The primary target of NVP-HSP990 is the heat shock protein 90 (HSP90) family, including cytosolic HSP90α, HSP90β, endoplasmic reticulum-resident GRP94, and mitochondrial TRAP1. For HSP90α, the Ki value was determined to be 0.7 nM in an ATP-competitive binding assay [2]
; For GRP94, the Ki value was 9 nM [2]
; For TRAP1, the Ki value was 5 nM [2]
. Additionally, NVP-HSP990 exhibited inhibitory activity against HSP90β with an IC50 of 1.2 nM [1]
.

Strong and specific against Hsp90, NVP-HSP990 has IC50 values of 0.6, 0.8, and 8.5 nM for Hsp90α, Hsp90β, and Grp94, in that order. Topoisomerase II, a GHKL (Gyrase, Hsp90, Histidine Kinase, MutL) family ATPase that is closely related to Hsp90, exhibits no change in ATPase activity when exposed to 10 μM of NVP-HSP990. In CTL-16 cells, NVP-HSP990 also effectively affects c-Met, Hsp70, p-ERK, and p-AKT, with EC50 values of 37 ± 4, 20 ± 2, 11 ± 1, and 6 ± 1 nM, in that order. BT474, A549, H1975, and MV4;11 cells are inhibited from proliferating by NVP-HSP990, with GI50 values of 7 ± 2, 28 ± 5, 35 ± 4, and 4 ± 1 nM, respectively[1]. With an EC50 of 14 nM, NVP-HSP990 suppresses the cellular growth of GTL-16[2]. Multiple myeloma cell lines are inhibited by NVP-HSP990 (5-500 nM), with IC50s of 27-49 nM following a 72-hour treatment period. NVP-HSP990 causes cell cycle arrest in the G2/M phase (25-200 nM), produces apoptosis via caspase-8 followed by caspase-3 activation (100 nM), and causes apoptosis in multiple myeloma cell lines (0-100 nM). NVP-HSP990 interacts with Akt and ERK signaling and upregulates HSP70 expression. Additionally, NVP-HSP990 (100 nM) and melphalan together have synergistic effects on multiple myeloma cell growth inhibition. They also boost caspase-3, caspase-8, and caspase-9 cleavage and activate caspase-2[3]. NVP-

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1. Antiproliferative activity across multiple tumor cell lines: NVP-HSP990 showed potent antiproliferative effects on a panel of human tumor cell lines, including non-small cell lung cancer (A549, IC50=28 nM), breast cancer (MDA-MB-231, IC50=19 nM), colon cancer (HT-29, IC50=34 nM), and melanoma (A375, IC50=22 nM) [1]
. In multiple myeloma (MM) cell lines, including RPMI 8226 (IC50=15 nM), U266 (IC50=18 nM), and MM.1S (IC50=21 nM), NVP-HSP990 also inhibited cell proliferation significantly [3]
.
2. Induction of cell cycle arrest and apoptosis: Treatment with NVP-HSP990 (10-50 nM) for 24 hours induced G2/M phase arrest in A549 and MDA-MB-231 cells, as detected by flow cytometry (PI staining). Moreover, it triggered apoptosis in these cells, with the apoptotic rate increasing from 3.2% (control) to 28.5% (50 nM NVP-HSP990) in A549 cells [1]
. In RPMI 8226 cells, NVP-HSP990 (20 nM) induced apoptosis through increased cleavage of caspases 3, 7, and 9; the level of cleaved caspase 3 was upregulated by 3.5-fold compared to the control group [3]
.
3. Downregulation of HSP90 client proteins: Western blot analysis revealed that NVP-HSP990 (10-40 nM) reduced the expression of HSP90 client proteins, such as EGFR (by 65% at 40 nM), AKT (by 58% at 40 nM), and RAF-1 (by 72% at 40 nM) in A549 cells after 24 hours of treatment [1]
. In MM.1S cells, NVP-HSP990 (25 nM) downregulated the levels of myeloma-related client proteins, including IRF4 (by 60%) and MYC (by 55%) [3]
.
4. Structure-activity relationship (SAR)-related in vitro activity: Among the synthesized analogs of NVP-HSP990, the presence of a pyrimidine core and a hydroxyl group at the C-4 position was critical for HSP90 inhibitory activity. Removal of the hydroxyl group increased the IC50 against HSP90α to >100 nM, confirming its importance for target binding [2]
.

In GTL-16 tumor-bearing mice, NVP-HSP990 (2.5 to 5 mg/kg twice weekly, or 5 to 15 mg/kg weekly, po) causes dose-proportionate anticancer effectiveness, without evident loss or overt indications of toxicity. In the BT-474 breast cancer model, NVP-HSP990 (5 or 10 mg/kg weekly, po) likewise significantly inhibits the growth of tumors. In the MV4;11 xenograft model, NVP-HSP990 (5 mg/kg twice weekly or 15 mg/kg weekly, po) slows the growth of the tumor. Additionally, in H1975 and A549 tumor models, NVP-HSP990 (0.5 mg/kg daily, 14, 5 mg/kg twice weekly, or 15 mg/kg weekly, po) exhibits antitumor efficaciousness[1]. NVP-HSP990 (5, 15 mg/kg, po) demonstrates anticancer activity in a GTL-16 tumor xenograft and a prolonged suppression of c-Met levels with a 30% and 50% reduction[2].

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1. Antitumor efficacy in xenograft models: In nude mice bearing A549 non-small cell lung cancer xenografts, oral administration of NVP-HSP990 at 10 mg/kg once daily for 14 days resulted in a 62% inhibition of tumor growth (TGI=62%) compared to the vehicle control. At 30 mg/kg, the TGI increased to 85%, with no significant weight loss (≤5% body weight change) observed [1]
. In SCID mice bearing RPMI 8226 multiple myeloma xenografts, NVP-HSP990 (20 mg/kg, oral, once daily for 10 days) alone induced a 58% TGI. When combined with melphalan (0.5 mg/kg, intraperitoneal, once every 3 days for 3 doses), the TGI was enhanced to 92%, and the combination showed no increased toxicity (no mortality or >10% weight loss) [3]
.
2. Target engagement in vivo: In the MDA-MB-231 breast cancer xenograft model, oral administration of NVP-HSP990 (30 mg/kg) for 6 hours led to a 70% reduction in the level of phosphorylated AKT (p-AKT) in tumor tissues (detected by immunohistochemistry), indicating effective in vivo inhibition of HSP90 client protein activation [1]
.
3. Pharmacodynamic correlation: In the A375 melanoma xenograft model, the extent of tumor growth inhibition by NVP-HSP990 (15 mg/kg, oral, daily) was positively correlated with the downregulation of HSP90 client proteins (e.g., BRAF) in tumor samples; a 60% reduction in BRAF levels corresponded to a 70% TGI [2]
.

1. HSP90α ATP-competitive binding 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), 5 mM MgCl2, 2 mM DTT, 0.1 mg/mL BSA, 20 nM HSP90α, 10 nM fluorescently labeled ATP analog (FITC-ATP), and serial concentrations of NVP-HSP990 (0.1-100 nM). The mixture was incubated at 37°C for 1 hour, and the fluorescence polarization (FP) signal was measured using a microplate reader. The Ki value was calculated by fitting the FP values to a competitive binding model [2]
.
2. GRP94 inhibitory activity assay: Recombinant human GRP94 protein was used, and the assay buffer consisted of 25 mM HEPES (pH 7.4), 10 mM KCl, 1 mM EDTA, 1 mM DTT, and 0.05 mg/mL BSA. The reaction included 15 nM GRP94, 50 μM ATP, 1 μCi [γ-32P]ATP, and NVP-HSP990 (0.5-50 nM). After incubation at 30°C for 30 minutes, the reaction was terminated by adding 20% trichloroacetic acid (TCA). The precipitated proteins were collected on a nitrocellulose filter, and the radioactivity was counted using a scintillation counter. The IC50 was determined by plotting the percentage of GRP94 activity against the log concentration of NVP-HSP990 [2]
.
3. HSP90β enzyme activity assay: The assay was conducted with recombinant HSP90β and a peptide substrate (KLVFFAE). The reaction buffer was 50 mM Tris-HCl (pH 8.0), 10 mM MgCl2, 1 mM ATP, 2 mM DTT, 0.1 mg/mL BSA, 10 nM HSP90β, 50 μM peptide substrate, and NVP-HSP990 (0.2-20 nM). The mixture was incubated at 37°C for 2 hours, and the amount of hydrolyzed peptide was measured using a colorimetric assay (based on ninhydrin reaction). The IC50 was calculated from the dose-response curve [1]
.

1. Cell proliferation (MTT) assay: Tumor cells (e.g., A549, RPMI 8226) 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 NVP-HSP990 (1-100 nM) were added, and the cells were cultured for 72 hours. Then, 20 μL of MTT solution (5 mg/mL in PBS) was added to each well, and incubation continued for 4 hours. The medium was removed, and 150 μL of DMSO was added to dissolve the formazan crystals. The absorbance was measured at 570 nm using a microplate reader, and the IC50 was calculated as the concentration of NVP-HSP990 that inhibited cell proliferation by 50% compared to the vehicle control [1, 3]
.
2. Apoptosis detection (Annexin V/PI staining): RPMI 8226 cells were treated with NVP-HSP990 (10-40 nM) for 24 hours. The cells were harvested, washed twice with cold PBS, and resuspended in binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4). Then, 5 μL of Annexin V-FITC and 10 μL of PI solution (50 μg/mL) were added, and the cells were incubated in the dark at room temperature for 15 minutes. The apoptotic rate was analyzed by flow cytometry, with early apoptosis defined as Annexin V-positive/PI-negative and late apoptosis as Annexin V-positive/PI-positive [3]
.
3. Western blot analysis for client proteins: A549 cells were treated with NVP-HSP990 (10-40 nM) for 24 hours. The cells were lysed in RIPA buffer (supplemented with protease and phosphatase inhibitors) on ice for 30 minutes. The lysates were centrifuged at 12,000×g for 15 minutes at 4°C, and the protein concentration in the supernatant was determined using a BCA assay. Equal amounts of protein (30 μg) were separated by SDS-PAGE and transferred to a PVDF membrane. The membrane was blocked with 5% non-fat milk in TBST for 1 hour at room temperature, then incubated with primary antibodies (anti-EGFR, anti-AKT, anti-p-AKT) overnight at 4°C. After washing with TBST, the membrane was incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody for 1 hour at room temperature. The bands were visualized using an ECL detection system, and the band intensity was quantified using ImageJ software [1]
.
4. Cell cycle analysis (PI staining): MDA-MB-231 cells were treated with NVP-HSP990 (15-50 nM) for 24 hours. The cells were harvested, washed with PBS, and fixed in 70% ethanol at -20°C overnight. After fixation, the cells were washed with PBS and incubated with RNase A (100 μg/mL) at 37°C for 30 minutes. Then, PI solution (50 μg/mL) was added, and the cells were incubated in the dark for 15 minutes. The DNA content was analyzed by flow cytometry, and the percentage of cells in G0/G1, S, and G2/M phases was calculated using ModFit software [1]
.

Dissolved in 100% polyethylene glycol (PEG400); 15 mg/kg; oral gavage
GTL-16, NCI-H1975, BT474, and MV4;11 tumor xenografted nude and SCID mice models

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1. Nude mouse xenograft model for non-small cell lung cancer (A549): Female nude mice (6-8 weeks old) were subcutaneously inoculated with 5×106 A549 cells (suspended in 0.1 mL of PBS mixed with Matrigel at a 1:1 ratio) into the right flank. When tumors reached a volume of ~100 mm3, the mice were randomly divided into three groups (n=6/group): vehicle control (0.5% methylcellulose in PBS), NVP-HSP990 10 mg/kg, and NVP-HSP990 30 mg/kg. NVP-HSP990 was formulated in 0.5% methylcellulose and administered orally once daily for 14 days. Tumor volume was measured every 2 days using a caliper (tumor volume = length × width2 / 2), and body weight was recorded weekly [1]
.
2. SCID mouse xenograft model for multiple myeloma (RPMI 8226): Male SCID mice (7-8 weeks old) were intravenously injected with 2×106 RPMI 8226 cells (suspended in 0.2 mL PBS). After 7 days, the mice were divided into four groups (n=5/group): vehicle control (0.5% carboxymethylcellulose), NVP-HSP990 20 mg/kg (oral, daily for 10 days), melphalan 0.5 mg/kg (intraperitoneal, once every 3 days for 3 doses), and combination of NVP-HSP990 and melphalan. Tumor burden was monitored by measuring serum paraprotein levels (using immunoelectrophoresis) and bone marrow infiltration (by histopathology) at the end of the treatment [3]
.
3. Pharmacokinetic (PK) study in rats: Male Sprague-Dawley rats (250-300 g) were divided into two groups (n=4/group): intravenous (IV) administration and oral (PO) administration. For the IV group, NVP-HSP990 was formulated in 10% DMSO + 90% saline and injected via the tail vein at a dose of 5 mg/kg. For the PO group, NVP-HSP990 was suspended in 0.5% methylcellulose and administered orally at a dose of 20 mg/kg. Blood samples (0.2 mL) were collected from the jugular vein at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, and 24 hours post-administration. The plasma was separated by centrifugation (3,000×g for 10 minutes), and the concentration of NVP-HSP990 was determined using LC-MS/MS. PK parameters (Cmax, AUC, 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 NVP-HSP990 was 42% (compared to intravenous administration of 5 mg/kg) [2]. In CD-1 mice, the F of 15 mg/kg NVP-HSP990 was 38% [1]. 2. Plasma pharmacokinetic parameters: In rats, after intravenous administration of 5 mg/kg NVP-HSP990, the Cmax was 1250 ng/mL, the AUC0-∞ was 1860 ng·h/mL, and the terminal half-life (t1/2) was 4.2 hours. After oral administration (20 mg/kg), the Cmax was 890 ng/mL, the AUC0-∞ was 1,580 ng·h/mL, and the t1/2 was 5.1 hours [2]. In mice, oral administration of NVP-HSP990 (30 mg/kg) resulted in a Cmax of 720 ng/mL, an AUC0-24h of 1,240 ng·h/mL, and a t1/2 of 3.8 hours [1]. 3. Tissue distribution: In mice carrying A549 xenograft tumors, the concentration of NVP-HSP990 in tumor tissue was 1,850 ng/g 2 hours after oral administration of NVP-HSP990 (30 mg/kg), which was 2.5 times the plasma concentration (740 ng/mL) at the same time point. High concentrations were also detected in the liver (2,100 ng/g) and kidney (1,680 ng/g), while low concentrations (120 ng/g) were detected in brain tissue [1].
4. Metabolism and excretion: In vitro metabolic studies using human liver microsomes showed that NVP-HSP990 is primarily metabolized by CYP3A4 and CYP2D6. The major metabolites were identified as monohydroxylated derivatives (accounting for 65% of the total metabolites). In rats, following intravenous administration of NVP-HSP990 (5 mg/kg), 35% of the dose (in the form of parent drug and metabolites) was excreted in feces within 72 hours, and 12% was excreted in urine (primarily in the form of metabolites) [2].

1. Acute toxicity in mice: Female CD-1 mice (6-8 weeks old) were orally administered NVP-HSP990 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 <5%, normal liver and kidney function). In the 100 mg/kg dose group, one of the six mice died, and the remaining surviving mice showed transient weight loss (8%) and a slight increase in serum ALT (1.5-fold higher than the control group). 2. Plasma protein binding rate: At the 200 mg/kg dose, four of the six mice died within 7 days with severe liver damage (4.2-fold increase in ALT) and kidney damage (2.1-fold increase in creatinine) [1]. 2. Plasma protein binding rate: In human plasma, the protein binding rate of NVP-HSP990 was 97.8% (measured by balanced dialysis). In rat and mouse plasma, the protein binding rates were 96.5% and 97.2%, respectively [2].
3. Chronic toxicity in rats: Male Sprague-Dawley rats were orally administered NVP-HSP990 once daily at doses of 5, 15, and 30 mg/kg for 28 days. No significant toxicity was observed at the 5 mg/kg dose. At the 15 mg/kg dose, mild myelosuppression (20% decrease in white blood cell count) and mild hepatic steatosis were observed. At the 30 mg/kg dose, severe myelosuppression (55% decrease in white blood cell count), moderate hepatic injury (3-fold increase in ALT) and renal tubular degeneration were observed. The no adverse event observed dose (NOAEL) was determined to be 5 mg/kg [2]
.
4. Drug interaction potential: In vitro studies have shown that NVP-HSP990 does not inhibit CYP1A2, CYP2C9, CYP2C19 or CYP2E1 (IC50 >100 μM), but has a weak inhibitory effect on CYP3A4 (IC50=25 μM) and CYP2D6 (IC50=32 μM), indicating that it is less likely to have drug interactions with CYP3A4 or CYP2D6 substrates [2]
.

[1]. The novel oral Hsp90 inhibitor NVP-HSP990 exhibits potent and broad-spectrum antitumor activities in vitro and in vivo. Mol Cancer Ther. 2012 Mar;11(3):730-9.

[2]. Design, structure-activity relationship, and in vivo characterization of the development candidate NVP-HSP990. J Med Chem. 2014 Nov 13;57(21):9124-9.

[3]. The novel, orally bioavailable HSP90 inhibitor NVP-HSP990 induces cell cycle arrest and apoptosis in multiple myeloma cells and acts synergistically with melphalan by increased cleavage of caspases. Eur J Haematol. 2012 May;88(5):406-15.

1. Background and Development: NVP-HSP990 is a novel, orally bioavailable HSP90 inhibitor developed through structure-based drug design, which has optimized efficacy and pharmacokinetic properties compared to earlier HSP90 inhibitors (e.g., gledycin analogs). Its design focuses on enhancing the affinity of the ATP-binding pocket by modifying the backbone, thereby improving oral absorption and reducing toxicity [2]. 2. Synergistic Mechanism with Melphalan: In multiple myeloma cells, NVP-HSP990 downregulates the expression of DNA repair proteins (e.g., RAD51) by inhibiting HSP90, thereby enhancing the sensitivity of cells to melphalan (an alkylating agent). This leads to increased DNA damage and enhanced apoptosis, as evidenced by a 4.0-fold higher level of γ-H2AX (a DNA damage marker) in the combination therapy group compared to the melphalan monotherapy group [3].
3. Broad-spectrum anti-tumor potential: NVP-HSP990 showed inhibitory activity against tumor cell lines with different genetic backgrounds, including cell lines carrying EGFR mutations (H1975, IC50=24 nM), BRAF mutations (A375, IC50=22 nM) and RAS mutations (HCT116, IC50=31 nM), which supports its potential as a broad-spectrum anti-tumor drug [1].

C20H18FN5O2

379.39

379.144

934343-74-5

46216556

Off-white to yellow solid powder

1.3±0.1 g/cm3

1.627

1.44

2

7

3

28

567

1

CC1=C2C(=NC(=N1)N)C[C@@H](NC2=O)C3=C(C=C(C=C3)F)C4=NC(=CC=C4)OC

WSMQUUGTQYPVPD-OAHLLOKOSA-N

InChI=1S/C20H18FN5O2/c1-10-18-16(26-20(22)23-10)9-15(25-19(18)27)12-7-6-11(21)8-13(12)14-4-3-5-17(24-14)28-2/h3-8,15H,9H2,1-2H3,(H,25,27)(H2,22,23,26)/t15-/m1/s1

(7R)-2-amino-7-[4-fluoro-2-(6-methoxypyridin-2-yl)phenyl]-4-methyl-7,8-dihydro-6H-pyrido[4,3-d]pyrimidin-5-one

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: ≥ 2.08 mg/mL (5.48 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 20.8 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: ≥ 2.08 mg/mL (5.48 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 20.8 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.)