ROCKII:12 nM (IC50); CaMKII:0.18 μM (IC50); PKG:0.36 μM (IC50); AuroraA:0.745 μM (IC50); MLCK:28.3 μM (IC50); EGFR:50 μM (IC50); MKK4:16.9 μM (IC50); GSK3α:60.7 μM (IC50); PKA:3.03 μM (IC50); Src:3.06 μM (IC50); PKC:5.68 μM (IC50); Abl:7.77 μM (IC50); AMPK:100 μM (IC50) P38α:100 μM (IC50)
The primary target of H-1152 is rho-kinase (ROCK), a serine/threonine kinase involved in the regulation of the actin cytoskeleton. It acts as an ATP-competitive inhibitor, binding to the ATP-binding site of ROCK and preventing its phosphorylation of downstream substrates. H-1152 has a Ki of 1.6 nM for ROCK, demonstrating high potency. It exhibits weaker affinity for other kinases, with a Ki of 630 nM for PKA, 9.27 µM for PKC, and 10.1 µM for MLCK.

The IC50 for ROCK2 of the Rho kinase inhibitor H-1152 is 12 nM. With IC50 values of 0.180, 0.360, 0.745, respectively, for CaMKII, PKG, AuroraA, PKA, Src, PKC, MLCK, Abl, EGFR, MKK4, GSK3α, AMPK, and P38α, H-1152 (H-1152P) likewise exhibits reduced inhibitory action [1]. H-1152 has a 1.6 nM Ki for Rho kinase potency and a 0.63, 9.27, and 10.1 μM Ki for PKA, PKC, and MLCK, respectively, for PKA, PKC, and MLCK moderate inhibition. H-1152 (0.1-10 µM) exhibits a strong inhibitory effect on MARCKS phosphorylation in LPA-treated cells, with an IC50 value of 2.5 µM; however, no discernible effect is observed in PDBu-treated cells [2]. H-1152 (0.5-10 μM) does not result in a decrease in neuronal viability. Additionally, H-1152 (1, 5, or 10 μM) did not change the proportion of various neuronal morphologies. Moreover, in BMP4 and LIF cultures, H-1152 (10 μM) lengthens neurites [3].

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In vitro, H-1152 is a potent and selective inhibitor of ROCK. It inhibits the phosphorylation of MARCKS in cells stimulated by lysophosphatidic acid and inhibits EP3-stimulated NO formation. The compound prevents fragmentation of apoptotic cells and has been shown to relieve neuropathic pain. Its high selectivity for ROCK over other kinases makes it a valuable tool for studying ROCK-mediated signaling pathways.

In vivo, H-1152 has been shown to relieve neuropathic pain. As a potent and selective ROCK inhibitor, it is used in animal models to study the role of ROCK in various physiological and pathological processes, including pain, inflammation, and cardiovascular function. Its effects are dose-dependent and related to its ability to inhibit ROCK activity in target tissues.

Protein kinase assay[2]To determine the effect of inhibitors on Rho-kinase activity in vitro, inhibitors was added at the indicated concentrations to 50 µL of the assay mixture 50 mm Tris-HCl (pH 7.5), 5 mm MgCl2, 1 mm EDTA, 1 mm EGTA, 1 mm dithiothreitol, 40 µm S6-peptide, various concentrations of [γ-32P]ATP and purified Rho-kinase, which was purified as described previously (Nagumo et al. 2000). The reactions were started by the addition of [γ-32P]ATP and carried out at 30°C for 5 min. The Michaelis–Menten equation was used to calculate the Michaelis constant (Km) and maximal velocity (Vmax) of Rho-kinase. Data were further analyzed with secondary plot to calculate the inhibitory constant (Ki). The effects of Rho-kinase inhibitors on protein kinase A (PKA) and C (PKC) were assayed according to our method with minor modifications (Tokumitsu et al. 1991). The reaction for PKA was carried out at 37°C for 5 min in a 20-µL mixture containing 25 mm Tris-HCl (pH 7.0), 10 mm MgCl2, 2 mm EGTA, 50 µm peptide of cyclic AMP response element binding protein and 10 µm[γ-32P]ATP (150 cpm/pmol). The reaction for PKC was carried out for 5 min in 30 µL of a mixture that contained 50 mm Tris-HCl (pH 7.0), 10 mm MgCl2, 0.5 mm CaCl2, 0.2 mg/mL histone type III-S, 50 µg/mL phosphatidyl serine and 10 µm[γ-32P]ATP (150 cpm/pmol). The effect of Rho-kinase inhibitors on myosin light chain kinase (MLCK) was assayed under the conditions previously described elsewhere (Asano et al. 1989), in 30 µL of a reaction mixture that contained 40 mm HEPES (pH 7.0), 5 mm magnesium acetate, 0.5 mm CaCl2, 0.1% (v/v) Tween-80, 5.3 nm calmodulin, 51 µm myosin light chain peptide, 1.95 nm MLCK and [γ-32P]ATP (150 cpm/pmol)[2].

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In vitro enzyme assays for H-1152 measure its inhibition of ROCK activity. The kinase activity is assessed using a substrate peptide and ATP, and the incorporation of phosphate is measured. The compound's Ki value of 1.6 nM is determined from these assays. Its selectivity over other kinases, such as PKA, PKC, and MLCK, is confirmed by testing its inhibitory activity against these enzymes.

ROCK inhibitor H-1152 was diluted in water and added in an additional 10 μl to cultures 24 h after plating. Water was added to controls. Eighteen hours after the addition of inhibitor, cultures were fixed in 4% paraformaldehyde (1 h at room temperature for peroxidase-linked labeling and 20 min at room temperature for fluorescence labeling). For ArrayScan/Cellomics automated analysis: Cells were plated in a total volume of 50 μl on 384 well plastic plates previously coated with poly-d-lysine/laminin, and cultured in the same medium[3].

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To investigate the effects of the protein kinase inhibitors (e.g. H-1152 ), the cells were preincubated with the inhibitors for 15 min and then stimulated with LPA for 1 min or with PDBu for 5 min. At various determination points after the stimulation, 5% (v/v) trichloroacetic acid was added to the cultures and cells were scraped off with a rubber scraper. After sedimentation, the precipitate was washed with acetone containing 6 mm dithiothreitol to remove trichloro-acetic acid and then dried. The dried cell powder was placed in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) sample buffer containing 125 mm Tris (pH 7.4), 8 m urea, 10% (w/v) sucrose, 0.02% (w/v) bromophenol blue, 4% (w/v) SDS and 2-mercaptoethanol, and the preparation was then extracted by heating at 95°C for 5 min. The samples were passed through a 0.22-µm centrifugal filter (Millipore, Bedford, MA, USA) to remove indissoluble materials. The samples (2.5 × 105 cells) were subjected to SDS–PAGE (7.5%)/western blotting analysis. Proteins in the gel were electrotransferred to a polyvinylidene difluoride membrane. After incubation with 5% (w/v) skimmed milk, the membrane was blotted with pS159-Mar-Ab, followed by anti-mouse IgG antibody. Immunoreactivity was detected by enhanced chemiluminescence-western blotting detection reagents, and its extent was measured by scanning densitometry of the X-ray film using KODAK 1D Image Analysis Software, and expressed as a percentage versus control under each condition[2].

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In vitro cell-based assays for H-1152 evaluate its effects on ROCK-mediated cellular functions. Cells are treated with the compound, and the phosphorylation of ROCK substrates, such as MARCKS, is measured by Western blot. The compound's ability to inhibit cell migration, proliferation, or apoptosis can also be assessed. These assays confirm its activity as a ROCK inhibitor in a cellular context.

In vivo animal studies for H-1152 are conducted to evaluate its pharmacological effects. The compound is administered via intraperitoneal or oral routes. Models of neuropathic pain, inflammation, or cardiovascular disease are used to assess its efficacy. Pharmacodynamic studies measure ROCK activity and the phosphorylation of downstream targets in tissues. Pharmacokinetic studies determine the compound's half-life and bioavailability.

The pharmacokinetic properties of H-1152 have been characterized in preclinical studies. The compound is soluble in water, ethanol (~20 mg/ml), DMSO (~12.5 mg/ml), DMF (~15 mg/ml), and methanol. Its molecular weight is 392.34. For research use, it is stored at -20°C. The compound has a melting point of 182-184°C. Further details on its half-life, Cmax, and bioavailability are available from the primary research literature.

The toxicity profile of H-1152 has not been extensively detailed in the available literature. It is classified for research use only and not for human consumption. As a potent kinase inhibitor, its potential side effects may include off-target effects at higher concentrations. Standard safety precautions for handling bioactive compounds should be followed.

[1]. Development of specific Rho-kinase inhibitors and their clinical application. Biochim Biophys Acta. 2005 Dec 30;1754(1-2):245-52. Epub 2005 Sep 12.

[2]. Inhibition of rho-kinase-induced myristoylated alanine-rich C kinase substrate (MARCKS) phosphorylation in human neuronal cells by H-1152, a novel and specific Rho-kinase inhibitor. J Neurochem. 2002 Apr;81(1):9-16.

[3]. Accelerated neurite growth from spiral ganglion neurons exposed to the Rho kinase inhibitor H-1152. Neuroscience. 2010 Aug 25;169(2):855-62.

(S)-2-methyl-1-(4-methylisoquinoline-5-sulfonyl)-1,4-diazacycloheptane belongs to the isoquinoline class of compounds and is a sulfonamide formed by the condensation of the sulfonyl group of 4-methylisoquinoline-5-sulfonic acid with the 1-amino group of (S)-2-methyl-1,4-diazacycloheptane. It is an EC 2.7.11.1 (non-specific serine/threonine protein kinase) inhibitor. It belongs to the isoquinoline class of compounds and is also an N-sulfonyldiazacycloheptane. It is the conjugate base of (S)-2-methyl-1-(4-methylisoquinoline-5-sulfonyl)-1,4-diazacycloheptane (2+).

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Additional information: H-1152 is also known as (S)-(+)-2-Methyl-1-[(4-methyl-5-isoquinolinyl)sulfonyl]homopiperazine. It is a more potent and selective ROCK inhibitor than Y-27632, with a Ki of 1.6 nM. The compound is cell-permeable and is used to study ROCK signaling in various cellular processes. This product is for research use only and is not approved for clinical or therapeutic applications.

C16H21N3O2S

319.423

319.1354

C, 60.16; H, 6.63; N, 13.16; O, 10.02; S, 10.04

451462-58-1

H-1152 dihydrochloride;871543-07-6;Glycyl H-1152 hydrochloride;913844-45-8

448043

Typically exists as Off-white to light yellow solids at room temperature

4.867

1

5

2

22

476

1

CC1=CN=CC2=C1C(S(=O)(N3[C@@H](C)CNCCC3)=O)=CC=C2

AWDORCFLUJZUQS-ZDUSSCGKSA-N

InChI=1S/C16H21N3O2S/c1-12-9-18-11-14-5-3-6-15(16(12)14)22(20,21)19-8-4-7-17-10-13(19)2/h3,5-6,9,11,13,17H,4,7-8,10H2,1-2H3/t13-/m0/s1

4-Methyl-5-[[(2S)-2-methyl-1,4-diazepan-1-yl]sulfonyl]isoquinoline

H 1152; H1152; 451462-58-1; (S)-2-METHYL-1-[(4-METHYL-5-ISOQUINOLINE)SULFONYL]-HOMOPIPERAZINE; H-1152P; CHEBI:88220; 4-methyl-5-[[(2S)-2-methyl-1,4-diazepan-1-yl]sulfonyl]isoquinoline; H1152; (S)-4-Methyl-5-((2-methyl-1,4-diazepan-1-yl)sulfonyl)isoquinoline; H-1152

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.)