Cell division control protein 42 (Cdc42), a Rho GTPase. The drug selectively inhibits Cdc42 activity without affecting Rac1 or RhoA from the same family.

AZA197 demonstrated selective, dose-dependent inhibition of Cdc42 activity in both SW620 and HT-29 human colon cancer cell lines, as measured by G-LISA assays. At concentrations of 1, 2, 5, and 10 µM, AZA197 reduced Cdc42 activity in SW620 cells by 56.7%, 75.2%, 76.0%, and 89.3%, respectively, compared to untreated controls. In HT-29 cells, Cdc42 activity was reduced by 18%, 48.5%, 52.9%, and 61.0% at the same concentrations. No inhibition of Rac1 or RhoA activity was observed.
AZA197 significantly suppressed proliferation of SW620 and HT-29 colon cancer cells in a dose-dependent manner, as determined by WST-1 assays over 72 hours. At 72 hours, all tested concentrations (1-10 µM) significantly reduced cell proliferation compared to controls (P < 0.001).
Flow cytometry analysis of SW620 cells treated with AZA197 (2, 5, and 10 µM for 24 hours) showed a dose-dependent reduction in cells in S and G2/M phases and an increase in cells with sub-G0/G1 DNA content, indicative of apoptosis.
AZA197 significantly reduced migration of SW620 and HT-29 colon cancer cells in Boyden chamber assays. Treatment with 2 or 5 µM AZA197 for 24 hours reduced SW620 cell migration by 47.4% and 43.5%, respectively (P < 0.05). HT-29 cell migration was reduced up to 77.1%.
AZA197 significantly reduced invasion of SW620 and HT-29 colon cancer cells in Matrigel invasion assays. Treatment with 1, 2, and 5 µM AZA197 for 24 hours decreased SW620 invasion by 61.3%, 71.0%, and 83.9%, respectively (P < 0.003). HT-29 invasion was reduced up to 84.6%.
Fluorescence microscopy of AZA197-treated colon cancer cells stained with phalloidin revealed dramatic changes in cell morphology, including cell rounding and loss of filopodia formation, consistent with Cdc42 inhibition.
Western blot analysis showed that AZA197 treatment (2-10 µM for 24 hours) did not alter total Cdc42, PAK1, or ERK protein levels but significantly and dose-dependently reduced phosphorylation of PAK1/2 and ERK1/2 in both SW620 and HT-29 cells. In SW620 cells, pPAK1/2 was reduced by up to 66.2% and pERK by up to 40.2%. Cyclin D1 protein expression was also significantly reduced following AZA197 treatment.
In a guanine nucleotide exchange assay, AZA197 (10 µM) inhibited the GEF (Dbs)-stimulated nucleotide exchange activity on Cdc42 by approximately 61%, indicating that the compound disrupts Cdc42-GEF interaction.
Cytotoxicity assays (LDH release) showed that AZA197 at concentrations up to 10 µM did not cause significant cell membrane damage in SW620, HT-29, or 3T3 fibroblasts after 24 hours. At concentrations of 20 µM and above, significant LDH release was observed in all cell lines.

In a xenograft mouse model, athymic nude mice bearing subcutaneous SW620 human colon cancer tumors were treated with daily intraperitoneal injections of AZA197 (100 µg/day in 30% DMSO) or vehicle control for 14 days (days 8-22 post-tumor cell injection). On day 22, mean tumor weight was significantly reduced in the AZA197-treated group (676.7 ± 106 mg) compared to the control group (968 ± 208 mg) (P = 0.006), indicating suppression of primary tumor growth.
Immunohistochemical analysis of tumor tissue from AZA197-treated mice showed a significant 27.4% reduction in Ki-67-positive proliferating cells (P = 0.046) and a significant 80.6% increase in TUNEL-positive apoptotic cells (P = 0.035) compared to controls.
Western blot analysis of tumor tissue lysates confirmed that AZA197 treatment did not alter total Cdc42, PAK1, or ERK protein levels but significantly reduced phosphorylation of PAK1/2 (by 48.5%) and ERK1/2 (by 59.2%) compared to controls, consistent with in vitro findings.
In a survival study, AZA197 treatment significantly prolonged the survival of SW620 tumor-bearing mice (P = 0.042). Median survival was 69 days in the treatment group versus 53 days in the control group. At the study endpoint (day 100), 50% of AZA197-treated mice were still alive, whereas all control mice had died.

Guanine Nucleotide Exchange Assay: The ability of AZA197 to inhibit GEF-stimulated nucleotide exchange on Cdc42 was assessed using a RhoGEF Exchange Assay Biochem Kit. Fluorescence spectroscopy was used to monitor the incorporation of N-methylanthraniloyl (mant)-labeled GTP into purified His-tagged Cdc42. Exchange reaction mixtures were prepared containing 20 mM Tris (pH 7.5), 50 mM NaCl, 10 mM MgCl₂, 50 µg/mL BSA, 0.8 µM mant-GTP, and 1 µM Cdc42 GTPase, in the presence or absence of 10 µM AZA197. After equilibration and baseline fluorescence readings, the GEF Dbs (or water as a negative control) was added to a concentration of 0.8 µM. Fluorescence measurements (excitation 360 nm, emission 440 nm) were taken every 30 seconds for a total of 30 minutes. The increase in fluorescence, representing mant-GTP incorporation, was monitored. The exchange activity in the presence of AZA197 was compared to the Dbs-stimulated control to calculate the percentage of inhibition.

Cytotoxicity Assay: Cells (SW620, HT-29, and 3T3 fibroblasts) were seeded in 96-well plates, cultured for 24 hours, and then incubated with 1-100 µM AZA197 for 24 hours. Culture supernatants were harvested and LDH release was measured using a CytoTox96 Non-Radioactive Cytotoxicity Assay kit according to the manufacturer's instructions. Absorbance was measured at 490 nm, and cytotoxicity was expressed as a percentage of maximum LDH release from lysed cells.
Rho GTPase Activation Assay: Colon cancer cells (SW620, HT-29) were seeded in 6-well plates and incubated with 1, 2, 5, and 10 µM AZA197 for 24 hours. Rac1, Cdc42, and RhoA activation were then measured using G-LISA activation assay kits (colorimetric format) according to the manufacturer's protocols. This assay specifically captures the active, GTP-bound form of each GTPase.
Cell Proliferation Assay: Human SW620 and HT-29 cells were seeded in 96-well plates at 1×10⁴ cells/well. Cells were incubated with 1, 2, 5, or 10 µM AZA197. Cell proliferation was determined at 24, 48, and 72 hours after treatment using the WST-1 reagent according to the manufacturer's protocol. Absorbance was measured, and relative cell density was calculated.
Flow Cytometry Cell Cycle Analysis: SW620 cells were seeded in 6-well plates and treated with 2, 5, or 10 µM AZA197 for 24 hours. Cells were then trypsinized, washed in PBS, fixed in 70% ethanol for 1 hour at 4°C, and stained in PBS supplemented with 800 µg/mL propidium iodide containing 50 µg/mL RNase A. 10⁴ events were analyzed on a FACScan flow cytometer with an argon laser tuned to 488 nm.
Migration Assay: Colon cancer cells (1×10⁵ in 1 mL DMEM with 10% FCS) were added to the top of Boyden migration chambers (8 µm pore size). Cells were incubated with 1, 2, or 5 µM AZA197. After 24 hours, membranes were washed, fixed in methanol at -20°C for 10 min, and stained with 1 µg/mL DAPI. Migrated cells on the lower side of the membrane were counted under a fluorescence microscope from five individual consecutive fields of view. Each experiment was performed in triplicate.
Invasion Assay: Colon cancer cells (5×10⁴ in 1 mL culture medium with 0.2% FCS) were added to the top of BioCoat Matrigel Invasion Chambers (8 µm pore size). DMEM containing 10% FCS was added to the lower chamber as a chemoattractant. AZA197 was added to final concentrations of 1, 2, and 5 µM. After 24 hours, membranes were processed and stained as described for the migration assay. Invaded cells were counted under a fluorescence microscope from five individual consecutive fields of view. Each experiment was performed in triplicate.
Actin Cytoskeleton Visualization: SW620 and HT-29 cells were grown on fibronectin/gelatin-coated chambered coverglasses and incubated with 5 or 10 µM AZA197 for 24 hours. Cells were then fixed, permeabilized, labeled with Atto-488 phalloidin to stain F-actin, and counterstained with DAPI. Fluorescence was observed with a Nikon Eclipse 80i microscope at 1000× magnification, and images were digitally acquired.
Western Blotting: Colon cancer cells were seeded in 100 mm plates and incubated with 2, 5, and 10 µM AZA197 for 24 hours. Cell lysates (50 µg/lane) were separated by 12% SDS-PAGE and transferred onto membranes. Blots were probed with antibodies against Cdc42, PAK1, phospho-PAK1 (Ser144)/PAK2(Ser141), ERK1/2, phospho-ERK1/2 (Thr202/Tyr204), Cyclin D1, and α-tubulin (loading control). Proteins were detected by chemiluminescence and quantified.

Xenograft Tumor Model:** Pathogen-free, 5-week-old male athymic nu/nu (nude) mice were used. Mice were anesthetized (ketamine/xylazine 55/7.5 mg/kg i.p.), and 8×10⁶ SW620 human colon cancer cells in 100 µL PBS were injected subcutaneously into the left flank. Eight days after cell injection (day 8), mice received daily intraperitoneal injections of 100 µg AZA197 in 100 µL of 30% DMSO for two weeks (until day 22). Control animals received 100 µL of 30% DMSO per day. Tumor volumes were measured using a caliper and calculated as length × width² / 2. On day 22, all animals were sacrificed, and tumors were excised, photographed, and weighed. Tumor tissue was processed for histology (Ki-67 IHC, TUNEL) and Western blotting.
**Survival Study:** In a separate cohort, tumor-bearing mice were treated with AZA197 (n=6) or vehicle control (n=6) as described above. The survival study was set for 100 days. Mice were euthanized when moribund, and survival curves were analyzed by the Kaplan-Meier test.

---

Xenograft Tumor Model: Pathogen-free, 5-week-old male athymic nu/nu (nude) mice were used. Mice were anesthetized (ketamine/xylazine 55/7.5 mg/kg i.p.), and 8×10⁶ SW620 human colon cancer cells in 100 µL PBS were injected subcutaneously into the left flank. Eight days after cell injection (day 8), mice received daily intraperitoneal injections of 100 µg AZA197 in 100 µL of 30% DMSO for two weeks (until day 22). Control animals received 100 µL of 30% DMSO per day. Tumor volumes were measured using a caliper and calculated as length × width² / 2. On day 22, all animals were sacrificed, and tumors were excised, photographed, and weighed. Tumor tissue was processed for histology (Ki-67 IHC, TUNEL) and Western blotting.
Survival Study: In a separate cohort, tumor-bearing mice were treated with AZA197 (n=6) or vehicle control (n=6) as described above. The survival study was set for 100 days. Mice were euthanized when moribund, and survival curves were analyzed by the Kaplan-Meier test.

In vitro, AZA197 showed no significant cytotoxicity (LDH release) in SW620, HT-29, or 3T3 fibroblasts at concentrations up to 10 µM. At concentrations of 20 µM and above, significant LDH release was observed in all cell lines, indicating cell membrane damage.
In vivo, AZA197 treatment (100 µg/day i.p. for 14 days) was well tolerated in athymic nude mice. No changes in body weight or gross indications of toxicity were observed during the treatment period.

[1]. Targeting Cdc42 with the small molecule drug AZA197 suppresses primary colon cancer growth and prolongs survival in a preclinical mouse xenograft model by downregulation of PAK1 activity. J Transl Med. 2013 Nov 27;11:295.

AZA197 (N2-(4-Diethylamino-1-methyl-butyl)-N4-[2-(1H-indol-3-yl)-ethyl]-6-methyl-pyrimidine-2,4-diamine) is a novel small-molecule inhibitor developed based on structural modifications of the Rac1 inhibitor NSC23766. It was identified through an in vitro screen for compounds with inhibitory activity in SW620 colon cancer cells.
AZA197 selectively targets the Rho GTPase Cdc42 by disrupting its interaction with guanine nucleotide exchange factors (GEFs), thereby preventing its activation. This specificity for Cdc42 over other Rho family members (Rac1, RhoA) makes it a valuable tool for studying Cdc42 biology and a promising therapeutic candidate.
The antitumor effects of AZA197 are mediated through inhibition of the Cdc42-PAK1-ERK signaling pathway, leading to reduced proliferation, migration, and invasion, and increased apoptosis in colon cancer cells.
This preclinical study demonstrates the therapeutic potential of targeting Cdc42 with AZA197 for the treatment of colorectal cancer, particularly in KRAS-mutant disease, which is often resistant to existing targeted therapies. The compound significantly suppressed tumor growth and prolonged survival in a xenograft mouse model.

C24H36N6

408.582844734192

408.3

1249398-09-1

46938132

Off-white to light yellow solid powder

5.2

3

5

12

30

471

0

N(CC)(CC)CCCC(C)NC1N=C(C)C=C(N=1)NCCC1=CNC2C=CC=CC1=2

SUXUDORZKKZLJK-UHFFFAOYSA-N

InChI=1S/C24H36N6/c1-5-30(6-2)15-9-10-18(3)27-24-28-19(4)16-23(29-24)25-14-13-20-17-26-22-12-8-7-11-21(20)22/h7-8,11-12,16-18,26H,5-6,9-10,13-15H2,1-4H3,(H2,25,27,28,29)

2-N-[5-(diethylamino)pentan-2-yl]-4-N-[2-(1H-indol-3-yl)ethyl]-6-methylpyrimidine-2,4-diamine

AZA197 AZA 197 AZA-197

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.

---

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).

View More

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)

---

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).

View More

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

---

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.

---

 (Please use freshly prepared in vivo formulations for optimal results.)