BF844 alleviates hearing loss associated with the USH3 mutation CLRN1N48K. BF844 induces CLRN1N48K translocation to the plasma membrane. BF844 displays significant hearing-sparing effects in vivo.

Physicochemical Properties

Molecular Formula	C21H19CLN4O
Molecular Weight	378.85
Exact Mass	378.124
CAS #	1404506-35-9
PubChem CID	66576257
Appearance	Typically exists as solid at room temperature
LogP	3.6
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	4
Heavy Atom Count	27
Complexity	493
Defined Atom Stereocenter Count	0
InChi Key	TVNSCKMVODVWRS-UHFFFAOYSA-N
InChi Code	InChI=1S/C21H19ClN4O/c1-21(2,27)13-26-20-16(18(25-26)14-9-5-3-6-10-14)17(22)19(23-24-20)15-11-7-4-8-12-15/h3-12,27H,13H2,1-2H3
Chemical Name	1-(4-chloro-3,5-diphenylpyrazolo[3,4-c]pyridazin-1-yl)-2-methylpropan-2-ol
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

ln Vitro	BF844 (Compound 3) (0.846 µM), inhibits HSP60 activity with potency (87.07±27.70% inhibition) and moderately (40.06±19.10% inhibition) [1]. About 6% of total CLRN1N48K is transported to the plasma membrane of C1, D1, and D6 cells by BF844 (2.90 µM; 24 hours) [1]. Non-glycosylated CLRN1 is readily transported to the plasma membrane in C1 and D1 cells, and BF844 (2.90 µM; 24 hours) effectively increases the amount of non-glycosylated CLRN1 [1].
ln Vivo	In vivo, BF844 exhibits strong penetration into the cochlea and retina [1]. In Tg;KI/KI mice, BF844 (10 mg/kg; i.p.) was demonstrated to significantly preserve hearing [1].
Animal Protocol	Animal/Disease Models: Juvenile mice[1] Doses: 3, 10 mg/kg (P6 (postnatal day 6) mice are 3 mg/kg, P20 (postnatal day 20) mice are 10 mg/kg) Dosing: IP Experimental Results: The measured AUC values were 1.76 μM.h and 1.98 μM.h respectively. Animal/Disease Models: P30 Tg; KI/KI C57BL/6J mice [1] Doses: 30 mg/kg Route of Administration: intraperitoneal (ip) injection; one time/day from P30 to P45 Experimental Results: Demonstrated significant hearing preservation, logarithmic scale The median sound intensity threshold was 57.5–67.5 dB SPL, resulting in an approximately 1,000-fold increase in hearing sensitivity compared with untreated controls at P55. Animal/Disease Models: P10 Tg; KI/KI C57BL/6J mice [1] Doses: 10 mg/kg Route of Administration: intraperitoneal (ip) injection; 10 mg/kg every other day, and gradually increase the dose to 20 mg/kg at P28 kg. From P30 to P45, mice received 30 mg/kg daily. Experimental Results: Median sound intensity thresholds showing logarithmic scale decreases at P55 by 55, 42.5 and 37.5 dB SPL at 8, 16 and 32 kHz, respectively.
References	[1]. A small molecule mitigates hearing loss in a mouse model of Usher syndrome III. Nat Chem Biol. 2016 Jun;12(6):444-51.

Solubility Data

Solubility (In Vitro)

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

Solubility (In Vivo)

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. Injection Formulations (e.g. IP/IV/IM/SC) 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). 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) Oral Formulations 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). 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 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.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	2.6396 mL	13.1978 mL	26.3957 mL
	5 mM	0.5279 mL	2.6396 mL	5.2791 mL
	10 mM	0.2640 mL	1.3198 mL	2.6396 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.