MET (wild-type IC50 = 1 nM; D1246N IC50 = 8 nM; Y1248C IC50 = 16 nM; D1246H IC50 = 11 nM; Y1248D IC50 = 17 nM; Y1248H IC50 = 1 nM; M1268T IC50 = 1 nM) [1];
AXL (IC50 = 7 nM) [1];
MER (IC50 = 2 nM) [1];
FGFR1 (wild-type IC50 = 18 nM; V561M IC50 = 23 nM) [1];
FGFR2 (wild-type IC50 = 17 nM; N549H IC50 = 19 nM) [1];
FGFR3 (IC50 = 15 nM) [1];
Binding affinity to MET: KD = 1.26 × 10^{-9} mol/L (ka = 1.27 × 10^6 M^{-1}s^{-1}, kd = 1.62 × 10^{-3} s^{-1}) measured by SPR [1].

This study describes the preclinical characterization of S49076, a novel, potent inhibitor of MET, AXL/MER, and FGFR1/2/3. S49076 potently blocked cellular phosphorylation of MET, AXL, and FGFRs and inhibited downstream signaling in vitro and in vivo. In cell models, S49076 inhibited the proliferation of MET- and FGFR2-dependent gastric cancer cells, blocked MET-driven migration of lung carcinoma cells, and inhibited colony formation of hepatocarcinoma cells expressing FGFR1/2 and AXL.[1]

---

S49076 potently inhibits autophosphorylation of MET in H441 NSCLC cells (constitutive active) with total inhibition at 10 nM and IC50 = 2 nM from densitometric analysis; corresponding inhibition of GAB1 phosphorylation on Tyr627 was also observed [1].
In GTL-16 gastric carcinoma cells, S49076 inhibited MET phosphorylation on Tyr1349 with IC50 = 3 nM and IC90 = 10 nM by ELISA [1].
In MEFs expressing human AXL, S49076 inhibited AXL phosphorylation with IC50 = 56 nM by ELISA [1].
In SNU-16 gastric cancer cells (FGFR2-amplified), S49076 inhibited FGFR2 autophosphorylation with complete inhibition at 300 nM [1].
S49076 inhibited downstream signaling: in GTL-16 cells, inhibited phosphorylation of AKT (Ser473) and p70S6K (Thr421/Ser424) with IC50 values consistent with MET inhibition; in SNU-16 cells, inhibited phosphorylation of the adaptor protein FRS2 (Tyr436) [1].
In cell viability assays, S49076 inhibited proliferation of MET-dependent GTL-16 cells (IC50 = 3 nM) and FGFR2-dependent SNU-16 cells (IC50 = 167 nM), but had no effect on EGFR-dependent MKN-7 cells (IC50 > 10 μM) [1].
In A549 lung carcinoma cells, S49076 blocked HGF-driven cell migration with IC50 = 14 nM (scratch wound assay) [1].
In soft agar colony formation assays, S49076 inhibited anchorage-independent growth of hepatocarcinoma cell lines HLE, JHH2, JHH4, and JHH6 (which overexpress activated AXL and FGFR2) in a concentration-dependent manner, whereas little effect was seen on a cell line with low AXL expression [1].
In MDA-MB-231 breast cancer cells, siRNA knockdown confirmed that S49076 specifically inhibits AXL-mediated AKT phosphorylation upon GAS6 stimulation [1].
S49076 did not inhibit VEGFR2 phosphorylation in cell assays (HUVEC stimulated with VEGF), showing selectivity over VEGFR2 [1].

In tumor xenograft models, a good pharmacokinetic/pharmacodynamic relationship for MET and FGFR2 inhibition following oral administration of S49076 was established and correlated well with impact on tumor growth.MET, AXL, and the FGFRs have all been implicated in resistance to VEGF/VEGFR inhibitors such as bevacizumab. Accordingly, combination of S49076 with bevacizumab in colon carcinoma xenograft models led to near total inhibition of tumor growth. Moreover, S49076 alone caused tumor growth arrest in bevacizumab-resistant tumors. On the basis of these preclinical studies showing a favorable and novel pharmacologic profile of S49076, a phase I study is currently underway in patients with advanced solid tumors.
In female balb/c nu/nu mice bearing subcutaneous GTL-16 gastric carcinoma xenografts (MET-driven), oral administration of S49076 once daily for 5 days a week for 4 weeks resulted in dose-dependent tumor growth inhibition (TGI): 69% at 3.125 mg/kg, 82% at 6.25 mg/kg, 91% at 12.5 mg/kg, 95% at 25 mg/kg, and 103% at 50 mg/kg (tumor stasis/regression) [1].
In U87-MG glioblastoma xenografts (HGF-MET autocrine loop), S49076 once daily for 5 days a week for 2 weeks gave TGI of 90% at 6.25 mg/kg, 78% at 12.5 mg/kg, 103% at 25 mg/kg, and tumor regression of 68% at 50 mg/kg [1].
In SNU-16 gastric xenografts (FGFR2-driven), S49076 administered once daily (QD) for 5 days a week for up to 4 weeks resulted in TGI of 61% at 50 mg/kg, 91% at 75 mg/kg, and 100% at 100 mg/kg; twice daily (BID) dosing gave TGI of 102% at 37.5 mg/kg and 103% at 50 mg/kg [1].
In HT-29 colon carcinoma xenografts, S49076 alone (100 mg/kg QD) induced 65% TGI, bevacizumab alone (10 mg/kg IP twice weekly) induced 71% TGI, and the combination of S49076 (100 mg/kg QD) with bevacizumab (10 mg/kg twice weekly) led to near-total inhibition of tumor growth [1].
In a bevacizumab-resistant LS-174T colon carcinoma xenograft model (where tumors ceased to respond to bevacizumab after 2-3 weeks), subsequent treatment with S49076 (50 mg/kg BID) led to complete tumor growth arrest [1].
In pharmacodynamic studies, oral S49076 inhibited MET phosphorylation in GTL-16 tumors by >80% at 2 and 6 h for doses from 3.125 mg/kg; at 6.25 mg/kg, inhibition was 95% at 6 h and 58% at 16 h. Dose-dependent inhibition of AKT and p70S6K phosphorylation was also observed at 6 h. For FGFR2 in SNU-16 tumors, S49076 at 12.5 mg/kg gave 93% inhibition of FGFR2 phosphorylation at 2 h, and at 50 mg/kg maintained >80% inhibition at 6 and 16 h; parallel inhibition of FRS2 phosphorylation was confirmed [1].

The binding mode of S49076 in the ATP pocket of MET was inferred from X-ray resolution of cocrystal complexes of analogs with a C-terminal portion of MET (aa1038-1346). Real-time label-free interaction analysis (SPR) was performed using a C-terminal fragment of MET (aa956-1390) captured via His6 tag on an NTA chip at pH 7.4, 25°C in 10 mmol/L HEPES, 150 mmol/L NaCl, 0.05% P20, 50 μmol/L EDTA, and 5% DMSO. A typical cycle consisted of 200 s MET capture (5 μL/min), 90 s sample injection (30 μL/min), 600 s buffer flow (dissociation), and 180 s EDTA (350 mmol/L) regeneration. GluR2 was used as control. The on-rate (ka = 1.27 × 10^6 M^{-1}s^{-1}), off-rate (kd = 1.62 × 10^{-3} s^{-1}), and dissociation constant (KD = 1.26 × 10^{-9} mol/L) were determined [1].
Radiometric biochemical assays for inhibition of AXL, MER, and wild-type and mutated isoforms of MET and FGFRs were conducted (Millipore). IC50 values were calculated from dose-response curves [1].
The kinase-binding selectivity of S49076 was determined on the KINOMEScan panel of 442 human wild-type and mutated kinases (DiscoverRx). At 100 nmol/L, apart from MET, AXL/MER, and FGFRs, only 6% of kinases were identified as hits [1].

For cell viability assays, GTL-16 and SNU-16 cells were seeded in 96-well plates in 10% FCS medium, and 48 h later serial dilutions of S49076 were added. After 96 h (GTL-16) or 120 h (SNU-16), MTT (5 mg/mL) was added for 4 h at 37°C; formazan was solubilized in SDS (SNU-16) or DMSO (GTL-16) and OD at 540 nm measured. For MKN-7 cells, a modified propidium iodide (PI) assay was used: after 96 h treatment, PI solution (7 μg/mL) was added for 2 h at 20°C and fluorescence measured (excitation 530 nm, emission 620 nm) [1].
For migration assays, A549 cells were seeded in 96-well plates, grown to confluence, and a 1 mm gap was introduced by scraping. Cultures were incubated for 40 h with 80 ng/mL HGF and serial dilutions of S49076. Gap width was measured using a high-content cell analyzer [1].
For colony formation assays, 5-10 × 10^3 cells were seeded in agar in 24-well dishes and cultured for 8-18 days in Iscove's modified Dulbecco's medium supplemented with 40 ng/mL HGF, 20% FCS, and serial dilutions of S49076. 24 h before evaluation, vital colonies were stained with 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-phenyltetrazolium chloride, and colonies were counted with an automatic image analysis system [1].
For Western blotting and ELISA, cells were starved in 0.1% FCS for 24 h, then treated with S49076 for 2 h, with growth factors added in the final 10-20 min (HGF 20 ng/mL, FGF2 50 ng/mL, GAS6 400 ng/mL, EGF 80 ng/mL, VEGF 30 ng/mL). Cells were lysed in RIPA buffer with protease and phosphatase inhibitors. Protein extracts were resolved by SDS-PAGE, transferred to nitrocellulose, and probed with specific antibodies. Chemiluminescence was detected and quantified by densitometry. ELISAs for phosphorylated proteins were performed using Meso Scale Discovery protocols [1].

Female balb/c nu/nu or swiss nu/nu mice (Charles River) were used. S49076 hydrochloride salt was administered orally in 1% (w/v) hydroxyethylcellulose in ammonium acetate buffer pH 4.5 at a volume of 200 μL per 20 g body weight. The maximal tolerated dose was determined to be 100 mg/kg/day (5 days a week for at least 3 weeks). Bevacizumab was dissolved in PBS and administered intraperitoneally at 10 mg/kg twice weekly in a volume of 200 μL per 20 g body weight [1].
For pharmacodynamic studies, female nude balb/c nu/nu mice were subcutaneously injected with 10^7 GTL-16 or 5×10^6 SNU-16 cells. When tumors reached ~150 mm^3, mice were randomized (n=3 per group) and given a single oral dose of S49076 (0.78 to 50 mg/kg). At 2, 6, and 16 h post-treatment, tumors were excised and lysed for Western blot or ELISA. Blood and tumor concentrations of S49076 were determined by LC/MS-MS analysis [1].
For efficacy studies, mice were injected subcutaneously with GTL-16 (10^7 cells), SNU-16 (5×10^6), U87-MG (10^6 cells suspended in Matrigel), HT-29 (10^7), or LS-174T (10^7) cells. When tumors reached ~100-150 mm^3, mice were randomized into groups of 8-12 and treated orally with S49076 (3.125 to 100 mg/kg) once or twice daily for 5 days a week for up to 4 weeks. Tumor sizes were measured twice weekly with calipers, and body weights recorded. TGI was calculated as [1 - RTV(treated)/RTV(control)] × 100. Statistical analysis used two-way ANOVA with repeated measures followed by Dunnett test on log tumor volumes. Body weight loss was <5% for all groups [1].
For combination studies with bevacizumab, HT-29 tumors were treated with S49076 (50 or 100 mg/kg QD) and/or bevacizumab (10 mg/kg IP twice weekly) for 3 weeks. For bevacizumab-resistant LS-174T model, mice were treated with bevacizumab twice weekly for 2 weeks, then randomized to continued bevacizumab or switched to S49076 (50 mg/kg BID) for 3 weeks [1].

In nude mice, S49076 showed linear pharmacokinetics up to 50 mg/kg. At 2 h post-dose, blood concentrations were: 0.78 mg/kg ~10 nM, 1.56 mg/kg ~20 nM, 3.125 mg/kg ~100 nM, 6.25 mg/kg ~200 nM, 12.5 mg/kg ~400 nM, 25 mg/kg ~1 μM, 50 mg/kg ~3 μM, 100 mg/kg ~7 μM (saturated absorption and possibly saturated elimination at 100 mg/kg). The free fraction of S49076 is estimated to be 20% [1].
The half-life of S49076 in tumors was approximately 7 hours (for 3.125 mg/kg dose) versus less than 2 hours in blood. At 3.125 mg/kg, intratumor concentration remained above 100 nM for at least 16 hours following treatment [1].
For near-complete MET inhibition (P-Tyr1349) at 2 h, a dose of 3.125 mg/kg corresponding to a blood concentration of 100 nM (20 nM free) was required, consistent with in vitro IC90 of 10 nM in GTL-16 cells. For FGFR2 inhibition, a blood concentration of ~1 μM (200 nM free) gave 93% inhibition at 2 h, consistent with in vitro complete inhibition at 300 nM [1].
Numerical pharmacokinetic parameters (such as AUC, Cmax, t1/2, Vd, Cl) are provided in Supplementary Table S1 but not detailed in the main text [1].

The maximal tolerated dose (MTD) of S49076 in mice was determined to be 100 mg/kg/day (administered orally 5 days a week for at least 3 weeks) [1].
In all efficacy studies, body weight loss was less than 5% for all groups, all time points, indicating good tolerability [1].
No other toxicity data (e.g., LD50, organ toxicity, hypercalcemia) are reported [1].

https://pubmed.ncbi.nlm.nih.gov/23804704/

S49076 is an ATP-competitive tyrosine kinase inhibitor with a unique selectivity profile: it potently inhibits MET, AXL/MER, and FGFR1/2/3 but does not potently inhibit VEGFR2 in cell assays, which may avoid dose-limiting side effects related to VEGFR2 inhibition [1].
MET, AXL, and FGFRs have been implicated in primary or acquired resistance to various anticancer therapies, including EGFR inhibitors, BRAF inhibitors, chemotherapy, and anti-angiogenic agents. S49076 is proposed to be of potential benefit in cancers where these receptors are activated individually or in combination, and in resistance settings [1].
The drug showed strong synergistic or additive activity with bevacizumab in colon carcinoma xenografts and was active in a bevacizumab-resistant model, suggesting a switch from VEGFR dependency to MET/AXL/FGFR dependency [1].
Based on these preclinical studies, a phase I study of S49076 is currently underway in patients with advanced solid tumors [1].
The chemical name: 3-[(3-{[4-(4-Morpholinylmethyl)-1H-pyrrol-2-yl]methylene}-2-oxo-2,3-dihydro-1H-indol-5-yl)methyl]-1,3-thiazolidine-2,4-dione. The binding mode shows two interactions in the hinge region (oxindole moiety with Pro1158/Met1160) and a third interaction between a carbonyl group of the thiazolidinedione and the N-H backbone of Asp1222 [1].

C22H23CLN4O4S

474.960422754288

474.112

1265966-31-1

101043621

Typically exists as solid at room temperature

3

6

5

32

769

0

Cl.S1CC(N(C1=O)CC1C=CC2=C(C=1)/C(/C(N2)=O)=C/C1=CC(=CN1)CN1CCOCC1)=O

FPSAJUOIYZSJJA-NAIZSXBXSA-N

InChI=1S/C22H22N4O4S.ClH/c27-20-13-31-22(29)26(20)12-14-1-2-19-17(8-14)18(21(28)24-19)9-16-7-15(10-23-16)11-25-3-5-30-6-4-25;/h1-2,7-10,23H,3-6,11-13H2,(H,24,28);1H/b18-9-;

3-[[(3Z)-3-[[4-(morpholin-4-ylmethyl)-1H-pyrrol-2-yl]methylidene]-2-oxo-1H-indol-5-yl]methyl]-1,3-thiazolidine-2,4-dione;hydrochloride

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

---

Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

View More

Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

---

Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

View More

Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

---

Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

---

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