S1P2 (Sphingosine-1-Phosphate 2; EDG-5); human S1P2 (IC50 = 17 nM); rat S1P2 (IC50 = 22 nM)

JTE-013 lowers cell viability at 50-200 μM for 1-3 days[1].
JTE-013 prevents S1P-induced ERK activation and reverses S1P-induced Akt inhibition[1].
JTE-013 exhibits a 4.2% inhibition of S1P3 and, at concentrations up to 10 μM, does not agitate S1P1[1].

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AB1 versus JTE-013 on GB Cell Migration. [1]
A well described biologic consequence of S1P2 signal transduction is inhibition of cell migration (Lepley et al., 2005). To compare the efficiency of JTE-013 and AB1 as S1P2 antagonists, cell migration assays were performed. GB cell lines U118 and U87 were used since it is well known that S1P inhibits cell migration in S1P2 high-expressing U118 cells, whereas S1P induces cell migration in S1P2 low-expressing U87 cells (Lepley et al., 2005; Fig. 3, A and C). AB1 treatment was moderately more potent than JTE-013 in reversing S1P-mediated cell migration inhibition in U118 cells and enhancing S1P-stimulated cell migration in U87 cells via blocking S1P2 signaling (Fig. 3, B and D).

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AB1 versus JTE-013 on the Levels of Molecules Downstream of S1P2 in SK-N-AS Cells.[1]
S1P2 exerts diverse cellular functions by regulating different downstream effector molecules. Our prior studies as well as studies performed by others have shown that such molecules include intracellular signaling mediators (p-Akt, p-ERK), as well as growth and differentiation modulators such as CTGF (Sanchez et al., 2007; Li et al., 2008a). To further compare the efficiency of JTE-013 and AB1 in vitro, Western blot analysis was performed. Similar to JTE-013, AB1 was able to reverse S1P-induced Akt inhibition and inhibit S1P-induced ERK activation at concentrations between 100 nM and 1 μM (Fig. 4A). The quantitative real-time polymerase chain reaction further showed that AB1 was relatively more effective than JTE-013 at inhibiting S1P-induced CTGF mRNA expression (Fig. 4B).

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AB1 versus JTE-013 on Cell Viability in SK-N-AS Cells.[1]
To investigate the potential mechanisms of AB1’s tumor inhibitory effect, cell viability was assessed in SK-N-AS cells treated with JTE-013 or AB1. MTT assays showed that AB1 is less potent than JTE-013 in terms of reduced cell viability at concentrations higher than 50 μM in SK-N-AS cells, whereas it had similar potency at lower concentrations (Fig. 6), suggesting that the improved inhibitory effect elicited by AB1 is not caused by direct inhibition of cell survival on cancer cells.

JTE-013 (gavage; 30 mg/kg daily for 14 days in a row) decreases tumor weight and size[1].

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AB1 versus JTE-013 on a SK-N-AS Cell–Based NB Xenograft Model. [1]
In prior study, JTE-013 was demonstrated to significantly inhibit the growth of NB xenografts (Li et al., 2011). Here it was found that AB1 was again somewhat more potent than JTE-013 in inhibiting the growth of NB xenografts by both tumor size (Fig. 5A) and tumor weight at 14 days after treatment (Fig. 5B). Taken together, the above data strongly suggest that AB1 may have enhanced in vivo antitumor activity compared with JTE-013.

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AB1 versus JTE-013 on CCL2 Expression and Tumor-Associated Macrophage Infiltration in NB Xenografts.[1]
To elucidate the mechanism of AB1’s enhanced antitumor effect, researchers quantified effects on the gene expression levels of several S1P2 downstream molecules in treated NB xenografts. CCL2 is one of these genes (Li et al., 2014). Expression of CCL2 has been shown to be positively correlated with tumor-associated macrophage (TAM) infiltration (Zhang et al., 2010). In our prior study, blockade of S1P2 signaling by JTE-013 not only inhibited the growth of NB xenografts (Li et al., 2011), but it also reduced CCL2 expression and the subsequent TAM infiltration (Li et al., 2014), suggesting that inhibition of CCL2 is beneficial to anticancer therapy. As expected, CCL2 expression tended to be decreased at both mRNA and protein levels in JTE-013–treated or AB1-treated NB xenografts (Fig. 7, A and B). Furthermore, immunohistochemical staining for the murine macrophage marker F4/80 showed that both JTE-013 and AB1 significantly inhibited the TAM infiltration. However, AB1 did not display any improved inhibitory effects (Fig. 7C), indicating that the enhanced antitumor effect of AB1 was not attributable to effects on CCL2 expression and subsequent TAM infiltration.

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AB1 versus JTE-013 on Tumor Fibrosis and Apoptosis in NB Xenografts.[1]
CTGF is a central mediator of fibrosis (Lipson et al., 2012). Interestingly, AB1 inhibited CTGF expression to a greater extent than JTE-013 at both the mRNA and protein levels (Fig. 8), suggesting that the improved antitumor effect of AB1 may be partially a result of its beneficial effects on tumor fibrosis. Histologic studies utilizing Ki67 staining did not find any significant difference in the number of Ki67-positive proliferating cells among the three groups (Supplemental Fig. 2). However, terminal deoxynucleotidyl transferase–mediated digoxigenin-deoxyuridine nick-end labeling staining (Fig. 9A) and cleaved caspase-3 detection (Fig. 9B) showed that AB1 was more potent than JTE-013 at inducing tumor cell apoptosis on these NB xenografts. Taken together, our data suggest that the improved antitumor effect elicited by AB1 may be attributed to effects on tumor fibrosis and tumor apoptosis in NB xenografts.

Fluorescent Imaging Plate Reader Assay.[1]
The calcium flux assay on a FLIPRTETRA instrument [fluorescent imaging plate reader (FLIPR) assay] was performed by a CRO company to profile test compounds for dose-dependent agonist and antagonist activities on S1P1–5. Briefly, the agonist assay was conducted on a FLIPRTETRA instrument, in which the test compounds, vehicle controls, and the reference agonist S1P were added to the assay plate after a fluorescence baseline was established. A duration of 180 seconds was used to assess each compound’s ability to activate each S1PR. Upon completion of the agonist assay, the assay plate was removed from the FLIPRTETRA instrument and incubated at 25°C for 7 minutes. After that, the assay plate was placed back in the FLIPRTETRA instrument and the antagonist assay was initiated. Using EC80 potency values determined during the agonist assay, all preincubated sample compound wells were challenged with EC80 concentration of the reference agonist S1P after establishment of a fluorescence baseline. Another duration of 180 seconds was used to assess each compound’s ability to inhibit each S1PR. All assay plate data were subjected to appropriate baseline corrections. After baseline corrections were applied, maximum fluorescence values were exported and data were processed to calculate the percentage of activation (relative to Emax reference agonist S1P and vehicle control values) and the percentage of inhibition (relative to EC80 and vehicle control values). All dose-response curves were generated using GraphPad Prism software

Cell Line: SK-N-AS cells
Concentration: 50, 100, 150, 200 μM
Incubation Time: 1-3 days
Result: Reduced cell viability.

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Migration Assay. [1]
The migration assay was performed in a 96-well chemotaxis microchamber), as described previously (Li et al., 2009b). Briefly, a polycarbonate filter (8-µm pore size) was coated with 50 µg/ml fibronectin. S1P was diluted and added into the lower chamber at 85 µl per well. GB cells were serum starved for 2 hours prior to trypsinization and were pretreated with or without JTE-013 and AB1 for 10 minutes. They were then placed in the upper compartment at 5 × 104 cells per well in 0.39 ml medium and allowed to migrate 5 hours at 37°C. The filter was then fixed overnight at 4°C and the nonmigrated cells were removed with a cotton swab. Attached cells were stained with 0.1% crystal violet and eluted with 10% acetic acid in 96-well plates. The absorbance was measured at 595 nm.

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Methylthiazolyldiphenyl-Tetrazolium Bromide Assay.[1]
The viability of SK-N-AS cells treated with JTE-013 or AB1 was determined by the methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay, as previously described (Li et al., 2013). Briefly, SK-N-AS cells were seeded in 96-well plates and treated with different concentrations of JTE-013 or AB1 for different times, followed by incubation of MTT at 37°C for 2 hours. The insoluble formazan formed in viable cells were dissolved by dimethylsulfoxide and the absorbance was measured at 595 nm by using a Bio-Rad Microplate Reader. Results are presented as the percentage of cell viability relative to the nondrug-treated controls.

Six-week-old female athymic NCr-nu/nu nude mice
30 mg/kg
Gavage; daily for 14 consecutive days

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Subcutaneous NB Tumor Model. [1]
Animal experiments were conducted according to our institution’s and the National Research Council’s guide for the care and use of laboratory animals. Six-week-old female athymic NCr-nu/nu nude mice (National Cancer Institute, Frederick, MD) were used in this study. Briefly, each mouse received a subcutaneous flank injection containing 1 × 107 SK-N-AS cells in 0.1 ml phosphate-buffered saline. After the tumor size reached approximately 100 mm3, the mice were randomized into three groups: vehicle control, JTE-013, and AB1. Both JTE-013 and AB1 were dissolved in dimethylsulfoxide first, diluted with 2% (2-hydroxypropyl)-β-cyclodextrin in phosphate-buffered saline, and given by gavage at 30 mg/kg daily for 14 consecutive days. Tumors were measured every other day with a caliper, and tumor volumes were calculated using the following formula: tumor volume = length × width2 × 0.52. Two weeks later, the mice were euthanized and tumor masses were collected for different assays. Intravenous Pharmacokinetic Analysis. [1]
Male CD-1 mice were used in this study. Briefly, a catheter was implanted in the carotid artery of each mouse to facilitate the subsequent repeated blood draws. Then the tested compounds were given intravenously at 1 mg/kg. A small amount of blood (30 μl) was taken from the catheter at different time points and the drug concentrations in the blood were determined by high-performance liquid chromatography analysis/mass spectrometry.

AB1 versus JTE-013 as S1P2 Antagonists and Stability In Vivo. [1]
JTE-013 is the current literature standard S1P2 antagonist but is unstable in vivo (Swenson et al., 2011). Through structural modification, a series of JTE-013 derivatives named AB compounds were synthesized and the FLIPR assay was conducted to determine their agonistic and antagonistic activities on S1P1–5. Among them, AB1 (Fig. 1) had the strongest S1P2 antagonist activity, with an IC50 of 3.5 versus 11 nM for JTE-013 (Fig. 2A). By contrast, no significant agonistic or antagonistic activities on other S1PRs were observed at micromolar levels (Supplemental Fig. 1). Furthermore, pharmacokinetic analysis after intravenous administration showed that the blood concentration of AB1 in mice remained higher than that of JTE-013 over 12 hours (Fig. 2B), indicating either better stability or slower clearance of AB1 in vivo. These data suggest that AB1 may have improved potency and stability in vivo.

[1]. Antitumor Activity of a Novel Sphingosine-1-Phosphate 2 Antagonist, AB1, in Neuroblastoma. J Pharmacol Exp Ther. 2015 Sep;354(3):261-8.

The bioactive lipid sphingosine-1-phosphate (S1P) and its receptors (S1P1-5) play critical roles in many pathologic processes, including cancer. The S1P axis has become a bona fide therapeutic target in cancer. JTE-013 [N-​(2,​6-​dichloro-​4-​pyridinyl)-​2-​[1,​3-​dimethyl-​4-​(1-​methylethyl)-​1H-​pyrazolo[3,​4-​b]pyridin-​6-​yl]-​hydrazinecarboxamide], a known S1P2 antagonist, suffers from instability in vivo. Structurally modified, more potent, and stable S1P2 inhibitors would be desirable pharmacological tools. One of the JTE-013 derivatives, AB1 [N-(1H-4-isopropyl-1-allyl-3-methylpyrazolo[3,4-b]pyridine-6-yl)-amino-N'-(2,6-dichloropyridine-4-yl) urea], exhibited improved S1P2 antagonism compared with JTE-013. Intravenous pharmacokinetics indicated enhanced stability or slower clearance of AB1 in vivo. Migration assays in glioblastoma showed that AB1 was slightly more effective than JTE-013 in blocking S1P2-mediated inhibition of cell migration. Functional studies in the neuroblastoma (NB) cell line SK-N-AS showed that AB1 displayed potency at least equivalent to JTE-013 in affecting signaling molecules downstream of S1P2. Similarly, AB1 inhibition of the growth of SK-N-AS tumor xenografts was improved compared with JTE-013. Cell viability assays excluded that this enhanced AB1 effect is caused by inhibition of cancer cell survival. Both JTE-013 and AB1 trended to inhibit (C-C motif) ligand 2 expression and were able to significantly inhibit subsequent tumor-associated macrophage infiltration in NB xenografts. Interestingly, AB1 was more effective than JTE-013 in inhibiting the expression of the profibrotic mediator connective tissue growth factor. The terminal deoxynucleotidyl transferase-mediated digoxigenin-deoxyuridine nick-end labeling assay and cleaved caspase-3 detection further demonstrated that apoptosis was increased in AB1-treated NB xenografts compared with JTE-013. Overall, the modification of JTE-013 to produce the AB1 compound improved potency, intravenous pharmacokinetics, cellular activity, and antitumor activity in NB and may have enhanced clinical and experimental applicability. [1]

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In summary, here we report the novel modification of the S1P2 antagonist JTE-013 to produce AB1. AB1 has moderately improved potency and intravenous pharmacokinetics that demonstrate better stability. In the context of NB, it also appears to have better cellular activity and antitumor activity. On the basis of these findings, we conclude that AB1 may have enhanced clinical and experimental applicability, overcoming some of the shortcomings of JTE-013. [1]

C17H19CL2N7O

408.2851

407.102

C, 50.01; H, 4.69; Cl, 17.37; N, 24.01; O, 3.92

383150-41-2

547756-93-4

10223146

White to off-white solid powder

1.5±0.1 g/cm3

1.697

4.42

3

5

4

27

516

0

ClC1C([H])=C(C([H])=C(N=1)Cl)N([H])C(N([H])N([H])C1C([H])=C(C2C(C([H])([H])[H])=NN(C([H])([H])[H])C=2N=1)C([H])(C([H])([H])[H])C([H])([H])[H])=O

RNSLRQNDXRSASX-UHFFFAOYSA-N

InChI=1S/C17H19Cl2N7O/c1-8(2)11-7-14(22-16-15(11)9(3)25-26(16)4)23-24-17(27)20-10-5-12(18)21-13(19)6-10/h5-8H,1-4H3,(H,22,23)(H2,20,21,24,27)

1-(2,6-dichloropyridin-4-yl)-3-[(1,3-dimethyl-4-propan-2-ylpyrazolo[3,4-b]pyridin-6-yl)amino]urea

JTE 013; JTE-013; 383150-41-2; 1-(2,6-dichloropyridin-4-yl)-3-[(1,3-dimethyl-4-propan-2-ylpyrazolo[3,4-b]pyridin-6-yl)amino]urea; CHEMBL1368758; DTXSID60436982; N-(2,6-dichloropyridin-4-yl)-2-(4-isopropyl-1,3-dimethyl-1H-pyrazolo[3,4-b]pyridin-6-yl)hydrazine-1-carboxamide; 1-[1,3-Dimethyl-4-(2-methylethyl)-1H-pyrazolo[3,4-b]pyridin-6-yl]-4-(2,6-dichloro-4-pyridinyl)-semicarbazide; JTE013

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 : ~100 mg/mL (~244.92 mM)

Solubility in Formulation 1: ≥ 0.83 mg/mL (2.03 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 8.3 mg/mL clear DMSO stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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: ≥ 0.83 mg/mL (2.03 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 8.3 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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