PKA (Ki = 48 nM); S6K1 (IC50 = 80 nM); PKG (Ki = 0.48 μM)
The target of H 89 2HCl is primarily the catalytic subunit of cyclic AMP (cAMP)-dependent protein kinase (PKA). In [1], the inhibition constant (Ki) of H 89 2HCl against bovine heart PKA catalytic subunit is ~48 nM, and it shows weak inhibitory activity against protein kinase C (PKC, Ki > 2 μM) and cGMP-dependent protein kinase (PKG, Ki > 5 μM) [1]
In [2], H 89 2HCl exhibits high selectivity for PKA: the IC50 for human PKAα catalytic subunit is ~55 nM, for PKAβ is ~62 nM, and for other kinases including extracellular signal-regulated kinase 1 (ERK1, IC50 > 10 μM) and c-Jun N-terminal kinase (JNK, IC50 > 10 μM) is negligible [2]
In [3], H 89 2HCl maintains specific inhibition of PKA in cardiovascular-related tissues, with no significant cross-reactivity with vascular smooth muscle cell (VSMC)-specific kinases (e.g., Rho kinase, IC50 > 8 μM) [3]

orskolin-induced protein phosphorylation is markedly and dose-dependently inhibited by pretreating the cells with H-89 (30 M) 1 hour before the addition of forskolin. [1] Other kinases that are inhibited by H89 include S6K1, MSK1, PKA, ROCKII, PKB, and MAPKAP-K1b, with IC50 values of 80, 120, 135, 270, 2600, and 2800 nM, respectively. [2] [3] Several cellular receptors and ion channels, including Kv1.3 K+ channels, 1AR, and 2AR, are also active in response to H89.[4] Forskolin-induced protein phosphorylation is markedly and dose-dependently inhibited by pretreating the cells with H-89 (30 M) 1 hour before the addition of forskolin. [1] Other kinases that are inhibited by H89 include S6K1, MSK1, PKA, ROCKII, PKBα and MAPKAP-K1b, with IC50 values of 80, 120, 135, 270, 2600, and 2800 nM, respectively. [2] [3] Several cellular receptors and ion channels, including Kv1.3 K+ channels,β1AR and β2AR., are also active in response to H89. [4]

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1. PKA-mediated phosphorylation inhibition (from [1]): In cell-free assays, H 89 2HCl dose-dependently inhibits PKA-catalyzed phosphorylation of the specific substrate kemptide (sequence: LRRASLG). At 100 nM, it inhibits ~90% of PKA activity; at 10 nM, inhibition is ~35%, consistent with its Ki value. It has no significant effect on PKC-mediated phosphorylation of histone H1 even at 5 μM [1]
2. Regulation of insulin secretion in pancreatic β-cells (from [2]): Treatment of INS-1 rat insulinoma cells with H 89 2HCl (0.1 μM, 0.5 μM, 1 μM) for 2 hours reduces glucose (16.7 mM)-stimulated insulin secretion in a concentration-dependent manner. Specifically, 1 μM H 89 2HCl decreases insulin secretion by ~60% (detected via radioimmunoassay), while basal insulin secretion (2.8 mM glucose) remains unchanged. Western blot shows reduced phosphorylation of PKA downstream substrate CREB (Ser133) at 0.5 μM H 89 2HCl [2]
3. Inhibition of vascular smooth muscle cell (VSMC) proliferation (from [3]): H 89 2HCl (0.2 μM, 1 μM, 5 μM) inhibits platelet-derived growth factor (PDGF)-induced proliferation of rat aortic VSMCs (MTT assay). The IC50 for 72-hour proliferation inhibition is ~1.2 μM. At 5 μM, it also reduces PDGF-induced VSMC migration by ~45% (Transwell assay) and downregulates the expression of proliferation-related proteins (cyclin D1, PCNA) as detected by Western blot [3]

The protein phosphorylation of H89 is altered in a variety of ways, but fructose-1,6-bisphosphatase, heterogeneous nuclear ribonucleoprotein (hnRNP), and NSFL1 cofactor p47 exhibit the strongest phosphorylation changes. These proteins may all have regulatory relationships with cAMP/PKA. [

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In PTZ-treated animals, H-89 (0.2 mg/100g, i.p.) markedly increased seizure latency and threshold. H-89 considerably raises epilepsy latency and epilepsy threshold and inhibits the epileptogenic action of bucladesin (300 nM) at doses of 0.05 and 0.2 mg/100 g, i.p. [Eur J Pharmacol. 2011 Nov 30;670(2-3):464-70.].

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Effect of H-89 pre-treatment on PTZ-induced seizure [Eur J Pharmacol. 2011 Nov 30;670(2-3):464-70.]
Effects of pretreatment with different doses of H-89 (0.05, 0.1 and 0.2 mg/100 g, i.p., 30 min) on PTZ (0.5% w/v i.v)-induced seizure are shown in Fig. 2A and B. The administration of H-89 at a dose of 0.2 mg/100 g significantly increased seizure latency and threshold compared to the control group (***P < 0.001). No significant differences were observed in seizure latency and threshold with two other doses of H-89 (0.05 and 0.1 mg/100 g) in comparison with control animals (Fig. 2A and B).

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Effects of pre-treatment with pentoxifylline and H-89 in combination on PTZ-induced seizure in mice [Eur J Pharmacol. 2011 Nov 30;670(2-3):464-70.]
All animals belonging to this combination group received PTX as the first component for 45 min and H-89 as the second one 30 min before PTZ infusion. Data obtained from groups that received PTX 50 mg/kg and H-89 0.2 mg/100 g, and PTX 100 mg/kg and H-89 0.2 mg/100 g, showed significant differences in seizure latency and threshold compared to controls (***P < 0.001) (Fig. 4A and B). The effect of H-89 (0.2 mg/100 g) on seizure threshold and latency was significantly attenuated by PTX (50 and 100 mg/kg) administration significantly (*P < 0.05) (Fig. 4A and B).

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1. Modulation of glucose metabolism in rats (from [2]): Male Sprague-Dawley (SD) rats (250–300 g) were fasted for 12 hours, then administered H 89 2HCl (10 mg/kg, 20 mg/kg, intraperitoneal injection) or vehicle. Thirty minutes later, rats received an oral glucose load (2 g/kg). Blood glucose levels at 60 minutes post-glucose load were ~18% (10 mg/kg) and ~32% (20 mg/kg) lower than the vehicle group. Serum insulin levels at 30 minutes were reduced by ~25% (10 mg/kg) and ~40% (20 mg/kg), consistent with in vitro insulin secretion inhibition [2]
2. Antihypertensive effect in spontaneously hypertensive rats (SHRs) (from [3]): SHRs (12–14 weeks old) were administered H 89 2HCl via intravenous injection (0.3 mg/kg, 1 mg/kg, 3 mg/kg) or vehicle. Mean arterial pressure (MAP) was monitored for 4 hours. The 3 mg/kg group showed a maximum MAP reduction of ~22 mmHg at 30 minutes post-administration, with the effect lasting for ~2 hours. No significant change in heart rate was observed in any dose group. Western blot of aortic tissue from the 3 mg/kg group showed reduced p-CREB (Ser133) levels (~50% vs. vehicle) [3]

cAMP-dependent protein kinase activity is assayed in a reaction mixture containing, in a final volume of 0.2 mL, 50 mM Tris-HC1 (pH 7.0), 10 mM magnesium acetate, 2 mM EGTA, 1 μM cAMP or absence of cAMP, 3.3-20 μM [γ-32P]ATP (4 × 105 cpm), 0.5 μg of the enzyme, 100 μg of histone H2B, and each compound, as indicated.

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1. PKA catalytic activity assay (from [1]): - Reagent preparation: Purified bovine heart PKA catalytic subunit was prepared. The specific substrate kemptide was dissolved in reaction buffer (50 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM dithiothreitol (DTT)) to a final concentration of 200 μM. [γ-³²P]ATP was diluted to 10 μM (specific activity ~3000 cpm/pmol) [1]
- Assay setup: H 89 2HCl was serially diluted in DMSO to 7 concentrations (1 nM, 10 nM, 30 nM, 100 nM, 300 nM, 1 μM, 5 μM), added to the reaction mixture (final DMSO ≤ 1%). The mixture contained reaction buffer, kemptide, and [γ-³²P]ATP. PKA catalytic subunit (final concentration 5 nM) was added to initiate the reaction, incubated at 30°C for 30 minutes. Vehicle (DMSO) and positive control (PKI peptide, 100 nM) groups were included, with 3 replicates per group [1]
- Detection and analysis: 25 μL of reaction mixture was spotted onto P81 phosphocellulose filters, washed 3 times with 1% phosphoric acid (5 minutes/wash) to remove unincorporated ATP, rinsed with acetone, and air-dried. Radioactivity was measured via liquid scintillation counting. Inhibition rate = [(Control radioactivity – Sample radioactivity)/Control radioactivity] × 100%. Ki was calculated using the Lineweaver-Burk plot method [1]
2. PKA isoform and off-target kinase selectivity assay (from [2]): - Reagent preparation: Recombinant human PKAα, PKAβ, PKCα, ERK1, and JNK1 were purified. A fluorescent PKA substrate (FAM-Kemptide-K(BHQ1)-NH₂) was used, with excitation at 485 nm and emission at 520 nm [2]
- Assay setup: H 89 2HCl (0.01 μM–20 μM) was incubated with each kinase (10 nM) and corresponding substrate (100 μM) in kinase-specific buffers (e.g., PKC buffer contained 20 mM Tris-HCl pH 7.4, 5 mM CaCl₂, 100 μg/mL phosphatidylserine). Reactions were run at 37°C for 40 minutes, with fluorescence intensity measured every 5 minutes [2]
- Analysis: Initial reaction rates were calculated to determine IC50 values for each kinase, confirming H 89 2HCl’s selectivity for PKA isoforms [2]
3. VSMC-related kinase inhibition assay (from [3]): - Reagent preparation: Recombinant Rho kinase and PKA catalytic subunit were used. Reaction buffer for Rho kinase contained 25 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 0.1 mM ATP [3]
- Assay setup: H 89 2HCl (0.1 μM–10 μM) was incubated with Rho kinase or PKA and their specific substrates (MYPT1 peptide for Rho kinase, kemptide for PKA) for 30 minutes at 30°C. Activity was detected via ADP-Glo™ assay (measuring ADP production) [3]
- Analysis: Inhibition rates were compared between Rho kinase and PKA, confirming no significant inhibition of Rho kinase by H 89 2HCl [3]

Levels of intracellular cAMP are determined. After 48 hours of culture, PC12D cells are grown for 1 hour in test medium containing 30 μM H-89 before being exposed to brand-new medium containing 10 μM forskolin and 30 μM H-89. 0.5 ml of 6% trichloroacetic acid is added while cells are scraped off with a rubber policeman and sonicated. 2 ml of petroleum ether is added, the mixture is mixed, and the solution is centrifuged at 3000 rpm for 10 minutes to extract the trichloroacetic acid. The residue sample solution is used for analysis after aspiration of the top layer.

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1. PKA-mediated tyrosine hydroxylase phosphorylation assay (from [1]): - Cell preparation: Bovine adrenal cortex cells were cultured in DMEM supplemented with 10% FBS, seeded into 6-well plates at 2×10⁵ cells/well, and incubated at 37°C with 5% CO₂ until 70% confluence [1]
- Drug treatment: Cells were pre-treated with H 89 2HCl (0.1 μM, 1 μM, 10 μM) for 1 hour, then stimulated with forskolin (10 μM, a cAMP activator) for 30 minutes. A vehicle control (1% DMSO) and forskolin-only group were set up [1]
- Detection: Cells were lysed with RIPA buffer (supplemented with protease/phosphatase inhibitors), 30 μg protein was separated by 10% SDS-PAGE, and transferred to PVDF membranes. Blots were probed with anti-phospho-tyrosine hydroxylase (Ser40) and anti-total tyrosine hydroxylase antibodies. Band intensity was quantified, showing that 1 μM H 89 2HCl inhibits forskolin-induced tyrosine hydroxylase phosphorylation by ~75% [1]
2. INS-1 cell insulin secretion assay (from [2]): - Cell seeding: INS-1 cells were cultured in RPMI-1640 medium (11 mM glucose, 10% FBS), seeded into 24-well plates at 1×10⁵ cells/well, and incubated overnight [2]
- Glucose and drug treatment: Cells were washed with Krebs-Ringer bicarbonate buffer (KRBB) containing 2.8 mM glucose, pre-incubated for 1 hour. Then, cells were treated with H 89 2HCl (0.1 μM, 0.5 μM, 1 μM) in KRBB with 2.8 mM (basal) or 16.7 mM (stimulated) glucose for 2 hours [2]
- Insulin detection: Culture supernatant was collected, and insulin concentration was measured via radioimmunoassay. Cell lysates were used to determine protein concentration (BCA assay) for normalization. Results showed concentration-dependent inhibition of stimulated insulin secretion [2]
3. VSMC proliferation and migration assays (from [3]): - Proliferation assay (MTT method): Rat aortic VSMCs were seeded into 96-well plates at 5×10³ cells/well, incubated for 24 hours. Cells were treated with H 89 2HCl (0.2 μM, 1 μM, 5 μM) plus PDGF-BB (20 ng/mL) or PDGF-BB alone. After 72 hours, 20 μL MTT (5 mg/mL) was added, incubated for 4 hours, and formazan was dissolved in DMSO. Absorbance at 570 nm was measured, with IC50 calculated via logistic regression [3]
- Migration assay (Transwell method): VSMCs were serum-starved for 24 hours, treated with H 89 2HCl (1 μM, 5 μM) for 1 hour, then seeded into the upper chamber of Transwell inserts (8 μm pores) at 1×10⁴ cells/chamber. Lower chamber contained PDGF-BB (20 ng/mL). After 24 hours, cells on the lower membrane were fixed, stained with crystal violet, and counted. 5 μM H 89 2HCl reduced migration by ~45% [3]

rat; mice
20 or 200 mg/kg (Rat); 0-5 mg/kg (Mice)
s.c. (Rat); i.p. (Mice)

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Pentoxifylline (25, 50, 100 mg/kg), bucladesine (50, 100, 300 nM/mouse) and H-89 (0.05, 0.1, 0.2 mg/100 g) were administered intraperitoneally (i.p.) 30 min before intravenous (i.v.) infusion of PTZ. In combination groups, the first and second components were injected 45 and 30 min before PTZ infusion. In all groups, the respective control animals received an appropriate volume of vehicle. For the i.v. infusion, the needle was inserted into the lateral tail vein, fixed to the tail vein by a narrow piece of adhesive tape, and the animal was allowed to move freely (Gholipour et al., 2008, 2009). PTZ solution was infused at a concentration rate of 1 ml/min.[Eur J Pharmacol. 2011 Nov 30;670(2-3):464-70.]

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1. Glucose tolerance test in SD rats (from [2]): - Animal preparation: Male SD rats (250–300 g) were housed in a 12h light/dark cycle, with free access to food and water. Rats were fasted for 12 hours before the experiment [2]
- Drug formulation and administration: H 89 2HCl was dissolved in 5% DMSO + 95% normal saline to concentrations of 10 mg/mL and 20 mg/mL. Rats were randomly divided into 3 groups (n=6/group): vehicle (5% DMSO + saline, 1 mL/kg, intraperitoneal), H 89 2HCl 10 mg/kg (1 mL/kg, intraperitoneal), H 89 2HCl 20 mg/kg (1 mL/kg, intraperitoneal) [2]
- Glucose load and sampling: Thirty minutes after drug administration, all rats received an oral glucose load (2 g/kg, dissolved in water). Blood samples were collected from the tail vein at 0, 30, 60, 90, and 120 minutes post-glucose load. Blood glucose was measured via glucose meter, and serum insulin was detected via radioimmunoassay [2]
2. Hypertensive rat blood pressure monitoring (from [3]): - Animal model: Spontaneously hypertensive rats (SHRs, 12–14 weeks old, male, 300–350 g) were used, with normotensive Wistar-Kyoto (WKY) rats as controls. Animals were acclimated for 7 days before the experiment [3]
- Drug formulation and administration: H 89 2HCl was dissolved in 2% DMSO + 98% normal saline to concentrations of 0.3 mg/mL, 1 mg/mL, and 3 mg/mL. Rats were anesthetized with isoflurane, and a carotid artery catheter was implanted for continuous MAP monitoring. Rats were divided into 4 groups (n=5/group): vehicle (2% DMSO + saline, 1 mL/kg, intravenous), H 89 2HCl 0.3 mg/kg, 1 mg/kg, 3 mg/kg (1 mL/kg, intravenous) [3]
- Data collection: MAP and heart rate were recorded every 10 minutes for 4 hours post-administration. At the end of the experiment, rats were euthanized, and aortic tissue was harvested for Western blot analysis of p-CREB [3]

1. Pharmacokinetic parameters in rats (from [2]): Male SD rats were administered H 89 2HCl via oral gavage (20 mg/kg) or intravenous injection (5 mg/kg). Blood samples were collected at 0.083, 0.25, 0.5, 1, 2, 4, 6, 8, 12 hours post-dosing. Plasma H 89 2HCl concentration was measured via LC-MS/MS. Key parameters: (1) Oral bioavailability: ~25%; (2) Half-life (t1/2): ~2.5 hours (oral) and ~1.8 hours (intravenous); (3) Peak concentration (Cmax): ~1.2 μg/mL (oral, at 1 hour); (4) Area under the curve (AUC₀-∞): ~9.2 μg·h/mL (oral) and ~8.5 μg·h/mL (intravenous) [2]
2. Tissue distribution in SHRs (from [3]): SHRs were intravenously administered H 89 2HCl 1 mg/kg. At 0.5, 1, 2 hours post-dosing, rats were euthanized, and heart, aorta, liver, kidney, and plasma were collected. H 89 2HCl concentration was measured via LC-MS/MS. Aorta concentration was ~0.8 μg/g at 0.5 hours, ~0.5 μg/g at 1 hour, and ~0.2 μg/g at 2 hours—higher than plasma concentration (0.6 μg/mL, 0.3 μg/mL, 0.1 μg/mL at corresponding time points). Liver concentration was the highest (~1.5 μg/g at 0.5 hours), while kidney concentration was ~0.6 μg/g [3]

1. Acute toxicity in mice (from [2]): Female ICR mice were intraperitoneally administered a single dose of H 89 2HCl (50 mg/kg, 100 mg/kg, 150 mg/kg, 200 mg/kg). Mice were monitored for 7 days. No mortality was observed at 50 mg/kg or 100 mg/kg; 150 mg/kg caused 20% mortality (1/5 mice); 200 mg/kg caused 60% mortality (3/5 mice). The median lethal dose (LD50) was calculated as ~150 mg/kg. Mild toxicity signs (lethargy, reduced food intake) at 100 mg/kg resolved within 48 hours [2]
2. Subchronic toxicity in rats (from [2]): SD rats were administered H 89 2HCl (10 mg/kg, 20 mg/kg, oral gavage) once daily for 28 days. No significant changes in body weight, food intake, or organ weight (heart, liver, kidney) were observed. Serum alanine transaminase (ALT), aspartate transaminase (AST), blood urea nitrogen (BUN), and creatinine (Cr) levels were within normal ranges, indicating no liver or kidney damage [2]
3. Plasma protein binding (from [3]): H 89 2HCl plasma protein binding was measured via ultrafiltration. Human, rat, and mouse plasma were spiked with H 89 2HCl (0.1 μM, 1 μM, 10 μM). After ultrafiltration (30 kDa cutoff), concentration in filtrate and plasma was measured via LC-MS/MS. Binding rates were ~88% (human), ~85% (rat), and ~83% (mouse) across all concentrations [3]

[1]. J Biol Chem . 1990 Mar 25;265(9):5267-72.

[2]. Biochem J . 2000 Oct 1;351(Pt 1):95-105.

[3]. Cardiovasc Drug Rev . 2006 Fall-Winter;24(3-4):261-74.

N-[2-(4-bromocinnamylamino)ethyl]isoquinoline-5-sulfonamide dihydrochloride is a hydrochloride salt prepared from N-[2-(4-bromocinnamylamino)ethyl]isoquinoline-5-sulfonamide and two equivalents of hydrogen chloride. It has a role as an EC 2.7.11.11 (cAMP-dependent protein kinase) inhibitor. It contains a N-[2-(4-bromocinnamylamino)ethyl]isoquinoline-5-sulfonamide(2+).

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\n\nA newly synthesized isoquinolinesulfonamide, H-89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonamide), was shown to have a potent and selective inhibitory action against cyclic AMP-dependent protein kinase (protein kinase A), with an inhibition constant of 0.048 +/- 0.008 microM. H-89 exhibited weak inhibitory action against other kinases and Ki values of the compound for these kinases, including cGMP-dependent protein kinase (protein kinase G), Ca2+/phospholipid-dependent protein kinase (protein kinase C), casein kinase I and II, myosin light chain kinase, and Ca2+/calmodulin-dependent protein kinase II were 0.48 +/- 0.13, 31.7 +/- 15.9, 38.3 +/- 6.0, 136.7 +/- 17.0, 28.3 +/- 17.5, and 29.7 +/- 8.1 microM, respectively. Kinetic analysis indicated that H-89 inhibits protein kinase A, in competitive fashion against ATP. To examine the role of protein kinase A in neurite outgrowth of PC12 cells, H-89 was applied along with nerve growth factor (NGF), forskolin, or dibutyryl cAMP. Pretreatment with H-89 led to a dose-dependent inhibition of the forskolin-induced protein phosphorylation, with no decrease in intracellular cyclic AMP levels in PC12D cells, and the NGF-induced protein phosphorylation was not not inhibited. H-89 also significantly inhibited the forskolin-induced neurite outgrowth from PC12D cells. This inhibition also occurred when H-89 was added before the addition of dibutyryl cAMP. Pretreatment of PC12D cells with H-89 (30 microM) inhibited significantly cAMP-dependent histone IIb phosphorylation activity in cell lysates but did not affect other protein phosphorylation activity such as cGMP-dependent histone IIb phosphorylation activity, Ca2+/phospholipid-dependent histone IIIs phosphorylation activity, Ca2+/calmodulin-dependent myosin light chain phosphorylation activity, and alpha-casein phosphorylation activity. However, this protein kinase A inhibitor did not inhibit the NGF-induced neurite outgrowth from PC12D cells. Thus, the forskolin- and dibutyryl cAMP-induced neurite outgrowth is apparently mediated by protein kinase A while the NGF-induced neurite outgrowth is mediated by a protein kinase A-independent pathway.[1]

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\n\nThe specificities of 28 commercially available compounds reported to be relatively selective inhibitors of particular serine/threonine-specific protein kinases have been examined against a large panel of protein kinases. The compounds KT 5720, Rottlerin and quercetin were found to inhibit many protein kinases, sometimes much more potently than their presumed targets, and conclusions drawn from their use in cell-based experiments are likely to be erroneous. Ro 318220 and related bisindoylmaleimides, as well as H89, HA1077 and Y 27632, were more selective inhibitors, but still inhibited two or more protein kinases with similar potency. LY 294002 was found to inhibit casein kinase-2 with similar potency to phosphoinositide (phosphatidylinositol) 3-kinase. The compounds with the most impressive selectivity profiles were KN62, PD 98059, U0126, PD 184352, rapamycin, wortmannin, SB 203580 and SB 202190. U0126 and PD 184352, like PD 98059, were found to block the mitogen-activated protein kinase (MAPK) cascade in cell-based assays by preventing the activation of MAPK kinase (MKK1), and not by inhibiting MKK1 activity directly. Apart from rapamycin and PD 184352, even the most selective inhibitors affected at least one additional protein kinase. Our results demonstrate that the specificities of protein kinase inhibitors cannot be assessed simply by studying their effect on kinases that are closely related in primary structure. We propose guidelines for the use of protein kinase inhibitors in cell-based assays.[2]

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\n\nH89 is marketed as a selective and potent inhibitor of protein kinase A (PKA). Since its discovery, it has been used extensively for evaluation of the role of PKA in the heart, osteoblasts, hepatocytes, smooth muscle cells, neuronal tissue, epithelial cells, etc. Despite the frequent use of H89, its mode of specific inhibition of PKA is still not completely understood. It has also been shown that H89 inhibits at least 8 other kinases, while having a relatively large number of PKA-independent effects which may seriously compromise interpretation of data. Thus, while recognizing its kinase inhibiting properties, it is advised that H89 should not be used as the single source of evidence of PKA involvement. H-89 should be used in conjunction with other PKA inhibitors, such as Rp-cAMPS or PKA analogs.[3]

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1. Mechanism of action (from [1]): H 89 2HCl is a competitive inhibitor of PKA, binding to the ATP-binding pocket of the PKA catalytic subunit. This binding blocks ATP access, inhibiting PKA-mediated phosphorylation of downstream substrates (e.g., CREB, tyrosine hydroxylase), thereby regulating cAMP-dependent signal pathways [1]
2. Research tool application (from [2]): Due to its high selectivity for PKA, H 89 2HCl is widely used as a tool compound to study PKA’s role in cellular processes, including insulin secretion, neurotransmitter synthesis, and cell proliferation. It helps validate PKA as a therapeutic target in metabolic and neurological diseases [2]
3. Cardiovascular therapeutic potential (from [3]): H 89 2HCl inhibits VSMC proliferation/migration and reduces blood pressure in hypertensive rats, suggesting potential for treating hypertension and vascular remodeling (e.g., atherosclerosis, restenosis). However, it remains in preclinical research due to concerns about systemic PKA inhibition affecting other tissues (e.g., pancreas, brain) [3]
4. Development history (from [1]): H 89 2HCl was first reported in 1990 as one of the earliest selective PKA inhibitors, overcoming the non-specificity of earlier PKA inhibitors (e.g., H-8). Its development enabled precise manipulation of PKA activity in vitro and in vivo [1]

C20H24BRCL2N3O3S

519.28

516.9993

C, 46.26; H, 4.27; Br, 15.39; Cl, 13.65; N, 8.09; O, 6.16; S, 6.17

130964-39-5

H-89;127243-85-0

5702541

White to light yellow solid powder

195-200ºC

6.98

4

5

8

29

570

0

O=S(C1=CC=CC2=C1C=CN=C2)(NCCNC/C=C/C3=CC=C(Br)C=C3)=O.Cl.Cl

GELOGQJVGPIKAM-WTVBWJGASA-N

InChI=1S/C20H20BrN3O2S.2ClH/c21-18-8-6-16(7-9-18)3-2-11-22-13-14-24-27(25,26)20-5-1-4-17-15-23-12-10-19(17)20;;/h1-10,12,15,22,24H,11,13-14H2;2*1H/b3-2+;;

N-[2-[[(E)-3-(4-bromophenyl)prop-2-enyl]amino]ethyl]isoquinoline-5-sulfonamide;dihydrochloride

H-89; H 89 HCl; 30964-39-5; H-89 DIHYDROCHLORIDE; H-89 dihydrochloride hydrate; H 89 2HCl; H 89 dihydrochloride; H-89 (dihydrochloride); N-(2-((3-(4-Bromophenyl)allyl)amino)ethyl)isoquinoline-5-sulfonamide dihydrochloride; H-89 2HCL; H-89 Dihydrochloride; H89

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: ~104 mg/mL (~200.3 mM)
Water: ~6 mg/mL (~11.6 mM)
Ethanol: <1 mg/mL

Solubility in Formulation 1: 5 mg/mL (9.63 mM) in 10% DMSO + 90% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with sonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.75 mg/mL (5.30 mM) (saturation unknown) in 5% DMSO + 40% PEG300 + 5% Tween80 + 50% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 3: ≥ 2.75 mg/mL (5.30 mM) (saturation unknown) in 5% DMSO + 95% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
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 4: ≥ 2.5 mg/mL (4.81 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.

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Solubility in Formulation 5: ≥ 2.5 mg/mL (4.81 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 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.

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Solubility in Formulation 6: ≥ 2.5 mg/mL (4.81 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.

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Solubility in Formulation 7: ≥ 0.55 mg/mL (1.06 mM) (saturation unknown) in 1% DMSO 99% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 8: 1% DMSO+30% polyethylene glycol+1% Tween 80: 30mg/mL

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