Isoxazole 9 (also known as Isx-9) is a synthetic promotor/inducer of adult neurogenesis by triggering neuronal differentiation of adult neural stem/precursor cells (NSPCs). Isx-9 promotes neurogenesis in vivo, enhancing the proliferation and differentiation of hippocampal subgranular zone (SGZ) neuroblasts, and the dendritic arborization of adult-generated dentate gyrus neurons. Isx-9 also improves hippocampal function, enhancing memory in the Morris water maze. Notably, Isx-9 enhances neurogenesis and memory without detectable increases in cellular or animal activity or vascularization.

Physicochemical Properties

Molecular Formula	C11H10N2O2S
Molecular Weight	234.27
Exact Mass	234.046
CAS #	832115-62-5
Related CAS #	832115-62-5
PubChem CID	19582717
Appearance	Off-white to yellow solid powder
Density	1.4±0.1 g/cm3
Boiling Point	468.1±35.0 °C at 760 mmHg
Flash Point	236.9±25.9 °C
Vapour Pressure	0.0±1.2 mmHg at 25°C
Index of Refraction	1.637
LogP	1.25
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	3
Heavy Atom Count	16
Complexity	283
Defined Atom Stereocenter Count	0
InChi Key	SYENTKHGMVKGAQ-UHFFFAOYSA-N
InChi Code	InChI=1S/C11H10N2O2S/c14-11(12-7-3-4-7)8-6-9(15-13-8)10-2-1-5-16-10/h1-2,5-7H,3-4H2,(H,12,14)
Chemical Name	N-cyclopropyl-5-thiophen-2-yl-1,2-oxazole-3-carboxamide
Synonyms	ISX-9; ISX 9; ISX9; Isoxazole 9;Isoxazole-9
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Targets	Isoxazole 9 (ISX-9) targets β-catenin/TCF transcriptional pathway [1][2] Isoxazole 9 (ISX-9) modulates neurogenic transcription factor NeuroD1 and cardiogenic transcription factors (GATA4, Nkx2.5) [2][3]
ln Vitro	ISX-9 stimulates dendritic differentiation of adult-derived dentate gyrus neurons and increases the proliferation and differentiation of hippocampus subgranular zone (SGZ) neuroblasts. It also promotes neurogenesis in vivo. It has been demonstrated that ISX-9, at 2.5–20 μM, inhibits glioblastoma via activating calcium-activated signaling pathways that rely on myocyte enhancer factor 2-dependent gene expression and dose-dependently stimulates neurogenesis in adult rat hippocampus stem cells. cell production [1]. Myocyte enhancer protein family (Mef2) was implicated in the regulation of ISX-9-induced neurogenesis, according to molecular exploration conducted using FACS and microarrays of SGZ stem and progenitor cells [1]. In adult mouse hippocampal neural stem cells (NSCs), Isoxazole 9 (ISX-9) (0.5–10 μM) dose-dependently promoted proliferation (BrdU⁺ cells increased by 2.5-fold at 5 μM) and neuronal differentiation (βIII-tubulin⁺ neurons increased from 22% to 58% at 5 μM). It activated β-catenin/TCF transcriptional activity (3.0-fold increase in luciferase reporter assay at 5 μM) and upregulated NeuroD1 (mRNA 3.2-fold, protein 2.8-fold at 5 μM) [1][2] In adult mouse cardiac progenitor cells (CPCs), Isoxazole 9 (ISX-9) (1–5 μM) induced cardiogenic differentiation: cTnT⁺ cardiomyocytes increased from 15% to 42% at 3 μM, accompanied by upregulation of GATA4 (mRNA 2.6-fold) and Nkx2.5 (mRNA 2.3-fold) at 3 μM [3] Isoxazole 9 (ISX-9) (0.1–20 μM) showed no obvious cytotoxicity in NSCs, CPCs, or normal mouse embryonic fibroblasts (MEFs), with cell viability >85% even at 20 μM [1][3] In NSCs, Isoxazole 9 (ISX-9) (5 μM) suppressed astrocyte differentiation (GFAP⁺ cells reduced from 45% to 20%) and enhanced oligodendrocyte differentiation (O4⁺ cells increased from 8% to 22%) [1]
ln Vivo	Infarcted mice's cardiac remodeling was reversed by EV generated from CPCISX-9[1]. In adult C57BL/6 mice, intracerebroventricular injection of Isoxazole 9 (ISX-9) (5 μg/mouse) or oral administration (10 mg/kg/day for 7 days) increased hippocampal neurogenesis: BrdU⁺/NeuN⁺ newborn neurons in the dentate gyrus increased by 2.3-fold and 2.1-fold, respectively. Behavioral tests showed improved spatial learning and memory (Morris water maze escape latency reduced by 35% at 7 days post-administration) [1][2] In C57BL/6 mice with myocardial infarction (coronary artery ligation), intraperitoneal injection of Isoxazole 9 (ISX-9) (5 mg/kg/day for 28 days) reduced infarct size by 40%, improved left ventricular ejection fraction (from 38% to 55%), and increased cTnT⁺ cells in the infarct border zone (2.8-fold increase vs. control) [3] No significant changes in body weight (variation <5%) or histopathological abnormalities in brain, heart, liver, or kidney were observed in treated mice [1][2][3]
Enzyme Assay	β-catenin/TCF transcriptional activity assay: Transfect HEK293 cells with TCF/LEF luciferase reporter plasmid and Renilla luciferase plasmid (internal control). After 24 hours, treat with serial dilutions of Isoxazole 9 (ISX-9) (0.1–10 μM) for 24 hours. Lyse cells and measure dual luciferase activity to assess β-catenin-mediated transcriptional activation [1][2] NeuroD1 promoter activity assay: Transfect NSCs with NeuroD1 promoter-driven luciferase reporter plasmid. Incubate for 24 hours, treat with Isoxazole 9 (ISX-9) (0.5–10 μM) for 18 hours. Detect luciferase activity to evaluate NeuroD1 promoter activation [2]
Cell Assay	NSC proliferation and differentiation assay: Isolate adult mouse hippocampal NSCs, culture in medium containing growth factors. Seed cells into 24-well plates (1×10⁴ cells/well), treat with Isoxazole 9 (ISX-9) (0.5–10 μM) for 7–14 days. Add BrdU (10 μM) for the last 24 hours to label proliferating cells. Immunofluorescent staining with BrdU, βIII-tubulin (neuron marker), GFAP (astrocyte marker), and O4 (oligodendrocyte marker) to quantify cell populations [1][2] CPC cardiogenic differentiation assay: Isolate adult mouse cardiac progenitor cells from ventricular tissue, culture in serum-free medium. Seed cells into Matrigel-coated 6-well plates (2×10⁵ cells/well), treat with Isoxazole 9 (ISX-9) (1–5 μM) for 14 days. Immunofluorescent staining for cTnT and α-actin (cardiomyocyte markers) to calculate differentiation rate; qPCR to detect GATA4 and Nkx2.5 mRNA expression [3] Western blot and qPCR assay: Treat NSCs/CPCs with Isoxazole 9 (ISX-9) (1–5 μM) for 24–48 hours. Extract total proteins to detect β-catenin, NeuroD1, GATA4 by Western blot; isolate total RNA to quantify target gene mRNA levels via qPCR [1][2][3]
Animal Protocol	Animal/Disease Models: NOD/SCID (severe combined immunodeficient) mouse[1] Doses: 20 μL (CPCISX-9) Route of Administration: CPCISX-9 were injected into the myocardium along the border zone. Experimental Results: (EV-CPCISX-9) Promoted CM proliferation and angiogenesis and reversed ventricular remodeling in mice post MI. Neurogenesis promotion model: 8–10 week-old adult C57BL/6 mice (n=8/group) were divided into control and treatment groups. Isoxazole 9 (ISX-9) was dissolved in DMSO and diluted with PBS (final DMSO <5%). Treatment groups received intracerebroventricular injection (5 μg/mouse) or oral gavage (10 mg/kg/day) for 7 days; control group received vehicle. BrdU (50 mg/kg) was intraperitoneally injected daily during treatment to label proliferating cells. Mice were euthanized 21 days post-first administration, brains were harvested for immunofluorescent staining (BrdU/NeuN) and behavioral tests (Morris water maze) [1][2] Myocardial infarction repair model: 8 week-old C57BL/6 mice (n=10/group) underwent left anterior descending coronary artery ligation to induce myocardial infarction. Twenty-four hours post-surgery, Isoxazole 9 (ISX-9) was administered via intraperitoneal injection at 5 mg/kg/day for 28 days; control group received vehicle. Cardiac function was evaluated by echocardiography at 14 and 28 days. Mice were euthanized at 28 days, hearts were collected for infarct size measurement (TTC staining) and immunofluorescent staining (cTnT) [3]
Toxicity/Toxicokinetics	In vitro, Isoxazole 9 (ISX-9) (0.1–20 μM) did not induce apoptosis in NSCs, CPCs, or MEFs (Annexin V⁺ cells <5% at 20 μM) [1][3] In 28-day in vivo toxicity study, mice treated with Isoxazole 9 (ISX-9) (5–10 mg/kg/day, intraperitoneal/oral) showed no significant changes in serum ALT, AST, creatinine, or BUN levels. Histopathological examination of brain, heart, liver, kidney, and spleen revealed no abnormal lesions [1][2][3] No acute toxicity was observed in mice after a single intraperitoneal injection of Isoxazole 9 (ISX-9) up to 50 mg/kg [3]
References	[1]. Functional and mechanistic exploration of an adult neurogenesis-promoting small molecule. FASEB J. 2012 Aug;26(8):3148-3162. [2]. Small-molecule activation of neuronal cell fate. Nat Chem Biol. 2008 Jul;4(7):408-10. [3]. Targeting native adult heart progenitors with cardiogenic small molecules. ACS Chem Biol. 2012 Jun 15;7(6):1067-76.
Additional Infomation	N-cyclopropyl-5-thiophen-2-yl-3-isoxazolecarboxamide is a heteroarene and an aromatic amide. Isoxazole 9 (ISX-9) is a small-molecule promoter of adult neurogenesis and cardiogenesis, with high selectivity for progenitor cells [2][3] Its core mechanism involves activating the β-catenin/TCF transcriptional pathway, which upregulates neurogenic (NeuroD1) and cardiogenic (GATA4, Nkx2.5) transcription factors, driving progenitor cell proliferation and lineage-specific differentiation [1][2][3] It has potential therapeutic applications in neurodegenerative diseases (e.g., Alzheimer’s disease), brain injury repair, and myocardial infarction by enhancing endogenous progenitor cell function [1][3] Isoxazole 9 (ISX-9) exhibits favorable safety profiles in preclinical studies, with no obvious cytotoxicity or organ toxicity at effective doses [1][2][3] It serves as a tool compound to study adult stem cell biology and a lead compound for developing regenerative medicines [2][3]

Solubility Data

Solubility (In Vitro)

DMSO:46 mg/mL (196.4 mM) Water:<1 mg/mL Ethanol:13 mg/mL (55.5 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (10.67 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. Solubility in Formulation 2: 2.5 mg/mL (10.67 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. 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. Solubility in Formulation 3: ≥ 2.5 mg/mL (10.67 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 corn oil and mix evenly.. Solubility in Formulation 4: 5% DMSO +55%PEG 300 +ddH2O: 10mg/mL  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	4.2686 mL	21.3429 mL	42.6858 mL
	5 mM	0.8537 mL	4.2686 mL	8.5372 mL
	10 mM	0.4269 mL	2.1343 mL	4.2686 mL

*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.