FGFR1 (IC50 = 25 nM); VEGFR2 (IC50 = 100 nM)

PD173074 is an FGFR1 ATP-competitive inhibitor with a Ki of approximately 40 nM. Another potent VEGFR2 inhibitor is PD173074. With 1000-fold or higher IC50 values, PD173074 weakly inhibits the activities of Src, InsR, EGFR, PDGFR, MEK, and PKC in comparison to FGFR1. With an IC50 of 1–5 nM and 100–200 nM, respectively, PD173074 inhibits the autophosphorylation of FGFR1 and VEGFR2 in a dose-dependent manner.[1] With an IC50 of 12 nM, PD173074 exhibits dose-dependent inhibition of FGF-2 promotion of granule neuron survival, 1,000 times more potent than SU 5402.[2] In oligodendrocyte (OL) lineage cells, PD173074 specifically blocks the effects of FGF-2 on proliferation, differentiation, and MAPK activation.[3] PD173074 exhibits efficacy against both FGFR3 mutations and the WT receptor in multiple myeloma (MM) cell lines. Moreover, PD173074 has an IC50 of approximately 5 nM and strongly suppresses the dose-dependent autophosphorylation of FGFR3. With an IC50 of less than 20 nM, PD173074 treatment significantly lowers the viability of FGFR3-expressing KMS11 and KMS18 cells. The expression of FGFR3 is closely associated with PD173074's inhibition of aFGF-stimulated MM cell growth. Treatment with PD173074 totally eliminates NIH 3T3 transformation caused by Y373C FGFR3, but not Ras V12. This indicates that PD173074 does not have a non-specific cytotoxic effect and instead targets FGFR3-mediated cell transformation. KMS11 and KMS18 cells are also induced to functionally mature by PD173074.[4]

PD173074 can be administered to mice at a dose-dependent rate of either 1 mg/kg/day or 2 mg/ka/day to effectively block angiogenesis induced by VEGF or FGF, with no apparent toxicity.[1] PD173074 prevents NIH 3T3 cells transfected with mutant FGFR3 from growing in vivo in nude mice. In a KMS11 xenograft myeloma model, inhibiting FGFR3 with PD173074 slows the growth of tumors and improves mice survival.[4] Compared to control sham-treated animals, oral administration of PD173074 in the H-510 xenograft increases median survival by blocking tumor growth in a manner similar to that observed with single-agent cisplatin administration. Fifty percent of the mice receiving PD173074 in H-69 xenografts experience full responses that last longer than six months. These effects do not result from disruption of the tumor vasculature; rather, they are correlated with increased apoptosis in removed tumors.[5]

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Oral administration of PD173074 inhibits H510 and H69 tumor growth and potentiates cisplatin effects in nude mice. [5]
[18F]FLT-PET is an early predictor of response to PD173074 in vivo[5]
To determine if we could predict response to PD173074 using an in vivo imaging technique applicable to patients in the clinic, we next used [18F]FLT-PET to monitor intratumoral proliferation. Animals bearing subcutaneous H69 xenografts in the neck were given diluent with or without PD173074 by oral gavage daily and injected with [18F]FLT-PET before imaging at day 8 and 14. Figure 5A shows representative [18F]FLT-PET imaging from one control and one PD173074-treated animal before and 14 days into administration of the treatment. Analysis of [18F]FLT-PET results by the fractional retention time, a parameter independent of tumor size, and less dependent on perfusion, showed that PD173074 administration reduced cellular proliferation (Fig. 5B). In the same tumors, growth inhibition was shown by calliper measurements (Fig. 5A , bottom). This suggests that [18F]FLT-PET might provide a noninvasive way to predict early tumor response in patients treated with an agent like PD173074.

In assays with the full-length FGFR-1 kinase, a total volume of 100 μL is used. It contains the following concentrations: 750 μg/mL of a random copolymer of glutamic acid and tyrosine (4:1), different concentrations of PD173074, 60 to 75 ng of enzyme, and 150 mM NaCl, 10 mM MnCl₂, 0.2 mM sodium orthovanadate. [γ-³²P]ATP (5 μM ATP containing 0.4 μCi of [γ-³²P]ATP per incubation) is added to start the reaction, and samples are incubated for 10 minutes at 25°C. Thirty percent trichloroacetic acid is added to stop the reaction, and the material precipitates onto glass-fiber filter mats. The incorporation of [³²P] into the glutamate tyrosine polymer substrate is measured by counting the radioactivity retained on the filters in a Wallac 1250 betaplate reader after the filters are cleaned three times with 15% trichloroacetic acid. The radioactivity that remains on the filters after samples without enzyme are incubated is known as nonspecific activity. Total activity (enzyme plus buffer) less nonspecific activity is the formula for calculating specific activity. An IC50 chart is used to calculate the concentration of PD173074 that inhibits FGFR-1 enzymatic activity by 50%.

There has been prior description of an NIH 3T3 cell line that overexpresses VEGFR2 (Flk-1). Additionally, this cell line naturally expresses FGFR1. In 10 cm² plates, 1×10⁶ cells grown in DMEM enhanced with 10% calf serum are seeded and given 48 hours to grow. The cells are then put in starvation medium (DMEM with 0.1% calf serum) to become quiescent after the medium is removed. PD 173074 prepared in starvation medium is added to the cells at different concentrations and incubated for 5 minutes after 18 hours. Next, for five minutes at 37°C, the cells are stimulated with a growth factor [100 ng/mL of VEGF or 100 ng/mL of aFGF and 10 µg/mL of heparin]. Following an ice-cold PBS wash, the cells are lysed in 1 mL of lysis buffer that contains phosphatase inhibitor (0.2 mM Na₃VO₄) and contains 25 mM HEPES pH 7.5, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 1 mM EGTA, 1.5 mM MgCl₂, 1 mM PMSF, and 10 µg/mL aprotonin and 10 µg/mL leupeptin. Cell lysates are immunoprecipitated using FGFR1 antibodies for FGFR1 inhibition studies. Phosphotyrosine-specific antibodies are then used for SDS-PAGE and immunoblotting analysis. Cell lysates (20 µL) are subjected to direct SDS-PAGE analysis and immunoblotting using phosphotyrosine-specific antibodies in order to study the inhibition of VEGFR2.

Subcutaneous inoculation with 3×10⁵ NIH 3T3 cells expressing Y373C FGFR3 and Ras V12 is performed on six-week-old athymic nude mice. The intraperitoneal injection of 0.05 mol/L lactic acid buffer or 20 mg/kg PD173074 is started on the day of the tumor injection and is administered for nine days. For every experiment, ten mice are used.

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Xenografts and immunohistochemistry[5]
H510 (1:1 cell suspension; Matrigel) or H69 cells were implanted into the flank of nude mice. When tumors became measurable, 50 mg/kg PD173074/mice or equivalent volume of buffer alone were administered daily for 14 or 28 d. In addition, mice received or did not receive two doses of 5 mg/kg cisplatin. Tumor volume was monitored using a calliper. Animals were sacrificed when tumor burden reached 15 mm in any dimension and survival recorded as a Kaplan-Meier plot. Tissues were formalin fixed and paraffin embedded before staining as indicated in the figure legends. For the endomucin experiments, pictures were acquired using a ×10 objective and analyzed using ImageJ. For activated Caspase 3 and cytokeratin 18 scoring, the number of positive cells in five high-power field views/tumor (five tumors per condition) was determined and results represented as bar graphs (Fig. 5C , bottom). The total number of nuclei per field was determined by manual counting using event flagging in Metamorph. Nuclei partly outside the field of view were excluded.

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[18F]FLT-PET imaging[5]
Animals with subcutaneous H-69 xenografts in the neck were used when the tumors reached ∼150 mm3. The tumor-bearing mice were given vehicle or PD173074 once daily by oral gavage and imaged with [18F]FLT-PET on days 0, 7, and 14 of treatment. Dynamic [18F]FLT-PET studies were carried out on a dedicated small animal PET scanner, quad-HIDAC (Oxford Positron Systems; ref. 15). Scanning was performed as previously described (16). [18F]FLT (80–100 μCi; 2.96–3.7 MBq) was injected into the tail veins of anesthetized mice positioned prone within the scanner. Dynamic scans were acquired in list-mode format over a 60-min period and sorted into 0.5-mm sinogram bins and 19 time frames (0.5 × 0.5 × 0.5 mm voxels; 4 × 15 s, 4 × 60 s, and 11 × 300 s) for image reconstruction. Cumulative images comprising of 30 to 60 min of the dynamic data were used for visualization of radiotracer uptake and to draw regions of interest. Regions of interest were defined on five tumor and five heart slices (each was 0.5 mm thick). Dynamic data from these slices were averaged for each tissue and at each of the 19 time points to obtain time versus radioactivity curves for these tissues. Tumor radioactivity was corrected for physical decay and normalized to that of heart to obtain a standardized uptake value. The fractional retention of tracer was calculated as the normalized uptake in tumors 60 min relative to that at 1.5 min

[1]. EMBO J . 1998 Oct 15;17(20):5896-904.

[2]. J Neurochem . 2000 Oct;75(4):1520-7.

[3]. J Neurosci Res . 2003 Nov 15;74(4):486-93.

[4]. Blood . 2004 May 1;103(9):3521-8.

[5]. Cancer Res . 2009 Nov 15;69(22):8645-51.

[6]. Int J Gynecol Cancer . 2012 Nov;22(9):1517-26.

PD173074 is a member of the class of ureas that is 1-tert-butylurea in which one of the hydrogens attached to N(3) is substituted by a pyrido[2,3-d]pyrimidin-7-yl group, which is itself substituted at positions 2 and 6 by a 4-(diethylamino)butyl]amino group and a 3,5-dimethoxyphenyl group, respectively. It is a FGF/VEGF receptor tyrosine kinase inhibitor. It has a role as a fibroblast growth factor receptor antagonist, an antineoplastic agent and an EC 2.7.10.1 (receptor protein-tyrosine kinase) inhibitor. It is a pyridopyrimidine, a member of ureas, a tertiary amino compound, a dimethoxybenzene, an aromatic amine and a biaryl. It is functionally related to a PD-166866.

C28H41N7O3

523.67

523.327

C, 64.22; H, 7.89; N, 18.72; O, 9.17

219580-11-7

1401

Off-white to yellow solid powder

1.2±0.1 g/cm3

82-85°C

1.599

3.33

3

8

13

38

690

0

O=C(NC1=NC2=NC(NCCCCN(CC)CC)=NC=C2C=C1C3=CC(OC)=CC(OC)=C3)NC(C)(C)C

DXCUKNQANPLTEJ-UHFFFAOYSA-N

InChI=1S/C28H41N7O3/c1-8-35(9-2)13-11-10-12-29-26-30-18-20-16-23(19-14-21(37-6)17-22(15-19)38-7)25(31-24(20)32-26)33-27(36)34-28(3,4)5/h14-18H,8-13H2,1-7H3,(H3,29,30,31,32,33,34,36)

1-tert-butyl-3-[2-[4-(diethylamino)butylamino]-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-7-yl]urea

PD 173074; PD-173074; 219580-11-7; 1-(tert-Butyl)-3-(2-((4-(diethylamino)butyl)amino)-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-7-yl)urea; 1-tert-butyl-3-[2-[4-(diethylamino)butylamino]-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-7-yl]urea; MFCD08705327; 1-tert-butyl-3-[2-{[4-(diethylamino)butyl]amino}-6-(3,5-dimethoxyphenyl)pyrido[2,3-d]pyrimidin-7-yl]urea; PD173074

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 (4.77 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.

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Solubility in Formulation 2: ≥ 2.5 mg/mL (4.77 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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (3.97 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.

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Solubility in Formulation 4: 5% DMSO+corn oil: 15mg/mL

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