Patent Application
20100144743
The present disclosure, according to specific example embodiments, generally relates to tyrosine kinases. In particular, the present disclosure relates to compositions useful in inhibiting catalytic activity of tyrosine kinases and associated methods of use.
Tyrosine kinases are a class of enzymes, which catalyze the transfer of the terminal phosphate of adenosine triphosphate to the phenolic hydroxyl group of a tyrosine residue present in the target protein. Tyrosine kinases play a critical role in signal transduction for several cellular functions including cell proliferation, angiogenesis, carcinogenesis, apoptosis, and cell differentiation. Therefore inhibitors of these enzymes would be useful for the treatment or prevention of proliferative diseases which are dependent on these enzymes. Strong epidemiologic evidence suggests that the overexpression or activation of receptor protein tyrosine kinases leading to constitutive mitogenic signaling is an important factor in a growing number of human malignancies, such as cancer. Tyrosine kinases that have been implicated in these processes include Abl, CDK's, EGF, EMT, FGF, FAK, Flk-1/KDR, HER-2, IGF-1R, IR, LCK, MET, PDGF, Src, ephrins, and VEGF.
Ephrin receptors represent the largest family of receptor tyrosine kinases and have emerged as essential regulators of angiogenesis. Angiogenesis refers to the formation of new capillaries from existing vasculature and it is a crucial element in tumor growth. It is required for nourishment and removal of metabolic waste. If neovascularization is insufficient to support the growth and development of tumors, then hypoxia may arise. Tumors with low oxygenation are generally associated with poor prognosis. A key element in the hypoxic response is the hypoxia induced factor (HIF). HIF-1 is a transcription factor that is activated in solid tumors, and is up-regulated along with ephrins and VEGF. As a result of this, ephrins have been described as good targets for anticancer therapies.
Some specific example embodiments of the disclosure may be understood by referring, in part, to the following description and the accompanying drawings.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
While the present disclosure is susceptible to various modifications and alternative forms, specific example embodiments have been shown in the figures and are herein described in more detail. It should be understood, however, that the description of specific example embodiments is not intended to limit the invention to the particular forms disclosed, but on the contrary, this disclosure is to cover all modifications and equivalents as illustrated, in part, by the appended claims.
The present disclosure, according to specific example embodiments, generally relates to tyrosine kinases. In particular, the present disclosure relates to compositions useful in inhibiting catalytic activity of tyrosine kinases and associated methods of use.
The compositions of the present disclosure may at least partially inhibit catalytic activity of tyrosine kinases. Examples of tyrosine kinases that may be suitable targets for the compositions of the present disclosure include, but are not limited to, receptor tyrosine kinases and cellular tyrosine kinases. Examples of receptor tyrosine kinases include, but are not limited to, Eph receptors, EGF receptors, insulin receptors, IGF receptor-1, Trk A, PDGF receptors, M-CSF receptors, FGR receptors, VEGF receptors. Examples of cellular tyrosine kinases include, but are not limited to, Src, Frk, Btk, Csk, Abl, ZAP70, Fes, Fps, Fak, Jak, Ack, Yes, Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk.
The compositions of the present disclosure may at least partially inhibit catalytic activity of a tyrosine kinase directly by interacting with the kinase itself; or indirectly, by interacting with another molecule on which the catalytic activity of the kinase is dependent.
The compositions of the present disclosure may also inhibit HIF-1 expression and HIF-1 signal transduction pathways.
In one embodiment, compositions of the present disclosure may comprise a compound of the following Formula (I):
In another embodiment, compositions of the present disclosure may comprise a compound of the following Formula (II):
In Formula (I) or in Formula (II), R1 may represent a group selected from the following:
The compounds of Formula (I) and (II) may be enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, prodrugs, or solvates thereof.
In another embodiment, the compositions of the present disclosure may comprise a compound of the following Formula (III):
In Formula (III), R1 may represent a group selected from the following:
In Formula (III), R2 may represent a group selected from the following:
The compounds of Formula (III) may be enantiomers, diastereomers, pharmaceutically acceptable salts, hydrates, prodrugs, or solvates thereof.
The compositions of the present disclosure generally may be synthesized using methods known in the art, including for example, suzuki coupling reactions.
The compositions of the present disclosure also may be provided as a pharmaceutical composition comprising a compound of Formula (I), (II), or (III) and a pharmaceutically acceptable carrier.
The compositions also may be used in a pharmaceutical composition comprising a compound represented by Formula (I), (II), or (III) in combination with pharmaceutically acceptable carrier and an anti-cancer or cytotoxic agent. In certain embodiments, the anti-cancer or cytotoxic agent may be chosen from one or more of linomide; inhibitors of integrin αVβ3 function; angiostatin; razoxane; tamoxifen; toremifene; raloxifene; droloxifene; iodoxifene; megestrol acetate; anastrozole; letrozole; borazole; exemestane; flutamide; nilutamide; bicalutamide; cyproterone acetate; gosereline acetate; leuprolide; finasteride; metalloproteinase inhibitors; inhibitors of urokinase plasminogen activator receptor function; growth factor antibodies; growth factor receptor antibodies such as Avastin® (bevacizumab) and Erbitu® (cetuximab); tyrosine kinase inhibitors; serine/threonine kinase inhibitors; methotrexate; 5-fluorouracil; purine; adenosine analogues; cytosine arabinoside; doxorubicin; daunomycin; epirubicin; idarubicin; mitomycin-C; dactinomycin; mithramycin; cisplatin; carboplatin; nitrogen mustard; melphalan; chlorambucil; busulphan; cyclophosphamide; ifosfamide nitrosoureas; thiotepa; vincristine; Taxol® (pacliatxel); Taxotere® (docetaxel); epothilone analogs; discodermolide analogs; eleutherobin analogs; etoposide; teniposide; amsacrine; topotecan; flavopyridols; biological response modifiers and proteasome inhibitors such as Velcade® (bortezomib).
The compounds represented by Formula (I), (II), or (III) or a pharmaceutically acceptable salt or hydrate or solvate thereof may be administered to a mammal, including a human, to treat cancers of that mammal. The administration method may include, for example, oral or parenteral. As used herein, the term “cancer” refers to an abnormal growth of cells which tend to proliferate in an uncontrolled way, including neoplasms, tumors, and leukemia.
In certain embodiments, the methods of the present disclosure may provide for control of neovascularization of tumor cells using compounds represented by Formula (I), (II), or (III). In these embodiments, the compounds represented by Formula (I), (II), or (III) may bind to a tyrosine kinase, thereby inhibiting its activity. In some embodiments, the angiogenesis of tumor cells may depend on the activation of tyrosine kinases, and thus, inhibition of tyrosine kinases may decrease tumor growth and development.
In certain embodiments, a compound represented by Formula (I), (II), or (III) or a pharmaceutically acceptable salt or hydrate or solvate thereof may be used to inhibit catalytic activity of tyrosine kinases in cancer cells in a dose dependent manner. It will be recognized by one of skill in the art that the optimal quantity and spacing of individual dosages of a compound represented by Formula (I), (II), or (III) will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the particular patient being treated, and that such optimums can be determined by conventional techniques. Similarly, the optimal course of treatment, for example, the number of doses of a compound represented by Formula (I), (II), or (III) given per day for a defined number of days, can be ascertained by those skilled in the art using conventional course of treatment determination tests.
The present disclosure also provides methods of inhibiting protein kinase activity of tyrosine kinases which may comprise administering to a mammal in need thereof, a therapeutically effective amount of a compound of Formula (I), (II), or (III). As used herein, the term “therapeutically effective amount” refers to the amount of an active compound or pharmaceutical agent that elicits the desired biological or medicinal response in a tissue, system, animal or human. In certain embodiments, the methods of the present disclosure may be useful in inhibiting tyrosine kinases of the Eph family of receptors.
In certain embodiments, the compositions of the present disclosure may be used to treat a proliferative disease, comprising administering to a mammal in need thereof, a therapeutically effective amount of a compound of Formula (I), (II), or (III). In certain embodiments, the proliferative disease may be cancer.
To facilitate a better understanding of the present invention, the following examples of specific embodiments are given. In no way should the following examples be read to limit or define the entire scope of the invention.
The design, synthesis, and preliminary results from the in-vitro testing of compounds designed as inhibitors of ephrin kinase, specifically ephrin A4 are hereinafter described. The design resulted from the in-silico screening of several targeted libraries via docking to a homology model of ephrin A4 constructed from the nearly identical ephrin A2 crystal structure, followed by post-filtering using several criteria. This filtering included consideration of the binding mode, docking score, and consensus docking score. This resulted in several structural cores and two of those cores were selected based on synthetic feasibility. Several compounds were synthesized based on the core and structures identified by in-silico screening. These were tested in an ephrin A4 kinase assay and found to have only marginal activity at the 10 uM level. Knowledge of a link between hypoxia induced factor-1 alpha (hif1alpha) and ephrin, led to the same compounds and additional analogs being tested in an assay used to gauge hif1alpha pathway inhibition. In these assays, several compounds were found to inhibit hif1alpha expression in the low uM range that was separate from a much higher general cytotoxicity value.
Structure-Based Design of Ephrin Inhibitors.
Since no crystal structure currently exists for ephA4, the crystal structure of ephA2 (PDB accession code 1MQB) was used as a template to construct a homology model. The primary amino acid sequence for human ephA4 was located with the tools available from NCBI (http://www.ncbi.nlm.nih.gov), accession code NP—004429. The sequence was truncated to 615-881 and this region was read in as a raw sequence to Swiss-PDB Viewer. A search for suitable templates, not surprisingly, led to ephB2 (1JPA) and the aforementioned ephA2 structure. A fit was completed to initially align the sequences and prepare for model building. Due to the high sequence identity, the alignment did not require any further modification; however, there was a missing structural segment corresponding to the activation loop, which is noted in the paper discussing the crystal structure. This alignment was submitted to the Swiss-Model server and it returned a viable structure to begin design work. Although the sequence is nearly identical, it was not until the homology model was constructed that the extent of differences in the ATP binding site region could be assessed. The crystal structure of ephA2 and the homology model of ephA4 were compared in the region within 5 Å of the ANP ligand. In this region, the sequence identity between these two receptors is nearly 100%, with only two residues differing between them. Several commercially available kinase targeted libraries were prepared for in-silico screening and the docking of these libraries led to the identification of several functionalized cores to use as “lead” structures.
The design of ephrin inhibitors provided herein includes the selection of screening compounds, in-silico screening via docking, analysis of docking results, preliminary selection of compounds with a degree of specificity toward ephrin A4 kinase, and final selection of candidate compounds.
Compounds from three vendors: Asinex (Winston-Salem, N.C.), BioFocus (Saffron Walden, Essex, UK), and LifeChem (Burlington, ON, Canada) were screened in-silico against ephrin A4 via docking of the individual ligands into the receptor binding site.
The traditional metric for this type of screening has been the individual docking scores, which aim to capture the binding affinity of the ligand and receptor. Though useful in ranking ligands, the scoring methods are imperfect, so the consensus of several scoring methods (Clark, et al., (2002) J Mol Graph Model 20, 281-95) is generally utilized in the final selection. In the present study, the consensus scoring was completed with the scoring methods provided in the FlexX module within Sybyl 7.1. Over 32,000 compounds were screened in this manner. While compounds with the highest scores were of interest, additional metrics were also considered. One additional element used in the selection of compounds was the evaluation of binding mode. Utilization of specific interactions along with the docking scores has been shown to increase the hit ratio of in-silico screening. Information on these binding interactions was derived from the inhibitor bound crystal structure. An example of such a structure is provided by
Preparation of Ephrin Inhibitors.
50 mg (0.13 mmol) N-(2-methoxyphenyl)-3-(6-Chloro-pyrimidine-4-yl)-benzenesulfonamide, 2′-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)acetanilide (0.13 mmol), 70 mg (0.5 mmol) of potassium carbonate and 10 mg palladium tetrakis triphenylphonsphine (0.01 mmol) was mixed in MeCN/water (1:1) 4 ml. After fill with argon, the reaction was performed under microwave at 150° C. for 30 min. Filter hot reaction solution was filtered and the product (b in
1H NMR(DMSO) δ 9.54 (s, 1H), 9.28 (s, 1H), 8.37 (s, 1H), 7.77 (br, 1H), 7.67 (br, 2H), 7.15 (t, J = 7.2 Hz, 1H), 7.08 (d, J = 8.4 Hz, 1H), 6.95 (t, J = 7.2 Hz, 1H), 3.85 (s, 3H).
For LG2-13 synthesis, to a solution of LG2-9 (98 mg, 0.307 mmol), 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-benzamide (116 mg, 0.469 mmol, 1.5 equiv.), and Tetrakis(triphenylphosphine) palladium(0) [Pd(PPh3)4, 41 mg, 0.035 mmol) in THF (2.0 ml) was added 1.0 ml of aqueous 1 N K2CO3 solution. The resultant mixture was heated at 160° C. under microwave irradiation for 10 min. The mixture was diluted with EtOAc (15 ml), and the organic phase was separated out. After removal of solvent, the residue was subjected to flash column chromatography on silica gel (elution with CH2Cl2-MeOH) to give LG2-13 as a white solid (67 mg, 61%). ESI-MS: m/z 360 (M+1); 1H NMR (600 MHz, MeOH-d4): δ 8.14 (1H, s), 7.97 (1H, d, J=7.8 Hz), 7.89 (1H, d, J=4.8 Hz), 7.84 (1H, d, J=7.8 Hz), 7.77 (1H, s), 7.72 (2H, d, J=6.6 Hz), 7.68 (1H, t, J=7.8 Hz), 7.42 (1H, d, J=4.8 Hz), 6.95 (2H, d, J=6.6 Hz), 3.81 (3H, s, OCH3); 13C NMR (150.9 MHz, CDCl3-MeOH-d4 85:15 v/v): δ 169.59, 155.80, 146.78, 134.51, 133.57, 132.09, 131.15, 130.65, 129.50, 128.92, 128.66, 127.66, 127.55, 127.23, 121.95, 114.21, 108.62, 55.43.
For synthesis of LG2-7, to a solution of LG2-3 (70 mg, 0.183 mmol), 5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-indole (67 mg, 0.275 mmol, 1.5 equiv.), and Tetrakis(triphenylphosphine)-palladium(0) (25 mg, 0.022 mmol) in THF (2.0 ml) was added 1.0 ml of aqueous 1 N K2CO3 solution. The resultant mixture was heated at 160° C. under microwave irradiation for 10 min. The mixture was diluted with EtOAc (15 ml), and the organic phase was separated out. After removal of solvent, the residue was subjected to flash column chromatography on silica gel (elution with CH2Cl2-MeOH) to yield LG2-7 as a white solid (46 mg, 61%). ESI-MS: m/z 418 (M+1); 1H NMR (600 MHz, CDCl3): δ 8.33 (1H, s), 7.99 (1H, s), 7.84 (3H, m), 7.76 (1H, d, J=4.8 Hz), 7.63 (1H, s), 7.56 (1H, d, J=7.8 Hz), 7.45 (1H, d, J=4.8 Hz), 7.38 (1H, dd, J=2.4, 8.4 Hz), 7.34-7.31 (3H, m), 7.09-7.06 (3H, m), 7.03 (2H, m), 6.66 (1H, m); 13C NMR (150.9 MHz, CDCl3): δ 152.28, 146.66, 135.82, 135.15, 130.48, 130.09, 129.66, 128.44, 128.22, 125.49, 122.79, 122.50, 121.23, 120.91, 120.00, 119.89, 118.23, 111.90, 109.48, 103.13.
For preparation of LG2-75, to a solution of LG2-73 (30 mg, 0.079 mmol), 143-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenylkethanone (29 mg, 0.118 mmol, 1.5 equiv.), and Tetrakis(triphenylphosphine)-palladium(0) (11 mg, 0.0096 mmol) in THF (0.8 ml) was added 0.4 ml of aqueous 1 N K2CO3 solution. The resultant mixture was heated at 160° C. under microwave irradiation for 10 min. The mixture was diluted with EtOAc (10 ml), and the organic phase was separated out. After removal of solvent, the residue was subjected to flash column chromatography on silica gel (elution with hexane-EtOAc) to yield LG2-75 as a white foam (35 mg). ESI-MS: m/z 419 (M+1); 1H NMR (600 MHz, CDCl3): δ 8.18 (1H, s), 8.04 (1H, d, J=7.8 Hz), 7.99 (1H, s), 7.78 (1H, d, J=7.8 Hz), 7.70 (1H, s), 7.69 (1H, d, J=4.8 Hz), 7.66 (1H, t, J=6.8 Hz), 7.52 (1H, d, J=4.8 Hz), 7.21 (2H, s), 3.92 (6H, s), 3.85 (3H, s), 2.69 (3H, s); 13C NMR (150.9 MHz, CDCl3): δ 197.41, 153.43, 146.73, 138.11, 135.32, 133.93, 133.90, 132.36, 132.15, 132.08, 131.33, 129.74, 129.13, 129.07, 128.61, 128.56, 127.56, 108.95, 97.43, 61.05, 56.17, 26.78.
For preparation of LG2-89, to a solution of LG2-87 (25 mg, 0.076 mmol), 2-(1-naphthylene)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (39 mg, 0.152 mmol, 2.0 equiv.), and Tetrakis(triphenylphosphine)-palladium(0) (11 mg, 0.0096 mmol) in THF (1.0 ml) was added 0.5 ml of aqueous 1 N K2CO3 solution. The resultant mixture was heated at 160° C. under microwave irradiation for 10 min. The mixture was diluted with EtOAc (10 ml), and the organic phase was separated out. After removal of solvent, the residue was subjected to flash column chromatography on silica gel (elution with hexane-EtOAc) to yield LG2-89 as a white foam (33 mg). ESI-MS: m/z 376 (M+1); 1H NMR (600 MHz, CDCl3): δ 8.23 (1H, s), 8.18 (1H, br. s), 8.11 (1H, s), 8.02 (1H, br. s), 7.98 (1H, d, J=7.8 Hz), 7.73 (1H, s), 7.62 (3H, m), 7.57 (1H, t, J=7.2 Hz), 7.52 (1H, d, J=8.4 Hz), 7.49 (1H, t, J=7.8 Hz), 7.42 (1H, d, J=9.0 Hz), 7.39 (1H, d, J=4.2 Hz), 7.23 (1H, s), 7.13 (1H, d, J=4.2 Hz), 6.59 (1H, s); 13C NMR (150.9 MHz, CDCl3): δ 147.36, 133.91, 133.84, 132.91, 132.22, 131.98, 131.78, 129.94, 129.10, 128.70, 128.63, 128.32, 127.13, 126.58, 126.51, 125.61, 125.49, 125.20, 124.93, 117.03, 112.68, 111.27, 109.27, 103.01.
To synthesize LG2-65, to a solution of LG2-60 (83 mg, 0.25 mmol), 3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-phenol (83 mg, 0.375 mmol, 1.5 equiv.), and Tetrakis(triphenylphosphine)-palladium(0) (35 mg, 0.03 mmol) in THF (2.5 ml) was added 1.2 ml of aqueous 1 N K2CO3 solution. The resultant mixture was heated at 160° C. under microwave irradiation for 10 min. The mixture was diluted with EtOAc (20 ml), and the organic phase was separated out. After removal of solvent, the residue was redissolved in dichloromethane, and the resultant white solids were collected to yield LG2-65 as a white solid (44 mg, 51%). ESI-MS: m/z 345 (M+1); 1H NMR (600 MHz, CDCl3-MeOH-d4 85:15 v/v): δ 7.85 (1H, d, J=4.8 Hz), 7.74 (2H, d, J=8.4 Hz), 7.65 (1H, s), 7.43 (1H, d, J=4.8 Hz), 7.38 (1H, t, J=7.8 Hz), 7.25 (2H, d, J=8.4 Hz), 7.08 (1H, d, J=7.2 Hz), 7.03 (1H, br. s), 6.91 (1H, dd, J=1.8, 8.4 Hz), 2.91 (1H, m), 1.27 (6H, d, J=7.2 Hz); 13C NMR (150.9 MHz, CDCl3-MeOH-d4 85:15 v/v): δ 157.87, 146.50, 143.97, 136.83, 133.20, 130.29, 130.00, 129.12, 128.92, 128.20, 126.60, 120.28, 119.05, 115.82, 114.70, 109.24, 33.55, 23.50.
To synthesize LG2-62, to a solution of LG2-55 (31 mg, 0.107 mmol), 2-methoxy-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-phenol (40 mg, 0.161 mmol, 1.5 equiv.), and Tetrakis(triphenylphosphine)-palladium(0) (15 mg, 0.013 mmol) in THF (1.0 ml) was added 0.5 ml of aqueous 1 N K2CO3 solution. The resultant mixture was heated at 160° C. under microwave irradiation for 10 min. The mixture was diluted with EtOAc (15 ml), and the organic phase was separated out. After removal of solvent, the residue was subjected to flash column chromatography on silica gel (elution with CH2Cl2-MeOH) to yield LG2-62 as a white solid (19 mg, 54%). ESI-MS: m/z 333 (M+1); 1H NMR (600 MHz, CDCl3): δ 8.03 (1H, s, NH), 7.87 (2H, d, J=7.8 Hz), 7.67 (1H, d, J=4.8 Hz), 7.57 (1H, s), 7.48 (1H, d, J=4.8 Hz), 7.39 (2H, t, J=7.8 Hz), 7.09 (1H, t, J=7.8 Hz), 7.08 (2H, s), 7.01 (1H, s), 5.83 (1H, s, OH), 3.97 (3H, s, OCH3); 13C NMR (150.9 MHz, CDCl3): δ 148.98, 147.12, 146.64, 146.38, 139.32, 130.45, 129.07, 128.46, 122.98, 121.69, 120.33, 119.56, 115.20, 110.92, 109.31, 56.18.
To synthesize LG2-98, to a solution of LG2-95 (34 mg, 0.110 mmol), N-(2-hydroxyethyl)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-benzamide (63 mg, 0.203 mmol, 1.85 equiv.) and Tetrakis(triphenylphosphine)-palladium(0) (147 mg, 0.127 mmol, 1.15 equiv) in THF (1.0 ml) was added 0.5 ml of aqueous 1 N K2CO3 solution. The resultant mixture was heated at 160° C. under microwave irradiation for 10 min. The mixture was diluted with EtOAc, and the organic phase was separated out. After removal of solvent, the residue was subjected to flash column chromatography on silica gel (elution with CH2Cl2-acetone) to yield LG2-98 as a white solid (8.8 mg, 20%). ESI-MS: m/z 394 (M+1); 1H NMR (600 MHz, CDCl3): δ 8.00 (1H, br. s), 7.81 (1H, d, J=7.8 Hz), 7.69 (1H, d, J=7.8 Hz), 7.60 (3H, m), 7.44 (1H, d, J=4.8 Hz), 7.23 (1H, dd, J=0.9, 5.1 Hz), 7.10 (1H, d, J=3.0 Hz), 6.97 (1H, dd, J=5.1, 3.0 Hz), 5.00 (2H, d, J=5.4 Hz), 3.88 (2H, dd, J=4.8, 9.6 Hz), 3.68 (2H, J=4.8, 9.6 Hz); 13C NMR (150.9 MHz, CDCl3): δ 167.58, 148.88, 141.20, 135.46, 133.75, 131.05, 130.80, 129.51, 129.25, 129.22, 127.34, 126.85, 126.74, 126.72, 126.10, 125.09, 107.94, 62.09, 42.85, 39.49.
To synthesize LG2-96, to a solution of LG2-91 (39 mg, 0.121 mmol), N-(2-hydroxyethyl)-3-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-benzamide (99 mg, 0.340 mmol, 2.8 equiv.) and Tetrakis(triphenylphosphine)-palladium(0) (49 mg, 0.042 mmol) in THF (1.0 ml) was added 0.5 ml of aqueous 1 N K2CO3 solution. The resultant mixture was heated at 160° C. under microwave irradiation for 10 min. The mixture was diluted with EtOAc, and the organic phase was separated out. After removal of solvent, the residue was subjected to flash column chromatography on silica gel (elution with CH2Cl2-MeOH) to yield LG2-96 as a pale brown foam (37 mg, 73%). ESI-MS: m/z 406 (M+1); 1H NMR (600 MHz, CDCl3): δ 8.01 (1H, s), 7.80 (1H, d, J=7.8 Hz), 7.68 (1H, d, J=7.2 Hz), 7.60 (3H, m), 7.40 (3H, m), 7.03 (2H, t, J=8.4 Hz), 6.67 (111, br. s, OH), 6.34 (1H, t, J=5.4 Hz, NH), 4.79 (2H, d, J=5.4 Hz), 3.87 (2H, t, J=5.4 Hz), 3.68 (2H, J=5.4, 10.2 Hz); 13C NMR (150.9 MHz, CDCl3): δ 167.61, 162.17 (d, 1JC-F=245 Hz), 149.14, 135.41, 134.29, 133.66, 130.82, 130.67, 129.47, 129.41, 129.30, 128.99, 127.38, 126.86, 126.69, 115.45 (d, 2JC-F=21 Hz), 107.76, 61.78, 43.97, 42.87.
To prepare LG2-102, to a solution of LG2-101 (32 mg, 0.119 mmol), 2-(3,5-dimethoxyphenyl)-(4,4,5,5-tetramethyl-1,3,2-dioxaborolane (47 mg, 0.178 mmol, 1.5 equiv.) and Tetrakis(triphenylphosphine)-palladium(0) (16 mg, 0.013 mmol) in THF (1.0 ml) was added 0.5 ml of aqueous 1 N K2CO3 solution. The resultant mixture was heated at 160° C. under microwave irradiation for 10 min. The mixture was diluted with EtOAc, and the organic phase was separated out. After removal of solvent, the residue was subjected to flash column chromatography on silica gel (elution with hexane-EtOAc) to yield LG2-102 as a white gel (16 mg, 41%). ESI-MS: m/z 325 (M+1); 1H NMR (600 MHz, CDCl3): δ 7.60 (1H, d, J=4.8 Hz), 7.55 (1H, s), 7.36 (1H, d, J=4.8 Hz), 6.673 (1H, s), 6.670 (1H, s), 6.53 (1H, s), 6.14 (1H, t, J=5.4 Hz, NH), 3.85 (6H, s), 3.46 (2H, dd, J=5.4, 12.0 Hz), 1.18 (1H, m), 0.59 (2H, m), 0.32 (2H, m); 13C NMR (150.9 MHz, CDCl3): δ 161.41, 149.54, 133.76, 130.59, 130.47, 129.07, 128.25, 107.78, 106.16, 100.37, 55.54, 45.84, 10.71, 3.62.
LG-2 Compounds and HIF-1GFP/DsRFP expression and WST-1 test in C6#4 cells.
In this experiment were used Ephrin compounds: LG2-7; LG2-11; LG2-13; LG2-62; LG2-65; LG2-75; LG2-81; LG2-85; LG2-89; LG2-96:LG2-98; LG2-102. Px-478 HIF-1 inhibitor was used as positive control, DMSO—as negative control.•C6#4 cell line (after additional sorting) was growing into flasks with D-MEM/F-12 medium with 10% FBS and antibiotics at 37° C. in humidified atmosphere with 5% CO2. Cells were kept in the Log phase proliferate activity. On the day of experiment, C6#4 cells were transfer into in 96 well Costar plates 20×10̂3 cell/well in 100 μl/well media and cultivated for 24 hours, than cultural media was replaced with 100 μl/well media, containing the drugs in different concentrations with logarithmic dilutions from 200 μM to 0.1 μM and cultivated for additional 24 hours. TECAN (SAFIRE plate reader) was used for determination of the level of expression GFP and RFP. Excitation wavelength: 484 nm; Emission wavelength: 510 nm. Cell viability (proliferation) was estimated by and WST-1 4 h testing. 11 μl/well of Cell Proliferation Reagent (WST-1) was added into wells with cells, growing in 100 ul media and plates were incubated at 37° C. for additional 4 hours. Measure the absorbance was made by SAFIRE plate reader with wavelength 440 nm for measuring the absorbance of the formazan product, and wavelength 600 nm for the reference.
To analyze possible influence drugs on HIF-1 expression, we first normalized level of the GFP by subdividing data of the GFP expression on data of RFP expression (G/R data) and then compared it to cell viability detected by WST-1 test. We classified the compound as possibly involved in HIF-1 pathways if drug concentration down-regulated GFP expression, stimulated by CoCl2, but did not influence on cell viability.
Statistical Analysis, Detection of the EC-50, and Graphics Made with Graph Pad Prism-4.
Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contain certain errors necessarily resulting from the standard deviation found in their respective testing measurements.
Therefore, the present invention is well adapted to attain the ends and advantages mentioned as well as those that are inherent therein. While numerous changes may be made by those skilled in the art, such changes are encompassed within the spirit of this invention as illustrated, in part, by the appended claims.
This application claims the benefit of U.S. Provisional Application Ser. No. 60/824,501 filed on Sep. 5, 2006, which is incorporated by reference.
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/US2007/077511 | 9/4/2007 | WO | 00 | 2/9/2010 |
| Number | Date | Country | |
|---|---|---|---|
| 60824501 | Sep 2006 | US |
