AMD3465 (AMD-3465) is a novel monomacrocyclic antagonist of CXCR4 with 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	C24H38N6
Molecular Weight	410.10
Exact Mass	410.315
Elemental Analysis	C, 70.20; H, 9.33; N, 20.47
CAS #	185991-24-6
Related CAS #	AMD 3465 hexahydrobromide; 185991-07-5
PubChem CID	483559
Appearance	Off-white to light yellow solid powder
Density	1.0±0.1 g/cm3
Boiling Point	571.3±50.0 °C at 760 mmHg
Flash Point	299.3±30.1 °C
Vapour Pressure	0.0±1.6 mmHg at 25°C
Index of Refraction	1.533
LogP	0.92
Hydrogen Bond Donor Count	4
Hydrogen Bond Acceptor Count	6
Rotatable Bond Count	6
Heavy Atom Count	30
Complexity	413
Defined Atom Stereocenter Count	0
SMILES	C1(CNCC2=CC=C(CN3CCNCCCNCCNCCC3)C=C2)=NC=CC=C1
InChi Key	CWJJHESJXJQCJA-UHFFFAOYSA-N
InChi Code	InChI=1S/C24H38N6/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
Chemical Name	N-(pyridin-2-ylmethyl)-1-[4-(1,4,8,11-tetrazacyclotetradec-1-ylmethyl)phenyl]methanamine
Synonyms	AMD3465; GENZ-644494; AMD-3465; GENZ644494; AMD 3465; GENZ 644494
HS Tariff Code	2934.99.03.00
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	12G5 mAb-CXCR4 ( IC50 = 0.75 nM ); CXCL12AF647-CXCR4 ( IC50 = 18 nM ); X4 HIV-1 (IIIB) ( IC50 = 12.3 nM ); X4 HIV-1 (NL4.3) ( IC50 = 6.1 nM ); X4 HIV-1 (NL4.3AMD3100) ( IC50 = 2822 nM ); X4 HIV-1 (RF) ( IC50 = 7.4 nM ); X4 HIV-1 (HE) ( IC50 = 9.8 nM ); HIV-2 (ROD) ( IC50 = 12.3 nM ); HIV-2 (EHO) ( IC50 = 12.3 nM )
ln Vitro	In vitro activity: AMD 3465 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: ~10 mM Water: N/A Ethanol: Insoluble

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	2.4384 mL	12.1921 mL	24.3843 mL
	5 mM	0.4877 mL	2.4384 mL	4.8769 mL
	10 mM	0.2438 mL	1.2192 mL	2.4384 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.