Intracellular chelatable iron (redox-active iron pool). LK-614 (3,4-dimethoxy-N-methyl-benzhydroxamic acid) is a newly developed lipophilic, membrane-permeable hydroxamic acid derivative that acts as an iron chelator. No specific binding affinity data (e.g., IC₅₀, Kᵢ) were reported in these studies. [1,2]
LK-614 functions as a membrane-permeable iron chelator, a classification that defines its primary mechanism of action. Rather than targeting a specific protein or enzyme, it exerts its biological effects by binding to and sequestering ferric iron (Fe³⁺) ions. Through this chelation, LK-614 reduces the availability of free iron, which is a critical catalyst in the production of highly reactive hydroxyl radicals via the Fenton reaction. By limiting this catalytic iron pool, the compound indirectly mitigates oxidative stress and the subsequent cellular damage caused by reactive oxygen species (ROS). This mechanism is distinct from enzyme inhibition, as it operates through metal ion sequestration, and it forms the basis for its protective effects in models of histidine-induced cytotoxicity and cold-induced injury.

Protection against histidine-induced injury in hepatocytes: In cultured rat hepatocytes exposed to 100 mM L-histidine in modified Krebs-Henseleit buffer at 37°C under normoxic conditions, the addition of LK-614 (1 mM) provided significant protection against cell injury. LDH release was reduced from 63 ± 26% (histidine alone) to 15 ± 5% (with LK-614), and thiobarbituric acid-reactive substances (TBARS), a marker of lipid peroxidation, decreased from 13.2 ± 6.3 nmol/10⁶ cells to 0.8 ± 0.8 nmol/10⁶ cells. The protection was comparable to that provided by the membrane-permeable iron chelator 2,2′-dipyridyl. [1]
Mechanism of action: LK-614, like other membrane-permeable iron chelators (1,10-phenanthroline, 2,2′-dipyridyl, deferoxamine), protected cells from histidine-induced injury, whereas the membrane-impermeable chelator diethylenetriaminepentaacetic acid (DTPA) did not. This indicates that LK-614 acts by chelating intracellular iron, preventing the formation of redox-active iron complexes that catalyze the production of reactive oxygen species. [1]
In vitro, LK-614 demonstrates its activity primarily as an iron chelator, effectively binding to ferric iron and reducing the labile iron pool within cells. Its biological activity is assessed by its ability to protect cells from L-histidine-induced damage, a model that relies on the generation of oxidative stress. By chelating iron, LK-614 inhibits the Fenton reaction, thereby reducing the production of damaging hydroxyl radicals and preserving cell viability. The compound's potency as a chelator and its protective effects are quantified in cell-based assays, where it shows a capacity to diminish the beneficial effects of modified HTK-1 solution in improving myocardial contractility after heart transplantation. In vitro, LK-614 is compared to other iron chelators like 1,10-phenanthroline, 2,2′-dipyridyl, and deferoxamine, against which it protects cells from histidine-induced injury.

Effect on myocardial function after heart transplantation: In a heterotopic rat heart transplantation model, donor hearts were preserved for 1 hour in modified HTK solution containing LK-614 (0.02 mmol/L) and deferoxamine (0.1 mmol/L) (HTK-2 group). After 1 hour of reperfusion, left ventricular systolic pressure (LVSP) was 60 ± 39 mmHg and minimum rate of pressure development (dP/dtmin) was 660 ± 446 mmHg/s in the HTK-2 group. In contrast, the HTK-1 group (containing N-α-acetyl-L-histidine but no iron chelators) showed significantly better functional recovery (LVSP: 106 ± 33 mmHg; dP/dtmin: 1388 ± 627 mmHg/s). The addition of LK-614 and deferoxamine did not improve myocardial contractility or relaxation compared to standard HTK solution. Energy charge potential was not significantly different between groups. [2]
Detailed in vivo activity data for LK-614 are limited in the available literature. However, its application in research models of cardiovascular disease and transplantation suggests potential for in vivo efficacy. The compound is used to study cold-induced injury, which is relevant to organ preservation and transplantation research. In these contexts, LK-614's ability to chelate iron and mitigate oxidative stress is hypothesized to confer protective effects against ischemia-reperfusion injury and other forms of cold-induced cellular damage. Further in vivo studies would be required to fully characterize its pharmacokinetic profile and therapeutic potential in animal models of cardiovascular and other iron-related diseases.

Non-cell-based assays for LK-614 involve standard in vitro iron chelation assays to quantify its metal-binding capacity. These assays typically employ spectrophotometric methods where the compound is incubated with a known concentration of ferric iron (Fe³⁺) in a buffered solution. The formation of the iron-chelate complex is then measured by changes in absorbance at specific wavelengths, or by using a competing chromogenic iron indicator such as Ferrozine or Calcein, whose signal is quenched upon iron binding. The chelation efficacy is determined by calculating the amount of iron bound by LK-614, often expressed as a molar ratio or as an IC50 value representing the concentration required to chelate 50% of the available iron. These cell-free assays provide a direct measure of the compound's fundamental chelating activity.

Hepatocyte injury assessment: Rat hepatocytes were cultured and exposed to modified Krebs-Henseleit buffer containing 100 mM L-histidine or 198 mM L-histidine at 37°C under normoxic conditions for 4 hours. LK-614 was added at a concentration of 1 mM to test its protective effect. Cell injury was assessed by measuring lactate dehydrogenase (LDH) release and thiobarbituric acid-reactive substances (TBARS) as a marker of lipid peroxidation. [1]
Mechanistic studies: To confirm the intracellular site of action, the membrane-permeable iron chelator LK-614 was compared with the membrane-impermeable chelator DTPA (100 μM). Only the membrane-permeable chelators provided protection, indicating that LK-614 acts by chelating intracellular iron. [1]
Cellular assays for LK-614 utilize various cell lines to assess its protective effects against iron-mediated oxidative damage. A key model involves treating cells with L-histidine, which induces cell damage through the generation of oxidative stress. Cells are pre-incubated with LK-614 at varying concentrations, typically in the micromolar range, before exposure to the damaging agent. Cell viability is then assessed using standard assays such as MTT, CCK-8, or LDH release. Additionally, intracellular reactive oxygen species (ROS) levels can be measured using fluorescent probes like DCFH-DA to directly demonstrate the compound's antioxidant effect. The compound's ability to protect cells from cold-induced injury is also evaluated in relevant cell culture models.

Heterotopic rat heart transplantation: Male Lewis rats (280-400 g) were used as donors and recipients. Donor hearts were arrested with 20 mL of cardioplegic solution (HTK, HTK-1, or HTK-2) and stored in cold (+4°C) preservation solution for 1 hour. Hearts were then transplanted heterotopically by anastomosing the donor aorta and pulmonary artery to the recipient abdominal aorta and vena cava, respectively. After 1 hour of reperfusion, hemodynamic functional assessment was performed using a Millar pressure catheter. The modified HTK-2 solution contained LK-614 at a concentration of 0.02 mmol/L and deferoxamine at 0.1 mmol/L. [2]
High-energy phosphate determination: After hemodynamic measurements, hearts were immediately frozen in liquid nitrogen. Adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) contents were assessed using an enzyme-kinetic assay, and energy charge potential was calculated. [2]
In vivo animal models for LK-614 would be required to fully assess its therapeutic potential. Based on its mechanism as an iron chelator and its use in cardiovascular research, relevant models could include rodent models of myocardial ischemia-reperfusion injury, where the compound would be administered prior to or during the ischemic event. Heart transplantation models, where LK-614 is used to study the effects of modified HTK-1 solution on myocardial contractility, are also relevant. In such studies, the compound would be administered via appropriate routes (e.g., intravenous or intraperitoneal), and endpoints would include infarct size, cardiac function, and biomarkers of oxidative stress. Detailed protocols are not extensively reported in the available literature.

Pharmacokinetic properties of LK-614 include a molecular weight of 211.21 g/mol and a molecular formula of C10H13NO4. The compound is characterized as a membrane-permeable iron chelator, indicating it can cross cellular membranes to access intracellular iron. The CAS number is 82461-57-2. The compound is supplied as an off-white to pink solid powder with a purity of ≥98%. Detailed PK parameters such as half-life, bioavailability, volume of distribution, and clearance are not extensively reported in the available literature. The compound is a small molecule with moderate lipophilicity, which may influence its absorption and distribution. Storage conditions: powder at -20°C.

In the heart transplantation study, the addition of LK-614 and deferoxamine (HTK-2 group) did not improve functional outcomes and was associated with numerically lower myocardial function compared to the HTK-1 group, suggesting that the combination may not be beneficial in this model. No overt signs of toxicity were described. [2]
Detailed toxicological data for LK-614 are not extensively reported in the available literature. The compound is noted to have low toxicity, but as a research chemical, its safety profile would need to be established through standard preclinical toxicity assessments. The compound is supplied for research use only and is not for human consumption. Given its mechanism of iron chelation, potential effects on iron homeostasis and erythropoiesis would be key safety considerations. Standard toxicity assessments would include evaluation of hematological parameters, as well as hepatic and renal function.

[1]. Histidine-induced injury to cultured liver cells, effects of histidine derivatives and of iron chelators. Cellular and molecular life sciences. 2007 Jan;64(2):192-205.
[2]. Deferoxamine, the newly developed iron chelator LK-614 and N-alpha-acetyl-histidine in myocardial protection. Interact Cardiovasc Thorac Surg. 2010 Feb;10(2):181-4.

Background and mechanism: LK-614 is a lipophilic, membrane-permeable hydroxamic acid derivative developed as an iron chelator. It was designed to penetrate cell membranes and chelate intracellular "chelatable iron," which becomes redox-active during cold storage and catalyzes the formation of hydroxyl radicals via the Fenton reaction. This process contributes to cold-induced injury in organ preservation. [1,2]
Chemical properties: LK-614 (3,4-dimethoxy-N-methyl-benzhydroxamic acid) is a lipophilic derivative of benzhydroxamic acid. Its membrane permeability allows it to access the intracellular pool of chelatable iron, unlike hydrophilic chelators such as deferoxamine (which has limited membrane permeability) or DTPA (which is impermeable). [1]
Clinical context: LK-614 was tested as an additive to histidine-tryptophan-ketoglutarate (HTK) solution (Custodiol) to reduce cold ischemic injury in organ preservation. However, in the rat heart transplantation model, the combination of LK-614 and deferoxamine did not improve myocardial functional recovery after 1 hour of cold storage and reperfusion. In contrast, partial replacement of histidine with N-α-acetyl-L-histidine (HTK-1 solution) significantly improved functional outcomes. [2]
Experimental context: In vitro studies in cultured rat hepatocytes demonstrated that LK-614 effectively protects against histidine-induced cell injury and lipid peroxidation, confirming its ability to chelate intracellular iron and prevent oxidative damage. This protective effect was observed at a concentration of 1 mM. [1]
LK-614 is a small-molecule research compound identified by CAS number 82461-57-2. It is a membrane-permeable iron chelator that is used to study the protective effects against L-histidine-induced cell damage and cold-induced injury. The compound has a molecular weight of 211.21 g/mol and a purity of ≥98%. It is also known by its IUPAC name, N-hydroxy-3,4-dimethoxy-N-methylbenzamide. LK-614 is exclusively intended for laboratory research applications and is not for human consumption. It is a valuable tool for investigating the role of iron in oxidative stress and related pathologies, particularly in cardiovascular research.**

C10H13NO4

211.21452

211.084

C, 56.87; H, 6.20; N, 6.63; O, 30.30

82461-57-2

22925579

Off-white to pink solid powder

1.165

1

4

3

15

222

0

ON(C)C(=O)C1C=CC(OC)=C(OC)C=1

CFAMAWCPWJCAFR-UHFFFAOYSA-N

InChI=1S/C10H13NO4/c1-11(13)10(12)7-4-5-8(14-2)9(6-7)15-3/h4-6,13H,1-3H3

N-hydroxy-3,4-dimethoxy-N-methylbenzamide

LK-614; LK 614; 82461-57-2; LK 614; Benzamide, N-hydroxy-3,4-dimethoxy-n-methyl-; UNII-83OI05F214; LK614

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)

DMSO: ~175 mg/mL (828.6 mM)

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.)