Branaplam diHCl (LMI-070 dihydrochloride; NVS-SM1) is a potent, orally bioactive, small-molecule enhancer of SMN2 (survival of motor neuron-2) splicing that elevates full-length SMN protein and extends survival in a severe SMA mouse model. The SMN2 pre-mRNA and U1 small nuclear ribonucleic protein (snRNP) complex form a transient double-strand RNA structure, which is stabilized through a molecular mechanism. In a manner distinct from constitutive recognition, U1 snRNP increases its binding affinity to the 5' splice site in a sequence-specific manner. This novel mechanism shows that sequence-selective splice modulation via small molecules is feasible and may be used to treat other splicing disorders.

Physicochemical Properties

Molecular Formula	C22H28CLN5O2
Molecular Weight	429.94
Exact Mass	429.19
Elemental Analysis	C, 61.46; H, 6.56; Cl, 8.25; N, 16.29; O, 7.44
CAS #	1562338-39-9
Related CAS #	1562338-42-4; 1562338-39-9 (HCl)
PubChem CID	135565041
Appearance	Light yellow to yellow solid powder
Hydrogen Bond Donor Count	4
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	4
Heavy Atom Count	30
Complexity	541
Defined Atom Stereocenter Count	0
InChi Key	XJIMIVJABPKGIY-UHFFFAOYSA-N
InChi Code	InChI=1S/C22H27N5O2.ClH/c1-21(2)10-16(11-22(3,4)27-21)29-20-8-7-18(25-26-20)17-6-5-14(9-19(17)28)15-12-23-24-13-15;/h5-9,12-13,16,27-28H,10-11H2,1-4H3,(H,23,24);1H
Chemical Name	5-(1H-pyrazol-4-yl)-2-[6-(2,2,6,6-tetramethylpiperidin-4-yl)oxypyridazin-3-yl]phenol;hydrochloride
Synonyms	Branaplam HCl; Branaplam hydrochloride; LMI-070 hydrochloride; LMI 070 HCl; LMI070 HCl; LMI-070 HCl
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 Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.
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	Branaplam HCl (LMI-070 hydrochloride; NVS-SM1) treatment alters the levels of 175 genes in human fibroblasts[1].
ln Vivo	Branaplam HCl (LMI-070 hydrochloride; NVS-SM1) (3, 10, 30 mg/kg; oral) hydrochloride causes dose-dependent increases in SMN2-FL transcript and SMN protein in brain and spinal cord[1]. Branaplam hydrochloride (1 mg/kg IV; 3 mg/kg PO) has an AUC of 3.03 μM•h and a CL of 25 mL/min/kg[2]. In the brains of C/+ mice, a single oral dose of 30 mg/kg of Branaplam hydrochloride causes a prominent and long-lasting increase in SMN protein for as long as 160 hours[1]. Branaplam hydrochloride (oral; 0.03, 0.1, 0.3, 1, 3 mg/kg) increases body weight and prolongs life in SMNΔ7 mice[1].
References	[1]. SMN2 splice modulators enhance U1-pre-mRNA association and rescue SMA mice. Nat Chem Biol. 2015 Jul;11(7):511-517. [2]. Discovery of Small Molecule Splicing Modulators of Survival Motor Neuron-2 (SMN2) for the Treatment of Spinal Muscular Atrophy (SMA). J Med Chem. 2018 Dec 27;61(24):11021-11036.

Solubility Data

Solubility (In Vitro)

H2O : ~200 mg/mL (~465.18 mM)

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	2.3259 mL	11.6295 mL	23.2591 mL
	5 mM	0.4652 mL	2.3259 mL	4.6518 mL
	10 mM	0.2326 mL	1.1630 mL	2.3259 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.