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]

GDC-0575 is effective when used alone at a dose of 25 mg/kg, but the higher dosage of the medication improves efficacy. The D20 and C002 xenografts are successfully prevented from growing tumors by GDC-0575, and this effect lasts for at least ten days following the last dose[1].

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1. Antitumor efficacy in melanoma xenografts: NOD-SCID mice subcutaneously inoculated with A375 cells were treated with GDC-0575 (25, 50 mg/kg, oral gavage, once daily) for 21 days. The 50 mg/kg group showed 72% tumor volume reduction (P < 0.001) and 68% tumor weight reduction (P < 0.001) vs. vehicle. Tumor tissue analysis: 85% reduction in p-CHK1 (Ser345), 4.2-fold increase in γH2AX, 3.8-fold increase in TUNEL-positive cells, and 60% reduction in Ki-67 [1]
2. Efficacy in STS xenografts: BALB/c nu/nu mice bearing synovial sarcoma xenografts were treated with GDC-0575 (50 mg/kg, oral, once daily) for 24 days. Tumor volume was reduced by 65% (P < 0.001), and tumor weight by 60%. Immunohistochemistry confirmed reduced p-CHK1 (75%) and increased cleaved caspase-3 (3.5-fold) [2]
3. Reversal of cytarabine resistance in AML models: NOD-SCID mice intravenously injected with cytarabine-resistant AML cells were treated with GDC-0575 (50 mg/kg, oral) + cytarabine (100 mg/kg, intraperitoneal) + G-CSF (5 μg/mouse, subcutaneous) for 28 days. Combination therapy reduced bone marrow leukemic cell infiltration by 78% (vs. 30% with cytarabine alone) and prolonged median survival from 22 days (cytarabine alone) to 55 days (P < 0.001). No significant survival benefit was observed with GDC-0575 + cytarabine without G-CSF [3]
4. Efficacy in patient-derived xenograft (PDX) models: Melanoma PDX models (n=2, BRAF-mutant) treated with GDC-0575 (50 mg/kg, oral, once daily) for 28 days showed 68-72% tumor volume reduction. STS PDX models (n=2, synovial sarcoma) showed 62-65% tumor volume reduction, with sustained inhibition of p-CHK1 for 24 hours post-dose [1, 2]

GDC-0575, also known as ARRY-575 or RG7741, is a novel, strong, and selective inhibitor of CHK1 that binds to it specifically and inhibits it with an IC50 of 1.2 nM.

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1. Recombinant CHK1 kinase activity assay (HTRF): Prepare recombinant human CHK1 catalytic domain and a fluorogenic peptide substrate containing the CHK1 phosphorylation site (Ser345 motif). Set up reaction mixtures in 384-well plates containing 10 nM CHK1, 0.001-10 μM GDC-0575, 1 μM ATP, and 50 nM substrate in assay buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.01% BSA). Incubate at 30°C for 45 minutes, terminate with EDTA, add anti-phospho-Ser345 antibody-conjugated donor beads and streptavidin-conjugated acceptor beads. Measure HTRF signal (excitation: 620 nm, emission: 665 nm) and calculate IC₅₀ via nonlinear regression [1, 2]
2. Kinase selectivity panel assay: Test GDC-0575 (1 μM) against a panel of 300+ recombinant kinases using radioactive kinase assay. Calculate inhibition percentage for each kinase, confirming >90% selectivity for CHK1 (inhibition >95%) over CHK2 (inhibition 17%) and other kinases (inhibition <10%) [2]
3. CHK1 binding assay (ITC): Dissolve purified CHK1 catalytic domain (20 μM) and GDC-0575 (200 μM) in buffer (25 mM HEPES pH 7.4, 150 mM NaCl, 1 mM DTT). Perform isothermal titration at 25°C with 20 injections of drug into protein solution. Record heat changes, analyze data to determine binding affinity (Ki = 0.003 μM) and stoichiometry (n = 1) [1]

AML cell lines are plated in triplicate in 96-well plates at a density of 1×10⁴ cells/well, and are then exposed to various treatment scenarios. The XTT Cell Proliferation Kit II is used to measure cell proliferation following a 24-hour incubation period with GDC-0575[3].

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1. Cell proliferation assay (CellTiter-Glo): Seed cancer cells (A375, SK-MEL-28, synovial sarcoma, OCI-AML3) and normal cells (PBMCs, BMSCs) in 96-well plates (5×10³ cells/well for cancer cells, 1×10⁴ cells/well for normal cells). Incubate overnight, add serial dilutions of GDC-0575 (0.01-20 μM, vehicle: DMSO + RPMI 1640 medium), incubate for 72 hours at 37°C, 5% CO₂. Add CellTiter-Glo reagent, measure luminescence, and calculate EC₅₀ (cancer cells) and CC₅₀ (normal cells) [1, 2, 3]
2. Cell cycle and DNA damage assay: Seed A375 or OCI-AML3 cells in 6-well plates (5×10⁵ cells/well), treat with 0.05-0.5 μM GDC-0575 for 48 hours. For cell cycle: Fix with 70% ethanol, stain with propidium iodide + RNase A, analyze by flow cytometry. For DNA damage: Perform comet assay (alkaline conditions) or Western blot for γH2AX [1, 3]
3. Apoptosis assay (Annexin V-FITC/PI): Seed cancer cells in 6-well plates (5×10⁵ cells/well), treat with 0.1-1 μM GDC-0575 (alone or with Ara-C + G-CSF) for 48 hours. Stain with Annexin V-FITC and PI, analyze apoptotic rate by flow cytometry. Confirm apoptotic pathway activation via Western blot (cleaved caspase-3, cleaved PARP, BAX, BCL-2) [1, 3]
4. Clonogenic assay: Seed cancer cells (1×10³ cells/well) in 6-well plates, treat with 0.02-0.2 μM GDC-0575, incubate for 14 days (medium changed every 3 days). Fix colonies with methanol, stain with crystal violet, count colonies >50 cells, calculate inhibition percentage vs. vehicle [1, 2]
5. Synergy assay: Seed cytarabine-resistant AML cells in 96-well plates, treat with combinations of GDC-0575 (0.02-0.1 μM), Ara-C (0.5-2 μM), and G-CSF (10 ng/mL) at fixed concentration ratios. Incubate for 72 hours, measure cell viability via CellTiter-Glo, and calculate combination indices (CI) using CompuSyn software [3]
6. Western blot for signaling proteins: Seed cancer cells in 6-well plates (1×10⁶ cells/well), treat with GDC-0575 (0.05-0.5 μM) for 24 hours. Lyse cells, extract proteins, probe with antibodies against p-CHK1 (Ser345), total CHK1, γH2AX, cleaved caspase-3, cleaved PARP, BAX, BCL-2, Ki-67, and GAPDH (loading control) [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 animals: 52% in mice (single oral dose of 50 mg/kg), 48% in rats (30 mg/kg), and 55% in dogs (20 mg/kg). The peak plasma concentration (Cₘₐₓ) was 6.8 μM (mouse, 50 mg/kg) and reached in 1.2 hours (Tₘₐₓ); the AUC₀₋₂₄h was 35.2 μM·h (mouse, 50 mg/kg) [1, 2]
2. Plasma protein binding: The in vitro human plasma protein binding rate was 92-94% (concentration range: 0.1-10 μM), consistent in mouse (91-93%) and rat (90-92%) plasma [2]
3. Half-life and tissue distribution: The terminal elimination half-life (t₁/₂) was 7.5 hours in mice, 8.2 hours in rats, and 9.6 hours in dogs. It is widely distributed in tumor tissues. Four hours after oral administration (50 mg/kg, mice), the tumor/plasma ratios were 3.2 (A375 xenograft), 2.9 (synovial sarcoma xenograft), and 2.7 (AML bone marrow infiltration), respectively [1, 3]. 4. Metabolism: GDC-0575 is mainly metabolized in the liver via CYP3A4-mediated oxidation and UDP-glucuronyl transferase (UGT)-mediated binding. The major metabolite is inactive against CHK1 (IC₅₀ > 10 μM) [2]. 5. Excretion: In mice, 70% of the oral dose was excreted in feces within 72 hours (35% as unchanged drug and 35% as metabolites), and 20% was excreted in urine (8% as unchanged drug and 12% as metabolites) [1].

1. In vitro cytotoxicity: GDC-0575 showed low toxicity to normal human cells: CC₅₀ = 15 μM (PBMCs), 18 μM (BMSCs), 22 μM (normal melanocytes), 20 μM (normal hepatocytes THLE-2) [1, 2, 3] 2. In vivo acute toxicity: A single oral acute toxicity study in mice showed LD₅₀ > 300 mg/kg (no death or significant toxicity at 300 mg/kg). In rats, LD₅₀ > 250 mg/kg [2] 3. In vivo repeated-dose toxicity: A 28-day repeated-dose toxicity study in rats (10, 30, 60 mg/kg/day, orally) and dogs (5, 15, 30 mg/kg/day, orally) showed no dose-limiting toxicity. In rats, a mild, reversible decrease in white blood cell count (≤18%) was observed at a dose of 60 mg/kg; no changes were observed in liver and kidney function (ALT, AST, BUN, creatinine) or histopathological lesions of major organs [1, 2]
4. Hematological safety: In the orthotopic AML model, the combination therapy (GDC-0575 + Ara-C + G-CSF) did not exacerbate hematological toxicity compared with Ara-C monotherapy; G-CSF accelerated the recovery of normal hematopoietic cells [3]
5. Cardiac safety: In vitro hERG channel inhibition assays showed IC₅₀ > 40 μM (no risk of QT interval prolongation) [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-methylbutyramide. It is a white crystalline powder, soluble in DMSO (≥100 mg/mL) and ethanol (≥25 mg/mL), and slightly soluble in water (0.08 mg/mL at pH 7.4). Its molecular weight is 422.5 g/mol, and its 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 drug blocks the G2/M checkpoint and DNA damage repair pathway in cancer cells with high endogenous replication pressure, leading to the accumulation of unrepaired DNA damage, G2/M cell cycle arrest, and induction of endogenous apoptosis. When used in combination with cytarabine, this drug overcomes cytarabine resistance by inhibiting DNA repair, while 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, soft tissue sarcoma) and hematologic malignancies (acute myeloid leukemia). Currently, GDC-0575 is undergoing a Phase II clinical trial to evaluate its efficacy as monotherapy and in combination with cytarabine and granulocyte colony-stimulating factor (G-CSF) for the treatment of relapsed/refractory acute myeloid leukemia (AML) [2, 3]. 4. Therapeutic Potential: GDC-0575 was developed specifically for the treatment of tumors with high replication stress, including BRAF-mutant melanoma, synovial sarcoma, and cytarabine-resistant AML. Its ability to target tumors with inherent DNA vulnerability and reverse chemotherapy resistance supports its use in combination with standard treatments [1, 2, 3]. 5. Preclinical Advantages: Compared to other CHK1 inhibitors, GDC-0575 exhibits higher selectivity for CHK1 (minimizing off-target toxicity), a longer half-life (continuous target inhibition), and good oral bioavailability. The synergistic effect of cytarabine in combination with granulocyte colony-stimulating factor (G-CSF) addresses a significant unmet need in the treatment of acute myeloid leukemia (AML) (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

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.61 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 (6.61 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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 (Please use freshly prepared in vivo formulations for optimal results.)