BTK; Ikaros (IKZF1); Aiolos (IKZF3)

- BTK Degradation: NX-2127 induced dose-dependent BTK degradation in multiple B-cell lymphoma cell lines (e.g., TMD8, MEC1) and primary human B cells, with DC50 values < 5 nM. Degradation was confirmed via Western blot analysis, showing >80% reduction in BTK protein levels within 24 hours [3]
- IMiD-like Activity: The compound demonstrated immunomodulatory effects by degrading IKZF1 and IKZF3 in primary human T cells, leading to increased IL-2 production and T-cell activation, comparable to lenalidomide [3]
- Cell Proliferation Inhibition: NX-2127 potently inhibited proliferation of BTK-dependent ABC-DLBCL cells (TMD8) with EC50 < 15 nM after 72 hours. Notably, it showed superior activity against BTK C481S mutant cells (EC50 < 30 nM) compared to ibrutinib (>1 μM) [3]
With an EC50 value of <30 nM, NX-2127 inhibits the proliferation of BTK-C481S mutant TMD8 cells[1]. Primary human T cells produce more IL-2 when exposed to NX-2127 [1].

- Tumor Growth Inhibition: Oral administration of NX-2127 in mouse xenograft models (TMD8 WT and C481S mutant) resulted in dose-dependent tumor growth inhibition (TGI). At 10 mg/kg daily, TGI exceeded 80% in both models, outperforming ibrutinib [3]
- BTK Degradation in Non-Human Primates: In cynomolgus monkeys, once-daily oral dosing (1–10 mg/kg) led to sustained BTK degradation in peripheral blood mononuclear cells (PBMCs), with levels suppressed to <10% of baseline for at least 24 hours [3]
Cynomolgus monkeys treated with NX-2127 (1 mg/kg; oral; once daily for 14 days) showed effective BTK degradation [1]. Oral dosing of NX-2127 causes BTK in plasma to degrade to less than 10% of baseline levels in circulating and splenic B cells, with exposure occurring in a dose-proportionate manner [1]. In mouse WT TMD8 and C481S mutant xenograft models, NX-2127 results in greater tumor growth inhibition (TGI) [1].

- BTK Binding and Degradation Assay: Recombinant BTK protein was incubated with NX-2127 and purified CRBN E3 ligase complex. Degradation was assessed via SDS-PAGE and immunoblotting, showing dose-dependent BTK ubiquitination and proteasomal degradation. The ternary complex formation between BTK, NX-2127, and CRBN was confirmed using surface plasmon resonance (SPR), with a binding affinity (KD) of 0.8 nM [3]
- IKZF1/3 Degradation Assay: Primary human T cells were treated with NX-2127, and IKZF1/3 levels were quantified by flow cytometry. The compound induced rapid degradation of both proteins, with maximal effect at 100 nM after 6 hours [3]

- BTK Degradation in B Cells: Human CLL cells isolated from patient samples were treated with NX-2127 (0.1–100 nM). BTK protein levels were measured by Western blot after 24 hours, demonstrating >90% degradation at 10 nM. Degradation was independent of BTK mutational status (e.g., C481S, L528W) [3]
- T-Cell Activation Assay: Primary human T cells were co-cultured with NX-2127 and anti-CD3/CD28 beads. IL-2 secretion was quantified by ELISA, showing a dose-dependent increase (EC50 10 nM) comparable to lenalidomide [3]

- Mouse Xenograft Model: NX-2127 was formulated as a suspension in 0.5% methylcellulose and administered orally (10–30 mg/kg daily) to NSG mice bearing TMD8 tumors. Tumor volume was measured twice weekly, with significant inhibition observed at all doses. Pharmacokinetic analysis revealed peak plasma concentrations (Cmax) at 1–2 hours post-dose, supporting once-daily dosing [3]
- Cynomolgus Monkey Study: Animals received oral doses of NX-2127 (1–10 mg/kg) for 14 days. Blood samples were collected at predefined time points to assess BTK degradation in PBMCs and plasma drug concentrations. The compound exhibited linear pharmacokinetics with a terminal half-life of ~24 hours [3]

Absorption: The oral bioavailability in mice is approximately 60%, with peak plasma concentration (Cmax) reached within 1-2 hours. The absolute bioavailability in cynomolgus monkeys is approximately 50%, supporting once-daily administration [3]
- Metabolism: NX-2127 is primarily metabolized in the liver via cytochrome P450 enzyme (CYP3A4), with the main metabolite being glucuronide conjugates. Less than 5% of the dose is excreted unchanged in the urine [3]
- Half-life: The terminal half-life in mice and cynomolgus monkeys is approximately 12 hours and 24 hours, respectively, enabling sustained BTK degradation [3]
NX-2127 exhibits potent in vivo target degradation activity in various species. After intravenous administration, the clearance rate of NX-2127 is low (Tables 4 and 5). Following oral administration of NX-2127 to mice, plasma exposure was dose-dependent (Figure 3A), oral bioavailability was moderate, and BTK degradation in mouse blood was also dose-dependent (Figure 3B). After single oral administration of 0.3, 3, 10, and 30 mg/kg of NX-2127 to mice, BTK levels in circulating B cells decreased to 81%, 36%, 21%, and 12% of baseline, respectively, 24 hours later. In addition to mice, we also conducted in vivo pharmacokinetic-pharmacodynamic (PK-PD) experiments in rats, dogs, and cynomolgus monkeys. NX-2127 showed moderate clearance and low oral bioavailability in rats. Although NX-2127 exhibited relatively low bioavailability and high clearance in dogs and cynomolgus monkeys (Table 5), it still effectively promoted BTK degradation, highlighting the powerful role of the targeted protein degradation (TPD) catalytic mechanism. Oral administration of 10 mg/kg to dogs and cynomolgus monkeys resulted in a reduction of BTK levels to 17% and 9% of baseline BTK levels, respectively. [3]

Acute toxicity: The median lethal dose (LD50) in mice was >2000 mg/kg (orally), indicating low acute toxicity. No dose-limiting toxicity was observed in cynomolgus monkeys at doses up to 10 mg/kg [3] - Safety: NX-2127 was generally well tolerated in the Phase I clinical trial. Common adverse events included neutropenia, fatigue, and diarrhea, all of grade 1-2 and reversible. No significant QT interval prolongation or cardiovascular toxicity was reported [2]

[1]. Nx-2127, a degrader of BTK and IMiD neosubstrates, for the treatment of B-cell malignancies. Blood, 2020, 136: 34.

[2]. A first-in-human phase 1 trial of NX-2127, a first-in-class oral BTK degrader with IMiD-like activity, in patients with relapsed and refractory B-cell malignancies. 2022.

[3].Discovery and Preclinical Pharmacology of NX-2127, an Orally Bioavailable Degrader of Bruton's Tyrosine Kinase with Immunomodulatory Activity for the Treatment of Patients with B Cell Malignancies. J Med Chem. 2024 Feb 22;67(4):2321-2336.

Mechanism of action: NX-2127 is a bifunctional molecule that recruits CRBN E3 ligases to degrade BTK and IMiD novel substrates (IKZF1/3). This dual activity can inhibit BCR signaling and enhance T cell-mediated anti-tumor immunity [3]
- Clinical development: In a phase 1 clinical trial (NCT04830137), NX-2127 showed good efficacy in relapsed/refractory B-cell malignancies, including chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). In 27 evaluable CLL patients, the overall response rate (ORR) was 40.7% (11 partial responses), with a duration of response exceeding 12 months [2]
- Overcoming resistance: NX-2127 can effectively degrade BTK mutants (C481S, L528W) that confer resistance to covalent BTK inhibitors, thus providing a treatment option independent of mutation type [3]
Bruton's tyrosine kinase (BTK) is a member of the TEC kinase family and an important effector molecule in B cell receptor (BCR) signaling. Chronic activation of BTK-mediated BCR signaling is a hallmark of many hematologic malignancies, making it a highly attractive therapeutic target. Currently, pharmacological inhibition of BTK enzyme activity has become a well-established strategy for treating patients with these malignancies. We report the discovery and characterization of NX-2127, an immunomodulatory BTK degrader. NX-2127 is designed to mediate the degradation of transcription factors IKZF1 and IKZF3 through molecular glue interaction with the cereblon E3 ubiquitin ligase complex. NX-2127 can degrade common BTK resistance mutants, including BTKC481S. NX-2127 has oral bioavailability, has shown in vivo degradation activity in different species, and has shown efficacy in preclinical tumor models. NX-2127 has entered the first human clinical trial phase, and 100 mg of oral administration per day can achieve deep and sustained degradation of BTK. [1]

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Zelebrudomide is a chimeric targeting molecule (CTM) with high oral bioavailability that targets and degrades Bruton's tyrosine kinase (BTK) and has potential immunomodulatory drug (IMiD) and antitumor activity. Zelebrudomide consists of a cereblon (CRBN) binding moiety linked to the BTK binding moiety via a linker. Following administration, zelebrudomide targets and binds to BTK via its BTK-targeting moiety. Upon binding, the CRBN-binding moiety recruits CRBN, a component of the CRL4-CRBN E3 ubiquitin ligase complex. The drug catalyzes the ubiquitination and proteasome-mediated degradation of BTK and inhibits activation of the B-cell antigen receptor (BCR) signaling pathway. This inhibits both B-cell activation and the activation of BTK-mediated downstream survival pathways. This results in the suppression of the growth of BTK-overexpressing malignant B cells. Furthermore, zelebrudomide also catalyzes the degradation of novel CRBN substrates Aiolos (IKZF3) and Ikaros (IKZF1), two transcription factors that regulate T-cell function. This modulates immune system activity and enhances T-lymphocyte activation, thereby enhancing T-cell-mediated antitumor activity. BTK is a member of the src-associated BTK/Tec family of cytoplasmic tyrosine kinases and is overexpressed in B-cell malignancies. It plays a crucial role in the development, activation, signal transduction, proliferation, and survival of B lymphocytes. CRBN is a substrate recognition component of the CRL4-CRBN E3 ubiquitin ligase complex and plays a key role in the ubiquitination of certain proteins. Compared to BTK inhibitors, zelebrutinib may overcome tumor resistance caused by BTK inhibitor-induced resistance mutations.

C39H45N9O5

719.83

719.354

C, 65.07; H, 6.30; N, 17.51; O, 11.11

3024312-52-2

2416131-46-7

167282486

Light yellow to yellow solid powder

1.371±0.06 g/cm3(Temp: 20 °C; Press: 760 Torr)(predicted)

949.0±65.0 °C(predicted)

3.7

3

11

9

53

1380

1

C1CCN(CC1)C2=CN=C(C(=N2)NC3=CC=C(C=C3)C4CCN(CC4)C[C@H]5CCN(C5)C6=CC7=C(C=C6)C(=O)N(C7=O)C8CCC(=O)NC8=O)C(=O)N

XLWJWCMQMBVNSG-LZDHLTRGSA-N

InChI=1S/C39H45N9O5/c40-35(50)34-36(43-32(21-41-34)46-15-2-1-3-16-46)42-27-6-4-25(5-7-27)26-13-17-45(18-14-26)22-24-12-19-47(23-24)28-8-9-29-30(20-28)39(53)48(38(29)52)31-10-11-33(49)44-37(31)51/h4-9,20-21,24,26,31H,1-3,10-19,22-23H2,(H2,40,50)(H,42,43)(H,44,49,51)/t24-,31?/m1/s1

3-[4-[1-[[(3R)-1-[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindol-5-yl]pyrrolidin-3-yl]methyl]piperidin-4-yl]anilino]-5-piperidin-1-ylpyrazine-2-carboxamide

(R)-NX-2127; Zelebrudomide (USAN); (R)-NX-2127; CHEMBL5435454; 3024312-52-2; Zelebrudomide (USAN); ZELEBRUDOMIDE [USAN]; Zelebrudomide; 3-((4-(1-(((3R)-1-(2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)pyrrolidin-3-yl)methyl)piperidin-4-yl)phenyl)amino)-5-(piperidin-1-yl)pyrazine-2-carboxamide; zelebrudomide [INN]; ZELEBRUDOMIDE [USAN]; Zelebrudomide; 3024312-52-2; NX2127; C9V5FHZ2L3; SCHEMBL24833161;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)