FIIN-3 exhibits strong inhibition of both gatekeeper mutants of FGFR2 (EC50 = 64 nM) and WT FGFR (EC50 = 1 to 41 nM). Also, EGFR is significantly inhibited by FIIN-3, with an EC50 of 43 nM. The gatekeeper mutant V564F was effectively inhibited by FIIN-3, while the gatewaykeeper-plus-1 mutant E565K was also successfully targeted by FIIN-3. Furthermore, Ba transformed with EGFR vIII fusion protein (containing the WT EGFR kinase domain) /F3 cells demonstrate antiproliferative activity (EC50 of 135 nM). FIIN-3 exhibited moderate action against the EGFR mutant L858R/T790M mutant, with an EC50 of 231 nM, and greater activity against the EGFR mutant L858R (EC50 of 17 nM). Even at dosages as low as 3 nM, FIIN-3 totally blocked FGFR2 autophosphorylation on Tyr656/657 in WT FGFR2 Ba/F3 cells. FIIN-3 has the ability to partially inhibit FGFR2 mutant V564M autophosphorylation in FGFR2 V564M Ba/F3 cells, and to completely inhibit it at 300 nM [1].

FIIN-3 exhibits strong inhibition of both gatekeeper mutants of FGFR2 (EC50 = 64 nM) and WT FGFR (EC50 = 1 to 41 nM). Also, EGFR is significantly inhibited by FIIN-3, with an EC50 of 43 nM. The gatekeeper mutant V564F was effectively inhibited by FIIN-3, while the gatewaykeeper-plus-1 mutant E565K was also successfully targeted by FIIN-3. Furthermore, Ba transformed with EGFR vIII fusion protein (containing the WT EGFR kinase domain) /F3 cells demonstrate antiproliferative activity (EC50 of 135 nM). FIIN-3 exhibited moderate action against the EGFR mutant L858R/T790M mutant, with an EC50 of 231 nM, and greater activity against the EGFR mutant L858R (EC50 of 17 nM). Even at dosages as low as 3 nM, FIIN-3 totally blocked FGFR2 autophosphorylation on Tyr656/657 in WT FGFR2 Ba/F3 cells. FIIN-3 has the ability to partially inhibit FGFR2 mutant V564M autophosphorylation in FGFR2 V564M Ba/F3 cells, and to completely inhibit it at 300 nM [1].

---

FIIN-3 potently inhibited the proliferation of Ba/F3 cells engineered to be dependent on wild-type (WT) FGFR1, FGFR2, FGFR3, or FGFR4 kinase activity, with EC₅₀ values in the single- to double-digit nanomolar range, and was especially potent against FGFR2 (EC₅₀ in the 1 nM range). [1]
FIIN-3 inhibited the proliferation of Ba/F3 cells transformed by the EGFR vIII fusion protein (EC₅₀ = 135 nM), EGFR L858R mutant (EC₅₀ = 17 nM), and the EGFR L858R/T790M double mutant which is resistant to first-generation EGFR inhibitors (EC₅₀ = 231 nM). [1]
FIIN-3 potently inhibited the proliferation of Ba/F3 cells dependent on the FGFR2 V564M gatekeeper mutant (EC₅₀ = 64 nM), whereas the first-generation inhibitor BGJ398 had an EC₅₀ of over 1.0 μM against this mutant. It also showed good potency against the FGFR2 V564F gatekeeper mutant and the E565K mutant. [1]
FIIN-3 exhibited antiproliferative activity against a panel of cancer cell lines harboring FGFR alterations, including H2077 and H1581 (FGFR1-amplified NSCLC, EC₅₀ = 5.3 nM and 2.5 nM, respectively), Kato III (FGFR2-amplified gastric carcinoma, EC₅₀ = 2.5 nM), AN3 CA (FGFR2 N549K-mutant endometrial adenocarcinoma, EC₅₀ = 26.2 nM), RT112 (FGFR3/TACC3 fusion bladder carcinoma, EC₅₀ = 15.9 nM), and A2780 (FGFR4-amplified ovarian carcinoma, EC₅₀ = 0.01 nM). [1]
FIIN-3 was active against cancer cell lines engineered to express the FGFR1 V561M gatekeeper mutation (H2077 V561M EC₅₀ = 1.4 nM; H1581 V561M EC₅₀ = 11.8 nM), overcoming resistance observed with BGJ398. [1]
FIIN-3 inhibited the proliferation of the SKOV-3 ovarian carcinoma cell line (which overexpresses both EGFR and FGFR) with an EC₅₀ of 499 nM, and was more potent than FIIN-2 or BGJ398 in this setting. Its activity was further evaluated in the presence of exogenous FGF or EGF ligands. [1]
In cellular wash-out experiments using Ba/F3 cells expressing WT FGFR2, treatment with FIIN-3 followed by extensive washing resulted in sustained inhibition of FGFR2 autophosphorylation, confirming its irreversible, covalent mechanism of action. This sustained inhibition was not observed with the reversible inhibitor BGJ398. [1]
FIIN-3 inhibited FGFR2 autophosphorylation and downstream signaling proteins (p-FRS2, p-AKT, p-ERK1/2) in FGFR2 V564M Ba/F3 cells, with complete inhibition observed at 300 nM. [1]
In H1581 cells expressing the FGFR1 V561M gatekeeper mutant, FIIN-3 inhibited autophosphorylation of FGFR1 V561M in a dose-responsive manner, with significant inhibition at 333 nM. [1]
In SKOV-3 cells, FIIN-3 (1.0 μM) uniquely inhibited phosphorylation of both EGFR and FGFR, and prevented the restoration of p-AKT and p-ERK1/2 signaling following FGF1 stimulation, unlike BGJ398 or FIIN-2. [1]
In a 3D microfluidic dispersion assay, FIIN-3 (1.0 μM) suppressed FGF1-induced dispersal of SKOV-3 cell spheroids and, uniquely among the tested compounds, also blocked EGF-induced dispersal. [1]

The in vivo efficacy of FIIN-3 was examined using a zebrafish (Danio rerio) embryonic developmental model. Treatment of embryos from 2 hours post-fertilization (hpf) with 25 μM FIIN-3 caused defects in posterior mesoderm and tail morphogenesis, phenotypes consistent with inhibition of FGFR signaling. The efficiency was reported to be lower than that of BGJ398 (tested at 5.0 μM) but higher than that of AZD4547 and PD173074. [1]

The kinase selectivity profile of FIIN-3 was assessed using an in vitro ATP-site competition binding assay (KinomeScan) against a diverse panel of 456 kinases at a concentration of 1.0 μM. FIIN-3 displayed strong binding to FGFRs and EGFR and exhibited good overall kinase selectivity with a selectivity score (S(1)) of 15. [1]
The IC₅₀ values of FIIN-3 against purified kinase domains were determined using a Z’-lyte enzyme activity assay. FIIN-3 inhibited FGFR1, FGFR2, FGFR3, and FGFR4 with IC₅₀s of 13, 21, 31, and 35 nM, respectively. It inhibited EGFR with an IC₅₀ of 43 nM. It also inhibited the FGFR1 V561M gatekeeper mutant with an IC₅₀ of 109 nM in this biochemical assay. [1]

For cell viability assays, TEL-FGFR2-transformed Ba/F3 cells or other kinase-transformed Ba/F3 cells were seeded in 96-well plates and treated with serially diluted compounds. After 72 hours (or 96 hours for cancer cell lines), cell viability was assessed using an MTS tetrazolium assay (for Ba/F3) or CellTiter-Glo luminescent cell viability assay (for cancer cell lines). Dose-response curves were generated and EC₅₀ values calculated using GraphPad Prism software. [1]
For immunoblotting (Western blot) analysis to assess signaling pathway inhibition, cells were treated with inhibitors under specified conditions (e.g., for 6 or 12 hours, with or without serum starvation/growth factor stimulation). Cells were then lysed, proteins were separated by SDS-PAGE, transferred to membranes, and probed with specific antibodies against total and phosphorylated forms of target proteins (e.g., p-FGFR, p-FRS2, p-AKT, p-ERK1/2, p-EGFR). [1]
Cellular wash-out experiments were performed to confirm covalent inhibition. Cells were incubated with inhibitors for 3 hours, washed extensively with PBS, then maintained in fresh medium for 4 hours before harvesting for immunoblotting analysis to check for sustained target inhibition. [1]
For the 3D dispersion assay, SKOV-3 cells were first cultured to form spheroids. Spheroids of defined size were embedded in a collagen gel within a microfluidic device. Medium containing FGF1 or EGF, with or without inhibitors, was perfused through channels flanking the gel. Spheroid dispersal (cell migration out of the spheroid) was monitored over 48 hours using microscopy, and the extent of inhibition was quantified. [1]

Zebrafish embryo study: Wild-type zebrafish (TübingeB strain) embryos were collected and incubated at 28°C. At 2 hours post-fertilization (hpf), 15 embryos were placed per well in a 24-well plate containing 1 mL of E3 embryo medium. FIIN-3 (or vehicle control DMSO, or other FGFR inhibitors) was added to the medium to a final concentration of 25 μM. The treated embryos were incubated at 28°C until 50 hpf, at which point they were scored for phenotypic defects in posterior mesoderm and tail morphogenesis. Images were captured using a dissecting microscope. [1]

[1]. Development of covalent inhibitors that can overcome resistance to first-generation FGFR kinase inhibitors. Proc Natl Acad Sci U S A, 2014 Nov 11, 111(45):E4869-77.

N-[4-[[[(2,6-dichloro-3,5-dimethoxyaniline)-oxymethyl]-[6-[4-(4-methyl-1-piperazinyl)aniline]-4-pyrimidinyl]amino]methyl]phenyl]-2-acrylamide belongs to the piperazine class of compounds. FIIN-3 is a new generation of irreversible (covalent) fibroblast growth factor receptor (FGFR) inhibitors. It was developed using a structure-based drug design approach to overcome resistance to first-generation FGFR inhibitors (such as NVP-BGJ398 and AZD4547) caused by gating mutations (e.g., V564M in FGFR2 and V561M in FGFR1). [1]
One unique feature of FIIN-3 is that it can act as a dual covalent inhibitor of FGFR and EGFR by targeting different cysteine residues in the ATP-binding pocket of FGFR and epidermal growth factor receptor (EGFR) with a single electrophilic acrylamide group (Cys477 in the P ring of FGFR4; Cys797 in EGFR). This is the first reported kinase inhibitor with this capability. [1]
The cocrystal structure of FIIN-3 with FGFR4 V550L and EGFR L858R reveals its binding mode. In FGFR4, FIIN-3 induces a unique “DFG-out” inactive conformation, which is stabilized by covalent binding to Cys477 and the resulting π-π stacking interaction. In EGFR, FIIN-3 binds in a “DFG-in” conformation and is covalently linked to Cys797. Its 4-acrylamidobenzyl group is flexible enough to adapt to different binding pockets. [1] The corresponding non-covalent analog FIIN-3 (FRIN-3) exhibits reduced inhibitory activity against both EGFR and FGFR-gated mutants, highlighting the necessity of covalent formation for its potent dual inhibitory activity, particularly against EGFR and its resistant mutants. [1] The dual inhibition of FGFR and EGFR by FIIN-3 is considered a strategy to overcome potential bypass signaling and resistance mechanisms that may arise when targeting only one pathway in cancers involving both receptors. [1]

C34H36CL2N8O4

691.606844902039

690.223

1637735-84-2

73707531

White to off-white solid powder

1.4±0.1 g/cm3

909.4±65.0 °C at 760 mmHg

503.8±34.3 °C

0.0±0.3 mmHg at 25°C

1.683

5.65

3

9

11

48

1020

0

SFLKJNSBBVSPFE-UHFFFAOYSA-N

InChI=1S/C34H36Cl2N8O4/c1-5-30(45)40-24-8-6-22(7-9-24)20-44(34(46)41-33-31(35)26(47-3)18-27(48-4)32(33)36)29-19-28(37-21-38-29)39-23-10-12-25(13-11-23)43-16-14-42(2)15-17-43/h5-13,18-19,21H,1,14-17,20H2,2-4H3,(H,40,45)(H,41,46)(H,37,38,39)

N-(4-((3-(2,6-dichloro-3,5-dimethoxyphenyl)-1-(6-((4-(4-methylpiperazin-1-yl)phenyl)amino)pyrimidin-4-yl)ureido)methyl)phenyl)acrylamide

FIIN3; FIIN 3; FIIN-3.

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 : ~10 mg/mL (~14.46 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (3.61 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 25.0 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: 2.5 mg/mL (3.61 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 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: ≥ 2.5 mg/mL (3.61 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

---

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