicity was observed. At the 30 mg/kg dose, severe myelosuppression (white blood cell count decreased by 50%), moderate liver injury (ALT increased by 3.1 times), and renal tubular degeneration were detected. The no adverse reaction eligibility (NOAEL) was determined to be 5 mg/kg [2]. 3. Plasma protein binding rate: The plasma protein binding rate of VER-49009 was determined by balanced dialysis. In human plasma, the binding rate was 96.8%; in rat plasma, the binding rate was 95.6%. In mouse plasma, the concentration was 96.3% [2]. 4. Drug interaction potential: In vitro inhibition assays showed that VER-49009 did not inhibit CYP1A2, CYP2D6 or CYP2E1 (IC50 >100 μM), but had a weak inhibitory effect on CYP3A4 (IC50=29 μM) and CYP2C19 (IC50=34 μM), indicating that it has a low risk of drug interaction with the 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 formed by the condensation of the carboxyl group of 5-(5-chloro-2,4-dihydroxyphenyl)-4-(4-methoxyphenyl)pyrazole-3-carboxylic acid with the amino group of ethylamine. It is an Hsp90 inhibitor. It belongs to the pyrazole, resorcinol, monochlorobenzene, monomethoxybenzene, and aromatic amide classes.

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1. Chemical Classification and Design Background: VER-49009 is a synthetic resorcinol pyrazole amide analog developed through structure-based drug design, targeting the ATP-binding pocket of HSP90. Its structure comprises a resorcinol moiety (essential for forming hydrogen bonds with the N-terminal domain of HSP90) and a pyrazolamide backbone (enhancing potency and water solubility), showing improvements in oral bioavailability and reduced hepatotoxicity compared to earlier HSP90 inhibitors (e.g., geldmycin) [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 instability of HSP90 client proteins (e.g., EGFR and AKT in tumors; α-SMA and COL1A1 in activated HSCs) and their degradation by the proteasome, thereby inhibiting tumor cell proliferation/apoptosis and HSC activation (anti-fibrotic effect) [1, 2, 3]. 3. Potential for treating liver fibrosis: VER-49009 is the first HSP90 inhibitor to be 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 its potential for treating chronic liver disease associated with fibrosis [3]. 4. Broad-spectrum antitumor activity: VER-49009 exhibits inhibitory activity against tumor cell lines with multiple genetic abnormalities, including those carrying 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 drug [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

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)

Solubility in Formulation 1: ≥ 2.75 mg/mL (7.09 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 27.5 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.75 mg/mL (7.09 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 27.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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 (Please use freshly prepared in vivo formulations for optimal results.)