ALKL1196 (IC50 = 15-43 nM); ALKG1269A (IC50 = 14-80 nM); ALK1151Tins (IC50 = 38-50 nM); ALKG1202R (IC50 = 77-113 nM); ALKWT (IC50 <0.07 nM); ALKL1996M (IC50 = 0.6 nM); ALKG1269A (IC50 = 0.9 nM); ALK1151Tins (IC50 = 0.1 nM); ALKL1152R (IC50 <0.1 nM); ALKS1206Y (IC50 = 0.2 nM); ALKC1156Y (IC50 <0.1 nM); ALKF1174L (IC50 <0.1nM)

De novoGBM tumorigenesis is initiated in LSL-FIG-ROS1;Cdkn2a−/−;LSL-Luc mice through intracranial stereotactic injections of Adeno-Cre as described previously. Tumor development is monitored using BLI as described below. Once tumors reach a given size (107 p-1·s-1·cm-2·sr-1), animals are randomLy enrolled into vehicle control or 3-, 7-, or 14-d treatment with the indicated doses of Lorlatinib. Drug is administered through s.c. implanted Alzet osmotic pumps. After treatment, mice are killed, GBM tumors are microdissected, and tissues are flash-frozen in liquid N2. The remaining brains are processed for histology. In rats, PF-06463922 displays low plasma clearance, a moderate volume of distribution, a reasonable half-life, low propensity for p-glycoprotein 1-mediated efflux and a bioavailability of 100%. In vivo, PF-06463922 shows cytoreductive antitumor efficacy in the NIH3T3 xenograft models expressing human CD74-ROS1 and Fig-ROS1 via inhibition in ROS1 phosphorylation and the downstream signaling molecules, as well as inhibition of the cell cycle protein Cyclin D1 in tumors. In vivo, PF-06463922 also demonstrates marked antitumor activity in mice bearing tumor xenografts expressing EML4-ALK, EML4-ALK-L1196M, EML4-ALK-G1269A, EML4-ALK-G1202R or NPM-ALK.

Microfluidic mobility shift assay is used to measure kinase activity in recombinant human wild-type and mutant ALK kinase domain proteins (amino acids 1093–1411), which are produced in-house via baculoviral expression and autophosphorylation with MgATP. The reactions contained 3 μM 5-FAM-KKSRGDYMTMQIG-CONH2), 5 mM MgCl2, 1.3 nM wild-type ALK or 0.5 nM mutant ALK (suitable to produce 15-20% phosphorylation of peptide substrate after 1 hour of reaction), and the Kmlevel of ATP in 25 mM Hepes, pH 7.1. The results of kinetic and crystallographic investigations demonstrate that the inhibitors are ATP-competitive. Fitting the conversion (%) to a competitive inhibition equation yields the Kivalues. The procedure for assaying ROS1 enzyme is the same as that for ALK, with the exception that 0.25 nM recombinant human ROS1 catalytic domain (amino acids 1883-2347) is used. A 206-kinase panel is utilized to assess the selectivity of kinase inhibitors.

In 96-well plates, cells are sown in growth medium with 10% FBS, and they are incubated at 37°C for the entire night. The cells are incubated at 37°C for 72 hours after serial dilutions of Lorlatinib or suitable controls are added to the assigned wells the following day. To ascertain the relative cell numbers, a CellTiter-Glo assay is conducted. A four-parameter analytical method is used to fit a concentration-response curve and determine IC50 values.

In LSL-FIG-ROS1;Cdkn2a−/−;LSL-Luc mice, de novoGBM tumorigenesis is induced by intracranial stereotactic injections of Adeno-Cre, as previously reported. BLI is used to track the development of tumors as will be discussed below. Animals are randomly assigned to either vehicle control or 3-, 7-, or 14-day treatments with the prescribed doses of lerlatinib once tumors reach a specific size (107 p -1·s -1·cm -2·sr -1). The medication is delivered via s.c. implanted Alzet osmotic pumps. Following therapy, GBM tumors are microdissected, tissues are flash-frozen in liquid N2, and mice are killed. For histology, the remaining brains are processed.

J Med Chem.2014 Jun 12;57(11):4720-44;Clin Cancer Res.2012 Sep 1;18(17):4570-9.

Despite the significant efficacy of crizotinib in patients with anaplastic lymphoma kinase (ALK)-positive non-small cell lung cancer, disease progression still occurs during treatment. Samples from resistant patients show multiple point mutations in the ALK kinase domain, including the L1196M gating mutation. Furthermore, some patients experience disease progression due to brain metastases. Through structure-based drug design, lipophilicity optimization, and physical property-based optimization, we have prepared a series of highly potent ALK macrocyclic inhibitors with favorable absorption, distribution, metabolism, and excretion (ADME) properties, low P-glycoprotein 1-mediated efflux tendency, and good passive permeability. These structurally unique cyclic inhibitors inhibit both wild-type ALK and clinically reported ALK kinase domain mutations. We overcame numerous challenges in the synthesis process and utilized novel transformation methods to enable these macrocyclic compounds for drug development. This work led to the discovery of compound 8k (PF-06463922), which possesses broad-spectrum activity, central nervous system ADME properties, and high kinase selectivity. [1]

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Oncogene ROS1 fusion (Figure - ROS1) was first discovered in glioblastoma cells more than two decades ago. In recent years, ROS1 gene rearrangements have been found in a variety of human cancers, including lung adenocarcinoma, cholangiocarcinoma, ovarian cancer, gastric adenocarcinoma, colorectal cancer, inflammatory myofibroblastoma, angiosarcoma, and epithelioid angioendothelioma, providing further evidence for ROS1 as an attractive cancer target. The first-generation Met/ALK/ROS1 inhibitor XALKORI® (crizotinib) has shown good clinical efficacy in ROS1 fusion-positive non-small cell lung cancer. However, similar to the acquired ALK secondary resistance mutations observed in XALKORI-resistant patients, a ROS1 kinase domain mutant—ROS1G2032R—was also found in a ROS1-positive non-small cell lung cancer (NSCLC) patient who developed resistance to XALKORI. Therefore, there is an urgent need to develop drugs that can overcome such resistance. PF-06463922 is a novel orally administered ATP-competitive small molecule inhibitor of ROS1/ALK with extremely high inhibitory activity against ROS1 kinase. PF-06463922 inhibited the catalytic activity of recombinant ROS1 at an average Ki < 0.005 nM concentration and, with IC50 values ranging from 0.1 nM to 1 nM, inhibited ROS1 autophosphorylation in a range of cell lines carrying oncogenic ROS1 fusion variants (including CD74-ROS1, SLC34A2-ROS1, and Fig-ROS1). Furthermore, PF-06463922 inhibited the proliferation of HCC78 human non-small cell lung cancer cells carrying the SLC34A2-ROS1 fusion gene and BaF3-CD74-ROS1 cells expressing human CD74-ROS1 and induced apoptosis at sub-nanomolar to low nanomolar concentrations. In BaF3 cells genetically engineered to express the XALKORI-resistant mutant CD74-ROS1G2032R, PF-06463922 exhibited nanomolar inhibitory efficacy against both ROS1G2032R cell viability and proliferation. In vivo experiments showed that in a NIH3T3 xenograft model expressing human CD74-ROS1 and Fig-ROS1, PF-06463922 demonstrated significant cytoreductive antitumor efficacy even at low nanomolar concentrations. The antitumor efficacy of PF-06463922 was dose-dependent and closely related to the inhibition of ROS1 phosphorylation and its downstream signaling molecules pSHP1, pSHP2, and pErk1/2, as well as the inhibition of the tumor cell cyclin Cyclin D1. To our knowledge, PF-06463922 is the first reported ROS1 inhibitor capable of blocking resistant ROS1G2032R mutants at predicted pharmacologically relevant concentrations. Our data suggest that PF-06463922 has great potential in treating ROS1 fusion-positive cancers, including patients who have relapsed from XALKORI treatment due to acquired ROS1 G2032R mutations. [2]

C23H23FN6O4

466.464927911758

466.176

1924207-18-0

2135926-03-1;2306217-6 (hydrate) ;1924207-18-0 (PF-06463922 acetate); 1454846-35-5;

124203822

Typically exists as solid at room temperature

2

9

0

34

731

1

FC1C=CC2C(N(C)CC3C(=C(C#N)N(C)N=3)C3=CN=C(C(=C3)O[C@H](C)C=2C=1)N)=O.OC(C)=O

BLNAIBLTPYGILH-RFVHGSKJSA-N

InChI=1S/C21H19FN6O2.C2H4O2/c1-11-15-7-13(22)4-5-14(15)21(29)27(2)10-16-19(17(8-23)28(3)26-16)12-6-18(30-11)20(24)25-9-12;1-2(3)4/h4-7,9,11H,10H2,1-3H3,(H2,24,25);1H3,(H,3,4)/t11-;/m1./s1

acetic acid;(16R)-19-amino-13-fluoro-4,8,16-trimethyl-9-oxo-17-oxa-4,5,8,20-tetrazatetracyclo[16.3.1.02,6.010,15]docosa-1(22),2,5,10(15),11,13,18,20-octaene-3-carbonitrile

Lorlatinib acetate; PF-06463922 acetate; 1924207-18-0; TE9WI16FEU; 2H-4,8-Methenopyrazolo(4,3-H)(2,5,11)benzoxadiazacyclotetradecine-3-carbonitrile, 7-amino-12-fluoro-10,15,16,17-tetrahydro-2,10,16-trimethyl-15-oxo-, (10R)-, acetate; acetic acid;(16R)-19-amino-13-fluoro-4,8,16-trimethyl-9-oxo-17-oxa-4,5,8,20-tetrazatetracyclo[16.3.1.02,6.010,15]docosa-1(22),2,5,10(15),11,13,18,20-octaene-3-carbonitrile; UNII-TE9WI16FEU; PF 06463922 acetate; .

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.)