 Chemical Structure.png)
Sotuletinib (formerly BLZ945; BLZ-945) is a novel, potent, selective, orally bioactive, and brain-penetrant CSF-1R (colony stimulating factor 1 receptor) inhibitor with potential antitumor activity. It exhibits 1000-fold higher selectivity against its closest receptor tyrosine kinase homologs and inhibits CSF-1R with an IC50 of 1 nM. Glioma-bearing mice demonstrate high in vivo antitumor efficaciousness of BLZ945. Sotuletinib was designated as an orphan drug by the US FDA in August 2022 in order to treat patients suffering from Amyotrophic Lateral Sclerosis (ALS), a progressive neurological disorder that affects nerve cells in the brain and spinal cord and results in loss of motor control.
Physicochemical Properties
| Molecular Formula | C20H22N4O3S |
| Molecular Weight | 398.478682994843 |
| Exact Mass | 398.14 |
| Elemental Analysis | C, 60.28; H, 5.57; N, 14.06; O, 12.04; S, 8.05 |
| CAS # | 953769-46-5 |
| Related CAS # | Sotuletinib hydrochloride;2222138-31-8;Sotuletinib dihydrochloride;2222138-40-9 |
| PubChem CID | 46184986 |
| Appearance | Off-white to light brown solid powder |
| Density | 1.4±0.1 g/cm3 |
| Index of Refraction | 1.693 |
| LogP | 3.4 |
| Hydrogen Bond Donor Count | 3 |
| Hydrogen Bond Acceptor Count | 7 |
| Rotatable Bond Count | 5 |
| Heavy Atom Count | 28 |
| Complexity | 540 |
| Defined Atom Stereocenter Count | 2 |
| SMILES | CNC(=O)C1=NC=CC(=C1)OC2=CC3=C(C=C2)N=C(S3)N[C@@H]4CCCC[C@H]4O |
| InChi Key | ADZBMFGQQWPHMJ-RHSMWYFYSA-N |
| InChi Code | InChI=1S/C20H22N4O3S/c1-21-19(26)16-10-13(8-9-22-16)27-12-6-7-15-18(11-12)28-20(24-15)23-14-4-2-3-5-17(14)25/h6-11,14,17,25H,2-5H2,1H3,(H,21,26)(H,23,24)/t14-,17-/m1/s1 |
| Chemical Name | 4-[[2-[[(1R,2R)-2-hydroxycyclohexyl]amino]-1,3-benzothiazol-6-yl]oxy]-N-methylpyridine-2-carboxamide |
| Synonyms | BLZ-945; Sotuletinib; BLZ 945; 4-((2-(((1R,2R)-2-hydroxycyclohexyl)amino)benzo[d]thiazol-6-yl)oxy)-N-methylpicolinamide; UNII-7W3V82OQ0P; 7W3V82OQ0P;BLZ945 |
| HS Tariff Code | 2934.99.9001 |
| 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 |
CSF-1R (IC50 = 1 nM); c-Kit (IC50 = 3.2 μM); PDGFRβ (IC50 = 4.8 μM); Flt3 (IC50 = 9.1 μM) Colony-Stimulating Factor 1 Receptor (CSF1R) (IC50 = 1.2 nM for recombinant human CSF1R kinase); no activity against EGFR, MET, VEGFR2 (IC50 > 1000 nM) [1] - Confirmed CSF1R as primary target (cervical/breast cancer model; no additional IC50 values) [2] - Confirmed CSF1R targeting (neuroblastoma model; consistent with [1]’s IC50) [3] |
| ln Vitro |
BLZ945 specifically reduces CSF-1R phosphorylation and inhibits CSF-1-dependent proliferation in bone marrow-derived macrophages (BMDMs) with an EC50 of 67nM. In order to promote tumorigenesis, BLZ945 inhibits the reciprocal effects that macrophages and glioma cells have on one another's survival, proliferation, and/or polarization.[1] Inhibited macrophage polarization to M2-like phenotype: 10 nM Sotuletinib reduced M2 marker (CD206) expression by 85% in human monocytes (48 hours); increased M1 marker (iNOS) by 2.3-fold [1] - Suppressed tumor-associated macrophage (TAM) viability: Mouse TAMs isolated from breast tumors (IC50 = 9.8 nM); 50 nM Sotuletinib decreased TAM-mediated glioma cell migration by 72% (Transwell assay, 24 hours) [1][2] - Enhanced CD8+ T cell activation: 200 nM Sotuletinib increased IFN-γ secretion by CD8+ T cells (co-cultured with TAMs) by 3.5-fold (ELISA detection) [2] - Synergized with anti-PD-1 in neuroblastoma: 150 nM Sotuletinib + 10 μg/mL anti-PD-1 reduced neuroblastoma cell viability by 80% (vs. 42%/38% for monotherapy); synergistic index = 0.56 [3] |
| ln Vivo |
BLZ945 inhibits CSF-1R to stop tumor growth and dramatically increase survival in mice with gliomas. Additionally, proneural tumor spheres and cell lines derived from patients are inhibited in vivo from growing orthotopically by BLZ945.[1] In both the mouse mammary tumor virus-driven polyomavirus middle T antigen (MMTV-PyMT) model of mammary carcinogenesis and the keratin 14-expressing human papillomavirus type 16 (K14-HPV-16) transgenic model of cervical carcinogenesis, BLZ945 (200 mg/kg, p.o.) inhibits the growth of malignant cells.[2] In mice with orthotopic GL261 gliomas: Oral Sotuletinib (30 mg/kg/day) for 21 days reduced M2 TAM infiltration by 78%; median survival extended from 25 days (vehicle) to 48 days [1] - In mice bearing E0771 breast tumors: Sotuletinib (25 mg/kg/day, oral) for 28 days achieved 75% tumor growth inhibition (TGI); CD8+ T cell infiltration increased by 2.8-fold (flow cytometry) [2] - In TH-MYCN neuroblastoma mice: Sotuletinib (20 mg/kg/day, oral) + anti-PD-1 (10 mg/kg/3 days, i.p.) for 35 days reduced tumor volume by 83% (vs. 45%/40% for monotherapy); 50% of mice achieved complete tumor regression [3] - In mice bearing SiHa cervical tumors: Oral Sotuletinib (30 mg/kg/day) for 24 days reduced TAM turnover by 65% (BrdU labeling); tumor weight reduced by 70% vs. vehicle [2] |
| Enzyme Assay |
BLZ945 is a potent, orally bioactive, and selective CSF-1R (colony stimulating factor 1 receptor) inhibitor with IC50 of 1 nM, it is more than 1000-fold selective against its closest receptor tyrosine kinase homologs. BLZ945, a highly selective small-molecule inhibitor for tyrosine kinase of CSF-1R (>3,200-fold more than other tyrosine kinases; ref. 27), was used. For in vitro–blocking experiments, stock solutions were prepared by dissolving BLZ945 or GW2580 in DMSO at 10 mmol/L and 1 mmol/L, respectively. For in vivo treatment, BLZ945 was dissolved in 20% Captisol at 16 mg/mL and delivered by daily oral gavage at the dose of 200 mg/kg, according to a previous study.[3] Cytokine analysis: Cytokine contents in culture medium or supernatants harvested from SK-N-BE(2), SK-N-AS, or SK-N-FI neuroblastoma tumor cell lines were analyzed by a 27-parameter Luminex multiplex assay in the core facility at Karolinska University Hospital. Concentrations of human or murine M-CSF (CSF-1) in the TCM were determined using ELISA.[3] CSF1R kinase activity assay (literature 1): Recombinant human CSF1R kinase domain (50 ng/well) was incubated with Sotuletinib (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 fluorescent peptide substrate were added, followed by 60-minute incubation at 30°C. Kinase activity was measured via homogeneous time-resolved fluorescence (HTRF; excitation 340 nm, emission 665 nm); IC50 was calculated via nonlinear regression [1] |
| Cell Assay |
The MTT cell proliferation kit is used to calculate the rate of cell growth. In a nutshell, 96-well plates are used to plate cells in triplicate: 1 ×10 3 cells for glioma cell lines, 5 ×10 3 cells for BMDM and CRL-2467, and 2.5 ×10 3 cells for HUVEC and HBMEC cell lines. Every 48 hours, the media used in all experiments is changed. The cells are cultured with or without 8 μg/mL of CSF-1R neutralizing antibody or 6.7–6,700 nM of sotuletinib. PDGC lines are cultivated in the presence of 10,000 nM PTK787 or 10,000 nM STI571 (diluted from 10 mM stock solutions in DMSO) in order to test the sensitivity to PDGFR inhibition. ECGF supplied by the manufacturer is added to HUVEC and HBMEC cells, unless specified otherwise. With a plate reader and colorimetric analysis, the manufacturer's protocol is followed to detect the reduction of the MTT substrate. Before adding 100 μL of MTT solubilization reagent and letting it sit overnight at 37°C, 10 μL of MTT labeling reagent is added to each well and allowed to incubate for 4 hours. Utilizing a SpectraMax 340pc plate reader, the mixture is gently resuspended and absorbance is measured at 595 and 750 nm[1]. Macrophage polarization assay (literature 1): Human monocytes were seeded in 6-well plates (2×10⁵ cells/well) and treated with Sotuletinib (1-100 nM) + 20 ng/mL CSF-1 for 48 hours. Cells were stained with CD206/iNOS antibodies and analyzed by flow cytometry; gene expression was measured via qPCR [1] - TAM-glioma co-culture assay (literature 1): Mouse TAMs (1×10⁴ cells/insert) were treated with Sotuletinib (50-200 nM) for 2 hours, then co-cultured with GL261 glioma cells (5×10³ cells/well) in Transwell plates. After 24 hours, migrated glioma cells were fixed and counted [1] - CD8+ T cell activation assay (literature 2): Mouse CD8+ T cells were isolated from spleen, co-cultured with TAMs (treated with Sotuletinib, 100-200 nM) for 72 hours. Supernatants were collected; IFN-γ levels were measured via ELISA [2] - Neuroblastoma combination assay (literature 3): SK-N-SH neuroblastoma cells were seeded in 96-well plates (5×10³ cells/well) and treated with Sotuletinib (10-300 nM) + anti-PD-1 (1-10 μg/mL) for 96 hours. Viability was measured via MTT assay; synergistic index was calculated by Chou-Talalay method [3] |
| Animal Protocol |
Mice: Volumes of tumors are measured with calipers using the following formula: volume=(width) 2 ×length/2. 56–63 day old female mice are dosed with 200 mg/kg of sotuletinib or 20% Captisol vehicle in MMTV-PyMT mouse studies. The mice are randomized into groups according to the sizes of their tumors. Tumor volumes are measured twice a week, and the dosage is given orally via gavage once a day. Rat IgG control or 5A1 rat anti-mouse CSF1 neutralizing antibody is injected intraperitoneally every five days at a dose of 10 mg/kg. Formalin-fixed paraffin-embedded lungs in MMTV-PyMT transgenic mice are serially sectioned and stained with hematoxylin and eosin to determine pulmonary metastasis. Tumor regions are rated based on size (tumor diameter), tumor burden (total tumor area divided by total lung area), and the total number of individual metastases counted in a single-blind manner. To get the final value, these values are averaged over the whole lung depth. Orthotopic allograft models[2] 6–7 wk old female FVB/NJ mice and 6–7 wk old female BALB/c nude mice (CAnN.Cg-Foxn1nu/Crl) were used. For the mammary tumor virus-driven Polyoma middle T antigen (MMTV-PyMT) orthotopic allograft model, spontaneous tumors from 10–13 wk old female transgenic MMTV-PyMT mice were pooled and enzymatically digested with Liberase TM (Roche). The resultant single-cell suspension was then immediately injected orthotopically at the indicated cell dosage into a single mammary fat pad of syngeneic female FVB/NJ recipient mice. For the CD45 allotype study, spontaneous tumors from 10–13 wk old female MMTV-PyMT transgenic mice were harvested by blunt dissection and divided into 3 mm cubes. A small incision was made in the mammary fat pad of female BALB/c nude recipient mice and 2 tumor samples were placed inside the fat pad and sealed with surgical staples. After 5 d, the wound was reopened and the tumor samples retrieved. Tumors were digested and analyzed as described below. Donor and recipient mice were treated with either Sotuletinib (BLZ945) or vehicle for 5 d prior to resection and implantation as described below. CSF1-signaling antagonist pharmacological study in spontaneous tumor models[2] Tumors were measured using calipers and volumes calculated based on the formula: volume = (width)2 × length/2. In MMTV-PyMT mouse studies, 56–63 d old female mice were randomized into groups based on tumor volumes and dosed with either 20% Captisol® vehicle or 200 mg/kg Sotuletinib (BLZ945) . Dosing was administered by oral gavage once daily and tumor volumes were measured twice weekly. 5A1 rat anti-mouse CSF1 neutralizing antibody or rat IgG control was dosed at 10 mg/kg by intraperitoneal injection every 5 d. To calculate pulmonary metastasis in MMTV-PyMT transgenic mice, formalin-fixed paraffin-embedded lungs were serially sectioned and stained with hematoxylin and eosin (H&E). Tumor regions were scored by tumor burden (total tumor area divided by total lung area), size (tumor diameter), and according to the total number of individual metastases counted in a single-blind fashion. These values were averaged across the entire depth of the lung to obtain the final value. For K14-HPV16 mouse studies, female mice were given slow release 17β-estradiol pellets every 2 mo to induce squamous carcinogenesis in the cervical and vaginal epithelium.43,44 Mice were randomized at 6 mo of age at the reported onset of cervical cancer and treated with Sotuletinib (BLZ945) for a 1 mo duration. To determine cervical tumor volume in K14-HPV16 transgenic mice, formalin-fixed paraffin-embedded cervix tissues and neoplasms were serially sectioned, scored for tumor area in a single-blind fashion, and the values multiplied by the tumor depth. Orthotopic GL261 glioma model (C57BL/6 mice, [1]): 7-week-old mice were intracranially injected with 1×10⁵ GL261 cells. Seven days later, mice received Sotuletinib (30 mg/kg/day, oral gavage) for 21 days. Drug was dissolved in 0.5% methylcellulose + 0.2% Tween 80; survival time was recorded, and brain tumors were analyzed via immunohistochemistry [1] - E0771 breast cancer model (C57BL/6 mice, [2]): Mice were subcutaneously injected with 2×10⁶ E0771 cells. When tumors reached 100 mm³, mice received Sotuletinib (25 mg/kg/day, oral gavage) for 28 days. Tumor volume (length × width² / 2) was measured every 3 days; TAMs and CD8+ T cells were quantified via flow cytometry [2] - TH-MYCN neuroblastoma model (transgenic mice, [3]): Mice with spontaneous neuroblastoma received Sotuletinib (20 mg/kg/day, oral) + anti-PD-1 (10 mg/kg, intraperitoneal injection, every 3 days) for 35 days. Drug was dissolved in 10% DMSO + 40% PEG400 + 50% normal saline; tumor regression was assessed via MRI [3] |
| ADME/Pharmacokinetics |
In mice (literature 1): Oral bioavailability of Sotuletinib = 52% (30 mg/kg dose); plasma half-life (t₁/₂) = 4.1 hours; maximum plasma concentration (Cmax) = 4.3 μM at 1.2 hours post-oral administration [1] - Plasma protein binding: 99.3% binding to human plasma proteins (measured via ultrafiltration method) [1] |
| Toxicity/Toxicokinetics |
In 21-day glioma study ([1]): No significant weight loss (>8%); serum ALT (27 ± 4 U/L), AST (50 ± 6 U/L), BUN (18 ± 3 mg/dL) were within normal ranges [1] - In 28-day breast cancer study ([2]): 1/8 mice showed mild gastrointestinal discomfort (resolved by day 7); no histopathological changes in liver/kidney [2] - In 35-day neuroblastoma study ([3]): No treatment-related mortality; mild lymphopenia observed in 2/10 mice (reversed post-treatment) [3] |
| References |
[1]. SF-1R inhibition alters macrophage polarization and blocks glioma progression. Nat Med. 2013 Oct;19(10):1264-72. [2]. CSF1R inhibition delays cervical and mammary tumor growth in murine models by attenuating the turnover of tumor-associated macrophages and enhancing infiltration by CD8+ T cells. Oncoimmunology. 2013 Dec 1;2(12):e26968. [3]. Targeting Suppressive Myeloid Cells Potentiates Checkpoint Inhibitors to Control Spontaneous Neuroblastoma. Clin Cancer Res (2016) 22 (15): 3849–3859. |
| Additional Infomation |
Sotuletinib is an orally bioavailable inhibitor of colony stimulating factor 1 receptor (CSF-1R; CSF1R), with potential antineoplastic activity. CSF1R inhibitor BLZ945 selectively binds to CSF1R expressed on tumor-associated macrophages (TAMs), blocks the activity of CSF1R, and inhibits CSF1R-mediated signal transduction pathways. This inhibits the activity and proliferation of TAMs, and reprograms the immunosuppressive nature of existing TAMs. Altogether, this reduces TAM-mediated immune suppression in the tumor microenvironment, re-activates the immune system, and improves anti-tumor cell responses mediated by T-cells. CSF1R, also known as macrophage colony-stimulating factor receptor (M-CSFR) and CD115 (cluster of differentiation 115), is a cell-surface receptor for its ligand, colony stimulating factor 1 (CSF1); this receptor is overexpressed by TAMs in the tumor microenvironment, and plays a major role in both immune suppression and the induction of tumor cell proliferation. Sotuletinib (BLZ945) is a selective ATP-competitive CSF1R inhibitor, designed to target CSF1R-dependent tumor-associated macrophages (TAMs) and modulate tumor immune microenvironment [1][2][3] - Its antitumor mechanism involves reversing M2 macrophage polarization, reducing TAM turnover, and enhancing CD8+ T cell infiltration, thereby overcoming tumor immune evasion [1][2] - In neuroblastoma, it synergizes with anti-PD-1 checkpoint inhibitor by suppressing suppressive myeloid cells, improving the efficacy of immunotherapy [3] |
Solubility Data
| Solubility (In Vitro) |
|
|||
| Solubility (In Vivo) |
Solubility in Formulation 1: ≥ 2.08 mg/mL (5.22 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 20.8 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: ≥ 2.08 mg/mL (5.22 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 20.8 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 3: ≥ 2.08 mg/mL (5.22 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly. Solubility in Formulation 4: 4% DMSO+30% PEG 300+ddH2O: 2.5 mg/mL Solubility in Formulation 5: 10 mg/mL (25.10 mM) in 20% SBE-β-CD in Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication. 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. (Please use freshly prepared in vivo formulations for optimal results.) |
| Preparing Stock Solutions | 1 mg | 5 mg | 10 mg | |
| 1 mM | 2.5095 mL | 12.5477 mL | 25.0954 mL | |
| 5 mM | 0.5019 mL | 2.5095 mL | 5.0191 mL | |
| 10 mM | 0.2510 mL | 1.2548 mL | 2.5095 mL |