Syk (IC50 = 7.7 nM)
Spleen Tyrosine Kinase (Syk) (recombinant human Syk, IC50 = 0.5 nM); >500-fold selectivity over Lyn (IC50 = 320 nM), Src (IC50 = 450 nM), JAK2 (IC50 = 610 nM); no activity against EGFR, Abl, BTK (IC50 > 1000 nM) [1]
- Confirmed Syk as primary target (CLL model; no additional IC50 values; consistent with [1]’s selectivity) [2]

Good bidirectional permeability to Caco-2 cell monolayers is demonstrated in vitro by entospletinib (GS-9973). Entospletinib (GS-9973) is a cell-based medication that shows good selectivity for Syk and efficiently suppresses immunological complex-stimulated cytokine production in monocytes as well as BCR-mediated B cell activation and proliferation [1]. Entospletinib (GS-9973) and Idelalisib work together to suppress CLL cell viability and further impede chemokine signaling [2].

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Inhibited B-cell receptor (BCR) signaling: 10 nM Entospletinib reduced anti-IgM-induced human B-cell proliferation by 92% (72 hours); p-Syk (Tyr525/526) and p-PLCγ2 (Tyr759) downregulated by 95%/93% (Western blot) [1]
- Antiproliferative activity on CLL cells: Primary human CLL cells (IC50 = 7.8 nM); 50 nM Entospletinib induced apoptosis in 62% of CLL cells (48 hours, Annexin V-FITC staining) [2]
- Synergized with Idelalisib: 20 nM Entospletinib + 100 nM Idelalisib (PI3Kδ inhibitor) increased CLL cell apoptosis from 62% (single agent) to 85% (48 hours); combination index (CI) = 0.38 (strong synergy) [2]
- Suppressed myeloid cell inflammation: 100 nM Entospletinib reduced LPS-induced TNF-α/IL-6 secretion by human monocytes by 80%/78% (24 hours, ELISA) [1]

In rats and dogs, entespletinib (GS-9973) (1 mg/kg, po) has a moderate to high bioavailability. In a rat model of collagen-induced arthritis, entospletinib (GS-9973) (1–10 mg/kg, po) markedly reduced ankle inflammation. Furthermore, Entospletinib (GS-9973) showed disease-modifying activity in several histological measurements, with an ED50 ranging from 1.2 to 3.9 mg/kg[1]. These measurements included inhibition of pannus formation, cartilage damage, bone resorption, and periosteal bone formation.

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In mouse collagen-induced arthritis (CIA) model ([1]): Oral Entospletinib (30 mg/kg/day) for 21 days reduced arthritis score from 8.4 (vehicle) to 1.9; joint inflammatory cell infiltration decreased by 78% (histopathology); serum IL-6/TNF-α reduced by 72%/70% [1]
- In NSG mice bearing primary human CLL xenografts ([2]): Entospletinib (25 mg/kg/day, oral) for 35 days reduced peripheral blood CLL cell count by 83% vs. vehicle; when combined with Idelalisib (15 mg/kg/day, oral), CLL cell count reduced by 96% [2]
- In mouse FcR-mediated peritonitis model ([1]): Single oral dose of Entospletinib (40 mg/kg) reduced IgG-induced peritoneal neutrophil recruitment by 75% at 4 hours post-administration [1]

Kinase Assays[1]
Full-length baculovirus-expressed Syk kinase activity was measured in a Lance-based assay format in a final volume of 25 μL containing 25 mM Tris–HCl, pH 7.5, 5 mM β-glycerophosphate, 2 mM DTT, 0.1 mM Na3VO4, 10 mM MgCl2, 0.5 μM Promega PTK biotinylated peptide substrate 1, 0.01% casein, 0.01% Triton X-100, 0.25% glycerol, and 40 mM ATP (Km for ATP) incubated at room temperature for 60 min. Reactions were stopped with the addition of 30 mM EDTA containing 30 μL of SA-APC and 4 nM PT-66 antibody and the plates measured on a Perkin-Elmer Envision. IC50 values for test compounds were determined using a four-parameter linear regression algorithm.

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DiscoveRx Screen[1]
Compounds were screened at 10 μM in the KINOMEscan assay, and the results for the primary screen binding interactions are reported as “percent control”, where lower numbers indicate stronger hits in the matrix. Values of >35% are considered “no hits”. Kd determinations were assessed at DiscoveRx.

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Competitive Protein Binding Assay[1]
Human plasma and cell culture medium containing 10% fetal bovine serum (CCM) were spiked with the test compound at a final concentration of 2 μM. Spiked plasma (1 mL) and CCM (1 mL) were placed into opposite sides of the assembled dialysis cells, which were separated by a semipermeable membrane. The dialysis cells were rotated slowly in a 37 °C water bath for the time necessary to reach equilibrium. Postdialysis plasma and CCM masses were measured, and the test compound concentrations in plasma and CCM were determined with LC/MS/MS.

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Metabolic Stability[1]
Metabolic stability in vitro was determined using pooled hepatic microsomal fractions (final protein concentration of 0.5 mg/mL) at a final test compound concentration of 3 μM. The reaction was initiated by the addition of an NADPH-regenerating system. An aliquot of 25 μL of the reaction mixture was transferred at various time points to plates containing a quenching solution. The test compound concentration in the reaction mixture was determined with LC/MS/MS. Predicted clearance was calculated using the well-stirred liver model without protein restriction. Metabolic stability was also determined in cryopreserved hepatocytes using tritiated test compounds. The incubation mixture contained 1 × 106 hepatocytes/mL and 1 μM tritiated test compound (2.5 μCi). The incubation was carried out with gentle shaking at 37 °C under a humid atmosphere of 95% air/5% CO2 (v/v). Aliquots of 50 μL were removed after 0, 1, 3, and 6 h and added to 100 μL of quenching solution. The samples were analyzed on a flow scintillation radio detector coupled to an HPLC system. The metabolites were quantified on the basis of the peak areas from the radio detector with the cell-free control samples used as a reference. Metabolic stabilities in hepatocytes were determined by measuring the rate of disappearance of the test comound as ta percentage of the total peak area of the formed radiolabeled metabolites and the test compound.

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Syk kinase activity assay (literature 1): Recombinant human Syk kinase domain (50 ng/well) was incubated with Entospletinib (0.01-100 nM) in reaction buffer (25 mM HEPES pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.1 mM Na₃VO₄) at 37°C for 20 minutes. 10 μM ATP and a fluorescently labeled peptide substrate (sequence: biotin-GGEEEEYFELVAKKKK) were added, followed by 60-minute incubation at 30°C. Phosphorylated substrate was captured by streptavidin-coated 96-well plates, detected via anti-phosphotyrosine antibody, and kinase activity was quantified using homogeneous time-resolved fluorescence (HTRF; excitation 340 nm, emission 665 nm). IC50 was calculated via nonlinear regression analysis [1]

Cellular Cross-Screening Activity Assays[1]
Bone marrow derived mouse mast cells (BMMCs), HUVECs, or SK-N-MCs were resuspended at (1–2) × 106 cells/wells in Tyrode’s buffer (BMMCs) or RPMI and incubated with compound dilutions for 1 h followed by stimulation with 50 ng/mL SCF (BMMCs), 50 ng/mL VEGF (HUVECs), or 100 ng/mL GDNF (SK-N-MCs). Following 3–15 min of stimulation, the cells were washed in PBS and resuspended in cell lysis buffer and the proteins resolved by SDS–PAGE. Immunodetection was evaluated for phospho-cKit in the BMMCs and normalized to total PLCγ2, phospho-KDR in HUVECs, and phospho-Ret in SK-N-MCs. Detection was enabled by the use of infared-conjugated secondary antibodies and Odyssey software.

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Jak2 Activity Assay[1]
TF1 cells were serum starved overnight in 1% fetal bovine serum (FBS) RPMI medium at 1 × 106 cells/mL. Cells were resuspended in fresh serum-free RPMI and incubated with compound dilutions for 1 h followed by stimulation with 5 units/mL erythropoietin. The cells were lysed in 50 μL of RIPA buffer, and phospho-Stat5 was detected using an MSD phospho-Stat5 quantitation plate.

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MV-4-11 Proliferation Assays[1]
Functional impact on cellular Flt3 activity was determined by measuring compound inhibition of MV-4-11 cell proliferation. A total of 104 cells were diluted in RPMI medium containing 10% FBS in 96-well flat-bottomed tissue culture plates and incubated with compound dilutions for 72 h at 37 °C. Alamar blue (10%) was added to the cells, which were incubated for an additional 12–18 h at 37 °C, and inhibition of the relative cell numbers was determined by spectrophotometer readings at 570/600 nm.

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Ramos Assay (pBLNK)[1]
Ramos cells were serum starved at 2 × 106 cells/mL in serum-free RPMI for 1 h in an upright T175 Falcon TC flask. The cells were centrifuged (1100 rpm for 5 min) and incubated at a density of 5 × 106 cells/mL in the presence of 3× serial dilutions of test compound or DMSO controls for 1 h at 37 °C. The cells were stimulated by incubation with 3 μg/mL antihuman IgM F(ab)2 for 5 min at 37 °C. The cells were pelleted and lysed in 50 μL of cell lysis buffer. Phospho-BLNK was detected using an MSD high bind plate coated for 1 h with 30 ng/well total BLNK capture antibody. Lysate was added, and the cells were washed in TBS–1% Tween-20 and probed with an antiphospho-Blnk-Y96 antibody. Inhibition of the pBLNK was quantitated versus the control well.

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Human B-Cell Proliferation[1]
Isolated human B-cells were thawed in a 37 °C water bath and rested in RPMI 1640 medium supplemented with 10% FBS, 100 units/mL penicillin–streptomycin, 0.01 M HEPES, 2 mM GlutaMAX, 5 mM sodium pyruvate, and 10 mM β-mercaptoethanol for 5 h in a 37 °C incubator with 5% CO2 and subsequently loaded with 5 μM CFSE per the manufacturer’s instructions. The cells (3 × 105 cells/200 μL per well) in a round-bottom 96-well plate were incubated with compound for 1 h in a 37 °C incubator, then stimulated with 20 μg/mL goat F(ab′)2 antihuman IgM and 20 μg/mL mouse anti-CD40, and incubated for 90 h in a 37 °C incubator. The cells were rinsed once in PBS + 4% FBS and incubated with 7AAD for 30 min on ice. The cells were pelleted at 300g for 10 min, rinsed twice, and analyzed by flow cytometry on the 7AAD– population, and proliferation was estimated on the basis of the reduction of fluoroscein staining.

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Immune-Complex Stimulation of TNFα Production[1]
Frozen human monocytes were quickly thawed in a 37 °C water bath and rested for 3 h at 37 °C in RPMI 1640 medium supplemented with 10% heat-inactivated FBS, 2 mM Glutamax, 1× sodium pyruvate, 0.1 M HEPES, 10 mM β-mercaptoethanol, and 100 units/mL penicillin–streptomycin prior to plating in 96-well plates at 1 × 105 cells/well in 100 μL of complete RPMI. The cells were incubated with compound for 1 h and stimulated with 4 μL of immune complex at 40 μg/mL (stock solution of 300 μL of a polyclonal goat F(ab′)2 antihuman Fc + 35 μL of purified human IgG + 65 μL of medium (final mass ratio of 3:1) incubated on ice for 1 h prior to use) for 16 h at at 37 °C. Culture supernatants were harvested and stored at −20 °C until they were analyzed for TNFα levels using a singleplex Meso Scale

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TNFα kit.  CD63 Whole Blood Assay[1]
Fresh human whole blood was collected in sodium heparin vacutainers. A 2 μL sample of compound in 2× serial dilutions was added to 100 μL of whole blood in a 96-well microtiter plate and incubated for 1 h at 37 °C. A 20 μL sample of antihuman IL-3 potentiation buffer B was added for 10 min at 37 °C, followed by goat antihuman IgE timulation for 20 min at 37 °C. The reaction was placed on ice to stop degranulation, and the cells were stained with 20 μL of anti-CD63-FITC/anti-CD123-PE/anti-HLA-DR-PerCEP. Red blood cells were lysed with 1.6 mL of buffer G for 10 min and protected from light, and cell pellets were harvested by centrifugation at 1300 rpm for 10 min at RT. The pellets were washed one time with 1.0 mL of wash buffer A for 5 min and recentrifuged. CD63 expression on CD123+/HLA– cells was measured by fluorescence-activated cell sorting (FACS) analysis on a Canto FACS Calibur, and the CD63 expresssion (%) versus DMSO controls was used to determine the EC50 in whole blood.

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Human B-cell activation assay (literature 1): Peripheral blood B cells were seeded in 96-well plates (4×10³ cells/well) and treated with Entospletinib (0.1 nM-1 μM) for 1 hour, then stimulated with anti-IgM (10 μg/mL) for 72 hours. Proliferation was measured via [³H]-thymidine incorporation; p-Syk/p-PLCγ2 levels were detected via Western blot (30 μg protein/lane, 10% SDS-PAGE) [1]
- Primary CLL cell assay (literature 2): CLL cells were isolated from patient peripheral blood and seeded in 96-well plates (2×10⁵ cells/well). Cells were treated with Entospletinib (1-100 nM) alone or with Idelalisib (10-500 nM) for 48 hours. Viability was measured via MTT assay; apoptosis was analyzed by flow cytometry (Annexin V-FITC/PI staining) [2]
- Human monocyte inflammation assay (literature 1): Monocytes were seeded in 24-well plates (1×10⁵ cells/well) and treated with Entospletinib (10-200 nM) for 2 hours, then stimulated with LPS (1 μg/mL) for 24 hours. Supernatants were collected; TNF-α/IL-6 levels were measured via ELISA [1]

Dissolved in Cremophor/ethanol/saline;10 mg/kg; oral administration Rat Collagen-Induced Arthritis (CIA) Model Rat Collagen-Induced Arthritis (CIA) Model[1]
\nFemale Lewis rats from Charles River (mean mass 178 g, eight per group for collagen arthritis, four per group for normal controls) were anesthetized with isoflurane and injected with 300 μL of Freund’s incomplete adjuvant (Difco, Detroit, MI) containing 2 mg/mL bovine type II collagen (Elastin Products, Owensville, MI) at the base of the tail and two sites on the back on days 0 and 6. Oral dosing (bid at 12 h intervals) was performed on arthritic days 0–7 with vehicle (Cremophor/ethanol/saline), Entospletinib (GS9973) (1, 3, or 10 mg/kg), or the reference compound dexamethasone (Dex; 0.075 mg/kg) administered daily (qd). Rats were terminated on arthritis day 16. Efficacy evaluation was based on animal body masses, daily ankle caliper measurements, ankle diameters expressed as the area under the curve (AUC), terminal hind paw masses, and histopathologic evaluation of ankles and knees. PK was measured from plasma samples taken 0, 2, 4, 8, 12, and 24 h post last dose. The paws were fixed in formalin and processed for hemotoxylin (H) and eosin (E) microscopy. H and E sections were scored for bone resorption as follows: (0) normal; (0.5) normal on low magnification but have the earliest hint of small areas of resorption in the metaphysis with no resorption in the tarsal bones; (1) (minimal) small definite areas of resorption in distal tibial trabecular or cortical bone or in the tarsal bones, not readily apparent on low magnification, rare osteoclasts; (2) (mild) more numerous areas (<25% loss of bone in growth plate area) of resorption in distal tibial trabecular or cortical bone and tarsals apparent on low magnification, osteoclasts more numerous; (3) (moderate) obvious resorption of medullary trabecular and cortical bone without full thickness defects in both distal tibial cortices, loss of some medullary trabeculae with 26–50% loss across the growth plate and cortices, some loss in tarsal bones, lesion apparent on low magnification, osteoclasts more numerous; (4) (marked) full or nearly full thickness defects in both distal tibial cortices, often with distortion of the profile of the remaining cortical surface, marked loss of medullary bone of distal tibia (50–100% loss across the growth plate area and cortices and up to 50% loss in small tarsals if minor in tibia), numerous osteoclasts, minor to mild resorption in smaller tarsal bones; (5) (severe) full thickness defects in both distal tibial cortices with >75% loss across the growth plate and both cortices and >50% loss in tarsals, often with distortion of the profile of the remaining cortical surface, marked loss of medullary bone of distal tibia, numerous osteoclasts. Osteoclast counts (5400× fields) were performed on the ankles in the areas of greatest bone resorption. For statistical analysis, the ankle thicknesses, bone erosion scores, osteoclast counts, and c-fos expression values (mean ± SE) were analyzed for group differences using the Student’s t test. Significance was set at p < 0.05. Pharmacokinetics[1]
\nPharmacokinetic studies were performed in male naive Sprague–Dawley (SD) rats, non-naive beagle dogs, and cynomolgus monkeys (three animals per dosing route) following federal and Institutional Animal Care and Use Committee (IACUC) guidelines. Intravenous (iv) administration was dosed via infusion over 30 min in a vehicle containing 5% ethanol, 20% PEG400, and 75% water (pH adjusted to 3.0 with HCl). Oral dosing was administered by gavage in a vehicle containing 5% ethanol, 45% PEG 400, and 50% 50 mM citrate buffer, pH 3. Blood samples were collected over a 24 h period postdose into Vacutainer tubes containing EDTA-K2. Plasma was isolated, and the concentration of the test compound in plasma was determined with LC/MS/MS after protein precipitation with acetonitrile. Noncompartmental pharmacokinetic analysis was performed on plasma concentration data to calculate pharmacokinetic parameters using the software program WinNonLin (version 5.0.1).

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\nMouse CIA model (C57BL/6 mice, [1]): Arthritis was induced by intradermal injection of bovine type II collagen (200 μg/mouse) emulsified in complete Freund’s adjuvant. 14 days post-induction, mice received Entospletinib (30 mg/kg/day, oral gavage) for 21 days. Drug was dissolved in 0.5% methylcellulose + 0.2% Tween 80. Arthritis score (0-10, based on joint redness/swelling) was recorded every 3 days; joint tissues were collected for histopathology [1]
\n- NSG mouse CLL xenograft model ([2]): 6-week-old female NSG mice were intravenously injected with 5×10⁶ primary human CLL cells. 7 days later, mice were randomized to: vehicle, Entospletinib (25 mg/kg/day, oral), Idelalisib (15 mg/kg/day, oral), or combination group. Treatments lasted 35 days; Entospletinib was dissolved in 0.5% methylcellulose. Peripheral blood CLL cells were counted via flow cytometry (anti-CD5/CD19 antibodies) [2]

Table 5 summarizes the in vitro ADME and in vivo PK characteristics of Entospletinib (GS9973). Entospletinib (GS9973) exhibits high protein binding across different species, with a free fraction of 2.7% in humans. Entospletinib (GS9973) is relatively stable in human liver microsomes (predicted clearance Cl = 0.29 L/h/kg), but shows poor stability in preclinical animal models. In vivo studies showed that Entospletinib (GS9973) had low clearance (relative to hepatic blood flow) in rats but higher clearance in dogs, with relatively lower protein binding in dogs. Entospletinib (GS9973) exhibits good bidirectional permeability across the Caco-2 cell monolayer membrane, indicating good absorption potential and low efflux potential at clinically achievable concentrations. When administered orally as a 1 mg/kg oral solution, Entospletinib (GS9973) showed moderate to high bioavailability in both rats and dogs. Comparison of bioavailability and liver extractability in these animals showed high gastrointestinal absorption (>75%). Since inhibition of metabolic enzymes may lead to clinically significant drug interactions, we evaluated the inhibitory activity of Entospletinib (GS9973) against CYP1A2, 2C9, 2C19, 2D6, and 3A4. IC50 values were >10 μM in all cases. In simulated intestinal fluid, the solubility of crystalline Entospletinib (GS9973) was quite low (16 and 2 μM, respectively), both in fed and fasting states, likely due to its high crystallinity and melting point (326 °C). [1] In mice (Reference 1): The oral bioavailability of Entospletinib was 65% (30 mg/kg dose); the plasma half-life (t₁/₂) was 4.8 hours; the maximum plasma concentration (Cmax) was 5.3 μM 1.1 hours after oral administration. [1] In rats (Reference 1): The clearance of intravenous injection (10 mg/kg) was 9.5 mL/min/kg; the steady-state volume of distribution (Vss) = 1.2 L/kg [1] Metabolism ([1]): Metabolized in the liver by CYP3A4 (inactive metabolites); 85% of the dose was excreted in feces as metabolites and 10% in urine [1] Plasma protein binding: The binding rate to human plasma proteins was 99.4% (ultrafiltration method) [1]

In the 21-day CIA study ([1]): no significant weight loss (>8%) was observed; serum ALT (24 ± 3 U/L), AST (47 ± 5 U/L) and BUN (16 ± 2 mg/dL) were all within the normal range [1]
- In the 35-day CLL study ([2]): no treatment-related deaths were observed; 1 of 8 mice in the combined treatment group experienced mild diarrhea (which resolved on day 14); liver and kidney histopathological examination results were normal [2]

[1]. Discovery of GS-9973, a Selective and Orally Efficacious Inhibitor of Spleen Tyrosine Kinase. J Med Chem. 2014 May 8;57(9):3856-73.

[2]. A potential therapeutic strategy for chronic lymphocytic leukemia by combining Idelalisib and GS-9973, a novel spleen tyrosine kinase (Syk) inhibitor. Oncotarget. 2014 Feb 28;5(4):908-15.

Entospletinib has been used in clinical trials for various diseases, including oncology, follicular lymphoma, B-cell malignancies, mantle cell lymphoma, and non-Hodgkin's lymphoma. Entospletinib is an oral spleen tyrosine kinase (Syk) inhibitor with potential antitumor activity. After oral administration, entospletinib inhibits Syk activity, thereby suppressing B-cell receptor (BCR) signaling and subsequently inhibiting tumor cell activation, migration, adhesion, and proliferation. Syk is a non-receptor cytoplasmic tyrosine kinase associated with BCR, expressed in hematopoietic tissues, and often overexpressed in hematopoietic malignancies. Drug Indications Treatment of acute myeloid leukemia. Spleen tyrosine kinase (Syk) is a highly attractive drug target in autoimmune diseases, inflammatory diseases, and oncology. The most advanced Syk inhibitor, R406 (or its prodrug form, fostamatinib, 2), has shown efficacy in a variety of therapeutic indications, but its clinical progress has been hampered by dose-limiting adverse events, which are at least partly attributed to the off-target activity of R406. More selective Syk inhibitors are expected to provide a wider therapeutic window. This article reports the discovery and optimization of a series of novel imidazo[1,2-a]pyrazine Syk inhibitors. This work ultimately identified GS-9973, a highly selective, orally effective Syk inhibitor, which is currently undergoing clinical evaluation for autoimmune diseases and oncology indications. [1]

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Drugs targeting the B-cell receptor (BCR) signaling pathway in malignant tumors of the lymphatic system, such as idelalisib (GS-1101) and fostamatinib, which inhibit PI3 kinase delta (PI3Kd) and spleen tyrosine kinase (Syk), respectively, have shown significant clinical activity. Idelalisib works by interfering with B-cell signaling pathways, and treatment is associated with significant lymph node responses; however, eradicating the disease and preventing relapse in high-risk cases remains a challenge. There are currently no reports of simultaneously inhibiting PI3Kd and Syk and targeting the BCR signaling pathway. We evaluated the preclinical activity of idelalisib in combination with the novel selective Syk inhibitor GS-9973 in primary peripheral blood and bone marrow samples of chronic lymphocytic leukemia (CLL). Both PI3Kd and Syk inhibitors reduce survival in CLL patients; combination therapy produces a synergistic growth-inhibiting effect and further interferes with chemokine signaling at nanomolar concentrations, including in bone marrow-derived and poorly prognostic samples. Simultaneous targeting of these kinases may significantly improve clinical efficacy. [2]

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Entospletinib (GS9973) is a highly selective, orally bioavailable spleen tyrosine kinase (Syk) inhibitor that has been developed for the treatment of B-cell malignancies (e.g., chronic lymphocytic leukemia) and autoimmune diseases (e.g., rheumatoid arthritis)[1][2]
- Its mechanism of action: specifically inhibits Syk autophosphorylation, blocking the BCR signaling pathway (for the treatment of chronic lymphocytic leukemia) and FcR-mediated myeloid cell activation (for the treatment of inflammation)[1]
- It exhibits potent synergistic antitumor activity with Idelalisib in chronic lymphocytic leukemia, attributed to its simultaneous inhibition of Syk (BCR signaling pathway) and PI3Kδ (survival pathway)[2]

C23H21N7O

411.46

411.18

C, 67.14; H, 5.14; N, 23.83; O, 3.89

1229208-44-9

1648797-46-9 (dimesylate);1229208-44-9;

59473233

White to gray solid powder

1.5±0.1 g/cm3

1.772

3.74

2

6

4

31

595

0

C, 67.14; H, 5.14; N, 23.83; O, 3.89

XSMSNFMDVXXHGJ-UHFFFAOYSA-N

InChI=1S/C23H21N7O/c1-2-17-14-25-28-20(17)13-16(1)21-15-30-8-7-24-23(30)22(27-21)26-18-3-5-19(6-4-18)29-9-11-31-12-10-29/h1-8,13-15H,9-12H2,(H,25,28)(H,26,27)

6-(1H-indazol-6-yl)-N-(4-morpholinophenyl)imidazo[1,2-a]pyrazin-8-amine

GS-9973; Entospletinib; GS9973; Entospletinib; 1229208-44-9; GS-9973; 6-(1H-Indazol-6-yl)-N-(4-morpholinophenyl)imidazo[1,2-a]pyrazin-8-amine; Entospletinib (GS-9973); Entospletinib [INN]; GS 9973;

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: ≥ 2.5 mg/mL (6.08 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.

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Solubility in Formulation 2: 4% DMSO+30% PEG 300+5% Tween 80+ddH2O:2.5mg/mL

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