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Purity: ≥98%
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.
| 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 ) C-X-C Chemokine Receptor 4 (CXCR4) (Ki = 1.2 nM for human recombinant CXCR4; IC50 = 2.7 nM for [125I]-SDF-1α binding to CXCR4; EC50 = 0.1-0.5 nM for inhibiting HIV-1 (X4-tropic strains) entry into T cells; >1000-fold selectivity over CCR5, CCR3, and other chemokine receptors) [1] |
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| ln Vitro |
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| ln Vivo |
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| 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.
CXCR4 radioligand binding assay: Membrane preparations from human recombinant CXCR4-expressing cells or Jurkat T cells were incubated with [125I]-SDF-1α (0.1 nM) and serial concentrations of AMD3465 hexahydrobromide (0.001-100 nM) at 25°C for 90 minutes. Bound and free ligands were separated by filtration, and radioactivity was quantified to calculate Ki and IC50 values for binding inhibition [1] - Chemokine receptor selectivity assay: Membrane preparations from cells expressing CCR5, CCR3, or CXCR7 were incubated with respective [125I]-labeled chemokines and AMD3465 hexahydrobromide (0.01-10 μM) under the same conditions as CXCR4 assay. Binding inhibition was measured to confirm selectivity over other chemokine receptors [1] |
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| 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].
HIV entry inhibition assay: Jurkat T cells were pretreated with AMD3465 hexahydrobromide (0.001-1 μM) for 1 hour, then infected with X4-tropic HIV-1 (NL4-3 or IIIB) at an MOI of 0.1. Viral replication was quantified by p24 antigen ELISA after 48 hours, and EC50 values were derived from dose-response curves [1] - Glioblastoma cell proliferation assay: U87MG and U251 cells were cultured in DMEM medium supplemented with fetal bovine serum. Cells were treated with AMD3465 hexahydrobromide (0.1-100 nM) for 72 hours, and cell viability was assessed by MTT assay; GI50 values were calculated [2] - cAMP detection assay: U87MG cells were serum-starved for 24 hours, pretreated with AMD3465 hexahydrobromide (0.1-50 nM) for 30 minutes, then stimulated with CXCL12 (100 nM) for 15 minutes. Intracellular cAMP levels were quantified by a competitive ELISA kit [2] - Cell migration assay: U87MG cells were seeded in the upper chamber of transwell inserts, and AMD3465 hexahydrobromide (0.1-50 nM) was added to both chambers. CXCL12 (100 nM) was added to the lower chamber as a chemoattractant. After 24 hours, migrated cells on the lower membrane were fixed, stained, and counted [2] |
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| Animal Protocol |
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| ADME/Pharmacokinetics |
The plasma protein binding rate of AMD3465 hexahydrobromide in human plasma is 85%, and that in rat plasma is 82% [1]. The terminal elimination half-life (t1/2) of the drug in rat plasma is 3.5 hours (iv) and 5.8 hours (sc); the peak plasma concentration (Cmax) is 850 ng/mL (intravenous injection, 10 mg/kg) and 420 ng/mL (subcutaneous injection, 20 mg/kg), respectively [1]. AMD3465 hexahydrobromide is widely distributed in tissues. The highest concentrations in rats were found in the liver (620 ng/g), kidney (580 ng/g), and brain (180 ng/g) 1 hour after subcutaneous administration [1]. Approximately 60% of the dose in rats is excreted in urine within 72 hours, and approximately 30% is excreted in feces (in unchanged form) [1].
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| Toxicity/Toxicokinetics |
AMD3465 hexahydrobromide (≤1 μM) showed no cytotoxicity to normal human astrocytes and Jurkat T cells, with cell survival >90% after 72 hours [1][2]
- Acute toxicity in mice: A single intraperitoneal injection of AMD3465 hexahydrobromide (up to 500 mg/kg) did not cause death or significant weight loss (<5%) [1] - In a rat subchronic toxicity study (28 days), administration of AMD3465 hexahydrobromide (20 mg/kg/day, subcutaneous injection) did not result in significant changes in serum ALT, AST, creatinine, or blood urea nitrogen levels; no pathological damage was observed in the liver, kidneys, heart, or lungs [1] - AMD3465 hexahydrobromide (5 mg/kg/day, intraperitoneal injection ×21) did not induce neurotoxicity or inflammation in mouse brain tissue [2] |
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| References | ||
| Additional Infomation |
AMD3465 hexahydrobromide is a monocyclic selective CXCR4 antagonist and potent HIV invasion inhibitor[1] - Its mechanism of action involves competitive binding to CXCR4, blocking its interaction with its ligand SDF-1α (CXCL12), thereby inhibiting CXCR4-mediated viral invasion (HIV-1 X4 tropism) and tumor cell proliferation/migration[1][2] - The drug can cross the blood-brain barrier and is detectable in rat brain tissue, supporting its development for the treatment of CXCR4-positive brain tumors[2] - It is highly selective for CXCR4 compared to other chemokine receptors, minimizing off-target effects associated with chemokine signaling[1] - Preclinical studies support its potential application in the treatment of X4 tropism HIV infection and CXCR4-positive malignancies such as glioblastoma[1][2]
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| Molecular Formula |
C24H44BR6N6
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| Molecular Weight |
896.07
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| Exact Mass |
889.872
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| Elemental Analysis |
C, 32.17; H, 4.95; Br, 53.50; N, 9.38
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| CAS # |
185991-07-5
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| Related CAS # |
AMD 3465; 185991-24-6
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| PubChem CID |
9897616
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| Appearance |
Light yellow to yellow solid powder
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| Density |
1.022g/cm3
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| Boiling Point |
571.3ºC at 760 mmHg
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| Flash Point |
299.3ºC
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| Vapour Pressure |
5.1E-13mmHg at 25°C
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| Index of Refraction |
1.533
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| LogP |
8.799
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| Hydrogen Bond Donor Count |
10
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| Hydrogen Bond Acceptor Count |
6
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| Rotatable Bond Count |
6
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| Heavy Atom Count |
36
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| Complexity |
413
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| Defined Atom Stereocenter Count |
0
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| SMILES |
C1(CNCC2=CC=C(CN3CCNCCCNCCNCCC3)C=C2)=NC=CC=C1.[6HBr]
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| InChi Key |
ARHBIBDGWDRBJH-UHFFFAOYSA-N
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| 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
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| Chemical Name |
N-(pyridin-2-ylmethyl)-1-[4-(1,4,8,11-tetrazacyclotetradec-1-ylmethyl)phenyl]methanamine;hexahydrobromide
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| Synonyms |
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| HS Tariff Code |
2934.99.9001
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| 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. |
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| Shipping Condition |
Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)
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| Solubility (In Vitro) |
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| 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. View More
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. |
| 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.
Calculation results
Working concentration： mg/mL；
Method for preparing DMSO stock solution： mg drug pre-dissolved in μL DMSO (stock solution concentration mg/mL). Please contact us first if the concentration exceeds the DMSO solubility of the batch of drug.
Method for preparing in vivo formulation:：Take μL DMSO stock solution, next add μL PEG300, mix and clarify, next addμL Tween 80, mix and clarify, next add μL ddH2O，mix and clarify.
(1) Please be sure that the solution is clear before the addition of next solvent. Dissolution methods like vortex, ultrasound or warming and heat may be used to aid dissolving.
(2) Be sure to add the solvent(s) in order.
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