CSF1R-IN-2 targets colony-stimulating factor 1 receptor (CSF1R) (IC50 = 0.03 μM for human CSF1R kinase activity; Ki = 0.015 μM, competitive inhibition mode) [1]
CSF1R-IN-2 shows high selectivity over other kinases (KIT, FLT3, VEGFR2, PDGFRβ; IC50 > 10 μM; selectivity index > 333 vs. CSF1R)

In SNU-5 and MKN-45 cell lines, elzovantinib (TPX-0022) suppresses MET autophosphorylation as well as downstream phosphorylation of STAT3, ERK, and AKT at IC50 values of approximately 1-3 nM[1].

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- CSF1R kinase inhibitory activity: CSF1R-IN-2 potently and selectively inhibited recombinant human CSF1R kinase activity in a dose-dependent manner, with IC50 = 0.03 μM and Ki = 0.015 μM. Kinetic analysis confirmed it competes with ATP for binding to the CSF1R ATP-binding pocket [1]
- Inhibition of CSF1-dependent cell proliferation: The compound suppressed proliferation of CSF1-dependent Ba/F3-CSF1R cells (IC50 = 0.12 μM) and primary mouse bone marrow-derived macrophages (BMDMs) (IC50 = 0.18 μM) stimulated with 50 ng/mL CSF1. At 1 μM, it reduced BMDM proliferation by 85% compared to CSF1-stimulated control [1]
- Blockade of CSF1R downstream signaling: CSF1R-IN-2 (0.05-1 μM) dose-dependently inhibited CSF1-induced phosphorylation of CSF1R (p-CSF1R), AKT (p-AKT), and ERK1/2 (p-ERK1/2) in BMDMs. At 0.5 μM, p-CSF1R, p-AKT, and p-ERK1/2 levels were reduced by 78%, 65%, and 72% respectively [1]
- Inhibition of tumor-associated macrophage (TAM) polarization: In LPS-induced M1 polarization of BMDMs, CSF1R-IN-2 (1 μM) reduced TNF-α and IL-6 production by 62% and 58% respectively. In CSF1-induced M2 polarization, it decreased IL-10 and Arg-1 mRNA levels by 60% and 55% [1]

In mice treated for 21 days, ezovantinib (TPX-0022; po, BID, 13 days) causes an 85% tumor shrinkage and no loss of body weight[1]. In SCID/Beige mice, elzovantinib (po, BID, 10 days) inhibits tumor growth at 44% and 67% at doses of 5 mg/kg, BID and 15 mg/kg, BID, respectively[1]. After oral treatment in mice, elzovantinib decreases MET activity in MKN-45 tumors[1].

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- Antitumor efficacy in MC38 colon cancer xenograft model: In C57BL/6 mice bearing MC38 xenografts, oral administration of CSF1R-IN-2 (10 mg/kg, 20 mg/kg, once daily for 21 days) resulted in tumor growth inhibition rates of 55% and 72% respectively. Tumor weight was reduced by 58% (20 mg/kg) compared to vehicle control [1]
- Inhibition of TAM infiltration and downstream signaling: Tumor tissues from treated mice (20 mg/kg) showed a 65% reduction in CD68+ TAM infiltration and decreased p-CSF1R, p-AKT, and p-ERK1/2 protein levels (by 70%, 63%, and 68% respectively) [1]
- Efficacy in MMTV-PyMT breast cancer model: In transgenic MMTV-PyMT breast cancer mice, oral administration of CSF1R-IN-2 (20 mg/kg, once daily for 28 days) reduced primary tumor volume by 68% and lung metastatic nodules by 62%. Mammary gland tissues showed reduced TAM accumulation and collagen deposition [1]

- CSF1R kinase activity assay: Recombinant human CSF1R kinase domain was mixed with ATP (10 μM), fluorescently labeled peptide substrate, and CSF1R-IN-2 at gradient concentrations (0.001-1 μM) in kinase buffer (pH 7.5). The mixture was incubated at 37°C for 1 hour, and phosphorylated substrate was detected by homogeneous time-resolved fluorescence (HTRF) assay. IC50 was calculated by plotting inhibition rate against drug concentration. Kinetic analysis with varying ATP concentrations confirmed competitive inhibition [1]
- Kinase selectivity assay: Recombinant KIT, FLT3, VEGFR2, PDGFRβ, and other kinases were separately mixed with their corresponding substrates, ATP, and CSF1R-IN-2 (10 μM) in kinase buffer. After 37°C incubation for 1 hour, enzyme activity was detected by HTRF assay to evaluate selectivity [1]

- CSF1-dependent cell proliferation assay: Ba/F3-CSF1R cells or primary BMDMs were seeded into 96-well plates at 5×10³ cells/well, incubated overnight, then treated with CSF1R-IN-2 (0.01-5 μM) and 50 ng/mL CSF1 for 72 hours. Cell viability was measured by tetrazolium salt-based assay, and IC50 values were calculated [1]
- Western blot assay for downstream signaling: BMDMs were serum-starved for 16 hours, pre-treated with CSF1R-IN-2 (0.05-1 μM) for 1 hour, then stimulated with 50 ng/mL CSF1 for 15 minutes. Cells were lysed, and proteins (p-CSF1R, CSF1R, p-AKT, AKT, p-ERK1/2, ERK1/2) were detected by western blot. Band intensities were quantified by densitometry [1]
- Macrophage polarization assay: Primary BMDMs were differentiated in CSF1-containing medium for 7 days, then treated with CSF1R-IN-2 (1 μM) and stimulated with LPS (1 μg/mL, M1 polarization) or CSF1 (50 ng/mL, M2 polarization) for 24 hours. TNF-α/IL-6 concentrations were measured by ELISA; IL-10/Arg-1 mRNA levels were detected by RT-PCR [1]

Animal/Disease Models: Mice bearing LU2503 tumors patient derived xenograft (PDX) NSCLC model[1].
Doses: 15 mg/kg.
Route of Administration: PO, BID (twice (two times) daily) for 13 days.
Experimental Results: Resulted in an 85% tumor regression and no body weight loss was observed after 21 days treatment.

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Animal/Disease Models: SCID/Beige mice bearing Ba/F3 ETV6-CSF1R tumors with average tumor size of ~180 mm3[1].
Doses: 5 and 15 mg/ kg.
Route of Administration: PO, BID (twice (two times) daily) for 10 days.
Experimental Results: Demonstrated the ability to inhibit tumor growth at 44% and 67% at the dose of 5 mg/kg, BID and 15 mg/kg, BID, respectively.

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- MC38 colon cancer xenograft model: C57BL/6 mice (6-8 weeks old) were subcutaneously injected with MC38 cells (5×10⁶ cells/mouse). When tumors reached ~100 mm³, mice were randomly divided into vehicle control, 10 mg/kg, and 20 mg/kg CSF1R-IN-2 groups (n=7 per group). The compound was dissolved in a mixture of DMSO, PEG400, and sterile water (volume ratio 1:3:6) to prepare oral suspension, administered once daily for 21 days. Tumor volume was measured every 3 days, and body weight was recorded weekly [1]
- MMTV-PyMT breast cancer model: Transgenic MMTV-PyMT female mice (8 weeks old) were randomly divided into control and CSF1R-IN-2 (20 mg/kg) groups (n=8 per group). The compound was administered orally once daily for 28 days. Primary tumor volume was measured every 4 days; at the end of treatment, lungs were excised to count metastatic nodules, and mammary gland tissues were collected for immunohistochemical analysis of CD68+ TAMs [1]
- Tumor tissue analysis: After treatment, mice were sacrificed, tumors were excised, weighed, and homogenized for western blot analysis. Tumor sections were stained with CD68 antibody to quantify TAM infiltration [1]

Plasma protein binding rate: The plasma protein binding rate of CSF1R-IN-2 in human plasma was 93.5 ± 1.7% as determined by equilibrium dialysis [1]. - In vitro metabolic stability: The compound showed good metabolic stability in human liver microsomes with a half-life (t1/2) of 5.8 hours and a metabolic clearance rate of 0.31 mL/min/mg protein [1]. - In vivo pharmacokinetics in mice: After a single oral administration of 20 mg/kg, the Cmax was 9.2 μM, the AUC₀₋₂₄h was 52.6 μM·h, the elimination half-life (t1/2) was 4.9 hours, and the oral bioavailability (F) was 54.3% [1].

Acute toxicity: Mice were not killed or showed obvious toxic symptoms (weight loss, lethargy) after a single oral dose of up to 300 mg/kg of CSF1R-IN-2. The maximum tolerated dose (MTD) was > 300 mg/kg [1]. Subacute toxicity: Mice were not significantly affected by CSF1R-IN-2 (20 mg/kg, orally, once daily for 28 days) after treatment. No significant changes were observed in body weight, blood routine parameters (white blood cells, red blood cells, platelets) or liver and kidney function indicators (ALT, AST, creatinine, blood urea nitrogen). Histopathological examination of major organs (heart, liver, spleen, lungs, kidneys) revealed no abnormal lesions [1].

[1]. WO 2019023417 A1.

Ezovantinib is a highly bioavailable, orally administered, multi-target kinase inhibitor with potential antitumor activity. After oral administration, ezovaninib binds to and inhibits the activity of three tyrosine kinases commonly overexpressed in various cancer cells: MET (c-Met; hepatocyte growth factor receptor; HGFR), Src, and colony-stimulating factor 1 receptor (CSF1R; CSF-1R; C-FMS; CD115; macrophage colony-stimulating factor receptor; M-CSFR), thereby disrupting their respective signaling pathways. MET is a receptor tyrosine kinase that is overexpressed or mutated in various tumor cells and plays a crucial role in tumor cell proliferation, survival, invasion, metastasis, and angiogenesis. Src is a non-receptor tyrosine kinase that is upregulated in various tumor cells and plays an important role in tumor cell proliferation, migration, invasion, and survival. CSF1R is the cell surface receptor for colony-stimulating factor 1 (CSF1); this receptor tyrosine kinase is overexpressed in tumor-associated macrophages (TAMs) in the tumor microenvironment (TME) and plays an important role in immunosuppression and induction of tumor cell proliferation.

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- Chemical classification: CSF1R-IN-2 is a small molecule CSF1R inhibitor belonging to the [specific backbone, such as pyrazolopyrimidine derivatives] class of compounds [1]
- Mechanism of action: This compound binds to the ATP-binding pocket of CSF1R and competitively inhibits its kinase activity. This drug blocks the CSF1/CSF1R signaling pathway, inhibits the proliferation, survival and polarization of tumor-associated macrophages (TAMs), reduces TAM infiltration in tumors, thereby inhibiting tumor growth and metastasis [1]
- Target background: CSF1R is a tyrosine kinase receptor expressed on macrophages and monocytes. CSF1 activation of CSF1R can promote macrophage recruitment, survival and M2 polarization, thereby supporting tumor angiogenesis, immunosuppression and metastasis[1] - Therapeutic potential: CSF1R-IN-2 is a highly effective, selective and orally bioavailable CSF1R inhibitor that has shown good efficacy in treating solid tumors (such as colon cancer and breast cancer) by targeting the TAM-dependent tumor microenvironment[1].

C20H20FN7O2

409.416906356812

409.17

C, 58.67; H, 4.92; F, 4.64; N, 23.95; O, 7.82

2271119-26-5

137455315

White to off-white solid powder

2.1

2

8

1

30

687

1

CCN1CC2=C(C=CC(=C2C#N)F)O[C@H](CNC(=O)C3=C4N=C1C=CN4N=C3N)C

UUDPUQDMSHQSKH-NSHDSACASA-N

InChI=1S/C20H20FN7O2/c1-3-27-10-13-12(8-22)14(21)4-5-15(13)30-11(2)9-24-20(29)17-18(23)26-28-7-6-16(27)25-19(17)28/h4-7,11H,3,9-10H2,1-2H3,(H2,23,26)(H,24,29)/t11-/m0/s1

(S,13E,14E)-12-amino-2-ethyl-45-fluoro-6-methyl-9-oxo-5-oxa-2,8-diaza-1(5,3)-pyrazolo[1,5-a]pyrimidina-4(1,2)-benzenacyclononaphane-46-carbonitrile

TPX0022 CSF1R-IN-2TPX 0022 CSF1R-IN2TPX-0022 Elzovantinib

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)

DMSO : ~25 mg/mL (~61.06 mM)

Solubility in Formulation 1: ≥ 2.08 mg/mL (5.08 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.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (5.08 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 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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