Patent Application
20060287381
The invention relates to protein kinase inhibitors and to their use in treating disorders related to abnormal protein kinase activities such as cancer and inflammation. More particularly, the invention relates to alpha-hydroxy-ω-(2-oxo-indolylidenemethyl-pyrrole-3′-carbonyl) amino alkanoic acid and amide derivatives and their pharmaceutically acceptable salts employable as protein kinase inhibitors.
Protein kinases are enzymes that catalyze the phosphorylation of hydroxyl groups of tyrosine, serine, and threonine residues of proteins. Many aspects of cell life (for example, cell growth, differentiation, proliferation, cell cycle and survival) depend on protein kinase activities. Furthermore, abnormal protein kinase activity has been related to a host of disorders such as cancer and inflammation. Therefore, considerable effort has been directed to identifying ways to modulate protein kinase activities. In particular, many attempts have been made to identify small molecules that act as protein kinase inhibitors.
Several pyrrolyl-indolinone derivatives have demonstrated excellent activity as inhibitors of protein kinases (Larid et al. FASEB J. 16, 681, 2002; Smolich et al. Blood, 97, 1413, 2001; Mendel et al. Clinical Cancer Res. 9, 327, 2003; Sun et al. J. Med. Chem. 46, 1116, 2003). The clinical utility of these compounds has been promising, but has been partially compromised due to the relatively poor aqueous solubility and/or other drug properties. What is needed is a class of modified pyrrolyl-indolinone derivatives having both inhibitory activity and enhanced drug properties.
The invention is directed to hydroxy carboxy pyrrolyl-indolinone derivatives and to their use as inhibitors of protein kinases. It is disclosed herein that hydroxy carboxy pyrrolyl-indolinone derivatives have enhanced and unexpected drug properties that advantageously distinguish this class of compounds over known pyrrolyl-indolinone derivatives having protein kinase inhibition activity. It is also disclosed herein that hydroxy carboxy pyrrolyl-indolinone derivatives are useful in treating disorders related to abnormal protein kinase activities such as cancer.
One aspect of the invention is directed to a compound represented by Formula (I):
In Formula I, R1 is selected from the group consisting of hydrogen, halo, (C1-C6) alkyl, (C3-C8) cycloalkyl, (C1-C6) haloalkyl, hydroxy, (C1-C6) alkoxy, amino, (C1-C6) alkylamino, amide, sulfonamide, cyano, substituted or unsubstituted (C6-C10) aryl; R2 is selected from the group consisting of hydrogen, halo, (C1-C6) alkyl, (C3-C8) cycloalkyl, (C1-C6) haloalkyl, hydroxy, (C1-C6) alkoxy, (C2-C8) alkoxyalkyl, amino, (C1-C6) alkylamino, (C6-C10) arylamino; R3 is selected from the group consisting of hydrogen, (C1-C6) alkyl, (C6-C10) aryl, (C5-C10) heteroaryl, and amide; R4, R5 and R6 are independently selected from the group consisting of hydrogen and (C1-C6) alkyl; each R7 is independently selected from the group consisting of hydrogen, (C1-C6) alkyl and hydroxyl; R8 is selected from the group consisting of hydroxy, (C1-C6) O-alkyl, (C3-C8) O-cycloalkyl, and NR9R10; where R9 and R10 are independently selected from the group consisting of hydrogen, (C1-C6) alkyl, (C1-C6) hydroalkyl, (C1-C6) dihydroxyalkyl, (C1-C6) alkoxy, (C1-C6) alkyl carboxylic acid, (C1-C6) alkyl phosphoric acid, (C1-C6) alkyl sulfuric acid, (C1-C6) hydroxyalkyl carboxylic acid, (C1-C6) alkyl amide, (C3-C8) cycloalkyl, (C5-C8) heterocycloalkyl, (C6-C8) aryl, (C5-C8) heteroaryl, (C3-C8) cycloalkyl carboxylic acid, or R9 and R10 together with N forms a (C5-C8) heterocyclic ring either unsubstituted or substituted with one or more hydroxyls, ketones, ethers, and carboxylic acids; and n and m are independently 0, 1, 2, or 3; p is 1, 2, or 3. Alternatively, this aspect of the invention may also be directed to a pharmaceutically acceptable salt, its tautomer, a pharmaceutically acceptable salt of its tautomer, or a prodrug of compounds represented by Formula (I). Key features of this aspect of the invention include the hydroxyl moiety or moieties between R6 and R7 and the carboxy moiety between R7 and R8. It is disclosed herein that these key features enhance the drug properties of the attached pyrrolyl-indolinone pharmacophore. Preferred species of this aspect of the invention include compounds represented by the following structures:
In the above structures, R2 is selected from the group consisting of hydrogen and fluoro.
As illustrated above, this first aspect of the invention may be divided into two categories. The first category includes acids and esters; the second category includes amides.
One preferred embodiment of this first category may be represented by Formula (II):
In Formula II, R8a is selected from the group consisting of hydrogen, (C1-C6) alkyl, and (C3-C8) cycloalkyl. Within preferred species of this embodiment, R1 and R2 are independently selected from the group consisting of hydrogen and fluoro; R3 and R4 are methyl; R5, R6, R7 and R8a are hydrogen; and n and m are independently 0, 1, or 2. Preferred species include compounds represented by the following structures:
Another preferred embodiment of this first category may be represented by Formula (III):
In Formula III, R8a is selected from the group consisting of hydrogen, (C1-C6) alkyl, and (C3-C8) cycloalkyl. Within preferred species of this embodiment, R1 and R2 are independently selected from the group consisting of hydrogen and fluoro; R3 and R4 are methyl; R5, R6, and R8a are hydrogen; and n and p are independently 1, or 2. Preferred species of this embodiment include compounds represented by the following structures:
Preferred enantiomeric species of this embodiment of the invention include compounds represented by the following structures:
Another preferred embodiment of this first category may be represented by Formula (IIIa):
In Formula IIIa, R1 and R2 are independently selected from the group consisting of hydrogen and fluoro; R3 and R4 are methyl; R5, R6, and R8a are hydrogen; and n and p are 2. Within this embodiment, each species may exist either as the acid or as the cyclic lactone and they may co-exist in solution or in vivo. Furthermore, in the above examples the stereochemistry at the carbon atom carrying a hydroxyl group is either RS, R, or S. The preferred stereochemistry is R.
An alternative of the above preferred embodiment of this first category may be represented by Formula (IIIb):
In Formula IIIb, R1 and R2 are independently selected from the group consisting of hydrogen and fluoro; and R3 and R4 are methyl. Preferred species of this embodiment include compounds represented by the following structures:
The second category of the first aspect of the invention is embodied by a compound, salt, tautomer, or prodrug according to claim 1 represented by Formula (IV):
wherein R8 is NR9R10. In preferred embodiments of this aspect of the invention, R1 and R2 are independently selected from the group consisting of hydrogen, halo, cyano; R3, R4, R5 and R6 are independently hydrogen or (C1-C6))alkyl; R7 is hydrogen, or hydroxyl; n, and p are independently 1, or 2; m is 0 or 1; and R9 and R10 are selected from the group consisting of hydrogen, (C1-C6) alkyl, (C1-C6) hydroxyalkyl, (C1-C6) dihydroxyalkyl, (C1-C6) alkoxy, (C1-C6) alkyl carboxylic acid, (C1-C6) alkyl phosphoric acid, (C1-C6) alkyl sulfuric acid, (C1-C6) hydroxyalkyl carboxylic acid, (C1-C6) alkyl amide, (C3-C8) cycloalkyl, (C5-C8) heterocycloalkyl, (C6-C8) aryl, (C5-C8) heteroaryl, (C3-C8) cycloalkyl carboxylic acid, or R9 and R10 together with N forms a (C5-C8) heterocyclic ring either unsubstituted or substituted with one or more hydroxyls, ketones, ethers, and carboxylic acids. Preferred examples of R8 include the following radicals:
Preferred species of this embodiment may be selected from the group represented by the following structures:
Further preferred species of this embodiment of the invention may be selected from the group represented by the following structures:
Further preferred species of this embodiment of the invention may be selected from the group represented by the following structures:
wherein n is 0, 1, or 2. Further preferred species of this embodiment of the invention may be selected from the group represented by the following structures:
Further preferred species of this embodiment of the invention may be selected from the group represented by the following structures:
Further preferred species of this embodiment of the invention may be selected from the group represented by the following structures:
Further preferred species of this embodiment of the invention may be selected from the group represented by the following structures:
Further preferred species of this embodiment of the invention may be selected from the group represented by the following structures:
wherein R2 is selected from the group consisting of hydrogen and fluoro; and R8 is selected from the group consisting of radicals represented by the following structures:
Another category within this first aspect of the invention is directed to alpha-hydroxy-omega-(2-oxo-indolylidenemethyl-pyrrole-3′-carbonyl) amino alkanoic acid and amide derivatives and to their use as inhibitors of protein kinases. It is disclosed herein that alpha-hydroxy-ω-(2-oxo-indolylidenemethyl-pyrrole-3′-carbonyl) amino alkanoic acid and amide derivatives have enhanced and unexpected drug properties that advantageously distinguish this class of compounds over known pyrrolyl-indolinone derivatives having protein kinase inhibition activity and over their corresponding beta hydroxy-ω-(2-oxo-indolylidenemethyl-pyrrole-3′-carbonyl) amino alkanoic acid and amide derivatives. It is also disclosed herein that alpha-hydroxy-ω(2-oxo-indolylidenemethyl-pyrrole-3′-carbonyl) amino alkanoic acid and amide derivatives are useful in treating disorders related to abnormal protein kinase activities such as cancer.
One such category within this first aspect of the invention is directed to a compound represented by Formula (V):
In Formula (V), R1 is selected from the group consisting of hydrogen, halo, (C1-C6) alkyl, (C3-C8) cycloalkyl, (C1-C6) haloalkyl, hydroxy, (C1-C6) alkoxy, amino, (C1-C6) alkylamino, amide, sulfonamide, cyano, substituted or unsubstituted (C6-C10) aryl; R2 is selected from the group consisting of hydrogen, halo, (C1-C6) alkyl, (C3-C8) cycloalkyl, (C1-C6) haloalkyl, hydroxy, (C1-C6) alkoxy, (C2-C8) alkoxyalkyl, amino, (C1-C6) alkylamino, (C6-C10) arylamino; R3 is selected from the group consisting of hydrogen, (C1-C6) alkyl, (C6-C10) aryl, (C5-C10) heteroaryl, and amide; R4, R5 and R6 are independently selected from the group consisting of hydrogen and (C1-C6) alkyl; R7 is selected from the group consisting of hydroxy, (C1-C6) O-alkyl, (C3-C8) O-cycloalkyl, and NR8R9; where R8 and R9 are independently selected from the group consisting of hydrogen, (C1-C6) alkyl, (C1-C6) hydroxyalkyl, (C1-C6) dihydroxyalkyl, (C1-C6) alkoxy, (C1-C6) alkyl carboxylic acid, (C1-C6) alkyl phosphonic acid, (C1-C6) alkyl sulfonic acid, (C1-C6) hydroxyalkyl carboxylic acid, (C1-C6) alkyl amide, (C3-C8) cycloalkyl, (C5-C8) heterocycloalkyl, (C6-C8) aryl, (C5-C8) heteroaryl, (C3-C8) cycloalkyl carboxylic acid, or R8 and R9 together with N forms a (C5-C8) heterocyclic ring either unsubstituted or substituted with one or more hydroxyls, ketones, ethers, and carboxylic acids; and n is 1, 2, or 3. Alternatively, this aspect of the invention may be directed to a pharmaceutically acceptable salt, its tautomer, a pharmaceutically acceptable salt of its tautomer, or a prodrug of the compound of Formula (V). Preferred species of the invention include compounds represented by the following structures:
In the above structures, R2 is selected from the group consisting of hydrogen and fluoro. More particularly, a preferred stereoisomer is represented by the following structure:
A first subgenus of this aspect of the invention is represented by Formula (VI):
In Formula (VI), R10 is selected from the group consisting of hydrogen, (C1-C6) alkyl, and (C3-C8) cycloalkyl. In preferred species of this first subgenus, R1 and R2 are independently selected from the group consisting of hydrogen and fluoro;
Provisos may apply to any of the above categories or embodiments wherein any one or more of the other above described embodiments or species may be excluded from such categories or embodiments.
A second aspect of the invention is directed to a method for the modulation of the catalytic activity of a protein kinase with a compound or salt represented by Formulas I-VII, above. In a preferred mode of the second aspect of the invention, said protein kinase is selected from the group of receptors consisting of VEGF, PDGF, c-kit, Flt-3, Axl, and TrkA.
Utility:
The present invention provides compounds capable of regulating and/or modulating protein kinase activities of, but not limited to, VEGFR and/or PDGFR. Thus, the present invention provides a therapeutic approach to the treatment of disorders related to the abnormal functioning of these kinases. Such disorders include, but not limited to, solid tumors such as glioblastoma, melanoma, and Kaposi's sarcoma, and ovarian, lung, prostate, pancreatic, colon and epidermoid carcinoma. In addition, VEGFR/PDGFR inhibitors may also be used in the treatment of restenosis and diabetic retinopathy.
Furthermore, this invention relates to the inhibition of vasculogenesis and angiogenesis by receptor-mediated pathways, including the pathways comprising VEGF receptors, and/or PDGF receptors. Thus the present invention provides therapeutic approaches to the treatment of cancer and other diseases which involve the uncontrolled formation of blood vessels.
The compounds of this invention can be synthesized by following the published general procedures (e.g. Sun et al., 2003, J. Med. Chem., 46:1116-119). But the following intermediates are specific to compounds of this invention and may be used in place of their respective counterparts in the above-mentioned general procedure: 4,5-difluoro-oxindole; (4R,6R)-t-butyl-6-(2-aminoethyl)-2,2-dimethyl-1,3-dioxane-4-acetate; t-Butyl(3R,5S)-6-hydroxy-3,5-O-isopropylidene-3,5-dihydroxyhexanoate, and 4-amino-3-hydroxy-butanic acid. These intermediates may be purchased from commercial sources (e.g. Fisher Scientific, Fairlawn, N.J., or Kaneka Corp., Japan). Another variation from the above-mentioned general procedure is that in the synthesis of 1/1a and 2/2a using (4R,6R)-t-butyl-6-(2-aminoethyl)-2,2-dimethyl-1,3-dioxane-4-acetate, the protecting groups need to be removed from the final product. Yet another variation from the above-mentioned general procedure is that in the synthesis of 3 and 4 using 4-amino-3-hydroxy-butanic acid, the acid needs to be protected before amidation and the protection group needs to be removed from the final product. These variations from the above-mentioned general procedure can be understood and carried out by those skilled in the art. Thus, the compounds of the present invention can be synthesized by those skilled in the art.
The compounds described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.
The synthesis of the title compound is summarized in Scheme 1. In the first step, 5-fluoro-1,3-dihydroindol-2-one (1A, purchased from Combi-Blocks, Inc.) was condensed with 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid in refluxing ethanol under the influence of catalytic amounts of pyrrolidine in analogy to the literature-known preparation of similar compounds (Li Sun, Chris Liang, Sheri Shirazian, Yong Zhou, Todd Miller, Jean Cui, Juri Y. Fukuda, Ji-Yu Chu, Asaad Nematalla, Xueyan Wang, Hui Chen, Anand Sistla, Tony C. Luu, Flora Tang, James Wei, and Cho Tang. Discovery of 5-[5-Fluoro-2-oxo-1,2-dihydroindol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylic Acid (2-Diethylaminoethyl)amide, a Novel Tyrosine Kinase Inhibitor Targeting Vascular Endothelial and Platelet-Derived Growth Factor Receptor Tyrosine Kinase. J. Med. Chem. 2003, 46, 1116-1119) to give pyrrole carboxylic acid 1B in 92% yield.
Amide coupling between carboxylic acid 1B and amine 1C (obtained from Acros) was affected by treatment with hydroxybenzotriazole, 1-(3-dimethylaminopropyl-3-ethylcarbodiimide hydrochloride, and triethylamine in DMF to afford 1D, after chromatographic purification, in 96% yield. Removal of the acetonide and tert-butyl ester protective groups was then conducted in a stepwise fashion (H. Jendralla, E. Granzer, B. Von Kerekjarto, R. Krause, U. Schacht, E. Baader, W. Bartmann, G. Beck, A. Bergmann, and et al. Synthesis and biological activity of new HMG-CoA reductase inhibitors. 3. Lactones of 6-phenoxy-3,5-dihydroxyhexanoic acids. J. Med. Chem. 1991, 34, 2962-2983). First, the acetonide protection in 1D was removed by treatment with aqueous HCl in a mixture of THF and ethanol to give an intermediary ester diol (not shown), which was isolated by extraction after neutralization of the reaction mixture with sodium bicarbonate. This intermediate was then treated with aqueous NaOH (1 equiv) in methanol to furnish the title compound: (3R,5R)-7-{[5-(5-Fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carbonyl]-amino}-3,5-dihydroxyheptanoic acid, sodium salt (87% yield over both steps) after concentration of the reaction mixture as a yellow solid.
A mixture of 5-fluoro-1,3-dihydroindol-2-one (0.81 g, 5.1 mmol), 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (0.98 g, 5.35 mmol), pyrrolidine (6 drops) and absolute ethanol (15 mL) was heated to reflux for 3 hours. The mixture was cooled to room temperature and the solids were collected by filtration. The solids were stirred with ethanol (14 mL) at 72° C. for 30 minutes. The mixture was cooled to room temperature. The solids were collected by filtration, washed with ethanol (3 mL), dried under vacuum at 54° C. overnight to give 5-(5-fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (1.4 g, 91.5% yield) as an orange solid. 1H NMR (300 MHz, DMSO-d6) δ 12.19 (br s, 1H), 10.95 (s, 1H), 7.90-7.70 (m, 2H), 7.00-6.80 (m, 2H), 2.54 (s, 3H), 2.51 (s, 3H). 13C NMR (75 MHz, DMSO-d6) δ 169.4, 165.7, 159.6, 156.5, 140.7, 134.6, 133.3, 128.9, 126.8, 125.9, 124.7, 115.5, 114.2, 110.9, 110.0, 106.3, 105.9, 14.6, 11.6.
To a stirred solution of 5-(5-fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (1.3 g, 4.33 mmol) in dimethylformamide (11.6 mL) at room temperature were added 1-(3-dimethylaminopropyl-3-ethylcarbodiimide hydrochloride (1.25 g, 6.39 mmol); hydroxybenzotriazole (0.88 g, 6.39 mmol), triethylamine (1.3 mL, 9.34 mmol), and (4R,6R)-[6-(2-aminoethyl)-2,2-dimethyl-[1,3]dioxan-4-yl]-acetic acid tert-butyl ester (1.38 g, 4.87 mmol). The reaction mixture was stirred at room temperature for 30 h, then filtered through a silica gel pad and washed with ethyl acetate (100 mL). The filtrate was concentrated and the residue was diluted with water (20 mL), saturated sodium bicarbonate solution (10 mL) and 10 N sodium hydroxide solution (5 mL). The mixture was extracted with a mixture of methylene chloride/methanol (9/1, 2×50 mL). The combined organic layers were concentrated to dryness. The residue was triturated with heptane/diethyl ether (3/1, 60 mL). The solids were collected by filtration and dried under vacuum at 34° C. overnight to obtain (4R,6R)-[6-(2-{[5-(fluoro-2-oxo-1,2-dihydro-indol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carbonyl]-amino}ethyl)-2,2-dimethyl-[1,3]dioxan-4-yl]-acetic acid tert-butyl ester (2.3 g, 95.6%) as a yellow solid. 1H NMR (300 MHz, DMSO-d6) δ 11.05 (br s, 1H), 7.94 (d, J=6.9 Hz, 1H), 7.85 (s, 1H), 7.14-6.90 (m, 2H), 4.35 (m, 1H), 4.12 (m, 1H), 3.51 (br s, 1H), 3.42 (m, 2H), 2.64 (m, 2H), 2.57 (s, 3H), 2.56 (s, 3H), 2.50-2.30 (m, 2H), 1.76 (m, 3H), 1.54 (s, 9H), 1.41 (s, 3H), 1.24 (m, 1H). 13C NMR (75 MHz, DMSO-d6) δ 169.4, 164.4, 159.6, 156.5, 136.2, 134.3, 129.9, 127.1, 126.9, 125.6, 124.7, 120.8, 114.4, 112.3, 112.0, 109.9, 109.8, 105.9, 105.6, 97.9, 79.6, 66.5, 65.9, 42.2, 35.9, 35.8, 35.1, 29.9, 27.7, 19.6, 13.3, 10.5.
Under argon atmosphere and exclusion of light, a solution of (4R,6R)-[6-(2-{[5-(fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carbonyl]-amino}-ethyl)-2,2-dimethyl-[1,3]dioxan-4-yl]-acetic acid tert-butyl ester (1.69 g, 3.04 mmol) in ethanol (15.2 mL), THF (7.6 mL) and 2 N hydrochloric acid (1.7 mL) was stirred at room temperature for 24 hours. The reaction mixture was neutralized with sodium bicarbonate solution (0.256 g NaHCO3 in 5 mL water) to pH 7 and concentrated to remove ethanol and THF. The residue was diluted with water (50 mL) and extracted with a mixture of methyl tert-butyl ether/methanol (9/1, 200 mL), and then with methyl tert-butyl ether (3×50 mL). The combined organic layers were dried over magnesium sulfate and concentrated to dryness to give (3R,5R)-7-{[5-(5-fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carbonyl]-amino}-3,5-dihydroxyheptanoic acid tert-butyl ester (1.57 g, 3 mmol). This ester (1.56 g, 3.0 mmol) was dissolved in methanol (33.4 mL) and a solution of sodium hydroxide (0.12 g, 3.0 mmol) in deionized water (8.3 mL) was added. The mixture was stirred at room temperature for 3 hours. The reaction mixture was concentrated to dryness. The residue was dissolved in methanol (66 mL) and the mixture was concentrated again. The mixture was triturated with isopropanol (40 mL). The solids were collected by filtration, washed with diethyl ether (100 mL) and dried under vacuum at 34° C. for 3 hours to furnish (3R,5R)-7-{[5-(5-fluoro-2-oxo-1,2-dihydroindol-3-ylidenemethyl)-2,4-dimethyl-1H-pyrrole-3-carbonyl]-amino}-3,5-dihydroxyheptanoic acid, sodium salt (1.28 g, 87.4% yield over two steps) as a yellow solid. Mp 256-258° C. (decomposition). 1H NMR (300 MHz, methanol-d4) δ 7.49 (s, 1H), 7.31 (d, J=8.4 Hz, 1H), 6.74 (d, J=6.6 Hz, 1H), 4.03 (m, 1H), 3.83 (m, 1H), 3.45 (m, 1H), 3.37 (m, 1H), 2.38 (s, 3H), 2.34 (s, 3H), 2.25 (m, 2H), 1.85-1.40 (m, 4H). 13C NMR (75 MHz, methanol-d4) δ 180.1, 171.4; 168.4, 161.8, 158.7, 137.7, 135.7, 131.4, 128.6, 128.5, 127.3, 125.2, 121.1, 116.4, 113.6, 113.3, 111.1, 110.9, 106.3, 106.0, 69.1, 68.9, 45.5, 44.9, 37.8, 37.7, 13.4, 10.8.
The advanced intermediate 4-Amino-2-ethyl-3-hydroxy-butyric acid ethyl ester can be made following published procedures (e.g. Seebach, Dieter; Chow, Hak-Fun; Jackson, Richard F. W.; Lawson, Kevin; Sutter, Marius A.; et al.; J. Am. Chem. Soc. 1985, 107, 18, 5292-5293. Itoh, Toshiyuki; Takagi, Yumiko; Fujisawa, Tamotsu; Tetrahedron Lett. 1989, 30; 29, 3811-3812). Subsequent amide coupling with 1B followed by deprotection afforded the title compound.
The title compound was prepared following the procedure described in the preparation of Example 1. In this synthesis, 4,5-difluoro-1,3-dihydroindol-2-one was used instead of 5-fluoro-1,3-dihydroindol-2-one as in Example 1. LC-MS: a single peak was observed at 254 nm, MH+ calcd for the free acid C23H25F2N3O6: 478, obtained 478. 1H NMR (400 MHz, methanol-d4) δ 7.71 (d, J=2.4 Hz, 1H), 7.00 (m, 1H), 6.65 (dd, J=3.2 Hz, J=8.4 Hz, 1H), 4.13 (m, 1H), 3.93 (m, 1H), 3.56 (m, 1H), 3.45 (m, 1H), 2.48 (s, 3H), 2.39 (s, 3H), 2.34 (m, 2H), 1.84 (m, 1H), 1.69 (m, 3H).
The title compound was prepared following the procedure described in the preparation of Example 1. In this synthesis, 4-amino-3-hydroxybutanoic acid was used instead of (4R,6R)-[6-(2-aminoethyl)-2,2-dimethyl-[1,3]dioxan-4-yl]-acetic acid tert-butyl ester as in Example 1. LC-MS: a single peak was observed at 254 nm, MH+ calcd for the free acid C20H20FN3O5: 402, obtained 402. 1H NMR (400 MHz, DMSO-d6) δ 13.68 (s, 1H), 11.40 (s, 1H), 10.90 (s, 1H), 7.76 (dd, J=3.2 Hz, J=8.4 Hz, 1H), 7.71 (s, 1H), 7.59 (t, J=4.8 Hz, 1H), 6.92 (m, 1H), 6.83 (dd, J=4.8 Hz, J=9.2 Hz, 1H), 4.00 (m, 1H), 3.33 (m, 2H, buried in water signals), 3.24 (m, 2H), 2.43 (s, 3H), 2.41 (s, 3H).
The title compound was prepared following the procedure described in the preparation of Example 1. In this synthesis, 4-amino-3-hydroxylbutanoic acid was used instead of (4R,6R)-[6-(2-aminoethyl)-2,2-dimethyl-[1,3]dioxan-4-yl]-acetic acid tert-butyl ester as in Example 1. LC-MS: a single peak was observed at 254 nm, MH+ calcd for the free acid C20H19F2N3O5: 420, obtained 420. 1H NMR (400 MHz, DMSO-d6) δ 13.55 (s, 1H), 12.10 (s, 1H), 11.15 (s, 1H), 7.67 (t, J=6.0 Hz, 1H), 7.59 (d, J=2.0 Hz, 1H), 7.14 (m, 1H), 6.68 (dd, J=3.2 Hz, J=8.4 Hz, 1H), 5.05 (b, 1H), 4.03 (m, 1H), 3.31 (m, 2H), 3.25 (m, 2H), 2.44 (s, 3H), 2.32 (s, 3H).
Preparation of ((4R,6S)-6-Aminomethyl-2,2-dimethyl-[1,3]dioxan-4-yl)-acetic acid tert-butyl ester: Triflic anhydride 1.4 mL (2.36 g, 8.345 mmol) was dropwise added at −78° C. to a solution of 2,6-lutidine 1.35 mL (11.63 mmol) and t-Butyl-(3R,5S)-6-hydroxy-3,5-O-isopropylidene-3,5-dihydroxy-hexanoate 1.981 g (7.609 mmol, obtained from Kaneka Corp.) in dichloromethane (anh., 50 mL) over 3 minutes. The mixture was stirred at −78° C. for 10 min, then placed on ice-slush bath and stirred at 0° C. for 45 min. The resulting pink mixture was transferred into ice-cooled solution of ammonia in methanol (7 M solution, 200 mL). The mixture was placed on ambient water bath and stirred at RT for 6 hours. The reaction mix was evaporated to dryness, the residue partitioned between ether (200 mL) and aqueous potassium carbonate (6 g in 200 mL of water), the aqueous phase re-extracted with ether (150 mL). Combined organic extracts were dried (magnesium sulfate) and evaporated. The crude product was purified on a column of silica (125 g) eluting with a mix of chloroform-methanol-conc. aq. ammonia 100:10:1 (v/v) (1.5 L) to give Y=1.777 g of a colorless liquid (90%), ((4R,6S)-6-Aminomethyl-2,2-dimethyl-[1,3]dioxan-4-yl)-acetic acid tert-butyl ester.
1H (dDMSO, 400 MHz): δ 4.167 (m, 1H), 3.741 (m, 1H), 2.484 (m, 2H), 2.384 (ddAB, J=15.2 Hz, 5.1 Hz, 1H), 2.201 (ddAB, J=15 Hz, 7.8 Hz, 1H), 1.533 (br d, J=12.5 Hz, 1H), 1.373 (s, 9H), 1.363 (s, 3H), 1.250 (br s, 2H), 1.223 (s, 3H).
5-[(Z)-(5-fluoro-2-oxo-1,2-dihydro-3H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-carboxylic acid 1-oxy-7-azabenztriazole ester 419 mg (1.00 mmol, prepared according to U.S. Pat. No. 6,653,308) was suspended in anh. dimethylacetamide (4 mL) and a solution of ((4R,6S)-6-Aminomethyl-2,2-dimethyl-[1,3]dioxan-4-yl)-acetic acid tert-butyl ester 310 mg (1.2 mmol) and diisopropylethyl amine 175 μL (1.0 mmol) in anh. DMAc (7 mL) was added to the slurry. The mixture was stirred for 20 min at RT. The obtained homogenous mixture was evaporated under high vacuum (0.5 Torr, 45° C.). The residue was taken up with methanol 10 mL, sonicated for 2 minutes, then allowed to crystallize at 5° C. for 3 hours. The precipitated intermediate (acetonide-tBu ester) was collected by filtration, washed with ice-cold methanol and dried under high vacuum. This intermediate (485 mg of an orange-yellow cryst. solid, 89.5% th.) was dissolved in neat TFA 20 mL and the obtained solution was kept at RT for 15 min, then evaporated. The residue was dried under high vacuum for 1 day. The residue was dissolved in a mixture of methanol 100 mL and THF 100 mL (with 15 min stirring). 40 mL of 1M NaOH was added and the mixture was kept at RT for 30 min. The mixture was acidified with 2M HCl to pH=3. The mixture was concentrated to a small volume on rotavap to remove organic solvents, the precipitate was collected by filtration, washed with water and dried by suction, then under high vacuum. This precipitate (consisting of the free acid with approx 5% of the corresponding lactone) was dissolved in a mixture of methanol (200 mL), water (30 mL) and 1M NaOH (0.96 mL) with stirring and gentle heating to reflux for 3 minutes. The mixture was stirred at RT for additional 15 minutes, then saturated with CO2 (g), evaporated to dryness and dried under high vacuum to give Y=376.5 mg (90%) of an orange solid, (3R,5S)-6-({5-[5-Fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carbonyl}-amino)-3,5-dihydroxy-hexanoic acid, sodium salt. LC/MS(+ESI): 446 (M+1)
1H (D2O, 400 MHz): 6.655 (br d, J=9.4 Hz, 1H), 6.594 (m, 2H), 6.292 (dd, J=8.2 Hz, 4.7 Hz, 1H), 4.155 (m, 1H), 3.891 (m, 1H), 3.405 (dd, J=14.1 Hz, 3.9 Hz, 1H), 3.195 (dd, J=15.7 Hz, 7.5 Hz, 1H), 2.429 (ddAB, J=14.9 Hz, 5.0 Hz, 1H), 2.329 (ddAB, J=14.9 Hz, 8.2 Hz, 1H), 1.782 (m, 2H).
The general procedure for the synthesis of amides of Examples 3 and 4 is shown in Scheme 2 below:
A corresponding amine (0.3 mmol) was added to a solution of compound 6A (80 mg, 0.2 mmol), EDC (0.25 mmol), HOBt (0.25 mmol), and DIEA (1 mmol) in DMF (3 mL). After the solution was stirred at 25° C. overnight, DMF was removed via evaporation under reduced pressure. The resulting residue was suspended in ethyl acetate (200 mL), washed by saturated NaHCO3 (3×) and brine (3×), and dried over Na2SO4. The ethyl acetate was removed under vacuum to give the crude product. This crude material was subjected to preparative HPLC to give the final product 6B, which was subsequently characterized by LC-MS and NMR spectroscopy.
Preparative HPLC gave 70 mg of the title compound (75%). LC-MS: single peak at 254 nm, MH+ calcd. for C24H26F2N4O5: 489, obtained: 489. 1H-NMR (DMSO-d6, 400 MHz), δ 13.55 (s, 1H), 11.20 (s, 1H), 7.64 (t, J=6.0 Hz, 1H), 7.58 (d, J=2.4 Hz, 1H), 7.13 (m, 1H), 6.70 (dd, J=3.2 Hz, J=8.4 Hz, 1H), 4.99 (s, 1H), 4.04 (m, 1H), 3.20-3.60 (m, 12H), 2.45 (s, 3H), 2.32 (s, 3H).
Preparative HPLC gave 50 mg of the title compound (53%). LC-MS: single peak at 254 nm, MH+ calcd. for C24H27FN4O5: 471, obtained: 471. 1H-NMR (DMSO-d6, 400 MHz), δ 13.69 (s, 1H), 10.91 (s, 1H), 7.76 (dd, J=3.2 Hz, J=9.2 Hz, 1H), 7.71 (s, 1H), 7.57 (t, J=6.0 Hz, 1H), 6.95 (m, 1H), 6.83 (dd, J=4.8 Hz, J=8.8 Hz, 1H), 4.98 (d, J=5.2 Hz, 1H), 4.04 (m, 1H), 3.53 (m, 5H), 3.45 (m, 4H), 3.28 (m, 3H), 2.43 (s, 3H), 2.41 (s, 3H).
Preparative HPLC gave 55 mg of the title compound (57%). LC-MS: single peak at 254 nm, MH+ calcd. for C25H30FN5O4: 484, obtained: 484. 1H-NMR (DMSO-d6, 400 MHz), δ 13.65 (s, 1H), 10.90 (s, 1H), 7.74 (m, 2H), 7.71 (m, 1H), 7.54 (m, 1H), 6.92 (m, 1H), 6.83 (m, 1H), 4.95 (s, 1H), 4.04 (m, 1H), 3.44 (m, 4H), 3.25 (m, 4H, buried in water signals), 2.43 (s, 3H), 2.41 (s, 3H), 2.25 (m, 4H), 2.16 (s, 3H).
The general procedure for the synthesis of amides of Examples 1 and 5 is shown in Scheme 3 below:
Method 1: EDC (1 mmol), and HOBt (0.6 mmol) were added to a solution of compound 9A (0.2 mmol) in DMF (3 mL). After the solution was stirred at 25° C. for 3 h, the corresponding amine (1.0 mmol) was added, and the solution was stirred at 25° C. overnight. If the reaction was not complete, the solution was stirred at 50° C. for another couple of hours. This DMF solution was directly subjected to preparative HPLC to obtain the final product 9B, which was subsequently characterized by LC-MS and proton NMR spectroscopy.
Method 2: TBDMS-Cl (1.0 mmol), and DMAP (1.0 mmol) were added to a solution of compound 9A (0.2 mmol) in DMF (3 mL). After the solution was stirred at 25° C. for 5 h (LC-MS demonstrated that a mixture of mono- and disilyl ether products was formed), EDC (1 mmol), HOBt (0.6 mmol), and the corresponding amine (0.4 mmol) were added to the solution. The solution was continuously stirred at 25° C. overnight (LC-MS demonstrated that a mixture of the amides of the corresponding mono- and di-silyl ether products was formed). After the solvent was removed via evaporation under reduced pressure, the resulting residue was suspended in ethyl acetate (100 mL), washed with saturated NaHCO3 (3×), and brine (3×). The organic solvent was then evaporated under vacuum to give the crude silyl ether amide products. TBAF (3 equiv, 1M in THF) was added to a solution of this crude material in THF. After stirring at 25° C. for 30 min., the THF was removed under reduced pressure. The residue was suspended in ethyl acetate (100 mL), washed with brine (3×). The organic solvent was then evaporated under reduced pressure, and the resulting residue was directly subjected to preparative HPLC to obtain the final product 9B, which was subsequently characterized by LC-MS and proton NMR spectroscopy.
This compound was prepared via Method 2. An amount of 65 mg (64%) product was obtained after preparative HPLC. LC-MS: single peak at 254 nm, MH+ calcd. for C25H31FN4O5: 487, obtained: 487. 1H-NMR (DMSO-d6, 400 MHz), δ 13.67 (s, 1H), 10.90 (s, 1H), 7.76 (dd, J=2.8 Hz, J=9.2 Hz, 1H), 7.71 (s, 1H), 7.63 (t, J=5.6 Hz, 1H), 6.92 (m, 1H), 6.83 (dd, J=4.8 Hz, J=8.8 Hz, 1H), 4.72 (d, J=4.4 Hz, 1H), 4.67 (d, J=4.8 Hz, 1H), 4.00 (m, 1H), 3.71 (m, 1H), 3.31 (m, 2H), 2.96 (s, 3H), 2.80 (s, 1H), 2.42 (s, 3H), 2.40 (s, 3H), 2.39 (m, 2H), 1.65 (m, 1H), 1.52 (m, 3H).
This compound was prepared via Method 1. An amount of 55 mg (61%) product was obtained after preparative HPLC. LC-MS: single peak at 254 nm, MH+ calcd. for C27H33FN4O5: 513, obtained: 513. 1H-NMR (DMSO-d6, 400 MHz), δ 13.66 (s, 1H), 10.90 (s, 1H), 7.76 (dd, J=2.4 Hz, J=9.2 Hz, 1H), 7.71 (s, 1H), 7.63 (t, J=5.6 Hz, 1H), 6.91 (m, 1H), 6.83 (dd, J=4.8 Hz, J=8.8 Hz, 1H), 4.75 (d, J=4.4 Hz, 1H), 4.68 (d, J=4.8 Hz, 1H), 4.03 (m, 1H), 3.70 (m, 1H), 3.40 (m, 2H), 3.26 (m, 4H), 2.42 (s, 3H), 2.40 (s, 3H), 2.34 (m, 2H), 1.83 (m, 2H), 1.74 (m, 2H), 1.65 (m, 1H), 1.52 (m, 3H).
This compound was prepared via Method 2. An amount of 72 mg (66%) product was obtained after preparative HPLC. LC-MS: single peak at 254 nm, MH+ calcd. for C27H33FN4O6: 529, obtained: 529. 1H-NMR (DMSO-d6, 400 MHz), δ 13.66 (s, 1H), 7.76 (dd, J=2.4 Hz, J=9.2 Hz, 1H), 7.71 (s, 1H), 7.63 (t, J=5.6 Hz, 1H), 6.91 (m, 1H), 6.83 (dd, J=4.8 Hz, J=8.8 Hz, 1H), 6.70 (b, 1H), 4.71 (d, J=4.4 Hz, 1H), 4.67 (d, J=4.8 Hz, 1H), 4.01 (m, 1H), 3.70 (m, 1H), 3.51 (m, 5H), 3.45 (m, 3H), 3.42-3.24 (m, 4H), 2.42 (s, 3H), 2.40 (s, 3H), 1.65 (m, 1H), 1.52 (m, 3H).
This compound was prepared via Method 2. An amount of 30 mg (27%) product was obtained after preparative HPLC. LC-MS: single peak at 254 nm, MH+ calcd. for C28H36FN5O5: 542, obtained: 542. 1H-NMR (DMSO-d6, 400 MHz), δ 13.66 (s, 1H), 7.76 (dd, J=2.4 Hz, J=9.2 Hz, 1H), 7.70 (s, 1H), 7.63 (t, J=5.6 Hz, 1H), 6.91 (m, 1H), 6.84 (dd, J=4.8 Hz, J=8.8 Hz, 1H), 4.70 (b, 2H), 4.01 (m, 1H), 3.70 (m, 1H), 3.43 (m, 4H), 3.30 (m, 4H), 2.42 (s, 3H), 2.40 (s, 3H), 2.26 (m, 2H), 2.21 (m, 2H), 2.15 (s, 3H), 1.65 (m, 1H), 1.52 (m, 3H).
This compound was prepared via Method 1. An amount of 66 mg (54%) product was obtained after preparative HPLC. LC-MS: single peak at 254 nm, MH+ calcd. for C24H29FN4O5: 473, obtained: 473. 1H-NMR (DMSO-d6, 400 MHz), δ 13.72 (s, 1H), 10.90 (s, 1H), 7.77 (dd, J=2.4 Hz, J=9.2 Hz, 1H), 7.72 (s, 1H), 7.49 (t, J=5.6 Hz, 1H), 6.92 (m, 1H), 6.83 (dd, J=4.4 Hz, J=8.4 Hz, 1H), 4.80 (s, 1H), 4.78 (s, 1H), 4.05 (m, 1H), 3.76 (m, 1H), 3.41 (m, 2H), 3.26 (m, 4H), 2.44 (s, 3H), 2.42 (s, 3H), 2.36 (m, 2H), 1.85 (m, 2H), 1.75 (m, 2H), 1.61 (m, 1H), 1.50 (m, 1H).
This compound was prepared via Method 2. An amount of 97 mg (75%) product was obtained after preparative HPLC. LC-MS: single peak at 254 nm, MH+ calcd. for C27H34FN5O5: 528, obtained: 528. 1H-NMR (DMSO-d6, 400 MHz), δ 13.72 (s, 1H), 10.90 (s, 1H), 7.75 (dd, J=2.4 Hz, J=9.6 Hz, 1H), 7.70 (s, 1H), 7.48 (t, J=5.6 Hz, 1H), 6.92 (m, 1H), 6.83 (dd, J=4.4 Hz, J=8.4 Hz, 1H), 4.82 (s, 1H), 4.72 (s, 1H), 4.05 (m, 1H), 3.77 (m, 1H), 3.43 (m, 4H), 3.25 (m, 2H), 3.15 (m, 4H), 2.44 (s, 3H), 2.41 (s, 3H), 2.27 (m, 2H), 2.20 (m, 2H), 2.15 (s, 3H).
This compound was prepared via Method 2. An amount of 40 mg (40%) product was obtained after preparative HPLC. LC-MS: single peak at 254 nm, MH+ calcd for C25H31FN4O5: 487, obtained: 487. 1H-NMR (DMSO-d6, 400 MHz), δ 13.68 (s, 1H), 10.90 (s, 1H), 7.78 (t, J=5.6 Hz, 1H), 7.75 (dd, J=2.8 Hz, J=9.2 Hz, 1H), 7.70 (s, 1H), 7.61 (t, J=5.6 Hz, 1H), 6.92 (m, 1H), 6.83 (dd, J=4.8 Hz, J=8.8 Hz, 1H), 4.76 (s, 1H), 4.67 (s, 1H), 3.96 (m, 1H), 3.69 (m, 1H), 3.28 (m, 2H), 3.04 (m, 2H), 2.42 (s, 3H), 2.40 (s, 3H), 2.16 (m, 2H), 1.65 (m, 1H), 1.52 (m, 3H), 0.99 (t, 7.6 Hz, 3H).
This compound was prepared via Method 2. An amount of 87 mg (69%) product was obtained after preparative HPLC. LC-MS: single peak at 254 nm, MH+ calcd for C26H31FN4O6: 515, obtained: 515. 1H-NMR (DMSO-d6, 400 MHz), δ 13.69 (s, 1H), 10.88 (s, 1H), 7.76 (dd, J=2.4 Hz, J=9.2 Hz, 1H), 7.71 (s, 1H), 7.48 (t, J=4.0 Hz, 1H), 6.91 (m, 1H), 6.83 (dd, J=4.8 Hz, J=9.2 Hz, 1H), 4.84 (d, J=4.4 Hz, 1H), 4.74 (d, J=4.4 Hz, 1H), 4.05 (m, 1H), 3.77 (m, 1H), 3.50 (m, 9H), 3.25 (m, 3H), 2.44 (s, 3H), 2.41 (s, 3H), 1.58 (m, 2H).
Further amide examples of Example 1. The following examples 17a-f can be made by those skilled in the art following the above procedure and/or known procedures.
Further amide examples of Example 3. The following examples 18a-f can be made by those skilled in the art following the above procedure and/or known procedures.
Further amide examples of Example 5. The following examples 19a-d can be made by those skilled in the art following the above procedure and/or known procedures.
The compounds described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.
Still further amide examples of Examples 1-5 are shown in the following table.
In the above table, R2 is selected from the following radicals:
These amide examples 20-269 can be made by those skilled in the art following the above procedure and/or known procedures.
The synthesis of acid (1-3) and amides (1-4) is shown in
Compound 1-1 was prepared by following a literature procedure used for similar compounds (Li Sun, Chris Liang, et al; Discovery of 5-[5-Fluoro-2-oxo-1,2-dihydroindol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carboxylic Acid (2-Diethylaminoethyl)amide, a Novel Tyrosine Kinase Inhibitor Targeting Vascular Endothelial and Platelet-Derived Growth Factor Receptor Tyrosine Kinase. J. Med. Chem. 2003, 46, 1116-1119). Compound 1-1 and DIEA (di-isopropyl ethylamine) (3 equiv) were suspended in dry DMF at room temperature (
The general procedure for the synthesis of amides of Example 301: An amine (2 equiv) was added to a solution of the acid from Example 301, HATU (1.05 mmol), and DIEA (5 equiv) in DMF (5 mL). After the solution was stirred at 25° C. for 2 h, aqueous HCl (2 mL, 1N) was added. This solution was subjected to preparative HPLC to obtain the pure amide product, which was subsequently characterized by LC-MS and NMR spectroscopy.
Preparative HPLC gave 32 mg of the title compound (34%) from 90 mg starting material (acid). LC-MS: single peak at 254 nm, MH+ calcd. for C24H27FN4O4: 455, obtained: 455.
Preparative HPLC gave 27 mg of the title compound (41%) from 61 mg starting material (acid). LC-MS: single peak at 254 nm, MH+ calcd. for C24H27FN4O5: 471, obtained: 471.
Preparative HPLC gave 22 mg of the title compound (37%) from 61 mg starting material (acid). LC-MS: single peak at 254 nm, MH+ calcd. for C22H25FN4O4: 429, obtained: 429.
Preparative HPLC gave 43 mg of the title compound (27%) from 140 mg starting material (acid). LC-MS: single peak at 254 nm, MH+ calcd. for C24H29FN4O4: 457, obtained: 457.
Preparative HPLC gave 15 mg of the title compound (20%) from 81 mg starting material (acid). LC-MS: single peak at 254 nm, MH+ calcd. for C20H21FN4O4: 401, obtained: 401.
Preparative HPLC gave 18 mg of the title compound (21%) from 81 mg starting material (acid). LC-MS: single peak at 254 nm, MH+ calcd. for C24H27FN4O5: 471, obtained: 471.
The synthesis of acid (2-2) and amides (2-3) is shown in
To a solution of compound 1-2 (1.0 mmol) and DIEA (3 equiv) in DMF, the HCl salt of methyl 3-amino-2-hydroxypropionate (1.2 equiv, prepared by refluxing the isoserine in dry methanol with 1.2 equiv HCl) was added. After stirring at 25° C. for 2 h (at which time LC-MS showed the completion of the reaction), KOH in water (5 equiv) was added, and the stirring was continued until the hydrolysis was complete (monitored by LC-MS). The solvents were removed by evaporation under reduced pressure. Aqueous HCl (1N) was added to the residue, and the precipitate was collected by filtration, washed with water, and dried under high vacuum to obtain compound 2-2 (0.33 g, 85%). LC-MS: single peak at 254 nm, MH+ calcd. for C19H18FN3O5: 388, obtained: 388.
The general procedure for the synthesis of amides of Example 308: An amine (2 equiv) was added to a solution of the acid, HATU (1.05 mmol), and DIEA (5 equiv) in DMF (5 mL). After the solution was stirred at 25° C. for 2 h, aqueous HCl (2 mL, 1N) was added. This solution was subjected to preparative HPLC to obtain the pure amide product, which was subsequently characterized by LC-MS and NMR spectroscopy.
Preparative HPLC gave 50 mg of the title compound (72%) from 65 mg starting material (acid). LC-MS: single peak at 254 nm, MH+ calcd. for C21H23FN4O4: 415, obtained: 415. 1H NMR (DMSO-d6, 400 MHz) δ 13.67 (s, 1H), 10.87 (s, 1H), 7.75 (dd, J=2.4 Hz, 9.6 Hz, 1H), 7.70 (s, 1H), 7.56 (t, J=6.0 Hz, 1H), 6.92 (m, 1H), 6.83 (dd, J=4.8 Hz, 8.4 Hz, 1H), 4.53 (t, J=5.6 Hz, 1H), 3.48-3.25 (m, 2H), 3.08 (s, 3H), 2.85 (s, 3H), 2.43 (s, 3H), 2.41 (s, 3H).
Preparative HPLC gave 14 mg of the title compound (18%) from 65 mg starting material (acid). LC-MS: single peak at 254 nm, MH+ calcd. for C23H25FN4O5: 457, obtained: 457. 1H NMR (DMSO-d6, 400 MHz) δ 13.68 (s, 1H), 10.90 (s, 1H), 7.75 (dd, J=2.4 Hz, 9.6 Hz, 1H), 7.71 (s, 1H), 7.60 (t, J=6.0 Hz, 1H), 6.92 (m, 1H), 6.83 (dd, J=4.4 Hz, 8.4 Hz, 1H), 5.2 (b, 1H), 4.51 (t, J=6.0 Hz, 1H), 3.65-3.35 (m, 10H), 2.43 (s, 3H), 2.41 (s, 3H).
Preparative HPLC gave 16 mg of the title compound (18%) from 80 mg starting material (acid). LC-MS: single peak at 254 nm, MH+ calcd. for C21H23FN4O5: 431, obtained: 431. 1H NMR (DMSO-d6, 400 MHz) δ 13.67 (s, 1H), 10.89 (s, 1H), 7.75 (dd, J=2.0 Hz, 9.2 Hz, 1H), 7.70 (s, 1H), 7.55 (t, J=5.6 Hz, 1H), 6.92 (m, 1H), 6.82 (dd, J=4.8 Hz, 8.8 Hz, 1H), 4.51 (t, J=6.0 Hz, 1H), 3.74 (s, 3H), 3.55-3.40 (m, 2H), 3.13 (s, 3H), 2.42 (s, 3H), 2.41 (s, 3H).
A general scheme for synthesizing chiral species of the invention is outline below:
Step 1:
A mixture of 5-fluoro-1,3-dihydroindol-2-one (1.62 g, 10.2 mmol), 5-formyl-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (1.96 g, 10.7 mmol), pyrrolidine (12 drops) and absolute ethanol was heated to reflux for 3 hours. The mixture was cooled to 25° C. and the solids were collected by filtration. The solids were stirred with ethanol (30 mL) at 72° C. for 30 min. The mixture was cooled to 25° C. and the solids were collected again by filtration, washed with ethanol (6 mL), and dried under vacuum overnight to give an orange solid (Z)-5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (3.094 g, 96%). LC-ESIMS observed [M+H]+ 300.95 (calculated for C16H13FN2O3 300.09).
Step 2:
(Z)-5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxylic acid (3.094 g, 10.3 mmol) was suspended in DMF (15 mL), and stirred for 5 minutes. DIEA (2.7 mL, 15.5 mmol) was then added and the mixture was stirred for 10 minutes. HATU (3.91 g, 10.28 mmol) was added and the reaction mixture was stirred at 25° C. for completion. LC/MS detected the completion of the reaction. Most of the DMF was removed and the residue was suspended in ACN and stirred for another 40 minutes. The solid was collected by filtration, washed with ACN, and dried under high vacuum overnight. (Z)-3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl 5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxylate (3.97 g, 92%) was obtained. LC-ESIMS observed [M+H]+ 418.68 (calculated for C21H15FN6O3 418.12).
Step 3:
To (Z)-3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl 5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxylate (1.0 eq) DMF solution was added amine (1.2 eq), the reaction mixture was stirred at 25° C. for 2 h. LC/MS was applied to detect the completion of the reaction. Remove DMF under reduced pressure and the crude was precipitated with 5% diethylamine/methanol (3 mL) under sonication, the solid was collected by filtration and washed with 5% diethylamine/methanol (1 mL) twice.
To the (S)-isoserine (921.6 mg, 8.77 mmol) in methanol (20 mL) was added concentrated HCl (0.5 mL), and the mixture was refluxed overnight. The mixture was cooled to 25° C. and the solvent was removed under reduced pressure. The crude material was dried and used directly in the next step.
To (S)-methyl 3-amino-2-hydroxypropanoate hydrochloride (172.3 mg, 1.11 mmol) DMF solution was added DIEA (0.48 mL, 2.76 mmol) and the mixture was stirred at 25° C. for 20 minutes. (Z)-3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl 5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxylate (174.8 mg, 0.418 mmol) was added, and the mixture was stirred at 25° C. for the completion. The solvent was removed under reduced pressure to afford (S)-3-({5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidene-methyl]-2,4-dimethyl-1H-pyrrole-3-carbonyl}-amino)-2-hydroxypropanoic acid methyl ester (quantitative yield). The product was used in the next step with no purification. LC-ESIMS observed [M+H]+ 401.98 (calculated for C20H20FN3O5 401.15).
(S)-3-({5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carbonyl}-amino)-2-hydroxypropanoic acid methyl ester (167 mg, 0.418 mmol) and LiOH.H2O (36 mg, 0.86 mmol) and methanol/water (10 ml/2 mL) was stirred at 25° C. overnight. Most of the solvent was removed under reduced pressure and excess 1N HCl was added to acidify the mixture. The orange solid was collected by filtration and washed with cold methanol to afford (S)-3-({5-[5-fluoro-2-oxo-1,2-dihydro-indol-(3Z)-ylidenemethyl]-2,4-dimethyl-1H-pyrrole-3-carbonyl}-amino)-2-hydroxypropanoic acid (yield 88%). LCESIMS observed [M+H]+ 387.96 (calculated for C19H18FN3O5 387.12); 1H NMR (400 MHz, DMSO-d6) δ 13.91 (s, 1H), 10.89 (s, 1H), 7.75 (dd, J=9.6 Hz, 2.4 Hz, 1H), 7.70 (s, 1H), 7.57 (t, J=6.2 Hz, 1H), 6.92 (td, J=9.2 Hz, 2.4 Hz, 1H), 6.85-6.82 (m, 1H), 4.17-4.14 (m, 1H), 3.64 (s, 1H), 3.55-3.49 (m, 1H), 3.45-3.39 (m, 1H), 2.43 (s, 3H), 2.41 (s, 3H).
The general procedure for the synthesis of amides: An amine (1.2 equiv) was added to a suspension of the (Z)-3H-[1,2,3]triazolo[4,5-b]pyridin-3-yl 5-((5-fluoro-2-oxoindolin-3-ylidene)methyl)-2,4-dimethyl-1H-pyrrole-3-carboxylate (1.0 eq) in DMF. The mixture was stirred at 25° C. for 2 h and LC/MS was applied to detect the completion of the reaction. The final solution was removed to get the crude solid, which was precipitated in 5% diethylamine/methanol, the solid was collected by filtration and washed with 5% diethylamine/methanol to afford the pure amide product, which was subsequently characterized by LC-MS and NMR spectroscopy.
To the THF/water (50 mL/50 mL) solution of (S)-isoserine (2.429 g, 23.12 mmol) was added K2CO3 (3.834 g, 27.74 mmol) and N-(Benzyloxy-carbonyloxy)-succinimide (5.76 g, 23.11 mmol). The reaction mixture was stirred at 25° C. overnight. The reaction mixture was concentrated and diluted with EtOAc and acidified with excess HCl. The aqueous layer was extracted with EtOAc, and the combined organic layers were washed with dilute HCl, water, brine and dried over sodium sulfate. The solvent was removed under reduced pressure to afford (S)-3-(benzyloxycarbonyl)-2-hydroxypropanoic acid (5.11 g, 92%), which was used in the next step with no further purification. LC-ESIMS observed [M+H]+ 239.91 (calculated for C11H13NO5 239.08).
To (S)-3-(benzyloxycarbonyl)-2-hydroxypropanoic acid (377.8 mg, 1.58 mmol) in DMF (5 mL) was added dimethylamine hydrogen chloride (193.2 mg, 2.37 mmol) and DIEA (0.9 mL, 5.17 mmol). The mixture was then stirred for 5 min and EDC (454.3 mg, 2.37 mmol) and HOBt (320.3 mg, 2.37 mmol) were added. The reaction mixture was stirred at 25° C. overnight. DMF was removed under reduced pressure and the crude material was diluted with EtOAc and washed with saturated NaHCO3. The aqueous layer was extracted twice with EtOAc and the combined organic layers were washed with water, 1N HCl and dried over NaSO4. The solution was concentrated and the crude material was purified by flash chromatography with 0-20% MeOH/DCM to obtain the (S)-benzyl 3-(dimethylamino)-2-hydroxy-3-oxopropylcarbamate (349.2 mg, 83%). LC-ESIMS observed [M+H]+ 266.96 (calculated for C13H18N2O4 266.13).
To the degassed (S)-benzyl 3-(dimethylamino)-2-hydroxy-3-oxopropylcarbamate (256.6 mg, 0.964 mmol) in ethanol (10 mL) was added Pd/C (10%, 30 mg) under argon protection, and then the mixture was degassed. The hydrogen balloon was used to provide the H2 source. The reaction was stirred at 50° C. overnight. The mixture was filtered with Celite 521. The filtrate was evaporated to afford (S)-3-amino-2-hydroxy-N,N-dimethylpropanamide (125.2 mg, 98%). 1H NMR (400 MHz, CDCl3) δ 4.65 (t, J=5.4 Hz, 1H), 3.71-3.59 (m, 2H), 3.07 (s, 3H), 3.04 (s, 3H), 1.94 (broad s, 2H).
The title compound was obtained following the general procedure for amide synthesis (79%). LC-ESIMS observed [M+H]+ 414.97 (calculated for C21H23FN4O4 414.17); 1H NMR (400 MHz, DMSO-d6) δ 13.68 (s, 1H), 10.89 (s, 1H), 7.76 (dd, J=9.6 Hz, 2.4 Hz, 1H), 7.71 (s, 1H), 7.59 (t, J=6.2 Hz, 1H), 6.92 (td, J=9.2 Hz, 2.4 Hz, 1H), 6.85-6.82 (m, 1H), 5.04 (d, J=7.6 Hz, 1H), 4.53 (q, J=6.2 Hz, 1H), 3.47-3.41 (m, 1H), 3.36-3.30 (m, 1H), 3.08 (s, 3H), 2.85 (s, 3H), 2.43 (s, 3H), 2.40 (s, 3H).
Similar method to synthesis of (S)-benzyl 3-(dimethylamino)-2-hydroxy-3-oxopropylcarbamate was applied and the title compound was obtained (yield 86%). LC-ESIMS observed [M+H]+ 408.96 (calculated for C15H20N2O5 308.96).
Similar method to synthesis of (S)-3-amino-2-hydroxy-N,N-dimethylpropanamide was applied and the title compound was obtained (yield 94%). 1H NMR (400 MHz, CDCl3) δ 4.36-4.34 (m, 1H), 3.75-3.54 (m, 8H), 3.50 (d, J=4.0 Hz, 1H), 2.96-2.79 (m, 2H), 1.94 (broad s, 2H).
The title compound was obtained following the general procedure for amide synthesis (75%). LC-ESIMS observed [M+H]+ 457.01 (calculated for C23H25FN4O5 456.18); 1H NMR (400 MHz, DMSO-d6) δ 13.68 (s, 1H), 10.89 (s, 1H), 7.76 (dd, J=9.6 Hz, 2.4 Hz, 1H), 7.71 (s, 1H), 7.59 (t, J=6.2 Hz, 1H), 6.92 (td, J=9.2 Hz, 2.4 Hz, 1H), 6.85-6.82 (m, 1H), 5.18 (d, J=8.8 Hz, 1H), 4.51 (q, J=6.0 Hz, 1H), 3.61-3.51 (m, 6H), 3.49-3.36 (m, 4H), 2.43 (s, 3H), 2.41 (s, 3H).
Sodium azide (5.487 g, 84.39 mmol) and ammonium chloride (2.257 g, 42.2 mmol) were added to a solution of methyl (2R)-glycidate (2.872 g, 28.13 mmol) in methanol (40 mL) and water (2 mL). After refluxing for 10 h, methanol was evaporated. The mixture was diluted in CHCl3, washed with 1N HCl (5 mL) and extracted. After drying over sodium sulfate, the organic phase was concentrated and purified by flash chromatography to give the (R)-methyl 3-azido-2-hydroxypropanoate (2.82 g, 69%). 1H NMR (400 MHz, CDCl3) δ 4.39-4.36 (m, 1H), 3.84 (s, 3H), 3.67-3.48 (m, 2H), 3.18 (d, J=4.0 Hz, 1H).
To a solution of (R)-methyl 3-azido-2-hydroxypropanoate (7.3 g, 50.3 mmol) in MeOH (150 mL) at 0° C. was added 1N NaOH (65 mL, 65 mmol). After being stirred at room temperature for 1 h, the mixture was acidified by 1N HCl and extracted with EtOAc. The organic layers were dried over sodium sulfate and concentrated in vacuo to give the acid as a white solid. The compound was used in the next step with no further purification.
Similar method to synthesis of (S)-benzyl 3-(dimethylamino)-2-hydroxy-3-oxopropylcarbamate was applied and the title compound was obtained (yield 93%). 1H NMR (400 MHz, CDCl3) δ 4.39-4.36 (m, 1H), 3.67-3.48 (m, 2H), 3.18 (d, J=4.0 Hz, 1H), 3.08 (s, 3H), 3.04 (s, 3H).
To the degassed (R)-3-azido-2-hydroxy-N,N-dimethylpropanamide (8.37 g, 46.6 mmol) in ethanol (150 mL) was added Pd/C (10%, 837 mg) under argon protection, and then the mixture was degassed. A hydrogen balloon was used to provide an H2 source. The reaction was stirred at 25° C. for 2 h, and TLC was applied to detect the completion of the reaction. The mixture was filtered with Celite 521. The filtrate was evaporated to afford the desired compound (5.38 g, 87%). 1H NMR (400 MHz, CDCl3) δ 4.65 (t, J=5.4 Hz, 1H), 3.71-3.59 (m, 2H), 3.07 (s, 3H), 3.04 (s, 3H).
The title compound was obtained following the general procedure for amide synthesis (yield 85%), LC-ESIMS observed [M+H]+ 414.97 (calculated for C21H23FN4O4 414.17); 1H NMR (400 MHz, DMSO-d6) δ 13.67 (s, 1H), 10.89 (s, 1H), 7.76 (dd, J=9.6 Hz, 2.4 Hz, 1H), 7.71 (s, 1H), 7.59 (t, J=6.2 Hz, 1H), 6.92 (td, J=9.2 Hz, 2.4 Hz, 1H), 6.85-6.82 (m, 1H), 5.04 (d, J=7.6 Hz, 1H), 4.53 (q, J=6.2 Hz, 1H), 3.47-3.41 (m, 1H), 3.36-3.30 (m, 1H), 3.08 (s, 3H), 2.85 (s, 3H), 2.43 (s, 3H), 2.40 (s, 3H).
Similar method to synthesis of (S)-benzyl 3-(dimethylamino)-2-hydroxy-3-oxopropylcarbamate was applied and the title compound was obtained (yield 90%), 1H NMR (400 MHz, CDCl3) δ 4.55 (t, J=5.2 Hz, 1H), 3.71-3.60 (m, 6H), 3.48-3.41 (m, 3H), 3.40-3.35 (m, 2H).
A similar method to synthesis of (R)-3-amino-2-hydroxy-N,N-dimethylpropanamide was used and the title compound was obtained in high yield (yield 95%). 1H NMR (400 MHz, CDCl3) δ 4.36-4.34 (m, 1H), 3.75-3.54 (m, 8H), 3.50 (d, J=4.0 Hz, 1H), 2.96-2.79 (m, 2H), 1.94 (broad s, 2H).
The title compound was obtained following the general procedure for amide synthesis (yield 75%). LC-ESIMS observed [M+H]+ 457.01 (calculated for C23H25FN4O5 456.18); 1H NMR (400 MHz, DMSO-d6) δ 13.68 (s, 1H), 10.89 (s, 1H), 7.76 (dd, J=9.6 Hz, 2.4 Hz, 1H), 7.71 (s, 1H), 7.59 (t, J=6.2 Hz, 1H), 6.92 (td, J=9.2 Hz, 2.4 Hz, 1H), 6.85-6.82 (m, 1H), 5.18 (d, J=6.4 Hz, 1H), 4.54-4.49 (m, 1H), 3.61-3.51 (m, 6H), 3.49-3.36 (m, 4H), 2.43 (s, 3H), 2.41 (s, 3H).
(R)-methyl 3-azido-2-hydroxypropanoate (505.4 mg, 3.48 mmol) and methylamine ethanol solution (15 mL) was sealed and stirred at 60° C. oil bath overnight. TLC analysis was applied to detect the reaction completion. The solvent was removed and the crude was purified by flash chromatography (0-20% Methanol/DCM) to afford (R)-3-azido-2-hydroxy-N-methylpropan-amide (385.2 mg, yield 77%), 1H NMR (400 MHz, CDCl3) δ 6.90-6.70 (broad s, 1H), 4.28-4.24 (m, 1H), 3.69-3.57 (m, 3H), 2.87 (d, J=5.6 Hz, 3H).
Similar method to synthesis of (R)-3-amino-2-hydroxy-N,N-dimethylpropan-amide was used and the title compound was obtained (yield 98%). 1H NMR (400 MHz, CDCl3) δ 7.05 (broad s, 1H), 3.97 (t, J=5.6 Hz, 1H), 3.12-2.96 (m, 2H), 2.85 (d, J=5.2 Hz, 3H), 1.90 (broad, 2H).
The title compound was obtained following the general procedure for amide synthesis (yield 86%), LC-ESIMS observed [M+H]+ 400.96 (calculated for C20H21FN4O4 400.15); 1H NMR (400 MHz, DMSO-d6) δ 13.69 (s, 1H), 10.89 (s, 1H), 7.87 (d, J=4.8 Hz, 1H), 7.76 (dd, J=9.6 Hz, 2.4 Hz, 1H), 7.71 (s, 1H), 7.52 (t, J=5.6 Hz, 1H), 6.95-6.90 (m, 1H), 6.85-6.82 (m, 1H), 5.83 (d, J=5.2 Hz, 1H), 4.07-4.03 (m, 1H), 3.57-3.51 (m, 1H), 3.37-3.30 (m, 1H), 2.62 (d, J=4.4 Hz, 3H) 2.45 (s, 3H), 2.42 (s, 3H).
The compounds described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. It will be readily apparent to one skilled in the art that varying substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention.
Still further amide examples are shown in the following table:
In the above core structures, R2 is selected from the group consisting of hydrogen and fluoro; and R8 is selected from the group consisting of hydroxyl or radicals represented by the following structures:
In the above table, R8 is selected from the following radicals:
These amide examples 320-515 can be made by those skilled in the art following the above procedure and/or known procedures.
VEGFR Biochemical Assay
The compounds were assayed for biochemical activity by Upstate Ltd at Dundee, United Kingdom, according to the following procedure. In a final reaction volume of 25 μl, KDR (h) (5-10 mU) is incubated with 8 mM MOPS pH 7.0, 0.2 mM EDTA, 0.33 mg/ml myelin basic protein, 10 mM MgAcetate and [γ-33P-ATP] (specific activity approx. 500 cpm/pmol, concentration as required). The reaction is initiated by the addition of the MgATP mix. After incubation for 40 minutes at room temperature, the reaction is stopped by the addition of 5 μl of a 3% phosphoric acid solution. 10 μl of the reaction is then spotted onto a P30 filtermat and washed three times for 5 minutes in 75 mM phosphoric acid and once in methanol prior to drying and scintillation counting.
Cellular Assay: HUVEC: VEGF Induced Proliferation
The compounds were assayed for cellular activity in the VEGF induced proliferation of HUVEC cells. HUVEC cells (Cambrex, CC-2517) were maintained in EGM (Cambrex, CC-3124) at 37° C. and 5% CO2. HUVEC cells were plated at a density 5000 cells/well (96 well plate) in EGM. Following cell attachment (1 hour) the EGM-medium was replaced by EBM (Cambrex, CC-3129)+0.1% FBS (ATTC, 30-2020) and the cells were incubated for 20 hours at 37° C. The medium was replaced by EBM+1% FBS, the compounds were serial diluted in DMSO and added to the cells to a final concentration of 0-5,000 nM and 1% DMSO. Following a 1 hour pre-incubation at 37° C. cells were stimulated with 10 ng/ml VEGF (Sigma, V7259) and incubated for 45 hours at 37° C. Cell proliferation was measured by BrdU DNA incorporation for 4 hours and BrdU label was quantitated by ELISA (Roche kit, 16472229) using 1M H2SO4 to stop the reaction. Absorbance was measured at 450 nm using a reference wavelength at 690 nm.
| Number | Date | Country | |
|---|---|---|---|
| 60754360 | Dec 2005 | US | |
| 60685144 | May 2005 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US04/39752 | Nov 2004 | US |
| Child | 11441537 | May 2006 | US |
