IGF-1R; ALK (Ki = 0.75 nM)

AZD3463 exhibits strong effects in preclinical models driven by ALK as well as in a range of crizotinib-resistant models. As evidenced by its capacity to reduce ALK autophosphorylation in tumor cell lines carrying ALK fusions, such as DEL (ALCL NPM-ALK), H3122 (NSCLC EML4-ALK), and H2228 (NSCLC EML4-ALK), AZD3463 inhibits ALK in cells. ALK inhibition is linked to disruptions in the ERK, AKT, and STAT3 signaling cascades, which specifically inhibit the proliferation of ALK-fusion-containing cell lines in vitro. AZD3463 continues to exhibit strong activity against several clinically significant crizotinib-resistant mutations, such as the gatekeeper mutant L1196M, which exhibits in vitro and in vivo equivalent potency to wild type ALK in BAF3 cell lines containing EML4-ALK. In order to evaluate AZD3463's potential to circumvent additional resistance mechanisms, antiproliferative activity is evaluated in both a patient-derived crizotinib relapsed model and multiple independently derived in vitro crizotinib resistant cell lines derived from H3122 cells. The L1196M gatekeeper and T115Ins mutations, ALK amplification, and/or secondary drivers like EGFR and IGF1R are just a few of the resistance mechanisms present in these resistant cell lines. In vitro, AZD3463 maintains its antiproliferative efficacy in 10 of the 12 acquired resistance models, four times that of the parental H3122 cells.[1]

AZD3463 exhibits the capacity to inhibit pALK in xenograft tumors in vivo in a dose-dependent manner, leading to either stasis (H3122) or regression (DEL, H2228).[1]

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AZD3463 also demonstrates the ability to dose dependently inhibit pALK in xenograft tumors in vivo resulting in stasis (H3122) or regression (DEL, H2228). AZD3463 retains good activity against a number of clinically relevant crizotinib resistant mutations including the gatekeeper mutant L1196M where equivalent potency to wild type ALK is observed in vivo in EML4-ALK containing BAF3 cell lines[1].

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AZD3463 shows anti-tumor efficacy in both ALK WT and F1174L mutant orthotropic xenograft mouse models of NB [3]
Based on the cytotoxic effects of AZD3463 on NB cells in vitro, we proceeded to assess the drug’s effect on inhibiting tumor growth in orthotopic xenograft mouse models of NB. In this set of in vivo experiments, NGP and SH-SY5Y cells with stable luciferase gene expression were separately implanted into the left kidneys of the nude mice. At the end of the AZD3463 treatment, the xenograft tumors from control and treatment groups were dissected and weighed. Significant tumor growth inhibition was observed in both AZD3463 treatment groups (SH-SY5Y and NGP) compared with the control groups. Treatment in SH-SY5Y xenograft mice with AZD3463 resulted in almost complete tumor regression and significant regression was observed in NGP xenograft mice (Fig. 5A,C). After the mice had been bearing the SH-SY5Y and NGP tumors for 4 weeks, the mice were treated with either AZD3463 or DMSO via intraperitoneal injection for 48 hours. Then we examined the effect of AZD3463 on the PI3K/AKT/mTOR signaling in the tumor tissues and found that AZD3463 efficiently blocked Akt and RPS6 phosphorylation and induced the cleavage of PARP, caspase 3, and LC3 A/BΙΙ (Fig. 5E) in vivo. These results suggest that AZD3463 can effectively induce apoptosis and autophagy as a single agent in orthotopic xenograft mouse models of NB.

Genomic rearrangement of Anaplastic Lymphoma Kinase (ALK) has been observed in several tumor types including 60-80% anaplastic large cell lymphoma (ALCL) and 3-6% of non small cell lung cancer (NSCLC). Although the ALK inhibitor crizotinib has clinical efficacy in selected ALK positive NSCLC patients, the majority of patients who show initial responses eventually relapse. Various mechanisms leading to resistance have been proposed and include ALK amplification and resistance mutations, as well as alternative pathway drivers including EGFR, cKIT and, more recently, IGF1R. We have discovered a novel and potent inhibitor of ALK, AZD3463 with a Ki value of 0.75nM which also inhibits additional receptor tyrosine kinases including insulin growth factor receptor (IGF1R) with equivalent potency. AZD3463 inhibits ALK in cells as demonstrated by its ability to decrease ALK autophosphorylation in tumor cell lines containing ALK fusions including DEL (ALCL NPM-ALK), H3122 (NSCLC EML4-ALK) and H2228 (NSCLC EML4-ALK). Inhibition of ALK is associated with perturbations in downstream signaling including ERK, AKT and STAT3 pathways leading to preferential inhibition of proliferation in the ALK fusion containing cell lines in vitro. [1]

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To further assess the potential ability of AZD3463 to overcome additional resistance mechanisms, antiproliferative activity was assessed in multiple crizotinib resistant cell lines independently derived in vitro from H3122 cells as well as a patient derived crizotinib relapsed model. These resistant cell lines contain multiple resistance mechanisms including the L1196M gatekeeper and T115Ins mutations, ALK amplification and/or secondary drivers including EGFR and IGF1R. AZD3463 retains antiproliferative potency within 4 fold of parental H3122 cells for 10 out of 12 of these acquired resistance models in vitro. In summary, AZD3463 is a potent ALK inhibitor which inhibits additional kinases including IGF1R and has activity in a number of crizotinib resistant models driven by multiple resistance mechanisms.[1]

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ZD-3463 is an ALK/IGF1R inhibitor that circumvents a number of mechanisms involved in the development of crizotinib resistance.

Cell proliferation and growth assays [2]
Cells were seeded in 96-well plates at a density of either 7000 cells (H3122, 24-H3122 and CR-H3122) and 4000 cells (A549) and allowed to adhere for 24 hours. Cells were treated with individual drugs alone or drug combinations for 72 hours before assay. Cytotoxicity and proliferation rate was evaluated using the sulforhodamine B (SRB) assay, as described by Skehan et al. (1990). Briefly, cells were fixed with 50 µL 10% trichloroacetic acid (TCA) for 30 minutes at 4 °C. Protein was stained with 50 µL of SRB and then the wells washed with 1% acetic acid. The plate was then dried and SRB solubilised in 100 µL of 100 μM TRIS buffer. Absorbance was read at 490 nm with a Spectromax plate reader, deducting the background of 630 nm; cell growth inhibition was evaluated as the ratio of the absorbance of the treated cells with the DMSO-treated control. All cytotoxicity assays were carried out in three independent experiments measured in technical triplicate. Cell proliferation time points were measured in hextuplicate.

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Cell cycle analysis [2]
To assess the effect of crizotinib on the cell cycle in our model of innate resistance H3122 cells were seeded in a 6-well plate at a density of 10,000 cells per well and incubated overnight to allow for cells adhesion prior to treatment. Cells were treated with crizotinib (10 μM) for 24 hours after which cells were washed with 0.01 M Phosphate buffered Saline and lysates collected via centrifugation and fixed with 70% ethanol over the following 12 days. For cell cycle analysis, cells were washed with 0.01 M PBS, treated with RNase (20 mg/mL), and then stained with propidium iodide. Cells were processed with the GalliosTM flow cytometer and analysed with FlowJo LLC version 10. Three independent experiments were carried out measured in technical triplicate.

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Immunoblotting [2]
For Western blot assays cells were first seeded at a density of 200,000 cells per well in 6 well plates. Following treatments cells were harvested, washed in 0.01 M PBS and lysed in a buffer consisting of 50 μM Tris-HCl (pH 8), 150 mM NaCl, 1 μM EDTA, 1 mM NaF, 1 mM sodium orthovanadate, 1 mM phenylmethylsulfonyl fluoride (PMSF), 1% TritonX-100, and 1% SDS. Cell extracts were clarified by centrifugation (12,500 RPM at 4 °C for 8 mins) and lysates then subjected to SDS-PAGE followed by transfer to a PVDF membrane which was probed for proteins of interest with primary antibodies, diluted in 0.01 mM PBS with 1% Bovine serum albumin.

5 to 6-week-old female athymic Ncr nude mice (SH-SY5Y and NGP xenograft tumors bearing mice)[1].
15 mg/kg
Intraperitoneal injection; once daily for 2 days

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Orthotopic Mouse Model of neuroblastoma (NB) [3]
Five to six-week-old female athymic Ncr nude mice were maintained under barrier conditions (pathogen-free conditions provided by plastic cages with sealed air filters). The preclinical mouse model of NB was established using orthotopic (intrarenal) implantation of the NB cells. Briefly, a transverse incision was created over the left flank of the nude mouse and 1.5 × 106 human luciferase-transduced SH-SY5Y or NGP cells suspended in 0.1 ml of PBS were surgically injected into the left renal capsule and towards the superior pole of the left kidney of the nude mice. After allowing the cells to engraft for 2 to 3 weeks, mice bearing tumors with similar sizes (using bioluminescent imaging to monitor tumor growth) were randomly divided into two groups: a DMSO control group and an AZD3463 treatment group. Both control and experimental groups contained six mice each. Three mice from each group were selected and treated with DMSO or AZD3463 over a 48 hour period via15 mg/kg intraperitoneal (i.p.) injection once daily for 2 days. Tumors were then harvested for WB analysis. The rest were treated for 21 days (15 mg/kg by intraperitoneal (i.p.) injection once daily). At the end of the treatment, all mice were sacrificed. Tumors and the right kidneys (control) were harvested and weighed.

[1]. Cancer Res (2013) 73 (8_Supplement): 919.

[2]. Sci Rep. 2017 Oct 24;7(1):13955.

[3]. Sci Rep. 2016 Jan 20;6:19423. Sci Rep. 2016 Jan 20;6:19423.

AZD3463 is a member of the class of indoles that is 1H-indole substituted by a 2-[4-(4-aminopiperidin-1-yl)-2-methoxyanilino]-5-chloropyrimidin-4-yl group at position 3. It is an orally bioavailable dual inhibitor of ALK and IGF1R with Ki value of 0.75 nM for ALK. It has a role as an apoptosis inducer, an antineoplastic agent, an EC 2.7.10.1 (receptor protein-tyrosine kinase) inhibitor and an autophagy inducer. It is a member of indoles, an aminopiperidine, a monomethoxybenzene, an aminopyrimidine, a secondary amino compound, a tertiary amino compound and an organochlorine compound.

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ALK positive non-small cell lung cancer is highly responsive to ALK inhibitors such as crizotinib, but drug resistance typically develops within a year of treatment. In this study we investigated whether IGF-1R is an independent druggable target in ALK-positive lung cancer cells. We confirmed that combination ALK and IGF-1R inhibitor treatment is synergistically cytotoxic to ALK-positive lung cancer cells and that this remains the case for at least 12 days after initial exposure to crizotinib. ALK-positive cells with acquired resistance to crizotinib did not acquire cross-resistance to IGF-1R inhibition, though combination treatment in the resistant cells gave additive rather than synergistic cytotoxicity. We concluded that IGF-1R is an independent druggable target in ALK-positive lung cancer and support the trial of combination treatment.[2]

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ALK receptor tyrosine kinase has been shown to be a therapeutic target in neuroblastoma. Germline ALK activating mutations are responsible for the majority of hereditary neuroblastoma and somatic ALK activating mutations are also frequently observed in sporadic cases of advanced NB. Crizotinib, a first-line therapy in the treatment of advanced non-small cell lung cancer (NSCLC) harboring ALK rearrangements, demonstrates striking efficacy against ALK-rearranged NB. However, crizotinib fails to effectively inhibit the activity of ALK when activating mutations are present within its kinase domain, as with the F1174L mutation. Here we show that a new ALK inhibitor AZD3463 effectively suppressed the proliferation of NB cell lines with wild type ALK (WT) as well as ALK activating mutations (F1174L and D1091N) by blocking the ALK-mediated PI3K/AKT/mTOR pathway and ultimately induced apoptosis and autophagy. In addition, AZD3463 enhanced the cytotoxic effects of doxorubicin on NB cells. AZD3463 also exhibited significant therapeutic efficacy on the growth of the NB tumors with WT and F1174L activating mutation ALK in orthotopic xenograft mouse models. These results indicate that AZD3463 is a promising therapeutic agent in the treatment of NB.[3]

C24H25CLN6O

448.9479

448.177

C, 64.21; H, 5.61; Cl, 7.90; N, 18.72; O, 3.56

1356962-20-3

1356962-20-3

56599293

Light green to green solid powder

1.3±0.1 g/cm3

723.6±70.0 °C at 760 mmHg

391.4±35.7 °C

0.0±2.3 mmHg at 25°C

1.693

3.11

3

6

5

32

605

0

ClC(C=NC(NC1=C(OC)C=C(N2CCC(N)CC2)C=C1)=N3)=C3C4=CNC5=C4C=CC=C5

GCYIGMXOIWJGBU-UHFFFAOYSA-N

InChI=1S/C24H25ClN6O/c1-32-22-12-16(31-10-8-15(26)9-11-31)6-7-21(22)29-24-28-14-19(25)23(30-24)18-13-27-20-5-3-2-4-17(18)20/h2-7,12-15,27H,8-11,26H2,1H3,(H,28,29,30)

N-[4-(4-aminopiperidin-1-yl)-2-methoxyphenyl]-5-chloro-4-(1H-indol-3-yl)pyrimidin-2-amine

AZD 3463; AZD-3463; 1356962-20-3; AZD3463; AZD-3463; ALK/IGF1R inhibitor; N-(4-(4-Aminopiperidin-1-yl)-2-methoxyphenyl)-5-chloro-4-(1H-indol-3-yl)pyrimidin-2-amine; N-[4-(4-aminopiperidin-1-yl)-2-methoxyphenyl]-5-chloro-4-(1H-indol-3-yl)pyrimidin-2-amine; AZD3463

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: 6~20 mg/mL (13.4~44.6 mM)

Solubility in Formulation 1: ≥ 2 mg/mL (4.45 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 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 20.0 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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 (Please use freshly prepared in vivo formulations for optimal results.)