Motolimod (also known as VTX-2337; VTX 378; VTX2337) is a novel, selective and potent small molecule agonist of the Toll-like receptor (TLR) 8 with immunostimulating/modulatory and anticancer effects. It activates TLR8 with EC50 of 100 nM, and displays > 50-fold selectivity over TLR7 and has potential immunostimulating and anticancer activities. Motolimod is currently under clinical development as an immunotherapy for multiple oncology indications such as ovarian cancer and squamous cell carcinoma of the head and neck. TLR8 is located in the endosome where it functions in the recognition of foreign nucleic acids from intracellular pathogens. TLR8 has emerged as a potential target for anticancer immunotherapies.

Physicochemical Properties

Molecular Formula	C28H34N4O2
Molecular Weight	458.6
Exact Mass	458.268
CAS #	926927-61-9
Related CAS #	926927-61-9
PubChem CID	16049404
Appearance	Light yellow to brown solid powder
Density	1.2±0.1 g/cm3
Boiling Point	718.7±70.0 °C at 760 mmHg
Flash Point	388.5±35.7 °C
Vapour Pressure	0.0±2.3 mmHg at 25°C
Index of Refraction	1.623
LogP	2.71
Hydrogen Bond Donor Count	1
Hydrogen Bond Acceptor Count	3
Rotatable Bond Count	7
Heavy Atom Count	34
Complexity	771
Defined Atom Stereocenter Count	0
InChi Key	QSPOQCXMGPDIHI-UHFFFAOYSA-N
InChi Code	InChI=1S/C28H34N4O2/c1-3-13-31(14-4-2)28(34)24-17-23-12-11-22(18-25(23)30-26(29)19-24)20-7-9-21(10-8-20)27(33)32-15-5-6-16-32/h7-12,17-18H,3-6,13-16,19H2,1-2H3,(H2,29,30)
Chemical Name	2-amino-N,N-dipropyl-8-[4-(pyrrolidine-1-carbonyl)phenyl]-3H-1-benzazepine-4-carboxamide
Synonyms	VTX-2337; VTX 2337;VTX 378; VTX2337;VTX-378; VTX378
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	Motolimod (VTX-2337) selectively activates TLR8, out of all the studied TLRs (TLR2, 3, 4, 5, 7, 8, and 9). Motolimod increases TNFα (EC50=140±30 nM, based on 10 donors) and IL-12 (EC50=120±30 nM, based on 10 donors) production in peripheral blood monolayers. Motolimod has an EC50 value of 60 nM for inducing MIP-1β[1].
ln Vivo	Motolimod (1 or 10 mg/kg) was given subcutaneously into the monkeys, and plasma was taken prior to the injection as well as six, twelve, twenty-four, and ninety-six hours later. Six hours after injection (10 mg/kg), mean plasma levels of IL-1β increased with 10 mg/kg Motolimod from a baseline level of 0.5 pg/mL to as high as 9.12±2.7 ng/mL (p<0.05, t-test). Additionally, circulating levels of IL-18 rose (p<0.05, t-test) from a baseline of approximately 1 pg/mL to 68.7±4.4 pg/mL. Since IL-6 is released in response to TLR8 activation but is not activated by NLRP3 inflammasome activation, IL-6 levels were observed. Additionally, IFNγ plasma levels were measured in response to Motolimod treatment-induced NK cell activation [2].
Animal Protocol	1 or 10 mg/kg, s.c. injection Mouse model
References	[1]. VTX-2337 is a novel TLR8 agonist that activates NK cells and augments ADCC. Clin Cancer Res. 2012 Jan 15;18(2):499-509. [2]. Coordinated Activation of Toll-Like Receptor8 (TLR8) and NLRP3 by the TLR8 Agonist, VTX-2337, Ignites Tumoricidal Natural Killer Cell Activity. PLoS One. 2016 Feb 29;11(2):e0148764.
Additional Infomation	Motolimod has been used in trials studying the treatment of Lymphoma, Tongue Cancer, Ovarian Cancer, Adult Solid Neoplasm, and Fallopian Tube Cancer, among others. Motolimod is a small-molecule Toll-like receptor 8 (TLR8) agonist with potential immunostimulating and antineoplastic activities. Motolimod binds to TLR8, present in cutaneous dendritic cells, monocytes/macrophages, and mast cells, which may result in the activation of the central transcription factor nuclear factor-B, the secretion of proinflammatory cytokines and other mediators, and a Th1-weighted antitumoral cellular immune response. Primarily localized to endosomal membranes intracellularly, TLR8, like other TLRs, recognizes pathogen-associated molecular patterns (PAMPs) and plays a key role in the innate immune system.

Solubility Data

Solubility (In Vitro)

DMSO:55 mg/mL (119.9 mM) Water:<1 mg/mL Ethanol:15 mg/mL (32.7 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.45 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 (5.45 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in 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 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly. 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. Solubility in Formulation 3: ≥ 2.5 mg/mL (5.45 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	2.1805 mL	10.9027 mL	21.8055 mL
	5 mM	0.4361 mL	2.1805 mL	4.3611 mL
	10 mM	0.2181 mL	1.0903 mL	2.1805 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.