EGFR (IC50 = 0.3 nM); EGFRL858R (IC50 = 0.2 nM); EGFRExon 19 deletion (IC50 = 0.2 nM)

AZD3759 inhibits EGFR phosphorylation in H3255 (L858R) cells at an IC50 of 7.2 nM. With an IC50 of 7.7 nM and 7 nM, respectively, AZD3759 exhibits mo activity on H838 cell proliferation as well as inhibitory effects on the pEGFR pathway and cell proliferation of EGFR mutation-derived cells PC-9 and H3255.[1]

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In Vitro Profile of Candidate Zorifertinib (AZD3759)/1m [1]
The inhibition of 1m/Zorifertinib (AZD3759) against EGFR tyrosine kinase (EGFR TK) wild type and mutant enzymes was tested at their corresponding Km and 2 mM ATP concentrations (Table 6). At Km ATP concentrations, the inhibition IC50 values were 0.3, 0.2, and 0.2 nM for EGFR TK wild-type, L858R mutant, and Exon 19Del enzymes, respectively. In cellular EGFR phosphorylation and proliferation studies, PC-9 (Exon 19Del), H3255 (L858R), and H838 (wild-type EGFR) cells were used (see details in the Supporting Information). The results are shown in Table 6. Compound 1m was an equally potent inhibitor of cellular phosphorylation or proliferation in EGFR-activating mutant cell lines (PC-9 and H3255 cell lines) in the range of 7.0–7.7 nM, suggesting a high level of potency against all these clinically relevant EGFR mutations. In cellular phosphorylation studies, 1m also demonstrated 9-fold inhibition selectivity in EGFR-activating mutant cell lines over EGFR wild-type cell lines (H838 cell line). This was consistent with the findings in biochemical assays when ATP concentration was increased from Km to 2 mM (the hypothesized cellular ATP concentration), suggesting ATP competitive binding mode of 1m with its possible differential binding affinities to mutant and wild-type EGFRs. Although 1m/Zorifertinib (AZD3759) showed some activity against pEGFR in H838 cells, as these cells do not rely on activation of the EGFR pathway for proliferation, we did not see activity of 1m on cell proliferation of H838 cells. In contrast, 1m demonstrated inhibitory effects on both the pEGFR pathway and cell proliferation of EGFR mutation-derived cells PC-9 and H3255, suggesting these cells rely on activation of the EGFR pathway for proliferation. To broadly evaluate the selectivity of the compound, we screened 1m in a kinase panel and in a secondary pharmacology panel (see details in the Supporting Information). The kinase panel, screened at Millipore, was comprised of 124 recombinant protein kinases and lipid kinases. The percent inhibition of 1m was tested at one single concentration (1 μM) across each of these kinases. At this concentration, 1m displayed <50% inhibition against 115 kinases and >50% inhibition against the other nine kinases, including EGFR kinase in the panel. The inhibition of those eight off targets were 83% for EphB4, 57% for Flt, 58% for Fyn, 62% for KDR, 61% for activated Lck, 74% for Lyn, 69% for Src, and 87% for Yes. A secondary pharmacology panel, performed at Cerep, covered 150 distinct molecular targets. Assays were run in concentration–response mode. It was found that 147 molecular targets exhibited >1 μM and 3 targets <1 μM of IC50s. These active molecular targets with an IC50 of <1 μM were KDR (156 nM), Src (622 nM), and D2 (797 nM). The results from these panel screenings indicated that 1m was a highly selective compound. Furthermore, 1m/Zorifertinib (AZD3759) was neither a direct inhibitor (IC50 > 50 μM) nor a time-dependent inhibitor for CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, or CYP3A4/5 isoforms. This agent exhibited <3-fold mRNA induction for CYP1A2, CYP2B6, and CYP3A4 at the predicted maximum total concentration (0.3 μM) of the compound, suggesting a low enzyme induction liability. Also, the candidate showed acceptable hERG inhibition activity with an IC50 of 13.3 μM in a conventional manual whole-cell patch clamp study.

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Zorifertinib (AZD3759) is a tyrosine kinase inhibitor and has an encouraging future in treating brain metastases of non-small cell lung cancer. Here, we determined that AZD3759 suppressed the viability of HepG2 cells, a hepatoma cell line, and induced their apoptosis, suggesting a new therapeutic potential of Zorifertinib (AZD3759) in hepatocellular carcinoma (HCC) treatment. Furthermore, we found that the activation of p53-SMAD family member 4 (SMAD4) positive feedback loop was involved in the induction of bulks of apoptosis in HepG2 cells in response to Zorifertinib (AZD3759) treatment. In this positive feedback loop, p53 induced the expression of SMAD4 by directly promoting its transcription as shown by p53 could bind to SMAD4 promoter; SMAD4, in turn, promoted the nuclear translocation of p53, which increased the transcription of pro-apoptotic genes, including PUMA and BAX (two p53 target genes) and finally resulted in apoptosis. To the best of our knowledge, p53-induced SMAD4 transcription and SMAD4-determined the sub-location of p53 have not been reported. Taken together, our results demonstrated that AZD3759 might be an alternative strategy for HCC treatment and activating p53-SMAD4 positive feedback loop might enhance its therapeutic effects on HCC [2].

AZD3759 penetrates the monkey brain deeply and exhibits good oral bioavailability in dogs. AZD3759 (15 mg/kg) significantly increases the antitumor efficacy in a dose-dependent manner in a brain metastasis PC-9 (Exon19Del) model.[1]

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In Vivo Antitumor Efficacy Study [1]
A brain metastasis model was generated by intracerebral injection of luciferase transfected PC-9 (Exon19Del) cells. Tumor growth was monitored by an IVIS Xenogen imaging system. Zorifertinib (AZD3759) demonstrated significant dose-dependent antitumor efficacy (∼78% tumor growth inhibition at 7.5 mg/kg qd and tumor regression at 15 mg/kg qd, respectively, 4 weeks after treatment) with <20% body weight loss, whereas erlotinib had a limited effect in this model (Figure 2A). At the end of the study, brain tissues were collected for histological assessment. Significantly decreased tumor area was observed by Zorifertinib (AZD3759) treatment at the doses of 7.5 and 15 mg/kg (Figure 2B). In addition, modulation of pEGFR was detected by a single dose of Zorifertinib (AZD3759) at 15 mg/kg 1h after dosing, which confirmed target engagement by Zorifertinib (AZD3759) (Figure 2C).

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Microdosing Positron Emission Tomography (PET) Studies [1]
Brain distribution of Zorifertinib (AZD3759) in monkeys was investigated by microdosing PET to substantiate the PK data in rats. Radiolabeled [11C]-Zorifertinib (AZD3759) (0.28 μg, 150 MBq and 0.35 μg, 155 MBq) was injected into two male cynomolgus monkeys (PET1 and PET2, respectively). The PET distribution volume (VT) was estimated for the whole brain region using the 2-tissue compartment model. The distribution volume and estimated free brain to plasma partition coefficient (Cu,brain/Cu,plasma) are summarized in Table 8. The partition coefficient values (0.50 and 0.53 for PET1 and PET2, respectively) indicated Zorifertinib (AZD3759) penetrated extensively into monkey brain. PET images (Figure 1) for both monkeys also suggested that the radiolabeled compound was well distributed throughout the brain.

Following the manufacturer's instructions, the CisBio homogenous time resolved fluorescence approach (HTRF, Cat. No. 62TK0PEJ) is used to evaluate the inhibitory potency of compounds against EGFR WT and mutant enzymes. The assay employs the following final enzyme concentrations: 0.1 nM, 0.03 nM, and 0.026 nM for EGFR wild type, L858R, and Exon19Del, respectively. The corresponding Km values of EGFR enzymes are applied to 0.8 μM, 4 μM, and 25 μM ATP. In summary, 384-well Greiner white polystyrene assay plates are incubated with 3 μL of ATP and 2 μM TK biotin-peptide substrate at room temperature, either with or without a serially diluted compound. The assay buffer includes 1 mM DTT, 5 mM MgCl₂, 1 mM MnCl₂, and 0.01% CHAPS. The reaction is started by adding 3 μL kinase, which has the ability to phosphorylate the substrate peptide. Once the reaction has been incubated for 30 minutes, it is stopped by adding 6 μl of a detection reagent mix that contains diluted TK Ab Europium Cryptate in detection buffer and 250 nM Strep-XL665. After giving the plates a one-hour incubation period, the EnVision Multilabel Reader from Perkin Elmer is used to measure the fluorescence at 615 and 665 nm, respectively, with an excitation wavelength of 320 nm and standard HTRF settings. The kinase activity is inversely correlated with the calculated signal ratio of 665 to 615 nanometers. The four-parameter logistic fit method is used to determine the concentration of compound that results in 50% inhibition of the corresponding kinase (IC50).[1]

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Kinase selectivity of 1m/Zorifertinib (AZD3759) [1]
1m/Zorifertinib (AZD3759) was tested at a single 1 μM concentration across each of 124 kinases from Millipore kinase panel at an ATP concentration that is within 15 μM of their corresponding apparent Km values. The detailed protocols could be obtained from Millipore. In brief, recombinant kinases were incubated within appropriate buffer containing peptide substrate and radiolabelled γ33P-ATP together with presence or absence of required inhibitor concentration. The reaction was initiated by adding ATP/Mg2+ mix. After incubation for 40 minutes at room temperature, the reaction was stopped by adding 3% phosphoric acid solution. A portion of reaction mix was spotted onto P30 filtermat to trap peptide, and washed three times for 5 minutes with phosphoric acid to remove non-specific γ33P-ATP. The phosphorylated substrate was then measured by scintillation counting, which determined the level of kinase activity inhibition compared to control reactions.

MTS techniques are used to determine the results of the cell proliferation test. In summary, cells are plated in 96-well plates at a density that permits logarithmic growth throughout the 72-hour experiment, and they are then incubated at 37 °C and 5% CO₂ for the entire night. Following that, compounds with concentrations ranging from 30 to 0.0003 mM are exposed to cells for a duration of 72 hours. The CellTiter AQueous Non-Radioactive Cell Proliferation Assay reagent is used to measure cell proliferation for the MTS endpoint in compliance with the manufacturer's protocol. A Tecan Ultra device is used to measure absorbance. Predose measurements are performed, and absorbance readings are used to calculate the concentration required to limit treated cell growth to half that of untreated cells (GI50) values.

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Cellular inhibition of EGFR phosphorylation [1]
100 µL of cells were seeded in 96 well cell culture plates and incubated at 37 °C with 5% CO2 overnight. Cells were exposed to compounds at concentrations ranging from 10 to 0.0003 mM. After a 4-hour treatment, the plates were aspirated of medium, (For H838, adding human recombinant epidermal growth factor to each well to make a final concentration of 100 ng/mL for another 10 minutes before aspirating the medium), 110 µL IP lysis buffer (1:100 phosphatase inhibitor cocktail 2&3), and 1:100 protease inhibitor cocktail. Pierce IP lysis buffer was then added to each well. After 1 hour of incubation, 100 µl/well of cell lysis was transferred to coated plates and incubated overnight at 4°C with rotation of 300 rpm. The plates were taken from 4 °C to 37 °C with rotation of 300 rpm for 1 hour. Following aspiration and S8 washing of the plates with 1 x wash buffer, 100 µl of detection antibody was added to each well. The plate was sealed with tape and incubated for 2 hours at 37 °C with rotation of 300 rpm. Following aspiration and washing of the plates with 1 x wash buffer, 100 µl of HRP-linked secondary antibody was added to each well. The plate was sealed with tape and incubated for 1 hour at 37 °C with rotation of 300 rpm. Following aspiration and washing of the plates with 1 x wash buffer, 100 µl of TMB substrate was added to each well. The plate was sealed with tape and incubated for 30 minutes at 37 °C with rotation of 300 rpm. 100 µl stop solution was added to the plates and absorbance read at 450 nm within 30 minutes on SpectraMax M5e plate reader.

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Cellular proliferation assay [1]
Cell proliferation assay was determined by MTS methods. Briefly, cells were seeded in 96-well plates (at a density to allow for logarithmic growth during the 72-hour assay) and incubated overnight at 37 °C and 5% CO2. Cells were then exposed to concentrations of compounds ranging from 30 to 0.0003 mM for 72 hours. For the MTS endpoint, cell proliferation was measured by the CellTiter AQueous Non-Radioactive Cell Proliferation Assay reagent in accordance with the manufacturer’s protocol. Absorbance was measured with a Tecan Ultra instrument. Predose measurements were made, and concentration needed to reduce the growth of treated cells to half that of untreated cells (GI50) values were determined using absorbance readings.

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Detection of cell viability, apoptosis and BAX+/PUMA+/SMAD4+ cells [2]
Cell viability was quantified using the MTT colorimetric assay kit as previously described. Flow cytometry was used to detect apoptotic cells (Annexin V+ cells) and BAX+/PUMA+/SMAD4+ cells as previously described. Fluorescein labeling antibodies, including Anti-Annxin V-PE, anti-BAX-FITC, anti-PUMA-FITC and anti-SMAD4-FITC, were used to stain cells.

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Luciferase activity assay [2]
The detailed methods have been described previously. Briefly, cells were transfected with promoter reporter plasmids that contain PUMA or BAX or SMAD4 promoter. Relative light unit values were normalized to the β-galactosidase signal. One microgram of each indicated promoter-reporter plasmid and pRSV β-galactosidase plasmid was used for all transfections.

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Detection of mRNA and protein levels [2]
Real-time PCR and Immunoblot assays were used to detect mRNA and protein levels as previously described. Briefly, the mRNA content was normalized to the expression of the housekeeping gene β-actin. The following specific primer sequences were used for real-time PCR: β-actin, 5′-GCCCTGAGGCACTCTTCCA-3’ (forward) and 5′-CGGATGTCCACGTCACACTT-3’ (reverse). The BAX, PUMA and SMAD4 primers were obtained from the PrimePCR TM SYBR Green ® Assay kit These antibodies, including anti-p5, anti-caspase 3, anti-cleaved caspase 3, anti-β-actin, anti-SMAD4 and anti-Lamin B1, were used for immunoblot assay.

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Chromatin immunoprecipitation (ChIP) and subcellular fractionation assays [2]
Detailed procedures for ChIP had been described in a previous study. The antibody for detecting p53 was used for immunoprecipitating the DNA fragments that interacted with the related proteins. The methods for isolation of nuclear and cytoplasmic extracts were described in our previous study.

In Vivo Animal Models [1]
PC-9 cells (EGFR Exon19 deletion) were cultured with RPMI-1640 medium supplemented with 10% fetal bovine serum (FBS) at 37 °C. To monitor tumor growth in the brain, we stably transfected PC-9 cells with pGL4.50[luc2/CMV/Hygro] vector containing luciferase, and the bioluminescence signals were measured by a Xenogen imaging system. A brain metastasis model was established by intracerebral (ICB) injection of PC-9_Luc cells using the method by Lal with modifications. In brief, after a sagittal incision over the parieto-occipital bone, a hole was punctured on the skull at 2.5 mm to the right of the bregma and 1 mm anterior to the coronal suture. Then, the syringe was placed perpendicular to the skull through the hole and placed 3 mm deep below the skull surface, and the PC-9_Luc cell suspension was slowly injected. After injection, a sterile bone gel was applied to the hole, the scalp was pulled back to cover the skull, and the wound was closed. The mouse was gently put on a heating pad to recover, and closely monitored after surgery.
Xenograft tissues were obtained from the PC-9 model after treatment with Zorifertinib (AZD3759) for assessment of histology and pEGFR modulation. Samples were harvested following formalin fixation and paraffin embedding (FFPE) for further study. pEGFR(Tyr1068) IHC was performed on 3 μm FFPE sections using a Ventana automation for staining.

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In each PET measurement a sterile solution of [11C]-Zorifertinib (AZD3759) was injected as a bolus into a sural vein during 5 sec with simultaneous start of PET-data acquisition. Injected radioactivity was 150 and 155 MBq. The unbound fraction for [11C]-Zorifertinib (AZD3759) in plasma was measured using a previously described ultrafiltration method (Varrone, A.; Stepanov, V.; Nakao, R.; Toth, M.; Gulyas. B.; Emond, P.; et al. Imaging of the striatal and extrastriatal dopamine transporter with 18F-LBT-999: quantification, biodistribution, and radiation dosimetry in nonhuman primates. J. Nucl. Med. 2011, 52, 1313- 1321).[1]

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Rats: Zorifertinib (AZD3759) is administered orally to male Han Wistar rats at a dose of 2 mg/kg in 1% methylcellulose. Cerebral spinal fluid (CSF) is extracted from the cisterna magna at 0.25, 0.5, 1, 2, 4, and 7 hours post-dose. Blood samples (>60 μL/time point/each site) are obtained by cardiac puncture, placed into individual EDTA-coagulated tubes, and then promptly diluted with three times the volume of water. After being removed, brain tissue is homogenized in three times the volume of 100 mM phosphate buffered saline (pH7.4). All samples are kept cold until they are analyzed using LC/MS/MS.

Agent 1m/Zorifertinib (AZD3759) exhibited the best pharmacokinetic profile with moderate blood clearance at 17 mL min–1 kg–1 and volume of distribution of 5.2 L/kg. The intrinsic clearance (Clint) of 1m/Zorifertinib (AZD3759) is 11.4 μL min–1 10–6 cells in human hepatocytes, and the predicted clearance of the agent in humans is 7.7 mL min–1 kg–1 by in vitro–in vivo extrapolation (IVIVe). Liver blood flow (LBF) method with blood protein binding correction was applied to generate individual estimates of unbound human hepatic clearance (CLhepatic) using available data from each preclinical species. The predicted human clearance by the LBF method from rat is 8.1 mL min–1 kg–1, which is very similar to that derived from IVIVe. Following oral dosing in rats at 2 mg/kg, absorption of 1m/Zorifertinib (AZD3759) was rapid with blood Cmax of 0.58 μM achieved at 1.0 h. Subsequently, blood concentrations of 1m/Zorifertinib (AZD3759) declined monoexponentially with a mean elimination half-life of 4.3 h, which was close to the same parameter obtained from intravenous dosing of 4.1 h. The bioavailability following an oral dose in rats was 91%. Given its promising PK properties, CNS penetration, and in vitro potency, 1m/Zorifertinib (AZD3759) was selected as the candidate for comprehensive biological profiling.[1]

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Dog Pharmacokinetics [1]
Blood pharmacokinetic parameters of 1m/Zorifertinib (AZD3759) in male dogs were determined following both a single dose intravenous infusion and oral administration. These data are summarized in Table 7. Following the IV dose in dogs, 1m/Zorifertinib (AZD3759) blood clearance was determined as 14 mL min–1 kg–1, and the volume of distribution was 6.4 L/kg. Its elimination half-life was 6.2 h. Absorption of 1m/Zorifertinib (AZD3759) was rapid with blood Cmax (698 nM) occurring between 0.5 and 1.5 h. The oral bioavailability of 1m/Zorifertinib (AZD3759) was excellent at 90%.

[1]. Discovery and Evaluation of Clinical Candidate AZD3759, a Potent, Oral Active, Central Nervous System-Penetrant, Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor. J Med Chem. 2015 Oct 22;58(20):8200-15.

[2]. AZD3759 induces apoptosis in hepatoma cells by activating a p53-SMAD4 positive feedback loop. Biochem Biophys Res Commun. 2019 Feb 5;509(2):535-540.

AZD-3759 is under investigation in clinical trial NCT03360929 (Evaluate Safety, Tolerability, Pharmacokinetics and Anti-tumor Activity of AZD3759).
Zorifertinib is an orally available inhibitor of the epidermal growth factor receptor (EGFR), with potential antineoplastic activity. Upon oral administration, zorifertinib binds to and inhibits the activity of EGFR as well as certain mutant forms of EGFR. This prevents EGFR-mediated signaling, and may lead to both induction of cell death and inhibition of tumor growth in EGFR-overexpressing cells. EGFR, a receptor tyrosine kinase mutated in many tumor cell types, plays a key role in tumor cell proliferation and tumor vascularization.

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Recent reports suggest that an increasing number of patients with lung cancer, especially those with activating mutations of the epidermal growth factor receptor (EGFR), also present with brain metastases and leptomeningeal metastases. These patients have poor prognosis as there are no approved drugs for these indications. Available agents have poor efficacy for these patients even at well above their standard dose. Herein, we report the discovery of (4-[(3-chloro-2-fluorophenyl)amino]-7-methoxyquinazolin-6-yl (2R)-2,4-dimethylpiperazine-1-carboxylate 1m/Zorifertinib (AZD3759), an investigational drug currently in Phase 1 clinical trial, which has excellent central nervous system penetration and which induces profound regression of brain metastases in a mouse model. [1]

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By correctly balancing physicochemical properties, such as permeability, solubility, and efflux ratio, we were able to identify compound 1a. This early lead has significantly improved CNS penetration without compromising the in vitro potency. Replacement of the methylene group present in 1a by carbamate linker led to compound 1e. This compound significantly improved metabolic stability. The terminal basic nitrogen in 1e was found to be an essential element to achieve good exposures in both brain and blood. By incorporating a methyl group into the piperazine moiety, we further improved PK properties of 1e, leading to successful discovery of clinical candidate 1m/Zorifertinib (AZD3759). 1m exhibited excellent free compound distribution in brain, CSF, and blood. The extensive in vitro evaluation, including biochemical, cellular, and panel screening, indicates that this agent is highly potent against EGFR-activating mutants and highly selective toward these targets. Importantly, 1m showed tumor regression in the mouse model with brain metastasis. The promising data package for 1m strongly supported its selection as a drug candidate for development. The results from additional in vivo studies are due to be published separately.[1]

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Our study demonstrated that SMAD4 was a p53 target gene when HepG2 cells were treated with AZD3759. We believe that other factors induced by Zorifertinib (AZD3759) treatment are required for p53 to induce SMAD4 transcription or this phenomenon is just a unique property of HepG2 cells. But no matter what the reason is, the finding that p53 cooperates with SMAD4 to form the positive loop, which amplifies p53-induced apoptosis, opens a new avenue for the research of p53-mediated cell death.
The finding that SMAD4 could induce p53 nuclear translocation in the presence of Zorifertinib (AZD3759)treatment also first uncovered a new role of SMAD4 on determining p53 sub-localization. The exact mechanisms by which SMAD4 induces p53 nuclear localization will be detected at the proper time in future, since nuclear p53-mediated transcriptional activation/inhibition results in dramatic effects on the physiological and pathological processes of cells [2].
Taken together, our results uncover a new potential usage of AZD3759 for HCC treatment, since this chemical compound can activate p53-SMAD4 positive feedback loop to induce apoptosis and suppress the cell viability in hepatoma cells.[2]

C22H24CL2FN5O3

496.36

495.124

C, 53.24; H, 4.87; Cl, 14.28; F, 3.83; N, 14.11; O, 9.67

1626387-81-2

Zorifertinib;1626387-80-1

78322366

Typically exists as solid at room temperature

2

8

5

33

649

1

ClC1=CC=CC(=C1F)NC1=C2C(C=C(C(=C2)OC(N2CCN(C)C[C@H]2C)=O)OC)=NC=N1.Cl

IFVBAZHARMVMRV-BTQNPOSSSA-N

InChI=1S/C22H23ClFN5O3.ClH/c1-13-11-28(2)7-8-29(13)22(30)32-19-9-14-17(10-18(19)31-3)25-12-26-21(14)27-16-6-4-5-15(23)20(16)24;/h4-6,9-10,12-13H,7-8,11H2,1-3H3,(H,25,26,27);1H/t13-;/m1./s1

[4-(3-chloro-2-fluoroanilino)-7-methoxyquinazolin-6-yl] (2R)-2,4-dimethylpiperazine-1-carboxylate;hydrochloride

AZD3759 hydrochloride; 1626387-81-2; AZD-3759 hydrochloride; Zorifertinib (hydrochloride); AZD3759hydrochloride; 05Z3WK3SO6; [4-(3-chloro-2-fluoroanilino)-7-methoxyquinazolin-6-yl] (2R)-2,4-dimethylpiperazine-1-carboxylate;hydrochloride; (R)-4-((3-Chloro-2-fluorophenyl)amino)-7-methoxyquinazolin-6-yl 2,4-dimethylpiperazine-1-carboxylate hydrochloride;

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)

May dissolve in DMSO (in most cases), if not, try other solvents such as H2O, Ethanol, or DMF with a minute amount of products to avoid loss of samples

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.)