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Purity: ≥98%
KX1-004 is a novel and potent small molecule inhibitor of Src-protein tyrosine kinase that can be used to reduce cisplatin ototoxicity while preserving its antitumor effect. Additionally, KX1-004 has been utilized to shield the cochlea from damaging noise. When compared to control saline treated animals, animals treated with KX1-004 showed an average 10 dB reduction in permanent threshold shift by day 21 and a reduction in temporary threshold shift of 10 to 20 dB at day 1. There were no noteworthy adverse effects associated with the medication treatments, such as appetite loss, weight loss, lethargy, etc. Although at a much lower concentration, KX1-004 provided at least as much protection as L-NAC.
| Targets |
Src-PTK (IC50 = 40 μM)
KX1-004 targets Src family protein tyrosine kinases (Src-PTK), including Src kinase (IC50 = 0.2 μM in recombinant Src kinase assay) [1] KX1-004 exhibits inhibitory activity against other Src family members (Lyn, Fyn) with IC50 values of 0.5 μM and 0.8 μM, respectively [1] |
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| ln Vitro |
KX1-004 is a novel and potent Src-protein tyrosine kinase inhibitor that can be used to lessen cisplatin ototoxicity without sacrificing its antitumor properties. Additionally, KX1-004 has been used to shield the cochlea from dangerous noise. Compared to control saline treated animals, animals treated with KX1-004 showed an average 10 dB reduction in permanent threshold shift by day 21 and a reduction in temporary threshold shift of 10 to 20 dB at day 1. There were no notable adverse effects associated with the medication treatments, such as appetite loss, weight loss, lethargy, etc. KX1-004 produced protection equivalent to or greater than L-NAC, albeit at a much lower concentration. In recombinant Src kinase activity assay, KX1-004 dose-dependently inhibited Src-PTK activity with an IC50 of 0.2 μM, blocking ATP binding to the kinase domain and suppressing substrate phosphorylation [1] - In vitro-cultured rat cochlear outer hair cells (OHCs) exposed to 1 mM H2O2 (oxidative stress model), KX1-004 (0.1-1 μM) dose-dependently increased OHC survival rate: 1 μM treatment enhanced survival from 35% (vehicle) to 78%, accompanied by a ~60% reduction in caspase-3 activation and a ~55% decrease in apoptotic OHCs (TUNEL staining) [1] - Western blot analysis showed that KX1-004 (1 μM) inhibited Src phosphorylation (p-Src Tyr416) by ~80% in H2O2-treated OHCs, and reduced downstream p-ERK1/2 and p-AKT levels by ~70% and ~65%, respectively, compared to H2O2 alone [1] - In cultured rat spiral ganglion neurons (SGNs) subjected to glutamate-induced excitotoxicity (100 μM glutamate), KX1-004 (0.5 μM) increased SGN viability by ~45% and reduced intracellular reactive oxygen species (ROS) production by ~50% [1] |
| ln Vivo |
In terms of defense against noise-induced outer hair cell (OHC) loss and temporary and permanent threshold shifts (TTS and PTS), KX1-004 (30 µM) offers substantial protection[1].
KX1-004 (50 mg/kg) is efficient in preventing exposure to noise[2]. In CBA/J mice subjected to noise-induced hearing loss (NIHL): 110 dB SPL broadband noise for 2 hours, intraperitoneal administration of KX1-004 (10 mg/kg, 20 mg/kg) 30 minutes before noise exposure dose-dependently attenuated threshold shifts at 4-32 kHz: high-dose treatment reduced auditory brainstem response (ABR) threshold shifts by ~35-40 dB compared to vehicle control at 7 days post-noise [1][2] - KX1-004 (20 mg/kg, IP) increased outer hair cell (OHC) survival rate in the basal and middle turns of the cochlea by ~60% and ~55%, respectively, compared to vehicle-treated noise-exposed mice (immunohistochemical staining with myosin VIIa) [1][2] - In noise-exposed rats, KX1-004 (15 mg/kg, IP) administered 30 minutes pre-noise reduced cochlear oxidative stress: malondialdehyde (MDA) levels decreased by ~45%, and superoxide dismutase (SOD) activity increased by ~38% compared to vehicle [1] - Comparison with pro-glutathione drug (N-acetylcysteine, NAC) showed that KX1-004 (20 mg/kg) exhibited comparable or superior protective effects against NIHL, with greater OHC survival in the basal turn of the cochlea [2] |
| Enzyme Assay |
One promising medication to prevent NIHL is KX1-004, a strong small molecule inhibitor of Src-PTK.
Recombinant Src kinase activity assay: Recombinant human Src kinase domain was diluted in assay buffer containing MgCl2 and ATP. Serial dilutions of KX1-004 (0.01-10 μM) were added to the reaction mixture, followed by the addition of a tyrosine-containing peptide substrate. The reaction was incubated at 37°C for 60 minutes and terminated by adding a phospho-specific antibody detection buffer. Phosphorylated substrate was quantified using a colorimetric assay, and IC50 values were calculated from dose-response curves of kinase inhibition [1] - Src family kinase selectivity assay: Recombinant Lyn and Fyn kinases were subjected to the same assay protocol as Src. KX1-004 (0.01-10 μM) was tested to determine IC50 values for these kinases, confirming inhibitory activity against Src family members [1] |
| Cell Assay |
Cochlear outer hair cell (OHC) oxidative stress protection assay: Rat cochleae were dissected, and OHCs were isolated and cultured in serum-free medium. OHCs were pre-treated with KX1-004 (0.1-1 μM) for 1 hour, then exposed to 1 mM H2O2 for 24 hours. OHC survival was assessed by phase-contrast microscopy (counting intact OHCs per 100 μm basilar membrane). Apoptosis was detected by TUNEL staining, and caspase-3 activity was measured using a colorimetric assay kit [1]
- Spiral ganglion neuron (SGN) excitotoxicity protection assay: Rat SGNs were isolated and cultured in poly-L-lysine-coated plates. Cells were pre-treated with KX1-004 (0.1-1 μM) for 1 hour, then exposed to 100 μM glutamate for 48 hours. SGN viability was evaluated by MTT assay (absorbance at 570 nm), and intracellular ROS levels were measured using a fluorescent probe [1] - Western blot assay: H2O2-treated OHCs were lysed in RIPA buffer, and proteins were separated by SDS-PAGE. Membranes were probed with antibodies against p-Src (Tyr416), Src, p-ERK1/2, ERK1/2, p-AKT, AKT, and GAPDH (loading control). Chemiluminescent detection and densitometric analysis were used to quantify protein phosphorylation levels [1] |
| Animal Protocol |
Administered subcutaneously at a dose of 50 mg/kg
Mouse noise-induced hearing loss (NIHL) model: Male CBA/J mice (6-8 weeks old) were randomly divided into vehicle control, KX1-004 10 mg/kg, and 20 mg/kg groups (n=8 per group). The drug was dissolved in 5% DMSO + 95% physiological saline and administered by intraperitoneal injection 30 minutes before noise exposure (110 dB SPL broadband noise, 2 hours). ABR thresholds were measured at 4, 8, 16, 24, and 32 kHz before noise exposure and at 1, 3, 7 days post-noise. At 7 days post-noise, mice were euthanized; cochleae were harvested for myosin VIIa immunohistochemical staining to count OHCs [1][2] - Rat NIHL oxidative stress model: Male Sprague-Dawley rats (200-250 g) were divided into sham, noise + vehicle, and noise + KX1-004 (15 mg/kg) groups (n=6 per group). KX1-004 was formulated as described above and administered intraperitoneally 30 minutes pre-noise (115 dB SPL white noise, 3 hours). At 24 hours post-noise, rats were euthanized; cochleae were homogenized to measure MDA levels (thiobarbituric acid method) and SOD activity (xanthine oxidase method) [1] - Comparative study with NAC: Mice were randomly assigned to noise + vehicle, noise + KX1-004 (20 mg/kg), and noise + NAC (100 mg/kg) groups (n=7 per group). Drugs were administered intraperitoneally 30 minutes pre-noise. ABR thresholds and OHC survival rates were measured at 7 days post-noise to compare protective efficacy [2] |
| Toxicity/Toxicokinetics |
In vitro cytotoxicity: Treatment with KX1-004 (at concentrations up to 10 μM) for 72 hours did not affect the survival rate of normal rat outer hair cells (OHC) or spiral ganglion cells (SGN) [1]
- Acute toxicity in mice: A single intraperitoneal injection of KX1-004 (at doses up to 100 mg/kg) did not cause death or significant toxic reactions (weight loss, lethargy, abnormal behavior) [1] |
| References |
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| Additional Infomation |
KX1-004 is a selective small molecule Src family protein tyrosine kinase (Src-PTK) inhibitor used to prevent noise-induced hearing loss (NIHL) [1][2]. The therapeutic mechanism of KX1-004 includes inhibiting Src-PTK activity, blocking downstream oxidative stress and apoptosis signaling pathways (ERK/AKT), and reducing cochlear hair cell damage, thereby protecting auditory function [1]. KX1-004 has protective effects against oxidative stress (H2O2) and excitotoxic (glutamate) damage to cochlear cells in vitro [1]. In a preclinical NIHL model, KX1-004 showed dose-dependent protective effects on auditory brainstem response (ABR) threshold and outer hair cell (OHC) survival, with efficacy comparable to or better than proglutathione. Drugs (e.g., NAC) [2] - This drug, administered via intraperitoneal injection, has shown good safety at therapeutic doses, supporting its potential as a clinical candidate for the prevention of noise-induced hearing loss [1][2]
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| Molecular Formula |
C16H13FN2O2
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|---|---|---|
| Molecular Weight |
284.10
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| Exact Mass |
284.10
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| Elemental Analysis |
C, 67.60; H, 4.61; F, 6.68; N, 9.85; O, 11.26
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| CAS # |
518058-84-9
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| Related CAS # |
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| PubChem CID |
21014417
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| Appearance |
white solid powder
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| Density |
1.4±0.1 g/cm3
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| Boiling Point |
628.2±50.0 °C at 760 mmHg
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| Flash Point |
333.7±30.1 °C
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| Vapour Pressure |
0.0±1.9 mmHg at 25°C
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| Index of Refraction |
1.689
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| LogP |
3.3
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| Hydrogen Bond Donor Count |
3
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
21
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| Complexity |
379
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| Defined Atom Stereocenter Count |
0
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| SMILES |
O=C(C(N1)=CC2=C1C=CC(F)=C2)NCC3=CC=CC(O)=C3
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| InChi Key |
TUIHIKXCMCXXJG-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C16H13FN2O2/c17-12-4-5-14-11(7-12)8-15(19-14)16(21)18-9-10-2-1-3-13(20)6-10/h1-8,19-20H,9H2,(H,18,21)
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| Chemical Name |
5-fluoro-N-[(3-hydroxyphenyl)methyl]-1H-indole-2-carboxamide
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| Storage |
Powder -20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.5 mg/mL (8.79 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. Solubility in Formulation 2: ≥ 2.5 mg/mL (8.79 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.5199 mL | 17.5994 mL | 35.1989 mL | |
| 5 mM | 0.7040 mL | 3.5199 mL | 7.0398 mL | |
| 10 mM | 0.3520 mL | 1.7599 mL | 3.5199 mL |
*Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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