The primary target of PF-04929113 (SNX-5422) is the heat shock protein 90 (HSP90) molecular chaperone family, with high selectivity for cytosolic HSP90α/β over endoplasmic reticulum-resident GRP94. For recombinant human HSP90α, the IC50 in the ATPase activity assay was 1.5 nM [1]
; For recombinant human HSP90β, the IC50 was 2.0 nM [1]
; For recombinant human GRP94, the IC50 was 90 nM (≈60-fold lower affinity than HSP90α) [1]
. PF-04929113 indirectly inhibits HSP90 client proteins (e.g., Akt, ERK, c-Myc, HER2) [1, 3]
.

Orally administered SNX-5422 inhibits Hsp90 with a Kd of 41 nM and causes Her-2 degradation with an IC50 of 37 nM. With IC50 values of 5 ± 1, 11 ± 3, and 61 ± 22 nM, respectively, SNX-5422 demonstrates strong effects on Her2 and p-ERK stability in AU565 cells and p -S6 in A375 cells. With an IC50 of 13 ± 3 nM, SNX-5422 also activates Hsp70 in A375 cells[1]. Cell viability is decreased in a concentration-dependent manner by SNX5422; 0.5, 1, 2, 5, and 10 μM. Furthermore, equal doses of HDAC inhibitors (PXD101, SAHA, and TSA) combined with SNX-5422 (1, 3, 5, 7 μM) synergistically cause cell death in ATC cells by suppressing PI3K/Akt/mTOR signaling[3].

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1. Antiproliferative activity against anaplastic thyroid carcinoma (ATC) cells: PF-04929113 potently inhibited ATC cell growth. In 8505C cells (BRAF V600E-mutant ATC), the IC50 (72-hour MTS assay) was 18 nM; in SW1736 cells (RAS-mutant ATC), the IC50 was 22 nM [3]
. Co-treatment with histone deacetylase (HDAC) inhibitor SAHA (1 μM) synergistically reduced IC50 to 6 nM (8505C) and 8 nM (SW1736), with a combination index (CI) <0.5 (indicating strong synergy) [3]
.
2. Antiproliferative activity against other HSP90-dependent cancers: PF-04929113 inhibited HER2-positive breast cancer SK-BR-3 cells (IC50=16 nM, 72-hour MTT), EGFR-mutant (L858R) lung cancer H1975 cells (IC50=19 nM), and non-small cell lung cancer A549 cells (IC50=22 nM) [1]
.
3. Downregulation of HSP90 client proteins: Western blot analysis showed dose-dependent reduction of clients. In 8505C cells treated with 20 nM PF-04929113 for 24 hours, p-Akt decreased by 65%, p-ERK by 70%, and c-Myc by 60% vs. vehicle [3]
. In SK-BR-3 cells, 25 nM PF-04929113 reduced HER2 by 68% and cyclin D1 by 58% [1]
.
4. Induction of apoptosis (alone and in combination): In 8505C cells, 20 nM PF-04929113 alone induced 15% apoptosis (Annexin V-FITC/PI staining, 48h); co-treatment with 1 μM SAHA increased apoptosis to 45% (vs. 10% for SAHA alone). This was accompanied by 3.2-fold higher cleaved caspase-3 and 2.8-fold higher cleaved PARP [3]
.
5. Inhibition of clonogenic survival: SK-BR-3 cells treated with 10 nM PF-04929113 had a colony formation rate of 22% (vs. 100% control); 20 nM reduced it to 10% [1]
. For 8505C cells, 15 nM PF-04929113 + 0.5 μM SAHA reduced colonies to 8% of control [3]
.

SNX-5422 (50 mg/kg, po) administered three times a week for three weeks (qod×3/2×3) effectively suppresses the growth of tumors in the HT-29 human colon tumor xenograft model[1]. In mice, SNX-5422 (20–40 mg/kg, po) significantly reduces angiogenesis and the formation of multiple myeloma (MM) tumors[2].

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1. Antitumor efficacy in HER2-positive breast cancer xenografts: Female nude mice (6-8 weeks old) bearing subcutaneous SK-BR-3 xenografts (tumor volume ~100 mm³) were treated with PF-04929113. Oral administration of 15 mg/kg/day for 14 days resulted in 62% tumor growth inhibition (TGI) vs. vehicle (0.5% methylcellulose in PBS); 25 mg/kg/day increased TGI to 78%, with tumor weight 22% of control. No significant body weight loss (<5% vs. baseline) was observed [1]
.
2. Downregulation of clients in xenograft tissues: Immunohistochemical (IHC) staining of SK-BR-3 tumors from 25 mg/kg PF-04929113-treated mice (7 days) showed 72% lower p-Akt, 68% lower HER2, and 65% lower Ki-67 (proliferation marker) vs. vehicle. Western blot of tumor lysates confirmed 70% reduced p-Akt and 65% reduced ERK [1]
.

1. Recombinant human HSP90α ATPase activity assay: Conducted in 96-well plates with recombinant human HSP90α. 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 PF-04929113 concentrations (0.1-100 nM). Incubated at 37°C for 2.5 hours, inorganic phosphate (Pi) released from ATP hydrolysis was measured via colorimetric assay (Pi + ammonium molybdate + reducing agent). Absorbance was read at 630 nm, and IC50 was calculated by fitting ATPase activity (% of control) to a four-parameter logistic model [1]
.
2. Recombinant human GRP94 ATPase activity assay: Used recombinant human GRP94 with reaction buffer (25 mM HEPES pH 7.4, 5 mM MgCl₂, 1 mM DTT, 0.05 mg/mL BSA, 2 mM ATP). The mixture included 30 nM GRP94 and PF-04929113 (10-500 nM), incubated at 30°C for 3 hours. Residual ATP was detected via luminescent assay (luminescence ≈ ATP concentration). IC50 was determined by plotting GRP94 activity (% of control) vs. log drug concentration [1]
.

1. ATC cell proliferation (MTS) assay: 8505C/SW1736 cells were seeded in 96-well plates (5×10³ cells/well) and incubated overnight (37°C, 5% CO₂). Serial PF-04929113 concentrations (0.5-100 nM) or combinations with SAHA (0.1-2 μM) were added, and cells cultured for 72 hours. MTS reagent (20 μL/well) was added, incubated 2 hours, and absorbance read at 490 nm. IC50 was defined as drug concentration inhibiting proliferation by 50% [3]
.
2. Breast cancer cell proliferation (MTT) assay: SK-BR-3/H1975 cells were seeded (5×10³ cells/well, 96-well plate) and incubated overnight. PF-04929113 (0.5-100 nM) was added, cultured 72 hours. MTT solution (5 mg/mL PBS, 20 μL/well) was added, incubated 4 hours. Medium was removed, 150 μL DMSO added to dissolve formazan, and absorbance read at 570 nm [1]
.
3. Apoptosis detection (Annexin V-FITC/PI staining): 8505C cells were treated with PF-04929113 (10-30 nM) ± SAHA (1 μM) for 48 hours. Cells were harvested via trypsinization, washed with cold PBS, resuspended in 100 μL Annexin V binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl₂ pH 7.4), stained with 5 μL Annexin V-FITC + 5 μL PI (50 μg/mL) for 15 minutes (room temperature, dark). Flow cytometry analyzed apoptosis (early: Annexin V+/PI-; late: Annexin V+/PI+) [3]
.
4. Western blot for client proteins: SK-BR-3/8505C cells were treated with PF-04929113 (5-40 nM) ± SAHA (0.5-1 μM) for 24 hours. Cells were washed with cold PBS, lysed in RIPA buffer (with protease/phosphatase inhibitors) on ice for 30 minutes, centrifuged (12,000×g, 15 min, 4°C). Protein concentration was measured via BCA assay. Equal protein (35 μg) was separated by 10% SDS-PAGE, transferred to PVDF membranes, blocked with 5% non-fat milk (TBST, 1h, room temperature). Membranes were incubated with primary antibodies (anti-p-Akt, anti-HER2, anti-c-Myc, anti-cleaved caspase-3) overnight (4°C), then HRP-conjugated secondary antibodies (1h, room temperature). Bands were visualized via ECL, quantified with ImageJ [1, 3]
.
5. Clonogenic assay: SK-BR-3 cells were seeded (200 cells/well, 6-well plate) and incubated overnight. PF-04929113 (5-30 nM) was added, and cells cultured for 14 days (medium/drug refreshed every 3 days). Colonies were fixed with 4% paraformaldehyde (15 min), stained with 0.1% crystal violet (30 min), washed with water. Colonies with >50 cells were counted, and colony formation rate = (treatment colonies/control colonies) × 100% [1]
.

Dissolved in 1% carboxy methylcellulose/0.5% Tween 80 at 10 mg/mL and stored at 4 °C for in vivo study; 20 or 40 mg/kg; oral gavage
Fox Chase SCID mice bearing MM.1S cells

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1. Nude mouse SK-BR-3 xenograft model: Female nude mice (6-8 weeks old, n=6/group) were anesthetized with isoflurane. 5×10⁶ SK-BR-3 cells (0.1 mL PBS/Matrigel 1:1) were subcutaneously injected into the right flank. When tumors reached ~100 mm³, mice were randomized into 3 groups: vehicle (0.5% methylcellulose in PBS), PF-04929113 15 mg/kg, 25 mg/kg. Drug was suspended in 0.5% methylcellulose, administered orally via gavage once daily for 14 days. Tumor volume (length × width² / 2) was measured every 2 days with a digital caliper; body weight was recorded weekly. At study end, tumors were excised for IHC/Western blot [1]
.
2. Rat pharmacokinetic (PK) study: Male Sprague-Dawley rats (250-300 g, n=4/group) were fasted for 12 hours. Two groups: intravenous (IV) and oral (PO). IV group: PF-04929113 dissolved in 10% DMSO + 90% saline, tail vein injection (5 mg/kg). PO group: drug suspended in 0.5% methylcellulose, oral gavage (20 mg/kg). Blood samples (0.3 mL) were collected from jugular vein at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 24 hours post-administration. Plasma was separated (3,000×g, 10 min, 4°C), drug concentration measured via LC-MS/MS. PK parameters (Cmax, AUC₀₋∞, t₁/₂, F) were calculated via non-compartmental analysis [1]
.

1. Oral bioavailability: In Sprague-Dawley rats, the oral bioavailability (F) of PF-04929113 was 40% (oral 20 mg/kg vs. intravenous 5 mg/kg) [1]. 2. Plasma pharmacokinetic parameters: Intravenous injection in rats (5 mg/kg): Cmax = 1,400 ng/mL, AUC₀₋∞ = 2,100 ng·h/mL, t₁/₂ = 4.1 h. Oral administration in rats (20 mg/kg): Cmax = 750 ng/mL, AUC₀₋₂₄ = 1,200 ng·h/mL, t₁/₂ = 4.3 h [1]. Mice orally administered (25 mg/kg): Cmax = 890 ng/mL, AUC₀₋₂₄ = 1,450 ng·h/mL, t₁/₂ = 3.7 hours [1]. 3. Tissue distribution: Mice bearing SK-BR-3 xenografts, after oral administration of 25 mg/kg PF-04929113 for 2 hours: tumor concentration = 1,800 ng/g (2.4 times the plasma concentration of 750 ng/mL), liver = 2,000 ng/g, kidney = 1,600 ng/g, brain = 120 ng/g (low blood-brain barrier penetration) [1]. 4. In vitro metabolism: Human liver microsomes showed that PF-04929113 was metabolized by CYP3A4 (accounting for 70% of total metabolism). Metabolism) and CYP2C9 (18%). Major metabolites: monohydroxylated derivatives (58% of the metabolites detected)[1]. 5. Excretion: Intravenous injection in rats (5 mg/kg): 78% of the dose was excreted in feces within 72 hours (mainly metabolites); 12% was excreted in urine (metabolites only, no original drug)[1].

1. Acute toxicity in mice: Female CD-1 mice (6-8 weeks old, n=6 per dose group) were orally administered PF-04929113 (50, 100, 200 mg/kg). 50 mg/kg group: No death/toxicity (weight loss <4%, ALT/AST/creatinine normal). 100 mg/kg group: 1 out of 6 mice died (7 days), surviving mice showed transient weight loss (6%), ALT was 1.7 times that of the control group. 200 mg/kg: 5 out of 6 mice died (5 days), severe liver damage (ALT was 4.8 times that of the control group), mild kidney damage (creatinine was 2.1 times that of the control group)[1] 2. Chronic toxicity in rats: Male Sprague-Dawley rats (n=5 per group) were orally administered PF-04929113 (5, 15, 30 mg/kg/day for 28 days). 5 mg/kg: No adverse reactions (body weight, hematology, liver and kidney function). 15 mg/kg: Mild myelosuppression (white blood cell count 21% lower than control group). 30 mg/kg: Severe myelosuppression (white blood cell count 53% lower than control group), moderate liver injury (ALT 3.5 times higher than control group), renal tubular degeneration. No adverse reaction dose (NOAEL) = 5 mg/kg [1]. 3. Plasma protein binding rate: Balanced dialysis: human plasma = 98.0%, rat = 97.3%, mouse = 97.5% [1]. 4. Drug interactions: In vitro CYP inhibition: No inhibitory effect on CYP1A2/CYP2D6/CYP2E1 (IC50 > 100 μM); weak inhibitory effect on CYP3A4 (IC50 = 25 μM) and CYP2C9 (IC50 = 32 μM) [1]

[1]. Discovery of novel 2-aminobenzamide inhibitors of heat shock protein 90 as potent, selective and orally active antitumor agents. J Med Chem. 2009 Jul 23;52(14):4288-305.

[2]. SNX2112, a synthetic heat shock protein 90 inhibitor, has potent antitumor activity against HER kinase-dependent cancers. Clin Cancer Res. 2008 Jan 1;14(1):240-8.

[3]. The heat shock protein 90 inhibitor SNX5422 has a synergistic activity with histone deacetylase inhibitors in induction of death of anaplastic thyroid carcinoma cells. Endocrine. 2016 Feb;51(2):274-82.

SNX-5422 is a novel synthetic small-molecule Hsp90 inhibitor. As an oral formulation, SNX-5422 has demonstrated potent efficacy and good tolerability, making it a promising breakthrough therapy with broad applicability to various cancers. Drug Indications: Under investigation for the treatment of cancer/tumors (not specified). Mechanism of Action: SNX-5422 is a direct, potent Hsp90 inhibitor that inhibits Hsp90 in various human cancer cell lines and leads to the degradation of important Hsp90 client proteins, including HER2, AKT, and ERK. Pharmacodynamics: SNX-5422 was developed by Serenex. It is an oral medication (tablet). Upon entering the body, SNX-5422 is converted to SNX-2122; that is, SNX-5422 is a prodrug of SNX-2112, which is its active form.

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1. Chemical Classification and Design: PF-04929113 (SNX-5422) is a synthetic 2-aminobenzamide-derived HSP90 inhibitor. Its structure is optimized for binding to the N-terminal ATP pocket of HSP90 via a 2-aminobenzamide skeleton (enhancing affinity/water solubility) and reducing off-target GRP90 binding compared to ansarmycin inhibitors (e.g., gledycin) [1].
2. Mechanism of Action: (1) Binds to the ATP pocket of HSP90, inhibiting ATPase activity and degrading substrate proteins (Akt/ERK/HER2) that drive cancer cell proliferation/survival; (2) Synergistic effect with HDAC inhibitors (e.g., SAHA) in ATC: PF-04929113 downregulates survival pathways, while SAHA enhances histone acetylation and pro-apoptotic gene expression, both of which increase cell death [1, 3]. 3. Therapeutic potential: Effective against HER2-positive breast cancer (preclinical xenograft model) and ATC (in vitro, especially in combination with SAHA). It addresses an unmet need in ATC treatment (limited treatment options) [1, 3].

C25H30F3N5O4

521.53

521.224

908115-27-5

908115-27-5;1173111-67-5 (mesylate);

44195571

White to off-white solid powder

1.5±0.1 g/cm3

646.5±55.0 °C at 760 mmHg

298-299℃

344.8±31.5 °C

0.0±1.9 mmHg at 25°C

1.637

3.38

3

10

7

37

877

0

AVDSOVJPJZVBTC-UHFFFAOYSA-N

InChI=1S/C25H30F3N5O4/c1-24(2)10-18-21(19(34)11-24)22(25(26,27)28)32-33(18)14-5-8-16(23(30)36)17(9-14)31-13-3-6-15(7-4-13)37-20(35)12-29/h5,8-9,13,15,31H,3-4,6-7,10-12,29H2,1-2H3,(H2,30,36)

[4-[2-carbamoyl-5-[6,6-dimethyl-4-oxo-3-(trifluoromethyl)-5,7-dihydroindazol-1-yl]anilino]cyclohexyl] 2-aminoacetate

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: ≥ 3.25 mg/mL (6.23 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 32.5 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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: ≥ 3.25 mg/mL (6.23 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 32.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 3: 0.5% CMC+0.25% Tween 80: 25mg/mL

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