c-FMS (IC50 = 60 nM)

GW2580 completely inhibits human cFMS kinase in vitro at a concentration of 0.06 μM. With an IC50 of 0.33, 13.5, 0.47, and 12 μM, respectively, GW2580 suppresses the growth of myeloid tumor cells stimulated by CSF-1, serum-stimulated NSO myeloid tumor cells, freshly isolated human monocytes stimulated by CSF-1, and human umbilical vein vascular endothelial cells stimulated by VEGF. A single microm Complete inhibition of bone degradation in cultures of human osteoclasts, rat calvaria, and rat fetal long bone is observed with GW2580. It also completely inhibits the growth of mouse M-NFS-60 myeloid cells and human monocytes induced by CSF-1.[1] With an IC50 of about 10 nM, GW2580 suppresses CSF1R phosphorylation in RAW264.7 murine macrophages stimulated with 10 ng/mL. With an IC50 of 0.88 μM, GW2580 likewise suppresses TRKA activity [2][3]

GW2580 (dosed orally at 40 mg/kg 0.5 h before the CSF-1-priming dose) suppresses the 63% increase in TNF-α production in mice induced by LPS when exogenous CSF-1 is added. GW2580 completely prevents CSF-1 from priming mice for enhanced IL-6 production when it is administered to them prior to CSF-1 priming. The peritoneal cavity's M-NFS-60 tumor cells that are dependent on CSF-1 are totally inhibited from growing by GW2580 (80 mg/kg p.o.). When taken orally twice a day the week prior to and for four days following thioglycolate injection, GW2580 (80 mg/kg) reduces (by 45%) the amount of macrophages that accumulate in the peritoneal cavity.[1] GW2580 (50 mg/kg), administered twice daily from days 0 to 21, 7 to 21, or 14 to 21, inhibits the breakdown of joint connective tissue and bone in a 21-day adjuvant arthritis model.[3] By preventing the tumor from recruiting myeloid cells from peripheral blood, Gw2580 (160 mg/kg) causes a more than two-fold decrease in total CD45⁺ CD11b⁺ myeloid cells, CD11b⁺ F4/80⁺ TAMs, and CD11b⁺ Gr-1⁺ MDSCs in implanted 3LL lung tumors. Treatment with GW2580 (80 mg/kg) can reduce tumor vascular density (CD31 staining) and the expression of Vegf-a (by 35%) and Mmp9 (by 70%). Combination therapy involving GW2580 and an anti-VEGFR-2 antibody reduces tumor growth in a synergistic manner. While DC101 by itself inhibits tumor growth by 35%, when combined with GW2580, there appears to be a synergistic reduction in tumor growth of about 70%.[2]

After 90 minutes at room temperature, 10 μM enzyme, 100 μM ATP, and 5 mM MgCl₂ are incubated in 50 mM Tris HCL to activate the enzyme through autophosphorylation. Round-bottom polystyrene 96-well plates on a Biomek 2000 are used to conduct 45 μL enzyme reactions. In each well, 30 mL of a 1.5-substrate reaction mix containing 50 mM Mops (3-[N-Morpholino]propanesulfonic acid), pH 7.5, 15 mM MgCl₂, 6 M peptide substrate, and biotin-EAIYAPFAKKK-NH2 is added, either alone or in 1 mL of DMSO. 0.5 μCi (1 Ci = 37 GBq) [³³P-γ] ATP, 75 mM NaCl, 10 μM ATP, and 7.5 mM DTT are needed for each assay. The final enzyme concentration is 20 nM, and the reaction is started by adding 15 μL of diluted enzyme solution. Background is determined by adding EDTA to the control wells. A 96-well phosphocellulose filter plate is prewet with 100 μL of 0.5% phosphoric acid, and 75 μL of the reaction is transferred to it after it has been allowed to proceed for 40 minutes and stopped by adding an equal volume of the acid. Following three rounds of washing with the phosphoric acid solution and filtering on a Millipore filter-plate vacuum manifold, 40 μL of scintillation solution is added. In a Packard Topcount NXT scintillation counter, the plates are sealed and tallied.

The cells are spun down and diluted media-infused with 2× 10⁶ cells/ml for 24 hours prior to the commencement of the cell growth assay. M-NSF60 cell depleted medium is devoid of MCSF. The following day, GW2580 at 10 mM in DMSO is serially diluted to produce a 10-point concentration curve, starting at 20 μM and 0.2% DMSO in medium containing 10% serum. After being resuspended in medium, the M-NFS-60 cells are added to 0.5× 10⁶ cells/mL along with 10% serum and 20 ng/mL mouse MCSF. Each well is filled with 50 μL of inhibitor-containing cells, and then, after three days, 10 μL of WST-1 reagent is added to each well. Growth is determined by measuring the difference between wells with full medium and wells with depleted medium after a 4-hour incubation period. The absorbance is measured at 440 nm.

Mouse myeloid carcinoma xenografts M-NFS-60
80 mg/kg
Orally twice a day

---

After oral administration, GW2580 blocked the ability of exogenous CSF-1 to increase LPS-induced IL-6 production in mice, inhibited the growth of CSF-1-dependent M-NFS-60 tumor cells in the peritoneal cavity, and diminished the accumulation of macrophages in the peritoneal cavity after thioglycolate injection. Unexpectedly, GW2580 inhibited LPS-induced TNF production in mice, in contrast to effects on monocytes and macrophages in vitro. In conclusion, GW2580's selective inhibition of monocyte growth and bone degradation is consistent with cFMS kinase inhibition. The ability of GW2580 to chronically inhibit CSF-1 signaling through cFMS kinase in normal and tumor cells in vivo makes GW2580 a useful tool in assessing the role of cFMS kinase in normal and disease processes.[1]

---

In the present study, the kinase selectivity of GW2580 was further characterized, and the effects of chronic treatment were evaluated in normal and arthritic rats. GW2580 selectively inhibited cFMS kinase compared with 186 other kinases in vitro and completely inhibited CSF-1-induced growth of rat monocytes, with an IC(50) value of 0.2 microM. GW2580 dosed orally at 25 and 75 mg/kg 1 and 5 h before the injection of lipopolysaccharide inhibited tumor necrosis factor-alpha production by 60 to 85%, indicating a duration of action of at least 5 h. In a 21-day adjuvant arthritis model, GW2580 dosed twice a day (b.i.d.) from days 0 to 21, 7 to 21, or 14 to 21 inhibited joint connective tissue and bone destruction as assessed by radiology, histology and bone mineral content measurements. In contrast, GW2580 did not affect ankle swelling in the adjuvant model nor did it affect ankle swelling in a model where local arthritis is reactivated by peptidoglycan polysaccharide polymers. GW2580 administered to normal rats for 21 days showed no effects on tissue histology and only modest changes in serum clinical chemistry and blood hematology. In conclusion, GW2580 was effective in preserving joint integrity in the adjuvant arthritis model while showing minimal effects in normal rats.[3]

[1]. Inhibition of colony-stimulating-factor-1 signaling in vivo with the orally bioavailable cFMS kinase inhibitor GW2580. Proc Natl Acad Sci U S A. 2005 Nov 1;102(44):16078-83.

[2]. Targeting distinct tumor-infiltrating myeloid cells by inhibiting CSF-1 receptor: combating tumor evasion of antiangiogenic therapy. Blood. 2010 Feb 18;115(7):1461-71

[3]. Effects of the cFMS kinase inhibitor 5-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)pyrimidine-2,4-diamine (GW2580) in normal and arthritic rats. J Pharmacol Exp Ther. 2008 Jul;326(1):41-50.

Colony-stimulating-factor-1 (CSF-1) signaling through cFMS receptor kinase is increased in several diseases. To help investigate the role of cFMS kinase in disease, we identified GW2580, an orally bioavailable inhibitor of cFMS kinase. GW2580 completely inhibited human cFMS kinase in vitro at 0.06 microM and was inactive against 26 other kinases. GW2580 at 1 microM completely inhibited CSF-1-induced growth of mouse M-NFS-60 myeloid cells and human monocytes and completely inhibited bone degradation in cultures of human osteoclasts, rat calvaria, and rat fetal long bone. In contrast, GW2580 did not affect the growth of mouse NS0 lymphoblastoid cells, human endothelial cells, human fibroblasts, or five human tumor cell lines. GW2580 also did not affect lipopolysaccharide (LPS)-induced TNF, IL-6, and prostaglandin E2 production in freshly isolated human monocytes and mouse macrophages. After oral administration, GW2580 blocked the ability of exogenous CSF-1 to increase LPS-induced IL-6 production in mice, inhibited the growth of CSF-1-dependent M-NFS-60 tumor cells in the peritoneal cavity, and diminished the accumulation of macrophages in the peritoneal cavity after thioglycolate injection. Unexpectedly, GW2580 inhibited LPS-induced TNF production in mice, in contrast to effects on monocytes and macrophages in vitro. In conclusion, GW2580's selective inhibition of monocyte growth and bone degradation is consistent with cFMS kinase inhibition. The ability of GW2580 to chronically inhibit CSF-1 signaling through cFMS kinase in normal and tumor cells in vivo makes GW2580 a useful tool in assessing the role of cFMS kinase in normal and disease processes.[1]

---

Tumor-infiltrating myeloid cells (TIMs) support tumor growth by promoting angiogenesis and suppressing antitumor immune responses. CSF-1 receptor (CSF1R) signaling is important for the recruitment of CD11b(+)F4/80(+) tumor-associated macrophages (TAMs) and contributes to myeloid cell-mediated angiogenesis. However, the impact of the CSF1R signaling pathway on other TIM subsets, including CD11b(+)Gr-1(+) myeloid-derived suppressor cells (MDSCs), is unknown. Tumor-infiltrating MDSCs have also been shown to contribute to tumor angiogenesis and have recently been implicated in tumor resistance to antiangiogenic therapy, yet their precise involvement in these processes is not well understood. Here, we use the selective pharmacologic inhibitor of CSF1R signaling, GW2580, to demonstrate that CSF-1 regulates the tumor recruitment of CD11b(+)Gr-1(lo)Ly6C(hi) mononuclear MDSCs. Targeting these TIM subsets inhibits tumor angiogenesis associated with reduced expression of proangiogenic and immunosuppressive genes. Combination therapy using GW2580 with an anti-VEGFR-2 antibody synergistically suppresses tumor growth and severely impairs tumor angiogenesis along with reverting at least one TIM-mediated antiangiogenic compensatory mechanism involving MMP-9. These data highlight the importance of CSF1R signaling in the recruitment and function of distinct TIM subsets, including MDSCs, and validate the benefits of targeting CSF1R signaling in combination with antiangiogenic drugs for the treatment of solid cancers.[2]

---

The cFMS (cellular homolog of the V-FMS oncogene product of the Susan McDonough strain of feline sarcoma virus) (Proc Natl Acad Sci U S A 83:3331-3335, 1986) kinase inhibitor 5-(3-methoxy-4-((4-methoxybenzyl)oxy)benzyl)pyrimidine-2,4-diamine (GW2580) inhibits colony-stimulating factor (CSF)-1-induced monocyte growth and bone degradation in vitro and inhibits CSF-1 signaling through cFMS kinase in 4-day models in mice (Proc Natl Acad Sci U S A 102:16078, 2005).[3]

C20H22N4O3

366.41

366.169

C, 65.56; H, 6.05; N, 15.29; O, 13.10

870483-87-7

11617559

white solid powder

1.3±0.1 g/cm3

617.5±65.0 °C at 760 mmHg

327.2±34.3 °C

0.0±1.8 mmHg at 25°C

1.635

2.66

2

7

7

27

433

0

N1C(N)=NC(N)=C(CC2C=C(OC)C(OCC3C=CC(OC)=CC=3)=CC=2)C=1

MYQAUKPBNJWPIE-UHFFFAOYSA-N

InChI=1S/C20H22N4O3/c1-25-16-6-3-13(4-7-16)12-27-17-8-5-14(10-18(17)26-2)9-15-11-23-20(22)24-19(15)21/h3-8,10-11H,9,12H2,1-2H3,(H4,21,22,23,24)

5-[[3-methoxy-4-[(4-methoxyphenyl)methoxy]phenyl]methyl]pyrimidine-2,4-diamine

GW 2580; SC-203877; SC 203877; GW 2580; GW-2580; 5-(3-Methoxy-4-((4-methoxybenzyl)oxy)benzyl)pyrimidine-2,4-diamine; 5-[[3-methoxy-4-[(4-methoxyphenyl)methoxy]phenyl]methyl]pyrimidine-2,4-diamine; GW632580X; GW 2580; SRV0JCF9LI; SC203877

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: ≥ 2.08 mg/mL (5.68 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 (5.68 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.

---

View More

Solubility in Formulation 3: ≥ 2.08 mg/mL (5.68 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: 5% DMSO+30% PEG 300+5% Tween 80+ddH2O: 5 mg/mL

---

Solubility in Formulation 5: 5 mg/mL (13.65 mM) in 0.5% CMC-Na/saline water (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

---

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