SMALL-MOLECULE INHIBITORS OF THE FRS2-FGFR INTERACTION

Abstract

The present invention relates to small-molecule inhibitors of the FRS2-FGFR interaction. The present invention relates the small-molecule inhibitors for use as a medicament and for use in cancer treatment or prevention.

Claims

1. A compound of the general formula (500) for use in treatment or prevention of metastasis ##STR00018## wherein X.sup.1 is selected from N, O, and S, particularly X.sup.1 is N, R.sup.1 is selected from a (linear or branched) C.sub.1-C.sub.16 alkyl, (linear or branched) C.sub.2-C.sub.16 alkene, heteroaryl, aryl, a C.sub.4-C.sub.7 cyclo-alkyl, and a C.sub.3-C.sub.6 heterocycle, wherein R.sup.1 is unsubstituted or substituted with OR.sup.O, CN, halogen, NR.sup.N1R.sup.N2, SO.sub.2R.sup.S, COOR.sup.A with R.sup.N1, R.sup.N2, R.sup.A, R.sup.O, and R.sup.S being independently selected from H, and unsubstituted or substituted C.sub.1-C.sub.5 alkyl or C.sub.2-C.sub.5 alkene, particularly R.sup.1 is substituted with one moiety selected from OR.sup.O, CN, halogen, NR.sup.N1R.sup.N2, SO.sub.2R.sup.S, COOR.sup.A with R.sup.N1, R.sup.N2, R.sup.A, R.sup.O, and R.sup.S being independently selected from H, and C.sub.1-C.sub.3 alkyl; each R.sup.2 and R.sup.3 is independently selected from C.sub.1-C.sub.3 alkyl, OR.sup.OH, NH.sub.2, CN, COOR.sup.COO and halogen, with R.sup.COO and R.sup.OH being independently selected from H, and C.sub.1-C.sub.3 alkyl; n is 0, 1, 2, or 3, particularly n is 1; m is 0, 1, 2, 3, or 4, particularly m is 1 or 2.

2. The compound for use according to claim 1, wherein R.sup.1 is —CH.sub.2—NH—CHR.sup.4R.sup.5, wherein R.sup.4 and R.sup.5 are independently selected from a C.sub.1-C.sub.5 alkyl, C.sub.2-C.sub.5 alkene, wherein R.sup.4 and R.sup.5 are unsubstituted or substituted with OR.sup.O, CN, halogen, NR.sup.N1R.sup.N2, SO.sub.2R.sup.S, COOR.sup.A with R.sup.N1, R.sup.N2, R.sup.A, R.sup.O, and R.sup.S being independently selected from H, and C.sub.1-C.sub.3 alkyl; or R.sup.4 and R.sup.5 together form an unsubstituted or OH—, halogen-, and/or CN-substituted cyclo-pentane or cyclo-hexane.

3. The compound for use according to claim 2 of the general formula (700) ##STR00019## wherein each R.sup.2 and R.sup.3 is independently selected from C.sub.1-C.sub.3 alkyl, OR.sup.OH, NH.sub.2, CN, COOR.sup.COO and halogen, with R.sup.COO and R.sup.OH being independently selected from H, and C.sub.1-C.sub.3 alkyl; X.sup.1 is selected from N, O, and S, particularly X.sup.1 is N.

4. The compound for use according to claim 2, wherein R.sup.4 is selected from unsubstituted C.sub.1-C.sub.5 alkyl and C.sub.2-C.sub.5 alkene and R.sup.5 is an electronegative moiety selected from C.sub.1-C.sub.5 alkyl and C.sub.2-C.sub.5 alkene substituted with OR.sup.O, CN, halogen, NR.sup.N1R.sup.N2, SO.sub.2R.sup.S, COOR.sup.A with R.sup.N1, R.sup.N2, R.sup.A, R.sup.O, and R.sup.S being independently selected from H, and C.sub.1-C.sub.3 alkyl; particularly R.sup.4 is selected from ethyl, iso-propyl, and tert-butyl.

5. The compound for use according to claim 2, wherein R.sup.5 is selected from OH—, halogen-, and/or CN-substituted methyl, ethyl, and isopropyl.

6. The compound for use according to claim 1, wherein R.sup.2 is selected from C.sub.1-C.sub.3 alkyl, OH, NH.sub.2, and halogen, particularly F or Cl, particularly R.sup.2 is selected from C.sub.1-C.sub.3 alkyl, and OH.

7. The compound for use according to claim 1, wherein R.sup.3 is selected from OH, NH.sub.2, and halogen, particularly R.sup.3 is halogen, more particularly R.sup.3 is F.

8. The compound for use according to claim 1, wherein X.sup.1 is N.

9. The compound for use according to claim 1, wherein said metastasis arises from a cancer selected from bladder cancer, pediatric brain tumour, medulloblastoma, multiple myeloma, colorectal cancer and gastric cancer.

10. The compound as described in claim 1 for use as an angiogenesis antagonist, particularly an angiogenesis antagonist in treatment or prevention of cancer, more particularly wherein said cancer is selected from bladder cancer, hepatocellular carcinoma, and prostate cancer.

11. The compound as described in claim 1 for use in prevention or treatment of an FGFR-driven disease.

12. A compound of the general formula (700) ##STR00020## wherein each R.sup.2 and R.sup.3 is independently selected from C.sub.1-C.sub.3 alkyl, OR.sup.OH, NH.sub.2, CN, COOR.sup.COO and halogen, with R.sup.COO and R.sup.OH being independently selected from H, and C.sub.1-C.sub.3 alkyl; R.sup.4 and R.sup.5 are independently selected from a C.sub.1-C.sub.5 alkyl, C.sub.2-C.sub.5 alkene, wherein R.sup.4 and R.sup.5 are unsubstituted or substituted with OR.sup.O, CN, halogen, NR.sup.N1R.sup.N2, SO.sub.2R.sup.S, COOR.sup.A with R.sup.N1, R.sup.N2, R.sup.A, R.sup.O, and R.sup.S being independently selected from H, and C.sub.1-C.sub.3 alkyl; or R.sup.4 and R.sup.5 together form an unsubstituted or OH—, halogen-, and/or CN-substituted cyclo-pentane or cyclo-hexane; X.sup.1 is selected from N, O, and S, particularly X.sup.1 is N, with the proviso that the compound is not characterized by the formula (001), ##STR00021##

13. The compound according to claim 12, wherein R.sup.4 is selected from unsubstituted C.sub.1-C.sub.5 alkyl and C.sub.2-C.sub.5 alkene and R.sup.5 is an electronegative moiety selected from C.sub.1-C.sub.5 alkyl and C.sub.2-C.sub.5 alkene substituted with OR.sup.O, CN, halogen, NR.sup.N1R.sup.N2, SO.sub.2R.sup.S, COOR.sup.A with R.sup.N1, R.sup.N2, R.sup.A, R.sup.O, and R.sup.S being independently selected from H, and C.sub.1-C.sub.3 alkyl; particularly R.sup.4 is selected from ethyl, iso-propyl, and tert-butyl, and R.sup.5 is selected from OH—, halogen-, and/or CN-substituted methyl, ethyl, and isopropyl.

14. The compound according to claim 12, wherein R.sup.2 is selected from C.sub.1-C.sub.3 alkyl, OH, NH.sub.2, and halogen, particularly F or Cl, particularly R.sup.2 is selected from C.sub.1-C.sub.3 alkyl, and OH.

15. The compound according to claim 12, wherein R.sup.3 is selected from OH, NH.sub.2, and halogen, particularly R.sup.3 is halogen, more particularly R.sup.3 is F.

16. The compound according to claim 12, wherein X.sup.1 is N.

17. A compound according to claim 12, for use as a medicament with the proviso that the compound includes the compound characterized by formula (001), ##STR00022##

18. A compound as described in claim 12 for use in treatment or prevention of cancer, particularly wherein said cancer is selected from ependymoma, prostate cancer, esophageal cancer, thyroid cancer, hepatocellular carcinoma, testicular cancer, pediatric brain tumour, medulloblastoma, rhabdomyosarcoma, gastric cancer, pulmonary pleomorphic carcinoma, breast cancer, non-small cell lung cancer, liposarcoma, cervical cancer, colorectal cancer, melanoma, multiple myeloma, endometrial cancer, bladder cancer, glioblastoma, squamous cell carcinoma of the lung, ovarian cancer, head and neck cancer, and pancreatic cancer, sarcoma, more particularly said cancer is selected from bladder cancer, multiple myeloma, gastric cancer, pediatric brain tumour, medulloblastoma, glioblastoma, ependymoma, colorectal cancer and sarcoma, most particularly said cancer is selected from bladder cancer, pediatric brain tumour, medulloblastoma, multiple myeloma, colorectal cancer and gastric cancer with the proviso that the compound includes the compound characterized by the formula (001).

Description

DESCRIPTION OF THE FIGURES

[0165] FIG. 1 shows the efficacy of the compound F3.14 determining its ability to inhibit cancer cell invasion. The graph represents the efficacy of F3.14 at 3 different concentrations—1 μM, 5 μM and 10 μM.

[0166] FIG. 2 shows the efficacy of F3.14 at 10 M.

[0167] FIG. 3 shows the binding affinities and dissociation constant (Kd) of F3.14. Nano diffraction scanning fluorimetry (nanoDSF) and Microscale thermophoresis (MST) are biophysical assays used to assess the binding of the compounds to the target protein. Any temperature shift above 1.5 degree Celsius is considered as indication for significant binding.

[0168] FIG. 4 shows the effective inhibitory concentration of F3.14—EC50 (μM).

[0169] FIG. 5 shows the biochemical specificity of F3.14 determining the ability of the compounds to inhibit FGF signalling pathway without affecting other signalling pathways. Lane 1: Control—DAOY LA-EGFP cells unstimulated, serum starved overnight and then lysed. Lane 2: bFGF (100 ng/ml)—Overnight serum starved DAOY LA-EGFP cells stimulated with bFGF for 10 minutes and then lysed. Lane 3: F3.14 (10 μM)—Overnight serum starved DAOY LA-EGFP cells treated with F3.14 for four hours, cells stimulated with bFGF for 10 minutes and then lysed.

[0170] FIG. 6 A) Binding site 1 is not involved in FGFR binding and located below the interaction site of FGFR's N-terminus with FRS2. B) Binding site 2 is the extended surface area interacting with FGFR's C-terminal end.

[0171] FIG. 7 Spheroid invasion assay using DAOY cells stimulated with bFGF+/−BGJ398 or F3-14 to determine the EC5o of F3.14.

[0172] FIG. 8 Cell titer glo assay performed with DAOY cells treated with BGJ398 or F3.14.

[0173] FIG. 9 Cell titer glo assay performed with AGS cells treated with BGJ398 or F3.14.

[0174] FIG. 10 Cell titer glo assay performed with M059K cells treated with BGJ398 or F3.14.

[0175] FIG. 11 Cell titer glo assay performed with RT112 cells treated with BGJ398 or F3.14.

[0176] FIG. 12 Cell titer glo assay performed with DMS114 cells treated with BGJ398 or F3.14.

[0177] FIG. 13 Cell titer glo assay performed with HCT116 cells treated with BGJ398 or F3.14.

[0178] FIG. 14 Cell titer glo assay performed with SKOV3 cells treated with BGJ398 or F3.14.

[0179] FIG. 15 Cell titer glo assay performed with SNU16 cells treated with BGJ398 or F3.14.

[0180] FIG. 16 Table showing the in vitro absorption, distribution, metabolism, elimination and toxicity (ADMET) properties of F3.14. Efflux ration represents the permeability of F3.14, Semi-thermodynamic solubility shows the solubility of F3.14 in aqueous solutions. Intrinsic clearance and t½ shows the metabolic stability of F3.14, MTT shows the toxicity of F3.14 and potency shows the efficacy of F3.14.

[0181] FIG. 17 In vivo pharmacokinetics, 3 mice/treatment, serum concentration of compounds in μM. Table showing the in vivo pharmacokinetic (PK) properties of F3.14.

[0182] FIG. 18 Immunoblots using various FGFR-driven cell lines treated with BGJ398 or F3.14 showing the effect of the treatment on the downstream effectors of FGF signalling.

EXAMPLES

[0183] The inventors designed an inhibitor of FRS2-FGFR interaction by screening a large library of fragments of small molecules. The inventors identified F3.14 as a putative small molecule inhibitor of FRS2-FGFR interaction. The inventors confirmed the binding of F3.14 to FRS2 using biophysical assays—nanoDSF, MST and NMR analysis. The inventors evaluated the efficacy of F3.14 in inhibiting cancer cell invasion and proliferation using FGFR-driven cancer cell models. Results from the spheroid invasion assay and cell titer glo assay show that F3.14 effectively inhibits cancer cell invasion and proliferation in all the FGFR-driven cancer cell lines tested. To test the effect of F3.14 on FGF signaling pathway, the inventors used immunoblotting. F3.14 inhibits the FGF signal transduction by inhibiting the phosphorylation of the downstream effectors of FGF signaling pathway. The inventors used in vitro ADMET studies and in vivo PK studies to determine the ‘drug-like’ properties of F3.14. Results from these assays demonstrate that F3.14 has good permeability, very good solubility, moderate intrinsic clearance, very low toxicities, and high potency. The in vivo PK studies show that F3.14 is well-tolerated in mice and could be safely administered via intravenous route to living organisms for the treatment of FGFR-driven diseases.

Methods and Instruments

[0184] Spheroid Invasion Assay (S/A) and Automated Cell Dissemination Counter (aCDc)

[0185] 1000 cells/100 μl per well were seeded in cell-repellent 96 well microplate (650790, Greiner Bio-one). The cells were incubated at 37° C. overnight to form spheroids. 70 μl of the medium were removed from each well, and remaining medium with spheroid overlaid with 2.5% bovine collagen 1. Following the polymerization of collagen, fresh medium was added to the cells and treated with growth factors and/or with inhibitors. The cells were allowed to invade the collagen matrix for 24 h, after which they were fixed with 4% PFA and stained with Hoechst. Images were acquired on an Axio Observer 2 mot plus fluorescence microscope (Zeiss, Munic, Germany) using a 5×objective. Cell invasion is determined as the average of the distance invaded by the cells from the centre of the spheroid as determined using automated cell dissemination counter (aCDc) with our cell dissemination counter software aSDlcs (Kumar et al., Sci Rep 5, 15338 (2015)).

Nano Differential Scanning Fluorimetry (nanoDSF)

[0186] Purified FRS2 protein tagged with 6× Histidine residues and Guanine nucleotide-binding protein subunit beta (GB1) was diluted in the protein buffer (100 mM sodium phosphate, 50 mM NaCl, 0.5 mM EDTA, 50 mM arginine, 1 mM TCEP, pH 7.0) to final concentration of 30 μM. The compounds were dissolved in 100% at 50 or 100 mM and further diluted to 1 mM with a final concentration of 100% DMSO. Compound and protein were mixed at 1:1 ration yielding final concentrations of 15 μM and 500 μM for the compounds. The mixture was incubated at room temperature for 15 minutes before measurement. The measurement was performed on a Prometheus system in high sensitivity capillaries. Samples were subjected to a temperature gradient of 20 to 95° C. with 1° C./min intervals.

Microscale Thermophoresis (MST)

[0187] Purified FRS2 protein tagged with 6× Histidine residues and Guanine nucleotide-binding protein subunit beta (GB1) was labelled with 2.sup.nd generation BLUE-NHS dye. The protein was labelled at a final concentration of 20 M with 60 μM dye. The labelling was performed in the protein buffer without arginine supplementation. Arginine was re-buffered to protein's buffer post-labelling. The compounds were dissolved in 100% at 50 or 100 mM and further diluted to 1 mM with a final concentration of 100% DMSO. The compounds were then diluted. In a 1:1 serial dilution from 1 mM to 61.04 nM in protein buffer supplemented with 10% DMSO. 10 μl of 50 nM labelled protein was added to 10 μl of each compound dilution for a final labelled protein concentration of 25 nM and DMSO-concentration of 5%. The samples were incubated at room temperature for 15 minutes. The experiments were performed in premium-coated capillaries. Excitation power was set at 20%, MST power to 40% (4 Kelvin temperature gradient) with a laser-on time of 20 seconds and a laser-off time of 3 seconds. Temperature was set to 25° C. Each measurement was repeated twice. The interaction was measured in two independent duplicates.

Immunoblotting (IB)

[0188] Cancer cells were treated with bFGF (100 ng/ml) and/or with compounds and lysed using Radioimmunoprecipitation assay (RIPA) buffer. RIPA buffer lysates were resolved by SDS-PAGE and transferred to a nitrocellulose membrane using a transfer apparatus according to the manufacturer's instructions (Bio-Rad). Membranes were probed with primary antibodies against phospho-FRS2, FRS2, ERK1/2, phospho-ERK1/2, AKT, phosphor-AKT, phospho-PKC and tubulin. HRP-linked secondary antibodies (1:5000) were used to detect the primary antibodies. Chemiluminescence detection was performed using ChemiDoc Touch Gel and Western Blot imaging system (BioRad).

Cell Titer Glo Assay

[0189] The metabolic activity and the proliferation of the cells were determined using the Cell Titer glo assay from Promega according to the manufacturer's instructions. In brief, 250 cells/100 μl/per well (for up to 72 h incubation) were seeded in Greiner Bio-One p-clear 384 well plates (655090, Greiner Bio-One) and incubated overnight at 37° C. The old media was then replaced with fresh serum-free media and the cells were treated with BGJ398 or F3.14 till the desired time point. Following appropriate incubation for each timepoint, 10 μl of the Cell titer glo reagent was added to each well (final concentration of cell titer glo reagent per well is 1:10) and incubated at 37° C. for 30 minutes. The luminescence was then measured with a signal integration time of 0.5 to 1 second per well.

In Vivo Pharmacokinetics

[0190] 3 Healthy non-SCID mice were intravenously treated with F3.14—Blood samples were collected at 2, 4, 6, 8 and 24 hours after treatment. Serum from the collected blood samples were isolated and the concentration of F3.14 in the serum was measure to determine the intrinsic clearance of F3.14.

Pathway Analysis

[0191] RIPA buffer FGFR-driven cell lysates were resolved by SDS-PAGE and transferred to a nitrocellulose membrane using a transfer apparatus according to the manufacturer's instructions (Bio-Rad). Membranes were probed with primary antibodies against phospho-FRS2, FRS2, ERK1/2, phospho-ERK1/2, AKT, phospho-AKT, phospho-PKC and tubulin. HRP-linked secondary antibodies (1:5000) were used to detect the primary antibodies. Chemiluminescence detection was performed using ChemiDoc Touch Gel and Western Blot imaging system (BioRad). Integrated density of Immuno-reactive bands was quantified using Adobe Photoshop CS5.

Availability of Compounds

[0192] The compound was purchased at ChemBridge under the following vendor ID: F3.14 24662310 (ChemBridge)