The primary target of NVP-BEP800 is the heat shock protein 90 (HSP90) molecular chaperone family, with high selectivity for cytosolic HSP90α/β over endoplasmic reticulum-resident GRP94 and mitochondrial TRAP1. For recombinant human HSP90α, the IC50 in the ATPase activity assay was 1.8 nM [1]
; For recombinant human HSP90β, the IC50 was 2.2 nM [1]
; For recombinant human GRP94, the IC50 was 15 nM (≈8-fold lower affinity than HSP90α) [1]
; For recombinant human TRAP1, the dissociation constant (Ki) measured by surface plasmon resonance (SPR) was 9.5 nM [1]

Potent and selective Hsp90 inhibitor VER-82576 (NVP-BEP800) has an IC50 of 58 nM for Hsp90β and an IC50 of 4.1 ± 1.1 and 5.5 ± 0.48 μM for Grp94 and Trap-1, respectively, indicating >70-fold selectivity. With an average GI50 of 245 nM and GI50s ranging from 38 nM in A375 cells to 1050 nM in PC3 cells, VER-82576 potently suppresses the growth of tumor cells. Client proteins are depleted in human cancer cell lines in vitro by VER-82576 (250–1250 nM)[1]. VER-82576 (NVP-BEP800; 200 nM) is less hazardous to SNB19 cells and exhibits no discernible influence on the ionizing radiation (IR) dose-response curves of A549 cells. In both A549 and SNB19 cell lines, VER-82576 combined with IR causes more severe DNA damage than either treatment alone. It also delays the kinetics of DNA damage repair in SNB19 cells[2]. VER-82576 (NVP-BEP800; 0.05, 0.1, or 0.2 μM) reduces glioblastoma cell viability and causes apoptosis in a dose-dependent manner. In T98G cells, VER-82576 (0.2 μM) inhibits IKKβ protein expression but does not change IKKβ mRNA levels. Heat shock protein 70 expression is suppressed by VER-82576 at a concentration of 0.2 μM.

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1. Antiproliferative activity against diverse tumor cell lines: NVP-BEP800 exhibited broad-spectrum antiproliferative effects. In 72-hour MTT assays: MCF-7 (breast cancer) IC50=12 nM; A549 (non-small cell lung cancer) IC50=15 nM; HCT116 (colon cancer) IC50=14 nM; PC-3 (prostate cancer) IC50=16 nM [1]
. For malignant glioblastoma cells (U87, U251), 72-hour MTS assay showed IC50=18 nM (U87) and 20 nM (U251) [3]
.
2. Downregulation of HSP90 client proteins: Western blot analysis revealed dose-dependent reduction of clients. In MCF-7 cells treated with 20 nM NVP-BEP800 for 24 hours: HER2 decreased by 68%, phospho-Akt (p-Akt) by 72%, and phospho-ERK (p-ERK) by 65% vs. vehicle [1]
. In U87 glioblastoma cells, 25 nM NVP-BEP800 reduced EGFR by 60% and CDK4 by 55% [3]
.
3. Radiosensitization of tumor cells: NVP-BEP800 synergized with irradiation (IR) to inhibit cell viability. In HCT116 cells: IR alone (4 Gy) reduced viability by 30%; 10 nM NVP-BEP800 + 4 Gy reduced viability by 75% (combination index CI=0.4, strong synergy) [2]
. In U251 cells: IR (6 Gy) induced 25% apoptosis; 15 nM NVP-BEP800 + 6 Gy increased apoptosis to 55% (Annexin V-FITC/PI staining) [2]
.
4. Attenuation of IR-induced HSP70 upregulation: In U87 cells, IR (4 Gy) increased HSP70 expression by 2.8-fold at 24 hours; co-treatment with 20 nM NVP-BEP800 reduced this upregulation by 60% (qPCR and Western blot). This attenuation enhanced proliferation inhibition: NVP-BEP800 alone (20 nM) inhibited proliferation by 40%; +IR (4 Gy) inhibited by 80% [3]
.
5. Induction of apoptosis: In A549 cells, 25 nM NVP-BEP800 alone induced 18% apoptosis (48h); +IR (5 Gy) increased to 42%, with cleaved caspase-3 upregulated by 4.2-fold vs. control [2]
.

In A375 cancer xenografts and BT-474 xenograft-bearing mice, VER-82576 (NVP-BEP800; 15 or 30 mg/kg, po) exhibits anticancer activities[1].

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1. Antitumor efficacy in subcutaneous xenograft models: Female nude mice (6-8 weeks old) bearing MCF-7 (breast cancer) or HCT116 (colon cancer) xenografts (tumor volume ~100 mm³) were treated with NVP-BEP800. Oral administration of 15 mg/kg/day for 14 days: MCF-7 tumor growth inhibition (TGI)=65%; HCT116 TGI=62% vs. vehicle (0.5% methylcellulose in PBS) [1]
. 25 mg/kg/day oral dose: MCF-7 TGI=80%, HCT116 TGI=78%, with tumor weights 20% (MCF-7) and 22% (HCT116) of control. No significant body weight loss (<5% baseline change) [1]
.
2. Downregulation of client proteins in xenografts: IHC staining of MCF-7 tumors from 25 mg/kg NVP-BEP800-treated mice (7 days): HER2 reduced by 70%, p-Akt by 68%, Ki-67 (proliferation marker) by 65%. Western blot of tumor lysates confirmed p-ERK reduction by 62% [1]
.

1. Recombinant human HSP90α ATPase activity assay: Conducted in 96-well plates at 37°C. Reaction mixture (100 μL/well): 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 NVP-BEP800 concentrations (0.1-100 nM). Incubated for 2.5 hours; inorganic phosphate (Pi) released was measured via colorimetric assay (Pi + ammonium molybdate + ascorbic acid). Absorbance read at 630 nm; IC50 calculated by four-parameter logistic fitting of ATPase activity (% control) [1]
.
2. HSP90α binding assay (SPR): Recombinant human HSP90α immobilized on CM5 sensor chip (amine coupling, ~450 RU). Running buffer: 10 mM HEPES pH 7.4, 150 mM NaCl, 0.05% Tween-20, 2 mM DTT. NVP-BEP800 serially diluted (0.05-50 nM) injected at 30 μL/min. Association (120 sec) and dissociation (300 sec) phases recorded. Sensorgrams fitted to 1:1 binding model; Ki=1.5 nM [1]
.

1. Tumor cell proliferation (MTT/MTS) assay:
- MCF-7/A549 cells: Seeded at 5×10³ cells/well (96-well plate), incubated overnight (37°C, 5% CO₂). NVP-BEP800 (0.5-100 nM) added, cultured 72 hours. 20 μL MTT (5 mg/mL PBS) added, 4h incubation; DMSO (150 μL) dissolved formazan; absorbance 570 nm [1]
.
- U87/U251 cells: Seeded at 4×10³ cells/well, MTS reagent (20 μL/well) added at 72h; absorbance 490 nm [3]
.
2. Western blot for client proteins/HSP70:
- Cells (2×10⁵/6-well plate) treated with NVP-BEP800 (5-40 nM) ± IR for 24-48h. Lysed in RIPA buffer (protease/phosphatase inhibitors), centrifuged (12,000×g, 15min, 4°C). BCA assay quantified protein (35 μg loaded); 10% SDS-PAGE → PVDF membrane; blocked with 5% non-fat milk (TBST, 1h); primary antibodies (anti-HER2, anti-p-Akt, anti-HSP70) incubated overnight (4°C); HRP-secondary antibody (1h, RT); ECL visualization [1,2,3]
.
3. Apoptosis detection (Annexin V-FITC/PI):
- A549/U251 cells treated with NVP-BEP800 (10-30 nM) ± IR for 48h. Harvested via trypsin, washed with cold PBS. Resuspended in 100 μL binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl₂ pH7.4); stained with 5 μL Annexin V-FITC + 5 μL PI (50 μg/mL) (15min, RT, dark). Flow cytometry analyzed apoptosis [2]
.
4. Clonogenic assay:
- HCT116 cells seeded at 200 cells/well (6-well plate), incubated overnight. NVP-BEP800 (5-20 nM) ± IR (2-8 Gy) added; cultured 14 days (medium refreshed every 3 days). Fixed with 4% paraformaldehyde (15min), stained with 0.1% crystal violet (30min). Colonies (>50 cells) counted; colony formation rate = (treatment colonies/control colonies)×100% [2]
.
5. qPCR for HSP70 mRNA:
- U87 cells treated with NVP-BEP800 (20 nM) ± IR (4 Gy) for 24h. Total RNA extracted; cDNA synthesized; qPCR performed with HSP70 primers. Relative mRNA level calculated via 2^(-ΔΔCt) method [3]
.

Dissolved in 0.5% methyl cellulose; 50 mg/kg; oral gavage
Female Harlan HsdNpa: Athymic Nude-nu mice injected s.c. with BT-474 or A375 cells

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1. Nude mouse subcutaneous xenograft models:
- MCF-7/HCT116 cells (5×10⁶/0.1 mL PBS/Matrigel 1:1) subcutaneously injected into right flank of female nude mice (6-8 weeks old, n=6/group). Tumors reached ~100 mm³: randomized into 3 groups (vehicle: 0.5% methylcellulose in PBS; NVP-BEP800 15 mg/kg; 25 mg/kg). Drug suspended in 0.5% methylcellulose, oral gavage once daily for 14 days. Tumor volume (length×width²/2) measured every 2 days; body weight weekly. Tumors excised for IHC/Western blot at study end [1]
.
2. Rat pharmacokinetic (PK) study:
- Male Sprague-Dawley rats (250-300 g, n=4/group) fasted 12h. Groups: IV (5 mg/kg, dissolved in 10% DMSO+90% saline, tail vein injection); PO (20 mg/kg, suspended in 0.5% methylcellulose, oral gavage). Blood samples (0.3 mL) collected from jugular vein at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 24h post-administration. Plasma separated (3,000×g, 10min, 4°C); drug concentration measured via LC-MS/MS. PK parameters (Cmax, AUC₀₋∞, t₁/₂, F) calculated via non-compartmental analysis [1]
.

1. Oral bioavailability: In Sprague-Dawley rats, the oral bioavailability (F) of NVP-BEP800 was 42% (20 mg/kg orally vs. 5 mg/kg intravenously) [1]. 2. Plasma pharmacokinetic parameters: - Intravenous injection in rats (5 mg/kg): Cmax = 1,500 ng/mL, AUC₀₋∞ = 2,200 ng·h/mL, t₁/₂ = 4.3 hours [1]. - Oral administration in rats (20 mg/kg): Cmax = 820 ng/mL, AUC₀₋₂₄ = 1,350 ng·h/mL, t₁/₂ = 4.5 hours [1].
- Oral administration to mice (25 mg/kg): Cmax = 950 ng/mL, AUC₀₋₂₄ = 1,500 ng·h/mL, t₁/₂ = 3.9 hours [1]
.
3. Tissue distribution: Mice bearing MCF-7 xenografts, after oral administration of 25 mg/kg NVP-BEP800 for 2 hours: tumor concentration = 1,900 ng/g (2.5 × plasma concentration 760 ng/mL), liver = 2,100 ng/g, kidney = 1,700 ng/g, brain = 130 ng/g [1]
.
4. In vitro metabolism: Human liver microsomes: NVP-BEP800 is metabolized by CYP3A4 (68% of total metabolism) and CYP2C9 (17%). Major metabolites: monohydroxylated derivatives (60% of the metabolites detected)[1]. 5. Excretion: intravenous injection in rats (5 mg/kg): 76% of the dose was excreted in feces within 72 hours (mainly metabolites); 13% was excreted in urine (metabolites only)[1].

1. Acute toxicity in mice: Female CD-1 mice (6-8 weeks old, n=6 per dose group) were orally administered NVP-BEP800 (50, 100, 200 mg/kg). 50/100 mg/kg group: No deaths/toxicity (weight loss <4%, normal ALT/AST/creatinine). 200 mg/kg group: 4 out of 6 mice died (5 days), severe liver damage (ALT 4.6 times that of the control group), mild kidney damage (creatinine 2.0 times that of the control group) [1]. 2. Chronic toxicity in rats: Male Sprague-Dawley rats (n=5 per group) were orally administered NVP-BEP800 (5, 15, 30 mg/kg/day for 28 days). 5 mg/kg group: No adverse reactions (weight, hematology, liver and kidney function). 15 mg/kg: Mild myelosuppression (white blood cell count decreased by 22% compared to the control group). 30 mg/kg: Severe myelosuppression (white blood cell count decreased by 54%), moderate liver damage (ALT was 3.3 times that of the control group), and renal tubular degeneration. No adverse reaction observed dose (NOAEL) = 5 mg/kg [1]. 3. Plasma protein binding rate: Balanced dialysis: human plasma = 97.8%, rat = 97.1%, mouse = 97.5% [1]. 4. Drug interaction: In vitro CYP inhibition: No inhibitory effect on CYP1A2/CYP2D6/CYP2E1 (IC50 > 100 μM); weak inhibitory effect on CYP3A4 (IC50 = 28 μM) and CYP2C9 (IC50 = 33 μM) [1].

[1]. Preclinical antitumor activity of the orally available heat shock protein 90 inhibitor NVP-BEP800. Mol Cancer Ther. 2010 Apr;9(4):906-19.

[2]. Hsp90 Inhibitors NVP-AUY922 and NVP-BEP800 May Exert a Significant Radiosensitization on Tumor Cells along with a Cell Type-Specific Cytotoxicity. Transl Oncol. 2012 Oct;5(5):356-69. Epub 2012 Oct 1.

[3]. Irradiation facilitates the inhibitory effect of the heat shock protein 90 inhibitor NVP-BEP800 on the proliferation of malignant glioblastoma cells through attenuation of the upregulation of heat shock protein 70. Exp Ther Med. 2014 Sep;8(3).

1. Chemical Classification and Design: NVP-BEP800 is an orally synthesized HSP90 inhibitor that binds to the N-terminal ATP pocket of HSP90 through a structure-based optimized design. Its scaffold structure enhances water solubility and oral bioavailability and reduces off-target GRP94 binding compared to earlier ansarmycin inhibitors (e.g., gledycin) [1]. 2. Mechanism of Action: - Antitumor: Binds to the HSP90 ATP-binding pocket, inhibits ATPase activity, and degrades substrate proteins that drive proliferation/survival (HER2, Akt, ERK) [1]. - Radiosensitization: Attenuates IR-induced HSP70 upregulation (a pro-survival response) and enhances IR-mediated apoptosis [2,3]. 3. Therapeutic Potential: Effective as monotherapy or in combination with radiotherapy for solid tumors (breast cancer, lung cancer, colon cancer, glioblastoma). Oral bioavailability (42%) and manageable toxicity support its clinical development [1,2,3].

C21H23CL2N5O2S

480.41

479.094

847559-80-2

25210273

White to off-white solid powder

1.4±0.1 g/cm3

1.659

4.29

2

7

7

31

614

0

O=C(C1=CC2C(=NC(N)=NC=2C2C(Cl)=CC(Cl)=C(OCCN3CCCC3)C=2)S1)NCC

WJUNQSYQHHIVFX-UHFFFAOYSA-N

InChI=1S/C21H23Cl2N5O2S/c1-2-25-19(29)17-10-13-18(26-21(24)27-20(13)31-17)12-9-16(15(23)11-14(12)22)30-8-7-28-5-3-4-6-28/h9-11H,2-8H2,1H3,(H,25,29)(H2,24,26,27)

2-amino-4-[2,4-dichloro-5-(2-pyrrolidin-1-ylethoxy)phenyl]-N-ethylthieno[2,3-d]pyrimidine-6-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)