EG01377 di-HCl is a potent, bioavailable and selective neuropilin-1 (NRP1) inhibitor (antagonist) with a Kd of 1.32 μM and IC50 of 609 nM for both NRP1-a1 and NRP1-b1. EG01377 di-HCl has anti-angiogenesis, anti-migration and anti-tumor activities.

Physicochemical Properties

Molecular Formula	C26H32CL2N6O6S2
Molecular Weight	659.604881286621
Exact Mass	658.12
CAS #	2749438-61-5
Related CAS #	EG01377;2227996-00-9
PubChem CID	146681179
Appearance	White to off-white solid powder
Hydrogen Bond Donor Count	8
Hydrogen Bond Acceptor Count	10
Rotatable Bond Count	12
Heavy Atom Count	42
Complexity	1010
Defined Atom Stereocenter Count	1
SMILES	C1COC2=C1C=C(C=C2S(=O)(=O)NC3=C(SC=C3)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)C4=CC=C(C=C4)CN.Cl.Cl
InChi Key	ZYFLTIBQQSYBQT-FJSYBICCSA-N
InChi Code	InChI=1S/C26H30N6O6S2.2ClH/c27-14-15-3-5-16(6-4-15)18-12-17-7-10-38-22(17)21(13-18)40(36,37)32-19-8-11-39-23(19)24(33)31-20(25(34)35)2-1-9-30-26(28)29;;/h3-6,8,11-13,20,32H,1-2,7,9-10,14,27H2,(H,31,33)(H,34,35)(H4,28,29,30);2*1H/t20-;;/m0../s1
Chemical Name	(2S)-2-[[3-[[5-[4-(aminomethyl)phenyl]-2,3-dihydro-1-benzofuran-7-yl]sulfonylamino]thiophene-2-carbonyl]amino]-5-(diaminomethylideneamino)pentanoic acid;dihydrochloride
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.
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	IC50: 609 nM (NRP1-a1 and NRP1-b)[1]
ln Vitro	Vascular endothelial growth factor A (VEGF-A) induced tyrosine phosphorylation of VEGF-R2/KDR is inhibited by EG01377 (3-30 μM; 30 minutes)[1]. VEGFA can considerably lessen HUVEC cell migration when EG01377 (30 μM) is applied[1]. EG01377 (30 μM; 5 days) can postpone the closure of wounds caused by VEGF[1]. The network area, length, and branching points are all decreased by EG01377 (30 μM)[1]. EG01377 (30 μM; 7 days) inhibits angiogenesis produced by VEGF [1]. When combined with VEGFA, EG01377 (30 μM; 7 days) decreases the growth of A375P (malignant melanoma) spheroid outgrowth[1]. When tumor cell-derived factors are present, EG01377 (500 nM; 2 hours) prevents Nrp1+ regulatory T-cell SMAD3/AKT (Tregs) from producing transforming growth factor beta (TGFβ)[1].
ln Vivo	EG01377 (2 mg/kg; iv) has a promising half-life of 4.29 h, long enough to support treatment once daily in mice[1].
Cell Assay	Western Blot Analysis[1] Cell Types: Human umbilical vein endothelial cells (HUVECs) Tested Concentrations: 3, 10 , 30 μM Incubation Duration: 30 minutes Experimental Results: Inhibited VEGF-A stimulated tyrosine phosphorylation of VEGF-R2/KDR with an IC50 of 30 μM.
Animal Protocol	Animal/Disease Models: 6-8 week-old BABL/c female mice[1] Doses: 2 mg/kg (pharmacokinetic/PK Analysis) Route of Administration: Iv administration Experimental Results: The half time (T1/2) of 4.29 h.
References	[1]. Small Molecule Neuropilin-1 Antagonists Combine Antiangiogenic and Antitumor Activity with Immune Modulation through Reduction of Transforming Growth Factor Beta (TGFβ) Production in Regulatory T-Cells. J Med Chem. 2018 May 10;61(9):4135-4154.

Solubility Data

Solubility (In Vitro)

DMSO: 200 mg/mL (303.21 mM)

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 5 mg/mL (7.58 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 50.0 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: ≥ 5 mg/mL (7.58 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 50.0 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: ≥ 5 mg/mL (7.58 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 50.0 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	1.5161 mL	7.5804 mL	15.1607 mL
	5 mM	0.3032 mL	1.5161 mL	3.0321 mL
	10 mM	0.1516 mL	0.7580 mL	1.5161 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.