FGFR1 (IC50 = 39.9 nM); FGFR1 (Ki = 42 nM); FGFR1 autophosphorylation (IC50 = 622 nM); PDGFRβ (IC50 = 262 nM); PDGFR (IC50 = 310 nM); EGFR (IC50 = 240 nM); c-Src (IC50 = 44 nM); TGF-β Receptor

In a dose-dependent manner, treatment with PD-161570 (Compound 6c; 0.1-1 µM; Days 1-8; VSMC) suppresses the proliferation of PDGF-stimulated vascular smooth muscle cells, with an IC50 of 0.3 µM on day 8 [1]. When human ovarian cancer cells (A121(p)) and Sf9 insect cells overexpress the human FGF-1 receptor, PD-161570 suppresses the constitutive phosphorylation of the FGF-1 receptor, preventing the A121(p) cells from growing in culture[2]. The angiogenesis mediated by basic fibroblast growth factor (bFGF) can be efficiently inhibited by PD-161570 [4].

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Through direct synthetic efforts we discovered a small molecule which is a 40 nanomolar inhibitor of the human FGF-1 receptor tyrosine kinase. 1-Tert-butyl-3-[6-(2,6-dichloro-phenyl)-2-(4-diethylamino-butylamino)-py rido[2,3-d]pyrimidin-7-yl]-urea (PD 161570) had about 5- and 100-fold greater selectivity toward the FGF-1 receptor (IC50 = 40 nM) compared with the PDGFbeta receptor (IC50 = 262 nM) or EGF receptor (IC50 = 3.7 microM) tyrosine kinases, respectively. In addition, PD 161570 suppressed constitutive phosphorylation of the FGF-1 receptor in both human ovarian carcinoma cells (A121(p)) and Sf9 insect cells overexpressing the human FGF-1 receptor and blocked the growth of A121(p) cells in culture. The results demonstrate a novel synthetic inhibitor with nanomolar potency and specificity towards the FGF-1 receptor tyrosine kinase.[2]

In Vivo Therapeutic Effects of AZD0530 and TAK 165 [3]
Next, the therapeutic effects of these drug candidates on FOP model mice were evaluated. We focused on AZD0530 and TAK 165 because they are applicable to in vivo experiments (Hennequin et al., 2006, Nagasawa et al., 2006). Previously, we generated FOP model mice that conditionally express hFOP-ACVR1 (R206H) by Dox administration and develop HO by muscle injury using cardiotoxin (CTX) (Hino et al., 2017). The intraperitoneal administration of AZD0530 or TAK 165 significantly suppressed the HO in these mice (Figures 5A–5C). In the CTX-injected site, we observed positive staining for safranin O (acidic proteoglycan, an extracellular matrix protein of chondrocytes), von Kossa (calcium deposition), and COL1 (bone marker) (Figure 5D, vehicle). On the other hand, mice administered AZD0530 or TAK 165 seemed to show less positive staining for von Kossa or COL1 (Figure 5D, AZD0530 and TAK 165). No apparent differences in body weight change was observed in mice administered AZD0530 or TAK 165 compared with vehicle (Figure 5E). These observations demonstrated that AZD0530 and TAK 165 are effective at suppressing HO in FOP model mice. These compounds' therapeutic effects were also confirmed in a BMP-7-induced HO model using WT mice (Figure S3). Furthermore, we validated whether AZD0530 and TAK 165 have the potential to suppress the HO of FOP patient-derived cells in vivo. We previously reported a human FOP-iPSC-based in vivo model (Hino et al., 2015, Hino et al., 2017). In this humanized FOP model, the transplantation of FOP-iMSCs and activin A-expressing cells into mice induces FOP patient-derived heterotopic bone in vivo. Notably, the administration of AZD0530 or TAK 165 significantly suppressed HO in these mice (Figures 6A–6C). Hypertrophic chondrocytes (based on safranin O and von Kossa staining) and von Kossa- and COL1-positive bone regions seemed to be fewer in mice administered AZD0530 or TAK 165 (Figure 6D). Because a large number of anti-human-specific vimentin-positive cells were observed in the AZD0530 and TAK 165-treated groups (Figure 6D), we could conclude that the therapeutic effect of these compounds was not due to the death of the human transplanted cells but rather the suppression of HO. In these experiments, neither AZD0530 nor TAK 165 administration decreased body weight (Figures 5E, 6E, and S3D), and the dosing used was comparable with that in previous studies (Hennequin et al., 2006, Nagasawa et al., 2006). TAK 165 in particular did not impair the chondrogenesis of normal chondrocytes (Figures S4A–S4C), normal skeletal development in vivo (Figures S4D and S4E), or wound healing in vitro (Figures S4F and S4G). Thus, we concluded the HO suppression was not primarily caused by toxicity, although further in vivo assessment might be preferable. Taken together, AZD0530 and TAK 165 are promising drug candidates since they suppressed the HO of FOP patient-derived cells in vivo in addition to the HO of FOP model mice.

Protein Tyrosine Kinase Assays [2]
Assays using the full length FGF-1, PDGFβ and EGF receptor tyrosine kinases were performed in a total volume of 100 μl containing 25 mM Hepes buffer (pH 7.4), 150 mM NaCl, 10 mM MnCl2, 0.2 mM sodium orthovanadate, 750 μg/ml of a random co-polymer of glutamic acid and tyrosine (4:1), various concentrations of inhibitor and 60–75 ng of enzyme as previously described 32, 33. The reaction was initiated by the addition of [γ-32P]ATP (50 μM ATP containing 0.4 μCi [γ-32P]ATP per incubation) and samples incubated at 25°C for 10 min. The reaction was terminated by the addition of 30% trichloroacetic acid (TCA) and the precipitation of material onto glass fiber filter mats. Filters were washed three times with 15% TCA and the incorporation of [32P] into the glutamate tyrosine polymer substrate was determined by counting the radioactivity retained on the filters in a Wallac 1250 betaplate reader. Nonspecific activity was defined as radioactivity retained on the filters following incubation of samples without enzyme. Specific activity was determined as total activity (enzyme plus buffer) minus nonspecific activity. The concentration of compound that inhibited specific enzymatic activity by 50% (IC50) was determined graphically. For determination of ATP kinetics, assay conditions were the same as above except that varying concentrations of ATP were added in the absence or presence of a single concentration of PD 161570 in order to generate ATP concentration curves. Ki determinations for PD 161570 were obtained by a non linear regression analysis to fit the inhibition data to equations which describe different types of inhibition.

Cell Proliferation Assay[1]
Cell Types: Vascular Smooth Muscle Cells (VSMC)
Tested Concentrations: 0.1 µM, 0.3 µM, 1 µM
Incubation Duration: 1 day, 3 days, 6 days, 8 days
Experimental Results: Inhibition of VSMC proliferation in a dose-dependent manner Chapter The 8-day IC50 is 0.3 µM.

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Cell Growth Assays [2]
A121(p) human ovarian carcinoma cells were plated at 10,000 cells per well in 24 well plates in 0.5 ml RPMI-1640 / 10 % FBS. After 24 hr serum supplemented medium was removed and cells were washed thoroughly and then maintained in a serum free medium as described above. PD 161570, PD 153035 or CGP 53716 were added every other day to triplicate cultures of cells together with either bFGF (25 ng/ml) or inhibitor vehicle. Cell number was measured by coulter counting on days 1, 3, 5 and 7 after drug exposure.

In Vivo Experiments [3]
hFOP-ACVR1 conditional transgenic mice (Beard et al., 2006, Hino et al., 2017, Ohnishi et al., 2014, Yamada et al., 2013), BMP-7-induced HO model mice (Hino et al., 2017), and activin A-induced HO model mice transplanted with FOP-iMSCs (Hino et al., 2017) were intraperitoneally administered 5 mg/kg AZD0530, TAK 165, or rapamycin (once daily, five times a week) and analyzed as previously described (Hino et al., 2017).

[1]. Structure-activity relationships for a novel series of pyrido[2,3-d]pyrimidine tyrosine kinase inhibitors. J Med Chem. 1997 Jul 18;40(15):2296-303.

[2]. Inhibition of FGF-1 receptor tyrosine kinase activity by PD 161570, a new protein-tyrosine kinase inhibitor. Life Sci. 1998;62(2):143-50.

[3]. An mTOR Signaling Modulator Suppressed Heterotopic Ossification of Fibrodysplasia Ossificans Progressiva. Stem Cell Reports. 2018 Nov 13;11(5):1106-1119.

[4]. Pharmacologic characterization of a kinetic in vitro human co-culture angiogenesis model using clinically relevant compounds. J Biomol Screen. 2013 Dec;18(10):1234-45.

Screening of a compound library for inhibitors of the fibroblast growth factor (FGFr) and platelet-derived growth factor (PDGFr) receptor tyrosine kinases led to the development of a novel series of ATP competitive pyrido[2,3-d]pyrimidine tyrosine kinase inhibitors. The initial lead, 1-[2-amino-6-(2,6-dichlorophenyl)pyrido[2,3-d]pyrimidin-7-yl]-3- tert-butylurea (4b, PD-089828), was found to be a broadly active tyrosine kinase inhibitor. Compound 4b inhibited the PDGFr, FGFr, EGFr, and c-src tyrosine kinases with IC50 values of 1.11, 0.13, 0.45, and 0.22 microM, respectively. Subsequent SAR studies led to the synthesis of new analogs with improved potency, solubility, and bioavailability relative to the initial lead. For example, the introduction of a [4-(diethylamino)butyl]amino side chain into the 2-position of 4b afforded compound 6c with enhanced potency and bioavailability. Compound 6c inhibited PDGF-stimulated vascular smooth muscle cell proliferation with an IC50 of 0.3 microM. Furthermore, replacement of the 6-(2,6-dichlorophenyl) moiety of 4b with a 6-(3',5'-dimethoxyphenyl) functionality produced a highly selective FGFr tyrosine kinase inhibitor 4e. Compound 4e inhibited the FGFr tyrosine kinase with an IC50 of 0.060 microM, whereas IC50s for the inhibition of the PDGFr, FGFr, EGFr, c-src, and InsR tyrosine kinases for this compound (4e) were all greater than 50 microM. [1]

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Fibrodysplasia ossificans progressiva (FOP) is a rare and intractable disorder characterized by extraskeletal bone formation through endochondral ossification. FOP patients harbor gain-of-function mutations in ACVR1 (FOP-ACVR1), a type I receptor for bone morphogenetic proteins. Despite numerous studies, no drugs have been approved for FOP. Here, we developed a high-throughput screening (HTS) system focused on the constitutive activation of FOP-ACVR1 by utilizing a chondrogenic ATDC5 cell line that stably expresses FOP-ACVR1. After HTS of 5,000 small-molecule compounds, we identified two hit compounds that are effective at suppressing the enhanced chondrogenesis of FOP patient-derived induced pluripotent stem cells (FOP-iPSCs) and suppressed the heterotopic ossification (HO) of multiple model mice, including FOP-ACVR1 transgenic mice and HO model mice utilizing FOP-iPSCs. Furthermore, we revealed that one of the hit compounds is an mTOR signaling modulator that indirectly inhibits mTOR signaling. Our results demonstrate that these hit compounds could contribute to future drug repositioning and the mechanistic analysis of mTOR signaling. [3]

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Angiogenesis, the formation of new vessels from preexisting vessels, involves multiple cell types acting in concert to cause endothelial cell proliferation, migration, and differentiation into microvascular arrays. Under pathologic conditions, microenvironment changes result in altered blood vessel production. Historically, in vitro angiogenesis assays study individual aspects of the process and tend to be variable, difficult to quantify, and limited in clinical relevance. Here, we describe a kinetic, quantitative, co-culture angiogenesis model and demonstrate its relevance to in vivo pharmacology. Similar to in vivo angiogenesis, a co-culture of human umbilical vein endothelial cells with normal human dermal fibroblasts remains sensitive to multiple cytokines, resulting in a concentration-dependent stimulation of tube formation over time. Treatment with axitinib, a selective vascular endothelial growth factor (VEGF) antagonist, inhibited VEGF-mediated tube length and branch point formation and was selective for inhibiting VEGF over basic fibroblast growth factor (bFGF), similar to previous studies. Conversely, an FGFR-1 selective compound, PD-161570, was more potent at inhibiting bFGF-mediated angiogenesis. These results demonstrate the cytokine dynamics, selective pharmacology, and translational application of this model system. Finally, combining quantitative angiogenic biology with kinetic, live-content imaging highlights the importance of using validated in vitro models in drug discovery research. [4]

C26H35CL2N7O

532.514

531.228

C, 58.64; H, 6.63; Cl, 13.31; N, 18.41; O, 3.00

192705-80-9

5328135

Light yellow to yellow solid powder

157 °C

6.151

3

6

11

36

665

0

MKVMEJKNLUWFSQ-UHFFFAOYSA-N

InChI=1S/C26H35Cl2N7O/c1-6-35(7-2)14-9-8-13-29-24-30-16-17-15-18(21-19(27)11-10-12-20(21)28)23(31-22(17)32-24)33-25(36)34-26(3,4)5/h10-12,15-16H,6-9,13-14H2,1-5H3,(H3,29,30,31,32,33,34,36)

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

PD-161570; 192705-80-9; PD-161,570; PD 161,570; N-[6-(2,6-Dichlorophenyl)-2-[[4-(diethylamino)butyl]amino]pyrido[2,3-d]pyrimidin-7-yl]-N'-(1,1-dimethylethyl)urea; pd161,570; CHEMBL45827; 1-(tert-butyl)-3-(6-(2,6-dichlorophenyl)-2-((4-(diethylamino)butyl)amino)pyrido[2,3-d]pyrimidin-7-yl)urea; 1-tert-butyl-3-[6-(2,6-dichlorophenyl)-2-[4-(diethylamino)butylamino]pyrido[2,3-d]pyrimidin-7-yl]urea; PD161570; PD 161570

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 : ~33.33 mg/mL (~62.59 mM)

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.

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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).

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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)

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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).

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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

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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.

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