Semapimod is an inhibitor (blocker/antagonist) of proinflammatory cytokine production and inhibits TNF-α, IL-1β and IL-6. Semapimod inhibits macrophage p38 MAPK and nitric oxide production. Semapimod inhibits TLR4 signaling (IC50≈0.3 μM). Semapimod has potential role in a variety of inflammatory and autoimmune diseases.

Physicochemical Properties

Molecular Formula	C34H52N18O2
Molecular Weight	744.90
Exact Mass	744.452
CAS #	352513-83-8
Related CAS #	Semapimod tetrahydrochloride;164301-51-3
PubChem CID	5745214
Appearance	Typically exists as solid at room temperature
Density	1.39
Index of Refraction	1.677
LogP	7.122
Hydrogen Bond Donor Count	10
Hydrogen Bond Acceptor Count	10
Rotatable Bond Count	19
Heavy Atom Count	54
Complexity	1290
Defined Atom Stereocenter Count	0
SMILES	C/C(/C1=CC(/C(/C)=N/NC(N)=N)=CC(NC(CCCCCCCCC(NC2=CC(/C(/C)=N\NC(N)=N)=CC(/C(/C)=N/NC(N)=N)=C2)=O)=O)=C1)=N/NC(N)=N
InChi Key	PWDYHMBTPGXCSN-VCBMUGGBSA-N
InChi Code	InChI=1S/C34H52N18O2/c1-19(45-49-31(35)36)23-13-24(20(2)46-50-32(37)38)16-27(15-23)43-29(53)11-9-7-5-6-8-10-12-30(54)44-28-17-25(21(3)47-51-33(39)40)14-26(18-28)22(4)48-52-34(41)42/h13-18H,5-12H2,1-4H3,(H,43,53)(H,44,54)(H4,35,36,49)(H4,37,38,50)(H4,39,40,51)(H4,41,42,52)/b45-19+,46-20+,47-21+,48-22+
Chemical Name	N,N'-bis[3,5-bis[(E)-N-(diaminomethylideneamino)-C-methylcarbonimidoyl]phenyl]decanediamide
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

Targets	CD40 IL-1β IL-6 p38 MAPK
ln Vitro	Semapimod causes pro-inflammatory gene expression in macrophages, including p38-MAPK phosphorylation, macrophage inflammatory protein-1alpha, interleukin-6, monocyte chemoattractant protein-1, and intercellular adhesion molecule-1. It also drastically lowers neutral granulocyte infiltration. Semapimod totally stops the myometrium from producing nitric oxide [2]. Semapimod acts on the TLR chaperone gp96, which desensitizes TLR signaling. In vitro, gp96's ATP binding and ATPase activity are inhibited by semapimod tetrahydrochloride (IC50 = 0.2–0.4 μM). By affecting the TLR chaperone gp96, semapimod desensitizes TLR signaling [3]. GL261 invasion induced by microglia is inhibited by semapimod (0-500 nM) [4]. Even at 10 µM, serum-stimulated glioblastoma cell invasion is unaffected by Semapimod (0-10 µM), highlighting Semapimod's selectivity for monocyte-lineage cells [4]. Glioblastoma cells activated by microglia do not proliferate when semapimod (200 nM) is present [4].
ln Vivo	Semapimod (5 mg/kg; intraperitoneal injection; once daily for 2 weeks) improves endothelial dysfunction in Obese Zucker (OZ) rats [1]. Semapimod rescues AM-induced Akt phosphorylation and cGMP synthesis in OZ rats [1]. Semapimod (6 mg/kg/day, intracranial injection for 1 week) inhibits glioblastoma cell invasion in vivo [4]. Semapimod (intracranial injection, 2 weeks) semapimos paired with radiotherapy considerably enhanced survival in GL261 tumor-bearing rats, but had no meaningful benefit in the absence of radiotherapy [4].
Animal Protocol	Animal/Disease Models: Male OZ rats[1] Doses: 5 mg/kg Route of Administration: Ip; daily for 2 weeks Experimental Results: Restored endothelium-dependent vasorelaxation in OZ rats . Animal/Disease Models: C57Bl/6 mice (GL261 cells were orthotopically implanted)[4] Doses: 6 mg/kg/day Route of Administration: Intracranially for 1 week, delivered via an osmotic pump Experimental Results: Inhibited tumor cell invasion by more than 75%.
References	[1]. Blockade of endogenous proinflammatory cytokines ameliorates endothelial dysfunction in obese Zucker rats. Hypertens Res. 2008;31(4):737‐743. [2]. Inhibition of p38 mitogen-activated protein kinase pathway as prophylaxis of postoperative ileus in mice. Gastroenterology. 2009;136(2):619‐629. [3]. Experimental Anti-Inflammatory Drug Semapimod Inhibits TLR Signaling by Targeting the TLR Chaperone gp96. J Immunol. 2016;196(12):5130‐5137. [4]. Semapimod sensitizes glioblastoma tumors to ionizing radiation by targeting microglia. PLoS One. 2014 May 9;9(5):e95885.
Additional Infomation	Semapimod has been used in trials studying the treatment of Crohn Disease.

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	1.3425 mL	6.7123 mL	13.4246 mL
	5 mM	0.2685 mL	1.3425 mL	2.6849 mL
	10 mM	0.1342 mL	0.6712 mL	1.3425 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.