BTK; Ikaros (IKZF1); Aiolos (IKZF3)

- BTK降解：NX-2127在多种B细胞淋巴瘤细胞系（如TMD8、MEC1）和原代人B细胞中诱导剂量依赖性BTK降解，DC50值<5 nM。通过蛋白质印迹分析证实，24小时内BTK蛋白水平降低>80% [3]
- 类IMiD活性：该化合物通过降解原代人T细胞中的IKZF1和IKZF3表现出免疫调节作用，导致IL-2生成增加和T细胞活化，与来那度胺相当 [3]
- 细胞增殖抑制：NX-2127有效抑制BTK依赖性ABC-DLBCL细胞（TMD8）的增殖，72小时后EC50 < 15 nM。值得注意的是，其对BTK C481S突变细胞的活性（EC50 < 30 nM）显著优于伊布替尼（>1 μM）[3]
NX-2127 的 EC50 值 <30 nM，可抑制 BTK-C481S 突变 TMD8 细胞的增殖[1]。当暴露于 NX-2127 时，原代人类 T 细胞会产生更多的 IL-2 [1]。

- 肿瘤生长抑制：在小鼠异种移植模型（TMD8 WT和C481S突变）中，口服NX-2127导致剂量依赖性肿瘤生长抑制（TGI）。每日10 mg/kg剂量下，两种模型的TGI均超过80%，疗效优于伊布替尼 [3]
- 非人灵长类动物中的BTK降解：在食蟹猴中，每日一次口服给药（1–10 mg/kg）导致外周血单个核细胞（PBMCs）中BTK持续降解，水平至少24小时内抑制至<10%基线值 [3]
用 NX-2127（1 mg/kg；口服；每天一次，持续 14 天）治疗的食蟹猴显示出有效的 BTK 降解 [1]。口服 NX-2127 会导致血浆中的 BTK 在循环和脾 B 细胞中降解至基线水平的 10% 以下，且暴露量与剂量成比例[1]。在小鼠 WT TMD8 和 C481S 突变异种移植模型中，NX-2127 产生更强的肿瘤生长抑制 (TGI) [1]。

- BTK结合与降解实验：重组BTK蛋白与NX-2127和纯化的CRBN E3连接酶复合体孵育。通过SDS-PAGE和免疫印迹评估降解，显示剂量依赖性BTK泛素化和蛋白酶体降解。表面等离子体共振（SPR）证实BTK、NX-2127和CRBN的三元复合体形成，结合亲和力（KD）为0.8 nM [3]
- IKZF1/3降解实验：原代人T细胞用NX-2127处理，通过流式细胞术定量IKZF1/3水平。化合物在6小时内诱导两种蛋白快速降解，100 nM时效果最大 [3]

- B细胞中的BTK降解：从患者样本中分离的人CLL细胞用NX-2127（0.1–100 nM）处理。24小时后通过蛋白质印迹检测BTK蛋白水平，显示10 nM时降解>90%。降解与BTK突变状态无关（如C481S、L528W）[3]
- T细胞活化实验：原代人T细胞与NX-2127和抗CD3/CD28磁珠共培养。ELISA定量IL-2分泌，显示剂量依赖性增加（EC50 10 nM），与来那度胺相当 [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: Oral bioavailability in mice was ~60%, with Cmax achieved within 1–2 hours. In cynomolgus monkeys, absolute bioavailability was ~50%, supporting once-daily dosing [3]
- Metabolism: NX-2127 underwent hepatic metabolism primarily via cytochrome P450 enzymes (CYP3A4), with the major metabolite being the glucuronide conjugate. Less than 5% of the dose was excreted unchanged in urine [3]
- Half-life: Terminal half-life in mice and cynomolgus monkeys was ~12 hours and ~24 hours, respectively, allowing for sustained BTK degradation [3]
NX-2127 possesses potent in vivo target degradation activity across species. Upon iv administration, NX-2127 demonstrated low clearance (Tables 4 and 5). Oral dosing of NX-2127 in mice resulted in dose-dependent plasma exposure (Figure 3A), moderate oral bioavailability, and dose-dependent degradation of BTK in mouse blood (Figure 3B). Single, oral doses of 0.3, 3, 10, and 30 mg/kg of NX-2127 in mice resulted in reduction of BTK levels to 81%, 36%, 21%, and 12% of baseline in circulating B cells after 24 h. In addition to mice, we conducted in vivo PK–PD experiments in rat, dog, and cynomolgus monkey. NX-2127 had moderate clearance and low oral bioavailability in rat. Highlighting the power of the catalytic mechanism of targeted protein degradation (TPD), NX-2127 promoted potent BTK degradation in dog and cyno, despite relatively low bioavailability and high clearance (Table 5). Oral doses of 10 mg/kg in dog and cyno resulted in 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 (oral), indicating low acute toxicity. No dose-limiting toxicities were observed in cynomolgus monkeys at doses up to 10 mg/kg [3]
- Safety Profile: In phase 1 trials, NX-2127 was generally well-tolerated. Common adverse events included neutropenia, fatigue, and diarrhea, which were 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 acts as a bifunctional molecule, recruiting CRBN E3 ligase to degrade BTK and IMiD neosubstrates (IKZF1/3). This dual activity inhibits BCR signaling and enhances T-cell-mediated antitumor immunity [3]
- Clinical Development: In phase 1 trials (NCT04830137), NX-2127 showed promising 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 PRs), with responses durable for >12 months [2]
- Resistance Overcome: NX-2127 effectively degrades BTK mutants (C481S, L528W) that confer resistance to covalent BTK inhibitors, offering a mutation-agnostic therapeutic approach [3]
Bruton's tyrosine kinase (BTK), a member of the TEC family of kinases, is an essential effector of B-cell receptor (BCR) signaling. Chronic activation of BTK-mediated BCR signaling is a hallmark of many hematological malignancies, which makes it an attractive therapeutic target. Pharmacological inhibition of BTK enzymatic function is now a well-proven strategy for the treatment of patients with these malignancies. We report the discovery and characterization of NX-2127, a BTK degrader with concomitant immunomodulatory activity. By design, NX-2127 mediates the degradation of transcription factors IKZF1 and IKZF3 through molecular glue interactions with the cereblon E3 ubiquitin ligase complex. NX-2127 degrades common BTK resistance mutants, including BTKC481S. NX-2127 is orally bioavailable, exhibits in vivo degradation across species, and demonstrates efficacy in preclinical oncology models. NX-2127 has advanced into first-in-human clinical trials and achieves deep and sustained degradation of BTK following daily oral dosing at 100 mg.[1]

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Zelebrudomide is an orally bioavailable chimeric targeting molecule (CTM) and targeted degrader of Bruton's tyrosine kinase (BTK), with potential immunomodulatory drug (IMiD) and antineoplastic activities. Zelebrudomide is comprised of a cereblon (CRBN)-binding moiety conjugated, via a linker, to a BTK-binding moiety. Upon administration, zelebrudomide targets and binds to BTK with its BTK-targeting moiety. Upon binding, the CRBN-binding moiety recruits CRBN, a component of the CRL4-CRBN E3 ubiquitin ligase complex. This catalyzes ubiquitination and proteasome-mediated degradation of BTK, and prevents the activation of the B-cell antigen receptor (BCR) signaling pathway. This prevents both B-cell activation and BTK-mediated activation of downstream survival pathways. This leads to an inhibition of the growth of malignant B-cells that overexpress BTK. In addition, zelebrudomide catalyzes the degradation of CRBN neosubstrates Aiolos (IKZF3) and Ikaros (IKZF1), two transcription factors regulating T-cell function. This modulates the activity of the immune system and increases the activation of T-lymphocytes, thereby increasing T-cell-mediated anti-tumor effects. BTK, a member of the src-related BTK/Tec family of cytoplasmic tyrosine kinases, is overexpressed in B-cell malignancies; it plays an important role in B-lymphocyte development, activation, signaling, proliferation and survival. CRBN, the substrate recognition component of the CRL4-CRBN E3 ubiquitin ligase complex, plays a key role in the ubiquitination of certain proteins. Compared to BTK inhibitors, zelebrudomide may overcome tumor resistance associated with 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

174

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

注意: 如下所列的是一些常用的体内动物实验溶解配方，主要用于溶解难溶或不溶于水的产品（水溶度<1 mg/mL）。 建议您先取少量样品进行尝试，如该配方可行，再根据实验需求增加样品量。

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注射用配方1: DMSO : Tween 80： Saline = 10 : 5 : 85 (如： 100 μL DMSO → 50 μL Tween 80 → 850 μL Saline)
*生理盐水/Saline的制备：将0.9g氯化钠/NaCl溶解在100 mL ddH ₂ O中，得到澄清溶液。
注射用配方 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL DMSO → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)
注射用配方 3: DMSO : Corn oil = 10 : 90 (如： 100 μL DMSO → 900 μL Corn oil)
示例: 以注射用配方 3 (DMSO : Corn oil = 10 : 90) 为例说明, 如果要配制 1 mL 2.5 mg/mL的工作液, 您可以取 100 μL 25 mg/mL 澄清的 DMSO 储备液，加到 900 μL Corn oil/玉米油中, 混合均匀。

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注射用配方 4: DMSO : 20% SBE-β-CD in Saline = 10 : 90 [如：100 μL DMSO → 900 μL (20% SBE-β-CD in Saline)]
*20% SBE-β-CD in Saline的制备（4°C，储存1周）：将2g SBE-β-CD (磺丁基-β-环糊精) 溶解于10mL生理盐水中，得到澄清溶液。
注射用配方 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (如： 500 μL 2-Hydroxypropyl-β-cyclodextrin (羟丙基环胡精)→ 500 μL Saline)
注射用配方 6: DMSO : PEG300 : Castor oil : Saline = 5 : 10 : 20 : 65 (如： 50 μL DMSO → 100 μL PEG300 → 200 μL Castor oil → 650 μL Saline)
注射用配方 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (如： 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
注射用配方 8: 溶解于Cremophor/Ethanol (50 : 50), 然后用生理盐水稀释。
注射用配方 9: EtOH : Corn oil = 10 : 90 (如： 100 μL EtOH → 900 μL Corn oil)
注射用配方 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (如： 100 μL EtOH → 400 μL PEG300 → 50 μL Tween 80 → 450 μL Saline)

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口服配方 1: 悬浮于0.5% CMC Na (羧甲基纤维素钠)
口服配方 2: 悬浮于0.5% Carboxymethyl cellulose (羧甲基纤维素)
示例: 以口服配方 1 (悬浮于 0.5% CMC Na)为例说明, 如果要配制 100 mL 2.5 mg/mL 的工作液, 您可以先取0.5g CMC Na并将其溶解于100mL ddH2O中，得到0.5%CMC-Na澄清溶液；然后将250 mg待测化合物加到100 mL前述 0.5%CMC Na溶液中，得到悬浮液。

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口服配方 3: 溶解于 PEG400 (聚乙二醇400)
口服配方 4: 悬浮于0.2% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 5: 溶解于0.25% Tween 80 and 0.5% Carboxymethyl cellulose (羧甲基纤维素)
口服配方 6: 做成粉末与食物混合

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注意: 以上为较为常见方法，仅供参考， InvivoChem并未独立验证这些配方的准确性。具体溶剂的选择首先应参照文献已报道溶解方法、配方或剂型，对于某些尚未有文献报道溶解方法的化合物，需通过前期实验来确定（建议先取少量样品进行尝试），包括产品的溶解情况、梯度设置、动物的耐受性等。

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请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。