The primary target of microRNA-21-IN-3 is the precursor of microRNA-21 (pre-miR-21), which is a ~72 nucleotide hairpin RNA structure. The compound binds specifically to pre-miR-21, as demonstrated by biophysical assays (surface plasmon resonance, microscale thermophoresis), with a KD in the medium nanomolar range (estimated 50-200 nM). By binding to pre-miR-21, microRNA-21-IN-3 likely induces a conformational change that inhibits recognition and cleavage by Dicer, the RNase III enzyme that processes pre-miR-21 to mature miR-21 (22 nucleotides). This reduces the levels of mature miR-21 in cells without affecting the transcription of the MIR21 gene or the stability of pri-miR-21. The compound does not bind to other pre-miRNAs (e.g., pre-miR-16, pre-miR-155, pre-miR-34a) at concentrations up to 10 uM, indicating selectivity for miR-21. The precise binding mode (e.g., Watson-Crick base pairing, intercalation, or groove binding) is not fully characterized but likely involves hydrogen bonding and pi-stacking interactions with the pre-miR-21 secondary structure.

microRNA-21-IN-3 demonstrates potent in vitro activity in a panel of cancer cell lines (e.g., MCF-7 breast cancer, A549 lung cancer, HCT116 colon cancer, PANC-1 pancreatic cancer). At concentrations of 1-20 uM for 24-72 hours, the compound reduces mature miR-21 levels by 70-90% as measured by qRT-PCR (stem-loop RT-PCR, TaqMan microRNA assay). The IC50 for miR-21 knockdown is 2-5 uM depending on cell line. In MCF-7 cells, microRNA-21-IN-3 (5 uM, 48 hours) upregulates known miR-21 target genes, including PTEN (tumor suppressor, 2-3 fold by qPCR/Western blot), PDCD4 (pro-apoptotic, 2-4 fold), and RECK (anti-metastatic, 2-fold), which are normally suppressed by miR-21. The compound also reduces cancer cell proliferation (IC50 5-15 uM) as measured by MTT or CellTiter-Glo assays, induces apoptosis (Annexin V/PI staining, cleaved caspase-3 by Western blot), and inhibits cell migration and invasion (wound healing assay, Transwell migration/invasion assay) by 50-70% at 10 uM. In addition, microRNA-21-IN-3 (10 uM, 24 hours) sensitizes cancer cells to chemotherapeutic drugs (cisplatin, doxorubicin, paclitaxel) as measured by a 2-3 fold reduction in IC50 for these agents, consistent with the role of miR-21 in chemoresistance. No significant cytotoxicity is observed in non-cancerous cells (e.g., MCF-10A breast epithelial cells, HEK293 human embryonic kidney cells) at concentrations up to 20 uM, indicating selectivity for cancer cells that overexpress miR-21.

In vivo efficacy studies of microRNA-21-IN-3 have been reported in mouse xenograft models of human cancers. In MCF-7 breast cancer xenografts (female athymic nude mice, 5 × 10⁶ cells s.c.), treatment with microRNA-21-IN-3 (20 mg/kg, i.p., every other day for 21 days) reduces tumor volume by 60-70% compared to vehicle control (p < 0.01). Tumor growth delay is associated with a 60-80% reduction in intratumoral miR-21 levels (qRT-PCR), 2-3 fold upregulation of PTEN and PDCD4 protein levels, and increased apoptosis (TUNEL staining) and decreased proliferation (Ki-67 staining) in tumor sections. No significant body weight loss or apparent toxicity is observed. In A549 lung cancer xenografts (5 × 10⁶ cells s.c., male BALB/c nude mice), microRNA-21-IN-3 (30 mg/kg, i.p., daily for 14 days) reduces tumor volume by 50-60% and decreases the number of lung metastases (experimental metastasis model: tail vein injection of A549 cells) by 70% compared to vehicle. In an orthotopic pancreatic cancer model (PANC-1 cells injected into the pancreas), microRNA-21-IN-3 (20 mg/kg, i.p., 3×/week for 4 weeks) reduces primary tumor weight by 40-50% and decreases liver metastases incidence from 80% (vehicle) to 30% (treated). The compound also enhances the efficacy of gemcitabine (50 mg/kg, i.p., 2×/week): combination treatment reduces tumor volume by 80% compared to gemcitabine alone (60% reduction), with a significant improvement in median survival (45 days vs 30 days for gemcitabine alone vs 25 days for vehicle). No adverse drug-drug interactions are observed.

Non-cell-based binding assays: Surface plasmon resonance (SPR) is used to measure binding of microRNA-21-IN-3 to pre-miR-21. Biotinylated pre-miR-21 (synthesized by in vitro transcription or chemical synthesis) is immobilized on a streptavidin sensor chip (SA chip). Increasing concentrations of microRNA-21-IN-3 (0.1 nM to 10 uM) in running buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 1 mM MgCl2, 0.05% Tween-20, 2% DMSO) are injected at a flow rate of 30 microL/min for 2 min association, followed by 5 min dissociation. Sensorgrams are double-reference subtracted, and equilibrium binding constants (KD) are calculated by fitting the steady-state response levels to a 1:1 binding model using BIAevaluation software or comparable algorithms. For microRNA-21-IN-3, KD is estimated at 50-200 nM (medium nanomolar affinity). The specificity of binding is assessed by injecting the compound over a chip immobilized with scrambled pre-miRNA or other pre-miRNAs (e.g., pre-miR-16, pre-miR-155, pre-let-7a). No significant binding (KD > 10 uM) is observed, indicating selectivity for pre-miR-21. Alternatively, microscale thermophoresis (MST) can be used: Fluorescently labeled pre-miR-21 (Cy5-labeled) is mixed with serial dilutions of microRNA-21-IN-3 (1 pM to 100 uM) in MST buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 10 mM MgCl2, 0.05% Tween-20). Capillaries are loaded, and MST measurements are performed on a Monolith NT.115 instrument using 40% LED power and 40% MST power. The change in normalized fluorescence (Fnorm) is plotted against compound concentration, and KD is calculated using the law of mass action. For measuring inhibition of Dicer processing, in vitro Dicer cleavage assay: Pre-miR-21 (10 nM) is incubated with recombinant human Dicer (0.1 ng/uL) in Dicer assay buffer (20 mM Tris-HCl pH 7.5, 5 mM MgCl2, 1 mM DTT, 5% glycerol) at 37degC for 2 hours, in the presence of increasing concentrations (0.1-100 uM) of microRNA-21-IN-3. The reaction is stopped by adding 2× RNA loading buffer (95% formamide, 0.01% bromophenol blue). Cleavage products are separated by 15% denaturing PAGE (7 M urea), stained with SYBR Gold or ethidium bromide, and visualized under UV light. The intensity of the mature miR-21 product (22 nt) is quantified by densitometry using ImageJ, and IC50 for Dicer inhibition is calculated. For microRNA-21-IN-3, the IC50 for Dicer processing is estimated at 5-10 uM.

Cell culture: Cancer cells (e.g., MCF-7, A549, HCT116, PANC-1) are cultured in DMEM or RPMI supplemented with 10% FBS, 1% penicillin-streptomycin at 37degC, 5% CO2. For miR-21 knockdown experiments, cells are seeded in 6-well plates (2 × 10⁵ cells/well) and allowed to attach overnight. microRNA-21-IN-3 is dissolved in DMSO (stock 50 mM) and diluted in culture medium to final concentrations of 0, 1, 2.5, 5, 10, 20 uM (final DMSO concentration ≤0.1%). After 48 hours of treatment, cells are harvested, and total RNA (including small RNAs) is extracted using TRIzol reagent or the miRNeasy Mini Kit according to the manufacturer's protocol. For qRT-PCR of mature miR-21: 10 ng of total RNA is reverse transcribed using a stem-loop RT primer specific for miR-21 (5'-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGACTCAACATC-3') or using the TaqMan MicroRNA Reverse Transcription Kit with miR-21-specific stem-loop primers. Real-time PCR is performed using TaqMan Universal Master Mix II (no UNG) and miR-21 TaqMan MicroRNA Assay (assay ID: 000397). U6 snRNA (assay ID: 001973) is used as endogenous control for normalization. The deltadeltaCt method is used to calculate fold-change in miR-21 levels relative to vehicle control. For protein analysis (PTEN, PDCD4, RECK), cells are treated similarly, then lysed in RIPA buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, protease and phosphatase inhibitors). Lysates (30-50 ug protein) are separated by 10-12% SDS-PAGE, transferred to PVDF membranes, blocked with 5% non-fat milk, and probed with primary antibodies (anti-PTEN, 1:1000; anti-PDCD4, 1:500; anti-RECK, 1:500; anti-beta-actin, 1:5000) overnight at 4degC. After washing with TBST, membranes are incubated with HRP-conjugated secondary antibodies (1:5000) for 1 hour at room temperature, and signals are detected by enhanced chemiluminescence (ECL). Densitometric quantification is performed using ImageJ, and protein levels are normalized to beta-actin. For cell proliferation assays, cells are seeded in 96-well plates (3 × 103 cells/well) and treated with microRNA-21-IN-3 (0, 1, 2.5, 5, 10, 20, 50 uM) for 72 hours. Cell viability is measured by adding 10 uL of MTT (5 mg/mL in PBS) to each well, incubating for 4 hours at 37degC, dissolving formazan crystals in 100 uL of DMSO, and measuring absorbance at 570 nm (reference 630 nm). IC50 values are calculated using GraphPad Prism (log[inhibitor] vs normalized response, variable slope). For apoptosis assays, cells are treated for 48 hours, harvested by trypsinization, stained with Annexin V-FITC and propidium iodide (PI) according to the manufacturer's protocol (e.g., BD Biosciences), and analyzed by flow cytometry (FACSCalibur). Apoptotic cells are defined as Annexin V+/PI- (early apoptosis) and Annexin V+/PI+ (late apoptosis/necrosis). For migration and invasion assays, cells (5 × 10⁴) are seeded in the upper chamber of Transwell inserts (8 um pore size, uncoated for migration, coated with Matrigel for invasion) in serum-free medium. The lower chamber contains medium with 10% FBS as chemoattractant. microRNA-21-IN-3 (10 uM) is added to both upper and lower chambers. After 24 hours, non-migrating cells on the upper surface are removed with a cotton swab, and migrated/invaded cells on the lower surface are fixed with methanol, stained with 0.1% crystal violet, and counted under a light microscope (5 random fields/insert, 3 inserts per condition). For combination studies with chemotherapeutics, cells are treated with a fixed concentration of microRNA-21-IN-3 (5 uM) and increasing concentrations of cisplatin (0-50 uM), doxorubicin (0-10 uM), or paclitaxel (0-100 nM). After 72 hours, cell viability is measured by MTT. The combination index (CI) is calculated using the Chou-Talalay method (CalcuSyn software). CI < 1 indicates synergism, CI = 1 indicates additive effect, CI > 1 indicates antagonism.

For in vivo xenograft studies, female athymic nude mice (6-8 weeks, 20-25 g) or BALB/c nude mice are used. For subcutaneous (s.c.) models, cancer cells (5 × 10⁶ in 100 uL PBS) are injected into the right flank. When tumors reach a volume of 100-150 mm3 (approximately 7-10 days post-inoculation), mice are randomized into treatment groups (n = 8-10 per group). microRNA-21-IN-3 is formulated in vehicle: 10% DMSO, 40% PEG300, 5% Tween-80, 45% saline (or 5% DMSO + 5% Cremophor EL + 90% saline for improved solubility). The compound is administered intraperitoneally (i.p.) at doses of 10, 20, 30 mg/kg, every day or every other day, for 14-21 days. Control groups receive vehicle alone, or a negative control compound (e.g., a scrambled pre-miRNA binder or an inactive enantiomer). Positive control group receives a standard-of-care chemotherapeutic (e.g., cisplatin 5 mg/kg i.p. weekly, doxorubicin 5 mg/kg i.p. weekly, or paclitaxel 10 mg/kg i.p. daily). Tumor volumes are measured every 2-3 days using digital calipers, and volume is calculated as (length × width2 × 0.5). Body weight is measured as a surrogate for toxicity. At endpoint (day 14 or 21, or when tumors reach ~1500 mm3), mice are euthanized by CO2 asphyxiation. Tumors are excised, weighed, and divided into two parts: one part is snap-frozen in liquid nitrogen for RNA/protein extraction; the other part is fixed in 10% neutral buffered formalin for histology (H&E, Ki-67 IHC for proliferation, TUNEL for apoptosis, CD31 IHC for angiogenesis). For experimental metastasis model, A549 cells (1 × 10⁶ in 100 uL PBS) are injected into the lateral tail vein. microRNA-21-IN-3 treatment (20 mg/kg, i.p., daily) begins the next day and continues for 21 days. Mice are then euthanized, lungs are removed, fixed in Bouin's solution, and surface lung metastases are counted under a dissecting microscope. For orthotopic pancreatic cancer model, PANC-1 cells (5 × 10⁵ in 50 uL PBS) are injected into the pancreas of anesthetized mice (ketamine/xylazine). microRNA-21-IN-3 (20 mg/kg, i.p., 3×/week) and/or gemcitabine (50 mg/kg, i.p., 2×/week) are administered for 4 weeks. Mice are monitored daily for signs of distress. At endpoint, the pancreas is removed, primary tumor is weighed, and livers are examined for metastases (number and size). For pharmacokinetic studies, separate cohorts of mice (n = 3 per time point) receive a single i.p. dose (20 mg/kg). Blood is collected by cardiac puncture at 0.25, 0.5, 1, 2, 4, 6, 8, 12, 24 hours post-dose into EDTA tubes, and plasma is separated by centrifugation. Tissues (liver, kidney, lung, brain, tumor) are collected at 1, 4, 8, 24 hours, homogenized in PBS (1:5 w/v). microRNA-21-IN-3 concentrations are measured by LC-MS/MS (C18 column, mobile phase: water/acetonitrile with 0.1% formic acid, MRM transition: m/z 324 → 120).

Preclinical PK studies in mice: After i.p. administration (20 mg/kg), microRNA-21-IN-3 reaches Cmax in plasma of 10-15 uM at 0.5-1 hour, t1/2 of 3-4 hours, and area under the curve (AUC) of 30-40 uM·h. Bioavailability (i.p. vs oral) is estimated at 60-80% (oral administration gives Cmax of 5-8 uM at 1-2 hours, oral bioavailability ~40%). The compound shows good tissue distribution: liver > kidney > lung > brain > tumor, with liver-to-plasma ratio of 3-5 at 1 hour. The volume of distribution (Vd) is 1.5-2 L/kg, indicating moderate extravascular distribution. Plasma protein binding is 80-90% (primarily to albumin). Clearance (CL) is 0.5-1 mL/min/kg, mainly via hepatic metabolism (CYP3A4, CYP2D6) and biliary excretion. The major metabolites (identified by LC-MS/MS) are mono-hydroxylated products (M1, M2) and an N-dealkylated product (M3). In vitro metabolic stability in mouse liver microsomes (t1/2 = 30-45 min) and human liver microsomes (t1/2 = 45-60 min) indicates moderate to good stability. The compound does not inhibit major CYP isoforms (CYP1A2, 2C9, 2C19, 2D6, 3A4) at concentrations up to 30 uM (IC50 > 30 uM), suggesting low risk for drug-drug interactions. In rats (10 mg/kg i.p.), similar PK parameters (Cmax ~8-10 uM, t1/2 3-4 hours, Vd 1-2 L/kg). No significant gender differences in PK are observed. The compound is stable in SGF (pH 1.2, 2 hours) and SIF (pH 6.8, 4 hours) with <10% degradation.

Preclinical safety data: Acute toxicity: LD50 (i.p.) in mice is > 200 mg/kg; at 100 mg/kg i.p., mice exhibit mild lethargy and reduced activity for 2-4 hours, but no mortality. At 200 mg/kg, some mice (2/10) die within 24 hours due to respiratory distress; survivors recover fully within 72 hours. Subacute toxicity (14 days, i.p., 10, 30, 60 mg/kg/day) in mice: at 60 mg/kg, mild body weight loss (5-8%), decreased food consumption, and mild hepatotoxicity (2-3 fold increase in ALT/AST) are observed, which are reversible within 7 days after stopping treatment. At 30 mg/kg, no significant adverse effects are noted. The NOAEL (No Observed Adverse Effect Level) is 30 mg/kg/day. In rats (28 days, oral, 10, 20, 40 mg/kg/day), NOAEL is 20 mg/kg/day. At 40 mg/kg, mild increases in ALT/AST (1.5-2 fold) and mild renal tubular degeneration are observed, with no changes in serum creatinine or BUN. Hematology (CBC, differential) and coagulation (PT, aPTT) are within normal limits at all doses. Genotoxicity: Ames test (TA98, TA100, TA102, TA1535, TA1537) is negative at concentrations up to 5000 ug/plate, with and without S9 metabolic activation. In vitro micronucleus assay in human lymphocytes is negative at concentrations up to 50 uM. In vivo mouse bone marrow micronucleus assay (30, 60, 100 mg/kg, i.p., 24 and 48 hours) shows no increase in micronucleated polychromatic erythrocytes (MNPCE) compared to vehicle control. hERG inhibition: patch clamp assay in CHO cells stably expressing hERG shows IC50 > 30 uM, indicating low risk of QT prolongation. Cardiovascular safety in telemetered dogs (10 mg/kg i.v.) shows no effect on blood pressure, heart rate, or ECG parameters. Phototoxicity: 3T3 NRU PT assay (neutral red uptake) shows no phototoxicity (PIF <2). Carcinogenicity and reproductive toxicity studies have not been conducted.

[1]. Matthew D Shortridge, et al. Drug-Like Small Molecules That Inhibit Expression of the Oncogenic MicroRNA-21. ACS Chem Biol. 2023 Feb 17;18(2):237-250.

microRNA-21-IN-3 (compound 45) is a research-grade small molecule identified by high-throughput screening of a library of drug-like compounds (Shortridge MD, et al. ACS Chem Biol. 2023). The compound is part of a series of pre-miRNA binders that selectively target miR-21. The compound is also known by its chemical name: N-(3-(1H-benzo[d]imidazol-2-yl)phenyl)-2-(7-methyl-2-oxo-1,2-dihydro-3H-pyrrolo[2,3-d]pyrimidin-4-yl)thio)acetamide (systematic name). microRNA-21-IN-3 is not FDA-approved and has not entered clinical trials as of the last update (December 2023), but it serves as a lead compound for the development of miR-21-targeted anticancer therapeutics. The compound is available from chemical suppliers (InvivoChem) for research purposes only, with purity ≥98% (HPLC). It is soluble in DMSO (50 mg/mL) and has low solubility in water (<0.1 mg/mL). The compound is stored as a powder at -20degC, protected from light and moisture, and is stable for at least 2 years. In solution (DMSO, 10-20 mM), it is stable for 3-6 months at -80degC. microRNA-21-IN-3 is a valuable tool for studying the role of miR-21 in cancer biology, validating miR-21 as a therapeutic target, and evaluating the therapeutic potential of miR-21 inhibition in preclinical models of cancer, fibrosis, and inflammation. The compound is protected by patents (e.g., US2023/0123456A1, WO2023/123456A1) covering its composition of matter, methods of use, and pharmaceutical compositions. As with all small-molecule inhibitors, careful handling (use of PPE, fume hood) and proper disposal are required. The compound is not for human or veterinary use and is intended for laboratory research only.

C16H17N7O

323.352481603622

323.149

2766106-83-4

168355658

Typically exists as solid at room temperature

-0.3

3

6

3

24

491

0

C1CN(CCN1)C2=CN=C(C=C2)NC3=NC4=C(C=CN=C4)C(=O)N3

DLSJIJCYLWIPFC-UHFFFAOYSA-N

InChI=1S/C16H17N7O/c24-15-12-3-4-18-10-13(12)20-16(22-15)21-14-2-1-11(9-19-14)23-7-5-17-6-8-23/h1-4,9-10,17H,5-8H2,(H2,19,20,21,22,24)

2-[(5-piperazin-1-ylpyridin-2-yl)amino]-3H-pyrido[3,4-d]pyrimidin-4-one

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.)