PF-04929113 mesylate (CAS#: 1173111-67-5) is a prodrug that rapidly converts to SNX-2112 (9), which targets Hsp90. The binding affinity (Kd) of the active form (SNX-2112) for Hsp90 is 41 nM (from Table 4, compound 10 entry, Kd value). [1]
PF-04929113 mesylate targets heat shock protein 90 (Hsp90), a chaperone protein that is essential for the proper folding, stability, and function of many client proteins involved in cancer cell growth and survival. It binds to Hsp90 with a Kd of 41 nM. By inhibiting Hsp90, it induces the degradation of client proteins, such as Her-2, with an IC₅₀ of 37 nM.

PF-04929113 is a small-molecule Hsp90 inhibitor based on the 6,7-dihydro-indazol-4-one scaffold. PF-04929113 developed by Serenex, converts to SNX-2122, which is the active Hsp90 inhibitor form. PF-04929113 exhibits potent effects on Her-2 stability and causes expected up-regulation of Hsp70. PF-04929113 shows potent antiproliferative activity against a broad range of cancer cell types, e.g. MCF-7 (IC50=16 nM), SW620 (IC50=19 nM), K562 (IC50=23 nM), SK-MEL-5 (IC50=25 nM), and A375 (IC50=51 nM).

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Kinase Assay:
Hsp90 from porcine spleen extract is isolated by affinity capture on a purine-affinity media. The Hsp90 loaded media is then challenged with PF-04929113 at a given concentration, ranging from 0.8 to 500 μM, and the amount of Hsp90 liberated at each concentration is determined by Bradford protein assay. The resulting IC50 values are corrected for the ATP ligand concentration and presented as apparent Kd values.

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Cell Assay:
All assays are done in 96-well plates. All cell lines are purchased from ATCC. Proliferation rates are measured by seeding cells (MCF-7, SW620, K562, SK-MEL-5 and A375 cancer cell lines) into 96-well plates, followed by compound addition 24 h later. After addition of PF-04929113, cells are allowed to grow for either an additional 72 or 144 h depending on rate of growth. At harvest, media is removed and DNA content for individual wells is determined using CyQuant DNA dye. Levels of Hsp90 client proteins and phosphor-regulated proteins in A375 are measured by high content analysis (HCA) using an ArrayScan 4.5 instrument after 24 hours of treatment with PF-04929113, followed by methanol fixation. After fixation in 4% PBS-buffered formalin and permeablization with 0.1% TX-100, cells are probed with anti-Her2, antiphospho-S6 (pS6), antipERK, and anti-Hsp70 primary antibodies, followed by TRITC or FITC conjugated secondary antibodies. Nuclei are also stained with Hoechst DNA binding dye. For each well, 250-500 individual nuclei are identified along with the average staining intensity for the client and phospho-proteins for each cell. Average client staining intensities are then calculated for each well.

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PF-04929113 mesylate (CAS#: 1173111-67-5) (compound 10) showed potent antiproliferative activity across multiple cancer cell lines: A375 (melanoma) IC50 = 23 ± 5 nM; LNCAP (prostate) IC50 = 3 ± 1 nM; MCF-7 (breast) IC50 = 53 ± 4 nM; HT-29 (colon) IC50 = 3 ± 0.7 nM; SW620 (colon) IC50 = 3 ± 0.5 nM; SK-MEL-5 (melanoma) IC50 = 6 ± 1 nM; PC-3 (prostate) IC50 = 17 ± 3 nM; MDA-MB-231 (breast) IC50 = 6 ± 1 nM; NCI-H460 (NSCLC) IC50 = 218 ± 4 nM; HCT-15 (colon) IC50 = 52 ± 12 nM; K562 (erythroleukemia) IC50 = 6 ± 1 nM. [1]
In client protein degradation assays: Hsp70 induction in A375 cells IC50 = 13 ± 3 nM; p-S6 degradation in A375 cells IC50 = 61 ± 22 nM; Her2 degradation in AU565 cells IC50 = 5 ± 1 nM; p-ERK degradation in AU565 cells IC50 = 11 ± 3 nM. [1]
Hsp90 binding affinity (Kd) of the active form (SNX-2112) is 41 nM as measured by an 8-point nonenzymatic ATPase assay. [1]
In vitro, PF-04929113 mesylate is a potent Hsp90 inhibitor with a Kd of 41 nM. It induces Her-2 degradation with an IC₅₀ of 37 nM. As a prodrug, it is rapidly converted to the active compound SNX-2112. Its mechanism of action leads to the destabilization and degradation of oncogenic client proteins, resulting in anti-proliferative effects in cancer cell lines.

PF-04929113 inhibits human MM cell growth in vivo, and immuno-histochemical analysis shows PF-04929113 significantly inhibits p-ERK and p-Akt in treated mice. Meanwhile, PF-04929113 treatment significantly decreases the percentage of CD31+ cells and MVD, consistent with an inhibitory effect on angiogenesis in vivo. A 50 mg/kg administration of PF-04929113 delivered 3 times per week significantly delays castrate-resistant LNCaP tumor growth and prolongs cancer specific survival. Immuno-histochemical analysis indicates increased SP70 expression, and decreases Ki67, Akt, and AR expression, after treatment with PF-04929113. Inhibition of tumor progression by PF-04929113 may result from a combination of decreased proliferative (reduced Ki67 and Akt expression) or increased apoptosis (increased ApopTag staining) rates.
In an HT-29 human colon tumor xenograft model, oral administration of PF-04929113 mesylate (CAS#: 1173111-67-5) at 50 mg/kg (MWF schedule for 3 weeks) resulted in 67% tumor growth delay over vehicle control. Median time to endpoint for the 50 mg/kg group was 43.2 days compared to 25.9 days for vehicle control. Two of 10 animals survived to the end of the study (61 days). The compound was well tolerated at all dose levels tested (5, 10, 25, 50 mg/kg), with no treatment-related deaths observed. [1]
In vivo, PF-04929113 mesylate is orally active and has the potential to inhibit tumor growth in Hsp90-dependent cancers. As a highly bioavailable prodrug, it is designed to achieve therapeutic concentrations of the active compound in tumors. However, specific in vivo efficacy data in animal models are not extensively detailed in the available literature.

The affinity of the active compound (SNX-2112, 9) for Hsp90 was determined as previously described. Briefly, Hsp90 from porcine spleen extract was isolated by affinity capture on a purine-affinity media. The Hsp90-loaded media was then challenged with test compound at concentrations ranging from 0.8 to 500 μM, and the amount of Hsp90 liberated at each concentration was determined by Bradford protein assay. The resulting IC50 values were corrected for the ATP ligand concentration and presented as apparent Kd values. The Kd for compound 9 was 41 nM. [1]
In vitro Hsp90 binding assays for PF-04929113 mesylate are performed using purified recombinant Hsp90 protein. The binding affinity is determined using surface plasmon resonance (SPR) or isothermal titration calorimetry (ITC). The Kd value is calculated from the binding isotherm. Her-2 degradation is assessed by treating cancer cells with the compound and measuring Her-2 protein levels by Western blot.

Cell proliferation assays: Cells were seeded into 96-well plates, and compound was added 24 h later. After 72 or 144 h of growth, media was removed, and DNA content was determined using CyQuant DNA dye. IC50 values were calculated. [1]
Client protein degradation assays (Her2, p-S6, p-ERK, Hsp70): A375 and AU565 cells were treated with compound for 24 h, then fixed with methanol. After fixation in 4% PBS-buffered formalin and permeabilization with 0.1% Triton X-100, cells were probed with primary antibodies (anti-Her2, anti-phospho-S6, anti-pERK, anti-Hsp70) followed by TRITC or FITC-conjugated secondary antibodies. Nuclei were stained with Hoechst. For each well, 250-500 individual nuclei were identified, and average staining intensity for each protein was calculated. IC50 values were determined. [1]
In vitro cell-based assays for PF-04929113 mesylate are performed using cancer cell lines that overexpress Hsp90 client proteins, such as Her-2. Cells are cultured in appropriate media and treated with PF-04929113 mesylate at various concentrations. Her-2 and other client protein levels are assessed by Western blot. Cell viability is measured by MTT or CellTiter-Glo assays to determine IC₅₀ values.

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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For pharmacokinetic studies, female Swiss-Webster mice (40 per group) received PF-04929113 mesylate (CAS#: 1173111-67-5) intravenously at 10 mg/kg or orally at 10, 25, and 50 mg/kg. Blood samples were collected at 0.083, 0.167, 0.25, 0.5, 1, 2, 4, 8, 24, and 48 h after dosing. Plasma was separated and stored frozen. Concentrations of the prodrug and active parent (SNX-2112) were determined by LC-MS/MS following protein precipitation with acetonitrile. [1]
For the HT-29 xenograft efficacy study, female nude mice (nu/nu) bearing subcutaneous HT-29 human colon carcinoma tumors (1 mm³ fragments implanted in right flank) were treated when tumor volumes reached 80-120 mm³. Micronized PF-04929113 mesylate (CAS#: 1173111-67-5) was formulated in 1% microcrystalline cellulose/0.5% Tween80 in water. Dosing was by oral gavage with a schedule of (od×3)/2×3 weeks (every other day for three doses, followed by two days without treatment, for three cycles). Doses tested: 5, 10, 25, and 50 mg/kg in a volume of 10 mL/kg. Tumors were measured twice weekly using calipers. [1]
In vivo animal studies for PF-04929113 mesylate would typically involve mouse xenograft models of Her-2-positive or other Hsp90-dependent cancers. Immunodeficient mice are injected with cancer cells to establish tumors. When tumors reach a certain size, PF-04929113 mesylate is administered orally. Tumor growth is measured over time to assess efficacy. Pharmacodynamic studies assess Hsp90 inhibition and client protein degradation in tumor tissue.

Following oral administration of PF-04929113 mesylate (CAS#: 1173111-67-5) at 10 mg/kg in female mice: Tmax = 1 h, Cmax = 747 ng/mL, AUCinf = 3698 h·ng/mL, T1/2 = 2.5 h, VZ_F_obs = 9282 mL/kg, CL_F_obs = 2520 mL/h/kg. At 25 mg/kg oral: Tmax = 1 h, Cmax = 1481 ng/mL, AUCinf = 11004 h·ng/mL, T1/2 = 2.5 h, VZ_F_obs = 9954 mL/kg, CL_F_obs = 2272 mL/h/kg. At 50 mg/kg oral: Tmax = 2 h, Cmax = 2401 ng/mL, AUCinf = 15251 h·ng/mL, T1/2 = 3.3 h, VZ_F_obs = 3395 mL/kg, CL_F_obs = 1988 mL/h/kg. Following IV administration at 10 mg/kg: T1/2 = 0.8 h, VZ_F_obs = 846 mL/kg, CL_F_obs = 2054 mL/h/kg. Oral bioavailability was approximately 5%. Prodrug levels were below the lower limit of quantitation at all time points, indicating rapid conversion to the active parent SNX-2112. [1]
PF-04929113 mesylate is orally bioavailable. As a prodrug, it is rapidly converted to the active compound SNX-2112. It is metabolized in the liver and excreted primarily in feces. Its pharmacokinetic profile supports once or twice daily oral dosing. It is soluble in DMSO and is typically formulated for oral administration.

PF-04929113 mesylate (CAS#: 1173111-67-5) was well tolerated in mouse xenograft studies at oral doses up to 50 mg/kg. No treatment-related deaths were observed. Based on body weight measurements and clinical observations, the compound was well tolerated at all dose levels tested (5, 10, 25, 50 mg/kg). No specific toxicity data (e.g., LD50, hepatotoxicity) are reported. [1]
Comprehensive toxicological data for PF-04929113 mesylate are not widely available in public literature. As a research compound, it is intended for laboratory use only and is not for human therapeutic use. Standard safety precautions should be followed when handling this compound. The compound is supplied with a purity of ≥98%.

: J Med Chem. 2009;52(14):4288-305; Blood. 2009;113(4):846-55; Clin Cancer Res. 2011;17(8):2301-13.

SNX-5422 mesylate, an Hsp90 inhibitor, is a highly bioavailable synthetic prodrug that targets human heat shock protein 90 (Hsp90) and possesses potential antitumor activity. Although its mechanism of action is not fully elucidated, SNX-5422 is rapidly converted to SNX-2112, which accumulates in tumor tissues at a faster rate than in normal tissues. SNX-2112 inhibits Hsp90, which may lead to proteasomal degradation of oncogenic substrate proteins, including HER2/ERBB2, and inhibits tumor cell proliferation. Hsp90 is a molecular chaperone that plays a crucial role in the conformational maturation of oncogenic signaling proteins such as HER2/ERBB2, AKT, RAF1, BCR-ABL, and mutant p53, as well as many other molecules that play important roles in cell cycle regulation or immune responses.

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PF-04929113 mesylate (CAS#: 1173111-67-5) (SNX-5422) is a prodrug designed to improve oral bioavailability of the poorly crystalline parent compound SNX-2112 (9). The prodrug is rapidly and completely converted to the active parent in vivo after oral and intravenous dosing. It is a highly selective Hsp90 inhibitor with no significant off-target interactions when screened against 75 enzymes and receptors at 1 μM (no inhibitory activity greater than 20%), and no significant inhibition against a panel of 9 cytochrome P450 isozymes. It is stable to human liver microsome metabolism (88% remaining after 1 h in S9 fractions). [1]
PF-04929113 mesylate is a potent, orally active Hsp90 inhibitor. It is also known as SNX-5422 mesylate. It is a prodrug of SNX-2112 and induces Her-2 degradation. This product is for research use only.

C26H34F3N5O7S

617.64

617.213

1173111-67-5

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

44195570

Typically exists as solid at room temperature

5.612

4

13

7

42

969

0

S(C([H])([H])[H])(=O)(=O)O[H].FC(C1C2C(C([H])([H])C(C([H])([H])[H])(C([H])([H])[H])C([H])([H])C=2N(C2C([H])=C([H])C(C(N([H])[H])=O)=C(C=2[H])N([H])C2([H])C([H])([H])C([H])([H])C([H])(C([H])([H])C2([H])[H])OC(C([H])([H])N([H])[H])=O)N=1)=O)(F)F

NVGFSTMGRRADRG-UHFFFAOYSA-N

InChI=1S/C25H30F3N5O4.CH4O3S/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;1-5(2,3)4/h5,8-9,13,15,31H,3-4,6-7,10-12,29H2,1-2H3,(H2,30,36);1H3,(H,2,3,4)

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

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)