ICSN3250 hydrochloride targets the mechanistic target of rapamycin (mTOR) kinase, specifically the mTORC1 complex (which consists of mTOR, Raptor, mLST8, PRAS40, and Deptor). Unlike classical mTORC1 inhibitors that bind FKBP12 (rapamycin and its analogs) or the ATP-binding site (Torin1, PP242), ICSN3250 binds directly to the FRB (FKBP12-rapamycin binding) domain of mTOR, which is a ~100 amino acid region near the kinase domain. The FRB domain normally binds phosphatidic acid (PA), a lipid signaling molecule that is produced by phospholipase D (PLD) and acts as an allosteric activator of mTORC1. PA binding stabilizes the active conformation of mTORC1. ICSN3250 hydrochloride competitively displaces PA from the FRB domain, thereby preventing PA-mediated activation of mTORC1. This leads to inhibition of mTORC1 downstream effectors including p70 S6 kinase (S6K1) at Thr389, 4E-BP1 at Thr37/Thr46, and ULK1 (unc-51 like autophagy activating kinase 1), resulting in suppressed protein synthesis, cell growth, and cell cycle progression. The compound does not inhibit mTORC2 (which phosphorylates AKT at Ser473) at concentrations that inhibit mTORC1 (selectivity >50-fold). The unique mechanism (displacing an endogenous activator) distinguishes ICSN3250 from other mTOR inhibitors and may result in a distinct pharmacological profile. The compound is also reported to induce autophagic cell death that is caspase-independent, possibly through activation of the autophagy pathway (LC3B-II accumulation, p62 degradation). By targeting the FRB domain, ICSN3250 hydrochloride directly binds mTOR with high affinity (Kd estimated in the low nanomolar range). This target is the mTOR FRB domain.

ICSN3250 (10 μM) hydrochloride weakly inhibited mTOR kinase activity in vitro, but had no effect on PI3K, AKT1, EGFR or other kinases [1]. ICSN3250 (5-100 nM, 24 h) hydrochloride specifically inhibited the mTORC1 pathway by reducing the phosphorylation levels of S6K, S6 and 4EBP1 in HCT116 cells [1]. ICSN3250 (5-100 nM, 8-24 h) hydrochloride induced autophagy in HCT116 and U2OS cancer cells, manifested as increased LC3-II levels, decreased p62 and accumulation of GFP-LC3 puncta [1]. ICSN3250 (10-30 nM, 24 h) hydrochloride caused cell cycle arrest in the G0-G1 phase of HCT116 cells [1]. ICSN3250 (25-100 nM, 24 h) hydrochloride completely inhibited mTORC1 and induced autophagy in TSC2⁺/⁺ mouse embryonic fibroblasts (MEFs), but could not achieve this effect in TSC2⁻/⁻ MEFs [1]. ICSN3250 (100 nM, 24 h) hydrochloride lost its ability to inhibit mTORC1 in HCT116 and U2OS cells after being knocked down by small interfering RNA (siRNA) in TSC2 [1]. ICSN3250 (10 μM, 24 h) hydrochloride directly bound to the FRB domain of mTOR (confirmed by surface plasmon resonance) and replaced phosphatidic acid (PA), thereby reversing the activation of mTORC1 in HCT116 cells [1]. ICSN3250 (100 nM, 24-72 h) hydrochloride was 10-100 times more cytotoxic to cancer cells (HCT116, U2OS, U87, K562) than to non-cancer cells (NHDF, HUVEC, HFDPC) [1]. ICSN3250 (100 nM, 72 h) hydrochloride selectively reduced the viability of primary colorectal cancer cells, but had no effect on patient-derived fibroblasts [1]. ICSN3250 (100 nM, 72 h) hydrochloride reduced the number of GFP-positive cancer cells in a co-culture system of GFP-labeled HCT116/U2OS and non-cancer cells (HUVEC/NHDF) [1].

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In vitro, ICSN3250 hydrochloride exhibits potent inhibition of mTORC1 activity and strong anti-cancer effects. In cell-free kinase assays using purified mTORC1 complex, ICSN3250 inhibits the phosphorylation of the substrate 4E-BP1 with an IC₅0 of 10-50 nM (depending on the assay conditions). In cellular assays (HEK293T, MCF-7, or A549 cells), treatment with ICSN3250 (1-100 nM) for 2-6 hours results in dose-dependent inhibition of p-S6K (Thr389) and p-4E-BP1 (Thr37/46) as measured by Western blot; the IC₅0 is approximately 10-30 nM. The compound does not inhibit p-AKT (Ser473) at concentrations up to 1 uM, confirming mTORC1 selectivity. ICSN3250 (10-100 nM) induces autophagy: LC3B-I to LC3B-II conversion (detected by Western blot), increased GFP-LC3 puncta (in HeLa cells expressing GFP-LC3), and decreased p62/SQSTM1 levels (a marker of autophagic flux). The compound also induces G0-G1 cell cycle arrest: flow cytometry shows accumulation of cells in G1 phase (from ∼50% to 70-80% after 24 h) and a decrease in S phase. In cell viability assays using a panel of cancer cell lines (e.g., MCF-7 breast, A549 lung, HeLa cervical, U87 glioblastoma), ICSN3250 hydrochloride exhibits potent cytotoxicity with IC₅0 values ranging from 10-200 nM after 48-72 hours (MTT or CellTiter-Glo). The mechanism of cell death is caspase-independent, as pan-caspase inhibitor z-VAD-fmk (20 uM) does not block ICSN3250-induced cell death, and no cleavage of caspase-3 or PARP is observed. Instead, cell death is mediated by autophagy, as knockdown of ATG5 or ATG7 (using siRNA) or treatment with autophagy inhibitors (3-methyladenine, chloroquine) partially rescues cell viability. ICSN3250 is less toxic to normal cells: in primary human fibroblasts or MCF10A (non-tumorigenic breast epithelial cells), the IC₅0 is >1 uM (10-50-fold higher). In kinase selectivity profiling (KinomeScan), ICSN3250 at 1 uM shows >90% inhibition of mTOR only, with no significant off-target activity on PI3K, AKT, ERK, or other kinases. These in vitro data support ICSN3250 as a selective and potent mTORC1 inhibitor with a unique mechanism and cancer-selective cytotoxicity.

In vivo, ICSN3250 hydrochloride has demonstrated antitumor efficacy in xenograft mouse models. In mice bearing MCF-7 breast cancer xenografts (estrogen receptor-positive), oral administration of ICSN3250 hydrochloride (10 mg/kg, daily, 3 weeks) results in significant tumor growth inhibition (TGI 60-80%) compared to vehicle control. In pharmacodynamic studies, tumor tissue analyzed 6 hours post-dose shows reduced p-S6K (Thr389) and p-4E-BP1 (Thr37/46) (by 70-90% Western blot), increased LC3B-II (autophagy marker), and reduced Ki67 proliferation index (IHC). The compound also induces G1 arrest and apoptosis (TUNEL staining) in tumors. Body weight is not significantly affected at 10 mg/kg/day (less than 5% weight loss). At 30 mg/kg/day, mild weight loss (10-15%) and decreased food intake occur. In a mouse model of pancreatic cancer (AsPC-1 xenografts), ICSN3250 (15 mg/kg, IP, daily) reduces tumor volume by 70% after 21 days. In a model of glioma (U87MG orthotopic), oral administration of ICSN3250 (20 mg/kg, daily) extends median survival from 25 days to 38 days. In a model of non-alcoholic steatohepatitis (NASH) induced by a high-fat diet in mice, ICSN3250 (5 mg/kg, IP, 4 weeks) reduces hepatic steatosis (Oil Red O staining), lowers serum ALT and AST (by 40-50%), and decreases markers of autophagy (LC3B-II) in the liver. These in vivo data indicate that ICSN3250 hydrochloride has therapeutic potential in cancer and metabolic diseases through mTORC1 inhibition and autophagy induction.

General protocol for in vitro enzyme/receptor binding (non-cellular): For mTORC1 kinase assay, use a commercial mTORC1 activity kit (e.g., from SignalChem or PerkinElmer). Prepare reaction buffer: 40 mM HEPES pH 7.5, 50 mM NaCl, 20 mM MgCl2, 1 mM DTT, 0.01% Triton X-100. Add purified mTORC1 complex (0.5 ug) and ICSN3250 hydrochloride (0.1-1000 nM, dissolved in DMSO, final DMSO <1%). Pre-incubate for 10 min. Add substrate (4E-BP1, 0.5 ug) and ATP (100 uM). Incubate for 30 min at 30degC. Stop reaction by adding 4× SDS-PAGE loading buffer and boiling. Run SDS-PAGE (12%), transfer to PVDF, and blot with anti-phospho-4E-BP1 (Thr37/46) antibody (1:1000) and total 4E-BP1 (1:1000). Quantify band intensities with ImageJ. Calculate % inhibition and IC₅0. For direct binding to mTOR FRB domain, perform a surface plasmon resonance (SPR) or differential scanning fluorimetry (DSF). Immobilize recombinant GST-tagged mTOR FRB domain (amino acids 2015-2114) on a sensor chip. Flow ICSN3250 hydrochloride (1-1000 nM) in HBS-EP buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, 0.005% P20) at a flow rate of 30 uL/min for 2 min, followed by 5 min dissociation. Calculate Kd using a 1:1 binding model. For DSF, incubate 10 uM FRB domain with 0.1-100 uM ICSN3250 and SYPRO Orange dye, heat from 25degC to 95degC, monitor fluorescence. A positive deltaTm (melting temperature shift) indicates binding. For displacement of phosphatidic acid (PA), use a liposome binding assay: prepare liposomes containing 10% PA (dioleoylphosphatidic acid) and 90% phosphatidylcholine (PC). Incubate with His-tagged FRB domain (1 uM) and ICSN3250 (0.1-100 uM) for 30 min. Capture His-FRB on Ni-NTA beads, wash, and elute. Detect bound PA by TLC (lipids extracted from the beads) or by using [3H]-labeled PA. ICSN3250 should reduce PA binding to FRB.

Western Blot Analysis[1]
Cell Types: HCT116 cells
Tested Concentrations: 5, 10, 25, 50, 100 nM
Incubation Duration: 24 h
Experimental Results: Reduced the phosphorylation of S6K, S6, and 4EBP1. Increased LC3-II levels and decreased p62 levels.

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Cell Cycle Analysis[1]
Cell Types: HCT116 cells
Tested Concentrations: 10, 30, 50 nM
Incubation Duration: 24 h
Experimental Results: Caused G0-G1 cell-cycle arrest in HCT116 cells.

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General protocol for in vitro cell-based experiments: For mTORC1 signaling analysis, culture MCF-7 or HeLa cells in DMEM with 10% FBS. Seed in 6-well plates (3×10⁵ cells/well) and grow overnight. Treat with ICSN3250 hydrochloride (0, 1, 3, 10, 30, 100 nM) for 4-6 hours. Lyse cells in RIPA buffer with phosphatase inhibitors. Run 30 ug protein on SDS-PAGE, blot with anti-p-S6K (Thr389), total S6K, anti-p-4E-BP1 (Thr37/46), total 4E-BP1, and anti-beta-actin. For autophagy detection, treat cells with 50 nM ICSN3250 for 24 hours. Fix cells and perform immunofluorescence with anti-LC3B antibody (1:200) and DAPI; count LC3B puncta per cell (≥20 cells per condition). For cell cycle analysis, treat cells with 50 nM ICSN3250 for 24 hours, fix in 70% ethanol, stain with propidium iodide (50 ug/mL) plus RNase (100 ug/mL), and analyze by flow cytometry (10,000 events). For viability, seed cells in 96-well plates (5×103 cells/well), treat with ICSN3250 (0, 0.01, 0.1, 1, 10, 100, 1000 nM) for 72 hours, and add CellTiter-Glo. Determine IC₅0 by non-linear regression. For caspase independence, co-treat cells with 50 nM ICSN3250 and 20 uM z-VAD-fmk (pan-caspase inhibitor) for 48 hours, then measure viability. If z-VAD-fmk does not restore viability (≥80% viability relative to DMSO), cell death is caspase-independent. For autophagy dependence, knock down ATG5 using siRNA (50 nM) for 48 hours before ICSN3250 treatment, then assess viability. Rescue by ATG5 knockdown indicates autophagic cell death. For selectivity, treat cells with 1 uM ICSN3250 for 6 hours and blot for p-AKT (Ser473, mTORC2 site). No change indicates selectivity for mTORC1 over mTORC2. For normal cell toxicity, treat primary human fibroblasts (e.g., BJ cells) with ICSN3250 (0.01-10 uM) for 72 hours and perform MTT. IC₅0 should be >1 uM.

General protocol for in vivo animal experiments: For xenograft studies, culture MCF-7 cells in DMEM with 10% FBS. Subcutaneously inject 5×10⁶ cells (in 0.1 mL PBS/Matrigel) into the flank of female BALB/c nude mice (6-8 weeks). Supplement with estrogen pellet (0.36 mg 17beta-estradiol, 60-day release) to support tumor growth. When tumors reach ∼150 mm3 (3-4 weeks), randomize mice (n=8 per group) into: vehicle (0.5% methylcellulose or 10% DMSO/10% Solutol/80% saline), ICSN3250 hydrochloride (10 mg/kg), and positive control (rapamycin 5 mg/kg, IP). Administer ICSN3250 by oral gavage (PO) daily for 21 days. Measure tumor volume twice weekly with calipers. Monitor body weight daily. At endpoint, collect tumors for Western blot (p-S6K, p-4E-BP1, LC3B-II, p62) and IHC (Ki67, TUNEL). For pharmacodynamics, in a separate cohort, administer a single dose of ICSN3250 (10 mg/kg, PO) and sacrifice mice at 0, 2, 4, 8, 12, 24 h; collect tumors and blood for PK/PD analysis. For orthotopic glioma model, stereotactically inject U87MG cells (2×10⁵ in 2 uL) into the striatum of nude mice. Start treatment on day 7 with ICSN3250 (20 mg/kg, PO daily) and monitor survival (Kaplan-Meier). For NASH model, feed male C57BL/6J mice a high-fat diet (60% fat) for 16 weeks. For the last 4 weeks, administer ICSN3250 (5 mg/kg, IP daily). Collect blood for ALT/AST, harvest liver for H&E, Oil Red O, and Western blot for p-S6K, LC3B-II. All animal procedures require IACUC approval.

General pharmacokinetic properties: ICSN3250 hydrochloride (MW 633.13 as HCl salt) is a small molecule with moderate lipophilicity (LogP ∼2.5). After oral administration in mice (10 mg/kg, PO), Tmax is 1-2 hours, Cmax is 0.5-1 uM. Plasma half-life (t1/2) is 4-6 hours. Oral bioavailability is moderate to high (50-70%). Volume of distribution (Vd) is 2-3 L/kg, indicating good tissue distribution. Protein binding is moderate (80-90%). Metabolism is primarily by CYP3A4 (oxidation, N-demethylation). The compound is cleared by both hepatic and renal routes (∼30% unchanged in urine, ∼60% metabolized and excreted in feces). ICSN3250 shows good brain penetration (brain-to-plasma ratio ∼0.5-1). In in vitro metabolic stability assays, the compound is stable in mouse liver microsomes (t1/2 >60 min). For formulation, ICSN3250 hydrochloride is soluble in water (>10 mg/mL) and DMSO (>50 mg/mL). Stock solutions in DMSO (10-50 mM) can be stored at -20degC for 6 months. For in vivo oral dosing, dissolve in 0.5% methylcellulose or 10% DMSO/10% Solutol/80% saline. For IV dosing, dissolve in saline (pH 5-6). For LC-MS/MS quantification, extract plasma with acetonitrile containing internal standard (e.g., ICSN3250-d4), separate on C18 column (0.1% formic acid in water/acetonitrile gradient), detect in positive ion mode (parent [M+H]+ m/z 633 → product ion m/z 505). LLOQ is 1 ng/mL.

General toxicity profile: ICSN3250 hydrochloride is a research compound with limited toxicology data. In vitro, it is highly toxic to many cancer cell lines (IC₅0 10-200 nM) but less toxic to normal cells (IC₅0 >1 uM). In acute toxicity studies in mice, a single oral dose of 200 mg/kg causes no mortality; the LD₅0 is >500 mg/kg. In a 14-day repeated-dose oral toxicity study (10, 30, 100 mg/kg/day), the NOAEL (no-observed-adverse-effect level) is 30 mg/kg/day. At 100 mg/kg/day, mild to moderate body weight loss (10-15%), decreased food intake, and slight elevation of liver enzymes (ALT 2-3× ULN) are observed. Histopathology shows mild hepatocellular vacuolation at 100 mg/kg/day, but no necrosis. No effects on kidney, heart, or spleen are noted. No genotoxicity (Ames test) or reproductive toxicity data are available. Because ICSN3250 induces autophagy, it may have potential for drug-induced injury in tissues with high basal autophagy (e.g., liver, kidney) at high doses. However, at therapeutically relevant doses (10 mg/kg/day), the compound is well tolerated. Standard safety precautions (gloves, lab coat, safety glasses) should be followed. The compound is not a controlled substance. Store at -20degC, protect from light. ICSN3250 hydrochloride is for research use only; not for human therapeutic use.

[1]. mTOR Inhibition via Displacement of Phosphatidic Acid Induces Enhanced Cytotoxicity Specifically in Cancer Cells. Cancer Res. 2018 Sep 15;78(18):5384-5397.

ICSN3250 hydrochloride is also known as ICSN3250 HCl, halitulin analog. The molecular formula is C31H41ClN4O₈ (HCl salt) or C31H42ClN4O₈ (free base). The compound is a white to off-white powder. Solubility: water >10 mg/mL (pH 5-6), DMSO >50 mg/mL. The chemical structure includes a spiro ring and a morpholino group. ICSN3250 was first reported as a novel mTORC1 inhibitor with a unique mechanism of action (displacing phosphatidic acid) in 2018 (e.g., published by the Institute of Cancer Research, London). It is a valuable tool for studying the role of mTORC1 in cancer, autophagy, and metabolism, especially for understanding the PA-mTORC1 axis. The compound is not approved for clinical use; for research use only.

C31H41CLN4O8

633.13

632.261

1561902-79-1

ICSN3250

134817233

Typically exists as solids at room temperature

5

6

44

808

0

C1CCCCCCN(CCCCC1)CCCN2C=C(C(=C2)C3=CC(=C(C(=C3)O)O)[N+](=O)[O-])C4=CC(=C(C(=C4)O)O)[N+](=O)[O-].Cl

VTTAAXQGVVGOFT-UHFFFAOYSA-N

InChI=1S/C31H40N4O8.ClH/c36-28-18-22(16-26(30(28)38)34(40)41)24-20-33(21-25(24)23-17-27(35(42)43)31(39)29(37)19-23)15-11-14-32-12-9-7-5-3-1-2-4-6-8-10-13-32;/h16-21,36-39H,1-15H2;1H

5-[1-[3-(azacyclotridec-1-yl)propyl]-4-(3,4-dihydroxy-5-nitrophenyl)pyrrol-3-yl]-3-nitrobenzene-1,2-diol;hydrochloride

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.

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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.)