AMD3465 hexahydrobromide (AMD-3465; GENZ-644494 6-HBr) is a novel monomacrocyclic antagonist of CXCR4 that has potential anticancer and anti-HIV activity. It effectively prevents CXCL12 from binding to SupT1 cells, with an IC50 of 18 nM. With an IC50 of 17 nM, AMD3465 blocks both MAPK phosphorylation and CXCL12-induced calcium signaling in SupT1 cells. However, in U87.CD4.CCR5 cells, AMD3465 was unable to inhibit the intracellular calcium fluxes induced by the CCR5 ligands RANTES, LD78β, and MIP-1β. AMD3465 inhibits the chemotaxis that human T-lymphoid SupT1 cells experience when exposed to CXCL12 and stops U87.CD4 cells from internalizing CXCR4 due to chemokines. Furthermore, AMD3465 exhibits activity against the X4 HIV-1 strains IIIB, NL4.3, RF, and HE, with an IC50 ranging from 6 to 12 nM. With an IC50 of 12.3 nM, AMD3465 inhibits the HIV-2 strains ROD and EHO.

Physicochemical Properties

Molecular Formula	C24H44BR6N6
Molecular Weight	896.07
Exact Mass	889.872
Elemental Analysis	C, 32.17; H, 4.95; Br, 53.50; N, 9.38
CAS #	185991-07-5
Related CAS #	AMD 3465; 185991-24-6
PubChem CID	9897616
Appearance	Light yellow to yellow solid powder
Density	1.022g/cm3
Boiling Point	571.3ºC at 760 mmHg
Flash Point	299.3ºC
Vapour Pressure	5.1E-13mmHg at 25°C
Index of Refraction	1.533
LogP	8.799
Hydrogen Bond Donor Count	10
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	6
Heavy Atom Count	36
Complexity	413
Defined Atom Stereocenter Count	0
SMILES	C1(CNCC2=CC=C(CN3CCNCCCNCCNCCC3)C=C2)=NC=CC=C1.[6HBr]
InChi Key	ARHBIBDGWDRBJH-UHFFFAOYSA-N
InChi Code	InChI=1S/C24H38N6.6BrH/c1-2-13-29-24(5-1)20-28-19-22-6-8-23(9-7-22)21-30-17-4-12-26-15-14-25-10-3-11-27-16-18-30;;;;;;/h1-2,5-9,13,25-28H,3-4,10-12,14-21H2;6*1H
Chemical Name	N-(pyridin-2-ylmethyl)-1-[4-(1,4,8,11-tetrazacyclotetradec-1-ylmethyl)phenyl]methanamine;hexahydrobromide
Synonyms	AMD3465 hexahydrobromide; GENZ644494 hexahydrobromide; AMD-3465 6HBr; GENZ-644494 hexahydrobromide; AMD 3465; GENZ 644494 hexahydrobromide
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, 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	12G5 mAb-CXCR4 ( IC50 = 1.1 nM ); CXCL12AF647-CXCR4 ( IC50 = 1.7 nM ); X4 HIV-1 (NL4.3) ( IC50 = 121 nM ); X4 HIV-1 (RF) ( IC50 = 1.1 nM ); X4 HIV-1 (HE) ( IC50 = 1.7 nM ); X4 HIV-1 (IIIB) ( IC50 = 121 nM ); X4 HIV-1 (NL4.3AMD3100) ( IC50 = 1.1 nM ); HIV-2 (ROD) ( IC50 = 1.7 nM ); HIV-2 (EHO) ( IC50 = 121 nM )
ln Vitro	In vitro activity: AMD 3465 hexahydrobromide is a potent antagonist of CXCR4 that prevents the binding of 12G5 mAb and CXCL12AF647 to CXCR4, with IC50s of 0.75 nM and 18 nM in SupT1 cells. With an IC50 of 17 nM, AMD 3465 (50 nM) completely inhibits the calcium mobilization induced by CXCL12, but it has no effect on the intracellular calcium fluxes that are induced in U87.CD4.CCR5 cells by the CCR5 ligands RANTES, LD78β, and MIP-1β. AMD 3465 has no effect on viruses that use CCR5 (R5), but it potently inhibits the replication of X4 HIV strains (IC50: 1-10 nM). With an IC50 ranging from 6 to 12 nM, AMD3465 is cytotoxic to the X4 HIV-1 strains IIIB, NL4.3, RF, and HE. The HIV-2 strains ROD and EHO can be suppressed at an IC50 of 12.3 nM[1]. AMD 3465 prevents U87 and Daoy cells from growing in response to CXCL-12. In U87 and Daoy cells, AMD 3465 treatment increases Erk1/2 phosphorylation[2].
ln Vivo	AMD 3465 (2.5 mg/kg/d, s.c. for 5 weeks) notably inhibits the proliferation of Daoy xenografts and U87 GBM[2].
Enzyme Assay	In order to conduct competition binding studies against CXCR4, a concentration range of AMD3465 is incubated for three hours at 4°C in binding buffer (PBS containing pH 7.4, 0.25% BSA, 1 mM CaCl2, and 5 ×105 CCRF-CEM cells) in Millipore DuraporeTM filter plates. Washing with cold 50 mM HEPES and 0.5 M NaCl pH 7.4 removes unbound 125I-SDF-1α. Membranes from CHO-S cells that express recombinant BLT1 are used for the competition binding assay against that protein. The membranes are prepared using mechanical cell lysis, high-speed centrifugation, resuspension in a buffer containing 50 mM HEPES and 5 mM MgCl2, and flash freezing. The assay mixture comprising 50 mM Tris, pH 7.4, 10 mM MgCl2, 10 mM CaCl2, 4 nM LTB4 combined with 1 nM 3H-LTB4, and 8 μg membrane is incubated with AMD3465 for 1 hour at room temperature. Filtration is used to separate the unbound 3H-LTB4 on Millipore Type GF-C filter plates. A Liquid Scintillation Counter (LKB Rackbeta 1209), is used to count the bound radioactivity. MCE has not independently verified these techniques' accuracy. They are merely meant to be used as references.
Cell Assay	After a 24-hour serum starvation period, 1 μg/mL CXCL12, 2.5 ng/mL AMD 3465, 200 μM rolipram, or 10 μM forskolin are administered to astrocytes, granule cells, U87 cells, and Daoy cells. After 24 and 48 hours of treatment, respectively, trypan blue exclusion is used to measure the growth of Daoy and U87 cells in culture[2].
Animal Protocol	Mice: After cells are implanted, mice are imaged at least twice in order to identify those with comparable tumor growth rates. Cohorts of mice with roughly equal tumor bioluminescence are split into equal control and treatment groups two weeks after tumor cell implantation. In AMD 3465 experiments, animals are given sterile PBS or PBS alone through s.c. osmotic pumps that are loaded with 10 mg/mL AMD 3465. The rate of infusion is 50 μg/d, or 0.25 μL/h. In the rolipram or caffeine experiments, 100 μg/g/d of caffeine or 5 μg/g/d of rolipram are given orally to the mice in the treatment groups. Over the course of seven days, daily measurements of each animal's water consumption are used to calculate the drug concentration in the water. The recommended dosage is provided by adjusting concentrations in accordance with water consumption[2].
References	[1]. AMD3465, a monomacrocyclic CXCR4 antagonist and potent HIV entry inhibitor. Biochem Pharmacol. 2005 Sep 1;70(5):752-61. [2]. Blocking CXCR4-mediated cyclic AMP suppression inhibits brain tumor growth in vivo. Cancer Res. 2007 Jan 15;67(2):651-8.

Solubility Data

Solubility (In Vitro)

DMSO: 2~50 mg/mL (2.2~55.8 mM) Water: ~98 mg/mL (~109.4 mM) Ethanol: Insoluble

Solubility (In Vivo)

Solubility in Formulation 1: ≥ 2.5 mg/mL (2.79 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 (2.79 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. Solubility in Formulation 3: 100 mg/mL (111.60 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.  (Please use freshly prepared in vivo formulations for optimal results.)

Preparing Stock Solutions		1 mg	5 mg	10 mg
	1 mM	1.1160 mL	5.5799 mL	11.1598 mL
	5 mM	0.2232 mL	1.1160 mL	2.2320 mL
	10 mM	0.1116 mL	0.5580 mL	1.1160 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.