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Purity: ≥98%
LIT-927 (LIT927 ) is a novel potent, locally and orally bioactive neutraligand for the chemokine CXCL12 with anti-inflammatory and anticancer activity. Its Ki for blocking CXCL12-TR binding is 267 nM. In a murine model of allergic airway hypereosinophilia, it has anti-inflammatory effects. LIT-927 neutralizes the biological activity of chemokine CXCL12 by binding to it instead of its cognate receptors, CXCR4 or CXCR7. The novel neutraligand of CXCL12, LIT-927, is no longer a Michael acceptor and has a higher solubility than the lead compound. In a murine model of allergic airway hypereosinophilia, LIT-927 decreases eosinophil recruitment. As such, it is a potent pharmacological tool for studying CXCL12 physiology in vivo and the role of chemokine neutralization in inflammatory and related diseases.
| Targets |
CXCR4; CXCL12( Ki = 267 nM )
LIT-927 has the ability to specifically bind CXCL12 and block the interaction between CXCL12 and CXCR4[1]. |
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| ln Vitro |
LIT-927 has the ability to specifically bind CXCL12 and block the interaction between CXCL12 and CXCR4[1].
LIT-927 selectively inhibits CXCL12 binding to its CXCR4 receptor in a FRET-based binding assay with a Ki of 267 ± 71 nM. [1] In a calcium mobilization assay, LIT-927 (10 µM) inhibits the intracellular calcium increase induced by CXCL12 (5 nM) in HEK cells expressing CXCR4-EGFP. [1] LIT-927 shows high selectivity for CXCL12. At 10 µM, it does not inhibit calcium responses triggered by other chemokines (CCL17, CCL22, CCL5, CCL2) on cells expressing their respective receptors (CCR4, CCR5, CCR2). [1] LIT-927 binds directly to CXCL12, as evidenced by a dose-dependent quenching of intrinsic tryptophan (Trp-57) fluorescence of CXCL12, yielding a Kd of 780 ± 320 nM. [1] LIT-927 is not a Michael acceptor. Incubation with glutathione (GSH) for 2 hours at 37°C does not lead to the formation of a GSH adduct, unlike the parent chalcone compound. [1] |
| ln Vivo |
LIT-927 exhibits a significant and statistically significant inhibition of eosinophil recruitment in a mouse model of hypereosinophilia. When given orally or locally, it reduces allergic eosinophilic airway inflammation in mice[1].
In a murine 8-day model of allergic airway hypereosinophilia, intranasal (i.n.) administration of LIT-927 (300 nmol/kg) 2 hours before each ovalbumin (OVA) challenge significantly reduces eosinophil recruitment into bronchoalveolar lavage (BAL) fluid by 48%. [1] The dose-response study for topical (i.n.) administration of LIT-927 in the same model yields an IC50 of 310 nmol/kg for inhibition of eosinophil recruitment. [1] Intraperitoneal (i.p.) administration of LIT-927 (350 µmol/kg) 2 hours before OVA challenge inhibits eosinophil infiltration by 55%. This anti-inflammatory activity is comparable to dexamethasone (2.5 µmol/kg) and the CXCR4 antagonist AMD3100 (12.6 µmol/kg), but without causing body weight or spleen weight loss observed with the comparator drugs. [1] Oral administration (by gavage) of LIT-927 (1400 µmol/kg) 2 hours before OVA challenge significantly inhibits eosinophil recruitment in the BAL fluid by 62%. This identifies LIT-927 as the first orally active CXCL12 neutraligand in this asthma model. [1] No signs of toxicity (e.g., body weight loss, prostration, bristly hair) were observed after 10 daily administrations of LIT-927. Histological examination (Gomori's staining) of liver, spleen, heart, and kidney showed no lesions. [1] |
| Cell Assay |
FRET-based CXCL12-CXCR4 Binding Assay: HEK-293 cells stably expressing EGFP-tagged human CXCR4 receptor are washed, detached, and resuspended in HEPES buffer containing bovine serum albumin. Cell suspension is transferred to a quartz cuvette in a spectrofluorometer. Fluorescence emission at 510 nm (excitation at 470 nm) is recorded. Binding of Texas Red-labeled CXCL12 (CXCL12-TR) to the receptor induces fluorescence resonance energy transfer (FRET), detected as a reversible decline in EGFP emission at 510 nm. For the "neutraligand protocol", CXCL12-TR is preincubated with the test compound for 1 hour before addition to cells. For the "antagonist protocol", the test compound is added to cells before CXCL12-TR. Dose-response curves of inhibition are generated to determine inhibitory constants (Ki). The CXCR4 antagonist T134 is used as a control. [1]
Time-Resolved Intracellular Calcium Recording (TRIC-r) Assay: This assay is used to assess selectivity. HEK cells expressing EGFP-tagged chemokine receptors (CXCR4, CCR4, CCR5, CCR2) are loaded with a calcium-sensitive dye. Changes in intracellular calcium concentration are monitored upon stimulation with respective chemokines (CXCL12, CCL17/CCL22, CCL5, CCL2 at 5 nM). Test compounds are preincubated with the cells ("neutraligand protocol") before chemokine addition to identify molecules that block the chemokine's action. [1] Tryptophan Fluorescence Binding Assay: The intrinsic fluorescence of the single tryptophan residue (Trp-57) in CXCL12 is monitored. CXCL12 (2 µM in HEPES buffer) is titrated with increasing concentrations of the test compound. The emission intensity at 340 nm (excitation at 285 nm) is measured. A decrease in fluorescence indicates binding, and data are fitted to determine the dissociation constant (Kd). [1] |
| Animal Protocol |
Murine Model of Allergic Airway Hypereosinophilia: Nine-week-old male Balb/c mice are sensitized by intraperitoneal injection of ovalbumin adsorbed on aluminium hydroxide on days 0, 1, and 2. Mice are challenged intranasally with ovalbumin in saline under anesthesia on days 5, 6, and 7. Control mice receive saline. Test compounds are administered either intranasally, intraperitoneally, or orally (by gavage) 2 hours before each challenge. Compounds are typically formulated in phosphate-buffered saline containing 10% (w/w) (2-hydroxypropyl)-β-cyclodextrin for intranasal and intraperitoneal studies, or in a vehicle containing methylcellulose and Tween 80 for oral studies. Twenty-four hours after the last challenge, mice are anesthetized, bronchoalveolar lavage is performed using ice-cold saline-EDTA. Lavage fluid is centrifuged, cells are resuspended, and total and differential cell counts (eosinophils, neutrophils, lymphocytes, macrophages) are determined on cytospin preparations stained with Diff-Quick. [1]
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| ADME/Pharmacokinetics |
LIT-927 has a thermodynamic solubility of 36.4 µM in phosphate buffer at pH 7.4, higher than its parent compound chalcone (9 µM). [1] Its solubility is significantly increased to over 5000 µM in PBS containing 10% (w/w) (2-hydroxypropyl)-β-cyclodextrin. [1] LIT-927 is chemically stable with 100% compound residue after being placed in PBS and PBS/cyclodextrin solutions at 37°C for 24 hours. [1]
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| Toxicity/Toxicokinetics |
In an 8-day mouse model of allergic inflammation, intraperitoneal injection of LIT-927 (350 µmol/kg) did not cause a decrease in body weight or spleen weight, unlike the positive control drugs dexamethasone and AMD3100. [1] No toxic reactions (such as weakness or piloerection) were observed after 10 consecutive days of administration. Histological examination of major organs (liver, spleen, heart, and kidneys) revealed no lesions. [1]
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| References | |
| Additional Infomation |
LIT-927 (compound 57) is a pyrimidinone derivative discovered through systematic structure-activity relationship optimization of the original chalcone CXCL12 neutralizing ligand chalcone-4 (1). It was designed to overcome the limitations of the lead compound, such as poor solubility, high Michael receptor reactivity, and lack of oral activity. [1]
Its mechanism of action involves direct binding to the chemokine CXCL12, thereby neutralizing its ability to activate the CXCR4 receptor, rather than acting as a receptor antagonist. [1] Molecular modeling shows that LIT-927 binds to a small druggable pocket near Trp57 on CXCL12. The 4-chlorobenzene ring occupies a hydrophobic subpocket, the pyrimidinone core forms a hydrogen bond with Arg20, and the 4-hydroxybenzene ring interacts with Lys64 and Gln37. [1] LIT-927 has been reported as the first orally effective selective CXCL12 neutralizing ligand, exhibiting anti-inflammatory properties in a mouse asthma model. It represents a novel pharmacological tool and may serve as a starting point for the development of anti-asthma drugs. [1] |
| Molecular Formula |
C17H13CLN2O3
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| Molecular Weight |
328.75
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| Exact Mass |
328.06
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| Elemental Analysis |
C, 62.11; H, 3.99; Cl, 10.78; N, 8.52; O, 14.60
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| CAS # |
2172879-52-4
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| Related CAS # |
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| PubChem CID |
137287575
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| Appearance |
Light yellow to yellow solid powder
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| LogP |
3.1
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| Hydrogen Bond Donor Count |
2
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| Hydrogen Bond Acceptor Count |
3
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| Rotatable Bond Count |
3
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| Heavy Atom Count |
23
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| Complexity |
512
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| Defined Atom Stereocenter Count |
0
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| InChi Key |
BYYRNPIGDRRGLJ-UHFFFAOYSA-N
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| InChi Code |
InChI=1S/C17H13ClN2O3/c1-23-16-8-11(4-7-15(16)21)14-9-13(19-17(22)20-14)10-2-5-12(18)6-3-10/h2-9,21H,1H3,(H,19,20,22)
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| Chemical Name |
6-(4-chlorophenyl)-4-(4-hydroxy-3-methoxyphenyl)-1H-pyrimidin-2-one
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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 |
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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) |
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
Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)(e.g. IP/IV/IM/SC) *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). View More
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)] 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). View More
Oral Formulation 3: Dissolved in PEG400 (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 3.0418 mL | 15.2091 mL | 30.4183 mL | |
| 5 mM | 0.6084 mL | 3.0418 mL | 6.0837 mL | |
| 10 mM | 0.3042 mL | 1.5209 mL | 3.0418 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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