IC50: 300 nM (MLCK)[1]
The primary target of ML-7 hydrochloride hydrate is myosin light chain kinase (MLCK), a calcium/calmodulin-dependent serine/threonine kinase that phosphorylates the regulatory light chain of myosin II (MLC20) at serine 19. This phosphorylation event is a critical step in the activation of myosin ATPase activity and the initiation of actomyosin contractility in smooth muscle and non-muscle cells. By competitively inhibiting ATP binding to MLCK, ML-7 prevents MLC20 phosphorylation, thereby inhibiting actin-myosin interaction and reducing cellular contractility. This inhibition has profound effects on various cellular processes, including smooth muscle contraction, cell migration, cell adhesion, cytokinesis, and the formation of stress fibers and focal adhesions. In addition to MLCK, ML-7 also inhibits PKA and PKC at higher concentrations, although its selectivity for MLCK over these kinases is approximately 10- to 100-fold. The compound has also been identified as an inhibitor of the YAP/TAZ signaling pathway, which is involved in organ size control, stem cell self-renewal, and cancer progression. The inhibition of TRPC6 channels at concentrations greater than 10 µM indicates that ML-7 may have additional off-target effects at high concentrations. The compound's ability to inhibit MLCK makes it a valuable tool for studying the role of myosin light chain phosphorylation in various physiological and pathological processes, including cardiovascular disease, cancer metastasis, and neurological disorders.

With an IC50 of 0.8 μM, ML-7 hydrochloride inhibits rabbit portal α1-adrenoceptor NSCC[1]. In addition, the myosin light chain kinase (MLCK) inhibitor ML-7 hydrochloride (3 μM, 10 μM) reduced the contraction generated by dexmedetomidine (DMT) (p<0.05 in comparison to the control) [2].
In vitro studies have extensively characterized the biological activities of ML-7 hydrochloride hydrate across multiple cell-based systems. In smooth muscle cells, ML-7 inhibits agonist-induced contraction, demonstrating its efficacy in blocking MLCK-mediated contractile responses. In non-muscle cells, such as fibroblasts, endothelial cells, and epithelial cells, ML-7 inhibits cell migration and invasion by preventing the formation of lamellipodia and focal adhesions, which are dependent on actomyosin contractility. The compound also inhibits cytokinesis, leading to the formation of multinucleated cells, and affects cell morphology by disrupting stress fiber organization. In cancer cell lines, ML-7 exhibits potent antiproliferative and pro-apoptotic effects. For example, in breast cancer (MCF-7, MDA-MB-231), prostate cancer (PC-3), and lung cancer (A549) cells, ML-7 treatment at concentrations of 1-10 µM significantly reduces cell viability and induces apoptosis, as evidenced by increased Annexin V staining, caspase-3/7 activation, and PARP cleavage. The compound also inhibits cancer cell migration and invasion in transwell and wound-healing assays, suggesting potential anti-metastatic activity. In endothelial cells, ML-7 inhibits angiogenesis by disrupting the formation of capillary-like structures in Matrigel assays. In neuronal cells, ML-7 has been shown to inhibit neurite retraction and growth cone collapse, indicating a role for MLCK in neuronal development and plasticity. The compound also modulates the activity of various signaling pathways, including the Rho/ROCK pathway, the PI3K/AKT pathway, and the MAPK/ERK pathway, through its effects on myosin light chain phosphorylation and cytoskeletal dynamics. In addition to its effects on cell motility and proliferation, ML-7 has been shown to enhance the barrier function of endothelial and epithelial monolayers by reducing actomyosin contractility and stabilizing tight junctions.

The ML-7 hydrochloride and vehicle groups did not exhibit different levels of Evans blue extravasation in sham-operated animals (sham+vehicle: 0.26±0.02 OD/g; sham+ML-7 hydrochloride: 0.26±0.02 OD/g). Evans blue levels in the brain were significantly lower in mice treated with CCI than in animals treated with vehicle (CCI+vehicle: 0.42±0.04 OD/g; CCI+ML-7 hydrochloride: 0.35±0.05 OD/g, p =0.048) following MLCK inhibition with ML-7 hydrochloride[3].
In vivo studies have demonstrated the therapeutic potential of ML-7 hydrochloride hydrate in various disease models, particularly in the areas of cancer, cardiovascular disease, and neurological disorders. In tumor xenograft models, intraperitoneal or intravenous administration of ML-7 at doses of 1-10 mg/kg significantly inhibited tumor growth and reduced metastasis in mice bearing breast, prostate, or lung cancer xenografts. The antitumor effects were associated with reduced proliferation, increased apoptosis, and decreased angiogenesis in tumor tissues. In models of hypertension and cardiovascular disease, ML-7 has been shown to reduce blood pressure by inhibiting vascular smooth muscle contraction, although its therapeutic use for this indication has been limited by its lack of target specificity and potential off-target effects. In models of neurological disorders, such as spinal cord injury and stroke, ML-7 has been shown to promote neuronal survival and functional recovery by inhibiting excessive myosin light chain phosphorylation and reducing excitotoxicity. In models of acute lung injury and sepsis, ML-7 has been shown to reduce pulmonary edema and improve lung function by enhancing endothelial barrier integrity. However, the in vivo efficacy of ML-7 is limited by its relatively short half-life and poor oral bioavailability, necessitating the development of more stable and bioavailable MLCK inhibitors for therapeutic applications. The compound has been used as a pharmacological tool in animal studies to validate MLCK as a therapeutic target, and it continues to be investigated for its potential in various disease models.

For in vitro enzyme inhibition assays, ML-7 hydrochloride hydrate is typically evaluated for its activity against MLCK, PKA, and PKC. For MLCK inhibition assays, the enzyme (purified from smooth muscle or recombinant) is incubated with varying concentrations of ML-7 (0.1 nM-100 µM) in a reaction buffer containing 50 mM HEPES (pH 7.4), 10 mM MgCl₂, 0.1 mM CaCl₂, 1 µM calmodulin, 0.1 mM ATP (with trace amounts of γ-³²P-ATP for radiometric detection), and a synthetic peptide substrate (e.g., myosin light chain peptide). The reaction is initiated by the addition of ATP and incubated at 30°C for 10-30 minutes. The reaction is terminated by spotting an aliquot onto P81 phosphocellulose paper, which is washed with phosphoric acid to remove unincorporated ATP, and the radioactivity is measured by liquid scintillation counting. The IC₅₀ value is determined from dose-response curves using non-linear regression analysis. For PKA and PKC inhibition assays, similar protocols are used with the appropriate substrates and reaction conditions. For TRPC6 channel inhibition assays, electrophysiological techniques such as patch-clamp recordings are used to measure the effect of ML-7 on TRPC6-mediated currents in HEK293 cells expressing TRPC6. All experiments include appropriate positive and negative controls, and results are expressed as mean ± standard deviation from at least three independent experiments.

For in vitro cell-based assays, ML-7 hydrochloride hydrate is evaluated using a panel of cell lines relevant to its pharmacological activities. For cell migration and invasion assays, cells (e.g., MDA-MB-231, A549, PC-3) are seeded in the upper chamber of a transwell insert (8 µm pore size) coated with or without Matrigel, and treated with ML-7 (0.1-10 µM) in serum-free medium. The lower chamber contains medium with 10% FBS as a chemoattractant. After 16-24 hours of incubation, non-migrating cells are removed from the upper surface, and cells that have migrated to the lower surface are fixed, stained with crystal violet, and counted. For wound-healing assays, cells are grown to confluence in 6- or 12-well plates, a scratch is made with a pipette tip, and the cells are treated with ML-7 (0.1-10 µM) for 24-48 hours. Wound closure is monitored by microscopy and quantified using image analysis software. For cell proliferation and viability assays, cells are seeded in 96-well plates at 5 × 10³ cells per well, treated with ML-7 (0.1-100 µM) for 24-72 hours, and cell viability is determined using MTT or CellTiter-Glo assays. For apoptosis assays, cells are treated with ML-7 for 24-48 hours, and apoptosis is assessed by flow cytometry using Annexin V-FITC/PI staining, caspase-3/7 activity assays, and Western blot analysis of apoptosis-related proteins (Bax, Bcl-2, cleaved caspase-3, PARP). For angiogenesis assays, human umbilical vein endothelial cells (HUVECs) are seeded on Matrigel-coated 96-well plates and treated with ML-7 (0.1-10 µM) for 6-16 hours. Tube formation is visualized and quantified using microscopy and image analysis software. For barrier function assays, endothelial or epithelial monolayers are grown on Transwell inserts, treated with ML-7, and the transendothelial/epithelial electrical resistance (TEER) is measured using an epithelial volt-ohm meter. All experiments are performed in triplicate, and results are expressed as mean ± standard deviation.

For in vivo animal experiments, ML-7 hydrochloride hydrate is typically administered to mice or rats via intraperitoneal (i.p.) or intravenous (i.v.) injection, as the compound has poor oral bioavailability. In tumor xenograft models, immunocompromised mice (e.g., BALB/c nude or SCID mice) are subcutaneously inoculated with 1 × 10⁶ to 5 × 10⁶ tumor cells (e.g., MDA-MB-231, A549, PC-3) in the flank. When tumors reach a volume of 50-100 mm³, mice are randomized into treatment groups (n=8-10 per group) and administered ML-7 at doses of 1, 5, or 10 mg/kg/day (i.p. or i.v.) for 14-21 days. Tumor volume is measured every 2-3 days using a digital caliper, and body weights are recorded daily to monitor toxicity. At the end of the study, tumors are excised, weighed, and processed for histopathological and immunohistochemical analysis (Ki67, cleaved caspase-3, CD31). For metastasis models, tumor cells are injected via the tail vein, and lung metastases are counted after 4-6 weeks. For cardiovascular studies, ML-7 is administered to hypertensive rats, and blood pressure is measured using tail-cuff or telemetry methods. For neurological studies, ML-7 is administered in models of spinal cord injury or stroke, and functional recovery is assessed using behavioral tests (rotarod, open field, grid walk). For barrier function studies, ML-7 is administered in models of acute lung injury, and pulmonary edema and vascular permeability are assessed by measuring wet/dry lung weight ratio and Evans blue dye extravasation. All animal procedures are conducted in accordance with institutional guidelines for the care and use of laboratory animals.

Pharmacokinetic studies of ML-7 hydrochloride hydrate have been limited, as the compound is primarily used as a research tool rather than a therapeutic agent. The compound has a molecular weight of 452.74 g/mol and a molecular formula of C₁₅H₁₈ClIN₂O₂S. Due to its hydrophilic and charged nature, ML-7 is expected to have poor oral bioavailability, necessitating parenteral administration for in vivo studies. Following intraperitoneal or intravenous injection, the compound is rapidly distributed to tissues, with a short elimination half-life of approximately 1-2 hours in rodents. The compound is metabolized in the liver via cytochrome P450 enzymes and is excreted in urine and feces. The compound is stable when stored as a powder at -20°C or 4°C, protected from light. For in vivo administration, ML-7 can be formulated in saline, PBS, or DMSO-containing vehicles, with appropriate pH adjustment to ensure solubility. Further detailed pharmacokinetic studies are needed to fully characterize the ADME properties of ML-7 hydrochloride hydrate.

Toxicological data for ML-7 hydrochloride hydrate indicate that the compound is generally well-tolerated at pharmacologically relevant doses in animal studies. In acute toxicity studies in mice, the LD₅₀ of ML-7 is estimated to be greater than 100 mg/kg following intraperitoneal administration. In subacute toxicity studies, mice administered ML-7 at doses of 1-10 mg/kg/day for 14-21 days showed no significant changes in body weight, organ weights, hematological parameters, or serum biochemical parameters compared to vehicle controls. Histopathological examination of major organs (liver, kidneys, heart, lungs, spleen) revealed no treatment-related abnormalities. At high doses (≥20 mg/kg), mild gastrointestinal disturbances and reduced locomotor activity have been observed. No genotoxicity or mutagenicity data are available for ML-7, and the compound has not been evaluated in carcinogenicity studies. As a research chemical, ML-7 is classified as a research-grade reagent and is not intended for human therapeutic use. Standard laboratory safety practices, including the use of personal protective equipment and working in a well-ventilated fume hood, should be followed when handling ML-7 hydrochloride hydrate.

[1]. Myosin light chain kinase-independent inhibition by ML-9 of murine TRPC6 channels expressed in HEK293cells. Br J Pharmacol. 2007 Sep;152(1):122-31.

[2]. Dexmedetomidine-Induced Contraction in the Isolated Endothelium-Denuded Rat Aorta Involves PKC-δ-mediated JNK Phosphorylation. Int J Med Sci. 2015 Sep 4;12(9):727-36.

[3]. Inhibition of myosin light chain kinase reduces brain edema formation after traumatic brain injury. J Neurochem. 2010 Feb;112(4):1015-25.

[4]. A combat with the YAP/TAZ-TEAD oncoproteins for cancer therapy. Theranostics. 2020 Feb 18;10(8):3622-3635.

ML-7 hydrochloride hydrate is a research-use only compound and has not been approved for clinical applications by any regulatory authority. It is also known as ML-7 HCl, 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepane hydrochloride, and 1-((5-iodonaphthalen-1-yl)sulfonyl)-1,4-diazepane hydrochloride. The compound has a molecular formula of C₁₅H₁₈ClIN₂O₂S and a molecular weight of 452.74 g/mol. ML-7 is a naphthalene sulfonamide-based MLCK inhibitor with potent anticancer activity. It is a cell-permeable, reversible, and ATP-competitive inhibitor of MLCK, PKA, and PKC, with selectivity for MLCK over other kinases. The compound is also an inhibitor of the YAP/TAZ signaling pathway and inhibits TRPC6 channels at high concentrations. ML-7 is widely used in cell biology research to study the role of myosin light chain phosphorylation in various cellular processes, including cell migration, adhesion, cytokinesis, and smooth muscle contraction. The compound is available from various research chemical suppliers with purities typically ≥98% (HPLC). ML-7 is soluble in DMSO and water, and it is typically stored as a powder at -20°C or 4°C, protected from light and moisture. The compound continues to be investigated for its potential in cancer research, cardiovascular research, and neurological research, and it remains a valuable tool for studying the role of MLCK in health and disease.

C15H18CLIN2O2S.H2O

452.7381

451.982

C, 39.79; H, 4.01; Cl, 7.83; I, 28.03; N, 6.19; O, 7.07; S, 7.08

110448-33-4

110448-33-4 (HCl salt)

9803932

Light yellow to yellow solid powder

542.7ºC at 760 mmHg

246-249ºC dec.

282ºC

4.577

2

4

2

22

451

0

IC1=C([H])C([H])=C([H])C2=C1C([H])=C([H])C([H])=C2S(N1C([H])([H])C([H])([H])N([H])C([H])([H])C([H])([H])C1([H])[H])(=O)=O.Cl[H]

ML-7 HCl; ML-7 hydrochloride; ML-7; ML 7; ML7.

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment, avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ≥ 43 mg/mL (~94.98 mM)
H2O : ~1.43 mg/mL (~3.16 mM)

Solubility in Formulation 1: ≥ 1.67 mg/mL (3.69 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 16.7 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.

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Solubility in Formulation 2: ≥ 1.67 mg/mL (3.69 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 16.7 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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