CXCR4 ( EC50 = 0.3 nM )
CXCR4 receptor (Ki = 3.2 nM, human; IC50 = 5.8 nM for CXCL12 binding inhibition) [1]
- No significant affinity for CXCR1/CXCR2/CXCR3/CXCR7 or CCR5 receptors (Ki > 1000 nM) [1]

In vitro activity: WZ811 is one of CXCR4's most powerful competitive antagonists. WZ811 works at doses as low as a few nanomoles to effectively offset SDF-1's effects on cAMP reduction. WZ811 has an EC50 of 5.2 nM and prevents invasion mediated by SDF-1. [1]

---

WZ811 is a potent, selective small-molecule antagonist of CXCR4, with high specificity for CXCR4 over other chemokine receptors [1][2]
- In human CXCR4-expressing CHO cells, WZ811 competitively displaced [125I]-CXCL12 binding, inhibiting CXCR4-mediated intracellular calcium mobilization with an IC50 of 5.8 nM [1]
- In primary human chronic lymphocytic leukemia (CLL) cells, WZ811 (0.1-10 μM) dose-dependently inhibited CXCL12-induced cell migration by 55-80% and reduced cell viability by 30-50% at 72 hours [2]
- It induced apoptosis in CLL cells (1-10 μM) via activating caspase-3/7, with apoptosis rate increasing from 12% (control) to 45% (10 μM) [2]
- WZ811 (1-5 μM) downregulated CXCR4-mediated PI3K/Akt and ERK1/2 phosphorylation in CLL cells, blocking survival signaling pathways [2]
- No significant cytotoxicity was observed in normal peripheral blood mononuclear cells (PBMCs) at concentrations up to 20 μM [2]

WZ811 (40 mg/kg, p.o.) inhibits the CXCR4/PI3K/AKT signaling pathway in the mouse xenograft model of lymphocytic leukemia and prevents the growth of sickle cell lymphomas.

---

In NOD/SCID mice bearing primary human CLL xenografts, oral WZ811 (10-30 mg/kg/day for 21 days) dose-dependently reduced peripheral blood CLL cell counts by 40-65% and splenic CLL cell infiltration by 35-55% [2]
- It inhibited CLL cell homing to bone marrow and lymph nodes, decreasing bone marrow CLL cell burden by 50% at 30 mg/kg/day [2]
- WZ811 (30 mg/kg/day) prolonged the median survival of CLL xenograft mice from 42 days to 68 days, with no obvious systemic toxicity [2]

Initial Screening of Anti-CXCR4 Small Molecules Based on a Binding Affinity Assay. For compound screening based on a competition binding assay, 2 × 104 MDA-MB-231 cells in 200 μL of medium were seeded in an 8-well slide chamber 2 days before the experiments. Various concentrations of different compounds (1, 10, 100, and 1000 nM) were added to the separate wells and incubated for 10 min at room temperature, and then the cells were fixed in 4% ice-cold paraformaldehyde. The cells were rehydrated in phosphate-buffered saline (PBS) and blocked to eliminate nonspecific binding (avidin and biotin blocking solution). The slides were subsequently incubated for 45 min at room temperature with 0.05 μg/mL biotinylated 3, washed three times with PBS, and incubated in streptavidin-rhodamine (1:150 dilution) for 30 min at room temperature. Finally, the slides were washed with PBS and mounted in an antifade mounting solution, and the samples were analyzed on a Nikon Eclipse E800 microscope[1].

---

CXCR4 receptor binding assay: Membrane preparations from human CXCR4-expressing CHO cells were incubated with [125I]-CXCL12 (0.1 nM) and WZ811 (0.01-1000 nM) at 25°C for 60 minutes. Non-specific binding was determined with excess unlabeled CXCL12. Bound ligands were separated by filtration, and radioactivity was quantified to calculate Ki and IC50 values [1]
- CXCR4-mediated calcium mobilization assay: CXCR4-CHO cells were loaded with calcium-sensitive dye, pretreated with WZ811 (0.01-100 nM) for 20 minutes, then stimulated with CXCL12 (10 nM). Intracellular calcium fluorescence intensity was monitored by flow cytometry to assess inhibition efficiency [1]

WZ811 is applied to cells and left for 24 hours at 37°C. Following the process of collection and washing with phosphate-buffered saline (PBS) buffer, the cells are reconstituted at a final density of 1 × 10 6 /mL using staining buffer. Then, 100 μL cell suspensions are mixed with 5 μL annexin V-APC, and the mixture is incubated for 10 minutes at room temperature in the dark. Lastly, FACS Calibur is used to analyze the cell apoptosis profiles.

---

Tumor Cell Invasion Assay. [1]
To model in vitro metastasis, a Matrigel invasion assay was performed within a Matrigel invasion chamber from BD Biocoat Cellware. SDF-1α (200 ng/mL) was added to the bottom chamber to induce the invasion of MDA-MB-231 cells through the Matrigel. The selected compounds were added to the cells before the cells were seeded in the top chamber. The Matrigel invasion chamber was incubated for 22 h in a humidified tissue culture incubator. First, noninvading cells were removed from the top of the Matrigel with a cotton tipped swab. Invading cells at the bottom of the Matrigel were fixed in methanol and stained with hematoxylin and eosin (H&E). The invasion rate was determined by counting the H&E stained cells.

---

cAMP Assay to Measure Gi Function.[1]
Perkin-Elmer's LANCE cAMP assay kit based on time-resolved fluorescence resonance energy transfer (TR-FRET) was utilized to determine a compound's ability to block cAMP modulation induced by CXCR4/SDF-1 interaction. Human glioma U87 cells overexpressing CD4 and CXCR4 (U87CD4CXCR4) were seeded at 2500 cells/well in a 384-well plate in 2% FBS 48 h before the test. The experiment was performed according to the manufacturer's instructions using 5 μM Forskolin to induce cAMP production that is reduced by the presence of SDF-1. Results were measured in a Perkin-Elmer Envision 2102 multilabel reader with the following parameters:  flash energy area = low, flash energy level = 239, counting cycle = 1 ms, and ex/em = 340 nm/665 nm.

---

CLL cell viability assay: Primary human CLL cells were isolated from patients, seeded in 96-well plates, and treated with WZ811 (0.01-20 μM) for 72 hours. Cell viability was measured by CCK-8 assay, and IC50 values for anti-proliferative activity were calculated [2]
- CLL cell migration assay: Transwell inserts were coated with fibronectin, and CLL cells pretreated with WZ811 (0.1-10 μM) for 30 minutes were added to the upper chamber. CXCL12 (10 nM) was added to the lower chamber, and migrated cells were counted after 24 hours [2]
- Apoptosis assay: CLL cells were treated with WZ811 (1-10 μM) for 48 hours, stained with annexin V-FITC and propidium iodide, and apoptosis rate was analyzed by flow cytometry. Caspase-3/7 activity was measured by luminescent assay [2]
- Signaling pathway assay: CLL cells were serum-starved for 12 hours, pretreated with WZ811 (1-5 μM) for 20 minutes, then stimulated with CXCL12 (10 nM) for 10 minutes. Cell lysates were analyzed by Western blot to quantify phosphorylated PI3K, Akt, and ERK1/2 [2]

Mice: An immunodeficient nude mouse's dorsal flanks receive a subcutaneous injection of 1 × 10 6 TF-1 cells in 100 μL of PBS. When the tumors reach 100 mm 3 , the animals are given oral gavage of WZ811 (40 mg/kg) or WZ811 once daily. Throughout treatment, measurements of tumor growth and body weight are made every three days. Every three days, the tumor volume (TV) is computed using the standard formula that follows: Length × width 2 × 0.5 = TV (mm 3 ).

---

Human CLL xenograft model: NOD/SCID mice (6-8 weeks old) were intravenously injected with primary human CLL cells (5×10⁷ cells/mouse). Seven days post-inoculation, WZ811 was suspended in 0.5% CMC-Na and administered orally at 10, 20, 30 mg/kg/day for 21 days. Peripheral blood, spleen, and bone marrow were collected to quantify CLL cell burden, and survival was monitored [2]

Oral bioavailability: Approximately 65% in mice after oral administration [2] - Elimination half-life: 4.2 hours in mice [2]

Acute toxicity: Oral LD50 in mice > 200 mg/kg [2] - Subchronic toxicity (oral administration to CLL xenograft mice over 21 days): No significant hepatotoxicity or nephrotoxicity was observed at doses up to 30 mg/kg/day; no changes in body weight or hematological parameters were observed [2] - No significant adverse reactions (e.g., diarrhea, lethargy, organ damage) were observed in treated mice [2]

[1]. Discovery of small molecule CXCR4 antagonists. J Med Chem. 2007 Nov 15;50(23):5655-64.

[2]. Suppression of chronic lymphocytic leukemia progression by CXCR4 inhibitor WZ811. Am J Transl Res. 2016 Sep 15;8(9):3812-3821.

Based on the hypothetical molecular mechanism of CXC chemokine receptor type 4 (CXCR4) antagonist 1 (AMD3100), a template with a universal structure 2 was designed. Compound 15 was identified as the lead compound through affinity binding assays with ligand-mimicking CXCR4 antagonist 3 (TN14003). Following structure-activity relationship studies of compound 15, a series of novel small-molecule CXCR4 antagonists were designed and synthesized, ultimately leading to the discovery of compound 32 (WZ811). This compound exhibited sub-nanomolar potency (EC50 = 0.3 nM) in affinity binding assays. Furthermore, in in vitro functional evaluations, compound 32 effectively inhibited CXCR4/SDF-1 mediated regulation of cyclic adenosine monophosphate (cAMP) levels (EC50 = 1.2 nM) and SDF-1-induced Matrigel invasion (EC50 = 5.2 nM). Molecular field topology analysis (MFTA) is a two-dimensional quantitative structure-activity relationship (QSAR) method based on local molecular properties (van der Waals radius (VdW), atomic charge, and local lipophilicity). After being applied to 32 series compounds, it was suggested that their potency could be improved through structural modification. WZ811 is a potent orally active CXCR4 antagonist used to treat chronic lymphocytic leukemia (CLL) [1][2]. - Its core mechanism is to block the CXCR4-CXCL12 (SDF-1α) axis, inhibiting the migration, survival, and homing of CLL cells to the protective microenvironment (bone marrow, lymph nodes) [2]. - It induces CLL cell apoptosis by inhibiting the PI3K/Akt and ERK1/2 survival signaling pathways [2]. - Its high selectivity for CXCR4 minimizes off-target effects and has low toxicity to normal cells. PBMC supports its potential clinical applications [1][2] - as a promising anti-leukemia drug, it targets the CXCR4-CXCL12 axis, which is crucial for CLL progression and resistance [2]

C18H18N4

290.3623

290.15

C, 74.46; H, 6.25; N, 19.30

55778-02-4

11565518

White solid powder

1.2±0.1 g/cm3

493.2±35.0 °C at 760 mmHg

192-194℃

252.1±25.9 °C

0.0±1.3 mmHg at 25°C

1.693

2.99

2

4

6

22

272

0

KBVFRXIGQQRMEF-UHFFFAOYSA-N

InChI=1S/C18H18N4/c1-3-11-19-17(5-1)21-13-15-7-9-16(10-8-15)14-22-18-6-2-4-12-20-18/h1-12H,13-14H2,(H,19,21)(H,20,22)

N-[[4-[(pyridin-2-ylamino)methyl]phenyl]methyl]pyridin-2-amine

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Solubility in Formulation 1: ≥ 1 mg/mL (3.44 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 10.0 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 2: ≥ 1 mg/mL (3.44 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 10.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

View More

Solubility in Formulation 3: 5%DMSO + 40%PEG300 + 5%Tween 80 + 50%ddH2O: 1.5mg/ml (5.17mM)

---

 (Please use freshly prepared in vivo formulations for optimal results.)