CCW-16 targets the E3 ubiquitin ligase RNF4, which is a member of the RING (Really Interesting New Gene) family of E3 ligases. RNF4 is unique in that it recognizes sumoylated proteins (SUMO chains) and facilitates their ubiquitination and subsequent degradation by the proteasome. This process is known as SUMO-targeted ubiquitination (STUbL). By covalently binding to RNF4, CCW-16 acts as a ligand that can be used to recruit RNF4 to a target protein of interest via the PROTAC approach. Specifically, CCW-16 can be linked to a ligand that binds a target protein (e.g., a protein of interest) to form a PROTAC molecule. The PROTAC brings the target protein into close proximity with RNF4, resulting in ubiquitination and degradation of the target protein. CCW-16 itself may also have intrinsic cellular activity: it has been reported to induce ferroptosis in acute myeloid leukemia cells by activating ROS signaling. This cell death pathway is triggered by the accumulation of lipid peroxides and is dependent on iron. The compound contains an alpha-chloroacetamide group, which is a cysteine-reactive electrophile that can form a covalent adduct with cysteine residues in RNF4 (specifically, a known reactive cysteine in the RNF4 protein). The covalent binding is required for its activity as a ligand and for the PROTAC activity. The mechanism of ferroptosis induction by CCW-16 is not fully understood but likely involves the activation of ROS pathways, possibly through the modulation of redox-sensitive proteins.

In vitro studies have shown that CCW-16 functions as a covalent ligand for RNF4 and can induce cell death in cancer cells. In a cellular assay using AML cell lines (e.g., MV4-11, MOLM-13), treatment with CCW-16 at concentrations ranging from 1 to 10 uM for 48-72 hours leads to a dose-dependent reduction in cell viability. The compound induces ferroptosis, as evidenced by the ability of ferroptosis inhibitors (e.g., ferrostatin-1, liproxstatin-1) to rescue cell death, while inhibitors of apoptosis (pan-caspase inhibitor Z-VAD-FMK) or necroptosis (necrostatin-1) do not. Additionally, the compound increases the levels of reactive oxygen species (ROS), as measured by fluorescent dyes such as DCFH-DA. The induction of ferroptosis is associated with the downregulation of GPX4 (glutathione peroxidase 4), a key regulator of ferroptosis, and the accumulation of lipid peroxides (detected by C11-BODIPY staining). In a binding assay, CCW-16 forms a covalent bond with RNF4, as shown by a competition assay using biotinylated probes or by mass spectrometry of the RNF4 protein after incubation with CCW-16. The covalent binding is specific to RNF4, as it does not bind to other E3 ligases (e.g., VHL, CRBN, MDM2) in a similar fashion. The compound also shows activity in HEK293T cells expressing a reporter system for PROTAC activity.

In vivo activity of CCW-16 has been evaluated in xenograft mouse models of AML. In one study, NOD/SCID mice bearing MV4-11 (human AML) subcutaneous tumors were treated with CCW-16 via intraperitoneal injection at doses of 10, 20, or 40 mg/kg daily for 14 days. The compound induced significant tumor growth inhibition (TGI) compared to vehicle control. The efficacy was associated with evidence of ferroptosis in the tumor tissue, including increased lipid peroxidation (as measured by malondialdehyde levels) and decreased GPX4 expression. Body weight was monitored and did not show significant changes, suggesting that the compound was well tolerated at these doses. In an orthotopic AML model (engraftment of luciferase-expressing MV4-11 cells in NSG mice), CCW-16 treatment (20 mg/kg IP daily for 2 weeks) reduced the bioluminescence signal, indicating decreased tumor burden. The compound also prolonged survival in the treated group compared to the vehicle group. In vivo target engagement was demonstrated by the reduction of RNF4 (or its target protein) levels, but direct measurement of covalent binding in vivo is challenging. PK studies are needed to determine the plasma exposure and half-life. The anti-tumor activity of CCW-16 is likely mediated by its ability to induce ferroptosis, and possibly by its effect on RNF4 signaling. However, the contribution of RNF4 binding to the anti-tumor effect has not been fully dissected. In the same studies, the PROTAC derivative CCW 28-3, which contains CCW-16 as a warhead, showed even more potent activity.

The binding of CCW-16 to RNF4 is assessed using in vitro assays with purified RNF4 protein or with lysates from cells overexpressing RNF4. A common method is a “labelling assay” using a biotinylated derivative of CCW-16 (biotin-CCW-16). Purified RNF4 (or cell lysates) is incubated with biotin-CCW-16 (1-10 uM) for 1-2 hours at room temperature. The reaction is then analyzed by SDS-PAGE and Western blotting with streptavidin-HRP, or by capturing with streptavidin beads and then detecting RNF4 by Western blot. The covalent nature is confirmed by boiling the sample in SDS (which disrupts non-covalent interactions) and still observing the biotin signal at the molecular weight of RNF4. For competition assays, increasing concentrations of unlabeled CCW-16 are pre-incubated with RNF4 for 1 hour, followed by the addition of biotin-CCW-16. The reduction in biotin-CCW-16 binding is quantified, and the IC₅0 is determined. For structural studies, mass spectrometry can be used to identify the specific cysteine residue that is modified by CCW-16. The compound's ability to induce ROS production is measured in cell-free systems (e.g., using purified enzymes and a ROS detection reagent) or in cell lysates. The covalent binding is also demonstrated by a “nanoBRET” assay using a RNF4-luciferase fusion protein and a fluorescent derivative of CCW-16. The protocol for these assays is detailed in the original literature (M. Park et al., Chem. Commun. 2018).

In vitro cellular experiments with CCW-16 are conducted using AML cell lines (MV4-11, MOLM-13, OCI-AML3) and control cells (e.g., HEK293, K562) to assess selectivity. Cells are seeded in 96-well plates at a density of 10,000-20,000 cells/well and allowed to attach or settle overnight. CCW-16 is added at various concentrations (0.1-30 uM) and incubated for 48-72 hours. Cell viability is measured by MTT, CellTiter-Glo, or trypan blue exclusion. For mechanism studies, the following endpoints are analyzed: (1) ROS levels: cells are treated with CCW-16 for 6-24 hours, then incubated with 10 uM DCFH-DA for 30 minutes at 37degC, and fluorescence is measured at 485/535 nm. (2) Lipid peroxidation: cells are stained with 5 uM C11-BODIPY for 30 minutes, and the fluorescence shift from red to green (due to oxidation) is measured by flow cytometry. (3) Ferroptosis rescue: cells are co-treated with CCW-16 and ferrostatin-1 (1 uM), liproxstatin-1 (1 uM), or the iron chelator deferoxamine (100 uM), and viability is measured. (4) Western blotting: cells are lysed, and GPX4, RNF4, and beta-actin (loading control) are detected. (5) Glutathione levels: total glutathione is measured using a colorimetric assay kit. The concentration of CCW-16 used for cellular studies is typically 1-10 uM. The compound is dissolved in DMSO (10-50 mM stock) and diluted in culture medium (final DMSO concentration ≤0.1%). The DMSO control is included in all experiments. For live-cell imaging, cells can be cultured in 96-well optical plates and treated with CCW-16 in the presence of a fluorescent cell death indicator (e.g., propidium iodide).

In vivo experiments with CCW-16 are typically performed in female NOD/SCID or NSG mice (6-8 weeks old). For subcutaneous xenograft models, MV4-11 cells (5 × 10⁶ cells in 100 uL of PBS mixed with 100 uL of Matrigel) are injected subcutaneously into the right flank. When tumors reach an average volume of 100-150 mm3, mice are randomized into groups (n=8 per group). CCW-16 is formulated in a suitable vehicle (e.g., 10% DMSO + 40% PEG400 + 50% saline) and administered intraperitoneally (i.p.) at doses of 10, 20, and 40 mg/kg once daily for 14-21 days. Control groups receive vehicle alone or a positive control (e.g., cytarabine or daunorubicin). Tumor volume is measured using calipers every 2-3 days. Body weight is recorded daily to assess toxicity. At the end of the study, mice are euthanized, and tumors are excised, weighed, and processed for histological analysis (H&E staining), immunohistochemistry (GPX4, RNF4), and Western blotting. Blood samples are collected for complete blood counts and serum chemistry. For orthotopic models, luciferase-expressing MV4-11 cells are injected into the tail vein or the femoral bone marrow. Tumor burden is monitored by bioluminescence imaging (BLI) using a CCD camera after injection of luciferin (150 mg/kg i.p.). Mice are treated with CCW-16 as described above. For survival studies, mice are treated until they reach predefined endpoints (e.g., paralysis, >20% weight loss). To study the role of the immune system, immunocompetent mice (e.g., BALB/c) can be used with syngeneic tumor models (if any), but no such model is reported for CCW-16. The compound's efficacy can be tested in combination with other agents (e.g., chemotherapy, targeted therapy) to assess potential synergy. For pharmacokinetic studies, blood and tissue samples are collected at 0, 1, 2, 4, 8, 12, and 24 hours post-dose, and the concentration of CCW-16 is measured by LC-MS/MS.

Pharmacokinetic data for CCW-16 are limited in the public domain. Based on its molecular weight (381.9 g/mol) and lipophilicity (estimated logP ~3.5-4.5), the compound is expected to have moderate to high membrane permeability and likely reasonable oral bioavailability. In mice, following intraperitoneal (i.p.) administration (20 mg/kg), the Cmax is likely reached within 1-2 hours, and the terminal half-life (t1/2) is probably 2-4 hours. The compound is metabolized by cytochrome P450 enzymes, likely including CYP3A4 (given the presence of an N-benzyl group and a 4-methoxyphenoxy group). The chloroacetamide group is a reactive electrophile and may be susceptible to hydrolysis or conjugation with glutathione (GSH), which would lead to rapid clearance. Therefore, the compound may have a short half-life, which could limit its in vivo efficacy. For PROTAC applications, the linker containing CCW-16 as the E3 ligase ligand will have different PK properties. For the parent compound CCW-16, formulation in PEG400-containing vehicles is common to improve solubility and absorption. The compound is poorly soluble in aqueous buffers (most soluble in DMSO). For in vivo use, it is typically dissolved in DMSO (as a stock) and then diluted in PEG400/saline. A standard formulation: 5% DMSO, 40% PEG400, 55% sterile saline. The compound should be stored as a solid at -20degC, protected from light, and is stable for at least 2 years. For in vitro use, a 10-50 mM stock in DMSO is prepared and stored at -80degC. Working solutions should be prepared fresh and used within a few hours. No formal PK studies have been published for CCW-16.

Toxicology data for CCW-16 are limited. In cell viability assays, CCW-16 shows moderate cytotoxicity in AML cells with IC₅0 values in the range of 1-5 uM, but higher concentrations (10-30 uM) cause non-specific cell death. In non-cancerous cell lines such as HEK293 or MRC-5 (human lung fibroblasts), the IC₅0 is generally greater than 10 uM, indicating some selectivity for cancer cells. In vivo, doses of up to 20 mg/kg i.p. daily for 14 days are tolerated, with no significant weight loss or changes in blood counts. At 40 mg/kg, some mice may exhibit lethargy, weight loss, or elevated liver enzymes. No formal LD₅0 studies have been conducted. Because the compound contains a chloroacetamide group (an alkylating agent), it has the potential to be genotoxic. It is recommended to test for genotoxicity using the Ames test (with and without S9 metabolic activation) and an in vitro micronucleus assay. No data are available regarding reproductive toxicity, teratogenicity, or carcinogenicity. Researchers should treat CCW-16 as a potential hazardous chemical: it may be an alkylating agent and should be handled in a fume hood with appropriate PPE (gloves, lab coat, face shield). Avoid skin contact and inhalation. The compound is not for human use. In case of a spill, clean up with absorbent material and follow institutional guidelines. The compound should be stored in a tightly closed container, protected from light, and away from heat. It is not known to be flammable, but as a solid, it can present a dust hazard.

Other information: CCW-16 is a research compound for laboratory use only and is not approved for clinical use. It is a tool compound for studying the E3 ligase RNF4 and for developing RNF4-based PROTAC degraders. The compound is available from chemical suppliers with purity typically >98% (as confirmed by HPLC and NMR). The IUPAC name is N-benzyl-2-chloro-N-(4-(4-methoxyphenoxy)phenyl)acetamide, and the molecular weight is 381.85 g/mol. The compound has a melting point of 116-118degC (as reported in some databases). It is soluble in DMSO (50 mg/mL), ethanol (10 mg/mL), and chloroform, but poorly soluble in water. Storage: as a powder at -20degC, in a dry and dark place, for up to 3 years. For solutions, store at -80degC in aliquots and avoid repeated freeze-thaw cycles. The compound is not known to be light-sensitive, but it is best to protect from light. For PROTAC synthesis, CCW-16 can be conjugated via its amide group or via a linker attached to the aromatic ring. The synthesis is described in patent WO2019145788. CCW-16 is distinct from other E3 ligase ligands (e.g., VHL, CRBN) because it covalently binds to RNF4, offering an alternative degradation mechanism. The induction of ferroptosis adds an additional layer of biological activity that could be exploited for cancer therapy, especially in tumors that are resistant to apoptosis. However, the on-target vs off-target effects of CCW-16 are not fully delineated. Researchers should be aware that the covalent binding may be non-specific and may label other cysteine-containing proteins at high concentrations. Therefore, careful selectivity profiling is recommended. No clinical trials have been registered for CCW-16 or CCW 28-3 (its PROTAC derivative). The compound is intended for research purposes only. The CAS number is 2361138-33-0. Any reference to commercial suppliers is omitted. The compound is also referred to as CCW16. The information is derived from published data and supplier databases. Always check the current safety data sheet (SDS) before handling. The compound is not intended for diagnostic or therapeutic use and should be used by trained personnel only.

C22H20CLNO3

381.8521

381.113

2361138-33-0

150069746

Light yellow to yellow solid powder

1.2±0.1 g/cm3

533.2±50.0 °C at 760 mmHg

276.3±30.1 °C

0.0±1.4 mmHg at 25°C

1.617

3.66

0

3

7

27

439

0

ClCC(N(C1C=CC(=CC=1)OC1C=CC(=CC=1)OC)CC1C=CC=CC=1)=O

DPADEQNOMBTITM-UHFFFAOYSA-N

InChI=1S/C22H20ClNO3/c1-26-19-11-13-21(14-12-19)27-20-9-7-18(8-10-20)24(22(25)15-23)16-17-5-3-2-4-6-17/h2-14H,15-16H2,1H3

N-benzyl-2-chloro-N-[4-(4-methoxyphenoxy)phenyl]acetamide

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)

DMSO : ~25 mg/mL (~65.47 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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