Substituted macrocycles useful as kinase inhibitors

Abstract

There are provided novel substituted macrocyclic compounds, their pharmaceutically acceptable salts, solvates and hydrates. The compounds and compositions have protein kinase inhibitory activity and are expected to be useful in the treatment of protein kinase-mediated diseases and conditions.

Description

[0001] Related Applications

[0002] This invention claims the benefit of U.S. Provisional Patent Application No. 62 / 920,732, filed on May 14, 2019, which is incorporated herein by reference in its entirety. Field of the Invention

[0003] This invention relates to inhibitors of kinases, and pharmaceutically acceptable salts, solvates, hydrates, prodrugs, and metabolites thereof, methods for their preparation, and the use of such compounds for treating kinase-mediated diseases and conditions such as cancer. Background of the Invention

[0005] Protein kinases represent a large family of enzymes that catalyze the phosphorylation of target protein substrates. Phosphorylation is generally a transfer reaction of a phosphate group from ATP to a protein substrate. Common attachment points of the phosphate group to the protein substrate include, for example, tyrosine, serine, or threonine residues. Examples of kinases in the protein kinase family include, but are not limited to, Abl1 (v-Abl Abelson murine leukemia viral oncogene homolog 1), Akt, Alk, Bcr-Abl1, Blk, Brk, Btk, c-Kit, c-Met, c-Src, c-Fms, CDK1-10, b-Raf, c-Raf1, CSF1R, CSK, EGFR, ErbB2, ErbB3, ErbB4, Erk, FGFR1, FGFR2, FGFR3, FGFR4, FGFR5, Flt-1, Fps, Frk, Jak, KDR, MEK, PDGFR, PIK, PKC, PYK2, Ros, Tie, Tie2, and Zap70. Due to their activity in many cellular processes, protein kinases have emerged as important therapeutic targets.

[0006] Anaplastic lymphoma kinase (ALK) is a 1,620 - amino - acid transmembrane protein, consisting of an extracellular domain with an amino - terminal signal peptide, an intracellular domain with a juxtamembrane segment having a binding site for insulin receptor substrate - 1, and a carboxy - terminal kinase domain. ALK is a member of the insulin receptor tyrosine kinase family. Echinoderm microtubule - associated protein - like 4 (EML4) is a 120KDa cytoplasmic protein that is involved in the formation of microtubules and microtubule - binding proteins. EML4 - ALK is a novel fusion gene caused by an inversion on the short arm of chromosome 2, which joins exon 1 - 13 of EML4 to exon 20 - 29 of ALK. The presence of the EML4 - ALK fusion has been identified in approximately 3% - 13% of patients with non - small - cell lung cancer (NSCLC). For this reason, attempts have been made to identify small molecules that act as PK inhibitors. For example, amino - heteroaryl compounds (WO2004 / 076412) have been described as ALK / c - MET inhibitors. Azaindole derivatives (WO2010 / 068292) have been described as ALK / EGFR kinase inhibitors.

[0007] Thus, compounds that can inhibit the activities of protein kinases such as ALK and other kinases, either alone or in combination, can be used to treat human diseases such as cancer.

[0008] However, there remains a significant unmet medical need to overcome resistance to ALK mutations and to improve the safety profile of ALK inhibitors. SUMMARY OF THE INVENTION

[0010] In one aspect, there is provided a compound of formula I:

[0011]

[0012] or a pharmaceutically acceptable salt, solvate, enantiomer, prodrug, or metabolite thereof, wherein

[0013] R 1 is C 1-6 alkyl or C 1-6 alkoxy.

[0014] In another aspect, the present application also provides a pharmaceutical composition comprising a compound of formula I as described above and a pharmaceutically acceptable carrier.

[0015] In another aspect, the present application also provides a method for treating or preventing kinase - mediated disorders, the method comprising administering to a mammalian subject a therapeutically effective amount of any compound of formula I as described above. DETAILED DESCRIPTION OF THE INVENTION

[0017] In some embodiments of the present invention, there is provided a compound of formula I:

[0018]

[0019] or a pharmaceutically acceptable salt, solvate, enantiomer, prodrug, or metabolite thereof, wherein

[0020] R 1 is C 1-6 alkyl or C 1-6 alkoxy.

[0021] In some embodiments of the present invention, there are provided compounds of Formula II:

[0022]

[0023] or a pharmaceutically acceptable salt, solvate, enantiomer, prodrug, or metabolite thereof, wherein

[0024] R 1 is C 1-6 alkyl or C 1-6 alkoxy.

[0025] In some embodiments of the present invention, R 1 is methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, or tert-butyl.

[0026] In one embodiment of the present invention, R 1 is methyl.

[0027] In some embodiments of the present invention, R 1 is methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, sec-butoxy, isobutoxy, or tert-butoxy.

[0028] In one embodiment of the present invention, R 1 is methoxy.

[0029] In one embodiment of the present invention, R 1 is ethoxy.

[0030] In certain embodiments, there are provided compounds not limited to those selected from the group consisting of:

[0031]

[0032] and similar compounds, or a pharmaceutically acceptable salt, solvate, prodrug, or metabolite thereof.

[0033] In certain embodiments, there are provided compounds not limited to those selected from the group consisting of:

[0034]

[0035] and similar compounds, or pharmaceutically acceptable salts, solvates, prodrugs, or metabolites thereof.

[0036] In other embodiments, the compounds of the invention are in the form of pharmaceutically acceptable salts. In some embodiments, the compounds of the invention are in the form of solvates. In other embodiments, the compounds of the invention are in the form of metabolites. In other embodiments, the compounds of the invention are in the form of prodrugs. In some embodiments, the compounds of the invention are enantiomers. In other embodiments, the compounds of the invention are diastereomers. In another embodiment, the deuterium enrichment in the compounds of the invention is at least about 1%.

[0037] In some embodiments, there are provided pharmaceutical compositions comprising a compound of the invention and a pharmaceutically acceptable carrier. In certain embodiments, the composition is for treating diseases regulated by protein kinases. In certain embodiments, the composition is for preventing or treating hyper-proliferative disorders and / or angiogenic disorders. In some embodiments, the pharmaceutical composition further comprises an anti-neoplastic agent, an immunosuppressant, an immunostimulant, or a combination thereof. In other embodiments, the pharmaceutical composition is suitable for oral administration, parenteral administration, or intravenous administration.

[0038] In some embodiments, the invention provides a method for modulating kinase signal transduction, the method comprising administering to a mammalian subject a therapeutically effective amount of any of the compounds of the invention described herein.

[0039] In other embodiments, there are provided methods for treating or preventing disorders mediated by ALK (including all fusion and / or mutant kinases), ROS1, and / or NTRK kinases, the methods comprising administering to a mammalian subject a therapeutically effective amount of any of the compounds of the invention described herein.

[0040] In other embodiments, there are provided methods for treating neoplasia, the methods comprising administering to a mammalian subject in need thereof a therapeutically effective amount of any of the compounds of the invention described herein. In certain embodiments, the neoplasia is selected from skin cancer, leukemia, colon cancer, renal cell carcinoma, gastrointestinal stromal cancer, solid tumor cancer, myeloma, breast cancer, pancreatic cancer, non-small cell lung cancer, non-Hodgkin lymphoma, hepatocellular carcinoma, thyroid cancer, bladder cancer, colorectal cancer, prostate cancer, and brain cancer. In some embodiments, the method further comprises administering one or more anti-cancer agents.

[0041] The following definitions should assist in understanding the present invention described herein.

[0042] The term "alkyl" is intended to include straight-chain, branched-chain, and cyclic hydrocarbon groups that contain only carbon-carbon single bonds and that may be unsubstituted or optionally substituted with one or more functional groups. The preferred chain length of the alkyl group is from 1 to 6 carbon atoms. C1-C6 alkyl is intended to include C1 alkyl groups (methyl), C2 alkyl groups (ethyl), C3 alkyl groups (n-propyl, isopropyl), C4 alkyl groups (e.g., n-butyl, isobutyl, sec-butyl, tert-butyl), C5 alkyl groups (e.g., n-pentyl), and C6 alkyl groups. The alkyl may be substituted or unsubstituted. Illustrative substituted alkyl groups include, but are not limited to, fluoromethyl, difluoromethyl, trifluoromethyl, aminomethyl, aminoethyl, hydroxymethyl, methoxymethyl, 2-fluoroethyl, and 2-methoxyethyl, etc.

[0043] The term "alkoxy" refers to an -O-(alkyl) group or an -O-(unsubstituted cycloalkyl) group. C1-C6 alkoxy is intended to include -O-C1-C6 alkyl groups, where C1-C6 alkyl is as defined above. Representative examples include, but are not limited to, methoxy, ethoxy, propoxy, butoxy, cyclopropoxy, cyclobutoxy, cyclopentyloxy, cyclohexyloxy, and the like.

[0044] When used in connection with the compounds of the present invention, the term "pharmaceutically acceptable" is intended to refer to forms of the compounds that are safe for administration to a subject. For example, the free base, salt forms, solvates, hydrates, prodrugs, or derivative forms of the compounds of the present invention that have been approved by a regulatory agency or regulatory bodies such as the U.S. Food and Drug Administration (FDA) for use in mammals via oral ingestion or any other route of administration are pharmaceutically acceptable.

[0045] The phrase "effective amount" is intended to quantify the amount of each agent that, relative to the treatment of each agent itself, will achieve the goal of improving the severity and frequency of occurrence of a disorder while avoiding the adverse side effects typically associated with alternative therapy. In one embodiment, the effective amount is administered in a single dosage form or in multiple dosage forms.

[0046] The starting materials of the present invention are known, commercially available, or can be synthesized in a manner similar to or in accordance with methods known in the art. Many starting materials can be prepared according to known processes, and in particular, the processes described in the examples can be used. When synthesizing starting materials, in certain cases, functional groups are protected with suitable protecting groups when necessary. Protecting groups, their introduction, and removal are described below.

[0047] When synthesizing the compounds of formula I according to the desired procedures, in some embodiments the steps are carried out in an order suitable for preparing the compounds, which order includes the procedures described herein or an alternative order of steps described herein, and in one embodiment, additional protecting / deprotecting steps are carried out before or after as necessary. In some embodiments, the intermediates are isolated or continued in situ with or without purification. Synthetic chemical transformations and protecting group methods (protection and deprotection) useful in synthesizing the inhibitor compounds described herein are known in the art and include, for example, methods such as those described in: R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 3rd Edition, John Wiley and Sons (1999); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); A. Katritzky and A. Pozharski, Handbook of Heterocyclic Chemistry, 2nd Edition (2001); M. Bodanszky, A. Bodanszky, The Practice of Peptide Synthesis, Springer-Verlag, Berlin Heidelberg (1984); J. Seyden-Penne, Reductions by the Alumino- and Borohydrides in Organic Synthesis, 2nd Edition, Wiley-VCH, (1997); and L. Paquette, editor, Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995).

[0048] The compounds of the present invention also exist in several tautomeric forms in some embodiments. The present invention expressly includes all tautomeric forms of the compounds described herein.

[0049] In one embodiment, the compounds also exist in cis- or trans- or E- or Z-double bond isomer forms. Such isomeric forms of all such compounds are expressly included in the present invention.

[0050] Indications

[0051] The present invention provides compounds that are capable of modulating one or more signal transduction pathways, including but not limited to ALK.

[0052] The term "modulate", which means that the functional activity of a pathway (or a component thereof) is altered compared to its normal activity in the absence of the compound. Such effects include modulation of any quality or degree, including increase, agonism, augmentation, enhancement, facilitation, stimulation, decrease, blockade, inhibition, reduction, attenuation, antagonism, etc.

[0053] The compounds of the present invention may also modulate one or more of the following processes, including but not limited to, for example, cell growth (including, for example, differentiation, cell survival, and / or proliferation), tumor cell growth (including, for example, differentiation, cell survival, and / or proliferation), tumor regression, endothelial cell growth (including, for example, differentiation, cell survival, and / or proliferation), angiogenesis (blood vessel growth), lymphangiogenesis (lymphatic vessel growth), and / or hematopoiesis (e.g., T cell and B cell development, dendritic cell development, etc.).

[0054] While not wishing to be bound by any theory or mechanism of action, it has been found that the compounds of the present invention have the ability to modulate kinase activity. However, the methods of the present invention are not limited to any particular mechanism or how the compound achieves its therapeutic effect. The phrase "kinase activity", which means the catalytic activity in which the γ-phosphate from adenosine triphosphate (ATP) is transferred to an amino acid residue (e.g., serine, threonine, or tyrosine) in a protein substrate. A compound may modulate kinase activity, for example, by directly competing with ATP for the ATP-binding pocket of the kinase to inhibit kinase activity, by producing a conformational change in the structure of the enzyme that affects its activity (e.g., by disrupting the three-dimensional structure of the biological activity), by binding to and locking the kinase in an inactive conformation, and so on.

[0055] Formulations and Methods of Use

[0056] The amount of the compound administered and the dosage regimen for treating cancer using the compounds and / or compositions of the present invention depend on a variety of factors, including the age, weight, sex and medical condition of the subject, the type of disease, the severity of the disease, the route and frequency of administration, and the specific compound used. Accordingly, the dosage regimen can vary widely, but can be routinely determined using standard methods. A daily dose of from about 0.01 mg / kg to 500 mg / kg, advantageously between about 0.01 mg / kg and about 50 mg / kg, more advantageously between about 0.01 mg / kg and about 30 mg / kg, and even more advantageously between about 0.1 mg / kg and about 10 mg / kg may be suitable and should be useful for all methods of use disclosed herein. The daily dose can be administered in one to four doses per day.

[0057] While the compounds of the present invention can be administered alone, in the methods described, the compounds administered will generally be present as the active ingredient in a pharmaceutical composition. Accordingly, in another embodiment of the present invention, there is provided a pharmaceutical composition comprising a compound of the present invention in combination with: a pharmaceutically acceptable carrier, which includes diluents, excipients, adjuvants and the like as described herein (collectively referred to herein as "carrier" materials), and, if desired, other active ingredients. The pharmaceutical compositions of the present invention can contain an effective amount of the compound of the present invention or an effective dose of the compound of the present invention. An effective dose of the compound of the present invention includes an amount that is less than, equal to or greater than the effective amount of the compound; for example, a pharmaceutical composition in which two or more unit doses (such as in the form of tablets, capsules and the like) are required to administer an effective amount of the compound, or alternatively a multi-dose pharmaceutical composition (such as a powder, a liquid and the like) in which an effective amount of the compound is administered by administering a portion of the composition.

[0058] Route of administration

[0059] Suitable routes of administration include, but are not limited to, oral administration, intravenous administration, rectal administration, aerosol administration, parenteral administration, ocular administration, pulmonary administration, transmucosal administration, transdermal administration, vaginal administration, otic administration, nasal administration and topical administration. In addition, by way of example only, parenteral delivery includes intramuscular injection, subcutaneous injection, intravenous injection, intramedullary injection, as well as intrathecal injection, direct intraventricular injection, intraperitoneal injection, lymphatic injection and intranasal injection.

[0060] The compounds of the present invention can be administered orally. Oral administration can include swallowing such that the compound enters the gastrointestinal tract, or buccal or sublingual administration can be used, by which the compound enters the bloodstream directly from the mouth. Formulations suitable for oral administration include solid formulations such as tablets, capsules containing granules, liquids or powders, lozenges (including liquid-filled), chewable tablets, multi-particulates and nanoparticles, gels, solid solutions, liposomes, membranes (including muco-adhesive), ovules, sprays and liquid formulations.

[0061] The compounds of the present invention can also be used in fast-dissolving dosage forms, fast-disintegrating dosage forms, such as those described by Liang and Chen in Expert Opinion in Therapeutic Patents, 11(6), 981 - 986 (2001), the disclosure of which is incorporated herein by reference in its entirety.

[0062] Formulations for parenteral administration can be formulated for immediate release and / or modified release. Modified release formulations include delayed release, sustained release, pulsatile release, controlled release, targeted release and programmed release. Thus, the compounds of the present invention can be formulated as solids, semi-solids or thixotropic liquids for administration as an implanted depot, providing modified release of the active compound. Examples of such formulations include drug-coated stents and PGLA microspheres.

[0063] Combination

[0064] Although the compounds of the present invention can be administered or applied as the sole active pharmaceutical agent, they can also be used in combination with one or more compounds of the present invention or in combination with other agents. When administered as a combination, the therapeutic agents can be formulated as separate compositions for simultaneous administration or sequentially at different times, or the therapeutic agents can be given as a single composition.

[0065] The synthesis of the compounds in the present invention is described in Scheme 1 below.

[0066] The synthesis of the compounds of formula I is described in Scheme 1. The starting material compound 1 is prepared according to procedures known from similar literature. The amide formation reaction of compound 1 and the substituted glycine provides the compounds of formula I.

[0067] Scheme 1

[0068]

[0069] The synthesis of the compound of formula II is described in Scheme 2. The starting material compound 2 is prepared according to known literature procedures. The acylation reaction of compound 2 provides the compound of formula II.

[0070] Scheme 2

[0071]

[0072] Proton NMR spectrum

[0073] Unless otherwise indicated, all 1 1H NMR spectra were recorded on a Varian series Mercury 300, 400, 500 MHz instrument or a Bruker series 400, 500 MHz instrument. In such a characterization, all observed protons are reported as parts per million (ppm) downfield from tetramethylsilane (TMS) or other internal standard in the indicated appropriate solvent.

[0074] Abbreviations

[0075] DCM means dichloromethane.

[0076] EA means ethyl acetate.

[0077] TLC means thin layer chromatography.

[0078] HATU means 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate.

[0079] DIPEA means diisopropylethylamine.

[0080] HPLC means high performance liquid chromatography.

[0081] LC-MS means liquid chromatography - mass spectrometry.

[0082] NMR means nuclear magnetic resonance.

[0083] Example 1: Synthesis of Compound 3

[0084]

[0085] At 0 °C, pyridine (263.1 mg, 1.5 equiv) was added to a solution of compound 2 (1 g) in DCM (15 mL), and the mixture was stirred for 0.5 h. Then acetyl chloride (208.9 mg, 1.2 equiv) was added to the reaction mixture in several portions. The reaction was stirred at 0 °C for 0.5 h, and TLC indicated the completion of the reaction. Then water (20 mL) was added to the reaction, the layers were separated, and the aqueous layer was extracted with DCM (2 × 10 mL). The combined organic layers were washed with brine (20 mL), dried over anhydrous sodium sulfate. The crude product was purified by preparative TLC plate to give compound 3, 782.3 mg as a solid.

[0086] 1 1H-NMR (400 MHz, CDCl3): 1.867 - 1.852 (d, J = 6, 3H), 2.139 (s, 3H), 3.160 (s, 3H), 4.130 (s, 3H), 4.396 - 4.489 (q, J1 = 14.4, J2 = 8.4, 2H), 4.613 (s, 2H), 5.796 - 5.816 (q, J1 = 4.8, J2 = 1.6, 1H), 6.499 - 6.522 (t, J1 = 4.8, J2 = 4.4, 1H), 7.230 - 7.285 (m 1H), 7.375 - 7.379 (d, J = 1.6, 1H), 8.139 (s, 1H), 8.143 (s, 1H); LC-MS (M+1 = 506).

[0087] Example 2: Synthesis of compound 4

[0088]

[0089] At room temperature, (methoxycarbonyl)glycine (78.6 mg, 1.2 equiv), HATU (280.8 mg, 1.5 equiv) and DIPEA (127 mg, 2 equiv) were added to a solution of compound 2 (200 mg) in toluene (20 mL), and the mixture was stirred. The reaction was stirred under reflux for 5 h, and water was removed using a water separator. TLC indicated the completion of the reaction, then water (30 mL) was added to the reaction, and the aqueous layer was extracted with EA (3 × 10 mL). The combined organic layers were washed with brine (20 mL), dried over anhydrous sodium sulfate. The crude product was purified by preparative TLC to give compound 4, 146.6 mg as a solid.

[0090] 1H-NMR (400 MHz, CDCl3): 1.840 - 1.856 (d, J = 6.4, 3H), 3.158 (s, 3H), 3.782 (s, 3H), 4.130 (s, 3H), 4.434 - 4.491 (m, 4H), 5.655 (s, 1H), 5.790 - 5.810 (q, J1 = 4.8, J2 = 1.6, 1H), 7.025 - 7.092 (m, 2H), 7.232 - 7.267 (m 1H), 7.334 - 7.355 (d, J = 8.4, 1H), 8.138 - 8.142 (s, 1H), 8.607 (s, 1H). LC-MS (M+1 = 522).

[0091] Example 3: Synthesis of Compound 5

[0092]

[0093] At room temperature, to a solution of Compound 2 (100 mg) in DCM (5 mL) was added (ethoxycarbonyl) glycine (54.3 mg, 1.5 equiv), HATU (187.2 mg, 2 equiv) and DIPEA (80 mg, 2.5 equiv), and the mixture was stirred. The reaction was stirred under reflux for 8 h, then water (5 mL) was added to the reaction. The aqueous layer was extracted with DCM (3 × 5 mL). The combined organic layers were washed with brine (10 mL) and dried over anhydrous sodium sulfate. The crude product was purified by preparative TLC to give Compound 5 as a solid, 40.8 mg. 1 H-NMR (400 MHz, CDCl3): 1.263 - 1.354 (m, 3H), 1.839 - 1.855 (d, J = 6.4, 3H), 3.159 (s, 3H), 4.117 - 4.131 (s, 3H), 4.199 - 4.252 (q, J1 = 7.2, J2 = 6.8, 2H), 4.399 - 4.522 (m, 4H), 5.58 (s, 1H), 5.795 - 5.811 (q, J1 = 4.8, J2 = 1.6 1H), 7.047 - 7.090 (m 2H), 7.232 - 7.267 (m, 1H), 7.324 - 7.344 (d, J = 8, 1H), 8.142 (s, 1H), 8.146 (s, 1H).

[0094] LC-MS (M+1 = 536).

[0095] Biological Assay:

[0096] As described above, the compounds defined in the present invention have anti-proliferative activity. These properties can be evaluated, for example, using one or more of the procedures set forth below:

[0097] An in vitro assay for determining the ability of a test compound to inhibit a kinase.

[0098] A kinase-labeled T7 phage strain is prepared in an Escherichia coli host derived from the BL21 strain. The Escherichia coli is grown to the logarithmic phase and infected with T7 phage, and incubated at 32 °C with shaking until lysis. The lysate is centrifuged and filtered to remove cell debris. The remaining kinase is produced in HEK-293 cells and then labeled with DNA for qPCR detection. Streptavidin-coated magnetic beads are treated with biotinylated small molecule ligand at room temperature for 30 minutes to produce affinity resin for kinase assay. The liganded beads are blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and reduce non-specific binding. The binding reaction is assembled by combining kinase, liganded affinity beads, and test compound in 1× binding buffer (20% SeaBlock, 0.17× PBS, 0.05% Tween 20, 6 mM DTT). All reactions are carried out in a polystyrene 96-well plate with a final volume of 0.135 ml. The assay plate is incubated with shaking at room temperature for 1 hour, and the affinity beads are washed with wash buffer (1× PBS, 0.05% Tween 20). The beads are then resuspended in elution buffer (1× PBS, 0.05% Tween 20, 0.5 μM non-biotinylated affinity ligand) and incubated with shaking at room temperature for 30 minutes. The kinase concentration in the eluate is measured by qPCR.

[0099] Determination of a representative number of compounds against the BAF3 cell line using a cell proliferation assay:

[0100] Cell culture and seeding:

[0101] 1. Harvest cells in the logarithmic growth phase and count them using a hemocytometer. The Trypan blue exclusion method is used to detect cell viability to ensure that the cell viability is higher than 90%.

[0102] 2. Adjust the cell concentration; add 90 μL of cell suspension to a 96-well plate respectively.

[0103] 3. Incubate the cells in the 96-well plate overnight at 37 °C, 5% CO2 and 95% humidity.

[0104] Drug dilution and administration:

[0105] 1. Prepare a 10-fold drug solution with a maximum concentration of 100 μM, 9 concentrations, and a 3.16-fold dilution. Add 10 μL of the drug solution to each well of a 96-well plate seeded with cells, and set up 3 replicate wells for each drug concentration.

[0106] 2. Incubate the cells in the 96-well plate with the added drug at 37 °C, 5% CO2, and 95% humidity for 72 hours, and then perform CTG analysis.

[0107] End reading board:

[0108] 1. Melt the CTG reagent and equilibrate the cell plate to room temperature for 30 minutes.

[0109] 2. Add an equal volume of CTG solution to each well.

[0110] 3. Vortex on a fixed-orbit shaker for 5 minutes to lyse the cells.

[0111] 4. Place the cell plate at room temperature for 20 minutes to stabilize the luminescence signal.

[0112] 5. Read the luminescence value.

[0113] Analyze the data using GraphPad Prism 5.0 software, and obtain a dose-response curve by fitting the data via non-linear S-curve regression, and calculate the IC 50 value accordingly.

[0114] Cell viability (%) = (Lum 待测药物 - Lum 培养液对照 ) / (Lum 细胞对照 - Lum 培养液对照 ) × 100%.

[0115] Table A below lists the compounds representative of the present invention and their activities in the cell assay.

[0116] Table A. Cell proliferation assay

[0117]

[0118] The representative number of compounds tested in the P450 3A4 assay in human liver microsomes to measure CYP inhibition:

[0119] Human liver microsomes (HLM) were stored at -80 °C. Before the study, the microsomes were thawed in a cold water bath and then immediately placed on ice. The test compound and the P450 3A4 specific inhibitor ketoconazole were dissolved in DMSO to obtain a stock solution of 10 mM. The stock solution was diluted with 50% acetonitrile to obtain a working solution with a concentration of 1.5 mM. The working solution was further diluted with 0.1 M potassium phosphate buffer to obtain a series of working solutions with concentrations of 150 μM, 50 μM, 15 μM, 5 μM, 1.5 μM, 0.5 μM, 0.15 μM, and 0.05 μM. The incubation mixtures in duplicate contained pooled human liver microsomes (0.1 mg / mL), 3.3 mM MgCl2, the CYP 3A4 probe substrate testosterone (50 μM), the specific inhibitor or the test compound (30 μM, 10 μM, 3 μM, 0.1 μM, 0.03 μM, 0.01 μM, 0.003 μM, 0.01 μM) in 0.1 M potassium phosphate buffer (total volume 0.1 mL). The negative control contained 0.1 M phosphate buffer instead of the specific inhibitor or the test compound. The final concentrations of DMSO and acetonitrile were equal to or less than 0.1%. The mixtures were pre-incubated at 37 °C for 10 min. Then, 1 mM NADPH was added to initiate the reaction. After a 10-min incubation at 37 °C, the reaction was terminated by adding 300 μL of acetonitrile containing the internal standard. The formation of the corresponding products was detected by LC / MS / MS.

[0120] LCMS method: An API 4000 Qtrap system coupled with a Waters ACQUITY UPLC system was used. The mass spectrometer was equipped with a Turbo ion spray (ESI) interface (Applied Biosystems, Concord, Ontario, Canada). The LC-MS / MS system was controlled using the Analyst 1.5 software package (Applied Biosystems) for data acquisition and processing. Chromatographic separation was achieved on a Waters ACQUITY UPLC BEH C18 column (50 × 2.1 mm ID, 1.7 μm). The column temperature was maintained at ambient temperature (25 °C). Mobile phase A was pure water supplemented with 0.1% formic acid (v / v). Mobile phase B was acetonitrile supplemented with 0.1% formic acid (v / v). The flow rate was maintained at 0.6 mL / min.

[0121] Sample preparation: The reaction was quenched by adding 3 volumes of ice-cold methanol / acetonitrile (1 / 1, v / v) mixture containing the internal standard. The mixture was centrifuged at 4000 rpm for 20 min. 100 μL of the supernatant was mixed with 200 μL of H2O and the final solution was injected for LC-MS / MS analysis.

[0122] Data analysis: The peak area ratio of the product (6β-hydroxy testosterone) to the internal standard was plotted as a percentage of the relevant negative control for each reaction to represent the residual enzyme activity. The IC 50 value of the test compound was determined by non-linear regression of the enzyme activity against the inhibitor concentration using GraphPad Prism software. The general criteria for evaluating the potential risk of drug-drug interaction (DDI) are as follows:

[0123] IC 50 > 10 μM CYP inhibition is low;

[0124] 3 μM < IC 50 < 10 μM CYP inhibition is moderate;

[0125] IC 50 < 3 μM CYP inhibition is high.

[0126] Table B below lists the compounds representative of the present invention and their activities in cytochrome P450 3A4 assays. Compound 3 showed much weaker inhibition of CYP 3A4, indicating a much smaller likelihood of drug-drug interaction.

[0127] Table B. CYP 3A4 inhibition

[0128] Compound <![CDATA[CYP 3A4(IC 50 )]]> Lorlatinib 7.8 μM 2 10 μM 3 279 μM

[0129] Solubility measurement:

[0130] Preparation of reference standard solution: 2 mg of the compound was added separately to each 100 mL volumetric flask. The compound was diluted to 100 mL with acetonitrile.

[0131] Preparation of sample solution: 2 mg of the compound was added separately to a 2 mL eppendorf tube (EP), and then 1 mL of buffer solution (20 mM) at pH 7.0 or pH 10.0 was added. The solution was shaken for 2 minutes and placed at 25 °C for 30 minutes. After standing for 30 minutes, a precipitate formed at the bottom of the EP. The solution was filtered through a 0.2 μm membrane filter and then diluted 50-fold with water.

[0132] Standard solutions and sample solutions of the same volume were injected into HPLC on a Shim-Pack CLC-ODS C18 column (150 mm × 6.0 mm, 5 μm). The mobile phase consisted of acetonitrile and 2% chloroform - 20 mM KH2PO4 buffer (pH = 7.0), and the flow rate was 1 mL / min (40:60). The detection wavelength was at 264 nm. Calculation: Solubility of the sample = Concentration of the standard × Area of the sample × 50 / Area of the standard. Compound 3 had a much higher solubility at pH = 7.0 than Compound 2 and lorlatinib.

[0133] Table C. Solubility of the compounds of the present invention

[0134] Lorlatinib (PF-06463922) Compound 2 Compound 3 Solubility at pH 7.0 0.029 mg / mL 0.091 mg / mL 0.772 mg / mL

[0135] In vivo xenograft assay:

[0136] A representative protocol for in vivo experiments is as follows to establish a subcutaneous BaF3-SLC34A2-ROS1 cell line xenograft model in nude mice and evaluate the in vivo therapeutic efficacy of the compounds: Animals: Male Balb / c nude mice (6 - 8 weeks old) were obtained from SLAC Laboratory Animal, Shanghai, China. The animals were maintained in sterile filter-top cages under SPF conditions and were housed on a HEPA-filtered ventilated rack. The animals were allowed free access to sterile rodent chow and water. Cell line: BaF3-SLC34A2-ROS1, xenograft model in athymic mice: Cells for implantation into athymic mice were harvested and pelleted by centrifugation at 1200 r / min for 5 min. The cells were washed once and resuspended in 200 μl of sterile PBS buffer at 5×10 6 . Then the cells were implanted s.c. into the right scapular region of each mouse, and the cells were allowed to grow to 200 mm 3 - 300 mm 3 before the administration of the compounds. Preparation of the dosing formulation: Each compound was suspended in 0.5% CMC-Na. Randomization: When the tumor volume was close to 200 mm 3 - 300 mm 3 , the mice were randomized into 5 groups according to the tumor volume. This day was designated as D1, and the treatment started on this day. Administration: The dose was administered once daily by oral gavage needle for several days. When the tumor volume was 200 mm 3 - 300 mm 3When starting, the treatment of the compound administered at 0.5% CMC-Na by p.o. gavage was initiated. Observation: After inoculation, the morbidity and mortality of the animals were examined daily. During routine monitoring, the tumor growth of the animals and any effects of the treatment on normal behavior, such as activity, weight gain / loss (weight will be measured twice a week or every other day), eye / hair matting, and any other abnormal effects, were examined. Deaths and observed clinical signs were recorded based on the number of animals in each subgroup. Tumor size measurement: The tumor volume was determined every 3 days by measurement with an electronic vernier caliper, and the tumor volume was calculated as the product of its length × width2 × 0.5. Effect study: The tumor volume was expressed as the mean tumor volume ± SD on the designated day. The percentage (%) inhibition value of the drug-treated mice was measured compared to the vehicle-treated mice, and calculated as follows: Tumor growth inhibition (TGI, %) = 100 - [MTV treated / MTV control] × 100. The t-test was used to determine the significant difference between the treatment group and the control group (p < 0.05). At the end of the study, after blood collection, the mice were euthanized by cervical dislocation. First, the tumor tissue was collected, then the abdominal cavity was opened, the liver and spleen were excised, and then weighed separately after removing the gallbladder. The organ weights and organ weight / body weight ratios were compared between the treatment group and the control group. The ratio was calculated as follows: Ratio = organ weight / (body weight - tumor weight). Both the organ weight and the organ weight / body weight ratio were also expressed as the mean ± SD, and the t-test was used to determine the significant difference between the treatment group and the control group (p < 0.05).

[0137] Table D below lists the compounds representative of the present invention and their activities in the subcutaneous BaF3-SLC34A2-ROS1 cell line xenograft model of nude mice described above. Compounds 1 and 3 were administered once daily at 5 mg / kg by oral gavage for several days. The tumor sizes were weighted and averaged. On day 20, Compound 3 showed a significantly better tumor growth inhibitory effect compared to lorlatinib. The remaining tumor weight of Compound 3 was much smaller than that of lorlatinib (PF-06463922).

[0138] Table D. Tumor weights on day 20

[0139] Control Lorlatinib 3 Tumor weight 2.118g 0.531g 0.327g

[0140] PK study:

[0141] Representative protocols for PK (pharmacokinetics) determination are as follows: Compounds are administered IV or orally to the same animals. The dose for each compound is 1 mg / kg (volume 5 ml / kg) IV and 10 mg / kg (volume 10 ml / kg) orally. The formulation for IV is 10% Solutol HS 15 + 90% phosphate buffered saline at 0.4 mg / mL. A suspension at 0.5 mg / mL in 100% (0.5% Tween 80 in 0.5% MC in water) is used for the oral formulation. Sample collection: For the IV route, plasma samples are collected at time points of 0.083 h, 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, and 24 h. For the oral route, plasma samples are collected at time points of 0.25 h, 0.5 h, 1 h, 2 h, 4 h, 8 h, and 24 h. Total bioavailability is calculated by adding the parent compound and major metabolites. Analysis: PK parameters are calculated using LC-MS / MS: t 1 / 2 、t 最大 、C 最大 、Vss, and AUC, etc.

[0142] Table E below lists the selected compounds and their bioavailability.

[0143] Compound Total F (%) 2 49 3 100 4 100

[0144] In rats, compound 2 showed an oral bioavailability of 49%. Compounds 3 and 4 had a greatly improved bioavailability (F = 100%).

[0145] Toxicity studies

[0146] In a typical one-month rat toxicity study, multiple representative compounds including lorlatinib (PF-06463922) and compound 3 were tested at a dose of 8 mg / kg / day for females and 16 mg / kg / day for males. Under the conditions of this experiment, at the end of the compound administration period and the end of the recovery period, in the group administered with the lorlatinib dose, some animals had reduced lymph and red blood cells in the blood, and platelets and / or %NEUT showed a tendency to increase. The corresponding animals showed a shortened prothrombin time. In contrast, no similar changes were observed in the compound 3 or vehicle control groups at the same dose.

[0147] Stability testing: The stability of the compounds was tested at 25 °C for one month, and HPLC was used to measure the purity. It was found that compound 2 decomposed significantly at 25 °C, where the main impurities A and B increased from 1.13% and 1.39% on day 0 to 3.98% and 8.34% on day 30, respectively. Compound 2 was unstable at -30 °C. On the other hand, compounds 3, 4, and 5 showed no significant changes at 0 °C or 25 °C (Table F).

[0148] HPLC method and conditions:

[0149]

[0150] Table F. Stability of the compound at 25 °C for one month.

[0151]

[0152]

Claims

1. A compound, or a pharmaceutically acceptable salt thereof.

2. A pharmaceutical composition comprising the compound according to claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

3. Use of the compound according to claim 1 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 2, in the preparation of a medicament for the treatment of a cancer disease selected from lung cancer and / or brain cancer.

Images