Rac1 (Rac1 GTPase). ZINC69391 is a Rac1-GEF interaction inhibitor that reduces Rac1 activation levels. [1]

ZINC69391 has an IC50 ranging from 41 to 54 μM, which inhibits the development of U937, HL-60, KG1A, and Jurkat cells[1]. The enzymatic activity of caspase 3 is enhanced in a concentration-dependent manner by ZINC69391 (50-100 μM; 24 hours) [1]. ZINC69391 (0-125 μM; 72h) inhibits human glioma cell growth [2]. ZINC69391 causes cell cycle arrest at 50–100 μM for 48 hours [2]. Human acute leukemia cells undergo apoptosis in response to ZINC69391 (50 μM; 24 hours) [1].

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ZINC69391 inhibited the proliferation of human acute leukemia cell lines U937, HL-60, KG1A, and Jurkat in a concentration-dependent manner after 48 hours of treatment, with IC50 values of 43.4 μM (95% CI: 38-49), 41.7 μM (95% CI: 37.4-46.6), 54.1 μM (95% CI: 41-71), and 41 μM (95% CI: 29.6-58.5), respectively. [1]
In HL-60 and KG1A cells, treatment with 50 μM ZINC69391 for 24 hours significantly increased the G2/M cell cycle subpopulation. A similar trend was observed in U937 and Jurkat cells. [1]
Treatment with 50 μM ZINC69391 for 24 hours significantly increased apoptosis (Annexin V positive) in HL-60, U937, and KG1A cell lines, while no significant change was observed in Jurkat cells. No necrosis was induced. [1]
ZINC69391 treatment (50 μM and 100 μM) increased caspase 3 activity in a concentration- and time-dependent manner, as measured by colorimetric assay, and increased cleaved caspase 3 protein levels in HL-60, U937, KG1A, and Jurkat cells, as shown by Western blot. [1]
In HL-60 cells, treatment with 50 μM ZINC69391 for 12 hours significantly decreased Mcl-1 mRNA levels, while Bcl-2 and Bcl-xL mRNA levels were not significantly changed. In Jurkat cells, mRNA levels of Bcl-2, Bcl-xL, and Mcl-1 were all increased. [1]
ZINC69391 treatment induced a shift in Bcl-2 protein mobility on Western blot in HL-60, KG1A, and Jurkat cells after 12 hours, consistent with Bcl-2 phosphorylation. [1]
In U937, HL-60, and KG1A cells, ZINC69391 (50 μM) induced a time-dependent loss of mitochondrial membrane potential (increased DiOC6-negative cells), with a maximum effect at 4 hours post-treatment. A modest, non-significant effect was observed in Jurkat cells. [1]
ZINC69391 induced time-dependent activation of caspase 9 (initiator caspase for the intrinsic pathway) in HL-60, U937, and KG1A cells, with a significant increase around 6 hours post-treatment. Caspase 8 activation was observed at later time points. [1]
In HL-60 cells, ZINC69391 (50 μM, 12 hours) significantly decreased IL-8 mRNA levels, while in Jurkat cells, IL-8 mRNA levels were increased. [1]
In HL-60 cells, ZINC69391 (50 μM, 24 hours) reduced Rac1-GTP levels as assessed by a pull-down assay. [1]
ZINC69391 (50 μM and 100 μM, 24 hours) did not induce significant apoptosis in normal peripheral blood mononuclear cells, including both unstimulated and phytohemagglutinin A-activated (proliferating) lymphocytes. [1]

In syngeneic animal models, ZINC69391 (25 mg/kg; i.p.; once daily for 21 days) inhibits the colonization of the lung that metastasizes [3].

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Rac1 activation assay (pull-down): HL-60 cells were treated with 50 μM ZINC69391 for 24 hours. Cell lysates were subjected to a pull-down assay to measure Rac1-GTP levels (the activated form of Rac1). [1]

Cell proliferation assay[2]
Cell Types: U-87 MG, LN229 Cell
Tested Concentrations: 0-125μM
Incubation Duration: 72 hrs (hours)
Experimental Results: Cell proliferation diminished in a concentration-dependent manner.

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Cell cycle analysis[2]
Cell Types: LN229 Cell
Tested Concentrations: 50, 100 μM
Incubation Duration: 48 hrs (hours)
Experimental Results: The percentage of sub-G0/G1 phase cells increased Dramatically in a concentration-dependent manner.

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Apoptosis analysis[1]
Cell Types: HL-60, U937 and KG1A Cell lines
Tested Concentrations: 50 μM
Incubation Duration: 24 hrs (hours)
Experimental Results: Resulted in a significant increase in apoptotic cells.

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Cell proliferation assay (MTS): Cells were seeded in 96-well plates and exposed to serial dilutions of ZINC69391 for 48 hours. MTS reagent was added, and after 2 hours of incubation, absorbance was measured at 490 nm to determine cell viability. IC50 values were calculated using sigmoidal dose-response function. [1]
Cell cycle analysis (PI staining): Synchronized cells were treated with 50 μM ZINC69391 for 24 hours. Cells were fixed in 70% ethanol, stained with propidium iodide (PI) containing RNase A, and analyzed by flow cytometry to determine cell cycle phase distribution. [1]
Apoptosis assay (Annexin V/PI): Cells were treated with ZINC69391 for 24 hours. Cells were stained with Annexin V-FITC and propidium iodide (PI) and analyzed by flow cytometry to detect early and late apoptotic cells. [1]
Caspase 3 activity assay (colorimetric): Cells were treated with ZINC69391 (50 and 100 μM) for 12 and 24 hours. Cell lysates were incubated with the caspase 3 substrate Ac-DEVD-pNA. Caspase 3 activity was determined by measuring absorbance at 405 nm. [1]
Caspase 9 activity assay (colorimetric): Cells were treated with ZINC69391 (50 μM) for 0, 2, 4, 6, 8, and 12 hours. Cell lysates were incubated with a caspase 9 substrate, and activity was measured at 405 nm. [1]
Western blotting: Cells were treated with ZINC69391, lysed, and subjected to SDS-PAGE. Proteins were transferred to nitrocellulose membranes and probed with antibodies against cleaved caspase 3, caspase 8, Bcl-2, and α-tubulin. [1]
Mitochondrial membrane potential assay (DiOC6): Cells were treated with 50 μM ZINC69391 for 0 to 7 hours. Cells were stained with the lipophilic cationic probe DiOC6 (10 mM) for 20 minutes, and fluorescence was analyzed by flow cytometry to assess mitochondrial membrane integrity. [1]
Quantitative real-time PCR (qPCR): Total RNA was extracted from HL-60 and Jurkat cells treated with 50 μM ZINC69391 for 12 hours. cDNA was synthesized, and qPCR was performed using primers for Bcl-2, Bcl-xL, Mcl-1, IL-8, and β-actin (housekeeping gene). Relative mRNA expression was calculated using the comparative ΔΔCt method. [1]
Primary cell culture: Peripheral blood mononuclear cells (PBMC) from healthy donors were isolated by Ficoll-Hypaque density gradient centrifugation. Cells were cultured in RPMI-1640 with 10% FBS. For T cell activation, cells were incubated with 1.0 μg/mL phytohemagglutinin A for 48 hours before ZINC69391 treatment. [1]

Animal/Disease Models: Specific pathogen-free female BALB/c inbred mice (carrying F3II cells) [3]
Doses: 25 mg/kg body weight
Route of Administration: intraperitoneal (ip) injection; one time/day for 21 days
Experimental Results: Total number of lung metastatic colonies The formation is Dramatically diminished by approximately 60%.

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ZINC69391 (50 μM and 100 μM, 24 hours) did not induce significant apoptosis in normal peripheral blood mononuclear cells (PBMC) from healthy donors, including both unstimulated and phytohemagglutinin A-activated (proliferating) lymphocytes. [1]

[1]. Pharmacological Rac1 inhibitors with selective apoptotic activity in human acute leukemic cell lines. Oncotarget. 2017;8(58):98509‐98523. Published 2017 Oct 4.

[2]. Proapoptotic and antiinvasive activity of Rac1 small molecule inhibitors on malignant glioma cells. Onco Targets Ther. 2014;7:2021‐2033. Published 2014 Oct 30.

[3]. Preclinical development of novel Rac1-GEF signaling inhibitors using a rational design approach in highly aggressive breast cancer cell lines. Anticancer Agents Med Chem. 2014;14(6):840‐851.

ZINC69391 is a small molecule identified as a Rac1-GEF interaction inhibitor using a docking-based virtual library screening approach. It is a first-generation Rac1 inhibitor. [1]
ZINC69391 has previously been shown to inhibit cell proliferation, cell cycle progression, migration, and lung metastasis in vivo in breast cancer models, and to induce apoptosis and decrease cell migration and invasion in glioblastoma cell lines. [1]
The pro-apoptotic activity of ZINC69391 in acute leukemia cells involves activation of the mitochondrial (intrinsic) apoptotic pathway, characterized by loss of mitochondrial membrane potential, activation of caspase 9, and subsequent activation of caspase 3 and caspase 8. [1]
ZINC69391 treatment induced a G2/M cell cycle arrest in HL-60 and KG1A cells. [1]

C14H14F3N5

309.289672374725

309.12

303094-67-9

5728890

White to off-white solid powder

2.7

2

6

3

22

388

0

CC1=CC(=NC(=N1)/N=C(\N)/NC2=CC=CC=C2C(F)(F)F)C

BEZGMANANMSUSQ-UHFFFAOYSA-N

InChI=1S/C14H14F3N5/c1-8-7-9(2)20-13(19-8)22-12(18)21-11-6-4-3-5-10(11)14(15,16)17/h3-7H,1-2H3,(H3,18,19,20,21,22)

2-(4,6-dimethylpyrimidin-2-yl)-1-[2-(trifluoromethyl)phenyl]guanidine

ZINC69391 ZINC-69391 ZINC 69391

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 (~80.83 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.)