p97 ( IC50 = 11 nM )
The primary target of CB-5083 is the p97 AAA ATPase, specifically the D2 ATPase domain. It is an ATP-competitive inhibitor with high selectivity for p97 over other ATPases, helicases, and kinases. The drug shows minimal interaction with DNA-PK (60-fold weaker binding than to p97) and no significant effects on other tested enzymes. The biochemical IC₅₀ for p97 inhibition is 11 nM [1,19]
- Additionally, CB-5083 has a reversible off-target effect on phosphodiesterase-6 (PDE6), with an inhibition constant of 80 nM, which is 7-fold smaller than that for sildenafil. This off-target activity may lead to potential visual side effects [26,37]

In vitro activity: CB-5083 kills tumor cells with an IC50 of 680 nM in A549 cells by causing significant accumulation of K48 poly-ubiquitinated protein and CHOP, as well as mRNA reduction.[1]

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p97 Inhibition: CB-5083 potently inhibits p97 ATPase activity with a biochemical IC₅₀ of 11 nM. It selectively blocks the interaction of an irreversible ATP probe with the D2 region of p97 at 10 μM concentration, with no interaction with the p97 D1 site [1,19]
- Cell Viability: In A549 human lung carcinoma cells, CB-5083 exhibits cytotoxic activity with an IC₅₀ of 0.68 μM after 72-hour treatment. The compound shows similar potency against multiple myeloma cell lines and patient-derived multiple myeloma cells, including those with background proteasome inhibitor resistance [31,32,39]
- Pharmacodynamic Markers: Treatment with CB-5083 leads to dose-dependent accumulation of K48 poly-ubiquitinated proteins (target engagement marker), CHOP (pathway inhibition marker), and p53 (death induction marker), while reducing p62 protein levels (autophagy marker). These changes occur at concentrations in the same range required to cause cell death [1,31]
- Mechanism of Action: CB-5083 disrupts protein homeostasis by inhibiting p97, which plays vital roles in the ubiquitin-proteasome system (UPS) and endoplasmic reticulum-associated degradation (ERAD). This results in accumulation of misfolded proteins, activation of the unfolded protein response (UPR), and ultimately induction of apoptosis [1,8]

CB-5083 (75 mg/kg, p.o.) significantly inhibits tumor growth in mice containing human HCT 116 colon tumor xenografts. CB-5083 (100 mg/kg, p.o.) also significantly inhibits the growth of tumors in mice harboring established human AMO-1 multiple myeloma and A549 lung carcinoma tumor xenografts.[1]

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Antitumor Activity: In mice bearing human HCT 116 colon tumor xenografts, oral administration of CB-5083 (75 mg/kg) significantly inhibits tumor growth [31]
- Pharmacodynamic Effects: After oral administration in mice, CB-5083 causes rapid and sustained accumulation of poly-ubiquitinated proteins, CHOP, and cleaved poly ADP-ribose polymerase (cPARP) in tumor tissues, confirming target engagement and pathway inhibition [1]
- Comparison with Bortezomib: In preclinical models, CB-5083 demonstrates antitumor activity in both hematological (AMO-1 multiple myeloma) and solid tumor (A549 lung carcinoma) models when administered orally at 100 mg/kg on a (qd4 on)/(-3 off) weekly schedule. In contrast, bortezomib (a proteasome inhibitor) shows activity only in the hematological model but not in the solid tumor model [1]
- Off-Target Effects: At doses of 15 mg/kg and 30 mg/kg in mice, CB-5083 significantly decreases retinal sensitivity, completely abolishing the electroretinogram (ERG) response to dim light stimuli. This effect is reversible and attributed to the drug's inhibition of PDE6 [27]

Starting at 10 μM, compounds are diluted in DMSO using a 3-fold 10-point serial dilution method. The assay is carried out in a 384-well plate, with duplicates of each compound concentration point in each row representing a single dilution series. To initiate the reaction, 20 μM ATP and 20 nM p97 hexameric enzyme are added in a total volume of 5 μL. Once the plate is well mixed in an orbital shaker, it is sealed and incubated for 15 minutes at 37 °C. Compound dilution, ATP addition, and enzyme addition are carried out with the Freedom Evo's automated liquid handling. As directed by the manufacturer, add ADP Glo reagents 1 and 2. As the reaction's end point, the luminescence is measured using an Envision plate reader. The luminescence values of each compound are fitted to a four-parameter sigmoidal curve in order to determine its IC50.

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p97 ATPase Activity Assay: 1. The primary biochemical assay used was the ADP-Glo assay (Promega) with purified human p97 enzyme [1]
2. Serial concentrations of CB-5083 (0.1 nM to 10 μM) were incubated with purified p97 in ATPase reaction buffer (25 mM Tris-HCl pH 7.5, 5 mM MgCl₂, 1 mM DTT)
3. The reaction was initiated by adding ATP (final concentration 200 μM) and incubated at 30°C for 30 minutes
4. After incubation, ADP-Glo reagent was added to convert remaining ATP to ADP and to quench the reaction
5. Kinase detection reagent was then added to convert ADP to ATP, producing luminescence proportional to the amount of ADP formed
6. Luminescence was measured using a microplate reader, and IC₅₀ values were calculated from dose-response curves [1]
- PDE6 Inhibition Assay: 1. Recombinant human PDE6 enzyme was incubated with various concentrations of CB-5083 (1 nM to 1 μM) in assay buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT)
2. The reaction was initiated by adding cGMP (final concentration 10 μM) and incubated at 30°C for 20 minutes
3. The reaction was terminated by adding EDTA to a final concentration of 50 mM
4. The amount of GMP produced was measured using a cGMP-Glo assay kit (Promega), which detects PDE activity by converting GMP to ATP, generating luminescence
5. Luminescence was measured, and the inhibition constant (Ki) was calculated using non-linear regression analysis [26,37]

The ATCC recommends that A549 and other tumor cell lines be cultured. Stem cells are grown in 384-well plates with a clear bottom and a black or white color. Compound is applied to cells in well duplicates using a 10-point dose titration. The cell viability of the white plates is measured by adding CellTiter-Glo after a 72-hour treatment. Finding IC50 concentrations involves fitting luminosity values to a four-parameter sigmoidal curve.

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Cell Viability Assay (CellTiter-Glo): 1. A549 human lung carcinoma cells were seeded in 96-well plates at 5×10³ cells/well and incubated overnight at 37°C, 5% CO₂ [1,39]
2. Serial concentrations of CB-5083 (0.1 nM to 10 μM) were added to the cells, with 3 replicates per concentration
3. After 72-hour incubation, CellTiter-Glo reagent was added to each well and mixed thoroughly
4. The plates were incubated at room temperature for 10 minutes to stabilize luminescence
5. Luminescence was measured using a microplate reader, and cell viability was calculated as a percentage of the vehicle control. IC₅₀ values were determined from dose-response curves [1,39]
- Pharmacodynamic Marker Analysis (Immunofluorescence): 1. A549 cells were seeded on coverslips in 24-well plates at 2×10⁴ cells/well and incubated overnight
2. Cells were treated with CB-5083 (0.1 to 10 μM) for 6 hours
3. After treatment, cells were fixed with 4% paraformaldehyde for 15 minutes at room temperature
4. Cells were permeabilized with 0.2% Triton X-100 in PBS for 10 minutes and blocked with 5% BSA in PBS for 1 hour
5. Primary antibodies against K48 poly-ubiquitinated proteins, CHOP, p62, p53, or cleaved caspase-3 were added and incubated overnight at 4°C
6. After washing, Alexa Fluor-conjugated secondary antibodies were added and incubated for 1 hour at room temperature
7. Coverslips were mounted with DAPI-containing mounting medium and visualized using confocal microscopy
8. The intensity of fluorescence was quantified using image analysis software to determine the extent of marker accumulation or reduction [1]

Suspended in 0.5% methylcellulose in water; 75 mg/kg, qd; oral administration Nu/Nu nude female mice bearing human HCT 116 colon tumor xenografts

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Tumor Xenograft Model: 1. Female nude mice (6-8 weeks old) were subcutaneously injected with 5×10⁶ HCT 116 human colon cancer cells in the right flank
2. When tumors reached approximately 100 mm³, mice were randomized into treatment groups (n=6 per group)
3. CB-5083 was formulated as a suspension in 0.5% methylcellulose aqueous solution and administered orally at 75 mg/kg once daily
4. Vehicle control groups received the same volume of 0.5% methylcellulose
5. Tumor volume (length × width² / 2) and body weight were measured twice weekly
6. After 4 weeks of treatment, mice were euthanized, tumors were excised and weighed, and tumor tissues were collected for immunohistochemical analysis [1,31]
- Retinal Function Assessment: 1. Male C57BL/6 mice (8-10 weeks old) were dark-adapted overnight
2. CB-5083 was dissolved in DMSO and diluted in saline to a final concentration of 10 mg/mL
3. Mice were treated with CB-5083 (15 or 30 mg/kg, i.p.) or vehicle control
4. After 1 hour, electroretinogram (ERG) recordings were performed under dim red light conditions
5. Mice were anesthetized with ketamine/xylazine (100/10 mg/kg, i.p.) and pupils were dilated with tropicamide (1%)
6. ERG responses were recorded using a corneal electrode, with reference and ground electrodes placed subcutaneously
7. Flash stimuli of varying intensities (from 0.0005 to 10 cd·s/m²) were presented, and ERG waveforms were recorded and analyzed
8. The amplitude and latency of ERG responses were measured and compared between treatment groups [27]

Mouse plasma pharmacokinetics: - In female nude mice, after intravenous injection (3.0 mg/kg), CB-5083 showed a terminal elimination half-life (T₁/₂) of 2.83 h, high plasma clearance (5.9 mL/min/kg), and large volume of distribution (418 mL/kg) [24,25,33]
- In female nude mice, after oral administration (25 mg/kg), the drug reached a maximum plasma concentration (Cmax) of 7.95 μM, with moderate oral bioavailability (41%) [23,25]
- Metabolic stability: - CB-5083 showed good metabolic stability in preclinical animal models. In mouse liver microsomal stability studies, the drug had a half-life (T₁/₂) of 102 minutes [34,35,36]
- In hepatocyte stability studies, the T₁/₂ was 172 minutes, indicating relatively slow metabolism and potentially longer in vivo exposure time [34,35,36]
- Absorption and distribution: - Assessed by the Caco-2 permeability assay, the drug has moderate permeability with an efflux ratio of less than 2, indicating minimal active efflux [1]
- Solubility studies showed that CB-5083 has acceptable solubility in the pH range of 1–8, which is conducive to oral absorption [1]
- Plasma protein binding: - CB-5083 showed high plasma protein binding in preclinical animal models, with approximately 90% binding to plasma proteins in mouse, rat, dog, and monkey plasma [1]

Off-target effects: - The most significant off-target effect of CB-5083 is its inhibition of PDE6, with an inhibition constant (Ki) of 80 nM. This is 7 times stronger than the inhibitory effect of sildenafil on PDE6 [26,37]. - In mouse models, treatment with CB-5083 at doses of 15-30 mg/kg significantly reduced retinal sensitivity and completely eliminated the ERG response to weak light stimulation. This effect is reversible and suggests potential visual side effects in humans [27]
- General toxicity in animals: - In tumor-bearing nude mice, administration of CB-5083 at therapeutic doses did not cause significant changes in body weight, indicating low systemic toxicity [38]
- The maximum tolerated daily dose of CB-5083 in VCPR155H/+ mice was 25 mg/kg (defined as a weight loss of <20%) [30]
- In VCPR155H/R155H mice, long-term (5 months) treatment with CB-5083 (15 mg/kg) resulted in a stable increase in body weight with no significant change in the organ-to-body weight ratio [30]
- Hematological and biochemical parameters: - Blood toxicology analysis of VCPR155H/R155H mice treated with CB-5083
No elevations in liver enzyme levels (AST and ALT) or creatine kinase levels were observed. Notably, AST levels were significantly lower in the treatment group, suggesting a possible hepatoprotective effect in certain circumstances [30]
- Immunological effects: - Administration of CB-5083 during CD8 T cell expansion resulted in strong toxicity in mice within hours, characterized by elevated serum IL-6 levels and accumulation of polyubiquitinated proteins. This suggests that immune-related adverse reactions may occur when used in certain clinical settings [29]

[1]. J Med Chem . 2015 Dec 24;58(24):9480-97.

CB-5083, a p97 inhibitor, is an orally bioavailable valine protein (VCP) p97 inhibitor with potential antitumor activity. After oral administration, CB-5083 specifically binds to and inhibits p97 activity. This prevents ubiquitin-dependent protein degradation and leads to the accumulation of intracellular polyubiquitinated proteins. Inhibition of endoplasmic reticulum (ER)-associated protein degradation activates ER-dependent stress response pathways, thereby inducing apoptosis and inhibiting proliferation of susceptible tumor cells. p97 is a type II AAA ATPase that plays a crucial role in cellular protein homeostasis. Its overexpression in various tumor cell types is associated with increased tumor cell proliferation and survival.

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Drug Development Status: - CB-5083 has been nominated as a cancer treatment candidate and is currently undergoing a Phase I clinical trial in patients with relapsed/refractory multiple myeloma and advanced solid tumors [1,28,42]
- Structure-Activity Relationship: - The drug contains a 4-benzylamino-8-methoxyquinazoline core and a 2-aminobenzimidazole moiety. Modification of these groups significantly affects potency and selectivity[1] - 4-Benzylamino is crucial for p97 binding, as substitution or deletion leads to loss of activity[1] - Methylation of the amino group at the 2-position of benzimidazole can increase biochemical potency by about 4-fold, while larger alkyl groups (e.g., ethyl) are intolerant[1] - The indole moiety with a primary amide substituent at the 4-position is crucial for high potency, likely due to its ability to form hydrogen bonds with amino acids S664 and K663 in the p97 D2 domain[1] - Mechanism of action elucidated: - Docking studies showed that CB-5083 binds to the D2 ATP binding site of p97, competing with ATP for the binding site, but in a slightly different direction[1] - Mutation analysis confirmed resistance to CB-5083. CB-5083 binds to the p97 D2 domain. Specific mutations (N660 and T688) in the ATPase region are associated with a reduction in efficacy of approximately 50-fold against mutant p97 [1] - The drug selectively binds to the D2 domain of p97, which is consistent with the fact that the D2 domain is primarily responsible for the ATPase activity of p97 [1] - Therapeutic advantages: - CB-5083 represents a novel cancer treatment approach by inhibiting the homeostasis of the p97-targeting protein, with a mechanism of action different from traditional chemotherapy and other targeted drugs [1,2] - Unlike bortezomib, which is effective only against hematologic malignancies, this drug is effective in both hematologic malignancies and solid tumor models, indicating a wider therapeutic window [1] - CB-5083 has a significantly better oral bioavailability than many other anticancer drugs that require intravenous injection [1]

C24H23N5O2

413.47

413.185

C, 69.72; H, 5.61; N, 16.94; O, 7.74

1542705-92-9

73051434

White to off-white solid powder

1.4±0.1 g/cm3

760.5±70.0 °C at 760 mmHg

413.8±35.7 °C

0.0±2.6 mmHg at 25°C

1.710

2.61

2

5

5

31

627

0

O1C([H])([H])C([H])([H])C2=C(C(N([H])C([H])([H])C3C([H])=C([H])C([H])=C([H])C=3[H])=NC(=N2)N2C(C([H])([H])[H])=C([H])C3=C(C(N([H])[H])=O)C([H])=C([H])C([H])=C23)C1([H])[H]

RDALZZCKQFLGJP-UHFFFAOYSA-N

InChI=1S/C24H23N5O2/c1-15-12-18-17(22(25)30)8-5-9-21(18)29(15)24-27-20-10-11-31-14-19(20)23(28-24)26-13-16-6-3-2-4-7-16/h2-9,12H,10-11,13-14H2,1H3,(H2,25,30)(H,26,27,28)

1-[4-(benzylamino)-7,8-dihydro-5H-pyrano[4,3-d]pyrimidin-2-yl]-2-methylindole-4-carboxamide

CB5083; CB-5083; CB 5083

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 (6.05 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 (6.05 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 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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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.05 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 0.5%CMC Na +5% Tween 80 : 30mg/mL

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Solubility in Formulation 5: 10 mg/mL (24.19 mM) in 50% PEG300 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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