FGFR2 (IC50 = 1.8 nM); RET (IC50 = 3 nM); DDR2 (IC50 = 3.6 nM); PDGFRβ (IC50 = 4.1 nM); PDGFRβ (IC50 = 4.1 nM)
Fibroblast Growth Factor Receptor 1 (FGFR1) (IC₅₀=4.5 nM, Kᵢ=2.7±0.2 nM) [1]
Fibroblast Growth Factor Receptor 2 (FGFR2) (IC₅₀=1.8 nM, Kᵢ=0.68±0.07 nM) [1]
Fibroblast Growth Factor Receptor 3 (FGFR3) (IC₅₀=4.5 nM) [1]
Fibroblast Growth Factor Receptor 4 (FGFR4) (IC₅₀=34 nM) [1]
RET (IC₅₀=3 nM) [1]
DDR2 (IC₅₀=3.6 nM) [1]
FMS (CSF1-R) (IC₅₀=3.8 nM) [1]
PDGFRβ (IC₅₀=4.1 nM) [1]
LCK (IC₅₀=6.2 nM) [1]
YES (IC₅₀=7.6 nM) [1]
ARG (IC₅₀=7.9 nM) [1]
KIT (IC₅₀=8.2 nM) [1]
PDGFRα (IC₅₀=9.5 nM) [1]
QIK (IC₅₀=9.7 nM) [1]
VEGFR1 (FLT1) (IC₅₀=11 nM) [1]
SRC (IC₅₀=11 nM) [1]
ABL (IC₅₀=14 nM) [1]
EPHA1 (IC₅₀=15 nM) [1]
CSK (IC₅₀=17 nM) [1]
FGR (IC₅₀=17 nM) [1]
LYN (IC₅₀=17 nM) [1]
VEGFR2 (KDR) (IC₅₀=21 nM) [1]
VEGFR3 (FLT4) (IC₅₀=31 nM) [1]
IGFR (IC₅₀>100 nM) [1]

ARQ-087 has anti-proliferative action against cell lines with mutations, fusions, and amplifications caused by FGFR dysregulation. A positive correlation has been observed between the induction of apoptosis and ARQ 087-induced G1 cell cycle arrest in cell lines expressing high levels of FGFR2 protein. It is observed that ARQ 087 has a dose-dependent effect on FGFR1 and FGFR2 auto-phosphorylation. Using EC50 values of less than 0.123 μM, 0.185 μM, 0.463 μM, and greater than 10 μM, ARQ 087 suppresses the phosphorylation of full-length FGFR1, FGFR2, FGFR3, and FGFR4 in Cos-1 cells overexpressing them. ARQ 087 selectively inhibits both the fully active and inactive forms of FGFR kinase through an ATP competitive mechanism. Thus, by preventing FGFR from autophosphorylating and by inhibiting phosphorylated active kinase, ARQ 087 postpones FGFR activation[1].

---

Derazantinib (ARQ-087) is an ATP-competitive inhibitor that binds to both inactive and active forms of FGFRs, inhibiting their autophosphorylation and kinase activity in a dose-dependent manner [1]
- In COS-1 cells ectopically expressing FGFRs, it inhibits phosphorylation of FGFR1 (EC₅₀<0.123 μM), FGFR2 (EC₅₀=0.185 μM), FGFR3 (EC₅₀=0.463 μM), and shows weak activity against FGFR4 (EC₅₀>10 μM) [1]
- It exhibits potent anti-proliferative activity in FGFR-dysregulated cell lines: NCI-H716 (FGFR2 amp/fusion, GI₅₀=0.10 μM), SNU-16 (FGFR2 amp/fusion, GI₅₀=0.10 μM), KG-1 (FGFR1 fusion, GI₅₀=0.13 μM), KATO-III (FGFR2 amp, GI₅₀=0.20 μM), J82 (FGFR3 mutation, GI₅₀=0.30 μM), etc. [1]
- In Ba/F3 transfected cell lines dependent on FGFR fusions or isoforms, it shows anti-proliferative effects: FGFR3-BAIAP2L1 (GI₅₀=34.9 nM), FGFR2-CIT (GI₅₀=39.5 nM), TEL-FGFR2 (GI₅₀=59.8 nM), FGFR2 (GI₅₀=232 nM), FGFR1 (GI₅₀=355 nM), etc. [1]
- It inhibits the FGFR signaling pathway in FGFR-dependent cancer cells, reducing phosphorylation of downstream proteins including FRS2α, AKT, MEK, and ERK [1]
- Treatment with 0.1–1 μM Derazantinib (ARQ-087) induces G1 cell cycle arrest in NCI-H716 and SNU-16 cells in a dose- and time-dependent manner; 1 μM treatment for 72 hours increases the sub-G1 population (apoptotic cells) to 17.5% in NCI-H716 cells [1]
- In SNU-16 cells, it downregulates XIAP and upregulates cleaved PARP, activated caspase 3, and phospho-p53, indicating induction of apoptosis [1]

ARQ 087 attenuates FGFR signaling in human xenograft tumors SNU-16, resulting in a decrease in phospho-FGFR, phospho-FRS2-α, and phospho-ERK, but has no effect on the total FGFR2 protein. In FGFR2 altered xenograft tumor models with gene amplifications and fusions (SNU-16 and NCI-H716), ARQ 087 effectively inhibits tumor growth in vivo. ARQ 087 was well tolerated at doses up to 75 mg/kg and showed effectiveness in several in vivo xenograft models[1].

---

In SNU-16 xenograft models (FGFR2 amp/fusion), oral administration of Derazantinib (ARQ-087) at 75 mg/kg and 50 mg/kg (qdx14) achieves tumor growth inhibition (TGI) of 83% (p=0.002) and 69% (p=0.013), respectively, with partial and complete tumor regressions observed [1]
- In NCI-H716 xenograft models (FGFR2 amp/fusion), doses of 75 mg/kg and 50 mg/kg (qdx14) result in TGI of 96% (p=0.0001) and 68% (p=0.0001), respectively [1]
- In Ba/F3-FGFR2 xenograft models, it potently inhibits tumor growth, while showing no efficacy in Ba/F3-INSR models (non-FGFR dependent) [1]
- A single oral dose of 25–75 mg/kg reduces phosphorylation of FGFR, FRS2α, and ERK in SNU-16 xenograft tumors without affecting total FGFR2 protein levels [1]

The recombinant FGFR1 or FGFR2 proteins' kinase inhibitory activity was assessed by ARQ 087 using ATP and a biotinylated PYK2 peptide substrate. ARQ 087 was diluted ten times more in deionized water after being titrated in DMSO using a three-fold dilution scheme, resulting in a final DMSO concentration of 10%. In each well of a reaction plate, a volume (2.5 μL) of these dilutions or vehicle was added. In each well, a volume of 17.5 μL was used to add FGFR1 or FGFR2 to the assay buffer (50 mM Tris, pH 8.0, 0.02 mg/mL BSA, 10 mM MgCl2, 1 mM EGTA, 10% glycerol, 0.1 mM Na3PO4, 1 mM DTT) at a final concentration of 0.50 or 0.25 nM, respectively. ATP and substrate were added to assay buffer (5 μL) for a final reaction volume of 25 μL, with final concentrations of 0–1,000 μM ATP and 80 nM biotinylated-PYK2, following a 30-minute pre-incubation period. After 60 minutes of room temperature incubation, 10 μL of a stop/detection mixture prepared in assay buffer containing EDTA, AlphaScreen^TM Streptavidin Donor, and P-TYR-100 Acceptor beads were added to the plates to stop them in the dark. The final concentrations of EDTA were 10 mM, and the amounts of both AlphaScreen^TM Donor and Acceptor beads were 500 ng/well. The assay plates were read on a Perkin Elmer Envision Multilabel plate reader after being incubated for 60 minutes at room temperature in the dark. (wavelength of emission: 570 nm, wavelength of excitation: 640 nm). For tight-binding inhibitors, the effect of enzyme concentration was used. If the enzyme concentration was higher than the IC50 values under the used assay conditions, the IC50 values were, if needed, converted into Ki values.

---

Kinase inhibitory activity assay: Serial 3-fold dilutions of Derazantinib (ARQ-087) in DMSO are prepared and further diluted in deionized water. Recombinant FGFR1 or FGFR2 is added to assay buffer (50 mM Tris pH 8.0, 0.02 mg/mL BSA, 10 mM MgCl₂, 1 mM EGTA, 10% glycerol, 0.1 mM Na₃PO₄, 1 mM DTT) with the drug dilutions and pre-incubated for 30 minutes. ATP (0–1000 μM) and biotinylated PYK2 peptide substrate (80 nM) are added to start the reaction, which is incubated at room temperature for 60 minutes. The reaction is stopped with a mixture containing EDTA, AlphaScreen™ Streptavidin Donor, and P-TYR-100 Acceptor beads. After 60 minutes of dark incubation, signal is detected at excitation 640 nm and emission 570 nm, and Kᵢ values are calculated using DynaFit software [1]
- FGFR auto-phosphorylation assay: The assay mixture contains 100 mM Tris pH 8.0, 10 mM MgCl₂, 1 mM phosphoenolpyruvate, 0.28 mM NADH, pyruvate kinase (89 U/mL), lactate dehydrogenase (124 U/mL), and 2% DMSO. Unphosphorylated FGFR1 or FGFR2 kinase domains are incubated with various concentrations of Derazantinib (ARQ-087), and the reaction is initiated with 1 mM ATP. Inhibition of auto-phosphorylation is monitored by measuring the decrease in NADH absorption at 340 nm using a plate reader at 30°C [1]

After plating and incubating the cells for a full night at 37°C, they are subjected to a 24- or 72-hour treatment with 0.1 μM or 1 μM of ARQ 087. A FACS Calibur flow cytometer was used to analyze the cell cycle profiles after the cells were fixed, stained, and treated with the Cycletest Plus Reagent Kit.

---

Cell proliferation assay: Cells are seeded at 3000–5000 cells/well in 96-well plates and incubated overnight. Serial 3-fold dilutions of Derazantinib (ARQ-087) (starting at 100 μM) are added, and cells are cultured for 72 hours. MTS/PMS reagent is added, and after 4 hours of incubation at 37°C, absorbance is measured at 490 nM to calculate GI₅₀ values [1]
- FGFR phosphorylation assay: COS-1 cells transfected with FGFR1-4 are pre-treated with Derazantinib (ARQ-087) for 2 hours, then stimulated with FGF1/FGF2/FGF7 (100 pM) for 15 minutes. Cells are lysed, and Western blot is performed to detect phospho-FGFR and total FGFR [1]
- FGFR pathway inhibition assay: FGFR-dependent cancer cells (NCI-H716, SNU-16, KATO-III) are treated with Derazantinib (ARQ-087) for 2 hours, or pre-treated for 2 hours followed by FGF stimulation. Cell lysates are analyzed by Western blot to detect phosphorylation of FGFR, FRS2α, AKT, MEK, and ERK [1]
- Cell cycle analysis: Cells are treated with 0.1 μM or 1 μM Derazantinib (ARQ-087) for 24 or 72 hours, fixed, stained with Cycletest Plus Reagent, and analyzed by flow cytometry to determine cell cycle distribution (sub-G1, G1, S, G2/M phases) [1]
- Apoptosis-related protein assay: SNU-16 cells are treated with 1 μM Derazantinib (ARQ-087) for 0, 24, 48, or 72 hours. Western blot is used to detect XIAP, cleaved PARP, activated caspase 3, and phospho-p53, with β-actin as a loading control [1]

Mice: Female CB17 SCID mice (NCI-H716) or NCr nu/nu mice (SNU-16) with well-established (400 mg) subcutaneous tumors are given either vehicle control or a single oral dosage of derazantineb. Four hours after a single dose, samples of plasma and tumor are obtained. It is oral to administer derazantinib. For every group, the dosage volume is 10 mL/kg, or 0.1 mL/10 g of body weight.

---

Xenograft tumor model establishment: Six-week-old female NCr nu/nu mice (for SNU-16) or CB-17 SCID female mice (for NCI-H716 and BaF3 models) are acclimated for >2 weeks. Tumor cells (5×10⁶ SNU-16, 8×10⁶ NCI-H716, or 2×10⁶ BaF3) are implanted subcutaneously into the upper right flank; SNU-16 cells are suspended in HBSS, while NCI-H716 cells are in 50% Matrigel/HBSS [1]
- Drug formulation and administration: Derazantinib (ARQ-087) is formulated in DMA:cremophor EL:propylene glycol:0.2 M acetate buffer pH 5 (10:10:30:50) and administered orally at a volume of 10 mL/kg (0.1 mL/10 g body weight). Treatment starts when tumor burden reaches 100–200 mg, with dosing schedules of qdx10 or qdx14 [1]
- In vivo pharmacodynamic assay: Mice bearing ~400 mg SNU-16 or NCI-H716 tumors receive a single oral dose of Derazantinib (ARQ-087) or vehicle. Plasma and tumor samples are collected 4 hours post-dose, and immunohistochemistry (IHC) is performed to detect phospho-FGFR, phospho-FRS2α, phospho-ERK, and total FGFR2 [1]
- Tumor and body weight monitoring: Tumor size is measured 2–3 times/week with electronic calipers, and tumor weight is calculated as length×(width)²/2. Body weight is monitored regularly; mice with >20% weight loss, moribund status, or tumor size exceeding 2000 mg are euthanized by CO₂ inhalation [1]

Drazatinib (ARQ-087) at a dose of 150 mg/kg was poorly tolerated, resulting in unacceptable weight loss and generalized drowsiness [1]
- At a dose of 100 mg/kg once daily, approximately 10% weight loss was observed [1]
- At doses up to 75 mg/kg, it was well tolerated with no significant toxicity [1]

[1].Published online 2016 Sep 14.

Derazantinib is being investigated in the clinical trial NCT03230318 (Derazantinib for patients with FGFR2 gene fusion-positive, unresectable or advanced intrahepatic cholangiocarcinoma). Derazantinib is an orally bioavailable fibroblast growth factor receptor (FGFR) inhibitor with potential antitumor activity. Derazantinib binds to FGFR subtypes 1, 2 and 3 and effectively inhibits their activity. This may lead to the suppression of FGFR-mediated signal transduction pathways, tumor cell proliferation, tumor angiogenesis and death of FGFR-overexpressing tumor cells. FGFR is a receptor tyrosine kinase that is upregulated in a variety of tumor cell types and plays a key role in tumor cell proliferation, differentiation, angiogenesis and survival. Derazantinib (ARQ-087) is a novel ATP-competitive multi-kinase inhibitor that targets FGFR family members and other kinases (RET, PDGFR, VEGFR, etc.) by blocking autophosphorylation and kinase activity to inhibit the inactive and active forms of FGFR [1].
- It is currently being evaluated in a phase I/II clinical trial (NCT01752920), which includes a subgroup of patients with intrahepatic cholangiocarcinoma who have been shown to have FGFR2 gene fusions[1].
- Preclinical studies have shown that Derazantinib is effective against cancers driven by FGFR dysregulation (amplification, mutation, gene fusion), including cholangiocarcinoma, gastric cancer, etc. Bladder cancer, endometrial cancer, and leukemia[1].
- The presence of FGFR gene fusions is associated with high sensitivity to Derazantinib (ARQ-087), which is attributed to its potent inhibition of ERK1/2 activation in cells carrying the fusion gene[1].

C29H29FN4O

468.58

468.232

C, 74.34; H, 6.24; F, 4.05; N, 11.96; O, 3.41

1234356-69-4

Derazantinib Racemate;2309668-44-6;Derazantinib dihydrochloride;1821329-75-2

46834118

White to yellow solid powder

1.2±0.1 g/cm3

615.1±65.0 °C at 760 mmHg

325.8±34.3 °C

0.0±1.8 mmHg at 25°C

1.632

5.66

2

6

9

35

638

1

COCCNCCC1=CC(NC2=NC=C3C[C@@H](C4=CC=CC=C4F)C5=CC=CC=C5C3=N2)=CC=C1

KPJDVVCDVBFRMU-AREMUKBSSA-N

InChI=1S/C29H29FN4O/c1-35-16-15-31-14-13-20-7-6-8-22(17-20)33-29-32-19-21-18-26(24-10-4-5-12-27(24)30)23-9-2-3-11-25(23)28(21)34-29/h2-12,17,19,26,31H,13-16,18H2,1H3,(H,32,33,34)/t26-/m1/s1

(6R)-6-(2-fluorophenyl)-N-[3-[2-(2-methoxyethylamino)ethyl]phenyl]-5,6-dihydrobenzo[h]quinazolin-2-amine

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: ≥ 2.5 mg/mL (5.34 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 (5.34 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.)