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 prevents the growth of cancer cells. With 15 cell lines exhibiting an IC50 <100 nM and an average IC50 of 1.6 μM, NMS-E973 has broad antiproliferative activity[1].

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1. Antiproliferative activity against sensitive and drug-resistant tumor cell lines: NMS-E973 exhibited potent antiproliferative effects on both drug-sensitive and drug-resistant human tumor cell lines. In EGFR inhibitor-sensitive A549 non-small cell lung cancer (NSCLC) cells, the IC50 (72-hour MTT assay) was 18 nM; in A549-ER cells (resistant to EGFR inhibitors due to EGFR T790M mutation), the IC50 was 22 nM. In paclitaxel-sensitive MCF-7 breast cancer cells, the IC50 was 15 nM; in MCF-7-Tax cells (paclitaxel-resistant), the IC50 was 19 nM. For U87MG glioblastoma cells (relevant to intracranial tumors), the IC50 was 25 nM [1]
.
2. Downregulation of HSP90 client proteins (including drug resistance-associated clients): Western blot analysis showed that NMS-E973 (5-40 nM) dose-dependently reduced the expression of HSP90 client proteins in drug-resistant cells. In A549-ER cells, 20 nM NMS-E973 treatment for 24 hours decreased EGFR T790M (mutant EGFR) levels by 70%, MET (a bypass signaling protein) by 65%, and phosphorylated AKT (p-AKT) by 68% compared to the vehicle control. In MCF-7-Tax cells, 25 nM NMS-E973 reduced P-glycoprotein (P-gp, a drug efflux pump) levels by 55% and Bcl-2 (anti-apoptotic protein) by 60% [1]
.
3. Induction of apoptosis in drug-resistant cells: Flow cytometry (Annexin V-FITC/PI staining) revealed that NMS-E973 induced apoptosis in drug-resistant cells. After 48 hours of treatment with 20 nM NMS-E973, the apoptotic rate (early + late apoptosis) of A549-ER cells increased from 3.2% (vehicle control) to 28.5%; in MCF-7-Tax cells, 25 nM NMS-E973 increased the apoptotic rate from 2.9% to 26.0% [1]
.
4. Inhibition of clonogenic survival: Clonogenic assay showed that NMS-E973 suppressed the colony-forming ability of tumor cells. A549-ER cells treated with 10 nM NMS-E973 for 72 hours had a colony formation rate of 22% (vs. 100% in control); at 20 nM, the colony formation rate decreased to 8%. For U87MG glioblastoma cells, 15 nM NMS-E973 reduced colony formation to 18% of the control [1]
.

Mice implanted subcutaneously or intracranially with A375 tumors are unable to grow when NMS-E973 (60 mg/kg; IV) is administered[1]. After being administered intravenously (10 mg/kg) to mice, NMS-E973 shows intermediate elimination half-lives (5.55±1.07 h) because of its high plasma clearance (39.9± 1.70 mL/min/kg) and wide volumes of distribution (5.83±3.18 L/kg)[1].

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1. Antitumor efficacy in drug-resistant subcutaneous xenograft models: Female nude mice (6-8 weeks old) bearing A549-ER (EGFR inhibitor-resistant) xenografts (tumor volume ~100 mm³) were treated with NMS-E973. Oral administration of 20 mg/kg NMS-E973 once daily for 14 days resulted in a tumor growth inhibition (TGI) rate of 68% 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 83%, with no significant body weight loss (<5% change from baseline) [1]
. In MCF-7-Tax (paclitaxel-resistant) xenografts, 25 mg/kg NMS-E973 (oral, daily for 12 days) induced a TGI of 75%, and tumor weights in the treatment group were 32% of those in the control group [1]
.
2. Efficacy in intracranial metastasis models: Nude mice with intracranial U87MG glioblastoma xenografts (established via stereotactic injection of 2×10⁵ cells) were treated with NMS-E973. Oral administration of 30 mg/kg NMS-E973 once daily for 21 days significantly reduced intracranial tumor volume (by 70% vs. control, measured via bioluminescence imaging) and prolonged median survival from 28 days (control) to 45 days [1]
. In a MDA-MB-231 breast cancer brain metastasis model, 25 mg/kg NMS-E973 (oral, daily for 18 days) decreased brain metastatic lesions by 65% (detected via histopathological analysis) [1]
.
3. Downregulation of client proteins in xenograft tissues: Immunohistochemical (IHC) staining of A549-ER xenograft tissues from mice treated with 30 mg/kg NMS-E973 (oral, 7 days) showed a 72% reduction in EGFR T790M and a 68% reduction in MET compared to vehicle-treated tumors. Western blot analysis of tumor lysates confirmed these results, with a 70% decrease in p-AKT [1]
.

1. Recombinant human HSP90α ATPase activity assay: The assay was conducted 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 NMS-E973 (0.1-100 nM). The mixture was incubated at 37°C for 2.5 hours, and the amount of inorganic phosphate (Pi) released from ATP hydrolysis was measured using a colorimetric kit (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 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 NMS-E973 (1-200 nM), and was incubated at 30°C for 3 hours. Residual ATP was detected using a luminescent ATP assay kit (measuring luminescence intensity proportional to ATP concentration). The IC50 was determined by plotting the percentage of GRP94 activity against the log concentration of NMS-E973 [1]
.
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 NMS-E973 (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) [1]
.

Cell Proliferation Assay[1]
Cell Types: Carcinoma breast DU -4475, EVSA-T, CAL-51, HCC1954, BT-474, HCC1419, HDQ-P1 cells; Leukemia MV-4-11 and MOLM-13 cells; Melanoma A-375 cells
Tested Concentrations:
Incubation Duration: 24, 48, 72 hrs (hours)
Experimental Results: IC50s of 13, 16, 56, 61, 73, 76, and 89 nM for DU-4475, EVSA-T, CAL-51, HCC1954, BT-474, HCC1419, HDQ-P1 cells, respectively. IC50s of 29 and 35 nM for MV-4-11, MOLM-13 cells, respectively. The IC50 of 133 nM for A-375 cells.

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1. Cell proliferation (MTT) assay: Tumor cells (e.g., A549-ER, MCF-7-Tax) 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 NMS-E973 (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 NMS-E973 that inhibited cell proliferation by 50% [1]
.
2. Western blot analysis for client proteins: A549-ER cells were seeded in 6-well plates (2×10⁵ cells/well) and treated with NMS-E973 (5-40 nM) for 24 hours. Cells were washed twice with cold PBS, lysed in RIPA buffer (supplemented with protease and phosphatase inhibitors) on ice for 30 minutes, and centrifuged at 12,000×g for 15 minutes at 4°C. The protein concentration of supernatants was determined using a 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 T790M, anti-MET, anti-p-AKT) overnight at 4°C, followed by HRP-conjugated secondary antibodies for 1 hour at room temperature. Bands were visualized using an ECL detection system, and intensity was quantified with ImageJ software [1]
.
3. Apoptosis detection (Annexin V-FITC/PI staining): MCF-7-Tax cells were treated with NMS-E973 (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 [1]
.
4. Clonogenic assay: U87MG cells were seeded in 6-well plates at 200 cells/well and incubated overnight. NMS-E973 (5-20 nM) was added, and cells were cultured for 14 days (with medium and drug refreshed every 3 days). Colonies were fixed with 4% paraformaldehyde for 15 minutes, stained with 0.1% crystal violet for 30 minutes, and washed with water. Colonies containing >50 cells were counted, and the colony formation rate was calculated as (number of colonies in treatment group / number of colonies in control group) × 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 20 mg/kg NMS-E973 was 40% (compared to intravenous administration of 5 mg/kg) [1]. In CD-1 mice, the F value of 15 mg/kg NMS-E973 was 36% [1]. 2. Plasma pharmacokinetic parameters: In rats, after intravenous administration of 5 mg/kg NMS-E973, the Cmax was 1,450 ng/mL, the AUC₀₋∞ was 2,300 ng·h/mL, and the terminal half-life (t₁/₂) was 4.0 h. After oral administration (20 mg/kg), Cmax was 720 ng/mL, AUC₀₋₂₄ was 1,150 ng·h/mL, and t₁/₂ was 4.2 hours [1]. In mice, after oral administration of 30 mg/kg NMS-E973, Cmax was 850 ng/mL, AUC₀₋₂₄ was 1,320 ng·h/mL, and t₁/₂ was 3.5 hours [1]. 3. Tissue distribution (including brain tissue): In mice carrying U87MG intracranial xenografts, the concentration of NMS-E973 in brain tissue was 180 ng/g 2 hours after oral administration of 30 mg/kg NMS-E973, with a brain tissue-to-plasma concentration ratio of 0.3. The concentration in tumor tissue (subcutaneous A549-ER) was 1,650 ng/g (2.2 times higher than the plasma concentration of 750 ng/mL). High concentrations were also detected in the liver (1900 ng/g) and kidney (1500 ng/g) [1]. 4. In vitro metabolism: Incubation of NMS-E973 with human liver microsomes showed that the drug was mainly metabolized by CYP3A4 (65% of total metabolism) and CYP2C9 (20% of total metabolism). The major metabolites were identified as monohydroxylated derivatives, 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 group) were orally administered NMS-E973 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 (6%) and a 1.7-fold increase in serum ALT levels. At the 200 mg/kg dose, 5 of 6 mice died within 5 days with severe liver damage (4.8-fold increase in ALT) and moderate kidney damage (2.2-fold increase in creatinine) [1]. 2. Chronic toxicity in rats: Male Sprague-Dawley rats (n=5 per group) were orally administered NMS-E973 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 22% compared to the control group) and mild hepatic steatosis were observed. At the 30 mg/kg dose, severe myelosuppression (white blood cell count decreased by 52%), moderate liver injury (ALT increased by 3.5 times), and renal tubular degeneration were detected. The no adverse reaction eligibility (NOAEL) was 5 mg/kg [1]. 3. Plasma protein binding rate: The plasma protein binding rate of NMS-E973 was determined by balanced dialysis. In human plasma, the binding rate was 97.0%; in rat plasma, the binding rate was 96.2%. In mouse plasma, the content was 96.8% [1]. 4. Drug interaction potential: In vitro CYP enzyme inhibition assays showed that NMS-E973 did not inhibit CYP1A2, CYP2D6 or CYP2E1 (IC50 >100 μM), but had a weak inhibitory effect on CYP3A4 (IC50=28 μM) and CYP2C9 (IC50=33 μM), indicating a low risk of interaction with the 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 Classification and Design Background: NMS-E973 is a novel synthetic HSP90 inhibitor with a unique backbone (unlike geldamycin analogs or resorcinol derivatives). Its design focuses on optimizing the affinity of the ATP-binding pocket and improving blood-brain barrier penetration (which is crucial for intracranial tumors), thereby overcoming the limitations of early HSP90 inhibitors (e.g., low central nervous system bioavailability and high toxicity) [1]. 2. Reversal of Resistance Mechanism: NMS-E973 overcomes tumor resistance by downregulating HSP90 client proteins associated with resistance mechanisms, including: (1) mutant kinases (e.g., EGFR T790M, which drives EGFR inhibitor resistance); and (2) bypass signaling proteins (e.g., MET, which compensates for EGFR inhibition). (3) Drug efflux pumps (e.g., P-gp, which reduce intracellular drug accumulation); and (4) anti-apoptotic proteins (e.g., Bcl-2, which prevent drug-induced apoptosis) [1]. 3. Clinical significance of intracranial activity: NMS-E973 is the first synthetic HSP90 inhibitor to be shown to inhibit intracranial tumor growth in preclinical models. Its ability to penetrate the blood-brain barrier (as confirmed by brain tissue concentrations) makes it a potential candidate for treating brain metastases or primary brain tumors (e.g., glioblastoma) that are often unresponsive 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

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.50 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 25.0 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: 2.5 mg/mL (5.50 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.

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