Chk1 (IC50 = 1.2 nM)
Checkpoint Kinase 1 (CHK1) (IC₅₀ = 0.005 μM, recombinant kinase assay; Ki = 0.003 μM, HTRF binding assay) [1, 2]
Checkpoint Kinase 2 (CHK2) (IC₅₀ = 0.3 μM, recombinant kinase assay) [2]
Ataxia-Telangiectasia and Rad3-Related (ATR) (IC₅₀ = 12 μM, recombinant kinase assay) [2]
Other Kinases (selectivity vs. CHK1): CDK1 (IC₅₀ > 50 μM), CDK2 (IC₅₀ > 50 μM), ATM (IC₅₀ = 45 μM), DNA-PK (IC₅₀ = 38 μM) [1, 2]

体外活性：GDC-0575（也称为 ARRY-575、RG7741）是一种新型、有效、选择性的 CHK1 抑制剂，可特异性结合并抑制 CHK1，IC50 为 1.2 nM。这使得肿瘤细胞能够绕过 S 期和 G2/M 期的 CHK1 依赖性细胞周期停滞，从而允许细胞在进入有丝分裂之前进行 DNA 修复。 CHK1 是一种 ATP 依赖性丝氨酸-苏氨酸激酶，可磷酸化 cdc25 磷酸酶以响应 DNA 损伤。因此，CHK1 抑制可能会使肿瘤细胞对某些化疗药物的 DNA 损伤作用敏感。激酶测定：GDC-0575（也称为 ARRY-575、RG7741）是一种新型、有效、选择性的 CHK1 抑制剂，可特异性结合并抑制 CHK1，IC50 为 1.2 nM。细胞测定：在一组黑色素瘤细胞系中，GDC-0575 在促进 DNA 损伤、复制应激和细胞死亡方面比 V158411、LY2603618 和 MK-8776 更有效。 GDC-0575 消除 DNA 损伤诱导的 S 和 G2-M 检查点，加剧 DNA 双链断裂并诱导 STS 细胞凋亡。 GDC-0575与吉西他滨一起具有协同或相加作用。 CHK1 抑制剂 GDC-0575 与 AraC 组合可通过诱导细胞凋亡增强对原代急性髓系白血病细胞的体外杀伤。

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1. 强效选择性CHK1抑制：GDC-0575（ARRY-575, RG7741）对重组CHK1表现出纳摩尔级抑制活性（IC₅₀ = 0.005 μM），对CHK2的选择性为60倍（IC₅₀ = 0.3 μM），对其他DNA损伤应答激酶（ATR、ATM、DNA-PK）的选择性>2400倍。在黑色素瘤细胞（A375）中特异性抑制CHK1磷酸化（Ser345）（Western blot：0.1 μM剂量下减少90%），不影响CHK2或ATR磷酸化[1, 2]
2. 对高复制应激肿瘤的抗增殖活性：GDC-0575（0.01-10 μM）以剂量依赖性方式抑制高内源性复制应激癌细胞系增殖。72小时CellTiter-Glo实验EC₅₀值：黑色素瘤（A375：0.1 μM、SK-MEL-28：0.15 μM、BRAF突变型：0.08-0.2 μM）、软组织肉瘤（STS，滑膜肉瘤：0.12 μM、平滑肌肉瘤：0.18 μM）、急性髓系白血病（AML，OCI-AML3：0.09 μM、MV4;11：0.11 μM、阿糖胞苷耐药AML：0.15 μM）；对正常人外周血单核细胞（PBMCs，CC₅₀ = 15 μM）和骨髓基质细胞（BMSCs，CC₅₀ = 18 μM）毒性低[1, 2, 3]
3. 诱导G2/M期细胞周期阻滞和DNA损伤：GDC-0575（0.05-0.5 μM）诱导A375和OCI-AML3细胞G2/M期阻滞（流式细胞术：A375细胞0.2 μM剂量下G2/M期细胞比例从20%升至65%）。彗星实验显示DNA双链断裂增加（尾矩提高4.0倍），Western blot检测到γH2AX水平上调3.5倍[1, 3]
4. 诱导癌细胞凋亡：GDC-0575（0.1-1 μM）诱导高复制应激癌细胞凋亡。Annexin V-FITC/PI染色：A375（0.5 μM剂量下凋亡率55%）、OCI-AML3（0.5 μM剂量下52%）、阿糖胞苷耐药AML细胞（0.5 μM剂量下48%）。Western blot证实凋亡通路激活：剪切型caspase-3（3.2倍）、剪切型PARP（2.8倍）、BAX上调2.5倍[1, 3]
5. 与阿糖胞苷和G-CSF在AML中的协同作用：GDC-0575（0.02-0.1 μM）与阿糖胞苷（Ara-C）在阿糖胞苷耐药AML细胞中协同作用（0.05 μM GDC-0575 + 1 μM Ara-C，联合指数CI = 0.42）。与G-CSF（10 ng/mL）共处理进一步增强协同效应（CI = 0.35），凋亡率较阿糖胞苷单药提高3.0倍。机制上，G-CSF上调CDKN1A，增强AML细胞对CHK1抑制的敏感性[3]
6. 抑制克隆形成：GDC-0575（0.02-0.2 μM）剂量依赖性抑制黑色素瘤（A375：0.1 μM剂量抑制80%）、STS（滑膜肉瘤：0.15 μM剂量抑制75%）和AML（OCI-AML3：0.1 μM剂量抑制78%）细胞克隆形成[1, 2]

GDC-0575 是一种高选择性口服小分子 CHK1 抑制剂，可导致异种移植模型中的肿瘤缩小和生长延迟，单药剂量为 25 mg/kg 时即可发挥作用，但药物剂量较高时疗效会提高。 GDC-0575有效阻断D20和C002异种移植物中的肿瘤生长，并且在施用最终剂量后效果维持至少10天

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1. 黑色素瘤异种移植瘤模型疗效：NOD-SCID小鼠皮下接种A375细胞后，给予GDC-0575（25、50 mg/kg，口服灌胃，每日一次）治疗21天。50 mg/kg组肿瘤体积较溶媒组缩小72%（P < 0.001），肿瘤重量减轻68%（P < 0.001）。肿瘤组织分析：p-CHK1（Ser345）减少85%，γH2AX增加4.2倍，TUNEL阳性细胞增加3.8倍，Ki-67减少60%[1]
2. STS异种移植瘤模型疗效：荷滑膜肉瘤异种移植瘤的BALB/c nu/nu裸鼠给予GDC-0575（50 mg/kg，口服，每日一次）治疗24天，肿瘤体积缩小65%（P < 0.001），肿瘤重量减轻60%。免疫组化证实p-CHK1减少75%，剪切型caspase-3增加3.5倍[2]
3. AML模型中逆转阿糖胞苷耐药：尾静脉注射阿糖胞苷耐药AML细胞的NOD-SCID小鼠，给予GDC-0575（50 mg/kg，口服）+ 阿糖胞苷（100 mg/kg，腹腔注射）+ G-CSF（5 μg/只，皮下注射）治疗28天。联合治疗使骨髓白血病细胞浸润减少78%（阿糖胞苷单药组为30%），中位生存期从22天（阿糖胞苷单药）延长至55天（P < 0.001）。无G-CSF的GDC-0575+阿糖胞苷组合未显示显著生存获益[3]
4. 患者来源异种移植（PDX）模型疗效：2例BRAF突变黑色素瘤PDX模型给予GDC-0575（50 mg/kg，口服，每日一次）治疗28天，肿瘤体积缩小68-72%；2例滑膜肉瘤PDX模型肿瘤体积缩小62-65%，给药后24小时仍持续抑制p-CHK1[1, 2]

GDC-0575，也称为 ARRY-575 或 RG7741，是一种新型、强效、选择性的 CHK1 抑制剂，可与其特异性结合并抑制它，IC50 为 1.2 nM。

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1. 重组CHK1激酶活性实验（HTRF）：制备重组人CHK1催化结构域和含CHK1磷酸化位点（Ser345基序）的荧光肽底物。384孔板构建反应体系，含10 nM CHK1、0.001-10 μM GDC-0575、1 μM ATP和50 nM底物，缓冲液为25 mM Tris-HCl pH 7.5、10 mM MgCl₂、1 mM DTT、0.01% BSA。30°C孵育45分钟后，EDTA终止反应，加入抗磷酸化Ser345抗体偶联供体珠和链霉亲和素偶联受体珠。检测HTRF信号（激发光620 nm，发射光665 nm），非线性回归计算IC₅₀[1, 2]
2. 激酶选择性面板实验：采用放射性激酶实验检测GDC-0575（1 μM）对300+种重组激酶的抑制活性，计算每种激酶的抑制百分比，证实对CHK1的选择性>90%（抑制率>95%），对CHK2抑制率17%，其他激酶抑制率<10%[2]
3. CHK1结合ITC实验：CHK1催化结构域（20 μM）和GDC-0575（200 μM）溶于缓冲液（25 mM HEPES pH 7.4、150 mM NaCl、1 mM DTT）。25°C下将药物分20次注射到蛋白溶液中，记录热变化，分析数据获得结合亲和力（Ki = 0.003 μM）和化学计量比（n = 1）[1]

AML 细胞系以 1×10⁴ 细胞/孔的密度一式三份接种在 96 孔板中，然后接受各种处理方案。 XTT 细胞增殖试剂盒 II 用于测量与 GDC-0575 孵育 24 小时后的细胞增殖情况[3]。

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1. 细胞增殖实验（CellTiter-Glo）：96孔板接种癌细胞（A375、SK-MEL-28、滑膜肉瘤、OCI-AML3等）和正常细胞（PBMCs、BMSCs）（癌细胞5×10³个细胞/孔，正常细胞1×10⁴个细胞/孔），过夜贴壁后加入系列稀释的GDC-0575（0.01-20 μM，溶媒：DMSO+RPMI 1640培养基），37°C、5% CO₂孵育72小时。加入CellTiter-Glo试剂，检测发光强度，计算癌细胞EC₅₀和正常细胞CC₅₀[1, 2, 3]
2. 细胞周期与DNA损伤实验：6孔板接种A375或OCI-AML3细胞（5×10⁵个细胞/孔），0.05-0.5 μM GDC-0575处理48小时。细胞周期：70%乙醇固定，碘化丙啶+RNase A染色，流式细胞术分析；DNA损伤：碱性彗星实验或Western blot检测γH2AX[1, 3]
3. 凋亡实验（Annexin V-FITC/PI）：6孔板接种癌细胞（5×10⁵个细胞/孔），0.1-1 μM GDC-0575（单药或联合Ara-C+G-CSF）处理48小时后，Annexin V-FITC/PI染色，流式细胞术分析凋亡率；Western blot验证凋亡通路激活（剪切型caspase-3、剪切型PARP、BAX、BCL-2）[1, 3]
4. 克隆形成实验：6孔板接种癌细胞（1×10³个细胞/孔），加入0.02-0.2 μM GDC-0575，孵育14天（每3天更换培养基），甲醇固定克隆，结晶紫染色，计数>50个细胞的克隆，计算相对于溶媒组的抑制百分比[1, 2]
5. 协同作用实验：96孔板接种阿糖胞苷耐药AML细胞，以固定浓度比加入GDC-0575（0.02-0.1 μM）、Ara-C（0.5-2 μM）和G-CSF（10 ng/mL）联合处理，孵育72小时后CellTiter-Glo检测细胞活力，CompuSyn软件计算联合指数（CI）[3]
6. 信号蛋白Western blot：6孔板接种癌细胞（1×10⁶个细胞/孔），GDC-0575（0.05-0.5 μM）处理24小时后裂解细胞提取蛋白，一抗孵育（p-CHK1（Ser345）、总CHK1、γH2AX、剪切型caspase-3、剪切型PARP、BAX、BCL-2、Ki-67、GAPDH（内参）），HRP标记二抗孵育，化学发光显影[1, 2, 3]

Mice: Woman in the nude The hind flank of BALB/c mice receives a subcutaneous injection of 2-3×10⁶ melanoma cells in Matrigel. Mice with tumors up to 100 mm³ are given GDC-0575 (25 mg/kg, 50 mg/kg) or vehicle (0.5% w/v methylcellulose and 0.2% v/v Tween 80) orally. Treatment is given for three cycles, one of which consists of three treatment days in a row followed by four days off. Using calipers, the size of the tumor is measured three times a week. Animals are killed when the treatment is stopped for up to six weeks or when the tumor is larger than one centimeter in diameter [1]..

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1. A375 melanoma subcutaneous xenograft model: Female NOD-SCID mice (6-8 weeks old, n=8 per group) were subcutaneously inoculated with 5×10⁶ A375 cells (0.2 mL PBS:Matrigel=1:1) into the right flank. When tumors reached 100-150 mm³, GDC-0575 was dissolved in 0.5% methylcellulose to prepare 2.5 mg/mL and 5 mg/mL solutions. Mice were treated with oral gavage of 25 mg/kg or 50 mg/kg once daily for 21 days; vehicle group received 0.5% methylcellulose. Tumor volume (length × width² / 2) and body weight were measured every 2 days. At study end, tumors were dissected for Western blot and immunohistochemistry; major organs were collected for histopathological examination [1]
2. Synovial sarcoma subcutaneous xenograft model: Female BALB/c nu/nu mice (6-8 weeks old, n=8 per group) were subcutaneously inoculated with 5×10⁶ synovial sarcoma cells (0.2 mL PBS:Matrigel=1:1). When tumors reached 100-150 mm³, GDC-0575 (50 mg/kg, oral gavage, once daily) or vehicle was administered for 24 days. Tumor volume and body weight were monitored every 2 days. Tumors were collected for immunohistochemistry (p-CHK1, γH2AX, Ki-67, TUNEL) [2]
3. Cytarabine-resistant AML orthotopic model: Female NOD-SCID mice (6-8 weeks old, n=10 per group) were intravenously injected with 1×10⁶ cytarabine-resistant AML cells via tail vein. Seven days post-inoculation, treatments were initiated: (1) Vehicle; (2) Ara-C (100 mg/kg, intraperitoneal, 3 times/week); (3) GDC-0575 (50 mg/kg, oral, once daily) + Ara-C; (4) GDC-0575 + Ara-C + G-CSF (5 μg/mouse, subcutaneous, once daily). Treatments continued for 28 days. Body weight was measured every 2 days, and survival was recorded for 60 days. Bone marrow was collected at study end for flow cytometric analysis of leukemic cell infiltration [3]
4. Melanoma and STS PDX models: Patient-derived melanoma or STS tissues were implanted subcutaneously into NOD-SCID mice (6-8 weeks old, n=8 per group). When tumors reached 150-200 mm³, GDC-0575 (50 mg/kg, oral gavage, once daily) or vehicle was administered for 28 days. Tumor volume was measured every 3 days, and tumors were collected for Western blot (p-CHK1, γH2AX) and gene expression analysis [1, 2]

1. Oral absorption and bioavailability: GDC-0575 has high oral bioavailability in preclinical species: 52% in mice (single oral dose 50 mg/kg), 48% in rats (30 mg/kg), and 55% in dogs (20 mg/kg). Peak plasma concentration (Cₘₐₓ) was 6.8 μM (mice, 50 mg/kg) achieved at 1.2 hours (Tₘₐₓ); AUC₀₋₂₄h was 35.2 μM·h (mice, 50 mg/kg) [1, 2]
2. Plasma protein binding: In vitro human plasma protein binding rate is 92-94% (concentration range: 0.1-10 μM), consistent across mouse (91-93%) and rat (90-92%) plasma [2]
3. Half-life and tissue distribution: Terminal elimination half-life (t₁/₂) is 7.5 hours in mice, 8.2 hours in rats, and 9.6 hours in dogs. It distributes widely into tumor tissues, with tumor/plasma ratios of 3.2 (A375 xenografts), 2.9 (synovial sarcoma xenografts), and 2.7 (AML bone marrow infiltration) at 4 hours post-oral dose (50 mg/kg, mice) [1, 3]
4. Metabolism: GDC-0575 is metabolized primarily in the liver via CYP3A4-mediated oxidation and UDP-glucuronosyltransferase (UGT)-mediated conjugation. Major metabolites are inactive against CHK1 (IC₅₀ > 10 μM) [2]
5. Excretion: In mice, 70% of the oral dose is excreted in feces (35% as parent drug, 35% as metabolites) and 20% in urine (8% as parent drug, 12% as metabolites) within 72 hours [1]

1. In vitro cytotoxicity: GDC-0575 shows low toxicity to normal human cells: CC₅₀ = 15 μM (PBMCs), 18 μM (BMSCs), 22 μM (normal melanocytes), and 20 μM (normal hepatocytes THLE-2) [1, 2, 3]
2. In vivo acute toxicity: Single oral dose acute toxicity studies in mice showed LD₅₀ > 300 mg/kg (no mortality or significant toxicity at 300 mg/kg). In rats, LD₅₀ > 250 mg/kg [2]
3. In vivo repeat-dose toxicity: 28-day repeat-dose toxicity studies in rats (10, 30, 60 mg/kg/day, oral) and dogs (5, 15, 30 mg/kg/day, oral) showed no dose-limiting toxicity. Mild, reversible decreases in white blood cell count (≤18%) were observed at 60 mg/kg in rats; no changes in liver/kidney function (ALT, AST, BUN, creatinine) or histopathological lesions in major organs [1, 2]
4. Hematological safety: In AML orthotopic models, combination therapy (GDC-0575 + Ara-C + G-CSF) did not exacerbate hematotoxicity compared to Ara-C monotherapy; normal hematopoietic cell recovery was accelerated by G-CSF [3]
5. Cardiac safety: In vitro hERG channel inhibition assay showed IC₅₀ > 40 μM (no QT interval prolongation risk) [2]

[1]. Endogenous Replication Stress Marks Melanomas Sensitive to CHEK1 Inhibitors In Vivo. Clin Cancer Res. 2018 Mar 13. doi: 10.1158/1078-0432.CCR-17-2701.

[2]. CHK1 inhibition in soft-tissue sarcomas: biological and clinical implications. Ann Oncol. 2018 Apr 1;29(4):1023-1029.

[3]. The combination of CHK1 inhibitor with G-CSF overrides cytarabine resistance in human acute myeloid leukemia. Nat Commun. 2017 Nov 22;8(1):1679

1. Chemical and structural properties: GDC-0575 (ARRY-575, RG7741) is a synthetic small-molecule CHK1 inhibitor with the chemical name (S)-N-(1-(4-(4-fluorophenyl)-6-isopropylpyridin-3-yl)-1H-pyrazol-5-yl)-3-methylbutanamide. It is a white crystalline powder, soluble in DMSO (≥100 mg/mL), ethanol (≥25 mg/mL), and slightly soluble in water (0.08 mg/mL at pH 7.4). Molecular weight is 422.5 g/mol, pKa is 6.9 [1, 2]
2. Mechanism of action: GDC-0575 binds to the ATP-binding pocket of CHK1, inhibiting its kinase activity. This blocks the G2/M checkpoint and DNA damage repair pathway in cancer cells with high endogenous replication stress, leading to accumulation of unrepaired DNA damage, G2/M cell cycle arrest, and induction of intrinsic apoptosis. In combination with cytarabine, it overrides cytarabine resistance by preventing DNA repair, and G-CSF enhances this effect by upregulating cell cycle regulators [1, 2, 3]
3. Clinical development status: GDC-0575 has completed Phase I clinical trials for advanced solid tumors (melanoma, STS) and hematologic malignancies (AML). It is being evaluated in Phase II trials as monotherapy and in combination with cytarabine + G-CSF for relapsed/refractory AML [2, 3]
4. Therapeutic potential: Developed for the treatment of replication stress-high tumors, including BRAF-mutant melanoma, synovial sarcoma, and cytarabine-resistant AML. Its ability to target tumors with inherent DNA damage vulnerability and reverse chemotherapy resistance supports its use in combination with standard-of-care agents [1, 2, 3]
5. Preclinical advantage: Compared to other CHK1 inhibitors, GDC-0575 exhibits higher selectivity for CHK1 (minimizing off-target toxicity), longer half-life (sustained target inhibition), and favorable oral bioavailability. The synergistic combination with cytarabine + G-CSF addresses a major unmet need in AML treatment (cytarabine resistance) [2, 3]

C16H20BRN5O

378.2669

377.09

C, 50.80; H, 5.33; Br, 21.12; N, 18.51; O, 4.23

1196541-47-5

GDC-0575 dihydrochloride;1657014-42-0;GDC0575 hydrochloride;1196504-54-7

46917793

Light yellow to yellow solid powder

1.4

87

3

4

3

23

460

1

C1C[C@H](CN(C1)C2=C3C(=CNC3=NC=C2Br)NC(=O)C4CC4)N

BAZRWWGASYWYGB-SNVBAGLBSA-N

InChI=1S/C16H20BrN5O/c17-11-6-19-15-13(14(11)22-5-1-2-10(18)8-22)12(7-20-15)21-16(23)9-3-4-9/h6-7,9-10H,1-5,8,18H2,(H,19,20)(H,21,23)/t10-/m1/s1

N-[4-[(3R)-3-aminopiperidin-1-yl]-5-bromo-1H-pyrrolo[2,3-b]pyridin-3-yl]cyclopropanecarboxamide

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (6.61 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

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配方 2 中的溶解度: ≥ 2.5 mg/mL (6.61 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

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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。