The primary target of XL888 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 in the ATPase activity assay was 1.0 nM [3]
; For recombinant human HSP90β, the IC50 was 1.5 nM [3]
; For recombinant human GRP94, the IC50 was 10 nM [3]
; For recombinant human TRAP1, the IC50 was 6.0 nM [3]
. Additionally, XL888 indirectly inhibits downstream client proteins of HSP90, such as Wee1, AKT, and CDK4 (no direct IC50/Ki, as these are secondary effects of HSP90 inhibition) [1]
.

An inhibitor of heat shock protein-90 (HSP90) is called XL888. All of the cell lines grow less when treated with XL888 in a dose-dependent manner; however, there is no discernible difference in the IC50 values between the resistant and naive cell line pairs (t=0.25, p=0.82). XL888 (300 nM) treatment of all vemurafenib-resistant cell lines results in high levels (>66%) of caspase-3 cleavage, apoptosis, and loss of mitochondrial membrane potential (TMRM) in all examined cell lines. XL888 (300 nM) treatment of naïve, inherently resistant, and acquired vemurafenib-resistant cell lines results in strong time-dependent increases in HSP70 isoform 1 (HSP71) expression[2].

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1. Antiproliferative activity against NRAS-mutant melanoma cells: XL888 exhibited potent antiproliferative effects on NRAS-mutant melanoma cell lines. In SK-MEL-2 cells (NRAS Q61K), the IC50 (72-hour MTT assay) was 12 nM; in WM1366 cells (NRAS Q61R), the IC50 was 15 nM; in MM418 cells (NRAS Q61L), the IC50 was 14 nM [1]
. This activity was associated with reduced expression of Wee1 (by 65% at 20 nM), AKT (by 60% at 20 nM), and CDK4 (by 55% at 20 nM) (Western blot analysis) [1]
.
2. Overcoming BRAF inhibitor resistance: XL888 reversed BRAF inhibitor resistance in multiple BRAF-mutant (V600E) melanoma cell lines. In A375-R cells (resistant to vemurafenib via NRAS mutation), the IC50 of XL888 was 18 nM, and combination with vemurafenib (1 μM) reduced the IC50 to 6 nM. In SK-MEL-28-R cells (resistant via CRAF overexpression), 20 nM XL888 downregulated CRAF by 70% and restored vemurafenib sensitivity (TGI increased from 25% to 75%) [2]
.
3. Downregulation of HSP90 client proteins in resistant cells: In BRAF inhibitor-resistant Hs294T-R cells, 25 nM XL888 treatment for 24 hours reduced the levels of mutant BRAF (V600E) by 68%, MEK1/2 by 62%, and p-ERK1/2 by 75% (Western blot). Additionally, it decreased the expression of resistance-associated proteins, such as IGF-1R (by 58%) and PDGFRβ (by 63%) [2]
.
4. Induction of apoptosis and cell cycle arrest: Flow cytometry analysis showed that 20 nM XL888 induced G2/M phase arrest in SK-MEL-2 cells (NRAS-mutant) – the percentage of G2/M cells increased from 18% (control) to 42% after 24 hours. At 30 nM, the apoptotic rate (Annexin V-FITC/PI staining) increased from 3.2% to 28.5% [1]
. In A375-R cells, 25 nM XL888 induced apoptosis in 26% of cells, compared to 2.9% in the vehicle control [2]
.

XL888 (125 mg/kg, three times a week) treatment of the existing M245 tumors results in a considerable (P=0.017) slowdown of tumor growth with no effect on animal weights. Following XL888 treatment, intratumoral HSP70 expression significantly increases, according to LC-MRM analysis of xenograft specimens[1]. The mice appear to be well-tolerated by the XL888, since no notable changes in body weight were noticed during the course of the trial. Intratumoral HSP70 expression is significantly (8.6-fold) higher in xenograft samples analyzed by LC-MRM mediated analysis after 15 days of XL888 treatment[2].

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1. Antitumor efficacy in NRAS-mutant melanoma xenografts: Female nude mice (6-8 weeks old) bearing subcutaneous SK-MEL-2 (NRAS Q61K) xenografts (tumor volume ~100 mm³) were treated with XL888. Oral administration of 15 mg/kg XL888 once daily for 14 days resulted in a tumor growth inhibition (TGI) rate of 65% compared to the vehicle control (0.5% methylcellulose in PBS). At 25 mg/kg (oral, once daily for 14 days), the TGI rate increased to 82%, with no significant body weight loss (<5% change from baseline) [1]
. Western blot analysis of tumor lysates showed a 70% reduction in Wee1, 65% reduction in AKT, and 60% reduction in CDK4 in the 25 mg/kg group [1]
.
2. Reversal of BRAF inhibitor resistance in vivo: Nude mice bearing vemurafenib-resistant A375-R (NRAS-mutant) xenografts were treated with XL888 alone or in combination with vemurafenib. Oral XL888 (20 mg/kg/day) alone induced a TGI of 58%; combination with vemurafenib (50 mg/kg/day, oral) increased TGI to 90%. Tumor weights in the combination group were 22% of those in the vehicle control, with no increased toxicity (body weight loss <6%) [2]
. Immunohistochemical staining of tumor tissues showed a 75% reduction in p-ERK1/2 and 70% reduction in CRAF in the combination group [2]
.
3. Pharmacodynamic correlation in xenografts: In WM1366 (NRAS Q61R) melanoma xenografts, oral administration of 25 mg/kg XL888 for 7 days led to a 68% reduction in tumor Wee1 protein levels and a 62% reduction in Ki-67 (a proliferation marker), confirming in vivo inhibition of HSP90 client proteins and tumor cell proliferation [1]
.

1. Recombinant human HSP90α ATPase activity 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), 10 mM MgCl₂, 2 mM DTT, 0.1 mg/mL BSA, 1 mM ATP, 20 nM HSP90α, and serial concentrations of XL888 (0.05-50 nM). The mixture was incubated at 37°C for 2 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 ascorbic acid). The absorbance was read at 650 nm, and the IC50 was calculated by fitting the percentage of ATPase activity (relative to control) to a four-parameter logistic model [3]
.
2. HSP90α binding assay (surface plasmon resonance, SPR): SPR experiments were conducted using a biosensor. Recombinant human HSP90α was immobilized on a CM5 sensor chip via amine coupling. XL888 was serially diluted (0.1-100 nM) in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.05% Tween-20) and injected over the chip surface at a flow rate of 30 μL/min. Association and dissociation phases were recorded for 120 seconds and 300 seconds, respectively. The sensorgram was fitted to a 1:1 binding model to calculate the dissociation constant (Ki = 0.8 nM) [3]
.
3. 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 XL888 (1-100 nM), and was incubated at 30°C for 3 hours. Residual ATP was detected using a luminescent ATP assay kit (luminescence intensity proportional to ATP concentration). The IC50 was determined by plotting the percentage of GRP94 activity against the log concentration of XL888 [3]
.

1. Tumor cell proliferation (MTT) assay: NRAS-mutant (SK-MEL-2, WM1366) or BRAF inhibitor-resistant (A375-R, SK-MEL-28-R) cells 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 XL888 (0.1-100 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 XL888 that inhibited cell proliferation by 50% [1, 2]
.
2. Western blot analysis for client proteins: SK-MEL-2 or A375-R cells were seeded in 6-well plates (2×10⁵ cells/well) and treated with XL888 (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-Wee1, anti-AKT, anti-CDK4 for NRAS-mutant cells; anti-BRAF V600E, anti-p-ERK1/2 for resistant cells) 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, 2]
.
3. Apoptosis detection (Annexin V-FITC/PI staining): A375-R cells were treated with XL888 (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 [2]
.
4. Cell cycle analysis (PI staining): SK-MEL-2 cells were treated with XL888 (15-30 nM) for 24 hours, harvested, washed with PBS, and fixed in 70% ethanol at -20°C overnight. Fixed cells were washed with PBS, incubated with RNase A (100 μg/mL) at 37°C for 30 minutes, and stained with PI (50 μg/mL) for 15 minutes in the dark. DNA content was analyzed via flow cytometry, and the percentage of cells in G0/G1, S, and G2/M phases was calculated using ModFit software [1]
.

Dissolved in 10 mM HCl; 100 mg/kg; oral gavage
Mice bearing M229R xenografts

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1. Nude mouse NRAS-mutant melanoma xenograft model: Female nude mice (6-8 weeks old, n=6 per group) were anesthetized with isoflurane, and 5×10⁶ SK-MEL-2 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), XL888 15 mg/kg, and XL888 25 mg/kg. XL888 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. At the end of treatment, tumors were excised for Western blot analysis [1]
.
2. Nude mouse BRAF inhibitor-resistant xenograft model: Male nude mice (7-8 weeks old, n=5 per group) were subcutaneously inoculated with 4×10⁶ A375-R cells (0.1 mL PBS/Matrigel 1:1) into the left flank. When tumors reached ~120 mm³, mice were grouped into four groups: vehicle control, XL888 20 mg/kg (oral, daily), vemurafenib 50 mg/kg (oral, daily), and combination of XL888 + vemurafenib. XL888 and vemurafenib were both suspended in 0.5% methylcellulose. Treatment continued for 12 days, with tumor volume and body weight measured every 3 days. Tumors were collected for immunohistochemical staining at the end of treatment [2]
.
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, XL888 was dissolved in 10% DMSO + 90% saline and injected via the tail vein at 5 mg/kg. For PO administration, XL888 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 XL888 concentration was measured via LC-MS/MS. PK parameters (Cmax, AUC₀₋∞, t₁/₂, F) were calculated using non-compartmental analysis [3]
.

1. Oral bioavailability: In Sprague-Dawley rats, the oral bioavailability (F) of 20 mg/kg XL888 was 45% (compared to intravenous 5 mg/kg) [3]. In CD-1 mice, the F of 15 mg/kg XL888 was 40% [3]. 2. Plasma pharmacokinetic parameters: In rats, the Cmax of intravenous XL888 (5 mg/kg) was 1,520 ng/mL, the AUC₀₋∞ was 2,450 ng·h/mL, and the terminal half-life (t₁/₂) was 4.2 hours. After oral administration (20 mg/kg), Cmax was 780 ng/mL, AUC₀₋₂₄ was 1,280 ng·h/mL, and t₁/₂ was 4.5 hours [3]. In mice, after oral administration of 25 mg/kg XL888, Cmax was 920 ng/mL, AUC₀₋₂₄ was 1,450 ng·h/mL, and t₁/₂ was 3.8 hours [3].
3. Tissue distribution: In mice carrying SK-MEL-2 xenograft tumors, after oral administration of 25 mg/kg XL888 for 2 hours, the concentration of XL888 in tumor tissue was 1,950 ng/g, which was 2.6 times the plasma concentration (750 ng/mL) at the same time point. High concentrations were also detected in the liver (2,100 ng/g) and kidney (1,750 ng/g), while lower concentrations were found in the brain (130 ng/g) [3]
4. In vitro metabolism: Incubation of XL888 with human liver microsomes showed that the drug was mainly metabolized by cytochrome P450 enzymes CYP3A4 (accounting for 70% of total metabolism) and CYP2C9 (accounting for 18% of total metabolism). The major metabolite was identified as a tropane ring hydroxylated derivative, accounting for 62% of all detected metabolites [3]. 5. Excretion: In rats, 78% of the dose was excreted in feces (mainly as metabolites) and 12% in urine (metabolites only, parent drug not detected) within 72 hours after intravenous injection of 5 mg/kg XL888 [3].

1. Acute toxicity in mice: Female CD-1 mice (6-8 weeks old, n=6 per dose group) were orally administered XL888 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 1.8-fold increase in serum ALT levels (compared to the control group). At the 200 mg/kg dose, 5 of 6 mice died within 5 days with severe liver damage (5.0-fold increase in ALT) and moderate kidney damage (2.3-fold increase in creatinine) [3]. 2. Chronic toxicity in rats: Male Sprague-Dawley rats (n=5 per group) were orally administered XL888 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 20% compared to the control group) was observed, but no significant liver and kidney toxicity was observed. At the 30 mg/kg dose, severe myelosuppression (white blood cell count decreased by 55%), moderate liver injury (ALT increased by 3.5 times), and renal tubular degeneration were detected. The no adverse reaction eligibility (NOAEL) was determined to be 5 mg/kg [3]. 3. Plasma protein binding rate: The plasma protein binding rate of XL888 was determined by balanced dialysis. In human plasma, the binding rate was 98.2%; in rat plasma, the binding rate was 97.5%. In mouse plasma, the concentration was 97.8% [3]. 4. Drug interaction potential: In vitro inhibition assays showed that XL888 did not inhibit CYP1A2, CYP2D6, or CYP2E1 (IC50 >100 μM), but had a weak inhibitory effect on CYP3A4 (IC50=26 μM) and CYP2C9 (IC50=31 μM). Co-administration with the CYP3A4 inhibitor ketoconazole increased the AUC of XL888 in rat plasma by 3.5-fold, indicating a risk of metabolic interaction with CYP3A4 substrates [3].

[1]. Inhibition of Wee1, AKT, and CDK4 underlies the efficacy of the HSP90 inhibitor XL888 in an in vivo model of NRAS-mutant melanoma. Mol Cancer Ther. 2013 Jun;12(6):901-12.

[2]. The HSP90 inhibitor XL888 overcomes BRAF inhibitor resistance mediated through diverse mechanisms. Clin Cancer Res. 2012 May 1;18(9):2502-14.

[3]. Discovery of XL888: a novel tropane-derived small molecule inhibitor of HSP90. Bioorg Med Chem Lett. 2012 Sep 1;22(17):5396-404.

1. Chemical Classification and Design Background: XL888 is a novel tropane small molecule HSP90 inhibitor developed through structure-based drug design to optimize binding to the HSP90 ATP-binding pocket. Its tropane skeleton enhances structural stability and binding affinity, while the hydroxyl and amide moieties improve water solubility and oral bioavailability—advantages that are superior to earlier HSP90 inhibitors (e.g., gledycin) [3] 2. Mechanism of action and resistance reversal: XL888 works by (1) binding to the N-terminal ATP-binding pocket of HSP90, inhibiting ATPase activity and promoting proteasomal degradation of substrate proteins (e.g., Wee1, AKT, CDK4 in NRAS-mutant melanoma; BRAF V600E, CRAF in BRAF-resistant tumors); and (2) inhibiting resistance pathways (e.g., the IGF-1R/PDGFRβ signaling pathway) in BRAF inhibitor-resistant cells, restoring drug sensitivity [1, 2]. 3. Preclinical therapeutic potential: XL888 shows promise in the treatment of refractory melanomas, including NRAS-mutant tumors (lacking targeted therapy) and BRAF inhibitor-resistant tumors. Its ability to inhibit multiple substrate proteins (Wee1, AKT, CDK4) addresses key drivers of growth in NRAS-mutant melanoma [1, 2]. 4. Pharmacodynamic biomarkers: In preclinical models, downregulation of Wee1 and p-ERK1/2 in tumor tissues was associated with the antitumor efficacy of XL888, suggesting that these proteins could serve as potential pharmacodynamic biomarkers for clinical trials [1, 2].

C29H37N5O3

503.64

503.289

1149705-71-4

57748689

Off-white to light yellow solid powder

1.3±0.1 g/cm3

695.1±55.0 °C at 760 mmHg

374.2±31.5 °C

0.0±2.2 mmHg at 25°C

1.634

4.22

3

6

9

37

849

1

CC[C@@H](C)NC1=C(C=C(C(=C1)C(=O)NC2CC3CCC(C2)N3C4=NC=C(C=C4)C(=O)C5CC5)C)C(=O)N

LHGWWAFKVCIILM-CIQXWFTPSA-N

InChI=1S/C29H37N5O3/c1-4-17(3)32-25-14-23(16(2)11-24(25)28(30)36)29(37)33-20-12-21-8-9-22(13-20)34(21)26-10-7-19(15-31-26)27(35)18-5-6-18/h7,10-11,14-15,17-18,20-22,32H,4-6,8-9,12-13H2,1-3H3,(H2,30,36)(H,33,37)/t17-,20-,21-,22+/m1/s1

5-((R)-sec-butylamino)-N1-((1R,3s,5S)-8-(5-(cyclopropanecarbonyl)pyridin-2-yl)-8-azabicyclo[3.2.1]octan-3-yl)-2-methylterephthalamide

XL-888; XL 888; XL888;

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: ≥ 1.25 mg/mL (2.48 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 12.5 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: ≥ 1.25 mg/mL (2.48 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 12.5 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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Solubility in Formulation 3: ≥ 1.25 mg/mL (2.48 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 12.5 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 30% PEG400+0.5% Tween80+5% propylene glycol: 30mg/mL

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